NZ715226B2 - 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, wherein the antibody or antigen-binding fragment thereof comprises: a) a heavy chain variable region comprising three complementary determining regions CDR-H1, CDR-H2 and CDR-H3; and b) 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:29; CDR-H2 comprises an amino acid sequence as set forth in SEQ ID NO:33; CDR-H3 comprises an amino acid sequence as set forth in SEQ ID NO:38; CDR-L1 comprises an amino acid sequence as set forth in SEQ ID NO:92; wherein X at position 2 is N or D and wherein X at position 4 is I or L and whrein X at position 6 is S or K and wherein X at position 8 is N or R CDR-L2 comprises an amino acid sequence as set forth in SEQ ID NO:49; CDR-L3 comprises an amino acid sequence as set forth in SEQ ID NO:94; wherein X at position 4 is T or I and wherein X at position 5 is T or A. ry determining regions CDR-H1, CDR-H2 and CDR-H3; and b) 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:29; CDR-H2 comprises an amino acid sequence as set forth in SEQ ID NO:33; CDR-H3 comprises an amino acid sequence as set forth in SEQ ID NO:38; CDR-L1 comprises an amino acid sequence as set forth in SEQ ID NO:92; wherein X at position 2 is N or D and wherein X at position 4 is I or L and whrein X at position 6 is S or K and wherein X at position 8 is N or R CDR-L2 comprises an amino acid sequence as set forth in SEQ ID NO:49; CDR-L3 comprises an amino acid sequence as set forth in SEQ ID NO:94; wherein X at position 4 is T or I and wherein X at position 5 is T or A.

Description

COMPLETE SPECIFICATION Compositions for ting MASP-Z dependent complement activation We, Omeros Corporation, of 201 Elliott Avenue West, Seattle, 98119, Washington, United States of America, hereby declare the invention, for which we pray that a patent may be granted to us and the method by which it is to be performed, to be particularly described in and by the following statement: (followed by page 1a) COMPOSITIONS FOR INHIBITING MASP-2 DEPENDENT COMPLEMENT ACTIVATION FIELD OF THE INVENTION The present invention relates to anti-MASP-2 inhibitory antibodies and compositions comprising such antibodies for use in inhibiting the e s of MASP-2 dependent complement activation.

REFERENCE TO RELATED APPLICATION This application claims the benefit of US. Provisional Application No. 61/482,567 filed May 4, 2011, which is incorporated herein by reference in its entirety.

This application was divided from NZ 617487 and NZ 733310 was d from the present application. The description of the present invention and the inventions ofNZ 617487 and NZ 733310 are retained herein for clarity and completeness.

STATEMENT REGARDING SEQUENCE LISTING The sequence listing associated with this application is ed 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_01l5_PCT_SequenceListingasFiled_20120504_ST25. The text file is 158 KB, was created on May 4, 2012; and is being ted via EFS-Web with the filing of the specification.

BACKGROUND The complement system provides an early acting mechanism to initiate, amplify and trate the immune response to microbial infection and other acute insults (M.K. Liszewski and JP. Atkinson, 1993, in Fundamental Immunology, Third Edition, edited by W.E. Paul, Raven Press, Ltd, New York) in humans and other rates. While complement activation provides a valuable first-line defense against ial pathogens, the activities of ment that promote a protective immune response can also represent a potential threat to the host (KR. 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 phils. While indispensable for host defense, activated neutrophils are indiscriminate in their release of destructive enzymes and may cause organ . In addition, complement -1a- (followed by page 2) PCT/U82012/036509 activation may cause the deposition of lytic complement components on nearby host cells as well as on microbial targets, resulting in host cell lysis.

The complement system has also been implicated in the pathogenesis of numerous acute and chronic e states, including: dial infarction, stroke, acute respiratory distress me (ARDS), reperfusion injury, septic shock, capillary leakage following thermal burns, post cardiopulmonary bypass inflammation, transplant rejection, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, and Alzheimer's disease. In almost all of these conditions, complement is not the cause but is one of l 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 e states.

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

Currently, it is widely accepted that the complement system can be activated through three distinct ys: the classical pathway, the lectin pathway, and the ative pathway. The classical pathway is usually triggered 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 c antibody response. Since activation of the classical pathway depends on a prior adaptive immune response by the host, the cal pathway is part of the ed 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, Clq is associated with the Clr and Cls serine protease proenzymes as a complex called Cl. Upon binding of Clq to an immune complex, autoproteolytic W0 2012/151481 2012/036509 cleavage of the Arg-Ile site of Clr is followed by Clr-mediated cleavage and activation of C13, which thereby acquires the ability to cleave C4 and C2. C4 is cleaved into two nts, 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 tase ) activates C3 by proteolytic cleavage into C3a and C3b subcomponents leading to generation 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 nes leading to cell lysis. The activated forms of C3 and C4 (C3b and C4b) are covalently deposited on the foreign target surfaces, which are ized by ment receptors on multiple phagocytes.

Independently, the first step in activation of the complement system h the lectin pathway is also the binding of specific ition molecules, which is followed by the activation of associated serine protease proenzymes. However, rather than the binding of immune complexes by Clq, the ition molecules in the lectin pathway comprise a group of carbohydrate-binding proteins (mannan-binding lectin (MBL), H-ficolin, in, L-ficolin and C-type lectin CL-ll), collectively referred to as lectins. See J. Lu et al., Biochz'm. Biophys. Acta 1572:387~400, 2002; Holmskov et al., Annu. Rev. 1mmzmol.21:547—578 (2003); Teh et al., In/zmzmology [01:225—232 (2000)).

See also J. Luet et al., Bz'ochz‘m Biophys Acta 1572:387-400 (2002); Holmskov et a1, 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 Clq, MBL could te the complement system upon binding to yeast mannan-eoated erythrocytes in a endent manner (Ikeda ct 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 undetectable 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 -digit millimolar range.

MBL achieves tight, specific binding to glycan ligands by avidity, i.e., by interacting PCT/U82012/036509 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 ydrate patterns that commonly decorate microorganisms such as bacteria, yeast, parasites and certain viruses. In st, MBL does not recognize D—galactose and sialic acid, the imate and ultimate sugars that usually te "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. 257:3788-3794, 1982). Therefore, damaged cells are potential targets for lectin pathway activation Via MBL binding.

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

The two serum ficolins, lin and H—ficolin, have in common a specificity for N-acetyl-D-glucosamine; however, H-ficolin also binds N-acetyl-D-galactosamine. The ence in sugar specificity of L-ficolin, H—ficolin, CL-ll and MBL means that the different lectins may be complementary and target different, though overlapping, glycoconjugates. This concept is supported by the recent report that, of the known lectins in the lectin pathway, only L~f1colin binds specifically to lipoteichoic acid, a cell wall glycoconjugate found on all Gram—positive bacteria , N.J.,et al., J. [71277211710]. 172:1198-1202, 2004). The collectins (i.e., MBL) and the ficolins bear no icant similarity in amino acid ce. However, the two groups of ns have similar domain organizations and, like Clq, le into oligomeric ures, which maximize the possibility of multisite binding.

The serum trations of MBL are highly variable in healthy populations and this is genetically controlled by the polymorphism/mutations in both the promoter and coding s of the MBL gene. As an acute phase protein, the expression of MBL is further upregulated during inflammation. L-ficolin is present in serum at concentrations similar to those of MBL. Therefore, the L-ficolin branch of the lectin pathway is potentially comparable to the MBL arm in strength. MBL and ficolins can also function as opsonins, which allow phagocytes to target MBL- and ficolin—decorated surfaces (see PCT/U82012/036509 Jack et al., J Leukoc Biol., 77(3):328—36 (2004); Matsushita and Fujita, biology, 205(4-5):490-7 (2002); Aoyagi et al., J Immzmol 174( l):418-25 (2005). This opsonization requires the interaction of these proteins with phagocyte receptors (Kuhlman, M., et al., J. Exp. Med. 16921733, 1989; hita, M., et al., J. Biol.

Chem. 271 :2448-54, 1996), the identity ofwhich 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 sociated serine proteases (MASPs). To date, three MASPs have been described. First, a single enzyme "MASP" was identified and characterized as the enzyme responsible for the initiation of the complement cascade (i.e., cleaving C2 and C4) (Matsushita M and Fujita T., JExp A/[ed l76(6):l497-1502 , Ji, Y.H., et al., 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 , S., et al., Nature 386:506—510, 1997). However, it was trated that the MBL—MASP—2 complex alone is sufficient for ment activation Worup-Jensen, T., et al., J. Immunol. 165:2093-2100, 2000). rmore, only MASP-2 cleaved C2 and C4 at high rates (Ambrus, G., et al., J. Immunol. 74-1382, 2003). Therefore, MASP-Z is the protease responsible for activating C4 and C2 to generate the C3 convertase, C4b2a. This is a significant difference from the Cl 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 on, a third novel protease, MASP-3, has been isolated (Dahl, M.R., et al., Immunity 15:127-35, 200]). MASP-l and MASP~3 are atively spliced products of the same gene.

MASPs share cal domain organizations with those of Clr and Cls, the enzymatic components of the Cl complex (Sim, RB, et al., Biochem. Soc. Trans. 28:545, 2000). These domains include an N—terminal Clr/Cls/sea urchin VEGF/bone morphogenic protein (CUB) domain, an epidermal growth factor—like domain, a second CUB domain, a tandem of complement control protein s, and a serine protease domain. As in the Cl proteases, tion of MASP-Z occurs through cleavage of an Arg-Ile bond adjacent to the serine protease domain, which splits the enzyme into disulfide-linked A and B chains, the latter consisting of the serine protease domain.

Recently, a genetically determined deficiency of MASP-2 was described (Stengaard—Pedersen, K., et al., New Eng. J. Med. 349:554-560, 2003). The mutation of a PCT/U82012/036509 single nucleotide leads to an Asp-Gly exchange in the CUBl domain and renders MASP-2 incapable of binding to MBL.

MBL can also associated with an alternatively d form of MASP-2, known as MEL-associated, protein of 19 kDa (MAp19) (Stover, C.M., J Immzmol. [62:3481-90, 1999) or small MEL-associated protein (SMAP) (Takahashi, M., et al., Int.

Immzmol. 11:859-863, 1999), which lacks the catalytic ty of MASP—Z. MAp19 comprises the first two domains of MASP-Z, followed by an extra sequence of four unique amino acids. The MASP 1 and MASP 2 genes are located on human chromosomes 3 and 1, respectively eble, W., et al., biology 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. Immzmol. 165:878-887, 2000). Both H— and L—ficolin bind to all MASPs and activate the lectin complement pathway, as does MBL (Dahl, M.R., eta1., Immunity 15:127-35, 2001; Matsushita, M., et al., J. l. 16823502-3506, 2002). Both the lectin and classical pathways form a common C3 convertase ) and the two pathways converge at this step.

The lectin pathway is widely thought to have a major role in host defense t 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 (Kilpatriek, D.C., Biochim. Bioplzys. Acta 1572:401-413, 2002). Such patients display susceptibility to recurrent bacterial and fungal infections. These symptoms are y 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 results from mutations at several sites in the collagenous portion of MBL, which interfere with proper formation of MBL oligomers. However, since MBL can function as an opsonin 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 y amplified on foreign or other abnormal es (bacteria, yeast, Virally infected cells, or PCT/U82012/036509 damaged tissue) that lack the proper molecular elements that keep neous complement activation in check. There are four plasma proteins directly involved in the activation of the alternative pathway: C3, factors B and D, and properdin. 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 pathway is just beginning to be evaluated. Recent studies e evidence that activation of the lectin pathway can be responsible for complement activation and related inflammation in ischemia/reperfusion injury. Collard et a1. (2000) reported that cultured endothelial cells subjected to oxidative stress bind MBL and show deposition of C3 upon exposure to human serum (Collard, C.D., et al., Am. J. Pathol. 156:1549-1556, 2000). In on, treatment of human sera with ng anti—MEL onal antibodies inhibited MBL binding and complement activation. These findings were extended to a rat model of dial ischemia—reperfusion 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., eta1., Circulation 104:1413-1418, 2001). The molecular mechanism of MBL binding to the vascular endothelium after oxidative stress is r; a recent study ts that activation of the lectin pathway after ive stress may be mediated by MBL binding to vascular endothelial cytokeratins, and not to glycoconjugates (Collard, C.D., et al., Am. J. Pathol. 159:]045-1054, 2001). Other studies have implicated the cal and alternative ys in the enesis of ischemia/reperfusion injury and the role of the lectin pathway in this disease s controversial (Riedermann, N.C., et al., Am. J. Pathol. 162:363-367, 2003).

A recent study has shown that MASP-l (and possibly also MASP—3) is required to convert the alternative pathway activation enzyme Factor D from its zymogen form into its enzymatically active form(See Takahashi M. et al., J Exp Med 207(1):29-37 (2010)).

The physiological importance of this s is underlined by the absence of alternative pathway functional activity 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 eonvertase (C3bBb) contains C3b as an ial subunit, the question regarding the origin of the first C3b via the alternative pathway has presented a puzzling problem and has ated considerable research.

PCT/U52012/036509 C3 belongs to a family of proteins (along with C4 and (1-2 macroglobulin) 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 thioester linkage with the sulfhydryl group of a cysteine three amino acids away. This bond is le and the electrophilic yl-thioester can react with philic moieties such as hydroxyl or amino groups and thus form a covalent bond with other molecules. The thioester bond is reasonably stable when sequestered within a hydrophobic pocket of intact C3. However, proteolytic cleavage of C3 to C3a and C3b s in exposure of the highly reactive thioester bond on C3b and, following nucleophilic attack by adjacent moieties comprising yl or amino groups, C3b becomes ntly linked to a target. In addition to its well-documented role in covalent attachment of C3b to complement targets, the C3 thioester is also thought to have a pivotal role in triggering the alternative pathway. According to the widely accepted "tick—over theory", the alternative pathway is initiated by the generation of a hase convertase, iC3Bb, which is formed from C3 with hydrolyzed thioester (iC3; C3(H20)) and factor B (Lachmann, P.J., et al., Springer Semin. Immzmopathol. 7:143-162, 1984).

The C3b-like C3(H20) is generated from native C3 by a slow spontaneous ysis of the internal thioester in the protein um, M.K., et al., J. Exp. Med. 154:856-867, 1981). Through the activity of the C3(H20)Bb convertase, C3b molecules are deposited on the target surface, thereby initiating the alternative y.

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

Activators are thought to include yeast cell walls (zymosan), many pure polysaccharides, rabbit erythrocytes, certain immunoglobulins, viruses, fungi, bacteria, animal tumor cells, tes, and damaged cells. The only feature common to these activators is the presence of carbohydrate, but the complexity and y of carbohydrate structures has made it lt to establish the shared lar 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):17-21 (1999)).

In addition to the ntly 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 W0 2012/]51481 PCT/U52012/036509 with factor B in g additional alternative pathway C3 convertase (C3bBb). The alternative pathway C3 convertase is stabilized by the binding of properdin. Properdin extends the ative pathway C3 tase ife six to ten fold. Addition of C3b to the alternative pathway C3 convertase leads to the formation of the ative y C5 convertase.

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

The converged pathway has been referred to as the terminal complement pathway. CSa 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. C5a—mcdiatcd cellular activation can significantly amplify inflammatory responses by inducing the release of multiple onal 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 deposition may play an important role in inflammation in addition to its role as a lytic pore-forming complex.

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

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

In one aspect, the invention es an isolated human monoclonal antibody, or antigen g fragment thereof, that binds to human MASP-2, comprising:(i) a heavy chain variable 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 sequence comprises an amino acid sequence set W0 20121151481 PCT/U82012/036509 forth as SEQ ID N0238 or SEQ ID N0290, and conservative sequence modifications thereof, wherein the light chain le region CDR-L3 sequence comprises an amino acid sequence set forth as SEQ ID NO:51 or SEQ ID N0:94, and conservative sequence modifications thereof, and wherein the ed antibody inhibits MASP-2 dependent complement activation.

In another , the present invention provides a human antibody that binds human MASP-Z, wherein the dy 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 N021; and ii) a heavy chain CDR-H2 comprising the amino acid sequence from 50—65 of SEQID N021; and iii) a heavy chain CDR-H3 comprising the amino acid sequence from 95-102 of SEQ ID N021; and b) a light chain le region comprising: i) a light chain CDR-LI comprising the amino acid sequence from 24-34 of either SEQ ID N025 or SEQ ID N027; and ii) a light chain CDR—L2 comprising the amino acid sequence from 50~56 of either SEQ ID N025 or SEQ ID N027; and iii) a light chain CDR-L3 comprising the amino acid sequence from 89-97 of either SEQ ID N025 or SEQ ID N027; 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 , wherein the dy or variant thereof inhibits MASP-2 dependent complement activation.

In another aspect, the t invention provides an isolated human monoclonal antibody, or n binding fragment thereof, that binds human MASP-Z, 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 N020; and ii) a heavy chain CDR-H2 comprising the amino acid sequence from 50-65 of SEQ ID N020; and iii) a heavy chain CDR-H3 comprising the amino acid sequence from 95-102 of either SEQ ID N0:18 or SEQ ID N020; and b) a light chain variable region comprising: i) a light chain CDR-L1 comprising the amino acid sequence from 24-34 of either SEQ ID N022 or SEQ ID N024; and ii) a light chain CDR-L2 comprising the amino acid sequence from 50-56 of either SEQ ID N022 or SEQ ID N024; and iii) a light chain PCT/U82012/036509 CDR—L3 comprising the amino acid sequence from 89-97 of either SEQ ID NO:22 or SEQ ID N024; or II) a variant f, 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 regions of said light chain variable region, wherein the antibody or variant f inhibits MASP-2 dependent complement activation.

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

In another aspect, the present invention es an isolated monoclonal antibody, or antigen-binding fragment thereof, that binds to human , comprising a light chain variable region comprising an one of the amino acid sequences set forth in SEQ ID N022, SEQ ID N024, SEQ ID N0225 or SEQ ID NO:27.

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

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

In another aspect, the invention provides a method of generating an isolated MASP-Z antibody comprising culturing 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 allowing for expression of the nucleic acid molecules encoding the ASP-2 antibody and isolating said anti-MASP-2 antibody.

In another aspect, the invention provides an isolated fully human monoclonal antibody or antigen-binding fragment thereof that dissociates from human MASP-2 with 21 KD of IOnM or less as determined by surface plasmon resonance and inhibits C4 W0 20]2/151481 PCT/U52012/036509 activation on a -coated substrate with an ICso of IOnM or less in 1% serum. In some embodiments, said antibody or antigen binding fragment thereof cally izes 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 NO:20 and a light chain variable region as set forth in SEQ ID NO:24.

In another aspect, the present ion provides itions comprising the fully human monoclonal anti—MASP-2 antibodies of the invention and a pharmaceutically acceptable excipient.

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

In another aspect, the present invention provides an article of manufacture comprising a unit dose of human monoclonal MASP-2 antibody of the invention suitable for therapeutic administration to a human subject, wherein the unit dose is the range of from 1mg to 1000mg.

DESCRIPTION OF THE DRAWINGS The foregoing aspects and many of the attendant ages of this ion will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein: FIGURE 1A is a diagram illustrating the genomic structure of human ; FIGURE 1B is a diagram illustrating the domain structure of human MASP-2 protein; FIGURE 2 graphically illustrates the results of an ELISA assay carried out on polyclonal populations selected from a schv phage library panned against s MASP-Z antigens, as described in Example 2; FIGURE 3A and 3B show results of testing of 45 candidate scFv clones for functional activity in the complement assay, as described in Example 3; WO 51481 PCT/U82012/036509 FIGURE 4 graphically illustrates the s of an experiment that was carried out to compare C30 levels in the three sera (human, rat and NHP), as described in Example 4; FIGURE 5A is an amino acid sequence alignment of the heavy chain region (residues 1-120) of the most active clones reveals two distinct groups belonging to VH2 and VH6 gene family, respectively, as described in Example 4; FIGURE SB is an amino acid sequence alignment of the scFv clones 17D20, 17NI6, I8L16 and 4D9, as described in Example 4; FIGURE 6 graphically illustrates the inhibitory activities of preparations of IgG4 converted mother clones in a C3b deposition assay using 90% human plasma, as described in Example 5; FIGURE 7A graphically illustrates the results of the ELISA assay on the I7N16 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 17D20 mother clone versus daughter clones ed on huMASPZA, as described in Example 6; FIGURE 8 is a protein sequence alignment of the mother clone I7Nl 6 and the 17N9 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 17D20 mother clone, as described in Example 7; FIGURE 10A is a protein sequence alignment of the CDR3 region of the 17D20 mother clone with the chain shuffled clone d21N11 and the mutagensis clone #35 CDR—H3 clone shown in FIGURE 9 combined with the VL of 17D20md21NII (VH35-VL21N11), as described in Example 7; FIGURE 10B is a protein sequence alignment of the VL and VH regions of the 17D20 mother clone and the daughter clone l7D20md21Nl l, as described in Example 7; FIGURE 11A graphically illustrates the results of the C3b deposition assay d out for the er clone isotype variants (MoAb#1—3), derived from the human anti-MASP-2 monoclonal antibody mother clone 17N16, as described in Example 8; FIGURE 11B cally illustrates the results of the C3b tion assay d out for the er clone isotype variants (MoAb#4-6), derived from the human anti-MASP-2 monoclonal antibody mother clone I7D20, as described in Example 8; 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 rates the inhibition of C4b deposition in 95% normal human serum, as described in Example 8; FIGURE 14 graphically illustrates the inhibition of C3b deposition in 95% African Green monkey serum, as described 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 Example 8; FIGURE 16 graphically illustrates the preferential binding ofMoAb#6 to human MASP2 as ed to Cls, as described in Example 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 bed 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- murinc MASP-2 antibody (mAle 1) or uman 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 dy (mAle 1) or anti—human MASP—2 antibody (mAbOMS646), as described in Example 11; FIGURE 18C is a Kaplan—Meier survival plot showing the t survival over time after exposure to 8.0 Gy radiation in control mice and in mice d with anti- human MASP—Z antibody (mAbOMS646), as described in e 11; FIGURE 19 graphically illustrates the results of e plasmon resonance (Biacore) analysis on anti-MASP—2 antibody OMS646 (response units ng) versus time in seconds), showing that immobilized OMS646 binds to recombinant MASP-2 with a Koff rate of about '4S'1 and a Kon rate of about 1.6-3x106M‘1S'1, as described in Example 12; FIGURE 20 cally illustrates the results of an ELISA assay to determine the binding affinity of anti-MASP-2 antibody OMS646 to immobilized human MASP-Z, showing that OMS646 binds to immobilized recombinant human MASP—2 with a KD of approximately 100 pM, as described in Example 12; PCT/U52012/036509 FIGURE 21A graphically illustrates the level of C4 tion on a mannan- coated surface in the presence or absence of anti-MASP-2 antibody (OMS646), demonstrating that OMS646 inhibits C4 activation on a mannan-coated surface with an ICso of approximately 0.5 nM in 1% human serum, as described 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 (OM8646) under lectin pathway—specific assay ions, demonstrating that OMS646 inhibits -mediated MAC deposition with an ICso value of imately 1 nM, as described in e 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 Example 12; FIGURE 22C graphically illustrates the level of MAC deposition in the presence or absence of anti-MASP—2 antibody 6) under alternative pathway—specific assay conditions, demonstrating that OM8646 does not inhibit alternative pathway-mediated MAC tion, as described in e 12; FIGURE 23A graphically illustrates the level of C3 deposition in the presence or absence of anti-MASP-Z 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 tion under logical conditions, as described in Example 12; FIGURE 24A graphically illustrates the level of C4 deposition in the absence or presence of anti-MASP-Z 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 esponsive manner with IC50 values in the range of 30 to 50nM, as described in Example 12; and WO 51481 PCT/U52012/036509 FIGURE 24B graphically illustrates the level of C4 deposition in the absence or presence of anti—MASP-Z 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 dose-responsive manner with IC50 values in the range of 15 to 30 nM, as bed in Example 12.

DESCRIPTION OF THE SEQUENCE LISTING 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 N026 rat MASP—2 protein (mature) ANTIGENS (in reference to human MASP-Z mature n) SEQ ID NO:7 CUBI domain of human MASP—2 (aa l-l2l) SEQ ID NO:8 CUBI/EGF domains of human MASP-2 (aa 1-166) SEQ ID NO:9 CUBI/EGF/CUBII domains ofhuman MASP-2 (aa 1-277) SEQ ID NO:10 EGF domain ofhuman MASP-2 (aa l22-l 66) SEQ ID NO:11 CCPI/CCPII/SP domains ofhuman MASP—Z (aa 1) SEQ ID NO:12 CCPI/CCPII domains of human MASP-Z (aa 278—429) SEQ ID NO:13 CCPI domain of human MASP-2 (aa 278-347) SEQ ID NOzl4 CCPII/SP domain of human MASP-Z (aa348-67l) SEQ ID NO:15 CCPII domain of human MASP-Z (aa 348-429) SEQ ID NO:16 SP domain ofhuman MASP-2 (aa 429-671) SEQ ID NO:17: Serine—protease vated mutant (aa 610-625 with mutated Ser 618) ANTI-MASP-Z MONOCLONAL ANTIBODIES VH chains SEQ ID NO:18 17D20mc heavy chain le region (VH) polypeptide SEQ ID NO:19 DNA encoding l7D20_dc35VH21N11VL (OMS646) heavy chain variable region (VH) (without signal peptide) SEQ ID NO:20 17D20_dc35VH21Nl IVL (OMS646) heavy chain le region (VH) polypeptide SEQ ID NO:21 17Nl6mc heavy chain variable region (VH) polypeptide —16- ' PCT/U52012/036509 ANTI-MASP-2 MONOCLONAL ANTIBODIES VL chains SEQ ID N022 c light chain variable region (VL) polypeptide SEQ ID NO:23 DNA encoding dc2 1N1 lVL (OMS644) light chain variable region (VL) (without signal peptide) SEQ ID NO:24 l7D20_dc2lNl lVL (OM8644) light chain variable region (VL) polypeptide SEQ ID N0225 l7N16mc light chain variable region (VL) polypeptide SEQ ID N0226 DNA encoding l7N16_dcl7N9 (OMS64I) light chain variable region (VL) ut signal peptide) SEQ ID NO:27 l7Nl6_dcl7N9 (OMS64I) light chain variable region (VL) ptide ANTI-MASP—2 MONOCLONAL ANTIBODIES HEAVY CHAIN CDRS SEQ ID NOS:28-3I CDR—Hl SEQ ID NOS:32-35 CDR—HZ SEQ ID NOS:36-40 CDR—H3 ANTI-MASP-2 MONOCLONAL ANTIBODIES LIGHT CHAIN CDRS SEQ ID NOS:41-45 CDR—Ll SEQ ID NOS:46-50 CDR-L2 SEQ ID NOS:51-54 CDR—L3 MASP-Z antibody Sequences SEQ ID NO:55: scFv mother clone 17D20 full length polypeptide SEQ ID NO:56: scFv mother clone 18Ll6 full length polypeptide SEQ ID NO:57: scFv mother clone 4D9 full length polypeptide SEQ ID NO:58: scFv mother clone 17L20 full length ptide SEQ ID NO:59: scFv mother clone l7N16 full length polypeptide SEQ ID NO:60: scFv mother clone 3F22 full length polypeptide SEQ ID NO:61: scFv mother clone 9P1 3 full length ptide SEQ ID NO:62: DNA encoding wild type IgG4 heavy chain constant region SEQ ID NO:63: wild type IgG4 heavy chain constant region polypeptide SEQ ID NO:64 DNA encoding IgG4 heavy chain constant region with mutant $228P SEQ ID NO:65: IgG4 heavy chain constant region with mutant 8228P polypeptide W0 2012/151481 PCT/U52012/036509 SEQ ID N0266: scFv daughter clone 17Nl6m_dl7N9 full length polypeptide SEQ ID NO:67: scFv daughter clone l7D20m_d21N11 full length polypeptide SEQ ID N0268: scFv daughter clone 17D20m_d3521Nll full length polypeptide SEQ ID NO:69: DNA encoding wild type IgGZ heavy chain constant region SEQ ID NO:70: wild type IgG2 heavy chain nt region polypeptide SEQ ID NO:71: 17Nl6m_dl7N9 light chain gene ce (with signal peptide encoded by nt 1-57)) SEQ ID NO:72: 17Nl6m_dl7N9 light chain protein sequence (with signal peptide aa1-19) SEQ ID NO:73: 17Nl6m_dl7N9 IgG2 heavy chain gene sequence (with signal peptide encoded by nt 1-57) SEQ ID NO:74: 17Nl6m_dl7N9 IgGZ heavy chain protein sequence (with signal peptide aa 1-19) SEQ ID NO:75: 17Nl6m_dl7N9 IgG4 heavy chain gene sequence (with signal peptide encoded by nt 1-57) SEQ ID NO:76: 17Nl6m_dl7N9 IgG4 heavy chain protein ce (with signal peptide aa 1-19) SEQ ID NO:77: 17Nl6m_dl7N9 IgG4 mutated heavy chain gene sequence (with signal peptide encoded by nt 1—57) SEQ ID NO:78: l7Nl 7m_dl7N9 IgG4 mutated heavy chain protein ce (with signal peptide aa 1-19) SEQ ID NO:79: 3521N11 light chain gene ce (with signal peptide encoded by nt 1-57) SEQ ID N0280: 17D20_3521N11 light chain protein sequence (with signal peptide aa 1-19) SEQ ID NO:81: 3521N11 IgGZ heavy chain gene sequence (with signal peptide encoded by nt 1-57) SEQ ID N0282: 17D20_3521N11 IgG2 heavy chain protein sequence (with signal peptide aa 1-19) SEQ ID NO:83: 17D20_3521Nll IgG4 heavy chain gene sequence (with signal peptide encoded by nt 1-57) SEQ ID NOc84: 17D20_3521N11 IgG4 heavy chain protein sequence (with signal peptide aa 1-19) -18— W0 2012f15148] PCT/U52012/036509 SEQ ID NO:85: 17D20_3521N11 IgG4 mutated heavy chain gene sequence (with signal peptide encoded by nt 1-57) SEQ ID NO:86: 3521N11 IgG4 mutated heavy chain protein sequence (with signal peptide aa 1-l9) SEQ ID NO:87: scFv daughter clone l7N16m_d17N9 DNA encoding full length polypeptide (without signal peptide) SEQ ID NO:88: scFv daughter clone l7D20m_d21Nl 1 DNA encoding full length polypeptide (without signal e) SEQ ID NO:89: scFv daughter clone l7D20m_d3521N11 DNA encoding full length polypeptide (without signal peptide) SEQ ID NO:90: consensus heavy chain CDR—H3 of l7D20m and ll SEQ ID N019l: consensus light chain CDR—Ll of 17D20m and d3521N11 SEQ ID N01922: consensus light chain CDR—Ll of 17N16m and dl7N9 SEQ ID NO:93: consensus light chain CDR-L2 of l7D20m, d3521Nll, 17Nl6m and d1 7N9 SEQ ID NO:94: consensus light chain CDR-L3 of 17Nl6m and dl7N9 DETAILED DESCRIPTION The present invention es fully human antibodies that bind to human MASP— 2 and inhibit lectin—mediated complement activation while leaving the classical (Clq- ent) y component of the immune system intact. The human anti-MASP~2 antibodies have been identified by screening a phage display library, as described in Examples 2-9. As described in Examples 10-12, high affinity anti-MASP—2 antibodies have been identified with the ability to inhibit lectin-mediated complement activation, as demonstrated in both in vitro assays and in. vivo. The variable light and heavy chain nts of the antibodies have been isolated in both a scFv format and in a full length IgG format. The human anti-MASP-2 antibodies are useful for inhibiting cellular injury associated with lectin—mediated complement pathway activation while leaving the classical (Cl q—dcpendent) pathway ent 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 ry skill in the art of the t invention. The W0 51481 PCT/U52012/036509 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 pathway, which occurs under physiological conditions (i.e., in the presence of Ca“) leading to the formation of the lectin pathway C3 convertase C4b2a and upon accumulation of the C3 cleavage product C3b subsequently to the C5 convertase C4b2a(C3b)n.

As used herein, the term "alternative y" refers to ment activation that is triggered, for example, by zymosan from fiingal and yeast cell walls, lipopolysaccharide (LPS) from Gram ve outer membranes, and rabbit erythrocytes, as well as from many pure ccharides, rabbit erythrocytes, viruses, bacteria, animal tumor cells, parasites and damaged cells, and which has traditionally been thought to arise from neous 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 ns including mannan-binding lectin (MBL), CL-ll and the ficolins (H-ficolin, M-ficolin, or in).

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

As used herein, the term "MASP-Z inhibitory antibody" refers to any anti—MASP- 2 antibody, or MASP-2 binding fragment thereof, that binds to or directly cts with MASP-2 and effectively inhibits -dependent complement activation. MASP-2 inhibitory antibodies useful in the method of the ion may reduce MASP-Z-dependent complement activation by greater than 20%, such as greater than %, or greater 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 "MASP—2 blocking antibody" refers to MASP—2 inhibitory antibodies that reduce MASP-Z-dependent ment activation by greater than 90%, such as greater than 95%, or greater than 98% (i.e., resulting in MASP-Z 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%).

PCT/U82012/036509 The terms ody" and "immunoglobulin" 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 n. One form of antibody tutes 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 s are together responsible for binding to an antigen, and the constant regions are responsible for the antibody effector functions.

As used herein, the term "antibody" encompasses antibodies and antibody fragments thereof, derived from any antibody—producing mammal (e.g., mouse, rat, rabbit, and e including human), or from a hybridoma, phage selection, inant expression or transgenic animals (or other methods of producing antibodies or antibody nts), that specifically bind to MASP—2 polypeptides or portions thereof. It is not intended that the term ody” be limited as regards to the source of the antibody or manner in which it is made (e.g., by hybridoma, phage selection, recombinant expression, transgenic animal, peptide synthesis, etc). Exemplary antibodies include polyclonal, monoclonal and inant antibodies; multispecific antibodies (e.g., bispecific antibodies); humanized antibodies; murine antibodies; chimeric, mouse-human, mouse—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 f (such as dAb, Fab, Fab”, F(ab’)2, FV), single chain (ScFv), tic variants thereof, naturally occurring variants, fusion ns comprising an antibody portion with an antigen—binding fragment of the required specificity, 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 fragment" refers to a polypeptide fragment that ns at least one CDR of an immunoglobulin heavy and/or light chains that binds to human MASP-Z. In this regard, an antigen-binding fragment of the herein described 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 e ofbinding to MASP-Z. In PCT/U52012/036509 certain embodiments, an antigen—binding fragment or an dy comprising an antigen- binding fragment, mediates inhibition ofMASP-2 dependent complement activation.

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

As used herein, the modifier "monoclonal" indicates the character of the antibody as being obtained from a ntially homogenous population of antibodies, and is not. ed 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 sion, transgenic animals, etc.). The term includes whole immunoglobulins as well as the fragments etc. described above under the definition of "antibody”. Monoclonal antibodies can be obtained using any technique that es for the production of antibody molecules by continuous cell lines in e, such as the oma method described by Kohler, G., et al., Nature 256:495, 1975, or they may be made by recombinant DNA s (see, e.g., US Patent No. 4,816,567 to Cabilly). onal antibodies may also be isolated from phage antibody ies using the techniques described in Clackson, T., et al., Nature 352:624—628, 1991, and Marks, J.D., et al., J. 11101. Biol. 222:581-597, 1991. Such antibodies can be of any immunoglobulin class including lgG, lgM, lgE, lgA, IgD and any subclass thereof.

The recognized 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 immunoglobulin "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.

PCT/U52012/036509 Full—length immunoglobulin "heavy chains" (of about 50 kDa or about 446 amino acids) rly se 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 antibodies can polymerize to form polyvalent 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 disulfide bonds, depending on the H chain isotype. 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), ed by three constant domains (CH) for each of the or and y , and four CH domains (CH) for u and aisotypes.

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 aligned with the first constant domain of the heavy chain (CH1). The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda (it), 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), immunoglobulins can be ed to ent classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, having heavy chains designated alpha (a), delta (8), n (a), gamma (y) and mu (u), respectively. The y and 0. classes are further divided into subclasses on the basis of minor differences in CH sequence and function, for example, humans express the following sses: IgGl, IgGZ, IgG3, 1gG4, IgAl and IgA2.

For the structure and properties of the different classes of antibodies, see, c.g., Basic and Clinical logy, 8th Edition, Daniel P. Stites, Abba I. Terr and am 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 2012/036509 g 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 s. Rather, the V regions consist of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme ility 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 forming 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 ion of the antigen-binding site of antibodies (see Kabat, et al., ces of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody ent cellular cytotoxicity (ADCC).

As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody that are responsible for n 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 of Proteins ofImmunological Interest, 5th Ed. Public Health Service, National Institutes 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 variable domain when numbered in accordance with the Chothia numbering system, as bed in Chothia and Lesk, J. Mol. Biol. [96:901— 917 (1987)); and/or those residues from a "hypervariable loop"/CDR (e.g., residues 27—38 (Ll), 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 numbered in accordance with the IMGT numbering system as described in Lefrane, J .P., et al., c Acids Res 272209-212; Ruiz, M., et al., Nucleic Acids Res 28:219-221 (2000)).

PCT/U82012/036509 As used herein, the term "antibody fragmen " refers to a portion derived from or related to a ength ASP-Z antibody, generally including the antigen binding or variable region thereof. rative es of antibody fragments include Fab, Fab', F(ab)2, F(ab‘)2 and Fv fragments, scFv fragments, ies, linear dies, single—chain antibody molecules, bispecific and multispecific antibodies formed from dy fragments.

Where bispecific 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.

Current Opinion Biotechnol. 4, 446-449 (1993)), e.g. ed chemically or from hybrid hybridomas, or may be any of the bispecific antibody fragments mentioned above.

As used herein, a "single-chain Fv" or "scFv" antibody fragment comprises the VH and VL s of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the FV polypeptide r comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for n binding. See Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

"Fv" is the minimum antibody fragment that contains a complete antigen-recognition and binding site. This fragment consists of a dimer of one heavy and one light chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (three loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an FV comprising only three CDRs c for an antigen) has the ability to ize and bind antigen, 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 ular analyte that is present in a homogeneous mixture of different analytes. In certain embodiments, a specific binding interaction will discriminate between desirable and rable analytes in a sample, in some embodiments more than about l0 to lOO-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 cally 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 WO 51481 PCT/U82012/036509 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 nt” ASP-2 antibody refers to a molecule which differs in amino acid sequence from a "parent" or nce antibody amino acid sequence by Virtue of addition, deletion, and/or substitution of one or more amino acid residue(s) in the parent dy sequence. in one embodiment, a variant anti—MASP—2 antibody refers to a molecule which contains variable s that are identical to the parent variable domains, 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 vative sequence modifications.

As used herein, the term "parent antibody" refers to an antibody 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 fully human antibody.

As used herein, the term ted 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, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody 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 ator; or (3) to homogeneity by SDS—PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated dy includes the antibody in situ within recombinant cells since at least one component of the dy's natural environment will not be present.

Ordinarily, however, isolated antibody will be ed by at least one purification step.

As used herein, the term "epitope" refers to the portion of an antigen to which a monoclonal antibody specifically binds. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side —26- W0 2012/]51481 PCT/U82012/036509 chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. More specifically, the term "MASP-2 epitope," 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 immunogenic.

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

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

As used herein, the ane attack complex" ) refers to a complex of the al five complement components (C5-C9) that inserts into and disrupts membranes. Also referred to as C5b-9.

As used , "a subject" includes all mammals, including without tion, humans, non-human primates, dogs, cats, horses, sheep, goats, cows, rabbits, pigs and rodents.

As used herein, the amino acid residues are abbreviated as s: alanine (Ala;A), asparagine (Asn;N), aspartic acid (AspgD), arginine (ArggR), cysteine (Cys;C), glutamic acid (Glu;E), glutamine (GlngQ), glycine (Gly;G), histidine (His;H), isoleucine (Ile;I), leueine (Leu;L), lysine (Lys;K), nine (Meth), phenylalanine (Phe;F), proline (Pro;P), serine (SergS), threonine (Thr;T), 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 lle, Leu, Met, Phe, Trp, Tyr, Val, Ala, Cys or Pro. By philic" amino acid is meant either Gly, Asn, Gln, Ser, Thr, Asp, Glu, Lys, Arg or His. This grouping of amino acids can be further subclassed as follows. By "uncharged hilic" 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, WO 2012115148] PCT/U52012/036509 valine, leucine, and isoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine, (4) aspartate and glutamate, (5) glutamine and asparagine, and (6) lysine, arginine and histidine.

As used herein, 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 ed DNA molecule. Another example of an isolated nucleic acid molecule is a ally-synthesized nucleic 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 smaller than the complete DNA molecule of a some from that species.

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

As used herein, an "expression 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 ator. Gene expression is usually placed under the control of a promoter, and such a gene is said to be "operably linked to" the er.

Similarly, a regulatory element and a core promoter are operably linked if the regulatory element tes the ty 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 (except where such number would exceed 100% of a possible .

Where ranges are stated, 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 aspects 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 agent" includes one agent, as well as two or more agents; reference to "an antibody" includes a plurality of such antibodies and reference to "a framework region" includes reference to one or more framework regions and equivalents thereof known to those skilled in the art, and so forth.

PCT/U52012/036509 Each ment in this cation is to be applied mutatis mutandz‘s to every other embodiment unless expressly stated otherwise. rd 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 . 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 nces that are cited and discussed throughout the present specification. See e.g., Sambrook et al., 2001, MOLECULAR CLONING: A LABORATORY MANUAL, 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; 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 ations and other like references. Unless specific definitions are provided, the nomenclature utilized in tion with, and the laboratory procedures and techniques of, molecular biology, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical try 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 t to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods ofthe invention. 11. Overview s (MBL, M-ficolin, H-ficolin, L-ficolin and CL-ll) are the specific recognition molecules that trigger the innate ment system and the system includes the lectin initiation pathway and the associated terminal pathway amplification loop that amplifies lectin-initiated activation of terminal complement effector molecules. Clq is the specific recognition molecule that triggers the ed complement system and the system includes the classical initiation pathway and associated terminal pathway W0 2012/]51481 2012/036509 amplification loop that amplifies Clq-initiated tion of terminal ment effector molecules. We refer to these two major complement activation systems as the lectin-dependent complement system and the Clq-dependent complement system, respectively.

In on to its essential role in immune defense, the complement system contributes to tissue damage in many clinical ions. Thus, there is a pressing need to develop therapeutically effective complement inhibitors to prevent these adverse As described in US. Patent No. 094, co—pending US. Patent Application Serial No. 12/905,972 (published as US 091450), 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 nce, it was determined through the use of a MASP/- mouse model that it is possible to inhibit 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 without completely shutting down the immune defense capabilities of complement. For example, in disease states in which complement tion is mediated predominantly by the lectin—dependent complement system, it would be advantageous to specifically inhibit only this system. This would leave the Clq—dependent complement activation system intact to handle immune complex processing and to aid in host defense against infection.

The preferred n component to target in the pment of therapeutic agents to specifically inhibit the lectin-dependent complement system is MASP—2. Of all the known protein components of the lectin-dependent complement system (MBL, H—fieolin, M-ficolin, L—ficolin, MASP-2, C2-C9, Factor B, Factor D, and properdin), only MASP-2 is both unique to the lectin-dependent complement system and required for the system to function. The lectins (MBL, H—ficolin, M-ficolin, L-ficolin and CL-l l) 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 lectin-dependent ment activation . Furthermore, 2012/036509 since MBL and the ficolins are also known to have opsonic activity independent of complement, inhibition of lectin fimction would result in the loss of this beneficial host defense mechanism against infection. In contrast, this complement-independent lectin opsonic activity would remain intact if MASP-Z was the inhibitory target. An added benefit of MASP-2 as the therapeutic target to t the lectin—dependent complement tion system is that the plasma concentration of MASP—Z is among the lowest of any complement protein (2 500 ng/ml); therefore, pondingly low concentrations of high-affinity inhibitors of MASP-2 is sufficient to obtain full inhibition, as demonstrated in the Examples .

In accordance with the foregoing, as described herein, the present invention provides onal fully human anti—MASP-Z antibodies that bind to human MASP-2 with high affinity and are capable of inhibiting lectin-mediated complement pathway activation. 11]. 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-Z and inhibits or blocks MASP-Z-dependent complement activation. MASP-2 inhibitory antibodies may effectively inhibit or effectively block the -dependent complement activation system by inhibiting or blocking the biological function of MASP-Z. For example, an inhibitory antibody may effectively inhibit or block MASP—2 protein-to-protein interactions, interfere with MASP-2 dimerization or assembly, block Ca2+ g, or interfere with the MASP-2 serine protease active site. 2 Epitopes The invention provides fully human antibodies that specifically bind to human MASP-2. The MASP-2 polypeptide exhibits a molecular structure similar to MASP-l, MASP-3, and Clr and Cls, the proteases of the Cl ment system. The cDNA molecule set forth in SEQ ID NO:1 encodes a representative example of MASP-2 sting of the amino acid ce set forth in SEQ ID N022) and provides the human MASP-2 polypeptide with a leader sequence (aa 1-15) that is cleaved after secretion, ing 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 PCT/U52012/036509 set forth in SEQ ID NO:5) and provides the rat MASP~2 polypeptide with a leader sequence that is cleaved after secretion, resulting in the mature form of rat MASP-2 (SEQ ID NO:6).

Those skilled in the art will recognize that the sequences disclosed in SEQ ID N021 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. Allelic ts of the nucleotide sequences shown in SEQ ID NO:1 and SEQ ID NO:4, ing those containing silent mutations and those in which mutations result in amino acid sequence changes, are within the scope of the present invention. c variants of the MASP-2 sequence can be cloned by probing cDNA or genomic libraries 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 factor-like domain, a second CUB domain (CUBII), as well as a tandem of complement control n domains CCPl and CCP2, and a serine protease domain. Alternative splicing of the MASP-2 gene s in MApl9. MApl9 is a nonenzymatic n containing the N-terminal CUB l -EGF region ofMASP-Z with four additional es (EQSL). l proteins have been shown to bind to, or interact with MASP-Z through protein-to—protein interactions. For example, MASP—2 is known to bind to, and form Ca2+ ent complexes with, the lectin ns MBL, H—ficolin and L—ficolin. Each MASP-Z/lectin complex has been shown to activate complement through the MASP-Z-dependent cleavage of proteins C4 and C2 (Ikeda, K., et al., J Biol.

Chem. 262:7451-7454, 1987; Matsushita, M., et al., J. Exp. Med. 176:1497—2284, 2000; Matsushita, M., et al., J. l. 168:3502—3506, 2002). Studies have shown that the CUBl-EGF domains of MASP-2 are essential for the association of MASP-2 with MBL (Thielens, N.M., et al., J. Immunol. 166:5068, 2001). It has also been shown that the CUBIEGFCUBII s mediate dimerization of MASP—Z, which is required for formation of an active MBL complex (Wallis, R., et al., J. Biol. Chem. 27530962-30969, 2000). Therefore, MASP-2 inhibitory antibodies can be identified that bind to or interfere with MASP-Z target regions known to be important for MASP-Z-dependent complement activation.

PCT/U82012/036509 TABLE I: MASP-Z Pol Hetide Domains SE. 11) No: an [D Noe SE 11) NO:3 m ID No:5 SEQ ID No:6 SEQ ID NO:7 CUBI domain ofhuman MASP—Z (aa1-121 ofSEO ID NO:3) SEQ ID NO:8 CUBI/EGF domains of human MASP-Z aa1-166 ofSEO ID NO:3) SEQ ID NO:9 CUBI/EGF/CUBII domains of human MASP-2 (aa 1—277 of SEQ ID NO:3) SEQ ID NO:10 EGF domain ofhuman MASP-Z (aa 122—166 of SEQ ID NO:3 SEQ ID NO:11 CCPI/CCPII/SP domains of human MASP-2 aa 278-671 aa of SEQ ID NO:3) SEQ ID NO: 12 CCPI/CCPII s of human MASP-Z aa 9 of SEO ID NO:3) SEQ ID NO:13 CCPI domain ofhuman MASP—2 aa 278—347 of SE 0 ID NO:3) SEQ ID NO:14 CCPII/SP domains ofhuman MASP-Z aa 348-671 of SEO ID NO:3 SEQ ID NOZIS CCPII domain of human MASP—2 aa 348—429 of SEQ ID NO:3) SEQ ID NO: 16 SP domain ofhuman MASP~2 (aa 429-671 of SEO ID NO:3) SEQ ID NO:17 . Serine-protease inactivated mutant form (GKDSCRGDAGGALVFL) (aa 610—625 of SEQ ID NO:3 with mutated Ser 618) In one embodiment, the anti-MASP-Z inhibitory antibodies 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—2. In some embodiments, the anti- MASP-Z inhibitory antibodies of the invention bind to an epitope in the CCP1 domain (SEQ ID NO:I3 (aa 278-347 of SEQ ID NO:3)). For e, anti-MASP-2 antibodies (e.g., OMS646) have been identified that only bind to MASP—2 fragments containing the CCP1 domain and inhibit MASP—2 dependent complement activation, as described in Example 9.

Binding Aflinily 0fMASP-2 Inhibitory Antibodies The ASP—Z inhibitory antibodies specifically bind to human MASP-2 (set forth as SEQ ID NO:3, encoded by SEQ ID NO:l), with an affinity of at least ten times greater than to other antigens in the complement system. In some embodiments, the MASP-2 inhibitory antibodies specifically bind to human MASP-Z with a binding affinity of at least 100 times greater than to other antigens in the complement system.

In some embodiments, the MASP—2 inhibitory dies 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 0.1nM. The binding affinity of the MASP-2 inhibitory antibodies can be ined using a suitable binding assay known in the art, such as an ELISA assay, as described in Examples 3-5 herein.

Potency -Z Inhibitory Antibodies In one embodiment, a MASP-2 inhibitory antibody 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 1% serum.

In one embodiment, a MASP-2 inhibitory dy is sufficiently potent to inhibit MASP—2 dependent ment activation at an IC50 S 30 nM, ably 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 ts C4a, C3a, C5a and/or C5b-9 (MAC) (measured, for example, as described in e 2 of US Patent No. 7,919,094) as well as their eatabolie degradation products (e.g., PCT/U52012/036509 C3desArg), the reduction of C4 cleavage and C4b deposition (measured, for example, as described in Example 5) and its uent catabolic degradation 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 uent catabolic degradation products (e.g., C3bc, C3d, etc).

In some embodiments, the MASP-Z inhibitory dies 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 tion.

In some embodiments, the MASP~2 inhibitory antibodies of the invention are capable of inhibiting 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 ASP-Z inhibitory antibodies selectively inhibit MASP-2 ment activation (z'.e., bind to MASP-2 with at least 100—fold or greater affinity than to Clr or Cls), leaving the Clq-dependent complement activation system functionally intact (i.e., at least 80%, or at least 90%, or at least 95%, or at least 98%, or 100% ofthe classical pathway activity is ed).

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

As described in Examples 2-12, fully human antibodies have been identified that bind with high affinity to MASP—2 and t 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, isolated and analyzed in both a scFv format and in a full length IgG format.

FIGURE 5A is an amino acid ce alignment of seven scFv anti-MASP-Z clones that were identified as having high binding affinity to MASP—2 and the ability to inhibit MASP-Z dependent activity. FIGURE 5B is an amino acid sequence alignment of four of W0 2012/]51481 PCT/U52012/036509 the scFv mother clones 17D20, l7N16, 18L16 and 4D9, showing the framework regions and the CDR regions. The scFv mother clones 17D20 and 17Nl6 were subjected to affinity maturation, leading to the tion of daughter clones with higher affinity and increased y as ed to the mother clones, as described in Examples 6 and 7.

The amino acid sequences of the heavy chain variable regions (VH) (aa 1—120) and the light chain variable regions (VL) (aa 0) of the scFv clones shown in FIGURES 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 ons, 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 antibodies. In one exemplary embodiment, the heavy and light chain amino acid sequences of FIGURES 5A and SB are d, 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 present at each position of the heavy and light chains of the exemplary MASP-Z inhibitory dies), several substitutable positions, as well as the amino acid residues that can be substituted 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: Se uences of re resentative anti-MASP-Z antibodies ID Reference: mother/daughter 17D20 mother clone SEQ ID SEQ ID N0222 NO: 18 -3 6— W0 51481 PCT/U52012/036509 ID Reference: mother/daughter VH 17D20_35VH- daughter clone SEQ ID SEQ ID NO: 24 IgG2 21N1 IVL NO:20 (10 aa changes (OMS644) (one aa change in VB (A to $1? parent R) at position 102 of SEQ ID NO: 1 8) 17D20n35VH- daughter clone SEQ ID SEQ ID NO: 24 21N1 1VL NO:20 (OMS645) (one aa change in VH (A to R) at position 102 of SEQ ID NO: 18) 17D20_35VH— daughter clone SEQ ID SEQ ID NO: 24 IgG4 (mutant 2IN11VL NO:20 IgG4 hinge (OMS646) (one aa change region) in VH (A to R) at position 102 of SEQ ID SEQ ID N0221, 17N9 daughter SEQ ID NO:21 (OMS641) (l 7aa changes from SEQ ID 17N16_17N9 daughter SEQ ID SEQ ID NO:27 IgG4 17N16_17N9 daughter SEQ ID SEQ ID NO:27 IgG4 (mutant N021 (OM8643) IgG4 hinge reion) In certain embodiments, a subject human ASP-2 monoclonal tory 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 PCT/U52012/036509 leastabout9596,oratleastabout9696idenncal,oratleastabout9796idenfical,oratleast about 98% identical, or at least 99% identical), to that of any of the heavy chain variable domain sequences set forth in TABLE 2.

In some embodiments, a subject human anti—MASP-2 monoclonal tory antibody has a heavy chain variable domain that is substantially cal (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:18. In some embodiments, the subject human anti—MASP-Z onal inhibitory antibody has a heavy chain variable domain that comprises, or consists of SEQ ID NO:l8.

In some embodiments, a t 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 17D20_cd35VH2Nll (VH), set forth as SEQ ID NO:20. 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:20.

In some embodiments, a subject human anti—MASP-Z onal inhibitory antibody has a heavy chain variable domain that is ntially identical (e.g., at least about 70%, at least 75%, at least about 80%, at least about 85%, at least about 90%, at leastabout9596,oratleastabout9696idenfical,oratleastabout9796idenfical,oratleast 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-Z onal inhibitory antibody has a heavy chain variable domain that comprises, or consists of SEQ ID IJCX21.

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,atleast7596,atleastabout8096,atleastabout8596,atleastabout9096,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), to that of any of the light chain variable domain sequences set forth in TABLE 2. —38— W0 2012/15148] 2012/036509 In some embodiments, a subject human anti—MASP-2 onal inhibitory antibody has a light chain variable domain that is substantially identical (e.g., at least about7096,atleast7596,atleastabout8096,atleastabout8596,atleastabout9096,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 (VL), set forth as SEQ ID N0222. In some embodiments, the subject human ASP-2 monoclonal inhibitory antibody has a light chain that comprises, or ts of SEQ ID N0222.

In some embodiments, a subject human anti-MASP-Z 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% identical, or at least about 97% identical, or at least about 98% identical, or at least 99% identical) to 17D20_35VH~21N11VL (VL), set forth as SEQ ID NO:24. In some embodiments, the subject human anti-MASP-Z monoclonal inhibitory antibody has a light chain that comprises, or ts of SEQ ID NO:24.

In some ments, a subject human ASP-2 monoclonal inhibitory dy has a light chain variable domain that is substantially identical (e.g., at least about7096,atleast7596,atleastabout8096,atleastabout8596,atleastabout9096,at 1eastabout9596,oratleastabout9696idenficai,oratleastabout9796idenfica],oratleast about 98% cal, or at least 99% cal) to 17Nl6 (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 comprises, or consists of SEQ ID NO:25.

In some embodiments, a subject human anti-MASP-Z monoclonal inhibitory antibody has a light chain variable domain that is substantially identical (e.g., at least about7096,atleast7596,atleastabout8096,atleastabout8596,atleastabout9096,at leastabout9596,oratleastabout9696idenfical,oratleastabout9796idenfical,oratleast about 98% identical, or at least 99% identical) to 17N16_17N9 (VL), set forth as SEQ ID N027. In some embodiments, the subject human anti-MASP-2 monoclonal inhibitory antibody has a light chain that comprises, 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).

PCT/U52012/036509 In some embodiments, the anti-MASP—Z inhibitory antibodies of the invention have a heavy chain variable region comprising one or more CDRs (CDRI, CDR2 and/or CDR3) that are substantially cal (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), or comprise or consist of the cal 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 ments, the anti—MASP-2 inhibitory dies 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% identical, 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 light chain vatiable sequences shown in FIGURES 5A or 5B, or described below in TABLES 4A—F and TABLE 5.

Hea Chain Variable Re ion TABLE 3A: Heav chain aa 1-20 17D20m (SEQ:18) d3521N11 P T (SEQ:21) dl7N9 (SEQ:21) -IIIII cum II PCT/U52012/036509 23 “min-lull!!! IIIIIIIIIIIIIIIIIIII II“IIIIIIIIIIIIIIIIIIII(SEQ220) 17N16m (SEQ:21) mmmm P P A L E L A _ _ _ (SEQ218) d3521N11 (SEQ220) 17N16m (SEQ:21) I'll—IIIIIMEM 17D20m (SEQ:18) IIIIIIIIIIIIIIIIIIII d3521N11 (SEQz20) IIIIIIIIIIIIII 17mm IBII-II-I IEIIIIIHII_IIIIIIIIIIIIIIIIII(SEQ:2 1) chain aa 81100 CDR—H3 ss 88mm 8) II: (SEQ220) 17N 16m (SEQ221) dl7N9 (SEQz2l) TABLE 3F: hea chain aa 101—118) Heavy CDR-H3 (cont'd) chain aa 101 m (SEQ:19) (SEQ220) III G v P F D I w (SEQ:21) III 6 v P F D (SEQ:21) 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 (H1), 50-65 (H2) and 95—102 (H3)) are bolded; and the Chothia CDRs (26-32 (H1), 52—56 (H2) and 95-101 (H3)) are underlined. 2012/036509 17D20 heavy chain variable region (VH2 {SEQ ID NO:18 1: QVTLKESGPVLVKPTETLTLTCTVSGFSLSRGKMGVSWIRQPPGKALEWL AHIFSSDEKSYRTSLKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCARIRAGG IDYWGQGTLVTVSS 17D20 35VH—21N11VL heafl chain variable region gVH) §SEQ ID N010} QVTLKESGPVLVKPTETLTLTCTVSGFSLSRGKMGVSWIRQPPGKALEWL AHIFSSDEKSYRTSLKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCARIRRGG IDYWGQGTLVTVSS l7N16 heam chain variable region (VH2 (SEQ ID N0221 1 QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSTSAAWNWIRQSPSRGLEWL GRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARDPF GVPFDIWGQGTMVTVSS TABLE 4: Heav Chain CDRS Clone Reference CDR mom 30 d3521N1 l CDR-H1 Chothia) GFSLSRG l7Nl6m CDR—H1 chothia) GDSVSST dl7N9 CDR-H1 chothia) T _—__ LGRTYYRSKWYNDYAV LGRTYYRSKWYNDYAV CDR-H2 chothia) HIFSS WO 51481 PCT/U52012/036509 Clone Reference CDR SEQ ID NO: d3521N1 1 CDR H2 chothla' HIFSS 17D20m CDR H3. kabat) YYCARIRA _36 11 CDR-H3 (kabat) YYCARIRR _37 17D20m and CDR-H3(kabat) YYCARIRX d3521N11 (wherein X at position 8 is consensus A Ala) or R Ar ) 17mm 0011-113 kabat) 117119 CDR-H1 11111) 17D20m CDR-H3 chothia) d3521N11 CDR-H3 a 17N16m CDR-H3 chothia mg CDR-H3 chothia Lioht Chain Variable Re ions TABLE 5A: Liht chain aa 1-20) Light chain aa "56 7 8—11 121114 1s...19 21 17D20m a-T Q P Pfls V S .I'II (SEQ:22) d3521N11 a (SEQ:24) -TQPPSL S VS-EIIII 17N16m S-TQPPSVVL s v A P GHIIII (SEQ225) d17N9 ”QEL 11 plls v A 11 Ga” (SEQ:27) TABLE 5B: Light chain (aa 21—40) PCT/U52012/036509 Light CDR—Ll chain aa 28 -I229 .3ssIIIII IIIIIIIIIII—I [’11 IIIIIIII (SEQ:22) IIIIIIIIIIII IR |I< (SEQ:24) III IW Iz |< m [2 IIIIII (SEQz25)”Wmllifififilfi II I IIIII— IW IFU |< M I? (SEQz27) __ ’IIIIII IIMII 17N16m (SEQ:25) d17N9 (SEQz27) IIIIIIIIIIIIIII 26 I2IIII 17D20m (SEQ:22)IIHII d3 521N1 1 (SEQ224) IIIIIIHII 2012/036509 Light CDR-LZ (cont'd) chain Mum-IIIIIIIMEMM (SEQ225) (SEQz27) TABLE 5E: Li; Light CDR-L3 chain mama 17D20m (SEQ222) d3521N1 1 (SEQ:24) 17N16m (SEQ225) d17N9 (SEQz27) TABLE 5F: Liht chain aa 101-120) Light CDR-L3 (cont'd) chain ms 106 109 no 112 us 114MI- 119 no 17D20m (SEQ:22) d3521N1 1 (SEQ:24) 17N16m (SEQ225) d17N9 (SEQ227) PCT/U52012/036509 Presented below are the light chain variable region (VL) sequences for the mother clones and daughter clones listed above in TABLE 2 and TABLES SA-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. 17D20m light chain variable region {VLQ (SEQ ID N022) PPSVSVSPGQTASITCSGDKLGDKFAYWYQQKPGHSPVLVIYQQ NKRPSGIPGRFSGSNSGNTATLTISGTQAMDEADYYCQAWDSSTAVFGTGTKVT l7D20m d3521Nll light chain variable region {VLQ (SEQ ID N024) QPVLTQPPSLSVSPGQTASITCSGEKLGDKYA Y W Y QQKPGQSPVLVMYQ DKS[RPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQAWDSSTAVFGGGTKL l7N16m light chain variable region (VL) (SEQ ID N02251 SYVLTQPPSVSVAPGQTARITCGGNNIGSKNVHWYQQKPGQAPVLVVYD DSDRPSGIPERFSGSNSGNTATLTVSRVEAGDEADYYCQVWDTTTDHVVFGGG TKLTVLAAAGSEQKLISE l7N16m dl 7N9 light chain variable region (VLl (SEQ ID NO:27[ SYELIQPPSVSVAPGQTATITCAGDNLGKKRVHWYQQRPGQAPVLVIYD DSDRPSGIPDRFSASNSGNTATLTITRGEAGDEADYYCQVWDIATDHVVFGGGT KLTVLAAAGSEQKLISE TABLE 6: Liht Chain CDRs /chothia) ___SE11)No 17D20m CDRL1 GDKLGDKFAYW d3521N11 CDRL1 GEKLGDKYAYW 42_ 17D20m and CDR-Ll KXAYW d352lNll wherein X at osition 2is D W0 51481 2012/036509 consensus (Asp) or E (Glu); and wherein X at position 8 is F Phe) or Y T r) 17N16m CDR-Ll GNNIGSKNVHW d17N9 CDR—Ll GDNLGKKRVHW 17N16m and CDR-L 1 GXNXGXKXVHW 92 d17N9 consensus (wherein X at position 2 is N (Asn) or D (Asp); wherein X at position 4 is I (He) or L (Leu); wherein X at position 6 is S (Ser) or K (Lys); and n X at position 8 is N (Asn) or R Ar)) d17N9 17D20m dsszmu asszmn 17N16m mm 49 17D20m, DXXRPSG 93 d3521N11, (wherein X at position 2 is N 17N16m, d17N9 (Asn), K (Lys) or S (Ser); COIlSensus and wherein X at position 3 is K (Lys), Q (Gln) or D 17D20m CDR—L3 AWDSSTAVF 51 d3521N1 1 CDR-L3 AWDSSTAVF _51 d3521N11 CDR—L3 (aa 89- AWDSSTAVFGGGTKLT 104) —48- W0 2012/]51481 PCT/U82012/036509 17N16m CDR—L3 VWDTTTDHV d17N9 CDR-L3 VWDIATDHV 17N16m and CDR-L3 VWDXXTDHV 94 d17N9 consensus (wherein X at position 4 is T (Thr) or I (He); and wherein X at position 5 is T (Thr) or A Ala)) In one aspect, the invention provides an isolated human monoclonal dy, or antigen binding fragment thereof, that binds to human MASH-2, comprising: (i) a heavy chain variable region comprising CDR-HI, CDR-HZ 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 sequence comprises an amino acid sequence set forth as SEQ ID N038 or SEQ ID NO:90, and conservative sequence ations 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 ce modifications f, and wherein the isolated dy inhibits MASP—2 dependent ment 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-HI sequence ses an amino acid sequence set forth as SEQ ID N028 or SEQ ID N029, and conservative modifications thereof. In one ment, 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 ce set forth as SEQ ID NO:91 or SEQ ID N092 and conservative modifications thereof. In one embodiment, the CDR-HI of the heavy chain variable region comprises SEQ ID N0128.

WO 2012151481 PCT/U82012/036509 In one embodiment, the CDR—H2 of the heavy chain variable region comprises SEQ ID N032. 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 contains an R (Arg) at position 8.

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

In one ment, 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 contains a K (Lys) at position 2. In one embodiment, the amino acid sequence set forth in SEQ ID NO:93 contains a Q (Gin) 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 n binding fragment thereof binds human MASP-2 with a KD of 10 nM or less. In one embodiment, said antibody or antigen binding fragment f 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 n binding fragment 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 contains variable s that are identical to the recited variable domain(s), except for a combined total of I, 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 I, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions with said CDR s 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 dy comprises: I) a) a heavy chain variable region comprising: i) a heavy chain CDR—H1 comprising the WO 51481 PCT/U82012/036509 amino acid sequence from 31-35 of SEQ ID N0221; and ii) a heavy chain CDR—H2 comprising the amino acid ce from 50-65 of SEQ ID N0:21; and iii) a heavy chain CDR—H3 comprising the amino acid sequence from 95-102 of SEQ ID N021; and b) a light chain le region comprising: i) a light chain CDR-L1 comprising the amino acid sequence from 24—34 of either SEQ ID N0:25 or SEQ ID N027; and ii) a light chain CDR~L2 comprising the amino acid sequence from 50-56 of either SEQ ID N0:25 or SEQ ID NO:27; and iii) a light chain CDR-L3 comprising the amino acid sequence from 89-97 of either SEQ ID N0:25 or SEQ ID N0227; 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 combined 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 ses an amino acid tution at one or more ons 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 or 102 of said heavy chain variable region. In one embodiment, said variant comprises an amino acid substitution at one or more positions ed 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 dy comprises SEQ ID N0221. In one embodiment, the light chain of said antibody comprises SEQ ID N0225. In one embodiment, the light chain of said antibody comprises SEQ ID N0227.

In another aspect, the invention provides an isolated human monoclonal antibody that binds to human MASP-2, wherein the antibody comprises: 1) a) a heavy chain variable region comprising: i) a heavy chain CDR-H1 comprising the amino acid sequence from 31-35 of SEQ ID N020; and ii) a heavy chain CDRH-2 comprising the amino acid sequence from 50-65 of SEQ ID N020; and iii) a heavy chain CDR-H3 comprising the amino acid sequence from 95-102 of either SEQ ID N0218 or SEQ ID PCT/U52012/036509 N020; and b) a light chain variable region comprising: i) a light chain CDR-L1 comprising the amino acid sequence from 24-34 of either SEQ ID N022 or SEQ ID N024; and ii) a light chain CDR-L2 comprising the amino acid sequence from 50—56 of either SEQ ID N022 or SEQ ID N024; and iii) a light chain CDR—L3 comprising the amino acid sequence from 89-97 of either SEQ ID N022 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 l, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid tutions 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 s of said light chain variable region, wherein the antibody or t 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 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 or 102 of said heavy chain variable region. In one embodiment, said variant ses an amino acid substitution at one or more ons 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 dy comprises SEQ ID N020, or a variant thereof comprising at least 80% identity to SEQ ID N020 (e.g., at least 85%, at least 90%, at least 95% or at least 98% identity to SEQ ID N020). In one embodiment, the heavy chain of said antibody comprises SEQ ID NO:18, or a variant thereof comprising at least 80% identity to SEQ ID NO:18 (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 N022, or a variant thereof comprising at least 80% identity to SEQ ID N022 (e.g., at least 85%, at least 90%, at least 95% or at least 98% ty to SEQ ID N022). In one embodiment, the light chain of said antibody comprises SEQ ID N024, or a variant thereof comprising at least 80% identity to SEQ ID N024 (e.g., at least 85%, at least 90%, at least 95% or at least 98% identity to SEQ ID N024).

PCT/U82012/036509 In one embodiment, said antibody binds to an e in the CCPl domain of In one ment, said antibody binds human MASP-Z 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 embodiment, said antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2 and F(ab')2. In one embodiment, said antibody 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 ment, said antibody is an IgG4 molecule. In one embodiment, said IgG4 molecule comprises a 8228? mutation.

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

In another aspect, the invention provides an isolated fully human monoclonal antibody or antigen—binding fragment thereof that dissociates from human MASP-Z with a KD of lOnM or less as determined by surface plasmon resonance and inhibits C4 activation on a mannan-coated substrate with an ICso of lOnM or less in 1% serum. In some embodiments, said antibody or antigen binding fragment thereof specifically izes at least part of an epitope recognized by a reference antibody comprising a heavy chain variable region as set forth in SEQ ID N0220 and, a light chain variable region as set forth in SEQ ID N024, 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—Z with any dy described herein which both (i) cally binds to the antigen and (ii) comprises a VH and/or VL domain disclosed herein, or comprises a CDR—H3 disclosed herein, or a variant of any of these.

Competition between g members may be assayed easily in vitro, for e using ELISA and/or by tagging a specific reporter molecule to one binding member which can PCTflJS2012/036509 be ed in the presence of other ed binding member(s), to enable identification of specific binding s which bind the same epitope or an overlapping epitope.

Thus, there is presently ed a specific antibody or n—binding fragment thereof, comprising a human antibody antigen-binding site which competes with an antibody bed herein that binds to human MASP-Z, such as any one of OMS641 to OMS646 as set forth in TABLE 24, for binding to human .

Variant MASP—Z Inhibitory Antibodies The above—described human monoclonal antibodies may be modified to provide variant antibodies that inhibit MASP-2 dependent complement activation. 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 n at least the CDRs of an above-described human antibody, or, in certain embodiments, CDRs that are very similar to the CDRs of an described human antibody.

In the red ment, 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 substitution 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 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 some embodiments, the variant antibodies have an amino acid sequence that is otherwise identical to the variable domain of a subject antibody 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 combined total of 1, 2, 3, 4, 5 or 6 amino acid substitutions within said CDR regions of said light chain variable PCT/U82012/036509 region, wherein the antibody or variant thereof inhibits MASP-2 ent complement activation.

Ordinarily, the variant will have an amino acid sequence having at least 75% amino acid sequence identity 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%,Ior at least 99% identity. Identity or homology with t 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 necessary, to achieve the m percent sequence identity.

None of N-tcrminal, C-terminal, or internal extensions, deletions, or insertions into the dy sequence (such as, for example, 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-2 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 inhibit or block MASP-2 dependent complement activation.

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 t to a full length form of the parent dy, for example, since it has been found that the format of the anti-MASP—2 antibody impacts its activity in the biological activity assays disclosed herein. The variant antibody of particular interest herein is one which ys at least about d, preferably at least about 20—fold, and most preferably at least about 50-fold, ement in biological ty when compared to the parent antibody.

The antibodies of the invention may be modified to enhance desirable properties, such as it may be ble 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 rapidly through the kidney. Hence, if long—term action of the MASP—Z antibody is desirable, the MASP-2 antibody is preferably a complete full length IgG antibody (such as IgG2 or IgG4), whereas if shorter action of the MASP-Z antibody is desirable, an dy fragment may be preferred. As bed in Example , it has been determined that an 8228P substitution in the hinge region of IgG4 increases _55_ PCT/U52012/036509 serum stability. Accordingly, in some embodiments, the subject MASP-2 antibody is a full length IgG4 dy with an SZ28P substitution.

Single Chain Antibodies In one embodiment of the present invention, the MASP-2 inhibitory dy is a single chain antibody, defined as a genetically 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 dies are also referred to as "single-chain Fv" or "scFV" antibody fragments.

Generally, the EV polypeptide further comprises a polypeptide linker between the VH and VL domains that s 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 yl 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 antibody are introduced directly 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 f set forth in TABLE 2. In some embodiments the invention provides a nucleic acid molecule comprising a nucleic acid sequence ed from the group consisting of SEQ ID NOzl9, SEQ ID N0223, SEQ ID N026, 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 N0283, SEQ ID N0285, SEQ ID N0197, SEQ ID NO:88 and SEQ ID N0289.

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

In some embodiments, the invention provides an expression cassette comprising a c acid molecule encoding an anti—MASP-Z dy of the ion.

In some ments, the invention provides a method of producing anti-MASP- 2 antibodies comprising culturing a cell comprising a nucleic acid molecule ng an anti-MASP-Z antibody of the invention. ~56— WO 51481 PCT/U52012/036509 According to certain related embodiments there is provided a inant host cell which comprises one or more constructs as described herein; a nucleic acid encoding any antibody, 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 therefor. Expression may conveniently be achieved by culturing under riate conditions 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, es of which are as s: monkey kidney cells (COS cells), monkey kidney CV1 cells transformed by SV40 (COS—7, 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. Acacl. Sci.

(USA) 6, (1980); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells 76, ATCC CRL-1587); 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 GZ, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL 51); TRI cells (Mather et al., Annals N. Y. Acad. Sci 383:44-68 (1982)); NIH/3T3 cells (ATCC CRL—l658); and mouse L cells (ATCC CCL-l). Additional 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 e Collection, 10801 University Boulevard, Manassas, Va. 20110-2209; Methods of ucing nucleic acids into cells are well known in the art. Suitable s include electroporation, particle gun technology, calcium ate 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 ormation is taking place (i.e., in vitro, ex vz‘vo, or in vivo). A general discussion of these methods can be found in Ausubel, et al., Short Protocols in lar Biology, 3d ed, Wiley & Sons, 1995. In some embodiments, lipofectamine and calcium mediated gene transfer technologies are used.

PCT/U82012/036509 After the subject nucleic acids have been introduced into a cell, the cell is typically incubated, normally at 37°C, sometimes 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 sion vector, the dy coding sequence of interest may be ligated to an adenovirus transcription/translation control x, e.g., the late promoter and tripartite leader sequence. This ic 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 (c.g., region El or E3) will result in a recombinant Virus that is Viable and capable of expressing the antibody molecule in infected hosts. (e.g., see Logan & Shenk, Proc. Natl.

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

For long-term, high—yield production of recombinant antibodies, stable expression may be used. For example, cell lines, which stably express the antibody molecule, may be engineered. Rather than using sion vectors which contain Viral origins of replication, host cells can be transformed with globulin expression cassettes and a selectable marker. Following the introduction of the foreign DNA, ered 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 integrate 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 tion of compounds that interact directly or indirectly with the dy molecule.

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

In many embodiments, antibodies are secreted from the cell into culture medium and harvested from the culture medium. For example, a nucleic acid sequence encoding a ~58- PCT/U82012/036509 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 adjacent to the 5' end of the amino acid sequences set forth herein for the subject dies in order to facilitate production of the subject antibodies.

Pharmaceutical Carriers and Delivegg es In another aspect, the invention provides compositions for inhibiting the adverse effects of MASP-Z-dependent complement activation comprising a therapeutically effective amount of a human anti-MASP-Z tory antibody and a pharmaceutically able carrier.

In general, the human MASP-2 inhibitory antibody compositions of the present invention, combined with any other selected therapeutic agents, are suitably contained in a pharmaceutically acceptable carrier. The carrier is non-toxic, biocompatible and is ed so as not to entally affect the biological activity of the MASP-2 inhibitory antibody (and any other therapeutic agents combined therewith). Exemplary ceutically acceptable rs for polypeptides are described in US. Patent No. 5,211,657 to Yamada. The anti-MASP-2 antibodies may be ated into preparations in solid, semi-solid, gel, liquid or gaseous forms such as tablets, capsules, powders, granules, ointments, ons, tories, inhalants and ions 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, infusion or irrigation and topical delivery include distilled water, physiological phosphate-buffered saline, normal or lactated Ringer's solutions, dextrose solution, Hank's solution, or propanediol. In addition, sterile, fixed oils may be employed as a t 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 nded as a liquid, suspension, polymerizable or non-polymerizable gel, paste or salve.

The carrier may also comprise a delivery vehicle to sustain (i.e., extend, delay or regulate) the delivery of the agent(s) or to e the delivery, uptake, stability or pharrnacokinetics of the therapeutic agent(s). Such a delivery vehicle may include, by way of non—limiting example, articles, microspheres, nanospheres or nanopartieles PCT/U52012/036509 composed of ns, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymeric or copolymeric hydrogels and polymeric micelles. Suitable hydrogel and micelle delivery s include the PEO:PHB:PEO copolymers and copolymer/cyclodextrin complexes disclosed, in A2 and the PEG and PEO/cyclodextrin xes disclosed in US. Patent Application Publication No. 2002/0019369 A1. Such hydrogels may be injected y at the site of intended action, or subcutaneously or intramuscularly to form a sustained release depot.

For intra—articular delivery, the MASP-2 inhibitory 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 administer the present invention.

The compositions of the present invention may also include patible excipients, such as sing or g agents, 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 uent injection, such as for intramuscular ion of a depot.

More specifically with respect to anti-MASP—Z antibodies, exemplary formulations can be erally administered as injectable dosages of a solution or suspension of the compound in a physiologically acceptable diluent with a ceutical r that can be a sterile liquid such as water, oils, saline, glycerol or ethanol. Additionally, auxiliary substances such as wetting or fying agents, surfactants, pH buffering substances and the like can be present in compositions comprising anti—MASP—2 antibodies. Additional components of pharmaceutical compositions include petroleum (such as of animal, vegetable or tic origin), for example, soybean oil and mineral oil. In general, glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers for injectable solutions. -60— PCT/U82012/036509 The anti-MASP-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 ile release of the active agents.

The pharmaceutical compositions comprising MASP—Z inhibitory antibodies may be administered in a number of ways depending on whether a local or systemic mode of administration is most appropriate for the ion being treated. Additionally, as described herein above with respect to orporeal reperfusion procedures, MASP—Z 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 red by coating or incorporating the compositions on or into an implantable medical device.

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

It will be iated that the exact systemic administration route for selected agents utilized in particular compositions of the present invention will be determined in part to t for the agent's tibility to metabolic transformation pathways associated with a given route of administration.

MASP-2 inhibitory antibodies and polypeptides can be delivered into a subject in need thereof by any suitable means. Methods of delivery of MASP-Z 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), ermal, 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 peptides can be introduced into a living body by application to a bodily membrane capable of absorbing the polypeptides, for e the nasal, gastrointestinal and rectal membranes. —6l— PCT/U52012/036509 The polypeptides are typically applied to the tive membrane in conjunction with a permeation enhancer. (See, e.g., Lee, V.H.L., Crit. Rev. Tlzer. Drug Carrier Sys. 5:69, 1988; Lee, V.H.L., J. Controlled Release 132213, 1990; Lee, V.H.L., Ed., Peptide and Protein Drug Delivery, Marcel Dekker, New York (1991); DeBoer, A.G., et al., J. Controlled Release , 1990.) For example, STDHF is a synthetic derivative of fusidic acid, a steroidal surfactant that is similar in structure to the bile salts, and has been used as a permeation enhancer 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 le, 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 prolong half—life (Fuertges, F., et al., J. lled Release 11:139, 1990). Many polymer systems have been reported for n delivery (Bae, Y.H., et al., J. Controlled Release 9:271, 1989; Hori, R., et al., Pharm. Res. 6:813, 1989; Yamakawa, 1., ct al., J. Pharm. Sci. 792505, 1990; Yoshihiro, 1., ct al.,J. Controlled Release 10:195, 1989; Asano, M., et al., J. Controlled Release 9:111, 1989; latt, J., et al., J. Controlled e 9:195, 1989; Makino, K., J. Controlled Release 12:235, 1990; Takakura, Y., et al., J. Pharm. Sci. 782117, 1989; Takakura, Y., et al., J. Pharm.

Sci. 78:219, 1989).

Recently, liposomes have been developed 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 ations 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 polypeptides may be combined with other suitable ingredients, such as carriers and/or adjuvants.

There are no limitations on the nature of such other ingredients, except that they must be pharmaceutically acceptable for their intended stration, and cannot degrade the ty of the active ingredients of the composition. Examples of suitable vehicles e ointments, creams, gels, or suspensions, with or without d collagen. The -62— W0 2012/]5148] PCT/U52012/036509 MASP-2 inhibitory antibodies and polypeptides may also be nated into transdermal patches, plasters, and bandages, preferably in liquid or semi-liquid form.

The compositions of the present invention may be systemically administered on a periodic basis at intervals determined to maintain a desired level of therapeutic .

For example, compositions may be administered, such as by subcutaneous injection, every two to four weeks or at less frequent intervals. The dosage regimen will be determined by the physician considering various factors that may influence the action of the combination of agents. These factors 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 function of the MASP-2 inhibitory antibody that is included in the composition, as well as the presence and nature of any drug ry 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).

LOCAL DELIVERY As used herein, the term ” 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, intratheeal (IT), intracerebrovcntricular (ICV), intra-articular, intracavity, ranial or intravesicular administration, placement or irrigation. Local administration may be red to enable stration of a lower dose, to avoid ic side s, and for more te control of the timing of delivery and concentration of the active agents at the site of local delivery. Local administration provides a known concentration at the target site, regardless of interpatient variability in metabolism, blood flow, etc. ed dosage control is also provided by the direct mode of delivery.

Local delivery of a MASP-Z inhibitory antibody may be achieved in the context of surgical methodsfor treating a disease or condition, such as for example during procedures such as arterial bypass y, atherectomy, laser ures, ultrasonic procedures, balloon angioplasty and stent placement. For example, a MASP-2 inhibitor can be administered to a subject in conjunction with a balloon angioplasty procedure. A balloon angioplasty ure involves inserting a catheter having a deflated balloon into an artery. The deflated balloon is positioned in proximity to the atherosclerotic plaque and is inflated such that the plaque is compressed t the vascular wall. As a result, PCT/U82012/036509 the balloon surface is in t 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 er in a manner that permits release 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 tment of the balloon catheter until the balloon is inflated, at which point it is released into the local environment. Alternatively, the agent may be impregnated on the balloon e, such that it contacts the cells of the arterial wall as the balloon is inflated.

The agent may also be delivered in a ated n catheter such as those disclosed in Flugelman, M.Y., et al., ation 85:1110—1117, 1992. See also published PCT Application WO 95/23161 for an exemplary ure for attaching a therapeutic protein to a balloon angioplasty er. Likewise, the MASP~2 inhibitory antibody may be included in a gel or polymeric coating applied to a stent, or may be incorporated into the material of the stent, such that the stent elutes the MASP—2 inhibitory antibody after vascular placement.

Treatment Regimes MASP-2 tory 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 further example of instances in which local ry may be desired, MASP-2 inhibitory antibody compositions used in the treatment of urogenital conditions may be suitably instilled intravesically or within r urogenital structure.

In prophylactic applications, the pharmaceutical compositions are administered to a subject susceptible to, or otherwise at risk of, a condition associated with MASPdependent complement activation in an amount sufficient to eliminate or reduce the risk of developing symptoms of the condition. In therapeutic applications, the pharmaceutical compositions are administered to a subject suspected of, or already suffering from, a condition ated with MASP-Z-dependent complement activation in a therapeutically effective amount sufficient to relieve, or at least partially reduce, the symptoms of the condition. In both prophylactic and therapeutic regimens, compositions comprising MASP—Z inhibitory antibodies may be stered in l dosages until a sufficient eutic outcome has been achieved in the subject. ation of the MASP-2 inhibitory antibody compositions of the present invention may be carried out by —64— W0 2012/]51481 PCT/U52012/036509 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. atively, the composition may be administered at periodic intervals over an extended period oftime for treatment of chronic conditions, e.g., arthritides or psoriasis.

MASP—2 inhibitory compositions used in the present invention 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 ischemia reperfusion, renal ischemia reperfusion, al ischemia reperfusion, 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 le following an acute event that activates the lectin pathway, preferably within twelve hours and more preferably within two to three hours of a triggering event, such as through ic delivery of the MASP—2 inhibitory composition.

The methods and itions of the present invention may be used to inhibit inflammation and related processes that typically result from diagnostic and therapeutic medical and surgical procedures. To inhibit such processes, the MASP-2 inhibitory composition of the present invention may be applied ocedurally. As used herein "periprocedurally" refers to administration of the inhibitory composition preprocedurally and/or intraprocedurally and/or postprocedurally, i.e., before the procedure, before and during the procedure, before and after the procedure, before, during and after the procedure, during the procedure, during and after the procedure, or after the procedure. ocedural application may be carried out by local stration of the composition to the surgical or procedural site, such as by ion or continuous or ittent irrigation of the site or by systemic administration. Suitable methods for local perioperative delivery of MASP-2 inhibitory antibody solutions are disclosed in US Patent Nos. 6,420,432 to Demopulos and 6,645,168 to Demopulos. Suitable methods for local delivery of chondroprotective compositions including MASP-2 inhibitory antibodies are disclosed in International PCT Patent Application WO 67 A2. Suitable methods and compositions for targeted systemic ry of chondroprotective compositions including MASP-2 inhibitory dies are disclosed in ational PCT Patent Application WO 03/063799 A2. -65— PCT/U52012/036509 Dosages The MASP-Z inhibitory dies can be administered to a subject in need thereof, at therapeutically effective doses to treat or ameliorate conditions associated 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.

Toxicity and therapeutic efficacy of MASP-Z inhibitory dies can be determined by standard pharmaceutical procedures employing experimental animal models, such as the African Green Monkey, as described herein. Using such animal models, the NOAEL (no ed adverse effect level) and the MED (the minimally 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 NOAEL/MED. MASP—2 tory antibodies that exhibit large therapeutic ratios or indices are most red. The data obtained from the cell culture assays and animal studies can be used in ating 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 ty. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.

For any nd formulation, the therapeutically effective dose can be estimated using animal models. For e, a dose may be formulated 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 ted based on the amount of MASP—2 protein present in a living subject and the binding affinity of the MASP-2 inhibitory antibody. It has been shown that MASP—2 levels in normal human ts is present in serum in low levels in the range of 500 ng/ml, and MASP—Z 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 orated herein by reference.

Generally, the dosage of administered compositions comprising MASP-2 inhibitory antibodies varies depending on such s as the subject's age, weight, height, —66- W0 51481 PCT/U82012/036509 sex, general medical condition, and previous medical history. As an ration, MASP-Z tory 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 0.1mg/kg of the subj eet body weight, Therapeutic efficacy of MASP-Z tory compositions and methods of the present invention in a given subject, and riate s, can be determined in accordance with complement assays well known to those of skill in the art. Complement generates numerous specific products. During the last decade, sensitive and specific assays have been developed and are available commercially for most of these activation products, including the small activation fragments C3a, C4a, and C5a and the large activation fragments iC3b, C4d, Bb, and sC5b-9. Most of these assays e 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 e both the unprocessed fragments and their 'desArg' fragments, which are the major forms found in the circulation. Unproccsscd fragments and Arg are y cleared by binding to cell surface receptors and are hence present in very low concentrations, whereas C3adesmg does not bind to cells and accumulates in plasma. Measurement of C3a provides a sensitive, pathway-independent indicator of complement activation. ative pathway activation can be assessed by measuring the Bb fragment. Detection of the fluid-phase product of membrane attack pathway activation, sC5b—9, provides 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 provide any information about which of these two ys has ted the activation products.

The tion of MASP-Z-dependent complement tion 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—Z antibody in accordance with the present invention: the inhibition of the generation or production of MASP-2—dependent complement activation system products C4b, VC3a, CSa and/or C5b-9 (MAC), the reduction of C4 cleavage and C4b deposition, or the reduction of C3 cleavage and C3b deposition.

Articles ofManufacture -67— W0 2012/]51481 PCT/U82012/036509 In another aspect, the t invention provides an article of manufacture containing a human MASP-2 inhibitory antibody, or antigen binding fragment thereof, as described herein in a unit dosage form le for therapeutic administration to a human subject, such as, for example, a unit dosage in the range of 1mg to SOOOmg, 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 e 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 binding fragment thereof of the invention. The label or e insert indicates that the composition is used for treating the ular condition. The label or package insert will further comprise instructions for administering the antibody composition to the patient. Articles of manufacture and kits comprising combinatorial therapies described herein are also plated.

Therapeutic Uses of the anti—MASP-Z inhibitory antibodies In r , the invention provides a method of inhibiting MASP—2 dependent complement activation in a human subject sing administering a human monoclonal anti—MASP—2 inhibitory antibody of the ion in an amount sufficient to inhibit MASP-Z ent complement activation in said human subject.

In accordance with this aspect of the ion, as described in Example 10, the MASP-Z inhibitory dies of the present invention are capable of inhibiting the lectin pathway in African Green Monkeys following intravenous administration. As shown in Table 24, Example 8, the antibody used in this study, OM8646, 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, ding 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 Corporation, the ee of the instant application), each of which is hereby incorporated by reference, MASP—2 dependent ~68- PCT/U82012/036509 complement activation 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 reperfusion injury, sclerosis, inflammatory gastrointestinal disorder, a pulmonary condition, an extracorporeal reperfusion procedure, a musculoskeletal condition, a renal condition, a skin condition, organ or tissue transplant, nervous system disorder or , a blood disorder, a urogenital condition, diabetes, chemotherapy or radiation therapy, ancy, an endocrine 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 es and conditions.

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

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

EXAMPLE 1 This e describes the recombinant expression and protein production of recombinant full-length human, rat and murine MASP-Z, MASP-Z derived polypeptides, and catalytically inactivated mutant forms of MASP-Z.

Expression of Full-length human and rat MASP~2: The full length cDNA sequence of human MASP-Z (SEQ ID NO: 1), encoding the human MASP-2 polypeptide with leader sequence (SEQ ID N022) was subcloned into the mammalian expression vector pCI—Neo (Promega), which drives eukaryotic expression under the control of the CMV enhancer/promoter region (described in Kaufman RJ. et al., Nucleic Acids Research 19:4485-90, 1991; Kaufman, Methods in Enzymology, 7—66 ). The full length rat MASP—2 cDNA (SEQ ID NO:4), encoding the rat MASP-2 polypeptide with leader sequence (SEQ ID N025) was subcloned into the pED expression vector. The MASP-2 expression vectors were then transfected into the adherent Chinese hamster ovary cell line DXB1 using the standard calcium phosphate transfection procedure described in is etal., 1989. Cells transfected with these constructs grew very slowly, implying that the d protease is —69- PCT/U52012/036509 cytotoxic. The mature form of the human MASP—Z protein (SEQ ID N03) and the mature form of the rat MASP-Z protein (SEQ ID NO:6) were secreted into the culture media and isolated as described below.

Expression of Full-length catalflically inactive MASP—Z: Rationale: MASP-2 is activated by autocatalytic ge after the recognition subcomponents MBL, C-type lectin CL—ll, or fieolins r L-fieolin, H—fieolin or M—ficolin), tively referred to as s, bind to their respective carbohydrate pattern. 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 , designed as MASP—ZA, was created by ing the serine residue that is present in the catalytic triad of the protease domain with an alanine residue in the mature rat MASP-2 protein (SEQ ID NO:6 Ser6l7 to Ala617); or in mature human MASP-2 protein (SEQ ID N023 Ser6l8 to Ala618).

In order to generate catalytically inactive human and rat MASP-ZA proteins, site-directed mutagenesis was carried out as described in USZOO7/Ol72483, hereby orated herein by reference. The PCR products were purified after agarose gel electrophoresis and band preparation and single adenosine overlaps were generated using a standard g procedure. The adenosine tailed MASP—2A was then cloned into the pGEM-T easy vector, ormed into E. coli. The human and rat MASP—ZA were each further subcloned into either of the mammalian expression s pED or pCl-Neo and transfected into the Chinese Hamster ovary cell line DXBl as described below.

Construction of Expression 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 N022) to secrete various domains of MASP—2. A construct expressing the human MASP-Z CUBI domain (SEQ ID N027) was made by PCR amplifying the region encoding residues 1—121 of MASP-2 (SEQ ID N03) (corresponding to the N-terminal CUBl domain). A construct expressing the human MASP-2 CUBI/EGF domain (SEQ ID N028) was made by PCR amplifying the region encoding residues 1—166 of MASP—Z (SEQ ID N013) (corresponding to the inal CUBl/EGF domain). A construct expressing the human MASP—2 CUBI/EGF/CUBII WO 51481 PCT/U82012/036509 domain (SEQ ID N029) was made by PCR amplifying the region encoding aa residues 1-277 of MASP-2 (SEQ ID N023) (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-Z (SEQ ID N023) (corresponding to the EGF ). A construct expressing the human MASP-2 CCPI/CCPII/SP domains (SEQ ID NO:ll) was made by PCR amplifying the region encoding aa residues 278-671 of MASP-2 (SEQ ID N013) (corresponding to the CCPI/CCPII/SP domains). A construct expressing the human MASP-2 CCPI/CCPII domains (SEQ ID NO:lZ) was made by PCR amplifying the region encoding aa residues 278—429 of MASP—2 (SEQ ID NO:3) (corresponding to the CPII 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 N023) (corresponding to the CCPI domain). A construct expressing the CCPII/SP domains of MASP-2 (SEQ ID NO: 14) was made by PCR amplifying the region encoding aa residues 348—671 of MASP-2 (SEQ ID N03) sponding to the CCPII/SP domains). A construct expressing the CCPII domain of MASP—2 (SEQ ID NO:lS) was made by PCR amplifying the region encoding aa residues 348-429of MASP-2 (SEQ ID NO:3) sponding to the CCPII domain). A uct expressing the SP domain of MASP—2 (SEQ ID NO:l6) was made by PCR amplifying the region ng aa residues 429—671 of MASP-Z (SEQ ID NO:3) (corresponding to the SP domain).

The above mentioned MASP—2 domains were amplified by PCR using VentR polymerase and pBS—MASP-2 as a template, according to established PCR methods. The ' primer sequence of the sense primer uced a BamHI ction site (underlined) at the 5' end of the PCR products. Antisense primers for each of the MASP-2 domains were designed to introduce 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 corresponding sites of the pFastBacl vector. The resulting constructs were characterized by restriction mapping and ed by dsDNA sequencing.

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

The MASP-2 and MASP-ZA expression constructs described above were transfected into DXBl cells using the standard m phosphate transfection procedure (Maniatis et al., 1989). MASP-ZA was produced in serum-free medium to ensure that PCT/U52012/036509 preparations were not contaminated with other serum proteins. Media was harvested from confluent cells every second day (four times in total). The level of recombinant MASP-ZA averaged approximately 1.5 mg/liter of culture medium for each of the two species.

MASP-ZA protein purification: The MASP-ZA (Ser—Ala mutant described above) was purified by y chromatography on MBP-A—agarose columns. This gy enabled rapid purification without the use of extraneous tags. MASP-2A (100-200 ml of medium diluted with an equal volume of loading buffer (50 mM Tris-Cl, pH 7.5, ning 150 mM NaCl and 25 mM CaClz) 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, A was purified r 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 equilibratcd in the same buffer. Following washing, bound MASP-ZA was eluted with a 0.05—1 M NaCl gradient over 10 ml.

Results: Yields of 025—05 mg of MASP-ZA protein were obtained from 200 ml of medium. The molecular mass of 77.5 kDa determined by MALDI—MS is greater than the ated 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. MASP-ZA migrates as a single band on SDS-polyacrylamide gels, demonstrating that it is not proteolytically processed during biosynthesis. 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 affinity fiilly human anti-MASP-2 scFv dy candidates that block MASP-2 onal activity for ssion into affinity maturation.

Background and Rationale: MASP-2 is a complex n with many separate functional s, ing: binding site(s) for MBL and ficolins, a serine protease catalytic site, a binding site for PCT/U52012/036509 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 determine if they were able to block MASP—Z onal activity.

To block MASP—Z functional activity, an antibody or scFV or Fab2 antibody fragment must bind and interfere with a structural epitope on MASP-2 that is required for MASP-2 functional activity. Therefore, many or all of the high affinity binding anti-MASP-2 scFvs or FabZS may not inhibit MASP-2 functional activity unless they bind to ural es on MASP-2 that are ly involved in MASP-2 functional activity.

A functional assay that measures inhibition of lectin y _C3 convertase formation was used to evaluate the "blocking activity" of anti-MASP-Z scFvs. It is known that the primary physiological role ofMASP-2 in the lectin pathway is to te the next functional 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 required in order to generate the two n components (C4b, C2a) that comprise the lectin pathway C3 convertase. Furthermore, all of the te functional ties of MASP-2 listed above appear to be required in order for MASP—Z to generate the lectin pathway C3 convertase. For these reasons, a preferred assay to use in ting the ing activity" of anti-MASP~2 FabZS and scFV antibody fragments is believed to be a functional 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 formats and in viva pharmacodynamics was validated mentally using odent MASP-Z antibodies.

The cn'teria for selection of first generation MASP-Z blocking antibodies for therapeutic use were as follows: high affinity to MASP-2 and functional IC50 values up W0 20] 2[]51481 PCT/U82012/036509 to ~25 nM. In addition, candidates were screened for reactivity with non-human primate serum, and with rat serum.

Methods: Screening of scFv phagemid library against MASP—2 antigen Antigens: Human A 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 supernatants by nickel—affinity chromatograph, as previously bed (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—2.

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

Panning Methods: Overview: Two panning strategies were used to isolate phages from the phagemid y that bound to MASP-2 in a total of three rounds of panning. Both strategies involved panning in on 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 alkaline elution (TEA), and the third panning round was first eluted competitively with MBL before a conventional alkaline (TEA) elution step. Negative selection was d out before rounds 2 and 3, and this was against the functional analogs, C15 and Clr of the classical complement pathway.

After panning, specific enrichment of phages with scFv fragments against MASP-2A was monitored, and it was determined that the panning gy 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 against MASP- 2A, as further described below.

PCT/U52012/036509 TABLE 7: Phae Pann1n; Methods biotm/stre tav1d1n Panning magnetic - Round Anti-_en( _) beads block ”_reannin elution biotin human avidin 4% blot nothing TEA (alkaline) A 10 block biotin rat MASP- streptavidin 4% blot Cls/C 11' TBA (alkaline) 2A block biotin human streptavidin 4% blot C ls/C lr Competition MASP-2A block w/MBL, followedby (1 Hg) TEA alkaline) TABLE 8: Pha e Pannin Methods HIS/NiNTA) Panning magnetic Round Anti _en- beads block I re 1 annino elution human MASP-2A NiNTA 4% milk nothing TEA (alkaline) His taued 10 ) in PBS rat MASP-ZA His NiNTA 4% milk Cls/Clr TEA (alkaline) tagged in PBS biotin human 4% milk C 1 s/C 1r Competitively MASP-ZA in PBS with MBL + TEA (alkaline) Panning Reagents: Human MASP-2A OMSlOO antibody (positive control) Goat anti-human lgG (H+L) (Pierce ) NiNTA beads (Qiagen #LB13267) Dynabeads® M—280 Streptavidin, 10 mg/ml 21) Normal human serum (LB 13294) Polyclonal rabbit anti—human C3c (LB13137) Goat anti-rabbit IgG, HRP can Qualex #AlOZPU) W0 20] 2/151481 PCT/U82012/036509 To test the tagged MASP-2A antigen, an experiment was carried out to capture the positive control OMSIOO antibody (200 ng/ml) preincubated with biotin-tagged MASP-2A or HIS-tagged MASP-ZA antigen (10 ug), with 50 ul NiNTA beads in 4% milk PBS or 200 pl Streptavidin beads, respectively. Bound MASP-2A-OMSIOO antibody was detected with Goat-anti—human IgG (H+L) HRP (115000) 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 ed saline (PBS) and incubated on a rotator wheel for 1 hour at room temperature. In el, 10 ug of MASP-ZA and OMSIOO 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 MASP—ZA pre—incubated with OMSIOO antibody was added to the washed beads. The mixture was incubated on a rotator wheel for I 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 d 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 1x 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 erred to a microtiter plate and the reaction was stopped with 100 pl 2M H2804. 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 instead, and non-biotinylated Results: The His-tagged and biotin-tagged MASP—ZA antigen, preineubated with the positive control OMSIOO antibody, were each successfully captured with NiNTA beads, or avidin beads, tively.

Panning ~76— PCT/U52012/036509 Three rounds of panning the scFv phage y against HIS-tagged or biotin— tagged MASP-ZA was carried out as shown in TABLE 7 or TABLE 8, respectively. The third round of g 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 polyclonal phage ELISA against immobilized MASP-2A was carried out as described below.

MASP—ZA ELISA on Polyclonal phage ed after Panning After three rounds of panning the scFv phage y against human MASP—2 as described above, specific ment of phages with scFv fragments against the target MASP-ZA was monitored by carrying out an ELISA assay on the ed polyclonal phage populations generated by panning against immobilized MASP-ZA as described below.

Methods: ng/ml MASP-ZA 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 control 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 y dy was Rabbit (x-fd (M13 coat protein), 1:5000 in 4% Milk-PBS (w/v). The conjugate was Rabbit (it—Goat —HRP at 1:10.000 in 4% Milk-PBS (w/V). The substrate was ABTS. All volumes, except washes and blocking, were 100 ul/well. All incubations were for 1 hour with shaking at room temperature.

The results of the phage ELISA showed a specific enrichment of scFv's t 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 t MASP-ZA after two rounds of panning, whereas 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-ZA at its lowest dilution. These results demonstrate that the signal observed is due to scFv specifically binding to MASP-2A.

Filter Screening: WO 51481 PCT/U82012/036509 Bacterial colonies containing plasmids encoding scFv fragments from the third round of panning were , 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 n and half from the subsequent TEA elution.

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

ScFvs that bound to MASP-ZA were detected via their c-Myc tag with Mouse 0t- cMye mAb and Rabbit (X-MOUSC 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.

RLults: Panning of the scFv phagemid library t MASP-ZA followed by scFv conversion and a filter screen yielded 137 positive clones. The ty 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 centrifugation step. The supernatant containing scFv secreted into the medium together with the contents of the periplasma was analyzed by two assays: ELISA using physically ed A, and binding analysis using amine coupled MASP-ZA to a CMS chip on the Biocore, as described in more detail below.

MASP-ZA ELISA on ScFv ate Clones identified by panning/scFv conversion and filter screening Methods: 4 rig/ml MASP—ZA was immobilized 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 al (100 pl medium-periplasma extract) from each of the 137 scFv candidates (generated as bed above) was added per well to the plate.

Next, anti—cMyc was added, and in the final step HRP-conjugated Rabbit anti-Mouse was 2012/036509 applied to detect bound scFv. The reaction was developed in peroxidase substrate l-step ABTS ochem). The positive control was OMSlOO (an ASP-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 PBS-Tween 0.05% (v/v) were carried out between every step.

The primary antibody was murine (x-cMyc, 125000 in PBS—Tween 0.05% (w/v).

The conjugate was rabbit d-Goat—HRP at 125000 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 til/well. All incubations were for 1 hour with shaking at room temperature.

RLults: 108/137 clones were ve in this ELISA assay (data not shown), of which 45 clones were further analyzed as described below. The positive control was OMSlOO Fab2 diluted to 10 rig/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 functional screening method used to e the high affinity fully human ASP-Z scFv antibody candidates for the ability to block MASP-2 activity in normal human serum.

Rationale/Background Assay to Measure Inhibition of Formation of Lectin Pathway C3 Convertase: A functional assay that measures inhibition of lectin y C3 tase formation was used to evaluate the "blocking activity" 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 pathway in terms of mediating inflammation. MASP-2 is not a structural component of the lectin pathway C3 convertase (C4b2a); therefore anti-MASP—2 antibodies (or Fab2) will not directly t activity of sting C3 tase. r, MASP—2 serine protease activity is required in order to generate the two protein components (C4b, C2a) that comprise the lectin pathway C3 convertase. Therefore, PCT/U52012/036509 anti-MASP-2 scFv which inhibit MASP-2 functional activity (i.e., blocking anti—MASP-2 scFv) will inhibit de novo formation of lectin pathway C3 convertase. 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 measure ion of C3 convertase, c wells coated with mannan were incubated with diluted human serum to activate the lectin y. The wells were then washed and d for C3b immobilized onto the wells using standard ELISA methods.

The amount of C3b generated in this assay is a direct reflection of the de novo formation of lectin pathway C3 convertase. Anti-MASP-2 scFv's at selected trations were tested in this assay for their ability to inhibit C3 convertase formation and consequent C3b generation.

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++ and Mg‘”r containing GVB buffer (4.0 mM al, 141 mM NaCl, 1.0 mM MgC12, 2.0 mM CaC12 , 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 OMSlOO Fab2 and was tested at 0.4 rig/ml. C30 formation was monitored in the presence and absence of the scFv/IgG clones.

Mannan was diluted to a concentration of 20 ug/ml (l 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 til 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 pl PBS, and stored on ice with 200 ul PBS until addition of the samples.

Normal human serum was diluted to 0.5% in CaMgGVB buffer, and scFV clones or the OMSlOO Fab2 positive l were added in triplicates at 0.01 ug/ml; 1 ug/ml (only OMSIOO control) and 10 ug/ml to this buffer and preincubated 45 minutes on ice before on to the blocked 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 ~80— PCT/U52012/036509 deposition was detected with a Rabbit (it-Mouse C3c antibody ed by Goat (ii-Rabbit HRP. The negative control was buffer without antibody (no OMSIOO = m C3b deposition), and the positive control 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 ve signals. A f criterion was set at half of the activity of an irrelevant scFv clone (VZV) and buffer alone.

Begging: 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% y ssion were selected 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 fimctional activity in the complement assay. All ten clones were found to have the same light chain subclass, 7L3, but three ent heavy chain subclasses, VH2, VH3 and VH6. The sequence identity of the clones to germline sequences is also shown in TABLE 9.

TABLE 9: 10 Uni ue Clones with Functional anti-MASP-2 Activit Germline Germline Bio— VH identity VL identity Clone name ELISA core Panning Elution subclass (%) subclass (%) 18P15 + Strep- X3 94.27 tavidin (13C24/6118) 4D9 + + 73 95 .34 ( 1 8L 16) 17D20 + + Strep— . )3 94.98 tavidin ( NP 1 0) 17L2O Strep- Comp VH6 96.3 X3 93 .55 tavidin + + Strep- . X3 98.21 tavidin (16L13X4F2) 18Ll6 Strep- Comp 100 73 93 .55 tavidin 21Bl7 9Pl3 ~81- PCT/U82012/036509 17N16 + Strep- TEA/Co VH6 99.66 793 97.85 tavidin mp 3F22 + Strep- VH6 100 )3 96.42 tavidin (18C 1 5) As shown above in TABLE 9, 10 different clones with acceptable functional ty 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 urification of ten stc Candidate Clones The ten ate clones shown in TABLE 9 were expressed in one liter scale and purified via ion exchange in Nickel tography. 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 monomer form, and this monomer fraction was isolated for further testing and ranking.

TABLE 10: Anal sis of Monomer Content 999, 3F22 86% ~82- WO 51481 PCT/U52012/036509 Testing Monomer Fraction for binding 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 K350 values and cross-reactivity.

Functional assay on 1140;20:11tefractions: The monomer fraction of the top ten clones, shown in TABLE 10, was purified and tested for functional 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 ons in triplicate. The positive control was OMSlOO Fab2.

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

Binding Assay: Binding affinity KD was determined in two different ways for purified r fractions of the ten candidate scFv clones. MASP—2A 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 a-cMyc antibody, and next a concentration series of MASP- 2A in solution was passed over the chip. The results are shown below in TABLE I 1.

Results: TABLE ll: Summary of functional inhibitory activity (IC50) and MASP-2 binding affinity (KD) for the ten candidate scFv clones assayed in the r state Binding Affinity to Inhibitory activity in human MASP—Z Binding y human Human Serum (immobilized) MASP-2 in solution Clone name IC50 (nM) KD (11M) KD (11M) 18P15 (13C24/6118) (18L16) 17D20 (17P10) PCT/U52012/036509 Binding Affinity to Inhibitory activity in human MASP-2 Binding Affinity human Human Serum (immobilized) MASP-Z in solution Clone name IC50 (nM) KD (nM) KD (nM) (mus/41:2)_ —_ 390 — _— 2200 35600 31722 206 ND 2 (18C15) Discussion of Results: As shown in TABLE 11, in the functional assay, five out of the ten candidate scFv clones gave IC50 nM values less than the 25 nM target criteria using 0.5% human serum.

As described below, these clones were further tested in the presence of non-human primate serum and rat serum to ine functional activity in other species. With regard to binding y, in solution, all binding affinities were in the range of low nM or better, whereas in the conventional assay with immobilized MASP-2, only two clenes (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 on. Also, when the target is immobilized on the chip (Via oriented coupling) the epitope may be masked, thereby reducing the observed ies in the immobilized assay.

EXAMPLE 4 This e 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 functional assay to determine their ability to inhibit MASP-2 dependent complement activation in human serum, non-human primate serum, and rat serum. -84— 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-ZA in a conventional ELISA assay against adsorbed rat MASP-ZA. Rat MASP-ZA was d to 4 ug/rnl 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 PBS-Tween (0.05%). The ScFV clones (100 ul) diluted in 20 ug/ml in PBS-Tween were added to the plate, and further titrated with 4-fold dilutions three times.

MASP-ZA specific stc clones (wells containing bound scFv) were detected with anti- cMyc and rabbit anti—mouse HRP secondary dy. The reaction was ped in peroxidase substrate TMB e). The positive control was OMS l 00 Fab2 diluted to 10 ug/ml in PBS—Tween. All the tested clones showed cross reaction with rat MASP-ZA, which was expected since the second panning round was with rat MASP-2 (data not shown).

Functional characterization of the ten candidate scFv clones in human serum non- human primate gEHP) serum and rat serum Determination ofbaselz'ne C30 levels in Different Sera First, an experiment was carried out to compare the baseline C3b levels in the three sera (human, rat and NHP) as follows.

Mannan was diluted to 20 34ng and coated on an ELISA plate overnight at 4°C.

The next day wells were blocked with 1% 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 d by transferring the plate to an ice bath. C3b deposition was detected with a rabbit anti-mouse C30 antibody, followed by goat anti-rabbit HRP. The negative control was buffer without antibody (no OMSlOO results in maximum C3b tion) and the ve l for tion was buffer with EDTA (no C3b deposition).

FIGURE 4 graphically illustrates the baseline C3c levels in the three sera (human, rat and NHP). As shown in FIGURE 4, the C30 levels were very different in the different sera tested. When comparing the C30 levels, it appeared that 1% human serum gave lent levels as 0.2% NHP and 0.375% rat serum. Based on these results, the WO 51481 concentrations of sera were normalized so that the scFv s could be directly compared in the three different types of sera.

Functional Assay 0fthe ScFv clones in Dz‘flerent Sera Purified monomer fractions ofthe ten candidate scFv clones were then tested for functional IC50 nM in human serum, rat serum and n 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 ; African Green Monkey serum diluted to 0.2% in CaMgGVB buffer; or Rat serum diluted 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 red in the presence and absence of scFv or Fab2 antibody.

The background signal of OMS100 at 100 ng/ml was subtracted from all signals.

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

TABLE: 12: Functional 1C50 (nM) activity of the scFv clones in three different types of sera.

Non— Non- human human primate primate Clone name Exp #1* Exp #1 Exp #2 123.1 207.5 198.9 81.92 407.1 249.6 ND (13C24/6118) 38.37 114.6 203.1 ND (18L16) 17D20 94.75 71.85 434.1 (17P10) 17L20 308 1 198.9 ambiguous 40.97 413 54.9 105.6 123.8 41.64 ND (16L13/4F2) 96.85 52.32 53.51 65.60 127.6 ND 21B17 ND 93.73 325 4 434.7 338.3 366.4 ND ~86— WO 2012/]51481 Non- Non- human human primate primate Exp #1 Exp #2 9P13 28.9 120.5 17.28 24.26 99.29 77.1 17N16 24.78 19.16 95.57 58.78 ND 3F22 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 ions started at identical concentrations.

Summa of results for functional activit in scFv candidate clones in different sera: All ten of the scFv clones showed function in both human and man primate (NHP) serum after the sera had been normalized with respect to C3b deposition levels.

The six most active clones in human serum were: 9Pl3>17N16>17D20>4D9>3F22>18L16, when ranked from best to worst. In NHP serum, the clones ranked (best to worst): 17L20>17N16>17D20>9P13>18L16>3F22.

Both 17N16 and 17D20 ranked in the top three for both human and NHP sera. 17D20 also showed some ty in rat serum.

Based on these results, the top three scFv clones were determined to be: 18L16, 17D20 and 17N16. These three clones were r 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 17D20 17N16 18L16 131.41 Ex. #2 Ex #3 65 nM 97 11M mean 36 +/— 15 67 +/- 15 non—human 17D20 17N16 18L16 -87— PCT/U32012/036509 I———— FIGURE 5A is an amino acid sequence ent of the full length scFv clones 17D20, 18Ll6, 4D9, l7L20, 17N16, 3F22 and 9Pl3. The scFv clones comprise a heavy chain variable region (aal-120), a linker region —l45), and a light chain variable region (aa 146~250). As shown in FIGURE 5A, alignment of the heavy chain region (residues 1-120) 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 Ge. 83% identity).

As fiirther shown in FIGURE 5A, the VH region with respect to the clones of the VH6 class: 17L20, 17N16, 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 ce alignment of the scFv clones 17D20, 17N16, 18Ll6 and 4D9.

TABLE 14: Seuence of ScFV Candidate clones shown in FIGURE 5A and 5B Clone Reference ID full len_th AA se o uence SEO ID NO:55 l8Ll6 SEO ID NO:56 SEQ ID NO:58 17N16 SEQ ID NO:59 3F22 SEO ID NO:60 9Pl3 SEO ID N026l The ranking priorities were (1) human serum functional potency and full blockage; (2) NHP cross-reactivity and (3) sequence diversity. l7D20 and 17N16 were selected as the best representatives from each gene family. 18Ll6 was selected as the third candidate with appreciable CDR3 ce diversity.

PCT/U82012/036509 17N16 and 17D20 were the top two choices due to complete onal blockage, with the best functional potencies against human; appreciable monkey cross-reactivity and ent VH gene families. 3F22 and 9P13 were eliminated due to VH sequences nearly identical to I7Nl6. 18P15, 4J9 and 21B17 were eliminated due to modest potency. 17L20 was not pursued e it was only partially blocking. 18Ll6 and 4D9 had similar activities and appreciable diversity ed to 17D20. 18L16 was chosen due to greater primate cross—reactivity than 4D9.

Therefore, based on these criteria: the following three mother clones: 17D20, I7N16 and 18Ll6 were advanced into affinity maturation as further bed below.

EXAMPLE 5 This Example describes the cloning of three mother clones 17D20, l7N16 and 18L16 (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 antibodies with moderate functional potency were identified using phage display as described in Examples 2-4. Three such mother clones, 17D20, l7Nl6 and 18Ll6 were selected for affinity maturation. To assess the onality of these mother clones as full length IgGs, IgG4 wild-type and SZZ8P hinge region IgG4 mutant forms of these antibodies were produced. The 8228P 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 N0262.

The amino acid sequence of IgG4 8228P is set forth as SEQ ID NO:65, d by SEQ ID NO:64.

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

Methods: Generating the clones intofull lengthformat -89— PCT/U52012/036509 The three mother clones were converted into wild type IgG4 format and into IgG4 mutant 8228P 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 harboring the appropriate heavy chain constant regions to create in- frame s to produce the desired antibody. The three mother clones in mutant lgG4 format were then cleaved with pepsin to generate F(ab')2 fragments and the latter were purified by fractionation on a size exclusion tography column.

Binding assay The candidate mother clones converted into IgG4 format were transiently transfected into HEK 293 cells and supernatants from the transient transfection were titrated in an ELISA assay. The clones showed excellent reactivity with physically adsorbed human MASP-ZA, and ranked in the ing order: 17Nl6>l7D20>l8L16 (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 ) and Fab'2 (30 ug/ml) were diluted in PBS-Tween to a starting concentration of 300 nM, and further with 3-fold ons. IgGs were detected with HRP conjugated Goat a—Human lgG (Southern Biotech) and the F(ab')2 were detected with HRP-eonjugated Goat (it—Human IgG H+L (Pierce 31412). The reaction was developed with TMB substrate and stopped with 2M H2804. The s are shown below in TABLE 15.

TABLE 15: Bindin- affini to human MASP-Z mmReference format M F ab' 2 M scFv nM Functional Assay The C3 tase assay using 1% normal human serum (NHS), as described in Example 4, was used to compare the functional ty of the mother scFv clones and full length IgG4 counterparts in 1% NHS. Mannan was diluted to a concentration of 20 W0 2012/]5148] PCT/U52012/036509 ug/ml and coated on ELISA plate ght 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 antibodies; scFv (900 nM), F(ab')2 (300 nM), IgG (300 nM) were added in duplicates at a series of different ons to the same amount of buffer, and preincubated for 45 minutes on ice before adding to the d ELISA plate. The reaction was initiated by incubation at 37°C for one hour and was d by placing the plate on ice.

C3b deposition was determined with a Rabbit u-Mouse C30 dy 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 Fold improvement scFv clone ID# (ICSO nM) (ICSO 11M) scFv to divalent form 171320 7392/1032 7305/1354 9827/1510 ~13.5x/~12.6x 17N16 5447/3088 5701/5092 36.18/77.60 ~6.6x/~19.3x 18L16 33.93/220 1602/1930 ~4.7x/~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 1gG4 hinge mutant (S228P) format for each clone. The numeric IC50 values for the C3b deposition assay using 1% NHS are shown below in TABLE 17.

TABLE 17: Wild type (IgG4) versus Hinge Mutant format (82281)) in C31) deposition assay in 1% human serum (IC50 nM) W0 2012/]51481 PCT/U52012/036509 As shown above in TABLE 17, in some cases, unexpected t pharmacology 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 further evaluated under more stringent assay conditions that more closely mimic physiological conditions. To estimate antibody activity under physiological ions, 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 minimally diluted (90%) human plasma.

The results of the C3b deposition assay in 90% human plasma are shown in FIGURE 6. Since MASP-2 and its ates are present in the assay mixture at approximately 100-fold higher concentration than in the dilute serum assay using 1% normal human serum, a right-shift of the antagonist dose-response curve is lly expected. As shown in FIGURE 6, as expected, a shift to lower apparent ies was observed for OMSlOO and all the MASP-2 antibodies tested. r, surprisingly, no reduction in apparent potency was observed for the hinge region mutant (S228P) of 17D20, 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 17D20 IgG4 ($228) was found to be modestly lower than OMSIOO Fab2, which is in contrast to the assay results in 1% NHS where OMSIOO was 50 to 100-fold more potent than 17D20 IgG4 SZ28P (data not shown). The wild type IgG4 form of 17N16 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 18L16 was less potent and only partially inhibitory, as shown in FIGURE 6.

Based on these findings, the ty of IgG4 ted mother clones was further evaluated by examining 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 specific and only two protein substrates for MASP-2 have been identified; C2 and C4. Cleavage of C4 generates C4a and C4b. Anti-MASP—Z Fab2 may bind to structural epitopes on MASP—Z that are directly involved in C4 ge (e.g., MASP-Z binding site for C4; MASP-2 PCT/U82012/036509 serine protease catalytic site) and thereby t the C4 cleavage functional ty of MASP-Z.

Yeast mannan is a known activator of the lectin y. In the following method to measure the C4 cleavage activity of MASP-Z, 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 ted in this assay is a measure of MASP—2 dependent C4 cleavage activity. Anti—MASP—Z 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 buffer, pH 9.5 at 1.0 rig/50 uL/well. Each well was washed 3X with 200 uL PBS. The wells were then blocked with 100 uL/weli 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. Anti-MASP-2 antibody samples were diluted to selected concentrations 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) at ° C. 90% human serum was added to the above samples at 5°C and 100 uL was erred to each well. The plates were covered and incubated for 90 min in an ice waterbath to allow complement activation. The reaction 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 ature with gentle mixing. Each well was washed x 200 uL PBS. 100 l of 0.1 ug/ml of peroxidase—conjugated avidin (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 rneasured.

Results: PCT/U52012/036509 In this format, both IgG4 forms of l7D20 inhibited Lectin pathway driven C4b deposition, although the IC50 values were :3 fold higher compared to the C3b deposition assay. Interestingly, 17Nl6 IgG4 wild type showed good activity in this assay with an IC50 value and dose-response profile comparable to the C3b deposition assay. l8L16 was considerably less potent and did not achieve complete inhibition in this format (data not shown).

Discussion: As described in Examples 2-5, fully human anti-MASP-Z scFv dies with functional blocking activity were fied using phage display. Three such clones, l7N16, l7D20 and 18Ll6, were selected for y maturation and r testing. To assess the fiinctionality of these mother clones as full 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 full length lgGs had improved functional activity as compared to their scFV counterparts when tested in a standard functional assay format with 1% human plasma. To estimate antibody activity under physiological conditions, testing of mother clone IgG4 preparations was ted under stringent assay conditions using 90% human . Under these conditions, several antibodies revealed functional potencies which were ntially better than expected based on their performance in standard (1%) plasma functional assays.

EXAMPLE 6 This e describes the chain shuffling and affinity maturation of mother clones l7D20, 17N16 and 18L16, and analysis of the resulting daughter clones.

Methods: To identify dies with improved potency, the three mother scFV clones, l7D20, 17Nl6 and 18Ll6, identified as described in Examples 2-5, were subjected to light chain shuffling. This process 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 healthy donors. This library was then screened for scFV clones with improved binding y and/or functionality. 9,000 light chain shuffled er 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 -94..

PCT/U52012/036509 human MASP—ZA were detected via their c-Myc tag. This initial screen resulted in the selection of a total of 119 clones, which included 107 daughter clones from the 17Nl6 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 columns, and tested for binding affinity in an ELISA assay against physically ed 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 illustrates the results of the ELISA assay on the 17Nl6 mother clone versus daughter clones titrated on - 2A. FIGURE 7B graphically illustrates the results of the ELISA assay on the l7D20 mother clone versus daughter clones titrated on huMASP-ZA.

As shown in FIGURE 7A, daughter clones l7Nl6m~dl6E12 and 17Nl6m_d17N9, derived from the 17N16 mother clone had affinities that were higher than the mother clone. Also, as shown in FIGURE 7B, one clone derived from the 17D20 mother clone, 17D20m_d18M24, had a higher y that the mother clone.

These three clones, and an additional three clones: l7Nl6m_dl3LlZ, _dl6K5, l7Nl6m_le5, and l7D20m_d1824 that had a low expression level were expressed in 0.5 L scale, purified into monomer fraction by size exclusion chromatography and were retested in an ELISA and functional assay. The 18Ll6 library did not e any daughter clones with the desired binding affinity.

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

TABLE 18: Com lement assa ofmother and dau hter clones PCT/U52012/036509 As shown above in TABLE 18, only one of the clones, 17N16m_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 17N16 (SEQ ID N059) and the 17N16m_d17N9 daughter clone (SEQ ID NO:66), Showing that the light chains (starting with SYE) have 17 amino acid residues that differ between the two clones.

Rescreening of the 17N16 lambda y resulted in several additional candidate daughter clones, of which 17N16m_d27E13 was identified in an ELISA and complement assay, and was included in the set of ate daughter clones for further analysis.

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

Mannan was diluted to 20 ug/ml and coated on an ELISA plate overnight at 4°C. The next day, the wells were blocked with 1% HSA. African Green monkey serum was diluted to 0.2%, rat serum was diluted to 0.375% and human serum was diluted to 1% in CaMgGVB buffer. Purified scFv fiom 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 on was initiated by incubation for one hour at 37°C, and was stopped by transferring the plate to an ice bath. C3c e was detected with a Rabbit e C3c antibody followed by a Goat a—Rabbit HRP. The background of OMS] 00 at 0.1 ug/ml on mannan negative plates was subtracted from these curves. The s are summarized below in TABLE 19.

TABLE 19: IC50 values for mother clone 17N16 and daughter clones 17N16m d17N9 and 17N16m d27E l 3 in ent sera.

African Green Serum Human Serum Rat Serum ScFv Clones IC50 (11M) ICSO (11M) I€50 (11M) l7Nl6mc 9293/8137 6531/7354 ND/195.8 17N16m d17N9 63.82/81.11 3990/5767 7932/1406 17N16m d27El3 ND/430.9 389.1/NA NA ~96— 2012/036509 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 17N16m_d17N9 has higher functional ty 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_d17N9 was selected for further analysis.

This Example describes the generation and analysis of daughter clone 17D20m_d3521N11, derived from mother clone 17D20. ound/Rationale: To e on affinity of the mother clone candidate l7D20mc, an additional "look-through-mutagenesis" was performed on the first three amino acids in the CDR3 of the heavy chain (CDR-H3). This was a mutagenesis gn in parallel with the normal light chain shuffling of 17D20mc. ore, three different scFv libraries were constructed by PCR where the amino acid positions 1, 2 and 3 were ized to the set of all le 20 amino acids using degenerate codons. After cloning the libraries, microscale expression was performed and scFv binding was monitored on a MASP—ZA coated CMS chip (not shown). BIAcore analysis of microscale expression was carried out on the three different libraries on chips coated with MASP-ZA, randomized at position 1, 2, or 3 and potentially interesting daughter clones were identified.

It was observed that for the amino acid positions 1 and 2 of CDR—H3, no clone was found having an improved off-rate in comparison with the mother candidate clone . However, a few candidates with mutations in amino acid position 3 in the CDR—H3 demonstrated improved off-rates in comparison with the mother clone 17D20m.

These clones (#35, #59 and #90) were sequenced to identify the mutation. Sequences of two "look-through—mutagenesis" d clones are compared with 17D20mc (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 comparison of the amino acid sequence of the heavy Chain region of the scFv mother clone 17D20m (aa 61—119 of SEQ ID NO:18) and the PCT/U82012/036509 amino acid sequence of the CRD-H3 region of scFv clones with ons in CDR—H3, clone #35 (aa 61-119 of SEQ ID NO:20, having a substitution of R for A at position 102 of SEQ ID NO:18), clone #59 (same sequence as clone #35), and clone #90 (substitution of P for A at position 102 of SEQ ID N018).

Analysis of Mutant clones #35 and #59 The mutant clones #35 and #59 were expressed in small scale and further tested in comparison with the mother ate clone 17D20 in a titration—ELISA on immobilized MASP—ZA (10 ug/ml). The scFvs were serially diluted 5-fold starting from 20 11le and binding was detected using anti-Myc (mouse)/anti~mouse HRP. Slightly improved g was observed in the ELISA assay for the candidate clones #35 and #59 in comparison with the mother candidate clone 17D20 (data not shown).

The improved clone #35 was combined with the best light chain shuffled clone _d21N11. The mutation in the VH of the candidate l7D20md35 (AIa-Arg) was combined with the light chain of the candidate l7D20m_d21Nll, thus resulting in the clone termed VH35-VL21N11, ise referred to as 3521Nl 1.

FIGURE 10A is an amino acid sequence alignment of sequence of the CDR3 region of mother clone 17D20 (aa 61-119 of SEQ ID N0218), the same region of er clone 17D20m_d21Nl l the same sequence, and the same region of the , having mutagenesis clone #35 combined with the VL of l7D20m_d21Nll, referred to " 3521N11" (aa 61—119 of SEQ ID NO:20). The highlighted VH sequence regions comprise the CDRH3, and d target residue region is underlined.

FIGURE 1013 is a protein sequence alignment of the full length scFV including VL and VH regions of the 17D20 mother clone (SEQ ID N0255) and the daughter clone l7D20m_d21NIl (SEQ ID NO:67). scFv daughter clone 17D20m_d3521Nll is set forth as SEQ ID NO:68. Note: it has been determined that the X residue in FIGURE 10B at position 220 is an "E", as set forth in SEQ ID NO:68.

A ion 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 Kn mm mm mm -98— W0 2012/]51481 PCT/U52012/036509 17D20m d3521N11 17mm monoonn l7D20md#35 (monomer The l7D20m_d3521Nll daughter clone was further analyzed for fiinetional activity as bed below in Example 8.

EXAMPLE 8 This Example describes the sion and analysis of the candidate daughter clones l7Nl6m_dl7N9 and l7D20m_d3521Nll into IgG4, IgG4/8228P and IgG2 format.

Rationale/Background The antibody screening methods described in Examples 2—7 have identified two mother clones, l7N16 and l7D20, with suitable functionality. Affinity maturation of these mother clones has yielded daughter clones that showed approximately 2-fold ements in potency as compared to the mother clones in surrogate functional assays at the scFv level. The daughter clones with the best activities are _dl7N9 and 17D20m_d3521N11. As described in Example 6, in a comparison of fiinctional activity of 17Nl6 mother clone with light chain shuffled daughter clones (scFv format, 1% NHS assay) it was determined that 17N16m_dl7N9 is ly more potent than the mother clone and has the best functional y 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 lC50 (nM) of the lead er clones and their res ective mother clones (all in scFv format) 1% human serum 90% human serum 90% human serum C3 assay scFv clone (IC50 11M) (1C50 nM) 17D20mc ——_ W0 2012/151481 PCT/USZ012/036509 mm mm 17D20md3521N1_ >1000 17mm “—— d1m9 17N16md2ms As shown above in TABLE 21, I7N16m_d17N9 has good activity when assayed in 90% normal human serum HS in the C3 assaY and is more Potent that the other daughter clones in this format.

Conversion of Candidate Clones into lgG4, IgG4/82281) and IgG2 format All of these candidate clones were converted to IgG4, IgG4/S228P and IgG2 format for further 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 SZ28P SEQ ID NO:65: IgG4 mutant 8228P polypeptide SEQ ID NO:69: CDNA encoding wild type IgG2 SEQ ID NO: 70: wild type IgG2 polypeptide TABLE 22: Summa of candidate clones: aauhtercone: Iformat vn #1 OMS641) 17N16m d17N9 SE ID\I0121 SE 11331027 _d17N9' IgG4 SEQIDNOQI SEQIDVO:27 (OMS642) I7N16m_d17N9 IgG4 (mutant) SEQ ID \IO:21 SEQ ID \IO:27 (OMS643) 17D20_3521N11 IgG2 SEQ ID \10120 SEQ ID NO:24 OMS644) 17D20_3521N11 IgG4 SEQ ID \10220 SEQ ID \10224 (OMS645 #6 17D20_3521N1 l IgG4 mutant SEQ ID NO:20 SEQ ID NO:24 OhdS646) —100— PCT/U52012/036509 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 . 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—Z MoAbs (IC50 nM) in 90% human serum Assa MOAb#l MoAb#2 MoAb#3 MoAb#4 MoAb#5 MOAb#6 Assa assa African nd 26 nd 18 16 14 Green Monkey C3 assa FIGURE 11A graphically illustrates the results of the C3b deposition assay carried out for the daughter clone isotype variants (MoAb#1-3), derived fiom the human anti-MASP—2 monoclonal antibody mother clone 17N16.

FIGURE 11B graphically illustrates the s of the C3b deposition assay carried out for the daughter clone isotype variants (MoAb#4-6), derived from the human anti-MASP-2 monoclonal dy mother clone 17D20.

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

The -6 were further analyzed in 95% human serum, 95% African Green serum. The results are summarized below in TABLE 24. —lOl- PCT/U52012/036509 TABLE 24 Antibody ID Binding to Functional Functional Functional lized inhibition of C3 inhibition of inhibition of hMASP-2 deposition in C3 deposition C4 deposition (average Kd) 95% human in 95% in 95% serum African human Green Serum serum (Average IC50; Average IC90) (Average (Average ICso) IC50) nM nM 111116 1G4) 0.1161 4.916113 1101111111 1.G21 0.291 1011041 110111111211 G4) 0.314 11.941210 MoAb#3 (IgG4 0.323 9.4; 61.0 9.2 19.8 mutant) 111320 1,114) 0.073 26119.0 110111114 1-G2> 0.085 0.9195 MoAb#5 I_G4) 0.067 2.6;122.0 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 cally illustrates the inhibition of C4b deposition in 95% normal human serum.

FIGURE 14 graphically illustrates the inhibition of C31) 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 embled MBL-MASP-2 complexes; classical pathway tion, and selectivity over Cls. The results are summarized in TABLE 25. —102— WO 51481 PCT/U52012/036509 TABLE 25: Summa of functional assa results Antibody ID Inhibition of Inhibition of cal ivity Rat C3b preassembled Pathway over Cls MBL-MASP—Z inhibition complexes ICSO (HM) 17m 164 Mom 162 >5000x 00 not detected not detected >5000x 1,200nM) not detected not detected >5000x mutant) @ZOOnM) >5000x Yes, IC50=17nM not detected >5000x MoAb#6 (IgG4 >500 Yes, IC50=24. lnM not detected >5000x FIGURE 15 graphically illustrates the inhibition of C4 cleavage activity of pre-assembled MBL-MASP-Z complex by MoAb#2, 3, 5 and 6.

FIGURE 16 graphically illustrates the preferential binding of MoAb#6 to human MASP-Z as compared to Cls. -lO3- PCT/U82012/036509 Table 26: Summa of se uences of er clones in various formats: Clone ID Descri tion SE I ID NO: 17N16m d17N9 li_ht chain _ene se uence 71 17N16m d17N9 li_ht chain n se uenee —72 17N16m d17N9 ene se-uence _73 17N16m d17N9 1G2 hea chain rotein seuence _74 m-75 “—76 m-77 ”-78 17N16m d17N9 ful 87 l7D20m d21Nll EXAMPLE 9 This Example describes the epitope mapping that was carried out for several of the blocking human anti-MASP-Z MoAbs.

Methods: The following recombinant proteins were produced as described in Example 1: Human MApl 9 Human MASPZA Human MASP-2 SP Human MASP-Z CCPZ-SP Human MASP—Z CCPl-CCPZ—SP -104— W0 2012/151481 PCT/U32012/036509 Human MASP-l/3 CUB 1 -EGF—CUB2 Human MASP-l CCP l—CCP2-SP The anti-MASP-2 antibodies OMSIOO and MoAb#6 (35VH—21N11VL), which have both been demonstrated to bind to human MASP-2 with high affinity and have the ability to block functional complement activity (see Examples 6-8) were analyzed with regard to epitope g by dot blot is.

Dot Blot Analysis Serial dilutions of the recombinant MASP-2 polypeptides described above were spotted onto a nitrocellulose membrane. The amount of protein d ranged from 50 ng to 5 pg, in ten-fold steps. In later ments, the amount of protein 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 rig/ml anti—MASP-2 FabZS in blocking buffer (containing 5.0 mM Ca2+). Bound Fab2s were detected using HRP-conjugated anti-human Fab erotec; diluted 1/10,000) and an ECL detection kit (Amersham). One membrane was incubated with polyclonal rabbit-anti human MASP-2 Ab (described in Stover et al., J Immunol 163:6848-59 (1999)) as a positive control. In this case, bound Ab was detected using HRP-eonjugated goat anti-rabbit IgG (Dako; diluted l/2,000), Results: The results are ized in TABLE 27.

TABLE 27: En-itoe Ma “in EX n construct OMSIOO human MA 5 19 —105— PCT/U52012/036509 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 Map19 and MASP-Z fragments not containing the CCPl domain of , leading to the conclusion that the binding sites encompass the CCPl domain.

EXAMPLE 10 This Example demonstrates that human anti-MASP—2 MoAb#6 inhibits the lectin pathway in n Green Monkeys following intravenous stration.

MoAb#6 was administered intravenously 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 ce 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 e 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 treatment, amelioration or prevention of acute radiation syndrome.

Rationale: Exposure to high doses of ionizing radiation causes mortality by two main mechanisms: toxicity to the bone marrow and gastrointestinal syndrome. Bone marrow toxicity results in a drop in all hematologic cells, predisposing the organism to death by infection and hemorrhage. The gastrointestinal syndrome is more severe and is driven by a loss of inal r function due to disintegration of the gut epithelial layer and a loss of inal 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 ation in response to tissue injury and exposure to foreign 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 -106— WO 51481 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 al in a mouse model of radiation injury by administering anti-murine MASP-2 antibodies.

Study #1 Methods and als: als. The test articles used in this study were (i) a high affinity anti-murine MASP—2 antibody (mAle 1) and (ii) a high affinity anti—human MASP-2 antibody (mAbOMS646) that block the MASP-2 n component of the lectin complement pathway which were produced in transfeeted mammalian cells. Dosing concentrations were 1 mg/kg of anti-murine MASP-2 antibody (mAle 1), 5mg/kg of anti—human MASP-2 antibody (mAbOM8646), or sterile saline. For each closing session, an adequate volume of fresh dosing solutions were prepared.

Animals. Young adult male Swiss-Webster mice were obtained from Harlan Laboratories (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 collimatdr 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. stration of anti-MASP-2 antibody mAlel and 646 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 —107— PCT/U82012/036509 Groups 7, 11 and 13 were sacrificed at post-irradiation day 7 and blood collected by cardiac re under deep anesthesia. ing 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 Grous Group Irradiation ID N Level (Gy) Treatment Dose Schedule 1 20 6.5 Vehicle 18 hr prior to irradiation, 2 hr post irradiation, weekly booster 2 20 6.5 anti—murine 18 hr prior to irradiation MASP-2 ab only (mAle 1) 3 20 6.5 anti-murine 18 hr prior to irradiation, 2 MASP—2 ab hr post irradiation, weekly (mAle 1) 4 20 6.5 anti-murine 2 hr post irradiation, MASP-2 ab weekly r (mAle 1) 20 6.5 anti~human 18 hr prior to irradiation, 2 MASP—Z ab hr post irradiation, weekly boos ert |_ S646) 6 20 7.0 Vehicle 18 hr prior to irradiation, 2 hr post irradiation, weekly booster 7 5 7.0 Vehicle 2 hr post irradiation only 8 20 7.0 anti—murine 18 hr prior to irradiation MASP-2 ab only (mAle 1) 9 20 7.0 anti-murine 18 hr prior to irradiation, 2 MASP-Z ab hr post irradiation, weekly boos ert (mAle 1) 20 7.0 anti—murine 2 hr post irradiation, MASP-2 ab weekly booster ~108- W0 51481 PCT/U52012/036509 Gioup Irradiation Level (Gy) Treatment Dose Schedule --_<mAan> anti—murine 2 hr post irradiation only . MASP-Z ab (mAle 1) 12 20 7.0 anti-human 18 hr prior to irradiation, 2 MASP-2 ab hr post ation, weekly boos ert (mAbOMSé46) 13 10 anti-human 18 hr prior to irradiation, 2 MASP—Z ab hr post irradiation, weekly boos er‘t (mAbOM8646) icle 2 hr post irradiation only Statistical Analysis. Kaplan-Meier survival curves were generated and used to e mean survival time n treatment groups using log-Rank and Wilcoxon methods. Averages with standard deviations, or means with standard error of the mean are reported. Statistical comparisons were made using a two—tailed unpaired t-test between controlled irradiated animals and individual treatment 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 ized below in Table 29.

Overall, treatment with anti-murine MASP-2 ab (mAle 1) pre-irradiation increased the survival of irradiated mice compared to vehicle d 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%) compared to vehicle control irradiated animals.

In comparison, all treated animals at the 7.0 Gy and 8.0 Gy exposure level showed an increase in al ed to vehicle treated irradiated control animals. The greatest change in survival ed in animals receiving mAbOMS646, 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. Further, at the 7.0 Gy exposure level, —109- 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 increase of 7 days over control s. Mean time to mortality for mice receiving mAbOMS646 (27.3 at 1.3 days) was significantly increased (p = 0.0087) ed to control animals (20.7 i 2.0 days) at the 7.0 Gy exposure level.

The percent change in body weight compared to pre-irradiation day (day -l) 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 exosed to radiation Time to Death re (Mean :1: SEM, First/Last Test Group Level Survival (%) Day) Death (Day) Control Irradiation 6.5 Gy 65 % 24.0 i 2.0 9/16 mAlel pre- 65 Gy 85 % 27.7 i- 1.5 13/17 mAlel pre + 6.5 Gy 65 % 24.0 i 2.0 9/15 post—exposure mAlel post— 6.5 Gy 80 % 26.3 i 1.9 9/13 exposure mAbOMS646 6.5 Gy 65 % 24.6 :1: 1.9 9/19 pre+post-exposure My 35 % mAlel pre- 7.0 Gy 65 % 23.0 i 2.3 mAlel pre+ 7.0 Gy 55 % 21.6i2.2 post-exposure mAlel post- 7.0 Gy 70 % 24.3 i 2.1 exposure mAbOMS646 7.0 Gy 8O % 27.3 :t 13* st-exposure mAb OMS646 8.0 Gy 32% pre+post-exposure 2012/036509 Time to Death Exposure (Mean i SEM, First/Last Test Group Level al (%) Death (Day) socy >"p = 0.0087 by two-tailed unpaired t—test between lled irradiated animals and treatment group at the same irradiation exposure level. sion Acute radiation syndrome consists of three defined, dromes: hematopoietic, gastrointestinal, and cerebrovascular. The syndrome observed depends on the radiation dose, with the hematopoietic effects observed in humans with cant l 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 , 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 uncontrollable hemorrhage lead to death.

In the present study, administration ofmAbH6 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 animals ing mAbOMS646 survived to 30 days compared to 35% of vehicle treated control irradiated animals.

Importantly, the first day of death in this treated group did not occur until post—irradiation day 15, a 7-day increase over that ed in vehicle treated control irradiated animals. sly, 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 multiple reasons for this difference in response between exposure levels, although verification of any hypothesis may require additional studies, including interim sample collection for microbiological culture and hematological parameters. One explanation may simply be that the number of s assigned to groups may have precluded seeing any subtle treatment-related differences.

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

On the other hand, the difference between 35% (vehicle control at 7.0 Gy exposure) and —lll- PCT/U52012/036509 80% (mAbOMS646 at 7.0 Gy exposure) is 9 animals, and es sound evidence of a treatment-related difference.

These results demonstrate that anti-MASP-2 antibodies are effective in ng a mammalian subject at risk for, or ing from, the detrimental effects of acute radiation syndrome.

Study #2 Swiss Webster mice (n=50) were exposed to ionizing radiation (8.0 Gy). The effect of anti-MASP-2 antibody 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 antibody OM8646 increased survival in mice exposed to 8.0 Gy, with an adjusted median survival rate from 4 to 6 days as ed 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 exposed to ionizing radiation (8.0 Gy) in the following groups: icle) saline control; (II: 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.

Results of Study #3 It was determined that administration of anti—MASP~2 antibody pre— and post- irraditaion ed the mean survival from 4 to 5 days in comparison to s that received vehicle control. ity in the anti-MASP~2 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 summary, the results in this Example trate that ASP-2 antibodies of the invention are effective in treating a mammalian subject at risk for, or suffering from the detrimental s of acute radiation syndrome. —112— W0 20121151481 PCT/U$2012/036509 EXJflflPLEIZ This Example describes further characterization of the OMS646 dy (17D20m_d3521N11), fully human anti-human MASP-2 IgG4 antibody with a mutation in the hinge region). hdethods: OMS646 (17D20m_d3521N1 I), fully human anti-human MASP-2 IgG4 antibody with a on in the hinge region) was generated as described above in Examples 2—8.

OMS646 antibody was purified from culture atants 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 exchange into PBS. 1. OMS646 binds to human MASP—2 with high y Surface Plasmon. Resonance re) Analysis ofImmobilized OMS646 Binding to recombinant human MASP-2 OM%%thmmwmmdMWMWs®MM%bymMmemgmaCM5mm 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 (Kori) and dissociation (K0132) rate constants. The equilibrium binding constant (K9) was calculated based on mental Kon and Koff values.

Results: FIGURE 19 graphically illustrates the results of the surface plasmon nce (Biocore) analysis on OMS646, showing that lized OM8646 binds to recombinant MASP-2 with a Kofi‘ rate of about 1-3x10'4 S”1 and a Kon rate of about 1.6-3x106M'IS'1, implying an affinity (KD of about 92pM) under these experimental conditions.

Depending on the density of OMS646 immobilized and the concentration of MASP-2 in solution, experimental KD values in the range of 50 to 250pM were determined.

ELISA Assay 0fOMS646 Binding to Immobilized recombinant human MASP—2 Methods: An ELISA assay was carried out to assess the dose-response of OMS646 binding to immobilized recombinant MASP—Z. Recombinant human MASP—2 (50 ng/well) was immobilized on maxisorp ELISA plates (Nunc) in PBS overnight at —113— PCT/U82012/036509 4°C. The next day, the plates were blocked by washing three times with PBS—Tween (0.05%). A serial dilution series of OM8646 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 binding to immobilized antigen, the wells were washed to remove unbound OMS646. 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 ill/well of 1.0 M H3PO4, and substrate conversion was quantified photometrically at 450nM using a 96 well plate reader ramax). A single g site curve fitting algorithm (Graphpad) was used to calculate KD values.

FIGURE 20 graphically illustrates the results of the ELISA assay to determine the binding affinity of OM8646 to immobilized human MASP-Z. As shown in FIGURE 20, it was determined that OMS646 binds to immobilized recombinant human MASP-2 with a KD of 85:6 pM, which is consistent with the results obtained in the Biocore analysis, as shown in FIGURE 19. These results demonstrate that OMS646 has high y to human MASP-Z, with a K1) of approximately 100pM. 2. OMS646 inhibits C4 Activation on a mannan—coated surface but not on an immune complex—coated surface Methods: C4 activation was measured on a mannan—coated surface or an immune complex- coated surface in the presence or e of OM8646 over the concentration range shown in FIGURES 21A and 21B, tively as follows.

In the following method to measure the C4 cleavage activity of MASP—2, plastic wells coated with mannan were incubated for 60 minutes at 37°C with 1% human serum to activate the lectin y. The wells were then washed and assayed for human C4b immobilized onto the wells using rd ELISA s. The amount of C4b generated in this assay is a measure of MASP-Z ent C4 cleavage ty.

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

Methods: C4 activation 0n. mannan—caated surfaces: «114— PCT/U52012/036509 In order to determine the effect of OMS646 on the —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 preineubated with 1% human serum in Ca++ and Mg“ containing GVB buffer (4.0 mM barbital, 141 mM NaCl, 1.0 mM MgC12, 2.0 mM CaClg, 0.1% gelatin, pH 7.4) for 1 hour at 5° C. These antibody- serum preincubation mixtures were subsequently transferred into the corresponding wells of the mannan-coated assay plate. Complement activation was initiated by transfer of the assay plate to a 37°C water bath. Following a 60 minute incubation, the reaction 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 1:100 on of —conj ugated n anti-human C4c system 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 5 x 200 uL PBS. 100 uL/well of 0.] ug/ml of peroxidase—conjugated streptavidin (Pierce Chemical #21126) was added in PBS containing 2.0 mg/ml BSA and ted 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 on was stopped by adding 100 uL/well of 1.0 M H3PO4 and the OD450 was measured.

C4 activation on immune—complex coated sulfaces In order to measure the effect of OMS646 on the classical pathway, the assay described above was modified to use -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 ce or e of . FIGURE 21B graphically illustrates the level of C4 activation on an IgG-eoated surface in the presence or absence ‘115— PCT/U82012/036509 of OMS646. As shown in FIGURE 21A, OMS646 inhibits C4 activation on - coated surface with an ICso of approximately 0.5nM in 1% human serum. The ICSO value obtained in 10 ndent ments was 0.52i0.28 nM (averageiSD). In st, as shown in FIGURE 21B, OM8646 did not inhibit C4 activation on an IgG-coated e.

These results demonstrate that OM8646 blocks lectin-dependent, but not classical pathway-dependent activation of complement component C4. 3. OMS646 cally blocks lectin-dependent activation of terminal complement components Methods: The effect of OMS646 on membrane attack complex (MAC) deposition was analyied using y-specific conditions for the lectin pathway, the classical pathway and the alternative pathway. For this purpose, the Wieslab Comp300 complement screening kit (Wieslab, Lund, Sweden) was used following the manufacturer’s instructions.

Results: FIGURE 22A graphically illustrates the level of MAC deposition in the ce or absence of anti-MASP-2 antibody 6) 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 rates 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, OM8646 blocks lectin pathway—mediated activation of MAC deposition with an ICso value of approximately InM. However, OMS646 had no effect on MAC deposition generated from cal pathway-mediated activation (FIGURE 22B) or from alternative pathway-mediated activation (FIGURE 22C). 4. OMS646 effectively inhibits lectin pathway activation under physiologic Lndingns Methods: The lectin dependent C3 and C4 activation was assessed in 90% human serum in the absence and in the presence of various concentrations of OMS646 as follows: C4 Activation Assay —116— PCT/U52012/036509 To assess the effect of OMS646 on lectin—dependent C4 activation, l 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 pl PBS and blocked with 100 l 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 ment 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 albumin (BSA) and incubated 1 hour at room temperature with gentle . 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 incubated 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 incubated at room temperature for 16 minutes. The dase reaction was stopped by adding 100 uL/well of 1.0M H3P04 and the OD450 was measured.

C3 Activation Assay To assess the effect of OMS646 on lectin—dependent C3 activation, assays were carried out in an cal 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 on of rabbit anti- human C30 dy (Dako) in PBS containing 2.0 mg/mL bovine serum albumin (BSA).

Each plate was washed five times with 200 uL PBS, and then ted for 1 hour at room temperature with 100 uL/well of HRP-labeled goat anti-rabbit IgG (American 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 (Kirkegaard & Perry Laboratories) was added and incubated at room ature for 10 minutes. The —117— W0 151481 peroxidase reaction was stopped by adding 100 uL/well of 1.0M H3PO4 and the OD450 was ed. ICso values were d by applying a sigmoidal dose-response curve fitting algorithm (GraphPad) to the experimental data sets.

Results: FIGURE 23A graphically illustrates the level of C3 deposition in the presence or absence of ASP—Z antibody (OM8646) over a range of concentrations in 90% human serum under lectin pathway 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 trations 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 = 3il.5 nM (n=6). As shown in FIGURE 23B, OM8646 blocked C4 deposition with an ICso = 2.8il .3 nM (n=6).

These results demonstrate that OMS646 provides potent, effective blockade of lectin y activation under physiological conditions, thereby ing support 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 ation of a patient at a plasma concentration 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 al 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 l0 mg administered intravenously will be effective at blocking the lectin pathway during an acute time period (i.e., a transient time period, such as from I 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 l 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 commonly observed for antibodies in , 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 lOOOmg), with have a longer duration of action (e.g., greater than 2 weeks).

. OMS646 blocks lectin pathway activation in monkeys —118— PCT/U82012/036509 As described above in Example 10 and shown in FIGURE 17, it was determined that OMS646 ablates systemic lectin pathway activity for a time period of about 72 hours following intravenous administration of OMS646 (3 mg/kg) into African Green monkeys, ed by recovery of lectin pathway activity.

This Example describes a follow up study in which lectin dependent C4 tion was assessed in 90% African Green monkey serum or in 90% glus 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 different non-human primate s, 96-well Costar medium binding plates were coated overnight at 5°C with 2 ug ofmannan (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 1 hour at room temperature with gentle mixing. In a separate preincubation plate, select concentrations of OMS646 were mixed with 90% serum ted 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 l of 1:1000 dilution of biotin—conjugated chicken anti-human C4c osystem AB, Uppsala, Sweden) was added in PBS containing 2.0 mg/mL BSA and incubated one hour at room temperature with gentle . 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 . 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 H3P04 and the OD450 was measured. ICso values were d by applying a sigmoidal dose-response curve fitting thm (GraphPad) to the experimental data sets.

Results: —119— WO 2012115148] 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 ICso values in the range of 30 nM to 50 nM, and 15 nM to 30 nM, respectively.

In summary, OMS646, a fully human anti-human MASP-2 IgG4 antibody (with a on in the hinge region) was observed to have the following advantageous properties: high y binding to human MASP-Z (KD in the range of 50 to 250 pM, with a Kory rate in the range of 1-3x10‘4 S’1 and a K(m rate in the range of 1.6-3x106M'IS‘1; functional potency in human serum with inhibition of C4 deposition with an ICso of 0.522t0.28 nM (n=10) in 1% human serum; and an ICso of 33:1.5nM in 90% serum); and cross-reactivity in monkey showing inhibition of C4 tion with an ICso 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 expected to be effective at y ng the lectin pathway in human circulation (e.g., for a period of at least I 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 patient 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 (so) or intramuscular (im) routes of administration can be employed to further extend the time window of effective lectin pathway 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 y for 1 day, and a 3 mg/kg dose of OMS646 resulted in inhibition of the lectin pathway for about 3 days (72 . It is therefore estimated that a larger dosage of 7 to 10mg/kg 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 potency t human MASP-Z 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: OM8646 dosin to inhibit the lectin athwa in viva —Monke 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 (25)

CLAIMS is:

1. An isolated human monoclonal antibody, or antigen binding nt thereof, that binds to human MASP—2 and inhibits MASP-Z dependent complement activation, wherein the antibody or antigen—binding fragment thereof comprises: a) a heavy chain le region comprising three complementary determining regions CDR-Hl, CDR-HZ and CDR—H3; and b) a light chain variable region comprising three CDRs CDR—Ll, CDR—L2 and CDR-L3; wherein: CDR-Hl comprises an amino acid sequence as set forth as STSAA (SEQ ID NO:29); CDR-H2 comprises an amino acid sequence as set forth as LGRTYYRSKWYNDYAV (SEQ ID N0233); CDR-H3 ses an amino acid sequence as set forth as RD (SEQ ID N038); CDR—Ll comprises an amino acid sequence as set forth as GXNXGXKXVI—IW (SEQ ID N0292), wherein X at position 2 is N or D and wherein X at position 4 is I or L and n X at position 6 is S or K and wherein X at position 8 is N or R; CDR—L2 comprises an amino acid sequence as set forth as DSDRPSG (SEQ ID ; and CDR—L3 comprises an amino acid sequence as set forth as VWDXXTDHV (SEQ ID NO:94), wherein X at position 4 is T or I and wherein X at position 5 is T or A, and wherein the isolated antibody or antigen—binding fragment f binds to human MASP—Z and inhibits MASP-2 dependent complement activation.

2. The antibody of claim 1, wherein the amino acid sequence set forth in SEQ ID NO:92 contains N at position 2, I at position 4, S at position 6, and N at position 8.

3. The antibody of claim I, wherein the amino acid ce set forth in SEQ ID NO:92 contains D at position 2, L at position 4, K at on 6, and R at position 8.

4. The antibody of claim 1, wherein at least one of the following applies: (i) wherein said antibody binds human MASP-2 with a KD of 10 nM or less; ~122- (ii) wherein said antibody inhibits C3b deposition in an in vitro assay in 1% human serum at an IC50 of 10 nM or less; (iii) wherein said antibody inhibits C3b deposition in 90% human serum with an ICso 0f30 nM or less; or (iv) wherein the antibody does not substantially inhibit the classical pathway.

5. The antibody of claim I, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of Fv, Fab, Fab', F(ab)2, F(ab')2, a single chain antibody, an ScFv, a univalent antibody lacking a hinge region and a whole antibody.

6. The antibody of claim 1, wherein said antibody is selected from the group consisting of an IgGZ le, and IgG1 le and an IgG4 molecule.

7. The antibody of Claim 6, wherein the IgG4 le comprises a S228P mutation.

8. The antibody of claim 1, wherein the heavy chain variable region comprises a CDR—Hl comprising SEQ ID NO:29, a CDR-H2 sing SEQ ID N033 and a CDR— H3 comprising SEQ ID N038; and wherein the light chain le region comprises a CDR-Ll comprising SEQ ID NOI43, a CDR-L2 comprising SEQ ID NO:49 and a CDR- L3 sing SEQ ID NO:53.

9. The antibody of claim I, wherein the heavy chain variable region comprises a CDR-Hl comprising SEQ ID N0229, a CDR-H2 comprising SEQ ID NO:33 and a CDR- H3 comprising SEQ ID N038 and wherein the light chain variable region comprises a CDR—Ll sing SEQ ID NO:44, a CDR-LZ comprising SEQ ID NO:49 and a CDR— L3 comprising SEQ ID NO:54.

10. The antibody or fragment thereof of claim 1, wherein CDR—H3 comprises amino acid residues 95-110 of SEQ ID NO:21.

11. An ed monoclonal antibody, or antigen-binding fragment thereof, that binds to human MASP—Z, comprising a heavy chain variable region set forth in SEQ ID NO:21 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:25 or SEQ ID N027.

12. The ed monoclonal antibody of claim _11, wherein said antibody is selected from the group consisting of an IgGZ molecule, and IgG1 molecule and an IgG4 molecule. -123—

13. The antibody of claim 11, wherein the IgG4 molecule comprises a 8228P mutation.

14. A monoclonal antibody or antigen binding fragment thereof that specifically binds to human MASP—2 and comprises: (a) a heavy chain variable region comprising SEQ ID N022l or a variant thereof comprising an amino acid sequence having at least 95% identity to SEQ ID N021; and (b) a light chain variable region sing SEQ ID NO:25 or a variant thereof sing an amino acid sequence having at least 95% ty to SEQ ID NO:25, wherein residue 23 is G or A, residue 24 is a G, residue 25 is an N or D, residue 26 is an N, residue 27 is an I or L, residue 28 is a G, residue 29 is an S or K, residue 30 is a K, residue 31 is a N or R, residue 32 is a V, residue 33 is an H, residue 49 is a D, e 50 is a D, residue 51 is an S, residue 52 is a D, residue 53 is an R, e 54 is a P, residue 55 is an S, e 56 is a G, residue 88 is a Q, residue 89 is a V, residue 90 is a W, residue 91 is a D, residue 92 is T or I, e 93 is T or A, residue 94 is a T, residue 95 is a D, residue 96 is an H and residue 97 is a V.

15. The isolated monoclonal antibody, or antigen—binding fragment thereof according to claim 14, wherein the light chain variable region comprises an amino acid sequence wherein e 23 is G, residue 25 is N, residue 27 is I, residue 29 is S, e 31 is N, residue 92 is T, and e 93 is T.

16. The isolated monoclonal antibody, or antigen-binding fragment thereof according to claim 14, wherein the light chain variable region comprises an amino acid sequence wherein residue 23 is A, e 25 is D, residue 27 is L, residue 29 is K, residue 31 is R, residue 92 is I, and residue 93 is A.

17. A nucleic acid molecule encoding the amino acid sequence of a MASP-2 antibody, or antigen—binding fragment thereof, as set forth in any one of claims 1, 11 or

18. An expression cassette comprising a nucleic acid molecule encoding a MASP-2 antibody of the invention according to Claim 17. -124—

19. A cell comprising at least one of the nucleic acid molecules ng a MASP—2 antibody of claim 1 according to claim 17 or claim 18, with the proviso that if the cell is a human cell it is ex vivo.

20. A method of generating an isolated anti-MASP-2 antibody sing culturing the cell of claim 19 under conditions allowing for expression of the nucleic acid molecules encoding the anti-MASP—2 antibody of claim 1 and isolating said anti-MASP-2 antibody.

21. A pharmaceutical composition comprising the human monoclonal anti— MASP-Z antibody, or fragment thereof, of any one of claims 1, 11 or 14, and a pharrnaceutically acceptable excipient.

22. The ition of claim 21, wherein the composition is formulated for intra-arterial, intravenous, ranial, uscular, inhalational, nasal or subcutaneous administration.

23. A composition of claim 21 comprising a unit dose of 1 mg to 1000 mg of said human monoclonal anti-MASP-2 dy or fragment thereof.

24. Use of the isolated MASP-2 antibody of any one of claims 1, 11 or 14 in the manufacture of a medicament for inhibiting the effects of MASP-Z-dependent complement activation in a subject in need thereof.

25. The antibody of any one of claims 1 to 16, substantially as herein described with reference to any one of the es and/or