BR112019001247B1 - ISOLATED MONOCLONAL ANTIBODY OR ANTIGEN-BINDING FRAGMENT THEREOF, ISOLATED ANTIBODY OR ANTIGEN-BINDING FRAGMENT THEREOF, COMPOSITION, AND, USE OF A COMPOSITION - Google Patents

Abstract

The present invention relates to MASP-3 inhibitory antibodies and compositions comprising such antibodies for use in inhibiting the adverse effects of MASP-3 dependent complement activation.

Description

[001] This application claims the benefit of U.S. Provisional Application No. 62/369,674, filed on August 1, 2016, and claims the benefit of U.S. Provisional Application No. 62/419,420, filed on November 8, 2016, and claims the benefit of U.S. Provisional Application No. 62/478,336, filed on March 29, 2017, all three of which are incorporated herein by reference in their entirety.

STATEMENT REGARDING SEQUENCE LISTING

[002] The sequence listing associated with this application is provided in text format rather than a paper copy and is incorporated herein by reference into the specification. The name of the text file containing the sequence listing is MP_1_0254_US_Sequence_Listing_20170628_ST25; the file is 191 KB; it was created on June 28, 2017, and is being submitted via EFS-Web with the filing of the specification.

FUNDAMENTALS

[003] The complement system provides an early-acting mechanism for initiating, amplifying, and orchestrating the immune response to microbial infection and other acute insults (M.K. Liszewski and J.P. Atkinson, 1993, in Fundamental Immunology, Third Edition, edited by W.E. Paul, Raven Press, Ltd., New York), in humans and other vertebrates. Although complement activation provides a valuable first-line defense against potential pathogens, complement activities that promote a protective immune response can also pose a potential threat to the host (K.R. Kalli, et al., Springer Semin. Immunopathol. 15:417-431, 1994; B.P. Morgan, Eur. J. Clinical Investig. 24:219-228, 1994). For example, the proteolytic products of C3 and C5 recruit and activate neutrophils. Although they are indispensable for host defense, activated neutrophils are indiscriminate in their release of destructive enzymes and can cause organ damage. Furthermore, complement activation can cause the deposition of lytic complement components on neighboring host cells as well as on microbial targets, resulting in host cell lysis.

[004] The complement 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 leak after thermal burns, cardiopulmonary bypass inflammation, transplant rejection, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, and Alzheimer's disease. In nearly all of these conditions, complement is not the cause but is one of several factors involved in the pathogenesis. However, complement activation can be an important pathological mechanism and represents an effective point of clinical control in many of these disease states. The growing recognition of the importance of complement-mediated tissue injury in a variety of disease states highlights the need for effective complement inhibitors. To date, eculizumab (Solaris®), an antibody against C5, is the only complement-targeting drug that has been approved for human use. However, C5 is one of several effector molecules located “downstream” in the complement system, and blocking C5 does not inhibit complement activation. Therefore, an inhibitor of the initiating steps of complement activation would have significant advantages over a “downstream” complement inhibitor.

[005] It is now widely accepted that the complement 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 foreign particle (i.e., an antigen) and thus requires prior exposure to an antigen for the generation of a specific antibody response. Since activation of the classical pathway depends on a prior adaptive immune response by the host, the classical pathway is part of the acquired immune system. In contrast, both the lectin and alternative pathways are independent of adaptive immunity and are part of the innate immune system.

[006] Activation of the complement system results in the sequential activation of serine protease zymogens. The first step in activation of the classical pathway is the binding of a specific recognition molecule, CIq, to antigen-bound IgG and IgM molecules. C1q is associated with the serine protease proenzymes C1r and Cls as a complex called CI. After binding of C1q to an immune complex, autoproteolytic cleavage of the Arg-Ile site of C1r is followed by C1r-mediated cleavage and activation of Cls, which thus 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 capable of forming covalent bonds with adjacent hydroxyl or amino groups and generate C3 convertase (C4b2a) through non-covalent interaction with the C2a fragment of activated C2. C3 convertase (C4b2a) activates C3 by proteolytic cleavage into C3a and C3b subcomponents leading to the generation of C5 convertase (C4b2a3b), which, upon cleavage of C5, leads to the formation of the membrane attack complex (C5b combined with C6, C7, C8, and C-9, also known as "MAC") that can disrupt cell membranes resulting in cell lysis. The activated forms of C3 and C4 (C3b and C4b) are covalently deposited on foreign target surfaces, where they are recognized by complement receptors on multiple phagocytes.

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

[008] Ikeda et al. first demonstrated that, like C1q, MBL could activate the complement system upon binding to mannan-coated yeast erythrocytes in a C4-dependent manner (Ikeda et al., J. Biol. Chem. 262:7451-7454, (1987)). MBL, a member of the collectin protein family, is a calcium-dependent lectin that binds carbohydrates with 3- and 4-hydroxy groups oriented in the equatorial plane of the pyranose ring. Thus, the prominent ligands for MBL are D-mannose and N-acetyl-D-glucosamine, whereas carbohydrates that do not fit this steric requirement have undetectable affinity for MBL (Weis, W. I., et al., Nature 360:127-134, 1992). The interaction between MBL and monovalent sugars is extremely weak, with dissociation constants typically in the single-digit millimolar range. MBL achieves tight and specific binding to glycan ligands by avidity, that is, by simultaneous interaction with multiple monosaccharide residues located in close proximity to each other (Lee et al., Archiv. Biochem. Biophys. 299:129-136, (1992)). MBL recognizes the carbohydrate motifs that commonly coat microorganisms such as bacteria, yeast, parasites, and certain viruses. In contrast, MBL does not recognize D-galactose and sialic acid, the penultimate and final sugars that commonly coat “mature” complex glycoconjugates present in mammalian plasma and cell surface glycoproteins. This binding specificity is thought to promote recognition of “foreign” surfaces and help protect against “self-activation.” However, MBL does not bind with high affinity to the high-mannose “precursor” glycan groups on N-linked glycoproteins and glycolipids sequestered in the endoplasmic reticulum and Golgi of mammalian cells (Maynard, Y., et al., J. Biol. Chem. 257:3788-3794, (1982)). Furthermore, it has been shown that MBL can bind polynucleotides, DNA, and RNA, which can be exposed on necrotic and apoptotic cells (Palaniyar et al., Ann. N.Y. Acad. Sci., 1010:467-470 (2003); Nakamura et al., J. Leuk. Biol. 86:737-748 (2009)). Therefore, damaged cells are potential targets for activation of the lectin pathway via MBL binding.

[009] Ficolins possess a different type of lectin domain than MBL, termed the fibrinogen-like domain. Ficolins bind sugar residues in a Ca++-independent manner. In humans, three types of ficolins (L-ficolin, M-ficolin, and H-ficolin) have been identified. The two serum ficolins, L-ficolin and H-ficolin, have in common a specificity for N-acetyl-D-glucosamine; however, H-ficolin also binds N-acetyl-D-galactosamine. The difference in sugar specificity of L-ficolin, H-ficolin, and MBL means that the different lectins may be complementary and target different, although 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 in all Gram-positive bacteria (Lynch et al., J. Immunol. 172:1198–1202, (2004)). In addition to acetylated sugar moieties, ficolins can also bind acetylated amino acids and polypeptides (Thomsen et al., Mol. Immunol. 48(4):369–81 (2011)). Collectins (i.e., MBL) and ficolins do not share any significant amino acid sequence similarities. However, the two groups of proteins have similar domain organizations and, like C1q, assemble into oligomeric structures that maximize the possibility of multiple binding sites.

[0010] Serum concentrations of MBL are highly variable in healthy populations and this is genetically controlled by polymorphisms/mutations in both the promoter and coding regions of the MBL gene. As an acute phase protein, MBL expression is further upregulated during inflammation. L-ficolin is present in serum in similar concentrations to MBL. Therefore, the L-ficolin branch of the lectin pathway is potentially comparable to the MBL branch in strength. MBL and ficolins can also function as opsonins, which allow phagocytes to target surfaces coated with MBL and ficolin (see Jack et al., J Leukoc Biol., 77(3):328-36 (2004), Matsushita and Fujita, Immunobiology, 205(4-5):490-7 (2002), Aoyagi et al., J Immunol, 174(1):418-25(2005). This opsonization requires the interaction of these proteins with phagocytic receptors (Kuhlman et al., J. Exp. Med. 169:1733, (1989); Matsushita et al., J. Biol. Chem. 271:2448-54, (1996)), the identity of which has not been established.

[0011] Human MBL forms a specific, high-affinity interaction through its collagen-like domain with unique C1r/C1s-type serine proteases, called MBL-associated serine proteases (MASPs). To date, three MASPs have been described. The first, a single “MASP” enzyme, was identified and characterized as the enzyme responsible for initiating the complement cascade (i.e., cleaving C2 and C4) (Matsushita et al., J Exp Med 176(6):1497-1502 (1992); Ji et al., J Immunol. 150:571-578, (1993)). It was later determined that MASP activity was actually a mixture of two proteases: MASP-1 and MASP-2 (Thiel et al., Nature 386:506-510, (1997)). However, it has been shown that the MBL-MASP-2 complex alone is sufficient for complement activation (Vorup-Jensen et al., J. Immunol. 165:2093-2100, (2000)). Furthermore, MASP-2 alone cleaved C2 and C4 at high rates (Ambrus et al., J. Immunol. 170:1374-1382, (2003)). Therefore, MASP-2 is the protease responsible for activating C4 and C2 to generate the C3 convertase, C4b2b. This is a significant difference from the CI complex of the classical pathway, where the coordinated action of two specific serine proteases (C1r and Cls) leads to activation of the complement system. Recently, a third novel protease, MASP-3, has been isolated (Dahl, M. R., et al., Immunity 15:127-35, 2001). MASP-1 and MASP-3 are alternatively spliced products of the same gene.

[0012] MASPs share identical domain organizations with those of C1r and Cls, the enzymatic components of the CI complex (Sim et al., Biochem. Soc. Trans. 28:545, (2000)). These domains include an N-terminal C1r/C1s/sea urchin Vegf/bone morphogenic protein (CUB) domain, an epidermal growth factor-like domain, a second CUB domain, a tandem of complement control protein domains, and a serine protease domain. As with CI proteases, activation of MASP-2 occurs through cleavage of an Arg-Ile bond adjacent to the serine protease domain, which divides the enzyme into disulfide-linked A and B chains, the latter consisting of the serine protease domain.

[0013] MBL can also associate with an alternatively spliced form of MASP-2, known as MBL-associated protein of 19 kDa (MAp19) or small MBL-associated protein (sMAP), which lacks the catalytic activity of MASP-2. (Stover, J. Immunol. 162:3481-90, (1999); Takahashi et al., Int. Immunol. 11:859-863, (1999)). MAp19 comprises the first two domains of MASP-2, followed by an extra sequence of four unique amino acids. The function of Map19 is unclear (Degn et al., J Immunol. Methods, 2011). The MASP-1 and MASP-2 genes are located on human chromosomes 3 and 1, respectively (Schwaeble et al., Immunobiology 205:455-466, (2002)).

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

[0015] The lectin pathway is widely considered to play an important role in host defense against infection in the naive host. Strong evidence for the involvement of MBL in host defense comes from analysis of patients with decreased serum levels of functional MBL (Kilpatrick, Biochim. Biophys. Acta 1572:401-413, (2002)). Such patients demonstrate susceptibility to recurrent bacterial and fungal infections. These symptoms are usually evident early in life, during a clear window of vulnerability when maternally derived antibody titers wane but before a full repertoire of antibody responses has developed. This syndrome often results from mutations at several sites in the collagenous portion of MBL, which interfere with the proper formation of MBL oligomers. However, since MBL can function as a complement-independent opsonin, it is not known to what degree the increased susceptibility to infection is due to impaired complement activation.

[0016] In contrast to the classical and lectin pathways, no initiator of the alternative pathway has previously been found to perform the recognition functions that C1q and lectins perform in the other two pathways. It is now widely accepted that the alternative pathway spontaneously undergoes a low level of activation turnover, which can be readily amplified on foreign or other abnormal surfaces (bacteria, yeast, virus-infected cells, or damaged tissues) that lack the appropriate molecular elements that keep spontaneous complement activation in check. There are four plasma proteins directly involved in the activation of the alternative pathway: C3, factors B and D, and properdin.

[0017] Although there is extensive evidence implicating both classical and alternative complement pathways in the pathogenesis of noninfectious human diseases, the role of the lectin pathway is only beginning to be appreciated. Recent studies provide evidence that activation of the lectin pathway may be responsible for complement activation and related inflammation in ischemia/reperfusion injury. Collard et al. (2000) reported that cultured endothelial cells subjected to oxidative stress bind MBL and show C3 deposition upon exposure to human serum (Collard et al., Am. J. Pathol. 156:1549-1556, (2000)). Furthermore, treatment of human sera with blocking anti-MBL monoclonal antibodies inhibited MBL binding and complement 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 injury in coronary artery occlusion than rats treated with a control antibody (Jordan et al., Circulation 104:1413-1418, (2001)). The molecular mechanism of MBL binding to vascular endothelium after oxidative stress is unclear; a recent study suggests that activation of the lectin pathway after oxidative stress may be mediated by MBL binding to vascular endothelial cytokeratins rather than glycoconjugates (Collard et al., Am. J. Pathol. 159:1045-1054, (2001)). Other studies have implicated both the classical and alternative pathways in the pathogenesis of ischemia/reperfusion injury, and the role of the lectin pathway in this disease remains controversial (Riedermann, N.C., et al., Am. J. Pathol. 162:363-367, 2003).

[0018] Recent studies have shown that MASP-1 and MASP-3 convert the alternative pathway activating enzyme factor D from its zymogen form to its enzymatically active form (see Takahashi M. et al., J Exp Med 207(1):29-37 (2010); Iwaki et al., J Immunol. 187:3751-58 (2011)). The physiological importance of this process is evidenced by the absence of functional activity of the alternative pathway in the plasma of MASP-1/3-deficient mice. Proteolytic generation of C3b from native C3 is required for the alternative pathway to function. Since the alternative pathway C3 convertase (C3bBb) contains C3b as an essential subunit, the question regarding the origin of the first C3b by the alternative pathway has presented a perplexing problem and has stimulated considerable research.

[0019] C3 belongs to a family of proteins (along with C4 and α-2 macroglobulin) that contain a rare post-translational modification known as a thioester bond. The thioester group is composed of a glutamine whose terminal carbonyl group forms a covalent thioester bond with the sulfhydryl group of a cysteine three amino acids away. This bond is unstable and the electrophilic glutamyl thioester can react with nucleophilic moieties such as hydroxyl or amino groups and thus form a covalent bond with other molecules. The thioester bond is reasonably stable when sequestered within a hydrophobic cavity of intact C3. However, proteolytic cleavage of C3 to C3a and C3b results in exposure of the highly reactive thioester bond in C3b and, upon nucleophilic attack by adjacent moieties comprising hydroxyl or amino groups, C3b covalently binds to a target. In addition to its well-documented role in the covalent binding of C3b to complement targets, the C3 thioester is also thought to play a key role in triggering the alternative pathway. According to the widely accepted “tickover theory,” the alternative pathway is initiated by the generation of a fluid-phase convertase, iC3Bb, which is formed from thioester-hydrolyzed C3 (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 the internal thioester in the protein (Pangburn, M. K., et al., J. Exp. Med. 154:856–867, 1981). Through the activity of C3(H2O)Bb convertase, C3b molecules are deposited on the target surface, thus initiating the alternative pathway.

[0020] Prior to the discovery described here, very little was known about the initiators of alternative pathway activation. Activators were thought to include yeast cell walls (zymosan), many pure polysaccharides, rabbit erythrocytes, certain immunoglobulins, viruses, fungi, bacteria, animal tumor cells, parasites, and damaged cells. The only common feature among these activators is the presence of carbohydrate, but the complexity and variety of carbohydrate structures have made it difficult to establish the shared molecular determinants that are recognized. It has been widely accepted that activation of the alternative pathway is controlled by a fine balance between the inhibitory regulatory components of this pathway, such as factor H, factor I, DAF and CR1, and properdin, the latter being the only positive regulator of the alternative pathway (see Schwaeble W.J. and Reid K.B., Immunol Today 20(1):17-21 (1999)).

[0021] In addition to the apparently unregulated activation mechanism described above, the alternative pathway may also provide a powerful amplification loop for the lectin/classical pathway C3 convertase (C4b2a), since any C3b generated can participate with factor B in the formation of additional alternative pathway C3 convertase (C3bBb). The alternative pathway C3 convertase is stabilized by the binding of properdin. Properdin prolongs the half-life of the alternative pathway C3 convertase six- to ten-fold. Addition of C3b to the alternative pathway C3 convertase leads to the formation of the alternative pathway C5 convertase.

[0022] All three pathways (i.e., classical, lectin, and alternative) are believed to converge on C5, which is cleaved to form products with multiple pro-inflammatory effects. The converged pathway has been referred to as the terminal complement pathway. C5a is the most potent anaphylatoxin, inducing alterations in smooth muscle and vascular tone, as well as vascular permeability. It is also a potent chemotaxin and activator of 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, arachidonic acid metabolites, and reactive oxygen species. Cleavage of C5 leads to the formation of C5b-9, also known as the membrane attack complex (MAC). There is now strong evidence that sublytic MAC deposition may play an important role in inflammation beyond its role as a lytic pore-forming complex.

[0023] In addition to its essential role in immune defense, the complement system contributes to tissue damage in many clinical conditions. Thus, there is an urgent need to develop therapeutically effective complement inhibitors to prevent these adverse effects.

Summary

[0024] In one aspect, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to the serine protease domain of human MASP-3 (amino acid residues 450-728 of SEQ ID NO:2) with high affinity (having a K of less than 500 pM), wherein the antibody or antigen-binding fragment thereof inhibits activation of the alternative complement pathway. In some embodiments, the antibody or antigen-binding fragment is characterized by at least one or more of the following properties: (a) inhibits profactor D maturation; (b) does not bind to human MASP-1 (SEQ ID NO:8); (c) inhibits the alternative pathway at a molar ratio of about 1:1 to about 2.5:1 (MASP-3 target to mAb) in a mammalian subject; (d) does not inhibit the classical pathway; (e) inhibits hemolysis and/or opsonization; (f) inhibits substrate-specific cleavage by MASP-3 serine protease; (g) reduces hemolysis or reduced cleavage of C3 and surface deposition of C3b; (h) reduces deposition of Factor B and/or Bb on an activating surface; (i) reduces resting levels (in circulation and without experimental addition of an activating surface) of active Factor D relative to pro-Factor D; (j) reduces the level of active Factor D relative to pro-Factor D in response to an activating surface; (k) reduces the production of resting and surface-inducing levels of Ba, Bb, C3b, or C3a in fluid phase; and/or (l) reduces factor P deposition. In some embodiments, the isolated antibody or antigen-binding fragment thereof of paragraph 1 or 2, wherein said antibody or antigen-binding fragment thereof specifically binds to an epitope located within the serine protease domain of human MASP-3, wherein the epitope is located in at least one or more of: VLRSQRRDTTVI (SEQ ID NO:9), TAAHVLRSQRRDTTV (SEQ ID NO:10), DFNIQNYNHDIALVQ (SEQ ID NO:11), PHAECKTSYESRS (SEQ ID NO:12), GNYSVTENMFC (SEQ ID NO:13), VSNYVDWVWE (SEQ ID NO:14), and/or VLRSQRRDTTV (SEQ ID NO:15). In some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope within at least one of: ECGQPSRSLPSLV (SEQ ID NO:16), RNAEPGLFPWQ (SEQ ID NO:17); KWFGSGALLSASWIL (SEQ ID NO:18); EHVTVYLGLH (SEQ ID NO:19); PVPLGPHVMP (SEQ ID NO:20); APHMLGL (SEQ ID NO:21); SDVLQYVKLP (SEQ ID NO:22); and/or AFVIFDDLSQRW (SEQ ID NO:23).

[0025] In another aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof that binds to MASP-3, comprising: (a) a heavy chain variable region comprising a HC-CDR1 defined as SEQ ID NO:209 (XXDIN, wherein X at position 1 is S or T and wherein X at position 2 is N or D); a HC-CDR2 defined as SEQ ID NO:210 (WIYPRDXXXKYNXXFXD, wherein X at position 7 is G or D; X at position 8 is S, T or R; X at position 9 is I or T; X at position 13 is E or D; X at position 14 is K or E and X at position 16 is T or K); and a HC-CDR3 defined as SEQ ID NO: 211 (XEDXY, where X at position 1 is L or V and where X at position 4 is T or S); and (b) a light chain variable region comprising a LC-CDR1 defined as SEQ ID NO: 212 (KSSQSLLXXRTRKNYLX, where X at position 8 is N, I, Q or A; where X at position 9 is S or T; and where X at position 17 is A or S); a LC-CDR2 defined as SEQ ID NO: 144 (WASTRES) and a LC-CDR3 defined as SEQ ID NO:146 (KQSYNLYT).

[0026] In another aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof that binds to MASP-3, comprising: (a) a heavy chain variable region comprising a HC-CDR1 defined as SEQ ID NO:213 (SYGXX, wherein X at position 4 is H or I and wherein X at position 5 is S or T); a HC-CDR2 defined as SEQ ID NO:74; and a HC-CDR3 defined as SEQ ID NO:214 (GGXAXDY, wherein X at position 3 is E or D and wherein X at position 5 is M or L); and (b) a light chain variable region comprising a LC-CDR1 defined as SEQ ID NO:215 (KSSQSLLDSXXKTYLX, wherein X at position 10 is D and or A; at position 16 is N or S); an LC-CDR2 defined as SEQ ID NO:155; and an LC-CDR3 defined as SEQ ID NO:216 (WQGTHFPXT, where X at position 8 is either W or Y).

[0027] In another aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof that binds to MASP-3, comprising: (a) a heavy chain variable region comprising a HC-CDR1 defined as SEQ ID NO:84 (GKWIE); a HC-CDR2 defined as SEQ ID NO:86 (EILPGTGSTNYNEKFKG) or SEQ ID NO:275 (EILPGTGSTNYAQKFQG); and a HC-CDR3 defined as SEQ ID NO:88 (SEDV); and (b) a light chain variable region comprising a LC-CDR1 defined as SEQ ID NO:142 (KSSQSLLNSRTRKNYLA), SEQ ID NO:257 (KSSQSLLQSRTRKNYLA); SEQ ID NO:258 (KSSQSLLASRTRKNYLA); or SEQ ID NO:259 (KSSQSLLNTRTRKNYLA), an LC-CDR2 presented as SEQ ID NO:144 (WASTRES); and an LC-CDR3 defined as SEQ ID NO:161 (KQSYNIPT).

[0028] In another aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof that binds to MASP-3, comprising: (a) a heavy chain variable region comprising a HC-CDR1 defined as SEQ ID NO:91 (GYWIE); a HC-CDR2 defined as SEQ ID NO:93 (EMLPGSGSTHYNEKFKG); and a HC-CDR3 defined as SEQ ID NO:95 (SIDY); and (b) a light chain variable region comprising a LC-CDR1 defined as SEQ ID NO:163 (RSSQSLVQSNGNTYLH), a LC-CDR2 defined as SEQ ID NO:165 (KVSNRFS), and a LC-CDR3 defined as SEQ ID NO:167 (SQSTHVPPT).

[0029] In another aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof that binds to MASP-3, comprising: (a) a heavy chain variable region comprising a HC-CDR1 defined as SEQ ID NO:109 (RVHFAIRDTNYWMQ), a HC-CDR2 defined as SEQ ID NO:110 (AIYPGNGDTSYNQKFKG), a HC-CDR3 defined as SEQ ID NO:112 (GSHYFDY); and a light chain variable region comprising a LC-CDR1 defined as SEQ ID NO:182 (RASQSIGTSIH), a LC-CDR2 defined as SEQ ID NO:184 (YASESIS), and a LC-CDR3 defined as SEQ ID NO:186 (QQSNSWPYT); or (b) a heavy chain variable region comprising a HC-CDR1 defined as SEQ ID NO:125 (DYYMN), a HC-CDR2 defined as SEQ ID NO:127 (DVNPNNDGTTYNQKFKG), a HC-CDR3 defined as SEQ ID NO:129 (CPFYYLGKGTHFDY); and a light chain variable region comprising a LC-CDR1 defined as SEQ ID NO:196 (RASQDISNFLN), a LC-CDR2 defined as SEQ ID NO:198 (YTSRLHS) and a LC-CDR3 defined as SEQ ID NO:200 (QQGFTLPWT); or (c) a heavy chain variable region comprising a HC-CDR1 defined as SEQ ID NO:137 a HC-CDR2 defined as SEQ ID NO:138, a HC-CDR3 defined as SEQ ID NO:140; and a light chain variable region comprising an LC-CDR1 defined as SEQ ID NO:206, an LC-CDR2 defined as SEQ ID NO:207 and an LC-CDR3 defined as SEQ ID NO:208: or (d) a heavy chain variable region comprising an HC-CDR1 defined as SEQ ID NO:98, an HC-CDR2 defined as SEQ ID NO:99, an HC-CDR3 defined as SEQ ID NO:101; and a light chain variable region comprising an LC-CDR1 defined as SEQ ID NO:169, an LC-CDR2 defined as SEQ ID NO:171 and an LC-CDR3 defined as SEQ ID NO:173; or (e) a heavy chain variable region comprising a HC-CDR1 defined as SEQ ID NO:103, a HC-CDR2 defined as SEQ ID NO:105, a HC-CDR3 defined as SEQ ID NO:107; and a light chain variable region comprising a LC-CDR1 defined as SEQ ID NO:176, a LC-CDR2 defined as SEQ ID NO:178 and a LC-CDR3 defined as SEQ ID NO:193; or (f) a heavy chain variable region comprising a HC-CDR1 defined as SEQ ID NO:114, a HC-CDR2 defined as SEQ ID NO:116, a HC-CDR3 defined as SEQ ID NO:118; and a light chain variable region comprising an LC-CDR1 defined as SEQ ID NO:188, an LC-CDR2 defined as SEQ ID NO:178, and an LC-CDR3 defined as SEQ ID NO:190; or (g) a heavy chain variable region comprising an HC-CDR1 defined as SEQ ID NO:114, an HC-CDR2 defined as SEQ ID NO:121, an HC-CDR3 defined as SEQ ID NO:123; and a light chain variable region comprising an LC-CDR1 defined as SEQ ID NO:191, an LC-CDR2 defined as SEQ ID NO:178, and an LC-CDR3 defined as SEQ ID NO:193.

[0030] In another aspect, the present invention provides a method of inhibiting alternative complement pathway activation in a mammal, the method comprising administering to a mammal in need thereof an amount of a composition comprising a high affinity MASP-3 inhibitory antibody or antigen-binding fragment thereof sufficient to inhibit alternative pathway complement activation in the mammal. In one embodiment of the method, the antibody or antigen-binding fragment thereof binds to MASP-3 with an affinity of less than 500 pM. In one embodiment of the method, as a result of administering the composition comprising the antibody or antigen-binding fragment, one or more of the following is present in the mammal: (a) inhibition of Factor D maturation; (b) inhibition of the alternative pathway when administered to the subject in a molar ratio of from about 1:1 to about 2.5:1 (MASP-3 target to mAb); (c) the classical pathway is not inhibited; (d) inhibition of hemolysis and/or opsonization; (e) reduction of hemolysis or reduction of C3 cleavage and surface deposition of C3b; (f) reduction of Factor B and Bb deposition on an activating surface; (g) a reduction of resting levels (in circulation and without the experimental addition of an activating surface) of active Factor D relative to pro-Factor D; (h) a reduction of active Factor D levels relative to pro-Factor D in response to an activating surface; and/or (1) a reduction of the production of resting and surface induction levels of fluid phase Ba, Bb, C3b, or C3a. In one embodiment of the method, the composition comprises a MASP-3 inhibitory antibody that inhibits the alternative pathway in a molar ratio of from about 1:1 to about 2.5:1 (MASP-3 target to mAb).

[0031] In another aspect, the present invention provides a method of inhibiting MASP-3-dependent complement activation in a subject suffering from paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy, asthma, dense deposit disease, pauci-immune crescentic necrotizing glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis optica, or Behçet's disease. The method includes the step of administering to the subject a composition comprising an amount of a high-affinity MASP-3 inhibitory agent effective to inhibit MASP-3-dependent complement activation. In some embodiments, the method further comprises administering to the subject a composition comprising a MASP-2 inhibitory agent.

[0032] In another aspect, the present invention provides a method of manufacturing a medicament for use in inhibiting the effects of MASP-3-dependent complement activation in living subjects in need thereof, comprising combining a therapeutically effective amount of a MASP-3 inhibitory agent in a pharmaceutical carrier. In some embodiments, the MASP-3 inhibitory agent is a high affinity MASP-3 inhibitory antibody. In some embodiments, the method according to this aspect of the invention comprises manufacturing a medicament for use in inhibiting the effects of MASP-3-dependent complement activation in a subject suffering from or at risk of developing a disease or disorder selected from the group consisting of paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy, asthma, dense deposit disease, pauci-immune necrotizing crescentic glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis optica, or Behçet's disease. In some embodiments, the method further comprises combining a therapeutically effective amount of a MASP-2 inhibitory agent in or with the medicament comprising the MASP-3 inhibitor.

[0033] In another aspect, the present invention provides a pharmaceutical composition comprising a physiologically acceptable carrier and a high affinity MASP-3 monoclonal antibody or antigen-binding fragment thereof that binds to human MASP-3 and inhibits alternative pathway complement activation. In one embodiment, said high affinity MASP-3 antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region comprising (i) VHCDR1 comprising SEQ ID NO:84, (ii) VHCDR2 comprising SEQ ID NO:86 or SEQ ID NO:275, and (iii) VHCDR3 comprising SEQ ID NO:88; and (b) a light chain variable region comprising (i) VLCDR1 comprising SEQ ID NO:142, SEQ ID NO:257, SEQ ID NO:258 or SEQ ID NO:259 (ii) VLCDR2 comprising SEQ ID NO:144 and (iii) VLCDR3 comprising SEQ ID NO:161.

[0034] In another aspect, the present invention provides a method of treating a subject suffering from or at risk of developing paroxysmal nocturnal hemoglobinuria (PNH), comprising administering to the subject a pharmaceutical composition comprising an effective amount of a high affinity monoclonal antibody or antigen-binding fragment thereof that binds to human MASP-3 and inhibits alternative pathway complement activation to treat or reduce the risk of PNH in the subject. In one embodiment, the antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region comprising (i) VHCDR1 comprising SEQ ID NO:84, (ii) VHCDR2 comprising SEQ ID NO:86 or SEQ ID NO:275, and (iii) VHCDR3 comprising SEQ ID NO:88; and (b) a light chain variable region comprising (i) VLCDR1 comprising SEQ ID NO:142, SEQ ID NO:257, SEQ ID NO:258 or SEQ ID NO:259 (ii) VLCDR2 comprising SEQ ID NO:144 and (iii) VLCDR3 comprising SEQ ID NO:161. In some embodiments, the pharmaceutical composition increases red blood cell survival in the individual suffering from PNH. In some embodiments, wherein the individual suffering from or at risk of developing PNH exhibits one or more symptoms selected from the group consisting of (i) below normal hemoglobin levels, (ii) below normal platelet levels; (iii) above normal reticulocyte levels and (iv) above normal bilirubin levels. In some embodiments, the pharmaceutical composition is administered systemically (e.g., subcutaneously, intramuscularly, intravenously, intra-arterially, or as an inhalant) to a subject suffering from or at risk of developing PNH. In some embodiments, the subject suffering from or at risk of PNH has previously undergone or is currently undergoing treatment with a terminal complement inhibitor that inhibits the cleavage of complement protein C5. In some embodiments, the method further comprises administering to the subject a terminal complement inhibitor that inhibits the cleavage of complement protein C5. In some embodiments, the terminal complement inhibitor is a humanized anti-C5 antibody or antigen-binding fragment thereof. In some embodiments, the terminal complement inhibitor is eculizumab.

[0035] In another aspect, the present invention provides a method for treating a subject suffering from or at risk of developing arthritis (inflammatory and non-inflammatory arthritis) comprising administering to the subject a pharmaceutical composition comprising an effective amount of a high affinity monoclonal antibody or antigen-binding fragment thereof that binds to human MASP-3 and inhibits alternative pathway complement activation to treat or reduce the risk of arthritis in the subject. In one embodiment, said antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region comprising (i) VHCDR1 comprising SEQ ID NO:84, (ii) VHCDR2 comprising SEQ ID NO:86 or SEQ ID NO:275, and (iii) VHCDR3 comprising SEQ ID NO:88; and (b) a light chain variable region comprising (i) VLCDR1 comprising SEQ ID NO:142, SEQ ID NO:257, SEQ ID NO:258, or SEQ ID NO:259 (ii) VLCDR2 comprising SEQ ID NO:144, and (iii) VLCDR3 comprising SEQ ID NO:161. In some embodiments, the subject has arthritis selected from the group consisting of osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, ankylosing spondylitis, Behcet's disease, infection-related arthritis, and psoriatic arthritis. In some embodiments, the pharmaceutical composition is administered systemically (i.e., subcutaneously, intramuscularly, intravenously, intraarterially, or as an inhalant). In some embodiments, the pharmaceutical composition is administered locally to a joint.

[0036] As described herein, various embodiments of high affinity MASP-3 inhibitory antibodies, optionally in combination with various embodiments of MASP-2 inhibitory agents, can be used in the pharmaceutical compositions of the invention.

[0037] As described herein, the pharmaceutical compositions of the invention can be used in accordance with the methods of the invention.

[0038] These and other aspects and embodiments of the invention described herein will be apparent with reference to the detailed description and drawings which follow. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referred to in this specification are hereby incorporated by reference in their entirety, as if each were incorporated individually.

Description of drawings

[0039] The foregoing aspects and many of the advantages of the present invention will be more readily appreciated as the same becomes better understood by reference to the following detailed description when considered in conjunction with the accompanying drawings, in which: FIGURE 1 illustrates a new understanding of the lectin and alternative pathways; FIGURE 2 is a schematic diagram adapted from Schwaeble et al., Immunobiol 205:455-466 (2002), as modified by Yongqing et al., BBA 1824:253 (2012), illustrating the protein domains of MASP-1, MASP-3, and MAp44 and the exons encoding the same; FIGURE 3 depicts the amino acid sequence of human MASP-3 (SEQ ID NO: 2) with the leader sequence shown underlined; FIGURE 4 shows an alignment of the full-length MASP-3 protein from multiple species; FIGURE 5 shows an alignment of the SP domain of the MASP-3 protein from multiple species; FIGURE 6 is a Kaplan-Meyer plot graphically illustrating the percent survival of WT and MASP-2 KO mice following administration of a 2.6 × 10 7 cfu infectious dose of N. meningitidis serogroup A Z2491, demonstrating that MASP-2-deficient mice are protected against N. meningitidis-induced mortality as described in Example 1; FIGURE 7 is a Kaplan-Meyer plot graphically illustrating the percent survival of WT and MASP-2 KO mice following administration of a 6 × 10 6 cfu infectious dose of N. meningitidis serogroup B strain MC58, demonstrating that MASP-2-deficient mice are protected against N. meningitidis-induced mortality as described in Example 1; FIGURE 8 graphically illustrates the log cfu/mL of N. meningitidis serogroup B strain MC58 per mL of blood recovered from WT and MASP-2 KO mice at different time points following i.p. infection with 6x106 cfu of N. meningitidis serogroup B strain MC58 (n = 3 at different time points for both groups of mice), demonstrating that although MASP-2 KO mice were infected with the same dose of N. meningitidis serogroup B strain MC58 as WT mice, MASP-2 KO mice had increased clearance of bacteremia compared to WT, as described in Example 1; FIGURE 9 graphically illustrates the mean disease score of WT and MASP-2 KO mice at 3, 6, 12, and 24 hours post-infection with 6x106 cfu of N. meningitidis serogroup B strain MC58, demonstrating that MASP-2-deficient mice showed much lower disease scores at 6 hours, 12 hours, and 24 hours post-infection compared to WT mice, as described in Example 1; FIGURE 10 is a Kaplan-Meyer plot graphically illustrating the percent survival of mice following administration of an infectious dose of 4x106 cfu of N. meningitidis serogroup B strain MC58 followed by administration 3 hours post-infection of either inhibitory MASP-2 antibody (1 mg/kg) or isotype control antibody, demonstrating that MASP-2 antibody is effective in treating and improving survival in individuals infected with N. meningitidis as described in Example 2; FIGURE 11 graphically illustrates the log cfu/mL viable counts of N. meningitidis serogroup B strain MC58 recovered at different time points in the human serum samples presented in TABLE 6 taken at various time points following incubation with N. meningitidis serogroup B strain MC58 as described in Example 3; FIGURE 12 graphically illustrates the log cfu/mL of viable counts of N. meningitidis serogroup B-MC58 recovered at different time points in the human serum samples presented in TABLE 8, showing that complement-dependent killing of N. meningitidis in 20% (v/v) human serum is dependent on MASP-3 and MBL, as described in Example 3; FIGURE 13 graphically illustrates the log cfu/mL of viable counts of N. meningitidis serogroup B-MC58 recovered at different time points in the mouse serum samples shown in TABLE 10, showing that MASP-2 -/- knockout mouse serum (referred to as “MASP-2 -/-”) has a higher level of bactericidal activity for N. meningitidis than WT mouse serum, while in contrast, MASP-1/3 -/- mouse serum has no bactericidal activity, as described in Example 3; FIGURE 14 graphically illustrates the kinetics of C3 activation under specific lectin pathway conditions (1% plasma) in sera from WT, C4 -/-, MASP-1/3 -/-, Factor B -/-, and MASP-2 -/- mice, as described in Example 4; FIGURE 15 graphically illustrates the level of alternative pathway-driven (AP-driven) C3b deposition in zymosan-coated microtiter plates under “traditional” alternative pathway-specific (AP-specific) conditions (i.e., BBS/EGTA/Mg++ without Ca++) as a function of serum concentration in serum samples obtained from MASP-3-deficient, C4-deficient, and MBL-deficient individuals as described in Example 4; FIGURE 16 graphically illustrates the level of AP-driven C3b deposition in zymosan-coated microtiter plates under “traditional” AP-specific conditions (i.e., BBS/EGTA/Mg++ without Ca++) as a function of time in 10% human serum samples from MASP-3-deficient, C4-deficient, and MBL-deficient individuals as described in Example 4; FIGURE 17A graphically illustrates the level of C3b deposition on mannan-coated microtiter plates as a function of serum concentration in serum samples obtained from WT, MASP-2-deficient, and MASP-1/3-deficient mice under specific “traditional” AP conditions (i.e., BBS/EGTA/Mg++ without Ca++) or under physiological conditions that allow both the lectin pathway and the alternative pathway (AP) to function (BBS/Mg++/Ca++), as described in Example 4; FIGURE 17B graphically illustrates the level of C3b deposition on zymosan-coated microtiter plates as a function of serum concentration in serum samples obtained from MASP-2-deficient and MASP-1/3-deficient WT mice under traditional AP-specific conditions (i.e., BBS/EGTA/Mg++ without Ca++) or under physiological conditions that allow both the lectin pathway and the alternative pathway to function (BBS/Mg++/Ca++), as described in Example 4; FIGURE 17C graphically illustrates the level of C3b deposition on S. pneumoniae D39-coated microtiter plates as a function of serum concentration in serum samples obtained from WT, MASP-2-deficient, and MASP-1/3-deficient mice under specific “traditional” AP conditions (i.e., BBS/EGTA/Mg++ without Ca++) or under physiological conditions that allow both the lectin pathway and the alternative pathway to function (BBS/Mg++/Ca++), as described in Example 4; FIGURE 18A graphically illustrates the results of a C3b deposition assay in highly diluted sera performed in mannan-coated microtiter plates under traditional AP-specific conditions (i.e., BBS/EGTA/Mg++ without Ca++) or under physiological conditions that allow both the lectin pathway and the alternative pathway to function (BBS/Mg++/Ca++), using serum concentrations ranging from 0% to 1.25%, as described in Example 4; FIGURE 18B graphically illustrates the results of a C3b deposition assay in highly diluted sera performed in zymosan-coated microtiter plates under traditional AP-specific conditions (i.e., BBS/EGTA/Mg++ without Ca++) or under physiological conditions that allow both the lectin pathway and the alternative pathway to function (BBS/Mg++/Ca++), using serum concentrations ranging from 0% to 1.25%, as described in Example 4; FIGURE 18C graphically illustrates the results of a C3b deposition assay performed on S. pneumoniae D39-coated microtiter plates under specific traditional AP conditions (i.e., BBS/EGTA/Mg++ without Ca++) or under physiological conditions that allow both the lectin pathway and the alternative pathway to function (BBS/Mg++/Ca++), using serum concentrations ranging from 0% to 1.25%, as described in Example 4; FIGURE 19 graphically illustrates the level of hemolysis (as measured by the release of hemoglobin from lysed rat erythrocytes (Crry/C3-/-) into the supernatant measured by photometry) of mannan-coated murine erythrocytes by human serum under physiological conditions (i.e., in the presence of Ca++) in a range of serum dilutions in MASP-3-/- serum, heat-inactivated normal human serum (HI NHS), MBL-/-, NHS+ MASP-2 monoclonal antibody, and NHS control, as described in Example 5; FIGURE 20 graphically illustrates the level of hemolysis (as measured by the release of hemoglobin from lysed rat erythrocytes (Crry/C3-/-) into the supernatant measured by photometry) of mannan-coated murine erythrocytes by human serum under physiological conditions (i.e., in the presence of Ca++) over a range of serum concentrations in MASP-3-/-, heat-inactivated (HI) NHS, MBL-/-, NHS+ MASP-2 monoclonal antibody, and NHS control serum, as described in Example 5; FIGURE 21 graphically illustrates the level of hemolysis (as measured by the release of hemoglobin from lysed WT mouse erythrocytes into the supernatant measured by photometry) of uncoated murine erythrocytes by human serum under physiological conditions (i.e., in the presence of Ca++) over a range of serum concentrations in the serum of 3MC (MASP-3-/-), heat-inactivated (HI) NHS, MBL-/-, NHS+MASP-2 monoclonal antibody, and NHS control, as described in Example 5; FIGURE 22 graphically illustrates the hemolysis (as measured by the release of hemoglobin from lysed mouse (CD55/59-/-) erythrocytes into the supernatant measured by photometry) of uncoated murine erythrocytes by human serum under physiological conditions (i.e., in the presence of Ca++) over a range of serum concentrations in heat-inactivated (HI) NHS serum, MBL-/-, NHS + MASP-2 monoclonal antibody, and control NHS, as described in Example 5; FIGURE 23 graphically illustrates the hemolysis (as measured by the release of hemoglobin from lysed rabbit erythrocytes into the supernatant measured by photometry) of mannan-coated rabbit erythrocytes by MASP-1/3-/- mouse serum and control WT mouse serum under physiological conditions (i.e., in the presence of Ca++) over a range of serum concentrations as described in Example 6; FIGURE 24A is a FACS histogram of MASP-3 antigen/antibody binding to clone M3J5, as described in Example 7; FIGURE 24B is a FACS histogram of MASP-3 antigen/antibody binding to clone M3M1, as described in Example 7; FIGURE 25 graphically illustrates a saturation binding curve of clone M3J5 (Clone 5) to MASP-3 antigen, as described in Example 7; FIGURE 26A is an amino acid sequence alignment of the VH regions of M3J5, M3M1, D14, and 1E10 to the chicken DT40 VH sequence, wherein dots represent amino acid identity to the DT40 sequence and dashes indicate gaps introduced to maximize alignment, as described in Example 7; FIGURE 26B is an amino acid sequence alignment of the VL regions of M3J5, M3M1, D14, and 1E10 to the chicken DT40 VL sequence, wherein dots represent amino acid identity to the DT40 sequence and dashes indicate gaps introduced to maximize alignment, as described in Example 7; FIGURE 27 is a bar graph showing the inhibitory activity of monoclonal antibody (mAb) 1E10 in the Wieslab Complement System Screen, MBL pathway compared to positive serum provided with the assay kit as well as an isotype control antibody, demonstrating that mAb1E10 partially inhibits LEA-2-dependent activation (via inhibition of MASP-1-dependent MASP-2 activation), whereas the isotype control antibody does not, as described in Example 7; FIGURE 28A provides the results of flow cytometric analysis of C3b deposition in heat-killed Staphylococcus aureus, demonstrating that in normal human serum in the presence of EDTA, which is known to inactivate both the lectin and alternative pathways, no C3b deposition was observed (panel 1), in normal human serum treated with Mg++/EGTA, alternative pathway-driven C3b deposition was observed (panel 2), and as shown in panels 3, 4, and 5, in factor B-depleted, factor D-depleted, and properdin (factor P)-depleted serum, respectively, no alternative pathway-driven C3b deposition was observed, as described in Example 8; FIGURE 28B provides the results of flow cytometric analysis of C3b deposition in heat-killed S. aureus, demonstrating that, as in EDTA-treated normal serum (panel 1), AP-driven C3b deposition is absent in 3MC serum in the presence of Mg++/EGTA (panel 3), whereas panels 4 and 5 show that full-length active rMASP-3 (panel 4) and active rMASP-3 (CCP1-CCP2-SP) (panel 5) restore AP-driven C3b deposition in 3MC serum to levels observed in Mg++/EGTA-treated normal serum (panel 2); neither inactive rMASP-3 (S679A) (panel 6) nor wild-type rMASP-1 (panel 7) can restore AP-driven C3b deposition in 3MC serum. 3MC, as described in Example 8; FIGURE 29 shows the results of a Western blot analysis to determine Factor B cleavage in response to S. aureus in 3MC serum in the presence or absence of rMASP-3, demonstrating that normal human serum in the presence of EDTA (negative control, lane 1) demonstrates very little Factor B cleavage relative to normal human serum in the presence of Mg++/EGTA, shown in lane 2 (positive control), as also shown in lane 3, 3MC serum demonstrates very little Factor B cleavage in the presence of Mg++/EGTA. However, as shown in lane 4, Factor B cleavage is restored by the addition and preincubation of full-length recombinant MASP-3 protein to 3MC serum, as described in Example 8; FIGURE 30 shows Comassie staining of a protein gel in which Factor B cleavage is analyzed, demonstrating that Factor B cleavage is most optimal in the presence of C3, MASP-3, and profactor D (lane 1) and as shown in lanes 4 and 5, MASP-3 or profactor D alone are capable of mediating Factor B cleavage, provided that C3 is present, as described in Example 8; FIGURE 31 graphically illustrates the mean fluorescence intensities (MFI) of S. aureus C3b staining obtained from mAbD14 (which binds to MASP-3), mAb1A5 (negative control antibody), and an isotype control antibody plotted as a function of mAb concentration in 3MC serum in the presence of rMASP-3, demonstrating that mAbD14 inhibits MASP-3-dependent C3b deposition in a concentration-dependent manner as described in Example 8; FIGURE 32 shows Western blot analysis of pro-factor D substrate cleavage, wherein compared to pro-factor D alone (lane 1) or full-length recombinant MASP-3 (S679A; lane 3) or MASP-1 (S646A; lane 4), full-length recombinant wild-type MASP-3 (lane 2) and MASP-1 (lane 5) completely or partially cleave pro-factor D to generate mature factor D, as described in Example 9; FIGURE 33 is a Western blot showing the inhibitory activity of the MASP-3-binding mAbs D14 (lane 2) and M3M1 (lane 3) on MASP-3-dependent cleavage of profactor D, compared with a control reaction containing only MASP-3 and profactor D (no mAb, lane 1), as well as a control reaction containing a mAb obtained from the DTLacO library that binds to MASP-1 but not MASP-3 (lane 4), as described in Example 9; FIGURE 34 graphically illustrates the level of AP-driven C3b deposition in zymosan-coated microtiter plates as a function of serum concentration in serum samples obtained from MASP-3 deficient (3MC), C4 deficient, and MBL deficient individuals, demonstrating that MASP-3 deficient serum from Patient 2 and Patient 3 have residual AP activity at high serum concentrations (serum concentrations of 25%, 12.5%, 6.25%) but a significantly higher AP50 (i.e., 8.2% and 12.3% of serum required to achieve 50% of maximal C3 deposition), as described in Example 10; FIGURE 35A graphically illustrates the level of AP-driven C3b deposition on zymosan-coated microtiter plates under “traditional” AP-specific conditions (i.e., BBS/EGTA/Mg++ without Ca++) as a function of time in 10% human serum samples from MASP-3-deficient, C4-deficient, and MBL-deficient individuals as described in Example 10; FIGURE 35B shows a Western blot with plasma obtained from 3MC patient #2 (MASP-3 (-/-), MASP-1 (+/+)), 3MC patient #3 (MASP-3 (-/-), MASP-1 (-/-)), and normal donor sera (W), in which human pro-factor D (25,040 Da) and/or mature factor D (24,405 Da) was detected with a human factor D-specific antibody as described in Example 10; FIGURE 35C graphically illustrates the results of Weislab classical, lectin, and alternative pathway assays with plasma obtained from 3MC patient #2, 3MC patient #3, and normal human serum, as described in Example 10; FIGURE 36 graphically illustrates the percentage hemolysis (as measured by the release of hemoglobin from lysed rabbit erythrocytes into the supernatant measured by photometry) of mannan-coated rabbit erythrocytes over a range of serum concentrations in serum from two normal human subjects (NHS) and two 3MC patients (Patient 2 and Patient 3), measured in the absence of Ca++, demonstrating that MASP-3 deficiency reduces the percentage of complement-mediated lysis of mannan-coated erythrocytes compared to normal human serum, as described in Example 10; FIGURE 37 graphically illustrates the level of AP-driven C3b deposition on zymosan-coated microtiter plates as a function of the concentration of recombinant full-length MASP-3 protein added to serum samples obtained from human 3MC patient 2 (MASP-3-/-), demonstrating that, compared to the negative control inactive recombinant MASP-3 (MASP-3A; S679A), active recombinant MASP-3 protein reconstitutes AP-driven C3b deposition on zymosan-coated plates in a concentration-dependent manner as described in Example 10; FIGURE 38 graphically illustrates the percentage hemolysis (measured by the release of hemoglobin from lysed rabbit erythrocytes into the supernatant measured by photometry) of mannan-coated rabbit erythrocytes across a range of serum concentrations in (1) normal human serum (NHS); (2) 3MC patient serum; (3) patient serum with 3MC plus active full-length recombinant MASP-3 (20 μg/ml); and (4) heat-inactivated human serum (HIS), measured in the absence of Ca++, demonstrating that the percentage of rabbit erythrocyte lysis is significantly increased in 3MC serum containing rMASP-3 compared to the percentage of lysis in 3MC serum without recombinant MASP-3 (p = 0.0006), as described in Example 10; FIGURE 39 graphically illustrates the percentage of rabbit erythrocyte lysis in 7% human serum from 3MC Patient 2 and 3MC Patient 3 containing active recombinant MASP-3 over a concentration range of 0 to 110 μg/ml (in BBS/Mg++/EGTA, demonstrating that the percentage of rabbit erythrocyte lysis increases with the amount of recombinant MASP-3 in a concentration-dependent manner, as described in Example 10; FIGURE 40 graphically illustrates the level of LEA-2-driven C3b deposition on mannan-coated ELISA plates as a function of the concentration of human serum diluted in BBS buffer, for serum from a normal human subject (NHS), two 3MC patients (Patient 2 and Patient 3), the parents of 3MC Patient 3, and an MBL-deficient individual, as described in Example 10; FIGURE 41 graphically illustrates a representative example of a binding experiment that was performed with human MASP-3 in which the M3-1 Fab (also referred to as 13B1) shows an apparent binding affinity (EC50) of about 0.117 nM to the human protein, as described in Example 11; FIGURE 42 graphically illustrates a representative example of a binding experiment that was performed with rat MASP-3 in which the M3-1 Fab (also referred to as 13B1) shows an apparent binding affinity (EC50) of about 0.214 nM to the rat protein, as described in Example 11; FIGURE 43 graphically illustrates the level of complement factor Bb deposition on zymosan particles (determined by cytometric detection measured in MFI units) in the presence of varying concentrations of mAb M3-1 (also referred to as 13B1) in CFD-depleted human serum, as described in Example 11; Figure 44 graphically illustrates the level of C3 deposition on zymosan particles at various time points following a single dose of mAb M3-1 (13B1) (10 mg/kg iv) in wild-type mice as described in Example 11; FIGURE 45 graphically illustrates the percent survival of donor erythrocytes (WT or Crry-) over a 14-day period in wild-type recipient mice treated with mAb M3-1 (13B1) (10 mg/kg on days -11, 04, -1, and +6), mAb BB5.1-treated, or carrier-treated mice as described in Example 12; FIGURE 46 graphically illustrates the percent survival of donor erythrocytes (WT or Crry-) over a 16-day period in wild-type recipient mice treated with a single dose of mAb M3-1 (13B1) (20 mg/kg on day -6) or carrier-treated mice as described in Example 12; Figure 47 graphically illustrates the clinical scores of mice treated with mAb M3-1 (13B1) (5 mg/kg or 20 mg/kg) or carrier-treated mice over a 14-day period in a collagen antibody-induced arthritis model as described in Example 13; Figure 48 graphically illustrates the percentage incidence of arthritis of mice treated with mAb M3-1 (13B1) (5 mg/kg or 20 mg/kg) or of mice treated with carrier over a 14-day period in a collagen antibody-induced arthritis model as described in Example 13; FIGURE 49A shows the amino acid sequences of the VH regions of high-affinity (<500 pM) anti-human MASP-3 inhibitory mAbs as described in Example 15; FIGURE 49B shows the amino acid sequences of the VL regions of high-affinity (<500 pM) anti-human MASP-3 inhibitory mAbs as described in Example 15; FIGURE 50A is a dendrogram of the VH regions of high-affinity anti-human MASP-3 inhibitory mAbs as described in Example 15; FIGURE 50B is a dendrogram of the VL regions of high affinity anti-human MASP-3 inhibitory mAbs as described in Example 15; FIGURE 51A graphically illustrates the results of a binding experiment in which representative purified recombinant anti-human MASP-3 inhibitory antibodies show an apparent binding avidity of less than 500 pM (e.g., from 240 pM to 23 pM) to human MASP-3 protein, as described in Example 16; FIGURE 51B graphically illustrates the results of a binding experiment in which representative purified recombinant anti-human MASP-3 inhibitory antibodies show an apparent binding avidity of less than 500 pM (e.g., from 91 pM to 58 pM) to human MASP-3 protein, as described in Example 16; FIGURE 51C graphically illustrates the results of a binding experiment in which representative purified recombinant high-affinity anti-human MASP-3 inhibitory antibodies are shown to be selective for binding to MASP-3 and do not bind to human MASP-1, as described in Example 16; FIGURE 51D graphically illustrates the results of a binding experiment in which representative purified recombinant high-affinity anti-human MASP-3 inhibitory antibodies are shown to be selective for binding to MASP-3 and do not bind to human MASP-2, as described in Example 16; FIGURE 52 graphically illustrates the results of a binding experiment in which representative purified recombinant anti-human MASP-3 inhibitory antibodies also show high binding avidity to rat MASP-3 protein, as described in Example 16; FIGURE 53 graphically illustrates the results of an experiment measuring the ability of representative high-affinity MASP-3 antibodies to inhibit fluorogenic tripeptide cleavage, as described in Example 16; FIGURE 54 shows a Western blot demonstrating the ability of high-affinity MASP-3 inhibitory mAbs to block recombinant MASP-3-mediated cleavage of profactor D to factor D in an in vitro assay, as described in Example 16; FIGURE 55A graphically illustrates the level of complement factor Bb deposition on zymosan particles (determined by flow cytometric detection measured in MFI units) in the presence of varying concentrations of high-affinity MASP-3 mAbs 1F3, 1G4, 2D7, and 4B6 in factor D-depleted human serum, as described in Example 16; FIGURE 55B graphically illustrates the level of complement factor Bb deposition on zymosan particles (determined by flow cytometric detection measured in MFI units) in the presence of varying concentrations of high affinity MASP-3 mAbs 4D5, 10D12, and 13B1 in factor D-depleted human serum as described in Example 16; FIGURE 56A graphically illustrates the level of inhibition of rabbit erythrocyte lysis in the presence of varying concentrations of high affinity MASP-3 mAbs 1A10, 1F3, 4B6, 4D5, and 2F2 as described in Example 16; FIGURE 56B graphically illustrates the level of inhibition of rabbit erythrocyte lysis in the presence of varying concentrations of high affinity MASP-3 mAbs 1B11, 1E7, 1G4, 2D7, and 2F5 as described in Example 16; FIGURE 57 shows a Western blot analyzing the level of pro-Factor D and Factor D in serum from patient with 3MC (Patient B) in the presence of active recombinant MASP-3 (rMASP-3), inactive rMASP-3, and active rMASP-3 plus high affinity MASP-3 mAb 4D5 as described in Example 16; Figure 58 graphically illustrates the level of C3/C3b/iC3b deposition on zymosan particles at various time points following a single dose of high-affinity mAbs ]M3-1 (13B1, 10 mg/kg) or 10D12 (10 mg/kg) in wild-type mice, as described in Example 17; FIGURE 59 shows a Western blot analyzing the status of the Factor B Ba fragment in mice treated with high-affinity MASP-3 mAb 10D12 (10 mg/kg) or mice treated with carrier control, as described in Example 17; FIGURE 60 graphically illustrates the level of inhibition of hemolysis in 20% serum from mice treated with high-affinity MASP-3 mAb 10D12 (10 mg/kg or 25 mg/kg), as described in Example 17; FIGURE 61A graphically illustrates the results of competition binding analysis to identify high-affinity MASP-3 mAbs that block the interaction between high-affinity MASP-3 mAb 4D5 and human MASP-3, as described in Example 18; FIGURE 61B graphically illustrates the results of competition binding analysis to identify high-affinity MASP-3 mAbs that block the interaction between high-affinity MASP-3 mAb 10D12 and human MASP-3, as described in Example 18; FIGURE 61C graphically illustrates the results of competition binding analysis to identify high-affinity MASP-3 mAbs that block the interaction between high-affinity MASP-3 mAb 13B1 and human MASP-3, as described in Example 18; FIGURE 61D graphically illustrates the results of competition binding analysis to identify high affinity MASP-3 mAbs that block the interaction between high affinity MASP-3 mAb 1F3 and human MASP-3, as described in Example 18; FIGURE 61E graphically illustrates the results of competition binding analysis to identify high affinity MASP-3 mAbs that block the interaction between high affinity MASP-3 mAb 1G4 and human MASP-3, as described in Example 18; FIGURE 62 provides a schematic diagram showing the contact regions on human MASP-3 by high affinity MASP-3 mAbs, as determined by Pepscan analysis, as described in Example 18; FIGURE 63A shows the contact regions between human MASP-3 and high-affinity MASP-3 mAbs 1F3, 4D5, and 1A10, including amino acid residues 498-509 (SEQ ID NO:9), amino acid residues 544-558 (SEQ ID NO:11), amino acid residues 639-649 (SEQ ID NO:13), and amino acid residues 704-713 (SEQ ID NO:14) of MASP-3, as described in Example 18; FIGURE 63B shows the contact regions between human MASP-3 and high-affinity MASP-3 mAb 10D12, including amino acid residues 498-509 (SEQ ID NO:9) of MASP-3, as described in Example 18; FIGURE 64 shows the contact regions between human MASP-3 and high-affinity MASP-3 mAb 13B1, including amino acid residues 494-508 (SEQ ID NO:10) and amino acid residues 626-638 (SEQ ID NO:12) of MASP-3, as described in Example 18; FIGURE 65 shows the contact regions between human MASP-3 and high-affinity MASP-3 mAb 1B11, including amino acid residues 435-447 (SEQ ID NO:16), amino acid residues 454-464 (SEQ ID NO:17), amino acid residues 583-589 (SEQ ID NO:21), and amino acid residues 614-623 (SEQ ID NO:22) of MASP-3, as described in Example 18; FIGURE 66 shows the contact regions between human MASP-3 and high-affinity MASP-3 mAbs 1E7, 1G4, and 2D7, including amino acid residues 454-464 (SEQ ID NO:17), amino acid residues 514-523 (SEQ ID NO:19), and amino acid residues 667-678 (SEQ ID NO:23) of MASP-3, as described in Example 18; FIGURE 67 shows the contact regions between human MASP-3 and high-affinity MASP-3 mAbs, 15D9 and 2F5, including amino acid residues 454-464 (SEQ ID NO:17), amino acid residues 479-493 (SEQ ID NO:18), amino acid residues 562-571 (SEQ ID NO:20), and amino acid residues 667-678 (SEQ ID NO:23) of MASP-3, as described in Example 18; FIGURE 68 graphically illustrates the results of the experimental mouse autoimmune encephalomyelitis (EAE) model treated with high-affinity MASP-3 inhibitory mAb 13B1 (10 mg/kg), Factor B mAb 1379 (30 mg/kg), or isotype control mAb (10 mg/kg) as described in Example 20; Figure 69 graphically illustrates the APC activity, as determined by mean MFI in a flow cytometric assay detecting complement factor Bb on the surface of zymosan particles, in serum samples obtained from a group of three cynomolgus monkeys over time after treatment with high-affinity MASP-3 mAb h13B1X, in the presence or absence of anti-factor D antibody added to the serum sample, as described in Example 21; FIGURE 70 graphically illustrates the APC activity, determined by Bb deposition on zymosan, in serum samples obtained from groups of cynomolgus monkeys (3 animals per group) treated with a single intravenous dose of 5 mg/kg of high-affinity MASP-3 inhibitory mAbs h4D5X, h10D12X, or h13B1X, as described in Example 21; FIGURE 71A graphically illustrates APC activity as determined by fluid-phase Ba in serum samples obtained from groups of cynomolgus monkeys (3 animals per group) over time following treatment with a single 5 mg/kg intravenous dose of mAbs h4D5X, h10D12X, and h13B1X as described in Example 21; FIGURE 71B graphically illustrates APC activity as determined by fluid-phase Ba in serum samples obtained from groups of cynomolgus monkeys (3 animals per group) over time following treatment with a single 5 mg/kg intravenous dose of mAbs h4D5X, h10D12X, and h13B1X as described in Example 21; FIGURE 71C graphically illustrates APC activity as determined by fluid-phase C3a in serum samples obtained from groups of cynomolgus monkeys (3 animals per group) over time following treatment with a single 5 mg/kg intravenous dose of mAbs h4D5X, h10D12X, and h13B1X as described in Example 21; Figure 72A graphically illustrates the molar ratio of target (MASP-3) to the high-affinity MASP-3 antibody h4D5X at the time points assessing complete APC inhibition as measured by fluid-phase Ba as described in Example 21; Figure 72B graphically illustrates the molar ratio of target (MASP-3) to the high-affinity MASP-3 antibody h10D12X at the time points assessing complete APC inhibition as measured by fluid-phase Ba as described in Example 21; Figure 72C graphically illustrates the molar ratio of target (MASP-3) to high affinity MASP-3 antibody h13B1X at the time points assessing complete APC inhibition, as measured by Ba in fluid phase, as described in Example 21; and FIGURE 73 shows a Western blot analyzing the level of profactor D and Factor D in the serum of a cynomolgus monkey over time (hours) after treatment with a single 5 mg/kg intravenous dose of mAb h13B1X, as described in Example 21.

SEQUENCE LISTING DESCRIPTION

[0040] SEQ ID NO:1 Human MASP-3 cDNA SEQ ID NO:2 Human MASP-3 protein (with leader) SEQ ID NO:3 Rat MASP-3 protein (with leader) SEQ ID NO:4 Rat MASP-3 protein SEQ ID NO:5 Chicken MASP-3 protein SEQ ID NO:6 Rabbit MASP-3 protein SEQ ID NO:7 Cynomolgus monkey MASP-3 protein SEQ ID NO:8 Human MASP-1 protein (with leader) Peptide fragments of the SP domain of human MASP-3: SEQ ID NO:9 (aa 498-509 of human MASP-3 w/leader) SEQ ID NO:10 (aa 494-508 of human MASP-3 w/leader) SEQ ID NO:11 (aa 544-558 of human MASP-3 w/leader) SEQ ID NO:12 (aa 626-638 of human MASP-3 w/leader) SEQ ID NO:13 (aa 639-649 of human MASP-3 w/leader) SEQ ID NO:14 (aa 704-713 of human MASP-3 w/leader) SEQ ID NO:15 (aa 498-508 of human MASP-3 w/leader) SEQ ID NO:16 (aa 435-447 of human MASP-3 w/leader) SEQ ID NO:17 (aa 454-464 of human MASP-3 w/leader) SEQ ID NO:18 (aa 479-493 of human MASP-3 w/leader) SEQ ID NO:19 (aa 514-523 of human MASP-3 w/leader) SEQ ID NO:20 (aa 562-571 of Human MASP-3 w/leader) SEQ ID NO:21 (aa 583-589 of human MASP-3 w/leader) SEQ ID NO:22 (aa 614-623 of human MASP-3 w/leader) SEQ ID NO:23 (aa 667-678 of human MASP-3 w/leader) SEQ ID NO:24-39: Heavy chain variable regions - rat precursors SEQ ID NO:24 4D5_VH SEQ ID NO:25 1F3_VH SEQ ID NO:26 4B6_VH SEQ ID NO:27 1A10_VH SEQ ID NO:28 10D12_VH SEQ ID NO:29 35C1_VH SEQ ID NO:30 13B1_VH SEQ ID NO:31 1G4_VH SEQ ID NO:32 1E7_VH SEQ ID NO:33 2D7_VH SEQ ID NO:34 49C11_VH SEQ ID NO:35 15D9_VH SEQ ID NO:36 2F5_VH SEQ ID NO:37 1B11_VH SEQ ID NO:38 2F2_VH SEQ ID NO:39 11B6_VH SEQ ID NO:40-54: light chain variables - mouse precursors SEQ ID NO:40 4D5_VL SEQ ID NO:41 1F3_VL SEQ ID NO:42 4B6/1A10_VL SEQ ID NO:43 10D12_VL SEQ ID NO:44 35C1_VL SEQ ID NO:45 13B1_VL SEQ ID NO:46 1G4_VL SEQ ID NO:47 1E7_VL SEQ ID NO:48 2D7_VL SEQ ID NO:49 49C11_VL SEQ ID NO:50 15D9_VL SEQ ID NO:51 2F5_VL SEQ ID NO:52 1B11_VL SEQ ID NO:53 2F2_VL SEQ ID NO:54 11B6_VL SEQ ID NO:55-140: heavy chain framework regions (FRs) and complementarity determining regions (CDRs) of MASP-3 mAbs from rat precursors SEQ ID NO:141-208: light chain FRs and CDRs of MASP-3 mAbs from rat precursors SEQ ID NO:209-216: CDR consensus sequences SEQ ID NO:217-232: DNA encoding heavy chain variable regions (rat precursors) SEQ ID NO:233-247: DNA encoding light chain variable regions (mouse precursor) SEQ ID NO:248: humanized 4D5_VH-14 (h4D5_VH-14) heavy chain variable region SEQ ID NO:249: humanized 4D5_VH-19 (h4D5_VH-19) heavy chain variable region SEQ ID NO:250: humanized 4D5_VL-1 (h4D5_VL-1) light chain variable region SEQ ID NO:251: humanized 10D12_VH-45 (h10D12_VH-45) heavy chain variable region SEQ ID NO:252: humanized 10D12_VH-49 (h10D12_VH-49) heavy chain variable region SEQ ID NO:253: humanized 4D5_VL-1 (h4D5_VL-1) light chain variable region 10D12_VL-21 (h10D12-VL-21) humanized SEQ ID NO:254: 13B1_VH-9 (h13B1-VH-9) heavy chain variable region humanized SEQ ID NO:255: 13B1_VH-10 (h13B1-VH-10) heavy chain variable region humanized SEQ ID NO:256: 13B1-VL-1 (h13B1-VL-1) light chain variable region humanized SEQ ID NO:257: 4D5 and 13B1 LC-CDR1-NQ SEQ ID NO:258: 4D5 and 13B1 LC-CDR1-NA SEQ ID NO:259: 4D5 and 13B1 LC-CDR1-ST SEQ ID NO:260: consensus LC-CDR1 for 4D5, 13B1 from precursors and variants SEQ ID NO:261: 10D12 LC-CDR1-DE SEQ ID NO:262: 10D12 LC-CDR1-DA SEQ ID NO:263: 10D12 LC-CDR1-GA SEQ ID NO:264-277: HC FR and CDRs for 4D5, 10D12 and 13B 1 humanized SEQ ID NO:278: h4D5_VL-1-NA SEQ ID NO:279: h10D12_VL-21-GA SEQ ID NO:280: h13B1_VL-1-NA SEQ ID NO:281-287 LC FR and CDRs for 4D5, 10D12 and 13B1 humanized SEQ ID NO:288-293: DNA that encoding humanized 4D5, 10D12, 13B1 heavy chain variable region and variants SEQ ID NO:294-299: DNA encoding humanized 4D5, 10D12, 13B1 light chain variable region and variants SEQ ID NO:300: DTLacO precursor heavy chain variable region (VH) polypeptide SEQ ID NO:301: MASP-3-specific clone M3J5 heavy chain variable region (VH) polypeptide SEQ ID NO:302: MASP-3-specific clone M3M1 heavy chain variable region (VH) polypeptide SEQ ID NO:303: DTLacO precursor light chain variable region (VL) polypeptide SEQ ID NO:304: MASP-3-specific clone M3J5 light chain variable region (VL) polypeptide SEQ ID NO:305: Light chain variable region polypeptide (VL) from MASP-3-specific clone M3M1 SEQ ID NO:306: Heavy chain variable region (VH) polypeptide from MASP-3 clone D14 SEQ ID NO:307: Light chain variable region (VL) polypeptide from MASP-3 clone D14 SEQ ID NO:308: Heavy chain variable region (VH) polypeptide from MASP-1 clone 1E10 SEQ ID NO:309: Light chain variable region (VL) polypeptide from MASP-1 clone 1E10 SEQ ID NO:310: Human IgG4 constant region SEQ ID NO:311: Human IgG4 constant region with S228P mutation SEQ ID NO:312: Human IgG4 constant region with S228P_X mutation SEQ ID NO:313: Human IgGK constant region

Detailed Description I. Definitions

[0041] Unless specifically defined herein, all terms used herein have the same meaning as would be understood by those skilled in the art of the present invention. The following definitions are provided in order to provide clarity with respect to the terms as they are used in the specification and claims to describe the present invention.

[0042] As used herein, lectin pathway effector arm 1 (“LEA-1”) refers to the lectin-dependent activation of factor B and factor D by MASP-3.

[0043] As used herein, lectin pathway effector arm 2 (“LEA-2”) refers to MASP-2-dependent complement activation.

[0044] As used herein, the term "MASP-3-dependent complement activation" comprises two components: (i) lectin-dependent MASP-3 factor B and factor D activation, encompassed by LEA-1-mediated complement activation, occurs in the presence of Ca++, commonly leading to the conversion of C3bB to C3bBb and profactor D to factor D; and (ii) lectin-independent factor B and factor D conversion, which can occur in the absence of Ca++, commonly leading to the conversion of C3bB to C3bBb and profactor D to factor D. LEA-1-mediated complement activation and lectin-independent factor B and factor D conversion have been determined to cause opsonization and/or lysis. Without wishing to be bound by any particular theory, it is believed that only when multiple C3b molecules associate and bind in close proximity does the C3bBb C3 convertase change its substrate specificity and cleaves C5 as the alternative pathway C5 convertase called C3bBb(C3b)n.

[0045] As used herein, the term “MASP-2-dependent complement activation”, also referred to herein as LEA-2-mediated complement activation, encompasses lectin-dependent activation of MASP-2 that occurs in the presence of Ca++, leading to the formation of the lectin pathway C3 convertase C4b2a and following accumulation of the C3 cleavage product C3b subsequently to the C5 convertase C4b2a(C3b)n, which has been determined to cause opsonization and/or lysis.

[0046] As used herein, the term "traditional understanding of the alternative pathway" also referred to as the "traditional alternative pathway" refers to the alternative pathway prior to the discovery described herein, i.e., complement activation that is triggered, for example, by zymosan from fungal and yeast cell walls, lipopolysaccharide (LPS) from Gram-negative outer membranes and rabbit erythrocytes, as well as from many pure polysaccharides, viruses, bacteria, animal tumor cells, parasites, and damaged cells, and that was traditionally believed to arise from the spontaneous proteolytic generation of C3b from complement factor C3. As used herein, activation of the "traditional alternative pathway", also referred to herein as the "alternative pathway", is measured in Mg++/EGTA buffer (i.e., in the absence of Ca++).

[0047] As used herein, the term "lectin pathway" refers to complement activation that occurs through the specific binding of serum and non-serum carbohydrate-binding proteins including mannan-binding lectin (MBL), CL-11, and ficolins (H-ficolin, M-ficolin, or L-ficolin). As described herein, the inventors have discovered that the lectin pathway is driven by two effector arms, lectin pathway effector arm 1 (LEA-1), which is now known to be dependent on MASP-3, and lectin pathway effector arm 2 (LEA-2), which is dependent on MASP-2. As used herein, activation of the lectin pathways is assessed using Ca++-containing buffers.

[0048] As used herein, the term "classical pathway" refers to complement activation that is triggered by antibody bound to a foreign particle and requires binding of the C1q recognition molecule.

[0049] As used herein, the term “HTRA-1” refers to the high temperature requirement serine protease serine peptidase A1.

[0050] As used herein, the term "MASP-3 inhibitory agent" refers to any agent that directly inhibits MASP-3-dependent complement activation, including agents that bind to or interact directly with MASP-3, including MASP-3 antibodies and MASP-3 binding fragments thereof, natural and synthetic peptides, competitive substrates, small molecules, expression inhibitors, and isolated natural inhibitors, and also encompasses peptides that compete with MASP-3 for binding to another recognition molecule (e.g., MBL, CL-11, H-ficolin, M-ficolin, or L-ficolin) in the lectin pathway. In one embodiment, the MASP-3 inhibitory agent is specific to MASP-3 and does not bind to MASP-1 or MASP-2. An inhibitory agent that directly inhibits MASP-3 may be referred to as a direct MASP-3 inhibitory agent (e.g., a MASP-3 antibody), while an inhibitory agent that indirectly inhibits MASP-3 may be referred to as an indirect MASP-3 inhibitory agent (e.g., a MASP-1 antibody that inhibits MASP-3 activation). An example of a direct MASP-3 inhibitory agent is a specific MASP-3 inhibitory agent, such as a MASP-3 inhibitory agent, that specifically binds to a portion of human MASP-3 (SEQ ID NO:2) with a binding affinity at least 10-fold greater than other components of the complement system. Another example of a direct MASP-3 inhibitory agent is a high-affinity MASP-3 antibody that specifically binds to the serine protease domain of human MASP-3 (SEQ ID NO:2) with an affinity of less than 500 pM and does not bind to human MASP-1 (SEQ ID NO:8). In one embodiment, a MASP-3 inhibitory agent indirectly inhibits MASP-3 activity, such as, for example, an inhibitor of MASP-3 activation, including an inhibitor of MASP-1-mediated MASP-3 activation (e.g., a MASP-1 antibody or MASP-1 binding fragments thereof, natural and synthetic peptides, small molecules, expression inhibitors, and isolated natural inhibitors, and also encompasses peptides that compete with MASP-1 for binding to MASP-3). In a preferred embodiment, a MASP-3 inhibitory agent, such as an antibody or antigen-binding fragment thereof or antigen-binding peptide, inhibits MASP-3-mediated maturation of factor D. In another embodiment, a MASP-3 inhibitory agent inhibits MASP-3-mediated activation of factor B. MASP-3 inhibitory agents useful in the method of the invention can reduce MASP-3-dependent complement activation by more than 10%, such as more than 20%, more than 50%, or more than 90%. In one embodiment, the MASP-3 inhibitory agent reduces MASP-3-dependent complement activation by more than 90% (i.e., resulting in MASP-3 complement activation of only 10% or less). Inhibition of MASP-3 is expected to block, in whole or in part, both LEA-1-related lysis and opsonization and the lectin-independent conversion of factor B and factor D-related lysis and opsonization.

[0051] In one embodiment, a high affinity MASP-3 inhibitory antibody binds to the serine protease domain of MASP-3 (amino acid residues 450-728 of SEQ ID NO:2) with an affinity of less than 500 pM (e.g., less than 250 pM, less than 100 pM, less than 50 pM, or less than 10 pM) and inhibits the alternative pathway of complement activation in the blood of a mammal by at least 50% (e.g., at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95% or more).

[0052] An “antibody” is an immunoglobulin molecule capable of specifically binding to a target, such as a polypeptide, through at least one epitope recognition site located in the variable region (also referred to herein as the variable domain) of the immunoglobulin molecule.

[0053] As used herein, the term “antibody” encompasses antibodies and antibody fragments thereof, derived from any antibody-producing mammal (e.g., mouse, rat, rabbit, and primate including human) or from a hybridoma, phage selection, recombinant expression, or transgenic animals (or other methods of producing antibodies or antibody fragments), that specifically bind to a target polypeptide, such as, for example, MASP-1, MASP-2, or MASP-3 polypeptides or portions thereof. The term “antibody” is not intended to be limited with respect to the source of the antibody or the manner in which it is generated (e.g., by hybridoma, phage selection, recombinant expression, transgenic animal, peptide synthesis, etc.). Exemplary antibodies include polyclonal, monoclonal, and recombinant antibodies; pan-specific multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies); humanized antibodies; murine antibodies; chimeric monoclonal antibodies, mouse-human, mouse-primate, primate-human; 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 a single variable region antibody (dAb) or other known antibody fragments such as Fab, Fab', F(ab')2, Fv and the like, single chain (ScFv), synthetic variants thereof, naturally occurring variants, fusion proteins comprising an antibody portion with an antigen-binding fragment of the required specificity, humanized antibodies, chimeric antibodies, bispecific antibodies, and any other modified configuration of the immunoglobulin molecule comprising an antigen-binding site or fragment (epitope recognition site) of the required specificity.

[0054] A “monoclonal antibody” refers to a population of homogeneous antibodies wherein the monoclonal antibody is comprised of amino acids (naturally occurring and non-naturally occurring) that are involved in the selective binding of an epitope. Monoclonal antibodies are highly specific 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 molecule that comprises an antigen-binding fragment (epitope recognition site) of the required specificity and the ability to bind to an epitope. It is not intended to be limiting with respect to the source of the antibody or the manner in which it is generated (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term includes whole immunoglobulins as well as the fragments, etc. described above under the definition of "antibody".

[0055] As used herein, the term "antibody fragment" refers to a portion derived from or related to a full-length antibody, such as, for example, a MASP1, MASP-2, or MASP-3 antibody, generally including the antigen-binding or variable region thereof. Illustrative examples of antibody fragments include Fab, Fab', F(ab)2, F(ab')2 fragments, scFv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0056] In certain embodiments, antibodies and antigen-binding fragments thereof as described herein include a set of heavy chain (VH) and light chain (VL) complementarity determining region (“CDR”), respectively, interposed between a set of heavy chain and light chain framework regions (FR) that provide support for the CDRs and define the spatial relationship of the CDRs to one another. As used herein, the term “CDR set” refers to the three hypervariable regions of a heavy or light chain V region. Proceeding from the N-terminus of a heavy or light chain, these regions are denoted as “CDR1”, “CDR2”, and “CDR3”, respectively. An antigen-binding site therefore includes six CDRs, comprising the set of CDRs of each of a heavy and light chain V region.

[0057] As used herein, the term "FR cluster" refers to the four flanking amino acid sequences that frame the CDRs of a heavy or light chain V region CDR cluster. Some FR residues may contact bound antigen; however, FRs are primarily responsible for folding the V region into the antigen-binding site, particularly FR residues directly adjacent to CDRs. Within FRs, certain amino acid residues and certain structural features are highly conserved. In this regard, all V region sequences contain an internal disulfide loop of about 90 amino acid residues. As V regions fold into a binding site, CDRs are displayed as projecting loop motifs that form an antigen-binding surface. It is generally recognized that there are conserved FR structural regions that influence the folded shape of CDR loops in certain “canonical” structures, regardless of the precise amino acid sequence of the CDR.

[0058] The structures and locations of immunoglobulin variable regions can be determined by reference to Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, 1987 and an update thereof, now available on the Internet (immuno.bme.nwu.edu.).

[0059] As used herein, a "single-chain Fv" or "scFv" antibody fragment comprises the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for binding to antigens.

[0060] As used herein, a "chimeric antibody" is a recombinant protein that contains the variable domains and complementarity determining regions derived from a non-human species (e.g., rodent) antibody, while the remainder of the antibody molecule is derived from a human antibody. In some embodiments, a chimeric antibody comprises an antigen-binding fragment of a MASP-3 inhibitory antibody operatively linked or otherwise fused to a heterologous Fc portion of a different antibody. In some embodiments, the heterologous Fc domain can be from a different Ig class than the parent antibody, including IgA (including IgA1 and IgA2 subclasses), IgD, IgE, IgG (including IgG1, IgG2, IgG3, and IgG4 subclasses), and IgM.

[0061] As used herein, a “humanized antibody” is a chimeric molecule, generally prepared using recombinant techniques, having an antigen-binding site derived from an immunoglobulin of a non-human species and the remaining immunoglobulin framework of the molecule based on the structure and/or sequence of a human immunoglobulin. The antigen-binding site may comprise either the complete variable regions fused to the constant domains or only the CDRs grafted to appropriate framework regions in the variable domains. The epitope-binding sites may be wild-type or may be modified by one or more amino acid substitutions. Another approach focuses not only on providing human-derived constant regions, but also on modifying the variable regions so as to remodel them as closely as possible to the human form. In some embodiments, humanized antibodies preserve all CDR sequences (e.g., a humanized mouse antibody that contains all six CDRs of the mouse antibodies). In other embodiments, humanized antibodies have one or more CDRs (one, two, three, four, five, six) that are altered from the original antibody, which are also referred to as one or more CDRs “derived from” one or more CDRs of the original antibody.

[0062] An antibody “specifically binds” to a target if it binds with greater affinity and/or avidity than it binds to other substances. In one embodiment, the antibody, or antigen-binding fragment thereof, specifically binds to the serine protease domain of human MASP-3 (amino acid residues 450-728 of SEQ ID NO:2). In one embodiment, the antibody, or antigen-binding fragment thereof, specifically binds to one or more of the epitopes described in TABLE 4, TABLE 28, or shown in FIGURE 62.

[0063] As used herein, the term "mannan-binding lectin" ("MBL") is equivalent to mannan-binding protein ("MBP").

[0064] As used herein, "membrane attack complex" ("MAC") refers to a complex of the five terminal complement components (C5b combined with C6, C7, C8, and C9) that inserts and disrupts membranes (also referred to as C5b-9).

[0065] As used herein, "an individual" includes all mammals, including without limitation humans, non-human primates, dogs, cats, horses, sheep, goats, cows, rabbits, pigs, and rodents.

[0066] As used herein, amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0067] In the broadest sense, naturally occurring amino acids can be divided into groups based on the chemical characteristics of the respective amino acid side chain. A "hydrophobic" amino acid is Ile, Leu, Met, Phe, Trp, Tyr, Val, Ala, Cys or Pro. A "hydrophilic" amino acid is Gly, Asn, Gln, Ser, Thr, Asp, Glu, Lys, Arg or His. This grouping of amino acids can be further subclassified as follows. "Uncharged hydrophilic" amino acids are Ser, Thr, Asn or Gln. An "acidic" amino acid is Glu or Asp. A "basic" amino acid is Lys, Arg or His.

[0068] As used herein, the term "conservative amino acid substitution" is illustrated by a substitution between amino acids within each of the following groups: (1) glycine, alanine, valine, leucine, and isoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine, (4) aspartate and glutamate, (5) glutamine and asparagine, and (6) lysine, arginine, and histidine.

[0069] 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 encompasses those oligonucleobases composed of naturally occurring nucleotides, sugars, and covalent internucleoside (backbone) linkages as well as oligonucleotides with naturally occurring modifications.

[0070] As used herein, an "epitope" refers to the site on a protein (e.g., a human MASP-3 protein) that is bound by an antibody. "Coincident epitopes" include at least one (e.g., two, three, four, five, or six) common amino acid residue(s), including both non-linear and linear epitopes.

[0071] As used herein, the terms "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-3 proteins described herein may contain either wild-type proteins or may be variants that have no more than 50 (e.g., no more than one, two, three, four, five, six, seven, eight, nine, ten, 12, 15, 20, 25, 30, 35, 40, or 50) conservative amino acid substitutions. Conservative substitutions typically include substitutions in the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine, and threonine; lysine, histidine, and arginine; and phenylalanine and tyrosine.

[0072] In some embodiments, the human MASP-3 protein can have an amino acid sequence that is, or is greater than, 70 (e.g., 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100) % identical to the human MASP-3 protein having the amino acid sequence set forth in SEQ ID NO: 2.

[0073] 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 in SEQ ID NO: 2). In some embodiments, an antigenic peptide fragment of a human MASP-3 protein has less than 500 (e.g., less than 450, 400, 350, 325, 300, 275, 250, 225, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 1 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., less than 500 contiguous amino acid residues in SEQ ID NO: 2).

[0074] In some embodiments, in the context of generating an antibody that binds to MASP-3, the peptide fragments are antigenic and retain at least 10% (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% or more) of the ability of the full-length protein to induce an antigenic response in a mammal (see below under "Methods of Producing an Antibody").

[0075] Percent (%) amino acid sequence identity is defined as the percentage of amino acids in a candidate sequence that are identical to amino acids in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve maximum percentage sequence identity. Alignment for purposes of determining percentage sequence identity can be achieved in a variety of ways that are within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared, can be determined by known methods.

[0076] In representative embodiments, the human MASP-3 protein (SEQ ID NO:2) is encoded by the cDNA sequence set forth as SEQ ID NO:1. Those skilled in the art will recognize that the cDNA sequence disclosed in SEQ ID NO:1 represents a single allele of human MASP-3 and that allelic variation and alternative splicing are expected to occur. Allelic variants of the nucleotide sequences set forth in SEQ ID NO:1, including those that contain silent mutations and those in which the mutations result in changes in the amino acid sequence, are within the scope of the present invention. Allelic variants of the MASP-3 sequence can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures or can be identified by homology comparison searching (e.g., BLAST searching) of databases containing such information.

[0077] As used herein, an "isolated nucleic acid molecule" is a nucleic acid molecule (e.g., a polynucleotide) that is not integrated into the genomic DNA of an organism. For example, a DNA molecule encoding a growth factor that has been separated from the genomic DNA of a cell is an isolated DNA molecule. Another example of an isolated nucleic acid molecule is a chemically synthesized nucleic acid molecule that is not integrated into the genome of an organism. A nucleic acid molecule that has been isolated from a particular species is smaller than the complete DNA molecule of a chromosome of that species.

[0078] As used herein, a "nucleic acid molecule construct" is a nucleic acid molecule, single or double stranded, that has been modified through human intervention to contain nucleic acid segments combined and juxtaposed in an arrangement that does not exist in nature.

[0079] As used herein, an "expression vector" is a nucleic acid molecule that encodes a gene that is expressed in a host cell. Typically, an expression vector comprises a transcription promoter, a gene, and a transcription terminator. Gene expression is generally placed under the control of a promoter, and such a gene is said to be "operably linked to" the promoter. Similarly, a regulatory element and a core promoter are operably linked if the regulatory element modulates the activity of the core promoter.

[0080] As used herein, the term "about" as used herein is intended to specify that the specific value provided may vary to some extent, such that a variation in the range of ±10%, preferably ±5%, more preferably ±2% is included in the value provided. Where ranges are stated, the endpoints

[0081] Where ranges are stated, endpoints are included within the range unless otherwise indicated or otherwise evident from the context.

[0082] As used herein, the singular forms "a", "an", and "the" include plural aspects unless the context clearly indicates otherwise. Thus, for example, reference to "an excipient" includes a plurality of such excipients and their equivalents known to those skilled in the art; reference to "an agent" includes one agent as well as two or more agents; reference to "an antibody" includes a plurality of such antibodies; and reference to "a framework region" includes reference to one or more framework regions and their equivalents known to those skilled in the art; and so on.

[0083] Each embodiment in this specification should be applied, mutatis mutandis, to any other embodiment, unless specifically indicated otherwise. It is contemplated that any embodiment discussed in this specification may be implemented in connection with any method, kit, reagent or composition of the invention and vice versa. Furthermore, compositions of the invention may be used to achieve methods of the invention.

II. THE LECTIN PATHWAY: A New Understanding Overview: The lectin pathway has been redefined

[0084] As described herein, the inventors have made the surprising discovery that the complement lectin pathway has two effector arms for activating complement, both driven by lectin pathway activation complexes formed by carbohydrate recognition components (MBL, CL-11, and ficolins): (i) the effector arm formed by the lectin pathway-associated serine proteases MASP-1 and MASP-3, referred to herein as “lectin pathway effector arm 1” or “LEA-1”; and (ii) the MASP-2-driven activation effector arm, referred to herein as “lectin pathway effector arm 2” or “LEA-2”. Both LEA-1 and LEA-2 can perform lysis and/or opsonization.

[0085] It has also been determined that lectin-independent conversion of factor B by MASP-3 and lectin-independent conversion of factor D by HTRA-1, MASP-1, and MASP-3, both of which can occur in the absence of Ca++, generally leads to the conversion of C3bB to C3bBb and of pro-factor D to factor D. Therefore, inhibition of MASP-3 may inhibit both LEA-1 and lectin-independent activation of factor B and/or factor D, which may result in inhibition of lysis and/or opsonization.

[0086] FIGURE 1 illustrates this new understanding of complement activation pathways. As shown in FIGURE 1, LEA-1 is driven by lectin-bound MASP-3, which can activate factor D zymogen to its active form and/or cleave factor Bb bound to C3b or C3b(H2O), leading to the conversion of the C3bB zymogen complex to its enzymatically active form C3bBb. Activated factor D, generated by MASP-3, can also convert C3bB or C3b(H2O) zymogen complexes to their enzymatically active form. MASP-1 is capable of rapid autoactivation, whereas MASP-3 is not. In many cases, MASP-1 is the activator of MASP-3.

[0087] While in many examples lectins (i.e., MBL, CL-11, or ficolins) can direct activity to cell surfaces, FIGURE 1 also depicts the lectin-independent functions of MASP-3, MASP-1, and HTRA-1 in factor B activation and/or factor D maturation. As with the lectin-associated form of MASP-3 in LEA-1, the lectin-independent form of MASP-3 is capable of mediating the conversion of C3bB or C3b(H2O) to C3bBb (see also FIGURES 29 and 30) and of pro-factor D to factor D (see FIGURE 32). MASP-1 (see also FIGURE 32) and the unrelated MASP protein HTRA-1 can also activate factor D (Stanton et al., Evidence That the HTRA1 Interactome Influences Susceptibility to Age-Related Macular Degeneration, presented at The Association for Research in Vision and Ophthalmology 2011 conference on May 4, 2011) in a manner in which no lectin component is required.

[0088] Thus, MASP-1 (via LEA-1 and lectin-independent forms), MASP-3 (via LEA-1 and lectin-independent forms), and HTRA-1 (lectin-independent only) are capable of direct or indirect activation at one or more points along the MASP-3-factor D-factor B axis. In doing so, they generate C3bBb, the alternative pathway C3 convertase, and stimulate the production and deposition of C3b on microbial surfaces. C3b deposition plays a critical role in opsonization by marking the surfaces of microbes for destruction by host phagocytic cells, such as macrophages. As an example here (FIGURES 28A and 28B), MASP-3 is critical for the opsonization of S. aureus. C3b deposition occurs rapidly in S. aureus exposed to human serum in a MASP-3-dependent manner (FIGURES 28A and 28B).

[0089] However, the contributions of LEA-1 and the lectin-independent functions of MASP-3, MASP-1, or HTRA-1 are not limited to opsonization. As diagrammed in FIGURE 1, these three components can also cause cell lysis by indirect or direct activation of factor B and the production of C3b. These components form complexes that generate the alternative pathway C5 convertase, C3bBb(C3b)n. As described later, the requirement for MASP-3 and MBL, but not MASP-2 (and thus not LEA-2 in this example), in the lysis of N. meningitidis (see FIGURES 11, 12, and 13) demonstrates a role for LEA-1 in lysis. In summary, the opsonization results obtained from the S. aureus studies and the lysis results observed in the N. meningitidis studies support a role for LEA-1 in both processes (as outlined in FIGURE 1). Furthermore, these studies demonstrate that both opsonization and lysis can result from the conversion of C3bB or C3b(H2O) and/or profactor D to factor D; therefore, both processes may result from the lectin-independent roles of MASP-3, MASP-1, or HTRA-1. Thus, the model developed by the inventors in FIGURE 1 supports the use of inhibitors of primarily MASP-3, but also MASP-1 and/or HTRA-1, to block opsonization and/or lysis and to treat pathologies caused by dysregulation of these processes.

1. Lectin Pathway Effector Arm (LEA-1)

[0090] The first effector arm of the lectin pathway, LEA-1, is formed by the lectin pathway-associated serine proteases MASP-1 and MASP-3. As described herein, the inventors have now shown that in the absence of MASP-3 and in the presence of MASP-1, the alternative pathway is not effectively activated at surface structures. These results demonstrate that MASP-3 plays a previously undescribed role in the initiation of the alternative pathway and this is confirmed using MASP-3-deficient 3MC serum obtained from patients with the rare autosomal recessive disorder 3MC (Rooryck C, et al., Nat Genet. 43(3):197-203 (2011)) with mutations that render the serine protease domain of MASP-3 dysfunctional. Based on these new findings, complement activation involving the alternative pathway, as conventionally defined, is expected to be dependent on MASP-3. Indeed, MASP-3 and its activation of LEA-1 may represent the hitherto elusive initiator of the alternative pathway.

[0091] As further described in Examples 1-4 herein, in MASP-2 deficient sera, the inventors observed increased lectin-dependent alternative pathway activation activity resulting in increased bactericidal activity (i.e., lytic activity) against N. meningitidis. Without wishing to be bound by any particular theory, it is believed that in the absence of MASP-1-containing carbohydrate recognition complexes, MASP-2 are more likely to bind near MASP-3-containing carbohydrate recognition complexes to activate MASP-3. It is known that in many cases, MASP-3 activation is dependent on MASP-1 activity, since MASP-3 is not a self-activating enzyme and often requires MASP-1 activity to be converted from its zymogenic form to its enzymatically active form. MASP-1 (like MASP-2) is a self-activating enzyme, whereas MASP-3 does not self-activate and in many cases requires the enzymatic activity of MASP-1 to be converted to its enzymatically active form. See, Zundel S, et al., J Immunol., 172(7):4342-50 (2004). In the absence of MASP-2, all lectin pathway recognition complexes are loaded with either MASP-1 or MASP-3. Therefore, the absence of MASP-2 facilitates MASP-1-mediated conversion of MASP-3 to its enzymatically active form. Once MASP-3 is activated, activated MASP-3 initiates the activation of alternative pathways, now termed “LEA-1” activation, through a MASP-3-mediated conversion of C3bB to C3bBb and/or conversion of profactor D to factor D. C3bBb, also referred to as the alternative pathway C3 convertase, cleaves additional C3 molecules producing deposition of opsonic C3b molecules. If multiple C3b fragments bind in proximity to the C3bBb convertase complex, this results in the formation of the alternative pathway C5 convertase C3bBb(C3b)n, which promotes MAC formation. Additionally, the surface-deposited C3b molecules form new sites for factor B binding, which can now be cleaved by factor D and/or MASP-3 to form additional sites where alternative pathway C3 and C5 convertase complexes can be formed. This latter process is necessary for efficient lysis and does not require lectins once the initial deposition of C3b has occurred. A recent publication (Iwaki D. et al., J Immunol 187(7):3751-8 (2011)) as well as data generated from the inventors (FIGURE 30) demonstrate that the alternative pathway C3 convertase zymogen complex C3bB is converted to its enzymatically active form by activated MASP-3. The inventors have now discovered that MASP-3-mediated cleavage of factor B represents a subcomponent of the recently described LEA-1, which promotes the lectin-dependent formation of the alternative pathway C3 convertase C3bBb.

2. Lectin Pathway Effector Arm (LEA-2)

[0092] The second effector arm of the lectin pathway, LEA-2, is formed by the lectin pathway-associated serine protease MASP-2. MASP-2 is activated upon binding of its respective pattern recognition components and can also be activated by MASP-1, and subsequently cleaves the complement component C4 into C4a and C4b. Following binding of the C4b cleavage product to plasma C2, the C4b-bound C2 becomes the substrate for a second MASP-2-mediated cleavage step that converts C4b-bound C2 into the enzymatically active complex C4bC2a and a small cleavage fragment of C2b. C4b2a is the C3-converting C3 convertase of the lectin pathway, converting the abundant plasma C3 component into C3a and C3b. C3b binds to any nearby surface via a thioester bond. If multiple C3b fragments bind in close proximity to the C3 convertase complex C4b2a, this convertase switches its specificity to convert C5 to C5b and C5a, forming the C5 convertase complex C4b2a(C3b)n. Although this C5 convertase can initiate MAC formation, this process is thought to be insufficiently effective in promoting lysis on its own. Instead, the initial C3b opsonins produced by LEA-2 form the nucleus for the formation of new C3 convertase and C5 convertase sites of the alternative pathway, which ultimately leads to abundant MAC formation and lysis. This latter event is mediated by factor D activation of C3b-associated factor B formed by LEA-2 and is therefore LEA-1 dependent because of the essential role of MASP-1 in factor D maturation. There is also a MASP-2-dependent C4 bypass pathway to activate C3 in the absence of C4, which plays an important role in the pathophysiology of ischemia-reperfusion injury, since C4-deficient mice are not protected from ischemia-reperfusion injury whereas MASP-2-deficient mice are (Schwaeble et al., PNAS, 2011 supra ). LEA-2 is also linked to the coagulation pathway, involving the cleavage of prothrombin to thrombin (common pathway) and also the cleavage of factor XII (Hageman factor) to convert to its enzymatically active form XIIa. Factor XIIa in turn cleaves factor XI to XIa (intrinsic pathway). Activation of the intrinsic pathway of the coagulation cascade leads to the formation of fibrin, which is of critical importance for thrombus formation.

[0093] FIGURE 1 illustrates the new understanding of the lectin pathway and the alternative pathway based on the results provided here. FIGURE 1 delineates the role of LEA-2 in both opsonization and lysis. While MASP-2 is the initiator of “downstream” C3b deposition (and resulting opsonization) in multiple physiologically lectin-dependent settings (FIGURES 18A, 18B, 18C), it also plays a role in the lysis of serum-sensitive bacteria. As illustrated in FIGURE 1, the proposed molecular mechanism responsible for the increased bactericidal activity of MASP-2-deficient or MASP-2-depleted serum/plasma for serum-sensitive pathogens such as N. meningitidis is that, for bacterial lysis, the lectin pathway recognition complexes associated with MASP-1 and MASP-3 must bind in close proximity to each other on the bacterial surface, thereby allowing MASP-1 to cleave MASP-3. In contrast to MASP-1 and MASP-2, MASP-3 is not a self-activating enzyme but, in many cases, requires activation/cleavage by MASP-1 to be converted to its enzymatically active form.

[0094] As further shown in FIGURE 1, activated MASP-3 can then cleave factor B bound to C3b on the pathogen surface to initiate the alternative activation cascade by formation of the enzymatically active alternative pathway C3 and C5 convertases C3bBb and C3bBb(C3b)n, respectively. Lectin pathway activation complexes containing MASP-2 do not participate in MASP-3 activation, and in the absence or following depletion of MASP-2, bulk lectin pathway activation complexes will be loaded with either MASP-1 or MASP-2. Therefore, in the absence of MASP-2, the probability is markedly increased that, on the microbial surface, lectin pathway activation complexes containing MASP-1 and MASP-3 will come into close proximity to each other, leading to more MASP-3 being activated and thus leading to an increased rate of MASP-3-mediated cleavage of factor B-bound C3b to form the alternative pathway C3 and C5 convertases C3bBb and C3bBb(C3b)n on the microbial surface. This leads to the activation of the C5b-C9 terminal activation cascades that form the Membrane Attack Complex, composed of surface-bound C5b associated with C6, C5bC6 associated with C7, C5bC6C7 associated with C8, and C5bC6C7C8, leading to the polymerization of C9 that inserts into the bacterial surface structure and forms a pore in the bacterial wall, which will lead to the osmolytic killing of the complement-targeted bacteria.

[0095] The core of this new concept is that the data provided here clearly show that lectin pathway activation complexes guide the following two distinct activation pathways, as illustrated in FIGURE 1: i) LEA-1: A MASP-3-dependent activation pathway that initiates and guides complement activation by generating the alternative pathway convertase C3bBb through the initial cleavage and activation of factor B on activating surfaces, which will then catalyze the deposition of C3b and the formation of the alternative pathway convertase C3bBb. The MASP-3-guided activation pathway plays an essential role in the opsonization and lysis of microbes and triggers the alternative pathway on the surface of bacteria, leading to optimal activation rates to generate membrane attack complexes; and ii) LEA-2: A MASP-2-dependent activation pathway leading to the formation of the lectin pathway C3 convertase C4b2a and, after accumulation of the C3 cleavage product C3b, subsequently to the C5 convertase C4b2a(C3b)n. In the absence of complement C4, MASP-2 can form an alternative C3 convertase complex involving C2 and coagulation factor XI.

[0096] In addition to its role in lysis, the MASP-2-driven activation pathway plays an important role in bacterial opsonization, leading to microbes being coated with covalently bound C3b and its cleavage products (i.e., iC3b and C3dg), which will be targeted for uptake and killing by C3 receptor-bearing phagocytes, such as granulocytes, macrophages, monocytes, microglia, and the reticuloendothelial system. This is the most effective route of killing bacteria and microorganisms that are resistant to complement lysis. These include most Gram-positive bacteria.

[0097] In addition to LEA-1 and LEA-2, there is the potential for lectin-independent activation of factor D by MASP-3, MASP-1 and/or HTRA-1 and there is also the potential for lectin-independent activation of factor B by MASP-3.

[0098] Without wishing to be bound by any particular theory, it is believed that each of (i) LEA-1, (ii) LEA-2 and (iii) lectin-independent factor B and/or factor D activation leads to opsonization and/or MAC formation with resultant lysis.

ii. Fundamentals of MASP-1, MASP-2 and MASP-3

[0099] Three mannan-binding lectin-associated serine proteases (MASP-1, MASP-2 and MASP-3) are currently known to associate with human serum mannan-binding lectin (MBL). Mannan-binding lectin is also called "mannose-binding protein" or "mannose-binding lectin" in recent literature. The MBL-MASP complex plays an important role in innate immunity by virtue of the binding of MBL to carbohydrate structures present in a wide variety of microorganisms. The interaction of MBL with specific arrangements of carbohydrate structures leads to the activation of MASP proenzymes, which in turn activate complement by cleaving complement components C4 and C2 to form the C3 convertase C3b2b (Kawasaki et al., J. Biochem 106:483-489 (1989); Matsushita & Fujita, J. Exp Med. 176:1497-1502 (1992); Ji et al., J. Immunol 150:571-578 (1993)).

[00100] The MASP-MBL proenzyme complex was, until recently, thought to contain only one type of protease (MASP-1), but it is now clear that there are two other distinct proteases (i.e., MASP-2 and MASP-3) associated with MBL (Thiel et al., Nature 386:506-510 (1997); Dahl et al., Immunity 15:127-135 (2001)), as well as an additional 19-kDa serum protein referred to as "MAp19" or "sMAP" (Stover et al., J. Immunol 162:3481-3490 (1999); Stover et al., J. Immunol 163:6848-6859 (1999); Takahashi et al., Int. Immunol 11:859-63 (1999)).

[00101] MAp19 is an alternatively spliced gene product of the structural gene for MASP-2 and lacks the four C-terminal domains of MASP-2, including the serine endopeptidase domain. The abundantly expressed truncated mRNA transcript encoding MAp19 is generated by an alternative splicing/polyadenylation event of the MASP-2 gene. By a similar mechanism, the MASP-1/3 gene gives rise to three major gene products, the two serine proteases MASP-1 and MASP-3 and a truncated 44 kDa gene product known as “MAp44” (Degn et al., J Immunol 183(11):7371-8 (2009); Skjoedt et al., J Biol Chem 285:8234-43 (2010)).

[00102] MASP-1 was first described as the P-100 protease component of serum Ra-reactive factor, which is now recognized to be a complex composed of MBL plus MASP (Matsushita et al., Collectins and Innate Immunity, (1996); Ji et al., J Immunol 150:571-578 (1993). The ability of an MBL-associated endopeptidase in the MBL-MASPs complex to act on complement components C4 and C2 in a manner apparently identical to that of the C1s enzyme in the C1q-(C1r)2-(C1s)2 complex of the classical complement pathway suggests that there is an MBL-MASPs complex that is functionally analogous to the C1q-(C1r)2-(C1s)2 complex. The C1q-(C1r)2-(C1s)2 complex is activated by the interaction of C1q with the Fc regions of IgG or IgM antibodies present in the immune complexes. This causes the autoactivation of the C1r proenzyme which, in turn, activates the C1s proenzyme which then acts on the C4 and C2 components of complement.

[00103] The stoichiometry of the MBL-MASPs complex differs from that found for the C1q-(C1r)2-(C1s)2 complex in that different MBL oligomers appear to associate with different proportions of MASP-1/MAp19 or MASP-2/MASP-3 (Dahl et al., Immunity 15:127-135 (2001). Most MASPs and MAp19 found in serum are not complexed with MBL (Thiel et al., J Immunol 165:878-887 (2000)) and may associate in part with ficolins, a recently described group of lectins with a fibrinogen-like domain capable of binding to N-acetylglucosamine residues on microbial surfaces (Le et al., FEBS Lett 425:367 (1998); Sugimoto et al., J. Biol Chem 273:20721 (1998)). Among these, human L-ficolin, H-ficolin, and M-ficolin associate with MASPs as well as MAp19 and can activate the lectin pathway by binding to specific carbohydrate structures recognized by ficolins (Matsushita et al., J Immunol 164:2281–2284 (2000); Matsushita et al., J Immunol 168:3502–3506 (2002)). In addition to ficolins and MBL, an MBL-like lectin collectin, called CL-11, has been identified as a recognition molecule of the lectin pathway (Hansen et al. J Immunol 185:6096–6104 (2010); Schwaeble et al. PNAS 108:75237528 (2011)). There is compelling evidence highlighting the physiological importance of these alternative carbohydrate recognition molecules, and it is therefore important to understand that MBL is not the only recognition component of the lectin activation pathway and that MBL deficiency should not be confused with lectin pathway deficiency. The existence of a possible array of alternative carbohydrate recognition complexes structurally related to MBL may broaden the spectrum of microbial structures that initiate a direct innate immune response via complement activation.

[00104] All lectin pathway recognition molecules are characterized by a specific MASP-binding motif within their collagen-homologous stalk region (Wallis et al. J. Biol Chem 279:14065-14073 (2004)). The MASP-binding site in MBLs, CL-11, and ficolins is characterized by a distinct motif within this Hyp-Gly-Lys-Xaa-Gly-Pro domain, where Hyp is hydroxyproline and Xaa is usually an aliphatic residue. Point mutations in this sequence disrupt MASP binding.

1. Respective structures, sequences, chromosomal location and splice variants of MASP-1 and MASP-3

[00105] FIGURE 2 is a schematic diagram illustrating the domain structure of the human MASP-1 polypeptide (SEQ ID NO:8), human MASP-3 polypeptide (SEQ ID NO:2), and human MAp44 polypeptide and the exons encoding them. As shown in FIGURE 2, the MASP-1 and MASP-3 serine proteases consist of six distinct domains arranged as found in C1r and C1s; that is, (I) an N-terminal C1r/C1s/sea urchin VEGF/bone morphogenic protein (CUB) domain; (II) an epidermal growth factor (EGF)-like domain; (III) a second CUB domain (CUBII); (IV and V) two complement control protein domains (CCP1 and CCP2); and (VI) a serine protease (SP) domain.

[00106] Amino acid sequences derived from human and mouse MASP-1 cDNA (Sato et al., Int Immunol 6:665-669 (1994); Takada et al., Biochem Biophys Res Commun 196:1003-1009 (1993); Takayama et al., J. Immunol 152:2308-2316 (1994)), human, rat and mouse MASP-2 (Thiel et al., Nature 386:506-510 (1997); Endo et al., J Immunol 161:4924-30 (1998); Stover et al., J. Immunol 162:3481-3490 (1999); 163:6848-6859 (1999)), as well as human MASP-3 (Dahl et al., Immunity 15:127-135 (2001)) indicate that these proteases are serine peptidases possessing the characteristic triad of His, Asp and Ser within their putative catalytic domains (GenBank accession numbers: human MASP-1: BAA04477.1 (SEQ ID NO:8); mouse MASP-1: BAA03944; rat MASP-1: AJ457084; MASP-3 human: AAK84071 (SEQ ID NO: 2); mouse MASP-3: AB049755, as accessed in Genbank on 2/15/2012 (SEQ ID NO: 3); rat MASP-3 (SEQ ID NO: 4); MASP -3 chicken (SEQ ID NO: 5); rabbit MASP-3 (SEQ ID NO: 6); and cynomolgus monkey (SEQ ID NO: 7).

[00107] As further shown in FIGURE 2, following conversion of the zymogen to the active form, the heavy chain (alpha or A chain) and light chain (beta or B chain) are cleaved to produce a disulfide-linked A chain and a smaller B chain representing the serine protease domain. The single-chain proenzyme MASP-1 is activated (like proenzyme C1r and C1s) by cleavage of an Arg-Ile bond located between the second CCP domain (domain V) and the serine protease domain (domain VI). The proenzymes MASP-2 and MASP-3 are considered to be activated in a manner similar to that of MASP-1. Each MASP protein forms homodimers and individually associates with MBL and ficolins in a Ca++-dependent manner.

[00108] The human MASP-1 polypeptide (SEQ ID NO: 8) and the MASP-3 polypeptide (SEQ ID NO: 2) arise from a structural gene (Dahl et al., Immunity 15: 127-135 (2001)), which has been mapped to the 3q27-28 region of the long arm of chromosome 3 (Takada et al., Genomics 25: 757-759 (1995)). The MASP-3 and MASP-1 mRNA transcripts are generated from the primary transcript by an alternative splicing/polyadenylation process. The MASP-3 translation product is composed of an alpha chain, which is common to both MASP-1 and MASP-3, and a beta chain (the serine protease domain), which is unique to MASP-3. As shown in FIGURE 2, the human MASP-1 gene encompasses 18 exons. The human MASP-1 cDNA is encoded by exons 2, 3, 4, 5, 6, 7, 8, 10, 11, 13, 14, 15, 16, 17, and 18. As further shown in FIGURE 2, the human MASP-3 gene spans twelve exons. The human MASP-3 cDNA (defined as SEQ ID NO: 1) is encoded by exons 2, 3, 4, 5, 6, 7, 8, 10, 11, and 12. Alternative splicing results in a protein called MBL-associated protein 44 ("MAp44"), originating from exons 2, 3, 4, 5, 6, 7, 8, and 9.

[00109] The human MASP-1 polypeptide (Genbank SEQ ID NO: 8 BAA04477.1) has 699 amino acid residues, which include a 19-residue leader peptide. When the leader peptide is omitted, the calculated molecular mass of MASP-1 is 76,976 Da. As shown in FIGURE 2, the amino acid sequence of MASP-1 contains four N-linked glycosylation sites. The human MASP-1 protein domains (with reference to SEQ ID NO: 8) are shown in Figure 2 and include an N-terminal C1r/C1s/sea urchin VEGF/bone morphogenic protein (CUB) domain (aa 25-137 of SEQ ID NO: 8), an epidermal growth factor-like domain (aa 139-181 of SEQ ID NO: 8), a second CUB (CUBII) domain (aa 185-296 of SEQ ID NO: 8), as well as a tandem of complement control protein domains (CCP1 aa 301-363 and CCP2 aa 367-432 of SEQ ID NO:8) and a serine protease domain (AA 449694 of SEQ ID NO:8).

[00110] The human MASP-3 polypeptide (SEQ ID NO:2, from Genbank AAK84071) has 728 amino acid residues (as shown in FIGURE 3), which includes a leader peptide of 19 residues (shown as the underlined amino acid residues in FIGURE 3).

[00111] When the leader peptides are omitted, the calculated molecular mass of MASP-3 is 81,873 Da. As shown in FIGURE 2, there are seven N-linked glycosylation sites in MASP-3. The human MASP-1 protein domains (with reference to SEQ ID NO:2) are shown in Figure 2 and include an N-terminal C1r/C1s/sea urchin VEGF/bone morphogenic protein (CUB) domain (aa 25-137 of SEQ ID NO:2), an epidermal growth factor-like domain (aa 139-181 of SEQ ID NO:2), a second CUB (CUBII) domain (aa 185-296 of SEQ ID NO:2), as well as a tandem of complement control protein domains (CCP1 aa 299-363 and CCP2 aa 367-432 of SEQ ID NO:2) and a serine protease domain (aa 450-728 of SEQ ID NO:2).

[00112] The translation product of MASP-3 is composed of an alpha chain (heavy chain), containing the CUB-1-EGF-CUB-2-CCP-1-CCP-2 domains (alpha chain: aa 1-448 of SEQ ID NO:2) that is common to both MASP-1 and MASP-3, and a light chain (beta chain: aa 449-728 of SEQ ID NO:2), containing the serine protease domain, which is unique to MASP-3. 2. Comparison of amino acid sequences of MASP-3 from various species

[00113] FIGURE 4 provides a multispecies alignment of MASP-3, showing a comparison of full-length MASP-3 protein from human (SEQ ID NO:2), cynomolgus monkey (SEQ ID NO:7), rat (SEQ ID NO:4), murine (SEQ ID NO:3), chicken (SEQ ID NO:5), and rabbit (SEQ ID NO:6). FIGURE 5 provides a multispecies alignment of the serine protease (SP) domain from human (aa 450-728 of SEQ ID NO: 2); rabbit (aa 450-728 of SEQ ID NO: 6); murine (aa aa455-733 of SEQ ID NO: 3); rat (aa 455-733 of SEQ ID NO: 4), and chicken (aa aa448-730 of SEQ ID NO: 5).

[00114] As shown in FIGURE 4, there is a high level of conservation of the amino acid sequence of the MASP-3 polypeptide between different species, particularly in the SP domain (FIGURE 5). As further shown in FIGURE 5, the catalytic triad (H at residue 497; D at residue 553; and S at residue 664 with reference to full-length human MASP-3 (SEQ ID NO: 2) is conserved between species. TABLE 1 summarizes the percent identity of the MASP-3 SP domain between species. TABLE 1: Percent Identity of the MASP-3 SP Domain in Various Species

[00115] MASP-3 has no proteolytic activity toward C4, C2, or C3 substrates. On the other hand, MASP-3 was initially reported to act as an inhibitor of the lectin pathway (Dahl et al., Immunity 15: 127-135 (2001)). This conclusion may have occurred because, in contrast to MASP-1 and MASP-2, MASP-3 is not an autoactivating enzyme (Zundel S. et al., J Immunol 172:4342-4350 (2004); Megyeri et al., J Biol. Chem. 288:8922-8934 (2013).

[00116] Recently, evidence for possible physiological functions of MASP-1 and MASP-3 has emerged from transgenic mouse studies using a mouse strain with combined deficiency of MASP-1 and MASP-3. While MASP-1/3 knockout mice have a functional lectin pathway (Schwaeble et al., PNAS 108:7523-7528 (2011)), they appear to lack alternative pathway activity (Takahashi et al., JEM 207(1):29-37 (2010)). The lack of alternative pathway activity appears to be due to a defect in processing of complement factor D, which is required for alternative pathway activity. In MASP-1/3 knockout mice, all factor D is circulating as a proteolytically inactive proform, whereas in the serum of normal mice, substantially all factor D is in the active form. Biochemical analysis has suggested that MASP-1 may be capable of converting complement factor D from its zymogenic form to its enzymatically active form (FIGURE 32; Takahashi et al., JEM 207(1):29-37 (2010)). MASP-3 also cleaves the profactor D zymogen and produces active factor D in vitro (FIGURE 32; Takahashi et al., JEM 207(1):29-37 (2010)). Factor D is present as an active enzyme in the circulation in normal individuals, and MASP-1 and MASP-3, as well as HTRA-1, may be responsible for this activation. Furthermore, mice with combined MBL and ficolin deficiencies still produce normal levels of factor D and have a fully functional alternative pathway. Thus, these physiological functions of MASP-1 and MASP-3 do not necessarily involve lectins and are therefore unrelated to the lectin pathway. Recombinant mouse and human MASP-3 also appear to cleave factor B and support C3 deposition in S. aureus in vitro (FIGURE 29; Iwaki D. et al., J Immunol 187(7):3751-8 (2011)).

[00117] An unexpected physiological role for MASP-3 has emerged from recent studies of patients with 3MC syndrome (previously referred to as Carnevale, Mingarelli, Malpuech, and Michels syndrome; OMIM no. 257920). These patients have severe developmental abnormalities, including cleft palate, cleft lip, cranial malformations, and mental retardation. Genetic analysis identified patients with 3MC who were homozygous for a dysfunctional MASP-3 gene (Rooryck et al., Nat Genet. 43(3):197-203 (2011)). Another group of 3MC patients was found to be homozygous for a mutation in the MASP-1 gene that leads to the absence of functional MASP-1 and MASP-3 proteins. Still another group of 3MC patients lacked a functional CL-11 gene. (Rooryck et al., Nat Genet. 43(3):197-203 (2011)). Thus, the CL-11 MASP-3 axis appears to play a role during embryonic development. The molecular mechanisms of this developmental pathway are unclear. It is unlikely, however, to be mediated by a conventional complement-driven process, since individuals with deficiencies of common complement C3 components do not develop this syndrome. Thus, prior to the present inventors' discovery as described herein, a functional role for MASP-3 in lectin-dependent complement activation had not previously been established.

[00118] The structures of the catalytic fragment of MASP-1 and MASP-2 have been determined by X-ray crystallography. Structural comparison of the MASP-1 protease domain with those of other complement proteases revealed the basis of its relaxed substrate specificity (Dobó et al., J. Immunol 183:1207-1214 (2009)). Although the accessibility of the MASP-2 substrate-binding cleft is restricted by surface loops (Harmat et al., JMol Biol 342:1533-1546 (2004)), MASP-1 has an open substrate-binding cavity that resembles that of trypsin rather than other complement proteases. A thrombin-like property of the MASP-1 structure is the unusually large 60-amino-acid loop (loop B) that can interact with substrates. Another interesting feature of the MASP-1 structure is the internal salt bridge between SI Asp189 and Arg224. A similar salt bridge can be found in the substrate-binding pocket of factor D, which may regulate its protease activity. C1s and MASP-2 have nearly identical substrate specificities. Surprisingly, some of the eight surface loops of MASP-2, which determine substrate specificities, have quite different conformations compared to those of C1s. This means that the two functionally related enzymes interact with the same substrates in a different manner. The structure of the MASP-2 zymogen shows an inactive protease domain with a disrupted oxyanion cleft and substrate-binding pocket (Gál et al., J Biol Chem 280:33435–33444 (2005)). Surprisingly, the MASP-2 zymogen displays considerable activity on a large protein substrate, C4. The structure of the MASP-2 zymogen is likely to be quite flexible, enabling the transition between inactive and active forms. This flexibility, which is reflected in the structure, may play a role in the self-activation process.

[00119] Northern blot analyses indicate that the liver is the major source of MASP-1 and MASP-2 mRNA. Using a 5'-specific cDNA probe for MASP-1, the major MASP-1 transcript was seen at 4.8 kb and a minor one at approximately 3.4 kb, present in both human and mouse liver (Stover et al., Genes Immunity 4:374-84 (2003)). MASP-2 mRNA (2.6 kb) and MAp19 mRNA (1.0 kb) are abundantly expressed in liver tissue. MASP-3 is expressed in the liver and also in many other tissues, including neuronal tissue (Lynch N. J. et al., J Immunol 174:4998-5006 (2005)).

[00120] A patient with a history of infections and chronic inflammatory disease was found to have a mutated form of MASP-2 that fails to form an active MBL-MASP complex (Stengaard-Pedersen et al., N Engl J Med 349:554-560 (2003)). Some investigators have determined that MBL deficiency leads to a tendency toward frequent infections in childhood (Super et al., Lancet 2:1236-1239 (1989); Garred et al., Lancet 346:941-943 (1995)) and a decreased resistance to HIV infection (Nielsen et al., Clin Exp Immunol 100:219-222 (1995); Garred et al., Mol Immunol 33 (suppl 1):8 (1996)). However, other studies have not demonstrated a significant correlation of low MBL levels with increased infections (Egli et al., PLoS One. 8(1):e51983 (2013); Ruskamp et al., J Infect Dis. 198(11):1707-13 (2008); Israels et al., Arch Dis Child Fetal Neonatal Ed. 95(6):F452-61 (2010)). Although the literature is mixed, deficiency or non-utilization of MASP may have an adverse effect on an individual's ability to mount an immediate, non-antibody-dependent defense against certain pathogens.

[00121] Supporting data for the new understanding, highlighting traditional assay conditions that are devoid of Ca++ and results obtained using a more physiological set of conditions that include Ca++.

[00122] Several independent lines of strong experimental evidence are provided here pointing to the conclusion that the complement lectin pathway activation pathway activates complement through two independent effector mechanisms: i) LEA-2: a MASP-2-driven pathway that mediates complement-driven opsonization; chemotaxis (Schwaeble et al., PNAS 108:7523-7528 (2011)) and cell lysis and ii) LEA-1: a novel MASP-3-dependent activation pathway that initiates complement activation by generating the alternative pathway C3bBb convertase through the cleavage and activation of factor B on activating surfaces, which will then catalyze the deposition of C3b and the formation of the alternative pathway C3bBb convertase, which can result in cell lysis as well as microbial opsonization. Furthermore, as described herein, separate lectin-independent activation of factor B and/or factor D by MASP-1, MASP-3, or htrA-1, or a combination of any three, can also lead to complement activation via the alternative pathway.

[00123] A MASP-3-driven alternative pathway activation dependent on the lectin pathway appears to contribute to the well-established factor D-mediated cleavage of C3b-bound factor B to achieve optimal activation rates for complement-dependent lysis via the terminal activation cascade for bacterial cell lysis via the formation of C5b-9 membrane attack complexes (MACs) on the cell surface (FIGURES 12-13). This rate-limited event appears to require optimal coordination, as it is deficient in the absence of MASP-3 functional activity as well as in the absence of factor D functional activity. As described in Examples 1-4 herein, the inventors discovered this function of the MASP-3-dependent lectin pathway when studying the phenotype of MASP-2 deficiency and MASP-2 inhibition in experimental mouse models of N. menigitidis infection. Targeted MASP-2-deficient mice and wild-type mice treated with antibody-based MASP-2 inhibitors were highly resistant to experimental infection with N. meningitidis (see FIGURES 6–10). When the infectious dose was adjusted to yield approximately 60% mortality in wild-type littermates, all MASP-2-deficient or MASP-2-depleted mice cleared the infection and survived (see FIGURE 6 and FIGURE 10). This high degree of resistance was reflected in a significant increase in serum bactericidal activity in sera from MASP-2-deficient or MASP-2-depleted mice. Further experiments showed that this bactericidal activity was dependent on alternative pathway-driven bacterial lysis. Sera from mice deficient in factor B or factor D or C3 showed no bactericidal activity against N. meningitidis, indicating that the alternative pathway is essential for activation of the terminal activation cascade. A surprising result was that sera from mice deficient in MBL-A and MBL-C (both of which are the lectin pathway recognition molecules that recognize N. meningitidis) as well as sera from mice deficient in the lectin pathway-associated serine proteases MASP-1 and MASP-3 had lost all bacteriolytic activity toward N. meningitidis (FIGURE 13). A recent paper (Takahashi M. et al., JEM 207:29-37 (2010)) and the work presented here (Figure 32) demonstrate that MASP-1 can convert the zymogen form of factor D to its enzymatically active form and may, in part, explain the loss of lytic activity by the absence of enzymatically active factor D in these sera. This does not explain the lack of bactericidal activity in MBL-deficient mice since these normal mice have enzymatically active factor D (Banda et al., Mol Immunol 49(1-2):281-9 (2011)). Remarkably, when testing human sera from patients with the rare autosomal recessive disorder 3MC (Rooryck C, et al., Nat Genet. 43(3):197-203) with mutations that render the serine protease domain of MASP-3 dysfunctional, no bactericidal activity against N. meningitidis was detectable (note: these sera have MASP-1 and factor D but no MASP-3).

[00124] The hypothesis that human serum requires MASP-3-dependent lectin pathway-mediated activity to develop bactericidal activity is further supported by the observation that MBL-deficient human sera also fail to lyse N. meningitidis (FIGURES 11-12). MBL is the only human lectin pathway recognition molecule that binds to this pathogen. Since MASP-3 does not autoactivate, the inventors hypothesized that the higher bacteriolytic activity in MASP-2-deficient sera could be explained by a favored activation of MASP-3 through MASP-1 since, in the absence of MASP-2, all lectin pathway activation complexes that bind to the bacterial surface will be loaded with either MASP-1 or MASP-3. Since activated MASP-3 cleaves both factor D (FIGURE 32) and factor B to generate the respective enzymatically active forms in vitro (FIGURE 30 and Iwaki D., et al., J. Immunol. 187(7):3751-3758 (2011)), the most likely function of MASP-3 is to facilitate the formation of the alternative pathway C3 convertase (i.e., C3bBb).

[00125] Although the data for a lectin-dependent role are compelling, several experiments suggest that MASP-3 and MASP-1 are not necessarily required to function in complex with lectin molecules. Experiments such as those shown in FIGURE 28B demonstrate the ability of MASP-3 to activate the alternative pathway (as demonstrated by C3b deposition in S. aureus) under conditions (i.e., the presence of EGTA) in which lectin complexes would not be present. FIGURE 28A demonstrates that deposition under these conditions is dependent on factor B, factor D, and factor P, all critical components of the alternative pathway. Additionally, factor D activation by MASP-3 and MASP-1 (FIGURE 32) and factor B activation by MASP-3 (FIGURE 30) can occur in vitro in the absence of lectin. Finally, hemolysis studies of mouse erythrocytes in the presence of human serum demonstrate a clear role for both MBL and MASP-3 in cell lysis. However, MBL deficiency does not fully replicate the severity of MASP-3 deficiency, in contrast to what would be expected if all functional MASP-3 were complexed with MBL. Thus, the inventors do not wish to be constrained by the notion that all roles for MASP-3 (and MASP-1) demonstrated here can be attributed solely to lectin-associated function.

[00126] The identification of the two effector arms of the lectin pathway, as well as the possible lectin-independent functions of MASP-1, MASP-3, and HTRA-1, represent new opportunities for therapeutic interventions to effectively treat defined human pathologies caused by excess complement activation in the presence of microbial pathogens or altered host cells or metabolic depots. As described herein, the inventors have discovered that in the absence of MASP-3 and in the presence of MASP-1, the alternative pathway is not activated at surface structures (see FIGURES 15-16, 28B, 34-35A,B, 38-39). Since the alternative pathway is important in driving the rate-limiting events that lead to bacterial lysis as well as cell lysis (Mathieson PW, et al., J Exp Med 177(6):1827-3 (1993)), our results demonstrate that activated MASP-3 plays an important role in the lytic activity of complement. As shown in FIGURES 12-13, 19-21, 36-37, and 39-40, in serum from 3MC patients lacking MASP-3 but with MASP-1, the terminal lytic complement activation cascade is defective. The data presented in FIGURES 12 and 13 demonstrate a loss of bacteriolytic activity in the absence of functional MASP-3 and/or MASP-1/MASP-3 activity. Similarly, the loss of hemolytic activity in MASP-3-deficient human serum (Figures 19-21, 36-37, and 39-40), coupled with the ability to reconstitute hemolysis by adding recombinant MASP-3 (Figures 39-40), strongly supports the conclusion that activation of the alternative pathway on target surfaces (which is essential for triggering complement-mediated lysis) is dependent on the presence of activated MASP-3. Based on the new understanding of the lectin pathway detailed above, activation of the alternative pathway on target surfaces is therefore dependent on activation of LEA-1, and/or activation of lectin-independent factor B and/or factor D, which is also mediated by MASP-3, and therefore agents that block MASP-3-dependent complement activation will prevent activation of alternative pathways on target surfaces.

[00127] The description of the essential role of MASP-3-dependent initiation of alternative pathway activation implies that the alternative pathway is not an autonomous, complement-independent pathway of activation, as described in essentially all current medical textbooks and recent review articles on complement. The current and widely accepted scientific view is that the alternative pathway is activated on the surface of certain particular targets (microbes, zymosan, and rabbit erythrocytes) through amplification of spontaneous "tick-over" C3 activation. However, the absence of any alternative pathway activation in serum from MASP-1 and MASP-3 double-deficient mice and serum from a human 3MC patient on both zymosan-coated plates and on two different bacteria (N. meningitidis and S. aureus), and the reduction in erythrocyte hemolysis in MASP-3-deficient serum from humans and mice indicate that initiation of alternative pathway activation on these surfaces requires functional MASP-3. The required role for MASP-3 may be lectin-dependent or -independent and leads to the formation of alternative pathway C3 convertase and C5 convertase complexes, i.e., C3bBb and C3bBb(C3b)n, respectively. Thus, the inventors herein describe the existence of a previously elusive initiation route for the alternative pathway. This initiation route is dependent on (i) LEA-1, a recently discovered activation arm of the lectin pathway, and/or (ii) lectin-independent roles of MASP-3, MASP-1, and HTRA-1 proteins. 3. The use of MASP-3 inhibitory agents for the treatment of alternative pathway-related diseases and conditions.

[00128] As described herein, high affinity MASP-3 inhibitory antibodies (e.g., with a binding affinity of less than 500 pM) have been shown to completely inhibit the alternative pathway in mammals such as rodents and non-primates at molar concentrations lower than the concentration of the target MASP-3 (e.g., at a molar ratio of about 1:1 to about 2.5:1 (MASP-3 target to mAb) (see Examples 11-21). As described in Example 11, a single dose administration of a high affinity MASP-3 inhibitory antibody, mAb 13B1, to mice led to complete or near complete ablation of systemic alternative pathway complement activity for at least 14 days. As further described in Example 12, in a study conducted in a well-established animal model associated with PNH, mAb 13B1 was shown to significantly improve PNH red blood cell survival and protect red blood cells red blood cell counts significantly better than did C5 inhibition. As described in Example 13, mAb 13B1 was further demonstrated to reduce disease incidence and severity in a mouse model of arthritis. The results in this example demonstrate that representative high-affinity MASP-3 inhibitory mAbs 13B1, 10D12, and 4D5 are highly effective in blocking the alternative pathway in primates. Administration of a single dose of mAb 13B1, 10D12, or 4D5 to cynomolgus monkeys resulted in sustained ablation of systemic alternative pathway activity lasting approximately 16 days. The extent of alternative pathway ablation in cynomolgus monkeys treated with high-affinity MASP-3 inhibitory antibodies was comparable to that achieved by factor D blockade in vitro and in vivo, indicating complete blockade of factor D conversion by MASP-3 inhibitory antibodies. Therefore, high-affinity MASP-3 inhibitory mAbs have therapeutic utility in the treatment of patients suffering from diseases related to alternative pathway hyperactivity.

[00129] Accordingly, in one aspect, the invention provides methods of inhibiting the alternative pathway in a mammalian subject in need thereof, comprising administering to the subject a composition comprising an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to the serine protease domain of human MASP-3 (amino acid residues 450-728 of SEQ ID NO: 2) with high affinity (having a KD of less than 500 pM), in an amount effective to inhibit complement activation of the alternative pathway in the subject. In some embodiments, the individual is suffering from an alternative pathway-related disease or disorder (i.e., a disease or disorder related to alternative pathway hyperactivity), such as paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD, including wet and dry AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy (including hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), thrombotic thrombocytopenic purpura (TTP), or transplant-associated TMA), asthma, dense deposit disease, pauci-immune crescentic necrotizing glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis optica, Behçet's disease, multiple sclerosis, Guillain Barre syndrome, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), lupus nephritis, lupus systemic erythematosus (SLE), diabetic retinopathy, uveitis, chronic obstructive pulmonary disease (COPD), C3 glomerulopathy, transplant rejection, graft-versus-host disease (GVHD), hemodialysis, sepsis, systemic inflammatory response syndrome (SIRS), acute respiratory distress syndrome (ARDS), ANCA vasculitis, antiphospholipid syndrome, atherosclerosis, IgA nephropathy, and myasthenia gravis, as described below.

a. THE ROLE OF MASP-3 IN PAROXYSMAL NOCTURNAL HEMOGLOBINURIA AND THERAPEUTIC METHODS USING MASP-3 INHIBITOR ANTIBODIES, OPTIONALLY IN COMBINATION WITH MASP-2 INHIBITOR AGENTS HPN Overview

[00130] Paroxysmal nocturnal hemoglobinuria (PNH), sometimes also referred to as Marchiafava-Micheli syndrome, is an acquired and potentially fatal blood disorder. PNH can develop on its own, referred to as "primary PNH", or in the context of other bone marrow disorders such as aplastic anemia, referred to as "secondary PNH". Most cases are primary PNH. PNH is characterized by complement-induced destruction of red blood cells (hemolysis), low red blood cell counts (anemia), thrombosis, and bone marrow failure. Laboratory findings in PNH show changes consistent with intravascular hemolytic anemia: low hemoglobin, elevated lactate dehydrogenase, elevated reticulocyte counts (immature red blood cells released by the bone marrow to replace destroyed cells), elevated bilirubin (a breakdown product of hemoglobin), absence of autoreactive red blood cell-binding antibodies as a possible cause.

[00131] The hallmark of PNH is chronic complement-mediated hemolysis caused by dysregulated activation of terminal complement components, including the membrane attack complex, on the surface of circulating red blood cells. PNH red blood cells are subject to uncontrolled complement activation and hemolysis due to the absence of the complement regulators CD55 and CD59 on their surface (Lindorfer, M.A., et al., Blood 115(11):2283-91 (2010), Risitano, et al., Mini-Reviews in Medicinal Chemistry, 11:528-535 (2011)). CD55 and CD59 are abundantly expressed on normal red blood cells and control complement activation. CD55 acts as a negative regulator of the alternative pathway by inhibiting the formation of the alternative pathway C3 convertase complex (C3bBb) and accelerating the disintegration of the preformed convertase, thereby blocking the formation of the membrane attack complex (MAC). CD59 inhibits the complement membrane attack complex by directly binding the C5b678 complex and preventing the binding and polymerization of C9.

[00132] While hemolysis and anemia are the dominant clinical features of PNH, the disease is a complex hematologic disorder that also includes thrombosis and bone marrow failure as part of the clinical findings (Risitano et al, Mini Reviews in Med Chem, 11:528-535 (2011)). At the molecular level, PNH is caused by the abnormal clonal expansion of hematopoietic stem cells lacking a functional PIG A gene. PIG A is an X-linked gene that encodes a glycosyl-phosphatidyl inositol transferase required for the stable surface expression of GPI-anchored class A glycoproteins, including CD55 and CD59. For reasons that are currently under investigation, hematopoietic stem cells with a dysfunctional PIG A gene that arise as a result of spontaneous somatic mutations may undergo clonal expansion to the point where their progeny constitute a significant portion of the peripheral hematopoietic cell pool. Although both erythrocyte and lymphocyte progeny of the mutant stem cell clone lack CD55 and CD59, only the red blood cells undergo evident lysis after they enter the circulation.

[00133] Current treatment for PNH includes blood transfusion for anemia, anticoagulation for thrombosis, and the use of the monoclonal antibody eculizumab (Soliris®), which protects blood cells against destruction by the immune system by inhibiting the complement system (Hillmen P. et al., N. Engl. J. Med. 350(6):552-559 (2004)). Eculizumab (Soliris®) is a humanized monoclonal antibody that targets the C5 component of complement, blocking its cleavage by C5 convertases, thus preventing the production of C5a and the formation of MAC. Treatment of patients with PNH with eculizumab resulted in a reduction in intravascular hemolysis, as measured by lactate dehydrogenase (LDH), leading to stabilization of hemoglobin and transfusion independence in about half of patients (Risitano et al, Mini-Reviews in Medicinal Chemistry, 11(6) (2011)). While almost all patients receiving eculizumab therapy achieve normal or near-normal LDH levels (due to control of intravascular hemolysis), only about one-third of patients achieve a hemoglobin value of 11 g/dL, and the remaining patients receiving eculizumab continue to have moderate to severe anemia (i.e., transfusion-dependent) in approximately equal proportions (Risitano A.M. et al., Blood 113:4094-100 (2009)). As described in Risitano et al., Mini-Reviews in Medicinal Chemistry 11: 528-535 (2011), it was demonstrated that PNH patients receiving eculizumab contained large amounts of C3 fragments bound to their PNH RBCs (whereas untreated patients did not). This finding leads to the recognition that in PNH patients treated with Soliris, PNH RBCs that are no longer hemolyzed due to C5 blockade can now accumulate large amounts of membrane-bound C3 fragments, which operate as opsonins, resulting in their trapping in reticuloendothelial cells via specific C3 receptors and subsequent extravascular hemolysis. Thus, while preventing intravascular hemolysis and the resulting sequelae, eculizumab therapy simply diverts the disposition of these red blood cells from intravascular to extravascular hemolysis, resulting in substantial untreated residual anemia in many patients (Risitano A.M. et al., Blood 113:4094-100 (2009)). Therefore, therapeutic strategies beyond the use of eculizumab are needed for those patients who develop C3 fragment-mediated extravascular hemolysis because they continue to require red blood cell transfusions. Such C3 fragment-targeting approaches have demonstrated utility in experimental systems (Lindorfer et al., Blood 115:2283-91, 2010).

Complement initiation mechanisms in PNH

[00134] The causal relationship between defective surface expression of the negative complement regulators CD55 and CD59 in PNH, combined with the efficacy of eculizumab in preventing intravascular hemolysis, clearly defines PNH as a complement-mediated condition. Although this paradigm is widely accepted, the nature of the events that initiate complement activation and the complement activation pathway(s) involved remain unresolved. Because CD55 and CD59 negatively regulate the terminal amplification steps in the complement cascade common to all complement initiation pathways, deficiency of these molecules will lead to exaggerated formation and membrane integration of membrane attack complexes, regardless of whether complement activation is initiated by the lectin pathway, the classical pathway, or by spontaneous turnover of the alternative pathway. Thus, in PNH patients, any complement activation events that lead to C3b deposition on the red blood cell surface may trigger subsequent amplification and pathological hemolysis (intravascular and/or extravascular) and precipitate a hemolytic crisis. A clear mechanistic understanding of the molecular events that trigger hemolytic crisis in PNH patients remains elusive. Because no complement priming events are clearly evident in PNH patients undergoing a hemolytic crisis, the prevailing view is that complement activation in PNH may occur spontaneously due to low-level “tick-over” activation of the alternative pathway, which is subsequently amplified by inadequate control of terminal complement activation due to the lack of CD55 and CD59.

[00135] However, it is important to note that in its natural history, PNH usually develops or is exacerbated following certain events, such as infection or injury (Risitano, Biologics 2: 205-222 (2008)), which have been shown to trigger complement activation. This complement activation response is not dependent on prior host immunity against the inciting pathogen and therefore likely does not involve the classical pathway. Rather, it appears that this complement activation response is initiated by lectin binding to foreign or “self-altered” carbohydrate patterns expressed on the surface of microbial agents or damaged host tissue. Thus, the events that led to the hemolytic crisis in PNH are intimately linked to complement activation initiated via lectins. This makes it very likely that lectin activation pathways provide the trigger for the initiation that ultimately leads to hemolysis in PNH patients.

[00136] Using well-defined pathogens that activate complement via lectins as experimental models to dissect the activation cascades at the molecular level, it has been demonstrated that, depending on the stimulatory microbe, complement activation can be initiated by either LEA-2 or LEA-1, leading to opsonization and/or lysis. This same principle of dual responses (i.e., opsonization and/or lysis) to lectin-priming events likely applies to other types of infectious agents or to complement activation by lectins following host tissue damage or other lectin-driven complement activation events that can cause PNH. Based on this duality in the lectin pathway, we infer that LEA-2- and/or LEA-1-primed complement activation in PNH patients promotes opsonization and/or lysis of red blood cells with C3b and subsequent extravascular and intravascular hemolysis. Therefore, in the setting of PNH, inhibition of both LEA-1 and LEA-2 would be expected to treat both intravascular and extravascular hemolysis, providing a significant advantage over the C5 inhibitor eculizumab.

[00137] It has been determined that exposure to S. pneumoniae preferentially triggers lectin-dependent activation of LEA-2, which leads to opsonization of this microbe with C3b. Because S. pneumoniae is resistant to MAC-mediated lysis, its clearance from the circulation occurs through opsonization with C3b. This opsonization and subsequent clearance from the circulation is LEA-2 dependent, as indicated by impaired bacterial control in MASP-2-deficient mice and in mice treated with MASP-2 monoclonal antibodies (PLOS Pathog., 8: e1002793. (2012)).

[00138] In exploring the role of LEA-2 in innate host responses to microbial agents, we tested additional pathogens. A dramatically different outcome was observed when Neisseria meningitidis was studied as a model organism. N. meningitidis also activates complement via lectins, and complement activation is required to contain N. meningitidis infections in the naïve host. However, LEA-2 plays no functional host-protective role in this response: As shown in FIGURES 6 and 7, blockade of LEA-2 by genetic ablation of MASP-2 does not reduce survival after infection with N. meningitidis. Conversely, blockade of LEA-2 by ablation of MASP-2 significantly improved survival (FIGURES 6 and 7) as well as disease scores (FIGURE 9) in these studies. Blocking LEA-2 by administration of MASP-2 antibody produced the same result (FIGURE 10), eliminating secondary or compensatory effects in the knockout mouse strain as a possible cause. These favorable outcomes in LEA-2-ablated animals were associated with a more rapid clearance of N. meningitidis from the blood (FIGURE 8). Also, as described here, incubation of N. meningitidis with normal human serum killed N. meningitidis (FIGURE 11). The addition of a functional monoclonal antibody specific for human MASP-2 that blocks LEA-2, but not the administration of an isotype control monoclonal antibody, can enhance this killing response. However, this process depends on lectins and at least a partially functional complement system, since MBL-deficient human serum or heat-inactivated human serum failed to kill N. meningitidis (FIGURE 11). Collectively, these new findings suggest that N. meningitidis infections in the presence of a functional complement system are controlled by a lectin-dependent, but LEA-2-independent, pathway of complement activation.

[00139] The hypothesis that LEA-1 may be the complement pathway responsible for lectin-dependent killing of N. meningitidis was tested using a serum sample from a patient with 3MC. This patient was homozygous for a nonsense mutation in exon 12 of the MASP-1/3 gene. As a result, this patient lacked a functional MASP-3 protein but had sufficient complement (exon 12 is specific for the MASP-3 transcript; the mutation has no effect on MASP-1 function or expression levels). (See Nat Genet 43(3):197-203 (2011)). Normal human serum efficiently kills N. meningitidis, but heat-inactivated serum deficient in MBL (one of the recognition molecules for the lectin pathway) and serum deficient in MASP-3 were unable to kill N. meningitidis (FIGURE 12). Thus, LEA-1 appears to mediate the killing of N. meningitidis. This finding was confirmed using serum samples from knockout mouse strains. Whereas complement-containing serum from normal mice readily killed N. meningitidis, serum from MBL-deficient or MASP-1/3-deficient mice was as ineffective as heat-inactivated serum that lacked functional complement (FIGURE 13). Conversely, MASP-2-deficient serum exhibited efficient killing of N. meningitidis.

[00140] These findings provide evidence for a previously unknown duality in the lectin pathway, revealing the existence of separate LEA-2 and LEA-1 pathways of lectin-dependent complement activation. In the examples detailed above, LEA-2 and LEA-1 are non-redundant and mediate distinct functional outcomes. The data suggest that certain types of lectin pathway activators (including but not limited to S. pneumonia) preferentially initiate complement activation via LEA-2 leading to opsonization, while others (exemplified by N. meningitidis) preferentially initiate complement activation via LEA-1 and promote cytolytic processes. However, the data do not necessarily limit LEA-2 to opsonization and LEA-1 to cytolytic processes, as both pathways in other settings can mediate opsonization and/or lysis.

[00141] In the context of lectin-dependent complement activation by N. meningitidis, the LEA-2 and LEA-1 arms appear to compete with each other, whereas blockade of LEA-2 increased LEA-1-dependent lytic killing of the organism in vitro (FIGURE 13). As detailed above, this finding can be explained by the increased likelihood of lectin-MASP-1 complexes residing in close proximity to lectin-MASP-3 complexes in the absence of MASP-2, which will increase LEA-1 activation and promote more effective lysis of N. meningitides. Because lysis of N. meningitidis is the primary protective mechanism in the naïve host, blockade of LEA-2 in vivo increases clearance of N. meningitidis and leads to increased killing.

[00142] Although the examples discussed above illustrate the opposing effects of LEA-2 and LEA-1 on outcomes following infection with N. meningitidis, there may be other settings where both LEA-2 and LEA-1 may synergize to produce a given outcome. As detailed below, in other settings of pathological complement activation via lectins such as those present in PNH, LEA-2 and LEA-1-driven complement activation may cooperate synergistically to contribute to the overall pathology of PNH. Furthermore, as described herein, MASP-3 also contributes to the lectin-independent conversion of factor B and factor D, which can occur in the absence of Ca++, commonly leading to the conversion of C3bB to C3bBb and profactor D to factor D, which may further contribute to the pathology of PNH. Biology and expected functional activity in PNH

[00143] This section describes the inhibitory effects of LEA-2 and LEA-1 blockade on hemolysis in an in vitro model of PNH. The findings support the utility of LEA-2 blocking agents (including, but not limited to, antibodies that bind to and block the function of MASP-2) and LEA-1 blocking agents (including, but not limited to, antibodies that bind to and block the function of MASP-3 activation mediated by MASP-1, MASP-3, or both) to treat individuals suffering from one or more aspects of PNH and also the use of inhibitors of LEA-2 and/or LEA-1, and/or MASP-3-dependent and lectin-independent complement activation (including MASP-2 inhibitors, MASP-3 inhibitors, dual or bispecific MASP-2/MASP-3 or MASP-1/MASP-2 inhibitors, and pan-specific MASP-1/MASP-2/MASP-3 inhibitors) to ameliorate the effects of C3 fragment-mediated extravascular hemolysis in patients with PNH undergoing therapy with a LEA-2 and/or LEA-1 inhibitor. C5 such as eculizumab. MASP-2 inhibitors to block opsonization and extravascular hemolysis of PNH red blood cells via the reticuloendothelial system

[00144] As detailed above, patients with PNH become anemic due to two distinct mechanisms of red blood cell clearance from the circulation: intravascular hemolysis via activation of the membrane attack complex (MAC) and extravascular hemolysis following opsonization with C3b and subsequent clearance following complement receptor binding and uptake by the reticuloendothelial system. Intravascular hemolysis is largely prevented when a patient is treated with eculizumab. Because eculizumab blocks the terminal lytic effector mechanism that occurs downstream of the complement priming activation event as well as subsequent opsonization, eculizumab does not block extravascular hemolysis (Risitano A.M. et al., Blood 113:4094-100 (2009)). Instead, red blood cells that would have undergone hemolysis in patients with untreated PNH can now accumulate activated C3b proteins on their surface, which increases their uptake by the reticuloendothelial system and increases their extravascular hemolysis. Thus, eculizumab treatment effectively shifts the disposition of red blood cells from intravascular hemolysis to potential extravascular hemolysis. As a result, some PNH patients treated with eculizumab remain anemic. Agents that block upstream complement activation and prevent opsonization of PNH red blood cells may be particularly suited to block the extravascular hemolysis occasionally observed with eculizumab.

[00145] The microbial data presented here suggest that LEA-2 is often the dominant route for lectin-dependent opsonization. Furthermore, when lectin-dependent opsonization (measured as C3b deposition) was assessed on three prototypical lectin-activating surfaces (mannan, FIGURE 17A; zymosan, FIGURE 17B; and S. pneumonia; FIGURE 17C), LEA-2 appears to be the dominant route for lectin-dependent opsonization under physiological conditions (i.e., in the presence of Ca++ in which all complement pathways are operational). Under these experimental conditions, MASP-2-deficient serum (which lacks LEA-2) is substantially less effective at opsonizing the test surfaces than WT serum. MASP-1/3-deficient serum (which lacks LEA-1) is also impaired, although this effect is much less pronounced compared to serum lacking LEA-2. The relative magnitude of the contributions of LEA-2 and LEA-1 to lectin-driven opsonization is also illustrated in FIGURES 18A–18C. Although the alternative complement pathway has been reported to support opsonization of lectin-activating surfaces in the absence of either the lectin or the classical pathway (Selander et al., J Clin Invest 116(5):1425–1434 (2006)), the alternative pathway alone (measured under Ca++-free assay conditions) appears to be substantially less effective than the LEA-2- and LEA-1-initiated processes described here. By extrapolation, these data suggest that PNH red cell opsonization may also be preferentially initiated by LEA-2 and, to a lesser extent, by LEA-1 (possibly amplified by the amplification loop of the alternative pathway), as opposed to the result of lectin-independent activation of the alternative pathway. Therefore, LEA-2 inhibitors might be expected to be more effective in limiting opsonization and preventing extravascular hemolysis in PNH. However, recognition of the fact that lectins other than MBL, such as ficolins, bind to different carbohydrate structures, such as acetylated proteins, and that MASP-3 preferentially associates with H-ficolin (Skjoedt et al., Immunobiol. 215: 921–931, 2010), leaves open the possibility of a significant role for LEA-1 in PNH-associated red cell opsonization. Therefore, LEA-1 inhibitors are expected to have additional antiopsonization effects, and the combination of LEA-1 and LEA-2 inhibitors is expected to be optimal and mediate the most robust treatment benefit in limiting opsonization and extravascular hemolysis in PNH patients. Thus, LEA-2 and LEA-1 act additively or synergistically to promote opsonization, and a cross-reactive or bispecific LEA-1/LEA-2 inhibitor is expected to be more effective in blocking opsonization and extravascular hemolysis in PNH.

Role of MASP-3 inhibitors in PNH

[00146] Using an in vitro model of PNH, we demonstrate that complement activation and resultant hemolysis in PNH are indeed initiated by activation of LEA-2 and/or LEA-1 and are not independent of the alternative pathway. These studies used mannan-sensitive red blood cells from several mouse strains, including red blood cells from mice deficient in Crry (a major negative regulator of the terminal complement pathway in mice) as well as red blood cells from mice deficient in CD55/CD59, which lack the same complement regulators that are absent in PNH patients. When mannan-sensitive Crry-deficient red blood cells were exposed to complement-sufficient human serum, the red blood cells effectively hemolyzed at a serum concentration of 3% (FIGURES 19 and 20) whereas complement-deficient (HI: heat-inactivated) serum was not hemolytic. Notably, complement-sufficient serum, where LEA-2 was blocked by the addition of MASP-2 antibody, reduced hemolytic activity, and 6% serum was required for effective hemolysis. Similar observations were made when CD55/CD59-deficient red blood cells were tested (FIGURE 22). Complement-sufficient human serum supplemented with MASP-2 monoclonal antibody (i.e., serum where LEA-2 is suppressed) was approximately two-fold less effective than untreated serum in supporting hemolysis. Furthermore, higher concentrations of LEA-2-blocked serum (i.e., treated with anti-MASP-2 monoclonal antibody) were required to promote effective hemolysis of untreated wild-type red blood cells compared with untreated serum (FIGURE 21).

[00147] Even more surprisingly, serum from a 3MC patient homozygous for a dysfunctional MASP-3 protein (and thus lacking LEA-1) was completely incapable of hemolyzing mannan-sensitive Crry-deficient red blood cells (FIGURE 20 and FIGURE 21). A similar outcome was observed when nonsensitive normal red blood cells were used: As shown in FIGURE 21, LEA-1-deficient serum isolated from a 3MC patient was completely ineffective in mediating hemolysis. Collectively, these data indicate that while LEA-2 contributes significantly to the intravascular hemolysis response, LEA-1 is the predominant pathway of complement initiation leading to hemolysis. Thus, while LEA-2 blocking agents are expected to significantly reduce intravascular red blood cell hemolysis in PNH patients, LEA-1 blocking agents are expected to have a more profound effect and largely eliminate complement-driven hemolysis.

[00148] It should be noted that the serum from the LEA-1-deficient 3MC patient used in this study had a diminished but functional alternative pathway when tested under conventional alternative pathway assay conditions (FIGURE 15). This finding suggests that LEA-1 contributes more to hemolysis than conventionally defined alternative pathway activity in this experimental PNH setting. By inference, it is implied that LEA-1 blocking agents will be at least as effective as agents that block other aspects of the alternative pathway in preventing or treating intravascular hemolysis in PNH patients. Role of MASP-2 Inhibitors in PNH

[00149] The data presented here suggest the following pathogenetic mechanisms for anemia in PNH: intravascular hemolysis due to dysregulated activation of terminal complement components and lysis of red blood cells by MAC formation, which is initiated predominantly, although not exclusively, by LEA-1, and extravascular hemolysis caused by opsonization of red blood cells by C3b, which appears to be initiated predominantly by LEA-2. Although a discernible role for LEA-2 in initiating complement activation and promoting MAC formation and hemolysis is evident, this process appears to be substantially less effective than LEA-1-initiated complement activation leading to hemolysis. Thus, LEA-2 blocking agents are expected to significantly reduce intravascular hemolysis in PNH patients, although this therapeutic activity is expected to be only partial. By comparison, a more substantial reduction in intravascular hemolysis in PNH patients is expected for LEA-1 blocking agents.

[00150] Extravascular hemolysis, a less dramatic but equally important mechanism of red blood cell destruction leading to anemia in PNH, is primarily the result of C3b opsonization, which appears to be predominantly mediated by LEA-2. Thus, LEA-2 blocking agents can be expected to preferentially block red blood cell opsonization and subsequent extravascular hemolysis in PNH. This unique therapeutic activity of LEA-2 blocking agents is expected to provide a significant treatment benefit for all PNH patients, since there is currently no treatment for PNH patients exhibiting this pathogenic process. LEA-2 inhibitors as adjunctive therapy to LEA-1 inhibitors or terminal complement blocking agents

[00151] The data presented detail two pathogenetic mechanisms for red blood cell clearance and anemia in PNH that may be targeted, separately or in combination, by distinct classes of therapeutic agents: intravascular hemolysis initiated predominantly, though not exclusively, by LEA-1 and thus expected to be effectively prevented by a LEA-1 blocking agent, and extravascular hemolysis due to C3b opsonization driven predominantly by LEA-2 and thus effectively prevented by a LEA-2 blocking agent.

[00152] It is well documented that both intravascular and extravascular mechanisms of hemolysis lead to anemia in patients with PNH (Risitano et al., Blood 113:4094-4100 (2009)). Therefore, it is expected that a LEA-1 blocking agent that prevents intravascular hemolysis in combination with a LEA-2 blocking agent that primarily prevents extravascular hemolysis will be more effective than either agent alone in preventing anemia from developing in patients with PNH. Indeed, the combination of LEA-1 and LEA-2 blocking agents is expected to prevent all relevant mechanisms of complement initiation in PNH and thus block all symptoms of anemia in PNH.

[00153] C5 blocking agents (such as eculizumab) are also known to effectively block intravascular hemolysis but do not interfere with opsonization. This leaves some PNH patients treated with anti-C5 with substantial residual anemia due to LEA-2-mediated extravascular hemolysis, which remains untreated. Therefore, it is expected that a C5 blocking agent (such as eculizumab) that prevents intravascular hemolysis in combination with a LEA-2 blocking agent that reduces extravascular hemolysis will be more effective than either agent alone in preventing anemia from developing in PNH patients.

[00154] Other agents that block the terminal amplification loop of the complement system leading to C5 activation and MAC deposition (including, but not limited to, agents that block properdin, factor B, or factor D or increase the inhibitory activity of factor I, factor H, or other complement inhibitory factors) are also expected to inhibit intravascular hemolysis. However, these agents are not expected to interfere with LEA-2-mediated opsonization in PNH patients. This leaves some PNH patients treated with such agents with substantial residual anemia due to LEA-2-mediated extravascular hemolysis that remains untreated. Therefore, it is expected that treatment with such agents that prevent intravascular hemolysis in combination with a LEA-2 blocking agent that minimizes extravascular hemolysis will be more effective than either agent alone in preventing anemia from developing in PNH patients. Indeed, the combination of such agents and a LEA-2 blocking agent is expected to prevent all relevant mechanisms of red blood cell destruction in PNH and thus block all symptoms of anemia in PNH.

Use of multiple, bispecific, or panspecific LEA-1 and LEA-2 antibodies to treat PNH

[00155] As detailed above, the use of a combination of pharmacological agents that individually block LEA-1 and LEA-2, and thus in combination block all complement activation events that mediate intravascular hemolysis as well as extravascular hemolysis, is expected to provide the best clinical outcome for patients with PNH. This outcome may be achieved, for example, by co-administration of an antibody that has LEA-1 blocking activity in conjunction with an antibody that has LEA-2 blocking activity. In some embodiments, the LEA-1- and LEA-2 blocking activities are combined in a single molecular entity and this entity with combined LEA-1- and LEA-2 blocking activity will effectively block intravascular hemolysis as well as extravascular hemolysis and will prevent anemia in PNH. Such an entity may comprise or consist of a bispecific antibody, wherein one antigen-combining site specifically recognizes MASP-1 and blocks LEA-1 and downregulates LEA-2 and the second antigen-combining site specifically recognizes MASP-2 and further blocks LEA-2. Alternatively, such an entity may consist of a bispecific monoclonal antibody, wherein one antigen-combining site specifically recognizes MASP-3 and thereby blocks LEA-1 and the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Such an entity may ideally consist of a bispecific monoclonal antibody, wherein one antigen-combining site specifically recognizes MASP-1 and MASP-3 and thereby blocks LEA-1 and downregulates LEA-2 while the second antigen-combining site specifically recognizes MASP-2 and further blocks LEA-2. Based on the similarities in overall protein sequence and architecture, it can also be envisaged that a conventional antibody with two identical binding sites can be developed that specifically binds to MASP-1 and MASP-2 and MASP-3 in a functional manner, thereby achieving functional blockade of LEA-1 and LEA-2. Such an antibody with pan-MASP inhibitory activity is expected to block both intravascular hemolysis and extravascular hemolysis and thus effectively treat anemia in PNH patients.

[00156] As described in Examples 11-21, high affinity MASP-3 inhibitory antibodies have been generated that have therapeutic utility for inhibition of the alternative pathway in PA-related diseases or conditions, such as PNH.

[00157] Suitably, in one embodiment, the present invention provides a method for treating a subject suffering from or at risk of developing PNH comprising an effective amount of a high affinity monoclonal antibody or antigen-binding fragment thereof, as described herein, that binds to human MASP-3 and inhibits alternative pathway complement activation to treat or reduce the risk of PNH in the subject.

[00158] In one embodiment, the present invention provides a method of treating a subject suffering from or at risk of developing paroxysmal nocturnal hemoglobinuria (PNH), comprising administering to the subject a pharmaceutical composition comprising an effective amount of a monoclonal antibody or antigen-binding fragment thereof as described herein that binds to human MASP-3 and inhibits alternative pathway complement activation to treat or reduce the risk of PNH in the subject, such as, wherein said antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region comprising (i) VHCDR1 comprising SEQ ID NO: 84, (ii) VHCDR2 comprising SEQ ID NO: 86 or SEQ ID NO: 275, and (iii) VHCDR3 comprising SEQ ID NO: 88; and (b) a light chain variable region comprising (i) VLCDR1 comprising SEQ ID NO: 142, SEQ ID NO: 257, SEQ ID NO: 258, or SEQ ID NO: 259, (ii) VLCDR2 comprising SEQ ID NO: 144, and (iii) VLCDR3 comprising SEQ ID NO: 161. In some embodiments, the pharmaceutical composition increases red blood cell survival in the individual suffering from PNH. In some embodiments, wherein the individual suffering from or at risk of developing PNH exhibits one or more symptoms selected from the group consisting of (i) below normal hemoglobin levels, (ii) below normal platelet levels; (iii) above normal reticulocyte levels, and (iv) above normal bilirubin levels. In some embodiments, the pharmaceutical composition is administered systemically (e.g., subcutaneously, intramuscularly, intravenously, intra-arterially, or as an inhalant) to a subject suffering from or at risk of developing PNH. In some embodiments, the subject suffering from or at risk of PNH has previously undergone or is currently undergoing treatment with a terminal complement inhibitor that inhibits cleavage of complement protein C5. In some embodiments, the method further comprises administering to the subject a terminal complement inhibitor that inhibits cleavage of complement protein C5. In some embodiments, the terminal complement inhibitor is a humanized anti-C5 antibody or antigen-binding fragment thereof. In some embodiments, the terminal complement inhibitor is eculizumab. B. THE ROLE OF MASP-3 IN AGE-RELATED MACULAR DEGENERATION AND THERAPEUTIC METHODS USING MASP-3 INHIBITOR ANTIBODIES, OPTIONALLY IN COMBINATION WITH MASP-2 INHIBITOR AGENTS

[00159] Age-related macular degeneration (AMD) is the leading cause of visual impairment and blindness in the elderly and accounts for up to 50% of blindness in developed countries. The prevalence of AMD is about 3% in adults and increases with age, such that nearly two-thirds of the population over 80 years of age will have some signs. It is estimated that over 1.75 million individuals in the United States have advanced AMD, and the prevalence is increasing as the population ages and is expected to reach nearly 3 million by 2020 (Friedman, D.S., et al., Arch Ophthalmol. 122:564-572, 2004). AMD is an abnormality of the retinal pigment epithelium (RPE) that results in degeneration of the photoreceptors in the overlying central retina or macula and loss of central vision. Early and intermediate forms of AMD are characterized by progressive deposits of drusen, a yellowish material containing lipids, proteins, lipoproteins, and cellular debris, in the subretinal space adjacent to the RPE, along with pigmentary irregularities in the retina. Advanced AMD consists of two clinical subtypes: non-neovascular geographic atrophic AMD (‘dry’) and neovascular exudative AMD (‘wet’). Although dry AMD accounts for 80–90% of advanced AMD, most sudden and severe vision loss occurs in patients with wet AMD. It is not known whether the two types of AMD represent different phenotypes arising from similar pathologies or are two distinct conditions. Currently, no therapies have been approved by the United States Food and Drug Administration (FDA) to treat dry AMD. FDA-approved treatment options for wet AMD include intravitreal injections of antiangiogenic drugs (ranibizumab, pegaptanib sodium, aflibercept), laser therapy, photodynamic laser therapy, and implantable telescope.

[00160] The etiology and pathophysiology of AMD are complex and incompletely understood. Several lines of evidence support the role of complement dysregulation in the pathogenesis of AMD. Genetic association studies have identified multiple genetic loci associated with AMD, including genes encoding a number of complement proteins, factors, and regulators. The strongest association is with polymorphisms in the complement factor H (CFH) gene, with homozygotes for the Y402H variant having approximately a 6-fold and heterozygotes approximately a 2.5-fold increased risk of developing AMD compared with the non-risk genotype (Khandhadia, S., et al., Immunobiol. 217:127-146, 2012). Mutations in other genes encoding the complement pathway have also been associated with increased or decreased risk of AMD, including complement factor B (CFB), C2, C3, factor I, and CFH-related proteins 1 and 3 (Khandhadia et al.). Immunohistochemical and proteomic studies in donor eyes of AMD patients have shown that complement cascade proteins are increased and localized to drusen (Issa, P.C., et al., Graefes. Arch. Clin. Exp. Ophthalmol. 249:163-174, 2011). In addition, AMD patients have increased systemic complement activation, as measured in peripheral blood (Issa et al., 2011, supra).

[00161] The alternative complement pathway appears to be more relevant than the classical pathway in the pathogenesis of AMD. C1q, the essential recognition component for activation of the classical pathway, was not detected in drusen by immunohistochemical analyses (Mullins et al., FASEB J. 14:835-846, 2000; Johnson et al., Exp. Eye Res. 70:441-449, 2000). Genetic association studies have involved CFH and CFB genes. These proteins are involved in the amplification loop of the alternative pathway, CFH being a fluid phase inhibitor and CFB being a protease activating component of the alternative pathway. The Y402H variant of CFH affects the interaction with ligand binding, including binding to C-reactive protein, heparin, M protein and glycosaminoglycans. This altered ligand binding may reduce binding to cell surfaces, which in turn may lead to reduced factor I-mediated degradation of the C3b activation fragment and impaired regulation of the alternative C3 convertase, resulting in excessive activation of the alternative pathway (Khandhadia et al., 2012, supra). Variations in the CFB gene are associated with a protective effect for the development of AMD. A functional variant fB32Q had 4-fold lower binding affinity for C3b than the risk variant fB32R, resulting in reduced formation of C3 convertase (Montes, T. et al., Proc. Natl. Acad. Sci. U.S.A. 106:4366-4371, 2009).

Complement initiation mechanisms in AMD

[00162] The human genetic linkage studies discussed above suggest an important role for the complement system in the pathogenesis of AMD. Furthermore, complement activation products are abundantly present in drusen (Issa, P.C., et al., Graefes. Arch. Clin. Exp. Ophthalmol. 249:163-174, 2011), a characteristic pathological lesion in both wet and dry AMD. However, the nature of the events that initiate complement activation and the complement activation pathway(s) involved remain incompletely understood.

[00163] It is important to note that drusen deposits are composed of cellular debris and oxidative waste products originating in the retina that accumulate beneath the RPE as the eye ages. Furthermore, oxidative stress appears to play an important role (Cai et al; Front Biosci., 17: 1976-95, 2012) and has been shown to cause complement activation in the RPE (J Biol Chem., 284(25): 16939-47, 2009). It is widely appreciated that both oxidative stress and cellular or tissue injury activate the lectins of the complement system. For example, Collard et al. have shown that endothelial cells exposed to oxidative stress elicit abundant lectin-mediated complement deposition (Collard C.D. et al., Mol Immunol., 36(13-14):941-8, 1999; Collard C.D. et al., Am J Pathol., 156(5):1549-56, 2000), and that blocking lectin binding and lectin-dependent complement activation improves outcomes in experimental models of oxidative stress injury (Collard C.D. et al., Am J Pathol., 156(5):1549-56, 2000). Thus, it seems likely that oxidative waste products present in drusen also activate complement through lectins. By inference, lectin-dependent complement activation may play a key role in the pathogenesis of AMD.

[00164] The role of the complement system has been evaluated in mouse models of AMD. In the light-damaged mouse model, an experimental model for oxidative stress-mediated photoreceptor degeneration, knockout mice with a deletion of the classical pathway (C1qa -/- on a C57BL/6 background) had the same sensitivity to light damage compared to wild-type littermates, whereas deletion of the alternative pathway complement factor D (CFD -/-) resulted in protection against light damage (Rohrer, B. et al., Invest. Ophthalmol. Vis. Sci. 48:5282-5289, 2007). In a mouse model of choroidal neovascularization (CNV) induced by laser photocoagulation of Bruch's membrane, knockout mice lacking complement factor B (CFB -/-) were protected against CNV compared with wild-type mice (Rohrer, B. et al., Invest. Ophthalmol. Vis. Sci. 50:30563064, 2009). In the same model, intravenous administration of a recombinant form of complement factor H targeted to complement activation sites (CR2-fH) reduced the extent of CNV. This protective effect was observed whether CR2-fH was administered at the time of laser injury or therapeutically (after laser injury). A human therapeutic version of CR2-fH (TT30) was also effective in the murine CNV model (Rohrer, B. et al. J. Ocul. Pharmacol. Ther., 28:402-409, 2012). Because fB is activated by LEA-1 and MASP-1 and MASP-3 contribute to factor D maturation, these findings suggest that LEA-1 inhibitors may have therapeutic benefit in patients with AMD. Furthermore, recently reported results from a Phase 2 study showed that monthly intravitreal injection with Lampalizumab (previously referred to as FCFD4514S and anti-factor D, which is an antigen-binding fragment of a humanized monoclonal antibody directed against Factor D) reduced the progression of the area of geographic atrophy in patients with geographic atrophy secondary to AMD (Yaspan B.L. et al., Sci Transl. Med. 9, Issue 395, June 21, 2017).

[00165] Initial experimental studies in a rodent model of AMD using MBL-deficient mice did not support a critical role for the lectin pathway in pathogenic complement activation (Rohrer et al., Mol Immunol. 48:e1-8, 2011). However, MBL is only one of several lectins, and lectins other than MBL can trigger complement activation in AMD. Indeed, our previous work has shown that MASP-2, the rate-limiting serine protease that is critically required for lectin pathway function, plays a critical role in AMD. As described in U.S. Patent No. 7,919,094 (assigned to Omeros Corporation), incorporated herein by reference, MASP-2-deficient mice and MASP-2 antibody-treated mice were protected in a mouse model of laser-induced CNV, a validated preclinical model of wet AMD (Ryan et al., Tr Am Opth Soc LXXVII:707-745, 1979). Thus, LEA-2 inhibitors are expected to effectively prevent CNV and improve outcomes in patients with AMD.

[00166] Thus, in view of the above, LEA-1 and LEA-2 inhibitors are expected to have independent therapeutic benefit in AMD. Furthermore, LEA-1 and LEA-2 inhibitors used together may achieve additional treatment benefit compared to either agent alone or may provide effective treatment for a broader spectrum of patient subsets. Combined inhibition of LEA-1 and LEA-2 may be accomplished by co-administration of a LEA-1 blocking agent and a LEA-2 blocking agent. Ideally, the inhibitory function of LEA-1 and LEA-2 may be encompassed in a single molecular entity, such as a bispecific antibody composed of MASP-1/3 and a specific MASP-2 binding site or a dual-specific antibody where each binding site can bind to and block either MASP-1/3 or MASP-2.

[00167] In accordance with the foregoing, one aspect of the invention thus provides a method of inhibiting LEA-1 dependent complement activation to treat age-related macular degeneration (wet and dry forms) by administering a composition comprising a therapeutically effective amount of a MASP 1 inhibitory agent, a MASP 3 inhibitory agent, or a combination of a MASP 1/3 inhibitory agent, in a pharmaceutical carrier to a subject suffering from such a condition. The MASP 1, MASP 3, or MASP 1/3 inhibitory composition may be administered locally to the eye, such as by irrigation, intravitreal administration, or application of the composition in the form of a gel, ointment, or drops. Alternatively, the MASP 1, MASP 3, or MASP 1/3 inhibitory agent may be administered to the individual systemically, such as by intra-arterial, intravenous, intramuscular, inhaled, nasal, subcutaneous, or other parenteral administration, or potentially by oral administration for non-peptidergic agents. Administration may be repeated as determined by a physician until the condition has resolved or is controlled.

[00168] In one embodiment, the method according to this aspect of the invention further comprises inhibiting LEA-2-dependent complement activation in a subject suffering from age-related macular degeneration, comprising administering a therapeutically effective amount of a MASP-2 inhibitory agent and a MASP-1, MASP-3, or MASP1/3 inhibitory agent to the subject in need thereof. As detailed above, the use of a combination of pharmacological agents that individually block LEA-1 and LEA-2 is expected to provide an improved therapeutic outcome in patients with AMD, compared to LEA-1 inhibition alone. This outcome can be achieved, for example, by co-administration of an antibody that has LEA-1 blocking activity in conjunction with an antibody that has LEA-2 blocking activity. In some embodiments, the LEA-1- and LEA-2-blocking activities are combined in a single molecular entity and that entity has both LEA-1- and LEA-2-blocking activity. Such an entity may comprise or consist of a bispecific antibody, wherein one antigen-combining site specifically recognizes MASP-1 and blocks LEA-1 and the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Alternatively, such an entity may consist of a bispecific monoclonal antibody, wherein one antigen-combining site specifically recognizes MASP-3 and thereby blocks LEA-1 and the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Such an entity may ideally consist of a bispecific monoclonal antibody, wherein one antigen-combining site specifically recognizes MASP-1 and MASP-3 and thereby blocks LEA-1 while the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2.

[00169] The MASP 2 inhibitory composition may be administered locally to the eye, such as by irrigation, intravitreal injection, or topical application of the composition in the form of a gel, ointment, or drops. Alternatively, the MASP 2 inhibitory agent may be administered to the subject systemically, such as by intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous, or other parenteral administration, or potentially by oral administration for non-peptidergic agents. Administration may be repeated as determined by a physician until the condition has resolved or is controlled.

[00170] Application of the MASP-3 inhibitory compositions and optional MASP2 inhibitory compositions of the present invention can be accomplished by a single administration of the composition (e.g., a single composition comprising MASP-2 and MASP-3 inhibitory agents or bispecific or dual inhibitory agents, or co-administration of separate compositions) or a limited sequence of administrations, for the treatment of AMD. Alternatively, the composition can be administered at periodic intervals, such as daily, biweekly, weekly, every other week, monthly, or bimonthly over an extended period of time for the treatment of AMD.

[00171] As described in Examples 11-21, high affinity MASP-3 inhibitory antibodies have been generated that have therapeutic utility for inhibition of the alternative pathway in PA-related diseases or conditions, such as AMD.

[00172] Suitably, in one embodiment, the present invention provides a method for treating a subject suffering from or at risk of developing AMD comprising an effective amount of a high affinity monoclonal antibody or antigen-binding fragment thereof, as described herein, that binds to human MASP-3 and inhibits alternative pathway complement activation to treat or reduce the risk of PNH in the subject. In one embodiment, the present invention provides a method of treating a subject suffering from or at risk of developing AMD, comprising administering to the subject a pharmaceutical composition comprising an effective amount of a monoclonal antibody or antigen-binding fragment thereof as described herein that binds to human MASP-3 and inhibits alternative pathway complement activation to treat or reduce the risk of AMD in the subject, such as, for example, wherein said antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region comprising (i) VHCDR1 comprising SEQ ID NO: 84, (ii) VHCDR2 comprising SEQ ID NO: 86 or SEQ ID NO: 275, and (iii) VHCDR3 comprising SEQ ID NO: 88; and (b) a light chain variable region comprising (i) VLCDR1 comprising SEQ ID NO: 142, SEQ ID NO: 257, SEQ ID NO: 258, or SEQ ID NO: 259, (ii) VLCDR2 comprising SEQ ID NO: 144, and (iii) VLCDR3 comprising SEQ ID NO: 161. C. THE ROLE OF MASP-3 IN ISCHEMIA-REPERFUSION INJURY AND THERAPEUTIC METHODS USING MASP-3 INHIBITOR ANTIBODIES, OPTIONALLY IN COMBINATION WITH MASP-2 INHIBITOR AGENTS

[00173] Tissue ischemia is the basis for a wide spectrum of clinical disorders. Although timely restoration of blood flow is essential for the preservation of ischemic tissue, it has long been recognized that reperfusion, which can occur spontaneously or by therapeutic intervention, can lead to additional tissue injury, a phenomenon termed ischemia-reperfusion (I/R) injury (Eltzschig, H.K. and Tobias, E., Nat. Med. 17:1391-1401, 2011). I/R injury can affect single organs, such as the heart (acute coronary syndrome), kidney (acute kidney injury), intestine (intestinal I/R), and brain (stroke). I/R injury can also affect multiple organs, including trauma and resuscitation (multiple organ failure), circulatory arrest (hypoxic brain injury, acute kidney injury), peripheral vascular disease, and sickle cell disease (acute thoracic syndrome, acute renal failure). Major surgery may be associated with I/R injury, including cardiac surgery (acute heart failure after cardiopulmonary bypass), thoracic surgery (acute lung injury), peripheral vascular surgery (compartment syndrome), vascular surgery (acute kidney injury), and solid organ transplantation (acute graft failure). Currently, there are no specific therapies that target I/R injury, and there is a need for effective treatments to maximize tissue recovery in the ischemic zone and improve functional outcome in these common scenarios.

[00174] The pathophysiology of I/R injury is complex and characterized by a robust inflammatory response following reperfusion. Activation of the complement system has been suggested as an important component of I/R injury, and inhibition of complement activity has been effective in a variety of animal models (Diepenhorst, G.M.P. et al., Ann. Surg. 249:889-899, 2009). The relative importance of the classical, lectin, and alternative pathways in I/R injury is largely fluid and may differ depending on the organs affected. Recently, the availability of knockout mice deficient in specific complement proteins and specific pathway inhibitors has generated data implicating both the lectin and alternative pathways in I/R injury.

[00175] The role of the alternative pathway in gastrointestinal I/R injury was investigated using factor D-deficient (-/-) and heterozygous (+/-) mice (Stahl, G. L., et al. Am. J. Pathol. 162:449-455, 2003). After transient gastrointestinal ischemia, intestinal and lung injury was reduced but not prevented in factor D-deficient mice compared with heterozygous mice, and addition of human factor D to factor D (-/-) mice restored I/R injury. The same model was evaluated in C1q-deficient and MBL-A/C-deficient mice, and the results showed that gastrointestinal I/R injury was independent of C1q and classical pathway activation, but that MBL and lectin pathway activation were required for intestinal injury (Hart, M. L. et al. J. Immunol. 174:63736380, 2005). In contrast, the classical pathway recognition molecule C1q was responsible for lung injury after intestinal I/R (Hart, M. L., et al. J. Immunol. 174:6373-6380, 2005). One hypothesis is that complement activation during I/R injury occurs through the natural binding of IgM to self-antigens present on the surface of ischemic (but not normal) tissue, e.g., nonmuscle myosin type II heavy chains. In a rat model of gastrointestinal I/R injury, immune complexes from intestinal tissue were evaluated for the presence of initiation factors in the classical (C1q), lectin (MBL), or alternative (Factor B) pathways (Lee, H., et al. Mol. Immunol. 47:972–981, 2010). The results showed that C1q and MBL were detected, whereas factor B was not detected in these immune complexes, indicating involvement of the classical and lectin pathways, but not the alternative pathway. In the same model, factor B-deficient mice were not protected from local tissue injury, providing further support for the lack of involvement of the alternative pathway. The role of the lectin pathway in gastrointestinal I/R injury was directly evaluated in MASP-2-deficient mice, and the results showed that gastrointestinal injury was reduced in these mice compared with wild-type controls; treatment with MASP-2 monoclonal antibody was similarly protective (Schwaeble, W. J., et al., Proc. Natl. Acad. Sci. 108:7523–7528, 2011). Taken together, these results provide support for the involvement of the lectin pathway in gastrointestinal I/R injury, with conflicting data regarding the involvement of the alternative pathway.

[00176] In a rat model of myocardial I/R injury, a pathogenic role was demonstrated for the lectin pathway, as MBL-deficient mice were protected from myocardial injury, whereas C1q-deficient and C2/fB-deficient mice were not protected (Walsh, M. C. et al. J. Immunol. 175:541-546, 2005). Protection from myocardial I/R injury was also observed in MASP-2-deficient mice (Schwaeble, W. J., et al., Proc. Natl. Acad. Sci. 108:7523-7528, 2011). Treatment of rats in a model of myocardial I/R with monoclonal antibodies against rat MBL resulted in reduced post-ischemic reperfusion injury (Jordan, J. E., et al., Circulation 104:1413–18, 2001). In a study of patients with myocardial infarction treated with angioplasty, MBL deficiency was associated with reduced 90-day mortality compared with MBL-sufficient counterparts (Trendelenburg M et al., Eur Heart J. 31:1181, 2010). Furthermore, patients with myocardial infarction who develop cardiac dysfunction after angioplasty have approximately threefold higher MBL levels compared with patients with functional recovery (Haahr-Pedersen S., et al., J Inv Cardiology, 21:13, 2009). MBL antibodies also reduced complement deposition on endothelial cells in vitro after oxidative stress, indicating a role for the lectin pathway in myocardial I/R injury (Collard, C. D., et al., Am. J. Pathol. 156:1549-56, 2000). In a rat model of heterotopic isograft cardiac transplantation of I/R injury, the role of the alternative pathway was investigated using the pathway-specific fusion protein CR2-fH (Atkinson, C., et al., J. Immunol. 185:7007-7013, 2010). Systemic administration of CR2-fH immediately after transplantation resulted in a reduction in myocardial I/R injury to an extent comparable to treatment with CR2-Crry, which inhibits all complement pathways, indicating that the alternative pathway is of critical importance in this model.

[00177] In a rat model of renal I/R injury, the alternative pathway was involved because factor B-deficient mice were protected from a decline in renal function and tubular injury compared with wild-type mice (Thurman, J. M., et al., J Immunol. 170:15171523, 2003). Treatment with a factor B inhibitory monoclonal antibody prevented complement activation and reduced murine renal I/R injury (Thurman, J. M., et al., J. Am. Soc. Nephrol. 17:707-715, 2006). In a bilateral renal I/R injury model, MBL-A/C-deficient mice were protected from renal damage compared with wild-type mice, and recombinant human MBL reversed the protective effect in MBL-A/C-deficient mice, suggesting a role for MBL in this model (Moller-Kristensen, M., et al., Scand. J. Immunol. 61:426-434, 2005). In a rat unilateral renal I/R injury model, inhibition of MBL with a monoclonal antibody to MBL-A preserved renal function after I/R (van der Pol, P., et al., Am. J. Transplant. 12:877-887, 2010). Interestingly, the role of MBL in this model does not appear to involve activation of terminal complement components, as treatment with an antibody to C5 was ineffective in preventing renal injury. Instead, MBL appeared to have a direct toxic effect on tubular cells, as human proximal tubular cells incubated with MBL internalized MBL in vitro with subsequent cell apoptosis. In a swine model of renal I/R, Castellano G. et al., (Am J Pathol, 176 (4): 1648-59, 2010) tested a C1 inhibitor, which irreversibly inactivates the C1r and C1s proteases in the classical pathway and also the MASP-1 and MASP-2 proteases in MBL complexes of the lectin pathway, and found that the C1 inhibitor reduced complement deposition in the peritubular capillaries and glomeruli and reduced tubular damage.

[00178] The alternative pathway appears to be involved in experimental traumatic brain injury, as factor B-deficient mice have reduced systemic complement activation as measured by serum C5a levels and reduced posttraumatic neuronal cell death compared with wild-type mice (Leinhase, I., et al., BMC Neurosci. 7:55-67, 2006). In human stroke, complement components C1q, C3c, and C4d have been detected by immunohistochemical staining in ischemic lesions, suggesting activation through the classical pathway (Pedersen, E. D., et al., Scand. J. Immunol. 69:555-562, 2009). Targeting the classical pathway in animal models of cerebral ischemia has produced mixed results, with some studies demonstrating protection while others showing no benefit (Arumugam, T. V., et al., Neuroscience 158:1074-1089, 2009). Experimental and clinical studies have provided strong evidence for the involvement of the lectin pathway. In experimental models of stroke, deficiency of MBL or MASP-2 results in reduced infarct sizes compared to wild-type mice (Cervera A, et al.; PLoS One 3;5(2):e8433, 2010; Osthoff M. et al., PLoS One, 6(6):e21338, 2011). Furthermore, stroke patients with low MBL levels have a better prognosis compared to their counterpart with sufficient MBL (Osthoff M. et al., PLoS One, 6(6):e21338, 2011).

[00179] In a baboon model of cardiopulmonary bypass, treatment with a factor D monoclonal antibody inhibited systemic inflammation, as measured by plasma levels of C3a, sC5b-9, and IL-6, and reduced myocardial tissue injury, indicating the involvement of the alternative pathway in this model (Undar, A., et al., Ann. Thorac. Surg. 74:355-362, 2002).

[00180] Thus, depending on the organ affected by I/R, all three complement pathways may contribute to the pathogenesis and adverse outcomes. Based on the clinical and experimental findings detailed above, LEA-2 inhibitors are expected to be protective in most I/R scenarios. Lectin-dependent LEA-1 activation may cause complement activation via the alternative pathway in at least some scenarios. Additionally, LEA-2-initiated complement activation may be further amplified by the amplification loop of the alternative pathway and thus exacerbate I/R-related tissue injury. Thus, LEA-1 inhibitors are expected to provide additional or complementary treatment benefits in patients suffering from an ischemia-related condition.

[00181] In view of the above, LEA-1 and LEA-2 inhibitors are expected to have independent therapeutic benefits in the treatment, prevention or reduction of the severity of ischemia-reperfusion related conditions. Furthermore, LEA-1 and LEA-2 inhibitors used together may achieve additional treatment benefits compared to either agent alone. An optimally effective treatment for an I/R related condition therefore comprises active pharmaceutical ingredients that, alone or in combination, block both LEA-1 and LEA-2. Combined inhibition of LEA-1 and LEA-2 may be accomplished by co-administration of a LEA-1 blocking agent and a LEA-2 blocking agent. Preferably, the inhibitory function of LEA-1 and LEA-2 can be encompassed in a single molecular entity, such as a bispecific antibody composed of MASP-1/3 and a specific MASP-2 binding site or a dual specificity antibody where each binding site can bind to and block either MASP-1/3 or MASP-2.

[00182] In accordance with the foregoing, one aspect of the invention thus provides a method of inhibiting LEA-1 dependent complement activation to treat, prevent or reduce the severity of ischemia-reperfusion injury by administering a composition comprising a therapeutically effective amount of a LEA-1 inhibiting agent comprising a MASP 1 inhibiting agent, a MASP 3 inhibiting agent or a combination of a MASP 1/3 inhibiting agent, in a pharmaceutical carrier to a subject experiencing ischemic reperfusion. The MASP 1, MASP 3 or MASP 1/3 inhibiting composition may be administered to the subject by intra-arterial, intravenous, intracranial, intramuscular, subcutaneous or other parenteral administration, and potentially orally for non-peptidergic inhibitors and most suitably by intra-arterial or intravenous administration. Administration of the LEA-1 inhibitory compositions of the present invention suitably begins immediately after or as soon as possible after an ischemia-reperfusion event. In cases where reperfusion occurs in a controlled environment (e.g., following aortic aneurysm repair, organ transplantation, or reattachment of severed or traumatized limbs or digits), the LEA-1 inhibitory agent may be administered prior to and/or during and/or after reperfusion. Administration may be repeated periodically, as determined by a physician, for optimal therapeutic effect.

[00183] In some embodiments, the methods are used to treat or prevent an ischemia-reperfusion injury associated with at least one aortic aneurysm repair, cardiopulmonary bypass, vascular reanastomosis in connection with organ transplants and/or extremity/digit reimplantation, stroke, myocardial infarction, and hemodynamic resuscitation following shock and/or surgical procedures.

[00184] In some embodiments, the methods are used to treat or prevent an ischemia-reperfusion injury in a subject who is about to undergo, is undergoing, or has undergone an organ transplant. In some embodiments, the methods are used to treat or prevent an ischemia-reperfusion injury in a subject who is about to undergo, provided that the organ transplant is not a kidney transplant.

[00185] In one embodiment, the method according to this aspect of the invention further comprises inhibiting LEA-2-dependent complement activation in a subject experiencing ischemic reperfusion, comprising administering a therapeutically effective amount of a MASP-2 inhibitory agent and a MASP-1, MASP-3, or MASP-1/3 inhibitory agent to the subject. As detailed above, the use of a combination of pharmacological agents that individually block LEA-1 and LEA-2 is expected to provide an improved therapeutic outcome in treating, preventing, or reducing the severity of ischemia-reperfusion injury, as compared to LEA-1 inhibition alone. This outcome can be achieved, for example, by co-administering an antibody that has LEA-1 blocking activity in conjunction with an antibody that has LEA-2 blocking activity. In some embodiments, the LEA-1- and LEA-2-blocking activities are combined in a single molecular entity and that entity has both LEA-1- and LEA-2-blocking activity. Such an entity may comprise or consist of a bispecific antibody, wherein one antigen-combining site specifically recognizes MASP-1 and blocks LEA-1 and the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Alternatively, such an entity may consist of a bispecific monoclonal antibody, wherein one antigen-combining site specifically recognizes MASP-3 and thereby blocks LEA-1 and the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Such an entity may ideally consist of a bispecific monoclonal antibody, wherein one antigen-combining site specifically recognizes MASP-1 and MASP-3 and thereby blocks LEA-1 while the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2.

[00186] The MASP-2 inhibitory composition may be administered to a subject in need thereof by intra-arterial, intravenous, intracranial, intramuscular, subcutaneous or other parenteral administration, and potentially orally for non-peptidergic inhibitors and most suitably by intra-arterial or intravenous administration. Administration of the MASP-2 inhibitory compositions of the present invention suitably begins immediately following or as soon as possible after an ischemia-reperfusion event. In cases where reperfusion occurs in a controlled environment (e.g., following aortic aneurysm repair, organ transplantation or reattachment of severed or traumatized limbs or digits), the MASP-2 inhibitory agent may be administered prior to and/or during and/or after reperfusion. Administration may be repeated periodically, as determined by a physician, for optimal therapeutic effect.

[00187] Application of the MASP-3 inhibitory compositions and optional MASP2 inhibitory compositions of the present invention can be accomplished by a single administration of the composition (e.g., a single composition comprising MASP-2 and MASP-3 inhibitory agents or bispecific or dual inhibitory agents, or co-administration of separate compositions) or a limited sequence of administrations, for treating or preventing ischemia-reperfusion injury. Alternatively, the composition can be administered at periodic intervals, such as daily, biweekly, weekly, every other week, monthly, or bimonthly over an extended period of time for the treatment of a subject experiencing ischemic reperfusion.

[00188] As described in Examples 11-21, high affinity MASP-3 inhibitory antibodies were generated that have therapeutic utility for inhibition of the alternative pathway in AP-related diseases or conditions, such as in an individual experiencing ischemic reperfusion.

[00189] Suitably, in one embodiment, the present invention provides a method of treating a subject suffering from or at risk of developing ischemia-reperfusion comprising an effective amount of a high affinity monoclonal antibody or antigen-binding fragment thereof, as described herein, that binds to human MASP-3 and inhibits alternative pathway complement activation to treat or reduce the risk of tissue injury associated with ischemia-reperfusion in the subject. D. THE ROLE OF MASP-3 IN INFLAMMATORY AND NON-INFLAMMATORY ARTHRITIS AND THERAPEUTIC METHODS USING MASP-3 INHIBITOR ANTIBODIES, OPTIONALLY IN COMBINATION WITH MASP-2 INHIBITOR AGENTS

[00190] Rheumatoid arthritis (RA) is a chronic inflammatory disease of the synovial joints that can also have systemic manifestations. RA affects approximately 1% of the world's population, with women being two to three times more likely to be affected. Joint inflammation manifests as swelling, pain, and stiffness. As the disease progresses, joint erosion and destruction may occur, resulting in impaired range of motion and deformities. The goals of treatment in RA include preventing or controlling joint damage, preventing loss of joint function and disease progression, relieving symptoms and improving quality of life, and achieving drug-free remission. Pharmacological treatment of RA includes disease-modifying antirheumatic drugs (DMARDs), analgesics, and anti-inflammatory agents (glucocorticoids and nonsteroidal anti-inflammatory drugs). DMARDs are the most important treatment because they can induce long-lasting remissions and slow or halt the progression of joint destruction, which is irreversible. Traditional DMARDs include small molecules such as methotrexate, sulfasalazine, hydroxychloroquine, gold salts, leflunomide, D-penicillamine, cyclosporine, and azathioprine. If traditional DMARDs are inadequate to control the disease, several biologic agents that target inflammatory cells or mediators are available treatment options, such as tumor necrosis factor inhibitors (etanercept, infliximab, adalimumab, certolizumab pegol, and golimumab), cytokine antagonists (anakinra and tocilizumab), rituximab, and abatacept.

[00191] Although adaptive immunity is clearly central to the pathogenesis of RA, as evidenced by genetic association with T-cell activation genes and the presence of autoantibodies, innate immune mechanisms have also been implicated (McInnes, I. B. and Schett, G. New Engl. J. Med. 365:2205-2219, 2011). In human RA, synovial fluid levels of the alternative pathway cleavage fragment Bb were several-fold higher than samples from patients with crystal-induced arthritis or degenerative joint disease, implicating preferential activation of the alternative pathway in RA patients (Brodeur, J. P., et al., Arthritis Rheum. 34:1531-1537, 1991). In the experimental model of passive transfer of anti-collagen type II antibody arthritis, factor B-deficient mice had decreased inflammation and joint damage compared with wild-type mice, whereas C4-deficient mice had similar activity to wild-type mice, indicating the requirement for the alternative pathway rather than the classical pathway in this model (Banda, N.K. et al., J. Immunol. 177:1904-1912, 2006). In the same experimental model of collagen antibody-induced arthritis (CAIA), mice with only the classical pathway active or only the lectin pathway active were unable to develop arthritis (Banda, N.K. et al., Clin. Exp. Immunol. 159:100-108, 2010). The data from this study suggested that either the classical or lectin pathways were capable of activating low levels of C3 in vitro. However, in the absence of the alternative pathway amplification loop, the level of C3 deposition in the joint was inadequate to produce clinical disease. A key step in alternative pathway activation is the conversion of factor D zymogen (profactor D) to mature factor D, which is mediated by MASP-1 and/or MASP-3 (Takahashi, M., et al. J. Exp. Med. 207:29-37, 2010) and/or HTRA1 (Stanton et al., Evidence That the HTRA1 Interactome Influences Susceptibility to Age-Related Macular Degeneration, presented at The Association for Research in Vision and Ophthalmology 2011 conference on May 4, 2011). The role of MASP-1/3 was evaluated in murine CAIA and the results showed that MASP-1/3-deficient mice were protected against arthritis compared with wild-type mice (Banda, N.K., et al., J. Immunol. 185:5598-5606, 2010). In MASP-1/3-deficient mice, profactor D, but not mature factor D, was detected in serum during the course of CAIA and the addition of human factor D reconstituted C3 activation in vitro and generation of C5a using sera from these mice. In contrast, in a murine model of the effector phase of arthritis, C3-deficient mice developed very mild arthritis compared with wild-type mice, whereas factor B-deficient mice still developed arthritis, indicating the independent contribution of the classical/lectin and alternative pathways (Hietala, M. A. et al., Eur. J. Immunol. 34:1208-1216, 2004). In the K/BxN T cell receptor transgenic mouse model of inflammatory arthritis, mice lacking C4 or C1q developed arthritis similar to wild-type mice whereas mice lacking factor B developed no arthritis or had mild arthritis, demonstrating the requirement for the alternative pathway rather than the classical pathway in this model (Ji H. et al., Immunity 16:157-168, 2002). In the K/BxN model, mice lacking MBL-A were not protected from serum-induced arthritis, but because the role of MBL-C was not investigated, a potential role for the lectin pathway cannot be ruled out (Ji et al., 2002, supra).

[00192] Two research groups have independently proposed that lectin-dependent complement activation promotes inflammation in RA patients through the interaction of MBL with specific IgG glycoforms (Malhotra et al., Nat. Med. 1:237-243, 1995; Cuchacovich et al., J. Rheumatol. 23:44-51, 1996). Notably, rheumatoid conditions are associated with a marked increase in IgG glycoforms that lack galactose (referred to as IgG0 glycoforms) in the Fc region of the molecule (Rudd et al., Trends Biotechnology 22:524-30, 2004). The percentage of IgG0 glycoforms increases with disease progression in rheumatoid conditions and returns to normal when patients go into remission. In vivo, IgG0 is deposited in synovial tissue and MBL is present at increased levels in synovial fluid in individuals with RA. RA-associated agalactosyl-aggregated IgG (IgG0) can bind to MBL and thus may initiate lectin-dependent complement activation via LEA-1 and/or LEA-2. Furthermore, results of a clinical study examining MBL allelic variants in RA patients suggest that MBL may have an inflammatory-enhancing role in the disease (Garred et al., J. Rheumatol. 27:26–34, 2000). Therefore, lectin-dependent complement activation via LEA-1 and/or LEA-2 may play an important role in the pathogenesis of RA.

[00193] Complement activation also plays an important role in juvenile rheumatoid arthritis (Mollnes, T. E., et al., Arthritis Rheum. 29:1359-64, 1986). Similar to adult RA, in juvenile rheumatoid arthritis, elevated serum and synovial fluid levels of the alternative pathway complement activation product Bb compared with C4d (a marker for classical or LEA-2 activation) indicate that complement activation is mediated predominantly by LEA-1 (El Ghobarey, A. F. et al., J. Rheumatology 7:453-460, 1980; Agarwal, A., et al., Rheumatology 39:189-192, 2000).

[00194] Similarly, complement activation plays an important role in psoriatic arthritis. Patients with this condition have increased complement activation products in their circulation, and their red blood cells appear to have lower levels of the complement regulator CD59 (Triolo, Clin Exp Rheumatol., 21(2):225-8, 2003). Complement levels are associated with disease activity and have a high predictive value for determining treatment outcomes (Chimenti et al., Clin Exp Rheumatol., 30(1):23-30, 2012). Indeed, recent studies suggest that the effect of anti-TNF therapy for this condition is attributable to complement modulation (Ballanti et al., Autoimmun Rev., 10(10):617-23, 2011). Although the precise role of complement in psoriatic arthritis has not been determined, the presence of complement activation products C4d and Bb in the circulation of these patients suggests an important role in the pathogenesis. Based on the products observed, it is believed that LEA-1, and possibly also LEA-2, are responsible for the pathological complement activation in these patients.

[00195] Osteoarthritis (OA) is the most common form of arthritis, affecting more than 25 million people in the United States. OA is characterized by the breakdown and eventual loss of articular cartilage, accompanied by new bone formation and synovial proliferation, leading to pain, stiffness, loss of joint function, and disability. Joints frequently affected by OA are the hands, neck, low back, knees, and hips. The disease is progressive, and current treatments are for symptomatic pain relief and do not alter the natural course of the disease. The pathogenesis of OA is unclear, but a role for complement has been suggested. In a proteomic and transcriptomic analysis of synovial fluid from OA patients, several complement components were aberrantly expressed compared with samples from healthy individuals, including the classical (C1s and C4A) and alternative (factor B) pathways, as well as C3, C5, C7, and C9 (Wang, Q., et al., Nat. Med. 17:1674-1679, 2011). Furthermore, in a mouse model of medial meniscectomy-induced OA, C5-deficient mice had less cartilage loss, osteophyte formation, and synovitis than C5-positive mice, and treatment of wild-type mice with CR2-fH, a fusion protein that inhibits the alternative pathway, attenuated the development of OA (Wang et al., 2011 supra).

[00196] Ross River virus (RRV) and chikungunya virus (CHIKV) belong to a group of mosquito-borne viruses that can cause acute and persistent arthritis and myositis in humans. In addition to causing endemic disease, these viruses can cause epidemics involving millions of infected individuals. Arthritis is believed to be initiated by viral replication and induction of the host inflammatory response in the joint, and the complement system has been invoked as a key component in this process. Synovial fluid from humans with RRV-induced polyarthritis contains higher levels of C3a than synovial fluid from humans with OA (Morrison, T. E., et al., J. Virol. 81:5132-5143, 2007). In a mouse model of RRV infection, C3-deficient mice developed less severe arthritis compared with wild-type mice, suggesting a role for complement (Morrison et al., 2007, supra). The specific complement pathway involved was investigated, and mice with the lectin pathway knockout (MBL-A-/- and MBL-C-/-) had attenuated arthritis compared with wild-type mice. In contrast, mice with the classical pathway knockout (C1q-/-) or alternative pathway knockout (factor B-/-) developed severe arthritis, indicating that the MBL-initiated lectin pathway played an essential role in this model (Gunn, B. M., et al., PLoS Pathog. 8:e1002586, 2012). Because arthritis involves joint damage, initial joint damage caused by various etiologies can trigger a secondary wave of complement activation via LEA-2. In support of this concept, our previous work demonstrated that MASP-2 KO mice had reduced joint damage compared with wild-type mice in the collagen-induced RA model.

[00197] In view of the body of evidence detailed above, inhibitors of LEA-1 and LEA-2, alone or in combination, are expected to be therapeutically useful for the treatment of arthritis. An ideal effective treatment for arthritis may therefore comprise active pharmaceutical ingredients that, alone or in combination, block both LEA-1 and LEA-2. Combined inhibition of LEA-1 and LEA-2 may be accomplished by co-administration of a LEA-1 blocking agent and a LEA2 blocking agent. Preferably, the LEA-1 and LEA-2 inhibitory function may be encompassed in a single molecular entity, such as a bispecific antibody composed of MASP-1/3 and a specific MASP-2 binding site or a dual specificity antibody where each binding site can bind to and block either MASP-1/3 or MASP-2. In accordance with the foregoing, one aspect of the invention thus provides a method of inhibiting LEA-1-dependent complement activation to treat, prevent or reduce the severity of inflammatory or non-inflammatory arthritis, including osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis and psoriatic arthritis, by administering a composition comprising a therapeutically effective amount of a LEA-1 inhibiting agent comprising a MASP 1 inhibiting agent, a MASP 3 inhibiting agent or a combination of a MASP 1/3 inhibiting agent, in a pharmaceutical carrier to a subject suffering from or at risk of developing inflammatory or non-inflammatory arthritis. The MASP 1, MASP 3 or MASP 1/3 inhibiting agent may be administered to the subject systemically, such as by intra-arterial, intravenous, intramuscular, subcutaneous or other parenteral administration or by oral administration. Alternatively, administration may be by local administration, such as by intra-articular injection. The LEA-1 inhibitor agent may be administered periodically over a long period of time for the treatment or control of a chronic condition or may be administered as a single or repeated administration in the period before, during and/or after acute trauma or injury, including procedures performed on the joint.

[00198] In one embodiment, the method according to this aspect of the invention further comprises inhibiting LEA-2-dependent complement activation in a subject suffering from or at risk of developing inflammatory or non-inflammatory arthritis (including osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, and psoriatic arthritis), by administering a therapeutically effective amount of a MASP-2 inhibitory agent and a MASP-1, MASP-3, or MASP1/3 inhibitory agent to the subject. As detailed above, the use of a combination of pharmacological agents that individually block LEA-1 and LEA-2 is expected to provide a therapeutic outcome in the treatment or prevention of arthritis compared to LEA-1 inhibition alone. This outcome can be achieved, for example, by co-administration of an antibody that has LEA-1 blocking activity in conjunction with an antibody that has LEA-2 blocking activity. In some embodiments, the LEA-1- and LEA-2-blocking activities are combined in a single molecular entity and that entity has both LEA-1- and LEA-2-blocking activity. Such an entity may comprise or consist of a bispecific antibody, wherein one antigen-combining site specifically recognizes MASP-1 and blocks LEA-1 and the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Alternatively, such an entity may consist of a bispecific monoclonal antibody, wherein one antigen-combining site specifically recognizes MASP-3 and thereby blocks LEA-1 and the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Such an entity may ideally consist of a bispecific monoclonal antibody, wherein one antigen-combining site specifically recognizes MASP-1 and MASP-3 and thereby blocks LEA-1 while the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2.

[00199] The MASP-2 inhibitory composition may be administered to the subject in need thereof systemically, such as by intra-arterial, intravenous, intramuscular, subcutaneous or other parenteral administration or potentially by oral administration for non-peptidergic inhibitors. Alternatively, administration may be by local administration, such as by intra-articular injection. The MASP-2 inhibitory agent may be administered periodically over an extended period of time for treatment or control of a chronic condition or may be by single or repeated administration in the period before, during and/or after acute trauma or injury, including procedures performed on the joint.

[00200] Application of the MASP-3 inhibitory compositions and optional MASP2 inhibitory compositions of the present invention can be accomplished by a single administration of the composition (e.g., a single composition comprising MASP-2 and MASP-3 inhibitory agents or bispecific or dual inhibitory agents or co-administration of separate compositions) or a limited sequence of administrations, to treat, prevent, or reduce the severity of inflammatory or non-inflammatory arthritis. Alternatively, the composition can be administered at periodic intervals, such as daily, biweekly, weekly, every other week, monthly, or bimonthly over an extended period of time for the treatment of a subject suffering from inflammatory or non-inflammatory arthritis.

[00201] As described in Examples 11-21, high affinity MASP-3 inhibitory antibodies have been generated that have therapeutic utility for inhibition of the alternative pathway in PA-related diseases or conditions, such as arthritis.

[00202] Accordingly, in one embodiment, the present invention provides a method for treating a subject suffering from or at risk of developing arthritis (inflammatory or non-inflammatory arthritis) comprising administering to the subject a pharmaceutical composition comprising an effective amount of a high affinity monoclonal antibody or antigen-binding fragment thereof as described herein that binds to human MASP-3 and inhibits alternative pathway complement activation to treat or reduce the risk of arthritis in the subject, such as, for example, wherein said antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region comprising (i) VHCDR1 comprising SEQ ID NO: 84, (ii) VHCDR2 comprising SEQ ID NO: 86 or SEQ ID NO: 275, and (iii) VHCDR3 comprising SEQ ID NO: 88; and (b) a light chain variable region comprising (i) VLCDR1 comprising SEQ ID NO: 142, SEQ ID NO: 257, SEQ ID NO: 258, or SEQ ID NO: 259 (ii) VLCDR2 comprising SEQ ID NO: 144, and (iii) VLCDR3 comprising SEQ ID NO: 161. In some embodiments, the subject has arthritis selected from the group consisting of osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, ankylosing spondylitis, Behcet's disease, infection-related arthritis, and psoriatic arthritis. In some embodiments, the pharmaceutical composition is administered systemically (i.e., subcutaneously, intramuscularly, intravenously, intraarterially, or as an inhalant). In some embodiments, the pharmaceutical composition is administered locally to a joint. E. THE ROLE OF MASP-3 IN DISSEMINATE INTRAVASCULAR COAGULATION (DIC) AND THERAPEUTIC METHODS USING MASP-3 INHIBITOR ANTIBODIES, OPTIONALLY IN COMBINATION WITH MASP-2 INHIBITOR AGENTS

[00203] Disseminated intravascular coagulation (DIC) is a syndrome of pathological overstimulation of the coagulation system that may manifest clinically as hemorrhage and/or thrombosis. DIC does not occur as a primary condition, but rather in association with a variety of pathological processes, including tissue damage (trauma, burns, heat stroke, transfusion reaction, acute transplant rejection), neoplasia, infections, obstetric conditions (placenta praevia, amniotic fluid embolism, toxemia of pregnancy), and various diseases such as cardiogenic shock, near-drowning due to fluid aspiration, fat embolism, aortic aneurysm. Thrombocytopenia is a common abnormality in intensive care unit patients, with an incidence of 35% to 44%; DIC is the etiology in approximately 25% of these cases, i.e., it occurs in approximately 10% of critically ill patients (Levi, M. and Opal, S.M. Crit. Care 10:222-231, 2006). The pathophysiology of DIC is that the underlying disease process initiates a physiological decoagulation response. However, prothrombotic substances overwhelm the normal counterbalancing mechanisms, such that there is inappropriate deposition of fibrin and platelets in the microcirculation, leading to organ ischemia, hypofibrinogenemia, and thrombocytopenia. The diagnosis of DIC is based on the clinical presentation of the appropriate underlying disease or process, along with abnormalities in laboratory parameters (prothrombin time, partial thromboplastin time, fibrin degradation products, D-dimer, or platelet count). The primary treatment of DIC is to address the underlying condition that is the responsible trigger. Blood product support in the form of red blood cells, platelets, fresh frozen plasma, and cryoprecipitate may be necessary to treat or prevent clinical complications.

[00204] The role of complement pathways in DIC has been investigated in several studies. Complement activation was evaluated in pediatric patients with meningococcal infection, comparing the clinical course in relation to MBL genotype (Sprong, T. et al., Clin. Infect. Dis. 49:13801386, 2009). At hospital admission, MBL-deficient patients had lower circulating levels of C3bc, terminal complement complex, C4bc, and C3bBbP than MBL-sufficient patients, indicating a lower extent of common complement, terminal complement, and alternative pathway activation. Furthermore, the extent of systemic complement activation correlated with disease severity and DIC parameters, and MBL-deficient patients had a milder clinical course than MBL-sufficient patients. Therefore, although MBL deficiency is a risk factor for susceptibility to infections, MBL deficiency during septic shock may be associated with reduced disease severity.

[00205] As demonstrated in Examples 1-4, experimental studies have highlighted the important contribution of MBL and MASP-1/3 in the innate immune response to Neisseria menigitidis, the etiological agent of meningococcal infection. MBL-deficient sera from mice or humans, MASP-3-deficient human serum, or MASP-1/3 knockout mice are less effective in activating complement and lysing meningococci in vitro compared to wild-type sera. Similarly, naïve MASP-1/3 knockout mice are more susceptible to neisserial infection than their wild-type counterparts. Thus, in the absence of adaptive immunity, the LEA-1 pathway contributes to host innate resistance to neisserial infection. Conversely, LEA-1 enhances pathological complement activation, triggering a detrimental host response, including DIC.

[00206] In a murine model of arterial thrombosis, MBL-null MASP-1/-3 knockout mice displayed decreased FeCl3-induced thrombogenesis compared with wild-type or C2/factor B null mice, and the defect was reconstituted with recombinant human MBL (La Bonte, L. R., et al., J. Immunol. 188:885-891, 2012). In vitro, sera from MBL-null MASP-1/-3 knockout mice displayed decreased thrombin substrate cleavage compared with sera from wild-type or C2/factor B null mice; addition of recombinant human MASP-1 restored thrombin substrate cleavage to sera from MASP-1/-3 knockout mice (La Bonte et al., 2012, supra). These results indicate that MBL/MASP complexes, in particular MASP-1, play a key role in thrombus formation. Thus, LEA-1 may play an important role in pathological thrombosis, including DIC.

[00207] Experimental studies have established an equally important role for LEA-2 in pathological thrombosis. In vitro studies further demonstrate that LEA-2 provides a molecular link between the complement system and the coagulation system. MASP-2 has factor Xa-like activity and activates prothrombin through cleavage to form thrombin, which can subsequently release fibrinogen and promote fibrin clot formation (see also Krarup et al., PLoS One, 18:2(7):e623, 2007).

[00208] Separate studies have shown that lectin-MASP complexes can promote clot formation, fibrin deposition, and fibrinopeptide release in a MASP-2-dependent process (Gulla et al., Immunology, 129(4):482-95, 2010). Thus, LEA-2 promotes the simultaneous activation of the lectin-dependent complement and coagulation systems.

[00209] In vitro studies have also shown that MASP-1 has thrombin-like activity (Presanis J.S., et al., Mol Immunol, 40(13):921-9, 2004) and cleaves fibrinogen and factor XIII (Gulla K. C. et la., Immunology, 129(4):482-95, 2010), suggesting that LEA-1 may activate coagulation pathways independently or in conjunction with LEA-2.

[00210] The data detailed above suggest that LEA-1 and LEA-2 provide independent links between lectin-dependent complement activation and coagulation. Thus, in light of the foregoing, inhibitors of LEA-1 and LEA-2 are expected to have independent therapeutic benefits in the treatment of a subject suffering from disseminated intravascular coagulation. In some embodiments, the subject is suffering from disseminated intravascular coagulation secondary to sepsis, trauma, infection (bacterial, viral, fungal, parasitic), malignancy, transplant rejection, transfusion reaction, obstetric complications, vascular aneurysm, liver failure, heat stroke, burn, radiation exposure, shock, or severe toxic reaction (e.g., snake bite, insect bite, transfusion reaction). In some embodiments, the trauma is neurological trauma. In some embodiments, the infection is a bacterial infection, such as a Neisseria meningitidis infection.

[00211] Furthermore, LEA-1 and LEA-2 inhibitors used together may achieve additional treatment benefits compared to either agent alone. Since both LEA-1 and LEA-2 are known to be activated by conditions that lead to DIC (e.g., infection or trauma), LEA-1 and LEA-2 blocking agents, separately or in combination, are expected to have therapeutic utility in the treatment of DIC. LEA-1 and LEA-2 blocking agents may prevent different mechanisms of cross-activation between complement and coagulation. LEA-1- and LEA-2 blocking agents may thus have complementary, additive, or synergistic effects in the prevention of DIC and other thrombotic disorders.

[00212] Furthermore, LEA-1 and LEA-2 inhibitors used together may achieve additional treatment benefit compared to either agent alone or may provide effective treatment for a broader spectrum of patient subsets. Combined inhibition of LEA-1 and LEA-2 may be accomplished by co-administration of a LEA-1 blocking agent and a LEA-2 blocking agent. Ideally, the inhibitory function of LEA-1 and LEA-2 may be encompassed in a single molecular entity, such as a bispecific antibody composed of MASP-1/3 and a specific MASP-2 binding site or a dual specificity antibody where each binding site binds to and blocks either MASP-1/3 or MASP-2.

[00213] In accordance with the foregoing, one aspect of the invention thus provides a method of inhibiting LEA-1 dependent complement activation to treat, prevent or reduce the severity of disseminated intravascular coagulation in a subject in need thereof comprising administering a composition comprising a therapeutically effective amount of a LEA-1 inhibiting agent comprising a MASP-1 inhibiting agent, a MASP-3 inhibiting agent or a combination of a MASP-1/3 inhibiting agent, in a pharmaceutical carrier to a subject having, or at risk of developing, disseminated intravascular coagulation. The MASP-1, MASP-3 or MASP-1/3 inhibiting composition may be administered to the subject systemically, such as by intra-arterial, intravenous, intramuscular, inhaled, nasal, subcutaneous or other parenteral administration or potentially by oral administration for non-peptidergic agents. Administration may be repeated as determined by a physician until the condition has resolved or is controlled. For the treatment or prevention of DIC secondary to trauma or other acute event, the LEA-1 inhibitory composition can be administered immediately following the traumatic injury or prophylactically before, during, immediately after, or within one to seven days or more, such as within 24 hours to 72 hours, following trauma-inducing injury or situations such as surgery in patients considered to be at risk for DIC. In some embodiments, the LEA-1 inhibitory composition can suitably be administered in a fast-acting dosage form, such as by intravenous or intra-arterial administration of a bolus of a solution containing the LEA-1 inhibitory agent composition.

[00214] In one embodiment, the method according to this aspect of the invention further comprises inhibiting LEA-2-dependent complement activation to treat, prevent, or reduce the severity of disseminated intravascular coagulation in a subject in need thereof, comprising administering a therapeutically effective amount of a MASP-2 inhibitory agent and a MASP-1, MASP-3, or MASP-1/3 inhibitory agent to the subject. As detailed above, the use of a combination of pharmacological agents that individually block LEA-1 and LEA-2 is expected to provide a therapeutic outcome in the treatment or prevention of disseminated intravascular coagulation compared to inhibition of LEA-1 alone. This outcome may be achieved, for example, by co-administration of an antibody that has LEA-1 blocking activity in conjunction with an antibody that has LEA-2 blocking activity. In some embodiments, the LEA-1- and LEA-2-blocking activities are combined in a single molecular entity and that entity has both LEA-1- and LEA-2-blocking activity. Such an entity may comprise or consist of a bispecific antibody, wherein one antigen-combining site specifically recognizes MASP-1 and blocks LEA-1 and the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Alternatively, such an entity may consist of a bispecific monoclonal antibody, wherein one antigen-combining site specifically recognizes MASP-3 and thereby blocks LEA-1 and the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Such an entity may ideally consist of a bispecific monoclonal antibody, wherein one antigen-combining site specifically recognizes MASP-1 and MASP-3 and thereby blocks LEA-1 while the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2.

[00215] The MASP-2 inhibitory agent can be administered to the subject in need thereof systemically, such as by intra-arterial, intravenous, intramuscular, inhaled, nasal, subcutaneous or other parenteral administration or potentially by oral administration for non-peptidergic agents. Administration can be repeated as determined by a physician until the condition has resolved or is controlled. For DIC secondary to trauma or other acute event, the MASP-2 inhibitory composition can be administered immediately following the traumatic injury or prophylactically before, during, immediately after or within one to seven days or more, such as within 24 hours to 72 hours, following trauma-inducing injury or situations such as surgery in patients considered to be at risk for DIC. In some embodiments, the MASP-2 inhibitory composition can suitably be administered in a rapid-acting dosage form, such as by intravenous or intra-arterial administration of a bolus of a solution containing the MASP-2 inhibitory agent composition.

[00216] Application of the MASP-3 inhibitory compositions and optional MASP2 inhibitory compositions of the present invention can be accomplished by a single administration of the composition (e.g., a single composition comprising MASP-2 and MASP-3 inhibitory agents or bispecific or dual inhibitory agents or co-administration of separate compositions) or a limited sequence of administrations, to treat, prevent, or reduce the severity of disseminated intravascular coagulation in a subject in need thereof. Alternatively, the composition can be administered at periodic intervals, such as daily, biweekly, weekly, every other week, monthly, or bimonthly over an extended period of time for the treatment of a subject having, or at risk of developing, disseminated intravascular coagulation.

[00217] As described in Examples 11-21, high affinity MASP-3 inhibitory antibodies have been generated that have therapeutic utility for inhibition of the alternative pathway in AP-related diseases or conditions, such as disseminated intravascular coagulation.

[00218] Accordingly, in one embodiment, the present invention provides a method for treating a subject suffering from or at risk of developing disseminated intravascular coagulation comprising an effective amount of a high affinity monoclonal antibody or antigen-binding fragment thereof as described herein that binds to human MASP-3 and inhibits alternative pathway complement activation to treat or reduce the risk of developing disseminated intravascular coagulation, such as, for example, wherein said antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region comprising (i) VHCDR1 comprising SEQ ID NO: 84, (ii) VHCDR2 comprising SEQ ID NO: 86 or SEQ ID NO: 275, and (iii) VHCDR3 comprising SEQ ID NO: 88; and (b) a light chain variable region comprising (i) VLCDR1 comprising SEQ ID NO: 142, SEQ ID NO: 257, SEQ ID NO: 258 or SEQ ID NO: 259 (ii) VLCDR2 comprising SEQ ID NO: 144 and (iii) VLCDR3 comprising SEQ ID NO: 161. F. THE ROLE OF MASP-3 IN THROMBOTIC MICROANGIOPATHY (TMA), INCLUDING HEMOLYTIC UREMIC SYNDROME (HUS), ATYPICAL HEMOLYTIC UREMIC SYNDROME (aHUS), AND THROMBOTIC THROMBOCYTOPENIC PURPURA (TTP), AND THERAPEUTIC METHODS USING MASP-3 INHIBITOR ANTIBODIES, OPTIONALLY IN COMBINATION WITH MASP-2 INHIBITOR AGENTS

[00219] Thrombotic microangiopathy (TMA) refers to a group of disorders characterized clinically by thrombocytopenia, microangiopathic hemolytic anemia, and variable organ ischemia. The characteristic pathological features of TMA are platelet activation and microthrombus formation in small arterioles and venules. The classic TMAs are hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). HUS is distinguished from TTP by the presence of acute renal failure. HUS occurs in two forms: diarrhea-associated (D+) or typical HUS and diarrhea-negative (D-) or atypical HUS (aHUS).

SHU

[00220] HUS+ is associated with a prodromal diarrheal illness usually caused by Escherichia coli O157 or another strain of Shiga toxin-producing bacteria, which accounts for more than 90% of HUS cases in children and is the most common cause of acute renal failure in children. Although human infection with Escherichia coli O157 is relatively common, the percentages of bloody diarrhea that progress to HUS+ have ranged from 3% to 7% in sporadic cases and 20% to 30% in some outbreaks (Zheng, X.L. and Sadler, J.E., Annu. Rev. Pathol. 3:249-277, 2008). HUS usually occurs 4 to 6 days after the onset of diarrhea, and approximately two-thirds of children require dialysis in the acute phase of the illness. Treatment of HUS+ is favorable, since no specific treatment has been shown to be effective. The prognosis for SHUD+ is favorable, with most patients recovering renal function.

[00221] The pathogenesis of SHUD+ involves bacterially produced Shiga toxins that bind to membranes on microvascular endothelial cells, monocytes, and platelets. The renal microvasculature is most commonly affected. After binding, the toxin is internalized, leading to the release of proinflammatory mediators and ultimately cell death. Endothelial cell damage is thought to trigger renal microvascular thrombosis by promoting activation of the coagulation cascade. There is evidence of complement activation in SHUD+. In children with SHUD+, plasma levels of Bb and SC5b-9 were increased at the time of hospitalization compared with normal controls, and by day 28 after hospital discharge, plasma levels had normalized (Thurman, J. M. et al., Clin J. Am. Soc. Nephrol. 4:1920-1924, 2009). Shiga toxin 2 (Stx2) has been shown to activate human complement in the fluid phase in vitro, predominantly through the alternative pathway as activation occurs in the presence of ethylene glycol tetraacetic acid which blocks the classical pathway (Orth, D. et al., J. Immunol. 182:6394-6400, 2009). Furthermore, Stx2 bound factor H but not factor I and delayed factor H cofactor activity on cell surfaces (Orth et al., 2009, supra). These results suggest that Shiga toxin may cause renal damage through multiple potential mechanisms, including a direct toxic effect and indirectly through complement activation or inhibition of complement regulators. Toxic effects on vascular endothelium are expected to activate complement via LEA-2, as evidenced by the efficacy of MASP-2 blockade in preventing complement-mediated reperfusion injury in multiple vascular beds as described in Schwaeble, W. J., et al., Proc. Natl. Acad. Sci. 108:7523-7528, 2011.

[00222] In a murine model of HUS induced by coinjection of Shiga toxin and lipopolysaccharide, factor B-deficient mice displayed reduced thrombocytopenia and were protected from renal failure compared with wild-type mice, suggesting LEA-1-dependent activation of the alternative pathway in microvascular thrombosis (Morigi, M. et al., J. Immunol. 187:172-180, 2011). As described herein, in the same model, administration of MASP-2 antibody was also effective and increased survival after STX challenge, implicating the LEA-2-dependent complement pathway in microvascular thrombosis.

[00223] Based on the foregoing, LEA-1 and LEA-2 inhibitors are expected to have independent therapeutic benefits in the treatment or prevention of HUS. Furthermore, LEA-1 and LEA-2 inhibitors used together may achieve additional treatment benefit compared to either agent alone or may provide effective treatment for a broader spectrum of patient subsets. Combined inhibition of LEA-1 and LEA-2 may be accomplished by co-administration of a LEA-1 blocking agent and a LEA-2 blocking agent. Ideally, the inhibitory function of LEA-1 and LEA-2 may be encompassed in a single molecular entity, such as a bispecific antibody composed of MASP-1/3 and a specific MASP-2 binding site or a dual-specific antibody where each binding site can bind to and block either MASP-1/3 or MASP-2.

HUS

[00224] Atypical HUS is a rare disease, with an estimated incidence of 2 per million in the United States (Loirat, C. and Fremeaux-Bacchi, V. Orphanet J. Rare Dis. 6:60-90, 2011). Atypical HUS can develop at any age, although most patients present with onset during childhood. Atypical HUS is heterogeneous: some cases are familial, others are recurrent, and some are triggered by an infectious disease, usually of the upper respiratory tract or gastroenteritis. The onset of aHUS is usually sudden, and most patients require dialysis with hospitalization. Extra renal manifestations are present in approximately 20% of patients and may involve the central nervous system, myocardial infarction, distal ischemic gangrene, or multiple organ failure. Treatment of aHUS includes supportive care for organ dysfunction, plasma infusion or plasma exchange, and eculizumab, a humanized monoclonal antibody targeting C5 that was recently approved for use in the United States and the European Union. The prognosis in aHUS is not as good as in UHS+, with approximately 25% mortality during the acute stage, and most survivors develop end-stage renal disease.

[00225] Atypical HUS has been characterized as a complement dysregulation disease in which approximately 50% of patients have mutations in genes encoding complement regulatory proteins (Zheng and Sadler, 2008 supra ). The majority of mutations are seen in factor H (FH); other mutations include membrane cofactor protein (MCP), factor I (FI), factor B, and C3. Functional studies have shown that mutations in FH, MCP, and FI lead to loss of function and thus increased complement activation, whereas mutations in factor B are gain of function. The effects of these mutations predominantly affect the alternative pathway. These genetic abnormalities are risk factors and not the sole cause of the disease, as approximately 50% of family members carrying the mutation do not have the disease by age 45 (Loirat and Fremeaux-Bacchi, 2011 supra ).

[00226] Factor H is a complement control protein that protects host tissue from attack by alternative pathway complement. FH regulates the alternative pathway amplification loop in three ways: it is a cofactor for IF, which cleaves C3b, it inhibits the formation of the alternative pathway C3 convertase, C3bBb, and it binds to polyanions on cell surfaces and tissue matrices and blocks C3b deposition (Atkinson, JP and Goodship, T.H.J., J. Exp. Med. 6:1245-1248, 2007). The majority of FH mutations in aHUS patients occur in the short C-terminal consensus repeat domains of the protein, which result in defective binding of FH to heparin, C3b, and endothelium, but do not alter the plasma C3 regulation that resides between the N-terminal domains (Pickering, MC et al., J. Exp. Med. 204:1249-1256, 2007). FH-deficient mice have uncontrolled plasma C3 activation and spontaneously develop type II membranoproliferative glomerulonephritis but not aHUS. However, FH-deficient mice transgenically expressing a rat FH protein functionally equivalent to aHUS-associated human FH mutants spontaneously develop HUS but not type II membranoproliferative glomerulonephritis, providing in vivo evidence that defective control of alternative pathway activation in the renal endothelium is a key event in the pathogenesis of HF-associated aHUS (Pickering et al., 2007 supra). Another form of HF-associated HUS occurs in patients who have anti-HF autoantibodies, resulting in loss of functional HF activity; most of these patients have deletions in genes encoding five FH-related proteins (Loirat and Fremeaux-Bacchi, 2011, supra).

[00227] Similar to FH, MCP inhibits complement activation by regulating C3b deposition on target cells. MCP mutations result in proteins with low C3b binding and cofactor activity, thus allowing dysregulated alternative pathway activation. FI is a serine protease that cleaves C3b and C4b in the presence of cofactors such as FH and MCP and thus prevents the formation of C3 and C5 convertases and inhibits both the alternative and classical complement pathways. Most FI-associated aHUS mutations result in reduced FI activity for C3b and C4b degradation (Zheng and Stadler, 2008, supra). FB is a zymogen that carries the catalytic sites of the alternative pathway C3bBb convertase. Functional analysis has shown that FB mutations associated with aHUS result in increased activation of the alternative pathway (Loirat and Fremeaux-Bacchi, 2011, supra). Heterozygous mutations in C3 are associated with aHUS. Most C3 mutations induce a defect of C3 to bind to MCP, leading to an increased ability of FB to bind C3b and increased formation of C3 convertase (Loirat and Fremeaux-Bacchi, 2011, supra). Thus, aHUS is a disease closely associated with mutations in complement genes that lead to inadequate control of the amplification loop of the alternative pathway. Because the amplification loop of the alternative pathway is dependent on the proteolytic activity of factor B and because LEA-1 is required for factor B activation (either by MASP-3-dependent cleavage or by factor D-mediated cleavage, in which MASP-1 contributes to factor D maturation), LEA-1 blocking agents are expected to prevent uncontrolled complement activation in susceptible individuals. As a result, LEA-1 blocking agents are expected to effectively treat aHUS.

[00228] Although the central role of a dysregulated alternative pathway amplification loop in aHUS is widely accepted, the activators that initiate complement activation and the molecular pathways involved are unresolved. Not all individuals carrying the above-described mutations develop aHUS. Indeed, family studies have suggested that the penetrance of aHUS is only ~50% (Sullivan M. et al., Ann Hum Genet 74: 17-26 2010). The natural history of the disease suggests that aHUS usually develops after an initiating event, such as an infectious episode or injury. Infectious agents are well known to activate the complement system. In the absence of pre-existing adaptive immunity, complement activation by infectious agents may be primarily initiated via LEA-1 or LEA-2. Thus, lectin-dependent complement activation triggered by an infection may represent the initial activator for subsequent pathological amplification of complement activation in individuals predisposed to aHUS, which may lead to disease progression. Accordingly, another aspect of the present invention comprises treating a patient suffering from aHUS secondary to an infection by administering an effective amount of a LEA-1 or LEA-2 inhibitory agent.

[00229] Other forms of host tissue injury will activate complement via LEA-2, in particular injury to the vascular endothelium. Human vascular endothelial cells subjected to oxidative stress, for example, respond by expressing surface moieties that bind lectins and activate the LEA-2 pathway of complement (Couve et al., Am J Pathol. 156(5):1549-1556, 2000). Vascular injury following ischemia/reperfusion also activates complement via LEA-2 in vivo (Moller-Kristensen et al., Scand J Immunol. 61(5):426-34, 2005). Activation of the lectin pathway in this setting has pathological consequences for the host, and as shown in Examples 22 and 23, inhibition of LEA-2 by blocking MASP-2 further prevents host tissue injury and adverse outcomes (see also Schwaeble PNAS, 2011, supra).

[00230] Thus, other processes that cause aHUS are also known to activate LEA-1 or LEA-2. Therefore, it is likely that the LEA-1 and/or LEA-2 pathway may represent the initial mechanism of complement activation that is inappropriately amplified in a dysregulated manner in individuals genetically predisposed to aHUS, thereby initiating the pathogenesis of aHUS. By inference, agents that block complement activation via LEA-1 and/or LEA-2 would be expected to prevent disease progression or reduce exacerbations in individuals susceptible to aHUS.

[00231] In further support of this concept, recent studies have identified Streptococcus pneumoniae as an important etiologic agent in pediatric cases of aHUS. (Lee, C.S. et al, Nephrology, 17(1):48-52 (2012); Banerjee R. et al., Pediatr Infect Dis J., 30(9):736-9 (2011)). This particular etiology appears to have a poor prognosis, with significant mortality and long-term morbidity. Notably, these cases involved nonenteric infections leading to manifestations of microangiopathy, uremia, and hemolysis, with no evidence of concurrent mutations in complement genes known to predispose to aHUS. Importantly, S. pneumoniae is particularly effective at activating complement and does so predominantly through LEA-2. Thus, in cases of non-enteric HUS associated with pneumococcal infection, the manifestations of microangiopathy, uremia, and hemolysis are expected to be predominantly caused by LEA-2 activation, and agents that block LEA-2, including MASP-2 antibodies, are expected to prevent the progression of aHUS or reduce the severity of the disease in such patients. Accordingly, another aspect of the present invention comprises the treatment of a patient suffering from non-enteric aHUS that is associated with S. pneumoniae infection by administering an effective amount of a MASP-2 inhibiting agent.

PTT

[00232] Thrombotic thrombocytopenic purpura (TTP) is a potentially fatal disorder of the blood clotting system caused by autoimmune or inherited dysfunctions that activate the coagulation cascade or complement system (George, JN, N Engl J Med; 354: 1927-35, 2006). This results in numerous microscopic clots, or thromboses, in small blood vessels throughout the body, which is a hallmark of TMAs. Red blood cells are subjected to shear stress, which damages their membranes, leading to intravascular hemolysis. The resulting reduced blood flow and endothelial injury results in damage to organs, including the brain, heart, and kidneys. TTP is characterized clinically by thrombocytopenia, microangiopathic hemolytic anemia, neurological changes, renal failure, and fever. In the pre-plasma exchange era, the fatality rate was 90% during acute episodes. Even with plasma exchange, survival at six months is about 80%.

[00233] TTP can arise from genetic or acquired inhibition of the enzyme ADAMTS-13, a metalloprotease responsible for the cleavage of large multimers of von Willebrand factor (vWF) into smaller units. Inhibition or deficiency of ADAMTS-13 ultimately results in increased coagulation (Tsai, H. J Am Soc Nephrol 14: 1072-1081, 2003). ADAMTS-13 regulates vWF activity; in the absence of ADAMTS-13, vWF forms large multimers that are more likely to bind to platelets and predisposes patients to platelet aggregation and thrombosis in the microvasculature.

[00234] Numerous mutations in ADAMTS13 have been identified in individuals with TTP. The disease can also develop due to autoantibodies against ADAMTS-13. Additionally, TTP can develop during breast, gastrointestinal, or prostate cancer (George JN., Oncology (Williston Park). 25:908-14, 2011), pregnancy (second trimester or postpartum), (George JN., Curr Opin Hematol 10:339-344, 2003), or is associated with diseases such as HIV or autoimmune diseases such as systemic lupus erythematosus (Hamasaki K, et al., Clin Rheumatol. 22:355-8, 2003). TTP can also be caused by certain drug therapies including heparin, quinine, immune-mediated inhibitors, cancer chemotherapeutic agents (bleomycin, cisplatin, cytosine arabinoside, daunomycin gemcitabine, mitomycin C, and tamoxifen), cyclosporine A, oral contraceptives, penicillin, rifampin, and antiplatelet drugs including ticlopidine and clopidogrel (Azarm, T. et al., J Res Med Sci., 16: 353357, 2011). Other factors or conditions associated with TTP are toxins such as bee venom, sepsis, splenic sequestration, transplantation, vasculitis, vascular surgery, and infections such as Streptococcus pneumoniae and cytomegalovirus (Moake JL., N Engl J Med., 347: 589–600, 2002). TTP due to transient functional deficiency of ADAMTS-13 may occur as a consequence of endothelial cell injury associated with S. pneumoniae infection (Pediatr Nephrol, 26: 631-5, 2011).

[00235] Plasma exchange is the standard treatment for TTP (Rock GA, et al., N Engl J Med 325:393-397, 1991). Plasma exchange replaces ADAMTS-13 activity in patients with genetic defects and removes ADAMTS-13 autoantibodies in those patients with acquired autoimmune TTP (Tsai, H-M, Hematol Oncol Clin North Am., 21(4):609-v, 2007). Additional agents, such as immunosuppressive drugs, are routinely added to therapy (George, JN, N Engl J Med, 354:1927-35, 2006). However, plasma exchange is unsuccessful in about 20% of patients, relapse occurs in more than one-third of patients, and plasmapheresis is expensive and technically complex. Furthermore, many patients are unable to tolerate plasma exchange. Consequently, there is still an essential need for additional and improved treatments for TTP.

[00236] Since TTP is a disorder of the blood coagulation cascade, treatment with complement antagonists may help stabilize and correct the disease. Although pathological activation of the alternative complement pathway is linked to aHUS, the role of complement activation in TTP is less clear. Functional deficiency of ADAMTS13 is important for susceptibility to TTP, but is not sufficient to cause acute episodes. Environmental factors and/or other genetic variations may contribute to the manifestation of TTP. For example, genes encoding proteins involved in the regulation of the coagulation cascade, vWF, platelet function, components of the endothelial vessel surface or the complement system may be involved in the development of acute thrombotic microangiopathy (Galbusera, M. et al., Haematologica, 94: 166-170, 2009). In particular, complement activation has been shown to play a critical role; ADAMTS-13 deficiency-associated thrombotic microangiopathy serum has been shown to cause C3 and MAC deposition and subsequent neutrophil activation that can be abrogated by complement abrogation (Ruiz-Torres MP et al., Thromb Haemost, 93: 443-52, 2005). Furthermore, it has recently been shown that during acute episodes of TTP, there are increased levels of C4d, C3bBbP, and C3a (M. Réti et al., J Thromb Haemost. 10(5):791-798, 2012), consistent with activation of the classical, lectin, and alternative pathways. This increased amount of complement activation in acute episodes may initiate activation of the terminal pathway and may be responsible for further exacerbation of TTP.

[00237] The role of ADAMTS-13 and vWF in TTP is clearly responsible for platelet activation and aggregation and their subsequent role in shear stress and deposition in microangiopathies. Activated platelets interact and trigger both the classical and alternative complement pathways. Platelet-mediated complement activation increases the inflammatory mediators C3a and C5a (Peerschke E. et al., Mol Immunol., 47: 2170-5 (2010)). Platelets may thus serve as targets of classical complement activation in hereditary or autoimmune TTP.

[00238] As described above, lectin-dependent complement activation, by virtue of the thrombin-like activity of MASP-1 and LEA-2-mediated activation of prothrombin, is the dominant molecular pathway linking endothelial injury to coagulation and the microvascular thrombosis that occurs in HUS. Similarly, activation of LEA-1 and LEA-2 may directly trigger the coagulation system in TTP. Activation of the LEA-1 and LEA-2 pathway may be initiated in response to the initial endothelial injury caused by ADAMTS-13 deficiency in TTP. Inhibitors of LEA-1 and LEA-2, including but not limited to antibodies that block MASP-2 function, MASP-1 function, MASP-3 function, or both MASP-1 and MASP-3 function, are therefore expected to mitigate microangiopathies associated with microvascular coagulation, thrombosis, and hemolysis in patients suffering from TTP.

[00239] Patients suffering from TTP typically present to the emergency room with one or more of the following symptoms: purpura, renal failure, low platelets, anemia, and/or thrombosis, including stroke. The current standard of care for TTP involves intracatheter (e.g., intravenous or other catheter form) administration of plasma exchange for a period of two weeks or longer, typically three times per week but up to daily. If the individual tests positive for the presence of an ADAMTS13 inhibitor (i.e., an endogenous antibody against ADAMTS13), then plasma exchange may be performed in combination with immunosuppressive therapy (e.g., corticosteroids, rituxan, or cyclosporine). Individuals with refractory TTP (approximately 20% of patients with TTP) do not respond to at least two weeks of plasma exchange therapy.

[00240] In accordance with the foregoing, in one embodiment, in the setting of an initial diagnosis of TTP or in a subject exhibiting one or more symptoms consistent with a diagnosis of TTP (e.g., central nervous system involvement, severe thrombocytopenia (a platelet count less than or equal to 5000/μL if not receiving aspirin, less than or equal to 20,000/μL if receiving aspirin), severe cardiac involvement, severe pulmonary involvement, gastrointestinal infarction, or gangrene), a method is provided for treating the subject with an effective amount of a LEA-2 inhibitory agent (e.g., a MASP-2 antibody) or a LEA-1 inhibitory agent (e.g., a MASP-1 or MASP-3 antibody) as a first-line therapy in the absence of plasmapheresis or in combination with plasmapheresis. As first-line therapy, the LEA-1 and/or LEA-2 inhibiting agent can be administered to the subject systemically, such as by intra-arterial, intravenous, intramuscular, inhaled, nasal, subcutaneous, or other parenteral administration. In some embodiments, the LEA-1 and/or LEA-2 inhibiting agent is administered to a subject as a first-line therapy in the absence of plasmapheresis to avoid the potential complications of plasmapheresis, such as hemorrhage, infection, and exposure to disorders and/or allergies inherent to the plasma donor or in a subject averse to plasmapheresis or in a setting where plasmapheresis is not available. In some embodiments, the LEA-1 and/or LEA-2 inhibiting agent is administered to the subject suffering from TTP in combination (including co-administration) with an immunosuppressive agent (e.g., corticosteroids, rituxan, or cyclosporine) and/or in combination with ADAMTS-13 concentrate.

[00241] In some embodiments, the method comprises administering a LEA-1 and/or LEA-2 inhibiting agent to a subject suffering from TTP via a catheter (e.g., intravenously) for a first period of time (e.g., an acute phase lasting at least one day to one week or two weeks) followed by administering a LEA-1 and/or LEA-2 inhibiting agent to the subject subcutaneously for a second period of time (e.g., a chronic phase of at least two weeks or more). In some embodiments, the administration in the first and/or second time period occurs in the absence of plasmapheresis. In some embodiments, the method is used to maintain the subject to prevent the subject from experiencing one or more symptoms associated with TTP.

[00242] In another embodiment, a method is provided for treating a subject suffering from refractory TTP (i.e., a subject who has not responded to at least two weeks of plasmapheresis therapy), by administering an amount of LEA-1 and/or LEA-2 inhibitor effective to reduce one or more symptoms of TTP. In one embodiment, the LEA-1 and/or LEA-2 inhibitor is administered to a subject with refractory TTP on a chronic basis, over a period of time of at least two weeks or more, via subcutaneous or other parenteral administration. Administration may be repeated as determined by a physician until the condition has resolved or is controlled.

[00243] In some embodiments, the method further comprises determining the level of at least one complement factor (e.g., C3, C5) in the subject prior to treatment, and optionally during treatment, wherein determining a reduced level of at least one complement factor compared to a standard value or healthy control subject is indicative of the need for continued treatment with the LEA-1 and/or LEA-2 inhibiting agent.

[00244] In some embodiments, the method comprises administering, subcutaneously or intravenously, a LEA-1 and/or LEA-2 inhibitory agent to a subject suffering from or at risk of developing TTP. Treatment is preferably daily, but may be as infrequent as monthly. Treatment is continued until the subject's platelet count is greater than 150,000/ml for at least two consecutive days.

[00245] In summary, LEA-1 and LEA-2 inhibitors are expected to have therapeutic benefits independent of TMAs, including HUS, aHUS, and TTP. Furthermore, LEA-1 and LEA-2 inhibitors used together are expected to achieve additional treatment benefit compared to either agent alone or may provide effective treatment for a broader spectrum of patient subsets suffering from variant forms of TMA. Combined inhibition of LEA-1 and LEA-2 can be accomplished by co-administration of a LEA-1 blocking agent and a LEA-2 blocking agent. Ideally, the inhibitory function of LEA-1 and LEA-2 can be encompassed in a single molecular entity, such as a bispecific antibody composed of MASP-1/3 and a specific MASP-2 binding site or a dual-specific antibody where each binding site can bind to and block either MASP-1/3 or MASP-2.

[00246] In accordance with the foregoing, one aspect of the invention thus provides a method of inhibiting LEA-1 dependent complement activation to treat, prevent or reduce the severity of thrombotic microangiopathy, such as hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS) or thrombotic thrombocytopenic purpura (TTP) comprising administering a composition comprising a therapeutically effective amount of a LEA-1 inhibiting agent comprising a MASP 1 inhibiting agent, a MASP 3 inhibiting agent or a combination of a MASP 1/3 inhibiting agent, in a pharmaceutical carrier to a subject suffering from, or at risk of developing, a thrombotic microangiopathy. The MASP 1, MASP 3 or MASP 1/3 inhibitor composition may be administered to the subject systemically, such as by intra-arterial, intravenous, intramuscular, inhaled, nasal, subcutaneous or other parenteral administration or potentially by oral administration for non-peptidergic agents. Administration may be repeated as determined by a physician until the condition has resolved or is controlled.

[00247] In one embodiment, the method according to this aspect of the invention further comprises inhibiting LEA-2-dependent complement activation to treat, prevent, or reduce the severity of thrombotic microangiopathy, such as hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), or thrombotic thrombocytopenic purpura (TTP) comprising administering a therapeutically effective amount of a MASP-2 inhibitory agent and a MASP-1, MASP 3, or MASP 1/3 inhibitory agent to a subject suffering from, or at risk of developing, a thrombotic microangiopathy. As detailed above, the use of a combination of pharmacological agents that individually block LEA-1 and LEA-2 is expected to provide an improved therapeutic outcome in treating or preventing or reducing the severity of a thrombotic microangiopathy compared to LEA-1 inhibition alone. This result can be achieved, for example, by co-administration of an antibody that has LEA-1 blocking activity in conjunction with an antibody that has LEA-2 blocking activity. In some embodiments, the LEA-1- and LEA-2 blocking activities are combined in a single molecular entity and that entity has both LEA-1- and LEA-2 blocking activity. Such an entity can comprise or consist of a bispecific antibody, wherein one antigen-combining site specifically recognizes MASP-1 and blocks LEA-1 and the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Alternatively, such an entity can consist of a bispecific monoclonal antibody, wherein one antigen-combining site specifically recognizes MASP-3 and thus blocks LEA-1 and the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Such an entity could ideally consist of a bispecific monoclonal antibody, in which one antigen-combining site specifically recognizes MASP-1 and MASP-3 and thus blocks LEA-1 while the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2.

[00248] The MASP-2 inhibitory agent may be administered to the subject systemically, such as by intra-arterial, intravenous, intramuscular, inhaled, nasal, subcutaneous, or other parenteral administration, or potentially by oral administration for non-peptidergic agents. Administration may be repeated as determined by a physician until the condition has resolved or is controlled.

[00249] Application of the MASP-3 inhibitory compositions and optional MASP2 inhibitory compositions of the present invention can be accomplished by a single administration of the composition (e.g., a single composition comprising MASP-2 and MASP-3 inhibitory agents or bispecific or dual inhibitory agents or co-administration of separate compositions) or a limited sequence of administrations, to treat, prevent, or reduce the severity of a thrombotic microangiopathy in a subject suffering from or at risk of developing a thrombotic microangiopathy. Alternatively, the composition can be administered at periodic intervals, such as daily, biweekly, weekly, every other week, monthly, or bimonthly over an extended period of time for the treatment of a subject in need thereof.

[00250] As described in Examples 11-21, high affinity MASP-3 inhibitory antibodies were generated that have therapeutic utility for inhibition of the alternative pathway in AP-related diseases or conditions, such as a thrombotic microangiopathy (e.g., hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), or thrombotic thrombocytopenic purpura (TTP).

[00251] Accordingly, in one embodiment, the present invention provides a method for treating a subject suffering from or at risk of developing a thrombotic microangiopathy (e.g., hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), or thrombotic thrombocytopenic purpura (TTP)) comprising an effective amount of a high affinity monoclonal antibody or antigen-binding fragment thereof as described herein that binds to human MASP-3 and inhibits alternative pathway complement activation to treat or reduce the risk of developing a thrombotic microangiopathy (e.g., hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), or thrombotic thrombocytopenic purpura (TTP) or transplant-related TMA (TA-TMA), such as, for example, wherein said antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region comprising (i) VHCDR1 comprising SEQ ID NO: 84, (ii) VHCDR2 comprising SEQ ID NO: 86 or SEQ ID NO: 275 and (iii) VHCDR3 comprising SEQ ID NO: 88; and (b) a light chain variable region comprising (i) VLCDR1 comprising SEQ ID NO: 142, SEQ ID NO: 257, SEQ ID NO: 258 or SEQ ID NO: 259 (ii) VLCDR2 comprising SEQ ID NO: 144 and (iii) VLCDR3 comprising SEQ ID NO: 161. G. THE ROLE OF MASP-3 IN ASTHMA AND THERAPEUTIC METHODS USING MASP-3 INHIBITOR ANTIBODIES, OPTIONALLY IN COMBINATION WITH MASP-2 INHIBITOR AGENTS

[00252] Asthma is a common chronic inflammatory disease of the airways. Approximately 25 million people in the United States have asthma, including seven million children under the age of 18, with more than half experiencing at least one asthma attack per year, leading to more than 1.7 million emergency room visits and 450,000 hospitalizations per year (world-wide-web at gov/health/prof/lung/asthma/naci/asthma-info/index.htm., accessed May 4, 2012). The disease is heterogeneous with multiple clinical phenotypes. The most common phenotype is allergic asthma. Other phenotypes include nonallergic asthma, aspirin-exacerbated respiratory disease, postinfectious asthma, occupational asthma, airborne irritant-induced asthma, and exercise-induced asthma. The hallmarks of allergic asthma include airway hyperresponsiveness (AHR) to a variety of specific and nonspecific stimuli, excessive airway mucus production, pulmonary eosinophilia, and elevated serum IgE concentrations. Symptoms of asthma include cough, wheezing, chest tightness, and shortness of breath. The goal of asthma treatment is to control the disease and minimize exacerbations, daily symptoms, and allow patients to be physically active. Current treatment guidelines recommend treatment in stages until asthma control is achieved. The first stage of treatment is, when necessary, inhaled short-acting β2-agonists, followed by the addition of controller medications such as inhaled corticosteroids, long-acting inhaled β2-agonists, leukotriene-modifying drugs, theophylline, oral glucocorticosteroids, and anti-IgE monoclonal antibodies.

[00253] Although asthma has a multifactorial origin, it is generally accepted that it arises as a result of inappropriate immune responses to common environmental antigens in genetically susceptible individuals. Asthma is associated with complement activation, and the anaphylatoxins (AT) C3a and C5a have proinflammatory and immunoregulatory properties that are relevant to the development and modulation of the allergic response (Zhang, X. and Kohl, J. Expert. Rev. Clin. Immunol., 6:269-277, 2010). However, the relative involvement of the classical, alternative, and lectin pathways of complement in asthma is not well understood. The alternative pathway can be activated on the surface of allergens, and the lectin pathway can be activated through recognition of allergenic polysaccharide structures, both processes leading to the generation of AT. Complement can be activated by different pathways depending on the causative allergen involved. Pollen from the highly allergic grass of the Parietaria family, for example, is very effective in promoting MBL-dependent C4 activation involving LEA-2. In contrast, house dust mite allergen does not require MBL for complement activation (Varga et al. Mol Immunol., 39(14):839-46, 2003).

[00254] Environmental activators of asthma may activate complement by the alternative pathway. For example, in vitro exposure of human serum to cigarette smoke or diesel exhaust particles resulted in complement activation, and the effect was unaffected by the presence of EDTA, suggesting that activation was by the alternative rather than the classical pathway (Robbins, R. A. et al., Am J. Physiol. 260:L254-L259, 1991; Kanemitsu, H., et al., Biol. Pharm. Bull. 21:129-132, 1998). The role of complement pathways in allergic airway inflammation was evaluated in a rat model of sensitization and ovalbumin challenge. Wild-type mice developed AHR and airway inflammation in response to aeroallergen challenge. A Crry-Ig fusion protein that inhibits all pathways of complement activation was effective in preventing AHR and lung inflammation when administered systemically or locally by inhalation in the rat ovalbumin model of allergic lung inflammation (Taube et al., Am J Respir Crit Care Med., 168(11):1333-41, 2003).

[00255] Compared with wild-type mice, factor B-deficient mice demonstrated less AHR and airway inflammation whereas C4-deficient mice had similar effects to wild-type mice (Taube, C., et al., Proc. Natl. Acad. Sci. USA 103:8084-8089, 2006). These results support a role for the alternative pathway rather than the involvement of the classical pathway in the murine aeroallergen challenge model. Additional evidence for the importance of the alternative pathway was provided in a study of factor H (FH) using the same mouse model (Takeda, K., et al., J. Immunol. 188:661667, 2012). HF is a negative regulator of the alternative pathway and acts to prevent autologous self-tissue injury. Endogenous HF was found to be present in the airways during allergen challenge, and inhibition of HF with a recombinant competitive antagonist increased the extent of AHR and airway inflammation (Takeda et al., 2012, supra). Therapeutic administration of CR2-fH, a chimeric protein that links the iC3b/C3d-binding region of CR2 to the complement regulatory region of FH that targets the complement regulatory activity of fH to existing complement activation sites, protected the development of AHR and eosinophil infiltration into the airways after allergen challenge (Takeda et al., 2012, supra). The protective effect was demonstrated with ovalbumin and ragweed allergen, which is a relevant allergen in humans.

[00256] The role of lectin-dependent complement activation in asthma was evaluated in a mouse model of fungal asthma (Hogaboam et al., J. Leukocyte Biol. 75:805-814, 2004). These studies utilized mice genetically deficient in mannan-binding lectin A (MBL-A), a carbohydrate-binding protein that functions as the recognition component for activation of lectin complement pathways. MBL-A(+/+) and MBL-A(-/-) mice sensitized to Aspergillus fumigatus were examined on days 4 and 28 after i.t. challenge with A. fumigatus conidia. AHR in sensitized MBL-A(-/-) mice was significantly attenuated at both time points after conidial challenge compared with the sensitized MBL-A(+/+) group. Lung TH2 cytokine (IL-4, IL-5, and IL-13) levels were significantly lower in A. fumigatus-sensitized MBL-A (-/-) mice compared with the wild-type group at day 4 post-conidia. These results indicate that MBL-A and the lectin pathway play an important role in the development and maintenance of AHR during chronic fungal asthma.

[00257] The findings detailed above suggest the involvement of lectin-dependent complement activation in the pathogenesis of asthma. Experimental data suggest that factor B activation plays a key role. In light of the key role of LEA-1 in lectin-dependent factor B activation and subsequent activation of the alternative pathway, LEA-1 blocking agents are expected to be beneficial for the treatment of certain forms of alternative pathway-mediated asthma. Such treatment may thus be particularly useful in house dust mite-induced asthma or asthma caused by environmental triggers such as cigarette smoke or diesel exhaust. Asthmatic responses triggered by grass pollen, on the other hand, are likely to invoke LEA-2-dependent complement activation. Therefore, LEA-2 blocking agents are expected to be particularly useful in the treatment of asthmatic conditions in this subset of patients.

[00258] In view of the data detailed above, the inventors believe that LEA-1 and LEA-2 mediate pathological complement activation in asthma. Depending on the triggering allergic agent, LEA-1 or LEA-2 may be preferentially engaged. Thus, a LEA-1 blocking agent combined with a LEA-2 blocking agent may have utility in the treatment of multiple forms of asthma, regardless of the underlying etiology. LEA-1 and LEA-2 blocking agents may have complementary, additive, or synergistic effects in preventing, treating, or reversing lung inflammation and asthma symptoms.

[00259] Combined inhibition of LEA-1 and LEA-2 can be accomplished by co-administration of a LEA-1 blocking agent and a LEA-2 blocking agent. Ideally, the inhibitory function of LEA-1 and LEA-2 can be encompassed in a single molecular entity, such as a bispecific antibody composed of MASP-1/3 and a specific MASP-2 binding site or a dual specificity antibody where each binding site can bind to and block either MASP-1/3 or MASP-2.

[00260] In accordance with the foregoing, one aspect of the invention thus provides a method of inhibiting LEA-1-dependent complement activation to treat, prevent, or reduce the severity of asthma, comprising administering a composition comprising a therapeutically effective amount of a LEA-1 inhibiting agent comprising a MASP-1 inhibiting agent, a MASP-3 inhibiting agent, or a combination of a MASP-1/3 inhibiting agent, in a pharmaceutical carrier to a subject suffering from, or at risk of developing, asthma. The MASP-1, MASP-3, or MASP-1/3 inhibiting composition may be administered to the subject systemically, such as by intraarterial, intravenous, intramuscular, inhalation, nasal, subcutaneous, or other parenteral administration, or potentially by oral administration for non-peptidergic agents. Administration may be repeated as determined by a physician until the condition has resolved or is controlled.

[00261] In one embodiment, the method according to this aspect of the invention further comprises inhibiting LEA-2-dependent complement activation to treat, prevent, or reduce the severity of asthma, comprising administering a therapeutically effective amount of a MASP-2 inhibitory agent and a MASP-1, MASP-3, or MASP-1/3 inhibitory agent to a subject suffering from, or at risk of developing, asthma. As detailed above, the use of a combination of pharmacological agents that individually block LEA-1 and LEA-2 is expected to provide an improved therapeutic outcome in the treatment or prevention or reduction of the severity of asthma compared to LEA-1 inhibition alone. This result can be achieved, for example, by co-administration of an antibody that has LEA-1 blocking activity in conjunction with an antibody that has LEA-2 blocking activity. In some embodiments, the LEA-1- and LEA-2-blocking activities are combined in a single molecular entity and that entity has both LEA-1- and LEA-2-blocking activity. Such an entity may comprise or consist of a bispecific antibody, wherein one antigen-combining site specifically recognizes MASP-1 and blocks LEA-1 and the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Alternatively, such an entity may consist of a bispecific monoclonal antibody, wherein one antigen-combining site specifically recognizes MASP-3 and thereby blocks LEA-1 and the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Such an entity may ideally consist of a bispecific monoclonal antibody, wherein one antigen-combining site specifically recognizes MASP-1 and MASP-3 and thereby blocks LEA-1 while the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2.

[00262] The MASP-2 inhibitory agent may be administered to the subject systemically, such as by intra-arterial, intravenous, intramuscular, inhaled, nasal, subcutaneous, or other parenteral administration, or potentially by oral administration for non-peptidergic agents. Administration may be repeated as determined by a physician until the condition has resolved or is controlled.

[00263] Application of the MASP-3 inhibitory compositions and optional MASP-2 inhibitory compositions of the present invention can be accomplished by a single administration of the composition (e.g., a single composition comprising MASP-2 and MASP-3 inhibitory agents or bispecific or dual inhibitory agents or co-administration of separate compositions) or a limited sequence of administrations, to treat, prevent, or reduce the severity of asthma in a subject suffering from, or at risk of developing, asthma. Alternatively, the composition can be administered at periodic intervals, such as daily, biweekly, weekly, every other week, monthly, or bimonthly over an extended period of time for the treatment of a subject in need thereof.

[00264] As described in Examples 11-21, high affinity MASP-3 inhibitory antibodies have been generated that have therapeutic utility for inhibition of the alternative pathway in AP-related diseases or conditions, such as asthma.

[00265] Accordingly, in one embodiment, the present invention provides a method of treating a subject suffering from or at risk of developing asthma comprising an effective amount of a high affinity monoclonal antibody or antigen-binding fragment thereof as described herein that binds to human MASP-3 and inhibits alternative pathway complement activation to treat or reduce the risk of developing asthma, such as, for example, wherein said antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region comprising (i) VHCDR1 comprising SEQ ID NO: 84, (ii) VHCDR2 comprising SEQ ID NO: 86 or SEQ ID NO: 275, and (iii) VHCDR3 comprising SEQ ID NO: 88; and (b) a light chain variable region comprising (i) VLCDR1 comprising SEQ ID NO: 142, SEQ ID NO: 257, SEQ ID NO: 258 or SEQ ID NO: 259 (ii) VLCDR2 comprising SEQ ID NO: 144 and (iii) VLCDR3 comprising SEQ ID NO: 161. H. THE ROLE OF MASP-3 IN DENSE DEPOSIT DISEASE AND THERAPEUTIC METHODS USING MASP-3 INHIBITOR ANTIBODIES, OPTIONALLY IN COMBINATION WITH MASP-2 INHIBITOR AGENTS

[00266] Membranoproliferative glomerulonephritis (MPGN) is a renal disorder characterized morphologically by mesangial cell proliferation and thickening of the glomerular capillary wall due to subendothelial extension of the mesangium. MPGN is classified as primary (also called idiopathic) or secondary, with underlying diseases such as infectious diseases, systemic immune complex diseases, neoplasms, chronic liver disease, and others. Idiopathic MPGN includes three morphological types. Type I, or classical MPGN, is characterized by subendothelial deposits of immune complexes and activation of the classical complement pathway. Type II, or dense deposit disease (DDD), is characterized by additional dense intramembranous deposits. Type III is characterized by additional subepithelial deposits. Idiopathic GPMN is rare, accounting for approximately 4% to 7% of primary renal causes of nephrotic syndrome (Alchi, B. and Jayne, D. Pediatr. Nephrol. 25:1409–1418, 2010). GPMN primarily affects children and young adults and may present as nephrotic syndrome, acute nephritic syndrome, asymptomatic proteinuria, and hematuria or recurrent gross hematuria. Renal dysfunction occurs in most patients, and the disease has a slowly progressive course, with approximately 40% of patients developing end-stage renal disease within 10 years of diagnosis (Alchi and Jayne, 2010, supra). Current treatment options include corticosteroids, immunosuppressants, antiplatelet regimens, and plasma exchange.

[00267] DDD is diagnosed by the absence of immunoglobulin and the presence of C3 by immunofluorescence staining of renal biopsies, and electron microscopy shows characteristic dense osmiophilic deposits along the glomerular basement membranes. DDD is caused by dysregulation of the alternative complement pathway (Sethi et al, Clin J Am Soc Nephrol. 6 (5):1009-17, 2011), which can arise from several different mechanisms. The most common complement abnormality in DDD is the presence of C3 nephritic factors that are autoantibodies to alternative pathway C3 convertase (C3bBb) that increase its half-life and thus activation of the pathway (Smith, R. J. H. et al., Mol. Immunol. 48:1604-1610, 2011). Other alternative pathway abnormalities include factor H autoantibody that blocks factor H function, gain of function mutations in C3, and genetic factor H deficiency (Smith et al., 2011, supra). Recent case reports show that treatment with eclizumab (anti-C5 monoclonal antibody) was associated with improvements in renal function in two patients with DDD (Daina, E. et al., New Engl. J. Med. Chem. 366:1161-1163, 2012; Vivarelli, M. et al., New Engl. J. Med. 366:1163-1165, 2012), suggesting a causal role for complement activation in renal outcomes.

[00268] Given the above genetic, functional, immunohistochemical and anecdotal clinical data, the central role of complement in the pathogenesis of DDD is well established. Thus, interventions that block the causative mechanisms of complement activation, or the subsequent products of complement activation, are expected to be therapeutically useful in treating this condition.

[00269] Although human genetic data suggest that inadequate control or excessive activation of the amplification loop of the alternative pathways plays a key role, the events that initiate complement have not been identified. Immunohistochemical studies on renal biopsies show evidence of MBL deposition in diseased tissue, suggesting involvement of lectin pathways in the initiation of pathological complement activation in DDD (Lhotta et al, Nephrol Dial Transplant., 14(4):881-6, 1999). The importance of the alternative pathway has been further corroborated in experimental models. Factor H-deficient mice develop progressive proteinuria and the pathological renal lesions characteristic of the human condition (Pickering et al., Nat Genet., 31(4):424, 2002). Pickering et al. further demonstrated that ablation of factor B, which mediates LEA-1-dependent activation of the alternative pathway, fully protects factor H-deficient mice from DDD (Pickering et al., Nat Genet., 31(4):424, 2002).

[00270] Thus, agents that block LEA-1 are expected to effectively block lectin-dependent activation of the alternative pathway and thus provide an effective treatment for DDD. Given that the amplification loop of the alternative pathway is dysregulated in patients with DDD, it can also be expected that agents that block the amplification loop will be effective. Since agents that target LEA-1 that block MASP-1 or both MASP-1 and MASP-3 inhibit factor D maturation, such agents are expected to effectively block the amplification loop of the alternative pathway.

[00271] As detailed above, pronounced MBL deposition was found in diseased renal samples, highlighting the likely involvement of lectin-driven activation events in the pathogenesis of DDD. Once initial tissue injury to the glomerular capillaries is established, further binding of MBL to the injured glomerular endothelium and underlying mesangial structures is likely to occur. Such tissue injury is known to lead to activation of LEA-2, which may then cause further complement activation. Therefore, LEA-2 blocking agents are also expected to have utility in preventing further complement activation in injured glomerular structures and thereby preventing further disease progression towards end-stage renal failure.

[00272] The data detailed above suggest that LEA-1 and LEA-2 promote separate pathological processes of complement activation in DDD. Thus, a LEA-1 blocking agent and a LEA-2 blocking agent, alone or in combination, are expected to be useful for the treatment of DDD.

[00273] When used in combination, LEA-1- and LEA-2-blocking agents are expected to be more effective than either agent alone or useful for treating different stages of the disease. LEA-1- and LEA-2-blocking agents may thus have complementary, additive, or synergistic effects in preventing, treating, or reversing renal dysfunction associated with DDD.

[00274] Combined inhibition of LEA-1 and LEA-2 can be accomplished by co-administration of a LEA-1 blocking agent and a LEA-2 blocking agent. Ideally, the LEA-1 and LEA-2 blocking agents with inhibitory function can be encompassed in a single molecular entity, such as a bispecific antibody composed of MASP-1/3 and a MASP-2-specific binding site or a dual-specific antibody where each binding site can bind to and block either MASP-1/3 or MASP-2.

[00275] In accordance with the foregoing, one aspect of the invention thus provides a method of inhibiting LEA-1-dependent complement activation to treat, prevent, or reduce the severity of dense deposit disease, comprising administering a composition comprising a therapeutically effective amount of a LEA-1 inhibitory agent comprising a MAPS 1 inhibitory agent, a MASP 3 inhibitory agent, or a combination of a MAPS 1/3 inhibitory agent, in a pharmaceutical carrier to a subject suffering from, or at risk of developing, dense deposit disease. The MASP-1, MASP-3, or MASP-1/3 inhibitory composition may be administered to the subject systemically, such as by intra-arterial, intravenous, intramuscular, inhaled, nasal, subcutaneous, or other parenteral administration, or potentially by oral administration for non-peptidergic agents. Administration may be repeated as determined by a physician until the condition has resolved or is controlled.

[00276] In another aspect, a method is provided for inhibiting LEA-2-dependent complement activation to treat, prevent, or reduce the severity of dense storage disease, comprising administering a therapeutically effective amount of a MASP-2 inhibitory agent to a subject suffering from, or at risk of developing, dense storage disease. In another aspect, a method is provided comprising inhibiting LEA-2- and LEA-1-dependent complement activation to treat, prevent, or reduce the severity of dense storage disease, comprising administering a therapeutically effective amount of a MASP-2 inhibitory agent and a MASP-1, MASP-3, or MASP-1/3 inhibitory agent to a subject suffering from, or at risk of developing, dense storage disease.

[00277] In some embodiments, the method comprises inhibiting both LEA-1-dependent complement activation and LEA-2-dependent complement activation. As detailed above, the use of a combination of pharmacological agents that individually block LEA-1 and LEA-2 is expected to provide an improved therapeutic outcome in the treatment, prevention, or reduction in severity of dense deposit disease, compared to inhibition of LEA-1 alone. This outcome can be achieved, for example, by co-administration of an antibody that has LEA-1-blocking activity in conjunction with an antibody that has LEA-2-blocking activity. In some embodiments, the LEA-1- and LEA-2-blocking activities are combined into a single molecular entity and that entity has both LEA-1- and LEA-2-blocking activity. Such an entity may comprise or consist of a bispecific antibody, wherein one antigen-combining site specifically recognizes MASP-1 and blocks LEA-1 and the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Alternatively, such an entity may consist of a bispecific monoclonal antibody, wherein one antigen-combining site specifically recognizes MASP-3 and thus blocks LEA-1 and the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Such an entity may ideally consist of a bispecific monoclonal antibody, wherein one antigen-combining site specifically recognizes MASP-1 and MASP-3 and thus blocks LEA-1 while the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2.

[00278] LEA-1 and/or LEA-2 inhibitory agents may be administered to the individual systemically, such as by intraarterial, intravenous, intramuscular, inhaled, nasal, subcutaneous, or other parenteral administration, or potentially by oral administration for nonpeptidergic agents. Administration may be repeated as determined by a physician until the condition has resolved or is controlled.

[00279] Application of the MASP-3 inhibitory compositions and/or MASP-2 inhibitory compositions of the present invention can be accomplished by a single administration of the composition (e.g., a single composition comprising MASP-2 and/or MASP-3 inhibitory agents or bispecific or dual inhibitory agents or co-administration of separate compositions) or a limited sequence of administrations, to treat, prevent, or reduce the severity of dense deposit disease in a subject in need thereof. Alternatively, the composition can be administered at periodic intervals, such as daily, biweekly, weekly, every other week, monthly, or bimonthly over an extended period of time for the treatment of a subject in need thereof.

[00280] As described in Examples 11-21, high affinity MASP-3 inhibitory antibodies have been generated that have therapeutic utility for inhibition of the alternative pathway in PA-related diseases or conditions, such as dense deposit disease.

[00281] Accordingly, in one embodiment, the present invention provides a method for treating a subject suffering from or at risk of developing dense deposit disease comprising an effective amount of a high affinity monoclonal antibody or antigen-binding fragment thereof as described herein that binds to human MASP-3 and inhibits alternative pathway complement activation to treat or reduce the risk of developing dense deposit disease, such as, for example, wherein said antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region comprising (i) VHCDR1 comprising SEQ ID NO: 84, (ii) VHCDR2 comprising SEQ ID NO: 86 or SEQ ID NO: 275, and (iii) VHCDR3 comprising SEQ ID NO: 88; and (b) a light chain variable region comprising (i) VLCDR1 comprising SEQ ID NO: 142, SEQ ID NO: 257, SEQ ID NO: 258 or SEQ ID NO: 259 (ii) VLCDR2 comprising SEQ ID NO: 144 and (iii) VLCDR3 comprising SEQ ID NO: 161. I. THE ROLE OF MASP-3 IN PAUCI-IMMUNE CRESCENT NECROTIZING GLOMERULONEPHRITIS and THERAPEUTIC METHODS USING MASP-3 INHIBITORY ANTIBODIES, OPTIONALLY IN COMBINATION WITH MASP-2 INHIBITORY AGENTS

[00282] Pauci-immune necrotizing crescentic glomerulonephritis (NCGN) is a rapidly progressive form of glomerulonephritis in which the glomerular capillary walls show signs of inflammation but lack detectable immune complex deposition or antibodies against the glomerular basement membrane. The condition is associated with a rapid decline in renal function. Most patients with NCGN have antineutrophil cytoplasmic autoantibodies (ANCA) and therefore belong to a group of diseases called ANCA-associated vasculitis. Vasculitis is a disorder of the blood vessels characterized by inflammation and fibrinoid necrosis of the vessel wall. Systemic vasculitides are classified based on vessel size: large, medium, and small. Several forms of small vessel vasculitis are associated with the presence of ANCA, notably Wegener's granulomatosis, microscopic polyangiitis, Churg-Strauss syndrome, and vasculitis limited to the kidney (NCGN). They may also be a manifestation of underlying conditions such as systemic lupus erythematosus. Target antigens for ANCA include proteinase-3 (PR3) and myeloperoxidase (MPO). Pauci-immune NCGN is rare, with a reported incidence of approximately 4 per million in Wessex, United Kingdom (Hedger, N. et al., Nephrol. Dial. Transplant. 15:1593–1599, 2000). In the Wessex series of 128 patients with pauci-immune NCGN, 73% were ANCA positive and initial dialysis was required in 59% of patients and 36% required long-term dialysis. Treatments for pauci-immune NCGN include corticosteroids and immunosuppressive agents such as cyclophosphamide and azathioprine. Other treatment options for ANCA-associated vasculitis include rituximab and plasma exchange (Chen, M. and Kallenberg, C.G.M. Nat. Rev. Rheumatol. 6:653-664, 2010).

[00283] Although NCGN is characterized by a lack of complement deposition, the alternative complement pathway has been implicated in its pathogenesis. An evaluation of renal biopsies from 7 patients with MPO-ANCA-associated pauci-immune NCGN detected the presence of the membrane attack complex, C3d, factor B, and factor P (which were not detected in biopsies from normal controls or patients with minimal change disease), whereas C4d-binding lectin and mannose were not detected, suggesting selective activation of the alternative pathway (Xing, G. Q. et al. J. Clin. Immunol. 29:282-291, 2009). Experimental NCGN can be induced by transfer of anti-MPO IgG into wild-type mice or anti-MPO splenocytes into immunodeficient mice (Xiao, H. et al. J. Clin. Invest. 110:955-963, 2002). In this mouse model of NCGN, the role of specific complement activation pathways was investigated using knockout mice. After injection of anti-MPO IgG, C4 -/- mice developed renal disease comparable to wild-type mice whereas C5 -/- and factor B -/- mice did not develop renal disease, indicating that the alternative pathway was involved in this model and the classical and lectin pathways were not (Xiao, H. et al. Am. J. Pathol. 170:52-64, 2007). Furthermore, incubation of MPO-ANCA or PR3-ANCA IgG from patients with TNF-primed human neutrophils caused the release of factors that resulted in complement activation in normal human serum, as detected by the generation of C3a; this effect was not observed with IgG from healthy individuals, suggesting the potential pathogenic role of ANCA in neutrophil and complement activation (Xiao et al., 2007, supra).

[00284] Based on the role outlined above for the alternative pathway in this condition, blocking alternative pathway activation is expected to have utility in the treatment of ANCA-positive NCGN. Given the requirement of fB activation for pathogenesis, LEA-1 inhibitors are expected to be particularly useful in treating this condition and preventing further decline in renal function in these patients.

[00285] Yet another subset of patients develops progressive renal vasculitis with crescent formation accompanied by a rapid decline in renal function in the absence of ANCA. This form of the condition is termed ANCA-negative NCGN and constitutes about one-third of all patients with pauci-immune NCGN (Chen et al, JASN 18(2):599-605, 2007). These patients tend to be younger, and renal outcomes tend to be particularly severe (Chen et al, Nat Rev Nephrol., 5(6):313-8, 2009). A discriminating pathologic feature of these patients is the deposition of MBL and C4d in the renal lesions (Xing et al, J Clin Immunol. 30(1):144-56, 2010). The staining intensity of MBL and C4d in renal biopsies was negatively correlated with renal function (Xing et al., 2010, supra). These findings suggest an important role for lectin-dependent complement activation in pathogenesis. The fact that C4d, but not factor B, is commonly found in diseased tissue samples indicates involvement of LEA-2.

[00286] Based on the role of lectin-dependent complement activation in ANCA-negative NCGN described above, blocking LEA-2 pathway activation is expected to have utility in the treatment of ANCA-negative NCGN.

[00287] The data detailed above suggest that LEA-1 and LEA-2 mediate pathological complement activation in ANCA-positive and ANCA-negative NCGNs, respectively. Thus, a LEA-1 blocking agent combined with a LEA-2 blocking agent is expected to have utility in the treatment of all forms of pauci-immune NCGN, regardless of the underlying etiology. LEA-1 and LEA-2 blocking agents may thus have complementary, additive, or synergistic effects in preventing, treating, or reversing renal dysfunction associated with NCGN.

[00288] LEA-1 and LEA-2 inhibitors used together may achieve additional treatment benefit compared to either agent alone or may provide effective treatment for a broader spectrum of patient subsets. Combined inhibition of LEA-1 and LEA-2 may be accomplished by co-administration of a LEA-1 blocking agent and a LEA-2 blocking agent. Ideally, the inhibitory function of LEA-1 and LEA-2 may be encompassed in a single molecular entity, such as a bispecific antibody composed of MASP-1/3 and a specific MASP-2 binding site or a dual specificity antibody where each binding site can bind to and block either MASP-1/3 or MASP-2.

[00289] In accordance with the foregoing, one aspect of the invention thus provides a method of inhibiting LEA-1-dependent complement activation to treat, prevent, or reduce the severity of pauci-immune crescentic necrotizing glomerulonephritis, comprising administering a composition comprising a therapeutically effective amount of a LEA-1 inhibitory agent comprising a MASP-1 inhibitory agent, a MASP-3 inhibitory agent, or a combination of a MASP-1/3 inhibitory agent, in a pharmaceutical carrier to a subject suffering from, or at risk of developing, pauci-immune crescentic necrotizing glomerulonephritis. The MASP-1, MASP-3, or MASP-1/3 inhibitory composition can be administered to the subject systemically, such as by intraarterial, intravenous, intramuscular, inhaled, nasal, subcutaneous, or other parenteral administration, or potentially by oral administration for non-peptidergic agents. Administration may be repeated as determined by a physician until the condition has resolved or is controlled.

[00290] In another aspect, a method is provided for inhibiting LEA-2-dependent complement activation to treat, prevent, or reduce the severity of pauci-immune crescentic necrotizing glomerulonephritis, comprising administering a therapeutically effective amount of a MASP-2 inhibitory agent to a subject suffering from, or at risk of developing, pauci-immune crescentic necrotizing glomerulonephritis. In another aspect, a method is provided comprising inhibiting LEA-2- and LEA-1-dependent complement activation to treat, prevent, or reduce the severity of pauci-immune crescentic necrotizing glomerulonephritis, comprising administering a therapeutically effective amount of a MASP-2 inhibitory agent and a MASP-1, MASP-3, or MASP-1/3 inhibitory agent to a subject in need thereof.

[00291] In some embodiments, the method comprises inhibiting both LEA-1-dependent complement activation and LEA-2-dependent complement activation. As detailed above, the use of a combination of pharmacological agents that individually block LEA-1 and LEA-2 is expected to provide an improved therapeutic outcome in the treatment or prevention or reduction in severity of pauci-immune crescentic necrotizing glomerulonephritis compared to inhibition of LEA-1 alone. This outcome can be achieved, for example, by co-administration of an antibody having LEA-1-blocking activity in conjunction with an antibody having LEA-2-blocking activity. In some embodiments, the LEA-1- and LEA-2-blocking activities are combined into a single molecular entity and that entity has both LEA-1- and LEA-2-blocking activity. Such an entity may comprise or consist of a bispecific antibody, wherein one antigen-combining site specifically recognizes MASP-1 and blocks LEA-1 and the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Alternatively, such an entity may consist of a bispecific monoclonal antibody, wherein one antigen-combining site specifically recognizes MASP-3 and thus blocks LEA-1 and the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Such an entity may ideally consist of a bispecific monoclonal antibody, wherein one antigen-combining site specifically recognizes MASP-1 and MASP-3 and thus blocks LEA-1 while the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2.

[00292] The MASP-2 inhibitory agent may be administered to the subject systemically, such as by intra-arterial, intravenous, intramuscular, inhaled, nasal, subcutaneous, or other parenteral administration, or potentially by oral administration for non-peptidergic agents. Administration may be repeated as determined by a physician until the condition has resolved or is controlled.

[00293] Application of the MASP-3 inhibitory compositions and/or MASP-2 inhibitory compositions of the present invention can be accomplished by a single administration of the composition (e.g., a single composition comprising MASP-2 and/or MASP-3 inhibitory agents or bispecific or dual inhibitory agents or co-administration of separate compositions) or a limited sequence of administrations, to treat, prevent, or reduce the severity of pauci-immune crescentic necrotizing glomerulonephritis. Alternatively, the composition can be administered at periodic intervals, such as daily, biweekly, weekly, every other week, monthly, or bimonthly for an extended period of time for treatment of a subject in need thereof.

[00294] As described in Examples 11-21, high affinity MASP-3 inhibitory antibodies were generated that have therapeutic utility for inhibition of the alternative pathway in AP-related diseases or conditions, such as pauci-immune crescentic necrotizing glomerulonephritis (NCGN).

[00295] Accordingly, in one embodiment, the present invention provides a method for treating a subject suffering from or at risk of developing pauci-immune crescentic necrotizing glomerulonephritis (NCGN) comprising an effective amount of a high affinity monoclonal antibody or antigen-binding fragment thereof as described herein that binds to human MASP-3 and inhibits alternative pathway complement activation to treat or reduce the risk of developing pauci-immune crescentic necrotizing glomerulonephritis (NCGN), such as, for example, wherein said antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region comprising (i) VHCDR1 comprising SEQ ID NO: 84, (ii) VHCDR2 comprising SEQ ID NO: 86 or SEQ ID NO: 275, and (iii) VHCDR3 comprising SEQ ID NO: 88; and (b) a light chain variable region comprising (i) VLCDR1 comprising SEQ ID NO: 142, SEQ ID NO: 257, SEQ ID NO: 258 or SEQ ID NO: 259 (ii) VLCDR2 comprising SEQ ID NO: 144 and (iii) VLCDR3 comprising SEQ ID NO: 161. J. THE ROLE OF MASP-3 IN TRAUMATIC BRAIN INJURY and THERAPEUTIC METHODS USING MASP-3 INHIBITORY ANTIBODIES, OPTIONALLY IN COMBINATION WITH MASP-2 INHIBITORY AGENTS

[00296] Traumatic brain injury (TBI) is a major global health problem that leads to at least 10 million deaths or hospitalizations annually (Langlois, J. A. et al., J. Head Trauma Rehabil. 21:375-378, 2006). In 2003, there were an estimated 1.6 million TBIs in the United States, including 1.2 million emergency department visits, 290,000 hospitalizations, and 51,000 deaths (Rutland-Brown, W. et al., J. Head Trauma Rehabil. 21:544-548, 2006). The majority of TBIs in the United States are caused by falls and motor vehicle traffic. TBI can result in long-term or permanent physical, cognitive, behavioral, and emotional consequences. More than 5 million Americans are living with long-term or permanent disability associated with a TBI (Langlois et al., 2006, supra).

[00297] TBI may involve penetration of the brain substance (“penetrating” injuries) or injuries that do not penetrate the brain (“closed” injuries). The injury profiles and associated neurobehavioral sequelae can be quite different between penetrating and closed TBI. Although each injury is unique, certain regions of the brain are particularly vulnerable to trauma-induced damage, including the frontal cortex and subfrontal white matter, the basal ganglia and diencephalon, the rostral brainstem, and the temporal lobes, including the hippocampus (McAllister, T.W. Dialogues Clin. Neurosci. 13:287-300, 2011). TBI can lead to alterations in several neurotransmitter systems, including release of glutamate and other excitatory amino acids during the acute phase and chronic alterations in the catecholaminergic and cholinergic systems, which may be associated with neurobehavioral disability (McAllister, 2011, supra). Survivors of significant TBI often suffer from cognitive defects, personality changes, and increased psychiatric disorders, particularly depression, anxiety, and posttraumatic stress disorder. Despite intensive research, no clinically effective treatment for TBI that can reduce mortality and morbidity and improve functional outcome has yet been found. Complementary Factors and TBI

[00298] Numerous studies have identified a relationship of complement proteins and neurological disorders, including Alzheimer's disease, multiple sclerosis, myasthenia gravis, Guillain-Barré syndrome, cerebral lupus, and stroke (reviewed in Wagner, E., et al., Nature Rev Drug Disc. 9:4356, 2010). Recently, a role for C1q and C3 in synapse elimination has been demonstrated, whereby complement factors are likely involved in normal CNS function and neurodegenerative disease (Stevens, B. et al., Cell 131:1164-1178, 2007). The gene for MASP-1 and MASP-3 is extensively expressed in the brain and also in a glioma cell line, T98G (Kuraya, M. et al., Int Immunol., 15: 109-17, 2003), consistent with a role for the lectin pathway in the CNS.

[00299] MASP-1 and MASP-3 are critical for immediate defense against pathogens and altered self-cells, but the lectin pathway is also responsible for severe tissue damage after stroke, heart attack, and other ischemia-reperfusion injuries. Similarly, MASP-1 and MASP-3 are likely mediators of tissue damage caused by TBI. Inhibition of factor B in the alternative pathway has been shown to attenuate TBI in two mouse models. Factor B knockout mice are protected from neuroinflammation and complement-mediated neuropathology after TBI (Leinhase I, et al., BMC Neurosci. 7:55, 2006). Furthermore, anti-factor B antibody attenuated brain tissue damage and neuronal cell death in mice induced by TBI (Leinhase I, et al., J Neuroinflammation 4:13, 2007). MASP-3 directly activates Factor B (Iwaki, D. et al., J Immunol. 187: 3751-8, 2011) and is therefore also a likely mediator in TBI. Similar to Factor B inhibition, LEA-1 inhibitors, such as antibodies against MASP-3, are expected to provide a promising strategy for the treatment of tissue damage and subsequent sequelae in TBI.

[00300] Thus, LEA-1 and LEA-2 inhibitors may have independent therapeutic benefit in TBI. Furthermore, LEA-1 and LEA-2 inhibitors used together may achieve additional treatment benefit compared to either agent alone or may provide effective treatment for a broader spectrum of patient subsets. Combined inhibition of LEA-1 and LEA-2 may be accomplished by co-administration of a LEA-1 blocking agent and a LEA-2 blocking agent. Ideally, the inhibitory function of LEA-1 and LEA-2 may be encompassed in a single molecular entity, such as a bispecific antibody composed of MASP-1/3 and a specific MASP-2 binding site or a dual-specific antibody where each binding site can bind to and block either MASP-1/3 or MASP-2.

[00301] In accordance with the foregoing, one aspect of the invention thus provides a method of inhibiting LEA-1-dependent complement activation to treat or reduce the severity of traumatic brain injury, comprising administering a composition comprising a therapeutically effective amount of a LEA-1 inhibitory agent comprising a MASP-1 inhibitory agent, a MASP-3 inhibitory agent, or a combination of a MASP-1/3 inhibitory agent, in a pharmaceutical carrier to a subject suffering from a traumatic brain injury. The MASP-1, MASP-3, or MASP-1/3 inhibitory composition may be administered to the subject systemically, such as by intra-arterial, intravenous, intramuscular, inhaled, nasal, intracranial, subcutaneous, or other parenteral administration, or potentially by oral administration for non-peptidergic agents. Administration may be repeated as determined by a physician until the condition has resolved or is controlled.

[00302] In another aspect, a method is provided for inhibiting LEA-2-dependent complement activation to treat or reduce the severity of traumatic brain injury, comprising administering a therapeutically effective amount of a MASP-2 inhibitory agent to a subject suffering from a traumatic brain injury. In another aspect, a method is provided comprising inhibiting LEA-2- and LEA-1-dependent complement activation to treat or reduce the severity of traumatic brain injury, comprising administering a therapeutically effective amount of a MASP-2 inhibitory agent and a MASP-1, MASP-3, or MASP-1/3 inhibitory agent to a subject suffering from a traumatic brain injury.

[00303] In some embodiments, the method comprises inhibiting both LEA-1-dependent complement activation and LEA-2-dependent complement activation. As detailed above, the use of a combination of pharmacological agents that individually block LEA-1 and LEA-2 is expected to provide an improved therapeutic outcome in treating or reducing the severity of traumatic brain injury, compared to inhibiting LEA-1 alone. This result can be achieved, for example, by co-administering an antibody that has LEA-1-blocking activity in conjunction with an antibody that has LEA-2-blocking activity. In some embodiments, the LEA-1- and LEA-2-blocking activities are combined into a single molecular entity and that entity has both LEA-1- and LEA-2-blocking activity. Such an entity may comprise or consist of a bispecific antibody, wherein one antigen-combining site specifically recognizes MASP-1 and blocks LEA-1 and the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Alternatively, such an entity may consist of a bispecific monoclonal antibody, wherein one antigen-combining site specifically recognizes MASP-3 and thus blocks LEA-1 and the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Such an entity may ideally consist of a bispecific monoclonal antibody, wherein one antigen-combining site specifically recognizes MASP-1 and MASP-3 and thus blocks LEA-1 while the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2.

[00304] The MASP-2 inhibitory agent may be administered to the subject systemically, such as by intra-arterial, intravenous, intramuscular, inhaled, nasal, subcutaneous, intracranial, or other parenteral administration, or potentially by oral administration for non-peptidergic agents. Administration may be repeated as determined by a physician until the condition has resolved or is controlled.

[00305] Application of the MASP-3 inhibitory compositions and/or MASP-2 inhibitory compositions of the present invention can be accomplished by a single administration of the composition (e.g., a single composition comprising MASP-2 and/or MASP-3 inhibitory agents or bispecific or dual inhibitory agents or co-administration of separate compositions) or a limited sequence of administrations, to treat or reduce the severity of traumatic brain injury. Alternatively, the composition can be administered at periodic intervals, such as daily, biweekly, weekly, every other week, monthly, or bimonthly over an extended period of time for the treatment of a subject in need thereof.

[00306] As described in Examples 11-21, high affinity MASP-3 inhibitory antibodies have been generated that have therapeutic utility for inhibition of the alternative pathway in AP-related diseases or conditions, such as traumatic brain injury.

[00307] Accordingly, in one embodiment, the present invention provides a method for treating a subject suffering from or at risk of developing traumatic brain injury comprising an effective amount of a high affinity monoclonal antibody or antigen-binding fragment thereof as described herein that binds to human MASP-3 and inhibits alternative pathway complement activation to treat or reduce the risk of developing traumatic brain injury, such as, for example, wherein said antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region comprising (i) VHCDR1 comprising SEQ ID NO: 84, (ii) VHCDR2 comprising SEQ ID NO: 86 or SEQ ID NO: 275, and (iii) VHCDR3 comprising SEQ ID NO: 88; and (b) a light chain variable region comprising (i) VLCDR1 comprising SEQ ID NO: 142, SEQ ID NO: 257, SEQ ID NO: 258 or SEQ ID NO: 259 (ii) VLCDR2 comprising SEQ ID NO: 144 and (iii) VLCDR3 comprising SEQ ID NO: 161. K. THE ROLE OF MASP-3 IN ASPIRATION PNEUMONIA and THERAPEUTIC METHODS USING MASP-3 INHIBITOR ANTIBODIES, OPTIONALLY IN COMBINATION WITH MASP-2 INHIBITOR AGENTS

[00308] Aspiration is defined as the inhalation of oropharyngeal or gastric contents into the lower airway. Aspiration may result in complications of aspiration (chemical) pneumonitis, primary bacterial aspiration pneumonia, or secondary bacterial infection due to chemical pneumonitis. Risk factors for aspiration include decreased levels of consciousness (e.g., head injury, alcohol- or drug-induced sensorium changes, stroke), various gastrointestinal and esophageal abnormalities, and neuromuscular disease. An estimated 5–15% of the 4.5 million cases of community-acquired pneumonia are due to aspiration pneumonia (Marik, P.E. New Engl. J. Med. 344:665–671, 2001). Treatment of chemical pneumonitis is primarily supportive, and the use of empirical antibiotics is controversial. Treatment of bacterial aspiration pneumonia is with appropriate antibiotics, based on whether the aspiration occurred in the community or in the hospital, since the likely causative organisms differ between these settings. Measures should be taken to prevent aspiration in high-risk patients, for example, elderly patients in nursing homes who have impaired gag reflexes. Measures that have been shown to be effective for prophylaxis include elevating the head of the bed during feeding, dental prophylaxis, and good oral hygiene. Prophylactic antibiotics have not been shown to be effective and are not recommended, as they may lead to the emergence of resistant organisms.

[00309] Modulation of complement components has been proposed for numerous clinical indications, including infectious diseases—sepsis, viral, bacterial, and fungal infections—and pulmonary conditions—respiratory distress syndrome, chronic obstructive pulmonary disease, and cystic fibrosis (reviewed in Wagner, E., et al., Nature Rev Drug Disc. 9:4356, 2010). Support for this proposal is provided by numerous clinical and genetic studies. For example, there is a significantly lower frequency of patients with low MBL levels with clinical tuberculosis (Soborg et al., Journal of Infectious Diseases 188:777-82, 2003), suggesting that low MBL levels are associated with protection against the disease.

[00310] In a murine model of acid aspiration injury, Weiser MR et al., J. Appl. Physiol. 83(4): 1090-1095, 1997, it was demonstrated that C3 knockout mice were protected from severe injury; whereas C4 knockout mice were not protected, indicating that complement activation is mediated by the alternative pathway. Consequently, blockade of the alternative pathway with LEA-1 inhibitors is expected to provide a therapeutic benefit in aspiration pneumonia.

[00311] Thus, LEA-1 and LEA-2 inhibitors may have independent therapeutic benefit in aspiration pneumonia. Furthermore, LEA-1 and LEA-2 inhibitors used together may achieve additional treatment benefit compared to either agent alone or may provide effective treatment for a broader spectrum of patient subsets. Combined inhibition of LEA-1 and LEA-2 may be accomplished by co-administration of a LEA-1 blocking agent and a LEA-2 blocking agent. Ideally, the inhibitory function of LEA-1 and LEA-2 may be encompassed in a single molecular entity, such as a bispecific antibody composed of MASP-1/3 and a specific MASP-2 binding site or a dual-specific antibody where each binding site binds to and blocks either MASP-1/3 or MASP-2.

[00312] One aspect of the invention thus provides a method of inhibiting LEA-1-dependent complement activation to treat aspiration pneumonia by administering a composition comprising a therapeutically effective amount of a MASP-1 inhibitory agent, a MASP-3 inhibitory agent, or a combination of a MASP-1/3 inhibitory agent, in a pharmaceutical carrier to a subject suffering from such a condition or other complement-mediated pneumonia. The MASP-1/3 inhibitory composition, MASP-1 or MASP-3, may be administered locally to the lung, such as by an inhaler. Alternatively, the MASP-1, MASP-3 or MASP-1/3 inhibitory agent may be administered to the subject systemically, such as by intra-arterial, intravenous, intramuscular, inhaled, nasal, subcutaneous or other parenteral administration, or potentially by oral administration. Administration may be repeated as determined by a physician until the condition has resolved or is controlled.

[00313] In accordance with the foregoing, one aspect of the invention thus provides a method of inhibiting LEA-1-dependent complement activation to treat, prevent, or reduce the severity of aspiration pneumonia, comprising administering a composition comprising a therapeutically effective amount of a LEA-1 inhibiting agent comprising a MASP-1 inhibiting agent, a MASP-3 inhibiting agent, or a combination of a MASP-1/3 inhibiting agent, in a pharmaceutical carrier to a subject suffering from, or at risk of developing, aspiration pneumonia. The MASP-1, MASP-3, or MASP-1/3 inhibiting composition may be administered to the subject systemically, such as by intra-arterial, intravenous, intramuscular, inhaled, nasal, subcutaneous, or other parenteral administration, or potentially by oral administration for non-peptidergic agents. Administration may be repeated as determined by a physician until the condition has resolved or is controlled.

[00314] In another aspect, a method is provided for inhibiting LEA-2-dependent complement activation to treat, prevent, or reduce the severity of aspiration pneumonia, comprising administering a therapeutically effective amount of a MASP-2-inhibiting agent to a subject suffering from, or at risk of developing, aspiration pneumonia. In another aspect, a method is provided comprising inhibiting LEA-2- and LEA-1-dependent complement activation to treat or reduce the severity of aspiration pneumonia, comprising administering a therapeutically effective amount of a MASP-2-inhibiting agent and a MASP-1, MASP-3, or MASP-1/3-inhibiting agent to a subject suffering from aspiration pneumonia. In some embodiments, the method comprises inhibiting both LEA-1-dependent complement activation and LEA-2-dependent complement activation. As detailed above, the use of a combination of pharmacological agents that individually block LEA-1 and LEA-2 is expected to provide an improved therapeutic outcome in the treatment or reduction in severity of aspiration pneumonia, compared to inhibition of LEA-1 alone. This result can be achieved, for example, by co-administering an antibody that has LEA-1-blocking activity in conjunction with an antibody that has LEA-2-blocking activity. In some embodiments, the LEA-1- and LEA-2-blocking activities are combined into a single molecular entity and that entity has both LEA-1- and LEA-2-blocking activity. Such an entity may comprise or consist of a bispecific antibody, wherein one antigen-combining site specifically recognizes MASP-1 and blocks LEA-1 and the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Alternatively, such an entity may consist of a bispecific monoclonal antibody, wherein one antigen-combining site specifically recognizes MASP-3 and thus blocks LEA-1 and the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Such an entity may ideally consist of a bispecific monoclonal antibody, wherein one antigen-combining site specifically recognizes MASP-1 and MASP-3 and thus blocks LEA-1 while the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2.

[00315] The MASP-2 inhibitory agent may be administered to the subject systemically, such as by intra-arterial, intravenous, intramuscular, inhaled, nasal, subcutaneous, or other parenteral administration, or potentially by oral administration for non-peptidergic agents. Administration may be repeated as determined by a physician until the condition has resolved or is controlled.

[00316] Application of the MASP-3 inhibitory compositions and/or MASP-2 inhibitory compositions of the present invention can be accomplished by a single administration of the composition (e.g., a single composition comprising MASP-2 and/or MASP-3 inhibitory agents or bispecific or dual inhibitory agents or co-administration of separate compositions) or a limited sequence of administrations, to treat, prevent, or reduce the severity of aspiration pneumonia in a subject in need thereof. Alternatively, the composition can be administered at periodic intervals, such as daily, biweekly, weekly, every other week, monthly, or bimonthly over an extended period of time for the treatment of a subject in need thereof.

[00317] As described in Examples 11-21, high affinity MASP-3 inhibitory antibodies were generated that have therapeutic utility for inhibition of the alternative pathway in AP-related diseases or conditions, such as aspiration pneumonia.

[00318] Accordingly, in one embodiment, the present invention provides a method of treating a subject suffering from or at risk of developing aspiration pneumonia comprising an effective amount of a high affinity monoclonal antibody or antigen-binding fragment thereof as described herein that binds to human MASP-3 and inhibits alternative pathway complement activation to treat or reduce the risk of developing aspiration pneumonia, such as, for example, wherein said antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region comprising (i) VHCDR1 comprising SEQ ID NO: 84, (ii) VHCDR2 comprising SEQ ID NO: 86 or SEQ ID NO: 275, and (iii) VHCDR3 comprising SEQ ID NO: 88; and (b) a light chain variable region comprising (i) VLCDR1 comprising SEQ ID NO: 142, SEQ ID NO: 257, SEQ ID NO: 258 or SEQ ID NO: 259 (ii) VLCDR2 comprising SEQ ID NO: 144 and (iii) VLCDR3 comprising SEQ ID NO: 161. L. THE ROLE OF MASP-3 IN ENDOPHTHALMITIS AND THERAPEUTIC METHODS USING MASP-3 INHIBITOR ANTIBODIES, OPTIONALLY IN COMBINATION WITH MASP-2 INHIBITOR AGENTS

[00319] Endophthalmitis is an inflammatory condition of the intraocular cavities and is usually caused by infection. Endophthalmitis may be endogenous, resulting from hematogenous spread of organisms from a distant source of infection (e.g., endocarditis), or exogenous, from direct inoculation of an organism from the external eye as a complication of ocular surgery, foreign bodies, and/or penetrating or blunt ocular trauma. Exogenous endophthalmitis is much more common than endogenous endophthalmitis, and most cases of exogenous endophthalmitis occur after ocular surgery. In the United States, cataract surgery is the leading cause of endophthalmitis, occurring in 0.1–0.3% of cataract surgery, with an apparent increase in incidence over the past decade (Taban, M. et al., Arch. Ophthalmol. 123:613–620, 2005). Post-surgical endophthalmitis may present acutely, within two weeks after surgery, or late, months after surgery. Acute endophthalmitis typically presents with pain, redness, swelling of the eyelid, and decreased visual acuity. Late-onset endophthalmitis is less common than the acute form, and patients may report only mild pain and photosensitivity. Treatment of endophthalmitis depends on the underlying cause and may include systemic and/or intravitreal antibiotics. Endophthalmitis may result in decreased or loss of vision.

[00320] As previously described for AMD, multiple complement pathway genes have been associated with ophthalmic disorders and these specifically include lectin pathway genes. For example, MBL2 has been identified with AMD subtypes (Dinu V, et al., Genet Epidemiol 31:22437, 2007). The LEA-1 and LEA-2 pathways are likely to be involved in ocular inflammatory conditions such as endophthalmitis (Chow SP et al., Clin Experiment Ophthalmol. 39:871-7, 2011). Chow et al. examined MBL levels from patients with endophthalmitis and demonstrated that MBL levels and functional lectin pathway activity are significantly elevated in inflamed human eyes but virtually undetectable in non-inflamed control eyes. This suggests a role for MBL and the lectin pathway in vision-threatening ocular inflammatory conditions, particularly endophthalmitis. Furthermore, in a murine model of corneal fungal keratitis, the MBL-A gene was one of five upregulated inflammatory pathway genes (Wang Y., et al., Mol Vis 13: 1226-33, 2007).

[00321] Thus, it is expected that LEA-1 and LEA-2 inhibitors may have independent therapeutic benefit in the treatment of endophthalmitis. Furthermore, LEA-1 and LEA-2 inhibitors used together may achieve additional treatment benefit compared to either agent alone or may provide effective treatment for a broader spectrum of patient subsets. Combined inhibition of LEA-1 and LEA-2 may be accomplished by co-administration of a LEA-1 blocking agent and a LEA-2 blocking agent. Ideally, the inhibitory function of LEA-1 and LEA-2 may be encompassed in a single molecular entity, such as a bispecific antibody composed of MASP-1/3 and a specific MASP-2 binding site or a dual-specific antibody where each binding site binds to and blocks either MASP-1/3 or MASP-2.

[00322] In accordance with the foregoing, one aspect of the invention thus provides a method of inhibiting LEA-1-dependent complement activation to treat, prevent or reduce the severity of endophthalmitis, comprising administering a composition comprising a therapeutically effective amount of a LEA-1 inhibiting agent comprising a MASP-1 inhibiting agent, a MASP-3 inhibiting agent or a combination of a MASP-1/3 inhibiting agent, in a pharmaceutical carrier to a subject suffering from, or at risk of developing, endophthalmitis. The MASP-1, MASP-3 or MASP-1/3 inhibiting composition may be administered locally to the eye, such as by irrigation or application of the composition in the form of a topical gel, ointment or drops or by intravitreal administration. Alternatively, the MASP-1, MASP-3, or MASP-1/3 inhibitory agent may be administered to the individual systemically, such as by intra-arterial, intravenous, intramuscular, inhaled, nasal, subcutaneous, or other parenteral administration, or potentially by oral administration for non-peptidergic agents. Administration may be repeated as determined by a physician until the condition has resolved or is controlled.

[00323] In another aspect, a method is provided for inhibiting LEA-2-dependent complement activation to treat, prevent, or reduce the severity of endophthalmitis, comprising administering a therapeutically effective amount of a MASP-2 inhibitory agent to a subject suffering from, or at risk of developing, endophthalmitis. In another aspect, a method is provided comprising inhibiting LEA-2- and LEA-1-dependent complement activation to treat or reduce the severity of endophthalmitis, comprising administering a therapeutically effective amount of a MASP-2 inhibitory agent and a MASP-1, MASP-3, or MASP-1/3 inhibitory agent to a subject suffering from endophthalmitis.

[00324] In some embodiments, the method comprises inhibiting both LEA-1-dependent complement activation and LEA-2-dependent complement activation. As detailed above, the use of a combination of pharmacological agents that individually block LEA-1 and LEA-2 is expected to provide an improved therapeutic outcome in the treatment or prevention or reduction in severity of endophthalmitis compared to inhibition of LEA-1 alone. This outcome can be achieved, for example, by co-administration of an antibody having LEA-1-blocking activity in conjunction with an antibody having LEA-2-blocking activity. In some embodiments, the LEA-1- and LEA-2-blocking activities are combined into a single molecular entity and that entity has both LEA-1- and LEA-2-blocking activity. Such an entity may comprise or consist of a bispecific antibody, wherein one antigen-combining site specifically recognizes MASP-1 and blocks LEA-1 and the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Alternatively, such an entity may consist of a bispecific monoclonal antibody, wherein one antigen-combining site specifically recognizes MASP-3 and thus blocks LEA-1 and the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Such an entity may ideally consist of a bispecific monoclonal antibody, wherein one antigen-combining site specifically recognizes MASP-1 and MASP-3 and thus blocks LEA-1 while the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2.

[00325] The MASP-2 inhibitory agent may be administered locally to the eye, such as by irrigation or application of the composition in the form of a topical gel, ointment, or drops, or by intravitreal injection. Alternatively, the MASP-2 inhibitory agent may be administered to the subject systemically, such as by intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous, or other parenteral administration, or potentially by oral administration for non-peptidergic agents. Administration may be repeated as determined by a physician until the condition has resolved or is controlled.

[00326] Application of the MASP-3 inhibitory compositions and/or MASP-2 inhibitory compositions of the present invention can be accomplished by a single administration of the composition (e.g., a single composition comprising MASP-2 and/or MASP-3 inhibitory agents or bispecific or dual inhibitory agents or co-administration of separate compositions) or a limited sequence of administrations, to treat, prevent, or reduce the severity of endophthalmitis in a subject in need thereof. Alternatively, the composition can be administered at periodic intervals, such as daily, biweekly, weekly, every other week, monthly, or bimonthly over an extended period of time for the treatment of a subject in need thereof.

[00327] As described in Examples 11-21, high affinity MASP-3 inhibitory antibodies have been generated that have therapeutic utility for inhibition of the alternative pathway in PA-related diseases or conditions, such as endophthalmitis.

[00328] Accordingly, in one embodiment, the present invention provides a method for treating a subject suffering from or at risk of developing endophthalmitis comprising an effective amount of a high affinity monoclonal antibody or antigen-binding fragment thereof as described herein that binds to human MASP-3 and inhibits alternative pathway complement activation to treat or reduce the risk of developing endophthalmitis, such as, for example, wherein said antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region comprising (i) VHCDR1 comprising SEQ ID NO: 84, (ii) VHCDR2 comprising SEQ ID NO: 86 or SEQ ID NO: 275, and (iii) VHCDR3 comprising SEQ ID NO: 88; and (b) a light chain variable region comprising (i) VLCDR1 comprising SEQ ID NO: 142, SEQ ID NO: 257, SEQ ID NO: 258 or SEQ ID NO: 259 (ii) VLCDR2 comprising SEQ ID NO: 144 and (iii) VLCDR3 comprising SEQ ID NO: 161. M. THE ROLE OF MASP-3 IN NEUROMYELITIS OPTICUS and THERAPEUTIC METHODS USING MASP-3 INHIBITOR ANTIBODIES, OPTIONALLY IN COMBINATION WITH MASP-2 INHIBITOR AGENTS

[00329] Neuromyelitis optica (NMO) is an autoimmune disease that affects the optic nerves and spinal cord. It results in inflammation of the optic nerve, known as optic neuritis, and of the spinal cord, known as myelitis. The spinal cord lesions in NMO can lead to weakness or paralysis in the legs or arms, blindness, bladder and bowel dysfunction, and sensory dysfunction.

[00330] NMO shares several similarities with multiple sclerosis (MS) in that both are due to immunological attack of CNS targets and both result in demyelination (Papadopoulos and Verkman, Lancet Neurol., 11(6):535-44, 2013). However, the molecular targets, treatments, and lesions for NMO are distinct from those of MS. While MS is largely T-cell mediated, patients with NMO typically have antibodies that target the water channel protein aquaporin 4 (AQP4), a protein found on astrocytes that surround the blood-brain barrier. Interferon beta is the most commonly used therapy for MS, but it is generally recognized as detrimental in NMO. The inflammatory lesions of NMO are found in the spinal cord and optic nerve and can progress to the brain, including white and gray matter. The demyelination that occurs in NMO lesions is complement-mediated (Papadopoulos and Verkman, Lancet Neurol., 11(6):535-44, 2013).

[00331] Complement-dependent cytotoxicity appears to be the primary mechanism causing the development of NMO. More than 90% of NMO patients have IgG antibodies against AQP4 (Jarius and Wildemann, Jarius S, Wildemann B., Nat Rev Neurol. 2010 Jul;6(7):383-92). These antibodies initiate the formation of a blood-brain barrier lesion. The initial antigen-antibody complex --- AQP4/AQP4-IgG --- on the surface of astrocytes activates the classical complement pathway. This results in the formation of the membrane attack complex on the astrocyte surface, leading to granulocyte infiltration, demyelination, and ultimately necrosis of astrocytes, oligodendrocytes, and neurons (Misu et al., Acta Neuropathol 125(6):815-27, 2013). These cellular events are reflected in tissue destruction and formation of cystic necrotic lesions.

[00332] The classical complement pathway is clearly critical for the pathogenesis of NMO. NMO lesions show vasculocentric deposition of immunoglobulin and activated complement components (Jarius et al., Nat Clin Pract Neurol. 4(4):202-14, 2008). Furthermore, complement proteins such as C5a have been isolated from cerebrospinal fluid of NMO patients (Kuroda et al., J Neuroimmunol., 254(1-2):178-82, 2013). Furthermore, serum IgG obtained from NMO patients can cause complement-dependent cytotoxicity in a rat model of NMO (Saadoun et al., Brain, 133(Pt 2):349-61, 2010). A monoclonal antibody against C1q prevents complement-mediated destruction of astrocytes and injury in a rat model of NMO (Phuan et al., Acta Neuropathol, 125(6):829-40, 2013).

[00333] The alternative complement pathway serves to amplify overall complement activity. Harboe and colleagues (2004) demonstrated that selective blockade of the alternative pathway inhibited >80% of classical pathway-induced membrane attack complex formation (Harboe et al., Clin Exp Immunol 138(3):439-46, 2004). Tiiziin and colleagues (2013) examined classical and alternative pathway products in NMO patients (Tüzün E, et al., J Neuroimmunol. 233(1-2): 211-5, 2011). C4d, the degradation product of C4, was measured to assess classical pathway activity and was increased in sera from NMO patients compared with controls (a 2.14-fold elevation). Furthermore, an increase in Factor Bb, the degradation product of Factor Ba of the alternative pathway, was observed in NMO patients compared with MS patients or normal control subjects (a 1.33-fold increase). This suggests that alternative pathway function is also increased in NMO. This activation would be expected to increase overall complement activation, and indeed, sC5b-9, the end product of the complement cascade, was significantly increased (a 4.14-fold increase).

[00334] Specific MASP-3 inhibitors are expected to provide benefits in the treatment of patients suffering from NMO. As demonstrated here, serum lacking MASP-3 is incapable of activating Factor B, an essential component of C5 convertase, or Factor D, the central activator of the alternative pathway. Therefore, blocking MASP-3 activity with an inhibitory agent, such as an antibody or small molecule, could also inhibit the activation of Factor B and Factor D. Inhibition of these two factors will prevent amplification of the alternative pathway, resulting in decreased overall complement activity. Inhibition of MASP-3 should therefore significantly improve therapeutic outcomes in NMO.

[00335] Thus, it is expected that LEA-1 and/or LEA-2 inhibitors may have independent therapeutic benefit in the treatment of NMO. Furthermore, LEA-1 and LEA-2 inhibitors used together may achieve additional treatment benefit compared to either agent alone or may provide effective treatment for a broader spectrum of patient subsets. Combined inhibition of LEA-1 and LEA-2 may be accomplished by co-administration of a LEA-1 blocking agent and a LEA-2 blocking agent. Ideally, the inhibitory function of LEA-1 and LEA-2 may be encompassed in a single molecular entity, such as a bispecific antibody composed of MASP-1/3 and a specific MASP-2 binding site or a dual-specific antibody where each binding site binds to and blocks either MASP-1/3 or MASP-2.

[00336] In accordance with the foregoing, one aspect of the invention thus provides a method of inhibiting LEA-1-dependent complement activation to treat, prevent or reduce the severity of NMO, comprising administering a composition comprising a therapeutically effective amount of a LEA-1 inhibitory agent comprising a MASP-1 inhibitory agent, a MASP-3 inhibitory agent or a combination of a MASP-1/3 inhibitory agent, in a pharmaceutical carrier to a subject suffering from, or at risk of developing, NMO. The MASP-1, MASP-3 or MASP-1/3 inhibitory composition may be administered locally to the eye, such as by irrigation or application of the composition in the form of a topical gel, ointment or drops or by intravitreal administration. Alternatively, the MASP-1, MASP-3, or MASP-1/3 inhibitory agent may be administered to the individual systemically, such as by intra-arterial, intravenous, intramuscular, inhaled, nasal, subcutaneous, or other parenteral administration, or potentially by oral administration for non-peptidergic agents. Administration may be repeated as determined by a physician until the condition has resolved or is controlled.

[00337] In another aspect, a method is provided for inhibiting LEA-2-dependent complement activation to treat, prevent, or reduce the severity of NMO, comprising administering a therapeutically effective amount of a MASP-2 inhibitory agent to a subject suffering from, or at risk of developing, NMO. In another aspect, a method is provided comprising inhibiting LEA-2- and LEA-1-dependent complement activation to treat or reduce the severity of NMO, comprising administering a therapeutically effective amount of a MASP-2 inhibitory agent and a MASP-1, MASP-3, or MASP-1/3 inhibitory agent to a subject suffering from NMO.

[00338] In some embodiments, the method comprises inhibiting both LEA-1-dependent complement activation and LEA-2-dependent complement activation. As detailed above, the use of a combination of pharmacological agents that individually block LEA-1 and LEA-2 is expected to provide an improved therapeutic outcome in the treatment or prevention or reduction in severity of NMO compared to inhibition of LEA-1 alone. This outcome can be achieved, for example, by co-administration of an antibody having LEA-1-blocking activity in conjunction with an antibody having LEA-2-blocking activity. In some embodiments, the LEA-1- and LEA-2-blocking activities are combined in a single molecular entity and that entity has both LEA-1- and LEA-2-blocking activity. Such an entity may comprise or consist of a bispecific antibody, wherein one antigen-combining site specifically recognizes MASP-1 and blocks LEA-1 and the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Alternatively, such an entity may consist of a bispecific monoclonal antibody, wherein one antigen-combining site specifically recognizes MASP-3 and thus blocks LEA-1 and the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Such an entity may ideally consist of a bispecific monoclonal antibody, wherein one antigen-combining site specifically recognizes MASP-1 and MASP-3 and thus blocks LEA-1 while the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2.

[00339] The MASP-2 inhibitory agent may be administered locally to the eye, such as by irrigation or application of the composition in the form of a topical gel, ointment, or drops, or by intravitreal injection. Alternatively, the MASP-2 inhibitory agent may be administered to the subject systemically, such as by intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous, or other parenteral administration, or potentially by oral administration for non-peptidergic agents. Administration may be repeated as determined by a physician until the condition has resolved or is controlled.

[00340] Application of the MASP-3 inhibitory compositions and/or MASP-2 inhibitory compositions of the present invention can be accomplished by a single administration of the composition (e.g., a single composition comprising MASP-2 and/or MASP-3 inhibitory agents or bispecific or dual inhibitory agents or co-administration of separate compositions) or a limited sequence of administrations, to treat, prevent, or reduce the severity of NMO in a subject in need thereof. Alternatively, the composition can be administered at periodic intervals, such as daily, biweekly, weekly, every other week, monthly, or bimonthly over an extended period of time for the treatment of a subject in need thereof.

[00341] As described in Examples 11-21, high affinity MASP-3 inhibitory antibodies have been generated that have therapeutic utility for inhibition of the alternative pathway in PA-related diseases or conditions, such as neuromyelitis optica (NMO).

[00342] Accordingly, in one embodiment, the present invention provides a method for treating a subject suffering from or at risk of developing neuromyelitis optica (NMO) comprising an effective amount of a high affinity monoclonal antibody or antigen-binding fragment thereof as described herein that binds to human MASP-3 and inhibits alternative pathway complement activation to treat or reduce the risk of developing neuromyelitis optica (NMO), such as, for example, wherein said antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region comprising (i) VHCDR1 comprising SEQ ID NO: 84, (ii) VHCDR2 comprising SEQ ID NO: 86 or SEQ ID NO: 275, and (iii) VHCDR3 comprising SEQ ID NO: 88; and (b) a light chain variable region comprising (i) VLCDR1 comprising SEQ ID NO: 142, SEQ ID NO: 257, SEQ ID NO: 258 or SEQ ID NO: 259 (ii) VLCDR2 comprising SEQ ID NO: 144 and (iii) VLCDR3 comprising SEQ ID NO: 161. N. THE ROLE OF MASP-3 IN BEHÇET'S DISEASE and THERAPEUTIC METHODS USING MASP-3 INHIBITOR ANTIBODIES, OPTIONALLY IN COMBINATION WITH MASP-2 INHIBITOR AGENTS

[00343] Behçet's disease, or Behçet's syndrome, is a rare immune-mediated systemic small vessel vasculitis that often presents with mucous membrane ulceration and ocular problems. Behçet's disease (BD) was named in 1937 after the Turkish dermatologist Hulusi Behçet, who first described the triple symptom complex of recurrent oral ulcers, genital ulcers, and uveitis. BD is a recurrent, systemic inflammatory disorder of unknown cause. The inflammatory perivasculitis of BD can involve the gastrointestinal, pulmonary, musculoskeletal, cardiovascular, and neurological systems. BD can be fatal due to ruptured vascular aneurysms or severe neurological complications. Optic neuropathy and atrophy may result from vasculitis and occlusion of the vessels supplying the optic nerve. See Al-Araji A, et al., Lancet Neurol., 8(2):192-204, 2009.

[00344] The highest incidence of BD is in the Middle East and Far East regions, but it is rare in Europe and North America. BD is usually controlled initially with corticosteroids and immunosuppressants, but many cases are refractory with severe morbidity and mortality. Biologic agents, including interferon-alpha, IVIG, anti-TNF, anti-IL-6, and anti-CD20, have shown benefit in some cases, but there is no consensus on the best treatment.

[00345] While BD is clearly an inflammatory disorder, its pathobiology is unclear. There are genetic associations with HLA antigens, and genome-wide association studies have implicated numerous cytokine genes (Kirino et al., Nat Genet, 45(2):202-7, 2013). Immune system hyperactivity appears to be regulated by the complement system. Increased levels of C3 have been observed in sera from patients with BD (Bardak and Aridogan, Ocul Immunol Inflamm 12(1):53-8, 2004), and elevated C3 and C4 in cerebrospinal fluid are correlated with disease (Jongen et al., Arch Neurol, 49(10):1075-8, 1992).

[00346] Tiiziin and colleagues (2013) examined classical and alternative pathway products in serum from BD patients (Tüzün E, et al., J Neuroimmunol, 233(1-2):211-5, 2011). 4d, the degradation product of C4, is generated upstream of the alternative pathway and was measured to assess baseline classical pathway activity. C4d was increased in sera from BD patients compared to controls (a 2.18-fold elevation). Factor Bb is the degradation product of factor B and was measured to determine alternative pathway activity. BD patients had an increase in factor Bb compared to normal control subjects (a 2.19-fold elevation) consistent with an increase in BD alternative pathway function. Since the alternative complement pathway serves to amplify overall complement activity, this activation would be expected to increase overall complement activation. Harboe et al. (2004) demonstrated that selective blockade of the alternative pathway inhibited over 80% of the membrane attack complex formation induced by the classical pathway (Harboe M, et al., Clin Exp Immunol, 138(3):439-46, 2004). Indeed, sC5b-9, the end product of the complement cascade, was significantly increased in BD patients (a 5.46-fold elevation). Specific MASP-3 inhibitors should provide benefit in BD. Blockade of MASP-3 should inhibit the activation of Factor B and Factor D. This will interrupt the amplification of the alternative pathway, resulting in a decreased response of overall complement activity. Inhibition of MASP-3 should therefore significantly improve therapeutic outcomes in BD. Thus, it is expected that inhibitors of LEA-1 and/or LEA-2 may have independent therapeutic benefit in the treatment of BD. Furthermore, LEA-1 and LEA-2 inhibitors used together may achieve additional treatment benefit compared to either agent alone or may provide effective treatment for a broader spectrum of patient subsets. Combined inhibition of LEA-1 and LEA-2 can be accomplished by coadministration of a LEA-1 blocking agent and a LEA-2 blocking agent. Ideally, the inhibitory function of LEA-1 and LEA-2 could be encompassed in a single molecular entity, such as a bispecific antibody composed of MASP-1/3 and a specific MASP-2 binding site or a dual-specific antibody where each binding site binds to and blocks either MASP-1/3 or MASP-2.

[00347] In accordance with the foregoing, one aspect of the invention thus provides a method of inhibiting LEA-1-dependent complement activation to treat, prevent, or reduce the severity of BD, comprising administering a composition comprising a therapeutically effective amount of a LEA-1 inhibiting agent comprising a MASP-1 inhibiting agent, a MASP-3 inhibiting agent, or a combination of a MASP-1/3 inhibiting agent, in a pharmaceutical carrier to a subject suffering from, or at risk of developing, BD. The MASP-1, MASP-3, or MASP-1/3 inhibiting composition may be administered locally to the eye, such as by irrigation or application of the composition in the form of a topical gel, ointment, or drops, or by intravitreal administration. Alternatively, the MASP-1, MASP-3, or MASP-1/3 inhibitory agent may be administered to the individual systemically, such as by intra-arterial, intravenous, intramuscular, inhaled, nasal, subcutaneous, or other parenteral administration, or potentially by oral administration for non-peptidergic agents. Administration may be repeated as determined by a physician until the condition has resolved or is controlled.

[00348] In another aspect, a method is provided for inhibiting LEA-2-dependent complement activation to treat, prevent, or reduce the severity of BD, comprising administering a therapeutically effective amount of a MASP-2 inhibitory agent to a subject suffering from, or at risk of developing, BD. In another aspect, a method is provided comprising inhibiting LEA-2- and LEA-1-dependent complement activation to treat or reduce the severity of BD, comprising administering a therapeutically effective amount of a MASP-2 inhibitory agent and a MASP-1, MASP-3, or MASP-1/3 inhibitory agent to a subject suffering from BD.

[00349] In some embodiments, the method comprises inhibiting both LEA-1-dependent complement activation and LEA-2-dependent complement activation. As detailed above, the use of a combination of pharmacological agents that individually block LEA-1 and LEA-2 is expected to provide an improved therapeutic outcome in the treatment or prevention or reduction in severity of BD compared to inhibition of LEA-1 alone. This outcome can be achieved, for example, by co-administration of an antibody that has LEA-1-blocking activity in conjunction with an antibody that has LEA-2-blocking activity. In some embodiments, the LEA-1- and LEA-2-blocking activities are combined in a single molecular entity and that entity has both LEA-1- and LEA-2-blocking activity. Such an entity may comprise or consist of a bispecific antibody, wherein one antigen-combining site specifically recognizes MASP-1 and blocks LEA-1 and the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Alternatively, such an entity may consist of a bispecific monoclonal antibody, wherein one antigen-combining site specifically recognizes MASP-3 and thus blocks LEA-1 and the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Such an entity may ideally consist of a bispecific monoclonal antibody, wherein one antigen-combining site specifically recognizes MASP-1 and MASP-3 and thus blocks LEA-1 while the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2.

[00350] The MASP-2 inhibitory agent can be administered locally to the eye, such as by irrigation or application of the composition in the form of a topical gel, ointment, or drops, or by intravitreal injection. Alternatively, the MASP-2 inhibitory agent can be administered to the subject systemically, such as by intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous, or other parenteral administration, or potentially by oral administration for non-peptidergic agents. Administration can be repeated as determined by a physician until the condition has resolved or is controlled.

[00351] Application of the MASP-3 inhibitory compositions and/or MASP-2 inhibitory compositions of the present invention can be accomplished by a single administration of the composition (e.g., a single composition comprising MASP-2 and/or MASP-3 inhibitory agents or bispecific or dual inhibitory agents or co-administration of separate compositions) or a limited sequence of administrations, to treat, prevent, or reduce the severity of BD in a subject in need thereof. Alternatively, the composition can be administered at periodic intervals, such as daily, biweekly, weekly, every other week, monthly, or bimonthly over an extended period of time for the treatment of a subject in need thereof.

[00352] As described in Examples 11-21, high affinity MASP-3 inhibitory antibodies have been generated that have therapeutic utility for inhibition of the alternative pathway in AP-related diseases or conditions, such as Behçet's disease (BD).

[00353] Accordingly, in one embodiment, the present invention provides a method for treating a subject suffering from or at risk of developing Behçet's disease (BD) comprising an effective amount of a high affinity monoclonal antibody or antigen-binding fragment thereof as described herein that binds to human MASP-3 and inhibits alternative pathway complement activation to treat or reduce the risk of developing Behçet's disease (BD), such as, for example, wherein said antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region comprising (i) VHCDR1 comprising SEQ ID NO: 84, (ii) VHCDR2 comprising SEQ ID NO: 86 or SEQ ID NO: 275, and (iii) VHCDR3 comprising SEQ ID NO: 88; and (b) a light chain variable region comprising (i) VLCDR1 comprising SEQ ID NO: 142, SEQ ID NO: 257, SEQ ID NO: 258 or SEQ ID NO: 259 (ii) VLCDR2 comprising SEQ ID NO: 144 and (iii) VLCDR3 comprising SEQ ID NO: 161.

MASP-3 Inhibiting Agents

[00354] With the recognition that the complement lectin pathway is composed of two major complement activation arms, LEA-1 and LEA-2, and that there is also a lectin-independent complement activation arm, comes the realization that it would be highly desirable to specifically inhibit one or more of these effector arms that cause a pathology associated with alternative pathway complement activation, such as at least one of paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD, including wet and dry AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy (including hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), thrombotic thrombocytopenic purpura (TTP), or transplant-associated TMA), asthma, dense deposit disease, pauci-immune crescentic necrotizing glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis optica, Behçet's disease, multiple sclerosis (MS), Guillain Barre syndrome, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), lupus nephritis, systemic lupus erythematosus (SLE), diabetic retinopathy, uveitis, chronic obstructive pulmonary disease (COPD), C3 glomerulopathy, transplant rejection, graft-versus-host disease (GVHD), hemodialysis, sepsis, systemic inflammatory response syndrome (SIRS), acute respiratory distress syndrome (ARDS), ANCA vasculitis, antiphospholipid syndrome, atherosclerosis, IgA nephropathy, and myasthenia gravis, without completely shutting down the immune defense capabilities of complement (i.e., leaving the classical pathway intact). This would leave the C1q-dependent complement activation system intact to handle immune complex processing and to aid in host defense against infection.

Compositions to inhibit LEA-1-mediated complement activation

[00355] As described herein, the inventors have unexpectedly discovered that LEA-1 activation, leading to lysis, is dependent on MASP-3. As further described herein, under physiological conditions, MASP-3-dependent LEA-1 activation also contributes to opsonization, thus providing an additive effect with LEA-2-mediated complement activation. As demonstrated herein, in the presence of Ca++, factor D is not required, since MASP-3 can lead to LEA-1 activation in factor D-/- sera. MASP-3, MASP-1 and HTRA-1 are capable of converting pro-factor D to active factor D. Thus, MASP-3 activation appears in many cases to be MASP-1 dependent, since MASP-3 (in contrast to MASP-1 and MASP-2) is not an autoactivating enzyme and is unable to convert to its active form without the help of MASP-1 (Zundel, S. et al., J. Immunol. 172: 4342–4350 (2004); Megyeri et al., J. Biol. Chem. 288:89228934 (2013). Since MASP-3 does not autoactivate and in many cases requires MASP-1 activity to be converted to its enzymatically active form, MASP-3-mediated activation of the alternative pathway C3 convertase C3Bb can be inhibited by targeting the MASP-3 zymogen or already activated MASP-3 or by targeting MASP-3-mediated activation by MASP-1 or both, since in many cases, in the absence of MASP-1 functional activity, MASP-3 remains in its zymogen form and is not able to trigger LEA-1 through direct formation of alternative pathway C3 convertase (C3bBb).

[00356] Therefore, in one aspect of the invention, the preferred protein component to target in the development of therapeutic agents to specifically inhibit LEA-1 is a MASP-3 inhibitor (including inhibitors of MASP-1-mediated MASP-3 activation (e.g., a MASP-1 inhibitor that inhibits MASP-3 activation)).

[00357] In accordance with the foregoing, in one aspect, the invention provides methods of inhibiting the adverse effects of LEA-1 (i.e., hemolysis and opsonization) by administering a MASP-3 inhibitory agent, such as a MASP-3 inhibitory antibody, to a subject suffering from, or at risk of developing, a disease or disorder selected from the group consisting of paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy (including hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), thrombotic thrombocytopenic purpura (TTP), asthma, dense deposit disease, pauci-immune crescentic necrotizing glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis optica, Behçet's disease, multiple sclerosis, Guillain Barre, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), lupus nephritis, systemic lupus erythematosus (SLE), diabetic retinopathy, uveitis, chronic obstructive pulmonary disease (COPD), C3 glomerulopathy, transplant rejection, graft-versus-host disease (GVHD), hemodialysis, sepsis, systemic inflammatory response syndrome (SIRS), acute respiratory distress syndrome (ARDS), ANCA vasculitis, antiphospholipid syndrome, atherosclerosis, IgA nephropathy, and myasthenia gravis, comprising administering to the subject a pharmaceutical composition comprising an amount of a MASP-3 inhibitory agent effective to inhibit MASP-3-dependent complement activation and a pharmaceutically acceptable carrier.

[00358] MASP-3 inhibitory agents are administered in an amount effective to inhibit MASP-3-dependent complement activation in a living subject who has, or is at risk of developing, paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy (including hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), thrombotic thrombocytopenic purpura (TTP), asthma, dense deposit disease, pauci-immune crescentic necrotizing glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis optica, Behçet's disease, multiple sclerosis, Guillain Barre syndrome, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), lupus nephritis, lupus erythematosus systemic lupus erythematosus (SLE), diabetic retinopathy, uveitis, chronic obstructive pulmonary disease (COPD), C3 glomerulopathy, transplant rejection, graft-versus-host disease (GVHD), hemodialysis, sepsis, systemic inflammatory response syndrome (SIRS), acute respiratory distress syndrome (ARDS), ANCA vasculitis, antiphospholipid syndrome, atherosclerosis, IgA nephropathy, and myasthenia gravis. In the practice of this aspect of the invention, representative MASP-3 inhibitory agents include: molecules that inhibit the biological activity of MASP-3, including molecules that inhibit at least one or more of the following: MASP-3 lectin-dependent factor B activation, MASP-3 lectin-dependent pro-factor D activation, MASP-3 lectin-independent and MASP-3-dependent factor B activation, and MASP-3 lectin-independent and MASP-3 pro-factor D activation (such as small molecule inhibitors, MASP-3 antibodies and fragments thereof, or blocking peptides that interact with MASP-3 or interfere with a protein-protein interaction), and molecules that decrease the expression of MASP-3 (such as MASP-3 antisense nucleic acid molecules, MASP-3-specific RNAi molecules, and MASP-3 ribozymes). A MASP-3 inhibitory agent may effectively block MASP-3 protein-protein interactions, interfere with MASP-3 dimerization or formation, block Ca++ binding, interfere with the MASP-3 serine protease active site, or reduce MASP-3 protein expression, thereby preventing MASP-3 from activating LEA-1-mediated or lectin-independent complement activation. MASP-3 inhibitory agents may be used alone as a primary therapy or in combination with other therapeutic agents as an adjunctive therapy to enhance the therapeutic benefits of other medical treatments, as further described herein. High-affinity monoclonal MASP-3 inhibitory antibodies

[00359] As described in Examples 11-21 and summarized in TABLES 2A, 2B and TABLE 3 below, the inventors have surprisingly generated high affinity (i.e., <500 pM) MASP-3 inhibitory antibodies that bind to an epitope in the serine protease domain of human MASP-3. As described herein, the inventors have demonstrated that these high affinity MASP-3 antibodies are capable of inhibiting alternative pathway complement activation in human, rodent, and non-human primate serum. The variable light and heavy chain regions of these antibodies were sequenced, isolated, and analyzed in a Fab format and in a full-length IgG format. As described in Example 15 and shown in the dendrograms depicted in FIGURES 50A and 50B, the antibodies can be grouped according to sequence similarity. A summary of the heavy chain variable regions and light chain variable regions of these antibodies is shown in FIGURES 49A and 49B and provided in TABLES 2A and 2B below. Humanized versions of representative high-affinity MASP-3 inhibitory antibodies were generated as described in Example 19 and are summarized in TABLE 3. TABLE 2A: Sequences of high-affinity inhibitory antibodies of Note: “SIN” refers to “SEQ ID NO:” TAB IELA 2B: Inhibitory antibodies of all affinity of MASP-3:CDRs TABLE 3: Representative high-affinity MASP-3 inhibitory antibodies: humanized and modified to remove post-translational modification sites

[00360] Accordingly, in one aspect, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to the serine protease domain of human MASP-3 (amino acid residues 450-728 of SEQ ID NO:2) with high affinity (having a K of less than 500 pM), wherein the antibody or antigen-binding fragment thereof inhibits activation of the alternative complement pathway. In some embodiments, the high affinity MASP-3 inhibitory antibody or antigen-binding fragment thereof inhibits the alternative pathway at a molar ratio of about 1:1 to about 2.5:1 of target MASP-3 to mAb in a mammalian subject.

[00361] Inhibition of alternative pathway complement activation is characterized by at least one or more of the following changes in a component of the complement system that occur as a result of administration of a high affinity MASP-3 inhibitory antibody according to various embodiments of the invention: inhibition of hemolysis and/or opsonization; inhibition of lectin-independent Factor B conversion; inhibition of lectin-independent Factor D conversion; inhibition of substrate-specific cleavage of MASP-3 serine protease; reduction of hemolysis or reduction of C3 cleavage and C3b surface deposition; reduction of Factor B and Bb deposition on an activating surface; reduction of resting (circulating and without experimental addition of an activating surface) levels of active Factor D relative to pro-Factor D; reduction of levels of active Factor D relative to pro-Factor D in response to an activating surface; and/or the production of resting and surface-induced levels of fluid-phase Ba, Bb, C3b or C3a.

[00362] For example, as described herein, high affinity MASP-3 inhibitory antibodies are antibodies or antigen-binding fragments thereof capable of inhibiting factor D maturation (i.e., cleavage of pro-factor D to factor D) in a mammalian subject. In some embodiments, high affinity MASP-3 inhibitory antibodies are capable of inhibiting factor D maturation in whole serum to a level less than 50% of that found in untreated control serum (such as less than 40%, e.g. less than 30%, such as less than 25%, e.g. less than 20%, such as less than 15%, e.g. less than 10%, such as less than 5% of untreated control serum not contacted with a MASP-3 inhibitory antibody).

[00363] In preferred embodiments, high affinity MASP-3 inhibitory antibodies selectively inhibit the alternative pathway, leaving the C1q-dependent complement activation system functionally intact.

[00364] In another aspect, the present disclosure features a nucleic acid molecule encoding one or both of the heavy and light chain polypeptides of any of the MASP-3 inhibitory antibodies or antigen-binding fragments described herein. Also featured is a vector (e.g., a cloning or expression vector) comprising the nucleic acid and a cell (e.g., an insect cell, bacterial cell, fungal cell, or mammalian cell) comprising the vector. The disclosure also provides a method for producing any of the MASP-3 inhibitory antibodies or antigen-binding fragments described herein. The methods include providing a cell containing an expression vector containing a nucleic acid encoding one or both of the heavy and light chain polypeptides of any of the antibodies or antigen-binding fragments described herein. The cell or cell culture is cultured under conditions and for a time sufficient to allow expression by the cell (or cell culture) of the antibody or antigen-binding fragment thereof encoded by the nucleic acid. The method may also include isolating the antibody or antigen-binding fragment thereof from the cell (or cell culture) or from the medium in which the cell or cells were cultured. MASP-3 peptides and epitopes

[00365] As described in Example 18, illustrated in FIGURE 62 and summarized in TABLE 4 below, high-affinity MASP-3 inhibitory antibodies and antigen-binding fragments thereof according to the present invention have been found to specifically recognize one or more epitopes within the serine protease domain of human MASP-3 (amino acid residues 450 to 728 of SEQ ID NO:2). “Specifically recognizes” means that the antibody binds to said epitope with significantly greater affinity than to any other molecule or portion thereof. TABLE 4: Representative High-Affinity MASP-3 Inhibitory Antibodies: MASP-3 Epitope-Binding Regions (see also FIGURE 62)

[00366] Accordingly, in some embodiments, the high affinity MASP-3 inhibitory antibody or antigen-binding fragment thereof specifically binds to an epitope located within the serine protease domain of human MASP-3, wherein the epitope is located within at least one or more of: VLRSQRRDTTVI (SEQ ID NO:9), TAAHVLRSQRRDTTV (SEQ ID NO:10), DFNIQNYNHDIALVQ (SEQ ID NO:11), PHAECKTSYESRS (SEQ ID NO:12), GNYSVTENMFC (SEQ ID NO:13), VSNYVDWVWE (SEQ ID NO:14), and/or VLRSQRRDTTV (SEQ ID NO:15). In some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope within SEQ ID NO:15. In some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope within SEQ ID NO:9. In some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope within SEQ ID NO:10. In some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope within SEQ ID NO:12. In some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope within both SEQ ID NO:10 and SEQ ID NO:12. In some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope within at least SEQ ID NO:11, SEQ ID NO:13, and/or SEQ ID NO:14.

[00367] In other embodiments, the high affinity MASP-3 inhibitory antibody or antigen-binding fragment thereof specifically binds to an epitope located within the serine protease domain of human MASP-3, wherein the epitope is located within at least one or more of: ECGQPSRSLPSLV (SEQ ID NO:16), RNAEPGLFPWQ (SEQ ID NO:17); KWFGSGALLSASWIL (SEQ ID NO:18); EHVTVYLGLH (SEQ ID NO:19); PVPLGPHVMP (SEQ ID NO:20); APHMLGL (SEQ ID NO:21); SDVLQYVKLP (SEQ ID NO:22); and/or AFVIFDDLSQRW (SEQ ID NO:23). In one embodiment, the antibody or antigen-binding fragment binds to an epitope within SEQ ID NO:17. In one embodiment, the antibody or antigen-binding fragment binds to an epitope within EHVTVYLGLH (SEQ ID NO:19) and/or AFVIFDDLSQRW (SEQ ID NO:23). In one embodiment, the antibody or antigen-binding fragment binds to an epitope within SEQ ID NO:18, SEQ ID NO:20, and/or SEQ ID NO:23. In one embodiment, the antibody or antigen-binding fragment binds to an epitope within at least SEQ ID NO:16, SEQ ID NO:21, and/or SEQ ID NO:22.

CDR Regions:

[00368] In one aspect of the present invention, the antibody or functional equivalent thereof comprises specific hypervariable regions, designated CDRs. Preferably, the CDRs are CDRs according to the Kabat CDR definition. The CDRs or hypervariable regions can, for example, be identified by sequence alignment to other antibodies. The CDR regions of the high affinity MASP-3 inhibitory antibodies are shown in TABLES 18-23. Group IA mAbs

[00369] In one aspect, the invention provides an isolated antibody or antigen-binding fragment thereof that binds to MASP-3, comprising: (a) a heavy chain variable region comprising a HC-CDR1 defined as SEQ ID NO:209 (XXDIN, wherein X at position 1 is S or T and wherein X at position 2 is N or D); a HC-CDR2 defined as SEQ ID NO:210 (WIYPRDXXXKYNXXFXD, wherein X at position 7 is G or D; X at position 8 is S, T or R; X at position 9 is I or T; X at position 13 is E or D; X at position 14 is K or E and X at position 16 is T or K); and a HC-CDR3 defined as SEQ ID NO: 211 (XEDXY, wherein X at position 1 is L or V and wherein X at position 4 is T or S); and and (b) a light chain variable region comprising a LC-CDR1 defined as SEQ ID NO: 212 (KSSQSLLXXRTRKNYLX, wherein X at position 8 is N, I, Q or A; wherein X at position 9 is S or T; and wherein X at position 17 is A or S); a LC-CDR2 defined as SEQ ID NO: 144 (WASTRES); and a LC-CDR3 defined as SEQ ID NO:146 (KQSYNLYT). In one embodiment, the HC-CDR1 of the heavy chain variable region according to (a) comprises SEQ ID NO:56 (TDDIN). In an embodiment, the HC-CDR1 of the heavy chain variable region according to (a) comprises SEQ ID NO:62 (SNDIN). In an embodiment, the HC-CDR2 of the heavy chain variable region according to (a) comprises SEQ ID NO:58 (WIYPRDDRTKYNDKFKD). In an embodiment, the HC-CDR2 of the heavy chain variable region according to (a) comprises SEQ ID NO:63 (WIYPRDGSIKYNEKFTD). In an embodiment, the HC-CDR2 of the heavy chain variable region according to (a) comprises SEQ ID NO:67 (WIYPRDGTTKYNEEFTD). In an embodiment, the HC-CDR2 of the heavy chain variable region according to (a) comprises SEQ ID NO:69 (WIYPRDGTTKYNEKFTD). In an embodiment, the heavy chain variable region HC-CDR3 according to (a) comprises SEQ ID NO:60 (LEDTY). In an embodiment, the heavy chain variable region HC-CDR3 according to (a) comprises SEQ ID NO:65 (VEDSY). In an embodiment, the light chain variable region LC-CDR1 comprises SEQ ID NO:142 (KSSQSLLNSRTRKNYLA); SEQ ID NO:257 (KSSQSLLQSRTRKNYLA), SEQ ID NO:258 (KSSQSLLASRTRKNYLA); or SEQ ID NO:259 (KSSQSLLNTRTRKNYLA). In an embodiment, the LC-CDR1 comprises SEQ ID NO:258 (KSSQSLLASRTRKNYLA). In an embodiment, the LC-CDR1 comprises SEQ ID NO:149 (KSSQSLLISRTRKNYLS).

[00370] In one embodiment, the HC-CDR1 comprises SEQ ID NO:56, the HC-CDR2 comprises SEQ ID NO:58, the HC-CDR3 comprises SEQ ID NO:60, and the LC-CDR1 comprises SEQ ID NO:142, SEQ ID NO:257, SEQ ID NO:258, or SEQ ID NO:259; the LC-CDR2 comprises SEQ ID NO:144, and the LC-CDR3 comprises SEQ ID NO:146.

[00371] In one embodiment, the HC-CDR1 comprises SEQ ID NO:62, the HC-CDR2 comprises SEQ ID NO:63, SEQ ID NO:67 or SEQ ID NO:69, the HC-CDR3 comprises SEQ ID NO:65, and the LC-CDR1 comprises SEQ ID NO:149, the LC-CDR2 comprises SEQ ID NO:144 and the LC-CDR3 comprises SEQ ID NO:146. Group IB mAbs

[00372] In another aspect, the invention provides an isolated antibody or antigen-binding fragment thereof that binds to MASP-3, comprising: (a) a heavy chain variable region comprising a HC-CDR1 defined as SEQ ID NO:213 (SYGXX, wherein X at position 4 is H or I and wherein X at position 5 is S or T); a HC-CDR2 defined as SEQ ID NO:74; and a HC-CDR3 defined as SEQ ID NO:214 (GGXAXDY, wherein X at position 3 is E or D and wherein X at position 5 is M or L); and (b) a light chain variable region comprising a LC-CDR1 defined as SEQ ID NO:215 (KSSQSLLDSXXKTYLX, wherein X at position 10 is D and or A; at position 16 is N or S); an LC-CDR2 defined as SEQ ID NO:155; and an LC-CDR3 defined as SEQ ID NO:216 (WQGTHFPXT, where X at position 8 is either W or Y).

[00373] In an embodiment, the HC-CDR1 of the heavy chain variable region according to (a) comprises SEQ ID NO:72 (SYGMS). In an embodiment, the HC-CDR1 comprises SEQ ID NO:79 (SYGIT). In an embodiment, the HC-CDR3 comprises SEQ ID NO:76 (GGEAMDY). In an embodiment, the HC-CDR3 comprises SEQ ID NO:82 (GGDALDY). In an embodiment, the LC-CDR1 comprises SEQ ID NO:153 (KSSQSLLDSDGKTYLN); SEQ ID NO:261 (KSSQSLLDSEGKTYLN), SEQ ID NO:262 (KSSQSLLDSAGKTYLN), or SEQ ID NO:263 (KSSQSLLDSDAKTYLN). In an embodiment, the LC-CDR1 comprises SEQ ID NO:263 (KSSQSLLDSDAKTYLN). In an embodiment, the LC-CDR1 comprises SEQ ID NO:152. In an embodiment, the LC-CDR3 comprises SEQ ID NO:159 (KSSQSLLDSDGKTYLS).

[00374] In one embodiment, the LC-CDR3 comprises SEQ ID NO:160 (WQGTHFPYT). In one embodiment, the HC-CDR1 comprises SEQ ID NO:72, the HC-CDR2 comprises SEQ ID NO:74, the HC-CDR3 comprises SEQ ID NO:76, and the LC-CDR1 comprises SEQ ID NO:153, SEQ ID NO:261, SEQ ID NO:262, or SEQ ID NO:263; the LC-CDR2 comprises SEQ ID NO:155, and the LC-CDR3 comprises SEQ ID NO:157.

[00375] In one embodiment, the HC-CDR comprises SEQ ID NO:72, the HC-CDR2 comprises SEQ ID NO:74, the HC-CDR3 comprises SEQ ID NO:76, and the LC-CDR1 comprises SEQ ID NO:153 or SEQ ID NO:263; the LC-CDR2 comprises SEQ ID NO:155, and the LC-CDR3 comprises SEQ ID NO:157.

[00376] In one embodiment, the HC-CDR1 comprises SEQ ID NO:79, the HC-CDR2 comprises SEQ ID NO:74, the HC-CDR3 comprises SEQ ID NO:82, and the LC-CDR1 comprises SEQ ID NO:159, the LC-CDR2 comprises SEQ ID NO:155, and the LC-CDR3 comprises SEQ ID NO:160. Group IC mAbs

[00377] In one aspect, the present invention provides an isolated antibody, or antigen-binding fragment thereof, that binds to MASP-3 comprising (a) a heavy chain variable region comprising a HC-CDR1 defined as SEQ ID NO:84 (GKWIE); a HC-CDR2 defined as SEQ ID NO:86 (EILPGTGSTNYNEKFKG) or SEQ ID NO:275 (EILPGTGSTNYAQKFQG); and a HC-CDR3 defined as SEQ ID NO:88 (SEDV); and (b) a light chain variable region comprising a LC-CDR1 defined as SEQ ID NO:142 (KSSQSLLNSRTRKNYLA), SEQ ID NO:257 (KSSQSLLQSRTRKNYLA); SEQ ID NO:258 (KSSQSLLASRTRKNYLA); or SEQ ID NO:259 (KSSQSLLNTRTRKNYLA), an LC-CDR2 set forth as SEQ ID NO:144 (WASTRES); and an LC-CDR3 set forth as SEQ ID NO:161 (KQSYNIPT). In one embodiment, the LC-CDR1 comprises SEQ ID NO:258. Group II mAbs

[00378] In one aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof that binds to MASP-3, comprising: (a) a heavy chain variable region comprising a HC-CDR1 defined as SEQ ID NO:91 (GYWIE); a HC-CDR2 defined as SEQ ID NO:93 (EMLPGSGSTHYNEKFKG) and a HC-CDR3 defined as SEQ ID NO:95 (SIDY); and (b) a light chain variable region comprising a LC-CDR1 defined as SEQ ID NO:163 (RSSQSLVQSNGNTYLH), a LC-CDR2 defined as SEQ ID NO:165 (KVSNRFS) and a LC-CDR3 defined as SEQ ID NO:167 (SQSTHVPPT). Group III mAbs

[00379] In another aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof that binds to MASP-3, comprising: (a) a heavy chain variable region comprising a HC-CDR1 defined as SEQ ID NO:109 (RVHFAIRDTNYWMQ), a HC-CDR2 defined as SEQ ID NO:110 (AIYPGNGDTSYNQKFKG), a HC-CDR3 defined as SEQ ID NO:112 (GSHYFDY); and a light chain variable region comprising a LC-CDR1 defined as SEQ ID NO:182 (RASQSIGTSIH), a LC-CDR2 defined as SEQ ID NO:184 (YASESIS), and a LC-CDR3 defined as SEQ ID NO:186 (QQSNSWPYT); or (b) a heavy chain variable region comprising a HC-CDR1 defined as SEQ ID NO:125 (DYYMN), a HC-CDR2 defined as SEQ ID NO:127 (DVNPNNDGTTYNQKFKG), a HC-CDR3 defined as SEQ ID NO:129 (CPFYYLGKGTHFDY); and a light chain variable region comprising a LC-CDR1 defined as SEQ ID NO:196 (RASQDISNFLN), a LC-CDR2 defined as SEQ ID NO:198 (YTSRLHS) and a LC-CDR3 defined as SEQ ID NO:200 (QQGFTLPWT); or (c) a heavy chain variable region comprising a HC-CDR1 defined as SEQ ID NO:137 a HC-CDR2 defined as SEQ ID NO:138, a HC-CDR3 defined as SEQ ID NO:140; and a light chain variable region comprising an LC-CDR1 defined as SEQ ID NO:206, an LC-CDR2 defined as SEQ ID NO:207 and an LC-CDR3 defined as SEQ ID NO:208: or (d) a heavy chain variable region comprising an HC-CDR1 defined as SEQ ID NO:98, an HC-CDR2 defined as SEQ ID NO:99, an HC-CDR3 defined as SEQ ID NO:101; and a light chain variable region comprising an LC-CDR1 defined as SEQ ID NO:169, an LC-CDR2 defined as SEQ ID NO:171 and an LC-CDR3 defined as SEQ ID NO:173; or (e) a heavy chain variable region comprising a HC-CDR1 defined as SEQ ID NO:103, a HC-CDR2 defined as SEQ ID NO:105, a HC-CDR3 defined as SEQ ID NO:107; and a light chain variable region comprising a LC-CDR1 defined as SEQ ID NO:176, a LC-CDR2 defined as SEQ ID NO:178 and a LC-CDR3 defined as SEQ ID NO:193; or (f) a heavy chain variable region comprising a HC-CDR1 defined as SEQ ID NO:114, a HC-CDR2 defined as SEQ ID NO:116, a HC-CDR3 defined as SEQ ID NO:118; and a light chain variable region comprising an LC-CDR1 defined as SEQ ID NO:188, an LC-CDR2 defined as SEQ ID NO:178, and an LC-CDR3 defined as SEQ ID NO:190; or (g) a heavy chain variable region comprising an HC-CDR1 defined as SEQ ID NO:114, an HC-CDR2 defined as SEQ ID NO:121, an HC-CDR3 defined as SEQ ID NO:123; and a light chain variable region comprising an LC-CDR1 defined as SEQ ID NO:191, an LC-CDR2 defined as SEQ ID NO:178, and an LC-CDR3 defined as SEQ ID NO:193; or (h) a heavy chain variable region comprising a HC-CDR1 defined as SEQ ID NO:132, a HC-CDR2 defined as SEQ ID NO:133, a HC-CDR3 defined as SEQ ID NO:135; and a light chain variable region comprising a LC-CDR1 defined as SEQ ID NO:203, a LC-CDR2 defined as SEQ ID NO:165 and a LC-CDR3 defined as SEQ ID NO:204. Heavy Chain and Light Chain Variable Regions

[00380] In one embodiment, the invention provides a high affinity MASP-3 inhibitory antibody comprising a heavy chain variable region that comprises or consists of a sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% identical to any of SEQ ID NOs: 24-39, 248-249, 251-252, 254-255 or wherein the antibody comprises a heavy chain variable region comprising SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:252, SEQ ID NO:254 or SEQ ID NO:255.

[00381] In one embodiment, the invention provides a high affinity MASP-3 inhibitory antibody comprising a light chain variable region comprising or consisting of a sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% identical to any of SEQ ID NOs: 40-54, 250, 253, 256, 278, 279, or 280 or wherein the antibody comprises a light chain variable region comprising SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:250, SEQ ID NO:253, SEQ ID NO:256, SEQ ID NO:278, SEQ ID NO:279 or SEQ ID NO:280.

[00382] In one embodiment, the MASP-3 monoclonal antibody comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:24, SEQ ID NO:248, or SEQ ID NO:249 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:40, SEQ ID NO:250, or SEQ ID NO:278.

[00383] In one embodiment, the MASP-3 monoclonal antibody comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:25 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:41.

[00384] In one embodiment, the MASP-3 monoclonal antibody comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:26 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:42.

[00385] In one embodiment, the MASP-3 monoclonal antibody comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:27 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:42.

[00386] In one embodiment, the MASP-3 monoclonal antibody comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:28, SEQ ID NO:251, or SEQ ID NO:252 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:43, SEQ ID NO:253, or SEQ ID NO:279.

[00387] In one embodiment, the MASP-3 monoclonal antibody comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:29 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:44.

[00388] In one embodiment, the MASP-3 monoclonal antibody comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:30, SEQ ID NO:254, or SEQ ID NO:255 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:45, SEQ ID NO:256, or SEQ ID NO:280.

[00389] In one embodiment, the MASP-3 monoclonal antibody comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:31 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:46.

[00390] In one embodiment, the MASP-3 monoclonal antibody comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:32 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:47.

[00391] In one embodiment, the MASP-3 monoclonal antibody comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:33 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:48.

[00392] In one embodiment, the MASP-3 monoclonal antibody comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:34 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:49.

[00393] In one embodiment, the MASP-3 monoclonal antibody comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:35 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:50.

[00394] In one embodiment, the MASP-3 monoclonal antibody comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:36 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:51.

[00395] In one embodiment, the MASP-3 monoclonal antibody comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:37 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:52.

[00396] In one embodiment, the MASP-3 monoclonal antibody comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:38 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:53.

[00397] In one embodiment, the MASP-3 monoclonal antibody comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:39 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:54.

Cross-competition of high-affinity MASP-3 antibodies

[00398] As described herein, the high affinity MASP-3 inhibitory antibodies described herein recognize overlapping epitopes within the serine protease domain of MASP-3. As described in Example 18, shown in FIGURES 61A-E and 62-67 and summarized in TABLES 4 and 28, cross-competition analysis and Pepscan binding analysis show that the high affinity MASP-3 inhibitory antibodies cross-compete and bind to epitopes located within the serine protease domain of MASP-3. Thus, in one embodiment, the invention provides high affinity MASP-3 inhibitory antibodies that specifically recognize an epitope or portion thereof within the serine protease domain of human MASP-3 recognized by one or more selected from the group consisting of: a monoclonal antibody comprising a heavy chain variable region defined as SEQ ID NO: 24 and a light chain variable region defined as SEQ ID NO: 40; a monoclonal antibody comprising a heavy chain variable region defined as SEQ ID NO: 25 and a light chain variable region defined as SEQ ID NO: 41; a monoclonal antibody comprising a heavy chain variable region defined as SEQ ID NO: 26 and a light chain variable region defined as SEQ ID NO: 42; a monoclonal antibody comprising a heavy chain variable region defined as SEQ ID NO: 27 and a light chain variable region defined as SEQ ID NO: 42; a monoclonal antibody comprising a heavy chain variable region defined as SEQ ID NO: 28 and a light chain variable region defined as SEQ ID NO: 43; a monoclonal antibody comprising a heavy chain variable region defined as SEQ ID NO: 29 and a light chain variable region defined as SEQ ID NO: 44; a monoclonal antibody comprising a heavy chain variable region defined as SEQ ID NO: 30 and a light chain variable region defined as SEQ ID NO: 45; a monoclonal antibody comprising a heavy chain variable region defined as SEQ ID NO: 31 and a light chain variable region defined as SEQ ID NO: 46; a monoclonal antibody comprising a heavy chain variable region defined as SEQ ID NO: 32 and a light chain variable region defined as SEQ ID NO: 47; a monoclonal antibody comprising a heavy chain variable region defined as SEQ ID NO: 33 and a light chain variable region defined as SEQ ID NO: 48; a monoclonal antibody comprising a heavy chain variable region defined as SEQ ID NO: 34 and a light chain variable region defined as SEQ ID NO: 49; a monoclonal antibody comprising a heavy chain variable region defined as SEQ ID NO: 35 and a light chain variable region defined as SEQ ID NO: 50; a monoclonal antibody comprising a heavy chain variable region defined as SEQ ID NO: 36 and a light chain variable region defined as SEQ ID NO: 51; a monoclonal antibody comprising a heavy chain variable region defined as SEQ ID NO:37 and a light chain variable region defined as SEQ ID NO:52; a monoclonal antibody comprising a heavy chain variable region defined as SEQ ID NO:38 and a light chain variable region defined as SEQ ID NO:53; and a monoclonal antibody comprising a heavy chain variable region defined as SEQ ID NO:39 and a light chain variable region defined as SEQ ID NO:54.

[00399] According to the present invention, when a given antibody recognizes at least part of an epitope recognized by another given antibody, these two antibodies recognize the same epitopes or coincident epitopes.

[00400] The different assays available to the person skilled in the art can be used to determine whether an antibody (also called test antibody) recognizes the same or a matching epitope as a specific monoclonal antibody (also called reference antibody). Preferably, the assay involves the steps of: • Providing MASP-3 or a fragment thereof comprising the epitope recognized by the reference antibody

[00401] • Adding the test antibody and the reference antibody to said MASP-3, wherein the test antibody or the reference antibody is labeled with a detectable marker. Alternatively, both antibodies may be labeled with different detectable markers • Detecting the presence of the detectable marker on MASP-3 • Thus, detecting whether the test antibody can displace the reference antibody

[00402] If the reference antibody is displaced, the test antibody recognizes the same or a matching epitope as the reference antibody. Thus, if the reference antibody is labeled with a detectable marker, then a low detectable signal on MASP-3 is indicative of displacement of the reference antibody. If the test antibody is labeled with a detectable marker, then a high detectable signal on MASP-3 is indicative of displacement of the reference antibody. The MASP-3 fragment may preferably be immobilized on a solid support that allows for easy handling. The detectable marker may be any directly or indirectly detectable marker, such as an enzyme, a radioactive isotope, a heavy metal, a colored compound or a fluorescent compound. Example 18, in the “Competition Binding Analysis” section herein below describes an exemplary method of determining whether a test antibody recognizes the same or a matching epitope as a reference antibody. One skilled in the art can readily adapt said method to the particular antibodies in question.

[00403] MASP-3 antibodies useful in this aspect of the invention include monoclonal or recombinant antibodies derived from any antibody-producing mammal and can be multispecific (i.e., bispecific or trispecific), chimeric, humanized, fully human, anti-idiotype, and antibody fragments. Antibody fragments include Fab, Fab', F(ab)2, F(ab')2, Fv fragments, scFv fragments, and single chain antibodies as further described herein.

[00404] MASP-3 antibodies can be screened for the ability to inhibit the alternative pathway complement activation system using the assays described herein. Inhibition of alternative pathway complement activation is characterized by at least one or more of the following changes in a component of the complement system that occurs as a result of administration of a high affinity MASP-3 inhibitory antibody according to various embodiments of the invention: inhibition of hemolysis and/or opsonization; inhibition of lectin-independent Factor B conversion; inhibition of lectin-independent Factor D conversion; inhibition of substrate-specific cleavage of MASP-3 serine protease; reduction of hemolysis or reduction of C3 cleavage and C3b surface deposition; reduction of Factor B and Bb deposition on an activating surface; reduction of resting levels (in circulation and without experimental addition of an activating surface) of active Factor D relative to pro-Factor D; the reduction of active Factor D levels relative to pro-Factor D in response to an activating surface; and/or the production of resting and surface-induced levels of fluid-phase Ba, Bb, C3b, or C3a. MASP-3 antibodies with reduced effector function

[00405] In some embodiments of this aspect of the invention, the high affinity MASP-3 inhibitory antibodies described herein have reduced effector function so as to reduce inflammation that may arise from classical pathway complement activation. The ability of IgG molecules to trigger the classical complement pathway has been shown to reside within the Fc portion of the molecule (Duncan, A.R., et al., Nature 332:738-740 (1988)). IgG molecules in which the Fc portion of the molecule has been removed by enzymatic cleavage are devoid of this effector function (see Harlow, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988). Accordingly, antibodies with reduced effector function may be generated as a result of the lack of the Fc portion of the molecule having a genetically engineered Fc sequence that minimizes effector function or being of the human IgG4 or IgG2 isotype.

[00406] Antibodies with reduced effector function can be produced by standard molecular biological manipulation of the Fc portion of IgG heavy chains as described in Jolliffe et al., Int'l Rev. Immunol. 10:241-250, (1993), and Rodrigues et al., J. Immunol. 151:6954-6961, (1998). Antibodies with reduced effector function also include human IgG2 and IgG4 isotypes that have a reduced ability to activate complement and/or interact with Fc receptors (Ravetch, J. V., et al., Annu. Rev. Immunol. 9:457-492, (1991); Isaacs, J. D., et al., J. Immunol. 148:3062-3071, 1992; van de Winkel, J. G., et al., Immunol. Today 14:215-221, (1993)). Humanized or fully human antibodies specific for human MASP-1, MASP-2, or MASP-3 (including dual, pan, bispecific, or trispecific antibodies) consisting of IgG2 or IgG4 isotypes can be produced by one of several methods known to one of ordinary skill in the art, as described in Vaughan, T. J., et al., Nature Biotechnical 16:535-539, (1998). Production of High-Affinity MASP-3 Inhibitors

[00407] MASP-3 antibodies can be produced using MASP-3 polypeptides (e.g., full-length MASP-3) or using peptides containing antigenic MASP-3 epitopes (e.g., a portion of the MASP-3 polypeptide), for example as described in Example 14 below. Immunogenic peptides can be as small as five amino acid residues. Peptides and polypeptides used to raise antibodies can be isolated as natural polypeptides or synthetic and recombinant peptides and catalytically inactive recombinant polypeptides. Antigens useful for producing MASP-3 antibodies also include fusion polypeptides, such as fusions of a MASP-3 polypeptide or a portion thereof with an immunoglobulin polypeptide or with maltose-binding protein. The polypeptide immunogen can be a full-length molecule or a portion thereof. If the polypeptide moiety is hapten-like, such moiety may advantageously be joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), or tetanus toxoid) for immunization. Monoclonal antibodies

[00408] As used herein, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. Monoclonal antibodies may be obtained using any technique that provides for the production of antibody molecules by continuous cell lines in culture, such as the hybridoma method described by Kohler, G., et al., Nature 256:495, (1975) or they may be prepared 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., et al., J. Mol. Biol. 222:581-597, (1991). Such antibodies may be of any class of immunoglobulins including IgG, IgM, IgE, IgA, IgD, and any subclass thereof.

[00409] For example, monoclonal antibodies can be obtained by injecting into a suitable mammal (e.g., a BALB/c mouse) a composition comprising a MASP-3 polypeptide or a portion thereof. After a predetermined period of time, splenocytes are removed from the mouse and suspended in a cell culture medium. The splenocytes 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-3. (See also Current Protocols in Immunology, Vol. 1., John Wiley & Sons, pages 2.5.1-2.6.7, 1991.)

[00410] Human monoclonal antibodies can be obtained through the use of transgenic mice that have been engineered to produce specific human antibodies in response to an antigenic challenge. In this technique, elements of the human immunoglobulin heavy and light chain locus are introduced into strains of mice derived from embryonic stem cell lines that contain disruptions of the endogenous immunoglobulin heavy and light chain loci. The transgenic mice can synthesize human antibodies specific for human antigens, such as the MASP2 antigens described herein, and the mice can be used to produce human MASP2 antibody-secreting hybridomas by fusing B cells from such animals to suitable myeloma cell lines using conventional Kohler-Milstein technology. 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 368:856, 1994; and Taylor, L.D., et al., Int. Immun. 6:579, 1994.

[00411] Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include Protein A Sepharose affinity chromatography, size exclusion chromatography, and ion exchange chromatography (see, e.g., Coligan pp. 2.7.1-2.7.12 and pp. 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, pp. 79-104, 1992).

[00412] Once produced, monoclonal antibodies are first tested for specific binding to MASP-3 or, when desired, binding to MASP-1/3, MASP-2/3, or dual MASP-1/2. Methods for determining whether an antibody binds to a protein antigen and/or the affinity for an antibody to a protein antigen are known in the art. For example, binding of an antibody to a protein antigen can be detected and/or quantified using a variety of techniques, such as, but not limited to, Western blot, dot blot, surface plasmon resonance method (e.g., BIAcore system; Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ) or enzyme-linked immunosorbent assays (ELISA). See, e.g., Harlow and Lane (1988) "Antibodies: A Laboratory Manual" Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Benny K. C. Lo (2004) "Antibody Engineering: Methods and Protocols," Humana Press (ISBN: 1588290921); Borrebaek (1992) "Antibody Engineering, A Practical Guide," W. H. Freeman and Co., NY; Borrebaek (1995) "Antibody Engineering," 2nd Edition, Oxford University Press, NY, Oxford; Johne et al. (1993), Immunol. Met. 160:191-198; Jonsson et al. (1993) Ann. Biol. Clinic. 51: 19-26; and Jonsson et al. (1991) Biotechniques 11:620-627. See, for example, US Patent No. 6,355,245.

[00413] The affinity of MASP-3 monoclonal antibodies can be readily determined by one of skill in the art (see, e.g., Scatchard, A., NY Acad. Sci. 51:660-672, 1949). In one embodiment, MASP-3 monoclonal antibodies useful for the methods of the invention bind to MASP-3 with a binding affinity of <100 nM, preferably <10 nM, preferably <2 nM, and most preferably with a high affinity of <500 pM.

[00414] Once antibodies that specifically bind to MASP-3 have been identified, the MASP-3 antibodies are tested for the ability to function as an alternative pathway inhibitor in one of several functional assays, such as, for example, inhibition of alternative pathway complement activation is distinguished by at least one or more of the following changes in a component of the complement system that occur as a result of administration of a high affinity MASP-3 inhibitory antibody in accordance with various embodiments of the invention: inhibition of hemolysis and/or opsonization; inhibition of lectin-independent factor B conversion; inhibition of lectin-independent factor D conversion; inhibition of substrate-specific cleavage of MASP-3 serine protease; reduction of hemolysis or reduction of C3 cleavage and C3b surface deposition; reduction of Factor B and Bb deposition on an activating surface; the reduction of resting levels (in circulation and without the experimental addition of an activating surface) of active Factor D relative to pro-Factor D; the reduction of active Factor D levels relative to pro-Factor D in response to an activating surface; the reduction in the production of resting and surface-induced levels of fluid-phase Ba, Bb, C3b, or C3a; and/or the reduction in factor P deposition. Chimeric/humanized antibodies

[00415] Monoclonal antibodies useful in the method of the invention include chimeric antibodies in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or corresponding subclass, while the remainder of the chain(s) is identical 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 (U.S. Patent No. 4,816,567, to Cabilly; and Morrison, S.L., et al., Proc. Nat'l Acad. Sci. USA 81:6851-6855, (1984)).

[00416] One form of a chimeric antibody useful in the invention is a humanized MASP-3 monoclonal 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., mouse) complementarity determining regions (CDRs) of the variable light and heavy chains of rat immunoglobulin to a human variable domain. Typically, residues from human antibodies are then substituted into the framework regions of the non-human counterparts. In addition, humanized antibodies may comprise residues that are not found in the recipient antibody or the donor antibody. Such modifications are made to further refine the performance of the antibody. In general, the humanized antibody will comprise substantially at least one, and typically two, variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the Fv framework regions are those of a human immunoglobulin sequence. The humanized antibody will optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones, P.T., et al., Nature 321:522-525, (1986); Reichmann, L., et al., Nature 332:323-329, (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596, (1992).

[00417] Humanized antibodies useful in the present invention include human monoclonal antibodies including at least one MASP-3 binding CDR3 region. In addition, the Fc portions may be substituted so as to produce human IgA or IgM as well as human IgG antibodies. Such humanized antibodies will have particular clinical utility because they will specifically recognize human MASP-3 but will not evoke an immune response in humans against the antibody itself. Consequently, they are best suited for in vivo administration in humans, especially when repeated or long-term administration is required.

[00418] Techniques for producing humanized monoclonal antibodies are also described by Jones, P. T., et al., Nature 321:522, (1986); Carter, P., et al., Proc. Nat'l. Acad. Sci. USA 89:4285, (1992); Sandhu, J. S., Crit. Rev. Biotech. 12:437, (1992); Singer, I. I., et al., J. Immun. 150:2844, (1993); Sudhir (ed.), Antibody Engineering Protocols, Humana Press, Inc., (1995); Kelley, "Engineering Therapeutic Antibodies," in Protein Engineering: Principles and Practice, Cleland et al. (eds.), John Wiley & Sons, Inc., pages 399-434, (1996); and by U.S. Patent No. 5,693,762, to Queen, 1997. In addition, there are commercial entities that will synthesize humanized antibodies from specific murine antibody regions, such as Protein Design Labs (Mountain View, CA). Recombinant Antibodies

[00419] MASP-3 antibodies can also be prepared using recombinant methods. For example, human antibodies can be produced using human immunoglobulin expression libraries (e.g., available from Stratagene, Corp., La Jolla, CA) to produce human antibody fragments (VH, VL, Fv, factor D, Fab, or F(ab')2). These fragments are then used to construct full-length human antibodies using techniques similar to those for producing chimeric antibodies. Immunoglobulin fragments

[00420] MASP-3 inhibitory agents useful in the method of the invention encompass not only intact immunoglobulin molecules, but also the well-known fragments, including Fab', F(ab)2, F(ab')2 and Fv fragments, scFv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific (e.g., bispecific and trispecific) antibodies formed from antibody fragments.

[00421] It is well known in the art that only a small portion of an antibody molecule, the paratope, is involved in binding of the antibody to its epitope (see, e.g., Clark, W.R., The Experimental Foundations of Modern Immunology, Wiley & Sons, Inc., NY, 1986). The pFc' and Fc regions of the antibody are effectors of the classical complement pathway but are not involved in antigen binding. An antibody from which the pFc' region has been enzymatically cleaved, or which has been produced without the pFc' region, is designated an F(ab')2 fragment and retains both 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 region has been enzymatically cleaved or that has been produced without the Fc region is called a Fab fragment and retains one of the antigen-binding sites of an intact antibody molecule.

[00422] 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 termed F(ab')2. This fragment can be further cleaved using a thiol reducing agent to produce monovalent 3.5S Fab' fragments. Optionally, the cleavage reaction can be carried out using a blocking group for the sulfhydryl groups that result from cleavage of disulfide bonds. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab fragments and an Fc fragment directly. Such methods are described, e.g., U.S. Pat. No. 4,331,647 to Goldenberg; Nisonoff, A., et al., Arch. Biochem. Biophys. 89:230, (1960); Porter, R.R., Biochem. J. 73:119, (1959); Edelman, et al., in Methods in Enzymology 1:422, Academic Press, (1967); and by Coligan on pages 2.8.1-2.8.10 and 2.10-2.10.4.

[00423] In some embodiments, the use of antibody fragments lacking the Fc region are preferred to avoid activation of the classical complement pathway, which is initiated upon binding of Fc to the Fey receptor. There are several methods by which one can produce a monoclonal antibody that avoids Fey receptor interactions. For example, the Fc region of a monoclonal antibody can be chemically removed using partial digestion by proteolytic enzymes (such as ficin digestion), thereby generating, for example, antigen-binding antibody fragments, such as Fab or F(ab)2 fragments (Mariani, M., et al., Mol. Immunol. 28:69-71, (1991)). Alternatively, human Y4 IgG isotype, which does not bind to Fey receptors, can be used during the construction of a humanized antibody, as described herein. Antibodies, single-chain antibodies, and antigen-binding domains lacking the Fc domain can also be engineered using recombinant techniques described herein. Single-chain antibody fragments

[00424] Alternatively, one can create single peptide chain binding molecules specific for MASP-3 in which the heavy and light chain Fv regions are connected. The Fv fragments can be linked by a peptide linker, to form a single chain antigen-binding protein (scFv). Such single chain antigen-binding proteins are prepared by constructing a structural gene comprising DNA sequences encoding the VH 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 that bridges the two V domains. Methods for producing scFvs are described for example, by Whitlow, et al., "Methods: A Companion to Methods in Enzymology" 2:97, (1991); Bird, et al., Science 242:423, (1988); U.S. Patent No. 4,946,778, to Ladner; Pack, P., et al., Bio/Technology 11:1271, (1993).

[00425] As an illustrative example, a MASP-3-specific scFv can be obtained by exposing lymphocytes to MASP-3 polypeptide in vitro and screening phage-displayed antibody libraries or similar vectors (e.g., through the use of immobilized or labeled MASP-3 protein or peptide). Genes encoding polypeptides having potential MASP3 polypeptide-binding domains can be obtained by screening random peptide libraries displayed on phage or in bacteria such as E. coli. Such random peptide display libraries can be used to screen for peptides that interact with MASP-3. 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 et al., Phage Display of Peptides and Proteins Academic Press, Inc., 1996), and random peptide display libraries and kits for screening such libraries are commercially available from, for example, Clontech Laboratories, Inc. (Palo Alto, Calif.), Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc. (Beverly, Mass.), and Pharmacia LKB Biotechnology Inc. (Piscataway, N.J.).

[00426] Another form of a MASP-3 antibody fragment useful in this aspect of the invention is a peptide that encodes a unique complementarity determining region (CDR) that binds to an epitope on a MASP-3 antigen and inhibits alternative pathway complement activation.

[00427] CDR ("minimal recognition units") peptides can be obtained by constructing genes that encode the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region of RNA from antibody-producing cells (see, for example, Larrick et al., Methods: A Companion to Methods in Enzymology 2:106, (1991); Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies," in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), page 166, Cambridge University Press, (1995); and Ward et al., "Genetic Manipulation and Expression of Antibodies,” in Monoclonal Antibodies: Principles and Applications, Birch et al. (eds.), page 137, Wiley-Liss, Inc., 1995).

[00428] The high-affinity MASP-3 inhibitory antibodies described herein are administered to a subject in need thereof to inhibit activation of the alternative pathway. In some embodiments, the high-affinity MASP-3 inhibitory antibody is a humanized monoclonal MASP-3 antibody, optionally with reduced effector function. Bispecific Antibodies

[00429] High affinity MASP-3 inhibitory antibodies useful in the method of the invention encompass multispecific (i.e., bispecific and trispecific) antibodies. Bispecific antibodies are monoclonal antibodies, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In one embodiment, the compositions and methods comprise the use of a bispecific antibody comprising a binding specificity for the serine protease domain of MASP-3 and a binding specificity for MASP-2 (e.g., binding to at least one of CCP1-CCP2 or the serine protease domain of MASP-2). In another embodiment, the method comprises the use of a bispecific antibody comprising a binding specificity for the serine protease domain of MASP-3 and a binding specificity for MASP-1 (e.g., binding to the serine protease domain of MASP-1). In another embodiment, the method comprises using a trispecific antibody comprising a binding specificity for MASP-3 (e.g., binding to the serine protease domain of MASP-3), a binding specificity for MASP-2 (e.g., binding to at least one of CCP1-CCP2 or the serine protease domain of MASP-2), and a binding specificity for MASP-1 (e.g., binding to the serine protease domain of MASP-1).

[00430] Methods for producing bispecific antibodies are within the scope of those skilled in the art. Traditionally, recombinant production of bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain/light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature 305:537539 (1983)). Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion is preferably with an immunoglobulin heavy chain constant domain, including at least part of the hinge, CH2, and CH3 regions. The DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors and are cotransfected into a suitable host organism. For further details of illustrative methods currently known for generating bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology 121:210 (1986); WO96/27011; Brennan et al., Science 229:81 (1985); Shalaby et al., J. Exp. Med. 175:217-225 (1992); Kostelny et al., J. Immunol. 148(5):1547-1553 (1992); Hollinger et al. Proc. Natl. Acad. Sci USA 90:6444-6448 (1993); Gruber et al., J. Immunol. 152:5368 (1994); and Tutt et al., J. Immunol. 147:60 (1991). Bispecific antibodies include cross-linked or heteroconjugate antibodies. Heteroconjugate antibodies may be produced using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Pat. No. 4,676,980, along with various cross-linking techniques.

[00431] Various techniques for preparing and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. (See, e.g., Kostelny et al. J. Immunol. 148(5):1547-1553 (1992)). The "diabody" technology, described by Hollinger et al. Proc. Natl. Acad. Sci USA 90:6444-6448 (1993), has provided an alternative mechanism for producing bispecific antibody fragments. The fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) by a linker that is too short to allow pairing between the two domains on the same chain. Consequently, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thus forming two antigen-binding sites. Bispecific diabodies, as opposed to bispecific whole antibodies, may also be particularly useful because they can be readily constructed and expressed in E. coli. Diabodies (and many other polypeptides such as antibody fragments) with appropriate binding specificities can be readily selected using phage display (WO94/13804) from libraries. If one arm of the diabody is to be kept constant, for example, with a specificity directed against antigen X, then a library can be prepared in which the other arm is varied and an antibody of appropriate specificity is selected.

[00432] Another strategy for producing bispecific antibody fragments by the use of single-chain Fv (scFv) dimers has also been reported. (See, e.g., Gruber et al. J. Immunol., 152:5368 (1994)). Alternatively, the antibodies may be "linear antibodies" as described in, e.g., Zapata et al., Protein Eng. 8(10):1057-1062 (1995). Briefly described, these antibodies comprise a pair of tandem Factor D segments (VH-CHI-VH-CHI) that form a pair of antigen-binding regions. Linear antibodies may be bispecific or monospecific. The methods of the invention also encompass the use of variant forms of bispecific antibodies such as the tetravalent dual variable domain immunoglobulin (DVD-Ig) molecules described in Wu et al., Nat Biotechnol 25:1290-1297 (2007). The DVD-Ig molecules are created such that two different light chain variable (VL) domains from two different precursor antibodies are linked in tandem either directly or via a short linker via recombinant DNA techniques, followed by the light chain constant domain. Methods for generating DVD-Ig molecules from two precursor antibodies are further described in, e.g., WO08/024188 and WO07/024715, each of the disclosures of which are incorporated herein by reference in their entirety.

XVIIi. PHARMACEUTICAL COMPOSITIONS AND METHODS OF DELIVERY DOSAGE

[00433] In another aspect, the invention provides compositions comprising high affinity MASP-3 inhibitory antibodies for inhibiting the adverse effects of alternative pathway complement activation in a subject in need thereof, such as, for example, a subject suffering from an alternative pathway-related disease or condition, such as, for example, a hemolytic disease such as PNH or a disease or disorder selected from the group consisting of age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy (including hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), thrombotic thrombocytopenic purpura (TTP), asthma, dense deposit disease, pauci-immune crescentic necrotizing glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis optica, Behçet's disease, multiple sclerosis (MS), Guillain Barre syndrome, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), lupus nephritis, systemic lupus erythematosus (SLE), diabetic retinopathy, uveitis, chronic obstructive pulmonary disease (COPD), C3 glomerulopathy, transplant rejection, graft-versus-host disease (GVHD), hemodialysis, sepsis, systemic inflammatory response syndrome (SIRS), acute respiratory distress syndrome (ARDS), ANCA vasculitis, antiphospholipid syndrome, atherosclerosis, IgA nephropathy, and myasthenia gravis.

[00434] The methods of this aspect of the invention comprise administering to the subject a composition comprising an amount of a high affinity MASP-3 inhibitory antibody effective to inhibit alternative pathway-dependent complement activation and a pharmaceutically acceptable carrier. In some embodiments, the method further comprises administering a composition comprising a MASP-2 inhibitory agent. The high affinity MASP-3 inhibitory antibodies and MASP-2 inhibitory agents can be administered to a subject in need thereof, in therapeutically effective doses to treat or ameliorate conditions associated with alternative pathway complement activation and optionally also MASP-2-dependent complement activation. A therapeutically effective dose refers to the amount of the MASP-3 inhibitory antibody or a combination of a MASP-3 inhibitory antibody and a MASP-2 inhibitory agent sufficient to result in improvement of symptoms of the condition. Inhibition of alternative pathway complement activation is characterized by at least one or more of the following changes in a component of the complement system that occur as a result of administration of a high affinity MASP-3 inhibitory antibody according to various embodiments of the invention: inhibition of hemolysis and/or opsonization; inhibition of lectin-independent Factor B conversion; inhibition of lectin-independent Factor D conversion; inhibition of substrate-specific cleavage of MASP-3 serine protease; reduction of hemolysis or reduction of C3 cleavage and C3b surface deposition; reduction of Factor B and Bb deposition on an activating surface; reduction of resting (circulating and without experimental addition of an activating surface) levels of active Factor D relative to pro-Factor D; reduction of levels of active Factor D relative to pro-Factor D in response to an activating surface; and/or the reduction in the production of resting and surface-induced levels of fluid-phase Ba, Bb, C3b or C3a.

[00435] The toxicity and therapeutic efficacy of MASP-3 and MASP-2 inhibitory agents can be determined by standard pharmaceutical procedures using experimental animal models. 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 the NOAEL and the MED effects is the therapeutic ratio, which is expressed as the NOAEL/MED ratio. MASP3 inhibitory agents and MASP-2 inhibitory agents that exhibit large therapeutic rates or indices are most preferred. Data obtained from cell culture assays and animal studies can be used to formulate a range of dosages for use in humans. The dosage of MASP3 inhibitory agents and MASP-2 inhibitory agents is preferably within a range of circulating concentrations that include the MED with little or no toxicity. The dosage may vary within this range according to the dosage form employed and the route of administration used.

[00436] For any compound formulation, the therapeutically effective dose can be estimated using animal models. For example, a dose can be formulated in an animal model to achieve a circulating plasma concentration range that includes the MED. Quantitative levels of the MASP-3 inhibitory agent or MASP-2 inhibitory agent in plasma can also be measured, for example, by high performance liquid chromatography.

[00437] In addition to toxicity studies, the effective dosage can also be estimated based on the amount of target MASP protein present in a living individual and the binding affinity of the inhibitory agent for MASP-3 or MASP2.

[00438] MASP-1 levels in normal human subjects have been reported to be present in serum at levels ranging from 1.48 to 12.83 μg/mL (Terai I. et al, Clin Exp Immunol 110:317-323 (1997); Theil et al., Clin. Exp. Immunol. 169:38 (2012)). Mean serum MASP-3 concentrations in normal humans have been reported to range from about 2.0 to 12.9 μg/mL (Skjoedt M et al., Immunobiology 215(11):921-31 (2010); Degn et al., J. Immunol Methods, 361-37 (2010); Csuka et al., Mol. Immunol. 54:271 (2013). MASP-2 levels in normal human subjects have been shown to be present in serum at levels as low as 500 ng/mL, and MASP-2 levels in a given individual can be determined using a quantitative assay for MASP-2 described in Moller-Kristensen M., et al., J. Immunol. Methods 282:159-167 (2003) and Csuka et al. al., Mol. Immunol. 54:271 (2013).

[00439] Generally, the dosage of administered compositions comprising MASP-3 inhibitory agents or MASP-2 inhibitory agents varies depending on factors such as the subject's age, weight, height, sex, general medical condition, and past medical history. As an illustration, MASP-3 inhibitory agents or MASP-2 inhibitory agents (such as MASP-3 antibodies, MASP-1 antibodies, or MASP-2 antibodies), can be administered in dosage ranges of about 0.010 to 100.0 mg/kg, preferably 0.010 to 10 mg/kg, preferably 0.010 to 1.0 mg/kg, more preferably 0.010 to 0.1 mg/kg of the subject's body weight. In some embodiments, MASP-2 inhibitory agents (such as MASP-2 antibodies), are administered in dosage ranges of about preferably 0.010 to 10 mg/kg, preferably 0.010 to 1.0 mg/kg, more preferably 0.010 to 0.1 mg/kg of the subject's body weight. In some embodiments, MASP-1 inhibitory agents (such as MASP-1 antibodies) or MASP-3 inhibitory agents (such as MASP-3 antibodies) are administered in dosage ranges of about 0.010 to 100.0 mg/kg, preferably 0.010 to 10 mg/kg, such as about 1 mg/kg to about 10 mg/kg, preferably 0.010 to 1.0 mg/kg, more preferably 0.010 to 0.1 mg/kg of the subject's body weight.

[00440] The therapeutic efficacy of MASP-3 inhibitory compositions, optionally in combination with MASP-2 inhibitory compositions, or of MASP-1 inhibitory compositions, optionally in combination with MASP-2 inhibitory compositions, and the methods of the present invention in a given individual and at appropriate dosages, can be determined according to complement assays well known to those skilled in the art. Complement generates several specific products. Over the past decade, sensitive and specific assays have been developed and are commercially available for most of these activation products, including the small activation fragments C3a, C4a, and C5a and the large activation fragments iC3b, C4d, Bb, and sC5b-9. Most of these assays utilize monoclonal antibodies that react with novel antigens (neoantigens) exposed on the fragment but not on the native proteins from which they are formed, making these assays very simple and specific. Most rely on ELISA technology, although radioimmunoassay is still sometimes used for C3a and C5a. The latter assays measure the unprocessed fragments and their 'desArg' fragments, which are the main forms found in the circulation. The unprocessed fragments and C5adesArg are rapidly cleared by binding to cell surface receptors and are therefore present in very low concentrations, whereas C3adesArg does not bind to cells and accumulates in plasma. Measurement of C3a provides a sensitive, pathway-independent indicator of complement activation. Activation of the alternative pathway can be assessed by measuring the Bb fragment and/or by measuring factor D activation. Detection of the fluid phase product of membrane attack pathway activation, sC5b-9, provides evidence that complement is being fully activated. Since both the lectin pathway and the classical pathway generate the same activation products, C4a and C4d, measurement of these two fragments does not provide any information about which of these two pathways generated the activation products.

[00441] Inhibition of the alternative pathway in a mammalian subject is characterized by at least one or more of the following events in the mammalian subject following treatment with a high affinity MASP-3 inhibitory antibody described herein: inhibition of Factor D maturation; inhibition of the alternative pathway when administered to the subject in a molar ratio of about 1:1 to about 2.5:1 (MASP-3 target to mAb); the classical pathway is not inhibited; inhibition of hemolysis and/or opsonization; reduction of hemolysis or reduction of C3 cleavage and C3b surface deposition; reduction of Factor B and Bb deposition on an activating surface; a reduction in resting (circulating and without the experimental addition of an activating surface) levels of active Factor D relative to pro-Factor D; a reduction in levels of active Factor D relative to pro-Factor D in response to an activating surface; and/or a reduction in the production of resting and surface induction levels of fluid phase Ba, Bb, C3b or C3a.

[00442] Inhibition of MASP-2-dependent complement activation is characterized by at least one of the following changes in a component of the complement system that occurs as a result of administration of a MASP-2 inhibitory agent according to the methods of the invention: inhibition of the generation or production of the MASP-2-dependent complement activation system (MAC) products C4b, C3a, C5a, and/or C5b-9 (measured, e.g., as described in Example 2 of U.S. Pat. No. 7,919,094), reduction of C4 cleavage and C4b deposition, or reduction of C3 cleavage and C3b deposition. Pharmaceutical Vehicles and Delivery Carriers

[00443] In general, MASP-3 inhibitory antibody compositions or compositions comprising a combination of MASP-2 and MASP-3 inhibitory agents, which may be combined with any other selected therapeutic agents, are suitably contained in a pharmaceutically acceptable carrier. The carrier is non-toxic, biocompatible, and is selected so as not to adversely affect the biological activity of the MASP-3 inhibitory antibody or 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 MASP-3 antibodies useful in the invention, as described herein, can be formulated into solid, semi-solid, gel, liquid or gaseous form preparations, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, inhalants and injections that allow oral, parenteral or surgical administration. The invention also contemplates local administration of the compositions by coating medical devices and the like.

[00444] Suitable vehicles for parenteral delivery via injectable delivery, infusion or irrigation, and topical administration include distilled water, phosphate buffered physiological saline, lactated or normal Ringer's solutions, dextrose solution, Hank's solution, or propanediol. Additionally, sterile fixed oils may be used as a solvent or suspending medium. For this purpose, any biocompatible oil may be used including synthetic mono- or diglycerides. Additionally, fatty acids such as oleic acid are used in the preparation of injectables. The vehicle and agent may be compounded as a liquid, suspension, gel, or polymerizable or non-polymerizable, paste, or ointment.

[00445] The vehicle may also comprise a delivery carrier to maintain (i.e., extend, delay, or regulate) the release of the agent(s) or to enhance the delivery, absorption, stability, or pharmacokinetics of the therapeutic agent(s). Such a delivery carrier may include, by way of non-limiting example, microparticles, microspheres, nanospheres, or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymeric or copolymeric hydrogels, and polymeric micelles. Suitable hydrogel and micelle delivery systems include the PEO:PHB:PEO copolymers and copolymer/cyclodextrin complexes described in WO 2004/009664 A2 and the PEO and PEO/cyclodextrin complexes described in US Patent Application Publication No. 2002/0019369 A1. Such hydrogels can be injected locally at the site of intended action or subcutaneously or intramuscularly to form a sustained release depot.

[00446] The compositions of the present invention may be formulated for delivery subcutaneously, intramuscularly, intravenously, intra-arterially or as an inhalant.

[00447] For intra-articular delivery, the MASP-3 inhibitory antibody, optionally in combination with a MASP-2 inhibitory agent, may be carried in liquid or gel vehicles described above that are injectable, sustained release delivery carriers described above that are injectable, or a hyaluronic acid or hyaluronic acid derivative.

[00448] For oral administration of non-peptidergic agents, the MASP-3 inhibitory antibody, optionally in combination with a MASP-2 inhibitory agent, may be carried in an inert filler or diluent such as sucrose, cornstarch, or cellulose.

[00449] For topical administration, the MASP-3 inhibitory antibody, optionally in combination with a MASP2 inhibitory agent, may be carried in an ointment, lotion, cream, gel, drop, suppository, spray, liquid or powder form or in gel or microcapsular delivery systems via a transdermal patch.

[00450] Various nasal and pulmonary delivery systems, including aerosols, metered dose inhalers, dry powder inhalers, and nebulizers, are being developed and may be suitably adapted for delivery of the present invention in an aerosol, inhalant, or nebulized delivery carrier, respectively.

[00451] For intrathecal (IT) or intracerebroventricular (ICV) delivery, appropriately sterile delivery systems (e.g., liquids; gels, suspensions, etc.) may be used to administer the present invention.

[00452] The compositions of the present invention may also include biocompatible excipients, such as dispersing or wetting agents, suspending agents, diluents, buffers, penetration enhancers, emulsifiers, binders, thickeners, flavoring agents (for oral administration). Pharmaceutical carriers for antibodies and peptides

[00453] More specifically with respect to high affinity MASP-3 inhibitory antibodies as described herein, exemplary formulations may be parenterally administered as injectable dosages of a solution or suspension of the compound in a physiologically acceptable diluent with a pharmaceutical carrier which may be a sterile liquid such as water, oils, saline, glycerol or ethanol. Additionally, auxiliary substances such as wetting or emulsifying agents, surfactants, pH buffering substances and the like may be present in compositions comprising MASP-3 antibodies. Additional components of pharmaceutical compositions include petroleum (such as animal, vegetable or synthetic origin), e.g. soybean oil and mineral oil. In general, glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers for injectable solutions.

[00454] MASP-3 antibodies may also be administered in the form of a depot injection or implant preparation which may be formulated in such a manner as to permit a sustained or pulsatile release of the active agents. XVix. Modes of Administration

[00455] The pharmaceutical compositions comprising the MASP-3 inhibitory antibodies, optionally in combination with MASP2 inhibitory agents, can be administered in a variety of ways, depending on whether a local or systemic mode of administration is most appropriate for the condition being treated. Furthermore, the compositions of the present invention can be delivered by coating or incorporating the compositions onto or into an implantable medical device. Systemic Delivery

[00456] As used herein, the terms "systemic delivery" and "systemic administration" are intended to include, but are not limited to, oral and parenteral routes, including intramuscular (IM), subcutaneous, intravenous (IV), intra-arterial, inhalation, sublingual, buccal, topical, transdermal, nasal, rectal, vaginal, and other routes of administration that effectively result in the dispersion of the delivered agent to a single or multiple sites of intended therapeutic action. Preferred routes of systemic delivery for the present compositions include intravenous, intramuscular, subcutaneous, intra-arterial, and inhalation. It will be appreciated that the exact route of systemic administration for selected agents utilized in particular compositions of the present invention will be determined in part to account for the susceptibility of the agent to the metabolic transformation pathways associated with a given route of administration. For example, peptidergic agents may be more suitably administered by routes other than oral.

[00457] MASP-3 inhibitory antibodies as described herein may be administered to a subject in need thereof by any suitable means. Methods of delivery of MASP-3 antibodies and polypeptides include administration by oral, pulmonary, parenteral (e.g., intramuscular, intraperitoneal, intravenous (IV), or subcutaneous), inhalation (such as via a fine powder formulation), transdermal, nasal, vaginal, rectal, or sublingual routes of administration, and may be formulated into dosage forms appropriate for each route of administration.

[00458] By way of representative example, MASP-3 inhibitory peptides and antibodies can be introduced into a living body by application to a body membrane capable of absorbing the polypeptides, e.g., the nasal, gastrointestinal, and rectal membranes. The polypeptides are typically applied to the absorbent membrane in conjunction with a permeation enhancer. (See, e.g., Lee, V. H. L., Crit. Rev. Ther. Drug Carrier Sys. 5:69, (1988); Lee, V. H. L., J. Controlled Release 13:213, (1990); Lee, V. H. L., Ed., Peptide and Protein Drug Delivery, Marcel Dekker, New York (1991); DeBoer, A. G., et al., J. Controlled Release 13:241, (1990). For example, STDHF is a synthetic derivative of fusidic acid, a steroidal surfactant that is similar in structure to bile salts and has been used as a permeation enhancer for nasal administration. (Lee, W. A., Biopharm. 22, Nov./Dec. 1990.)

[00459] MASP-3 inhibitory antibodies, as described herein, can be introduced in association with another molecule, such as a lipid, to protect the polypeptides from enzymatic degradation. For example, covalent attachment of polymers, especially polyethylene glycol (PEG), has been used to protect certain proteins from enzymatic hydrolysis in the body and thus prolong half-life (Fuertges, F., et al., J. Controlled Release 11:139, (1990)). Many polymeric systems have been reported for protein delivery (Bae, Y.H., et al., J. Controlled Release 9:271, (1989); Hori, R., et al., Pharm. Res. 6:813, (1989); Yamakawa, I., et al., J. Pharm. Sci. 79:505, (1990); Yoshihiro, I., et al., J. Controlled Release 10:1 95, (1989); Asano, M., et al., J. Controlled Release 9:111, (1989); Rosenblatt, J., et al., J. Controlled Release 9:195, (1989); 78:117, (1989); Takakura, Y., et al., J. Pharm. Sci. 78:219, (1989)).

[00460] Recently, liposomes have been developed with improved serum stability and circulating half-lives (see, e.g., U.S. Patent No. 5,741,516 to Webb). In addition, various methods of liposomes and liposome-like preparations as potential drug carriers have 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).

[00461] For transdermal applications, MASP-3 inhibitory antibodies as described herein may be combined with other suitable ingredients, such as carriers and/or adjuvants. There are no limitations as to the nature of such other ingredients, except that they must be pharmaceutically acceptable for their intended administration and must not degrade the activity of the active ingredients of the composition. Examples of suitable carriers include ointments, creams, gels or suspensions, with or without purified collagen. MASP-3 inhibitory antibodies may also be impregnated into transdermal patches, adhesive dressings and bandages, preferably in liquid or semi-liquid form.

[00462] The compositions of the present invention may be systemically administered on a periodic basis at intervals determined to maintain a desired level of therapeutic effect. For example, the compositions may be administered, such as by subcutaneous injection, every two to four weeks or at less frequent intervals. The dosage regimen will be determined by the physician considering various factors that may influence the action of the combination of agents. Such factors will include the extent of progression of the condition being treated, the patient's age, sex and weight, and other clinical factors. The dosage for each individual agent will vary depending on the MASP-3 inhibitory antibody or MASP-2 inhibitory agent that is included in the composition, as well as the presence and nature of any drug delivery carrier (e.g., a sustained release delivery vehicle). Additionally, the dosage amount may be adjusted to account for variation in frequency of administration and the pharmacokinetic behavior of the delivered agent(s).

Local delivery

[00463] As used herein, the term "local" encompasses the application of a drug within or around a site of intended localized action and may include, for example, topical administration to the skin or other affected tissues, ophthalmic delivery, intrathecal (IT), intracerebroventricular (ICV), intra-articular, intracavitary, intracranial or intravesicular administration, placement or irrigation. Local administration may be preferred to allow for administration of a lower dose, to avoid systemic side effects and for more precise control of the delivery time and concentration of the active agents at the local delivery site. Local administration provides a known concentration at the target site, regardless of inter-patient variability in metabolism, blood flow, etc. Improved dosage control is also provided by the direct mode of delivery.

[00464] Local delivery of a MASP-3 inhibitory antibody or a MASP-2 inhibitory agent can be achieved in the context of surgical methods for treating a disease or condition, such as, for example, during procedures such as arterial bypass surgery, atherectomy, laser procedures, ultrasound procedures, balloon angioplasty, and stenting. For example, a MASP-3 inhibitory antibody or a MASP2 inhibitory agent can be administered to an individual in conjunction with a balloon angioplasty procedure. A balloon angioplasty procedure involves the insertion of a catheter with a deflated balloon into an artery. The deflated balloon is positioned in proximity to the atherosclerotic plaque and is inflated such that the plaque is compressed against the vascular wall. As a result, the surface of the balloon is in contact with the vascular endothelial cell layer on the surface of the blood vessel. The MASP-3 inhibitory antibody or MASP2 inhibitory agent can be attached to the balloon angioplasty catheter in a manner that allows release of the agent at the site of the atherosclerotic plaque. The agent can be attached to the balloon catheter according to standard procedures known in the art. For example, the agent can be stored in a compartment of the balloon catheter until the balloon is inflated, at which time it is released into the local environment. Alternatively, the agent can be impregnated into the surface of the balloon such that it contacts the cells of the arterial wall as the balloon is inflated. The agent can also be delivered in a perforated balloon catheter such as those described in Flugelman, M.Y., et al., Circulation 85:1110-1117, (1992). See also published PCT Application WO 95/23161 for an exemplary procedure for attaching a therapeutic protein to a balloon angioplasty catheter. Similarly, the MASP-3 inhibiting agent or MASP-2 inhibiting agent may be included in a gel or polymeric coating applied to a stent or may be incorporated into the stent material, such that the stent elutes the MASP-3 inhibiting agent or MASP-2 inhibiting agent after vascular placement.

[00465] MASP-3 inhibitory antibodies used in the treatment of arthritis and other musculoskeletal disorders can be administered locally by intra-articular injection. Such compositions can suitably include a sustained-release delivery carrier. As another example of instances where local delivery may be desired, MASP-3 inhibitory compositions used in the treatment of urogenital conditions can be conveniently instilled intravesically or into another urogenital structure.

XX. TREATMENT REGIMES

[00466] In prophylactic applications, the pharmaceutical compositions are administered to a subject susceptible to, or otherwise at risk for, an alternative pathway-associated disease or disorder, e.g., an alternative pathway disease or disorder selected from the group consisting of paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy (including hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), and thrombotic thrombocytopenic purpura (TTP), asthma, dense deposit disease, pauci-immune crescentic necrotizing glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis optica, Behçet's disease, multiple sclerosis, Guillain Barre syndrome, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), lupus nephritis, systemic lupus erythematosus (SLE), diabetic retinopathy, uveitis, chronic obstructive pulmonary disease (COPD), C3 glomerulopathy, transplant rejection, graft-versus-host disease (GVHD), hemodialysis, sepsis, systemic inflammatory response syndrome (SIRS), acute respiratory distress syndrome (ARDS), ANCA vasculitis, antiphospholipid syndrome, atherosclerosis, IgA nephropathy, and myasthenia gravis, in an amount sufficient to eliminate or reduce the risk of developing symptoms of the condition. In therapeutic applications, the pharmaceutical compositions are administered to an individual suspected of, or already suffering from, an alternative pathway-related disease or disorder, such as an alternative pathway disease or disorder selected from the group consisting of paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy (including hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), thrombotic thrombocytopenic purpura (TTP)), asthma, dense deposit disease, pauci-immune necrotizing glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis optica, Behçet's disease, multiple sclerosis, Guillain Barre syndrome, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), lupus nephritis, lupus erythematosus systemic lupus erythematosus (SLE), diabetic retinopathy, uveitis, chronic obstructive pulmonary disease (COPD), C3 glomerulopathy, transplant rejection, graft-versus-host disease (GVHD), hemodialysis, sepsis, systemic inflammatory response syndrome (SIRS), acute respiratory distress syndrome (ARDS), ANCA vasculitis, antiphospholipid syndrome, atherosclerosis, IgA nephropathy, and myasthenia gravis, in a therapeutically effective amount sufficient to alleviate, or at least partially reduce, the symptoms of the condition.

[00467] In one embodiment, the pharmaceutical composition comprising a high affinity MASP-3 inhibitory antibody is administered to a subject suffering from, or at risk of developing, PNH. Accordingly, the subject's red blood cells are opsonized by C3 fragments in the absence of the composition, and administration of the composition to the subject increases red blood cell survival in the subject. In one embodiment, the subject exhibits one or more symptoms, in the absence of the composition selected from the group consisting of (i) below normal hemoglobin levels, (ii) below normal platelet levels; (iii) above normal levels of reticulocytes and (iv) above normal levels of bilirubin and administration of the composition to the subject improves at least one or more of the symptoms, resulting in (i) increased, normal or near normal levels of hemoglobin (ii) increased, normal or near normal levels of platelets, (iii) decreased, normal or near normal levels of reticulocytes, and/or (iv) decreased, normal or near normal levels of bilirubin.

[00468] In both therapeutic and prophylactic regimens for the treatment, prevention, or reduction of the severity of a disease or condition selected from the group consisting of paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy (including hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), or thrombotic thrombocytopenic purpura (TTP)), asthma, dense deposit disease, pauci-immune necrotizing glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis optica, Behçet's disease, compositions comprising high affinity MASP-3 inhibitory antibodies and optionally MASP-2 inhibitory agents may be administered at various dosages until a sufficient therapeutic result is achieved in the subject. In one embodiment of the invention, the high affinity MASP-3 inhibitory antibody and/or MASP-2 inhibitory agent may be administered to an adult patient (e.g., an average adult weight of 70 kg) at a dose of 0.1 mg to 10,000 mg, more suitably 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 even more suitably 50.0 mg to 500 mg or 10 to 200 mg. For pediatric patients, the dosage may be adjusted in proportion to the patient's weight.

[00469] The application of the high affinity MASP-3 inhibitory antibodies and optional MASP-2 inhibitory compositions of the present invention can be carried out by a single administration of the composition (e.g., a single composition comprising MASP-3 and optionally MASP-2 inhibitory agents or bispecific or dual inhibitory agents or co-administration of separate compositions) or a limited sequence of administrations, for the treatment of an alternative pathway-related disease or disorder, such as a form of disease or disorder selected from the group consisting of paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy (including hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), thrombotic thrombocytopenic purpura (TTP), asthma, dense deposit disease, pauci-immune necrotizing glomerulonephritis, crescentic brain injury traumatic, aspiration pneumonia, endophthalmitis, neuromyelitis optica, Behçet's disease, multiple sclerosis, Guillain Barre syndrome, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), lupus nephritis, systemic lupus erythematosus (SLE), diabetic retinopathy, uveitis, chronic obstructive pulmonary disease (COPD), C3 glomerulopathy, transplant rejection, graft-versus-host disease (GVHD), hemodialysis, sepsis, systemic inflammatory response syndrome (SIRS), acute respiratory distress syndrome (ARDS), ANCA vasculitis, antiphospholipid syndrome, atherosclerosis, IgA nephropathy, and myasthenia gravis.

[00470] Alternatively, the composition may be administered at periodic intervals, such as daily, biweekly, weekly, every other week, monthly, or bimonthly for an extended period of time as determined by a physician for optimal therapeutic effect.

[00471] In some embodiments, a first composition comprising at least one high affinity MASP-3 inhibitory antibody and a second composition comprising at least one MASP-2 inhibitory agent are administered to a subject suffering from, or at risk of developing, a disease or condition selected from the group consisting of paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy (including hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), thrombotic thrombocytopenic purpura (TTP), asthma, dense deposit disease, pauci-immune necrotizing glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis optica, Behçet's disease, multiple sclerosis, Guillain Barre syndrome, Alzheimer's disease, lateral sclerosis amyotrophic lateral sclerosis (ALS), lupus nephritis, systemic lupus erythematosus (SLE), diabetic retinopathy, uveitis, chronic obstructive pulmonary disease (COPD), C3 glomerulopathy, transplant rejection, graft-versus-host disease (GVHD), hemodialysis, sepsis, systemic inflammatory response syndrome (SIRS), acute respiratory distress syndrome (ARDS), ANCA vasculitis, antiphospholipid syndrome, atherosclerosis, IgA nephropathy, and myasthenia gravis.

[00472] In one embodiment, the first composition comprising at least one high affinity MASP-3 inhibitory antibody and a second composition comprising at least one MASP-2 inhibitory agent are administered simultaneously (i.e., within a time interval of no more than about 15 minutes or less, such as no more than any of 10, 5, or 1 minute). In one embodiment, the first composition comprising at least one high affinity MASP-3 inhibitory antibody and a second composition comprising at least one MASP-2 inhibitory agent are administered sequentially (i.e., the first composition is administered before or after administration of the second composition, wherein the time interval of administration is greater than 15 minutes). In some embodiments, the first composition comprising at least one high affinity MASP-3 inhibitory antibody and a second composition comprising at least one MASP-2 inhibitory agent are administered concomitantly (i.e., the period of administration of the first composition coincides with administration of the second composition). For example, in some embodiments, the first composition and/or the second composition are administered over a period of at least one, two, three, or four weeks or more. In one embodiment, at least one high affinity MASP-3 inhibitory antibody and at least one MASP-2 inhibitory agent are combined in a unitary dosage form. In one embodiment, a first composition comprising at least one high affinity MASP-3 inhibitory antibody and a second composition comprising at least one MASP-2 inhibitory agent are packaged together in a kit for use in treating an alternative pathway-related disease or condition, such as paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy (including hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), thrombotic thrombocytopenic purpura (TTP), asthma, dense deposit disease, pauci-immune crescentic necrotizing glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis optica, Behçet's disease, multiple sclerosis, Guillain Barre syndrome, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), nephritis lupus, systemic lupus erythematosus (SLE), diabetic retinopathy, uveitis, chronic obstructive pulmonary disease (COPD), C3 glomerulopathy, transplant rejection, graft-versus-host disease (GVHD), hemodialysis, sepsis, systemic inflammatory response syndrome (SIRS), acute respiratory distress syndrome (ARDS), ANCA vasculitis, antiphospholipid syndrome, atherosclerosis, IgA nephropathy, or myasthenia gravis.

[00473] In some embodiments, the individual suffering from PNH, age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy (including hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), thrombotic thrombocytopenic purpura (TTP)), asthma, dense deposit disease, pauci-immune necrotizing glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis optica, Behçet's disease, multiple sclerosis, Guillain Barre syndrome, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), lupus nephritis, systemic lupus erythematosus (SLE), diabetic retinopathy, uveitis, chronic obstructive pulmonary disease (COPD), C3 glomerulopathy, transplant rejection, graft-versus-host disease (GVHD), hemodialysis, sepsis, systemic inflammatory response syndrome (SIRS), acute respiratory distress syndrome (ARDS), ANCA vasculitis, antiphospholipid syndrome, atherosclerosis, IgA nephropathy, and myasthenia gravis, has previously undergone or is currently undergoing treatment with a terminal complement inhibitor that inhibits the cleavage of complement protein C5. In some embodiments, the method comprises administering to the subject a composition of the invention comprising a high-affinity MASP-3 inhibitory antibody and optionally a MASP-2 inhibitor and further administering to the subject a terminal complement inhibitor that inhibits the cleavage of complement protein C5. In some embodiments, the terminal complement inhibitor is a humanized anti-C5 antibody or antigen-binding fragment thereof. In some embodiments, the terminal complement inhibitor is eculizumab.

Xxi . Examples

[00474] The following examples merely illustrate the best mode now contemplated for practicing the invention, but are not to be construed as limiting the invention. All literature citations are expressly incorporated by reference.

EXAMPLE 1

[00475] This Example demonstrates that MASP-2-deficient mice are protected from Neisseria meningitidis-induced mortality following infection with N. meningitidis serogroup A or N. meningitidis serogroup B. Methods:

[00476] MASP-2 knockout mice (MASP-2 KO mice) were generated as described in Example 1 of US 7,919,094, incorporated herein by reference. 10-week-old MASP-2 KO mice (n=10) and wild-type (WT) C57/BL6 mice (n=10) were inoculated by intraperitoneal (ip) injection with a dosage of 2.6 x 107 CFU of N. meningitidis serogroup A Z2491 in a volume of 100 μL. The infectious dose was administered to mice in conjunction with iron dextran at a final concentration of 400 mg/kg. Survival of mice post-infection was monitored over a 72-hour time period.

[00477] In a separate experiment, 10-week-old MASP-2 KO mice (n=10) and WT C57/BL6 mice (n=10) were challenged by i.p. injection with a dosage of 6 x 106 CFU of N. meningitidis serogroup B strain MC58 in a volume of 100 μL. The infective dose was administered to mice in conjunction with iron dextran at a final dose of 400 mg/kg. Survival of mice post-infection was monitored over a 72-hour time period. A disease score was also determined for the WT and MASP-2 KO mice over the 72-hour post-infection period based on the disease scoring parameters described below in TABLE 5, which is based on the scheme of Fransen et al. (2010) with minor modifications. TABLE 5: Disease scores associated with clinical signs in infected mice <DRAW-CODE>>

[00478] Blood samples were collected from the mice at one-hour intervals after infection and analyzed to determine the serum level (log cfu/mL) of N. meningitidis to verify infection and to determine the rate of clearance of bacteria from the serum. Results:

[00479] FIGURE 6 is a Kaplan-Meyer plot graphically illustrating the percent survival of WT and MASP-2 KO mice following administration of an infectious dose of 2.6 x 10 7 cfu of N. meningitidis serogroup A Z2491. As shown in FIGURE 6, 100% of the MASP-2 KO mice survived through the 72-hour post-infection period. In contrast, only 80% of the WT mice (p=0.012) were still alive at 24 hours post-infection, and only 50% of the WT mice were still alive at 72 hours post-infection. These results demonstrate that MASP-2-deficient mice are protected from N. meningitidis serogroup A Z2491-induced mortality.

[00480] FIGURE 7 is a Kaplan-Meyer plot graphically illustrating the percent survival of WT and MASP-2 KO mice following administration of an infectious dose of 6 x 106 cfu of N. meningitidis serogroup B strain MC58. As shown in FIGURE 7, 90% of the MASP-2 KO mice survived through the 72-hour post-infection period. In contrast, only 20% of the WT mice (p = 0.0022) were still alive 24 hours post-infection. These results demonstrate that MASP-2-deficient mice are protected from mortality induced by N. meningitidis serogroup B strain MC58.

[00481] FIGURE 8 graphically illustrates the log cfu/mL of N. meningitidis serogroup B strain MC58 recovered at different time points in blood samples collected from MASP-2 KO and WT mice following i.p. infection with 6x106 cfu of N. meningitidis serogroup B strain MC58 (n = 3 at different time points for both groups of mice). Results are expressed as means ± SEM. As shown in FIGURE 8, in WT mice the level of N. meningitidis in blood peaked at approximately 6.0 log cfu/mL at 24 hours post-infection and declined to approximately 4.0 log cfu/mL by 36 hours post-infection. In contrast, in MASP-2 KO mice, the blood level of N. meningitidis peaked at approximately 4.0 log cfu/mL at 12 h postinfection and declined to approximately 1.0 log cfu/mL by 36 h postinfection (the symbol "*" indicates p < 0.05; the symbol "**" indicates p = 0.0043). These results demonstrate that although MASP-2 KO mice were infected with the same dose of N. meningitidis serogroup B strain MC58 as WT mice, MASP-2 KO mice had increased clearance of bacteremia compared with WT.

[00482] FIGURE 9 graphically illustrates the mean disease score of MASP-2 KO and WT mice at 3, 6, 12, and 24 hours post-infection with 6x106 cfu of N. meningitidis serogroup B strain MC58. As shown in FIGURE 9, MASP-2-deficient mice showed high resistance to infection, with much lower disease scores at 6 hours (the symbol "*" indicates p = 0.0411), 12 hours (the symbol "**" indicates p = 0.0049), and 24 hours (the symbol "***" indicates p = 0.0049) post-infection, compared to WT mice. Results in FIGURE 9 are expressed as means ± SEM.

[00483] In summary, the results in this Example demonstrate that MASP-2-deficient mice are protected from N. meningitides-induced mortality following infection with N. meningitidis serogroup A or N. meningitidis serogroup B.

EXAMPLE 2

[00484] This Example demonstrates that administration of MASP-2 antibody following infection with N. meningitidis increases the survival of N. meningitidis-infected mice. Rationale:

[00485] As described in Example 24 of U.S. Patent 7,919,094, incorporated herein by reference, rat MASP-2 protein was used to screen a Fab phage display library, from which Fab2 #11 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 MASP-2 antibody of the mouse IgG2a isotype was characterized for pharmacodynamic parameters (as described in Example 38 of U.S. Patent 7,919,094).

[00486] In this Example, Fab2-derived mouse full-length MASP-2 antibody #11 was analyzed in the mouse model of N. meningitidis infection. Methods:

[00487] The Fab2-derived mouse IgG2a full-length MASP-2 antibody isotype #11, generated as described above, was tested in the mouse model of N. meningitidis infection as follows. 1. Administration of mouse MASP-2 monoclonal antibodies (MoAb) following infection

[00488] 9-week-old Charles River C57 BL6 mice were treated with rat MASP-2 inhibitory antibody (1.0 mg/kg) (n = 12) or isotype control antibody (n = 10) 3 hours after i.p. injection with a high dose (4x106 cfu) of N. meningitidis serogroup B strain MC58. Results:

[00489] FIGURE 10 is a Kaplan-Meyer plot graphically illustrating the percent survival of mice following administration of an infectious dose of 4x106 cfu of N. meningitidis serogroup B strain MC58, followed by administration 3 hours post-infection of either MASP-2 inhibitory antibody (1.0 mg/kg) or isotype control antibody. As shown in FIGURE 10, 90% of the mice treated with MASP-2 antibody survived through the 72-hour post-infection period. In contrast, only 50% of the mice treated with isotype control antibody survived through the 72-hour post-infection period. The symbol "*" indicates p=0.0301, as determined by comparing the two survival curves.

[00490] These results demonstrate that administration of a MASP-2 antibody is effective in treating and improving survival in individuals infected with N. meningitidis.

[00491] As demonstrated herein, the use of MASP-2 antibody in the treatment of an individual infected with N. meningitidis is effective when administered within 3 hours of infection and is expected to be effective within 24 hours to 48 hours of infection. Meningococcal disease (meningococcemia or meningitis) is a medical emergency and therapy will typically be initiated immediately if meningococcal disease is suspected (i.e., before N. meningitidis is positively identified as the etiologic agent).

[00492] In view of the results in the MASP-2 KO mouse demonstrated in EXAMPLE 1, it is believed that administration of MASP-2 antibody prior to infection with N. meningitidis would also be effective in preventing or ameliorating the severity of the infection.

EXAMPLE 3

[00493] This Example demonstrates that complement-dependent killing of N. meningitidis in human sera is dependent on MASP-3. Rationale:

[00494] Patients with decreased serum levels of functional MBL have been shown to exhibit increased susceptibility to recurrent bacterial and fungal infections (Kilpatrick et al., Biochim Biophys Acta 1572:401-413 (2002)). N. meningitidis is known to be recognized by MBL, and MBL-deficient sera have been shown not to lyse N. meningitidis.

[00495] In view of the results described in Examples 1 and 2, a series of experiments were performed to determine the efficacy of administering MASP-2 antibody to treat N. meningitidis infection in control and complement-deficient human sera. The experiments were performed in a high serum concentration (20%) in order to preserve the complement pathway. Methods: 1. Serum bactericidal activity in various complement-deficient human sera and in human sera treated with human MASP-2 antibody

[00496] The following control human sera and complement-deficient human sera were used in this experiment: TABLE 6: Human serum samples tested (as shown in FIGURE 11)

[00497] A recombinant antibody against human MASP-2 was isolated from a combinatorial antibody library (Knappik, A., et al., J. Mol. Chem. Biol. 296:57-86 (2000)), using recombinant human MASP-2A as an antigen (Chen, C.B. and Wallis, J. Biol. Chem. 276:25894-25902 (2001)). An anti-human scFv fragment that potently inhibited lectin-mediated activation of C4 and C3 in human plasma (IC50 ~20 nM) was identified and converted to a full-length human IgG4 antibody.

[00498] N. meningitidis serogroup B MC58 was incubated with the different sera presented in TABLE 6, each at a serum concentration of 20%, with or without the addition of human MASP-2 inhibitory antibody (3 μg in 100 μl total volume) at 37°C with shaking. Samples were collected at the following evaluation time points: 0, 30, 60 and 90 minute intervals, transferred to plates and the viable content was determined. Heat-inactivated human serum was used as a negative control. Results:

[00499] FIGURE 11 graphically illustrates the log cfu/mL of viable N. meningitidis serogroup B MC58 titer recovered at different time points in the human serum samples shown in TABLE 6. TABLE 7 provides the Student's t-test results for FIGURE 11; TABLE 7: Student's t-test results for FIGURE 11 (60 minute time point)

[00500] As shown in FIGURE 11 and TABLE 7, complement-dependent killing of N. meningitidis in 20% human serum was significantly increased by the addition of the human MASP-2 inhibitory antibody.

2. Serum bactericidal activity in various complement-deficient human sera

[00501] The following control human sera and complement-deficient human sera were used in this experiment: TABLE 8: Human serum samples tested (as shown in FIGURE 12)

[00502] Note: The MASP-3 -/- (MASP-1 +) serum in sample D was collected from an individual with 3MC syndrome, which is a unifying term for the coincidental Carnevale, Mingarelli, Malpuech, and Michels syndromes. As further described in Example 4, mutations in exon 12 of the MASP-1/3 gene render the serine protease domain of MASP-3, but not MASP-1, dysfunctional. As described in Example 10, profactor D is preferentially present in 3MC serum, whereas activated factor D is preferentially present in normal human serum.

[00503] N. meningitidis serogroup B MC58 was incubated with different complement-deficient human sera, each at a serum concentration of 20%, at 37°C with shaking. Samples were collected at the following evaluation time points: 0, 15, 30, 15, 60, 90 and 120 minute intervals, transferred to plates and the viable content was determined. Heat-inactivated human serum was used as a negative control. Results:

[00504] FIGURE 12 graphically illustrates the log cfu/mL of viable N. meningitidis serogroup B MC58 recovered at different time points in the human serum samples shown in TABLE 8. As shown in FIGURE 12, WT (NHS) serum has the highest level of bactericidal activity for N. meningitidis. In contrast, MBL -/- and MASP-3 -/- (which is MASP-1 sufficient) human sera have no bactericidal activity. These results indicate that complement-dependent killing of N. meningitidis in 20% (v/v) human serum is dependent on MASP-3 and MBL. TABLE 9 provides the results of Student's t-test for FIGURE 12; TABLE 9: Student's t-test results for FIGURE 12

[00505] In summary, the results shown in FIGURE 12 and TABLE 9 demonstrate that complement-dependent killing of N. meningitidis in 20% human serum is dependent on MASP-3 and MBL. 3. Complement-dependent killing of N. meningitidis in 20% (v/v) MASP-2, MASP-1/3, or MBL A/C-deficient mouse serum.

[00506] The following control mouse sera and complement-deficient mouse sera were used in this experiment: TABLE 10: Mouse Serum Samples Tested (as shown in FIGURE 13)

[00507] N. meningitidis serogroup B MC58 was incubated with different complement-deficient mouse sera, each at a serum concentration of 20%, at 37°C with shaking. Samples were collected at the following evaluation time points: 15, 30, 60, 90 and 120 minute intervals, transferred to plates and the viable content was determined. Heat-inactivated human serum was used as a negative control. Results:

[00508] FIGURE 13 graphically illustrates the log cfu/mL of viable N. meningitidis serogroup B MC58 content recovered at different time points in the mouse serum samples shown in TABLE 10. As shown in FIGURE 13, MASP-2 -/- mouse serum has a higher level of bactericidal activity for N. meningitidis than WT mouse serum. In contrast, MASP-1/3 -/- mouse sera do not possess any bactericidal activity. The symbol "**" indicates p = 0.0058, the symbol "***" indicates p = 0.001. TABLE 11 provides the results of Student's t-test for FIGURE 13. TABLE 11: Student's t-test results for FIGURE 13

[00509] In summary, the results in this Example demonstrate that MASP-2 -/- serum has a higher level of bactericidal activity for N. meningitidis than WT serum and that complement-dependent killing of N. meningitidis in 20% serum is dependent on MBL and MASP-3.

EXAMPLE 4

[00510] This Example describes a series of experiments that were performed to determine the mechanism of MASP-3-dependent resistance to N. meningitidis infection observed in MASP-2 KO mice, as described in Examples 1-3. Rationale:

[00511] In order to determine the mechanism of MASP-3-dependent resistance to N. meningitidis infection observed in MASP-2 KO mice (described in Examples 1-3 above), a series of experiments were performed as follows. 1. MASP-1/3-deficient mice exhibit no deficiency in the functional activity of the lectin pathway (also known as “LEA-2”) Methods:

[00512] To determine whether MASP-1/3-deficient mice are deficient in the functional activity of the lectin pathway (also referred to as LEA-2), an assay was performed to measure the kinetics of C3 convertase activity in plasma from various complement-deficient mouse strains tested under specific lectin activation pathway assay conditions (1% plasma), as described in Schwaeble W. et al., PNAS vol 108(18):7523-7528 (2011), incorporated herein by reference.

[00513] Plasma was tested from WT, C4-/-, MASP-1/3-/-; Factor B-/- and MASP-2-/- mice as follows.

[00514] To measure C3 activation, microtiter plates were coated with mannan (1 μg/well), zymosan (1 μg/well) in coating buffer (15 mM Na2Co3, 35 mM NaHCO3), or immune complexes, generated in situ by coating with 1% human serum albumin (HSA) in coating buffer then addition of sheep anti-HAS serum (2 μg/mL) in TBS (10 mM Tris, 140 mM NaCl, pH 7.4) with 0.05% Tween 20 and 5 mM Ca++. Plates were blocked with 0.1% HSA in TBS and washed three times with TBS/Tween20/Ca++. Plasma samples were diluted in 4 mM barbital, 145 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4, added to the plates and incubated for 1.5 h at 37°C. After washing, bound C3b was detected using rabbit anti-human C3c (Dako), followed by goat anti-rabbit IgG conjugated with alkaline phosphatase and p-nitrophenyl-phosphate. Results:

[00515] The kinetics of C3 activation (measured by deposition of C3b on mannan-coated plates with 1% serum) under lectin pathway-specific conditions are shown in FIGURE 14. No C3 cleavage was observed in MASP-2 -/- plasma. Factor B -/- (Factor B -/-) plasma cleaved C3 at half the rate of WT plasma, likely due to loss of the amplification loop. A significant lectin pathway-dependent delay in the conversion of C3 to C3b was observed in C4 -/- (T1/2=33min) as well as in MASP-1/3 -/- deficient plasma (T1/2=49min). This delay in C3 activation in MASP-1/3 -/- plasma was shown to be MASP-1-dependent rather than MASP-3-dependent. (See Takahashi M. et al., J Immunol 180:6132-6138 (2008).) These results demonstrate that MASP-1/3-deficient mice do not exhibit impairment of the functional activity of the lectin pathway (also known as “LEA-2”) 2. Effect of inherited MASP-3 deficiency on alternative pathway activation. Rationale:

[00516] The effect of inherited MASP-3 deficiency on alternative pathway activation was determined by testing serum from a MASP-3-deficient patient with 3MC syndrome caused by a frameshift mutation in the exon encoding the MASP-3 serine protease. 3MC syndrome is a unifying term for the coincidental Carneavale, Mingarelli, Malpuech, and Michels syndromes. These rare autosomal recessive disorders exhibit a spectrum of developmental features, including characteristic facial dysmorphism, cleft lip and/or palate, craniosynostosis, learning disability, and genital, limb, and vesicorenal abnormalities. Rooryck et al., Nature Genetics 43:197-203 (2011) studied 11 families with 3MC syndrome and identified two mutated genes, COLEC11 and MASP-1. Mutations in the MASP-1 gene render the exon encoding the MASP-3 serine protease domain, but not the exons encoding the MASP-1 serine protease, dysfunctional. Therefore, 3MC patients with mutations in the exon encoding the MASP-3 serine protease are deficient in MASP-3 but sufficient in MASP-1. Methods:

[00517] MASP-3-deficient serum was obtained from a 3MC patient, the mother and father of the 3MC patient (both heterozygous for the allele carrying a mutation that renders the exon encoding the serine protease domain of MASP-3 dysfunctional), as well as from a C4-deficient patient (deficient in both human C4 genes) and an MBL-deficient individual. An alternative pathway assay was performed under specific traditional AP conditions (BBS/Mg++/EGTA, without Ca++, where BBS = barbital-buffered saline containing sucrose), as described in Bitter-Suermann et al., Eur. J. Immunol 11:291-295 (1981)), in zymosan-coated microtiter plates at serum concentrations ranging from 0.5 to 25%, and C3b deposition was measured over time. Results:

[00518] FIGURE 15 graphically illustrates the level of alternative pathway-driven C3b deposition in zymosan-coated microtiter plates as a function of serum concentration in serum samples obtained from MASP-3-deficient, C4-deficient, and MBL-deficient individuals. As shown in FIGURE 15, the serum from the MASP-3-deficient patient has residual alternative pathway (AP) activity at high serum concentrations (serum concentrations of 25%, 12.5%, 6.25%), but a significantly higher AP50 (i.e., 9.8% of serum required to achieve 50% of maximal C3 deposition).

[00519] FIGURE 16 graphically illustrates the level of alternative pathway-driven C3b deposition in zymosan-coated microtiter plates under “traditional” (AP-specific) alternative pathway-specific conditions (i.e., BBS/EGTA/Mg++ without Ca++) as a function of time in 10% human serum samples obtained from MASP-3-deficient, C4-deficient, and MBL-deficient individuals.

[00520] TABLE 12 below summarizes the AP50 results shown in FIGURE 15 and the time halves for C3b deposition shown in FIGURE 16. TABLE 12: Summary of results shown in FIGURES 15 and 16

[00521] Note: In BBS/Mg++/EGTA buffer, lectin pathway-mediated effects are deficient due to the absence of Ca++ in this buffer.

[00522] In summary, under the conditions of these assays, the alternative pathway is significantly compromised in the 3MC patient. 3. Measurement of C3b deposition on mannan, zymosan, and S. pneumonia D39 in MASP-2 or MASP-1/3-deficient mouse sera. Methods:

[00523] C3b deposition was measured in mannan-, zymosan-, and S. pneumonia D39-coated microtiter plates using mouse serum concentrations ranging from 0% to 20% obtained from MASP-2-/-, MASP-1/3-/-, and WT mice. C3b deposition assays were performed under “traditional” alternative pathway-specific conditions (i.e., BBS/EGTA/Mg++ without Ca++) or under physiological conditions allowing both the lectin and alternative pathways to function (i.e., BBS/Mg++/Ca++). Results:

[00524] FIGURE 17A graphically illustrates the level of C3b deposition on mannan-coated microtiter plates as a function of serum concentration in serum samples obtained from MASP-2-deficient and MASP-1/3-deficient WT mice under “traditional” alternative pathway-specific conditions (i.e., BBS/EGTA/Mg++ without Ca++) or under physiological conditions that allow both the lectin pathway and the alternative pathway to function (BBS/Mg++/Ca++). FIGURE 17B graphically illustrates the level of C3b deposition on zymosan-coated microtiter plates as a function of serum concentration in serum samples from MASP-2-deficient and MASP-1/3-deficient WT mice under traditional AP-specific conditions (i.e., BBS/EGTA/Mg++ without Ca++) or under physiological conditions that allow both the lectin pathway and the alternative pathway to function (BBS/Mg++/Ca++). FIGURE 17C graphically illustrates the level of C3b deposition on S. pneumoniae D39-coated microtiter plates as a function of serum concentration in serum samples from MASP-2-deficient and MASP-1/3-deficient WT mice under specific “traditional” AP conditions (i.e., BBS/EGTA/Mg++ without Ca++) or under physiological conditions that allow both the lectin pathway and the alternative pathway to function (BBS/Mg++/Ca++).

[00525] Figure 18A graphically illustrates the results of a C3b deposition assay in highly diluted sera performed in mannan-coated microtiter plates under specific traditional AP conditions (i.e., BBS/EGTA/Mg++ without Ca++) or under physiological conditions that allow both the lectin pathway and the alternative pathway to function (BBS/Mg++/Ca++), using serum concentrations ranging from 0% to 1.25%. Figure 18B graphically illustrates the results of a C3b deposition assay in highly diluted sera performed in zymosan-coated microtiter plates under specific traditional AP conditions (i.e., BBS/EGTA/Mg++ without Ca++) or under physiological conditions that allow both the lectin pathway and the alternative pathway to function (BBS/EGTA/Mg++/Ca++), using serum concentrations ranging from 0% to 1.25%. Figure 18C graphically illustrates the results of a C3b deposition assay performed on S. pneumoniae D39-coated microtiter plates under specific traditional AP conditions (i.e., BBS/EGTA/Mg++ without Ca++) or under physiological conditions that allow both the lectin pathway and the alternative pathway to function (BBS/EGTA/Mg++/Ca++), using serum concentrations ranging from 0% to 1.25%.

[00526] As shown in FIGURES 18A-C, C3b deposition assays were also performed under traditional alternative pathway-specific conditions (i.e., BBS/EGTA/Mg++ without Ca++) or under physiological conditions allowing both the lectin pathway and the alternative pathway to function (BBS/Mg++/Ca++), using higher dilutions ranging from 0% to 1.25% serum on mannan-coated plates (FIGURE 18A); zymosan-coated plates (FIGURE 18B); and S. pneumoniae D39-coated plates (FIGURE 18C). The alternative pathway disappears at higher serum dilutions, whereby the activity observed in MASP-1/3-deficient serum in the presence of Ca++ is MASP-2-mediated LP activity and the activity in MASP-2-deficient serum in the presence of Ca++ is residual MASP-1/3-mediated activation of the AP. Discussion:

[00527] The results described in this Example demonstrate that a MASP-2 inhibitor (or MASP-2 knockout) provides significant protection from N. meningitidis infection by promoting activation of the alternative MASP-3-driven pathway. The results of the mouse serum bacteriolysis assays and the human serum bacteriolysis assays further show, by monitoring the bactericidal activity of the serum against N. meningitidis, that bactericidal activity against N. meningitidis is absent in MBL-deficient (human MBL-deficient and mouse MBL A and MBL C double-deficient serum).

[00528] FIGURE 1 illustrates the new understanding of the lectin pathway and the alternative pathway based on the results provided here. FIGURE 1 delineates the role of LEA-2 in both opsonization and lysis. While MASP-2 is the initiator of “downstream” C3b deposition (and resulting opsonization) in multiple physiologically lectin-dependent settings (FIGURES 18A, 18B, 18C), it also plays a role in the lysis of serum-sensitive bacteria. As illustrated in FIGURE 1, the proposed molecular mechanism responsible for the increased bactericidal activity of MASP-2-deficient or MASP-2-depleted serum/plasma for serum-sensitive pathogens such as N. meningitidis is that, for bacterial lysis, the lectin pathway recognition complexes associated with MASP-1 and MASP-3 must bind in close proximity to each other on the bacterial surface, thereby allowing MASP-1 to cleave MASP-3. In contrast to MASP-1 and MASP-2, MASP-3 is not a self-activating enzyme but, in many cases, requires activation/cleavage by MASP-1 to be converted to its enzymatically active form.

[00529] As further shown in FIGURE 1, activated MASP-3 can then cleave factor B bound to C3b on the pathogen surface to initiate the alternative pathway activation cascade by formation of the enzymatically active alternative pathway C3 and C5 convertases C3bBb and C3bBb(C3b)n, respectively. Lectin pathway activation complexes containing MASP-2 do not participate in MASP-3 activation, and in the absence or following depletion of MASP-2, bulk lectin pathway activation complexes will be loaded with either MASP-1 or MASP-2. Therefore, in the absence of MASP-2, the probability is markedly increased that, on the microbial surface, the lectin pathway activation complexes containing MASP-1 and MASP-3 will come into close proximity to each other, leading to more MASP-3 being activated and thus leading to an increased rate of MASP-3-mediated cleavage of factor B-bound C3b to form the alternative pathway C3 and C5 convertases C3bBb and C3bBb(C3b)n on the microbial surface. This leads to the activation of the C5b-C9 terminal activation cascades that form the Membrane Attack Complex, composed of surface-bound C5b associated with C6, C5bC6 associated with C7, C5bC6C7 associated with C8, and C5bC6C7C8, leading to the polymerization of C9 that inserts into the bacterial surface structure and forms a pore in the bacterial wall, which will lead to the osmolytic killing of the complement-targeted bacteria.

[00530] The core of this new concept is that the data provided here clearly show that lectin pathway activation complexes guide the two distinct activation routes, as illustrated in FIGURE 1.

EXAMPLE 5

[00531] This Example demonstrates the inhibitory effect of MASP-2 deficiency and/or MASP-3 deficiency on red blood cell lysis of blood samples obtained from a rat model of paroxysmal nocturnal hemoglobinuria (PNH). Rationale:

[00532] Paroxysmal nocturnal hemoglobinuria (PNH), also referred to as Marchiafava-Micheli syndrome, is an acquired and potentially fatal blood disorder characterized by complement-induced intravascular hemolytic anemia. The hallmark of PNH is chronic complement-mediated intravascular hemolysis, which is a consequence of dysregulated activation of the alternative complement pathway due to the absence of the complement regulators CD55 and CD59 on PNH erythrocytes, with subsequent hemoglobinuria and anemia. Lindorfer, M.A., et al., Blood 115(11) (2010), Risitano, A.M, Mini-Reviews in Medicinal Chemistry, 11:528-535 (2011). Anemia in PNH is due to the destruction of red blood cells in the bloodstream. Symptoms of PNH include red urine due to the appearance of hemoglobin in the urine, back pain, fatigue, shortness of breath, and thrombosis. PNH can develop on its own, referred to as "primary PNH," or in the context of other bone marrow disorders, such as aplastic anemia, referred to as "secondary PNH." Treatment for PNH includes blood transfusion for anemia, anticoagulation for thrombosis, and the use of the monoclonal antibody eculizumab (Soliris®), which protects blood cells from destruction by the immune system by inhibiting the complement system (Hillmen P. et al., N. Engl. J. Med. 350(6):552-9 (2004)). Eculizumab (Soliris®) is a humanized monoclonal antibody that targets the C5 component of complement, blocking its cleavage by C5 convertases, thereby preventing the production of C5a and the formation of MAC. Treatment of patients with PNH with eculizumab resulted in a reduction in intravascular hemolysis, as measured by lactate dehydrogenase (LDH), leading to stabilization of hemoglobin and transfusion independence in about half of patients (Hillmen P, et al., Mini-Reviews in Medicinal Chemistry, vol 11(6) (2011)). While almost all patients undergoing eculizumab therapy achieve normal or near-normal LDH levels (due to control of intravascular hemolysis), only about one-third of patients achieve a hemoglobin value of 11 g/dL, and the remaining patients receiving eculizumab continue to have moderate to severe anemia (i.e., transfusion-dependent) in approximately equal proportions (Risitano A.M. et al., Blood 113:4094-100 (2009)). As described in Risitano et al., Mini-Reviews in Medicinal Chemistry 11:528-535 (2011), it was demonstrated that PNH patients receiving eculizumab contained C3 fragments bound to a substantial portion of their PNH erythrocytes (whereas untreated patients did not), leading to the conclusion that membrane-bound C3 fragments function as opsonins in PNH erythrocytes, resulting in their trapping in reticuloendothelial cells via specific C3 receptors and subsequent extravascular hemolysis. Therefore, therapeutic strategies other than eculizumab are needed for those patients who develop C3 fragment-mediated extravascular hemolysis because they continue to require red blood cell transfusions.

[00533] This Example describes methods for evaluating the effect of MASP-2 and MASP-3 deficient serum on red blood cell lysis of blood samples obtained from a mouse model of PNH and demonstrates the efficacy of MASP-2 inhibition and/or MASP-3 inhibition to treat subjects suffering from PNH and also supports the use of MASP-2 inhibitors and/or MASP-3 inhibitors (including dual or bispecific MASP-2/MASP-3 inhibitors) to ameliorate the effects of C3 fragment-mediated extravascular hemolysis in subjects with PNH undergoing therapy with a C5 inhibitor, such as eculizumab. Methods: PNH Animal Model:

[00534] Blood samples were obtained from gene-targeted mice with deficiencies in C3-deficient (Crry/C3-/-) and CD55/CD59 mice. These mice lack the respective surface complement regulators on their erythrocytes and therefore these erythrocytes are susceptible to spontaneous complement autolysis, as are human PNH blood cells.

[00535] In order to further sensitize these erythrocytes, these cells were used with and without mannan coating and then tested for hemolysis in WT C56/BL6 plasma, MBL null plasma, MASP-2 -/- plasma, MASP-1/3 -/- plasma, human NHS, human MBL -/- plasma, and human MASP-2 antibody-treated NHS. 1. Hemolysis assay of Crry/C3 and CD55/CD59 double-deficient murine erythrocytes in MASP-2 deficient/depleted serum and controls Day 1. Preparation of murine red blood cells (± mannan coating).

[00536] Materials included: fresh rat blood, BBS/Mg++/Ca++ (4.4 mM barbituric acid, 1.8 mM sodium barbitone, 145 mM NaCl, pH 7.4, 5mM Mg++, 5mM Ca++), chromium chloride, CrCly6H20 (0.5mg/mL in BBS/Mg++/Ca++), and mannan, 100 μg/mL in BBS/Mg++/Ca++.

[00537] Whole blood (2 mL) was centrifuged for 1-2 min at 2000xg in a refrigerated centrifuge at 4°C. Plasma and buffy coat were aspirated. The sample was then washed 3x by resuspending the red blood cell pellet in 2 mL of ice-cold BBS/gelatin/Mg++/Ca++ and repeating the centrifugation step. After the third wash, the pellet was resuspended in 4 mL of BBS/Mg++/Ca++. A 2 mL aliquot of the red blood cells was reserved as an uncoated control. To the remaining 2 mL, 2 mL of CrCl3 and 2 mL of mannan were added, and the sample was incubated with gentle shaking at room temperature for 5 min. The reaction was terminated by adding 7.5 mL of BBS/gelatin/Mg++/Ca++. The sample was centrifuged as above, resuspended in 2 mL BBS/gelatin/Mg++/Ca++ and washed twice more as above, then stored at 4°C. Day 2. Hemolysis assay

[00538] Materials included BBS/gelatin/Mg++/Ca++ (as above), test sera, 96-well round-bottom and flat-bottom plates, and a spectrophotometer that reads 96-well plates at 410-414 nm.

[00539] The red blood cell concentration was first determined and the cells were adjusted to 109/mL and stored at this concentration. Prior to use, the cells were diluted in assay buffer to 108/mL and then 100 μL per well was used. Hemolysis was measured at 410-414 nm (allowing greater sensitivity than 541 nm). Dilutions of test sera were prepared in ice-cold BBS/gelatin/Mg++/Ca++. 100 μL of each serum dilution was pipetted into a round bottom plate. 100 μL of appropriately diluted red blood cell preparation was added (i.e., 108/mL), incubated at 37°C for approximately 1 hour and observed for lysis. (Plates may be photographed at this time.) The plate was then centrifuged at maximum speed for 5 minutes. 100 μL of the fluid phase was aspirated, transferred to flat bottom plates and the OD was recorded at 410-414 nm. Red blood cell pellets were retained (these can subsequently be lysed with water to obtain a reverse result). Experiment #1

[00540] Fresh blood was obtained from CD55/CD59 double-deficient mice and blood from Crry/C3 double-deficient mice and erythrocytes were prepared as described in detail in the protocol above. The cells were split and half of the cells were coated with mannan and the other half were left untreated, adjusting the final concentration to 10A/mL, of which 100 μL were used in the hemolysis assay, which was performed as described above. Results of Experiment #1: The lectin pathway is involved in erythrocyte lysis in the animal model of PNH

[00541] In an initial experiment, it was determined that erythrocytes from uncoated WT mice did not lyse in any rat serum. It was also determined that erythrocytes from mannan-coated Crry-/- mice were slowly lysed (over 3 hours at 37 degrees) in WT rat serum, but were not lysed in MBL-null serum. (Data not shown.)

[00542] It was determined that erythrocytes from mannan-coated Crry-/- mice were rapidly lysed in human serum but not in heat-inactivated NHS. Importantly, erythrocytes from mannan-coated Crry-/- mice were lysed in NHS diluted to 1/640 (i.e., dilutions of 1/40, 1/80, 1/160, 1/320, and 1/640 all lysed). (Data not shown). At this dilution, the alternative pathway does not function (AP functional activity is significantly reduced below 8% of the serum concentration). Conclusions of Experiment #1

[00543] Erythrocytes from mannan-coated Crry-/- mice are very well lysed in highly diluted human serum with MBL, but not in the same serum without MBL. Efficient lysis at all serum concentrations tested implies that the alternative pathway is not involved or is required for this lysis. The inability of MBL-deficient mouse serum and human serum to lyse erythrocytes from mannan-coated Crry-/- mice indicates that the classical pathway also has nothing to do with the observed lysis. Since recognition molecules of the lectin pathway are required (i.e., MBL), this lysis is mediated by the lectin pathway. Experiment #2

[00544] Fresh blood was obtained from Crry/C3 and CD55/CD59 double-deficient mice and mannan-coated Crry-/- mouse erythrocytes were analyzed in the hemolysis assay as described above in the presence of the following human serum: MASP-3 -/-; MBL null; WT; NHS pretreated with human MASP-2 antibody; and heat-inactivated NHS as a control. Results of Experiment #2: MASP-2 Inhibitors and MASP-3 Deficiency Prevent Erythrocyte Lysis in the Animal Model of PNH

[00545] With mannan-coated Crry-/- mouse erythrocytes, NHS was incubated at dilutions diluted up to 1/640 (i.e., 1/40, 1/80, 1/160, 1/320, and 1/640), human MBL-/- serum, human MASP-3-deficient serum (from 3MC patient), and NHS pretreated with MASP-2 mAb and heat-inactivated NHS as a control.

[00546] The ELISA microtiter plate was centrifuged and the unlysed erythrocytes were collected at the bottom of the round well plate. The supernatant from each well was collected and the amount of hemoglobin released from the lysed erythrocytes was measured by reading OD415 nm on an ELISA reader.

[00547] It was observed that MASP-3 -/- serum did not lyse mannan-coated mouse erythrocytes. In the control heat-inactivated NHS (negative control), as expected, no lysis was observed. MBL -/- human serum lysed mannan-coated mouse erythrocytes at 1/8 and 1/16 dilutions. NHS pretreated with MASP-2 antibody lysed mannan-coated mouse erythrocytes at 1/8 and 1/16 dilutions while WT human serum lysed mannan-coated mouse erythrocytes up to 1/32 dilutions.

[00548] FIGURE 19 graphically illustrates the hemolysis (measured by the release of hemoglobin from lysed mouse erythrocytes (Crry/C3-/-) into the supernatant measured by photometry) of mannan-coated murine erythrocytes by human serum over a range of serum dilutions in the serum of MASP-3 -/-, heat-inactivated (HI) NHS, MBL -/-, MASP-2 antibody-pretreated NHS, and NHS control.

[00549] FIGURE 20 graphically illustrates the hemolysis (measured by the release of hemoglobin from lysed mouse erythrocytes (Crry/C3-/-) into the supernatant measured by photometry) of mannan-coated murine erythrocytes by human serum over a range of serum concentrations in the serum of MASP-3-/-, heat-inactivated (HI) NHS, MBL-/-, MASP-2 antibody-pretreated NHS, and control NHS.

[00550] From the results shown in Figures 19 and 20, it is demonstrated that inhibiting MASP-3 will prevent any complement-mediated lysis of sensitized erythrocytes with poor protection from autologous complement activation. Inhibition of MASP-2 with MASP-2 antibody significantly altered CH50 and was protective to some extent, but inhibition of MASP-3 was more effective. Experiment #3

[00551] Uncoated Crry-/- mouse erythrocytes obtained from fresh blood of Crry/C3 and CD55/CD59 double-deficient mice were analyzed in the hemolysis assay as described above in the presence of the following sera: MASP-3 -/-; MBL -/-; WT; NHS pretreated with human MASP-2 antibody and heat-inactivated NHS as a control. Results:

[00552] FIGURE 21 graphically illustrates the hemolysis (measured by the release of hemoglobin from lysed WT mouse erythrocytes into the supernatant measured by photometry) of uncoated murine erythrocytes across a range of serum concentrations in human serum from a patient with 3MC (MASP-3-/-), heat-inactivated (HI) NHS, MBL-/-, MASP-2 antibody-pretreated NHS, and NHS control. As shown in FIGURE 21 and summarized in TABLE 13, it is demonstrated that inhibition of MASP-3 inhibits complement-mediated lysis of unsensitized WT mouse erythrocytes.

[00553] FIGURE 22 graphically illustrates the hemolysis (as measured by the release of hemoglobin from lysed mouse erythrocytes (CD55/59 -/-) into the supernatant measured by photometry) of uncoated murine erythrocytes by human serum over a range of serum concentrations in human serum from heat-inactivated (HI) NHS, MBL -/-, NHS pretreated with MASP-2 antibody, and control NHS. As shown in FIGURE 22 and summarized in TABLE 13, it is demonstrated that inhibition of MASP-2 was protective to some extent. TABLE 13: CH50 Values Expressed as Serum Concentrations

[00554] Note: “CH50” is the point at which complement-mediated hemolysis reaches 50%.

[00555] In summary, the results in this Example demonstrate that inhibiting MASP-3 prevents any complement lysis of both sensitized and non-sensitized erythrocytes with poor protection from autologous complement activation. Inhibition of MASP-2 is also protective to some extent. Thus, MASP-2 and MASP-3 inhibitors alone or in combination (i.e., co-administered, administered sequentially) or bispecific or dual MASP-2/MASP-3 inhibitors can be used to treat individuals suffering from PNH and can also be used to ameliorate (i.e., inhibit, prevent, or reduce the severity of) extravascular hemolysis in PNH patients undergoing treatment with a C5 inhibitor such as eculizumab (Soliris®).

EXAMPLE 6

[00556] This Example describes a hemolysis assay testing mannan-coated rabbit erythrocytes for lysis in the presence of WT or MASP-1/3-/- mouse serum. Methods: 1. Hemolysis assay of rabbit red blood cells (mannan-coated) in mouse MASP-1/3-deficient sera and control WT serum Day 1. Preparation of rabbit red blood cells.

[00557] Materials included: fresh rabbit blood, BBS/Mg++/Ca++ (4.4 mM barbituric acid, 1.8 mM sodium barbitone, 145 mM NaCl, pH 7.4, 5 mM Mg++, 5 mM Ca++), BBS/Mg++/Ca++ with 0.1% gelatin, chromium chloride contained in buffer, i.e., CrCl3.6 H2O (0.5 mg/mL in BBS/Mg++/Ca++), and mannan, 100 μg/mL in BBS/Mg++/Ca++.

[00558] 1. Rabbit whole blood (2 mL) was divided into two 1.5 mL eppendorf tubes and centrifuged for 3 minutes at 8000 rpm (approximately 5.9 rcf) in a refrigerated eppendorf centrifuge at 4°C. The red blood cell pellet was washed three times after resuspension in ice-cold BBS/Mg++/Ca++. After the third wash, the pellet was resuspended in 4 mL of BBS/Mg++/Ca++. Two mL of this aliquot was added to a 15 mL falcon tube to be used as an uncoated control. The remaining 2 mL of the red blood cell aliquot was diluted in 2 mL of CrCl3 buffer, 2 mL of the mannan solution was added, and the suspension was incubated at room temperature for 5 minutes with gentle shaking. The reaction was terminated by adding 7.5 mL of BBS/0.1% gelatin/Mg++/Ca++ to the mixture. The erythrocytes were pelleted and the red blood cells were washed twice with BBS/0.1% gelatin/Mg++/Ca++ as described above. The red blood cell suspension was stored in BBS/0.1% gelatin/Mg++/Ca++ at 4°C.

[00559] 2. 100 μL of suspended red blood cells were diluted with 1.4 mL of water and centrifuged at 8000 rpm (approximately 5.9 rcf) for 3 minutes and the OD of the supernatant was adjusted to 0.7 at 541nm (an OD of 0.7 at 541nm corresponds to approximately 109 erythrocytes/mL).

[00560] 3. Resuspended erythrocytes were diluted with BBS/0.1% gelatin/Mg++/Ca++ to a concentration of 108/mL.

[00561] 4. Dilutions of the test sera were prepared in ice-cold BBS/gelatin/Mg++/Ca++ and 100 μL of each serum dilution was pipetted into the corresponding well of the round bottom plate. 100 μL of appropriately diluted red blood cells (108/mL) was added to each well. As a control for complete lysis, purified water (100 μL) was mixed with the diluted red blood cells (100 μL) to cause 100% lysis, while BBS/0.1% gelatin/Mg++/Ca++ without serum (100 uL) was used as a negative control. The plate was then incubated for 1 hour at 37°C.

[00562] 5. The round bottom plate was centrifuged at 3250 rpm for 5 minutes. The supernatant from each well (100 μL) was transferred to the corresponding wells of a flat bottom plate and the OD was read in an ELISA reader at 415-490 nm. The results are presented as the ratio of OD at 415 nm to OD at 490 nm. Results:

[00563] FIGURE 23 graphically illustrates the hemolysis (as measured by the release of hemoglobin from lysed rabbit erythrocytes into the supernatant measured by photometry) of mannan-coated rabbit erythrocytes by mouse serum over a range of serum concentrations in MASP-1 -/- and control WT serum. As shown in FIGURE 23, it is demonstrated that inhibition of MASP-3 prevents complement-mediated lysis of mannan-coated WT rabbit erythrocytes. These results also support the use of MASP-3 inhibitors for the treatment of one or more aspects of PNH, as described in Example 5.

EXAMPLE 7

[00564] This example describes the generation of MASP-1 and MASP-3 monoclonal antibodies using an in vitro system comprising a modified DT40 cell line, DTLacO. Background/Rationale:

[00565] Antibodies against human MASP-1 and MASP-3 were generated using an in vitro system comprising a modified DT40 cell line, DTLacO, which allows for the reversible induction of diversification of a particular polypeptide, as further described in WO2009029315 and US2010093033. DT40 is a chicken B cell line that is known to constitutively mutate its heavy and light chain immunoglobulin (Ig) genes in culture. Like other B cells, this constitutive mutagenesis targets mutations in the V region of the Ig genes and thus in the CDRs of the expressed antibody molecules. Constitutive mutagenesis in DT40 cells occurs by gene conversion using as donor sequences a variety of non-functional V gene segments (pseudo-V genes; yV) situated upstream of each of the functional V regions. Deletion of the yV region has previously been shown to cause a shift in the diversification mechanism from gene conversion to somatic hypermutation, the mechanism commonly observed in human B cells. The chicken B-cell lymphoma line DT40 has been shown to be a promising starting point for ex vivo antibody evolution (Cumbers, S. J. et al. Nat Biotechnol 20, 1129-1134 (2002); Seo, H. et al. Nat Biotechnol 23, 731-735 (2005)). DT40 cells proliferate robustly in culture, with a doubling time of 8-10 hours (compared to 20-24 hours for human B-cell lines), and they support very efficient targeting of homologous genes (Buerstedde, J. M. et al. Embo J 9, 921-927 (1990)). DT40 cells command an enormous diversity of potential V region sequences because they can access two distinct physiological pathways for diversification, gene conversion and somatic hypermutation, which create DNA template-specified and DNA template-unspecified mutations, respectively (Maizels, N. Annu Rev Genet 39, 23-46 (2005)). Diversified heavy and light chain immunoglobulins (Igs) are expressed as a cell surface-displayed IgM. Surface IgM is a bivalent form, structurally similar to an IgG molecule. Cells displaying IgM with specificity for a given antigen can be isolated by binding to immobilized membrane-displayed and soluble versions of the antigen. However, the utility of DT40 cells for antibody evolution has been limited in practice because—as in other transformed B cell lines—diversification occurs at less than 1% of the physiological rate.

[00566] In the system used in this example, as described in WO2009029315 and US2010093033, DT40 cells were engineered to accelerate the rate of Ig gene diversification without sacrificing the capacity for further genetic modification or the potential for gene conversion and somatic hypermutation to contribute to mutagenesis. Two important modifications to DT40 were made to increase the rate of diversification and, consequently, the complexity of binding specificities in our cell library. First, Ig gene diversification was placed under the control of the potent E. coli lactose operator/repressor regulatory network. Multimers consisting of approximately 100 polymerized repeats of the potent E. coli lactose operator (PolyLacO) were inserted upstream of the rearranged Ig and IgH genes and expressed by targeting homologous genes. Regulatory factors fused to the lactose repressor protein (LacI) can then be tethered to LacO regulatory elements to regulate diversification, taking advantage of the high affinity (kD = 10-14 M) of the lactose repressor for operator DNA. DT40 PolyLacO-ÀR cells, in which PolyLacO was integrated with Ig alone, exhibited a 5-fold increase in the rate of Ig gene diversification relative to the parent DT40 cells prior to any manipulation (Cummings, W. J. et al. PLoS Biol 5, e246 (2007)). Diversification was further enhanced in cells engineered to carry PolyLacO targeting both the Ig and IgH genes ("DTLacO"). DTLacO cells have been shown to have diversification rates 2.5- to 9.2-fold elevated relative to the 2.8% characteristic of the parent DT40 PolyLacO-R LacI-HP1 strain. Thus, targeting PolyLacO elements to both the heavy and light chain genes accelerated diversification 21.7-fold relative to the parent DT40 strain. Fixation of regulatory factors at Ig loci not only alters the frequency of mutagenesis but can also alter the mutagenesis pathway, creating a larger collection of unique sequence changes (Cummings et al. 2007; Cummings et al. 2008). Second, a diverse collection of sequence starting points for factor-accelerated Ig gene diversification was generated. These diverse sequence starting points were added to DTLacO targeting rearranged Ig heavy chain variable regions isolated from a two-month-old chick to the heavy chain locus. The addition of these heavy chain variable regions created a repertoire of 107 new starting points for antibody diversification. Construction of these new starting points in the DTLacO cell line allows identification of clones that bind to a particular target, and then rapid affinity maturation by the fixed factors. After affinity maturation, a recombinant full-length chimeric IgG is made by cloning the mature rearranged heavy and light chain variable sequences (VH and VA; consisting of chicken framework regions and the complementarity determining regions or CDRs) into expression vectors containing human lambda and IgG1 constant regions. These recombinant mAbs are suitable for in vitro and in vivo applications and serve as a starting point for humanization. Methods: Selection for antigen binding of MASP-1 and MASP-3.

[00567] Initial selections were performed by binding gene-diversified DTLacO populations targeting antigen-complexed granules of human MASP-1 (SEQ ID NO: 8) and MASP-3 (SEQ ID NO: 2); and subsequent FACS selections using fluorescently labeled soluble antigen (Cumbers, S. J. et al. Nat Biotechnol 20, 1129-1134 (2002); Seo, H. et al. Nat Biotechnol 23, 731-735 (2005). Because of the conserved amino acid sequence in the alpha chain that is shared between MASP-1 and MASP-3 (shown in FIGURE 2) and the distinct sequences in the beta chain (shown in FIGURE 2), separate parallel screens for MASP-1 and MASP-3 binders were performed to identify MASP-1-specific mAbs, MASP-3-specific mAbs, and also mAbs capable of binding to both MASP-1 and MASP-3 (dual specific). Both forms of antigen were used to select and screen for binders. First, recombinant MASP-1 or MASP-3, Full-length or a fragment, fused to an Fc domain were bound to Dynal Protein G magnetic beads or used in FACS-based selections using a PECy5-labeled anti-human IgG(Fc) secondary antibody. Alternatively, recombinant versions of MASP-1 or MASP-3 proteins were directly labeled with DyLight Fluor and used for selections and screenings. Binding and affinity.

[00568] Recombinant antibodies were generated by cloning PCR-amplified V regions into a vector that supported expression of human IgG1 in 293F cells (Yabuki et al., PLoS ONE, 7(4):e36032 (2012)). Saturation binding kinetics were determined by staining DTLacO cells expressing antibody-binding MASP-1 or MASP-3 with various concentrations of fluorescently labeled soluble antigen. Functional assays for MASP-3-specific activity including MASP-3-dependent C3b deposition and MASP-3-dependent factor D cleavage were performed as described in Examples 8 and 9, respectively. A functional assay for MASP-1-specific activity, namely inhibition of MASP-1-dependent C3b deposition, was performed as described below. Results:

[00569] Numerous MASP-1 and MASP-3 binding antibodies were generated using the methods described above. Binding, as demonstrated by FACS analysis, is described for representative clones M3J5 and M3M1, which were isolated in screens for MASP-3 ligands.

[00570] FIGURE 24A is a FACS histogram of MASP-3 antigen/antibody binding for DTLacO clone M3J5. FIGURE 24B is a FACS histogram of MASP-3 antigen/antibody binding for DTLacO clone M3M1. In FIGURES 24A and 24B, the gray filled curves are negative control stained with IgG1 and the thick black curves are stained with MASP-3.

[00571] FIGURE 25 graphically illustrates a saturation binding curve of clone M3J5 (Clone 5) for MASP-3 antigen. As shown in FIGURE 25, the apparent binding affinity of the M3J5 antibody for MASP-3 is about 31 nM.

[00572] Sequence analysis of identified clones was performed using standard methods. All clones were compared to common VH and VL sequences (DT40) and to each other. The sequences for the two aforementioned clones, M3J5 and M3M1 are provided in an alignment with two additional representative clones, D14 and 1E10, which were identified in screens for the CCP1-CCP2-SP fragments of MASP-1 and MASP-3, respectively. D14 and 1E10 bind regions common to MASP-1 and MASP-3.

[00573] FIGURE 26A is an amino acid sequence alignment of the VH regions of M3J5, M3M1, D14, and 1E10 to the chicken DT40 VH sequence.

[00574] FIGURE 26B is an amino acid sequence alignment of the VL regions of M3J5, M3M1, D14, and 1E10 to the chicken DT40 VL sequence.

[00575] The VH and VL amino acid sequence of each clone is provided below. Heavy Chain Variable Region (VH) Sequences

[00576] FIGURE 26A shows an amino acid alignment of the heavy chain variable region (VH) sequences for the precursor DTLaco (SEQ ID NO: 300), the MASP-3 binding clones M3J5 (SEQ ID NO: 301) and M3M1 (SEQ ID NO: 302), and the MASP-1/MASP-3 dual binding clones D14 (SEQ ID NO: 306) and 1E10 (SEQ ID NO: 308).

[00577] The Kabat CDRs in the VH sequences below are located at the following amino acid positions: H1:aa 31-35; H2:aa 5062; and H3:aa 95-102.

[00578] The Chothia CDRs in the VH sequences below are located at the following amino acid positions: H1: aa 26-32; H2: aa 5256; and H3: aa 95-101. DTLacO VH precursor: (SEQ ID NO: 300) AVTLDESGGGLQTPGGALSLVCKASGFTFSSNAMGWVRQAPGKGLE WVAGIDDDGSGTRYAPAVKGRATISRDNGQSTLRLQLNNLRAEDTGT YYCTKCAYSSGCDYEGGYIDAWGHGTEVIVSS Clone M3J5 VH: (SEQ ID NO:301) AVTLDESGGGLQTPGGGL SLVCKASGFTFSSYAMGWMRQAPGKGLE YVAGIRSDGSFTLYATAVKGRATISRDNGQSTVRLQLNNLRAEDTAT YFCTRSGNVGDIDAWGHGTEVIVSS Clone M3M1 VH: (SEQ ID NO:302) AVTLDESGGGLQTPGGGLSLVCKASGFDFSSYQMNWIRQAPGKGLEF VAAINRFGNSTGHGAAVKGRVTISRDDGQSTVRLQLSNLRAEDTATY YCAKGVYGYCGSYSCCGVDTIDAWGHGTEVIVSS Clone D14 VH: (SEQ ID NO:306) AVTLDESGGGLQTPGGALSLVCKASGFTFSSYAMHWVRQAPGKGLE WVAGIYKSGAGTNYAPAVKGRATISRDNGQSTVRLQLNNLRAEDTG TYYCAKTTGSGCSSGYRAEYIDAWGHGTEVIVSS Clone 1E10 VH: (SEQ ID NO:308) AVTLDESGGGLQTPGGALSLVCKASGFTFSSYDMVWVRQAPGKGLE FVAGISRNDGRYTEYGSAVKGRATISRDNGQSTVRLQLNNLRAEDTA TYYCARDAGGSAYWFDAGQIDAWGHGTEVIVSS Light Chain Variable Region Sequences (VL)

[00579] FIGURE 26B shows an amino acid alignment of the light chain variable region (VL) sequences for the precursor DTLac (SEQ ID NO:303) and the MASP-3 binding clones M3J5 (SEQ ID NO:304) and M3M1 (SEQ ID NO:305) and the MASP-1/MASP-3 dual-binding clones D14 (SEQ ID NO:307) and 1E10 (SEQ ID NO:309). Precursor DTLacO VL: (SEQ ID NO:303)

[00580] ALTQPASVSANLGGTVKITCSGGGSYAGSYYYGWYQQ KSPGSAPVTVIYDNDKRPSDIPSRFSGSLSGSTNTLTITGVRADDEAVY FCGSADNSGAAFGAGTTLTVL Clone M3J5 VL: (SEQ ID NO: 304)

[00581] ALTQPASVSANPGETVKITCSGGYSGYAGSYYYGWYQ QKAPGSAPVTLIYYNNKRPSDIPSRFSGSLSGSTNTLTITGVRADDEAV YFCGSADNSGAAFGAGTTLTVL Clone M3M1 VL: (SEQ ID NO: 305)

[00582] ALTQPASVSANPGETVKITCSGGGSYAGSYYYGWYQQ KAPGSAPVTLIYYNNKRPSDIPSRFSGSLSGSTNTLTITGVRADDEAVY FCGSADNSGAAFGAGTTLTVL Clone D14 VL: (SEQ ID NO:307)

[00583] ALTQPASVSANPGETVKITCSGGGSYAGSYYYGWYQQ KAPGSAPVTLIYYNNKRPSDIPSRFSGSLSGSTNTLTITGVRADDEAVY FCGSADNSGAAFGAGTTLTVL Clone 1E10 VL: (SEQ ID NO:309)

[00584] ALTQPASVSANPGETVKITCSGGGGSYAGSYYYGWYQQ KAPGSAPVTLIYYNNKRPSDIPSRFSGSLSGSTNTLTITGVRADDEAVY FCGSADNSGAAFGAGTTLTVL Functional Assay of LEA-2 (MASP-2-dependent)

[00585] MASP-1 contributes to LEA-2 through its ability to activate MASP-2 (see FIGURE 1). The Wieslab® complement system screening MBL assay (Euro Diagnostica, Malmo, Sweden) measures C5b-C9 deposition under conditions that isolate LEA-2-dependent activation (i.e., traditional lectin pathway activity). The assay was performed according to the manufacturer's instructions with the representative clone 1E10 tested at a final concentration of 400 nM.

[00586] FIGURE 27 is a bar graph showing the inhibitory activity of mAb 1E10 compared to positive serum provided with the assay kit, as well as an isotype control antibody. As shown in FIGURE 27, mAb 1E10 demonstrates partial inhibition of LEA-2-dependent activation (via inhibition of MASP-1-dependent activation of MASP-2), while the isotype control antibody does not. Stronger inhibition should be achieved by continued affinity maturation of this antibody for MASP-1 binding using the factors set in the DTLacO system.

[00587] LEA-1 (MASP-3 dependent) assays for representative mAbs are described below in Examples 8 and 9. Summary of Results:

[00588] The above results showed that the DTLacO platform enabled rapid ex vivo discovery of MASP-1 and MASP-3 monoclonal antibodies with inhibitory properties on LEA-1 (as shown below in Examples 8 and 9) and on LEA-2 (as shown in this Example).

EXAMPLE 8

[00589] Complement pathway analysis in serum of 3MC with S. aureus Background/Rationale:

[00590] It was determined that MASP-3 is not activated by exposure to non-fluid phase immobilized mannan, zymosan A, or N-acetylcysteine in the presence or absence of normal human serum. However, it was determined that recombinant and native MASP-3 is activated on the surface of heat-inactivated S. aureus in the presence and absence of normal human serum (NHS) or heat-inactivated human serum (HIS) (data not shown). It was also determined that C3b deposition occurs on the surface of S. aureus in the presence of normal human serum and that deposition can be monitored using a flow cytometer. Therefore, the alternative pathway (AP) response to S. aureus was measured as described in this Example as a means of assessing the contribution of MASP-3 to LEA-1. Methods:

[00591] Recombinant MASP-3: Polynucleotide sequences encoding recombinant full-length human MASP-3, a truncated serine protease (SP)-active version of MASP-3 (CCP1-CCP2-SP), and an SP-inactivated form of MASP-3 (S679A) were cloned into the mammalian expression vector pTriEx7 (Invivogen). The resulting expression constructs encode full-length MASP-3 or the CCP1-CCP2-SP fragment with an amino-terminal streptag and a carboxy-terminal His6 tag. The expression constructs were transfected into Freestyle 293-F or Expi293F cells (Invitrogen) according to the protocols provided by the manufacturer. After three to four days of cultivation in 5% CO2 at 37°C, the recombinant proteins were purified using streptactin affinity chromatography.

[00592] Recombinant MASP-1: Full-length or truncated CCP1-CCP2-SP forms of recombinant MASP-1 with or without the stabilizing R504Q (Dobo et al., J. Immunol 183:1207, 2009) or SP-inactivating (S646A) mutations and having an amino-terminal Steptag and a carboxy-terminal His6 tag were generated as described for recombinant MASP-3 above. 6. C3b deposition and factor B cleavage in S. aureus in 3MC (human) serum

[00593] An initial experiment was performed to demonstrate that the flow cytometry assay is capable of detecting the presence or absence of AP-driven C3b deposition (AP-C3b) as follows. Five percent of the following sera: normal human serum, factor B (Factor B)-depleted human serum, factor D-depleted human serum, and properdin-depleted human serum (obtained from Complement Technology, Tyler, Texas, USA) were mixed with test antibody in Mg++/EGTA or EDTA buffer at 4°C overnight. Heat-killed S. aureus (108/reaction) was added to each mixture to a total volume of 100 μL and centrifuged at 37°C for 40 minutes. Bacteria were washed in wash buffer, the bacterial pellet was resuspended in wash buffer and an 80 μl aliquot of each sample was analysed for C3b deposition on the bacterial surface, which was detected with anti-human C3c (Dako, UK) using flow cytometry.

[00594] The results of C3b detection by flow cytometry are shown in FIGURE 28A. As shown in FIGURE 28A, panel 1, in normal human serum in the presence of EDTA, which is known to inactivate AP, no C3b deposition was observed (negative control). In normal human serum treated with Mg++/EGTA, only the lectin-independent complement pathways can function. In panel 2, Mg++/EGTA buffer is used, therefore AP is active and AP-driven C3b deposition is observed (positive control). As shown in panels 3, 4, and 5, in factor B-depleted, factor D-depleted, and properdin-depleted serum, respectively, no alternative pathway-driven C3b deposition is observed, as expected. These results demonstrate that the assay is capable of detecting AP-dependent C3b deposition.

[00595] A C3b deposition in S. aureus assay was performed as described above to assess the ability of recombinant MASP-3 to reconstitute AP (LEA-1) in serum from human 3MC, which is deficient in MASP-3 (Rooryck C, et al. Nat Natet 43(3):197-203 (2011)). The following reagent combinations were tested.

[00596] 1. 5% normal human serum + EDTA 7. 5% normal human serum + Mg/EGTA 8. 5% human serum 3MC (MASP-3-/-) + Mg++/EGTA 9. 5% human serum 3MC (MASP-3-/-) + Mg++/EGTA plus active full-length rMASP-3 10. 5% human serum 3MC (MASP-3-/-) + Mg++/EGTA plus active truncated rMASP-3 (CCP1/CCP2/SP) 11. 5% human serum 3MC (MASP-3-/-) + Mg++/EGTA plus inactive rMASP-3 (S679A) 12. 5% human serum 3MC (MASP-3-/-) + Mg++/EGTA plus active full-length rMASP-1

[00597] The various 5% serum and recombinant protein mixtures (5 μg each) as shown above were incubated under the specified buffer conditions (Mg++/EGTA or EDTA buffer) at 4°C overnight. After overnight incubation, 108 heat-killed S. aureus were added to each mixture in a total volume of 100 μL and centrifuged at 37°C for 40 minutes. The bacteria were washed and resuspended in wash buffer and an 80 μL aliquot of each sample was analyzed for C3b deposition by FACS. The remaining 20 μL aliquot of each sample was used to measure factor B cleavage by Western blot using anti-factor B antibody as described below.

[00598] The results of C3b detection by flow cytometry are shown in FIGURE 28B. The panel numbers correspond to the numbers designated for each reagent combination described above. The negative control (panel 1) and the positive control (panel 2) show the absence and presence of C3b deposition, as expected. Panel 3 shows that AP-driven C3b deposition is absent in 3MC serum. Panels 4 and 5 show that full-length active rMASP-3 (panel 4) and active rMASP-3 (CCP1-CCP2-SP) (panel 5) restore AP-driven C3b deposition in 3MC serum. Panel 6 shows that inactive rMASP-3 (S679A) does not restore AP-driven C3b deposition in 3MC serum. Panel 7 shows that rMASP-1 does not restore AP-driven C3b deposition in 3MC serum.

[00599] Together, these results demonstrate that MASP-3 is required for AP-driven C3b deposition in S. aureus in human serum.

MASP-3-dependent Factor B activation

[00600] In order to analyze MASP-3-dependent activation of Factor B, the various mixtures of 5% serum (normal human serum or 3MC patient serum) and recombinant proteins as shown above were assayed as described above. From each reaction mixture, 20 μL was removed and added to the protein sample loading buffer. The samples were heated at 70°C for 10 minutes and loaded onto an SDS-PAGE gel. Western blot analysis was performed using a polyclonal Factor B antibody (R&D Systems). Factor B activation was apparent by the formation of two low molecular weight cleavage products (Bb and Ba) derived from the higher molecular weight pro-Factor B protein.

[00601] FIGURE 29 shows the results of a Western blot analysis to determine Factor B cleavage in response to S. aureus in 3MC serum in the presence or absence of rMASP-3. As shown in lane 1, normal human serum in the presence of EDTA (negative control) demonstrates very little Factor B cleavage relative to normal human serum in the presence of Mg++/EGTA, shown in lane 2 (positive control). As shown in lane 3, 3MC serum demonstrates very little Factor B cleavage in the presence of Mg++/EGTA. However, as shown in lane 4, Factor B cleavage is restored by the addition and preincubation of full-length recombinant MASP-3 protein (5 μg) to 3MC serum. Assay to Determine the Effect of rMASP-3 on the Cleavage of Profactor D to Factor B/C3 (H2O)

[00602] The following assay was performed to determine the minimal requirement for MASP-3-dependent factor B3 activation/cleavage.

[00603] C3(H2O) (200 ng), purified plasma factor B (20 μg), recombinant profactor D (200 ng), and recombinant human MASP-3 (200 ng) were mixed in various combinations (as shown in FIGURE 30) in a total volume of 100 μL in BBS/Ca++/Mg++ and incubated at 30°C for 30 min. The reaction was stopped by the addition of 25 μL of SDS loading dye containing 5% 2-mercaptoethanol. After boiling at 95°C for 10 min with shaking (300 rpm), the mixture was centrifuged at 1400 rpm for 5 min, and 20 μL of the supernatant was loaded and separated on a 10% SDS gel. The gel was stained with Coomassie brilliant blue. Results:

[00604] FIGURE 30 shows a Comassie-stained SDS-PAGE gel in which factor B cleavage is analyzed. As shown in lane 1, factor B cleavage is most optimal in the presence of C3, MASP-3, and profactor D. As shown in lane 2, C3 is absolutely required; however, as shown in lanes 4 and 5, either MASP-3 or profactor D are capable of mediating factor B cleavage as long as C3 is present. Analysis of the Ability of MASP-3 mAbs to Inhibit MASP-3-Dependent AP-Driven C3b Deposition

[00605] As described in this Example, MASP-3 has been shown to be required for AP-driven C3b deposition in S. aureus in human serum. Therefore, the following assay was performed to determine whether a representative MASP-3 mAb identified as described in Example 7 could inhibit MASP-3 activity. Active recombinant MASP-3 fragment protein (CCP1-CCP2-SP) (250 ng) was pre-incubated with an isotype control mAb, mAb1A5 (control obtained from the DTLacO platform that does not bind to MASP-3 or MASP-1) or mAbD14 (binds to MASP-3) at three different concentrations (0.5, 2, and 4 μM) for 1 hour on ice. The enzyme-mAb mixture was exposed to 5% 3MC serum (MASP-3 deficient) and 5x107 heat-killed S. aureus in a final reaction volume of 50 μL. Reactions were incubated at 37°C for 30 min and then stained for C3b deposition. Stained bacterial cells were analyzed by a flow cytometer.

[00606] FIGURE 31 graphically illustrates the mean fluorescent intensities (MFI) of C3b staining obtained from the three antibodies plotted as a function of mAb concentration in 3MC serum in the presence of rMASP-3. As shown in FIGURE 31, mAbD14 demonstrates inhibition of C3b deposition in a concentration-dependent manner. In contrast, none of the control mAbs inhibited C3b deposition. These results demonstrate that mAbD14 is capable of inhibiting MASP-3-dependent C3b deposition. Enhanced inhibitory activity for mAbD14 is expected following continued affinity maturation of this antibody for MASP-3 binding using the factors set in the DTLacO system. Summary of Results:

[00607] In summary, the results in this Example demonstrate a clear AP defect in MASP-3-deficient serum. Thus, MASP-3 has been shown to make a critical contribution to AP, using Factor B activation and C3b deposition as functional endpoints. Furthermore, addition of functional recombinant MASP-3, including the catalytically active C-terminal portion of MASP-3, corrects the defect in Factor B activation and C3b deposition in 3MC patient serum. Conversely, as further demonstrated in this Example, addition of a MASP-3 antibody (e.g., mAbD14) to 3MC serum with rMASP-3 inhibits AP-driven C3b deposition. A direct role for MASP-3 in Factor B activation, and thus AP, is demonstrated by the observation that recombinant MASP-3, together with C3, is sufficient to activate recombinant Factor B. EXAMPLE 9

[00608] This example demonstrates that MASP-1 and MASP-3 activate factor D. Methods:

[00609] Recombinant MASP-1 and MASP-3 were tested for their ability to cleave two different recombinant versions of profactor D. The first version (profactor D-His) lacks an N-terminal tag but does have a C-terminal His tag. Thus, this version of profactor D contains the 5 amino acid propeptide that is removed by cleavage during activation. The second version (ST-profactor D-His) has a Strep-TagII sequence at the N-terminus, thus increasing the cleaved N-terminal fragment to 15 amino acids. ST-profactor D also contains a His6 tag at the C-terminus. Increasing the propeptide length of ST-profactor D-His improves the resolution between the cleaved and uncleaved forms by SDS-PAGE compared to the resolution possible with the profactor D-HIS form.

[00610] Recombinant MASP-1 or MASP-3 proteins (2 μg) were added to profactor D-His or ST-profactor D-His substrates (100 ng) and incubated for 1 hour at 37°C. Reactions were electrophoresed on a 12% Bis-Tris gel to resolve profactor D and the active factor D cleavage product. Resolved proteins were transferred to a PVDF membrane and analyzed by Western blot by detection with a biotinylated factor D antibody (R&D Systems). Results:

[00611] FIGURE 32 shows Western blot analysis of profactor D substrate cleavage. TABLE 14: Lane Description for the Western Blot Shown in FIGURE 32

[00612] As shown in FIGURE 32, only the full-length MASP-3 (lane 2) and CCP1-CCP2-SP MASP-1 (lane 5) fragments cleaved ST-profactor D-His6. The catalytically inactive full-length MASP-3 (S679A; lane 3) and MASP-1 (S646A; lane 3) failed to cleave either substrate. Identical results were obtained with the profactor D-His6 polypeptide (not shown). Comparison of a molar excess of MASP-1 (CCP1-CCP2-SP) to MASP-3 suggests that MASP-3 is a more effective catalyst for profactor D cleavage than MASP-1, at least under the conditions described here.

[00613] Conclusions: Both MASP-1 and MASP-3 are capable of cleaving and activating factor D. This activity directly links LEA-1 with AP activation. More specifically, activation of factor D by MASP-1 or MASP-3 will lead to factor B activation, C3b deposition, and likely opsonization and/or lysis. Assay for inhibition of MASP-3-dependent cleavage of profactor D with MASP-3 antibodies

[00614] An assay was performed to determine the inhibitory effect of representative MASP-3 and MASP-1 mAbs, identified as described in Example 7, on MASP-3-dependent factor D cleavage as follows. Active recombinant MASP-3 protein (80 ng) was preincubated with 1 μg of representative mAbs D14, M3M1, and a control antibody (which specifically binds to MASP-1 but not MASP-3) at room temperature for 15 minutes. Pro-Factor D with an N-terminal Strep-tag (ST-pro-factor D-His, 70 ng) was added, and the mixture was incubated at 37°C for 75 minutes. The reactions were then electrophoresed, blotted, and stained with anti-factor D as described above.

[00615] FIGURE 33 is a Western blot showing the partial inhibitory activity of the D14 mAbs and M3M1 compared to a control reaction containing only MASP-3 and ST-pro-factor D-His (no mAb, lane 1), as well as a control reaction containing a mAb obtained from the DTLacO library that binds to MASP-1 but not MASP-3 (lane 4). As shown in FIGURE 33, in the absence of an inhibitory antibody, MASP-3 cleaves approximately 50% of the pro-factor D into factor D (lane 1). The control MASP-1-specific antibody (lane 4) does not alter the ratio of pro-factor D to factor D. In contrast, as shown in lanes 2 and 3, both mAb D14 and mAb M3M1 inhibit the MASP-3-dependent cleavage of pro-factor D to factor D, resulting in a reduction in factor D generated.

[00616] Conclusions: These results demonstrate that MASP-3 mAbs D14 and M3M1 are capable of inhibiting MASP-3-dependent factor D cleavage. Enhanced inhibitory activity for mAb D14 and mAb M3M1 is expected following continued affinity maturation of these antibodies for MASP-3 binding using the factors fixed in the DTLacO system. EXAMPLE 10

[00617] This example shows that MASP-3 deficiency prevents complement-mediated lysis of mannan-coated WT rabbit erythrocytes. Rationale:

[00618] As described in Examples 5 and 6 herein, the effect of MASP-2 and MASP-3 deficient serum on red blood cell lysis of blood samples obtained from a rat model of PNH demonstrated the efficacy of MASP-2 inhibition and/or MASP-3 inhibition to treat subjects suffering from PNH and also supported the use of MASP-2 inhibitors and/or MASP-3 inhibitors (including dual or bispecific MASP-2/MASP-3 inhibitors) to ameliorate the effects of C3 fragment-mediated extravascular hemolysis in subjects with PNH undergoing therapy with a C5 inhibitor, such as eculizumab.

[00619] As described in this Example, C3b deposition experiments and hemolysis experiments were performed on MASP-3 deficient serum from additional 3MC patients, confirming the results obtained in Examples 5 and 6. Additionally, experiments were performed which demonstrated that the addition of rMASP-3 to 3MC serum was able to reconstitute C3b deposition and hemolytic activity. Methods:

[00620] MASP-3 deficient serum was obtained from three different 3MC patients as follows: 3MC Patient 1: Contains an allele carrying a mutation that renders the exon coding for the serine protease domain of MASP-3 dysfunctional, provided together with the 3MC patient's mother and father (both heterozygous for the allele carrying a mutation that renders the exon coding for the serine protease domain of MASP-3 dysfunctional), 3MC Patient 2: Has a C1489T (H497Y) mutation in MASP-1 exon 12, the exon encoding the serine protease domain of MASP-3, resulting in non-functional MASP-3 proteins but functional MASP-1 proteins.

[00621] 3MC Patient 3: Has a confirmed defect in the MASP-1 gene, resulting in nonfunctional MASP-3 and nonfunctional MASP-1 proteins. Experiment #1: C3b deposition assay

[00622] An AP assay was performed under specific traditional AP conditions (BBS/Mg++/EGTA, without Ca++, where BBS = barbital-buffered saline containing sucrose), as described in Bitter-Suermann et al., Eur. J. Immunol 11:291-295 (1981)), on zymosan-coated microtiter plates at serum concentrations ranging from 0.5 to 25%, and C3b deposition was measured over time. Results:

[00623] FIGURE 34 graphically illustrates the level of AP-driven C3b deposition on zymosan-coated microtiter plates as a function of serum concentration in serum samples obtained from MASP-3 deficient (3MC), C4 deficient, and MBL deficient individuals. As shown in FIGURE 34 and summarized below in TABLE 15, the serum from the MASP-3 deficient patient Patient 2 and Patient 3 has residual AP activity at high concentrations (serum concentrations of 25%, 12.5%, 6.25%), but a significantly higher AP50 (i.e., 8.2% and 12.3% of serum required to achieve 50% of maximal C3 deposition).

[00624] FIGURE 35A graphically illustrates the level of AP-driven C3b deposition in zymosan-coated microtiter plates under specific “traditional” AP conditions (i.e., BBS/EGTA/Mg++ without Ca++) as a function of time in 10% human serum samples from MASP-3-deficient, C4-deficient, and MBL-deficient individuals.

[00625] TABLE 15 below summarizes the AP50 results shown in FIGURE 34 and the time halves for C3b deposition shown in FIGURE 35A.

[00626] Note: In BBS/Mg++/EGTA buffer, lectin pathway-mediated effects are deficient due to the absence of Ca++ in this buffer. Experiment #2: Analysis of profactor D cleavage in sera from 3MC patients by Western Blot

[00627] Methods: Serum was obtained from 3MC patient #2 (-/-), MASP-1 (+/+)) and 3MC patient #3 (MASP-3 (MASP-3 (-/-), MASP-1 (-/-)). Patient sera, together with sera from normal donors (W), were separated by SDS-polyacrylamide gel and the resolved proteins were transferred to a polyvinylidine fluoride membrane. Human pro-factor D (25,040 Da) and/or mature factor D (24,405 Da) were detected with an antibody specific for human factor D.

[00628] Results: Western blot results are shown in FIGURE 35B. As shown in FIGURE 35B, in sera from normal donors (W), factor D antibody detected a protein of a size consistent with mature factor D (24,405 Da). As further shown in FIGURE 35B, factor D antibody detected a slightly larger protein in sera from 3MC patient #2 (P2) and 3MC patient #3 (P3), consistent with the presence of pro-factor D (25,040 Da) in these 3MC patients. Experiment #3: Wieslab Complement Assays with 3MC Patient Sera

[00629] Methods: Sera obtained from 3MC patient #2 (MASP-3 (-/-), MASP-1 (+/+)) and from 3MC patient #3 (MASP-3 (-/-), MASP-1 (-/-)) were also tested for classical, lectin and alternative pathway activity using the Wieslab Complement System Screen (Euro-Diagnostica, Malmo, Sweden) according to the manufacturer's instructions. Normal human serum was tested in parallel as a control.

[00630] Results: FIGURE 35C graphically illustrates the results of the Weislab classical, lectin, and alternative pathway assays with plasma obtained from 3MC patient #2, 3MC patient #3, and normal human serum. As shown in FIGURE 35C, under Wieslab assay conditions, the classical, alternative, and MBL (lectin) pathways are all functional in normal human serum. In serum from 3MC patient #2 (MASP-3(-/-), MASP-1(+/+)), the classical pathway and lectin pathway are functional, however, there is no detectable alternative pathway activity. In serum from 3MC patient #3 (MASP-3(-/-), MASP-1(+/+)), the classical pathway and lectin pathway are functional, however, there is no detectable alternative pathway activity.

[00631] The result in FIGURES 35B and 35C further supports our understanding of the role of MASP-1 and MASP-3 in the LEA-1 and LEA-2 pathways. Specifically, the absence of the alternative pathway with a nearly fully functional lectin pathway in the serum of Patient 2, which lacks only MASP-3, confirms that MASP-3 is essential for activation of the alternative pathway. The serum of Patient 3, which lacks both MASP-1 and MASP-3, has lost the ability to activate the lectin pathway as well as the alternative pathway. This result confirms the requirement of MASP-1 for a functional LEA-2 pathway and is consistent with Example 7 and the literature demonstrating that MASP-1 activates MASP-2. The apparent inability of both sera to activate profactor D is also consistent with the data described in Example 9, demonstrating that MASP-3 cleaves profactor D. These observations are consistent with the LEA-1 and LEA-2 pathways as diagrammed in Figure 1. Experiment #4: Hemolysis assay testing mannan-coated rabbit erythrocytes for lysis in the presence of normal human serum or 3MC (in the absence of Ca++) Methods: Preparation of rabbit red blood cells in the absence of Ca++ (i.e., using EGTA)

[00632] Rabbit whole blood (2 mL) was divided into two 1.5 mL eppendorf tubes and centrifuged for 3 minutes at 8000 rpm (approximately 5.9 rcf) in a refrigerated eppendorf centrifuge at 4°C. The red blood cell pellet was washed three times after resuspension in ice-cold BBS/Mg++/Ca++ (4.4 mM barbituric acid, 1.8 mM sodium barbitone, 145 mM NaCl, pH 7.4, 5 mM Mg++, 5 mM Ca++). After the third wash, the pellet was resuspended in 4 mL of BBS/Mg++/Ca++. Erythrocytes were pelleted and the red blood cells were washed with BBS/0.1% gelatin/Mg++/Ca++ as described above. The red blood cell suspension was stored in BBS/0.1% gelatin/Mg++/Ca++ at 4°C. Then, 100 μL of suspended red blood cells were diluted with 1.4 mL of water and centrifuged at 8000 rpm (approximately 5.9 rcf) for 3 min, and the OD of the supernatant was adjusted to 0.7 at 541 nm (an OD of 0.7 at 541 nm corresponds to approximately 109 erythrocytes/mL). Then, 1 mL of red blood cells resuspended at OD 0.7 was added to 9 mL of BBS/Mg++/EGTA to reach a concentration of 108 erythrocytes/mL. Dilutions of test sera or plasma were prepared in ice-cold BBS, Mg++, EGTA and 100 μL of each serum or plasma dilution was pipetted into the corresponding well of a round-bottom plate. 100 μL of appropriately diluted red blood cells (108 erythrocytes/ml) were added to each well. Nano-water was used to produce the positive control (100% lysis), while a dilution with BBS/Mg++/EGTA without serum or plasma was used as a negative control. The plate was then incubated for 1 h at 37°C. The round-bottom plate was centrifuged at 3750 rpm for 5 min. Then, 100 μL of the supernatant from each well was transferred into the corresponding wells of a flat-bottom plate and OD was read at 415–490 nm. Results:

[00633] FIGURE 36 graphically illustrates the percentage hemolysis (as measured by the release of hemoglobin from lysed rabbit erythrocytes into the supernatant measured by photometry) of mannan-coated rabbit erythrocytes over a range of serum concentrations in the serum of normal individuals and two 3MC patients (Patient 2 and Patient 3), measured in the absence of Ca++. As shown in FIGURE 36, it is demonstrated that MASP-3 deficiency reduces the percentage of complement-mediated lysis of mannan-coated erythrocytes compared to normal human serum. The differences between the two curves from normal human serum and the two curves from 3MC patients are significant (p = 0.013, Friedman test).

[00634] TABLE 16 below summarizes the AP50 results shown in FIGURE 36. TABLE 16: The summary of results is shown in FIGURES 36

[00635] Note that when the serum samples presented in TABLE 16 were pooled, the AP50 value for normal human serum = 7.9 and the AP50 value for 3MC serum = 12.8 (p = 0.031, Wilcox matched-pairs signed rank test). Experiment #5: Reconstitution of human 3MC serum by recombinant MASP-3 restores AP-driven C3b deposition on zymosan-coated plates Methods:

[00636] An AP assay was performed under specific traditional AP conditions (BBS/Mg++/EGTA, without Ca++, where BBS = barbital-buffered saline containing sucrose), as described in Bitter-Suermann et al., Eur. J. Immunol 11:291-295 (1981)), in zymosan-coated microtiter plates on the following serum samples: (1) 5% human serum from 3MC Patient No. 2 with full-length active rMASP-3 spiked in a range of 0 to 20 μg/mL; (2) 10% human serum from 3MC Patient No. 2 with full-length active rMASP-3 spiked in a range of 0 to 20 μg/mL; and (3) 5% human serum from 3MC Patient #2 with inactive rMASP-3A (S679A) added at a range of 0 to 20 μg/mL. Results:

[00637] FIGURE 37 graphically illustrates the level of AP-driven C3b deposition on zymosan-coated microtiter plates as a function of the concentration of rMASP-3 protein spiked into serum samples obtained from human 3MC patient No. 2 (MASP-3 deficient). As shown in FIGURE 37, active recombinant MASP-3 protein reconstitutes AP-driven C3b deposition on zymosan-coated plates in a concentration-dependent manner. As further shown in FIGURE 37, no C3b deposition was observed in 3MC serum containing inactive rMASP-3 (S679A). Experiment #6: Reconstitution of Human 3MC Serum with Recombinant MASP-3 Restores Hemolytic Activity in 3MC Patient Serum Methods:

[00638] A hemolytic assay was performed using rabbit red blood cells using the methods described above in Experiment #2 with the following test sera at a range of 0 to 12% serum: (1) normal human serum; (2) patient serum with 3MC; (3) patient serum with 3MC plus full-length active rMASP-3 (20 μg/ml); and (4) heat-inactivated human serum. Results:

[00639] FIGURE 38 graphically illustrates the percentage hemolysis (as measured by the release of hemoglobin from lysed rabbit erythrocytes into the supernatant measured by photometry) of mannan-coated rabbit erythrocytes over a range of serum concentrations in (1) normal human serum; (2) 3MC patient serum; (3) 3MC patient serum plus full-length active rMASP-3 (20 μg/ml); and (4) heat-inactivated human serum, measured in the absence of Ca++. As shown in FIGURE 38, the percentage of rabbit red blood cell lysis is significantly increased in 3MC serum including rMASP-3 compared to the percentage of lysis in 3MC serum without rMASP-3 (p = 0.0006).

[00640] FIGURE 39 graphically illustrates the percent lysis of rabbit erythrocytes in 7% human serum from 3MC Patient 2 and 3MC Patient 3 containing active rMASP-3 at a range of concentrations from 0 to 110 μg/ml in BBS/Mg++/EGTA. As shown in FIGURE 39, the percent lysis of rabbit red blood cells is restored with increasing rMASP-3 in a concentration-dependent manner up to 100% activity. Experiment #7: MASP-3-deficient patient (3MC) serum has functional MASP-2 if MBL is present Methods:

[00641] A C3b deposition assay was performed using mannan-coated ELISA plates to examine whether 3MC serum is deficient in LEA-2. Plasma citrate was diluted in BBS buffer in serial dilutions (starting with 1:80, 1:160, 1:320, 1:640, 1:1280, 1:2560) and transferred to mannan-coated plates. C3b deposition was detected using a chicken anti-human C3b assay. LEA-2-driven C3b deposition (plasma dilutions are high for AP and LEA-1 to function) on mannan-coated ELISA plates was assessed as a function of human serum concentration in serum from a normal human subject (NHS), two patients with 3MC (Patient 2 and Patient 3), the parents of Patient 3, and an individual with MBL deficiency. Results:

[00642] FIGURE 40 graphically illustrates the level of LEA-2-driven C3b deposition on mannan-coated ELISA plates as a function of human serum concentration diluted in BBS buffer, for serum from a normal human subject (NHS), two 3MC patients (Patient 2 and Patient 3), Patient 3's parents, and an MBL-deficient individual. These data indicate that Patient 2 is MBL sufficient. However, Patient 3 and Patient 3's mother are MBL deficient and therefore their serum does not deposit C3b in the mother via LEA-2. Replacement of MBL in these sera restores LEA-2-mediated C3b deposition in the serum of Patient 3 (who is homozygous for the SNP leading to MASP-3 deficiency) and his mother (who is heterozygous for the mutant MASP-3 allele) (data not shown). This finding demonstrates that 3MC serum is not deficient in LEA-2 but appears to have functional MASP-2. Overall summary and conclusions:

[00643] These results demonstrate that MASP-3 deficiency in human serum results in loss of AP activity, as manifested by reduced C3b deposition in zymosan-coated wells and reduced lysis of rabbit erythrocytes. AP can be restored in both assays by supplementing the sera with functional recombinant human MASP-3.

EXAMPLE 11

[00644] This Example demonstrates that a human IgG4 constant region/V region anti-human MASP-3 monoclonal chimeric mouse antibody (mAb M3-1, also referred to as mAb 13B1) is a potent inhibitor of MASP-3-mediated Alternative Pathway Complement Activation (APC). Methods:

[00645] Generation of human IgG4 constant region/V region anti-human MASP-3 chimeric mouse monoclonal antibody (mAb M3-1)

[00646] A murine anti-human MASP-3 inhibitory antibody (mAb M3-1) was generated by immunizing MASP-1/3 knockout mice with the human MASP-3 CCP1-CCP2-SP domain (aa 301-728 of SEQ ID NO: 2) (see also Example 14). Briefly described, splenocytes from the immunized mice were fused with P3/NS1/1-Ag4-1 and supernatants from resulting hybridoma clones were screened for the production of antibodies that bind to human MASP-3 and for the ability to block MASP-3-mediated cleavage of pro-factor D (pro-CFD) from complement factor D (CFD). Monoclonal antibody (mAb) variable regions were isolated by RT-PCR, sequenced, and cloned into human IgG4 expression vectors. Chimeric monoclonal antibodies were expressed in transiently transfected HEK293T cells, purified, and tested for binding affinity to mouse and human MASP-3 and for the ability to inhibit MASP-3-mediated cleavage of pro-CFD to CFD.

[00647] The MASP-3 inhibitory monoclonal antibody M3-1 (13B1) comprises a heavy chain variable region (VH) defined as SEQ ID NO:30 and a light chain variable region defined as SEQ ID NO:45. The sequences of the variable regions of the monoclonal antibody M3-1 are provided below: Heavy Chain Variable Region

[00648] The heavy chain variable region (VH) sequence is presented below for mAb M3-1. The Kabat CDRs (31-35 (H1), 50-65 (H2), and 95-102 (H3) are underlined, which correspond to amino acid residues 31-35 (H1), 50-66 (H2), and 99-102 (H3) of SEQ ID NO:30. mAb M3-1 heavy chain variable region (VH) (SEQ ID NO:30)

[00649] QVQLKQSGAELMKPGASVKLSCKATGYTFTGKWIEWV KQRPGHGLEWIGEILPGTGSTNYNEKFKGKATFTADSSSNTAYMQLSS LTTEDSAMYYCLRSEDVWGTGTTVTVSS Light Chain Variable Region

[00650] The sequence of the light chain variable region (VL) is presented below for mAb M3-1. The Kabat CDRs (24-34 (H1), 50-56 (H2), and 89-97 (H3) are underlined, which correspond to amino acid residues 24-40 (L1); 56-62 (L2), and 95-102 (L3) of SEQ ID NO:45. These regions are the same as numbered by the Kabat or Chothia system. mAb M3-1 light chain variable region (VL) (SEQ ID NO:45)

[00651 ] DIVMTQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNY LAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQA EDLAVYYCKQSYNIPTTFGGGTKLEIKR mAb M3-1 VH CDRs VHCDR1; GKWIE (SEQ ID NO:84) VHCDR2; EILPGTGSTNYNEKFKG (SEQ ID NO:86) VHCDR3; SEDV (SEQ ID NO:88) mAb M3-1 VL CDRs VLCDR1; KSSQSLLNSRTRKNYLA (SEQ ID NO:142) VLCDR2; WASTRES (SEQ ID NO:144) VLCDR3; KQSYNIPT (SEQ ID NO:161)

[00652] As shown above, the MASP-3 monoclonal antibody M3-1 comprises (a) a heavy chain variable region comprising (i) VHCDR1 comprising SEQ ID NO:84, (ii) VHCDR2 comprising SEQ ID NO:86, and (iii) VHCDR3 comprising SEQ ID NO:88; and (b) a light chain variable region comprising (i) VLCDR1 comprising SEQ ID NO:142, (ii) VLCDR2 comprising SEQ ID NO:144, and (iii) VLCDR3 comprising SEQ ID NO:161. Binding of mAb M3-1 to Recombinant Forms of Human and Mouse MASP-3

[00653] A monovalent Fab version of M3-1 was tested for binding to recombinant full-length human or mouse MASP-3 protein in an ELISA experiment. Binding affinity determinations were made by coating 96-well plates with an anti-MASP-3 capture antibody that binds to the protein from multiple species. The capture antibody was shown to bind to the CCP1-CCP2 region of MASP-1 and MASP-3. Full-length human and mouse protein versions were immobilized on ELISA plates coated with the capture antibody, and varying concentrations of M3-1 Fab were bound to the target protein in separate wells. Bound M3-1 was detected using an anti-kappa light chain antibody that is conjugated to HRP (Novus Biologicals NBP1-75064) and was visualized with the TMB substrate reagent set (BD Biosciences 555214).

[00654] FIGURE 41 graphically illustrates a representative example of a binding experiment that was performed with human MASP-3 in which the M3-1 Fab (also referred to as 13B1) shows an apparent binding affinity (EC50) of about 0.117 nM to the human protein.

[00655] FIGURE 42 graphically illustrates a representative example of a binding experiment that was performed with mouse MASP-3 in which the M3-1 Fab shows an apparent binding affinity (EC50) of about 0.214 nM to the mouse protein.

[00656] These results demonstrate that mAb M3-1 (13B1) has high binding affinity for human and mouse MASP-3.

[00657] Demonstration that mAb M3-1 is capable of inhibiting Alternative Pathway Complement (APC) activation and measurement of the in vitro potency of mAb M3-1

[00658] As described herein, MASP-3 has been determined to be a key regulator of APC, at least in part due to its requirement for activation of CFD, a core APC enzyme. As also described herein, MASP-3 circulates in the body at a relatively low concentration and has a slow catabolic rate, allowing for long-lasting inhibition of the pro-inflammatory pathway through intravenous, subcutaneous, and oral routes of MASP-3 antibody administration. The following experiment was performed to determine the efficacy of mAb M3-1 to inhibit MASP-3-mediated CFD maturation and APC inhibition in human serum. Normal human serum contains predominantly activated or processed (i.e., mature) CFD, so experiments were performed in which human serum depleted of CFD (Complement Technology A336) was reconstituted with a recombinant unprocessed form of CFD (pro-CFD). Thus, in this experimental system, APC activation requires the processing of pro-CFD into active CFD.

[00659] APC was induced by the addition of zymosan particles, which function as an activation surface for complement deposition. Varying concentrations of mAb M3-1 were added to the serum prior to the addition of recombinant pro-CFD and zymosan. The mixtures were incubated at 37°C for 75 minutes and APC activity was measured by flow cytometric detection of complement factor Bb (Quidel A252) on the surface of the zymosan particles.

[00660] FIGURE 43 graphically illustrates the level of complement factor Bb deposition on zymosan particles (determined by flow cytometric detection measured in MFI units) in the presence of varying concentrations of mAb M3-1 in CFD-depleted human serum. As shown in FIGURE 43, mAb M3-1 exhibits potent inhibition of APC in 10% human serum, with an IC50 of 0.311 nM in this experimental example.

[00661] These results demonstrate that MASP-3 plays a key role in APC activation in an in vitro human serum model and further demonstrate that mAb M3-1 is a potent inhibitor of APC. Inhibition of APC by mAb M3-1 in vivo:

[00662] To determine the efficacy of M3-1 for APC inhibition in vivo, a group of mice (n = 4) received a single intravenous injection of 10 mg/kg mAb M3-1 into the tail vein. Blood collected from the animals was used to prepare serum, providing a matrix for flow cytometric assessment of APC activity in an ex vivo assay measuring the level of C3 (also C3b and iC3b) deposition on zymosan particles. Serum prepared from blood collected at a pre-dose assessment time point and several post-dose assessment time points (96 hours, 1 week, and 2 weeks) was diluted to 7.5% and zymosan particles were added to induce APC. The antibody-treated mice were compared to a group of control mice (n = 4) given a single intravenous dose of vehicle.

[00663] Figure 44 graphically illustrates the level of C3 deposition on zymosan particles at various time points following a single dose of mAb M3-1 (10 mg/kg i.v.) in wild-type mice. As shown in FIGURE 44, at the pre-dose time point the two conditions show comparable levels of APC activity. At 96 hours and two subsequent time points, the mAb M3-1-treated group displays essentially complete APC inhibition, while the carrier-treated group's APC remains undiminished. As shown in FIGURE 44, a single dose of mAb M3-1 administered intravenously to mice led to nearly complete ablation of systemic APC activity for at least 14 days.

[00664] These results demonstrate that mAb M3-1 is a potent inhibitor of APC in vivo in a mouse model.

EXAMPLE 12

[00665] This Example demonstrates that the chimeric human IgG4 constant region/mouse V region anti-human MASP-3 monoclonal antibody (mAb M3-1, also referred to as mAb 13B1) provides a clear benefit to the survival of Crry-depleted red blood cells in a mouse model associated with paroxysmal nocturnal hemoglobinuria (PNH). Methods:

[00666] The chimeric human IgG4 constant region/mouse V region anti-human MASP-3 monoclonal antibody (mAb M3-1) was generated as described in Example 11 and Example 14. As further described in Example 11, mAb M3-1 was determined to be a potent inhibitor of APC in an in vivo mouse model. This Example describes the analysis of mAb M3-1 for efficacy in a murine model of PNH. Analysis of mAb M3-1 for efficacy in a murine model of PNH

[00667] In a mouse model of PNH, red blood cells from Crry-deficient mice lacking the major cell surface repressor of APC in the rat were obtained for use as donor cells. Red blood cells obtained from a wild-type (WT) donor mouse were assayed in parallel. These donor red blood cells were differentially labeled with lipophilic fluorescent dyes (Sigma): WT (red) and Crry- (green). In two separate experiments, the labeled WT and Crry- donor cells were mixed 1:1 and injected intravenously into wild-type recipient mice, and the percentage of red blood cell survival (relative to the previous time point) in the recipient mice was determined by flow cytometric evaluation of 20,000 live cell events. In the first experiment, multiple pre-dose treatments of mAb M3-1 were given and the effect of mAb M3-1 was compared with that of another complement inhibitory antibody mAb BB5.1 (available from Hycult Biotech), which is a C5 inhibitory antibody that has shown efficacy in multiple mouse studies (Wang et al., PNAS vol 92:8955-8959, 1995; Hugen et al., Kidney Int 71(7):646-54, 2007). Administration of a C5 inhibitor is the current standard of care for human patients with PNH. In the second experiment, a single pre-treatment dose of mAb M3-1 was evaluated.

[00668] In the first experiment, three different groups of mice (n = 4 per condition) were evaluated: vehicle-treated condition, mAb M3-1-treated condition, and mAb BB5.1 (rat C5 blocking mAb)-treated condition. Labeled cells were injected into mice on “day 0,” and multiple doses of M3-1 and BB5.1 were administered as follows: mAb M3-1 was administered intravenously (10 mg/kg) on days -11, -4, -1, and +6. mAb BB5.1 was administered by intraperitoneal injection (40 mg/kg) on days -1, +3, +6, and +10. Carrier treatment followed the same dosing schedule as mAb M3-1.

[00669] FIGURE 45 graphically illustrates the percent survival of donor red blood cells (WT or Crry-) over a 14-day period in WT recipient mice treated with mAb 13B1 (10 mg/kg on days -11, 04, WT, and +6), mAb BB5.1-treated, or vehicle-treated mice. As shown in FIGURE 45, compared to WT red blood cells that showed typical mouse red blood cell survival in vehicle-treated animals, Crry-deficient red blood cells had rapid clearance (greater than 75% cleared within 24 hours). Treatment of mice with mAb BB5.1 provided no improvement over vehicle treatment in the survival of Crry-deficient red blood cells. In contrast, treatment with mAb M3-1 caused a dramatic improvement in the survival of Crry-deficient red blood cells in both mAb BB5.1- and carrier-treated animals. The protective effect of mAb M3-1 was observed throughout the duration of the experiment.

[00670] In the second study, differentially labeled WT (red) and Crry (green) red blood cells were evaluated in two different groups of WT mice (n = 4 per condition): carrier-treated and M3-1 mAb-treated. A single dose of carrier or antibody (20 mg/kg) was given to recipient mice via intravenous administration six days (day -6) before labeled donor cells were injected into the recipient mice. The labeled red blood cells were then analyzed for percent survival in the recipient mice at incremental assessment times after injection over a 16-day period.

[00671] FIGURE 46 graphically illustrates the percent survival of donor red blood cells (WT or Crry-) over a 16-day period in WT recipient mice treated with a single dose of mAb M3-1 (20 mg/kg on day -6) or carrier-treated mice. As shown in FIGURE 46, a single pretreatment dose of mAb M3-1 demonstrated improved survival of Crry-red blood cells compared to survival of Crry-red blood cells in carrier-treated mice. At 96 hours post-injection, approximately 90% of vehicle-treated WT red blood cells survived under control conditions, while only 5% of Crry-red blood cells survived in carrier-treated WT mice. In contrast to vehicle-treated mice, 40% of CrRy red blood cells survived in mice treated with mAb M3-1.

[00672] Together, these results demonstrate that the MASP-3 inhibitory antibody mAb M3-1 provides a clear survival benefit to red blood cells lacking Crry, a key surface complement inhibitor in a mouse model of PNH.

EXAMPLE 13

[00673] This Example describes a study demonstrating that a chimeric MASP-3 inhibitory monoclonal antibody (mAb M3-1, also referred to as mAb 13B1) reduces clinical scores in collagen antibody-induced arthritis (CAIA), a murine model of rheumatoid arthritis (RA). Background/Rationale:

[00674] CAIA is a well-established animal model of arthritis. In addition to providing insight into RA, the pathology of the CAIA model has an established link to the APC. Banda and colleagues demonstrated improved outcomes in the CAIA model in mice deficient in components of the APC, such as factor B and factor D (Banda et al., J. Immunol vol 177:1904-1912, 2006 and Banda et al., Clinical & Exp Immunol vol 159:100-108, 2009). APC knockout mice demonstrate lower arthritis (disease) scores, lower incidence, and decreased deposition of C3 and factor H in the synovium and adjacent tissues relative to WT controls. Furthermore, disease activity scores, tissue deposition of complement C3 in the joint, and histopathological lesion scores were markedly decreased in MASP1/3 knockout mice. Therefore, the MASP-3 inhibitory antibody mAb M3-1 was analyzed for efficacy in CAIA. Methods:

[00675] The chimeric MASP-3 monoclonal antibody (mAb M3-1) was generated as described in Example 11 and Example 14. As further described in Example 11, mAb M3-1 was determined to be a potent inhibitor of APC in an in vivo mouse model.

[00676] The mAb M3-1 was tested in the CAIA model as follows. Wild-type mice (n = 7) were injected intravenously with 3 mg of an anti-collagen antibody mixture on day 0. Mice were dosed intraperitoneally with E. coli lipopolysaccharide (LPS) (25 μg/mouse) on day +3. As described in Nandakumar et al. (Am J Pathol 163(5):1827-1837, 2003), arthritis typically occurs in this model on days +3 to +10. Terminal serum samples were collected on day +14. The mAb M3-1 (5 mg/kg and 20 mg/kg) was administered on days -12, -5, +1, and day +7. Carrier (PBS) was used as a negative control.

[00677] Clinical scores were assessed for each rat on all 4 paws on study days 0 through 14 using the following scoring standards: 0=normal

[00678] 1 = 1 hind and/or forepaw joint affected or minimal diffuse erythema and swelling 2 = 2 hind and/or forepaw joints affected or mild diffuse erythema and swelling 3 = 3 hind and/or forepaw joints affected or moderate diffuse erythema and swelling 4 = marked diffuse erythema and swelling or 4 toe joints affected 5 = severe diffuse erythema and severe swelling of entire paw, unable to flex toes.

[00679] The incidence = % of mice within a treatment group showing arthritic symptoms was also determined.

[00680] The results are presented in Figure 47 (clinical scores) and Figure 48 (arthritis incidence). Figure 47 graphically illustrates the clinical scores of mice treated with mAb M3-1 (5 mg/kg or 20 mg/kg) or carrier over a 14-day period. Figure 48 graphically illustrates the percentage arthritis incidence of mice treated with mAb M3-1 (5 mg/kg or 20 mg/kg) or carrier over a 14-day period. As shown in FIGURE 47, mAb M3-1 demonstrates a clear therapeutic benefit for both endpoints, beginning on Day 5 and lasting throughout the duration of the study. As shown in FIGURE 48, while disease incidence reached 100% in vehicle-treated animals, two-thirds of the animals in the mAb M3-1 5 mg/kg condition remained disease free. Additionally, only one of the animals (i.e., only one out of a total n = 7) demonstrated any arthritic symptoms in the 20 mg/kg mAb M3-1 condition.

[00681] The results of this study demonstrate that the MASP-3 inhibitory antibody mAb M3-1 provides a clear therapeutic benefit in the CAIA model, a well-established murine model of rheumatoid arthritis (RA) and a model strongly linked to APC activation. As shown in Example 44, a single dose of mAb M3-1 administered intravenously to mice led to a near-complete ablation of systemic APC activity for at least 14 days. As shown in this Example, in the animal model induced by administration of autoantibodies against rat connective tissue, mAb M3-1 reduced the incidence and severity of clinical arthritis scores in a dose-dependent manner. Compared to control-treated animals, mAb M3-1 reduced disease incidence and severity by approximately 80% at the highest dose tested. Therefore, it is expected that administration of a MASP-3 inhibitory antibody such as mAb M3-1 will be an effective therapy in patients suffering from arthritis such as rheumatoid arthritis, osteoarthritis, juvenile rheumatoid arthritis, infection-related arthritis, psoriatic arthritis, as well as ankylosing spondylitis and Bechçet's disease.

EXAMPLE 14

[00682] This example describes the generation of high affinity anti-human MASP-3 inhibitory antibodies. Background/Rationale:

[00683] A limited number of antibodies specific for MASP-3 have been described (Thiel et al., Mol. Immunol. 43:122, 2006; Moller-Kristensen et al., Int. Immunol. 19:141, 2006; Skjoedt et al., Immunobiol 215:921, 2010). These antibodies were useful for detection assays such as Western blotting, immunoprecipitation, and as capture or detection reagents in ELISA assays. However, the antibodies described in Thiel et al., 2006, Moller-Kristensen et al., 2006, and Skjoedt et al., 2010 did not inhibit the catalytic activity of MASP-3.

[00684] MASP-3 antibodies were also previously generated as described herein in Example 7 (also published as Example 15 in WO2013/192240) by screening a chicken antibody library on a modified DT40 cell line, DTLacO, for MASP-3 binding molecules. These antibodies bound to human MASP-3 in the nanomolar range with an EC50 between 10 nM and 100 nM and partially inhibited pro-CFD cleavage by MASP-3.

[00685] This Example describes the generation of anti-human MASP-3 inhibitory antibodies with exceptionally strong binding affinity (i.e., subnanomolar binding affinity, ranging from <500 pM to 20 pM). The antibodies described in this Example specifically bind to human MASP-3 with high affinity (e.g., <500 pM), inhibit Factor D maturation, and do not bind to human MASP-1 (SEQ ID NO: 8). Methods: 1. Generation of human IgG4 constant region/V region anti-human MASP-3 monoclonal chimeric mouse antibodies

[00686] Seven- to fourteen-week-old C57BL/6, MASP-1/3 knockout mice were immunized with the CCP1/CCP2/SP polypeptide of human MASP-3 (amino acid residues 299-728 of SEQ ID NO:2) including a StrepTag II epitope tag at the N-terminus; or were immunized with the SP domain of human MASP-3 (amino acid residues 450-728 of SEQ ID NO:2) including StrepTagII at the N-terminus using the Sigma Adjuvant System (Sigma-Aldrich, St Louis, MO). Mice were injected intraperitoneally with 50 μg of immunogen per mouse. Immunized mice were boosted 14 days later with additional immunogen in adjuvant. Thereafter, for several weeks, mice were boosted every 14 to 21 days with immunogen in PBS. Serum samples from mice were prepared periodically from tail bleeds and tested by ELISA for the presence of antigen-specific antibodies. Mice with a significant antibody titer received a prefusion immunogen boost in PBS 4 days before splenic fusion. Three days before fusion, mice were treated subcutaneously at the base of the tail with 50 μg of an anti-CD40 agonist mAb in PBS (R&D Systems, Minneapolis, MN) to increase B cell numbers (see Rycyzyn et al., Hybridoma 27:2530, 2008). Mice were sacrificed and spleen cells were harvested and fused to a selected murine myeloma cell line P3/NSI/1-AG4-1 (NS-1) (ATCC No. TIB18) using 50% polyethylene glycol or 50% polyethylene glycol plus 10% DMSO. Fusions generated hybridoma cells that were transferred to 96-well tissue culture plates containing HAT (hypoxanthine, aminopterin, and thymidine) medium to inhibit proliferation of unfused cells, myeloma hybrids, and spleen hybrids. After hybridoma selection, culture supernatants were assayed for MASP-3 binding (ELISA) and inhibition of profactor D activation. Positive hybridomas were identified and subcloned by serial dilution methods. TABLE 17: Summary of Fusion Experiments

[00687] Note: "ND" means that this fusion was selected only for functional inhibition of pro-CFD activation. Results:

[00688] As shown in TABLE 17, a total of 3328 hybridomas from immunized MASP1/3 KO mice were screened, of which >303 bound to MASP-3 and of which 16 bound to MASP-3 and inhibited pro-CFD activation. The mAb M3-1 (13B1) described in Example 11 is one of the 16 functional MASP-3 inhibitory antibodies described in TABLE 17. As described in Example 15, all 16 functional MASP-3 inhibitory antibodies were determined to bind to human MASP-3 with exceptionally strong binding affinity (<500 pM). Discussion:

[00689] This Example describes the generation of antibodies that inhibit human MASP-3 with exceptionally strong binding affinity (i.e., subnanomolar binding affinity, ranging from <500 pM to 20 pM) by immunization of MASP1/3 knockout mice. The antibodies described in this Example specifically bind to human MASP-3 with high affinity (e.g., <500 pM), inhibit Factor D maturation, and do not bind to human MASP-1. As described herein, the amino acid sequences of human, rat, and chicken MASP-3 revealed that the SP domain of MASP-3 is highly conserved, especially in the active site (see FIGURES 4 and 5). It is likely that the ability to generate MASP-3 inhibitory antibodies with exceptionally strong binding affinity in MASP1/3 KO mice, as described in this example, is due in part to the prevention of immune tolerance that can hamper the generation of MASP-3 catalytic site-specific antibodies in wild-type animals.

EXAMPLE 15

[00690] This example describes the cloning and sequence analysis of high-affinity anti-human MASP-3 inhibitory mAbs. Methods: Cloning and purification of recombinant antibodies:

[00691] The light chain and heavy chain variable regions were cloned from the hybridomas described in Examples 11 and 14 using RT-PCR and sequenced. Mouse-human chimeric mAbs consisting of the mouse mAb variable regions fused to human IgG4 heavy chain (SEQ ID NO: 311) and kappa light chain (SEQ ID NO: 313) constant regions were produced as recombinant proteins in Expi293F cells. The IgG4 constant hinge region (SEQ ID NO: 311) contains the stabilizing S228P amino acid substitution. In one embodiment, the chimeric mAbs were fused to the human IgG4 constant hinge region (SEQ ID NO: 312) that contains the S228P amino acid substitution and also a mutation that promotes FcRn interactions at low pH.

[00692] The sequences of the heavy chain variable regions and light chain variable regions are shown in FIGURES 49A and 49B, respectively (“SIN” = “SEQ ID NO:” in FIGURE 49A and FIGURE 49B) and are included below. The complementarity regions (CDRs) and framework regions (FRs) of each are provided in TABLES 18-22 below.

[00693] FIGURE 50A is a dendrogram of the VH regions of high-affinity anti-human MASP-3 inhibitory mAbs generated in MASP1/3 KO mice. FIGURE 50B is a dendrogram of the VL regions of high-affinity anti-human MASP-3 inhibitory mAbs generated in MASP1/3 KO mice. As shown in FIGURES 50A and 50B, several groups of related antibodies have been identified.

[00694] The heavy chain variable region (VH) sequence for each high affinity MASP-3 inhibitory antibody is presented below. The Kabat CDRs are underlined. Heavy Chain Variable Regions: 4D5 VH: SEQ ID NO:24

[00695] QVQLKQSGPELVKPGASVKLSCKASGYTFTTDDINWVK QRPGQGLEWIGWIYPRDDRTKYNDKFKDKATLTVDTSSNTAYMDLH SLTSEDSAVYFCSSLEDTYWGQGTLVAVSS 1F3 VH: SEQ ID NO:25

[00696] QVQLKQSGPELVKPGASVKLSCKASGYTFTSNDINWVK QRPGQGLEWIGWIYPRDGSIKYNEKFTDKATLTVDVSSSTAYMELHS LTSEDSAVYFCSGVEDSYWGQGTLVTVSS 4B6 VH: SEQ ID NO:26

[00697] QVQLKQSGPELVKPGASVKLSCKASGYTFTSNDINWVK QRPGQGLEWIGWIYPRDGTTKYNEEFTDKATLTVDVSSSTAFMELHS LTSEDSAVYFCSSVEDSYWGQGTLVTVSS 1A10 VH: SEQ ID NO:27

[00698] QVQLKQSGPELVKPGASVKLSCKASGYTFTSNDINWVK QRPGQGLEWIGWIYPRDGTTKYNEKFTDKATLTVDVSSSTAFMELHR LTSEDSAVYFCSSVEDSYWGQGTLVTVSS 10D12 VH: SEQ ID NO:28

[00699] QIQLVQSGPELKKPGETVKISCKASGYIFTSYGMSWVRQ APGKGLKWMGWINTYSGVPTYADDFKGRFAFSLETSARTPYLQINNL KNEDTATYFCARGGEAMDYWGQGTSVTVSS 35C1 VH: SEQ ID NO:29

[00700] QIQLVQSGPELKTPGETVKISCKASGYIFTSYGITWVKQ APGKGLKWMGWINTYSGVPTYADDFKGRFAFSLETSASTAYLQINNL KNEDTTTYFCTRGGDALDYWGQGTSVTVSS 13B1 VH: SEQ ID NO:30

[00701 ] QVQLKQSGAELMKPGASVKLSCKATGYTFTGKWIEWV KQRPGHGLEWIGEILPGTGSTNYNEKFKGKATFTADSSSNTAYMQLSS LTTEDSAMYYCLRSEDVWGTGTTVTVSS 1G4 VH: SEQ ID NO:31

[00702] QVQLKQSGAELMKPGASVKLACKATGYTFTGYWIEWI KQRPGQGLEWIGEMLPGSGSTHYNEKFKGKATFTADTSSNTAYMQLS GLTTEDSAIYYCVRSIDYWGQGTTLTVSS 1E7 VH: SEQ ID NO: 32

[00703] QVQLKQSGPELARPWASVKISCQAFYTFSRRVHFAIRDT NYWMQWVKQRPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKS SSTAYMQLSSLTSEDSAVYYCASGSHYFDYWGQGTTLTVSS 2D7 VH: SEQ ID NO:33

[00704] EVQLQQSGPELVKPGASVKVSCKASGYTLTDYYMNWV KQSHGKSLEWIGDVNPNNDGTTYNQKFKGRATLTVDKSSNTASMEL RSLTSEDSAVYYCAICPFYYLGKGTHFDYWGQGTSLTVSS 49C11 VH: SEQ ID NO: 34

[00705] EVQLQQSGPVLVKPGASGKMSCKASGYKFTDYYMIWV KQSHGKSLEWIGVIKIYNGGTSYNQKFKGKATLTVDKSSSTAYMELN SLTSEDSAVYYCARGPSLYDYDPYWYFDVWGTGTTVTVSS 15D9 VH: SEQ ID NO:35

[00706] QVQLKQSGTELMKPGASVNLSCKASGYTFTAYWIEWV KQRPGHGLEWIGEILPGSGTTNYNENFKDRATFTADTSSNTAYMQLSS LTSEDSAIYYCARSYYYASRWFAFWGQGTLVTVSS 2F5 VH: SEQ ID NO:36

[00707] EVQLQQPGAELVKPGASVKMSCKASGYTFTSYWITWV KQRPGQGLEWIGDIYPGSGSTNYNEKFKSKATLTVDTSSSTAYMQLSS LTSEDSAVYYCARRRYYATAWFAYWGQGTLVTVSS 1B11 VH: SEQ ID NO:37

[00708] QVQLKQSGAELVRPGASVKLSCKASGYTFTDYYINWV KQRPGQGLEWIARIYPGSGNTYYNEKFKGKATLTAEKSSSTAYMQLS SLTSEDSAVYFCARNYYISSPWFAYWGQGTLVTVSS 2F2 VH: SEQ ID NO:38

[00709] QVQLKQSGAELVTPGASVKMSCKASGYTFTTYPIEWM KQNHGKSLEWIGNFHPYNDDTKYNEKFKGKATLTVEKSSNTVYLELS RLTSDDSAVYFCARRVYYSYFWFGYWGHGTLVTVSS 11B6 VH: SEQ ID NO:39

[00710] QVQLKQSGAELVKPGASVKMSCKASGYTFTTYPIEWM KQNHGKSLEWIGNFHPYNGDSKYNEKFKGKATLTVEKSSSTVYLELS RLPSADSAIYYCARRHYAASPWFAHWGQGTLVTVSS TABLE 18: MASP-3 antibody VH sequence (CDRs and FR regions, Kabat)

[00711] The light chain variable region (VL) sequences for high-affinity MASP-3 inhibitory antibodies are presented below. The Kabat CDRs are underlined. These regions are the same, numbered by the Kabat or Chothia system. Light Chain Variable Regions: 4D5 VL; SEQ ID NO:40

[00712] DIVMTQSPSSLAVSAGEKVTMTCKSSQSLLNSRTRKNY LAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFSLTISSVQA EDLAVYYCKQSYNLYTFGGGTKLEIKR 1F3 VL: SEQ ID NO:41

[00713] DIVMTQSPSSLAVSAGERVTMSCKSSQSLLISRTRKNYL SWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAE DLAVYYCKQSYNLYTFGGGTKLEIKR 4B6 VL: SEQ ID NO:42 (SAME for 1A10 VL)

[00714] DIVMTQSPSSLAVSAGEKVTMSCKSSQSLLISRTRKNYL SWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAE DLAVYYCKQSYNLYTFGGGTKLEIKR 10D12 VL: SEQ ID NO:43

[00715] DVLMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLN WLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAED LGVYYCWQGTHFPWTFGGGTKLEIKR 35C1 VL: SEQ ID NO:44

[00716] DIVMTQAPLTLSVTIGQPASISCKSSQSLLDSDGKTYLS WLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAED LGVYYCWQGTHFPYTFGGGTKLEIKR 13B1 VL: SEQ ID NO:45

[00717] DIVMTQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNY LAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQA EDLAVYYCKQSYNIPTFGGGTKLEIKR 1G4 VL: SEQ ID NO:46

[00718] DVLMTQTPLSLPVSLGEQASISCRSSQSLVQSNGNTYLH WYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAED LGVYFCSQSTHVPPTFGGGTKLEIKR 1E7 VL: SEQ ID NO:47

[00719] DIQLTQSPAILSVSPGERVSFSCRASQSIGTSIHWYQQRT NGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQS NSWPYTFGGGTKLEIKR 2D7 VL: SEQ ID NO:48

[00720] DIQMTQTPASLSASLGDRVTISCRASQDISNFLNWYQQK PNGTVKLLVFYTSRLHSGVPSRFSGSGSGAEHSLTISNLEQEDVATYFC QQGFTLPWTFGGGTKVEIKR 49C11 VL:SEQ ID NO:49

[00721 ] DVLMTQTPL SLPVSLGDQASFSCRSSQSLIHSNGNTYLH WYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAED LGVYFCSQSTHVPWTFGGGTKLEIKR 15D9 VL: SEQ ID NO: 50

[00722] DIVMTQSQKFMSTSIGDRVSVTCRASQNVGPNLAWYQ QKPGQSPKALIYSASYRFSGVPDRFTGSGSGTDFTLTISNVQSEDLAEY FCQQYNRYPFTFGSGTKLEIKR 2F5 VL: SEQ ID NO: 51

[00723] DIVMTQSQKFMSTSVGDRVSITCKASQNVGTAVAWYQ QKPGQSPKLLISSASNRYTGVPDRFTGSGSGTDFTLTISNMQSEDVAD YFCQQYNSYPLTFGAGTKLELKR 1B11 VL: SEQ ID NO:52

[00724] DIVMTQSQKFMSTSVGDRVSVTCKASQNVGPNVAWYQ QKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAD YFCQQYNRYPLTFGAGTKLELKR 2F2 VL: SEQ ID NO:53

[00725] DIVMTQSQKFMSTSVGDRVNVTCKASQNVGTHVAWY QQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAE YFCQQYNSYPRALTFGAGTKLELKR 11B6 VL: SEQ ID NO:54

[00726] DIVMTQSQKFMSTSVGDRVNVTCKASQNVGPTVAWY QQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVHSEDLAE YFCQQYNSYPFTFGSGTKLEIKR TABLE 19: MASP-3 antibody VL sequence (CDRs and FR regions, Kabat and Chothia) *Note: The light chain for mAb 1A10 was not identified, so the light chain of 4B6 was used with the 1A10 HC. TABLE 20: Consensus sequences for HC CDRs of group IA: TABLE 21: Consensus sequences for LC CDRs of group IA: *Note: The CDR-L1 consensus includes variants generated as described in Example 19. TABLE 22 Consensus sequences for group IB HC CDRs: TABLE 23 Consensus sequences for group IB LC CDRs: *Note: The consensus CDR-L1 includes variants generated as described in Example 19. DNA encoding mouse mAb light and heavy chains: SEQ ID NO:217: DNA encoding 4D5 heavy chain variable region (precursor)

[00727] CAGGTGCAGCTGAAGCAGTCTGGACCTGAGCTGGTG AAGCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGCTACA CCTTCACAACCGACGATATAAACTGGGTGAAGCAGAGGCCTGGAC AGGGACTTGAGTGGATTGGATGGATTTATCCTAGAGATGATAGAA CTAAGTACAATGACAAGTTCAAGGACAAGGCCACATTGACTGTAG ACACATCTTCCAACACAGCGTACATGGACCTCCACAGCCTGACATC TGAGGACTCTGCGGTCTATTTCTGTTCAAGCCTCGAGGATACTTAC TGGGGCCAAGGGACTCTGGTCGCTGTCTCTTCA SEQ ID NO:218: DNA encoding heavy chain variable region 1F3 (precursor)

[00728] CAGGTGCAGCTGAAGCAGTCTGGACCTGAGCTGGTG AAGCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGCTACA CCTTCACAAGTAACGATATAAACTGGGTGAAGCAGAGGCCTGGAC AGGGACTTGAGTGGATTGGATGGATTTATCCTAGAGATGGGAGTA TTAAATATAATGAGAAATTCACGGACAAGGCCACATTGACAGTTG ACGTATCCTCCAGCACAGCGTACATGGAGCTCCACAGCCTGACAT CTGAGGACTCTGCGGTCTATTTCTGTTCAGGTGTCGAGGATTCTTA CTGGGGCCAAGGGACTCTGGTCACTGTCTCTTCA SEQ ID NO:219: DNA encoding heavy chain variable region 4B6 (precursor)

[00729] CAGGTGCAGCTGAAGCAGTCTGGACCTGAACTGGTG AAGCCTGGGGCTTCAGTGAAATTGTCCTGCAAGGCTTCTGGCTACA CCTTCACAAGTAACGATATAAACTGGGTGAAACAGAGGCCTGGAC AGGGACTTGAGTGGATTGGATGGATTTATCCTAGAGATGGTACTAC TAAGTACAATGAGGAGTTCACGGACAAGGCCACATTGACTGTTGA CGTATCCTCCAGCACAGCGTTCATGGAGCTCCACAGCCTGACATCT GAGGACTCTGCTGTCTATTTCTGTTCAAGTGTCGAGGATTCTTACT GGGGCCAAGGGACTCTGGTCACTGTCTCTTCA SEQ ID NO:220: DNA encoding heavy chain variable region 1A10 (precursor)

[00730] CAGGTGCAGCTGAAGCAGTCTGGACCTGAGCTGGTG AAGCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGCTACA CCTTCACAAGTAACGATATAAACTGGGTGAAGCAGAGGCCTGGAC AGGGACTTGAGTGGATTGGATGGATTTATCCTAGAGATGGTACTAC TAAGTACAATGAGAAGTTCACGGACAAGGCCACATTGACTGTTGA CGTATCCTCCAGCACAGCGTTCATGGAGCTCCACAGGCTGACATCT GAGGACTCTGCGGTCTATTTCTGTTCAAGTGTCGAGGATTCTTACT GGGGCCAAGGGACTCTGGTCACTGTCTCTTCA SEQ ID NO:221: DNA encoding heavy chain variable region 10D12 (precursor)

[00731] CAGATCCAGTTGGTACAGTCTGGACCTGAGCTGAAG AAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGTAT ATTTTCACAAGCTATGGAATGAGCTGGGTGAGACAGGCTCCAGGA AAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACTCTGGAGTG CCAACATATGCTGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGG AAACCTCTGCCAGAACTCCCTATTTGCAGATCAACAACCTCAAAAA ATGAGGACACGGCTACATATTTCTGCGCAAGAGGGGGCGAAGCTA TGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA SEQ ID NO:222: DNA encoding heavy chain variable region 35C1 (precursor)

[00732] CAGATCCAGTTGGTACAGTCTGGACCTGAGCTGAAG ACGCCAGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGTAT ATCTTCACATCCTATGGAATTACCTGGGTGAAACAGGCTCCAGGA AAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACTCTGGAGTG CCAACATATGCTGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGG AAACGTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAA ATGAGGACACGACTACATATTTCTGTACAAGAGGGGGTGATGCTT TGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA SEQ ID NO:223: DNA encoding heavy chain variable region 13B1 (precursor)

[00733] CAGGTGCAGCTGAAGCAGTCTGGAGCTGAGCTGATG AAGCCTGGGGCCTCAGTGAAGCTTTCCTGCAAGGCTACTGGCTAC ACATTCACTGGCAAGTGGATAGAGTGGGTAAAACAGAGGCCTGGA CATGGCCTAGAGTGGATTGGAGAGATTTTACCTGGAACTGGTAGT ACTAACTACAATGAGAAGTTCAAGGGCAAGGCCACATTCACTGCA GACTCATCCTCCAACACAGCCTACATGCAACTCAGCAGCCTGACA ACTGAAGACTCTGCTATGTATTATTGTTTAAGATCCGAGGATGTCT GGGGCACAGGGACCACGGTCACCGTCTCCTCA SEQ ID NO:224: DNA encoding heavy chain variable region 1G4 (precursor)

[00734] CAGGTGCAGCTGAAGCAGTCTGGAGCTGAGCTGATG AAGCCTGGGGCCTCAGTGAAGCTTGCCTGCAAGGCTACTGGCTAC ACATTCACTGGCTACTGGATAGAGTGGATAAAGCAGAGGCCTGGA CAAGGCCTTGAGTGGATTGGAGAGATGTTACCTGGAAGTGGTAGT ACTCACTACAATGAGAAGTTCAAGGGTAAGGCCACATTCACTGCA GATACATCCTCCAACACAGCCTACATGCAACTCAGCGGCCTGACA ACTGAGGACTCTGCCATCTATTACTGTGTAAGAAGCATAGACTACT GGGGCCAAGGCACCACTCTCACAGTCTCCTCA SEQ ID NO:225: DNA encoding heavy chain variable region 1E7 (precursor)

[00735] CAGGTGCAGCTGAAGCAGTCTGGGCCTGAGCTGGCA AGGCCTTGGGCTTCAGTGAAGATATCCTGCCAGGCTTTCTACACCT TTTCCAGAAGGGTGCACTTTGCCATTAGGGATACCAACTACTGGAT GCAGTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATCGG GGCTATTTATCCTGGAAATGGTGATACTAGTTACAATCAGAAGTTC AAGGGCAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCC TACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATT ACTGTGCATCCGGTAGCCACTACTTTGACTGGGGCCAAGGCAC CACTCTCACAGTCTCCTCA SEQ ID NO:226: DNA encoding heavy chain variable region 2D7 (precursor)

[00736] GAGGTCCAGCTGCAACAATCTGGGCCTGAGCTGGTG AAGCCTGGGGCTTCAGTGAAGGTATCCTGTAAGGCTTCTGGATAC ACGCTCACTGACTACTACATGAACTGGGTGAAGCAGAGCCATGGA AAGAGCCTTGAGTGGATTGGAGATGTTAATCCTAACAATGATGGT ACTACCTACAACCAGAAATTCAAGGGCAGGGCCACATTGACTGTA GACAAGTCTTCCAACACAGCCTCCATGGAGCTCCGCAGCCTGACA TCTGAGGACTCTGCAGTCTACTACTGTGCAATATGCCCCTTTTATT ACCTCGGTAAAGGGACCCACTTTGACTGGGGCCAAGGCACCT CTCTCACAGTCTCCTCA SEQ ID NO:227: DNA encoding heavy chain variable region 49C11 (precursor)

[00737] GAGGTCCAGCTGCAACAATCTGGACCTGTGCTGGTG AAGCCTGGGGCTTCAGGGAAGATGTCCTGTAAGGCTTCTGGATAC AAATTCACTGACTACTATATGATCTGGGTGAAGCAGAGCCATGGA AAGAGCCTTGAGTGGATTGGAGTTATTAAAATTTATAACGGTGGTA CGAGCTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTTG ACAAGTCCTCCAGCACAGCCTACATGGAGCTCAACAGCCTGACAT CTGAGGACTCTGCAGTCTATTACTGTGCAAGAGGGCCATCTCTCTA TGATTACGACCCTTACTGGTACTTCGATGTCTGGGGCACAGGGACC ACGGTCACCGTCTCCTCA SEQ ID NO:228: DNA encoding heavy chain variable region 15D9 (precursor)

[00738] CAGGTGCAGCTGAAGCAGTCTGGAACTGAGCTGATG AAGCCTGGGGCCTCAGTGAACCTTTCCTGCAAGGCTTCTGCTACA CATTCACTGCCTACTGGATAGAGTGGGTAAAGCAGAGGCCTGGAC ATGGCCTTGAGTGGATTGGAGAGATTTTACCTGGAAGTGGTACTAC TAACTACAATGAGAACTTCAAGGACAGGGCCACATTCACTGCAGA TACATCCTCCAACACAGCCTACATGCAACTCAGCAGCCTGACAAG TGAGGACTCTGCCATCTATTACTGTGCAAGATCCTATTACTACGCT AGTAGATGGTTTGCTTTCTGGGGCCAAGGGACTCTGGTCACTGTCT CTTCA SEQ ID NO:229: DNA encoding 2F5 heavy chain variable region (precursor)

[00739] GAGGTCCAGCTGCAGCAGCCTGGGGCTGAGCTTGTG AAGCCTGGGGCTTCAGTGAAGATGTCCTGTAAGGCTTCTGCTACA CCTTCACCAGCTACTGGATAACCTGGGTGAAGCAGAGGCCTGGAC AAGGCCTTGAGTGGATTGGAGATATTTATCCTGGTAGTGGTAGTAC TAACTACAATGAGAAGTTCAAGAGCAAGGCCACACTGACTGTAGA CACATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCT GAGGACTCTGCGGTCTATTACTGTGCAAGAAGGAGATACTACGCT ACGGCCTGGTTTGCTTACTGGGGCCAAGGACTCTGGTCACTGTCT CTTCA SEQ ID NO:230: DNA encoding heavy chain variable region 1B11 (precursor)

[00740] CAGGTGCAGCTGAAGCAGTCTGGGGCTGAGCTGGTG AGGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACA CTTTCACTGACTACTATATAAACTGGGTGAAGCAGAGGCCTGGAC AGGGACTTGAGTGGATTGCAAGGATTTATCCTGGAAGTGGTAATA CTTACTACAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGCAG AAAAATCCTCCAGCACTGCCTACATGCAGCTCAGCAGCCTGACAT CTGAGGACTCTGCTGTCTATTTCTGTGCAAGAAATTACTACATTAG TAGTCCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTC TCTTCA SEQ ID NO:231: DNA encoding heavy chain variable region 2F2 (precursor)

[00741] CAGGTGCAGCTGAAGCAGTCTGGGGCTGAGCTAGTG ACGCCTGGAGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTAC ACCTTCACTACCTATCCTATAGAGTGGATGAAACAGAATCATGGA AAGAGCCTAGAGTGGATTGGAAATTTTCATCCTTACAATGATGATA CTAAGTACAATGAAAAGTTCAAGGGCAAGGCCACATTGACTGTAG AAAAATCCTCTAACACAGTCTACTTGGAGCTCAGCCGATTAACATC TGATGACTCTGCTGTTTATTTCTGTGCAAGGAGGGTCTACTATAGT TACTTCTGGTTTGGTTACTGGGGCCACGGGACTCTGGTCACTGTCT CTTCA SEQ ID NO:232: DNA encoding heavy chain variable region 11B6 (precursor)

[00742] CAGGTGCAGCTGAAGCAGTCTGGGGCTGAGCTAGTG AAACCTGGAGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTAC ACCTTCACTACCTATCCTATAGAGTGGATGAAGCAGAATCATGGG AAGAGCCTAGAGTGGATTGGAAATTTTCATCCTTACAATGGTGATT CTAAGTACAATGAAAAGTTCAAGGGCAAGGCCACCTTGACTGTAG AAAAATCCTCTAGCACAGTCT ACTTAGAACTCAGCCGATTACCATC TGCTGACTCTGCTATTTATTACTGTGCAAGGAGGCACTACGCTGCT AGTCCCTGGTTTGCTCACTGGGGCCAAGGGACTCTGGTCACTGTCT CTTCA

[00743] DNA encoding light chain variable region (rat mAbs): SEQ ID NO:233: DNA encoding 4D5 light chain variable region (precursor)

[00744] GACATTGTGATGACCCAGTCTCCATCCTCCCTGGCTG TGTCAGCAGGAGAGAAGGTCACTATGACCTGCAAATCCAGTCAGA GTCTGCTCAACAGTAGAACCCGAAAGAACTACTTGGCTTGGTACC AGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTACTGGGCAT CCACTAGGGAATCTGGGGTCCCTGATCGCTTCACCAGGCAGTGGAT CTGGGACAGATTTCTCTCTCACCATCAGCAGTGTGCAGGCTGAAGA CCTGGCAGTTTATTACTGCAAGCAATCTTATAATCTGTACACGTTC GGAGGGGGGACCAAGCTGGAAATAAAACGG SEQ ID NO:234: DNA encoding light chain variable region 1F3 (precursor)

[00745] GACATTGTGATGACCCAGTCTCCATCCTCCCTGGCTG TGTCAGCAGGAGAGAGGGTCACTATGAGCTGCAAATCCAGTCAGA GTCTGCTCATCAGTAGAACCCGAAAGAACTATTTGTCTTGGTACCA GCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTACTGGGCATC CACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGGGATCT GGGACAGATTTCACTCTCACCATCAGCAGTGTACAGGCTGAAGAC CTGGCAGTTTATTACTGCAAGCAATCTTATAATCTGTACACGTTCG GCGGGGGGACCAAGCTGGAAATAAAACGG SEQ ID NO:235: DNA encoding light chain variable region 4B6/1A10 (precursor)

[00746] GACATTGTGATGACCCAGTCTCCATCCTCCCTGGCTG TGTCAGCAGGAGAGAAGGTCACTATGAGCTGCAAATCCAGTCAGA GTCTGCTCATCAGTAGAACCCGAAAGAACTATTTGTCTTGGTACCA GCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTATTGGGCATC CACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGGGATCT GGGACAGATTTCACTCTCACCATCAGCAGTGTACAGGCTGAAGAC CTGGCAGTTTATTACTGCAAACAATCTTATAATCTGTACACGTTCG GCGGGGGGACCAAGCTGGAAATCAAACGG SEQ ID NO:236: DNA encoding light chain variable region 10D12 (precursor)

[00747] GATGTTTTGATGACCCAAACTCCACTCACTTTGTCGG TTACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAG CCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGGTTGTTACAG AGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAAC TGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGA CAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGG GAGTTTATTATTGCTGGCAAGGTACACATTTTCCGTGGACGTTCGG TGGAGGCACCAAGCTGGAAATCAAACGG SEQ ID NO:237: DNA encoding light chain variable region 35C1 (precursor)

[00748] GATATTGTGATGACGCAGGCTCCACTCACTTTGTCGG TTACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAG CCTCTTAGATAGTGATGGAAAGACATATTTGAGTTGGTTGTTACAG AGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAAC TGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGA CAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGG GAGTTTATTATTGCTGGCAAGGTACACATTTTCCGTACACGTTCGG AGGGGGGACCAAGCTGGAAATAAAACGG SEQ ID NO:238: DNA encoding light chain variable region 13B1 (precursor)

[00749] GACATTGTGATGACCCAGTCTCCATCCTCCCTGGCTG TGTCAGCAGGAGAGAAGGTCACTATGAGCTGCAAATCCAGTCAGA GTCTGCTCAACAGTAGAACCCGAAAGAACTACTTGGCTTGGTACC AGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTACTGGGCAT CCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGGGAT CTGGAACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAG ACCTGGCAGTTTATTACTGCAAGCAATCTTATAATATTCCGACGTT CGGTGGAGGCACCAAGCTGGAAATCAAACGG SEQ ID NO:239: DNA encoding light chain variable region 1G4 (precursor)

[00750] GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTG TCAGTCTTGGAGAACAAGCCTCCATCTCTTGCAGATCAAGTCAGAG CCTTGTACAAAGTAATGGAAACACCTATTTACATTGGTACCTGCAG AAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACC GATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGA CAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGG GAGTTTATTTCTGCTCTCAAAGTACACATGTTCCTCCGACGTTCGG TGGAGGCACCAAGCTGGAAATCAAACGG SEQ ID NO:240: DNA encoding light chain variable region 1E7 (precursor)

[00751] GACATCCAGCTGACTCAGTCTCCAGCCATCCTGTCTG TGAGTCCAGGAGAAAGAGTCAGTTTCTCCTGCAGGGCCAGTCAGA GCATTGGCACAAGCATACACTGGTATCAGCAAAGAACAAATGGTT CTCCAAGGCTTCTCATAAAGTATGCTTCTGAGTCTATCTCTGGGAT CCCTTCCAGGTTTAGTGGCAGTGGATCAGGGACAGATTTTACTCTT AGCATCAACAGTGTGGAGTCTGAAGATATTGCAGATTATTACTGTC AACAAAGTAATAGCTGGCCGTACACGTTCGGAGGGGGGACCAAGC TGGAAATAAAACGG SEQ ID NO:241: DNA encoding light chain variable region 2D7 (precursor)

[00752] GATATCCAGATGACACAGACTCCAGCCTCCCTGTCTG CCTCTCTGGGAGACAGAGTCACCATCAGTTGTAGGGCAAGTCAGG ACATTAGCAATTTTTTAAACTGGTATCAACAGAAACCGAATGGAA CTGTTAAACTCCTAGTCTTCTACACATCAAGATTACACTCAGGAGT CCCATCAAGGTTCAGTGGCAGTGGGTCTGGAGCAGAGCATTCTCTC ACCATTAGCAACCTGGAGCAGGAAGATGTTGCCACTTACTTTTGCC AACAGGGTTTTACGCTTCCGTGGACGTTCGGTGGGGGCACCAAGG TGGAAATCAAACGG SEQ ID NO:242: DNA encoding light chain variable region 49C11 (precursor)

[00753] GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTG TCAGTCTTGGAGATCAAGCCTCCTTCTCTTGCAGATCTAGTCAGAG CCTTATACACAGTAATGGAAACACCTATTTACATTGGTACCTGCAG AAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACC GATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGA CAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGG GAGTTTATTTCTGCTCTCAAAGTACACATGTTCCGTGGACGTTCGG TGGAGGCACCAAGCTGGAAATCAAACGG SEQ ID NO:243: DNA encoding light chain variable region 15D9 (precursor)

[00754] GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCA CATCAATAGGAGACAGGGTCAGCGTCACCTGCAGGGCCAGTCAGA ATGTGGGTCCCAATTTAGCCTGGTATCAACAGAAACCAGGGCAAT CTCCTAAAGCACTGATTTACTCGGCATCCTACCGATTCAGTGGAGT CCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTC ACCATCAGCAATGTGCAGTCTGAAGACTTGGCAGAGTATTTCTGTC AGCAATATAACAGGTATCCATTCACGTTCGGCTCGGGGACAAAGT TGGAAATAAAACGG SEQ ID NO:244: DNA encoding 2F5 light chain variable region (precursor)

[00755] GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCA CATCAGTAGGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAGA ATGTGGGTACTGCTGTAGCCTGGTATCAACAGAAACCAGGACAAT CTCCTAAACTACTGATTTCCTCGGCATCCAATCGGTACACTGGAGT CCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTC ACCATCAGTAATATGCAGTCTGAAGACGTGGCAGATTATTTCTGCC AGCAATATAACAGCTATCCTCTCACGTTCGGTGCTGGGACCAAGCT GGAGCTGAAACGG SEQ ID NO:245: DNA encoding light chain variable region 1B11 (precursor)

[00756] GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCA CTTCAGTAGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGA ATGTGGGTCCTAATGTAGCCTGGTATCAACAGAAACCAGGGCAAT CTCCTAAAGCACTGATTTACTCGGCATCCTACCGGTACAGTGGAGT CCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTC ACCATCAGCAATGTGCAGTCTGAAGACTTGGCAGACTATTTCTGTC AGCAATATAACCGCTATCCTCTCACGTTCGGTGCTGGGACCAAACT GGAGCTGAAACGG SEQ ID NO:246: DNA encoding light chain variable region 2F2 (precursor)

[00757] GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCA CATCAGTAGGAGACAGGGTCAACGTCACCTGCAAGGCCAGTCAGA ATGTGGGTACTCATGTAGCCTGGTATCAACAGAAACCAGGGCAAT CTCCTAAAGCACTGATTTACTCGGCATCCTACCGGTACAGTGGCGT CCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTC ACCATCAGCAATGTGCAGTCTGAAGACCTGGCAGAGTATTTCTGTC AGCAATATAACAGCTATCCTCGAGCGCTCACGTTCGGTGCTGGGA CCAAGCTGGAGCTGAAACGG SEQ ID NO:247: DNA encoding light chain variable region 11B6 (precursor)

[00758] GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCA CATCAGTAGGAGACAGGGTCAACGTCACCTGCAAGGCCAGTCAGA ATGTGGGTCCTACTGTAGCCTGGTATCAACAGAAACCAGGGCAAT CTCCTAAAGCACTAATTTACTCGGCATCCTACCGGTACAGTGGAGT CCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTC ACCATCAGCAATGTGCACTCTGAAGACTTGGCAGAGTATTTCTGTC AGCAATATAACAGCTATCCATTCACGTTCGGCTCGGGGACAAAGT TGGAAATAAAACGG SEQ ID NO:310: Human IgG4 constant region

[00759] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH KPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS LGK SEQ ID NO:311: human IgG4 constant region with S228P mutation

[00760] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH KPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS LGK SEQ ID NO:312: Human IgG4 constant region with S228P mutation and also a mutation (Xtend) that promotes interactions of FcRn at low pH

[00761] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH KPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQKSLSLSL GK SEQ ID NO:313: Human IgK constant region

[00762] TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC

EXAMPLE 16

[00763] This Example describes the functional characterization of purified recombinant high-affinity MASP-3 inhibitory antibodies in various in vitro assays. Methods:

[00764] Recombinant MASP-3 mAbs generated as described in Examples 11 and 14 were characterized for (i) binding to human MASP-3 and MASP-3 from other species; (ii) the ability to inhibit cleavage of an artificial substrate; (iii) the ability to inhibit cleavage of profactor D to factor D; (iv) inhibition of complement deposition in human serum; and (v) inhibition of lysis of rabbit erythrocytes in human serum as follows: I. Assays to Determine Binding to Mouse or Human MASP-3 ELISA Assays: MASP-3 Binding Assay with Purified Recombinant MASP-3 mAbs: Human MASP-3:

[00765] A sandwich ELISA assay was performed to measure the binding of 16 purified recombinant MASP-3 antibodies to human MASP-3 (CCP1-CCP2-SP fragment) as follows. An ELISA plate was coated in carbonate/bicarbonate buffer overnight at 4°C with the capture antibody aM3-259 at 4 μg/mL. aM3-259 is a high-avidity recombinant chimeric chicken-human MASP-3 mAb from chickens immunized with the CCP1-CCP2-SP region of human MASP-3. Domain mapping studies revealed that aM3-259 binds the CCP1-CCP2 region of MASP-3 from multiple species, including humans, cynomolgus monkey, mouse, rat, and dog. As shown in FIGURE 51C, aM3-259 also binds to MASP-1.

[00766] The plate was subsequently blocked with 1% BSA/PBS, washed in PBS and then incubated for one hour at room temperature with MASP-3 CCP1-CCP2-SP (2 μg/mL). The plate was then washed (PBS-T, 0.05%) and candidate MASP-3 antibodies were added and then incubated for one hour at room temperature. The plate was washed (PBS-T, 0.05%) and a detection antibody (rat anti-human kappa-HRP, SouthernBiotech #9230-05) was added for one hour at room temperature. After another wash (PBS-T, 0.05%) the plate was developed (5 minutes) with OPT EIA TMB (BD Biosciences #555214). Absorbance reading at A450 was measured using Spectramax M5e plate reader. Results:

[00767] FIGURE 51A and FIGURE 51B graphically illustrate the avidities of MASP-3 mAb (purified recombinant) for human MASP-3 (CCP1-CCP2-SP). As shown in FIGURES 51A, FIGURE 51B and Table 24, MASP-3 mAbs exhibit high avidity for human MASP-3, ranging from 0.241 nM to 0.023 nM. These values are 10- to 100-fold lower than those reported for previously described MASP-3 mAbs (see Example 7 herein, also published as Example 15 in WO2013/192240). MASP-3 mAb Binding Specificity:

[00768] To determine the specificity of the high-affinity MASP-3 MAbs for MASP-3, binding experiments were performed to measure the binding of 16 purified recombinant MASP-3 antibodies to human MASP-1 and human MASP-2. Binding was determined as described for the MASP-3 binding ELISA, except that recombinant MASP-1A (S646A, CCP1-CCP2-SP fragment) and MASP-2 (CCP1-CCP2-SP fragment) were immobilized directly to the plate. Results:

[00769] FIGURE 51C graphically illustrates the results of a binding experiment in which representative purified recombinant high affinity human MASP-3 inhibitory antibodies are shown to be selective for binding to MASP-3 and do not bind to human MASP-1.

[00770] FIGURE 51D graphically illustrates the results of a binding experiment in which representative purified recombinant high-affinity human MASP-3 inhibitory antibodies are shown to be selective for binding to MASP-3 and do not bind to human MASP-2. Mouse MASP-3:

[00771] Binding of MASP-3 mAbs to mouse MASP-3 was measured as described above for human MASP-3 except that recombinant full-length rat MASP-3 (SEQ ID NO:3) was captured on the plate with aM3-259. The negative control mAb used in both experiments was mAb77, a recombinant chimeric chicken-human mAb obtained from the same immunized chickens as aM3-259, however, mAb 77 did not bind to mouse MASP-3. Results:

[00772] FIGURE 52 graphically illustrates the avidities of representative MASP-3 mAbs (purified recombinants) for mouse full-length MASP-3. As shown in FIGURE 52, most of the MASP-3 mAbs tested also have high avidity for mouse MASP-3.

[00773] The avidity (EC50) values of the 16 recombinant chimeric MASP-3 mAbs for human and mouse MASP-3 are summarized in TABLE 24. TABLE 24: Binding Avidity of MASP-3 mAbs for Human and Mouse MASP-3 (FIGURES 51A, 51B, and 52)

[00774] Three of the MASP-3 mAbs—13B1, 10D12, and 4D5—were also tested for binding to recombinant MASP-3 from cynomolgus monkey, dog, and rat. These results are summarized below in Table 25. TABLE 25: Summary of MASP-3 mAb Binding Experiments Across Species

[00775] As shown in Table 25, MASP-3 mAbs 13B1, 10D12, and 4D5 bind to all five species of MASP-3 tested (human, mouse, rat, dog, and cynomolgus monkey). Although these mAbs bind to human with high avidity (<500 pM), they bind to other species of MASP-3 with varying avidity. 2. Fluorogenic Tripeptide Cleavage Assay Background/Rationale:

[00776] In addition to its known natural substrates (Iwaki et al., J. Immunol. 187:3751, 2011; Cortesio and Jiang, Arch. Biochem. Biophys. 449:164-170, 2006), MASP-3 has been shown to hydrolyze several tripeptide substrates (Cortesio and Jiang, Ibid.). As very small substrates, these molecules can be used to map the catalytic site of the protease. Inhibition of tripeptide cleavage is an indication that an inhibitory agent, such as an antibody, either directly blocks access of the small substrate to the catalytic site or causes a conformational shift in the SP domain that similarly denies access. Therefore, the antibody might also be expected to block catalysis of the large natural substrates by interfering with the active site of the enzyme. Functionally, this would closely approximate the MASP-3 null mouse or the 3MC (MASP-3 deficient) patient. Methods:

[00777] Titrations of recombinant mAbs (3-fold dilution from 666 nM to 0.91 nM) were incubated with MASP-3 CCP1-CCP2-SP (197 nM) for 15 minutes at room temperature. The tripeptide substrate BOC-V-P-R-AMC (t-Butyloxycarbonyl-Val-Pro-Arg-7-Amino-4-methylcoumarin) (R&D Systems, Cat. No. ES011) was added to a final concentration of 0.2 mM. Hydrolysis of the Arg-AMC amide bond releases AMC, a highly fluorescent group. The 380 nm excitation/460 nm emission kinetic values were recorded every 5 minutes at 37°C for 70 minutes using the Spectramax M5e fluorescence plate reader. Results:

[00778] FIGURE 53 graphically illustrates the results of the assay measuring the inhibition of fluorogenic tripeptide-dependent cleavage of MASP-3 with MASP-3 monoclonal antibodies.

[00779] As shown in FIGURE 53, the MASP-3 mAbs tested belong to three distinct groups: 1. MASP-3 mAbs that are strong inhibitors of peptide cleavage by MASP-3: 1A10 (29.77 nM), 1G4 (29.64 nM), 1F3 (32.99 nM), 4B6 (26.03 nM), 4D5 (27.54 nM), 10D12 (30.94 nM), and 13B1 (30.13 nM).

[00780] 2. MASP-3 mAbs that are weak or very weak inhibitors of peptide cleavage by MASP-3: 15D9, 11B6, 2F5, 1E7, and 2D7 3. MASP-3 mAbs that are neutral or appear to stimulate peptide cleavage by MASP-3: 1B11; 2F2; 77 (control mAb) 3. Inhibition of Pro-Factor D Cleavage to Factor D Methods:

[00781] Active recombinant human MASP-3 protein (240 ng per reaction) was preincubated with representative MASP-3 mAbs and a control mAb (which binds to MASP-1 but not MASP-3) in GVB++ buffer with a total volume of 9 μL at room temperature for 15 minutes. 70 ng of pro-factor D with an N-terminal Strep-tag II epitope tag (ST-pro-factor D-His) was then added to each tube to the final volume per reaction of 10 μL. Reactions were incubated in a thermal cycler at 37°C for 6 hours. One-tenth of each reaction was then electrophoresed on a 12% Bis-Tris gel to resolve the pro-factor D cleavage product and active factor D. The resolved proteins were transferred to a PVDF membrane and analyzed by Western blot using detection with a biotinylated factor D antibody (R&D Systems). Results:

[00782] FIGURE 54 shows a Western blot analysis demonstrating the ability of MASP-3 mAbs to block recombinant MASP-3-mediated cleavage of pro-CFD to CFD in an in vitro assay. As shown in Figure 54, representative high-affinity MASP-3 inhibitory mAbs 13B1, 4B6, 1G4, 2D7, 10D12, 1A10, 4D5, 1E7, and 1F3 mouse-human chimeric mAbs showed partial to complete inhibition of pro-CFD cleavage in this assay. 4. Factor Bb Deposition Assay on Zymosan Methods:

[00783] Varying concentrations of MASP-3 mAbs were added to 10% CFD-depleted human serum (Complement Technology A336) and GVB + Mg/EGTA (20 nM) and incubated for 30 min on ice prior to the addition of ST-profactor D-His (2 μg/mL final) and zymosan (0.1 mg/mL final). Zymosan particles function as an activation surface for complement deposition. The mixtures were incubated at 37°C and APC activity was measured by flow cytometric detection of complement factor Bb (Quidel A252 antibody) on the surface of zymosan particles. Results:

[00784] FIGURE 55A graphically illustrates the level of factor Bb deposition on zymosan particles (determined by flow cytometric detection measured in MFI units) in the presence of varying concentrations of MASP-3 mAbs 1F3, 1G4, 2D7, and 4B6 in factor D-depleted human serum at 37°C for 70 minutes.

[00785] FIGURE 55B graphically illustrates the level of factor Bb deposition on zymosan particles (determined by flow cytometric detection measured in MFI units) in the presence of varying concentrations of MASP-3 mAbs 4D5, 10D12, and 13B1 in CFD-depleted human serum at 37°C for 70 minutes.

[00786] The results shown in FIGURES 55A and 55B are summarized below in TABLE 26. TABLE 26: Inhibition of factor Bb deposition on zymosan by MASP-3 mAbs (FIGURE 55A and FIGURE 55B)

[00787] As shown in FIGURES 55A, FIGURE 55B, and Table 26, MASP-3 mAbs exhibit potent inhibition of APC in human serum, with IC50 values ranging from 0.1 nM to 3.5 nM. These results demonstrate that MASP-3 plays a key role in APC activation in an in vitro model in human serum and further demonstrate that MASP-3 inhibitory antibodies are potent inhibitors of APC. 5. Assay to Measure the Ability of Representative MASP-3 mAbs to Inhibit Rabbit Erythrocyte Lysis Methods:

[00788] To monitor APC inhibition in another experimental context, the ability of representative MASP-3 mAbs to block rabbit erythrocyte lysis in human serum was assessed. Varying concentrations of MASP-3 mAbs were added to 10% factor D-depleted human serum and RBCs + Mg/EGTA (20 nM) and incubated for 30 min on ice prior to the addition of ST-pro-factor B-His (2 μg/mL final) and erythrocytes (2.5x108 cells/mL final). The mixtures were incubated at 37°C for 70 min and APC-mediated hemolysis was measured by diluting the reactions and measuring absorbance (A405), which indicates free hemoglobin levels. Results:

[00789] FIGURE 56A graphically illustrates the level of inhibition of rabbit erythrocyte lysis in the presence of varying concentrations of MASP-3 mAbs 1A10, 1F3, 4B6, 4D5, 1G4, and 2F2 in CFD-depleted human serum. FIGURE 56B graphically illustrates the level of inhibition of rabbit erythrocyte lysis in the presence of varying concentrations of MASP-3 mAbs 1B11, 1E7, 1G4, 2D7, and 2F5 in CFD-depleted human serum. The results are summarized in TABLE 27. TABLE 27: Inhibition of Rabbit Erythrocyte Lysis by MASP-3 mAbs (FIGURE 56A and FIGURE 56B)

[00790] As shown in FIGURES 56A, FIGURE 56B, and Table 27, MASP-3 mAbs exhibit inhibition of APC-driven hemolysis of rabbit erythrocytes, with IC50 values ranging from 0.1 nM to 5.4 nM. These results corroborate the observations for MASP-3 antibodies in the zymosan assay and further demonstrate that MASP-3 inhibitory antibodies are potent inhibitors of APC. 6. Inhibition of profactor D cleavage in 3MC patient serum Methods:

[00791] A representative recombinant MASP-3 mAb (4D5) was tested for the ability to block the cleavage (and activation) of recombinant MASP-3 pro-factor D derived from normal human serum and serum from 3MC Patient B (“Pat B”), an individual who has no detectable MASP-3 in serum and manifests an APC deficiency.

[00792] Normal human serum and patient B serum (10% final) and GVB + Mg/EGTA (30 nM) were incubated without enzyme or with recombinant active MASP-3 (rMASP-3; 0.5 μg/mL), inactive rMASP-3, or active rMASP-3 plus MASP-3 mAb 4D5 (500 nM final) on ice for 1 hour. Zymosan (0.1 mg/mL final) was added, and mixtures were incubated at 37°C. After 2 hours, samples were centrifuged and supernatants were collected. Samples were immunoprecipitated with goat antibody formulated against human Factor D (R&D Systems AF1824), heat denatured, and treated with Peptide-N-Glycosidase (New England Biolabs P0704L). Captured and deglycosylated proteins were resolved with SDS-PAGE and gels were electroblotted for Western blot analysis with a biotinylated anti-CFD (R&D Systems BAF1824) and high-sensitivity Streptavidin-HRP (Thermo Fischer Scientific 21130). Results:

[00793] FIGURE 57 shows a Western blot analyzing the level of pro-Factor D and Factor D in the serum of Patient B with 3MC in the presence of active rMASP-3, inactive rMASP-3, and active rMASP-3 plus mAb 4D5. As shown in FIGURE 57, normal human serum contains predominantly the mature form, whereas Patient B's serum contains primarily the zymogenic form of Factor D. As shown in Figure 57, active rMASP-3 in the presence of zymosan causes cleavage of pro-Factor D in Patient 3's serum, whereas the inactive (zymogen) form of MASP-3 does not. Finally, as shown in FIGURE 57, the MASP-3 mAb 4D5 blocks cleavage of pro-Factor D in Patient 3's serum in the presence of active rMASP-3. These results further demonstrate the role of MASP-3 in profactor D cleavage in APC activation and demonstrate that a MASP-3 inhibitory mAb is able to block MASP-3-mediated profactor D cleavage and thereby block APC.

EXAMPLE 17

[00794] Analysis of representative MASP-3 inhibitory mAbs 10D12 and 13B1 for the ability to inhibit APC in vivo. 1. Inhibition of APC by mAb M3-1 (13B1) and 10D12 in vivo: Methods:

[00795] To determine the efficacy of MASP-3 mAbs 13B1 (M3-1) and 10D12 for APC inhibition in vivo, one group of mice (n = 4) received a single intravenous tail vein injection of 10 mg/kg mAb 13B1 and a second group of mice (n = 4) received a single intravenous tail vein injection of 10 mg/kg mAb 10D12. Blood collected from the animals was used to prepare serum, providing a matrix for flow cytometric assessment of APC activity in an ex vivo assay measuring the level of C3 (also C3b and iC3b, Dako F020102-2) deposition on zymosan particles. Serum prepared from blood collected at a pre-dose assessment time point and multiple post-dose assessment time points (96 hours, 1 week, and 2 weeks) was diluted to 7.5%, and zymosan particles (0.1 mg/mL final) were added to induce APC. Antibody-treated mice were compared with a group of control mice (n = 4) given a single intravenous dose of vehicle. Results:

[00796] Figure 58 graphically illustrates the level of C3 deposition on zymosan particles at various time points following a single dose of mAb M3-1 (13B1), mAb 10D12, or carrier in wild-type mice. As shown in FIGURE 58, at the pre-dose time point all three conditions show comparable levels of APC activity. At 96 hours and two subsequent time points, both mAb-treated groups show a near-complete ablation of systemic APC activity, while the carrier-treated group's APC activity remains undiminished.

[00797] These results demonstrate that MASP-3 mAb M3-1 (13B1) and mAb 10D12 are potent inhibitors of APC in vivo in mice. 2. Factor B status in mice treated with MASP-3 mAb 10D12 Methods:

[00798] During the conversion of the Factor B zymogen to an active proteolytic enzyme, Factor B is cleaved into the Ba (~30 kDa) and Bb (~60 kDa) fragments by Factor D. The status of the Ba fragment in mouse serum obtained from mice treated with the MASP-3 mAb 10D12 was determined as follows.

[00799] Mice (n = 4) received two intravenous tail vein injections of 10 mg/kg mAb 10D12. Treatments occurred seven days apart, and blood was collected from the animals three days after the second injection. A second set of four mice received a single intravenous dose of carrier (PBS). Blood collected from both groups was used to prepare serum, providing a matrix for complement activation. Zymosan particles (0.1 mg/mL final) were added to the diluted serum (7.5% final) and incubated for 35 minutes at 37°C. Results:

[00800] As a measure of APC activation, FIGURE 59 shows a Western blot analysis of the Ba fragment status in mouse serum obtained from mice treated with mAb 10D12 or PBS and stimulated with zymosan. Each lane in FIGURE 59 represents a different mouse, and the lanes alternate to show serum from a representative carrier mouse adjacent to a MASP-3 mAb-treated mouse for comparison purposes. Two control conditions, from mice treated with carrier or mAb 10D12, are shown in lanes 1 and 2, respectively (from the left side of the blot) as representative of the basal level of Ba present in the serum samples in the absence of zymosan. Lanes 3 through 10 show the level of Ba fragment present following incubation with zymosan. In all cases, mice treated with MASP-3 mAb demonstrate a reduced level of the Ba fragment compared with carrier-treated animals. 3. Serum from mice treated with 10D12 mAb inhibits hemolysis Methods:

[00801] As a further measure of APC inhibition by MASP-3 inhibitory antibodies, the ability of MASP-3 antibodies to block rabbit erythrocyte lysis was assessed in serum from mice treated with a representative MASP-3 mAb 10D12, compared to serum from mice treated with a carrier control.

[00802] Mice (n = 4/group) received three intravenous tail vein injections of carrier control (PBS), 10 mg/kg MASP-3 mAb 10D12, or 25 mg/kg MASP-3 mAb 10D12. Treatments were performed 7 days apart, and blood was collected from animals 3 days after the third injection. The blood was used to prepare serum, providing a matrix for hemolysis reactions. Red blood cells (2.5 x 108 cells/mL final) were added to 20% pooled serum from four mice in GVB + Mg/EGTA (20 nM). The mixtures were incubated at 37°C, and APC-mediated hemolysis was measured by diluting the reactions and measuring absorbance (A405). Results:

[00803] FIGURE 60 graphically illustrates the level of hemolysis inhibition in 20% serum from mice treated with MASP-3 10D12 mAb (10 mg/kg or 25 mg/kg) or carrier control-treated mice. As shown in FIGURE 60, serum from mice treated with MASP-3 10D12 mAb at 10 mg/kg and 25 mg/kg demonstrated less overall hemolysis over the 1-hour test period compared to carrier-treated mice. Overall Summary of Results:

[00804] As described in this Example, representative high-affinity MASP-3 inhibitory mAbs 13B1 and 10D12 inhibit APC in vivo. As described in Example 12, the MASP-3 monoclonal antibody 13B1 (mAb M3-1, also referred to as mAb M3-1) was determined to provide a clear benefit to the survival of Crry-free red blood cells in a mouse model associated with paroxysmal nocturnal hemoglobinuria (PNH). As described in Example 13, the MASP-3 mAb M3-1 was determined to reduce the incidence and severity of clinical arthritis scores in a dose-dependent manner. EXAMPLE 18

[00805] This Example describes the results of epitope binding analysis of high-potency MASP-3 inhibitory mAbs. 1. Competition Binding Analysis Methods:

[00806] 96-well ELISA assay plates were coated with the capture antibody, aM3-259, an IgG4 isotype mAb that has been shown to bind the CCP1-CCP2 region of MASP-1 and MASP-3. Full-length human MASP-3 protein was immobilized on the plate via the capture antibody aM3-259. In separate, uncoated wells, a 2-fold dilution series of a test MASP-3 mAb of an IgG4 isotype was mixed with a constant concentration of another test MASP-3 antibody of an IgG1 isotype. The mixture was added to the coated wells and bound to the captured MASP-3. Potential competition between the two antibodies was determined by detecting the IgG1 isoform using an HRP-conjugated antibody against the hinge region of human IgG1 (Southern Biotech 9052-05) and a TMB substrate reagent set (BD Biosciences 555214). Results:

[00807] Figures 61A-61E graphically illustrate the results of the competition binding analysis.

[00808] Figure 61A graphically illustrates the results of competition binding analysis to identify MASP-3 (IgG4) mAbs that block the interaction between mAb 4D5 (IgG1) and human MASP-3.

[00809] Figure 61B graphically illustrates the results of competition binding analysis to identify MASP-3 (IgG4) mAbs that block the interaction between mAb 10D12 (IgG1) and human MASP-3.

[00810] Figure 61C graphically illustrates the results of competition binding analysis to identify MASP-3 (IgG4) mAbs that block the interaction between mAb 13B1 (IgG1) and human MASP-3.

[00811] Figure 61D graphically illustrates the results of competition binding analysis to identify MASP-3 (IgG4) mAbs that block the interaction between mAb 1F3 (IgG1) and human MASP-3.

[00812] Figure 61E graphically illustrates the results of competition binding analysis to identify MASP-3 (IgG4) mAbs that block the interaction between mAb 1G4 (IgG1) and human MASP-3.

[00813] The data from FIGURES 61A through 61E are summarized below in TABLE 28.

[00814] These data indicate that MASP-3 mAbs 4D5, 10D12, 13B1, 1A10, 1F3, and 1G4 share a common epitope or overlapping epitopes on human MASP-3. Surprisingly, 1G4 has very limited ability to block the binding of the other five mAbs to MASP-3, but these mAbs almost completely block the binding of 1G4 itself to MASP-3. 2. Analysis of mAb binding to peptides representing linear and discontinuous MASP-3 epitopes Methods:

[00815] Fourteen of the 16 MASP-3 mAbs were evaluated by Pepscan to identify the regions of MASP-3 to which they bind. To reconstruct both linear and potential discontinuous epitopes of the target molecule, a peptide library corresponding to amino acid residues 299 to 728 of SEQ ID NO:2 (human MASP-3) was synthesized. Amino acid residues 1-298 of MASP-3 were not present in the immunogen and were not included in this analysis.

[00816] Pepscan epitope analysis includes the use of CLIPS technology, which structurally fixes peptides into defined three-dimensional structures (see Timmerman et al., J Mol Recog. 20:283-299, 2007 and Langedijk et al., Analytical Biochemistry 417:149-155, 2011). The binding of each antibody to each of the synthesized peptides was tested in PEPSCAN-based ELISA. Results:

[00817] Pepscan peptide binding results for each antibody analyzed are described below and summarized in TABLE 4, TABLE 28, and FIGURES 62-67. Antibodies 1F3, 4B6, 4D5, and 1A10 (Group IA)

[00818] When tested under conditions of moderate stringency, the bound discontinuous epitopes 1F3, 4B6, 4D5, and 1A10 mimic and also bind to simple, linear restricted mimics. Analysis of the data demonstrates that antibodies 1F3, 4B6, 4D5, and 1A10 dominantly recognize the peptide stretch VLRSQRRDTTVI509 (SEQ ID NO:9) of MASP-3. This peptide is immediately adjacent to the active site histidine, H497. The data obtained for these discontinuous mimic antibodies suggest that the peptide stretches (544DFNIQNYNHDIALVQ558 (SEQ ID NO:11), 639GNYSVTENMFC649 (SEQ ID NO:13), and 704VSNYVDWVWE713 (SEQ ID NO:14) of MASP-3 also contribute to binding. The 544DFNIQNYNHDIALVQ558 (SEQ ID NO:11) peptide contains the aspartate active site (D553). Antibody 10D12 (Group IB)

[00819] When tested under moderate stringency conditions, antibody 10D12 binds to peptides with the MASP-3 core sequence 498VLRSQRRDTTVI509 (SEQ ID NO:9), the active site histidine adjacent sequence, H497. Antibody 13B1 (Group IC)

[00820] When tested under moderate stringency conditions, antibody 13B1 recognizes a discontinuous epitope comprising the peptide stretches 494TAAHVLRSQRRDTTV508 (SEQ ID NO:10) and 626PHAECKTSYESRS638 (SEQ ID NO:12) of MASP-3, wherein the peptide stretch 626PHAECKTSYESRS638 (SEQ ID NO:12) appears to be the dominant part of the epitope since it can also be bound in single restricted form. The peptide 494TAAHVLRSQRRDTTV508 (SEQ ID NO:10) contains the active site histidine, H497. Antibody 1G4 (Group II) When tested under low stringency conditions, antibody 1G4 recognizes a discontinuous epitope comprising the peptide stretches 54RNAEPGLFPWQ464 (SEQ ID NO:17), 514EHVTVYLGLH523 (SEQ ID NO:19), and 667AFVIFDDLSQRW678 (SEQ ID NO:23) of MASP-3, where peptide stretch 667AFVIFDDLSQRW678 (SEQ ID NO:23) is the dominant part of the epitope. The dominant peptide is within three amino acids of the active site serine, S664. Antibodies 1E7 and 2D7 (Group IIIA)

[00821] When tested under low or high stringency conditions, respectively, antibodies 1E7 and 2D7 recognize a discontinuous epitope comprising the peptide stretches 54RNAEPGLFPWQ464 (SEQ ID NO:17), 514EHVTVYLGLH523 (SEQ ID NO:19), and 667AFVIFDDLSQRW678 (SEQ ID NO:23) of MASP-3, where the peptide stretch 667AFVIFDDLSQRW678 (SEQ ID NO:23) is the dominant part of the epitope and lies within three amino acids of the active site serine, S664. Antibodies 2F5 and 15D9 (Group IIIB)

[00822] When tested under low stringency conditions, antibodies 2F5 and 15D9 dominantly recognize a discontinuous epitope comprising the peptide stretches 454RNAEPGLFPWQ464 (SEQ ID NO:17), 479KWFGSGALLSASWIL493 (SEQ ID NO:18), 562PVPLGPHVMP571 (SEQ ID NO:20), and 667AFVIFDDLSQRW678 (SEQ ID NO:23) of MASP-3. Peptides 479KWFGSGALLSASWIL493 (SEQ ID NO:18) and 667AFVIFDDLSQRW678 (SEQ ID NO:23) are located within four or three amino acids of the active site residues H497 and S664, respectively. Antibody 1B11 (Group IIIC)

[00823] When tested under moderate stringency conditions, antibody 1B11 recognizes a discontinuous epitope comprising the peptide stretches 435ECGQPSRSLPSLV447 (SEQ ID NO:16), 454RNAEPGLFPWQ464 (SEQ ID NO:17), 583APHMLGL589 (SEQ ID NO:21), and 614SDVLQYVKLP623 (SEQ ID NO:22) of MASP-3. TABLE 28: Summary of Epitope Binding Analysis

[00824] FIGURE 62 provides a schematic diagram showing the contact regions on human MASP-3 by MASP-3 mAbs, as determined by Pepscan analysis. As shown in FIGURE 62, all MASP-3 mAbs have contact regions on the beta chain containing the MASP-3 SP domain. One mAb, 1B11, also has a contact region between the CCP2 and SP domains on the MASP-3 alpha chain.

[00825] FIGURES 63A through 67 show 3-D models illustrating the contact regions of high-affinity MASP-3 mAbs on the CCP1/2/SP domains of human MASP-3, where the MASP-3 SP domain active site is facing forward and the catalytic triad is shown as side chains.

[00826] FIGURE 63A shows the contact regions between human MASP-3 and high affinity MASP-3 mAbs 1F3, 4D5, and 1A10, including aa residues 498-509 (SEQ ID NO:9), aa residues 544-558 (SEQ ID NO:11), aa residues 639-649 (SEQ ID NO:13), and aa residues 704-713 (SEQ ID NO:14).

[00827] FIGURE 63B shows the contact regions between human MASP-3 and high affinity MASP-3 mAb 10D12, including aa residues 498 to 509 (SEQ ID NO:9).

[00828] FIGURE 64 shows the contact regions between human MASP-3 and high affinity MASP-3 mAb 13B1, including aa residues 494 to 508 (SEQ ID NO:10) and aa residues 626 to 638 (SEQ ID NO:12).

[00829] FIGURE 65 shows the contact regions between human MASP-3 and high affinity MASP-3 mAb 1B11, including aa residues 435-447 (SEQ ID NO:16), aa residues 454-464 (SEQ ID NO:17), aa residues 583-589 (SEQ ID NO:21), and aa residues 614-623 (SEQ ID NO:22).

[00830] FIGURE 66 shows the contact regions between human MASP-3 and high affinity MASP-3 mAbs 1E7, 1G4, and 2D7, including aa residues 454 to 464 (SEQ ID NO:17), aa residues 514 to 523 (SEQ ID NO:19), and aa residues 667 to 678 (SEQ ID NO:23).

[00831] FIGURE 67 shows the contact regions between human MASP-3 and high affinity MASP-3 mAbs 15D9 and 2F5, including aa residues 454-464 (SEQ ID NO:17), aa residues 479-493 (SEQ ID NO:18), aa residues 562-571 (SEQ ID NO:20), and aa residues 667-678 (SEQ ID NO:23).

[00832] In summary, conclusive binding profiles were obtained for 12 of the 14 antibodies. All 12 mapped antibodies recognized solvent-exposed epitopes within the peptidase SI domain. The proximity of several epitope determinants to residues within the active site catalytic triad (H497, D553, S664) is consistent with a model in which high-affinity inhibitory MASP-3 mAbs block enzymatic activity by interfering with the enzyme-substrate interaction.

EXAMPLE 19

[00833] This example describes the humanization of representative MASP-3 mAbs and the engineering of potential post-translational modification sites. Methods: 1. Humanization of representative high-affinity MASP-3 mAbs Methods:

[00834] To reduce the risk of immunogenicity, representative high-affinity MASP-3 inhibitory antibodies 4D5, 10D12, and 13B1 were humanized by a CDR grafting method. The CDRs of each MASP-3 antibody were grafted to the closest human consensus framework sequences. Some of the Vernier zone residues were modified by Quickchange site-directed mutagenesis (Agilent Technologies). The resulting humanized VH and VL regions were transferred to pcDNA3.1-based human IgG1 or IgG4 and IgGK expression constructs, and recombinant antibodies were expressed and purified as described above. The affinity of the humanized antibodies was determined by ELISA using monovalent Fab fragments, and potency was assessed by C3 deposition assay using intact IgG4 formats. Results:

[00835] The amino acid sequences of representative humanized versions of the heavy chain variable regions and light chain variable regions for mAbs 4D5, 10D12, and 13B1 are provided below. The CDRs (Kabat) are underlined.

[00836] 4D5; h4D5 VH-14 (SEQ ID NO:248) ASVKVSCKASGYTFTTDDINWV RQAPGQGLEWIGWIYPRDDRTKYNDKFKDRATLTVDTSSNTAYMEL SSLRSEDTAVYYCSSLEDTYWGQGTLVTVSS h4D5 VL-1 (SEQ ID NO:250) DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYL AWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAE DVAVYYCKQSYNLYTFGQGTKVEIKR 10D12; h10D12 VH-45 (SEQ ID NO:251) PGASVKVSCKASGYIFTSYGMSWVR QAPGKGLKWMGWINTYSGVPTYADDFKGRFVFSLDTSVRTPYLQISS LKAEDTATYFCARGGEAMDYWGQGTLVTVSS h10D12 VL-21 (SEQ ID NO:253) DVLMTQTPLSLSVTPGQPASISCKSSQSLLDSDGKTYLN WLLQRPGQSPKRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAED VGVYYCWQGTHFPWTFGQGTKVEIKR 13B1 h13B1 VH-9 (SEQ ID NO:254) YMELSS LRSEDTAVYYCLRSEDVWGQGTLVTVSS h13B1 VH-10 (SEQ ID NO:255) VL-1 (SEQ ID NO:256) DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYL AWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAE DVAVYYCKQSYNIPTFGQGTKVEIKR

[00837] The affinity of representative humanized antibodies 4D5, 10D12, and 13B1 for human MASP-3 is presented below in TABLE 29. TABLE 29: Binding of Representative Humanized MASP-3 mAbs to MASP-3

[00838] The percentage identity of humanized framework sequences to that of human germline framework sequences.

[00839] h4D5_VH-14=90%; h4D5_VH-19=91%; h4D5_VL-1=100%; h10D12_VH-45=92%; h10D12_VH-49=91%; h10D12_VL- 21=93%; h13B1_VH-9=95%; h13B1_VH-10=94%; h13B1_VL- 1=100% 2. Mutagenesis of representative MASP-3 mAbs to remove Asn/Asp modification sites in CDR-1 of the light chain variable region of 4D5, 10D12, and 13B1

[00840] Representative high-affinity MASP-3 inhibitory mAbs 4D5, 10D12, and 13B1 were analyzed for post-translational modification. Asparagine residues with subsequent glycine, serine, histidine, alanine, or asparagine (“NG”, “NS”, “NH”, “NA”, or “NN” motif) are often susceptible to hydrolysis of the asparagine side chain amide group, or “deamidation.” Aspartic acid residues with subsequent glycine or proline (“DG” or “DP” motif) are often susceptible to interconversion or “isomerization.” Such modifications result in charge heterogeneity and may affect antibody function if they occur at a binding interface. They may also increase the risks of fragmentation, immunogenicity, and aggregation.

[00841] Potential post-translational modification motifs have been identified in CDR-1 of the light chain variable regions of 4D5, 10D12, and 13B1.

[00842] 4D5 and 13B1 contained a possible Asn deamidation site in light chain CDR1 (shown as "NS" at positions 8 and 9 of underlined SEQ ID NO:142 in TABLE 30 below. As further shown below in Table 30, 10D12 contained a possible Asp isomerization site in light chain CDR1.

[00843] Humanized version variants of these MASP-3 mAbs were generated by site-directed mutagenesis as shown in TABLE 30. The variants were expressed and purified as described above. Affinity was determined by ELISA using monovalent Fab fragments and potency was assessed by C3 deposition assay using intact IgG4 formats as described above. TABLE 30: CDR-L1 variants for 4D5, 10D12, and 13B1 TABLE 31: Binding of mutagenized humanized mAbs 4D5, 10D12, and 13B1 candidates to human MASP-3 TABLE 32: Humanized VH sequences of MASP-3 antibody (CDRs and FR regions, Kabat)

Representative Humanized Light Chain Variable Regions with Variants:

[00844] h4D5 VL-1-NA (SEQ ID NO:278) VLMTQTPLSLSVTPGQPASISCKSSQSLLDSDAKTYLN WLLQRPGQSPKRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAED VGVYYCWQGTHFPWTFGQGTKVEIKR h13B1 VL-1-NA (SEQ ID NO:280) DIVMTQSPDSLAVSLGERATINCKSSQSLLASRTRKNYL AWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAE DVAVYYCKQSYNIPTFGQGTKVEIKR TABLE 33: Humanized MASP-3 antibody VL sequences (CDRs and FR regions, Kabat) [more variants in LC-CDR1]

EXAMPLE 20

[00845] Analysis of a representative MASP-3 inhibitory mAb 13B1 in a mouse model of multiple sclerosis.

[00846] Background/Rationale: Experimental autoimmune encephalomyelitis (EAE), an acquired autoimmune inflammatory and demyelinating disorder, is an established animal model of multiple sclerosis (MS). Evidence suggesting that APC plays a significant role in the development/progression of EAE was provided by reports that the disease is attenuated in mice treated with a FactorB neutralizing antibody (Hu et al., Mol. Immunol. 54:302, 2013). This Example describes the analysis of a representative high-affinity MASP-3 inhibitory antibody, 13B1, in the EAE model. Methods: Induction of EAE:

[00847] An EAE induction kit purchased from Hooke Laboratories (Lawrence, MA) was used to induce EAE in this study. This kit contained the neuroantigen MOG35-55 in complete Freund's adjuvant (CFA) as well as pertussis toxin.

[00848] Thirty wild-type female C57 Bl/6J mice were used for this study and were acclimated to the facility for at least one week prior to EAE induction. Mice were approximately 10 weeks of age at the time of induction. As shown in TABLE 34 below, at the time of induction, each mouse received two subcutaneous (sc) injections of 100 μL of MOG35-55 and one intraperitoneal (ip) injection of 100 μL (400 ng) of pertussis toxin. A second injection of pertussis toxin was administered 24 hours after the first.

[00849] Treatment: The 30 mice were divided into three groups of 10 and treated with either an irrelevant isotype control mAb 10 mg/kg iv); mAb 13B1 (anti-MASP-3, 10 mg/kg iv); or mAb 1379 (anti-Factor B (Hu et al., Mol. Immunol. 54:302, 2013) 40 mg/kg ip). As shown in TABLE 34, dosing with the isotype control mAb and MASP-3 mAb 13B1 occurred weekly on Day -16 and ended on Day +12. Dosing with mAb 1379 occurred every other day from Day +3 to Day +11 according to the dosing schedule described in Hu et al., Mole Immunol 54:302-308, (2013). TABLE 34: Experimental methods for the EAE experiment with MASP-3 mAb 13B1

[00850] Scoring: The mice were checked daily until symptoms appeared, then they were checked daily. The first signs of disease appeared 7 to 12 days after immunization, as expected. The mice were scored according to the scale shown below in TABLE 35. TABLE 35: EAE Model Scoring Criteria Results:

[00851] FIGURE 68 graphically illustrates the results of the EAE model in mice treated with MASP-3 inhibitory mAb 13B1 (10 mg/kg), Factor B mAb 1379 (40 mg/kg), or isotype control mAb (10 mg/kg), where downward pointing arrows indicate the anti-factor B antibody dosage and upward pointing arrows indicate the last dose of mAb 13B1. As shown in FIGURE 68, mice treated with MASP-3 inhibitory mAb 13B1 and Factor B mAb 1379 exhibited an improvement in clinical symptoms scored according to the parameters shown in TABLE 35, compared to the isotype control.

[00852] In accordance with the foregoing, MASP-3 inhibitory antibodies, such as the high affinity MASP-3 inhibitory antibodies described herein, are expected to be beneficial (neuroprotective or neuroregenerative) in the treatment and/or rehabilitation of an individual suffering from multiple sclerosis, Balo's concentric sclerosis, neuromyelitis optica, Marburg multiple sclerosis, Schilder's disease, tumefactive multiple sclerosis, and acute disseminated encephalomyelitis (ADM).

EXAMPLE 21

[00853] Pharmacodynamic study with representative high-affinity MASP-3 mAbs in cynomolgus monkeys.

[00854] Background/Rationale: As demonstrated in rodent studies (FIGURE 44), a high-affinity MASP-3 inhibitory antibody was able to inhibit steady-state (resting) profactor D maturation in vivo. This Example describes a study that was performed in cynomolgus monkeys to determine whether representative high-affinity MASP-3 inhibitory mAbs are capable of inhibiting APC activity in a non-human primate.

[00855] Methods: To confirm that MASP-3 functions at the APC in a non-human primate and that high-affinity MASP-3 antibodies are capable of inhibiting the APC in a non-human primate, 9 cynomolgus monkeys (3 animals per mAb condition) were administered a single 5 mg/kg intravenous dose of one of three representative high-affinity MASP-3 inhibitory antibodies: h4D5X, h10D12X, or h13B1X. (“H” refers to humanized, “X” refers to the IgG4 constant hinge region (SEQ ID NO: 312) containing the stabilizing amino acid substitution S228P and a human IgG4 constant region mutation with S228P mutation and also a mutation that promotes FcRn interactions at low pH). Plasma (EDTA) and serum samples were collected at regular intervals over a period of three weeks or longer.

[00856] Two assays were employed to measure APC activity in the serum of treated monkeys. The first assay assessed the levels of complement factor Bb deposited on zymosan beads added to diluted serum. The second assay measured the fluid phase products of zymosan-activated APC, complement factors Ba and Bb, as well as C3a.

[00857] Flow cytometry using factor Bb antibody A252 (Quidel) was used to detect factor Bb deposited on zymosan. As a means of determining the background signal in the assay after complete inhibition of APC, an aliquot of serum (5% final, diluted in GVB + Mg/EGTA) prepared from treated cynomolgus monkeys was spiked with MASP-3 mAb containing 300 nM of a Factor D inhibitory antibody. To determine the degree of APC inhibition by the MASP-3 mAb delivered intravenously to the monkey, another aliquot of the diluted serum was spiked with 300 nM of a neutral isotype control antibody (which has no APC inhibitory activity) prior to assaying for factor Bb deposition on zymosan. The spiked serum and antibody mixtures were incubated for 30 minutes on ice prior to the addition of zymosan (0.1 mg/mL final). The mixtures were incubated at 37°C for 65 minutes and APC activity was measured by flow cytometric detection of complement factor Bb (Quidel A252 antibody) on the surface of zymosan particles.

[00858] To determine the generation of the fluid phase markers Ba, Bb, and C3a, APC was induced in ex vivo assays by incubating zymosan (1 mg/mL final) in serum (5% final, diluted in GVB + Mg/EGTA) prepared from cynomolgus monkeys treated with anti-MASP-3 mAb. The mixtures were incubated at 37°C for 40 minutes, and APC activity was measured by ELISA-based detection of complement endpoints. Ba, Bb, and C3a were detected in the reaction supernatants using commercially available ELISA kits (Quidel). Absorbance values for all assays were normalized by setting pretreatment values as 100% activity and a pretreatment sample incubated with, but not exposed to, zymosan at 0%.

[00859] To relate the degree of APC inhibition to the antibody-to-target ratio in MASP-3 mAb-treated monkeys, serum MASP-3 and inhibitory MASP-3 mAb levels were quantified. Serum MASP-3 was measured by a sandwich ELISA assay. MASP-3 protein was captured on a plate with aM3-259 (described in Example 16). Serum samples (diluted 1:40) were first incubated with unlabeled (non-biotinylated) MASP-3 mAb, corresponding to the treating mAb, at 37°C for 1 hour, then further diluted 1:250 (final 1:10,000) and added to the plate and incubated at 37°C for an additional hour. The plate was washed and a biotinylated version of mAb 10D12 was used as the detection antibody. Large dilution of serum prior to detection steps was used to uncouple the target and treatment mAb and to prevent competition between the treatment antibody and the detection antibody. After the plate was washed several times, streptavidin-HRP was used for the final detection step. Absorbance values were collected on A450 with a plate reader. Serum MASP-3 concentrations were extrapolated from a standard curve generated by testing recombinant full-length cynomolgus MASP-3 protein. The amount of anti-MASP-3 antibody present in serum was detected using the Therapeutic Human IgG4 ELISA kit (Cayman Chemicals) following the manufacturer's instructions.

[00860] Western blot analysis was used to analyze the level of pro-factor D and Factor D in the serum of a cynomolgus monkey over time (hours) after treatment with a single intravenous dose of 5 mg/kg of mAb h13B1X. In short, Western blot analysis was performed by mixing 20 μL of cynomolgus plasma obtained at different evaluation time points before treatment (-120hr, -24hr) and after treatment (72hr, 168hr, 336 h, 504hr, 672hr and 840 h) with PBS and 11.2 μL of anti-CFD antibody (0.5 μg/μL) in a total volume of 400 μL at 4°C for 1 hour. 12 μL of Protein A/G Plus Agarose (Santa Cruz Biotech) was added and the mixture was incubated overnight at 4°C. Immunoprecipitates were collected by centrifugation at 1000 x g for 5 min at 4°C. The pellets were washed five times with PBS. After the final wash, the pellets were resuspended in 30 μL of 1x glycoprotein denaturation buffer and the glycoprotein was denatured by heating the reaction at 100°C for 10 min. 10X G2 reaction buffer, 10% NP-40 and 2.5 µM peptide-N-glycosidase (New England Biolabs, P0704L) were added to each tube and the reaction was incubated at 37°C for 2 h. Agarose beads were pelleted by centrifugation at 1000 x g for 5 min and 20 μL of supernatant was collected in fresh tubes. Captured and deglycosylated proteins were resolved with SDS-PAGE (NuPAGE 12% Bis-Tris Mini Gel) and gels were electroblotted for Western blot analysis with a biotinylated anti-CFD (R&D Systems BAF1824) and Pierce™ High Sensitivity Streptavidin-HRP (Thermo Fischer Scientific 21130). Results:

[00861] FIGURE 69 graphically illustrates APC activity in serum samples obtained from a group of three cynomolgus monkeys over time following a single treatment at time = 0 with the high-affinity MASP-3 mAb h13B1X. The figure shows the mean MFI in a flow cytometry assay detecting complement factor Bb on the surface of zymosan particles in 5% serum spiked with either the APC-inhibiting Factor D mAb or neutral isotype control mAb. As shown in FIGURE 69, the animals demonstrate a decrease in APC activity as early as 4 hours. If MASP-3 antibody treatment blocks APC as effectively as Factor D inhibition, the two spiked antibody conditions will demonstrate identical levels of inhibition of Bb deposition in post-dose samples, but not in the pre-dose (or time = 0; FIGURE 69) condition. As shown in FIGURE 69, 72 h after treatment, APC activity is decreased to approximately that achieved by addition of factor D mAb to serum samples. The near-complete inhibition due to h13B1X treatment, as experimentally determined by comparison with spiked factor D antibody, persists up to 336 h (14 days) postdose. Thus, these results demonstrate that treatment with a high-affinity MASP-3 inhibitory mAb provides complete and sustained inhibition of APC in a nonhuman primate.

[00862] FIGURE 70 graphically illustrates APC activity, as determined by Bb deposition on zymosan, in serum samples obtained from groups of cynomolgus monkeys (3 animals per group) treated with a single intravenous dose of 5 mg/kg of high-affinity MASP-3 inhibitory mAbs h4D5X, h10D12X, or h13B1X. Bb deposition data were collected as described above. APC activity for the treatment assessment time points was normalized by setting pretreatment MFIs of samples spiked with the non-inhibitory isotype control antibody at 100% activity and a pretreatment sample incubated with 50 mM EDTA (to inhibit all complement activity) at 0%. The h13BX treatment data used for FIGURE 70 are also reflected in FIGURE 69. As shown in FIGURE 70, treatment with all three high-affinity MASP-3 inhibitory antibodies resulted in greater than 95% inhibition of APC. Animals treated with h4D5X, h10D12X, and h13B1X maintained at least 90% inhibition of APC for 6.7, 11.7, and 16 days, respectively. Thus, these results demonstrate that treatment with these representative high-affinity MASP-3 inhibitory mAbs provides sustained inhibition of APC in a nonhuman primate at a single 5 mg/kg dose.

[00863] FIGURE 71A-C graphically illustrates additional measures of APC activity. Fluid-phase Ba (FIGURE 71A), Bb (FIGURE 71B), and C3a (FIGURE 71C) were measured in diluted, zymosan-treated serum samples obtained from groups of cynomolgus monkeys (3 animals per group) over time following treatment with a single 5 mg/kg intravenous dose of h4D5X, h10D12X, and h13B1X as described above.

[00864] As shown in FIGURE 71A-C, single administrations of all three high-affinity MASP-3 inhibitory antibodies resulted in inhibition of the APC, as defined by three different fluid phase endpoints. These data are consistent with the level of APC inhibition demonstrated in the Bb deposition study of FIGURE 70 and further illustrate the efficacy of these mAbs to inhibit the pathway for several weeks.

[00865] FIGURE 72A-C graphically illustrates the relationship of APC activity, as determined by fluid-phase Ba production, to the molar ratio of MASP-3 mAb antibody and monomeric MASP-3 detected in the serum of monkeys treated with h4D5X (FIGURE 72A), h10D12X (FIGURE 72B), or h13B1X (FIGURE 72C). Each panel in FIGURE 72A-C represents data from one monkey. The monkeys used and the serum (or plasma) obtained in this study are the same as those described above (FIGURES 69, 70, and 71).

[00866] FIGURE 72A-C graphically illustrates the molar ratio of target (MASP-3) to the high affinity MASP-3 inhibitory antibodies h4D5X (FIGURE 72A), h10D12X (FIGURE 72B), and h13B1X (FIGURE 72C) at the time points of complete inhibition of APC, as measured by fluid phase Ba. For reference purposes, the 1:1 molar ratio of target to antibody is shown as a dotted line in each graph. As shown in FIGURES 72A-C, the target (MASP-3) to the high affinity MASP-3 inhibitory mAbs h4D5X, h10D12X, and h13B1X at a molar ratio in the range of about 2:1 to about 2.5:1 (target to antibody) are sufficient to completely inhibit APC. These data demonstrate that these three representative MASP-3 inhibitory mAbs are high-affinity and potent MASP-3 inhibitory antibodies that are capable of inhibiting APC when present at molar concentrations below the target concentration. These potency levels strongly indicate that the mAbs have the potential to be used clinically to treat diseases or indications caused by APC.

[00867] FIGURE 73 shows a Western blot analyzing the level of pro-Factor D and Factor D in the serum of a cynomolgus monkey over time (hours) before and after treatment with a single 5 mg/kg intravenous dose of mAb h13B1X, as described in Example 21. As shown in FIGURE 73, Factor D is present in the plasma as pro-Factor D for at least 336 hours (14 days) after a single dose of mAb h13B1X. Summary of Results

[00868] As described in Example 11, a single dose administration of a high affinity MASP-3 inhibitory antibody, mAb 13B1, to mice led to complete or near complete ablation of systemic alternative pathway complement activity for at least 14 days. As further described in Example 12, in a study conducted in a well-established animal model associated with PNH, mAb 13B1 was shown to significantly improve PNH red blood cell survival and protect PNH red blood cells significantly better than did C5 inhibition. As described in Example 13, mAb 13B1 was further shown to reduce disease incidence and severity in a mouse model of arthritis. The results in this example demonstrate that representative high affinity MASP-3 inhibitory mAbs 13B1, 10D12, and 4D5 are highly effective in blocking the alternative pathway in primates. Administration of a single dose of mAb 13B1, 10D12, or 4D5 to cynomolgus monkeys resulted in prolonged ablation of systemic alternative pathway activity lasting approximately 16 days. The extent of alternative pathway ablation in cynomolgus monkeys treated with high-affinity MASP-3 inhibitory antibodies was comparable to that achieved by factor D blockade in vitro, indicating complete blockade of factor D conversion by MASP-3 inhibitory antibodies. Therefore, high-affinity MASP-3 inhibitory mAbs have therapeutic utility in the treatment of patients suffering from diseases associated with alternative pathway hyperactivity, such as, for example, paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD, including wet and dry AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy (including hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), thrombotic thrombocytopenic purpura (TTP) or transplant-associated TMA), asthma, dense deposit disease, pauci-immune crescentic necrotizing glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis optica, Behçet's disease, multiple sclerosis, Guillain-Barre syndrome, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), lupus nephritis, lupus systemic erythematosus (SLE), diabetic retinopathy, uveitis, chronic obstructive pulmonary disease (COPD), C3 glomerulopathy, transplant rejection, graft-versus-host disease (GVHD), hemodialysis, sepsis, systemic inflammatory response syndrome (SIRS), acute respiratory distress syndrome (ARDS), ANCA vasculitis, antiphospholipid syndrome, atherosclerosis, IgA nephropathy, and myasthenia gravis.

VII. OTHER MODALITIES

[00869] All publications, patent applications and patents mentioned in this specification are incorporated herein by reference.

[00870] Various modifications and variations of the methods, compositions and compounds of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific desired embodiments, it is to be understood that the invention as claimed is not to be unduly limited to such specific embodiments. Indeed, various modifications of the described modes of carrying out the invention that are obvious to those skilled in the fields of medicine, immunology, pharmacology, oncology or related fields are intended to be within the scope of the following claims.

[00871] In accordance with the foregoing, the invention features the following embodiments. High affinity MASP-3 inhibitory antibodies that bind one or more epitopes within the SP domain

[00872] 1A. An isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to the serine protease domain of human MASP-3 (amino acid residues 450-728 of SEQ ID NO:2) with high affinity (having a K of less than 500 pM), wherein the antibody or antigen-binding fragment thereof inhibits activation of the alternative complement pathway.

[00873] 2A. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the antibody or antigen-binding fragment is characterized by at least one or more of the following properties: (a) inhibits profactor D maturation; (b) does not bind to human MASP-1 (SEQ ID NO: 8); (c) inhibits the alternative pathway at a molar ratio of about 1:1 to about 2.5:1 (MASP-3 target to mAb) in a mammalian subject; (d) does not inhibit the classical pathway.

[00874] (e) inhibition of hemolysis and/or opsonization; (f) inhibition of substrate-specific cleavage by the serine protease of MASP-3; (g) a reduction in hemolysis or a reduction in cleavage of C3 and surface deposition of C3b; (h) a reduction in the deposition of Factor B and Bb on an activating surface; (i) a reduction in resting levels (in circulation and without the experimental addition of an activating surface) of active Factor D relative to pro-Factor D; (j) a reduction in levels of active Factor D relative to pro-Factor D in response to an activating surface; (k) a reduction in the production of resting and/or surface-induced levels of fluid-phase Ba, Bb, C3b, or C3a and/or

[00875] (l) a reduction in factor P deposition.

[00876] 3A. The isolated antibody or antigen-binding fragment thereof of paragraph 1 or 2, wherein said antibody or antigen-binding fragment thereof specifically binds to an epitope located within the serine protease domain of human MASP-3, wherein the epitope is located within at least one or more of: VLRSQRRDTTVI (SEQ ID NO:9), TAAHVLRSQRRDTTV (SEQ ID NO:10), DFNIQNYNHDIALVQ (SEQ ID NO:11), PHAECKTSYESRS (SEQ ID NO:12), GNYSVTENMFC (SEQ ID NO:13), VSNYVDWVWE (SEQ ID NO:14), and/or VLRSQRRDTTV (SEQ ID NO:15). [Group I]

[00877] 4A. The antibody or antigen-binding fragment thereof of paragraph 1 or 2, wherein said antibody or antigen-binding fragment binds to an epitope in SEQ ID NO:15. [Includes all group I Abs]

[00878] 5A. The antibody or antigen-binding fragment of paragraph 3, wherein said antibody or antigen-binding fragment binds to an epitope in SEQ ID NO:9. [10D12]

[00879] 6A. The antibody or antigen-binding fragment of paragraph 3, wherein said antibody or antigen-binding fragment binds to an epitope in SEQ ID NO:10. [13B1]

[00880] 7A. The antibody or antigen-binding fragment of paragraph 6, wherein said antibody or antigen-binding fragment also binds to an epitope in SEQ ID NO:12. [13B1]

[00881] 8A. The antibody or antigen-binding fragment of paragraph 3, wherein said antibody or antigen-binding fragment also binds to an epitope in SEQ ID NO:10 and/or SEQ ID NO:12. [13B1]

[00882] 9A. The antibody or antigen-binding fragment thereof of paragraph 3, wherein said antibody or antigen-binding fragment binds to an epitope in SEQ ID NO:9. [1F3, 4B6, 4D5, 1A10]

[00883] 10A. The antibody or antigen-binding fragment thereof of paragraph 7, wherein said antibody or antigen-binding fragment also binds to an epitope in at least one of SEQ ID NO:11, SEQ ID NO:13, and/or SEQ ID NO:14. [1F3, 4B6, 4D5, 1A10]

[00884] 11A. The antibody or antigen-binding fragment thereof of paragraph 7, wherein the antibody or antigen-binding fragment thereof also binds to an epitope in at least one of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, and/or SEQ ID NO:14. [1F3, 4B6, 4D5, 1A10]

[00885] 12A. The antibody or antigen-binding fragment of paragraph 1 or 2, wherein said antibody binds to an epitope on at least one of: ECGQPSRSLPSLV (SEQ ID NO:16), RNAEPGLFPWQ (SEQ ID NO:17); KWFGSGALLSASWIL(SEQ ID NO:18); EHVTVYLGLH (SEQ ID NO:19); PVPLGPHVMP (SEQ ID NO:20); APHMLGL (SEQ ID NO:21); SDVLQYVKLP (SEQ ID NO:22); and/or AFVIFDDLSQRW(SEQ ID NO:23). [group II and III]

[00886] 13A. The antibody or antigen-binding fragment of paragraph 12, wherein said antibody or antigen-binding fragment binds to an epitope in SEQ ID NO:17. [All group II and III abs]

[00887] 14A. The antibody or antigen-binding fragment of paragraph 13, wherein said antibody or antigen-binding fragment also binds to an epitope on EHVTVYLGLH (SEQ ID NO:19) and/or AFVIFDDLSQRW (SEQ ID NO:23). [1G4, 1E7, 2D7 15D9]

[00888] 15A. The antibody or antigen-binding fragment thereof of paragraph 14, wherein said antibody or antigen-binding fragment also binds to an epitope in SEQ ID NO:23. [1G4, 1E7, 2D7, 15D9, 2F5]

[00889] 16A. The antibody or antigen-binding fragment thereof of paragraph 14, wherein said antibody or antigen-binding fragment also binds to an epitope in SEQ ID NO:19 and/or SEQ ID NO:23. [Ig4, 1E7, 2D7]

[00890] 17A. The antibody or antigen-binding fragment of paragraph 14, wherein said antibody or antigen-binding fragment also binds to an epitope in SEQ ID NO:18, SEQ ID NO:20, and/or SEQ ID NO:23. [15D9, 2F5]

[00891] 18A. The antibody or antigen-binding fragment thereof of paragraph 14, wherein said antibody or antigen-binding fragment also binds to an epitope in at least one of SEQ ID NO:18, SEQ ID NO:20, and/or SEQ ID NO:23 [15D9, 2F5].

[00892] 19A. The antibody or antigen-binding fragment thereof of paragraph 14, wherein said antibody or antigen-binding fragment also binds to an epitope in at least one of SEQ ID NO:16, SEQ ID NO:21, and/or SEQ ID NO:22. [1B11]

[00893] 20A. The antibody or antigen-binding fragment thereof of paragraph 14, wherein said antibody or antigen-binding fragment also binds to an epitope in at least one of SEQ ID NO:16, SEQ ID NO:21, and/or SEQ ID NO:22 [1B11].

[00894] 21A. The antibody or antigen-binding fragment thereof of any of paragraphs 1-20, wherein the antibody or antigen-binding fragment is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a murine antibody, and an antigen-binding fragment of any of the foregoing.

[00895] 22A. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-21, wherein said antibody or antigen-binding fragment thereof is selected from the group consisting of a single chain antibody, a ScFv, a Fab fragment, a Fab’ fragment, an F(ab’)2 fragment, a univalent antibody lacking a hinge region, and a full-length antibody.

[00896] 23A. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-22, further comprising an immunoglobulin constant region.

[00897] 24A. The antibody or antigen-binding fragment thereof of any of paragraphs 1-23, wherein the antibody or antigen-binding fragment is humanized.

[00898] 25A. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-24, wherein said antibody binds to the serine protease domain of human MASP-3 with an affinity of less than 500 pM.

[00899] 26A. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-25, wherein said antibody inhibits activation of the alternative pathway in mammalian blood.

[00900] 27A. A composition comprising the antibody or antigen-binding fragment of any one of paragraphs 1A-26A and a pharmaceutically acceptable excipient. A. Group IA high-affinity MASP-3 inhibitory antibodies that bind one or more epitopes within the SP domain (variants of 4D5, 4B6, 1A10 plus 4D5)

[00901] 1B. An isolated antibody or antigen-binding fragment thereof, that binds to MASP-3, comprising: (a) a heavy chain variable region comprising a HC-CDR1 defined as SEQ ID NO:209 (XXDIN, wherein X at position 1 is S or T and wherein X at position 2 is N or D); a HC-CDR2 defined as SEQ ID NO:210 (WIYPRDXXXKYNXXFXD, wherein X at position 7 is G or D; X at position 8 is S, T or R; X at position 9 is I or T; X at position 13 is E or D; X at position 14 is K or E and X at position 16 is T or K); and a HC-CDR3 defined as SEQ ID NO:211 (XEDXY, wherein X at position 1 is L or V and wherein X at position 4 is T or S); and (b) a light chain variable region comprising an LC-CDR1 defined as SEQ ID NO: 212 (KSSQSLLXXRTRKNYLX, wherein X at position 8 is N, I, Q or A; wherein X at position 9 is S or T; and wherein X at position 17 is A or S); an LC-CDR2 defined as SEQ ID NO: 144 (WASTRES) and an LC-CDR3 defined as SEQ ID NO:146 (KQSYNLYT).

[00902] 2B. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the HC-CDR1 of the heavy chain variable region according to (a) comprises SEQ ID NO: 56 (TDDIN). [4D5 and variants]

[00903] 3B. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the HC-CDR1 of the heavy chain variable region according to (a) comprises SEQ ID NO: 62 (SNDIN). [1F3, 4B6 and 1A10]

[00904] 4B. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the HC-CDR2 of the heavy chain variable region according to (a) comprises SEQ ID NO: 58 (WIYPRDDRTKYNDKFKD) [4D5 and variants].

[00905] 5B. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the HC-CDR2 of the heavy chain variable region according to (a) comprises SEQ ID NO: 63 (WIYPRDGSIKYNEKFTD). [1F3]

[00906] 6B. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the HC-CDR2 of the heavy chain variable region according to (a) comprises SEQ ID NO: 67 (WIYPRDGTTKYNEEFTD). [4B6]

[00907] 7B. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the HC-CDR2 of the heavy chain variable region according to (a) comprises SEQ ID NO: 69 (WIYPRDGTTKYNEKFTD). [1A10]

[00908] 8B. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the HC-CDR3 of the heavy chain variable region according to (a) comprises SEQ ID NO: 60 (LEDTY) [4D5 and variants].

[00909] 9B. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the HC-CDR3 of the heavy chain variable region according to (a) comprises SEQ ID NO: 65 (VEDSY). [1F3, 4B6 and 1A10]

[00910] 10B. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the LC-CDR1 of the light chain variable region according to (b) comprises SEQ ID NO:142 (KSSQSLLNSRTRKNYLA); SEQ ID NO:257 (KSSQSLLQSRTRKNYLA), SEQ ID NO:258 (KSSQSLLASRTRKNYLA); or SEQ ID NO:259 (KSSQSLLNTRTRKNYLA). [4D5 and variants]

[00911] 11B. The isolated antibody or antigen-binding fragment thereof of paragraph 10, wherein the HC-CDR1 of the light chain variable region according to (b) comprises SEQ ID NO: 258 (KSSQSLLASRTRKNYLA). [4D5 NA Mutant]

[00912] 12B. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the LC-CDR1 of the light chain variable region according to (b) comprises SEQ ID NO: 149 (KSSQSLLISRTRKNYLS). [1F3 and 4B6]

[00913] 13B. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the HC-CDR1 comprises SEQ ID NO: 56, the HC-CDR2 comprises SEQ ID NO: 58, the HC-CDR3 comprises SEQ ID NO: 60, and wherein the LC-CDR1 comprises SEQ ID NO: 142, SEQ ID NO: 257, SEQ ID NO: 258, or SEQ ID NO: 259; wherein the LC-CDR2 comprises SEQ ID NO: 144, and wherein the LC-CDR3 comprises SEQ ID NO: 146. [all 6 CDRs from 4D5 with variants in the LC-CDR1].

[00914] 14B. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the HC-CDR1 comprises SEQ ID NO: 62, the HC-CDR2 comprises SEQ ID NO: 62, the HC-CDR2 comprises SEQ ID NO: 63, SEQ ID NO: 67 or SEQ ID NO: 69, the HC-CDR3 comprises SEQ ID NO: 65, and wherein the LC-CDR1 comprises SEQ ID NO: 149, the LC-CDR2 comprises SEQ ID NO: 144, and the LC-CDR3 comprises SEQ ID NO: 146. [all 6 CDRs from 1F3, 4B6, and 1A10]

[00915] 15B. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-14, wherein the antibody or antigen-binding fragment is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a murine antibody, and an antigen-binding fragment of any one of the foregoing.

[00916] 16B. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-15, wherein said antibody or antigen-binding fragment thereof is selected from the group consisting of a single chain antibody, a ScFv, a Fab fragment, a Fab’ fragment, an F(ab’)2 fragment, a univalent antibody lacking a hinge region, and a full-length antibody.

[00917] 17B. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-16, further comprising an immunoglobulin constant region.

[00918] 18B. The antibody or antigen-binding fragment thereof of any of paragraphs 1-17, wherein the antibody or antigen-binding fragment is humanized.

[00919] 19B. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:24, SEQ ID NO:248, or SEQ ID NO:249 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:40, SEQ ID NO:250, or SEQ ID NO:278 [parent, humanized, and modified versions of 4D5].

[00920] 20B. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:25 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:41 [1F3].

[00921] 21B. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:26 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:42 [4B6].

[00922] 22B. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:27 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:42 [1A10].

[00923] 23B. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-22, wherein said antibody binds to a human MASP-3 with an affinity of less than 500 pM.

[00924] 24B. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-23, wherein said antibody inhibits activation of the alternative pathway in mammalian blood.

[00925] 25B. A composition comprising the antibody or antigen-binding fragment of any one of paragraphs 1B-24B and a pharmaceutically acceptable excipient. B. Group IB high-affinity MASP-3 inhibitory antibodies that bind one or more epitopes within the SP domain (10D12, 35C1, and 10D12 variants)

[00926] 1C. An isolated antibody, or antigen-binding fragment thereof, that binds to MASP-3, comprising: a heavy chain variable region comprising a HC-CDR1 defined as SEQ ID NO:213 (SYGXX, wherein X at position 4 is M or I, and wherein X at position 5 is S or T); a HC-CDR2 defined as SEQ ID NO:74; and a HC-CDR3 defined as SEQ ID NO:214 (GGXAXDY, wherein X at position 3 is E or D, and wherein X at position 5 is M or L); and a light chain variable region comprising a LC-CDR1 defined as SEQ ID NO:215 (KSSQSLLDSXXKTYLX, wherein X at position 10 is D and or A; wherein X at position 11 is G or A; and wherein X at position 16 is N or S); an LC-CDR2 defined as SEQ ID NO: 155; and an LC-CDR3 defined as SEQ ID NO:216 (WQGTHFPXT, where X at position 8 is either W or Y).

[00927] 2C. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the HC-CDR1 of the heavy chain variable region according to (a) comprises SEQ ID NO: 72 (SYGMS). [10D12 and variants]

[00928] 3C. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the HC-CDR1 of the heavy chain variable region according to (a) comprises SEQ ID NO: 79 (SYGIT). [35C1]

[00929] 4C. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the HC-CDR3 of the heavy chain variable region according to (a) comprises SEQ ID NO: 76 (GGEAMDY). [10D12 and variants]

[00930] 5C. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the HC-CDR3 of the heavy chain variable region according to (a) comprises SEQ ID NO: 82 (GGDALDY). [35C1]

[00931] 6C. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the LC-CDR1 of the light chain variable region according to (b) comprises SEQ ID NO:153 (KSSQSLLDSDGKTYLN); SEQ ID NO:261 (KSSQSLLDSEGKTYLN), SEQ ID NO:262 (KSSQSLLDSAGKTYLN), or SEQ ID NO:263 (KSSQSLLDSDAKTYLN) [10D12 and variants]

[00932] 7C. The isolated antibody or antigen-binding fragment thereof of paragraph 6, wherein the LC-CDR1 of the light chain variable region comprises SEQ ID NO: 263 (KSSQSLLDSDAKTYLN). [Variant 10D12 GA]

[00933] 8C. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the LC-CDR1 of the light chain variable region comprises SEQ ID NO: 152. [35C1]

[00934] 9C. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the LC-CDR3 of the light chain variable region according to (b) comprises SEQ ID NO: 159 (KSSQSLLDSDGKTYLS). [10D12]

[00935] 10C. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the LC-CDR3 of the light chain variable region according to (b) comprises SEQ ID NO: 160 (WQGTHFPYT). [35C1]

[00936] 11C. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the HC-CDR1 comprises SEQ ID NO: 72, the HC-CDR2 comprises SEQ ID NO: 74, the HC-CDR3 comprises SEQ ID NO: 76, the LC-CDR1 comprises SEQ ID NO: 153, SEQ ID NO: 261, SEQ ID NO: 262, or SEQ ID NO: 263; the LC-CDR2 comprises SEQ ID NO: 155, and the LC-CDR3 comprises SEQ ID NO: 157. [all 6 CDRs from 10D12 with variants in the LC-CDR1].

[00937] 12C. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the HC-CDR1 comprises SEQ ID NO: 79, the HC-CDR2 comprises SEQ ID NO: 74, the HC-CDR3 comprises SEQ ID NO: 82, the LC-CDR1 comprises SEQ ID NO: 159, the LC-CDR2 comprises SEQ ID NO: 155, and the LC-CDR3 comprises SEQ ID NO: 160. [all 6 CDRs from 35C1]

[00938] 13C. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-12, wherein the antibody or antigen-binding fragment is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a murine antibody, and an antigen-binding fragment of any one of the foregoing.

[00939] 14C. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-13, wherein said antibody or antigen-binding fragment thereof is selected from the group consisting of a single chain antibody, a ScFv, a Fab fragment, a Fab’ fragment, an F(ab’)2 fragment, a univalent antibody lacking a hinge region, and a full-length antibody.

[00940] 15C. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-14, further comprising an immunoglobulin constant region.

[00941] 16C. The antibody or antigen-binding fragment thereof of any of paragraphs 1-15, wherein the antibody or antigen-binding fragment is humanized.

[00942] 17C. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:28, SEQ ID NO:251, or SEQ ID NO:252 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:43, SEQ ID NO:253, or SEQ ID NO:279 [10D12 parent, humanized, and variant antibodies].

[00943] 18C. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:29 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:44 [35C1].

[00944] 19C. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-18, wherein said antibody binds to a human MASP-3 with an affinity of less than 500 pM.

[00945] 20C. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-19, wherein said antibody inhibits activation of the alternative pathway in mammalian blood.

[00946] 21C. A composition comprising the antibody or antigen-binding fragment of any one of paragraphs 1C-20C and a pharmaceutically acceptable excipient. C. Group IC high-affinity MASP-3 inhibitory antibodies that bind one or more epitopes within the SP domain (13B1 and variants)

[00947] 1D. An isolated antibody, or antigen-binding fragment thereof, that binds to MASP-3, comprising: (a) a heavy chain variable region comprising a HC-CDR1 defined as SEQ ID NO: 84 (GKWIE); a HC-CDR2 defined as SEQ ID NO: 86 (EILPGTGSTNYNEKFKG) or SEQ ID NO: 275 (EILPGTGSTNYAQKFQG); and a HC-CDR3 defined as SEQ ID NO: 88 (SEDV); and (b) a light chain variable region comprising a LC-CDR1 defined as SEQ ID NO: 142 (KSSQSLLNSRTRKNYLA), SEQ ID NO: 257 (KSSQSLLQSRTRKNYLA); SEQ ID NO: 258 (KSSQSLLASRTRKNYLA); or SEQ ID NO: 259 (KSSQSLLNTRTRKNYLA), an LC-CDR2 defined as SEQ ID NO: 144 (WASTRES); and an LC-CDR3 defined as SEQ ID NO: 161 (KQSYNIPT). [all 6 CDRs of 13B1 and variants in LC-CDR1].

[00948] 2D. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the LC-CDR1 comprises SEQ ID NO: 258. [13B1 LC-CDR1 NA variant]

[00949] 3D. The antibody or antigen-binding fragment thereof of any of paragraphs 1-2, wherein the antibody or antigen-binding fragment is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a murine antibody, and an antigen-binding fragment of any of the foregoing.

[00950] 4D. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-3, wherein said antibody or antigen-binding fragment thereof is selected from the group consisting of a single chain antibody, a ScFv, a Fab fragment, a Fab' fragment, an F(ab')2 fragment, a univalent antibody lacking a hinge region, and a full-length antibody.

[00951] 5D. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-4, further comprising an immunoglobulin constant region.

[00952] 6D. The antibody or antigen-binding fragment thereof of any of paragraphs 1-5, wherein the antibody or antigen-binding fragment is humanized.

[00953] 7D. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:30, SEQ ID NO:254, or SEQ ID NO:255 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:45, SEQ ID NO:256, or SEQ ID NO:280 [parent, humanized, and variant 13B1 antibodies].

[00954] 8D. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-7, wherein said antibody binds to a human MASP-3 with an affinity of less than 500 pM.

[00955] 9D. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-8, wherein said antibody inhibits activation of the alternative pathway in mammalian blood.

[00956] 10D. A composition comprising the antibody or antigen-binding fragment of any one of paragraphs 1D-9D and a pharmaceutically acceptable excipient. D. Group II high-affinity MASP-3 inhibitory antibodies that bind one or more epitopes within the SP domain (1G4)

[00957] 1E. An isolated antibody, or antigen-binding fragment thereof, that binds to MASP-3, comprising: (a) a heavy chain variable region comprising a HC-CDR1 defined as SEQ ID NO:91 (GYWIE); a HC-CDR2 defined as SEQ ID NO:93 (EMLPGSGSTHYNEKFKG); and a HC-CDR3 defined as SEQ ID NO:95 (SIDY); and (b) a light chain variable region comprising a LC-CDR1 defined as SEQ ID NO:163 (RSSQSLVQSNGNTYLH), a LC-CDR2 defined as SEQ ID NO:165 (KVSNRFS), and a LC-CDR3 defined as SEQ ID NO:167 (SQSTHVPPT).

[00958] 2E. The antibody or antigen-binding fragment thereof of paragraph 1, wherein the antibody or antigen-binding fragment is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a murine antibody, and an antigen-binding fragment of any of the foregoing.

[00959] 3E. The antibody or antigen-binding fragment thereof of any of paragraphs 1-2, wherein said antibody or antigen-binding fragment thereof is selected from the group consisting of a single chain antibody, a ScFv, a Fab fragment, a Fab’ fragment, an F(ab’)2 fragment, a univalent antibody lacking a hinge region, and a full-length antibody.

[00960] 4E. The antibody or antigen-binding fragment thereof of any of paragraphs 1-3, further comprising an immunoglobulin constant region.

[00961] 5E. The antibody or antigen-binding fragment thereof of any of paragraphs 1-4, wherein the antibody or antigen-binding fragment is humanized.

[00962] 6E. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:31 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:46 [1G4].

[00963] 7E. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-6, wherein said antibody binds to a human MASP-3 with an affinity of less than 500 pM.

[00964] 8E. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-7, wherein said antibody inhibits activation of the alternative pathway in mammalian blood.

[00965] 9E. A composition comprising the antibody or antigen-binding fragment of any one of paragraphs 1E-8E and a pharmaceutically acceptable excipient. F. Group III high-affinity MASP-3 inhibitory antibodies that bind one or more epitopes within the SP domain (1E7, 2D7, 15D9, 2F5, 1B11, 2F2, 11B6)

[00966] 1F. An isolated antibody, or antigen-binding fragment thereof, that binds to MASP-3, comprising: (a) a heavy chain variable region comprising a HC-CDR1 defined as SEQ ID NO:109 (RVHFAIRDTNYWMQ), a HC-CDR2 defined as SEQ ID NO:110 (AIYPGNGDTSYNQKFKG), a HC-CDR3 defined as SEQ ID NO:112 (GSHYFDY); and a light chain variable region comprising a LC-CDR1 defined as SEQ ID NO:182 (RASQSIGTSIH), a LC-CDR2 defined as SEQ ID NO:184 (YASESIS), and a LC-CDR3 defined as SEQ ID NO:186 (QQSNSWPYT) [1E7]; or (b) a heavy chain variable region comprising a HC-CDR1 defined as SEQ ID NO:125 (DYYMN), a HC-CDR2 defined as SEQ ID NO:127 (DVNPNNDGTTYNQKFKG), a HC-CDR3 defined as SEQ ID NO:129 (CPFYYLGKGTHFDY); and a light chain variable region comprising a LC-CDR1 defined as SEQ ID NO:196 (RASQDISNFLN), a LC-CDR2 defined as SEQ ID NO:198 (YTSRLHS) and a LC-CDR3 defined as SEQ ID NO:200 (QQGFTLPWT) [2D7]; or (c) a heavy chain variable region comprising a HC-CDR1 defined as SEQ ID NO:132, a HC-CDR2 defined as SEQ ID NO:133, a HC-CDR3 defined as SEQ ID NO:135; and a light chain variable region comprising a LC-CDR1 defined as SEQ ID NO:203, a LC-CDR2 defined as SEQ ID NO:165 and a LC-CDR3 defined as SEQ ID NO:204 [49C11]; or (d) a heavy chain variable region comprising a HC-CDR1 defined as SEQ ID NO:137 a HC-CDR2 defined as SEQ ID NO:138, a HC-CDR3 defined as SEQ ID NO:140; and a light chain variable region comprising an LC-CDR1 defined as SEQ ID NO:206, an LC-CDR2 defined as SEQ ID NO:207, and an LC-CDR3 defined as SEQ ID NO:208 [15D9]; or (e) a heavy chain variable region comprising an HC-CDR1 defined as SEQ ID NO:98, an HC-CDR2 defined as SEQ ID NO:99, an HC-CDR3 defined as SEQ ID NO:101; and a light chain variable region comprising an LC-CDR1 defined as SEQ ID NO:169, an LC-CDR2 defined as SEQ ID NO:171, and an LC-CDR3 defined as SEQ ID NO:173. [2F5]; or (f) a heavy chain variable region comprising a HC-CDR1 defined as SEQ ID NO:103, a HC-CDR2 defined as SEQ ID NO:105, a HC-CDR3 defined as SEQ ID NO:107; and a light chain variable region comprising a LC-CDR1 defined as SEQ ID NO:176, a LC-CDR2 defined as SEQ ID NO:178 and a LC-CDR3 defined as SEQ ID NO:193 [1B11]; or (g) a heavy chain variable region comprising a HC-CDR1 defined as SEQ ID NO:114, a HC-CDR2 defined as SEQ ID NO:116, a HC-CDR3 defined as SEQ ID NO:118; and a light chain variable region comprising an LC-CDR1 defined as SEQ ID NO:188, an LC-CDR2 defined as SEQ ID NO:178, and an LC-CDR3 defined as SEQ ID NO:190 [2F2]; or (h) a heavy chain variable region comprising an HC-CDR1 defined as SEQ ID NO:114, an HC-CDR2 defined as SEQ ID NO:121, an HC-CDR3 defined as SEQ ID NO:123; and a light chain variable region comprising an LC-CDR1 defined as SEQ ID NO:191, an LC-CDR2 defined as SEQ ID NO:178, and an LC-CDR3 defined as SEQ ID NO:193. [11B6]

[00967] 2F. The antibody or antigen-binding fragment thereof of paragraph 1(a)-(g), wherein the antibody or antigen-binding fragment is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a murine antibody, and an antigen-binding fragment of any of the foregoing.

[00968] 3F. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-2, wherein said antibody or antigen-binding fragment thereof is selected from the group consisting of a single chain antibody, a ScFv, a Fab fragment, a Fab’ fragment, an F(ab’)2 fragment, a univalent antibody lacking a hinge region, and a full-length antibody.

[00969] 4F. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-3, further comprising an immunoglobulin constant region.

[00970] 5F. The antibody or antigen-binding fragment thereof of any of paragraphs 1-4, wherein the antibody or antigen-binding fragment is humanized.

[00971] 6F. The antibody or antigen-binding fragment thereof of paragraph 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:32 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:47 [1E7].

[00972] 7F. The antibody or antigen-binding fragment thereof of paragraph 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:33 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:48 [2D7].

[00973] 8F. The antibody or antigen-binding fragment thereof of paragraph 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:34 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:49 [49C11].

[00974] 9F. The antibody or antigen-binding fragment thereof of paragraph 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:35 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:50 [15D9].

[00975] 10F. The antibody or antigen-binding fragment thereof of paragraph 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:36 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:51 [2F5].

[00976] 11F. The antibody or antigen-binding fragment thereof of paragraph 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:37 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:52 [1B11].

[00977] 12F. The antibody or antigen-binding fragment thereof of paragraph 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:38 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:53 [2F2].

[00978] 13F. The antibody or antigen-binding fragment thereof of paragraph 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:39 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:54 [11B6].

[00979] 14F. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-13, wherein said antibody binds to a human MASP-3 with an affinity of less than 500 pM.

[00980] 15F. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-14, wherein said antibody inhibits activation of the alternative pathway in mammalian blood.

[00981] 16F. A composition comprising the antibody or antigen-binding fragment of any one of paragraphs 1F-15F and a pharmaceutically acceptable excipient. Use of MASP-3 Inhibitory Antibodies for Treatment of PsA Diseases

[00982] 1. A method of inhibiting activation of the alternative complement pathway in a mammal, the method comprising administering to a mammal in need thereof an amount of a composition comprising a high affinity MASP-3 inhibitory antibody or antigen-binding fragment thereof sufficient to inhibit activation of the alternative complement pathway in the mammal.

[00983] 2. The method of claim 1, wherein the antibody or antigen-binding fragment thereof binds to MASP-3 with an affinity of less than 500 pM.

[00984] 3. The method of paragraph 1, wherein as a result of administering the composition comprising the antibody or antigen-binding fragment of one or more of the following is present in the mammalian subject: (a) inhibition of Factor D maturation; (b) inhibition of the alternative pathway when administered to the subject in a molar ratio of about 1:1 to about 2.5:1 (MASP-3 target to mAb); (c) the classical pathway is not inhibited; (d) inhibition of hemolysis and/or opsonization; (e) a reduction in hemolysis or the reduction in C3 cleavage and C3b surface deposition; (f) a reduction in Factor B and Bb deposition on an activating surface; (g) a reduction in resting levels (in circulation and without the experimental addition of an activating surface) of active Factor D relative to pro-Factor D; (h) a reduction in active Factor D levels relative to pro-Factor D in response to an activating surface; and/or (i) a reduction in the production of resting and surface-induced levels of fluid phase Ba, Bb, C3b or C3a.

[00985] 4. The method of paragraph 1, wherein the antibody inhibits the alternative pathway in a molar ratio of about 1:1 to about 2.5:1 (MASP-3 target to mAb).

[00986] 5. The method of any one of paragraphs 1-3, wherein the high affinity MASP-3 antibody, characterized according to any of claims 27A, 25B, 21C, 10D, 9E, or 16F.

[00987] 6. The method of any of paragraphs 1-4, wherein the antibody or antigen-binding fragment thereof selectively inhibits the alternative pathway without affecting activation of the classical pathway.

[00988] 7. The method of any one of paragraphs 1-6, wherein the mammalian subject suffers from or is at risk of developing an alternative pathway disease or disorder selected from the group consisting of paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD, including wet and dry AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy (including hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), thrombotic thrombocytopenic purpura (TTP), or transplant-associated TMA), asthma, dense deposit disease, pauci-immune crescentic necrotizing glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis optica, Behçet's disease, multiple sclerosis, Guillain Barre syndrome, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), nephritis lupus, systemic lupus erythematosus (SLE), diabetic retinopathy, uveitis, chronic obstructive pulmonary disease (COPD), C3 glomerulopathy, transplant rejection, graft-versus-host disease (GVHD), hemodialysis, sepsis, systemic inflammatory response syndrome (SIRS), acute respiratory distress syndrome (ARDS), ANCA vasculitis, antiphospholipid syndrome, atherosclerosis, IgA nephropathy, and myasthenia gravis.

[00989] Although the preferred embodiment of the invention has been illustrated and described, it will be appreciated that various changes may be made without departing from the spirit and scope of the invention.

Claims (18)

1. An isolated antibody or antigen-binding fragment thereof that binds to MASP-3 with high affinity, comprising: a heavy chain variable region comprising a HC-CDR1 defined as SEQ ID NO: 84 (GKWIE); a HC-CDR2 defined as SEQ ID NO: 86 (EILPGTGSTNYNEKFKG) or SEQ ID NO: 275 (EILPGTGSTNYAQKFQG); and a HC-CDR3 defined as SEQ ID NO:88 (SEDV); and a light chain variable region comprising a LC-CDR1 defined as SEQ ID NO:142 (KSSQSLLNSRTRKNYLA), SEQ ID NO:257 (KSSQSLLQSRTRKNYLA); SEQ ID NO:258 (KSSQSLLASRTRKNYLA); or SEQ ID NO:259 (KSSQSLLNTRTRKNYLA), an LC-CDR2 presented as SEQ ID NO:144 (WASTRES); and an LC-CDR3 defined as SEQ ID NO:161 (KQSYNIPT); wherein the antibody or antigen-binding fragment thereof inhibits alternative pathway complement activation. 2. The antibody or antigen-binding fragment thereof of claim 1, wherein the HC-CDR1 comprises SEQ ID NO:84, the HC-CDR2 comprises SEQ ID NO:86, the HC-CDR3 comprises SEQ ID NO:88, the LC-CDR1 comprises SEQ ID NO:258, the LC-CDR2 comprises SEQ ID NO:144, and the LC-CDR3 comprises SEQ ID NO:161. 3. The antibody or antigen-binding fragment thereof of claim 1, wherein the HC-CDR1 comprises SEQ ID NO:84, the HC-CDR2 comprises SEQ ID NO:275, the HC-CDR3 comprises SEQ ID NO:88; the LC-CDR1 comprises SEQ ID NO:258, the LC-CDR2 comprises SEQ ID NO:144, and the LC-CDR3 comprises SEQ ID NO:161. 4. The antibody or antigen-binding fragment thereof according to claim 1, wherein the variable region of the heavy chain comprises the amino acid sequence set forth as SEQ ID NO:30. 5. Isolated antibody or antigen-binding fragment thereof, according to claim 1, characterized by the fact that the variable region of the light chain comprises the amino acid sequence set forth as SEQ ID NO:45. 6. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO:30 and the light chain variable region comprises the amino acid sequence set forth as SEQ ID NO:45. 7. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO:254 or SEQ ID NO:255. 8. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the variable region of the light chain comprises the amino acid sequence set forth as SEQ ID NO:256 or SEQ ID NO:280. 9. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO:254 or SEQ ID NO:255 and the light chain variable region comprises the amino acid sequence set forth as SEQ ID NO:256 or SEQ ID NO:280. 10. Antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, characterized in that the antibody or antigen-binding fragment is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a murine antibody and an antigen-binding fragment of any of the preceding. 11. Antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, characterized in that said antibody or antigen-binding fragment thereof is selected from the group consisting of a single chain antibody, a ScFv, a Fab fragment, a Fab' fragment, an F(ab')2 fragment, a univalent antibody lacking a hinge region and a full-length antibody. 12. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, further comprising an immunoglobulin constant region. 13. Antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, characterized in that the antibody or antigen-binding fragment is humanized. 14. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein said antibody binds to the serine protease domain of human MASP-3 with an affinity of less than 500 pM. 15. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein said antibody inhibits activation of the alternative pathway in the blood of mammals. 16. Composition, characterized by the fact that it comprises the antibody or antigen-binding fragment as defined in any one of claims 1 to 9 and a pharmaceutically acceptable excipient. 17. Use of a composition as defined in claim 16 comprising a high affinity MASP-3 inhibitory antibody or an antigen-binding fragment thereof, characterized in that it is for manufacturing a medicament for inhibiting alternative pathway complement activation in a mammal in need thereof, wherein the composition is in a quantity sufficient to inhibit alternative pathway complement activation in the mammal. 18. Use according to claim 17, characterized in that the mammal suffers from, or is at risk of developing, an alternative pathway disease or disorder selected from the group consisting of paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD, including wet and dry AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy (including hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), thrombotic thrombocytopenic purpura (TTP) or transplant-associated TMA), asthma, dense deposit disease, pauci-immune necrotizing crescentic glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis optica, Behçet's disease, multiple sclerosis, Guillain Barre syndrome, Alzheimer's disease, amylotrophic lateral sclerosis (ALS), lupus nephritis, systemic lupus erythematosus (SLE), diabetic retinopathy, uveitis, chronic obstructive pulmonary disease (COPD), C3 glomerulopathy, transplant rejection, graft-versus-host disease (GVHD), hemodialysis, sepsis, systemic inflammatory response syndrome (SIRS), acute respiratory distress syndrome (ARDS), ANCA vasculitis, antiphospholipid syndrome, atherosclerosis, IgA nephropathy, and myasthenia gravis.