Compositions for inhibiting MASP-2 dependent complement activation

Abstract

To provide therapeutically effective complement inhibitors to prevent tissue damage in many clinical conditions involving the complement system.SOLUTION: The present invention provides fully human antibodies that bind to human MASP-2 and inhibit lectin-mediated complement activation while leaving the classical (C1q-dependent) pathway component of the immune system intact. The human anti-MASP-2 antibodies are useful for inhibiting cellular injury associated with lectin-mediated complement pathway activation while leaving the classical (C1q-dependent) pathway component of the immune system intact.SELECTED DRAWING: None

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to anti-MASP-2 inhibitory antibodies and compositions comprising such antibodies for use in inhibiting the deleterious effects of MASP-2-dependent complement activation.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of Provisional Application No. 61 / 482,567, filed May 4, 2011, which is incorporated herein by reference in its entirety.

[0003] SEQUENCE LISTING STATEMENT The sequence listing accompanying this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference herein. The name of the text file containing the sequence listing is MP_1_0115_PCT_SequenceListingasFiled_20120504_ST25. This text file is 158KB; was created on May 4, 2012; and has been submitted via EFS-Web with the filing of this application. [Background technology]

[0004] background The complement system provides an early-acting mechanism to initiate, amplify and direct immune responses to microbial infections and other acute insults in humans and other vertebrates (Non-Patent Document 1). Complement activation provides an important first line of defense against potential pathogens, but complement activity in promoting protective immune responses can also be a potential threat to the host (Non-Patent Document 2; Non-Patent Document 3). For example, C3 and C5 proteolytic products recruit and activate neutrophils. Activated neutrophils are essential for host defense, but can be indiscriminate in the release of destructive enzymes and cause organ damage. Furthermore, complement activation can cause deposition of lytic complement components in the vicinity of host cells, as well as on microbial targets, resulting in host cell lysis.

[0005] The complement system has also been implicated in the pathogenesis of many acute and chronic disease states, including myocardial infarction, stroke, acute respiratory distress syndrome (ARDS), reperfusion injury, septic shock, capillary leakage after burn injury, inflammation after cardiopulmonary bypass, graft rejection, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, and Alzheimer's disease. In almost all of these conditions, complement is one of several factors involved in the pathogenesis, although it is not the cause. Nevertheless, complement activation may be the primary pathological mechanism and is an effective issue for clinical management in many of these disease states.

[0006] Increasing recognition of the importance of complement-mediated tissue damage in various disease states highlights the need for effective complement-inhibiting drugs. To date, Eculizumab (Soliris®), an antibody against C5, is the only complement-targeting drug approved for use in humans. However, C5 is one of several effector molecules located "downstream" of the complement system, and blocking C5 does not inhibit activation of the complement system. Thus, inhibitors of the initiation step of complement activation would have a significant advantage over "downstream" complement inhibitors.

[0007] It is now widely accepted that the complement system can be activated through three separate pathways: the classical pathway, the lectin pathway, and the alternative pathway. Usually, the classical pathway is triggered by a complex composed of a host antibody bound to a foreign particle (i.e., an antigen), and thus requires prior exposure to the antigen to mount a specific antibody response. Since activation of the classical pathway depends on a prior adaptive immune response by the host, the classical pathway is part of the adaptive immune system. In contrast, both the lectin pathway and the alternative pathway are independent of adaptive immunity and are part of the innate immune system.

[0008] Activation of the complement system leads to the sequential activation of serine protease zymogens. The first step in activation of the classical pathway is the binding of a specific recognition molecule, C1q, to ​​antigen-bound IgG and IgM complexes. C1q associates with C1r and C1s serine protease proenzymes as a complex called C1. Binding of C1q to immune complexes results in autoproteolytic cleavage at the Arg-Ile site of C1r, followed by C1r-mediated cleavage and activation of C1s, thereby acquiring the ability to cleave C4 and C2. C4 is cleaved into two fragments, designated C4a and C4b, and similarly, C2 is cleaved into C2a and C2b. The C4b fragment is now capable of forming a covalent bond with an adjacent hydroxyl or amino group, and can generate the C3 convertase (C4b2a) by noncovalent interaction with the C2a fragment of activated C2. C3 convertase (C4b2a) activates C3 by proteolytic cleavage into the C3a and C3b subcomponents to generate C5 convertase (C4b2a3b), which cleaves C5 to form the membrane attack complex (C5b combined with C6, C7, C8, and C9, also called "MAC"), which can disrupt cell membranes and result in cell lysis. The activated forms of C3 and C4 (C3b and C4b) are covalently deposited on foreign target surfaces where they are recognized by complement receptors on multiple phagocytes.

[0009] Independently, the first step in the activation of the complement system by the lectin pathway is also the binding of specific recognition molecules followed by the activation of associated serine protease proenzymes. However, the recognition molecules in the lectin pathway are not the binding of immune complexes by C1q, but include a group of carbohydrate-binding proteins (mannan-binding lectin (MBL), H-ficolin, M-ficolin, L-ficolin and C-type lectin CL-11) (collectively called lectins). See Non-Patent Document 4; Non-Patent Document 5; Non-Patent Document 6. See also Non-Patent Document 4; Non-Patent Document 5; Non-Patent Document 6; Hansen S. et al., J.Immunol 185(10):6096-6104(2010).

[0010] Ikeda et al. were the first to demonstrate that, like C1q, MBL can activate the complement system in a C4-dependent manner when bound to yeast mannan-coated erythrocytes (Non-Patent Document 7). MBL, a member of the collectin protein family, is a calcium-dependent lectin that binds carbohydrates through the equatorially oriented 3- and 4-hydroxy groups of the pyranose ring. Thus, the predominant ligands for MBL are D-mannose and N-acetyl-D-glucosamine, while carbohydrates that do not meet this steric requirement have no detectable affinity for MBL (Non-Patent Document 8). The interaction of MBL with monovalent sugars is very weak, with dissociation constants typically in the single-digit millimolar range. MBL achieves tight and specific binding to glycan ligands by avidity with multiple monosaccharide residues that are closely positioned to each other, i.e., by interacting simultaneously (Non-Patent Document 9). MBL normally recognizes carbohydrate patterns that are borne by microorganisms (e.g., bacteria, yeast, parasites, and certain viruses). In contrast, MBL does not recognize the penultimate and terminal sugars D-galactose and sialic acid normally found on "mature" complex glycoconjugates present on mammalian plasma glycoproteins and cell surface glycoproteins. This binding specificity is thought to facilitate recognition of "foreign" surfaces and help protect against autoactivation. However, MBL binds with high affinity to a population of high-mannose "precursor" glycans on N-linked glycoproteins and glycolipids sequestered within the endoplasmic reticulum and Golgi of mammalian cells (10). Thus, damaged cells can be targeted for lectin pathway activation via MBL binding.

[0011] Ficolin has a type of lectin domain called a fibrinogen-like domain, which is different from that of MBL. Ficolin binds Ca ++They bind to sugar residues in a C-dependent manner. In humans, three types of ficolins (L-ficolin, M-ficolin, and H-ficolin) have been identified. Two serum ficolins, L-ficolin and H-ficolin, share a common specificity for N-acetyl-D-glucosamine; however, H-ficolin also binds to N-acetyl-D-galactosamine. The difference in sugar specificity between L-ficolin, H-ficolin, CL-11, and MBL means that the various lectins may be complementary and target different but overlapping glycoconjugates. This concept is supported by a recent report of known lectins in the lectin pathway, in which only L-ficolin specifically binds to lipoteichoic acid, a cell wall glycoconjugate found on all gram-positive bacteria (Non-Patent Document 11). Collectins (i.e., MBL) and ficolins do not share significant similarity in amino acid sequence. However, the two groups of proteins have similar domain composition and, like C1q, are organized into oligomeric structures that maximize the potential for multiple site binding.

[0012] The serum concentration of MBL is highly variable in healthy populations and is genetically controlled by polymorphisms / mutations in both the promoter and coding regions of the MBL gene. As an acute phase protein, the expression of MBL is further upregulated during inflammation. L-ficolin is present in serum at concentrations similar to MBL. Thus, the L-ficolin branch of the lectin pathway is potentially comparable in strength to the MBL arm. MBL and ficolin can also function as opsonins, allowing phagocytes to target MBL- and ficolin-bearing 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, M. et al., J. Exp. Med. 169:1733, 1989; Matsushita, M. et al., J. Biol. Chem. 271:2448-54, 1996), but their identities have not been established.

[0013] Human MBL forms a specific and high affinity interaction with a unique Clr / Cls-like serine protease, called MBL-associated serine protease (MASP), via its collagen-like domain. To date, three MASPs have been reported. Initially, a single enzyme, "MASP," was identified and characterized as the enzyme involved in the initiation of the complement cascade (i.e., cleavage of C2 and C4) (Matsushita M and Fujita T., J Exp Med 176(6):1497-1502 (1992), Ji, YH et al., J. Immunol. 150:571-578, 1993). Later, it was determined that MASP activity is actually a mixture of two proteases: MASP-1 and MASP-2 (Thiel, S. et al., Nature 386:506-510, 1997). However, it was demonstrated that the MBL-MASP-2 complex alone was sufficient for complement activation (Vorup-Jensen, T. et al., J. Immunol. 165:2093-2100, 2000). Furthermore, only MASP-2 cleaved a high percentage of C2 and C4 (Ambrus, G. et al., J. Immunol. 170:1374-1382, 2003). Thus, MASP-2 is the protease responsible for the activation of C4 and C2 to generate the C3 convertase C4b2a. This is in marked contrast to the C1 complex of the classical pathway, in which activation of the complement system is brought about by the concerted action of two specific serine proteases (C1r and C1s). Furthermore, a third novel protease, MASP-3, was isolated (Dahl, MR et al., Immunity 15:127-35, 2001). MASP-1 and MASP-3 are alternatively spliced ​​products of the same gene.

[0014] MASPs share a domain composition identical to that of the enzyme components of the C1 complex, C1r and C1s (Sim, RB et al., Biochem. Soc. Trans., 2000, vol. 28, p. 545). These domains include an N-terminal C1r / C1s / sea urchin VEGF / bone morphogenetic protein (CUB) domain, an epidermal growth factor-like domain, a second CUB domain, tandem complement control protein domains, and a serine protease domain. As in the C1 protease, activation of MASP-2 occurs by cleavage of an Arg-Ile bond adjacent to the serine protease domain, which separates the enzyme into disulfide-linked A and B chains, the latter of which consists of the serine protease domain. Recently, a genetic defect in MASP-2 has been reported (Stengaard-Pedersen, K. et al., New Eng. J. Med., 2003, vol. 349, pp. 554-560). A single nucleotide mutation causes an Asp-Gly exchange in the CUB1 domain, rendering MASP-2 unable to bind to MBL.

[0015] MBL may also be associated with an alternatively spliced ​​form of MASP-2 known as the 19 kDa MBL-associated protein (MAp19) (Stover, C. M., J. Immunol. 162:3481-90, 1999) or the small MBL-associated protein (sMAP) (Takahashi, M. et al., Int. Immunol. 11:859-863, 1999), which lacks the catalytic activity of MASP-2. MAp19 contains a unique extra sequence of four amino acids following the first two domains of MASP-2. The MASP1 and MASP2 genes are located on human chromosome 3 and human chromosome 1, respectively (Schwaeble, W. et al., Immunobiology 205:455-466, 2002).

[0016] Several lines of evidence suggest that different MBL-MASP complexes exist and that the majority of MASPs in serum are not complexed with MBL (Thiel, S. et al., J. Immunol., 2000, vol. 165, pp. 878-887). Both H-ficolin and L-ficolin bind to all MASPs and, like MBL, activate the lectin complement pathway (Dahl, MR et al., Immunity, 2001, vol. 15, pp. 127-35; Matsushita, M. et al., J. Immunol., 2002, vol. 168, pp. 3502-3506). Both the lectin and classical pathways form a common C3 convertase (C4b2a) and the two pathways merge at this stage.

[0017] The lectin pathway is widely believed to have a major role in host defense against infection in naive hosts. Strong evidence for the involvement of MBL in host defense comes from the analysis of patients with low serum levels of functional MBL (Kilpatrick, DC, Biochim. Biophys. Acta, 2002, vol. 1572, p. 401-413). Such patients are susceptible to recurrent bacterial and fungal infections. These symptoms usually become evident shortly after birth, during an apparently vulnerable period when maternally derived antibody titers wane but before a full repertoire of antibody responses has developed. This syndrome often results from mutations at several sites in the collagenous portion of MBL that interfere with the proper formation of MBL oligomers. However, since MBL can function as a complement-independent opsonin, the extent to which the increased susceptibility to infections is due to impaired complement activation is unclear.

[0018] In contrast to the classical and lectin pathways, no initiators of the alternative pathway have been found that perform the recognition functions that C1q and lectins perform in the other two pathways. It is now widely recognized that the alternative pathway spontaneously activates low levels of turnover that can be easily amplified on foreign or other abnormal surfaces (bacteria, yeast, virus-infected cells or damaged tissues) that lack the proper molecular elements to suppress spontaneous complement activation. There are four plasma proteins that are directly involved in the activation of the alternative pathway: C3, factors B and D, and properdin. Although there is extensive evidence that the pathogenesis of non-infectious human diseases involves both the classical and alternative complement pathways, the role of the lectin pathway is only beginning to be evaluated. Recent studies have provided evidence that activation of the lectin pathway may be involved in complement activation and associated inflammation in ischemia / reperfusion injury. Collard et al. (2000) reported that cultured endothelial cells subjected to oxidative stress bind MBL and show deposition of C3 when exposed to human serum (Collard, CD et al., Am. J. Pathol., 2000, vol. 156, p. 1549-1556). Furthermore, treatment of human serum with a blocking anti-MBL monoclonal antibody inhibited MBL binding and complement activation. These findings were extended to a rat model of myocardial ischemia-reperfusion, in which rats treated with a blocking antibody raised against rat MBL showed significantly less myocardial damage during coronary artery occlusion than rats treated with a control antibody (Jordan, JE et al., Circulation, 2001, vol. 104, p. 1413-1418). The molecular mechanism of MBL binding to vascular endothelium after oxidative stress is unclear; recent studies suggest that activation of the lectin pathway after oxidative stress may be mediated by MBL binding to endothelial cytokeratins but not to glycoconjugates (Collard, CD et al., Am. J. Pathol., 2001, vol. 159, pp. 1045-1054).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, NC et al., Am. J. Pathol., 2003, Vol. 162, p. 363-367).

[0019] Recent studies have shown that MASP-1 (and possibly also MASP-3) are required to convert the alternative pathway activating enzyme, factor D, from its zymogen form to its enzymatically active form (see Takahashi M. et al., J Exp Med 207(1):29-37(2010)). The physiological importance of this process is underscored by the absence of alternative pathway functional activity in the plasma of MASP-1 / 3-deficient mice. For the alternative pathway to function, proteolytic generation of C3b from native C3 is required. Because the alternative pathway C3 convertase (C3bBb) contains C3b as an essential subunit, the question regarding the origin of the initial C3b in the alternative pathway has posed a puzzling problem and stimulated considerable research.

[0020] C3 belongs to a family of proteins (along with C4 and α-2 macroglobulin) that contain an unusual post-translational modification known as a thioester bond. The thioester group consists of a glutamine with a terminal carbonyl group that forms a covalent thioester bond with the sulfhydryl group of a cysteine ​​three amino acids away. This bond is unstable, and the electrophilic glutamyl-thioester can react with nucleophilic moieties such as hydroxyl or amino groups, and thus form covalent bonds with other molecules. The thioester bond is naturally stable when sequestered within the hydrophobic pocket of intact C3. However, proteolytic cleavage of C3 to C3a and C3b exposes the highly reactive thioester bond on C3b, which becomes covalently bound to targets after nucleophilic attack by adjacent moieties containing hydroxyl or amino groups. In addition to its well-documented role in covalently binding C3b to complement targets, the C3 thioester is also thought to play a central role in triggering the alternative pathway. According to the widely accepted "tickover theory," the alternative pathway is initiated by the generation of a fluid-phase convertase, iC3Bb, which is formed from C3 together with hydrolyzed thioesters (iC3; C3(H2O)) and factor B (Lachmann, PJ et al., Springer Semin. Immunopathol., 1984, vol. 7, pp. 143-162). The C3b-like C3(H2O) is generated from native C3 by slow spontaneous hydrolysis of an internal thioester in the protein (Pangburn, MK et al., J. Exp. Med., 1981, vol. 154, pp. 856-867). The activity of the C3(H2O)Bb convertase deposits C3b molecules onto target surfaces, thereby initiating the alternative pathway.

[0021] Very little is known about the initiators of alternative pathway activation. Activators are thought to include yeast cell walls (zymosan), many pure polysaccharides, rabbit erythrocytes, certain immunoglobulins, viruses, fungi, bacteria, animal tumor cells, parasites and damaged cells. The only common feature of these activators is the presence of carbohydrates, but the complexity and diversity of carbohydrate structures makes it difficult to establish a recognized shared molecular determinant. It is widely accepted that alternative pathway activation is controlled by a fine balance between the inhibitory regulatory components of this pathway, such as factor H, factor I, DAF, CR1 and properdin (the only positive regulator of the alternative pathway). See Schwaeble WJ and Reid KB, Immunol Today 20(1):17-21(1999)).

[0022] In addition to the apparently unregulated activation mechanism described above, the alternative pathway may also provide a powerful amplification loop for the lectin / classical pathway C3 convertase (C4b2a) since any C3b produced may participate with factor B in forming additional alternative pathway C3 convertase (C3bBb). The alternative pathway C3 convertase is stabilized by binding to properdin, which extends the half-life of the alternative pathway C3 convertase by 6-10 fold. The alternative pathway C5 convertase is formed by the addition of C3b to the alternative pathway C3 convertase.

[0023] All three pathways (i.e., classical, lectin and alternative) are believed to converge at C5, which is cleaved to form products with multiple proinflammatory effects. The convergent pathway is called the terminal complement pathway. C5a is the most potent anaphylatoxin that induces alterations in smooth muscle and vascular tone as well as vascular permeability. C5a is also a potent chemotaxin and activator of both neutrophils and monocytes. C5a-mediated cell activation can significantly amplify the inflammatory response by inducing the release of multiple additional inflammatory mediators, including cytokines, hydrolytic enzymes, arachidonic acid metabolites and reactive oxygen species. C5 cleavage leads to the formation of C5b-9, also known as the membrane attack complex (MAC). There is now strong evidence that sublytic MAC deposits may play an important role in inflammation in addition to their role as soluble pore-forming complexes. [Prior art documents] [Non-patent literature]

[0024] [Non-Patent Document 1] M. K. Liszewski and J. P. Atkinson, Fundamental Immunology, 1993, 3rd ed., edited by W. E. Paul, Raven Press, Ltd., New York [Non-Patent Document 2] K. R. Calli et al., Springer Semin. Immunopathol., 1994, Vol. 15, p. 417-431 [Non-Patent Document 3] BPMorgan, Eur. J. Clinical Investig., 1994, Vol. 24, p. 219-228 [Non-Patent Document 4] J. Lu et al., Biochim. Biophys. Acta, 2002, vol. 1572, p. 387-400 [Non-Patent Document 5] Holmskov et al., Annu. Rev. Immunol., 2003, vol. 21, pp. 547-578 [Non-Patent Document 6] Teh et al., Immunology, 2000, Vol. 101, pp. 225-232 [Non-Patent Document 7] K. Ikeda et al., J. Biol. Chem., 1987, Vol. 262, pp. 7451-7454 [Non-Patent Document 8] Weis, WI et al., Nature, 1992, Vol. 360, p. 127-134 [Non-Patent Document 9] Lee, RT et al., Archiv. Biochem. Biophys., 1992, Vol. 299, pp. 129-136 [Non-Patent Document 10] Maynard, Y. et al., J. Biol. Chem., 1982, Vol. 257, pp. 3788-3794 [Non-Patent Document 11] Lynch, NJ et al., J. Immunol., 2004, Vol. 172, p. 1198-1202 Summary of the Invention [Problem to be solved by the invention]

[0025] In addition to its essential role in immune defense, the complement system is involved in tissue damage in many clinical conditions, therefore there is an urgent need to develop therapeutically effective complement inhibitors that prevent these deleterious effects. [Means for solving the problem]

[0026] Abstract This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0027] In one aspect, the present invention provides an isolated human monoclonal antibody or antigen-binding fragment thereof that binds to human MASP-2, comprising: (i) a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3 sequences; and (ii) a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3, wherein the CDR-H3 sequence of the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 38 or SEQ ID NO: 90 and conservative sequence modifications thereof, and the CDR-L3 sequence of the light chain variable region comprises the amino acid sequence shown as SEQ ID NO: 51 or SEQ ID NO: 94 and conservative sequence modifications thereof, and the isolated antibody inhibits MASP-2-dependent complement activation.

[0028] In another aspect, the present invention provides a human antibody that binds to human MASP-2, comprising: I) a) a heavy chain variable region comprising i) a heavy chain CDR-H1 comprising the amino acid sequence of 31 to 35 of SEQ ID NO: 21; and ii) a heavy chain CDR-H2 comprising the amino acid sequence of 50 to 65 of SEQ ID NO: 21; and iii) a heavy chain CDR-H3 comprising the amino acid sequence of 95 to 102 of SEQ ID NO: 21; and b) a light chain CDR-L1 comprising the amino acid sequence of 24 to 34 of SEQ ID NO: 25 or SEQ ID NO: 27; and ii) a light chain CDR-L2 comprising the amino acid sequence of 25 to 34 of SEQ ID NO: 25 or SEQ ID NO: 27; 0 to 56 of SEQ ID NO: 25 or SEQ ID NO: 27; and iii) a light chain variable region comprising a light chain CDR-L3 comprising an amino acid sequence of 89 to 97 of SEQ ID NO: 25 or SEQ ID NO: 27; or II) a variant thereof which is identical to the variable domain except for a total of up to 10 amino acid substitutions in the CDR region of the heavy chain variable region and a total of up to 10 amino acid substitutions in the CDR region of the light chain variable region, and the antibody or its variant inhibits MASP-2-dependent complement activation.

[0029] In another aspect, the present invention provides an isolated human monoclonal antibody or antigen-binding fragment thereof that binds to human MASP-2, the antibody comprising: I) a) a heavy chain variable region comprising i) a heavy chain CDR-H1 comprising an amino acid sequence of 31 to 35 of SEQ ID NO: 20; and ii) a heavy chain CDR-H2 comprising an amino acid sequence of 50 to 65 of SEQ ID NO: 20; and iii) a heavy chain CDR-H3 comprising an amino acid sequence of 95 to 102 of SEQ ID NO: 18 or SEQ ID NO: 20; and b) i) a light chain CDR-L1 comprising an amino acid sequence of 24 to 34 of SEQ ID NO: 22 or SEQ ID NO: 24; and ii) a light chain CDR-L2 having an amino acid sequence of 50 to 56 of SEQ ID NO: 22 or SEQ ID NO: 24; and iii) a light chain variable region having a light chain CDR-L3 having an amino acid sequence of 89 to 97 of SEQ ID NO: 22 or SEQ ID NO: 24; or II) a variant thereof which is identical to the variable domain except for a total of up to 10 amino acid substitutions in the CDR region of the heavy chain and a total of up to 10 amino acid substitutions in the CDR region of the light chain variable region, and the antibody or its variant inhibits MASP-2-dependent complement activation.

[0030] In another aspect, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof that binds to human MASP-2, comprising a heavy chain variable region comprising any one of the amino acid sequences shown in SEQ ID NO:18, SEQ ID NO:20 or SEQ ID NO:21.

[0031] In another aspect, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof that binds to human MASP-2, comprising a light chain variable region comprising any one of the amino acid sequences shown in SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25 or SEQ ID NO:27.

[0032] In another aspect, the present invention provides a nucleic acid molecule encoding the amino acid sequence of an anti-MASP-2 antibody or fragment thereof of the present invention (e.g., those shown in Table 2).

[0033] In another aspect, the present invention provides a cell comprising at least one of the nucleic acid molecules encoding the amino acid sequence of an anti-MASP-2 antibody or fragment thereof of the present invention (e.g., those shown in Table 2).

[0034] In another aspect, the present invention provides a method for producing an isolated MASP-2 antibody, the method comprising culturing a cell containing at least one of the nucleic acid molecules encoding the amino acid sequence of the anti-MASP-2 antibody of the present invention under conditions allowing expression of the nucleic acid molecule encoding the anti-MASP-2 antibody, and isolating the anti-MASP-2 antibody.

[0035] In another embodiment, the present invention provides a method for the treatment of cancer comprising administering to a patient a therapeutically effective amount of a cancer cell comprising administering to the ... D It dissociates from human MASP-2 at 100 ng / mL and activates C4 on mannan-coated substrates with an IC of 10 nM or less in 1% serum. 50 In some embodiments, the antibody or antigen-binding fragment thereof specifically recognizes at least a portion of an epitope recognized by a reference antibody, wherein the reference antibody comprises a heavy chain variable region as set forth in SEQ ID NO:20 and a light chain variable region as set forth in SEQ ID NO:24.

[0036] In another aspect, the present invention provides a composition comprising a fully human monoclonal anti-MASP-2 antibody of the present invention and a pharma- ceutically acceptable excipient.

[0037] In another aspect, the present invention provides a method for inhibiting MASP-2-dependent complement activation in a human subject, comprising administering a human monoclonal antibody of the present invention in an amount sufficient to inhibit MASP-2-dependent complement activation in the human subject.

[0038] In another aspect, the present invention provides a product comprising a unit dose of a human monoclonal anti-MASP-2 antibody of the present invention suitable for therapeutic administration to a human subject, wherein the unit dose ranges from 1 mg to 1000 mg.

[0039] The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, in which: For example, the present invention provides the following: (Item 1) (i) a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3 sequences; and (ii) a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3; An isolated human monoclonal antibody or antigen-binding fragment thereof that binds to human MASP-2, comprising: a CDR-H3 sequence of the heavy chain variable region comprising the amino acid sequence shown as SEQ ID NO: 38 or SEQ ID NO: 90 and conservative sequence modifications thereof; and a CDR-L3 sequence of the light chain variable region comprising the amino acid sequence shown as SEQ ID NO: 51 or SEQ ID NO: 94 and conservative sequence modifications thereof, wherein the isolated antibody inhibits MASP-2-dependent complement activation. (Item 2) 2. The isolated antibody or antigen-binding fragment of item 1, wherein the CDR-H2 sequence of the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 32 or 33 and conservative sequence modifications thereof. (Item 3) 3. The isolated antibody or antigen-binding fragment according to item 2, wherein the CDR-H1 sequence of the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 28 or SEQ ID NO: 29 and conservative modifications thereof. (Item 4) 4. The isolated antibody or antigen-binding fragment according to item 3, wherein the CDR-L2 sequence of the light chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 93 and conservative modifications thereof. (Item 5) 5. The isolated antibody or antigen-binding fragment according to item 4, wherein the CDR-L1 sequence of the light chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 91 or SEQ ID NO: 92 and conservative modifications thereof. (Item 6) 4. The isolated antibody or antigen-binding fragment of item 3, wherein CDR-H1 of the heavy chain variable region comprises SEQ ID NO:28. (Item 7) 3. The isolated antibody or antigen-binding fragment of item 2, wherein CDR-H2 of the heavy chain variable region comprises SEQ ID NO: 32. (Item 8) 2. The isolated antibody or antigen-binding fragment of item 1, wherein the CDR-H3 of the heavy chain variable region comprises SEQ ID NO: 90. (Item 9) 9. The isolated antibody according to item 8, wherein the amino acid sequence shown in SEQ ID NO: 90 comprises an R at position 8. (Item 10) 6. The isolated antibody of item 5, wherein CDR-L1 of the light chain variable region comprises SEQ ID NO: 91. (Item 11) 11. The isolated antibody of item 10, wherein the amino acid sequence shown in SEQ ID NO: 91 includes E at position 2. (Item 12) 11. The isolated antibody described in item 10, wherein the amino acid sequence shown in SEQ ID NO: 91 contains Y at position 8. (Item 13) 5. The isolated antibody according to item 4, wherein CDR-L2 of the light chain variable region comprises SEQ ID NO: 93, and the amino acid sequence shown in SEQ ID NO: 93 comprises K at position 2. (Item 14) 5. The isolated antibody according to item 4, wherein CDR-L2 of the light chain variable region comprises SEQ ID NO: 93, and the amino acid sequence shown in SEQ ID NO: 93 comprises Q at position 3. (Item 15) 2. The isolated antibody of item 1, wherein CDR-L3 of the light chain variable region comprises SEQ ID NO:51. (Item 16) 2. The isolated antibody of claim 1, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a Fab, a Fab' fragment, a F(ab')2 fragment and a whole antibody. (Item 17) 2. The isolated antibody of claim 1, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a single-chain antibody, an ScFv, and a monovalent antibody lacking a hinge region. (Item 18) The antibody has a K D An isolated antibody described in item 1, which binds to human MASP-2. (Item 19) The antibody inhibits C4 activation in an in vitro assay in 1% human serum with an IC of 10 nM or less. 50 2. The antibody according to item 1, which inhibits (Item 20) The above antibody has an IC value of 30 nM or less to activate C4 in 90% human serum. 50 2. The antibody according to item 1, which inhibits (Item 21) An isolated human monoclonal antibody or antigen-binding fragment thereof that binds to human MASP-2, said antibody comprising: I)a) i) a heavy chain CDR-H1 comprising an amino acid sequence of 31 to 35 of SEQ ID NO: 20; and ii) a heavy chain CDR-H2 comprising an amino acid sequence of 50 to 65 of SEQ ID NO: 20; and iii) A heavy chain CDR-H3 comprising an amino acid sequence of 95 to 102 of SEQ ID NO: 18 or SEQ ID NO: 20 a heavy chain variable region comprising b) i) a light chain CDR-L1 comprising an amino acid sequence of 24 to 34 of SEQ ID NO: 22 or SEQ ID NO: 24; and ii) a light chain CDR-L2 comprising an amino acid sequence of 50 to 56 of SEQ ID NO: 22 or SEQ ID NO: 24; and iii) Light chain CDR-L3 comprising the amino acid sequence of 89 to 97 of SEQ ID NO: 22 or SEQ ID NO: 24 a light chain variable region comprising: II) A variant thereof in which the remaining parts are identical to the variable domain except for up to a total of 10 amino acid substitutions in the CDR regions of the heavy chain and up to a total of 10 amino acid substitutions in the CDR regions of the light chain variable region. An isolated human monoclonal antibody or an antigen-binding fragment thereof, wherein the antibody or a variant thereof inhibits MASP-2-dependent complement activation. (Item 22) The antibody has a K D An antibody described in item 21, which binds to human MASP-2. (Item 23) The antibody described in item 21, wherein the antibody binds to an epitope in the CCP1 domain of MASP-2. (Item 24) The above antibodies inhibit C3b deposition in an in vitro assay in 1% human serum with an IC of 10 nM or less. 50 22. The antibody according to item 21, which inhibits (Item 25) The above antibodies inhibited C3b deposition in 90% human serum with IC of 30 nM or less. 50 22. The antibody according to item 21, which inhibits (Item 26) 22. The antibody of item 21, wherein the antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2 and F(ab')2. (Item 27) 22. The antibody of claim 21, wherein the antibody is a single chain molecule. (Item 28) 22. The antibody according to item 21, wherein the antibody is an IgG2 molecule. (Item 29) 22. The antibody of item 21, wherein the antibody is an IgG1 molecule. (Item 30) 22. The antibody according to item 21, wherein the antibody is an IgG4 molecule. (Item 31) 31. The antibody of item 30, wherein the IgG4 molecule comprises an S228P mutation. (Item 32) 22. The antibody of item 21, wherein the antibody does not substantially inhibit the classical pathway. (Item 33) 22. The antibody of claim 21, wherein the heavy chain variable region comprises SEQ ID NO: 20 or a variant thereof comprising at least 80% identity to SEQ ID NO: 20. (Item 34) 22. The antibody of claim 21, wherein the heavy chain variable region comprises SEQ ID NO: 18 or a variant thereof comprising at least 80% identity to SEQ ID NO: 18. (Item 35) 22. The antibody of claim 21, wherein the light chain variable region comprises SEQ ID NO:24 or a variant thereof comprising at least 80% identity to SEQ ID NO:24. (Item 36) 22. The antibody of claim 21, wherein the light chain variable region comprises SEQ ID NO:22 or a variant thereof comprising at least 80% identity to SEQ ID NO:22. (Item 37) 22. The antibody according to item 21, wherein the variant is identical to the heavy chain variable region except for a total of up to 1, 2, 3, 4, 5, or 6 amino acid substitutions in the CDR regions of the heavy chain variable region. (Item 38) 22. The antibody of item 21, wherein the variant comprises an amino acid substitution at one or more positions selected from the group consisting of positions 31, 32, 33, 34, 35, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 95, 96, 97, 98, 99, 100, or 102 of the heavy chain variable region. (Item 39) 22. The antibody according to item 21, wherein the variant is identical to the light chain variable region except for a total of up to 1, 2, 3, 4, 5, or 6 amino acid substitutions in the CDR regions of the light chain variable region. (Item 40) 22. The antibody of claim 21, wherein the variant comprises an amino acid substitution at one or more positions selected from the group consisting of positions 25, 26, 27, 29, 31, 32, 33, 51, 52, 89, 92, 93, 95, 96, or 97 of the light chain variable region. (Item 41) An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human MASP-2, comprising a heavy chain variable region comprising any one of the amino acid sequences shown in SEQ ID NO:18, SEQ ID NO:20 or SEQ ID NO:21. (Item 42) 42. The isolated antibody of claim 41, further comprising a light chain variable region comprising an amino acid sequence having at least 95% identity to the amino acid sequence set forth in SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, or SEQ ID NO:27. (Item 43) An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human MASP-2, comprising a light chain variable region comprising any one of the amino acid sequences shown in SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 27. (Item 44) 44. The isolated antibody of claim 43, further comprising a heavy chain variable region comprising an amino acid sequence having at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 21. (Item 45) A nucleic acid molecule encoding the amino acid sequence of an anti-MASP-2 antibody or fragment thereof as shown in items 1, 21, 41 or 43. (Item 46) An expression cassette comprising a nucleic acid molecule encoding the anti-MASP-2 antibody of the present invention described in item 45. (Item 47) A cell comprising at least one of the nucleic acid molecules encoding the anti-MASP-2 antibody of the present invention described in item 45 or item 46. (Item 48) A method for producing an isolated anti-MASP-2 antibody, comprising culturing the cell described in item 47 under conditions allowing expression of a nucleic acid molecule encoding the anti-MASP-2 antibody, and isolating the anti-MASP-2 antibody. (Item 49) K below 10 nM as measured by surface plasmon resonance DIt dissociates from human MASP-2 at 100 ng / mL and activates C4 on mannan-coated substrates with an IC of 10 nM or less in 1% serum. 50 4. An isolated fully human monoclonal antibody or antigen-binding fragment thereof which inhibits (Item 50) 50. The isolated human antibody of claim 49, wherein the antibody or antigen-binding fragment thereof specifically recognizes at least a portion of the epitope recognized by a reference antibody comprising a heavy chain variable region as set forth in SEQ ID NO: 20 and a light chain variable region as set forth in SEQ ID NO: 24. (Item 51) A composition comprising a fully human monoclonal anti-MASP-2 antibody or a fragment thereof described in items 1, 21, 41, 43 or 49 and a pharma- ceutically acceptable excipient. (Item 52) 52. The composition of claim 51, wherein the composition is formulated for systemic delivery. (Item 53) 53. The composition according to item 52, wherein the composition is formulated for intra-arterial, intravenous, intracranial, intramuscular, inhalation, nasal or subcutaneous administration. (Item 54) 53. The composition of claim 52, wherein the composition is formulated for subcutaneous administration. (Item 55) A method for inhibiting MASP-2-dependent complement activation in a human subject, comprising administering a human monoclonal antibody described in items 1, 21, 41, 43 or 49 in an amount sufficient to inhibit MASP-2-dependent complement activation in the human subject. (Item 56) A product comprising a unit dose of a human monoclonal anti-MASP-2 antibody suitable for therapeutic administration to a human subject, said unit dose being in the range of 1 mg to 1000 mg. [Brief description of the drawings]

[0040] [Figure 1A] FIG. 1A is a diagram illustrating the genomic structure of human MASP-2. [Figure 1B] FIG. 1B is a diagram illustrating the domain structure of human MASP-2 protein. [Diagram 2] Figure 2 graphically illustrates the results of an ELISA assay performed on polyclonal populations selected from an scFcv phage library panned against various MASP-2 antigens as described in Example 2. [Figure 3A] 3A and 3B show the results of testing 45 candidate scFv clones for functional activity in a complement assay, as described in Example 3. [Figure 3B] 3A and 3B show the results of testing 45 candidate scFv clones for functional activity in a complement assay, as described in Example 3. [Figure 4] FIG. 4 graphically illustrates the results of an experiment performed to compare C3c levels in three types of serum (human, rat and NHP) as described in Example 4. [Figure 5A] FIG. 5A is an alignment of the amino acid sequences of the heavy chain regions (residues 1-120) of the most active clones, revealing two distinct groups belonging to the VH2 and VH6 gene families, respectively, as described in Example 4. [Figure 5B] FIG. 5B is an alignment of the amino acid sequences of scFv clones 17D20, 17N16, 18L16 and 4D9 as described in Example 4. [Figure 6] FIG. 6 graphically illustrates the inhibitory activity of preparations of IgG4 converted parental clones in a C3b deposition assay using 90% human plasma, as described in Example 5. [Figure 7A] FIG. 7A graphically illustrates the results of an ELISA assay on 17N16 mother versus daughter clones titrated against huMASP2A as described in Example 6. [Figure 7B]FIG. 7B graphically illustrates the results of an ELISA assay on 17D20 mother versus daughter clones titrated against huMASP2A as described in Example 6. [Figure 8] FIG. 8 is an alignment of the protein sequences of the mother clone 17N16 and the daughter clone 17N9, showing that the light chain (starting with SYE) has 17 amino acid residues that differ between the two clones, as described in Example 6. [Figure 9] FIG. 9 is a protein sequence alignment of the CDR-H3 region of the sequences of clones #35, #59 and #90 resulting from mutagenesis compared to the 17D20 parent clone, as described in Example 7. [Figure 10] Figure 10A is a protein sequence alignment of the CDR3 regions of the 17D20 mother clone plus the chain shuffled clone 17D20md21N11, and the mutagensis clone #35 CDR-H3 clone (VH35-VL21N11) shown in Figure 9 combined with the VL of 17D20md21N11, as described in Example 7. Figure 10B is a protein sequence alignment of the VL and VH regions of the 17D20 mother clone and the daughter clone 17D20md21N11, as described in Example 7. [Figure 11] Figure 11A graphically illustrates the results of a C3b deposition assay performed on daughter clone isotype variants (MoAb #1-3) derived from human anti-MASP-2 monoclonal antibody mother clone 17N16, as described in Example 8. Figure 11B graphically illustrates the results of a C3b deposition assay performed on daughter clone isotype variants (MoAb #4-6) derived from human anti-MASP-2 monoclonal antibody mother clone 17D20, as described in Example 8. [Figure 12A] FIG. 12A graphically illustrates testing of the parent clones and MoAbs #1-6 in a C3b deposition assay in 95% serum, as described in Example 8. [Figure 12B]FIG. 12B graphically illustrates testing of the parent clones and MoAbs #1-6 in a C3b deposition assay in 95% serum, as described in Example 8. [Figure 13] FIG. 13 graphically illustrates the inhibition of C4b deposition in 95% normal human serum, as described in Example 8. [Figure 14] FIG. 14 graphically illustrates the inhibition of C3b deposition in 95% African green monkey serum, as described in Example 8. [Figure 15] FIG. 15 graphically illustrates the inhibition of C4 cleavage activity of preassembled MBL-MASP2 complexes by MoAbs #2-6, as described in Example 8. [Figure 16] FIG. 16 graphically illustrates the preferential binding of MoAb#6 to human MASP2 compared to C1s, as described in Example 8. [Figure 17] FIG. 17 graphically illustrates complete inhibition of the lectin pathway following intravenous administration of anti-human MoAb #OMS646 to African green monkeys, as described in Example 10. [Figure 18-1] Figure 18A is a Kaplan-Meier survival plot showing the percentage survival over time after exposure to 7.0 Gy of radiation in control mice and mice treated with anti-mouse MASP-2 antibody (mAbM11) or anti-human MASP-2 antibody (mAbOMS646), as described in Example 11. Figure 18B is a Kaplan-Meier survival plot showing the percentage survival over time after exposure to 6.5 Gy of radiation in control mice and mice treated with anti-mouse MASP-2 antibody (mAbM11) or anti-human MASP-2 antibody (mAbOMS646), as described in Example 11. [Figure 18-2]Figure 18C is a Kaplan-Meier survival plot showing the percentage survival over time after exposure to 8.0 Gy of radiation in control mice and mice treated with anti-human MASP-2 antibody (mAbOMS646), as described in Example 11. [Figure 19] Figure 19 graphically illustrates the results of surface plasmon resonance (Biacore) analysis of anti-MASP-2 antibody OMS646 (response units (binding) vs. seconds) showing that immobilized OMS646 binds to recombinant MASP-2 with a Koff rate of approximately 1-3 x 10-4 S-1 and a Kon rate of approximately 1.6-3 x 106 M-1 S-1, as described in Example 12. [Figure 20] Figure 20 graphically illustrates the results of an ELISA assay measuring the binding affinity of anti-MASP-2 antibody OMS646 to immobilized human MASP-2, as described in Example 12, showing that OMS646 binds to immobilized recombinant human MASP-2 with a KD of approximately 100 pM. [Figure 21] Figure 21A graphically illustrates the level of C4 activation on mannan-coated surfaces in the presence or absence of anti-MASP-2 antibody (OMS646), demonstrating that OMS646 inhibits C4 activation on mannan-coated surfaces with an IC50 of approximately 0.5 nM in 1% human serum, as described in Example 12. Figure 21B graphically illustrates the level of C4 activation on IgG-coated surfaces in the presence or absence of anti-MASP-2 antibody (OMS646), demonstrating that OMS646 does not inhibit classical pathway-dependent activation of complement component C4, as described in Example 12. [Figure 22-1]Figure 22A graphically illustrates the levels of MAC deposition in the presence or absence of an anti-MASP-2 antibody (OMS646) under lectin pathway-specific assay conditions, demonstrating that OMS646 inhibits lectin-mediated MAC deposition with an IC50 value of approximately 1 nM, as described in Example 12. Figure 22B graphically illustrates the levels of MAC deposition in the presence or absence of an anti-MASP-2 antibody (OMS646) under classical pathway-specific assay conditions, demonstrating that OMS646 does not inhibit classical pathway-mediated MAC deposition, as described in Example 12. [Figure 22-2] Figure 22C graphically illustrates the levels of MAC deposition in the presence or absence of anti-MASP-2 antibody (OMS646) under alternative pathway-specific assay conditions, demonstrating that OMS646 does not inhibit alternative pathway-mediated MAC deposition, as described in Example 12. [Diagram 23] Figure 23A graphically illustrates the levels of C3 deposition in the presence or absence of anti-MASP-2 antibody (OMS646) over a range of concentrations in 90% human serum under lectin pathway specific conditions, demonstrating that OMS646 inhibits C3 deposition under physiological conditions, as described in Example 12. Figure 23B graphically illustrates the levels of C4 deposition in the presence or absence of anti-MASP-2 antibody (OMS646) over a range of concentrations in 90% human serum under lectin pathway specific conditions, demonstrating that OMS646 inhibits C4 deposition under physiological conditions, as described in Example 12. [Figure 24]Figure 24A graphically illustrates the levels of C4 deposition in the presence or absence of anti-MASP-2 antibody (OMS646) in 90% cynomolgus monkey serum under lectin pathway specific conditions, demonstrating that OMS646 inhibits lectin pathway C4 deposition in a dose-response manner in Cynomuglus monkey serum with IC50 values ​​in the range of 30-50 nM, as described in Example 12. Figure 24B graphically illustrates the levels of C4 deposition in the absence or presence of anti-MASP-2 antibody (OMS646) in 90% African green monkey serum under lectin pathway specific conditions, demonstrating that OMS646 inhibits lectin pathway C4 deposition in a dose-response manner in African green monkey serum with IC50 values ​​in the range of 15-30 nM, as described in Example 12. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] Description of sequence listing SEQ ID NO: 1 Human MASP-2 cDNA SEQ ID NO:2 Human MASP-2 protein (including leader) SEQ ID NO: 3 Human MASP-2 protein (mature) SEQ ID NO: 4 Rat MASP-2 cDNA SEQ ID NO: 5 Rat MASP-2 protein (including leader) Sequence number 6 Rat MASP-2 protein (mature).

[0042] Antigen (for human MASP-2 mature protein) SEQ ID NO: 7 CUBI domain of human MASP-2 (aa1-121) SEQ ID NO: 8 CUBI / EGF domain of human MASP-2 (aa1-166) SEQ ID NO: 9 CUBI / EGF / CUBII domain of human MASP-2 (aa1-277) SEQ ID NO: 10 EGF domain of human MASP-2 (aa122-166) SEQ ID NO: 11 CCPI / CCPII / SP domain of human MASP-2 (aa278-671) SEQ ID NO: 12 CCPI / CCPII domain of human MASP-2 (aa 278-429) SEQ ID NO: 13 CCPI domain of human MASP-2 (aa278-347) SEQ ID NO: 14 CCPII / SP domain of human MASP-2 (aa 348-671) SEQ ID NO: 15 CCPII domain of human MASP-2 (aa348-429) SEQ ID NO: 16 Human MASP-2 SP domain (aa429-671) SEQ ID NO: 17: Serine protease inactivating mutant (aa 610-625 including mutated Ser618).

[0043] Anti-MASP-2 monoclonal antibody VH chain SEQ ID NO: 18 17D20mc heavy chain variable region (VH) polypeptide SEQ ID NO: 19: DNA encoding 17D20_dc35VH21N11VL (OMS646) heavy chain variable region (VH) (without signal peptide) SEQ ID NO: 20 17D20_dc35VH21N11VL (OMS646) Heavy chain variable region (VH) polypeptide SEQ ID NO:21 17N16mc heavy chain variable region (VH) polypeptide.

[0044] Anti-MASP-2 monoclonal antibody VL chain SEQ ID NO: 22 17D20mc light chain variable region (VL) polypeptide SEQ ID NO: 23: DNA encoding 17D20_dc21N11VL (OMS644) light chain variable region (VL) (without signal peptide) SEQ ID NO: 24 17D20_dc21N11VL (OMS644) light chain variable region (VL) polypeptide SEQ ID NO: 25 17N16mc light chain variable region (VL) polypeptide SEQ ID NO: 26 DNA encoding 17N16_dc17N9 (OMS641) light chain variable region (VL) (without signal peptide) SEQ ID NO: 27 17N16_dc17N9 (OMS641) light chain variable region (VL) polypeptide.

[0045] Anti-MASP-2 monoclonal antibody heavy chain CDR SEQ ID NOs: 28 to 31 CDR-H1 SEQ ID NOs: 32 to 35 CDR-H2 Sequence numbers 36 to 40 CDR-H3.

[0046] Anti-MASP-2 monoclonal antibody light chain CDR SEQ ID NOs: 41 to 45 CDR-L1 SEQ ID NO: 46-50 CDR-L2 SEQ ID NOs: 51 to 54 CDR-L3 MASP-2 antibody sequence SEQ ID NO: 55: scFv parent clone 17D20 full length polypeptide SEQ ID NO: 56: scFv parent clone 18L16 full length polypeptide SEQ ID NO: 57: scFv parent clone 4D9 full length polypeptide SEQ ID NO: 58: scFv parent clone 17L20 full length polypeptide SEQ ID NO: 59: scFv parent clone 17N16 full length polypeptide SEQ ID NO: 60: scFv parent clone 3F22 full length polypeptide SEQ ID NO: 61: scFv parent clone 9P13 full length polypeptide SEQ ID NO: 62: DNA encoding the wild-type IgG4 heavy chain constant region SEQ ID NO: 63: Wild-type IgG4 heavy chain constant region polypeptide SEQ ID NO: 64: DNA encoding the IgG4 heavy chain constant region with the mutation S228P SEQ ID NO: 65: IgG4 heavy chain constant region with mutant S228P polypeptide SEQ ID NO: 66: scFv daughter clone 17N16m_d17N9 full length polypeptide SEQ ID NO: 67: scFv daughter clone 17D20m_d21N11 full length polypeptide SEQ ID NO: 68: scFv daughter clone 17D20m_d3521N11 full length polypeptide SEQ ID NO: 69: DNA encoding the wild-type IgG2 heavy chain constant region SEQ ID NO: 70: Wild-type IgG2 heavy chain constant region polypeptide SEQ ID NO: 71: 17N16m_d17N9 light chain gene sequence (including the signal peptide encoded by nt 1 to 57) SEQ ID NO: 72: 17N16m_d17N9 light chain protein sequence (including signal peptide aa1 to 19) SEQ ID NO: 73: 17N16m_d17N9 IgG2 heavy chain gene sequence (including signal peptide encoded by nt 1 to 57) SEQ ID NO: 74: 17N16m_d17N9 IgG2 heavy chain protein sequence (including signal peptide aa1 to 19) SEQ ID NO: 75: 17N16m_d17N9 IgG4 heavy chain gene sequence (including signal peptide encoded by nt 1 to 57) SEQ ID NO: 76: 17N16m_d17N9 IgG4 heavy chain protein sequence (including signal peptide aa1 to 19) SEQ ID NO: 77: 17N16m_d17N9 IgG4 mutant heavy chain gene sequence (including the signal peptide encoded by nt 1 to 57) SEQ ID NO: 78: 17N17m_d17N9 IgG4 mutant heavy chain protein sequence (including signal peptide aa1 to 19) SEQ ID NO: 79: 17D20_3521N11 light chain gene sequence (including signal peptide encoded by nt 1 to 57) SEQ ID NO: 80: 17D20_3521N11 light chain protein sequence (including signal peptide aa1-19) SEQ ID NO: 81: 17D20_3521N11 IgG2 heavy chain gene sequence (including signal peptide encoded by nt 1 to 57) SEQ ID NO: 82: 17D20_3521N11 IgG2 heavy chain protein sequence (including signal peptide aa1-19) SEQ ID NO: 83: 17D20_3521N11 IgG4 heavy chain gene sequence (including signal peptide encoded by nt 1 to 57) SEQ ID NO: 84: 17D20_3521N11 IgG4 heavy chain protein sequence (including signal peptide aa1-19) SEQ ID NO: 85: 17D20_3521N11 IgG4 mutant heavy chain gene sequence (including signal peptide encoded by nt 1 to 57) SEQ ID NO: 86: 17D20_3521N11 IgG4 mutant heavy chain protein sequence (including signal peptide aa1-19) SEQ ID NO: 87: scFv daughter clone 17N16m_d17N9 DNA encoding the full-length polypeptide (without signal peptide) SEQ ID NO: 88: scFv daughter clone 17D20m_d21N11 DNA encoding the full-length polypeptide (without signal peptide) SEQ ID NO: 89: scFv daughter clone 17D20m_d3521N11 DNA encoding the full-length polypeptide (without signal peptide) SEQ ID NO: 90: Consensus heavy chain CDR-H3 of 17D20m and d3521N11 SEQ ID NO: 91: Consensus light chain CDR-L1 of 17D20m and d3521N11 SEQ ID NO: 92: Consensus light chain CDR-L1 of 17N16m and d17N9 SEQ ID NO: 93: Consensus light chain CDR-L2 of 17D20m, d3521N11, 17N16m and d17N9 SEQ ID NO:94: Consensus light chain CDR-L3 of 17N16m and d17N9.

[0047] Detailed Description The present invention provides a fully human antibody that binds to human MASP-2 and inhibits lectin-mediated complement activation while leaving components of the classical (C1q-dependent) pathway of the immune system intact. The human anti-MASP-2 antibody was identified by screening a phage display library as described in Examples 2-9. As described in Examples 10-12, a high-affinity anti-MASP-2 antibody was identified that has the ability to inhibit lectin-mediated complement activation, as demonstrated in both in vitro assays and in vivo. The variable light and heavy chain fragments of the antibody were isolated as both scFv and full-length IgG formats. The human anti-MASP-2 antibody is useful for inhibiting cellular damage associated with activation of the lectin-mediated complement pathway while leaving components of the classical (C1q-dependent) pathway of the immune system intact.

[0048] (I. Definition) Unless otherwise defined herein, all terms used herein have the same meaning as would be understood by one of ordinary skill in the art in the field of the invention. The following definitions are provided for clarity regarding terms used in the specification and claims describing the invention.

[0049] As used herein, the term "MASP-2-dependent complement activation" includes MASP-2-dependent activation of the lectin pathway, which occurs under physiological conditions (i.e., Ca ++ This occurs in the presence of lectin pathway C4b2a, leading to the formation of the lectin pathway C3 convertase, C4b2a, and accumulation of the C3 cleavage product, C3b, subsequently leading to the C5 convertase, C4b2a(C3b)n.

[0050] As used herein, the term "alternative pathway" refers to complement activation triggered, for example, by zymosan from fungal and yeast cell walls, lipopolysaccharide (LPS) from gram-negative outer membranes and rabbit erythrocytes, as well as from many pure polysaccharides, rabbit erythrocytes, viruses, bacteria, animal tumor cells, parasites and damaged cells, and complement activation traditionally thought to result from the spontaneous proteolytic generation of C3b from complement factor C3.

[0051] As used herein, the term "lectin pathway" refers to complement activation that occurs through the specific binding of serum and non-serum carbohydrate-binding proteins, including mannan-binding lectin (MBL), CL-11 and ficolins (H-ficolin, M-ficolin or L-ficolin).

[0052] As used herein, the term "classical pathway" refers to complement activation that is triggered by antibodies bound to foreign particles and requires the binding of the recognition molecule C1q.

[0053] As used herein, the term "MASP-2 inhibitory antibody" refers to any anti-MASP-2 antibody or MASP-2-binding fragment thereof that binds to or directly interacts with MASP-2 and effectively inhibits MASP-2-dependent complement activation. MASP-2 inhibitory antibodies useful in the methods of the present invention may reduce MASP-2-dependent complement activation by more than 20% (e.g., more than 30%, or more than 40%, or more than 50%, or more than 60%, or more than 70%, or more than 80%, or more than 90%, or more than 95%).

[0054] As used herein, the term "MASP-2 blocking antibody" refers to a MASP-2 inhibitory antibody that reduces MASP-2-dependent complement activation by more than 90% (e.g., more than 95% or more than 98%) (i.e., resulting in only 10% (e.g., no more than 9%, or no more than 8%, or no more than 7%, or no more than 6%, e.g., no more than 5%, or no more than 4%, or no more than 4%, or no more than 3%, or no more than 2%, or no more than 1%).

[0055] The terms "antibody" and "immunoglobulin" are used interchangeably herein. These terms are well understood by those skilled in the art and refer to a protein consisting of one or more polypeptides that specifically bind to an antigen. One form of antibody constitutes the basic unit of antibody structure. This form is a tetramer, consisting of two identical pairs of antibody chains, each pair having one light chain and one heavy chain. In each pair, the light chain variable region and the heavy chain variable region are together involved in binding to the antigen, and the constant region is involved in the effector function of the antibody.

[0056] As used herein, the term "antibody" includes antibodies and antibody fragments thereof that specifically bind to a MASP-2 polypeptide or a portion thereof, from any antibody-producing mammal (e.g., mouse, rat, rabbit and primate (including human)) or from hybridoma, phage selection, recombinant expression or transgenic animals (or other methods of generating antibodies or antibody fragments). The term "antibody" is not intended to be limited with respect to the origin of the antibody or the manner in which it is produced (e.g., hybridoma, phage selection, recombinant expression, transgenic animals, peptide synthesis, etc.). Exemplary antibodies include polyclonal, monoclonal and recombinant antibodies; multispecific antibodies (e.g., bispecific antibodies); humanized antibodies; murine antibodies; chimeric monoclonal antibodies, mouse-human monoclonal antibodies, mouse-primate monoclonal antibodies, primate-human monoclonal antibodies; and anti-idiotypic antibodies, which may be any intact molecule or fragment thereof. As used herein, the term "antibody" includes not only intact polyclonal or monoclonal antibodies, but also fragments thereof (e.g., dAbs, Fab, Fab', F(ab')2, Fv), single chains (ScFv), synthetic variants thereof, naturally occurring variants, fusion proteins containing an antibody portion containing an antigen-binding fragment with the required specificity, humanized antibodies, chimeric antibodies, and any other modified configuration of an immunoglobulin molecule that contains an antigen-binding site or a fragment (epitope recognition site) with the required specificity.

[0057] As used herein, the term "antigen-binding fragment" refers to a polypeptide fragment that contains at least one CDR of an immunoglobulin heavy and / or light chain that binds to human MASP-2. In this regard, the antigen-binding fragment of the antibody described herein may contain one, two, three, four, five or all six CDRs of the VH and VL sequences shown herein from an antibody that binds to MASP-2. The antigen-binding fragment of the MASP-2-specific antibody described herein can bind to MASP-2. In certain embodiments, the antigen-binding fragment, or an antibody that contains the antigen-binding fragment, mediates inhibition of MASP-2-dependent complement activation.

[0058] As used herein, the term "anti-MASP-2 monoclonal antibody" refers to a homogenous antibody population, in which the monoclonal antibody contains amino acids involved in selective binding of an epitope on MASP-2. Anti-MASP-2 monoclonal antibodies are highly specific for the MASP-2 target antigen. The term "monoclonal antibody" encompasses not only intact and full-length monoclonal antibodies, but also fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), single chain (ScFv), variants thereof, fusion proteins containing antigen-binding portions, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of an immunoglobulin molecule containing an antigen-binding fragment (epitope recognition site) having the required specificity and ability to bind to the epitope.

[0059] As used herein, the modifier "monoclonal" indicates the character of the antibody being obtained from a substantially homogenous population of antibodies and is not intended to be limited as to the source of the antibody or the manner in which it is made (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term includes whole immunoglobulins, as well as fragments such as those described in the definition of "antibody" above. Monoclonal antibodies may be obtained using any technique which provides for the production of antibody molecules by continuous cell lines in culture (e.g., the hybridoma method described in Kohler, G. et al., Nature 256:495, 1975) or may be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567 to Cabilly). Monoclonal antibodies can also be isolated from phage antibody libraries using the techniques described in Clackson, T. et al., Nature 352:624-628, 1991 and Marks, JD et al., J. Mol. Biol. 222:581-597, 1991. Such antibodies can be of any immunoglobulin class, including IgG, IgM, IgE, IgA, IgD and any subclass thereof.

[0060] Recognized immunoglobulin polypeptides include kappa and lambda light chains, as well as alpha, gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon and mu heavy chains or equivalents in other species. A full-length immunoglobulin "light chain" (about 25 kDa or about 214 amino acids) contains a variable region of about 110 amino acids at the NH2-terminus, and a kappa or lambda constant region at the COOH-terminus. A full-length immunoglobulin "heavy chain" (about 50 kDa or about 446 amino acids) similarly contains a variable region (about 116 amino acids) and one of the heavy chain constant regions mentioned above, e.g., gamma (about 330 amino acids).

[0061] The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of five of these basic heterotetrameric units, plus an additional polypeptide called the J chain, and therefore contain ten antigen-binding sites. Secreted IgA antibodies can polymerize to form polyvalent assemblies containing two to five basic four-chain units along with the J chain. Each L chain is linked to an H chain by one covalent disulfide bond, and the two H chains are linked to each other by one or more disulfide bonds, depending on the H chain isotype. Each of the H and L chains also has regularly spaced intrachain disulfide bridges. A single antigen-binding site is formed by pairing together a VH and a VL.

[0062] Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) in the case of each of the α and γ chains, and four CH domains (CH) in the case of the μ and ε isotypes.

[0063] Each L chain has a variable domain (VL) at the N-terminus followed by a constant domain (CL) at its other end. The VL aligns with the VH, and the CL aligns with the first constant domain (CH1) of the heavy chain. The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain (CL).

[0064] Depending on the amino acid sequence of the constant domain (CH) of the heavy chain, immunoglobulins can be assigned to various classes or isotypes. There are five classes of immunoglobulins with heavy chains designated alpha (α), delta (δ), epsilon (ε), gamma (γ) and mu (μ), respectively: IgA, IgD, IgE, IgG and IgM. The gamma and alpha classes are further divided into subclasses based on slight differences in the CH sequence and function, for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.

[0065] For the structure and properties of various classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th Edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds); Appleton and Lange, Norwalk, Conn., 1994, page 71 and Chapter 6.

[0066] The term "variable" refers to the fact that certain segments of the V domains vary widely in sequence among antibodies. The V domains mediate antigen binding and define the specificity of a particular antibody for a particular antigen. However, the variability is not evenly distributed across the 110 amino acid length of the variable domains. Instead, the V regions consist of relatively invariant stretches of 15-30 amino acids called framework regions (FRs), separated by shorter regions (each 9-12 amino acids long) that are highly variable, called "hypervariable regions". Each of the naturally occurring heavy and light chain variable domains contains four FRs, mostly in a beta-sheet configuration, connected by three hypervariable regions (which form loops connecting, and in some cases form part of, the n-sheet structure). The hypervariable regions in each chain are held in close proximity to the FRs and together with the hypervariable regions from the other chain, and contribute to the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md (1991)). The constant domains are not directly involved in binding the antibody to an antigen, but exhibit various effector functions (e.g., participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC)).

[0067] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody that are involved in antigen binding. A hypervariable region generally comprises amino acid residues from the "complementarity determining regions" or "CDRs" (i.e., about residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain, and about residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain, when numbered according to the Kabat numbering system as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md (1991)); and / or residues from the "hypervariable loops" (i.e., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain, and about residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain, when numbered according to the Kabat numbering system as described in Chothia and residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 26-32 (H1), 52-56 (H2) and 95-101 (H3) in the heavy chain variable domain when numbered according to the Chothia numbering system as described in Lesk, J. Mol. Biol. 196:901-917 (1987); and / or residues from the "hypervariable loops" / CDRs (e.g., Lefranc, JP et al., Nucleic Acids Res 27:209-212; Ruiz, M. et al., Nucleic Acids Res When numbered according to the IMGT numbering system as described in J. Med. 28:219-221 (2000), these include residues 27-38 (L1), 56-65 (L2), and 105-120 (L3) in VL and 27-38 (H1), 56-65 (H2), and 105-120 (H3) in VH.

[0068] As used herein, the term "antibody fragment" refers to a portion derived from or related to a full-length anti-MASP-2 antibody (generally including the antigen-binding region or its variable region). Examples of antibody fragments include Fab fragments, Fab' fragments, F(ab)2 fragments, F(ab')2 fragments and Fv fragments, scFv fragments, diabodies, linear antibodies, single-chain antibody molecules, bispecific and multispecific antibodies formed from antibody fragments.

[0069] When bispecific antibodies are used, they may be conventional bispecific antibodies which may be produced in a variety of ways (Holliger, P. and Winter G. Current Opinion Biotechnol. 4, 446-449 (1993)), e.g., prepared chemically or from hybrid hybridomas, or may be any of the bispecific antibody fragments mentioned above.

[0070] As used herein, a "single-chain Fv" or "scFv" antibody fragment refers to a single-chain Fv of an antibody. H Domains and V L Fv polypeptides generally comprise a V domain, where these domains are present in a single polypeptide chain. H Domain and V L The domains may further comprise a polypeptide linker which enables the scFv to form the desired structure for antigen-binding. See The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). "Fv" is the minimum antibody fragment that contains a complete antigen recognition and binding site. This fragment consists of a dimer of one heavy chain variable region domain and one light chain variable region domain in tight non-covalent association. The folding of these two domains results in six hypervariable loops (three loops each from the H and L chains) that provide the amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind antigen (albeit with lower affinity than the entire binding site).

[0071] As used herein, the term "specific binding" refers to the ability of an antibody to preferentially bind to a particular analyte present in a homogenous mixture of various analytes. In certain embodiments, the specific binding interaction discriminates between desired and undesired analytes in a sample, in some embodiments by about 10-100 fold or more (e.g., by about 1000 or 10,000 fold or more). In certain embodiments, the affinity between the capture agent and the analyte, when they are specifically bound to the capture agent / analyte complex, is less than about 100 nM, or less than about 50 nM, or less than about 25 nM, or less than about 10 nM, or less than about 5 nM, or less than about 1 nM, K D (dissociation constant).

[0072] As used herein, the term "variant" anti-MASP-2 antibody refers to a molecule whose amino acid sequence differs from the "parent" or reference antibody amino acid sequence by the addition, deletion and / or substitution of one or more amino acid residues in the parent antibody sequence. In one embodiment, a variant anti-MASP-2 antibody refers to a molecule that comprises a variable region identical to the parent variable domain except for a total of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions in total within the CDR regions of the heavy chain variable region, and / or up to a total of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions in total within the CDR regions of the light chain variable region. In some embodiments, the amino acid substitutions are conservative sequence modifications.

[0073] As used herein, the term "parent antibody" refers to an antibody encoded by an amino acid sequence used to prepare a variant. Preferably, the parent antibody has human framework regions and, if present, human antibody constant regions. For example, the parent antibody can be a humanized antibody or a fully human antibody.

[0074] As used herein, the term "isolated antibody" refers to an antibody that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that may interfere with the diagnostic or therapeutic use of the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In a preferred embodiment, the antibody is purified (1) to greater than 95% by weight, most preferably greater than 99% by weight, as measured by the Lowry method; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by using a spinning cup sequencer; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions, using Coomassie blue or preferably silver staining. An isolated antibody includes an antibody in situ in a recombinant cell, since at least one component of the antibody's natural environment is absent. Ordinarily, however, an isolated antibody is prepared by at least one purification step.

[0075] As used herein, the term "epitope" refers to the portion of an antigen to which a monoclonal antibody specifically binds. Epitope determinants usually consist of chemically active surface groups of molecules (e.g., amino acids or sugar side chains) and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. More specifically, the term "MASP-2 epitope" as used herein refers to the portion of a corresponding polypeptide to which an antibody immunospecifically binds as determined by any method well known in the art, for example, by immunoassay. An antigenic epitope is not necessarily immunogenic. Such an epitope may be essentially linear or may be a discontinuous epitope. Thus, as used herein, the term "conformational epitope" refers to a discontinuous epitope formed by spatial relationships between amino acids of an antigen other than a continuous string of amino acids.

[0076] As used herein, the term "mannan-binding lectin" ("MBL") is equivalent to mannan-binding protein ("MBP").

[0077] As used herein, "membrane attack complex" ("MAC") refers to the complex of the last five complement components (C5-C9) that inserts into and destroys membranes. Also called C5b-9.

[0078] As used herein, a "subject" includes all mammals, including, but not limited to, humans, non-human primates, dogs, cats, horses, sheep, goats, cows, rabbits, pigs and rodents.

[0079] As used herein, amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0080] In the broadest sense, naturally occurring amino acids can be divided into groups based on the chemical properties of the side chain of each amino acid. By "hydrophobic" amino acid we mean either Ile, Leu, Met, Phe, Trp, Tyr, Val, Ala, Cys or Pro. By "hydrophilic" amino acid we mean either Gly, Asn, Gln, Ser, Thr, Asp, Glu, Lys, Arg or His. This group of amino acids can be further classified as follows: By "uncharged hydrophilic" amino acid we mean either Ser, Thr, Asn or Gln. By "acidic" amino acid we mean either Glu or Asp. By "basic" amino acid we mean either Lys, Arg or His.

[0081] As used herein, the term "conservative amino acid substitutions" describes substitutions between amino acids within each of the following groups: (1) glycine, alanine, valine, leucine, and isoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine, (4) aspartic acid and glutamic acid, (5) glutamine and asparagine, and (6) lysine, arginine, and histidine.

[0082] As used herein, an "isolated nucleic acid molecule" is a nucleic acid molecule (e.g., a polynucleotide) that is not integrated into the genomic DNA of an organism. For example, a DNA molecule encoding a growth factor that is separated from the genomic DNA of a cell is an isolated DNA molecule. Another example of an isolated nucleic acid molecule is a chemically synthesized nucleic acid molecule that is not integrated into the genome of an organism. A nucleic acid molecule that is isolated from a particular species is smaller than a complete DNA molecule of a chromosome of that species.

[0083] As used herein, a "nucleic acid molecule construct" is a single- or double-stranded nucleic acid molecule that has been engineered by human intervention to contain segments of nucleic acid combined and arranged in an arrangement that does not occur in nature.

[0084] As used herein, an "expression vector" is a nucleic acid molecule that encodes a gene to be expressed in a host cell. Typically, an expression vector contains a transcription promoter, a gene, and a transcription terminator. Gene expression is usually under the control of a promoter, and such a gene is said to be "operably linked to" that promoter. Similarly, a regulatory element and a core promoter are operably linked if the regulatory element regulates the activity of the core promoter.

[0085] As used herein, the terms "approximately" or "about" in reference to numerical values ​​are generally deemed to include numerical values ​​that fall within a range of 5% (greater or less) in either direction of that numerical value, unless otherwise stated or otherwise clear from the context (except where such numerical values ​​may exceed 100% of the possible values). When a range is stated, the endpoints are included within that range, unless otherwise stated or otherwise clear from the context.

[0086] As used herein, the singular forms "a," "an," and "the" include plural embodiments unless the context clearly dictates otherwise. Thus, for example, "a cell" is a reference to "a cell" includes a single cell as well as two or more cells; reference to "an agent" includes one agent as well as two or more agents; reference to "an antibody" includes a plurality of such antibodies, reference to "a framework region" includes reference to one or more framework regions and equivalents thereof known to those skilled in the art, and so forth.

[0087] Each embodiment herein applies mutatis mutandis to every other embodiment unless expressly stated otherwise.

[0088] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly performed in the art or as described herein. These and related techniques and procedures may be performed according to conventional methods well known in the art and generally as described in various general and more specific references cited and described throughout this specification. See, for example, Sambrook et al., 2001, MOLECULAR CLONING: A LABORATORY MANUAL, 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., NY, NY); Current Protocols in Immunology (Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober 2001 John Wiley & Sons, NY, NY); or other relevant Current Protocol publications and other similar references. Unless specific definitions are provided, the nomenclature used in connection with, and the laboratory procedures and techniques thereof, of molecular biology, analytical chemistry, organic synthetic chemistry, and pharmaceutical and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for recombinant techniques, molecular biological synthesis, microbiological synthesis, chemical synthesis, chemical analysis, pharmaceutical formulation and delivery, and treatment of patients.

[0089] It is contemplated that any embodiment described herein may be fulfilled with respect to any method, kit, reagent, or composition of the invention, and vice versa. Further, a composition of the invention may be used to achieve a method of the invention.

[0090] II. Overview Lectins (MBL, M-ficolin, H-ficolin, L-ficolin and CL-11) are specific recognition molecules that trigger the innate complement system, which includes the lectin-initiation pathway and an associated terminal pathway amplification loop that amplifies the activation of terminal complement effector molecules initiated by lectins. C1q is a specific recognition molecule that triggers the acquired complement system, which includes the classical initiation pathway and an associated terminal pathway amplification loop that amplifies the activation of terminal complement effector molecules initiated by C1q. We refer to these two major complement activation systems as the lectin-dependent and C1q-dependent complement systems, respectively.

[0091] In addition to its essential role in immune defense, the complement system is involved in tissue damage in many clinical conditions, therefore there is an urgent need to develop therapeutically effective complement inhibitors that prevent these deleterious effects.

[0092] As described in U.S. Patent No. 7,919,094, co-pending U.S. Patent Application No. 12 / 905,972 (published as US2011 / 0091450) and co-pending U.S. Patent Application No. 13 / 083,441 (published as US2011 / 0311549), each of which is assigned to the present application's assignee, Omeros Corporation, and each of which is hereby incorporated by reference, it has been found by using the MASP-2- / - mouse model that the lectin-mediated MASP-2 pathway can be inhibited while leaving the classical pathway intact. With the realization that the lectin-mediated MASP-2 pathway can be inhibited while leaving the classical pathway intact, it is understood that it is highly desirable to specifically inhibit only the complement activation system that causes a particular pathology without completely shutting down the immune defense ability of complement. For example, in disease states in which complement activation is primarily mediated by the lectin-dependent complement system, it may be beneficial to specifically inhibit only this system. This would allow for the processing of immune complexes without compromising the C1q-dependent complement activation system and aid in host defense against infection.

[0093] A preferred protein component to target in the development of therapeutics that specifically inhibit the lectin-dependent complement system is MASP-2. Of all known proteins of the lectin-dependent complement system (MBL, H-ficolin, M-ficolin, L-ficolin, MASP-2, C2-C9, factor B, factor D and properdin), only MASP-2 is unique to the lectin-dependent complement system and is necessary for the system to function. The lectins (MBL, H-ficolin, M-ficolin, L-ficolin and CL-11) are also unique components in the lectin-dependent complement system. However, the loss of any one of the lectin components does not necessarily inhibit the activation of the system due to lectin redundancy. In order to ensure inhibition of the lectin-dependent complement activation system, it is necessary to inhibit all five lectins. Furthermore, MBL and ficolins are known to have complement-independent opsonic activity, so inhibition of lectin function may result in the loss of this beneficial host defense mechanism against infection. In contrast, this complement-independent lectin opsonic activity can remain intact even if MASP-2 is the inhibitory target. An added advantage of MASP-2 as a therapeutic target for inhibiting the lectin-dependent complement activation system is that the plasma concentration of MASP-2 is the lowest of any complement protein (approximately 500 ng / ml); therefore, correspondingly, low concentrations of high-affinity inhibitors of MASP-2 are sufficient to effect complete inhibition, as demonstrated in the Examples herein.

[0094] In accordance with the foregoing, as described herein, the present invention provides a monoclonal, fully human anti-MASP-2 antibody that binds to human MASP-2 with high affinity and can inhibit activation of the lectin-mediated complement pathway.

[0095] III. MASP-2 inhibitor antibody In one embodiment, the present invention provides a monoclonal fully human anti-MASP-2 antibody or an antigen-binding fragment thereof that specifically binds to human MASP-2 and inhibits or blocks MASP-2-dependent complement activation. A MASP-2 inhibitory antibody may effectively inhibit or effectively block the MASP-2-dependent complement activation system by inhibiting or blocking the biological function of MASP-2. For example, an inhibitory antibody may effectively inhibit or block protein-protein interactions of MASP-2, may interfere with the dimerization or assembly of MASP-2, may inhibit or effectively block Ca. 2+ It may block binding or interfere with the MASP-2 serine protease active site.

[0096] MASP-2 epitopes The present invention provides a fully human antibody that specifically binds to human MASP-2. MASP-2 polypeptides exhibit a molecular structure similar to MASP-1, MASP-3, and the proteases C1r and C1s of the C1 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:1) and provides a human MASP-2 polypeptide (aa1-15) with a leader sequence that is cleaved after secretion to give the mature form of human MASP-2 (SEQ ID NO:3). As shown in FIG. 1A, the human MASP2 gene encompasses 12 exons. The human MASP-2 cDNA is encoded by exons B, C, D, F, G, H, I, J, K and L. The cDNA molecule shown in SEQ ID NO:4 encodes rat MASP-2 (consisting of the amino acid sequence shown in SEQ ID NO:5) and provides a rat MASP-2 polypeptide with a leader sequence that is cleaved after secretion to give the mature form of rat MASP-2 (SEQ ID NO:6).

[0097] Those skilled in the art will recognize that the sequences disclosed in SEQ ID NO:1 and SEQ ID NO:4 are single alleles of human and rat MASP-2, respectively, and that allelic variations and alternative splicing are expected to occur. Allelic variants of the nucleotide sequences shown in SEQ ID NO:1 and SEQ ID NO:4, including sequences containing silent mutations and sequences in which the amino acid sequence is altered by mutation, are within the scope of the present invention. Allelic variants of the MASP-2 sequences can be cloned by probing cDNA or genomic libraries from various individuals according to standard procedures.

[0098] The domains of human MASP-2 protein (SEQ ID NO:3) are shown in FIG. 1B and Table 1 below, and include the N-terminal C1r / C1s / sea urchin VEGF / bone morphogenetic protein (CUBI) domain, the epidermal growth factor-like domain, the second CUB domain (CUBII) and the tandem complement control protein domains CCP1 and CCP2 and a serine protease domain. Alternative splicing of the MASP-2 gene results in MAp19. MAp19 is a non-enzymatic protein that contains the N-terminal CUB1-EGF region of MASP-2 with four additional residues (EQSL).

[0099] Several proteins have been shown to bind to MASP-2 or interfere with it through protein-protein interactions. For example, MASP-2 binds to the lectin proteins MBL, H-ficolin, and L-ficolin and interacts with them to mediate Ca 2+It is known that MASP-2 / lectin complexes form a complement-dependent complex. Each MASP-2 / lectin complex has been shown to activate complement by MASP-2-dependent cleavage of proteins C4 and C2 (Ikeda, K. et al., J. Biol. Chem. 262: 7451-7454, 1987; Matsushita, M. et al., J. Exp. Med. 176: 1497-2284, 2000; Matsushita, M. et al., J. Immunol. 168: 3502-3506, 2002). Studies have shown that the CUB1-EGF domain of MASP-2 is essential for the association of MASP-2 with MBL (Thielens, NM et al., J. Immunol. 166: 5068, 2001). It has also been shown that the CUB1EGFCUBII domain mediates the dimerization of MASP-2 required for the formation of active MBL complexes (Wallis, R. et al., J. Biol. Chem. 275:30962-30969, 2000). Thus, MASP-2 inhibitory antibodies can be identified that bind to or interfere with MASP-2 target regions known to be important for MASP-2-dependent complement activation.

[0100] [Table 1] In one embodiment, the anti-MASP-2 inhibitory antibody of the present invention binds to a portion of the full-length human MASP-2 protein (SEQ ID NO: 3) (e.g., the CUBI, EGF, CUBII, CCPI, CCPII or SP domain of MASP-2). In some embodiments, the anti-MASP-2 inhibitory antibody of the present invention binds to an epitope within the CCP1 domain (SEQ ID NO: 13 (aa 278-347 of SEQ ID NO: 3)). For example, as described in Example 9, an anti-MASP-2 antibody (e.g., OMS646) has been identified that binds only to a MASP-2 fragment containing the CCP1 domain and inhibits MASP-2-dependent complement activation.

[0101] Binding affinity of MASP-2 inhibitor antibodies The anti-MASP-2 inhibitory antibody specifically binds to human MASP-2 (shown as SEQ ID NO:3, encoded by SEQ ID NO:1) with an affinity that is at least 10 times higher than other antigens in the complement system. In some embodiments, the MASP-2 inhibitory antibody specifically binds to human MASP-2 with a binding affinity that is at least 100 times higher than other antigens in the complement system.

[0102] In some embodiments, the MASP-2 inhibitory antibody has a K of less than about 100 nM, or less than about 50 nM, or less than about 25 nM, or less than about 10 nM, or less than about 5 nM, or about 1 nM or less, or 0.1 nM or less. D It specifically binds to human MASP-2 with a dissociation constant (A). The binding affinity of the MASP-2 inhibitory antibody can be measured using a suitable binding assay known in the art, such as an ELISA assay as described in Examples 3 to 5 herein.

[0103] Efficacy of MASP-2 inhibitory antibodies In one embodiment, the MASP-2 inhibitory antibody has an IC of ≦30 nM, preferably less than or about 20 nM, or less than about 10 nM, or less than about 5 nM, or about 3 nM or less, or about 1 nM or less, when measured in 1% serum. 50 is sufficiently potent to inhibit MASP-2-dependent complement activation at 100-fold.

[0104] In one embodiment, the MASP-2 inhibitory antibody has an IC of ≦30 nM, preferably less than or about 20 nM, or less than about 10 nM, or less than about 5 nM, or about 3 nM or less, or about 1 nM or less, when measured in 90% serum. 50 is sufficiently potent to inhibit MASP-2-dependent complement activation at 100-fold.

[0105] Inhibition of MASP-2-dependent complement activation is characterized by at least one of the following changes in components of the complement system that result from administration of a MASP-2 inhibitory antibody: inhibition of the production or generation of MASP-2-dependent complement activation system products C4a, C3a, C5a and / or C5b-9 (MAC) (measured, for example, as described in Example 2 of U.S. Patent No. 7,919,094) and their catabolic degradation products (e.g., C3desArg), a decrease in C4 cleavage and C4b deposition (measured, for example, as described in Example 5) and their subsequent catabolic degradation products (e.g., C4bc or C4d), or a decrease in C3 cleavage and C3b deposition (measured, for example, as described in Example 5) or their subsequent catabolic degradation products (e.g., C3bc, C3d, etc.).

[0106] In some embodiments, the MASP-2 inhibitory antibodies of the present invention are capable of inhibiting C3 deposition in full serum to less than 80%, such as less than 70%, such as less than 60%, such as less than 50%, such as less than 40%, such as less than 30%, such as less than 20%, such as less than 15%, such as less than 10% of control C3 deposition.

[0107] In some embodiments, the MASP-2 inhibitory antibodies of the present invention are capable of inhibiting C4 deposition in all serum to less than 80%, such as less than 70%, such as less than 60%, such as less than 50%, such as less than 40%, such as less than 30%, such as less than 20%, such as less than 15%, such as less than 10% of control C4 deposition.

[0108] In some embodiments, the MASP-2 inhibitory antibody selectively inhibits MASP-2 complement activation (i.e., binds to MASP-2 with at least 100-fold greater affinity than C1r or C1s) while leaving the C1q-dependent complement activation system functionally intact (i.e., at least 80% or at least 90% or at least 95% or at least 98% or 100% of the classical pathway activity is retained).

[0109] In some embodiments, the subject anti-MASP-2 inhibitory antibodies have the following characteristics: (a) high affinity for human MASP-2 (e.g., a K of 10 nM or less); D , preferably a K of 1 nM or less D ) and (b) IC50 of 30 nM or less to inhibit MASP-2-dependent complement activation in 90% human serum. 50 , preferably with an IC of 10 nM or less 50 and inhibits.

[0110] As described in Examples 2-12, a fully human antibody was identified that binds to MASP-2 with high affinity and inhibits lectin-mediated complement activation while leaving components of the classical (C1q-dependent) pathway of the immune system intact. The variable light and heavy fragments of the antibody were sequenced, isolated and analyzed, both as scFv and full-length IgG formats. Figure 5A is an alignment of the amino acid sequences of seven scFv anti-MASP-2 clones identified as having high binding affinity to MASP-2 and the ability to inhibit MASP-2-dependent activity. Figure 5B is an alignment of the amino acid sequences of four scFv mother clones 17D20, 17N16, 18L16 and 4D9, showing the framework and CDR regions. As described in Examples 6 and 7, the scFv mother clones 17D20 and 17N16 were subjected to affinity maturation, which generated daughter clones with higher affinity and enhanced potency compared to their mother clones. The amino acid sequences of the heavy chain variable region (VH) (aa 1-120) and light chain variable region (VL) (aa 148-250) of the scFv clones shown in Figures 5A and 5B and the resulting daughter clones are provided in Table 2 below.

[0111] By aligning the heavy and light chain amino acid sequences of the anti-MASP-2 inhibitory antibodies described above and determining which amino acids are present at which positions in those antibodies, the substitutable positions of the human anti-MASP-2 inhibitory antibody, as well as the selection of amino acids that can be substituted at those positions, are identified. In one representative embodiment, the heavy and light chain amino acid sequences of Figures 5A and 5B were aligned and the identity of the amino acid at each position of the representative antibody was determined. As illustrated in Figures 5A and 5B (which illustrate the amino acids present at each position of the heavy and light chains of a representative MASP-2 inhibitory antibody), several substitutable positions and the amino acid residues that can be substituted at those positions are easily identified. In another representative embodiment, to determine the substitutable positions and the amino acid residues that can be substituted at those positions, the light chain amino acid sequences of the mother and daughter clones are aligned and the identity of the amino acid at each position of the representative antibody is determined.

[0112] [Table 2] In certain embodiments, the subject human anti-MASP-2 monoclonal inhibitory antibodies have a heavy chain variable domain that is substantially identical (e.g., at least about 70%, at least 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 96% identical, or at least about 97% identical, or at least about 98% identical, or at least 99% identical) to any of the heavy chain variable domain sequences shown in Table 2.

[0113] In some embodiments, the subject human anti-MASP-2 monoclonal inhibitory antibody has a heavy chain variable domain that is substantially identical (e.g., at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 96% identical, or at least about 97% identical, or at least about 98% identical, or at least 99% identical) to 17D20(VH) shown as SEQ ID NO: 18. In some embodiments, the subject human anti-MASP-2 monoclonal inhibitory antibody has a heavy chain variable domain that includes or consists of SEQ ID NO: 18.

[0114] In some embodiments, the subject human anti-MASP-2 monoclonal inhibitory antibody has a heavy chain variable domain that is substantially identical (e.g., at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least 99% identical) to 17D20_cd35VH2N11(VH) set forth as SEQ ID NO: 20. In some embodiments, the subject human anti-MASP-2 monoclonal inhibitory antibody has a heavy chain variable domain that includes or consists of SEQ ID NO: 20.

[0115] In some embodiments, the subject human anti-MASP-2 monoclonal inhibitory antibody has a heavy chain variable domain that is substantially identical (e.g., at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 96% identical, or at least about 97% identical, or at least about 98% identical, or at least 99% identical) to 17N16(VH) shown as SEQ ID NO: 21. In some embodiments, the subject human anti-MASP-2 monoclonal inhibitory antibody has a heavy chain variable domain that includes or consists of SEQ ID NO: 21.

[0116] In some embodiments, the subject human anti-MASP-2 monoclonal inhibitory antibodies have a light chain variable domain that is substantially identical (e.g., at least about 70%, at least 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 96% identical, or at least about 97% identical, or at least about 98% identical, or at least 99% identical) to any of the light chain variable domain sequences shown in Table 2.

[0117] In some embodiments, the subject human anti-MASP-2 monoclonal inhibitory antibody has a light chain variable domain that is substantially identical (e.g., at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 96% identical, or at least about 97% identical, or at least about 98% identical, or at least 99% identical) to 17D20(VL) shown as SEQ ID NO: 22. In some embodiments, the subject human anti-MASP-2 monoclonal inhibitory antibody has a light chain that comprises or consists of SEQ ID NO: 22.

[0118] In some embodiments, the subject human anti-MASP-2 monoclonal inhibitory antibody has a light chain variable domain that is substantially identical (e.g., at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 96% identical, or at least about 97% identical, or at least about 98% identical, or at least 99% identical) to 17D20_35VH-21N11VL(VL) shown as SEQ ID NO: 24. In some embodiments, the subject human anti-MASP-2 monoclonal inhibitory antibody has a light chain that comprises or consists of SEQ ID NO:24.

[0119] In some embodiments, the subject human anti-MASP-2 monoclonal inhibitory antibody has a light chain variable domain that is substantially identical (e.g., at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 96% identical, or at least about 97% identical, or at least about 98% identical, or at least 99% identical) to 17N16(VL) shown as SEQ ID NO: 25. In some embodiments, the subject human anti-MASP-2 monoclonal inhibitory antibody has a light chain that comprises or consists of SEQ ID NO:25.

[0120] In some embodiments, the subject human anti-MASP-2 monoclonal inhibitory antibody has a light chain variable domain that is substantially identical (e.g., at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 96% identical, or at least about 97% identical, or at least about 98% identical, or at least 99% identical) to 17N16_17N9(VL) shown as SEQ ID NO: 27. In some embodiments, the subject human anti-MASP-2 monoclonal inhibitory antibody has a light chain that comprises or consists of SEQ ID NO:27.

[0121] In some embodiments, the anti-MASP-2 antibody of the present invention comprises a heavy or light chain encoded by a nucleotide sequence that hybridizes under high stringency conditions to a nucleotide sequence encoding a heavy or light chain as shown in Table 2. High stringency conditions include incubation in 0.1xSSC (15 mM saline / 0.15 mM sodium citrate) at 50°C or higher.

[0122] In some embodiments, the anti-MASP-2 inhibitory antibody of the present invention has a heavy chain variable region comprising one or more CDRs (CDR1, CDR2 and / or CDR3) that are substantially identical (e.g., at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 96% identical, or at least about 97% identical, or at least about 98% identical, or at least 99% identical) to the amino acid sequence of the CDR of any heavy chain variable sequence shown in Figure 5A or 5B or described in Tables 3A-F and Table 4 below, or that comprise or consist of the same sequence.

[0123] In some embodiments, the anti-MASP-2 inhibitory antibody of the present invention has a light chain variable region comprising one or more CDRs (CDR1, CDR2 and / or CDR3) that are substantially identical (e.g., at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 96% identical, or at least about 97% identical, or at least about 98% identical, or at least 99% identical) to the amino acid sequence of the CDR of any light chain variable sequence shown in Figure 5A or 5B or described in Tables 4A-F and Table 5 below, or that comprise or consist of the same sequence.

[0124] Heavy chain variable region

[0125] [Table 3A]

[0126] [Table 3B]

[0127] [Table 3C]

[0128] [Table 3D]

[0129] [Table 3E]

[0130] [Table 3F] The heavy chain variable region (VH) sequences for the mother and daughter clones listed in Tables 2 and 3A-F above are shown below.

[0131] The Kabat CDRs (31-35 (H1), 50-65 (H2) and 95-102 (H3)) are in bold; the Chothia CDRs (26-32 (H1), 52-56 (H2) and 95-101 (H3)) are underlined.

[0132] [ka]

[0133] [Table 4] Light chain variable region

[0134] [Table 5A]

[0135] [Table 5B]

[0136] [Table 5C]

[0137] [Table 5D]

[0138] [Table 5E]

[0139] [Table 5F] The light chain variable region (VL) sequences for the mother and daughter clones listed in Tables 2 and 5A-F above are shown below.

[0140] The Kabat CDRs (24-34 (L1); 50-56 (L2); and 89-97 (L3) are in bold; the Chothia CDRs (24-34 (L1); 50-56 (L2) and 89-97 (L3) are underlined. These regions are the same whether numbered by the Kabat or Chothia system.

[0141] [ka]

[0142] [Table 6] In one embodiment, the present invention provides an isolated human monoclonal antibody or antigen-binding fragment thereof that binds to human MASP-2, comprising: (i) a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3 sequences; and (ii) a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3, wherein the CDR-H3 sequence of the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 38 or SEQ ID NO: 90 and conservative sequence modifications thereof, and the CDR-L3 sequence of the light chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 51 or SEQ ID NO: 94 and conservative sequence modifications thereof, and the isolated antibody inhibits MASP-2-dependent complement activation.

[0143] In one embodiment, the CDR-H2 sequence of the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 32 or 33, and conservative sequence modifications thereof. In one embodiment, the CDR-H1 sequence of the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 28 or SEQ ID NO: 29, and conservative modifications thereof. In one embodiment, the CDR-L2 sequence of the light chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 93, and conservative modifications thereof. In one embodiment, the CDR-L1 sequence of the light chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 91 or SEQ ID NO: 92, and conservative modifications thereof. In one embodiment, the CDR-H1 of the heavy chain variable region comprises SEQ ID NO: 28.

[0144] In one embodiment, the CDR-H2 of the heavy chain variable region comprises SEQ ID NO: 32. In one embodiment, the CDR-H3 of the heavy chain variable region comprises SEQ ID NO: 90 (as shown in Table 4). In one embodiment, the amino acid sequence shown in SEQ ID NO: 90 comprises an R (Arg) at position 8.

[0145] In one embodiment, CDR-L1 of the light chain variable region comprises SEQ ID NO: 91 (as shown in Table 6). In one embodiment, the amino acid sequence set forth in SEQ ID NO: 91 comprises E (Glu) at position 2. In one embodiment, the amino acid sequence set forth in SEQ ID NO: 91 comprises Y (Tyr) at position 8.

[0146] In one embodiment, the CDR-L2 of the light chain variable region comprises SEQ ID NO:93 (as shown in Table 6), wherein the amino acid sequence set forth in SEQ ID NO:93 comprises K (Lys) at position 2. In one embodiment, the amino acid sequence set forth in SEQ ID NO:93 comprises Q (Gln) at position 3.

[0147] In one embodiment, the CDR-L3 of the light chain variable region comprises SEQ ID NO:51.

[0148] In one embodiment, the antibody or antigen-binding fragment thereof has a K D In one embodiment, the antibody or antigen-binding fragment thereof binds to human MASP-2 with an IC of 10 nM or less in an in vitro assay in 1% human serum. 50 In one embodiment, the antibody or antigen-binding fragment thereof has an IC of 30 nM or less in 90% human serum. 50 In one embodiment, the conservative sequence modifications comprise or consist of a molecule comprising a variable region identical to the recited variable domains except for a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in total in the CDR regions of the heavy chain variable region, and / or up to a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in total in said CDR regions of the light chain variable region.

[0149] In another aspect, the present invention provides an isolated human antibody or antigen-binding fragment thereof that binds to human MASP-2, wherein the antibody comprises: I) a) a heavy chain variable region comprising i) a heavy chain CDR-H1 comprising the amino acid sequence of 31 to 35 of SEQ ID NO:21; and ii) a heavy chain CDR-H2 comprising the amino acid sequence of 50 to 65 of SEQ ID NO:21; and iii) a heavy chain CDR-H3 comprising the amino acid sequence of 95 to 102 of SEQ ID NO:21; and b) a light chain CDR-L1 comprising the amino acid sequence of 24 to 34 of SEQ ID NO:25 or SEQ ID NO:27; and ii) a light chain CDR-L2 comprising the amino acid sequence of 50 to 56 of SEQ ID NO:25 or SEQ ID NO:27. and iii) a light chain variable region comprising a light chain CDR-L3 comprising an amino acid sequence of 89 to 97 of SEQ ID NO: 25 or SEQ ID NO: 27; or II) a variant thereof which is identical to the variable domain except for a total of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions in total in the CDR region of the heavy chain variable region and a total of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions in total in the CDR region of the light chain variable region, wherein the antibody or its variant inhibits MASP-2-dependent complement activation. In one embodiment, the variants comprise an amino acid substitution at one or more positions selected from the group consisting of positions 31, 32, 33, 34, 35, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 95, 96, 97, 98, 99, 100, or 102 of the heavy chain variable region. In one embodiment, the variants comprise an amino acid substitution at one or more positions selected from the group consisting of positions 25, 26, 27, 29, 31, 32, 33, 51, 52, 89, 92, 93, 95, 96, or 97 of the light chain variable region. In one embodiment, the antibody heavy chain comprises SEQ ID NO:21. In one embodiment, the antibody light chain comprises SEQ ID NO:25. In one embodiment, the antibody light chain comprises SEQ ID NO:27.

[0150] In another aspect, the present invention provides an isolated human monoclonal antibody that binds to human MASP-2, wherein the antibody comprises: I) a) a heavy chain variable region comprising i) a heavy chain CDR-H1 comprising the amino acid sequence of 31 to 35 of SEQ ID NO: 20; and ii) a heavy chain CDRH-2 comprising the amino acid sequence of 50 to 65 of SEQ ID NO: 20; and iii) a heavy chain CDR-H3 comprising the amino acid sequence of 95 to 102 of SEQ ID NO: 18 or SEQ ID NO: 20; and b) a light chain CDR-L1 comprising the amino acid sequence of 24 to 34 of SEQ ID NO: 22 or SEQ ID NO: 24; and ii) a light chain CDR-L2 comprising the amino acid sequence of 50 to 56 of SEQ ID NO: 22 or SEQ ID NO: 24. and iii) a light chain variable region comprising a light chain CDR-L3 comprising an amino acid sequence of 89 to 97 of SEQ ID NO: 22 or SEQ ID NO: 24; or II) a variant thereof which is identical to the variable domain except for a total of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions in total in the CDR region of the heavy chain variable region and a total of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions in total in the CDR region of the light chain variable region, wherein the antibody or its variant inhibits MASP-2-dependent complement activation. In one embodiment, the variant comprises an amino acid substitution at one or more positions selected from the group consisting of positions 31, 32, 33, 34, 35, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 95, 96, 97, 98, 99, 100, or 102 of the heavy chain variable region. In one embodiment, the variant comprises an amino acid substitution at one or more positions selected from the group consisting of positions 25, 26, 27, 29, 31, 32, 33, 51, 52, 89, 92, 93, 95, 96, or 97 of the light chain variable region. In one embodiment, the heavy chain of the antibody comprises SEQ ID NO:20, or a variant thereof comprising at least 80% identity to SEQ ID NO:20 (e.g., at least 85%, at least 90%, at least 95%, or at least 98% identity to SEQ ID NO:20).In one embodiment, the heavy chain of the antibody comprises SEQ ID NO: 18, or a variant thereof comprising at least 80% identity to SEQ ID NO: 18 (e.g., at least 85%, at least 90%, at least 95%, or at least 98% identity to SEQ ID NO: 18). In one embodiment, the light chain of the antibody comprises SEQ ID NO: 22, or a variant thereof comprising at least 80% identity to SEQ ID NO: 22 (e.g., at least 85%, at least 90%, at least 95%, or at least 98% identity to SEQ ID NO: 22). In one embodiment, the light chain of the antibody comprises SEQ ID NO: 24, or a variant thereof comprising at least 80% identity to SEQ ID NO: 24 (e.g., at least 85%, at least 90%, at least 95%, or at least 98% identity to SEQ ID NO: 24).

[0151] In one embodiment, the antibody binds to an epitope in the CCP1 domain of MASP-2.

[0152] In one embodiment, the antibody has a K D In one embodiment, the antibody binds to human MASP-2 with an IC of 10 nM or less in an in vitro assay in 1% human serum. 50 In one embodiment, the antibody has an IC of 30 nM or less in 90% human serum. 50 inhibits C3b deposition.

[0153] In one embodiment, the antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)2 and F(ab')2. In one embodiment, the antibody is a single chain molecule. In one embodiment, the antibody is an IgG2 molecule. In one embodiment, the antibody is an IgG1 molecule. In one embodiment, the antibody is an IgG4 molecule. In one embodiment, the IgG4 molecule comprises an S228P mutation.

[0154] In one embodiment, the antibody does not substantially inhibit the classical pathway (ie, classical pathway activity is at least 80%, or at least 90%, or at least 95%, or at least 95% intact).

[0155] In another embodiment, the present invention provides a method for the treatment of cancer comprising administering to a patient a therapeutically effective amount of a cancer cell comprising administering to the ... D It dissociates from human MASP-2 at 100 ng / mL and activates C4 on mannan-coated substrates with an IC of 10 nM or less in 1% serum. 50 The present invention provides an isolated fully human monoclonal antibody or antigen-binding fragment thereof that inhibits MASP-2. In some embodiments, the antibody or antigen-binding fragment thereof specifically recognizes at least a portion of the epitope recognized by a reference antibody (e.g., reference antibody OMS646 (see Table 22)) comprising a heavy chain variable region as set forth in SEQ ID NO: 20 and a light chain variable region as set forth in SEQ ID NO: 24. In accordance with the above, an antibody or antigen-binding fragment thereof according to certain preferred embodiments of the present application (i) specifically binds to human MASP-2 and (ii) may compete for binding to its antigen with any antibody described herein comprising a VH and / or VL domain disclosed herein or comprising a CDR-H3 or any variant thereof disclosed herein. Competition between binding members can be easily assayed in vitro, for example, using ELISA and / or by tagging one binding member with a specific reporter molecule that can be detected in the presence of other untagged binding members that allow for the discrimination of specific binding members that bind to the same or overlapping epitopes. Thus, provided herein are certain antibodies or antigen-binding fragments thereof that contain the antigen-binding site of a human antibody that competes for binding to human MASP-2 with the antibodies described herein (e.g., any one of OMS641 to OMS646 as shown in Table 24) that bind to human MASP-2.

[0156] Modified MASP-2 inhibitor antibody The human monoclonal antibodies described above can be modified to provide modified antibodies that inhibit MASP-2-dependent complement activation. The modified antibodies can be produced by substituting, adding or deleting at least one amino acid of the human monoclonal antibodies described above. Usually, these modified antibodies have the general characteristics of the human antibodies described above and contain at least the CDRs of the human antibodies described above, or in certain embodiments, CDRs that closely resemble the CDRs of the human antibodies described above.

[0157] In a preferred embodiment, the variant comprises one or more amino acid substitutions in one or more hypervariable regions of the parent antibody. For example, the variant may comprise at least one, for example, about 1 to about 10, for example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 substitutions, and preferably about 2 to about 6 substitutions, in one or more CDR regions of the parent antibody. In one embodiment, the variant comprises an amino acid substitution at one or more positions selected from the group consisting of positions 31, 32, 33, 34, 35, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 95, 96, 97, 98, 99, 100, or 102 of the heavy chain variable region. In one embodiment, the variant comprises an amino acid substitution at one or more positions selected from the group consisting of positions 25, 26, 27, 29, 31, 32, 33, 51, 52, 89, 92, 93, 95, 96 or 97 of the light chain variable region.

[0158] In some embodiments, the above-mentioned variant antibody has an amino acid sequence identical to the variable domain of the subject antibody shown in Table 2, except for a total of up to 1, 2, 3, 4, 5 or 6 amino acid substitutions in the CDR regions of the heavy chain variable region and / or a total of up to 1, 2, 3, 4, 5 or 6 amino acid substitutions in the CDR regions of the light chain variable region, wherein the antibody or variant thereof inhibits MASP-2-dependent complement activation.

[0159] Typically, the variant may have an amino acid sequence with at least 75% amino acid sequence identity with the heavy or light chain variable domain sequence of the parent antibody, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identity. Identity or homology with respect to this sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical to those of the parent antibody after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. N-terminal, C-terminal or internal extensions, deletions or insertions into the antibody sequence (e.g., signal peptide sequences, linker sequences, or tags such as HIS tags) shall not be construed as affecting sequence identity or homology. The variant retains the ability to bind to human MASP-2 and preferably has properties superior to the parent antibody. For example, the variant may have stronger binding affinity and / or enhanced ability to inhibit or block MASP-2-dependent complement activation.

[0160] Since the type of anti-MASP-2 antibody has been found to affect its activity in the biological activity assays disclosed herein, one should compare, for example, a Fab type variant with a Fab type parent antibody or a full-length type variant with a full-length type parent antibody to analyze such characteristics. Variant antibodies of particular interest herein are those that exhibit an enhancement in biological activity of at least about 10-fold, preferably at least about 20-fold, and most preferably at least about 50-fold, when compared to the parent antibody.

[0161] The antibodies of the present invention may be modified to enhance desirable properties (e.g., where it is desirable to control the serum half-life of the antibody). Typically, intact antibody molecules have a very long serum persistence, whereas fragments (<60-80 kDa) are filtered very quickly by the kidney. Thus, where a long-term action of the MASP-2 antibody is desirable, the MASP-2 antibody is preferably a complete full-length IgG antibody (e.g., IgG2 or IgG4), whereas where a shorter-term action of the MASP-2 antibody is desirable, an antibody fragment may be preferred. As described in Example 5, it has been confirmed that the S228P substitution in the hinge region of IgG4 increases serum stability. Thus, in some embodiments, the subject MASP-2 antibody is a full-length IgG4 antibody comprising an S228P substitution.

[0162] single chain antibody In one embodiment of the present invention, the MASP-2 inhibitory antibody is a single chain antibody, defined as a genetically engineered molecule comprising a light chain variable region and a heavy chain variable region linked by a suitable polypeptide linker as a genetically fused single chain molecule. Such single chain antibodies are also referred to as "single chain Fv" or "scFv" antibody fragments. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. scFv antibodies that bind to MASP-2 may have the variable light chain region oriented amino-terminal or carboxyl-terminal to the variable heavy chain region. Representative scFv antibodies of the present invention are set forth herein as SEQ ID NOs: 55-61 and 66-68.

[0163] Methods for generating antibodies In many embodiments, nucleic acids encoding a subject monoclonal antibody are introduced directly into host cells, and the cells are incubated under conditions sufficient to induce expression of the encoded antibody.

[0164] In some embodiments, the present invention provides a nucleic acid molecule encoding an anti-MASP-2 antibody or fragment thereof of the present invention (e.g., an antibody or fragment thereof shown in Table 2). In some embodiments, the present invention provides a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:97, SEQ ID NO:88 and SEQ ID NO:89.

[0165] In some embodiments, the present invention provides a cell comprising a nucleic acid molecule encoding an anti-MASP-2 antibody of the present invention.

[0166] In some embodiments, the present invention provides an expression cassette comprising a nucleic acid molecule encoding an anti-MASP-2 antibody of the present invention.

[0167] In some embodiments, the present invention provides a method for producing an anti-MASP-2 antibody, comprising culturing a cell containing a nucleic acid molecule encoding an anti-MASP-2 antibody of the present invention.

[0168] According to certain related embodiments, there are provided recombinant host cells comprising one or more constructs as described herein; nucleic acids encoding any antibody, CDR, VH or VL domain or antigen-binding fragment thereof; and methods of producing the encoded product, which methods include expression from the encoding nucleic acid therefor. Expression may conveniently be achieved by culturing recombinant host cells comprising the nucleic acid under suitable conditions. After production by expression, the antibody or antigen-binding fragment thereof may be isolated and / or purified using any suitable technique and then used as desired.

[0169] For example, any cell suitable for expressing the expression cassette as a host cell can be used, such as yeast, insect, plant, etc. In many embodiments, a mammalian host cell line that does not normally produce antibodies is used, examples of which are: monkey kidney cells (COS cells), monkey kidney CVI cells transformed with SV40 (COS-7, ATCC CRL1651); human embryonic kidney cells (HEK-293, Graham et al., J. Gen. Virol. 36:59 (1977); baby hamster kidney cells (BHK, ATCC CCL10); Chinese hamster ovary cells (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. (USA) 77:4216, (1980); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CVI ATCC CCL70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL2); dog kidney cells (MDCK, ATCC CCL34); buffalo rat liver cells (BRL 3A, ATCC CRL1442); human lung cells (W138, ATCC CCL75); human liver cells (hep G2, HB8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals NYAcad.Sci 383:44-68 (1982)); NIH / 3T3 cells (ATCC CRL-1658); and mouse L cells (ATCC CCL-1). Additional cell lines will be apparent to those of skill in the art. A wide variety of cell lines are available from the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209.

[0170] Methods for introducing nucleic acid into cells are well known in the art. Suitable methods include electroporation, particle gun technology, calcium phosphate precipitation, direct microinjection, and the like. The choice of method generally depends on the type of cell to be transformed and the context in which the transformation is performed (i.e., in vitro, ex vivo, or in vivo). A general discussion of these methods can be found in Ausubel et al., Short Protocols in Molecular Biology, 3d ed., Wiley & Sons, 1995. In some embodiments, lipofectamine and calcium-mediated gene transfer techniques are used.

[0171] After a subject nucleic acid is introduced into the cells, the cells are typically incubated, usually at 37° C., sometimes under selection, for a suitable period of time to allow expression of the antibody. In most embodiments, the antibody is typically secreted into the supernatant of the medium in which the cells are growing.

[0172] In mammalian host cells, several virus-based expression systems can be utilized to express the subject antibodies. When adenovirus is used as an expression vector, the antibody coding sequence of interest can be linked to the adenovirus transcription / translation control complex, such as the late promoter and tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) can result in a recombinant virus that is viable and capable of expressing antibody molecules in infected hosts (see, e.g., Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Expression efficiency can be increased by including appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., Methods in Enzymol. 153:51-544 (1987)).

[0173] For long-term, high-yield production of recombinant antibodies, stable expression can be used. For example, cell lines can be engineered that stably express an antibody molecule. Rather than using an expression vector containing a viral origin of replication, host cells can be transformed with an immunoglobulin expression cassette and a selectable marker. After introduction of the foreign DNA, engineered cells can be grown in rich medium for 1-2 days and then switched to selective medium. The selectable marker in the recombinant plasmid confers resistance to selection, allowing the cells to stably integrate the plasmid into their chromosomes and grow to form foci that can then be cloned and expanded into cell lines. Such engineered cell lines can be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.

[0174] Once an antibody molecule of the invention has been generated, it may be purified by any method known in the art for purifying immunoglobulin molecules, such as, for example, chromatography (e.g., ion exchange chromatography, affinity chromatography (particularly by affinity for a particular antigen following Protein A), and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for purifying proteins. In many embodiments, the antibody is secreted from the cells into the culture medium and recovered from the culture medium. For example, a nucleic acid sequence encoding a signal peptide may be included adjacent to the coding region of the antibody or fragment, such as, for example, as provided in nucleotides 1-57 of SEQ ID NO:71, which encodes a signal peptide as provided in amino acids 1-19 of SEQ ID NO:72. Such a signal peptide may be incorporated adjacent to the 5' end of the amino acid sequence set forth herein for the subject antibodies to facilitate production of the subject antibodies.

[0175] Pharmaceutical Carriers and Delivery Vehicles In another aspect, the present invention provides a composition for inhibiting the deleterious effects of MASP-2-dependent complement activation, comprising a therapeutically effective amount of a human anti-MASP-2 inhibitory antibody and a pharma- ceutically acceptable carrier.

[0176] Typically, the human MASP-2 inhibitory antibody composition of the present invention in combination with any other selected therapeutic agent is suitably contained in a pharma- ceutically acceptable carrier. The carrier is selected to be non-toxic, biocompatible, and not to adversely affect the biological activity of the MASP-2 inhibitory antibody (and any other therapeutic agent used in combination therewith). Representative pharma- ceutically acceptable carriers for polypeptides are described in U.S. Patent No. 5,211,657 to Yamada. The anti-MASP-2 antibody may be formulated into preparations in solid, semi-solid, gel, liquid or gaseous form (e.g., tablets, capsules, powders, granules, ointments, solutions, depositories, inhalants, and injections for oral, parenteral or surgical administration). The present invention also contemplates local administration of the composition by coating medical devices and the like.

[0177] Suitable carriers for parenteral delivery, injection or infusion and local delivery via injectable substances include distilled water, physiological phosphate buffered saline, normal Ringer's solution or lactated Ringer's solution, dextrose solution, Hank's solution or propanediol.In addition, sterile non-volatile oils can be used as solvents or suspension media.For this purpose, any biocompatible oils can be used, including synthetic monoglycerides or synthetic diglycerides.In addition, fatty acids such as oleic acid find use in the preparation of injectable substances.The above carriers and agents can be formulated as liquids, suspensions, polymerizable or non-polymerizable gels, pastes or ointments.

[0178] The carrier may also include a delivery vehicle to sustain (i.e., extend, delay, or control) the delivery of the agent or to improve the delivery, uptake, stability, or pharmacokinetics of the therapeutic agent. Such delivery vehicles may include, but are not limited to, microparticles, microspheres, nanospheres, or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymeric or copolymeric hydrogels, and polymeric micelles. Suitable hydrogel and micelle delivery systems include PEO:PHB:PEO copolymers and copolymer / cyclodextrin complexes disclosed in WO2004 / 009664A2 and PEO and PEO / cyclodextrin complexes disclosed in US Patent Application Publication No. 2002 / 0019369. Such hydrogels may be injected locally at the intended site of action or subcutaneously or intramuscularly to form sustained release depots.

[0179] For intra-articular delivery, the MASP-2 inhibitory antibody may be carried in an injectable liquid or gel carrier as described above, an injectable sustained release delivery vehicle as described above, or hyaluronic acid or a hyaluronic acid derivative.

[0180] For intrathecal (IT) or intracerebroventricular (ICV) delivery, the present invention can be administered using an appropriately sterile delivery system (eg, liquid; gel, suspension, etc.).

[0181] The compositions of the present invention may also contain biocompatible excipients (e.g., dispersing or wetting agents, suspending agents, diluents, buffers, penetration enhancers, emulsifying agents, binders, thickening agents, flavoring agents (for oral administration)).

[0182] To achieve high concentrations of anti-MASP-2 antibody for local delivery, the antibody may be formulated as a suspension of particles or crystals in solution for subsequent injection (e.g., intramuscular injection of a depot).

[0183] More specifically, with respect to anti-MASP-2 antibodies, representative formulations may be parenterally administered as injectable doses of a solution or suspension of the compound in a physiologically acceptable diluent, including pharmaceutical carriers, which may be sterile liquids (e.g., water, oil, saline, glycerol or ethanol). In addition, auxiliary agents (e.g., wetting or emulsifying agents, surfactants, pH buffering substances, etc.) may be present in compositions containing anti-MASP-2 antibodies. Additional components of pharmaceutical compositions include petroleum oils (e.g., of animal, vegetable or synthetic origin), such as soybean oil and mineral oil. Typically, glycols (e.g., propylene glycol or polyethylene glycol) are preferred liquid carriers for injectable solutions.

[0184] Anti-MASP-2 antibodies may also be administered in the form of a depot injection or implant preparation, which may be formulated in such a manner as to permit sustained or pulsatile release of the active agent.

[0185] Pharmaceutical compositions containing MASP-2 inhibitory antibodies can be administered in a number of ways, depending on whether a local or systemic mode of administration is most suitable for the condition being treated. Also, as described herein above with respect to extracorporeal reperfusion procedures, MASP-2 inhibitory antibodies can be administered via introduction of the compositions of the present invention into recirculating blood or plasma. Furthermore, the compositions of the present invention can be delivered by coating or incorporating the compositions on or into implantable medical devices.

[0186] systemic delivery As used herein, the terms "systemic delivery" and "systemic administration" are intended to include, but are not limited to, oral and parenteral routes, including intramuscular (IM), subcutaneous, intravenous (IV), intra-arterial, inhalation, sublingual, buccal, topical, transdermal, nasal, rectal, vaginal and other routes of administration that effectively result in the distribution of the delivered antibody to the single site or sites where therapeutic effect is intended. Preferred routes of systemic delivery for the present compositions include intravenous, intramuscular, subcutaneous and inhalation. It will be understood that the correct systemic administration route for a selected agent utilized in a particular composition of the present invention is determined in part to account for the agent's susceptibility to metabolic conversion pathways associated with a given administration route.

[0187] MASP-2 inhibitory antibodies and MASP-2 inhibitory polypeptides may be delivered to a subject in need of such inhibition by any suitable means, including oral, pulmonary, parenteral (e.g., intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), inhalation (e.g., inhalation via fine powder formulation), transdermal, nasal, vaginal, rectal or sublingual administration routes, and may be formulated into dosage forms suitable for each administration route.

[0188] By way of example, MASP-2 inhibitory antibodies and MASP-2 inhibitory peptides can be introduced into the living body by application to a bodily membrane capable of absorbing the polypeptide, such as the nasal, gastrointestinal and rectal membranes. The polypeptide is typically applied to the absorbent membrane in combination with a permeation enhancer (e.g., Lee, VHL, Crit. Rev. Ther. Drug Carrier Sys. 5:69, 1988; Lee, VHL, J. Controlled Release 13:213, 1990; Lee, VHL, Ed., Peptide and Protein Drug Delivery, Marcel Dekker, New York (1991); DeBoer, AG, et al., J. Controlled Release, 13:241, 1990. For example, STDHF is a synthetic derivative of fusidic acid, a steroidal surfactant similar in structure to bile salts, and has been used as a permeation enhancer for nasal delivery (Lee, WA, Biopharm. 22, Nov. / Dec. 1990).

[0189] MASP-2 inhibitory antibodies and MASP-2 inhibitory polypeptides may be introduced in conjunction with another molecule, such as a lipid, that protects the polypeptide from enzymatic degradation. For example, covalently attached polymers, particularly polyethylene glycol (PEG), have been used to protect certain proteins from enzymatic hydrolysis in the body, thereby extending their half-life (Fuertges, F. et al., J. Controlled Release 11:139, 1990). Many polymer systems have been reported for protein delivery (Bae, YH, et al., J. Controlled Release 9:271, 1989; Hori, R., et al., Pharm. Res. 6:813, 1989; Yamakawa, I., et al., J. Pharm. Sci. 79:505, 1990; Yoshihiro, I., et al., J. Controlled Release 10:195, 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. Sci. 78:117,1989; Takakura, Y. et al., J. Pharm. Sci. 78:219,1989).

[0190] Recently, liposomes with improved serum stability and circulation half-time have been developed (see, for example, U.S. Patent No. 5,741,516 to Webb). In addition, various methods of liposomes and liposome-like preparations as potential drug carriers have been reviewed (see, for example, U.S. Patent No. 5,567,434 to Szoka; U.S. Patent No. 5,552,157 to Yagi; U.S. Patent No. 5,565,213 to Nakamori; U.S. Patent No. 5,738,868 to Shinkarenko; and U.S. Patent No. 5,795,587 to Gao).

[0191] For transdermal application, the MASP-2 inhibitory antibodies and MASP-2 inhibitory polypeptides may be combined with other suitable ingredients (e.g., carriers and / or adjuvants). There are no limitations on the nature of such other ingredients, except that they must be pharma- ceutically acceptable for the intended administration and cannot reduce the activity of the active ingredients of the composition. Examples of suitable vehicles include ointments, creams, gels or suspensions, with or without purified collagen. The MASP-2 inhibitory antibodies and MASP-2 inhibitory polypeptides may also be impregnated into transdermal patches, plasters and bandages, preferably in liquid or semi-liquid form.

[0192] The compositions of the present invention may be administered systemically periodically at intervals determined to maintain a desired level of therapeutic effect. For example, the compositions may be administered by subcutaneous injection, for example, every 2-4 weeks or less. The administration regimen is determined by the physician, taking into consideration various factors that may affect the action of the combined agents. 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 the individual agents may vary as a function of the MASP-2 inhibitory antibody contained in the composition, as well as the presence and nature of any drug delivery vehicle (e.g., sustained release delivery vehicle). Furthermore, the dosage may be adjusted to compensate for variations in the frequency of administration and pharmacokinetic behavior of the delivered agent.

[0193] Local delivery As used herein, the term "local" encompasses application of a drug at or near the site of intended localized action, and may include, for example, local delivery to the skin or other affected tissue, delivery to the eye, intrathecal (IT), intracerebroventricular (ICV), intraarticular, intracavitary, intracranial or intravesicular administration, placement or irrigation.Local administration may be preferred to allow low dose administration to avoid systemic side effects, and to more precisely control the timing and concentration of delivery of active agent at the site of local delivery.Local administration provides a known concentration at the target site, regardless of interpatient variability in metabolism, blood flow, etc.Direct delivery modes also provide improved dosage control.

[0194] Local delivery of MASP-2 inhibitory antibodies can be achieved in the context of surgical methods for treating a disease or condition, for example, during procedures such as arterial bypass surgery, atherectomy, laser procedures, ultrasound procedures, balloon angioplasty and stent placement. For example, a MASP-2 inhibitor can be administered to a subject in combination with a balloon angioplasty procedure. A balloon angioplasty procedure involves the insertion of a catheter having a deflated balloon into an artery. The deflated balloon is placed proximal to an atherosclerotic plaque and the balloon is inflated so that the plaque is compressed against the vessel wall. As a result, the surface of the balloon contacts the vascular endothelial cell layer on the surface of the blood vessel. A MASP-2 inhibitory antibody can be attached to a balloon angioplasty catheter in a manner that allows the agent to be released at the site of the atherosclerotic plaque. The agent can be attached to the balloon catheter according to standard procedures known in the art. For example, the agent can be stored in a compartment of the balloon catheter until the balloon is inflated at a location where the agent is released into the local environment. Alternatively, the drug may be impregnated onto the surface of the balloon so that the drug contacts the cells of the arterial wall when the balloon is inflated. The drug may also be delivered in a perforated balloon catheter as disclosed in Flugelman, MY et al., Circulation 85:1110-1117, 1992. See also published PCT application WO95 / 23161 for a representative procedure for attaching therapeutic proteins to balloon angioplasty catheters. Similarly, the MASP-2 inhibitory antibody may be included in a gel or polymer coating applied to the stent, or may be incorporated into the material of the stent to elute the MASP-2 inhibitory antibody after the stent is placed in the blood vessel.

[0195] Treatment regimen MASP-2 inhibitory antibody compositions used in the treatment of arthritis and other musculoskeletal disorders may be delivered locally by intra-articular injection. Such compositions may suitably include a sustained release delivery vehicle. As a further example of when local delivery may be desired, MASP-2 inhibitory antibody compositions used in the treatment of urogenital conditions may suitably be instilled into the bladder or another urogenital structure.

[0196] In preventive applications, the pharmaceutical composition is administered to a subject susceptible to or otherwise at risk of a condition associated with MASP-2-dependent complement activation in an amount sufficient to eliminate or reduce the risk of developing the symptoms of the condition. In therapeutic applications, the pharmaceutical composition is administered to a subject suspected of or already suffering from a condition associated with MASP-2-dependent complement activation in a therapeutically effective amount sufficient to alleviate or at least partially reduce the symptoms of the condition. In both preventive and therapeutic regimens, the composition comprising the MASP-2 inhibitory antibody may be administered in several doses until a sufficient therapeutic result is achieved in the subject. The application of the MASP-2 inhibitory antibody composition of the present invention may be performed by a single administration or a limited series of administrations of the composition to treat acute conditions, such as reperfusion injury or other traumatic injuries. Alternatively, the composition may be administered at periodic intervals over an extended period of time to treat chronic conditions, such as arthritis or psoriasis.

[0197] The MASP-2 inhibitory composition used in the present invention can be delivered immediately or shortly after an acute event that results in activation of the lectin pathway (e.g., after an ischemic event and reperfusion of ischemic tissue). Examples include myocardial ischemia reperfusion, renal ischemia reperfusion, cerebral ischemia reperfusion, organ transplantation, and finger / limb reattachment. Other acute examples include sepsis. The MASP-2 inhibitory composition of the present invention can be administered as soon as possible after an acute event that activates the lectin pathway, preferably within 12 hours, more preferably within 2 to 3 hours of the triggering event, for example, by systemic delivery of the MASP-2 inhibitory composition.

[0198] The method and composition of the present invention can be used to inhibit inflammation and related processes that typically result from diagnostic and therapeutic medical and surgical procedures.To inhibit such processes, the MASP-2 inhibitory composition of the present invention can be applied periprocedurally.As used herein, "periprocedurally" refers to administering the inhibitory composition before and / or during and / or after the procedure, i.e., before the procedure, before and during the procedure, before and after the procedure, before, during and after the procedure, during, during and after the procedure, or after the procedure.Periprocedural application can be performed by local administration of the composition to the surgical or procedural site (e.g., by injection of the site or continuous or intermittent irrigation of the site, or by systemic administration). Methods suitable for localized perioperative delivery of MASP-2 inhibitory antibody solutions are disclosed in U.S. Patent Nos. 6,420,432 to Demopulos and 6,645,168 to Demopulos. Methods suitable for localized delivery of chondroprotective compositions comprising MASP-2 inhibitory antibodies are disclosed in International PCT Patent Application WO01 / 07067A2. Methods and compositions suitable for targeted systemic delivery of chondroprotective compositions comprising MASP-2 inhibitory antibodies are disclosed in International PCT Patent Application WO03 / 063799A2.

[0199] Dosage MASP-2 inhibitory antibodies may be administered to a subject in need of such inhibition in a therapeutically effective dose to treat or ameliorate a condition associated with MASP-2-dependent complement activation, a therapeutically effective dose being the amount of MASP-2 inhibitory antibody sufficient to result in amelioration of the symptoms of the condition.

[0200] The toxicity and therapeutic efficacy of MASP-2 inhibitory antibodies can be measured by standard pharmaceutical procedures using experimental animal models (e.g., African green monkeys as described herein). Using such animal models, NOAEL (no observed adverse effect level) and MED (minimal effective dose) can be measured using standard methods. The dose ratio between the NOAEL effect and the MED effect is the healing ratio, expressed as the ratio NOAEL / MED. MASP-2 inhibitory antibodies that show a large healing ratio or index are most preferred. Data obtained from cell culture assays and animal studies can be used in formulating a range of dosages for use in humans. The dosage of MASP-2 inhibitory antibodies is preferably set within a range of circulating concentrations that includes the MED with little or no toxicity. The dosage can vary within this range depending on the dosage form used and the route of administration utilized.

[0201] For the formulation of any compound, animal model can be used to estimate therapeutically effective dose.For example, dose can be formulated in animal model to achieve circulating plasma concentration range including MED.Quantitative level of MASP-2 inhibitory antibody in plasma can be measured, for example, by high performance liquid chromatography.

[0202] In addition to toxicity studies, effective dosage can also be estimated based on the amount of MASP-2 protein present in living subjects and the binding affinity of MASP-2 inhibitory antibodies.It has been shown that MASP-2 levels in normal human subjects are present in serum at low levels in the range of 500ng / ml, and that MASP-2 levels in certain subjects can be measured using the quantitative assay for MASP-2 described in Moller-Kristensen M. et al., J.Immunol.Methods 282:159-167,2003 (hereby incorporated by reference).

[0203] In general, the dosage of an administration composition containing a MASP-2 inhibitory antibody will vary depending on factors such as the subject's age, weight, height, sex, general medical condition and previous medical history. By way of example, the MASP-2 inhibitory antibody may be administered at a dosage in the range of approximately 0.010 to 10.0 mg / kg, preferably 0.010 to 1.0 mg / kg, and more preferably 0.010 to 0.1 mg / kg of the subject's body weight.

[0204] The therapeutic efficacy and appropriate dosage of the MASP-2 inhibitory compositions and methods of the present invention in a given subject can be determined according to complement assays well known to those skilled in the art. Complement produces a number of specific products. In 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 utilize antibodies that react with neoantigens that are not exposed on the native protein that forms the new antigens (neoantigens) but are exposed on the fragments, making these assays very simple and specific. Most of these rely on ELISA technology, although radioimmunoassays are still occasionally used for C3a and C5a. These latter assays measure both the unprocessed fragments and their "desArg" fragments, which are the major forms found in the circulation. The unprocessed fragments and C5a desArg is rapidly cleared by binding to cell surface receptors and is therefore present in very low concentrations, whereas C3a desArgdoes not bind to cells and accumulates in plasma. Measurement of C3a provides a sensitive, pathway-independent index of complement activation. Alternative pathway activation can be assessed by measuring the Bb fragment. Detection of the fluid phase product of membrane attack pathway activation, sC5b-9, provides evidence that complement activation is complete. Because both the lectin and classical pathways generate the same activation products, C4a and C4d, measurement of these two fragments does not provide any information about which of the two pathways generated the activation product.

[0205] Inhibition of MASP-2-dependent complement activation is characterized by at least one of the following changes in the components of the complement system that result from administration of an anti-MASP-2 antibody according to the present invention: inhibition of the generation or production of C4b, C3a, C5a and / or C5b-9 (MAC), which are products of the MASP-2-dependent complement activation system, a decrease in C4 cleavage and C4b deposition or a decrease in C3 cleavage and C3b deposition.

[0206] product In another aspect, the present invention provides an article of manufacture comprising a human MASP-2 inhibitory antibody or antigen-binding fragment thereof as described herein in a unit dosage form suitable for therapeutic administration to a human subject (e.g., in the range of 1 mg to 5000 mg, e.g., 1 mg to 2000 mg, e.g., 1 mg to 1000 mg, e.g., a unit dosage of 5 mg, 10 mg, 50 mg, 100 mg, 200 mg, 500 mg or 1000 mg). In some embodiments, the article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The containers can be formed from a variety of materials, such as glass or plastic. The container holds a composition effective for treating the above-mentioned condition and can have a sterile access port (e.g., the container can be an intravenous solution bag or vial having a stopper pierceable by a hypodermic needle). At least one active agent in the composition is a MASP-2 inhibitory antibody or antigen-binding fragment thereof of the present invention. The label or package insert indicates that the composition is used for treating a particular condition. The label or package insert may further include instructions for administering the antibody composition to a patient. Articles of manufacture and kits comprising the combination therapeutic agents described herein are also contemplated.

[0207] Therapeutic Uses of Anti-MASP-2 Inhibitory Antibodies In another aspect, the present invention provides a method for inhibiting MASP-2-dependent complement activation in a human subject, comprising administering a human monoclonal anti-MASP-2 inhibitory antibody of the present invention in an amount sufficient to inhibit MASP-2-dependent complement activation in the human subject.

[0208] According to this aspect of the invention, as described in Example 10, the MASP-2 inhibitory antibody of the present invention can inhibit the lectin pathway in African green monkeys after intravenous administration. As shown in Table 24, Example 8, the antibody OMS646 used in this study was found to be more potent in human serum. As known to those skilled in the art, non-human primates are often used as models to evaluate antibody therapy.

[0209] As described in U.S. Patent No. 7,919,094, co-pending U.S. Patent Application No. 13 / 083,441 and co-pending U.S. Patent Application No. 12 / 905,972 (each of which is assigned to the assignee of the present application, Omeros Corporation) (each of which is hereby incorporated by reference), MASP-2-dependent complement activation has been implicated as being involved in the pathogenesis of many acute and chronic disease conditions, including MASP-2-dependent complement-mediated vascular conditions, ischemia-reperfusion injury, atherosclerosis, inflammatory gastrointestinal disorders, pulmonary conditions, extracorporeal reperfusion procedures, musculoskeletal conditions, renal conditions, skin conditions, organ or tissue transplants, nervous system disorders or injuries, blood disorders, genitourinary conditions, diabetes, chemotherapy or radiation therapy, malignant diseases, endocrine disorders, coagulation disorders or ophthalmological conditions. Thus, the MASP-2 inhibitory antibodies of the present invention can be used to treat the diseases and conditions mentioned above.

[0210] As further described in Example 11, the MASP-2 inhibitory antibodies of the present invention are effective in treating mammalian subjects at risk of or suffering from the harmful effects of acute radiation syndrome, thereby demonstrating in vivo therapeutic efficacy.

[0211] The following examples are merely illustrative of the best mode contemplated herein for carrying out the invention and are not to be construed as limiting the invention. EXAMPLES

[0212] Example 1 This example describes the recombinant expression and protein production of recombinant full-length human, rat and mouse MASP-2, MASP-2-derived polypeptides, and catalytically inactive mutant forms of MASP-2.

[0213] Expression of full-length human and rat MASP-2: The full-length cDNA sequence of human MASP-2 (SEQ ID NO:1), encoding the human MASP-2 polypeptide (SEQ ID NO:2) including the leader sequence, was subcloned into the mammalian expression vector pCI-Neo (Promega), which drives eukaryotic expression under the control of the CMV enhancer / promoter region (Kaufman RJ et al., Nucleic Acids Research 19:4485-90, 1991; Kaufman, Methods in Enzymology, 185:537-66 (1991)). The full-length rat MASP-2 cDNA (SEQ ID NO:4), encoding the rat MASP-2 polypeptide (SEQ ID NO:5) including the leader sequence, was subcloned into the pED expression vector. These MASP-2 expression vectors were then transfected into the adherent Chinese hamster ovary cell line DXB1 using the standard calcium phosphate transfection procedure described in Maniatis et al., 1989. Cells transfected with these constructs grew very slowly, implying that the encoded proteases were cytotoxic. The mature human MASP-2 protein (SEQ ID NO: 3) and the mature rat MASP-2 protein (SEQ ID NO: 6) were secreted into the culture medium and isolated as described below.

[0214] Expression of catalytically inactive full-length MASP-2: Rationale: After the binding of the recognition subcomponents MBL, C-type lectin CL-11 or ficolins (either L-ficolin, H-ficolin or M-ficolin) (collectively referred to as lectins) to their respective carbohydrate patterns, MASP-2 is activated by autocatalytic cleavage. Autocatalytic cleavage leading to activation of MASP-2 often occurs during the isolation procedure of 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 generated by replacing the serine residues present in the catalytic triad of the protease domain with alanine residues in the mature rat MASP-2 protein (SEQ ID NO:6 Ser617 replaced by Ala617); or in the mature human MASP-2 protein (SEQ ID NO:3 Ser618 replaced by Ala618).

[0215] To generate catalytically inactive human and rat MASP-2A proteins, site-directed mutagenesis was performed as described in US2007 / 0172483, which is hereby incorporated by reference. After agarose gel electrophoresis and band preparation, the PCR products were purified and a single adenosine overlap was generated using a standard tailing procedure. The adenosine-tailed MASP-2A was then cloned into pGEM-T easy vector and transformed into E. coli. Each of the human and rat MASP-2A was further subcloned into either the mammalian expression vector pED or pCI-Neo, as described below, and transfected into the Chinese hamster ovary cell line DXB1.

[0216] Construction of an expression plasmid containing the polypeptide domain derived from human Masp-2 The following constructs were made using the MASP-2 signal peptide (residues 1-15 of SEQ ID NO:2) which directs the secretion of various domains of MASP-2. A construct expressing the human MASP-2 CUBI domain (SEQ ID NO:7) was made by PCR amplifying the region encoding residues 1-121 of MASP-2 (SEQ ID NO:3) (corresponding to the N-terminal CUB1 domain). A construct expressing the human MASP-2 CUBI / EGF domain (SEQ ID NO:8) was made by PCR amplifying the region encoding residues 1-166 of MASP-2 (SEQ ID NO:3) (corresponding to the N-terminal CUB1 / EGF domain). A construct expressing the human MASP-2 CUBI / EGF / CUBII domain (SEQ ID NO:9) was made by PCR amplifying the region encoding aa residues 1-277 of MASP-2 (SEQ ID NO:3) (corresponding to the N-terminal CUBI EGF CUBII domain). A construct expressing the human MASP-2 EGF domain (SEQ ID NO: 10) was generated by PCR amplifying the region encoding aa residues 122-166 (corresponding to the EGF domain) of MASP-2 (SEQ ID NO: 3). A construct expressing the human MASP-2 CCPI / CCPII / SP domain (SEQ ID NO: 11) was generated by PCR amplifying the region encoding aa residues 278-671 (corresponding to the CCPI / CCPII / SP domain) of MASP-2 (SEQ ID NO: 3). A construct expressing the human MASP-2 CCPI / CCPII domain (SEQ ID NO: 12) was generated by PCR amplifying the region encoding aa residues 278-429 (corresponding to the CCPI / CCPII domain) of MASP-2 (SEQ ID NO: 3). A construct expressing the CCPI domain of MASP-2 (SEQ ID NO: 13) was generated by PCR amplifying the region encoding aa residues 278-347 (corresponding to the CCPI domain) of MASP-2 (SEQ ID NO: 3). A construct expressing the CCPII / SP domain of MASP-2 (sequence number 14) was generated by PCR amplifying the region encoding aa residues 348 to 671 (corresponding to the CCPII / SP domain) of MASP-2 (sequence number 3).A construct expressing the CCPII domain of MASP-2 (SEQ ID NO: 15) was generated by PCR amplifying the region encoding aa residues 348-429 (corresponding to the CCPII domain) of MASP-2 (SEQ ID NO: 3). A construct expressing the SP domain of MASP-2 (SEQ ID NO: 16) was generated by PCR amplifying the region encoding aa residues 429-671 (corresponding to the SP domain) of MASP-2 (SEQ ID NO: 3).

[0217] The MASP-2 domain mentioned above was R The PCR products were amplified according to established PCR methods using polymerase and pBS-MASP-2 as template. The 5' primer sequence of the sense primer introduced a BamHI restriction enzyme recognition site (underlined) at the 5' end of the PCR product. Antisense primers for each of the MASP-2 domains were designed to introduce an EcoRI site after the stop codon at the end of each PCR product. Once amplified, the DNA fragments were digested with BamHI and EcoRI and cloned into the corresponding sites of the pFastBac1 vector. The resulting constructs were characterized by restriction mapping and verified by dsDNA sequencing.

[0218] Recombinant eukaryotic expression of MASP-2 and protein production of enzymatically inactive rat and human MASP-2A.

[0219] The MASP-2 and MASP-2A expression constructs described above were transfected into DXB1 cells using standard calcium phosphate transfection procedures (Maniatis et al., 1989). MASP-2A was produced in serum-free medium to ensure that the preparation was not contaminated by other serum proteins. Medium was harvested from confluent cells every two days (a total of four times). The levels of recombinant MASP-2A for each of the two species averaged approximately 1.5 mg per liter of culture medium.

[0220] Purification of MASP-2A protein: MASP-2A (Ser-Ala mutant as described above) was purified by affinity chromatography on an MBP-A-agarose column. This strategy allowed for rapid purification without the use of exogenous tags. MASP-2A (100-200 ml of medium diluted with an equal volume of loading buffer (50 mM Tris-Cl, pH 7.5, containing 150 mM NaCl and 25 mM CaCl2)) was loaded onto an MBP-agarose affinity column (4 ml) that had been pre-equilibrated with 10 ml of loading buffer. After washing with an additional 10 ml of loading buffer, the protein was eluted in 1 ml fractions with 50 mM Tris-Cl, pH 7.5, containing 1.25 M NaCl and 10 mM EDTA. Fractions containing MASP-2A were identified by SDS-polyacrylamide gel electrophoresis. If necessary, MASP-2A was further purified by ion-exchange chromatography on a MonoQ column (HR5 / 5). The protein was dialyzed against 50 mM Tris-Cl pH 7.5 containing 50 mM NaCl and loaded onto a column equilibrated with the same buffer. After washing, bound MASP-2A was eluted with a 0.05-1 M NaCl gradient over 10 ml.

[0221] Results: Yields of 0.25-0.5 mg of MASP-2A protein were obtained from 200 ml of culture medium. The molecular weight of 77.5 kDa determined by MALDI-MS is greater than that calculated for the unmodified polypeptide (73.5 kDa) due to glycosylation. Attachment of glycans at each of the N-glycosylation sites explains the observed mass. MASP-2A migrates as a single band on SDS-polyacrylamide gels, indicating that it is not proteolytically processed during biosynthesis. The weight-average molecular weight determined by equilibrium ultracentrifugation is consistent with that calculated for the glycosylated polypeptide homodimer.

[0222] Example 2 This example describes the screening method used to identify high-affinity fully human anti-MASP-2 scFv antibody candidates that block the functional activity of MASP-2 for progression to affinity maturation.

[0223] Background and Rationale: MASP-2 is a complex protein with many distinct functional domains, including binding sites for MBL and ficolin, a serine protease catalytic site, a binding site for the proteolytic substrate C2, a binding site for the proteolytic substrate C4, a MASP-2 cleavage site for autoactivation of the MASP-2 zymogen, and two Ca ++ The binding sites include: scFv antibody fragments that bind to MASP-2 with high affinity, and the identified Fab2 fragments were tested in functional assays to determine whether they can block MASP-2 functional activity.

[0224] In order to block MASP-2 functional activity, an antibody or scFv antibody fragment or Fab2 antibody fragment must bind to and interfere with a structural epitope on MASP-2 that is required for MASP-2 functional activity. Thus, unless many or all of the high affinity binding anti-MASP-2 scFvs or Fab2s bind to a structural epitope on MASP-2 that is directly involved in MASP-2 functional activity, they may not inhibit MASP-2 functional activity.

[0225] The "blocking activity" of anti-MASP-2 scFv was evaluated using a functional assay that measures inhibition of lectin pathway C3 convertase formation. The primary physiological role of MASP-2 in the lectin pathway is known to generate the next functional component of the lectin-mediated complement pathway, namely, the lectin pathway C3 convertase. The lectin pathway C3 convertase is a key enzymatic complex (C4bC2a) that proteolytically cleaves C3 into C3a and C3b. MASP-2 is not a structural component of the lectin pathway C3 convertase (C4bC2a); however, MASP-2 functional activity is required to generate the two protein components (C4b, C2a) that comprise the lectin pathway C3 convertase. Furthermore, all of the separate functional activities of MASP-2 listed above appear to be required for MASP-2 to generate the lectin pathway C3 convertase. For these reasons, the preferred assay to use in assessing the "blocking activity" of anti-MASP-2 Fab2 and scFv antibody fragments is believed to be a functional assay that measures inhibition of lectin pathway C3 convertase formation.

[0226] The target profile for a therapeutic anti-MASP-2 antibody predicted to result in >90% lectin pathway cleavage in vivo after 1 mg / kg administration in humans was IC 50 <5 nM. The relationship between in vitro pharmacological activity in these assay formats and in vivo pharmacodynamics was experimentally confirmed using anti-rodent MASP-2 antibodies.

[0227] The selection criteria for first generation MASP-2 blocking antibodies for therapeutic use were: high affinity for MASP-2 and a functional IC of approximately 25 nM. 50 Additionally, candidates were screened for cross-reactivity with non-human primate and rat sera.

[0228] method: Screening of scFv phagemid library against MASP-2 antigen antigen: Human MASP-2A with an N-terminal 5xHis tag and rat MASP-2A with an N-terminal 6xHis tag were produced using the reagents described in Example 1 and purified from culture supernatants by nickel affinity chromatography as previously described (Chen et al., J. Biol. Chem. 276:25894-02 (2001)).

[0229] OMS100, a human anti-MASP-2 antibody of Fab2 type, was used as a positive control for binding to MASP-2.

[0230] Phagemid library description: High-affinity scFv antibodies against rat and human MASP-2 proteins were identified by subjecting a phage display library of human immunoglobulin light and heavy chain variable region sequences to antigen panning followed by automated antibody screening and selection.

[0231] Panning Method: Summary: Two panning strategies were used to isolate phages that bound to MASP-2 from a phagemid library, for a total of three rounds of panning. Both strategies involved panning in solution and fishing out phages that bound to MASP-2. MASP-2 was immobilized on magnetic beads either via the His-tag on the target (using NiNTA beads) or via biotin (using streptavidin beads).

[0232] The first two panning rounds involved alkaline elution (TEA), while the third panning was first competitively eluted with MBL followed by a conventional alkaline (TEA) elution step. Negative selection was performed before the second and third rounds, against functional analogs of the classical complement pathway, C1s and C1r. After panning, specific enrichment of phages with scFv fragments against MASP-2A was monitored, determining that the panning strategy was successful (data not shown).

[0233] The scFv genes from the third panning were cloned into the pHOG expression vector, as described further below, and subjected to small-scale filter screening to search for specific clones against MASP-2A.

[0234] [Table 7]

[0235] [Table 8] Panning Reagents: Human MASP-2A OMS100 antibody (positive control) Goat anti-human IgG (H+L) (Pierce #31412) NiNTA beads (Qiagen #LB13267) Dynabeads® M-280 Streptavidin, 10 mg / ml (LB12321) Normal human serum (LB13294) Polyclonal rabbit anti-human C3c (LB13137) Goat anti-rabbit IgG, HRP (American Qualex #A102PU) To test tagged MASP-2A antigen, experiments were performed in which positive control OMS100 antibody (200 ng / ml) preincubated with biotin-tagged MASP-2A or HIS-tagged MASP-2A antigen (10 μg) was captured with 50 μl NiNTA beads or 200 μl streptavidin beads in 4% milk PBS, respectively. Bound MASP-2A-OMS100 antibody was detected with goat anti-human IgG(H+L)HRP (1:5000) and TMB (3,3',5,5'-tetramethylbenzidine) substrate.

[0236] NiNTA bead ELISA assay 50 μl of NiNTA beads were blocked with 1 ml of 4% milk in phosphate-buffered saline (PBS) and incubated for 1 h at room temperature on a rotator wheel. In parallel, 10 μg of MASP-2A and OMS100 antibodies (diluted to 200 ng / ml in 4% milk-PBS) were pre-incubated for 1 h. The beads were then washed 3 times with 1 ml of PBS-T using a magnet between each step. MASP-2A pre-incubated with OMS100 antibody was added to the washed beads. The mixture was incubated for 1 h at RT on a rotator wheel and then washed 3 times with 1 ml of PBS-T using a magnet as described above. The tube was incubated for 1 h at RT with goat anti-human IgG (H+L) HRP diluted 1:5000 in 4% milk in PBS. For the negative control, goat anti-human IgG(H+L)HRP (1:5000) was added to the washed, blocked, Ni-NTA beads in a separate tube.

[0237] The samples were incubated for 1 hour at room temperature on a rotator wheel, then washed 3 times with 1 ml PBS-T and once with 1×PBS using a magnet as described above. 100 μl of TMB substrate was added and incubated for 3 minutes at room temperature. The tubes were placed on a magnetic rack for 2 minutes to concentrate the beads, then the TMB solution was transferred to a microtiter plate and the reaction was stopped with 100 μl of 2M H2SO4. The absorbance at 450 nm was read in an ELISA reader.

[0238] Streptavidin bead ELISA assay This assay was performed as described above for the NiNTA bead ELISA assay, except that 200 μl of streptavidin beads and non-biotinylated antigen were used instead per sample.

[0239] Results: Each of the His-tagged and biotin-tagged MASP-2A antigens preincubated with the positive control OMS100 antibody was successfully captured using NiNTA or streptavidin beads, respectively.

[0240] Panning Three rounds of panning of the scFv phage library against HIS-tagged or biotin-tagged MASP-2A were performed as shown in Table 7 or Table 8, respectively. The third round of panning was first eluted with MBL and then with TEA (alkaline). To monitor the specific enrichment of phages displaying scFv fragments against the target MASP-2A, a polyclonal phage ELISA against immobilized MASP-2A was performed as described below.

[0241] MASP-2A ELISA for polyclonal phages enriched after panning After three rounds of panning of the scFv phage library against human MASP-2 as described above, specific enrichment of phages with scFv fragments against the target MASP-2A was monitored by performing an ELISA assay on the enriched polyclonal phage population generated by panning against immobilized MASP-2A, as described below.

[0242] method: 5 ng / ml MASP-2A was immobilized on maxisorp ELISA plates in PBS overnight at 4° C. Packaged phage from all three pannings were diluted 1:3 in 4% milk-PBS and titered at 3-fold dilutions. The negative control was M13 helper phage.

[0243] Blocking was 4% milk in PBS. The plates were washed 3 times in 200 μl PBS-Tween 0.05% (v / v) between all steps. Primary antibody was rabbit α-fd (M13 coat protein) at 1:5000 in 4% milk-PBS (w / v). Conjugate was rabbit α-goat-HRP at 1:10.000 in 4% milk-PBS (w / v). Substrate was ABTS. All volumes except washes and blocking were 100 μl / well. All incubations were for 1 hour at room temperature with shaking.

[0244] result: Phage ELISA results showed specific enrichment of scFv against MASP-2A for both panning strategies. See Figure 2. As shown in Figure 2, the strategy involving capture with NiNTA magnetic beads resulted in enrichment of scFv on phages against MASP-2A after two rounds of panning, whereas both strategies had good enrichment in both competitive and TEA elution after the third round of panning. The negative control phage was M13 helper phage, which showed no cross-reactivity against MASP-2A at its lowest dilution. These results demonstrate that the observed signal is due to scFv that specifically binds to MASP-2A.

[0245] Filter Screening: Bacterial colonies containing plasmids encoding the scFv fragments from the third panning were picked, gridded onto nitrocellulose membranes, and grown overnight on non-inducing medium to create a master plate. A total of 18,000 colonies were picked from the third panning, half of which were analyzed from the competitive elution and half from the subsequent TEA elution.

[0246] The nitrocellulose membrane containing the above bacterial colonies was induced with IPTG to express and secrete soluble scFv protein and contacted with a second nitrocellulose membrane coated with MASP-2A antigen, along with a corresponding membrane coated with 4% milk in PBS (blocking solution).

[0247] ScFvs bound to MASP-2A were detected via their c-Myc tag using mouse α-cMyc mAb and rabbit α-mouse HRP. Hits corresponding to scFv clones positive for MASP-2A and negative for milk-PBS were selected for further expression and subsequent ELISA analysis.

[0248] Results: Panning of the scFv phagemid library against MASP-2A followed by scFv conversion and filter screening yielded 137 positive clones. The majority of the positive clones were derived from competitive elution with MBL using both NiNTA and streptavidin strategies. All positive clones proceeded to microexpression (200 μl scale) and subsequent extraction. ScFvs were isolated from the bacterial periplasm by incubating the bacterial suspension with sucrose lysis buffer and lysozyme for 1 h and then isolating the supernatant by a centrifugation step. The supernatant containing the scFvs secreted into the medium together with the contents of the periplasm were analyzed by two assays: an ELISA using physically adsorbed MASP-2A, as described in detail below, and a binding analysis using amine-coupled MASP-2A to a CM5 chip in Biocore.

[0249] MASP-2A ELISA for ScFv candidate clones identified by panning / scFv conversion and filter screening method: MASP-2A at 4 μg / ml was immobilized on maxisorp ELISA plates (Nunc) in PBS overnight at 4° C. The next day the plates were blocked by washing three times with PBS-Tween (0.05%). Crude scFv material (100 μl of medium-periplasmic extract) from each of the 137 scFv candidates (generated as described above) was added per well of the plate. Anti-cMyc was then added and in a final step bound scFv was detected by applying HRP-conjugated rabbit anti-mouse. The reaction was developed in the peroxidase substrate 1-step ABTS (Calbiochem). The positive control was OMS100 (anti-MASP-2 antibody in Fab2 form) diluted to 10 μg / ml in PBS-Tween 0.05%. The negative control was medium-periplasmic from XL1-Blue without plasmid.

[0250] 3×200 μl PBS-Tween 0.05% (v / v) washes were performed between all steps.

[0251] The primary antibody was mouse α-cMyc, 1:5000 in PBS-Tween 0.05% (w / v).

[0252] Conjugates were rabbit α-goat-HRP or goat anti-human IgG (H+L, Pierce 31412) at 1:5000 in PBS-Tween 0.05% (w / v). Substrate was ABTS with 15 min incubation at room temperature. All volumes except washes and blocking were 100 μl / well. All incubations were for 1 h at room temperature with shaking.

[0253] Results: 108 / 137 clones were positive in this ELISA assay (data not shown), of which 45 clones were further analyzed as described below. The positive control was OMS100 Fab2 diluted to 10 μg / ml in PBS-Tween, this clone was positive. The negative control was medium-periplasm from XL1-Blue without plasmid, this was negative.

[0254] Example 3 This example describes a MASP-2 functional screening method that was used to analyze high affinity fully human anti-MASP-2 scFv antibody candidates for their ability to block MASP-2 activity in normal human serum.

[0255] Rationale / Background Assay to measure inhibition of lectin pathway C3 convertase formation: The "blocking activity" of anti-MASP-2 scFv candidate clones was evaluated using a functional assay measuring inhibition of lectin pathway C3 convertase formation. The lectin pathway C3 convertase is an enzymatic complex (C4bC2a) that proteolytically cleaves C3 into two potent proinflammatory fragments, the anaphylatoxin C3a and the opsonin C3b. Formation of the C3 convertase appears to be a critical step in the lectin pathway with respect to mediating inflammation. MASP-2 is not a structural component of the lectin pathway C3 convertase (C4bC2a); therefore, anti-MASP-2 antibodies (or Fab2) do not directly inhibit the activity of existing C3 convertase. However, MASP-2 serine protease activity is required to generate the two protein components (C4b, C2a) that comprise the lectin pathway C3 convertase. Therefore, anti-MASP-2 scFvs that inhibit MASP-2 functional activity (i.e., blocking anti-MASP-2 scFvs) inhibit the de novo formation of lectin pathway C3 convertase. C3 contains a highly reactive unique thioester group as part of its structure. When the C3 convertase cleaves C3 in this assay, the thioester group on C3b can form a covalent bond via an ester or amide bond with a hydroxyl or amino group on a polymer immobilized on the bottom of a plastic well, facilitating detection of C3b in an ELISA assay.

[0256] Yeast mannan is a known activator of the lectin pathway. In the following method for measuring the formation of C3 convertase, mannan-coated plastic wells are incubated with diluted human serum to activate the lectin pathway. The wells are then washed and assayed for C3b immobilized on the wells using standard ELISA methods. The amount of C3b generated in this assay directly reflects the de novo formation of lectin pathway C3 convertase. Anti-MASP-2 at selected concentrations The scFvs were tested in this assay for their ability to inhibit C3 convertase formation and subsequent C3b generation.

[0257] method: Forty-five candidate clones identified as described in Example 2 were expressed, purified, and diluted to the same stock concentration, which was then reinjected into the Ca ++ and Mg ++ The clones were diluted in GVB buffer (4.0 mM barbital, 141 mM NaCl, 1.0 mM MgCl2, 2.0 mM CaCl2, 0.1% gelatin, pH 7.4) to ensure that all clones had the same amount of buffer. The scFv clones were each tested in triplicate at a concentration of 2 μg / ml. The positive control was OMS100 Fab2, tested at 0.4 μg / ml. C3c formation was monitored in the presence and absence of the scFv / IgG clones.

[0258] Mannan was diluted to a concentration of 20 μg / ml (1 μg / well) in 50 mM carbonate buffer (15 mM Na2CO3 + 35 mM NaHCO3 + 1.5 mM NaN3), pH 9.5, and coated onto ELISA plates overnight at 4°C. The next day, the mannan-coated plates were washed three times with 200 μl of 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 three times with 200 μl of PBS and stored on ice with 200 μl of PBS until the addition of samples.

[0259] Normal human serum was diluted to 0.5% in CaMgGVB buffer and scFv clones or OMS100 Fab2 positive controls were added to this buffer in triplicate at 0.01 μg / ml; 1 μg / ml (OMS100 control only) and 10 μg / ml and pre-incubated on ice for 45 min before being added to blocked ELISA plates. The reaction was started by incubation at 37° C. for 1 h and stopped by transferring the plate to an ice bath. C3b deposition was detected using rabbit α-mouse C3c antibody followed by goat α-rabbit HRP. The negative control was buffer without antibody (no OMS100=maximal C3b deposition) and the positive control was buffer with EDTA (no C3b deposition). Background was measured by performing the same assay but with mannan-negative wells. The background signal for the mannan-free plate was subtracted from the mannan-positive signal. The cut-off criterion was set at half the activity of an irrelevant scFv clone (VZV) and buffer alone.

[0260] Results: Based on the cutoff criteria, a total of 13 clones were found to block the activity of MASP-2 as shown in Figures 3A and 3B. All 13 clones resulting in >50% pathway inhibition were selected and sequenced, resulting in 10 unique clones, as shown in Table 9 below. The 10 different clones shown in Table 9 were found to result in acceptable functional activity in the complement assay. All 10 clones were found to have the same light chain subclass λ3 but three different heavy chain subclasses VH2, VH3 and VH6. The sequence identity of those clones to the germline sequence is also shown in Table 9.

[0261] [Table 9] As shown above in Table 9, 10 different clones with acceptable functional activity and unique sequences were selected for further analysis. As noted in Table 9, some of those clones were detected two or three times based on the same sequence (see the first column of Table 9 including the clone names).

[0262] Expression and purification of 10 svFc candidate clones The ten candidate clones shown in Table 9 were expressed at a 1 liter scale and purified by ion exchange on nickel chromatography. A sample of each clone was then run on a size exclusion chromatography column to assess the monomer and dimer content. As shown in Table 10 below, almost all scFv clones were present in monomeric form, and this monomeric fraction was isolated for further testing and ranking.

[0263] [Table 10] Testing monomeric fractions for binding and functional activity The clones shown in Table 10 were expressed at 1 L scale, purified by metal chromatography and ion exchange, separated into monomeric fractions by size exclusion chromatography (SEC), and subjected to repeated functional assays to confirm the IC 50 The antibody titers and cross-reactivity were determined.

[0264] Functional assays on monomeric fractions: Monomer fractions of the top 10 clones shown in Table 10 were purified and showed functional IC in a dilution series (each of which received the same concentrations of GVB buffer containing calcium and magnesium and human serum). 50 The scFv clones were tested in triplicate at 12 dilutions. The positive control was OMS100 Fab2. C3b deposition was monitored in the presence and absence of antibody. The results are shown in Table 11 below.

[0265] Binding assay: The binding affinity K for purified monomer fractions of 10 candidate scFv clones was measured in two different methods. D was measured. MASP-2A was either immobilized by amine coupling to a CM5 chip, or a fixed concentration of scFv (50 nM) was first captured with an amine-coupled high affinity α-cMyc antibody, and then a range of concentrations of MASP-2A in solution were passed over the chip. The results are shown in Table 11 below.

[0266] result:

[0267] [Table 11] Discussion of results: As shown in Table 11, in functional assays, 5 of 10 candidate scFv clones had IC below the target criterion of 25 nM when using 0.5% human serum. 50 The results yielded nM values. As described below, these clones were further tested in the presence of non-human primate and rat serum to measure functional activity in other species. With regard to binding affinity in solution, all binding affinities were in the low nM or better range, whereas in conventional assays using immobilized MASP-2, only two clones (4D9 and 17D20) had affinities in the low nM range. The observation of higher affinity in solution-based assays is probably a result of the fact that antigens multimerize in solution. Also, when targets are immobilized on chips (by oriented coupling), epitopes are masked, which may reduce the observed affinity in immobilized assays.

[0268] Example 4 In this example, ten candidate human anti-MASP-2 scFv clones were tested for cross-reactivity to rat MASP-2 and the IC of these scFv clones in a functional assay measuring their ability to inhibit MASP-2-dependent complement activation in human serum, non-human primate serum, and rat serum is reported. 50 The results of measuring the values ​​are explained below.

[0269] method: Cross-reactivity to rat MASP-2 Ten candidate scFv clones shown in Table 9 of Example 3 were tested for cross-reactivity against rat MASP-2A in a conventional ELISA assay against adsorbed rat MASP-2A. Rat MASP-2A was diluted to 4 μg / ml in PBS and coated on Maxisorp ELISA plates (Nunc) overnight at 4° C. The next day, the plates were blocked by washing three times with PBS-Tween (0.05%). ScFv clones (100 μl) diluted to 20 μg / ml in PBS-Tween were added to the plates and titrated in triplicate with further 4-fold dilutions. MASP-2A-specific svFc clones (wells containing bound scFv) were detected with anti-cMyc and rabbit anti-mouse HRP secondary antibodies. The reaction was developed in the peroxidase substrate TMB (Pierce). The positive control was OMS100 Fab2 diluted to 10 μg / ml in PBS-Tween. All clones tested showed cross-reactivity to rat MASP-2A, which was expected since rat MASP-2 was used in the second panning (data not shown).

[0270] Functional characterization of 10 candidate scFv clones in human, non-human primate (NHP) and rat serum Measurement of baseline C3c levels in various serum samples First, an experiment was performed to compare baseline C3b levels in the three types of serum (human, rat and NHP) as follows.

[0271] Mannan was diluted to 20 μg / ml and coated on ELISA plates overnight at 4° C. The next day, wells were blocked with 1% HSA. Normal human, rat and African green monkey sera (non-human primate "NHP") sera were diluted in CaMgGVB buffer in two-fold dilutions starting at 2%. Reactions were initiated by incubation at 37° C. for 1 hour and stopped by transferring plates to an ice bath. C3b deposition was detected using rabbit anti-mouse C3c antibody followed by goat anti-rabbit HRP. Negative control was buffer without antibody (no OMS100 results in maximum C3b deposition) and positive control for inhibition was buffer with EDTA (no C3b deposition).

[0272] Figure 4 graphically illustrates the baseline C3c levels in three types of serum (human, rat and NHP). As shown in Figure 4, the C3c levels were very different in the various sera tested. When comparing the C3c levels, 1% human serum appeared to provide equivalent levels to 0.2% NHP and 0.375% rat serum. Based on these results, the serum concentrations were normalized so that the scFv results could be directly compared in the three different types of serum.

[0273] Functional assay of ScFv clones in different sera The purified monomer fractions of the 10 candidate scFv clones were then assayed for functional IC in human serum, rat serum, and African green monkey serum (non-human primate "NHP"). 50nM. The assay was performed as described in Example 3 using 1000 nM scFv purified protein and normal human serum diluted to 0.9% in CaMgGVB buffer; African green monkey serum diluted to 0.2% in CaMgGVB buffer; or rat serum diluted to 0.375% in CaMgGVB buffer. All 10 scFv clones were tested in a dilution series given the same concentrations of GVB buffer and serum containing calcium and magnesium. The scFv clones were tested in triplicate at 12 dilutions. The positive control was 100 ng / ml OMS100 Fab2 or the addition of EDTA to the reaction. The negative control was an irrelevant scFv control or PBS without scFv. C3b deposition was monitored in the presence and absence of scFv or Fab2 antibodies. The background signal of 100 ng / ml OMS100 was subtracted from all signals. Table 12 summarizes the results of the functional assays for all three sera.

[0274] [Table 12] Summary of results on functional activity of scFv candidate clones in various sera: After normalizing the sera for C3b deposition levels, all ten of the scFv clones showed function in both human and non-human primate (NHP) sera. The six most active clones in human sera were ranked from best to worst: 9P13>17N16>17D20>4D9>3F22>18L16. In NHP sera, the clones were ranked (best to worst): 17L20>17N16>17D20>9P13>18L16>3F22. Both 17N16 and 17D20 ranked in the top three for both human and NHP sera. 17D20 also showed some activity in rat sera.

[0275] Based on these results, the top three scFv clones were determined to be 18L16, 17D20 and 17N16. These three clones were further analyzed in dilute human serum (1% serum) as shown in Table 13 below.

[0276] [Table 13] Figure 5A is an alignment of the amino acid sequences of full-length scFv clones 17D20, 18L16, 4D9, 17L20, 17N16, 3F22 and 9P13. These scFv clones contain a heavy chain variable region (aa1-120), a linker region (aa121-145) and a light chain variable region (aa146-250). As shown in Figure 5A, alignment of the heavy chain regions (residues 1-120) of the most active clones reveals two distinct groups that belong to the VH2 and VH6 gene families, respectively. As shown in Figure 5A, the VH regions for the VH2 class clones: 17D20, 18L16 and 4D9 had variability at 20 aa positions (i.e., 83% identity) in a total of 120 amino acid regions.

[0277] As further shown in FIG. 5A, the VH regions for the VH6 class of clones: 17L20, 17N16, 3F22 and 9P13 are variable at 18 aa positions (ie, 85% identity) in a total of 120 amino acid regions.

[0278] FIG. 5B is an alignment of the sequences of scFv clones 17D20, 17N16, 18L16 and 4D9.

[0279] [Table 14] The ranking priorities were: (1) human serum functional potency and complete blockade; (2) NHP cross-reactivity; and (3) sequence diversity. 17D20 and 17N16 were selected as the best representatives of each gene family. 18L16 was selected as a third candidate with significant CDR3 sequence diversity.

[0280] 17N16 and 17D20 were the top two choices due to complete functional blocking, as well as best functional potency in humans; significant cross-reactivity to monkeys, and different VH gene families. 3F22 and 9P13 were excluded due to nearly identical VH sequences to 17N16. 18P15, 4J9, and 21B17 were excluded due to moderate potency. 17L20 was not pursued as it only partially blocked.

[0281] 18L16 and 4D9 had similar activity and significant diversity compared to 17D20. 18L16 was chosen because of its greater cross-reactivity in primates than 4D9.

[0282] Therefore, based on these criteria: the following three mother clones were advanced into affinity maturation as further described below: 17D20, 17N16 and 18L16.

[0283] Example 5 This example describes the cloning of three maternal clones, 17D20, 17N16 and 18L16 (identified as described in Examples 2-4), into wild-type IgG4 format and evaluation of the functionality of the three maternal clones as full-length IgG.

[0284] Rationale: Fully human anti-MASP-2 scFv antibodies with moderate functional potency were identified using phage display as described in Examples 2-4. Three such mother clones, 17D20, 17N16 and 18L16, were selected for affinity maturation. To evaluate the functionality of these mother clones as full-length IgGs, IgG4 wild-type and S228P hinge region IgG4 mutants of these antibodies were generated. The S228P hinge region mutation was included to increase serum stability (see Labrijn AF et al., Nature Biotechnology 27:767 (2009)).

[0285] The amino acid sequence of IgG4 wild type is shown as SEQ ID NO:63, which is encoded by SEQ ID NO:62.

[0286] The amino acid sequence of IgG4 S228P is shown as SEQ ID NO:65, which is encoded by SEQ ID NO:64.

[0287] To directly compare the parent clones with the OMS100 control antibody, which is a F(ab)2 molecule, the IgG4 molecules were cleaved into F(ab')2 form by pepsin digestion and fractionated by size exclusion chromatography.

[0288] method: Creation of full-length clones The above three mother clones were converted to wild type IgG4 and IgG4 mutant S228P by PCR isolating the appropriate VH and VL regions from the mother clones and cloning them into a pcDNA3 expression vector with the appropriate heavy chain constant region to generate an in-frame fusion to generate the desired antibody. The three mother clones of mutant IgG4 were then digested with pepsin to generate F(ab')2 fragments, and the latter were purified by fractionation on a size-exclusion chromatography column.

[0289] Binding assays Candidate IgG4-converted mother clones were transiently transfected into HEK293 cells and supernatants from the transient transfections were titrated in an ELISA assay. The clones showed excellent reactivity with physically adsorbed human MASP-2A and were ranked in the following order: 17N16>17D20>18L16 (data not shown).

[0290] The clones were then purified and tested again in ELISA and activity assays as follows: Human MASP-2A was coated on maxisorp plates at 3 μg / ml in PBS, IgG (45 μg / ml) and Fab'2 (30 μg / ml) were diluted in PBS-Tween to a starting concentration of 300 nM and further diluted in 3-fold dilutions. IgG was detected with HRP-conjugated goat α-human IgG (Southern Biotech) and F(ab')2 was detected with HRP-conjugated goat α-human IgG H+L (Pierce 31412). The reaction was developed with TMB substrate and stopped with 2M H2SO4. The results are shown in Table 15 below.

[0291] [Table 15] Functional assays The functional activity of the parent scFv clones and full-length IgG4 counterparts in 1% normal human serum (NHS) was compared using a C3 convertase assay using 1% NHS as described in Example 4. Mannan was diluted to a concentration of 20 μg / ml and coated on ELISA plates overnight at 4° C. The next day, wells were blocked with 1% human serum. Human serum was diluted to 1% in CaMgGVB buffer and purified antibodies; scFv (900 nM), F(ab')2 (300 nM), IgG (300 nM) were added in duplicate to the same amount of buffer in a series of different dilutions and pre-incubated on ice for 45 minutes before being added to the blocked ELISA plate. The reaction was started by incubation at 37° C. for 1 hour and stopped by placing the plate on ice. C3b deposition was determined using rabbit α-mouse C3c antibody followed by goat α-rabbit HRP. The background of 50 nM OMS100 on the mannan positive plate was subtracted from the curve. A summary of the results of this analysis is shown below in Table 16.

[0292] [Table 16] The functional potency of the IgG4 parent clones was also compared to the IgG4 hinge mutant (S228P) version for each clone. IC against C3b deposition assay using 1% NHS 50 The figures are shown in Table 17 below.

[0293] [Table 17] As shown above in Table 17, in some cases unexpected agonist pharmacology was evident for the antagonistic scFv-derived IgGs. The mechanistic basis for this observation is not understood.

[0294] The activity of the mother clones converted to IgG4 with inhibitory function in 1% NHS was further evaluated under more stringent assay conditions that more closely mimic physiological conditions.To estimate antibody activity under physiological conditions, mother clone IgG4 preparations were tested for their ability to inhibit lectin pathway (LP)-dependent C3b deposition onto mannan-coated plates under stringent assay conditions using minimally diluted (90%) human plasma.

[0295] The results of the C3b deposition assay in 90% human plasma are shown in Figure 6. Since MASP-2 and its substrate are present in the assay mixture at approximately 100-fold higher concentrations than in the dilute serum assay using 1% normal human serum, a rightward shift in the antagonist dose-response curve is usually expected. As shown in Figure 6, as expected, a rightward shift to lower apparent potency was observed for OMS100 and all MASP-2 antibodies tested. Surprisingly, however, no reduction in apparent potency was observed for the hinge region mutant of 17D20 (S228P), and potency in this format was comparable to that measured in 1% plasma (see Table 17). In the 90% NHS assay format, the functional potency of 17D20 IgG4(S228) was found to be moderately lower than OMS100 Fab2, in contrast to the assay results in 1% NHS, where OMS100 was 50-100 times more potent than 17D20 IgG4 S228P (data not shown). As shown in Figure 6, the wild-type IgG4 version of 17N16 also showed complete inhibition in 90% NHS, but was somewhat less potent in this assay format (IC of approximately 15 nM). 50 ), whereas wild-type IgG4 18L16 was less potent and only partially inhibitory.

[0296] Based on these findings, we further assessed the activity of the IgG4-converted parental clones by examining C4b deposition under stringent assay conditions (90% NHS). This assay format provides a direct measure of antibody activity against the enzymatic reaction catalyzed by MASP-2.

[0297] Assay measuring inhibition of MASP-2-dependent C4 cleavage Background: The serine protease activity of MASP-2 is highly specific, and only two protein substrates for MASP-2 have been identified; C2 and C4. Cleavage of C4 generates C4a and C4b. Anti-MASP-2 Fab2 can bind to structural epitopes on MASP-2 that are directly involved in C4 cleavage (e.g., MASP-2 binding site for C4; MASP-2 serine protease catalytic site), thus inhibiting the C4 cleavage functional activity of MASP-2.

[0298] Yeast mannan is a known activator of the lectin pathway. In the following method for measuring the C4 cleavage activity of MASP-2, mannan-coated plastic wells were incubated with 90% human serum at 4°C for 90 minutes to activate the lectin pathway. The wells were then washed and assayed for human C4b immobilized on the wells using standard ELISA methods. The amount of C4b produced in this assay is a measure of MASP-2-dependent C4 cleavage activity. In this assay, anti-MASP-2 antibodies at selected concentrations were tested for their ability to inhibit C4 cleavage.

[0299] Method: 96-well Costar Medium Binding plates were incubated overnight at 5°C with mannan diluted in 50 mM carbonate buffer (pH 9.5) at 1.0 μg / 50 μl / well. Each well was washed 3 times with 200 μl PBS. The wells were then blocked with 100 μl / well of 1% bovine serum albumin in PBS and incubated for 1 hour at room temperature with gentle mixing. Each well was washed 3 times with 200 μl PBS. Anti-MASP-2 antibody samples were incubated at 5°C with Ca ++ and Mg ++The antibodies were diluted to the selected concentration in GVB buffer (4.0 mM barbital, 141 mM NaCl, 1.0 mM MgCl2, 2.0 mM CaCl2, 0.1% gelatin, pH 7.4) containing 0.1% glycerol, ... A 1:700 dilution of biotin-conjugated chicken anti-human C4c (Immunsystem AB, Uppsala, Sweden) in PBS containing 2.0 mg / ml bovine serum albumin (BSA) was added at 100 μl / well and incubated for 1 hour at room temperature with gentle mixing. Each well was washed five times with 200 μl PBS. Peroxidase-conjugated streptavidin (Pierce Chemical #21126) at 0.1 μg / ml in PBS containing 2.0 mg / ml BSA was added at 100 μl / well and incubated for 1 hour at room temperature with gentle mixing on a shaker. Each well was washed five times with 200 μl PBS. Peroxidase substrate TMB (Kirkegaard & Perry Laboratories) was added at 100 μl / well and incubated for 16 minutes at room temperature. The peroxidase reaction was stopped by adding 100 μl / well of 1.0 M H3PO4, and the OD 450 was measured.

[0300] result: In this format, both IgG4 forms of 17D20 inhibited C4b deposition driven by the lectin pathway, but not IC 50 The IC values ​​were approximately 3-fold higher compared to the C3b deposition assay. Interestingly, the IgG4 wild type of 17N16 showed comparable IC values ​​in this assay to the C3b deposition assay. 50 18L16 was not significantly more potent and did not achieve complete inhibition in this format (data not shown).

[0301] Consideration: As described in Examples 2-5, fully human anti-MASP-2 scFv antibodies with functional blocking activity were identified using phage display. Three such clones, 17N16, 17D20 and 18L16, were selected for affinity maturation and further testing. To evaluate the functionality of these parent clones as full-length IgGs, IgG4 wild type and IgG4 S228P hinge region mutant versions of these antibodies were generated. As described in this Example, the majority of the full-length IgGs had improved functional activity compared to their scFv counterparts when tested in a standard functional assay format using 1% human plasma. To estimate antibody activity under physiological conditions, parent clone IgG4 preparations were tested under stringent assay conditions using 90% human plasma. Under these conditions, some antibodies demonstrated functional potency substantially better than expected based on their performance in a standard (1%) plasma functional assay.

[0302] Example 6 This example describes chain shuffling and affinity maturation of mother clones 17D20, 17N16 and 18L16, and analysis of the resulting daughter clones.

[0303] method: To identify antibodies with improved potency, the three mother scFv clones 17D20, 17N16 and 18L16 identified as described in Examples 2-5 were subjected to light chain shuffling. This process involved the creation of a combinatorial library consisting of the VH of each mother clone paired with a library of naive human lambda light chains (VL) from six healthy donors. This library was then screened for scFv clones with improved binding affinity and / or functionality.

[0304] A total of 27,000 light chain shuffled daughter clones were analyzed, 9,000 per mother clone. Each daughter clone was induced to express and secrete soluble scFv and screened for the ability to bind human MASP-2A. ScFvs that bound to human MASP-2A were detected via their c-Myc tag. A total of 119 clones were selected from this initial screening, including 107 daughter clones from the 17N16 library, 8 daughter clones from the 17D20 library, and 4 daughter clones from the 18L16 library.

[0305] The 119 clones were expressed on a small scale, purified on a NiNTA column and tested for binding affinity in an ELISA assay against physically adsorbed human MASP-2A.

[0306] result: The results of ELISA assays for a representative subset of the 119 daughter clones are shown in Figures 7A and B. Figure 7A graphically illustrates the results of an ELISA assay of 17N16 mother clones versus daughter clones titrated against huMASP-2A. Figure 7B graphically illustrates the results of an ELISA assay of 17D20 mother clones versus daughter clones titrated against huMASP-2A.

[0307] As shown in Figure 7A, daughter clones 17N16m_d16E12 and 17N16m_d17N9 derived from the 17N16 mother clone had higher affinity than the mother clone. Also, as shown in Figure 7B, one clone 17D20m_d18M24 derived from the 17D20 mother clone had higher affinity than the mother clone. These three clones, as well as three more clones: 17N16m_d13L12, 17N16m_d16K5, 17N16m_d1G5 and 17D20m_d1824 with low expression level, were expressed at 0.5L scale, purified into monomer fractions by size exclusion chromatography, and tested again in ELISA and functional assays. The 18L16 library did not produce any daughter clones with the desired binding affinity.

[0308] After purification, the six daughter clones were tested for inhibitory activity in the complement assay, and the results are shown in Table 18.

[0309] [Table 18] As shown in Table 18 above, only one clone, 17N16m_d17N9, had affinity and activity in the same range as the parent clones.

[0310] FIG. 8 is an alignment of the amino acid sequences of the full-length scFv mother clone 17N16 (sequence number 59) and the 17N16m_d17N9 daughter clone (sequence number 66) showing that the light chain (starting with SYE) has 17 amino acid residues that differ between these two clones.

[0311] By rescreening the 17N16 lambda library, several additional candidate daughter clones were obtained, of which 17N16m_d27E13 was identified in ELISA and complement assays and included in the set of candidate daughter clones for further analysis.

[0312] Assay of daughter clones in various sera The above candidate daughter clones were analyzed in various sera as follows: Mannan was diluted to 20 μg / ml and coated on ELISA plates overnight at 4° C. The next day, wells were blocked with 1% HSA. African green monkey serum was diluted to 0.2%, rat serum was diluted to 0.375%, and human serum was diluted to 1% in CaMgGVB buffer. Purified scFv from each candidate daughter clone was added to the same volume of buffer in duplicate at a series of different concentrations and pre-incubated on ice for 45 minutes before being added to the blocked ELISA plate. The reaction was started by incubation at 37° C. for 1 hour and stopped by transferring the plate to an ice bath. C3c release was detected using rabbit α-mouse C3c antibody followed by goat α-rabbit HRP. The background of 0.1 μg / ml OMS100 on mannan-negative plates was subtracted from these curves. The results are summarized in Table 19 below.

[0313] [Table 19] Discussion of results: As shown in Table 19, the daughter clone 17N16m_d17N9 has higher functional activity than the mother clone. The sequence difference of 17 amino acids in the light chain compared to the mother clone plus improved function in rat serum makes this clone a positive candidate. Based on this data, the daughter clone 17N16m_d17N9 was selected for further analysis.

[0314] Example 7 This example describes the generation and analysis of a daughter clone, 17D20m_d3521N11, derived from the mother clone, 17D20.

[0315] Background / Rationale: To improve the affinity of the mother clone candidate 17D20mc, further "look-through-mutagenesis" was performed on the first three amino acids in the CDR3 of the heavy chain (CDR-H3). This was a parallel mutagenesis campaign to the regular light chain shuffling of 17D20mc. Thus, three different scFv libraries were constructed by PCR randomizing the amino acids at positions 1, 2 and 3 against a set of all 20 possible amino acids using degenerate codons. After cloning the libraries, microscale expression was performed and scFv binding was monitored on MASP-2A-coated CM5 chips (not shown). BIAcore analysis of microscale expression was performed on the three different libraries on MASP-2A-coated chips randomized at positions 1, 2 or 3 to identify potentially interesting daughter clones.

[0316] It was observed that no clones were found with improved off-rates for amino acids at positions 1 and 2 of CDR-H3 compared to the mother candidate clone 17D20m. However, several candidates with mutations at amino acid position 3 in CDR-H3 showed improved off-rates compared to the mother clone 17D20m. The mutations were identified by sequencing these clones (#35, #59 and #90). The sequences of the two "look-through mutagenesis" derived clones are compared to 17D20mc (the original sequence). Interestingly, all sequenced clones except one (#90) had Ala-Arg substitutions compared to the mother candidate.

[0317] FIG. 9 shows a comparison of the amino acid sequence of the heavy chain region of scFv parent clone 17D20m (aa 61 to 119 of SEQ ID NO: 18) and the amino acid sequence of the CDR-H3 region of clone #35, an scFv clone containing a mutation in CDR-H3 (aa 61 to 119 of SEQ ID NO: 20, with a substitution of A for R at position 102 of SEQ ID NO: 18), clone #59 (same sequence as clone #35), and clone #90 (substitution of A for P at position 102 of SEQ ID NO: 18).

[0318] Analysis of mutant clones #35 and #59 Mutant clones #35 and #59 were expressed on a small scale and further tested in a titration-ELISA against immobilized MASP-2A (10 μg / ml) compared to the mother candidate clone 17D20. The scFvs were serially diluted 5-fold starting at 20 μg / ml and binding was detected using anti-Myc (mouse) / anti-mouse HRP. Slightly improved binding was observed in the ELISA assay for candidate clones #35 and #59 compared to the mother candidate clone 17D20 (data not shown).

[0319] The improved clone #35 was combined with the best light chain shuffled clone 17D20m_d21N11. The mutations (Ala-Arg) in the VH of candidate 17D20md35 were combined with the light chain of candidate 17D20m_d21N11 to generate a clone called VH35-VL21N11, otherwise called 3521N11.

[0320] 10A is an amino acid sequence alignment of the sequence of the CDR3 region of mother clone 17D20 (aa 61-119 of SEQ ID NO: 18), the same region of daughter clone 17D20m_d21N11 having the same sequence, and the same region of mutagenized clone #35 (aa 61-119 of SEQ ID NO: 20) combined with the VL of 17D20m_d21N11 designated "3521N11." The VH sequence region highlighted includes CDRH3, and the mutated target residue region is underlined.

[0321] Figure 10B is an alignment of the protein sequences of the full-length scFv including the VL and VH regions of the 17D20 mother clone (SEQ ID NO:55) and the daughter clone 17D20m_d21N11 (SEQ ID NO:67). The scFv daughter clone 17D20m_d3521N11 is shown as SEQ ID NO:68. Note: The X residue at position 220 in Figure 10B was determined to be "E" as shown in SEQ ID NO:68.

[0322] Titration ELISA assays of the scFv sets shown in Figure 10 were performed against MASP-2 (10 μg / ml) and the results are shown in Table 20.

[0323] [Table 20] 17D20m_d3521N11 daughter clones were further analyzed for functional activity as described in Example 8 below.

[0324] Example 8 This example describes the conversion and analysis of candidate daughter clones 17N16m_d17N9 and 17D20m_d3521N11 into IgG4, IgG4 / S228P and IgG2 types.

[0325] Rationale / Background The antibody screening methods described in Examples 2-7 identified two mother clones with suitable functionality, 17N16 and 17D20. Affinity maturation of these mother clones yielded daughter clones that showed approximately 2-fold improvement in potency compared to their mother clones in a surrogate functional assay at the scFv level. The daughter clones with the best activity are 17N16m_d17N9 and 17D20m_d3521N11. In a comparison of the functional activity of the 17N16 mother clone and the light chain shuffled daughter clones (scFv format, 1% NHS assay) as described in Example 6, 17N16m_d17N9 was determined to be slightly more potent than the mother clone and to have the best functional potency of all daughter clones tested in this assay.

[0326] method: Comparison of the functional potency of candidate scFv clones was performed in C3 (1% human serum and 90% human serum) and C4 conversion assays (90% human serum) performed as described in Example 5.

[0327] The results are shown in Table 21 below.

[0328] [Table 21] As shown in Table 21 above, 17N16m_d17N9 has good activity when assayed in 90% normal human serum (NHS) in the C3 assay and is more potent than other daughter clones of this type.

[0329] Conversion of candidate clones to IgG4, IgG4 / S228P, and IgG2 types All of these candidate clones were converted to IgG4, IgG4 / S228P and IgG2 types for further analysis. SEQ ID NO: 62: cDNA encoding wild type IgG4 SEQ ID NO: 63: Wild type IgG4 polypeptide SEQ ID NO: 64 cDNA encoding the IgG4 mutation S228P SEQ ID NO: 65: IgG4 mutant S228P polypeptide SEQ ID NO: 69: cDNA encoding wild type IgG2 SEQ ID NO: 70: Wild type IgG2 polypeptide

[0330] [Table 22] These antibodies were tested for their ability to cross-react with non-human MASP-2 protein (African green (AG) monkey) in a C3 assay to determine whether monoclonal antibodies #1-6 could be used to test for toxicity in animal models that may be predictive of humans. Monoclonal antibodies #1-6 were tested in a C3b deposition assay and a C4 assay in 90% human serum. The results are shown in Table 23 below.

[0331] [Table 23] Figure 11A graphically illustrates the results of a C3b deposition assay performed on daughter clone isotype variants (MoAb #1-3) derived from the human anti-MASP-2 monoclonal antibody mother clone 17N16.

[0332] Figure 11B graphically illustrates the results of a C3b deposition assay performed on daughter clone isotype variants (MoAb #4-6) derived from the human anti-MASP-2 monoclonal antibody mother clone 17D20.

[0333] As shown in Table 23 and Figures 11A and 11B, human anti-MASP-2 monoclonal antibodies (MoAb #1-6) bind to MASP-2 with high affinity and inhibit the function of C3 and C4 activity in 90% human serum. It is also noted that human anti-MASP-2 MoAbs cross-react with non-human MASP-2 protein (African green monkey), providing an animal model for toxicity studies that may be predictive for humans.

[0334] MoAbs #1-6 were further analyzed in 95% human serum, 95% African green monkey serum, and the results are summarized in Table 24 below.

[0335] [Table 24] 12A and 12B graphically illustrate testing of the parent clones and MoAbs #1-6 in a C3b deposition assay in 95% normal human serum.

[0336] FIG. 13 graphically illustrates the inhibition of C4b deposition in 95% normal human serum.

[0337] FIG. 14 graphically illustrates the inhibition of C3b deposition in 95% African Green Monkey serum.

[0338] MoAbs #1-6 were further tested for their ability to selectively inhibit the lectin pathway by assaying for inhibition of rat C3b, inhibition of preassembled MBL-MASP-2 complexes; inhibition of the classical pathway and selectivity for C1s. The results are summarized in Table 25.

[0339] [Table 25] FIG. 15 graphically illustrates the inhibition of C4 cleavage activity of preassembled MBL-MASP-2 complexes by MoAbs #2, 3, 5 and 6.

[0340] FIG. 16 graphically illustrates the preferential binding of MoAb#6 to human MASP-2 compared to C1s.

[0341] [Table 26] Example 9 This example describes epitope mapping performed on some of the blocking human anti-MASP-2 MoAbs.

[0342] method: The following recombinant proteins were produced as described in Example 1: Human MAp19 Human MASP2A Human MASP-2 SP Human MASP-2 CCP2-SP Human MASP-2 CCP1-CCP2-SP Human MASP-1 / 3 CUB1-EGF-CUB2 Human MASP-1 CCP1-CCP2-SP Anti-MASP-2 antibodies OMS100 and MoAb#6 (35VH-21N11VL), both of which have been shown to bind human MASP-2 with high affinity and have the ability to block functional complement activity (see Examples 6-8), were analyzed for epitope binding by dot blot analysis.

[0343] Dot blot analysis Serial dilutions of recombinant MASP-2 polypeptides as described above were spotted onto nitrocellulose membranes. The amount of protein spotted ranged from 50 ng to 5 pg in 10-fold steps. In later experiments, the amount of protein spotted ranged from a lower amount of 50 ng to 16 pg, again in 5-fold steps. The membranes were blocked with 5% skim milk powder in TBS (blocking buffer) and then blocked with blocking buffer (5.0 mM Ca). 2+ The membranes were incubated with 1.0 μg / ml anti-MASP-2 Fab2 in 100 mM NaCl (containing 1 mM NaCl). Bound Fab2 was detected using HRP-conjugated anti-human Fab (AbD / Serotec; 1 / 10,000 dilution) and an ECL detection kit (Amersham). One membrane was incubated with polyclonal rabbit anti-human MASP-2 Ab (described in Stover et al., J Immunol 163:6848-59 (1999)) as a positive control. In this case, bound Ab was detected using HRP-conjugated goat anti-rabbit IgG (Dako; 1 / 2,000 dilution).

[0344] result: The results are summarized in Table 27.

[0345] [Table 27] The above results indicate that MoAb#6 and OMS100 antibodies are highly specific for MASP-2 and do not bind to MASP-1 or MASP-3. Neither antibody bound to Map19 or to the MASP-2 fragment that does not contain the CCP1 domain of MASP-2, leading to the conclusion that their binding sites contain the CCP1 domain.

[0346] Example 10 This example demonstrates that human anti-MASP-2 MoAb #6 inhibits the lectin pathway in African green monkeys following intravenous administration.

[0347] method: MoAb#6 was administered intravenously at a dose of 1 mg / kg to three African green monkeys in Group 1 and at a dose of 3 mg / kg to three African green monkeys in Group 2. Blood samples were obtained 2, 4, 8, 10, 24, 48, 72, and 98 hours after administration and tested for the presence of lectin pathway activity.

[0348] As shown in FIG. 17, the lectin pathway was completely inhibited following intravenous administration of anti-human MoAb#6.

[0349] Example 11 In this example, it is demonstrated that MASP-2 inhibitors, such as anti-MASP-2 antibodies, are effective in treating radiation exposure and / or treating, ameliorating or preventing acute radiation syndrome.

[0350] Rationale: Exposure to high doses of ionizing radiation causes death by two main mechanisms: bone marrow toxicity and gastrointestinal syndrome. Bone marrow toxicity reduces all hematologic cells, predisposing the organism to death by infection and bleeding. The gastrointestinal syndrome is more severe and is driven by loss of intestinal barrier function due to the breakdown of the intestinal epithelial layer and loss of intestinal endocrine function. This leads to sepsis and an associated systemic inflammatory response syndrome that can be fatal.

[0351] The lectin pathway of complement is an innate immune mechanism that initiates inflammation in response to tissue injury and exposure to foreign surfaces (i.e., bacteria). Blocking this pathway leads to better outcomes in mouse models of ischemic intestinal tissue injury or septic shock. It is hypothesized that the lectin pathway may cause excessive and harmful inflammation in response to tissue injury induced by irradiation. Thus, blocking the lectin pathway may reduce secondary injury and increase survival after acute radiation exposure.

[0352] The aim of the study performed as described in this example was to evaluate the effect of lectin pathway blockade on survival in a mouse model of radiation injury by administering anti-mouse MASP-2 antibody.

[0353] Research #1 Methods and Materials: Materials. The test substances used in this study were (i) a high affinity anti-mouse MASP-2 antibody (mAbM11) and (ii) a high affinity anti-human MASP-2 antibody (mAbOMS646), which block the MASP-2 protein component of the lectin complement pathway produced in transfected mammalian cells. The dosing concentrations were 1 mg / kg for anti-mouse MASP-2 antibody (mAbM11), 5 mg / kg for anti-human MASP-2 antibody (mAbOMS646) or sterile saline. For each dosing session, an appropriate volume of fresh dosing solution was prepared.

[0354] Animals. Young adult male Swiss-Webster mice were obtained from Harlan Laboratories (Houston, TX). Animals were housed in solid bottom cages with Alpha-Dri bedding and provided certified PMI 5002 Rodent Diet (Animal Specialties, Inc., Hubbard OR) and water ad libitum. Temperature was monitored and the rooms housing the animals were operated on a 12-hour light / 12-hour dark photocycle.

[0355] After 2 weeks of acclimation in the above facility, the patient was treated with a Therapax X-ray machine equipped with a 320 kV highly stable X-ray generator, a metal-ceramic X-ray tube, a variable X-ray beam collimator and filters. Mice were irradiated by total body irradiation to 6.5, 7.0, and 8.0 Gy in groups of 10 at a dose rate of 0.78 Gy / min using an X-RAD 320 system (Precision X-ray Incorporated, East Haven, CT).

[0356] Drug formulation and administration. According to the protocol, dosing solutions of 0.2 mg / ml anti-mouse MASP-2 antibody (mAbM11) or 0.5 mg / ml anti-human MASP-2 antibody (mAbOMS646) were prepared by diluting the appropriate volume of concentrated stock solution with ice-cold saline. Administration of anti-MASP-2 antibodies mAbM11 and mAbOMS646 was via IP injection using a 25-gauge needle to deliver 1 mg / kg mAbM11, 5 mg / kg mAbOMS646 or saline vehicle based on the animal's body weight.

[0357] Study design. Mice were randomly assigned to groups as described in Table 28. Body weight and temperature were measured and recorded daily. Mice in groups 7, 11 and 13 were sacrificed 7 days after irradiation and blood was collected by cardiac puncture under deep anesthesia. Animals that survived 30 days after irradiation were sacrificed in the same manner and blood was collected. Plasma was prepared from the collected blood samples according to the protocol and returned to Sponsor for analysis.

[0358] [Table 28] Statistical analysis. Kaplan-Meier survival curves were constructed and used to compare mean survival times between treatment groups using the log-rank and Wilcoxon methods. Means with standard deviations or means with standard errors of the means are reported. Statistical comparisons between control irradiated animals and individual treatment groups were made using unpaired two-tailed t-tests.

[0359] Study #1 Results Kaplan-Meier survival plots for the 6.5, 7.0 and 8.0 Gy exposure groups are provided in Figures 18A, 18B and 18C, respectively, and summarized in Table 29 below. Overall, pre-irradiation treatment with anti-mouse MASP-2ab (mAbM11) increased survival of irradiated mice compared to vehicle-treated irradiated control animals at both the 6.5 Gy (20% increase) and 7.0 Gy (30% increase) exposure levels. At the 6.5 Gy exposure level, post-irradiation treatment with anti-mouse MASP-2ab resulted in a modest increase in survival (15%) compared to vehicle control irradiated animals.

[0360] In comparison, all treated animals at the 7.0 Gy and 8.0 Gy exposure levels showed increased survival compared to vehicle-treated irradiated control animals. The greatest changes in survival occurred in animals administered mAbOMS646, with a 45% increase in survival compared to control animals at the 7.0 Gy exposure level and a 12% increase in survival at the 8.0 Gy exposure level. Furthermore, at the 7.0 Gy exposure level, death in the mAbOMS646-treated group first occurred on day 15 post-irradiation versus day 8 post-irradiation for vehicle-treated irradiated control animals, 7 days later than control animals. The mean time to death for mice administered mAbOMS646 (27.3±1.3 days) was significantly increased (p=0.0087) compared to control animals (20.7±2.0 days) at the 7.0 Gy exposure level.

[0361] Percent change in body weight compared to the day before irradiation (day -1) was recorded throughout the study. Transient weight loss occurred in all irradiated animals, but there was no evidence of differential changes due to mAbM11 or mAbOMS646 treatment compared to controls (data not shown). At the end of the study, all surviving animals showed an increase in body weight from their starting weight (day -1).

[0362] [Table 29] Consideration The acute radiation syndrome consists of three distinct subsyndromes: hematopoietic, gastrointestinal and cerebrovascular. The syndrome observed depends on the radiation dose, with effects on hematopoiesis observed in humans who received significant partial or total body radiation exposure exceeding 1 Gy. The hematopoietic syndrome is characterized by an extreme depression of bone marrow function leading to pancytopenia with alterations in blood counts, red and white blood cells and platelets, accompanied by damage to the immune system. The development of a nadir with almost no neutrophils and platelets in the peripheral blood leads to neutropenia, fever, septic complications and death from uncontrolled bleeding.

[0363] In this study, administration of mAbH6 was found to enhance survival from total body X-irradiation in Swiss-Webster male mice irradiated at 7.0 Gy. Notably, at the 7.0 Gy exposure level, 80% of animals administered mAbOMS646 survived to 30 days compared to 35% of vehicle-treated control-irradiated animals. Importantly, the first day of death in this treatment group did not occur until day 15 after irradiation, a 7-day extension to that observed in vehicle-treated control-irradiated animals. Curiously, at the lower X-ray exposure (6.5 Gy), administration of mAbOMS646 did not appear to affect survival or delay in death compared to vehicle-treated control-irradiated animals. There could be multiple reasons for this difference in response between exposure levels, but further studies, including collection of intermediate samples for microbial cultures and hematological parameters, may be required to test either hypothesis. One interpretation could simply be that the number of animals assigned to groups may have hindered the understanding of any subtle differences related to treatment. For example, with a group size of n=20, the difference in survival between 65% (mAbOMS646 at 6.5 Gy exposure) and 80% (mAbH6 at 7.0 Gy exposure) is 3 mice. On the other hand, the difference between 35% (vehicle control at 7.0 Gy exposure) and 80% (mAbOMS646 at 7.0 Gy exposure) is 9 mice, providing reliable evidence of a treatment-related difference.

[0364] These results demonstrate that anti-MASP-2 antibodies are effective in treating mammalian subjects at risk of or suffering from the deleterious effects of acute radiation syndrome.

[0365] Research #2 Swiss Webster mice (n=50) were exposed to ionizing radiation (8.0 Gy). The effect of treatment with an anti-MASP-2 antibody (OMS646 5 mg / kg), administered 18 hours before and 2 hours after radiation exposure and weekly thereafter, on mortality was evaluated.

[0366] Study #2 Results Administration of the anti-MASP-2 antibody OMS646 increased the survival rate of mice exposed to 8.0 Gy compared to mice receiving vehicle control (adjusted median survival 4 to 6 days) and reduced mortality by 12% compared to mice receiving vehicle control (log-rank test, p=0.040).

[0367] Research #3 Swiss Webster mice (n=50) were exposed to ionizing radiation (8.0 Gy) in the following groups: (I: vehicle) saline control; (II: low) anti-MASP-2 antibody OMS646 (5 mg / kg) administered 18 hours before and 2 hours after irradiation, (III: high) OMS646 (10 mg / kg) administered 18 hours before and 2 hours after irradiation; and (IV: post-high) OMS646 (10 mg / kg) administered only 2 hours after irradiation.

[0368] Study #3 Results It was determined that administration of anti-MASP-2 antibodies before and after irradiation adjusted the mean survival time from 4 to 5 days compared to animals that received a vehicle control. Mortality in mice treated with anti-MASP-2 antibodies was reduced by 6-12% compared to vehicle control mice. It is further noted that no significant adverse effects of the treatment were observed.

[0369] In summary, the results in this example demonstrate that the anti-MASP-2 antibodies of the present invention are effective in treating mammalian subjects at risk of or suffering from the deleterious effects of acute radiation syndrome.

[0370] Example 12 This example describes further characterization of the OMS646 antibody (17D20m_d3521N11), a fully human anti-human MASP-2 IgG4 antibody with mutations in the hinge region.

[0371] method: OMS646 (17D20m_d3521N11), a fully human anti-human MASP-2 IgG4 antibody with mutations in the hinge region, was generated as described in Examples 2-8 above. OMS646 antibody was purified from culture supernatants of a CHO expressing cell line stably transfected with expression constructs encoding the heavy and light chains of OMS646. Cells were grown in PF-CHO medium for 16-20 days and cell-free supernatants were harvested when cell viability fell below 50%. OMS646 was purified by Protein A affinity chromatography followed by ion exchange, concentration and buffer exchange into PBS.

[0372] 1. OMS646 binds to human MASP-2 with high affinity Surface plasmon resonance (Biocore) analysis of binding between immobilized OMS646 and recombinant human MASP-2 method: OMS646 was immobilized at various densities by amine coupling to a CM5 chip, and the association and dissociation of recombinant human MASP-2 dissolved at 9 nM, 3 nM, 1 nM, or 0.3 nM was recorded over time to determine the association rate constant (K on ) and dissociation rate constant (K off ) was measured. on and K. off Based on the value of the equilibrium binding constant (K D ) was calculated.

[0373] result: Figure 19 shows that the immobilized OMS646 was approximately 1–3 × 10 -4 S -1 K off Velocity and approximately 1.6 to 3 × 10 6 M -1 S -1 K on We show that it binds to recombinant MASP-2 with a high affinity under these experimental conditions (K of approximately 92 pM). DThe graph shows the results of surface plasmon resonance (Biocore) analysis for OMS646, which means the K = 0.01, which is the K = 0.01, which is the K = 0.01. The experimental K ranges from 50 to 250 pM, depending on the density of immobilized OMS646 and the concentration of MASP-2 in solution. D The value was determined.

[0374] ELISA assay of OMS646 binding to immobilized recombinant human MASP-2 Methods: An ELISA assay was performed to assess the dose response of OMS646 binding to immobilized recombinant MASP-2. Recombinant human MASP-2 (50 ng / well) was immobilized on maxisorp ELISA plates (Nunc) in PBS overnight at 4°C. The next day, the plates were blocked by washing three times with PBS-Tween (0.05%). A serial dilution series of OMS646 (concentration range 0.001-10 nM) in blocking buffer was then added to the MASP-2-coated wells. After 1 h of incubation to allow binding of OMS646 to the immobilized antigen, unbound OMS646 was removed by washing the wells. Bound OMS646 was detected using an HRP-conjugated goat anti-human IgG antibody (Qualex; diluted 1:5000 in blocking buffer) followed by development with TMB peroxidase substrate (Kirkegaard & Perry Laboratories). The peroxidase reaction was stopped by adding 100 μl / well of 1.0 M H3PO4, and the conversion of the substrate was quantified photometrically at 450 nM using a 96-well plate reader (Spectramax). A single-binding site curve-fitting algorithm (Graphpad) was used to determine the K D values ​​were calculated.

[0375] result: Figure 20 graphically illustrates the results of an ELISA assay to determine the binding affinity of OMS646 to immobilized recombinant human MASP-2. As shown in Figure 20, OMS646 binds immobilized recombinant human MASP-2 with a K DThese results indicate that OMS646 binds to human MASP-2 with a K of approximately 100 pM, which is consistent with the results obtained in Biocore analysis as shown in Figure 19. D It is demonstrated that the β-glucosidase has a high affinity.

[0376] 2. OMS646 inhibits C4 activation on mannan-coated surfaces but not on immune complex-coated surfaces method: C4 activation was measured on mannan-coated or immune complex-coated surfaces in the presence or absence of OMS646 over the range of concentrations shown in Figures 21A and 21B, respectively, as follows.

[0377] In the following method for measuring the C4 cleavage activity of MASP-2, mannan-coated plastic wells were incubated with 1% human serum for 60 minutes at 37° C. to activate the lectin pathway. The wells were then washed and assayed for human C4b immobilized on the wells using standard ELISA. The amount of C4b generated in this assay is a measure of MASP-2-dependent C4 cleavage activity. In this assay, selected concentrations of anti-MASP-2 antibodies were tested for their ability to inhibit C4 cleavage.

[0378] method: C4 activation on mannan-coated surfaces: To measure the effect of OMS646 on the lectin pathway, 96-well Costar A Medium Binding plate was coated with mannan by overnight incubation at 5° C. with 50 μL of a 40 μg / mL mannan solution diluted in 50 mM carbonate buffer, pH 9.5. Each well was washed three times with 200 μL of PBS. The wells were then blocked with 100 μL / well of 1% bovine serum albumin in PBS and incubated for 1 hour at room temperature with gentle mixing. Each well was washed three times with 200 μL of PBS. In a separate 96-well plate, serial dilutions of MASP-2 antibody (OMS646) were incubated with Ca ++ and Mg ++The wells were preincubated with 1% human serum in GVB buffer (4.0 mM barbital, 141 mM NaCl, 1.0 mM MgCl2, 2.0 mM CaCl2, 0.1% gelatin, pH 7.4) at 5°C for 1 hour. These antibody-serum preincubation mixtures were then transferred to the corresponding wells of the mannan-coated assay plate. Complement activation was initiated by transferring the assay plate to a 37°C water bath. After 60 minutes of incubation, the reaction was stopped by adding EDTA to the reaction mixture. Each well was washed five times with 200 μL of PBS-Tween20 (0.05% Tween20 in PBS), and then each well was washed twice with 200 μL of PBS. 100 μL / well of a 1:100 dilution of biotin-conjugated chicken anti-human C4c (Immunsystem AB, Uppsala, Sweden) was added in PBS containing 2.0 mg / ml bovine serum albumin (BSA) and incubated for 1 hour at room temperature with gentle mixing. Each well was washed 5 times with 200 μL PBS. 100 μL / well of 0.1 μg / ml peroxidase-conjugated streptavidin (Pierce Chemical #21126) was added in PBS containing 2.0 mg / ml BSA and incubated for 1 hour at room temperature with gentle mixing on a shaker. Each well was washed 5 times with 200 μL PBS. 100 μL / well of peroxidase substrate TMB (Kirkegaard & Perry Laboratories) was added and incubated for 10 minutes at room temperature. The peroxidase reaction was stopped by adding 100 μL / well of 1.0 M H3PO4, and the OD 450 was measured.

[0379] C4 activation on immune complex-coated surfaces To measure the effect of OMS646 on the classical pathway, the assay described above was modified to use immune complex-coated plates. The assay was performed as detailed for the lectin pathway above, with the difference that the wells were coated with purified sheep IgG, which is used to stimulate C4 activation via the classical pathway.

[0380] result: Figure 21A graphically illustrates the levels of C4 activation on mannan-coated surfaces in the presence or absence of OMS646. Figure 21B graphically illustrates the levels of C4 activation on IgG-coated surfaces in the presence or absence of OMS646. As shown in Figure 21A, OMS646 inhibited C4 activation on mannan-coated surfaces with an IC of approximately 0.5 nM in 1% human serum. 50 IC obtained in 10 independent experiments 50 The value was 0.52±0.28 nM (mean±SD). In contrast, as shown in FIG 21B, OMS646 did not inhibit C4 activation on IgG-coated surfaces. These results demonstrate that OMS646 blocks lectin-dependent activation of complement component C4 but does not inhibit classical pathway-dependent activation.

[0381] 3. OMS646 specifically blocks lectin-dependent activation of terminal complement components method: The effect of OMS646 on the deposition of the membrane attack complex (MAC) was analyzed using pathway-specific conditions for the lectin, classical and alternative pathways. For this purpose, the Wieslab Comp300 Complement Screening Kit (Wieslab, Lund, Sweden) was used according to the manufacturer's instructions.

[0382] result: Figure 22A graphically illustrates the levels of MAC deposition in the presence or absence of anti-MASP-2 antibody (OMS646) under lectin pathway-specific assay conditions. Figure 22B graphically illustrates the levels of MAC deposition in the presence or absence of anti-MASP-2 antibody (OMS646) under classical pathway-specific assay conditions. Figure 22C graphically illustrates the levels of MAC deposition in the presence or absence of anti-MASP-2 antibody (OMS646) under alternative pathway-specific assay conditions.

[0383] As shown in Figure 22A, OMS646 inhibited the lectin pathway-mediated activation of MAC deposition with an IC of approximately 1 nM. 50 However, OMS646 did not affect MAC deposition resulting from classical pathway-mediated activation (FIG. 22B) or alternative pathway-mediated activation (FIG. 22C).

[0384] 4. OMS646 effectively inhibits lectin pathway activation under physiological conditions method: Lectin-dependent C3 and C4 activation was assessed in 90% human serum in the absence and presence of various concentrations of OMS646 as follows: C4 activation assay To evaluate the effect of OMS646 on lectin-dependent C4 activation, 96-well Costar medium binding plates were coated with 2 μg of mannan (50 μl of 40 μg / mL solution in 50 mM carbonate buffer, pH 9.5) overnight at 5° C. The plates were then washed three times with 200 μl of PBS and blocked with 100 μL / well of 1% bovine serum albumin in PBS for 1 h at room temperature with gentle mixing. In separate preincubation plates, selected concentrations of OMS646 were mixed with 90% human serum and incubated on ice for 1 h. These antibody-serum preincubation mixtures were then transferred to the mannan-coated wells of the assay plate on ice. The assay plate was then incubated in an ice-water bath for 90 min to activate complement. The reaction was stopped by adding EDTA to the reaction mixture. Each well was washed five times with 200 μL of PBS-Tween20 (0.05% Tween20 in PBS), then each well was washed twice with 200 μL of PBS. 100 μL / well of a 1:1000 dilution of biotin-conjugated chicken anti-human C4c (Immunsystem AB, Uppsala, Sweden) was added in PBS containing 2.0 mg / ml bovine serum albumin (BSA) and incubated for 1 hour at room temperature with gentle mixing. Each well was washed five times with 200 μL of PBS. 100 μL / well of 0.1 μg / mL peroxidase-conjugated streptavidin (Pierce Chemical #21126) was added in PBS containing 2.0 mg / ml BSA and incubated for 1 hour at room temperature with gentle mixing on a shaker. Each well was washed five times with 200 μL of PBS. 100 μL / well of peroxidase substrate TMB (Kirdegaard & 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 the OD 450 was measured.

[0385] C3 activation assay To evaluate the effect of OMS646 on lectin-dependent C3 activation, the assay was performed in the same manner as the C4 activation assay described above, except that C3 deposition was evaluated at the end point. C3 deposition was quantified as follows. At the end of the complement deposition reaction, the plates were washed as described above, followed by incubation for 1 hour with 100 μL / well of a 1:5000 dilution of rabbit anti-human C3c antibody (Dako) in PBS containing 2.0 mg / mL bovine serum albumin (BSA). Each plate was washed five times with 200 μL of PBS, and then incubated for 1 hour at room temperature with 100 μL / well of HRP-labeled goat anti-rabbit IgG (American Qualex Antibodies) in PBS containing 2.0 mg / mL BSA. The plates were washed five times with 200 μL of PBS, then 100 μL / well of peroxidase substrate TMB (Kirkegaard & Perry Laboratories) was added and incubated at room temperature for 10 min. The peroxidase reaction was stopped by adding 100 μL / well of 1.0 M H3PO4, and the OD 450 The IC was determined by applying a sigmoidal dose-response curve fitting algorithm (GraphPad) to the experimental data set. 50 Got the value.

[0386] result: Figure 23A graphically illustrates the levels of C3 deposition in the presence or absence of anti-MASP-2 antibody (OMS646) across a range of concentrations in 90% human serum under lectin pathway specific conditions. Figure 23B graphically illustrates the levels of C4 deposition in the presence or absence of anti-MASP-2 antibody (OMS646) across a range of concentrations in 90% human serum under lectin pathway specific conditions. As shown in Figure 23A, OMS646 inhibits C3 deposition in 90% human serum at IC 50 As shown in Figure 23B, OMS646 blocked C4 deposition at IC 50 =2.8±1.3 nM (n=6).

[0387] These results demonstrate that OMS646 provides potent and effective blockade of lectin pathway activation under physiological conditions, thereby providing support for the use of low therapeutic doses of OMS646. Based on these data, it is expected that OMS646 may block >90% of the lectin pathway in the patient circulation at plasma concentrations of 20 nM (3 μg / mL) or less. Based on the knowledge that a typical human plasma volume is approximately 3 L and that the majority of the antibody material administered is retained in plasma (Lin YS et al., JPET 288:371 (1999)), it is expected that doses as low as 10 mg of OMS646 administered intravenously may be effective in blocking the lectin pathway in the short term (i.e., transient periods such as 1-3 days). In chronic disease situations, it may be beneficial to block the lectin pathway for extended periods of time to achieve the maximum benefit of treatment. Therefore, for such chronic conditions, a dose of 100mg of OMS646 may be preferred, which is expected to be effective in blocking lectin pathway in adult human subjects for at least one week or more.Taking into account the slow clearance and long half-life usually observed for human antibodies, it is possible that a dose of 100mg of OMS646 can be effective for more than one week (e.g., two or even four weeks).Higher doses of antibody (i.e., more than 100mg, e.g., 200mg, 500mg or more, e.g., 700mg or 1000mg) are expected to have a longer duration of action (e.g., more than two weeks).

[0388] 5. OMS646 blocks activation of the lectin pathway in monkeys As described above in Example 10 and shown in FIG. 17, OMS646 ablated systemic lectin pathway activity for approximately 72 hours following intravenous administration of OMS646 (3 mg / kg) to African green monkeys, after which lectin pathway activity was measured to be restored. This example describes a follow-up study in which lectin-dependent C4 activation was assessed in 90% African green monkey serum or 90% Cynomoglus monkey serum across a range of concentrations of OMS646 and in the absence of OMS646, as follows: To measure the effect of OMS646 on lectin-dependent C4 activation in various non-human primate species, 96-well Costar medium binding plates were coated with 2 μg of mannan (50 μl of 40 μg / mL solution in 50 mM carbonate buffer, pH 9.5) overnight at 5° C. The plates were then washed three times with 200 μL of PBS and blocked with 100 μL / well of 1% bovine serum albumin in PBS for 1 hour at room temperature with gentle mixing. In separate preincubation plates, selected concentrations of OMS646 were mixed with 90% serum collected from African green monkeys or cynomolgus monkeys and incubated on ice for 1 hour. These antibody-serum preincubation mixtures were then transferred to mannan-coated wells of the assay plate on ice. Complement was then activated by incubating the assay plate in an ice-water bath for 90 minutes. The reaction was stopped by adding EDTA to the reaction mixture. Each well was washed five times with 200 μL of PBS-Tween20 (0.05% Tween20 in PBS), then each well was washed twice with 200 μL of PBS. 100 μL / well of a 1:1000 dilution of biotin-conjugated chicken anti-human C4c (Immunosystem AB, Uppsala, Sweden) was added in PBS containing 2.0 mg / mL of BSA and incubated for 1 hour at room temperature with gentle mixing. Each well was washed five times with 200 μL of PBS. 100 μL / well of 0.1 μg / mL of peroxidase-conjugated streptavidin (Pierce Chemical #21126) was added in PBS containing 2.0 mg / mL of BSA and incubated for 1 hour at room temperature with gentle mixing on a shaker. Each well was washed five times with 200 μL of PBS. 100 μL / well of peroxidase substrate TMB (Kirdegaard & Perry Laboratories) was added and incubated at room temperature for 10 min. The peroxidase reaction was stopped by adding 100 μL / well of 1.0 M H3PO4, and the OD 450The IC was determined by applying a sigmoidal dose-response curve fitting algorithm (GraphPad) to the experimental data set. 50 Got the value.

[0389] result: Dose responses for lectin pathway inhibition in 90% cynomolgus monkey serum (Figure 24A) and 90% African green monkey serum (Figure 24B) showed IC values ​​in the range of 30 nM to 50 nM and 15 nM to 30 nM, respectively. 50 values ​​were observed.

[0390] In summary, OMS646, a fully human anti-human MASP-2 IgG4 antibody (with mutations in the hinge region), was observed to have the following beneficial properties: high affinity binding to human MASP-2 (K in the range of 50–250 pM); D , 1 to 3 × 10 -4 S -1 K in the range off Speed ​​and 1.6 to 3 × 10 6 M -1 S -1 K in the range on rate); functional potency in human serum (IC of 0.52 ± 0.28 nM (n = 10) in 1% human serum) 50 and an IC of 3 ± 1.5 nM in 90% serum 50 inhibition of C4 deposition in the 15-50 nM range; and IC 50 Cross-reactivity in monkeys showing inhibition of C4 deposition in (90% monkey serum).

[0391] As noted above, doses as low as 10 mg of OMS646 (corresponding to 0.15 mg / kg for an average human) are expected to be effective in acutely blocking the lectin pathway in the human circulation (e.g., for at least 1-3 days), while a dose of 100 mg of OMS646 (corresponding to 1.5 mg / kg for an average human) is expected to block the lectin pathway in the patient's circulation for at least a week or more. By using higher doses of OMS646 (e.g., doses greater than 100 mg, e.g., at least 200 mg, at least 300 mg, at least 400 mg, at least 500 mg or more), and preferably subcutaneous (sc) or intramuscular (im) routes of administration, the time frame for effective lectin pathway disruption can be further extended to 2 weeks, preferably 4 weeks.

[0392] For example, as shown in the experimental data herein, in primates, a dose of 1 mg / kg of OMS646 inhibited the lectin pathway for 1 day, and a dose of 3 mg / kg of OMS646 inhibited the lectin pathway for about 3 days (72 hours). It is therefore estimated that a higher dose of 7-10 mg / kg may be effective in inhibiting the lectin pathway for about 7 days. As shown herein, OMS646 is 5-10 times more potent against human MASP-2 than monkey MASP-2. Assuming comparable pharmacokinetics, the range of doses expected to achieve effective lectin pathway cleavage in humans is shown in Table 30 below.

[0393] [Table 30] While the preferred embodiment of the invention has been illustrated and described, it will be understood that various changes can be made therein without departing from the spirit and scope of the invention.

[0394] The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

Claims

[Claim 1] A composition or method as described in the specification.

Images