AU2018200437A1 - Methods for treating conditions associated with MASP-2 dependent complement activation - Google Patents

Abstract

The invention relates to methods of inhibiting the effects of MASP-2 dependent complementation in a living subject.

Description

The invention relates to methods of inhibiting the effects of MASP-2 dependent complementation in a living subject.

1002049519

2018200437 19 Jan 2018

METHODS FOR TREATING CONDITIONS ASSOCIATED WITH MASP-2 DEPENDENT

COMPLEMENT ACTIVATION

CROSS-REFERENCES TO RELATED APPLICATION This application is a divisional application of AU 2016200531, which is a divisional of

AU 2013201606, which is a divisional of AU 2012239889, which in turn claims the benefit of Provisional Application No. 61/473,698 filed April 8, 2011, the entire disclosures of which are incorporated herein by reference.

STATEMENT REGARDING SEQUENCE LISTING The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the text file containing the sequence listing is MP_l_0126_US2_SequenceListingasFiled.txt. The text file is 110 KB; was created on March 30, 2012; and is being submitted via EFS-Web with the filing of the specification.

BACKGROUND

Reference to any prior art in the specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other jurisdiction or that this prior art could reasonably be expected to be ascertained, understood and regarded as relevant by a person skilled in the art.

As used herein, except where the context requires otherwise the term ‘comprise’ and variations of the term, such as ‘comprising’, ‘comprises’ and ‘comprised’, are not intended to exclude other additives, components, integers or steps.

The complement system provides an early acting mechanism to initiate, amplify and orchestrate the immune response to microbial infection and other acute insults (M.K. Liszewski and J.P. Atkinson, 1993, in Fundamental Immunology, Third Edition, edited by W.E. Paul,

Raven Press, Ltd., New York), in humans and other vertebrates. While complement activation provides a valuable first-line defense against potential pathogens, the activities of complement that promote a protective immune response can also represent a potential threat to the host (K.R. Kalli, etal., Springer Semin. Immunopathol. 15:411-431, 1994; B.P. Morgan, Eur. J. Clinical Investig. 24:219-228, 1994). For example, C3 and C5 proteolytic products recruit and activate neutrophils. While indispensable for host defense, activated neutrophils are indiscriminate in their release of destructive enzymes and may cause organ damage. In addition, complement activation may cause the deposition of lytic complement components on nearby host cells as well as on microbial targets, resulting in host cell lysis.

The complement system has also been implicated in the pathogenesis of numerous acute and chronic disease states, including: myocardial infarction, stroke, ARDS,

2018200437 19 Jan 2018 reperfusion injury, septic shock, capillary leakage following thermal burns, postcardiopulmonary bypass inflammation, transplant rejection, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, and Alzheimer's disease. In almost all of these conditions, complement is not the cause but is one of several factors involved in pathogenesis. Nevertheless, complement activation may be a major pathological mechanism and represents an effective point for clinical control in many of these disease states. The growing recognition of the importance of complement-mediated tissue injury in a variety of disease states underscores the need for effective complement inhibitory drugs. To date, Bculizumab (Solaris®), an antibody against C5, is the only complement10 targeting drug that has been approved for human use. Yet, C5 is one of several effector molecules located “downstream” in the complement system, and blockade of C5 does not inhibit activation of the complement system. Therefore, an inhibitor of the initiation steps of complement activation would have significant advantages over a “downstream” complement inhibitor.

Currently, it is 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.

The activation of the complement system results in the sequential activation of serine protease zymogens. The first step in activation of the classical pathway is the binding of a specific recognition molecule, Clq, to antigen-bound IgG andlgM molecules. Clqis associated with theClr and Cis serine protease proenzymes as a complex called CL Upon binding ofClq to an immune complex, autoproteolytic cleavage of the Arg-Ile site of Cir is followed by Clr-mediated cleavage and activation of Cis, which thereby acquires the ability to cleave C4 and C2. C4 is cleaved into two fragments, designated C4a and C4b, and, similarly, C2 is cleaved into C2a and C2b. C4b fragments are able to form covalent bonds with adjacent hydroxyl or amino groups and generate the C3 convertase (C4b2a) through noncovalent interaction with the C2a

-22018200437 19 Jan 2018 fragment of activated C2. C3 convertase (C4b2a) activates C3 by proteolytic cleavage into C3a and C3b subcomponents leading to generation of the C5 convertase (C4b2a3b). which, by cleaving C5 leads to the formation of the membrane attack complex (C5b combined with C6, C7, C8 and C~9, also referred to as “MAC”) that can disrupt cellular membranes leading to cell lysis. The activated forms ofC3 andC4 (C3b and C4b) are covalently deposited on the foreign target surfaces, which are recognized by complement receptors on multiple phagocytes.

Independently, the first step in activation of the complement system through the lectin pathway is also the binding of specific recognition molecules, which is followed by the activation of associated serine protease proenzymes. However, rather than the binding of immune complexes byClq, the recognition molecules in the lectin pathway comprise a group of carbohydrate-binding proteins (mannan-binding lectin (MBL), H-ficolin, M-ficolin, L-ficoiin and C-type lectin CL-11). collectively referred to as lectins. See J. Lu et af, Biochim. Biophys. Acta 7572:387-400, (2002); Holmskov et af,

Antrn. Rev, 21:547-578 (2003); Teh etaf, Immunology 101:225-232 (2000)).

See also J. Lust et al., Biochim Biophys Acta 1572:387-400 (2002); Holmskov et ai, Amu Rev Immunol 21:547-578 (2003); Teh et af. Immunology 101:225-232 (2000); Hansen et al, J Immunol 185(i 0):6096-6104 (20i 0).

Ikeda et af first demonstrated that, like Clq, MBL could activate the complement system upon binding io yeast mannan-coated erythrocytes in a C4-dependent manner (Ikeda etaf, J Biol. Chem. 242:7451-7454, (1987)). MBL, a member of the col lectin protein family, is a calcium-dependent lectin that binds carbohydrates with 3- and 4-hydroxv groups oriented in the equatorial plane of the pyranose ring. Prominent ligands for MBL are thus D-mannose and N-acetyl-D-glucosamine, while carbohydrates not fitting this steric requirement have undetectable affinity for MBL (Weis etaf, Nature 360:127-134, (1992)), The interaction between MBL and monovalent sugars is extremely weak, with dissociation constants typically in the single-digit millimolar range. MBL achieves tight, specific binding to glycan ligands by avidity, i.e., by interacting simultaneously 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 patterns that commonly decorate microorganisms such as bacteria, yeast, parasites and certain viruses, in contrast, MBL does not recognize D-galactose and sialic acid, the penultimate and ultimate sugars that usually decorate mature'' complex

2018200437 19 Jan 2018 glycoconjugates present on mammalian plasma and cell surface glycoproteins. This binding specificity is thought to promote recognition of “foreign” surfaces and help protect from “seif-activation,” However, MBL does bind with high affinity to clusters of high-mannose precursor glycans on N-linked glycoproteins and glycolipids sequestered in the endoplasmic reticulum and Golgi of mammalian cells (Maynard et al., J Biol. Chem. 257:3788-3794, (1982)). Therefore, damaged cells are potential targets for lectin padway activation via MBL binding.

The fieoiins possess a different type of lectin domain than MBL, called the fibrinogen-like domain. Fieoiins bind sugar residues in a Ca~^H'-independent manner, in humans, three kinds of fieoiins (L-ficolin, M-ficolin and H-ficolin) have been identified. The two serum fieoiins, 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 hi sugar specificity of L-ficolin, H-ficolin, CL-11, and MBL means that the different lectins may be complementary and target different, though overlapping, ! 5 glycoconjugates. This concept is supported by the recent report that, of the known lectins in the lectin pathway, only L-ficolin binds specifically to lipoteichoic acid, a cell wall glycoconjugate found on all Gram-positive bacteria (Lynch et ah, J Immunol. 172:1 198-1202, (2004)). The collectins (i.e., MBL) and the fieoiins bear no significant similarity in amino acid sequence. However, the two groups of proteins have similar domain organizations and, like Clq, assemble into oligomeric structures, which maximize the possibility of multisite binding,

The serum concentrations of MBL are highly variable in healthy populations and this is genetically controlled by polymorphisms/mutacions in both the promoter and coding regions of the MBL gene. As an acute phase protein, the expression of MBL is

2.5 further upregulated during inflammation. L-ficolin is present in serum at concentrations similar to those of MBL. Therefore, the L-ficolin branch of the lectin pathway is potentially comparable to the MBL arm in strength. MBL and fieoiins can also function as opsonins, which allow phagocytes to target MBL- and ficolin-decorated surfaces (see Jack et al., J Leukoc Biol., 77(3):328-36 (2004), Matsushita and Fujita, Immunobiology,

205(4-5):490-7 (2002), Aoyagi et al., J Immunol, 174(1):43 8-25(2005). This opsonization requires the interaction of these proteins with phagocyte receptors (Kuhlman eta!., J. Exp. Med. 169:1733, (1989); Matsushita et al., J. Biol. Chem, 271:2448-54, (1996)), the indentity of which has not been established.

.4.

2018200437 19 Jan 2018

Human MBL forms a specific and high-affinity interaction through its collagen-like domain with unique Clr/Cls-like serine proteases, termed MBL-associated serine proteases (MASPs), To date, three MASPs have been described, First, a singie enzyme MASP was identified and characterized as the enzyme responsible for the initiation of the complement cascade (i.e,, cleaving C2 and C4) (Matsushita et ah, J Exp Med 176(6):1497-1502 (1992); Ji etal., J. Immunol, 150:571-578, (1993)). it was subsequently determined that the MASP activity was, in fact, a mixture of two proteases: MASP-i and MASP-2 (Thiel etai., Nature 3X5:506-510, (1997)). However, it was demonstrated that the MBL-MASP-2 complex alone is sufficient for complement activation (Vorup-Jensen etai., J. Immunol 765:2.093-2100, (2000)). Furthermore, only MASP-2 cleaved C2 and C4 at high rates (Ambrus etal., J. Immunol, /70:1374-1382, (2003)). Therefore, MASP-2 is the protease responsible for activating C4 and C2 to generate the C3 convertase, C4b2a. This is a significant difference from the Cl complex of the classical pathway, where the coordinated action of two specific serine proteases (Or and Cis) leads to the activation of the complement system. In addition, a third novel protease, MASP-3, has been isolated (Dahl, M.R., etal., Immunity /5:127-35, 2001), MASP-1 and MASP-3 are alternatively spliced products of the same gene.

MASPs share identical domain organizations with those of Clr and Cis, the enzymatic components of the Cl complex (Sim etal., Biochem. Soc. Trans. 28:545, (2000)). These domains include an N-terminal Clr/Cis/sea urchin VEGF/bone morpbogenic protein (CUB) domain, an epidermal growth factor-like domain, a second CUB domain, a tandem of complement control protein domains, and a serine protease domain. As in the Cl proteases, activation of MASP-2 occurs through cleavage of an Arg-Iie bond adjacent to the serine protease domain, which splits the enzyme into disulfide-iinked A and B chains, the latter consisting of the serine protease domain,

MBL can also associate with an alternatively sliced form of MASP-2, known as

MBL-associated protein of 19 kDa (MApI9) or small MBL-associated protein (sMAP), which Sacks the catalytic acivity of MASP2. (Stover, J. Immunol. /62:3481-90, (1999); Takahashi et al., Int, Immunol. I /:859-863, (1999)). MApl9 comprises the first two domains of MASP-2, followed by an extra sequence of four unique amino acids. The function of Map 19 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 ah, Immunobiology 265:455-466, (2002)).

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Several lines of evidence suggest that there are different MBL-MASP complexes and a large fraction of the MASPs in serum Is not complexed with MBL (Thiel, et al., J. Immunol. /65:878-887, (2000)). Both H- and L-ficoIin bind to al! MASPs and activate the lectin complement pathway, as does MBL (Dahl etal., Immunity /5:127-35, (2001):

Matsushita etal., J. Immunol. /68:3502-3506, (2002)), Both the lectin and classical pathways form a common C3 eonvertase (C4b2a) and the two pathways converge at this step.

The lectin pathway Is widely thought to have a major role in host defense against infection in the na'ive 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 /572:401-413, (2002)), Such patients display susceptibility to recurrent bacterial and fungal infections. These symptoms are usually evident early in life, during an apparent window of vulnerability' as maternally derived antibody titer wanes, but before a full repertoire of antibody responses develops. This syndrome often results from mutations at several sites in the collagenous portion of MBL, which interfere with proper formation of MBL oligomers. However, since MBL can function as an opsonin independent of complement, it is not known to what extent the increased susceptibility to infection is due to impaired complement activation.

In contrast to the classical and lectin pathways, no initiators of the alternative pathway have been found to fulfill the recognition functions that Clq and lectins perform in the other two pathways. Currently if is widely accepted that the alternative pathway spontaneously undergoes a low level of turnover activation, which can be readily amplified on foreign or other abnormal surfaces (bacteria, yeast, viraily infected cells, or damaged tissue) that lack the proper molecular elements that keep spontaneous complement activation in check. There are four plasma proteins directly Involved in the activation ofthe alternative pathway: C3, factors B and D, and properdin.

Although there is extensive evidence implicating both the classical and alternative complement pathways in the pathogenesis of non-mtectious human diseases, the role of the lectin pathway is just beginning to be evaluated. Recent studies provide evidence that activation of the lectin pathway can be responsible for complement activation and related inflammation in ischemia/reperfusion injury. Collard etal. (2000) reported that cultured endothelial cells subjected to oxidative stress bind MBL and show deposition of C3 upon exposure to human serum (Collard etal., Am. J. Pathol, /56)1549-1556, (2000)). in

-62018200437 19 Jan 2018 addition, 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 damage upon occlusion of a coronary artery than rats treated with a control antibody (Jordan et ah, Circulation 104:1413-1418, (2001)). The molecular mechanism of MBL binding to the 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, and not to glycoconjugates (Coiiard etah, Am. J.

Pathol. 759:1045-1054, (2001)), Other studies have implicated the classical and alternative pathways in the pathogenesis of ischemia/reperfusion injury and the role of the lectin pathway in this disease remains controversial (Riedermann, N.C., et ah, Am. J. Pathol. /62:363-367, 2003),

A recent study has shown that MASP-i (and possibly also MASP-3) is required to convert the alternative pathway activation enzyme Factor D from its zymogen form into its enzymatically active form (see Takahashi M. et ah, J Exp .Med 207( 3):29-37 (2010)), The physiological importance of this process is underlined by the absence of alternative pathway functional activity in plasma of MASP-l/3-deficient mice. Proteolytic generation of C3b from native C3 is required for the alternative pathway to function.

Since the alternative pathway C3 convertase (C3bBb) contains C3b as an essential subunit, the question regarding the origin of the first C3b via the alternative pathway has presented a puzzling problem and has stimulated considerable research.

C3 belongs to a family of proteins (along with C4 and a-2 macroglobulin) that contain a rare posttranslationai modification known as a thioester bond. The thioester group is composed of a glutamine whose terminal carbonyl group forms a covalent thioester linkage with the sulfhydryl group of a cysteine three amino acids away. This bond is 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 pocket of intact C3. However, proteolytic cleavage of C3 to C3a and C3b results in exposure of the highly reactive thioester bond on C3b and, following nucleophilic attack by adjacent moieties comprising hydroxyl or amino groups, C3b becomes covalently linked to a target. In addition to its well-documented role in covalent

-72018200437 19 Jan 2018 attachment of C3b to complement targets, the C3 thioester is aiso thought to have a pivotal role in triggering the alternative pathway. According to the widely accepted tick-over theory, the alternative pathway is initiated by the generation of a fluid-phase convertase, iC3Bb, which is formed from C3 with hydrolyzed thioester (iC3; CAfHjO)) and factor B (Lachmann, P.J., et ah. Springer Semin. Immunopathol. 7:143-162, (1984)). The C3b-Hke C3{H₂Q) is generated from native C3 by a slow spontaneous hydrolysis of the internal thioester in the protein (Pangburn, M.K., etai., J. Exp. Med. 154:856-867, 1981), Through the activity of the C3(H₂O)Bb convertase, C3b molecules are deposited on the target surface thereby initiating the alternative pathway,

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

Activators are thought to include yeast cell walls (zymosan), many pure polysaccharides, rabbit erythrocytes, certain immunoglobulins, viruses, 'fungi, bacteria, animal tumor cells, parasites, and damaged cells. The only feature common to these activators is the presence of carbohydrate, but the complexity and variety of carbohydrate structures has made it difficult to establish the shared molecular determinants which are recognized, it has been widely accepted that alternative pathway activation is controlled through the fine balance between inhibitory regulatory components of this pathway, such as Factor H, Factor I, DAF, and CR1, and properdin, which is the only positive regulator of the alternative pathway (see Schwaeble W.J. and Reid K.B., Immunol Today 20(1):17-21 (1999)).

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

Ail three pathways (i.e., the classical, lectin and alternative) have been thought to converge at C5, which is cleaved to form products with multiple proinflammatory effects. The converged pathway has been referred to as the terminal complement pathway. C5a is the most potent anaphylatoxin, inducing alterations in smooth muscle and vascular tone, as well as vascular permeability. It is also a powerful chemotaxin and activator of both

-82018200437 19 Jan 2018 neutrophils and monocytes, CSa-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. C5 cleavage leads to the formation of C5b-9, also known as the membrane attack complex (MAC). There is now strong evidence that sublytic MAC deposition may play an important role in inflammation in addition to its role as a lytic pore-forming complex.

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

SUMMARY

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

In one aspect, the present invention provides a method of inhibiting the adverse effects of MASP-2-dependent complement activation in a living subject. The method includes the step of administering to a subject in need thereof, an amount of a MASP-2 inhibitory agent effective to inhibit MASP-2-dependent complement activation. In another aspect of the invention, the MASP-2 inhibitory agent inhibits complement activation via the lectin-dependent MASP-2 system without substantially inhibiting complement activation via the classical or Clq-dependent system, such that the CI q-dependent system remains functional.

In some embodiments of these aspects of the invention, the MASP-2 inhibitoryagent is an anti-MASP-2 antibody or fragment thereof, In further embodiments, the anti-MASP-2 antibody has reduced effector function, in some embodiments, the MASP-2 inhibitory agent is a MASP-2 inhibitory peptide or a non-peptide MASP-2 inhibitor.

In another aspect, the present invention provides compositions for inhibiting the adverse effects of MASP-2-dependent complement activation, comprising a therapeutically effective amount of a MASP-2 inhibitory agent and a pharmaceutically acceptable carrier. Methods are also provided for manufacturing a medicament for use in

92018200437 19 Jan 2018 inhibiting the adverse effects of MASP-2-dependent complement activation in living subjects in need thereof, comprising a therapeutically effective amount of a MASP-2 inhibitory agent in a pharmaceutical carrier. Methods are also provided for manufacturing medicaments for use in inhibiting MASP-2-dependent complement activation for treatment of each of the conditions, diseases and disorders described herein below.

The methods, compositions and medicaments of the invention are useful for inhibiting the adverse effects of MASP-2-dependent complement activation in vivo in mammalian subjects, including humans suffering from an acute or chronic pathological condition or injury as further described herein,

In another aspect of the invention, methods are provided for inhibiting MASP-2 dependent complement activation in a subject suffering from paroxysmal nocturnal hemoglobinuria, comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory agent effective to inhibit MASP-2 dependent complement activation.

In another aspect, the invention provides a method of inhibiting MASP-2dependent complement activation in a subject suffering from or at risk for developing non-Factor H-dependent atypical hemolytic uremic syndrome (aHUS), comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory agent effective to inhibit MASP-2 dependent complement activation.

In another aspect, the invention provides a method for reducing the likelihood that a subject at risk for developing atypical hemolytic uremic syndrome (aHUS) will suffer clinical symptoms associated with aHUS comprising:(a) determining the presence of a genetic marker in the subject known to be associated with aHtJS; (b) periodically monitoring the subject to determine the presence or absence of at least one symptom selected from the group consisting of anemia, thrombocytopenia, renal insufficiency and rising creatinine; and (c) administering to the subject a composition comprising an amount of a MASP-2 inhibitory agent effective to inhibit MASP-2-dependent complement acti vation upon the determination of the presence of at least one of anemia, thrombocytopenia, renal insufficiency or rising creatinine, wherein the composition is

-102018200437 19 Jan 2018 administered in an effective amount and for a sufficient time period to improve said one or more symptoms.

In another aspect, the invention provides a method of inhibiting MASP-2dependent complement activation in a subject suffering from, or at risk tor developing, atypical hemolytic uremic syndrome (aHUS) secondary to an infection, comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory agent effective to inhibit MASP-2 complement activation.

in another aspect, the invention provides a method of treating a subject suffering from atypical hemolytic uremic syndrome (aHUS) comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory agent effective to inhibit MASP-2 dependent complement activation, wherein the administration of the MASP-2 inhibitory agent is administered via an intravenous catheter or other catheter ddiyery method.

In another aspect, the invention provides a method for decreasing the likelihood of developing impaired renal function in a subject at risk for developing hemolytic uremic syndrome (HUS) comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory agent effective to inhibit MASP-2 dependent complement activation.

In another aspect, the invention provides a method of treating a subject suffering from hemolytic uremic syndrome (HUS) comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory agent effective to inhibit MASP-2-dependent complement activation, wherein the administration of the MASP-2 inhibitory agent is administered to the subject via an intravenous catheter or other catheter delivery method.

In another aspect, the invention provides a method of treating a subject suffering from thrombotic thrombocytopenic purpura (TTP), or exhibiting symptoms consistent with a diagnosis of TTP, comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory agent effective to inhibit MASP-2-112018200437 19 Jan 2018 dependent complement activation, wherein the administration of the MASP-2 inhibitory agent is administered to the subject via an intravenous catheter or other catheter delivery method.

In another aspect, the invention provides a method of treating a subject suffering 5 from refractory thrombotic thrombocytopenic purpura (TTP) comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory agent effective to inhibit MASP-2 dependent complement activation.

In another aspect of the invention, methods are provided for inhibiting MASP-2dependent complement activation in a subject suffering from cryoglobulinemia, comprising administering to the subject a composition comprising an amount of a MASP2 inhibitory agent effective to inhibit MASP-2 dependent complement activation.

in another aspect of the invention, methods are provided for inhibiting MASP-2dependent complement activation in a subject suffering from cold agguitinin disease, comprising administering to the subject a composition comprising an amount of a MASP! 5 2 inhibitory agent effective to inhibit MASP-2 dependent complement activation,

In another aspect of the invention, methods are provided for inhibiting MASP-2 dependent complement activation in a subject suffering from glaucoma, comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory agent effective to inhibit MASP-2 dependent complement activation.

In another aspect of the invention, methods are provided for inhibiting MASP-2 dependent complement activation in a subject at risk for developing or suffering from acute radiation syndrome comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory agent effective to inhibit MASP-2 dependent complement activation. In some embodiments, the anti-MASP-2 inhibitory agent is an anti-MASP-2 antibody. In some embodiments, the MASP-2 inhibitory agent is administered prophylacticaiiy to the subject prior to radiation exposure (such as prior to treatment with radiation, or prior to an expected exposure to radiation). In some embodiments, the MASP-2 inhibitory agent is administered within 24 to 48 hours after exposure to radiation. In some embodiments, the MASP-2 inhibitory agent is administered prior to and/or after exposure to radiation in an amount sufficient to ameliorate one or more symptoms associated with acute radiation syndrome.

2018200437 19 Jan 2018

DESCRIFHON OF THE DRAWINGS

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

FIGURE 1 is a diagram illustrating the genomic structure of human MASP-2; FIGURE 2A is a schematic diagram illustrating the domain structure of human

MASP-2 protein;

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

MApl9 protein:

FIGURE 3 is a diagram illustrating the murine MASP-2 knockout strategy; FIGURE 4 is a diagram illustrating the human MASP-2 minigene construct; FIGURE 5A presents results demonstrating that MASP-2-deficiency leads to the loss of lectin-pathway-mediated C4 activation as measured by lack of C4b deposition on mannan, as described in Example 2;

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

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

FIGURE 6 presents results demonstrating that the addition of murine recombinant MASP-2 io MASP-2-/- serum samples recovers lectin-path way-mediated C4 activation in a protein concentration dependant manner, as measured by C4b deposition on mannan, as described in Example 2;

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

FIGURE 8A presents results demonstrating that anti-MASP-2 Fab2 antibody #11 inhibits C3 convertase formation, as described in Example 10;

FIGURE SB presents results demonstrating that anti-MASP-2 Fab2 antibody #11 binds to native rat MASP-2, as described in Example 10;

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

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

FIGURE 10 is a diagram illustrating the recombinant polypeptides derived from rat MASP-2 that were used for epitope mapping of the anti-MASP-2 blocking Fab2 antibodies, as described in Example 11;

FIGURE 1Ϊ presents results demonstrating the binding of anti-MASP-2 Fab2 #40 and #60 to rat MASP-2 polypeptides, as described in Example 1 1;

FIGURE 12 presents results demonstrating die blood urea nitrogen clearance for wild type (+/+) and MASP-2 (-/-) mice at 24 and 48 hours after reperfusion in a renal ischemia/reperfusion injury model, as described in Example 12;

FIGURE 13A presents results showing the baseline VEGF protein levels in

RPE-choroid complex isolated from wild type (+/+) and MASP-2 (-/-) mice, as described in Example 13;

FIGURE I3B presents resuits showing the VEGF protein levels in RPE-choroid complex at day 3 in wild type (+/+) and MASP-2 (-/-) mice following laser induced injury in a macular degeneration model, as described in Example 13;

FIGURE 14 presents results showing the mean choroidal neovascularization (CNV) volume at day seven following laser induced injury in wild type (+/+) and MASP-2 (-/-) mice, as described in Example 13;

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

FIGURES J6A and 16B present measured platelet aggregation (expressed as aggregate area) in MASP-2 (-/-) mice (FIG, 16B) as compared to platelet aggregation in • untreated wild type mice and wild type mice in which the complement pathway is inhibited by depletory agent cobra venom factor (CVF) and a terminal pathway inhibitor (C5aR antagonist) (FIGURE 16A) in a localized Schwartzman reaction model of disseminated intravascular coagulation, as described in Example 15;

-142018200437 19 Jan 2018

FIGURE 17 graphically illustrates the blood urea nitrogen (BUN) levels measured in either WT (+/+) (B6) or MASP-2 (-/-) transplant recipient mice of WT (+/+) donor kidneys, as described in Example 16;

FIGURE 18 graphically illustrates the percentage survival of WT (+/+) and 5 MASP-2 (-/-) mice as a function of the number of days after microbial infection in the cecal ligation and puncture (CLP) model, as described in Example 17;

FIGURE 19 graphically illustrates the number of bacteria measured in WT (+/+) and MASP-2 (-/-) after microbial infection in the cecal ligation and puncture (CLP) model, as described in Example 17;

FIGURE 20 is a Kaplan-Mayer plot illustrating the percent survival of WT (+/+),

MASP-2 (-/-) and C3 (-/-) mice six days after challenge with intranasai administration of Pseudomonas aeruginosa, as described in Example 18;

FIGURE 21 graphically illustrates the level of C4b deposition, measured as % of control, in samples taken at various time points after subcutaneous dosing of either 0.3 mg/kg or 1.0 mg/kg of mouse anti-MASP-2 monoclonal antibody in WT mice, as described in Example 19;

FIGURE; 22 graphically illustrates the level of C4b deposition, measured as % of control, in samples taken at various time points after ip dosing of 0.6 mg/kg of mouse anti-MASP-2 monoclonal antibody in WT mice, as described in Example 19;

FIGURE 23 graphically illustrates the mean choroidal neovascularization (CNV) volume at day seven following laser induced injury in WT (+/+) mice pre-treated with a single ip injection of 0.3 mg/kg or 1.0 mg/kg mouse anti-MASP-2 monoclonal antibody; as described in Example 20;

FIGURE 24A graphically illustrates the percent survival of MASP-2 (-/-) and WT (+/+) mice after infection with 5xlO⁸/iOO μΐ efu ΛΙ meningitidis, as described in

Example 21;

FIGURE 24B graphically illustrates the Jog efu/mi of .V. meningitidis recovered at different time points in blood samples taken from the MASP-2 KO (-/-) and WT (+/+) mice infected with 5x10⁸ cfu/l 00 μΐ N. meningitidis, as described in Example 21;

FIGURE 25A graphically illustrates the percent survival of MASP-2 KO (-/-) and

WT (+/+) mice after infection with 2x10⁸ cfu/l00 μΐ N. meningitidis, as described in Example 21;

-152018200437 19 Jan 2018

FIGURE 25B graphically illustrates the log cfu/ml of IV. meningitidis recovered at different time points in blood samples taken tram the WT (+/+) mice infected with 2x1 ()⁸ cfu/100 μΐ N. meningitidis, as described in Example 21;

FIGURE 25C graphically illustrates the log cfu/ml of N. meningitidis recovered at 5 different time points in blood samples taken from the MASP-2 (-/-) mice infected with

2x10⁸ cfu/100 μΐ /V. meningitidis, as described in Example 21;

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

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

FIGURE 27A graphically illustrates myocardial ischemia/reperfusion injury (MIRJ)-induced tissue loss following ligation of the left anterior descending branch of the coronary artery (LAD) and reperfusion in C4 (-/-) mice (n==6) and matching WT littermate controls (n=7), showing area at risk (AAR) and infarct size (INF) as described in Example 22;

FIGURE 27B graphically illustrates infarct size (INF) as a function of area at risk (AAR) in C4 (-/-) and WT mice treated as describe in FIGURE 42A, demonstrating that

C4 (-/-) mice are as susceptible to M1R1 as WT controls (dashed line), as described in Example 22;

FIGURE 28A graphically illustrates the results of a C3b deposition assay using serum from WT mice, C4 (-/-) mice and serum from C4 (-/-) mice pre-incubated with mannan, as described in Example 22;

FIGURE· 28B graphically illustrates the results of a C3b deposition assay on serum from WT, C4 (-/-), and MASP-2 (-/-) mice mixed with various concentrations of an anti-murine MASP-2 mAb (mAbMll), as described in Example 22;

FIGURE 28C graphically illustrates the results of a C3b deposition assay on human serum from WT (C4 sufficient) and C4 deficient serum, and serum from C4 deficient subjects pre-incubated with mannan, as described in Example 22;

FIGURE 28D graphically illustrates the results of a C3b deposition assay on human serum from WT (C4 sufficient) and C4 deficient subjects mixed with anti-human MASP-2 mAb (mAbH3), as described in Example 22;

-162018200437 19 Jan 2018

FIGURE 29A graphically illustrates a comparative analysis of C3 convertase activity in plasma from various complement deficient mouse strains tested either under lectin activation pathway specific assay conditions, or under classical activation pathway specific assay conditions, as described in Example 22;

FIGURE 29B graphically illustrates the time-resolved kinetics of C3 convertase activity in plasma from various complement deficient mouse strains tested under lectin activation pathway specific conditions, as described in Example 22;

FIGURE 30 illustrates the results of a Western blot analysis showing activation of human C3, shown by the presence of the a’ chain, by thrombin substrates FXIa and FXa, as described in Example 23;

FIGURE 31 shows the results of the C3 deposition assay on serum samples obtained from WT, MASP-2 (-/-), FI 1(-/-), FI 1 (-/-)/C4 (-/-) and C4 (-/-), as described in Example 23;

FIGURE 32A is a Kaplain-Meier survival plot showing the percent survival over 15 time after exposure to 7.0 Gy radiation in control mice and in mice treated with antimurine MASP-2 antibody (mAbMll) or anti-human MASP-2 antibody (mAbH6) as described in Example 29;

FIGURE 32B is a Kaplain-Meier survival plot showing the percent survival over time after exposure to 6.5 Gy radiation in control mice and in mice treated with anti20 murine MASP-2 antibody (mAbMll) or anti-human MASP-2 antibody (mAbH6), as described in Example 29;

FIGURE 33 is a Kaplan-Meyer plot graphically illustrating the percent survival of MASP-2 KO and WT mice after administration of an infective dose of 2.6 χ 10⁷ cfu of N. meningitidis serogroup A 22491, demonstrating that MASP-2 deficient mice arc protected from N. meningitidis induced mortality, as described in Example 30;

FIGURE 34 is a Kaplan-Meyer plot graphically illustrating the percent survival of MASP-2 KO and WT mice after administration of an infective dose of 6 χ 10⁶ cfu of A. meningitidis serogroup B strain MC58, demonstrating that MASP-2-deficient mice are protected from Al meningitidis serogroup B strain MC58 induced mortality, as described in Example 30;

FIGURE 35 graphically illustrates the log cfu/ml of N. meningitidis serogroup B strain MC58 recovered at different time points in blood samples taken from the MASP-2 KO and WT mice after i.p. infection with 6x10⁶ cfu of Al meningitidis serogroup B strain

2018200437 19 Jan 2018

MC58 (n=3 at different time points for both groups of mice, results are expressed as Means±SEM) demonstrating that although the MASP-2 KO mice were infected with the same dose of A meningitidis serogroup B strain MC58 as the WT mice, the MASP-2 KO mice have enhanced clearance of bacteraemia as compared to WT, as described in

Example 30;

FIGURE 36 graphically illustrates the average illness score of MASP-2 and WT mice at 3, 6, 12 and 24 hours after infection with 6xI0⁶ cfu/100 μί N. meningitidis Serogroup Serogroup B strain MC58, demonstrating that the MASP-2 deficient mice showed high resistance to the infection, with much lower illness scores at 6 hours, as described in Example 30;

FIGURE 37 is a Kaplan-Meyer plot graphically Illustrating the percent survival of mice after administration of an infective dose of 4 χ 10⁶/100 μί efu N. meningitidis Serogroup B strain MC58, followed by administration 3 hours post infection of either inhibitory anti-MASP-2 antibody (1 mg/kg) or control isotype antibody, demonstrating that anti-MASP-2 antibody is effective to treat and improve survival in subjects infected with N, meningitidis, as described in Example 31;

FIGURE 38 graphically illustrates the log cfu/ml of viable counts of A, meningitidis serogroup B-MC58 recovered at different time points in 20% human serum concentration after i.p. infection with 6,5x10⁶ cfu/100 μ] A'. meningitidis serogroup B strain MC58 at 0, 30, 60 and 90 minutes after incubation in the presence of: (A) normal human serum (NHS) plus human anti-MASP-2 antibody; (B) normal human serum (NHS) plus isotype control antibody; (C) MBL-/- human serum; (D) normal human serum (NHS) and (E) heat inactivated normal human serum (NHS), showing that complement dependent killing of A, meningitidis in human serum was significantly enhanced by the addition of the human anti-MASP-2 antibody, as described in Example 32;

FIGURE 39 graphically illustrates the log cfu/ml of viable counts of N meningitidis serogroup B-MC58 recovered at different time points In the mouse sera samples, demonstrating MASP-2 -/- mouse sera has a higher level of bactericidal activity for N. meningitidis than WT mouse sera, as described in Example 32;

FIGURE 40 graphically illustrates hemolysis (as measured by hemoglobin release of lysed mouse erythrocytes (Crry/C3-/-) into the supernatant measured by photometry)

-182018200437 19 Jan 2018 of mannan-coated murine erythrocytes by human serum over a range of serum concentrations The sera tested included heat-inactivated (Hl) NHS, MBL-/-, NHS +antiMASP-2 antibody and NHS control, as described in Example 33;

FIGURE 41 graphically illustrates hemolysis (as measured by hemoglobin release 5 of lysed WT mouse erythrocytes into the supernatant measured by photometry) of noncoated murine erythrocytes by human serum over a range of serum concentrations. The sera tested included heat-inactivated (HI) NHS, MBL-/-, NHS +anti-MASP-2 antibody and NHS control, demonstrating that inhibiting MASP-2 inhibits complement-mediated lysis of non-sensitized WT mouse erythrocytes, as described in Example 33;

FIGURE 42 graphically illustrates hemolysis (as measured by hemoglobin release of lysed mouse erythrocytes (CD55/59 -/-) into the supernatant measured by photometry) of non-coated murine erythrocytes by human serum over a range of serum concentrations. The sera tested included heat-inactivated (HS) NHS, MBL-/-, NHS +anti-MASP-2 antibody and NHS control, as described in Example 33;

FIGURE. 43 graphically illustrates the percent survival over time (days) after exposure to 8,0 Gy radiation in control mice and in mice treated with anti-human MASP2 antibody (mAbH6), as described in Example. 34;

FIGURE 44 graphically illustrates the time to onset of mierovaseuiar occlusion following LPS injection in MASP-2 -/- and WT mice, showing the percentage of mice with thrombus formation measured over 60 minutes, demonstrating that thrombus formation is detected after 15 minutes in WT mice, with up to 80% of the WT mice demonatrated thrombus formation at 60 minutes; in contrast, none of the MASP-2 -/mice showed any thrombus formation during the 60 minute period (log rank: p-0.0005), as described in Example 35; and

FIGURE 45 graphically illustrates the percent survival of saline treated control mice (n=5) and anti-MASP-2 antibody treated mice (n=5) in the STX/LPS-induced model of HUS over time (hours), demonstrating that all of the control mice died by 42 hours, whereas, in contrast, 100 % of the anti-MASP-2 antibody-treated mice survived throughout the time course ofthe experiment, as described in Example 36,

-192018200437 19 Jan 2018

DESCRIPTION OF THE SEQUENCE LISTING SEQ SD NO: I human MApl9 cDNA SEQ ID NO:2 human MApS9 protein (with leader)

SEQ ID NOG human MApl9 protein (mature)

SEQ ID NO:4 human MASP-2 cDNA

SEQ ID NOG human MASP-2 protein (with leader)

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

SEQ ID NO:7 human MASP-2 gDNA (exons 1-6)

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

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

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

SEQ ID NO:IO CUBEGFCUBII (aa 1-293)

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

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

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

SEQ ID NO: 14 TPLGPKWPEPVFGRL (CUBI peptide)

SEQ ID NO: 15

TAPPGYRLRLYFTHFDLELSHLCEYDFVKLSSGAKVLATLC

GQ (CUBI peptide)

SEQ ID NO: 16 TFRSDYSN (MBL binding region core)

SEQ ID NO: 17 FYSLGSSLD1TFRSDYSNEKPFTGF (MBL binding region) SEQ ID NO: 18 1DECQVAPG (EGF PEPTIDE)

SEQ ID NO: 19 ANMLCAGLESGGKDSCRGDSGGALV (serine protease binding core)Detai!ed Description

PEPTIDE INHIBITORS:

SEQ ID NO:20 MBL full length cDNA SEQ ID NO:21 MBL full length protein

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

SEQ ID NOGS OGKLG

SEQ ID NO:24 GLR GLQ GPO GKL GPO G

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

-202018200437 19 Jan 2018

SEQ ID NO:26 GKDGRDGTK.GEKGEPGQGLRGLQGPOGKLGPOG SEQ ID NO:27 GAOGSOGEKGAOGPQGPOGPOGKMGPKGEOGDO (human h-ficolin)

SEQ ID NO:28

GCOGLOGAOGDKGEAGTNGKRGERGPOGPQGKAGPOGPN

GAOGEO (human ficolin p35)

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

SEQ ID NO:30 cDNA of CUB1-EGF domain (nucleotides 22-680 of SEQ

ID NO :4)

SEQ iD NO:31

5’ CGGGCACACCATGAGGCTGCTGACCCTi t 1GGGC 3' Nucleotides 12-45 of SEQ ID NO:4 including the MASP-2 translation start site (sense)

SEQIDNO;32

5OACATTACCTTCCGCTCCGACTCCAACGAGAAG3' Nucleotides 361-396 of SEQ ID NO:4 encoding a region comprising the MASP-2 MBL binding site (sense)

SEQ ID NO:33

5’AGCAGCCCTGAATACCCACGGCCGTATCCCAAA3’

Nucleotides 610-642 of SEQ ID NO:4 encoding a region comprising the CIJB1I domain

CLONING PRIMERS;

SEQ ID NO:34 CGGGATCCATGAGGCTGCTGACCCTC (5' PCR for

CUB)

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

SEQ ID NO:36 GGAATTCCTA.CAGGGCGCT (3' PCR FOR CUBIEGF) SEQ ID NO:37 GGAATTCCTAGTAGTGGAT (3' PCR FOR

CUBIEGFCUBII)

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

SEQ ID NO:48 is 9 aa peptide bond

EXPRESSION VECTOR:

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

-212018200437 19 Jan 2018

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

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

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

SEQ ID NO: 53 the rat MASP-2 cDNA 5 SEQ ID NO: 54 is the rat MASP-2 protein (w/ leader)

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

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

SEQ ID NO: 60-63 are the oligonucleotides for site-directed mutagenesis 10 of murine MASP-2 used to generate murine MASP-2 A

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

DETAILED DESCRIPTION

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

I. DEFINITIONS

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

As used herein, the term “MASP-2-dependent complement activation” comprises MASP-2- dependent activation of the lectin pathway, which occurs under physiological conditions (i.e., in the presence of Ca⁴*) leading to the formation of the lectin pathway C3 convertase C4b2a and upon accumulation of the C3 cleavage product C3b subsequently to the C5 convertase C4b2a(C3b)n, which has been determined to primarily cause opsonization.

As used herein, the term ''alternative pathway refers to complement activation that is triggered, for example, by zymosan from fungal and yeast cell walls,

-222018200437 19 Jan 2018 lipopolysaccharide (LPS) from Gram negative outer membranes, and rabbit erythrocytes, as well as from many pure polysaccharides, rabbit erythrocytes, viruses, bacteria, animal tumor ceils, parasites and damaged ceils, and which has traditionally been thought to arise from spontaneous proteolytic generation of C3b from complement factor C3.

As used herein, the term lectin pathway refers to complement activation that occurs via the specific· binding of serum and non-serum carbohydrate-binding proteins including mannan-binding lectin (MBL), CL-11 and the fscolins (H-fscolin, M-ficolin, or L-fieolin).

As used herein, the term classical pathway refers to complement activation that 10 is triggered by antibody bound to a foreign particle and requires binding of the recognition molecule Clq.

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

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-2, polypeptides or portions thereof. It is not intended that the term “antibody” limited as regards to the source of the antibody or the manner in which it is made (e.g., by hybridoma, phage selection, recombinant expression, transgenic animal, peptide synthesis, etc), Exemplary antibodies include polyclonal, monoclonal and recombinant

-232018200437 19 Jan 2018 antibodies; pan-specific, muldspeeific antibodies (e.g., bispecific antibodies, irispecific antibodies); humanized antibodies; murine antibodies; chimeric, mouse-human, mouse-primate, primate-human monoclonal antibodies; and anti-idiotype antibodies, and may be any intact antibody or fragment thereof. As used herein, the term “antibody” encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as dAb, Fab, Fab¹, F(ab')2, Fv), 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, and any other modified configuration of the immunoglobulin molecule that iO comprises an antigen-binding site or fragment (epitope recognition site) of the required specificity.

A “monoclonal antibody refers to a homogeneous antibody population wherein the monoclonal antibody is comprised of amino acids (naturally occurring and nonnaturaliy 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 antigenbinding fragment (epitope recognition site) of the required specificity and the ability to bind to an epitope. It is not intended to be limited as regards the source of the antibody or the manner in which it is made (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term includes whole immunoglobulins as well as the fragments etc. described above under the definition of antibody.

As used herein, the term antibody fragment” refers to a portion derived from osrelated to a full-length antibody, such as, for example, an anti-MASP-2 antibody, generally including the antigen binding or variable region thereof. Illustrative examples of antibody fragments inciude. Fab, Fab', F(ab)2, F(ab')2 and Fv fragments, scFv fragments, diabodies, linear antibodies, sirsgle-cbain antibody molecules and multispecific antibodies formed from antibody fragments.

-242018200437 19 Jan 2018

As used herein, a single-chain Fv or scFv antibody fragment comprises the Vjj and Vp 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 and Vp domains, which enables the scFv to form the desired structure for antigen binding.

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

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

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

As used herein, the membrane attack complex (MAC) refers to a complex of the terminal five complement components (C5b combined with C6, C7, C8 and C-9) that inserts into and disrupts membranes (also referred to as CSb-9), .As used herein, a subject includes ail mammals, including without limitation humans, non-human primates, dogs, cats, horses, sheep, goats, cows, rabbits, pigs and rodents.

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

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

-252018200437 19 Jan 2018 follows. By uncharged hydrophilic amino acid is meant either Ser, Thr, Asn or Gin.

By acidic amino acid is meant either Glu or Asp. By basic amino acid is meant either

Lys, Arg or His.

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

The term oligonucleotide as used herein refers to an oligomer or polymer of 10 ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof, This term also covers those oligonucleobases composed of naturally-occurring nucleotides, sugars and covalent inlemucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring modifications.

As used herein, an ''epitope refers to the site on a protein (e.g., a human MASP-2 15 protein) that is bound by an antibody. Overlapping epitopes include at least one (e.g., two, three, four, five, or six) common amino acid residue(s), including linear and nonlinear epitopes.

As used herein, the 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-2 protein described herein can contain or be wild-type proteins or can be variants that have not more than 50 (e.g., not more than one, two, three, four, five, six, seven, eight, nine, ten, 12, 15, 20, 25, 30, 35, 40, or 50) conservative amino acid substitutions. Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine and threonine; lysine, histidine and arginine; and phenylalanine and tyrosine.

In some embodiments, the human MASP-2 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-2 protein having the amino acid sequence set forth in SEQ ID NO: 5.

In some embodiments, peptide fragments can be at least 6 (e.g., at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90,

-26»

95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, or 600 or more) amino acid residues in length (e.g., at least 6 contiguous amino acid residues of SEQ ID NO: 5). In some embodiments, an antigenic peptide fragment of a

2018200437 19 Jan 2018 human MASP-2 protein is fewer than 500 (e.g., fewer than 450, 400, 350, 325, 300, 275, 250, 225, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6) amino acid residues in length (e.g., fewer than 500 contiguous amino acid residues in any one of SEQ ID NOS: 5).

Percent (%) amino acid sequence identity is defined as the percentage of amino acids in a candidate sequence that are identical to the amino acids in a reference sequence, after aligning the sequences and introducing gaps, if necessary, io achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, 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 maximal alignment over the fulllength of the sequences being compared can be determined by known methods.

II. Overview of the Invention

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

in addition to its essential role in immune defense, the complement system contributes to tissue damage in many clinical conditions. Thus, there is a pressing need to develop therapeutically effective complement inhibitors to prevent these adverse effects. With the recognition that it is possible to inhibit the lectin mediated MASP-2

-272018200437 19 Jan 2018 pathway while leaving the classical pathway intact comes the realization that it would be highly desirable to specifically inhibit only the complement activation system causing a particular pathology without completely shutting down the immune defense capabilities of complement. For example, in disease states in which complement activation is mediated predominantly by the lectin-dependent complement system, it would be advantageous to specifically inhibit only this system. This would leave the C1 q-dependent complement activation system intact to handle immune complex processing and to aid in host defense against infection.

The preferred protein component to target in the development of therapeutic agents to specifically inhibit the lectin-dependent complement system is MASP-2. Of all the known protein components of the lectin-dependent complement system (MBL, H-ficolin, M-ficolin, L-ficoiin, MASP-2, C2-C9, Factor B, Factor D, and properdin), only MASP-2 is both unique to the lectin-dependent complement system and required for the system to function. The lectins (MBL, H-ficolin, M-ficolin,L-ficoiin and CL-1I) are also unique components in the lectin-dependent complement system. However, loss of any one of the lectin components would not necessarily inhibit activation of the system due to lectin redundancy. It would be necessary to inhibit all five lectins in order to guarantee inhibition of the lectin-dependent complement activation system. Furthermore, since MBL and the ficolins are also known to have opsonic activity independent of complement, inhibition of lectin function would result in the loss of this beneficial hostdefense mechanism against infection. In contrast, this complement-independent lectin opsonic activity would remain intact if MASP-2 was the inhibitory target. An added benefit of MASP-2 as the therapeutic target to inhibit the lectin-dependent complement activation system is that the plasma concentration of MASP-2 is among the lowest of any complement protein (~ 500 ng/ml); therefore, correspondingly low concentrations of high-affinity inhibitors of MASP-2 may be sufficient to obtain full inhibition (Moller-Kristensen, M., et at, J. Immunol Methods 282; 159-167, 2003).

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I1L ROLE OF MASP-2 IN VARIOUS DISEASES AND CONDITIONS AND THERAPEUTIC METHODS USING MASP-2 INHIBITORY AGENTS

RENAL CONDITIONS

Activation of the complement system has been implicated in the pathogenesis of a wide variety of renal diseases; including, mesangioproliferative glomerulonephritis (IgA-nephropathy, Berger's disease) (Endo, M., etal., Clin. Nephrology 55:185-191, 2001), membranous glomerulonephritis (Keijashki, D., Arch B Cell Pathol. 55:253-71, 1990; Brenchiey, P.E., etal.. Kidney Int., 41:933-7, 1992; Salant, D.J., etal., Kidney /«1,35:976-84, 1989), membranoproliferative glomerulonephritis (mesangiocapillary glomerulonephritis) (Bartlow, B.G., et al., Kidney Int. 75:294-300, 1979; Meri, S., et a!., ,/. Exp. Med. /75:939-50, 1992), acute postinfectious glomerulonephritis (poststreptococcal glomerulonephritis), cryoglobulinemic glomerulonephritis (Ohsawa, L, etal., Clin Immunol. 101:59-66, 2001), lupus nephritis (Gatenby, P.A.,

Autoimmunity /7:61-6, 1991), and Henoch-Schonlein purpura nephritis (Endo, M., et al., Am, J, Kidney Dis. 35:401-407, 2000), The involvement, of complement in renal disease has been appreciated for several decades but there is still a major discussion on its exact role in the onset, the development and the resolution phase of renal disease. Under normal conditions the contribution of complement is beneficial to the host, but inappropriate activation and deposition of complement may contribute to tissue damage.

There is substantial evidence that glomerulonephritis, inflammation of the glomeruli, is often initiated by deposition of immune complexes onto glomerular or tubular structures which then triggers complement activation, inflammation and tissue damage. Kahn and Sinniah demonstrated increased deposition of C5b-9 in tubular basement membranes in biopsies taken from patients with various forms of glomerulonephritis (Kahn. T.N., et ah, ffistopalh. 26:351-6, 1995). In a study of patients with IgA nephrology (Alexopoulos, A., etal, Nephrol. Dial. Transplant 76:1166-1172, 1995), C5b-9 deposition in the tubular epithelial/basement membrane structures correlated with plasma creatinine levels. Another study of membranous nephropathy demonstrated a relationship between clinical outcome and urinary sC5b-9 levels (Kon, S.P., etal., Kidney Ini. 45:1953-58, 1995). Elevated sC5b-9 levels were correlated positively with poor prognosis. Lchto et al,, measured elevated levels of CD59, a complement regulatory factor that inhibits the membrane attack complex in plasma

-292018200437 19 Jan 2018 membranes, as well as C5b-9 in urine from patients with membranous glomerulonephritis (Lehto, T., etal., Kidney Int. 47:1403-11, 1995), Histopathological analysis of biopsy samples taken from these same patients demonstrated deposition of C3 and C9 proteins in the glomeruli, whereas expression of CD59 in these tissues was diminished compared to that of normal kidney tissue. These various studies suggest that ongoing complement-mediated glomerulonephritis results in urinary excretion of complement proteins that correlate with the degree of tissue damage and disease prognosis.

inhibition of complement activation in various animal models of glomerulonephritis has also demonstrated the importance of complement activation in the etiology of the disease, in a model of membranoproliterative glomerulonephritis (MPGN), infusion of anti-Thyl antiserum in C6-deficienl rats (that cannot form C5b-9) resulted in 90% less glomerular cellular proliferation, 80% reduction in platelet and macrophage infiltration, diminished collagen type IV synthesis (a marker for mesangial matrix expansion), and 50% less proteinuria than in C6+ normal rats (Brandt, J., etal.,

Kidney Ini. 49:335-343, 1996). These results Implicate C5b-9 as a major mediator of tissue damage by complement in this rat anti-thymocyte serum model, in another model of glomerulonephritis, infusion of graded dosages of rabbit anti-rat glomerular basement membrane produced a dose-dependent influx of polymorphonuclear leukocytes (PMN) that was attenuated by prior treatment with cobra venom factor (to consume complement) (Scandretf, AX., etal., Am. J. Physiol. 268:F256~F265, 1995). Cobra venom factor-treated rats also showed diminished histopathology, decreased long-term proteinuria, and lower creatinine levels than control rats. Employing three models of GN in rats (anti-thymocyte serum, Con A anti-Con A, and passive Heymann nephritis), Couser et al., demonstrated the potential therapeutic efficacy of approaches to inhibit complement by using the recombinant sCRl protein (Couser, W.G., etal., J. Am. Soc. Nephrol, 5:1888-94, 1995). Rats treated with sCRl showed significantly diminished PMN, platelet and macrophage influx, decreased mesangiolysss, and proteinuria versus control rats. Further evidence for the importance of complement activation in glomerulonephritis has been provided by the use of an anti-C5 MoAb in the NZB/W FI mouse model. The anti-C5 MoAb inhibits cleavage of C5, thus blocking generation of C5a and C5b~9, Continuous therapy with anti-C5 MoAb for 6 months resulted in significant amelioration of the course of glomerulonephritis. A humanized anti-C5 MoAb monoclonal antibody (5G1.1) that prevents the cleavage of human complement

-302018200437 19 Jan 2018 component C5 into its pro-inflammatory components is under development by Alexion Pharmaceuticals, Inc., New Haven, Connecticut, as a potential treatment for glomerulonephritis.

Direct evidence for a pathological role of complement in renal injury is provided 5 by studies of patients with genetic deficiencies in specific complement components. A number of reports have documented an association of renal disease with deficiencies of complement regulatory factor H (Ault, B.H., Nephrol. /4:1045-1053, 2000; Levy, M., et al., Kidney Ini: 3(5:949-56, 1986; Pickering, M.C., etal., Nat. Genet. 3/:424-8, 2002). Factor H deficiency results in low plasma levels of factor B and C3 and in consumption of C5b-9. Both atypical membranoproliferative glomerulonephritis (MPGN) and idiopathic hemolytic uremic syndrome (HUS) are associated with factor H deficiency. Factor H deficient pigs (Jansen, J.H., et al., Kidney Ini. 55:331-49, 1998) and factor H knockout mice (Pickering, M.C., 2002) display MPGN-like symptoms, confirming the importance of factor H in complement regulation. Deficiencies of other complement components are associated with renal disease, secondary to the development of systemic lupus erythematosus (SLE) (Walport, M.J., Davies, et al., Ann. N.Y. Acad. 5c7, 57.5:267-81, 1997). Deficiency for Clq, C4 and C2 predispose strongly to the development of SLE via mechanisms relating to defective clearance of immune complexes and apoptotic material. In many of these SLE patients lupus nephritis occurs, characterized by the deposition of immune complexes throughout the glomerulus.

Further evidence linking complement activation and renal disease has been provided by the identification in patients of autoants bodies directed against complement components, some of which have been directly related to renal disease (Trouw, L.A., et al., Moi. Immunol. 55:199-206, 2001), .A number of these autoantibodies show such a high degree of correlation with renal disease that the term nephritic factor (NeF) was introduced to indicate this activity. In clinical studies, about 50% of the patients positive for nephritic factors developed MPGN (Spitzer, R.E„ et al., Clin. Immunol. Immunopathoi. 64:177-83, 1992). C3NeF is an autoantibody directed against the alternative pathway C3 convertase (C3bBb) and it stabilizes this convertase, thereby promoting alternative pathway activation (Daha, M.R., et ah, J. Immunol. //6:1-7, 1976). Likewise, autoantibody with a specificity for the classical pathway C3 convertase (C4b2a), called C4NeF, stabilizes this convertase and thereby promotes classical pathway activation (Daha, M.R. etal., J. Immunol, /25:2051-2054, 1980; Halbwachs, L,, et al,,

-312018200437 19 Jan 2018

J, Clin. Invest. 65:1249-56, 1980). Anti-Clq autoantibodies have been described to be related to nephritis in SLE patients (Hovath, L., etal., Clin. Exp. Rheumatol. /9:667-72,

2001; Siegert, C., etal., J. Rheumatol. 18:230-34, 1991; Siegert, C., etal., Clin. Exp.

Rheumatol. /0:19-23, 1992), and a rise in the titer of these anti-Clq autoantibodies was reported to predict a flare of nephritis (Coremans, I.E,, et al., Am. J Kidney Dis. 26:595-601, 1995). Immune deposits eluted from postmortem kidneys of SLE patients revealed the accumulation of these anti-Clq autoantibodies (Mannick, M., et ak, Arthritis Rheumatol. 40:1504-11, 1997). .All these facts point to a pathological role for these autoantihodies. However, not all patients with anti-Clq autoantibodies develop renal disease and also some healthy individuals have low titer anti-Clq autoantibodies (Siegert, C.E,, et ak, Clin. Immunol. Immunopathol. 67:204-9, 1993).

In addition to the alternative and classical pathways of complement activation, the lectin pathway may also have an important pathological role in renal disease. Elevated levels of MBL, MBL-associated serine protease and complement activation products have been detected by immunohistochemical techniques on renal biopsy material obtained from patients diagnosed with several different renal diseases, including Henoch-Schonlein purpura nephritis (Endo, M., etal., Am. J. Kidney Dis. 35:401-407, 2000), cryoglobulinemic glomenikmephritis (Ohsawa, I., etai., Clin. Immunol. 101:59-66, 2001) and IgA neuropathy (Endo, M„ etal., Clin.

Nephrology 55:185-191, 2001). Therefore, despite the fact that an association between complement and renal diseases has been known tor several decades, data on how complement exactly influences these renal diseases is far from complete.

One aspect of the invention is thus directed to the treatment of renal conditions including but not limited to mesangioproliferative glomerulonephritis, membranous glomerulonephritis, membranoproliferative glomerulonephritis (mesangiocaplllary glomerulonephritis), acute postinfeetious glomerulonephritis (poststreptococcal glomerulonephritis), cryoglobulinemic glomerulonephritis, lupus nephritis, Henoch-Schonlein purpura nephritis or IgA nephropathy, by administering a composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent in a pharmaceutical carrier to a subject suffering from such a disorder. The MASP-2 inhibitory' agent may be administered to the subject systemically, such as by intra-arterial, intravenous, intramuscular, subcutaneous or other parenteral administration, or potentially by oral administration for non-peptidergic agents. The MASP-2 inhibitory

-322018200437 19 Jan 2018 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 or following acute trauma or injury.

BLOOD DISORDERS

Sepsis is caused by an overwhelming reaction of the patient to invading microorganisms. A major function of the complement system is to orchestrate the inflammatory response to invading bacteria and other pathogens. Consistent with this physiological role, complement activation has been shown in numerous studies to have a major role in the pathogenesis of sepsis (Bone, R.C., Annals. Internal. Med. //5:457-469, 1991). The definition of the clinical manifestations of sepsis is ever evolving. Sepsis is usually defined as the systemic host response to an infection. However, on many occasions, no clinical evidence for infection (e.g., positive bacterial blood cultures) is found in patients with septic symptoms. This discrepancy was first taken into account at a Consensus Conference in 1992 when the term systemic inflammatory response syndrome (SIRS) was established, and for which no definable presence of bacterial infection was required (Bone, R.C., et ah, Crit. Care Med. 20:724-726, 1992). There is now general agreement that sepsis and SIRS are accompanied by the inability to regulate the inflammatory response. For the purposes of this brief review, we will consider the clinical definition of sepsis to also include severe sepsis, septic shock, and SIRS.

The predominant source of infection in septic patients before the late 1980s was

Oram-negative bacteria. Lipopolysaccharide (EPS), the main component of the Oram-negative bacterial ceil wall, was known to stimulate release of inflammatory mediators from various cell types and induce acute infectious symptoms when injected into animals (Haeney, M.R., etal., Antimicrobial Chemotherapy 4/(Suppl. A):41-6, 1998), Interestingly, the spectrum of responsible microorganisms appears to have shifted from predominantly Gram-negative bacteria In the late 1970s and 1980s to predominantly Gram-positive bacteria at present, for reasons that are currently unclear (Martin, G.S., et ah, A Eng. J. Med. 5/5:1546-54, 2003).

Many studies have shown the importance of complement activation in mediating inflammation and contributing to the features of shock, particularly septic and hemorrhagic shock. Both Gram-negative and Gram-positive organisms commonly precipitate septic shock. EPS is a potent activator of complement, predominantly via the

-332018200437 19 Jan 2018 alternative pathway, although classical pathway activation mediated by antibodies also occurs (Fearon, D.T., et al., M Engl. J. Med. 292:937-400, 1975). The major components of the Gram-positive cell wall are peptidoglycan and Hpoteichoic acid, and both components are potent activators of the alternative complement pathway, although in the presence of specific antibodies they can also activate the classical complement pathway (Joiner, K.A., et at, Ann. Rev. Immunol. 2:461-2, 1984).

The complement system was initially implicated in the pathogenesis of sepsis when it was noted by researchers that anaphyiatoxins C3a and C5a mediate a variety of inflammatory reactions that might also occur during sepsis. These anaphyiatoxins evoke vasodilation and an increase in microvascular permeability, events that play a central role hi septic shock (Schumacher, W.A., eta'., Agents Actions 34:345-349, 1991). In addition, the anaphyiatoxins induce bronchospasm, histamine release from mast cells, and aggregation of platelets. Moreover, they exert numerous effects on granulocytes, such as chemotaxis, aggregation, adhesion, release of lysosomal enzymes, generation of toxic

5 super oxide anion and formation of leukotrienes (Shin, H.S., et ah, Science /62:361 -363, 1968; Vogt, W., Complement 5:3 77-86, 1986). These biologic effects are thought to play a role in development of complications of sepsis such as shock or acute respiratory distress syndrome (ARDS) (Hammerschmidt, D.E., etai., Lancet 7:947-949, 1980; Siotman, G.T., etai., 5«rgery 99:744-50, 1986). Furthermore, elevated levels of the anaphyiatoxin C3a is associated with a fatal outcome in sepsis (Hack, C.E., et al., Am. J Med 86:20-26, 1989). In some animal models of shock, certain complement-deficient strains (e.g., C5-deficient ones) are more resistant to the effects of EPS infusions (Hseuh, W, et ah, Immunol. 70:309-14, 1990).

Blockade of C5a generation with antibodies during the onset of sepsis in rodents has been shown to greatly improve survival (Czerniak, B.J., etai., Nai, Med. 5:788-792, 1999). Similar findings were made when the C5a receptor (C5aR) was blocked, either with antibodies or with a small molecular inhibitor (Huber-Lang, M.S., et ah, FASEB J. 76:1567-74, 2002; Riedemann, N.C., et al., J. Clin. Invest. /70:101-8, 2002). Earlier experimental studies in monkeys have suggested that antibody blockade of C5a attenuated E. co/Mnduced septic shock and adult respiratory distress syndrome (Hangen, D.H., et al., J. Sung. Res. 46:195-9, 1989; Stevens, J.H., et ah, J. Clin. Invest. 77:1812-16, 1986), In humans with sepsis, C5a was elevated and associated with significantly reduced survival rates together with multiorgan failure, when compared with that in less

-342018200437 19 Jan 2018 severely septic patients and survivors (Nakae, H,, etak, Res. Commun. Chem. Pathol. Pharmacol. 54:189-95, 1994; Nakae, etah, Swg. Today 26:225-29, 1996; Bengtson, A., et al., Arch. Surg. /25:645-649, 1988). The mechanisms by which C5a exerts its harmful effects during sepsis are yet to be investigated in greater detail, but recent data suggest the generation of C5a during sepsis significantly compromises innate immune functions of blood neutrophils (Huber-Lang, M.S., et ah, J. Immunol. I69:3223-31, 2002), their ability to express a respiratory burst, and their ability to generate cytokines (Riedemann, N.C., et ah, Immunity /9:193-202, 2003). Jn addition, C5a generation during sepsis appears to have procoagulant effects (Laudes, I J., eta!., Am. J, Pathol. /66/1867-75, 2002), The complement-modulating protein Ci INH has also shown efficacy in animal models of sepsis and ARDS (Dickneite, G., Behring Ins. Mitt. 93:299-305, 1993).

The lectin pathway may also have a role in pathogenesis of sepsis, MBL has been shown to bind to a range of clinically important microorganisms including both Gram-negative and Gram-positive bacteria, and to activate the lectin pathway (Neth, 0., et ah, Infect. Immun. 65:688, 2000), Lipoteichoic acid (LTA) is increasingly regarded as the Gram-positive counterpart of LPS. It is a potent immunostimuiant that induces cytokine release from mononuclear phagocytes and whole blood (Morath, S,, et ah, J. Exp. Med. 195:1635, 2002; Morath, S„ eta!., Infect. Immun. 70:95%, 2002). Recently it was demonstrated that L-ficoiin specifically binds to LTA isolated from numerous

Gram-positive bacteria species, including Staphylococcus aureus, and activates the lectin pathway (Lynch, NJ., et ah,,/ Immunol. 172:} 198-02, 2004). MBL also has been shown to bind to LTA from Enterococcus spp in which the polyglycerophosphate chain is substituted with glycosyl groups), but not to LTA from nine other species including S. aureus (Polotsky, V.Y., et ah, Infect. Immun. 64:380, 1996).

An aspect of the invention thus provides a method for treating sepsis or a condition resulting from sepsis, by administering a composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent in a pharmaceutical carrier to a subject suffering from sepsis or a condition resulting from sepsis including without limitation severe sepsis, septic shock, acute respiratory distress syndrome resulting from sepsis, and systemic inflammatory response syndrome. Related methods are provided for the treatment of other blood disorders, including hemorrhagic shock, hemolytic anemia, autoimmune thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), or other

-352018200437 19 Jan 2018 marrow/blood destructive conditions, by administering a composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent in a pharmaceutical carrier to a subject suffering from such a condition, The MASP-2 inhibitory agent is administered to the subject systemically, such as by intra-arterial, intravenous, intramuscular, inhalational (particularly in the case of ARDS), subcutaneous or other parenteral administration, or potentially by oral administration for non-peptidergic agents, The MASP-2 inhibitory agent composition may be combined with one or more additional therapeutic agents to combat the sequelae of sepsis and/or shock. For advanced sepsis or shock or a distress condition resulting therefrom, the MASP-2 inhibitory composition may suitably be administered in a fast-acting dosage form, such as by intravenous or intra-arterial delivery of a bolus of a solution containing the MASP-2 inhibitory agent composition. Repeated administration may be carried out as determined by a physician until the condition has beers resolved.

THE ROLE OF MASP-2 IN PAROXYSMAL NOCTURNAL HEMOOLOBINUR1NA

AND THERAPEUTIC METHODS USING MASP-2 INHIBITORY AGENTS

OyaiYipy/ of PNH

Paroxysmal nocturnal hemoglobinuria (PNH), sometimes also referred to as Marchiafava-Micheli syndrome, is an acquired, potentially life-threatening disease ofthe blood. PNH may 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,” The majority of cases are primary PNH, PNH is characterized by complement-induced destruction of red blood cells (hemolysis), low red blood ceil counts (anemia), thrombosis and bone marrow failure. Laboratory findings in PNH show changes consistent with intravascular hemolytic anemia: low hemoglobin, raised lactate dehydrogenase, raised reticulocyte counts (immature red cells released by the bone marrow to replace the destroyed cells), raised bilirubin (a breakdown product of hemoglobin), in the absence of autoreactive RBC-binding antibodies as a possible cause,

The hallmark of PNH is the chronic complement-mediated hemolysis caused by the unregulated activation of terminal complement components, including the membrane attack complex, on the surface of circulating RBCs, PNH RBCs are subject to

-362018200437 19 Jan 2018 uncontrolled complement activation and hemolysis due to the absence of the complement regulators CD55 and CDS9 on their surface (Lindorfer, M.A., et al., Blood 115(11):228391 (2010), Risitano, et al., Mini-Reviews in Medicinal Chemistry, 11:528-535 (2011)).

CDS5 and CD59 are abundantly expressed on normal RBCs and control complement activation. CD55 acts as a negative regulator of the alternative pathway, inhibiting the assembly of the alternative pathway C3 convertase (C3bBb) complex and accelerating the decay of preformed convertase, thus blocking the formation of the membrane attack complex (MAC). CD59 inhibits the complement membrane attack complex directly by binding the C5b678 complex and preventing C9 from binding and polymerizing.

While hemolysis and anemia are the dominant clinical features of PNH, the disease is a complex hematologic disorder that further includes thrombosis and bone marrow failure as part of the clinical findings (Risitano et al, Mini Reviews in Med ChewA 1:528-535 (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 X15 linked gene encoding a glycosyl-phosphatidyl inositol (GPI) transferase required for the stable surface expression of GPI-anchored class A glycoproteins, including CD55 and CDS9. For reasons that are presently under investigation, hematopoietic stem ceils with a dysfunctional PIG A gene that arise as the result of spontaneous somatic mutations can undergo clonal expansion to the point where their progeny constitute a significant portion of the peripheral hematopoietic cell pool. While both erythrocyte and lymphocyte progeny of the mutant stem ceil clone lack CD55 and CD59, only the RBCs undergo overt lysis after they enter the circulation.

Current treatment for PNH includes blood transfusion for anemia, anticoagulation for thrombosis and the use of the monoclonal antibody eculizumab (Soiiris®'), which protects blood cells against immune destruction by inhibiting the complement system (Hilimen P. et al., N. Engl. J. Med. 350(6):552.-559 (2004)). Eculizumab (Soiiris®) is a humanized monoclonal antibody that targets the complement component C5, blocking its cleavage by C5 convertases, thereby preventing the production of C5a and the assembly

-372018200437 19 Jan 2018 of the MAC. Treatment of PNH patients with eculizumab has resulted in a reduction of intravascular hemolysis, as measured by lactate dehydrogenase (LDH), leading to hemoglobin stabilization and transfusion independence in about half of the patients (Risitano et al, Mini-Reviews in Medicinal Chemistry, 11(6) (2011)). While nearly all patients undergoing therapy with eculizumab achieve normal or almost normal LDH levels (due to control of intravascular hemolysis), only about one third of the patients reach a hemoglobin value about 11 gr/d’L, and the remaining patients ors eculizumab continue to exhibit moderate to severe (/.e.,transfusion-dependent) anemia, in about equal proportions (Risitano A.M. et ah, Blood 133:4094-100 (2009)). As described in Risitano et ah, Mini-Reviews in Medicinal Chemistty 11:528-535 (2011), it was demonstrated that PNH patients on eculizumab contained large amounts of C3 fragments bound to their PNH erythrocytes (while untreated patients did not). This finding led to the recognition that in eculizumab-ireated PNH patients, PNH RBCs that are no longer hemolyzed due to C5 blockade now can accumulate copious amounts of membrane-bound C3 fragments, which operate as opsonins, resulting In their entrapment in the reticuloendothelial ceils through specific C3 receptors and subsequent extravascular hemolysis. Thus, while preventing intravascular hemolysis and the resulting sequelae, eculizumab therapy simply diverts the disposition of these RBCs from intravascular to extravascular hemolysis, resulting in substantial residual untreated anemia in many patients (Risitano A.M. et ah,

Blood 113:4094-100 (2009)). Therefore, therapeutic strategies in addition to the use of eculizumab are needed for those patients developing Co-fragment mediated extravascular hemolysis, because they continue to require red cell transfusions. Such C3 fragment targeting approaches have demonstrated utility in experimental systems (Lindorfer et ah, Blood 115:2283-91,2010).

CqmpjepNtifrinitiating mechanisms in PNH

The causal link between defective expression of the negative complement regulators CD55 and CD59 in PNH, combined with the effectiveness of eculizumab in preventing intravascular hemolysis, clearly define PNH as a condition caused by the

-382018200437 19 Jan 2018 complement system. While this paradigm is widely accepted, the nature of the events initiating complement activation, and the complement activation pathway(s) involved remain unresolved. Because CDS5 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 terminal complement activation regardless of whether complement activation is initiated by the lectin pathway, by 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 RBC surface can trigger subsequent amplification and pathological hemolysis (intravascular and/or extravascular) and precipitate a hemolytic crisis. A. clear mechanistic understanding of the molecular events triggering hemolytic crisis in PNH patients has remained elusive. Because no complement initiating event is overtly evident in PNH patients undergoing a hemolytic crisis, the prevailing view is that complement activation in PNH may occur spontaneously owing to low level tick-over activation of the alternative pathway, which is subsequently magnified by inappropriate control of terminal complement activation due to lack of CD55 and CD59.

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

-392018200437 19 Jan ,__i MASP-2 inhibitors to block opsoruzgifett and extravascular hemolysis of PNH

O

CM RBCs through the reticuloendothelia! system

This section describes the inhibitory effects of MASP-2 inhibitory agents on hemolysis in an in vitro model of PNH. The findings support the utility of MASP-25 blocking agents (including, but not limited to, antibodies that bind to and block the function of MASP-2) to treat subjects suffering from aspects of PNH, and also the use of inhibitors of MASP-2 to ameliorate the effects of C3-fragment-mediated extravascular hemolysis in PNH patients undergoing therapy with a C5-inhibitor such as eculizumab.

As detailed above, PNH patients become anemic owing to two distinct 10 mechanisms of RBC clearance from 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, The intravascular hemolysis is largely prevented when a patient is treated with eculizumab. Because eculizumab blocks the 15 terminal lytic effector mechanism that occurs downstream of both the complementinitiating activation event as well as the ensuing opsonization, eculizumab does not block extravascular hemolysis (Risitano A.M. et ah, Blood 113:4094-100 (2009)), instead, RBCs that would have undergone hemolysis in untreated PNH patients now can accumulate activated C3b proteins on their surface, which augments uptake by the 20 reticuloendothelial system and enhances their extravascular hemolysis. Thus, eculizumab treatment effectively diverts RBC disposition from intravascular hemolysis to extravascular hemolysis. As a result, some eculizumab-treated PNH patients remain anemic. It follows that agents that block complement activation upstream and prevent the opsonization of PNH RBCs may be particularly suitable to block the extravascular 25 hemolysis not prevented by eculizumab.

The data presented here demonstrate that MASP-2 dependent complement activation is the dominant route for lectin-dependent opsonization. Therefore, MASP-2

-402018200437 19 Jan 2018 inhibitory agents are expected to be effective at limiting opsonization and inhibiting extravascular hemolysis in PNH.

Using an in vitro model of PNH, we demonstrated that complement activation and the resulting hemolysis in PNH are indeed initiated, at least in part, by MASP-2 dependent complement activation and that it is not an independent function of the alternative pathway. These studies used mannan-sensitized RBCs of various mouse strains, including RBCs from Crry-deficient mice (an important negative regulator ofthe terminal complement pathway In mice) as well as RBCs from CD55/CD59-deficient mice, which lack the same complement regulators that are absent in PNH patients),

When Mannan-sensitlzed Crry-deficient RBCs were exposed to complement-sufficient human scrum, the RBCs effectively hemolysed at a serum concentration of 3% (FIGURE 40) while complement-deficient serum (Hl: heat-inactivated) was not hemolytic. Remarkably, complement-sufficient serum in the presence of anti-MASP-2 antibody had reduced hemolytic activity, and 6% serum was needed for effective hemolysis (FIGURE

40). Similar observations were made when CD55/CD59-deficient RBCs were tested (FIGURE 42). Complement-sufficient human serum supplemented with anti-MASP-2 monoclonal antibody was about, two-fold less effective than untreated serum in supporting hemolysis. Furthermore, higher concentrations of serum treated with antiMASP-2 monoclonal antibody were needed to promote effective hemolysis of untreated

WT RBCs compared to untreated serum (FIGURE 40). Collectively, these data indicate that MASP-2 dependent complement activation contributes significantly to the hemolysis response. The data presented herein reveals the following pathogenic mechanisms tor anemia in PNH: intravascular hemolysis due to unregulated activation of terminal complement components and lysis of RBC by formation of MAC, and extravascular hemolysis caused by opsonization of RBCs by C3b, which is initiated predominately by MASP-2 dependent complement activation. Thus, MASP-2-inhibitory agents are expected to significantly reduce intravascular hemolysis in PNH patients.

-412018200437 19 Jan 2018

Extravascular hemolysis, a less dramatic, yet equally important mechanism of

RBC destruction that leads to anemia in PNH, is primarily the result of opsonization by

C3b. which is predominantly mediated by MASP-2 dependent complement activation.

Thus, MASP-2-inhibitory agents will preferentially block RBC opsonization and C3b and the ensuing extravascular hemolysis in PNH. This unique therapeutic activity of MASP2-inhibitory agents is expected to provide a significant treatment benefit to all PNH patients as no treatment currently exists for this pathogenic process.

The data presented herein detail two pathogenic mechanisms for RBC clearance 10 and anemia in PNH: the intravascular hemolysis initiated, at least in part, by MASP-2 dependent complement activation, and thus expected to be effectively inhibited by a MASP-2-inhibitory agent, and extravascular hemolysis due to C3b opsonization driven by MASP-2, and thus effectively prevented by a MASP-2-inhibitoiy agent.

It is well documented that both intravascular and extravascular mechanisms of 15 hemolysis lead to anemia in PNH patients (Risitano et al., Blood 113:4094-4100 (2009)). Therefore, in the setting of PNH, inhibition of MASP-2 would be expected to address both intravascular and extravascular hemolysis, providing a significant advantage oyer the C5 inhibitor eculizumab. Accordingly, it is expected that a MASP-2-blocking agent that inhibits intravascular hemolysis and prevents extravascular hemolysis is expected to be effective in preventing the degree of anemia that develops in PNH patients.

It is also known that C5-blocking agents (such as eculizumab) effectively block intravascular hemolysis but do not interfere with opsonization. This leaves anti-C5treated PNH patients with substantial residual anemia due to extravascular hemolysis mediated by MASP-2 dependent complement activation that remains untreated.

Therefore, it is expected that a C5-block!ng agent (such as eculizumab) that prevents intravascular hemolysis in combination with a MASP-2 inhibitory agent that prevents extravascular hemolysis will be more effective than either agent alone in preventing the anemia that develops in PNH patients. In fact, the combination of an anli-C5 and a

-422018200437 19 Jan 2018

MASP-2-inhibitory agent is expected to prevent all relevant mechanisms of RBC destruction in PNH and thus reduce or block all symptoms of anemia in PNH.

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 enhance 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 MASP-2-mediated opsonization in PNH patients. This leaves PNH patients treated with such agents with substantial residual anemia due to extravascuiar hemolysis mediated by MASP-2 dependent complement activation that remains untreated. Therefore, it is expected that treatment with such agents that prevent intravascular hemolysis in combination with a MASP-2-inhibitory agent that prevents extravascuiar hemolysis will be more effective than either agent alone in preventing the anemia that develops in PNH patients. In fact, the combination of such agents and a MASP-2 inhibitory agent is expected to prevent all or a large majority of the relevant mechanisms of RBC destruction in PNH and thus block ail or a large majority of the symptoms of anemia in PNH.

Inhibition of MASP-2 Improves survival in subjects infected with Neisseria meningitidis

As described in Examples 30-32 and shown in FIGURES 33-37, inhibition of

MASP-2 does not reduce survival following infection with Neisseria meningitidis. To the contrary, it was surprisingly discovered that MASP-2 inhibition significantly improved survival (FIGURES 33 and 34) as well as illness scores (FIGURE 36) in these studies. Administration of anti-MASP2 antibody yielded the same result (FIGURE 37), eliminating secondary or compensatory effects in the knockout-mouse strain as a possible cause. These favorable outcomes in MASP-2-ablated animals were associated with a more rapid elimination of Neisseria from the blood (FIGURE 35). Also, as described herein, incubation of Neisseria with human serum killed Neisseria (FIGURE 38). Furthermore, addition of a functional monoclonal antibody specific for human MASP-2

-432018200437 19 Jan 2018 that blocks MASP-2-dependent lectin pathway complement activation, but not administration of an isotype control monoclonal antibody, enhanced this killing response.

In the context of lectin-dependent complement activation by Neisseria, blockade of

MASP-2-enhanced lytic destruction of the organism in vitro (FIGURE 38). Because lysis of Neisseria is the main protective mechanism in the naive host, blockade of MASP-2 in vivo increases Neisseria clearance and leads to enhanced killing. These results are surprising, and provide a significant advantage over treatment with eculizumab, which has been shown to increase susceptibility to life-threatening and fetal meningococcal infections (Dmytrijuk A., et ah, The Oncologist 13:993-1000 (2008)).

in accordance with the foregoing, in one aspect, the invention provides a method for treating paroxysmal nocturnal hemoglobinuria (PNH) by administering a composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent in a pharmaceutical carrier ίο a subject suffering from PNH or a condition resulting from PNH (e.g., anemia, hemoglobin in the urine and thrombosis). The MASP-2 inhibitory agent is administered systemically io the subject suffering from PNH or a condition resulting from PNH, such as by intra-arterial, intravenous, intramuscular, inhalational, subcutaneous or other parenteral administration, or potentially by oral administration for non-peptidergic agents.

THE ROLE OF MASP-2 IN THROMBOTIC MICROANGIOPATHIES, INCLUDING

HEMOLYTIC UREMIC SYNDROME (HUS), ATYPICAL HEMOLYTIC

UREMIC SYNDROME (AHUS) AND THROMBOTIC THROMBOCYTOPENIC PURPURA (TTP), AND THERAPEUTIC METHODS USING MASP-2 INHIBITORY AGENTS Qxeryieyy

Thrombotic microangiopathy (TMA) is a pathology characterized by blood clots in small blood vessels (Benz, K.; et ah, Curr Opin Nephrol Hypertens 19(3):242-7 (2010)). Stress or injury to the underlying vascular endothelium is believed to be a primary driver. Clinical and laboratory findings of TMA include thrombocytopenia, anemia, purpura, and renal failure. The classic TMAs are hemolytic uremic syndrome .44.

2018200437 19 Jan 2018 (HUS) and thrombotic thrombocytopenic purpura (TTP). The characteristic underlying pathological feature of TMAs are platelet activation and the formation of microthrombi in the small arterioles and venules.

Direct evidence for a pathological role of complement in a host of nephritides is 5 provided by studies of patients with genetic deficiencies in specific complement components. A number of reports have documented an association of renal injury with deficiencies of complement regulatory factor H (.Ault, B.H., Nephrol. 14:1045-1053, 2000; Levy, M., eta!., Kidney Int. 50:949-56, 1986; Pickering, M.C., etah, Nat. Genet. 37:424-8, 2002). Factor H deficiency results in low plasma levels of factor B and C3 due to activation-related consumption of these components. Circulating levels of C5b-9 are also elevated in the serum of these patients, implying complement activation. Membranoprohferative glomerulonephritis (MPGN) and idiopathic hemolytic uremic syndrome (HUS) arc associated with factor H deficiency or mutations of factor H. Factor H-deficient pigs (Jansen, J.H., et al., Kidney Int. 53:331-49, 1998) and factor-H knockout mice (Pickering, M.C., 2002) display MPGN-like symptoms, confirming the importance of factor H in complement regulation. Deficiencies of other complement components are associated with renal disease, secondary to the development of systemic lupus erythematosus (SLE) (Walport, MJ., Davies, et al., Ann. N.Y. Acad. Afo. 5/5:267-81, 1997). Deficiency for Clq, C4 and C2 predispose strongly to the development of SLE via mechanisms relating to defective clearance of immune complexes and apoptotic material. In many of these SLE patients lupus nephritis occurs, characterized by the deposition of immune complexes throughout the glomerulus.

aHUS

Atypical hemolytic uremic syndrome (aHUS) is part of a group of conditions termed “Thrombotic microangiopathies.” In the atypical form of HUS (aHUS), the disease is associated with defective complement regulation and can be either sporadic or familial. Familial eases of aHUS are associated with mutations in genes coding for complement activation or complement regulatory proteins, including complement factor H, factor I, factor B, membrane cofactor CD46 as well as complement factor H-related protein 1 (CFHR1) and complement factor H-related protein 3 (CFHR3). (Zipfel, P.F., el al., PloS Genetics 3(3):e41 (2007)). The unifying feature of this diverse array of genetic mutations associated with aHUS is a predisposition to enhanced complement activation on cellular or tissue surfaces. Therefore, one aspect of the present invention

-452018200437 19 Jan 2018 comprises treating a patient suffering with aHUS that is associated with a factor H defiency by administering an effective amount of a MASP-2 inhibitory agent. Another aspect of the present invention comprises treating a patient suffering with HUS that is associated with a factor I, factor B, membrane cofactor CD46, CFHR1 or CFHR3 deficiency by administering an effective amount of a MASP-2 inhibitory agent.

Significant progress has been made recently toward the understanding of the molecular pathophysiology underlying enhanced complement activation in aHUS caused by the diverse set of mutant complement factors. This mechanism is best understood for factor H mutations. Factor H is an abundant serum protein comprising 20 short consensus repeat (SCR) domains that acts as a negative regulator of complement activation both in solution as well as on host cell surfaces. It targets the activated form of C3 and. together with factor i and other cofactors, promotes its inactivation, forestalling further complement activation. To effectively control complement activation on host cell surfaces, factor H needs to interact with host ceils, which is mediated by SCR domains

16-20. All factor H mutations associated with aHUS described io date are clustered in the

C-tsrminal region encompassing (SCR) domains 16-20. These mutant factor H proteins are fully functional in controlling C3 activation in solution, but are unable to interact with host cell surfaces and consequently cannot control C3 activation on cellular surfaces (Exp Med 204(6):1249-56 (2007)). Thus, certain mutations of factor H are associated with aHUS because the mutant factor H protein fails to interact with host cell surfaces and thus cannot effectively down modulate complement activation on host cell surfaces, including the microvascular endothelium. As a result, once initial C3 activation has occurred, subsequent complement activation on microvascular endothelial surfaces proceeds unabated in patients with factor H mutations. This uncontrolled activation of complement ultimately leads to progressive injury to the vascular endothelium, subsequent platelet aggregation and microvascular coagulation, and hemolysis caused by sheer stress of RBC passage through partially occluded microvessels. Thus, aHUS disease manifestations and clinical and laboratory findings are directly linked to a defect in the negative regulation of complement on the surface of the microvascular endothelium.

Analogous to factor H mutation, loss-of-function mutations in the negative complement modulators factor I and membrane cofactor protein (CD46) are also linked to aHUS. The opposite has been observed for factor B the C3 protein in that aHUS was found to be associated with gain-of-function mutations in these proteins (Pediatr Nephrol

-462018200437 19 Jan 2018

25(12):2431-42 (2010)). Thus, a host of converging data implicates complement activation in aHUS pathogenesis. This notion is most convincingly supported by the therapeutic efficacy ofeculizumab, a monoclonal antibody that blocks the terminal complement protein C5 in the treatment of aHUS.

While the central role of complement as an effector mechanism in aHUS is widely accepted, the triggers initiating complement activation and the molecular pathways involved are unresolved. Not ail individuals carrying the above described mutations develop aHUS. In fact, familial studies have suggested that the penetrance of aHUS is only --50% (.Ann Hum Genet 74(1):17-26 (2010)). The natural history' of the disease suggests that aHUS most often develops after an initiating event such as an infectious episode or an 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 the lectin pathway. Thus, lectin pathway activation triggered by an infection may represent the initiating trigger for subsequent pathological amplification of complement activation in aHUS-predisposed individuals, which may ultimately lead to disease progression. Accordingly, another aspect of the present invention comprises treating a patient suffering with aHUS secondary to an infection by administering an effective amount of a MASP-2 inhibitory agent.

Other forms of injury io host tissue will activate complement via the lectin pathway, in particular injury to the vascular endothelium. Human vascular endothelial cells subject to oxidative stress for example respond by expressing surface moieties that bind lectins and activate the lectin pathway of complement (Am J. Pathol 156(6): 1549-56 (2000)). Vascular injury following ischemia/reperfuston also activates complement via the lectin pathway in vivo (Scand J Immunol 61(5):426-34 (2005)). Lectin pathway activation in this setting has pathological consequences for the host, and inhibition of the lectin pathway by blocking MASP-2 prevents further host tissue injury and adverse outcomes (Sehwaebfe PNAS 2011).

Thus, other processes that precipitate aHUS are also known to activate the lectin pathway of complement. It is therefore likely that the iec-tin pathway may represent the initial complement activating mechanism that is inappropriately amplified in a deregulated fashion in individuals genetically predisposed io aHUS, thus initiating aHUS pathogenesis. By inference, agents that biock activation of complement via the lectin

-472018200437 19 Jan 2018 pathway, including anti-MASP-2 antibodies, are expected to prevent disease progression or reduce exacerbations in aHUS susceptible individuals.

In further support of this concept, recent studies have identified 5. pneumonia as an important etiological agent in pediatric cases of aHUS. (Nephrology (Carlton), 17:485 52 (2012); Pediatr Infect Dis J. 30(9):736-9 (2011)). This particular etiology appears to have an unfavorable prognosis, with significant mortality and long-term morbidity'. Notably, these eases involved non-enteric infections leading to manifestations of microangiopathy, uremia and hemolysis without evidence of concurrent mutations in complement genes known to predispose to aHUS, It is important to note that S', pneumonia is particularly effective at activating complement, and does so predominantly through the lectin pathway. Thus, in cases of non-enteric HUS associated with pneumococcal infection, manifestations of microangiopathy, uremia and hemolysis are expected to be driven predominantly by activation of the lectin pathway, and agents that block the lectin pathway, including anti-MASP-2 antibodies, are expected to prevent progression of aHUS or reduce disease severity in these patients. Accordingly, another aspect of the present invention comprises treating a patient suffering with non-enteric aHUS that is associated with S. pneumonia infection by administering an effective amount of a MASP-2 inhibitory agent.

In accordance with the foregoing, in some embodiments, in the setting of a subject at risk for developing renal failure associated with aHUS, a method is provided for decreasing the likelihood of developing aHUS, or of developing renal failure associated with aHUS, comprising administering an amount of an MASP-2 inhibitory agent for a time period effective to ameliorate or prevent renal failure in the subject. In some embodiments, the method further comprises the step of determining whether a subject is at risk for developing aHUS prior to the onset of any symptoms associated with aHUS. in other embodiments, the method comprises determining whether a subject is a risk for developing aHUS upon the onset of at least one or more symptoms indicative of aHUS (e.g., the presence of anemia, thrombocytopenia and/or renal insufficiency) and/or the presence of thrombotic rnscroangiopathy in a biopsy obtained from the subject. The determination of whether a subject is at risk tor developing aHUS comprises determining whether the subject has a genetic predisposition to developing aHUS, which may be carried out by assessing genetic information (e.g, from a database containing the genotype of the subject), or performing at least one genetic screening test on the subject

-482018200437 19 Jan 2018 to determine the presence or absence of a genetic marker associated with aHUS (i.e., determining the presence or absence of a genetic mutation associated with aHUS in the genes encoding complement factor H (CFH), factor 1 (CFI), factor B (CFB), membrane cofactor CD46, €3, complement factor H-related protein 1 (CFHR1), or THBD (encoding the anticoagulant protein thrombodulin) or complement factor H-related protein 3 (CFHR3), or complement factor H-related protein 4 (CFHR4)) either via genome sequencing or gene-specific analysis (e.g,, PCR analysis), and/or determining whether the subject has a family history of aHUS. Methods of genetic screening for the presence or absence of a genetic mutation associated with aHUS are well established, for example, see Noris M et ah “Atypical Hemolytic-Uremic Syndrome,” 2007 Nov 16 [Updated 2011 Mar 10]. in: Paeon RA, Bird TD, Dolan CR, et al,, editors, GeneReviews™, Seattle (WA): University of Washington, Seattle,

For example, overall the penetrance of the disease in those with mutations of complement factor H (CFH) is 48%, and the penetrance for mutations in CD46 is 53%, for mutations in CFI is 50%, for mutations in C3 is 56% and for mutations in THBD is 64% (Caprioli J. et al., Blood, 108:1267-79 (2006); Noris ef ah, Clin J Am Soc Nephrol 5:1844-59 (2010)). As described in Caprioli et ah, (2006), supra, a substantial number of individuals with a mutation in complement Factor H (CFH) never develop aHUS, and if is postulated that suboptimal CFH activity in these individuals is sufficient to protect the host from the effects of complement activation in physiological conditions, however, suboptimal CFH activity is not sufficient to prevent C3b from being deposited on vascular endothelial cells when exposure to an agent that activates complement produces higher than normal amounts of C3b. Accordingly, in one embodiment, a method is provided for inhibiting MASP-2-dependent complement activation In a subject suffering from, or at risk for developing non-Factor H-dependcnt atypical hemolytic uremic syndrome, comprising administering to the subject a composition comprising art amount of a MASP-2 inhibitory agent effective to inhibit MASP-2-dependent complement activation. In another embodiment, a method is provided for inhibiting MASP-2 dependent complement activation in a subject at risk for developing Factor H-dependent atypical hemolytic uremic syndrome, comprising periodically monitoring the subject to determine the presence or absence of anemia, thrombocytopenia or rising creatinine, and treating with a MASP-2 inhibitory agent upon the determination of the presence of anemia thrombocytopenia, or rising creatinine. In another embodiment, a method is

-492018200437 19 Jan 2018 provided for reducing the likelihood that a subject at risk for developing Factor iddependent aHUS will suffer clinical symptoms associated with aHUS, comprising administering a MASP-2 inhibitory·' agent prior to, or during, or after an event known to be associated with triggering aHUS clinical symptoms, for example, drug exposure (e.g,, chemotherapy), infection (e.g., bacterial infection), malignancy, an injury', organ or tissue transplant, or pregnancy.

In one embodiment, a method is provided for reducing the likelihood that a subject at risk for developing aHUS will suffer clinical symptoms associated with aHUS, comprising periodically monitoring the subject to determine the presence or absence of anemia, thrombocytopenia or rising creatinine, and treating with a MASP-2 inhibitoryagent upon the determination of the presence of anemia, thrombocytopenia, or rising creatinine,

In another embodiment, a method is provided for reducing the likelihood that a subject at risk for developing aHUS will suffer clinical symptoms associated with aHUS comprising administering a MASP-2 inhibitory agent prior to, or during, or after an event known to be associated with triggering aHUS clinical symptoms, for example, drug exposure (e.g., chemotherapy), infection (e.g., bacteria! infection), malignancy, an injury, organ or tissue transplant, or pregnancy,

In some embodiments, the MASP-2 inhibitory agent is administered for a time period of at least one, two, three, four days, or longer, prior to, during, or after the event associated with triggering aHUS clinical symptoms and may be repeated as determined by a physician until the condition has been resolved or is controlled. In a pre-aHUS setting, the MASP-2 inhibitory agent may be administered to the subject systemically. such as by intra-arterial, intravenous, intramuscular, inhalational, nasal, subcutaneous or other parenteral administration.

in some embodiments, in the setting of initial diagnosis of aHUS, or in a subject exhibiting one or more symptoms consistent with a diagnosis of aHUS (e.g,, the presence of anemia, thrombocytopenia and/or renal insufficiency), the subject is treated with an effective amount of a MASP-2 inhibitory agent (e.g., an anti-MASP-2 antibody) as a first line therapy in the absence of plasmapheresis, or in combination with plasmapheresis, As a first line therapy, the MASP-2 inhibitory agent may be administered to the subject systemically, such as by intra-arterial, intravenous, intramuscular, inhalational, nasal, subcutaneous or other parenteral administration, In some embodiments, the MASP-2

-502018200437 19 Jan 2018 inhibitory agent is administered to a subject as a first fine therapy in the absence of plasmaphersis to avoid the potential complications of plasmaphersis including hemorrhage, infection, and exposure to disorders and/or allergies inherent in the plasma donor, or in a subject otherwise averse to plasmapheresis, or in a setting where plasmapheresis is unavailable.

In some embodiments, the method comprises administering a MASP-2 inhibitory agent to a subject suffering from aHUS via a catheter (e.g., intravenously) for a first time period (e.g., at least one day to a week or two weeks) followed by administering a MASP-2 inhibitory agent to the subject subcutaneously for a second time period (e.g., a chronic phase of at least two weeks or longer). In some embodiments, the administration in the first and/or second time period occurs in the absence of plasmapheresis, 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 the determination of a reduced level of at least one complement factor in comparison to a standard value or healthy control subject is indicative of the need for continued treatment with the MASf-'I inhibitory ageist.

in some embodiments, the method comprises administering a MASP-2 inhibitoryagent, such as an anti-MASP-2 antibody, to a subject suffering from, or at risk for developing, aHUS either intravenously, intramuscularly, or preferably, subcutaneously.

Treatment may be chronic and administered daily to monthly, but preferably every two weeks. The anti-MASP-2 antibody may be administered alone, or in combination with a C5 inhibitor, such as eculizamab.

HUS

Like atypical HUS, the typical form of HUS displays all the clinical and laboratory findings of a TMA. Typical HUS, however, is often a pediatric disease and usually has no familial component or direct association with mutations in complement genes. The etiology of typical HUS is tightly linked to infection with certain intestinal pathogens. The patients typically present with acute renal failure, hemoglobinuria, and thrombocytopenia, which typically follows an episode of bloody diarrhea. The condition is caused by an enteric infection with Shigella dissenferia, Salmonella or shiga toxin-like producing enterohemorrhagic strains of E. Coli. such as E.Coli O157:H7. The pathogens are acquired from contaminated food or water supply. HUS is a medical emergency and

-512018200437 19 Jan 2018 carries a 5-10% mortality. A significant portion of survivors develop chronic kidney disease (Corrigan and Boineau, Pediair Rev 22 (11): 365-9 (2011)) and may require kidney transplantation.

The mierovaseuiar coagulation in typical HUS occurs predominantly, though not 5 exclusively, in the renal microvasculature. The underlying pathophysiology is mediated by Shiga toxin (STX). Excreted by enteropathic microbes into the intestinal lumen, STX crosses the intestinal barrier, enters the bloodstream and binds to vascular endothelial cells via the blobotriaosyl ceramide receptor CD77 (Boyd and Lingwood Nephron 51:207 (1989)), which is preferentially expressed on glomerular endothelium and mediates the toxic effect of STX. Once bound to the endothelium, STX induces a series of events that damage vascular endothelium, activate leukocytes and cause vWF-dependent thrombus formation (Forsyth et al., Lancet 2: 413-414 (1989); Zoja et al., Kidney In!. 62: 846-856 (2002); Zanchi et at, J Immunol. 181:1460--1469 (2008); Morigi et al., Blood 98: 1828— 1835 (2001); Guessou et ah, Infect. Immun., 73: 8306-8316 (2005)). These microthrombi obstruct or occlude the arterioles and capillaries of the kidney and other organs. The obstruction of blood flow in arterioles and capillaries by microthrombi increases the shear force applied to RBCs as they squeeze through the narrowed blood vessels. This can result in destruction of RBC by shear force and the formation of RBC fragments called schistocytes. The presence of schistocytes is a characteristic finding in HUS. This mechanism is known as microangiopathic hemolysis. In addition, obstruction of blood flow results in ischemia, initiating a complement-mediated inflammatory response that causes additional damage to the affected organ.

The lectin pathway of complement contributes to the pathogenesis of HUS by two principle mechanisms: 1) MASP-2-mediated direct activation ofthe coagulation cascade caused by endothelial injury, and 2) lectin-mediated subsequent complement activation induced by the ischemia resulting from the initial occlusion of mierovaseuiar blood flow.

STX injures mierovaseuiar endothelial ceils, and injured endothelial cells are known to activate the complement system. As detailed above, complement activation following endothelial cell injury is driven predominantly by the lectin pathway. Human vascular endothelial cells subject to oxidative stress respond by expressing surface moieties that bind lectins and activate the lectin pathway of complement (Collard et al.,

2018200437 19 Jan 2018

Am J Pathol. 156(5):5549-56 (2000)). Vascular injury following ischemia reperfiision also activates complement via the lectin pathway in vivo (Scand J Immunol 61(5):426-34 (2005)).Lectin pathway activation in this setting has pathological consequences for the host, and inhibition of the lectin pathway by blockade of MASP-2 prevents further host tissue injury and adverse outcomes (Schwaeble et al., PNAS (2011)), in addition to complement activation, lectin-dependent activation of MASP-2 has been shown to result in cleavage of prothrombin to form thrombin and to promote coagulation. Thus, activation of the lectin pathway of complement by injured endothelial cells can directly activate the coagulation system. The lectin pathway of complement, by virtue of MASP10 2-mediated prothombin activation, therefore is likely the dominant molecular pathway linking the initial endothelial injury by STX to the coagulation and microvascular thrombosis that occurs in HUS, It is therefore expected that lectin pathway inhibitors, including, but not limited to, antibodies that block MASP-2 function, will prevent or mitigate microvascular coagulation, thrombosis and hemolysis in patients suffering from

HUS. Indeed, administration of anii-MASP-2 antibody profoundly protects mice in a model of typical HUS. As described in Example 36 and shown in FIGURE 45, all control mice exposed to STX and EPS developed severe HUS and became moribund or died within 48 hours, On the other hand, as further shown in FIGURE 45, ail mice treated with an anti-MASP-2 antibody and then exposed to STX and LPS survived (Fisher’s exact p<0.01; N=5). Thus, anti-MASP-2 therapy profoundly protects mice in this model of HUS. it is expected that administration of a MASP-2 inhibitory agent, such as a MASP-2 antibody, will be effective in the treatment of HUS patients and provide protection from microvascular coagulation, thrombosis, and hemolysis caused by infection with enteropathie E. coli or other STX-producing pathogens.

While shown here for HUS caused by STX, it is expected that anti-MASP-2 therapy will also be beneficial for HUS-like syndromes due to endothelial injury caused by other toxic agents. This includes agents such as mitomycin, ticlopidine, cycplatin, quinine, cyclosporine, bleomycin as well as other chemotherapy drugs and immunosuppresssive drugs. Thus, it is expected that anti-MASP-2 antibody therapy, or other modalities that inhibit MASP-2 activity, will effectively prevent or limit coagulation, thrombus formation, and RBC destruction and prevent renal failure in HUS and other TMA related diseases (i.e., aHUS and TTP),

-532018200437 19 Jan 2018

Patients suffering from HUS often present with diarrhea and vomiting, their platelet counts are usually reduced (thrombocytopenia), and RBCs are reduced (anemia),

A pre-HUS diarrhea phase typically lasts for about four days, during which subjects at risk for developing HUS typically exhibit one or more of the following symptoms in addition to severe diarrhea: a hematocrit level below' 30% with smear evidence of intravascular erythrocyte destruction, thrombocytopenia (platelet count <150 x lOVmm’), and/or the presence of impaired renal function (serum creatinine concentration greater than the upper limit of reference range for age). The presence of oligoanuria (urine output <0.5 mL/kg/h for >1 day) can be used as a measure for progression towards developing HUS (see C. Hickey et al., Arch Pediatr Adolesc Med 165(10):884-889 (2011)). Testing is typically carried out for the presence of infection with E, coli bacteria (E.coli O157:H7), or Shigella or Salmonella species. In a subject testing positive for infection with enterogenic E. coli (e.g.. £. coli 0157:117), the use of antibiotics is contraindicated because the use of antibiotics may increase the risk of developing HUS through increased STX production (See Wong C. et ah, N Engl J Med 342:1930-1936 (2000). For subjects testing positive for Shigella or Salmonella, antibiotics are typically administered to clear the infection. Other well established first-line therapy for HUS includes volume expansion, dialysis and plasmapheresis.

In accordance with the foregoing, in some embodiments, in the setting of a subject suffering from one or more symptoms associated with a pre-HUS phase and at risk for developing HUS (i.e., the subject exhibits one or more of the following: diarrhea, a hematocrit level less than 30% with smear evidence of intravascular erythrocyte destruction, thrombocytopenia (platelet count less than 150 x lG7mra³), and/or the presence of impaired renal function (serum creatinine concentration greater than the upper limit of reference range for age)), a method is provided for decreasing the risk of developing HUS, or of decreasing the likelihood of renal failure in the subject, comprising administering an amount of an MASP-2 inhibitory agent for a time period effective to ameliorate or prevent impaired renai function. In some embodiments, the MASP-2 inhibitory agent is administered for a time period of at least one, two, three, four or more days, and may be repeated as determined by a physician until the condition has been resolved or is controlled. In a pre-HUS setting, the MASP-2 inhibitory agent may be administered to the subject systemically, such as by intra-arterial, intravenous, intramuscular, inhalational, nasal, oral, subcutaneous or other parenteral administration.

-542018200437 19 Jan 2018

The treatment of E. coli 0157:117 infection with bactericidal antibiotics, particularly β-lactams, has been associated with an increased risk of developing HUS (Smith et al., Pediatr Infect Dis J 31(1):37-41 (2012).In some embodiments, in the setting of a subject suffering from symptoms associated with a pre-HUS phase, wherein the subject is known to have an infection with enterogenic co// for which the use of antibiotics is contra-indicated (e.g., E. coli 0157:H7), a method is provided for decreasing the risk of developing HUS, or of decreasing the likelihood of renal failure in the subject, comprising administering an amount of a MASP-2 inhibitory agent for a first time period effective to inhibit or prevent the presence of oligoanuria in the subject (e.g., at least, one, two, three or tour days), wherein the administration of the MASP-2 inhibitory agent during the first time period occurs in the absence of an antibiotic. In some embodiments, the method further comprises administering the MASP-2 inhibitory agent to the subject In combination with an antibiotic for a second time period (such as at least one to two weeks), in other embodiments, in the setting of a subject suffering from symptoms associated with a pre-HUS phase, wherein the subject is known to have an infection with Shigella or Salmonella, a method is provided for decreasing the risk of developing HUS, or of decreasing the likelihood of renal failure in the subject, comprising administering an amount of a MASP-2 inhibitory agent and for a time period effective to inhibit or prevent the presence of oligoanuria in the subject, wherein the administration of the MASP-2 inhibitory agent is either in the presence or in the absence of a suitable antibiotic.

In some embodiments, in the setting of an initial diagnosis of HUS, or in a subject exhibiting one or more symptoms consistent with a diagnosis of HUS (e.g., the presence of renal failure, or microangiopathic hemolytic anemia in the absence of low fibrinogen, or thrombocytopenia) the subject is treated with an effective amount of a MASP-2 inhibitory agent (e.g, a anti-MASP-2 antibody) as a first-line therapy in the absence of plasmapheresis, or in combination with plasmapheresis. As a first-line therapy, the MASP-2 inhibitory agent may be administered to the subject systemically, such as by intra-arterial, intravenous, intramuscular, inhaiationai, nasal, subcutaneous or other parenteral administration. In some embodiments, the MASP-2 inhibitory agent is administered to a subject as a first line therapy in the absence of plasmapheresis to avoid the complications of plasmapheresis such as hemorrhage, infection, and exposure to

-552018200437 19 Jan 2018 disorders and/or allergies inherent in the plasma donor, or in a subject otherwise averse to plasmaphoresis, or in a setting where plasmapheresis is unavailable.

In some embodiments, the method comprises administering a MASP-2 inhibitory agent to a subject suffering from HUS via a catheter (e.g., intravenously) for a first time period (e.g., an acute phase lasting at least one day to a week or two weeks) followed by administering a MASP-2 inhibitory agent to the subject subcutaneously for a second time period (e.g,, a chronic phase of at least two weeks or longer). In some embodiments, the administration in the first and/or second time period occurs in the absence of plasmapheresis. 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 the determination of a reduced level of the at least one complement factor in comparison to a standard value or healthy control subject is indicative of the need for treatment, and wherein the determination of a normal level is indicative of improvement.

In some embodiments, the method comprises administering a MASP-2 inhibitory agent, such as an anti-MASP-2 antibody, to a subject suffering from, or at risk for developing, HUS either subcutaneously or intravenously. Treatment is preferably daily, but can be as infrequent as weekly or monthly. Treatment will continue for at least one week and as long as 3 months. The anti-MASP-2 antibody may be administered alone, or in combination with a C5 inhibitor, such as eeulizamab.

TTP:

Thrombotic thrombocytopenic purpura (TTP) is a life threatening disorder of the blood-coagulation system, caused by autoimmune or hereditary dysfunctions that activate the coagulation cascade or the complement system (George, JN, jV Engl J Med; 354:1927-35 (2006)), This results in numerous microscopic clots, or thomboses, in small blood vessels throughout the body. 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 organ damage, including brain, heart, and kidneys. TTP is clinically characterized by thrombocytopenia, microangiopathic hemolytic anemia, neurological changes, renal failure and fever. In the era before plasma exchange, the fatality rate was 90% during acute episodes. Even with plasma exchange, survival at six months is about 80%.

-562018200437 19 Jan 2018

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

Numerous mutations in ADAMTS13 have been identified in individuals with TTP. The disease can also develop due to auto-antibodies against ADAMTS-13. In

0 addition, TTP can develop during breast, gastrointestinal tract, or prostate cancer (George IN., 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 like systemic lupus erythematosis (Hamasaki K, et al., Clin Rheumatol.22:355-8 (2003)). ΓΓΡ can also be caused by certain drug therapies, including heparin, Quinine, immunemediated ingredient, cancer chemotherapeutic agents (bleomycin, cisplatin, cytosine arabinoside, daunomycin gemcitabine, mitomycin C, and tamoxifen), cyclosporine A, oral contraceptives, penicillin, rifampin and anti-platelet drugs including tielopidine and clopidogrel (Azarm, Τ', et al., J Res Med Sci., 16: 353-357 (201 1)). Other factors or conditions associated with TTP are toxins such as bee venoms, sepsis, splenic sequestration, transplantion, vasculitis, vascular surgery, and infections like Streptococcus pneumonia and cytomegalovirus (Moake JL., N Engl J Med., 347:589-600 (2002)). TTP due to transient functional ADAMTS-13 deficiency can occur as a consequence of endothelial cell injury associated with A. pneumonia infection (Pediatr Nephrol., 26:631-5 (2011)).

Plasma exchange is the standard treatment for TTP (Rock GA, st ah, 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 not successful for about 20% of patients, relapse occurs in more than a third of patients, and plasmapheresis is costly and technically demanding. Furthermore, many patients are

-3 72018200437 19 Jan 2018 unable to tolerate plasma exchange. Consequently there remains a critical need for additional and better treatments for TTP.

Because TTP is a disorder of the blood coagulation cascade, treatment with antagonists of the complement system may aid in stabilizing and correcting the disease.

While pathological activation of the alternative complement pathway is linked to aHUS, the role of complement activation in TTP is less clear. The functional deficiency of ADAMTS13 is important for the susceptibility of ΤΓΡ, however it 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 ofthe coagulation cascade, vWF, platelet function, components of the endothelial vessel surface, or the complement system may be implicated in the development of acute thrombotic microangiopathy (Galbusera, M, et ai., Haematologica, 94: 166-170 (2009)). In particular, complement activation has been shown to play a critical role; serum from thrombotic microangiopathy associated with ADAMTS-13 deficiency has been shown to cause C3 and MAC deposition and subsequent neutrophil activation which could be abrogated by complement inactivation (Ruiz-Torres MP, et al., Thromb Haemost, 93:443-52 (2005)). In addition, it has recently been shown that during acute episodes of TTP there are increased levels of C4d, C3bBbP, and C3a (M. Red et al,, J Thromb Haemost. Feb 28,(2012) doi: 10,111 l/j,1538-7836.2012.04674.x. [Epub ahead of print]), consistent with activation ofthe classicaldectin and alternative pathways. This increased amount of complement activation in acute episodes may initiate the terminal pathway activation and be responsible for further exacerbation of TTP,

The role of ADAMTS-13 and vWF in TTP clearly is responsible for activation and aggregation of platelets and their subsequent role in shear stress and deposition in microangiopathies. Activated platelets interact with and trigger both the classicai and alternative pathways of complement. Platelet mediated complement activation increases the inflammatory mediators G3a and C5a (Peerschke E et al,, Moi Immunol, 47:2170-5 (2010)), Platelets may thus serve as targets of classical complement activation in inherited or autoimmune TTP,

As described above, the lectin pathway of complement, by virtue of MASP-2 mediated prothombin activation, is the dominant molecular pathway Sinking endothelial injury to the coagulation and microvascular thrombosis that occurs in HUS, Similarly, activation ofthe lectin pathway of complement may directly drive the coagulation system

-582018200437 19 Jan 2018 in TTP. Lectin pathway activation may be initiated in response to the initial endothelium injury caused by ADAMTS-13 deficiency in TTP. It is therefore expected that lectin pathway inhibitors, including but not limited to antibodies that block MASP-2 function, will mitigate the microangiopathies associated with microvascular coagulation, thrombosis, and hemolysis in patients suffering from TTP.

Patients suffering from TTP typically present in the emergency room with one or more of the following: purpura, renal failure, low' platelets, anemia and/or thrombosis, including stroke. The current standard of care for TTP involves inlra-catheter delivery (e.g., intravenous or other form of catheter) of replacement plasmapheresis for a period of two weeks or longer, typically three times a week, but up to daily. If the subject tests positive for the presence of an inhibitor of ADAMTS13 (i.e., an endogenous antibody against ADAMTS13), then the plasmapheresis may be carried out in combination with immunosuppressive therapy (e.g., corticosteroids, rituxan, or cyclosporine). Subjects with refractory TTP (approximately 20% of TTP patients) do not respond to at least two weeks of plasmapheresis therapy.

In accordance with the foregoing, in 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 of iess that or equal to 5000/pL if off aspirin, iess than or equal to

20,0()()/pL if on aspirin), severe cardiac involvement, severe pulmonary involvement, gastro-intestinai infarction or gangrene), a method is provided for treating the subject with an effective amount of a MASP-2 inhibitory agent (e.g., a anti-MASP-2 antibody) as a first line therapy in the absence of plasmapheresis, or in combination with plasmapheresis. As a first-line therapy, the MASP-2 inhibitory agent may be administered to the subject systemlcally, such as by intra-arterial, intravenous, intramuscular, Inhalational, nasal, subcutaneous or other parenteral administration. In some embodiments, the MASP-2 inhibitory agent is administered to a subject as a firstline 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 in the plasma donor, or in a subject otherwise averse to plasmapheresis, or in a setting where plasmapheresis is unavailable. In some embodiments, the MASP-2 inhibitory agent is administered to the subject suffering from TTP in combination

-592018200437 19 Jan 2018 (including co-administration) with an immunosuppressive agent (e.g,, corticosteroids, rituxan or cyclosporine) and/or in combination with concentrated ADAMTS-13.

In some embodiments, the method comprises administering a MASP-2 inhibitory agent to a subject suffering from TTP via a catheter (e.g,, intravenously) for a first time period (e.g., an acute phase lasting at least one day to a week or two weeks) followed by administering a MASP-2 inhibitory agent to the subject subcutaneously for a second time period (e.g., a chronic phase of at least two weeks or longer). 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 suffering one or more symptoms associated with TTP.

In another embodiment, a method is provided for treating a subject suffering from refractory TTP (i.e., a subject that has not responded to at ieast two weeks of plasmaphoresis therapy), by administering an amount of a MASP-2 inhibitor effective to reduce one or more symptoms of TTP. In one embodimen t, the MASP-2 inhibitor (e.g., an anti-MASP-2 antibody) is administered to a subject with refractory TTP on a chronic basis, over a time period of at ieast two weeks or longer via subcutaneous or other parenteral administration. Administration may be repeated as determined by a physician until file condition has been resolved or is controlled.

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

In some embodiments, the method comprises administering a MASP-2 inhibitory agent, such as an anti-MASP-2 antibody, to a subject suffering from, or at risk for developing, TTP either subcutaneously or intravenously. Treatment is preferably daily, but can be as Infrequent as biweekly. Treatment is continued until the subject’s platelet count is greater than 150,000/ml for at least two consecutive days. The anti-MASP-2 antibody may be administered alone, or in combination with a C5 inhibitor, such as eculizamab.

Another aspect of the invention provides methods for treating Cryoglobulinemia by administering a composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent in a pharmaceutical carrier to a subject suffering from

-602018200437 19 Jan 2018

Cryoglobulinemia or a condition resulting from Cryoglobulinemia. Cryoglobulinemia is characterized by the presence of cryoglobulins in the serum, which are single or mixed immmunoglobulins (typically IgM antibodies) that undergo reversible aggregation at low temperatures. Conditions resulting from Cryoglobulinemia include vasculitis, glomeruionepthritis, and systemic inflammation. The MASP-2 inhibitory agent is administered systemically to the subject suffering front Cryoglobulinemia or a condition resulting from Cryoglobulinemia, such as by intra-arterial, intravenous, intramuscular, inhalational, subcutaneous or other parenteral administration, or potentially by oral administration for non-peptidergic agents,

In another aspect, the invention provides methods tor treating Cold Agglutinin disease (CAD) by administering a composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent in a pharmaceutical carrier to a subject suffering from CAD or a condition resulting from CAD. CAD disease manifests as anemia and can be caused by an underlying disease or disorder, referred to as Secondary CAD such as an infectious disease, lymphoproliferative disease or connective tissue disorder. These patients develop IgM antibodies against their red blood cells that trigger an agglutination reaction at low temperatures. The MASP-2 inhibitory agent is administered systemically to the subject suffering from CAD or a condition resulting from CAD, such as by intra-arterial, intravenous, intramuscular, inhalational, subcutaneous or other parenteral administration, or potentially by oral administration for non-peptidergic agents.

COAGULOPATHIES

Evidence has been developed for the role of the complement system in disseminated intravascular coagulation (DIC”), such as DIG secondary to significant bodily trauma.

Previous studies have shown that C4-/- mice are not protected from renal reperfusion injury. (Zhou, W., et al, Predominant role for C5b-9 in renal ischemia/reperfusion injury, J Clin Invest /05:1363-1373 (2000)) In order to investigate whether C4-/- mice may still be able to activate complement via either the classical or the lectin pathway, C3 turn-over in C4-/- plasma was measured in assays specific for either the classical, or the lectin pathway activation route. While no C3 cleavage could be observed when triggering activation via the classical, a highly efficient lectin pathwaydependent activation of C3 in C4 deficient serum was observed (FIGURE 30). It can be

-612018200437 19 Jan 2018 seen that C3b deposition on mannan and zymosan is severely compromised in MASP-2-/mice, even under experimental conditions, that according to many previously published papers on alternative pathway activation, should be permissive for all three pathways.

When using the same sera in wells coated with immunoglobulin complexes instead of mannan or zymosan, C3b deposition and Factor B cleavage are seen in MASP-2+/+ mouse sera and MASP-2-/- sera, but not in Clq depleted sera. This indicates that alternate pathway activation is facilitated in MASP-2-/- sera when the initial C3b is provided via classical activity. FIGURE 30C depicts the surprising finding that C3 can efficiently be activated in a lectin pathway-dependent fashion in C4 deficient plasma.

This C4 bypass” is abolished by the inhibition of lectin pathway-activation through preincubation of plasma with soluble mannan or mannose.

Aberrant non-immune, activation of the complement system is potentially hazardous to man and may also play an Important role in hematological pathway activation, particularly in severe trauma situations wherein both inflammatory and hematological pathways are activated, In normal health, C3 conversion is <5% of the total plasma C3 protein. In rampant infection, including septicaemia and immune complex disease, C3 conversion re-establishes itself at about 30% with complement levels frequently lower than normal, due to increased utilization and changes in pool distribution, immediate C3 pathway activation of greater than 30% generally produces obvious clinical evidence of vasodilatation and of fluid loss to the tissues. Above 30% C3 conversion, the initiating mechanisms are predominantly non-immune and the resulting clinical manifestations are harmful to the patient, Complement C5 levels in health and in controlled disease appear much more stable than C3, Significant decreases and or conversion of C5 levels are associated with the patient's response to abnormal polytrauma (e.g., road traffic accidents) and the likely development of shock lung syndromes. Thus, any evidence of either complement C3 activation beyond 30% of the vascular pool or of any C5 involvement, or both, may be considered likely to be a harbinger of a harmful pathological change in the patient.

Both C3 and C5 liberate anaphylatoxins (C3a and C5a) that act on mast cells and basophils releasing vasodilators' chemicals. They set up chemotactic gradients to guide polymorphonuclear ceils (PMN) to the center of immunological disturbances (a beneficial response), but here they differ because C5a has a specific clumping (aggregating) effect on these phagocytic cells, preventing their random movement away from the reaction site,

-622018200437 19 Jan 2018

In normal control of infection, C3 activates C5, However, in polytrauma, C5 appears to be widely activated, generating C5a anaphyiatoxins systemically. This uncontrolled activity causes polymorphs to clump within the vascular system, and these clumps are then swept into the capillaries of the lungs, which they occlude and generate local damaging effects as a result of superoxide liberation. While not wishing to be limited by theory, the mechanism Is probably important in the pathogenesis of acute respiratory distress syndrome (ARDS), although this view' has recently been challenged. The C3a anaphyiatoxins in vitro can be shown to be potent platelet aggregators, but their involvement in vivo is less defined and the release of platelet substances and plasmin in

SO wound repair may only secondarily involve complement C3. It is possible that prolonged elevation of C3 activation is necessary to generate DIG,

In addition to cellular and vascular effects of activated complement component outlined above that could explain the link between trauma and DIG, emerging scientific discoveries have identified direct molecular links and functional cross-talk between complement and coagulation systems. Supporting data has been obtained from studies in C3 deficient mice. Because €3 is the shared component for each of the complement pathways, C3 deficient mice are predicted to lack all complement function. Surprisingly, however, C3 deficient mice are perfectly capable of activating terminal complement components. (Huber-Lang, M., et ah, Generation of C5a in the absence of C3: a new complement activation pathway, Nat. Med /2:682-687 (2006)) In depth studies revealed that Co-independent activation of terminal complement components is mediated by thrombin, the rate limiting enzyme of the coagulation cascade. (Huber et at, 2006) The molecular components mediating thrombin activation following initial complement activation remained elusive.

The present inventors have elucidated what is believed to be the molecular basis for cross-talk between complement and clotting cascades and identified MASP-2 as a central control point linking the two systems. Biochemical studies into the substrate specificity of MASP-2 have identified prothrombin as a possible substrate, in addition to the well known C2 and C4 complement proteins. MASP-2 specifically cleaves prothrombin at functionally relevant sites, generating thrombin, the rate limiting enzyme of the coagulation cascade. (Krarup, A., et al., Simultaneous Activation of Complement and Coagulation by MBL-Associated Serine Protease 2, PLoS. ONE. 2:e623 (2007)) MASP-2-generated thrombin is capable of promoting fibrin deposition in a defined

-632018200437 19 Jan 2018 reconstituted in vitro system, demonstrating the functional relevance of MASP-2 cleavage. (Krarup et al., 2007) As discussed in the examples herein below, the inventors have further corroborated the physiological significance of this discovery by documenting thrombin activation in normal rodent serum following lectin pathway activation, and demonstrated that this process is blocked by neutralizing MASP-2 monoclonal antibodies.

MASP-2 may represent a central branch point in the lectin pathway, capable of promoting activation of both complement and coagulation systems. Because lectin pathway activation is a physiologic response to many types of traumatic injury, the present inventors believe that concurrent systemic inflammation (mediated by complement components) and disseminated coagulation (mediated via the clotting pathway) can be explained by the capacity of MA.SP-2 to activate both pathways. These findings clearly suggest a role for MASP-2 in DIG generation and therapeutic benefit of MASP-2 inhibition in treating or preventing DIG. MASP-2 may provide the molecular link between complement and coagulation system, and activation of the lectin pathway as it occurs in settings of trauma can directly initiate activation of the clotting system via the MASP-2-thrombin axis, providing a mechanistic link between trauma and DIG. In accordance with an aspect of the present invention, inhibition of MASP-2 would inhibit lectin pathway activation and reduce the generation of both anaphylatoxins C3a and C5a,

It is believed that prolonged elevation of C3 activation is necessary to generate DIG.

Therefore, an aspect of the invention thus provides a method for inhibiting

MASP-2-dependent complement activation to treat disseminated intravascular coagulation or other complement mediated coagulation disorder by administering a composition comprising a therapeutically effective amount of a MASP-2 inhibitory' agent (e.g., anti-MASP-2 antibody or fragment thereof, peptide inhibitors or small molecule inhibitors) in a pharmaceutical carrier to a subject suffering from or at risk for developing such a condition. In some embodiments, the MASP-2 inhibitory agents can block MASP-2 that has already been activated. The MASP-2 inhibitory composition is suitably administered to the subject systemically, such as by intra-arterial, intravenous, intramuscular, inhalational, 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 been resolved or is controlled. The methods of this aspect of the present invention may be utilized for

-642018200437 19 Jan 2018 treatment of DIG secondary to sepsis, severe trauma, including neurological trauma (e.g., acute bead injury, see Kumura, E., et at, Ada Neurochirurgica 55:23-28 (1987), infection (bacterial, viral, fungal, parasitic), cancer, obstetrical complications, liver disease, severe toxic reaction (e.g,, snake bite, insect bite, transfusion reaction), shock, heat stroke, transplant rejection, vascular aneurysm, hepatic failure, cancer treatment by chemotherapy or radiation therapy, burn, accidental radiation exposure, and other causes. See e.g., Becker j.U. and Wira C.R. Disseminated Intravascular Coagulation emedicine.medscape.com/9/10/2009. For DIG secondary to trauma or other acute event, the MASP-2 inhibitory composition may be administered immediately following the traumatic injury or prophylacticaiiy prior to, during, immediately following, or within one to seven days or longer, such as within 24 hours to 72 hours, after trauma-inducing injury or situations such as surgery in patients deemed at risk of DIG. In some embodiments, the MASP-2 inhibitory composition may suitably be administered in a fast-acting dosage form, such as by intravenous or intra-arterial delivery of a bolus of a solution containing j 5 the MASP-2 inhibitory agent composition.

in another aspect, the present invention provides methods of treating a subject suffering from or at risk for developing thrombosis, microcirculatory coagulation or multi-organ failure subsequent to microcireulatory coagulation. Physiological thrombus (blood clot) forms in response to vascular insult to prevent leakage of blood from a damaged blood vessel.

The lectin pathway may play a role in pathological thrombosis triggered by an underlying vascular inflammation linked ίο various etiologies. For example, a thrombus can form around atherosclerotic plaques, which is a known initiator of the lectin pathway. Thus, treatment with a MASP-2 inhibitor may be used to block thrombus formation in patients with underlying atheroscelorsis.

Microcirculatory coagulation (blot clots in capillaries and small blood vessels) occurs in settings such a septic shock. A role of the lectin pathway in septic shock is established, as evidenced by the protected phenotype of MASP-2 (-/-) mouse models of sepsis, described in Example 17 and FIGURES 18 and 19. Furthermore, as demonstrated in Example 15 and FIGURES I6A and 16B, MASP-2 (-/-) mice are protected in the localized Schwartzman reaction model of disseminated intravascular coagulation (DIG), a model of localized coagulation in microvessels.

-652018200437 19 Jan 2018

PERICHEMOTHERAPEUTIC .ADMINISTRATION AND TREATMENT OF

MALIGNANCIES

Activation of the complement system may also be implicated in the pathogenesis of malignancies. Recently, the neoantigens of the C5b~9 complement complex, IgG, C3,

C4, S-protein/vitronectin, fibronectin, and macrophages were localized on 17 samples of breast cancer and on 6 samples of benign breast tumors using polyclonal or monoclonal antibodies and the streptavidin-biotin-peroxidase technique. All the tissue samples with carcinoma in each the TNM stages presented C5b-9 deposits on the membranes of tumor cells, thin granules on cell remnants, and diffuse deposits in the necrotic areas (Niculescu,

E, et ah, Am. J, Pathol. 740:1039-1043, 1992).

In addition, complement activation may be a consequence of chemotherapy or radiation therapy and thus inhibition of complement activation would be useful as an adjunct in the treatment of malignancies to reduce iatrogenic inflammation. When chemotherapy and radiation therapy preceded surgery, C5b-9 deposits were more intense and extended. The C5b-9 deposits were absent in all the samples with benign lesions. S-protein/vitronectin was present as fibrillar deposits in the connective tissue matrix and as diffuse deposits around the tumor cells, less intense and extended than fibronectin. IgG, C3, and C4 deposits were present only in carcinoma samples. The presence of C5b-9 deposits is indicative of complement activation and its subsequent pathogenetic effects in breast cancer (Niculescu, F., et ah, Am. J. Pathol. 146:1039-1043, 1992).

Pulsed tunable dye laser (577 nm) (PTDL) therapy induces hemoglobin coagulation and tissue necrosis, which is mainly limited to blood vessels. In a PTDL-irradiated normal skin study, the main findings were as follows: 1) C3 fragments, C8, C9, and MAC were deposited in vessel wails; 2) these deposits were not due to denaturation of the proteins since they became apparent only 7 min after irradiation, contrary to immediate deposition of transferrin at the sites of erythrocyte coagulates; 3) the C3 deposits were shown to amplify complement activation by the alternative pathway, a reaction which was specific since tissue necrosis itself did not lead to such amplification; and 4) these reactions preceded the local accumulation of polymorphonuclear leucocytes. Tissue necrosis was more pronounced in the hemangiomas. The larger angiomatous vessels in the center of the necrosis did not fix complement significantly, By contrast, complement deposition in the vessels situated at the periphery was similar to that observed in normal skin with one exception: C8, C9,

-662018200437 19 Jan 2018 and MAC were detected In some blood vessels immediately after laser treatment, a finding consistent with assembly of the MAC occurring directly without the formation of a C.S convertase. These results indicate that complement Is activated in PTDL-induced vascular necrosis, and might be responsible for the ensuing inflammatory response,

Photodynamic therapy (PDT) of tumors elicits a strong host immune response, and one of its manifestations is a pronounced neutrophilia. In addition to complement fragments (direct mediators) released as a consequence of PDT-induced complement activation, there are at least a dozen secondary mediators that ail arise as a result of complement activity, The latter include cytokines IL-ί beta, TNF-alpha, IL-6, IL-10,

G-CSF and KC, thromboxane, prostaglandins, leukotrienes, histamine, and coagulation factors (Cede, I,, etal., Cancer Lett. /53:43-51, 2002).

Finally, the use of inhibitors of MASP-2-dependent complement activation may be envisioned in conjunction with the standard therapeutic regimen for the treatment of cancer, For example, treatment with rituximab, a chimeric anti-CD20 monoclonal antibody, can be associated with moderate to severe first-dose side-effects, notably in patients with high numbers of circulating tumor cells. Recent studies during the first infusion of rituximab measured complement activation products (C3b/c and C4b/e) and cytokines (tumour necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6) and IL-8) in five relapsed low-grade .non-Hodgkin's lymphoma (NHL) patients. Infusion of rituximab induced rapid complement activation, preceding the release of TNF-alpha, IL-6 and IL-8, Although the study group was small, the level of complement activation appeared to be correlated both with the number of circulating B ceils prior to the infusion (r = 0.85; P == 0,07), and with the severity of the side-effects. The results indicated that complement plays a pivotal role in the pathogenesis of side-effects of rituximab treatment, As complement activation cannot be prevented bv corticosteroids, it may be relevant to study the possible role of complement inhibitors during the first administration of rituximab (van der Kolk, L.E„ et al., Br. J. Haematol. 7 75:807-811,2001).

In another aspect of the invention, methods are provided for inhibiting MASP-2-dependent complement activation in a subject being treated with chemotherapeutics and/or radiation therapy, including without limitation for the treatment of cancerous conditions. This method includes administering a composition comprising a therapeutically effective amount of a MASP-2 inhibitor in a pharmaceutical carrier ίο a patient perichemotherapeatically, i.e., before and/or during and/or after the administration

-672018200437 19 Jan 2018 of chemotherapeirtic(s) and/or radiation therapy. For example, administration of a

MASP-2 inhibitor composition of the present invention may be commenced before or concurrently with the administration of chemo- or radiation therapy, and continued throughout the course of therapy, to reduce the detrimental effects of the chemo- and/or radiation therapy in the non-targeted, healthy tissues. In addition, the MASP-2 inhibitor composition can be administered following chemo- and/or radiation therapy. It is understood that chemo- and radiation therapy regimens often entail repeated treatments and, therefore, it is possible that administration of a MASP-2 inhibitor composition would aiso be repetitive and relatively coincident with the chemotherapeutic and radiation treatments. It is also believed that MASP-2 inhibitory agents may be used as chemotherapeutic agents, alone or in combination with other chemotherapeutic agents and/or radiation therapy, to treat patients suffering from malignancies. Administration may suitably be via oral (for non-peptidcrgic), intravenous, intramuscular or other parenteral route.

In another embodiment, MASP-2 inhibitory agents may be used to treat a subject for acute radiation syndrome (also known as radiation sickness or radiation poisoning) to reduce the detrimental effects of exposure to ionizing radiation (accidental or otherwise). Symptoms associated with acute radiation syndrome include nausea, vomiting, diarrhea, skin damage, hair loss, fatigue, fever, seizures and coma, For treatment of acute radiation syndrome, the MASP-2 inhibitory composition may be administered immediately following the radiation exposure or prophylactically prior to, during, immediately following, or within one to seven days or longer, such as within 24 hours to 72 hours, after exposure. In some embodiments, the methods may be used to treat a subject prior to or after exposure to a dosage of ionizing radiation sufficient io cause acute radiation syndrome (i.e. a whole body dosage of ionizing radiation of at least 1 Gy, or at least 2 Gy, or at least 3 Gy, or at least 4 Gy, or at least 5 Gy, or at least 6 Gy, or at least 7 Gy, or higher). In some embodiments, the MASP-2 inhibitory composition may suitably be administered in a fast-acting dosage form, such as by intravenous or intra-arterial delivery of a bolus of a solution containing the MASP-2 inhibitory agent composition.

OPHTHALMOLOGIC CONDITIONS

Age-related macular degeneration (AMD) is a blinding disease that afflicts millions of adults, yet the sequelae of biochemical, cellular, and/or molecular events leading to the development of AMD are poorly understood. AMD results in the

-682018200437 19 Jan 2018 progressive destruction of the macula which has been correlated with the formation of extracellular deposits called drusen located in and around the macula, behind the retina and between the retina pigment epithelium (RPE) and the choroid. Recent studies have revealed that proteins associated with inflammation and immune-mediated processes are prevalent among drusen-associated constituents. Transcripts that encode a number of these molecules have been detected in retinal, RPE, and choroidal ceils. These data also demonstrate that dendritic cells, which are potent antigen-presenting cells, are intimately associated with drusen development, and that complement activation is a key pathway that is active both within drusen and along the RPE-ehoroid interface (Hageman, G.S..

et at, Prog. Retin. Eye Res. 20:705-7.32, 2001).

Several independent studies have shown a strong association between AMD and a genetic polymorphism in the gene for complement factor H (CFH) in which the likelihood of AMD is increased by a factor of 7.4 in individuals homozygous for the risk allele (Klein, R.J, etal., Science 305:362-364, 2005; Haines etal., Science 305:362-364,

2005: Edwards etal,, Science 308:263-264, 2005). The CFH gene has been mapped to chromosome lq31 a region that had been implicated in AMD by six. independent linkage scans (see, e.g., Schultz, D.W., et al., Hum. Mol. Genet: 12:3315, 2003). CFH is known to be a key regulator of the complement system. It has been shown that CFH on cells and in circulation regulates complement activity by inhibiting the activation of C3 to C3a and

C3b, and by inactivating existing C3b, Deposition of C5b-9 has been observed in Brusch's membrane, the intercapillary pillars and within drusen in patients with AMD (Klein et al,). Immunofluorescence experiments suggest that in AMD, the polymorphism of CFH may give rise to complement deposition in chorodial capillaries and chorodial vessels (Klein et ah).

The membrane-associated complement inhibitor, complement receptor 1, is also localized in drusen, but it is not detected in RPE ceils immunohistochemicaUy. In contrast, a second membrane-associated complement inhibitor, membrane cofactor protein, is present in drusen-associated RPE cells, as well as in small, spherical substructural elements within drusen. These previously unidentified elements also show strong immunoreactivity for proteolytic fragments of complement component C3 that are characteristically deposited at sites of complement activation. It is proposed that these structures represent residual debris from degenerating RPE cells that are the targets of complement attack (Johnson, L.V,, et ah, Exp. Eye Res. 73:887-896, 2001).

-692018200437 19 Jan 2018

Identification and localization of these multiple complement regulators as well as complement activation products (C3a, C5a, C3b, C5b-9) have led investigators to conclude that chronic complement activation plays an important role in the process of drusen biogenesis and the etiology of AMD (Hageman et al., Progress Retinal Eye

Res. 20:705-32, 2001). Identification of C3 and C5 activation products in drusen provides no insight into whether complement is activated via the classical pathway, the lectin pathway or the alternative amplification loop, as understood in accordance with the present invention, since both C3 and C5 are common to all three. However, two studies have looked for drusen immuno-iabeltng using antibodies specific to Clq, the essential recognition component for activation of the classical pathway (Mullins etai., EASES J. /4:835-846, 2000; Johnson eta!., Exp. Eye Res. 70:441-449, 2000). Both studies concluded that Clq inimuno-labelfing in drusen was not generally observed. These negative results with Clq suggest that complement activation in drusen does not occur via the classical pathway. In addition, immuno-labeling of drusen for immune-complex constituents (IgG light chains, IgM) is reported in the Mullins et ah, 2000 study as being weak to variable, further indicating that the classical pathway plays a minor role in the complement activation that occurs in this disease process.

Two recent published studies have evaluated the role of complement in the development of laser-induced choroidal neovascularization (CNV) in mice, a model of human CNV. Hsing immunohistological methods, Bora and colleagues (2005) found significant deposition of the complement activation products C3b and C5b-9 (MAC) in the neovascular complex following laser treatment (Bora et ah, J. Immunol. 174·Α9\-Ί, 2005), importantly, CNV did not develop in mice genetically deficient in C3 (C3-/mice), the essential component required in all complement activation pathways, RNA message levels for VEGF, TGF-Pj, and β-FGF, three angiogenic factors implicated in CNV, were elevated in eye tissue from mice after laser-induced CNV, Significantly, complement depletion resulted in a marked reduction in the RNA levels of these angiogenic factors.

Using ELISA methods, Nozaki and colleagues demonstrated that the potent anaphylaloxins C3a and C5a are generated early in the course of laser-induced CNV (Nozaki et ah, Proc. Natl. Acad. Sci. U.S. A. /03:2328-33, 2006). Furthermore, these two bioactive fragments of C3 and C5 induced VEGF expression following intravitreal injection in wild-type mice. Consistent with these results Nozaki and colleagues also

-702018200437 19 Jan 2018 showed that genetic ablation of receptors for C3a and C5a reduces VEGF expression and

CNV formation after laser injury, and that antibody-mediated neutralization of C3a or

C5a or pharmacologic blockade of their receptors also reduces CNV. Previous studies have established that recruitment of leukocytes, and macrophages in particular, plays a pivotal role in laser-induced CNV (Sakurai et ah, Invest. Opthomol. Vis. Sci. 44:3578-85, 2003; Espinosa-Heidmann, etah, Invest. Opthomol. Vis. Sci. 44:3586-92, 2003), In their 2006 paper, Nozaki and colleagues report that leukocyte recruitment is markedly reduced in C3aR(-/“) and C5aR(-/-) mice after laser injury.

An aspect of the Invention thus provides a method for inhibiting 10 MASP-2-dependent complement activation to treat age-related macular degeneration or other complement mediated ophthalmologic condition by administering a composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent in a pharmaceutical carrier to a subject suffering from such a condition or other complement-mediated ophthalmologic condition. The MASP-2 inhibitory composition may be administered locally to the eye, such as by Irrigation or application of the composition in the form of a gel, salve or drops. /Alternately, the MASP-2 inhibitory agent may be administered to the subject systemically, such as by intra-arterial, intravenous, intramuscular, inhalational, nasal, subcutaneous or other parenteral administration, or potentially by oral administration for non-peptidergic agents. The

MASP-2 inhibitory agent composition may be combined with one or more additional therapeutic agents, such as are disclosed hi U.S. Patent Application Publication No. 2004-0072809-A1, Administration may be repeated as determined by a physician until the condition has been resolved or is controlled.

In another aspect, the invention provides a method for inhibiting

MASP-2-dependent complement activation to treat a subject suffering from or at risk for developing glaucoma. It has been shown that uncontrolled complement activation contributes to the progression of degenerative injury to retinal ganglion cells (RGCs), their synapses and axons in glaucoma. See Tezel G. et ah, Invest Ophthalmol Vis Sci 57:5071-5082 (2010), For example, histopathologic studies of human tissues and in vivo studies using different animal models have demonstrated that complement components, including Ciq and C3, are synthesized and terminal complement complex is formed in the glaucomatous retina (see Stasi K. et ah. Invest Ophthalmol Vis Sci 47:1024-1029 (2006), Kuehn M.H. et al., Exp Eye Res 53:620-628 (2006)). As described in Tezel G.

-71“

2018200437 19 Jan 2018 et al., it has been determined that in addition to the classical pathway, the lectin pathway is likely to be involved in complement activation during glaucomatous neurodegeneration, thereby facilitating the progression of neurodegenerative injury by collateral ceil lysis, inflammation and autoimmunity. As described in Tezel G, et al., proteomic analysis of human retinal samples obtained from donor eyes with or without glaucoma detected the expression and differential regulation of several complement components. Notably, expression levels of complement components from the lectin pathway were higher, or only detected, in glaucomatous samples than controls, including MASP-1 and MASP-2, and C-type lectin. As further described in Kuehn M,H. et ah,

Experimental Eye Research §7:89-95 (2008), complement synthesis and deposition is induced by retinal I/R and the disruption of the complement cascade delays RGC degeneration. In this study, mice carrying a targeted disruption of the complement component C3 were found to exhibit delayed RGC degeneration after transient retinal I/R when compared to normal animals.

The findings of these studies suggest that alterations in the physiological balance between complement activation and intrinsic regulation under glaucomatous stress consitions may have an important impact on the progression of neurodegenerative injury, indicating that inhibition of complement activation, such as through the administration of anti-MASP-2 antibodies, can be used as a therapeutic for glaucoma patients.

An aspect of the invention thus provides a method for inhibiting

MASP-2-dependent complement activation to treat glaucoma by administering a composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent in a pharmaceutical carrier to a subject suffering from glaucoma. The MASP-2 inhibitory composition may be administered locally to the eye, such as by irrigation or application of the composition in the form of a gel, salve or drops. Alternately, the MASP-2 inhibitory agent may be administered to the subject systemicaily, such as by intra-arterial, intravenous, intramuscular, inhalational, 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 been resolved or is controlled.

IV, MASP-2 INHIBITORY AGENTS

In one aspect, the present invention provides methods of inhibiting the adverse effects of MASP-2-dependent complement activation, MASP-2 inhibitory agents are

2018200437 19 Jan 2018 administered in an amount effective to inhibit MASP-2-dependent complement activation in a living subject. In the practice of this aspect of the invention, representative MASP-2 inhibitory agents include: molecules that inhibit tire biological activity of MASP-2 (such as small molecule inhibitors, anti-MASP-2 antibodies or blocking peptides which interact with MASP-2 or interfere with a protein-protein interaction), and molecules that decrease the expression of MASP-2 (such as MASP-2 antisense nucleic acid molecules, MASP-2 specific RNAi molecules and MASP-2 ribozymes), thereby preventing MASP-2 from activating the lectin complement pathway. The MASP-2 inhibitory agents can be used alone as a primary therapy or in combination with other therapeutics as an adjuvant therapy to enhance the therapeutic benefits of other medical treatments.

The 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 in accordance with the methods of the invention: the inhibition of the generation or production of

MASP-2-dependent complement activation system products C4b, C3a, C5a and/or C5b-9 (MAC) (measured, for example, as described in Example 2), the reduction of complement activation assessed in a hemolytic assay using unsensitized rabbit or guinea pig red blood cells (measured, for example as described in Example 33), the reduction of C4 cleavage and C4b deposition (measured, for example as described in Example 2), or the reduction of C3 cleavage and C3b deposition (measured, for example, as described in Example 2).

According to the present invention, MASP-2 inhibitory agents are utilized that are effective in inhibiting the MASP-2-dependcnt complement activation system, MASP-2 inhibitory agents useful in the practice of this aspect of the invention include, for example, anti-MASP-2 antibodies and fragments thereof, MASP-2 inhibitory peptides, small molecules, MASP-2 soluble receptors and expression inhibitors. MASP-2 inhibitory agents may inhibit the MASP-2-dependeni complement activation system by blocking the biological function of MASP-2, For example, an inhibitory agent may effectively block MASP-2 protein-to-protein interactions, interfere with MASP-2 dimerization or assembly, block Ca2+ binding, interfere with the MASP-2 serine protease active site, or may reduce MASP-2 protein expression.

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

-732018200437 19 Jan 2018

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

The MASP-2 polypeptide exhibits a molecular structure similar to MASP-1, MASP-3, and Clr and Cis, the proteases of the Cl complement system. The cDNA molecule set forth in SEQ ID NO:4 encodes a representative example of MASP-2 (consisting of the amino acid sequence set forth in SEQ ID NO:5) and provides the human MASP-2 polypeptide with a leader sequence (aa 1-15) that is cleaved after secretion, resulting in the mature form of human MASP-2 (SEQ ID NO:6). As shown in FIGURE 2, the human MASP 2 gene encompasses twelve exons. The human MASP-2 cDNA is encoded by exons B, C, D, F, G, Η, I, J, K AND L. An alternative splice results in a 20 kDa protein termed MBL-associated protein 19 (MApl9”, also referred to as sMAP) (SEQ ID NO:2), encoded by (SEQ ID NO: I) arising from exons B, C, D and E as shown in FIGURE 2. The cDNA molecule set forth in SEQ ID NO.AO encodes the murine MASP-2 (consisting of the amino acid sequence set forth in SEQ ID NO:51) and provides the murine MASP-2 polypeptide with a leader sequence that is cleaved after secretion, resulting in the mature form of murine MASP-2 (SEQ ID NO:52). The cDNA molecule set forth in SEQ ID NO:53 encodes the rat MASP-2 (consisting of the amino acid sequence set forth in SEQ ID NO:54) and provides the rat MASP-2 polypeptide with a leader sequence that is cleaved after secretion, resulting in the mature form of rat

MASP-2 (SEQ ID NO:55).

Those skilled in the art will recognize that the sequences disclosed in SEQ ID NO:4, SEQ ID NO:50 and SEQ ID NO:53 represent single alleles of human, murine and rat MASP-2 respectively, and that allelic variation and alternative splicing are expected to occur. Allelic variants of the nucleotide sequences shown in SEQ ID NO:4, SEQ ID

NO:50 and SEQ ID NO:53, including those containing silent mutations and those in which mutations result in amino acid sequence changes, are within the scope of the present invention. Allelic variants of the MASP-2 sequence can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures.

-742018200437 19 Jan 2018

The domains of the human MASP-2 protein (SEQ ID NO:6) are shown in

FIGURE 1 and 2A and include an N-terminal Clr/Cls/sea urchin Vegf/bone morphogenic protein (CUB!) domain (aa 1-121 of SEQ ID NO:6), an epidermal growth factor-like domain (aa 122-166), a second CUBI domain (aa 167-293), as well as a tandem of complement control protein domains and a serine protease domain. Alternative splicing ofthe MASP 2 gene results in MApl9 shown in FIGURE 1. MApl9 is a nonenzymatic protein containing (he N-terminal CUB 1-EOF region of MASP-2 with four additional residues (EQSL) derived from exon E as shown in FIGURE 1.

Several proteins have been shown to bind to, or interact with MASP-2 through protein-to-protein interactions. For example, MASP-2 is known to bind to, and form Ca2⁺ dependent complexes with, the iectin proteins MBL, H-ficolin and L-ficoiin. Each MASP-2/lectin complex has been shown to activate complement through the MASP-2-dependent cleavage of proteins C4 and C2 (Ikeda, K., et al., J. Biol, Chem. 262:7451-7454, 1987: Matsushita, M„ etal., J. Exp. Med. /76:1497-2284, 2000;

Matsushita, M., etal., J. Immunol. /66:3502-3506, 2002). Studies have shown that the CUB1-EGF domains of MASP-2 are essential for the association of MASP-2 with MBL (Thieiens, N.M., etal,, J. Immunol. /66:5068, 2001). It. has also been shown that the CUB1EGFCUBH domains mediate dimerization of MASP-2, which is required for formation of an active MBL complex (Wallis, R,, et at, J. Biol. Chem. 275:30962-30969,

2000). Therefore, MASP-2 inhibitory agents can be identified that bind to or interfere with MASP-2 target regions known to be Important for MASP-2-dependent complement activation.

ANT1-MA SP-2 ANTIBODIES

In some embodiments of this aspect of the invention, the MASP-2 inhibitory agent comprises an anti-MASP-2 antibody that inhibits the MASP-2-dependent complement activation system. The anti-MASP-2 antibodies useful in this aspect of the invention include polyclonal, monoclonal or recombinant antibodies derived from any antibody producing mammal and may be multispecific, chimeric, humanized, 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.

Several anti-MASP-2 antibodies have been described in the literature, some of which are listed below in TABLE 1. These previously described anti-MASP-2 antibodies

-75can be screened for the ability to inhibit the MASP-2-dependent complement activation system using the assays described herein. For example, anti rat MASP-2 Fab2 antibodies have been identified that block MASP-2 dependent complement activation, as described in more detail in Examples 10 and II herein. Once ars anti-MASP-2 antibody is identified that functions as a MASP-2 inhibitory agent, it can be used to produce anti-idiotype antibodies and used to identify other MASP-2 binding molecules as further described below.

2018200437 19 Jan 2018

TABLE 1: MASP-2 SPECIFIC AN’iΊΒΟΙ.)1	ES FROM THE LITERATURE
ANTIGEN	ANTIBODY TYPE	REFERENCE
Recombinant MASP-2	Rat Polyclonal	Peterson, S.V., et al., Mol. Immunol. 37:803-8 ! 1,2000
Recombinant human CCP1/2-SP fragment (MoAb 8B5)	Rat MoAb (subclass IgG l)	Moller-Kristensen, M., et a!,, J. of Immunol. Methods 282:159-167, 2003
Recombinant human MApl9 (MoAb 6G12) (cross reacts with MASP-2)	Rat MoAb (subclass IgGl)	Moller-Kristensen, M., et ah, J. of Immunol. Methods 282:159-167, 2003
hMASP-2	Mouse MoA b (SZP) Mouse MoAb (N-term)	Peterson, S.V., et al., Mol. Immunol. 35:409, April 1998
hMASP-2 (CCP1-CCP2-SP domain	rat MoAb: Nirooabl 01, produced by hybridoma cell line 03050904 (ECACC)	WO 2004/106384
hMASP-2 (full length-his tagged)	murine MoAbs: NimoAbl04, produced by hybridoma cell line M0545YM035 (DSMZ) NimoAb 108, produced by hybridoma cell line M0545YM029 (DSMZ) NimoAb 109 produced by hybridoma cell line M0545YM046 (DSMZ) NimoAb 110 produced by hybridoma cell line M054SYM048 (DSMZ)	WO 2004/106384

-762018200437 19 Jan 2018

ANTI-MASP-2 ANTIBODIES WITH REDUCED EFFECTOR FUNCTION

In some embodiments of this aspect of the invention, the anti-MASP-2 antibodies have reduced effector function in order to reduce inflammation that may arise from the activation of the classical complement pathway. 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.. eta!,, 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 can be generated as the result of lacking the Fc portion of the molecule by having a genetically engineered Fc sequence that minimizes effector function, or being of either the human IgG2 or IgG4 isotype.

Antibodies with reduced effector function can be produced by standard molecular biological manipulation of the Fc portion of the IgG heavy chains as described in

Example 9 herein and also described in Jolliffe etai., Inti Rev. Immunol. /6:241-250, 1993, and Rodrigues etak, 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. 6:457-492, 1991; Isaacs, J.D., etak, J Immunol. /95:3062-3071, 1992; van de

Winkel, J.G., etak, Immunol. Today 14:215-221, 1993). Humanized or fully human antibodies specific to human MASP-2 comprised of lgG2 or IgG4 isotypes can be produced by one of several methods known to one of ordinary skilled in the art, as described in Vaughan, T.J., et al,, Nature Bioteckmcal /6:535-539, 1998.

PRODUCTION OF ANTI-MASP-2 ANTIBODIES

Anti-MASP-2 antibodies can be produced using MASP-2 polypeptides (e.g,, full length MASP-2) or using antigenic: MASP-2 epitope-bearing peptides (e.g., a portion of the MASP-2 polypeptide). Immunogenic peptides may be as small as five amino acid residues. For example, the MASP-2 polypeptide including the entire amino acid sequence of SEQ ID NO:6 may be used to induce anti-MASP-2 antibodies useful in the method of the invention. Particular MASP-2 domains known to be involved in protein-protein interactions, such as the CUBI, and CUBIEGF domains, as well as the region encompassing the serine-protease active site, may be expressed as recombinant polypeptides as described in Example 3 and used as antigens. In addition, peptides

2018200437 19 Jan 2018 comprising a portion of at least 6 amino acids of the MASP-2 polypeptide (SEQ ID NO:6) are also useful to induce MASP-2 antibodies. Additional examples of MASP-2 derived antigens useful to induce MASP-2 antibodies are provided below in TABLE 2. The MASP-2 peptides and polypeptides used to raise antibodies may be isolated as natural polypeptides, or recombinant or synthetic peptides and catalytically inactive recombinant polypeptides, such as MASP-2A, as further described in Examples 5-7. in some embodiments of this aspect of the invention, anti-MASP-2 antibodies are obtained using a transgenic mouse strain as described in Examples 8 and 9 and further described below.

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

TABLE 2: MASP-2 DERIVED ANTIGENS

SEQ ID NO:	Amino Acid Sequence
SEQ ID NO:6	Human MASP-2 protein
SEQ ID NO 51	Murine MASP-2 protein
SEQ ID NO:8	CUBI domain of human MASP-2 (aa 1-121 of SEQ ID NO:6)
SEQ ID NO:9	CUBIEGF domains of human MASP-2 (aa 1-166 of SEQ ID NO :6)
SEQ ID NO: 10	CUB1FGFCUBII domains of human MASP-2 .('aa 1-293 of SEQ ID NOto)
SEQ ID NO:11	EGF domain of human MASP-2 (aa 122-166 of SEQ1DNO:6)
SEQ ID NO: 12	Serine-Protease domain of human MASP-2 (aa 429-671 of SEQ ID Nto.6)
SEQ ID NO:13 GKDSCRODAGGALVFL	Serine-Protease inactivated mutant form (aa 610-625 ot M Q if) NO:6 with mutated Ser 63 8)
SEQ ID NO: 14 TPLGPKWPEPVFGRL	Human CUBI peptide

-782018200437 19 Jan 2018

|. SEQ ID NO:	Amino Acid Sequence
SEQ ID NOT5:	Human CUBI peptide 1
TA PPG YRLRL YFTHFDLEL
SHLCEYDFVKLSSGAKVL
s ATLCGQ
SEQ ID NO: 16:	.MBL binding region in human CUBI domain i
ί TFRSDYSN
i SEQ ID NOT 7:	MBL binding region in human CUBI domain |
1 FYSLGSSLDITFRSDYSNEK	:
iPFTGF
SEQ ID NOT 8	EGF peptide
| iDECQYAPG
SEQ ID NO: 19	Peptide from serine-protease active site i
ANMLCAGLESGGKDSCRG	5
DSGGALV

POLYCLONAL ANTIBODIES

Polyclonal antibodies against MASP-2 can be prepared by immunizing an animal with MASP-2 polypeptide or an immunogenic portion thereof using methods well known to those of ordinary skill in the art. See, for example, Green eta!,, Production of Polyclonal Antisera, in Immunochemical Protocols (Manson, ed.), page 105, and as further described in Example 6, The immunogenicity of a MASP-2 polypeptide can be increased through the use of an adjuvant, including mineral gels, such as aluminum hydroxide or Freund's adjuvant (complete or incomplete), surface active substances such as lysolecithin, pluronic polyols, polyanions, oil emulsions, keyhole limpet hemocyanin and dinitrophenol. Polyclonal antibodies are typically raised in animals such as horses, cows, dogs, chicken, rats, mice, rabbits, guinea pigs, goats, or sheep. Alternatively, an anti-MASP-2 antibody useful in the present invention may also be derived from a subhuman primate. General techniques for raising diagnostically and therapeutically useful antibodies in baboons may be found, tor example, in Goldenberg etal., international Patent Publication No. WO 91/11465, and in Losman, MJ., etal., Int J Cancer 46:310, 1990, Sera containing iminunologicaily active antibodies are then produced from the blood of such immunized animals using standard procedures well known in the art.

-79MONOCLONAL ANTIBODIES

O co

2018200437 19 Jan

In some embodiments, the MASP-2 inhibitory agent is an anti-MASP-2 monoclonal antibody, Anti-MASP-2 monoclonal antibodies are highly specific, being directed against a single MASP-2 epitope. As used herein, the modifier monoclonal indicates the character of the antibody as being obtained from a substantially homogenous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. Monoclonal antibodies can be obtained using any technique that provides for the production of antibody 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 made by recombinant DNA methods (see, e.g., U.S. Patent

No. 4,816,567 io Cabilly). Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson, T., et al., Nature 5.52:624-628, 1991, and Marks, J.D., et ah, J Mol. Biol. 222:581-597, 1991. Such antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.

For example, monoclonal antibodies can be obtained by injecting a suitable mammal (e.g., a BALB/c mouse) with a composition comprising a MASP-2. polypeptide or portion thereof. After a predetermined period of time, splenocytes are removed from the mouse and suspended in a ceil 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-2, An example further describing the production of anti-MASP-2 monoclonal antibodies is provided in Example 7. (See also Current Protocols in Immunology, Vol. 1., John Wiley & Sons, pages 2.5.1-2.6.7, 1991.)

Human monoclonal antibodies may be obtained through the use of transgenic mice that have been engineered to produce specific human antibodies in response to 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 targeted disruptions of the endogenous immunoglobulin heavy chain and light chain loci, The transgenic mice can synthesize human antibodies specific for human antigens, such as the MASP-2 antigens described herein, and the mice can be used to produce human MASP-2 antibody-secreting hybridomas by fusing B-cells from such animals to suitable myeloma cell lines using conventional Kohler-Milstein technology as

802018200437 19 Jan 2018 further described in Example 7. Transgenic mice with a human immunoglobulin genome are commercially available (e.g., from Abgenix, Inc., Fremont, CA, and Medarex, Inc.,

Annandale, N.J.). Methods for obtaining human antibodies from transgenic mice are described, for example, by Green. L.L., etal,, Nature Genet. 7:13, 1994; Lonberg, N., et ah, Nature 368:856, 1994; and Taylor, L.D., et ah, Int. Immun, 6:579, 3 994.

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

Molecular Biology, The Humana Press, Inc,, Vol. 10, pages 79-104, 1992).

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

The affinity of anti-MASP-2 monoclonal antibodies can be readily determined by one of ordinary skill in the art (see, e.g., Scatchard, A., NY Acad. Sci. 52:660-672, 1949). In one embodiment, the anti-MASP-2 monoclonal antibodies useful for the methods of the invention bind to MASP-2 with a binding affinity of <100 nM, preferably <10 nM and most preferably <2 nM.

CHIMERIC/HUMANIZED ANTIBODIES

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 with or homologous to corresponding sequences irs antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s)

-812018200437 19 Jan 2018 is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies (U.S. Patent No, 4,816,567, to Cabilly; and Morrison, S.L., etal,,

Proc. Natl Acad Sci. USA 81:6851-6855, 1984),

One form of a chimeric antibody useful in the invention is a humanized monoclonal anti-MASP-2 antibody. Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies, which contain minima! sequence derived from non-human immunoglobulin. Humanized monoclonal antibodies are produced by transferring the non-human (e.g., mouse) complementarity determining regions (CDR), from the heavy and light variable chains of the mouse immunoglobulin into a human variable domain. Typically, residues of human antibodies are then substituted in the framework regions of the non-human counterparts. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two variable domains, in which al! or substantially all ofthe hypervariable loops correspond to those of a non-human immunoglobulin and ail or substantially all ofthe Fv framework regions are those of a human immunoglobulin sequence, The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fe), typically that of a human immunoglobulin. For further details, see Jones, P.T., et al., Nature 327:522-525, 1986; Reicbmann, L., et ai., Nature 332:323-329, 1988; and Presta, Curr. Op. Struct. Bio/. 2:593-596, 1992,

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

An example of the generation of a humanized anti-MASP-2 antibody from a murine anti-MASP-2 monoclonal antibody is provided herein in Example 6. Techniques for producing humanized monoclonal antibodies are also described, for example, by Jones, P.T., etal., Nature 327:522, 1986; Carter, P,, etal,, Proc. Nat'l. Acad Sci.

-822018200437 19 Jan 2018

USA 69:4285, 1992; Sandhu, J.S., Crit. Rev. Biotech. /2:437, 1992; Singer, Ι.Ϊ., etal., J.

Immun. /50:2844, 1993; Sudhir (ed.), Antibody Engineering Protocols, Humana Press,

Inc., 1995; Kelley, Engineering Therapeutic Antibodies, in Protein Engineering:

Principles and Practice, Cleland etal. (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

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

ANTI-IDIO'TYPE ANTIBODIES

Once anti-MASP-2 antibodies are identified with the desired inhibitory activity.

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

IMMUNOGLOBULIN FRAGMENTS

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

It is well known in the art that only a small portion of an antibody molecule, the paratope, is involved in the 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

-832018200437 19 Jan 2018 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 of the 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 which has been produced without the Fc region. Is designated a Fab fragment, and retains one of the antigen binding sites of an intact antibody molecule.

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

2.8.1-2.8.10 and 2.10,-2.10.4.

In some embodiments, the use of antibody fragments lacking the Fc region are preferred to avoid activation of the classical complement pathway which is initiated upon binding Fc to the Fey receptor. There are several methods by which one can produce a MoAb that avoids Fey receptor interactions. For example, the Fc region of a monoclonal antibody can be removed chemically 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 (Mariant, M., et al., Moi. Immunol. 28:69-l\, 1991). Alternatively, the human y4 IgG isotype, which does not bind Fey receptors, can be used during construction of a humanized antibody as described herein. Antibodies, single chain antibodies and antigen-binding domains that lack the Fc domain can also be engineered using recombinant techniques described herein.

-842018200437 19 Jan 2018

SINGLE-CHAIN ANTIBOD Y FRAGMENTS

Alternatively, one can ereale single peptide chain binding molecules specific for

MASP-2. in which the heavy and light chain Fv regions are connected. The Fv fragments may be connected by a peptide linker to form a single-chain antigen binding protein (scFv). These single-chain antigen binding proteins are prepared by constructing a structural gene comprising DNA sequences encoding the Vjf 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 ceil, such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing scFvs are described for example, by Whitlow', etah, Methods: A Companion to Methods in Enzymology 2:97, 1991; Bird, etal., Science 242:423, 1988; U.S. Patent No. 4,946,778, to Ladner; Pack, P., etah, Bio/Technology 77:1271,1993.

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

Another form of an anti-MASP-2 antibody fragment useful in this aspect of the invention is a peptide coding for a single complementarity-determining region (CDR) that binds to an epitope on a MASP-2 antigen and inhibits MASP-2-dependent complement activation. CDR peptides (minimal recognition units”) can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for

-852018200437 19 Jan 2018 example, by using the polymerase chain reaction to synthesize the variable region from

RNA of antibody-producing cells (see, for example, Larrick et ah, 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 etal. (eds.), page 166, Cambridge University Press, 1995; and Ward etal., Genetic Manipulation and Expression of Antibodies, in Monoclonal Antibodies: Principles and Applications, Birch et ah (eds,), page 137, Wiley-Liss, Inc., 1995),

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

PEPTIDE INHIBITORS

In some embodiments of this aspect of the invention, the MASP-2 inhibitory agent comprises isolated MASP-2 peptide inhibitors, including isolated natural peptide inhibitors and synthetic peptide inhibitors that inhibit the MASP-2-dependent complement activation system. As used herein, the term isolated MASP-2 peptide Inhibitors refers to peptides that inhibit MASP-2 dependent complement activation by binding to, competing with MASP-2 for binding to another recognition molecule (e.g.,

MBL, H-ficolin, M-ficolin, or L-ficolin) in the lectin pathway, and/or directly interacting with MASP-2 to inhibit MASP-2-dependent complement activation that are substantially pure and are essentially free of other substances with which they may be found in nature to an extent practical and appropriate for their intended use.

Peptide inhibitors have been used successfully in vivo to interfere with protein-protein interactions and catalytic sites. For example, peptide inhibitors to adhesion molecules structurally related to LFA-l have recently been approved for clinical use in coagulopathies (Ohman, E.M., etal,, European Heart J. 16:50-55, 1995). Short linear peptides (<30 amino acids) have been described that prevent or interfere with integrin-dependent adhesion (Murayama, O., etal., J Biochem. /29:445-51, 1996).

Longer peptides, ranging in length from 25 to 200 amino acid residues, have also been used successfully to block integrin-dependent adhesion (Zhang, L., etal., J. Biol. Chem. 277(47):29953-57, 1996), In general, longer peptide inhibitors have higher affinities and/or slower off-rates than short peptides and may therefore be more potent

-862018200437 19 Jan 2018 inhibitors, Cyclic peptide inhibitors have also been shown to be effective inhibitors of integrins in vivo for the treatment of human inflammatory disease (Jackson, D.Y., et al.,

J, Med. Chem. 49:3359-68. 1997). One method of producing cyclic peptides involves the synthesis of peptides in winch the terminal amino acids of the peptide are cysteines, thereby allowing the peptide to exist in a cyclic form by disulfide bonding between the terminal amino acids, which has been shown to improve affinity and hall-life in vivo for the treatment of hematopoietic neoplasms (e.g,, U.S. Patent No, 6,649,592, to Larson),

SYNTHETIC MASP-2 PEPTIDE INHIBITORS

MASP-2 inhibitory peptides useful in the methods of this aspect of the invention 10 are exemplified by amino acid sequences that mimic the target regions important for MASP-2 function. The inhibitory peptides useful in the practice of the methods of the invention range in size from about 5 amino acids to about 300 amino acids, TABLE 3 provides a list of exemplary inhibitory peptides that may be useful in the practice of this aspect of the present invention. A candidate MASP-2 inhibitory peptide may be tested for the ability to function as a MASP-2 inhibitory agent in one of several assays including, for example, a lectin specific C4 cleavage assay (described in Example 2), and a C3b deposition assay (described in Example 2).

In some embodiments, the MASP-2 inhibitory peptides are derived from MASP-2 polypeptides and are selected from the full length mature MASP-2 protein (SEQ ID

N():6), or from a particular domain of the MASP-2 protein such as, for example, the

CUBI domain (SEQ ID NO:8), the CUBIEGF domain (SEQ ID NO:9), the EGF domain (SEQ ID NO:11), and the serine protease domain (SEQ ID NO:12). As previously described, the CUBEGFCUBII regions have been shown to be required for dimerization and binding with MBL (Thielens et al., supra}. In particular, the peptide sequence

TFRSDYN (SEQ ID NO: 16) in the CUBI domain of MASP-2 has been shown to be involved in binding to MBL in a study that identified a human carrying a homozygous mutation at Aspl05 to Glyl 05, resulting in the loss of MASP-2 from the MBL complex (Stengaard-Pedersen, K., et ah. New England J. Med. 349:554-560, 2003),

In some embodiments, MASP-2 inhibitory peptides are derived from the lectin proteins that bind to MASP-2 and are involved in the lectin complement pathway, Several different lectins have been identified that are involved in this pathway, including mannan-binding lectin (MBL), L-ficolin, M-ficolin and H-ficolin. (Ikeda, K., etal., J. Biol. Chem. 262:7451-7454, 1987; Matsushita, M., etal., J Exp. Med. /76:1497-2284,

-872018200437 19 Jan 2018

2000; Matsushita, M., et al., J. Immunol. 768:3502-3506,2002), These lectins are present in serum as oligomers of homotrimeric subunits, each having N-terminal coilagen-like fibers with carbohydrate recognition domains. These different lectins have been shown to bind to MASP-2, and the lectin/MASP-2 complex activates complement through cleavage of proteins C4 and C2. H-ficolin has an amino-terminal region of 24 amino acids, a coilagen-like domain with 11 Gly-Xaa-Yaa repeats, a neck domain of 12 amino acids, and a fibrinogen-like domain of 207 amino acids (Matsushita, M., et ah, J Immunol. /68:3502-3506, 2002). H-ficolin binds to GlcNAc and agglutinates human erythrocytes coated with LPS derived from .S’, typhimurium, S. Minnesota and E. coli.

H-ficolin has been shown to be associated with MASP-2 arid MApl9 and activates the lectin pathway. Id. L-ficolin/P35 also binds to GlcNAc and has been shown to be associated with MASP-2 and MApl9 in human serum and this complex has been shown to activate the lectin pathway (Matsushita, M., etab, J. Immunol. /69:2281, 2000). Accordingly, MASP-2 inhibitory peptides useful in the present invention may comprise a region of at least 5 amino acids selected from the MBL protein (SEQ ID NO:21), the H-ficolin protein (Genbank accession number NM__173452), the M-ficolin protein (Genbank accession number 000602) and the L-ficolin protein (Genbank accession number NM 015838),

More specifically, scientists have identified the MASP-2 binding site on MBL to be within the 12 Giy-X-Y triplets GKD GRD GTK GEK GEP GQG LRG LQG POG KLG POG NOG PSG SOG PKG QKG DOG KS (SEQ ID NO:26) that lie between the hinge and the neck in the C-terminal portion of the coilagen-like domain of MBP (Wallis, R,, et al,, J. Biol. Chem. 279:14065, 2004). This MASP-2 binding site region is also highly conserved in human H-ficolin and human L-ficolin. A consensus binding site has been described that is present in all three lectin proteins comprising the amino acid sequence GGK-X-GP (SEQ ID NO:22) where the letter Ό represents hydroxyproline and the letter X is a hydrophobic residue (Wallis et ah, 2004, supra). Accordingly, in some embodiments, MASP-2 inhibitory peptides useful in this aspect of the invention are at least 6 amino acids in length and comprise SEQ ID NO:22. Peptides derived from

MBL that include the amino acid sequence GLR GLQ GPO GKL GPO G (SEQ ID NO:24) have been shown to bind MASP-2 in vitro (Wallis, et al., 2004, supra). To enhance binding to MASP-2, peptides can be synthesized that are flanked by two GPO triplets at each end (GPO GPO GLR GLQ GPO GKL GPO GGP OGP O SEQ ID

-882018200437 19 Jan 2018

NO:25) io enhance the formation of triple helices as found in the native MBL- protein (as further described in Wallis, R., et al., J. Biol Chem. 279:14065, 2004).

MASP-2 inhibitory peptides may also be derived from human H-ficolin that include the sequence GAO GSO GEK GAO GPQ GPO GPO GKM GPK GEO GDO (SEQ ID NO:27) from the consensus MASP-2 binding region in H-ficoiin. Also included are peptides derived from human L-ficolin that include the sequence GCO GLO GAO GDK GEA GTN GKR GER GPO GPO GKA GPO GPN GAO GEO (SEQ ID 140:28) from the consensus MASP-2 binding region in L-ficolin.

MASP-2 inhibitory peptides may also be derived from the C4 cleavage site such as LQRALEILPNRVTIKANRPFLVFI (SEQ ID NO:29) which is the C4 cleavage site linked to the C-terminal portion of antithrombin ill (Glover, G.I., et al., Moi. Immunol. 25:1261 (1988)),

TABLE 3:.. EXEMPLARY MASP-2 INHIBITORY PEPTIDES

SEQ ID NO	Source
SEQ ID NO;6	Human MASP-2 protein
SEQ ID NO:S	CUBI domain of MASP-2 (aa I-121 of SEQ ID NQ:S)
SEQ ID NON	CUBIEGF domains of MASP-2 (aa 1-166 of SRQ ID NO'6)
SEQ ID NO: 10	CUBIEGFCUBIl domains of MASP-2 (aa 1-293 ot SEQ 1:) NO:6)
SEQ ID NO: 11	EGF domain, of MASP-2 (aa 122-166)
SEQ ID NO: 12	Serin? pt «tease domain of MASP-2 .(aa 429-671)
SEQ ID NO: 16	....... MBL binding region in MASP-2
SEQ ID NOG	Human MAjJ 19
SEQIDNO:21	Human MBL protein
SEQ ID NO:22 OGK-X-GP, Where ”0” = hydroxy proline and X is a hydrophobic amino acid residue	Synthetic peptide Consensus binding site from Human MBL and Human ficoiins

-892018200437 19 Jan 2018

SEQ ID NO	Source
SEQ ID NO:23 OGKLG	Human MBL core binding site
SEQ 1DNO:24 GLR GLQ GPO GKL GPO G	Human MBP Triplets 6-10- demonstrated binding to MASP-2
SEQ ID NO:25 GPOGPOGLRGLQGPO GKLGPOGGPQGPO	Human MBP Triplets with GPO added to enhance formation of triple helices
SEQ ID NO:26 GKDGRDGTKGEKGEP GQGLRGLQGPOGKLG POGNOGPSGSOGPKG QKGDOGKS	Human MBP Triplets 1-17
SEQ ID NO:27 GAOGSOGEKGAOGPQ GPOGPOGKMGPKGEO GDO	Human H-Ficolin (Hataka)
SEQ ID NO:28 GCOGLOGAOGDKGE AGTNGKRGERGPOGP OGKAGPOGPNG AOG E O	Human L-Ficolin P35
SEQ ID NO:29 LQRALEILPNRVTI KA NRPFLVEI	Human C4 cleavage site

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

Peptides derived from the C4 cleavage site as well as other peptides that inhibit the MASP-2 serine protease site can be chemically modified so that they are irreversible protease inhibitors. For example, appropriate modifications may include, but are not necessarily limited to, halomethyl ketones (Br, Cl, 1, F) at the C-terminus, Asp or Glu, or appended to functional side chains: haloacetyl (or other α-haioacetyl) groups on amino groups or other functional side chains; epoxide or imine-containing groups on the amino or carboxy termini or on functional side chains; or imidate esters on the amino or carboxy termini or on functional side chains. Such modifications would afford the advantage of

-902018200437 19 Jan 2018 permanently inhibiting the enzyme by covalent attachment of the peptide. This could result in lower effective doses and/or the need for less frequent administration of the peptide inhibitor.

In addition to the inhibitory peptides described above, MASP-2 inhibitory 5 peptides useful in the method of the invention include peptides containing the MASP-2-binding CDR3 region of anti-MASP-2 MoAb obtained as described herein. The sequence of the CDR regions for use in synthesizing the peptides may be determined by methods known in the art. The heavy chain variable region is a peptide that generally ranges from 300 to 150 amino acids in length. The light chain variable region is a peptide that generally ranges from SO to 130 amino acids in length. The CDR sequences within the heavy and light chain variable regions include only approximately 3-25 amino acid sequences that may be easily sequenced by one of ordinary skill in the art.

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

MASP-2 inhibitory peptides may be modified to increase solubility and/or to maximize the positive or negative charge in order to more closely resemble the segment in the intact protein. The derivative may or may not have the exact primary amino acid structure of a peptide disclosed herein so long as the derivative functionally retains the desired property of MASP-2 inhibition. The modifications can include amino acid substitution with one ofthe commonly known twenty amino acids or with another amino acid, with a derivatized or substituted amino acid with ancillary desirable characteristics, such as resistance to enzymatic degradation or with a D-amino acid or substitution with another molecule or compound, such as a carbohydrate, which mimics the natural confirmation and function ofthe amino acid, amino acids or peptide; amino acid deletion; amino acid insertion with one of the commonly known twenty' amino acids or with

-912018200437 19 Jan 2018 another amino acid, with a derivatized or substituted amino acid with ancillary desirable characteristics, such as resistance to enzymatic degradation or with a D-amino acid or substitution with another molecule or compound, such as a carbohydrate, which mimics the natural confirmation and function of the amino acid, amino acids or peptide; or substitution with another molecule or compound, such as a carbohydrate or nucleic acid monomer, which mimics the natural conformation, charge distribution and function of the parent peptide. Peptides may also be modified by acetylation or amidation.

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

SCREENING FOR MASP-2 INHIBITORY PEPTIDES

One may also use molecular modeling and rational molecular design to generate and screen for peptides that mimic the molecular structures of key binding regions of MASP-2 and inhibit the complement activities of MASP-2, The molecular structures used for modeling include the CDR regions of anti-MASP-2 monoclonal antibodies, as well as the target regions known to be important for MASP-2 function including the region required for dimerization, the region involved in binding to MBL, and the serine protease active site as previously described. Methods for identifying peptides that bind to a particular target are well known in the art. For example, molecular imprinting may be used for the de novo construction of macromolecular structures such as peptides that bind to a particular molecule. See, for example, Shea, KJ., Molecular Imprinting of Synthetic Network Polymers: The DeNovo synthesis of Macromolecular Binding and

Catalytic Sties, TRIP 2(5) 1994.

As an illustrative example, one method of preparing mimics of MASP-2 binding peptides is as follows. Functional monomers of a known MASP-2 binding peptide or the binding region of an anti-MASP-2 antibody that exhibits MASP-2 inhibition (the

-922018200437 19 Jan 2018 template) are polymerized. The template is then removed, followed by polymerization of a second class of monomers In the void left by the template, to provide a new molecule that exhibits one or more desired properties that are similar to the template, in addition to preparing peptides in this manner, other MASP-2 binding molecules that are MASP-2 inhibitory agents such as polysaccharides, nucleosides, drugs, nucieoproteins, lipoproteins, carbohydrates, glycoproteins, steroid, lipids and other biologically active materials can also be prepared. This method is useful for designing a wide variety of biological mimics that are more stable than their natural counterparts because they are typically prepared by free radical polymerization of fimction monomers, resulting in a compound with a nonbiodegradable backbone.

PEPTIDE SYNTHESIS

The MASP-2 inhibitory peptides can be prepared using techniques well known in the art, such as the solid-phase synthetic technique initially described by Merrifield, in J Amer. Chem. Soc. 85:2149-2154, 1963. Automated synthesis may be achieved, for example, using Applied Biosystems 431A Peptide Synthesizer (Foster City, Calif.) in accordance with the instructions provided by the manufacturer. Other techniques may be found, for example, in Bodanszky, M., et ah, Peptide Synthesis, second edition, John Wiley & Sons, 1976, as well as in other reference works known to those skilled in the art.

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

The MASP-2 inhibitory peptides that are useful in the method of the invention can also be produced in recombinant host cells following conventional techniques, To express a MASP-2 inhibitory peptide encoding sequence, a nucleic acid molecule encoding the

-932018200437 19 Jan 2018 peptide must be operably linked to regulatory sequences that control transcriptional expression in an expression vector and then introduced into a host cell, in addition to transcriptional regulatory sequences, such as promoters and enhancers, expression vectors can include translational regulatory sequences and a marker gene, which are suitable for selection of cells that carry the expression vector.

Nucleic acid molecules that encode a MASP-2 inhibitory peptide can be synthesized with gene machines using protocols such as the phosphoramidite method. If chemically synthesized double-stranded DNA is required for an application such as the synthesis of a gene or a gene fragment, then each complementary' strand is made separately. The production of short genes (60 to 80 base pairs) is technically straightforward and can be accomplished by synthesizing the complementary strands and then annealing them. For the production of longer genes, synthetic genes (double-stranded) are assembled in modular form from single-stranded fragments that are from 20 to 100 nucleotides in length. For reviews on polynucleotide synthesis, see, for example, Glick and Pasternak, Molecular Biotechnology, Principles and Applications of Recombinant DNA”, ASM Press, 1994; itakura, K., etai,, Amu. Rev. Biochem. 55:323, 1984; and Clirnie, S., et al., Proc. Nat'lAcad. Sci. USA 87:633, 1990.

SMALL MOLECULE INHfBiTORS

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

Smail molecule inhibitors may also be designed and generated based on the

MASP-2 crystal structure using computational drug design (Kuntz I.D., etai,, Science 257:1078, 1992). The crystal structure of rat MASP-2 has been described (Feinberg, H., etai., EMBO J. 22:2348-2359, 2003), Using the method described by Kuntz et ah, the MASP-2 crystal structure coordinates are used as an input for a computer program such as DOCK, which outputs a list of small molecule structures that are expected to bind to MASP-2, Use of such computer programs is well known to one of skill in the art. For example, the crystal structure of the HIV-1 protease inhibitor was used to identify unique nonpeptide ligands that are HiV-i protease inhibitors by

-942018200437 19 Jan 2018 evaluating the fit of compounds found in the Cambridge Crystallographic database to the binding site of the enzyme using the program DOCK (Kuntz, LD., etal., J. Mol,

Biol. ./6./:269-288, 1982; DesJarlais, R.L., etal,, PAAS 87:6644-6648, 1990).

The list of small molecule structures that are identified by a computational method 5 as potential MASP-2 inhibitors are screened using a MASP-2 binding assay such as described in Example 10. The small molecules that are found to bind ίο MASP-2 are then assayed in a functional assay such as described in Example 2 to determine if they inhibit MASP-2-dependent complement activation.

MASP-2 SOLUBLE RECEPTORS

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

EXPRESSION INHIBITORS OF MASP-2

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

Anti-sense RNA and DNA molecules act to directly block the translation of

MASP-2 mRNA by hybridizing to MASP-2 mRNA and preventing translation of MASP-2 protein, An antisense nucleic acid molecule may be constructed in a number of different ways provided that it is capable of interfering with the expression of MASP-2. For example, an antisense nucleic acid molecule can be constructed by inverting the coding region (or a portion thereof) of MASP-2 cDNA (SEQ ID NO:4) relative to its normal orientation for transcription to allow for the transcription of its complement.

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

-952018200437 19 Jan 2018 identity of more than about 80% typically is preferred, though about 95% to absolute identity is typically most preferred,

The antisense nucleic acid molecule need not have the same intron or exon pattern as the target gene, and non-coding segments of the target gene may be equally effective in achieving antisense suppression of target gene expression as coding segments. A DNA sequence of at least about 8 or so nucleotides may be used as the antisense nucleic acid molecule, although a longer sequence is preferable. In the present invention, a. representative example of a useful inhibitory agent of MASP-2 is an antisense MASP-2 nucleic acid molecule which is at least ninety percent identical to the complement of the

MASP-2 cDNA consisting ofthe nucleic acid sequence set forth in SEQ ID NO:4. The nucleic acid sequence set forth in SEQ ID NO:4 encodes the MASP-2 protein consisting ofthe amino acid sequence set forth in SEQ ID N0:5.

The targeting of antisense oligonucleotides to bind MASP-2 mRNA is another mechanism that may be used to reduce the level of MASP-2 protein synthesis. For example, the synthesis of polygalacturonase and the muscarine type 2 acetylcholine receptor is inhibited by antisense oligonucleotides directed to their respective mRNA sequences (U.S, Patent No. 5,739,119, to Cheng, and U.S, Patent No. 5,759,829, to Shewmaker). Furthermore, examples of antisense inhibition have been demonstrated with the nuclear protein cyclin, the multiple drug resistance gene (MDG1), ICAM-I,

E-selectin, STK-1, striatal GABA_a receptor and human EGF (see, e.g., U.S. Patent No. 5,801,154, to Baracchini; U.S. Patent No. 5,789,573, to Baker; U.S. Patent No. 5,718,709, to Considine; and U.S, Patent No, 5,610,288, to Reubenstein).

A system has been described that allows one of ordinary sk ill to determine which oligonucleotides are useful in the invention, which involves probing for suitable sites in the target mRNA using Rnase H cleavage as an indicator for accessibility of sequences within the transcripts, Scherr, M., etal., Nucleic Acids Res. 26:5079-5085, 1998: Lloyd, etal., Nucleic Acids Res. 29:3665-3673, 2001, A mixture of antisense oligonucleotides that are complementary to certain regions of the MASP-2 transcript is added to cell extracts expressing MASP-2, such as hepatocytes, and hybridized in order to create an RNAseH vulnerable site. This method cars be combined with computer-assisted sequence selection that can predict optimal sequence selection for antisense compositions based upon their relative ability to form dimers, hairpins, or other secondary structures that would reduce or prohibit specific binding to the target mRNA in

-962018200437 19 Jan 2018 a host cell. These secondary structure analysis and target site selection considerations may be performed using the OLIGO primer analysis software (Rychlik, 1., 1997) and the BLASTN 2.0.5 algorithm software (AJtsehul, S.F., et al., Nucl. Acids Res. 25:3389-3402, 1997). The antisense compounds directed towards the target sequence preferably comprise from about 8 to about 50 nucleotides in length. Antisense oligonucleotides comprising from about 9 to about 35 or so nucleotides are particularly preferred. The inventors contemplate all oligonucleotide compositions in the range of 9 to 35 nucleotides (i.e., those of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 or so bases in length) are highly preferred for the practice of antisense oligonucleotide-based methods of the invention. Highly preferred target regions of the MASP-2 mRNA are those that, are at or near the AUG translation initiation codon, and those sequences that are substantially complementary to 5’ regions of the mRNA, e.g., between the —10 and +10 regions of the MASP-2 gene nucleotide sequence (SEQ ID NO:4). Exemplary MASP-2 expression inhibitors are provided in TABLE 4.

TABLE 4:

EXEMPLARY EXPRESSION INHIBITORS OF MASP· .9

SEQ ID NO:30 (nucleotides 22-680 of SEQ ID NO:4)

SEQ ID NOG 1 ί 5'CGGGCACACCATGAGGCTGCTG jACCCTCCTGGGC3____ | SEQ ID NO:32

5'GACATTACCTTCCGCTCCGACTC j C.AACGAGAAG3' ΐ SEQ ID NO:33

I 5 AGCAGCCCTGAATACCCACGGCC j GT A'IGGGAA AT t Nucleic acid sequence of MASP-2 cDNA i (SEQ ID NO:4) encoding CUBIEGF j:

Nucleotides 12-45 of SEQ ID NO:4 including the MASP-2 translation start site (sense) ...... ..

Nucleotides 361-396 of SEQ ID NO:4 encoding a region comprising the MASP-2 MBL binding site (sense)

Nucleotides 610-642 of SEQ ID NO:4 encoding a region comprising the CUBII domain

As noted above, the term oligonucleotide as used herein refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof.

This term also covers those oligonucleobases composed of naturally occurring nucleotides, sugars and covalent intemucleoside (backbone) linkages as well as oligonucleotides having non-naturally occurring modifications. These modifications allow one to introduce certain desirable properties that are not offered through naturally

-972018200437 19 Jan 2018 occurring oligonucleotides, such as reduced toxic properties, increased stability against nuclease degradation and enhanced cellular uptake. In illustrative embodiments, the antisense compounds of the invention differ from native DNA by the modification of the phosphodiester backbone to extend the life of the antisense oligonucleotide in which the phosphate substituents are replaced by phospborothioates. Likewise, one or both ends of the oligonucleotide may br: substituted by one or more acridine derivatives that Intercalate between adjacent basepairs within a strand of nucleic acid.

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

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

Ribozymes can also be utilized to decrease the amount and/or biological activity of MASP-2, such as ribozymes that target MASP-2 mRNA. Ribozymes are catalytic RNA molecules that can cleave nucleic acid molecules having a sequence that is completely or partially homologous to the sequence of the ribozyme. It is possible to

-982018200437 19 Jan 2018 design ribozyme transgenes that encode RNA ribozymes that specifically pair with a target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA. In carrying out this cleavage, the ribozyme is not itself altered, and is thus capable of recycling and cleaving other molecules. The inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the antisense constructs.

Ribozymes useful in the practice of the invention typically comprise a hybridizing region of at least about nine nucleotides, which is complementary in nucleotide sequence to at least part of the target MASP-2 mRNA, and a catalytic region that is adapted to cleave the target MASP-2 mRNA (see generally, EPA No, 0 321 201; W088/04300; Haseloff, J., etai., Nature 534:585-591, 1988; Fedor, M,J., etai., Proc. Nail. Acad, Sci. USA 67:1668-1672, 1990; Cech, T.R., etal., Rev, Biochem. 55:599-629, 1986).

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

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

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

.00.

2018200437 19 Jan 2018

V, PHARMACEUTICAL COMPOSITIONS AND DELIVERY METHODS

DOSING

In another aspect, the invention provides compositions for inhibiting the adverseeffects of MASP-2-dependent complement activation in a subject suffering from a disease or condition as disclosed herein, comprising administering to the subject a composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent and a pharmaceutically acceptable carrier. The MASP-2 inhibitory agents can be administered to a subject in need thereof, at therapeutically effective doses to treat or ameliorate conditions associated with MASP-2-dependent complement activation. A therapeutical ly effective dose refers to the amount of the MASP-2 inhibitory agent sufficient to result in amelioration of symptoms associated with the disease or condition.

Toxicity and therapeutic efficacy of MASP-2 inhibitory agents can be determined by standard pharmaceutical procedures employing experimental animal models, such as the murine MASP-2 -/- mouse model expressing the human MASP-2 transgene described in Example 1. Using such animat models, the NOAEL (no observed adverse effect level) and the MED (the minimally effective dose) can be determined using standard methods. The dose ratio between NOAEL and MED effects is the therapeutic ratio, which is expressed as the ratio NOAEL/MED. MASP-2 inhibitory agents that exhibit large therapeutic ratios or indices are most preferred. The data obtained from the cell culture assays and animal studies cars be used in formulating a range of dosages for use in humans. The dosage of the MASP-2 inhibitory agent preferably lies within a range of circulating concentrations that include the MED with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.

For any compound formulation, the therapeutically effective dose can be estimated using animal models. For example, a dose may be formulated in an animal model to achieve a circulating plasma concentration range that includes the MED. Quantitative levels of the MASP-2 inhibitory agent in plasma may also be measured, for example, by high performance liquid chromatography, in addition to toxicity studies, effective dosage may also be estimated based on the amount of MASP-2 protein present in a living subject and the binding affinity of the MASP-2 inhibitory agent. It has been shown that MASP-2 levels in normal human subjects is present in serum in low levels in the range of 500 ng/ml, and MASP-2 levels

-1002018200437 19 Jan 2018 in a particular subject can be determined using a quantitative assay for MASP-2 described in Moller-Kristensen M., el al., J. Immunol. Methods 282:159-167, 2003.

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

Therapeutic efficacy of MASP-2 inhibitory compositions and methods of the present invention in a given subject, and appropriate dosages, ean be determined in accordance with complement assays well known to those of skill in the art. Complement generates numerous specific products. During the last decade, sensitive and specific assays have been developed and are available commercially for most of these activation products, including the small activation fragments C3a, C4a, and C5a and the large activation fragments iC3 b, C4d, Bb, and sC5b-9. Most of these assays utilize monoclonal antibodies that react with new antigens (neoantigens) exposed on the fragment, but not on the native proteins from which they are formed, making these assays very simple and specific. Most rely on ELISA technology, although radioimmunoassay is still sometimes used for C3a and C5a. These latter assays measure both the unprocessed fragments and their 'desArg' fragments, which are the major forms found in the circulation. Unprocessed fragments and C5a<te,Arg are rapidly cleared by binding to ceil surface receptors and are hence present in very low concentrations, whereas C3a<iesArg does not bind to cells and accumulates in plasma. Measurement of C3a provides a sensitive, pathway-independent indicator of complement activation. Alternative pathway activation can be assessed by measuring the Bb fragment. Detection of the fluid-phase product of membrane attack pathway activation, sC5b-9, provides evidence that complement is being activated to completion. Because both the lectin and classical pathways generate the same activation products, C4a and C4d, measurement of these two fragments does not provide any information about which of these two pathways has generated the activation products.

The 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

-1012018200437 19 Jan 2018 occurs as a result of administration of a MASP-2 inhibitory agent in accordance with the methods of the invention; the inhibition of the generation or production of

MASP-2-dependent complement activation system products C4b, C3a, C5a and/or C5b-9 (MAC.) (measured, for example, as described in measured, for example, as described in

Example 2, the reduction of C4 cleavage and C4b deposition (measured, for example as described in Example 10), or the reduction of C3 cleavage and C3b deposition (measured, for example, as described in ExampielO).

ADDITIONAL AGENT'S

The compositions and methods comprising MASP-2 inhibitory agents may optionally comprise one or more additional therapeutic agents, which may augment the activity of the MASP-2 inhibitory agent or that provide related therapeutic functions in an additive or synergistic fashion. For example, in the context of treating a subject suffering from TTP, wherein the subject is positive for an inhibitor of ADAM-TS13, one or more

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

The inclusion and selection of additional agent(s) will be determined to achieve a desired therapeutic result. In some embodiments, the MASP-2 inhibitory agent may be administered in combination with one or more anti-inflammatory and/or analgesic agents. Suitable anti-inflammatory and/or analgesic agents include: serotonin receptor antagonists; serotonin receptor agonists; histamine receptor antagonists; bradykinin receptor antagonists; kallikrein inhibitors; tachykinin receptor antagonists, including neurokinin! and neurokinin? receptor subtype antagonists; calcitonin gene-related peptide (CORP) receptor antagonists; interleukin receptor antagonists; inhibitors of enzymes

-1022018200437 19 Jan 2018 active in the synthetic pathway for arachidonic acid metabolites, including phospholipase inhibitors, including PLA2 tsotbrm inhibitors and PLCy isoform inhibitors, cyclooxygenase (COX) inhibitors (which may be either COX-3, COX-2, or nonselective COX-1 and -2 inhibitors), lipooxygenase inhibitors; prostanoid receptor antagonists including eicosanoid EP-1 and EP-4 receptor subtype antagonists and thromboxane receptor subtype antagonists; leukotriene receptor antagonists including leukotriene B4 receptor subtype antagonists and leukotriene D4 receptor subtype antagonists; opioid receptor agonists, including μ-opioid, δ-opioid, and κ-opioid receptor subiype agonists; purinoceptor agonists and antagonists including Ρρχ receptor antagonists and ?2γ receptor agonists; adenosine triphosphate (ATP)-sensitive potassium channel openers; MAP kinase inhibitors; nicotinic acetylcholine inhibitors; and alpha adrenergic receptor agonists (including alpha-1, alpha-2, and nonselective alpha-1 and 2 agonists).

The MASP-2 inhibitory agents of the present invention may also be administered in combination with one or more other complement inhibitors, such as an inhibitor of C5. To date, Eculizumab (Solaris®), an antibody against C5, is the only complementtargeting drug that has been approved for human use. However some pharmacological agents have been shown to block complement m vivo. K76COOH and nafamstat mesilate are two agersts that have shown some effectiveness in animal models of transplantation (Miyagawa, S,, et al., Transplant Proc, 24:483-484, 1992), Low molecular weight heparins have also been shown to be effective in regulating complement activity (Edens, R.E., et al., Complement Today, pp, 96-120, Basel: Karger, 1993), ft is believed that these small molecule inhibitors may be useful as agents to use in combination with the MASP-2 inhibitory agents of the present invention,

Other naturally occurring complement inhibitors may be useful in combination with the MASP-2 inhibitory agents of the present invention. Biological inhibitors of complement include soluble complement factor 1 (sCRl). This is a naturally-occurring inhibitor that can be found on the outer membrane of human cells. Other membrane inhibitors include DAF, MCP, and CD59. Recombinant forms have been tested for their anti-complement activity in vitro and in vivo. sCRl has been shown io be effective in xenotransplantation, wherein the complement system (both alternative and classical) provides the trigger for a hyperactive rejection syndrome within minutes of perfusing blood through the newly transplanted organ (Platt, J.L., et al., Immunol. Today 2/:450-6,

-1032018200437 19 Jan 2018

1990; Marino, I.R., etal., Transplant Proc. 1071:6, 1990; Johnstone, P.S., etal.,

Transplantation 54:573-6, 1992). The· use of sCRl protects and extends the survival time of the transplanted organ, implicating the complement pathway in the pathogenesis of organ survival (Leventhal, J.R., etah, Transplantation 55:857-66, 1993; Pruitt, S.K., et al., Transplantation 57:363-70, 1994),

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

PHARMACEUTICAL CARRIERS AND DELIVERY VEHICLES

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

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

The carrier may also comprise a delivery vehicle to sustain (I.e,, extend, delay or regulate) the delivery of the agent(s) or to enhance the delivery, uptake, stability or pharmacokinetics of the therapeutic agent(s). Such a delivery vehicle may include, by

-1042018200437 19 Jan 2018 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 disclosed in WO 2004/009664 A2 and the PEO and PEO/cyclodextrin complexes disclosed in U.S. Patent Application Publication No. 2002/0039369 A1. Such hydrogels may be injected locally at the site of intended action, or subcutaneously or intramuscularly to form a sustained release depot,

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

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

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

Various nasal and pulmonary delivery systems, including aerosols, metered-dose inhalers, dry powder inhalers, and nebulizers, are being developed and may suitably be adapted for delivery ofthe present invention in an aerosol, inhalant, or nebulized delivery vehicle, respectively.

For irstrathecal (IT) or intracerebroventricular (1CV) delivery, appropriately sterile delivery systems (e.g,, liquids; gels, suspensions, etc.) can be used to administer the present invention.

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

PHARMACEUTICAL CARRIERS FOR ANTIBODIES AND PEPTIDES

More specifically with respect to anti-MASP-2 antibodies and inhibitory peptides, exemplary formulations can be parenterally administered as injectable dosages of a solution or suspension of the compound in a physiologically acceptable diluent with a pharmaceutical carrier that can be a sterile liquid such as water, oils, saline, glycerol or

-1052018200437 19 Jan 2018 ethanol. Additionally, auxiliary substances such as wetting or emulsifying agents, surfactants, pH buffering substances and the like can be present in compositions comprising anti-MASP-2 antibodies and inhibitory peptides. Additional components of pharmaceutical compositions Include petroleum (such as of animal, vegetable or synthetic origin), for example, soybean oil and mineral oil. In general, glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers for injectable solutions.

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

PHARMACEUTICALLY ACCEPTABLE CARRIERS FOR. EXPRESSION

INHIBITORS

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

Pharmaceutical compositions that include expression inhibitors may include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emuisifying solids and self-emulsifying semisolids.

The preparation of such compositions typically involves combining the expression inhibitor with one or more of the following: buffers, antioxidants, low molecular weight polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with non-specific serum albumin are examples of suitable diluents.

In some embodiments, the compositions may be prepared and formulated as emulsions which are typically heterogeneous systems of one liquid dispersed in another in the form of droplets (see, Idson, in Pharmaceutical Dosage Forms, Vol. 1, Rieger and Banker (eds.), Marcek Dekkcr, Inc,, N.Y., 1988). Examples of naturally occurring emulsifiers used In emulsion formulations include acacia, beeswax, lanolin, lecithin and phosphatides.

-1062018200437 19 Jan 2018 in one embodiment, compositions including nucleic acids can be formulated as microemulsions, A microemulsion, as used herein refers to a system of water, oil, and atnphiphiie, which is a single optically isotropic and thermodynamically stable liquid solution {see Rosoff in Pharmaceutical Dosage Forms, Vol. 1), The method of the invention may also use liposomes for the transfer and delivery of antisense oligonucleotides to the desired site.

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

MODES OF ADMINISTRATION

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

SYSTEMIC DELIVERY

As used herein, the terms systemic delivery¹ and systemic administration are intended to include but are not limited to oral and parenteral routes including intramuscular (IM), subcutaneous, intravenous (IV), intra-arterial, inhalational, sublingual, buccal, topical, transdermal, nasal, rectal, vaginal and other routes of administration that effectively result in dispersement 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 and inhalational. It will be appreciated that the exact systemic administration route for selected agents utilized in particular compositions of the present invention will be determined in part to account for the agent's susceptibility to metabolic transformation pathways associated with a given route of administration. For example, peptidergic agents may be most suitably administered by routes other than orai.

-1072018200437 19 Jan 2018

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

By way of representative example, MASP-2 inhibitory antibodies and peptides can be introduced into a living body by application to a bodily membrane capable of absorbing the polypeptides, for example the nasal, gastrointestinal and rectal membranes. The polypeptides are typically applied to the absorptive 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 /3:213, 1990; Lee, V.H.L., Ed., Peptide and Protein Drug Delivery, Marcel Dekker, New York (1991); DeBoer, A.G., etal.,

J. Controlled Release /5:241, 1990.) For example, STDHF is a synthetic derivative of fusidic acid, a steroidal surfactant that is similar in structure to the bile salts, and has been used as a permeation enhancer for nasal delivery. (Lee, W.A., Biopharm. 22, Nov ./Dec. 1990.)

The MASP-2 inhibitory antibodies and polypeptides may be introduced in association with another molecule, such as a lipid, to protect the polypeptides from enzymatic degradation. For example, the covalent attachment of polymers, especially polyethylene glycol (PEG), has been used to protect certain proteins from enzymatic hydrolysis in the body and thus prolong half-life (Fuertges, F., etal., J. Controlled Release //:139, 1990). Many polymer systems have been reported for protein delivery (Bae, Y.H., et al., J, Controlled Release 9:271. 1989; Hori. R., et ah, Pham. Res. 6:813, 1989: Yamakawa, I., et ah, J. Pharm. Sci. 79:505,1990; Yoshihiro, I., et ah, J. Controlled Release /6:195, 1989; Asano, M,, etal., J. Controlled Release 9:111, 1989; Rosenblatt, J., etah, J. Controlled Release 9:195, 1989; Makino, K., J. Controlled Release 12:235, 1990; Takakura, Y., etah, J. Pharm. Sci. 78:117, 1989; Takakura, ¥., etah, 3. Pharm.

&?. 75:219, 1989).

Recently, liposomes have been developed with improved serum stability and circulation half-times (see, e.g., U.S. Patent No. 5,741,516, to Webb), Furthermore, various methods of liposome and liposome-like preparations as potential drug carriers

-1082018200437 19 Jan 2018 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),

For transdermal applications, the MASP-2 inhibitory antibodies and polypeptides 5 may be combined with other suitable ingredients, such as carriers and/or adjuvants. There are no limitations on the nature of such other ingredients, except that they must be pharmaceutically acceptable for their intended administration, and cannot degrade the activity' of the active ingredients of the composition. Examples of suitable vehicles include ointments, creams, gels, or suspensions, with or without purified collagen. The

MASP-2 inhibitory antibodies and polypeptides may also be impregnated into transdermal patches, plasters, and bandages, preferably in liquid or semi-liquid form.

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

LOCAL DELIVERY

As used herein, the term local encompasses application of a drug in or around a site of intended localized action, and may include for example topical delivery to the skin or other affected tissues, ophthalmic delivery, intrathecal (IT), intracerebroventricular (ICY), intra-articnlar, Intracavity, intracranial or intravesicular administration, placement or irrigation, Local administration may be preferred to enable administration of a lower dose, to avoid systemic side effects, and for more accurate control of the timing of delivery and concentration of the active agents at the site of local delivery. Local administration provides a known concentration at the target site, regardless of interpatient

-1092018200437 19 Jan 2018 variability in metabolism, blood flow, etc. Improved dosage control is also provided by the direct mode of delivery.

Local delivery of a MASP-2 inhibitory agent may be achieved in the context of surgical methods for treating a disease or condition, such as for example during procedures such as arterial bypass surgery, atherectomy, laser procedures, ultrasonic procedures, balloon angioplasty and stent placement. For example, a MASP-2 inhibitor can be administered to a subject in conjunction with a balloon angioplasty procedure. A balloon angioplasty procedure involves inserting a catheter having a deflated balloon into an artery. The deflated balloon is positioned in proximity to the atherosclerotic plaque and is inflated such that the plaque is compressed against the vascular wall. As a result, the balloon surface is in contact with the layer of vascular endothelial cells on the surface of the blood vessel. The MASP-2 inhibitory agent may be attached to the balloon angioplasty catheter in a manner that permits release of the agent at the site of the atherosclerotic plaque, The agent may be attached to the balloon catheter in accordance with standard procedures known in the art. For example, the agent may be stored in a compartment of the balloon catheter until the balloon is inflated, at which point it is released into the local environment. Alternatively, the agent may be impregnated on the balloon surface, such that it contacts the cells of the arterial wall as the balloon is inflated. The agent may also be delivered in a perforated balloon catheter such as those disclosed in Flugelman, M.Y., etal., Circulation55:1110-1117, 1992, See also published PCT Application WO 95/23161 for an exemplary procedure for attaching a therapeutic protein to a balloon angioplasty catheter. Likewise, the MASP-2 inhibitory agent may be included in a gel or polymeric coating applied to a stent, or may be incorporated into the material of the stent, such that the stent elutes the MASP-2 inhibitory agent after vascular placement.

MASP-2 inhibitory compositions used in the treatment of arthritides and other musculoskeletal disorders may be locally delivered by intra-articular injection. Such compositions may suitably include a sustained release delivery vehicle. As a further example of instances in which local delivery may be desired, MASP-2 inhibitory compositions used in the treatment of urogenital conditions may be suitably instilled intravesically or within another urogenital structure.

-1102018200437 19 Jan 2018

COATINGS ON A MEDICAL DEVICE

MASP-2 inhibitory agents such as antibodies and inhibitory peptides may be immobilized onto (or within) a surface of an implantable or attachable medical device.

The modified surface will typically be in contact with living tissue after implantation into an animal body. By implantable or attachable medical device is intended any device that is implanted into, or attached to, tissue of an animal body, during the normal operation of the device (e.g., stents and implantable drug delivery' devices). Such implantable or attachable medical devices can be made from, for example, nitrocellulose, diazoceliuiose, glass, polystyrene, polyvinylchloride, polypropylene, polyethylene, dextran, Sepharose, agar, starch, nylon, stainless steel, titanium and biodegradable and/or biocompatible polymers. Linkage of the protein to a device can be accomplished by any technique that does not destroy the biological activity of the linked protein, for example by attaching one or both of the N- C-terminal residues of the protein to the device. Attachment may also be made at one or more internal sites in the protein. Multiple attachments (both internal and at the ends of the protein) may also be used. A surface of an implantable or attachable medical device can be modified to include functional groups (e.g,, carboxyl, amide, amino, ether, hydroxyl, cyano, nitride, sulfanamido, acetylinic, epoxide, siianic, anhydrsc, succinimic, azido) for protein immobilization thereto. Coupling chemistries include, but are not limited to, the formation of esters, ethers, amides, azido and sulfanamido derivatives, cyanate and other linkages to the functional groups available on MASP-2 antibodies or inhibitory peptides. MASP-2 antibodies or inhibitory fragments can also be attached non-covalently by the addition of an affinity tag sequence to the protein, such as GST (D.B. Smith and K.S. Johnson, Gene 67:31, 1988), polyhistidines (E. Hochuli etal., J. Chromcitog. 411:17, 19S7), or biotin. Such affinity tags may be used for the reversible attachment of the protein to a device,

Proteins can also be covalently attached to the surface of a device body, for example, by covalent activation of the surface of the medical device. By way of representative example, matrieellular protein(s) can be attached to the device body by any of the following pairs of reactive groups (one member of the pair being present on the surface of the device body, and the other member of the pair being present on the matrieellular protein(s)): hydroxyI/carboxylie acid to yield an ester linkage;

hydroxyl/anhydride to yield an ester linkage; hydroxyl/isocyanate to yield a urethane linkage. A surface of a device body that does not possess useful reactive groups can be

-1112018200437 19 Jan 2018 treated with radio-frequency discharge plasma (RFGD) etching to generate reactive groups in order to allow deposition of matricellular protein(s) (e.g.. treatment with oxygen plasma to introduce oxygen-containing groups; treatment with propyl amino plasma to introduce amine groups).

MASP-2 inhibitory agents comprising nucleic acid molecules such as antisense,

RNAi-or DNA-encoding peptide inhibitors can be embedded in porous matrices attached to a device body. Representative porous matrices useful for making the surface layer are those prepared from tendon or derma! collagen, as may be obtained from a variety of commercial sources (e.g., Sigma and Collagen Corporation), or collagen matrices prepared as described in U.S. Patent Nos. 4,394,370, to Jefferies, and 4,975,527, to Koezuka. One collagenous materia! is termed UltraFiber™ and is obtainable from Norian Corp, (Mountain View, California).

Certain polymeric matrices may also be employed if desired, and include acrylic ester polymers and lactic acid polymers, as disclosed, for example, in U.S. Patent

Nos. 4,526,909 and 4,563,489, to Urist. Particular examples of useful polymers are those of orthoesters, anhydrides, propyiene-cofumarates, or a polymer of one or more α-hydroxy carboxylic acid monomers, (e.g., α-hydroxy acetic acid (glycolic acid) and/or α-hydroxy propionic acid (lactic acid)).

TREATMENT REGIMENS

In prophylactic applications, the pharmaceutical compositions are administered to a subject susceptible to, or otherwise at risk of, a condition associated with MASP-2-dependem complement activation in an amount sufficient to eliminate or reduce the risk of developing symptoms of the condition. In therapeutic applications, the pharmaceutical compositions are administered to a subject suspected of, or already suffering from, a condition associated with MASP-2-dependent complement activation in a therapeutically effective amount sufficient to relieve, or at least partially reduce, the symptoms of the condition, in both prophylactic and therapeutic regimens, compositions comprising MASP-2 inhibitory agents may be administered in several dosages until a sufficient therapeutic outcome has been achieved in the subject. Application of the

MASP-2 inhibitory compositions of the present invention may be earned out by a single administration of the composition, or a limited sequence of administrations, for treatment of an acute condition, e.g., reperfuslon injury or other traumatic in jury. Alternatively, the

2018200437 19 Jan 2018 composition may be administered at periodic intervals over an extended period of time for treatment of chronic conditions, e.g., arthritides or psoriasis.

The methods and compositions of the present invention may be used to inhibit inflammation and related processes that typically result from diagnostic and therapeutic medical and surgical procedures. To inhibit such processes, the MASP-2 inhibitory composition of the present invention may be applied periproeeduraily. As used herein periproeeduraily refers to administration of the inhibitory composition preprocedurally and/or intraprocedurally and/or postprocedurally, i.e., before the procedure, before and during the procedure, before and after the procedure, before, during and after the procedure, during the procedure, during and after the procedure, or after the procedure. Peri procedural application may be carried out by local administration of the composition to the surgical or procedural site, such as by injection or continuous or intermittent irrigation of the site or by systemic administration. Suitable methods for local perioperative delivery of MASP-2 inhibitory agent solutions are disclosed in US Patent

Nos, 6,420,432 to Demopulos and 6,645,168 to Demopulos. Suitable methods for local delivery of chondroprotective compositions including MASP-2 inhibitory agent(s) are disclosed in International PCT Patent Application WO 01/07067 A2, Suitable methods and compositions for targeted systemic delivery of chondroprotective compositions including MASP-2 inhibitory agent(s) are disclosed in International PCT Patent

Application WO 03/063799 A2.

In one aspect ofthe invention, the pharmaceutical compositions are administered to a subject susceptible to, or otherwise at risk of, PNH in an amount sufficient to eliminate or reduce the risk of developing symptoms of the condition. In therapeutic applications, the pharmaceutical compositions are administered to a subject suspected of, or already suffering from, PNH in a therapeutically effective amount sufficient to relieve, or at least partially reduce, the symptoms of the condition.

In one embodiment, the subject's red blood cells are opsonized by fragments of C3 in the absence of the composition, and administration of the composition comprising a A3 ASP-2 inhibitory agent to the subject increases the survival of red blood cells 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 levels of hemoglobin, (ii) below normal levels of platelets; (iii) above normal levels of

-1132018200437 19 Jan 2018 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 nearly normal levels of hemoglobin (ii) increased, normal or nearly normal levels of platelets, (iii) decreased, normal or nearly normal levels of reticulocytes, and/or (iv) decreased, normal or nearly normal levels of bilirubin.

In both prophylactic and therapeutic regimens, compositions comprising MASP-2 inhibitory agents may be administered in several dosages until a sufficient therapeutic outcome has been achieved in the subject. In one embodiment of the invention, the MASP-2 inhibitory agent comprises an anti-MASP-2 antibody, which suitably may be administered to an adult patient (e.g., an average adult weight of 70 kg) in a dosage of from 0.1 mg to 10,000 mg, more suitably from 1.0 mg to 5,000 mg, more suitably 10.0 mg to 2,000 mg, more suitably 10.0 mg to 1,000 mg and still more suitably from 50.0 mg to 500 mg. For pediatric patients, dosage can be adjusted in proportion to the patient’s weight. Application of the MASP-2 inhibitory compositions of the present invention may be carried out by a single administration of the composition, or a limited sequence of administrations, for treatment of PNH. Alternatively, the composition may be administered ar periodic intervals such as daily, biweekly, weekly, every other week, monthly or bimonthly over an extended period of time for treatment of PNH.

In some embodiments, the subject suffering from 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 comprises administering to the subject a composition of the invention comprising a MASP-2 inhibitor and further 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.

In one aspect of the invention, the pharmaceutical compositions arc administered to a subject susceptible to, or otherwise at risk of, aHUS in an amount sufficient to eliminate or reduce the risk of developing symptoms of the condition, in therapeutic applications, the pharmaceutical compositions are administered to a subject suspected of, or already suffering from, aHUS in a therapeutically effective amount sufficient to

-1142018200437 19 Jan 2018 relieve, or at least partially reduce, the symptoms of the condition. In one aspect of the invention, prior to administration, the subject may be examined to determine whether the subject exhibits one or more symptoms of aHUS, including (i) anemia, (ii) thrombocytopenia (iii) renal insufficiency and (iv) rising creatinine, and the composition of the present invention is then administered in an effective amount and for a sufficient time period to improve these symptom(s).

In another aspect of the invention, the MASP-2 inhibitory compositions of the present invention may be used to prophylacticaiiy treat a subject that has an elevated risk of developing aHUS and thereby reduce the likelihood that the subject will deliver aHUS,

The presence of a genetic marker in the subject known to be associated with aHUS is first determined by performing a genetic screening test on a sample obtained from the subject anti identifying the presence of at least one genetic marker associated with aHUS, complement factor H (CFH), factor 1 (CFI), factor B (CFB), membrane cofactor CD46, C3, complement factor H-related protein (CFHR1), anticoagulant protein thrombodulin (THBD), complement factor H-related protein 3 (CFHR3) or complement factor Hrelated protein 4 (CFHR4). The subject is then periodically monitored (e.g,, monthly, quarterly, twice annually or annually) to determine the presence or absence of at least one symptom of aHUS, such as anemia, thrombocytopenia, renal insufficiency and rising creatinine. Upon the determination of the presence of at least one of these symptoms, the subject can be administered an amount of a MASP-2 inhibitory agent effective to inhibit MASP-2 dependent complement activation, in an effective amount and for a sufficient time period to improve said one or more symptoms. In a still further aspect of the present invention, a subject at increased risk of developing aHUS due to having been screened and determined to have one of the genetic markers associated with aHUS may be monitored for the occurrence of an event associated with triggering aHUS clinical symptoms, including drug exposure, infection (e.g., bacterial infection), malignancy, injury, organ or tissue transplant and pregnancy.

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

-1152018200437 19 Jan 2018

In a still further aspect of the present invention, a subject suffering from aHUS may initially be treated with a MASP-2 inhibitory composition of the present invention that is administered through a catheter line, such as an intravenous catheter line or a subcutaneous catheter line, for a first period of time such as one hour, twelve hours, one day, two days or three days. The subject may then be treated for a second period of time with the MASP-2 inhibitory composition administered through regular subcutaneous injections, such as daily, biweekly, weekly, every other week, monthly or bimonthly, injections.

In a still further aspect of the present invention, a MASP-2 inhibitory composition 10 of the present invention may be administered to a subject suffering from aHUS in the absence of plasmapheresis (i.e., a subject whose aHUS symptoms have not been treated with plasmapheresis and are not treated with plasmapheresis at the time of treatment with the MASP-2 inhibitory composition), to avoid the potential complications of plasmaphersis including hemorrhage, infection, and exposure to disorders and/or allergies inherent in the plasma donor, or in a subject otherwise averse to plasmapheresis, or in a setting where plasmapheresis is unavailable.

In a. still further aspect of the present invention, a MASP-2 inhibitory composition of the present invention may be administered to a subject suffering from aHUS coincident with treating the patient with plasmapheresis. For example, a subject receiving plasmapheresis treatment can then be administered the MASP-2 inhibitory composition following or alternating with plasma exchange.

In a still further aspect of the present invention, a subject suffering from or at risk of developing aHUS and being treated with a MASP-2 inhibitory composition of the present invention can be monitored by periodically determining, such as every twelve hours or on a daily basis, the level of at least one complement factor, wherein the determination of a reduced level of the at least one complement factor in comparison to a standard value or to a healthy subject is indicative of the need for continued treatment with the composition.

In both prophylactic and therapeutic regimens, compositions comprising MASP-2 inhibitory agents may be administered in several dosages until a sufficient therapeutic outcome has been achieved in the subject. In one embodiment of the invention, the MASP-2 inhibitory' agent comprises an anti-MASP-2 antibody, which suitably may be administered to an adult patient (e.g., an average adult weight of 70 kg) in a dosage of

-1162018200437 19 Jan 2018 from 0.1 mg to 10,000 mg, more suitably from 1.0 mg to 5,000 mg, more suitably 10.0 mg to 2,000 mg, more suitably 10.0 mg to 1,000 mg and still more suitably from 50.0 mg to 500 mg. For pediatric patients, dosage can be adjusted in proportion to the patient’s weight. Application of the MASP-2 inhibitory compositions of the present invention may be carried out by a single administration of the composition, or a limited sequence of administrations, for treatment of aHUS. Alternatively, the composition may be administered at periodic intervals, such as daily, biweekly, weekly, every other week, monthly or bimonthly, over an extended period of time for treatment of aHUS.

In some embodiments, the subject suffering from aHUS 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 comprises administering to the subject a composition of the invention comprising a MASP-2 inhibitor and further administering to the subject a terminal complement inhibitor that inhibits cleavage of complement protein CS, 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.

In one aspect of the invention, the pharmaceutical compositions are administered to a subject susceptible to, or otherwise at risk of, HUS in an amount sufficient to eliminate or reduce the risk of developing symptoms of the condition, in therapeutic applications, the pharmaceutical compositions are administered to a subject suspected of, or already suffering from, HUS in a therapeutically effective amount sufficient to relieve, or at least partially reduce, the symptoms of the condition.

In another aspect of the present invention, the likelihood of developing impaired renal function in a subject at risk for developing HUS can be reduced by administering to the subject a MASP-2 inhibitory composition of the present invention in an amount effective to inhibit MASP-2 dependent complement activation. For example, a subject at risk for developing HUS and to be treated with a MASP-2 inhibitory composition of the present invention may exhibit one or more symptoms associated with HUS, including diarrhea, a hematocrit level of less than 30% with smear evidence of intravascular erythrocyte destruction, thrombocytopenia and rising creatinine levels. As a further example, a subject at risk for developing HUS and to be treated with the MASP-2 inhibitory compositions of the present invention may be infected with E, coli, shigella or

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2018200437 19 Jan 2018 salmonella. Such subjects infected with E. coli, shigella or salmonella may be treated with a MASP-2 inhibitory composition of the present invention concurrent with antibiotic treatment, or alternately may be treated with a MASP-2 inhibitory composition without concurrent treatment with an antibiotic, particularly for enterogenic E. coli tor which antibiotic treatment is contra-indicated. A subject infected with enterogenic E. coli that has been treated with an antibiotic may be at elevated risk of developing HUS, and may be suitably treated with a MASP-2 inhibitory composition of the present invention to reduce that risk. A subject infected with enterogenic E. coli may be treated for a first period of time with a MASP-2 inhibitory composition of the present invention in the absence of an antibiotic and then for a second period of time with both a MASP-2 inhibitory composition of the present invention and an antibiotic.

in a still further aspect ofthe present invention, a subject suffering from HUS may initially be treated with a MASP-2 inhibitory composition ofthe present invention that is administered through a catheter line, such as an intravenous catheter line or a subcutaneous catheter sine, for a first period of time such as one hour, twelve hours, one day, two days or three days. The subject may then be treated for a second period of time with the MASP-2 inhibitory composition administered through regular subcutaneous injections, such as daily, biweekly, weekly, every other week, monthly or bimonthly, injections.

In a still further aspect of the present invention, a MASP-2 inhibitory composition of the present invention may be administered to a subject suffering from HUS in the absence of plasmapheresis (i.e., a subject whose HUS symptoms have not been treated with plasmapheresis and are not treated with plasmapheresis at the time of treatment with the MASP-2 inhibitory composition), to avoid the potential complications of plasmaphersis including hemorrhage, infection, and exposure to disorders and/or allergies inherent in the plasma donor, or in a subject otherwise averse to plasmapheresis, or in a setting where plasmapheresis is unavailable.

In a still further aspect ofthe present invention, a MASP-2 inhibitory composition of the present invention may be administered to a subject suffering from HUS coincident with treating the patient with plasmapheresis. For example, a subject receiving plasmapheresis treatment can then be administered the MASP-2 inhibitory composition following or alternating with plasma exchange.

-1182018200437 19 Jan 2018

In a still further aspect of the present invention, a subject suffering from or at risk of developing HUS and being treated with a MASP-2 inhibitory composition of the present invention can be monitored by periodically determining, such as every twelve hours or on a daily basis, the level of at least one complement factor, wherein the determination of a reduced level of the at least one complement factor in comparison to a standard value or to a healthy subject is indicative of the need for continued treatment with the composition, in both prophylactic and therapeutic regimens, compositions comprising MASP-2 inhibitory agents may be administered in several dosages until a sufficient therapeutic outcome has been achieved in the subject. In one· embodiment of the invention, the MASP-2 inhibitory agent comprises an anti-MASP-2 antibody, which suitably may be administered to an adult patient (e.g., an average adult weight of 70 kg) in a dosage of from 0,1 mg to 10,000 mg, more suitably from 1.0 mg to 5,000 mg, more suitably 10.0 mg to 2,000 mg, more suitably 10.0 rug to 1,000 mg and still more suitably from 50.0 mg to 500 mg. For pediatric patients, dosage can be adjusted in proportion to the patient’s weight. Application of the MASP-2 inhibitory compositions of the present invention may be carried out by a single administration of the composition, or a limited sequence of administrations, for treatment of HUS, Alternatively, the composition may be administered at periodic intervals, such as daily, biweekly, weekly, every other week, monthly or bimonthly, over an extended period of time for treatment of HUS.

In some embodiments, the subject suffering from HUS 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 comprises administering to the subject a composition of the invention comprising a MASP-2 inhibitor and further 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.

In one aspect of the invention, the pharmaceutical compositions are administered to a subject susceptible to, or otherwise at risk of, TTP in an amount sufficient to eliminate or reduce the risk, of developing symptoms of the condition. In therapeutic applications, the pharmaceutical compositions are administered to a subject suspected of,

-1192018200437 19 Jan 2018 or already suffering from, TTP in a therapeutically effective amount sufficient to relieve, or at least partially reduce, the symptoms of the condition.

In another aspect of the present invention, a subject exhibiting one or more of the symptoms of TTP, including central nervous system involvement, thrombocytopenia, severe cardiac Involvement, severe pulmonary involvement, gastro-intestinai infarction and gangrene, may be treated with a MASP-2 inhibitory composition of the present invention, in another aspect of the present invention, a subject determined to have a depressed level of ADAMTS13 and also testing positive for the presence of an inhibitor of (i.e., an antibody) ADAMTS13 may be treated with a MASP-2 Inhibitory composition of the present invention. In a still further aspect of the present invention, a subject testing positive for the presence of an inhibitor of ADAMTS13 may be treated with an immunosupressant (e.g., corticosteroids, rituxan, or cyclosporine) concurrently with treatment with a MASP-2 inhibitory composition of the present invention. In a still further aspect of the present invention, a subject determined to have a reduced level of

ADAMTSI3 and testing positive for the presence of an inhibitor of ADA MI’S 13 may be treated with ADAMTS13 concurrently with treatment with a MASP-2 inhibitory composition of the present invention.

In a still further aspect of the present invention, a subject suffering from TTP may initially be treated with a MASP-2 Inhibitory composition of the present invention that is administered through a catheter line, such as an intravenous catheter line or a subcutaneous catheter line, for a first period of time such as one hour, twelve hours, one day, two days or three days. The subject may then be treated for a second period of time with the MASP-2 inhibitory composition administered through regular subcutaneous injections, such as daily, biweekly, weekly, every other week, monthly or bimonthly, injections.

In a stili further aspect of the present invention, a MASP-2 inhibitory composition of the present invention may be administered to a subject suffering from HUS in the absence of plasmapheresis (i.e., a subject whose TTP symptoms have not been treated with plasmapheresis and are not treated with plasmapheresis at the time of treatment with the MASP-2 inhibitory composition), to avoid the potential complications of plasmaphersis including hemorrhage, infection, and exposure to disorders and/or allergies inherent in the plasma donor, or in a subject otherwise averse to plasmapheresis, or in a setting where plasmapheresis is unavailable.

-1202018200437 19 Jan 2018

In a still further aspect of the present invention, a MASP-2 inhibitory composition of the present invention may be administered to a subject suffering from TTP coincident with treating the patient with plasmapheresis. For example, a subject receiving plasmapheresis treatment can then be administered the MASP-2 inhibitory composition following or alternating with plasma exchange.

In a still further aspect of the present invention, a subject suffering from refractory TTP, i.e.. symptoms of TTP that have not responded adequately to other treatment such as plasmapheresis, may be treated with a MASP-2 inhibitory composition of the present invention, with or without additional plasmapheresis.

hi a still further aspect of the present invention, a subject suffering from or at risk of developing TTP and being treated with a MASP-2 inhibitory composition of the present invention can be monitored by periodically determining, such as every twelve hours or on a daily basis, the level of at least one complement factor, wherein the determination of a reduced level of the at least one complement factor in comparison to a standard value or to a healthy subject is indicative of the need for continued treatment with the composition.

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

MASP-2 inhibitory agent comprises an anti-MASP-2 antibody, which suitably may be administered to an adult patient (e.g., an average adult weight of 70 kg) in a dosage of from 0.1 mg to 10,000 mg, more suitably from 1,0 mg to 5,000 mg, more suitably 10.0 mg to 2,000 nig, more suitably 10,0 mg to ί ,000 nig and still more suitably from 50,0 mg to 500 mg. For pediatric patients, dosage can be adjusted in proportion to the patient’s weight. Application of the MASP-2 inhibitory compositions of the present invention may be carried out by a single administration of the composition, or a limited sequence of administrations, for treatment of TTP. Alternatively, the composition may be administered at periodic intervals, such as daily, biweekly, weekly, every other week, monthly or bimonthly, over an extended period of time tor treatment of TTP.

In some embodiments, the subject suffering from TTP 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 comprises administering to the subject a composition of the invention comprising a MASP-2

-1212018200437 19 Jan 2018 inhibitor and further 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.

In one aspect of the invention, the pharmaceutical compositions are administered to a subject susceptible to, or otherwise at risk of, cold aggultinin disease or cryoglobulinemia in an amount sufficient to eliminate or reduce the risk of developing symptoms of the condition. In therapeutic applications, the pharmaceutical compositions are administered to a subject suspected of, or already suffering from, coid aggultinin disease or cryoglobulinemia in a therapeutically effective amount sufficient to relieve, or at least partially reduce, the symptoms of the condition.

In a still further aspect of the present invention, a subject suffering from or at risk of developing cold aggultinin disease or cryoglobulinemia and being treated with a MASP-2 inhibitory composition of the present invention can be monitored by periodically determining, such as every twelve hours or on a daily basis, the level of at least, one complement factor, wherein the determination of a reduced level of the at least one complement factor in comparison to a standard value or to a healthy subject is indicative of the need for continued treatment with the composition.

in both prophylactic and therapeutic regimens, compositions comprising MASP-2 inhibitory agents may be administered in several dosages until a sufficient therapeutic outcome has been achieved in the subject. In one embodiment of the invention, the MASP-2 inhibitory agent, comprises an anti-MASP-2 antibody, which suitably may be administered to an adult patient (e.g., an average adult weight of 70 kg) in a dosage of from 0,1 mg to 10,000 mg, more suitably from 1.0 mg to 5,000 mg, more suitably 10.0 mg to 2,000 mg, more suitably 10.0 mg to 1,000 mg and still more suitably from 50,0 mg to 500 mg. For pediatric patients, dosage can be adjusted in proportion to the patient’s weight. Application of the MASP-2 inhibitory compositions ofthe present invention may be carried out by a single administration of the composition, or a limited sequence of administrations, for treatment, of cold aggultinin disease or cryoglobulinemia.

Alternatively, the composition may be administered at periodic intervals, such as daily, biweekly, weekly, every other week, monthly or bimonthly, over an extended period of time for treatment of cold aggultinin disease or cryoglobulinemia.

2018200437 19 Jan 2018

In some embodiments, the subject suffering from cold aggultinin disease or cryoglobulinemia 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 comprises administering to the subject a composition of the invention comprising a MASP-2 inhibitor and further 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 ccuiizumab.

In one aspect of the invention, the pharmaceutical compositions are administered to a subject susceptible to, or otherwise at risk of, glaucoma in an amount sufficient to eliminate or reduce the risk of developing symptoms of the condition. In therapeutic applications, the pharmaceutical compositions are administered to a subject suspected of, or already suffering from, glaucoma in a therapeutically effective amount sufficient to relieve, or at least partially reduce, the symptoms of the condition.

In a still further aspect of the present invention, a subject suffering from or at risk of developing glaucoma and being treated with a MASP-2 inhibitory composition of the present invention can be monitored by periodically determining, such as every twelve hours or on a daily basis, the level of at least one complement factor, wherein the determination of a reduced level of the at least one complement factor in comparison to a standard value or to a healthy subject is indicative of the need for continued treatment with the composition.

in both prophylactic, and therapeutic regimens, compositions comprising MASP-2 inhibitory agents may be administered in several dosages until a sufficient therapeutic outcome has been achieved in the subject. In one embodiment of the invention, the MASP-2. inhibitory agent comprises an anti-MASP-2 antibody, which suitably may be administered to an adult patient (e.g., an average adult weight of 70 kg) in a dosage of from 0.1 mg to 10,000 mg, more suitably from 1.0 mg to 5,000 mg, more suitably 10.0 mg to 2,000 mg, more suitably 10.0 mg to 1,000 mg and still more suitably from 50.0 mg to 500 mg. For pediatric patients, dosage can be adjusted in proportion to the patient’s weight. Application of the MASP-2 inhibitory compositions of the present invention may be carried out by a single administration of the composition, or a limited sequence of administrations, for treatment of glaucoma. Alternatively, the composition may be

-1232018200437 19 Jan 2018 administered at periodic intervals, such as daily, biweekly, weekly, every other week, monthly or bimonthly, over an extended period of time for treatment of cold agguitinin disease.

In some embodiments, the subject suffering from glaucoma has previously 5 undergone, or is currently undergoing treatment with a terminal complement inhibitor that inhibits cleavage of complement protein C5. In some embodiments, the method comprises administering to the subject a composition of the invention comprising a MASP-2 inhibitor and further 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 ecuiizumab.

In one aspect of the invention, the pharmaceutical compositions are administered to a subject suffering from or ai risk of developing acute radiation syndrome, in an amount sufficient to eliminate or reduce the risk of developing symptoms of the condition, in therapeutic applications, the pharmaceutical compositions are administered to a subject suspected of, or already suffering from, acute radiation syndrome in a therapeutically effective amount sufficient to relieve, or at least partially reduce, the symptoms of the condition. A subject may be treated with a MASP-2 inhibitory composition of the present invention prior to or after exposure to radiation, such as radiation exposure for the treatment of cancerous conditions, while cleaning up a site contaminated with radiation, in working with radioactive materials in an energy generation plant or laboratory, or due to radiation exposure resulting from a nuclear accident, terrorist action or warfare. In one embodiment of the present invention, the

MASP-2 inhibitory composition is administered within 24 to 48 hours after radiation exposure,

In a still further aspect of the present invention, a subject suffering from or at risk of developing acute radiation syndrome and being treated with a MASP-2 inhibitory composition of the present invention can be monitored by periodically determining, such as every twelve hours or on a daily basis, the level of at least one complement factor, wherein the determination of a reduced level of the at least one complement factor in comparison to a standard value or to a healthy subject is indicative of the need for continued treatment with the composition.

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

MASP-2 inhibitory agent comprises an anti-MASP-2 antibody, which suitably may be administered to an adult patient (e.g., an average adult weight of 70 kg) in a dosage of from 0.1 mg to 10,000 mg, more suitably from 1.0 mg to 5,000 mg, more suitably 10,0 mg to 2,000 mg, more suitably 10,0 mg, to 1,000 mg and still more suitably from 50.0 mg to 500 mg. For pediatric patients, dosage can be adjusted in proportion to the patient’s weight. Application of the MASP-2 inhibitory compositions of the present invention may be carried out by a single administration of the composition, or a limited sequence of administrations, for treatment of glaucoma. Alternatively, the composition may be administered at periodic intervals, such as daily, biweekly, weekly, every other week, monthly or bimonthly, over an extended period of time for treatment of acute radiation syndrome.

In some embodiments, the subject suffering from acute radiation syndrome 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 comprises administering to the subject a composition of the invention comprising a MASP-2 inhibitor and further 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.

VI. EXAMPLES

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

EXAMPLE 1

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

1252018200437 19 Jan 2018

Materials and Methods: The targeting vector pKO-NTKV 1901 was designed to disrupt the three exons coding for the C-terminal end of murine MASP-2, including the exon that encodes the serine protease domain, as shown in FIGURE 3.

PKO-NTKV 1901 was used io transfect the murine ES ceil fineE14.la (SV129O!a).

Neomycin-resistant and Thymidine Kinase-sensitive clones were selected. 600 ES clones were screened and, of these, four different clones were identified and verified by southern blot to contain the expected selective targeting and recombination event as shown in FIGURE 3. Chimeras were generated from these four positive clones by embryo transfer. The chimeras were then backcrossed in the genetic background C57/BL6 to create transgenic males. The transgenic males were crossed with females to generate FIs with 50% of the offspring showing heterozygosity for the disrupted MASP-2 gene. The heterozygous mice were intercrossed to generate homozygous MASP-2 deficient offspring, resulting in heterozygous and wild-type mice in the ration of 1:2:1, respectively,

Results and Phenotype: The resulting homozygous MASP-2-/- deficient mice were found to be viable and fertile and were verified io be MASP-2 deficient by southern blot to confirm the correct targeting event, by Northern blot to confirm the absence of MASP-2 mRNA, and by Western blot to confirm the absence of MASP-2 protein (data not shown). The presence of MAp 19 mRNA and the absence of MASP-2 mRNA were further confirmed using time-resolved RT-PCR on a LightCycler machine. The MASP-2-/- mice do continue to express MAp 19, MASP-1, and MASP-3 mRNA and protein as expected (data not shown), The presence and abundance of mRNA in the MASP-2-/- mice for Properdin, Factor B, factor D, C4, €2, and C3 was assessed by LightCycler analysis and found to be identical to that of the wild-type littermate controls (data not shown). The plasma from homozygous MASP-2-/- mice is totally deficient of lectin-pathway-mediated complement activation as further described in Example 2,

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

A transgenic mouse strain that is murine MASP-2-/-, MAp 19+/+ and that expresses a human MASP-2 transgene (a murine MASP-2 knock-out and a human MASP-2 knock-in) was also generated as follows:

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Materials and Methods; A msnigene encoding human MASP-2 called mini hMASP-2 (SEQ ID NO:49) as shown in FIGURE 4 was constructed which includes the promoter region of the human MASP 2 gene, including the first 3 exons (exon 1 to exon 3) followed by the cDNA sequence that represents the coding sequence of the foliowing 8 exons, thereby encoding the full-length MASP-2 protein driven by its endogenous promoter. The mini hMASP-2 construct was injected into fertilized eggs of MASP-2-/- in order to replace the deficient murine MASP 2 gene by transgenlcally expressed human MASP-2.

EXAMPLES

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

Methods and Materials:

Lectin pathway specific C4 Cleavage Assay: A C4 cleavage assay has been described by Petersen, et al,, J. Immunol. Methods 257:191 (2001) that measures lectin pathway activation resulting from lipoteichoic acid (LTA) from S’, aureus, which binds L-ficolin. The assay described by Petersen et al., (2001) was adapted to measure lectin pathway activation via MBL by coating the plate with LPS and mannan or zymosan prior to adding serum from MASP-2 -/- mice as described below. The assay was also modified to remove the possibility of C4 cleavage due to the classical pathway. This was achieved by using a sample dilution buffer containing 1 M NaCl, which permits high affinity binding of lectin pathway recognition components to their ligands but prevents activation of endogenous C4, thereby excluding the participation of the classical pathway by dissociating the Cl complex, Briefly described, in the modified assay serum samples (diluted in high salt (1 M NaCl) buffer) are added to ligand-coated plates, followed by the addition of a constant amount of purified C4 in a buffer with a physiological concentration of salt. Bound recognition complexes containing MASP-2 cleave the C4. resulting in C4b deposition.

Assay Methods;

I) Nunc Maxisorb microtiter plates (Maxisorb, Nunc, Cat. No. 442404,

Fisher Scientific) were coated with 1 gg/mJ mannan (M7504 Sigma) or any other ligand (e.g., such as those listed below) diluted in coating buffer (15 mM N^COj, 35 mM NaHC.03, pH 9.6).

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The following reagents were used in the assay:

a. mannan (i .ug/weil mannan (M7504 Sigma) in 100 μΐ coating buffer):

b. zymosan (1 pg/weil zymosan (Sigma) in 100 μΐ coating buffer);

c. LTA (1 pg/well in 100 μΐ coating buffer or 2 pg/'well in 20 μΐ methanol)

d. 1 με of the H-ficolin specific Mab 4H5 in coating buffer

e. PSA from Aerococcus viridans (2 gg/weli in 100 μ I coating buffer)

f. 100 μΙ/well of formalin-fixed S. aureus DSM20233 (€®55θ=-Ό.5) in coating buffer.

2) The plates were incubated overnight at 4^CC.

3) After overnight incubation, the residual protein binding sites were saturated by incubated the plates with 0.1% HSA-TBS blocking buffer (0.1% (w/v) HSA in 10 mM Tris-CL, 140 mM NaCl, 1.5 mM NaNj, pH 7.4) for 1-3 hours, then washing the plates 3X with TBS/tween/Ca^⁴' (TBS with 0.05% Tween 20 and 5 mM CaCfo, 1 mM MgCQ, pH 7.4).

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

5) Following incubation overnight at 4°C, the plates were washed 3X with TBS/tween/Ca2+. Human C4 (100 μΐ/wet! of 1 ug/rnl diluted in BBS (4 mM barbital, 145 mM NaCl, 2 mM CaCl?, I mM MgCb, pH 7.4)) was then added ίο the plates and incubated for 90 minutes at 37°C. The plates were washed again 3X with TB S/tween/Ca2⁺,

6) C4b deposition was detected with an alkaline phosphatase-conjugated chicken anti-human C4c (diluted 1:1000 in TBS/tween/Ca^⁴), which was added to the plates and incubated tor 90 minutes at room temperature. The plates were then washed again 3X with TBS/tween/Ca^'L

7) Alkaline phosphatase was detected by adding 100 μΐ of p-nitrophenyl phosphate substrate solution, incubating at room temperature for 20 minutes, and reading the OD405 in a microtiter plate reader.

Results: FIGURES 5A-B show the amount ofC4b deposition on mannan (FIGURE 5A) and zymosan (FIGURE 5B) in serum dilutions from MASP-2+/+ (crosses), MASP-2+/- (closed circles) and MASP-2-/- (closed triangles). FIGURE 5C shows the relative C4 convertase activity on plates coated with zymosan (white bars) or

-1282018200437 19 Jan 2018 mannan (shaded bars) from MASP-2-/+ mice (n=5) and MASP-2-/- mice (n=4) relative to wild-type mice (n=5) based on measuring the amount of C4b deposition normalized to wild-type serum. The error bars represent the standard deviation. As shown in

FIGURES 5A-C, plasma from MASP-2-/- mice is totally deficient in iectin-pathway-mediated complement activation on mannan and on zymosan coated plates, These results clearly demonstrate that MASP-2 is an effector component of the lectin pathway,

Recombinant MASP-2 reconstitutes Lectin Pathway-Dependent C4 10 Activation in serum from the MASP-2-/- mice

In order to establish that the absence of MASP-2 was the direct cause of the loss of lectin pathway-dependent C4 activation in the MASP-2-/- mice, the effect of adding recombinant MASP-2 protein to serum samples was examined in the C4 cleavage assay described above. Functionally active murine MASP-2 and catalytically inactive murine

MASP-2A (in which the active-site serine residue in the serine protease domain was substituted for the alanine residue) recombinant proteins were produced and purified as described below in Example 3. Pooled serum from 4 MASP-2 -/- mice was pre-incubated with increasing protein concentrations of recombinant murine MASP-2 or inactive recombinant murine MASP-2A and C4 convertase activity was assayed as described above.

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

EXAMPLE 3

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

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Expression of FnH-Ieegth hsnoosi, murine and rat MASP-2:

The Kill length cDNA sequence of human MASP-2 (SEQ ID NO: 4) was also subcloned into the mammalian expression vector pCI-Neo (Promega), which drives eukaryotic expression under the control ofthe CMV enhancer/promoter region (described in Kaufman RJ. etal., Nucleic Acids Research 79:4485-90, 1991; Kaufman, Methods in Enzymology, 185:537-66 (3991)). The full length mouse cDNA (SEQ ID NO:50) and rat MASP-2 cDNA (SEQ ID NO:53) were each subcloned into the pED expression vector. The MASP-2 expression vectors were then transfected into the adherent Chinese hamster ovary ceil line DXBi using the standard calcium phosphate transfection procedure described in Maniatis et al., 1989, Cells transfected with these constructs grew' very slowly, implying that the encoded protease is cytotoxic.

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

Expression of Full-length catalytically inactive MASP-2:

Rationale: MASP-2 is activated by autocatalytic cleavage after the recognition subcomponents MBL or ficolins (either L-ficolin, H-ficolin or M-ficolin) bind to their respective carbohydrate pattern. Autocatalytic cleavage resulting in activation of

MASP-2 often occurs during the isolation procedure of MASP-2 from serum, or during the purification following recombinant expression. In order to obtain a more stable protein preparation for use as an antigen, a catalytically inactive form of MASP-2, designed as MASP-2A was created by replacing the serine residue that is present in the catalytic triad of the protease domain with an alanine residue in rat (SEQ ID NO:55

Serb 17 to Ala617); in mouse (SEQ ID NO:52 Ser617 to Ala617); or in human (SEQ ID

NOD Serb IS to Ala618).

In order to generate catalytically inactive human and murine MASP-2A proteins, site-directed mutagenesis was carried out using the oligonucleotides shown in TABLE 5. The oligonucleotides in TABLE 5 were designed to anneal to the region of the human and murine cDNA encoding the enzymatically active serine and oligonucleotide contain a mismatch in order ίο change the serine codon into an alanine codon. For example, PCR oligonucleotides SEQ ID NOS:56-59 were used in combination with human MASP-2 cDNA (SEQ ID NO:4) to amplify the region from the start codon to the enzymatically

-1302018200437 19 Jan 2018 active serine and from the serine to the stop codon to generate the complete open reading from of the mutated MASP-2A containing the Ser618 to Ala618 mutation, The PCR products were purified after agarose gel electrophoresis and band preparation and single adenosine overlaps were generated using a standard tailing procedure, The adenosine tailed MASP-2A was then cloned into the pGEM-T easy vector, transformed into £. coli.

A catalytically inactive rat MASP-2A protein was generated by kinasing and annealing SEQ ID NO:64 and SEQ ID NO:65 by combining these two oligonucleotides in equal molar amounts, heating at 100°C for 2 minutes and slowly cooling to room temperature. The resulting annealed fragment has Pstl and Xbal compatible ends and was inserted in place of the Pstl-Xbal fragment of the wild-type rat MASP-2 cDNA (SEQ ID NO:53) to generate rat MASP-2A.

'GAGGTGACGCAGGAGGGGCATTAGTGTTT 3' (SEQ ID NO :64)

5' CTAGAAACACTAATGCCCCTCCTGCGTCACCTCTGCA 3' (SEQ ID

NO;65)

The human, murine and rat MASP-2A were each further subcfoned into either of the mammalian expression vectors pED or pCI-Neo and transfected into the Chinese Hamster ovary cell line DXB1 as described below.

In another approach, a catalytically inactive form of MASP-2 is constructed using the method described in Chen et al,, J, Biol. Chem., 276(28):25894-25902, 2001. Briefly, the plasmid containing the full-length human MASP-2 cDNA (described in Thiel et al., Nature 386:566, 1997) is digested with Xhol and RcoRl and the MASP-2 cDNA (described herein as SEQ ID NO:4) is cloned into the corresponding restriction sites of the pFastBacl baculovirus transfer vector (Life Technologies, NY). The MASP-2 serine protease active site at Serb! 8 is then altered to Ala618 by substituting the double-stranded oligonucleotides encoding the peptide region amino acid 610-625 (SEQ ID NO: 13) with the native region amino acids 610 to 625 to create a MASP-2 full length polypeptide with an inactive protease domain. Construction of Expression Plasmids Containing Polypeptide Regions Derived from Human Masp-2.

The following constructs are produced using the MASP-2 signal peptide (residues 1-15 of SEQ ID NO:5) to secrete various domains of MASP-2, A construct expressing the human MASP-2 CUBI domain (SEQ ID NO:8) is made by PCR amplifying the region encoding residues 1-121 of MASP-2 (SEQ ID NO:6) (corresponding to the N-terminal CUBI domain), A construct expressing the human

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MASP-2 CUBIEGF domain (SEQ ID NO:9) is made by PCR amplifying the region encoding residues 1-166 of MASP-2 (SEQ ID NO:6) (corresponding io the N-terminal

CUBIEGF domain). A construct expressing the human MASP-2 CUBIEGFCUBii domain (SEQ ID NO: 10) is made by PCR amplifying the region encoding residues 1-293 of MASP-2 (S EQ ID NO:6) (corresponding to the N-terminal CUBIEGFCUBII domain). The above mentioned domains are amplified by PCR using VentR polymerase and pBS-MASP-2 as a template, according to established PCR methods. The 5’ primer sequence of the sense primer (5'-CGGGATCCATGAGGCTGCTGACCCTC-3' SEQ ID NO:34) introduces a BowHI restriction site (underlined) at the 5’ end of the PCR products. Antisense primers for each of the MASP-2 domains, shown below in TABLE 5, are designed to introduce a stop codon (boldface) followed by an EcoRl site (underlined) at the end of each PCR product. Once amplified, the DNA fragments are digested with BomHI and E'eoRI and cloned into the corresponding sites of the pFastBael vector. The resulting constructs are characterized by restriction mapping and confirmed by dsDNA sequencing.

TABLE 5: MASP-2 PCR PRIMERS ........................

MASP-2 domain	5' PCR Primer	3' PCR Primer
SEQ ID NO:8 CUBI (aa 1-121 of SEQ ID NO:6)	5’CGGGATCCATGA GGCTGCTGACCCT C-3'(SEQ ID NO:34)	5 'GGAA TTC CTAGGCTGC AT' A (SEQ ID NC ):35-
SEQ ID NON CUBIEGF (aa 1-166 of SEQ ID NO: 6)	5'CGGGATCCATGA GGCTGCTGACCCT' C-3’ (SEQ ID NO:34)	5 'GGA ATTCCTAC A GGGCGC T-3' (SEQ ID NQ:36)
SEQ ID NO: 10 CUBIEGFCUBII (aa i-293 of SEQ ID NO:6)	5'cgqgatccatga GGCTGCTGACCCT C-3! (SEQ ID NO:34)	5'GGAATTCCTAGTAGTGGA T 3' (SEQ ID NO:37)
SEQ ID NQ:4 human MASP-2	5'ATGAGGCTGCTG ACCCTCCTGGGCC TTC 3' (SEQ ID NO: 56) hMASP-2 forward	5 'TTAAAATC ACT AATTATG TTCTCGATC 3’ (SEQ ID NO: 59) hMASP-2 reverse

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MASP-2 domain	5’ PCR Primer 3' PCR Primer ΐ
SEQ ID NO:4 human MASP-2 cDNA	1 ........i 5'CAGAGGTGACGC i 5'GTGCCCCTCCTGCGTCAC | AGGAGGGGCAC 3' | CTCTG 3' (SEQ ID NO; 57) j (SEQ ID NO: 58) | hMASP-2 ala reverse i hMASP-2 ala forwar j j
SEQ IDNO’,50 Murine MASP-2 cDNA	5'ATGAGGCTACTC 1 S’TTAGAAATTACTI'ATTAT ATCTTCCTGG3' 1 GTTCTCAATCC3’ (SEQ ID (SEQ ID NO: 60) j NO: 63) raMASP-2 reverse mMASP-2. forward i
SEQ ID NO:SO Murine MASP-2 cDNA	5’CCCCCCCTGCGT 5‘CTGCAGAGGTGACGCAG | CACCTCTGCAG3* | GGGGGG 3' (SEQ ID NO: 61) | (SEQ ID NO: 62) i mMASP-2.jda jeverse | m.MASP-2_alaQbrwa j j rd i :

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

The MASP-2 and MASP-2A expression constructs described above were 5 transfected into DXB1 cells using the standard calcium phosphate transfection procedure (Maniatis et al., 1989). MASP-2A was produced in serum-free medium to ensure that preparations were not contaminated with other serum proteins. Media was harvested from confluent cells every second day (four times in total). The level of recombinant MASP-2 A averaged approximately 1.5 mg/iiter of culture medium for each of the three species.

MASP-2A protein purification; The MASP-2A (Ser-Ala mutant described above) was purified by affinity chromatography on MBP-A-agarose columns. This strategy enabled rapid purification without the use of extraneous tags. MASP-2 A (100-200 ml of medium diluted with an equal volume of loading buffer (50 mM Tris-Ci, pH 7.5, containing 150 mM NaCl and 25 mM CaCh) was loaded onto an MBP-agarose affinity column (4 ml) pre-equilibrated with 10 ml of loading buffer. Following washing with a further 10 ml of loading buffer, protein was eluted in 1 ml fractions with 50 mM Tris-Ci, pH 7,5, containing 1.25 M NaCl and lOmM EDTA. Fractions containing the MASP-2A were identified by SDS-polyacrylamide gel electrophoresis. Where necessary,

MASP-2A was purified further by ion-exchange chromatography on a MonoQ column

-1332018200437 19 Jan 2018 (HR 5/5). Protein was dialysed with 50 mM Tris-Ci pH 7.5, containing 50 mM NaCI and loaded onto the column equilibrated in the same buffer. Following washing, bound

MASP-2A was eluted with a 0.05-1 M NaCI gradient over 30 ml.

Results: Yields of 0.25-0,5 mg of MASP-2A protein were obtained from 200 ml 5 of medium. The molecular mass of 77.5 kDa determined by MALDI-MS is greater than the calculated value of the unmodified polypeptide (73.5 kDa) due to glycosylation. .Attachment of glycans at each of the TV-glycosylation sites accounts for the observed mass. MASP-2A migrates as a single band on SDS-polyacrylamide gels, demonstrating that It is not proteolytically processed during biosynthesis, The weight-average molecular mass determined by equilibrium ultracentrifugation is in agreement with the calculated value for homodimers of the glycosylated polypeptide.

PRODUCTION OF RECOMBINANT HUMAN MASP-2 POLYPEPTIDES

Another method for producing recombinant MASP-2 and MASP2A derived polypeptides is described in Thielens, N.M., etai., J. Immunol. 166:5068-5077, 2001.

Briefly, the Spodoptera frugiperda insect ceils (Ready-Plaque SIR cells obtained from Novagen, Madison, Wl) are grown and maintained in Sf900II serum-free medium (Life Technologies) supplemented with 50 lU/ml penicillin and 50 mg/ml streptomycin (Life Technologies). The Trichoplusia ni (High Five) insect ceils (provided by Jadwiga Chroboczek, Institut de Biologic Structurale, Grenoble, France) are maintained in TCI 00 medium (Life Technologies) containing 10% FCS (Dominique Dutscher, Brumath, France) supplemented with 50 lU/ml penicillin and 50 mg/ml streptomycin. Recombinant baculoviruses are generated using the Bac-to-Bac system (Life Technologies). The bacmid DNA is purified using the Qiagen midlprep purification system (Qiagen) and is used to transfect Sf9 insect ceils using eellfectin in Sf900 11 SFM medium (Life Technologies) as described in the manufacturer's protocol. Recombinant virus particles are collected 4 days later, titrated by virus plaque assay, and amplified as described by King and Possee, in The Baculovirus Expression System: Λ Laboratory Guide, Chapman and Hall Ltd., London, pp. 111-114, 1992,

High Five cells (1.75 x 10⁷ cells/175-cm² tissue culture flask) are infected with the recombinant viruses containing MASP-2 polypeptides at a multiplicity of infection of 2 in SfDOQ II SFM medium at 28°C for 96 h. The supernatants are collected by centrifugation and diisopropyl phosphorofluoridate is added to a final concentration of 1 mM.

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The MASP-2 polypeptides are secreted in the culture medium. The culture supernatants are dialyzed against 50 mM NaCl, 1 mM CaCh, 50 mM triethanolamine hydrochloride, pH 8.1, and loaded at 1.5 ml/min onto a Q-Sepharose Fast Flow column (Amersham Pharmacia Biotech) (2.8 x 12 cm) equilibrated in the same buffer. Elution is conducted by applying al .2 liter linear gradient to 350 mM NaCl in the same buffer. Fractions containing the recombinant MASP-2 polypeptides are identified by Western blot analysis, precipitated by addition of (NH^SC^ to 60% (w/v), and left overnight at4°C. The pellets are resuspended in 145 mM NaCl, 1 mM CaCl₂, 50 mM triethanolamine hydrochloride, pH 7.4, and applied onto a TSK G3000 SWG column (7.5 x 600 mm) (Tosohaas, Montgomeryville, PA) equilibrated in the same buffer. The purified polypeptides are then concentrated to 0.3 mg/ml by ultrafiltration on Microsep microconcentrators (m.w. cut-off == 10,000) (Filtron, Karistein, Germany),

EXAMPLE 4

This example describes a method of producing polyclonal antibodies against

MASP-2 polypeptides.

Materials and Methods:

MASP-2 Antigens: Polyclonal anti-human MASP-2 antiserum is produced by immunizing rabbits with the following isolated MASP-2 polypeptides: human MASP-2 (SEQ ID NO:6) isolated from serum; recombinant human MASP-2 (SEQ ID NQ:6), MASP-2A containing the inactive protease domain (SEQ ID NO: 13), as described in Example 3; and recombinant CUB1 (SEQ ID NO:8), CUBEGFI (SEQ ID NO:9), and CUBEGFCUBH (SEQ ID NO: 10) expressed as described above in Example 3.

Polyclonal antibodies; Six-week old Rabbits, primed with BCG (bacillus

Calmette-Guerin vaccine) are immunized by injecting 100 pg of MASP-2 polypeptide at 100 pg/ml in sterile saline solution. Injections are done every 4 weeks, with antibody titer monitored by ELISA assay as described in Example 5. Culture supernatants are collected for antibody purification by protein A affinity chromatography.

EXAMPLE S

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

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Materials and Methods:

.Male A/J mice (Harlan, Houston, Tex.), 8-12 weeks old, are injected subcutaneously with 100 pg human or rat rMASP-2 or rMASP-2A polypeptides (made as described in Example 3) in complete Freund's adjuvant (Difco Laboratories, Detroit,

Mich.) in 200 μί of phosphate buffered saline (PBS) pH 7.4. At two-week intervals the mice are twice injected subcutaneously with 50 pg of human or rat rMASP-2 or rMASP-2A polypeptide in incomplete Freund's adjuvant. On the fourth week the mice are injected with 50 pg of human or rat rMASP-2 or rMASP-2A polypeptide in PBS and are fused 4 days later.

For each fusion, single cell suspensions are prepared from the spleen of an immunized mouse and used for fusion with Sp2/0 myeloma ceils. 5x1of the Sp2/0 and 5x108 _Spi_een cells are fused in a medium containing 50% polyethylene glycol (M.W. 1450) (Kodak, Rochester, N.Y.) and 5% dimethylsulfoxide (Sigma Chemical Co., St. Louis, Mo.). The cells are then adjusted to a concentration of 1.5x16° spleen cells per

200 μ! of the suspension in lscove medium (Gibco, Grand island, NY,}, supplemented with 10% fetal bovine serum, 100 units/ml of penicillin, 100 pg/ml of streptomycin, 0.1 mM hypoxanthine, 0.4 μΜ aminopterin and 16 μΜ thymidine. Two hundred microliters of the cell suspension are added to each well of about twenty 96-weii microculture plates. After about ten days culture supernatants are withdrawn for screening for reactivity with purified factor MASP-2 in an ELISA assay,

ELISA Assay: Wells of Immulon 2 (Dynatech Laboratories, Chantilly, Va.) microtest plates are coated by adding 50 μί of purified hMASP-2 at 50 ng/ml or rat rMASP-2 (or rMASP-2A) overnight at room temperature. The low concentration of MASP-2 for coating enables the selection of high-affinity antibodies. After the coating solution is removed by flicking the plate, 200 μ! of BLOTTO (non-fat dry milk) in PBS is added to each well for one hour to block the non-specific sites. An hour later, the wells are then washed with a buffer PBST (PBS containing 0.05% Tween 20). Fifty microliters of culture supernatants from each fusion well is collected and mixed with 50 μί of BLOTTO and then added to the individual wells of the microtest plates. After one hour of incubation, the wells are washed with PBST. The bound murine antibodies are then detected by reaction with horseradish peroxidase (HRP) conjugated goat anti-mouse IgG (Fc specific) (Jackson ImmunoResearch Laboratories, West Grove, Pa.) and diluted at 1:2.000 in BLOTTO, Peroxidase substrate solution containing 0.1% 3,3,5,5 tetramethyl

-1362018200437 19 Jan 2018 benzidine (Sigma, St. Louis, Mo.) and 0.0003% hydrogen peroxide (Sigma) is added to the wells for color development for 30 minutes. The reaction is terminated by addition of μί of 2M H2SO4 per well. The Optical Density at 450 nm of the reaction mixture is read with a BioTek ELISA Reader (BioTek instruments, Winooski. Vt.).

MASP-2 Binding Assay:

Culture supernatants that test positive in the MASP-2 ELISA assay described above can be tested in a binding assay to determine the binding affinity the MASP-2 inhibitory agents have for MASP-2. A similar assay can also be used to determine if the inhibitory agents hind to other antigens in the complement system.

Polystyrene microtiter plate wells (96-well medium binding plates, Corning

Costar, Cambridge, MA) are coated with MASP-2 (20 ng/100 μί/well, Advanced Research Technology, San Diego, CA) in phosphate-buffered saline (PBS) pH 7.4 overnight at 4°C. After aspirating the MASP-2 solution, wells are blocked with PBS containing 1% bovine serum albumin (BSA; Sigma Chemical) for 2h at room temperature. Wells without MASP-2 coating serve as the background controls. Aiiquots of hybridoma supernatants or purified anti-MASP-2 MoAbs, at varying concentrations in blocking solution, are added to the wells, Following a 2 h incubation at room temperature, the wells are extensively rinsed with PBS. MASP-2-bound anti-MASP-2 MoAb is detected by the addition of peroxidase-conjugated goat anti-mouse IgG (Sigma

Chemical) in blocking solution, which is allowed to incubate for Ih at room temperature. The plate is rinsed again thoroughly with PBS, and 100 μ! of 3,3',5.5'-tetramethyi benzidine (TMB) substrate (Kirkegaard and Perry Laboratories, Gaithersburg, MD) is added. The reaction of TMB is quenched by the addition of 100 μ! of 1M phosphoricacid, and the plate is read at 450 nm in a microplate reader (SPECTRA MAX 250,

Molecular Devices, Sunny vale, CA).

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

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EXAMPLE 6

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

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

1, Cloning of anti-MASP-2 variable region genes from murine hybridoma ceils. Total RNA is isolated from the hybridoma cells secreting anti-MASP-2 MoAb (obtained as described in Example 7) using RNAzol following the manufacturer's protocol (Biotech, Houston, Tex.). First strand cDNA is synthesized from the total RNA using oiigo d'T as the primer. PCR is performed using the immunoglobulin constant C region-derived 3' primers and degenerate primer sets derived from the leader peptide or the first framework region of murine Vjq or Vjj genes as the 5' primers.

Anchored PCR is carried out as described by Chen and Platsucas (Chen, P.F., Scand. J. Immunol. 35:539-549, 1992). For cloning the V« gene, double-stranded cDNA is prepared using a Notl-MAKl primer (5MTGCGGCCGCTGTAGGTGCTGTCTTT-3'

SEQ ID NO:38). Annealed adaptors ADI (5'-GGAATTCACTCGTI'AlTCTCGGA-3' SEQ ID NO:39) and AD2 (5-TCCGAGAATAACGAGTG-3' SEQ ID NO:40) are ligated to both 5' and 3' termini of the double-stranded cDNA. Adaptors at the 3' ends are removed by Notl digestion. The digested product is then used as the template in PCR with the ADI oligonucleotide as the 5' primer and MAK2 (5'-CATTGAAAGCTTTGGGGTAGAAGTTGTTC-3' SEQ ID NO:41) as the 3’ primer. DNA fragments of approximately 500 bp are cloned into pUC19. Several clones are selected for sequence analysis to verify that the cloned sequence encompasses the expected murine immunoglobulin constant region. The Notl-MAK! and MAK2 oligonucleotides are derived from the Vfy region and are 182 and 84 bp, respectively, downstream from the first base pair of the C kappa gene. Clones are chosen that include the complete and leader peptide.

For cloning the Vjq gene, double-stranded cDNA is prepared using the Notl MAGI primer (5'-CGCGGCCGCAGCTGCTCAGAGTGTAGA-3' SEQ ID NO:42).

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Annealed adaptors ADI and AD2 are iigated to both 5' and 3' termini of the double-stranded cDNA. Adaptors at the 3’ ends are removed by Not! digestion. The digested product are used as the template in PCR with the .ADI oligonucleotide and

MAG2 (5-CGGTAAGCTTCACTGGCTCAGGGAAATA-3' SEQ 1DNO:43) as primers.

DNA fragments of 500 to 600 bp in iength are cloned into pUC19, The Notl-MAGl and MAG2 oligonucleotides are derived from the murine Cy.7.1 region, and are 180 and 93 bp, respectively, downstream from the first bp of the murine Cy.7.1 gene. Clones are chosen that encompass the complete Vjq and leader peptide.

2. Construction of Expression Vectors for Chimeric MASP-2 IgG and 10 Fab. The cloned Vjq and Vj; genes described above are used as templates in a PCR reaction to add the Kozak consensus sequence to the 5' end and the splice donor to the 3' end of the nucleotide sequence. After the sequences are analyzed to confirm the absence of PCR errors, the Vjq and Vj£ genes are inserted into expression vector cassettes containing human C.yl and C. kappa respectively, to give pSV2neoVpj~huCyl and

IS pSV2aeoV-huCy. CsCi gradient-purified plasmid DNAs ofthe heavy- and light-chain vectors are used to transfect COS cells by electroporation. After 48 hours, the culture supernatant is tested by ELISA to confirm the presence of approximately 200 ng/ml of chimeric IgG. The cells are harvested and total RNA Is prepared. First strand cDNA is synthesized from the total RNA using oligo dT as the primer, This cDNA is used as the template in PCR to generate the Fd and kappa DNA fragments. For the Fd gene, PCR is carried out usi n g 5 '-AAG AAGCTTGCCGCC ACC A TG G ATTGGCTGTGG AA CT-3' (SEQ ID NQ-,44) as the 5' primer and a CHI-derived 3’ primer (5'-CGGGATCCTCAAACTTTCTTGTCCACCTTGG-3' SEQ ID NO:45). The DNA sequence is confirmed to contain the complete Vyj and the CHI domain of human IgGl.

After digestion with the proper enzymes, the Fd DNA fragments are inserted at the Hindi!! and BamHI restriction sites of the expression vector cassette pSV2dhfr-TUS to give pSV2dhfrFd. The pSV2 plasmid is commercially available and consists of DNA segments from various sources: pBR322 DNA (thin line) contains the pBR.322 origin of DNA replication (pBR ori) and the lactamase arapicillin resistance gene (Amp); SV40

DNA, represented by wider hatching and marked, contains the SV40 origin of DNA replication (SV40 ori), early promoter (5' to the dhfr and neo genes), and polyadenylation signal (3’ to the dhfr and neo genes). The SV40-derived polyadenylation signal (pA) is also placed at the 3' end of the Fd gene,

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For the kappa gene, PCR is carried out using 5'AAGAAAGCTTGCCGCCACCATGTTCTCACTAGCTCT-3' (SEQ ID NO:46) as the 5' primer and a C_K-derived 3’ primer (5'-CGGGATCCTTCTCCCTCTAACACTCT-3' SEQ

ID NO:47). DNA sequence is confirmed io contain the complete and human regions. After digestion with proper restriction enzymes, the kappa DNA fragments are inserted at the Hindi!!! and BamHI restriction sites of the expression vector cassette pSV2neo-TUS to give pSV2neoK, The expression of both Fd and .kappa genes are driven by the HCMV-derived enhancer and promoter elements. Since the Fd gene does not include the cysteine amino acid residue involved in the inter-chain disulfide bond, this recombinant chimeric Fab contains non-covalentiy linked heavy- and light-chains. This chimeric Fab is designated as cFab.

To obtain recombinant Fab with an inter-heavy and light chain disulfide bond, the above Fd gene may be extended to include the coding sequence for additional 9 amino acids (EPKSCDKTH SEQ ID NO:48) from the hinge region of human IgGl. The

BstEII-BamHI DNA segment encoding 30 amino acids at the 3' end of the Fd gene may be replaced with DNA segments encoding the extended Fd, resulting in pSV2dhfrFd/9aa.

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

To generate cell lines secreting chimeric anti-MASP-2 IgG, NSO cells are transfected with purified plasmid DNAs of pSV2neoVf]-huC.yl and pSV2neoV-huC kappa by electroporation. Transfected ceils are selected in the presence of 0.7 mg/ml G418. Cells are grown in a 250 ml spinner flask using serum-containing medium.

Culture supernatant of 100 ml spinner culture is loaded on a 10-ml PROSEP-A column (Bioprocessing, Inc., Princeton, N.J.). The column is washed with 10 bed volumes of PBS. The bound antibody is eluted with 50 mM citrate buffer, pH 3.0. Equal volume of 1 M Hepes, pH 8.0 is added to the fraction containing the purified antibody to adjust the pH to 7.0. Residual salts are removed by buffer exchange with PBS by Millipore membrane ultrafiltration (M.W. cut-off: 3,000). The protein concentration of the purified antibody is determined by the BCA method (Pierce).

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

To generate cell lines secreting chimeric anti-MASP-2 Fab, CHO ceils are transfected with purified plasmid DNAs of pSV2dhfrFd (or pSV2dhfrFd/9aa) and pSVlneokappa, by electroporation. Transfected cells are selected in the presence of G418 and methotrexate. Selected cell Hues are amplified in increasing concentrations of

-1402018200437 19 Jan 2018 methotrexate, Cells are single-cell subcloned by limiting dilution. High-producing single-cell subcloned cell lines are then grown in 100 mi spinner culture using serum-tree medium.

Chimeric anti-MASP-2 Fab is purified by affinity chromatography using a mouse 5 anti-idiotypic MoAb to the MASP-2 MoAb. An anti-idiotypic MASP-2 MoAb can be made by immunizing mice with a murine anti-MASP-2 MoAb conjugated with keyhole limpet hemocyanin (KLHj and screening for specific MoAb binding that can be competed with human MASP-2, For purification, 100 ml of supernatant from spinner cultures of CHO cells producing cFab or cFab/9aa are loaded onto the affinity column coupled with an anti-idiotype MASP-2 MoAb. The column is then washed thoroughly with PBS before the bound Fab is eluted with 50 mM diethyiamine, pH 11,5, Residual salts are removed by buffer exchange as described above. The protein concentration of the purified Fab is determined by the BCA method (Pierce).

The ability of the chimeric MASP-2 IgG, cFab, and cFAb/9aa to inhibit

MASP-2-dependent complement pathways may be determined by using the inhibitory assays described in Example 2 or Example 7.

EXAMPLE 7

This example describes an in vitro C4 cleavage assay used as a functional screen to identify MASP-2 inhibitory agents capable of blocking MASP-2-dependent complement activation via L-ficolin/P35₅ H-ficolin, M-ficolin or mannan.

C4 Cleavage Assay: A C4 cleavage assay has been described by Petersen,

S.V., etai., J, Immunol. Methods 257:107, 2001, which measures lectin pathway activation resulting from Iipoteichoic acid (LTA) from A aureus which binds L-ficolin.

Reagents: Formalin-fixed S'. aureous (DSM20233) is prepared as follows:

bacteria is grown overnight at 37°C in tryptic soy blood medium, washed three times with PBS, then fixed for 1 h at room temperature in PBS/0.5% formalin, and washed a further three times with PBS, before being resuspended in coating buffer (15 mM NajCvyy 35 mM NaHCOj, pH 9.6).

Assay: The wells of a Nunc Maxi Sorb microtiter plate (Nalgene Nunc

International, Rochester, NY) are coated with: 100 μί of formalin-fixed S', aureus DSM20233 (00559 = 0.5) in coating buffer with 1 ug of L-ficolin in coating buffer. After overnight incubation, wells are blocked with 0.1% human serum albumin (HSA) in

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TBS (IOmM Tris-HCl, 140mM NaCl, pH 7.4), then are washed with TBS containing

0.05% Tween 20 and 5 mM CaCH (wash buffer). Human serum samples are diluted in mM Tris-HCl, ί M NaCl, IOmM CaCI₂, 0,05% Triton X-100, 0.1% HSA, pH 7.4, which prevents activation of endogenous C4 and dissociates the Ci complex (composed of Clq, Clr and Cis). MASP-2 inhibitory agents, including anti-MASP-2 MoAbs and inhibitory peptides are added to the serum samples in varying concentrations, Tire diluted samples are added ίο the plate and incubated overnight at 4C. /After 24 hours, the plates are washed thoroughly with wash buffer, then 0.1 p.g of purified human C4 (obtained as described in Dodds, A.W., Methods Enzymol. 223:46, 1993) in 100 μί of 4 mM barbital,

145 mM NaCl, 2 mM CaCQ, 1 mM MgClj, pH 7.4 is added to each well. After 1.5 h at

37°C, the plates are washed again and C4b deposition is detected using alkaline phosphatase-conjugated chicken anti-human C4c (obtained from lmmunsystem, Uppsala, Sweden) and measured using the colorimetric substrate p-nitrophenyl phosphate.

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

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

EXAMPLE 8

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

Methods: Immune complexes were generated in situ by coating microtiter plates 25 (Maxisorb, Nunc, cat. No. 442404, Fisher Scientific) with 0,1% human serum albumin in IOmM Tris, 140 mM NaCl, pH 7.4 for 1 hours at room temperature followed by overnight incubation at 4°C with sheep anti whole serum antiserum (Scottish Antibody Production Unit, Carluke, Scotland) diluted 1:1000 in TBS/tween/Ca2⁺, Serum samples w'ere obtained from wild-type and MASP-2-/- mice and added to the coated plates.

Control samples were prepared in which Clq was depleted from wild-type and MASP-2-/- serum samples. Clq-depleted mouse serum was prepared using protein-A-coupled Dvnabeads (Dynal Biotech, Oslo, Norway) coated with rabbit anti-human Clq IgG (Dako, Giostrup, Denmark), according to the supplier's instructions.

-1422018200437 19 Jan 2018

The plates were incubated for 90 minutes at 37°C. Bound C3b was detected with a polyclonal anti-human-C3c Antibody (Dako A 062) diluted in TBS/tw/ Ca⁺⁺' at 1:1000.

The secondary antibody is goat anti-rabbit IgG.

Results: FIGURE 7 shows the relative C3b deposition levels on plates coated 5 with IgG in wild-type serum, MASP-2-/- serum, Clq-depleied wild-type and Clq-depleted MASP-2-/- serum. These results demonstrate that the classical pathway is intact in the MASP-2-/- mouse strain.

EXAMPLE 9

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

Methods: To test the effect of a MASP-2 inhibitory agent on conditions of complement activation where the classical pathway is initiated by immune, complexes, triplicate 50 μΐ samples containing 90% NHS are incubated at 37°C in the presence of 10 gg/nil immune complex (IC) or PBS, and parallel triplicate samples (+/-IC) are also included which contain 200 nM anti-properdin monoclonal antibody during the 37°C incubation. After a two hour incubation at 37°C, 13 mM EDTA is added to all samples to stop further complement activation and the samples are immediately cooled to 5°C. The samples are then stored at -7()°C prior to being assayed for complement activation products (C3a and sC5b-9) using ELISA kits (Quidel, Catalog Nos. AO 15 and A009) following the manufacturer's instructions,

EXAMPLE 10

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

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

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To block MASP-2 functional activity, an antibody or Fab2 antibody fragment must bind and interfere with a structural epitope on MASP-2 that is required for MASP-2 functional activity. Therefore, many or all of the high affinity binding anti-MASP-2

Fab2s may not inhibit MASP-2 functional activity unless they bind to structural epitopes on MASP-2 that are directly involved in MASP-2 functional activity,

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

Generation of High Affinity Fal>2s: A phage display library of human variable light and heavy chain antibody sequences and automated antibody selection technology for identifying Fab2s that react with selected ligands of interest was used to create high affinity Fab2s to rat MASP-2 protein (SEQ ID NO:55), A known amount of rat MASP-2 (~1 mg, >85% pure) protein was utilized for antibody screening. Three rounds of amplification were utilized for selection of the antibodies with the best affinity'.

Approximately 250 different hits expressing antibody fragments were picked for ELISA screening. High affinity hits were subsequently sequenced to determine uniqueness of the different antibodies,

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

.144.

2018200437 19 Jan 2018

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

Fab2s

L Assay to Measure Inhibition of Formation of Lectin Pathway C3

Coavertase:

Background: The lectin pathway C3 convertase is the enzymatic complex (C4bC2a) that proteolytically cleaves C3 into the two potent proinfiammatory fragments, anaphylatoxin C3a and opsonic C3b. Formation of C3 convertase appears to a key step in the lectin pathway in terms of mediating inflammation. MASP-2 is not a structural component of the lectin pathway C3 convertase (C4bC2a); therefore anti-MASP-2 antibodies (or Fab2) will not directly inhibit activity of preexisting C3 convertase. However, MASP-2 serine protease activity is required in order to generate the two protein components (C4b, C2a) that comprise the lectin pathway ¢3 convertase. Therefore, anti-MASP-2 Fab2 which inhibit MASP-2 functional activity (i.e., blocking anti-MASP-2 Fab2) will inhibit de novo formation of lectin pathway C3 convertase, C3 contains art unusual and highly reactive thioester group as part of its structure, Upon cleavage of C3 by C3 convertase in this assay, the thioester group on C3b can form a covalent bond with hydroxyl or amino groups on macromoleeules immobilized on the bottom of the plastic wells via ester or amide linkages, thus facilitating detection of C3b in the ELISA assay.

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

Methods:

96-well Costar Medium Binding plates were incubated overnight at 5°C with mannan diluted in 50 mM carbonate buffer, pH 9.5 at 1 ug/50 Ti/'well, After overnight incubation, each well was washed three times with 200 T3 PBS. The wells were then blocked with 100 TJ/weli of 1% bovine serum albumin in PBS and incubated for one hour at room temperature with gentle mixing. Each well was then washed three times with 200 T1 of PBS, The anti-MASP-2 Fab2 samples were diluted to selected concentrations

-1452018200437 19 Jan 2018 in Ca^ and Mg⁺⁺ containing GVB buffer (4.0 mM barbital, 141 mM NaCl, 1.0 mM

MgCb, 2.0 mM CaClj, 0.1% gelatin, pH 7,4) at 5 C. A 0,5% rat serum was added to the above samples at 5 C and 100 71 was transferred to each well. Plates were covered and incubated for 30 minutes in a 37 C waterbath to allow complement activation, The reaction was stopped by transferring the plates from the 37 C waterbath to a container containing an ice-water mix, Each well was washed five times with 200 Tl with PBS-Tween 20 (0.05% Tween 20 in PBS), then washed two times with 200 Tl PBS. A 100 Tl/well of 3:10,000 dilution of the primary antibody (rabbit anti-human C3c, DAKO A0062) was added in PBS containing 2,0 mg/ml bovine serum albumin and incubated 1 hr at room temperature with gentle mixing, Each well was washed 5 x 200 Tl PBS. 100 Τί/weIJ of 1:10,000 dilution of the secondary antibody (peroxidase-conjugated goat anti-rabbit IgG, American Quaiex A102PU) was added in PBS containing 2.0 mg/ml bovine scrum albumin and incubated for one hour at room temperature on a shaker with gentle mixing. Each well was washed five times with 200 Tl with PBS. 100 ΐΐ/weli of the peroxidase substrate TMB (Kirkegaard & Perry Laboratories) was added and incubated at room temperature for 10 min. The peroxidase reaction was stopped by adding 100 Tl/well of 1.0 Μ H3PO4 and the OD453. was measured,

2. Assay to Measure Inhibition of MASP-2-dependent C4 Cleavage Background: The serine protease activity of MASP-2 is highly specific and only two protein substrates for MASP-2 have been identified; C2 and C4. Cleavage of' €4 generates C4a and C4b. Anti-MASP-2 Fab2 may bind to structural epitopes on MASP-2 that are directly involved in C4 cleavage (e.g., MASP-2 binding site for C4; MASP-2 serine protease catalytic site) and thereby inhibit the C4 cleavage functional activity' of MASP-2.

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

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Methods: 96-well Costar Medium Binding plates were incubated overnight at

C with mannan diluted in 50 mM carbonate buffer, pH 9.5 at 1.0 Tg/50 Tl/well. Each wed was washed 3X with 200 Tl PBS. The wells were then blocked with 100 Tl/well of

1% bovine serum albumin in PBS and incubated for one hour at room temperature with gentle mixing. Each well was washed 3X with 200 Tl of PBS, Anti-MASP-2 Fab2 samples were diluted to selected concentrations in Ca⁺⁺ and Mg⁺⁺ containing GVB buffer (4.0 mM barbital, 141 inM NaCl, 1.0 mM MgCT, 2.0 tnM CaCl₂, 0,1% gelatin, pH 7.4) at 5 C. 1.0 Tg/ml human C4 (Quidel) was also included in these samples. 0.5% rat serum was added to the above samples at 5 C and 100 Tl was transferred to each well. The plates were covered and incubated for 30 min in a 37 C waterbath to allow complement activation. The reaction was stopped by transferring the plates from the 37 C waterbath to a container containing an ice-water mix. Each well was washed 5 x200 Tl with PBS-Tween 20 (0.05% Tween 20 in PBS), then each well was washed with 2X with 2.00 Tl PBS, 100 Tl/well of 1:700 dilution of biotin-conjugated chicken anti-human C4c (Immunsystem AB, Uppsala, Sweden) was added in PBS containing 2.0 mg/ml bovine serum albumin (BSA) and incubated one hour at room temperature with gentle mixing. Each well was washed 5 x 200 Tl PBS. 100 Tl/well of 0.1 Tg/ml of peroxidase-conjugated streptavidin (Pierce Chemical #21126) was added in PBS containing 2.0 mg/ml BSA and incubated for one hour at room temperature on a shaker with gentle mixing. Each well was washed 5 x200 Tl with PBS. 100 Tl/well of the peroxidase substrate TMB (Kirkegaard & Perry Laboratories) was added and Incubated at room temperature for 16 min. The peroxidase reaction was stopped by adding 100 Tl/well of 1.0 Μ H3PO4 and the GD450 -was measured.

3. Binding Assay of anti-rat MASP-2 Fah2 to ’Native' rat MASP-2

Background: MASP-2 is usually present in plasma as a MASP-2 dimer complex that also includes specific lectin molecules (mannose-binding protein (MBL) and ficolins). Therefore, if one is interested in studying the binding of anti-MASP-2 Fab2 to the physiologically relevant form of MASP-2, It is important to develop a binding assay in which the interaction between the Fab2 and 'native* MASP-2 in plasma is used, rather than purified recombinant MASP-2. In this binding assay the 'native' MASP-2-MBL complex from 10% rat serum was first immobilized onto mannan-coated wells. The binding affinity of various anti-MASP-2 Fab2s to the immobilized 'native' MASP-2 was then studied using a standard ELISA methodology.

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Methods: 96-well Costar High Binding plates were incubated overnight at 5°C with mannan diluted in 50 mM carbonate buffer, pH 9.5 at I Tg/50 'Π/weli. Each well was washed 3X with 200 Tl PBS. The wells were blocked with 100 Tl/well of 0.5% nonfat dry milk in PBST (PBS with 0.05% Tween 20) and incubated for one hour at room temperature with gentle mixing. Each well was washed 3X with 200 Tl of TBS/Tween/Ca^r+ Wash Buffer (Tris-buffered saline, 0,05% Tween 20, containing 5,0 mM CaCL, pH 7.4. 10% rat serum in High Sait Binding Buffer (20 mM Tris, 1.0 M NaCl, 10 mM CaCfe, 0.05% Triton-X100, 0.1% (w/v) bovine serum albumin, pH 7.4) was prepared on ice, 100 Ti/well was added and incubated overnight at 5°C, Weils were washed 3X with 200 Tl of TBS/Tween/Ca*⁴ Wash Buffer. Wells were then washed 2X with 200 Tl PBS. 100 Tl/weli of selected concentration of anti-MASP-2 Fab2 diluted in Ca⁺⁺ and Mg'^H' containing GVB Buffer (4.0 mM barbital, 141 mM NaCi, 1.0 mM MgCL, 2.0 mM CaCfi, 0.1% gelatin, pH 7.4) was added and incubated for one hour at room temperature with gentle mixing. Each well was washed 5 x 200 Tl PBS. 100 Tl/weli of

HRP-conj ugated goat anti-Fab2 (Biogenesis Cat No 0500-0099) diluted 1:5000 in 2,0 mg/ml bovine serum albumin in PBS was added and incubated for one hour at room temperature with gentle mixing. Each well was washed 5 x 200 Ti PBS, 100 Tl/well of the peroxidase substrate TMB (Kirkegaard & Perry Laboratories) was added and incubated at room temperature for 70 min. The peroxidase reaction was stopped by adding 100 Tl/wel! of 1,0 M H₃PO₄ and OD₄₅o, was measured.

RESULTS:

Approximately 250 different Fab2s that reacted with high affinity to the rat MASP-2 protein were picked for ELISA screening. These high affinity Fab2s were sequenced fo determine the uniqueness of the different antibodies, and 50 unique anti-MASP-2 antibodies were purified for further analysis. 250 ug of each purified Fab2 antibody was used for characterization of MASP-2 binding affinity and complement pathway functional testing. The results of this analysis is shown below' in TABLE 6.

TABLE 6: ANTI-MASP-2 FAB2 THAT BLOCK LECTIN PATHWAY

COMPLEMENT ACTIVATION

: Pab2 antibody #	j C3 Convertase (IC5() (nM)	Γ'''	k-d	; C4 Cleavage 1 j 1C5O (nM) |
i 88	1 0.32		4.1	ΐ ND

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Fab2 antibody #	C3 Convertase (IC5o (nM)		C4 Cleavage IC50 (nM)
41 : j '	0.35	0.30	0.81
11 1	0.46	0.86	<2 nM
86 1	0.53	1,4	ND
81	0.54	2.0	ND
66	0.92	4.5	ND
57 1	0.95	3.6	<2 nM
40 1	El	i J	0.68
58	1.3	2.6	ND
60 |	1.6	3.1	ND
52	1.6	5.8	<2 nM
63 |	2.0	6.6	ND
49 ί	2.8	8,5	<2 nM

i 89		3.0	2.5			ND |
i 71	u	3.0	10,5			ND I
1 87	.........'1.........	6.0	2.5			ND
Ϊ 67		10.0	7.7			ND }

As shown above in TABLE 6, of the 50 anti-MASP-2 Fab2s tested, seventeen Fab2s were identified as MASP-2 blocking Fab2 that potently inhibit C3 convertase formation with IC50 equal to or less than 10 nM Fab2s (a. 34% positive hit rate). Eight of the seventeen Fab2s identified have ICsqs in the subnanomolar range. Furthermore, all seventeen of the MASP-2 blocking Fab2s shown in TABLE 6 gave essentially complete inhibition of C3 convertase formation in the lectin pathway C3 convertase assay. FIGURE 8A graphically illustrates the results of the C3 convertase formation assay for Fab2 antibody #-11, which is representative of the other Fab2 antibodies tested, the results of which are shown in TABLE 6. This is an important consideration, since it is theoretically possible that a blocking Fab2 may only fractionally inhibit MASP-2 function even when each MASP-2 molecule is bound by the Fab2.

Although mannan is a known activator of the lectin pathway, it is theoretically possible that the presence of anti-mannan antibodies in the rat serum might also activate the classical pathway and generate C3b via the classical pathway C3 convertase. However, each of the seventeen blocking anti-MASP-2 Fab2s listed in this example

-1492018200437 19 Jan 2018 potently inhibits C3b generation (>95 %), thus demonstrating the specificity of this assay for iectin pathway C3 convertase.

Binding assays were also performed with all seventeen of the blocking Fab2s in order to calculate an apparent K4 for each. The results of the binding assays of anti-rat

MASP-2 Fab2s to native rat MASP-2 for six of the blocking Fab2s are also shown in TABLE 6, FIGURE 8B graphically illustrates the results of a binding assay with the Fab2 antibody #11. Similar binding assays were also carried out for the other Fab2s, the results of which are shown in TABLE 6, In general, the apparent Kjs obtained for binding of each of the six Fab2s to 'native' MASP-2 corresponds reasonably well with the

IC5Q for the Fab2 in the C3 convertase functional assay. There is evidence that MASP-2 undergoes a conformational change from an ‘inactive' to an 'active' form upon activation of its protease activity (Feinberg et ah, EMBO J 22:2348-59 (2003); Gal et ah, J. Biol. Chem. 250:33435-44 (2005)). In the normal rat plasma used in the C3 convertase formation assay, MASP-2 is present primarily in the 'inactive' zymogen conformation. In contrast, in the binding assay, MASP-2 is present as part of a complex with MBL bound to immobilized mannan; therefore, the MASP-2 would be in the 'active' conformation (Petersen et al., J. Immunol Methods 257:107-16, 2001). Consequently, one would not necessarily expect an exact correspondence between the ICsq and Kjj for each of the seventeen blocking Fab2 tested in these two functional assays since in each assay the

Fab2 would be binding a different conformational form of MASP-2. Never-the-less, with the exception of Fab2 #88, there appears to be a reasonably close correspondence between the IC50 and apparent Kd for each of the other sixteen Fab2 tested in the two assays (see TABLE 6).

Several of the blocking Fab2s were evaluated for inhibition of MASP-2 mediated cleavage of C4. FIGURE 8C graphically illustrates the results of a C4 cleavage assay, showing inhibition with Fab2 #4i, with an ICjqMI.SI 11M (see TABLE 6). As shown in FIGURE 9, all of the Fab2s tested were found to inhibit C4 cleavage with IC5QS similar to those obtained in the C3 convertase assay (see TABLE 6),

Although mannan is a known activator of the lectin pathway, it is theoretically possible that the presence of anti-mannan antibodies in the rat serum might also activate the classical pathway and thereby generate C4b by Cls-mediated cleavage of C4. However, several anti-MASP-2 Fab2s have been identified which potently inhibit C4b generation (>95 %), thus demonstrating the specificity of this assay for MASP-2

-1502018200437 19 Jan 2018 mediated C4 cleavage. C4, like C3, contains an unusual and highly reactive thioester group as part of its structure. Upon cleavage of C4 by MASP-2 in this assay, the thioester group on C4b can form a covalent bond with hydroxyl or amino groups on macromolecules immobilized on the bottom of the plastic weds via ester or amide linkages, thus facilitating detection of C4b in the ELISA assay.

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

EXAMPLE 11

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

Methods:

As shown in FIGURE 10, the following proteins, all with N-terminal 6X His tags 15 were expressed in CHO ceils using the pED4 vector:

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

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

CUBI-II, an N-terminal fragment of rat MASP-2 that contains the CUBI,

EGF-like and CUBII domains only: and

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

These proteins were purified from culture supernatants by nickel-affinity 25 chromatography, as previously described (Chen etal,, J. Biol. Chem. 276:25894-02 (2001)).

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

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

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DOT BLOT ANALYSIS:

Serial dilutions of the five recombinant MASP-2 polypeptides described above and shown in FIGURE W (and the thioredoxin polypeptide as a negative control for

CCPil-serine protease polypeptide) were spotted onto a nitrocellulose membrane. The amount of protein spotted ranged from 100 ng to 6.4 pg, in five-foid steps. In later experiments, the amount of protein spotted ranged from 50 ng down to 16 pg, again in five-fold steps. Membranes were blocked with 5% skimmed milk powder in TBS (blocking buffer) then incubated with 1,0 pg/ml anti-MASP-2 Fab2s in blocking buffer (containing 5.0 mM Ca²). Bound Fab2s were detected using HRP-conjugated anti-human Fab (AbD/Serotec; diluted 1/10,000) and an ECL detection kit (Amersham). One membrane was incubated with poiyclonai rabbit-anti human MASP-2 Ab (described in Stover et ah, J Immunol /63:6848-59 (1999)) as a positive control, in this case, bound Ab was detected using HRP-conjugated goat anti-rabbit IgG (Dako; diluted 1/2,000).

MASP-2 Binding Assay

ELISA plates were coated with 1.0 pg/weil of recombinant MASP-2A or CUB I-11 polypeptide in carbonate buffer (pH 9.0) overnight at 4°C. Wells were blocked with 1% BSA in TBS, then serial dilutions of the anti-MASP-2 Fab2s were added in TBS containing 5.0 mM Ca²⁺. The plates were incubated for one hour at RT, After washing three times with TBS/tween/Ca²⁺, HRP-conjugated anti-human Fab (AbD/Serotec) diluted 1/10,000 in TBS/ Ca²⁺ was added and the plates incubated lor a further one hour at RT. Bound antibody was detected using a TMB peroxidase substrate kit (Biorad).

RESULTS:

Results of the dot blot analysis demonstrating the reactivity of the Fab2s with various MASP-2 polypeptides are provided below in TABLE 7. Tire numerical values provided in TABLE 7 indicate the amount of spotted protein required to give approximately haif-maximal signal strength, As shown, ah of the polypeptides (with the exception of the thioredoxin fusion partner alone) were recognized by the positive control Ab (polyclonal anti-human MASP-2 sera, raised in rabbits).

TABLE 7: REACTIVITY WITH VARIOUS RECOMBINANT' RAT MASP-2

POLYPEPTIDES ON DOT' BLOTS

Fab2 MASP-2A CUBI-Π 1 CUBl/EGF-like Antibody # :

CCPII-SP Thioredoxin

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Fab2 i	MASP-2 A
Antibody #	.... — ____________
40	0.16 ng
41 1	0,16 ng
11	0.16 ng
49	0.16 ng
52	0.16 ng
57	0,032 ng
58	0.4 ng
60 ;	0.4 ng
63	0.4 ng
66	0.4 ng
67	0.4 ng
71	0,4 ng
81 4	0.4 ng
86	0.4 ng
87	0.4 ng.

Positive

Control

CUBI-II i CUBl/EGF-like CCPH-SP Thioredox in

NR |	NR NR	0.8 ng 3 0.8 ng ΐ	NR
NR	NR
NR 1	NR	0.8 ng 1 NR
w f...	NR	r >20 na 1	NR	j
NR II	NR	0,8 ng 1	NR
NR	NR	NR 1.	NR
NR |	NR	! 2.0 ng 3	NR	ί

i().O32 ng

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

All of the Fab2s reacted with MASP-2A as well as MASP-2K (data not shown). The majority of the Fab2s recognized the CCPH-SP polypeptide but not the N-terminal fragments. The two exceptions are Fab2 #60 and Fab2 #57. Fab2 #60 recognizes MASP-2A and the CUBI-II fragment, but not the CUBl/EGF-like polypeptide or the CCPH-SP polypeptide, suggesting it binds to an epitope in CUBI1, or spanning the CUBII and the EGF-like domain. Fab2 # 57 recognizes MASP-2A but not any of the MASP-2 fragments tested, indicating that this Fab2 recognizes an epitope in CCP1. Fab2 #40 and #49 bound only to complete MASP-2A. In the ELISA binding assay shown in

FIGURE 11, Fab2 #60 also bound to the CUBI-II polypeptide, albeit with a slightly lower apparent affinity.

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

-1532018200437 19 Jan 2018

EXAMPLE 12

This Example describes the analysis of MASP-2-/- mice in a Murine Renal

Ischemia/Reperfusion Model.

Background/Ratkmale: Ischemia-Reperfusion (l/'R) injury in kidney at body temperature has relevance in a number of clinical conditions, including hypovolaemic shock, renal artery occlusion and cross-clamping procedures,

Kidney ischemia-reperfusion (I/R) is an important cause of acute renal failure, associated with a mortality rate of up to 50% (Levy etaf, JAMA 275:1489-94, 1996; Thadhani et al., N. Engl. J. Med. 554:1448-60, 1996). Post-transplant renal failure is a common and threatening complication after renal transplantation (Nicholson et al., Kidney lnt. 55:2585-91, 2000). Effective treatment for renal I/R injury is currently not available and hemodialysis is the only treatment available. The pathophysiology of renal ί/R injury is complicated. Recent studies have shown that the lectin pathway of complement activation may have an important role in the pathogenesis of renal I/R injury (deVries et ai., Am. J. Path. 165Ά6ΊΊ-^, 2004).

Methods:

A MAS P-2 (-/-) mouse was generated as described in Example 1 and backcrossed for at least 10 generations with C57B1/6. Six male MASP-2(-/-) and six wildtype (+/+) mice weighing between 22-25 g were administered an intraperitoneal injection of

Hypnovel (6.64 mg/kg; Roche products Ltd. Welwyn Garden City, UK), and subsequently anaesthetized by inhalation of isoflurane (Abbott Laboratories Ltd., Kent, UK). Isoflurane was chosen because it is a mild inhalation anaesthetic with minimal liver toxicity: the concentrations are produced accurately and the animal recovers rapidly, even after prolonged anaesthesia. Hypnovel was administered because it produces a condition of neuroleptanalgesia in the animal and means that less isoflurane needs to be administered. A warm pad was placed beneath, the animal in order to maintain a constant body temperature. Next, a midline abdominal incision was performed and the body cavity held open using a pair of retractors. Connective tissue was cleared above and below the renal vein and artery of both right and left kidneys, and the renal pedicle was clamped via the application of microaneurysm clamps for a period of 55 minutes. This period of ischemia was based initially on a previous study performed in this laboratory (Zhou etal., J Clin. Invest. 105:1363-71 (2000)). In addition, a standard ischemic time of 55 minutes was chosen following ischemic titration and it was

-1542018200437 19 Jan 2018 found that 55 minutes gave consistent injury that was also reversible, with low mortality, less than 5%. After occlusion, 0,4 ml of warm saline (37°C) was placed in the abdominal cavity and then the abdomen was closed for the period of ischemia, Following removal of tlie microaneurysm clamps, the kidneys were observed until color change, an indication of blood re-flow to the kidneys, A further 0.4 mi of warm saline was placed in the abdominal cavity and the opening was sutured, whereupon animals were returned to their cages. Tail blood samples were taken at 24 hours after removing the clamps, and at 48 hours the mice were sacrificed and an additional blood sample was collected.

Assessment of Renal injury: Renal function was assessed at 24 and 48 hours 10 after reperfusion in six male MASP-2(-/-) and six WT (+/+) mice. Blood creatinine measurement was determined by mass spectrometry, which provides a reproducible index of renal function (sensitivity < 1.0 μηιοΙ/L), FIGURE 12 graphically illustrates the blood urea nitrogen clearance for wildtype C57B1/6 controls and MASP-2 (-/-) at 24 hours and 48 hours after reperfusion. As shown in FIGURE 12, MASP-2(-/-) mice displayed a significant reduction in the amount of blood urea at 24 and 48 hours, in comparison to wildtype control mice, indicating a protective functional effect from renal damage in the ischemia reperfusion injury model.

Overall, increased blood urea was seen in both the WT (+/+) and MASP-2 (-/-) mice at 24 and 48 hours following the surgical procedure and ischemic insult. Levels of blood urea in a non-ischemsc WT (+/+) surgery animal was separately determined to be 5.8 mmof/L. In addition to the data presented in FIGURE 12, one MASP-2 (-/-) animal showed nearly complete protection from the ischemic insult, with values of 6.8 and 9.6 mmol/L at 24 and 48 hours, respectively. This animal was excluded from the group analysis as a potential outlier, wherein no ischemic injury may have been present.

Therefore, the final analysis shown in FIGURE 12 included 5 MASP-2(-/-) mice and 6 WT (+/+) mice and a statistically significant reduction in blood urea was seen at 24 and 48 hours in the MASP-2 (-/-) mice (Student t-test p<0.05). These findings indicate inhibition of MASP-2 activity would be expected to have a protective or therapeutic effect from renal damage due to ischemic injury.

EXAMPLE 13

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

-1552018200437 19 Jan ,-1 Background/Rationale: Age-related macular degeneration (AMD) is the leading fX| cause of blindness after age 55 in the industrialized world. AMD occurs in two major forms: neovascufar (wet) AMD and atrophic (dry) AMD. The neovascular (wet) form accounts for 90% of severe visual loss associated with AMD, even though only ~20% of 5 individuals with AMD develop the wet form, Clinical hallmarks of AMD include multiple drusen, geographic atrophy, and choroidal neovascularization (CNV). In December, 2004, the FDA approved Macugen (pegaptanib), a new' class of ophthalmic drugs to specifically target and block the effects of vascular endothelial growth factor (VEGF), for treatment of the wet (neovascular) form of AMD (Ng et ak, Nat Rev. Drug 10 Discov »5:123-32 (2006)). Although Macugen represents a promising new therapeutic option for a subgroup of AMD patients, there remains a pressing need to develop additional treatments for this complex disease. Multiple, independent lines of investigation implicate a central role for complement activation in the pathogenesis of AMD. The pathogenesis of choroidal neovascularization (CNV), the most serious form 15 of AMD. may involve activation of complement pathways.

Over twenty-five years ago, Ryan described a laser-induced injury model of CNV in animals (Ryan, S.J., Tr. Am. Opth, Soc. A¥Ak7/:707-745, 1979). The model was initially developed using rhesus monkeys, however, the same technology has since been used to develop similar models of CNV in a variety of research animals, including the 20 mouse (Tobe etal,, Am, J. Pathol. 755:1641-46, 1998). In this model, laser photocoagulation is used to break Bruch’s membrane, an act which results in the formation of CNV-like membranes. The laser-induced model captures many of the important features of the human condition (for a recent review, see Ambati et ah, Survey Ophthalmology 46:257-293, 2003). The laser-induced mouse model is now' well 25 established, and is used as an experimental basis in a large, and ever Increasing, number of research projects. It is generally accepted that the laser-induced model shares enough biological similarity with CNV in humans that preclinical studies of pathogenesis and drug inhibition using this model are relevant to CNV in humans.

Methods:

A MASP-2-/- mouse was generated as described in Example 1 and backcrossed for 10 generations with C57B1/6. The current study compared the results when MASP-2 (-/-) and MASP-2 (+/+) male mice were evaluated in the course of laser-induced CNV, an accelerated model of neovascular AMD focusing on the volume of laser-induced CNV by

-1562018200437 19 Jan 2018 scanning laser confocal microscopy as a measure of tissue injury and determination of levels of VEGF, a potent angiogenic factor implicated in CNV, in the retinal pigment epithelium (RPE)/choroids by ELISA after laser injury.

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

Fluorescein Angiography: Fluorescein angiography was performed with a camera and imaging system (TRC 50 1A camera; ImageNei 2.01 system; Topcon, Paramus , NJ) at 1 week after laser photocoagulation. The photographs were captured with a 20-D lens in contact with the fundus camera lens after intraperitoneal injection of 0.1 mi of 2.5% fluorescein sodium. A retina expert not involved in the laser photocoaguiation or angiography evaluated the fluorescein angiograms at a single sitting in masked fashion.

Volume of choroidal neovascularization (CNV): One week after laser injury, eyes were enucleated and fixed with 4% paraformaldehyde for 30 min at 4°C. Eye cups were obtained by removing anterior segments and were washed three times in PBS, followed by dehydration and rehydration through a methanol series. .After blocking twice with buffer (PBS containing 1% bovine serumalbumin and 0.5% Triton X-100) for 30 minutes at room temperature, eye cups were incubated overnight at 4°C with 0.5%

FITC-isolectin B4 (Vector laboratories, Burlingame, CA), diluted with PBS containing 0,2% BSA and 0.1% Triton X-100, which binds terminal β-D-galactose residues on the surface of endothelial ceils and selectively labels the murine vasculature. After two washings with PBS containing 0.1% Triton X-100, the neurosensory retina was gently detached and severed from the optic nerve. Four relaxing radial incisions were made, and the remaining RPE -choroid-sclera complex was flatmounted in antifade medium (Immu-Mount Vectashield Mounting Medium; Vector Laboratories) and cover-slipped.

Flatmounts were examined with a scanning laser confocal microscope (TCS SP; Leica, Heidelberg, Germany). Vessels were visualized by exciting with blue argon

-1572018200437 19 Jan 2018 wavelength (488 nm) and capturing emission between 515 and 545 nm. A 40X oil-immersion objective was used for all imaging studies. Horizontal optical sections (1 pm step) were obtained from the surface of the RPE-choroid-sciera complex. The deepest focal plane in which the surrounding choroidal vascular network connecting to the lesion could be identified was judged to be the floor of the lesion. Any vessel in the laser-targeted area and superficial to this reference plane was judged as CNV. Images of each section were digitally stored. The area of CNV-related fluorescence was measured by computerized image analysis with the microscope software (TCS SP; Leica). The summation of whole fluorescent area in each horizontal section was used as an index for the volume of CNV. Imaging was performed by an operator masked to treatment group assignment.

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

VEGF ELISA. At three days after injury by 12 laser spots, the RPE-choroid complex was sonicated in lysis buffer (20 mM imidazole HC1, IOmM KC1, 1 mM MgCL?, 10 mM EGTA, 1% Triton X-100, 10 mM NaF, 1 mM Na molybdate, and 1 mM EDTA with protease inhibitor) on ice for 15 min. VEGF protein levels in the supernatant were determined by an ELISA kit (R&D Systems, Minneapolis, MN) that recognizes all splice variants, at 450 to 570 nm (Emax; Molecular Devices, Sunnyvale, CA), and normalized to total protein. Duplicate measurements were performed in a masked fashion by an operator not involved in photocoagulation, imaging, or angiography. VEGF numbers were represented as the mean +/- SEM of at least three independent experiments and compared using the Mann-Whitney U test. The null hypothesis was rejected at P<0.05.

RESULTS:

Assessment of VEGF Levels:

FIGURE 13A graphically illustrates the VEGF protein levels in RPE-choroid complex isolated from C57B16 wildtype and MASP-2(-/-) mice at day zero. As shown in

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FIGURE 13A, the assessment of VEGF levels indicate a decrease in baseline levels for

VEGF in the MASP-2 (-/-) mice versus the C57bl wikitype control mice. FIGURE 13B graphically illustrates VEGF protein levels measured at day three following laser induced injury. As shown in FIGURE 13B VEGF levels were significantly increased in the wildtype (+/+) mice three days following laser induced injury, consistent with published studies (Nozaki etah, Prac. Natl. Acad. Sci. USA 103:2328-33 (2006)). However, surprisingly very low levels of VEGF were seen in the MASP-2 (-/-) mice.

Assessment of choroidal neovascularization (CNV):

In addition to the reduction in VEGF levels following laser induced macular 10 degeneration, CNV area was determined before and after laser injury. FIGURE 14 graphically illustrates the CNV volume measured in C57bl wildtype mice and MASP-2(-/-) mice at day seven following laser induced injury. As shown in FIGURE 14, the MASP-2 (-/-) mice displayed about a 30% reduction in the CNV area following laser induced damage at day seven in comparison to the wildtype control mice.

These findings indicate a reduction in VEGF and CNV as seen in the MASP (-/-) mice versus the wildtype (-j-/-:-) control and that blockade of MASP-2 with an inhibitor would have a preventive or therapeutic effect in the treatment of macular degeneration.

EXAMPLE 14

This Example demonstrates that thrombin activation can occur following lectin pathway activation under physiological conditions, and demonstrates the extent of MASP-2 involvement. In normal rat semm, activation of the lectin pathway leads to thrombin activation (assessed as thrombin deposition) concurrent with complement activation (assessed as C4 deposition). As can be seers in FIGURES 15A and 15B, thrombin activation in this system is inhibited by a MASP-2 blocking antibody (Fab2 format), exhibiting an inhibition concentration-response curve (FIGURE 15B) that parallels that for complement activation (FIGURE ISA). These data suggest that activation of the lectin pathway as it occurs in trauma will lead to activation of both complement and coagulation systems in a process that is entirely dependent on MASP-2,

By inference, MASP2 blocking antibodies may prove efficacious in mitigating cases of excessive systemic coagulation, e.g,, disseminated intravascular coagulation, which Is one of the hallmarks leading to mortality in major trauma cases.

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EXAMPLE 15

This Example provides results generated using a localized Schwartzman reaction model of disseminated intravascular coagulation (DIC) in MASP-2 -/- deficient and

MASP-2 +/+ sufficient mice to evaluate the roie of lectin pathway in DIC.

Baekground/Rationale:

As described supra, blockade of MASP-2 inhibits lectin pathway activation and reduces the generation of both anaphylatoxins C3a and C5a. C3a anaphylatoxins can be shown to be potent platelet aggregators in vitro, but their involvement in vivo is less well defined and the release of platelet substances and plasmin in wound repair may only secondarily involve complement C3. In this Example, the role of the lectin pathway was analyzed in MASP-2 (-/-) and WT (+/+) mice in order to address whether prolonged elevation of C3 activation is necessary to generate disseminated intravascular coagulation.

Methods:

The MASP-2 (-/-) mice used in this study were generated as described in

Example 1 and backcrossed for at least 10 generations with C57B1/6.

The localized Schwartzman reaction mode' was used in this experiment. The localized Schwartzman reaction (LSR) is a Iipopolysaccharide (EPS) -induced response with well-characterized contributions from cellular and humoral elements of the innate immune system. Dependent of the LSR on complement is well established (Polak, L., et al., Nature 223:738-739 (1969); Fong J.S. et al., J Exp Med /34:642-655 (1971)). in the LSR model, the mice were primed for 4 hours with TNF alpha (500 ng, intrascrotal), then the mice were anaesthetized and prepared for intravital microscopy of the cremaster muscle. Networks of post-capillary venules (15-60 pm diameter) with good blood flow (1-4 mm/s) were selected for observation. Animals were treated with fluorescent antibodies to selectively label neutrophils, or platelets. The network of vessels was sequentially scanned and images of all vessels were digitally recorded of later analysis. After recording the basal state of the microcirculation, mice received a single intravenous injection of LPS (100 pg), either alone or with the agents listed below. The same network of vessels was then scanned every 10 minutes for 1 hour. Specific accumulation of fluorophores was identified by subtraction of background fluorescence and enhanced by thresholding the image. The magnitude of reactions was measured from recorded images. The primary measure of Schwartzman reactions was aggregate data.

-3602018200437 19 Jan 2018

The studies compared the MASP-2 +/+ sufficient, or wild type, mice exposed io either a known complement pathway depletory agent, cobra venom factor (CVF), or a terminal pathway inhibitor (C5aR antagonist). The results (FIGURE 16A) demonstrate that CVF as well as a C5aR antagonist both prevented the appearance of aggregates in the vasculature. In addition, the MASP-2 -/- deficient mice (FIGURE 16B) also demonstrated complete inhibition of the localized Schwartzman reaction, supporting lectin pathway involvement. These results clearly demonstrate the role of MASP-2 in DIG generation and support the use of MASP-2 inhibitors for the treatment and prevention of DIG.

EXAMPLE 16

This Example describes the analysis of MASP-2 (-/-) mice in a Murine Renal Transplantation Model,

Background/Rntionale;

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

Methods:

The functional outcome of kidney transplantation was assessed using a single kidney isograft into uninephrecomized recipient mice, with six WT (+/+) transplant recipients (B6), and six MASP-2 (-/-) transplant recipients. To assess the function of the transplanted kidney, the remaining native kidney was removed from the recipient 5 days after transplantation, and renal function was assessed 24 hours later by measurement of blood urea nitrogen (BUN) levels.

Results:

FIGURE 17 graphically illustrates the blood urea nitrogen (BUN) levels of the kidney at 6 days post kidney transplant in the WT (+/+) recipients and the MASP-2 (-/-) recipients. As shown in FIGURE 17, strongly elevated BUN levels were observed in the WT (+/+) (B6) transplant recipients (norma! BUN levels in mice are < 5 mM), indicating renal failure, in contrast, MASP-2 (-/-) isograft recipient mice showed substantially lower BUN levels, suggesting improved renal function, it is noted that these results were obtained using grafts from WT (+/+) kidney donors, suggesting that the absence of a functional lectin pathway in the transplant recipient alone is sufficient to achieve a therapeutic benefit.

-1612018200437 19 Jan 2018

Taken together, these results indicate that transient inhibition of' the lectin pathway via MASP-2 inhibition provides a method of reducing morbidity and delayed graft function in renal transplantation, and that this approach is likely to be useful in other transplant settings.

EXAMPLE 17

This Example demonstrates that .MASP-2 (-/-) mice are resistant to septic shock in a Murine Polymicrobial Septic Peritonitis Model.

Background,/Rationale:

To evaluate the potential effects of MASP-2 (-/-) in infection, the cecal ligation and puncture (CLP) model, a mode! of polymicrobial septic peritonitis was evaluated. This model is thought to most accurately mimic the course of human septic peritonitis. The cecal ligation and puncture (CLP) mode! is a model in which the cecum is ligated and punctured by a needle, leading io continuous leakage of the bacteria into the abdominal cavity which reach the blood through the lymph drainage and are then distributed into all the abdominal organs, leading to multi-organ failure and septic shock (Eskandari et al, J Immunol /45(9):2724-2730 (1992)). The CLP model mimics the course of sepsis observed in patients and induces an early hyper-inflammatory response followed by a pronounced hypo-inflammatory phase. During this phase, the animals are highly sensitive to bacterial challenges (Wichterman et al, J. Surg. Res. 29(2): 189-20! (1980)).

Methods;

The mortality of polymicrobial infection using the cecal ligation and puncture (CLP) model was measured in WT (+/+) (n=18) and MASP-2 (-/-) (n=16) mice. Briefly described, MASP-2 deficient mice and their wild-type littermates were anaesthetized and the cecum was exteriorized and ligated 30% above the distal end. After that, the cecum was punctured once with a needle of 0.4 mm diameter. The cecum was then replaced into the abdominal cavity and the skin was closed with elamps. The survival of the mice subjected to CLP w'as monitored over a period of 14 days after CLP. A peritoneal lavage was collected in mice 16 hours post CLP to measure bacteria! load. Serial dilutions of the peritoneal lavage were prepared in PBS and inoculated in Mueller Hinton plates with subsequent incubation at 37°C under anaerobic conditions for 24 hours after which bacteria! load was determined.

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The TNF-alpha cytokine response to the bacterial infection was also measured in the WT (+/+) and MASP-2 (-/-) mice 16 hours after CLP in lungs and spleens via quantitative real time polymerase chain reaction (qRT-PCR.). The serum level of TNFalpha 16 hours after CLP in the WT (+/+) and MASP-2 (-/-) mice was also quantified by sandwich ELISA.

Results:

FIGURE 18 graphically illustrates the percentage survival of the CLP treated animals as a function of the days after the CLP procedure. As shown in FIGURE 18, the lectin pathway deficiency in the MASP-2 (-/-) mice does not increase the mortality of mice after polymicrobial infection using the cecal ligation and puncture model as compared to WT (+/+) mice. However, as shown in FIGURE 19, MASP-2 (-/-) mice showed a significantly higher bacterial load (approximately a 1000-fold increase in bacterial numbers) in peritoneal lavage after CLP when compared to their WT (+/+) littermates. These results Indicate that MASP-2 (-/-) deficient mice are resistant to septic shock. The reduced bacterial clearance in MASP-2 deficient mice in this model may be due to an impaired C3b mediated phagocytosis, as it was demonstrated that C3 deposition is MASP-2 dependent, it was determined that the TNF-alpha cytokine response to the bacterial infection was not elevated in the MASP-2 (-/-) mice as compared to the WT (+/+) controls (data not shown), it was also determined that there was a significantly higher serum concentration of'TNF-alpha in WT (+/+) mice 16 hours after CLP in contrast to MASP-2 (-/-) mice, where the serum level of TNF-alpha remained nearly unaltered. These results suggest that the intense inflammatory response to the septic condition was tempered in MASP-2 (-/-) mice and allowed the animals to survive in the presence of higher bacterial counts.

Taken together, these results demonstrate the potential deleterious effects of lectin pathway complement activation in the case of septicemia and the increased mortality in patients wdth overwhelming sepsis. These results further demonstrate that MASP-2 deficiency modulates the inflammatory immune response and reduces the expression levels of inflammatory mediators during sepsis, Therefore, it is believed that inhibition of MASP-2 (-/-) by administration of inhibitory monoclonal antibodies against MASP-2 would be effective to reduce the inflammatory response in a subject suffering from septic shock.

-1632018200437 19 Jan 2018

EXAMPLE 18

This Example describes analysis of MASP-2 (-/-) mice in a Murine Intranasal

Infectivity Model,

Baekgroijnd/RaOoaak·:

Pseudomonas aeruginosa is a Gram negative opportunistic human bacterial pathogen that causes a wide range of infections, particularly in immune-compromised individuals, It is a major source of acquired nosocomial infections, in particular hospitalacquired pneumonia. It is also responsible for significant morbidity and mortality in cystic fibrosis (CF) patients. P. aeruginosa pulmonary infection is characterized by strong neutrophil recruitment and significant lung inflammation resulting in extensive tissue damage (Palanki M.S. et ah, J. Med. Chem 57:1546-1559 (2008)).

In this Example, a study was undertaken to determine whether the removal of the lectin pathway in MASP-2 (-/-) mice increases the susceptibility of the mice to bacterial infections.

Methods:

Twenty-two WT (+/+) mice, twenty-two MASP-2 (-/-) mice, and eleven C3 (-/-) mice were challenged with intranasal administration of P. aeruginosa bacterial strain. The mice were monitored over the six days post-infection and Kaplan-Mayer plots were constructed showing percent survival.

Results:

FIGURE 20 is a Kaplan-Mayer plot of the percent survival of WT (+/+), MASP2 (-/-) or C3 (-/-) mice six days post-infection. As shown in FIGURE 20, no differences were observed in the MASP-2 (-/-) mice versus the WT (+/+) mice. However, removal of the classical (CIq) pathway in the C3 (-/-) mice resulted in a severe susceptibility to bacterial infection. These results demonstrate that MASP-2 inhibition does not increase susceptibility to bacterial infection, indicating that it is possible to reduce undesirable inflammatory complications in trauma patients by inhibiting MASP-2 without compromising the patient’s ability' to fight infections using the classical complement pathway.

-1642018200437 19 Jan 2018

EXAMPLE 19

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

Backgroend/Rationaie:

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

As shown in TABLE 6 of Example 10, 17 anti-MASP-2 Fab2s with functional blocking activity were identified as a result of this analysis (a 34% hit rate for blocking antibodies). Functional inhibition of the lectin complement pathway by Fab2s was apparent at the level of C4 deposition, which is a direct measure of C4 cleavage by MASP-2. Importantly, inhibition was equally evident when C3 convertase activity was assessed, demonstrating functional blockade of the lectin complement pathway. The 17

MASP-2 blocking Fab2s identified as described in Example 10 potently inhibit C3 convertase formation with IC₅₀ values equal to or less than 10 nM. Eight of the 17 Fab2s identified have IC₅₀ values in the sub-nanomolar range, Furthermore, ah 17 of the MASP-2 blocking Fab2s gave essentially complete inhibition of the €3 convertase formation in the lectin pathway C3 convertase assay, as shown in FIGURES 8A-C, and summarized in TABLE 6 of Example 10. Moreover, each of the 17 blocking anti-MASP2 Fab2s shown in TABLE 6 potently inhibit G3b generation (>95%), thus demonstrating the specificity of this assay tor lectin pathway C3 convertase.

Rat IgG2c and mouse lgG2a full-length antibody isotype variants were derived from Fab2 #11. This Example describes the in vivo characterization of these isotypes for pharmacodynamic parameters.

As described in Example 10, rat MASP-2 protein was utilized to pan a Fab phage display library, from which Fab2#il was identified. Rat IgG2c and mouse lgG2a fuli-1652018200437 19 Jan 2018 length antibody isotype variants were derived from Fab2 #11. Both rat lgG2c and mouse

IgG2a full length antibody isotypes were characterized in vivo for pharmacodynamic parameters as follows.

In vivo study in mice:

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

FIGURE 21 graphically illustrates lectin pathway specific C4b deposition, measured ex vivo in undiluted serum samples taken from mice (n=3 mice/group) at various time points after subcutaneous dosing of either 0.3 mg/kg or 1.0 mg/kg ofthe mouse anti-MASP-2 MoAb. Serum samples from mice collected prior to antibody dosing served as negative controls (100% activity), while serum supplemented in vitro with 100 nM of the same blocking anti-MASP-2 antibody was used as a positive control (0% activity).

The results shown in FIGURE 21 demonstrate a rapid and complete inhibition of C4b deposition following subcutaneous administration of 1,0 mg/kg dose of mouse anti20 MASP-2 MoAb. A partial inhibition of C4b deposition was seen following subcutaneous administration of 0.3 mg/kg dose of mouse anti-MASP-2 MoAb.

The time course of lectin pathway recovery was followed for three weeks following a single ip administration of mouse anti-MASP-2 MoAb at 0.6 mg/kg in mice. As shown in FIGURE 22, a precipitous drop in lectin pathway activity occurred post antibody dosing followed by complete lectin pathway inhibition that lasted for about 7 days after ip administration. Siow restoration of lectin pathway activity was observed over the second and third weeks, with complete lectin pathway restoration in the mice by 17 days post anti-MASP-2 MoAb administration.

These results demonstrate that the mouse anti-MASP-2 Moab derived from Fab2 #11 inhibits the lectin pathway of mice in a dose-responsive manner when delivered systemically.

-1662018200437 19 Jan 2018

EXAMPLE 20

This Example describes analysis of the mouse anti-MASP-2 Moab derived from

Fab2 #11 for efficacy in a mouse model for age-related macular degeneration.

Backgrouiid/Rationaie;

As described in Example 10, rat MASP-2 protein was utilized to pan a Fab phage display library, from which Fab2#ll was identified as a functionally active antibody. Full length antibodies of the rat IgG2c and mouse igG2a isotypes were generated from Fab2 #11. The full length anti-MASP-2 antibody of the mouse lgG2a isotype was characterized for pharmacodynamic parameters as described in Example 19, in this

Example, the mouse anti-MASP-2 full-length antibody derived from Fab2 #11 was analyzed in the mouse model of age-related macular degeneration (AMD), described by Bora P.S, et al. J Immunol 174:491-491 (2005).

Methods;

The mouse IgG2a full-length anti-MASP-2 antibody isotype derived from Fab2 #11 as described in Example 19, was tested in the mouse model of age-related macular degeneration (AMD) as described in Example 13 with the following modifications.

Administration of mouse-anii~MASP~2 MoAhs

Two different doses (0.3 mg/kg and 1.0 mg/kg) of mouse anti-MASP-2 MoAb along with an isotype control MoAb treatment were injected ip into WT (-+7-+-) mice (n= 8 mice per group) 16 hours prior to CNV induction

Induction of choroidal neovascularization (CNl⁷)

The induction of choroidal neovascularization (CNV) and measurement of the volume of CNV was carried out using laser photocoagulation as described in Example 13.

Results;

FIGURE 23 graphically illustrates the CNV area measured at 7 days post laser injury in mice treated with either isotype control MoAb, or mouse anti-MASP-2 MoAb (0,3 mg/kg and 1.0 mg/kg). As shown in FIGURE 23, in the mice pre-treated with 1.0 mg/kg anti-MASP-2 MoAb, a statistically significant (p <0,01) approximately 50% reduction in CNV was observed seven days post-laser treatment. As further shown in

FIGURE 23, it was observed that a 0.3 mg/kg dose of anti-MASP-2 MoAb was not efficacious in reducing CNV. it is noted that the 0.3 mg/kg dose of anti-MASP-2 MoAb was shown to have a partial and transient inhibition of C4b deposition following subcutaneous administration, as described in Example 19 and shown in FIGURE 21.

-1672018200437 19 Jan 2018

The results described in this Example demonstrate that blockade of MASP-2 with an inhibitor, such as anti-MASP-2 MoAb, has a preventative and/or therapeutic effect in the treatment of macular degeneration. It is noted that these results are consistent with the results observed in the study carried out in the MASP-2 (-/-) mice, described in

Example 13, in which a 30% reduction in the CNV 7 days post-laser treatment was observed in MASP-2 (-/-) mice in comparison to the wild-type control mice. Moreover, the results in this Example further demonstrate that systemically delivered anti-MASP-2 antibody provides local therapeutic benefit in the eye, thereby highlighting the potential for a systemic route of administration to treat AMD patients. In summary, these resuits provide evidence supporting the use of MASP-2 MoAb in the treatment of .AMD,

EXAMPLE 21

This Example demonstrates that MASP-2 deficient mice are protected from Neisseria meningitidis induced mortality after infection with N meningitidis and have enhanced clearance of bacteraemia as compared to wild type control mice.

Rationale: Neisseria meningitidis is a heterotrophic gram-negative diplococcai bacterium known for its role in meningitis and other forms of meningococcal disease such as meningococcemia. V. meningitidis is a major cause of morbidity and mortality during childhood. Severe complications include septicaemia, Waterhouse-Friderichsen syndrome, adrenal insufficiency and disseminated intravascular coagulation (DiC). See e.g., Rintaia E. et al., Critical Care Medicine 25(7):2373-2378 (2000). In this Example, the role of the lectin pathway was analyzed in MASP-2 (-/-) and WT (+/+) mice in order ίο address whether MASP-2 deficient mice would be susceptible to N. meningitidis induced mortality.

Methods:

MASP-2 knockout mice were generated as described in Example 1 and backcrossed for at least 10 generations with C57B1/6. 10 week old MASP-2 KO mice (n~10) and wild type C57/B6 mice (n~10) were innoculated by intravenous injection with either a dosage of 5x10^s cfii/100 μί, 2x10⁸ cfo/100 μΐ or 3x10 cfo/100 μΐ of Neisseria meningitidis Serogroup A Z2491 in 400 mg/kg iron dextran. Survival of the mice after infection was monitored over a 72 hour time period. Blood samples were taken from the mice at hourly intervals after infection and analyzed to determine the serum level (log

-168cfu/ml) of TV. meningitidis in order to verify infection and determine the rate of clearance of the bacteria from the serum.

2018200437 19 Jan 2018

FIGURE 24A graphically illustrates the percent survival of MASP-2 KO and WT 5 mice after administration of an infective dose of 5xl0⁸/100 μΐ cfu N. meningitidis. As shown in FIGURE 24A, after infection with the highest dose of 5xI0^s/100 μί cfu N. meningitidis, 100% of the MASP-2 KO mice survived throughout the 72 hour period after infection. In contrast, only 20% of the WT mice were still alive 24 hours after infection. These results demonstrate that MASP-2 deficient mice are protected from N. meningitidis induced mortality.

FIGURE 24B graphically illustrates the log cfu/ml of N. meningitidis recovered at different time points in blood samples taken from the MASP-2 KO and WT mice infected with 5x10⁸ cfu/100 μ] N. meningitidis. As shown in FIGURE 24B, in WT mice the level of N. meningitidis in the blood reached a peak of about 6.5 log cfu/ml at 24 hours after infection and dropped to zero by 48 hours after infection. In contrast, in the MASP-2 KO mice, the level of TV. meningitidis reached a peak of about 3.5 log cfu/ml at 6 hours after infection and dropped to zero by 36 hours after infection.

FIGURE 25A graphically illustrates the percent survival of MASP-2 KO and WT mice after infection with 2x10⁸ cfu/100 μΐ /V. meningitidis. As shown in FIGURE 25A, after infection with the dose of 2x10^s cfu/i 00 μΐ TV meningitidis, 100% of the MASP-2 KO mice survived throughout the 72 hour period after infection. In contrast, only 80% of the WT mice were still alive 24 hours after infection. Consistent with the results shown in FIGURE 24 A, these results further demonstrate that MASP-2 deficient mice are protected from N. meningitidis induced mortality.

FIGURE 25B graphically illustrates the log cfu/ml of As meningitidis recovered at different time points in blood samples taken from the WT mice infected with 2x10⁸ cfu/100 μί N. meningitidis. As shown in FIGURE 25B, the level of TV. meningitidis in the blood of WT mice infected with 2x10⁸ cfu reached a peak of about 4 log cfu/ml at 12 hours after infection and dropped to zero by 24 hours after infection, FIGURE 25C graphically illustrates the log cfu/ml of TV. meningitidis recovered at different time points in blood samples taken from the MASP-2 KO mice infected with 2x10^s cfu/100 μί TV. meningitidis. As shown in FIGURE 25G, the level of N. meningitidis in the blood of MASP-2 KO mice infected with 2x10⁸ cfu reached apeak level of about 3.5 log cfu/ml at

-1692018200437 19 Jan 2018 hours after infection and dropped to zero at 3 hours after infection. Consistent with the results shown in FIGURE 24B, these results demonstrate that although the MASP-2 KO mice were infected with the same dose of A', meningitidis as the WT mice, the MASP-2

KO mice have enhanced clearance of bacteraemia as compared to WT.

The percent survival of MASP-2 KO and WT mice after infection with the lowest dose of 3x10⁷ efts/100 μΐ N. meningitidis was 100% at the 72 hour time period (data not shown).

Discussion

These results show that MASP-2 deficient mice are protected from jY 10 meningitidis induced mortality and have enhanced clearance of bacteraemia as compared to the WT mice. Therefore, in view of these results, it is expected that therapeutic application of MASP-2 inhibitors, such as MASP-2 MoAb, would be expected to be efficacious to treat, prevent or mitigate the effects of infection with N. meningitidis bacteria (i.e., sepsis and D1C). Further, these results indicate that therapeutic application of MASP-2 inhibitors, such as MASP-2 MoAb would not predispose a subject to an increased risk to contract N. meningitidis infections.

EXAMPLE 22

This Example describes the discovery of novel lectin pathway mediated and 20 MASP-2. dependent C4-bypass activation of complement C3.

Rationale:

The principal therapeutic benefit of utilizing inhibitors of complement activation to limit myocardial ischemia/reperfusion injury (MIRI) was convincingly demonstrated in an experimental rat model of myocardial infarction two decades ago: Recombinant sCRl, a soluble truncated derivative of the cell surface complement receptor type-1 (CR1), was given intravenously and its effect assessed in a rat in vivo model of MIRI. Treatment with sCRl reduced infarct volume by more than 40% (Weisman, H.F., et ah, Science 249:146-151 (1990)). The therapeutic potential of this recombinant inhibitor was subsequently demonstrated in a clinical trial showing that the administration of sCRl in patients with MI prevented contractile failure in the post-ischemic heart (Shandelya, S,, et al., Circulation 87:536-546 (1993)). 'The primary mechanism leading to the activation of complement in ischemic tissue, however, has not been ultimately defined, mainly due to the lack of appropriate experimental models, the limited understanding of the molecular

-1702018200437 19 Jan 2018 processes that lead to complement activation of oxygen-deprived cells, and the cross-talk and synergisms between the different complement activation pathways.

As a fundamental component of the immune response, the complement system provides protection against invading microorganisms through both antibody-dependent and -independent mechanisms, if orchestrates many cellular and humoral interactions within the immune response, including chemotaxis, phagocytosis, ceil adhesion, and Bcell differentiation, Three different pathways initiate the complement cascade: the classical pathway, the alternative pathway, and the lectin pathway. The classical pathway recognition subcomponent Clq binds to a variety of targets - most prominently immune complexes - to initiate the step-wise activation of associated serine proteases, Clr and Cis, providing a major mechanism for pathogen and immune complex clearance following engagement by the adaptive immune system. Binding of Clq to immune complexes converts the Clr zymogen dimer into its active form to cleave and thereby activate Cis. Cis translates Clq binding into complement activation in two cleavage steps: It first converts C4 into C4a and C4b and then cleaves C4b-bound C2 to form the C3 convertase C4b2a, This complex converts the abundant plasma component C3 into C3a and C3b. Accumulation of C3b in close proximity of the C4b2a complex shifts the substrate specificity for C3 to C5 to form the C5 convertase C4b2a(C3b)„. The C3 and C5 convertase complexes generated via classical pathway activation are identical to those generated through the lectin pathway activation route, in the alternative pathway, spontaneous low-level hydrolysis of component C3 results in deposition of protein fragments onto cell surfaces, triggering complement activation on foreign cells, while cell-associated regulatory proteins on host tissues avert activation, thus preventing selfdamage, Like the alternative pathway, the lectin pathway may be activated in the absence of immune complexes. Activation is initiated by the binding of a multi-molecular lectin pathway activation complex to Pathogen-Associated Molecular Patterns (PAMPs), mainly carbohydrate structures present on bacterial, fungal or viral pathogens or aberrant glycosylation patterns on apoptotic, necrotic, malignant or oxygen-deprived cells (Collard, C.D., et ah, Am. J. Pathol. /56:1549-1556 (2000); Waiport, MJ., N. Engl. J.

Med. .344:1058-1066 (2001); Schwaeble, W., et al., Immunobiology 205:455-466 (2002); and Fujita, T., Nat. Rev. Immunol. 2:346-353 (2002)).

Mannan-binding lectin (MBL) was the first carbohydrate recognition subcomponent shown to form complexes with a group of novel serine proteases, named

-1712018200437 19 Jan 2018

MBL-assoeiaied Serine Proteases (MASPs) and numbered according to the sequence of their discovery (i.e., MASP-1, MASP-2 and MASP-3). In man, lectin pathway activation complexes can be formed with four alternative carbohydrate recognition subcomponents with different carbohydrate binding specificities, i.e,, MBL 2, and three different members of the ficolin family, namely L-Ficolin, H-ficolin and M-ficolin and MASPs. Two forms of MBL. MBL A and MBL C, and ficolin-A form lectin activation pathway complexes with MASPs in mouse and rat plasma. We have previously cloned and characterised MASP-2 and an additional truncated MASP-2 gene product of 19 kDa, termed MApl9 or sMAP, in human, mouse and rat (Thiel, S., et ai„ Nature 386:506-510 (1997);, Stover, C.M., et al., J, Immunol. /62:3481-3490 (1999); Takahashi, M., et al.,

Int. Immunol. //:859-863 (1999): and Stover, C.M., et al., J. Immunol. /65:6848-6859 (1999)), MApl9/ sMAP is devoid of protease activity, but may regulate lectin pathway activation by competing for the binding of MASPs to carbohydrate recognition complexes (Iwaki, D. et al., J. Immunol. 7 77:8626-8632 (2006)),

There is evidence suggesting that of the three MASPs, only MASP-2 is required to translate binding of the lectin pathway recognition complexes into complement activation (Thiel, S., et al. (1997); Vorup-Jensen, T., et ah,,/. Immunol. /65:2093-2100 (2000); Thiel, S., et al., J. Immunol. 165:313-831 (2000); Rossi, V., et al., J. Biol. Chem. 276:40880-40887 (2001)). This conclusion is underlined by the phenotype of a most recently described mouse strain deficient in MASP-1 and MASP-3. Apart from a delay in the onset of lectin pathway mediated complement activation in vitro -MASP-1/3 deficient mice retain lectin pathway functional activity. Reconstitution of MASP-1 and MASP-3 deficient serum with recombinant MASP-ί overcomes this delay in lectin pathway activation implying that MASP-1 may facilitate MASP-2 activation (Takahashi,

M., et al., J. Immunol. I50:6132-6138 (2008)), A most recent study has shown that

MASP-1 (and probably also MASP-3) are required to convert the alternative pathway activation enzyme Factor D from its zymogen form into its enzymatically active form (Takahashi, M,, et ah, 7. Exp. Med 207:29-31 (2010)). The physiological importance of this process is underlined by the absence of alternative pathway functional activity in plasma of MASP-1/3 deficient mice.

The recently generated mouse strains with combined targeted deficiencies of the lectin pathway carbohydrate recognition subcomponents MBL A and MBL C may still initiate lectin pathway activation via the remaining murine lectin pathway recognition

-1722018200437 19 Jan 2018 subcomponent ficolin A (Takahashi, K., et at, Microbes Infect. 4:773-784 (2002)). The absence of any residual lectin pathway functional activity in MASP-2 deficient mice delivers a conclusive model to study the role of this effector arm of innate humoral immunity' in health and disease·,

The availability of C4 and MASP-2 deficient mouse strains allowed us to define a novel lectin pathway specific, but MASP-2 dependent, C4-bypass activation route of complement C3. The essential contribution of this novel lectin pathway mediated €4bypass activation route towards post-ischemic tissue loss is underlined by the prominent protective phenotype of MASP-2 deficiency in MIRI while C4-deficient mice tested in the same model show no protection,

In this Example, we describe a novel lectin pathway mediated and MASP-2 dependent C4-bypass activation of complement C3. The physiological relevance of this new activation route is established by the protective phenotype of MASP-2 deficiency in an experimental model of myocardial ischemia/reperfusion injury (MIRI), where C4 deficient animals were not protected.

Methods:

MASP-2 deficient mice show no gross abnormalities. MASP-2 deficient mice were generated as described in Example 1. Both heterozygous (^IA) and homozygous (’^Λ) MASP-2 deficient mice are healthy and fertile, and show no gross abnormalities. Their life expectancy is similar to that of their WT littermates (>18 months). Prior to studying the phenotype of these mice in experimental models of disease, our MASP-2'^/' line was baekcrossed for eleven generations onto a C57BL/6 background. The total absence of MASP-2 mRNA was confirmed by Northern blotting of poly A+ selected liver RNA preparations, whiie the 1.2kb mRNA encoding MApl9 or sMAP (a truncated alternative splicing product of the MASP2 gene) is abundantly expressed.

qRT-PCR analysis using primer pairs specific for either the coding sequence for the serine protease domain of MASP-2 (B chain) or the remainder of the coding sequence for the A-chain showed that no B chain encoding mRNA is detectable in MASP-2 mice while the abundance of the disrupted A chain mRNA transcript was significantly increased, Likewise, the abundance of MApl9/sMAP encoding mRNA is increased in MASP-2 ^+/' and MASP-2 mice. Plasma MASP-2 levels, determined by ELISA for 5 animals of each genotype, were 300ng/ml for WT controls (range 260-33Ong/mf), 360ng/ml for heterozygous mice (range 330-395ng/ml) and undetectable inMASP-2

-1732018200437 19 Jan 2018 mice. Using qRT-PCR, mRNA expression profiles were established demonstrating that

MASP-2⁷mice express mRNA for MBL A, MBL C, ficolin A, MASP-I, MASP-3, Clq,

ClrA, ClsA, Factor B, Factor D. C4, and C3 at an abundance similar to that of their

MASP-2 sufficient littermates (data not shown),

Plasma C3 levels of MASP-2⁷ (n=8) and MASP-2(n=7) littermates were measured using a commercially available mouse C3 ELISA kit (Kamiya, Biomedical, Seattle, WA). C3 levels of MASP-2 deficient mice (average 0,84 mg/ml, +/- 0,34) were similar to those of the WT controls (average 0.92, +/- 0,37).

Results:

MASP-2 is essential for lectin pathway functional activity.

As described in Example 2 and shown in FIGURE 5, the in vitro analyses of

MASP-2''plasma showed a total absence of lectin pathway functional activity on activating Mannan- and Zymosan-coated surfaces for the activation of C4. Likewise, neither lectin pathway-dependent C4 nor C3 cleavage was detectable in MASP-2'^z'plasma on surfaces’ coated with N-acelyl glucosamine, which binds and triggers activation via MBL A, MBL C and ficolin A (data not shown).

The analyses of sera and plasma of MASP-2-/-mice clearly demonstrated that MASP-2 is essentially required to activate complement via the lectin pathway. The total deficiency of lectin pathway functional activity, however, leaves the other complement activation pathways intact: MASP-2.-/-plasma can still activate complement via the classical (FIGURE 26A) and the alternative pathway (FIGURE 26B). In FIGURE 26A and 26B, the symbol symbol indicates serum from WT (MASP-2 (+/+)); the symbol ® indicates serum from WT (Clq depleted); the symbol indicates serum from MASP-2 (-A); and the symbol Δ indicates serum from MASP-2 (-/-) (Clq depleted),

FIGURE 26A graphically illustrates that MASP-2-/- mice retain a functional classical pathway: C3b deposition was assayed on microtiter plates coated with immune complexes (generated in situ by coating with BSA then adding goat anti-BSA IgG). FIGURE 2fsB graphically illustrates MASP-2 deficient mice retain a functional alternative pathway: C3b deposition was assayed on Zymosan coated microtiter plates under conditions that permit only alternative pathway activation (buffer containing Mg²⁺ and EGTA). Results shown in FIGURE 26A and FIGURE 26B are means of duplicates and are typical of three independent experiments. Same symbols for plasma sources were

-1742018200437 19 Jan 2018 used throughout. These results show that a functional alternative pathway Is present in

MASP-2 deficient mice, as evidenced in the results shown in FIGURE 26B under experimental conditions designed to directly trigger the alternative pathway, while inactivating both the classical pathway and lectin pathway.

The lectin pathway of complement activation critically contributes to inflammatory tissue loss in myocardial ischemia/reperfusion injury (MIRI).

hi order to study the contribution of lectin pathway functional activity to MIRi, we compared MASP-2''mice and WT littermate controls in a. model of MIRi following transient ligation and reperfusion of the left anterior descending branch of the coronary artery (LAD). The presence or absence of complement C4 has no impact on the degree of ischemic tissue ioss in MIRI, We assessed the impact of C4 deficiency on infarct sizes following experimental MIRI. As shown in FIGURE 27A and FIGURE 27B, identical infarct sizes were observed in both C4-deficient mice and their WT littermates. FIGURE 27A graphically illustrates MIRI-induced tissue loss following LAD ligation and reperfusion in ¢4-/- mice (n=6) and matching WT littermate controls (n=7). FIGURE 27B graphically illustrates INF as a function of AAR, clearly demonstrating that C4-/mice are as susceptible to MIRI as their WT controls (dashed line).

These results demonstrate that C4 deficient mice are not protected from MIRI. This result was unexpected, as it is in conflict with the widely accepted view that the major C4 activation fragment, C4b, is an essential component of the classical and the lectin pathway C3 convertase C4b2a. We therefore assessed whether a residual lectin pathway specific activation of complement C3 can be detected in C4-deficient mouse and human plasma.

The lectin pathway can activate complement C3 in absence of C4 via a novel

MASP-2 dependent C4-bypass activation route.

Encouraged by historical reports indicating the existence of a C4-bypass activation route in C4-deficient guinea pig serum (May, J.E., and M. Frank. J. Immunol. //.1:1671-1677 (1973)), we analyzed whether C4-deficient mice may have residual classical or lectin pathway functional activity and monitored activation of C3 under pathway-specific assay conditions that exclude contributions of the alternative pathway.

C3b deposition was assayed on Mannan-coated microtiter plates using re-calcified plasma at plasma concentrations prohibitive for alternative pathway activation (1.25% and below). While no cleavage of C3 was detectable in C4-deficient plasma tested for

-1752018200437 19 Jan 2018 classical pathway activation (data not shown), a strong residual C3 cleavage activity was observed in C4-deficient mouse plasma when initiating complement activation via the lectin pathway. The lectin pathway dependence is demonstrated by competitive inhibition of C3 cleavage following preincubation of C4-deficient plasma dilutions with soluble

S Mannan (see FIGURE 28A), As shown in FIGURE 28.A-D. MASP-2 dependent activation of C3 was observed in the absence ofC4. FIGURE 28A graphically illustrates C3b deposition by C4+/+ (crosses) and C4-Z- (open circles) mouse plasma, Preinc abating the C4-A plasma with excess (1 gg/ml) fluid-phase Mannan prior to the assay completely inhibits C3 deposition (filled circles), Results are typical of 3 independent experiments, FIGURE 28B graphically illustrates the results of an experiment in which wild-type, MASP-2 deficient (open squares) and C4-/-tnouse plasma (1%) was mixed with various concentrations of anti-rat MASP-2 mAbMll (abscissa) and C3b deposition assayed on Mannan-coated plates. Results are means (± SD) of 4 assays (duplicates of 2 of each type of plasma). FIGURE 28C graphically illustrates the results of an experiment in which Human plasma: pooled NHS (crosses), C4-/- plasma (open circles) and C4-/plasma pre-incubated with 1 gg/ml Mannan (filled circles). Results are representative of three independent experiments. FIGURE 28D graphically illustrates that inhibition of C3b deposition in C4 sufficient and C4 deficient human plasma (1%) by anti-human MASP-2 rnAbH3 (Means ± SD of triplicates). As shown in FIGURE 28B, no lectin pathway-dependent C3 activation was detected in MASP-2-/-plasma assayed in parallel, implying that this C4-bypass activation route of C3 is MASP-2 dependent.

To further corroborate these findings, we established a series of recombinant inhibitory niAbs isolated from phage display antibody libraries by affinity screening against recombinant human and rat MASP-2A (where the serine residue of the active protease domain was replaced by an alanine residue by site-directed mutagenesis to prevent autolytic degradation of the antigen). Recombinant antibodies against MASP-2 (AbH3 and AbMll) were isolated from Combinatorial Antibody Libraries (Knappik, A,, st ah, ,J Mol. Biol. 296:57-86 (2000)), using recombinant human and rat MASP-2A as antigens (Chen, C.B, and Wallis, J Biol. Chem. 276:25894-25902 (2001)). An anti-rat

Fab2 fragment that potently inhibited lectin pathway-mediated activation of C4 and C3 in mouse plasma (IC50~l nM) was converted to a full-length IgG2a antibody. Polyclonal anti-murine MASP-2A antiserum was raised in rats. These tools allowed us to confirm

-1762018200437 19 Jan 2018

MASP-2 dependency of this novel lectin pathway specific C4-bypass activation route of

C3, as further described below.

As shown in FIGURE 28B, M211, an inhibitory monoclonal antibody which selectively binds to mouse and rat MASP-2 inhibited the C4-bypass activation of C3 in

C4-deficient mouse as well as C3 activation of WT mouse plasma via the lectin pathway in a concentration dependent fashion with similar 1C_5O values. Ail assays were carried out at high plasma dilutions rendering the alternative pathway activation route dysfunctional (with the highest plasma concentration being 1.2.5%),

In order to investigate the presence of an analogous lectin pathway specific C410 bypass activation of C3 in humans, we analyzed the plasma of a donor with an inherited deficiency of both human C4 genes (i.e., C4A and C4B), resulting in total absence of C4 (Yang, Y„ et al., J. Immunol. 773:2803-2814 (2004)). FIGURE 28C shows that this patient's plasma efficiently activates C3 in high plasma dilutions (rendering the alternative activation pathway dysfunctional). The lectin pathway specific mode of C3 activation on Marman-coated plates is demonstrated in murine C4-deficient plasma (FIGURE 28A) and human C4 deficient plasma (FIGURE 28C) by adding excess concentrations of fluid-phase Mannan. The MASP-2 dependence of this activation mechanism of C3 in human C4-deficient plasma was assessed using AbH3, a monoclonal antibody that specifically binds to human MASP-2 and ablates MASP-2 functional activity. As shown in FIGURE 28D, AbII3 inhibited the deposition of C3b (and C3dg) in both C4-sufficient and C4-deficient human plasma with comparable potency.

In order to assess a possible role of other complement components in the C4bypass activation of C3, we tested plasma of MASP-l/3-/-and Bf/C2-/-mice alongside MASP-2-/-, G4-/- and Clq-Z- plasma (as controls) under both lectin pathway specific and classical pathway specific assay conditions. The relative amount of C3 cleavage was plotted against the amount of C3 deposited when using WT plasma.

FIGURE 29A graphically illustrates a comparative analysis of €3 convertase activity in plasma from various complement deficient mouse strains tested either under lectin activation pathway or classical activation pathway specific assay conditions.

Diluted plasma samples (1%) of WT mice (n=6), MASP-2-/-mice (n=4), MASP-1/3-/roiee (n-2). C4-/- mice (n=8), C4/MASP-1/3-/- mice (n=8), Bf/C2-/- (n-2) and Op-Amice (n-2) were tested in parallel. Reconstitution of Bf/C2-/- plasma with 2.5pg/ml recombinant rat C2 (Bf7C2-/- +C2) restored C3b deposition. Results are means (±SD).

-1772018200437 19 Jan 2018 **p<0.01 (compared to WT plasma). As shown in FIGURE 29A, substantial C3 deposition is seen in C4-/- plasma tested under lectin pathway specific assay conditions, but not under classical pathway specific conditions. Again, no C3 deposition was seen in

MASP-2 deficient plasma via the lectin pathway activation route, while the same plasma deposited C3 via the classical pathway, in MASP-1/3-/- plasma, C3 deposition occurred in both lectin and classical pathway specific assay conditions. No C3 deposition was seen in plasma with a combined deficiency of C4 and MASP-1/3, either using lectin pathway or classical pathway specific conditions. No C3 deposition is detectable in C2/Bf-/- plasma, either via the lectin pathway, or via. the classical pathway,

Reconstitution of C2/Bf-/~ mouse plasma with recombinant C2, however, restored both lectin pathway and classical pathway-mediated C3 cleavage. The assay conditions were validated using Ciq-/- plasma.

FIGURE 29B graphically illustrates time-resolved kinetics of C3 eonvertase activity in plasma from various complement deficient mouse strains WT, fB-/-, C4-/-,

MASP-1/3-/-, and MASP-2-/-plasnia» tested under lectin activation pathway specific assay conditions (1% plasma, results are typical of three Independent experiments). As shown in FIGURE 29B, while no C3 cleavage was seen in MASP-2-/-plasma, fB-/plasma cleaved C3 with similar kinetics to the WT plasma. A significant delay in the lectin pathway-dependent conversion of C3 to C3b (and C3dg) was seen in C4-/-as well as in MASP-1/3 deficient plasma. This delay of C3 activation in MASP-1/3-/- plasma was recently shown to be MASP-1, rather than MAS P-3 dependent (Takahashi, M., et a!., ,/. Immunol. 180:6132-6} 38 (2008)).

Discussion:

The results described in this Example strongly suggest that MASP-2 -functional activity is essential for the activation of C3 via the lectin pathway both in presence and absence of C4, Furthermore, C2 and MASP-1 are required for this novel lectin pathwayspecific C4-bypass activation route of C3 to work, The comparative analysis of lectin pathway functional activity in MASP-2-/-as well as C4-/- plasma revealed the existence of a previously unrecognized C4-independent, but MASP-2-dependent activation route of complement C3 and showed that C3 can be activated in a lectin pathway-dependent mode in total absence of C4. While the detailed molecular composition and the sequence of activation events of this novel MASP-2 dependent C3 eonvertase remains to be elucidated, our results imply that this C4-bypass activation route additionally requires the

2018200437 19 Jan 2018 presence of complement C2 as well as MASP-1. The loss of lectin pathway-mediated C3 cleavage activity in plasma of mice with combined C4 and MASP-1/3 deficiency may be explained by a most recently described role of MASP-1 to enhance MASP-2 dependent complement activation through direct cleavage and activation of MASP-2 (Takahashi,

M<, et ah, J. Immunol. 159:6132-6138 (2008)). Likewise, MASP-1 may aid MASP-2 functional activity' through its ability to cleave C2 (Moiler-Kristensen, et ah, In!, Immunol. /9:141-149 (2007)). Both activities may explain the reduced rate by which MASP-1/3 deficient plasma cleaves C3 via the lectin activation pathway and why MASP1 may be required to sustain C3 conversion via the C4-bypass activation route.

The inability of C2/fB-/- plasma to activate C3 via the lectin pathway was shown to be C2-dependent as the addition of recombinant rat C2 to C2/IB-/- plasma restored the ability of the reconstituted plasma to activate C3 on Mannan-coated plates.

The finding that C4 deficiency specifically disrupts the classical complement activation pathway while the lectin pathway retains a physiologically critical level of C3 convertase activity via a MASP-2 dependent C4-bypass activation route calls for a reassessment of the role of the lectin pathway in various disease models, including experimental 6. pneumoniae infection (Brown, J. S., et ah, Proc. Natl. Acad Sci. U. S. A. 99:16969-16974 (2002); Experimental -Allergic Encephalomyelitis (Boos, L.A., et ah, Glia 49:158-160 (2005); and models of C3 dependent murine liver regeneration (Clark.

A., et ah, Moi. Immunol. 45:3125-3132. (2008)). The latter group demonstrated that C4deficient mice can activate C3 in an alternative pathway independent fashion as in vivo inhibition of the alternative pathway by an antibody-mediated depletion of factor B functional activity did not effect C3 cleavage-dependent liver regeneration in C4-/- mice (Clark, A., et ah (2008)). This lectin pathway mediated C4-bypass activation route of C3 may also explain the lack of a protective phenotype of C4 deficiency in our model of MIR1 as well as in a previously described model of renal allograft rejection (Lin, T., et ah, Am. J. Pathol. 168:1241-1248 (2006)), In contrast, our recent results have independently demonstrated a significant protective phenotype of MASP-2-/-mice in models of renal transplantation (Farrar. C.A., et ah, Mol. Immunol. 46:2832 (2009)).

In summary, the results of this Example support the view that MASP-2 dependent

C4-bypass activation of C3 is a physiologically relevant mechanism that may he important under conditions where availability of C4 is limiting C3 activation.

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EXAMPLE 23

This Example describes activation of C3 by thrombin substrates and C3 deposition on mannan in WT (+/+), MASP-2 (-/-), FI I (-/-), FI1/C4 (-/-) and C4 (-/-) mice.

Rationale:

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

Methods:

C3 Activation by thrombin substrates

Activation of C3 was measured in the presence of the following activated forms 15 of thrombin substrates; human FCXla, human FVIla, bovine FXa, human FXa, human activated protein C, and human thrombin. C3 was incubated with the various thrombin substrates, then separated under reducing conditions on 10% SDS-polyacrylamide gels. After electrophoretic transfer using cellulose membrane, the membrane was incubated with monoclonal biotin-coupled rat anti-mouse C3, detected with a streptavidin-HRP kit and developed using ECL reagent,

Results:

Activation of C3 involves cleavage of the intact a-chain into the truncated a' chain and soluble C3a (not shown in FIGURE 30). FIGURE 30 shows the results of a Western blot analysis on the activation of human C3 by thrombin substrates, wherein the uncleaved C3 alpha chain, and the activation product a' chain are shown by arrows. As shown in FIGURE 30, incubation of C3 with the activated forms of human clotting factor XI and factor X, as well as activated bovine clotting factor X, can cleave C3 in vitro in the absence of any complement pro teases.

C3 deposition on mannan

C3 deposition assays were carried out on serum samples obtained from WT,

MASP-2 (-/-), FI 1(-/-), FI 1(-/-)/64(-/-) and C4(-/-). F11 is the gene encoding coagulation factor XI. To measure C3 activation, microtiter plates were coated with mannan (1 pg/well), then adding sheep anti-HSA serum (2 ug/ml) in TBS/tween/Ca²*.

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Plates were blocked with 0.1% HSA in TBS and washed as above. Plasma samples were diluted in 4 mM barbital, 145 mM NaCi, 2 mM CaCh, 1 mM MgCl·?, 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 alkaline phosphatase-conjugated goat anti5 rabbit IgG and pNPP.

Results:

FIGURE 31 shows the results of the C3 deposition assay on serum samples obtained from WT, MASP-2 (-/-), Fl 1 (-/-), Fl 1(-/-),C4 (-/-) and C4 (-/-). As shown in FIGURE 31, there is a functional lectin pathway even in the complete absence of C4. As further shown in FIGURE 31, this novel lectin pathway dependent complement activation requires coagulation factor XI.

Discussion:

Prior to the results obtained in this experiment, it was believed by those in the art that the lectin pathway of complement required C4 for activity. Hence, data from C4 knockout mice (and C4 deficient humans) were interpreted with the assumption that such organisms were lectin pathway deficient (in addition to classical pathway deficiency). The present results demonstrate that this notion is false. Thus, conclusions of past studies suggesting that the lectin pathway was not important in certain disease settings based on the phenotype of C4 deficient animals may be false. The data described in this Example also show that, in the physiological context of whole serum the lectin pathway can activate components of the coagulation cascade. Thus, it is demonstrated that there is cross-talk between complement and coagulation involving MASP-2.

EXAMPLE 24

This Example describes methods to assess the effect of an anti-MASP-2 antibody on lysis of red blood cells from blood samples obtained from Paroxysmal nocturnal hemoglobinuria (PNH) patients.

Background/Rationale:

Paroxysmal nocturnal hemoglobinuria (PNH), also referred to as Marchiafava30 Micheli syndrome, is an acquired, potentially life-threatening disease of the blood, characterized by complement-induced intravascular hemolytic anemia. The hallmark of PNH is chronic intravascular hemolysis that is a consequence of unregulated activation of the alternative pathway of complement. Lindorfer, M.A., et a!., Blood //5(11) (2010).

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Anemia in PNH is due to destruction of red biood ceils in the bloodstream. Symptoms of

PNH include red urine, due to appearance of hemoglobin in the urine, and thrombosis.

PNH may 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 against immune destruction by inhibiting the complement system (Hillmen P. et ah, N. Engl. J. Med. 556(6):552-9 (2004)). However, a significant portion of PNH patients treated with eculizumab are left with clinically significant immune-mediated hemolytic anemia because the antibody does not block activation of the alternative pathway of complement.

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

Methods:

Erythrocytes from normal donors and from patients suffering from PNH (not treated with Soliris) are obtained by venipuncture, and prepared as described in Wilcox, L.A., et al., Blood 75:820-829 (1991), hereby incorporated herein by reference. Anti20 MASP-2 antibodies with functional blocking activity of the lectin pathway may be generated as described in Example 10.

Hemolysis Analysis:

The method for determining the effect of anti-MASP-2 antibodies on the ability to block hemolysis of erythrocytes from PNH patients is carried out using the methods described in Lindorfer, M.A., et al., Biood /5(11):2283-91 (2010) and Wilcox, L.A., et ah, Blood 75:820-829 (1991), both references hereby incorporated herein by reference. As described in Lindorfer et ah, erythrocytes from PNH patient samples are centrifuged, the huffy coat is aspirated and the cells are washed in gelatin veronal buffer (GVB) before each experiment, The erythrocytes are tested for susceptibility to APC-mediated lysis as follows. ABO-matched normal human sera are diluted with GVB containing 0.15 mM CaCfy and 0.5 mM MgCfy (GVB⁺²) and acidified to pH 6.4 (acidified NHS, aNHS) and used to reconstitute the erythrocytes to a hematocrit of 1.6% in 50% aNHS. The mixtures are then incubated at 37°C, and after 1 hour, the erythrocytes are pelleted by

-1822018200437 19 Jan 2018 centrifugation. The optical density of an aliquot of the recovered supernate is measured at 405 nM and used to calculate the percent lysis. Samples reconstituted in acidified serum-EDTA are processed similarly and used to define background noncomplementmediated lysis (typically less than 3%). Complete lysis (100%) is determined after incubating the erythrocytes in distilled water.

In order to determine the effect of anti-MASP-2 antibodies on hemolysis of PNH erythrocytes, erythrocytes from PNH patients are incubated in aNHS in the presence of incremental concentrations of the anti-MASP-2 antibodies, and the presence/amount of hemolysis is subsequently quantified.

In view of the fact that anti-MASP-2 antibodies have been shown to block subsequent activation of the alternative complement pathway, it is expected that antiMASP-2 antibodies will be effective in blocking alternative pathway-mediated hemolysis of PNH erythrocytes, and will be useful as a therapeutic to treat patients suffering from PNH.

EXAMPLE 25

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

Baekgrouml/Rafsonak:

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

Cryoglobulinemia may be classified as follows based on cryoglobulin composition: Type I cryoglobulinemia, or simple cryoglobulinemia, is the result of a monoclonal immunoglobulin, usually immunoglobulin M (IgM); Types II and Hi cryoglobulinemia (mixed cryoglobulinemia) contain rheumatoid factors (RFs), which are usually IgM in complexes with the Fc portion of polyclonal IgG.

Conditions associated with cryoglobulinemia include hepatitis C infection, lymphoproliferative disorders and other autoimmune diseases. Cryoglobulin-containing

-1832018200437 19 Jan 2018 immune complexes result in a clinical syndrome of systemic inflammation, possibly due to their ability to activate complement. While IgG immune complexes normally activate the classical pathway of complement, IgM containing complexes can also activate complement via the lectin pathway (Zhang, M., et at, Moi Immunol 44(1-3):103-110 (2007) and Zhang. M„ et ah, J. Immunol. /77(7):4727-34 (2006)).

Immunohistochemical studies have further demonstrated the cryoglobulin immune complexes contain components of the lectin pathway, and biopsies from patients with cryoglobulinemic glomerulonephritis showed immunohistochemical evidence of lectin pathway activation in situ (Ohsawa, I., et al„ Clin Immunol /67(1):59-66 (2001)).

These results suggest that the lectin pathway may contribute to inflammation and adverse outcomes in cryoglobulemic diseases.

Methods:

The method for determining the effect of anti-MASP-2 antibodies on the ability to block the adverse effects of Cryoglobulinemia is carried out using the assay for fluid phase C3 conversion as described in Ng Y.C. et ah, Arthritis and Rheumatism 7/(1):99107 (1988), hereby incorporated herein by reference. As described in Ng et ah, in essential mixed cryoglobulinemia (EMC), monoclonal rheumatoid factor (mRF), usually IgM, complexes with polyclonal IgG to form the characteristic cryoprecipitate immune complexes (IC) (type II cryoglobulin). Immunoglobulins and C3 have been demonstrated in vessel walls in affected tissues such as skin, nerve and kidney, As described in Ng et ah, ¹²⁵l-iabeled mRF is added to serum (normal human serum and serum obtained from patients suffering from cryoglobulinemia), incubated at 37°C, and binding to erythrocytes is measured.

Fluid phase C3 conversion is determined in serum (normal human serum and serum obtained from patients suffering from cryoglobulinemia) in the presence or absence of the following IC: BSA-anti BSA, mRF, mRF plus IgG, or cryoglobulins, in the presence or absence of anti-MASP-2 antibodies. The fixation of‘C3 and C4 to IC is measured using a coprecipitation assay with F(ab')2 anti-C3 and F(ab^!)2 anti-C4.

In view of the fact that anti-MASP-2 antibodies have been shown to block activation of the lectin pathway it is expected that anti-MASP-2 antibodies will be effective in blocking complement mediated adverse effects associated with cryoglobulinemia, and will be useful as a therapeutic to treat patients suffering from cryoglobulinemia.

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EXAMPLE 26

This Example describes methods to assess the effect of an anti-MASP-2 antibody on blood samples obtained from patients with Cold Agglutinin Disease, which manifests as anemia.

Bae kgrou n d/Ration ale:

Cold Agglutinin Disease (CAD), is a type of autoimmune hemolytic anemia. Cold agglutinins antibodies (usually IgM) are activated by cold temperatures and bind to and aggregate red blood cells. The cold agglutinin antibodies combine with complement and attack the antigen on the surface of red blood cells. This leads to opsonialion of red blood cells (hemolysis) which triggers their clearance by the reticuloendothelial system. The temperature at which the agglutination takes place varies from patient to patient.

CAD manifests as anemia. When the rate of destruction of red blood cell destruction exceeds the capacity of the bone marrow to produce an adequate number of oxygen-carrying cells, then anemia occurs. CAD can be caused by an underlying disease or disorder, referred to as Secondary CAD, such as an infectious disease (mycoplasma pneumonia, mumps, mononucleosis), lymphoproliferative disease (lymphoma, chronic lymphocytic leukemia), or connective tissue disorder. Primary CAD patients are considered to have a low' grade lymphoproliferative bone marrow' disorder. Both primary and secondary CAD are acquired conditions.

Methods:

Reagents:

Erythrocytes from normal donors and from patients suffering from CAD are obtained by venipuncture. Anti-MASP-2 antibodies with functional blocking activity of the lectin pathway may be generated as described in Example 10.

The effect of anti-MASP-2 antibodies to block cold aggultinin-mediated activation of the lectin pathway may be determined as follows. Erythrocytes from blood group I positive patients are sensitized with cold aggultinins (i.e., IgM antibodies), in the presence or absence of anti-MASP-2 antibodies. The erythrocytes are then tested for the ability to activate the lectin pathway by measuring C3 binding.

In view of the fact that anti-MASP-2 antibodies have been shown to block activation of the lectin pathway, it is expected that anti-MASP-2 antibodies will be effective in blocking complement mediated adverse effects associated with Cold

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Agglutinin Disease, and will be useful as a therapeutic to treat patients suffering from

Cold Agglutinin Disease,

EXAMPLE 27

This Example describes methods to assess the effect of an anti-MASP-2 antibody on lysis of red blood cells in blood samples obtained from mice with atypical hemolytic uremic syndrome (aHUS).

Background/Ratkmale:

Atypical hemolytic uremic syndrome (aHUS) is characterized by hemolytic 10 anemia, thrombocytopenia, and renal failure caused by platelet thrombi in the microcirculation of the kidney and other organs. aHUS is associated with detective complement regulation and can be either sporadic or familial. aHUS is associated with mutations in genes coding for complement activation, including complement factor H, membrane cofactor B and factor I, and well as complement factor H-related 1 (CFHR1) and complement factor H-related 3 (CFHR.3), Zipfel, P.F., et ah, PloS Genetics 3(3):e41 (2007). This Example describes methods to assess the effect of an anti-MASP-2 antibody on lysis of red blood cells from blood samples obtained from aHUS mice,

Methods:

The effect of anti-MASP-2 antibodies to treat aHUS may be determined in a mouse model of this disease in which the endogenou.se mouse fH gene has been replaced with a human homologue encoding a mutant form of fH frequently found in aHUS patients. See Pickering M.C. et al,, J. Exp. Med. 204(6): 1249-1256 (2007), hereby incorporated herein by reference. As described in Pickering et al., such mice develop an aHUS like pathology. In order to assess the effect of an anti-MASP-2 antibody for the treatment of aHUS, anti-MASP-2 antibodies are administered to the mutant aHUS mice and lysis of red blood cells obtained from anti-MASP-2 ab treated and untreated controls is compared. In view of the fact that anti-MASP-2 antibodies have been shown to block activation of the lectin pathway it is expected that anti-MASP-2 antibodies will be effective in blocking lysis of red blood cells in mammalian subjects suffering from aHUS.

This Example describes methods to assess the effect of an anti-MASP-2 antibody for the treatment of glaucoma.

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Rationale/Backgroand:

it has been shown that uncontrolled complement activation contributes to the progression of degenerative injury to retinal ganglion cells (RGCs), their synapses and axons in glaucoma. See Tezel G. et al,, Invest Ophthalmol Vis Sci 5/:5071-5082 (2010).

For example, histopathologic studies of human tissues and in vivo studies using different animal models have demonstrated that complement components, including Clq and C3, are synthesized and terminal complement complex is formed in the glaucomatous retina (see Stasi K. et al., Invest Ophthalmol Vis Sci 47:1024-1029 (2006). Kuehn M.H. et al., Exp Eye Res 53:620-628 (2006)). .As further described in Kuehn M.H. et ah,

Experimental Eye Research 57:89-95 (2008), complement synthesis and deposition is induced by retinal Ϊ/R and the disruption of the complement cascade delays RGC degeneration. In this study, mice carrying a targeted disruption of the complement component C3 were found to exhibit delayed RGC degeneration after transient retinal i/R when compared to normal animals.

Methods:

The method for determining the effect of anti-MASP-2 antibodies on RGC degeneration is carried out in an animal model of retinal l/R as described in Kuehn M.H, et al., Experimental Eye Research 57:89-95 (2008), hereby incorporated herein by reference. As described in Kuehn et al., retinal ischemia is induced by anesthetizing the animals, then inserting a 30-gauge needle connected to a reservoir containing phosphate buffered saline through the cornea into the anterior chamber of the eye. The saline reservoir is then elevated to yield an intraocular pressure of 104 mmHg, sufficient to completely prevent circulation through the retinal vasculature. Elevated intraocular ischemia is confirmed by blanching of the iris and retina and ischemia is maintained for

45 minutes in the left eye only; the right eye serves as a control and does not receive cannulation. Mice are then euthanized either 1 or 3 weeks after the ischemic insult, AntiMASP-2 antibodies are administered to the mice either locally to the eye or systemically to assess the effect of an anti-MASP antibody administered prior to ischemic insult.

Immunohistochemistry of the eyes is carried out using antibodies against Clq and

C3 to detect complement deposition. Optic nerve damage can also be assessed using standard electron microscopy methods. Quantitation of surviving retinal RGCs is performed using gamma synuclein labeling.

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Results:

As described in Kuehn et al., in normal control mice, transient retinal ischemia results in degenerative changes of the optic nerve and retinal deposits of Ciq and C3 detectable by immunohistochemistry. In contrast, C3 deficient mice displayed a marked reduction in axonal degeneration, exhibiting only minor levels of optic nerve damage 1 week after induction. Based on these results, it is expected that similar results would be observed when this assay is carried out in a MASP-2 knockout mouse, and when antiMASP-2 antibodies are administered to a normal mouse prior to ischemic insult.

EXAMPLE 29

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

Rationale:

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

The lectin pathway of complement is an innate immune mechanism that initiates inflammation in response to tissue injury and exposure to foreign surfaces (i.e., bacteria). Blockade of this pathway leads to better outcomes in mouse models of ischemic intestinal tissue injury or septic shock. It is hypothesized that the lectin pathway may trigger excessive and harmful inflammation in response to radiation-induced tissue injury. Blockade of the lectin pathway may thus reduce secondary injury and increase survival following acute radiation, exposure.

The objective of the study carried out as described in this Example was to assess the effect of iectin pathway blockade on survival in a mouse model of radiation injury by administering anti-murine MASP-2 antibodies.

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Methods and Materials:

Materials. The test articles used in this study were (i) a high affinity anti-murine

MASP-2 antibody (rnAbMll) and (ii) a high affinity anti-human MASP-2 antibody (mAbH6) that block the MASP-2 protein component of the lectin complement pathway which were produced in transfected mammalian ceils. Dosing concentrations were 1 mg/kg of anti-murine MASP-2 antibody (rnAbMll), 5mg/kg of anti-human MASP-2 antibody (mAbH6), or sterile saline. For each dosing session, an adequate volume of fresh dosing solutions were prepared.

Animals. Young adult male Swiss-Webster mice were obtained from Harlan 10 Laboratories (Houston, TX). Animals were housed in solid-bottom cages with Alpha-Dri bedding and provided certified PMI 5002 Rodent Diet (Animal Specialties, inc,, Hubbard OR) and water ad libitum, Temperature was monitored and the animal holding room operated with a 12 hour light/12 hour dark ligTt cycle.

Irradiation. After a 2-week acclimation in the facility, mice were irradiated at 6.5 15 and 7.0 Gy by whole-body exposure in groups of 10 at a dose rate of 0.78 Gy/min using a Therapax X-RAD 320 system equipped with a 320-kV high stability X-ray generator, metal ceramic X-ray tube, variable x-ray beam collimator and filter (Precision X-ray Incorporated, East Haven, CT). Dose levels were selected based on prior studies conducted with the same strain of mice indica ting the LD5O/30 was between 6.5 and 7.0 Gy (data not shown).

Drug Formulation and Administration. The appropriate volume of concentrated stock solutions were diluted with ice cold saline to prepare dosing solutions of 0.2 mg/rnl anti-murine MASP-2 antibody (rnAbMll) or 0,5 mg/rnl anti-human MASP-2 antibody (mAbH6) according to protocol. Administration of anti-MASP-2 antibody mAbMl 1 and mAbH6 was via IP injection using a 25-gauge needle base on animal weight to deliver 1 mg/kg mAbMl 1, 5mg/kg mAbH6, or saline vehicle.

Study Design. Mice were randomly assigned to the groups as described in Table 8. Body weight and temperature were measured and recorded daily. Mice in Groups 7, 11 and 13 were sacrificed at post-irradiation day 7 and blood collected by cardiac puncture under deep anesthesia. Surviving animals at post-irradiation day 30 were sacrificed in the same manner and blood collected, Plasma was prepared from collected blood samples according to protocol and returned to Sponsor for analysis.

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TABLE 8: Study Groups _ | Group j 1 irradiation

ID	N	Level (Gy)	Treatment	Dose Schedule
1	20	6.5	Vehicle	18 hr prior to irradiation, 2 hr post irradiation, weekly booster
2	20	6.5	anti-murine MASP-2 ab (mAbMl 1)	18 hr prior to irradiation only
3	20	6.5	anti-murine MASP-2 ab (mAbMl 1)	18 hr prior to irradiation, 2 hr post irradiation, weekly booster
4	20	6.5	anti-murine MASP-2 ab (mAbMl 1)	2 hr post irradiation, weekly booster
5	20	6.5	anti-human MASP-2 ab (mAbH6)	18 hr prior to irradiation, 2 hr post irradiation, weekly booster
6	20	7.0	Vehicle	18 hr prior to irradiation, 2 hr post irradiation, weekly booster
7	5	7.0	Vehicle	2 hr post irradiation only
8	20	7.0	anti-murine MASP-2 ab (mAbMl 1)	38 hr prior to irradiation only
9	20	7.0	anti-murine MASP-2 ab (mAbMl 1)	18 hr prior to irradiation, 2 hr post irradiation, weekly booster
10	20	7.0	anti-murine MASP-2 ab (mAbM i 1)	2 hr post irradiation, weekly booster
13	5	7.0	anti-murine MASP-2 ab (mAbMl 1)	2 hr post irradiation only
12	20	7.0	anti-human MASP-2 ab (mAbH6)	3 8 hr prior to irradiation, 2 hr post irradiation, weekly booster
13	5	None	None	None

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Statistical Analysis. Kaplan-Meier survival curves were generated and used to compare mean survival time between treatment groups using log-Rank and Wticoxon methods. Averages with standard deviations, or means with standard error of the mean are reported. Statistical comparisons were made using a two-tailed unpaired t-test between controlled irradiated animals and individual treatment groups.

Results

Kaplan-Meier survival plots for 7.0 and 6.5 Gy exposure groups are provided in FIGURES 32A and 32B, respectively, and summarized below in Table 9. Overall, treatment with anti-murine MASP-2 ab (mAbMl 1) pre-irradiation increased the survival of irradiated mice compared to vehicle treated irradiated control animals at both 6.5 (20% increase) and 7.0 Gy (30% increase) exposure levels. At the 6.5 Gy exposure level, postirradiation treatment with anti-murine MASP-2 ab resulted in a modest increase in survival (15%) compared to vehicle control irradiated animals.

In comparison, all treated animals at the 7.0 Gy exposure level showed an increase in survival compared to vehicle treated irradiated control animals. The greatest change in survival occurred in animals receiving mAbHd, with a 45% increase compared to control animals. Further, at the 7.0 Gy exposure level, mortalities in the mAbH6 treated group first occurred at post-irradiation day 15 compared to post-irradiation day 8 for vehicle treated irradiated control animals, an increase of 7 days over control animals.

Mean time to mortality for mice receiving mAbH6 (27.3 ± 1.3 days) was significantly increased (p = 0.0087) compared to control animals (20.7 ± 2.0 days) at the 7.0 Gy exposure level.

The percent change in body weight compared to pre-irradiation day (day -1) was recorded throughout the study. A transient weight loss occurred in all irradiated animals, with no evidence of differential changes due to mAbM 11 or mAbH6 treatment compared to controls (data not shown). At study termination, ail surviving animals showed an increase in body weight from starting (day -1) body weight.

TABLE 9: Survival rates of test animals exposed to radiation

Test Group	Exposure Level	Survival (%)	Time to Death (Mean ± SEM, Day)	First/Last Death (Day)
Control irradiation	6.5 Gy	65 %	24.0 A 2.0	9/16
mAbMl 1 pre-	6.5 Gy	85%	27.7 A 1.5	13/17

-1912018200437 19 Jan 2018

Test Group	Exposure Level	Survival (%)	Time to Death (Mean ± SEM, Dav)	First/Last Death (Day)
exposure
mAbMll pre + post-exposure	6.5 Gy	65 %	24.0 ± 2.0	9/15
mAbMl I postexposure	6.5 Gy	80 %	26.3 ±1.9	9/13
mAbH6 pre+postexposure	6.5 Gy	65 %	24.6+ T9	9/19
Control irraditation	7.0 Gy	35 %	20.7 + 2.0	8/17
mAbMll preexposure	7.0 Gy	65%	23.0 + 2.3	7/13
mAbMl 1 pre + post-exposure	7.0 Gy	55 %	21.6 + 2.2	7716
mAbMl 1 post-	7,0 Gy	70 %	24.3 + 2, i	9/14
exposure
mAbH6 pre+post- exposure	7.0 Gy	80 %	27.3 ±1.3*	15/20

*p = 0.0087 by two-tailed unpaired t-test between controlled irradiated animals and treatment group at the same irradiation exposure level.

Discussion

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

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

Notably, at the 7,0 Gy exposure level, 80% of the animals receiving mAbH6 survived to 30 days compared to 35% of vehicle treated control irradiated animals. Importantly, the first day of death in this treated group did not occur until post-irradiation day 15, a 7-day increase over that observed in vehicle treated control irradiated animals. Curiously, at the

-1922018200437 19 Jan 2018 lower X-ray exposure (6.5 Gy), administration of niAbH6 did not appear to impact survivability or delay in mortality compared to vehicle treated control irradiated animals.

There could be multiple reasons for this difference in response between exposure levels, although verification of any hypothesis may require additional studies, including interim sample collection for microbiological culture and hematological parameters. One explanation may simply be that the number of animals assigned to groups may have precluded seeing any subtle treatment-related differences. For example, with groups sizes of' n—20, the difference in survival between 65% fmAbH6 at 6.5 Gy exposure) and 80% (mAbH6 at 7.0 Gy exposure) is 3 animals. On the other hand, the difference between 35% (vehicle control at 7.0 Gy exposure) and 80% (m.AbHd at 7.0 Gy exposure) is 9 animals, and provides sound evidence of a treatment-related difference.

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

EXAMPLE 30

This Example demonstrates that MASP-2 deficient mice are protected from Neisseria meningitidis induced mortality after infection with either N. meningitidis serogroup A or Neisseria meningitidis serogroup B.

Methods:

MASP-2 knockout mice (MASP-2 KO mice) were generated as described in Example 1. 10-week-old MASP-2 KO mice (n=10) and wild-type (WT) C57/BL6 mice (n~10) were inoculated by intraperitoneal (i.p.) injection with a dosage of 2.6 x 10⁷ CPU of Neisseria meningitidis serogroup A Z2491 in a volume of 100 μί. The infective dose was administered to mice in conjunction with Iron dextran at a final concentration of 400 mg/kg. Survival of the mice after infection was monitored over a 72-hour time period.

In a separate experiment, 10-week-old MASP-2 KO mice (n=10) and wild-type C57/BL6 mice (n=l0) were inoculated by i.p. injection with a dosage of 6 x 10⁶ CPU of

Neisseria meningitidis serogroup 8 strain MC58 in a volume of 100 μί. The infective dose was administered to mice in conjunction with iron dextran at a final dose of 400 mg/kg. Survival of the mice after infection was monitored over a 72-hour time

-193period. An illness score was also determined for the WT and MASP-2 KO mice during the 72-hour time period after infection, based on the illness scoring parameters described below in TABLE 10, which is based on the scheme of Fransen et al, (2010) with slight modifications.

2018200437 19 Jan 2018

TABLE 10: Illness Seortag associated with e-liaicai signs in infected mice

Signs	Score
Normal	0
SSightiy ruffled fur	1
Ruffled fur, slow and sticky eyes	2
Ruffled fur, lethargic and eyes shut	3
Very' sick and no movement after stimulation	4
Dead	5

Blood samples were taken from the mice at hourly intervals after infection and analyzed to determine the serum level (log cfii/mL) of N. meningitidis in order to verily infection and determine the rate of clearance of the bacteria from the serum.

Results:

FIGURE 33 is a Kaplan-Meyer plot graphically illustrating the percent survival of MASP-2 KO and WT mice after administration of an infective dose of 2.6 x 10⁷ cfu of N. meningitidis serogroup A Z2491. As shown in FIGURE 33, 100% of the MASP-2

KO mice survived throughout, the 72-hour period after infection, in contrast, only 80% of the WT mice (p—0.012) were still alive 24 hours after infection, and only 50% of the WT mice were still alive at 72 hours after infection. These results demonstrate that MASP-2deficient mice are protected from N. meningitidis serogroup A Z2491 -induced mortality.

FIGURE 34 is a Kaplan-Meyer plot graphically illustrating the percent survival of MASP-2 KO and WT mice after administration of an infective dose of 6 x 10⁶ cfu of N. meningitidis serogroup B strain MC58. As shown in FIGURE 34, 90% of the MASP2 KO mice survived throughout the 72-hour period after infection. In contrast, only 20% of the WT mice (p=0.0022) were stili alive 24 hours after infection. These results

-1942018200437 19 Jan 2018 demonstrate that MASP-2-deficient mice are protected from N. meningitidis serogroup B strain MC58-induced mortality.

FIGURE 35 graphically illustrates the log cfu/mL of N. meningitidis serogroup B strain MC58 recovered at different time points in blood samples taken from the MASP-2

KO and WT mice after i.p. infection with 6x10⁶ efu of N. meningitidis serogroup B strain MC58 (n=3 at different time points for both groups of mice). The results are expressed as Means+SEM. As shown in FIGURE. 35, in WT mice the level of A. meningitidis in the blood reached a peak of about 6.0 log cfu/mL at 24 hours after infection and dropped to about 4.0 log cfu/mL by 36 hours after infection. In contrast, in the MASP-2 KO mice, the level of At meningitidis reached a peak of about 4.0 log cfu/mL at 12 hours after infection and dropped to about 1.0 log efu/mL by 36 hours after infection (the symbol indicates p<0.05; the symbol ”** indicates p=0.0043). These results demonstrate that although the MASP-2 KQ mice were infected with the same dose of N. meningitidis serogroup B strain MC58 as the WT mice, the MASP-2 KO mice have enhanced clearance of bacteraemia as compared to WT.

FIGURE 36 graphically illustrates the average illness score of MASP-2. KO and

WT mice at 3, 6, 12 and 24 hours after infection with 6x10° efu of N. meningitidis serogroup B strain MC58. As shown in FIGURE 36, the MASP-2-deficient mice showed high resistance to the infection, with much lower illness scores at 6 hours (symbol indicates p=0.0411), 12 hours (symbol ** indicates p=0.0049) and 24 hours (symbol ***» indicates p=0.0049) after infection, as compared to WT mice. The results in FIGURE 36 are expressed as mean&fcSEM.

In summary, the results in this Example demonstrate that MASP-2-deficient mice are protected from Neisseria meningitides-vcviwxd mortality after infection with either At meningitidis serogroup A or A', meningitidis serogroup B,

EXAMPLE 31

This Example demonstrates that the administration of anti-MASP-2 antibody after infection with N. meningitidis increases the survival of mice infected with N.

meningitidis.

-1952018200437 19 Jan 2018

RaeRground/Rationaie:

As described in Example 10, rat MASP-2 protein was utilized to pan 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 lgG2a isotypes were generated from

Fab2 #11. The full-length anti-MASP-2 antibody of the mouse lgG2a isotype was characterized for pharmacodynamic parameters (as described in Example 19).

In this Example, the mouse anti-MASP-2 full-length antibody derived from Fab2 # 11 was analyzed in the mouse model of Λ^Γ. meningitidis infection.

Methods:

The mouse IgG2a full-length anti-MASP-2 antibody isotype derived from Fab2 #11, generated as described above, was tested in the mouse model of A', meningitidis infection as follows.

Administration of mouse-anti-MASP-2 Monoclonal antibodies (MoAb) after infection

J 5 9-week-oId C57/BL6 Charles River mice were treated with inhibitory mouse antiMASP-2 antibody (1.0 mg/kg) (n=l2) or control isotype antibody (n=10) at 3 hours after i,p. injection with a high dose (4x10⁶ efu) of TV. meningitidis serogroup B strain MC58.

Results:

FIGURE 37 is a Kaplan-Meyer plot graphically illustrating the percent survival of mice after administration of an infective dose of 4x10⁶ cfu of N. meningitidis serogroup B strain MC58, followed by administration 3 hours post-infection of either Inhibitory anti-MASP-2 antibody (3.0 mg/kg) or control isotype antibody. As shown in FIGURE 37, 90% of the mice treated with anti-MASP-2 antibody survived throughout the 72-hour period after infection. In contrast, only 50% of the mice treated with isotype control antibody survived throughout the 72-hour period after infection, The symbol indicates p=0.0301, as determined by comparison of the two survival curves.

These results demonstrate that administration of anti-MASP-2 antibody is effective to treat and improve survival in subjects infected with N. meningitidis,

As demonstrated herein, the use of anti-MASP-2 antibody in the treatment of a subject infected with N. meningitidis is effective when administered within 3 hours postinfection, and is expected to be effective within 24 hours to 48 hours after infection. Meningococcal disease (either meningococcemia or meningitis) is a medical emergency,

-196and therapy will typically be initiated immediately if meningococcal disease is suspected (i.e., before At meningitidis is positively identified as the etiological agent).

In view of the results in the MASP-2 KO mouse demonstrated in EXAMPLE 30.

it is believed that administration of anti-MASP-2 antibody prior to infection with N.

meningitidis would also be effective to prevent or ameliorate the severity of infection.

2018200437 19 Jan 2018

EXAMPLE 32

This Example demonstrates that administration of anti-MASP-2 antibody is 10 effective to treat N. meningitidis infection in human serum.

Rationale:

Patients with decreased serum levels of functional MBL display increased susceptibility to recurrent bacterial and fungal infections (Kilpatrick et ah, Biochim Biophys Acta 1572:401-413 (2002)). It is known that N. meningitidis is recognized by

MBL, and it has been shown that MBL-deficient sera do not lyse Neisseria.

In view of the results described in Examples 30 and 31, a series of experiments were carried out to determine the efficacy of administration of anti-MASP-2 antibody to treat N. meningitidis infection in complement-deficient and control human sera. Experiments were carried out in a high concentration of serum (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 anti-MASP-2 antibody

The following complement-deficient human sera and control human sera were 25 used in this experiment;

TABLE Π: Human sera samples tested (as shown in FIGURE 38) | Sample | Scram.................... ....._:.............1 j A i Normal human sera (NMSf + human anti-MASP-2 Ab ; B | NHS + L'Otype control Ab (c p

I E

MBL -/- human serum NHS

Heat-Inactivated (Hi) NHS

-1972018200437 19 Jan 2018

A recombinant antibody against human MASP-2 was isolated from a

Combinatorial Antibody Library (Knappik, A., et al., J. Mol, Biol. 296:51-86 (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 pathway-mediated activation of C4 and C3 in human plasma (IC50~20 nM) was identified and converted to a full-length human igG4 antibody.

N. meningitidis serogroup B-MC58 was incubated with the different sera show in TABLE 11, each at a serum concentration of 20%, with or without the addition of inhibitory human anti-MASP-2 antibody (3 pg in 100 μΐ total volume) at 37°C with shaking. Samples were taken at the following time points: 0-, 30-, 60- and 90-minute intervals, plated out and then viable counts were determined. Heat-inactivated human serum was used as a negative control.

Results:

FIGURE 38 graphically illustrates the log cft/raL of viable counts of N.

meningitidis serogroup B-MC58 recovered at different time points in the human sera samples shown in TABLE 11. TABLE 12 provides the Student’s t-test results for FIGURE 38.

TABLE 12: Student's t-test Results for FIGURE 38 (time point 60 minutes)

	Mean Diff. (Log)	Significant? P<0.05?	P value summary
A vs B	-0.3678	Yes	***(0.0002)
A vs C........	-1.1053	Yes	***(p<0.0001)
A vs D	-0.2111	Yes	**(0.0012)
C vs D	1.9	Yes	***(p<0.0001)

As shown in FIGURE 38 and TABLE 12, complement-dependent killing of A meningitidis in human 20% serum was significantly enhanced by the addition of the human anti-MASP-2 inhibitory antibody.

2. Complement-dependent killing of N. meningitidis in 20% (v/v) mouse sera 25 deficient of MASP-2.

-1982018200437 19 Jan 2018

The foilowing complement-deficient mouse sera and control mouse sera were used in this experiment:

TABLE 13: Mouse sera sample»» tested (as shown inFIGURE 39)

Sample	Serum Type
A	WT
B	MASP-2 -/-
c	MBL A/C -/-
D	WT' heat-inactivated (HIS)

A/ meningitidis serogroup B-MC58 was incubated with different complement5 deficient mouse sera, each at a serum concentration of 20%, at 37°C with shaking. Samples were taken at the following time points: 0-, 15-, 30-, 60-, 90- and 120-minute intervals, plated out and then viable counts were determined. Heat-inactivated human serum was used as a negative control.

Results:

FIGURE 39 graphically illustrates the log ciu/mL of viable counts of Ai meningitidis serogroup B-MC58 recovered at different time points in the mouse sera samples shown in TABLE 13, As shown in FIGURE 39, the MASP-2 -/- mouse sera have a higher level of bactericidal activity for N. meningitidis than WT mouse sera. The symbol ** indicates p=0,0058, the symbol *** indicates p=0.001, TABLE 14 provides the Student's t-test results for FIGURE 39.

TABLE 14: Student's t-test Results for FIGURE 39

Comparison	Time point	Meats Diff. (LOG)	Significant? (p<0.05)?	P value summary
A vs. B	60 min.	0.39	yes	** (0.0058)
A vs, B	90 min.	0.6741	yes	***(0.001)

In summary, the results in this Example demonstrate that MASP-2 -/- sera has a 20 higher level of bactericidal activity for A/ meningitidis than WT sera.

-199This Example demonstrates the inhibitory effect of MASP-2 deficiency on lysis of red blood cells from blood samples obtained from a mouse model of paroxysmal nocturnal hemoglobinuria (PNH).

2018200437 19 Jan 2018

Paroxysmal nocturnal hemoglobinuria (PNH), also referred to as MarchiafavaMicheli syndrome, is an acquired, potentially life-threatening disease of the blood, characterized by complement-induced intravascular hemolytic anemia. The hallmark of PNH is the chronic complement-mediated intravascular hemolysis that is a consequence of unregulated activation of the alternative pathway of complement 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 /75(11) (2010), Risitano, A.M, Mini-Reviews in Medicinal Chemistry, 11:528-535 (2011). Anemia in PNH is due to destruction of red blood cells in the bloodstream. Symptoms of PNH include red urine, due to appearance of hemoglobin in the urine, back pain, fatigue, shortness of breath and thrombosis, PNH may 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, anticoaguiation for thrombosis and the use of the monoclonal antibody eculizumab (Soliris®’), which protects blood cells against immune destruction by inhibiting the complement system (Hillmen P. et al., N.

Engl, J. Med. 350(6):552-9 (2004)). Eculizumab (Soliris®) is a humanized monoclonal antibody that targets the complement component C5, blocking its cleavage by C5 convertases, thereby preventing tbs production of C5a and the assembly of the MAC, Treatment of PNH patients with eculizumab has resulted in a reduction of intravascular hemolysis, as measured by lactate dehydrogenase (LDH), leading to hemoglobin stabilization and transfusion independence in about half of the patients (Hillmen P, et ah, Mini-Reviews in Medicinal Chemistry, vol 11(6) (2011)), While nearly all patients undergoing therapy with eculizumab achieve normal or almost normal LDH levels (due to control of intravascular hemolysis), only about one third of the patients reach a hemoglobin value above 1 lgr/dL, and the remaining patients on eculizumab continue to exhibit moderate to severe (/.<?.,transfusion-dependent) anemia, in about equal proportions (Risitano A.M, et ah, Blood 113:4094-100 (2009)). As described in Risitano et ah, MiniReviews in Medicinal Chemistry 11:528-535 (2011), it was demonstrated that PNH patients on eculizumab contained C3 fragments bound to a substantial portion of their

-2002018200437 19 Jan 2018

PNH erythrocytes (while untreated patients did not), leading to the conclusion that membrane-bound C3 fragments work as opsonins on PNH erythrocytes, resulting in their entrapment in the reticuloendothelia! cells through specific C3 receptors and subsequent extravascular hemolysis. Therefore, therapeutic strategies in addition to the use of eculizumab are needed for those patients developing C3 fragment-mediated extravascular hemolysis because they continue to require red cell transfusions.

This Example describes methods to assess the effect of MASP-2- deficient serum and serum treated with MASP-2 inhibitory agent on lysis of red blood cells from blood samples obtained from a mouse model of PNH and demonstrates the efficacy of MASP-2 inhibition to treat subjects suffering from PNH, and also supports the use of inhibitors of MASP-2 to ameliorate the effects of C3 fragment-mediated extravascular hemolysis in PNH subjects undergoing therapy with a C5 inhibitor such as eculizumab.

Methods:

PNH aalmai model:

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

In order to sensitize these erythrocytes even more, these cells were used with and without coating by mannan and then tested for hemolysis in WT C56/BL6 plasma, MBL null plasma, MASP-2 -/- plasma, human NHS, human MBL -/- plasma, and NHS treated with human anti-MASP-2 antibody.

/. Hemolysis assay ofCrry/C3 and CD55/CD59 double-deficient murine erythrocytes in

MASP-2-deficient/depleted sera and controls

Day 1, Preparation of murine RBC (A mannan coating)

Materials included: fresh mouse blood, BBS/Mg^2+/Ca²⁺ (4.4 mM barbituric acid,

1,8 mM sodium barbitone, 145 mM NaCl, pH7.4, 5mM Mg^2t, 5mM Ca²⁺), chromium

-2012018200437 19 Jan 2018 chloride, CrClrbHiO (0.5mg/mL in BBS/Mg2+/Ca2+) and mannan, 100 pg/mL in BBS /Mg2-b''Ca2+,

Whole blood (2m L) was spun down for 1-2 min at 2000xg in a refrigerated centrifuge at 4‘'C. The plasma and huffy coat were aspirated off. The sample was then washed three times by re-suspending the RBC pellet in 2 mL ice-cold BBS/gelatin/Mg2+/Ca2+ and repeating centrifugation step. After the third wash, the pellet was re-suspended In 4mL BBS/Mg2+/Ca2+. A 2 mL aliquot of the RBC was set aside as an uncoated control. To the remaining 2 mL, 2 ml. CrC13 and 2 mL mannan were added and the sample was incubated with gentle mixing at room temperature for 5 minutes. The reaction was terminated by adding 7.5mL BBS/gelatin/Mg2+/Ca2+, The sample was spun down as above, re-suspended in 2 mL BBS/gelatin/Mg2+/Ca2+ and washed a further two times as above, then stored at 4°C.

Day 2, Hemolysis assay

Materials included BBS/gelatin/Mg^z+/Ca²'^r (as above), test sera, 96-well round15 bottomed and flat-bottomed plates and a spectrophotometer that reads 96-well plates at 410-414 nm.

The concentration of the RBC was first determined and the ceils were adjusted to I0⁹/mL, and stored at this concentration. Before use, the assay buffer was diluted to 10^s/mL, and then lOOul per well was used. Hemolysis was measured at 430-414 nm (allowing for greater sensitivity then 541nm). Dilutions of test sera were prepared in icecold BBS/geiatin/Mg2+/Ca2+. 100μ1 of each serum dilution was pipetted into roundbottomed plate (see plate layout). 100μ1 of appropriately diluted RBC preparation was added (i.e., 10’ /mL) (see plate layout), incubated at 37°C for about 1 hour, and observed for lysss. (The plates may be photographed at this point.) The plate was then spun down at maximum speed for 5 minutes. ίΟΟμΙ was aspirated of the fluid-phase, transferred to flat-bottom plates, and the OD was recorded at 410-414 nm. The RBC pellets were retained (these can be subsequently lysed with water to obtain an inverse result).

Experiment #1:

-202

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Fresh blood was obtained from CD55/CD59 double-deficient mice and blood of

Crry/C3 double-deficient mice and erythrocytes were prepared as described in detail in the above protocol. 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 lx iO⁸ per mL, of which 100 μ! was used in the hemolysis assay, which was carried out as described above,

Results of Experiment #1; Thd lectin pathway is IftVfflycdJs crytfarpcvte fow in the PNH animal model

In an initial experiment, it was determined that non-coated WT mouse 10 erythrocytes were not lysed in any mouse serum. It was further determined that mannancoated Crry-/- mouse erythrocytes were slowly lysed (more than 3 hours at 37 degrees) in

WT mouse serum, but they were not lysed in MBL null serum, (Data not shown).

It was determined that mannan-coated Crry-/- mouse erythrocytes were rapidly lysed in human serum but not in beat-inactivated NHS. Importantly, mannan-coated

Crry-/- mouse erythrocytes were lysed in NHS diluted down to 1/640 (i.e., I/40, 1/80, 1/160, 1/320 and 1/640 dilutions all lysed). (Data not shown), in this dilution, the alternative pathway does not work (AP functional activity is significantly reduced below 8% serum concentration).

Cotttfuskffls frdtft Experiment #1

Mannan-coated Crry-/- mouse erythrocytes are very well lysed In highly diluted human serum with MBL but not in that without MBL. The efficient lysis in every serum concentration tested implies that the alternative pathway is not involved or needed for this lysis. The inability of MBL-deficient mouse serum and human serum to lyse the mannan-coated Crry-/- mouse erythrocytes indicates that the classical pathway also has nothing to do with the lysis observed. As lectin pathway recognition molecules are required (i.e., MBL), this lysis is mediated by the lectin pathway.

Experiment #2:

Fresh blood was obtained from the Crry/C3 and CD55/CD59 double-deficient mice and mannan-coated Crry-/- mouse erythrocytes were analyzed in the haemolysis assay as described above in the presence ofthe following human serum: MBL null; WT;

-2032018200437 19 Jan 2018

NHS pretreated with human anti-MASP-2 antibody; and heat-inactivated NHS as a control.

PNff animal piodel

With the Mannan-coated Crry-/- mouse erythrocytes, NHS was incubated in the dilutions diluted down to 1Z640 (i.e,, 1/40, 1/80, 1/160, 1/320 and 1/640), human MBL-Zserum, NHS pretreated with anti-MASP-2 mAh, and heat-inactivated NIIS as a control.

The ELISA microtiter plate was spun down and the non-lysed erythrocytes were collected on the bottom of the round-well plate. The supernatant of each well was collected and the amount of hemoglobin released from the lysed erythrocytes was measured by reading the OD415 nm in an ELISA reader.

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, Anti-MASP-2-antibody-pretreated NHS lysed mannan-coated mouse erythrocytes at 1/8 and 1/16 dilutions while WT human serum lysed mannan-coated mouse erythrocytes down to dilutions of 1/32.

FIGURE 40 graphically illustrates hemolysis (as measured by hemoglobin release of lysed mouse erythrocytes (Cryy/C3-/-) into the supernatant measured by photometry) of mannan-coated murine erythrocytes by human serum over a range of serum concentrations in serum from heat-inactivated (HI) NHS, MBL-/-, NHS pretreated with anti-MASP-2 antibody, and NHS control.

From the results shown in FIGURE 40, it is demonstrated that MASP-2 inhibition with anti-MASP-2 antibody significantly shifted the CHso and inhibited complementmediated lysis of sensitized erythrocytes with deficient protection from autologous complement activation.

Fresh blood obtained from the Crry/C3 and CD55/CD59 double-deficient mice in non-coated Crry-/- mouse erythrocytes was analyzed in the hemolysis assay as described

2042018200437 19 Jan 2018 above in the presence of the following serum: MBL -/-: WT sera; NHS pretreated with human anti-MASP-2 antibody and heat-inactivated NHS as a control,

Results:

FIGURE 41 graphically illustrates hemolysis (as measured by hemoglobin release 5 of lysed WT mouse erythrocytes into the supernatant measured by photometry) of noneoated murine erythrocytes by human serum over a range of serum concentrations in serum from heat inactivated (Hi) NHS, MBL-/-, NHS pretreated with anti-MASP-2 antibody, and NHS control. As shown in FIGURE 41, it is demonstrated that inhibiting

MASP-2 inhibits complement-mediated lysis of non-sensitized WT mouse erythrocytes, 10 FIGURE. 42 graphically illustrates hemolysis (as measured by hemoglobin release of lysed mouse erythrocytes (CD55/59 -/-) into the supernatant measured by photometry) of non-coated murine erythrocytes by human serum over a range of serum concentration in serum from heat-inactivated (HI) NHS, MBL-/-, NHS pretreated with anti-MASP-2 antibody, and NHS control.

TABLE 12: CHso values expressed as scrum concentrations

----------------— Scrum	........... ............................... WT	................................................. CD55/59 -/-
Heat-inactivated NHS	No lysis	No lysis
MBL AO/XX donor ; (MBL deficient)	7.2%	2.1%
ί NHS < anti-MASP-2 antibody	5.4%	1.5%
NHS	3.1%	0.73%

Note: “CH50” is the point at which complement mediated hemolysis reaches 50%,

In summary, the results in this Example demonstrate that inhibiting MASP-2 20 inhibits complement-mediated lysis of sensitized and non-sensitized erythrocytes with deficient protection from autologous complement activation. Therefore, MASP-2 inhibitors may be used to treat subjects suffering from PNH, and may also be used to ameliorate (/.e., inhibit, prevent or reduce the severity of) extravascular hemolysis in

PNH patients undergoing treatment with a C5 inhibitor such as eculizumab (Soliris®).

-2052018200437 19 Jan 2018

EXAMPLE 34

This Example describes a follow on study to the study described above in Example 29, providing further evidence confirming that a MASP-2 inhibitor, such as a

MASP-2 antibody, is effective for the treatment of radiation exposure and/or for the treatment, amelioration or prevention of acute radiation sy ndrome.

Rationale: In the initial study described in Example 29, it was demonstrated that preirradiation treatment with an anti-MASP-2 antibody In mice increased the survival of irradiated mice as compared to vehicle treated irradiated control animals at both 6,5 Gy and 7.0 Gy exposure levels, it was further demonstrated in Example 29 that at the 6.5 G) exposure level, post-irradiation treatment with anti-MASP-2 antibody resulted in a modest increase in survival as compared to vehicle control irradiated animals. This Example describes a second radiation study that was carried out to confirm the results of the first study.

Methods:

Design of Study A:

Swiss Webster mice (n=50) were exposed to ionizing radiation (8,0 Gy). The effect of anti-MASP-2 antibody therapy (mAbH6 5mg/kg), administered 18 hours before and 2 hours after radiation exposure, and weekly thereafter, on mortality was assessed.

Results of Study A:

As shown in FIGURE 43, it was determined that administration of the anti-MASP-2 antibody mAbH6 increased survival in mice exposed to 8.0 Gy, with an adjusted median survival rate increased from 4 to 6 days as compared to mice that received vehicle control, and a mortality reduced by 12% when compared to mice that received vehicle control (log-rank test, p-0.040).

Design of Study B:

Swiss Webster mice (n^:;;:50) were exposed to ionizing radiation (8.0 Gy) in the following groups (1: vehicle) saline control; (IE low) anti-MASP-2 antibody mAbH6 (5 mg/kg) administered 18 hours before irradiation and 2 hours after irradiation; (HE high) mAbH6 (10 mg/kg) administered 18 hours before irradiation and 2 hours post irradiation; and (lV:high post) mAbH6 (lOmg/kg) administered 2 hours post irradiation only.

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Administration of anti-MASP-2 antibody pre- and post-irradiation adjusted the mean survival from 4 to 5 days in comparison to animals that received vehicle control·

Mortality in the anti-MASP-2 antibody-treated mice was reduced by 6-12% in comparison to vehicle control mice. It is further noted that no significant detrimental treatment effects were observed (data not shown).

In summary, the results shown in this Example are consistent with the results shown in Example 29 and further demonstrate that anti-MASP-2 antibodies are effective in treating a mammalian subject at risk for, or suffering from the detrimental effects of acute radiation syndrome.

EXAMPLE 35

This study investigates the effect of MASP-2-deficiency in a mouse model of LPS (bpopo3ysaccharide)~induced thrombosis,

Rationale;

Hemolytic uremic syndrome (HUS), which is caused by Shiga toxin-producing E. eol: infection, is the leading cause of acute renal failure in children. In this Example, a Schwartzman model of LPS-induced thrombosis (microvascular coagulation) was carried out in MASP-2-/- (KO) mice to determine whether MASP-2 inhibition is effective to inhibit or prevent the formation of intravascular thrombi,

Methods:

MASP-2-/- (n=9) and WT (n=l 0) mice were analyzed in a Schwarztraan model of LPS-induced thrombosis (microvascular coagulation). Mice were administered Serratia LPS and thrombus formation was monitored over time. A comparison of the incidence of microthromi and LPS-induced microvascular coagulation was carried out.

Results:

Notably, all MASP-2 -/- mice tested (9/9) did not form intravascular thrombi after Serratia LPS administration. In contrast, microthrombi were detected in 7 of 10 of the WT mice tested in parallel (p=0.0031, Fischer’s exact). As shown in FIGURE 44, the time to onset of microvascular occlusion following LPS infection was measured in

-2072018200437 19 Jan 2018

MASP-2-/- and WT mice, showing the percentage of WT mice with thrombus formation measured over 60 minutes, with thrombus formation detected as early as about 15 minutes. Up to 80% of the WT mice demonstrated thrombus formation at 60 minutes, in contrast, as shown in FIGURE 44, none of the MASP-2 -/- had thrombus formation at 60 minutes (log rank: p=0.0005).

These results demonstrate that MASP-2 inhibition is protective against the development of intravascular thrombi in an HUS model.

EXAMPLE 36

This Example describes the effect of anti-MASP-2 antibodies in a mouse model of

HUS using intraperitoneal co-injection of puri fied Shiga toxin 2 (STX2) plus LPS,

Background:

A mouse model of HUS was developed using intraperitoneal co-injection of purified Shiga toxin 2 (STX2) plus LPS. Biochemical and microarray analysis of mouse kidneys revealed the STX2 plus LPS challenge to be distinct from the effects of either agent alone. Blood and serum analysis of these mice showed neutrophilia, thrombocytopenia, red cell hemolysis, and increased serum creatinine and blood urea nitrogen, in addition, histologic analysis and electron microscopy of mouse kidneys demonstrated glomerular fibrin deposition, red cell congestion, microthrombi formation, and glomerular ultrastructural changes. It was established that this mode! of HUS induces al! clinical symptoms of human HUS pathology in C57BL/6 mice including thrombocytopenia, hemolytic anemia, and renal failure that define the human disease. (J. Immunol 187( 1): 172-80 (2013))

Methods:

C57BL/6 female mice that weighed between 18 to 20 g were purchased from

Charles River Laboratories and divided in to 2 groups (5 mice in each group). One group of mice was pretreated by intraperitoneal (i.p.) injection with the recombinant antiMASP-2 antibody mAbMIl (100 gg per mouse; corresponding to a final concentration

-208

2018200437 19 Jan 2018 of 5 mg/kg body weight) diluted in a total volume of 150 μΐ saline. The control group received saline without arty antibody. Six hours after i.p injection of anti-MASP-2 antibody mAbMl 1, all mice received a combined i.p. injection of a sublethal dose (3 pg/'animai; corresponding to 150 pg/kg body weight) of LPS of Serralia tnarcescens (L6136; Sigma-Aldrich, St. Louis, MO) and a dose of 4.5 ng/animal (corresponding to

2.2,5 ng/kg) of STX2 (two times the LD50 dose) in a total volume of 150 μ|, Saline injection was used lor control.

Survival of the mice was monitored every 6 hours after dosing, Mice were culled as soon as they reached the lethargic stage of HUS pathology. After 36 hours, all mice were culled and both kidneys were removed for immunohistochemistry and scanning electron microscopy. Blood samples were taken at the end of the experiment by cardiac puncture. Serum was separated and kept frozen at -80°C for measuring BUN and serum Creatinine levels in both treated and control groups.

Immunohistochemistry

One-third of each mouse kidney was fixed in 4% paraformaldehyde lor 24 h, processed, and embedded in paraffin. Three-micron-thick sections were cut and placed onto charged slides for subsequent staining with Η & E stain.

Electron Microscopy

The middle section of the kidneys was cut into blocks of approximately 1 to 2 mm³, and fixed overnight at 4°C in 2.5% glutaraldehyde in lx PBS. The fixed tissue subsequently was processed by the University of Leicester Electron Microscopy Facility

Cryostat sections

The other third of the kidneys was, cut into blocks approximately 1 to 2 nun and snap frozen in liquid nitrogen and kept at ~80°C for cryostat sections and mRNA analysis.

Results:

-2092018200437 19 Jan 2018

FIGURE 45 graphically illustrates the percent survival of saline-treated control mice (o^;=5) and anti-MASP-2 antibody-treated mice (n==5) in the STX/LP-S-induced model over time (hours), Notably, as shown in FIGURE 45, all of the control mice died by 42 hours. In sharp contrast, 100 % of the anti-MASP-2 antibody-treated mice survived throughout the time course of the experiment, Consistent with the results shown in FIGURE 45, it was observed that all the untreated mice that either died or had to be culled with signs of severe disease had significant glomerular injuries, whiie the glomeruli of all anti-MASP-2-treated mice looked normal (data not shown). These results demonstrate that MASP-2 inhibitors, such as anti-MASP-2 antibodies, may be used to treat subjects suffering from, or at risk for developing a thrombotic microangiopathy (TMA), such as hemolytic uremic syndrome (HUS), atypical HUS (aHUS), or thrombotic thrombocytopenic purpura (TTP),

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

-2101001362562

2018200437 19 Jan 2018

Claims (25)

1. THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

   1. A method of inhibiting MASP-2 dependent complement activation in a subject suffering from glaucoma, comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory agent effective to inhibit MASP-2 dependent complement activation.

   5 2. The method of claim 1, wherein the MASP-2 inhibitory agent is an anti-MASP-2 antibody, or fragment thereof.

   3. The method of claim 2, wherein the MASP-2 inhibitory agent is an anti-MASP-2 monoclonal antibody, or fragment thereof.

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    Page 1

    2018200437 19 Jan 2018

    SEQUENCE LISTING <110> Demopulos, G.A.

    Dudler, T.

    Schwaeble, H.-W.

    <120> METHODS FOR TREATING CONDITIONS ASSOCIATED WITH MASP2 DEPENDENT

    COMPLEMENT ACTIVATION <130> MP.1.0126.PCT <140> PCT/US2012/032650 <141> 2012-04-06 <150> US 61/473,698 <151> 2011-04-08 <160> 65 <170> PatentIn version 3.5 <210> 1 <211> 725 <212> DNA <213> Homo sapiens <220>

    <221> CDS <222> (27)..(584) <400> 1 ggccaggcca gctggacggg cacacc atg agg ctg ctg acc ctc ctg ggc ctt 53 Met Arg Leu Leu Thr Leu Leu Gly Leu 1 5 ctg tgt ggc tcg gtg gcc acc ccc ttg ggc ccg aag tgg cct gaa cct 101 Leu Cys Gly Ser Val Ala Thr Pro Leu Gly Pro Lys Trp Pro Glu Pro 10 15 20 25 gtg ttc ggg cgc ctg gca tcc ccc ggc ttt cca ggg gag tat gcc aat 149 Val Phe Gly Arg Leu Ala Ser Pro Gly Phe Pro Gly Glu Tyr Ala Asn

    30 35 40 gac cag gag cgg cgc tgg acc ctg act gca ccc ccc ggc tac cgc ctg 197 Asp Gln Glu Arg Arg Trp Thr Leu Thr Ala Pro Pro Gly Tyr Arg Leu

    45 50 55 cgc ctc tac ttc acc cac ttc gac ctg gag ctc tcc cac ctc tgc gag 245

    Page 2

    2018200437 19 Jan 2018

    Arg Leu Tyr Phe Thr His Phe Asp Leu Glu Leu Ser His Leu Cys Glu 60 65 70 tac gac ttc gtc aag ctg agc tcg ggg gcc aag gtg ctg gcc acg ctg 293

    Tyr Asp Phe Val Lys Leu Ser Ser Gly Ala Lys Val Leu Ala Thr Leu

    75 80 85 tgc ggg cag gag agc aca gac acg gag cgg gcc cct ggc aag gac act 341 Cys Gly Gln Glu Ser Thr Asp Thr Glu Arg Ala Pro Gly Lys Asp Thr 90 95 100 105 ttc tac tcg ctg ggc tcc agc ctg gac att acc ttc cgc tcc gac tac 389 Phe Tyr Ser Leu Gly Ser Ser Leu Asp Ile Thr Phe Arg Ser Asp Tyr

    110 115 120 tcc aac gag aag ccg ttc acg ggg ttc gag gcc ttc tat gca gcc gag 437 Ser Asn Glu Lys Pro Phe Thr Gly Phe Glu Ala Phe Tyr Ala Ala Glu

    125 130 135 gac att gac gag tgc cag gtg gcc ccg gga gag gcg ccc acc tgc gac 485 Asp Ile Asp Glu Cys Gln Val Ala Pro Gly Glu Ala Pro Thr Cys Asp

    140 145 150 cac cac tgc cac aac cac ctg ggc ggt ttc tac tgc tcc tgc cgc gca 533 His His Cys His Asn His Leu Gly Gly Phe Tyr Cys Ser Cys Arg Ala

    155 160 165 ggc tac gtc ctg cac cgt aac aag cgc acc tgc tca gag cag agc ctc 581 Gly Tyr Val Leu His Arg Asn Lys Arg Thr Cys Ser Glu Gln Ser Leu 170 175 180 185 tag cctcccctgg agctccggcc tgcccagcag gtcagaagcc agagccagcc 634 tgctggcctc agctccgggt tgggctgaga tggctgtgcc ccaactccca ttcacccacc 694 atggacccaa taataaacct ggccccaccc c 725 <210> 2 <211> 185 <212> PRT <213> Homo sapiens <400> 2

    Met Arg Leu Leu Thr Leu Leu Gly Leu Leu Cys Gly Ser Val Ala Thr 1 5 10 15

    Pro Leu Gly Pro Lys Trp Pro Glu Pro Val Phe Gly Arg Leu Ala Ser

    Page 3

    2018200437 19 Jan 2018
20. 20 25 30

    Pro Gly Phe Pro Gly Glu Tyr Ala Asn Asp Gln Glu Arg Arg Trp Thr 35 40 45

    Leu Thr Ala Pro Pro Gly Tyr Arg Leu Arg Leu Tyr Phe Thr His Phe 50 55 60

    Asp Leu Glu Leu Ser His Leu Cys Glu Tyr Asp Phe Val Lys Leu Ser 65 70 75 80

    Ser Gly Ala Lys Val Leu Ala Thr Leu Cys Gly Gln Glu Ser Thr Asp 85 90 95

    Thr Glu Arg Ala Pro Gly Lys Asp Thr Phe Tyr Ser Leu Gly Ser Ser 100 105 110

    Leu Asp Ile Thr Phe Arg Ser Asp Tyr Ser Asn Glu Lys Pro Phe Thr 115 120 125

    Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp Ile Asp Glu Cys Gln Val 130 135 140

    Ala Pro Gly Glu Ala Pro Thr Cys Asp His His Cys His Asn His Leu 145 150 155 160

    Gly Gly Phe Tyr Cys Ser Cys Arg Ala Gly Tyr Val Leu His Arg Asn 165 170 175

    Lys Arg Thr Cys Ser Glu Gln Ser Leu 180 185 <210> 3 <211> 170 <212> PRT <213> Homo sapiens <400> 3

    Page 4

    2018200437 19 Jan 2018

    Thr Pro Leu Gly Pro Lys Trp Pro Glu Pro Val Phe Gly Arg Leu Ala 15 10 15

    Ser Pro Gly Phe Pro Gly Glu Tyr Ala Asn Asp Gln Glu Arg Arg Trp 20 25 30

    Thr Leu Thr Ala Pro Pro Gly Tyr Arg Leu Arg Leu Tyr Phe Thr His 35 40 45

    Phe Asp Leu Glu Leu Ser His Leu Cys Glu Tyr Asp Phe Val Lys Leu 50 55 60

    Ser Ser Gly Ala Lys Val Leu Ala Thr Leu Cys Gly Gln Glu Ser Thr 65 70 75 80

    Asp Thr Glu Arg Ala Pro Gly Lys Asp Thr Phe Tyr Ser Leu Gly Ser 85 90 95

    Ser Leu Asp Ile Thr Phe Arg Ser Asp Tyr Ser Asn Glu Lys Pro Phe 100 105 110

    Thr Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp Ile Asp Glu Cys Gln 115 120 125

    Val Ala Pro Gly Glu Ala Pro Thr Cys Asp His His Cys His Asn His 130 135 140

    Leu Gly Gly Phe Tyr Cys Ser Cys Arg Ala Gly Tyr Val Leu His Arg 145 150 155 160

    Asn Lys Arg Thr Cys Ser Glu Gln Ser Leu 165 170 <210> 4 <211> 2460 <212> DNA <213> Homo sapiens

    Page 5

    2018200437 19 Jan 2018 <220>

    <221 > CDS <222> (22)..(2082) <400> 4 ggccagctgg acgggcacac c atg agg ctg ctg acc etc ctg ggc ett ctg 51 Met Arg Leu Leu Thr Leu Leu Gly Leu Leu 1 5 10 tgt ggc teg gtg gee acc ccc ttg ggc ccg aag tgg cct gaa cct gtg 99 Cys Gly Ser Val Ala Thr Pro Leu Gly Pro Lys Trp Pro Glu Pro Val

    15 20 25 ttc ggg ege ctg gca tec ccc ggc ttt cca ggg gag tat gee aat gac 147 Phe Gly Arg Leu Ala Ser Pro Gly Phe Pro Gly Glu Tyr Ala Asn Asp

    30 35 40 cag gag egg ege tgg acc ctg act gca ccc ccc ggc tac ege ctg ege 195 Gln Glu Arg Arg Trp Thr Leu Thr Ala Pro Pro Gly Tyr Arg Leu Arg

    45 50 55 etc tac ttc acc cac ttc gac ctg gag etc tee cac etc tgc gag tac 243 Leu Tyr Phe Thr His Phe Asp Leu Glu Leu Ser His Leu Cys Glu Tyr

    60 65 70 gac ttc gtc aag ctg age teg ggg gee aag gtg ctg gee aeg ctg tgc 291 Asp Phe Val Lys Leu Ser Ser Gly Ala Lys Val Leu Ala Thr Leu Cys 75 80 85 90 ggg cag gag age aca gac aeg gag egg gee cct ggc aag gac act ttc 339 Gly Gln Glu Ser Thr Asp Thr Glu Arg Ala Pro Gly Lys Asp Thr Phe

    95 100 105 tac teg ctg ggc tee age ctg gac att acc ttc ege tee gac tac tee 387 Tyr Ser Leu Gly Ser Ser Leu Asp Ile Thr Phe Arg Ser Asp Tyr Ser

    110 115 120 aac gag aag ccg ttc aeg ggg ttc gag gee ttc tat gca gee gag gac 435 Asn Glu Lys Pro Phe Thr Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp

    125 130 135 att gac gag tgc cag gtg gee ccg gga gag geg ccc acc tgc gac cac 483 Ile Asp Glu Cys Gln Val Ala Pro Gly Glu Ala Pro Thr Cys Asp His

    140 145 150 cac tgc cac aac cac ctg ggc ggt ttc tac tgc tee tgc ege gca ggc 531 His Cys His Asn His Leu Gly Gly Phe Tyr Cys Ser Cys Arg Ala Gly 155 160 165 170 tac gtc ctg cac cgt aac aag ege acc tgc tea gee ctg tgc tee ggc 579

    Page 6

    2018200437 19 Jan 2018

    Tyr Val Leu His Arg Asn Lys Arg Thr Cys Ser Ala Leu Cys Ser Gly 175 180 185 cag gtc ttc acc cag agg tct ggg gag etc age age cct gaa tac cca 627 Gln Val Phe Thr Gln Arg Ser Gly Glu Leu Ser Ser Pro Glu Tyr Pro

    190 195 200 egg ccg tat ccc aaa etc tcc agt tgc act tac age ate age ctg gag 675 Arg Pro Tyr Pro Lys Leu Ser Ser Cys Thr Tyr Ser Ile Ser Leu Glu

    205 210 215 gag ggg ttc agt gtc att ctg gac ttt gtg gag tcc ttc gat gtg gag 723 Glu Gly Phe Ser Val Ile Leu Asp Phe Val Glu Ser Phe Asp Val Glu

    220 225 230 aca cac cct gaa acc ctg tgt ccc tac gac ttt etc aag att caa aca 771 Thr His Pro Glu Thr Leu Cys Pro Tyr Asp Phe Leu Lys Ile Gln Thr 235 240 245 250 gac aga gaa gaa cat ggc cca ttc tgt ggg aag aca ttg ccc cac agg 819 Asp Arg Glu Glu His Gly Pro Phe Cys Gly Lys Thr Leu Pro His Arg

    255 260 265 att gaa aca aaa age aac aeg gtg acc ate acc ttt gtc aca gat gaa 867 Ile Glu Thr Lys Ser Asn Thr Val Thr Ile Thr Phe Val Thr Asp Glu

    270 275 280 tea gga gac cac aca ggc tgg aag ate cac tac aeg age aca gcg cag 915 Ser Gly Asp His Thr Gly Trp Lys Ile His Tyr Thr Ser Thr Ala Gln

    285 290 295 cct tgc cct tat ccg atg gcg cca cct aat ggc cac gtt tea cct gtg 963 Pro Cys Pro Tyr Pro Met Ala Pro Pro Asn Gly His Val Ser Pro Val

    300 305 310 caa gcc aaa tac ate ctg aaa gac age ttc tcc ate ttt tgc gag act 1011 Gln Ala Lys Tyr Ile Leu Lys Asp Ser Phe Ser Ile Phe Cys Glu Thr 315 320 325 330 ggc tat gag ett ctg caa ggt cac ttg ccc ctg aaa tcc ttt act gca 1059 Gly Tyr Glu Leu Leu Gln Gly His Leu Pro Leu Lys Ser Phe Thr Ala

    335 340 345 gtt tgt cag aaa gat gga tct tgg gac egg cca atg ccc gcg tgc age 1107 Val Cys Gln Lys Asp Gly Ser Trp Asp Arg Pro Met Pro Ala Cys Ser

    350 355 360 att gtt gac tgt ggc cct cct gat gat eta ccc agt ggc ega gtg gag 1155 Ile Val Asp Cys Gly Pro Pro Asp Asp Leu Pro Ser Gly Arg Val Glu

    365 370 375

    Page 7

    2018200437 19 Jan 2018 tac ate aca ggt cct gga gtg acc acc tac aaa get gtg att cag tac 1203 Tyr Ile Thr Gly Pro Gly Val Thr Thr Tyr Lys Ala Val Ile Gln Tyr

    380 385 390 age tgt gaa gag acc ttc tac aca atg aaa gtg aat gat ggt aaa tat 1251 Ser Cys Glu Glu Thr Phe Tyr Thr Met Lys Val Asn Asp Gly Lys Tyr 395 400 405 410 gtg tgt gag get gat gga ttc tgg aeg age tee aaa gga gaa aaa tea 1299 Val Cys Glu Ala Asp Gly Phe Trp Thr Ser Ser Lys Gly Glu Lys Ser

    415 420 425 etc cca gtc tgt gag cct gtt tgt gga eta tea gcc ege aca aca gga 1347 Leu Pro Val Cys Glu Pro Val Cys Gly Leu Ser Ala Arg Thr Thr Gly

    430 435 440 ggg cgt ata tat gga ggg caa aag gca aaa cct ggt gat ttt cct tgg 1395 Gly Arg Ile Tyr Gly Gly Gln Lys Ala Lys Pro Gly Asp Phe Pro Trp

    445 450 455 caa gtc ctg ata tta ggt gga acc aca gca gca ggt gca ett tta tat 1443 Gln Val Leu Ile Leu Gly Gly Thr Thr Ala Ala Gly Ala Leu Leu Tyr

    460 465 470 gac aac tgg gtc eta aca get get cat gcc gtc tat gag caa aaa cat 1491 Asp Asn Trp Val Leu Thr Ala Ala His Ala Val Tyr Glu Gln Lys His 475 480 485 490 gat gca tee gcc ctg gac att ega atg ggc acc ctg aaa aga eta tea 1539 Asp Ala Ser Ala Leu Asp Ile Arg Met Gly Thr Leu Lys Arg Leu Ser

    495 500 505 cct cat tat aca caa gcc tgg tet gaa get gtt ttt ata cat gaa ggt 1587 Pro His Tyr Thr Gln Ala Trp Ser Glu Ala Val Phe Ile His Glu Gly

    510 515 520 tat act cat gat get ggc ttt gac aat gac ata gca ctg att aaa ttg 1635 Tyr Thr His Asp Ala Gly Phe Asp Asn Asp Ile Ala Leu Ile Lys Leu

    525 530 535 aat aac aaa gtt gta ate aat age aac ate aeg cct att tgt ctg cca 1683 Asn Asn Lys Val Val Ile Asn Ser Asn Ile Thr Pro Ile Cys Leu Pro

    540 545 550 aga aaa gaa get gaa tee ttt atg agg aca gat gac att gga act gca 1731 Arg Lys Glu Ala Glu Ser Phe Met Arg Thr Asp Asp Ile Gly Thr Ala 555 560 565 570 tet gga tgg gga tta acc caa agg ggt ttt ett get aga aat eta atg 1779

    Page 8

    2018200437 19 Jan 2018

    Ser Gly Trp Gly Leu Thr Gln Arg Gly Phe Leu Ala Arg Asn Leu Met 575 580 585 tat gtc gac ata ccg att gtt gac cat caa aaa tgt act get gca tat 1827 Tyr Val Asp Ile Pro Ile Val Asp His Gln Lys Cys Thr Ala Ala Tyr

    590 595 600 gaa aag cca ccc tat cca agg gga agt gta act get aac atg ctt tgt 1875 Glu Lys Pro Pro Tyr Pro Arg Gly Ser Val Thr Ala Asn Met Leu Cys

    605 610 615 get ggc tta gaa agt ggg ggc aag gac age tgc aga ggt gac age gga 1923 Ala Gly Leu Glu Ser Gly Gly Lys Asp Ser Cys Arg Gly Asp Ser Gly

    620 625 630 ggg gca ctg gtg ttt eta gat agt gaa aca gag agg tgg ttt gtg gga 1971 Gly Ala Leu Val Phe Leu Asp Ser Glu Thr Glu Arg Trp Phe Val Gly 635 640 645 650 gga ata gtg tec tgg ggt tec atg aat tgt ggg gaa gca ggt cag tat 2019 Gly Ile Val Ser Trp Gly Ser Met Asn Cys Gly Glu Ala Gly Gln Tyr

    655 660 665 gga gtc tac aca aaa gtt att aac tat att ccc tgg ate gag aac ata 2067 Gly Val Tyr Thr Lys Val Ile Asn Tyr Ile Pro Trp Ile Glu Asn Ile

    670 675 680 att agt gat ttt taa cttgcgtgtc tgcagtcaag gattcttcat ttttagaaat 2122 Ile Ser Asp Phe

    685 gcctgtgaag accttggcag cgacgtggct cgagaagcat tcatcattac tgtggacatg 2182 gcagttgttg ctccacccaa aaaaacagac tccaggtgag gctgctgtca tttctccact 2242 tgccagttta attccagcct tacccattga ctcaagggga cataaaccac gagagtgaca 2302 gtcatctttg cccacccagt gtaatgtcac tgctcaaatt acatttcatt accttaaaaa 2362 gccagtctct tttcatactg gctgttggca tttctgtaaa ctgcctgtcc atgctctttg 2422 tttttaaact tgttcttatt gaaaaaaaaa aaaaaaaa 2460 <210> 5 <211> 686 <212> PRT <213> Homo sapiens <400> 5

    Page 9

    2018200437 19 Jan 2018

    Met Arg Leu Leu Thr Leu Leu Gly Leu Leu Cys Gly Ser Val Ala Thr 15 10 15

    Pro Leu Gly Pro Lys Trp Pro Glu Pro Val Phe Gly Arg Leu Ala Ser 20 25 30

    Pro Gly Phe Pro Gly Glu Tyr Ala Asn Asp Gln Glu Arg Arg Trp Thr 35 40 45

    Leu Thr Ala Pro Pro Gly Tyr Arg Leu Arg Leu Tyr Phe Thr His Phe 50 55 60

    Asp Leu Glu Leu Ser His Leu Cys Glu Tyr Asp Phe Val Lys Leu Ser 65 70 75 80

    Ser Gly Ala Lys Val Leu Ala Thr Leu Cys Gly Gln Glu Ser Thr Asp 85 90 95

    Thr Glu Arg Ala Pro Gly Lys Asp Thr Phe Tyr Ser Leu Gly Ser Ser 100 105 110

    Leu Asp Ile Thr Phe Arg Ser Asp Tyr Ser Asn Glu Lys Pro Phe Thr 115 120 125

    Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp Ile Asp Glu Cys Gln Val 130 135 140

    Ala Pro Gly Glu Ala Pro Thr Cys Asp His His Cys His Asn His Leu 145 150 155 160

    Lys Arg Thr Cys Ser Ala Leu Cys Ser Gly Gln Val Phe Thr Gln Arg 180 185 190

    Ser Gly Glu Leu Ser Ser Pro Glu Tyr Pro Arg Pro Tyr Pro Lys Leu

    Gly Gly Phe Tyr Cys Ser Cys Arg Ala Gly Tyr Val Leu His Arg Asn

    165 170 175

    Page 10

    2018200437 19 Jan 2018

    195 200 205

    Ser Ser Cys Thr Tyr Ser Ile Ser Leu Glu Glu Gly Phe Ser Val Ile 210 215 220

    Leu Asp Phe Val Glu Ser Phe Asp Val Glu Thr His Pro Glu Thr Leu 225 230 235 240

    Cys Pro Tyr Asp Phe Leu Lys Ile Gln Thr Asp Arg Glu Glu His Gly 245 250 255

    Pro Phe Cys Gly Lys Thr Leu Pro His Arg Ile Glu Thr Lys Ser Asn 260 265 270

    Thr Val Thr Ile Thr Phe Val Thr Asp Glu Ser Gly Asp His Thr Gly 275 280 285

    Trp Lys Ile His Tyr Thr Ser Thr Ala Gln Pro Cys Pro Tyr Pro Met 290 295 300

    Ala Pro Pro Asn Gly His Val Ser Pro Val Gln Ala Lys Tyr Ile Leu 305 310 315 320

    Lys Asp Ser Phe Ser Ile Phe Cys Glu Thr Gly Tyr Glu Leu Leu Gln 325 330 335

    Gly His Leu Pro Leu Lys Ser Phe Thr Ala Val Cys Gln Lys Asp Gly 340 345 350

    Ser Trp Asp Arg Pro Met Pro Ala Cys Ser Ile Val Asp Cys Gly Pro 355 360 365

    Pro Asp Asp Leu Pro Ser Gly Arg Val Glu Tyr Ile Thr Gly Pro Gly 370 375 380

    Val Thr Thr Tyr Lys Ala Val Ile Gln Tyr Ser Cys Glu Glu Thr Phe 385 390 395 400

    Page 11

    2018200437 19 Jan 2018

    Tyr Thr Met Lys Val Asn Asp Gly Lys Tyr Val Cys Glu Ala Asp Gly 405 410 415

    Phe Trp Thr Ser Ser Lys Gly Glu Lys Ser Leu Pro Val Cys Glu Pro 420 425 430

    Val Cys Gly Leu Ser Ala Arg Thr Thr Gly Gly Arg Ile Tyr Gly Gly 435 440 445

    Gln Lys Ala Lys Pro Gly Asp Phe Pro Trp Gln Val Leu Ile Leu Gly 450 455 460

    Gly Thr Thr Ala Ala Gly Ala Leu Leu Tyr Asp Asn Trp Val Leu Thr 465 470 475 480

    Ala Ala His Ala Val Tyr Glu Gln Lys His Asp Ala Ser Ala Leu Asp 485 490 495

    Ile Arg Met Gly Thr Leu Lys Arg Leu Ser Pro His Tyr Thr Gln Ala 500 505 510

    Trp Ser Glu Ala Val Phe Ile His Glu Gly Tyr Thr His Asp Ala Gly 515 520 525

    Phe Asp Asn Asp Ile Ala Leu Ile Lys Leu Asn Asn Lys Val Val Ile 530 535 540

    Asn Ser Asn Ile Thr Pro Ile Cys Leu Pro Arg Lys Glu Ala Glu Ser 545 550 555 560

    Gln Arg Gly Phe Leu Ala Arg Asn Leu Met Tyr Val Asp Ile Pro Ile 580 585 590

    Val Asp His Gln Lys Cys Thr Ala Ala Tyr Glu Lys Pro Pro Tyr Pro

    Phe Met Arg Thr Asp Asp Ile Gly Thr Ala Ser Gly Trp Gly Leu Thr

    565 570 575

    Page 12

    2018200437 19 Jan 2018

    595 600 605

    Arg Gly Ser Val Thr Ala Asn Met Leu Cys Ala Gly Leu Glu Ser Gly 610 615 620

    Gly Lys Asp Ser Cys Arg Gly Asp Ser Gly Gly Ala Leu Val Phe Leu 625 630 635 640

    Asp Ser Glu Thr Glu Arg Trp Phe Val Gly Gly Ile Val Ser Trp Gly 645 650 655

    Ser Met Asn Cys Gly Glu Ala Gly Gln Tyr Gly Val Tyr Thr Lys Val 660 665 670

    Ile Asn Tyr Ile Pro Trp Ile Glu Asn Ile Ile Ser Asp Phe 675 680 685 <210> 6 <211> 671 <212> PRT <213> Homo sapiens <400> 6

    Thr Pro Leu Gly Pro Lys Trp Pro Glu Pro Val Phe Gly Arg Leu Ala 15 10 15

    Ser Pro Gly Phe Pro Gly Glu Tyr Ala Asn Asp Gln Glu Arg Arg Trp 20 25 30

    Thr Leu Thr Ala Pro Pro Gly Tyr Arg Leu Arg Leu Tyr Phe Thr His 35 40 45

    Ser Ser Gly Ala Lys Val Leu Ala Thr Leu Cys Gly Gln Glu Ser Thr 65 70 75 80

    Phe Asp Leu Glu Leu Ser His Leu Cys Glu Tyr Asp Phe Val Lys Leu

    50 55 60

    Page 13

    2018200437 19 Jan 2018

    Asp Thr Glu Arg Ala Pro Gly Lys Asp Thr Phe Tyr Ser Leu Gly Ser 85 90 95

    Ser Leu Asp Ile Thr Phe Arg Ser Asp Tyr Ser Asn Glu Lys Pro Phe 100 105 110

    Thr Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp Ile Asp Glu Cys Gln 115 120 125

    Val Ala Pro Gly Glu Ala Pro Thr Cys Asp His His Cys His Asn His 130 135 140

    Leu Gly Gly Phe Tyr Cys Ser Cys Arg Ala Gly Tyr Val Leu His Arg 145 150 155 160

    Asn Lys Arg Thr Cys Ser Ala Leu Cys Ser Gly Gln Val Phe Thr Gln 165 170 175

    Arg Ser Gly Glu Leu Ser Ser Pro Glu Tyr Pro Arg Pro Tyr Pro Lys 180 185 190

    Leu Ser Ser Cys Thr Tyr Ser Ile Ser Leu Glu Glu Gly Phe Ser Val 195 200 205

    Ile Leu Asp Phe Val Glu Ser Phe Asp Val Glu Thr His Pro Glu Thr 210 215 220

    Leu Cys Pro Tyr Asp Phe Leu Lys Ile Gln Thr Asp Arg Glu Glu His 225 230 235 240

    Gly Pro Phe Cys Gly Lys Thr Leu Pro His Arg Ile Glu Thr Lys Ser 245 250 255

    Asn Thr Val Thr Ile Thr Phe Val Thr Asp Glu Ser Gly Asp His Thr 260 265 270

    Gly Trp Lys Ile His Tyr Thr Ser Thr Ala Gln Pro Cys Pro Tyr Pro 275 280 285

    Page 14

    2018200437 19 Jan 2018

    Met Ala Pro Pro Asn Gly His Val Ser Pro Val Gln Ala Lys Tyr Ile 290 295 300

    Leu Lys Asp Ser Phe Ser Ile Phe Cys Glu Thr Gly Tyr Glu Leu Leu 305 310 315 320

    Gln Gly His Leu Pro Leu Lys Ser Phe Thr Ala Val Cys Gln Lys Asp 325 330 335

    Gly Ser Trp Asp Arg Pro Met Pro Ala Cys Ser Ile Val Asp Cys Gly 340 345 350

    Pro Pro Asp Asp Leu Pro Ser Gly Arg Val Glu Tyr Ile Thr Gly Pro 355 360 365

    Gly Val Thr Thr Tyr Lys Ala Val Ile Gln Tyr Ser Cys Glu Glu Thr 370 375 380

    Phe Tyr Thr Met Lys Val Asn Asp Gly Lys Tyr Val Cys Glu Ala Asp 385 390 395 400

    Gly Phe Trp Thr Ser Ser Lys Gly Glu Lys Ser Leu Pro Val Cys Glu 405 410 415

    Pro Val Cys Gly Leu Ser Ala Arg Thr Thr Gly Gly Arg Ile Tyr Gly 420 425 430

    Gly Gln Lys Ala Lys Pro Gly Asp Phe Pro Trp Gln Val Leu Ile Leu 435 440 445

    Thr Ala Ala His Ala Val Tyr Glu Gln Lys His Asp Ala Ser Ala Leu 465 470 475 480

    Gly Gly Thr Thr Ala Ala Gly Ala Leu Leu Tyr Asp Asn Trp Val Leu

    450 455 460

    Page 15

    2018200437 19 Jan 2018

    Asp Ile Arg Met Gly Thr Leu Lys Arg Leu Ser Pro His Tyr Thr Gln 485 490 495

    Ala Trp Ser Glu Ala Val Phe Ile His Glu Gly Tyr Thr His Asp Ala 500 505 510

    Gly Phe Asp Asn Asp Ile Ala Leu Ile Lys Leu Asn Asn Lys Val Val 515 520 525

    Ile Asn Ser Asn Ile Thr Pro Ile Cys Leu Pro Arg Lys Glu Ala Glu 530 535 540

    Ser Phe Met Arg Thr Asp Asp Ile Gly Thr Ala Ser Gly Trp Gly Leu 545 550 555 560

    Thr Gln Arg Gly Phe Leu Ala Arg Asn Leu Met Tyr Val Asp Ile Pro 565 570 575

    Ile Val Asp His Gln Lys Cys Thr Ala Ala Tyr Glu Lys Pro Pro Tyr 580 585 590

    Pro Arg Gly Ser Val Thr Ala Asn Met Leu Cys Ala Gly Leu Glu Ser 595 600 605

    Gly Gly Lys Asp Ser Cys Arg Gly Asp Ser Gly Gly Ala Leu Val Phe 610 615 620

    Leu Asp Ser Glu Thr Glu Arg Trp Phe Val Gly Gly Ile Val Ser Trp 625 630 635 640

    Gly Ser Met Asn Cys Gly Glu Ala Gly Gln Tyr Gly Val Tyr Thr Lys 645 650 655

    Val Ile Asn Tyr Ile Pro Trp Ile Glu Asn Ile Ile Ser Asp Phe 660 665 670 <210> 7 <211> 4900

    Page 16

    2018200437 19 Jan 2018 <212> DNA <213> Homo sapiens <400> 7 cctgtcctgc ctgcctggaa ctctgagcag gctggagtca tggagtcgat tcccagaatc 60 ccagagtcag ggaggctggg ggcaggggca ggtcactgga caaacagatc aaaggtgaga 120 ccagcgtagg actgcagacc aggccaggcc agctggacgg gcacaccatg aggtaggtgg 180 gcgccacagc ctccctgcag ggtgtggggt gggagcacag gcctgggcct caccgcccct 240 gccctgccca taggctgctg accctcctgg gccttctgtg tggctcggtg gccaccccct 300 taggcccgaa gtggcctgaa cctgtgttcg ggcgcctggc atcccccggc tttccagggg 360 agtatgccaa tgaccaggag cggcgctgga ccctgactgc accccccggc taccgcctgc 420 gcctctactt cacccacttc gacctggagc tctcccacct ctgcgagtac gacttcgtca 480 aggtgccgtc agacgggagg gctggggttt ctcagggtcg gggggtcccc aaggagtagc 540 cagggttcag ggacacctgg gagcaggggc caggcttggc caggagggag atcaggcctg 600 ggtcttgcct tcactccctg tgacacctga ccccacagct gagctcgggg gccaaggtgc 660 tggccacgct gtgcgggcag gagagcacag acacggagcg ggcccctggc aaggacactt 720 tctactcgct gggctccagc ctggacatta ccttccgctc cgactactcc aacgagaagc 780 cgttcacggg gttcgaggcc ttctatgcag ccgagggtga gccaagaggg gtcctgcaac 840 atctcagtct gcgcagctgg ctgtgggggt aactctgtct taggccaggc agccctgcct 900 tcagtttccc cacctttccc agggcagggg agaggcctct ggcctgacat catccacaat 960 gcaaagacca aaacagccgt gacctccatt cacatgggct gagtgccaac tctgagccag 1020 ggatctgagg acagcatcgc ctcaagtgac gcagggactg gccgggcgcg gcagctcacg 1080 cctgtaattc cagcactttg ggaggccgag gctggcttga taatttgagg gtcaggagtt 1140 caaggccagc cagggcaaca cggtgaaact ctatctccac taaaactaca aaaattagct 1200 gggcgtggtg gtgcgcacct ggaatcccag ctactaggga ggctgaggca ggagaattgc 1260 ttgaacctgc gaggtggagg ctgcagtgaa cagagattgc accactacac tccacctggg 1320 cgacagacta gactccgtct caaaaaacaa aaaacaaaaa ccacgcaggg ccgagggccc 1380

    Page 17

    2018200437 19 Jan 2018 atttacaagc tgacaaagtg ggccctgcca gcgggagcgc tgcaggatgt ttgattttca 1440 gatcccagtc cctgcagaga ccaactgtgt gacctctggc aagtggctca atttctctgc 1500 tccttagaag ctgctgcaag ggttcagcgc tgtagccccg ccccctgggt ttgattgact 1560 cccctcatta gctgggtgac ctcggccgga cactgaaact cccactggtt taacagaggt 1620 gatgtttgca tctttctccc agcgctgctg ggagcttgca gcgaccctag gcctgtaagg 1680 tgattggccc ggcaccagtc ccgcacccta gacaggacct aggcctcctc tgaggtccac 1740 tctgaggtca tggatctcct gggaggagtc caggctggat cccgcctctt tccctcctga 1800 cggcctgcct ggccctgcct ctcccccaga cattgacgag tgccaggtgg ccccgggaga 1860 ggcgcccacc tgcgaccacc actgccacaa ccacctgggc ggtttctact gctcctgccg 1920 cgcaggctac gtcctgcacc gtaacaagcg cacctgctca ggtgagggag gctgcctggg 1980 ccccaacgca ccctctcctg ggatacccgg ggctcctcag ggccattgct gctctgccca 2040 ggggtgcgga gggcctgggc ctggacactg ggtgcttcta ggccctgctg cctccagctc 2100 cccttctcag ccctgcttcc cctctcagca gccaggctca tcagtgccac cctgccctag 2160 cactgagact aattctaaca tcccactgtg tacctggttc cacctgggct ctgggaaccc 2220 ctcatgtagc cacgggagag tcggggtatc taccctcgtt ccttggactg ggttcctgtt 2280 ccctgcactg ggggacgggc cagtgctctg gggcgtgggc agccccaccc tgtggcgctg 2340 accctgctcc cccgactcgg tttctcctct cggggtctct ccttgcctct ctgatctctc 2400 ttccagagca gagcctctag cctcccctgg agctccggct gcccagcagg tcagaagcca 2460 gagccaggct gctggcctca gctccgggtt gggctgagat gctgtgcccc aactcccatt 2520 cacccaccat ggacccaata ataaacctgg ccccacccca cctgctgccg cgtgtctctg 2580 gggtgggagg gtcgggaggc ggtggggcgc gctcctctct gcctaccctc ctcacagcct 2640 catgaacccc aggtctgtgg gagcctcctc catggggcca cacggtcctt ggcctcaccc 2700 cctgttttga agatggggca ctgaggccgg agaggggtaa ggcctcgctc gagtccaggt 2760 ccccagaggc tgagcccaga gtaatcttga accaccccca ttcagggtct ggcctggagg 2820 agcctgaccc acagaggaga caccctggga gatattcatt gaggggtaat ctggtccccc 2880

    Page 18

    2018200437 19 Jan 2018 gcaaatccag gggtgattcc cactgcccca taggcacagc cacgtggaag aaggcaggca 2940 atgttggggc tcctcacttc ctagaggcct cacaactcaa atgcccccca ctgcagctgg 3000 gggtggggtg gtggtatggg atggggacca agccttcctt gaaggataga gcccagccca 3060 acaccccgcc ccgtggcagc agcatcacgt gttccagcga ggaaggagag caccagactc 3120 agtcatgatc actgttgcct tgaacttcca agaacagccc cagggcaagg gtcaaaacag 3180 gggaaagggg gtgatgagag atccttcttc cggatgttcc tccaggaacc agggggctgg 3240 ctggtcttgg ctgggttcgg gtaggagacc catgatgaat aaacttggga atcactgggg 3300 tggctgtaag ggaatttagg ggagctccga aggggccctt aggctcgagg agatgctcct 3360 ctcttttccc gaattcccag ggacccagga gagtgtccct tcttcctctt cctgtgtgtc 3420 catccacccc cgccccccgc cctggcagag ctggtggaac tcagtgctct agcccctacc 3480 ctggggttgc gactctggct caggacacca ccacgctccc tgggggtgtg agtgagggcc 3540 tgtgcgctcc atcccgagtg ctgcctgttt cagctaaagc ctcaaagcaa gagaaacccc 3600 ctctctaagc ggcccctcag ccatcgggtg ggtcgtttgg tttctgggta ggcctcaggg 3660 gctggccacc tgcagggccc agcccaaccc agggatgcag atgtcccagc cacatccctg 3720 tcccagtttc ctgctcccca aggcatccac cctgctgttg gtgcgagggc tgatagaggg 3780 cacgccaagt cactcccctg cccttccctc cttccagccc tgtgctccgg ccaggtcttc 3840 acccagaggt ctggggagct cagcagccct gaatacccac ggccgtatcc caaactctcc 3900 agttgcactt acagcatcag cctggaggag gggttcagtg tcattctgga ctttgtggag 3960 tccttcgatg tggagacaca ccctgaaacc ctgtgtccct acgactttct caaggtctgg 4020 ctcctgggcc cctcatcttg tcccagatcc tcccccttca gcccagctgc accccctact 4080 tcctgcagca tggcccccac cacgttcccg tcaccctcgg tgaccccacc tcttcaggtg 4140 ctctatggag gtcaaggctg gggcttcgag tacaagtgtg ggaggcagag tggggagggg 4200 caccccaatc catggcctgg gttggcctca ttggctgtcc ctgaaatgct gaggaggtgg 4260 gttacttccc tccgcccagg ccagacccag gcagctgctc cccagctttc atgagcttct 4320 ttctcagatt caaacagaca gagaagaaca tggcccattc tgtgggaaga cattgcccca 4380

    Page 19

    2018200437 19 Jan 2018 caggattgaa acaaaaagca acacggtgac catcaccttt gtcacagatg aatcaggaga 4440 ccacacaggc tggaagatcc actacacgag cacagtgagc aagtgggctc agatccttgg 4500 tggaagcgca gagctgcctc tctctggagt gcaaggagct gtagagtgta gggctcttct 4560 gggcaggact aggaagggac accaggttta gtggtgctga ggtctgaggc agcagcttct 4620 aaggggaagc acccgtgccc tcctcagcag cacccagcat cttcaccact cattcttcaa 4680 ccacccattc acccatcact catcttttac ccacccaccc tttgccactc atccttctgt 4740 ccctcatcct tccaaccatt catcaatcac ccacccatcc atcctttgcc acacaaccat 4800 ccacccattc ttctacctac ccatcctatc catccatcct tctatcagca tccttctacc 4860 acccatcctt cgttcggtca tccatcatca tccatccatc 4900 <210> 8 <211> 136 <212> PRT <213> Homo sapiens <400> 8

    Met Arg Leu Leu Thr Leu Leu Gly Leu Leu Cys Gly Ser Val Ala Thr 15 10 15

    Pro Leu Gly Pro Lys Trp Pro Glu Pro Val Phe Gly Arg Leu Ala Ser 20 25 30

    Pro Gly Phe Pro Gly Glu Tyr Ala Asn Asp Gln Glu Arg Arg Trp Thr 35 40 45

    Leu Thr Ala Pro Pro Gly Tyr Arg Leu Arg Leu Tyr Phe Thr His Phe 50 55 60

    Ser Gly Ala Lys Val Leu Ala Thr Leu Cys Gly Gln Glu Ser Thr Asp 85 90 95

    Asp Leu Glu Leu Ser His Leu Cys Glu Tyr Asp Phe Val Lys Leu Ser

    65 70 75 80

    Page 20

    2018200437 19 Jan 2018

    Thr Glu Arg Ala Pro Gly Lys Asp Thr Phe Tyr Ser Leu Gly Ser Ser 100 105 110

    Leu Asp Ile Thr Phe Arg Ser Asp Tyr Ser Asn Glu Lys Pro Phe Thr 115 120 125

    Gly Phe Glu Ala Phe Tyr Ala Ala 130 135 <210> 9 <211> 181 <212> PRT <213> Homo sapiens <400> 9

    Met Arg Leu Leu Thr Leu Leu Gly Leu Leu Cys Gly Ser Val Ala Thr 15 10 15

    Pro Leu Gly Pro Lys Trp Pro Glu Pro Val Phe Gly Arg Leu Ala Ser 20 25 30

    Pro Gly Phe Pro Gly Glu Tyr Ala Asn Asp Gln Glu Arg Arg Trp Thr 35 40 45

    Leu Thr Ala Pro Pro Gly Tyr Arg Leu Arg Leu Tyr Phe Thr His Phe 50 55 60

    Asp Leu Glu Leu Ser His Leu Cys Glu Tyr Asp Phe Val Lys Leu Ser 65 70 75 80

    Ser Gly Ala Lys Val Leu Ala Thr Leu Cys Gly Gln Glu Ser Thr Asp 85 90 95

    Thr Glu Arg Ala Pro Gly Lys Asp Thr Phe Tyr Ser Leu Gly Ser Ser 100 105 110

    Leu Asp Ile Thr Phe Arg Ser Asp Tyr Ser Asn Glu Lys Pro Phe Thr 115 120 125

    Page
21. 21

    2018200437 19 Jan 2018

    Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp Ile Asp Glu Cys Gln Val 130 135 140

    Ala Pro Gly Glu Ala Pro Thr Cys Asp His His Cys His Asn His Leu 145 150 155 160

    Gly Gly Phe Tyr Cys Ser Cys Arg Ala Gly Tyr Val Leu His Arg Asn 165 170 175

    Lys Arg Thr Cys Ser 180 <210> 10 <211> 293 <212> PRT <213> Homo sapiens <400> 10

    Met Arg Leu Leu Thr Leu Leu Gly Leu Leu Cys Gly Ser Val Ala Thr 15 10 15

    Pro Leu Gly Pro Lys Trp Pro Glu Pro Val Phe Gly Arg Leu Ala Ser 20 25 30

    Pro Gly Phe Pro Gly Glu Tyr Ala Asn Asp Gln Glu Arg Arg Trp Thr 35 40 45

    Leu Thr Ala Pro Pro Gly Tyr Arg Leu Arg Leu Tyr Phe Thr His Phe 50 55 60

    Asp Leu Glu Leu Ser His Leu Cys Glu Tyr Asp Phe Val Lys Leu Ser 65 70 75 80

    Ser Gly Ala Lys Val Leu Ala Thr Leu Cys Gly Gln Glu Ser Thr Asp 85 90 95

    Thr Glu Arg Ala Pro Gly Lys Asp Thr Phe Tyr Ser Leu Gly Ser Ser 100 105 110

    Page
22. 22

    2018200437 19 Jan 2018

    Leu Asp Ile Thr Phe Arg Ser Asp Tyr Ser Asn Glu Lys Pro Phe Thr 115 120 125

    Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp Ile Asp Glu Cys Gln Val 130 135 140

    Ala Pro Gly Glu Ala Pro Thr Cys Asp His His Cys His Asn His Leu 145 150 155 160

    Gly Gly Phe Tyr Cys Ser Cys Arg Ala Gly Tyr Val Leu His Arg Asn 165 170 175

    Lys Arg Thr Cys Ser Ala Leu Cys Ser Gly Gln Val Phe Thr Gln Arg 180 185 190

    Ser Gly Glu Leu Ser Ser Pro Glu Tyr Pro Arg Pro Tyr Pro Lys Leu 195 200 205

    Ser Ser Cys Thr Tyr Ser Ile Ser Leu Glu Glu Gly Phe Ser Val Ile 210 215 220

    Leu Asp Phe Val Glu Ser Phe Asp Val Glu Thr His Pro Glu Thr Leu 225 230 235 240

    Cys Pro Tyr Asp Phe Leu Lys Ile Gln Thr Asp Arg Glu Glu His Gly 245 250 255

    Pro Phe Cys Gly Lys Thr Leu Pro His Arg Ile Glu Thr Lys Ser Asn 260 265 270

    Trp Lys Ile His Tyr 290

    Thr Val Thr Ile Thr Phe Val Thr Asp Glu Ser Gly Asp His Thr Gly

    275 280 285

    Page
23. 23

    2018200437 19 Jan 2018 <210> 11 <211> 41 <212> PRT <213> Homo sapiens <400> 11

    Glu Asp Ile Asp Glu Cys Gln Val Ala Pro Gly Glu Ala Pro Thr Cys 15 10 15

    Asp His His Cys His Asn His Leu Gly Gly Phe Tyr Cys Ser Cys Arg 20 25 30

    Ala Gly Tyr Val Leu His Arg Asn Lys 35 40 <210> 12 <211> 242 <212> PRT <213> Homo sapiens <400> 12

    Ile Tyr Gly Gly Gln Lys Ala Lys Pro Gly Asp Phe Pro Trp Gln Val 15 10 15

    Leu Ile Leu Gly Gly Thr Thr Ala Ala Gly Ala Leu Leu Tyr Asp Asn 20 25 30

    Trp Val Leu Thr Ala Ala His Ala Val Tyr Glu Gln Lys His Asp Ala 35 40 45

    Ser Ala Leu Asp Ile Arg Met Gly Thr Leu Lys Arg Leu Ser Pro His 50 55 60

    His Asp Ala Gly Phe Asp Asn Asp Ile Ala Leu Ile Lys Leu Asn Asn 85 90 95

    Tyr Thr Gln Ala Trp Ser Glu Ala Val Phe Ile His Glu Gly Tyr Thr

    65 70 75 80

    Page
24. 24

    2018200437 19 Jan 2018

    Lys Val Val Ile Asn Ser Asn Ile Thr Pro Ile Cys Leu Pro Arg Lys 100 105 110

    Glu Ala Glu Ser Phe Met Arg Thr Asp Asp Ile Gly Thr Ala Ser Gly 115 120 125

    Trp Gly Leu Thr Gln Arg Gly Phe Leu Ala Arg Asn Leu Met Tyr Val 130 135 140

    Asp Ile Pro Ile Val Asp His Gln Lys Cys Thr Ala Ala Tyr Glu Lys 145 150 155 160

    Pro Pro Tyr Pro Arg Gly Ser Val Thr Ala Asn Met Leu Cys Ala Gly 165 170 175

    Leu Glu Ser Gly Gly Lys Asp Ser Cys Arg Gly Asp Ser Gly Gly Ala 180 185 190

    Leu Val Phe Leu Asp Ser Glu Thr Glu Arg Trp Phe Val Gly Gly Ile 195 200 205

    Val Ser Trp Gly Ser Met Asn Cys Gly Glu Ala Gly Gln Tyr Gly Val 210 215 220

    Tyr Thr Lys Val Ile Asn Tyr Ile Pro Trp Ile Glu Asn Ile Ile Ser 225 230 235 240

    Asp Phe <210> 13 <211> 16 <212> PRT <213> Artificial Sequence <220>

    <223> Synthetic <400> 13

    Page 25

    2018200437 19 Jan 2018

    Gly Lys Asp Ser Cys Arg Gly Asp Ala Gly Gly Ala Leu Val Phe Leu 15 10 15 <210> 14 <211> 15 <212> PRT <213> Artificial Sequence <220>

    <223> Synthetic <400> 14

    Thr Pro Leu Gly Pro Lys Trp Pro Glu Pro Val Phe Gly Arg Leu 15 10 15 <210> 15 <211> 43 <212> PRT <213> Artificial Sequence <220>

    <223> Synthetic <400> 15

    Thr Ala Pro Pro Gly Tyr Arg Leu Arg Leu Tyr Phe Thr His Phe Asp 15 10 15

    Leu Glu Leu Ser His Leu Cys Glu Tyr Asp Phe Val Lys Leu Ser Ser 20 25 30

    Gly Ala Lys Val Leu Ala Thr Leu Cys Gly Gln 35 40 <210> 16 <211> 8 <212> PRT <213> Artificial Sequence <220>

    <223> Synthetic <400> 16

    Page 26

    2018200437 19 Jan 2018

    Thr Phe Arg Ser Asp Tyr Ser Asn 1 5 <210> 17 <211> 25 <212> PRT <213> Artificial Sequence <220>

    <223> Synthetic <400> 17

    Phe Tyr Ser Leu Gly Ser Ser Leu Asp Ile Thr Phe Arg Ser Asp Tyr 15 10 15

    Ser Asn Glu Lys Pro Phe Thr Gly Phe 20 25 <210> 18 <211> 9 <212> PRT <213> Artificial Sequence <220>

    <223> Synthetic <400> 18

    Ile Asp Glu Cys Gln Val Ala Pro Gly 1 5 <210> 19 <211> 25 <212> PRT <213> Artificial Sequence <220>

    <223> Synthetic <400> 19

    Ala Asn Met Leu Cys Ala Gly Leu Glu Ser Gly Gly Lys Asp Ser Cys 15 10 15

    Page 27

    2018200437 19 Jan 2018

    Arg Gly Asp Ser Gly Gly Ala Leu Val 20 25 <210> 20 <211> 960 <212> DNA <213> Homo sapiens <220>

    <221 > CDS <222> (51)..(797) <400> 20 attaactgag attaaccttc cctgagtttt ctcacaccaa ggtgaggacc atg tcc 56 Met Ser 1 ctg ttt cca tea etc cct etc ett etc ctg agt atg gtg gca gcg tet 104 Leu Phe Pro Ser Leu Pro Leu Leu Leu Leu Ser Met Val Ala Ala Ser

    5 10 15 tac tea gaa act gtg acc tgt gag gat gcc caa aag acc tgc cct gca 152 Tyr Ser Glu Thr Val Thr Cys Glu Asp Ala Gln Lys Thr Cys Pro Ala

    20 25 30 gtg att gcc tgt age tet cca ggc ate aac ggc ttc cca ggc aaa gat 200 Val Ile Ala Cys Ser Ser Pro Gly Ile Asn Gly Phe Pro Gly Lys Asp 35 40 45 50 ggg cgt gat ggc acc aag gga gaa aag ggg gaa cca ggc caa ggg etc 248 Gly Arg Asp Gly Thr Lys Gly Glu Lys Gly Glu Pro Gly Gln Gly Leu

    55 60 65 aga ggc tta cag ggc ccc cct gga aag ttg ggg cct cca gga aat cca 296 Arg Gly Leu Gln Gly Pro Pro Gly Lys Leu Gly Pro Pro Gly Asn Pro

    70 75 80 ggg cct tet ggg tea cca gga cca aag ggc caa aaa gga gac cct gga 344 Gly Pro Ser Gly Ser Pro Gly Pro Lys Gly Gln Lys Gly Asp Pro Gly

    85 90 95 aaa agt ccg gat ggt gat agt age ctg get gcc tea gaa aga aaa get 392 Lys Ser Pro Asp Gly Asp Ser Ser Leu Ala Ala Ser Glu Arg Lys Ala

    100 105 110 ctg caa aca gaa atg gca cgt ate aaa aag tgg etc acc ttc tet ctg 440 Leu Gln Thr Glu Met Ala Arg Ile Lys Lys Trp Leu Thr Phe Ser Leu 115 120 125 130

    Page 28

    2018200437 19 Jan 2018 ggc aaa caa gtt ggg aac aag ttc ttc ctg acc aat ggt gaa ata atg 488 Gly Lys Gln Val Gly Asn Lys Phe Phe Leu Thr Asn Gly Glu Ile Met

    135 140 145 acc ttt gaa aaa gtg aag gcc ttg tgt gtc aag ttc cag gcc tct gtg 536 Thr Phe Glu Lys Val Lys Ala Leu Cys Val Lys Phe Gln Ala Ser Val

    150 155 160 gcc acc ccc agg aat get gca gag aat gga gcc att cag aat etc ate 584 Ala Thr Pro Arg Asn Ala Ala Glu Asn Gly Ala Ile Gln Asn Leu Ile

    165 170 175 aag gag gaa gcc ttc ctg ggc ate act gat gag aag aca gaa ggg cag 632 Lys Glu Glu Ala Phe Leu Gly Ile Thr Asp Glu Lys Thr Glu Gly Gln

    180 185 190 ttt gtg gat ctg aca gga aat aga ctg acc tac aca aac tgg aac gag 680 Phe Val Asp Leu Thr Gly Asn Arg Leu Thr Tyr Thr Asn Trp Asn Glu 195 200 205 210 ggt gaa ccc aac aat get ggt tct gat gaa gat tgt gta ttg eta ctg 728 Gly Glu Pro Asn Asn Ala Gly Ser Asp Glu Asp Cys Val Leu Leu Leu

    215 220 225 aaa aat ggc cag tgg aat gac gtc ccc tgc tcc acc tcc cat ctg gcc 776 Lys Asn Gly Gln Trp Asn Asp Val Pro Cys Ser Thr Ser His Leu Ala

    230 235 240 gtc tgt gag ttc cct ate tga agggtcatat cactcaggcc ctccttgtct 827 Val Cys Glu Phe Pro Ile

    245 ttttactgca acccacaggc ccacagtatg ettgaaaaga taaattatat caatttcctc 887 atatccagta ttgttccttt tgtgggcaat cactaaaaat gatcactaac agcaccaaca 947 aagcaataat agt 960 <210> 21 <211> 248 <212> PRT <213> Homo sapiens <400> 21

    Met Ser Leu Phe Pro Ser Leu Pro Leu Leu Leu Leu Ser Met Val Ala 15 10 15

    Page 29

    2018200437 19 Jan 2018

    Ala Ser Tyr Ser Glu Thr Val Thr Cys Glu Asp Ala Gln Lys Thr Cys 20 25 30

    Pro Ala Val Ile Ala Cys Ser Ser Pro Gly Ile Asn Gly Phe Pro Gly 35 40 45

    Lys Asp Gly Arg Asp Gly Thr Lys Gly Glu Lys Gly Glu Pro Gly Gln 50 55 60

    Gly Leu Arg Gly Leu Gln Gly Pro Pro Gly Lys Leu Gly Pro Pro Gly 65 70 75 80

    Asn Pro Gly Pro Ser Gly Ser Pro Gly Pro Lys Gly Gln Lys Gly Asp 85 90 95

    Pro Gly Lys Ser Pro Asp Gly Asp Ser Ser Leu Ala Ala Ser Glu Arg 100 105 110

    Lys Ala Leu Gln Thr Glu Met Ala Arg Ile Lys Lys Trp Leu Thr Phe 115 120 125

    Ser Leu Gly Lys Gln Val Gly Asn Lys Phe Phe Leu Thr Asn Gly Glu 130 135 140

    Ile Met Thr Phe Glu Lys Val Lys Ala Leu Cys Val Lys Phe Gln Ala 145 150 155 160

    Ser Val Ala Thr Pro Arg Asn Ala Ala Glu Asn Gly Ala Ile Gln Asn 165 170 175

    Gly Gln Phe Val Asp Leu Thr Gly Asn Arg Leu Thr Tyr Thr Asn Trp 195 200 205

    Asn Glu Gly Glu Pro Asn Asn Ala Gly Ser Asp Glu Asp Cys Val Leu

    Leu Ile Lys Glu Glu Ala Phe Leu Gly Ile Thr Asp Glu Lys Thr Glu

    180 185 190

    Page 30

    2018200437 19 Jan 2018

    210 215 220

    Leu Leu Lys Asn Gly Gln Trp Asn Asp Val Pro Cys Ser Thr Ser His 225 230 235 240

    Leu Ala Val Cys Glu Phe Pro Ile 245 <210> 22 <211> 6 <212> PRT <213> Artificial Sequence <220>

    <223> Synthetic <220>

    <221 > MISC_FEATURE <222> (1)..(1) <223> Wherein X at position 1 represents hydroxyproline <220>

    <221 > MISC_FEATURE <222> (4)..(4) <223> Wherein X at position 4 represents hydrophobic residue <400> 22

    Xaa Gly Lys Xaa Gly Pro

    1 5 <210> 23 <211> 5 <212> PRT <213> Artificial Sequence <220>

    <223> Synthetic <220>

    <221 > MISC_FEATURE <222> (1)..(1) <223> Wherein X represents hydroxyproline

    Page 31

    2018200437 19 Jan 2018 <400> 23

    Xaa Gly Lys Leu Gly 1 5 <210> 24 <211> 16 <212> PRT <213> Artificial Sequence <220>

    <223> Synthetic <220>

    <221> MISC_FEATURE <222> (9)..(15) <223> Wherein X at positions 9 and 15 represents hydroxyproline <400> 24

    Gly Leu Arg Gly Leu Gln Gly Pro Xaa Gly Lys Leu Gly Pro Xaa Gly 15 10 15 <210> 25 <211> 27 <212> PRT <213> Artificial Sequence <220>

    <223> Synthetic <220>

    <221> MISC_FEATURE <222> (3)..(27) <223> Wherein X at positions 3, 6, 15, 21, 24, 27 represents hydroxyproline <400> 25

    Gly Pro Xaa Gly Pro Xaa Gly Leu Arg Gly Leu Gln Gly Pro Xaa Gly 15 10 15

    Lys Leu Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa 20 25

    Page 32

    2018200437 19 Jan 2018 <210> 26 <211> 53 <212> PRT <213> Artificial Sequence <220>

    <223> Synthetic <220>

    <221 > misc_feature <222> (26)..(26) <223> Xaa can be any naturally occurring amino acid <220>

    <221> misc_feature <222> (32)..(32) <223> Xaa can be any naturally occurring amino acid <220>

    <221 > misc_feature <222> (35)..(35) <223> Xaa can be any naturally occurring amino acid <220>

    <221> misc_feature <222> (41)..(41) <223> Xaa can be any naturally occurring amino acid <220>

    <221 > misc_feature <222> (50)..(50) <223> Xaa can be any naturally occurring amino acid <400> 26

    Gly Lys Asp Gly Arg Asp Gly Thr Lys Gly Glu Lys Gly Glu Pro Gly 15 10 15

    Gly Asn Xaa Gly Pro Ser Gly Ser Xaa Gly Pro Lys Gly Gln Lys Gly 35 40 45

    Asp Xaa Gly Lys Ser

    Gln Gly Leu Arg Gly Leu Gln Gly Pro Xaa Gly Lys Leu Gly Pro Xaa

    20 25 30

    Page 33

    2018200437 19 Jan 2018 <210> 27 <211> 33 <212> PRT <213> Artificial Sequence <220>

    <223> Synthetic <220>

    <221 > MISC_FEATURE <222> (3)..(33) <223> Wherein X at positions 3, 6, 12, 18, 21, 30, 33 represents hydroxyproline <400> 27

    Gly Ala Xaa Gly Ser Xaa Gly Glu Lys Gly Ala Xaa Gly Pro Gln Gly 15 10 15

    Pro Xaa Gly Pro Xaa Gly Lys Met Gly Pro Lys Gly Glu Xaa Gly Asp 20 25 30

    Xaa <210> 28 <211> 45 <212> PRT <213> Artificial Sequence <220>

    <223> Synthetic <220>

    <221> MISC_FEATURE <222> (3)..(45) <223> Wherein X at positions 3, 6, 9, 27, 30, 36, 42, 45 represents hydroxyproline <400> 28

    Gly Cys Xaa Gly Leu Xaa Gly Ala Xaa Gly Asp Lys Gly Glu Ala Gly

    Page 34

    2018200437 19 Jan 2018

    15 10 15

    Thr Asn Gly Lys Arg Gly Glu Arg Gly Pro Xaa Gly Pro Xaa Gly Lys 20 25 30

    Ala Gly Pro Xaa Gly Pro Asn Gly Ala Xaa Gly Glu Xaa 35 40 45 <210> 29 <211> 24 <212> PRT <213> Artificial Sequence <220>

    <223> Synthetic <400> 29

    Leu Gln Arg Ala Leu Glu Ile Leu Pro Asn Arg Val Thr Ile Lys Ala 15 10 15

    Asn Arg Pro Phe Leu Val Phe Ile 20 <210> 30 <211> 559 <212> DNA <213> Homo sapiens <400> 30 atgaggctgc tgaccctcct gggccttctg tgtggctcgg tggccacccc cttgggcccg 60 aagtggcctg aacctgtgtt cgggcgcctg gcatcccccg gctttccagg ggagtatgcc 120 aatgaccagg agcggcgctg gaccctgact gcaccccccg gctaccgcct gcgcctctac 180 ttcacccact tcgacctgga gctctcccac ctctgcgagt acgacttcgt caagctgagc 240 tcgggggcca aggtgctggc cacgctgtgc gggcaggaga gcacagacac ggagcgggcc 300 cctggcaagg acactttcta ctcgctgggc tccagcctgg acattacctt ccgctccgac 360 tactccaacg agaagccgtt cacggggttc gaggccttct atgcagccga ggacattgac 420 gagtgccagg tggccccggg agaggcgccc acctgcgacc accactgcca caaccacctg 480

    Page 35

    2018200437 19 Jan 2018 ggcggtttct actgctcctg ccgcgcaggc tacgtcctgc accgtaacaa gcgcacctgc tcagccctgt gctccggcc 559

    540 <210> 31 <211> 34 <212> DNA <213> Artificial Sequence <220>

    <223> Synthetic <400> 31 cgggcacacc atgaggctgc tgaccctcct gggc 34 <210> 32 <211> 33 <212> DNA <213> Artificial Sequence <220>

    <223> Synthetic <400> 32 gacattacct tccgctccga ctccaacgag aag 33 <210> 33 <211> 33 <212> DNA <213> Artificial Sequence <220>

    <223> Synthetic <400> 33 agcagccctg aatacccacg gccgtatccc aaa 33 <210> 34 <211> 26 <212> DNA <213> Artificial Sequence <220>

    <223> Synthetic

    Page 36

    2018200437 19 Jan 2018 <400> 34 cgggatccat gaggctgctg accctc 26 <210> 35 <211> 19 <212> DNA <213> Artificial Sequence <220>

    <223> Synthetic <400> 35 ggaattccta ggctgcata 19 <210> 36 <211> 19 <212> DNA <213> Artificial Sequence <220>

    <223> Synthetic <400> 36 ggaattccta cagggcgct 19 <210> 37 <211> 19 <212> DNA <213> Artificial Sequence <220>

    <223> Synthetic <400> 37 ggaattccta gtagtggat 19 <210> 38 <211> 25 <212> DNA <213> Artificial Sequence <220>

    <223> Synthetic <400> 38 tgcggccgct gtaggtgctg tcttt

    Page 37

    2018200437 19 Jan 2018 <210> 39 <211> 23 <212> DNA <213> Artificial Sequence <220>

    <223> Synthetic <400> 39 ggaattcact cgttattctc gga 23 <210> 40 <211> 17 <212> DNA <213> Artificial Sequence <220>

    <223> Synthetic <400> 40 tccgagaata acgagtg 17 <210> 41 <211> 29 <212> DNA <213> Artificial Sequence <220>

    <223> Synthetic <400> 41 cattgaaagc tttggggtag aagttgttc 29 <210> 42 <211> 27 <212> DNA <213> Artificial Sequence <220>

    <223> Synthetic <400> 42 cgcggccgca gctgctcaga gtgtaga

    Page 38

    2018200437 19 Jan 2018 <210> 43 <211> 28 <212> DNA <213> Artificial Sequence <220>

    <223> Synthetic <400> 43 cggtaagctt cactggctca gggaaata 28 <210> 44 <211> 37 <212> DNA <213> Artificial Sequence <220>

    <223> Synthetic <400> 44 aagaagcttg ccgccaccat ggattggctg tggaact 37 <210> 45 <211> 31 <212> DNA <213> Artificial Sequence <220>

    <223> Synthetic <400> 45 cgggatcctc aaactttctt gtccaccttg g 31 <210> 46 <211> 36 <212> DNA <213> Artificial Sequence <220>

    <223> Synthetic <400> 46 aagaaagctt gccgccacca tgttctcact agctct 36 <210> 47 <211> 26

    Page 39

    2018200437 19 Jan 2018 <212> DNA <213> Artificial Sequence <220>

    <223> Synthetic <400> 47 cgggatcctt ctccctctaa cactct 26 <210> 48 <211> 9 <212> PRT <213> Artificial Sequence <220>

    <223> Synthetic <400> 48

    Glu Pro Lys Ser Cys Asp Lys Thr His 1 5 <210> 49 <211> 4960 <212> DNA <213> Homo Sapiens <400> 49 ccggacgtgg tggcgcatgc ctgtaatccc agctactcgg gaggctgagg caggagaatt 60 gctcgaaccc cggaggcaga ggtttggtgg ctcacacctg taatcccagc actttgcgag 120 gctgaggcag gtgcatcgct ttggctcagg agttcaagac cagcctgggc aacacaggga 180 gacccccatc tctacaaaaa acaaaaacaa atataaaggg gataaaaaaa aaaaaaagac 240 aagacatgaa tccatgagga cagagtgtgg aagaggaagc agcagcctca aagttctgga 300 agctggaaga acagataaac aggtgtgaaa taactgcctg gaaagcaact tctttttttt 360 tttttttttt tttgaggtgg agtctcactc tgtcgtccag gctggagtgc agtggtgcga 420 tctcggatca ctgcaacctc cgcctcccag gctcaagcaa ttctcctgcc tcagcctccc 480 gagtagctgg gattataagt gcgcgctgcc acacctggat gatttttgta tttttagtag 540 agatgggatt tcaccatgtt ggtcaggctg gtctcaaact cccaacctcg tgatccaccc 600

    Page 40

    2018200437 19 Jan 2018 accttggcct cccaaagtgc tgggattaca ggtataagcc accgagccca gccaaaagcg 660 acttctaagc ctgcaaggga atcgggaatt ggtggcacca ggtccttctg acagggttta 720 agaaattagc cagcctgagg ctgggcacgg tggctcacac ctgtaatccc agcactttgg 780 gaggctaagg caggtggatc acctgagggc aggagttcaa gaccagcctg accaacatgg 840 agaaacccca tccctaccaa aaataaaaaa ttagccaggt gtggtggtgc tcgcctgtaa 900 tcccagctac ttgggaggct gaggtgggag gattgcttga acacaggaag tagaggctgc 960 agtgagctat gattgcagca ctgcactgaa gccggggcaa cagaacaaga tccaaaaaaa 1020 agggaggggt gaggggcaga gccaggattt gtttccaggc tgttgttacc taggtccgac 1080 tcctggctcc cagagcagcc tgtcctgcct gcctggaact ctgagcaggc tggagtcatg 1140 gagtcgattc ccagaatccc agagtcaggg aggctggggg caggggcagg tcactggaca 1200 aacagatcaa aggtgagacc agcgtagggc tgcagaccag gccaggccag ctggacgggc 1260 acaccatgag gtaggtgggc gcccacagcc tccctgcagg gtgtggggtg ggagcacagg 1320 cctgggccct caccgcccct gccctgccca taggctgctg accctcctgg gccttctgtg 1380 tggctcggtg gccaccccct tgggcccgaa gtggcctgaa cctgtgttcg ggcgcctggc 1440 atcccccggc tttccagggg agtatgccaa tgaccaggag cggcgctgga ccctgactgc 1500 accccccggc taccgcctgc gcctctactt cacccacttc gacctggagc tctcccacct 1560 ctgcgagtac gacttcgtca aggtgccgtc aggacgggag ggctggggtt tctcagggtc 1620 ggggggtccc caaggagtag ccagggttca gggacacctg ggagcagggg ccaggcttgg 1680 ccaggaggga gatcaggcct gggtcttgcc ttcactccct gtgacacctg accccacagc 1740 tgagctcggg ggccaaggtg ctggccacgc tgtgcgggca ggagagcaca gacacggagc 1800 gggcccctgg caaggacact ttctactcgc tgggctccag cctggacatt accttccgct 1860 ccgactactc caacgagaag ccgttcacgg ggttcgaggc cttctatgca gccgagggtg 1920 agccaagagg ggtcctgcaa catctcagtc tgcgcagctg gctgtggggg taactctgtc 1980 ttaggccagg cagccctgcc ttcagtttcc ccacctttcc cagggcaggg gagaggcctc 2040 tggcctgaca tcatccacaa tgcaaagacc aaaacagccg tgacctccat tcacatgggc 2100

    Page 41

    2018200437 19 Jan 2018 tgagtgccaa ctctgagcca gggatctgag gacagcatcg cctcaagtga cgcagggact 2160 ggccgggcgc agcagctcac gcctgtaatt ccagcacttt gggaggccga ggctggctga 2220 tcatttgagg tcaggagttc aaggccagcc agggcaacac ggtgaaactc tatctccact 2280 aaaactacaa aaattagctg ggcgtggtgg tgcgcacctg gaatcccagc tactagggag 2340 gctgaggcag gagaattgct tgaacctgcg aggtggaggc tgcagtgaac agagattgca 2400 ccactacact ccagcctggg cgacagagct agactccgtc tcaaaaaaca aaaaacaaaa 2460 acgacgcagg ggccgagggc cccatttaca gctgacaaag tggggccctg ccagcgggag 2520 cgctgccagg atgtttgatt tcagatccca gtccctgcag agaccaactg tgtgacctct 2580 ggcaagtggc tcaatttctc tgctccttag gaagctgctg caagggttca gcgctgtagc 2640 cccgccccct gggtttgatt gactcccctc attagctggg tgacctcggg ccggacactg 2700 aaactcccac tggtttaaca gaggtgatgt ttgcatcttt ctcccagcgc tgctgggagc 2760 ttgcagcgac cctaggcctg taaggtgatt ggcccggcac cagtcccgca ccctagacag 2820 gacgaggcct cctctgaggt ccactctgag gtcatggatc tcctgggagg agtccaggct 2880 ggatcccgcc tctttccctc ctgacggcct gcctggccct gcctctcccc cagacattga 2940 cgagtgccag gtggccccgg gagaggcgcc cacctgcgac caccactgcc acaaccacct 3000 gggcggtttc tactgctcct gccgcgcagg ctacgtcctg caccgtaaca agcgcacctg 3060 ctcagccctg tgctccggcc aggtcttcac ccagaggtct ggggagctca gcagccctga 3120 atacccacgg ccgtatccca aactctccag ttgcacttac agcatcagcc tggaggaggg 3180 gttcagtgtc attctggact ttgtggagtc cttcgatgtg gagacacacc ctgaaaccct 3240 gtgtccctac gactttctca agattcaaac agacagagaa gaacatggcc cattctgtgg 3300 gaagacattg ccccacagga ttgaaacaaa aagcaacacg gtgaccatca cctttgtcac 3360 agatgaatca ggagaccaca caggctggaa gatccactac acgagcacag cgcacgcttg 3420 cccttatccg atggcgccac ctaatggcca cgtttcacct gtgcaagcca aatacatcct 3480 gaaagacagc ttctccatct tttgcgagac tggctatgag cttctgcaag gtcacttgcc 3540 cctgaaatcc tttactgcag tttgtcagaa agatggatct tgggaccggc caatgcccgc 3600

    Page 42

    2018200437 19 Jan 2018 gtgcagcatt gttgactgtg gccctcctga tgatctaccc agtggccgag tggagtacat 3660 cacaggtcct ggagtgacca cctacaaagc tgtgattcag tacagctgtg aagagacctt 3720 ctacacaatg aaagtgaatg atggtaaata tgtgtgtgag gctgatggat tctggacgag 3780 ctccaaagga gaaaaatcac tcccagtctg tgagcctgtt tgtggactat cagcccgcac 3840 aacaggaggg cgtatatatg gagggcaaaa ggcaaaacct ggtgattttc cttggcaagt 3900 cctgatatta ggtggaacca cagcagcagg tgcactttta tatgacaact gggtcctaac 3960 agctgctcat gccgtctatg agcaaaaaca tgatgcatcc gccctggaca ttcgaatggg 4020 caccctgaaa agactatcac ctcattatac acaagcctgg tctgaagctg tttttataca 4080 tgaaggttat actcatgatg ctggctttga caatgacata gcactgatta aattgaataa 4140 caaagttgta atcaatagca acatcacgcc tatttgtctg ccaagaaaag aagctgaatc 4200 ctttatgagg acagatgaca ttggaactgc atctggatgg ggattaaccc aaaggggttt 4260 tcttgctaga aatctaatgt atgtcgacat accgattgtt gaccatcaaa aatgtactgc 4320 tgcatatgaa aagccaccct atccaagggg aagtgtaact gctaacatgc tttgtgctgg 4380 cttagaaagt gggggcaagg acagctgcag aggtgacagc ggaggggcac tggtgtttct 4440 agatagtgaa acagagaggt ggtttgtggg aggaatagtg tcctggggtt ccatgaattg 4500 tggggaagca ggtcagtatg gagtctacac aaaagttatt aactatattc cctggatcga 4560 gaacataatt agtgattttt aacttgcgtg tctgcagtca aggattcttc atttttagaa 4620 atgcctgtga agaccttggc agcgacgtgg ctcgagaagc attcatcatt actgtggaca 4680 tggcagttgt tgctccaccc aaaaaaacag actccaggtg aggctgctgt catttctcca 4740 cttgccagtt taattccagc cttacccatt gactcaaggg gacataaacc acgagagtga 4800 cagtcatctt tgcccaccca gtgtaatgtc actgctcaaa ttacatttca ttaccttaaa 4860 aagccagtct cttttcatac tggctgttgg catttctgta aactgcctgt ccatgctctt 4920 tgtttttaaa cttgttctta ttgaaaaaaa aaaaaaaaaa 4960 <210>

    <211>

    <212>

    2090

    DNA

    Page 43

    2018200437 19 Jan 2018 <213> Murine <220>

    <221> CDS <222> (33)..(2090) <400> 50 ggcgctggac tgcagagcta tggtggcaca cc atg agg eta etc ate ttc ctg 53 Met Arg Leu Leu Ile Phe Leu 1 5 ggt ctg ctg tgg agt ttg gtg gcc aca ett ctg ggt tea aag tgg cct 101 Gly Leu Leu Trp Ser Leu Val Ala Thr Leu Leu Gly Ser Lys Trp Pro

    10 15 20 gaa cct gta ttc ggg cgc ctg gtg tec cct ggc ttc cca gag aag tat 149 Glu Pro Val Phe Gly Arg Leu Val Ser Pro Gly Phe Pro Glu Lys Tyr
25. 25 30 35 get gac cat caa gat ega tec tgg aca ctg act gca ccc cct ggc tac 197 Ala Asp His Gln Asp Arg Ser Trp Thr Leu Thr Ala Pro Pro Gly Tyr 40 45 50 55 cgc ctg cgc etc tac ttc acc cac ttt gac ctg gaa etc tet tac cgc 245 Arg Leu Arg Leu Tyr Phe Thr His Phe Asp Leu Glu Leu Ser Tyr Arg

    60 65 70 tgc gag tat gac ttt gtc aag ttg age tea ggg acc aag gtg ctg gcc 293 Cys Glu Tyr Asp Phe Val Lys Leu Ser Ser Gly Thr Lys Val Leu Ala

    75 80 85 aca ctg tgt ggg cag gag agt aca gac act gag cag gca cct ggc aat 341 Thr Leu Cys Gly Gln Glu Ser Thr Asp Thr Glu Gln Ala Pro Gly Asn

    90 95 100 gac acc ttc tac tea ctg ggt ccc age eta aag gtc acc ttc cac tcc 389 Asp Thr Phe Tyr Ser Leu Gly Pro Ser Leu Lys Val Thr Phe His Ser

    105 110 115 gac tac tcc aat gag aag ccg ttc aca ggg ttt gag gcc ttc tat gca 437 Asp Tyr Ser Asn Glu Lys Pro Phe Thr Gly Phe Glu Ala Phe Tyr Ala 120 125 130 135 geg gag gat gtg gat gaa tgc aga gtg tet ctg gga gac tea gtc cct 485 Ala Glu Asp Val Asp Glu Cys Arg Val Ser Leu Gly Asp Ser Val Pro

    140 145 150 tgt gac cat tat tgc cac aac tac ttg ggc ggc tac tat tgc tcc tgc 533 Cys Asp His Tyr Cys His Asn Tyr Leu Gly Gly Tyr Tyr Cys Ser Cys

    Page 44

    2018200437 19 Jan 2018

    155 160 165 aga gcg ggc tac att etc cac cag aac aag cac aeg tgc tea gcc ett 581 Arg Ala Gly Tyr Ile Leu His Gln Asn Lys His Thr Cys Ser Ala Leu

    170 175 180 tgt tea ggc cag gtg ttc aca gga aga tct ggg tat etc agt age cct 629 Cys Ser Gly Gln Val Phe Thr Gly Arg Ser Gly Tyr Leu Ser Ser Pro

    185 190 195 gag tac ccg cag cca tac ccc aag etc tcc age tgc acc tac age ate 677 Glu Tyr Pro Gln Pro Tyr Pro Lys Leu Ser Ser Cys Thr Tyr Ser Ile 200 205 210 215 ege ctg gag gac ggc ttc agt gtc ate ctg gac ttc gtg gag tcc ttc 725 Arg Leu Glu Asp Gly Phe Ser Val Ile Leu Asp Phe Val Glu Ser Phe

    220 225 230 gat gtg gag aeg cac cct gaa gcc cag tgc ccc tat gac tcc etc aag 773 Asp Val Glu Thr His Pro Glu Ala Gln Cys Pro Tyr Asp Ser Leu Lys

    235 240 245 att caa aca gac aag ggg gaa cac ggc cca ttt tgt ggg aag aeg ctg 821 Ile Gln Thr Asp Lys Gly Glu His Gly Pro Phe Cys Gly Lys Thr Leu

    250 255 260 cct ccc agg att gaa act gac age cac aag gtg acc ate acc ttt gcc 869 Pro Pro Arg Ile Glu Thr Asp Ser His Lys Val Thr Ile Thr Phe Ala

    265 270 275 act gac gag teg ggg aac cac aca ggc tgg aag ata cac tac aca age 917 Thr Asp Glu Ser Gly Asn His Thr Gly Trp Lys Ile His Tyr Thr Ser 280 285 290 295 aca gca egg ccc tgc cct gat cca aeg gcg cca cct aat ggc age att 965 Thr Ala Arg Pro Cys Pro Asp Pro Thr Ala Pro Pro Asn Gly Ser Ile

    300 305 310 tea cct gtg caa gcc aeg tat gtc ctg aag gac agg ttt tct gtc ttc 1013 Ser Pro Val Gln Ala Thr Tyr Val Leu Lys Asp Arg Phe Ser Val Phe

    315 320 325 tgc aag aca ggc ttc gag ett ctg caa ggt tct gtc ccc ctg aaa tea 1061 Cys Lys Thr Gly Phe Glu Leu Leu Gln Gly Ser Val Pro Leu Lys Ser

    330 335 340 ttc act get gtc tgt cag aaa gat gga tct tgg gac egg ccg atg cca 1109 Phe Thr Ala Val Cys Gln Lys Asp Gly Ser Trp Asp Arg Pro Met Pro

    345 350 355

    Page 45

    2018200437 19 Jan 2018 gag tgc age att att gat tgt ggc cct ccc gat gac eta ccc aat ggc 1157 Glu Cys Ser Ile Ile Asp Cys Gly Pro Pro Asp Asp Leu Pro Asn Gly 360 365 370 375 cat gtg gac tat ate aca ggc cct caa gtg act acc tac aaa get gtg 1205 His Val Asp Tyr Ile Thr Gly Pro Gln Val Thr Thr Tyr Lys Ala Val

    380 385 390 att cag tac age tgt gaa gag act ttc tac aca atg age age aat ggt 1253 Ile Gln Tyr Ser Cys Glu Glu Thr Phe Tyr Thr Met Ser Ser Asn Gly

    395 400 405 aaa tat gtg tgt gag get gat gga ttc tgg aeg age tee aaa gga gaa 1301 Lys Tyr Val Cys Glu Ala Asp Gly Phe Trp Thr Ser Ser Lys Gly Glu

    410 415 420 aaa etc ccc ccg gtt tgt gag cct gtt tgt ggg ctg tee aca cac act 1349 Lys Leu Pro Pro Val Cys Glu Pro Val Cys Gly Leu Ser Thr His Thr

    425 430 435 ata gga gga ege ata gtt gga ggg cag cct gca aag cct ggt gac ttt 1397 Ile Gly Gly Arg Ile Val Gly Gly Gln Pro Ala Lys Pro Gly Asp Phe 440 445 450 455 cct tgg caa gtc ttg ttg ctg ggt caa act aca gca gca gca ggt gca 1445 Pro Trp Gln Val Leu Leu Leu Gly Gln Thr Thr Ala Ala Ala Gly Ala

    460 465 470 ett ata cat gac aat tgg gtc eta aca gcc get cat get gta tat gag 1493 Leu Ile His Asp Asn Trp Val Leu Thr Ala Ala His Ala Val Tyr Glu

    475 480 485 aaa aga atg gca geg tee tee ctg aac ate ega atg ggc ate etc aaa 1541 Lys Arg Met Ala Ala Ser Ser Leu Asn Ile Arg Met Gly Ile Leu Lys

    490 495 500 agg etc tea cct cat tac act caa gcc tgg ccc gag gaa ate ttt ata 1589 Arg Leu Ser Pro His Tyr Thr Gln Ala Trp Pro Glu Glu Ile Phe Ile

    505 510 515 cat gaa ggc tac act cac ggt get ggt ttt gac aat gat ata gca ttg 1637 His Glu Gly Tyr Thr His Gly Ala Gly Phe Asp Asn Asp Ile Ala Leu 520 525 530 535 att aaa etc aag aac aaa gtc aca ate aac gga age ate atg cct gtt 1685 Ile Lys Leu Lys Asn Lys Val Thr Ile Asn Gly Ser Ile Met Pro Val

    540 545 550 tgc eta ccg ega aaa gaa get gca tee tta atg aga aca gac ttc act 1733 Cys Leu Pro Arg Lys Glu Ala Ala Ser Leu Met Arg Thr Asp Phe Thr

    Page 46

    2018200437 19 Jan 2018

    555 560 565 gga act gtg get ggc tgg ggg tta acc cag aag ggg ett ett get aga 1781 Gly Thr Val Ala Gly Trp Gly Leu Thr Gln Lys Gly Leu Leu Ala Arg

    570 575 580 aac eta atg ttt gtg gac ata cca att get gac cac caa aaa tgt acc 1829 Asn Leu Met Phe Val Asp Ile Pro Ile Ala Asp His Gln Lys Cys Thr

    585 590 595 acc gtg tat gaa aag etc tat cca gga gta aga gta age get aac atg 1877 Thr Val Tyr Glu Lys Leu Tyr Pro Gly Val Arg Val Ser Ala Asn Met 600 605 610 615 etc tgt get ggc tta gag act ggt ggc aag gac age tgc aga ggt gac 1925 Leu Cys Ala Gly Leu Glu Thr Gly Gly Lys Asp Ser Cys Arg Gly Asp

    620 625 630 agt ggg ggg gca tta gtg ttt eta gat aat gag aca cag ega tgg ttt 1973 Ser Gly Gly Ala Leu Val Phe Leu Asp Asn Glu Thr Gln Arg Trp Phe

    635 640 645 gtg gga gga ata gtt tee tgg ggt tee att aat tgt ggg geg gca ggc 2021 Val Gly Gly Ile Val Ser Trp Gly Ser Ile Asn Cys Gly Ala Ala Gly

    650 655 660 cag tat ggg gtc tac aca aaa gtc ate aac tat att ccc tgg aat gag 2069 Gln Tyr Gly Val Tyr Thr Lys Val Ile Asn Tyr Ile Pro Trp Asn Glu

    665 670 675 aac ata ata agt aat ttc taa 2090

    Asn Ile Ile Ser Asn Phe

    680 685

    Leu Leu Gly Ser Lys Trp Pro Glu Pro Val Phe Gly Arg Leu Val Ser 20 25 30 <210> 51 <211> 685 <212> PRT <213> Murine <400> 51

    Met Arg Leu Leu Ile Phe Leu Gly Leu Leu Trp Ser Leu Val Ala Thr

    15 10 15

    Page 47

    2018200437 19 Jan 2018

    Pro Gly Phe Pro Glu Lys Tyr Ala Asp His Gln Asp Arg Ser Trp Thr 35 40 45

    Leu Thr Ala Pro Pro Gly Tyr Arg Leu Arg Leu Tyr Phe Thr His Phe 50 55 60

    Asp Leu Glu Leu Ser Tyr Arg Cys Glu Tyr Asp Phe Val Lys Leu Ser 65 70 75 80

    Ser Gly Thr Lys Val Leu Ala Thr Leu Cys Gly Gln Glu Ser Thr Asp 85 90 95

    Thr Glu Gln Ala Pro Gly Asn Asp Thr Phe Tyr Ser Leu Gly Pro Ser 100 105 110

    Leu Lys Val Thr Phe His Ser Asp Tyr Ser Asn Glu Lys Pro Phe Thr 115 120 125

    Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp Val Asp Glu Cys Arg Val 130 135 140

    Ser Leu Gly Asp Ser Val Pro Cys Asp His Tyr Cys His Asn Tyr Leu 145 150 155 160

    Gly Gly Tyr Tyr Cys Ser Cys Arg Ala Gly Tyr Ile Leu His Gln Asn 165 170 175

    Lys His Thr Cys Ser Ala Leu Cys Ser Gly Gln Val Phe Thr Gly Arg 180 185 190

    Ser Gly Tyr Leu Ser Ser Pro Glu Tyr Pro Gln Pro Tyr Pro Lys Leu 195 200 205

    Ser Ser Cys Thr Tyr Ser Ile Arg Leu Glu Asp Gly Phe Ser Val Ile 210 215 220

    Leu Asp Phe Val Glu Ser Phe Asp Val Glu Thr His Pro Glu Ala Gln 225 230 235 240

    Page 48

    2018200437 19 Jan 2018

    Cys Pro Tyr Asp Ser Leu Lys Ile Gln Thr Asp Lys Gly Glu His Gly 245 250 255

    Pro Phe Cys Gly Lys Thr Leu Pro Pro Arg Ile Glu Thr Asp Ser His 260 265 270

    Lys Val Thr Ile Thr Phe Ala Thr Asp Glu Ser Gly Asn His Thr Gly 275 280 285

    Trp Lys Ile His Tyr Thr Ser Thr Ala Arg Pro Cys Pro Asp Pro Thr 290 295 300

    Ala Pro Pro Asn Gly Ser Ile Ser Pro Val Gln Ala Thr Tyr Val Leu 305 310 315 320

    Lys Asp Arg Phe Ser Val Phe Cys Lys Thr Gly Phe Glu Leu Leu Gln 325 330 335

    Gly Ser Val Pro Leu Lys Ser Phe Thr Ala Val Cys Gln Lys Asp Gly 340 345 350

    Ser Trp Asp Arg Pro Met Pro Glu Cys Ser Ile Ile Asp Cys Gly Pro 355 360 365

    Pro Asp Asp Leu Pro Asn Gly His Val Asp Tyr Ile Thr Gly Pro Gln 370 375 380

    Val Thr Thr Tyr Lys Ala Val Ile Gln Tyr Ser Cys Glu Glu Thr Phe 385 390 395 400

    Trp Thr Ser Ser Lys Gly Glu Lys Leu Pro Pro Val Cys Glu Pro Val 420 425 430

    Tyr Thr Met Ser Ser Asn Gly Lys Tyr Val Cys Glu Ala Asp Gly Phe

    405 410 415

    Page 49

    2018200437 19 Jan 2018

    Cys Gly Leu Ser Thr His Thr Ile Gly Gly Arg Ile Val Gly Gly Gln 435 440 445

    Pro Ala Lys Pro Gly Asp Phe Pro Trp Gln Val Leu Leu Leu Gly Gln 450 455 460

    Thr Thr Ala Ala Ala Gly Ala Leu Ile His Asp Asn Trp Val Leu Thr 465 470 475 480

    Ala Ala His Ala Val Tyr Glu Lys Arg Met Ala Ala Ser Ser Leu Asn 485 490 495

    Ile Arg Met Gly Ile Leu Lys Arg Leu Ser Pro His Tyr Thr Gln Ala 500 505 510

    Trp Pro Glu Glu Ile Phe Ile His Glu Gly Tyr Thr His Gly Ala Gly 515 520 525

    Phe Asp Asn Asp Ile Ala Leu Ile Lys Leu Lys Asn Lys Val Thr Ile 530 535 540

    Asn Gly Ser Ile Met Pro Val Cys Leu Pro Arg Lys Glu Ala Ala Ser 545 550 555 560

    Leu Met Arg Thr Asp Phe Thr Gly Thr Val Ala Gly Trp Gly Leu Thr 565 570 575

    Gln Lys Gly Leu Leu Ala Arg Asn Leu Met Phe Val Asp Ile Pro Ile 580 585 590

    Ala Asp His Gln Lys Cys Thr Thr Val Tyr Glu Lys Leu Tyr Pro Gly 595 600 605

    Val Arg Val Ser Ala Asn Met Leu Cys Ala Gly Leu Glu Thr Gly Gly 610 615 620

    Lys Asp Ser Cys Arg Gly Asp Ser Gly Gly Ala Leu Val Phe Leu Asp 625 630 635 640

    Page 50

    2018200437 19 Jan 2018

    Asn Glu Thr Gln Arg Trp Phe Val Gly Gly Ile Val Ser Trp Gly Ser 645 650 655

    Ile Asn Cys Gly Ala Ala Gly Gln Tyr Gly Val Tyr Thr Lys Val Ile 660 665 670

    Asn Tyr Ile Pro Trp Asn Glu Asn Ile Ile Ser Asn Phe 675 680 685 <210> 52 <211> 670 <212> PRT <213> Murine <400> 52

    Thr Leu Leu Gly Ser Lys Trp Pro Glu Pro Val Phe Gly Arg Leu Val 15 10 15

    Ser Pro Gly Phe Pro Glu Lys Tyr Ala Asp His Gln Asp Arg Ser Trp 20 25 30

    Thr Leu Thr Ala Pro Pro Gly Tyr Arg Leu Arg Leu Tyr Phe Thr His 35 40 45

    Phe Asp Leu Glu Leu Ser Tyr Arg Cys Glu Tyr Asp Phe Val Lys Leu 50 55 60

    Ser Ser Gly Thr Lys Val Leu Ala Thr Leu Cys Gly Gln Glu Ser Thr 65 70 75 80

    Ser Leu Lys Val Thr Phe His Ser Asp Tyr Ser Asn Glu Lys Pro Phe 100 105 110

    Thr Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp Val Asp Glu Cys Arg

    Asp Thr Glu Gln Ala Pro Gly Asn Asp Thr Phe Tyr Ser Leu Gly Pro

    85 90 95

    Page 51

    2018200437 19 Jan 2018

    115 120 125

    Val Ser Leu Gly Asp Ser Val Pro Cys Asp His Tyr Cys His Asn Tyr 130 135 140

    Leu Gly Gly Tyr Tyr Cys Ser Cys Arg Ala Gly Tyr Ile Leu His Gln 145 150 155 160

    Asn Lys His Thr Cys Ser Ala Leu Cys Ser Gly Gln Val Phe Thr Gly 165 170 175

    Arg Ser Gly Tyr Leu Ser Ser Pro Glu Tyr Pro Gln Pro Tyr Pro Lys 180 185 190

    Leu Ser Ser Cys Thr Tyr Ser Ile Arg Leu Glu Asp Gly Phe Ser Val 195 200 205

    Ile Leu Asp Phe Val Glu Ser Phe Asp Val Glu Thr His Pro Glu Ala 210 215 220

    Gln Cys Pro Tyr Asp Ser Leu Lys Ile Gln Thr Asp Lys Gly Glu His 225 230 235 240

    Gly Pro Phe Cys Gly Lys Thr Leu Pro Pro Arg Ile Glu Thr Asp Ser 245 250 255

    His Lys Val Thr Ile Thr Phe Ala Thr Asp Glu Ser Gly Asn His Thr 260 265 270

    Gly Trp Lys Ile His Tyr Thr Ser Thr Ala Arg Pro Cys Pro Asp Pro 275 280 285

    Thr Ala Pro Pro Asn Gly Ser Ile Ser Pro Val Gln Ala Thr Tyr Val 290 295 300

    Leu Lys Asp Arg Phe Ser Val Phe Cys Lys Thr Gly Phe Glu Leu Leu 305 310 315 320

    Page 52

    2018200437 19 Jan 2018

    Gln Gly Ser Val Pro Leu Lys Ser Phe Thr Ala Val Cys Gln Lys Asp 325 330 335

    Gly Ser Trp Asp Arg Pro Met Pro Glu Cys Ser Ile Ile Asp Cys Gly 340 345 350

    Pro Pro Asp Asp Leu Pro Asn Gly His Val Asp Tyr Ile Thr Gly Pro 355 360 365

    Gln Val Thr Thr Tyr Lys Ala Val Ile Gln Tyr Ser Cys Glu Glu Thr 370 375 380

    Phe Tyr Thr Met Ser Ser Asn Gly Lys Tyr Val Cys Glu Ala Asp Gly 385 390 395 400

    Phe Trp Thr Ser Ser Lys Gly Glu Lys Leu Pro Pro Val Cys Glu Pro 405 410 415

    Val Cys Gly Leu Ser Thr His Thr Ile Gly Gly Arg Ile Val Gly Gly 420 425 430

    Gln Pro Ala Lys Pro Gly Asp Phe Pro Trp Gln Val Leu Leu Leu Gly 435 440 445

    Gln Thr Thr Ala Ala Ala Gly Ala Leu Ile His Asp Asn Trp Val Leu 450 455 460

    Thr Ala Ala His Ala Val Tyr Glu Lys Arg Met Ala Ala Ser Ser Leu 465 470 475 480

    Ala Trp Pro Glu Glu Ile Phe Ile His Glu Gly Tyr Thr His Gly Ala 500 505 510

    Gly Phe Asp Asn Asp Ile Ala Leu Ile Lys Leu Lys Asn Lys Val Thr

    Asn Ile Arg Met Gly Ile Leu Lys Arg Leu Ser Pro His Tyr Thr Gln

    485 490 495

    Page 53

    2018200437 19 Jan 2018

    515 520 525

    Ile Asn Gly Ser Ile Met Pro Val Cys Leu Pro Arg Lys Glu Ala Ala 530 535 540

    Ser Leu Met Arg Thr Asp Phe Thr Gly Thr Val Ala Gly Trp Gly Leu 545 550 555 560

    Thr Gln Lys Gly Leu Leu Ala Arg Asn Leu Met Phe Val Asp Ile Pro 565 570 575

    Ile Ala Asp His Gln Lys Cys Thr Thr Val Tyr Glu Lys Leu Tyr Pro 580 585 590

    Gly Val Arg Val Ser Ala Asn Met Leu Cys Ala Gly Leu Glu Thr Gly 595 600 605

    Gly Lys Asp Ser Cys Arg Gly Asp Ser Gly Gly Ala Leu Val Phe Leu 610 615 620

    Asp Asn Glu Thr Gln Arg Trp Phe Val Gly Gly Ile Val Ser Trp Gly 625 630 635 640

    Ser Ile Asn Cys Gly Ala Ala Gly Gln Tyr Gly Val Tyr Thr Lys Val 645 650 655

    Ile Asn Tyr Ile Pro Trp Asn Glu Asn Ile Ile Ser Asn Phe 660 665 670 <210> 53 <211> 2091 <212> DNA <213> Rat <220>

    <221> CDS <222> (10)..(2067) <400> 53

    Page 54

    2018200437 19 Jan 2018 tggcacaca atg agg eta ctg ate gtc ctg ggt ctg ett tgg agt ttg gtg 51 Met Arg Leu Leu Ile Val Leu Gly Leu Leu Trp Ser Leu Val 1 5 10 gcc aca ett ttg ggc tcc aag tgg cct gag cct gta ttc ggg ege ctg 99 Ala Thr Leu Leu Gly Ser Lys Trp Pro Glu Pro Val Phe Gly Arg Leu 15 20 25 30 gtg tcc ctg gcc ttc cca gag aag tat ggc aac cat cag gat cga tcc 147 Val Ser Leu Ala Phe Pro Glu Lys Tyr Gly Asn His Gln Asp Arg Ser

    35 40 45 tgg aeg ctg act gca ccc cct ggc ttc ege ctg ege etc tac ttc acc 195 Trp Thr Leu Thr Ala Pro Pro Gly Phe Arg Leu Arg Leu Tyr Phe Thr

    50 55 60 cac ttc aac ctg gaa etc tet tac ege tgc gag tat gac ttt gtc aag 243 His Phe Asn Leu Glu Leu Ser Tyr Arg Cys Glu Tyr Asp Phe Val Lys

    65 70 75 ttg acc tea ggg acc aag gtg eta gcc aeg ctg tgt ggg cag gag agt 291 Leu Thr Ser Gly Thr Lys Val Leu Ala Thr Leu Cys Gly Gln Glu Ser

    80 85 90 aca gat act gag egg gca cct ggc aat gac acc ttc tac tea ctg ggt 339 Thr Asp Thr Glu Arg Ala Pro Gly Asn Asp Thr Phe Tyr Ser Leu Gly 95 100 105 110 ccc age eta aag gtc acc ttc cac tcc gac tac tcc aat gag aag cca 387 Pro Ser Leu Lys Val Thr Phe His Ser Asp Tyr Ser Asn Glu Lys Pro

    115 120 125 ttc aca gga ttt gag gcc ttc tat gca gcg gag gat gtg gat gaa tgc 435 Phe Thr Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp Val Asp Glu Cys

    130 135 140 aga aca tcc ctg gga gac tea gtc cct tgt gac cat tat tgc cac aac 483 Arg Thr Ser Leu Gly Asp Ser Val Pro Cys Asp His Tyr Cys His Asn

    145 150 155 tac ctg ggc ggc tac tac tgc tcc tgc cga gtg ggc tac att ctg cac 531 Tyr Leu Gly Gly Tyr Tyr Cys Ser Cys Arg Val Gly Tyr Ile Leu His

    160 165 170 cag aac aag cat acc tgc tea gcc ett tgt tea ggc cag gtg ttc act 579 Gln Asn Lys His Thr Cys Ser Ala Leu Cys Ser Gly Gln Val Phe Thr 175 180 185 190 ggg agg tet ggc ttt etc agt age cct gag tac cca cag cca tac ccc 627 Gly Arg Ser Gly Phe Leu Ser Ser Pro Glu Tyr Pro Gln Pro Tyr Pro

    Page 55

    2018200437 19 Jan 2018

    195 200 205 aaa etc tee age tgc gee tac aac ate ege ctg gag gaa ggc ttc agt 675 Lys Leu Ser Ser Cys Ala Tyr Asn Ile Arg Leu Glu Glu Gly Phe Ser

    210 215 220 ate ace ctg gac ttc gtg gag tee ttt gat gtg gag atg cac cct gaa 723 Ile Thr Leu Asp Phe Val Glu Ser Phe Asp Val Glu Met His Pro Glu

    225 230 235 gee cag tgc ccc tac gac tee etc aag att caa aca gac aag agg gaa 771 Ala Gln Cys Pro Tyr Asp Ser Leu Lys Ile Gln Thr Asp Lys Arg Glu

    240 245 250 tac ggc ccg ttt tgt ggg aag aeg ctg ccc ccc agg att gaa act gac 819 Tyr Gly Pro Phe Cys Gly Lys Thr Leu Pro Pro Arg Ile Glu Thr Asp 255 260 265 270 age aac aag gtg ace att ace ttt ace acc gac gag tea ggg aac cac 867 Ser Asn Lys Val Thr Ile Thr Phe Thr Thr Asp Glu Ser Gly Asn His

    275 280 285 aca ggc tgg aag ata cac tac aca age aca gca cag ccc tgc cct gat 915 Thr Gly Trp Lys Ile His Tyr Thr Ser Thr Ala Gln Pro Cys Pro Asp

    290 295 300 cca aeg geg cca cct aat ggt cac att tea cct gtg caa gee aeg tat 963 Pro Thr Ala Pro Pro Asn Gly His Ile Ser Pro Val Gln Ala Thr Tyr

    305 310 315 gtc ctg aag gac age ttt tet gtc ttc tgc aag act ggc ttc gag ett 1011 Val Leu Lys Asp Ser Phe Ser Val Phe Cys Lys Thr Gly Phe Glu Leu

    320 325 330 ctg caa ggt tet gtc ccc ctg aag tea ttc act get gtc tgt cag aaa 1059 Leu Gln Gly Ser Val Pro Leu Lys Ser Phe Thr Ala Val Cys Gln Lys 335 340 345 350 gat gga tet tgg gac egg ccg ata cca gag tgc age att att gac tgt 1107 Asp Gly Ser Trp Asp Arg Pro Ile Pro Glu Cys Ser Ile Ile Asp Cys

    355 360 365 ggc cct ccc gat gac eta ccc aat ggc cac gtg gac tat ate aca ggc 1155 Gly Pro Pro Asp Asp Leu Pro Asn Gly His Val Asp Tyr Ile Thr Gly

    370 375 380 cct gaa gtg acc acc tac aaa get gtg att cag tac age tgt gaa gag 1203 Pro Glu Val Thr Thr Tyr Lys Ala Val Ile Gln Tyr Ser Cys Glu Glu

    385 390 395

    Page 56

    2018200437 19 Jan 2018 act ttc tac aca atg age age aat ggt aaa tat gtg tgt gag get gat 1251 Thr Phe Tyr Thr Met Ser Ser Asn Gly Lys Tyr Val Cys Glu Ala Asp

    400 405 410 gga ttc tgg aeg age tee aaa gga gaa aaa tee etc ccg gtt tgc aag 1299 Gly Phe Trp Thr Ser Ser Lys Gly Glu Lys Ser Leu Pro Val Cys Lys 415 420 425 430 cct gtc tgt gga ctg tee aca cac act tea gga ggc cgt ata att gga 1347 Pro Val Cys Gly Leu Ser Thr His Thr Ser Gly Gly Arg Ile Ile Gly

    435 440 445 gga cag cct gca aag cct ggt gac ttt cct tgg caa gtc ttg tta ctg 1395 Gly Gln Pro Ala Lys Pro Gly Asp Phe Pro Trp Gln Val Leu Leu Leu

    450 455 460 ggt gaa act aca gca gca ggt get ett ata cat gac gac tgg gtc cta 1443 Gly Glu Thr Thr Ala Ala Gly Ala Leu Ile His Asp Asp Trp Val Leu

    465 470 475 aca geg get cat get gta tat ggg aaa aca gag geg atg tee tee ctg 1491 Thr Ala Ala His Ala Val Tyr Gly Lys Thr Glu Ala Met Ser Ser Leu

    480 485 490 gac ate ege atg ggc ate etc aaa agg etc tee etc att tac act caa 1539 Asp Ile Arg Met Gly Ile Leu Lys Arg Leu Ser Leu Ile Tyr Thr Gln 495 500 505 510 gee tgg cca gag get gtc ttt ate cat gaa ggc tac act cac gga get 1587 Ala Trp Pro Glu Ala Val Phe Ile His Glu Gly Tyr Thr His Gly Ala

    515 520 525 ggt ttt gac aat gat ata gca ctg att aaa etc aag aac aaa gtc aca 1635 Gly Phe Asp Asn Asp Ile Ala Leu Ile Lys Leu Lys Asn Lys Val Thr

    530 535 540 ate aac aga aac ate atg ccg att tgt cta cca aga aaa gaa get gca 1683 Ile Asn Arg Asn Ile Met Pro Ile Cys Leu Pro Arg Lys Glu Ala Ala

    545 550 555 tee tta atg aaa aca gac ttc gtt gga act gtg get ggc tgg ggg tta 1731 Ser Leu Met Lys Thr Asp Phe Val Gly Thr Val Ala Gly Trp Gly Leu

    560 565 570 ace cag aag ggg ttt ett get aga aac cta atg ttt gtg gac ata cca 1779 Thr Gln Lys Gly Phe Leu Ala Arg Asn Leu Met Phe Val Asp Ile Pro 575 580 585 590 att gtt gac cac caa aaa tgt get act geg tat aca aag cag ccc tac 1827 Ile Val Asp His Gln Lys Cys Ala Thr Ala Tyr Thr Lys Gln Pro Tyr

    Page 57

    2018200437 19 Jan 2018

    595 600 605 cca gga gca aaa gtg act gtt aac atg etc tgt get ggc eta gac cgc 1875 Pro Gly Ala Lys Val Thr Val Asn Met Leu Cys Ala Gly Leu Asp Arg

    610 615 620 ggt ggc aag gac agc tgc aga ggt gac agc gga ggg gca tta gtg ttt 1923 Gly Gly Lys Asp Ser Cys Arg Gly Asp Ser Gly Gly Ala Leu Val Phe

    625 630 635 eta gac aat gaa aca cag aga tgg ttt gtg gga gga ata gtt tcc tgg 1971 Leu Asp Asn Glu Thr Gln Arg Trp Phe Val Gly Gly Ile Val Ser Trp

    640 645 650 ggt tet att aac tgt ggg ggg tea gaa cag tat ggg gtc tac acg aaa 2019 Gly Ser Ile Asn Cys Gly Gly Ser Glu Gln Tyr Gly Val Tyr Thr Lys 655 660 665 670 gtc acg aac tat att ccc tgg att gag aac ata ata aat aat ttc taa 2067 Val Thr Asn Tyr Ile Pro Trp Ile Glu Asn Ile Ile Asn Asn Phe

    675 680 685 tttgcaaaaa aaaaaaaaaa aaaa 2091 <210> 54 <211> 685 <212> PRT <213> Rat <400> 54

    Met Arg Leu Leu Ile Val Leu Gly Leu Leu Trp Ser Leu Val Ala Thr 15 10 15

    Leu Leu Gly Ser Lys Trp Pro Glu Pro Val Phe Gly Arg Leu Val Ser 20 25 30

    Leu Ala Phe Pro Glu Lys Tyr Gly Asn His Gln Asp Arg Ser Trp Thr 35 40 45

    Leu Thr Ala Pro Pro Gly Phe Arg Leu Arg Leu Tyr Phe Thr His Phe 50 55 60

    Asn Leu Glu Leu Ser Tyr Arg Cys Glu Tyr Asp Phe Val Lys Leu Thr 65 70 75 80

    Page 58

    2018200437 19 Jan 2018

    Ser Gly Thr Lys Val Leu Ala Thr Leu Cys Gly Gln Glu Ser Thr Asp 85 90 95

    Thr Glu Arg Ala Pro Gly Asn Asp Thr Phe Tyr Ser Leu Gly Pro Ser 100 105 110

    Leu Lys Val Thr Phe His Ser Asp Tyr Ser Asn Glu Lys Pro Phe Thr 115 120 125

    Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp Val Asp Glu Cys Arg Thr 130 135 140

    Ser Leu Gly Asp Ser Val Pro Cys Asp His Tyr Cys His Asn Tyr Leu 145 150 155 160

    Gly Gly Tyr Tyr Cys Ser Cys Arg Val Gly Tyr Ile Leu His Gln Asn 165 170 175

    Lys His Thr Cys Ser Ala Leu Cys Ser Gly Gln Val Phe Thr Gly Arg 180 185 190

    Ser Gly Phe Leu Ser Ser Pro Glu Tyr Pro Gln Pro Tyr Pro Lys Leu 195 200 205

    Ser Ser Cys Ala Tyr Asn Ile Arg Leu Glu Glu Gly Phe Ser Ile Thr 210 215 220

    Leu Asp Phe Val Glu Ser Phe Asp Val Glu Met His Pro Glu Ala Gln 225 230 235 240

    Pro Phe Cys Gly Lys Thr Leu Pro Pro Arg Ile Glu Thr Asp Ser Asn 260 265 270

    Cys Pro Tyr Asp Ser Leu Lys Ile Gln Thr Asp Lys Arg Glu Tyr Gly

    245 250 255

    Page 59

    2018200437 19 Jan 2018

    Lys Val Thr Ile Thr Phe Thr Thr Asp Glu Ser Gly Asn His Thr Gly 275 280 285

    Trp Lys Ile His Tyr Thr Ser Thr Ala Gln Pro Cys Pro Asp Pro Thr 290 295 300

    Ala Pro Pro Asn Gly His Ile Ser Pro Val Gln Ala Thr Tyr Val Leu 305 310 315 320

    Lys Asp Ser Phe Ser Val Phe Cys Lys Thr Gly Phe Glu Leu Leu Gln 325 330 335

    Gly Ser Val Pro Leu Lys Ser Phe Thr Ala Val Cys Gln Lys Asp Gly 340 345 350

    Ser Trp Asp Arg Pro Ile Pro Glu Cys Ser Ile Ile Asp Cys Gly Pro 355 360 365

    Pro Asp Asp Leu Pro Asn Gly His Val Asp Tyr Ile Thr Gly Pro Glu 370 375 380

    Val Thr Thr Tyr Lys Ala Val Ile Gln Tyr Ser Cys Glu Glu Thr Phe 385 390 395 400

    Tyr Thr Met Ser Ser Asn Gly Lys Tyr Val Cys Glu Ala Asp Gly Phe 405 410 415

    Trp Thr Ser Ser Lys Gly Glu Lys Ser Leu Pro Val Cys Lys Pro Val 420 425 430

    Cys Gly Leu Ser Thr His Thr Ser Gly Gly Arg Ile Ile Gly Gly Gln 435 440 445

    Pro Ala Lys Pro Gly Asp Phe Pro Trp Gln Val Leu Leu Leu Gly Glu 450 455 460

    Thr Thr Ala Ala Gly Ala Leu Ile His Asp Asp Trp Val Leu Thr Ala 465 470 475 480

    Page 60

    2018200437 19 Jan 2018

    Ala His Ala Val Tyr Gly Lys Thr Glu Ala Met Ser Ser Leu Asp Ile 485 490 495

    Arg Met Gly Ile Leu Lys Arg Leu Ser Leu Ile Tyr Thr Gln Ala Trp 500 505 510

    Pro Glu Ala Val Phe Ile His Glu Gly Tyr Thr His Gly Ala Gly Phe 515 520 525

    Asp Asn Asp Ile Ala Leu Ile Lys Leu Lys Asn Lys Val Thr Ile Asn 530 535 540

    Arg Asn Ile Met Pro Ile Cys Leu Pro Arg Lys Glu Ala Ala Ser Leu 545 550 555 560

    Met Lys Thr Asp Phe Val Gly Thr Val Ala Gly Trp Gly Leu Thr Gln 565 570 575

    Lys Gly Phe Leu Ala Arg Asn Leu Met Phe Val Asp Ile Pro Ile Val 580 585 590

    Asp His Gln Lys Cys Ala Thr Ala Tyr Thr Lys Gln Pro Tyr Pro Gly 595 600 605

    Ala Lys Val Thr Val Asn Met Leu Cys Ala Gly Leu Asp Arg Gly Gly 610 615 620

    Lys Asp Ser Cys Arg Gly Asp Ser Gly Gly Ala Leu Val Phe Leu Asp 625 630 635 640

    Ile Asn Cys Gly Gly Ser Glu Gln Tyr Gly Val Tyr Thr Lys Val Thr 660 665 670

    Asn Glu Thr Gln Arg Trp Phe Val Gly Gly Ile Val Ser Trp Gly Ser

    645 650 655

    Page 61

    2018200437 19 Jan 2018

    Asn Tyr Ile Pro Trp Ile Glu Asn Ile Ile Asn Asn Phe 675 680 685 <210> 55 <211> 670 <212> PRT <213> Rat <400> 55

    Thr Leu Leu Gly Ser Lys Trp Pro Glu Pro Val Phe Gly Arg Leu Val 15 10 15

    Ser Leu Ala Phe Pro Glu Lys Tyr Gly Asn His Gln Asp Arg Ser Trp 20 25 30

    Thr Leu Thr Ala Pro Pro Gly Phe Arg Leu Arg Leu Tyr Phe Thr His 35 40 45

    Phe Asn Leu Glu Leu Ser Tyr Arg Cys Glu Tyr Asp Phe Val Lys Leu 50 55 60

    Thr Ser Gly Thr Lys Val Leu Ala Thr Leu Cys Gly Gln Glu Ser Thr 65 70 75 80

    Asp Thr Glu Arg Ala Pro Gly Asn Asp Thr Phe Tyr Ser Leu Gly Pro 85 90 95

    Ser Leu Lys Val Thr Phe His Ser Asp Tyr Ser Asn Glu Lys Pro Phe 100 105 110

    Thr Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp Val Asp Glu Cys Arg 115 120 125

    Thr Ser Leu Gly Asp Ser Val Pro Cys Asp His Tyr Cys His Asn Tyr 130 135 140

    Leu Gly Gly Tyr Tyr Cys Ser Cys Arg Val Gly Tyr Ile Leu His Gln 145 150 155 160

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    Asn Lys His Thr Cys Ser Ala Leu Cys Ser Gly Gln Val Phe Thr Gly 165 170 175

    Arg Ser Gly Phe Leu Ser Ser Pro Glu Tyr Pro Gln Pro Tyr Pro Lys 180 185 190

    Leu Ser Ser Cys Ala Tyr Asn Ile Arg Leu Glu Glu Gly Phe Ser Ile 195 200 205

    Thr Leu Asp Phe Val Glu Ser Phe Asp Val Glu Met His Pro Glu Ala 210 215 220

    Gln Cys Pro Tyr Asp Ser Leu Lys Ile Gln Thr Asp Lys Arg Glu Tyr 225 230 235 240

    Gly Pro Phe Cys Gly Lys Thr Leu Pro Pro Arg Ile Glu Thr Asp Ser 245 250 255

    Asn Lys Val Thr Ile Thr Phe Thr Thr Asp Glu Ser Gly Asn His Thr 260 265 270

    Gly Trp Lys Ile His Tyr Thr Ser Thr Ala Gln Pro Cys Pro Asp Pro 275 280 285

    Thr Ala Pro Pro Asn Gly His Ile Ser Pro Val Gln Ala Thr Tyr Val 290 295 300

    Leu Lys Asp Ser Phe Ser Val Phe Cys Lys Thr Gly Phe Glu Leu Leu 305 310 315 320

    Gly Ser Trp Asp Arg Pro Ile Pro Glu Cys Ser Ile Ile Asp Cys Gly 340 345 350

    Pro Pro Asp Asp Leu Pro Asn Gly His Val Asp Tyr Ile Thr Gly Pro

    Gln Gly Ser Val Pro Leu Lys Ser Phe Thr Ala Val Cys Gln Lys Asp

    325 330 335

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    2018200437 19 Jan 2018

    355 360 365

    Glu Val Thr Thr Tyr Lys Ala Val Ile Gln Tyr Ser Cys Glu Glu Thr 370 375 380

    Phe Tyr Thr Met Ser Ser Asn Gly Lys Tyr Val Cys Glu Ala Asp Gly 385 390 395 400

    Phe Trp Thr Ser Ser Lys Gly Glu Lys Ser Leu Pro Val Cys Lys Pro 405 410 415

    Val Cys Gly Leu Ser Thr His Thr Ser Gly Gly Arg Ile Ile Gly Gly 420 425 430

    Gln Pro Ala Lys Pro Gly Asp Phe Pro Trp Gln Val Leu Leu Leu Gly 435 440 445

    Glu Thr Thr Ala Ala Gly Ala Leu Ile His Asp Asp Trp Val Leu Thr 450 455 460

    Ala Ala His Ala Val Tyr Gly Lys Thr Glu Ala Met Ser Ser Leu Asp 465 470 475 480

    Ile Arg Met Gly Ile Leu Lys Arg Leu Ser Leu Ile Tyr Thr Gln Ala 485 490 495

    Trp Pro Glu Ala Val Phe Ile His Glu Gly Tyr Thr His Gly Ala Gly 500 505 510

    Phe Asp Asn Asp Ile Ala Leu Ile Lys Leu Lys Asn Lys Val Thr Ile 515 520 525

    Asn Arg Asn Ile Met Pro Ile Cys Leu Pro Arg Lys Glu Ala Ala Ser 530 535 540

    Leu Met Lys Thr Asp Phe Val Gly Thr Val Ala Gly Trp Gly Leu Thr 545 550 555 560

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    Gln Lys Gly Phe Leu Ala Arg Asn Leu Met Phe Val Asp Ile Pro Ile 565 570 575

    Val Asp His Gln Lys Cys Ala Thr Ala Tyr Thr Lys Gln Pro Tyr Pro 580 585 590

    Gly Ala Lys Val Thr Val Asn Met Leu Cys Ala Gly Leu Asp Arg Gly 595 600 605

    Gly Lys Asp Ser Cys Arg Gly Asp Ser Gly Gly Ala Leu Val Phe Leu 610 615 620

    Asp Asn Glu Thr Gln Arg Trp Phe Val Gly Gly Ile Val Ser Trp Gly 625 630 635 640

    Ser Ile Asn Cys Gly Gly Ser Glu Gln Tyr Gly Val Tyr Thr Lys Val 645 650 655

    Thr Asn Tyr Ile Pro Trp Ile Glu Asn Ile Ile Asn Asn Phe 660 665 670 <210> 56 <211> 28 <212> DNA <213> Artificial Sequence <220>

    <223> Homo Sapiens <400> 56 atgaggctgc tgaccctcct gggccttc 28 <210> 57 <211> 23 <212> DNA <213> Artificial Sequence <220>

    <223> Homo Sapiens <400> 57

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    2018200437 19 Jan 2018 gtgcccctcc tgcgtcacct ctg 23 <210> 58 <211> 23 <212> DNA <213 > Artificial Sequence <220>

    <223> Homo Sapiens <400> 58 cagaggtgac gcaggagggg cac 23 <210> 59 <211> 27 <212> DNA <213 > Artificial Sequence <220>

    <223> Homo Sapiens <400> 59 ttaaaatcac taattatgtt ctcgatc 27 <210> 60 <211> 22 <212> DNA <213 > Artificial Sequence <220>

    <223> Murine <400> 60 atgaggctac tcatcttcct gg 22 <210> 61 <211> 23 <212> DNA <213> Artificial Sequence <220>

    <223> Murine <400> 61 ctgcagaggt gacgcagggg ggg

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    2018200437 19 Jan 2018 <210> 62 <211> 23 <212> DNA <213> Artificial Sequence <220>

    <223> Murine <400> 62 ccccccctgc gtcacctctg cag 23 <210> 63 <211> 29 <212> DNA <213> Artificial Sequence <220>

    <223> Murine <400> 63 ttagaaatta cttattatgt tctcaatcc 29 <210> 64 <211> 29 <212> DNA <213> Artificial Sequence <220>

    <223> Rat <400> 64 gaggtgacgc aggaggggca ttagtgttt 29 <210> 65 <211> 37 <212> DNA <213> Artificial Sequence <220>

    <223> Rat <400> 65 ctagaaacac taatgcccct cctgcgtcac ctctgca 37