NZ617487B2 - Compositions for inhibiting masp-2 dependent complement activation - Google Patents

Abstract

Disclosed is an isolated human monoclonal antibody, or antigen binding fragment thereof, that binds to human MASP-2 and inhibits MASP-2 dependent complement activation, comprising: (i) a heavy chain variable region comprising three complementary determining regions CDR-H1, CDR-H2 and CDR-H3; and (ii) a light chain variable region comprising three CDRs CDR-L1, CDR-L2 and CDR-L3, wherein; CDR-H1 comprises an amino acid sequence as set forth in RGKMG; CDR-H2 comprises an amino acid sequence as set forth in LAHIFSSDEKSYRTSL; CDR-H3 comprises an amino acid sequence set forth as YYCARIRX wherein X at position 8 is A or R; and wherein CDR-L1 comprises an amino acid sequence as set forth in GXKLGDKXAY, wherein X at position 2 is D or E and wherein X at position 8 is F or Y; CDR-L2 comprises an amino acid sequence as set forth in DXXRPSG, wherein X at position 2 is N or K and wherein X at position 3 is K or Q; and CDR-L3 comprises an amino acid sequence set forth as AWDSSTAVF, and wherein the isolated antibody inhibits MASP-2 dependent complement activation. ii) a light chain variable region comprising three CDRs CDR-L1, CDR-L2 and CDR-L3, wherein; CDR-H1 comprises an amino acid sequence as set forth in RGKMG; CDR-H2 comprises an amino acid sequence as set forth in LAHIFSSDEKSYRTSL; CDR-H3 comprises an amino acid sequence set forth as YYCARIRX wherein X at position 8 is A or R; and wherein CDR-L1 comprises an amino acid sequence as set forth in GXKLGDKXAY, wherein X at position 2 is D or E and wherein X at position 8 is F or Y; CDR-L2 comprises an amino acid sequence as set forth in DXXRPSG, wherein X at position 2 is N or K and wherein X at position 3 is K or Q; and CDR-L3 comprises an amino acid sequence set forth as AWDSSTAVF, and wherein the isolated antibody inhibits MASP-2 dependent complement activation.

Description

COMPOSITIONS FOR INHIBITING MASP-2 DEPENDENT MENT TION FIELD OF THE INVENTION The present invention relates to anti-MASP-2 tory antibodies and compositions comprising such antibodies for use in inhibiting the e effects of MASP-2 dependent complement activation.

CROSS-REFERENCE TO RELATED APPLICATION This application claims the benefit of U.S. Provisional Application No. 61/482,567 filed May 4, 2011, which is incorporated herein by nce in its ty.

NZ 715226 was divided from the t application. The description of the present invention and the invention of NZ 715226 is retained herein for clarity and completeness.

STATEMENT REGARDING SEQUENCE LISTING The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the text file containing the sequence listing is MP_1_0115_PCT_SequenceListingasFiled_20120504_ST25. The text file is 158 KB, was created on May 4, 2012; and is being submitted via b with the filing of the specification.

BACKGROUND The complement system provides an early acting mechanism to initiate, amplify and orchestrate 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 a valuable first-line defense against potential pathogens, the activities of complement that promote a tive immune response can also represent 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 C3 and C5 proteolytic products recruit and activate neutrophils. While indispensable for host defense, activated phils are indiscriminate in their release of destructive enzymes and may cause organ damage. In addition, complement tion may cause the deposition of lytic complement components on nearby host cells as well as on ial targets, resulting in host cell lysis.

The complement system has also been implicated in the pathogenesis of numerous acute and chronic disease states, including: myocardial infarction, stroke, acute respiratory distress syndrome , reperfusion injury, septic shock, capillary leakage following thermal burns, post cardiopulmonary bypass inflammation, transplant rejection, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, and mer's disease. In almost all of these conditions, complement is not the cause but is one of several factors involved in pathogenesis. Nevertheless, complement activation may be a major pathological mechanism and represents an effective point for clinical control in many of these disease states.

The growing recognition of the ance of complement-mediated tissue injury in a variety of disease states underscores the need for effective complement inhibitory drugs. To date, Eculizumab (Soliris®), an antibody against C5, is the only complement- targeting drug that has been approved for human use. Yet, C5 is one of l effector molecules located “downstream” in the complement system, and blockade of C5 does not inhibit activation of the ment system. Therefore, an tor of the initiation steps of ment activation would have significant advantages over a “downstream” ment inhibitor.

Currently, it is widely accepted that the complement system can be activated h three distinct pathways: the classical pathway, the lectin pathway, and the alternative pathway. The classical pathway is usually red by a complex composed of host antibodies bound to a foreign particle (i.e., an antigen) and thus requires prior exposure to an antigen for the generation of a specific antibody response. Since activation of the classical pathway depends on a prior adaptive immune se by the host, the cal pathway is part of the acquired immune system. In contrast, both the lectin and alternative pathways are independent of adaptive immunity and are part of the innate immune system.

The activation of the complement system results in the sequential activation of serine protease zymogens. The first step in activation of the classical pathway is the binding of a specific recognition molecule, Clq, to antigen-bound IgG and IgM complexes. Clqis associated with the Clr and Cls serine protease proenzymes as a complex called Cl. Upon binding of Clq to an immune complex, autoproteolytic cleavage of the Arg-Ile site of Clr is followed by Clr-mediated cleavage and activation of Cls, which thereby es the y to cleave C4 and C2. C4 is cleaved into two fragments, designated C4a and C4b, and, similarly, C2 is cleaved into C2a and C2b. C4b fragments are able to form covalent bonds with adjacent hydroxyl or amino groups and generate the C3 convertase (C4b2a) through noncovalent interaction with the C2a fragment of activated C2. C3 convertase (C4b2a) activates C3 by proteolytic ge into C3a and C3b subcomponents leading to tion of the C5 convertase (C4b2a3b), which, by cleaving C5 leads to the formation of the membrane attack complex (C5b combined with C6, C7, C8 and C9, also referred to as “MAC”) that can disrupt cellular membranes leading to cell lysis. The ted forms of C3 and C4 (C3b and C4b) are covalently deposited on the foreign target surfaces, which are recognized by complement receptors on multiple phagocytes.

Independently, the first step in activation of the complement system through the lectin pathway is also the g of specific ition molecules, which is followed by the activation of associated serine protease proenzymes. However, rather than the g of immune complexes by Clq, the recognition molecules in the lectin pathway se a group of carbohydrate-binding proteins (mannan-binding lectin (MBL), in, M-ficolin, L-ficolin and C-type lectin CL-11), collectively referred to as lectins. See J. Lu eta1., m. Biophys. Acta 1572:387-400, 2002; Holmskov eta1., Annu. Rev. Immunol. 21:547-578 (2003); Teh et al., Immunology [01:225-232 (2000)).

See also J. Luet et al., m s Acta 1572:387-400 (2002); Holmskov et al, Annu Rev Immunol 21:547-578 (2003); Teh et al., Immunology 101:225-232 (2000); Hansen S. et al., J. Immunol 185(10):6096-6104 (2010).

Ikeda et al. first demonstrated that, like C1q, MBL could activate the complement system upon binding to yeast mannan-coated erythrocytes in a C4-dependent manner (Ikeda et al., J. Biol. Chem. 262:7451-7454, 1987). MBL, a member of the collectin protein family, is a calcium-dependent lectin that binds carbohydrates with 3- and oxy groups oriented in the equatorial plane of the pyranose ring. Prominent ligands for MBL are thus D-mannose and N—acetyl-D-glucosamine, while carbohydrates not fitting this steric requirement have ctable affinity for MBL (Weis, W.I., et al., Nature 360:127-134, 1992). The interaction between MBL and monovalent sugars is extremely weak, with dissociation constants typically in the single-digit millimolar range.

MBL achieves tight, specific binding to glycan ligands by avidity, z'.e., by interacting simultaneously with multiple monosaccharide residues located in close proximity to each other (Lee, R.T., et al., Archiv. Biochem. Biophys. 299:129-136, 1992). MBL recognizes the carbohydrate patterns that commonly decorate microorganisms such as ia, yeast, parasites and certain viruses. In contrast, MBL does not recognize D-galactose and sialic acid, the penultimate and ultimate sugars that usually decorate "mature" complex glycoconjugates present on mammalian plasma and cell surface glycoproteins. This binding specificity is thought to promote recognition of “foreign” surfaces and help protect from “self-activation.” However, MBL does bind with high affinity to clusters of annose "precursor" glycans on N—linked glycoproteins and glycolipids sequestered in the endoplasmic reticulum and Golgi of mammalian cells (Maynard, Y., et al., J. Biol.

Chem. 25 3794, 1982). Therefore, damaged cells are potential targets for lectin y activation via MBL binding.

The f1colins possess a different type of lectin domain than MBL, called the f1brinogen-like domain. Ficolins bind sugar residues in a Ca++-independent manner. In humans, three kinds of f1colins (L-ficolin, M-ficolin and H-ficolin), have been fied.

The two serum f1colins, L-ficolin and H-ficolin, have in common a specificity for N—acetyl-D-glucosamine; however, H-ficolin also binds N—acetyl-D-galactosamine. The difference in sugar specificity of L-ficolin, lin, CL-ll and MBL means that the different lectins may be complementary and target different, though pping, glycoconjugates. This concept is supported by the recent report that, of the known s in the lectin pathway, only L-ficolin binds specifically to lipoteichoic acid, a cell wall glycoconjugate found on all ositive bacteria (Lynch, N.J., et al., J. Immunol. 1 8-1202, 2004). The collectins (i.e., MBL) and the f1colins bear no significant similarity in amino acid sequence. r, the two groups of proteins have similar domain organizations and, like Clq, assemble into oligomeric structures, which maximize the possibility of multisite binding.

The serum concentrations of MBL are highly variable in healthy populations and this is genetically controlled by the polymorphism/mutations in both the promoter and coding regions of the MBL gene. As an acute phase protein, the expression of MBL is further upregulated during inflammation. lin is present in serum at concentrations r to those of MBL. Therefore, the L-ficolin branch of the lectin pathway is potentially comparable to the MBL arm in th. MBL and f1colins can also function as opsonins, which allow ytes to target MBL- and f1colin-decorated surfaces (see Jack et a1., J Leukoc Biol., 77(3):328-36 (2004); Matsushita and Fujita, Immunobz'ology, 205(4-5):490-7 (2002); Aoyagi et a1., J Immunol :418-25 (2005). This opsonization requires the interaction of these proteins with phagocyte receptors (Kuhlman, M., et a1., J Exp. Med. 33, 1989; Matsushita, M., et 211., J. Biol.

Chem. 271:2448-54, 1996), the identity of which has not been established.

Human MBL forms a specific and high-affinity interaction through its collagen-like domain with unique Clr/Cls-like serine proteases, termed MBL-associated serine proteases (MASPs). To date, three MASPs have been described. First, a single enzyme "MASP" was identified and characterized as the enzyme responsible for the initiation of the complement e (i.e., ng C2 and C4) (Matsushita M and Fujita T., JExp Med 176(6):1497-1502 (1992), Ji, Y.H., et a1., J. Immunol. [50:571-578, 1993).

It was subsequently determined that the MASP actiVity was, in fact, a mixture of two proteases: MASP-l and MASP-2 (Thiel, S., et a1., Nature 386:506-510, 1997). However, it was demonstrated that the SP-2 complex alone is sufficient for complement tion (Vorup-Jensen, T., et a1., J. Immunol. [65:2093-2100, 2000). Furthermore, only MASP-2 d C2 and C4 at high rates (Ambrus, G., et a1., J. Immunol. 170:1374-1382, 2003). Therefore, MASP-2 is the protease responsible for activating C4 and C2 to generate 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 (Clr and Cls) leads to the activation of the complement system. In addition, a third novel se, MASP-3, has been isolated (Dahl, M.R., et a1., Immunity 15:127-35, 2001). MASP-l and MASP-3 are alternatively spliced products of the same gene.

MASPs share identical domain zations with those of Clr and Cls, the enzymatic components of the C1 complex (Sim, R.B., et a1., Biochem. Soc. Trans. 28:54.5, 2000). These s include an N—terminal Clr/Cls/sea urchin VEGF/bone morphogenic protein (CUB) , an epidermal growth factor-like domain, a second CUB domain, a tandem of complement control protein domains, and a serine protease domain. As in the Cl proteases, activation of MASP-2 occurs through cleavage of an e bond adjacent to the serine protease , which splits the enzyme into disulfide-linked A and B chains, the latter consisting of the serine protease domain.

Recently, a cally determined deficiency of MASP-2 was described (Stengaard-Pedersen, K., et a1., New Eng. J. Med. 349:554-560, 2003). The mutation of a single nucleotide leads to an Asp-Gly exchange in the CUBl domain and renders MASP-2 incapable of g to MBL.

MBL can also associated with an alternatively spliced form of MASP-2, known as MBL-associated protein of 19 kDa (MApl9) (Stover, C.M., J. Immunol. [62:3481-90, 1999) or small MBL-associated protein (sMAP) (Takahashi, M., et al., Int.

Immunol. 11:859-863, 1999), which lacks the catalytic activity of MASP-2. MApl9 comprises the first two domains of MASP-2, followed by an extra sequence of four unique amino acids. The MASP I and MASP 2 genes are located on human chromosomes 3 and 1, respectively eble, W., et al., Immunobz’ology 205:455-466, 2002).

Several lines of evidence suggest that there are different SPs complexes and a large fraction of the MASPs in serum is not complexed with MBL (Thiel, S., et al., J. Immunol. [65:878-887, 2000). Both H— and L-ficolin bind to all MASPs and activate the lectin complement pathway, as does MBL (Dahl, M.R., et al., Immunity 15:127-35, 2001; Matsushita, M., et al., J. Immunol. [68:3502-3506, 2002). Both the lectin and classical pathways form a common C3 convertase (C4b2a) and the two pathways converge at this step.

The lectin y is widely thought to have a major role in host defense against infection in the naive host. Strong evidence for the involvement of MBL in host defense comes from analysis of patients with decreased serum levels of onal MBL trick, D.C., Biochim. s. Acta 1572:401-413, 2002). Such patients y susceptibility to recurrent bacterial and fungal infections. These symptoms are usually evident early in life, during an apparent window of vulnerability as maternally derived antibody titer wanes, but before a full repertoire of antibody responses develops. This syndrome often s from mutations at several sites in the enous portion of MBL, which interfere with proper formation of MBL oligomers. However, since MBL can function as an n independent of complement, it is not known to what extent the increased susceptibility to infection is due to impaired ment activation.

In contrast to the classical and lectin pathways, no initiators of the alternative pathway have been found to fulfill the recognition functions that Clq and lectins perform in the other two pathways. Currently it is widely accepted that the alternative pathway spontaneously undergoes a low level of turnover activation, which can be readily ed on foreign or other abnormal surfaces (bacteria, yeast, virally infected cells, or damaged tissue) that lack the proper molecular elements that keep neous complement activation in check. There are four plasma ns directly involved in the activation of the alternative pathway: C3, s B and D, and din. Although there is extensive evidence implicating both the classical and alternative complement pathways in the pathogenesis of non-infectious human diseases, the role of the lectin y is just beginning to be evaluated. Recent studies provide evidence that activation of the lectin y can be responsible for complement activation and related inflammation in ischemia/reperfusion injury. Collard et al. (2000) reported that cultured endothelial cells ted to oxidative stress bind MBL and show deposition of C3 upon re to human serum (Collard, C.D., et al., Am. J. Pat/ml. 49-1556, 2000). In addition, treatment of human sera with blocking anti-MBL monoclonal antibodies inhibited MBL g and complement activation. These findings were extended to a rat model of myocardial ischemia-reperfilsion in which rats treated with a blocking antibody directed against rat MBL showed significantly less myocardial damage upon occlusion of a coronary artery than rats treated with a control antibody (Jordan, J.E., et al., Circulation 13-1418, 2001). The molecular mechanism of MBL binding to the vascular elium 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, and not to glycoconjugates (Collard, C.D., et al., Am. J. Pathol. [59:1045-1054, 2001). Other studies have implicated the classical and alternative ys 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. [62:363-367, 2003).

A recent study has shown that MASP-l (and possibly also MASP-3) is required to convert the alternative y activation enzyme Factor D from its zymogen form into its enzymatically active form(See Takahashi M. et al., J Exp Med 207(l):29-37 (2010)).

The physiological importance of this process is underlined by the absence of alternative pathway functional ty in plasma of MASP-l/3 deficient mice. Proteolytic generation of C3b from native C3 is required for the alternative pathway to function.

Since the alternative pathway C3 convertase (C3bBb) contains C3b as an essential t, the question regarding the origin of the first C3b Via the alternative pathway has presented a puzzling problem and has stimulated considerable research. 2012/036509 C3 belongs to a family of ns (along with C4 and (x-2 lobulin) that contain a rare posttranslational modification known as a thioester bond. The thioester group is composed of a glutamine whose terminal carbonyl group forms a covalent ter linkage with the sulfhydryl group of a cysteine three amino acids away. This bond is unstable and the ophilic glutamyl-thioester can react with nucleophilic es such as hydroxyl or amino groups and thus form a covalent bond with other molecules. The thioester bond is ably stable when sequestered within a hydrophobic pocket of intact C3. However, lytic cleavage of C3 to C3a and C3b results in exposure of the highly reactive thioester bond on C3b and, following nucleophilic attack by adjacent moieties comprising hydroxyl or amino groups, C3b becomes covalently linked to a target. In on to its well-documented role in covalent attachment of C3b to complement targets, the C3 ter is also thought to have a pivotal role in triggering the alternative pathway. ing to the widely accepted over theory", the alternative pathway is initiated by the tion of a fluid-phase convertase, iC3Bb, which is formed from C3 with hydrolyzed thioester (iC3; C3(H20)) and factor B (Lachmann, P.J., et al., Springer Semin. Immunopathol. 7:143-162, 1984).

The C3b-like C3(H20) is generated from native C3 by a slow spontaneous hydrolysis of the internal thioester in the protein (Pangbum, M.K., et al., J. Exp. Med. [54:856-867, 1981). Through the activity of the C3(H20)Bb tase, C3b molecules are deposited on the target surface, thereby initiating the alternative pathway.

Very little is known about the initiators of activation of the alternative pathway.

Activators are thought to include yeast cell walls (zymosan), many pure polysaccharides, rabbit erythrocytes, certain immunoglobulins, viruses, fungi, ia, animal tumor cells, parasites, and damaged cells. The only feature common to these activators is the presence of carbohydrate, but the complexity and variety of carbohydrate structures has made it difficult to establish the shared molecular determinants which are recognized. It is widely accepted that alternative pathway activation is controlled through the fine balance between inhibitory regulatory components of this pathway, such as Factor H, Factor I, DAF, CR1 and properdin, which is the only positive regulator of the alternative pathway. See Schwaeble W.J. and Reid K.B., Immunol Today 20(1):l7-21 (1999)).

In addition to the apparently unregulated activation mechanism described above, the alternative pathway can also provide a powerful amplification loop for the lectin/classical pathway C3 convertase (C4b2a) since any C3b generated can participate with factor B in forming additional alternative pathway C3 convertase (C3bBb). The alternative pathway C3 convertase is stabilized by the binding of properdin. Properdin extends the alternative pathway C3 convertase half-life six to ten fold. Addition of C3b to the alternative pathway C3 tase leads to the formation of the alternative pathway C5 convertase.

All three pathways (i.e., the cal, lectin and alternative) have been thought to converge at C5, which is cleaved to form products with multiple proinflammatory effects.

The ged pathway has been referred to as the terminal ment pathway. C5a is the most potent anaphylatoxin, inducing alterations in smooth muscle and vascular tone, as well as vascular permeability. It is also a powerful chemotaxin and activator of both neutrophils and monocytes. CSa-mediated cellular activation can significantly amplify inflammatory responses by inducing the release of le additional inflammatory mediators, including cytokines, hydrolytic enzymes, arachidonic acid metabolites and reactive oxygen species. C5 cleavage leads to the formation of C5b-9, also known as the membrane attack complex (MAC). There is now strong eVidence that sublytic MAC tion may play an important role in ation in addition to its role as a lytic pore-forming complex.

In addition to its essential role in immune e, the complement system contributes to tissue damage in many al conditions. Thus, there is a pressing need to develop therapeutically effective complement inhibitors to prevent these adverse effects.

SUMMARY This summary is ed to introduce a selection of ts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features of the d t matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

In one aspect, the invention provides an isolated human monoclonal antibody, or antigen binding fragment thereof, that binds to human MASP-2, comprising:(i) a heavy chain le region comprising CDR-Hl, CDR-H2 and CDR—H3 sequences; and (ii) a light chain variable region comprising CDR—Ll, CDR-L2 and CDR—L3, wherein the heavy chain variable region CDR—H3 ce comprises an amino acid sequence set forth as SEQ ID NO:38 or SEQ ID NO:90, and conservative sequence modifications thereof, wherein the light chain variable region CDR-L3 sequence comprises an amino acid sequence set forth as SEQ ID NO:51 or SEQ ID NO:94, and conservative sequence modifications thereof, and wherein the isolated dy inhibits MASP-2 dependent complement activation.

In another aspect, the present invention provides a human antibody that binds human MASP-2, wherein the dy comprises: I) a) a heavy chain variable region comprising: i) a heavy chain CDR-Hl comprising the amino acid sequence from 31-35 of SEQ ID NO:21; and ii) a heavy chain CDR-H2 comprising the amino acid sequence from 50-65 of SEQ ID NO:21; and iii) a heavy chain CDR-H3 comprising the amino acid ce from 95-102 of SEQ ID NO:21; and b) a light chain variable region comprising: i) a light chain CDR-Ll comprising the amino acid ce from 24-34 of either SEQ ID NO:25 or SEQ ID NO:27; and ii) a light chain CDR-L2 comprising the amino acid sequence from 50-56 of either SEQ ID NO:25 or SEQ ID NO:27; and iii) a light chain CDR-L3 comprising the amino acid ce from 89-97 of either SEQ ID NO:25 or SEQ ID NO:27; or II) a variant thereof that is otherwise identical to said variable domains, except for up to a combined total of 10 amino acid substitutions within said CDR regions of said heavy chain variable region and up to a combined total of 10 amino acid substitutions within said CDR regions of said light chain variable region, wherein the dy or variant f inhibits MASP-2 dependent complement activation.

In another aspect, the t invention provides an isolated human monoclonal antibody, or antigen binding fragment thereof, that binds human MASP-2, wherein the antibody comprises: I) a) a heavy chain variable region comprising: i) a heavy chain CDR-Hl comprising the amino acid sequence from 31-35 of SEQ ID NO:20; and ii) a heavy chain CDR-H2 comprising the amino acid sequence from 50-65 of SEQ ID NO:20; and iii) a heavy chain CDR-H3 comprising the amino acid sequence from 95-102 of either SEQ ID NO:18 or SEQ ID NO:20; and b) a light chain le region comprising: i) a light chain CDR-Ll comprising the amino acid sequence from 24-34 of either SEQ ID NO:22 or SEQ ID NO:24; and ii) a light chain CDR-L2 comprising the amino acid sequence from 50-56 of either SEQ ID NO:22 or SEQ ID NO:24; and iii) a light chain CDR-L3 comprising the amino acid sequence from 89-97 of either SEQ ID NO:22 or SEQ ID N024; or II) a variant thereof, that is otherwise identical to said variable domains, except for up to a combined total of 10 amino acid substitutions within said CDR regions of said heavy chain and up to a combined total of 10 amino acid substitutions within said CDR s of said light chain le region, wherein the antibody or variant thereof inhibits MASP-2 ent complement activation.

In r aspect, the present invention provides an isolated monoclonal antibody, or antigen-binding nt thereof, that binds to human MASP-2, comprising a heavy chain variable region comprising any one of the amino acid sequences set forth in SEQ ID NO:18, SEQ ID NO:20 or SEQ ID NO:21.

In another aspect, the t invention provides an isolated monoclonal antibody, or antigen-binding fragment thereof, that binds to human MASP-2, comprising a light chain variable region comprising an one of the amino acid sequences set forth in SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25 or SEQ ID NO:27.

In another aspect, the present invention provides nucleic acid molecules encoding the amino acid sequences of the anti-MASP-2 dies, or fragments thereof, of the present invention, such as those set forth in TABLE 2.

In another aspect, the present invention es a cell comprising at least one of the nucleic acid molecules encoding the amino acid sequences of the anti-MASP-2 antibodies, or fragments thereof, of the present invention, such as those set forth in TABLE 2.

In another aspect, the invention provides a method of generating an isolated MASP-Z antibody sing ing cells comprising at least one of the nucleic acid molecules ng the amino acid sequences of the anti-MASP-2 antibodies of the present invention under conditions ng for expression of the nucleic acid molecules encoding the anti-MASP-2 antibody and isolating said anti-MASP-2 antibody.

In another aspect, the invention provides an ed fully human monoclonal antibody or antigen-binding fragment thereof that dissociates from human MASP-2 with a KD of lOnM or less as determined by surface plasmon resonance and inhibits C4 activation on a mannan-coated substrate with an IC50 of lOnM or less in 1% serum. In some embodiments, said antibody or n g fragment thereof specifically recognizes at least part of an epitope recognized by a reference antibody, wherein said reference antibody comprises a heavy chain variable region as set forth in SEQ ID N020 and a light chain variable region as set forth in SEQ ID NO:24.

In r aspect, the present invention provides compositions comprising the fully human monoclonal anti-MASP-2 antibodies of the invention and a pharmaceutically acceptable excipient.

In another aspect, the present invention provides methods of ting MASP-2 dependent complement activation in a human subject comprising administering a human monoclonal antibody of the invention in an amount sufficient to inhibit MASP-Z dependent ment activation in said human subject.

In another aspect, the present invention provides an article of manufacture comprising a unit dose of human monoclonal MASP-Z antibody of the invention suitable for therapeutic stration to a human t, wherein the unit dose is the range of from lmg to lOOOmg.

DESCRIPTION OF THE DRAWINGS The foregoing aspects and many of the attendant advantages of this invention will become more y appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein: FIGURE 1A is a m illustrating the genomic structure of human MASP-2; FIGURE 1B is a diagram illustrating the domain structure of human MASP-2 protein; FIGURE 2 cally illustrates the results of an ELISA assay carried out on polyclonal populations ed from a schv phage library panned against various MASP-Z antigens, as described in Example 2; FIGURE 3A and 3B show results of testing of 45 candidate scFv clones for fianctional activity in the complement assay, as described in Example 3; FIGURE 4 graphically rates the results of an ment that was carried out to compare C3c levels in the three sera , rat and NHP), as described in e 4; FIGURE 5A is an amino acid sequence alignment of the heavy chain region (residues l-120) of the most active clones reveals two distinct groups belonging to VH2 and VH6 gene family, respectively, as described in Example 4; FIGURE 5B is an amino acid sequence alignment of the scFv clones l7D20, l7Nl6, l8Ll6 and 4D9, as described in Example 4; FIGURE 6 graphically illustrates the inhibitory activities of ations of IgG4 converted mother clones in a C3b deposition assay using 90% human plasma, as described in Example 5; FIGURE 7A graphically illustrates the s of the ELISA assay on the l7Nl6 mother clone versus daughter clones titrated on huMASPZA, as described in Example 6; FIGURE 7B graphically rates the results of the ELISA assay on the l7D20 mother clone versus er clones titrated on huMASPZA, as described in Example 6; FIGURE 8 is a protein sequence ent of the mother clone l7Nl6 and the l7N9 daughter clone showing that the light chains (starting with SYE) has 17 amino acid residues that differ between the two clones, as described in Example 6; FIGURE 9 is a protein sequence alignment of the CDR—H3 region of the sequences of the Clones #35, #59 and #90 ing from mutagenesis in comparison with the l7D20 mother clone, as described in e 7; FIGURE 10A is a protein sequence alignment of the CDR3 region of the l7D20 mother clone with the chain shuffled clone l7D20md21Nll and the nsis clone #35 CDR—H3 clone shown in FIGURE 9 combined with the VL of l7D20md21Nll (VH35-VL21Nl l), as described in Example 7; FIGURE 10B is a protein sequence alignment of the VL and VH regions of the l7D20 mother clone and the daughter clone l7D20md21Nl l, as described in Example 7; FIGURE llA graphically illustrates the results of the C3b deposition assay carried out for the daughter clone isotype variants (MoAb#l-3), derived from the human anti-MASP-2 monoclonal antibody mother clone l7Nl6, as described in Example 8; FIGURE 11B graphically illustrates the results of the C3b deposition assay carried out for the daughter clone isotype variants (MoAb#4-6), derived from the human anti-MASP-2 monoclonal antibody mother clone l7D20, as described in Example 8; 2012/036509 FIGURE 12A and 12B graphically illustrate the testing of the mother clones and MoAb#l-6 in a C3b deposition assay in 95% serum, as described in Example 8; FIGURE 13 graphically illustrates the inhibition of C4b deposition in 95% normal human serum, as described in e 8; FIGURE 14 graphically illustrates the tion of C3b deposition in 95% African Green monkey serum, as bed in Example 8; FIGURE 15 graphically illustrates the inhibition of C4 cleavage activity of pre- assembled MBL-MASP2 complex by MoAb#2-6, as described in e 8; FIGURE 16 graphically illustrates the preferential binding of MoAb#6 to human MASP2 as compared to Cls, as described in e 8; FIGURE 17 graphically illustrates that the lectin pathway was completely inhibited following intravenous administration of anti-human MoAb#OMS646 into African Green Monkeys, as described in Example 10; FIGURE 18A is a Kaplan-Meier survival plot showing the percent survival over time after exposure to 7.0 Gy radiation in control mice and in mice treated with anti- murine MASP-2 antibody (mAle 1) or anti-human MASP-2 antibody S646) as described in Example 11; FIGURE 18B is a Kaplan-Meier survival plot showing the percent survival over time after exposure to 6.5 Gy radiation in control mice and in mice treated with anti- murine MASP-2 antibody (mAle 1) or anti-human MASP-2 antibody (mAbOMS646), as described in Example 11; FIGURE 18C is a Kaplan-Meier survival plot showing the percent survival over time after exposure to 8.0 Gy radiation in control mice and in mice d with anti- human MASP-2 antibody (mAbOMS646), as described in e 11; FIGURE 19 graphically illustrates the results of surface plasmon resonance re) analysis on anti-MASP-2 antibody OMS646 (response units (binding) versus time in seconds), showing that immobilized OMS646 binds to recombinant MASP-2 with a Koff rate of about l-3x10'4S'1 and a K011 rate of about 1.6-3xlO6M'1S'1, as described in Example 12; FIGURE 20 graphically illustrates the s of an ELISA assay to determine the binding affinity of anti-MASP-2 antibody OMS646 to immobilized human MASP-2, g that OMS646 binds to immobilized recombinant human MASP-2 with a KD of imately 100 pM, as described in Example 12; FIGURE 21A graphically illustrates the level of C4 activation on a mannan- coated surface in the presence or absence of anti-MASP-2 antibody 6), demonstrating that OMS646 ts C4 activation on a mannan-coated e with an IC50 of approximately 0.5 nM in 1% human serum, as bed in Example 12; FIGURE 21B graphically illustrates the level of C4 activation on an IgG-coated surface in the presence or absence of anti-MASP-2 antibody 6), showing that OMS646 does not inhibit classical pathway-dependent activation of complement component C4, as described in Example 12; FIGURE 22A graphically illustrates the level of MAC tion in the presence or absence of anti-MASP-2 antibody 6) under lectin pathway-specific assay conditions, demonstrating that OMS646 inhibits lectin-mediated MAC deposition with an IC50 value of approximately 1 nM, as described in Example 12; FIGURE 22B graphically illustrates the level of MAC deposition in the presence or absence of anti-MASP-2 antibody (OMS646) under classical pathway-specific assay conditions, demonstrating that OMS646 does not inhibit classical pathway-mediated MAC deposition, as described in e 12; FIGURE 22C graphically rates the level of MAC deposition in the presence or absence of anti-MASP-2 dy (OMS646) under alternative pathway-specific assay ions, demonstrating that OMS646 does not inhibit alternative pathway-mediated MAC deposition, as bed in Example 12; FIGURE 23A graphically illustrates the level of C3 deposition in the presence or absence of anti-MASP-2 antibody (OMS646) over a range of concentrations in 90% human serum under lectin pathway-specific conditions, demonstrating that OMS646 blocks C3 deposition under physiological conditions, as described in Example 12; FIGURE 23B graphically illustrates the level of C4 deposition in the presence or absence of anti-MASP-2 antibody (OMS646) over a range of concentrations in 90% human serum under lectin pathway-specific conditions, demonstrating that OMS646 blocks C4 deposition under physiological ions, as described in Example 12; FIGURE 24A graphically illustrates the level of C4 deposition in the absence or presence of anti-MASP-2 antibody (OMS646) in 90% Cynomuglus monkey serum under lectin pathway-specific conditions, demonstrating that OMS646 inhibits lectin pathway C4 deposition in Cynomuglus monkey serum in a dose-responsive manner with IC50 values in the range of 30 to SOnM, as described in Example 12; and FIGURE 24B graphically illustrates the level of C4 deposition in the absence or presence of anti-MASP-2 antibody (OMS646) in 90% African Green monkey serum under lectin pathway-specific conditions, demonstrating that OMS646 inhibits lectin pathway C4 deposition in African Green monkey serum in a esponsive manner with IC50 values in the range of 15 to 30 nM, as described in Example 12.

DESCRIPTION OF THE SEQUENCE G SEQ ID NO:1 human MASP-2 cDNA SEQ ID NO:2 human MASP-2 protein (with leader) SEQ ID NO:3 human MASP-2 protein (mature) SEQ ID NO:4 rat MASP-2 cDNA SEQ ID NO:5 rat MASP-2 protein (with leader) SEQ ID NO:6 rat MASP-2 protein (mature) ANTIGENS (in reference to human MASP-Z mature protein) SEQ ID \O:7 CUBI domain of human MASP-2 (aa 1-121) SEQ ID \O:8 CUBI/EGF domains of human MASP-2 (aa 1-166) SEQ ID \O:9 CUBI/EGF/CUBII domains of human MASP-2 (aa 1-277) SEQ ID \O: 10 EGF domain of human MASP-2 (aa 122-166) SEQ ID \O:11 CPII/SP domains of human MASP-2 (aa 278-671) SEQ ID \O: 12 CCPI/CCPII domains of human MASP-2 (aa 278-429) SEQ ID \O: 13 CCPI domain of human MASP-2 (aa 278-347) SEQ ID \O: 14 CCPII/SP domain of human MASP-2 (aa348-671) SEQ ID \O: 15 CCPII domain of human MASP-2 (aa 348-429) SEQ ID \O: 16 SP domain of human MASP-2 (aa 429-671) SEQ ID NO: 17: Serine-protease inactivated mutant (aa 610-625 with mutated Ser 618) ANTI-MASP-2 MONOCLONAL ANTIBODIES VH chains SEQ ID NO:18 17D20mc heavy chain le region (VH) polypeptide SEQ ID NO: 19 DNA encoding 17D20_dc35VH21N11VL 6) heavy chain le region (VH) (without signal peptide) SEQ ID NO:20 17D20_dc35VH21N11VL (OMS646) heavy chain variable region (VH) polypeptide SEQ ID NO:21 17N16mc heavy chain variable region (VH) polypeptide -l6- ANTI-MASP-Z MONOCLONAL ANTIBODIES VL chains SEQ ID NO:22 l7D20rnc light chain variable region (VL) polypeptide SEQ ID NO:23 DNA encoding l7D20_dc21NllVL 4) light chain variable region (VL) (Without signal peptide) SEQ ID NO:24 l7D20_dc21NllVL (OMS644) light chain variable region (VL) ptide SEQ ID NO:25 l7Nl6rnc light chain variable region (VL) ptide SEQ ID NO:26 DNA encoding l7Nl6_dcl7N9 (OMS64l) light chain variable region (VL) (Without signal peptide) SEQ ID NO:27 dcl7N9 (OMS64l) light chain variable region (VL) polypeptide ANTI-MASP-Z ONAL ANTIBODIES HEAVY CHAIN CDRS SEQ ID NOS:28-3l CDR-Hl SEQ ID NOS:32-35 CDR—H2 SEQ ID NOS:36-40 CDR—H3 ASP-Z MONOCLONAL ANTIBODIES LIGHT CHAIN CDRS SEQ ID NOS:4l-45 CDR—Ll SEQ ID NOS:46-50 CDR—L2 SEQ ID NOS:51-54 CDR-L3 MASP-Z antibody Sequences SEQ ID \O:55: scFv rnother clone 17D20 fiJll length polypeptide SEQ ID \O:56: scFv rnother clone 18Ll6 fiJll length polypeptide SEQ ID \O:57: scFv rnother clone 4D9 full length polypeptide SEQ ID \O:58: scFv rnother clone 17L20 fiJll length polypeptide SEQ ID \O:59: scFv rnother clone l7Nl6 fiJll length polypeptide SEQ ID \O:60: scFv rnother clone 3F22 full length polypeptide SEQ ID \O:6l: scFv rnother clone 9P13 full length polypeptide SEQ ID \02622 DNA encoding Wild type IgG4 heavy chain constant region SEQ ID \02632 Wild type IgG4 heavy chain constant region polypeptide SEQ ID NO:64 DNA encoding IgG4 heavy chain constant region with mutant S228P SEQ ID NO:65: IgG4 heavy chain constant region with mutant S228P polypeptide SEQ ID NO:66: scFv daughter clone l7Nl6n1_dl7N9 full length polypeptide SEQ ID NO:67: scFv daughter clone l7D20n1_d21Nll full length polypeptide SEQ ID NO:68: scFv daughter clone 1_d3521Nll full length polypeptide SEQ ID NO:69: DNA encoding wild type IgG2 heavy chain constant region SEQ ID NO:70: wild type IgG2 heavy chain nt region polypeptide SEQ ID NO:7l: l7Nl6rn_dl7N9 light chain gene sequence (with signal peptide encoded by nt 1-57)) SEQ ID NO:72: l7Nl6rn_dl7N9 light chain n sequence (with signal peptide aal-l9) SEQ ID NO:73: l7Nl6rn_dl7N9 IgG2 heavy chain gene sequence (with signal peptide encoded by nt 1-57) SEQ ID NO:74: l7Nl6rn_dl7N9 IgG2 heavy chain protein sequence (with signal peptide aa l-l9) SEQ ID NO:75: l7Nl6rn_dl7N9 IgG4 heavy chain gene sequence (with signal peptide encoded by nt 1-57) SEQ ID NO:76: n_dl7N9 IgG4 heavy chain protein sequence (with signal peptide aa l-l9) SEQ ID NO:77: l7Nl6n1_dl7N9 IgG4 mutated heavy chain gene sequence (with signal peptide encoded by nt 1-57) SEQ ID NO:78: l7Nl7n1_dl7N9 IgG4 mutated heavy chain protein sequence (with signal peptide aa l-l9) SEQ ID NO:79: 17D20_3521N11 light chain gene sequence (with signal e encodedbyrule57) SEQ ID NO:80: 17D20_3521N11 light chain protein sequence (with signal peptide aa l-l9) SEQ ID NO:81: 17D20_3521N11 IgG2 heavy chain gene sequence (with signal peptide encoded by nt 1-57) SEQ ID NO:82: 17D20_3521N11 IgG2 heavy chain protein sequence (with signal peptide aa l-l9) SEQ ID NO:83: 17D20_3521N11 IgG4 heavy chain gene sequence (with signal peptide d by nt 1-57) SEQ ID NO:84: 17D20_3521N11 IgG4 heavy chain n sequence (with signal peptide aa 1-19) SEQ ID NO:85: 3521N11 IgG4 mutated heavy chain gene sequence (with signal peptide encoded by nt 1-57) SEQ ID NO:86: 17D20_3521N11 IgG4 mutated heavy chain protein sequence (with signal peptide aa l-l9) SEQ ID NO:87: scFv daughter clone _dl7N9 DNA encoding full length polypeptide (without signal peptide) SEQ ID NO:88: scFv daughter clone l7D20m_d21Nll DNA encoding full length polypeptide (without signal peptide) SEQ ID NO:89: scFv daughter clone _d3521Nll DNA encoding fiall length polypeptide (without signal peptide) SEQ ID NO:90: consensus heavy chain CDR—H3 of l7D20m and d3521Nll SEQ ID NO:9l: consensus light chain CDR—Ll of l7D20m and d3521Nll SEQ ID NO:92: consensus light chain CDR-Ll of l7Nl6m and dl7N9 SEQ ID NO:93: consensus light chain CDR-L2 of l7D20m, d3521Nl l, l7Nl6m and dl7N9 SEQ ID NO:94: consensus light chain CDR-L3 of l7Nl6m and dl7N9 DETAILED DESCRIPTION The present invention provides fully human antibodies that bind to human MASP- 2 and inhibit lectin-mediated complement activation while leaving the classical (Clq- dependent) pathway component of the immune system intact. The human anti-MASP-2 antibodies have been identified by screening a phage display library, as bed in Examples 2-9. As bed in Examples 10-12, high y anti-MASP-2 dies have been identified with the ability to inhibit lectin-mediated ment activation, as demonstrated in both in vitro assays and in vivo. The variable light and heavy chain fragments of the antibodies have been isolated in both a scFv format and in a filll length IgG format. The human anti-MASP-2 antibodies are useful for inhibiting cellular injury associated with lectin-mediated complement y activation while leaving the classical (Clq-dependent) pathway component of the immune system intact. 1. DEFINITIONS Unless specifically defined herein, all terms used herein have the same meaning as would be understood by those of ordinary skill in the art of the present invention. The following definitions are provided in order to provide clarity with respect to the terms as they are used in the specification and claims to describe the present invention.

As used herein, the term "MASP-Z-dependent complement activation" comprises MASP-Z-dependent activation of the lectin y, which occurs under physiological conditions (i.e., in the presence of Ca++) g to the formation of the lectin pathway C3 convertase C4b2a and upon accumulation of the C3 cleavage product C3b subsequently to the C5 tase C4b2a(C3b)n.

As used herein, the term "alternative pathway" refers to complement activation that is triggered, for example, by zymosan from filngal and yeast cell walls, lysaccharide (LPS) from Gram negative outer membranes, and rabbit erythrocytes, as well as from many pure polysaccharides, rabbit erythrocytes, viruses, bacteria, animal tumor cells, parasites and damaged cells, and which has traditionally been thought to arise from spontaneous proteolytic generation of C3b from complement factor C3.

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

As used herein, the term ical pathway" refers to complement activation that is triggered by an antibody bound to a foreign particle and es binding of the recognition molecule C l q.

As used , the term "MASP-Z inhibitory antibody" refers to any ASP- 2 antibody, or MASP-2 binding fragment thereof, that binds to or ly interacts with MASP-2 and effectively inhibits MASP-Z-dependent complement activation. MASP-2 inhibitory antibodies useful in the method of the invention may reduce MASP-Z-dependent complement activation by greater than 20%, such as greater than %, or r than 40%, or greater than 50%, or greater than 60%, or greater than 70%, or greater than 80%, or greater than 90%, or greater than 95%.

As used herein, the term Z blocking antibody" refers to MASP-2 inhibitory antibodies that reduce MASP-Z-dependent complement activation by r than 90%, such as greater than 95%, or greater than 98% (i.e., resulting in MASP-2 complement activation of only 10%, such as only 9%, or only 8%, or only 7%, or only 6%, such as only 5% or less, or only 4%, or only 4%, or only 3% or only 2% or only 1%).

The terms "antibody" and oglobulin" are used interchangeably herein.

These terms are well understood by those in the field, and refer to a protein consisting of one or more polypeptides that specifically binds an antigen. One form of antibody constitutes the basic structural unit of an antibody. This form is a tetramer and consists of two identical pairs of antibody chains, each pair having one light and one heavy chain. In each pair, the light and heavy chain variable regions are together responsible for binding to an antigen, and the constant s are responsible for the antibody effector fianctions.

As used herein, the term "antibody" encompasses antibodies and antibody fragments thereof, derived from any antibody-producing mammal (e.g., mouse, rat, rabbit, and primate including human), or from a hybridoma, phage selection, inant expression or transgenic animals (or other methods of producing antibodies or antibody fragments), that specifically bind to MASP-2 polypeptides or portions thereof. It is not intended that the term “antibody” be limited as s to the source of the antibody or manner in which it is made (e.g., by hybridoma, phage selection, inant expression, transgenic animal, peptide synthesis, etc). Exemplary antibodies include polyclonal, monoclonal and recombinant antibodies; multispecific antibodies (e. g., bispecific dies); zed antibodies; murine antibodies; chimeric, mouse-human, primate, primate-human monoclonal antibodies; and anti-idiotype antibodies, and may be any intact molecule or fragment thereof As used herein, the term “antibody” encompasses not only intact polyclonal or monoclonal dies, but also fragments thereof (such as dAb, Fab, Fab', F(ab')2, Fv), single chain (ScFv), synthetic variants thereof, naturally occurring variants, fusion proteins comprising an antibody portion with an n-binding fragment of the required city, humanized antibodies, chimeric antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen-binding site or fragment (epitope recognition site) of the required specificity.

As used herein, the term "antigen-binding fragmen " refers to a polypeptide fragment that contains at least one CDR of an globulin heavy and/or light chains that binds to human MASP-Z. In this , an n-binding fragment of the herein bed antibodies may se l, 2, 3, 4, 5, or all 6 CDRs of a VH and VL sequence set forth herein from antibodies that bind MASP-Z. An antigen-binding fragment of the herein described MASP-Z-specific antibodies is capable of binding to MASP-2. In certain embodiments, an antigen-binding fragment or an dy comprising an antigen- binding fragment, mediates inhibition of MASP-2 dependent complement activation.

As used herein the term "anti-MASP-2 monoclonal antibodies" refers to a homogenous antibody population, wherein the onal antibody is comprised of amino acids that are involved in the selecting binding of an epitope on MASP-2. Anti- MASP-2 monoclonal antibodies are highly specific for the MASP-2 target antigen. The term "monoclonal antibody" encompasses not only intact monoclonal antibodies and full- length onal antibodies, but also fragments f (such as Fab, Fab', F(ab')2, Fv), single chain (ScFv), ts thereof, fusion proteins comprising an antigen-binding portion, zed monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen- binding fragment (epitope recognition site) of the required specificity and the ability to bind to an epitope.

As used herein, the er "monoclonal" indicates the character of the antibody as being obtained from a substantially homogenous population of antibodies, and is not. intended to be limited as regards the source of the antibody or the manner in which it is made (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term includes whole immunoglobulins as well as the fragments etc. described above under the definition of ody". Monoclonal antibodies can be obtained using any technique that provides for the production of antibody molecules by uous cell lines in e, such as the hybridoma method described by Kohler, G., et al., Nature 256:495, 1975, or they may be made by recombinant DNA methods (see, e.g., US. Patent No. 4,816,567 to Cabilly). Monoclonal dies may also be isolated from phage antibody libraries using the techniques described in Clackson, T., et al., Nature 352:624-628, 1991, and Marks, J.D., et al., J. M01. Biol. 222:581-597, 1991. Such antibodies can be of any immunoglobulin class ing IgG, IgM, IgE, IgA, IgD and any subclass thereof.

The ized immunoglobulin polypeptides include the kappa and lambda light chains and the alpha, gamma (IgGl, IgG2, IgG3, IgG4), delta, epsilon and mu heavy chains or equivalents in other species. Full-length globulin "light chains" (of about 25 kDa or about 214 amino acids) comprise a variable region of about 110 amino acids at the NHZ-terminus and a kappa or lambda constant region at the COOH-terminus.

WO 51481 Full-length immunoglobulin "heavy chains" (of about 50 kDa or about 446 amino acids) similarly comprise a variable region (of about 116 amino acids) and one of the aforementioned heavy chain constant regions, e. g., gamma (of about 330 amino acids).

The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. An IgM antibody consists of 5 of the basic heterotetramer units along with an additional polypeptide called the J chain, and therefore ns 10 antigen binding sites. Secreted IgA dies can polymerize to form lent assemblages comprising 2-5 of the basic 4-chain units along with J chain. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more by one or more de bonds, depending on the H chain e. Each H and L chain also has regularly spaced intrachain disulfide bridges. The pairing of a VH and VL together forms a single antigen- binding site.

Each H chain has at the N—terminus, a variable domain (VH), followed by three constant domains (CH) for each of the or and y chains, and four CH domains (CH) for u and s isotypes.

Each L chain has at the N—terminus, a variable domain (VL) followed by a constant domain (CL) at its other end. The VL is aligned with the VH and the CL is d with the first constant domain of the heavy chain (CHl). The L chain from any vertebrate s can be assigned to one of two clearly distinct types, called kappa (K) and lambda (9»), based on the amino acid sequences of their constant domains (CL).

Depending on the amino acid sequence of the constant domain of their heavy chains (CH), globulins can be assigned to different classes or isotypes. There are five s of immunoglobulins: IgA, IgD, IgE, IgG and IgM, haVing heavy chains designated alpha (or), delta (8), epsilon (a), gamma (y) and mu (u), respectively. The y and or classes are fiarther divided into subclasses on the basis of minor differences in CH sequence and function, for example, humans express the following subclasses: IgGl, IgG2, IgG3, IgG4, IgAl and IgA2.

For the structure and properties of the different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th Edition, Daniel P. , Abba I. Terr and Tristram G. Parslow (eds); Appleton and Lange, Norwalk, Conn., 1994, page 71 and Chapter 6.

The term "variable" refers to that fact that certain segments of the V domains differ extensively in sequence among antibodies. The V domain mediates antigen binding and defines city of a particular antibody for its particular antigen.

However, the variability is not evenly distributed across the 110 amino acid span of the variable domains. Rather, the V s consist of relatively invariant stretches called framework s (FRs) of 15-30 amino acids separated by shorter s of extreme variability called "hypervariable regions" that are each 9-12 amino acids long. The variable domains of native heavy and light chains each comprise four FRs, largely adopting a beta-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases g part of, the n-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat, et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of , Bethesda, Md (1991)). The constant s are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC).

As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region generally comprises amino acid residues from a ementary determining region" or "CDR" (i.e., from around about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain, and around about 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain when numbering in accordance with the Kabat numbering system as described in Kabat, et al., Sequences ofProteins ofImmunological Interest, 5th Ed. Public Health Service, National utes of Health, Bethesda, Md (1991)); and/or those residues from a "hypervariable loop" (i.e., residues 24-34 (L1), 50- 56 (L2) and 89-97 (L3) in the light chain variable domain, and 26-32 (H1), 52-56 (H2) and 95-101 (H3) in the heavy chain le domain when numbered in ance with the Chothia numbering system, as described in Chothia and Lesk, J. Mol. Biol. [96:901- 917 ); and/or those residues from a "hypervariable loop"/CDR (e. g., residues 27-38 (L1), 56-65 (L2) and 105-120 (L3) in the VL, and 27-38 (H1), 56-65 (H2), and 105-120 (H3) in the VH when ed in accordance with the IMGT numbering system as described in Lefranc, JP, et al., Nucleic Acids Res 27:209-212; Ruiz, M., et al., Nucleic Acids Res 28:219-221 (2000)).

As used herein, the term "antibody fragment" refers to a portion derived from or related to a fiJll-length anti-MASP-Z antibody, generally including the antigen binding or variable region thereof. Illustrative examples of antibody fragments include Fab, Fab', F(ab)2, F(ab')2 and Fv fragments, scFv nts, diabodies, linear antibodies, single-chain antibody molecules, bispecif1c and multispecific dies formed from antibody fragments.

Where bispecif1c antibodies are to be used, these may be conventional bispecific antibodies, which can be manufactured in a variety of ways (Holliger, P. and Winter G. t Opinion Biotechnol. 4, 446-449 (1993)), e.g. prepared chemically or from hybrid hybridomas, or may be any of the bispecific dy fragments mentioned above.

As used herein, a "single-chain Fv" or "scFv" antibody fragment comprises the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide fiarther comprises a ptide linker between the VH and VL domains, which enables the scFv to form the d structure for antigen binding. See Pluckthun in The Pharmacology of Monoclonal dies, Vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 5 (1994).

"Fv" is the minimum antibody fragment that ns a complete antigen-recognition and binding site. This fragment consists of a dimer of one heavy and one light chain le region domain in tight, non-covalent association. From the folding of these two domains e six hypervariable loops (three loops each from the H and L chain) that contribute the amino acid residues for n binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to ize and bind n, although at a lower affinity than the entire binding site.

As used herein, the term "specific binding" refers to the ability of an antibody to preferentially bind to a particular analyte that is present in a homogeneous mixture of different analytes. In certain embodiments, a specific binding interaction will discriminate between desirable and undesirable analytes in a sample, in some embodiments more than about 10 to 100-fold or more (e.g., more than about 1000- or ,000-fold). In certain embodiments, the affinity between a capture agent and analyte when they are specifically bound in a capture agent/analyte complex is characterized by a KD (dissociation constant) of less than about 100 nM, or less than about 50 nM, or less than about 25 nM, or less than about 10 nM, or less than about 5 nM, or less than about 1 As used herein, the term "variant" anti-MASP-2 antibody refers to a molecule which differs in amino acid sequence from a "parent" or reference antibody amino acid sequence by virtue of addition, deletion, and/or tution of one or more amino acid residue(s) in the parent antibody ce. In one embodiment, a variant anti-MASP-2 dy refers to a molecule which contains variable regions that are identical to the parent variable s, except for a combined total of l, 2, 3, 4, 5, 6, 7, 8 9 or 10 amino acid tutions within the CDR regions of the heavy chain variable region, and/or up to a combined total of l, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions with said CDR regions of the light chain variable region. In some embodiments, the amino acid substitutions are conservative sequence modifications.

As used herein, the term "parent antibody" refers to an dy which is encoded by an amino acid sequence used for the preparation of the variant. Preferably, the parent antibody has a human framework region and, if present, has human antibody constant region(s). For example, the parent antibody may be a humanized or fillly human antibody.

As used herein, the term "isolated antibody" refers to an antibody that has been identified and separated and/or recovered from a component of its natural environment.

Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, es, and other proteinaceous or teinaceous solutes. In red embodiments, the dy will be purified (l) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N—terminal or internal amino acid sequence by use of a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within inant cells since at least one component of the antibody's natural environment will not be present.

Ordinarily, r, isolated antibody will be prepared by at least one purification step.

As used herein, the term "epitope" refers to the portion of an antigen to which a monoclonal dy specifically binds. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have c three ional structural teristics, as well as specific charge characteristics. More specifically, the term "MASP-Z e," as used herein refers to a portion of the corresponding polypeptide to which an antibody immunospecifically binds as determined by any method well known in the art, for example, by immunoassays. Antigenic epitopes need not necessarily be genic.

Such epitopes can be linear in nature or can be a discontinuous epitope. Thus, as used herein, the term "conformational epitope" refers to a discontinuous epitope formed by a spatial relationship between amino acids of an antigen other than an unbroken series of amino acids.

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

As used herein, the "membrane attack complex" ("MAC") refers to a complex of the terminal five ment components (C5-C9) that inserts into and ts membranes. Also referred to as C5b-9.

As used herein, a t" includes all mammals, including t limitation, humans, non-human primates, dogs, cats, horses, sheep, goats, cows, rabbits, pigs and rodents.

As used herein, the amino acid residues are abbreviated as follows: alanine (Ala;A), asparagine (AsngN), aspartic acid (Asp;D), arginine (Arg;R), ne (CysgC), glutamic acid (Glu;E), glutamine ), glycine (Gly;G), histidine ), isoleucine (Ile;I), leucine (Leu;L), lysine (Lys;K), nine (Meth), phenylalanine (PhegF), proline (ProgP), serine (Ser;S), threonine (ThrgT), tryptophan (Trp;W), tyrosine (TyrgY), and valine (ValgV).

In the broadest sense, the naturally occurring amino acids can be divided into groups based upon the chemical characteristic of the side chain of the respective amino acids. By "hydrophobic" amino acid is meant either Ile, Leu, Met, Phe, Trp, Tyr, Val, Ala, Cys or Pro. By "hydrophilic" amino acid is meant either Gly, Asn, Gln, Ser, Thr, Asp, Glu, Lys, Arg or His. This grouping of amino acids can be fiarther subclassed as follows. By "uncharged hydrophilic" amino acid is meant either Ser, Thr, Asn or Gln.

By "acidic" amino acid is meant either Glu or Asp. By "basic" amino acid is meant either Lys, Arg or His.

As used herein the term "conservative amino acid substitution" is illustrated by a substitution among 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, ne and histidine.

As used herein, an "isolated nucleic acid molecule" is a c acid molecule (e.g., a polynucleotide) that is not integrated in the genomic DNA of an organism. For example, a DNA molecule that encodes a growth factor that has been separated from the genomic DNA of a cell is an isolated DNA molecule. Another example of an isolated c acid molecule is a chemically-synthesized c acid molecule that is not integrated in the genome of an organism. A nucleic acid molecule that has been isolated from a particular species is r than the complete DNA molecule of a chromosome from that species.

As used herein, a "nucleic acid molecule construct" is a nucleic acid molecule, either single- or double-stranded, that has been modified through human intervention to contain segments of nucleic acid combined and juxtaposed in an arrangement not existing in nature.

As used herein, an ssion vector" is a nucleic acid molecule encoding a gene that is expressed in a host cell. lly, an expression vector comprises a transcription promoter, a gene, and a transcription terminator. Gene expression is usually placed under the control of a er, and such a gene is said to be "operably linked to" the promoter.

Similarly, a regulatory element and a core promoter are operably linked if the regulatory element modulates the activity of the core promoter.

As used herein, the terms "approximately" or "about" in reference to a number are generally taken to include numbers that fall within a range of 5% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context t where such number would exceed 100% of a possible value).

Where ranges are , the endpoints are included within the range unless otherwise stated or otherwise evident from the context.

As used herein the singular forms "a", "an" and "the" include plural s unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a single cell, as well as two or more cells; reference to "an agen " includes one agent, as well as two or more agents; reference to "an antibody" includes a plurality of such antibodies and nce to "a framework region" includes nce to one or more framework regions and lents thereof known to those skilled in the art, and so forth.

Each embodiment in this specification is to be applied mutatis mutandis to every other ment unless expressly stated ise.

Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection).

Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. These and related techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present cation. See e.g., Sambrook et al., 2001, MOLECULAR CLONING: A LABORATORY MANUAL, 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., NY, NY); Current Protocols in Immunology (Edited by: John E.

Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober 2001 John Wiley & Sons, NY, NY); or other relevant Current Protocol publications and other like nces. Unless specific ions are provided, the nomenclature ed in tion with, and the laboratory procedures and techniques of, molecular biology, analytical chemistry, synthetic organic chemistry, and medicinal and ceutical chemistry described herein are those well known and commonly used in the art. Standard ques may be used for inant technology, molecular biological, microbiological, chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention. 11. Overview Lectins (MBL, M-ficolin, H-ficolin, L-ficolin and CL-ll) are the specific recognition molecules that trigger the innate complement system and the system es the lectin initiation y and the associated al pathway cation loop that amplifies lectin-initiated activation of terminal complement effector molecules. Clq is the specific ition molecule that triggers the acquired complement system and the system includes the classical initiation pathway and associated terminal pathway amplification loop that amplifies C1q-initiated activation of terminal ment effector molecules. We refer to these two major complement activation systems as the lectin-dependent complement system and the pendent complement system, respectively.

In addition to its essential role in immune defense, the complement system contributes to tissue damage in many clinical conditions. Thus, there is a ng need to p therapeutically effective complement inhibitors to prevent these adverse effects.

As described in US. Patent No. 7,919,094, co-pending US. Patent Application Serial No. 12/905,972 (published as US 2011/0091450), and co-pending US. Patent Application Serial No. 13/083,441 (published as US2011/0311549), each of which is assigned to Omeros Corporation, the assignee of the instant application, and each of which is hereby incorporated by reference, it was determined h the use of a MASP/- mouse model that it is possible to t the lectin mediated MASP-2 pathway while leaving the classical pathway intact. With the recognition that it is possible to inhibit the lectin mediated MASP-2 pathway while leaving the classical pathway intact comes the realization that it would be highly desirable to specifically inhibit only the complement activation system causing a particular pathology t completely shutting down the immune defense capabilities of complement. For example, in e states in which complement activation is mediated inantly by the lectin-dependent complement system, it would be advantageous to specifically inhibit only this system. This would leave the C1q-dependent complement activation system intact to handle immune complex processing and to aid in host defense against infection.

The preferred protein component to target in the pment of therapeutic agents to specifically inhibit the -dependent complement system is MASP-2. Of all the known protein components of the lectin-dependent ment system (MBL, H-ficolin, M-ficolin, L-ficolin, MASP-2, C2-C9, Factor B, Factor D, and din), only MASP-2 is both unique to the lectin-dependent ment system and required for the system to filnction. The 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 one of the lectin components would not necessarily inhibit activation of the system due to lectin redundancy. It would be necessary to inhibit all five lectins in order to guarantee inhibition of the -dependent complement activation system. Furthermore, since MBL and the ficolins are also known to have opsonic activity independent of complement, tion of lectin filnction would result in the loss of this beneficial host defense mechanism against ion. In contrast, this complement-independent lectin opsonic actiVity would remain intact if MASP-2 was the inhibitory target. An added benefit of MASP-2 as the therapeutic target to inhibit the lectin-dependent complement tion system is that the plasma concentration of MASP-2 is among the lowest of any complement protein (z 500 ng/ml); therefore, correspondingly low trations of high-affinity inhibitors of MASP-2 is sufficient to obtain full inhibition, as demonstrated in the Examples herein.

In accordance with the foregoing, as described , the present invention provides monoclonal fillly human anti-MASP-Z antibodies that bind to human MASP-2 with high affinity and are capable of inhibiting lectin-mediated ment pathway activation.

III. MASP-2 INHIBITORY ANTIBODIES In one aspect, the invention provides a monoclonal fully human anti-MASP-2 antibody, or antigen binding fragment thereof, that specifically binds to human MASP-2 and inhibits or blocks MASP-Z-dependent ment activation. MASP-2 tory antibodies may effectively inhibit or effectively block the MASP-Z-dependent complement activation system by inhibiting or blocking the biological function of MASP-2. For example, an inhibitory antibody may ively inhibit or block MASP-2 protein-to-protein interactions, interfere with MASP-2 dimerization or assembly, block Ca2+ binding, or interfere with the MASP-2 serine protease active site.

MASP-2 Epitopes The invention provides fillly human antibodies that specifically bind to human . The MASP-2 polypeptide exhibits a molecular structure similar to MASP-l, MASP-3, and Clr and Cls, the proteases of the Cl complement . The cDNA molecule set forth in SEQ ID NO:1 encodes a representative example of MASP-2 sting of the amino acid sequence set forth in SEQ ID N02) and provides the human MASP-2 polypeptide with a leader sequence (aa l-lS) that is cleaved after ion, resulting in the mature form of human MASP-2 (SEQ ID NO:3). As shown in FIGURE 1A, the human MASP 2 gene encompasses twelve exons. The human MASP-2 cDNA is encoded by exons B, C, D, F, G, H, I, J, K and L. The cDNA molecule set forth in SEQ ID NO:4 encodes the rat MASP-2 (consisting of the amino acid sequence set forth in SEQ ID NO:5) and provides the rat MASP-2 polypeptide with a leader sequence that is cleaved after ion, resulting in the mature form of rat MASP-2 (SEQ ID NO:6).

Those d in the art will recognize that the sequences disclosed in SEQ ID NO:1 and SEQ ID NO:4 represent single alleles of human and rat MASP-2, respectively, and that allelic variation and alternative splicing are expected to occur. c variants of the nucleotide sequences shown in SEQ ID NO:1 and SEQ ID NO:4, including those ning silent mutations and those in which mutations result in amino acid sequence s, are within the scope of the present invention. Allelic variants of the MASP-2 sequence can be cloned by probing cDNA or genomic ies from different individuals according to standard procedures.

The domains of the human MASP-2 protein (SEQ ID NO:3) are shown in FIGURE 1B and TABLE 1 below, and include an N—terminal Clr/Cls/sea urchin VEGF/bone morphogenic protein (CUBI) domain, an epidermal growth -like domain, a second CUB domain (CUBII), as well as a tandem of complement control protein domains CCPl and CCP2, and a serine protease domain. Alternative splicing of the MASP-2 gene results in MApl9. MApl9 is a nonenzymatic protein containing the inal CUBl-EGF region of MASP-2 with four additional residues (EQSL).

Several proteins have been shown to bind to, or interact with MASP-2 through protein-to-protein interactions. For example, MASP-2 is known to bind to, and form Ca2+ dependent complexes with, the lectin proteins MBL, H-ficolin and L-ficolin. Each MASP-2/lectin complex has been shown to activate complement through the MASPdependent cleavage of proteins C4 and C2 (Ikeda, K., et al., J. Biol.

Chem. 262:7451-7454, 1987; hita, M., et al., J. Exp. Med. [76:1497-2284, 2000; Matsushita, M., et al., J. l. [68:3502-3506, 2002). Studies have shown that the CUBl-EGF domains of MASP-2 are ial for the association of MASP-2 with MBL (Thielens, N.M., et al., J. Immunol. [66:5068, 2001). It has also been shown that the CUBlEGFCUBII s mediate dimerization of MASP-2, which is required for formation of an active MBL complex (Wallis, R., et al., J. Biol. Chem. 275:30962-30969, 2000). Therefore, MASP-2 inhibitory antibodies can be identified that bind to or interfere with MASP-2 target regions known to be important for MASPdependent complement activation.

TABLE 1: MASP-2 Pol Hetide Domains aa 1-121 ofSEQ ID NO:3 aa1-166 ofSEQ ID NO:3 SEQ ID NO:9 CUBI/EGF/CUBII domains of human MASP-Z aa 1-277 of SEQ ID NO:3 SEQ ID NO:10 EGF domain of human MASP-2 aa122-166 of SEQ ID NO:3 SEQ ID NO:11 CCPI/CCPII/SP domains of human MASP-Z aa 278-671 aa of SEQ ID NO:3 aa 278-429 of SEQ ID NO:3 aa 278-347 of SEQ ID NO:3 aa 348-671 of SEQ ID NO:3 aa 348-429 of SEQ ID NO:3 aa 429-671 of SEQ ID NO:3 SEQ ID NO:17 Serine-protease inactivated mutant form (GKDSCRGDAGGALVFL) (aa 610-625 of SEQ ID N03 with mutated In one embodiment, the anti-MASP-2 inhibitory dies of the invention bind to a portion of the full length human MASP-2 protein (SEQ ID NO:3), such as CUBI, EGF, CUBII, CCPI, CCPII, or SP domain of MASP-Z. In some embodiments, the anti- MASP-Z inhibitory antibodies of the invention bind to an epitope in the CCPl domain (SEQ ID NO:l3 (aa 278-347 of SEQ ID . For example, anti-MASP-2 antibodies (e.g., OMS646) have been identified that only bind to MASP-2 fragments containing the CCPl domain and inhibit MASP-Z dependent complement activation, as described in Example 9.

Binding y -2 Inhibitory Antibodies The ASP-2 inhibitory antibodies specifically bind to human MASP-2 (set forth as SEQ ID NO:3, encoded by SEQ ID NO:1), with an affinity of at least ten times greater than to other antigens in the ment system. In some embodiments, the MASP-2 tory antibodies specifically bind to human MASP-2 with a binding affinity of at least 100 times greater than to other antigens in the ment system.

In some embodiments, the MASP-Z inhibitory antibodies specifically bind to human MASP-2 with a KD (dissociation constant) of less than about 100 nM, or less than about 50 nM, or less than about 25 nM, or less than about 10 nM, or less than about 5 nM, or less than or equal to about 1 nM, or less than or equal to 0.lnM. The binding y of the MASP-Z inhibitory antibodies can be determined using a suitable binding assay known in the art, such as an ELISA assay, as described in Examples 3-5 herein.

Potency ofMASP-2 Inhibitory Antibodies In one embodiment, a MASP-2 inhibitory antibody is sufficiently potent to inhibit MASP-Z ent complement activation at an IC50 S 30 nM, preferably less than or about 20 nM, or less than about 10 nM or less than about 5 nM, or less than or equal to about3nh4,orlessthan(n?equalu)aboutl,nhdvvhenrneasuredin,l96serunr In one embodiment, a MASP-2 inhibitory dy is sufficiently potent to inhibit MASP-Z dependent complement activation at an IC50 S 30 nM, preferably less than or about 20 nM, or less than about 10 nM or less than about 5 nM, or less than or equal to about 3nM, or less than or equal to about 1 nM, when measured in 90% serum.

The inhibition of MASP-Z-dependent complement activation is characterized by at least one of the following changes in a component of the ment system that occurs as a result of administration of a MASP-2 inhibitory antibody: the inhibition of the generation or production of MASP-Z-dependent complement activation system products C4a, C3a, C5a and/or C5b-9 (MAC) (measured, for example, as described in Example 2 of US Patent No. 7,919,094) as well as their catabolic degradation products (e.g., 2012/036509 C3desArg), the reduction of C4 ge and C4b deposition (measured, for example, as described in e 5) and its subsequent lic ation products (e.g., C4bc or C4d), or the reduction of C3 cleavage and C3b deposition (measured, for example, as described in Example 5), or its subsequent catabolic degradation products (e.g., C3bc, C3d, etc).

In some embodiments, the MASP-2 inhibitory antibodies of the invention are capable of inhibiting C3 deposition in full serum to less than 80%, such as less than 70%, such as less than 60%, such as less than 50%, such as less than 40%, such as less than %, such as less than 20%, such as less than 15%, such as less than 10% of control C3 deposition.

In some embodiments, the MASP-2 inhibitory antibodies of the invention are capable of ting C4 deposition in full serum to less than 80%, such as less than 70%, such as less than 60%, such as less than 50%, such as less than 40%, such as less than %, such as less than 20%, such as less than 15%, such as less than 10% of control C4 deposition.

In some embodiments, the anti-MASP-Z inhibitory dies selectively inhibit MASP-Z complement activation (z'.e., bind to MASP-2 with at least 100-fold or greater affinity than to C1r or Cls), leaving the pendent complement activation system functionally intact (i.e., at least 80%, or at least 90%, or at least 95%, or at least 98%, or 100% of the classical pathway activity is retained).

In some embodiments, the subject anti-MASP-2 inhibitory antibodies have the following characteristics: (a) high affinity for human MASP-Z (e.g., a KD of 10 nM or less, preferably a KD of 1nM or less), and (b) inhibit MASP-2 ent complement activity in 90% human serum with an IC50 of 30 nM or less, preferably an IC50 of 10nM or less).

As described in Examples 2-12, fillly human antibodies have been identified that bind with high affinity to MASP-2 and inhibit lectin-mediated complement activation while leaving the classical (Clq-dependent) pathway component of the immune system intact. The variable light and heavy chain fragments of the antibodies have been sequenced, ed and analyzed in both a scFv format and in a fill length IgG format.

FIGURE 5A is an amino acid sequence alignment of seven scFv anti-MASP-2 clones that were fied as having high binding affinity to MASP-Z and the ability to inhibit MASP-Z dependent activity. FIGURE 5B is an amino acid sequence alignment of four of the scFv mother clones 17D20, 17N16, 18Ll6 and 4D9, showing the framework regions and the CDR regions. The scFv mother clones 17D20 and l7Nl6 were subjected to affinity maturation, leading to the generation of daughter clones with higher affinity and increased potency as compared to the mother clones, as described in Examples 6 and 7.

The amino acid sequences of the heavy chain variable regions (VH) (aa l-120) and the light chain variable regions (VL) (aa 148-250) of the scFv clones shown in S 5A and 5B and the resulting daughter clones, is provided below in TABLE 2.

Substitutable positions of a human anti-MASP-2 inhibitory antibody, as well the choice of amino acids that may be substituted into those positions, are revealed by aligning the heavy and light chain amino acid ces of the anti-MASP-2 inhibitory antibodies discussed above, and determining which amino acids occur at which positions of those dies. In one exemplary ment, the heavy and light chain amino acid sequences of FIGURES 5A and 5B are aligned, and the identity of amino acids at each position of the exemplary antibodies is determined. As illustrated in FIGURES 5A and 5B (illustrating the amino acids t at each position of the heavy and light chains of the exemplary MASP-2 inhibitory antibodies), several tutable positions, as well as the amino acid residues that can be tuted into those positions, are readily identified.

In another exemplary embodiment, the light chain amino acid sequences of the mother and daughter clones are aligned and the identity of amino acids at each position of the exemplary antibodies is determined in order to determine substitutable positions, as well as the amino acid residues that can be substituted into these positions.

TABLE 2: es of re resentative anti-MASP-2 antibodies 17D20 mother clone SEQ ID SEQ ID NO:22 NO: 18 ID Reference: mother/daughter VH 17D20_35VH- daughter clone SEQ ID SEQ ID NO: 24 IgG2 21N11VL NO:20 (10 aa changes from parent (OMS644) (one aa change in VH (A to VL) R) at position 102 of SEQ ID NO: 1 8) 17D20_35VH- er clone SEQ ID SEQ ID NO: 24 21N11VL NO:20 (OMS645) (one aa change in VH (A to R) at position 102 of SEQ ID 17D20_35VH- daughter clone SEQ ID SEQ ID NO: 24 IgG4 (mutant 21N11VL NO:20 IgG4 hinge region) (OMS646) (one aa change in VH (A to R) at on 102 of SEQ ID SEQ ID NO:21, 17N16_17N9 daughter SEQ ID NO:27 IgG2 (OMS641) (17aa changes from SEQ ID l7N l 6_l 7N9 daughter SEQ ID SEQ ID NO:27 IgG4 NO:21 OMS642 17N16_17N9 daughter SEQ ID NO:27 IgG4 (mutant IgG4 hinge (OMS643) In n embodiments, a t human anti-MASP-2 monoclonal inhibitory antibody has a heavy chain variable domain that is substantially identical (e.g., at least about 70%, at least 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 96% cal, or at least about 97% identical, or at least about 98% identical, or at least 99% cal), to that of any of the heavy chain variable domain sequences set forth in TABLE 2.

In some ments, a subject human anti-MASP-2 monoclonal inhibitory antibody has a heavy chain variable domain that is substantially identical (e.g., at least about 70%, at least 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 96% identical, or at least about 97% identical, or at least about 98% identical, or at least 99% identical) to l7D20 (VH), set forth as SEQ ID NO:l8. In some ments, the subject human ASP-2 onal inhibitory antibody has a heavy chain variable domain that comprises, or consists of SEQ ID NO:l8.

In some embodiments, a subject human anti-MASP-2 monoclonal inhibitory antibody has a heavy chain variable domain that is substantially identical (eg. at least about 70%, at least 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least 99% identical) to l7D20_cd35VH2Nll (VH), set forth as SEQ ID NO:20. In some ments, the subject human anti-MASP-2 monoclonal tory antibody has a heavy chain variable domain that comprises, or consists of SEQ ID NO:20.

In some embodiments, a subject human anti-MASP-2 monoclonal inhibitory antibody has a heavy chain variable domain that is substantially identical (e.g., at least about 70%, at least 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 96% identical, or at least about 97% identical, or at least about 98% identical, or at least 99% identical) to l7Nl6 (VH), set forth as SEQ ID NO:21. In some embodiments, the subject human anti-MASP-2 monoclonal inhibitory antibody has a heavy chain variable domain that comprises, or consists of SEQ ID NO:21.

In some embodiments, a subject human anti-MASP-2 monoclonal inhibitory antibody has a light chain variable domain that is substantially identical (e.g., at least about 70%, at least 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 96% cal, or at least about 97% identical, or at least about 98% identical, or at least 99% identical), to that of any of the light chain variable domain sequences set forth in TABLE 2.

In some embodiments, a subject human anti-MASP-2 monoclonal inhibitory antibody has a light chain variable domain that is substantially identical (e.g., at least about7096,atleam:7596,atleastabout8096,atleam:about8596,atleam:about9096,at leastabout9596,oratleastabout9696idenfical,oratleastabout9796idenfical,oratleast about 98% cal, or at least 99% identical) to 17D20 (VL), set forth as SEQ ID NO:22. In some embodiments, the subject human anti-MASP-2 monoclonal inhibitory antibody has a light chain that comprises, or consists of SEQ ID NO:22.

In some embodiments, a subject human anti-MASP-2 monoclonal inhibitory antibody has a light chain variable domain that is substantially identical (e.g., at least about7096,atleam:7596,atleastabout8096,atleam:about8596,atleam:about9096,at leastabout9596,oratleastabout9696idenfical,oratleastabout9796idenfical,oratleast about 98% identical, or at least 99% identical) to l7D20_35VH-21N11VL (VL), set forth as SEQ ID NO:24. In some embodiments, the subject human anti-MASP-2 monoclonal inhibitory antibody has a light chain that ses, or consists of SEQ ID NO:24.

In some embodiments, a subject human anti-MASP-2 monoclonal inhibitory antibody has a light chain variable domain that is ntially identical (e.g., at least 096,atleam:7596,atleastabout8096,atleam:about8596,atleam:about9096,at leastabout9596,oratleastabout9696idenfical,oratleastabout9796idenfical,oratleast about 98% cal, or at least 99% identical) to l7Nl6 (VL), set forth as SEQ ID NO:25. In some embodiments, the subject human anti-MASP-2 monoclonal inhibitory antibody has a light chain that ses, or consists of SEQ ID NO:25.

In some embodiments, a subject human anti-MASP-2 monoclonal inhibitory antibody has a light chain le domain that is substantially identical (e.g., at least about7096,atleam:7596,atleastabout8096,atleam:about8596,atleam:about9096,at leastabout9596,oratleastabout9696idenfical,oratleastabout9796idenfical,oratleast about 98% identical, or at least 99% identical) to l7Nl6_l7N9 (VL), set forth as SEQ ID NO:27. In some embodiments, the t human anti-MASP-2 monoclonal inhibitory antibody has a light chain that ses, or consists of SEQ ID NO:27.

In some embodiments, the anti-MASP-2 antibodies of the invention contain a heavy or light chain that is encoded by a nucleotide sequence that hybridizes under high stringency conditions to a nucleotide sequence encoding a heavy or light chain, as set forth in TABLE 2. High stringency conditions include incubation at 50°C or higher in 0.1XSSC (15 mM saline/0.15mM sodium citrate).

In some embodiments, the anti-MASP-2 inhibitory antibodies of the invention have a heavy chain le region comprising one or more CDRs (CDRl, CDR2 and/or CDR3) that are substantially identical (e.g., at least about 70%, at least 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 96% identical, or at least about 97% identical, or at least about 98% cal, or at least 99% identical), or comprise or consist of the identical sequence as compared to the amino acid sequence of the CDRs of any of the heavy chain variable sequences shown in FIGURES 5A or 5B, or described below in TABLES 3A-F and TABLE 4.

In some embodiments, the anti-MASP-2 inhibitory antibodies of the invention have a light chain variable region comprising one or more CDRs (CDRl, CDR2 and/or CDR3) that are substantially identical (e.g., at least about 70%, at least 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 96% identical, or at least about 97% identical, or at least about 98% cal, or at least 99% identical), or se or consist of the identical sequence as compared to the amino acid sequence of the CDRs of any of the light chain variable sequences shown in FIGURES 5A or 5B, or bed below in TABLES 4A-F and TABLE 5.

Chain Variable Re ion 17D20m 17N16m (SEQ:21) d17N9 (SEQ:21) -IIIII mm II chain aa 21--- 17D20m (SEQ:18) d3521N11 (SEQ:20) 17N16m (SEQ:21) d17N9 (SEQ:21) 17D20m 17N16m (SEQ:21) d17N9 (SEQ:21) chain -IIIII IIIIIII aa maul-m (SEQ:18) lIIIIIIIIII (SEQ:20) lIlInlIIIIIIIIII 17mm Y ml...“- -IIIIIIIIIIIIIIIIIIII d17N9N SRITIN TSKN (SEQ:21) TABLE 3E'Hea chain aa 81-100 Heavy CDR-H3 chain aa II--_ 17D20mVLTMTNM PVDTATXXQAE; (SEQ:18) d3521N11V L T M T N M (SEQ:20) 17N16m (SEQ:21) d17N9 17D20m (SEQ: 19) d3521N11 IDYWG SS (SEQz20) 17N16m FGVPF IWG GTMVTVS (SEQz21) d17N9 FGVPF IWG GTMVTVS (SEQz21) Presented below are the heavy chain variable region (VH) sequences for the mother clones and daughter clones listed above in TABLE 2 and TABLES 3A-F.

The Kabat CDRs (31-35 (Hl), 50-65 (H2) and 95-102 (H3)) are bolded; and the Chothia CDRs (26-32 (Hl), 52-56 (H2) and 95-101 (H3)) are underlined. 17D20 hea chain variable re ion VH SE ID NO:18 : QVTLKESGPVLVKPTETLTLTCTVSGFSLSRGKMGVSWIRQPPGKALEWL AHIFSSDEKSYRTSLKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCARIRAGG GTLVTVSS 17D20 35VH-21N11VL hea chainvariable re ion VH SE ID NO:20 QVTLKESGPVLVKPTETLTLTCTVSGFSLSRGKMGVSWIRQPPGKALEWL AHIFSSDEKSYRTSLKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCARIRRGG IDYWGQGTLVTVSS 17N16 hea chain le re ion VH SE ID NO:21 QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSTSAAWNWIRQSPSRGLEWL GRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARDPF GVPFDIWGQGTMVTVSS TABLE 4: Hea Chain CDRs RGKMG 28 RGKMG 28 STSAA 29 STSAA 29 GFSLSRG 20 GFSLSRG 3o LAHIFSSDEKSYRTSL 32 LAHIFSSDEKSYRTSL 32 LGRTYYRSKWYNDYAV 33 LGRTYYRSKWYNDYAV 33 ms 34 Clone nce CDR aa Se uence SEQ ID NO: d3521N11 CDR-H2 chothia HIFSS 34 17N16rn CDR-H2 chothia RTYYR 35 d17N9 CDR-H2 chothia RTYYR 35 17D20m CDR-H3 kabat YYCARIRA 36 d3521N11 CDR-H3 (kabat) YYCARIRR 37 17D20m and CDR-H3 (kabat) RX 90 d3521N11 (wherein X at position 8 is consensus A Ala orR Ar CDR-H3 kabat CDR-H3 kabat CDR-H3 chothia CDR-H3 chotma CDR-H3 chothia CDR-H3 chothia Li ht Chain Variable Re ions TABLE 5A: Li_ht chain aa 1-20 Light chain aa "anal-13 16 17D20m (SEQ:22) “III.” d3521N11 (SEQ:24) “III.” 17N16m (SEQ:25) d17N9 (SEQ:27) TABLE 5B: Light chain (aa 21-40) Light chain 17D20m (SEQ:22) d3521N11 (SEQ:24) 17N16m (SEQ:25) d17N9 (SEQ:27) 17D20m 17N16m (SEQ:25) d17N9 Light chain 17D20m iifliiiEiT (SEQ:22) d3521N11 T (SEQ:24) Light chain aa EEEMEEHMEEHEEEEEHEEM 17N16m (SEQ:25) d17N9 (SEQ:27)I Light chain w Hwflflflflflkflflflflflflflflw IIIA — — —IX (SEQ:22) (SEQ:24) IIIII III— 17N16m (SEQ:25) III: (SEQ:27) TABLE 5F: L15 0 am (SEQz22) lIIlIIlII (SEQz24) 17N16m (SEQz25) d17N9 (SEQz27) Presented below are the light chain variable region (VL) sequences for the mother clones and daughter clones listed above in TABLE 2 and TABLES 5A-F.

The Kabat CDRs (24-34 (Ll); 50-56 (L2); and 89-97 (L3) are bolded; and the Chothia CDRs (24-34 (Ll); 50-56 (L2) and 89-97 (L3) are underlined. These regions are the same whether numbered by the Kabat or Chothia system. l7D20m light chain variable region (VL) (SEQ ID NO:22( QPVLTQPPSVSVSPGQTASITCSGDKLGDKFAYWYQQKPGHSPVLVIYQQ NKRPSGIPGRFSGSNSGNTATLTISGTQAMDEADYYCQAWDSSTAVFGTGTKVT l7D20m_d352lNll light chain variable region (VL) (SEQ ID NO:241 QPVLTQPPSLSVSPGQTASITCSGEKLGDKYAY W Y QQKPGQSPVLVMYQ SGIPERFSGSNSGNTATLTISGTQAMDEADYYCQAWDSSTAVFGGGTKL l7Nl6m light chain variable region (VL) (SEQ ID NO:251 PPSVSVAPGQTARITCGGNNIGSKNVHWYQQKPGQAPVLVVYD DSDRPSGIPERFSGSNSGNTATLTVSRVEAGDEADYYCQVWDTTTDHVVFGGG TKLTVLAAAGSEQKLISE l7Nl6m dl7N9 light chain variable region (VL) (SEQ ID NO:27) PPSVSVAPGQTATITCAGDNLGKKRVHWYQQRPGQAPVLVIYD DSDRPSGIPDRFSASNSGNTATLTITRGEAGDEADYYCQVWDIATDHVVFGGGT KLTVLAAAGSEQKLISE TABLE 6: Liht Chain CDRs Kabat/chothia SEQ ID No: 17D20m emu GDKLGDKFAYW _ d3521N1 1 CDR—Ll GEKLGDKYAYW _ l7D20m and CDR-Ll GXKLGDKXAYW d3521Nll wherein X at .osition 2 is D COIlSGIlSllS (Asp) or E (Glu); and n X at position 8 is F 17N16n1 CDR-Ll GNNIGSKNVHW 43 d17N9 CDR-Ll GDNLGKKRVHW 44 17N16n1 and CDR-Ll KXVHW 92 d17N9 consensus (wherein X at position 2 is N (Asn) or D (Asp); wherein X at on 4 is I (lie) or L (Leu); wherein X at position 6 is S (Ser) or K (Lys); and wherein X at position 8 is N Asn orR Ar d17N9 cnR-m aazs-as 17D20n1 cnR-Lz d3521N11 cnR-Lz d3521N11 cnR-Lz aaso-éo 17mm cnR-Lz d17N9 cnR-Lz 17D20n1, CDR-L2 DXXRPSG 93 d3521N11, (wherein X at position 2 is N 17N16n1, d17N9 (Asn), K (Lys) or S (Ser); IlSllS and wherein X at position 3 is K (Lys), Q (Gln) or D AS 0 d17N9 CDR-L2 aa 50-63 DSDRPSGIPDRFSA 50 17D20n1 CDR-L3 AWDSSTAVF 5 1 d3521N11 CDR-L3 AWDSSTAVF 5 1 d3521N11 CDR-L3 (aa 89- AWDSSTAVFGGGTKLT 52 l7N 1 6m CDR-L3 VWDTTTDHV dl 7N9 CDR-L3 VWDIATDHV l7N16m and CDR-L3 VWDXXTDHV d17N9 consensus (wherein X at on 4 is T (Thr) or I (Ile); and wherein X at position 5 is T (Thr) or In one aspect, the invention provides an ed human monoclonal antibody, or antigen binding nt thereof, that binds to human MASP-Z, comprising: (i) a heavy chain le region comprising CDR-Hl, CDR-H2 and CDR—H3 sequences; and (ii) a light chain variable region comprising CDR-Ll, CDR-L2 and CDR—L3, wherein the heavy chain le region CDR—H3 sequence comprises an amino acid sequence set forth as SEQ ID N038 or SEQ ID NO:90, and conservative sequence modifications thereof, wherein the light chain variable region CDR—L3 sequence comprises an amino acid sequence set forth as SEQ ID NO:51 or SEQ ID NO:94, and vative sequence modifications thereof, and wherein the isolated antibody ts MASP-2 dependent complement activation.

In one embodiment, the heavy chain variable region CDR-H2 sequence comprises an amino acid sequence set forth as SEQ ID NO:32 or 33, and conservative sequence modifications thereof. In one embodiment, the heavy chain variable region CDR-Hl sequence comprises an amino acid sequence set forth as SEQ ID NO:28 or SEQ ID N029, and conservative modifications thereof. In one embodiment, the light chain variable region CDR-L2 sequence comprises an amino acid sequence set forth as SEQ ID NO:93 and conservative modifications thereof. In one embodiment, the light chain variable region CDR—Ll sequence comprises an amino acid sequence set forth as SEQ ID NO:9l or SEQ ID NO:92 and conservative modifications thereof. In one embodiment, the CDR—Hl of the heavy chain variable region comprises SEQ ID NO:28.

In one ment, the CDR-H2 of the heavy chain variable region comprises SEQ ID NO:32. In one embodiment, the CDR-H3 of the heavy chain variable region comprises SEQ ID NO:90, (as shown in TABLE 4). In one embodiment, the amino acid sequence set forth in SEQ ID NO:90 ns an R (Arg) at position 8.

In one embodiment, the CDR-Ll of the light chain variable region comprises SEQ ID NO:9l (as shown in TABLE 6). In one embodiment, the amino acid set forth in SEQ ID NO:9l contains an E (Glu) at position 2. In one embodiment, the amino acid sequence set forth in SEQ ID NO:91 contains a Y (Tyr) at on 8.

In one embodiment, the CDR-L2 of the light chain variable region comprises SEQ ID NO: 93 (as shown in TABLE 6), and wherein the amino acid sequence set forth in SEQ ID NO:93 ns a K (Lys) at position 2. In one embodiment, the amino acid sequence set forth in SEQ ID NO:93 contains a Q (Gln) at position 3.

In one embodiment, the CDR-L3 of the light chain variable region comprises SEQ ID NO:51.

In one embodiment, said antibody or antigen binding nt thereof binds human MASP-2 with a KD of 10 nM or less. In one embodiment, said antibody or antigen binding fragment thereof inhibits C4 activation in an in vitro assay in 1% human serum at an IC50 of 10 nM or less. In one embodiment, said antibody or antigen binding nt thereof inhibits C4 activation in 90% human serum with an IC50 of 30 nM or less. In one embodiment, the conservative sequence modifications thereof comprise or consist of a molecule which ns variable regions that are identical to the recited variable domain(s), except for a combined total of l, 2, 3, 4, 5, 6, 7, 8 9 or 10 amino acid substitutions within the CDR regions of the heavy chain variable region, and/or up to a combined total of l, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions with said CDR regions of the light chain variable region.

In another aspect, the invention es an isolated human antibody, or antigen binding fragment thereof, that binds to human MASP-2 wherein the antibody comprises: I) a) a heavy chain le region comprising: i) a heavy chain CDR-Hl comprising the amino acid ce from 31-35 of SEQ ID NO:21; and ii) a heavy chain CDR-H2 comprising the amino acid sequence from 50-65 of SEQ ID NO:21; and iii) a heavy chain CDR-H3 comprising the amino acid sequence from 95-102 of SEQ ID NO:21; and b) a light chain le region sing: i) a light chain CDR-L1 comprising the amino acid sequence from 24-34 of either SEQ ID NO:25 or SEQ ID NO:27; and ii) a light chain CDR-L2 comprising the amino acid sequence from 50-56 of either SEQ ID NO:25 or SEQ ID NO:27; and iii) a light chain CDR-L3 comprising the amino acid sequence from 89-97 of either SEQ ID NO:25 or SEQ ID NO:27; or II) a variant thereof that is otherwise identical to said variable domains, except for up to a combined total of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions within said CDR regions of said heavy chain variable region and up to a combined total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions within said CDR regions of said light chain le region, wherein the antibody or variant thereof inhibits MASP-2 dependent complement activation. In one ment, said variant comprises an amino acid tution at one or more positions selected from the group consisting of position 31, 32, 33, 34, 35, 51, 52, 53, 54, 55, 56, 57,58,59,60,61,62,63,64,65,95,96,97,98,99,100 or102 heavy dnun variable region. In one embodiment, said variant comprises an amino acid substitution at one or more positions selected from the group consisting of position 25, 26, 27, 29, 31, 32, 33, 51, 52, 89, 92, 93, 95, 96 or 97 of said light chain variable region. In one embodiment, the heavy chain of said antibody comprises SEQ ID NO:21. In one embodiment, the light chain of said antibody ses SEQ ID NO:25. In one embodiment, the light chain of said antibody comprises SEQ ID NO:27.

In another aspect, the invention provides an isolated human monoclonal antibody that binds to human MASP-2, wherein the antibody comprises: I) a) a heavy chain variable region comprising: i) a heavy chain CDR-H1 comprising the amino acid sequence from 31-35 of SEQ ID NO:20; and ii) a heavy chain CDRH-2 comprising the amino acid sequence from 50-65 of SEQ ID NO:20; and iii) a heavy chain CDR-H3 comprising the amino acid sequence from 95-102 of either SEQ ID NO:18 or SEQ ID N020; and b) a light chain variable region comprising: i) a light chain CDR-Ll comprising the amino acid sequence from 24-34 of either SEQ ID NO:22 or SEQ ID N024; and ii) a light chain CDR-L2 comprising the amino acid sequence from 50-56 of either SEQ ID NO:22 or SEQ ID N024; and iii) a light chain CDR-L3 comprising the amino acid sequence from 89-97 of either SEQ ID NO:22 or SEQ ID NO:24; or II) a variant thereof that is otherwise identical to said variable domains, except for up to a combined total of l, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions within said CDR regions of said heavy chain variable region and up to a ed total of l, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions within said CDR regions of said light chain variable region, wherein the antibody or variant thereof inhibits MASP-2 dependent complement activation. In one embodiment, said variant comprises an amino acid substitution at one or more positions selected from the group ting of position 31, 32,33,34,35,5l,52,53,54,55,56,57,58,59,60,61,62,63,64,65,95,96,97,98,99, 100 or 102 of said heavy chain variable region. In one embodiment, said variant comprises an amino acid substitution at one or more positions selected from the group conmsfing(fl?posfinn125,26,27,29,3l,32,33,5l,52,89,92,93,95,96(n?97'ofsak1 light chain variable region. In one embodiment, the heavy chain of said dy comprises SEQ ID NO:20, or a variant thereof sing at least 80% identity to SEQ ID NO:20 (e.g., at least 85%, at least 90%, at least 95% or at least 98% identity to SEQ ID . In one embodiment, the heavy chain of said antibody ses SEQ ID NO:l8, or a variant thereof comprising at least 80% identity to SEQ ID NO:l8 (e.g., at least 85%, at least 90%, at least 95% or at least 98% identity to SEQ ID NO:18). In one embodiment, the light chain of said antibody comprises SEQ ID NO:22, or a variant f comprising at least 80% identity to SEQ ID NO:22 (e.g., at least 85%, at least 90%, at least 95% or at least 98% identity to SEQ ID NO:22). In one embodiment, the light chain of said antibody comprises SEQ ID NO:24, or a t thereof comprising at least 80% identity to SEQ ID NO:24 (e.g., at least 85%, at least 90%, at least 95% or at least 98% identity to SEQ ID NO:24).

In one embodiment, said antibody binds to an epitope in the CCPl domain of MASP-2.

In one embodiment, said antibody binds human MASP-2 with a KD of 10 nM or less. In one embodiment, said antibody inhibits C3b deposition in an in vitro assay in 1% human serum at an IC50 of 10 nM or less. In one embodiment, said antibody inhibits C3b deposition in 90% human serum with an IC50 of 30 nM or less.

In one ment, said antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2 and 2. In one embodiment, said dy is a single chain molecule. In one embodiment, said antibody is an IgG2 molecule. In one embodiment, said antibody is an IgGl molecule. In one embodiment, said antibody is an IgG4 le. In one embodiment, said IgG4 molecule ses a S228P mutation.

In one embodiment, said antibody does not substantially inhibit the classical pathway (i.e., the classical pathway activity is at least 80%, or at least 90% or at least 95%, or at least 95% intact).

In r aspect, the invention provides an isolated fully human monoclonal antibody or antigen-binding fragment thereof that dissociates from human MASP-2 with a KD of lOnM or less as determined by surface plasmon resonance and inhibits C4 activation on a mannan-coated substrate with an IC50 of lOnM or less in 1% serum. In some ments, said antibody or antigen binding fragment thereof specifically recognizes at least part of an epitope recognized by a reference antibody comprising a heavy chain variable region as set forth in SEQ ID N020 and a light chain variable region as set forth in SEQ ID NO:24, such as reference antibody OMS646 (see TABLE 22). In accordance with the foregoing, an antibody or antigen-binding fragment thereof according to certain preferred embodiments of the present application may be one that competes for binding to human MASP-2 with any antibody described herein which both (i) specifically binds to the antigen and (ii) comprises a VH and/or VL domain sed herein, or comprises a CDR—H3 disclosed herein, or a variant of any of these.

Competition between binding s may be assayed easily in vitro, for example using ELISA and/or by g a specific reporter molecule to one binding member which can be detected in the ce of other untagged binding member(s), to enable identification of specific g members which bind the same epitope or an overlapping epitope.

Thus, there is presently ed a specific antibody or antigen-binding fragment thereof, comprising a human antibody antigen-binding site which competes with an antibody described herein that binds to human MASP-2, such as any one of OMS64l to OMS646 as set forth in TABLE 24, for binding to human MASP-2.

Variant MASP-2 Inhibitory Antibodies The described human monoclonal antibodies may be modified to provide variant antibodies that inhibit MASP-2 dependent complement tion. The variant dies may be made by substituting, adding, or deleting at least one amino acid of an above-described human monoclonal antibody. In general, these variant antibodies have the general characteristics of the above-described human antibodies and contain at least the CDRs of an above-described human antibody, or, in certain ments, CDRs that are very similar to the CDRs of an above-described human antibody.

In the red embodiment, the variant comprises one or more amino acid substitution(s) in one or more hypervariable region(s) of the parent antibody. For example, the variant may comprise at least one, e. g., from about one to about ten, such as at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 substitutions, and preferably from about two to about six, substitutions in one or more CDR regions of the parent antibody. In one embodiment, said variant comprises an amino acid tution at one or more positions selected from the group consisting ofposition 31, 32, 33, 34, 35, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 95, 96, 97, 98, 99, 100 or 102 of said heavy chain variable region. In one embodiment, said variant comprises an amino acid substitution at one or more positions selected from the group ting of position 25, 26, 27, 29, 31, 32, 33, 51, 52, 89, 92, 93, 95, 96 or 97 of said light chain variable region.

In some embodiments, the variant antibodies have an amino acid sequence that is otherwise identical to the variable domain of a subject dy set forth in TABLE 2, except for up to a combined total of l, 2, 3, 4, 5 or 6 amino acid substitutions within said CDR regions of said heavy chain variable region and/or up to a ed total of l, 2, 3, 4, 5 or 6 amino acid substitutions within said CDR regions of said light chain variable region, wherein the antibody or variant thereof inhibits MASP-2 dependent complement activation.

Ordinarily, the variant will have an amino acid sequence having at least 75% amino acid sequence ty with the parent antibody heavy or light chain variable domain sequences, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identity. Identity or homology with respect to this sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the parent antibody residues, after aligning the sequences and introducing gaps, if ary, to e the maximum percent sequence identity.

None of inal, C-terminal, or internal extensions, deletions, or insertions into the antibody sequence (such as, for e, signal peptide sequences, linker sequences, or tags, such as HIS tags) shall be construed as affecting sequence identity or homology.

The variant retains the ability to bind human MASP-Z and preferably has properties which are superior to those of the parent antibody. For example, the variant may have a stronger binding affinity and/or an enhanced ability to t or block MASP-2 ent complement tion.

To analyze such properties, one should compare a Fab form of the variant to a Fab form of the parent antibody or a full length form of the variant to a full length form of the parent antibody, for example, since it has been found that the format of the anti-MASP-Z antibody impacts its activity in the biological activity assays disclosed herein. The variant dy of particular st herein is one which displays at least about lO-fold, preferably at least about 20-fold, and most preferably at least about 50-fold, enhancement in biological activity when compared to the parent antibody.

The antibodies of the invention may be modified to enhance desirable properties, such as it may be desirable to control serum half-life of the antibody. In general, complete antibody molecules have a very long serum persistence, whereas fragments (<60-80 kDa) are filtered very y through the kidney. Hence, if long-term action of the MASP-2 dy is desirable, the MASP-Z antibody is preferably a complete full length IgG antibody (such as IgG2 or IgG4), whereas if r action of the MASP-2 antibody is desirable, an antibody fragment may be preferred. As bed in e , it has been determined that an S228P substitution in the hinge region of IgG4 increases serum stability. Accordingly, in some ments, the t MASP-2 antibody is a full length IgG4 antibody with an S228P substitution.

Single Chain Antibodies In one embodiment of the present invention, the MASP-2 inhibitory antibody is a single chain antibody, defined as a cally engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule. Such single chain antibodies are also referred to as "single-chain Fv" or "scFv" antibody fragments.

Generally, the EV polypeptide filrther comprises a polypeptide linker between the VH and VL s that enables the scFv to form the desired structure for antigen binding. The scFv antibodies that bind MASP-Z can be oriented with the variable light region either amino terminal to the variable heavy region or carboxyl terminal to it. Exemplary scFv antibodies of the invention are set forth herein as SEQ ID NOS: 55-61 and SEQ ID NOS: 66-68.

Methodsfor Producing Antibodies In many embodiments, the nucleic acids encoding a subject monoclonal dy are introduced ly into a host cell, and the cell ted under conditions sufficient to induce expression of the encoded antibody.

In some embodiments, the invention provides a nucleic acid molecule encoding an anti-MASP-2 antibody, or fragment thereof, of the invention, such as an antibody or fragment thereof set forth in TABLE 2. In some embodiments the invention es a nucleic acid molecule comprising a nucleic acid ce selected from the group consisting of SEQ ID NO:l9, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:97, SEQ ID NO:88 and SEQ ID NO:89.

In some embodiments, the invention provides a cell comprising a c acid molecule encoding an anti-MASP-Z antibody of the invention.

In some embodiments, the invention provides an expression cassette comprising a nucleic acid molecule encoding an anti-MASP-Z antibody of the invention.

In some embodiments, the invention provides a method of producing anti-MASP- 2 dies comprising culturing a cell comprising a nucleic acid molecule encoding an anti-MASP-2 dy of the invention.

According to certain related embodiments there is provided a recombinant host cell which comprises one or more constructs as described ; a nucleic acid ng any dy, CDR, VH or VL domain, or antigen-binding fragment thereof; and a method of production of the encoded product, which method comprises expression from encoding nucleic acid or. Expression may conveniently be ed by culturing under appropriate ions recombinant host cells containing the nucleic acid.

Following production by expression, an antibody or antigen-binding fragment thereof, may be isolated and/or purified using any suitable technique, and then used as desired.

For example, any cell suitable for expression of expression cassettes may be used as a host cell, for example, yeast, insect, plant, etc., cells. In many embodiments, a mammalian host cell line that does not ordinarily produce antibodies is used, examples of which are as follows: monkey kidney cells (COS cells), monkey kidney CV1 cells transformed by SV40 , ATCC CRL 165 1); human embryonic kidney cells (HEK- 293, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary-cells (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci.

(USA) 77:4216, (1980); mouse i cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC 87); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL 51); TRI cells (Mather et al., Annals NY. Acad. Sci 383 :44-68 (1982)); NIH/3T3 cells (ATCC CRL-1658); and mouse L cells (ATCC CCL-1). onal cell lines will become apparent to those of ordinary skill in the art. A wide variety of cell lines are available from the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209.

Methods of ucing nucleic acids into cells are well known in the art. Suitable methods include electroporation, particle gun technology, calcium phosphate precipitation, direct microinjection, and the like. The choice of method is generally dependent on the type of cell being transformed and the circumstances under which the transformation is taking place (i.e., in vitro, ex vivo, or in vivo). A general discussion of these s can be found in Ausubel, et al., Short Protocols in Molecular Biology, 3d ed., Wiley & Sons, 1995. In some embodiments, lipofectamine and m mediated gene transfer technologies are used.

WO 51481 After the subject nucleic acids have been introduced into a cell, the cell is typically incubated, normally at 37°C, mes under selection, for a suitable time to allow for the expression of the antibody. In most embodiments, the antibody is typically secreted into the supernatant of the media in which the cell is growing in.

In mammalian host cells, a number of viral-based expression systems may be utilized to express a subject antibody. In cases where an adenovirus is used as an expression vector, the antibody coding ce of interest may be ligated to an adenovirus transcription/translation control complex, e. g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in viva recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of sing the dy molecule in infected hosts. (e.g., see Logan & Shenk, Proc. Natl.

Acad. Sci. USA 81:355-359 (1984)). The ncy of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., Methods in Enzymol. 153:5 1-544 (1987)).

For long-term, high-yield production of inant antibodies, stable expression may be used. For example, cell lines, which stably express the antibody molecule, may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with immunoglobulin expression cassettes and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably ate the plasmid into a chromosome and grow to form foci which in turn can be cloned and expanded into cell lines. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.

Once an dy le of the invention has been produced, it may be ed by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the cation of ns.

In many embodiments, antibodies are ed from the cell into e medium and harvested from the culture medium. For example, a nucleic acid sequence encoding a 2012/036509 signal peptide may be included adjacent the coding region of the antibody or fragment, for example as provided in nucleotides 1-57 of SEQ ID NO:71, encoding the signal peptide as provided in amino acids 1-19 of SEQ ID NO:72. Such a signal peptide may be incorporated nt to the 5' end of the amino acid sequences set forth herein for the subject antibodies in order to facilitate production of the subject antibodies.

Pharmaceutical Carriers and Delivery Vehicles In another aspect, the invention provides compositions for ting the adverse effects of MASP-Z-dependent ment activation comprising a therapeutically effective amount of a human anti-MASP-2 inhibitory antibody and a pharmaceutically acceptable carrier.

In general, the human MASP-Z inhibitory antibody itions of the t invention, combined with any other selected therapeutic agents, are ly contained in a pharmaceutically acceptable carrier. The carrier is non-toxic, patible and is selected so as not to detrimentally affect the biological activity of the MASP-2 inhibitory antibody (and any other eutic agents ed therewith). Exemplary pharmaceutically acceptable carriers for polypeptides are described in US. Patent No. 657 to Yamada. The anti-MASP-2 antibodies may be formulated into preparations in solid, semi-solid, gel, liquid or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, depositories, nts and injections allowing for oral, parenteral or surgical administration. The invention also contemplates local administration of the compositions by coating medical devices and the like.

Suitable carriers for parenteral delivery via injectable, infilsion or irrigation and topical delivery include led water, physiological phosphate-buffered saline, normal or lactated 's solutions, dextrose solution, Hank's solution, or propanediol. In addition, sterile, fixed oils may be employed as a solvent or suspending medium. For this purpose, any biocompatible oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. The carrier and agent may be compounded as a liquid, suspension, polymerizable or non-polymerizable gel, paste or salve.

The r may also comprise a delivery vehicle to sustain (i.e., extend, delay or regulate) the delivery of the agent(s) or to enhance the delivery, uptake, stability or pharmacokinetics of the eutic agent(s). Such a delivery vehicle may include, by way of non-limiting example, articles, microspheres, nanospheres or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymeric or copolymeric hydrogels and polymeric es. Suitable hydrogel and micelle delivery systems include the PEO:PHB:PEO copolymers and copolymer/cyclodextrin complexes disclosed in A2 and the PEG and PEO/cyclodextrin complexes sed in US. Patent Application Publication No. 2002/0019369 Al. Such hydrogels may be injected locally at the site of intended action, or subcutaneously or intramuscularly to form a sustained e depot.

For intra-articular delivery, the MASP-2 tory antibody may be carried in above-described liquid or gel carriers that are injectable, above-described sustained-release delivery vehicles that are injectable, or a hyaluronic acid or hyaluronic acid derivative.

For intrathecal (IT) or intracerebroventricular (ICV) delivery, appropriately sterile delivery systems (e.g., liquids; gels, suspensions, etc.) can be used to ster the present invention.

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

To achieve high concentrations of anti-MASP-2 antibodies for local delivery, the antibodies may be formulated as a suspension of particulates or crystals in solution for subsequent injection, such as for intramuscular injection of a depot.

More specifically with respect to anti-MASP-2 antibodies, exemplary formulations can be parenterally administered as injectable s of a solution or suspension of the compound in a physiologically acceptable diluent with a pharmaceutical r that can be a sterile liquid such as water, oils, saline, glycerol or ethanol. Additionally, auxiliary substances such as wetting or emulsifying agents, surfactants, pH buffering substances and the like can be t in compositions comprising anti-MASP-2 antibodies. Additional components of ceutical compositions include petroleum (such as of animal, vegetable or synthetic origin), for example, soybean oil and l oil. In general, glycols such as propylene glycol or polyethylene glycol are red liquid carriers for able solutions.

The ASP-2 antibodies can also be administered in the form of a depot injection or implant preparation that can be formulated in such a manner as to permit a sustained or pulsatile e of the active agents.

The pharmaceutical compositions comprising MASP-Z inhibitory antibodies may be administered in a number of ways depending on r a local or systemic mode of administration is most appropriate for the condition being treated. Additionally, as described herein above with respect to extracorporeal reperfiJsion procedures, MASP-2 inhibitory antibodies can be administered Via introduction of the compositions of the present invention to recirculating blood or plasma. Further, the compositions of the present invention can be delivered by coating or incorporating the compositions on or into an implantable medical .

SYSTEMIC DELIVERY As used herein, the terms mic delivery" and "systemic administration" are intended to include but are not limited to oral and parenteral routes including intramuscular (1M), subcutaneous, enous (IV), intra-arterial, inhalational, sublingual, buccal, topical, transdermal, nasal, , vaginal and other routes of administration that effectively result in dispersal of the red antibody to a single or multiple sites of intended therapeutic action. Preferred routes of systemic delivery for the present compositions include intravenous, intramuscular, subcutaneous and inhalational.

It will be appreciated that the exact systemic stration route for ed agents utilized in particular compositions of the present invention will be determined in part to account for the agent's susceptibility to metabolic transformation pathways associated with a given route of stration.

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

By way of representative example, MASP-2 inhibitory antibodies and es can be introduced into a liVing body by application to a bodily membrane capable of ing the polypeptides, for example the nasal, gastrointestinal and rectal membranes.

The polypeptides are typically applied to the absorptive membrane in conjunction with a tion enhancer. (See, e.g., Lee, V.H.L., Crit. Rev. Ther. Drag r Sys. 5:69, 1988; Lee, V.H.L., J. Controlled Release 13:213, 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 derivative of fusidic acid, a dal surfactant that is r in structure to the bile salts, and has been used as a permeation er for nasal delivery. (Lee, W.A., Biopharm. 22, Nov./Dec. 1990.) The MASP-2 inhibitory antibodies and polypeptides may be introduced in association with another molecule, such as a lipid, to protect the polypeptides from enzymatic degradation. For example, the covalent attachment of polymers, especially polyethylene glycol (PEG), has been used to protect certain proteins from enzymatic hydrolysis in the body and thus g half-life (Fuertges, F., et al., J. lled Release 11:139, 1990). Many polymer systems have been reported for protein delivery (Bae, Y.H., et al., J. Controlled Release 9:271, 1989; Hori, R., et al., Pharm. Res. 6:813, 1989; Yamakawa, 1., et al., J. Pharm. Sci. 79:505, 1990; Yoshihiro, 1., et al., J. Controlled Release 10:195, 1989; Asano, M., et al., J. Controlled e 9:111, 1989; Rosenblatt, J., et al., J. Controlled Release 9:195, 1989; Makino, K., J. Controlled Release 12:235, 1990; Takakura, Y., et al., J Pharm. Sci. 78:117, 1989; Takakura, Y., et al., J. Pharm.

Sci. 78:219, 1989).

Recently, liposomes have been ped with improved serum stability and circulation half-times (see, e.g., US. Patent No. 5,741,516, to Webb). Furthermore, various methods of liposome and liposome-like preparations as potential drug carriers have been reviewed (see, e.g., US. Patent No. 5,567,434, to Szoka; US. Patent No. 5,552,157, to Yagi; US. Patent No. 5,565,213, to Nakamori; US. Patent No. 5,738,868, to Shinkarenko; and US. Patent No. 5,795,587, to Gao).

For transdermal applications, the MASP-2 inhibitory antibodies and ptides may be combined with other suitable ingredients, such as carriers and/or adjuvants.

There are no limitations on the nature of such other ients, except that they must be pharmaceutically acceptable for their intended administration, and cannot e the activity of the active ingredients of the composition. Examples of suitable vehicles include ointments, creams, gels, or suspensions, with or t purified collagen. The 2012/036509 MASP-Z inhibitory antibodies and polypeptides may also be impregnated into transdermal patches, plasters, and bandages, preferably in liquid or semi-liquid form.

The compositions of the present ion may be systemically administered on a periodic basis at als ined to maintain a desired level of therapeutic effect.

For example, compositions may be administered, such as by subcutaneous injection, eveny hvo to four vveeks or atless frequent ls. 'The dosage regnnen,vvfll be determined by the ian considering various factors that may influence the action of the combination of agents. These s will include the extent of progress of the condition being treated, the patient's age, sex and weight, and other clinical factors. The dosage for each individual agent will vary as a fianction of the MASP-Z inhibitory antibody that is included in the composition, as well as the presence and nature of any drug delivery vehicle (e.g., a sustained release delivery vehicle). In addition, the dosage quantity may be adjusted to account for variation in the frequency of administration and the pharmacokinetic behavior of the delivered agent(s).

HHJVERY As used herein, the term "local" encompasses application of a drug in or around a site of intended localized action, and may include for example topical delivery to the skin or other affected tissues, ophthalmic delivery, intrathecal (IT), intracerebroventricular (ICV), intra-articular, intracavity, intracranial or intravesicular administration, placement or irrigation. Local administration may be preferred to enable administration of a lower dose, to avoid systemic side effects, and for more accurate l of the timing of delivery and concentration of the active agents at the site of local delivery. Local administration es a known concentration at the target site, regardless of interpatient variability in metabolism, blood flow, etc. Improved dosage control is also provided by the direct mode of delivery.

Local delivery of a MASP-2 tory antibody may be achieved in the context of surgical methods for treating a disease or condition, such as for example during procedures such as arterial bypass surgery, atherectomy, laser procedures, ultrasonic procedures, balloon angioplasty and stent placement. For example, a MASP-2 inhibitor can be stered to a subject in ction with a balloon angioplasty procedure. A balloon angioplasty procedure involves inserting a catheter having a deflated balloon into an artery. The d balloon is positioned in proximity to the atherosclerotic plaque and is inflated such that the plaque is compressed against the vascular wall. As a result, the balloon surface is in contact with the layer of vascular endothelial cells on the surface of the blood vessel. The MASP-2 inhibitory antibody may be attached to the balloon angioplasty catheter in a manner that permits e of the agent at the site of the atherosclerotic plaque. The agent may be attached to the balloon catheter in accordance with standard procedures known in the art. For example, the agent may be stored in a compartment of the balloon catheter until the balloon is inflated, at which point it is released into the local environment. atively, the agent may be impregnated on the balloon surface, such that it contacts the cells of the al wall as the balloon is d.

The agent may also be delivered in a perforated balloon catheter such as those disclosed in Flugelman, M.Y., et al., Circulation 0-1117, 1992. See also published PCT Application WO 95/23161 for an ary procedure for attaching a therapeutic n to a balloon angioplasty catheter. Likewise, the MASP-2 inhibitory antibody may be included in a gel or ric coating d to a stent, or may be orated into the material of the stent, such that the stent elutes the MASP-2 inhibitory dy after vascular placement.

Treatment Regimes MASP-2 inhibitory antibody compositions used in the treatment of arthritides and other musculoskeletal disorders may be locally delivered by intra-articular injection.

Such compositions may suitably include a sustained release delivery vehicle. As a fiarther example of instances in which local delivery may be desired, MASP-2 inhibitory antibody compositions used in the treatment of urogenital conditions may be suitably instilled intravesically or within another urogenital structure.

In prophylactic applications, the pharmaceutical compositions are administered to a subject tible to, or otherwise at risk of, a condition associated with MASP-Z-dependent complement activation in an amount ent to eliminate or reduce the risk of developing ms of the condition. In therapeutic applications, the pharmaceutical compositions are administered to a subject suspected of, or already suffering from, a condition associated with MASP-Z-dependent ment activation in a therapeutically effective amount sufficient to relieve, or at least partially reduce, the ms of the condition. In both prophylactic and therapeutic regimens, compositions comprising MASP-2 inhibitory antibodies may be administered in several dosages until a sufficient therapeutic outcome has been achieved in the subject. Application of the MASP-2 inhibitory antibody compositions of the present invention may be carried out by a single administration of the composition, or a limited sequence of administrations, for treatment of an acute condition, e.g., reperfusion injury or other traumatic injury.

Alternatively, the composition may be administered at periodic intervals over an extended period of time for treatment of chronic conditions, e.g., arthritides or psoriasis.

MASP-2 tory compositions used in the present ion may be delivered immediately or soon after an acute event that results in activation of the lectin pathway, such as following an ischemic event and reperfusion of the ischemic tissue. Examples include myocardial ia reperfusion, renal ia lsion, cerebral ischemia reperfilsion, organ transplant and digit/extremity reattachment. Other acute examples include sepsis. A MASP-2 inhibitory composition of the present invention may be administered as soon as possible following an acute event that activates the lectin pathway, preferably within twelve hours and more preferably within two to three hours of a ring event, such as through systemic delivery of the MASP-2 inhibitory composition.

The methods and compositions of the present invention may be used to inhibit inflammation and related processes that typically result from diagnostic and therapeutic medical and al procedures. To inhibit such processes, the MASP-2 inhibitory composition of the present invention may be applied periprocedurally. As used herein "periprocedurally" refers to administration of the inhibitory composition preprocedurally and/or rocedurally and/or postprocedurally, i.e., before the procedure, before and during the ure, before and after the procedure, before, during and after the procedure, during the procedure, during and after the procedure, or after the procedure.

Periprocedural ation may be carried out by local administration of the composition to the surgical or procedural site, such as by injection or continuous or intermittent irrigation of the site or by ic administration. Suitable methods for local perioperative delivery of MASP-2 inhibitory dy solutions are disclosed in US Patent Nos. 6,420,432 to Demopulos and 6,645,168 to Demopulos. Suitable methods for local ry of chondroprotective compositions including MASP-2 inhibitory dies are disclosed in International PCT Patent ation WO 01/07067 A2. Suitable methods and compositions for targeted systemic delivery of chondroprotective compositions including MASP-2 inhibitory antibodies are disclosed in International PCT Patent Application WO 03/063799 A2.

Dosages The MASP-2 inhibitory antibodies can be administered to a subject in need thereof, at therapeutically effective doses to treat or ameliorate conditions ated with MASP-Z-dependent complement activation. A therapeutically effective dose refers to the amount of the MASP-2 inhibitory antibody sufficient to result in amelioration of symptoms of the condition. ty and therapeutic efficacy of MASP-2 tory antibodies can be ined by standard pharmaceutical procedures employing mental animal , such as the African Green Monkey, as described herein. Using such animal models, the NOAEL (no observed adverse effect level) and the MED (the lly effective dose) can be determined using standard methods. The dose ratio between NOAEL and MED effects is the therapeutic ratio, which is expressed as the ratio MED. MASP-2 inhibitory antibodies that exhibit large therapeutic ratios or indices are most preferred. The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosages for use in humans. The dosage of the MASP-2 inhibitory antibody preferably lies within a range of circulating concentrations that include the MED with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.

For any compound formulation, the therapeutically ive dose can be estimated using animal models. For example, a dose may be ated in an animal model to achieve a circulating plasma concentration range that includes the MED.

Quantitative levels of the MASP-2 inhibitory antibody in plasma may also be measured, for example, by high performance liquid chromatography.

In addition to toxicity studies, effective dosage may also be estimated based on the amount of MASP-Z protein present in a liVing t and the binding affinity of the MASP-Z inhibitory antibody. It has been shown that MASP-2 levels in normal human subjects is present in serum in low levels in the range of 500 ng/ml, and MASP-2 levels in a particular subject can be determined using a quantitative assay for MASP-2 described in Moller-Kristensen M., et al., J. Immunol. Methods 282:159-167, 2003, hereby incorporated herein by reference.

Generally, the dosage of administered compositions comprising MASP-2 inhibitory dies varies depending on such factors as the subject's age, weight, height, sex, general medical condition, and previous medical history. As an illustration, MASP-2 inhibitory antibodies, can be administered in dosage ranges from about 0.010 to .0 mg/kg, preferably 0.010 to 1.0 mg/kg, more preferably 0.010 to 0.1 mg/kg of the t body weight.

Therapeutic efficacy of MASP-2 inhibitory itions and methods of the present invention in a given subject, and appropriate dosages, can be determined in accordance with complement assays well known to those of skill in the art. ment generates numerous specific ts. During the last decade, sensitive and c assays have been developed and are available commercially for most of these activation products, ing the small activation fragments C3a, C4a, and C5a and the large activation fragments iC3b, C4d, Bb, and sC5b-9. Most of these assays utilize antibodies that react with new antigens (neoantigens) exposed on the fragment, but not on the native proteins from which they are formed, making these assays very simple and specific.

Most rely on ELISA technology, although radioimmunoassay is still sometimes used for C3a and C5a. These latter assays measure both the unprocessed fragments and their 'desArg' fragments, which are the major forms found in the circulation. Unprocessed fragments and CSadeSArg are rapidly cleared by binding to cell surface ors and are hence t in very low trations, whereas C3adeSArg does not bind to cells and accumulates in plasma. Measurement of C3a provides a sensitive, pathway-independent indicator of complement activation. Alternative y activation can be assessed by measuring the Bb fragment. Detection of the fluid-phase product of membrane attack pathway activation, sC5b-9, es evidence that complement is being activated to completion. Because both the lectin and classical pathways generate the same activation products, C4a and C4d, measurement of these two fragments does not e any information about which of these two pathways has generated the tion products.

The inhibition of MASP-Z-dependent complement activation is characterized by at least one of the following changes in a component of the complement system that occurs as a result of administration of an anti-MASP-2 antibody in accordance with the present ion: the inhibition of the generation or production of MASPdependent complement activation system products C4b, C3a, C5a and/or C5b-9 (MAC), the reduction of C4 cleavage and C4b deposition, or the reduction of C3 cleavage and C3b deposition.

Articles ofManufacture In another aspect, the present invention provides an e of manufacture containing a human MASP-2 inhibitory antibody, or n binding fragment thereof, as described herein in a unit dosage form le for therapeutic administration to a human t, such as, for example, a unit dosage in the range of 1mg to 5000mg, such as from 1 mg to 2000mg, such as from 1mg to 1000 mg, such as 5mg, 10mg, 50mg, 100mg, 200mg, 500mg, or 1000mg. In some embodiments, the article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is the MASP-2 inhibitory antibody or antigen g fragment thereof of the ion. The label or package insert indicates that the composition is used for treating the particular condition. The label or package insert will further comprise instructions for administering the antibody composition to the patient. Articles of manufacture and kits comprising atorial therapies described herein are also contemplated.

Therapeutic Uses of the anti-MASP-2 inhibitory antibodies In another aspect, the invention provides a method of inhibiting MASP-2 dependent ment activation in a human subject comprising stering a human onal anti-MASP-2 inhibitory antibody of the invention in an amount sufficient to inhibit MASP-2 dependent complement activation in said human subject.

In accordance with this aspect of the invention, as described in Example 10, the MASP-2 tory antibodies of the present invention are capable of ting the lectin pathway in African Green Monkeys following intravenous administration. As shown in Table 24, Example 8, the antibody used in this study, OMS646, was found to be more potent in human serum. As known by those of skill in the art, non-human primates are often used as a model for evaluating antibody therapeutics.

As described in US Patent No. 7,919,094, co-pending US. Patent Application Serial No. 13/083,441, and co-pending US. Patent Application Serial No. 12/905,972 (each of which is assigned to Omeros ation, the assignee of the instant application), each of which is hereby orated by reference, MASP-2 dependent complement tion has been implicated as contributing to the pathogenesis of numerous acute and chronic disease states, including MASP-Z-dependent complement mediated vascular condition, an ischemia usion injury, atherosclerosis, inflammatory gastrointestinal disorder, a pulmonary condition, an extracorporeal reperfiJsion procedure, a musculoskeletal condition, a renal condition, a skin condition, organ or tissue transplant, nervous system disorder or injury, a blood disorder, a urogenital condition, diabetes, chemotherapy or radiation y, malignancy, an ine disorder, a coagulation disorder, or an ophthalmologic condition. Therefore, the MASP-2 inhibitory antibodies of the present ion may be used to treat the above- referenced diseases and conditions.

As further described in Example 11, the MASP-2 inhibitory antibodies of the present ion are effective in treating a mammalian subject at risk for, or suffering from the ental effects of acute radiation syndrome, thereby demonstrating therapeutic efficacy in vivo.

The following examples merely illustrate the best mode now plated for practicing the invention, but should not be construed to limit the invention.

EXAMPLE 1 This Example describes the recombinant expression and protein production of recombinant ength human, rat and murine , MASP-Z d polypeptides, and catalytically inactivated mutant forms of MASP-2.

Expression of ength human and rat MASP-2: The full length cDNA sequence of human MASP-2 (SEQ ID NO: 1), encoding the human MASP-2 polypeptide with leader sequence (SEQ ID NO:2) was subcloned into the mammalian expression vector pCI-Neo (Promega), which drives eukaryotic sion under the control of the CMV enhancer/promoter region (described in Kaufman R.J. et al., Nucleic Acids Research 19:4485-90, 1991; Kaufman, Methods in Enzymology, [85:537-66 (1991)). The full length rat MASP-Z cDNA (SEQ ID NO:4), encoding the rat MASP-2 polypeptide with leader sequence (SEQ ID NO:5) was subcloned into the pED expression vector. The MASP-Z sion vectors were then transfected into the adherent Chinese hamster ovary cell line DXBl using the standard calcium phosphate transfection procedure described in Maniatis et al., 1989. Cells transfected with these constructs grew very slowly, implying that the encoded protease is 2012/036509 cytotoxic. The mature form of the human MASP-Z protein (SEQ ID N03) and the mature form of the rat MASP-2 protein (SEQ ID NO:6) were secreted into the culture media and isolated as described below.

Expression of Full-length cataly_tically inactive MASP-2: Rationale: MASP-Z is activated by autocatalytic cleavage after the recognition ponents MBL, C-type lectin CL-ll, or ns (either L-ficolin, H-ficolin or M-ficolin), collectively ed to as lectins, bind to their respective carbohydrate n. Autocatalytic cleavage resulting in activation of MASP-2 often occurs during the isolation procedure of MASP-Z from serum, or during the purification following recombinant expression. In order to obtain a more stable protein ation for use as an antigen, a catalytically inactive form of MASP-2, designed as MASP-ZA, was d by replacing the serine residue that is present in the catalytic triad of the protease domain with an alanine residue in the mature rat MASP-Z n (SEQ ID NO:6 Ser6l7 to Ala6l7); or in mature human MASP-2 protein (SEQ ID NO:3 Ser618 to Ala618).

In order to te catalytically inactive human and rat MASP-ZA ns, site-directed mutagenesis was carried out as described in US2007/0172483, hereby incorporated herein by reference. The PCR ts were purified after e gel electrophoresis and band preparation and single adenosine overlaps were generated using a standard tailing procedure. The adenosine tailed MASP-ZA was then cloned into the pGEM-T easy vector, transformed into E. coli. The human and rat MASP-ZA were each further subcloned into either of the mammalian expression vectors pED or pCI-Neo and transfected into the Chinese Hamster ovary cell line DXBl as described below.

Construction of sion Plasmids Containing Polypeptide Regions Derived from Human Masp-Z.

The following constructs were produced using the MASP-2 signal peptide (residues 1-15 of SEQ ID N02) to secrete various domains of MASP-Z. A construct expressing the human MASP-2 CUBI domain (SEQ ID NO:7) was made by PCR amplifying the region encoding residues 1—121 of MASP-2 (SEQ ID NO:3) (corresponding to the N—terminal CUBl domain). A construct expressing the human MASP-Z CUBI/EGF domain (SEQ ID NO:8) was made by PCR amplifying the region encoding residues 1—166 of MASP-Z (SEQ ID NO:3) (corresponding to the N—terminal CUBl/EGF domain). A construct expressing the human MASP-2 CUBI/EGF/CUBII domain (SEQ ID NO:9) was made by PCR ying the region encoding aa residues 1-277 of MASP-2 (SEQ ID NO:3) (corresponding to the N—terminal CUBIEGFCUBII domain). A construct expressing the human MASP-2 EGF domain (SEQ ID NO: 10) was made by PCR amplifying the region encoding aa residues 122-166 of MASP-2 (SEQ ID NO:3) sponding to the EGF domain). A construct expressing the human MASP-2 CCPI/CCPII/SP domains (SEQ ID NO:ll) was made by PCR amplifying the region encoding aa es 278-671 of MASP-2 (SEQ ID NO:3) (corresponding to the CCPI/CCPII/SP domains). A construct expressing the human MASP-2 CCPI/CCPII domains (SEQ ID NO:12) was made by PCR amplifying the region encoding aa residues 278-429 of MASP-2 (SEQ ID NO:3) (corresponding to the CCPI/CCPII domains). A construct expressing the CCPI domain of MASP-2 (SEQ ID NO:l3) was made by PCR amplifying the region encoding aa residues 278-347 of MASP-2 (SEQ ID NO:3) (corresponding to the CCPI domain). A construct expressing the CCPII/SP domains of MASP-2 (SEQ ID NO:14) was made by PCR ying the region encoding aa residues 348-671 of MASP-2 (SEQ ID NO:3) sponding to the CCPII/SP domains). A construct expressing the CCPII domain of MASP-2 (SEQ ID NO:15) was made by PCR amplifying the region encoding aa residues 348-429of MASP-2 (SEQ ID NO:3) (corresponding to the CCPII domain). A construct expressing the SP domain of MASP-2 (SEQ ID NO:l6) was made by PCR amplifying the region encoding aa residues 1 of MASP-2 (SEQ ID NO:3) (corresponding to the SP domain).

The above mentioned MASP-2 domains were amplified by PCR using VentR polymerase and SP-2 as a template, according to ished PCR methods. The ' primer sequence of the sense primer introduced a BamHI restriction site lined) at the 5' end of the PCR products. Antisense primers for each of the MASP-2 domains were designed to uce a stop codon followed by an ECORI site at the end of each PCR product. Once amplified, the DNA fragments were digested with BamHI and EcoRI and cloned into the ponding sites of the pFastBacl vector. The resulting constructs were characterized by ction mapping and confirmed by dsDNA sequencing.

Recombinant eukagotic expression of MASP-2 and protein production of enzymatically inactive rat and human MASP-2A.

The MASP-2 and MASP-2A expression constructs described above were transfected into DXBl cells using the standard calcium phosphate transfection procedure (Maniatis et al., 1989). MASP-2A was produced in serum-free medium to ensure that preparations were not contaminated with other serum proteins. Media was ted from confluent cells every second day (four times in . The level of recombinant MASP-ZA averaged approximately 15 mg/liter of e medium for each of the two species.

MASP-ZA p_rotein purification: The MASP-ZA (Ser-Ala mutant described above) was d by affinity chromatography on agarose s. This strategy enabled rapid purification without the use of extraneous tags. MASP-ZA (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 CaClg) was loaded onto an MBP-agarose affinity column (4 ml) pre-equilibrated with 10 ml of loading buffer. Following washing with a further 10 ml of loading buffer, protein was eluted in 1 ml fractions with 50 mM Tris-Cl, pH 7.5, containing 1.25 M NaCl and 10 mM EDTA. Fractions containing the MASP-ZA were identified by SDS-polyacrylamide gel electrophoresis. Where necessary, MASP-ZA was purified further by ion-exchange chromatography on a MonoQ column (HR 5/5).

Protein was dialysed with 50 mM Tris-Cl pH 7.5, containing 50 mM NaCl and loaded onto the column equilibrated in the same buffer. Following washing, bound MASP-ZA was eluted with a 0.05—l M NaCl gradient over 10 ml.

Results: Yields of 0.25—0.5 mg of A protein were obtained from 200 ml of . The molecular mass of 77.5 kDa determined by MALDI-MS is greater than the calculated value of the unmodified polypeptide (73.5 kDa) due to glycosylation.

Attachment of glycans at each of the N-glycosylation sites accounts for the observed mass. A migrates as a single band on lyacrylamide gels, demonstrating that it is not proteolytically processed during thesis. The weight-average molecular mass determined by equilibrium ultracentrifugation is in agreement with the calculated value for homodimers of the glycosylated polypeptide.

EXAMPLE 2 This Example describes the screening method used to identify high y fully human anti-MASP-2 scFv antibody candidates that block MASP-2 functional activity for progression into affinity maturation.

Background and Rationale: MASP-Z is a complex protein with many separate filnctional domains, including: binding site(s) for MBL and ficolins, a serine protease catalytic site, a binding site for proteolytic substrate C2, a binding site for proteolytic substrate C4, a MASP-2 cleavage site for autoactivation of MASP-2 zymogen, and two Ca++ binding sites. scFv antibody fragments were identified that bind with high affinity to MASP-2, and the identified Fab2 fragments were tested in a functional assay to ine if they were able to block MASP-Z functional activity.

To block MASP-2 filnctional activity, an antibody or scFv or Fab2 antibody fragment must bind and interfere with a structural epitope on MASP-Z that is required for MASP-Z filnctional activity. Therefore, many or all of the high affinity binding ASP-Z scFvs or Fab2s may not inhibit MASP-2 filnctional activity unless they bind to structural epitopes on MASP-2 that are directly involved in MASP-2 filnctional activity.

A onal assay that measures tion of lectin pathway C3 convertase formation was used to evaluate the "blocking activity" of anti-MASP-Z scFvs. It is known that the y physiological role of MASP-2 in the lectin pathway is to generate the next onal component of the lectin-mediated complement pathway, namely the lectin pathway C3 convertase. The lectin pathway C3 convertase is a critical enzymatic complex (C4b2a) that proteolytically cleaves C3 into C3a and C3b. MASP-2 is not a structural component of the lectin pathway C3 convertase (C4b2a); however, MASP-2 functional activity is ed in order to generate the two protein components (C4b, C2a) that comprise the lectin pathway C3 tase. Furthermore, all of the separate functional activities of MASP-2 listed above appear to be required in order for MASP-2 to te the lectin y C3 convertase. For these reasons, a preferred assay to use in evaluating the "blocking activity" of anti-MASP-Z Fab2s and scFv antibody fragments is believed to be a filnctional assay that measures inhibition of lectin pathway C3 convertase formation.

The target profile for therapeutic anti-MASP-2 antibodies predicted to yield >90% lectin pathway ablation in viva following administration of 1 mg/kg to a human is an IC50 <5nM in 90% plasma. The relationship between in vitro pharmacological activity in these assay s and in viva pharmacodynamics was validated experimentally using anti-rodent MASP-2 dies.

The criteria for selection of first generation MASP-Z blocking antibodies for therapeutic use were as follows: high affinity to MASP-Z and filnctional IC50 values up WO 51481 to ~25 nM. In addition, candidates were screened for cross-reactivity with non-human primate serum, and with rat serum.

Methods: Screening of scFv phagemid library against MASP-Z antigen Human MASP-ZA with an N—terminal 5X His tag, and rat MASP-ZA with an N- terminal 6X His tags were generated using the reagents described in Example 1 and purified from culture supematants by nickel-affinity chromatograph, as previously described (Chen et al., J. Biol. Chem. 276:25894-02 (2001)).

OMSlOO, a human anti-MASP-2 antibody in Fab2 format, was used as a positive control for binding MASP-Z.

Phagemid Library Description: A phage display library of human immunoglobulin light and heavy chain le region sequences was subjected to antigen panning followed by ted antibody screening and selection to identify high affinity scFv antibodies to rat MASP-2 protein and human MASP-2 protein.

Panning Methods: ew: Two panning strategies were used to isolate phages from the phagemid library that bound to MASP-2 in a total of three rounds of panning. Both gies involved g in solution and fishing out phage bound to MASP-Z. MASP- 2 was immobilized on magnetic beads either via the His-tag (using NiNTA beads) or via a biotin (using Streptavidin beads) on the target.

The first two panning rounds involved ne elution (TEA), and the third panning round was first eluted competitively with MBL before a conventional alkaline (TEA) elution step. Negative selection was carried out before rounds 2 and 3, and this was against the filnctional analogs, Cls and Clr of the classical complement pathway.

After panning, specific enrichment of phages with scFv fragments against MASP-ZA was monitored, and it was ined that the panning strategy had been successful (data not shown).

The scFv genes from panning round 3 were cloned into a pHOG expression vector, and run in a small-scale filter screening to look for specific clones t MASP- 2A, as further described below.

TABLE 7: Phae Pannin Methods biotin/streotaVidin g magnetic Round n ( _) beads block re n annin_ elution l biotin human streptaVidin 4% blot nothing TEA (alkaline) MASP-2A lO block 2 biotin rat MASP- streptaVidin 4% blot C l s/C lr TEA (alkaline) 2A block 3 biotin human streptaVidin 4% blot C l s/C lr Competition MASP-2A block w/MBL, followed by (1 Mg) TEA alkaline TABLE 8: Phae Pannin Methods HIS/NiNTA Panning magnetic Round Anti 1 en 1 beads block I re n annin_ elution human MASP-2A NiNTA 4% milk nothing TEA (alkaline) His taed 10 g in PBS 2 rat MASP-2A His 4% milk Cls/Clr TEA ine) tagged in PBS 3 biotin human 4% milk C l s/C lr Competitively MASP-2A in PBS with MBL + TEA (alkaline) Panning Reagents: Human MASP-2A OMSlOO antibody (positive control) Goat anti-human IgG (H+L) e #31412) NiNTA beads (Qiagen #LBl3267) Dynabeads® M-280 StreptaVidin, 10 mg/ml (LB12321) Normal human serum (LB 13294) Polyclonal rabbit anti-human C3c (LB 1 3 137) Goat anti-rabbit IgG, HRP (American Qualex #AlO2PU) To test the tagged MASP-2A antigen, an ment was carried out to capture the positive control OMSlOO antibody (200 ng/ml) preincubated with biotin-tagged MASP-2A or gged MASP-2A antigen (10 ug), with 50 ul NiNTA beads in 4% milk PBS or 200 ul Streptavidin beads, respectively. Bound MASP-2A-OMSlOO antibody was detected with Goat-anti-human IgG (H+L) HRP (1:5000) and TMB (3 ,3',5 ,5 '-tetramethylbenzidine) substrate.

NiNTA beads ELISA Assay 50 ul NiNTA beads were blocked with 1 ml 4% milk in phosphate buffered saline (PBS) and incubated on a rotator wheel for 1 hour at room temperature. In parallel, 10 ug of MASP-2A and OMSlOO antibody (diluted to 200 ng/ml in 4% milk-PBS) were pre- incubated for one hour. The beads were then washed three times with 1 ml PBS-T using a magnet between each step. The A pre-incubated with OMSlOO antibody was added to the washed beads. The mixture was incubated on a rotator wheel for 1 h at RT, then washed three times with 1 ml PBS-T using a magnet as described above. The tubes were incubated for 1 hr at RT with Goat anti-human IgG (H+L) HRP diluted 1:5000 in 4% milk in PBS. For negative controls, nti-human IgG (H+L) HRP (1:5000) was added to washed and blocked Ni-NTA beads in a separate tube.

The samples were incubated on rotator wheel for 1 hour at room temperature, then washed three times with 1 ml PBS-T and once with lx PBS using the magnet as described above. 100 ul TMB substrate was added and incubated for 3 min at room temperature. The tubes were placed in a magnetic rack for 2 min to concentrate the beads, then the TMB solution was transferred to a microtiter plate and the reaction was stopped with 100 ul 2M H2S04. Absorbance at 450nm was read in the ELISA .

Streptavidin beads ELISA Assay This assay was carried out as described above for the NiNTA beads ELISA Assay, but using 200 ul Streptavidin beads per sample d, and non-biotinylated antigens.

Results: The His-tagged and biotin-tagged MASP-2A antigen, ubated with the positive control OMSlOO antibody, were each successfully captured with NiNTA beads, or Streptavidin beads, tively.

Panning Three rounds of g the scFv phage library against HIS-tagged or biotin- tagged MASP-2A was carried out as shown in TABLE 7 or TABLE 8, respectively. The third round of panning was eluted first with MBL, then with TEA (alkaline). To monitor the specific enrichment of phages displaying scFv fragments against the target MASP- 2A, a onal phage ELISA against immobilized MASP-2A was carried out as described below.

MASP-ZA ELISA on Polyclonal phage enriched after Panning After three rounds of panning the scFv phage library against human MASP-2 as described above, specific enrichment of phages with scFv fragments against the target MASP-2A was monitored by ng out an ELISA assay on the enriched polyclonal phage populations generated by g against immobilized MASP-2A as described below.

Methods: ng/ml MASP-2A was immobilized on maxisorp ELISA plates in PBS overnight at 4°C. The packaged phages from all three panning rounds were diluted 1:3 in 4% Milk- PBS and titrated with 3-fold dilutions. The negative l was M13 helper phage.

The block was 4% Milk in PBS. The plates were washed 3X in 200 ul PBS- Tween 0.05% (v/v) between every step. The primary antibody was Rabbit u-fd (M13 coat protein), 1:5000 in 4% Milk-PBS (w/v). The conjugate was Rabbit u-Goat -HRP at 1:10.000 in 4% Milk-PBS (w/v). The substrate was ABTS. All volumes, except washes and ng, were 100 ul/well. All incubations were for 1 hour with shaking at room temperature.

The s of the phage ELISA showed a c enrichment of scFv's against MASP-2A for both panning strategies. See FIGURE 2. As shown in FIGURE 2, the strategy involving capture by NiNTA magnetic beads gave enrichment of scFv on phages against MASP-2A after two rounds of panning, s both strategies had good enrichments both in competitive and TEA elution, after the third round of panning. The negative control phage was M13 helper phage, which showed no cross reaction against MASP-2A at its lowest dilution. These results demonstrate that the signal observed is due to scFv specifically binding to MASP-2A.

Filter Screening: ial colonies containing plasmids encoding scFv fragments from the third round of panning were picked, gridded onto nitrocellulose membranes and grown overnight on non-inducing medium to produce master plates. A total of 18,000 colonies were picked and analyzed from the third panning round, half from the competitive elution and half from the subsequent TEA n.

The nitrocellulose membranes with bacterial colonies were d with IPTG to express and secrete a soluble scFv protein and were brought into t with a secondary nitrocellulose membrane coated with MASP-2A antigen along with a parallel membrane coated with 4% milk in PBS (blocking solution).

ScFvs that bound to MASP-2A were detected via their c-Myc tag with Mouse 0L- cMyc mAb and Rabbit d-Mouse HRP. Hits corresponding to scFv clones that were positive on MASP-2A and negative on Milk-PBS were selected for further expression, and subsequent ELISA analysis.

Results: Panning of the scFv phagemid library t MASP-2A followed by scFv conversion and a filter screen yielded 137 ve . The majority of the positive clones came from competitive elution with MBL, using both NiNTA and Streptavidin strategies. All the positive clones were continued with micro expression (200 ul scale) and subsequent extraction. ScFv were isolated from the periplasma of the bacteria by incubating the bacteria suspension with sucrose lysis buffer and lysozyme for one hour, after which the supernatant was isolated by a filgation step. The atant containing scFv secreted into the medium together with the contents of the periplasma was analyzed by two assays: ELISA using physically adsorbed MASP-2A, and binding analysis using amine coupled MASP-2A to a CMS chip on the Biocore, as described in more detail below.

MASP-ZA ELISA on ScFv Candidate Clones identified by panning/scFv conversion and filter screening Methods: 4 ug/ml MASP-2A was lized on maxisorp ELISA plates (Nunc) in PBS overnight at 4°C. The next day, the plates were blocked by washing three times with PBS- Tween (0.05%). Crude scFv material (100 ul medium-periplasma extract) from each of the 137 scFv candidates ated as described above) was added per well to the plate.

Next, anti-cMyc was added, and in the final step HRP-conjugated Rabbit anti-Mouse was applied to detect bound scFv. The reaction was developed in peroxidase substrate l-step ABTS (Calbiochem). The positive control was OMSlOO (an anti-MASP-Z antibody in Fab2 format) diluted to 10 ug/ml in PBS-Tween 0.05%. The negative control was medium-periplasma from XLl-Blue without plasmid.

Washes of 3x 200 ul een 0.05% (v/v) were d out between every step.

The y antibody was murine u-cMyc, 1:5000 in PBS-Tween 0.05% (w/v).

The conjugate was rabbit u-Goat-HRP at 1:5000 in PBS-Tween 0.05% (w/v) or Goat anti-human IgG (H+L, Pierce 31412). The substrate was ABTS, with 15 minutes incubation at room temperature. All volumes, except washes and blocking, were 100 ul/well. All tions were for 1 hour with shaking at room temperature.

Results: 108/137 clones were positive in this ELISA assay (data not shown), of which 45 clones were fithher analyzed as described below. The positive control was OMSlOO Fab2 d to 10 ug/ml in PBS-Tween, and this clone was positive. The negative control was medium-periplasma from XLl-Blue without plasmid, which was negative.

EXAMPLE 3 This Example describes the MASP-2 fianctional screening method used to analyze the high affinity fully human anti-MASP-2 scFv antibody candidates for the ability to block MASP-Z activity in normal human serum.

Rationale/Background Assay to Measure Inhibition of Formation of Lectin Pathway C3 Convertase: A fianctional assay that measures inhibition of lectin pathway C3 convertase formation was used to evaluate the "blocking ty" of the anti-MASP-2 scFv candidate clones. The lectin pathway C3 convertase is the tic complex (C4b2a) that proteolytically cleaves C3 into the two potent proinflammatory fragments, anaphylatoxin C3a and opsonic C3b. Formation of C3 convertase appears to a key step in the lectin y in terms of mediating inflammation. MASP-2 is not a ural component of the lectin pathway C3 convertase (C4b2a); therefore anti-MASP-2 antibodies (or Fab2) will not directly t activity of preexisting C3 convertase.

However, MASP-2 serine protease ty is required in order to generate the two protein components (C4b, C2a) that se the lectin y C3 convertase. Therefore, ASP-2 scFV which inhibit MASP-2 fianctional activity (i.e., blocking anti-MASP-2 scFV) will inhibit de novo formation of lectin pathway C3 tase. C3 contains an unusual and highly reactive thioester group as part of its structure. Upon cleavage of C3 by C3 convertase in this assay, the thioester group on C3b can form a covalent bond with hydroxyl or amino groups on macromolecules immobilized on the bottom of the plastic wells Via ester or amide linkages, thus facilitating detection of C3b in the ELISA assay.

Yeast mannan is a known activator of the lectin pathway. In the following method to e formation of C3 convertase, plastic wells coated with mannan were incubated with diluted human serum to te the lectin pathway. The wells were then washed and assayed for C3b immobilized onto the wells using standard ELISA methods.

The amount of C3b generated in this assay is a direct ion of the de novo formation of lectin pathway C3 convertase. Anti-MASP-2 scFv's at selected concentrations were tested in this assay for their ability to t C3 convertase formation and consequent C3b generation.

Methods: The 45 candidate clones identified as described in Example 2 were expressed, purified and diluted to the same stock concentration, which was again diluted in Ca+Jr and Mg++ containing GVB buffer (4.0 mM barbital, 141 mM NaCl, 1.0 mM MgC12, 2.0 mM CaClZ, 0.1% gelatin, pH 7.4) to assure that all clones had the same amount of buffer. The scFv clones were each tested in triplicate at the concentration of 2 ug/ml. The positive control was OMS100 Fab2 and was tested at 0.4 ug/ml. C3c formation was monitored in the presence and absence of the scFv/IgG .

Mannan was diluted to a concentration of 20 ug/ml (1 ug/well) in 50mM carbonate buffer (15mM Na2C03 + 35mM NaHCO3 + 1.5 mM NaN3), pH 9.5 and coated on an ELISA plate overnight at 4°C. The next day, the mannan coated plates were washed 3X with 200 ul PBS. 100 ul of 1% HSA blocking solution was then added to the wells and incubated for 1 hour at room temperature. The plates were washed 3X with 200 ul PBS, and stored on ice with 200 ul PBS until on of the samples.

Normal human serum was diluted to 0.5% in CaMgGVB buffer, and scFv clones or the OMS100 Fab2 positive control were added in triplicates at 0.01 ug/ml; 1 ug/ml (only OMS100 l) and 10 ug/ml to this buffer and preincubated 45 minutes on ice before addition to the d ELISA plate. The reaction was initiated by incubation for one hour at 37°C and was stopped by transferring the plates to an ice bath. C3b deposition was detected with a Rabbit u-Mouse C3c antibody followed by Goat 01-Rabbit HRP. The negative control was buffer t dy (no OMSlOO = maximum C3b deposition), and the positive l was buffer with EDTA (no C3b deposition). The background was determined by carrying out the same assay, but in mannan negative wells. The background signal against plates without mannan was subtracted from the mannan positive signals. A cut-off criterion was set at half of the activity of an irrelevant scFv clone (VZV) and buffer alone.

Results: Based on the cut-off criteria, a total of 13 clones were found to block the activity of MASP-2 as shown in FIGURES 3A and 3B. All 13 clones producing > 50% pathway suppression were ed and sequenced, yielding 10 unique clones, as shown below in TABLE 9. The ten different clones shown in TABLE 9 were found to result in acceptable onal activity in the complement assay. All ten clones were found to have the same light chain ss, k3, but three different heavy chain subclasses, VH2, VH3 and VH6. The sequence ty of the clones to germline sequences is also shown in TABLE 9.

TABLE 9: 10 Uniue Clones with Functional anti-MASP-2 Activit Germline Germline Bio- VH identity VL identity Clone name ELISA core Panning Elution subclass subclass 18P15 Strep- Comp/T VH6 95.62 )3 tavidin EA (13C24/6118) Strep- TEA/Co VH2 . X3 . tavidin mp (18L16) Strep- Comp VH2 . X3 tavidin (171310) 17L20 Strep- Comp VH6 96.3 tavidin 4J3 -Strep- Comp/T VH2 98.97 )3 tavidin EA (l6Ll3/4F2) 18Ll6 Strep- Comp VH2 100 )3 tavidin 21Bl7 NiNTA TEA VH3 99.31 9Pl3 WA Comp VH6 17N16 Strep- TEA/Co VH6 99.66 97.85 taVidin mp 3F22 Strep- VH6 100 )3 96.42 taVidin (18C15) As shown above in TABLE 9, 10 different clones with acceptable functional activity and unique sequences were chosen for further analysis. As noted in TABLE 9, some of the clones were detected two or three times, based on identical sequences (see first column of TABLE 9 with clone names).

Ex ression and urif1cation of ten stc Candidate Clones The ten candidate clones shown in TABLE 9 were expressed in one liter scale and purified Via ion exchange in Nickel chromatography. After that a sample of each clone was run on a size exclusion chromatography column to assess the monomer and dimer content. As shown below in TABLE 10, nearly all of the scFV clones were present in the r form, and this monomer on was isolated for r testing and ranking.

TABLE 10: Anal sis of Monomer Content Clone Name Monomer 4D9 97% l8PlS 98% l7D20 95% l7Nl6 93% 3F22 86% 4J3 81% l7L20 98% l8Ll6 92% 9Pl3 89% 21Bl7 91% -82— Testing Monomer Fraction for g and functional activity The clones shown in TABLE 10 were expressed in l L scale, purified on metal chromatography and ion exchange, separated into monomer fraction by size exclusion chromatography (SEC) and functional assays were repeated to determine IC50 values and cross-reactivity.

Functional assay on Monomerfractions: The monomer fraction of the top ten clones, shown in TABLE 10, was d and tested for fimctional IC50 nM in a dilution series in which each received the same concentration of GVB buffer with Calcium and Magnesium and human serum. The scFv clones were tested in 12 dilutions in triplicate. The positive control was OMSlOO Fab2.

C3b deposition was monitored in the ce and absence of antibody. The results are shown below in TABLE 11.

Binding Assay: Binding affinity KD was determined in two ent ways for purified monomer fractions of the ten candidate scFv . MASP-ZA was either lized by amine coupling to a CMS chip, or a fixed concentration of scFv (50 nM) was first captured with amine coupled high affinity c antibody, and next a concentration series of MASP- 2A in solution was passed over the chip. The results are shown below in TABLE 11.

Results: TABLE 11: Summary of functional inhibitory activity (IC50) and MASP-2 binding affinity (KD) for the ten candidate scFv clones assayed in the monomer state Binding Affinity to Inhibitory activity in human MASP-Z Binding Affinity human Human Serum (immobilized) MASP-Z in solution Clone name IC50 (11M) KD (11M) KD (11M) 1 8P 15 (13C24/6118) (18L16) 171320 (171310) 2012/036509 g Affinity to Inhibitory activity in human MASP-Z Binding Affinity human Human Serum (immobilized) MASP-Z in solution Clone name IC50 (11M) KD (11M) KD (11M) /4F2) —— mm 6.1 211317 ND 9Pl3 28.9 220.0 2.4E-ll mm 15.4 3560.0 3F22 (18C15) Discussion of Results: As shown in TABLE ll, in the functional assay, five out of the ten candidate scFv clones gave IC50 nM values less than the 25 nM target ia using 0.5% human serum.

As described below, these clones were fiarther tested in the presence of non-human primate serum and rat serum to determine filnctional ty in other species. With regard to binding affinity, in on, all binding affinities were in the range of low nM or better, whereas in the conventional assay with immobilized MASP-2, only two clones (4D9 and l7D20) had affinities in the low nM range. The observation of higher affinities in the solution based assay is likely a result of the fact that the antigen multimerizes when in solution. Also, when the target is immobilized on the chip (via oriented coupling) the epitope may be masked, thereby reducing the observed affinities in the immobilized assay.

EXAMPLE 4 This Example describes the results of testing the ten candidate human anti-MASP- 2 scFv clones for reactivity with rat MASP-2 and determining the IC50 values of these scFv clones in a fimctional assay to determine their ability to inhibit MASP-2 dependent complement activation in human serum, non-human primate serum, and rat serum.

Methods: Cross-Reactivity with rat MASP-2 The ten candidate scFV clones, shown in TABLE 9 of Example 3, were tested for cross-reactivity against rat MASP-2A in a conventional ELISA assay against adsorbed rat MASP-2A. Rat MASP-2A was diluted to 4 ug/ml in PBS and coated on a Maxisorp ELISA plate (Nunc) overnight at 4°C. The next day, the plate was blocked by washing three times in een (0.05%). The ScFV clones (100 ul) d in 20 ug/ml in PBS-Tween were added to the plate, and further titrated with 4-fold ons three times.

MASP-2A specific stc clones (wells containing bound scFv) were detected with anti- cMyc and rabbit anti-mouse HRP secondary antibody. The reaction was developed in peroxidase substrate TMB (Pierce). The positive control was OMS100 Fab2 diluted to 10 ug/ml in PBS-Tween. All the tested clones showed cross reaction with rat MASP-2A, which was expected since the second panning round was with rat MASP-2 (data not shown).

Functional terization of the ten candidate scFV clones in human serum non- human rimate HP serum and rat serum Determination ofbaseline C36 levels in Dzflerent Sera First, an experiment was carried out to compare the ne C3b levels in the three sera (human, rat and NHP) as follows.

Mannan was diluted to 20 ug/ml and coated on an ELISA plate ght at 4°C.

The next day wells were blocked with l% HSA. Normal human, rat and African Green Monkey serum (non-human primate "NHP") serum was diluted starting at 2% with two- fold dilutions in CaMgGVB buffer. The reaction was initiated by incubation for one hour at 37°C, and was stopped by transferring the plate to an ice bath. C3b deposition was detected with a rabbit anti-mouse C3c antibody, followed by goat anti-rabbit HRP. The negative control was buffer without antibody (no OMS100 results in maximum C3b deposition) and the ve control for inhibition was buffer with EDTA (no C3b tion).

FIGURE 4 cally rates the baseline C3c levels in the three sera (human, rat and NHP). As shown in FIGURE 4, the C3c levels were very different in the different sera tested. When comparing the C3c levels, it appeared that 1% human serum gave equivalent levels as 0.2% NHP and 0.375% rat serum. Based on these results, the concentrations of sera were normalized so that the scFv results could be ly compared in the three different types of sera.

Functional Assay oft/w SCFV clones in Dzflerent Sera d monomer fractions of the ten candidate scFv clones were then tested for functional IC50 nM in human serum, rat serum and African green monkey serum (non- human primate "NHP"). The assay was performed as described in Example 3, using 1000 nM scFv purified protein and either normal human serum that was diluted to 0.9% in CaMgGVB buffer; African Green Monkey serum d to 0.2% in CaMgGVB ; or Rat serum d to 0.375% in CaMgGVB buffer. All ten scFv clones were tested in a dilution series in which they received the same concentration of GVB buffer with calcium and magnesium and serum. The scFv clones were tested in twelve dilutions in triplicates. The positive control was OMSlOO Fab2 at 100 ng/ml or addition of EDTA to the reaction. The negative control was an irrelevant scFv control or PBS with no scFv.

C3b deposition was monitored in the presence and absence of scFv or Fab2 antibody.

The ound signal of OMSlOO at 100 ng/ml was subtracted from all signals.

TABLE 12 summarizes the results of the fianctional assays in all three sera.

TABLE: 12: Functional IC50 (nM) activity of the scFv clones in three different types of sera.

Non- Non- human human primate primate Clone name Exp #1 Exp #2 (13C24/6118) (18L16) (171310) 17L20 ND 104.3 308.1 198.9 ambiguous 71.74 40 97 4J3 54.9 105.6 123.8 41.64 180.9 168.3 ND (16L13/4F2) mm 6.1 21317 ND ---—-ND Non- Non- human human primate primate Clone name Exp #1 Exp #2 9P13 28.9 120.5 17.28 24.26 99.29 77.1 ND 17N16 15.4 65.42 24.78 19.16 95.57 58.78 ND 3F22 20.6 36.73 41.40 68.81 114.2 172.8 ND (18C15) Note: * the first set of data on human serum (Exp #1) was done on scFv samples that were not concentrated, therefore, clones with low concentration could not be titrated fully. In the remaining experiments, all clones were concentrated and titrations started at identical concentrations.

Summa of results for onal activit in scFv ate clones in different sera: All ten of the scFv clones showed function in both human and non-human primate (NHP) serum after the sera had been normalized with t to C3b deposition levels.

The six most active clones in human serum were: 9Pl3>l7Nl6>l7D20>4D9>3F22>l8Ll6, when ranked from best to worst. In NHP serum, the clones ranked (best to worst): l7L20>l7Nl6>l7D20>9Pl3>l8L16>3F22.

Both l7Nl6 and 17D20 ranked in the top three for both human and NHP sera. 17D20 also showed some activity in rat serum.

Based on these results, the top three scFv clones were determined to be: l8Ll6, 17D20 and l7Nl6. These three clones were fiarther analyzed in dilute human serum (1% serum) as shown below in TABLE 13.

TABLE 13: C3 Assay of the three candidate clones: (IC50 nM) in dilute serum human serum 17D20 17N16 Ex #1 19 #14 Ex #2 24 #14 Ex #3 65 #14 mean 29 +/- 5 36 +/- 15 mmm mm I———— 154m 110m FIGURE 5A is an amino acid sequence alignment of the filll length scFv clones 17D20, 18Ll6, 4D9, l7L20, 17Nl6, 3F22 and 9P13. The scFv clones comprise a heavy chain variable region (aal-lZO), a linker region (aalZl-l45), and a light chain variable region (aa 146-250). As shown in FIGURE 5A, ent of the heavy chain region (residues l-120) of the most active clones reveals two distinct groups belonging to VH2 and VH6 gene family, respectively. As shown in FIGURE 5A, the VH region with respect to the clones of the VH2 class: 17D20, 18Ll6 and 4D9 has a variability in 20 aa positions in the total 120 amino acid region (i.e. 83% identity).

As further shown in FIGURE 5A, the VH region with t to the clones of the VH6 class: l7L20, l7Nl6, 3F22, and 9Pl3, has a variability in 18 aa positions in the total 120 amino acid region (i.e. 85% identity).

FIGURE 5B is a sequence alignment of the scFv clones 17D20, l7Nl6, 18Ll6 and 4D9.

TABLE 14: Seuence of ScFv Candidate clones shown in FIGURE 5A and 5B Clone Reference ID full len_th AA se u uence 17D20 SEQ ID \O:55 18L16 SEQ ID \O:56 4D9 SEQ ID \O:57 l7L20 SEQ ID \0258 l7Nl6 SEQ ID \O:59 3F22 SEQ ID \O:60 9Pl3 SEQ ID \O:6l The ranking ties were (1) human serum functional potency and full blockage; (2) NHP cross-reactivity and (3) sequence diversity. 17D20 and l7Nl6 were selected as the best representatives from each gene family. l8Ll6 was selected as the third candidate with appreciable CDR3 sequence diversity.

WO 51481 2012/036509 l7Nl6 and l7D20 were the top two choices due to complete functional blockage, with the best fimctional potencies against human; appreciable monkey cross-reactivity and different VH gene families. 3F22 and 9Pl3 were eliminated due to VH sequences nearly identical to l7Nl6. 18P15, 4J9 and 21Bl7 were eliminated due to modest potency. l7L20 was not pursued because it was only partially blocking. 18Ll6 and 4D9 had similar actiVities and appreciable diversity compared to l7D20. 18Ll6 was chosen due to greater primate cross-reactivity than 4D9.

Therefore, based on these criteria: the following three mother clones: l7D20, l7Nl6 and 18Ll6 were ed into affinity maturation as further described below.

EXAMPLE 5 This Example describes the cloning of three mother clones l7D20, l7Nl6 and 18Ll6 (identified as described in Examples 2-4) into wild-type IgG4 format, and assessing the functionality of three mother clones as full length IgGs.

Rationale: Fully human anti-MASP-Z scFV dies with moderate onal potency were identified using phage y as described in Examples 2-4. Three such mother clones, l7D20, l7Nl6 and 18Ll6 were selected for affinity maturation. To assess the functionality of these mother clones as full length IgGs, IgG4 wild-type and S228P hinge region IgG4 mutant forms of these antibodies were produced. The S228P hinge region mutant was included to increase serum stability (see Labrijn A.F. et al., Nature Biotechnology 27:767 (2009)).

The amino acid sequence of IgG4 wild-type is set forth as SEQ ID NO:63, encoded by SEQ ID NO:62.

The amino acid ce of IgG4 S228P is set forth as SEQ ID NO:65, encoded by SEQ ID NO:64.

The IgG4 molecules were also cleaved into F(ab')2 formats with pepsin digestion and fractionated by size exclusion tography in order to compare the mother clones directly to the OMSlOO control dy, which is a F(ab)2 molecule.

Methods: Generating the clones intofull length format WO 51481 The three mother clones were converted into wild type IgG4 format and into IgG4 mutant S228P format. This was accomplished by PCR isolation of the appropriate VH and VL regions from the above-referenced mother clones and cloning them into pcDNA3 expression vectors ing the appropriate heavy chain nt regions to create in- frame fusions to produce the desired antibody. The three mother clones in mutant IgG4 format were then cleaved with pepsin to generate F(ab')2 fragments and the latter were purified by fractionation on a size exclusion chromatography column.

Binding assay The candidate mother clones converted into IgG4 format were transiently transfected into HEK 293 cells and supematants from the transient transfection were titrated in an ELISA assay. The clones showed excellent reactivity with physically adsorbed human MASP-2A, and ranked in the following order: 17N16>17D20>18L16 (data not shown).

The clones were then purified and re-tested in an ELISA and activity assay as follows. Human MASP-2A was coated at 3 ug/ml in PBS on a maxisorp plate, IgG (45 ug/ml) and Fab'2 (30 ug/ml) were diluted in PBS-Tween to a ng concentration of 300 nM, and fiarther with 3-fold dilutions. IgGs were detected with HRP conjugated Goat u-Human IgG (Southern Biotech) and the F(ab')2 were detected with HRP-conjugated Goat (x-Human IgG H+L (Pierce 31412). The reaction was developed with TMB substrate and stopped with 2M H2S04. The results are shown below in TABLE 15.

TABLE 15: Bindin affinit to human MASP-2 Antibody Clone IgG4 mutated Reference format M F ab' 2 M scFv nM OMSIOO control 18m 178.7 171320 181.5 FunctionalAssay The C3 convertase assay using 1% normal human serum (NHS), as described in Example 4, was used to compare the functional activity of the mother scFv clones and full length IgG4 rparts in 1% NHS. Mannan was d to a concentration of 20 ug/ml and coated on ELISA plate overnight at 4°C. The next day, the wells were blocked with 1% human serum. Human serum was diluted to 1% in CaMgGVB buffer and the purified dies; scFv (900 nM), 2 (300 nM), IgG (300 nM) were added in duplicates at a series of different dilutions to the same amount of buffer, and preincubated for 45 minutes on ice before adding to the blocked ELISA plate. The reaction was initiated by incubation at 37°C for one hour and was stopped by placing the plate on ice.

C3b deposition was determined with a Rabbit 01-Mouse C3c antibody followed by a Goat (x-Rabbit HRP. The background of OMSlOO at 50 nM on mannan positive plates was subtracted from the curves. A summary of the results of this analysis are shown below in TABLE 16.

TABLE 16: C3 convertase assay using 1% human serum (IC50 nM) F(ab')2 scFv Fold improvement scFv clone ID# (IC50 nM) (IC50 nM) (scFv to divalent form) 17D20 7392/1032 7305/1354 9827/1510 ~13.5X/~12.6X 17N16 5447/3088 5701/5092 3618/7760 ~6.6X/~l9.3x 18L16 33.93/220 160.2/193.0 ~8.7X Note: The two values shown in columns 2-4 of Table 16 refer to the results of two separate experiments.

The functional potency of the IgG4 mother clones were also compared to the IgG4 hinge mutant ) format for each clone. The numeric IC50 values for the C3b tion assay using 1% NHS are shown below in TABLE 17.

TABLE 17: Wild type (IgG4) versus Hinge Mutant format (SZ28P) in C3b deposition assay in 1% human serum (IC50 nM) Clone ID WT format (I_G4) I_G4 hin_e mutant (SZZSP) mm 11/27 1114 2012/036509 As shown above in TABLE 17, in some cases, unexpected agonist cology was noted for IgG's derived from antagonistic scFv's. The mechanistic basis for this observation is not understood.

The activities of IgG4 converted mother clones with inhibitory function in 1% NHS were r evaluated under more ent assay conditions that more closely mimic physiological conditions. To estimate antibody activity under physiological conditions, testing of mother clone IgG4 preparations was conducted for their ability to inhibit Lectin-pathway (LP) dependent C3b deposition onto Mannan-coated plates under stringent assay conditions using lly diluted (90%) human .

The results of the C3b deposition assay in 90% human plasma are shown in FIGURE 6. Since MASP-2 and its substrates are t in the assay mixture at approximately lOO-fold higher concentration than in the dilute serum assay using 1% normal human serum, a right-shift of the antagonist dose-response curve is generally ed. As shown in FIGURE 6, as expected, a right-shift to lower apparent potencies was observed for OMSlOO and all the MASP-2 antibodies tested. However, surprisingly, no reduction in apparent potency was observed for the hinge region mutant (SZ28P) of l7D20, and the potency in this format was comparable to that measured in 1% plasma (see TABLE 17). In the 90% NHS assay format the functional potency of l7D20 IgG4 ($228) was found to be modestly lower than OMSlOO Fab2, which is in contrast to the assay results in 1% NHS where OMSlOO was 50 to lOO-fold more potent than 17D20 IgG4 8228P (data not shown). The wild type IgG4 form of l7Nl6 also showed full inhibition in 90% NHS but was somewhat less potent in this assay format (IC50 of z lSnM) while the wild type IgG4 form of 18Ll6 was less potent and only partially inhibitory, as shown in FIGURE 6.

Based on these findings, the activity of IgG4 converted mother clones was fiarther evaluated by ing C4b deposition under stringent assay conditions (90% NHS).

This assay format provides for a direct measure of antibody activity on the enzymatic reaction catalyzed by MASP-Z.

Assay to Measure Inhibition of MASP-Z-dependent C4 Cleavage Background: The serine protease activity of MASP-2 is highly ic and only two protein substrates for MASP-2 have been identified; C2 and C4. Cleavage of C4 generates C4a and C4b. Anti-MASP-2 Fab2 may bind to 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 C4 cleavage functional activity of MASP-2.

Yeast mannan is a known activator of the lectin y. In the following method to measure the C4 cleavage activity of , plastic wells coated with mannan were incubated for 90 minutes at 4°C with 90% human serum to activate the lectin pathway. The wells were then washed and assayed for human C4b immobilized onto the wells using standard ELISA methods. The amount of C4b generated in this assay is a e of MASP-2 dependent C4 cleavage activity. Anti-MASP-2 antibodies at selected concentrations were tested in this assay for their ability to inhibit C4 cleavage.

Methods: 96-well Costar Medium Binding plates were incubated overnight at °C with mannan diluted in 50 mM carbonate , pH 9.5 at 1.0 ug/50 uL/well. Each well was washed 3X with 200 uL PBS. The wells were then d with 100 uL/well of 1% bovine serum albumin in PBS and incubated for one hour at room temperature with gentle . Each well was washed 3X with 200 uL of PBS. Anti-MASP-2 antibody samples were diluted to selected concentrations in Ca++ and Mg++ containing GVB buffer (4.0 mM barbital, 141 mM NaCl, 1.0 mM MgC12, 2.0 mM CaClz, 0.1% gelatin, pH 7.4) at ° C. 90% human serum was added to the above samples at 5°C and 100 uL was transferred to each well. The plates were covered and incubated for 90 min in an ice waterbath to allow complement activation. The on was stopped by adding EDTA to the reaction mixture. Each well was washed 5 X 200 uL with PBS-Tween 20 (0.05% Tween 20 in PBS), then each well was washed with 2X with 200 uL PBS. 100 uL/well of 1:700 dilution of biotin-conjugated chicken anti-human C4c (Immunsystem AB, Uppsala, Sweden) was added in PBS containing 2.0 mg/ml bovine serum albumin (BSA) and incubated one hour at room temperature with gentle mixing. Each well was washed x 200 uL PBS. 100 l of 0.1 ug/ml of peroxidase-conjugated streptavidin (Pierce Chemical #21126) was added in PBS containing 2.0 mg/ml BSA and incubated for one hour at room temperature on a shaker with gentle . Each well was washed 5 x 200 uL with PBS. 100 uL/well of the peroxidase substrate TMB (Kirkegaard & Perry Laboratories) was added and incubated at room temperature for 16 min. The peroxidase reaction was stopped by adding 100 uL/well of 1.0 M H3PO4 and the OD450 was measured.

Results: In this format, both IgG4 forms of l7D20 inhibited Lectin pathway driven C4b deposition, although the IC50 values were z3 fold higher compared to the C3b deposition assay. Interestingly, l7Nl6 IgG4 wild type showed good activity in this assay with an IC50 value and dose-response profile comparable to the C3b deposition assay. l8Ll6 was considerably less potent and did not achieve complete inhibition in this format (data not shown).

Discussion: As described in es 2-5, fully human anti-MASP-2 scFV dies with functional blocking activity were identified using phage display. Three such clones, l7Nl6, l7D20 and 18Ll6, were ed for affinity maturation and further testing. To assess the fianctionality of these mother clones as filll length IgGs, IgG4 wild type and IgG4 8228P hinge region mutant forms of these antibodies were produced. As described in this Example, the majority of filll length IgGs had improved flinctional actiVity as compared to their scFv counterparts when tested in a standard functional assay format with 1% human . To estimate antibody actiVity under physiological conditions, g of mother clone IgG4 preparations was conducted under stringent assay ions using 90% human plasma. Under these conditions, several antibodies ed fianctional potencies which were substantially better than expected based on their mance in standard (1%) plasma functional assays.

EXAMPLE 6 This Example describes the chain shuffling and affinity maturation of mother clones l7D20, l7Nl6 and 18Ll6, and analysis of the resulting er clones.

To identify antibodies with improved potency, the three mother scFv clones, l7D20, l7Nl6 and 18Ll6, identified as described in Examples 2-5, were subjected to light chain shuffling. This s involved the generation of a combinatorial library consisting of the VH of each of the mother clones paired up with a library of naive, human lambda light chains (VL) derived from six y donors. This library was then screened for scFv clones with improved binding affinity and/or functionality. 9,000 light chain shuffled daughter clones were analyzed per mother clone, for a total of 27,000 clones. Each daughter clone was induced to express and secrete soluble scFv, and was screened for the ability to bind to human MASP-ZA. ScFvs that bound to human MASP-2A were detected via their c-Myc tag. This l screen resulted in the selection of a total of 119 clones, which included 107 daughter clones from the 17N16 library, 8 daughter clones from the 17D20 library, and 4 daughter clones from the 18L16 library.

The 119 clones were expressed in small scale, purified on NiNTA s, and tested for binding affinity in an ELISA assay against physically adsorbed human MASP- Results: The results of the ELISA assay on a representative subset of the 119 daughter clones is shown in FIGURES 7A and B. FIGURE 7A graphically rates the results of the ELISA assay on the 17N16 mother clone versus daughter clones ed on huMASP- 2A. FIGURE 7B graphically illustrates the results of the ELISA assay on the 17D20 mother clone versus er clones titrated on -2A.

As shown in FIGURE 7A, daughter clones 17N16m_d16E12 and 17N16m_dl7N9, derived from the 17N16 mother clone had affinities that were higher than the mother clone. Also, as shown in FIGURE 7B, one clone d from the 17D20 mother clone, 17D20m_d18M24, had a higher affinity that the mother clone.

These three clones, and an additional three clones: 17N16m_d13L12, 17N16m_d16K5, 17N16m_d1G5, and 17D20m_d1824 that had a low expression level were expressed in 0.5 L scale, purified into r fraction by size exclusion chromatography and were retested in an ELISA and functional assay. The 18L16 library did not produce any daughter clones with the desired binding affinity.

After purification, the six daughter clones were tested in a complement assay for inhibitory activity. The results are shown in TABLE 18.

TABLE 18: Comolement assa of mother and dau_hter clones F1101111111 1111161111 17111611 117119 11111611 116112 101.2 — 1111211111 11111124 112.1 — As shown above in TABLE 18, only one of the clones, _dl7N9, had affinity and activity in the same range as the mother clone.

FIGURE 8 is a amino acid sequence alignment of the full length scFv mother clone l7Nl6 (SEQ ID N059) and the l7Nl6m_dl7N9 er clone (SEQ ID NO:66), showing that the light chains (starting with SYE) have 17 amino acid es that differ between the two clones.

Rescreening of the l7Nl6 lambda library resulted in l additional candidate daughter clones, of which l7Nl6m_d27El3 was identified in an ELISA and complement assay, and was included in the set of candidate daughter clones for further analysis.

Assaying daughter clones in different sera The ate daughter clones were ed in different sera as follows.

Mannan was d to 20 ug/ml and coated on an ELISA plate overnight at 4°C. The next day, the wells were blocked with l% HSA. n Green monkey serum was diluted to 0.2%, rat serum was diluted to 0.375% and human serum was diluted to l% in CaMgGVB buffer. Purified scFv from each of the candidate daughter clones was added in duplicates at a series of different concentrations to the same amount of buffer and preincubated for 45 minutes on ice prior to addition to the blocked ELISA plate. The reaction was initiated by incubation for one hour at 37°C, and was stopped by transferring the plate to an ice bath. C3c release was detected with a Rabbit u-Mouse C3c antibody followed by a Goat u-Rabbit HRP. The background of OMSlOO at 0.1 ug/ml on mannan negative plates was subtracted from these curves. The results are summarized below in TABLE 19.

TABLE 19: IC50 values for mother clone l7Nl6 and daughter clones l7N 1 6m dl 7N9 and l7N 1 6m d27E l 3 in different sera.

African Green Serum Human Serum Rat Serum ScFv Clones IC50 (11M) IC50 (11M) IC50 (11M) l7Nl6mc 9293/8137 6531/7354 ND/l95.8 l7Nl6m dl7N9 63.82/8l.ll 39.90/57.67 79.32/l40.6 l7Nl6m d27El3 ND/430.9 389.l/NA NA Note: The two values shown in columns 2-4 of Table 19 refer to the results of two separate experiments.

Discussion of results: As shown in TABLE 19, daughter clone l7Nl6m_dl7N9 has higher functional activity than the mother clone. The improved function in rat serum in addition to the seventeen amino acid sequence difference in the light chain as compared to the mother clone makes this clone a positive candidate. Based on this data, daughter clone l7Nl6m_dl7N9 was selected for r analysis.

EXAMPLE 7 This Example describes the generation and analysis of daughter clone l7D20m_d3521Nl 1, derived from mother clone l7D20. ound/Rationale: To improve on affinity of the mother clone candidate l7D20mc, an additional through-mutagenesis" was performed on the first three amino acids in the CDR3 of the heavy chain (CDR-H3). This was a mutagenesis campaign in el with the normal light chain shuffling of l7D20mc. Therefore, three different scFv libraries were constructed by PCR where the amino acid positions 1, 2 and 3 were randomized to the set of all possible 20 amino acids using degenerate codons. After cloning the libraries, microscale expression was performed and scFv g was monitored on a MASP-ZA coated CMS chip (not . BIAcore is of microscale sion was carried out on the three different libraries on chips coated with MASP-ZA, ized at position 1, 2, or 3 and potentially interesting daughter clones were identified.

It was observed that for the amino acid positions I and 2 of CDR-H3, no clone was found having an improved off-rate in comparison with the mother candidate clone l7D20m. However, a few ates with mutations in amino acid position 3 in the CDR-H3 demonstrated improved off-rates in comparison with the mother clone l7D20m.

These clones (#35, #59 and #90) were sequenced to identify the mutation. Sequences of two "look-through-mutagenesis" derived clones are compared with l7D20mc (original sequence). Interestingly, all sequenced clones except one (#90), harbored an Ala-Arg substitution in comparison with the mother candidate.

FIGURE 9 is a sequence ison of the amino acid sequence of the heavy chain region of the scFv mother clone l7D20m (aa 61-119 of SEQ ID NO:18) and the amino acid sequence of the CRD-H3 region of scFv clones with ons in CDR—H3, clone #35 (aa 61-1 19 of SEQ ID NO:20, having a substitution of R for A at position 102 of SEQ ID NO:l8), clone #59 (same sequence as clone #35), and clone #90 (substitution ofP for A at position 102 of SEQ ID NOilS).

Analysis of Mutant clones #35 and #59 The mutant clones #35 and #59 were expressed in small scale and fithher tested in ison with the mother ate clone l7D20 in a titration-ELISA on immobilized MASP-2A (10 ug/ml). The scFvs were serially diluted 5-fold starting from 20 ug/ml and binding was detected using anti-Myc (mouse)/anti-mouse HRP. Slightly improved binding was observed in the ELISA assay for the candidate clones #35 and #59 in comparison with the mother candidate clone l7D20 (data not shown).

The improved clone #35 was combined with the best light chain shuffled clone l7D20m_d21Nll. The mutation in the VH of the ate l7D20md35 (Ala-Arg) was combined with the light chain of the candidate l7D20m_d21Nll, thus resulting in the clone termed VH35-VL21Nl 1, otherwise referred to as 3521Nl 1.

FIGURE 10A is an amino acid sequence alignment of sequence of the CDR3 region of mother clone l7D20 (aa 61-1 19 of SEQ ID NO:18), the same region of daughter clone _d21Nl 1, having the same sequence, and the same region of the mutagenesis clone #35 combined with the VL of l7D20m_d21Nll, referred to " 3521Nl 1" (aa 61-1 19 of SEQ ID NO:20). The highlighted VH sequence regions se the CDRH3, and mutated target e region is ined.

FIGURE 10B is a protein sequence alignment of the full length scFv including VL and VH regions of the l7D20 mother clone (SEQ ID NO:55) and the daughter clone l7D20m_d21Nll (SEQ ID NO:67). scFv daughter clone l7D20m_d3521Nll is set forth as SEQ ID NO:68. Note: it has been determined that the X residue in FIGURE lOB at position 220 is an "E", as set forth in SEQ ID NO:68.

A titration ELISA assay of the set of scFvs shown in FIGURE 10 was run on MASP-2 (10 ug/ml). The results are shown in TABLE 20.

TABLE 20: ELISA on human MASP-2 Clone ID KD (11M) mom mm mono ooszmu nozonno nonoennn 17D20md#35 r The 17D20m_d3521N11 daughter clone was fiarther analyzed for functional activity as described below in Example 8.

EXAMPLE 8 This Example describes the conversion and analysis of the candidate daughter clones l7Nl6m_dl7N9 and 17D20m_d3521N11 into IgG4, 228P and IgG2 format.

Rationale/Background The antibody screening methods bed in Examples 2-7 have identified two mother clones, 17N16 and 17D20, with le functionality. Affinity maturation of these mother clones has yielded daughter clones that showed approximately 2-fold improvements in potency as compared to the mother clones in surrogate functional assays at the scFv level. The daughter clones with the best activities are 17N16m_d17N9 and 17D20m_d3521N11. As described in Example 6, in a comparison of fianctional activity of 17N16 mother clone with light chain shuffled daughter clones (scFv format, 1% NHS assay) it was determined that 17N16m_d17N9 is slightly more potent than the mother clone and has the best onal potency of all daughter clones tested in this assay.

Methods: A comparison of the functional potency of the candidate scFv clones was carried out in the C3 conversion assay (1% human serum and 90% human , and in a C4 conversion assay (90% human serum), carried out as described in Example 5.

The results are shown below in TABLE 21.

TABLE 21: Comparison of functional potency in IC50 (nM) of the lead daughter clones and their res oective mother clones all in scFv format 1% human serum 90% human serum 90% human serum C3 assay scFv clone (IC50 nM) mono 2012/036509 l7D20m leNll l7D20m d3521Nll l7Nl6mc 17N16m d17N9 l7N16m d27E13 As shown above in TABLE 21, l7Nl6m_dl7N9 has good activity when assayed in 90% normal human serum (NHS) in the C3 assay and is more potent that the other daughter clones in this format.

Conversion of Candidate Clones into IgG4= IgG4/S228P and IgG2 format All of these candidate clones were converted to IgG4, IgG4/S228P and IgG2 format for fiarther is.

SEQ ID NO:62: cDNA encoding wild type IgG4 SEQ ID NO:63: wild type IgG4 polypeptide SEQ ID NO:64 cDNA encoding IgG4 mutant S228P SEQ ID NO:65: IgG4 mutant S228P polypeptide SEQ ID NO:69: cDNA encoding wild type IgG2 SEQ ID NO: 70: wild type IgG2 polypeptide TABLE 22: Summa of ate clones: #1 OMS641 17N16m d17N9 SEQID\O:21 SEQID \0272 ----VIS642#2 : ----VIS643#3 O:27 # 24 OVIS644 ----VIS645#5 \O:24 6 : 24 OVIS646 -lOO- Monoclonal antibodies #1-6 were tested for the ability to cross-react with a non- human MASP-2 protein (African Green (AG) monkey) in a C3 assay to determine if these antibodies could be used to test for toxicity in an animal model that would be predictive for humans. Monoclonal antibodies #1-6 were also tested in a C3b deposition assay and a C4 assay in 90% human serum. The results are shown below in TABLE 23.

TABLE 23: Human anti-MASP-2 MoAbs (IC50 nM) in 90% human serum Assa MOAb#l MOAb#2 MOAb#3 MOAb#4 MOAb#S Human C3 20 3 12 2 3 Assa Human C4 30 30 30 5 5 assa African nd 26 nd 1 8 l 6 Green Monkey C3 assa FIGURE 11A graphically rates the results of the C3b tion assay carried out for the daughter clone e variants (MoAb#l-3), derived from the human ASP-2 monoclonal antibody mother clone 17Nl6.

FIGURE 11B graphically illustrates the results of the C3b deposition assay carried out for the daughter clone e variants (MoAb#4-6), d from the human anti-MASP-2 monoclonal antibody mother clone l7D20.

As shown in TABLE 23 and FIGURES 11A and 11B, the human anti-MASP-2 monoclonal antibodies (MoAb#l-6) bind MASP-2 with high affinity, and inhibit the function of C3 and C4 ty in 90% human serum. It is also noted that the human anti- MASP-2 MoAbs cross-react with the non-human MASP-2 protein (African Green monkey), which provides an animal model for toxicity studies that would be predictive for humans.

The MoAb#l-6 were further analyzed in 95% human serum, 95% African Green serum. The results are summarized below in TABLE 24. -lOl- TABLE 24 Antibody ID Binding to Functional Functional Functional immobilized tion of C3 inhibition of inhibition of hMASP-2 tion in C3 tion C4 deposition (average Kd) 95% human in 95% in 95% serum African human Green Serum serum (Average IC50; Average IC90) ge (Average IC50) 17N16 IG4 0.067 4.9;60.3 3.3 MoAb#1 I_G2 0.291 10;104.1 nd 25.6 MoAb#2 I_G4 0.314 11.9;118.0 19.5 MoAb#3 (IgG4 0.323 9.4; 61.0 9.2 19.8 mutant 17D20 1G4 0.073 2.6;19.0 8.5 MoAb#4 1G2 0.085 099.5 12.4 MoAb#5 1G4 0.067 2.6;122.0 7.2 MoAb#6 (IgG4 0.067 1.5; 7.0 13.2 4.5 mutant FIGURE 12A and 12B graphically illustrate the testing of the mother clones and MoAb#1-6 in a C3b deposition assay in 95% normal human serum.

FIGURE 13 graphically illustrates the inhibition of C4b deposition in 95% normal human serum.

FIGURE 14 graphically illustrates the inhibition of C3b deposition in 95% African Green monkey serum.

The MoAb#1-6 were further tested for the ability to selectively inhibit the lectin y by assaying for inhibition of Rat C3b, inhibition of preassembled MBL-MASP-2 complexes; classical pathway inhibition, and selectivity over Cls. The results are summarized in TABLE 25.

TABLE 25: Summa of functional assa results Antibody ID Inhibition of Inhibition of Classical Selectivity Rat C3b embled Pathway over Cls MBL-MASP-2 inhibition complexes IC50 (nM) l 7N1 6 I _G4 nd MoAb#l I_G2 >5000x MoAb#2 (IgG4) not detected not detected >5000x I 200nM MoAb#3 (IgG4 not detected not detected >5000X mutant I 200nM l7D20 I_G4 _d MoAb#4 I_G2 n >5000X MoAb#5 IG4 Yes, IC50= l 7nM not detected >5000x MoAb#6 (IgG4 >500 Yes, IC50=24.lnM not ed >5000X mutant FIGURE 15 graphically illustrates the inhibition of C4 cleavage actiVity of sembled MBL-MASP-Z complex by MoAb#2, 3, 5 and 6.

FIGURE 16 graphically illustrates the preferential binding of MoAb#6 to human MASP-2 as compared to Cls. -lO3- Table 26: Summa of seuences of r clones in various formats: Clone ID Descrition SEQ ID NO: l7\16m dl7\O liht chain ene seuence 71 l7\16m dl7\O liht chain rotein seuence 72 l7\16m dl7\O IG2 hea chain ene seuence 73 l7\16m dl7/ \o I_G2 hea chain orotein seuence 74 l7\16m dl7\O IG4 hea chain ene seuence 75 l7\16m dl7\O I_G4 hea chain orotein seuence 76 l7\16m dl7\O I_G4 mutated hea chain _ene seuence \l \] l 7\ 17m dl7 I_G4 mutated hea chain orotein seuence \I 00 l7D20 3521\ ll liht chain ene e l7D20 3521\ ll liht chain rotein seuence l7D20 3521\ ll IG2 hea chain ene seuence l7D20 3521\ ll IG2 hea chain rotein seuence l7D20 3521\ ll IG4 hea chain ene seuence l7D20 3521\ ll IG4 hea chain rotein seuence l7D20 3521\ ll IG4 mutated hea chain ene seuence 000000000000 kh-hUJNO l7D20 3521\ ll IG4 mutated hea chain rotein seuence l7Nl6m dl7N9 cDNA n filll lenth scFV ol Hetide l7D20m leNll cDNA encodin fiJll lenth scFV ool ooetide l7D20m d3521Nll cDNA encodin fiJll lenth scFV ool ooetide 00000000 OOO\10\ EXAMPLE 9 This Example describes the epitope g that was carried out for several of the blocking human anti-MASP-2 MoAbs.

Methods: The following recombinant proteins were produced as described in Example 1: Human MApl9 Human MASP2A Human MASP-2 SP Human MASP-2 CCPZ-SP Human MASP-2 CCPl-CCPZ-SP —104— Human MASP-l/3 CUBl-EGF-CUB2 Human MASP-l CCPl-CCPZ-SP The anti-MASP-2 antibodies OMSlOO and MoAb#6 (35VH-21NllVL), which have both been demonstrated to bind to human MASP-2 with high affinity and have the ability to block fianctional complement ty (see Examples 6-8) were analyzed with regard to epitope binding by dot blot analysis.

Dot Blot is Serial dilutions of the recombinant MASP-2 polypeptides described above were spotted onto a nitrocellulose ne. The amount of protein spotted ranged from 50 ng to 5 pg, in ten-fold steps. In later experiments, the amount of n spotted ranged from 50 ng down to 16 pg, again in five-fold steps. Membranes were blocked with 5% skimmed milk powder in TBS (blocking buffer) then incubated with 1.0 ug/ml anti-MASP-2 Fab2s in blocking buffer (containing 5.0 mM Ca2+). Bound Fab2s were detected using HRP-conjugated anti-human Fab (AbD/Serotec; diluted 1/10,000) and an ECL detection kit ham). One membrane was incubated with polyclonal rabbit-anti human MASP-2 Ab ibed in Stover et al., J Immunol 48-59 (1999)) as a positive control. In this case, bound Ab was detected using HRP-conjugated goat anti-rabbit IgG (Dako; diluted l/2,000).

Results: The results are summarized in TABLE 27.

TABLE 27: Eooitoe Ma. o_in MoAb#6 OMsmo humanMm _ human MASP-2A Human MASP-2 SP human MASP-2 CCP2-SP _ human MASP-2 CCPl-CCPZ-SP human MASP-l/3 CUB-EGF-CUBII human MASP-l CCP 1 -CCP2-SP human MBL/MASP2 comlexes -lOS- The results show that MoAb#6 and OMSlOO antibodies are highly specific for MASP-2 and do not bind to MASP-l or MASP-3. Neither antibody bound to Mapl9 and MASP-2 fragments not containing the CCPl domain of MASP-2, leading to the conclusion that the binding sites encompass the CCPl domain.

E 10 This Example demonstrates that human anti-MASP-2 MoAb#6 inhibits the lectin pathway in African Green Monkeys following intravenous administration.

Methods: MoAb#6 was administered enously to a first group of three African Green Monkeys at a dosage of 1 mg/kg and to a second group of three African Green Monkeys at a dosage of 3 mg/kg. Blood samples were obtained 2, 4, 8, 10, 24, 48, 72 and 98 hours after administration and were tested for the presence of lectin pathway activity.

As shown in FIGURE 17, the lectin pathway was completely inhibited following intravenous administration of anti-human MoAb#6.

EXAMPLE 11 This Example demonstrates that a MASP-2 inhibitor, such as an anti-MASP-2 antibody, is effective for the treatment of ion exposure and/or for the ent, amelioration or prevention of acute ion syndrome.

Rationale: Exposure to high doses of ng radiation causes mortality by two main mechanisms: toxicity to the bone marrow and gastrointestinal syndrome. Bone marrow toxicity results in a drop in all logic cells, predisposing the sm to death by infection and hemorrhage. The gastrointestinal syndrome is more severe and is driven by a loss of intestinal r function due to disintegration of the gut lial layer and a loss of intestinal endocrine function. This leads to sepsis and associated systemic inflammatory response syndrome which can result in death.

The lectin pathway of complement is an innate immune mechanism that initiates inflammation in response to tissue injury and exposure to n surfaces (i.e., bacteria).

Blockade of this pathway leads to better outcomes in mouse models of ischemic intestinal tissue injury or septic shock. It is hypothesized that the lectin pathway may trigger -lO6- excessive and harmful inflammation in response to radiation-induced tissue injury.

Blockade of the lectin pathway may thus reduce secondary injury and increase survival following acute radiation exposure.

The objective of the study carried out as described in this Example was to assess the effect of lectin y de on survival in a mouse model of radiation injury by administering anti-murine MASP-2 dies.

Study #1 Methods and Materials: Materials. The test es used in this study were (i) a high ty urine MASP-2 antibody (mAle 1) and (ii) a high ty anti-human MASP-2 antibody (mAbOMS646) that block the MASP-2 protein component of the lectin ment y which were produced in transfected ian cells. Dosing concentrations were 1 mg/kg of anti-murine MASP-2 antibody (mAle 1), 5mg/kg of anti-human MASP-2 antibody (mAbOMS646), or sterile saline. For each dosing session, an adequate volume of fresh dosing solutions were prepared.

Animals. Young adult male Swiss-Webster mice were obtained from Harlan tories (Houston, TX). Animals were housed in solid-bottom cages with Alpha-Dri bedding and provided certified PMI 5002 Rodent Diet (Animal Specialties, Inc., Hubbard OR) and water ad libitum. Temperature was monitored and the animal holding room operated with a 12 hour light/12 hour dark light cycle.

Irradiation. After a 2-week acclimation in the facility, mice were irradiated at 6.5, 7.0 and 8.0 Gy by body exposure in groups of 10 at a dose rate of 0.78 Gy/min using a Therapax X-RAD 320 system equipped with a 320-kV high stability X-ray generator, metal ceramic X-ray tube, variable x-ray beam collimator and filter (Precision X-ray Incorporated, East Haven, CT).

Drug Formulation and Administration. The appropriate volume of concentrated stock solutions were diluted with ice cold saline to prepare dosing solutions of 0.2 mg/ml anti-murine MASP-2 antibody (mAlel) or 0.5 mg/ml anti-human MASP-2 antibody (mAbOMS646) according to protocol. Administration of anti-MASP-2 antibody mAlel and mAbOMS646 was via IP injection using a 25-gauge needle base on animal weight to deliver 1 mg/kg mAle 1, 5mg/kg mAbOMS646, or saline vehicle.

Study Design. Mice were randomly assigned to the groups as described in Table 28. Body weight and temperature were measured and recorded daily. Mice in Groups 7, 11 and 13 were sacrificed at post-irradiation day 7 and blood collected by cardiac puncture under deep anesthesia. Surviving animals at post-irradiation day 30 were sacrificed in the same manner and blood collected. Plasma was prepared from collected blood samples according to protocol and returned to Sponsor for analysis.

TABLE 28: Stud Grouos Irradiation HU Level (Gy) Treatment Dose le 0 6.5 Vehicle 18 hr prior to irradiation, 2 hr post irradiation, weekly booster anti-murine 18 hr prior to irradiation MASP-2 ab only (mAle 1) anti-murine 18 hr prior to irradiation, 2 MASP-2 ab hr post irradiation, weekly booster (mAle 1) urine 2 hr post irradiation, MASP-2 ab weekly booster (mAle 1) 6.5 anti-human 18 hr prior to irradiation, 2 MASP-2 ab hr post ation, weekly booster S646) II Vehicle 18 hr prior to ation, 2 hr post irradiation, weekly booster 7.0 Vehicle 2 hr post irradiation only .-N 7.0 anti-murine 18 hr prior to irradiation MASP-2 ab only (mAle 1) .- anti-murine 18 hr prior to irradiation, 2 MASP-2 ab hr post irradiation, weekly booster (mAle 1) )_A anti-murine 2 hr post irradiation, MASP-2 ab weekly booster -lO8- Group Irradiation I--_—Level (Gy) Treatment Dose Schedule anti-murine 2 hr post irradiation only MASP-2 ab (mAbM 1 1 ) 12 20 7.0 anti-human 18 hr prior to ation, 2 MASP-2 ab hr post irradiation, weekly boos ert (mAbOMS646) 13 10 anti-human 18 hr prior to irradiation, 2 MASP-2 ab hr post irradiation, weekly boos ert (mAbOMS646) icle 2 hr post irradiation only Statistical Analysis. Kaplan-Meier survival curves were generated and used to compare mean al time between treatment groups using log-Rank and Wilcoxon methods. Averages with standard deviations, or means with rd error of the mean are reported. Statistical comparisons were made using a two-tailed unpaired t-test between controlled irradiated animals and individual ent groups.

Results of Study #1 Kaplan-Meier survival plots for 6.5, 7.0 and 8.0 Gy exposure groups are provided in FIGURES 18A, 18B and 18C, respectively, and summarized below in Table 29.

Overall, treatment with anti-murine MASP-2 ab (mAle 1) pre-irradiation increased the al of irradiated mice compared to vehicle treated irradiated control animals at both 6.5 (20% increase) and 7.0 Gy (30% increase) exposure levels. At the 6.5 Gy exposure level, post-irradiation treatment with anti-murine MASP-2 ab resulted in a modest increase in survival (15%) ed to vehicle control irradiated animals.

In comparison, all treated animals at the 7.0 Gy and 8.0 Gy exposure level showed an se in survival compared to vehicle treated irradiated control animals. The greatest change in survival occurred in animals receiving 646, with a 45% increase in survival compared to control animals at the 7.0 Gy exposure level, and a 12% increase in survival at the 8.0 Gy exposure level. r, at the 7.0 Gy exposure level, mortalities in the mAbOMS646 treated group first occurred at post-irradiation day 15 compared to post-irradiation day 8 for vehicle treated irradiated control animals, an se of 7 days over l animals. Mean time to ity for mice receiving mAbOMS646 (27.3 :: 1.3 days) was significantly increased (p = 0.0087) compared to l animals (20.7 :: 2.0 days) at the 7.0 Gy exposure level.

The percent change in body weight compared to pre-irradiation day (day -1) was recorded throughout the study. A transient weight loss occurred in all irradiated animals, with no eVidence of differential changes due to mAlel or mAbOMS646 treatment compared to controls (data not shown). At study termination, all surviving animals showed an increase in body weight from starting (day -1) body weight.

TABLE 29: al rates of test animals ex.osed to radiation Time to Death Exposure (Mean i SEM, First/Last Test Group Level Survival (%) Day) Death (Day) Control Irradiation 6.5 Gy 24.0 :: 2.0 9/16 mAlel pre- 6.5 Gy 85 % 27.7 :: 1.5 13/17 mAlel pre + 6.5 Gy 65 % 24.0 :: 2.0 9/15 post-exposure mAlel post— 6.5 Gy 80 % 26.3 :: 1.9 9/13 exposure mAbOMS646 6.5 Gy 65 % 24.6 :: 1.9 9/19 pre+post-exposure Control irradiation 7.0 Gy 20.7 :: 2.0 8/17 mAlel pre- 7.0 Gy 65 % 23.0 :: 2.3 7/13 exposure mAlel pre + 7.0 Gy 55 % 21.6 :: 2.2 7/16 post-exposure mAbM11 post— 7.0 Gy 70 % 24.3 :: 2.1 9/14 exposure mAbOMS646 7.0 Gy 80 % 27.3 :: 1.3* 15/20 st-exposure mAb OMS646 8.0 Gy 32% pre+post-exposure Time to Death Exposure (Mean i SEM, First/Last Test Group Level Survival (%) Day) Death (Day) my ___ *p = 0.0087 by two-tailed unpaired t—test between controlled irradiated animals and treatment group at the same ation exposure level.

Discussion Acute radiation syndrome consists of three defined subsyndromes: hematopoietic, gastrointestinal, and cerebrovascular. The syndrome observed depends on the radiation dose, with the hematopoietic effects observed in humans with significant partial or whole-body radiation exposures exceeding 1 Gy. The hematopoietic syndrome is characterized by severe depression of bone-marrow function leading to pancytopenia with changes in blood counts, red and white blood cells, and platelets occurring concomitant with damage to the immune system. As nadir occurs, with few neutrophils and platelets present in peripheral blood, neutropenia, fever, complications of sepsis and rollable hemorrhage lead to death.

In the present study, administration of mAbH6 was found to increase survivability of whole-body x-ray irradiation in Swiss-Webster male mice irradiated at 7.0 Gy.

Notably, at the 7.0 Gy exposure level, 80% of the s receiving mAbOMS646 survived to 30 days compared to 35% of vehicle treated control irradiated animals. lmportantly, the first day of death in this treated group did not occur until post-irradiation day 15, a 7-day increase over that observed in vehicle treated control irradiated animals.

Curiously, at the lower X-ray exposure (6.5 Gy), administration of 646 did not appear to impact survivability or delay in mortality compared to vehicle treated control irradiated animals. There could be le reasons for this difference in response between exposure , gh verification of any hypothesis may require additional studies, including interim sample collection for microbiological culture and logical parameters. One explanation may simply be that the number of animals assigned to groups may have precluded seeing any subtle treatment-related differences.

For example, with groups sizes of n=20, the ence in survival n 65% (mAbOMS646 at 6.5 Gy exposure) and 80% (mAbH6 at 7.0 Gy exposure) is 3 s.

On the other hand, the difference between 35% le control at 7.0 Gy exposure) and -lll- WO 51481 80% (mAbOMS646 at 7.0 Gy exposure) is 9 animals, and provides sound evidence of a treatment-related difference.

These results demonstrate that anti-MASP-Z antibodies are effective in treating a mammalian subject at risk for, or suffering from, the detrimental effects of acute ion syndrome.

Study #2 Swiss Webster mice (n=50) were exposed to ionizing radiation (8.0 Gy). The effect of anti-MASP-Z dy therapy (OMS646 5mg/kg), administered 18 hours before and 2 hours after radiation exposure, and weekly thereafter, on mortality was assessed.

Results of Study #2 It was determined that administration of anti-MASP-2 dy OMS646 increased survival in mice exposed to 8.0 Gy, with an adjusted median survival rate from 4 to 6 days as compared to mice that received vehicle control, and a mortality reduced by 12% when compared to mice that received vehicle control (log-rank test, p=0.040).

Study #3 Swiss Webster mice (n=50) were d to ionizing radiation (8.0 Gy) in the ing groups: (I:vehicle) saline control; (11: low) anti-MASP-2 antibody OMS646 (5mg/kg) administered 18 hours before irradiation and 2 hours after irradiation, (III:high) OMS646 (lOmg/kg) administered 18 hours before irradiation and 2 hours post irradiation; and (IV: high post) OMS646 (lOmg/kg) administered 2 hours post irradiation only. s of Study #3 It was determined that administration of anti-MASP-Z antibody pre- and post- irraditaion adjusted the mean survival from 4 to 5 days in comparison to animals that received vehicle control. ity in the anti-MASP-Z antibody-treated mice was reduced by 6-12% in comparison to vehicle control mice. It was further noted that no significant detrimental treatment effects were observed.

In y, the results in this Example demonstrate that anti-MASP-Z antibodies of the ion are ive in treating a mammalian subject at risk for, or suffering from the detrimental effects of acute radiation syndrome. -llZ- EXAMPLE 12 This Example describes further characterization of the OMS646 antibody (l7D20m_d3521Nl 1), fully human anti-human MASP-2 IgG4 antibody with a mutation inthelnngeregnnfi.

Methods: OMS646 m_d3521Nl l), fillly human anti-human MASP-2 IgG4 antibody with a mutation in the hinge region) was generated as described above in Examples 2-8.

OMS646 antibody was purified from culture supematants of a CHO expression cell line stably transfected with expression constructs encoding the heavy and light chains of OMS646. Cells were grown in PF-CHO media for 16 to 20 days and cell free supernatant was collected when cell viability dropped below 50%. OMS646 was purified by Protein A affinity chromatograph ed by ion exchange, concentration and buffer exchangeinK)PBS. l. OMS646 binds to human MASP-2 with high affinity Surface Plasmon Resonance (Biocore) Analysis of Immobilized OMS646 Binding to recondfinanthanunzfll45fh2 Methods: OMS646 was lized at various ies by amine ng to a CMS chip and the association and disassociation of recombinant human MASP-2 ved at 9 nM, 3 nM, 1 nM or 0.3 nM was recorded over time to determine the association (Ken) and dissociation (K03) rate constants. The equilibrium binding nt (KD) was calculated based on experimental K011 and Koff values.

Results: FIGURE 19 graphically illustrates the results of the surface plasmon resonance (Biocore) analysis on OMS646, showing that immobilized OMS646 binds to recombinant MASP-2 with a K03 rate of about l-3xlO'4 S'1 and a K011 rate of about l.6-3xlO6M'1S'1, implying an y (KD of about 92pM) under these experimental ions.

Depending on the density of OMS646 immobilized and the concentration of MASP-2 in solution, experimental KD values in the range of 50 to 25OpM were determined.

ELISA Assay ofOMS646 Binding to Immobilized recombinant human MASP—2 s: An ELISA assay was carried out to assess the dose-response of OMS646 g to immobilized recombinant MASP-2. Recombinant human MASP-2 (50 ng/well) was immobilized on maxisorp ELISA plates (Nunc) in PBS overnight at -ll3- 4°C. The next day, the plates were blocked by washing three times with een (0.05%). A serial dilution series of OMS646 in blocking buffer (concentration range from 0.001 to 10 nM) was then added to the MASP-2 coated wells. After a 1 hour incubation to allow OMS646 g to immobilized antigen, the wells were washed to remove unbound . Bound OMS646 was detected using HRP-labeled goat anti- human IgG antibody (Qualex; diluted 1:5000 in blocking buffer) followed by development with TMB peroxidase substrate (Kirkegaard & Perry Laboratories). The peroxidase reaction was stopped by adding 100 ul/well of 1.0 M H3P04, and substrate conversion was quantified etrically at 450nM using a 96 well plate reader (Spectramax). A single binding site curve fitting algorithm (Graphpad) was used to calculate KD values.

Results: FIGURE 20 graphically illustrates the results of the ELISA assay to determine the g affinity of OMS646 to immobilized human MASP-2. As shown in FIGURE 20, it was determined that OMS646 binds to lized recombinant human MASP-2 with a KD of 85:5 pM, which is consistent with the results obtained in the Biocore analysis, as shown in FIGURE 19. These results demonstrate that OMS646 has high affinity to human MASP-2, with a KD of approximately 100pM. 2. OMS646 inhibits C4 Activation on a mannan-coated surface but not on an immune comp_lex-coated surface Methods: C4 activation was measured on a mannan-coated surface or an immune complex- coated surface in the presence or absence of OMS646 over the tration range shown in FIGURES 21A and 21B, respectively as s.

In the following method to measure the C4 ge activity of MASP-2, plastic wells coated with mannan were ted for 60 minutes at 37°C with 1% human serum to activate the lectin pathway. The wells were then washed and assayed for human C4b immobilized onto the wells using standard ELISA methods. The amount of C4b ted in this assay is a measure of MASP-2 dependent C4 cleavage activity.

Anti-MASP-2 antibodies at selected concentrations were tested in this assay for their ability to inhibit C4 cleavage.

Methods: C4 activation 0n mannan-coated surfaces: —114— In order to determine the effect of OMS646 on the lectin-pathway, 96-well Costar Medium Binding plates were coated with mannan by overnight incubation at 5°C with 50 uL of a 40 ug/mL solution of mannan diluted in 50 mM carbonate buffer, pH 9.5. Each well was washed 3X with 200 uL PBS. The wells were then blocked with 100 uL/well of 1% bovine serum albumin in PBS and incubated for one hour at room temperature with gentle mixing. Each well was washed 3X with 200 uL of PBS. In a separate 96 well plate, serial dilutions of MASP-2 antibody (OMS646) were preincubated with 1% human serum in Ca++ and Mg++ containing GVB buffer (4.0 mM al, 141 mM NaCl, 1.0 mM MgC12, 2.0 mM CaClz, 0.1% gelatin, pH 7.4) for 1 hour at 5° C. These antibody- serum preincubation mixtures were uently transferred into the ponding wells of the mannan-coated assay plate. Complement activation was initiated by transfer of the assay plate to a 37°C water bath. ing a 60 minute incubation, the reaction was stopped by adding EDTA to the reaction e. Each well was washed 5 x 200 uL with PBS-Tween 20 (0.05% Tween 20 in PBS), then each well was washed with 2X with 200 uL PBS. 100 uL/well of 1:100 dilution of -conjugated chicken uman C4c (Immunsystem AB, Uppsala, Sweden) was added in PBS containing 2.0 mg/ml bovine serum albumin (BSA) and ted one hour at room temperature with gentle mixing.

Each well was washed 5 x 200 uL PBS. 100 uL/well of 0.1 ug/ml of peroxidase-conjugated streptavidin (Pierce Chemical #21126) was added in PBS containing 2.0 mg/ml BSA and incubated for one hour at room temperature on a shaker with gentle mixing. Each well was washed 5 x 200 uL with PBS. 100 uL/well of the peroxidase substrate TMB (Kirkegaard & Perry Laboratories) was added and incubated at room temperature for 10 minutes. The peroxidase reaction was stopped by adding 100 uL/well of 1.0 M H3PO4 and the OD450 was ed.

C4 tion 0n immune-complex coated surfaces In order to measure the effect of OMS646 on the classical pathway, the assay described above was modified to use immune-complex coated plates. The assay was carried out as detailed for the lectin pathway above, with the difference that wells were coated with purified sheep IgG used to stimulate C4 activation via the classical pathway.

Results: FIGURE 21A graphically illustrates the level of C4 activation on a mannan- coated surface in the presence or absence of OMS646. FIGURE 21B graphically illustrates the level of C4 activation on an IgG-coated surface in the presence or absence of OMS646. As shown in FIGURE 21A, OMS646 inhibits C4 activation on - coated surface with an IC50 of approximately 0.5nM in 1% human serum. The IC50 value obtained in 10 independent experiments was 0.52:0.28 nM (average::SD). In contrast, as shown in FIGURE 21B, OMS646 did not inhibit C4 activation on an IgG-coated surface.

These results demonstrate that OMS646 blocks lectin-dependent, but not classical pathway-dependent activation of complement component C4. 3. OMS646 cally blocks lectin-dependent activation of terminal complement ents Methods: The effect of OMS646 on membrane attack complex (MAC) deposition was analyzed using pathway-specific conditions for the lectin pathway, the classical pathway and the alternative pathway. For this purpose, the Wieslab Comp300 complement screening kit ab, Lund, Sweden) was used following the manufacturer’s instructions.

Results: FIGURE 22A graphically illustrates the level of MAC tion in the presence or absence of anti-MASP-2 antibody (OMS646) under lectin pathway-specific assay conditions. FIGURE 22B graphically illustrates the level of MAC deposition in the presence or absence of anti-MASP-2 antibody (OMS646) under classical pathway- specific assay conditions. FIGURE 22C graphically illustrates the level of MAC deposition in the presence or absence of anti-MASP-2 antibody (OMS646) under alternative pathway-specific assay conditions.

As shown in FIGURE 22A, OMS646 blocks lectin pathway-mediated activation of MAC tion with an IC50 value of approximately 1nM. However, OMS646 had no effect on MAC deposition generated from cal pathway-mediated tion E 22B) or from alternative pathway-mediated tion (FIGURE 22C). 4. OMS646 effectively inhibits lectin pathway activation under physiologic Methods: The lectin dependent C3 and C4 activation was assessed in 90% human serum in the e and in the presence of various concentrations of OMS646 as follows: C4 Activation Assay WO 51481 To assess the effect of OMS646 on lectin-dependent C4 activation, 96-well Costar medium binding plates were coated overnight at 5°C with 2 ug of mannan (50 ul of a 40 ug/mL solution in 50 mM carbonate buffer, pH 9.5. Plates were then washed three times with 200 ul PBS and blocked with 100 uL/well of 1% bovine serum albumin in PBS for one hour at room temperature with gentle mixing. In a separate preincubation plate, select concentrations of OMS646 were mixed with 90% human serum and incubated for 1 hour on ice. These antibody-serum preincubation mixtures were then transferred into the mannan-coated wells of the assay plates on ice. The assay plates were then incubated for 90 minutes in an ice water bath to allow complement activation. The reaction was stopped by adding EDTA to the reaction mixture. Each well was washed 5 times with 200 uL of PBS-Tween 20 (0.05% Tween 20 in PBS), then each well was washed two times with 200 uL PBS. 100 uL/well of 1:1000 dilution of biotin-conjugated chicken anti-human C4c (Immunsystem AB, Uppsala, Sweden) was added in PBS containing 2.0 mg/ml bovine serum n (BSA) and incubated 1 hour at room temperature with gentle mixing. Each well was washed 5 times with 200 uL PBS. 100 uL/well of 0.1 ug/mL of peroxidase-conjugated streptavidin (Pierce Chemical #21126) was added in PBS containing 2.0 mg/ml BSA and ted for 1 hour at room temperature on a shaker with gentle mixing. Each well was washed five times with 200 uL PBS. 100 uL/well of the peroxidase substrate TMB (Kirdegaard & Perry Laboratories) was added and ted at room temperature for 16 minutes. The peroxidase reaction was stopped by adding 100 l of 1.0M H3PO4 and the OD450 was ed.

C3 Activation Assay To assess the effect of OMS646 on lectin-dependent C3 activation, assays were d out in an identical manner to the C4 activation assay described above, except that C3 deposition was assessed as the endpoint. C3 deposition was quantified as follows.

At the end of the complement deposition reaction, plates were washed as described above and subsequently incubated for 1 hour with 100 uL/well of 1:5000 dilution of rabbit anti- human C3c antibody (Dako) in PBS containing 2.0 mg/mL bovine serum albumin (BSA).

Each plate was washed five times with 200 uL PBS, and then incubated for 1 hour at room ature with 100 uL/well of HRP-labeled goat anti-rabbit IgG can Qualex Antibodies) in PBS containing 2.0 mg/mL BSA. Plates were washed five times with 200 uL PBS and then 100 uL/well of the peroxidase substrate TMB gaard & Perry Laboratories) was added and incubated at room temperature for 10 minutes. The peroxidase reaction was stopped by adding 100 uL/well of 1.0M H3PO4 and the OD450 was ed. IC50 values were derived by applying a sigmoidal dose-response curve fitting thm (GraphPad) to the experimental data sets.

Results: FIGURE 23A graphically illustrates the level of C3 deposition in the presence or absence of anti-MASP-2 antibody (OMS646) over a range of trations in 90% human serum under lectin y specific conditions. FIGURE 23B graphically illustrates the level of C4 deposition in the presence or absence of anti-MASP-2 antibody (OMS646) over a range of concentrations in 90% human serum under lectin pathway specific conditions. As shown in FIGURE 23A, OMS646 blocked C3 deposition in 90% human serum with an IC50 = 3::1.5 nM (n=6). As shown in FIGURE 23B, OMS646 blocked C4 deposition with an IC50 = 28:13 nM (n=6).

These results demonstrate that OMS646 es potent, effective blockade of lectin pathway activation under physiological conditions, thereby providing t for the use of low therapeutic doses of OMS646. Based on these data, it is expected that OMS646 will block >90% of the lectin pathway in the circulation of a patient at a plasma tration of 20 nM (3 ug/mL) or less. Based on a plasma volume of a typical human of approximately 3L, and the knowledge that the bulk of antibody material administered is retained in plasma (Lin Y.S. et al., JPET 288:371 (1999)), it is expected that a dose of OMS646 as low as 10 mg administered enously will be effective at blocking the lectin pathway during an acute time period (i.e., a transient time period, such as from 1 to 3 days). In the context of a chronic illness, it may be advantageous to block the lectin pathway for an extended period of time to achieve maximal treatment benefit. Thus, for such chronic conditions, an OMS646 dose of 100mg may be preferred, which is expected to be effective at blocking the lectin pathway in an adult human subject for at least one week or longer. Given the slow clearance and long half-life that is ly observed for antibodies in humans, it is possible that a 100 mg dose of OMS646 may be effective for longer than one week, such as for 2 weeks, or even 4 weeks. It is expected that a higher dose of antibody (i.e., greater than 100 mg, such as 200 mg, 500 mg or greater, such as 700mg or 1000mg), with have a longer on of action (e.g., r than 2 weeks).

. OMS646 blocks lectin pathway activation in monkeys As described above in Example 10 and shown in FIGURE 17, it was determined that OMS646 s systemic lectin pathway ty for a time period of about 72 hours following intravenous administration of OMS646 (3 mg/kg) into African Green monkeys, followed by recovery of lectin y activity.

This Example describes a follow up study in which lectin dependent C4 activation was assessed in 90% African Green monkey serum or in 90% Cynomoglus monkey serum over a range of concentrations of OMS646 and in the absence of OMS646, as follows: To assess the effect of OMS646 on lectin-dependent C4 activation in ent non-human primate species, 96-well Costar medium binding plates were coated overnight at 5°C with 2 ug of mannan (50 ul of a 40 ug/mL solution in 50 mM ate buffer, pH 9.5). Plates were then washed three times with 200 uL PBS and blocked with 100 uL/well of 1% bovine serum n in PBS for 1 hour at room temperature with gentle . In a separate preincubation plate, select concentrations of OMS646 were mixed with 90% serum collected from African Green Monkeys or Cynomoglus Monkeys, and incubated with 1 hour on ice. These antibody-serum preincubation mixtures were then transferred into the mannan-coated wells of the assay plates on ice. The assay plates were then incubated for 90 minutes in an ice water bath to allow complement activation. The reaction was stopped by adding EDTA to the reaction mixture. Each well was washed five times with 200 uL PBS-Tween 20 (0.05% Tween 20 in PBS), then each well was washed two times with 200 uL PBS. 100 uL/well of 1:1000 on of biotin-conjugated chicken uman C4c (Immunosystem AB, Uppsala, ) was added in PBS containing 2.0 mg/mL BSA and incubated one hour at room temperature with gentle mixing. Each well was washed five times with 200 uL PBS. 100 uL/well of 0.1 ug/mL of peroxidase-conjugated streptavidin (Pierce Chemical #21126) was added in PBS containing 2.0 mg/mL BSA and incubated for one hour at room temperature on a shaker with gentle mixing. Each well was washed five times with 200 uL PBS. 100 uL/well of the peroxidase substrate TMB (Kirkegaard & Perry Laboratories) was added and incubated at room temperature for 10 minutes. The peroxidase reaction was stopped by adding 100 uL/well of 1.0 M H3PO4 and the OD450 was measured. IC50 values were derived by applying a sigmoidal dose-response curve fitting algorithm (GraphPad) to the experimental data sets.

Results: -ll9- A dose response of lectin pathway inhibition in 90% Cynomoglus monkey serum (FIGURE 24A) and in 90% African Green monkey serum (FIGURE 24B) was observed with IC50 values in the range of 30 nM to 50 nM, and 15 nM to 30 nM, respectively.

In summary, OMS646, a fillly human anti-human MASP-2 IgG4 antibody (with a mutation in the hinge region) was observed to have the following advantageous properties: high ty binding to human MASP-2 (KD in the range of 50 to 250 pM, with a Koff rate in the range of 1-3x10'4 S"1 and a K011 rate in the range of 1 .6-3x106M'IS'1; functional potency in human serum with tion of C4 deposition with an IC50 of 0.52::0.28 nM (n=10) in 1% human serum; and an IC50 of 3::1.5nM in 90% serum); and cross-reactivity in monkey showing inhibition of C4 tion with an IC50 in the range of 15 to 50 nM (90% monkey serum).

As described above, doses as low as 10 mg OMS646 (corresponding to 0.15 mg/kg for an average human) are ed to be effective at acutely blocking the lectin pathway in human circulation (e.g., for a period of at least 1 to 3 days), while doses of 100 mg OMS646 (corresponding to 1.5 mg/kg for an average human) are expected to block the lectin pathway in the circulation of a t for at least one week or longer.

Larger doses of OMS646 (e.g., doses greater than 100mg, such as at least 200mg, at least 300mg, at least 400mg, at least 500mg, or greater), and preferably subcutaneous (sc) or intramuscular (im) routes of administration can be employed to r extend the time window of effective lectin y ablation to two weeks and preferably four weeks.

For example, as shown in the experimental data herein, in primates a dose of 1 mg/kg OMS646 resulted in inhibition of the lectin pathway for 1 day, and a 3 mg/kg dose of OMS646 resulted in inhibition of the lectin pathway for about 3 days (72 hours). It is therefore estimated that a larger dosage of 7 to g would be effective to inhibit the lectin pathway for a time period of about 7 days. As shown herein, the OMS646 has a 5- fold greater y against human MASP-2 as compared to monkey MASP-2.

Assuming comparable pharmacokinetics, the expected dosages ranges to achieve effective lectin pathway ablation in humans is shown in TABLE 30 below.

TABLE 30: OMS646 dosin_ to inhibit the lectin oathwa in viva —___ 00/0 0.1 to 0.2 Inn/k 0.3 to 0.0 Inn/k 1-2 Inn/k While the preferred embodiment of the invention has been illustrated and described, it will be appreciated that various s can be made therein without departing from the range and scope of the invention.

Claims (41)

CLAIMS is:

1. An isolated human onal antibody, or antigen binding fragment thereof, that binds to human MASP-2 and ts MASP-2 dependent complement activation, comprising: (i) a heavy chain variable region sing three complementary determining regions CDR-H1, CDR-H2 and CDR-H3; and (ii) a light chain variable region comprising three CDRs CDR-L1, CDR-L2 and CDR-L3, wherein; CDR-H1 comprises an amino acid sequence as set forth in SEQ ID NO:28; CDR-H2 comprises an amino acid sequence as set forth in SEQ ID NO:32; CDR-H3 comprises an amino acid sequence set forth as SEQ ID NO:90 wherein X at position 8 is A or R; and wherein CDR-L1 comprises an amino acid sequence as set forth in SEQ ID NO:91, wherein X at position 2 is D or E and wherein X at on 8 is F or Y; CDR-L2 comprises an amino acid sequence as set forth in SEQ ID NO:93, wherein X at position 2 is N or K and wherein X at position 3 is K or Q; and CDR-L3 comprises an amino acid sequence set forth as SEQ ID NO:51, and wherein the isolated antibody inhibits MASP-2 dependent complement activation.

2. The isolated antibody or antigen binding fragment of claim 1, wherein the CDR-H3 sequence comprises amino acid residues 95-107 of SEQ ID NO:20.

3. The isolated dy of claim 1, wherein the amino acid sequence set forth in SEQ ID NO:91 ns an E at position 2.

4. The isolated antibody of claim 1, wherein the amino acid sequence set forth in SEQ ID NO:91 contains a Y at on 8.

5. The isolated antibody of claim 1, wherein the amino acid ce set forth in SEQ ID NO:93 contains a K at position 2.

6. The isolated dy of claim 1, wherein the amino acid sequence set forth in SEQ ID NO:93 ns a Q at position 3.

7. The ed antibody of claim 1, n the antibody or antigen-binding fragment thereof is selected from the group ting of a Fab, a Fab’ fragment, a F(ab’)2 nt and a whole antibody.

8. The isolated antibody of claim 1, wherein the antibody or antigen-binding nt thereof is selected from the group consisting of a single chain antibody, an ScFv, and a univalent antibody lacking a hinge .

9. The isolated dy of claim 1, wherein said antibody binds human MASP-2 with a KD of 10 nM or less.

10. The antibody of claim 1, wherein said antibody inhibits C4 activation in an in vitro assay in 1% human serum at an IC50 of 10 nM or less.

11. The antibody of claim 1, wherein said antibody inhibits C4 activation in 90% human serum with an IC50 of 30 nM or less.

12. An isolated human monoclonal antibody, or antigen binding fragment thereof, that binds human MASP-2, wherein the antibody comprises: a) a heavy chain le region comprising SEQ ID NO:20 or a variant thereof comprising an amino acid sequence having at least 95% identity to SEQ ID NO:20, wherein residue 31 is an R, residue 32 is a G, residue 33 is a K, residue 34 is an M, residue 35 is a G, residue 36 is a V, residue 37 is an S, residue 50 is an L, residue 51 is an A, residue 52 is an H, residue 53 is an I, residue 54 is an F, residue 55 is an S, residue 56 is an S, e 57 is a D, residue 58 is an E, residue 59 is a K, residue 60 is an S, residue 61 is a Y, residue 62 is an R, residue 63 is a T, residue 64 is an S, residue 65 is an L, residue 66 is a K, residue 67 is an S, residue 95 is a Y, residue 96 is a Y, residue 97 is a C, residue 98 is an A, residue 99 is an R, residue 100 is an I, residue 101 is an R, residue 102 is an R or A, residue 103 is a G, residue 104 is a G, residue 105 is an I, residue 106 is a D and residue 107 is a Y; and b) a light chain variable region comprising SEQ ID NO:24 or a variant thereof comprising an amino acid sequence having at least 95% identity to SEQ ID NO:24, wherein residue 23 is an S, residue 24 is a G, residue 25 is an E or D, residue 26 is a K, residue 27 is an L, residue 28 is a G, residue 29 is a D, residue 30 is a K, residue 31 is a Y or F, residue 32 is an A, residue 33 is a Y, residue 49 is a Q, residue 50 is a D, residue 51 is a K or N, residue 52 is a Q or K, residue 53 is an R, residue 54 is a P, residue 55 is an S, residue 56 is a G, residue 88 is a Q, e 89 is an A, residue 90 is a W, residue 91 is a D, e 92 is an S, residue 93 is an S, residue 94 is a T, residue 95 is an A, e 96 is a V and residue 97 is an F; wherein the antibody or variant thereof inhibits MASP-2 dependent complement activation.

13. The antibody of claim 12, wherein said antibody binds human MASP-2 with a KD of 10 nM or less.

14. The antibody of claim 12, wherein said antibody binds an epitope in the CCP1 domain of MASP-2.

15. The dy of claim 12, n said dy inhibits C3b deposition in an in vitro assay in 1% human serum at an IC50 of 10 nM or less.

16. The antibody of claim 12, wherein said antibody inhibits C3b deposition in 90% human serum with an IC50 of 30 nM or less.

17. The antibody of claim 12, wherein the antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2 and F(ab')2.

18. The dy of claim 12, wherein the antibody is a single chain molecule.

19. The antibody of claim 12, wherein said antibody is an IgG2 molecule.

20. The antibody of claim 12, wherein said antibody is an IgG1 molecule.

21. The antibody of claim 12, wherein said antibody is an IgG4 molecule.

22. The antibody of claim 21, wherein the IgG4 molecule comprises a S228P mutation.

23. The antibody of claim 12, wherein the antibody does not substantially inhibit the cal pathway.

24. The antibody of claim 12, wherein the heavy chain variable region comprises SEQ ID NO:20.

25. The antibody of claim 12, wherein the light chain variable region comprises SEQ ID NO: 24.

26. The antibody of claim 12, wherein said variant comprises a heavy chain variable region comprising an amino acid sequence wherein residue 102 is an R.

27. The antibody of claim 12, n said variant comprises a light chain le region comprising an amino acid sequence wherein residue 25 is an E, residue 31 is a Y, residue 51 is a K, and residue 52 is a Q.

28. An isolated monoclonal antibody, or antigen-binding fragment thereof, that binds to human MASP-2, comprising a heavy chain variable region comprising any one of the amino acid sequences set forth in SEQ ID NO:18, or SEQ ID NO:20 and a light chain variable region comprising an amino acid sequence having at least 95% identity to an amino acid sequence set forth in SEQ ID NO:22 or SEQ ID NO:24.

29. The ed antibody of claim 28, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:20 and a light chain variable region sing the amino acid sequence set forth in SEQ ID NO:24.

30. An isolated monoclonal antibody, or antigen-binding fragment thereof, that binds to human MASP-2, sing a light chain variable region comprising any one of the amino acid sequences set forth in SEQ ID NO:22 or, SEQ ID NO:24 and a heavy chain variable region comprising an amino acid sequence having at least 95% identity to an amino acid sequence set forth in SEQ ID NO:18 or SEQ ID NO:20.

31. A nucleic acid molecule ng the amino acid sequence of an anti-MASP- 2 antibody, or fragment thereof, as set forth in any one of claims 1, 12, 28 or 30.

32. An expression cassette comprising a c acid le encoding an anti- MASP-2 antibody of the invention according to claim 31.

33. A cell comprising at least one of the nucleic acid molecules encoding an anti- MASP-2 dy of the invention according to claim 31 or claim 32, with the proviso that if the cell is a human cell it is ex vivo.

34. A method of generating an isolated anti-MASP-2 antibody comprising culturing the cell of claim 33 under conditions ng for expression of the c acid molecules encoding the anti-MASP-2 antibody and isolating said anti-MASP-2 antibody.

35. A composition comprising the fully human monoclonal anti-MASP-2 antibody, or fragment thereof, of any one of claims 1, 12, 28 or 30 and a pharmaceutically able ent.

36. The composition of claim 35, wherein the composition is formulated for ic delivery.

37. The composition of claim 36, wherein the composition is formulated for intra-arterial, intravenous, intracranial, intramuscular, inhalational, nasal or subcutaneous administration.

38. The composition of claim 36, wherein the composition is formulated for subcutaneous administration.

39. Use of the isolated monoclonal antibody of any one of claims 1, 12, 28 or 30 in the manufacture of a medicament for inhibiting MASP-2 dependent complement activation in a subject in need thereof.

40. An article of manufacture comprising a unit dose of a human monoclonal anti-MASP-2 antibody of any one of claims 1 to 30, wherein the unit dose is the range of from 1mg to 1000mg.

41. The antibody of any one of claims 1, 12, 28 or 30, substantially as herein described with nce to any one of the Examples and/or