OA19004A - Methods for inhibiting angiogenesis in a subject in need thereof. - Google Patents

Abstract

In one aspect, the present invention provides methods for preventing, treating, reverting and/or delaying angiogenesis in mammalian subject suffering from, or at risk for developing, an angiogenesis-dependent disease or condition, comprising administering to the subject an amount of a MASP-2 inhibitory agent effective to inhibit angiogenesis. In some embodiments of these aspects of the invention , the MASP-2 inhibitory agent is a MASP-2 antibody or fragment thereof.

Description

METHODS FOR INHIBITING ANGIOGENESIS IN A SUBJECT IN NEED THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

This application daims the benefit of Provisional Application No. 62/315,857, filed March 31, 2016, ali of which are hereby încorporated by reference in theîr entirety.

STATEMENT REGARD ING SEQUENCE LISTING

The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby încorporated by reference into the spécification. The name of the text file containîng the sequence listing is MP_l_0239_PCT_Sequence_Listing_20170321_ST25. The text file is 115 KB, was created on March 21, 2017, and is being submitted via EFS-Web with the filing of the spécification.

BACKGROUND

The complément System provides an early acting mechanism to initiate, amplify and orchestrate the immune response to microbial infection and other acute insults 20 (M.K. Liszewski and J.P. Atkinson, 1993, in Fundamental Immunology, Third Edition, edited by W.E. Paul, Raven Press, Ltd., New York), in humans and other vertebrates. While complément activation provides a valuable first-line defense against potential pathogens, the activities of complément that promote a protective immune response can also represent a potential threat to the host (K.R. Kalli, et al., Springer Semin.

Immunopathol. 15:4]7-43], 1994; B.P. Morgan, Eur. J Clinical Investig. 24:219-228, 1994). For example, C3 and C5 proteolytic products recruit and activate neutrophils. While indispensable for host defense, activated neutrophils are indiscriminate in their release of destructive enzymes and may cause organ damage. In addition, complément activation may cause the déposition of lytic complément components on nearby host cells as well as on microbial targets, resulting in host cell lysis.

The complément System has also been implicated in the pathogenesis of numerous acute and chronic disease states, including: myocardial infarction, stroke, ARDS, reperfusion injury, septic shock, capillary leakage following thermal bums, postcardiopulmonary bypass inflammation, transplant rejection, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, and Alzheimer's disease. In almost ail of these conditions, complément is not the cause but is one of several factors involved in pathogenesis. Nevertheless, complément activation may be a major pathological mechanism and represents an effective point for clinical control in many of these disease states. The growing récognition of the importance of complement-mediated tissue injury in a variety of disease states underscores the need for effective complément inhibitory drugs. To date, Eculizumab (Solaris®), an antibody against complément component C5, is the only complement-targeting drug that has been approved for use in man. Yet, C5 ts one of several effector molécules located “downstream” in the complément activation cascade, and blockade of C5 does not inhibit activation of the complément System. Therefore, an inhibitor of the initiation steps of complément activation would hâve significant advantages over a “downstream” complément inhibitor.

Currently, it is widely accepted that the complément System can be activated through three distinct pathways: the classical pathway, the lectin pathway, and the alternative pathway. The classical pathway is usually triggered by a complex composed of host antibodies bound to a foreîgn particle (ï.e., an antigen) and thus requires prior exposure to an antigen for the génération of a spécifie antibody response. Since activation of the classical pathway dépends on a prior adaptîve immune response by the host, the classical pathway is part of the acquired immune System. In contrasL both the lectin and alternative pathways are independent of adaptive immunity and are part of the innate immune System.

The activation of the complément System results in the sequential activation of serine protease zymogens. The first step in activation of the classical pathway is the binding of a spécifie récognition molécule, Clq, to antigen-bound IgG and IgM molécules. Clq is associated with the Clr andCls serine protease proenzymes as a complex called CL Upon binding ofClq to an immune complex, autoproteolytic cleavage of the Arg-Ile site ofClr is followed by Clr-mediated cleavage and activation ofCls, which thereby acquires the ability to cleave C4 and C2. C4 is cleaved into two fragments, designated C4a and C4b, and, similarly, C2 is cleaved into C2a and C2b. C4b fragments are able to form covalent bonds with adjacent hydroxyl or amino groups and generate the C3 convertase (C4b2a) through noncovalent interaction with the C2a fragment of activated C2. C3 convertase (C4b2a) activâtes C3 by proteolytic cleavage into C3a and C3b subcomponents leading to génération 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 C-9 polymers, also referred to as “MAC”) that can disrupt cellular membranes leading to cell lysîs. The activated forms ofC3 and C4 (C3b and C4b) are covalently deposited on the foreîgn target surfaces, which are recognized by complément receptors on multiple phagocytes.

The first step in activation of the complément System through the lectin pathway is the binding of lectin pathway-specific pattern récognition molécules to theÎr target ligands. This process initiâtes the activation of lectin pathway-specific serine protease proenzymes that in turn initiate the complément cascade. The pattern récognition molécules in the lectin pathway comprise a group of carbohydrate-binding C-type lectins, Le., mannan-bînding lectin (MBL), collectin-ll (CL-ll, also known as CL-Kl), collectin-10 (CL-10, also known as CL-Ll), and three different ficolins, i.e., H-ficolin, M-ficolin and L-ficolin that bind to acetylated structures of carbohydrates and proteins through fibrinogen-like binding domains (J. Lu et al., Biochim. Biophys. Acta 1572:387-400, (2002); Holmskov étal, Annu. Rev. Immunol. 21:547-578 (2003); Teh étal., Immunology 101:225-232 (2000), J. Luet et al., Biochim Biophys Acta 1572:387-400 (2002); Hansen et al, J. Immunol 185(10):6096-6104 (2010), and Hendriksen et al., J Immunol 191(12) :6117-27, 2013).

Ikeda et al. first demonstrated that like Clq, MBL could activate the complément System upon binding to yeast mannan-coated erythrocytes in a C4-dependent manner (Ikeda étal., J. Biol. Chem. 262:7451-7454, (1987)). MBL, a member of the collectîn protein family, is a calcium-dependent lectin that binds carbohydrates with 3- and 4-hydroxy groups oriented in the équatorial plane of the pyranose ring. Prominent ligands for MBL are thus D-mannose and N-acetyl-D-glucosamîne, while carbohydrates not fitting this steric requirement hâve undetectable affinity for MBL (Weis et al., Nature 360:127-134, (1992)). The interaction between MBL and monovalent sugars is extremely weak, with dissociation constants typicaîly in the single-digit millimolar range.

MBL achieves tight, spécifie binding to glycan ligands by avidity, i.e., by interacting simultaneously with multiple monosaccharide residues located in close proximity to each other (Lee étal., Archiv. Biochem. Biophys. 299:129-136, (1992)). MBL recognizes the carbohydrate patterns that commonly decorate microorganisms such as bacteria, yeast, parasites and certain viruses. In contrast, MBL does not recognize D-galactose and sîalïc acid, the penultimate and ultimate sugars that usually decorate mature complex glycoconjugates présent on mammalian plasma and cell surface glycoproteins. This binding specificity is thought to promote récognition of “foreign” surfaces and help protect from “self-activation.” However, MBL does bind with hîgh affmity to clusters of high-mannose precursor glycans on N-linked glycoproteins and glycolipids sequestered in the endoplasmic réticulum and Golgi of mammalian cells (Maynard et al., J. Biol. Chem. 257:3788-3794, (1982)). Therefore, damaged cells are potential targets for lectin pathway activation via MBL binding and more recent work has shown that CL-11 is another lectin pathway récognition subcomponent that initiâtes lectin pathway activation on distressed or damaged cells (Farar et al., J Clin Invest 126:1911-1925, 2016).

The ficolins possess a different type of lectin domain than MBL, called the fibrinogen-like domain. Ficolins bind sugar residues in a Ca++-independent manner. In humans, three kinds of ficolins (L-ficolin, M-ficolin and H-ficolîn) hâve been identified. The two sérum ficolins, L-ficolin and H-ficolin, hâve in common a specificity for N-acetyl-D-glucosamine; however, H-ficolin also binds N-acetyl-D-galactosamine. The différence in sugar specificity of L-ficolin, H-ficolin, CL-11, 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-ficolin binds specifically to lipoteichoic acid, a cell wall glycoconjugate found on ali Gram-positive bacteria (Lynch et al., J. Immunol. 772:1198-1202, (2004)). The collectins (i.e., MBL, CL-11, CL-10 and CL-ll/CL-10 complexes) and the ficolins bear no significant similarity in amino acid sequence. However, the two groups of proteins hâve similar domain organizations and, like Clq, assemble into olîgomeric structures, which maximize the possibility of multisite binding.

The sérum concentrations of MBL are highly variable in healthy populations and this îs genetically controlled by polymorphisms/mutations in both the promoter and coding régions of the MBL gene. As an acute phase protein, the expression of MBL is further upregulated during inflammation. L-ficolin is présent in sérum at concentrations similar to those of MBL. Therefore, the L-ficolin branch of the lectin pathway is potentially comparable to the MBL arm in physiological importance. MBL and fîcolins can also fonction as opsonins, which allow phagocytes to target MBL- and ficolindecorated surfaces (see Jack et al., J Leukoc Biol., 77(3):328-36 (2004), Matsushita and Fujita, Immunobiology, 205(4-5):490-7 (2002), Aoyagi et al., J Immunol, 174(1):41825(2005). This opsonization requires the interaction of these proteins with phagocyte receptors (Kuhlman étal., J. Exp. Med. Ι69:\Ί?>3, (1989); Matsushita et al., J. Biol. Chem. 277:2448-54, (1996)), the identity of which has not been established.

Human MBL forms a spécifie and high-affinity interaction through its collagen-like domain with unique Clr/Cls-like serine proteases, termed MBL-associated serine proteases (MASPs). To date, three MASPs hâve been described. First, a single enzyme MASP was identified and characterized as the enzyme responsible for the initiation of the complément cascade (i.e., cleaving C2 and C4) (Matsushita et al., J Exp Med 176(6):1497-1502 (1992); Ji étal., J. Immunol. 756:571-578, (1993)). It was subsequently determined that the MASP activity was, in fact, a mixture of two proteases: MASP-1 and MASP-2 (Thiel étal., Nature 356:506-510, (1997)). However, it was demonstrated that the MBL-MASP-2 complex alone is sufficîent for complément activation (Vorup-Jensen étal., J. Immunol. 765:2093-2100, (2000)). Furthermore, only MASP-2 cleaved C2 and C4 at high rates (Ambrus et al., J. Immunol. 770:1374-1382, (2003)). Therefore, MASP-2 is the protease responsible for activating C4 and C2 to generate the C3 convertase, C4b2a. This is a significant différence from the C1 complex of the classical pathway, where the coordinated action of two spécifie serine proteases (Clr and Cl s) leads to the activation of the complément System. In addition, a third novel protease, MASP-3, has been isolated (Dahl, M.R., étal., Immunity 75:127-35, 2001). MASP-1 and MASP-3 are altematively spliced products of the same gene.

MASPs share identical domain organizations with those of Clr and Cls, the enzymatic components of the CI complex (Sim et al., Biochem. Soc. Trans. 25:545, (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 complément control protein domains, and a serine protease domain. As in the Cl proteases, activation of MASP-2 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.

MBL can also associate with an alternatively spliced form of MASP-2, known as MBL-associated protein of 19 kDa (MApl9) or small MBL-associated protein (sMAP), which lacks the catalytic activity of MASP-2. (Stover, J. Immunol. /62:3481-90, (1999); Takahashi et al., Int. Immunol. /7:859-863, (1999)). MApl9 comprises the first two domains of MASP-2, followed by an extra sequence of four unique amino acids. The fonction of Mapl9 ts unclear (Degn et al., JImmunol. Methods, 2011). The MASP-1 and MASP-2 genes are located on human chromosomes 3 and 1, respectively (Schwaeble et al., Immunobiology 205:455-466, (2002)).

Several fines of evidence suggest that there are different MBL-MASP complexes and a large fraction of the MASPs in sérum is not complexed with MBL (Thiel, et al., J. Immunol. 165:878-887, (2000)). Both H- and L-ficolin bind to ail MASPs and activate the iectin complément pathway, as does MBL (Dahl étal., Immunity /5:127-35, (2001); Matsushita et ai., J. Immunol. /65:3502-3506, (2002)). Both the Iectin and classical pathways form a common C3 convertase (C4b2a) and the two pathways converge at this step.

The Iectin pathway is widely thought to hâve a major rôle in host defense against infection in the naïve host. Strong evidence for the involvement of MBL in host defense cornes from analysis of patients with decreased sérum levels of fonctional MBL (Kîlpatrick, Biochim. Biophys. Acta /572:401-413, (2002)). Such patients display susceptibilîty to récurrent bacterial and fongal infections. These symptoms are usuaily évident early in life, during an apparent window of vulnerability as matemaily derived antibody titer wanes, but before a foll répertoire of antibody responses develops. This syndrome often results from mutations at several sites in the collagenous portion of MBL, which interfère with proper formation of MBL oligomers. However, since MBL can fonction as an opsonin independent of complément, it is not known to what extent the increased susceptibilîty to infection is due to împaired complément activation.

In contrast to the classical and Iectin pathways, no initiators of the alternative pathway hâve been found to folfill the récognition fonctions 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 readiiy amplified on foreign or other abnormal surfaces (bacteria, yeast, vïrally înfected cells, or damaged tissue) that lack the proper molecular éléments that keep spontaneous complément 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 complément pathways in the pathogenesis of non-infectious human diseases, the rôle of the lectin pathway is just beginning to be evaluated. Recent studies provide evidence that activation of the lectin pathway can be responsible for complément activation and related inflammation in ischemia/reperfusion injury. Collard et al. (2000) reported that cultured endothélial cells subjected to oxidative stress bind MBL and show déposition of C3 upon exposure to human sérum (Collard et al., Am. J. Pathol. /5(5:1549-1556, (2000)). In addition, treatment of human sera with blocking anti-MBL monoclonal antibodies inhibîted MBL binding and complément activation. These findings were extended to a rat model of myocardial 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 et al., Circulation 704:1413-1418, (2001)). The molecular mechanism of MBL binding to the vascular endothélium after oxidative stress is unclear; a recent study suggests that activation of the lectin pathway after oxidative stress may be mediated by MBL binding to vascular endothélial cytokeratins, and not to glycoconjugates (Collard et al., Am. J. Pathol. /59:1045-1054, (2001)). Other studies hâve implîcated the classical and alternative pathways in the pathogenesis of ischemia/reperfusion injury and the rôle ofthe lectin pathway in this disease remains controversial (Riedermann, N.C., et al., Am. J. Pathol. /62:363-367, 2003).

A recent study has shown that MASP-1 (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 process is underlined by the absence of alternative pathway functional activity in plasma of MA SP-1/3-déficient mice. Proteolytic génération of C3b from native C3 is required for the alternative pathway to function. Since the alternative pathway C3 convertase (C3bBb) contains C3b as an essential subunif the question regarding the origin of the first C3b via the alternative pathway has presented a puzzling problem and has stimulated considérable research.

C3 belongs to a family of proteins (along with C4 and a-2 macroglobulin) that contain a rare posttranslational modification known as a thioester bond. The thioester 5 group is composed of a glutamine whose terminal carbonyl group forms a covalent thioester linkage with the sulfhydryl group of a cysteîne three amino acids away. This bond is unstable and the electrophilic glutamyl-thioester can react with nucleophilic moieties such as hydroxyl or amino groups and thus form a covalent bond with other molécules. The thioester bond is reasonably stable when sequestered within a 10 hydrophobie pocket of intact C3. However, proteolytic cleavage of C3 to C3a and C3b results in exposure of the highly reactive thioester bond on C3b and, following nucleophilic attack by adjacent moieties comprising hydroxyl or amino groups, C3b becomes covalently linked to a target. In addition to its well-documented rôle in covalent attachment of C3b to complément targets, the C3 thioester is also thought to hâve a 15 pivotai rôle in triggering the alternative pathway. According to the widely accepted tick-over theory, the alternative pathway is initiated by the génération of a fluid-phase convertase, iC3Bb, which is formed from C3 with hydrolyzed thioester (iC3; C3(H₂O)) and factor B (Lachmann, P.J., et al., Springer Semin. Immunopathol. 7:143-162, (1984)). The C3b-like C3(H₂O) is generated from native C3 by a slow spontaneous hydrolysis of 20 the internai thioester in the protein (Pangburn, M.K., étal., J. Exp. Med /54:856-867, 1981). Through the activity of the C3(H₂O)Bb convertase, C3b molécules are deposited on the target surface thereby initiatïng the alternative pathway.

Very little is known about the initiators of activation of the alternative pathway. Activators are thought to include yeast cell walls (zymosan), many pure polysaccharides, 25 rabbit érythrocytes, certain immunoglobulins, viruses, fungi, bacteria, animal tumor cells, parasites, and damaged cells. The only feature common to these activators is the presence of carbohydrate, but the complexity and variety of carbohydrate structures has made it difficult to establish the shared molecular déterminants which are recognized. It has been widely accepted that alternative pathway activation is controlled through the 30 fine balance between înhibitory regulatory components of this pathway, such as Factor H, Factor I, DAF, and CRI, and properdin, which is the only positive regulator of the alternative pathway (see Schwaeble W.J. and Reid K.B., Immunoi Today 20(1):17-21 (1999)).

In addition to the apparently unregulated activation mechanism described above, the alternative pathway can also provide a powerfiii amplification loop for the 5 lectin/classical pathway C3 convertase (C4b2a) since any C3b generated can participate with factor B in forming additional alternative pathway C3 convertase (C3bBb). The alternative pathway C3 convertase is stabilized by the binding of properdin. Properdîn extends the alternative pathway C3 convertase half-life six to ten fold. Addition of C3b to the alternative pathway C3 convertase leads to the formation of the alternative pathway 10 C5 convertase.

Ail three pathways (Le., the classical, lectin and alternative) hâve 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 complément pathway. C5a is the most potent anaphylatoxin, inducing alterations in smooth muscle and vascular tone, 15 as well as vascular permeability. It is also a powerfiii chemotaxin and activator of both neutrophils and monocytes. C5a-mediated cellular activation can significantly amplify inflammatory responses by inducing the release of multiple additional inflammatory mediators, including cytokines, hydrolytic enzymes, arachidonîc acid métabolites, and reactive oxygen species. C5 cleavage leads to the formation of C5b-9, also known as the 20 membrane attack complex (MAC). There is now strong evidence that sublytic MAC déposition may play an important rôle in inflammation in addition to its rôle as a lytic pore-forming complex.

In addition to its essential rôle in immune defense, the complément System contributes to tissue damage in many clinical conditions. Thus, there is a pressing need 25 to develop therapeutically effective complément inhibitors to prevent these adverse effects.

It is well established that angiogenesis is implicated in the pathogenesis of a variety of disorders including solid tumors and métastasés, and ocular neovascuiar diseases such as age-related macular degeneration (AMD), proliférative diabetic 30 retinopathy and neovascuiar glaucoma.

In view of the rôle of angiogenesis in many diseases and disorders, there is also a pressing need to develop therapeutically effective angiogenesis inhibitors.

ΙΟ SUMMARY

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

In one aspect, the présent invention provides methods for preventing, treating, reverting and/or delaying angiogenesis in a mammalian subject sufferîng from, or at risk for developing, an angiogenesis-dependent disease or condition, comprising administering to the subject an amount of a MASP-2 inhibitory agent effective to inhibit angiogenesis. In some embodiments of these aspects of the invention, the MASP-2 inhibitory agent is a MASP-2 antibody or fragment thereof. In further embodiments, the MASP-2 antibody has reduced effector fonction. In some embodiments, the MASP-2 inhibitory agent is a MASP-2 inhibitory peptide or a non-peptide MASP-2 inhibitor.

In another aspect, the présent invention provides compositions for inhibiting the adverse effects of angiogenesis, comprising a therapeutically effective amount of a MASP-2 inhibitory agent and a pharmaceutically acceptable carrier. Methods are also provided for manufacturing a médicament for use in inhibiting the adverse effects of angiogenesis in living subjects in need thereof, comprising a therapeutically effective amount of a MASP-2 inhibitory agent in a pharmaceutical carrier. Methods are also provided for manufacturing médicaments for use in inhibiting angiogenesis for treatment of each of the conditions, diseases and disorders described herein below.

The methods, compositions and médicaments of the invention are usefol for inhibiting the adverse effects of angiogenesis in vivo in mammalian subjects, including humans suffering from an acute or chronic pathological condition or injury as forther described herein.

In another aspect of the invention, methods are provided for inhibiting angiogenesis in a mammalian subject suffering from an angiogenesis-dependent disease or condition comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory agent effective to inhibit angiogenesis. In some embodiments, the angiogenesis-dependent disease or condition is an angiogenesis-dependent cancer, such as, for example, an angiogenesis-dependent cancer selected from the group consisting of solid tumor(s), blood borne tumors, high-risk carcinoîd tumors, and tumor métastasés. In some embodiments, the angiogenesis-dependent disease or condition is an angiogenesis-dependent benign tumor, such as, for example, an angiogenesis-dependent benign tumor selected from the group consisting of hemangiomas, acoustic neuromas, neurofibromas, trachomas, carcinoîd tumors, and pyogénie granulomas. In some embodiments, the angiogenesis-dependent disease or condition is an ocular angiogenic disease or condition, such as, for example, an ocular angiogenic disease or condition selected from the group consisting of age-related macular degeneration (AMD), uveitis, ocular melanoma, comeal neovascularization, primary pterygium, HSV stromal keratitis, HSV-l-induced comeal lymphangiogenesis, proliférative diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, comeal graft rejection, neovascular glaucoma, and rubeosis.

In another aspect, the présent invention provides methods of treating a subject suffering from an ocular angiogenic disease or condition selected from the group consisting of AMD, uveitis, ocular melanoma, comeal neovascularization, primary pterygium, HSV stromal keratitis, HSV-l-induced comeal lymphangiogenesis, proliférative diabetic retinopathy, diabetic macular edema, retinopathy of prematurity, retinal vein occlusion, comeal graft rejection, neovascular glaucoma, vitreous hemorrhage secondary to proliférative diabetic retinopathy, neuromyelitis optica and rubeosis, comprising administering to the subject an amount of a MASP-2 inhibitory agent effective to inhibit angiogenesis.

In another aspect, the présent invention provides methods of inhibiting tumor angiogenesis comprising administering to a subject with cancer an amount of a MASP-2 inhibitory agent effective to inhibit angiogenesis.

DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the saine become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:

FIGURE l is a diagram illustratîng the genomic structure of human MASP-2;

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

FIGURE 2B is a schematic diagram illustrating the domain structure of human MApl9 protein;

FIGURE 3 is a diagram illustrating the murine MASP-2 knockout strategy;

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

FIGURE 5A présents results demonstrating that MASP-2-deficiency leads to the loss of lectin-pathway-mediated C4 activation as measured by lack of C4b déposition on mannan, as described in Example 2;

FIGURE 5B présents results demonstrating that MASP-2-deficiency leads to the loss of lectin-pathway-mediated C4 activation as measured by lack of C4b déposition on zymosan, as described in Example 2;

FIGURE 5C présents results demonstrating the relative C4 activation levels of sérum samples obtained from MASP-2+/-; MASP-2-/- and wild-type strains as measure by C4b déposition on mannan and on zymosan, as described in Example 2;

FIGURE 6 présents results demonstrating that the addition of murine recombinant MASP-2 to MASP-2-/- sérum samples recovers lectin-pathway-mediated C4 activation in a protein concentration dépendant manner, as measured by C4b déposition on mannan, as described in Example 2;

FIGURE 7 présents results demonstrating that the classical pathway is ftinctional in the MASP-2-/- strain, as described in Example 8;

FIGURE 8A présents results demonstrating that anti-MASP-2 Fab2 antibody #l l inhibits C3 convertase formation, as described in Example 10;

FIGURE 8B présents results demonstrating that anti-MASP-2 Fab2 antibody #l l binds to native rat MASP-2, as described in Example 10;

FIGURE 8C présents results demonstrating that anti-MASP-2 Fab2 antibody #41 inhibits C4 cleavage, as described in Example 10;

FIGURE 9 présents results demonstrating that ail of the anti-MASP-2 Fab2 antibodies tested that inhibited C3 convertase formation also were found to inhibit C4 cleavage, as described in Example 10;

FIGURE 10 îs a diagram illustrating the recombinant polypeptides derîved from rat MASP-2 that were used for epitope mapping of the MASP-2 biocking Fab2 antibodies, as described in Example 1l;

FIGURE 11 présents results demonstrating the binding of anti-MASP-2 Fab2 #40 and #60 to rat MASP-2 polypeptides, as described in Example 11 ;

FIGURE 12A présents results showing the baseline VEGF protein levels in RPE-choroid complex isolated from wild type (+/+) and MASP-2 (-/-) mice, as described in Example 12;

FIGURE 12B présents results showing the VEGF protein levels in RPE-choroid complex at day 3 in wild type (+/+) and MASP-2 (-/-) mice following laser induced injury in a macular degeneratîon model, as described in Example 12;

FIGURE 13 présents results showing the mean choroidal neovascularïzation (CNV) volume at day seven following laser induced injury in wild type (+/+) and MASP-2 (-/-) mice, as described in Example I2;

FIGURE 14 graphically illustrâtes the level of C4b déposition, measured as % of control, în samples taken at various time points after subcutaneous (SC) dosing of either 0.3 mg/kg or LO mg/kg of mouse anti-MASP-2 monoclonal antibody in WT mice, as described in Example 13;

FIGURE 15 graphically illustrâtes the level of C4b déposition, measured as % of control, in samples taken at various time points after intraperitoneal (IP) dosing of 0.6 mg/kg of mouse anti-MASP-2 monoclonal antibody in WT mice, as described in Example 13;

FIGURE 16 graphically illustrâtes the mean choroidal neovascularïzation (CNV) volume at day seven following laser induced injury in WT (+/+) mice pre-treated with a single IP injection of 0.3 mg/kg or l.O mg/kg mouse anti-MASP-2 monoclonal antibody; as described in Example 14;

FIGURE 17A graphically illustrâtes the level of MAC déposition in the presence or absence of human MASP-2 monoclonal antibody (OMS646) under lectin pathway19004 spécifie assay conditions, demonstrating that OMS646 Lnhibits lectin-medîated MAC déposition with an IC50 value of approximately l nM, as described in Example 15;

FIGURE 17B graphically illustrâtes the level of MAC déposition in the presence or absence of human MASP-2 monoclonal antibody (OMS646) under classical pathwayspecific assay conditions, demonstrating that OMS646 does not inhibit classical pathway mediated MAC déposition, as described in Example 15;

FIGURE 17C graphically illustrâtes the level of MAC déposition in the presence or absence of human MASP-2 monoclonal antibody (OMS646) under alternative pathway-specific assay conditions, demonstrating that OMS646 does not inhibit alternative pathway-mediated MAC déposition, as described in Example 15;

FIGURE 18 graphically illustrâtes the pharmacokinetic (PK) profile of human MASP-2 monoclonal antibody (OMS646) in mice, showing the OMS646 concentration (mean of n=3 animals/groups) as a function of time after administration at the indicated dose, as described in Example 15;

FIGURE 19A graphically illustrâtes the pharmacodynamie (PD) response of human MASP-2 monoclonal antibody (OMS646), measured as a drop in systemic lectin pathway activity, in mice following întravenous administration, as described in Example I5;

FIGURE 19B graphically illustrâtes the pharmacodynamie (PD) response of human MASP-2 monoclonal antibody (OMS646), measured as a drop in systemic lectin pathway activity, in mice following subeutaneous administration, as described in Example 15; and

FIGURE 20 graphically illustrâtes the choroîdal neovascularization (CNV) area as a percentage of the area of laser-induced lésions at day seven following injury in WT (+/+) mice pre-treated with 2mg/kg, 5mg/kg or 20mg/kg human MASP-2 monoclonal antibody (OMS646) administered SC, or anti-VEGF antibody administered IP, as described in Example 16.

DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO:l human MApl9 cDNA

SEQ ID NO:2 human MApl9 protein (with leader)

SEQ ID NO:3 human MApl9 protein (mature)

SEQ ID NO:4 human MASP-2 cDNA

SEQ ID NO:5 human MASP-2 protein (with leader)

SEQ ID NO:6 human MASP-2 protein (mature)

SEQ ID NO:7 human MASP-2 gDNA (exons I-6)

ANTIGENS: (IN REFERENCE TO THE MASP-2 MATURE PROTEIN)

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

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

SEQ ID NO:lO CUBEGFCUBII (aa i-293)

SEQ ID NO:l l EGF région (aa 122-166)

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

SEQ ID NO:l3 serine protease domain inactive (aa 610-625 with Ser618 to Ala mutation)

SEQ IDNO:14 TPLGPKWPEPVFGRL (CUBI peptide)

SEQ IDNO:15

TAPPGYRLRLYFTHFDLELSHLCEYDFVKLSSGAKVLATLC

GQ (CUBI peptide)

SEQ IDNO:16 TFRSDYSN (MBL binding région core)

SEQ ID NO: 17 FYSLGSSLDITFRSDYSNEKPFTGF (MBL binding région)

SEQ ID NO: 18 IDECQVAPG (EGF PEPTIDE)

SEQ ID NO: 19 ANMLCAGLESGGKDSCRGDSGGALV (serine protease binding core)Detailed Description

PEPTIDE INHIBITORS:

SEQ ID NO:20 MBL full length cDNA

SEQ IDN0:21 MBL full length protein

SEQ ID N0:22 OGK-X-GP (consensus binding)

SEQ ID NO:23 OGKLG

SEQ ID NO:24 GLR GLQ GPO GKL GPO G

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

SEQ ID NO:26 GKDGRDGTKGEKGEPGQGLRGLQGPOGKLGPOG

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

SEQ ID NO:28 GCOGLOGAOGDKGEAGTNGKRGERGPOGPOGKAGPOGPN GAOGEO (human ficolin p35)

SEQ ID NO:29 LQRALEILPNRVTIKANRPFLVFI (C4 cleavage site) EXPRESSION INHIBITORS:

SEQ ID NO:30 cDNA of CUBI-EGF domain (nucléotides 22-680 of SEQ ID NO:4)

SEQ IDNO:31

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

SEQ ID NO:32 5'GACATTACCTTCCGCTCCGACTCCAACGAGAAG3' Nucléotides 361-396 of SEQ ID NO:4 encoding a région comprising the MASP-2 MBL binding site (sense)

SEQ IDNO:33 5AGCAGCCCTGAATACCCACGGCCGTATCCCAAA3' Nucléotides 610-642 of SEQ ID NO:4 encoding a région comprising the CUBII domain

CLONING PR1MERS:

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

SEQ ID NO:35 GGAATTCCTAGGCTGCATA (3’ PCR FOR CUB)

SEQ ID NO:36 GGAATTCCTACAGGGCGCT (3’ PCR FOR CUBIEGF)

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

SEQ ID NOS:38-47 are cloning primers for humanized antibody

SEQ ID NO:48 is 9 aa peptide bond

EXPRESSION VECTOR:

SEQ ID NO:49 is the MASP-2 minigene insert

SEQ ID NO: 50 is the murine MASP-2 cDNA

SEQ ID NO: 51 is the murine MASP-2 protein (w/leader)

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

SEQ ID NO: 53 the rat MASP-2 cDNA

SEQ ID NO: 54 is the rat MASP-2 protein (w/ leader)

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

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

SEQ ID NO; 60-63 are the oligonucleotides for site-directed mutagenesis of murine MASP-2 used to generate murine MASP-2A

SEQ ID NO: 64-65 are the oligonucleotides for site-directed mutagenesis of rat MASP-2 used to generate rat MASP-2A

SEQ ID NO:66 DNA encoding l7D20_dc35VH2lNl l VL (OMS646) heavy chain variable région (VH) (without signal peptide)

SEQ ID NO:67 17D20_dc35VH2lNlIVL (OMS646) heavy chain variable région (VH) polypeptide

SEQ ID NO:68 !7Nl6mc heavy chain variable région (VH) polypeptide

SEQ ID NO:69 !7D20_dc2lNl IVL (OMS644) light chain variable région (VL) polypeptide

SEQ ID NO:70 DNA encoding !7Nl6_dcl7N9 (OMS641) light chain variable région (VL) (without signal peptide)

SEQ ID NO:71 l7Nl6_dcl7N9 (OMS641) light chain variable région (VL) polypeptide

DETAILED DESCRIPTION

The présent invention is based upon the surprising discovery by the présent inventors that it is possible to inhibit the lectin mediated MASP-2 pathway while leaving the classical pathway intact. The présent invention also describes the use of MASP-2 as a therapeutic target for inhibiting cellular injury associated with lectin-mediated complément pathway activation while leaving the classical (Clq-dependent) pathway component of the immune System intact.

I. DEFINITIONS

Unless specifically defined herein, ail terms used herein hâve the same meaning as would be understood by those of ordinary skill in the art of the présent invention. The following définitions are provided in order to provide clarîty with respect to the terms as they are used in the spécification and daims to describe the présent invention.

As used herein, the term “MASP-2-dependent complément activation” comprises MASP-2- dépendent activation of the lectin pathway, which occurs under physiological conditions (Le., 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, which has been determined to primarily cause opsonization.

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

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

As used herein, the term classical pathway refers to complément activation that is triggered by antibody bound to a foreign particle and requîtes binding of the récognition molécule Clq.

As used herein, the term MASP-2 inhibitory agent refers to any agent that binds to or directly interacts with MASP-2 and effectively inhibits MASP-2-dependent complément activation, including anti-MASP-2 antibodies and MASP-2 binding fragments thereof, naturel and synthetic peptides, small molécules, soluble MASP-2 receptors, expression inhibhors and isolated naturel inhibitors, and also encompasses peptides that compete with MASP-2 for binding to another récognition molécule (e.g., MBL, H-ficolin, M-ficolin, or L-ficolin) in the lectin pathway, but does not encompass antibodies that bind to such other récognition molécules. MASP-2 inhibitory agents useful in the method of the invention may reduce MASP-2-dependent complément activation by greater than 20%, such as greater than 50%, such as greater than 90%. In one embodiment, the MASP-2 inhibitory agent reduces MASP-2-dependent complément activation by greater than 90% (i.e., resulting in MASP-2 complément activation of only 10% or less).

As used herein, the term “angiogenesis” refers to the growth of new microvessels out of pre-exîsting blood vessels.

As used herein, the term neo-angiogenesis” refers to angiogenesis when it is involved in a disease or condition that is not physiological or is pathological.

As used herein, the term antibody encompasses antibodies and antibody fragments thereof, derived from any antibody-producing mammal (e.g., mouse, rat, rabbit, and primate including human), or from a hybridoma, phage sélection, recombinant expression or transgenic animais (or other methods of producing antibodies or antibody fragments”), that specifically bind to a target polypeptide, such as, for example, MASP-2, polypeptides or portions thereof. It is not intended that the term “antibody” limited as regards to the source of the antibody or the manner in which it is made (e.g., by hybridoma, phage sélection, recombinant expression, transgenic animal, peptide synthesis, etc). Exemplary antibodies include polyclonal, monoclonal and recombinant antibodies; pan-specîfic, multispecific antibodies (e.g., bispecific antibodies, trispecîfic antibodies); humanized antibodies; murine antibodies; chimeric, mouse-human, mouse-primate, primate-human monoclonal antibodies; and anti-idiotype antibodies, and may be any intact antibody or fragment thereof. As used herein, the term antibody encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as dAb, Fab, Fab', F(ab% Fv), single chain (ScFv), synthetic variants thereof, naturally occurrîng variants, fusion proteins 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 molécule that comprises an antigen-binding site or fragment (epîtope récognition site) of the required specificity.

A “monoclonal antibody refers to a homogeneous antibody population wherein the monoclonal antibody is comprised of amino acids (naturally occurrîng and nonnaturally occurrîng) that are involved in the sélective binding of an epîtope. Monoclonal antibodies are highly spécifie for the 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 modified configuration of the immunoglobulin molécule that comprises an antigenbinding fragment (epitope récognition site) ofthe required specificîty and the ability to bind to an epitope. It is not intended to be limited as regards the source of the antibody or the manner in which it is made (e.g., by hybridoma, phage sélection, recombinant expression, transgenic animais, etc.). The term includes whole immunoglobulins as well as the fragments etc. described above under the définition of antibody.

As used herein, the term antibody fragment refers to a portion derived from or related to a full-length antibody, such as, for example, an antî-MASP-2 antibody, generally including the antigen binding or variable région thereof. Illustrative examples of antibody fragments include Fab, Fab', F(ab)2, F(ab')2 and Fv fragments, scFv fragments, diabodies, linear antibodies, single-chain antibody molécules and multispecific antibodies formed from antibody fragments.

As used herein, a single-chain Fv or scFv antibody fragment comprises the Vh and Vl domains of an antibody, wherein these domains are présent in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the Vjq and domains, which enables the scFv to form the desired structure for antigen binding.

As used herein, a chimeric antibody is a recombinant protein that contains the variable domains and complementarity-determining régions derived from a non-human species (e.g., rodent) antibody, while the remainder of the antibody molécule is derived from a human antibody.

As used herein, a humanized antibody is a chimeric antibody that comprises a minimal sequence that conforms to spécifie complementarity-determining régions derived from non-human immunoglobulin that is transplanted into a human antibody framework. Humanized antibodies are typically recombinant proteins in which only the antibody complementarity-determining régions are of non-human origin.

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

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

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

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

In the broadest sense, 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 hydrophobie amino acid is meant either Ile, Leu, Met, Phe, Trp, Tyr, Val, Ala, Cys or Pro. By hydrophilic amino acid is meant either Gly, Asn, Gin, Ser, Thr, Asp, Glu, Lys, Arg or His. This grouping of amino acids can be further subclassed as follows. By uncharged hydrophilic amino acid is meant either Ser, Thr, Asn or Gin. 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 ïs illustrated by a substitution among amino acids within each of the following groups: (l) glycine, alanine, valine, leucine, and isoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine, (4) aspartate and glutamate, (5) glutamine and asparagine, and (6) lysine, arginine and histidine.

The term oligonucleotide as used herein refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term also covers those oligonucleobases composed of naturally-occurring nucléotides, sugars and covalent intemucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring modifications.

As used herein, an epitope refers to the site on a protein (e.g·, a human MASP-2 protein) that is bound by an antibody. Overlapping epîtopes' include at least one (e.g., two, three, four, five, or six) common amino acid residue(s), including linear and nonlinear epitopes.

As used herein, the ternis polypeptide, peptide, and protein are used interchangeably and mean any peptide-linked chain of amino acids, regardless of length or post-translational modification. The MASP-2 protein described herein can contain or be wild-type proteins or can be variants that hâve not more than 50 (e.g., not more than one, two, three, four, five, six, seven, eight, nine, ten, 12, 15, 20, 25, 30, 35, 40, or 50) conservative amino acid substitutions. Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine and threonine; lysine, histidine and arginine; and phenylalanîne and tyrosine.

In some embodiments, the human MASP-2 protein can hâve an amino acid sequence that is, or is greater than, 70 (e.g., 7I, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100) % identical to the human MASP-2 protein having the amino acid sequence set forth in SEQ ID NO: 5.

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

Percent (%) amino acid sequence identîty is defined as the percentage of amino acids in a candidate sequence that are identical to the amino acids in a référencé sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determîning percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publïcly available computer software such as BLAST, BLAST-2, ALIGN, ALlGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the fulllength of the sequences being compared can be determined by known methods.

II. Overview of the Invention

Lectins (MBL, M-ficolin, H-ficolin, L-ficolin and CL-ll) are the spécifie récognition molécules that trigger the innate complément System and the System includes the lectin initiation pathway and the assocîated terminal pathway amplification loop that amplifies lectin-initiated activation of terminal complément effector molécules. Clq is the spécifie récognition molécule that triggers the acquired complément system and the System includes the classical initiation pathway and assocîated terminal pathway amplification loop that amplifies Clq-initiated activation of terminal complément effector molécules. We refer to these two major complément activation Systems as the lectin-dependent complément system and the C Iq-dependent complément System, respecti vely.

In addition to its essential rôle in immune defense, the complément System contributes to tissue damage in many clinical conditions. Thus, there îs a pressing need to develop therapeutically effective complément inhibîtors to prevent these adverse effects. With the récognition that it is possible to inhibit the lectin mediated MASP-2 pathway while leaving the classical pathway intact cornes the realization that it would be highly désirable to specifically inhibit only the complément activation system causing a particular pathology without completely shutting down the immune defense capabilities of complément. For example, in disease states in which complément activation is mediated predominantly by the lectin-dependent complément system, it would be advantageous to specifically inhibit only this system. This would leave the Clq-dependent complément activation system intact to handle immune complex processing and to aid in host defense against infection.

The preferred protein component to target in the development of therapeutic agents to specifically inhibit the lectin-dependent complément system is MASP-2. Of ail the known protein components of the lectin-dependent complément System (MBL, H-ficolin, M-ficolin, L-ficolÎn, MASP-2, C2-C9, Factor B, Factor D, and properdin), only MASP-2 is both unique to the lectin-dependent complément System and required for the System to fonction. The lectins (MBL, H-ficolin, M-ficolin,L-ficol in and CL-11) are also unique components in the lectin-dependent complément System. However, loss of any one of the Iectin components would not necessarily inhibit activation of the System due to Iectin redundancy. It would be necessary to inhibit ali five lectins in order to guarantee inhibition of the lectin-dependent complément activation System. Furthermore, since MBL and the ficolins are also known to hâve opsonic activity independent of complément, inhibition of Iectin fonction would resuit in the loss of this bénéficiai host defense mechanism against infection. In contrast, this complement-independent Iectin opsonic activity would remain intact if MASP-2 was the inhibitory target. An added benefit of MASP-2 as the therapeutic target to inhibit the lectin-dependent complément activation System is that the plasma concentration of MASP-2 is among the lowest of any complément protein (~ 500 ng/ml); therefore, correspondingly low concentrations of high-affinity inhibitors of MASP-2 may be sufficient to obtain foll inhibition (Moller-Kristensen, M., étal., J. Immunol Methods 282:159-167, 2003).

As described herein, it was unexpectedly determined that a MASP-2 inhibitor, such as a human MASP-2 antibody (OMS646), is at least as effective as an anti-VEGF antibody at reducing chorodial neovascularization (CNV) in a mouse mode! of agerelated macular degeneration (AMD) when delivered systemically to mice. Therefore, it is expected that a MASP-2 inhibitory agent such as a MASP-2 inhibitory antibody will also be effective as an anti-angiogenesis agent for use in inhibiting an angiogenesisdependent cancer, such as, for example, an angiogenesis-dependent cancer selected from the group consisting of solid tumor(s), blood borne tumors, high-risk carcinoid tumors, and tumor métastasés. It is also expected that a MASP-2 inhibitory agent, such as MASP-2 inhibitory antibody will be effective as an anti-angiogenesis agent for inhibiting an angiogenesis-dependent benign tumor, such as, for example, an angiogenesisdependent benign tumor selected from the group consisting of hemangiomas, acoustic neuromas, neurofibromas, trachomas, carcinoid tumors, and pyogénie granulomas. It is also expected that a MASP-2 inhibitory agent such as a MASP-2 inhibitory antibody will be effective as an anti-angiogenesis agent for use in inhibiting angiogenesis in AMD and other ocular angiogenic diseases or disorders such as uveitis, ocular melanoma, comeal neovascularization, primary (comeal) pterygium, HSV stromal keratitis, HSV-l-induced comeal lymphangiogenesis, proliférative diabetic retinopathy, retinopathy of prematunty, retinal vein occlusion, comeal graft rejection, neovascuiar glaucoma, and rubeosis.

ΙΠ. ROLE OF MASP-2 IN ANGIOGENESIS-DEPENDENT DISEASES AND CONDITIONS AND THERAPEUTIC METHODS USING MASP-2 INHIBITORY AGENTS

Angiogenesis-dependent diseases or conditions resuit when new blood vessels grow excessively at inappropriate locations (such as retinal pigmented epithelium) or when new blood vessels hâve undesirable characteristics such as leakiness and include diseases such as cancer and diseases of the eye. In these conditions, new blood vessels feed diseased tissue, may destroy new tissue and, in the case of cancer, new blood vessels allow the tumor to grow and the tumor cells to enter the circulation and metastasîze to other organs. Excessive angiogenesis may occur when diseased cells produce abnormal amounts of angiogenic growth factors, thereby overwhelming the effects of naturally occurring angiogenesis inhibitors.

The potential rôle for complément activation in angiogenesis has been shown in age-related macular degeneration (AMD). AMD is a blindîng disease that afflicts millions of adults, yet the sequelae of biochemical, cellular, and/or molecular events leading to the development of AMD are poorly understood. AMD results in the progressive destruction of the macula, which has been correlated with the formation of protein and lipid-rich extracellular deposits called drusen located in and around the macula, behind the retina and between the retina pigment epithelium (RPE) and the choroid. Drusen are characteristic of early and intermediate AMD. Many patients progress to advanced AMD, which includes two forms, géographie atrophy and neovascuiar or “wet” AMD. The terni “dry AMD” commonly refers to early and intermediate AMD, as well as géographie atrophy. While présent and potentially pathologie in early and intermediate forms of the disease, drusen persists in both advanced forms as well (van Lookeren-Campagne et al., J. Pathol. 232:151, 2014;

Ambatî et al., Nat. Rev. Immunol. 13:438, 2013). Recent studies hâve revealed that proteins associated with inflammation and immune-mediated processes are prévalent among drusen-associated constituents. Transcripts that encode a number of these molécules hâve been detected in retinal, RPE, and choroidal cells. These data also demonstrate that dendritic cells, which are potent antigen-presenting cells, are intimately associated with drusen development, and that complément activation is a key pathway that is active both within drusen and along the RPE-choroid interface (Hageman, G.S., étal., Prog. Retin. Eye Res. 20:705-732, 2001); Ebrahimi and Handa, J. Lipid 2011:802059, 2011). These observations indicate that local inflammation is likely a significant factor in the early pathogenesis of AMD.

Several independent studies hâve shown a strong association between AMD and a genetîc polymorphism in the gene for complément factor H (CFH) in which the likelihood of AMD is increased by a factor of 7.4 in individuals homozygous for the risk allele (Klein, R.J. et al., Science 308:362-364, 2005; Haines et al., Science 305:362-364. 2005; Edwards et al., Science 308:263-264, 2005). The CFH gene has been mapped to chromosome lq31, a région that had been implicated in AMD by six independent linkage scans (see, e.g., Schultz, D.W., étal., Hum. Mol. Genet. 72:3315, 2003). CFH is known to be a key regulator of the complément System. It has been shown that CFH on cells and in circulation régulâtes complément activity by inhibiting the activation of C3 to C3a and C3b, and by inactivating existing C3b. Déposition of C5b-9 has been observed in Bruch's membrane, the intercapillary pillars and within drusen in patients with AMD (Klein et al., Science 308:362-364, 2005). Immunofluorescence experiments suggest that in AMD the polymorphism of CFH may give rise to complément déposition in chorodial capillaries and choroidal vessels (Klein et al., Science 308:362-364, 2005).

The membrane-associated complément receptor 1 is also localized in drusen, but it is not detected in RPE cells immunohistochemically. In contrast, a second membrane-associated complément inhibitor, membrane cofactor protein, is présent in drusen-associated RPE cells as well as in small, spherical substructural éléments within drusen. These prevîously unidentîfied éléments also show strong immunoreactivity for proteolytîc fragments of complément component C3 that are characteristically deposited at sites of complément activation. It is proposed that these structures represent residual débris from degenerating RPE cells that are the targets of complément attack (Johnson,

L.V., et al., Exp. Eye Res. 73:887-896, 2001).

Identification and localization of these multiple complément regulators as well as complément activation products (C3a, C5a, C3b, C5b-9) hâve led investigators to conclude that chronic complément activation plays an important rôle in the process of drusen biogenesis and the etiology of AMD (Hageman et al., Progress Retinal Eye Res. 20:705-32, 2001). Identification of C3 and C5 activation products in drusen provides no insîght into whether complément is activated via the classical pathway, the lectin pathway or the alternative amplification loop, as understood in accordance with the présent invention, since both C3 and C5 are common to ail three. However, two studies hâve looked for drusen immuno-labeling using antibodies spécifie to Clq, the essential récognition component for activation of the classical pathway (Mullins et al., FASEBJ. /4:835-846, 2000; Johnson étal., Exp. Eye Res. 70:441-449, 2000). Both studies concluded that Clq immuno-labelling in drusen was not generally observed. These négative results with Clq suggest that complément activation in drusen does not occur via the classical pathway. In addition, immuno-labeling of drusen for immune-complex constituents (IgG light chains, IgM) is reported in the Mullins étal., 2000 study as being weak to variable, further indicatîng that the classical pathway plays a minor rôle in the complément activation that occurs in this disease process. Therefore, the lectin and/or alternative pathways are lîkely to account for most if not ail of the complement-mediated drusen biogenesis associated with AMD,

The relationship between drusen and complément activation is strong, particularly in early and intermediate AMD as well as in géographie atrophy. In fact, large and confluent drusen represent a significant risk factor for géographie atrophy (van LookerenCampagne et al., ibid). However, complément activation is not limited to the drusen environment. Two recent published studies hâve evaluated the rôle of complément in the development of laser-induced choroidal neovascularization (CNV) in mice, a model of human CNV. Using immunohistological methods, Bora and colleagues (2005) found significant déposition of the complément activation products C3b and C5b-9 (MAC) in the neovascular complex following laser treatment (Bora étal., J. Immunol. 174Ά9Χ-Ί, 2005). Important^, CNV did not develop in mice genetically déficient in C3 (C3-/mice), the essential component required in ail complément activation pathways. RNA message levels for VEGF, TGF-βΐ, and β-FGF, three angiogenic factors implicated m CNV, were elevated in eye tissue from mice after laser-induced CNV. Significantly, complément déplétion resulted in a marked réduction in the RNA levels of these angiogenic factors.

Using ELISA methods, Nozaki and colleagues demonstrated that the potent anaphylatoxins C3a and C5a are generated early in the course of laser-induced CNV (Nozaki et al., Proc. Natl. Acad. Sci. U.S.A. 103:232^-33, 2006). Furthermore, these two bioactive fragments of C3’and C5 induced VEGF expression following intravitreal injection in wild-type mice. Consistent with these results, Nozaki and colleagues also showed that genetic ablation of receptors for C3a and C5a reduces VEGF expression and CNV formation after laser injury and that antibody-mediated neutrahzation of C3a or C5a or pharmacologie blockade of their receptors also reduces CNV. Previous studies hâve established that recruitment of leukocytes, and macrophages in particular, plays a pivotai rôle in laser-induced CNV (Sakurai étal., Invest. Opthomol. Vis. Sci. 44:3578-85, 2003; Espinosa-Hetdmann, étal., Invest. Opthomol. Vis. Sci. 44:3586-92, 2003). In their 2006 paper, Nozaki and colleagues report that leukocyte recruitment is markedly reduced m C3aR(-/-) and C5aR(-/-) mice after laser injury.

The lectin pathway appears responsible for initiating the complément cascade in the CNV model following naturel antibody récognition of oxidatively modified phospholipids on the retinal pigment epithelium (Joseph et al. J. Biol. Chem. 288:12753, 2013). The alternative pathway is also critical for the retinal injury in this model, but it is not alone sufficient (Rohrer et al., Mol Immunol. 48:el, 2011). Importantly, Kunchithapautham and Rohrer (J. Biol. Chem. 286:23717, 2011) demonstrated that this complément activation triggers VEGF sécrétion by the retinal pigment épithélial cells. The VEGF is a key mediator of the neovascularization.

As described herein in Example 12, in a murine macular degeneration model in MASP-2(-/-) mice it was determined that there was a decrease in baseline levels of VEGF in the MASP-2 (-/-) mice versus the wild-type control mice and, further, that while VEGF levels were significantly increased in the wild-type mice following laser induced injury, surprisingly low levels of VEGF were seen in the MASP-2 (-/-) mice following laser induced injury. In addition, it was determined that the MASP-2 (-/-) mice displayed about a 30% réduction in the CNV area following laser induced damage at day 7 in comparison to the wild-type mice. As further described in Example 14, in mice pretreated with an anti-MASP-2 monoclonal antibody that specifically blocks the lectin pathway of complément activation, a statistically significant (p<0.01 ) approximately 50% réduction in CNV was observed seven days post-laser treatment as compared to untreated mice, demonstrating that blockade of MASP-2 with an inhibitor, such as MASP2monoclonal antibody, has a preventative and/or therapeutic effect in the treatment of macular degeneration. As further described in Example 16, in mice pre-treated with a human MASP-2 monoclonal antibody that specifically blocks the lectin pathway of complément activation, a statistically significant réduction in CNV was observed at ail dose levels tested with relative CNV area réductions ranging from 20% to 50%, whereas the VEGF antibody showed a modest (approximately 15%) relative réduction in CNV area. In view of the unexpected results disclosed in Example 16 that a MASP-2 inhibitor, such as a MASP-2 antibody, is at least as effective as VEGF antibody at reducing CNV in a mouse model of AMD when delivered systemically, it is expected that a MASP-2 inhibitory agent will be effective as an anti-angiogenesis agent for use in treating angiogenesis-dependent diseases and conditions, such as ocular angiogenic diseases or disorders, angiogenesis-dependent cancers, and angiogenesis-dependent benign tumors, as described below.

MASP-2 INHIBITORS FOR THE TREATMENT OF OCULAR ANGIOGENIC DISEASES OR DISORDERS

An ocular angiogenic disease or disorder is an eye disease or disorder wherein abnormal or excessive angiogenesis occurs in the eye, which may contribute to loss of vision, hemorrhage, or other fùnctional disorders of the eye, such as, for example, AMD, or an ocular angiogenic disease or disorder selected from the group consisting of uveitis, ocular melanoma, corneal neovascularization, primary (corneal) pterygium, HSV stromal keratitis, HSV-l-induced corneal lymphangiogenesis, proliférative diabetic retinopathy, diabetic macular edema, retinopathy of prematurity, retinal vein occlusion, corneal graft rejection, neovascular glaucoma, vitreous hemorrhage secondary to prohferative diabetic retinopathy, neuromyelitis optica, and rubeosis (see for example Rivera et al., Neonatology 100(4):343-53, 2011; Hosseinî et al., Cornea 31:322-34, 2012; Leyvraz et al., Curr Opin Oncol 162-9 (2012); Bock et al., Prog Retin Eye Res 34:89-124, 2013 and Kim et al., Am J Pathol 181(2):376-9, 2012).

As described in Examples 14 and 16, the présent application demonstrates that systemic administration of a MASP-2 antibody that specifîcally inhibits the lectin pathway of complément activation provides an effective therapy for treating neovascular AMD. Presently approved anti-angiogenic thérapies for ophthalmic conditions are biologie agents that inhibit VEGF. There are currently three approved anti-angiogenic therapeutics for ophthalmic diseases: an anti-VEGF aptamer (pegaptanib, Macugen®), a Fab fragment of a monoclonal antibody directed against VEGF-A (ranibizumab, Lucentis®), and a fusion protein that binds to VEGF-A, VEGF-B and Placental Growth Factor (aflibercept, Eylea®), ail of which are administered via intravitreal injection. Therefore, unlike current and emerging therapeutics for AMD and other ocular angiogenic diseases and disorders, which require intravitreal injection, MASP-2 antibody treatment is effective upon subeutaneous administration.

An aspect of the invention thus provides a method for inhibiting angiogenesis to treat an ocular angiogenic disease or dîsorder comprising administering a composition comprising a therapeutically effective amount of a MASP-2 ïnhibitory agent in a pharmaceutical carrier to a subject in need thereof. In some embodiments, the ocular angiogenic disease or dîsorder is selected from the group consisting of AMD, uveitis, ocular melanoma, comeal neovascularization, primary pterygium, HSV stromal keratîtis, HSV-l-induced comeal lymphangiogenesis, proliférative diabetic retinopathy, diabetic macular edema, retinopathy of prematurity, retînal veîn occlusion, comeal graft rejection, neovascular glaucoma, vitreous hemorrhage secondary to proliférative diabetic retinopathy, neuromyelitis optica and rubeosis. The MASP-2 ïnhibitory composition may be administered locally to the eye, such as by direct injection, irrigation or application of the composition in the form of a gel, salve or drops. Altemately, the MASP-2 ïnhibitory agent may be administered to the subject systemically, such as by intra-arterial, întravenous, intramuscular, inhalational, nasal, subeutaneous or other parentéral administration, or potentially by oral administration for non-peptidergic agents. The MASP-2 ïnhibitory agent composition may be combined with one or more additional therapeutîc agents, such as an additional anti-angiogenic agent. Administration may be repeated as determined by a physician untii the condition has been resoived or is controlled.

MASP-2 INHIBITORS FOR THE TREATMENT OF ANGIOGENESISDEPENDENT CANCER

It is well established that angiogenesis plays a critical rôle in the development of cancer. Tumors produce pro-angiogenic factors to stimulate neovascularization, which is one of the main mechanîsms for the progression of solid tumors and also allows for the migration of tumor cells to establish distant métastasés by accessing the systemic circulation. The process of tumor angiogenesis is prîmarily activated when a growing tumor mass surpasses the maximal volume that can be maîntained by diffusion of oxygen and nutrients. A corrélation between increased angiogenesis and tumor aggressiveness has been observed (Ferrara et al., Curr Top Microbiol Immunol 237:1-30, 1999). Angiogenesis is also known to play a rôle in the growth and survival of leukemias and other hematological malignancies (Ribatti et al., Neoplasia 15(3):231-238, 2013; Vacca et al., Br J Haematol 87:503-508, 1994). While different cell types contribute to neovascularization, the endothélial cell is generally acknowledged to be the central player in the angiogenesis process.

It is well established that VEGF plays an important rôle in tumor angiogenesis. VEGF was identified as a vascular permeability factor secreted by tumor cells (Mattéi et al., Genomics 32:168-169, 1996), and has been demonstrated to play a rôle in angiogenesis by stimulating endothélial cell migration and prolifération, as well as by stimulating expression of angiogenesis-related genes in endothélial cells. For example, soluble VEGF isoform 189 expression in human colon, rénal and lung cancers hâve been strongly associated with increased microvessels, cancer métastasés and poor prognoses (Tokunaga et al., Br J Cancer 77:998-1002, 1998; Yuan et al., J Clin Oncol 19:432-441, 2001). High levels of VEGF isoform 165 hâve been associated with poor survival rates in ovarian cancer (Mahner et al., BMC Cancer 10:139, 2010). In a phase 3 clinical trial, it was demonstrated that bevacizumab, a humanized monoclonal antibody that inhibits VEGF-A, improved progression-free survival in women with ovarian cancer (Perren et al., JVEMg/JÀfee/365:2484-2496, 2011).

In the context of cancer, researchers hâve traditionally focused on the rôle of complément in taggîng and élimination of tumor cells. However, recent studies hâve challenged this view. For example, Markiewski et al. (Nature Immunol vol 9:1225-1235, 2008), reported the unexpected findîng that complément proteins C3, C4 and C5a may aid tumor growth by promoting an immunosuppressive microenvironment. As described in Markiewski et al., the génération of complément C5a in a tumor microenvironment enhanced tumor growth by suppressing the anti-tumor CD8+ T cell-mediated response. As further described in Markiewski et al., a C5aR antagonist, the hexapeptîde AcF(OP(D)ChaWr), was as effective as paclitaxel (Taxol) in impairing tumor growth in wild type mice, thereby establishing a therapeutic fonction for complément inhibition in the treatment of cancer. As described in Gunn et al. (J Immunol 189:2985, 2012), wildtype mice with high C5a-producing syngeneic lymphoma cells had significantly accelerated tumor progression with more myeloid-derived suppressor cells (MDSC) in the spleen and overall decreased CD4+ and CD8+ T cells in the tumor, tumor-draining lymph nodes, and the spleen. In contrast, tumor-bearing mice with low C5a-producmg lymphoma cells had a significantly reduced tumor burden with increased interferon-γproducing CD4+ and CD8+ T cells in the spleen and tumor-draining lymph nodes. As further described in Corrales et al. (J Immunol 189:4674-4683, 2012) a significant increase in C5a in plasma from patients with non-small cell lung cancer (NSCLC) was found as compared to healthy subjects. It was also determined that C5a induced endothélial cell chemotaxis and blood-vessel formation. In a Lewis lung cancer model, syngeneic tumors of mouse Lewis lung carcinoma (3LL) cells grew slower in mice treated with an antagonist of the C5a receptor.

As further described in Nunez-Cruz et al. (Neoplasia 14:994-1004, 2012), to assess the rôle of complément during ovarîan cancer progression, a strain of mice with a complément deficiency in C3, or a strain of mice with a complément deficiency in C5a receptor (C5aR) were crossed with a strain of mice that develop épithélial ovarîan cancer (TgMISIIR-TAg). The TgMISIIR-Tag mice that were folly or partially déficient in C3 or folly déficient for C5aR either developed no ovarîan tumors or tumors that were small and poorly vascularized as compared to wild-type TgMISIlR-TAg littermates, thereby demonstrating that deficiency of C3 or C5aR significantly attenuated the ovarîan tumor phenotype. It was further demonstrated that CD31+ endothélial cell function in angiogenesis was impaired in both the C3 (-/-) and the C5aR (-/-) mice.

Activation of the complément System may also be implicated in the pathogenesis of malignancies. The neoantîgens of the C5b-9 complément complex, IgG, C3, C4, 5 S-protein/vitronectin, fibronectin, and macrophages were localized on 17 samples of breast cancer and on 6 samples of benign breast tumors using polyclonal or monoclonal antibodies and the streptavidin-biotin-peroxidase technique. Ail the tissue samples with carcinoma in each the TNM stages presented C5b-9 deposits on the membranes of tumor cells, thin granules on cell remuants, and diffuse deposits in the necrotic areas (Niculescu, I0 F., et al., Am. J. Pathol. /-/0:1039-1043, 1992). As further described in Rutkowski et al.

(Mol Cancer Res 8:1453, 2010), potential oncogenic rôles hâve been described for complément proteins C3, C3a, C5a and MAC, including tumor angiogenesis, invasion and migration. The lectin pathway of complément activation was found to be significantly elevated in the sérum of colorectal cancer patients when compared to 15 healthy subjects (Ytting et al., 2004, Scand J Gastroenterol 39:674) and high levels of

MASP-2 activity has been reported to be an independent prognostic biomarker predictîng colon cancer récurrence and poor survival (Ytting et al., Clin Cancer Res 11:1441, 2005).

It has also been determined that sérum MBL and/or MASP-2 are elevated in certain pédiatrie cancers, including acute lymphoblastic leukaemia (ALL), non-Hodgkin 20 lymphoma, CNS-tumors, and solid tumors outside the CNS (Fisch et al., 2011, Swiss Med Wkly 141 :w 13191). It has also been determined that MASP-2 is overexpressed in esophageal squamous cell carcinoma (ESCC) and dysplasia (premalignant) tissue samples (Verma et al., Int J Cancer 118:2930, 2006).

In addition to the above-mentioned studies, numerous studîes hâve reported an 25 association of MBL polymorphisms and cancer. For example, as summarized in Swîerzko et al., Mol Immunol 55:16, 2013, an association of MBL and MBL2 gene polymorphisms hâve been reported for gastric cancer (Baccarelli et al, International J Cancer 119:1970-1975, 2006; Scudiero et al., Clin Chem 52:1625-1626, 2006; Wang et al., Digestive Diseases and Sciences 53:2904-2908, 2008); hepatic cancer (Eurich et al., 30 Liver International 31:1006-1012, 2011); pancreatic cancer (Rong et al., BMC Gastroenterology 10:68, 2010); colon/colorectal cancer (Ytting et al., Scan J Gastroenterology 39:670-674, 2004; Ytting et aL, Scan J Gastroenterology 73:122-127,

2011; Zanetti et al., Cancer Res 72:1467-1677, 2012); ovarian cancer (Swieizko et al., Immunotherapy 56:959-971, 2007); Nevadunsky et al., European J of Obstetrics and Gynecology and Reproductive Biology 163:216-218, 2012); breast cancer (Bemig et al., Carcinogenesis 28:828-836, 2007); lung cancer (Pine et al., Journal of NCI 99:14015 1409, 2007; Olivo-Marston et al., Cancer Epidemiology, Biomarkers and Prévention

3375-3383, 2009); and acute lymphoblastic leukaemia (Schmiegelow et al., Blood 100:3757-3760, 2002).

It has also been determined that complément components are upregulated in human cancer patient biofluids, as shown below in TABLE L

TABLE 1 : Complément Components Upregulated in Human Cancer Patient Biofluids

lADLL· l.VUiHpivii Complément Cnmponent	Cancer 1	Biospecimen	Référencé
C3a/C3a(desArg)	Breast	Sérum	Fan et al., J Can Res Clin Oncol 136:1243,2010; Solassol et al., Oncogene 29:550, 2010; Li et al., Clin Chem 51:2229, 2005
C3a/C3a(desArg)	HCV-related Hepatocellular Carcinoma	Sérum	Kanmura et al., J Gastroenterol 45:459, 2010; Lee et al., Proteomics 6:2865, 2006
C3a/C3a(desArg)	Colorectal	Sérum	Fenz et al., Proteomics Clin Appi 1:536. 2007; Habermann et al., Gastroenterol 131:1020,2006
C3a	Chronic Lymphocytic leukemia (CLL)	Sérum	Miguet et al., J Proteome Res 5:2258, 2006;
C4a	CLL	Sérum	Miguet et al-, J Proteome Res 5:2258, 2006;
C3a	Ovarian	Ascites vs. sérum	Bjorge et al., Br J Cancer 92(5):895905,2005
C5b-9	Ovarian	Ascites vs. sérum	Bjorge et al., Br J Cancer 92(5):895905, 2005
C5a	Non-small cell lung cancer (NSCLC)	Sérum	Corrales et al., J Immunol 189:4674, 2012
Cl inhibitor, CD59, CD46 Factor H	Ovarian	Ascites vs. sérum	Bjorge et al., Br J Cancer 92(5):895905, 2005
Factor H	Acute myeloid leukemia	Sérum	Lee et al., Electrophoresis 33:1863, 2012
Factor H	Lung	Bronchoaveolar lavage (BAL),	Pio et al., Cancer Epidemiol Biomarkers P rev 19:2665,2010________

| sputum

In addition, complément activation may be a conséquence of chemotherapy or radiation therapy and thus inhibition of complément activation would be useful as an adjunct in the treatment of malignancies to reduce iatrogénie inflammation. When chemotherapy and radiation therapy preceded surgery, C5b-9 deposits were more intense and extended. The C5b-9 deposits were absent in ail the samples with benign lésions.

S-protein/vitronectin was présent as fibrillar deposits in the connective tissue matrix and as diffuse deposits around the tumor cells, less intense and extended than fibronectin. IgG, C3, and C4 deposits were présent only in carcinoma samples. The presence of C5b-9 deposits is indicative of complément activation and its subséquent pathogenetic effects in breast cancer (Niculescu, et al., Am. J. Pathol. 740:1039-1043, 1992).

In view of the data described in Example 16 that systemic administration of a MASP-2 antibody that specifîcally inhibits the lectin pathway of complément activation inhibits neovascularization at least as effectively as an anti-VEGF antibody, it is expected that systemic delivery of a MASP-2 inhibitory agent will be effective in inhibiting tumor angiogenesis, thereby reducing tumor growth and/or métastasés in a subject suffering from angiogenesis-dependent cancer.

Angiogenesis-dependent cancers include a cancer of épithélial origin or neuronal origin or a carcinoma or a solid tumor or a sarcoma or a liquîd tumor such as aleukemia or a lymphoma. Any cancer that is already known to be treated with, or in development to be treated with, an angiostatic compound (e.g., a VEGF antagonist) is encompassed within the scope of the methods of the invention. Preferred cancers in this context include; colorectal, breast (including metastatic breast cancer, inflammatory breast carcinoma), lung, rénal, hepatic, esophageal, ovarian, pancreatic, prostate and gastric cancers, as well as glioma, gastrointestinal stromal tumors, lymphoma, melanoma and carcinoid tumors (NCI clinical trials database; found at www cancer_gov_clînicaltrials/search, accessed on 3/25/2014). Many of these cancers hâve been shown to be responsive to treatment with bevacizumab (Avastin®), a humanized monoclonal antibody that blocks the binding of VEGF to its receptors and inhibits tumor angiogenesis (e.g., Amit et al., PLoS One 8(l):e51780 (2013).

In accordance with the foregoîng, in another aspect of the invention, methods are provided for inhibiting tumor angiogenesis and/or tumor métastasés in a subject suffering from an angiogenesis-dependent cancer. This method includes administering a composition comprising an amount of a MASP-2 inhibitor effective to inhibit tumor angiogenesis and/or tumor métastasés to a subject suffering from an angiogenesisdependent cancer. In some embodiments, the subject is suffering from an angiogenesisdependent cancer selected from the group consisting of colorectal, breast, lung, rénal, hepatic, esophageal, ovarian, pancreatic, prostate and gastric cancers, as well as glioma, gastrointestinal stromal tumors, lymphoma, melanoma and carcînoid tumor. In some embodiments, the angiogenesis-dependent cancers are cancer types that are expected to benefit by treatment by an anti-VEGF agent, such as the anti-VEGF antibody Avastin® (bevacizumab, Genentech, CA), such as, for example, any cancer that is already known to be treated with, or in development to be treated with, an angiostatic compound (e.g., a VEGF antagonist), including advanced cancers metastatitic to liver, melanoma, ovarian cancer, neuroblastoma, pancreatic cancer, hepatocellular carcinoma, endométrial cancer, prostate cancer, angiosarcoma, metastatic or unresectable angiosarcoma, relapsed ovarian sex-cord stromal tumours, esophageal cancer, gastric cancer, non-Hodgkin’s lymphoma, Hodgkin lymphoma, diffuse large B-cell lymphoma, récurrent or metastatic head and neck cancer, neoplastic meningitis, cervical cancer, uterine cancer, advanced peritoneal carcinomatosis, gliosarcoma, neuroendocrine carcinoma, extracranial Ewing sarcoma, acute myeloid leukemia, chronic myelogenous leukemia, întracranial meningioma, advanced Kaposi’s sarcoma, mesothelioma, biliary tract cancer, metastatic carcînoid tumors, and advanced urinary tract cancer. Preferred cancers in this context include: colorectal, breast (including metastatic breast cancer, inflammatory breast carcinoma), lung, rénal, hepatic, esophageal, ovarian, pancreatic, prostate and gastric cancers, as well as glioma, gastrointestinal stromal tumors, lymphoma, melanoma and carcînoid tumors.

The MASP-2 inhibitory composition may be administered locally to the région of tumor(s), such as by local application of the composition during surgery or local injection, either directly or remotely, for example, by cathéter. Altemately, the MASP-2 inhibitory agent may be administered to the subject systemically, such as by intra-arterial, intravenous, intramuscular, inhalational, nasal, subcutaneous or other parentéral administration, or potentially by oral administration for non-peptidergic agents. The

MASP-2 inhibitory agent composition may be combined with one or more additional therapeutic agents, such as an additional anti-angiogenic agent and/or an additional chemotherapeutic agent. Administration may be repeated as determined by a physician untii the condition has been resolved or is controlled.

In view of the data in the présent study demonstrating that OMS646 is at least as effective as the anti-VEGF antibody at reducing CNV when delivered systemically to mice at ail dose levels tested, it is also expected that a MASP-2 inhibitory agent such as OMS646 will also be effective as an anti-angiogenesis agent for use in inhîbiting an angiogenesis-dependent condition such as myelofibrosis and hereditary hémorrhagie telangiesctasia.

IV. MASP-2 INHIBITORY AGENTS

In various aspects, the présent invention provides methods of inhîbiting the adverse effects of angiogenesis by administering a MASP-2 inhibitory agent to a subject in need thereof. MASP-2 inhibitory agents are administered in an amount effective to inhibit MASP-2-dependent complément activation in a lîving subject. In the practice of this aspect of the invention, représentative MASP-2 inhibitory agents include: molécules that inhibit the biological activity of MASP-2 (such as small molécule inhibîtors, anti-MASP-2 antibodies or blockîng peptides which interact with MASP-2 or interfère with a protein-protein interaction), and molécules that decrease the expression of MASP-2 (such as MASP-2 antisense nucleic acid molécules, MASP-2 spécifie RNAi molécules and MASP-2 ribozymes), thereby preventing MASP-2 from activating the lectin complément pathway. The MASP-2 inhibitory agents can be used alone as a primary therapy or in combination wîth other therapeutics as an adjuvant therapy to enhance the therapeutic benefits of other medical treatments.

The inhibition of MASP-2-dependent complément activation is characterized by at least one of the followîng changes in a component of the complément system that occurs as a resuit of administration of a MASP-2 inhibitory agent in accordance with the methods of the invention: the inhibition of the génération or production of MASP-2-dependent complément activation system products C4b, C3a, C5a and/or C5b-9 (MAC) (measured, for example, as described in Example 2), the réduction of C4 cleavage and C4b déposition (measured, for example as described in Example 2), or the réduction of C3 cleavage and C3b déposition (measured, for example, as described in Example 2).

According to the présent invention, MASP-2 inhibitory agents are utilized that are effective in inhibiting angiogenesis and exhibit a détectable anti-angiogenesîs activity and/or induce a decrease of neo-angiogenesis. Within the context of the invention, an anti-angiogenic activity may comprise at least one or more of the following: réduction or decrease of neo-angiogenesis, normalization of vessels, and/or réduction in the number of vessels în a pathogenic area.

Neo-angiogenesis and assessment of an anti-angiogenic agent, such as a MASP-2 inhibitory agent, may be detected using any technique known to the skilled person. For example, neo-angiogenesis and assessment of an anti-angiogenic agent may be assessed in a laser-înduced injury model of CNV în animais (as described in Examples 12, 14 and 16 herein), or in situ in a patient or in a tumor by non-invasive techniques such as PET (Positron Emission Tomography), MRI (Magnetic Résonance Imaging), DCE-MRI (Dynamic Contrast Enhanced, MRI) or CT (Computed Tomography) imaging. These techniques may be used to monitor tumor burden based on increased leakage of the vasculature in tumors. Using MRI or PET, one could follow the presence of angiogenesis markers such as, for example, a5p3-integrin, plasma VEGF or bFGF.

Altematively, neo-angiogenesis may be assessed using a tumor biopsy or section taken from a pathogenic area of a patient suffering from an angiogenesis-dependent condition and subséquent immune-histochemical analyses on endothélial cells to assess their activity and compare it to the activity of normal endothélial cells from a healthy subject or from endothélial cells from the patient but isolated at a different place in the body. Such immune-histochemical analyses may be done using pan-endothelial cell antibodies such as anti-CD3l and anti-CD34 to assess microvessel density. Tissue sections can be stained with markers for endothélial cells, combined with prolifération markers, to explore the ratio between tumor endothélial cells and tumor proliferating cells in the tissue. Examples of endothélial markers are CD3l and CD34. An example of a prolifération marker is Ki67, which is an excellent marker to détermine the growth fraction of a given cell population. The fraction of Ki-67-positive tumor cells (the Ki-67 labelling index) is often correlated with the clinical course of cancer. The microvessel density (MVD) may be assessed, for example, in a tumor section stained with an antiCD31 and using the intensity of the staining to quantity MVD. Quantification of MVD is preferably done by counting the positively stained luminal structures in four to five représentative images per tumor section. A decrease, preferably a statistically significant decrease, of the MVD assessed in at least four to five représentative images per tumor section is preferably seen as an indication that the molécule administered has an antiangiogenesis activity or is able to induce a decrease of neo-angiogenesis.

Neo-angîogenesis may also be assessed using cells, preferably endothélial cells from a tumor, a healthy subject, or endothélial cell lines. Endothélial cells from a tumor are preferably designated as tumor endothélium. Tumor endothélial cells may be isolated by FACS (Fluoresence Activated Cell Sortîng) of tumor tissue using CD3l as an endothélial marker. This could be carried out as described in van Beijnum et al., Nat Protoc. 3(6):1085-91, 2008. Preferred endothélial cell to assess neo-angiogenesis in vitro are HUVEC and RF24. The assessment of neo-angiogenesis activity în vitro may be carried out using a MTS (3-(4,5-dîmethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl-)-2H-tetrazolium) assay for the assessment of the proliferative activity of endothélial cells. Alternatively, other viability assays known to the skilled person may be used such as MTT (3-(4,5-Dîmethylthiazol-2-yl)-2,5-diphenyltetrazolium bromîde), Crystal Violet and WST-1 (Water Soluble Tétrazolium).

In addition, other types of angiogenesis activity assays could be used such as spheroid sprouting assay and matrigel tube formation assay. In the matrigel tube formation assay, cells, especially endothélial cells, are seeded on a synthetic semi-naturel gel matrix (such as Matrigel from BD Biosciences or collagen-gel, or in some cases fibrin gels). In both assays, endothélial cells, preferably HUVECs, are being used. After a certain period of time, depending on cell culture conditions, cells begin to form tube-like structures. The formation of tube-like structures is regarded as a first step towards the génération of new vessels. The read-out parameter is the number of vessel-knots per area unit. For the spheroid sprouting assay, cell spheroids (e.g., endothélial cells) are placed on a gel (e.g., matrigel and collagen gels). After a certain period of time sprout formation can be observed. The extent of sprouting is consîdered as a criterion for the évaluation of the angiogenic potential of cells. The read-out parameter is the number of sprouts per spheroid. An anti-angiogenic activity may be présent when the number of sprouts per spheroid is reduced or decreased in treated cells for a given period of time by comparison to the number of sprouts per spheroid in untreated cells A decrease or a réduction may be a decrease of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. An antiangiogenic activity in a tumor tissue may also be présent when a normalization of vessels is visualized and/or when the number of vessels in the pathogenic area is reduced.

In a preferred embodiment, as soon as the number of vessels in the pathogenic area is found to be decreased by comparison to the number of vessels at the onset of the treatment, there is a détectable anti-angiogenic activity. A decrease may be a détectable decrease in the number of vessels in the pathogenic area or a decrease of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%ofthe vessels in the pathogenic area. Pathogenic area is the area of the tumor încluding the surrounding tissue, located close to the tumor area. Close in this context may mean up to a few centimètres.

A normalization of vessels is preferably a change in the three-dimensional structure of a vessel or microvessel. For example, a pathological vessel or microvessel associated with neo-angiogenesis activity in a tumor endothélium may be less regular and/or may appear more tortuous and/or may appear more leaky than a control vessel or microvessel. A control vessel may be a vessel from a healthy individual or a vessel from the patient but not located in the pathogenic area from said patient. In a preferred embodiment, as soon as the three-dimensional structure of a vessel appears more regular, less tortuous and/or less leaky than a control vessel, an anti-angiogenic activity is said to hâve been detected. Preferably, less irregular, tortuous and/or leaky vessels are detected in the pathogenic area than at the onset of the treatment. More preferably, less means 5% less, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% less. Most preferably, no irregular, tortuous and/or leaky vessels are detected in the pathogenic area. A normalization of vessels and/or the number of vessels in the pathogenic area may be assessed using a non-invasive imaging technique such as PET, MRI or CT imaging.

In the case of an eye disease or condition associated with neo-angiogenesis, several assays hâve been developed for assessing a détectable anti-angiogenesis activity and/or a réduction or decrease of neo-angiogenesis induced by a drug to be tested, such as a MASP-2 inhibitory agent. In these different disease models, the angiogenesis can be triggered by different stimuli such as physical injury (laser induced rupture of Bruch's membrane) (Shen et al, 2006 Gene therapy 13: 225-234) or by the overexpression of spécifie blood vessel growth factors such as VEGF in transgenic mice (Miki et al, 2009, Ophthalmology 2009 September 116(9): 1748-1754). If a détectable anti-angiogenesis activity and/or a réduction or decrease of angiogenesis is assessed using a MASP-2 inhibitory agent, such MASP-2 inhibitory agent is said to be used as a médicament for preventing, treating, reverting, curing and/or delaying angiogenesis or a disease or a condition associated with angiogenesis.

The assessment of neo-angiogenesis and/or anti-angiogenic activity may be carried out periodically, e.g., each week or each month. The increase/decrease of neoangiogenesis and/or presence of an anti-angiogenic activity may therefore be assessed periodically, e.g., each week or month. This assessment is preferably carried out at several time points for a given subject or at one or several time points for a given subject and a healthy control. The assessment may be carried out at regular time Întervals, e.g. each week, or each month. When one assessment of neo-angiogenesis or angiogenic activity related to a MASP-2 inhibitory agent has led to the finding of a decrease of neoangiogenesis or to the presence of an anti-angiogenic activity, a MASP-2 inhibitory agent, such as an anti-MASP-2 antibody, is said is exhibit a détectable anti-angiogenesis activity and/or inducing a réduction or decrease of neo-angiogenesis.

A détectable decrease of neo-angiogenesis activity and/or the presence of an antiangiogenic activity has been preferably detected when, for at least one time point, a decrease of neo-angiogenesis and/or the presence of an anti-angiogenic activity has been detected. Preferably, a decrease of neo-angiogenesis and/or the presence of an antiangiogenic activity has been detected for at least two, three, four, five time points.

MASP-2 inhibitory agents useful in the practice of this aspect of the invention include, for example, MASP-2 antibodies and fragments thereof, MASP-2 inhibitory peptides, small molécules, MASP-2 soluble receptors and expression inhibitors. MASP-2 inhibitory agents may inhibit the MASP-2-dependent complément activation System by blocking the biological function of MASP-2. For example, an inhibitory agent may effectively block MASP-2 protein-to-protein interactions, interfère with MASP-2 dimerization or assembly, block Ca2⁺ binding, interfère with the MASP-2 serine protease active site, or may reduce MASP-2 protein expression.

In some embodiments, the MASP-2 inhibitory agents selectively inhibit MASP-2 complément activation, leaving the Clq-dependent complément activation system functionally intact.

In one embodiment, a MASP-2 inhibitory agent useful in the methods of the invention is a spécifie MASP-2 inhibitory agent that specifically bînds to a polypeptide comprising SEQ ID NO:6 with an affinïty of at least ten times greater than to other antigens in the complément System. In another embodimenζ a MASP-2 inhibitory agent specîfically binds to a polypeptide comprising SEQ ID NO:6 with a binding affinity of at least 100 times greater than to other antigens in the complément System. In one embodiment, the MASP-2 inhibitory agent specîfically binds to at least one of (i) the CCPI-CCP2 domain (aa 300-431 of SEQ ID NO:6) or the serine protease domain of MASP-2 (aa 445-682 of SEQ ID NO:6) and inhibits MASP-2-dependent complément activation. In one embodiment, the MASP-2 inhibitory agent is a MASP-2 monoclonal antibody, or fragment thereof that specîfically binds to MASP-2. The binding affinity of the MASP-2 inhibitory agent can be determined using a suitable binding assay.

The MASP-2 polypeptide exhibits a molecular structure similar to MASP-1, MASP-3, and Cir and Cl s, the proteases ofthe Cl complément system. The cDNA molécule set forth in SEQ ID NO:4 encodes a représentative example of MASP-2 (consisting of the amino acid sequence set forth in SEQ ID NO:5) and provides the human MASP-2 polypeptide with a leader sequence (aa 1-15) that is cleaved after sécrétion, resulting in the mature form of human MASP-2 (SEQ ID NO:6). As shown in FIGURE 2, 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. An alternative splice results in a 20 kDa protein termed MBL-associated protein 19 (MApl9, also referred to as sMAP) (SEQ ID NO:2), encoded by (SEQ ID NO:1) arising from exons B, C, D and E as shown in FIGURE 2. The cDNA molécule set forth in SEQ ID NO:50 encodes the murine MASP-2 (consisting of the amino acid sequence set forth in SEQ ID NO:51) and provides the murine MASP-2 polypeptide with a leader sequence that is cleaved after sécrétion, resulting in the mature form of murine MASP-2 (SEQ ID NO:52). The cDNA molécule set forth in SEQ ID NO:53 encodes the rat MASP-2 (consisting of the amino acid sequence set forth in SEQ ID NO:54) and provides the rat MASP-2 polypeptide with a leader sequence that is cleaved after sécrétion, resulting in the mature form of rat MASP-2 (SEQ IDNO:55).

Those skilled in the art will recognize that the sequences disclosed in SEQ ID NO:4, SEQ DD NO:50 and SEQ ID NO:53 represent single alleles of human, murine and rat MASP-2 respectîvely, and that aile lie variation and alternative splicing are expected to occur. Allelic variants of the nucléotide sequences shown in SEQ ID NO:4, SEQ ID NO:50 and SEQ ID NO:53, including those containing silent mutations and those in which mutations resuit in amino acid sequence changes, are wîthin the scope of the présent invention. Allelic 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:6) are shown in FIGURE l and 2A and include an N-terminal Clr/Cls/sea urchin Vegf/bone morphogenic protein (CUBI) domain (aa 1-121 of SEQ ID NO:6), an epidermal growth factor-like domain (aa 122-166), a second CUBI domain (aa 167-293), as well as a tandem of complément control protein domains and a serine protease domain. Alternative splicing of the MASP 2 gene results in MApl9 shown in FIGURE 1. MApl9 is a nonenzymatic protein containing the N-termina! CUBI-EGF région of MASP-2 with four additional residues (EQSL) derîved from exon E as shown in FIGURE 1.

Several proteins hâve been shown to bind to, or interact with MASP-2 through protein-to-protein interactions. For example, MASP-2 is known to bind to, and form Ca~⁺ dépendent complexes with, the lectin proteins MBL, H-ficolin and L-ficolin. Each MASP-2/lectin complex has been shown to activate complément through the MASP-2-dependent cleavage of proteins C4 and C2 (Ikeda, K., et al., J. Biol. Chem. 262:7451-7454, 1987; Matsushita, M., étal., J. Exp. Med. / 76:1497-2284, 2000; Matsushita, M., et al., J. Immunol. 165:3502-3506, 2002). Studies hâve shown that the CUBI-EGF domains of MASP-2 are essential for the association of MASP-2 with MBL (Thielens, N.M., et al., J. Immunol. /66:5068, 2001). It has also been shown that the CUB1EGFCUBII domains médiate dimerization of MASP-2, which is required for formation of an active MBL complex (Wallis, R., et al., J. Biol. Chem. 275:30962-30969, 2000). Therefore, MASP-2 inhibitory agents can be identified that bind to or interféré with MASP-2 target régions known to be important for MASP-2-dependent complément activation.

ANTI-MASP-2 ANTIBODIES

In some embodiments of this aspect of the invention, the MASP-2 ïnhibitory agent comprises an anti-MASP-2 antibody that inhibits the MASP-2-dependent complément activation System. The anti-MASP-2 antibodies usefiil in this aspect of the invention include polyclonal, monoclonal or recombinant antibodies derived from any antibody producing mammal and may be multispecific, chîmeric, humanized, anti-idiotype, and antibody fragments. Antibody fragments include Fab, Fab’, F(ab)2, Ffab^, Fv fragments, scFv fragments and single-chain antibodies as further described herein.

MASP-2 antibodies can be screened for the ability to inhibit MASP-2-dependent complément activation System and for anti-angiogenic activity using the assays described herein. Several MASP-2 antibodies hâve been described in the literature and some hâve been newly generated, some of which are listed below in TABLE 2. For example, as described in Examples 10 and il herein, anti-rat MASP-2 Fab2 antibodies hâve been identified that block MASP-2-dependent complément activation, and as shown in Example 14, a monoclonal antibody derived from the anti-rat MASP-2 Fab2 antibody has anti-angiogenic activity in the mouse mode! of laser-induced CNV. As further described in Exampie 15, and as further described in US2012/0282263 which is hereby încorporated herein by référencé, fûlly human MASP-2 scFv antibodies hâve been identified that block MASP-2-dependent complément activation, and as described in Example 16, a représentative human MASP-2 monoclonal antibody (OMS646) that blocks the function of the lectin pathway has anti-angiogenic activity in the mouse model of laser-induced CNV. Accordingly, in one embodiment, the MASP-2 ïnhibitory agent for use in the methods of the invention comprises a human antibody such as, for example OMS646. Accordingly, in one embodiment, a MASP-2 ïnhibitory agent for use in the compositions and methods of the claîmed invention comprises a human antibody that binds a polypeptide consisting of human MASP-2 (SEQ ID NO:6), wherein the antibody comprises: I) a) a heavy chain variable région comprising: i) a heavy chain CDRl comprising the amino acid sequence from 31-35 of SEQ ID NO: 67 or SEQ ID NO:68; and iî) a heavy chain CDR2 comprising the amino acid sequence from 50-65 of SEQ ID

NO: 67 or SEQ ID NO:68; and iii) a heavy chain CDR3 comprising the amino acid sequence from 95-102 of SEQ ID NO:67 or SEQ ID NO:68; and

b) a light chain variable région comprising: i) a light chain CDRl comprising the amino acid sequence from 24-34 of either SEQ ID NO:69 or SEQ ID NO:7l; and ii) a 5 light chain CDR2 comprising the amino acid sequence from 50-56 of either SEQ ID NO:69 or SEQ ID NO:7l; and iii) a light chain CDR3 comprising the amino acid sequence from 89-97 of either SEQ ID NO:69 or SEQ ID NO:7l; or II) a variant thereof that is otherwise identical to said variable domains, except for up to a combîned total of 6 amino acid substitutions within said CDR régions of said heavy-chain variable région and 10 up to a combîned total of 6 amino acid substitutions within said CDR régions of said light-chain variable région, wherein the antibody or variant thereof inhibits MASP-2dependent complément activation. In one embodiment, the MASP-2 inhibitory agent for use in the methods of the invention comprises the human antibody OMS646.

TABLE 2: EXEMPLARY MASP-2 SPECIFIC ANTIBODIES__________________

ANTIGEN	ANTIBODY TYPE	REFERENCE
Recombinant MASP-2	Rat Polyclonal	Peterson, S.V., et al., Mol. Immunol. 37:803-811, 2000
Recombinant human CCPl/2-SP fragment (MoAb 8B5)	Rat MoAb (subclass IgGl)	Moller-Kristensen, M., et al., J. of Immunol. Methods 282:159-167, 2003
Recombinant human MApi9(MoAb 6G12) (cross reacts with MASP-2)	Rat MoAb (subclass IgGl)	Moller-Kristensen, M., et al., J. of Immunol. Methods 282:159-167, 2003
hMASP-2	Mouse MoAb (S/P) Mouse MoAb (N-term)	Peterson, S.V., et al., Mol. Immunol. 35:409, April 1998
hMASP-2 (CCP1-CCP2-SP domain	rat MoAb: NimoablOl, produced by hybridoma cell line 03050904 (ECACC)	WO 2004/106384

ANTIGEN	ANTIBODY TYPE	REFERENCE
hMASP-2 (foll length-his tagged)	murine MoAbs: NimoAbl04, produced by hybridoma cell line M0545YM035 (DSMZ) NimoAbl08, produced by hybridoma cell line M0545YM029 (DSMZ) NimoAbl09 produced by hybridoma cell line M0545YM046 (DSMZ) NimoAbl 10 produced by hybridoma cell line M0545YM048 (DSMZ)	WO 2004/106384
Rat MASP-2 (fulllength)	MASP-2 Fab2 antibody fragments	Example 10
hMASP-2 (folllength)	Fully human scFv clones	Example 15 and US2012/0282263

ANTI-MASP-2 ANTIBODIES WITH REDUCED EFFECTOR FUNCTION

In some embodiments of this aspect of the invention, the anti-MASP-2 antibodies hâve reduced effector fonction in order to reduce inflammation that may anse from the 5 activation of the classical complément pathway. The ability of IgG molécules to trigger the classical complément pathway has been shown to résidé within the Fc portion of the molécule (Duncan, A.R., et al., Nature 332-.13^-1 Ad 1988). IgG molécules in which the Fc portion of the molécule has been removed by enzymatic cleavage are devoid of this effector fonction (see Harlow, Antibodies: A Laboratory Manual, Cold Spring Harbor 10 Laboratory, New York, 1988). Accordingly, antibodies with reduced effector fonction can be generated as the resuit of lacking the Fc portion of the molécule by having a genetically engineered Fc sequence that minimizes effector fonction, or being of either the human IgG2 or IgG 4 isotype.

Antibodies with reduced effector fonction can be produced by standard molecular 15 biological manipulation of the Fc portion of the IgG heavy chains as described in Example 9 herein and also described in Jolliffe étal., Int'l Rev. Immunol. 70:241-250, 1993, and Rodrigues étal., J. Immunol. 757:6954-6961, 1998. Antibodies with reduced effector fonction also include human IgG2 and IgG4 isotypes that hâve a reduced ability to activate complément and/or interact with Fc receptors (Ravetch, J.V., étal., Annu. Rev. Immunol. 9:457-492, 1991 ; Isaacs, J.D., étal., J. Immunol. 745:3062-3071, 1992; van de Winkel, J.G., étal., Immunol. Today 14:2}5-221, 1993). Humanized or folly human antibodies spécifie to human MASP-2 comprised of IgG2 or lgG4 isotypes can be produced by one of several methods known to one of ordînary skilled in the art, as described in Vaughan, T.J., et al., Nature Biotechnical /6:535-539, 1998.

PRODUCTION OF ANTI-MASP-2 ANTIBODIES

Anti-MASP-2 antibodies can be produced using MASP-2 polypeptides (e.g., full length MASP-2) or using antigenic MASP-2 epitope-bearing peptides (e.g., a portion of the MASP-2 polypeptide). Immunogenic peptides may be as small as five amino acid residues. For example, the MASP-2 polypeptide including the entire amino acid sequence of SEQ ID NO:6 may be used to induce anti-MASP-2 antibodies usefol in the method of the invention. Particular MASP-2 domains known to be învolved in protein-protein interactions, such as the CUB1, and CUBIEGF domains, as well as the région encompassing the serine-protease active site, may be expressed as recombinant polypeptides as described in Example 3 and used as antigens. In addition, peptides comprising a portion of at least 6 amino acids of the MASP-2 polypeptide (SEQ ID N 0:6) are also usefol to induce MASP-2 antibodies. Additional examples of MASP-2 derived antigens usefol to induce MASP-2 antibodies are provided below in TABLE 2. The MASP-2 peptides and polypeptides used to raise antibodies may be isolated as naturel polypeptides, or recombinant or synthetic peptides and catalytically inactive recombinant polypeptides, such as MASP-2A, as forther described in Examples 5-7. In some embodiments of this aspect of the invention, anti-MASP-2 antibodies are obtained using a transgenic mouse strain as described in Examples 8 and 9 and forther described below.

Antigens usefol for producing anti-MASP-2 antibodies also include fosion polypeptides, such as fusions of MASP-2 or a portion thereof with an immunoglobulin polypeptide or with maltose-binding protein. The polypeptide immunogen may be a full-length molécule or a portion thereof. If the polypeptide portion is hapten-like, such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine sérum albumîn (BSA) or tetanus toxoîd) for immunization.

TABLE 3: MASP-2 DERIVED ANTIGENS

SEQ ID NO:	Ami no Acid Sequence
SEQ IDN0:6	Human MASP-2 protein
SEQ IDN0:5l	Murine MASP-2 protein
SEQlDNO:8	CUBI domain of human MASP-2 (aa 1-121 ofSEQIDNO:6)
SEQ IDN0:9	CUBIEGF domains of human MASP-2 (aa 1-166 of SEQ ID NO:6)__
SEQ IDNOJO	CUBIEGFCUBII domains of human MASP-2 (aa l -293 of SEQ ID NO:6)
SEQ IDNO:ll	EGF domain of human MASP-2 (aa 122-166 of SEQ ID NO:6)
SEQ ID NO:l2	Serine-Protease domain of human MASP-2 (aa 429-671 of SEQ ID NO:6) ______________
SEQ IDNO:l3 GKDSCRGDAGGALVFL	Serine-Protease înactivated mutant form (aa 610-625 of SEQ ID NO:6 with mutated Ser 618)
SEQ IDNO:l4 TPLGPKWPEPVFGRL	Human CUBI peptide
SEQ ID NO:l5: TAPPGYRLRLYFTHFDLEL SHLCEYDFVKLSSGAKVL ATLCGQ	Human CUBI peptide
SEQ IDN0:l6: TFRSDYSN	MBL binding région in human CUBI domain
SEQ IDNO:l7: FYSLGSSLDITFRSDYSNEK PFTGF	MBL binding région in human CUBI domain
SEQ IDNO:18 IDECQVAPG	EGF peptide
SEQ ID NO:l9 ANMLCAGLESGGKDSCRG DSGGALV	Peptide from serine-protease active site

POLYCLONAL ANTIBODIES

Polyclonal antibodies against MASP-2 can be prepared by itnmunîzing an animal with MASP-2 polypeptide or an tmmunogenic portion thereof using methods well known to those of ordinary skill in the art. See, for example, Green étal., Production of Polyclonal Antisera, in Immunochemical Protocols (Manson, ed.), page 105. The immunogenicity of a MASP-2 polypeptide can be increased through the use of an adjuvant, including minerai gels, such as aluminum hydroxide or Freund's adjuvant (complété or incomplète), surface active substances such as lysolecithin, pluronic polyols, polyanions, oil émulsions, keyhole limpet hemocyanin and dinîtrophenol. Polyclonal antibodies are typically raised in animais such as horses, cows, dogs, chicken, rats, mice, rabbits, guinea pigs, goats, or sheep. Altematively, an anti-MASP-2 antibody useful in the présent invention may also be derîved from a subhuman primate. General techniques for raising diagnostically and therapeutically useful antibodies in baboons may be found, for example, in Goldenberg et al., International Patent Publication No. WO 9l/l 1465, and in Losman, M.J., étal., Int. J. Cancer -/6:310, 1990. Sera containing immunologically active antibodies are then produced from the blood of such immunized animais using standard procedures well known in the art.

MONOCLONAL ANTIBODIES

In some embodiments, the MASP-2 inhibitory agent is an anti-MASP-2 monoclonal antibody. Anti-MASP-2 monoclonal antibodies are highly spécifie, being directed against a single MASP-2 epitope. As used herein, the modifier monoclonal indicates the character of the antibody as being obtained from a substantially homogenous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. Monoclonal antibodies can be obtained using any technique that provides for the production of antibody molécules by continuons cell fines în culture, such as the hybridoma method described by Kohler, G., et al., Nature 256:495, 1975, or they may be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567 to Cabilly). Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson, T., et al., Nature 352:624-628, 1991, and Marks, J.D., étal., J. Mol. Biol. 222:581-597, 1991. Such antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.

For example, monoclonal antibodies can be obtained by injecting a suitable mammal (e.g., a BALB/c mouse) with a composition comprising a MASP-2 polypeptide or portion thereof. After a predetermined period of time, splénocytes are removed from the mouse and suspended in a cell culture medium. The splénocytes are then fused with an immortal cell line to form a hybridoma. The formed hybridomas are grown in cell culture and screened for their ability to produce a monoclonal antibody against MASP-2. Examples further describing the production of anti-MASP-2 monoclonal antibodies are provided herein (e.g., Examples 10 and 13). (See also Current Protocols in Immunology, Vol. I., John Wiley & Sons, pages 2.5.1-2.6.7, 1991.)

Human monoclonal antibodies may be obtained through the use of transgenic mice that hâve been engineered to produce spécifie human antibodies in response to antigenîc challenge. In this technique, éléments of the human immunoglobulin heavy and light chain locus are întroduced into strains of mice derived from embryonic stem cell lines that contain targeted dîsruptions of the endogenous immunoglobulin heavy chain and light chain loci. The transgenic mice can synthesize human antibodies spécifie for human antigens, such as the MASP-2 antigens described herein, and the mice can be used to produce human MASP-2 antibody-secreting hybridomas by fusing B-cells from such animais to suitable myeloma cell fines using conventional Kohler-M liste in technology as further described in Example 7. Transgenic mice with a human immunoglobulin genome are commercially available (e.g., from Abgenix, Inc., Fremont, CA, and Medarex, Inc., Annandale, N.J.). Methods for obtaining human antibodies from transgenic mice are described, for example, by Green, L.L., et al., Nature Genet. 7:13, 1994; Lonberg, N., et al., Nature 365:856, 1994; and Taylor, L.D., et al., Int. Immun. 6:579, 1994.

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

Once produced, polyclonal, monoclonal or phage-derived antibodies are first tested for spécifie MASP-2 binding. A variety of assays known to those skilled in the art may be utilized to detect antibodies which specifically bind to MASP-2. Exemplary assays include Western blot or immunoprécipitation analysis by standard methods (e.g., as described in Ausubel étal.), immunoelectrophoresis, enzyme-linked immuno-sorbent assays, dot blots, inhibition or compétition assays and sandwich assays (as described in Harlow and Land, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988). Once antibodies are identified that specîfically bind to MASP-2, the anti-MASP-2 antibodies are tested for the ability to function as a MASP-2 inhibitory agent in one of several assays such as, for example, a lectin-specifîc C4 cleavage assay (described in Example 2), a C3b déposition assay (described in Example 2) or a C4b déposition assay (described in Example 2).

The affmity of anti-MASP-2 monoclonal antibodies can be readily determined by one of ordinary skill in the art (see, e.g., Scatchard, A., NY Acad. Sci. 57:660-672, 1949). In one embodiment, the anti-MASP-2 monoclonal antibodies usefui for the methods of the invention bînd to MASP-2 with a binding affmity of <100 nM, preferably <10 nM and most preferably <2 nM.

CHIMERIC/HUMANIZED ANTIBODIES

Monoclonal antibodies usefui in the method of the invention include chimeric antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies (LJ.S. Patent No. 4,816,567, to Cabilly; and Morrison, S.L., étal., Proc. Nat'lAcad. Sci. USA 57:6851-6855, 1984).

One form of a chimeric antibody usefui in the invention is a humanized monoclonal anti-MASP-2 antibody. Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies, which contain minimal sequence derived from non-human immunoglobulin. Humanized monoclonal antibodies are produced by transferring the non-human (e.g., tnouse) complementarity determining régions (CDR), from the heavy and light variable chains of the mouse immunoglobulin into a human variable domain. Typically, residues of human antibodies are then substituted in the framework régions of the non-human counterparts. Furthermore, humanized antibodies may comprise residues that are not found in the récipient antibody or in the donor antibody. These modifications are made to further refîne antibody performance. In general, the humanîzed antibody will comprise substantially ail of at least one, and typically two variable domains, in which ail or substantially ail of the hypervariable loops correspond to those of a non-human immunoglobulîn and ail or substantially ail of the Fv framework régions are those of a human immunoglobulîn sequence. The humanîzed antibody optionally also will comprise at least a portion of an immunoglobulîn constant région (Fc), typically that of a human immunoglobulîn. For further details, see Jones, P.T., et al., Nature 321:522-525, 1986; Reichmann, L., et al., Nature 332:323-329, 1988; and Presta, Curr. Op. Struct. Biol. 2:593-596, 1992.

The humanîzed antibodies useful in the invention include human monoclonal antibodies including at least a MASP-2 binding CDR3 région. In addition, the Fc portions may be replaced so as to produce IgA or IgM as well as human IgG antibodies. Such humanîzed antibodies will hâve particular clinical utîlity because they will specifically recognize human MASP-2 but will not evoke an immune response in humans against the antibody itself. Consequently, they are better suited for in vivo administration in humans, especially when repeated or long-term administration is necessary.

An example of the génération of a humanîzed anti-MASP-2 antibody from a murine anti-MASP-2 monoclonal antibody is provided herein in Example 6. Techniques for producing humanîzed monoclonal antibodies are also described, for example, by Jones, P.T., étal., Nature 321:522, 1986; Carter, P., étal., Proc. Nat'l. Acad. Sci. USA 59:4285, 1992; Sandhu, J.S., Crit. Rev. Biotech. 12:431, 1992; Singer, I.I., étal., J. Immun. /50:2844, 1993; Sudhir (ed.), Antibody Engineering Protocols, Humana Press, Inc., 1995; Kelley, Engineering Therapeutic Antibodies, in Protein Engineering: Principles and Practice, Cleland et al. (eds.), John Wiley & Sons, Inc., pages 399-434, 1996; and by U.S. Patent No. 5,693,762, to Queen, 1997. In addition, there are commercial entities that will synthesize humanîzed antibodies from spécifie murine antibody régions, such as Protein Design Labs (Mountain View, CA).

RECOMBINANT ANTIBODIES

Anti-MASP-2 antibodies can also be made using recombinant methods. For example, human antibodies can be made using human immunoglobulîn expression libraries (available for example, from Stratagene, Corp., La Jolla, CA) to produce fragments of human antibodies (Vpp Vl, Fv, Fd, Fab or F(ab')2). These fragments are then used to construct whole human antibodies using techniques similar to those for producing chimeric antibodies.

ANTI-ÏDIOTYPE ANTIBODIES

Once anti-MASP-2 antibodies are identifîed with the desired inhibitory activity, these antibodies can be used to generate anti-idiotype antibodies that resemble a portion of MASP-2 using techniques that are well known in the art. See, e.g., Greenspan, N.S., étal., FASEBJ. 7:437, 1993. For example, antibodies that bind to MASP-2 and competitîvely inhibit a MASP-2 protein interaction required for complément activation can be used to generate anti-idiotypes that resemble the MBL binding site on MASP-2 protein and therefore bind and neutralize a binding ligand of MASP-2 such as, for example, MBL.

IMMUNOGLOBUL1N FRAGMENTS

The MASP-2 inhibitory agents usefiii in the method of the invention encompass not only intact immunoglobulin molécules but also the well known fragments încluding Fab, Fab’, F(ab)2, F(ab^r)2 and Fv fragments, scFv fragments, diabodies, linear antibodies, single-chain antibody molécules and multispecific antibodies formed from antibody fragments.

It is well known în the art that only a small portion of an antibody molécule, the paratope, is involved in the binding of the antibody to its epitope (see, e.g., Clark, W.R., The Experimental Foundations of Modem Immunology, Wiley & Sons, Inc., NY, 1986). The pFc' and Fc régions of the antibody are effectors of the classical complément pathway, but are not involved in antigen binding. An antibody from which the pFc' région has been enzymatically cleaved, or which has been produced without the pFc' région, is designated an F(ab')2 fragment and retains both ofthe antigen binding sites of an intact antibody. An isolated F(ab')2 fragment is referred to as a bivalent monoclonal fragment because of its two antigen binding sites. Similarly, an antibody from which the Fc région has been enzymatically cleaved, or which has been produced without the Fc région, is designated a Fab fragment, and retains one of the antigen binding sites of an intact antibody molécule.

Antibody fragments can be obtained by proteolytic hydrolysis, such as by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab’)2- This fragment can be further cleaved using a thiol reducing agent to produce 3.5S Fab' monovalent fragments. Optionally, the cleavage reaction can be performed using a blocking group for the sulfhydryl groups that resuit from cleavage of disulfide linkages. As an alternative, an enzymatic cleavage using pepsin produces two monovalent Fab fragments and an Fc fragment directly. These methods are described, for example, U.S. Patent No. 4,331,647 to Goldenberg; Nisonoff, A., et al., Arch. Biochem. Biophys. 89:230, 1960; Porter, R.R., Biochem. J. 73:119, 1959; Edelman, et al., in Methods in Enzymology 1:422, Academie Press, 1967; and by Coligan at pages 2.8.1-2.8.10 and 2.10.-2.10.4.

In some embodiments, the use of antibody fragments lacking the Fc région are preferred to avoid activation ofthe classical complément pathway which is initiated upon binding Fc to the Fcy receptor. There are several methods by which one can produce a MoAb that avoids Fcy receptor interactions. For example, the Fc région of a monoclonat antibody can be removed chemically using partial digestion by proteolytic enzymes (such as ficîn digestion), thereby generating, for example, antigen-binding antibody fragments such as Fab or F(ab)2 fragments (Marîani, M., étal., Mol. Immunol. 28:69-71, 1991). Altematively, the human γ4 IgG isotype, which does not bind Fcy receptors, can be used during construction of a humanîzed antibody as described herein. Antibodies, single chain antibodies and antigen-binding domains that lack the Fc domain can also be engineered using recombinant techniques described herein.

SINGLE-CHAIN ANTIBODY FRAGMENTS

Altematively, one can create single peptide chain binding molécules spécifie for MASP-2 in which the heavy and light chain Fv régions are connected. The Fv fragments may be connected by a peptide linker to form a single-chain antigen binding protein (scFv). These single-chain antigen binding proteins are prepared by constructing a structural gene comprising DNA sequences encodîng the Vjq and Vl domains which are connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell, such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing scFvs are described for example, by

Whitlow, étal., Methods: A Companion to Methods in Enzymology 2:97, 1991; Bird, étal., Science 242:423, 1988; U.S. Patent No. 4,946,778, to Ladner; Pack, P., étal., BiofPechnology 77:1271, 1993.

As an illustrative example, a MASP-2 spécifie scFv can be obtained by exposing lymphocytes to MASP-2 polypeptide in vitro and selecting antibody display libraries in phage or similar vectors (for example, through the use of immobilized or labeled MASP-2 protein or peptide). Genes encoding polypeptides having potential MASP-2 polypeptide binding domains can be obtained by screening random peptide libraries displayed on phage or on bacteria such as E. coli. These random peptide display libraries can be used to screen for peptides which interact with MASP-2. Techniques for creating and screening such random peptide display libraries are well known in the art (U.S. Patent No. 5,223,409, to Lardner; U.S. Patent No. 4,946,778, to Ladner; U.S. Patent No. 5,403,484, to Lardner; U.S. Patent No. 5,571,698, to Lardner; and Kay étal., Phage Display of Peptides and Proteins Academie Press, Inc., 1996) and random peptide display libraries and kits for screening such libraries are available commercially, for instance from CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Invitrogen Inc. (San Diego, Calif.), New England Bîolabs, Inc. (Beverly, Mass.), and Pharmacia LKB Biotechnology Inc. (Piscataway, N.J.).

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

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

PEPTIDE INHIBITORS

In some embodiments of this aspect of the invention, the MASP-2 inhibitory agent comprises isolated MASP-2 peptide inhibitors, including isolated naturel peptide inhibitors and synthetic peptide inhibitors that inhibit the MASP-2-dependent complément activation System. As used herein, the term isolated MASP-2 peptide inhibitors refers to peptides that inhibit MASP-2 dépendent complément activation by binding to, competing with MASP-2 for binding to another récognition molécule (e.g., MBL, H-ficolin, M-ficolin, or L-ficolin) in the lectin pathway, and/or directly interacting with MASP-2 to inhibit MASP-2-dependent complément activation that are substantially pure and are essentially ffee of other substances with which they may be found in nature to an extent practical and appropriate for their intended use.

Peptide inhibitors hâve been used successfully in vivo to interfère with protein-proteîn interactions and catalytic sites. For example, peptide inhibitors to adhesion molécules structurally related to LFA-l hâve recently been approved for clinical use in coagulopathies (Ohman, E.M., et al., European Heart J. 76:50-55, 1995). Short linear peptides (<30 amino acids) hâve been described that prevent or interféré with întegrin-dependent adhesion (Murayama, O., étal., J. Biochem. /20:445-51, 1996). Longer peptides, ranging in length from 25 to 200 amino acid residues, hâve also been used successfully to block întegrin-dependent adhesion (Zhang, L., et al., J. Biol. Chem. 277(47):29953-57, 1996). In general, longer peptide inhibitors hâve higher affinltles and/or slower off-rates than short peptides and may therefore be more potent inhibitors. Cyclic peptide inhibitors hâve also been shown to be effective inhibitors of integrins in vivo for the treatment of human inflammatory disease (Jackson, D.Y., et aL,

J. Med. Chem. 40:3359-68, 1997). One method of producing cyclic peptides involves the synthesis of peptides In which the terminal amino acids of the peptide are cysteines, thereby allowing the peptide to exist in a cyclic form by disulfide bonding between the terminal amino acids, which has been shown to improve affinity and half-life in vivo for the treatment of hematopoietic neoplasms (e.g., U.S. Patent No. 6,649,592, to Larson).

SYNTHETIC MASP-2 PEPTIDE INHIBITORS

MASP-2 inhibitory peptides useful in the methods of this aspect of the invention are exemplified by amino acid sequences that mimic the target régions important for MASP-2 function. The inhibitory peptides useful in the practice of the methods of the invention range in size from about 5 amino acids to about 300 amino acids. TABLE 4 provides a list of exemplary inhibitory peptides that may be useful in the practice of this aspect of the présent invention. A candidate MASP-2 inhibitory peptide may be tested for the ability to function as a MASP-2 inhibitory agent in one of several assays including, for example, a lectin spécifie C4 cleavage assay (described in Example 2), and a C3b déposition assay (described in Example 2).

In some embodiments, the MASP-2 inhibitory peptides are derived from MASP-2 polypeptides and are selected from the full length mature MASP-2 protein (SEQ ID NO:6), or from a particular domain of the MASP-2 protein such as, for example, the CUBI domain (SEQ ID NO:8), the CUBIEGF domain (SEQ ID NO:9), the EGF domain (SEQ ID NO:ll), and the serine protease domain (SEQ ID NO:l2). As previously described, the CUBEGFCUBII régions hâve been shown to be required for dimerization and binding with MBL (Thielens et al., supra). In particular, the peptide sequence TFRSDYN (SEQ ID NO:l6) in the CUBI domain of MASP-2 has been shown to be involved in binding to MBL in a study that identifïed a human carrying a homozygous mutation at Asp 105 to Gly 105, resulting in the loss of MASP-2 from the MBL complex (Stengaard-Pedersen, K., et al., New EnglandJ. Med. 349:554-560, 2003).

In some embodiments, MASP-2 inhibitory peptides are derived from the lectin proteins that bind to MASP-2 and are involved in the lectin complément pathway. Several different lectins hâve been identifïed that are involved in this pathway, including mannan-binding lectin (MBL), L-ficolin, M-fïcolin and H-ficolin. (Ikeda, K., et al., J. Biol. Chem. 262:7451-7454, 1987; Matsushita, M., et al., J. Exp. Med. /76:1497-2284, 2000; Matsushita, M., et al., J. Immunol. /65:3502-3506, 2002). Thèse lectins are présent in sérum as oligomers of homotrimeric subunits. each having N-terminal collagen-like fibers with carbohydrate récognition domains. These different lectins hâve been shown to bind to MASP-2, and the lectîn/MASP-2 complex activâtes complément through cleavage of proteins C4 and C2. H-ficolin has an amino-terminal région of 24 amino acids, a collagen-like domain with 11 Gly-Xaa-Yaa repeats, a neck domain of 12 amino acids, and a fibrînogen-like domain of 207 amino acids (Matsushita, M., et al., J. Immunol. 765:3502-3506, 2002). H-ficolin binds to GlcNAc and agglutinâtes human erythrocytes coated with LPS derived from S. typhimurium, S. minnesota and E. coli. H-ficolin has been shown to be associated with MASP-2 and MApl9 and activâtes the lectin pathway. Id. L-ficoIin/P35 also binds to GlcNAc and has been shown to be associated with MASP-2 and MApl9 in human sérum and this complex has been shown to activate the lectin pathway (Matsushita, M., étal., J. Immunol. 164:2281, 2000). Accordingly, MASP-2 inhibitory peptides useful in the present invention may comprise a région of at least 5 amino acids selected from the MBL protein (SEQ ID NO:2l), the H-ficolin protein (Genbank accession number NM_l 73452), the M-ficolin protein (Genbank accession number 000602) and the L-ficolin protein (Genbank accession number NM_015838).

More specîfically, scientists hâve identified the MASP-2 binding site on MBL to be within the 12 Gly-X-Y triplets GKD GRD GTK GEK GEP GQG LRG LQG POG

K.LG POG NOG PSG SOG PKG QKG DOG KS (SEQ ID NO:26) that lie between the hinge and the neck in the C-terminal portion of the collagen-like domain of MBP (Wallis, R., étal., J. Biol. Chem. 279:14065, 2004). This MASP-2 binding site région is also highly conserved in human H-ficolin and human L-ficolin. A consensus binding site has been described that is present in ail three lectin proteins comprising the amino acid sequence OGK-X-GP (SEQ ID NO:22) where the letter O represents hydroxyproline and the letter X is a hydrophobie residue (Wallis étal., 2004, supra). Accordingly, in some embodiments, MASP-2 inhibitory peptides useful in this aspect of the invention are at least 6 amino acids in length and comprise SEQ ID NO:22. Peptides derived from MBL that include the amino acid sequence GLR GLQ GPO GKL GPO G (SEQ ID NO:24) hâve been shown to bind MASP-2 in vitro (Wallis, et al., 2004, supra). To enhance binding to MASP-2, peptides can be synthesized that are flanked by two GPO triplets at each end (GPO GPO GLR GLQ GPO GKE GPO GGP OGP O SEQ ID NO:25) to enhance the formation of triple helices as found in the native MBL protein (as further described in Wallis, R., et al., J. Biol. Chem. 279:14065, 2004).

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

MASP-2 inhibitory peptides may also be derived from the C4 cleavage site such 5 as LQRALEILPNRVTIKANRPFLVFI” (SEQ ID NO:29) which is the C4 cleavage site linked to the C-terminal portion of antithrombin III (Glover, G.L, étal., Mol. Immunol. 25:1261 (1988)).

TABLE 4: EXEMPLARY MASP-2 INHIBITORY PEPTIDES

SEQ ID NO	Source
SEQ ID NO:6	Human MASP-2 protein
SEQ ID NO:8	CUBI domain of MASP-2 (aa 1-121 of SEQ ID NO:6)
SEQ ID NO:9	CUBIEGF domains of MASP-2 (aa 1-166 of SEQ ID NO:6)
SEQ ID NO: 10	CUBIEGFCUBII domains of MASP-2 (aa 1-293 of SEQ ID NO:6)
SEQ IDNO:11	EGF domain of MASP-2 (aa 122-166)
SEQIDNO:12	Serine-protease domain of MASP-2 (aa 429-671)
SEQ ID NO: 16	MBL binding région in MASP-2
SEQ ID NO:3	Human MApl9
SEQ ID NO:21	Human MBL protein
SEQ ID NO:22 OGK-X-GP, Where O = hydroxyproline and X is a hydrophobie amino acid residue	Synthetic peptide Consensus binding site from Human MBL and Human ficolins
SEQ ID NO:23 OGKLG	Human MBL core binding site
SEQ ID NO:24 GLR GLQ GPO GKL GPO G	Human MBP Triplets 6-10- demonstrated binding to MASP-2

SEQ ID NO	Source
SEQ ID NO:25 GPOGPOGLRGLQGPO GKLGPOGGPOGPO	Human MBP Triplets with GPO added to enhance formation of triple helices
SEQ ID NO:26 GKDGRDGTKGEKGEP GQGLRGLQGPOGKLG POGNOGPSGSOGPKG QKGDOGKS	Human MBP Triplets 1-17
SEQ ID NO:27 GAOGSOGEKGAOGPQ GPOGPOGKMGPKGEO GDO	Human H-Ficolin (Hataka)
SEQ ID NO:28 GCOGLOGAOGDKGE AGTNGKRGERGPOGP OGKAGPOGPNGAOGE O	Human L-Ficolin P35
SEQ ID NO:29 LQRALEILPNRVTIKA NRPFLVFl	Human C4 cleavage site

Note: The letter O represents hydroxyprolîne. The letter X is a hydrophobie residue.

Peptides derived from the C4 cleavage site as well as other peptides that inhibit the MASP-2 serine protease site can be chemically modified so that they are irréversible 5 protease inhibitors. For example, appropriate modifications may include, but are not necessarily limited to, halomethyl ketones (Br, CI, I, F) at the C-terminus, Asp or Glu, or appended to functional side chaîns; haloacetyl (or other α-haloacetyl) groups on amino groups or other functional side chaîns; epoxide or imine-containing groups on the amino or carboxy termini or on functional side chains; or imîdate esters on the amino or carboxy 10 termini or on functional side chains. Such modifications would afford the advantage of permanently inhibiting the enzyme by covalent attachment of the peptide. This could resuit in lower effective doses and/or the need for less frequent administration of the peptide inhibitor.

6l

In addition to the inhibitory peptides described above, MASP-2 inhibitory peptides useful in the method of the invention include peptides containing the MASP-2-binding CDR3 région of anti-MASP-2 MoAb obtained as described herein. The sequence of the CDR régions for use in synthesizing the peptides may be determined by methods known in the art. The heavy chain variable région is a peptide that generally ranges from 100 to 150 amino acids in length. The light chain variable région is a peptide that generally ranges from 80 to 130 amino acids in length. The CDR sequences within the heavy and light chain variable régions include only approximately 3-25 amino acid sequences that may be easily sequenced by one of ordinary skiil in the art.

Those skilled in the art will recognize that substantially homologous variations of the MASP-2 inhibitory peptides described above will also exhibit MASP-2 inhibitory activity. Exemplary variations include, but are not necessarily limited to, peptides having insertions, délétions, replacements, and/or additional amino acids on the carboxy-terminus or amino-terminus portions of the subject peptides and mixtures thereof. Accordingly, those homologous peptides having MASP-2 inhibitory activity are considered to be useful in the methods of this invention. The peptides described may also include duplicating motifs and other modifications with conservative substitutions. Conservative variants are described elsewhere herein, and include the exchange of an amino acid for another of iike charge, size or hydrophobicity and the like.

MASP-2 inhibitory peptides may be modified to increase solubility and/or to maximize the positive or négative charge in order to more closely resemble the segment in the intact protein. The dérivative may or may not hâve the exact primary amino acid structure of a peptide disclosed herein so long as the dérivative functîonally retains the desired property of MASP-2 inhibition. The modifications can include amino acid substitution with one of the commonly known twenty amino acids or with another amino acid, with a derivatized or substituted amino acid with ancillary désirable characteristics, such as résistance to enzymatic dégradation or with a D-amino acid or substitution with another molécule or compound, such as a carbohydrate, which mimics the naturel confirmation and function of the amino acid, amino acids or peptide; amino acid délétion; amino acid insertion with one of the commonly known twenty amino acids or with another amino acid, with a derivatized or substituted amino acid with ancillary désirable characteristics, such as résistance to enzymatic dégradation or with a D-amino acid or substitution with another molécule or compound, such as a carbohydrate, which mimics the naturai confirmation and function of the amino acid, amino acids or peptide; or substitution with another molécule or compound, such as a carbohydrate or nucleic acid monomer, which mimics the naturai conformation, charge distribution and function of the parent peptide. Peptides may also be modified by acétylation or amidation.

The synthesis of dérivative inhibitory peptides can rely on known techniques of peptide biosynthests, carbohydrate biosynthesis and the like. As a starting ροΐηζ the artisan may rely on a suitable computer program to détermine the conformation of a peptide of interest. Once the conformation of peptide disclosed herein is known, then the artisan can détermine in a rational design fashion what sort of substitutions can be made at one or more sites to fashion a dérivative that retains the basic conformation and charge distribution of the parent peptide but which may possess characteristics which are not présent or are enhanced over those found in the parent peptide. Once candidate dérivative molécules are identified, the dérivatives can be tested to détermine if they function as MASP-2 inhibitory agents using the assays described herein.

SCREENING FOR MASP-2 INHIBITORY PEPTIDES

One may also use molecular modeling and rational molecular design to generate and screen for peptides that mimic the molecular structures of key binding régions of MASP-2 and inhibit the complément activitîes of MASP-2. The molecular structures used for modeling include the CDR régions of anti-MASP-2 monoclonal antibodies, as well as the target régions known to be important for MASP-2 function including the région required for dimerization, the région involved in binding to MBL, and the serine protease active site as prevîously described. Methods for identifying peptides that bind to a particular target are well known in the art. For example, molecular împrinting may be used for the de novo construction of macromolecular structures such as peptides that bind to a particular molécule. See, for example, Shea, K.J., Molecular Imprinting of Synthetic Network Polymers: The De Novo synthesis of Macromolecular Binding and Catalytic Sties, TRIP 2(5) 1994.

As an illustrative example, one method of preparîng mimics of MASP-2 binding peptides is as follows. Functional monomers of a known MASP-2 binding peptide or the binding région of an anti-MASP-2 antibody that exhibits MASP-2 inhibition (the template) are polymerized. The template is then removed, followed by polymerization of a second class of monomers in the void left by the template, to provide a new molécule that exhibîts one or more desired properties that are similar to the template. In addition to preparing peptides in this manner, other MASP-2 binding molécules that are MASP-2 inhibitory agents such as polysaccharides, nucleosides, drugs, nue leopro teins, lipoproteins, carbohydrates, glycoproteins, steroid, lipids and other biologically active materials can also be prepared. This method is useful for designing a wide variety of biological mîmics that are more stable than their naturel counterparts because they are typically prepared by free radical polymerization of fonction monomers, resulting in a compound with a non biodégradable backbone.

PEPTIDE SYNTHESIS

The MASP-2 inhibitory peptides can be prepared using techniques well known in the art, such as the solid-phase synthetic technique initially described by Merrifield, in J. Amer. Chem. Soc. 55:2149-2154, 1963. Automated synthesis may be achieved, for example, using Applied Biosystems 431A Peptide Synthesizer (Foster City, Calif.) in accordance with the instructions provided by the manufacturer. Other techniques may be found, for example, in Bodanszky, M., et al., Peptide Synthesis, second édition, John Wiley & Sons, 1976, as well as in other reference works known to those skilled in the art.

The peptides can also be prepared using standard genetic engineering techniques known to those skilled in the art. For example, the peptide can be produced enzymatically by inserting nucleic acid encoding the peptide into an expression vector, expressing the DNA, and translating the DNA into the peptide in the presence of the required amino acids. The peptide is then purified using chromatographie or electrophoretic techniques, or by means of a carrier protein that can be fosed to, and subsequently cleaved from, the peptide by inserting into the expression vector in phase with the peptide encoding sequence a nucleic acid sequence encoding the carrier protein. The fusion protein-peptide may be isolated using chromatographie, electrophoretic or immunological techniques (such as binding to a resin via an antibody to the carrier protein). The peptide can be cleaved using chemical methodology or enzymatically, as by, for example, hydrolases.

The MASP-2 inhibitory peptides that are usefol in the method of the invention can also be produced in recombinant host cells following conventional techniques. To express a MASP-2 inhibitory peptide encoding sequence, a nucleic acid molécule encoding the peptide must be operably linked to regulatory sequences that control transcriptional expression in an expression vector and then introduced into a host cell. In addition to transcriptional regulatory sequences, such as promoters and enhancers, expression vectors can include translational regulatory sequences and a marker gene, which are suitable for sélection of cells that carry the expression vector.

Nucleic acid molécules that encode a MASP-2 inhibitory peptide can be synthesized with gene machines using protocols such as the phosphoramidite method. If chemically synthesized double-stranded DNA is required for an application such as the synthesis of a gene or a gene fragment, then each complementary strand is made separately. The production of short genes (60 to 80 base pairs) is technically straîghtforward and can be accomplished by synthesizing the complementary strands and then armealing them. For the production of longer genes, synthetic genes (double-stranded) are assembled in modular form from single-stranded fragments that are from 20 to 100 nucléotides in length. For reviews on polynucleotide synthesis, see, for example, Glick and Pasternak, Molecular Biotechnology, Principles and Applications of Recombinant DNA, ASM Press, 1994; Itakura, K., étal., Annu. Rev. Biochem. 53:323, 1984; and Climie, S., et al., Proc. Nat'l Acad. Sci. USA 57:633, 1990.

SMALL MOLECULE INHIBITORS

In some embodiments, MASP-2 inhibitory agents are small molécule inhibitors încluding natural and synthetic substances that hâve a low molecular weight, such as for example, peptides, peptidomimetics and nonpeptide inhibitors (Încluding oligonucleotides and organic compounds). Small molécule inhibitors of MASP-2 can be generated based on the molecular structure of the variable régions of the anti-MASP-2 antibodies.

Small molécule inhibitors may also be designed and generated based on the MASP-2 crystal structure using computational drug design (Kuntz I.D., et al., Science 257:1078, 1992). The crystal structure of rat MASP-2 has been described (Feinberg, H., étal., EMBO J. 22:2348-2359, 2003). Using the method described by Kuntz et aL, the MASP-2 crystal structure coordinates are used as an input for a computer program such as DOCK, which outputs a list of small molécule structures that are expected to bind to MASP-2. Use of such computer programs is well known to one of skill in the art. For example, the crystal structure of the HIV-1 protease inhibitor was used to identify unique nonpepûde ligands that are HIV-l protease inhibitors by evaluating the fit of compounds found in the Cambridge Crystallographic database to the binding site of the enzyme using the program DOCK (Kuntz, I.D., étal., J. Mol. Biol. 161:269-288, 1982; DesJarlais, R.L., et al., PNAS 57:6644-6648, 1990).

The list of small molécule structures that are identifîed by a computational method as potential MASP-2 inhibitors are screened using a MASP-2 binding assay such as described in Example 10. The small molécules that are found to bind to MASP-2 are then assayed in a functional assay such as described in Example 2 to détermine if they inhibit MASP-2-dependent complément activation.

MASP-2 SOLUBLE RECEPTORS

Other suitable MASP-2 inhibitory agents are believed to include MASP-2 soluble receptors, which may be produced using techniques known to those of ordinary skill in the art.

EXPRESSION INHIBITORS OF MASP-2

In another embodiment of this aspect of the invention, the MASP-2 inhibitory agent is a MASP-2 expression inhibitor capable of inhibiting MASP-2-dependent complément activation. In the practice of this aspect of the invention, représentative MASP-2 expression inhibitors include MASP-2 antisense nucleic acid molécules (such as antisense mRNA, antisense DNA or antisense oligonucleotides), MASP-2 ribozymes and MASP-2 RNAi molécules.

Anti-sense RNA and DNA molécules act to directly block the translation of MASP-2 mRNA by hybridizing to MASP-2 mRNA and preventing translation of MASP-2 protein. An antisense nucleic acid molécule may be constructed in a number of different ways provided that it is capable of interfering with the expression of MASP-2. For example, an antisense nucleic acid molécule can be constructed by înverting the coding région (or a portion thereof) of MASP-2 cDNA (SEQ ID NO:4) relative to its normal orientation for transcription to allow for the transcription of its complément.

The antisense nucleic acid molécule is usually substantially identical to at least a portion of the target gene or genes. The nucleic acid, however, need not be perfectly identical to inhibit expression. Generally, higher homology can be used to compensate for the use of a shorter antisense nucleic acid molécule. The minimal percent identity is typically greater than about 65%, but a higher percent identity may exert a more effective repression of expression of the endogenous sequence. Substantially greater percent identity of more than about 80% typically is preferred, though about 95% to absolute identity is typically most preferred.

The antisense nucleic acid molécule need not hâve the same intron or exon pattern as the target gene, and non-coding segments of the target gene may be equally effective in achieving antisense suppression of target gene expression as coding segments. A DNA sequence of at least about 8 or so nucléotides may be used as the antisense nucleic acid molécule, although a longer sequence is préférable. In the présent invention, a représentative example of a useful inhibitory agent of MASP-2 is an antisense MASP-2 nucleic acid molécule which is at least ninety percent identical to the complément of the MASP-2 cDNA consisting of the nucleic acid sequence set forth in SEQ ID NO:4. The nucleic acid sequence set forth in SEQ ID NO:4 encodes the MASP-2 protein consisting of the amino acid sequence set forth in SEQ ID NO:5.

The targeting of antisense oligonucleotides to bind MASP-2 mRNA is another mechanism that may be used to reduce the level of MASP-2 protein synthesis. For example, the synthesis of polygalacturonase and the muscarine type 2 acétylcholine receptor is inhibited by antisense oligonucleotides directed to their respective mRNA sequences (U.S. Patent No. 5,739,119, to Cheng, and U.S. Patent No. 5,759,829, to Shewmaker). Furthermore, examples of antisense inhibition hâve been demonstrated with the nuclear protein cyclin, the multiple drug résistance gene (MDG1), ICAM-1, E-selectin, STK-1, striatal GABAa receptor and human EGF (see, e.g., U.S. Patent No. 5,801,154, to Baracchini; U.S. Patent No. 5,789,573, to Baker, U.S. Patent No. 5,718,709, to Considine; and U.S. Patent No. 5,610,288, to Reubensteîn).

A System has been described that allows one of ordinary skill to détermine which oligonucleotides are useful in the invention, which involves probing for suitable sites in the target mRNA using Rnase H cleavage as an indicator for accessibility of sequences within the transcrîpts. Scherr, M., et al., Nucleic Acids Res. 26:5079-5085, 1998; Lloyd, étal., Nucleic Acids Res. 29:3665-3673, 2001. A mixture of antisense oligonucleotides that are complementary to certain régions of the MASP-2 transcript is added to cell extracts expressing MASP-2, such as hépatocytes, and hybridized in order to create an RNAseH vulnérable site. This method can be combined with computer-assisted sequence sélection that can predict optimal sequence sélection for antisense compositions based upon their relative ability to form dimers, hairpins, or other secondary structures that woutd reduce or prohibit spécifie binding to the target mRNA in a host cell. These secondary structure analysis and target site sélection considérations may be performed using the OLIGO primer analysis software (Rychlik, I., 1997) and the BLASTN 2.0.5 algorithm software (Altschul, S.F., étal., Nucl. Acids Res. 25:3389-3402, 1997). The antisense compounds directed towards the target sequence preferably comprise from about 8 to about 50 nucléotides in length. Antisense oligonucleotides comprising from about 9 to about 35 or so nucléotides are particularly preferred. The inventors contemplate ail oligonucleotide compositions in the range of 9 to 35 nucléotides (Le., those of 9, 10, H, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23,24,25, 26, 27,28, 29, 30, 31, 32, 33, 34, or 35 or so bases in length) are highly preferred for the practice of antisense oligonucleotide-based methods of the invention. Highly preferred target régions of the MASP-2 mRNA are those that are at or near the AUG translation initiation codon, and those sequences that are substantially complementary to 5' régions of the mRNA, e.g., between the -10 and +10 régions of the MASP-2 gene nucléotide sequence (SEQ ID NO:4). Exemplary MASP-2 expression inhibitors are provided in TABLE 5.

TABLE 5: EXEMPLARY EXPRESSION INHIBITORS OF MASP-2

SEQ ID NO:30 (nucléotides 22-680 of SEQ1DNO:4)	Nucleic acid sequence of MASP-2 cDNA (SEQ ID NO:4) encoding CUBIEGF
SEQ ID NO:31 5'CGGGCACACCATGAGGCTGCTG ACCCTCCTGGGC3	Nucléotides 12-45 ofSEQ IDNO:4 including the MASP-2 translation start site (sense)
SEQ ID NO:32 5'GACATTACCTTCCGCTCCGACTC CAACGAGAAG3'	Nucléotides 361-396 ofSEQ ID NO:4 encoding a région comprising the MASP-2 MBL binding site (sense)
SEQ IDNO:33 5’AGCAGCCCTGAATACCCACGGCC GTATCCCAAA3'	Nucléotides 610-642 ofSEQ ID NO:4 encoding a région comprising the CUBH domain

As noted above, the term oligonucleotide as used herein refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term also covers those oligonucleobases composed of naturally occurring nucléotides, sugars and covalent înternucleoside (backbone) lînkages as well as oligonucleotides having non-naturally occurring modifications. These modifications allow one to introduce certain désirable properties that are not offered through naturally occurring oligonucleotides, such as reduced toxic properties, increased stability against nuclease dégradation and enhanced cellular uptake. In illustrative embodiments, the antisense compounds of the invention differ from native DNA by the modification of the phosphodiester backbone to extend the life of the antisense oligonucleotide in which the phosphate substituents are replaced by phosphorothîoates. Likewise, one or both ends of the oligonucleotide may be substituted by one or more acridine dérivatives that intercalate between adjacent basepairs within a strand of nucleic acid.

Another alternative to antisense is the use of RNA interférence (RNAi). Double-stranded RNAs (dsRNAs) can provoke gene silencing in mammals in vivo. The naturel fonction of RNAi and co-suppression appears to be protection of the genome against invasion by mobile genetic éléments such as retrotransposons and viruses that produce aberrant RNA or dsRNA in the host cell when they become active (see, e.g., Jensen, J., et al., Nat. Genet. 2/:209-12, 1999). The double-stranded RNA molécule may be prepared by synthesizing two RNA strands capable of forming a double-stranded RNA molécule, each having a length from about 19 to 25 (e.g., 19-23 nucléotides). For example, a dsRNA molécule usefol in the methods of the invention may comprise the RNA corresponding to a sequence and its complément iisted in TABLE 4. Preferably, at least one strand of RNA has a 3' overhang from 1-5 nucléotides. The synthesized RNA strands are combined under conditions that form a double-stranded molécule. The RNA sequence may comprise at least an 8 nucléotide portion of SEQ ID NO:4 with a total length of 25 nucléotides or less, The design of stRNA sequences for a given target is within the ordînary skill of one in the art. Commercial services are available that design siRNA sequence and guarantee at least 70% knockdown of expression (Qiagen, Valencia, Calif).

The dsRNA may be administered as a pharmaceutical composition and carried out by known methods, wherein a nucleic acid is introduced into a desired target cell. Commonly used gene transfer methods include calcium phosphate, DEAE-dextran, electroporation, microinjection and viral methods. Such methods are taught in Ausubel et al., Carrent Protocols in Molecular Biology, John Wiley & Sons, Inc., 1993.

Ribozymes can also be utilized to decrease the amount and/or bîological activity of MASP-2, such as ribozymes that target MASP-2 mRNA. Ribozymes are catalytic RNA molécules that can cleave nucieic acid molécules having a sequence that is completely or partially homologous to the sequence of the ribozyme. It is possible to design ribozyme transgenes that encode RNA ribozymes that specifically pair with a target RNA and cleave the phosphodiester backbone at a spécifie location, thereby functionally inactivating the target RNA. In carrying out this cleavage, the ribozyme is not itself altered, and is thus capable of recycling and cleaving other molécules. The inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasîng the activity of the antisense constructs.

Ribozymes use fui in the practice of the invention typically comprise a hybridizing région of at least about nîne nucléotides, which is complementary in nucléotide sequence to at least part of the target MASP-2 mRNA, and a catalytic région that is adapted to cleave the target MASP-2 mRNA (see generally, EPA No. 0 321 201; W088/04300; Haseloff, J., et al., Nature 334:585-591, 1988; Fedor, M.J., étal., Proc. Natl. Acad. Set. USA 37:1668-1672, 1990; Cech. T.R., etal.,rtnn. Rev. Biochem. 55:599-629, 1986).

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

Anti-sense RNA and DNA, ribozymes and RNAi molécules useiul in the methods of the invention may be prepared by any method known in the art for the synthesis of DNA and RNA molécules. These include techniques for chemically synthesizing oligodeoxyribonucleotîdes and oligoribonucleotides well known in the art, such as for example solid phase phosphoramidite chemical synthesis. Alternatively, RNA molécules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molécule. Such DNA sequences may be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.

Various well known modifications of the DNA molécules may be introduced as a means of increasing stability and half-life. Usefui modifications include, but are not limited to, the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molécule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the oligodeoxyribonucleotide backbone.

V. PHARMACEUTICAL COMPOSITIONS AND DELIVERY METHODS

DOSING

In another aspect, the invention provides compositions for inhibitîng the adverse effects of MASP-2-dependent complément activation in a subject sufferîng from a disease or condition as disciosed herein, comprising administering to the subject a composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent and a pharmaceutically acceptable carrier. The MASP-2 inhibitory agents can be administered to a subject in need thereof, at therapeutically effective doses to treat or ameliorate conditions associated with MASP-2-dependent complément activation. A therapeutically effective dose refers to the amount of the MASP-2 inhibitory agent sufficient to resuit in amelioration of symptoms associated with the disease or condition.

Toxicity and therapeutic efficacy of MASP-2 inhibitory agents can be determined by standard pharmaceutical procedures employing experimental animal models, such as the murine MASP-2 -/- mouse model expressing the human MASP-2 transgene described in Example l. Using such animal models, the NOAEL (no observed 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 inhibitory agents that exhibit large therapeutic ratios or indices are most preferred. The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosages for use in humans. The dosage of the MASP-2 inhibitory agent preferably lies within a range of circulatîng concentrations that include the MED with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilîzed.

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

In addition to toxicity studies, effective dosage may also be estimated based on the amount of MASP-2 protein présent in a living subject and the binding affînity of the MASP-2 inhibitory agent. It has been shown that MASP-2 levels in normal human subjects is présent în sérum in low levels in the range of 500 ng/ml, and MASP-2 levels in a particular subject can be determined using a quantitative assay for MASP-2 described in Moller-Kristensen M., et al., J. Immunol. Methods 252:159-167, 2003.

Generally, the dosage of administered compositions comprising MASP-2 inhibitory agents varies depending on such factors as the subject's âge, weight, height, sex, general medical condition, and previous medical history. As an illustration, MASP-2 inhibitory agents, such as anti-MASP-2 antibodies, can be administered in dosage ranges from about 0.010 to 10.0 mg/kg, preferably 0.010 to 1.0 mg/kg, more preferably 0.010 to 0.1 mg/kg of the subject body weight. In some embodiments the composition comprises a combination of anti-MASP-2 antibodies and MASP-2 inhibitory peptides.

Therapeutic efficacy of MASP-2 inhibitory compositions and methods of the présent invention in a gîven subject, and appropriate dosages, can be determined în accordance with complément assays well known to those of skill in the art. Complément generates numerous spécifie products. During the iast decade, sensitive and spécifie assays hâve 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 utilize monoclonal 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 spécifie. Most rely on ELISA technology, aithough radioimmunoassay is still sometimes used for C3a and C5a. These latter assays measure both the unprocessed fragments and their 'desArg' fragments, which are the major forms found in the circulation. Unprocessed fragments and C5a<iesArg are rapidly cleared by binding to cell surface receptors and are hence présent in very low concentrations, whereas C3adesArg does not bind to cells and accumulâtes in plasma. Measurement of C3a provides a sensitive, pathway-independent indicator of complément activation. Alternative pathway activation can be assessed by measuring the Bb fragment. Détection of the fluid-phase product of membrane attack pathway activation, sC5b-9, provides evidence that complément 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 5 provide any information about which of these two pathways has generated the activation products.

The inhibition of MASP-2-dependent complément activation is characterized by at least one of the following changes in a component of the complément System that occurs as a resuit of administration of a MASP-2 inhibitory agent in accordance with the I0 methods of the invention: the inhibition of the génération or production of MASP-2-dependent complément activation system products C4b, C3a, C5a and/or C5b-9 (MAC) (measured, for example, as described in measured, for example, as described in Exampie 2, the réduction of C4 cleavage and C4b déposition (measured, for example as described in Example 10), or the réduction of C3 cleavage and C3b déposition (measured, 15 for example, as described in Example 10).

ADDITIONAL AGENTS

The compositions and methods comprising MASP-2 inhibitory agents may optionally comprise one or more additional therapeutic agents, which may augment the 20 activity of the MASP-2 inhibitory agent or that provide related therapeutic fonctions in an additive or synergistic fashion. For example, in the context of treating a subject suffering from an angiogenesis-dependent disease or condition, one or more MASP-2 inhibitory agents may be administered in combination (including co-administration) with one or more additional anti-angiogenîc (also referred to as angîostatic) agents and/or one or 25 more chemotherapeutic agents.

MASP-2 inhibitory agents can be used in combination with other anti-angiogenic agents, such as, for example, VEGF antagonists, such as antibodies that bind to VEGF, such as the antibody known as “bevacizumab (BV)” (also known as AVASTIN®), antibodies that bind to VEGF-A, or VEGF-C, or to the VEGF-A receptor (e.g., KDR 30 receptor or Flt-l receptor), anti-PDGFR inhibitors such as Gleevec® (Imatinib Mesylate), small molécules that block VEGF receptor signaling (e.g., PTK787/ZK2284, SU6668, SUTENT.RTM./SUl 1248 (sunitinib malate)), AMG706, or those described in, e.g., international patent application WO 2004/113304. Anti-angiogensis agents also include native angiogenesis inhibitors, e.g., angiostatin, endostatin, etc. See, e.g., Klagsbrun and D'Amore (1991) Annu. Rev. Physîol. 53:217-39; Streit and Detmar (2003) Oncogene 22.3172-3179 (e.g., Table 3 listing anti-angiogenic thérapies in maiignant melanoma); Ferrara & Alitalo (1999) Nature Medicine 5(12): 1359-1364; Tonini et al. (2003) Oncogene 22:6549-6556 (e.g., Table 2 listing known antiangiogenic factors); and, Sato (2003) Int. J. Clin. Oncol. 8:200-206 (e.g., Table 1 listing anti-angiogenic agents used in clinical trials).

MASP-2 ïnhibitory agents can be used in combination with other anti-cancer and/or chemotherapeutic agents, such as, for example, abarelix, actinomycin D, adriamycin, aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, amifostine, anakînra, anastrozole, arsenic trioxide, asparaginase, azacitidine, BCG Live, bevacuzimab, bexarotene, bleomycin, bortezomib, busulfan, calusterone, capecitabine, carboplatin, carmustine, celecoxib, cetuximab, chlorambucil, cisplatin, cladribine, clofarabine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, dalteparin (e.g., sodium), darbepoetin alfa, dasatinib, daunorubicin, daunomycin, decitabine, denileukin, denileukin diftitox, dexrazoxane, docetaxel, doxorubicin, dromostanolone propionate, eculizumab, epirubicin (e.g., HCl), epoetin alfa, erlotinib, estramustine, etoposide (e.g., phosphate), exemestane, fentanyl (e.g., citrate), filgrastim, floxuridine, fludarabine, fluorouracil, 5-FLJ, fiilvestrant, gefitinib, gemcitabine (e.g., HCl), gemtuzumab ozogamicin, goserelin (e.g., acetate), histrelîn (e.g., acetate), hydroxyurea, ibritumomab tiuxetan, idarubicin, ifosfamide, imatinib (e.g., mesylate), Interferon alfa-2b, irinotecan, lapatînib ditosylate, lenalidomide, letrozole, leucovorin, leuprolide (e.g., acetate), levamisole, lomustine, CCNU, meclorethamine (nitrogen mustard), megestrol, melphalan (L-PAM), mercaptopurine (6-MP), mesna, methotrexate, methoxsalen, mitomycin C, mitotane, mitoxantrone, nandrolone phenpropionate, nelarabine, nofetumomab, oprelvekin, oxaliplatin, paclitaxel, palifermin, pamidronate, panitumumab, pegademase, pegaspargase, pegfilgrastim, peginterferon alfa-2b, pemetrexed (e.g., disodium). pentostatin, pipobroman, plicamycin (mîthramycin), porfimer (e.g., sodium), procarbazine, quinacrine, rasburicase, rituximab, sargramostim, sorafenib, streptozocin, sunitinib (e.g., maleate), talc, tamoxifen, temozolomide, teniposide (VM-26), testolactone, thalidomide, thioguanine (6-TG), thiotepa, thiotepa, thiotepa, topotecan (e.g., hcl), toremifene, Tositumomab/I-I3l (tositumomab), trastuzumab, tretinoln (ATRA), uracil mustard, valrubicin, Vinblastine, vincristine, vinorelbine, vorinostat, zolédronate, and zoledronic acid.

PHARMACEUTICAL CARRIERS AND DELIVERY VEHICLES

In general, the MASP-2 inhibitory agent compositions of the présent invention, combined with any other selected therapeutic agents, are suitably contained in a pharmaceutically acceptable carrier. The carrier is non-toxic, biocompatîbie and is selected so as not to detrimentally affect the biological activity of the MASP-2 inhibitory agent (and any other therapeutic agents combined therewith). Exemplary pharmaceutically acceptable carriers for peptides are described in U.S. Patent No. 5,211,657 to Yamada. The anti-MASP-2 antibodies and inhibitory peptides useful in the invention may be formulated into préparations in solid, semi-solid, gel, liquid or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, depositories, inhalants and injections allowing for oral, parentéral or surgical administration. The invention also contemplâtes local administration of the compositions by coating medical de vices and the like.

Suitable carriers for parentéral 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, stérile, fixed oils may be employed as a solvent or suspending medium. For this purpose any biocompatible oil may be employed including synthetîc mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the préparation of injectables. The carrier and agent may be compounded 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 enhance the delivery, uptake, stability or pharmacokinetics of the therapeutic agent(s). Such a delivery vehicle may include, by way of non-limiting example, microparticles, microspheres, nanospheres or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymerîc or copolymeric hydrogels and polymeric micelles. Suitable hydrogel and micelle delivery Systems include the PEO:PHB:PEO copolymers and copolymer/cyclodextrin complexes disclosed in WO 2004/009664 A2 and the PEO and PEO/cyclodextrin complexes disclosed in U.S. Patent Application Publication No. 2002/0019369 Al. Such hydrogels may be injected locally at the site of intended action, or subcutaneously or intramuscularly to form a sustained release depot.

For intra-articular delivery, the MASP-2 inhibitory agent may be carried in above-described liquid or gel carriers that are injectable, abo ve-descri bed sustained-release delivery vehicles that are injectable, or a hyaluronic acid or hyaluronic acid dérivative.

For oral administration of non-peptidergic agents, the MASP-2 inhibitory agent may be carried in an inert Aller or diluent such as sucrose, comstarch, or cellulose.

For topical administration, the MASP-2 inhibitory agent may be carried in ointment, lotion, cream, gel, drop, suppository, spray, liquid or powder, or in gel or microcapsular deliveiy Systems via a transdermal patch.

Various nasal and pulmonary delivery Systems, including aérosols, metered-dose inhalers, dry powder inhalers, and nebulizers, are being developed and may suitably be adapted for delivery of the present invention in an aérosol, inhalant, or nebulized delivery vehicle, respect!vely.

For intrathecal (IT) or întracerebroventricular (ICV) delivery, appropriately stérile 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 biocompatible excipients, such as dispersing or wetting agents, suspending agents, dîluents, buffers, pénétration enhancers, emulsifiers, binders, thickeners, flavouring agents (for oral administration).

PHARMACEUTICAL CARRIERS FOR ANTIBODIES AND PEPTIDES

More specîfically with respect to anti-MASP-2 antibodies and inhibitory peptides, exemplary formulations can be parenterally administered as injectable dosages of a solution or suspension of the compound in a physiologically acceptable diluent with a pharmaceutical carrier that can be a stérile liquid such as water, oils, saline, glycerol or éthanol. Additionally, auxiliary substances such as wetting or emulsifying agents, surfactants, pH buffering substances and the like can be present in compositions comprising anti-MASP-2 antibodies and inhibitory peptides. Additional components of pharmaceutical compositions include petroleum (such as of animal, vegetable or synthetic origin), for example, soybean oil and minerai oil. In general, glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers for injectable solutions.

The antî-MASP-2 antibodies and inhibitory peptides can also be administered in the form of a depot injection or implant préparation that can be formulated in such a manner as to permit a sustained or pulsatile release of the active agents.

PHARMACEUTICALLY ACCEPTABLE CARRIERS FOR EXPRESSION INHIBITORS

More specifically with respect to expression inhibitors useful in the methods of the invention, compositions are provided that comprise an expression inhibitor as described above and a pharmaceutically acceptable carrier or diluent. The composition may further comprise a colloïdal dispersion system.

Pharmaceutical compositions that include expression inhibitors may include, but are not limited to, solutions, émulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semîsolids. The préparation of such compositions typically involves combining the expression inhibitor with one or more of the following: buffers, antioxidants, low molecular weight polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with non-specific sérum album in are examples of suitable diluents.

In some embodiments, the compositions may be prepared and formulated as émulsions which are typically heterogeneous Systems of one liquid dispersed in another in the form of droplets (see, Idson, in Pharmaceutical Dosage Forms, Vol. I, Rieger and Banker (eds.), Marcek Dekker, Inc., N.Y., 1988). Examples of naturally occurrîng emulsifîers used in émulsion formulations include acacia, beeswax, lanolin, lecithin and phosphatides.

In one embodiment, compositions including nucleic acids can be formulated as microemulsions. A microemulsion, as used herein refers to a system of water, oil, and amphiphile, which is a single optically isotropie and thermodynamically stable liquid solution (see Rosoff in Pharmaceutical Dosage Forms, Vol. I). The method of the invention may also use liposomes for the transfer and delivery of antisense oligonucleotides to the desired site.

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

MODES OF ADMINISTRATION

The pharmaceutical compositions comprising MASP-2 inhibitory agents may be administered in a number of ways depending on whether a local or systemic mode of administration is most appropriate for the condition being treated. Further, the compositions of the présent invention can be delivered by coating or incorporating the compositions on or înto an implantable medical device.

SYSTEMIC DELIVERY

As used herein, the terms systemic delivery and systemic administration are intended to include but are not limited to oral and parentéral routes including intramuscular (IM), subcutaneous, intravenous (IV), intra-arterial, inhalational, sublingual, buccal, topical, transdermal, nasal, rectal, vaginal and other routes of administration that effectively resuit in dispersement of the delivered agent to a single or multiple sites of intended therapeutic action. Preferred routes of systemic delivery for the présent compositions include intravenous, intramuscular, subcutaneous and inhalational. It will be appreciated that the exact systemic administration route for selected agents utilized in particular compositions of the présent invention will be determined in part to account for the agent's susceptibility to metabolic transformation pathways associated with a given route of administration. For example, peptidergic agents may be most suîtably administered by routes other than oral.

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

By way of représentative 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 example the nasal, gastrointestinal and rectal membranes. The polypeptides are typically applied to the absorptive membrane in conjunction with a perméation enhancer. (See, e.g., Lee, V.H.L., Crit. Rev. Ther. Drug Carrier Sys. 5:69, 1988; Lee, V.H.L., J. Controlled Release 73:213, 1990; Lee, V.H.L., Ed„ Peptide and Protein Drug Delivery, Marcel Dekker, New York (1991); DeBoer, A.G., étal., J. Controlled Release 13ûA\, 1990.) For example, STDHF is a synthetic dérivative of fusidic acid, a stéroïdal surfactant that is similar in structure to the bile salts, and has been used as a perméation 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 molécule, such as a lipid, to protect the polypeptides from enzymatîc dégradation. For example, the covalent attachment of polymers, especially polyethylene glycol (PEG), has been used to protect certain proteins from enzymatîc hydrolysis in the body and thus prolong half-life (Fuertges, F., et al., J. Controlled Release 77:139, 1990). Many polymer Systems hâve been reported for protein delivery (Bae, Y.H., et al., J. Controlled Release 9:271, 1989; Hori. R., et al., Pharm. Res. 6:813, 1989; Yamakawa, L, et al., J. Pharm. Sci. 79:505, 1990; Yoshihiro, I., et al., J. Controlled Release I0:\95, 1989; Asano, M., étal., J. Controlled Release 9:111, 1989; Rosenblatt, J„ étal., J. Controlled Release 9:195, 1989; Makino, K., J. Controlled Release 12:235, 1990; Takakura, Y., étal., J. Pharm. Sci. 75:117, 1989; Takakura, Y., étal., J. Pharm. Sci. 75:219, 1989).

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

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

The compositions of the présent invention may be systemically administered on a periodic basis at intervals determined to maintain a desired level of therapeutic effect. 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 consîdering 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 patîent's âge, sex and weight, and other clinical factors. The dosage for each individual agent will vary as a fonction of the MASP-2 inhibitory agent that is included in the composition, as well as the presence and nature of any drug delivery vehicle (e.g., a sustained release delivery vehicle). In addition, the dosage quantity may be adjusted to account for variation in the frequency of administration and the pharmacokinetic behavior of the delivered agent(s).

LOCAL DELIVERY

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

Local delivery of a MASP-2 inhibitory agent may be achieved in the context of surgical methods for treating an angiogenesis-dependent disease or condition, such as for example during procedures such as eye surgeiy or cancer-related surgery.

TREATMENT REGIMENS

In prophylactic applications, the pharmaceutîcal compositions comprising a MASP-2 inhibitory agent are admînistered to a subject susceptible to, or otherwise at risk of, developing an angiogenesis-dependent disease or condition in an amount sufficient to inhibit angiogenesis and thereby eliminate or reduce the risk of developing symptoms of the condition. In some embodiments, the pharmaceutîcal compositions are admînistered to a subject suspected of, or already suffering from, an angiogenesis-dependent disease or condition in a therapeutically effective amount sufficient to relieve, or at least partiaily reduce, the symptoms of the condition. In both prophylactic and therapeutic regîmens, compositions comprising MASP-2 inhibitory agents may be admînistered in several dosages until a sufficient therapeutic outcome has been achieved in the subject. Application of the MASP-2 inhibitory compositions of the présent invention may be carried out by a single administration of the composition, or a limited sequence of administrations, for treatment of an acute condition associated with angiogenesis. Altematively, the composition may be admînistered at periodic intervals over an extended period oftime for treatment of chronic conditions associated with angiogenesis.

In both prophylactic and therapeutic regîmens, compositions comprising MASP-2 inhibitory agents may be admînistered in several dosages until a sufficient therapeutic outcome has been achieved in the subject. In one embodiment of the invention, the MASP-2 inhibitory agent comprises a MASP-2 antibody, which suitably may be admînistered to an adult patient (e.g., an average adult weight of 70 kg) in a dosage of from 0.1 mg to 10,000 mg, more suitably from 1.0 mg to 5,000 mg, more suitably 10.0 mg to 2,000 mg, more suitably 10.0 mg to 1,000 mg and still more suitably from 50.0 mg to 500 mg. For pédiatrie patients, dosage can be adjusted in proportion to the patient’s weight. Application of the MASP-2 inhibitory compositions of the présent invention may be carried out by a single administration of the composition, or a limited sequence of administrations, for treatment of a subject suffering from or at risk for developing an angiogenesis-dependent disease or condition, such as an angiogenesis-dependent cancer, an angiogenesis-dependent benign tumor or an ocular angiogenic disease or condition.

8I

Altematively, the composition may be administered at periodic intervals such as daily, twice weekly, weekly, every other week, monthly or bîmonthly over an extended period of time for treatment of a subject suffering from or at risk for deveioping an angiogenesis-dependent disease or condition, such as an angiogenesîs-dependent cancer, an angiogenesis-dependent benign tumor or an ocular angiogenic disease or condition.

In both prophylactic and therapeutic regimens, compositions comprising MASP-2 inhibitory agents may be administered in several dosages until a sufficient therapeutic outcome has been achieved in the subject.

In one embodiment, the pharmaceutical composition comprising a MASP-2 inhibitory agent is administered to a subject suffering from an ocular angiogenic disease or condition in an amount effective to inhibit angiogenesis. In one embodiment, the ocular angiogenic disease or condition is selected from the group consisting of AMD, uveitis, ocular melanoma, comeal neovascularization, primary pterygium, HSV stromal keratitis, HSV-l-induced comeal lymphangiogenesis, proliférative diabetic retinopathy, retînopathy of prematurity, retinal vein occlusion, comeal graft rejection, neovascular glaucoma, and rubeosis.

In another embodiment, the pharmaceutical composition comprising a MASP-2 inhibitory agent is administerd to a subject suffering from an angiogenesis-dependent cancer in an amount effective to inhibit angiogenesis. In one embodiment, the angiogenesis-dependent cancer is selected from the group consisting of solid tumor(s), blood borne tumors, high-risk carcinoîd tumors, and tumor métastasés. In one embodiment, the composition is administered in an amount effective to inhibit tumor angiogenesis. In one embodiment, the subject is suffering from or at risk for tumor métastasés and the composition is administered tn an amount effective to inhibit tumor métastasés. In one embodimenL the subject is suffering from an angiogenesis-dependent cancer selected from the group consisting of colorectal, breast, lung, rénal, hepatic, esophageal, ovarian, pancreatic, prostate, gastric, glioma, gastrointestinal stromal tumor, lymphoma, melanoma and carcinoîd tumor. In one embodiment, the subject is suffering from a benign tumor and the composition is administered in an amount effective to inhibit angiogenesis of the benign tumor.

VI. EXAMPLES

The following examples merely illustrate the best mode now contemplated for practicing the invention, but should not be construed to limit the invention. Ail literature citations herein are expressly incorporated by reference.

EXAMPLE 1

This example describes the génération of a mouse strain déficient in MASP-2 (MASP-2-/-) but sufficient of MApl9 (MApl9+/+),

Materials and Methods: The targeting vector pKO-NTKV 1901 was designed to disrupt the three exons coding for the C-terminal end of murine MASP-2, încluding the exon that encodes the serine protease domain, as shown in FIGURE 3. PKO-NTKV 1901 was used to transfect the murine ES cell line E14.1a (SV129 Ola). Neomycin-resistant and Thymidine Kinase-sensitive clones were selected. 600 ES clones were screened and, of these, four different clones were identified and verified by southern blot to contain the expected sélective targeting and recombination event as shown in FIGURE 3. Chimeras were generated from these four positive clones by embryo transfer. The chimeras were then backcrossed in the genetic background C57/BL6 to create transgenic males. The transgenîc males were crossed with females to generate Fis with 50% of the offspring showing heterozygosity for the disrupted MASP-2 gene. The heterozygous mice were întercrossed to generate homozygous MASP-2 déficient offspring, resulting in heterozygous and wild-type mice in the ration of 1:2:1, respectively.

Results and Phenotype: The resulting homozygous MASP-2-/- (i.e., genetargeted-deficîent) mice were found to be viable and fertile and were verified to be MASP-2 déficient by southern blot to confirm the correct targeting event, by Northern blot to confirm the absence of MASP-2 mRNA, and by Western blot to confirm the absence of MASP-2 protein (data not shown). The presence of MApl9 mRNA and the absence of MASP-2 mRNA were forther confîrmed using time-resolved RT-PCR on a LightCycler machine. The MASP-2-/- mice do continue to express MApl9, MASP-1, and MASP-3 mRNA and protein as expected (data not shown). The presence and abundance of mRNA in the MASP-2-/- mice for Properdin, Factor B, Factor D, C4, C2, and C3 was assessed by LightCycler analysis and found to be identical to that of the wild-type littermate controls (data not shown). The plasma from homozygous MASP-2-/19004 mîce is totally déficient of iectin-pathway-mediated complément activation as further described in Example 2.

Génération of a MASP-2-/- strain on a pure C57BL6 Background: The MASP-2-/- mice were back-crossed with a pure C57BL6 line for nîne générations prior to use of the MASP-2-/- strain as an experimental animal model.

A transgenic mouse strain that is murine MASP-2-/-, MApl9+/+ and that expresses a human MASP-2 transgene (a murine MASP-2 knock-out and a human MASP-2 knock-in) was also generated as follows:

Materials and Methods: A minigene encoding human MASP-2 called mini hMASP-2 (SEQ ID NO:49) as shown in FIGURE 4 was constructed which includes the promoter région of the human MASP 2 gene, including the first 3 exons (exon l to exon 3) followed by the cDNA sequence that represents the coding sequence of the following 8 exons, thereby encoding the full-length MASP-2 protein driven by its endogenous promoter, The mini hMASP-2 construct was injected into fertilized eggs of MASP-2-/- in order to replace the déficient murine MASP 2 gene by transgenically expressed human MASP-2.

EXAMPLE 2

This example demonstrates that MASP-2 is requîred for complément activation via the lectin pathway.

Methods and Materials:

Lectin pathway spécifie C4 Cleavage Assay: A C4 cleavage assay has been described by Petersen, étal., J. Immunol. Methods 237:107 (2001) that measures lectin pathway activation resulting from lipoteichoic acid (LTA) from S. aureus, which binds L-ficolin. The assay described by Petersen et al., (2001) was adapted to measure lectin pathway activation via MBL by coating the plate with LPS and mannan or zymosan prior to adding sérum from MASP-2 -/- mice as described below. The assay was also modified to remove the possibility of C4 cleavage due to the classical pathway. This was achteved by using a sample dilution buffer containing 1 M NaCl, which permits high affïnity binding of lectin pathway récognition components to their ligands but prevents activation of endogenous C4, thereby excluding the participation of the classical pathway by dissociating the Cl complex. Briefly described, in the modified assay sérum samples (diluted in high sait (1 M NaCl) buffer) are added to ligand-coated plates, followed by the addition of a constant amount of purified C4 in a buffer with a physiological concentration of sait. Bound récognition complexes containing MASP-2 cleave the C4, resulting in C4b déposition.

Assay Methods:

1) Nunc Maxisorb microtiter plates (Maxisorb, Nunc, Cat. No. 442404, Fisher Scientific) were coated with I gg/ml mannan (M7504 Sigma) or any other ligand (e.g., such as those Itsted below) diluted in coating buffer (15 mM Na₂CO₃, 35 mM NaHCO₃, pH 9.6).

The following reagents were used in the assay:

a. mannan (l gg/well mannan (M7504 Sigma) in 100 μΐ coating buffer):

b. zymosan (l gg/well zymosan (Sigma) in I00 μΙ coating buffer);

c. LTA (l gg/well in 100 μΐ coating buffer or 2 gg/well in 20 μΐ methanol)

d. I gg of the H-ficolin spécifie Mab 4H5 in coating buffer

e. PSA from Aerococcus viridans (2 gg/well in 100 μΙ coating buffer)

f. lOOgl/well of formalin-fixed S. aureus DSM20233 (OD55(j=0.5) in coating buffer.

2) The plates were incubated ovemight at 4°C.

3) After ovemight incubation, the residual protein binding sites were saturated by incubated the plates with 0.1% HSA-TBS blocking buffer (0.1% (w/v) HSA in 10 mM Tris-CL, 140 mM NaCl, 1.5 mM NaN₃, pH 7.4) for 1-3 hours, then washing the plates 3X with TBS/tween/Ca2⁺ (TBS with 0.05% Tween 20 and 5 mM CaCl₂, 1 mM MgCI₂, pH 7.4).

4) Sérum samples to be tested were diluted in MBL-binding buffer (1 M NaCl) and the diluted samples were added to the plates and incubated ovemight at 4°C. Wells receiving buffer only were used as négative controls.

5) Following incubation ovemight at 4°C, the plates were washed 3X with TBS/tween/Ca2+. Human C4 (100 glAvell of 1 gg/ml diluted in BBS (4 mM barbîtal, 145 mM NaCl, 2 mM CaCl₂, 1 mM MgCl₂, pH 7.4)) was then added to the plates and incubated for 90 minutes at 37°C. The plates were washed again 3X with TBS/tween/Ca2⁺.

6) C4b déposition was detected with an alkaline phosphatase-conjugated chicken anti-human C4c (diiuted l : 1000 in TBS/tween/Ca2⁺), which was added to the plates and încubated for 90 minutes at room température. The plates were then washed again 3X with TBS/tween/Ca2⁺.

7) Alkaline phosphatase was detected by adding 100 μΐ of p-nitrophenyl phosphate substrate solution, incubating at room température for 20 minutes, and reading the Οθ4θ5 in a microtiter plate reader.

Results: FIGURES 5A-B show the amount of C4b déposition on mannan (FIGURE 5A) and zymosan (FIGURE 5B) in serum dilutions from MASP-2+/+ (crosses), MASP-2+/- (closed circles) and MASP-2-/- (closed triangles). FIGURE 5C shows the relative C4 convertase activity on plates coated with zymosan (white bars) or mannan (shaded bars) from MASP-2-/+ mice (n=5) and MASP-2-/- mice (n=4) relative to wild-type mice (n—5) based on measuring the amount of C4b déposition normalized to wild-type serum. The error bars represent the standard déviation. As shown in FIGURES 5A-C, plasma from MASP-2-/- mice is totally déficient in lectin-pathway-mediated complément activation on mannan and on zymosan coated plates. These results clearly demonstrate that MASP-2 is an effector component of the lectin pathway.

Recombinant MASP-2 reconstitutes Lectin Pathway-Dependent C4 Activation in serum from the MASP-2-/- mice

In order to establish that the absence of MASP-2 was the direct cause of the loss of lectin pathway-dependent C4 activation in the MASP-2-/- mice, the effect of adding recombînant MASP-2 protein to serum samples was examined in the C4 cieavage assay described above. Functionaily active murine MASP-2 and catalytically inactive murine MASP-2A (în which the active-site serine residue in the serine protease domain was substituted for the alanine residue) recombinant proteins were produced and purified as described below În Example 3. Pooled serum from 4 MASP-2 -/- mice was pre-incubated with increasing protein concentrations of recombinant murine MASP-2 or inactive recombinant murine MASP-2A and C4 convertase activity was assayed as described above.

Results: As shown in FIGURE 6, the addition of functionally active murine recombinant MASP-2 protein (shown as open triangles) to sérum obtained from the MASP-2 -/- mice restored lectin pathway-dependent C4 activation in a protein concentration dépendent manner, whereas the catalytically inactive murine MASP-2A protein (shown as stars) did not restore C4 activation. The results shown in FIGURE 6 are normal ized to the C4 activation observed with pooled wild-type mouse sérum (shown as a dotted line).

EXAMPLE 3

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

Expression of Full-length human, murine and rat MASP-2:

The full length cDNA sequence of human MASP-2 (SEQ ID NO: 4) was also subcloned into the mammalian expression vector pCl-Neo (Promega), which drives eukaryotic expression under the control of the CMV enhancer/promoter région (described în Kaufman R.J. et al., Nucleic Acids Research /9:4485-90, 1991 ; Kaufman, Methods in Enzymology, /35:537-66 (I99l)). The full length mouse cDNA (SEQ ID NO:50) and rat MASP-2 cDNA (SEQ ID NO:53) were each subcloned into the pED expression vector. The MASP-2 expression vectors were then transfected into the adhèrent Chinese hamster ovary cell line DXBl using the standard calcium phosphate transfection procedure described in Maniatis et al., 1989. Cells transfected with these constructs grew very slowly, implying that the encoded protease is cytotoxic.

In another approach, the minigene construct (SEQ ID NO:49) containing the human cDNA of MASP-2 driven by its endogenous promoter is transiently transfected into Chinese hamster ovary cells (CHO). The human MASP-2 protein is secreted into the culture media and isolated as described below.

Expression of Full-length catalytically inactive MASP-2:

Rationale: MASP-2 is activated by autocatalytic cleavage after the récognition subcomponents MBL or ficolins (either L-ficolin, H-ficolin or M-ficolin) bind to their respective carbohydrate pattern. Autocatalytic cleavage resulting in activation of MASP-2 often occurs durîng the isolation procedure of MASP-2 from sérum, or during the purification following recombinant expression. In order to obtain a more stable protein préparation for use as an antigen, a catalytically inactive form of MASP-2, designed as MASP-2A was created by replacing the serine residue that is présent in the catalytic triad of the protease domain with an alanine residue in rat (SEQ ID NO;55 Ser617 to Aia6l7); in mouse (SEQ ID NO:52 Ser6l7 to Ala6l7); or in human (SEQ ID NO:3 Ser6l8 to Aia6l8).

In order to generate catalytically inactive human and murine MASP-2A proteins, sîte-directed mutagenesis was carried out using the oligonucleotides shown in TABLE 6. The oligonucleotides in TABLE 6 were designed to anneal to the région of the human and murine cDNA encoding the enzymatically active serine and oligonucleotide contain a mismatch in order to change the serine codon into an alanine codon. For example, PCR oligonucleotides SEQ ID NOS:56-59 were used in combination with human MASP-2 cDNA (SEQ ID NO:4) to amplify the région from the start codon to the enzymatically active serine and from the serine to the stop codon to generate the complété open reading from of the mutated MASP-2A containing the Ser6l8 to Ala6l8 mutation. The PCR products were purified after agarose gel electrophoresis and band préparation and single adenosine overlaps were generated using a standard tailing procedure. The adenosine tailed MASP-2A was then cloned into the pGEM-T easy vector, transformed into E. coli.

A catalytically inactive rat MASP-2A protein was generated by kinasing and annealing SEQ ID NO:64 and SEQ ID NO:65 by combining these two oligonucleotides in equal molar amounts, heating at !00°C for 2 minutes and slowly cooling to room température. The resulting annealed fragment has Pst î and Xbal compatible ends and was inserted in place of the Pstl-Xbal fragment of the wild-type rat MASP-2 cDNA (SEQ ID NO:53) to generate rat MASP-2A.

'GAGGTGACGCAGGAGGGGCATTAGTGTTT 3' (SEQ ID NO:64)

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

The human, murine and rat MASP-2A were each further subcloned into either of the mammalian expression vectors pED or pCI-Neo and transfected into the Chinese Hamster ovary cell line DXBl as described below.

In another approach, a catalytically inactive form of MASP-2 is constructed using the method described in Chen et al., J. Biol. Chem., 276(28):25894-25902, 2001. Briefly, the plasmîd containing the full-length human MASP-2 cDNA (described in Thiel étal., Nature 356:506, 1997) is digested with Xho\ and EcoRA and the MASP-2 cDNA (described herein as SEQ ID NO:4) is cloned into the corresponding restriction sites of the pFastBacl baculovirus transfer vector (Life Technologies, NY). The MASP-2 serine protease active site at Ser6l8 is then altered to Ala6l8 by substituting the double-stranded oligonucleotides encoding the peptide région amino acid 610-625 (SEQ ID NO: 13) with the native région amino acids 610 to 625 to create a MASP-2 full length polypeptide with an inactive protease domain. Construction of Expression Plasmids Containing Polypeptide Régions Derived from Human Masp-2.

The following constructs are produced using the MASP-2 signal peptide (residues 1-15 of SEQ ID NO:5) to secrete various domains of MASP-2. A construct expressing the human MASP-2 CUBI domain (SEQ ID NO:8) is made by PCR amplifying the région encoding residues 1-121 of MASP-2 (SEQ ID NO:6) (corresponding to the N-terminal CUBI domain). A construct expressing the human MASP-2 CUBIEGF domain (SEQ ID NO:9) is made by PCR amplifying the région encoding residues 1-166 of MASP-2 (SEQ ID NO:6) (corresponding to the N-terminal CUBIEGF domain). A construct expressing the human MASP-2 CUBIEGFCUBII domain (SEQ ID NO: 10) is made by PCR amplifying the région encoding residues 1-293 of MASP-2 (SEQ ID NO:6) (corresponding to the N-terminal CUBIEGFCUBII domain). The above mentioned domains are amplified by PCR using VentR polymerase and pBS-MASP-2 as a template, accordîng to establîshed PCR methods. The 5' primer sequence of the sense primer (5'-CGGGATÇÇATGAGGCTGCTGACCCTC-3^r SEQ ID NO:34) introduces a BamHl restriction site (underlined) at the 5' end of the PCR products. Antisense primers for each of the MASP-2 domains, shown below in TABLE 6, are designed to introduce a stop codon (boldface) followed by an Eco RI site (underlined) at the end of each PCR product. Once amplified, the DNA fragments are digested with EamHI and EcoRl and cloned into the corresponding sites of the pFastBacl vector. The resulting constructs are characterized by restriction mapping and confirmed by dsDNA sequencing.

TABLE 6: MASP-2 PCR PRIMERS

MASP-2 domain	5' PCR Primer	3' PCR Primer
SEQ ID NO:8 CUBI (aa 1-I2I ofSEQ ID NO:6)	5 ’CGGGATCCATGA GGCTGCTGACCCT C-3' (SEQ ID NO:34)	5'GGAATTCCTAGGCTGCAT A (SEQ IDNO:35)
SEQ ID NO:9 CUBIEGF (aa 1-I66 of SEQ ID NO:6)	5'CGGGATCCATGA GGCTGCTGACCCT C-3' (SEQ ID NO:34)	5’GGAATTCCTACAGGGCC.C T-3’ (SEQ ID NO:36)
SEQ IDNO:lO CUBIEGFCUBII (aa l-293 ofSEQ IDNO:6)	5'CGGGATCCATGA GGCTGCTGACCCT C-3' (SEQ ID NO:34)	5’GGAATTÇCTAGTAGTGGA T 3' (SEQ ID NO:37)
SEQ ID NO:4 human MASP-2	5'ATGAGGCTGCTG ACCCTCCTGGGCC TTC 3’ (SEQ ID NO: 56) hMASP-2 forward	5TTAAAATCACTAATTATG TTCTCGATC 3' (SEQ ID NO: 59) hMASP-2_reverse
SEQ ID NO:4 human MASP-2 cDNA	5'CAGAGGTGACGC AGGAGGGGCAC 3' (SEQ ID NO: 58) hMASP-2_ala_forwar d	5'GTGCCCCTCCTGCGTCAC CTCTG 3' (SEQ ID NO: 57) hMASP-2_ala_reverse
SEQ IDNO:50 Murine MASP-2 cDNA	5'ATGAGGCTACTC ATCTTCCTGG3' (SEQ ID NO: 60) mMASP-2 forward	5TTAGAAATTACTTATTAT GTTCTCAATCC3' (SEQ ID NO: 63) mMASP-2_reverse
SEQ ID NO:50 Murine MASP-2 cDNA	5'CCCCCCCTGCGT CACCTCTGCAG3' (SEQ ID NO: 62) mMASP-2_ala_forwa rd	5'CTGCAGAGGTGACGCAG GGGGGG 3' (SEQIDNO:6l) mMASP-2_ala_reverse

Recombinant eukaryotic expression of MASP-2 and protein production of enzymatically inactive mouse, rat, and human MASP-2A.

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

MASP-2A protein purification: The MASP-2A (Ser-Ala mutant described above) was purified by affinity chromatography on MBP-A-agarose columns. This strategy 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-CI, pH 7.5, containing 150 mM NaCl and 25 mM CaCI₂) was loaded onto an MBP-agarose I0 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 l ml fractions with 50 mM Tris-CI, pH 7.5, containing 1.25 M NaCl and lOmM EDTA. Fractions containing the MASP-2A were identified by SDS-polyacrylamide gel electrophoresis. Where necessary, MASP-2A was purified further by ion-exchange chromatography on a MonoQ column 15 (HR 5/5). Protein was dialysed with 50 mM Tris-CI pH 7.5, containing 50 mM NaCl and loaded onto the column equilibrated in the same buffer. Following washing, bound MASP-2A was eluted with a 0.05-1 M NaCl gradient over 10 ml.

Results: Yields of 0.25-0.5 mg of MASP-2A protein were obtained from 200 ml of medium. The molecular mass of 77.5 kDa determined by MALD1-MS is greater than 20 the calculated value of the unmodified polypeptide (73.5 kDa) due to glycosylation. Attachaient of glycans at each of the A-glycosylation sites accounts for the observed mass. MASP-2A migrâtes as a single band on SDS-polyacrylamide gels, demonstrating that it is not proteoiytically processed during biosynthesis. The weight-average molecular mass determined by equilibrium ultracentrifugation is in agreement with the calculated 25 value for homodimers of the giycosylated polypeptide.

PRODUCTION OF RECOMBINANT HUMAN MASP-2 POLYPEPTIDES

Another method for producing recombinant MASP-2 and MASP2A derived polypeptides is described in Thielens, N.M., et al., J. Immunol. 166:5068-5077, 2001. Briefly, the Spodoptera frugiperda insect cells (Ready-Plaque Sf9 cells obtained from 30 Novagen, Madison, WI) are grown and maintained in Sf900II serum-free medium (Life Technologies) supplemented with 50 lU/ml penicillin and 50 mg/ml streptomycin (Life Technologies). The Trichoplusia ni (High Five) insect cells (provided by Jadwiga

9l

Chroboczek, Institut de Biologie Structurale, Grenoble, France) are maîntained in TCI00 medium (Life Technologies) containing 10% FCS (Dominique Dutscher, Brumath. France) supplemented with 50 IU/ml penicillin and 50 mg/ml streptomycin. Recombinant baculoviruses are generated using the Bac-to-Bac System (Life Technologies), The bacmid DNA îs purified using the Qiagen midiprep purification System (Qiagen) and is used to transfert Sf9 insect cells using cellfectîn in Sf900 il SFM medium (Life Technologies) as described in the manufacturées protocol. Recombinant virus particles are collected 4 days later, titrated by virus plaque assay, and amplified as described by King and Possee, in The Baculovirus Expression System: A Laboratory Guide, Chapman and Hall Ltd., London, pp. 11 l-l 14, 1992.

High Five cells (1.75 x IO⁷cells/I75-cm² tissue culture flask) are infected with the recombinant viruses containing MASP-2 polypeptides at a multiplicity of infection of 2 in Sf900 II SFM medium at 28°C for 96 h. The supematants are collected by centrifugation and diisopropyl phosphorofluoridate îs added to a final concentration of l mM,

The MASP-2 polypeptides are secreted in the culture medium. The culture supematants are dialyzed against 50 mM NaCI, l mM CaCh, 50 mM triethanolamine hydrochloride, pH 8.1, and loaded at 1.5 mFmîn onto a Q-Sepharose Fast Flow column (Amersham Pharmacia Biotech) (2.8 x 12 cm) equiiibrated in the same buffer. Elution is conducted by applying al.2 liter lînear gradient to 350 mM NaCI in the same buffer. Fractions containing the recombinant MASP-2 polypeptides are identified by Western blot analysis, precipitated by addition of (NH^SCL to 60% (w/v), and left ovemight at 4°C. The pellets are resuspended in 145 mM NaCI, l mM CaCI₂, 50 mM triethanolamine hydrochloride, pH 7.4, and applied onto a TSK G3000 SWG column (7.5 x 600 mm) (Tosohaas, Montgomery vil le, PA) equiiibrated in the same buffer. The purified polypeptides are then concentrated to 0.3 mg/ml by ultrafiltration on Microsep microconcentrators (m.w. cut-off= 10,000) (Filtron, Karlstein, Germany).

EXAMPLE 4

This example describes a method of producing polyclonal antibodies against MASP-2 polypeptides.

Materials and Methods:

MASP-2 Antigens: Polyclonal anti-human MASP-2 antiserum is produced by immunizing rabbits with the following isolated MASP-2 polypeptides: human MASP-2 (SEQ ID NO:6) isolated from sérum; recombinant human MASP-2 (SEQ ID NO:6), MASP-2A containîng the inactive protease domain (SEQ ID NO:l3), as described in Example 3; and recombinant CUBI (SEQ ID NO:8), CUBEGFI (SEQ ID NO:9), and CUBEGFCUBII (SEQ ID NO:IO) expressed as described above in Example 3.

Polyclonal antibodies: Six-week old Rabbits, primed with BCG (bacillus Calmette-Guerin vaccine) are immunized by injecting 100 pg of MASP-2 polypeptide at 100 pg/ml in stérile saline solution. Injections are done every 4 weeks, with antibody titer monitored by ELISA assay as described in Example 5. Culture supematants are collected for antibody purification by protein A affinity chromatography.

EXAMPLE 5

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

Materials and Methods:

Male A/J mice (Harlan, Houston, Tex.), 8-12 weeks old, are injected subcutaneously with 100 pg human or rat rMASP-2 or rMASP-2A polypeptides (made as described in Example 3) in complété Freund's adjuvant (Difco Laboratories, Detroit, Mich.) in 200 pl of phosphate buffered saline (PBS) pH 7.4. At two-week intervals the mice are twice injected subcutaneously with 50 pg of human or rat rMASP-2 or rMASP-2A polypeptide in incomplète Freund’s adjuvant. On the fourth week the mice are injected with 50 pg of human or rat rMASP-2 or rMASP-2A polypeptide in PBS and are fused 4 days later.

For each fusion, single cell suspensions are prepared from the spleen of an immunized mouse and used for fusion with Sp2/0 myeloma cells. 5xl0⁸ ofthe Sp2/0 and 5xl0⁸ spleen cells are fused in a medium containîng 50% polyethylene glycol (M.W. 1450) (Kodak, Rochester, N.Y.) and 5% dimethylsulfoxide (Sigma Chemical Co., St. Louis, Mo.). The cells are then adjusted to a concentration of l.5xl0⁵ spleen cells per 200 pl of the suspension in Iscove medium (Gibco, Grand Island, N.Y.), supplemented with 10% fêtai bovine sérum, lOOunits/ml of penicillin, l00pg/ml of streptomycîn, 0.1 mM hypoxanthine, 0.4 μΜ aminopterin and 16 μΜ thymidine. Two hundred microliters of the cell suspension are added to each well of about twenty 96-well microculture plates. After about ten days culture supematants are withdrawn for screening for reactivity with purified factor MASP-2 in an ELISA assay.

ELISA Assay: Wells of Immulon 2 (Dynatech Laboratories, Chantilly, Va.) microtest plates are coated by adding 50 μΐ of purified hMASP-2 at 50 ng/ml or rat rMASP-2 (or rMASP-2A) overnight at room température. The low concentration of MASP-2 for coating enables the sélection of high-affinity antibodies. After the coating solution is removed by flicking the plate, 200 μΐ of BLOTTO (non-fat dry milk) in PBS is added to each well for one hour to block the non-specific sites. An hour later, the wells are then washed with a buffer PBST (PBS containing 0.05% Tween 20). Fifty microliters of culture supematants from each fusion well is collected and mîxed with 50 μΐ of BLOTTO and then added to the individual wells of the microtest plates. After one hour of incubation, the wells are washed with PBST. The bound murine antibodies are then detected by reaction with horseradish peroxidase (HRP) conjugated goat anti-mouse IgG (Fc spécifie) (Jackson ImmunoResearch Laboratories, West Grove, Pa.) and diluted at 1:2,000 in BLOTTO. Peroxidase substrate solution containing 0.1% 3,3,5,5 tetramethyl benzidine (Sigma, St. Louis, Mo.) and 0.0003% hydrogen peroxide (Sigma) is added to the wells for color development for 30 minutes. The reaction is terminated by addition of 50 μΐ of 2M H2SO4 per well. The Optical Density at 450 nm of the reaction mixture is read with a BioTek ELISA Reader (BioTek Instruments, Winooskî, Vt.).

MASP-2 Binding Assay:

Culture supematants that test positive in the MASP-2 ELISA assay described above can be tested in a binding assay to détermine the binding affinîty the MASP-2 inhibitory agents hâve for MASP-2. A similar assay can also be used to détermine if the inhibitory agents bind to other antigens in the complément system.

Polystyrène microtiter plate wells (96-well medium binding plates, Coming Costar, Cambridge, MA) are coated with MASP-2 (20 ng/100 μΙ/well, Advanced Research Technology, San Diego, CA) in phosphate-buffered saline (PBS) pH 7.4 overnight at 4°C. After aspirating the MASP-2 solution, wells are blocked with PBS containing l% bovine sérum albumin (BSA; Sigma Chemical) for 2 h at room température. Wells without MASP-2 coating serve as the background controls. Aliquots of hybridoma supematants or purified anti-MASP-2 MoAbs, at varyîng concentrations in blocking solution, are added to the wells. Following a 2 h incubation at room température, the wells are extensively rinsed with PBS. MASP-2-bound anti-MASP-2 MoAb is detected by the addition of peroxidase-conjugated goat anti-mouse IgG (Sigma Chemical) in blocking solution, which is allowed to incubate for Ih at room température. The plate is rinsed again thoroughly with PBS, and ΙΟΟμΙ of 3,3‘,5,5'-tetramethyl benzidine (TMB) substrate (Kirkegaard and Perry Laboratories, Gaithersburg, MD) is added. The reaction of TMB is quenched by the addition of 100 μΐ of IM phosphoric acid, and the plate is read at 450 nm in a microplate reader (SPECTRA MAX 250, Molecular Devices, Sunnyvale, CA).

The culture supematants from the positive wells are then tested for the ability to inhibit complément activation in a functional assay such as the C4 cleavage assay as described in Example 2. The cells in positive wells are then cloned by limiting dilution. The MoAbs are tested again for reactivity with hMASP-2 in an ELISA assay as described above. The selected hybridomas are grown in spînner flasks and the spent culture supematant collected for antibody purification by protein A afifinity chromatography.

EXAMPLE 6

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

A murine anti-MASP-2 monoclonal antibody is generated in Male A/J mice as described in Example 5. The murine antibody is then humanized as described below to reduce its immunogenicity by replacmg the murine constant régions with their human counterparts to generate a chimeric IgG and Fab fragment of the antibody, which is useful for inhibiting the adverse effects of MASP-2-dependent complément activation in human subjects in accordance with the présent invention.

1. Cloning of anti-MASP-2 variable région genes from murine hybridoma cells. Total RNA is isolated from the hybridoma cells secreting anti-MASP-2 MoAb (obtained as described in Example 7) using RNAzol following the manufacturées protocol (Biotech, Houston, Tex.). First strand cDNA is synthesized from the total RNA using oligo dT as the primer. PCR is performed using the immunoglobulin constant C region-derived 3' primers and degenerate primer sets derived from the leader peptide or the first framework région of murine Vjq or V|< genes as the 5' primers. Anchored PCR is carried out as described by Chen and Platsucas (Chen, P.F., Scand. J. Immunol. 35:539-549, 1992). For cloning the Vj^ gene, double-stranded cDNA is prepared using a Notl-MAKI primer (5'-TGCGGCCGCTGTAGGTGCTGTCTTT-3' SEQ ID NO:38). Annealed adaptors ADI (5'-GGAATTCACTCGTTATTCTCGGA-3’ SEQ ID NO:39) and AD2 (5'-TCCGAGAATAACGAGTG-3' SEQ ID NO:40) are ligated to both 5’ and 3' termini of the double-stranded cDNA. Adaptors at the 3' ends are removed by Notl digestion. The digested product is then used as the template in PCR with the ADI oligonucleotide as the 5' primer and MAK2 (5'-CATTGAAAGCTTTGGGGTAGAAGTTGTTC-3· SEQ ID NO:41) as the 3' primer. DNA fragments of approximately 500 bp are cloned into pUCl9. Several clones are selected for sequence analysis to verify that the cloned sequence encompasses the expected murine immunoglobulîn constant région. The Notl-MAKI and MAK2 oligonucleotides are derived from the V« région and are 182 and 84 bp, respectively, downstream from the first base pair of the C kappa gene. Clones are chosen that include the complété V[<; and leader peptide.

For cloning the Vjq gene, double-stranded cDNA is prepared using the Notl MAGl primer (5'-CGCGGCCGCAGCTGCTCAGAGTGTAGA-3‘ SEQ ID NO:42). Annealed adaptors ADI and AD2 are ligated to both 5' and 3' termini of the double-stranded cDNA. Adaptors at the 3' ends are removed by Notl digestion. The digested product are used as the template in PCR with the ADI oligonucleotide and MAG2 (5'-CGGTAAGCTTCACTGGCTCAGGGAAATA-3' SEQ ID NO:43) as primers. DNA fragments of 500 to 600 bp in length are cloned into pUCl9. The Notl-MAGl and MAG2 oligonucleotides are derived from the murine Cy.7.l région, and are 180 and 93 bp, respectively, downstream from the first bp of the murine Cy.7.l gene. Clones are chosen that encompass the complété Vjq and leader peptide.

2. Construction of Expression Vectors for Chimeric MASP-2 IgG and Fab. The cloned Vh and Vj< genes described above are used as templates in a PCR reaction to add the Kozak consensus sequence to the 5' end and the splice donor to the 3' end of the nucléotide sequence. After the sequences are analyzed to confirm the absence of PCR errons, the V]_j and genes are inserted into expression vector cassettes containing human C.yl and C. kappa respectively, to give pSV2neoVH-huCyl and pSV2neoV-huCy. CsCl gradient-purified plasmid DNAs of the heavy- and light-chain vectors are used to transfect COS cells by electroporation. After 48 hours, the culture supematant is tested by ELISA to confirm the presence of approxîmately 200 ng/ml of chimeric IgG. The cells are harvested and total RNA is prepared. First strand cDNA is synthesized from the total RNA using oligo dT as the primer. This cDNA is used as the template in PCR to generate the Fd and kappa DNA fragments. For the Fd gene, PCR is carried out using 5'-AAGAAGCTTGCCGCCACCATGGATTGGCTGTGGAACT-3' (SEQ LD NO:44) as the 5' primer and a CHl-derived 3' primer (5’-CGGGATCCTCAAACTTTCTTGTCCACCTTGG-3' SEQ ID NO:45). The DNA sequence is confirmed to contain the complété Vpj and the CHI domain of human IgGl. After digestion with the proper enzymes, the Fd DNA fragments are inserted at the HindlII and BamHI restriction sites of the expression vector cassette pSV2dhfr-TUS to give pSV2dhfrFd. The pSV2 plasmid is commercially available and consists of DNA segments from varîous sources: pBR322 DNA (thin line) contains the pBR322 origin of DNA réplication (pBR ori) and the lactamase ampicillin résistance gene (Amp); SV40 DNA, represented by wider hatching and marked, contains the SV40 origin of DNA réplication (SV40 ori), early promoter (5' to the dhfr and neo genes), and polyadenylation signal (3' to the dhfr and neo genes). The SV40-derived polyadenylation signal (pA) is also placed at the 3’ end of the Fd gene.

For the kappa gene, PCR is carried out using 5'AAGAAAGCTTGCCGCCACCATGTTCTCACTAGCTCT-3' (SEQ ID NO:46) as the 5' primer and a C£-derived 3' primer (5'-CGGGATCCTTCTCCCTCTAACACTCT-3' SEQ ID NO:47). DNA sequence is confirmed to contain the complété Vp; and human C« régions. After digestion with proper restriction enzymes, the kappa DNA fragments are inserted at the HindlII and BamHI restriction sites of the expression vector cassette pSV2neo-TUS to give pSV2neoK. The expression of both Fd and .kappa genes are driven by the HCMV-derived enhancer and promoter éléments. Since the Fd gene does not include the cysteine amino acid residue invoived in the inter-chain disulfide bond, this recombinant chimeric Fab contains non-covalently linked heavy- and lîght-chains. This chimeric Fab is designated as cFab.

To obtain recombinant Fab with an inter-heavy and light chain disulfide bond, the above Fd gene may be extended to include the coding sequence for additional 9 amino 5 acids (EPKSCDKTH SEQ ID NO:48) from the hinge région of human IgGl. The BstEII-BamHI DNA segment encoding 30 amino acids at the 3' end of the Fd gene may be replaced with DNA segments encoding the extended Fd, resulting in pSV2dhfrFd/9aa.

3. Expression and Purification of Chimeric Anti-MASP-2 IgG

To generate cell lînes secreting chimeric anti-MASP-2 IgG, NSO cells are I0 transfected with purified plasmid DNAs of pSV2neoVH-huC.yl and pSV2neoV-huC kappa by eiectroporation. Transfected cells are selected in the presence of 0.7 mg/ml G418. Cells are grown in a 250 ml spinner flask using serum-containing medium.

Culture supernatant of 100 ml spinner culture is loaded on a ΙΟ-ml PROSEP-A column (Bioprocessing, Inc., Princeton, N.J.). The column is washed with 10 bed 15 volumes ofPBS. The bound antibody is eluted with 50 mM citrate buffer, pH 3.0. Equal volume of l M Hepes, pH 8.0 is added to the fraction containing the purified antibody to adjust the pH to 7.0. Residual salts are removed by buffer exchange with PBS by Millipore membrane ultrafiltration (M.W. cut-off: 3,000). The protein concentration of the purified antibody is determined by the BCA method (Pierce).

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

To generate cell lines secreting chimeric anti-MASP-2 Fab, CHO cells are transfected with purified plasmid DNAs of pSV2dhfrFd (or pSV2dhfrFd/9aa) and pSV2neokappa, by eiectroporation. Transfected cells are selected in the presence of G418 and methotrexate. Selected cell lines are amplified in increasîng concentrations of 25 methotrexate. Cells are single-cell subcloned by limiting dilution. High-producing single-cell subcloned cell lines are then grown in 100 ml spinner culture using sérum-ffee medium.

Chimeric anti-MASP-2 Fab is purified by affinity chromatography using a mouse anti-idiotypic Mo Ab to the MASP-2 MoAb. An anti-idiotypic MASP-2 MoAb can be 30 made by immuntzing mice with a murine anti-MASP-2 MoAb conjugated with keyhole Hmpet hemocyanin (KLH) and screening for spécifie MoAb binding that can be competed with human MASP-2. For purification, 100 ml of supematant from spinner cultures of CHO cells producing cFab or cFab/9aa are loaded onto the affinity column coupled with an anti-idiotype MASP-2 MoAb. The column is then washed thoroughly with PBS before the bound Fab is eluted with 50 mM diethylamine, pH 11.5. Residual salts are removed by buffer exchange as described above. The protein concentration of the purified Fab is determined by the BCA method (Pierce).

The ability of the chimeric MASP-2 IgG, cFab, and cFAb/9aa to inhibit MASP-2-dependent complément pathways may be determined by using the inhibitory assays described in Example 2 or Example 7.

EXAMPLE 7

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

C4 Cleavage Assay: A C4 cleavage assay has been described by Petersen, S.V., et al., J Immunol. Methods 257:107, 2001, which measures lectin pathway activation resulting from lipoteichoic acid (LTA) from 5. aureus which binds L-ficolin.

Reagents: Formalin-fixed 5. aureous (DSM20233) is prepared as follows: bacteria is grown overnight at 37°C in tryptic soy blood medium, washed three times with PBS, then fixed for 1 h at room température in PBS/0.5% formai in, and washed a further three times with PBS, before being resuspended in coating buffer (15 mM Na2Co3, 35 mM NaHCOj, pH 9.6).

Assay: The wells of a Nunc MaxiSorb microtiter plate (Nalgene Nunc International, Rochester, NY) are coated with: 100 μΙ of formalin-fixed S. aureus DSM20233 (Οϋ55θ ⁼ θ·5) in coating buffer with l ug of L-ficolin in coating buffer. After overnight incubation, wells are blocked with 0.1% human sérum albumin (HSA) in TBS (10 mM Tris-HCl, 140 mM NaCl, pH 7.4), then are washed with TBS containing 0.05% Tween 20 and 5 mM CaC12 (wash buffer). Human sérum samples are diluted tn 20 mM Tris-HCI, 1 M NaCl, 10 mM CaC12, 0.05% Triton X-100, 0.1% HSA, pH 7.4, which prevents activation of endogenous C4 and dissociâtes the Cl complex (composed of Clq, Clr and Cl s). MASP-2 inhibitory agents, including anti-MASP-2 MoAbs and inhibitory peptides are added to the serum samples in varying concentrations. The diluted sampies are added to the plate and încubated ovemight at 4°C. After 24 hours, the plates are washed thoroughly with wash buffer, then 0.1 gg of purified human C4 (obtained as described in Dodds, A.W., Methods Enzymol. 223:46, 1993) in 100 μΐ of 4 mM barbital. 145 mM NaCI, 2 mM CaCQ, I mM MgCQ, pH 7.4 is added to each well. After 1.5 h at 37°C, the plates are washed again and C4b déposition is detected using alkaline phosphatase-conjugated chicken anti-human C4c (obtained from Immunsystem, Uppsala, Sweden) and measured using the colorimétrie substrate p-nitrophenyl phosphate.

C4 Assay on mannan: The assay described above is adapted to measure lectin pathway activation via MBL by coating the plate with LSP and mannan prior to adding serum mixed with various MASP-2 inhibitory agents.

C4 assay on H-fïcolin (Hakata Ag): The assay described above is adapted to measure lectin pathway activation via H-ficolin by coating the plate with LPS and H-fïcolin prior to adding serum mixed with various MASP-2 inhibitory agents.

EXAMPLE 8

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

Methods: Immune complexes were generated in situ by coating microtiter plates (Maxisorb, Nunc, cat. No. 442404, Fisher Scientific) with O.I% human serum albumin in lOmM Tris, 140 mM NaCI, pH 7.4 for l hours at room température followed by ovemight incubation at 4°C with sheep anti whole serum antîserum (Scottish Antibody Production Unit, Carluke, Scotland) diluted L1000 in TBS/tween/Ca2+. Serum samples were obtained from wild-type and MASP-2-/- mice and added to the coated plates. Control samples were prepared in which Clq was depleted from wild-type and MASP-2-/- serum samples. Clq-depleted mouse serum was prepared using protein-A-coupled Dynabeads (Dynal Biotech, Oslo, Norway) coated with rabbit anti-human Clq IgG (Dako, Glostrup, Denmark), according to the supplieris instructions. The plates were încubated for 90 minutes at 37°C. Bound C3b was detected with a polyclonal anti-human-C3c Antibody (Dako A 062) diluted în TBS/tw/ Ca^-1-7 at 1:1000. The secondary antibody is goat anti-rabbit IgG.

ιοο

Results: FIGURE 7 shows the relative C3b déposition levels on plates coated with IgG in wild-type sérum, MASP-2-/- sérum, Clq-depleted wild-type and Clq-depleted MASP-2-/- sérum, These results demonstrate that the classical pathway is intact in the MASP-2-/- mouse strain.

EXAMPLE 9

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

Methods: To test the effect of a MASP-2 inhibitory agent on conditions of complément activation where the classical pathway is inîtiated by immune complexes, triplicate 50 μΐ samples containing 90% NHS are incubated at 37°C in the presence of I0 gg/ml immune complex (IC) or PBS, and parallel triplicate samples (+/-IC) are also încluded which contain 200 nM anti-properdin monoclonal antibody during the 37°C incubation. After a two hour incubation at 37°C, 13 mM EDTA is added to ali samples to stop further complément activation and the samples are immediately cooled to 5°C. The samples are then stored at -70°C prier to being assayed for complément activation products (C3a and sC5b-9) using ELISA kits (Quidel, Catalog Nos. A015 and A009) following the manufacturer's instructions.

EXAMPLE 10

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

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

To block MASP-2 functional activity, an antibody or Fab2 antibody fragment must bind and interfère with a structural epitope on MASP-2 that is required for MASP-2

ΙΟΙ functional activity. Therefore, many or ail of the high affïnîty binding anti-MASP-2 Fab2s may not inhibit MASP-2 functional activity unless they bind to structural epitopes on MASP-2 that are directly involved in MASP-2 functional activity.

A functional assay that measures inhibition of lectin pathway C3 convertase formation was used to evaluate the blocking activity of anti-MASP-2 Fab2s. It is known that the primary physiological rôle of MASP-2 In the lectin pathway is to generate the next functional component of the lectin-mediated complément pathway, namely the lectin pathway C3 convertase. The lectin pathway C3 convertase is a critical enzymatic complex (C4bC2a) that proteolytically cleaves C3 into C3a and C3b. MASP-2 is not a structural component of the lectin pathway C3 convertase (C4bC2a); however, MASP-2 functional activity is required in order to generate the two protein components (C4b, C2a) that comprise the lectin pathway C3 convertase. Furthermore, ail of the separate functional activities of MASP-2 listed above appear to be required in order for MASP-2 to generate the lectin pathway C3 convertase. For these reasons, a preferred assay to use in evaluating the blocking activity of anti-MASP-2 Fab2s is believed to be a functional assay that measures inhibition of lectin pathway C3 convertase formation.

Génération of High Affinity Fab2s: A phage display library of human variable light and heavy chain antibody sequences and automated antibody sélection technology for identifying Fab2s that react with selected ligands of interest was used to create high affmity Fab2s to rat MASP-2 protein (SEQ ID NO:55). A known amount of rat MASP-2 (~l mg, >85% pure) protein was utilized for antibody screening. Three rounds of amplification were utilized for sélection of the antibodies with the best affmity. Approximately 250 different hits expressing antibody fragments were picked for ELISA screening. High affinity hits were subsequently sequenced to détermine uniqueness of the different antibodies.

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

I02

Assays used to Evaluate the Inhibitory (blocking) Activity of Anti-MASP-2 Fab2s

1. Assay to Measure Inhibition of Formation of Lectin Pathway C3 Convertase:

Background: The lectin pathway C3 convertase is the enzymatic complex (C4bC2a) 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 ternis of medîating inflammation. MASP-2 is not a structural component of the iectin pathway C3 convertase (C4bC2a); therefore anti-MASP-2 antibodies (or Fab2) will not directly inhibit activity of preexîsting C3 convertase. However, 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, anti-MASP-2 Fab2 which inhibit MASP-2 functional activity (î.e., blocking anti-MASP-2 Fab2) 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 îmmobilized on the bottom of the plastic wells via ester or amide linkages, thus facilîtating détection of C3b in the ELISA assay.

Yeast mannan is a known activator of the lectin pathway. In the following method to measure formation of C3 convertase, plastic wells coated with mannan were incubated for 30 min at 37°C with diluted rat sérum to activate the lectin pathway. The wells were then washed and assayed for C3b îmmobilized onto the wells using standard ELISA methods. The amount of C3b generated in this assay is a direct reflectîon of the de novo formation of lectin pathway C3 convertase. Anti-MASP-2 Fab2s at selected concentrations were tested in this assay for their ability to inhibit C3 convertase formation and conséquent C3b génération.

Methods:

96-welI Costar Medium Binding plates were incubated ovemight at 5°C with mannan diluted in 50 mM carbonate buffer, pH 9.5 at 1 ug/50 Tl/well. After ovemight incubation, each well was washed three times with 200 Tl PBS. The wells were then blocked with 100 Tl/well of 1% bovine sérum albumin in PBS and incubated for one hour

103 at room température with gentle mixing. Each well was then washed three times with 200 Tl of PBS. The anti-MASP-2 Fab2 samples were diluted to selected concentrations in Ca^ and Mg⁺⁺ containing GVB buffer (4.0 mM barbital, I4l mM NaCi, LO mM MgCh, 2.0 mM CaCh, 0.1% gelatin, pH 7.4) at 5 C. A 0.5% rat sérum was added to the above samples at 5 C and 100 Tl was transferred to each well. Plates were covered and incubated for 30 minutes in a 37 C waterbath to allow complément activation. The reaction was stopped by transferring the plates from the 37 C waterbath to a container containing an ice-water mix. Each well was washed five times with 200 Tl with PBS-Tween 20 (0.05% Tween 20 in PBS), then washed two times with 200 Tl PBS. A 100 Tl/well of 1:10,000 dilution of the primary antibody (rabbit anti-human C3c, DAK.0 A0062) was added in PBS containing 2.0 mg/ml bovine sérum albumin and incubated 1 hr at room température with gentle mixing. Each well was washed 5 x 200 Tl PBS. 100 Tl/well of 1:10,000 dilution of the secondary antibody (peroxidase-conjugated goat anti-rabbit IgG, Amerîcan Qualex A102PU) was added in PBS containing 2.0 mg/ml bovine sérum albumin and incubated for one hour at room température on a shaker with gentle mixing. Each well was washed five times with 200 Tl with PBS. 100 Tl/well of the peroxidase substrate TM B (Kirkegaard & Perry Laboratoires) was added and incubated at room température for 10 min. The peroxidase reaction was stopped by adding 100 Tl/well of 1.0 Μ H3PO4 and the OD450. was measured.

2. Assay to Measure Inhibition of MASP-2-dépendent C4 Cleavage

Background: The serine protease activity of MASP-2 is highly spécifie and only two protein substrates for MASP-2 hâve been identified; C2 and C4. Cleavage of C4 generates C4a and C4b. Anti-MASP-2 Fab2 may bind to structural epitopes on MASP-2 that are directly involved in C4 cleavage (e.g., MASP-2 binding site for C4; MASP-2 serine protease catalytic site) and thereby inhibit the C4 cleavage functional activity of MASP-2.

Yeast mannan is a known activator of the lectin pathway. In the following method to measure the C4 cleavage activity of MASP-2, plastic wells coated with mannan were incubated for 30 minutes at 37 C with diluted rat sérum to activate the lectin pathway. Sînce the primary antibody used in this ELISA assay only recognîzes human C4, the diluted rat sérum was also supplemented with human C4 (1.0 Tg/ml). The

104 wells were then washed and assayed for human C4b immobilized onto the wells using standard ELISA methods. The amount of C4b generated in this assay is a measure of MASP-2 dépendent C4 cleavage activity. Anti-MASP-2 Fab2 at selected concentrations were tested in this assay for their ability to inhibit C4 cleavage.

Methods: 96-well Costar Medium Binding plates were incubated ovemight at 5 C with mannan diluted in 50 mM carbonate buffer, pH 9.5 at l.O Tg/50 Tl/well. Each well was washed 3X with 200 Tl PBS. The wells were then blocked with 100 Tl/well of l% bovine serum albumîn in PBS and incubated for one hour at room température with gentle mixing. Each well was washed 3X with 200 Tl of PBS. Anti-MASP-2 Fab2 samples were diluted to selected concentrations in Ca^ and Mg^ containing GVB buffer (4.0 mM barbital, I4l mM NaCl, l.O mM MgCh, 2.0 mM CaCl₂, 0.1% gelatin, pH 7.4) at 5 C. l.O Tg/ml human C4 (Quidel) was also included in these samples. 0.5% rat serum was added to the above samples at 5 C and 100 Tl was transferred to each well. The plates were covered and incubated for 30 min in a 37 C waterbath to allow complément activation. The reaction was stopped by transferring the plates from the 37 C waterbath to a container containing an ice-water mix, Each well was washed 5 x 200 Tl with PBS-Tween 20 (0.05% Tween 20 in PBS), then each well was washed with 2X with 200 Tl PBS. 100 Tl/well of 1:700 dilution of biotin-conjugated chîcken anti-human C4c (Immunsystem AB, Uppsaia, Sweden) was added in PBS containing 2.0 mg/ml bovine serum albumin (BSA) and incubated one hour at room température with gentle mixing. Each well was washed 5 x200 Tl PBS. 100 Tl/well of 0.1 Tg/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 température on a shaker with gentle mixing. Each well was washed 5 x200 Tl with PBS. 100 Tl/well of the peroxidase substrate TMB (Kirkegaard & Perry Laboratories) was added and incubated at room température for 16 min. The peroxidase reaction was stopped by adding 100 Tl/well of 1.0 Μ H3PO4 and the OD450 .was measured.

3. Binding Assay of anti-rat MASP-2 Fab2 to 'Native' rat MASP-2

Background: MASP-2 is usually présent in plasma as a MASP-2 dimer complex that also includes spécifie lectin molécules (mannose-binding protein (MBL) and ficolins). Therefore, if one is interested in studying the binding of anti-MASP-2 Fab2 to

105 the physiologically relevant form of MASP-2, it is important to develop a binding assay in which the interaction between the Fab2 and 'native' MASP-2 in plasma is used, rather than purified recombinant MASP-2. In this binding assay the 'native' MASP-2-MBL complex from 10% rat sérum was first immobilized onto mannan-coated wells. The binding affinity of various anti-MASP-2 Fab2s to the immobilized 'native' MASP-2 was then studied using a standard ELISA methodology.

Methods: 96-well Costar High Binding plates were incubated ovemight at 5°C with mannan diluted in 50 mM carbonate buffer, pH 9.5 at l Tg/50 Tl/well. Each well was washed 3X with 200 Tl PBS. The wells were blocked with 100 Tl/well of 0.5% nonfat dry milk in PBST (PBS with 0.05% Tween 20) and incubated for one hour at room température with gentle mixing. Each well was washed 3X with 200 Tl of TBS/Tween/Ca⁴^ Wash Buffer (Tris-buffered saline, 0.05% Tween 20, containing 5.0 mM CaCh, pH 7.4. 10% rat sérum in High Sait Binding Buffer (20 mM Tris, l.O M NaCl, iOmM CaCh, 0.05% Triton-XlOO, 0.1% (w/v) bovine sérum albumin, pH 7.4) was prepared on ice. 100 Tl/well was added and incubated ovemight at 5°C. Wells were washed 3X with 200 Tl of TBS/Tween/Ca^4-4 Wash Buffer. Wells were then washed 2X with 200 TI PBS. 100 Tl/well of selected concentration of anti-MASP-2 Fab2 diluted in Ca~ and Mg⁴^ containing GVB Buffer (4.0 mM barbital, I4l mM NaCl, l.O mM MgCh, 2.0 mM CaCh, 0.1% gelatin, pH 7.4) was added and incubated for one hour at room température with gentle mixing. Each well was washed 5 x 200 Tl PBS. 100 Tl/well of HRP-conjugated goat anti-Fab2 (Biogenesis Cat No 0500-0099) diluted 1:5000 in 2.0 mg/ml bovine sérum albumin in PBS was added and incubated for one hour at room température with gentle mixing. Each well was washed 5 x 200 Tl PBS. 100 Tl/well of the peroxidase substrate TMB (Kirkegaard & Perry Laboratories) was added and incubated at room température for 70 min. The peroxidase reaction was stopped by adding 100 Tl/well of 1.0 Μ H3PO4 and OD450. was measured.

RESULTS:

Approximately 250 different Fab2s that reacted with high affinity to the rat MASP-2 protein were picked for ELISA screening. These high affinity Fab2s were sequenced to déterminé the uniqueness of the different antibodies, and 50 unique anti-MASP-2 antibodies were purified for further analysis. 250 ug of each purified Fab2

106 antibody was used for characterization of MASP-2 binding affmîty and complément pathway functional testing. The results of this analysis is shown below in TABLE 7.

TABLE 7: ANTI-MASP-2 FAB2 THAT BLOCK LECTIN PATHWAY

COMPLEMENT ACTIVATION
Fab2 antibody #	C3 Convertase (IC50 (nM)	Kd	C4 Cleavage IC50 (nM)
88	0.32	4.1	ND
41	0.35	0.30	0.8l
11	0.46	0.86	<2 nM
86	0.53	1.4	ND
8l	0.54	2.0	ND
66	0.92	4.5	ND
57	0.95	3.6	<2 nM
40	l.l	7.2	0.68
58	1.3	2.6	ND
60	1.6	3.I	ND
52	1.6	5.8	<2 nM
63	2.0	6.6	ND
49	2.8	8.5	<2 nM
89	3.0	2.5	ND
71	3.0	10.5	ND
87	6.0	2.5	ND
67	IO.O	7.7	ND

As shown above in TABLE 7, of the 50 anti-MASP-2 Fab2s tested, seventeen Fab2s were identified as MASP-2 blocking Fab2 that potently inhibit C3 convertase formation with IC50 equal to or less than IO nM Fab2s (a 34% positive hit rate). Eight of IO the seventeen Fab2s identified hâve IC50S in the subnanomolar range. Furthermore, ail seventeen of the MASP-2 blocking Fab2s shown in TABLE 7 gave essentially complété inhibition of C3 convertase formation in the lectin pathway C3 convertase assay. FIGURE 8A graphically illustrâtes the results of the C3 convertase formation assay for Fab2 antibody #l l, which is représentative of the other Fab2 antibodies tested, the results

107 of which are shown in TABLE 7. This is an important considération, since it is theoretically possible that a blocking Fab2 may only fractionally inhibit MASP-2 functîon even when each MASP-2 molécule is bound by the Fab2.

Aithough mannan is a known activator of the lectin pathway, it is theoretically possible that the presence of anti-mannan antibodies in the rat sérum might also activate the classical pathway and generate C3b via the classical pathway C3 convertase. However, each of the seventeen blocking anti-MASP-2 Fab2s listed in this example potently inhibits C3b génération (>95 %), thus demonstrating the specificity of this assay for lectin pathway C3 convertase.

Binding assays were also performed with ail seventeen of the blocking Fab2s in order to calculate an apparent Kj for each. The results of the binding assays of anti-rat MASP-2 Fab2s to native rat MASP-2 for six of the blocking Fab2s are also shown in TABLE 7. FIGURE 8B graphically illustrâtes the results of a binding assay with the Fab2 antibody #11. Similar binding assays were also carried out for the other Fab2s, the results of which are shown in TABLE 7. In general, the apparent K(js obtained for binding of each of the six Fab2s to 'native' MASP-2 corresponds reasonably well with the IC50 for the Fab2 in the C3 convertase functional assay. There is evidence that MASP-2 undergoes a conformational change from an 'inactive' to an 'active' form upon activation of its protease activity (Feinberg et al., EMBO J 22:2348-59 (2003); Gai et al., J. Biol. Chem. 250:33435-44 (2005)). In the normal rat plasma used in the C3 convertase formation assay, MASP-2 is présent primarily in the 'inactive' zymogen conformation. In contrast, in the binding assay, MASP-2 is présent as part of a complex with MBL bound to immobilized mannan; therefore, the MASP-2 would be in the 'active' conformation (Petersen et al., J. Immunol Methods 257:107-16, 2001). Consequently, one would not necessarily expect an exact correspondence between the IC50 and Kj for each of the seventeen blocking Fab2 tested in these two functional assays since in each assay the Fab2 would be binding a different conformational form of MASP-2. Never-the-less, with the exception of Fab2 #88, there appears to be a reasonably close correspondence between the IC50 and apparent Kd for each of the other sixteen Fab2 tested in the two assays (see TABLE 7).

108

Several of the blocking Fab2s were evaiuated for inhibition of MASP-2 mediated cleavage of C4. FIGURE 8C graphically illustrâtes the results of a C4 cleavage assay, showing inhibition with Fab2 #41, with an IC50=0.8l nM (see TABLE 7). As shown in FIGURE 9, ail of the Fab2s tested were found to inhibit C4 cleavage with IC50s similar to those obtained in the C3 convertase assay (see TABLE 7).

Although mannan is a known activator of the lectin pathway, it is theoretically possible that the presence of anti-mannan antibodies in the rat sérum might also activate the classical pathway and thereby generate C4b by Cls-mediated cleavage of C4. However, several anti-MASP-2 Fab2s hâve been identified which potently inhibit C4b génération (>95 %), thus demonstrating the specificity of this assay for MASP-2 mediated C4 cleavage. C4, like C3, contains an unusual and highly reactive thioester group as part of its structure. Upon cleavage of C4 by MASP-2 in this assay, the thioester group on C4b can form a covalent bond with hydroxyl or amino groups on macromolecules immobiiized on the bottom of the plastic wells via ester or amide linkages, thus facilitating détection of C4b in the ELISA assay.

These studies clearly demonstrate the création of high affinity FAB2s to rat MASP-2 protein that ftinctionally block both C4 and C3 convertase activity, thereby preventing lectin pathway activation.

EXAMPLE 11

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

Methods:

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

rat MASP-2A, a full length MASP-2 protein, inactivated by alterîng the serine at the active center to alanine (S613A);

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

CUBI-II, an Ν-termînal fragment of rat MASP-2 that contains the CUBI, EGF-Iike and CUBII domains only; and

109

CUBI/EGF-like, an N-terminal fragment of rat MASP-2 that contaîns the CUB1 and EGF-like domains only.

These proteins were purified from culture supematants by nickel-affinity chromatography, as previously described (Chen et al., J. Biol. Chem. 276.25894-02 (2001)).

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

Ail recombinant proteins were dialyzed into TBS buffer and their concentrations determined by measuring the OD at 280 nm.

DOT BLOT ANALYSIS:

Serial dilutions of the five recombinant MASP-2 polypeptides described above and shown in FIGURE 10 (and the thioredoxin polypeptide as a négative control for CCPII-serine protease polypeptide) were spotted onto a nîtrocellulose membrane. The amount of protein spotted ranged from 100 ng to 6.4 pg, in five-fold steps. In later experiments, 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 LO pg/ml anti-MASP-2 Fab2s in blocking buffer (containing 5.0 mM Ca²⁺). Bound Fab2s were detected using HRP-conjugated anti-human Fab (AbD/Serotec; diluted 1/10,000) and an ECL détection kit (Amersham). One membrane was incubated with polyclonal rabbît-anti human MASP-2 Ab (described in Stover et al., JImmunol I63-.(&tf>-59 (1999)) as a positive control. In this case, bound Ab was detected using HRP-conjugated goat anti-rabbit IgG (Dako; diluted 1/2,000).

MASP-2 Binding Assay

ELISA plates were coated with 1.0 pg/well of recombinant MASP-2A or CUBI-II polypeptide in carbonate buffer (pH 9.0) overnight at 4°C. Wells were blocked with 1% BSA in TBS, then serial dilutions of the anti-MASP-2 Fab2s were added in TBS containing 5.0 mM Ca²⁺. The plates were incubated for one hour at RT. After washing three times with TBS/tween/Ca²⁺, HRP-conjugated anti-human Fab (AbD/Serotec)

HO diluted l/l0,000 in TBS/ Ca²⁺ was added and the plates incubated for a further one hour at RT, Bound antibody was detected using a TMB peroxidase substrate kit (Biorad).

RESULTS:

Results of the dot blot analysis demonstrating the reactivity of the Fab2s with 5 various MASP-2 polypeptides are provided below in TABLE 8. The numerical values provided in TABLE 8 indicate the amount of spotted protein required to give approximately half-maximal signal strength. As shown, ail of the polypeptides (with the exception of the thioredoxin fusion partner alone) were recognized by the positive control Ab (polyclonal anti-human MASP-2 sera, raîsed in rabbits).

TABLE 8: REACTIVITY WITH VARIOUS RECOMBINANT RAT MASP-2 ______________POLYPEPTIDES ON DOT BLOTS______________

Fab2 Antibody #	MASP-2A	CUBI-II	CUBI/EGF-like	CCPII-SP	Thioredoxin
40	0.16 ng	NR	NR	0.8 ng	NR
4I	0.16 ng	NR	NR	0.8 ng	NR
H	0.16 ng	NR	NR	0.8 ng	NR
49	0.16 ng	NR	NR	>20 ng	NR
52	0.16 ng	NR	NR	0.8 ng	NR
57	0.032 ng	NR	NR	NR	NR
58	0.4 ng	NR	NR	2.0 ng	NR
60	0.4 ng	0.4 ng	NR	NR	NR
63	0.4 ng	NR	NR	2.0 ng	NR
66	0.4 ng	NR	NR	2.0 ng	NR
67	0.4 ng	NR	NR	2.0 ng	NR
71	0.4 ng	NR	NR	2.0 ng	NR
81	0.4 ng	NR	NR	2.0 ng	NR
86	0.4 ng	NR	NR	10 ng	NR
87	0.4 ng	NR	NR	2.0 ng	NR
Positive Control	<0.032 ng	0.16 ng	0.16 ng	<0.032 ng	NR

111

NR = No reaction. The positive control antibody is polyclonal anti-human MASP-2 sera, raised in rabbits.

Ail of the Fab2s reacted with MASP-2A as well as MASP-2K (data not shown). The majority of the Fab2s recognized the CCPII-SP polypeptide but not the N-terminal fragments. The two exceptions are Fab2 #60 and Fab2 #57. Fab2 #60 recognizes MASP-2A and the CUBI-ll fragment, but not the CUBI/EGF-like polypeptide or the CCPII-SP polypeptide, suggesting it binds to an epitope in CUBII, or spanning the CUBII and the EGF-like domain. Fab2 # 57 recognizes MASP-2A but not any ofthe MASP-2 fragments tested, indicating that this Fab2 recognizes an epitope in CCPI. Fab2 #40 and #49 bound only to complété MASP-2A. In the ELISA binding assay shown in FIGURE 11, Fab2 #60 also bound to the CUBI-II polypeptide, albeit with a slightly lower apparent affinity.

These finding demonstrate the identification of unique blocking Fab2s to multiple régions of the MASP-2 protein

EXAMPLE 12

This Example describes the results of MASP-2-/- in a Murine Macular Degeneration Model.

Background/Rationale: Age-related macular degeneration (AMD) is the leading cause of blindness after âge 55 in the indu striai ized world. AMD occurs in two major forms: neovascular (wet) AMD and atrophie (dry) AMD. The neovascular (wet) form accounts for 90% of severe visual loss associated with AMD, even though only -20% of individuals with AMD develop the wet form. Clinical hallmarks of AMD include multiple drusen, géographie atrophy, and choroidal neovascularization (CNV). In December, 2004, the FDA approved Macugen (pegaptanib), a new class of ophthalmic drugs to specîfically target and block the effects of vascular endothélial growth factor (VEGF), for treatment of the wet (neovascular) form of AMD (Ng et al., Nat Rev. Drug Discov 5:123-32 (2006)). Although Macugen represents a promising new therapeutic option for a subgroup of AMD patients, there remains a pressing need to develop additional treatments for this complex disease. Multiple, independent fines of investigation implicate a central rôle for complément activation in the pathogenesis of

H2

AMD. The pathogenesis of choroidal neovascularization (CNV), the most serious form of AMD, may invoive activation of complément pathways.

Over twenty-five years ago, Ryan described a laser-induced injury model of CNV în animais (Ryan, SJ., Tr. Am. Opth. Soc. LXXVlI:707-745, 1979). The model was inltially developed using rhésus monkeys, however, the same technology has since been used to develop similar models of CNV in a variety of research animais, including the mouse (Tobe et al., Am. J. Pathol. /55:1641-46, 1998). In this model, laser photocoagulation is used to break Bruch's membrane, an act which results in the formation of CNV-lîke membranes. The laser-induced model captures many of the important features of the human condition (for a recent review, see Ambati et al., Survey Ophthalmology 48:257-293, 2003). The laser-induced mouse model is now well established, and is used as an experimental basis in a large, and ever increasing, number of research projects. It is generally accepted that the laser-induced model shares enough biological similarity with CNV in humans that preclinical studies of pathogenesis and drug inhibition using this model are relevant to CNV in humans.

Methods:

A MASP-2-/- mouse was generated as described in Example 1 and backcrossed for 10 générations with C57B1/6. The current study compared the results when MASP-2 (-/-) and MASP-2 (+/+) male mice were evaluated in the course of laser-induced CNV, an accelerated model ofneovascular AMD focusing on the volume of laser-induced CNV by scanning laser confocal microscopy as a measure of tissue injury and détermination of levels of VEGF, a potent angiogenic factor implîcated in CNV, in the retinal pigment epithelium (RPE)/choroids by ELISA after laser injury.

Induction of choroidal neovascularization (CNV): Laser photocoagulation (532 nm, 200 mW, 100 ms, 75pm; Ocullght GL, Iridex, Mountain View, CA) was performed on both eyes ofeach animal on day zéro by a single îndividual masked to drug group assignment. Laser spots were applîed in a standardized fashion around the optic nerve, using a slit lamp delivery System and a coverslip as a contact lens. The morphologie end point of the laser injury was the appearance of a cavitation bubble, a sign thought to correlate with the disruption of Bruch's membrane. The detailed methods and endpoints that were evaluated are as follows.

H3

Fluorescein Angiography: Fluoresceîn angiography was performed with a caméra and imaging System (TRC 50 IA caméra; ImageNet 2.01 System; Topcon, Paramus , NJ) at l week after laser photocoagulation. The photographs were captured with a 20-D lens in contact with the fundus caméra lens after intraperitoneal injection of 0.1 inl of 2.5% fluorescein sodium. A retina expert not involved in the laser photocoagulation or angiography evaluated the fluorescein angiograms at a single sitting in masked fashion.

Volume of choroidal neovascularîzation (CNV): One week after laser injury, eyes were enucleated and fixed with 4% paraformaldéhyde for 30 min at 4°C. Eye cups were obtained by removing anterior segments and were washed three times in PBS, followed by déhydration and rehydration through a methanol sériés. After blocking twice with buffer (PBS containing l% bovine serumalbumin and 0.5% Triton X-100) for 30 minutes at room température, eye cups were incubated ovemight at 4°C with 0.5% FITC-isolectin B4 (Vector laboratories, Burlingame, CA), diluted with PBS containing 0.2% BSA and 0.l% Triton X-100, which bînds terminal β-D-galactose residues on the surface of endothélial cells and selectively labels the murine vasculature. After two washings with PBS containing 0.l% Triton X-100, the neurosensory retina was gently detached and severed from the optic nerve. Four relaxing radial incisions were made, and the remaining RPE -choroid-sclera complex was flatmounted in antifade medium (Immu-Mount Vectashield Mounting Medium; Vector Laboratories) and cover-slipped.

Flatmounts were examined with a scanning laser confocal microscope (TCS SP; Leica, Heidelberg, Germany). Vessels were visualîzed by exciting with blue argon wavelength (488 nm) and capturing émission between 515 and 545 nm. A 40X oil-immersion objective was used for ail imaging studies. Horizontal optical sections (l pm step) were obtained from the surface of the RPE-choroid-sclera complex. The deepest focal plane in which the surrounding choroidal vascular network connecting to the lésion could be identîfied was judged to be the floor of the lésion. Any vessel in the laser-targeted area and superfîcial to this reference plane was judged as CNV. Images of each section were digitally stored. The area of CNV-related fluorescence was measured by computerized image analysis with the microscope software (TCS SP; Leica). The summation of whole fluorescent area in each horizontal section was used as an index for

114 the volume of CNV. Imaging was performed by an operator masked to treatment group assîgnment.

Because the probability of each laser lésion developing CNV is influenced by the group to which it belongs (mouse, eye, and laser spot), the mean lésion volumes were compared using a linear mixed model with a splît plot repeated-measures design, The whole plot factor was the genetic group to which the animal belongs, whereas the split plot factor was the eye. Statistical significance was determined at the 0.05 level. Post hoc comparisons of means were constructed with a Bonferroni adjustment for multiple comparisons.

VEGF ELISA. At three days after injury by 12 laser spots, the RPE-choroid complex was sonicated in lysis buffer (20 mM imidazole HCl, lOmM KCl. I mM MgCLi, 10 mM EGTA, 1% Triton X-100, 10 mM NaF, 1 mM Na molybdate, and 1 mM EDTA with protease inhïbitor) on ice for 15 min. VEGF protein levels in the supematant were determined by an ELISA kit (R&D Systems, Minneapolis, MN) that recognizes ail splice variants, at 450 to 570 nm (Emax; Molecular Devices, Sunnyvale, CA), and normalized to total protein. Duplicate measurements were performed in a masked fashion by an operator not involved in photocoagulation, imaging, or angiography. VEGF numbers were represented as the mean +/- SEM of at least three independent experiments and compared using the Mann-Whitney U test. The null hypothesis was rejected at P<0.05.

RESULTS:

Assessment of VEGF Levels:

FIGURE 12A graphically illustrâtes the VEGF protein levels in RPE-choroid complex isolated from C57B16 wildtype and MASP-2(-/-) mice at day zéro. As shown in FIGURE 12A, the assessment of VEGF levels indicate a decrease in baseline levels for VEGF in the MASP-2 (-/-) mice versus the C57bl wildtype control mice. FIGURE 12B graphically illustrâtes VEGF protein levels measured at day three following laser induced injury. As shown in FIGURE 12B VEGF levels were significantly increased in the wildtype (+/+) mice three days following laser induced injury, consistent with published studies (NozakietaL, Proc. Natl. Acad. Sci. USA 103:2328-33 (2006)). However, surprisingly very low levels of VEGF were seen in the MASP-2 (-/-) mice.

H5

Assessment of choroidal neovascularizatïon (CNV):

In addition to the réduction in VEGF levels following laser induced macular degeneration, CNV area was determined before and after laser înjury. FIGURE 13 graphically illustrâtes the CNV volume measured in C57bl wîldtype mice and MASP-2(-/-) mice at day seven following laser induced injury. As shown in FIGURE 13, the MASP-2 (-/-) mice displayed about a 30% réduction in the CNV area following laser induced damage at day seven în comparison to the wîldtype control mice.

These findings indicate a réduction in VEGF and CNV as seen in the MASP (-/-) mice versus the wîldtype (+/+) control and that blockade of MASP-2 with an inhibitor would hâve a préventive or therapeutic effect in the treatment of macular degeneration.

EXAMPLE 13

This Example describes the pharmacodynamie analysis of représentative high affïnity anti-MASP-2 Fab2 antibodies that were identîfïed as described in Example 10.

Background/Rationale:

As described in Example 10, in order to identifÿ high-affïnity antibodies that block the rat Iectin pathway, rat MASP-2 protein was utilized to pan a phage display library. This library was designed to provide for high immunological diversity and was constructed using entirely human immunoglobin gene sequences. As described in Example 10, approximately 250 individual phage clones were identîfïed that bound with high affïnity to the rat MASP-2 protein by ELISA screening. Sequencing of these clones identîfïed 50 unique MASP-2 antibody encoding phage. Fab2 protein was expressed from these clones, purified and analyzed for MASP-2 binding affïnity and lectin complément pathway functional inhibition.

As shown în TABLE 7 of Example 10, 17 anti-MASP-2 Fab2s with functional blocking activity were identîfïed as a resuit of this analysis (a 34% hit rate for blocking antibodies). Functional inhibition of the lectin complément pathway by Fab2s was apparent at the level of C4 déposition, which is a direct measure of C4 cleavage by MASP-2. Importantly, inhibition was equally évident when C3 convertase activity was assessed, demonstratîng functional blockade of the lectin complément pathway. The 17 MASP-2 blocking Fab2s identîfïed as described in Example 10 potently inhibit C3 convertase formation with IC5Q values equai to or less than 10 nM. Eight of the 17 Fab2s

116 identifïed hâve ICjq values in the sub-nanomolar range. Furthermore, ali I7 of the MASP-2 blocking Fab2s gave essentially complété inhibition of the C3 convertase formation in the lectîn pathway C3 convertase assay, as shown in FIGURES 8A-C, and summarized in TABLE 7 of Example 10. Moreover, each of the 17 blocking anti-MASP2 Fab2s shown in TABLE 7 potently inhibit C3b génération (>95%), thus demonstratîng the specificity of this assay for lectin pathway C3 convertase.

Rat IgG2c and mouse IgG2a full-length antibody isotype variants were derived from Fab2 #l I. This Example describes the in vivo characterization of these isotypes for pharmacodynamie parameters.

Methods:

As described in Example 10, rat MASP-2 protein was utilized to pan a Fab phage display library, from which Fab2#ll was identifïed. Rat IgG2c and mouse lgG2a fiilllength antibody isotype variants were derived from Fab2 #11. Both rat IgG2c and mouse lgG2a full length antibody isotypes were characterized in vivo for pharmacodynamie parameters as follows.

In vivo study in mice:

A pharmacodynamie study was carried out in mice to investigate the effect of anti-MASP-2 antibody dosing on the plasma lectin pathway activity in vivo. In this study, C4 déposition was measured ex vivo in a lectin pathway assay at various time points following subeutaneous (SC) and intraperitoneal (IP) administration of 0.3 mg/kg or 1.0 mg/kg of the mouse anti-MASP-2 MoAb (mouse IgG2a full-length antibody isotype derived from Fab2#l 1).

FIGURE 14 graphically illustrâtes lectin pathway spécifie C4b déposition, measured ex vivo in undiluted sérum samples taken from mice (n=3 mice/group) at various time points after subeutaneous dosing of either 0.3 mg/kg or 1.0 mg/kg of the mouse anti-MASP-2 MoAb. Sérum samples from mice collected prior to antibody dosing served as négative controls (100% activity), while sérum supplemented in vitro with 100 nM of the same blocking anti-MASP-2 antibody was used as a positive control (0% activity).

The results shown in FIGURE 14 demonstrate a rapid and complété inhibition of C4b déposition following subeutaneous administration of 1.0 mg/kg dose of mouse anti19004

117

MASP-2 MoAb. A partial inhibition of C4b déposition was seen following subcutaneous administration of0.3 mg/kg dose of mouse anti-MASP-2 MoAb.

The time course of lectin pathway recovery was followed for three weeks following a single IP administration of mouse anti-MASP-2 MoAb at 0.6 mg/kg in mice. As shown in FIGURE 15, a precipitous drop in lectin pathway activity occurred post antibody dosing followed by complété lectin pathway inhibition that lasted for about 7 days after IP administration. Slow restoration of lectin pathway activity was observed over the second and third weeks, with complété lectin pathway restoration in the mice by 17 days post anti-MASP-2 MoAb administration.

These results demonstrate that the mouse anti-MASP-2 Moab derived from Fab2 #ll inhibits the lectin pathway of mice in a dose-responsive manner when delivered systemically.

EX AMPLE 14

This Example describes analysis of the mouse anti-MASP-2 Moab derived from Fab2 #11 for efïïcacy in a mouse model for age-related macular degeneration.

Background/Rationale:

As described in Example 10, rat MASP-2 protein was utilized to pan a Fab phage display library, from which Fab2#Il was identified as a functionally active antibody. Full length antibodies of the rat IgG2c and mouse IgG2a isotypes were generated from Fab2 #11. The full length anti-MASP-2 antibody of the mouse IgG2a isotype was characterized for pharmacodynamie parameters as described in Example 13. In this Example, the mouse anti-MASP-2 full-length antibody derived from Fab2 #11 was analyzed in the mouse model of age-related macular degeneration (AMD), described by Bora P.S. et al, JImmunol /74:491-497 (2005).

Methods:

The mouse IgG2a full-length anti-MASP-2 antibody isotype derived from Fab2 #11 as described in Example 13, was tested in the mouse model of age-related macular degeneration (AMD) as described in Example 12 with the following modifications.

lis

Administration of mouse-anti-MASP-2 MoAbs

Two different doses (0.3 mg/kg and l.O mg/kg) of mouse anti-MASP-2 MoAb along with an isotype control MoAb treatment were injected IP into WT (+/+) mice (n= 8 mice per group) 16 hours prior to CNV induction

Induction of choroidal neovascularization (CNV)

The induction of choroidal neovascularization (CNV) and measurement of the volume of CNV was carried out using laser photocoagulation as described in Example 12.

Results:

FIGURE 16 graphicaliy illustrâtes the CNV area measured at 7 days post laser injury in mice treated with either isotype control MoAb, or mouse anti-MASP-2 MoAb (0.3 mg/kg and l.O mg/kg). As shown in FIGURE 16, in the mice pre-treated with l.O mg/kg anti-MASP-2 MoAb, a statistically significant (p <0.01) approximately 50% réduction in CNV was observed seven days post-laser treatment. As further shown in FIGURE 16, it was observed that a 0.3 mg/kg dose of anti-MASP-2 MoAb was not efficacious in reducing CNV. It is noted that the 0.3 mg/kg dose of anti-MASP-2 MoAb was shown to hâve a partial and transient inhibition of C4b déposition following subcutaneous administration, as described in Example I3 and shown in FIGURE 14.

The results described in this Example demonstrate that blockade of MASP-2 with an inhibitor, such as anti-MASP-2 MoAb, has a preventative and/or therapeutic effect in the treatment of macular degeneratîon. It is noted that these results are consistent with the results observed in the study carried out in the MASP-2 (-/-) mice, described in Example I2, in which a 30% réduction in the CNV 7 days post-laser treatment was observed in MASP-2 (-/-) mice in comparison to the wîld-type control mice. Moreover, the results in this Example further demonstrate that systemically delivered anti-MASP-2 antibody provides local therapeutic benefit in the eye, thereby hîghlighting the potential for a systemic route of administration to treat AMD patients. In summary, these results provide evidence supporting the use of MASP-2 MoAb in the treatment of AMD.

EXAMPLE 15

This example describes the identification, using phage display, of fully human scFv antibodies that bind to MASP-2 and inhibit lectin-mediated complément activation

H9 while leaving the classical (Clq-dependent) pathway component of the immune System intact.

Overview:

Fully human, high-affinity MASP-2 antibodies were identified by screening a phage display library. The variable light and heavy chain fragments of the antibodies were isolated in both a scFv format and in a full-length IgG format. The human MASP-2 antibodies are useful for inhibiting cellular injury assocîated with lectin pathwaymediated alternative complément pathway activation while leaving the classical (Clq-dependent) pathway component of the immune System intact. In some embodiments, the subject MASP-2 inhibitory antibodies hâve the following characteristics: (a) high afïïnity for human MASP-2 (e.g., a K_D of 10 nM or less), and (b) inhibit MASP-2-dependent complément activity in 90% human sérum with an ICjq of 30 nM or less.

Methods:

Expression of full-length catalytically inactive MASP-2:

The full-length cDNA sequence of human MASP-2 (SEQ ID NO: 4), encoding the human MASP-2 polypeptide with leader sequence (SEQ ID NO:5) was subcloned into the mammalian expression vector pCl-Neo (Promega), which drives eukaryotic expression under the control of the CMV enhancer/promoter région (described in Kaufman R.J. et al., Nucleic Acids Research 79:4485-90, I99l; Kaufman, Methods in Enzymology, 755:537-66 ( 1991 )).

In order to generate catalytically inactive human MASP-2A protein, site-directed mutagenesis was carried out as described in US2007/0172483, hereby incorporated herein by reference. The PCR products were purified after agarose gel electrophoresis and band préparation and single adenosine overlaps were generated using a standard tailing procedure. The adenosine-tailed MASP-2A was then cloned into the pGEM-T easy vector and transformed into E. coli. The human MASP-2A was further subcloned into either of the mammalian expression vectors pED or pCI-Neo.

The MASP-2A expression construct described above was transfected into DXBl cells using the standard calcium phosphate transfection procedure (Maniatis et al., 1989). MASP-2A was produced in serum-free medium to ensure that préparations were not

120 contamînated with other serum proteins. Media was harvested from confluent ceils every second day (four times in total). The level of recombinant MASP-2A averaged approximately 1.5 mg/liter of culture medium. The MASP-2A (Ser-Ala mutant described above) was purified by affinity chromatography on MBP-A-agarose columns

MASP-2A EL ISA on ScFv Candidate Clones identified by panning/scFv conversion andfilter screening

A phage display library of human immunoglobulin light- and heavy-chain variable région sequences was subjected to antigen pannîng followed by automated antibody screening and sélection to identify high-affinity scFv antibodies to human MASP-2 protein. Three rounds of pannîng the scFv phage library against HIS-tagged or biotin-tagged MASP-2A were carried out. The third round of pannîng was eluted first with MBL and then with TEA (alkaline). To monitor the spécifie enrichment of phages displaying scFv fragments against the target MASP-2A, a polyclonal phage ELISA against immobilized MASP-2A was carried out. The scFv genes from pannîng round 3 were cloned into a pHOG expression vector and run in a small-scale filter screening to look for spécifie clones against MASP-2A.

Bacterîal colonies containing plasmids encoding scFv fragments from the third round of pannîng were pîcked, gridded onto nîtrocellulose membranes and grown overnight on non-inducing medium to produce master plates. A total of 18,000 colonies were pîcked and analyzed from the third pannîng round, half from the compétitive elution and half from the subséquent TEA elution. Pannîng of the scFv phagemid library against MASP-2A followed by scFv conversion and a filter screen yîelded 137 positive clones. 108/137 clones were positive in an ELISA assay for MASP-2 binding (data not shown), of which 45 clones were further analyzed for the ability to block MASP-2 activity in normal human serum.

Assay to Measure Inhibition of Formation of Lectin Pathway C3 Convertase

A functional assay that measures inhibition of lectin pathway C3 convertase formation was used to evaluate the blocking activity of the MASP-2 scFv candidate clones. MASP-2 serine protease activity is required in orderto generate the two protein components (C4b. C2a) that comprise the lectin pathway C3 convertase. Therefore, a MASP-2 scFv that inhibits MASP-2 functional activity (i.e., a blocking MASP-2 scFv), will inhibit de novo formation of lectin pathway C3 convertase. C3 contains an unusual

I2l and highly reactive thîoester group as part of its structure. Upon cleavage of C3 by C3 convertase in this assay, the thîoester 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 détection of C3b in the ELISA assay.

Yeast mannan is a known activator of the lectin pathway. In the following method to measure formation of C3 convertase, plastic wells coated with mannan were incubated with diluted human sérum to activate the lectin pathway. The wells were then washed and assayed for C3b immobilized onto the wells using standard ELISA methods. The amount of C3b generated in this assay is a direct reflection of the de novo formation of lectin pathway C3 convertase. MASP-2 scFv clones at selected concentrations were tested in this assay for their ability to inhibit C3 convertase formation and conséquent C3b génération.

Methods:

The 45 candidate clones identifïed as described above were expressed, purified and diluted to the same stock concentration, which was again diluted in Ca^4-1- and Mg^-4· containing GVB buffer (4.0 mM barbital, 141 mM NaCI, 1.0 mM MgCl₂, 2.0 mM CaC12, 0.1% gelatin, pH 7.4) to assure that ail clones had the same amount of buffer. The scFv clones were each tested in triplicate at the concentration of 2 pg/mL. The positive control was OMS 100 Fab2 and was tested at 0.4 pg/mL. C3c formation was monitored in the presence and absence of the scFv/IgG clones.

Mannan was diluted to a concentration of 20 pg/mL (1 pg/well) in 50mM carbonate buffer (15mM Na2CÛ3 + 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 3 times with 200 pl PBS. 100 pi of 1% HSA blocking solution was then added to the wells and incubated for 1 hour at room température. The plates were washed 3 times with 200 pl PBS, and stored on ice with 200 pl PBS until addition of the samples.

Normal human sérum was diluted to 0.5% in CaMgGVB buffer, and scFv clones or the OMS 100 Fab2 positive control were added in triplÎcates at 0.01 pg/mL; 1 pg/mL

122 (only OMSIOO control) and 10 pg/mL to this buffer and preincubated 45 minutes on ice before addition 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 déposition was detected with a Rabbit α-Mouse C3c antibody followed by Goat a-R.abbit H RP. The négative control was buffer without antibody (no antibody = maximum C3b déposition), and the positive control was buffer with EDTA (no C3b déposition). The background was determined by carrying out the same assay except that the wells were mannan-free. The background signal against plates without mannan was subtracted from the signais in the mannan-containing wells. A cut-off criterion was set at half of the activity of an irrelevant scFv clone (VZV) and buffer alone.

Results: Based on the cut-off criterion, a total of 13 clones were found to block the activity of MASP-2. Ail 13 clones producing > 50% pathway suppression were selected and sequenced, yielding 10 unique clones. Ail ten clones were found to hâve the same light chain subclass, A3, but three different heavy chain subclasses: VH2, VH3 and VH6. In the functîonal assay, five out of the ten candidate scFv clones gave IC50 nM values less than the 25 nM target criteria using 0.5% human sérum.

To identifÿ antibodies with improved potency, the three mother scFv clones, identified as described above, were subjected to light-chain shuffling. This process involved the génération of a combinatorial library consisting of the VH of each of the mother clones paired up with a library of naïve, human lambda light chains (VL) derived from six healthy donors. This library was then screened for scFv clones with improved binding affinity and/or functionality.

TABLE 9: Comparïson of functîonal potency in IC50 (nM) of the lead daughter

clones and their respective mother clones (al	in scFv format)
scFv clone	1% human sérum C3 assay (IC50 nM)	90% human sérum C3 assay (IC50 nM)	90% human sérum C4 assay (IC5q nM)
l7D20mc	38	nd	nd
!7D20m d352lNll	26	>1000	140
!7Nl6mc	68	nd	nd

123 !7Nl6m d!7N9 48 | 15__230________

Presented below are the heavy-chain variable région (VH) sequences for the mother clones and daughter clones shown above in TABLE 9.

The Kabat CDRs (31-35 (H l ), 50-65 (H2) and 95-107 (H3)) are bolded; and the Chothia CDRs (26-32 (Hl), 52-56 (H2) and 95-101 (H3)) are underlined.

17D20 35VH-21NHVL heavy chain variable région (VH) (SEQ ID NO:67, encoded by SEQ ID NO:66)

QVTLKESGPVLVKPTETLTLTCTVSGFSLSRGKMGVSWIRQPPGKALEW lahifssdeksyrtslksrltiskdtsknqwltmtnmdpvdtatyycarirrg GIDYWGQGTLVTVSS d!7N9 heaw chain variable région (VH) (SEQ ID NO:68)

QVQI .QQSGPGI VK PSOTI SLTCAISGDSVSSTSAAWN WIRQSPSRGLE WLGRTY YRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYÇARDPFGVPF DIWGQGTMVTVSS

Presented below are the light-chain variable région (VL) sequences for the mother clones and daughter clones.

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 régions are the same whether numbered by the Kabat or Chothia System.

l7D20m d352lNl l light chain variable région (VL) (SEQ ID NO:69)

QPVT TQPPSI SVSPGQTASITCSGEKLGDKYAYWYOQKPGQSPVLVMYQ DKQRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQAWDSSTAVFGGGTKL TVL !7Nl6m d!7N9 light chain variable région (VL) (SEQ ID NO:7l. encoded by

SEQ ID NQ:70)

124

SYELIQPPSVSVAPGQTATITCAGDNLGKKRVHWYQQRPGQAPVLVIYD dsdrpsgipdrfsasnsgntatltitrgeagdeadyycqvwdiatdhvvfgggt

KLTVLAAAGSEQKLISE

The MASP-2 antibodies OMS 100 and MoAb_d3521N 11 VL, (comprising a heavy chain variable région set forth as SEQ ID NO:67 and a light chain variable région set forth as SEQ ID NO:70, also referred to as “OMS646”), which hâve both been demonstrated to bind to human MASP-2 with high affinity and hâve the ability to block functional complément activity, were analyzed with regard to epitope binding by dot blot analysis. The results show that OMS646 and OMS 100 antibodies are highly spécifie for MASP-2 and do not bind to MASP-1/3. Neither antibody bound to MApl9 nor to MASP-2 fragments that did not contain the CCP1 domain of MASP-2, leading to the conclusion that the binding sites encompass CCPL

The MASP-2 antibody OMS646 was determined to avidly bind to recombinant MASP-2 (Kd 60-250pM) with >5000 fold selectivity when compared to Cls, Clr or MASP-1 (see TABLE 10 below):

TABLE 10: Affinity and Specificity of OMS646 MASP-2 antibody-MASP-2 interaction as assessed by solid phase ELISA studies

Anti gen	Kd (pM)__
MASP-1	>500.000
MASP-2	62±23*
MASP-3	>500,000
Purified human Clr	>500,000___
Purified human Cls	| -500,000

*Mean±SD; n—12

OMS646 specifically blocks lectin-dependent activation of terminal complément components

Methods:

125

The effect of OMS646 on membrane attack complex (MAC) déposition was analyzed using pathway-specific conditions for the lectin pathway, the classical pathway and the alternative pathway. For this purpose, the Wieslab Comp300 complément screening kit (Wieslab, Lund, Sweden) was used following the manufacturées instructions.

Results:

FIGURE 17A graphically illustrâtes the level of MAC déposition in the presence or absence of anti-MASP-2 antibody (OMS646) under lectin pathway-specific assay conditions. FIGURE 17B graphically illustrâtes the level of MAC déposition in the presence or absence of anti-MASP-2 antibody (OMS646) under classical pathwayspecific assay conditions. FIGURE 17C graphically illustrâtes the level of MAC déposition in the presence or absence of anti-MASP-2 antibody (OMS646) under alternative pathway-specific assay conditions.

As shown în FIGURE 17A, OMS646 blocks lectin pathway-mediated activation of MAC déposition with an IC50 value of approximately InM. However, OMS646 had no effect on MAC déposition generated from classical pathway-mediated activation (FIGURE 17B) or from alternative pathway-mediated activation (FIGURE 17C).

Pharmacokinetîcs and Pharmacodynamies of OMS646 following Intravenous (IV) or Subcutaneous (SC) Administration to Mice

The pharmacokinetîcs (PK) and pharmacodynamies (PD) of OMS646 were evaluated in a 28 day single dose PK/PD study in mice. The study tested dose levels of 5mg/kg and I5mg/kg of OMS646 admînistered subcutaneously (SC), as well as a dose level of 5mg/kg OMS646 admînistered intravenously (IV).

With regard to the PK profile ofOMS646, FIGURE 18 graphically illustrâtes the OMS646 concentration (mean of n=3 animals/groups) as a fonction of time after administration of OMS646 at the indicated dose. As shown in FIGURE 18, at 5mg/kg SC, OMS646 reached the maximal plasma concentration of 5-6 ug/mL approximately l-2 days after dosing. The bioavailability of OMS646 at 5 mg/kg SC was approximately 60%. As forther shown in FIGURE I8, at 15 mg/kg SC, OMS646 reached a maximal plasma concentration of 10-12 ug/mL approximately l to 2 days after dosing. For ail groups, the OMS646 was cleared slowly from systemic circulation with a terminal half19004

126 life of approximately 8-10 days. The profile of OMS646 is typîcal for human antibodies in mice.

The PD activity ofOMS646 is graphically illustrated in FIGURES 19A and 19B. FIGURES 19A and 19B show the PD response (drop in systemic lectîn pathway activity) for each mouse in the 5mg/kg IV (FIGURE 19A) and 5mg/kg SC (FIGURE 19B) groups. The dashed line indicates the baseline of the assay (maximal inhibition; naïve mouse sérum spiked in vitro with excess OMS646 prier to assay). As shown in FIGURE 19A, following IV administration of 5mg/kg of OMS646. systemic lectin pathway activity immediately dropped to near undetectable levels, and lectin pathway activity showed only a modest recovery over the 28 day observation period. As shown in FIGURE 19B, in mice dosed with 5mg/kg of OMS646 SC, time-dependent inhibition of lectin pathway activity was observed. Lectin pathway activity dropped to near-undetectable levels within 24 hours of drug administration and remained at low levels for at least 7 days. Lectin pathway activity gradually increased with time, but did not revert to pre-dose levels within the 28 day observation period. The lectin pathway activity versus time profile observed after administration of I5mg/kg SC was similar to the 5 mg/kg SC dose (data not shown), indicating saturation of the PD endpoint. The data further indicated that weekly doses of 5mg/kg of OMS646, administered either IV or SC, is suflficient to achieve continuons suppression of systemic lectin pathway activity in mice.

EX AMPLE 16

This Example describes analysis of the efficacy of MASP-2 monoclonal antibody (OMS646), a human IgG4 antibody that blocks the function of the lectin pathway, ίη a mouse model of age-related macular degeneration.

Background/Rationale:

As described in Example 15, a fully human monoclonal MASP-2 antibody (OMS646) was generated that specifically blocks the function of the human lectin pathway. In this example, OMS646 was analyzed in the mouse model of laser-induced chorodial neovascularization (CNV), a commonly used model of age-related macular degeneration (AMD), described by Bora et al. (J Immunol 174:491-497, 2005) along with an anti-VEGF antibody as a comparator.

127

Methods:

This study evaluated the effect of three dose levels of OMS646 (2mg/kg; 5mg/kg and 20mg/kg SC) compared to vehicle treatment. Anti-mouse MASP-2 mAb derived from Fab2 #11 (3mg/kg SC), generated as described in Example 14, and a rat monoclonal antibody that binds to mouse VEGF-A and blocks VEGF-A fonction (5mg/kg IP, clone 2G11-2A05, purchased from BioLegend®, San Diego, CA) were included as positive control and comparator treatments, respectively. The study included 9-10 mice per experimental group and was conducted in a blinded fashion. To assess efficacy at consistent and predictable drug levels, ail treatments were administered eight days prior to, and then again one day prior to laser induction, except for anti-VEGF antibody which was injected one day before and three days after laser induction. Seven days after laser injury, mice were anesthetized, perfosed System ically with 0.75 ml of FITC-dextran and sacrificed. Eyes were fixed in formalin, the posterîor part of the eyes containing the injured areas were dissected and fiat mounted in ProLong antifade reagent (Invitrogen). Confocal microscopy of injured areas was performed and images were captured from each area. Measurements of CNV and injured areas were performed with the ImageJ program (National Instîtutes of Health, Bethesda, Maryland USA). The CNV area was normalized with respect to the injured spot size for each eye, where % CNV represents the mean neovascularized area per injured spot, calculated as (CNV area/spot area) X 100. The study was conducted in a blinded fashion using coded test article solutions.

Results: The outcome of this study is shown in FIGURE 20. As shown in FIGURE 20, compared to the vehicle treated group, OMS646-treated mice showed appréciable inhibition of CNV at ail dose levels tested, with relative CNV réductions ranging from 29% to 50%. Anti-VEGF treatment showed a lesser (approxtmately 15%) réduction in CNV réduction. The anti-mouse MASP-2 mAb derived from Fab2 #11 also reduced CNV by approximately 30% compared to vehicle treatment (data not shown), which is consistent with the results observed in the study carried out in the MASP-2 (-/-) mice, described in Example 12, in which a 30% réduction in the CNV 7 days post-laser treatment was observed in MASP-2 (-/-) mice in comparison to the wild-type control mice.

The results of this study provide evidence that systemic administration of OMS646 provides an effective therapy for treating neovascular AMD. Unlike current

128 and emerging therapeutics for AMD and other ocular angiogenic diseases and disorders, which require intravitreal injection, OMS646 is also effective when administered subcutaneously.

It is further noted that the VEGF-A antibody used in this study (clone 2Gi I-2A05 from BioLegend®, San Diego, CA), has prevîously been shown to reduce vessei extension into the comea in a mouse model of HSV-l-induced comeal lymphangiogenesis when administered by subconjunctival injection at a concentration of lOOug/mL, as described in Wuest et al. (J Exp Med 207:101, 2009). In another study by Lu et al. (Cancer Res 72:2239-50, 2012), anti-VEGF antibody (clone 2G11-2A05) treatment of Ceacam 1-/- mice bearing B16 tumors significantly reduced tumor size as well as tumor vasculature in a colon tumor model when administered IP at approximately 3mg/kg twice a week. In view of the data in the présent study demonstrating that OMS646 is at least as effective as the anti-VEGF antibody at reducing CNV when delivered systemically to mice at ail dose levels tested, it is expected that a MASP-2 inhibitory agent such as OMS646 will also be effective as an anti-angiogenesis agent for use in inhibitîng an angiogenesis-dependent cancer, such as, for example, an angiogenesis-dependent cancer selected from the group consisting of solid tumor(s), blood borne tumors, high-risk carcinoid tumors, and tumor métastasés. Examples of angiogenesis-dependent cancers are cancer types that hâve been approved for treatment by an anti-VEGF agent, such as the anti-VEGF antibody Avastin® (bevacizumab, Genentech, CA). For example, bevacizumab has been approved for treatment of the following angiogenic-dependent cancers: metastatic colorectal cancer, non-squamous non-small cell lung cancer, metastatic rénal cell carcinoma, and glioblastoma.

Additional examples of angiogenesis-dependent cancers are cancer types that are expected to benefit by treatment by an anti-VEGF agent, such as the anti-VEGF antibody Avastin® (bevacizumab, Genentech, CA), such as, for example, any cancer that is already known to be treated with, or in development to be treated with, an angiostatic compound (e.g., a VEGF antagonist), including advanced cancers metastatitic to liver, melanoma, ovarian cancer, neuroblastoma, pancreatic cancer, hepatocellular carcinoma, endométrial cancer, prostate cancer, angîosarcoma, metastatic or unresectable

129 angiosarcoma, relapsed ovarian sex-cord stromal tumours, esophageal cancer, gastric cancer, non-Hodgkin’s lymphoma, Hodgkin lymphoma, diffuse large B-cell lymphoma, récurrent or metastatic head and neck cancer, neoplastic meningitis, cervical cancer, uterîne cancer, advanced peritoneal carcinomatosis, gliosarcoma, neuroendocrine carcinoma, extracranial Ewing sarcoma, acute myeloid leukemia, chronic myelogenous leukemia, intracranial meningioma, advanced Kaposi’s sarcoma, mesothelioma, biliary tract cancer, metastatic carcinoid tumors, and advanced urinary tract cancer. Preferred cancers in this context include: colorectal, breast (including metastatic breast cancer, inflammatory breast carcinoma), lung, rénal, hepatic, esophageal, ovarian, pancreatîc, prostate and gastric cancers, as well as glioma, gastrointestinal stromal tumors, lymphoma, melanoma and carcinoid tumors.

It is also expected that a MASP-2 inhibitory agent, such as OMS646 will be effective as an anti-angiogenesis agent for inhibiting an angiogenesis-dependent benign tumor, such as, for example, an angiogenesis-dependent benign tumor selected from the group consisting of hemangiomas, acoustic neuromas, neurofibromas, trachomas, carcinoid tumors, and pyogénie granulomas. It is also expected that a MASP-2 inhibitory agent such as OMS646 will be effective as an anti-angiogenesis agent for use in inhibiting angiogenesis in AMD and other ocular angiogenic diseases or disorders such as uveitis, ocular melanoma, corneal neovascularization, primary pterygium, HSV stromal keratitis, HSV-l-induced corneal lymphangiogenesis, proliférative diabetic retinopathy, diabetic macular edema, retinopathy of prematurity, retînal vein occlusion, corneal graft rejection, neovascular glaucoma, vitreous hemorrhage secondary to proliferative diabetic retinopathy, neuromyelitis optica and rubeosis.

In view of the data in the present study demonstrating that OMS646 is at least as effective as the anti-VEGF antibody at reducïng CNV when delivered systemically to mice at ail dose levels tested, it is also expected that a MASP-2 inhibitory agent such as OMS646 wtll also be effective as an anti-angiogenesis agent for use in inhibiting an angiogenesis-dependent condition such as myelofibrosis and hereditary hémorrhagie telangiesctasia.

130

EXAMPLE 17

This Example describes the use of a MASP-2 (-/-) straîn and MASP-2 inhibitory antibodies to confirm that inhibition of the MASP-2 dépendent lectin pathway of complément activation induces an anti-angiogenic effect in an animal model of fémoral artery ligation.

Background/Rationale: In view of the suprising results described in Example 16 that the human MASP-2 mAb OMS646 inhibits CNV in a model of AMD to at least an equal if not greater extent than a VEGF-A antibody, the following studies are carried out to confirm that angiogenesis is reduced in a MASP-2 déficient mouse, and also that a MASP-2 antibody that blocks the lectin pathway, such as OMS646, is effective for use in vivo as an angiogenesis inhibitory agent when administered systemically.

Methods:

Study # 1 : Arteriogenesis is induced in MASP-2 (-/-) mice, wild-type control mice, and wild-type mice pre-treated with MASP-2 inhibitory antibody, by fémoral artery ligation, and Laser Doppler perfusion measurements are performed in vivo to see whether the process of collateral artery growth is influenced by MASP-2 deficiency. The perfusion measurements are performed untii day 21 after fémoral artery ligation,

Immunohistochemistry is performed on day 3 after fémoral artery ligation to analyze:

(a) In the upper leg, wherein arteriogenesis occurs, for the influence of MASP-2 deficiency on perivascular leukocyte infiltration (arteriogenesis is strongly dépendent on leukocyte infiltration given that leukocytes provide the growing collaterals with growth factors, cytokines); and (b) In the lower leg, which gets ischémie due to fémoral artery ligation, the severity of ischémie tissue damage, leukocyte infiltration and angiogenesis in the MASP-2 (-/-) mice, anti-MASP-2 antibody-treated wild-type mice, and control wild-type mice.

(c) Gene expression studies on RNA and protein levels is also carried out on isolated collaterals 12h or 24h after fémoral artery ligation in the MASP-2 (-/-) mice, anti-MASP-2 antibody-treated wild-type mice, and control wild-type mice.

I3l

On the basis of the anti-angiogenic effect described above, it is expected that MASP-2 inhibition will prevent or reduce arteriogenesis by 25 to 50% in the upper leg. In addition, MASP-2 inhibition has been demonstrated to reduce post-ischemic complément 5 driven pathologie response by 25% to 50% (Schwaeble et al., PNAS 108(18):7523-7528).

Thus, it can also be expected that MASP-2 inhibition will inhibit vasculogenesis in the lower leg to a similar degree.

While illustrative embodiments hâve been illustrated and described, it will be 10 appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

Claims (18)

1. The embodiments of the invention in which an exclusive property or privilège is claimed are defined as follows:

   1. A MASP-2 inhibitory agent for use in a method for preventing, treating, reverting and/or delaying angiogenesis in a mammalian subject suffering from an angiogenesis-dependent disease or condition, wherein the MASP-2 inhibitory agent is a MASP-2 monoclonal antibody, or fragment thereof that specifically binds to a portion of SEQ ID NO:6.
2. 2. Use of a MASP-2 inhibitory agent in the manufacture of a pharmaceutical composition or a médicament for preventing, treating, reverting and/or delaying angiogenesis in a mammalian subject suffering from an angiogenesis-dependent disease or condition, wherein the MASP-2 inhibitory agent is a MASP-2 monoclonal antibody, or fragment thereof that specifically binds to a portion of SEQ ID NO:6.
3. 3. The MASP-2 inhibitory agent for use of Claim l or the use of Claim 2, wherein the angiogenesis-dependent disease or condition is an angiogenesis-dependent cancer.
4. 4. The MASP-2 inhibitory agent for use of Claim l or the use of Claim 2, wherein the angiogenesis-dependent disease or condition is a benign tumor.
5. 5. The MASP-2 inhibitory agent for use of Claim 1 or the use of Claim 2, wherein the angiogenesis-dependent disease or condition is an ocular angiogenic disease or condition.
6. 6. The MASP-2 inhibitory agent for the use of Claim 3, wherein the subject is suffering from an angiogenesis-dependent cancer selected from the group consisting of: solid tumor(s), blood borne tumors, high-risk carcinoîd tumors, and tumor métastasés.

   133
7. 7. The MASP-2 inhibitory agent for the use of Claim 6, wherein the subject is suffering from one or more solid tumors and the method comprises administering an amount of MASP-2 inhibitor effective to inhibit tumor angiogenesis.
8. 8. The MASP-2 inhibitory agent for the use of Claim 6, wherein the subject is suffering from or at risk for tumor métastasés and the method comprises administering an amount of MASP-2 inhibitor effective to inhibit tumor métastasés.
9. 9. The MASP-2 inhibitory agent for the use of Claim 4, wherein the subject is suffering from an angiogenesis-dependent benign tumor(s) selected from the group consisting of hemangiomas, acoustic neuromas, neurofibromas, trachomas, carcinoid tumors, and pyogénie granulomas.
10. 10. The MASP-2 inhibitory agent for the use of Claim 5, wherein the ocular angiogenic disease or condition is not AMD.
11. 11. The MASP-2 inhibitory agent for the use of Claim 5, wherein the ocular angiogenic disease or condition is selected from the group consisting of uveitis, ocular melanoma, corneal neovascularization, primary pterygium, HSV stromal keratitis, HSVl-induced corneal lymphangiogenesis, proliférative diabetic retinopathy, diabetic macular edema, retinopathy of prematurity, retinal vein occlusion, corneal graft rejection, neovascular glaucoma, vitreous hemorrhage secondary to proliférative diabetic retinopathy, neuromyelitis optica and rubeosis.
12. 12. The MASP-2 inhibitory agent for the use of Claim 1 or the use of Claim 2, wherein the antibody or fragment thereof is selected from the group consisting of a recombinant antibody, an antibody having reduced effector fiinction, a chimeric antibody, a humanized antibody and a human antibody.
13. 13. The MASP-2 inhibitory agent for use of Claim 1 or the use of Claim 2, wherein the composition is for subeutaneous, intraperitoneal, intra-muscular, intraarterial, or intravenous administration, or for administration as an inhalant.
14. 14. A MASP-2 inhibitory agent for use in a method of treating a subject suffering from an ocular angiogenic disease or condition selected from the group

    134 consisting of uveitis, ocular melanoma, comeal neovascularization, primary pterygium, HSV stromal keratitis, HSV-l-induced corneal lymphangiogenesis, proliférative diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, corneal graft rejection, neovascular glaucoma, and rubeosis, wherein the MASP-2 inhibitory agent is a MASP-2 5 monoclonal antibody, or fragment thereof thaï specifically binds to a portion of SEQ ID N0:6.
15. 15. Use of a MASP-2 inhibitory agent in the manufacture of a pharmaceutical composition or a médicament for treating a subject suffering from an ocular angiogenic disease or condition selected from the group consisting of uveitis, ocular melanoma, I0 comeal neovascularization, primary pterygium, HSV stromal keratitis, HSV-l-induced comeal lymphangiogenesis, proliférative diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, comeal graft rejection, neovascular glaucoma, and rubeosis, wherein the MASP-2 inhibitory agent is a MASP-2 monoclonal antibody, or fragment thereof that specifically binds to a portion ofSEQ ID N0:6.

    15
16. 16. The MASP-2 inhibitory agent for use of Claim 14 or the use of Claim 15, wherein the antibody or fragment thereof is selected from the group consisting of a recombinant antibody, an antibody having reduced effector function, a chimeric antibody, a humanized antibody and a human antibody,
17. 17. A MASP-2 inhibitory antibody or fragment thereof that specifically binds 20 to a portion of SEQ ID NO:6 for use in a method of inhibiting tumor angiogenesis in a subject.
18. 18. Use of a MASP-2 inhibitory antibody or fragment thereof that specifically binds to a portion of SEQ ID NO:6 in the manufacture of a pharmaceutical composition or a médicament for inhibiting tumor angiogenesis in a subject,

    25 19. The MASP-2 inhibitory agent for use of Claim 17 or the use of Claim 18 , wherein the antibody or fragment thereof is selected from the group consisting of a recombinant antibody, an antibody having reduced effector function, a chimeric antibody, a humanized antibody and a human antibody.