EA045598B1 - METHODS FOR INHIBITION OF ANGIOGENESIS IN A PATIENT - Google Patents

Description

Cross reference to related applications

This patent application claims priority to provisional patent application No. 62/315857, filed March 31, 2016, the entire contents of which are therefore incorporated by reference.

State of the art

The complement system provides an early mechanism for initiating, enhancing, and coordinating the immune response to infection by microorganisms 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 significant first-line protection against potential pathogens, types of complement activity that stimulate a protective immune response may also pose a potential threat to the host (K. R. Kalli, et al., Springer Semin. Immunopathol. 15:417 431, 19 94; B. P. Morgan, Eur J Clinical Investig 24:219 228, 1994). For example, the proteolytic products C3 and C5 attract and activate neutrophils. While they are an integral part of the host's defense, activated neutrophils are indiscriminate in their release of destructive enzymes and can cause organ damage. In addition, complement activation can cause the accumulation of lytic complement components in the vicinity of host cells, as well as on target microorganisms, leading to 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, reperfusion injury, septic shock, hypercapillary burn syndrome, inflammation after cardiopulmonary bypass surgery, graft 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. However, complement activation may be a major pathological mechanism and represents an effective point of clinical control in many of these disease states. Growing understanding of the importance of complement-mediated tissue damage in a variety of disease states underscores the need for effective complement-inhibiting drugs. To date, eculizumab (Solaris®), an antibody against complement component C5, is the only complement-targeting drug approved for use in humans. However, C5 is one of several effector molecules located downstream in the complement activation cascade, and blocking C5 does not inhibit complement activation. Thus, an inhibitor of the initial stages of complement activation may have significant advantages over a downstream complement inhibitor.

It is now generally 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 typically initiated by a complex consisting of host antibodies bound to a foreign particle (i.e., antigen) and thus requires prior exposure to the antigen to elicit a specific antibody response. Because activation of the classical pathway depends on the host's prior adaptive immune response, 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.

Activation of the complement system leads to the sequential activation of zymogens - serine proteases. The first step in the activation of the classical pathway is the binding of a specific recognition molecule, C1q, to antigen bound IgG and IgM molecules. C1q is associated with the serine protease proenzymes Clr and Cls in the form of a complex called Cl. Following binding of C1q to the immune complex, autoproteolytic cleavage of the Arg-Ile site of Clr is followed by Clr-mediated cleavage and activation of Cls, which thereby acquires the ability to cleave C4 and C2. C4 splits into two fragments, designated C4a and C4b, and similarly, C2 splits into C2a and C2b. C4b fragments are capable of forming covalent bonds with adjacent hydroxyl groups or amino groups and form C3 convertase (C4b2a) through non-covalent interaction with the C2a fragment of activated C2. C3 convertase (C4b2a) activates C3 through proteolytic cleavage into subcomponents C3a and C3b, resulting in the formation of C5 convertase (C4b2a3b), which, through cleavage of C5, leads to the formation of the membrane attack complex (C5b in combination with polymers C6, C7, C8 and C9 , also referred to as MAC), which can disrupt cell membranes, leading to cell lysis. Activated forms of C3 and C4 (C3b and C4b) accumulate covalently on the surfaces of foreign targets that are recognized by complement receptors on a variety of phagocytes.

The first step in activation of the complement system through the lectin pathway is the binding of molecules that recognize a lectin pathway-specific pattern to their target ligands. This process initiates the activation of lectin pathway-specific serine protease proenzymes, which in turn initiate a cascade of complement reactions. The pattern recognition molecules in the lectin pathway contain a group of carbohydrate-binding C-type lectins, i.e. mannan binding lectin (MBL), collectin-11 (CL-11, also known as CL-K1), collectin-10 (CL-10, also known as CL-L1), and three different ficolins, i.e. H-ficolin, M-ficolin and L-ficolin, which bind to acetylated carbohydrate and protein structures via fibrinogen-like binding domains (J. Lu et al., Biochim. Biophys. Acta 1572:387 400, (2002); Holmskov et al. ., Annu. Rev. Immunol. 21:547 578 (2003); Teh et al., Immunology 101:225 232 (2000); J. Luet et al., Biochim Biophys Acta 1572: 387400 (2002); Hansen et al. ., J. Immunol 185(10):6096–6104 (2010), and Hendriksen et al., J Immunol 191(12):611727, 2013).

Ikeda et al. first showed that, like C1q, MBL can activate the complement system when binding to yeast mannan-coated erythrocytes in a C4-dependent manner (Ikeda et al., J. Biol. Chem. 262:7451 7454, (1987)). MBL, a member of the collectin protein family, is a calcium-dependent lectin that binds carbohydrates with 3 and 4 hydroxy groups oriented in the equatorial plane of the pyranose ring. The prominent ligands for MBL are thus D-mannose and N-acetyl-O-glucosamine, while carbohydrates that do not meet these steric requirements have undetectable affinity for MBL (Weis et al., Nature 360:127 134, (1992)). The interaction between MBL and monovalent sugars is unusually weak, with dissociation constants typically in the single digit millimolar range. MBL achieves tight, specific binding to glycan ligands through avidity, i.e. through simultaneous interaction with numerous monosaccharide residues located in close proximity to each other (Lee et al., Archiv. Biochem. Biophys. 299:129 136, (1992)). MBL recognizes carbohydrate patterns commonly found on microorganisms such as bacteria, yeast, parasites and certain viruses. In contrast, MBL does not recognize D-galactose and sialic acid, the penultimate and final sugars typically capping mature complex glycoconjugates present in glycoproteins in the plasma and on the surface of mammalian cells. This binding specificity is thought to promote recognition of foreign surfaces and help protect against autoactivation. However, MBL does not bind with high affinity to high-mannose glycan precursor clusters on N-linked glycoproteins and glycolipids sequestered in the endoplasmic reticulum and Golgi apparatus of mammalian cells (Maynard et al., J. Biol. Chem. 257:3788 3794, (1982)). Thus, damaged cells are potential targets for lectin pathway activation through MBL binding, and more recent work has shown that CL-11 is another lectin pathway recognition subcomponent that initiates lectin pathway activation on stressed or damaged cells (Farar et al ., J Clin Invest 126:1911–1925, 2016).

Ficolins have a different type of lectin domain from MBL, called fibrinogen-like domain. Ficolins bind sugar residues in a Ca^-independent manner. Three types of ficolins have been identified in humans (L-ficolin, M-ficolin and H-ficolin). Two serum ficolins, L-ficolin and H-ficolin, share specificity for N-acetyl-O-glucosamine; however, H-ficolin also binds N-acetyl-O-galactosamine. The difference in specificity for the sugars L-ficolin, H-ficolin, CL11 and MBL means that different lectins may be complementary to and target different, albeit overlapping, glycoconjugates. This concept is supported by the recent report that of the known lectins in the lectin pathway, only L-ficolin binds specifically to lipoteichoic acid, a cell wall glycococonjugate found on all gram-positive bacteria (Lynch et al., J. Immunol. 172:1198 1202, (2004)). Collectins (i.e., MBL, CL-11, CL-10, and CL-11/CL-10 complexes) and ficolins do not share significant amino acid sequence similarity. However, these two groups of proteins have similar domain organizations and, like C1q, assemble into oligomeric structures that maximize the ability to bind at multiple sites.

Serum MBL concentrations are highly variable in healthy populations, and this is genetically controlled through polymorphisms/mutations in both the promoter and coding regions of the MBL gene. As an acute phase protein, MBL expression is further upregulated during inflammation. L-ficolin is present in serum at concentrations similar to those of MBL. Thus, the L-ficolin branch of the lectin pathway is potentially comparable to the MBL branch in physiological importance. MBL and ficolins may also function as opsonins that allow phagocytes to target MBL and ficolin-coated 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):418-25 (2005) This opsonization requires the interaction of these proteins with phagocyte receptors (Kuhlman et al., J Exp. Med. 169:1133, (1989); Matsushita et al., J. Biol. Chem. 271:2448 54, (1996)), the identity of which has not been established.

Human MBL forms a specific and high-affinity interaction through its collagen-like domain with unique Clr/Cls-like serine proteases called MBL-associated serine proteases (MASPs). To date, three MASPs have been described. First, a single MASP enzyme was identified and characterized as the enzyme responsible

- 2 045598 responsible for the initiation of the complement cascade (i.e., cleavage of C2 and C4) (Matsushita et al., J Exp Med 176(6):1497-1502 (1992); Ji et al., J. Immunol. 150:571578, (1993)). It was subsequently determined that MASP activity was actually a mixture of two proteases: MASP-1 and MASP-2 (Thiel et al., Nature 386:506 510, (1997)). However, the MBL-MASP-2 complex by itself has been shown to be sufficient to activate complement (Vorup Jensen et al., J. Immunol. 165:2093 2100, (2000)). In addition, only MASP-2 degraded C2 and C4 with high efficiency (Ambrus et al., J. Immunol. 170:1374 1382, (2003)). Thus, MASP-2 is the protease responsible for activating C4 and C2 to produce the C3 convertase, C4b2a. This is its significant difference from the C1 complex of the classical pathway, where the coordinated activity of two specific serine proteases (Clr and Cls) leads to activation of the complement system. In addition, a third novel protease, MASP-3, has been isolated (Dahl, M.R., et al., Immunity 15:121 35, 2001). MASP-1 and MASP-3 are products of the same gene resulting from alternative splicing.

MASPs share identical domain organization with the Clr and Cls domains, enzyme components of the Cl complex (Sim et al., Biochem. Soc. Trans. 28:545, (2000)). These domains include the sea urchin N-terminal Clr/Cls/VEGF/bone morphogenetic 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 Cl proteases, activation of MASP-2 occurs through cleavage of the Arg-I1e bond adjacent to the serine protease domain, resulting in cleavage of the enzyme into disulfide-linked chains A and B, the latter of which consists of the serine protease domain.

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

Several lines of evidence suggest that there are different MBL-MASP complexes and that a large fraction of MASP in serum is not complexed with MBL (Thiel, et al., J. Immunol. 165:878 887, (2000)). Both H- and L-ficolin bind to all MASPs and activate the complement lectin pathway, as does MBL (Dahl et al., Immunity 15:127 35, (2001); Matsushita et al., J. Immunol. 158 :3502 3506, (2002)). Both the lectin and classical pathways form a common C3 convertase (C4b2a), and both activation pathways converge at this stage.

The lectin complement pathway is widely believed to play a leading role in host defense against infection in the naïve host. Strong evidence for the involvement of MBL in host defense comes from analyzes of patients with reduced levels of functional MBL in serum (Kilpatrick, Biochim. Biophys. Acta 1572:401 413, (2002)). For such patients, sensitivity to recurrent bacterial and fungal infections is indicated. These symptoms usually appear early in life, during a marked window of vulnerability when the titer of maternally derived antibodies declines but before the development of the full repertoire of antibody responses. This syndrome often results from mutations at several sites in the collagen portion of the MBL that prevent the proper formation of MBL oligomers. However, because MBL can function as a complement-independent opsonin, it is unknown to what extent the increased susceptibility to infection is due to impaired complement activation.

In contrast to the classical and lectin pathways, no alternative pathway initiators have been identified to perform the recognition functions performed by C1q and lectins in the other two pathways. It is now generally accepted that the alternative pathway spontaneously undergoes low levels of variable activation, which can easily be enhanced on foreign or other abnormal surfaces (bacteria, yeast, virus-infected cells or damaged tissue) lacking the proper molecular elements to keep spontaneous complement activation under control. . There are four plasma proteins directly involved in alternative pathway activation: C3, factors B and D, and properdin.

Despite extensive evidence for the involvement of both the classical and alternative complement pathways in the pathogenesis of human non-infectious diseases, the role of the lectin pathway is only beginning to be appreciated. Recent studies provide evidence that activation of the lectin pathway may be responsible for complement activation and associated inflammation during ischemia/reperfusion injury. Collard et al. (2000) published that cultured endothelial cells subjected to oxidative stress bind to MBL and exhibit accumulation of C3 when exposed to human serum (Collard et al., Am. J. Pathol. 156:1549 1556, (2000)). In addition, treatment of human serum with blocking monoclonal antibodies against MBL inhibited MBL binding and complement activation. These discoveries extended to

- 3045598 a rat model of myocardial ischemia-reperfusion in which rats treated with a blocking antibody directed against rat MBL showed significantly less myocardial damage after coronary artery occlusion than rats treated with a control antibody (Jordan et al., Circulation 104:1413 1418, (2001)). The molecular mechanism of MBL binding to the vascular endothelium after oxidative stress remains unclear; a recent study suggests that activation of the lectin pathway after oxidative stress may be mediated by binding of MBL to vascular endothelial cytokeratins rather than to glycoconjugates (Collard et al., Am. J. Pathol. 159:1045 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 al., Am. J. Pathol. 162:363-367, 2003).

A recent study shows that MASP-1 (and possibly also MASP-3) is required for the conversion of the factor D alternative pathway activating enzyme from its zymogenic form to its enzymatically active form (see Takahashi M. et al., J Exp Med 207(1):29-37 (2010)). The physiological importance of this process is emphasized by the lack of functional activity of the alternative pathway in the plasma of MASP-1/3-deficient mice. Proteolytic production of C3b from native C3 is required for the alternative pathway to function. Because the alternative pathway C3 convertase (C3bBb) contains C3b as a required subunit, the question regarding the origin of the first C3b through the alternative pathway is a puzzling problem and prompts significant research.

C3 belongs to a family of proteins (together with C4 and α2-macroglobulin) that has a rare post-translational modification known as a thioester linkage. The thioester group consists of glutamine, in which the terminal carbonyl group forms a thioester bond with the sulfhydryl group of cysteine, which is three amino acids away. This bond is unstable and the electrophilic glutamyl thioether can react with nucleophilic groups such as the hydroxyl group or amino group and thus form a covalent bond with other molecules. The thioester group is quite stable when sequestered in the hydrophobic pocket of intact C3. However, proteolytic cleavage of C3 to C3a and C3b results in the exposure of a highly reactive thioether bond on C3b and, following nucleophilic attack by adjacent groups containing hydroxyl groups or amino groups, C3b becomes covalently attached to the target. In addition to its well-documented role in the covalent attachment of C3b to complement targets, the C3 thioester is also thought to play a major role in triggering the alternative pathway. According to the generally accepted theory of idly activation, the alternative pathway is initiated by the formation of a soluble convertase, iC3Bb, which is formed from C3 with a hydrolyzed thioester (iC3;C3( _H2O )) and factor B (Lachmann, PJ, et al., Springer Semin. Immunopathol. 7 :143 162, (1984)). The C3b-like C3(H ₂ O) is formed from native C3 by slow spontaneous hydrolysis of the internal thioester in the protein (Pangburn, MK, et al., J. Exp. Med. 154:856 867, 1981). Due to the activity of C3(H2O)Bb convertase, C3b molecules accumulate on the target surface, thus initiating an alternative pathway.

Very little is known about the initiators of alternative pathway activation. Activators are believed to include yeast cell walls (zymosan), a variety of pure polysaccharides, rabbit red blood cells, certain immunoglobulins, viruses, fungi, bacteria, animal tumor cells, parasites and damaged cells. The only common feature of these activators is the presence of carbohydrate, but the complexity and diversity of carbohydrate structures make it difficult to identify the common molecular determinants that are recognized. It is generally accepted that activation of the alternative pathway is controlled by a fine balance between the inhibitory regulatory components of this pathway, such as factor H, factor I, DAF and CR1, and properdin, 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 seemingly unregulated activation mechanism described above, the alternative pathway may also provide a powerful reinforcing loop for the lectin/classical pathway C3 convertase (C4b2a), since any C3b generated can co-locate with factor B to form an additional alternative pathway C3 convertase (C3bBb) . Alternative pathway convertase C3 is stabilized by binding to properdin. Properdin increases the half-life of alternative pathway convertase C3 by six to tenfold. The addition of C3b to the alternative pathway convertase C3 results in the formation of the alternative pathway convertase C5.

All three pathways (i.e., classical, lectin, and alternative) were thought to converge on C5, which is cleaved to form products with multiple proinflammatory effects. The combined pathway is designated the terminal complement activation pathway. C5a is the most potent anaphylatoxin, inducing changes in smooth muscle and vascular tone, as well as vascular permeability. It is also a potent chemotaxin and activator of both neutrophils and monocytes. C5a-mediated cellular activation can significantly enhance inflammatory responses by inducing the release of multiple additional mediators

- 4 045598 inflammation, including cytokines, hydrolytic enzymes, arachidonic acid metabolites and reactive oxygen species. Cleavage of C5 leads to the formation of C5b-9, also known as the membrane attack complex (MAC). There is now strong evidence that sublytic accumulation of MAC may play an important role in inflammation in addition to its role as a lytic pore-forming complex.

In addition to its important role in immune defense, the complement system contributes to tissue damage in a variety of clinical conditions. Thus, there is an urgent need to develop therapeutically effective complement inhibitors to prevent these adverse effects.

It is well established that angiogenesis is involved in the pathogenesis of a variety of disorders, including solid tumors and metastasis, and neovascular ophthalmic diseases such as age-related macular degeneration (AMD), proliferative diabetic retinopathy, and neovascular glaucoma.

Given the role of angiogenesis in a variety of diseases and disorders, there is also an urgent need to develop therapeutically effective angiogenesis inhibitors.

The essence of the invention

This summary is provided to present in simplified form a set of concepts that are further described below in the detailed description. This summary of the invention is neither intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid to defining the scope of the claimed subject matter.

In one aspect, the present invention provides methods for preventing, treating, reversing, and/or delaying angiogenesis in a patient who is a mammal suffering from or at risk of developing an angiogenesis-dependent disease or condition, comprising administering to the patient an amount of a MASP-2 inhibitory agent , effective for inhibiting angiogenesis. In some embodiments of these aspects of the invention, the MASP-2 inhibitory agent 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 angiogenesis comprising a therapeutically effective amount of a MASP-2 inhibitory agent and a pharmaceutically acceptable carrier. The invention also relates to methods of making a medicament for use in inhibiting the adverse effects of angiogenesis in living patients in need thereof, comprising a therapeutically effective amount of a MASP-2 inhibitory agent in a pharmaceutical carrier. The invention also relates to methods of making medicaments for use in inhibiting angiogenesis for the treatment of each of the conditions, diseases and disorders described herein below.

The methods, compositions and medicaments of the invention can be used to inhibit the adverse effects of angiogenesis in vivo in a patient who is a mammal, including humans suffering from an acute or chronic pathological condition or injury, as described later herein.

In another aspect, the invention provides methods for inhibiting angiogenesis in a patient who is a mammal suffering from an angiogenesis-dependent disease or condition, comprising administering to the individual a composition containing an amount of a MASP-2 inhibitory agent effective to inhibit angiogenesis. In some embodiments, the angiogenesis-dependent disease or condition is an angiogenesis-dependent cancer, such as an angiogenesis-dependent cancer selected from the group consisting of solid tumor(s), blood-borne tumors, high-risk carcinoid tumors and tumor metastases. In some embodiments, the angiogenesis-dependent disease or condition is an angiogenesis-dependent benign tumor, such as an angiogenesis-dependent benign tumor selected from the group consisting of hemangiomas, acoustic neuromas, neurofibromas, trachomas, carcinoid tumors, and pyogenic granulomas . In some embodiments, the angiogenesis-dependent disease or condition is an ophthalmic angiogenic disease or condition, such as an ophthalmic angiogenic disease or condition selected from the group consisting of age-related macular degeneration (AMD), uveitis, ocular melanoma, corneal neovascularization, primary pterygium, HSV stromal keratitis, HSV-1-induced corneal lymphangiogenesis, proliferative diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, corneal graft rejection, neovascular glaucoma and iris redness.

In another aspect, the present invention relates to methods of treating an individual suffering from

- 5 045598 ophthalmic angiogenic disease or condition selected from the group consisting of AMD, uveitis, ocular melanoma, corneal neovascularization, primary pterygium, HSV stromal keratitis, HSV-1 induced corneal lymphangiogenesis, proliferative diabetic retinopathy, diabetic macular edema, retinopathy of prematurity , retinal vein occlusion, corneal graft rejection, neovascular glaucoma, vitreous hemorrhage secondary to proliferative diabetic retinopathy, neuromyelitis optica, and iris redness, comprising administering to the individual an amount of a MASP-2 inhibitory agent effective to inhibit angiogenesis.

In another aspect, the present invention provides methods for inhibiting tumor angiogenesis, comprising administering to an individual with a cancer an amount of a MASP2 inhibitory agent effective to inhibit angiogenesis.

Description of drawings

The foregoing aspects and many attendant advantages of this invention will be more readily appreciated and made easier to understand by reference to the following detailed description taken in conjunction with the accompanying drawings, wherein:

fig. 1 is a diagram illustrating the genomic structure of human MASP-2;

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

fig. 2B is a schematic diagram illustrating the domain structure of human MAp19 protein;

fig. 3 is a diagram illustrating a strategy for knocking out the murine MASP2 gene;

fig. 4 is a diagram illustrating the design of the human MASP-2 minigene;

in fig. 5A presents results showing that MASP-2 deficiency results in loss of lectin pathway-mediated C4 activation, as measured by the absence of C4b accumulation on mannan as described in Example 2;

in fig. 5B presents results showing that MASP-2 deficiency results in loss of lectin pathway-mediated C4 activation as measured by the lack of C4b accumulation on zymosan as described in Example 2;

in fig. 5C presents results showing the relative levels of C4 activation in serum samples obtained from MASP-2+/- lines; MASP-2-/- and wild-type lines, as measured by C4b accumulation on mannan and on zymosan, as described in Example 2;

in fig. 6 presents results showing that addition of murine recombinant MASP-2 to MASP-2-/- serum samples restores lectin pathway-mediated C4 activation in a protein concentration-dependent manner, as measured by C4b accumulation on mannan as described in Example 2;

in fig. 7 presents results showing that the classical pathway is functional in the MASP-2-/- line, as described in example 8;

in fig. 8A shows results showing that anti-MASP-2 antibody Fab2 #11 inhibits the formation of C3 convertase as described in Example 10;

in fig. 8B shows results showing that anti-MASP-2 antibody Fab2 #11 binds to native rat MASP-2 as described in Example 10;

in fig. 8C shows results showing that anti-MASP-2 antibody Fab2 #41 inhibits C4 cleavage as described in Example 10;

in fig. 9 presents results showing that all anti-MASP-2 Fab2 antibodies tested that inhibit C3 convertase formation also inhibit C4 cleavage as described in Example 10;

fig. 10 is a diagram illustrating the use of recombinant rat MASP-2-derived polypeptides to map anti-MASP-2 Fab2 blocking antibody epitopes as described in Example 11;

in fig. 11 presents results showing the binding of anti-MASP-2 antibodies Fab2 #40 and #60 to rat MASP-2 polypeptides as described in Example 11;

in fig. 12A presents results showing baseline levels of VEGF protein in the RPEchoroidal complex isolated from wild-type (+/+) and MASP-2(-/-) mice as described in Example 12;

in fig. 12B shows results showing VEGF protein levels in the RPE-choroidal complex in wild-type (+/+) mice and MASP-2(-/-) mice on day 3 after laser-induced injury in the macular degeneration model as described in the Example. 12;

in fig. 13 presents results showing the average choroidal neovascularization volume (CNV) at day seven after laser-induced injury in wild-type (+/+) and MASP-2(-/-) mice as described in Example 12;

in fig. 14 graphically illustrates the level of C4b accumulation, measured as % of control,

- 6 045598 in samples collected at various time points after subcutaneous (SC) dosing of either 0.3 mg/kg or 1.0 mg/kg mouse monoclonal antibody against MASP-2 in WT mice as described in Example 13;

in fig. 15 graphically illustrates the level of C4b accumulation, measured as % of control, in samples collected at various time points following intraperitoneal (IP) dosing of 0.6 mg/kg murine anti-MASP-2 monoclonal antibody in WT mice as described in Example 13;

in fig. 16 graphically illustrates the mean choroidal neovascularization (CNV) volume on day seven after laser-induced injury in WT (+/+) mice pretreated with a single IP injection of 0.3 mg/kg or 1.0 mg/kg mouse monoclonal anti-MASP- 2; as described in example 14;

in fig. 17A graphically illustrates the level of MAC accumulation in the presence or absence of anti-human MASP-2 monoclonal antibody (OMS646) under lectin pathway-specific assay conditions, showing that OMS646 inhibits lectin-mediated MAC accumulation with an IC ₅₀ value of approximately 1 nM, as described in example 15;

in fig. 17B graphically illustrates the level of MAC accumulation in the presence or absence of anti-human MASP-2 monoclonal antibody (OMS646) under classical pathway-specific assay conditions, showing that OMS646 does not inhibit classical pathway-mediated MAC accumulation as described in Example 15;

in fig. 17C graphically illustrates the level of MAC accumulation in the presence or absence of anti-human MASP-2 monoclonal antibody (OMS646) under alternative pathway-specific assay conditions, showing that OMS646 does not inhibit alternative pathway-mediated MAC accumulation as described in Example 15;

in fig. 18 graphically illustrates the pharmacokinetic (PK) profile of anti-human MASP-2 monoclonal antibody (OMS646) in mice, showing the concentration of OMS646 (average of n=3 animals/group) as a function of time after administration at the indicated dose, as described in Example 15;

in fig. 19A graphically illustrates the pharmacodynamic (PD) response of anti-human MASP-2 monoclonal antibody (OMS646), measured as a drop in systemic lectin pathway activity, in mice following intravenous administration as described in Example 15;

in fig. 19B graphically illustrates the pharmacodynamic (PD) response of anti-human MASP-2 monoclonal antibody (OMS646), measured as a drop in systemic lectin pathway activity, in mice following subcutaneous administration as described in Example 15; and in fig. 20 graphically illustrates the area of choroidal neovascularization (CNV) as a percentage of the area of laser-induced lesions on day seven post-injury in WT (+/+) mice pretreated with 2 mg/kg, 5 mg/kg, or 20 mg/kg monoclonal anti-human MASP-2 antibody (OMS646) administered by SC or anti-VEGF antibody administered by IP as described in Example 16.

Description of the sequence list

SEQ ID NO: 1 human MAp19 cDNA

SEQ ID NO: 2 human MAp19 protein (with leader sequence)

SEQ ID NO: 3 human MAp19 protein (mature)

SEQ ID NO: 4 human MASP-2 cDNAs

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

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

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

Antigens: (relative to mature MASP-2 protein)

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

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

SEQ ID NO: 10 CUBEGFCUBII (ak 1-293)

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

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

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

SEQ ID NO: 14 TPLGPKWPEPVFGRL (CUBI peptide)

SEQ ID NO: 15 TAPPGYRLRLYFTHFDLELSHLCEYDFVKLSSGAKVLATLCGQ (CUBI peptide)

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

SEQ ID NO: 17 FYSLGSSLDITFRSDYSNEKPFTGF (MBL binding region)

SEQ ID NO: 18 IDECQVAPG (EGF peptide)

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

Detailed description

Peptide inhibitors:

SEQ ID NO: 20 full length MBL cDNA

- 7 045598

SEQ ID NO: 21 full-length MBL proteins

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

SEQ ID NO: 23 OGKLG

SEQ ID NO: 24 GLR GLQ GPO GKL GPO G

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

SEQ ID NO: 26 GKDGRDGTKGEKGEPGQGLRGLQGPOGKLGPOG

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

SEQ ID NO: 28 GCOGLOGAOGDKGEAGTNGKRGERGPOGPOGKAGPOGPNGAOGEO (human ficolin p35)

SEQ ID NO: 29 LQRALEILPNRVTIKANRPFLVFI (C4 cleavage site)

Expression inhibitors:

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

SEQ ID NO: 31

5' CGGGCACACCATGAGGCTGCTGACCCTCCTGGGC 3'

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

SEQ ID NO: 32

5'GACATTACCTTCCGCTCCGACTCCAACGAGAAG3'

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

SEQ ID NO: 33

5'AGCAGCCCTGAATACCCACGGCCGTATCCCAAA3'

Nucleotides 610-642 of SEQ ID NO: 4, encoding the region containing the CUBII domain

Primers for cloning:

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

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

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

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

SEQ ID NO: 38-47 are cloning primers for the humanized antibody

SEQ ID NO: 48 is a 9 aa peptide bond

Expression vector:

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

SEQ ID NO: 50 is mouse MASP-2 cDNA

SEQ ID NO: 51 is murine MASP-2 protein (with leader sequence)

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

SEQ ID NO: 53 is rat MASP-2 cDNA

SEQ ID NO: 54 is rat MASP-2 protein (with leader sequence)

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

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

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

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

SEQ ID NO: 66 DNA encoding heavy chain variable region (VH) 17D20_dc35VH21N11VL (OMS646) (no signal peptide)

SEQ ID NO: 67 heavy chain variable region (VH) polypeptide 17D20_dc35VH21N11VL (OMS646)

SEQ ID NO: 68 heavy chain variable region (VH) polypeptide 17N16mc

SEQ ID NO: 69 Light chain variable region (VL) polypeptide 17D20_dc21N11VL (OMS644)

SEQ ID NO: 70 DNA encoding light chain variable region (VL) 17N16_dc17N9 (OMS641) (no signal peptide)

SEQ ID NO: 71 light chain variable region (VL) polypeptide 17N16_dc17N9 (OMS641)

Detailed description

The present invention is based on the unexpected 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 relates to the use of MASP-2 as a therapeutic target for inhibiting cellular damage associated with activation of the lectin-mediated complement pathway, while leaving the classical (C1q-dependent) immune system component pathway intact.

I. Definitions.

Unless specifically defined otherwise, all terms used in this specification have the meaning

- 8 045598 the same meaning as would be understood by one skilled in the field of the present invention. The following definitions are presented for the purpose of providing clarity with respect to terms as used in the specification and claims to describe the present invention.

As used herein, the term MASP-2-dependent complement activation refers to MASP-2-dependent lectin pathway activation that occurs under physiological conditions (i.e., in the presence of Ca++) leading to the formation of the C4 lectin pathway convertase C3 and following accumulation the C3 cleavage product C3b, sequentially to the C5 convertase C4b2a(C3b)n, which was identified as the primary cause of opsonization.

Within the scope of the invention, the term alternative pathway refers to the activation of complement, which is triggered, for example, by zymosan from the cell walls of fungi and yeast, lipopolysaccharide (LPS) from the outer membrane of gram-negative bacteria, and rabbit red blood cells, as well as from a variety of pure polysaccharides, rabbit red blood cells , viruses, bacteria, animal tumor cells, parasites and damaged cells, and which is traditionally thought to arise from the spontaneous proteolytic formation of C3b from complement factor C3.

As used herein, the term lectin pathway refers to complement activation that occurs through the specific binding of serum and non-serum carbohydrate-binding proteins, including mannan binding lectin (MBL), CL-11, and ficolins (H-ficolin, Mficolin, or L-ficolin).

As used herein, the term classical pathway refers to complement activation triggered by an antibody bound to a foreign particle and requiring binding of the C1q recognition molecule.

As used herein, the term MASP-2 inhibitory agent refers to any agent that binds or directly interacts with MASP-2 and effectively inhibits MASP-2-dependent complement activation, including anti-MASP-2 antibodies and their naturally occurring MASP-2 binding fragments. and synthetic peptides, small molecules, soluble MASP-2 receptors, expression inhibitors, and isolated natural inhibitors, and also refers to peptides that compete with MASP-2 for binding to another recognition molecule (e.g., MBL, H-ficolin, Mficolin, or L -ficolin) in the lectin pathway, but is not an antibody that binds other recognition molecules. MASP-2 inhibitory agents that can be used in the method of the invention can reduce MASP-2-dependent complement activation by more than 20%, for example by more than 50%, for example by more than 90%. In one embodiment, the MASP-2 inhibitory agent reduces MASP-2-dependent complement activation by more than 90% (ie, resulting in MASP-2 complement activation of only 10% or less).

As used herein, the term angiogenesis refers to the growth of new microvessels from pre-existing blood vessels.

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

As used herein, the term antibody refers to antibodies and antibody fragments thereof derived from any antibody-producing mammal (e.g., mouse, rat, rabbit, and primate, including humans), or from hybridoma, phage selection, recombinant expression, or transgenic animals (or other methods for producing antibodies or antibody fragments) that specifically bind to a target polypeptide, such as MASP-2, polypeptides or parts thereof. The term antibody is not intended to be limited to the source of the antibody or the method in which it is produced (eg, by hybridoma, phage selection, recombinant expression, transgenic animal, peptide synthesis, etc.). Exemplary antibodies include polyclonal, monoclonal and recombinant antibodies; panspecific, multispecific antibodies (eg, bispecific antibodies, trispecific antibodies); humanized antibodies; mouse antibodies; chimeric, mouse-human, primate-mouse, primate-human monoclonal antibodies; and anti-idiotypic antibodies, and can be any intact antibody or fragment thereof. Within the scope of the invention, the term antibody includes not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as dAb, Fab, Fab', F(ab') ₂ , Fv), single chain antibodies (ScFv), their synthetic variants, natural variants, fusion proteins containing a portion of an antibody with an antigen-binding fragment of the required specificity, humanized antibodies, chimeric antibodies, and any other modified configuration of an immunoglobulin molecule that contains an antigen-binding region or fragment (epitope recognition region) of the required specificity.

A monoclonal antibody refers to a homogeneous population of antibodies, where the monoclonal antibody is composed of amino acids (natural and non-natural) involved in the selective binding of an epitope. Monoclonal antibodies are highly specific for the target antigen. The term monoclonal antibody includes not only intact monoclonal antibodies and full-length monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab') ₂ , Fv), single chain antibodies (ScFv), variants thereof, fusion proteins containing antigen-binding part, humanized

- 9 045598 monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of an immunoglobulin molecule that contains an antigen-binding fragment (epitope recognition region) of the required specificity and is capable of binding to the epitope. It is not limiting as to the source of the antibody or the manner in which it is produced (eg, through hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term includes full-length immunoglobulins, as well as fragments, etc., described above under the definition of antibody.

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

Within the scope of the invention, a single chain Fv or scFv antibody fragment comprises the VH and _VL domains of the antibody, where these domains are present on the same polypeptide chain. Typically, the Fv polypeptide further includes a polypeptide linker between the VH and _VL domains, which allows the scFv to form the desired structure for antigen binding.

Within the scope of the invention, a chimeric antibody is a recombinant protein containing variable domains and complementarity determining regions derived from an antibody from a non-human species (eg, rodents), while the remainder of the antibody molecule is derived from a human antibody.

Within the scope of the invention, a humanized antibody is a chimeric antibody containing a minimal sequence corresponding to specific complementarity determining regions derived from a non-human immunoglobulin transplanted into a human antibody framework. Humanized antibodies are typically recombinant proteins in which only the complementarity-determining region of the antibody is derived from a non-human source.

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 five terminal complement components (C5b in combination with C6, C7, C8 and C-9) that penetrates and disrupts the membrane (also referred to as C5b-9).

For purposes of the invention, an individual includes all mammals, including, without limitation, humans, non-human primates, dogs, cats, horses, sheep, goats, cows, rabbits, pigs and rodents.

Within the framework of the invention, amino acid residues have the following abbreviations: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid ( Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met ; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y) and valine (Val; V).

In the broadest sense, naturally occurring amino acids can be divided into groups based on the chemical characteristics of the side chains of the corresponding amino acids. By hydrophobic amino acid is meant Ile, Leu, Met, Phe, Trp, Tyr, Val, Ala, Cys or Pro. By hydrophilic amino acid is meant Gly, Asn, Gln, Ser, Thr, Asp, Glu, Lys, Arg or His. This grouping of amino acids can be further divided into subclasses as follows. By uncharged hydrophilic amino acid is meant Ser, Thr, Asn or Gln. By acidic amino acid we mean Glu or Asp. By basic amino acid we mean Lys, Arg or His.

For the purposes of the invention, the term conservative amino acid substitution is illustrated by 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 ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), or mimetics thereof. The term also refers to oligonucleotide bases consisting of natural nucleotides, sugars and covalent internucleoside bonds (backbones), as well as oligonucleotides having non-natural modifications.

As used herein, an epitope refers to a region of a protein (eg, human MASP2 protein) to which an antibody binds. Overlapping epitopes include at least one (eg, two, three, four, five or six) common amino acid residue(s), including linear and non-linear epitopes.

Within the scope of the invention, the terms polypeptide, peptide and protein are used interchangeably and mean any chain of amino acids linked by peptide bonds, regardless of length or post

- 10 045598 translational modification. The MASP-2 protein described herein may contain either wild-type proteins or may be variants having no more than 50 (e.g., no more than one, two, three, four, five, six, seven, eight, nine, ten, 12, 15, 20, 25, 30, 35, 40 or 50) conservative amino acid substitutions. Conservative substitutions generally 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 may have an amino acid sequence that is at or 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 human MASP-2 protein having the amino acid sequence indicated to SEQ ID NO: 5.

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

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

II. Overview of the invention.

Lectins (MBL, M-ficolin, H-ficolin, L-ficolin and CL-11) are specific recognition molecules that trigger the innate complement system, and this system includes the lectin initiation pathway and the associated terminal pathway reinforcing loop that enhances lectin-initiated activation terminal effector complement molecules. C1q is a specific recognition molecule that triggers the acquired complement system, and this system includes the classical initiation pathway and an associated terminal pathway reinforcing loop that enhances C1q-initiated activation of terminal effector complement molecules. The present inventors have designated these two major complement activation systems as the lectin-dependent complement system and the C1q-dependent complement system, respectively.

In addition to its important role in immune defense, the complement system contributes to tissue damage in many clinical conditions. Thus, there is an urgent need to develop therapeutically effective complement inhibitors to prevent these adverse effects. With the understanding that it is possible to inhibit the lectin-mediated MASP-2 pathway while leaving the classical pathway intact, it is realized that it is highly desirable to specifically inhibit only the complement activation system causing a particular pathology, without completely disabling the immunoprotective abilities of complement. For example, in disease states in which complement activation is mediated predominantly by the lectin-dependent complement system, specific inhibition of only that system may provide benefits. This may leave the C1q-dependent complement activation system intact to drive immune complex activity and promote host defense against infection.

A preferred protein moiety to target in drug development for specific inhibition of the lectin-dependent complement system is MASP-2. Of all the known protein components of the lectin-dependent complement system (MBL, H-ficolin, ficolin M, L-ficolin, MASP-2, C2 C9, factor B, factor D, and properdin), only MASP-2 is unique to lectin-dependent complement. lectin of the complement system, and necessary for the function

- 11 045598 system leveling. Lectins (MBL, H-ficolin, M-ficolin, L-ficolin, and CL-11) are also unique components in the lectin-dependent complement system. However, loss of any one of these lectin components does not necessarily inhibit activation of the system due to lectin redundancy. It would be necessary to inhibit all five lectins to ensure inhibition of the lectin-dependent complement activation system. In addition, since MBL and ficolins are also known to have complement-independent opsonic activity, inhibition of lectin function may result in loss of this beneficial host defense mechanism against infection. In contrast, this complement-independent lectin opsonic activity may remain intact if MASP-2 is the target of inhibition. An additional advantage of MASP-2 as a therapeutic target for inhibition of the lectin-dependent complement activation system is that plasma concentrations of MASP-2 are among the lowest compared to other complement proteins (~500 ng/ml); thus, accordingly, low concentrations of high affinity MASP-2 inhibitors may be sufficient to obtain complete inhibition (Moller Kristensen, M., et al., J. Immunol Methods 282:159 167, 2003).

As described herein, it has been unexpectedly determined that a MASP-2 inhibitor, such as anti-human MASP-2 antibody (OMS646), is at least as effective as an anti-VEGF antibody in reducing choroidal neovascularization (CNV) in a model of associated with age-related macular degeneration (AMD) in mice when delivered systemically to mice. Thus, it is expected that a MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody, may also be effective as an antiangiogenic agent for use in inhibiting an angiogenesis-dependent cancer, such as an angiogenesis-dependent cancer selected from group consisting of solid tumor(s), blood-borne tumors, high-risk carcinoid tumors and metastatic tumors. It is also expected that a MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody, may be effective as an antiangiogenic agent for inhibiting an angiogenesis-dependent benign tumor, such as an angiogenesis-dependent benign tumor selected from the group consisting of hemangiomas , acoustic neuromas, neurofibromas, trachomas, carcinoid tumors and pyogenic granulomas. It is also expected that a MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody, may be effective as an antiangiogenic agent for use in inhibiting angiogenesis in AMD and other ophthalmic angiogenic diseases or disorders such as uveitis, ocular melanoma, corneal neovascularization, primary pterygium (cornea), HSV stromal keratitis, HSV-1 induced corneal lymphangiogenesis, proliferative diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, corneal graft rejection, neovascular glaucoma and iris redness.

III. The role of MASP-2 in angiogenesis-dependent diseases and conditions, and therapeutic options using MASP-2 inhibitory agents.

Angiogenesis-dependent diseases or conditions result from new blood vessels growing excessively in inappropriate locations (such as the retinal pigment epithelium), or new blood vessels having undesirable characteristics such as leakage, and include diseases such as cancer tumor and eye diseases. In these conditions, new blood vessels feed the diseased tissue, can destroy new tissue and, in the case of a malignant tumor, the new blood vessels allow the tumor to grow and tumor cells to enter the bloodstream and metastasize to other organs. Excess angiogenesis can occur when diseased cells produce abnormal amounts of angiogenic growth factors, thereby overcoming the effects of natural angiogenesis inhibitors.

The potential role of complement activation in angiogenesis has been demonstrated in age-related macular degeneration (AMD). AMD is a vision loss disorder affecting millions of adults, although the consequences of the biochemical, cellular and/or molecular events leading to the development of AMD are poorly understood. AMD results in the progressive destruction of the macula, which is associated with the formation of extracellular deposits rich in proteins and lipids, called drusen, located in and around the macula, behind the retina, and between the retinal pigment epithelium (RPE) and the choroid. Drusen are characteristic of early and intermediate AMD. Many patients are susceptible to progression to advanced AMD, which includes two forms, geographic atrophy and neovascular or wet AMD. The term dry AMD generally refers to early and intermediate AMD, as well as geographic atrophy. While they are present and potentially pathological in early and intermediate forms of the disease, drusen also persist in both late forms (van Lookeren-Campagne et al., J. Pathol. 232:151, 2014; Ambati et al., Nat. Rev Immunol 13:438, 2013). Recent studies have revealed that proteins associated with inflammation and immune-mediated processes predominate among drusen-associated constituents. Transcripts encoding a number of these molecules have been detected in retinal, RPE and choroid cells. These data also show that dendritic cells, which are active antigen-presenting cells, are closely related to the development

- 12 045598 drusen, and that complement activation is a key pathway that is active both within drusen and along the RPE-choroid interface (Hageman, G.S., et al., Prog. Retin. Eye Res. 20:705

732, 2001); Ebrahimi and Handa, J. Lipid 2011:802059, 2011). These observations indicate that local inflammation is likely a significant factor in the early pathogenesis of AMD.

Several independent studies have shown a strong association between AMD and a genetic polymorphism in the complement factor H (CFH) gene, where the likelihood of AMD is increased 7.4-fold in individuals homozygous for the risk allele (Klein, R.J. et al., Science 308:362 364 , 2005; Haines et al., Science 308:362-364. 2005; Edwards et al., Science 308:263-264, 2005). The CFH gene has been mapped to chromosome 1q31, in a region implicated in AMD in six independent linkage scans (see, eg, Schultz, D.W., et al., Hum. Mol. Genet. 12:3315, 2003). CFH is known to be a key regulator of the complement system. CFH on cells and in the bloodstream has been shown to regulate complement activity by inhibiting the activation of cleavage of C3 to C3a and C3b, and by inactivating existing C3b. Accumulation of C5b-9 has been observed in Bruch's membrane, intercapillary constrictions, and within drusen in patients with AMD (Klein et al., Science 308:362 364, 2005). Experiments using immunofluorescence suggest that in AMD, the CFH polymorphism may cause complement accumulation in choroidal capillaries and choroidal vessels (Klein et al., Science 308:362-364, 2005).

Membrane-associated complement receptor 1 is also localized in drusen, but it was not detected in RPE cells by immunohistochemistry. In contrast, a second membrane-associated complement inhibitor, membrane cofactor protein, is present in drusen-associated RPE cells as well as in small spherical structural elements within drusen. These previously unidentified elements have also been shown to have strong immunoreactivity towards proteolytic fragments of complement component C3, which characteristically accumulate at sites of complement activation. It has been suggested that these structures represent residual debris from degenerating RPE cells that are targets of complement attack (Johnson, L.V., et al., Exp. Eye Res. 73:887-896, 2001).

The identification and localization of these multiple complement regulators, as well as complement activation products (C3a, C5a, C3b, C5b-9), led researchers to conclude that chronic complement activation plays an important role in drusen biogenesis and the etiology of AMD (Hageman et al., Progress Retinal Eye Res 20:105 32, 2001). Identification of the activation products C3 and C5 in drusen does not provide information as to whether complement is activated through the classical pathway, the lectin pathway, or the alternative reinforcing loop, since, as understood in accordance with the present invention, both C3 and C5 are common to all three. However, two immunolabeling studies of drusen have been conducted using antibodies specific for C1q, an important recognition component for activation of the classical pathway (Mullins et al., FASEB J. 14:835 846, 2000; Johnson et al., Exp. Eye Res. 70 :441 449, 2000). Both studies concluded that C1q immunolabeling was largely undetectable in drusen. These negative results for C1q suggest that complement activation in drusen does not occur through the classical pathway. In addition, a study by Mullins et al., 2000, reported that immunolabeling of immune complex components (IgG, IgM light chains) in drusen changed little, further indicating that the classical pathway plays a minor role in the complement activation that occurs during of this disease. Thus, the lectin and/or alternative pathways appear to be responsible for most, if not all, of the complement-mediated drusen biogenesis associated with AMD.

The relationship between drusen and complement activation is strong, particularly in early and intermediate AMD, as well as in geographic atrophy. In fact, large and confluent drusen represent a significant risk factor for geographic atrophy (van Lookeren-Campagne et al., ibid.). However, complement activation is not limited to the surrounding drusen. Two recently published studies assessed the role of complement in the development of laser-induced choroidal neovascularization (CNV) in a mouse model of human CNV. Using immunohistological techniques, Bora et al. (2005) found significant accumulation of complement activation products C3b and C5b 9 (MAC) in the neovascular complex after laser therapy (Bora et al., J. Immunol. 174:491 7, 2005). Importantly, CNV did not develop in mice genetically deficient in C3 (C3 −/− mice), an essential component required for all complement pathways. Messenger RNA levels for VEGF, TGF-e2, and β-FGF, three angiogenic factors involved in CNV, were increased in eyeball tissue of mice following laser-induced CNV. Importantly, complement depletion resulted in a significant reduction in RNA levels of these angiogenic factors.

Using ELISA methods, Nozaki and colleagues showed that the potent anaphylatoxins C3a and C5a are formed early during laser-induced CNV (Nozaki et al., Proc. Natl. Acad. Sci. U.S.A. 103:2328 33, 2006). Moreover, these two bioactive fragments C3 and C5 induced VEGF expression after their intravitreal injection in wild-type mice. Consistent with these results, Nozaki et al also showed that genetic ablation of the receptors for C3a and C5a reduces VEGF expression and CNV formation after laser injury, and that antibody-mediated neutralization of C3a or C5a or pharmacological blockade of their receptors also reduces CNV . Previous studies have established that the recruitment of leukocytes, and especially macrophages, plays a major role in laser-induced CNV (Sakurai et al., Invest. Opthomol. Vis. Sci. 44:3578 85, 2003; Espinosa Heidmann, et al., Invest. Opthomol Vis Sci 44:3586 92, 2003). In their 2006 work, Nozaki and colleagues published that leukocyte recruitment was significantly reduced in C3aR(-/-) and C5aR(/-) mice.

The lectin pathway appears to be responsible for initiating the complement cascade in the CNV model following recognition of oxidatively modified phospholipids on the retinal pigment epithelium by natural antibodies (Joseph et al. J. Biol. Chem. 288:12753, 2013). The alternative pathway is also critical for retinal damage in this model, but it alone is not sufficient (Rohrer et al., Mol Immunol. 48:el, 2011). Importantly, Kunchithapautham and Rohrer (J. Biol. Chem. 286:23717, 2011) showed that this complement activation triggers the secretion of VEGF by retinal pigment epithelial cells. VEGF is a key mediator of neovascularization.

As described herein in Example 12, in the MASP-2(-/-) mouse model of macular degeneration, it was determined that background VEGF levels were reduced in MASP-2(-/-) mice compared to wild-type and control mice. furthermore, while VEGF levels were significantly increased in wild-type mice after laser-induced injury, unexpectedly low VEGF levels were observed in MASP-2(-/-) mice after laser-induced injury. In addition, we determined that MASP-2 (-/-) mice showed a 30% reduction in CNV area after laser-induced injury on day 7 compared to wild-type mice. As further described in Example 14, mice pretreated with an anti-MASP-2 monoclonal antibody that specifically blocks the lectin pathway of complement activation had a statistically significant (p<0.01) reduction in CNV of approximately 50% seven days after laser treatment compared to untreated mice, showing that blocking MASP-2 using an inhibitor such as an anti-MASP-2 monoclonal antibody has a preventive and/or therapeutic effect in the treatment of macular degeneration. As further described in Example 16, in mice pretreated with an anti-human MASP-2 monoclonal antibody that specifically blocks the lectin pathway of complement activation, a statistically significant reduction in CNV was observed at all dosage levels tested, with a relative reduction in CNV area ranging from 20 % to 50%, while the anti-VEGF antibody showed a moderate (approximately 15%) relative reduction in CNV area. In view of the unexpected results described in Example 16 showing that a MASP-2 inhibitor, such as an anti-MASP-2 antibody, is at least as effective as an anti-VEGF antibody in reducing CNV in a mouse model of AMD, with systemic delivery, it is expected that a MASP-2 inhibitory agent may be effective as an antiangiogenic agent for use in the treatment of angiogenesis-dependent diseases and conditions, such as ophthalmic angiogenic diseases or disorders, angiogenesis-dependent malignancies, and angiogenesis-dependent benign tumors, such as described below.

MASP-2 inhibitors for the treatment of ophthalmic angiogenic diseases or disorders.

An ophthalmic angiogenic disease or disorder is an eye disease or disorder in which abnormal or excessive angiogenesis occurs in the eye, which may contribute to vision loss, hemorrhage, or other functional ocular disorders such as AMD, or an ophthalmic angiogenic disease or disorder. selected from the group consisting of uveitis, ocular melanoma, corneal neovascularization, primary pterygium (cornea), HSV stromal keratitis, HSV-1 induced corneal lymphangiogenesis, proliferative diabetic retinopathy, diabetic macular edema, retinopathy of prematurity, retinal vein occlusion, corneal graft rejection , neovascular glaucoma, vitreous hemorrhage secondary to proliferative diabetic retinopathy, neuromyelitis optica, and iris redness (see, for example, Rivera et al., Neonatology 100(4):343-53, 2011; Hosseini et al., Cornea 31:322-34, 2012; Leyvraz et al., Curr Opin Oncol 162-9 (2012); Bock et al., Prog Retin Eye Res 34:89-124, 2013 and Kim et al., Am J Pathol 181(2):376-9, 2012).

As described in Examples 14 and 16, this application demonstrates that systemic administration of an anti-MASP-2 antibody that specifically inhibits the lectin pathway of complement provides an effective therapy for the treatment of neovascular AMD. Currently approved antiangiogenic drugs for ophthalmic conditions are biological agents that inhibit VEGF. There are currently three approved antiangiogenic drugs for ophthalmic diseases: an anti-VEGF aptamer (pegaptanib, Macugen®), a Fab fragment of a monoclonal antibody directed against VEGF-A (ranibizumab, Lucentis®), and a fusion protein that binds to VEGF-A , VEGF-B and placental growth factor (aflibercept, Eylea®), all of which are administered by intravitreal injection. So

- 14 045598 manner, unlike current and emerging drugs against AMD and other ophthalmic angiogenic diseases and disorders that require intravitreal injection, anti-MASP-2 antibody treatment is effective when administered subcutaneously.

In one aspect, the invention thus provides a method of inhibiting angiogenesis for treating an ophthalmic angiogenic disease or disorder, comprising administering a composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent in a pharmaceutical carrier to an individual in need thereof. In some embodiments, the angiogenic ocular disease or disorder is selected from the group consisting of AMD, uveitis, ocular melanoma, corneal neovascularization, primary pterygium, HSV stromal keratitis, HSV-1 induced corneal lymphangiogenesis, proliferative diabetic retinopathy, diabetic macular edema, retinopathy prematurity, retinal vein occlusion, corneal graft rejection, neovascular glaucoma, vitreous hemorrhage secondary to proliferative diabetic retinopathy, optic neuromyelitis and iris redness. The MASP-2 inhibitory composition can be administered topically to the eye, for example, by direct injection, irrigation, or application of the composition in the form of a gel, ointment, or drops. Alternatively, the MASP-2 inhibitory agent can be administered to an individual systemically, for example, via intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous or other parenteral administration, or potentially via oral administration for non-peptidergic agents. The MASP-2 inhibitory agent composition can be combined with one or more additional drugs, such as an additional antiangiogenic agent. Administration may be repeated as determined by the physician until the condition is resolved or controlled.

MASP-2 inhibitors for the treatment of angiogenesis-dependent malignancies.

It is well established that angiogenesis plays a critical role in the development of malignancies. Tumors produce proangiogenic factors to stimulate neovascularization, which is one of the main mechanisms of progression of solid tumors, and also allows the migration of tumor cells to form distant metastases through access to the systemic circulation. The process of tumor angiogenesis is first activated when the growing tumor mass exceeds the maximum volume that can be supported by the diffusion of oxygen and nutrients. A correlation has been observed between increased angiogenesis and tumor aggressiveness (Ferrara et al., Curr Top Microbiol Immunol 237:1-30, 1999). Angiogenesis is also known to play a role in the growth and survival of leukemias and other hematological malignancies (Ribatti et al., Neoplasia 15(3):231-238, 2013; Vacca et al., Br J Haematol 87:503-508, 1994 ). While various cell types contribute to neovascularization, endothelial cells are generally known to play a central role in the process of angiogenesis.

It is well established that VEGF plays an important role in tumor angiogenesis. VEGF has been identified as a vascular permeability factor secreted by tumor cells (Mattei et al., Genomics 32:168-169, 1996) and has been shown to play a role in angiogenesis by stimulating the migration and proliferation of endothelial cells, as well as by stimulating the expression of associated with angiogenesis genes in endothelial cells. For example, expression of soluble VEGF isoform 189 in human colon, kidney, and lung cancers is strongly associated with increased microvessel numbers, cancer metastasis, and poor prognosis (Tokunaga et al., Br J Cancer 77:998-1002, 1998; Yuan et al. ., J Clin Oncol 19:432-441, 2001). High levels of VEGF isoform 165 are associated with poor survival rates in ovarian cancer (Manner et al., BMC Cancer 10:139, 2010). Bevacizumab, a humanized monoclonal antibody that inhibits VEGF-A, improved progression-free survival in women with ovarian cancer in a phase 3 clinical trial (Perren et al., N Engl J Med 365:2484-2496, 2011).

In the context of malignancies, researchers have traditionally focused on the role of complement in labeling and killing tumor cells. However, recent studies have questioned this view. For example, Markiewski et al. (Nature Immunol vol 9:1225-1235, 2008), published the unexpected discovery that complement proteins C3, C4 and C5a can promote tumor growth by stimulating an immunosuppressive microenvironment. As described by Markiewski et al., complement C5a production in the tumor microenvironment enhances tumor growth by suppressing the CD8+ T cell-mediated antitumor response. As further described by Markiewski et al., the C5aR antagonist, hexapeptide AcF(OP(D)ChaWr), was as effective as paclitaxel (Taxol) in impairing tumor growth in wild-type mice, thereby establishing a therapeutic function of complement inhibition in treatment of malignant tumors. As described in Gunn et al. (J Immunol 189:2985, 2012), wild-type mice with syngeneic lymphoma cells with high C5a production had significantly accelerated tumor progression with more myeloid-derived suppressor cells (MDSCs) in the spleen and an overall increase in the number of CD4+ and CD8+ T cells in the tumor , lymph nodes draining the tumor and in the spleen. In contrast, tumor-bearing mice with lymphoma cells with low C5a production had significantly

- 15 045598 reduced tumor burden with CD4+ and CD8+ T cells producing increased amounts of interferon-γ in the spleen and tumor-draining lymph nodes. As further described in Corrales et al. (J Immunol 189:4674–4683, 2012), found a significant increase in the amount of C5a in plasma from patients with non-small cell lung cancer (NSCLC) compared to healthy individuals. It was also determined that C5a induced endothelial cell chemotaxis and blood vessel formation. In the Lewis lung cancer model, syngeneic Lewis mouse lung carcinoma (3LL) tumor cells grew more slowly in mice treated with a C5a receptor antagonist.

As further described in Nunez-Cruz et al. (Neoplasia 14:994-1004, 2012), to evaluate the role of complement during the progression of ovarian cancer, a line of mice deficient in C3 complement, or a line of mice deficient in the C5a receptor (C5aR) in complement, was crossed with a line of mice that develop epithelial ovarian cancer (TgMISIIR-TAg). TgMISIIR-Tag mice that had complete or partial C3 deficiency or complete C5aR deficiency either did not develop ovarian tumors or the tumors were small and poorly vascularized compared to wild-type TgMISIIR-TAg littermates, thus indicating that C3 deficiency or C5aR significantly attenuated the ovarian tumor phenotype. In addition, it was shown that the function of CD31+ endothelial cells in angiogenesis was impaired in both C3 (-/-) and C5aR (-/-) mice.

Activation of the complement system may also be involved in the pathogenesis of malignancies. Complement complex neoantigens C5b-9, IgG, C3, C4, S protein/vitronectin, fibronectin and macrophages were localized on 17 malignant breast tumors and 6 benign breast tumors using polyclonal or monoclonal antibodies and the streptavidin method -biotin peroxidase. All carcinoma tissue samples at each TNM stage showed accumulations of C5b-9 on tumor cell membranes, small granules on cell debris, and diffuse accumulations in areas of necrosis (Niculescu, F., et al., Am. J. Pathol. 140:1039 1043, 1992). As further described in Rutkowski et al. (Mol Cancer Res 8:1453, 2010), potential oncogenic roles have been described for complement proteins C3, C3a, C5a, and MAC, including angiogenesis, tumor invasion, and migration. The lectin pathway of complement activation was found to be significantly upregulated in the serum of patients with colorectal cancer compared with healthy individuals (Ytting et al., 2004, Scand J. Gastroenterol 39:674), and it was reported that high levels of MASP-2 activity are independent a prognostic biomarker predicting relapse and poor survival in colon cancer (Ytting et al., Clin Cancer Res 11: 1441, 2005).

Serum levels of MBL and/or MASP-2 have also been found to be elevated in certain pediatric malignancies, including acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma, CNS tumors, and non-CNS solid tumors (Fisch et al., 2011, Swiss Med Wkly 141:w13191). MASP-2 was also found to be overexpressed in esophageal squamous cell carcinoma (ESCC) and dysplasia (premalignant) tissue samples (Verma et al., Int J Cancer 118:2930, 2006).

In addition to the above studies, numerous studies have been published on the association of MBL polymorphisms and malignancies. For example, as summarized in Swierzko et al., Mol Immunol 55:16, 2013, the association of MBL and MBL2 gene polymorphisms with gastric cancer has been published (Baccarelli et al., International J Cancer 119:1970-1975, 2006; Scudiero et al., Clin Chem 52:1625–1626, 2006; Wang et al., Digestive Diseases and Sciences 53:2904–2908, 2008); liver cancer (Eurich et al., Liver International 31:1006-1012, 2011); pancreatic cancer (Rong et al., BMC Gastroenterology 10:68, 2010); colon/colorectal cancer (Ytting et al., Scan J Gastroenterology 39:670-674, 2004; Ytting et al., Scan J Gastroenterology 73:122-127, 2011; Zanetti et al., Cancer Res 72:14 67 -1677, 2012); ovarian cancer (Swierzko et al., Immunotherapy 56:959-971, 2007); Nevadunsky et al., European J of Obstetrics and Gynecology and Reproductive Biology 163:216-218, 2012); breast cancer (Bernig et al., Carcinogenesis 28:828-836, 2007); lung cancer (Pine et al., Journal of NCI 99:1401-1409, 2007; OlivoMarston et al., Cancer Epidemiology, Biomarkers and Prevention 18:3375-3383, 2009); and acute lymphoblastic leukemia (Schmiegelow et al., Blood 100:3757-3760, 2002).

Complement components have also been determined to be upregulated in body fluids from human cancer patients, as shown in Table 1 below. 1.

- 16 045598

Table 1

Complement components up-regulated in body fluids from human patients with malignant tumors

Complement component SZA/SZA(desArg) SZA/SZA(desArg) SZA/SZA(desArg) Malignant tumor Mammary gland HCV related Hepatocellular carcinoma Colorectal Biological sample Serum Serum Serum Link Fan et al., J Can Res Clin Oncol 136:1243, 2010; Solassol et al., Oncogene 29:550, 2010; Li et al., Clin Chern 51:2229, 2005 Kanmura et al. , J Gastroenterol 45:459, 2010; Lee et al., Proteomics 6:2865, 2006 Fenz et al., Proteomics Clin Appl 1:536, 2007; Habermann et al., Gastroenterol 131:1020, 2006 NZA Chronic lymphocytic leukemia (CLL) Serum Miguet et al., 5:2258, 2006; J Proteome Res C4a CLL Serum Miguet et al., 5:2258, 2006; J Proteome Res NZA Ovarian Ascites versus serum Bj orge et al., 92(5):895-905, Br J Cancer 2005 S5b-9 Ovarian Ascites versus serum Bj orge et al., 92(5):895-905, Br J Cancer 2005 C5a Non-small cell lung cancer (NSCLC) Serum Corrales et al 189:4674, 2012 ., J Immunol C1 CD59, Factor inhibitor, CD46, H Ovarian Ascites versus serum Bj orge et al., 92(5):895-905, Br J Cancer 2005 Factor N Acute myeloid leukemia Serum Lee et al. El 33:1863, 2012 ectroporesis Factor N Lung Bronchoalveolus bright lavage (BAL), sputum Pio et al., Cancer Epidemiol Biomarkers Prev 19:2665, 2010

In addition, complement activation may be a consequence of chemotherapy or radiotherapy, and thus inhibition of complement activation may be used as an adjuvant therapy in the treatment of malignancies to reduce iatrogenic inflammation. When chemotherapy and radiotherapy preceded surgery, C5b-9 accumulations were more intense and extensive. C5b-9 accumulations were absent in all benign tumor samples. Protein S/vitronectin was present in the form of fibrillar deposits in the connective tissue matrix and in the form of diffuse deposits around tumor cells, less intense and extensive than fibronectin. Accumulations of IgG, C3, and C4 were present only in carcinoma samples. The presence of C5b-9 accumulations is an indicator of complement activation and its subsequent pathogenetic effects in breast cancer (Niculescu, et al., Am. J. Pathol. 140:1039 1043, 1992).

Given the data described in Example 16 that systemic administration of an anti-MASP2 antibody that specifically inhibits the lectin pathway of complement activation inhibits neovascularization at least as effectively as an anti-VEGF antibody, it is expected that systemic delivery of a MASP-2 inhibitory agent , may be effective for inhibiting tumor angiogenesis, thereby reducing the growth and/or metastasis of tumors in an individual suffering from an angiogenesis-dependent cancer.

Angiogenesis-dependent malignancies include a malignant tumor of epithelial origin or neuronal origin, or carcinoma, or a solid tumor or sarcoma, or a liquid tumor such as leukemia or lymphoma. Any cancer for which treatment is known or is in development using an angiostatic compound (eg, a VEGF antagonist) is included within the scope of the methods of the invention. Preferred malignancies in this context include: colorectal cancers, breast cancers (including metastatic breast cancer, inflammatory breast carcinoma), lung, kidney, liver, esophagus, ovary, pancreas, prostate and stomach, as well as glioma, gastrointestinal stromal tumors, lymphoma, melanoma and carcinoid tumors (NCI clinical trials database: found at www_cancer_gov_clinicaltrials/search, accessed 3/25/2014). Many of these malignancies have been shown to respond to treatment with bevacizumab (Avastin®), a humanized monoclonal antibody that blocks VEGF binding to its receptors and inhibits tumor angiogenesis (eg, Amit et al., PLoS One 8(1):e51780 (2013 ).

In accordance with the foregoing, another aspect of the invention relates to methods of inhibiting tumor angiogenesis and/or tumor metastasis in an individual suffering from an angiogenesis-dependent malignancy. This method involves administering a composition containing an amount of a MASP-2 inhibitor effective to inhibit tumor angiogenesis and/or tumor metastasis to an individual suffering from an angiogenesis-dependent cancer. In some embodiments, the individual suffers from an angiogenesis-dependent cancer selected from the group consisting of colorectal, breast, lung, kidney, liver, esophagus, ovarian, pancreatic, prostate and gastric cancer, as well as gliomas , gastrointestinal stromal tumors, lymphoma, melanoma and carcinoid tumor. In some embodiments, angiogenesis-dependent cancers are those types of cancers that are expected to benefit from treatment with an anti-VEGF agent, such as the anti-VEGF antibody Avastin® (bevacizumab, Genentech, CA), for example, such as any cancer tumor for which treatment is known or is in development using an angiostatic compound (eg, VEGF antagonist), including advanced malignancies metastatic to the liver, melanoma, ovarian cancer, neuroblastoma, pancreatic cancer, hepatocellular carcinoma, endometrial cancer, prostate cancer, angiosarcoma, metastatic or unresectable angiosarcoma, recurrent ovarian sex cord stromal tumors, esophageal cancer, gastric cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, diffuse large B-cell lymphoma, recurrent or metastatic head and neck cancer, neoplastic meningitis , cervical cancer, uterine body cancer, peritoneal carcinomatosis in advanced stages, gliosarcoma, neuroendocrine carcinoma, extracranial Ewing sarcoma, acute myeloid leukemia, chronic myelogenous leukemia, intracranial meningioma, Kaposi's sarcoma in advanced stages, mesothelioma, malignant tumor of the biliary tract, metastatic carcinoid tumors and malignant tumors of the urinary tract in late stages. Preferred malignancies in this context include: colorectal cancers, breast cancers (including metastatic breast cancer, inflammatory breast carcinoma), lung, kidney, liver, esophagus, ovary, pancreas, prostate and stomach, as well as glioma, gastrointestinal stromal tumors, lymphoma, melanoma and carcinoid tumors.

The MASP-2 inhibitory composition can be administered locally to the area of the tumor(s), for example, by local administration of the composition during surgery or local injection, either directly or remotely, for example, through a catheter. Alternatively, the MASP-2 inhibitory agent can be administered to a patient systemically, for example, through intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous or other parenteral administration, or potentially through oral administration for non-peptidergic agents. The MASP-2 inhibitory agent composition can be combined with one or more additional drugs, such as an additional antiangiogenic agent and/or an additional chemotherapeutic agent. Administration may be repeated as determined by the physician until the condition is resolved or controlled.

Given the data from the present study showing that OMS646 is at least as effective as an anti-VEGF antibody in reducing CNV when systemically delivered to mice at all dosage levels tested, it is also expected that a MASP-2 inhibitory agent such as OMS646 may also be effective as an antiangiogenic agent for use in inhibiting angiogenesis-dependent conditions such as myelofibrosis and hereditary hemorrhagic telangiectasia.

IV. MASP-2 inhibitory agents.

In various aspects, the present invention provides methods for inhibiting the adverse effects of angiogenesis by administering a MASP-2 inhibitory agent to a patient in need thereof. MASP-2 inhibitory agents are administered in amounts effective to inhibit MASP-2-dependent complement activation in a living subject. To practice this aspect of the invention, representative MASP-2 inhibitory agents include: molecules that inhibit the biological activity of MASP-2 (such as small molecule inhibitors, anti-MASP-2 antibodies, or blocking peptides that interact with MASP-2 or interfere with protein-protein interaction ), and molecules that reduce MASP-2 expression (such as MASP-2 antisense nucleic acid molecules, MASP-2-specific RNAi molecules, and MASP-2 ribozymes), thereby preventing activation by MASP-2 of the lectin pathway. tribe MASP-2 inhibitory agents can be used alone as primary therapy or in combination with other therapeutic agents as adjuvant therapy to enhance the therapeutic benefit of other medical treatment.

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: inhibition of the formation or production of MASP-2-dependent activation system products complement C4b, C3a, C5a and/or C5b 9 (MAC) (measured, e.g., as described in Example 2), decreased C4 cleavage and C4b accumulation (measured, e.g., as described in Example 2), or decreased C3 cleavage and accumulation C3b (measured, for example, as described in example 2).

In accordance with the present invention, MASP-2 inhibitory agents are used that are effective in inhibiting angiogenesis and have detectable antiangiogenic activity and/or induce a reduction in neoangiogenesis. In the context of the invention, antiangiogenic activity may include at least one or more of the following: reduction or reduction of neoangiogenesis, normalization of blood vessels, and/or reduction of the number of vessels in the pathogenic area.

Neoangiogenesis and assessment of an antiangiogenic agent, such as a MASP-2 inhibitory agent, can be detected using any method known to one skilled in the art. For example, neoangiogenesis and evaluation of an antiangiogenic agent can be assessed in a CNV animal model of laser-induced injury (as described in Examples 12, 14 and 16 herein), or in situ in a patient or tumor via non-invasive methods such as PET imaging (positron emission tomography), MRI (magnetic resonance imaging), DCE-MRI (dynamic contrast-enhanced MRI) or CT (computed tomography). These methods can be used to monitor tumor burden based on increased leakage of the blood vessel network in tumors. Using MRI or PET, the presence of angiogenesis markers such as α5β3 integrin, VEGF or bFGF in plasma can be monitored.

Alternatively, neoangiogenesis can be assessed using tumor biopsies or sections obtained from a pathogenic area from a patient suffering from an angiogenesis-dependent condition, and subsequent immunohistochemical assays of endothelial cells to assess the activity and compare it with the activity of normal endothelial cells from a healthy individual or endothelial cells from patient, but isolated from another part of the body. Such immunohistochemical assays can be performed using anti-pan-endothelial cell antibodies, such as anti-CD31 and anti-CD34 antibodies, to assess microvascular density. Tissue sections can be stained using endothelial cell markers, in combination with proliferation markers, to examine the relationship between tumor endothelial cells and proliferating tumor cells in the tissue. Examples of endothelial markers are CD31 and CD34. One example of a proliferation marker is Ki67, which is an excellent marker for determining the growing fraction from a given cell population. The fraction of Ki-67 positive tumor cells (Ki-67 labeling index) often correlates with the clinical course of a malignant tumor. Microvessel density (MVD) can be assessed, for example, in a tumor section stained with anti-CD31 antibody and using staining intensity to quantify MVD. Quantification of MVD is preferably performed by counting positively stained luminal structures in four to five representative images per tumor section. A reduction, preferably a statistically significant reduction, in MVD assessed in at least four to five representative images per tumor section is preferably observed as an indication that the administered molecule has antiangiogenic activity or is capable of reducing neoangiogenesis.

Neoangiogenesis can also be assessed using cells, preferably endothelial cells, from a tumor, from a healthy individual or from endothelial cell lines. Endothelial cells from a tumor are preferably referred to as tumor endothelium. Tumor endothelial cells can be isolated by FACS (Fluorescence Activated Cell Sorting) from tumor tissue using CD31 as an endothelial marker. This can be carried out as described in van Beijnum et al., Nat Protoc. 3(6):1085-91, 2008. The preferred endothelial cells for assessing neoangiogenesis in vitro are HUVEC and RF24. In vitro assessment of neoangiogenesis activity can be performed using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl-)-2H-tetrazolium) assay to assess proliferative activity endothelial cells. Alternatively, other viability assays known to one skilled in the art may be used, such as MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), crystal violet, and WST-1 (water soluble) assays. tetrazolium).

In addition, other types of angiogenesis activity assays can be used, such as the spheroid sprouting assay and the Matrigel tube formation assay. In the Matrigel tube formation assay, cells, especially endothelial cells, are plated on a synthetic semi-natural

- 19 045598 gel matrix (such as Matrigel from BD Biosciences or collagen gel, or in some cases fibrin gels). In both assays, endothelial cells, preferably HUVEC, are used. After a certain period of time, depending on the cell culture conditions, the cells begin to form tube-like structures. The formation of tube-like structures is considered the first stage in the formation of new vessels. The read parameter is the number of node vessels per unit area. To assay spheroid germination, cellular spheroids (eg, endothelial cells) are embedded in a gel (eg, Matrigel and collagen gels). After a certain period of time, the formation of processes can be observed. The degree of germination is considered as a criterion for assessing the angiogenic potential of cells. The parameter read is the number of processes per spheroid. Antiangiogenic activity may be present when the number of processes per spheroid is reduced or reduced in treated cells over a given period of time compared to the number of processes per spheroid in untreated cells. The reduction or reduction may be a reduction of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. Antiangiogenic activity in tumor tissue may also be present when vascular normalization is visualized and/or when the number of vessels in the pathogenic area is reduced.

In a preferred embodiment, once a decrease in the number of vessels in the pathogenic area is detected compared to the number of vessels at the start of treatment, detectable antiangiogenic activity is present. The reduction may be a detectable decrease in the number of vessels in the pathogenic region or a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% reduction in the number of vessels in the pathogenic region. The pathogenic region is the tumor region, including surrounding tissue located close to the tumor region. Close in this context can mean up to a few centimeters.

Vascular normalization is preferably a change in the three-dimensional structure of the vessel or microvessel. For example, abnormal vessels or microvessels associated with neoangiogenesis activity in the tumor endothelium may be less uniform and/or may appear more tortuous and/or may appear more leaky than a control vessel or microvessel. The control vessel may be a vessel from a healthy individual or a vessel from a patient, but not located in the pathogenic region of the specified patient. In a preferred embodiment, once the three-dimensional structure of the vessel begins to appear more uniform, less tortuous and/or less leaky than the control vessel, antiangiogenic activity is said to be detected.

Preferably, fewer irregular, tortuous and/or leaking vessels are detected in the pathogenic area than at the start of treatment. More preferably, less means 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% less, or 100% less. Most preferably, irregular, tortuous and/or leaking vessels are not detected in the pathogenic area. Vascular normalization and/or the number of vessels in the pathogenic area can be assessed using a non-invasive imaging modality such as PET, MRI or CT imaging.

In the case of an ocular disease or condition associated with neoangiogenesis, several assays have been developed to evaluate detectable antiangiogenic activity and/or reduction or reduction of neoangiogenesis induced by the drug being tested, such as a MASP-2 inhibitory agent. In these different disease models, angiogenesis can be triggered by various stimuli, such as physical injury (laser-induced rupture of Bruch's membrane) (Shen et al., 2006 Gene therapy 13:225-234), or by overexpression of specific blood vessel growth factors such as as VEGF in transgenic mice (Miki et al., 2009, Ophthalmology 2009 September 116(9):1748-1754). If detectable anti-angiogenic activity and/or reduction or reduction of angiogenesis is assessed using a MASP-2 inhibitory agent, such MASP-2 inhibitory agent is said to be used as a drug for the prevention, treatment, reversion, cure and/or delay angiogenesis or a disease or condition associated with angiogenesis.

Evaluation of neoangiogenesis and/or antiangiogenic activity can be performed periodically, for example every week or every month. The increase/decrease in neoangiogenesis and/or the presence of antiangiogenic activity can thus be assessed periodically, for example every week or month. This assessment is preferably conducted at multiple time points for a given individual or at one or more time points for a given individual and a healthy control individual. Assessments can be carried out at regular intervals, for example every week, or every month. When one of the assessments of neoangiogenesis or angiogenic activity associated with a MASP-2 inhibitory agent results in the detection of a decrease in neoangiogenesis or the presence of antiangiogenic activity, the MASP-2 inhibitory agent, such as an anti-MASP2 antibody, is said to have detectable antiangiogenic activity and/or induces a decrease or decrease in neoangiogenesis.

A detectable decrease in neoangiogenesis activity and/or the presence of antiangiogenic activity is preferably detected when, for at least one time point, a decrease in neoangiogenesis and/or the presence of antiangiogenic activity is detected. Preferably, a decrease in neoangiogenesis and/or the presence of antiangiogenic activity is detected for at least two, three, four, five time points.

MASP-2 inhibitory agents that may be used 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, soluble receptors, and inhibitors of MASP-2 expression. MASP-2 inhibitory agents can inhibit the MASP-2-dependent complement activation system by blocking the biological function of MASP-2. For example, the inhibitory agent may effectively block MASP-2 protein-protein interactions, interfere with MASP-2 dimerization or assembly, block Ca ²⁺ binding, interfere with the active site of MASP-2 serine protease, or may reduce MASP-2 protein expression .

In some embodiments, MASP-2 inhibitory agents selectively inhibit MASP-2 complement activation, leaving the C1q-dependent complement activation system functionally intact.

In one embodiment, the MASP-2 inhibitory agent that can be used in the methods of the invention is a specific MASP-2 inhibitory agent that specifically binds to the polypeptide containing SEQ ID NO: 6 with an affinity of at least tenfold higher than for other antigens in the complement system. In another embodiment, the MASP-2 inhibitory agent specifically binds to the polypeptide comprising SEQ ID NO: 6 with a binding affinity at least 100 times higher than other antigens in the complement system. In one embodiment, the MASP-2 inhibitory agent specifically binds to at least one of (i) a CCP1-CCP2 domain (aa 300-431 of SEQ ID NO: 6) or a MASP-2 serine protease domain (aa 445-682 of SEQ ID NO: 6) and inhibits MASP-2 dependent complement activation. In one embodiment, the MASP-2 inhibitory agent is an anti-MASP-2 monoclonal antibody, or fragment thereof, that specifically binds MASP-2. The binding affinity of a MASP-2 inhibitory agent can be determined using a suitable binding assay.

The MASP-2 polypeptide has a molecular structure similar to MASP-1, MASP-3, and Clr and Cls, the Cl proteases of the complement system. The cDNA molecule shown in SEQ ID NO: 4 encodes a representative example of MASP-2 (consisting of the amino acid sequence shown in SEQ ID NO: 5), and provides the formation of a human MASP-2 polypeptide with a leader sequence (aa 1-15), which is cleaved after secretion, resulting in the formation of the mature form of human MASP-2 (SEQ ID NO: 6). As shown in FIG. 2, the human MASP-2 gene contains twelve exons. The human MASP-2 cDNA is encoded by exons B, C, D, F, G, H, I, J, K, and L. Alternative splicing results in the formation of a 20 kDa protein called MBL-associated protein 19 (MAp19, also designated sMAP) (SEQ ID NO: 2), encoded by (SEQ ID NO: 1), formed from exons B, C, D and E, as shown in FIG. 2. The cDNA molecule set forth in SEQ ID NO: 50 encodes murine MASP-2 (consisting of the amino acid sequence set forth in SEQ ID NO: 51) and produces a murine MASP-2 polypeptide with a leader sequence that is cleaved off after secretion, resulting in to the formation of the mature form of mouse MASP-2 (SEQ ID NO: 52). The cDNA molecule shown in SEQ ID NO:53 encodes rat MASP2 (consisting of the amino acid sequence shown in SEQ ID NO: 54) and provides the formation of a rat MASP-2 polypeptide with a leader sequence that is cleaved after secretion, resulting in the formation of the mature form rat MASP-2 (SEQ ID NO: 55).

One skilled in the art will appreciate that the sequences described in SEQ ID NO: 4, SEQ ID NO: 50 and SEQ ID NO: 53 represent single allelic variants of human, murine and rat MASP-2, respectively, and that one would expect allelic variants and alternative splicing. Allelic variants of the nucleotide sequences presented in SEQ ID NO: 4, SEQ ID NO: 50 and SEQ ID NO: 53, including sequences containing silent mutations and sequences in which mutations cause changes in amino acid sequences, are included within the scope of the present invention. Allelic variants of the MASP-2 sequence can be cloned using probes from cDNA libraries or genomic libraries from different individuals, according to standard methods.

The human MASP-2 protein domains (SEQ ID NO: 6) are shown in FIG. 1 and 2A, and include the sea urchin/bone morphogenetic protein (CUBI) N-terminal Clr/Cls/Vegf domain (aa 1-121 of SEQ ID NO: 6), an epidermal growth factor-like domain (aa 122,166), a second CUBI domain (ac 167 293), also a tandem of complement control protein domains and a serine protease domain. Alternative splicing of the MASP-2 gene results in the formation of MAp19 shown in FIG. 1. MAp19 is a nonenzymatic protein containing the N-terminal CUB1-EGF region of MASP-2 with four additional residues (EQSL) derived from exon E, as shown in FIG. 1.

Several proteins have been shown to bind or interact with MASP-2 via

- 21 045598 protein interactions. For example, MASP-2 is known to bind and form Ca ²⁺ -dependent complexes with the lectin proteins MBL, H-ficolin and L-ficolin. Each MASP-2/lectin complex has been shown to activate complement through MASP-2-dependent cleavage of the C4 and C2 proteins (Ikeda, K., et al., J. Biol. Chem. 262:7451 7454, 1987; Matsushita, M. , et al., J. Exp. Med. 176:1497 2284, 2000; Matsushita, M., et al., J. Immunol. 168:3502 3506, 2002). Studies have shown that the CUB1EGF domains of MASP-2 are required for the association of MASP-2 with MBL (Thielens, NM, et al., J. Immunol. 166:5068, 2001). The CUB1EGFCUBII domains have also been shown to mediate the dimerization of MASP-2 required for the formation of the active MBL complex (Wallis, R., et al., J. Biol. Chem. 275:30962 30969, 2000). Thus, MASP-2 inhibitory agents can be identified that bind to or interfere with MASP-2 target sites known to be important for MASP-2-dependent complement activation.

Antibodies against MASP-2.

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. Anti-MASP-2 antibodies that can be used in this aspect of the invention include polyclonal, monoclonal or recombinant antibodies derived from any antibody-producing mammal and can be multispecific, chimeric, humanized, anti-idiotypic antibodies and antibody fragments. Antibody fragments include Fab fragments, Fab', F(ab) ₂ , F(ab') ₂ , Fv fragments, scFv fragments and single chain antibodies, as described later herein.

Anti-MASP-2 antibodies can be screened for their ability to inhibit the MASP-2-dependent complement activation system and for antiangiogenic activity using the assays described herein. Several antibodies against MASP-2 have been described in the literature, and several have been newly developed, some of which are listed below in the table. 2. For example, as described in Examples 10 and 11 herein, anti-rat MASP-2 Fab2 antibodies have been identified that block MASP-2-dependent complement activation, and as shown in Example 14, a monoclonal antibody derived from the anti-MASP Fab2 antibody -2 rats, has antiangiogenic activity in a mouse model of laser-induced CNV. As further described in Example 15, and as further described in US2012/0282263, the contents of which are incorporated herein by reference, fully human anti-MASP-2 scFv antibodies have been identified that block MASP-2-dependent complement activation, and as described in Example 16, a representative anti-human MASP-2 monoclonal antibody (OMS646), which blocks lectin pathway function, has antiangiogenic activity in a mouse model of laser-induced CNV. Accordingly, in one embodiment, the MASP-2 inhibitory agent for use in the methods of the invention comprises a human antibody, for example, such as OMS646. Accordingly, in one embodiment, a MASP-2 inhibitory agent for use in the compositions and methods of the claimed invention comprises a human antibody that binds a polypeptide consisting of human MASP-2 (SEQ ID NO: 6), wherein the antibody comprises: I) a) a heavy chain variable region comprising i) a heavy chain CDR1 containing the amino acid sequence 31-35 of SEQ ID NO: 67 or SEQ ID NO: 68; and ii) a heavy chain CDR2 comprising the amino acid sequence 50-65 of SEQ ID NO: 67 or SEQ ID NO: 68; and iii) a heavy chain CDR3 comprising the amino acid sequence 95-102 of SEQ ID NO: 67 or SEQ ID NO: 68; and b) a light chain variable region comprising: i) a light chain CDR1 comprising the 24-34 amino acid sequence of either SEQ ID NO: 69 or SEQ ID NO: 71; and ii) a light chain CDR2 comprising the amino acid sequence 50-56 of either SEQ ID NO: 69 or SEQ ID NO: 71; and iii) a light chain CDR3 comprising the amino acid sequence 89-97 of either SEQ ID NO: 69 or SEQ ID NO: 71; or ii) a variant thereof otherwise identical to said variable domains, except for up to 6 total amino acid substitutions within said CDR regions of said heavy chain variable region and up to 6 total amino acid substitutions within said CDR regions of said light chain variable region, wherein the antibody or a variant thereof inhibits MASP-2-dependent complement activation. In one embodiment, the MASP-2 inhibitory agent for use in the methods of the invention comprises the human antibody OMS646.

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table 2

Exemplary MASP-2 Specific Antibodies

ANTIGEN ANTIBODY TYPE LINK Recombinant MASP-2 Rat polyclonal Peterson, S.V., et al., Mol. Immunol. 37:803811, 2000 Recombinant Rat MoAb (subclass Moller Kristensen, M., et al., J. human fragment CCP1/2 SP (MoAb 8B5) IgGl) of Immunol. Methods 282:159-167, 2003 Recombinant Rat MoAb (subclass Moller Kristensen, M., et al., J. human MAp19 (MoAb 6G12) (enters in cross reaction with MASP-2) IgGl) of Immunol. Methods 282:159-167, 2003 hMASP-2 Mouse MoAb (S/P) Mouse MoAb (N-terminus) Peterson, S.V., et al., Mol. Immunol. 35:409, April 1998 hMASP-2 (CCP1CCP2-SP domain) rat MoAb: NimoablOl, produced by hybridoma cell line 03050904 (ECACC) WO 2004/106384 hMASP-2 (full size, with his tag) mouse MoAb: NimoAbl04 produced by a hybridoma cell line M0545YM035 (DSMZ) NimoAbl08 produced by a hybridoma cell line M0545YM029 (DSMZ) NimoAbl09 produced by hybridoma cell line M0545YM046 (DSMZ) NimoAbllO produced by a hybridoma cell line M0545YM048 (DSMZ) WO 2004/106384 Rat MASP-2 (full size) anti-MASP-2 Fab2 antibody fragments Example 10 hMASP-2 (full size) Fully human scFv clones Example 15 and US2012/0282263

Antibodies against MASP-2 with reduced effector function.

In some embodiments of this aspect of the invention, anti-MASP-2 antibodies have a reduced effector function to reduce inflammation that can occur when the classical complement pathway is activated. The ability of IgG molecules to trigger the classical pathway of complement activation has been shown to be associated with the Fc region of the molecule (Duncan, AR, et al., Nature 332:738 740 1988). IgG molecules in which the Fc region has been removed by enzymatic cleavage lack this effector function (see Harlow, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988). Accordingly, antibodies with reduced effector function can be obtained as a result of the absence of the Fc region of the molecule, as a result of the presence of a genetically engineered Fc sequence that minimizes effector function, or as a result of belonging to the human IgG ₂ or IgG ₄ isotype.

Antibodies with reduced effector function can be produced by standard molecular biological manipulation of the Fc region of the heavy chains of IgG, as described in Example 9 herein, and also described in Jolliffe et al., Int'l Rev. Immunol. 10:241250, 1993, and Rodrigues et al., J. Immunol. 151:6954 6961, 1998. Antibodies with reduced effector function also include human IgG2 and IgG4 isotypes, which have a reduced ability to activate complement and/or interact with Fc receptors (Ravetch, J.V., et al., App. Rev. Immunol. 9:457 492, 1991; Isaacs, J.D., et al., J. Immunol. 148:3062 3071, 1992; van de Winkel, J. G., et al., Immunol. Today 14:215 221, 1993). Humanized or fully human antibodies specific for human MASP-2, consisting of IgG2 or IgG4 isotypes, can be produced by one of several methods known to one skilled in the art, as described in Vaughan, T. J., et al., Nature Biotechnical 16:535 539 , 1998.

Obtaining antibodies against MASP-2.

Antibodies against MASP-2 can be generated using MASP-2 polypeptides (eg, full-length MASP-2) or using peptides bearing a MASP-2 antigenic epitope (eg, part of a MASP-2 polypeptide). Immunogenic peptides can be so small

- 23 045598 as five amino acid residues. For example, a MASP-2 polypeptide comprising the entire amino acid sequence of SEQ ID NO: 6 can be used to induce the formation of anti-MASP-2 antibodies that can be used in the method of the invention. Specific domains of MASP-2 known to be involved in protein-protein interactions, such as the CUBI, and CUBIEGF domains, as well as the region including the serine protease active site, can be expressed in the form of recombinant polypeptides, as described in Example 3, and used as antigens. In addition, peptides containing at least 6 amino acids of the MASP-2 polypeptide (SEQ ID NO: 6) can also be used to induce the formation of anti-MASP-2 antibodies. Additional examples of MASP-2-derived antigens that can be used to induce the formation of antibodies against MASP-2 are presented below in table. 2. MASP-2 peptides and polypeptides used to induce antibody formation can be isolated in the form of 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 generated using a transgenic mouse strain as described in Examples 8 and 9 and further described below.

Antigens that can be used to generate antibodies against MASP-2 also include fusion polypeptides, such as MASP-2 or a portion thereof fused to an immunoglobulin polypeptide or maltose binding protein. The polypeptide immunogen may be a full-length molecule or a portion thereof. If a portion of the polypeptide is hapten-like, such portion may advantageously be attached or linked to a macromolecular carrier (such as limpet hemocyanin (KLH), bovine serum albumin (BSA), or tetanus toxoid) for immunization.

Table 3 MASP-2-derived antigens

SEQ ID NO: Amino acid sequence SEQ ID NO:6 Human MASP-2 protein SEQ ID NO:51 Mouse MASP-2 protein SEQ ID NO:8 Tsemen CUBI human MASP-2 (AK 1121 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 CUBIEGFCUBII domains of human MASP2 (aa 1-293 of SEQ ID NO:6) SEQ ID NO:11 Human MASP-2 EGF comene (aa 122,166 of SEQ ID NO:6) SEQ ID NO:12 Human MASP-2 serine protease comene (aa 429,671 of SEQ ID NO:6) SEQ ID NO:13 GKDSCRGDAGGALLVFL Mutant form with inactivated serine protease domain (aa 610 625 of SEQ ID NO:6 with mutant Ser 618) SEQ ID NO:14 TPLGPKWPEPVFGRL Human peptide CUBI SEQ ID NO:15: TAPPGYRLRLYFTHFDLELSHLCEYDFVKLSSGAKVLA TLCGQ Human peptide CUBI SEQ ID NO:16: TFRSDYSN MBL binding site in the human CUBI domain SEQ ID NO:17: FYSLGSSLDITFRSDYSNEKPFTGF MBL binding site in the human CUBI domain SEQ ID NO:18 IDECQVAPG EGF peptide SEQ ID NO:19 ANMLCAGLESGGKDSCRGDSGGALV Serine protease active site peptide

Polyclonal antibodies.

Polyclonal antibodies against MASP-2 can be obtained by immunizing animals with the MASP-2 polypeptide or an immunogenic portion thereof using methods well known to one skilled in the art. See, for example, Green et al., Production of Polyclonal Antisera, in Immunochemical Protocols (Manson, ed.), page 105. The immunogenicity of the MASP-2 polypeptide can be increased by the use of an adjuvant including mineral gels such as aluminum hydroxide or Freund's adjuvant (complete or incomplete), surfactants such as lysolecithin, pluronyl polyols, polyanions, oil emulsions, limpet hemocyanin and dinitrophenol. Polyclonal antibodies are typically produced in animals such as horses, cows, dogs, chickens, rats, mice, rabbits, guinea pigs, goats, or sheep. Alternatively, an anti-MASP-2 antibody that can be used in the present invention can also be obtained from an ape. General methods for producing diagnostically and therapeutically useful antibodies in baboons can be found, for example, in Goldenberg et al., International publication

- 24 045598 patent no. WO 91/11465, and in Losman, M.J., et al., Int. J. Cancer 46:310, 1990. Serum containing immunologically active antibodies is then obtained from the blood of such immunized animals using standard methods well known in the art.

Monoclonal antibodies.

In some embodiments, the MASP-2 inhibitory agent is an anti-MASP-2 monoclonal antibody. Anti-MASP-2 monoclonal antibodies are highly specific, being directed against a single epitope of MASP-2. As used herein, the designation monoclonal refers to the nature of the antibody as being derived from a substantially homogeneous population of antibodies, and does not imply that the antibody must be produced by any particular method. Monoclonal antibodies can be produced using any method that relates to the production of antibody molecules in culture of stable cell lines, such as the hybridoma method described in Kohler, G., et al., Nature 256:495, 1975, or they can be produced by recombinant methods. DNA (see, for example, US Patent No. 4816567 from Cabilly). Monoclonal antibodies can also be isolated from phage antibody libraries using methods described in Clackson, T., et al., Nature 352:624 628, 1991, and Marks, J.D., et al., J. Mol. Biol. 222:581 597, 1991. Such antibodies may belong to any class of immunoglobulin, including IgG, IgM, IgE, IgA, IgD, and any subclass thereof.

For example, monoclonal antibodies can be produced by injecting a suitable mammal (eg, BALB/c mouse) with a composition containing a MASP-2 polypeptide or a portion thereof. After a predetermined period of time, splenocytes are isolated from the mouse and resuspended in culture medium. The splenocytes are then fused with an immortalized cell line to form a hybridoma. The formed hybridomas are grown in cell culture and screened for their ability to produce monoclonal antibodies against MASP-2. Examples further describing the production of anti-MASP-2 monoclonal antibodies are provided herein (e.g., Examples 10 and 13) (see also Current Protocols in Immunology, Vol. 1., John Wiley & Sons, pages 2, 5, 12, 6, 7, 1991).

Human monoclonal antibodies can be produced using transgenic mice engineered to produce specific human antibodies in response to an antigenic stimulus. In this method, elements of the human immunoglobulin heavy and light chain locus are introduced into mouse strains derived from embryonic stem cell lines containing targeted lesions of the endogenous immunoglobulin heavy and light chain loci. Transgenic mice can synthesize human antibodies specific for human antigens, such as the MASP-2 antigens described herein, and these mice can be used to produce hybridomas that secrete human anti-MASP-2 antibodies by fusing B cells from such animals with suitable myeloma cell lines using conventional Koehler-Milstein technology, as described further in Example 7. Transgenic mice with the human immunoglobulin genome are commercially available (for example, from Abgenix, Inc., Fremont, CA, and Medarex, Inc., Annandale, N.J.) . Methods for obtaining human antibodies from transgenic mice are described, for example, in Green, L.L., et al., Nature Genet. 7:13, 1994; Lonberg, N., et al., Nature 368:856, 1994; and Taylor, L.D., et al., Int. Immun. 6:579, 1994.

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

Once produced, polyclonal, monoclonal or phage-derived antibodies are first tested for specific binding to MASP-2. A variety of assays known to those skilled in the art can be used to detect antibodies that specifically bind to MASP-2. Exemplary assays include Western blot or immunoprecipitation assays by standard methods (eg, as described in Ausubel et al.), immunoelectrophoresis, enzyme-linked immunosorbent assay, dot blot, inhibition or competition assays, and sandwich assays (as described in Harlow and Land, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988). After identifying antibodies that specifically bind to MASP-2, anti-MASP-2 antibodies are tested for their ability to function as a MASP-2 inhibitory agent in one of several assays, such as the C4 lectin-specific cleavage assay (described in Example 2 ), a C3b accumulation assay (described in Example 2) or a C4b accumulation assay (described in Example 2).

The affinity of monoclonal antibodies against MASP-2 can be easily determined by one skilled in the art (see, for example, Scatchard, A., NY Acad. Sci. 51:660 672, 1949). In one embodiment, anti-MASP-2 monoclonal antibodies that can be used in 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.

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Monoclonal antibodies that can be used in the method of the invention include chimeric antibodies in which part of the heavy and/or light chain is identical or homologous to the corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibodies, while how the remainder of the chain(s) are identical or homologous to corresponding sequences in antibodies derived from other species or belonging to another class or subclass of antibodies, as well as fragments of such antibodies (U.S. Patent No. 4,816,567, from Cabilly; and Morrison, S.L., et al., Proc. Nat'1 Acad. Sci. USA 81:6851 6855, 1984).

One form of chimeric antibody that can be used in the invention is a humanized monoclonal antibody against MASP-2. Humanized forms of non-human (eg, murine) antibodies are chimeric antibodies that contain minimal sequence derived from a non-human immunoglobulin. Humanized monoclonal antibodies are produced by transferring non-human (eg, murine) complementarity determining regions (CDRs) from mouse immunoglobulin heavy and light variable chains into the human variable domain. Typically, substitutions are then made with human antibody residues in the framework regions of the non-human equivalents. In addition, humanized antibodies may contain residues not found in the recipient antibody or the donor antibody. These modifications are made to further refine the activity of the antibody. Typically, a humanized antibody contains substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to loops from a non-human immunoglobulin, and all or substantially all of the framework Fv regions are regions from the human immunoglobulin sequence. The humanized antibody optionally also contains at least a portion of an immunoglobulin constant region (Fc), typically from human immunoglobulin. For further details, see Jones, P.T., et al., Nature 321:522525, 1986; Reichmann, L., et al., Nature 332:323-329, 1988; and Presta, Curr. Op. Struct. Biol. 2:593596, 1992.

Humanized antibodies that can be used in the invention include human monoclonal antibodies containing at least the MASP-2 binding CDR3 region. In addition, the Fc portions can be replaced to produce IgA or IgM, just like human IgG antibodies. Such humanized antibodies may be of particular clinical utility because they specifically recognize human MASP-2 but do not provoke an immune response in the person against the antibody itself. Therefore, they are better suited for in vivo administration to humans, especially when repeated or long-term administration is required.

An example of the preparation of a humanized anti-MASP-2 antibody from a mouse monoclonal anti-MASP-2 antibody is presented herein in Example 6. Methods for the preparation of humanized monoclonal antibodies are also described, for example, in Jones, P.T., et al., Nature 321:522, 1986; Carter, P., et al., Proc. Nat'l. Acad. Sci. USA 89:4285, 1992; Sandhu, J. S., Crit. Rev. Biotech. 12:437, 1992; Singer, I.I., et al., J. Immun. 150:2844, 1993; Sudhir (ed.), Antibody Engineering Protocols, Humana Press, Inc., 1995; Kelley, Engineering Therapeutic Antibodies, in Protein Engineering: Principles and Practice, Cleland et al. (eds.), John Wiley & Sons, Inc., pages 399-434, 1996; and US Patent No. 5693762, from Queen, 1997. In addition, there are commercial organizations that synthesize humanized antibodies from specific regions of the mouse antibody, such as Protein Design Labs (Mountain View, CA).

Recombinant antibodies.

Antibodies against MASP-2 can also be produced using recombinant methods. For example, human antibodies can be produced using human immunoglobulin expression libraries (available, for example, from Stratagene, Corp., La Jolla, Calif.) to produce human antibody fragments ( _VH , VL, Fv, Fd, Fab or F(ab') ₂ ). These fragments are then used to construct fully human antibodies using methods similar to those for producing chimeric antibodies.

Anti-idiotypic antibodies.

Once anti-MASP-2 antibodies with the desired inhibitory activity have been identified, these antibodies can be used to produce anti-idiotypic antibodies resembling a portion of MASP-2 using methods well known in the art. See, for example, Greenspan, N.S., et al., FASEB J. 7:437, 1993. For example, antibodies that bind to MASP-2 and competitively inhibit MASP-2 protein interactions required for complement activation can be used to produce anti-idiotypic antibodies that resemble the MBL binding site on the MASP2 protein and thus bind and neutralize a MASP-2 binding ligand, such as MBL.

Fragments of immunoglobulins.

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

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It is well known in the art that only a small region of the 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 pathway but are not involved in antigen binding. An antibody from which the pFc' region has been enzymatically excised, or which has been produced without the pFc' region, is designated an F(ab')2 fragment and retains both antigen-binding regions of the intact antibody. The isolated F(ab') ₂ fragment is classified as a bivalent monoclonal fragment due to its two antigen binding sites. Likewise, an antibody from which the Fc region has been enzymatically excised, or which is produced without the Fc region, is designated a Fab fragment, and retains one of the antigen-binding regions of the intact antibody molecule.

Antibody fragments can be produced by proteolytic hydrolysis, such as digestion of full-length antibodies with pepsin or papain by conventional methods. For example, antibody fragments can be produced by enzymatic digestion of antibodies using pepsin to produce a 5S fragment designated F(ab')2. This fragment can be further cleaved using a thiol reducing agent to produce 3.5S monovalent Fab' fragments. Optionally, the cleavage reaction can be carried out using a blocking group for sulfhydryl groups that are formed by cleavage of disulfide bonds. Alternatively, enzymatic cleavage using pepsin directly produces two monovalent Fab fragments and an Fc fragment. These methods are described, for example, in US Patent No. 4331647 from Goldenberg; Nisonoff, A., et al., Arch. Biochem. Biophys. 89:230, 1960; Porter, R.R., Biochem. J. 73:119, 1959; Edelman, et al., in Methods in Enzymoyogy 1:422, Academic Press, 1967; and in 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 is preferred to avoid activation of the classical complement pathway, which is initiated by Fc binding to the Fcy receptor. There are several methods by which MoAbs can be produced that avoid Fcy receptor interactions. For example, the Fc region of a monoclonal antibody can be removed chemically using partial digestion with proteolytic enzymes (such as ficin digestion), thereby producing, for example, antigen binding antibody fragments such as Fab or F(ab) ₂ fragments (Mariani, M., et al. al., Mol. Immunol. 28:69 71, 1991). Alternatively, human IgG isotype y4, which does not bind Fcy receptors, can be used in the construction of a humanized antibody as described herein. Antibodies, single chain antibodies and antigen binding domains lacking an Fc domain can also be produced using recombinant methods described herein.

Single chain antibody fragments.

Alternatively, single polypeptide chain binding molecules specific for MASP-2 can be prepared in which the heavy and light chain Fv regions are connected. Fv fragments can be joined via a peptide linker to form a single chain antigen binding protein (scFv). These single-stranded antigen-binding proteins are produced by constructing a structural gene containing DNA sequences encoding the VH and _VL domains linked by an oligonucleotide. The structural gene is inserted into an expression vector, which is then introduced into a host cell such as E. coli. Recombinant host cells synthesize a single polypeptide chain with a peptide linker connecting the two V domains. Methods for preparing scFv are described, for example, by Whitlow, et al., Methods: A Companion to Methods in Enzymology 2:97, 1991; Bird, et al., Science 242:423, 1988; US Patent No. 4946778, to Ladner; Pack, P., et al., Bio/Technology 11:1271, 1993.

As an illustrative example, a MASP-2-specific scFv can be generated by exposing lymphocytes to a MASP-2 polypeptide in vitro and selecting from antibody display libraries in phage or similar vectors (eg, through the use of immobilized or tagged MASP-2 proteins or peptides). Genes encoding polypeptides having potential MASP-2 polypeptide binding domains can be obtained by screening libraries of random peptides displayed on phage or bacteria such as E. coli. These random peptide display libraries can be used to screen for peptides that interact with MASP-2. Methods for generating and screening such random peptide display libraries are well known in the art (U.S. Patent No. 5,223,409, from Lardner; U.S. Patent No. 4,946,778, from Ladner; U.S. Patent No. 5,403,484, from Lardner; U.S. Patent No. 5,571,698, from Lardner ; and Kay et al., Phage Display of Peptides and Proteins Academic Press, Inc., 1996) and random peptide display libraries and screening kits for such libraries are commercially available, for example, 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 anti-MASP-2 antibody fragment that can be used in this aspect of the invention is a peptide encoding a single complementarity determining region

- 27 045598 (CDR), which binds to an epitope on the MASP-2 antigen and inhibits MASP-2-dependent complement activation. CDR (minimum recognition unit) peptides can be generated by engineering genes encoding the CDR of an antibody of interest. Such genes are obtained, for example, using polymerase chain reaction to synthesize the variable region from RNA from antibody-producing cells (see, for example, Larrick et al., Methods: A Companion to Methods in Enzymology 2:106, 1991; Courtenay Luck, Genetic Manipulation of Monoclonal Antibodies, in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), page 166, Cambridge University Press, 1995; and Ward et al., Genetic Manipulation and Expression of Antibodies, in Monoclonal Antibodies : Principles and Applications, Birch et al. (eds.), page 137, Wiley Liss, Inc., 1995).

The anti-MASP-2 antibodies described herein are administered to a patient to inhibit MASP-2-dependent complement activation. In some embodiments, the MASP-2 inhibitory agent is a high affinity human or humanized anti-MASP-2 monoclonal 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 MASP2-dependent complement activation system. As used herein, the term isolated peptide MASP-2 inhibitors refers to peptides that inhibit MASP-2-dependent complement activation by binding to compete with MASP-2 for binding to another recognition molecule (eg, 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, which are substantially pure and are substantially free of other substances with which they may be found in nature, to the extent practicable and suitable for their intended use.

Peptide inhibitors have been successfully used in vivo to interfere with protein-protein interactions and catalytic sites. For example, peptide adhesion molecule inhibitors, structurally related to LFA-1, have recently been approved for clinical use in coagulopathies (Ohman, E.M., et al., 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., et al., J. Biochem. 120:445 51, 1996). Longer peptides, ranging in length from 25 to 200 amino acid residues, have also been successfully used to block integrin-dependent adhesion (Zhang, L., et al., J. Biol. Chem. 271(47):29953 57, 1996) . In general, longer peptide inhibitors have higher affinity and/or lower dissociation rates than shorter peptides and may thus be more potent inhibitors. Cyclic peptide inhibitors have also been shown to be effective integrin inhibitors in vivo for the treatment of human inflammatory diseases (Jackson, D.Y., et al., J. Med. Chem. 40:3359 68, 1997). One method for producing cyclic peptides involves peptide synthesis in which the terminal amino acids of the peptide are cysteines, thereby allowing the peptide to exist in a cyclic form via a disulfide bond between the terminal amino acids, which has been shown to improve affinity and half-life in vivo for the treatment of hematopoietic neoplasias ( for example, US Patent No. 6649592, from Larson).

Synthetic peptide inhibitors of MASP-2.

MASP-2 inhibitory peptides that can be used in the methods of this aspect of the invention are illustrated with amino acid sequences that mimic target regions important for MASP-2 function. Inhibitory peptides that can be used in the practice of the methods of the invention range in size from about 5 amino acids to about 300 amino acids. In table 4 provides a list of illustrative inhibitory peptides that may be used in the practice of this aspect of the present invention. A candidate MASP-2 inhibitory peptide can be tested for its ability to function as a MASP-2 inhibitory agent in one of several assays, including, for example, the lectin-specific C4 cleavage assay (described in Example 2) and the C3b accumulation assay (described in Example 2 ).

In some embodiments, the MASP-2 inhibitory peptides are derived from MASP-2 polypeptides and are selected from the full-length mature MASP-2 protein (SEQ ID NO: 6), or from a specific domain of the MASP-2 protein, such as the CUBI domain (SEQ ID NO: 8), CUBIEGF domain (SEQ ID NO: 9), EGF domain (SEQ ID NO: 11) and serine protease domain (SEQ ID NO: 12). As described previously, the CUBEGFCUBII regions have been shown to be essential for dimerization and binding to MBL (Thielens et al., supra). In particular, the peptide sequence TFRSDYN (SEQ ID NO: 16) in the CUBI domain of MASP-2 was shown to be involved in MBL binding in a study that identified the presence of a homozygous Asp105 to Gly105 mutation in humans resulting in loss of MASP-2 from the complex MBL (Stengaard Pedersen, K., et al., New England J. Med. 349:554 560, 2003).

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In some embodiments, MASP-2 inhibitory peptides are derived from lectin proteins that bind to MASP-2 and are involved in the lectin complement pathway. Several different lectins have been identified to be involved in this pathway, including mannan-binding lectin (MBL), L-ficolin, M-ficolin, and H-ficolin. (Ikeda, K., et al., J. Biol. Chem. 262:7451 7454, 1987; Matsushita, M., et al., J. Exp. Med. 176:1497 2284, 2000; Matsushita, M., et al., J. Immunol. 168:3502 3506, 2002). These lectins are present in serum in the form of oligomers of homotrimeric subunits, each of which has N-terminal collagen-like fibers with carbohydrate recognition domains. These various lectins have been shown to bind to MASP-2, and the lectin/MASP-2 complex activates complement through cleavage of the C4 and C2 proteins. H-ficolin has an amino-terminal region of 24 amino acids, a collagen-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 al., J. Immunol. 168:3502 3506, 2002). Hphycolin binds to GlcNAc and causes agglutination of human erythrocytes coated with LPS derived from S. typhimurium, S. minnesota and E. coli. H-ficolin has been shown to associate with MASP-2 and MAp19 and activate the lectin pathway. The aforementioned L-ficolin/P35 also binds to GlcNAc and has been shown to associate with MASP-2 and MAp19 in human serum, and this complex has been shown to activate the lectin pathway (Matsushita, M., et al., J. Immunol. 164 :2281, 2000). Accordingly, MASP-2 inhibitory peptides that can be used in the present invention may contain a region of at least 5 amino acids selected from MBL protein (SEQ ID NO: 21), H-ficolin protein (Genbank accession number NM_173452), protein M-ficolin (Genbank accession number O00602) and L-ficolin protein (Genbank accession number NM_015838).

More specifically, the scientists identified a MASP-2 binding site on MBL located within 12 Gly-X-Y triplets GKD-GRD-GTK-GEK-GEP-GQG-LRG-LQG-POG-KLG-POG-NOG-PSGSOG-PKG-QKG- DOG-KS (SEQ ID NO: 26), which are located between the hinge and neck regions in the C-terminal part of the collagen-like domain of MBP (Wallis, R., et al., J. Biol. Chem. 279:14065, 2004). This region of the MASP-2 binding site is also highly conserved in human H-ficolin and human L-ficolin. A consensus binding site that is present in all three lectin proteins has been described, containing the amino acid sequence OGK-X-GP (SEQ ID NO: 22), where O represents hydroxyproline and X represents a hydrophobic residue (Wallis et al., 2004, supra ). Accordingly, in some embodiments, MASP-2 inhibitory peptides that can be used in this aspect of the invention are at least 6 amino acids in length and contain SEQ ID NO: 22. Peptides derived from MBL that include the amino acid sequence of GLR are shown to be -GLQ-GPO-GKL-GPO-G (SEQ ID NO: 24) binds MASP-2 in vitro (Wallis, et al., 2004, supra). To enhance binding to MASP-2, peptides flanked by two GPO triplets at each end (GPO-GPO-GLR-GLQGPO-GKL-GPO-GGP-OGP-O SEQ ID NO: 25) can be synthesized to enhance triple helix formation in the native MBL protein (as further described in Wallis, R., et al., J. Biol. Chem. 279:14065, 2004).

MASP-2 inhibitory peptides can also be prepared from human H-ficolin, comprising the sequence GAO-GSO-GEK-GAO-GPQ-GPO-GPO-GKM-GPK-GEO-GDO (SEQ ID NO: 27) from the MASP-2 consensus binding region 2 in H-ficolin. Also included are peptides derived from human Lficolin, including the sequence GCO-GLO-GAO-GDK-GEA-GTN-GKR-GERGPO-GPO-GKA-GPO-GPN-GAO-GEO (SEQ ID NO: 28) from the MASP consensus binding region -2 in L-ficolin.

MASP-2 inhibitory peptides can also be derived from a C4 cleavage site, such as LQRALEILPNRVTIKANRPFLVFI (SEQ ID NO: 29), which is a C4 cleavage site associated with the C-terminal portion of antithrombin III (Glover, G.I., et al., Mol. Immunol 25:1261 (1988)).

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Table 4

Exemplary MASP-2 inhibitory peptides

SEQ ID NO Source SEQ ID NO:6 Human MASP-2 protein SEQ ID NO:8 CUBI MASP-2 domain (ac 1-121 from SEQ ID NO:6) SEQ ID NO:9 CUBIEGF MASP-2 domains (ac 1-166 of SEQ ID NO:6) SEQ ID NO:10 CUBIEGFCUBII MASP-2 domains (ac 1-293 of SEQ ID NO: 6) SEQ ID NO:11 EGF MASP-2 domain (ac 122 166) SEQ ID NO:12 MASP-2 serine protease domain (aa 429 671) SEQ ID NO:16 MBL binding site in MASP-2 SEQ ID NO:3 Human MAr19 SEQ ID NO:21 Human MBL protein SEQ ID NO:22 OGK X GP, where "O"=hydroxyproline and "X" represents a hydrophobic amino acid residue Synthetic peptide, consensus binding site for human MBL and human ficolins SEQ ID NO:23 OGKLG Human MBL binding site core SEQ ID NO:24 GLR GLQ GPO GKL GPO G Human MBP, triplets 6-10, which have been shown to bind MASP-2 SEQ ID NO:25 GPOGPOGLRGLQGPOGKLGPOGPOGP 0 Human MBP triplets with GPO added to improve triple helix formation SEQ ID NO:26 GKDGRDGTKGEKGEPGQGLRGLQGPOG KLGPOGNOGPSGSOGPKGQKGDOGKS Human MBP triplets 1-17 SEQ ID NO:27 GAOGSOGEKGAOGPQGPOGPOGKMGPK GEOGDO Human Ficolin N (Hataka) SEQ ID NO:28 GCOGLOGAOGDKGEAGTNGKRGERGPO GPOGKAGPOGPNGAOGEO Human Ficolin L P35 SEQ ID NO:29 LQRALEILPNRVTIKANRPFLVFI Human C4 cleavage site

Note: The letter O represents hydroxyproline. The letter X represents 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 to become irreversible protease inhibitors. For example, suitable modifications may include, but are not necessarily limited to, halomethyl ketones (Br, Cl, I, F) at the C-terminus, Asp or Glu, or attached to functional side chains; haloacetyl (or other α-haloacetyl) groups on amino groups or other functional side chains; containing epoxide or imine groups at 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 provide the advantage of permanently inhibiting the enzyme by covalently attaching the peptide. This may 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 peptides that can be used in the method of the invention include peptides containing the MASP-2 binding region CDR3 of an anti-MASP-2 MoAb prepared as described herein. The sequence of CDR regions for use in peptide synthesis can be determined by methods known in the art. The heavy chain variable region is a peptide whose length typically ranges from 100 to 150 amino acids. The light chain variable region is a peptide whose length typically ranges from 80 to 130 amino acids. The CDR sequences within the heavy and light chain variable regions include only sequences of about 3-25 amino acids that can be easily sequenced by one of ordinary skill in the art.

One skilled in the art will recognize that substantially homologous variants of the MASP-2 inhibitory peptides described above also have MASP-2 inhibitory activity.

Exemplary embodiments include, but are not necessarily limited to, peptides having insertions, deletions, substitutions and/or additions of amino acids in the carboxy-terminal or amino-terminal regions of the subject peptides and mixtures thereof. Accordingly, it is believed that these homologous peptides having MASP-2 inhibitory activity can be used in the methods of this invention. The described peptides may also include duplicated motifs and other modifications with conservative substitutions. Conservative variations are described elsewhere herein and include substitution of one amino acid for another with similar charge, size or hydrophobicity, or the like.

MASP-2 inhibitory peptides can be modified to increase solubility and/or to maximize positive or negative charge so that they more closely resemble an intact protein fragment. The derivative may or may not have the exact primary amino acid structure of the peptide described herein, so long as the derivative functionally retains the desired MASP-2 inhibitory property. Modifications may include replacing an amino acid with one of the commonly known twenty amino acids, or with another amino acid, with a derivatized or substituted amino acid with additional desirable characteristics such as resistance to enzymatic degradation, or with a D-amino acid, or with another molecule or compound such as a carbohydrate , which mimics the natural conformation and function of an amino acid, amino acids or peptide; amino acid deletion; insertion of an amino acid from one of the commonly known twenty amino acids or another amino acid, a derivatized or substituted amino acid with additional desirable characteristics such as resistance to enzymatic degradation, or D-amino acids, or substitution with another molecule or compound, such as a carbohydrate, that mimics the natural conformation and the function of an amino acid, amino acids or peptide; or substitution with another molecule or compound, such as a carbohydrate or nucleic acid monomer, that mimics the natural conformation, charge distribution, and function of the parent peptide. Peptides can also be modified by acetylation or amidation.

The synthesis of inhibitory peptide derivatives can be based on known methods for peptide biosynthesis, carbohydrate biosynthesis, and the like. As a starting point, one skilled in the art can rely on a suitable computer program to determine the conformation of the peptide of interest. Once the conformation of the peptide described herein has been determined, one skilled in the art can determine a rational design method that sorts substitutions that can be made at one or more sites to produce a derivative that retains the basic conformation and charge distribution of the parent peptide, but which may have the characteristics , which it lacks, or which enhances characteristics found in the original peptide. Once candidate derivative molecules have been identified, the derivatives can be tested to determine whether they function as MASP-2 inhibitory agents using the assays described herein.

Screening for MASP-2 inhibitory peptides.

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

As an illustrative example, one method for producing MASP-2 binding peptide mimetics is as follows. Functional monomers of a known MASP-2 binding peptide or anti-MASP-2 antibody binding region shown to inhibit MASP-2 (template) are polymerized. The matrix is then removed, followed by polymerization of a second class of monomers in the space left by the matrix to produce a new molecule having one or more desirable properties similar to those of the matrix. In addition to producing peptides by this method, MASP-2 binding molecules that are MASP-2 inhibitory agents such as polysaccharides, nucleosides, drugs, nucleoproteins, lipoproteins, carbohydrates, glycoproteins, steroids, lipids and other biologically active materials can also be produced. . This method can be used to develop a wide variety of biomimetics that are more stable than their natural equivalents because they are typically produced by polymerizing functional monomers using free radicals, resulting in a compound with a non-biodegradable backbone.

Peptide synthesis.

MASP-2 inhibitory peptides can be prepared using methods well known in the art, such as the solid phase synthesis method originally described by Merrifield, in J. Amer. Chem. Soc. 85:2149 2154, 1963. Automated synthesis can be performed, for example, using an Applied Biosystems 431A peptide synthesizer (Foster City, Calif.), according to the manufacturer's instructions. Other methods can be found, for example, in Bodanszky, M., et al., Peptide Synthesis, second edition, John Wiley & Sons, 1976, as well as in references to other works known to those skilled in the art.

Peptides can also be produced using standard genetic engineering techniques known to those skilled in the art. For example, a peptide can be produced enzymatically by inserting a nucleic acid encoding the peptide into an expression vector expressing DNA and translating the DNA into the peptide in the presence of the necessary amino acids. The peptide is then purified using chromatographic or electrophoretic methods, or through a carrier protein, which can be fused to the peptide and subsequently cleaved from it by insertion into an expression vector, in phase with the peptide coding sequence, to the nucleic acid sequence encoding the carrier protein . The peptide fusion protein can be isolated using chromatographic, electrophoretic or immunological methods (such as binding to a resin via an antibody against the carrier protein). The peptide can be cleaved using chemical methods or enzymatically, for example, through hydrolases.

MASP-2 inhibitory peptides that can be used in the method of the invention can also be produced in recombinant host cells following conventional methods. For expression of sequences encoding MASP-2 inhibitory peptides, a nucleic acid molecule encoding the peptide must be operably linked to transcriptional expression control sequences in an expression vector and then introduced into a host cell. In addition to transcriptional control sequences such as promoters and enhancers, expression vectors may include translational control sequences and a marker gene useful for selecting cells harboring the expression vector.

Nucleic acid molecules encoding the MASP-2 inhibitory peptide can be synthesized using gene machines using methods such as the phosphoramidite method. If chemically synthesized double-stranded DNA is needed for an application such as the synthesis of a gene or gene fragment, then each complementary strand is prepared separately. Obtaining short genes (60-80 base pairs) does not cause technical difficulties, and can be achieved through the synthesis of complementary chains and then their hybridization. To obtain longer genes, synthetic genes (double-stranded) are assembled into a modular form from single-stranded fragments 20-100 nucleotides in length. For a review of polynucleotide synthesis, see, for example, Glick and Pasternak, Molecular Biotechnology, Principles and Applications of Recombinant DNA, ASM Press, 1994; Itakura, K., et al., Annu. Rev. Biochem. 53:323, 1984; and Climie, S., et al., Proc. Nat'1 Acad. Sci. USA 87:633, 1990.

Low molecular weight inhibitors.

In some embodiments, MASP-2 inhibitory agents are small molecule inhibitors, including natural and synthetic substances having low molecular weight, such as peptides, peptidomimetics, and non-peptide inhibitors (including oligonucleotides and organic compounds). Small molecule inhibitors of MASP-2 can be prepared based on the molecular structure of the variable regions of anti-MASP-2 antibodies.

Small molecule inhibitors can also be designed and prepared based on the crystal structure of MASP-2 using computer-aided drug design (Kuntz I.D., et al., Science 257:1078, 1992). The crystal structure of rat MASP-2 has been reported (Feinberg, H., et al., EMBO J. 22:2348 2359, 2003). Using the method described in Kuntz et al., the MASP-2 crystal structure coordinates are used as input to a computer program such as DOCK, which outputs a list of small molecule structures with expected MASP-2 binding ability. The use of such computer programs is well known to one skilled in the art. For example, the crystal structure of the HIV-1 protease inhibitor was used to identify unique non-peptide ligands that are inhibitors of HIV-1 protease by assessing the compatibility of compounds found in the Cambridge Crystallographic database with the enzyme's binding site using the program DOCK (Kuntz, I.D., et al. al., J. Mol. Biol. 161:269-288, 1982; DesJarlais, R.L., et al., PNAS 87:6644-6648, 1990).

The list of small molecules computationally identified as potential MASP-2 inhibitors was screened using the MASP-2 binding assay as described in Example 10. The small molecules found to bind MASP-2 were then analyzed functionally. assay, such as described in Example 2, to determine whether they inhibit MASP-2-dependent complement activation.

Soluble MASP-2 receptors.

Other suitable MASP-2 inhibitory agents are believed to include soluble MASP-2 receptors, which can be prepared using methods known to one of ordinary skill in the art.

Inhibitors of MASP-2 expression.

In another embodiment of this aspect of the invention, the MASP-2 inhibitory agent is an inhibitor of MASP-2 expression 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.

Antisense RNA and DNA molecules act to directly block translation of MASP-2 mRNA by hybridizing to MASP-2 mRNA and preventing translation of MASP-2 protein. An antisense nucleic acid molecule can be constructed through a number of different methods, provided that it is capable of interfering with MASP-2 expression. For example, an antisense nucleic acid molecule can be constructed by inverting the coding region (or part thereof) of the MASP-2 cDNA (SEQ ID NO: 4) relative to its normal orientation for transcription, allowing transcription of its complement.

Typically, an antisense nucleic acid molecule is substantially identical to at least a portion of the target gene or genes. However, the nucleic acid does not have to be exactly identical to inhibit expression. Generally, higher homology can be used to compensate for the use of a shorter antisense nucleic acid molecule. The minimum percentage identity is typically greater than about 65%, however a higher percentage identity may be more effective in repressing expression of the endogenous sequence. As such, higher percent identity greater than about 80% is generally preferred, although values from about 95% to complete identity are generally most preferred.

An antisense nucleic acid molecule need not have the same intronic or exonic pattern as the target gene, and non-coding fragments of the target gene may be equally effective in achieving antisense suppression of target gene expression as coding fragments. A DNA sequence of at least about 8 or a similar number of nucleotides can be used as the antisense nucleic acid molecule, although a longer sequence is preferred. According to the present invention, a representative example of a MASP-2 inhibitory agent that can be used is a MASP-2 antisense nucleic acid molecule that is at least ninety percent sequence identical to the complementary MASP-2 cDNA consisting of the nucleic acid sequence presented in SEQ ID NO: 4. The nucleic acid sequence shown in SEQ ID NO: 4 encodes a MASP-2 protein consisting of the amino acid sequence shown in SEQ ID NO: 5.

Targeting antisense oligonucleotides to bind to MASP-2 mRNA is another mechanism that can be used to reduce the level of MASP-2 protein synthesis. For example, the synthesis of polygalacturonase and muscarinic acetylcholine receptor type 2 is inhibited by antisense oligonucleotides targeting their corresponding mRNA sequences (US Pat. No. 5,739,119 to Cheng, and US Pat. No. 5,759,829 to Shewmaker). In addition, examples of antisense inhibition for the nuclear protein cyclin, multidrug resistance gene (MDG1), ICAM-1, selectin E, STK-1, striatal GABA _a receptor and human EGF are shown (see, for example, US Pat. No. 5801154, from Baracchini; US Patent No. 5,789,573 from Baker; US Patent No. 5,718,709 from Considine; and US Patent No. 5,610,288 from Reubenstein).

A system is described that allows one skilled in the art to determine which oligonucleotides can be used according to the invention, involving probe analysis of suitable regions in the target mRNA using RNase H digestion as an indicator of accessibility of sequences within transcripts. Scherr, M., et al., Nucleic Acids Res. 26:5079 5085, 1998; Lloyd, et al., Nucleic Acids Res. 29:3665 3673, 2001. A mixture of antisense oligonucleotides that are complementary to specific regions of the MASP-2 transcript are added to extracts of MASP-2 expressing cells, such as hepatocytes, and hybridized to produce an RNase H sensitive region. This method can be combined with computerized sequence selection that can predict optimal sequence selection for antisense compositions based on their relative ability to form dimers, hairpins, or other secondary structures that interfere with specific binding to target mRNA in the host cell. These secondary structure analyzes and target site selection can be performed using OLIGO primer analysis software (Rychlik, I., 1997) and the BLASTN 2.0.5 computer algorithm (Altschul, S.F., et al., Nucl. Acids Res 25:3389 3402, 1997). Antisense compounds targeting the target sequence are preferably between about 8 and about 50 nucleotides in length. Antisense oligonucleotides containing from about 9 to about 35 or more nucleotides are particularly preferred. The present inventors consider all oligonucleotide compositions in the range from 9 to 35 nucleotides (i.e., compositions 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 more) as being highly preferred in the practice of the antisense oligonucleotide methods of the invention. Highly preferred target regions of MASP-2 mRNA are those located at or near the translation initiation codon AUG and sequences that are substantially complementary to the 5' regions of the mRNA, for example, between the -10 and +10 regions of the MASP gene nucleotide sequence -2 (SEQ ID NO: 4). Exemplary inhibitors of MASP-2 expression are presented in table. 5.

Table 5 _____________Illustrative Inhibitors of MASP-2 Expression_____________

SEQ ID NO:30 (nucleotides 22,680 of SEQ ID NO:4) MASP-2 cDNA nucleic acid sequence (SEQ ID NO:4) encoding CUBIEGF SEQ ID NO:31 5'CGGGCACACCATGAGGCTGCTGACCCTCCTGGGC3 Nucleotides 12-45 from SEQ ID NO:4, including the MASP-2 translation start site (sense) SEQ ID NO:32 5'GACATTACCTTCCGCTCCGACTCCAACGAGAAG3' Nucleotides 361,396 of SEQ ID NO:4 encoding the region containing the MBL MASP-2 binding site (sense) SEQ ID NO:33 5'AGCAGCCCTGAATACCCACGGCCGTATCCCAAA3' Nucleotides 610-642 of SEQ ID NO:4, encoding the region containing 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. The term also includes oligonucleotide bases consisting of natural nucleotides, sugars and covalent internucleoside linkages (backbones), as well as oligonucleotides having non-natural modifications. These modifications allow the introduction of certain desirable properties not provided by natural oligonucleotides, such as reduced toxicity properties, increased stability to nuclease degradation, and improved cellular uptake. In illustrative embodiments, the antisense compounds of the invention differ from native DNA due to modification of the phosphodiester backbone to increase the lifetime of the antisense oligonucleotide in which the phosphate substituents are replaced by phosphorothioate ones. Likewise, one or both ends of the oligonucleotide may be replaced by one or more acridine derivatives that intercalate between adjacent base pairs in the nucleic acid chain.

Another alternative to using antisense sequences is the use of RNA interference (RNAi). Double-stranded RNAs (dsRNAs) can induce gene silencing in mammals in vivo. The natural function of RNAi and cosuppression appears to be to protect the genome from penetration of mobile genetic elements such as retrotransposons and viruses that produce aberrant RNA or dsRNA in the host cell when activated (see, for example, Jensen, J., et al ., Nat. Genet. 21:209 12, 1999). A double-stranded RNA molecule can be produced by synthesizing two strands of RNA capable of forming a double-stranded RNA molecule, each having a length of approximately 19 to 25 (eg, 19 to 23 nucleotides). For example, a dsRNA molecule that can be used in the methods of the invention may contain RNA corresponding to the sequence and its complementary sequence listed in table. 4. Preferably, at least one RNA strand has a 3' overhang of 1-5 nucleotides. The synthesized RNA strands are combined under conditions that allow the formation of a double-stranded molecule.

The RNA sequence may comprise at least a portion of the 8 nucleotides of SEQ ID NO: 4 with a total length of 25 nucleotides or less. The development of siRNA sequences for a given target is within the purview of one skilled in the art. Commercial siRNA sequence engineering services are available and guarantee at least 70% expression knockdown (Qiagen, Valencia, Calif).

The dsRNA can be administered in the form of a pharmaceutical composition and administered by known methods wherein the nucleic acid is introduced into the desired target cell. Commonly known gene transfer methods include those using calcium phosphate, DEAE-dextran, electroporation, microinjection and viruses. Such methods are explained in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., 1993.

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Ribozymes, such as ribozymes targeting MASP-2 mRNA, can also be used to reduce the amount and/or biological activity of MASP-2. Ribozymes are catalytic RNA molecules capable of cleaving nucleic acid molecules having a sequence that is completely or partially homologous to the ribozyme sequence. It is possible to design ribozyme transgenes encoding RNA ribozymes that specifically pair with a target RNA and cleave the phosphodiester backbone at a specific position, thereby functionally inactivating the target RNA. During this cleavage, the ribozyme itself does not change, and is thus capable of repeating the cycle and cleaving other molecules. Incorporation of ribozyme sequences into antisense RNAs imparts RNA cleavage activity to them, thereby increasing the activity of the antisense constructs.

Ribozymes that can be used in the practice of the invention typically contain a hybridizing region of at least about nine nucleotides that is complementary in nucleotide sequence to at least a portion of the MASP-2 target mRNA, and a catalytic region adapted to cleave the mRNA. targets of MASP-2 (see generally EPA No. 0 321 201; WO88/04300; Haseloff, J., et al., Nature 334:585 591, 1988; Fedor, M. J., et al., Proc. Natl. Acad. Sci. USA 87:1668 1672, 1990; Cech, T. R., et al., Ann. Rev. Biochem. 55:599 629, 1986).

Ribozymes can also be targeted directly to cells in the form of RNA oligonucleotides comprising ribozyme sequences, or introduced into the cell in the form of an expression vector encoding the desired ribozyme RNA. Ribozymes can be used and administered in substantially the same manner as described for antisense polynucleotides.

Antisense RNA and DNA, ribozymes and RNAi molecules that can be used in the methods of the invention can be prepared by any method known in the art for the synthesis of DNA and RNA molecules. These methods include methods for the chemical synthesis of oligodeoxyribonucleotides and oligoribonucleotides well known in the art, such as, for example, solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules can be produced by in vitro and in vivo transcription of DNA sequences encoding an antisense RNA molecule. Such DNA sequences can be inserted into a wide variety of vectors incorporating suitable RNA polymerase promoters, such as T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesize antisense RNA in a constitutive or inducible manner, depending on the promoter used, can be stably introduced into cell lines.

Various well-known modifications to DNA molecules can be introduced as means to increase stability and half-life. Modifications that can be used include, but are not limited to, the addition of ribonucleotide or deoxyribonucleotide flanking sequences to the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2'-O methyl instead of phosphodiester bonds within the oligodeoxyribonucleotide backbone.

V. Pharmaceutical compositions and delivery methods.

Dosing.

In another aspect, the invention provides compositions for inhibiting the adverse effects of MASP-2-dependent complement activation in a patient suffering from a disease or condition as described herein, including administering to the patient a composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent and a pharmaceutically acceptable carrier. . MASP-2 inhibitory agents can be administered to a patient, in therapeutically effective doses, to treat or alleviate conditions associated with MASP-2-dependent complement activation. A therapeutically effective dose refers to an amount of a MASP-2 inhibitory agent sufficient to relieve symptoms associated with the disease or condition.

The toxicity and therapeutic efficacy of MASP-2 inhibitory agents can be determined by standard pharmaceutical methods using experimental animal models, such as the MASP-2-/- mouse model expressing the human MASP-2 transgene described in Example 1. Using such animal models, NOAEL (no observed adverse effect level) and MED (minimum effective dose) can be determined using standard methods. The dose relationship between the effects of NOAEL and MED represents the therapeutic index, which is expressed as the NOAEL/MED ratio. MASP-2 inhibitory agents having large therapeutic indices or indicators are most preferred. Data obtained from cell culture assays and animal studies can be used to develop dosage ranges for human use. The dose of the MASP-2 inhibitory agent preferably lies within a circulating concentration range that includes MEDs with little or no toxicity. The dose may vary within this range depending on the dosage form used and the route of administration used.

To obtain the formulation of any compound, the therapeutically effective dose can be assessed using animal models. For example, the dose can be adjusted in an animal model to achieve a circulating plasma concentration range that includes MED. Quantitative

- 35 045598 plasma levels of a MASP-2 inhibitory agent can also be measured, for example, by high performance liquid chromatography.

In addition to toxicity studies, the effective dose can also be estimated based on the amount of MASP-2 protein present in a living individual and the binding affinity of the MASP-2 inhibitory agent. MASP-2 has been shown to be present in serum in healthy individuals at low levels in the range of about 500 ng/mL, and MASP-2 levels in a given individual can be determined using the quantitative MASP-2 assay described in Moller Kristensen M., et al. , J. Immunol. Methods 282:159 167, 2003.

In general, the dosage of compositions containing MASP-2 inhibitory agents administered will vary depending on factors such as the age, weight, height, sex, general medical condition and previous medical history of the patient. By way of illustration, MASP-2 inhibitory agents, such as anti-MASP-2 antibodies, can be administered at a dose ranging from about 0.010 to 10.0 mg/kg, preferably 0.010 to 1.0 mg/kg, more preferably from 0.010 to 0.1 mg/kg body weight of the individual. In some embodiments, the composition contains a combination of anti-MASP-2 antibodies and MASP-2 inhibitory peptides.

The therapeutic efficacy of the MASP-2 inhibitory compositions and methods of the present invention for a given individual, and suitable dosages, can be determined in accordance with complement assays well known to one skilled in the art. Complement forms numerous specific products. Over the last decade, sensitive and specific assays have been developed and are commercially available for most of these activation products, including the small activation fragments C3a, C4a and C5a, and the large activation fragments iC3b, C4d, Bb and sC5b 9. Most of these assays use monoclonal antibodies that react with new antigens (neoantigens) exposed on these fragments, but not on the native proteins from which they are formed, which makes these tests very simple and specific. Most are based on ELISA technology, although radioimmunoassay is still sometimes used for C3a and C5a. These latter assays measure both full-length and desArg fragments, which are the major forms found in the bloodstream. Full-length fragments and C5a _desArg are rapidly cleared by binding to cell surface receptors and are thus present in very low concentrations, whereas C3a _desArg does not bind to cells and accumulates in the plasma. Measurement of C3a provides a sensitive, pathway-independent measure of complement activation. Activation of the alternative pathway can be assessed by measuring the amount of Bb fragment. Detection of a soluble activation product of the membrane attack pathway, sC5b 9, provides evidence that complement is fully activated. Because both the lectin and classical pathways produce the same activation products, C4a and C4d, measuring the amount of these two fragments does not provide any information about which of the two pathways produces the activation products.

Inhibition of MASP-2-dependent complement activation is characterized by at least one of the following changes in a component of the complement system resulting from administration of a MASP-2 inhibitory agent in accordance with the methods of the invention: inhibition of the formation or production of MASP-2-dependent complement activation system products C4b , C3a, C5a and/or C5b 9 (MAC) (measured, e.g., as described in Example 2, decreased C4 degradation and C4b accumulation (measured, e.g., as described in Example 10), or decreased C3 degradation and C3b accumulation (measured , for example, as described in example 10).

Additional funds.

Compositions and methods comprising MASP-2 inhibitory agents may optionally contain one or more additional drugs that can enhance the activity of the MASP-2 inhibitory agent, or that provide related therapeutic functions in an additive or synergistic manner. For example, in the context of treating an individual suffering from an angiogenesis-dependent disease or condition, one or more MASP-2 inhibitory agents may be administered in combination (including co-administration) with one or more additional anti-angiogenic (also referred to as angiostatic) agents and/or one or multiple chemotherapeutic agents.

MASP-2 inhibitory agents can be used in combination with other anti-angiogenic agents, such as VEGF antagonists, such as VEGF-binding antibodies, such as the antibody known as bevacizumab (BV) (also known as AVASTIN®), antibodies that bind with VEGF-A, or VEGF-C, or with the VEGF-A receptor (eg, KDR receptor or Flt-1 receptor), anti-PDGFR inhibitors such as Gleevec® (imatinib mesylate), small molecules that block VEGF receptor signaling ( for example PTK787/ZK2284, SU6668,

SUTENT.RTM./SU11248 (sunitinib malate)), AMG706, or described, for example, in International Patent Application WO 2004/113304. Antiangiogenic agents also include natural inhibitors of angiogenesis, for example, angiostatin, endostatin, etc. See, for example, Klagsbrun and D'Amore (1991) An- 36 045598 nu. Rev. Physiol. 53:217-39; Streit and Detmar (2003) Oncogene 22:3172–3179 (eg, Table 3 listing antiangiogenic drugs for malignant melanoma); Ferrara & Alitalo (1999)

Nature Medicine 5(12):1359–1364; Tonini et al. (2003) Oncogene 22:6549-6556 (eg, Table 2 listing known antiangiogenic factors); and Sato (2003) Int. J. Clin. Oncol. 8:200-206 (for example, table.

listing antiangiogenic agents used in clinical studies).

MASP-2 inhibitory agents can be used in combination with other anticancer and/or chemotherapeutic agents, for example, abarelix, actinomycin D, adriamycin, aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, amifostine, anakinra, anastrozole, arsenic trioxide, asparaginase, azacitidine, BCG Live, bevacizumab, bexarotene, bleomycin, bortezomib, busulfan, calusterone, capecitabine, carboplatin, carmustine, celecoxib, cetuximab, chlorambucil, cisplatin, cladribine, clofarabine, cyclophosphamide, cytarabine, decarbazine, dactinomycin, dalteparin (eg sodium ), darbepoetin alfa, dasatinib, daunorubicin, daunomycin, decitabine, denileukin, denileukin-diftitox, dexrazoxane, docetaxel, doxorubicin, dromostanolone propionate, eculizumab, epirubicin (eg, HCl), epoetin alfa, erlotinib, estramustine, etoposide (eg, phosphate) , exemestane, fentanyl (eg, citrate), filgrastim, floxuridine, fludarabine, fluorouracil, 5-FU, fulvestrant, gefitinib, gemcitabine (eg, HCl), gemtuzumab-ozogamicin, goserelin (eg, acetate), histrelin (eg, acetate) , hydroxyurea, ibritutomab tiuxetan, idarubicin, ifosfamide, imatinib (eg, mesylate), interferon-alpha-2b, irinotecan, lapatinib ditosylate, lenalidomide, lectrozole, leucovorin, leuprolide (eg, acetate), levamisole, lomustine, CCNU, mechlorethamine (nitrous mustard gas), magestrol, melphalan (L-PAM), mercaptopurine (6-MP), mesna, methotrexate, methoxalen, mitomycin C, mitotane, mitoxantrone, nandrolone phenpropionate, nelarabine, nofetumomab, oprelvekin, oxaliplatin, paclitaxel, palifermin, pamidronate, panitumumab , pegademase, pegaspargase, pegfilgrastim, peginterferon-alpha-2b, pemetrexed (eg, disodium), pentostatin, pipobroman, plicamycin (mithramycin), porfimer (eg, sodium), procarbazine, quinacrine, rasburicase, rituximab, sargramostim, sorafenib, streptozocin, sunitinib (eg maleate), talc, tamoxifen, temozololmide, teniposide (VM-26), testolactone, thalidomide, thioguanine (6-TG), thiotepa, topotecan (eg hcl), toremifene, TositumomabL-131 (tositumomab), trastuzumab , tretinoin (ATRA), uramustine, valrubicin, vinblastine, vincristine, vinorelbine, vorinostat, zoledronate and zoledronic acid.

Pharmaceutical carriers and delivery devices.

Typically, the MASP-2 inhibitory agent compositions of the present invention, in combination with any other selected drugs, are suitably contained in a pharmaceutically acceptable carrier. The carrier is non-toxic, biocompatible, and selected so as not to adversely affect the biological activity of the MASP-2 inhibitory agent (and any other drugs in combination therewith). Exemplary pharmaceutically acceptable carriers for peptides are described in US Patent No. 5211657 from Yamada. Anti-MASP-2 antibodies and inhibitory peptides that can be used in the invention can be formulated in solid, semi-solid, gel, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, deposits, inhalants and injections, allowing oral, parenteral or surgical administration. The invention also relates to topical administration of compositions by coating medical instruments, etc.

Suitable carriers for parenteral delivery by injection, infusion or irrigation and topical delivery include distilled water, phosphate buffered saline, normal or lactated Ringer's solutions, dextrose solution, Hank's solution or propanediol. Additionally, sterile, fatty oils can be used as a solvent or suspending medium. Any biocompatible oil can be used for this purpose, including synthetic or diglycerides. In addition, fatty acids such as oleic acid can be used to prepare injectables. The carrier and agent may be combined in the form of a liquid, suspension, polymerizable or non-polymerizable gel, paste or balm.

The carrier may also contain a delivery agent to slow (ie, prolong, delay or regulate) delivery of the drug(s) or to improve the delivery, absorption, stability or pharmacokinetics of the drug(s). Such delivery vehicle may include, by way of non-limiting example, microparticles, microspheres, nanospheres or nanoparticles consisting of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymer or copolymer hydrogels and polymer micelles. Suitable hydrogel and micelle delivery systems include PEO:PHB:PEO copolymers and copolymer/cyclodextrin complexes described in WO 2004/009664 A2, and PEO and PEO/cyclodextrin complexes described in US Patent Application Publication No. 2002/0019369 A1. Such hydrogels can be injected locally at the site of intended treatment, or subcutaneously or intramuscularly to form a sustained release depot.

For intra-articular delivery, the carrier for MASP-2 inhibitory agents can be

- 37 045598 the above-described liquid or gel carriers suitable for injection, the above-described sustained release delivery agents suitable for injection, or hyaluronic acid or a hyaluronic acid derivative.

For oral administration of non-peptidergic agents, the carrier for the MASP-2 inhibitory agent may be an excipient or diluent such as sucrose, corn starch or cellulose.

For topical administration, the carrier for the MASP-2 inhibitory agent may be an ointment, lotion, cream, gel, drops, suppository, spray, liquid or powder, or gel or microcapsule delivery systems via transdermal patch.

Various nasal and pulmonary delivery systems, including aerosols, metered dose inhalers, dry powder inhalers and nebulizers, are under development and can be suitably adapted for delivery of the present invention in an aerosol, inhalation or nebulized delivery vehicle, respectively.

For intrathecal (IT) or intracerebroventricular (ICV) delivery, appropriate sterile delivery systems (eg, liquids; gels, suspensions, etc.) can be used to deliver the present invention.

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

Pharmaceutical carriers for antibodies and peptides.

More specifically, with respect to anti-MASP-2 antibodies and inhibitory peptides, the exemplary formulations can be administered parenterally in the form of injectable doses of a solution or suspension of the compound in a physiologically acceptable diluent with a pharmaceutical carrier, which can be a sterile liquid such as water, oils, saline solution, glycerin or ethanol. In addition, excipients such as wetting agents or emulsifiers, surfactants, pH buffering agents, and the like may be present in compositions containing anti-MASP-2 antibodies and inhibitory peptides. Additional components of the pharmaceutical compositions include fuels and lubricants (eg, animal, vegetable or synthetic origin), such as soybean oil and mineral oil. In general, glycols such as propylene glycol or polyethylene glycol are the preferred liquid carriers for injectable solutions.

Anti-MASP-2 antibodies and inhibitory peptides can also be administered in the form of a depot injection or implantable formulation, which can be formulated to allow sustained or pulsatile release of the active agents.

Pharmaceutically acceptable carriers for expression inhibitors.

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

Pharmaceutical compositions containing expression inhibitors may include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be prepared from a variety of components, including, but not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semi-solids. The preparation of such compositions typically involves combining the expression inhibitor with one or more of the following: buffers, antioxidants, small molecule polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and fillers. Neutral buffered saline or saline mixed with nonspecific serum albumin are examples of suitable diluents.

In some embodiments, the compositions can be prepared and formulated in the form of emulsions, typically being 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 Dekker, Inc., N.Y., 1988). Examples of natural emulsifiers used in emulsion formulations include gum arabic, beeswax, lanolin, lecithin and phosphatides.

In one embodiment, compositions containing nucleic acids can be formulated in the form of microemulsions. Microemulsion, as used herein, refers to a system of water, oil, and amphiphilic substance that is a single optically homogeneous and thermodynamically stable liquid composition (see Rosoff in Pharmaceutical Dosage Forms, Vol. 1). The method of the invention can also use liposomes to carry and deliver antisense oligonucleotides to the desired site.

Pharmaceutical compositions and expression inhibitor formulations for topical administration may

- 38 045598 include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers may be used, as well as aqueous, powdery or oily bases and thickeners, and the like.

Methods of administration.

Pharmaceutical compositions containing MASP-2 inhibitory agents can be administered in a variety of ways, depending on whether a local or systemic route of administration is most appropriate for the condition being treated. In addition, the compositions of the present invention can be delivered by coating an implantable medical device with the compositions or incorporating the compositions into an implantable medical device.

System delivery.

As used herein, the terms systemic delivery and systemic administration 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 distribution of the delivered agent at one or multiple sites of intended therapeutic action. Preferred methods of systemic delivery of the present compositions include intravenous, intramuscular, subcutaneous and inhalation. It should be appreciated that the precise route of systemic administration for selected agents used in particular compositions of the present invention may be determined in part by taking into account the sensitivity of the agent to the metabolic pathways associated with that route of administration. For example, peptidergic agents may most suitably be administered by routes other than orally.

MASP-2 inhibitory antibodies and polypeptides can be delivered to a patient by any suitable means. Methods of delivery of MASP-2 antibodies and polypeptides include oral, pulmonary, parenteral (eg, intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), inhalation (eg, fine powder), transdermal, nasal, vaginal, rectal, or sublingual routes of administration, and dosage form formulations suitable for each route of administration can be prepared.

As a representative example, MASP-2 inhibitory antibodies and peptides can be administered to a living body by application to a body membrane capable of absorbing the polypeptides, such as the mucous membranes of the nose, gastrointestinal tract, and rectum. The polypeptides are typically applied to the absorbent shell in combination with a penetration enhancing agent. (See, for example, Lee, V.H.L., Crit. Rev. Ther. Drug Carrier Sys. 5:69, 1988; Lee, V.H.L., J. Controlled Release 13:213, 1990; Lee, V.H.L., Ed., Peptide and Protein Drug Delivery, Marcel Dekker, New York (1991); DeBoer, A. G., et al., J. Controlled Release 13:241, 1990.) For example, STDHF is a synthetic derivative of fusidic acid, a steroidal surfactant that is similar in structure to bile salts of bile acids and which has been used as a penetration enhancer for nasal delivery. (Lee, W.A., Biopharm. 22, Nov./Dec. 1990.)

MASP-2 inhibitory antibodies and polypeptides can be administered in combination with another molecule, such as a lipid, to protect the polypeptides from enzymatic degradation. For example, covalent attachment of polymers, especially polyethylene glycol (PEG), has been used to protect specific proteins from enzymatic hydrolysis in the body and thus extend half-life (Fuertges, F., et al., J. Controlled Release 11:139, 1990). Many polymer systems for protein delivery have been published (Bae, Y.H., et al., J. Controlled Release 9:271, 1989; Hori, R., et al., Pharm. Res. 5:813, 1989; Yamakawa, I., et al., J. Pharm. Sci. 79:505, 1990; Yoshihiro, I., et al., J. Controlled Release 10:195, 1989; Asano, M., et al., J. Controlled Release 9: 111, 1989; Rosenblatt, J., et al., J. Controlled Release 9:195, 1989; Makino, K., J. Controlled Release 12:235, 1990; Takakura, Y., et al., J. Pharm J. Sci. 78:117, 1989; Takakura, Y., et al., J. Pharm. Sci. 78:219, 1989).

Recently, liposomes have been developed with improved serum stability and circulation half-life (see, eg, US Pat. No. 5,741,516, from Webb). In addition, a review of various methods using liposomes and liposome-like preparations as potential drug carriers is provided (see, for example, US Patent No. 5567434, from Szoka; US Patent No. 5552157, from Yagi; US Patent No. 5565213 , from Nakamori; US Patent No. 5738868, from Shinkarenko; and US Patent No. 5795587, from Gao).

For transdermal administration, MASP-2 inhibitory antibodies and polypeptides can be combined with other suitable ingredients, such as carriers and/or adjuvants. There are no restrictions as to the nature of such other ingredients, except that they must be pharmaceutically acceptable for their intended administration and cannot interfere with the activity of the active ingredients of the composition. Examples of suitable agents include ointments, creams, gels or suspensions, whether or not containing purified collagen. MASP-2 inhibitory antibodies and polypeptides can also be incorporated into transdermal patches, patches and bandages, preferably in liquid or semi-liquid form.

- 39 045598

The compositions of the present invention can be administered systemically on a periodic basis at intervals determined to maintain the desired level of therapeutic effect. For example, the compositions can be administered, for example, by subcutaneous injection, every two to four weeks or at less frequent intervals. The dosage regimen is determined by the therapist, taking into account various factors that may influence the effect of the combination of drugs. These factors include the degree of progression of the condition being treated, the age, sex and weight of the patient, and other clinical factors. The dosage for each individual agent may be varied depending on the MASP-2 inhibitory agent included in the composition, as well as the presence and nature of any drug delivery agent (eg, sustained release delivery agent). In addition, the dosage level can be adjusted to accommodate changes in the frequency of administration and the pharmacokinetic behavior of the agent(s) being delivered.

Local delivery.

As used herein, the term topical includes administration of a drug at or near the site of intended local action, and may include, for example, local delivery to the skin or other affected tissues, ophthalmic delivery, intrathecal (IT), intracerebroventricular (ICV), intra-articular, intracavitary, intracranial or intravesical administration, application or irrigation. Local administration may be preferred to allow a lower dose to be administered, to avoid systemic side effects, and to more accurately control the timing of delivery and the concentration of active ingredients at the local delivery site. Local administration provides a known concentration at the target site, regardless of inter-patient variability in metabolism, blood flow, etc. Improved dosing control is also provided through the direct delivery method.

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

Treatment regimens.

In prophylactic applications, pharmaceutical compositions containing a MASP-2 inhibitory agent are administered to a patient susceptible to an angiogenesis-dependent disease or condition, or otherwise at risk of developing an angiogenesis-dependent disease or condition, in an amount sufficient to inhibit angiogenesis and thereby, eliminating or reducing the risk of developing symptoms of this condition. In some embodiments, the pharmaceutical compositions are administered to an individual suspected of having, or already suffering from, an angiogenesis-dependent disease or condition, in a therapeutically effective amount sufficient to alleviate, or at least partially reduce, the symptoms of that condition. In both prophylactic and therapeutic regimens, compositions containing MASP-2 inhibitory agents can be administered in multiple doses until a satisfactory therapeutic outcome is achieved for the patient. The use of the MASP-2 inhibitory compositions of the present invention can be accomplished through a single administration of the composition, or a limited series of administrations, for the treatment of an acute condition associated with angiogenesis. Alternatively, the composition can be administered at periodic intervals over an extended period of time to treat a chronic condition associated with angiogenesis.

In both prophylactic and therapeutic regimens, compositions containing MASP-2 inhibitory agents can be administered in multiple doses until a satisfactory therapeutic outcome is achieved for the patient. In one embodiment of the invention, the MASP-2 inhibitory agent comprises an anti-MASP-2 antibody that can suitably be administered to an adult patient (e.g., an average adult weighing 70 kg) at a dose of 0.1 mg to 10,000 mg, more suitably from 1.0 mg to 5000 mg, more suitably from 10.0 mg to 2000 mg, more suitably from 10.0 mg to 1000 mg and even more suitably from 50.0 mg to 500 mg. For pediatric patients, the dose can be adjusted in proportion to the patient's weight. Use of the MASP-2 inhibitory compositions of the present invention can be accomplished by a single administration of the composition, or a limited series of administrations, for the treatment of a patient suffering from or at risk of developing an angiogenesis-dependent disease or condition, such as an angiogenesis-dependent malignant tumor, angiogenesis-dependent benign tumor or an angiogenic disease or eye condition. Alternatively, the composition may be administered at periodic intervals, for example, daily, twice a week, once a week, every other week, once a month, or once every two months over an extended period of time to treat an individual suffering from or at risk of developing a drug addiction. an angiogenesis disease or condition, such as an angiogenesis-dependent malignant tumor, angiogenesis-dependent benign tumor, or an angiogenic ocular disease or condition.

In both prophylactic and therapeutic regimens, compositions containing MASP-2 inhibitory agents can be administered in multiple doses until satisfactory therapeutic outcome is achieved for the individual.

In one embodiment, a pharmaceutical composition containing a MASP-2 inhibitory agent is administered to an individual suffering from an ophthalmic angiogenic disease or condition in an amount effective to inhibit angiogenesis. In one embodiment, the ophthalmic angiogenic disease or eye condition is selected from the group consisting of AMD, uveitis, ocular melanoma, corneal neovascularization, primary pterygium, HSV stromal keratitis, HSV-1 induced corneal lymphangiogenesis, proliferative diabetic retinopathy, retinopathy of prematurity, vein occlusion retina, corneal graft rejection, neovascular glaucoma and iris redness.

In another embodiment, a pharmaceutical composition containing a MASP-2 inhibitory agent is administered to an individual suffering from angiogenesis-dependent cancer in an amount effective to inhibit angiogenesis. In one embodiment, the angiogenesis-dependent cancer is selected from the group consisting of solid tumor(s), blood-borne tumors, high-risk carcinoid tumors, and metastatic tumors. In one embodiment, the composition is administered in an amount effective to inhibit tumor angiogenesis. In one embodiment, the patient suffers from or is at risk of tumor metastasis, and the composition is administered in an amount effective to inhibit tumor metastasis. In one embodiment, the patient suffers from an angiogenesis-dependent cancer selected from the group consisting of colorectal cancer, breast, lung, kidney, liver, esophagus, ovarian, pancreatic, prostate, gastric, glioma, stromal gastrointestinal tumors, lymphoma, melanoma and carcinoid tumor. In one embodiment, the patient is suffering from a benign tumor, and the composition is administered in an amount effective to inhibit angiogenesis of the benign tumor.

VI. Examples.

The following examples are only illustrative of the best way to practice the invention, however, they should not be construed as limiting the invention. The contents of all references cited are expressly incorporated herein by reference.

Example 1.

This example describes the generation of a mouse strain that is MASP-2 deficient (MASP-2-/-) but MAp19-complete (MAp19+/+).

Materials and Methods: The pKO NTKV 1901 targeting vector is designed to disrupt the three exons encoding the C terminus of murine MASP-2, including the exon that encodes the serine protease domain, as shown in FIG. 3. PKO NTKV 1901 was used to transfect the mouse ES cell line E14.1a (SV129 Ola). Clones resistant to neomycin and sensitive to thymidine kinase were selected. 600 ES clones were screened and from these four different clones were identified and verified by Southern blotting to contain the expected selective targeting and recombinant events as shown in FIG. 3. From these four positive clones, chimeras were obtained through embryo transfer. The chimeras were then backcrossed onto the C57/BL6 genetic background to produce transgenic males. Transgenic males were crossed with females to produce F1 offspring with 50% of the offspring expressing heterozygosity for the disrupted MASP-2 gene. Heterozygous mice were cross-crossed to produce homozygous MASP-2-deficient offspring, resulting in heterozygous and wild-type mice in a 1:2:1 ratio, respectively.

Results and Phenotype: The resulting homozygous MASP-2-/(i.e., target gene deficient) mice were found to be viable and fertile, and were tested for MASP-2 deficiency by Southern blotting to confirm correct targeting event, by Northern blotting to confirm the absence of MASP-2 mRNA, and by Western blotting to confirm the absence of MASP-2 protein (data not shown). The presence of MAp19 mRNA and absence of MASP-2 mRNA was further confirmed using time-resolved RT-PCR on a LightCycler device. As expected, MASP-2 −/− mice did continue to express MAp19, MASP-1, and MASP-3 mRNA and protein (data not shown). The presence and relative abundance of properdin, factor B, factor D, C4, C2, and C3 mRNAs in MASP-2 −/− mice were assessed by LightCycler analysis and found to be identical to those of wild-type control littermates (data not shown). . Plasma from homozygous MASP-2 −/− mice is completely deficient in lectin pathway-mediated complement activation, as described further in Example 2.

Establishment of the MASP-2-/- line on a pure C57BL6 background: MASP-2-/- mice were backcrossed to the pure C57BL6 line for nine generations before using the MASP2-/- line as an experimental animal model.

A transgenic mouse line that has mouse MASP-2-/-, mouse MAp19+/+, and which

- 41 045598 expresses human MASP-2 transgene (with mouse MASP-2 knockout and human knockin

MASP-2) was also prepared as follows:

Materials and methods. A minigene encoding human MASP-2, named mini-hMASP-2 (SEQ ID NO: 49), was constructed as shown in FIG. 4, comprising the promoter region of the human MASP-2 gene, including the first 3 exons (exon 1 - exon 3), followed by a cDNA sequence representing the coding sequence of the next 8 exons, thus encoding the full-length MASP-2 protein under the control of its endogenous promoter . The minihMASP-2 construct was injected into fertilized MASP-2 −/− oocytes to replace the inactivated mouse MASP-2 gene with transgenically expressed human MASP-2.

Example 2.

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

Methods and materials.

Analysis of lectin pathway-specific C4 cleavage: in Petersen, et al., J. Immunol. Methods 257:107 (2001) describes a C4 cleavage assay that measures lectin pathway activation resulting from lipoteichoic acid (LTA) from S. Aureus, which binds L-ficolin. The assay described in Petersen et al., (2001) is adapted to measure the level of lectin pathway activation by MBL by coating the plate with LPS and mannan or zymosan before adding serum from MASP-2-/- mice, as described below. The assay was also modified to exclude the possibility of C4 cleavage via the classical pathway. This was achieved by using a sample dilution buffer containing 1 M NaCl, which allows high-affinity binding of recognition components of the lectin activation pathway to their ligands but prevents activation of endogenous C4, thus eliminating the participation of the classical pathway through dissociation of the C1 complex. Briefly, in a modified assay, serum samples (diluted in high salt buffer (1 M NaCl)) are added to ligand-coated plates, followed by the addition of a constant amount of purified C4 in physiological salt concentration buffer. Bound recognition complexes containing MASP-2 cleave C4, resulting in the accumulation of C4b.

Methods of analysis.

7) Nunc Maxisorb microtiter plates (Maxisorb, Nunc, Cat. No. 442404, Fisher Scientific) were coated using 1 μg/ml mannan (M7504, Sigma) or any other ligand (eg, such as those listed below) diluted in coating buffer (15 mM Na ₂ CO ₃ , 35 mM NaHCO ₃ , pH 9.6).

The following reagents were used in the analysis:

a) mannan (1 µg/well mannan (M7504, Sigma) in 100 µl coating buffer):

b) zymosan (1 µg/well zymosan (Sigma) in 100 µl coating buffer);

c) LTA (1 µg/well in 100 µl coating buffer or 2 µg/well in 20 µl methanol)

d) 1 µg H-ficolin specific Mab 4H5 in coating buffer

e) PSA from Aerococcus viridans (2 µg/well in 100 µl coating buffer)

f) 100 µl/well of formalin-fixed S. Aureus DSM20233 (OD ₅₅₀ =0.5) in coating buffer.

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

3) After overnight incubation, residual protein binding sites were saturated by incubating 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 NaN ₃ , pH 7.4) for 1-3 hours, then plates were washed 3X using TBS/tween/Ca ²⁺ (TBS with 0.05% Tween 20 and 5 mM CaCl ₂ , 1 mM MgCl ₂ , 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 containing only buffer were used as negative controls.

5) After incubation overnight at 4°C, plates were washed 3X using TBS/tween/Ca ²⁺ . Human C4 (100 µl/well of 1 µg/ml diluted in BBS (4 mM barbital, 145 mM NaCl, 2 _mM CaCl2, 1 mM _MgCl2 , pH 7.4)) was then added to the plates and incubated for 90 minutes at 37°C. The plates were again washed 3X using TBS/tween/Ca ²⁺ .

6) C4b accumulation was detected using alkaline phosphatase-conjugated chicken anti-human C4c antibodies (diluted 1:1000 in TBS/tween/Ca ²⁺ ), which were added to the plates and incubated for 90 min at room temperature. The plates were then washed again 3X using TBS/tween/Ca ²⁺ .

7) Alkaline phosphatase was detected by adding 100 μl of pnitrophenyl phosphate substrate solution, incubating at room temperature for 20 minutes, and reading OD ₄₀₅ in a microtiter plate reader.

Results. In fig. 5A B shows the level of C4b accumulation on mannan (Fig. 5A) and zymosan (Fig. 5B) in dilutions of sera from MASP-2+/+ (crosses), MASP-2+/- (solid circles), and MASP-2-/ - 42 045598 (solid triangles). In fig. 5C shows the relative activity of C4 convertase in plates coated with zymosan (white bars) or mannan (shaded bars) for MASP-2 -/+ mice (n=5) and MASP-2 -/- mice (n=4) compared to wild-type mice (n=5), based on measurements of C4b accumulation normalized to wild-type serum. Error bars represent standard deviation. As shown in FIG. 5A C, plasma from MASP-2-/- mice is completely devoid of lectin pathway-mediated complement activation in mannan-coated and zymosan-coated plates. These results clearly indicate that MASP-2 is an effector component of the lectin pathway.

Recombinant MASP-2 restores lectin pathway-dependent C4 activation in serum from MASP-2 −/− mice.

To determine that the absence of MASP-2 was the direct cause of the loss of lectin pathway-dependent C4 activation in 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 is replaced by an alanine residue) recombinant proteins were prepared and purified as described below in Example 3. Pooled serum from 4 MASP-2- mice /pre-incubated with increasing concentrations of recombinant mouse MASP-2 or inactive recombinant mouse MASP-2A, and C4 convertase activity was assessed as described above.

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

Example 3.

This example describes recombinant expression and protein production for recombinant full-length human, rat and mouse MASP-2, MASP-2-derived polypeptides, and catalytically inactive mutant forms of MASP-2.

Expression of full-length human, mouse, and rat MASP-2.

The full-length cDNA sequence of human MASP-2 (SEQ ID NO: 4) was also subcloned into the mammalian expression vector pCI Neo (Promega), which drives eukaryotic expression under the control of the CMV enhancer/promoter region (described in Kaufman R.J. et al., Nucleic Acids Research 19:4485 90, 1991; Kaufman, Methods in Enzymology, 185:537 66 (1991)). Each of the full-length mouse cDNA (SEQ ID NO: 50) and rat cDNA (SEQ ID NO: 53) MASP-2 was subcloned into the pED expression vector. MASP-2 expression vectors were then transfected into the adherent Chinese hamster ovary cell line DXB1 using the standard calcium phosphate transfection method described in Maniatis et al., 1989. Cells transfected with these constructs grew very slowly, due to the cytotoxicity of the encoded protease.

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

Expression of full-length catalytically inactive MASP-2.

Rationale: MASP-2 is activated through autocatalytic cleavage after MBL recognition subcomponents or ficolins (L-ficolin, H-ficolin, or M-ficolin) bind to their corresponding carbohydrate patterns. Autocatalytic cleavage leading to activation of MASP-2 often occurs during the procedure for isolating MASP-2 from serum, or during purification after recombinant expression. To obtain a more stable protein preparation for use as an antigen, a catalytically inactive form of MASP-2, designated MASP-2A, was prepared by replacing the serine residue present in the catalytic triad of the protease domain with an alanine residue in rat (SEQ ID NO: 55 , Ser617 to Ala617); in mouse (SEQ ID NO: 52, Ser617 to Ala617); or in humans (SEQ ID NO: 3, Ser618 to Ala618).

To obtain catalytically inactive human and mouse MASP-2A proteins, site-directed mutagenesis was performed using the oligonucleotides shown in table. 6.

Oligonucleotides in table. 6 are designed to hybridize to a region in human and mouse cDNAs encoding enzymatically active serine, and the oligonucleotide contains a mismatch to change the serine codon to an alanine codon. For example, PCR oligonucleotides SEQ ID NO: 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 active serine and from the serine to the stop codon to obtain the complete

- 43 045598 open reading frame of mutant MASP-2A containing mutation Ser618 to Ala618. Products

The PCR was purified after agarose gel electrophoresis and band acquisition, and single adenosine overlap was obtained using a standard tailing method. MASP-2A, equipped with an adenosine tail, was then cloned into the pGEM T easy vector and transformed into E. coli.

Catalytically inactive rat MASP-2A protein was prepared by kinase treatment and hybridization of SEQ ID NO: 64 and SEQ ID NO: 65 by combining the two oligonucleotides in equivalent molar amounts, heating at 100°C for 2 minutes and slowly cooling to room temperature. The resulting fragment had compatible ends for Pst1 and Xba1, and was inserted into the Pst1-Xba1 fragment of 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)

Human, mouse and rat MASP-2A were each further subcloned into the mammalian expression vectors pED or pCI Neo and transfected into the Chinese hamster ovary cell line DXB1 as described below.

In another method, a catalytically inactive form of MASP-2 was constructed using the method described in Chen et al., J. Biol. Chem., 276(28):25894 25902, 2001. Briefly, a plasmid containing the full-length human MASP-2 cDNA (described in Thiel et al., Nature 386:506, 1997) was digested with Xho1 and EcoR1, and the MASP cDNA -2 (described herein as SEQ ID NO: 4) was cloned into the appropriate restriction sites of the baculovirus transfer vector pFastBac1 (Life Technologies, NY). The active site of the MASP-2 serine protease at Ser618 was then changed to Ala618 by replacing the native amino acid region 610-625 using double-stranded oligonucleotides encoding the peptide amino acid region 610-625 (SEQ ID NO: 13) to produce a full-length MASP-2 polypeptide with inactive protease domain. Construction of expression plasmids containing polypeptide regions derived from human Masp2.

The following constructs are generated using the MASP-2 signal peptide (residues 1-15 of SEQ ID NO: 5) to secrete different domains of MASP-2. A construct expressing the CUBI domain of human MASP-2 (SEQ ID NO: 8) was generated by PCR amplification of the region encoding residues 1-121 of MASP-2 (SEQ ID NO: 6) (corresponding to the N-terminal domain of CUB1). A construct expressing the CUBIEGF domain of human MASP-2 (SEQ ID NO: 9) was generated by PCR amplification of the region encoding residues 1-166 of MASP-2 (SEQ ID NO: 6) (corresponding to the N-terminal domain of CUB1EGF). A construct expressing the CUBIEGFCUBII domain of human MASP-2 (SEQ ID NO: 10) was generated by PCR amplification of the region encoding residues 1-293 of MASP-2 (SEQ ID NO: 6) (corresponding to the N-terminal domain of CUBIEGFCUBII). The above domains are amplified by PCR using VentR polymerase and pBSMASP-2 as a template according to established PCR methods.

The 5' primer sequence for the sense primer (5' - CGGGATCCATGAGGCTGCTGACCCTC-3, seq id NO: 34) introduces a BamHI restriction site (underlined) at the 5' end of the PCR product. Antisense primers for each of the MASP-2 domains are shown in Table 1 below. 6 are designed to introduce a stop codon (bold) followed by an EcoRI site (underlined) at the end of each PCR product. After amplification, the DNA fragments are digested using BamHI and EcoRI, and cloned into the corresponding sections of the pFastBac1 vector. The resulting constructs are characterized by restriction mapping and confirmed by dsDNA sequencing.

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Table 6 Primers for MASP-2 PCR

MASP-2 domain SEQ ID NO:8, CUBI (aka 1121 from SEQ ID NO:6) SEQ ID NO:9, CUBIEGF (aka 1-166 of SEQ ID NO:6) 5' primer for PCR 5'CGGGATCCATGAGGCTGCTGACCCTC-3' (SEQ ID NO:34) 5'CGGGATCCATGAGGCTGCTGACCCTC-3' (SEQ ID NO:34) 3' primer for PCR 5'GGAATTCCTAGGCTGCATA (SEQ ID NO:35) 5'GGAATTCCTACAGGGCGCT-3' (SEQ ID NO:36) SEQ ID NO:10, CUBIEGFCUBII (aka 1-293 from SEQ ID NO:6) 5'CGGGATCCATGAGGCTGCTGACCCTC-3' (SEQ ID NO:34) 5'GGAATTCCTAGTAGTGGAT-3' (SEQ ID NO:37) SEQ ID NO:4, MASP-2 people 5'ATGAGGCTGCTGACCCTCCTGGGCCTTC- 3' (SEQ ID NO: 56) hMASP- 2 straight 5'TTAAAATCACTAATTATGTTCTCGATC 3' (SEQ ID NO: 59) NMAZR-2_reverse SEQ ID NO:4, human MASP-2 cDNA 5'CAGAGGTGACGCAGGAGGGGCAC 3' (SEQ ID NO: 58) hMASP2 ala direct 5'GTGCCCCTCCTGCGTCACCTCTG 3' (SEQ ID NO: 57) CMAZR-2_a1a_reverse SEQ ID NO:50 Mouse MASP-2 cDNA 5'ATGAGGCTACTCATCTTCCTGG3' (SEQ ID NO: 60) tMAZR-2_direct 5'TTAGAAATTACTTATTATGTTCTCAATCC3' (SEQ ID NO: 63) tMAZR-2_reverse SEQ ID NO:50 Mouse MASP-2 cDNA 5'CCCCCCCTGCGTCACCTCTGCAG3' (SEQ ID NO: 62) mMASP- 2 ala straight 5'CTGCAGAGGTGACGCAGGGGGGG-3' (SEQ ID NO: 61) tMAZR-2_a1a_reverse

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

The MASP-2 and MASP-2A expression constructs described above were transfected into DXB1 cells using the standard calcium phosphate transfection method (Maniatis et al., 1989). MASP-2A was prepared in serum-free medium to avoid contamination of the preparations with other serum proteins. The medium was collected from confluent cells every second day (four times in total). The average level of recombinant MASP-2A was approximately 1.5 mg/liter of culture medium for each of the three species.

Purification of MASP-2A protein: MASP-2A (the Ser-Ala mutant described above) was purified by affinity chromatography on MBP-A agarose columns. This method allows for quick cleaning without the use of external tags. MASP-2A (100-200 ml of medium diluted in an equal volume of loading buffer (50 mM Tris-Cl, pH 7.5, containing 150 mM NaCl and 25 mM CaCl ₂ ) was applied to an MBP-agarose affinity column (4 ml), pre-equilibrated using 10 ml loading buffer. After washing with a further 10 ml loading buffer, the protein was eluted in 1 ml fractions using 50 mM Tris Cl, pH 7.5, containing 1.25 M NaCl and 10 mM EDTA. Fractions containing MASP-2A were identified by SDS-polyacrylamide gel electrophoresis. If necessary, MASP-2A was further purified by ion exchange chromatography on a MonoQ column (HR 5/5). The protein was dialyzed using 50 mM Tris-Cl pH 7.5 containing 50 mM NaCl and applied to a column equilibrated with the same buffer.After washing, bound MASP-2A was eluted using a gradient of 0.05-1 M NaCl in 10 ml.

Results: Yields of 0.25-0.5 mg of MASP-2A protein were obtained from 200 ml of medium. The molecular weight of 77.5 kDa determined by MALDI-MS is higher than the calculated value for the unmodified polypeptide (73.5 kDa) due to glycosylation. The attachment of glycans to each of the Nglycosylation sites explains the observed mass. MASP-2A migrates as a single band on SDS-polyacrylamide gels, indicating that it is not subject to proteolytic processing during biosynthesis. The weight average molecular weight determined by equilibrium ultracentrifugation is consistent with the calculated value for homodimers of the glycosylated polypeptide.

Production of recombinant human MASP-2 polypeptides.

Another method for producing recombinant polypeptides derived from MASP-2 and MASP2A is

- 45 045598 described in Thielens, N.M., et al., J. Immunol. 166:5068 5077, 2001. Briefly, Spodoptera frugiperda insect cells (Ready Plaque Sf9 cells obtained from Novagen, Madison, WI) were grown and maintained in serum-free Sf900II medium (Life Technologies) supplemented with 50 IU/ml penicillin and 50 mg/ml streptomycin (Life Technologies). Trichoplusia ni (High Five) insect cells (obtained from Jadwiga Chroboczek, Institut de Biologie Structurale, Grenoble, France) were maintained in TC100 medium (Life Technologies) containing 10% FCS (Dominique Dutscher, Brumath, France) supplemented with 50 IU/ml penicillin and 50 mg/ml streptomycin. Recombinant baculoviruses were produced using the Bac-toBac system (Life Technologies). Bacmid DNA was purified using the Qiagen midiprep purification system (Qiagen) and used to transfect Sf9 insect cells using cellfectin in Sf900 II SFM medium (Life Technologies) as described in the manufacturer's protocol. Recombinant virus particles were collected after 4 days, titrated by viral plaque assay and amplified as described by King and Possee, in The Baculovirus Expression System: A Laboratory Guide, Chapman and Hall Ltd., London, pp. 111 114, 1992.

Five cells (1.75 x 107 cells/175 cm ² culture flask) were infected with recombinant viruses containing MASP-2 polypeptides at a multiplicity of infection of 2 in Sf900 II SFM medium at 28°C for 96 hours. Supernatants were collected by centrifugation and diisopropylphosphorofluoridate was added to a final concentration of 1 mM.

MASP-2 polypeptides are secreted into the culture medium. Culture supernatants were dialyzed against 50 mM NaCl, 1 mM CaCl ₂ , 50 mM triethanolamine hydrochloride, pH 8.1, and applied at 1.5 ml/min to a Q-Sepharose Fast Flow column (Amersham Pharmacia Biotech) (2.8 x 12 cm ), balanced by the same buffer. Elution was performed by applying a 1.2 liter linear gradient to 350 mM NaCl in the same buffer. Fractions containing recombinant MASP-2 polypeptides were identified by Western blot analysis, precipitated by adding (NH ₄ ) ₂ SO ₄ to 60% (wt/vol) and left overnight at 4°C. The pellets were resuspended in 145 mM NaCl, 1 mM CaCl ₂ , 50 mM triethanolamine hydrochloride, pH 7.4, and applied to a TSK G3000 SWG (7.5 x 600 mm) column (Tosohaas, Montgomeryville, PA) equilibrated with the same buffer. The purified polypeptides are then concentrated to 0.3 mg/ml by ultrafiltration in Microsep microconcentrators (cut-off mw=10000) (Filtron, Karlstein, Germany).

Example 4.

This example describes a method for producing polyclonal antibodies against MASP-2 polypeptides. Materials and methods.

MASP-2 antigens. Polyclonal antiserum against human MASP-2 is prepared 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 NO: 6), MASP-2A containing an inactive protease domain (SEQ ID NO: 13), as described in example 3; and recombinant CUBI (SEQ ID NO: 8), CUBEGFI (SEQ ID NO: 9) and CUBEGFCUBII (SEQ ID NO: 10) expressed as described above in Example 3.

Polyclonal Antibodies: Six-week-old rabbits primed with BCG (Bacillus Calmette-Guerin vaccine) were immunized by injection of 100 μg of MASP-2 polypeptide at 100 μg/ml in sterile saline. Injections were performed every 4 weeks, with antibody titer monitored by ELISA assay as described in Example 5. Culture supernatants were collected for antibody purification by protein A affinity chromatography.

Example 5.

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

Materials and methods.

Male A/J mice (Harlan, Houston, Tex.), 8-12 weeks of age, were injected subcutaneously with 100 μg of human or rat rMASP-2 or rMASP-2A polypeptides (prepared as described in Example 3) in Freund's complete adjuvant ( Difco Laboratories, Detroit, Mich.) in 200 µl phosphate-buffered saline (PBS) pH 7.4. At two-week intervals, mice are injected twice subcutaneously with 50 μg of human or rat rMASP-2 or rMASP-2A polypeptide in Freund's incomplete adjuvant. At the fourth week, mice are injected with 50 μg of human or rat rMASP-2 or rMASP2A polypeptide in PBS, and fusion is performed after 4 days.

For each fusion, single cell suspensions of immunized mouse spleen were prepared and used for fusion with Sp2/0 myeloma cells. 5x108 Sp2/0 cells and 5x108 spleen cells were confluent in a medium containing 50% polyethylene glycol (M.W. 1450) (Kodak, Rochester, N.Y.) and 5% dimethyl sulfoxide (Sigma Chemical Co., St. Louis, Mo.). The spleen cell concentration was then adjusted to 1.5 x 105 spleen cells per 200 μl suspension in Iscove's medium (Gibco, Grand Island, N.Y.) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, 0.1 mM hypoxanthine, 0.4 µM aminopterin and 16 µM thymidine. Two hundred microliters of cell suspension is added to each well of approximately twenty 96-well microplates. After approximately ten days, culture supernatants are screened for reactivity with purified MASP-2 factor in an ELISA assay.

ELISA assay. Immulon 2 microplate wells (Dynatech Laboratories, Chantilly, Va.) were coated by adding 50 μl 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 allows the selection of high-affinity antibodies. After removing the coating solution by tapping the plates, 200 µl of BLOTTO (non-fat dry milk) in PBS is added to each well for one hour to block non-specific sites. After one hour, the wells are then washed with PBST buffer (PBS containing 0.05% Tween 20). Fifty microliters of culture supernatants from each post-confluence well are collected and mixed with 50 µl of BLOTTO, and then added to individual microplate wells. After one hour of incubation, the wells are washed with PBST. Bound mouse antibodies were then detected by reaction with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Fc-specific) (Jackson ImmunoResearch Laboratories, West Grove, Pa.) and diluted to 1:2000 in BLOTTO. A peroxidase substrate solution containing 0.1% 3,3,5,5-tetramethylbenzidine (Sigma, St. Louis, Mo.) and 0.0003% hydrogen peroxide (Sigma) was added to the wells to allow color development for 30 minutes. The reaction is stopped by adding 50 μl of 2M H ₂ SO ₄ per well. The absorbance of the reaction mixture at 450 nm was read using 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 that MASP-2 inhibitory agents have for MASP-2. A similar assay can be used to determine whether inhibitory agents bind to other antigens in the complement system.

Wells of polystyrene 96-well microtiter plates (96-well medium binding plates, Corning Costar, Cambridge, MA) are coated using MASP-2 (20 ng/100 μl/well, Advanced Research Technology, San Diego, CA) in phosphate buffered saline (PBS), pH 7.4, overnight at 4°C. After removing the MASP-2 solution, the wells were blocked using PBS containing 1% bovine serum albumin (BSA; Sigma Chemical) for 2 h at room temperature. Wells without MASP-2 coating serve as background controls. Aliquots of hybridoma supernatants or purified anti-MASP-2 MoAb, at various concentrations in blocking solution, are added to the wells. After incubation for 2 hours at room temperature, the wells are washed extensively with PBS. MASP-2-bound anti-MASP-2 MoAb is detected by adding peroxidase-conjugated goat anti-mouse IgG (Sigma Chemical) to a blocking solution that is incubated for 1 hour at room temperature. The plate is again thoroughly washed with PBS, and 100 μl of 3,3',5,5'-tetramethylbenzidine (TMB) substrate (Kirkegaard and Perry Laboratories, Gaithersburg, MD) is added. The TMB reaction is stopped by adding 100 μl of 1 M phosphoric acid, and the plate is read at 450 nm in a microplate reader (SPECTRA MAX 250, Molecular Devices, Sunnyvale, CA).

Culture supernatants from positive wells are then tested for their ability to inhibit complement activation in a functional assay, such as a C4 cleavage assay, as described in Example 2. Cells from positive wells are then cloned by the limiting dilution method. The MoAb is again tested for reactivity with hMASP-2 in an ELISA assay as described above. Selected hybridomas are grown in shake flasks, and the depleted culture supernatant is collected for antibody purification by protein A affinity chromatography.

Example 6.

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

Mouse anti-MASP-2 monoclonal antibody is obtained from a male A/J mouse as described in Example 5. The mouse antibody is then humanized as described below to reduce its immunogenicity by replacing the mouse constant regions with their human counterparts to produce a chimeric IgG antibody and Fab fragment antibodies that can be used to inhibit the adverse effects of MASP-2 dependent complement activation in a human patient of the present invention.

1. Cloning of genes for variable regions of antibodies against MASP-2 from mouse hybridoma cells. Total RNA was isolated from hybridoma cells secreting anti-MASP-2 MoAb (prepared as described in Example 7) using RNAzol following the manufacturer's protocol (Biotech, Houston, Tex.). First-strand cDNA is synthesized from total RNA using oligo-dT as a primer. PCR is carried out using 3' primers derived from the immunoglobulin constant region and sets of degenerate primers derived from the leader peptide or first framework region of the mouse V _H or VK genes as 5' primers. Anchored PCR was performed as described in Chen and Platsucas (Chen, PF, Scand. J. Immunol. 35:539-549, 1992). To clone the VK gene, double-stranded cDNA was prepared using the primer Not1-MAK1 (5'-

- 47 045598

TGCGGCCGCTGTAGGTGCTGTCTTT-3' SEQ ID NO: 38). Hybridization adapters AD1 (5'GGAATTCACTCGTTATTCTCGGA-3' SEQ ID NO: 39) and AD2 (5'-TCCGAGAATAACGAGTG-3' SEQ ID NO: 40) are ligated to both the 5' and 3' ends of the double-stranded cDNA. Adapters at the 3' ends are removed by Not1 cleavage. The digestion product is then used as a template in a PCR with oligonucleotide AD1 as the 5' primer and MAK2 (5'-CATTGAAAGCTTTGGGGTAGAAGTTGTTC-3' SEQ ID NO: 41) as the 3' primer. DNA fragments of approximately 500 bp were cloned into pUC19. Several clones are selected for sequence analysis to confirm that the cloned sequence spans the expected murine immunoglobulin constant region. The Not1-MAK1 and MAK2 oligonucleotides are derived from the V _K region and are located 182 and 84 base pairs, respectively, downstream of the first base pair of the C kappa gene. Clones containing full-length VK and leader peptide are selected.

To clone the V _H gene, double-stranded cDNA was prepared using the primer Not1 MAG1 (5'-CGCGGCGCAGCTGCTCAGAGTGTAGA-3' SEQ ID NO: 42). Hybridization adapters AD1 and AD2 are ligated to both the 5' and 3' ends of the double-stranded cDNA. Adapters at the 3' ends are removed by Not1 cleavage. The digestion product is used as a template in PCR with oligonucleotide AD1 and MAG2 (5'-CGGTAAGCTTCACTGGCTCAGGGAAATA-3' SEQ ID NO: 43) as primers. DNA fragments 500-600 bp long. cloned into pUC19. The Not1 oligonucleotides MAG1 and MAG2 are derived from the mouse Cy.7.1 region, and are located 180 and 93 bp, respectively, downstream of the first bp. mouse gene Cy.7.1. Clones containing full-length VH and leader peptide are selected.

2. Construction of expression vectors for chimeric IgG and Fab against MASP-2. The cloned V _H and V _K genes described above are used as templates in a PCR reaction to add a Kozak consensus sequence to the 5' end and a splice donor to the 3' end of the nucleotide sequence. After sequence analysis to confirm the absence of PCR errors, the VH and V _K genes are inserted into expression vector cassettes containing human C. y1 and C. kappa, respectively, to produce pSV2neoV _H huCy1 and pSV2neoV huCy. CsCl gradient purified plasmid DNAs of the heavy and light chain vectors are used to transfect COS cells by electroporation. After 48 hours, the culture supernatant is tested by ELISA to confirm the presence of approximately 200 ng/ml chimeric IgG. Cells are harvested and total RNA is obtained. First-strand cDNA is synthesized from total RNA using oligo-dT as a primer. This cDNA is used as a template for PCR to produce Fd and kappa DNA fragments. For the Fd gene, PCR was performed using 5'AAGAAGCTTGCCGCCACCATGGATTGGCTGTGGAACT-3' (SEQ ID NO: 44) as the 5' primer and a CH1-derived 3' primer (5'-CGGGATCCTCAAACTTTCTTGTCCACCTTGG-3' SEQ ID NO: 45). The DNA sequence is confirmed to contain the complete V _H and CH1 domain of human IgG1. After digestion with appropriate enzymes, the Fd DNA fragments are inserted into the HindIII and BamHI restriction sites of the pSV2dhfr TUS expression vector cassette to produce pSV2dhfrFd. Plasmid pSV2 is commercially available and consists of DNA fragments from various sources: pBR322 DNA (thin line) contains the pBR322 DNA origin of replication (pBR ori) and the lactamase gene for ampicillin resistance (Amp); The SV40 DNA, represented by broader streaks and labeling, contains the SV40 DNA origin of replication (SV40 ori), the early promoter (5' of the dhfr and neo genes), and the polyadenylation signal (3' of the dhfr and neo genes). An SV40-derived polyadenylation (pA) signal is also placed at the 3' end of the Fd gene.

For the kappa gene, PCR was performed using 5'AAGAAAGCTTGCCGCCACCATGTTCTCACTAGCTCT-3' (SEQ ID NO: 46) as the 5' primer and a CK-derived 3' primer (5'-CGGGATCCTTCTCCCTCTAACACTCT-3' SEQ ID NO: 47). The DNA sequence was confirmed to contain full-length human _VK and _CK regions. After digestion with appropriate restriction enzymes, kappa DNA fragments are inserted into the HindIII and BamHI restriction sites of the pSV2neo TUS expression vector cassette to generate pSV2neoK. Expression of the Fd and kappa genes is controlled through HCMV-derived enhancer and promoter elements. Because the Fd gene does not include a cysteine amino acid residue involved in an interchain disulfide bond, this recombinant chimeric Fab contains non-covalently linked heavy and light chains. This chimeric Fab is designated cFab.

To produce a recombinant Fab with a disulfide bond between the heavy and light chains, the above Fd gene can be extended to include a sequence encoding 9 additional amino acids (EPKSCDKTH, SEQ ID NO: 48) from the hinge region of human IgG1. The BstEII-BamHI DNA fragment encoding 30 amino acids at the 3' end of the Fd gene can be replaced with DNA fragments 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 were transfected with pSV2neoV _H huC.y1 and pSV2neoV huC kappa plasmid DNAs by electroporation. Transfected cells were selected in the presence of 0.7 mg/ml G418. Grow cells

- 48 045598 pour into a 250 ml flask with stirring using serum-containing medium.

Culture supernatant from 100 ml of culture from the stir flask was applied to a 10 ml PROSEP A column (Bioprocessing, Inc., Princeton, N.J.). The column is washed with 10 bed volumes of PBS. Bound antibody is eluted using 50 mM citrate buffer, pH 3.0. An 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 were removed by buffer exchange to PBS via ultrafiltration with a Millipore membrane (M.W. cutoff: 3000). The protein concentration of the purified antibody was 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 Fabs, CHO cells were transfected with purified pSV2dhfrFd (or pSV2dhfrFd/9aa) and pSV2neokappa plasmid DNAs by electroporation. Transfected cells are selected in the presence of G418 and methotrexate. Selected cell lines are propagated at increasing concentrations of methotrexate. The cells are single cells subcloned by limiting dilution. Subcloned high-producing cell lines are then grown in 100 ml culture in a stir flask using serum-free medium.

The chimeric anti-MASP-2 Fab is purified by affinity chromatography using a mouse anti-idiotypic MoAb to anti-MASP-2 MoAb. Anti-idiotypic anti-MASP-2 MoAb antibody can be generated by immunizing mice with mouse anti-MASP-2 MoAb conjugated to limpet hemocyanin (KLH) and screening for specific binding of the MoAb that human MASP-2 can compete with. For purification, 100 ml of supernatant from stir flask cultures of CHO cells producing cFab or cFab/9aa is applied to an affinity column with an anti-idiotypic anti-MASP-2 MoAb attached. The column is then washed thoroughly with PBS until bound Fab is eluted using 50 mM diethylamine, pH 11.5. Residual salts are removed by buffer exchange as described above. The protein concentration of the purified Fab was determined by the BCA method (Pierce).

The ability of chimeric MASP-2 IgG, cFab, and cFAb/9aa to inhibit MASP-2-dependent complement pathways can be determined using the inhibition 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 by L-ficolin/P35, H-ficolin, M-ficolin, or mannan.

C4 cleavage assay. B Petersen, S.V., et al., J. Immunol. Methods 257:107, 2001, describes a C4 cleavage assay that measures lectin pathway activation caused by lipoteichoic acid (LTA) from S. aureus, which binds L-ficolin.

Reagents: Formalin-fixed S. aureous (DSM20233) is prepared as follows: bacteria are grown overnight at 37°C in tryptone-soy blood medium, washed three times with PBS, then fixed for 1 hour at room temperature in PBS/0 .5% formaldehyde, and washed three more times with PBS, before resuspending in coating buffer (15 mM Na ₂ Co ₃ , 35 mM NaHCO ₃ , pH 9.6).

Analysis. Wells of a Nunc MaxiSorb microtiter plate (Nalgene Nunc International, Rochester, NY) were coated using: 100 μl of formaldehyde-fixed S. aureus DSM20233 (OD ₅₅₀ =0/5) in coating buffer with 1 μg of L-ficolin in coating buffer. After overnight incubation, wells are blocked using 0.1% human serum albumin (HSA) in TBS (10 mM Tris HCl, 140 mM NaCl, pH 7.4), then washed using TBS containing 0.05% Tween 20 and 5 mM CaCl ₂ (wash buffer). Human serum samples are diluted in 20 mM Tris HCl, 1 M NaCl, 10 mM CaCl ₂ , 0.05% Triton X 100, 0.1% HSA, pH 7.4, which prevent activation of endogenous C4 and cause dissociation of the C1 complex (consisting from C1q, Clr and Cls). MASP-2 inhibitory agents, including anti-MASP-2 MoAbs and inhibitory peptides, are added to serum samples at various concentrations. Diluted samples are added to the plate and incubated overnight at 4°C. After 24 hours, the plates are washed thoroughly with wash buffer, then 0.1 μg of purified human C4 (prepared as described in Dodds, AW, Methods Enzymol. 223:46, 1993) in 100 μl of 4 mM barbital, 145 mM NaCl, 2 mM CaCl ₂ , 1 mM MgCl ₂ , pH 7.4 is added to each well. After 1.5 h at 37°C, the plates are washed again and C4b accumulation is detected using alkaline phosphatase-conjugated chicken anti-human C4c antibody (obtained from Immunsystem, Uppsala, Sweden) and measured using the colorimetric substrate pnitrophenyl phosphate.

C4 analysis on mannan. The above assay is adapted to measure lectin pathway activation using MBL by coating the plate with LSP and mannan before adding serum mixed with various MASP-2 inhibitory agents.

Ficolin H C4 Assay (Hakata Ag): The above assay is adapted to measure lectin pathway activation using H-ficolin by coating the plate with LPS and ficolin

H before adding serum mixed with various MASP-2 inhibitory agents.

Example 8.

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

Ways. Immune complexes were prepared in situ by coating microtiter plates (Maxisorb, Nunc, Cat. No. 442404, Fisher Scientific) with 0.1% human serum albumin in 10 mM Tris, 140 mM NaCl, pH 7.4 for 1 h at room temperature. 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/Ca ²⁺ . Serum samples were obtained from wild-type and MASP-2 −/− mice and added to coated plates. Control samples in which C1q was depleted were prepared from wild-type and MASP-2 −/− serum samples. C1q-depleted mouse serum was processed using protein A-coupled Dynabeads (Dynal Biotech, Oslo, Norway) coated with rabbit anti-human C1q IgG (Dako, Glostrup, Denmark), according to the supplier's instructions. The plates were incubated for 90 minutes at 37°C. Bound C3b was detected using polyclonal anti-human C3c antibody (Dako A 062) diluted in TBS/tw/Ca ⁺⁺ to 1:1000. The secondary antibody is a goat anti-rabbit IgG antibody.

Results. In fig. Figure 7 shows the relative levels of C3b accumulation on IgG-coated plates in wild-type serum, MASP-2−/− serum, C1q-depleted wild-type serum, and C1q-depleted MASP-2−/− serum. These results indicate that the classical pathway is intact in MASP-2 −/− mice.

Example 9.

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

Ways. To test the effect of a MASP-2 inhibitory agent under conditions of complement activation, where the classical activation pathway is initiated by immune complexes, 50 μl samples containing 90% NHS, in triplicate, were incubated at 37°C in the presence of 10 μg/ml immune complex (IC ) or PBS, and in parallel also include samples (+/- IC) in triplicate that contain 200 nM anti-properdin monoclonal antibody during incubation at 37°C. After two hours of incubation at 37°C, 13 mM EDTA was added to all samples to stop further complement activation, and the samples were immediately cooled to 5°C. Samples were then stored at 70°C until analysis of complement activation products (C3a and sC5b-9) using ELISA kits (Quidel, catalog no. A015 and A009), following the manufacturer's instructions.

Example 10.

This example describes the identification of high affinity anti-MASP2 Fab2 antibody fragments that block MASP-2 activity.

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

To block the functional activity of MASP-2, the Fab2 antibody or antibody fragment must bind to and interfere with a structural epitope on MASP-2 required for MASP-2 functional activity. Thus, many or all of the high-affinity anti-MASP-2 Fab2 binders may not inhibit the functional activity of MASP-2 unless they bind to structural epitopes on MASP-2 that are directly involved in the functional activity of MASP-2.

A functional assay that measures inhibition of C3 convertase formation in the lectin pathway was used to evaluate the anti-MASP-2 blocking activity of Fab2. It is known that the primary physiological role of MASP-2 in the lectin pathway is to obtain the next functional component of the lectin-mediated complement pathway, namely the C3 convertase of the lectin pathway. The C3 convertase of the lectin pathway is a critical enzyme complex (C4bC2a) that proteolytically cleaves C3 into C3a and C3b. MASP-2 is not a structural component of lectin pathway C3 convertase (C4bC2a); however, the functional activity of MASP-2 is required to produce the two protein components (C4b, C2a) that the C3 convertase of the lectin pathway contains. In addition, all of the individual MASP-2 functional activities listed above appear to be required for MASP-2 to produce lectin pathway C3 convertase. For this reason, the preferred assay to use to evaluate the anti-MASP-2 blocking activity of Fab2 is considered to be a functional assay that measures inhibition of lectin pathway C3 convertase formation.

Preparation of high affinity Fab2. A phage display library of human antibody light and heavy chain variable sequences and automated antibody selection technology to identify Fab2s that interact with selected ligands of interest were used to generate high-affinity Fab2s against rat MASP-2 protein (SEQ ID NO: 55). A known amount of rat MASP-2 protein (~1 mg, >85% purity) was used for antibody screening. Three cycles of amplification were used to select antibodies with the best affinity. Approximately 250 different best candidates expressing antibody fragments were selected for ELISA screening. High affinity candidates were subsequently sequenced to determine the uniqueness of the different antibodies.

Fifty unique anti-MASP-2 antibodies were purified, and 250 μg of each purified Fab2 antibody was used to characterize MASP-2 binding affinity and functional testing of the complement activation pathway, as described in more detail below.

Assays used to evaluate the inhibitory (blocking) activity of Fab2 against MASP2.

1. Assay to measure inhibition of lectin pathway C3 convertase formation.

State of the art. The lectin pathway C3 convertase is an enzyme complex (C4bC2a) that proteolytically cleaves C3 into two active proinflammatory fragments, anaphylatoxin C3a and opsonin C3b. Formation of C3 convertase appears to be a key step in the lectin pathway in mediating inflammation. MASP-2 is not a structural component of lectin pathway C3 convertase (C4bC2a); thus, antibodies against MASP-2 (or Fab2) do not directly inhibit the activity of preexisting C3 convertase. However, the activity of the serine protease MASP-2 is necessary for the production of two protein components (C4b, C2a) that the C3 convertase of the lectin pathway contains. Thus, anti-MASP-2 Fab2, which inhibits the functional activity of MASP-2 (ie, blocking anti-MASP-2 Fab2), inhibits de novo formation of the lectin pathway C3 convertase. C3 contains an atypical and highly reactive thioether group as part of the structure. When C3 is cleaved by C3 convertase in this assay, the thioester group on C3b can form a covalent bond with the hydroxyl group or amino group on the macromolecules immobilized on the bottom of the plastic wells through ester or amide bonds, thus facilitating the detection of C3b in the ELISA assay.

Yeast mannan is a known activator of the lectin pathway. In the following method to measure C3 convertase formation, mannan-coated plastic wells were incubated for 30 min at 37°C with diluted rat serum to activate the lectin pathway. The wells were then washed and the C3b immobilized in the wells was analyzed by standard ELISA methods. The amount of C3b formed in this assay is a direct reflection of de novo formation of the lectin pathway C3 convertase. Anti-MASP-2 Fab2 at selected concentrations were tested in this assay for their ability to inhibit C3 convertase formation and subsequent C3b formation.

Ways.

Costar Medium Binding 96-well plates were incubated overnight at 5°C with mannan diluted in 50 mM carbonate buffer, pH 9.5, at 1 μg/50 μl/well. After overnight incubation, each well was washed three times with 200 μl PBS. The wells were then blocked with 100 μl/well 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 μl PBS. Anti-MASP-2 Fab2 samples were diluted to selected concentrations in GVB buffer containing Ca~ and Mg~ (4.0 mM barbital, 141 mM NaCl, 1.0 mM MgCl ₂ , 2.0 mM CaCl ₂ , 0.1% gelatin, pH 7.4) at 5°C. 0.5% rat serum was added to the above samples at 5°C, and 100 μl was transferred to each well. The plates were coated and incubated for 30 minutes at 37°C in a water bath to ensure complement activation. The reaction was stopped by transferring the plates from a 37°C water bath to a container containing a mixture of ice and water. Each well was washed five times with 200 μl PBS Tween 20 (0.05% Tween 20 in PBS), then washed twice with 200 μl PBS. 100 μl/well of a 1:10,000 dilution of primary antibody (rabbit anti-human C3c antibody, DAKO A0062) was added to PBS containing 2.0 mg/ml bovine serum albumin and incubated for 1 hour at room temperature with gentle mixing. Each well was washed with 5x200 μl PBS. 100 μl/well of a 1:10,000 dilution of secondary antibody (peroxidase-conjugated goat anti-rabbit IgG, American Qualex A102PU) was added to PBS containing 2.0 mg/ml bovine serum albumin and incubated for 1 h at room temperature under shaker with gentle mixing. Each well was washed five times with 200 μl PBS. 100 μl/well of TMB peroxidase substrate (Kirkegaard & Perry Laboratories) was added and incubated at room temperature in

- 51 045598 within 10 minutes. The peroxidase reaction was stopped by adding 100 μl/well of 1.0 M H ₃ PO ₄ and the OD ₄₅₀ was measured.

2. Assay to measure inhibition of MASP-2 dependent C4 cleavage.

State of the art. The serine protease activity of MASP-2 is highly specific, and only two protein substrates for MASP-2 have been identified; C2 and C4. Cleavage of C4 produces C4a and C4b. Anti-MASP-2 Fab2 can bind to structural epitopes on MASP-2 directly involved in C4 cleavage (e.g., MASP-2 binding site for C4; MASP-2 serine protease catalytic site), and thus inhibits the functional activity of MASP-2 for C4 cleavage.

Yeast mannan is a known activator of the lectin pathway. In the following method to measure MASP-2 C4 cleavage activity, mannan-coated plastic wells were incubated for 30 minutes at 37°C with diluted rat serum to activate the lectin pathway. Because the primary antibody used in this ELISA recognizes only human C4, diluted rat serum was also supplemented with human C4 (1.0 μg/ml). The wells were then washed and the human C4b immobilized in the wells was analyzed using standard ELISA methods. The amount of C4b obtained in this assay is an indicator 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.

Ways. Costar Medium Binding 96-well plates were incubated overnight at 5°C with mannan diluted in 50 mM carbonate buffer, pH 9.5, at 1.0 μg/50 μl/well. Each well was washed 3X with 200 µl PBS. The wells were then blocked with 100 μl/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 µl PBS. Anti-MASP-2 Fab2 samples were diluted to selected concentrations in Ca~ and Mg ⁺⁺ containing GVB buffer (4.0 mM barbital, 141 mM NaCl, 1.0 mM MgCl ₂ , 2.0 mM CaCl ₂ , 0.1% gelatin , pH 7.4) at 5°C. 1.0 μg/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 μl was transferred to each well. The plates were covered and incubated for 30 min in a water bath at 37°C to ensure complement activation. The reaction was stopped by transferring the plates from a 37°C water bath to a container containing a mixture of ice and water. Each well was washed with 5x200 µl PBS Tween 20 (0.05% Tween 20 in PBS), then each well was washed 2X with 200 µl PBS. 100 μl/well of biotin-conjugated chicken anti-human C4c antibody at a dilution of 1:700 (Immunsystem AB, Uppsala, Sweden) was added to PBS containing 2.0 mg/ml bovine serum albumin (BSA) and incubated for one hour at room temperature. with gentle stirring. Each well was washed with 5x200 μl PBS. 100 μl/well of 0.1 μg/ml peroxidase-conjugated streptavidin (Pierce Chemical #21126) was added to PBS containing 2.0 mg/ml BSA and incubated for one hour at room temperature in a shaker with gentle agitation. Each well was washed with 5x200 μl PBS. 100 μl/well of TMB peroxidase substrate (Kirkegaard & Perry Laboratories) was added and incubated at room temperature for 16 min. The peroxidase reaction was stopped by adding 100 μl/well of 1.0 M H3PO4, and OD450 was measured.

3. Anti-rat MASP-2 Fab2 binding assay to native rat MASP-2.

State of the art. MASP-2 is usually present in plasma in the form of the MASP-2 dimmer complex, which also includes specific lectin molecules (mannose-binding protein (MBL) and ficolins). Thus, given the interest in investigating the binding of anti-MASP-2 Fab2 to a physiologically relevant form of MASP-2, it is important to develop a binding assay that utilizes the interaction between Fab2 and native MASP-2 in plasma rather than purified recombinant MASP-2. In this binding assay, native MASP-2-MBL complex from 10% rat serum was first immobilized onto mannan-coated wells. The binding affinities of various anti-MASP-2 Fab2s to immobilized native MASP-2 were then examined using a standard ELISA method.

Ways. Costar High Binding 96-well plates were incubated overnight at 5°C with mannan diluted in 50 mM carbonate buffer, pH 9.5, at 1 μg/50 μl/well. Each well was washed 3X with 200 µl PBS. wells were blocked with 100 μl/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 using 200 µl TBS/Tween/Ca ⁺⁺ wash buffer (Tris-buffered saline, 0.05% Tween 20, containing 5.0 mM CaCl ₂ , pH 7.4. 10% rat serum in binder high salt buffer (20 mM Tris, 1.0 M NaCl, 10 mM CaCl ₂ , 0.05% Triton X100, 0.1% (w/v) bovine serum albumin, pH 7.4) was prepared at ice. 100 µl/well was added and incubated overnight at 5°C. Wells were washed 3X using 200 µl TBS/Tween/Ca ⁺⁺ wash buffer. Wells were then washed 2X using 200 µl PBS. 100 µl/well Fab2 against

- 52 045598

MASP-2 at the selected concentration, diluted in GVB buffer containing Ca ⁺⁺ and Mg ⁺⁺ (4.0 mM barbital, 141 mM NaCl, 1.0 mM MgCl ₂ , 2.0 mM CaCl ₂ , 0.1% gelatin, pH 7.4) was added and incubated for one hour at room temperature with gentle mixing. Each well was washed with 5x200 μl PBS. 100 μl/well of HRP-conjugated goat anti-Fab2 antibody (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 with 5x200 μl PBS. 100 μl/well of TMB peroxidase substrate (Kirkegaard & Perry Laboratories) was added and incubated at room temperature for 70 min. The peroxidase reaction was stopped by adding 100 μl/well of 1.0 M H3PO ₄ and the OD ₄₅₀ was measured.

Results.

Approximately 250 different Fab2s that reacted with high affinity with the rat MASP-2 protein were selected for ELISA screening. These high-affinity Fab2s were sequenced to determine the uniqueness of the different antibodies, and 50 unique anti-MASP-2 antibodies were purified for further analysis. 250 μg of each purified Fab2 antibody was used for MASP-2 binding affinity characterization and complement pathway functional testing. The results of this analysis are shown in the table below. 7.

Table 7

FAB2 against MASP-2, blocking the lectin pathway of complement activation

Antibody Fab2# Convertase SZ, 1C ₅₀ (nM) K _d Cleavage C4, 1C ₅₀ (nM) 88 0.32 4.1 ND 41 0.35 0.30 0.81 eleven 0.46 0.86 <2 nM 86 0.53 1.4 ND 81 0.54 2.0 ND 66 0.92 4.5 ND 57 0.95 3, 6 <2 nM 40 1.1 7.2 0.68 58 1.3 2, 6 ND 60 16 3, 1 ND 52 16 5, 8 <2 nM 63 2.0 6, 6 ND 49 2.8 8.5 <2 nM 89 thirty 2.5 ND 71 thirty 10.5 ND 87 6, 0 2.5 ND 67 100 7.7 ND

As shown in the table above. 7, of 50 anti-MASP-2 Fab2s tested, seventeen Fab2s were identified as MASP-2 blocking Fab2s that potentially inhibit C3 convertase formation with an IC ₅₀ equal to or less than 10 nM Fab2 (positive best candidate selection rate 34%). Eight of the seventeen identified Fab2s have _IC50s in the subnanomolar range. In addition, for all seventeen MASP-2 blocking Fab2s shown in Table. 7 obtained substantially complete inhibition of C3 convertase formation in a lectin pathway C3 convertase assay. In fig. 8A graphically illustrates the results of a C3 convertase formation assay for Fab2 antibody #11, which is representative of the other Fab2 antibodies tested, the results for which are shown in Table. 7. This is important information because it is theoretically possible that a Fab2 blocker may only partially inhibit MASP-2 function, even when every MASP-2 molecule binds to Fab2.

Although mannan is a known activator of the lectin pathway, there is a theoretical possibility that the presence of anti-mannan antibodies in rat serum may also activate the classical pathway and lead to the formation of C3b via the classical pathway C3 convertase. However, each of the seventeen anti-MASP-2 Fab2 blockers listed in this example has the potential to inhibit C3b formation (>95%), thus demonstrating the specificity of this assay for lectin pathway C3 convertase.

Binding assays were also performed on all seventeen Fab2 blockers to calculate the apparent K _d for each. The results of anti-rat MASP-2 Fab2 binding assays with native rat MASP-2 for six of the blocking Fab2s are also shown in Table 1. 7. In FIG. 8B graphically illustrates the results of a binding assay with Fab2 antibody #11. Similar binding assays were also performed for other Fab2s, the results of which are shown in Table 1. 7. In general, the apparent Kds obtained for the binding of each of the six Fab2s to native MASP-2 correspond relatively closely to the IC ₅₀ for Fab2 in the C3 convertase functional assay. There is evidence that MASP-2

- 53 045598 undergoes a conformational change from an inactive to an active form upon activation of its protease activity (Feinberg et al., EMBO J 22:2348 59 (2003); Gal et al., J. Biol. Chem. 280:33435 44 (2005) ). In 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; thus, MASP-2 is in the active conformation (Petersen et al., J. Immunol Methods 257:107 16, 2001). Therefore, an exact match between the IC ₅₀ and K _d for each of the seventeen Fab2 blockers tested in these two functional assays should not be expected because Fab2 binds to different conformational forms of MASP-2 in each assay. However, with the exception of Fab2 #88, there appears to be an exact match between the IC ₅₀ and the apparent Kd for each of the other sixteen Fab2s tested in the two assays (see Table 7).

Several Fab2 blockers were evaluated for inhibition of MASP-2-dependent C4 cleavage. In fig. 8C graphically illustrates the results of the C4 cleavage assay showing inhibition using Fab2 #41, with IC ₅₀ =0.81 nM (see Table 7). As shown in FIG. 9, all of the tested Fab2s were found to inhibit C4 cleavage with _IC50s similar to those obtained in the C3 convertase assay (see Table 7).

Although mannan is a known activator of the lectin pathway, there is a theoretical possibility that the presence of anti-mannan antibodies in rat serum may also activate the classical pathway and thus lead to the formation of C4b via C1-mediated cleavage of C4. However, several anti-MASP-2 Fab2s have been identified that strongly inhibit C4b formation (>95%), thus demonstrating the specificity of this assay for MASP-2-mediated C4 cleavage. C4, like C3, contains unusual and highly reactive thioether groups as part of the structure. Upon cleavage of C4 by MASP-2 in this assay, the thioester group on C4b can form a covalent bond with hydroxyl groups or amino groups on macromolecules immobilized at the bottom of plastic wells via ester or amide bonds, thereby facilitating detection of C4b in the ELISA assay.

These studies clearly show the production of high-affinity FAB2 against the rat MASP-2 protein, which functionally blocks both C4 and C3 convertase activity, thereby preventing lectin pathway activation.

Example 11.

This example describes epitope mapping of several Fab2 anti-rat MASP-2 blocking antibodies prepared as described in Example 10.

Ways.

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

rat MASP-2A, full-length MASP-2 protein inactivated by replacing the active site serine with alanine (S613A);

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

CUBI II, N-terminal fragment of rat MASP-2 containing only the CUBI domain, EGF-like domain and CUBII domain; And

CUBI/EGF-like domain, an N-terminal fragment of rat MASP-2 containing only the CUBI domain and the EGF-like domain.

These proteins were purified from culture supernatants by nickel affinity chromatography as described previously (Chen et al., J. Biol. Chem. 276:25894 02 (2001)).

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

All recombinant proteins were dialyzed in TBS buffer and their concentrations were determined by measuring OD at 280 nm.

Dot blot analysis.

Serial dilutions of the five recombinant MASP-2 polypeptides described above and shown in FIG. 10 (and thioredoxin polypeptide as a negative control for the CCPII serine protease polypeptide) were spotted onto a nitrocellulose membrane. The amount of protein spotted ranged from 100 ng to 6.4 pg, in fivefold dilutions. In later experiments, the amount of protein spotted ranged from 50 ng, decreasing to 16 pg, again in fivefold dilutions. Membranes were blocked using 5% skim milk powder in TBS (blocking buffer), then incubated with 1.0 μg/ml Anti-MASP-2 Fab2 in blocking buffer (containing 5.0 mM Ca ²⁺ ). Bound Fab2 was detected using HRP-conjugated anti-human Fab antibody (AbD/Serotec; diluted 1/10,000) and an ECL detection kit (Amersham). One membrane was incubated with rabbit polyclonal Ab against

- 54 045598

Human MASP-2 (described in Stover et al., J Immunol 163:6848 59 (1999)) as a positive control. In this case, bound Ab was detected using HRP-conjugated goat anti-rabbit IgG (Dako; diluted 1/2000).

MASP-2 Binding Assay

ELISA plates were coated with 1.0 μg/well of recombinant MASP-2A or CUBI II polypeptide in carbonate buffer (pH 9.0) overnight at 4°C. Wells were blocked using 1% BSA in TBS, then serial dilutions of anti-MASP-2 Fab2 in TBS containing 5.0 mM Ca ²⁺ were added. The plates were incubated for one hour at RT. After washing three times with TBS/tween/Ca ²⁺ , HRP-conjugated anti-human Fab antibody (AbD/Serotec) diluted 1/10,000 in TBS/Ca ²⁺ was added and the plates were incubated for another hour at RT. Bound antibody was detected using the TMB peroxidase substrate kit (Biorad).

Results.

The results of dot blot analysis showing the reactivity of Fab2 towards various MASP-2 polypeptides are presented below in table. 8. Numerical values given in table. 8 indicate the amount of protein spotted required to obtain approximately half the maximum signal strength. As shown, all polypeptides (with the exception of only one thioredoxin binding partner) were recognized by a positive control Ab (polyclonal anti-human MASP-2 antiserum obtained from rabbits).

Table 8

Reactivity towards various recombinant _______rat MASP-2 polypeptides in dot blot assays_______

Antibody Fab2 # MASP-2A CUBI II CUBI/EGF-like CCII SP Thioredoxin 40 0.16 ng NR NR 0.8 ng NR 41 0.16 ng NR NR 0.8 ng NR eleven 0.16 ng NR NR 0.8 ng NR 49 0.16 ng NR NR >20 ng NR 52 0.16 ng NR NR 0.8 ng NR 57 0.032 ng NR NR NR NR 58 0.4 ng NR NR 2.0 ng NR 60 0.4 ng 0.4 ng NR NR NR 63 0.4 ng NR NR 2.0 ng NR 66 0.4 ng NR NR 2.0 ng NR 67 0.4 ng NR NR 2.0 ng NR 71 0.4 ng NR NR 2.0 ng NR 81 0.4 ng NR NR 2.0 ng NR 86 0.4 ng NR NR 10 ng NR 87 0.4 ng NR NR 2.0 ng NR Positive control <0.032 ng 0.16 ng 0.16 ng <0.032 ng NR

NR - no response. The positive control antibody is a polyclonal anti-human MASP-2 antiserum obtained from rabbits.

All of the Fab2s reacted with MASP-2A as well as with MASP-2K (data not shown). Most of the Fab2s recognized the CCPII-SP polypeptide, but not the N-terminal fragments. The two exceptions are Fab2 #60 and Fab2 #57. Fab2 #60 recognizes MASP-2A and a fragment of CUBI-II, but not CUBI/EGF-like polypeptide or CCPII-SP polypeptide, suggesting its binding to an epitope in CUBII, or overlap between the CUBII domain and the EGF-like domain. Fab2 #57 recognizes MASP-2A but none of the MASP-2 fragments tested, indicating that Fab2 recognizes an epitope in CCP1. Fab2 #40 and #49 bind only to full MASP-2A. In the ELISA binding assay shown in FIG. 11, Fab2 #60 also bound to the CUBI-II polypeptide, although with slightly lower apparent affinity.

These findings reveal the identification of unique blocking Fab2s with multiple regions of the MASP-2 protein.

Example 12.

This example describes the results for MASP-2-/- in a mouse model of macular degeneration.

State of the Art/Rationale. Age-related macular degeneration (AMD) is the leading cause of blindness after age 55 in industrialized countries. AMD occurs in two main forms: neovascular (wet) AMD and atrophic (dry) AMD. Neovascular (wet) form

- 55 045598 is responsible for 90% of severe vision loss associated with AMD, even though only ~20% of individuals with AMD develop the wet form. Characteristic clinical features 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 the treatment of wet (neovascular) AMD (Ng et al., Nat Rev Drug Discov 5:123 32 (2006). Although Macugen represents a promising new therapeutic option for a subset of patients with AMD, there remains an urgent need to develop additional treatments for this complex disease. Multiple independent pathways of investigation point to a central role of complement activation in the pathogenesis of AMD. The pathogenesis of choroidal neovascularization (CNV), the most severe form of AMD, may involve activation of the complement pathways.

More than twenty-five years ago, Ryan described an animal model of CNV with laser-induced injury (Ryan, S.J., Tr. Am. Opth. Soc. LXXVII:707 745, 1979). This model was originally developed using rhesus monkeys, however, the same technology has since been used to develop similar CNV models in a variety of laboratory animals, including mice (Tobe et al., Am. J. Pathol. 153:1641 46, 1998) . In this model, laser photocoagulation is used to rupture Bruch's membrane, an action that results in the formation of CNV-like membranes. The laser-induced model depicts many of the important features of the condition in humans (for a recent review, see Ambati et al., Survey Ophthalmology 48:257 293, 2003). The laser-induced injury mouse model is now well developed and is being used as the experimental basis for a large, and even increasing, number of research projects. It is generally accepted that the laser-induced model shares sufficient biological similarity with human CNVs that preclinical pathogenesis and drug inhibition studies using this model are relevant to human CNVs.

Ways.

A MASP-2-/- mouse was generated as described in Example 1 and backcrossed to C57Bl/6 for 10 generations. The present study compared results when male MASP-2 (-/-) and MASP-2 (+/+) mice were assessed during laser-induced CNV, an improved model of neovascular AMD, focusing on the volume of laser-induced CNV by scanning laser confocal microscopy , as an indicator of tissue damage, and determining levels of VEGF, a potent angiogenic factor involved 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, 75 μm; Oculight GL, Iridex, Mountain View, CA) was performed on both eyes of each animal on day zero by one individual, blinded to drug allocation. Laser spots were applied in a standardized manner around the optic nerve using a slit lamp mounting system and a coverslip as a contact lens. The morphological end point of laser damage was the appearance of a cavitation bubble, which was considered a sign correlating with damage to Bruch's membrane. A detailed description of the methods and endpoints assessed follows.

Angiography with fluorescein. Fluorescein angiography was performed using a camera and imaging system (TRC 50 1A camera; ImageNet 2.01 system; Topcon, Paramus, NJ) 1 week after laser photocoagulation. Photographs were taken using a 20-D lens in contact with a camera lens with a fundus camera lens after intraperitoneal injection of 0.1 ml of 2.5% sodium fluorescein. A retinal specialist not involved in laser photocoagulation or angiography scored fluorescein fluorescein angiograms in a single session in a blinded manner.

Choroidal neovascularization volume (CNV). One week after laser injury, the eyes were excised and fixed using 4% paraformaldehyde for 30 min at 4°C. The fundus was obtained by removing anterior fragments and washed three times in PBS, followed by dehydration and rehydration using a series of dilutions of methanol. After blocking twice with buffer (PBS containing 1% bovine serum albumin and 0.5% Triton X 100) for 30 minutes at room temperature, the fundus was incubated overnight at 4°C with 0.5% FITC-isolectin B4 ( Vector laboratories, Burlingame, CA) diluted in PBS containing 0.2% BSA and 0.1% Triton X 100, which binds terminal e-D-galactose residues on the surface of endothelial cells and selectively labels the mouse vasculature. After two washes with PBS containing 0.1% Triton X 100, the neurosensory retina was carefully peeled off and separated from the optic nerve. Four radial weakening cuts were then made, and the remaining RPE-choroid-sclera complex was immersed in anti-fluorescence fading medium (Immu Mount Vectashield Mounting Medium; Vector Laboratories) and covered with a coverslip.

The immersed samples were examined using a confocal laser scanning

- 56 045598 microscope (TCS SP; Leica, Heidelberg, Germany). Vessels were imaged by excitation with blue argon wavelength (488 nm) and capture of emission between 515 and 545 nm. A 40X oil immersion objective was used in all imaging studies. Horizontal optical sections (1 μm increments) were obtained from the surface of the RPE-choroid-sclera complex. The deepest focal plane in which the surrounding choroidal vasculature associated with the lesion could be identified was considered as the base of the lesion. Any vessel located in the laser target area and located on the surface of this reference plane was scored as a CNV. Images of each section were digitally saved. The area of CNV-associated fluorescence was measured by computerized image analysis using electron microscope software (TCS SP; Leica). The summation of the entire fluorescent region in each horizontal section was used as a measure of CNV volume. Imaging was performed by an operator blinded to the drug group assignment.

Because the probability of developing a CNV for each laser lesion is influenced by the group to which it belongs (mouse, eye, and laser spot), mean lesion volumes were compared using a linear mixed model method divided into repeated measures plots. The whole field factor represented the genetic group to which the animals belonged, while the plot factor represented the eye. Statistical significance was determined at the 0.05 level. Post hoc comparisons of means were performed using the Bonferroni correction for multiple comparisons.

VEGF ELISA. Three days after lesioning with 12 laser spots, the RPE choroid complex was sonicated in lysis buffer (20 mM imidazole HCl, 10 mM KCl, 1 mM MgCL ₂ , 10 mM EGTA, 1% Triton X 100, 10 mM NaF, 1 mM molybdate Na, and 1 mM EDTA with protease inhibitors) on ice for 15 min. VEGF protein levels in the supernatant were determined using an ELISA kit (R&D Systems, Minneapolis, MN) that recognizes all splice variants at 450–570 nm (Emax; Molecular Devices, Sunnyvale, CA) and normalized to total protein content. Measurements in duplicate were performed in a blinded manner by an operator not involved in photocoagulation, imaging, or angiography. VEGF amounts were presented 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.

In fig. 12A graphically illustrates the levels of VEGF protein in the RPE-choroid complex isolated from wild-type C57Bl6 mice and MASP-2(-/-) mice on day zero. As shown in FIG. 12A, assessment of VEGF levels shows a decrease in background VEGF levels in MASP-2 (-/-) mice compared to control wild-type C57bl mice. In fig. 12B graphically illustrates VEGF protein levels measured at day three after laser-induced injury. As shown in FIG. 12B, VEGF levels increased significantly in wild-type (+/+) mice three days after laser-induced injury, which is consistent with published studies (Nozaki et al., Proc. Natl. Acad. Sci. USA 103:2328 33 (2006) ). However, unexpectedly, very low levels of VEGF were observed in MASP-2 (-/-) mice.

Assessment of choroidal neovascularization (CNV).

In addition to the reduction in VEGF levels after laser-induced macular degeneration, CNV area was determined before and after laser injury. In fig. 13 graphically illustrates the volume of CNV measured in wild-type C57bl mice and MASP-2(-/-) mice on day seven after laser-induced injury. As shown in FIG. 13, MASP-2 (-/-) mice showed an approximately 30% reduction in CNV area after laser-induced injury on day seven compared with control wild-type mice.

These findings indicate a reduction in VEGF and CNV, as observed in MASP (-/-) mice compared to control wild-type (+/+) mice, and that blocking MASP-2 using an inhibitor may have a prophylactic or therapeutic effect in the treatment of macular degeneration.

Example 13.

This example describes a pharmacodynamic analysis of representative high-affinity anti-MASP-2 antibodies Fab2 identified as described in Example 10.

State of the Art/Rationale.

As described in Example 10, to identify high-affinity antibodies that block the lectin pathway in rats, rat MASP-2 protein was used to pan a phage display library. This library was designed to provide high immunological diversity and was constructed using fully human immunoglobulin gene sequences. As described in Example 10, approximately 250 individual phage clones were identified that bind with high affinity to rat MASP-2 protein through ELISA screening. Sequencing of these clones identified 50 unique phages encoding an antibody against MASP-2. Fab2 protein was expressed from these clones, purified, and analyzed for MASP-2 binding affinity and functional inhibition of the lectin complement pathway.

As shown in table. 7 from Example 10, identified 17 anti-MASP-2 Fab2s with functional blocking activity identified from this analysis (best hit rate for blocking antibodies 34%). Functional inhibition of the lectin complement pathway by Fab2 was noticeable at the level of C4 accumulation, which is a direct indicator of C4 degradation by MASP-2. Importantly, the inhibition was equally evident when assessing C3 convertase activity, indicating a functional blockade of the lectin complement pathway. The 17 identified MASP-2 blocking Fab2s, identified as described in Example 10, potently inhibit C3 convertase formation with IC ₅₀ values equal to or less than 10 nM. Eight of the 17 identified Fab2s have IC ₅₀ values in the subnanomolar range. Moreover, all 17 MASP-2 blocking Fab2s obtained substantially complete inhibition in the lectin pathway C3 convertase formation assay, as shown in FIG. 8A-C, and summarized in Table. 7 from Example 10. Moreover, each of the 17 anti-MASP-2 Fab2 blockers shown in Table. 7 strongly inhibits C3b formation (>95%), thus demonstrating the specificity of this assay for lectin pathway C3 convertase.

Rat IgG2c and mouse IgG2a full length antibody isotype variants were obtained from Fab2 #11. This example describes the in vivo characterization of these isotypes based on pharmacodynamic parameters.

Ways.

As described in Example 10, rat MASP-2 protein was used to pan a Fab phage display library from which Fab2#ll was identified. Rat IgG2c and mouse IgG2a full length antibody isotype variants were obtained from Fab2 #11. Both rat IgG2c and mouse IgG2a full-length antibody isotypes were characterized in vivo by pharmacodynamic parameters as follows.

In vivo study in mice.

A pharmacodynamic study was performed in mice to investigate the effect of anti-MASP-2 antibody dosing on plasma lectin pathway activity in vivo. In this study, C4 accumulation was measured ex vivo in a lectin pathway assay at various time points following subcutaneous (SC) and intraperitoneal (IP) administration of 0.3 mg/kg or 1.0 mg/kg mouse anti-MASP-2 MoAb (mouse full-length antibodies of the IgG2a isotype, derived from Fab2#11).

In fig. 14 graphically illustrates lectin pathway-specific accumulation of C4b measured ex vivo in undiluted serum samples collected from mice (n=3 mice/group) at various time points following subcutaneous dosing of either 0.3 mg/kg or 1.0 mg/kg. kg mouse anti-MASP-2 MoAb. Mouse serum samples collected before antibody dosing served as a negative control (100% activity), while serum supplemented in vitro with 100 nM of the same anti-MASP-2 blocking antibody was used as a positive control (0% activity). .

The results shown in FIG. 14 demonstrate rapid and complete inhibition of C4b accumulation following subcutaneous administration of a 1.0 mg/kg dose of murine anti-MASP-2 MoAb. Partial inhibition of C4b accumulation was observed following subcutaneous administration of a 0.3 mg/kg dose of murine anti-MASP-2 MoAb.

The time course of lectin pathway recovery was monitored over three weeks following a single IP administration of mouse anti-MASP-2 MoAb at 0.6 mg/kg to mice. As shown in FIG. 15, a sharp decrease in lectin pathway activity occurred after antibody dosing followed by complete inhibition of the lectin pathway, which lasted for approximately 7 days after IP administration. A slow recovery of lectin pathway activity was observed during the second and third weeks, with complete restoration of the lectin pathway in mice 17 days after administration of anti-MASP-2 MoAb.

These results indicate that mouse anti-MASP-2 Moab, derived from Fab2 #11, inhibits the lectin pathway in mice in a dose-dependent manner when delivered systemically.

Example 14.

This example describes the assay of a mouse anti-MASP-2 MoAb derived from Fab2 #11 for efficacy in a mouse model of age-related macular degeneration.

State of the Art/Rationale.

As described in Example 10, rat MASP-2 protein was used to pan a Fab phage display library from which Fab2#11 was identified as a functional antibody. Full-length antibodies of the rat IgG2c and mouse IgG2a isotypes were obtained from Fab2 #11. A full-length mouse anti-MASP-2 antibody of the IgG2a isotype was characterized pharmacodynamically as described in Example 13. In this example, a full-length mouse anti-MASP-2 antibody derived from Fab2 #11 was analyzed in a mouse model of age-related macular degeneration ( AMD), described in Bora P.S. et al., J Immunol 174:491–497 (2005).

- 58 045598

Ways.

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

Administration of mouse anti-MASP-2 MoAb.

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 IP injected into WT (+/+) mice (n=8 mice per group) over 16 hours before CNV induction.

Induction of choroidal neovascularization (CNV).

Induction of choroidal neovascularization (CNV) and measurement of CNV volume were performed using laser photocoagulation as described in Example 12.

Results.

In fig. 16 graphically illustrates CNV area measured 7 days after 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 FIG. 16, mice pretreated with 1.0 mg/kg anti-MASP-2 MoAb showed a statistically significant (p<0.01) reduction in CNV of approximately 50% 7 days after laser treatment. As further shown in FIG. 16 observed that a dose of 0.3 mg/kg anti-MASP-2 MoAb was not effective in reducing CNV. It is noted that the 0.3 mg/kg dose of anti-MASP-2 MoAb showed partial and transient inhibition of C4b accumulation after subcutaneous administration, as described in Example 13 and shown in FIG. 14.

The results described in this example demonstrate that blocking MASP-2 with an inhibitor, such as anti-MASP-2 MoAb, has a preventive 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 conducted on MASP-2 (-/-) mice described in Example 12, which observed a 30% reduction in CNV 7 days after laser treatment in MASP-2 mice (-/-) compared to wild-type control mice. In addition, the results in this example further demonstrate that the anti-MASP-2 antibody, when delivered systemically, provides a local therapeutic benefit to the eye, thus highlighting the potential of the systemic route of administration for the treatment of AMD patients. Overall, these results provide evidence supporting the use of MASP-2 MoAb in the treatment of AMD.

Example 15.

This example describes the identification, using phage display, of fully human scFv antibodies that bind to MASP-2 and inhibit lectin-mediated complement activation while leaving a component of the classical (C1q-dependent) immune system pathway intact .

Review.

Fully human, high affinity antibodies against MASP-2 were identified by screening a phage display library. Variable fragments of the light and heavy chain of antibodies were isolated in both scFv and full-length IgG formats. Human anti-MASP-2 antibodies can be used to inhibit cellular damage associated with lectin pathway-mediated activation of the alternative complement pathway, while leaving the classical (C1q-dependent) immune system pathway component intact. In some embodiments, subject MASP-2 inhibitory antibodies have the following characteristics: (a) high affinity for human MASP-2 (e.g., K _D of 10 nM or less), and (b) inhibits MASP-2-dependent complement activity in 90 % human serum with IC ₅₀ 3 0 nM or less.

Ways.

Expression of full-length catalytically inactive MASP-2.

The full-length cDNA sequence of human MASP-2 (SEQ ID NO: 4) encoding the human MASP-2 polypeptide with the leader sequence (SEQ ID NO: 5) was subcloned into the mammalian expression vector pCI Neo (Promega), driving eukaryotic expression under the control of an enhancer/ promoter region of CMV (described in Kaufman R. J. et al., Nucleic Acids Research 19:4485 90, 1991; Kaufman, Methods in Enzymology, 185:537 66 (1991)).

To obtain catalytically inactive human MASP-2A protein, site-directed mutagenesis was performed as described in US2007/0172483, the contents of which are hereby incorporated by reference. The PCR products were purified after agarose gel electrophoresis and band generation, and single adenosine overlap was obtained using a standard tailing method. The adenosine-tailed MASP-2A was then cloned into the pGEM T easy vector and transformed into E. coli. Human MASP-2A was then subcloned into either of the mammalian expression vectors pED or pCI Neo.

The MASP-2A expression construct described above was transfected into DXB1 cells using the standard calcium phosphate transfection method (Maniatis et al., 1989). MASP-2A

- 59 045598 were produced in serum-free medium to ensure that the preparations were not contaminated with other serum proteins. Media were collected from confluent cells every second day (four times in total). The average level of recombinant MASP-2A was approximately 1.5 mg/liter of culture medium. MASP-2A (the Ser-Ala mutant described above) was purified by affinity chromatography on MBP-A agarose columns.

MASP-2A ELISA for candidate ScFv clones identified by scFv panning/conversion and filter screening.

A phage display library of human immunoglobulin light and heavy chain variable region sequences was subjected to antigen panning followed by automated screening and antibody selection to identify high-affinity scFv antibodies against human MASP-2 protein. Three cycles of panning the phage scFv library against HIS-tagged or biotin-tagged MASP-2A were performed. After the third panning cycle, elution was carried out first using MBL and then using TEA (alkali). To monitor the specific enrichment of phages exhibiting scFv fragments against the target MASP-2A, a polyclonal phage ELISA was performed against immobilized MASP-2A. The scFv genes from round 3 panning were cloned into the pHOG expression vector and screened on small scale filters to look for specific anti-MASP-2A clones.

Bacterial colonies containing plasmids encoding scFv fragments from the third round of panning were selected, plated on nitrocellulose membrane grids, and grown overnight on non-inducing medium to obtain stock plates. A total of 18,000 were selected and analyzed from the third round of panning, half after competitive elution and half after subsequent TEA elution. Panning the scFv phagemid library against MASP-2A followed by scFv conversion and filter screening identified 137 positive clones. 108/137 clones were positive in the MASP-2 binding ELISA (data not shown), of which 45 were further assayed for their ability to block MASP-2 activity in normal human serum.

Assay to measure inhibition of lectin pathway C3 convertase formation.

A functional assay that measures inhibition of lectin pathway C3 convertase formation was used to evaluate the blocking activity of candidate scFv clones against MASP2. The activity of the serine protease MASP-2 is necessary for the formation of two protein components (C4b, C2a) that the C3 convertase of the lectin pathway contains. Thus, an anti-MASP-2 scFv that inhibits the functional activity of MASP-2 (ie, a blocking MASP-2 scFv) inhibits de novo formation of the lectin pathway C3 convertase. C3 contains an atypical and highly reactive thioether group as part of the structure. When C3 is cleaved by C3 convertase in this assay, the thioester group on C3b can form a covalent bond with the hydroxyl group or amino group on the macromolecules immobilized on the bottom of the plastic wells through ester or amide bonds, thus facilitating the detection of C3b in the ELISA assay.

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

Ways.

candidate clones identified as described above were expressed, purified and diluted to the same storage concentration, which was again diluted in Ca++ and Mg~ containing GVB buffer (4.0 mM barbital, 141 mM NaCl, 1.0 mM MgCl ₂ , 2.0 mM CaCl ₂ , 0.1% gelatin, pH 7.4) to ensure that all clones have the same amount of buffer. Each scFv clone was tested in triplicate at a concentration of 2 μg/ml. The positive control was Fab2 OMS100 and was tested at 0.4 μg/ml. C3c formation was monitored in the presence and absence of scFv/IgG clones.

Mannan was diluted to a concentration of 20 μg/ml (1 μg/well) in 50 mM carbonate buffer (15 mM Na ₂ CO ₃ +35 mM NaHCO ₃ +1.5 mM NaN ₃ ), pH 9.5, and coated the ELISA plate overnight at 4°C. The next day, mannan-coated plates were washed 3 times with 200 μl PBS. Then, 100 μl of 1% HSA blocking solution was added to the wells and incubated for 1 hour at room temperature. The plates were washed 3 times with 200 μl PBS, and kept on ice with 200 μl PBS until samples were added.

Normal human serum was diluted to 0.5% in CaMgGVB buffer, and scFv clones or Fab2 OMS100 positive control were added in triplicate at 0.01 μg/ml; 1 µg/ml (OMS100 control only) and 10 µg/ml into this buffer and pre-incubated for 45 minutes on ice before adding to the blocked ELISA plate. The reaction was initiated by incubating for one hour at 37°C and stopped by transferring the plates to an ice bath. C3b accumulation was detected using a rabbit anti-mouse C3c antibody, followed by a goat anti-rabbit antibody with HRP. The negative control was buffer without antibody (no antibody=maximum accumulation of C3b), and the positive control was buffer with EDTA (no accumulation of C3b). Background was determined by performing the same assay, except that the wells did not contain mannan. The background signal for plates without mannan was subtracted from the signals in wells containing mannan. The cutoff criterion was set to half the activity of the unrelated scFv clone (VZV) and buffer alone.

Results. Based on the cutoff criterion, a total of 13 clones were found to block MASP-2 activity. All 13 clones resulting in >50% pathway suppression were selected and sequenced, yielding 10 unique clones. All ten clones were found to have the same light chain subclass, λ3, but three different heavy chain subclasses: VH2, VH3 and VH6. In functional analysis, five of the ten candidate scFv clones obtained IC ₅₀ nM values less than the target criterion of 25 nM using 0.5% human serum.

To identify antibodies with improved activity, the three original scFv clones identified as described above were subjected to light chain shuffling. This method involves generating a combinatorial library consisting of VH from each of the original clones paired with a library of naive, human lambda light chains (VL) obtained from six healthy donors. This library was then screened for scFv clones with improved binding affinity and/or functionality.

Table 9

Comparison of functional activity according to IC ₅₀ (nM) of the leading daughter clones and their corresponding parent clones (all in scFv format)

scFv clone 1% human serum analysis S3 (1C ₅₀ nM) 90% human serum analysis S3 (1C ₅₀ nM) 90% human serum analysis C 4 (1C ₅₀ nM) 17D2 Omc 38 nd nd 17D20m d3521Nll 26 >1000 140 17N16mc 68 nd nd 17N16m d!7N9 48 15 230

Below are the heavy chain variable region (VH) sequences for the parent clones and the daughter clones shown in Table 1 above. 9.

Kabat CDRs (31-35 (H1), 50-65 (H2) and 95-107 (H3)) are in bold; and Chothia CDRs (26-32 (H1), 52-56 (H2) and 95-101 (H3)) are underlined.

Heavy chain variable region (VH) 17D20_35VH-21N11VL (SEQ ID NO: 67, encoded by SEQ ID NO: 66)

OVTLKESGPVLVKPTETLTLTCTVSGFSLSRGKMGVSWIROPPGKALEWLAHIFSSDEK

SYRTSLKSRLTISKDTSKNOWL·TMTNMDPVDTATYYCARIRRGGIDYWGOGTLVTVSS

Heavy chain variable region (VH) d17N9 (SEQ ID NO: 68)

OVOLOQSGPGLVKPSOTLSLTCAISGDSVSSTSAAWNWIRQSPSRGLEWLGRTYYRSKW

YNDYAVSVKSRITINPDTSKNOFSLOLNSVTPEDTAVYYCARDPFGVPFDIWGOGTMVTVSS

Below are the variable light chain (VL) sequences for the parent clones and daughter clones.

Kabat CDRs (24-34 (L1); 50-56 (L2); and 89-97 (L3) are in bold; and Chothia CDRs (24-34 (L1); 50-56 (L2) and 89- 97 (L3) are underlined.These areas are the same regardless of numbering through the Kabat or Chothia system.

Light chain variable region (VL) 17D20m_d3521N11 (SEQ ID NO: 69)

OPVLTOPPSLSVSPGOTASITCSGEKLGDKYAYWYOOKPGOSPVLVMYQDKQRPSGIPE

RFSGSNSGNTATLTISGTOAMDEADYYCOAWDSSTAVFGGGTKLTVL

Light chain variable region (VL) 17N16m_d17N9 (SEQ ID NO: 71, encoded by SEQ ID NO: 70)

SYELIQPPSVSVAPGQTATITCAGDNLGKKRVHWYOORPGOAPVLVIYDDSDRPSGIPD

RFSASNSGNTATLTITRGEAGDEADYYCQVWDIATDHVVFGGGTKLTVLAAAGSEQKLISE

Anti-MASP-2 antibodies OMS100 and MoAb_d3521N11VL, (containing the heavy chain variable region shown as SEQ ID NO: 67 and the light chain variable region shown as SEQ ID NO: 69, also designated OMS646), both of which are shown to bind to Human MASP-2's high affinity and ability to block complement functional activity were analyzed for epitope binding by dot blot analysis. The results show that antibodies OMS646 and OMS100 are highly specific for MASP-2 and do not bind to MASP-1/3. None of the antibodies bound to any fragments

MAp19, nor with MASP-2 fragments that did not contain the CCP1 domain of MASP-2, leading to the conclusion that the binding sites include CCP1.

Anti-MASP-2 antibody OMS646 was determined to bind avidly to recombinant MASP-2 (Kd 60-250 pM) with >5000-fold selectivity over Cls, Clr or MASP-1 (see Table 10 below).

Table 10

Affinity and specificity of the OMS646 anti-MASP-2 MASP-2 antibody interaction, as assessed by ELISA studies

Antigen K _D (pm) MASP-1 >500000 MASP-2 62+23* MASP-3 >500000 Purified human Clr >500000 Purified human Cls -500000

*Mean±SD; n=12.

OMS646 specifically blocks lectin-dependent activation of terminal complement components.

Ways.

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

Results.

In fig. 17A graphically illustrates the level of MAC accumulation in the presence or absence of anti-MASP-2 antibody (OMS646) under lectin pathway-specific assay conditions. In fig. 17B graphically illustrates the level of MAC accumulation in the presence or absence of anti-MASP-2 antibody (OMS646) under classical pathway-specific assay conditions. In fig. 17C graphically illustrates the level of MAC accumulation in the presence or absence of anti-MASP-2 antibody (OMS646) under alternative pathway-specific assay conditions.

As shown in FIG. 17A, OMS646 blocks lectin pathway-mediated activation of MAC accumulation with an IC50 value of approximately 1 nM. However, OMS646 had no effect on MAC accumulation resulting from classical pathway-mediated activation (Figure 17B) or alternative pathway-mediated activation (Figure 17C).

Pharmacokinetics and pharmacodynamics of OMS6 4 6 following intravenous (IV) or subcutaneous (SC) administration in mice.

The pharmacokinetics (PK) and pharmacodynamics (PD) of OMS646 were assessed in a 28-day single-dose PK/PD study in mice. The study tested 5 mg/kg and 15 mg/kg OMS646 dosing levels administered subcutaneously (SC), as well as 5 mg/kg OMS646 dosing levels administered intravenously (IV).

In relation to the PK OMS646 profile, FIG. 18 graphically illustrates the concentration of OMS646 (average of n=3 animals/group) as a function of time after administration of OMS646 at the indicated dose. As shown in FIG. 18, at 5 mg/kg SC, OMS646 reached a maximum plasma concentration of 5-6 μg/ml approximately 1-2 days after dosing. The bioavailability of OMS646 at 5 mg/kg SC was approximately 60%. As further shown in FIG. 18, at 15 mg/kg SC, OMS646 reached a maximum plasma concentration of 10-12 μg/ml approximately 1-2 days after dosing. For all groups, OMS646 was slowly cleared from the systemic circulation with a terminal half-life of approximately 8-10 days. The OMS646 profile is typical of human antibodies in mice.

The PD activity of OMS646 is graphically illustrated in FIG. 19A and 19B. In fig. 19A and 19B show the PD response (decline in systemic lectin pathway activity) for each mouse in the 5 mg/kg IV (FIG. 19A) and 5 mg/kg SC (FIG. 19B) groups. The dotted line shows the background of the assay (maximum inhibition; serum from naïve mice supplemented in vitro with excess OMS646 prior to assay). As shown in FIG. 19A, following IV administration of 5 mg/kg OMS646, systemic lectin pathway activity immediately dropped to almost undetectable levels, and lectin pathway activity showed only a modest recovery over the 28 day observation period. As shown in FIG. 19B, time-dependent inhibition of lectin pathway activity was observed in mice after dosing with 5 mg/kg OMS646 SC. Lectin pathway activity dropped to almost undetectable levels in

- 62 045598 within 24 hours after administration of the drug and remained at low levels for at least 7 days. Lectin pathway activity gradually increased over time but did not return to pre-dosing levels over the 28-day observation period. The observed lectin pathway activity over time profile following administration of 15 mg/kg SC was similar to that observed for the 5 mg/kg SC dose (data not shown), indicating saturation of the PD endpoint. These data further demonstrate that weekly doses of 5 mg/kg OMS646 administered either IV or SC are sufficient to achieve sustained suppression of systemic lectin pathway activity in mice.

Example 16.

This example describes an analysis of the effectiveness of an anti-MASP-2 monoclonal antibody (OMS646), a human IgG4 antibody that blocks lectin pathway function, in a mouse model of age-related macular degeneration.

State of the Art/Rationale.

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

Ways.

This study assessed the effect of three dosage levels of OMS646 (2 mg/kg; 5 mg/kg and 20 mg/kg SC) compared to vehicle treatment. Anti-mouse MASP-2 mAb derived from Fab2 #11 (3 mg/kg SC), prepared as described in Example 14, and a rat monoclonal antibody that binds to mouse VEGF-A and blocks VEGF-A function (5 mg/kg kg IP, clone 2G11-2A05, purchased from BioLegend®, San Diego, CA) were included as a positive control treatment and a comparative treatment, respectively. The study included 9-10 mice per experimental group and was conducted in a blinded manner. To evaluate efficacy at constant and predictable drug levels, all treatments were administered eight days before and then again one day before laser induction, with the exception of anti-VEGF antibody, which was injected one day before and three days after laser induction. Seven days after laser injury, mice were anesthetized, systemically perfused with 0.75 ml FITC-dextran, and sacrificed. Eyes were fixed in formaldehyde, and sections from the back of the eyes containing the affected areas were obtained and immersed in ProLong anti-fluorescence fading reagent (Invitrogen). Confocal microscopy of the damaged areas was performed and images were obtained from each area. CNV and lesion measurements were performed using ImageJ software (National Institutes of Health, Bethesda, Maryland USA). CNV area was normalized to lesion size for each eye, where %CNV is the average area of neovascularization per lesion calculated as (CNV area/spot area) X 100. The study was performed in a blinded manner using coded test solutions.

Results: The outcome of this study is shown in FIG. 20. As shown in FIG. 20, compared with the vehicle-treated group, OMS646-treated mice showed measurable inhibition of CNV at all dosage levels tested, with relative reductions in CNV ranging from 29% to 50%. Treatment with anti-VEGF antibody showed a smaller (approximately 15%) reduction in CNV. Anti-mouse MASP-2 mAb derived from Fab2 #11 also reduced CNV by approximately 30% compared to vehicle treatment (data not shown), which is consistent with the results observed in a study conducted in MASP-2 (-/-) mice ) described in Example 12, in which a 30% reduction in CNV was observed 7 days after laser treatment in MASP-2 (-/-) mice compared to control wild-type mice.

The results of this study provide evidence that systemic administration of OMS646 provides an effective therapy for the treatment of neovascular AMD. Unlike current and emerging drugs for AMD and other ophthalmic angiogenic diseases and disorders that require intravitreal injection, OMS646 is also effective when administered subcutaneously.

It may be additionally noted that the anti-VEGF-A antibody used in this study (clone 2G11-2A05 from BioLegend®, San Diego, CA) has previously been shown to reduce vascular proliferation into the cornea in a mouse model of HSV-1-induced corneal lymphangiogenesis in administration by subconjunctival injection at a concentration of 100 μg/ml, as described in Wuest et al. (J Exp Med 207:101, 2009). In another study, Lu et al. (Cancer Res 72:2239-50, 2012), treatment with anti-VEGF antibody (clone 2G11-2A05) of Ceacam 1 -/- mice bearing B16 tumors significantly reduced tumor size, as well as tumor vasculature in a colon tumor model with administration of IP at approximately 3 mg/kg twice weekly. Based on the data from this study, impressions

- 63 045598 finding that OMS646 is at least as effective as an anti-VEGF antibody in reducing CNV when systemically delivered to mice at all dosage levels tested, expects that a MASP-2 inhibitory agent such as OMS646 may also be effective as an antiangiogenic agent for use in inhibiting an angiogenesis-dependent cancer, such as an angiogenesis-dependent cancer selected from the group consisting of solid tumor(s), blood-borne tumors, high-risk carcinoid tumors, and metastatic tumors. Examples of angiogenesis-dependent malignancies are cancer types for which treatment with an anti-VEGF agent, such as the anti-VEGF antibody Avastin® (bevacizumab, Genentech, CA), is approved. For example, bevacizumab is approved for the treatment of the following angiogenesis-dependent malignancies: metastatic colorectal cancer, non-squamous non-small cell lung cancer, metastatic renal cell carcinoma, and glioblastoma.

Additional examples of angiogenesis-dependent cancers include those types of cancers that are expected to benefit from treatment with an anti-VEGF agent, such as the anti-VEGF antibody Avastin® (bevacizumab, Genentech, CA), for example, such as any cancer that is already treatment known or in development using an angiostatic compound (e.g., VEGF antagonist), including advanced malignancies metastasizing to the liver, melanoma, ovarian cancer, neuroblastoma, pancreatic cancer, hepatocellular carcinoma, endometrial cancer, prostate cancer, angiosarcoma, metastatic or inoperable angiosarcoma, recurrent ovarian sex cord stromal tumors, esophageal cancer, gastric cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, diffuse large B-cell lymphoma, recurrent or metastatic head and neck cancer, neoplastic meningitis, cervical cancer, body cancer uterus, peritoneal carcinomatosis at the Chosha stages, gliosarcoma, neuroendocrine carcinum, Extracranial sunga saginal, acute myeloid leukemia, chronic myelogenic leukemia, intracranial meningioma, capes' sarcoma, mesotheloma, malignant tumor of the bile, metallic circuits, meta -stand -up Cars butter tumors and a malignant tumor of the urinary tract on later stages. Preferred malignancies in this context include: colorectal cancers, breast cancers (including metastatic breast cancer, inflammatory breast carcinoma), lung, kidney, liver, esophagus, ovary, pancreas, prostate and stomach, as well as glioma, gastrointestinal stromal tumors, lymphoma, melanoma and carcinoid tumors.

It is also expected that a MASP-2 inhibitory agent, such as OMS646, may be effective as an antiangiogenic agent for inhibiting an angiogenesis-dependent benign tumor, such as an angiogenesis-dependent benign tumor selected from the group consisting of hemangiomas, acoustic neuromas nerve, neurofibromas, trachomas, carcinoid tumors and pyogenic granulomas. It is also expected that a MASP-2 inhibitory agent such as OMS646 may be effective as an antiangiogenic agent for use in inhibiting angiogenesis in AMD and other ophthalmic angiogenic diseases or disorders such as uveitis, ocular melanoma, corneal neovascularization, primary pterygium, stromal HSV keratitis, HSV-1 induced corneal lymphangiogenesis, proliferative diabetic retinopathy, diabetic macular edema, retinopathy of prematurity, retinal vein occlusion, corneal graft rejection, neovascular glaucoma, vitreous hemorrhage secondary to proliferative diabetic retinopathy, optic neuromyelitis and redness irises.

Given the data from the present study showing that OMS646 is at least as effective as an anti-VEGF antibody in reducing CNV when systemically delivered to mice at all dosage levels tested, it is also expected that a MASP-2 inhibitory agent such as OMS646 may also be effective as an antiangiogenic agent for use in inhibiting angiogenesis-dependent conditions such as myelofibrosis and hereditary hemorrhagic telangiectasia.

Example 17.

This example describes the use of a MASP-2 (-/-) line and MASP-2 inhibitory antibodies to confirm that inhibition of the MASP-2-dependent lectin pathway of complement activation induces an antiangiogenic effect in an animal model of femoral artery ligation.

BACKGROUND/BACKGROUND: In view of the unexpected results described in Example 16 showing that the human anti-MASP-2 mAb OMS646 inhibits CNV in an AMD model to at least equal, if not greater, extent than the anti-VEGF-A antibody, the following Studies were conducted to confirm that angiogenesis is reduced in MASP-2-deficient mice and that an anti-MASP-2 antibody that blocks the lectin pathway, such as OMS646, is effective for use in vivo as an angiogenesis inhibitory agent. when administered systemically.

Ways.

Study #1. Arteriogenesis was induced in MASP-2 (-/-) mice, wild-type control mice, and wild-type mice pretreated with an inhibitory anti-MASP-2 antibody by femoral artery ligation, and laser Doppler perfusion measurements were performed in vivo to observe whether Does MASP-2 deficiency affect the process of collateral artery growth? Perfusion measurements were performed until day 21 after femoral artery ligation.

Immunohistochemistry was performed on day 3 after ligation of the femoral artery.

(a) In the upper limb, where arteriogenesis occurs, to study the effect of MASP-2 deficiency on perivascular leukocyte infiltration (arteriogenesis is highly dependent on leukocyte infiltration, taking into account that leukocytes provide growing collateral vessels with growth factors, cytokines); and (b) In the lower extremity, where ischemia occurs due to femoral artery ligation, to examine the severity of ischemic tissue damage, leukocyte infiltration, and angiogenesis in MASP-2 (-/-) mice, in wild-type mice treated with antibody against MASP-2 and wild-type control mice.

(c) Gene expression studies at RNA and protein levels were also performed on isolated collateral vessels 12 h or 24 h after femoral artery ligation in MASP-2 (-/-) mice. In wild-type mice treated with anti-MASP-2 antibody and in control wild-type mice.

Based on the antiangiogenic effect described above, it is expected that inhibition of MASP-2 may prevent or reduce arteriogenesis in the upper limb by 25-50%. In addition, MASP-2 inhibition has been shown to reduce the complement-driven pathological response after ischemia by 25%-50% (Schwaeble et al., PNAS 108(18):7523-7528). Thus, it would also be expected that inhibition of MASP-2 could inhibit vasculogenesis in the lower limb to a similar extent.

While exemplary embodiments have been illustrated and described, it should be understood that various changes can be made without departing from the scope and scope of the invention.

Sequence List <110> Omeros Corporation

University of Leicester

Demopulos, Gregory A.

Schwaeble, Hans-Wilhelm

Dudler, Thomas Tjoelker, Larry W.

<120> Methods of inhibiting angiogenesis in a subject in need thereof <130> MP.1.0239.PCT <150> US 62/315,857 <151> 2016-03-31 <160>71 <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 ctt53

Met Arg Leu Leu Thr Leu Leu Gly Leu 15 ctg tgt ggc tcg gtg gcc acc ccc ttg ggc ccg aag tgg cct gaa cct101

- 65 045598

Leu 10 Cys Gly Ser Val Ala 15 Thr Pro Leu Gly Pro 20 Lys Trp Pro Glu Pro 25 gtg ttc ggg cgc ctg gca tcc ccc ggc ttt cca ggg gag tat gcc aat Val Phe Gly Arg Leu 30 Ala Ser Pro Gly Phe 35 Pro Gly Glu Tyr Ala 40 Asn gac cag gag cgg cgc tgg acc ctg act gca ccc ccc ggc tac cgc ctg Asp Gln Glu Arg 45 Arg Trp Thr Leu Thr 50 Ala Pro Pro Gly Tyr 55 Arg Leu cgc ctc tac ttc acc cac ttc gac ctg gag ctc tcc cac ctc tgc gag Arg Leu Tyr 60 Phe Thr His Phe Asp 65 Leu Glu Leu Ser His 70 Leu Cys Glu tac gac ttc gtc aag ctg agc tcg ggg gcc aag gtg ctg gcc acg ctg Tyr Asp 75 Phe Val Lys Leu Ser 80 Ser Gly Ala Lys Val 85 Leu Ala Thr Leu tgc ggg cag gag agc aca gac acg gag cgg gcc cct ggc aag gac act Cys 90 Gly Gln Glu Ser Thr 95 Asp Thr Glu Arg Ala 100 Pro Gly Lys Asp Thr 105 ttc tac tcg ctg ggc tcc agc ctg gac att acc ttc cgc tcc gac tac Phe Tyr Ser Leu Gly 110 Ser Ser Leu Asp Ile 115 Thr Phe Arg Ser Asp 120 Tyr tcc aac gag aag ccg ttc acg ggg ttc gag gcc ttc tat gca gcc gag Ser Asn Glu Lys 125 Pro Phe Thr Gly Phe 130 Glu Ala Phe Tyr Ala 135 Ala Glu gac att gac gag tgc cag gtg gcc ccg gga gag gcg ccc acc tgc gac Asp Ile Asp 140 Glu Cys Gln Val Ala 145 Pro Gly Glu Ala Pro 150 Thr Cys Asp cac cac tgc cac aac cac ctg ggc ggt ttc tac tgc tcc tgc cgc gca His His 155 Cys His Asn His Leu 160 Gly Gly Phe Tyr Cys 165 Ser Cys Arg Ala ggc tac gtc ctg cac cgt aac aag cgc acc tgc tca gag cag agc ctc Gly 170 Tyr Val Leu His Arg 175 Asn Lys Arg Thr Cys 180 Ser Glu Gln Ser Leu 185

tag cctcccctgg agctccggcc tgcccagcag gtcagaagcc agagccagcc tgctggcctc agctccgggt tgggctgaga tggctgtgcc ccaactccca ttcacccacc atggacccaa taataaacct ggccccaccc c

149

197

245

293

341

389

437

485

533

581

634

694

725 <210>2 <211>185 <212> PRT <213> Homo sapiens <400>2

Met Arg Leu Leu Thr Leu Leu Gly Leu 1 5

Leu Cys Gly Ser Val Ala 10 15

Thr

Pro Leu Gly Pro Lys Trp Pro Glu Pro 20 25

Val Phe Gly Arg Leu Ala

Ser

Pro Gly Phe Pro Gly Glu 35

Tyr Ala

Asn Asp Gln Glu Arg Arg Trp Thr 45

Leu Thr Ala Pro Pro Gly Tyr Arg Leu Arg Leu Tyr

Phe Thr His Phe

- 66 045598

55

Asp Leu Glu Leu Ser His Leu Cys Glu Tyr 65 70

Ser Gly Ala Lys Val Leu Ala Thr Leu Cys

90

Thr Glu Arg Ala Pro Gly Lys Asp Thr Phe

100 105

Leu Asp Ile Thr Phe Arg Ser Asp Tyr Ser

115 120

Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp

130 135

Ala Pro Gly Glu Ala Pro Thr Cys Asp His 145 150

Gly Gly Phe Tyr Cys Ser Cys Arg Ala Gly

165 170

Lys Arg Thr Cys Ser Glu Gln Ser Leu

180 185 <210>3 <211>170 <212> PRT <213> Homo sapiens <400>3

Thr Pro Leu Gly Pro Lys Trp Pro Glu Pro

510

Ser Pro Gly Phe Pro Gly Glu Tyr Ala Asn

25

Thr Leu Thr Ala Pro Pro Gly Tyr Arg Leu

40

Phe Asp Leu Glu Leu Ser His Leu Cys Glu

55

Ser Ser Gly Ala Lys Val Leu Ala Thr Leu 65 70

Asp Thr Glu Arg Ala Pro Gly Lys Asp Thr

90

Ser Leu Asp Ile Thr Phe Arg Ser Asp Tyr

100 105

Phe Val Lys Leu Ser 80

Gln Glu Ser Thr Asp 95

Ser Leu Gly Ser Ser 110

Glu Lys Pro Phe Thr 125

Asp Glu Cys Gln Val 140

Cys His Asn His Leu

160

Val Leu His Arg Asn

175

Phe Gly Arg Leu Ala 15

Gln Glu Arg Arg Trp 30

Leu Tyr Phe Thr His 45

Asp Phe Val Lys Leu 60

Gly Gln Glu Ser Thr

Tyr Ser Leu Gly Ser

Asn Glu Lys Pro Phe 110

- 67 045598 <210> 4 <211> 2460 <212> DNA <213> Homo sapiens <220>

<221>

<222>

CDS (22) .

(2082) <400> 4 ggccagctgg acgggcacac c tgt ggc tcg gtg gcc acc Cys Gly Ser Val Ala Thr 15 ttc ggg cgc ctg gca tcc Phe Gly Arg Leu Ala Ser 30 cag gag cgg cgc tgg acc Gln Glu Arg Arg Trp Thr 45 ctc tac ttc acc cac ttc Leu Tyr Phe Thr His Phe 60 gac ttc gtc aag ctg agc Asp Phe Val Lys Leu Ser 75 80 ggg cag gag agc aca gac Gly Gln Glu Ser Thr Asp 95 tac tcg ctg ggc tcc agc Tyr Ser Leu Gly Ser Ser 110 aac gag aag ccg ttc acg Asn Glu Lys Pro Phe Thr 125 att gac gag tgc cag gtg Ile Asp Glu Cys Gln Val 140 cac tgc cac aac cac ctg His Cys His Asn His Leu 155 160

Thr Gly Phe Glu Ala Phe

115

Val Ala Pro Gly Glu Ala

130

Leu Gly Gly Phe Tyr Cys

145 150

Asn Lys Arg Thr Cys Ser

165

Tyr Ala 120 Ala Glu Asp Ile Asp 125 Glu Cys Gln Pro 135 Thr Cys Asp His His 140 Cys His Asn His Ser Cys Arg Ala Gly 155 Tyr Val Leu His Arg 160 Glu Gln Ser Leu 170

atg agg ctg ctg acc Met Arg Leu Leu Thr 1 5 ccc ttg ggc ccg aag Pro Leu Gly Pro Lys 20 ccc ggc ttt cca ggg Pro Gly Phe Pro Gly 35 ctg act gca ccc ccc Leu Thr Ala Pro Pro 50 gac ctg gag ctc tcc Asp Leu Glu Leu Ser 65 tcg ggg gcc aag gtg Ser Gly Ala Lys Val 85 acg gag cgg gcc cct Thr Glu Arg Ala Pro 100 ctg gac att acc ttc Leu Asp Ile Thr Phe 115 ggg ttc gag gcc ttc

Gly Phe Glu Ala Phe 130 gcc ccg gga gag gcg Ala Pro Gly Glu Ala 145 ggc ggt ttc tac tgc Gly Gly Phe Tyr Cys 165 ctc ctg ggc ctt ctg Leu Leu Gly Leu Leu 10 tgg cct gaa cct gtg Trp Pro Glu Pro Val 25 gag tat gcc aat gac Glu Tyr Ala Asn Asp 40 ggc tac cgc ctg cgc Gly Tyr Arg Leu Arg 55 cac ctc tgc gag tac His Leu Cys Glu Tyr 70 ctg gcc acg ctg tgc Leu Ala Thr Leu Cys 90 ggc aag gac act ttc Gly Lys Asp Thr Phe 105 cgc tcc gac tac tcc Arg Ser Asp Tyr Ser 120 tat gca gcc gag gac Tyr Ala Ala Glu Asp 135 ccc acc tgc gac cac Pro Thr Cys Asp His 150 tcc tgc cgc gca ggc Ser Cys Arg Ala Gly 170

147

195

243

291

339

387

435

483

531

- 68 045598 tac gtc ctg Tyr Val Leu cag gtc ttc

Gln Val Phe cgg ccg tat Arg Pro Tyr 205 cac cgt aac aag His Arg Asn Lys 175 acc cag agg tct Thr Gln Arg Ser 190 ccc aaa ctc tcc Pro Lys Leu Ser cgc acc tgc tca gcc Arg Thr Cys Ser Ala 180 ggg gag ctc agc agc Gly Glu Leu Ser Ser 195 agt tgc act tac agc Ser Cys Thr Tyr Ser 210 ctg tgc

Leu Cys cct gaa Pro Glu 200 atc agc Ile Ser 215 tcc ggc Ser Gly 185 tac cca Tyr Pro ctg gag

Leu Glu gag ggg ttc agt gtc att ctg gac ttt gtg gag tcc ttc gat gtg gag

Glu Gly Phe Ser Val Ile Leu Asp Phe Val Glu Ser Phe Asp Val Glu

220 225230 aca cac cct gaa acc ctg tgt ccc tac gac ttt ctc aag att caaaca

Thr His Pro Glu Thr Leu Cys Pro Tyr Asp Phe Leu Lys Ile GlnThr

235 240 245250 gac aga gaa gaa cat ggc cca ttc tgt ggg aag aca ttg ccc cacagg

Asp Arg Glu Glu His Gly Pro Phe Cys Gly Lys Thr Leu Pro HisArg

255 260265 att gaa aca aaa agc aac acg gtg acc atc acc ttt gtc aca gatgaa

Ile Glu Thr Lys Ser Asn Thr Val Thr Ile Thr Phe Val Thr AspGlu

270 275280 tca gga gac cac aca ggc tgg aag atc cac tac acg agc aca gcgcag

Ser Gly Asp His Thr Gly Trp Lys Ile His Tyr Thr Ser Thr AlaGln

285 290295 cct tgc cct tat ccg atg gcg cca cct aat ggc cac gtt tca cctgtg

Pro Cys Pro Tyr Pro Met Ala Pro Pro Asn Gly His Val Ser ProVal

300 305310 caa gcc aaa tac atc ctg aaa gac agc ttc tcc atc ttt tgc gagact

Gln Ala Lys Tyr Ile Leu Lys Asp Ser Phe Ser Ile Phe Cys GluThr

315 320 325330 ggc tat gag ctt ctg caa ggt cac ttg ccc ctg aaa tcc ttt actgca

Gly Tyr Glu Leu Leu Gln Gly His Leu Pro Leu Lys Ser Phe ThrAla

335 340345 gtt tgt cag aaa gat gga tct tgg gac cgg cca atg ccc gcg tgcagc

Val Cys Gln Lys Asp Gly Ser Trp Asp Arg Pro Met Pro Ala CysSer

350 355360 att gtt gac tgt ggc cct cct gat gat cta ccc agt ggc cga gtggag

Ile Val Asp Cys Gly Pro Pro Asp Asp Leu Pro Ser Gly Arg ValGlu

365 370375 tac atc aca ggt cct gga gtg acc acc tac aaa gct gtg att cagtac

Tyr Ile Thr Gly Pro Gly Val Thr Thr Tyr Lys Ala Val Ile GlnTyr

380 385390 agc tgt gaa gag acc ttc tac aca atg aaa gtg aat gat ggt aaatat

Ser Cys Glu Glu Thr Phe Tyr Thr Met Lys Val Asn Asp Gly LysTyr

395 400 405410 gtg tgt gag gct gat gga ttc tgg acg agc tcc aaa gga gaa aaatca

Val Cys Glu Ala Asp Gly Phe Trp Thr Ser Ser Lys Gly Glu LysSer

415 420425 ctc cca gtc tgt gag cct gtt tgt gga cta tca gcc cgc aca acagga

Leu Pro Val Cys Glu Pro Val Cys Gly Leu Ser Ala Arg Thr ThrGly

430 435440 ggg cgt ata tat gga ggg caa aag gca aaa cct ggt gat ttt cct tgg

579

627

675

723

771

819

867

915

963

1011

1059

1107

1155

1203

1251

1299

1347

1395

- 69 045598

Gly Arg Ile Tyr Gly Gly Gln Lys Ala Lys Pro Gly Asp Phe Pro Trp 445 450455 caa gtc ctg ata tta ggt gga acc aca gca gca ggt gca ctt tta tat Gln Val Leu Ile Leu Gly Gly Thr Thr Ala Ala Gly Ala Leu Leu Tyr 460 465470 gac aac tgg gtc cta aca gct gct cat gcc gtc tat gag caa aaacat

Asp Asn Trp Val Leu Thr Ala Ala His Ala Val Tyr Glu Gln LysHis

475 480 485490 gat gca tcc gcc ctg gac att cga atg ggc acc ctg aaa aga ctatca

Asp Ala Ser Ala Leu Asp Ile Arg Met Gly Thr Leu Lys Arg LeuSer

495 500505 cct cat tat aca caa gcc tgg tct gaa gct gtt ttt ata cat gaaggt

Pro His Tyr Thr Gln Ala Trp Ser Glu Ala Val Phe Ile His GluGly

510 515520 tat act cat gat gct ggc ttt gac aat gac ata gca ctg att aaattg

Tyr Thr His Asp Ala Gly Phe Asp Asn Asp Ile Ala Leu Ile LysLeu

525 530535 aat aac aaa gtt gta atc aat agc aac atc acg cct att tgt ctgcca

Asn Asn Lys Val Val Ile Asn Ser Asn Ile Thr Pro Ile Cys LeuPro

540 545550 aga aaa gaa gct gaa tcc ttt atg agg aca gat gac att gga actgca

Arg Lys Glu Ala Glu Ser Phe Met Arg Thr Asp Asp Ile Gly ThrAla

555 560 565570 tct gga tgg gga tta acc caa agg ggt ttt ctt gct aga aat ctaatg

Ser Gly Trp Gly Leu Thr Gln Arg Gly Phe Leu Ala Arg Asn LeuMet

575 580585 tat gtc gac ata ccg att gtt gac cat caa aaa tgt act gct gcatat

Tyr Val Asp Ile Pro Ile Val Asp His Gln Lys Cys Thr Ala AlaTyr

590 595600 gaa aag cca ccc tat cca agg gga agt gta act gct aac atg ctttgt

Glu Lys Pro Pro Tyr Pro Arg Gly Ser Val Thr Ala Asn Met LeuCys

605 610615 gct ggc tta gaa agt ggg ggc aag gac agc tgc aga ggt gac agcgga

Ala Gly Leu Glu Ser Gly Gly Lys Asp Ser Cys Arg Gly Asp SerGly

620 625630 ggg gca ctg gtg ttt cta gat agt gaa aca gag agg tgg ttt gtggga

Gly Ala Leu Val Phe Leu Asp Ser Glu Thr Glu Arg Trp Phe ValGly

635 640 645650 gga ata gtg tcc tgg ggt tcc atg aat tgt ggg gaa gca ggt cagtat

Gly Ile Val Ser Trp Gly Ser Met Asn Cys Gly Glu Ala Gly GlnTyr

655 660665 gga gtc tac aca aaa gtt att aac tat att ccc tgg atc gag aacata

Gly Val Tyr Thr Lys Val Ile Asn Tyr Ile Pro Trp Ile Glu AsnIle

670 675680 att agt gat ttt taa cttgcgtgtc tgcagtcaag gattcttcat ttttagaaat Ile Ser Asp Phe 685 gcctgtgaag accttggcag cgacgtggct cgagaagcat tcatcattac tgtggacatg gcagttgttg ctccacccaa aaaaacagac tccaggtgag gctgctgtca tttctccact tgccagttta attccagcct tacccattga ctcaagggga cataaaccac gagagtgaca gtcatctttg cccacccagt gtaatgtcac tgctcaaatt acatttcatt accttaaaaa

1443

1491

1539

1587

1635

1683

1731

1779

1827

1875

1923

1971

2019

2067

2122

2182

2242

2302

2362

- 70 045598 gccagtctct tttttaaact tttcatactg tgttcttatt gctgttggca gaaaaaaaaa tttctgtaaa aaaaaaaa ctgcctgtcc atgctctttg

2422

2460 <210>5 <211>686 <212> PRT <213> Homo sapiens <400>5

Met

Arg

Leu

Leu

Thr 5

Leu

Leu

Gly

Leu

Leu 10

Cys

Gly

Ser

Val

Ala 15

Thr

Pro

Leu

Gly

Pro 20

Lys

Trp

Pro

Glu

Pro 25

Val

Phe

Gly

Arg

Leu 30

Ala

Ser

Pro

Gly

Phe 35

Pro

Gly

Glu

Tyr

Ala 40

Asn

Asp

Gln

Glu

Arg 45

Arg

Trp

Thr

Leu

Thr 50

Ala

Pro

Pro

Gly

Tyr 55

Arg

Leu

Arg

Leu

Tyr 60

Phe

Thr

His

Phe

Asp 65

Leu

Glu

Leu

Ser

His 70

Leu

Cys

Glu

Tyr

Asp 75

Phe

Val

Lys

Leu

Ser 80

Ser

Gly

Ala

Lys

Val 85

Leu

Ala

Thr

Leu

Cys 90

Gly

Gln

Glu

Ser

Thr 95

Asp

Thr

Glu

Arg

Ala

100

Pro

Gly

Lys

Asp

Thr

105

Phe

Tyr

Ser

Leu

Gly

110

Ser

Ser

Leu

Asp

Ile

115

Thr

Phe

Arg

Ser

Asp 120

Tyr

Ser

Asn

Glu

Lys

125

Pro

Phe

Thr

Gly

Phe

130

Glu

Ala

Phe

Tyr

Ala

135

Ala

Glu

Asp

Ile

Asp 140

Glu

Cys

Gln

Val

Ala

145

Pro

Gly

Glu

Ala

Pro

150

Thr

Cys

Asp

His

His

155

Cys

His

Asn

His

Leu

160

Gly

Gly

Phe

Tyr

Cys

165

Ser

Cys

Arg

Ala

Gly 170

Tyr

Val

Leu

His

Arg 175

Asn

Lys

Arg

Thr

Cys

180

Ser

Ala

Leu

Cys

Ser

185

Gly

Gln

Val

Phe

Thr

190

Gln

Arg

Ser

Gly

Glu

195

Leu

Ser

Ser

Pro

Glu

200

Tyr

Pro

Arg

Pro

Tyr 205

Pro

Lys

Leu

Ser

Ser

210

Cys

Thr

Tyr

Ser

Ile

215

Ser

Leu

Glu

Glu

Gly 220

Phe

Ser

Val

Ile

Leu

Asp

Phe

Val

Glu

Ser

Phe

Asp

Val

Glu

Thr

His

Pro

Glu

Thr

Leu

- 71 045598

240

225

230

235

Cys

Pro

Tyr

Asp

Phe

245

Leu

Lys

Ile

Gln

Thr

250

Asp

Arg

Glu

Glu

His

255

Gly

Pro

Phe

Cys

Gly

260

Lys

Thr

Leu

Pro

His

265

Arg

Ile

Glu

Thr

Lys

270

Ser

Asn

Thr

Val

Thr

275

Ile

Thr

Phe

Val

Thr

280

Asp

Glu

Ser

Gly

Asp 285

His

Thr

Gly

Trp

Lys 290

Ile

His

Tyr

Thr

Ser

295

Thr

Ala

Gln

Pro

Cys 300

Pro

Tyr

Pro

Met

Ala

305

Pro

Pro

Asn

Gly

His

310

Val

Ser

Pro

Val

Gln

315

Ala

Lys

Tyr

Ile

Leu

320

Lys

Asp

Ser

Phe

Ser

325

Ile

Phe

Cys

Glu

Thr

330

Gly

Tyr

Glu

Leu

Leu

335

Gln

Gly

His

Leu

Pro

340

Leu

Lys

Ser

Phe

Thr

345

Ala

Val

Cys

Gln

Lys 350

Asp

Gly

Ser

Trp

Asp 355

Arg

Pro

Met

Pro

Ala

360

Cys

Ser

Ile

Val

Asp 365

Cys

Gly

Pro

Pro

Asp 370

Asp

Leu

Pro

Ser

Gly 375

Arg

Val

Glu

Tyr

Ile

380

Thr

Gly

Pro

Gly

Val

385

Thr

Thr

Tyr

Lys

Ala

390

Val

Ile

Gln

Tyr

Ser

395

Cys

Glu

Glu

Thr

Phe

400

Tyr

Thr

Met

Lys

Val

405

Asn

Asp

Gly

Lys

Tyr 410

Val

Cys

Glu

Ala

Asp

415

Gly

Phe

Trp

Thr

Ser

420

Ser

Lys

Gly

Glu

Lys

425

Ser

Leu

Pro

Val

Cys 430

Glu

Pro

Val

Cys

Gly

435

Leu

Ser

Ala

Arg

Thr

440

Thr

Gly

Gly

Arg

Ile

445

Tyr

Gly

Gly

Gln

Lys

450

Ala

Lys

Pro

Gly

Asp

455

Phe

Pro

Trp

Gln

Val

460

Leu

Ile

Leu

Gly

Gly 465

Thr

Thr

Ala

Ala

Gly 470

Ala

Leu

Leu

Tyr

Asp

475

Asn

Trp

Val

Leu

Thr

480

Ala

Ala

His

Ala

Val

485

Tyr

Glu

Gln

Lys

His

490

Asp

Ala

Ser

Ala

Leu

495

Asp

Ile

Arg

Met

Gly

500

Thr

Leu

Lys

Arg

Leu

505

Ser

Pro

His

Tyr

Thr

510

Gln

Ala

- 72 045598

Trp Ser Glu Ala Val Phe Ile His Glu Gly

515 520

Phe Asp Asn Asp Ile Ala Leu Ile Lys Leu

530 535

Asn Ser Asn Ile Thr Pro Ile Cys Leu Pro 545 550

Phe Met Arg Thr Asp Asp Ile Gly Thr Ala

565 570

Gln Arg Gly Phe Leu Ala Arg Asn Leu Met

580 585

Val Asp His Gln Lys Cys Thr Ala Ala Tyr

595 600

Arg Gly Ser Val Thr Ala Asn Met Leu Cys

610 615

Gly Lys Asp Ser Cys Arg Gly Asp Ser Gly 625 630

Asp Ser Glu Thr Glu Arg Trp Phe Val Gly

645 650

Ser Met Asn Cys Gly Glu Ala Gly Gln Tyr

660 665

Ile Asn Tyr Ile Pro Trp Ile Glu Asn Ile

675 680 <210>6 <211>671 <212> PRT <213> Homo sapiens <400>6

Thr Pro Leu Gly Pro Lys Trp Pro Glu Pro

510

Ser Pro Gly Phe Pro Gly Glu Tyr Ala Asn

25

Thr Leu Thr Ala Pro Pro Gly Tyr Arg Leu

40

Phe Asp Leu Glu Leu Ser His Leu Cys Glu

55

Ser Ser Gly Ala Lys Val Leu Ala Thr Leu 65 70

Thr His Asp Ala Gly

525

Asn Lys Val Val Ile 540

Lys Glu Ala Glu Ser

560

Gly Trp Gly Leu Thr

575

Val Asp Ile Pro Ile 590

Lys Pro Pro Tyr Pro 605

Gly Leu Glu Ser Gly 620

Ala Leu Val Phe Leu

640

Ile Val Ser Trp Gly

655

Val Tyr Thr Lys Val

670

Ser Asp Phe

685

Phe Gly Arg Leu Ala 15

Gln Glu Arg Arg Trp 30

Leu Tyr Phe Thr His 45

Asp Phe Val Lys Leu 60

Gly Gln Glu Ser Thr

- 73 045598

Asp Thr Glu Arg Ala Pro Gly Lys Asp Thr 85 90

Ser Leu Asp Ile Thr Phe Arg Ser Asp Tyr

100 105

Thr Gly Phe Glu Ala Phe Tyr Ala Ala Glu

115 120

Val Ala Pro Gly Glu Ala Pro Thr Cys Asp

130 135

Leu Gly Gly Phe Tyr Cys Ser Cys Arg Ala

145 150

Asn Lys Arg Thr Cys Ser Ala Leu Cys Ser

165 170

Arg Ser Gly Glu Leu Ser Ser Pro Glu Tyr

180 185

Leu Ser Ser Cys Thr Tyr Ser Ile Ser Leu

195 200

Ile Leu Asp Phe Val Glu Ser Phe Asp Val

210 215

Leu Cys Pro Tyr Asp Phe Leu Lys Ile Gln

225 230

Gly Pro Phe Cys Gly Lys Thr Leu Pro His

245 250

Asn Thr Val Thr Ile Thr Phe Val Thr Asp

260 265

Gly Trp Lys Ile His Tyr Thr Ser Thr Ala

275 280

Met Ala Pro Pro Asn Gly His Val Ser Pro

290 295

Leu Lys Asp Ser Phe Ser Ile Phe Cys Glu

305 310

Gln Gly His Leu Pro Leu Lys Ser Phe Thr

325 330

Gly Ser Trp Asp Arg Pro Met Pro Ala Cys

340 345

Tyr Ser Leu Gly Ser 95

Asn Glu Lys Pro Phe 110

Ile Asp Glu Cys Gln

125

His Cys His Asn His 140

Tyr Val Leu His Arg

160

Gln Val Phe Thr Gln

175

Arg Pro Tyr Pro Lys 190

Glu Gly Phe Ser Val

205

Thr His Pro Glu Thr 220

Asp Arg Glu Glu His

240

Ile Glu Thr Lys Ser

255

Ser Gly Asp His Thr 270

Pro Cys Pro Tyr Pro

285

Gln Ala Lys Tyr Ile 300

Gly Tyr Glu Leu Leu

320

Val Cys Gln Lys Asp

335

Ile Val Asp Cys Gly 350

- 74 045598

Pro Pro Asp Asp Leu Pro Ser Gly Arg Val

355 360

Gly Val Thr Thr Tyr Lys Ala Val Ile Gln

370 375

Phe Tyr Thr Met Lys Val Asn Asp Gly Lys

385 390

Gly Phe Trp Thr Ser Ser Lys Gly Glu Lys

405 410

Pro Val Cys Gly Leu Ser Ala Arg Thr Thr

420 425

Gly Gln Lys Ala Lys Pro Gly Asp Phe Pro

435 440

Gly Gly Thr Thr Ala Ala Gly Ala Leu Leu

450 455

Thr Ala Ala His Ala Val Tyr Glu Gln Lys

465 470

Asp Ile Arg Met Gly Thr Leu Lys Arg Leu

485 490

Ala Trp Ser Glu Ala Val Phe Ile His Glu

500 505

Gly Phe Asp Asn Asp Ile Ala Leu Ile Lys

515 520

Ile Asn Ser Asn Ile Thr Pro Ile Cys Leu

530 535

Ser Phe Met Arg Thr Asp Asp Ile Gly Thr

545 550

Thr Gln Arg Gly Phe Leu Ala Arg Asn Leu

565 570

Ile Val Asp His Gln Lys Cys Thr Ala Ala

580 585

Pro Arg Gly Ser Val Thr Ala Asn Met Leu

595 600

Gly Gly Lys Asp Ser Cys Arg Gly Asp Ser

610 615

Leu Asp Ser Glu Thr Glu Arg Trp Phe Val

625 630

Tyr Ile Thr Gly Pro

365

Ser Cys Glu Glu Thr 380

Val Cys Glu Ala Asp

400

Leu Pro Val Cys Glu

415

Gly Arg Ile Tyr Gly 430

Gln Val Leu Ile Leu

445

Asp Asn Trp Val Leu 460

Asp Ala Ser Ala Leu

480

Pro His Tyr Thr Gln

495

Tyr Thr His Asp Ala 510

Asn Asn Lys Val Val 525

Arg Lys Glu Ala Glu 540

Ser Gly Trp Gly Leu

560

Tyr Val Asp Ile Pro

575

Glu Lys Pro Pro Tyr 590

Ala Gly Leu Glu Ser 605

Gly Ala Leu Val Phe 620

Gly Ile Val Ser Trp

640

- 75 045598

Gly Ser Met Asn Cys Gly Glu Ala Gly Gln Tyr Gly Val

645

Tyr Thr Lys

655

650

Val Ile Asn Tyr Ile Pro Trp Ile Glu Asn Ile

660665

Ile Ser Asp Phe

670 <210>7 <211>4900 <212> DNA <213> Homo sapiens <400> 7

cctgtcctgc ctgcctggaa ctctgagcag gctggagtca tggagtcgat tccagaatc 60 ccagagtcag ggaggctggg ggcaggggca ggtcactgga caaacagatc aaaggtgaga 120 ccagcgtagg actgcagacc aggccaggcc agctggacgg gcacaccatg aggtaggtgg 180 gcgccacagc ctccctgcag ggtgtggggt ggggagcacag 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 caccaccacttc 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 gtgcggggcag gagagcacag acacggagcg ggcccctggc aaggaactt 720 tctactcgct gggctccagc ctggacatta ccttccgctc cgactactcc aacgagaagc 780 cgttcacggg gttcgaggcc ttctatgcag ccgaggtga gccaagagg gtcctgcaac 840 atctcagtct gcgcagctgg ctgtggggggt aactctgtct taggccaggc agccctgcct 900 tcagtttccc cacctttccc agggcagggg agaggcctct ggcctgacat catccacaat 960 gcaaagacca aaacagccgt gacctcatt cacatggggct gagtgccaac tctgagccag 1020 ggatctgagg acagcatcgc ctcaagtgac gcaggactg 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 atttacaagc tgacaaagtg ggccctgcca gcggggagcgc tgcaggatgt ttgatttttca 1440 gatccagtc cctgcagaga ccaactgtgt gacctctggc aagtggctca atttctctgc 1500 tccttagaag ctgctgcaag ggttcagcgc tgtagccccg ccccctggggt ttgattgact 1560 cccctcatta gctgggtgac ctcggccgga cactgaaact cccactggtt taacagaggt 1620

- 76 045598

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 ccccggggaga 1860 ggcgcccacc tgcgaccacc actgccacaa ccacctggggc ggtttctact gctcctgccg 1920 cgcaggctac gtcctgcacc gtaacaagcg cacctgctca ggtgagggag gctgcctgggg 1980 ccccaacgca ccctctcctg ggatacccgg ggctcctcag ggccattgct gctctgccca 2040 ggggtgcgga gggcctggggc ctggacactg ggtgcttcta ggccctgctg cctccagctc 2100 cccttctcag ccctgcttcc cctctcagca gccaggctca tcagtgccac cctgccctag 2160 cactgagact aattctaaca tcccactgtg tacctggttc cacctggggct ctgggaaccc 2220 ctcatgtagc caggggagag tcggggtatc taccctcgtt ccttggactg ggttcctgtt 2280 ccctgcactg ggggacggggc cagtgctctg gggcgtgggc agccccacc tgtggcgctg 2340 accctgctcc cccgactcgg tttctcctct cggggtctct ccttgcctct ctgatctctc 2400 ttccagagca gagcctctag cctcccctgg agctccggct gccagcagg tcagaagcca 2460 gagccaggct gctggcctca gctccggggtt gggctgagat gctgtgcccc aactcccatt 2520 caccaccaccat ggaccata aaaacctgg 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 accacccca ttcagggtct ggcctggagg 2820 agcctgaccc acagaggaga caccctggga gatattcatt gaggggtaat ctggtccccc 2880 gcaaatccag gggtgattcc cactgcccca taggcacagc cacgtggaag aaggcaggca 2940 atgttggggc tcctcacttc ctagaggcct cacaactcaa atgcccccca ctgcagctgg 3000 gggtggggtg gtggtatggg atggggacca agccttcctt gaaggataga gccagccca 3060 acaccccgcc ccgtggcagc agcatcacgt gttccagcga ggaaggagag caccagactc 3120 agtcatgatch actgttgcct tgaacttcca agaacagccc caggcaagg gtcaaaacag 3180 gggaaagggg gtgatgagag atccttcttc cggatgttcc tccaggaacc aggggctgg 3240 ctggtcttgg ctggggttcgg gtagggacc catgatgaat aaacttggga atcactgggg 3300 tggctgtaag ggaatttagg ggagctccga aggggccctt aggctcgagg agatgctcct 3360 ctcttttccc gaattccag ggaccagga gagtgtccct tcttcctctt cctgtgtgtc 3420 catccacccc cgccccccgc cctggcagag ctggtggaac tcagtgctct agcccctacc 3480 ctggggttgc gactctggct caggacacca ccacgctccc tggggggtgtg agtgaggcc 3540 tgtgcgctcc atcccgagtg ctgcctgttt cagctaaagc ctcaaagcaa gagaaacccc 3600 ctctctaagc ggcccctcag ccatcgggtg ggtcgtttgg tttctgggta ggcctcaggg 3660 gctggccacc tgcagggccc agcccaacc agggatgcag atgtcccagc cacatccctg 3720

- 77 045598

tccagtttc ctgctcccca aggcatccac cctgctgttg gtgcgagggc tgatagaggg 3780 cagccaagt cactcccctg cccttccctc cttccagccc tgtgctccgg ccaggtcttc 3840 acccagaggt ctggggagct cagcagccct gaataccacc ggccgtatcc caaactctcc 3900 agttgcactt acagcatcag cctggagggag gggttcagtg tcattctgga ctttgtggag 3960 tccttcgatg tggagacaca ccctgaaacc ctgtgtccct acgactttct caaggtctgg 4020 ctcctggggcc cctcatcttg tccgatcc tcccccttca gccagctgc accccctact 4080 tcctgcagca tggccccacc 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 caggattgaa acaaaaagca acacggtgac catcaccttt gtcacagatg aatcaggaga 4440 ccacacaggc tggaagatcc actacacgag cacagtgagc aagtgggctc agatccttgg 4500 tggaagcgca gagctgcctc tctctggagt gcaagggagct gtagagtgta gggctcttct 4560 gggcaggact aggaagggac accaggttta gtggtgctga ggtctgaggc agcagcttct 4620 aaggggaagc acccgtgccc tcctcagcag cacccagcat cttcaccact cattcttcaa 4680 ccacccattc acccatcact catcttttac ccaccccacc tttgccactc atccttctgt 4740 ccctcatcct tccaaccatt catcaatcac ccacccatcc atcctttgcc acacaaccat 4800 ccacccattc ttctacctac ccatcctatc catccacatcct tctatcagca tccttctacc 4860 acccatcctt cgttcggtca tccacatca tccaccacatc 4900

<210>8 <211>136 <212> PRT <213> Homo sapiens <400>8

Met Arg Leu Leu Thr Leu Leu Gly Leu 15

Leu Cys Gly Ser Val Ala Thr

15

Pro Leu Gly Pro Lys Trp Pro 20

Glu Pro Val Phe Gly Arg Leu Ala

thirty

Ser

Pro Gly Phe Pro Gly Glu Tyr 35

Ala Asn Asp Gln Glu Arg Arg Trp 40 45

Thr

Leu Thr Ala

Pro Pro

Gly Tyr Arg Leu Arg Leu Tyr Phe Thr His Phe

60

Asp Leu Glu 65

Leu Ser

His Leu Cys Glu Tyr Asp Phe Val Lys Leu Ser 70 75 80

Ser Gly Ala

Lys Val 85

Leu Ala Thr Leu Cys Gly Gln Glu Ser Thr Asp

95

- 78 045598

Thr Glu Arg Ala Pro Gly Lys Asp Thr Phe

100 105

Leu Asp Ile Thr Phe Arg Ser Asp Tyr Ser

115 120

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

510

Pro Leu Gly Pro Lys Trp Pro Glu Pro Val

25

Pro Gly Phe Pro Gly Glu Tyr Ala Asn Asp

40

Leu Thr Ala Pro Pro Gly Tyr Arg Leu Arg

55

Asp Leu Glu Leu Ser His Leu Cys Glu Tyr 65 70

Ser Gly Ala Lys Val Leu Ala Thr Leu Cys

90

Thr Glu Arg Ala Pro Gly Lys Asp Thr Phe

100 105

Leu Asp Ile Thr Phe Arg Ser Asp Tyr Ser

115 120

Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp

130 135

Ala Pro Gly Glu Ala Pro Thr Cys Asp His

145 150

Gly Gly Phe Tyr Cys Ser Cys Arg Ala Gly

165 170

Ser Leu Gly Ser Ser 110

Glu Lys Pro Phe Thr 125

Gly Ser Val Ala Thr 15

Gly Arg Leu Ala Ser 30

Glu Arg Arg Trp Thr 45

Tyr Phe Thr His Phe 60

Phe Val Lys Leu Ser 80

Gln Glu Ser Thr Asp 95

Ser Leu Gly Ser Ser 110

Glu Lys Pro Phe Thr 125

Asp Glu Cys Gln Val 140

Cys His Asn His Leu

160

Val Leu His Arg Asn

175

Lys Arg Thr Cys Ser

180 <210> 10

- 79 045598 <211> 293 <212> PRT <213> Homo <400> 10

Met Arg Leu 1 sapiens

Leu

Thr 5

Leu

Leu

Gly

Leu

Leu 10

Cys

Gly

Ser

Val

Ala 15

Pro Leu Gly

Pro

Lys

Trp

Pro

Glu

Pro

Val

Phe

Gly

Arg

Leu 30

Ala

Pro Gly Phe

Pro

Gly

Glu

Tyr

Ala 40

Asn

Asp

Gln

Glu

Arg 45

Arg

Trp

Leu Thr Ala

Pro

Pro

Gly

Tyr 55

Arg

Leu

Arg

Leu

Tyr 60

Phe

Thr

His

Asp Leu Glu 65

Leu

Ser

His 70

Leu

Cys

Glu

Tyr

Asp 75

Phe

Val

Lys

Leu

Ser Gly Ala

Lys

Val 85

Leu

Ala

Thr

Leu

Cys 90

Gly

Gln

Glu

Ser

Thr 95

Thr Glu Arg

Ala

100

Pro

Gly

Lys

Asp

Thr

105

Phe

Tyr

Ser

Leu

Gly

110

Ser

Leu Asp Ile

115

Thr

Phe

Arg

Ser

Asp

120

Tyr

Ser

Asn

Glu

Lys

125

Pro

Phe

Gly Phe Glu

130

Ala

Phe

Tyr

Ala

135

Ala

Glu

Asp

Ile

Asp

140

Glu

Cys

Gln

Ala Pro Gly 145

Glu

Ala

Pro

150

Thr

Cys

Asp

His

His

155

Cys

His

Asn

His

Gly Gly Phe

Tyr

Cys 165

Ser

Cys

Arg

Ala

Gly 170

Tyr

Val

Leu

His

Arg 175

Lys Arg Thr

Cys 180

Ser

Ala

Leu

Cys

Ser

185

Gly

Gln

Val

Phe

Thr

190

Gln

Ser Gly Glu

195

Leu

Ser

Ser

Pro

Glu

200

Tyr

Pro

Arg

Pro

Tyr 205

Pro

Lys

Ser Ser Cys

210

Thr

Tyr

Ser

Ile

215

Ser

Leu

Glu

Glu

Gly

220

Phe

Ser

Val

Leu Asp Phe

225

Val

Glu

Ser

230

Phe

Asp

Val

Glu

Thr

235

His

Pro

Glu

Thr

Cys Pro Tyr

Asp

Phe

245

Leu

Lys

Ile

Gln

Thr

250

Asp

Arg

Glu

Glu

His

255

Thr

Ser

Thr

Phe

Ser 80

Asp

Ser

Thr

Val

Leu 160

Asn

Arg

Leu

Ile

Leu 240

Gly

- 80 045598

Pro Phe Cys Gly Lys Thr Leu Pro His Arg

260 265

Thr Val Thr Ile Thr Phe Val Thr Asp Glu

275 280

Trp Lys Ile His Tyr 290 <210>11 <211>41 <212> PRT <213> Homo sapiens <400>11

Glu Asp Ile Asp Glu Cys Gln Val Ala Pro

510

Asp His His Cys His Asn His Leu Gly Gly

25

Ala Gly Tyr Val Leu His Arg Asn Lys

40 <210>12 <211>242 <212> PRT <213> Homo sapiens <400>12

Ile Tyr Gly Gly Gln Lys Ala Lys Pro Gly

510

Leu Ile Leu Gly Gly Thr Thr Ala Ala Gly

25

Trp Val Leu Thr Ala Ala His Ala Val Tyr

40

Ser Ala Leu Asp Ile Arg Met Gly Thr Leu

55

Tyr Thr Gln Ala Trp Ser Glu Ala Val Phe 65 70

His Asp Ala Gly Phe Asp Asn Asp Ile Ala 85 90

Lys Val Val Ile Asn Ser Asn Ile Thr Pro

100 105

Glu Ala Glu Ser Phe Met Arg Thr Asp Asp

115 120

Glu Thr Lys Ser Asn 270

Gly Asp His Thr Gly 285

Glu Ala Pro Thr Cys

Tyr Cys Ser Cys Arg 30

Phe Pro Trp Gln Val

Leu Leu Tyr Asp Asn 30

Gln Lys His Asp Ala 45

Arg Leu Ser Pro His 60

His Glu Gly Tyr Thr

Ile Lys Leu Asn Asn 95

Cys Leu Pro Arg Lys

110

Gly Thr Ala Ser Gly 125

- 81 045598

Trp Gly Leu Thr Gln Arg Gly Phe

130 135

Leu Ala Arg Asn Leu Met Tyr Val

140

Asp Ile Pro Ile Val Asp His Gln

145 150

Lys Cys Thr Ala Ala Tyr Glu Lys

155 160

Pro Pro Tyr Pro Arg Gly Ser Val

165

Thr Ala Asn Met Leu Cys Ala Gly

170 175

Leu Glu Ser Gly Gly Lys Asp Ser

180

Cys Arg Gly Asp Ser Gly Gly Ala

185 190

Leu Val Phe Leu Asp Ser Glu Thr

195 200

Glu Arg Trp Phe Val Gly Gly Ile

205

Val Ser Trp Gly Ser Met Asn Cys

210 215

Gly Glu Ala Gly Gln Tyr Gly Val

220

Tyr Thr Lys Val Ile Asn Tyr Ile

225 230

Pro Trp Ile Glu Asn Ile Ile Ser

235 240

Asp Phe <210> 13 <211> 16 <212> PRT <213> Artificial sequence <220>

< 223> Synthetic < 400>13

Gly Lys Asp Ser Cys Arg Gly Asp Ala 15

Gly Gly Ala Leu Val Phe Leu

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

5 1015 <210>15 <211>43 <212> PRT <213> Artificial sequence <220>

<223> Synthetic <400> 15

- 82 045598

Thr Ala Pro Pro 1

Gly Tyr Arg Leu Arg Leu Tyr Phe Thr His Phe 5 10 15

Asp

Leu Glu Leu Ser

His Leu Cys Glu Tyr Asp Phe Val Lys Leu Ser

thirty

Ser

Gly Ala Lys Val 35

Leu Ala Thr Leu Cys Gly Gln 40 <210> 16 <211> 8 <212> PRT <213> Artificial sequence <220>

<223> Synthetic <400> 16

Thr Phe Arg Ser Asp Tyr Ser Asn

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

5 10 15

Tyr

Ser Asn Glu Lys Pro Phe Thr Gly Phe

25 <210> 18 <211> 9 <212> PRT <213> Artificial sequence <220>

<223> Synthetic <400> 18

Ile Asp Glu Cys Gln Val Ala Pro Gly

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 1 5 10 15

Cys

- 83 045598

Arg Gly Asp Ser Gly Gly Ala Leu Val

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 Met Ser 1 ctg ttt cca tca ctc cct ctc ctt ctc ctg agt atg gtg gca gcg tct Leu Phe Pro Ser Leu Pro Leu Leu Leu Leu Ser Met Val Ala Ala Ser 5 1015 tac tca gaa act gtg acc tgt gag gat gcc caa aag acc tgc cctgca

Tyr Ser Glu Thr Val Thr Cys Glu Asp Ala Gln Lys Thr Cys ProAla

2530 gtg att gcc tgt agc tct cca ggc atc aac ggc ttc cca ggc aaagat

Val Ile Ala Cys Ser Ser Pro Gly Ile Asn Gly Phe Pro Gly LysAsp

40 4550 ggg cgt gat ggc acc aag gga gaa aag ggg gaa cca ggc caa gggctc

Gly Arg Asp Gly Thr Lys Gly Glu Lys Gly Glu Pro Gly Gln GlyLeu

6065 aga ggc tta cag ggc ccc cct gga aag ttg ggg cct cca gga aatcca

Arg Gly Leu Gln Gly Pro Pro Gly Lys Leu Gly Pro Pro Gly AsnPro

7580 ggg cct tct ggg tca cca gga cca aag ggc caa aaa gga gac cctgga

Gly Pro Ser Gly Ser Pro Gly Pro Lys Gly Gln Lys Gly Asp ProGly

9095 aaa agt ccg gat ggt gat agt agc ctg gct gcc tca gaa aga aaagct

Lys Ser Pro Asp Gly Asp Ser Ser Leu Ala Ala Ser Glu Arg LysAla

100 105110 ctg caa aca gaa atg gca cgt atc aaa aag tgg ctc acc ttc tctctg

Leu Gln Thr Glu Met Ala Arg Ile Lys Lys Trp Leu Thr Phe SerLeu

115 120 125130 ggc aaa caa gtt ggg aac aag ttc ttc ctg acc aat ggt gaa ataatg

Gly Lys Gln Val Gly Asn Lys Phe Phe Leu Thr Asn Gly Glu IleMet

135 140145 acc ttt gaa aaa gtg aag gcc ttg tgt gtc aag ttc cag gcc tctgtg

Thr Phe Glu Lys Val Lys Ala Leu Cys Val Lys Phe Gln Ala SerVal

150 155160 gcc acc ccc agg aat gct gca gag aat gga gcc att cag aat ctcatc

Ala Thr Pro Arg Asn Ala Ala Glu Asn Gly Ala Ile Gln Asn LeuIle

165 170175 aag gag gaa gcc ttc ctg ggc atc act gat gag aag aca gaa gggcag

Lys Glu Glu Ala Phe Leu Gly Ile Thr Asp Glu Lys Thr Glu GlyGln

180 185190 ttt gtg gat ctg aca gga aat aga ctg acc tac aca aac tgg aac gag

104

152

200

248

296

344

392

440

488

536

584

632

680

- 84 045598

Phe 195 Val Asp Leu Thr Gly 200 Asn Arg Leu Thr Tyr 205 Thr Asn Trp Asn Glu 210 ggt gaa ccc aac aat gct ggt tct gat gaa gat tgt gta ttg cta ctg 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 Lys Asn Gly Gln Trp Asn Asp Val Pro Cys Ser Thr Ser His Leu Ala 230 235 240

gtc tgt gag ttc cct atc tga agggtcatat cactcaggcc ctccttgtct

Val Cys Glu Phe Pro Ile

245 ttttactgca atatccagta aagcaataat acccacaggc ttgttccttt agt ccacagtatg tgtgggcaat cttgaaaaga cactaaaaat taaattatat gatcactaac caatttcctc agcaccaaca

728

776

827

887

947

960 <210>21 <211>248 <212> PRT <213> Homo sapiens <400>21

Met Ser

Leu Phe

Ala Ser

Tyr Ser 20

Pro Ala

Val Ile 35

Lys Asp 50

Gly Arg

Gly Leu 65

Arg Gly

Asn Pro

Gly Pro

Pro Gly

Lys Ser

100

Lys Ala

Leu Gln

115

Ser Leu

130

Gly Lys

Ile Met

145

Thr Phe

Ser Val

Ala Thr

Pro Ser 5

Glu Thr

Ala Cys

Asp Gly

Leu Gln 70

Ser Gly 85

Pro Asp

Thr Glu

Gln Val

Glu Lys

150

Pro Arg

Leu Pro

Leu Leu

Leu Leu

SerMet

Val Thr

Cys Glu 25

Asp Ala

Gln Lys 30

Ser Ser

Thr Lys 55

Gly Pro

Ser Pro

Gly Asp

Met Ala

120

Gly Asn 135

Val Lys

Asn Ala

Pro Gly

Gly Glu

Pro Gly

Gly Pro 90

Ser Ser 105

Arg Ile

Lys Phe

Ala Leu

Ala Glu

Ile Asn

Gly Phe 45

Lys Gly 60

Glu Pro

Lys Leu 75

Gly Pro

Lys Gly

Gln Lys

Leu Ala

Lys Lys

Phe Leu

140

Cys Val 155

Asn Gly

Ala Ser

110

Trp Leu 125

Thr Asn

Lys Phe

Ala Ile

Val Ala 15

Thr Cys

Pro Gly

Gly Gln

Pro Gly 80

Gly Asp 95

Glu Arg

Thr Phe

Gly Glu

Gln Ala

160

Gln Asn

- 85 045598

165 170 175

Leu Ile Lys Glu

180

Glu Ala Phe Leu Gly Ile Thr Asp Glu

185

Gly Gln Phe Val

195

Asp Leu Thr Gly Asn Arg Leu Thr Tyr

200 205

Lys Thr Glu 190

Thr Asn Trp

Asn Glu Gly Glu 210

Pro Asn Asn Ala Gly Ser Asp Glu Asp

215 220

Cys Val Leu

Leu Leu Lys Asn 225

Gly Gln Trp Asn Asp Val Pro Cys Ser

230 235

Thr Ser His

240

Leu Ala Val Cys Glu Phe Pro Ile 245 <210> 22 <211> 6 <212> PRT <213> Artificial sequence <220>

<223> Synthetic <220>

< 221> UNDEFINED CHARACTER < 222> (1)..(1) < 223> Where X at position 1 is hydroxyproline <220>

< 221> UNDEFINED FEATURE < 222> (4)..(4) < 223> Where X at position 4 is a hydrophobic residue <400> 22

Xaa Gly Lys Xaa Gly Pro <210> 23 <211> 5 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic <220>

< 221> UNDEFINED SIGN < 222> (1)..(1) < 223> Where X represents hydroxyproline <400> 23

Xaa Gly Lys Leu Gly

5 <210> 24 <211> 16

- 86 045598 <212> PRT <213> Artificial sequence <220>

<223> Synthetic <220>

< 221> UNDEFINED FEATURE < 222> (9)..(15) < 223> Where X at positions 9 and 15 is hydroxyproline < 400> 24

Gly Leu Arg Gly Leu Gln Gly Pro Xaa Gly Lys Leu Gly Pro Xaa 1 5 10 15

Gly <210> 25 <211> 27 <212> PRT <213> Artificial sequence <220>

<223> Synthetic <220>

< 221> UNDEFINED SIGN < 222> (3)..(27) < 223> Where X is in positions 3, 6, hydroxyproline < 400> 25

Gly Pro Xaa Gly Pro Xaa Gly Leu 1 5

15, 21, 24, 27 represents

Arg Gly Leu Gln Gly Pro Xaa Gly

15

Lys Leu Gly Pro Xaa Gly Pro Xaa 20

Gly Pro Xaa 25 <210> 26 <211> 53 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic <220>

<221> undefined <222> (26)..(26) <223> Xaa can be any naturally occurring amino acid <220>

<221> undefined <222> (32)..(32) <223> Xaa can be any naturally occurring amino acid <220>

<221> undefined <222> (35)..(35) <223> Xaa can be any naturally occurring amino acid <220>

< 221> undefined < 222> (41)..(41)

- 87 045598 <223> Xaa can be any naturally occurring amino acid <220>

<221> undefined <222> (50).. (50) <223> Xaa can be any naturally occurring amino acid <400> 26

Gly Lys Asp Gly Arg Asp Gly Thr 1 5

Lys Gly Glu Lys Gly Glu Pro Gly

15

Gln Gly Leu Arg Gly Leu Gln Gly 20

Pro Xaa Gly Lys Leu Gly Pro Xaa 25 30

Gly Asn Xaa Gly Pro Ser Gly Ser

40

Xaa Gly Pro Lys Gly Gln Lys Gly 45

Asp Xaa Gly Lys Ser <210> 27 <211> 33 <212> PRT <213> Artificial sequence <220>

<223> Synthetic <220>

< 221> UNDEFINED CHARACTER < 222> (3)..(33) < 223> Where 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

5 1015

Pro Xaa Gly Pro Xaa Gly Lys Met Gly Pro Lys Gly Glu Xaa Gly Asp 20 2530

Xaa < 210>28 < 211>45 < 212> PRT < 213> Artificial sequence <220>

<223> Synthetic <220>

<221> UNDEFINED CHARACTER <222> (3)..(45) <223> Where X at positions 3, 6, 9, 27, 30, 36, 42, 45 represents hydroxyproline <400> 28

- 88 045598

Gly Cys Xaa Gly Leu Xaa Gly Ala Xaa 15

Thr Asn Gly Lys Arg Gly Glu Arg Gly

2025

Ala Gly Pro Xaa Gly Pro Asn Gly Ala

3540

Gly Asp Lys Gly Glu Ala Gly

15

Pro Xaa Gly Pro Xaa Gly Lys 30

Xaa Gly Glu Xaa 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 1 510

Ile Lys Ala 15

Asn Arg Pro Phe Leu Val Phe Ile <210>30 <211>559 <212> DNA <213> Homo sapiens

<400> 30 atgaggctgc tgaccctcct gggccttctg tgtggctcgg tggccacccc cttgggcccg 60 aagtggcctg aacctgtgtt cggggcgcctg gcatcccccg gctttccagg ggagtatgcc 120 aatgaccagg agcggcgctg gaccctgact gcaccccccg gctaccgcct gcgcctctac 180 ttcaccact tcgacctgga gctctcccac ctctgcgagt acgacttcgt caagctgagc 240 tcggggggcca aggtgctggc cacgctgtgc gggcaggaga gcacagacac ggagcggggcc 300 cctggcaagg acactttcta ctcgctgggc tccagcctgg acattacctt ccgctccgac 360 tactccaacg agaagccgtt cacggggttc gaggccttct atgcagccga ggacattgac 420 gagtgccagg tggccccggg agaggcgccc acctgcgacc accactgcca caaccacctg 480 ggcggtttct actgctcctg ccgcgcaggc tacgtcctgc accgtaacaa gcgcacctgc 540 tcagccctgt gctccggcc 559

<210>31 <211>34 <212> DNA <213> Artificial sequence <220>

<223> Synthetic <400> 31 cggggcacacc atgaggctgc tgaccctcct gggc 34

- 89 045598 <210> 32 <211> 33 <212> DNA <213> Artificial sequence <220>

<223> Synthetic <400>32 gacattacct tccgctccga ctccaacgag aag33 <210>33 <211>33 <212> DNA <213> Artificial sequence <220>

<223> Synthetic <400>33 agcagccctg aatacccacg gccgtatccc aaa33 <210>34 <211>26 <212> DNA <213> Artificial sequence <220>

<223> Synthetic <400>34 cgggatccat gaggctgctg accctc <210>35 <211>19 <212> DNA <213> Artificial sequence <220>

<223> Synthetic <400>35 ggaattccta ggctgcata19 <210>36 <211>19 <212> DNA <213> Artificial sequence <220>

<223> Synthetic <400>36 ggaattccta cagggcgct <210>37 <211>19 <212> DNA <213> Artificial sequence <220>

<223> Synthetic <400> 37 ggaattccta gtagtggat

- 90 045598 <210> 38 <211> 25 <212> DNA <213> Artificial sequence <220>

<223> Synthetic <400>38 tgcggccgct gtaggtgctg tcttt <210>39 <211>23 <212> DNA <213> Artificial sequence <220>

<223> Synthetic <400>39 ggaattcact cgttattctc gga23 <210>40 <211>17 <212> DNA <213> Artificial sequence <220>

<223> Synthetic <400>40 tccgagaata acgagtg17 <210>41 <211>29 <212> DNA <213> Artificial sequence <220>

<223> Synthetic <400>41 cattgaaagc tttggggtag aagttgttc29 <210>42 <211>27 <212>DNA <213>Artificial sequence <220>

<223> Synthetic <400>42 cgcggccgca gctgctcaga gtgtaga27 <210>43 <211>28 <212> DNA <213> Artificial sequence <220>

< 223> Synthetic

- 91 045598 <400>43 cggtaagctt cactggctca gggaaata28 <210>44 <211>37 <212> DNA <213> Artificial sequence <220>

<223> Synthetic <400>44 aagaagcttg ccgccaccat ggattggctg tggaact37 <210>45 <211>31 <212> DNA <213> Artificial sequence <220>

<223> Synthetic <400>45 cgggatcctc aaactttctt gtccaccttg g31 <210>46 <211>36 <212> DNA <213> Artificial sequence <220>

<223> Synthetic <400>46 aagaaagctt gccgccacca tgttctcact agctct36 <210>47 <211>26 <212> DNA <213> Artificial sequence <220>

<223> Synthetic <400>47 cgggatcctt ctccctctaa cactct26 <210>48 <211>9 <212> PRT <213> Artificial sequence <220>

< 223> Synthetic < 400>48

Glu Pro Lys Ser Cys Asp Lys Thr His <210>49 <211>4960 <212> DNA <213> Homo Sapiens

- 92 045598

<400> 49 ccggacgtgg tggcgcatgc ctgtaatccc agctactcgg gaggctgagg caggagaatt 60 gctcgaaccc cggaggcaga ggtttggtgg ctcacacctg taatcccagc actttgcgag 120 gctgaggcag gtgcatcgct ttggctcagg agttcaagac cagcctggggc 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 agatggatt tcaccatgtt ggtcaggctg gtctcaaact cccaacctcg tgatccacc 600 accttggcct cccaaagtgc tgggattaca ggtataagcc accgagccca gccaaaagcg 660 acttctaagc ctgcaaggga atcgggaatt ggtggcacca ggtccttctg acagggttta 720 agaaattagc cagcctgagg ctggggcacgg tggctcacac ctgtaatccc agcactttgg 780 gaggctaagg caggtggatc acctgaggc aggagttcaa gaccagcctg accaacatgg 840 agaaacccca tccctaccaa aaataaaaaa ttagccaggt gtggtggtgc tcgcctgtaa 900 tccagctac ttgggaggct gaggtggggag gattgcttga acacaggaag tagaggctgc 960 agtgagctat gattgcagca ctgcactgaa gccggggcaa cagaacaaga tccaaaaaaa 1020 agggaggggt gagggcaga gccaggattt gtttccaggc tgttgttacc taggtccgac 1080 tcctggctcc cagagcagcc tgtcctgcct gcctggaact ctgagcaggc tggagtcatg 1140 gagtcgattc ccagaatccc agagtcagg aggctggggg caggggcagg tcactggac 1200 aacagatcaa aggtgagacc agcgtagggc tgcagaccag gccaggccag ctggacggggc 1260 acaccatgag gtaggtggggc gccccacagcc tccctgcagg gtgtggggtg ggagcacagg 1320 cctggggccct caccgcccct gccctgccca taggctgctg accctcctgg gccttctgtg 1380 tggctcggtg gccaccccct tggggcccgaa gtggcctgaa cctgtgttcg ggcgcctggc 1440 atcccccggc tttccagggg agtatgccaa tgaccaggag cggcgctgga ccctgactgc 1500 accccccggc taccgcctgc gcctctactt caccaccacttc gacctggagc tctcccacct 1560 ctgcgagtac gacttcgtca aggtgccgtc aggacggggag ggctggggtt tctcagggtc 1620 ggggggtccc caagggagtag ccagggttca gggaacctg ggagcagggg ccaggcttgg 1680 ccaggagagga gatcaggcct gggtcttgcc ttcactccct gtgacacctg accccacagc 1740 tgagctcggg ggccaaggtg ctggccacgc tgtgcgggca ggagagcaca gacacggagc 1800 gggcccctgg caaggacact ttctactcgc tggggctccag cctggacatt accttccgct 1860 ccgactactc caacgagaag ccgttcacgg ggttcgaggc cttctatgca gccgaggtg 1920 agccaagagg ggtcctgcaa catctcagtc tgcgcagctg gctgtggggg taactctgtc 1980 ttaggccagg cagccctgcc ttcagtttcc ccacctttcc cagggcaggg gagaggcctc 2040

- 93 045598 tggcctgaca tcatccacaa tgcaaagacc aaaacagccg tgacctccat tcacatgggc2100 tgagtgccaa ctctgagcca gggatctgag gacagcatcg cctcaagtga cgcagggact2160 ggccgggcgc agcagctcac gcctgtaatt ccagcacttt gggagg ccga ggctggctga2220 tcatttgagg tcaggagttc aaggccagcc agggcaacac ggtgaaactc tatctccact2280 aaaactacaa aaattagctg ggcgtggtgg tgcgcacctg gaatcccagc tactagggag2340 gctgaggcag gagaattgct tgaacctgcg aggtggaggc tg cagtgaac agagattgca2400 ccactacact ccagcctggg cgacagagct agactccgtc tcaaaaaaca aaaaacaaaa2460 acgacgcagg ggccgagggc cccatttaca gctgacaaag tggggccctg ccagcgggag2520 cgctgccagg atgtttgatt tcagatccca gtccctgcag agaccaactg tgtgacctct2580 ggcaagtggc tcaatttctc tgctccttag gaagctgctg caagggttca gcgctgtagc2640 cccgccccct gggtttgatt gactcccctc attagctggg tgacct cggg ccggacactg2700 aaactcccac tggtttaaca gaggtgatgt ttgcatcttt ctcccagcgc tgctgggagc2760 ttgcagcgac cctaggcctg taaggtgatt ggcccggcac cagtcccgca ccctagacag2820 gacgaggcct cctctgaggt ccactctgag gt catggatc tcctgggagg agtccaggct2880 ggatcccgcc tctttccctc ctgacggcct gcctggccct gcctctcccc cagacattga2940 cgagtgccag gtggccccgg gagaggcgcc cacctgcgac caccactgcc acaaccacct3000 gggcggtttc tactgctcct gccgcgcagg ctacgtcctg caccgtaaca agcgcacctg3060 ctcagccctg tgctccggcc aggtcttcac ccagaggtct ggggagctca gcagccctga3120 atacccacgg ccgtatccca aactctccag ttgcacttac agcatcagcc tggagggagg3180 gtt cagtgtc attctggact ttgtggagtc cttcgatgtg gagacacacc ctgaaaccct3240 gtgtccctac gactttctca agattcaaac agacagagaa gaacatggcc cattctgtgg3300 gaagacattg ccccacagga ttgaaacaaa aagcaacacg gtgaccatca cctttgtcac3 360 agatgaatca ggagaccaca caggctggaa gatccactac acgagcacag cgcacgcttg3420 cccttatccg atggcgccac ctaatggcca cgtttcacct gtgcaagcca aatacatcct3480 gaaagacagc ttctccatct tttgcgagac tggctatgag cttctgcaag gtcacttgcc3540 cctgaaatcc tttactgcag tttgtcagaa agatggatct tgggaccggc caatgcccgc3600 gtgcagcatt gttgactgtg gccctcctga tgatctaccc agtggccgag tggagtacat3660 cacaggtcct ggagtgacca cctacaaagc tgtgattcag tacagctgtg aagagacctt3720 ctacacaatg aaagtgaatg atggtaaata tgtgtgtgag gctgatggat tctggacgag3780 ctccaaagga gaaaaatcac tcccagtctg tgagcctgtt tgtggactat cagcccgcac3840 aacaggaggg cgtatatatg gagggcaaaa ggcaaaacct ggtgattttc cttggcaagt3900 cctgatatta ggtggaacca cagcagcagg tgcactttta tatgacaact gggtcctaac3960 agctgctcat gccgtctatg agcaaaaaca tgatgcatcc gccctggaca ttcgaatggg4020 caccctgaaa agactatcac ctcattatac acaagcctgg tctgaagctg tttttataca4080 tgaaggttat actcatgatg ctggctttga caatgacata gcactgatta aattgaataa4140

- 94 045598

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 acagaggt ggtttgtggg aggaatagtg tcctggggtt ccatgaattg 4500 tggggaagca ggtcagtatg gagtctacac aaaagttatt aactatattc cctggatcga 4560 gaacataatt agtgattttt aacttgcgtg tctgcagtca aggattcttc atttttagaa 4620 atgcctgtga agaccttggc agcgacgtgg ctcgagaagc attcatcatt actgtggac 4680 tggcagttgt tgctccacc aaaaaaacag actccaggtg aggctgctgt catttctcca 4740 cttgccagtt taattccagc cttacccatt gactcaaggg gacataaacc acgagagtga 4800 cagtcatctt tgccaccca gtgtaatgtc actgctcaaa ttacatttca ttaccttaaa 4860 aagccagtct cttttcatac tggctgttgg catttctgta aactgcctgt ccatgctctt 4920 tgtttttaaa cttgttctta ttgaaaaaaa aaaaaaaaaa 4960

<210> 50 <211> 2090 <212> DNA <213> Mouse <220>

<221> CDS <222> (33) .. (2090) <400> 50 ggcgctggac tgcagagcta tggtggcaca cc atg agg cta ctc atc ttc ctg 53

Met 1 Arg Leu Leu Ile 5 Phe Leu ggt ctg ctg tgg agt ttg gtg gcc aca ctt ctg ggt tca aag tgg cct 101 Gly Leu Leu Trp Ser Leu Val Ala Thr Leu 10 15 Leu Gly Ser 20 Lys Trp Pro gaa cct gta ttc ggg cgc ctg gtg tcc cct ggc ttc cca gag aag tat 149 Glu Pro 25 Val Phe Gly Arg Leu Val Ser Pro 30 Gly Phe 35 Pro Glu Lys Tyr gct gac cat caa gat cga tcc tgg aca ctg act gca ccc cct ggc tac 197 Ala Asp 40 His Gln Asp Arg Ser Trp Thr Leu 45 Thr 50 Ala Pro Pro Gly Tyr 55 cgc ctg cgc ctc tac ttc acc cac ttt gac ctg gaa ctc tct tac cgc 245 Arg Leu Arg Leu Tyr Phe Thr His Phe Asp 60 65 Leu Glu Leu Ser Tyr 70 Arg tgc gag tat gac ttt gtc aag ttg agc tca ggg acc aag gtg ctg gcc 293 Cys Glu Tyr Asp Phe Val Lys Leu Ser Ser 75 80 Gly Thr Lys Val 85 Leu Ala 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 90 95 Glu Gln Ala 100 Pro Gly Asn gac acc ttc tac tca ctg ggt ccc agc cta aag gtc acc ttc cac tcc 389 Asp Thr Phe Tyr Ser Leu Gly Pro Ser Leu Lys Val Thr Phe His Ser

- 95 045598

437 gac Asp 120 gcg Ala tgt

Cys yeah

Arg tgt

Cys gag Glu 200 cgc Arg gat

Asp att

Ile cct

Pro act Thr 280 aca Thr tca

Ser tgc

Cys ttc

Phe gag Glu 360 cat His

105 110115 tac tcc aat gag aag ccg ttc aca ggg ttt gag gcc ttc tatgca

Tyr Ser Asn Glu Lys Pro Phe Thr Gly Phe Glu Ala Phe TyrAla

125 130135 gag gat gtg gat gaa tgc aga gtg tct ctg gga gac tca gtccct

Glu Asp Val Asp Glu Cys Arg Val Ser Leu Gly Asp Ser ValPro

140 145150 gac cat tat tgc cac aac tac ttg ggc ggc tac tat tgc tcctgc

Asp His Tyr Cys His Asn Tyr Leu Gly Gly Tyr Tyr Cys SerCys

155 160165 gcg ggc tac att ctc cac cag aac aag cac acg tgc tca gccctt

Ala Gly Tyr Ile Leu His Gln Asn Lys His Thr Cys Ser AlaLeu

170 175180 tca ggc cag gtg ttc aca gga aga tct ggg tat ctc agt agccct

Ser Gly Gln Val Phe Thr Gly Arg Ser Gly Tyr Leu Ser SerPro

185 190195 tac ccg cag cca tac ccc aag ctc tcc agc tgc acc tac agcatc

Tyr Pro Gln Pro Tyr Pro Lys Leu Ser Ser Cys Thr Tyr SerIle

205 210215 ctg gag gac ggc ttc agt gtc atc ctg gac ttc gtg gag tccttc

Leu Glu Asp Gly Phe Ser Val Ile Leu Asp Phe Val Glu SerPhe

220 225230 gtg gag acg cac cct gaa gcc cag tgc ccc tat gac tcc ctcaag

Val Glu Thr His Pro Glu Ala Gln Cys Pro Tyr Asp Ser LeuLys

235 240245 caa aca gac aag ggg gaa cac ggc cca ttt tgt ggg aag acgctg

Gln Thr Asp Lys Gly Glu His Gly Pro Phe Cys Gly Lys ThrLeu

250 255260 ccc agg att gaa act gac agc cac aag gtg acc atc acc tttgcc

Pro Arg Ile Glu Thr Asp Ser His Lys Val Thr Ile Thr PheAla

265 270275 gac gag tcg ggg aac cac aca ggc tgg aag ata cac tac acaagc

Asp Glu Ser Gly Asn His Thr Gly Trp Lys Ile His Tyr ThrSer

285 290295 gca cgg ccc tgc cct gat cca acg gcg cca cct aat ggc agcatt

Ala Arg Pro Cys Pro Asp Pro Thr Ala Pro Pro Asn Gly SerIle

300 305310 cct gtg caa gcc acg tat gtc ctg aag gac agg ttt tct gtcttc

Pro Val Gln Ala Thr Tyr Val Leu Lys Asp Arg Phe Ser ValPhe

315 320325 aag aca ggc ttc gag ctt ctg caa ggt tct gtc ccc ctg aaatca

Lys Thr Gly Phe Glu Leu Leu Gln Gly Ser Val Pro Leu LysSer

330 335340 act gct gtc tgt cag aaa gat gga tct tgg gac cgg ccg atgcca

Thr Ala Val Cys Gln Lys Asp Gly Ser Trp Asp Arg Pro MetPro

345 350355 tgc agc att att gat tgt ggc cct ccc gat gac cta ccc aatggc

Cys Ser Ile Ile Asp Cys Gly Pro Pro Asp Asp Leu Pro AsnGly

365 370375 gtg gac tat atc aca ggc cct caa gtg act acc tac aaa gctgtg

Val Asp Tyr Ile Thr Gly Pro Gln Val Thr Thr Tyr Lys AlaVal

380 385390

485

533

581

629

677

725

773

821

869

917

965

1013

1061

1109

1157

1205

- 96 045598 att cag tac

Ile Gln Tyr aaa tat gtg Lys Tyr Val 410 aaa ctc ccc Lys Leu Pro 425 ata gga gga Ile Gly Gly 440 cct tgg caa Pro Trp Gln ctt ata cat

Leu Ile His aaa aga atg Lys Arg Met 490 agg ctc tca Arg Leu Ser 505 cat gaa ggc His Glu Gly 520 att aaa ctc Ile Lys Leu tgc cta ccg Cys Leu Pro gga act gtg Gly Thr Val 570 aac cta atg Asn Leu Met 585 acc gtg tat Thr Val Tyr 600 ctc tgt gct Leu Cys Ala agt ggg ggg

Ser Gly Gly gtg gga gga Val Gly Gly 650 cag tat ggg Gln Tyr Gly agc tgt gaa gag act Ser Cys Glu Glu Thr 395 tgt gag gct gat gga Cys Glu Ala Asp Gly 415 ccg gtt tgt gag cct Pro Val Cys Glu Pro 430 cgc ata gtt gga ggg Arg Ile Val Gly Gly

445 gtc ttg ttg ctg ggt

Val Leu Leu Leu Gly 460 gac aat tgg gtc cta Asp Asn Trp Val Leu 475 gca gcg tcc tcc ctg Ala Ala Ser Ser Leu 495 cct cat tac act caa Pro His Tyr Thr Gln 510 tac act cac ggt gct Tyr Thr His Gly Ala 525 aag aac aaa gtc aca Lys Asn Lys Val Thr 540 cga aaa gaa gct gca Arg Lys Glu Ala Ala 555 gct ggc tgg ggg tta Ala Gly Trp Gly Leu 575 ttt gtg gac ata cca Phe Val Asp Ile Pro 590 gaa aag ctc tat cca Glu Lys Leu Tyr Pro 605 ggc tta gag act ggt Gly Leu Glu Thr Gly 620 gca tta gtg ttt cta Ala Leu Val Phe Leu 635 ata gtt tcc tgg ggt Ile Val Ser Trp Gly 655 gtc tac aca aaa gtc Val Tyr Thr Lys Val ttc tac aca Phe Tyr Thr 400 ttc tgg acg Phe Trp Thr gtt tgt ggg

Val Cys Gly cag cct gca Gln Pro Ala 450 caa act aca Gln Thr Thr 465 aca gcc gct Thr Ala Ala 480 aac atc cga Asn Ile Arg gcc tgg ccc Ala Trp Pro ggt ttt gac Gly Phe Asp 530 atc aac gga Ile Asn Gly 545 tcc tta atg Ser Leu Met 560 acc cag aag Thr Gln Lys att gct gac

Ile Ala Asp gga gta aga Gly Val Arg 610 ggc aag gac Gly Lys Asp 625 gat aat gag Asp Asn Glu 640 tcc att aat

Ser Ile Asn atc aac tat Ile Asn Tyr atg agc agc aat Met Ser Ser Asn 405 agc tcc aaa gga Ser Ser Lys Gly 420 ctg tcc aca cac Leu Ser Thr His 435 aag cct ggt gac Lys Pro Gly Asp gca gca gca ggt Ala Ala Ala Gly 470 cat gct gta tat His Ala Val Tyr 485 atg ggc atc ctc Met Gly Ile Leu 500 gag gaa atc ttt Glu Glu Ile Phe 515 aat gat ata gca Asn Asp Ile Ala agc atc atg cct Ser Ile Met Pro 550 aga aca gac ttc Arg Thr Asp Phe 565 ggg ctt ctt gct Gly Leu Leu Ala 580 cac caa aaa tgt His Gln Lys Cys 595 gta agc gct aac

Val Ser Ala Asn agc tgc aga ggt Ser Cys Arg Gly 630 aca cag cga tgg Thr Gln Arg Trp 645 tgt ggg gcg gca Cys Gly Ala Ala 660 att ccc tgg aat Ile Pro Trp Asn ggt 1253

Glygaa 1301

Glu act 1349

Thr ttt 1397

Phe

455 gca 1445

Ala gag 1493

Glu aaa 1541

Lys ata 1589

Ile ttg 1637

Leu

535 GT 1685

Val act 1733

Thr aga 1781

Arg acc 1829

Thr atg 1877

Met

615 gac 1925

Asp ttt 1973

Pheggc 2021

Gly gag 2069

Glu

- 97 045598

2090

665 aac ata ata agt Asn Ile Ile Ser 680

670 aat ttc taa Asn Phe

685

675 <210>51 <211>685 <212> PRT <213> Mouse <400>51

Met Arg Leu Leu 1

Leu Leu Gly Ser

Pro Gly Phe Pro 35

Leu Thr Ala Pro

Asp Leu Glu Leu 65

Ser Gly Thr Lys

Thr Glu Gln Ala

100

Ile Phe Leu Gly Leu 5

Lys Trp Pro Glu Pro

Glu Lys Tyr Ala Asp

Pro Gly Tyr Arg Leu 55

Ser Tyr Arg Cys Glu 70

Val Leu Ala Thr Leu 85

Pro Gly Asn Asp Thr

105

Leu 10 Trp Ser Leu Val Ala 15 Thr Val Phe Gly Arg Leu 30 Val Ser His Gln Asp Arg 45 Ser Trp Thr Arg Leu Tyr 60 Phe Thr His Phe Tyr Asp 75 Phe Val Lys Leu Ser 80 Cys 90 Gly Gln Glu Ser Thr 95 Asp Phe Tyr Ser Leu Gly 110 Pro Ser

Leu Lys Val Thr

115

Phe His Ser Asp Tyr Ser Asn Glu Lys Pro Phe Thr

120 125

Gly Phe Glu Ala 130

Phe Tyr Ala Ala Glu Asp Val Asp Glu Cys Arg Val

135 140

Ser Leu Gly Asp 145

Ser Val Pro Cys Asp His Tyr Cys His Asn Tyr Leu

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

180

Ser Ala Leu Cys Ser Gly Gln Val Phe Thr Gly Arg

185 190

Ser Gly Tyr Leu

195

Ser Ser Pro Glu Tyr Pro Gln Pro Tyr Pro Lys Leu

200 205

Ser Ser Cys Thr 210

Tyr Ser Ile Arg Leu Glu Asp Gly Phe Ser Val Ile

215 220

- 98 045598

Leu Asp Phe Val Glu Ser Phe Asp Val Glu

225 230

Cys Pro Tyr Asp Ser Leu Lys Ile Gln Thr

245 250

Pro Phe Cys Gly Lys Thr Leu Pro Pro Arg

260 265

Lys Val Thr Ile Thr Phe Ala Thr Asp Glu

275 280

Trp Lys Ile His Tyr Thr Ser Thr Ala Arg

290 295

Ala Pro Pro Asn Gly Ser Ile Ser Pro Val 305 310

Lys Asp Arg Phe Ser Val Phe Cys Lys Thr

325 330

Gly Ser Val Pro Leu Lys Ser Phe Thr Ala

340 345

Ser Trp Asp Arg Pro Met Pro Glu Cys Ser

355 360

Pro Asp Asp Leu Pro Asn Gly His Val Asp

370 375

Val Thr Thr Tyr Lys Ala Val Ile Gln Tyr 385 390

Tyr Thr Met Ser Ser Asn Gly Lys Tyr Val

405 410

Trp Thr Ser Ser Lys Gly Glu Lys Leu Pro

420 425

Cys Gly Leu Ser Thr His Thr Ile Gly Gly

435 440

Pro Ala Lys Pro Gly Asp Phe Pro Trp Gln

450 455

Thr Thr Ala Ala Ala Gly Ala Leu Ile His

465 470

Ala Ala His Ala Val Tyr Glu Lys Arg Met

485 490

Ile Arg Met Gly Ile Leu Lys Arg Leu Ser

500 505

His Pro Glu Ala Gln

240

Lys Gly Glu His Gly

255

Glu Thr Asp Ser His

270

Gly Asn His Thr Gly 285

Cys Pro Asp Pro Thr 300

Ala Thr Tyr Val Leu

320

Phe Glu Leu Leu Gln

335

Cys Gln Lys Asp Gly 350

Ile Asp Cys Gly Pro

365

Ile Thr Gly Pro Gln 380

Cys Glu Glu Thr Phe

400

Glu Ala Asp Gly Phe

415

Val Cys Glu Pro Val

430

Ile Val Gly Gly Gln

445

Leu Leu Leu Gly Gln 460

Asn Trp Val Leu Thr

480

Ala Ser Ser Leu Asn

495

His Tyr Thr Gln Ala

510

- 99 045598

Trp Pro Glu Glu

515

Ile

Phe

Ile

His

520

Glu

Gly

Tyr

Thr

His

525

Gly

Ala

Gly

Phe Asp Asn Asp 530

Ile

Ala

Leu

535

Ile

Lys

Leu

Lys

Asn

540

Lys

Val

Thr

Ile

Asn Gly Ser Ile 545

Met

Pro

550

Val

Cys

Leu

Pro

Arg 555

Lys

Glu

Ala

Ala

Ser

560

Leu Met Arg Thr

Asp

565

Phe

Thr

Gly

Thr

Val

570

Ala

Gly

Trp

Gly

Leu

575

Thr

Gln Lys Gly Leu

580

Leu

Ala

Arg

Asn

Leu

585

Met

Phe

Val

Asp

Ile

590

Pro

Ile

Ala Asp His Gln

595

Lys

Cys

Thr

Thr

600

Val

Tyr

Glu

Lys

Leu

605

Tyr

Pro

Gly

Val Arg Val Ser

610

Ala

Asn

Met

615

Leu

Cys

Ala

Gly

Leu

620

Glu

Thr

Gly

Gly

Lys Asp Ser Cys 625

Arg

Gly

630

Asp

Ser

Gly

Gly

Ala

635

Leu

Val

Phe

Leu

Asp

640

Asn Glu Thr Gln

Arg

645

Trp

Phe

Val

Gly

Gly 650

Ile

Val

Ser

Trp

Gly 655

Ser

Ile Asn Cys Gly

660

Ala

Ala

Gly

Gln

Tyr 665

Gly

Val

Tyr

Thr

Lys 670

Val

Ile

Asn Tyr Ile Pro

675

Trp

Asn

Glu

Asn

680

Ile

Ile

Ser

Asn

Phe

685 <210>52 <211>670 <212> PRT <213> Mouse <400>52

Thr Leu Leu Gly 1

Ser 5

Lys

Trp

Pro

Glu

Pro 10

Val

Phe

Gly

Arg

Leu 15

Val

Ser Pro Gly Phe 20

Pro

Glu

Lys

Tyr

Ala 25

Asp

His

Gln

Asp

Arg 30

Ser

Trp

Thr Leu Thr Ala

Pro

Pro

Gly

Tyr 40

Arg

Leu

Arg

Leu

Tyr 45

Phe

Thr

His

Phe Asp Leu Glu 50

Leu

Ser

Tyr 55

Arg

Cys

Glu

Tyr

Asp 60

Phe

Val

Lys

Leu

Ser Ser Gly Thr

Lys

Val

Leu

Ala

Thr

Leu

Cys

Gly

Gln

Glu

Ser

Thr

- 100 045598

70

Asp Thr Glu Gln Ala Pro Gly Asn Asp Thr

90

Ser Leu Lys Val Thr Phe His Ser Asp Tyr

100 105

Thr Gly Phe Glu Ala Phe Tyr Ala Ala Glu

115 120

Val Ser Leu Gly Asp Ser Val Pro Cys Asp

130 135

Leu Gly Gly Tyr Tyr Cys Ser Cys Arg Ala 145 150

Asn Lys His Thr Cys Ser Ala Leu Cys Ser

165 170

Arg Ser Gly Tyr Leu Ser Ser Pro Glu Tyr

180 185

Leu Ser Ser Cys Thr Tyr Ser Ile Arg Leu

195 200

Ile Leu Asp Phe Val Glu Ser Phe Asp Val

210 215

Gln Cys Pro Tyr Asp Ser Leu Lys Ile Gln

225 230

Gly Pro Phe Cys Gly Lys Thr Leu Pro Pro

245 250

His Lys Val Thr Ile Thr Phe Ala Thr Asp

260 265

Gly Trp Lys Ile His Tyr Thr Ser Thr Ala

275 280

Thr Ala Pro Pro Asn Gly Ser Ile Ser Pro

290 295

Leu Lys Asp Arg Phe Ser Val Phe Cys Lys

305 310

Gln Gly Ser Val Pro Leu Lys Ser Phe Thr

325 330

Gly Ser Trp Asp Arg Pro Met Pro Glu Cys

340 345

Tyr Ser Leu Gly Pro 95

Asn Glu Lys Pro Phe 110

Val Asp Glu Cys Arg

125

Tyr Cys His Asn Tyr 140

Tyr Ile Leu His Gln

160

Gln Val Phe Thr Gly

175

Gln Pro Tyr Pro Lys 190

Asp Gly Phe Ser Val 205

Thr His Pro Glu Ala 220

Asp Lys Gly Glu His

240

Ile Glu Thr Asp Ser

255

Ser Gly Asn His Thr 270

Pro Cys Pro Asp Pro

285

Gln Ala Thr Tyr Val 300

Gly Phe Glu Leu Leu

320

Val Cys Gln Lys Asp

335

Ile Ile Asp Cys Gly 350

- 101 045598

Pro Pro Asp Asp Leu Pro Asn Gly His Val

355 360

Gln Val Thr Thr Tyr Lys Ala Val Ile Gln

370 375

Phe Tyr Thr Met Ser Ser Asn Gly Lys Tyr

385 390

Phe Trp Thr Ser Ser Lys Gly Glu Lys Leu

405 410

Val Cys Gly Leu Ser Thr His Thr Ile Gly

420 425

Gln Pro Ala Lys Pro Gly Asp Phe Pro Trp

435 440

Gln Thr Thr Ala Ala Ala Gly Ala Leu Ile

450 455

Thr Ala Ala His Ala Val Tyr Glu Lys Arg

465 470

Asn Ile Arg Met Gly Ile Leu Lys Arg Leu

485 490

Ala Trp Pro Glu Glu Ile Phe Ile His Glu

500 505

Gly Phe Asp Asn Asp Ile Ala Leu Ile Lys

515 520

Ile Asn Gly Ser Ile Met Pro Val Cys Leu

530 535

Ser Leu Met Arg Thr Asp Phe Thr Gly Thr

545 550

Thr Gln Lys Gly Leu Leu Ala Arg Asn Leu

565 570

Ile Ala Asp His Gln Lys Cys Thr Thr Val

580 585

Gly Val Arg Val Ser Ala Asn Met Leu Cys

595 600

Gly Lys Asp Ser Cys Arg Gly Asp Ser Gly

610 615

Asp Asn Glu Thr Gln Arg Trp Phe Val Gly

Tyr Ile Thr Gly Pro

365

Ser Cys Glu Glu Thr 380

Cys Glu Ala Asp Gly

400

Pro Val Cys Glu Pro

415

Arg Ile Val Gly Gly 430

Val Leu Leu Leu Gly

445

Asp Asn Trp Val Leu 460

Ala Ala Ser Ser Leu

480

Pro His Tyr Thr Gln

495

Tyr Thr His Gly Ala 510

Lys Asn Lys Val Thr

525

Arg Lys Glu Ala Ala 540

Ala Gly Trp Gly Leu

560

Phe Val Asp Ile Pro

575

Glu Lys Leu Tyr Pro

590

Gly Leu Glu Thr Gly 605

Ala Leu Val Phe Leu 620

Ile Val Ser Trp Gly

- 102 045598

625 630 635640

Ser Ile Asn Cys Gly Ala Ala Gly Gln

645

Ile Asn Tyr Ile Pro Trp Asn Glu Asn

660665

Tyr Gly Val 650

Tyr Thr Lys Val

655

Ile Ile Ser Asn Phe

670 <210>53 <211>2091 <212> DNA <213> Rat <220>

<221> CDS <222> (10).. (2067) <400>53 tggcacaca atg agg cta ctg atc gtc ctg ggt ctg ctt tgg agt ttg gtg51

Met Arg Leu Leu Ile Val Leu Gly Leu Leu Trp Ser Leu Val 1 510 gcc aca ctt ttg ggc tcc aag tgg cct gag cct gta ttc ggg cgc ctg99

Ala Thr Leu Leu Gly Ser Lys Trp Pro Glu Pro Val Phe Gly ArgLeu

20 2530 gtg tcc ctg gcc ttc cca gag aag tat ggc aac cat cag gat cga tcc147

Val Ser Leu Ala Phe Pro Glu Lys Tyr Gly Asn His Gln Asp ArgSer

4045 tgg acg ctg act gca ccc cct ggc ttc cgc ctg cgc ctc tac ttc acc195

Trp Thr Leu Thr Ala Pro Pro Gly Phe Arg Leu Arg Leu Tyr PheThr

5560 cac ttc aac ctg gaa ctc tct tac cgc tgc gag tat gac ttt gtc aag243

His Phe Asn Leu Glu Leu Ser Tyr Arg Cys Glu Tyr Asp Phe ValLys

7075 ttg acc tca ggg acc aag gtg cta gcc acg ctg tgt ggg cag gag agt291

Leu Thr Ser Gly Thr Lys Val Leu Ala Thr Leu Cys Gly Gln GluSer

8590 aca gat act gag cgg gca cct ggc aat gac acc ttc tac tca ctg ggt339

Thr Asp Thr Glu Arg Ala Pro Gly Asn Asp Thr Phe Tyr Ser LeuGly

100 105110 ccc agc cta aag gtc acc ttc cac tcc gac tac tcc aat gag aag cca387

Pro Ser Leu Lys Val Thr Phe His Ser Asp Tyr Ser Asn Glu LysPro

115 120125 ttc aca gga ttt gag gcc ttc tat gca gcg gag gat gtg gat gaa tgc435

Phe Thr Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp Val Asp GluCys

130 135140 aga aca tcc ctg gga gac tca gtc cct tgt gac cat tat tgc cac aac483

Arg Thr Ser Leu Gly Asp Ser Val Pro Cys Asp His Tyr Cys HisAsn

145 150155 tac ctg ggc ggc tac tac tgc tcc tgc cga gtg ggc tac att ctg cac531

Tyr Leu Gly Gly Tyr Tyr Cys Ser Cys Arg Val Gly Tyr Ile LeuHis

160 165170 cag aac aag cat acc tgc tca gcc ctt tgt tca ggc cag gtg ttc act579

Gln Asn Lys His Thr Cys Ser Ala Leu Cys Ser Gly Gln Val PheThr

175 180 185190

- 103 045598 ggg agg tct

Gly Arg Ser aaa ctc tcc

Lys Leu Ser atc acc ctg Ile Thr Leu 225 gcc cag tgc Ala Gln Cys 240 tac ggc ccg Tyr Gly Pro 255 agc aac aag Ser Asn Lys aca ggc tgg

Thr Gly Trp cca acg gcg Pro Thr Ala 305 gtc ctg aag

Val Leu Lys 320 ctg caa ggt Leu Gln Gly 335 gat gga tct Asp Gly Ser ggc cct ccc

Gly Pro Pro cct gaa gtg Pro Glu Val 385 act ttc tac Thr Phe Tyr 400 gga ttc tgg Gly Phe Trp 415 cct gtc tgt Pro Val Cys gga cag cct Gly Gln Pro ggt gaa act Gly Glu Thr ggc ttt ctc agt agc Gly Phe Leu Ser Ser 195 agc tgc gcc tac aac Ser Cys Ala Tyr Asn 210 gac ttc gtg gag tcc Asp Phe Val Glu Ser 230 ccc tac gac tcc ctc Pro Tyr Asp Ser Leu 245 ttt tgt ggg aag acg Phe Cys Gly Lys Thr 260 gtg acc att acc ttt

Val Thr Ile Thr Phe 275 aag ata cac tac aca Lys Ile His Tyr Thr 290 cca cct aat ggt cac Pro Pro Asn Gly His 310 gac agc ttt tct gtc Asp Ser Phe Ser Val 325 tct gtc ccc ctg aag Ser Val Pro Leu Lys 340 tgg gac cgg ccg ata Trp Asp Arg Pro Ile 355 gat gac cta ccc aat Asp Asp Leu Pro Asn 370 acc acc tac aaa gct

Thr Thr Tyr Lys Ala 390 aca atg agc agc aat Thr Met Ser Ser Asn 405 acg agc tcc aaa gga Thr Ser Ser Lys Gly 420 gga ctg tcc aca cac Gly Leu Ser Thr His 435 gca aag cct ggt gac Ala Lys Pro Gly Asp 450 aca gca gca ggt gct Thr Ala Ala Gly Ala cct gag tac Pro Glu Tyr 200 atc cgc ctg Ile Arg Leu 215 ttt gat gtg Phe Asp Val aag att caa

Lys Ile Gln ctg ccc ccc

Leu Pro Pro 265 acc acc gac

Thr Thr Asp 280 agc aca gca

Ser Thr Ala 295 att tca cct

Ile Ser Pro ttc tgc aag

Phe Cys Lys tca ttc act Ser Phe Thr 345 cca gag tgc Pro Glu Cys 360 ggc cac gtg Gly His Val 375 gtg att cag

Val Ile Gln ggt aaa tat Gly Lys Tyr gaa aaa tcc Glu Lys Ser 425 act tca gga Thr Ser Gly 440 ttt cct tgg Phe Pro Trp 455 ctt ata cat

Leu Ile His cca cag cca tac Pro Gln Pro Tyr 205 gag gaa ggc ttc Glu Glu Gly Phe 220 gag atg cac cct Glu Met His Pro 235 aca gac aag agg Thr Asp Lys Arg 250 agg att gaa act Arg Ile Glu Thr gag tca ggg aac Glu Ser Gly Asn 285 cag ccc tgc cct Gln Pro Cys Pro 300 gtg caa gcc acg

Val Gln Ala Thr 315 act ggc ttc gag Thr Gly Phe Glu 330 gct gtc tgt cag Ala Val Cys Gln agc att att gac Ser Ile Ile Asp 365 gac tat atc aca Asp Tyr Ile Thr 380 tac agc tgt gaa Tyr Ser Cys Glu 395 gtg tgt gag gct

Val Cys Glu Ala 410 ctc ccg gtt tgc Leu Pro Val Cys ggc cgt ata att Gly Arg Ile Ile 445 caa gtc ttg tta Gln Val Leu Leu 460 gac gac tgg gtc Asp Asp Trp Val ccc 627

Pro agt 675

Sergaa 723

Glugaa 771

Glu gac 819

Asp 270 ca 867

His gat 915

Asp tat 963

Tyr ctt 1011

Leu aaa 1059

Lys

350 tgt 1107

Cys ggc 1155

Gly gag 1203

Glu gat 1251

Asp aag 1299

Lys

430 gga 1347

Gly ctg 1395

Leucta 1443

Leu

- 104 045598

465

470

475 aca gcg Thr Ala 480 gac atc Asp Ile 495 gcc tgg Ala Trp ggt ttt

Gly Phe atc aac

Ile Asn gct cat gct gta tat ggg aaa aca Ala His Ala Val Tyr Gly Lys Thr 485 cgc atg ggc atc ctc aaa agg ctc

Arg Met Gly Ile Leu Lys Arg Leu

500 cca gag gct gtc ttt atc cat gaa

Pro Glu Ala Val Phe Ile His Glu

515520 gac aat gat ata gca ctg attaaa

Asp Asn Asp Ile Ala Leu IleLys

530535 aga aac atc atg ccg att tgtcta

Arg Asn Ile Met Pro Ile CysLeu

545550 gag gcg atg tcc Glu Ala Met Ser 490 tcc ctc att tac Ser Leu Ile Tyr 505 ggc tac act cac Gly Tyr Thr His ctc aag aac aaa Leu Lys Asn Lys 540 cca aga aaa gaa Pro Arg Lys Glu 555 tcc ctg Ser Leu act caa Thr Gln 510 gga gct Gly Ala 525 gtc aca

Val Thr gct gca

Ala Ala

1491

1539

1587

1635

1683 tcc tta atg aaa aca gac ttc gtt gga

Ser Leu Met Lys Thr Asp Phe Val Gly

560 565 acc cag aag ggg ttt ctt gct aga aac

Thr Gln Lys Gly Phe Leu Ala Arg Asn

575 580 act gtg gct Thr Val Ala 570 cta atg ttt Leu Met Phe 585 ggc tgg

Gly Trp ggg tta

Gly Leu

1731 gtg gac

Val Asp ata cca Ile Pro 590

1779 att gtt gac cac caa aaa tgt gct Ile Val Asp His Gln Lys Cys Ala 595 act gcg tat aca aag cag ccc tac Thr Ala Tyr Thr Lys Gln Pro Tyr 600 605

1827 cca gga gca aaa gtg act Pro Gly Ala Lys Val Thr 610 ggt ggc aag gac agc tgc Gly Gly Lys Asp Ser Cys 625 cta gac aat gaa aca cag Leu Asp Asn Glu Thr Gln 640 ggt tct att aac tgt ggg Gly Ser Ile Asn Cys Gly 655 660 gtc acg aac tat att ccc Val Thr Asn Tyr Ile Pro 675 gtt aac atg ctc tgt gct ggc cta

Val Asn Met Leu Cys Ala Gly Leu 615 620 aga ggt gac agc gga ggg gca tta Arg Gly Asp Ser Gly Gly Ala Leu 630635 aga tgg ttt gtg gga gga atagtt

Arg Trp Phe Val Gly Gly IleVal

645650 ggg tca gaa cag tat ggg gtc tac

Gly Ser Glu Gln Tyr Gly Val Tyr

665 tgg att gag aac ata ata aat aat

Trp Ile Glu Asn Ile Ile Asn Asn

680 tttgcaaaaa aaaaaaaaaa aaaa <210> 54 <211> 685 <212> PRT <213> Rat <400> 54

Met Arg Leu Leu 1

Ile Val Leu Gly Leu Leu

10

Leu Leu Gly Ser Lys Trp Pro Glu Pro Val

25 gac cgc

Asp Arg gtg ttt

Val Phe tcc tgg Ser Trp acg aaa Thr Lys 670 ttc taa

Phe

685

Trp Ser Leu Val Ala Thr

Phe Gly Arg Leu Val

Ser

1875

1923

1971

2019

2067

2091

- 105 045598

Leu Ala Phe Pro Glu Lys Tyr Gly Asn His

40

Leu Thr Ala Pro Pro Gly Phe Arg Leu Arg

55

Asn Leu Glu Leu Ser Tyr Arg Cys Glu Tyr 65 70

Ser Gly Thr Lys Val Leu Ala Thr Leu Cys

90

Thr Glu Arg Ala Pro Gly Asn Asp Thr Phe

100 105

Leu Lys Val Thr Phe His Ser Asp Tyr Ser

115 120

Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp

130 135

Ser Leu Gly Asp Ser Val Pro Cys Asp His

145 150

Gly Gly Tyr Tyr Cys Ser Cys Arg Val Gly

165 170

Lys His Thr Cys Ser Ala Leu Cys Ser Gly

180 185

Ser Gly Phe Leu Ser Ser Pro Glu Tyr Pro

195 200

Ser Ser Cys Ala Tyr Asn Ile Arg Leu Glu

210 215

Leu Asp Phe Val Glu Ser Phe Asp Val Glu

225 230

Cys Pro Tyr Asp Ser Leu Lys Ile Gln Thr

245 250

Pro Phe Cys Gly Lys Thr Leu Pro Pro Arg

260 265

Lys Val Thr Ile Thr Phe Thr Thr Asp Glu

275 280

Trp Lys Ile His Tyr Thr Ser Thr Ala Gln

290 295

Asp Arg Ser Trp Thr 45

Tyr Phe Thr His Phe 60

Phe Val Lys Leu Thr 80

Gln Glu Ser Thr Asp

Ser Leu Gly Pro Ser 110

Glu Lys Pro Phe Thr 125

Asp Glu Cys Arg Thr 140

Cys His Asn Tyr Leu

160

Ile Leu His Gln Asn

175

Val Phe Thr Gly Arg

190

Pro Tyr Pro Lys Leu

205

Gly Phe Ser Ile Thr 220

His Pro Glu Ala Gln

240

Lys Arg Glu Tyr Gly

255

Glu Thr Asp Ser Asn 270

Gly Asn His Thr Gly 285

Cys Pro Asp Pro Thr 300

- 106 045598

Ala Pro Pro Asn Gly His Ile Ser Pro Val 305 310

Lys Asp Ser Phe Ser Val Phe Cys Lys Thr

325 330

Gly Ser Val Pro Leu Lys Ser Phe Thr Ala

340 345

Ser Trp Asp Arg Pro Ile Pro Glu Cys Ser

355 360

Pro Asp Asp Leu Pro Asn Gly His Val Asp

370 375

Val Thr Thr Tyr Lys Ala Val Ile Gln Tyr

385 390

Tyr Thr Met Ser Ser Asn Gly Lys Tyr Val

405 410

Trp Thr Ser Ser Lys Gly Glu Lys Ser Leu

420 425

Cys Gly Leu Ser Thr His Thr Ser Gly Gly

435 440

Pro Ala Lys Pro Gly Asp Phe Pro Trp Gln

450 455

Thr Thr Ala Ala Gly Ala Leu Ile His Asp

465 470

Ala His Ala Val Tyr Gly Lys Thr Glu Ala

485 490

Arg Met Gly Ile Leu Lys Arg Leu Ser Leu

500 505

Pro Glu Ala Val Phe Ile His Glu Gly Tyr

515 520

Asp Asn Asp Ile Ala Leu Ile Lys Leu Lys

530 535

Arg Asn Ile Met Pro Ile Cys Leu Pro Arg

545 550

Met Lys Thr Asp Phe Val Gly Thr Val Ala

565 570

Lys Gly Phe Leu Ala Arg Asn Leu Met Phe

580 585

Ala Thr Tyr Val Leu

320

Phe Glu Leu Leu Gln

335

Cys Gln Lys Asp Gly 350

Ile Asp Cys Gly Pro

365

Ile Thr Gly Pro Glu 380

Cys Glu Glu Thr Phe

400

Glu Ala Asp Gly Phe

415

Val Cys Lys Pro Val

430

Ile Ile Gly Gly Gln

445

Leu Leu Leu Gly Glu 460

Trp Val Leu Thr Ala

480

Ser Ser Leu Asp Ile

495

Tyr Thr Gln Ala Trp 510

His Gly Ala Gly Phe

525

Lys Val Thr Ile Asn 540

Glu Ala Ala Ser Leu

560

Trp Gly Leu Thr Gln

575

Asp Ile Pro Ile Val 590

- 107 045598

Asp His Gln Lys

595

Cys

Ala

Thr

Ala

600

Tyr

Thr

Lys

Gln

Pro

605

Tyr

Pro

Gly

Ala Lys Val Thr 610

Val

Asn

Met

615

Leu

Cys

Ala

Gly

Leu

620

Asp

Arg

Gly

Gly

Lys Asp Ser Cys 625

Arg

Gly

630

Asp

Ser

Gly

Gly

Ala

635

Leu

Val

Phe

Leu

Asp

640

Asn Glu Thr Gln

Arg

645

Trp

Phe

Val

Gly

Gly

650

Ile

Val

Ser

Trp

Gly 655

Ser

Ile Asn Cys Gly

660

Gly

Ser

Glu

Gln

Tyr 665

Gly

Val

Tyr

Thr

Lys

670

Val

Thr

Asn Tyr Ile Pro

675

Trp

Ile

Glu

Asn

680

Ile

Ile

Asn

Asn

Phe

685 <210>55 <211>670 <212> PRT <213> Rat <400>55

Thr Leu Leu Gly 1

Ser 5

Lys

Trp

Pro

Glu

Pro 10

Val

Phe

Gly

Arg

Leu 15

Val

Ser Leu Ala Phe

Pro

Glu

Lys

Tyr

Gly 25

Asn

His

Gln

Asp

Arg 30

Ser

Trp

Thr Leu Thr Ala

Pro

Pro

Gly

Phe 40

Arg

Leu

Arg

Leu

Tyr 45

Phe

Thr

His

Phe Asn Leu Glu

Leu

Ser

Tyr 55

Arg

Cys

Glu

Tyr

Asp 60

Phe

Val

Lys

Leu

Thr Ser Gly Thr 65

Lys

Val 70

Leu

Ala

Thr

Leu

Cys 75

Gly

Gln

Glu

Ser

Thr 80

Asp Thr Glu Arg

Ala 85

Pro

Gly

Asn

Asp

Thr 90

Phe

Tyr

Ser

Leu

Gly 95

Pro

Ser Leu Lys Val

100

Thr

Phe

His

Ser

Asp

105

Tyr

Ser

Asn

Glu

Lys

110

Pro

Phe

Thr Gly Phe Glu

115

Ala

Phe

Tyr

Ala

120

Ala

Glu

Asp

Val

Asp 125

Glu

Cys

Arg

Thr Ser Leu Gly 130

Asp

Ser

Val

135

Pro

Cys

Asp

His

Tyr 140

Cys

His

Asn

Tyr

Leu Gly Gly Tyr

Tyr

Cys

Ser

Cys

Arg

Val

Gly

Tyr

Ile

Leu

His

Gln

- 108 045598

160

145 150

Asn Lys His Thr Cys Ser Ala Leu Cys Ser

165 170

Arg Ser Gly Phe Leu Ser Ser Pro Glu Tyr

180 185

Leu Ser Ser Cys Ala Tyr Asn Ile Arg Leu

195 200

Thr Leu Asp Phe Val Glu Ser Phe Asp Val

210 215

Gln Cys Pro Tyr Asp Ser Leu Lys Ile Gln

225 230

Gly Pro Phe Cys Gly Lys Thr Leu Pro Pro

245 250

Asn Lys Val Thr Ile Thr Phe Thr Thr Asp

260 265

Gly Trp Lys Ile His Tyr Thr Ser Thr Ala

275 280

Thr Ala Pro Pro Asn Gly His Ile Ser Pro

290 295

Leu Lys Asp Ser Phe Ser Val Phe Cys Lys

305 310

Gln Gly Ser Val Pro Leu Lys Ser Phe Thr

325 330

Gly Ser Trp Asp Arg Pro Ile Pro Glu Cys

340 345

Pro Pro Asp Asp Leu Pro Asn Gly His Val

355 360

Glu Val Thr Thr Tyr Lys Ala Val Ile Gln

370 375

Phe Tyr Thr Met Ser Ser Asn Gly Lys Tyr 385 390

Phe Trp Thr Ser Ser Lys Gly Glu Lys Ser

405 410

Val Cys Gly Leu Ser Thr His Thr Ser Gly

420 425

Gln Val Phe Thr Gly

175

Gln Pro Tyr Pro Lys 190

Glu Gly Phe Ser Ile

205

Met His Pro Glu Ala 220

Asp Lys Arg Glu Tyr

240

Ile Glu Thr Asp Ser

255

Ser Gly Asn His Thr 270

Pro Cys Pro Asp Pro

285

Gln Ala Thr Tyr Val 300

Gly Phe Glu Leu Leu

320

Val Cys Gln Lys Asp

335

Ile Ile Asp Cys Gly 350

Tyr Ile Thr Gly Pro

365

Ser Cys Glu Glu Thr 380

Cys Glu Ala Asp Gly

400

Pro Val Cys Lys Pro

415

Arg Ile Ile Gly Gly 430

- 109 045598

Gln Pro Ala Lys Pro Gly Asp Phe Pro Trp

435 440

Glu Thr Thr Ala Ala Gly Ala Leu Ile His

450 455

Ala Ala His Ala Val Tyr Gly Lys Thr Glu 465 470

Ile Arg Met Gly Ile Leu Lys Arg Leu Ser

485 490

Trp Pro Glu Ala Val Phe Ile His Glu Gly

500 505

Phe Asp Asn Asp Ile Ala Leu Ile Lys Leu

515 520

Asn Arg Asn Ile Met Pro Ile Cys Leu Pro

530 535

Leu Met Lys Thr Asp Phe Val Gly Thr Val 545 550

Gln Lys Gly Phe Leu Ala Arg Asn Leu Met

565 570

Val Asp His Gln Lys Cys Ala Thr Ala Tyr

580 585

Gly Ala Lys Val Thr Val Asn Met Leu Cys

595 600

Gly Lys Asp Ser Cys Arg Gly Asp Ser Gly

610 615

Asp Asn Glu Thr Gln Arg Trp Phe Val Gly

625 630

Ser Ile Asn Cys Gly Gly Ser Glu Gln Tyr

645 650

Thr Asn Tyr Ile Pro Trp Ile Glu Asn Ile

660 665

Val Leu Leu Leu Gly

445

Asp Trp Val Leu Thr 460

Met Ser Ser Leu Asp

480

Ile Tyr Thr Gln Ala

495

Thr His Gly Ala Gly 510

Asn Lys Val Thr Ile 525

Lys Glu Ala Ala Ser 540

Gly Trp Gly Leu Thr

560

Val Asp Ile Pro Ile

575

Lys Gln Pro Tyr Pro

590

Gly Leu Asp Arg Gly 605

Ala Leu Val Phe Leu 620

Ile Val Ser Trp Gly

640

Val Tyr Thr Lys Val

655

Asn Asn Phe

670 <210> 56 <211> 28 <212> DNA <213> Artificial sequence <220>

< 223> Homo Sapiens < 400> 56

- 110 045598 atgaggctgc tgaccctcct gggccttc <210> 57 <211> 23 <212> DNA <213> Artificial sequence <220>

< 223> Homo Sapiens < 400>57 gtgcccctcc tgcgtcacct ctg23 < 210>58 < 211>23 < 212> DNA < 213> Artificial sequence <220>

< 223> Homo Sapiens < 400>58 cagaggtgac gcaggagggg cac23 < 210>59 < 211>27 < 212> DNA < 213> Artificial sequence <220>

< 223> Homo Sapiens < 400>59 ttaaaatcac taattatgtt ctcgatc27 < 210>60 < 211>22 < 212> DNA < 213> Artificial sequence <220>

<223> Mouse <400>60 atgaggctac tcatcttcct gg22 <210>61 <211>23 <212> DNA <213> Artificial sequence <220>

<223> Mouse <400>61 ctgcagaggt gacgcagggg ggg23 <210>62 <211>23 <212> DNA <213> Artificial sequence <220>

<223> Mouse

- 111 045598 <400> 62 ccccccctgc gtcacctctg cag <210> 63 <211> 29 <212> DNA <213> Artificial sequence <220>

<223> Mouse <400> 63 ttagaaatta cttattatgt tctcaatcc <210> 64 <211> 29 <212> DNA <213> Artificial sequence <220>

<223> Rat <400> 64 gaggtgacgc aggaggggca ttagtgttt <210> 65 <211> 37 <212> DNA <213> Artificial sequence <220>

<223> Rat <400> 65 ctagaaacac taatgcccct cctgcgtcac ctctgca <210> 66 <211> 354 <212> DNA <213> Artificial sequence <220>

<223> Synthetic <400> 66 caggtcacct acctgcaccg cagcccccag tacaggacat gtccttacaa cgacgtggag tgaaggagtc tctctgggtt ggaaggccct cgctgaagag tgaccaacat gaattgacta tggtcctgtg ctcactcagc ggagtggctt caggctcacc ggaccctgt g ctggggccag ctggtgaaac aggggtaaaa gcacacattt atctccaagg gacacagcca ggaaccctgg ccacagagac tgggtgtgag tttcgagtga acacctccaa cgtattactg tcactgtctc cctcacgctg ctggatccgt cgaaaaatcc aaaccaggtg tgcacggata ctca

120

180

240

300

354 <210> 67 <211> 118 <212> PRT <213> Artificial sequence <220>

< 223> Synthetic

- 112 045598 <400> 67

Gln Val Thr Leu Lys Glu Ser Gly Pro Val

5 10

Thr Leu Thr Leu Thr Cys Thr Val Ser Gly

25

Lys Met Gly Val Ser Trp Ile Arg Gln Pro

40

Trp Leu Ala His Ile Phe Ser Ser Asp Glu

55

Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp 65 70

Val Leu Thr Met Thr Asn Met Asp Pro Val 85 90

Cys Ala Arg Ile Arg Arg Gly Gly Ile Asp

100 105

Leu Val Thr Val Ser Ser

115 <210> 68 <211> 121 <212> PRT <213> Artificial sequence <220>

<223> Synthetic <400> 68

Gln Val Gln Leu Gln Gln Ser Gly Pro Gly

5 10

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly

25

Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser

40

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys

55

Val Ser Val Lys Ser Arg Ile Thr Ile Asn 65 70

Gln Phe Ser Leu Gln Leu Asn Ser Val Thr

90

Tyr Tyr Cys Ala Arg Asp Pro Phe Gly Val

Val Lys Pro Thr Glu

Ser Leu Ser Arg Gly 30

Gly Lys Ala Leu Glu 45

Ser Tyr Arg Thr Ser 60

Ser Lys Asn Gln Val 80

Thr Ala Thr Tyr Tyr 95

Trp Gly Gln Gly Thr 110

Val Lys Pro Ser Gln

Ser Val Ser Ser Thr

Ser Arg Gly Leu Glu 45

Tyr Asn Asp Tyr Ala 60

Asp Thr Ser Lys Asn 80

Glu Asp Thr Ala Val 95

Phe Asp Ile Trp Gly

- 113 045598

100

105

110

Gln Gly Thr Met Val Thr Val Ser Ser

115

120 <210> 69 <211> 106 <212> PRT <213> Artificial sequence <220>

<223> Synthetic <400>69

Gln 1 Pro Val Leu Thr 5 Gln Pro Pro Ser Leu 10 Ser Val Ser Pro Gly 15 Gln Thr Ala Ser Ile 20 Thr Cys Ser Gly Glu 25 Lys Leu Gly Asp Lys 30 Tyr Ala Tyr Trp Tyr 35 Gln Gln Lys Pro Gly 40 Gln Ser Pro Val Leu 45 Val Met Tyr Gln Asp 50 Lys Gln Arg Pro Ser 55 Gly Ile Pro Glu Arg 60 Phe Ser Gly Ser Asn 65 Ser Gly Asn Thr Ala 70 Thr Leu Thr Ile Ser 75 Gly Thr Gln Ala Met 80 Asp Glu Ala Asp Tyr 85 Tyr Cys Gln Ala Trp 90 Asp Ser Ser Thr Ala 95 Val Phe Gly Gly Gly 100 Thr Lys Leu Thr Val 105 Leu

<210>70 <211>324 <212> DNA <213> Artificial sequence <220>

< 223> Synthetic <400> 70 tcctatgagc tgatacagcc accctcggtg tcagtggccc caggacagac ggccaccatt acctgtgcgg gagacaacct tgggaagaaa cgtgtgcact ggtaccagca gaggccaggc caggcccctg tgttggtcat ctatgatgat agcgaccggc cctcagg gat ccctgaccga ttctctgcct ccaactctgg gaacacggcc accctgacca tcactagggg cgaagccggg gatgaggccg actattattg tcaggtgtgg gacattgcta ctgatcatgt ggtcttcggc ggagggacca agctcaccgt ccta

120

180

240

300

324 <210> 71 <211> 120 <212> PRT

- 114 -

Claims (6)

<213> Artificial sequence <220> <223> Synthetic <400>71 Ser Tyr Glu Leu Ile Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 1015 Thr Ala Thr Ile Thr Cys Ala Gly Asp Asn Leu Gly Lys Lys Arg Val 20 2530 His Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 4045 Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Ala Ser 50 5560 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Thr Arg Gly Glu Ala Gly 65 70 7580 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ile Ala Thr Asp His 85 9095 Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ala Ala Ala Gly 100 105110 Ser Glu Gln Lys Leu Ile Ser Glu 115120 CLAIM 1. A method of treating a mammalian patient suffering from an angiogenesis-dependent malignancy selected from the group consisting of colorectal cancer, breast cancer, lung cancer, kidney cancer, liver cancer, esophageal cancer, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, glioma, stromal tumor gastrointestinal tract, lymphoma, melanoma and carcinoid tumor, or an angiogenesis-dependent benign tumor selected from the group consisting of hemangioma, acoustic neuroma, neurofibroma, trachoma, carcinoid tumors and pyogenic granulomas, comprising administering to the patient a composition containing a monoclonal antibody or a MASP-2 inhibitory fragment that specifically binds to a portion of SEQ ID NO: 6, wherein the MASP-2 inhibitory monoclonal antibody or fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 67 and a light chain variable region comprising SEQ ID NO: 69. 2. The method of claim 1, wherein the patient has an angiogenesis-dependent malignant tumor selected from the group consisting of: solid tumor(s), blood-borne tumors, high-risk carcinoid tumors, and metastatic tumors. 3. The method of claim 2, wherein the patient has one or more solid tumors, and the method includes administering a MASP-2 inhibitor in an amount effective to inhibit tumor angiogenesis. 4. The method of claim 3, wherein the patient suffers from or is at risk of tumor metastasis, and the method includes administering a MASP-2 inhibitor in an amount effective to inhibit tumor metastasis. 5. The method of claim 1, wherein the antibody or fragment thereof is selected from the group consisting of a recombinant antibody, an antibody with reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody. 6. The method according to claim 1, where the composition is administered subcutaneously, intraperitoneally, intramuscularly, intraarterially or intravenously. - 115 -