EA040888B1 - COMPOSITIONS AND METHODS FOR MASP-3 INHIBITION USED TO TREAT VARIOUS DISEASES AND DISORDERS - Google Patents

Description

(57) The invention relates to MASP-3 inhibitory antibodies and compositions containing said antibodies for use in inhibiting the side effects of MASP-3 dependent complement activation.

040888 B1

040888 Bl

Cross-reference to related applications

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

Sequence listing hint

The sequence listing attached to this application is not presented in a paper copy, but in the form of a text file, and therefore it is incorporated into the present description by reference. The text file containing the sequence listing is named MP_l_0254_US_Sequence_Listing_20170628_ST25; this 191 kB file was created on June 28, 2017 and submitted via EFS-Web along with this application submission.

Prior Art

The complement system acts early on as a mechanism to initiate, amplify, and distribute the immune response to microbial infection and other acute infections (M.K. Liszewski and J.P. Atkinson, 1993, in Fundamental Immunology, Third Edition, edited by W.E. Paul, Raven Press, Ltd., New York) in humans and other vertebrates. Although complement activation provides effective first-line protection against potential pathogens, however, complement activation, which stimulates a protective immune response, may also pose a potential threat to the host (K. R. Kalli et al., Springer Semin. Immunopathol. 75:417-431, 1994 ; B.P. Morgan, Eur. J. Clinical Investig. 24:219-228, 1994). For example, proteolysis products C3 and C5 stimulate the recruitment and activation of neutrophils. Although neutrophils are essential for host defense, however, activated neutrophils cannot be identified by their release of destructive enzymes and can cause organ damage. In addition, complement activation can cause the deposition of lytic complement components at sites located in the vicinity of host cells, as well as on microbial targets, leading to host cell lysis.

The complement system is also involved in the pathogenesis of various acute and chronic pathological conditions, including myocardial infarction, stroke, acute respiratory distress syndrome (ARDS), reperfusion injury, septic shock, capillary permeability after heat burns, inflammation after cardiopulmonary bypass surgery, transplant rejection, rheumatoid arthritis, multiple sclerosis, myasthenia gravis and Alzheimer's disease. In almost all of these conditions, complement itself does not cause these conditions, but is one of several factors involved in their pathogenesis. However, complement activation may represent a major pathological mechanism and serve as an effective point of clinical control in many of these pathological conditions. The growing recognition of the importance in understanding the mechanisms of complement-mediated tissue damage in various pathological conditions once again indicates the need to develop effective complement-inhibiting drugs. Currently, eculizumab (Soliris®), i.e. the anti-C5 antibody is the only drug that targets complement and has been approved for medical use. In addition, C5 is one of several effector molecules that act late in the reaction of the complement system, and blockade of C5 does not result in inhibition of the activation of the complement system. Therefore, an inhibitor of the stages of initiation of complement activation would have a significant advantage over an inhibitor of a single component acting at later stages in the cascade of reactions of the complement system.

It is now well known that the complement system can be activated in three different ways: the classical pathway, the lectin pathway, and the alternative pathway. The classical pathway is usually triggered by a complex of host antibodies bound to a foreign particle (i.e., an antigen), and prior stimulation with the antigen is required to elicit a specific humoral response. Since the activation of the classical pathway depends on a pre-adaptive immune response in the host, such a classical pathway is part of the acquired immune system. In contrast, the lectin and alternative pathways are independent of the adaptive immune response and are part of the natural immune system.

Activation of the complement system leads to the subsequent activation of serine-protease zymogens. The first step in classical pathway activation is the binding of a specific recognition molecule, C1q, to antigen-bound IgG and IgM molecules. C1q associates with the serine protease proenzymes C1r and C1s as a complex, designated C1. After binding of C1q to immune complexes, autoproteolytic cleavage of the C1r Arg-Ile site is followed by C1r-mediated cleavage and activation of C1s, giving it the ability to cleave C4 and C2. C4 is cleaved into two fragments, designated C4a and C4b, and, similarly, C2 is cleaved into components C2a and C2b. C4b fragments can form covalent bonds with adjacent hydroxyl groups or amino groups and produce C3 convertase (C4b2a) through non-covalent interaction with the C2a fragment of activated C2. C3 convertase (C4b2a) activates C3

- 1 040888 by proteolytic cleavage into subcomponents C3a and C3b, which leads to the production of C5 convertase (C4b2a3b), which, through cleavage of C5, promotes the formation of a membrane attack complex (C5b together with C6, C7, C8 and C9 are also referred to as MAC), and this complex can disrupt cell membranes and cause cell lysis. Activated forms of C3 and C4 (C3b and C4b) are covalently deposited on the surfaces of foreign targets, which are recognized by complement receptors on a variety of phagocytes.

Regardless of this, the first step in the activation of the complement system via the lectin pathway is also the binding of specific recognition molecules, after which the activation of associated serine-protease proenzymes occurs. However, unlike C1q, which binds to the immune complex, recognition molecules in the lectin pathway constitute 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 J. Lu et al., Biochim. Biophys. Acta 7572:387-400 (2002); Holmskov et al., Annu. Rev. Immunol. 21:547-578 (2003); Teh et al., Immunology 101:225-232 (2000)). See also J. Luet et al., Biochim Biophys Acta 1572:387-400 (2002); Holmskov et al., Annu. Rev Immunol 21:547-578 (2003); Teh et al., Immunology 101:225-232 (2000); Hansen S. et al., J. Immunol. 185 (10): 6096-6104 (2010).

It was first demonstrated by Ikeda and co-workers that MBL, like C1q, can activate the complement system after binding to yeast-mannan-coated erythrocytes in a C4-dependent manner (Ikeda et al., J. Biol. Chem. 262:7451-7454 (1987)) . MBL, a member of the collectin protein family, is a calcium-dependent lectin that binds to carbohydrates at 3- and 4-hydroxy groups oriented in the equatorial plane of the pyranose ring. Thus, the most common ligands for MBL are D-mannose and N-acetyl-D-glucosamine, and carbohydrates that do not meet steric requirements have an undetectable affinity for MBL (Weis W.I. et al., Nature 360:127-134 (1992)). The interaction between MBL and monovalent sugars is very weak, and their dissociation constants are usually in the one digit millimolar range. MBL achieves tight specific binding to glycan ligands through avidity, i.e. through simultaneous interaction with multiple monosaccharide residues located in close proximity to each other (Lee et al., Archiv. Biochem. Biophys.

299:129-136 (1992)). MBL recognizes carbohydrate patterns that commonly surround microorganisms such as bacteria, yeast, parasites, and some viruses. In contrast, MBL does not recognize D-galactose and sialic acid, the penultimate and final sugars that normally surround the mature complex of glycoconjugates present on mammalian plasma and cell surface glycoproteins. This binding specificity is believed to promote recognition of foreign surfaces and contribute to protection against autoactivation. However, MBL does not bind with high affinity to high-mannose glycan precursor clusters on N-linked glycoproteins and glycolipids sequestered in the endoplasmic reticulum and Golgi apparatus of mammalian cells (Maynard et al., J. Biol. Chem. 257:3788- 3794 (1982)). In addition, it has been shown that MBL can bind to DNA and RNA polynucleotides that can be found on necrotic and apoptotic cells (Palaniyar et al., Ann. N.Y. Acad. Sci., 1010:467-470 (2003); Nakamura et al. ., J. Leuk Biol 86:737-748 (2009)). Therefore, damaged cells are potential targets for activation along the lectin pathway through binding to MBL.

Ficolins have a lectin domain that is different from the MBL lectin domain and is called the fibrinogen-like domain. Ficolins bind to sugar residues via a Ca ⁺⁺ -independent mechanism. Three types of ficolins have been identified in humans (L-ficolin, M-ficolin, and Nficolin). Two serum ficolins, L-ficolin and N-ficolin, share a common specificity for N-acetyl-O-glucosamine, however, N-ficolin also binds to N-acetyl-D-galactosamine. Differences in sugar specificity of L-ficolin, H-ficolin, CL-11, and MBL mean that different lectins can be complementary to different glycoconjugates and target different, albeit overlapping, glycoconjugates. This concept is supported by recent reports in the literature indicating that of all known lectins involved in the lectin pathway, only Lficolin specifically binds to lipoteichoic acid, i.e. a cell wall glycoconjugate present on all Gram-positive bacteria (Lynch et al., J. Immunol. 772: 1198-1202 (2004)). In addition, ficolins, in addition to acetylated sugar molecules, can also bind to acetylated amino acids and polypeptides (Thomsen et al., Mol. Immunol. 48(4):369-81 (2011)). Collectins (ie MBL) and ficolins do not show any significant similarity in their amino acid sequences. However, these two groups of proteins have the same domain organization and, like C1q, are involved in the assembly of oligomeric structures, which maximizes the likelihood of their binding at many sites.

Serum MBL concentrations in healthy individuals are highly variable and genetically regulated by polymorphisms/mutations in the promoter and coding regions of the MBL gene. The expression level of MBL, as an acute phase protein, further increases during inflammation. Lficolin is present in serum at concentrations similar to those of MBL. Therefore L

- 2 040888 the ficolin branch of the lectin pathway is possibly comparable in structure to the MBL branch. MBL and ficolins can also act as opsonins, allowing phagocytes to target MBL- and ficolin-surrounding 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)). Such opsonization requires the interaction of these proteins with phagocyte receptors (Kuhlman et al., J. Exp. Med. 169: 1733 (1989), Matsushita et al., J. Biol. Chem. 277:2448-54 (1996)), identity which have not yet been established.

Human MBL stimulates a specific and high affinity interaction through its collagen-like domain with unique C1r/C1s-like serine proteases called MBL-associated serine proteases (MASPs). Three MASPs have been described so far. First, one MASP enzyme was identified and this enzyme was characterized as the enzyme responsible for initiating the complement cascade (i.e., C2 and C4 cleavage reactions) (Matsushita et al., J. Exp. Med. 176(6):1497 -1502 (1992), Ji et al., J. Immunol 750:571-578 (1993)). It was then determined that MASP activity is actually a mixture of two proteases: MASP-1 and MASP-2 (Thiel et al., Nature 556:506-510 (1997)). However, it has been demonstrated that the MBL-MASP-2 complex alone is sufficient for complement activation (Vorup-Jensen et al., J. Immunol. 7(55:20932100 (2000)). C2 and C4 (Ambrus et al., J. Immunol. 170:1374 1382, (2003)). Therefore, MASP-2 is the protease responsible for the activation of C4 and C2 and the generation of the C3 convertase, C4b2a. This mechanism is significantly different from the C1 complex mechanism of the classical pathway, where the coordinated action of two specific serine proteases (C1r and C1s) leads to activation of the complement system.In addition, a third novel protease, MASP-3, has been isolated (Dahl M.R. et al., Immunity 15:127 35, 2001) MASP-1 and MASP-3 are alternative splicing products of the same gene.

MASPs have the same domain organization as the C1r and C1s domain organizations, i.e. enzyme components of complex C1 (Sim et al., Biochem. Soc. Trans. 28:545, (2000)). These domains include the C1r/C1s N-terminal domain/sea urchin VEGF domain/bone morphogenesis protein (CUB) domain; an epidermal growth factor-like domain; second CUB domain; a tandem of complement regulation protein domains and a serine protease domain. As in C1 proteases, MASP-2 activation is mediated by cleavage of the Arg-Ile bond directly at the serine protease domain, resulting in the cleavage of the enzyme into A and B chains linked by disulfide bonds, where these chains are composed of serine protease domains.

MBL can also be associated with an alternatively spliced form of MASP-2 known as 19 kDa MBL-associated protein (MAp19) or small MBL-associated protein (sMAP), which lacks MASP-2 catalytic activity (Stover, J. Immunol. 162 :3481 90 (1999); Takahashi et al., Int Immunol 11:859 863 (1999)). MApl9 contains the first two MASP-2 domains followed by an additional sequence of four unique amino acids. The function of Mar19 is unknown (Degn et al., J Immunol. Methods, 2011). The MASP-1 and MASP-2 genes are located on human chromosomes 3 and 1, respectively (Schwaeble et al., Immunobiology 205:455 466, (2002)).

There is some evidence to suggest that various MBL-MASP complexes and a large fraction of serum MASP do not complex with MBL (Thiel et al., J. Immunol. 165:818 887, (2000)). H- and L-ficolin bind to all MASPs and, like MBL, activate the complement lectin pathway (Dahl et al., Immunity 15:127 35, (2001); Matsushita et al., J. Immunol. 168:3502 3506, (2002)). The lectin and classical pathways form a common C3 convertase (C4b2a), and at this stage, the two pathways converge.

There is a widespread belief that the lectin pathway plays an important role in protecting the uninfected host from infection. Strong evidence for the involvement of MBL in host defense has been obtained from assays performed in patients with reduced serum levels of functional MBL (Kilpatrick, Biochim. Biophys. Acta 1572:401 413, (2002)). These patients are susceptible to recurrent bacterial and fungal infections. Such symptoms usually appear at an early age in a clear time window of susceptibility to infections as maternal antibody titers decrease, but before the development of a humoral response with the formation of a full antibody repertoire. This syndrome is often the result of mutations at several sites in the collagenous portion of the MBL that prevent proper formation of MBL oligomers. However, since MBL can function as an opsonin independent of complement, it is not known to what extent the increased susceptibility to infection is due to decreased complement activation.

In contrast to the classical and lectin pathways, no alternative pathway initiators have been found that provide the recognition functions inherent in C1q and lectins in the other two pathways. It is now well known that the alternative pathway spontaneously undergoes low-level cyclic activation, which can easily be amplified on foreign or other abnormal surfaces (bacteria, yeast, virus-infected cells, or damaged tissue) that do not have their own molecular elements that support the existing spontaneous complement activation. There are four plasma proteins directly involved

- 3 040888 in the activation of an alternative pathway, namely C3, factors B and D, and properdin.

Despite the existence of a wealth of data indicating the involvement of classical and alternative complement pathways in the pathogenesis of noncommunicable diseases in humans, the role of the lectin pathway in such pathogenesis has only just begun to be investigated. Recent studies have shown that activation of the lectin pathway may be responsible for complement activation and associated inflammation in ischemia/reperfusion injury. Collard et al. (2000) reported that cultured endothelial cells subjected to oxidative stress bind to MBL and exhibit C3 precipitation after exposure to human serum (Collard et al., Am. J. Pathol. 156:1549 1556, (2000) ). In addition, treatment of human sera with blocking anti-MBL monoclonal antibodies results in inhibition of MBL binding and complement activation. These data extend to a rat model of myocardial ischemia-reperfusion in which rats treated with blocking antibody against rat MBL had a more pronounced reduction in myocardial injury after coronary artery occlusion than rats treated with a control antibody (Jordan et al., Circulation 104: 1413 1418, (2001)). The molecular mechanism of MBL binding to vascular endothelium after oxidative stress remains unclear, however, recent studies suggest that activation of the lectin pathway after oxidative stress may be mediated by MBL binding to vascular endothelial cytokeratins rather than glycoconjugates (Collard et al., Am. J Pathol 159:1045 1054 (2001)). Other studies have pointed to the role of classical and alternative pathways in the pathogenesis of ischemia/reperfusion injury, and the role of the lectin pathway in the pathogenesis of this disease is still a matter of debate (Riedermann, N.C. et al., Am. J. Pathol. 162:363 367, 2003).

Recent studies have shown that MASP-1 and MASP-3 are required for factor D conversion, i.e. an alternative pathway activation enzyme, from its zymogenic form to an enzymatically active form (see Takahashi M. et al., J. Exp. Med. 207(1):29-37 (2010); Iwaki et al., J. Immunol. 187:3751-58 (2011)). The physiological importance of this process is indicated by the absence of functional activity of the alternative pathway in the plasma of MASP-1/3-deficient mice. The alternative pathway requires proteolytic production of C3b from native C3. Since the C3 convertase of the alternative pathway (C3bBb) contains C3b as the main subunit, the question concerning the origin of the first C3b in the alternative pathway seems to be difficult to resolve and provides an incentive for fundamental research.

C3 belongs to a family of proteins (along with C4 and macroglobulin α-2) that contain a rare post-translational modification known as a thioether bond. The thioether group consists of glutamine, in which the terminal carbonyl group forms a covalent thioether bond with the sulfhydryl group of cysteine without three amino acids. This bond is unstable and the electrophilic glutamyl thioether can react with nucleophilic groups such as hydroxyl groups or amino groups and thereby form a covalent bond with other molecules. The thioether bond is sufficiently stable if it is sequestered in the hydrophobic pocket of intact C3. However, proteolytic cleavage of C3 into C3a and C3b exposes a highly reactive thioether bond at C3b, and after nucleophilic attack by adjacent molecules containing hydroxyl groups or amino groups, C3b becomes covalently bound to the target. In addition to the well documented role of the C3 thioester in the covalent binding of C3b to complement targets, the C3 thioester is also believed to play a major role in triggering the alternative pathway. According to the widely accepted open circuit theory, the alternative pathway is initiated by the production of a liquid phase convertase, iC3Bb, which is formed from C3 by the action of a hydrolyzed thioester (iC3; C3(H ₂ O)) and factor B (Lachmann PJ et al., Springer Semin Immunopathol 7:143 162 (1984)). The C3b-sub6 C3(H ₂ O) is produced from native C3 by slow spontaneous hydrolysis of an internal thioester present in the protein (Pangburn MK et al., J. Exp. Med. 154:856 867, 1981). Owing to the activity of C3 (H ₂ O) Bb-convertase, C3b molecules are deposited on the target surface and thereby initiate an alternative pathway.

Prior to the disclosure of the invention described here, very little was known about initiators of alternative pathway activation. It is believed that yeast cell walls (zymosan), many pure polysaccharides, rabbit erythrocytes, some immunoglobulins, viruses, fungi, bacteria, animal tumor cells, parasites and damaged cells can be activators. The only common feature of these activators is the presence of a carbohydrate, however, due to the complexity and diversity of carbohydrate structures, it is difficult to identify common molecular determinants for recognition. It is widely believed that alternative pathway activation is regulated through a delicate balance between the inhibitory regulatory components of this pathway, such as factor H, factor I, DAF, CR1, and properdin, which is the only positive regulator of the alternative pathway (See Schwaeble W.J. and Reid K.B ., Immunol.Today 20(1):17-21 (1999)).

In addition to the apparently unregulated activation mechanism described above, the alternative pathway may also give rise to an enhanced amplification loop for the C3 convertase (C4b2a) of the lectin/classic pathway, since any C3b produced may be involved, along with factor B, in the formation of additional C3- convertase (C3bcbbb) alternative route. The alternative pathway C3 convertase is stabilized by properdin binding. Properdin promotes a 6-10-fold increase in the half-life of the C3 convertase of the alternative pathway. The addition of C3b to the alternative pathway C3 convertase promotes the formation of the alternative pathway C5 convertase.

All three pathways (i.e. classical, lectin and alternative) are thought to converge at C5 which, when cleaved, produces products with multiple pro-inflammatory effects. The pathway where all three pathways converge has been defined as the terminal complement pathway. C5a is an exceptionally strong anaphylotoxin that induces a change in smooth muscle and vascular tone, as well as vascular permeability. It is also a potent chemotaxin and an activator of neutrophils and monocytes. C5a-mediated cellular activation can significantly enhance inflammatory responses by inducing the release of a variety of additional inflammatory mediators, including cytokines, hydrolytic enzymes, arachidonic acid metabolites, and reactive oxygen molecules. C5 cleavage results in the formation of C5b-9, also known as the membrane attack complex (MAC). There is now strong evidence that the deposition of sublytic MAC, in addition to its role as a lytic pore-forming complex, may play an important role in the inflammatory process.

The complement system, in addition to its important role in immune defense, also contributes to the tissue damage seen in many clinical conditions. Therefore, it is imperative to develop therapeutically effective complement inhibitors that could prevent these side effects.

The essence of the invention

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

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

- 5 040888

In another aspect, the present invention provides an isolated antibody, or antigen-binding fragment thereof, that binds to MASP-3 and contains: (a) a heavy chain variable region comprising HC-CDR1 represented as SEQ ID NO: 213 (SYGXX where X in position 4 is M or I and where X in position 5 is S or T); HC-CDR2 shown as SEQ ID NO: 74; and HC-CDR3 shown as SEQ ID NO: 214 (GGXAXDY where X at position 3 is E or D and where X at position 5 is M or L); and (b) a light chain variable region comprising LC-CDR1 represented as SEQ ID NO: 215 (KSSQSLLDSXXKTYLX, where X at position 10 is D, E, or A; where X at position 11 is G or A; and where X at position 16 is N or S); LC-CDR2 shown as SEQ ID NO: 155; and LC-CDR3 shown as SEQ ID NO: 216 (WQGTHFPXT where X at position 8 is W or Y).

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

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

In another aspect, the present invention provides an isolated antibody, or antigen-binding fragment thereof, that binds to MASP-3 and contains:

(a) a heavy chain variable region comprising HC-CDR1 shown as SEQ ID NO: 109 (RVHFAIRDTNYWMQ), HC-CDR2 shown as SEQ ID NO: 110 (AIYPGNGDTSYNQKFKG), HC-CDR3 shown as SEQ ID NO: 112 (GSHYFDY); and a light chain variable region comprising LC-CDR1, shown as SEQ ID NO: 182 (RASQSIGTSIH), LC-CDR2, shown as SEQ ID NO: 184 (YASESIS), and LC-CDR3, shown as SEQ ID NO: 186 ( QQSNSWPYT); or (b) a heavy chain variable region comprising HC-CDR1 shown as SEQ ID NO: 125 (DYYMN), HC-CDR2 shown as SEQ ID NO: 127 (DVNPNNDGTTYNQKFKG), HCCDR3 shown as SEQ ID NO: 129 ( CPFYYLGKGTHFDY); and a light chain variable region comprising LC-CDR1, shown as SEQ ID NO: 196 (RASQDISNFLN), LC-CDR2, shown as SEQ ID NO: 198 (YTSRLHS), and LC-CDR3, shown as SEQ ID NO: 200 ( QQGFTLPWT); or (c) a heavy chain variable region comprising HC-CDR1, shown as SEQ ID NO: 137, hC-CDR2, shown as SEQ ID NO: 138, HC-CDR3, shown as SEQ ID NO: 140; and a light chain variable region comprising LC-CDR1, shown as SEQ ID NO: 206, LCCDR2, shown as SEQ ID NO: 207, and LC-CDR3, shown as SEQ ID NO: 208; or (d) a heavy chain variable region comprising HC-CDR1, shown as SEQ ID NO: 98, HC-CDR2, shown as SEQ ID NO: 99, HC-CDR3, shown as SEQ ID NO: 101; and a light chain variable region comprising LC-CDR1, shown as SEQ ID NO: 169, LCCDR2, shown as SEQ ID NO: 171, and LC-CDR3, shown as SEQ ID NO: 173; or (e) a heavy chain variable region comprising HC-CDR1, shown as SEQ ID NO: 103, hC-CDR2, shown as SEQ ID NO: 105, HC-CDR3, shown as SEQ ID NO: 107; and a light chain variable region comprising LC-CDR1, shown as SEQ ID NO: 176, LCCDR2, shown as SEQ ID NO: 178, and LC-CDR3, shown as SEQ ID NO: 193: or (f) heavy chain variable region, comprising HC-CDR1, shown as SEQ ID NO: 114, HC-CDR2, shown as SEQ ID NO: 116, HC-CDR3, shown as SEQ ID NO: 118; and a light chain variable region comprising LC-CDR1, shown as SEQ ID NO: 188, LCCDR2, shown as SEQ ID NO: 178, and LC-CDR3, shown as SEQ ID NO: 190; or (g) a heavy chain variable region comprising HC-CDR1, shown as SEQ ID NO: 114, HC-CDR2, shown as SEQ ID NO: 121, HC-CDR3, shown as SEQ ID NO: 123; and a light chain variable region comprising LC-CDR1, shown as SEQ ID NO: 191, LCcDr2, shown as SEQ ID NO: 178, and LC-CDR3, shown as SEQ ID NO: 193.

In another aspect, the present invention relates to a method for inhibiting the activation

- 6 040888 alternative pathway complement in a mammal, wherein said method comprises administering to a mammal in need thereof a composition containing a high affinity MASP-3 inhibitory antibody or antigen-binding fragment thereof in an amount sufficient to inhibit alternative pathway complement activation in the mammal. In one embodiment of this method, the antibody, or antigen-binding fragment thereof, binds to MASP-3 with an affinity of less than 500 pM. In one embodiment of this method, as a result of administration of a composition containing an antibody or antigen-binding fragment, one or more of the following properties are observed in mammals: (a) inhibition of factor D maturation; (b) inhibition of the alternative pathway when administered to an individual in a molar ratio of about 1:1 to about 2.5:1 (MASP-3 target: mAb); (c) no inhibition of the classical pathway; (d) inhibition of hemolysis and/or opsonization; (e) reducing the degree of hemolysis or degree of C3 cleavage and deposition of surface C3b; (f) reducing the deposition of factor B and Bb on the activating surface; (g) reduction of residual levels (in the circulation and without experimental additional surface activation) of active factor D compared to pro-factor D; (h) a decrease in levels of active factor D relative to profactor D in response to surface activation; and/or (i) reducing the level of production of residual and surface-induced levels of liquid phase Ba, Bb, C3b or C3a. In one embodiment of this method, the composition comprises a MASP-3 inhibitory antibody that inhibits the alternative pathway in a molar ratio of about 1:1 to about 2.5:1 (MASP-3 target: mAb).

In another aspect, the present invention relates to a method for inhibiting MASP-3 dependent complement activation in an individual suffering from paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy, asthma, hard deposit disease, oligoimmune necrosing crescentic glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, optic neuromyelitis, or Behçet's disease. The method includes the step of administering to the subject a composition comprising a high affinity MASP-3 inhibitory agent in an amount effective to inhibit MASP-3 dependent complement activation. In some embodiments of the invention, this method also includes administering to the individual a composition containing a MASP2 inhibitory agent.

In another aspect, the present invention relates to a method of preparing a medicinal product for use in inhibiting the effects of MASP-3-dependent complement activation in living individuals in need of it, which method includes combining a therapeutically effective amount of a MASP-3 inhibitory agent in pharmaceutical carrier. In some embodiments, the MASP-3 inhibitory agent is a high affinity MASP-3 inhibitory antibody. In some embodiments, the method of this aspect of the invention comprises formulating a medicament for use in inhibiting the effects of MASP-3-dependent complement activation in an individual suffering from a disease or disorder, or an individual at risk of developing a disease or disorder, selected from the group consisting of from paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy, asthma, hard deposit disease, oligoimmune necrosing crescentic glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis of the optic nerve or Behçet's disease. In some embodiments, the method also includes combining a therapeutically effective amount of a MASP-3 inhibitory agent in or with a drug product containing a MASP-3 inhibitor.

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

In another aspect, the present invention relates to a method of treating an individual suffering from paroxysmal nocturnal hemoglobinuria (PNH), or an individual at risk of developing paroxysmal nocturnal hemoglobinuria, which method includes administering to the individual a pharmaceutical composition containing an effective amount of a high affinity monoclonal antibody or antigen-binding fragment, that bind to human MASP-3 and inhibition

- 7 040888 ruyut complement activation alternative pathway, for the treatment of PNH or reduce the risk of developing PNH in an individual. In one embodiment, the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region comprising (i) VHCDR1 comprising SEQ ID NO: 84, (ii) VHCDR2 comprising SEQ ID NO: 86 or SEQ ID NO : 275, and (iii) VHCDR3 comprising SEQ ID NO: 88; and (b) a light chain variable region comprising (i) VLCDR1 comprising SEQ ID NO: 142, SEQ ID NO: 257, SEQ ID NO: 258, or SEQ ID NO: 259, (ii) VLCDR2 comprising SEQ ID NO: 144, and (iii) VLCDR3 comprising SEQ ID NO: 161. In some embodiments of the invention, the pharmaceutical composition increases the viability of red blood cells in an individual suffering from PNH. In some embodiments, an individual suffering from PNH, or an individual at risk of developing PNH, exhibits one or more symptoms selected from the group consisting of: (i) decrease in hemoglobin to below normal levels, (ii) decrease in platelet count to levels below the norm; (iii) an increase in the number of reticulocytes to levels above normal; and (iv) increasing bilirubin to above normal levels. In some embodiments of the invention, the pharmaceutical composition is administered to an individual suffering from PNH, or an individual at risk of developing PNH, systemically (eg, subcutaneously, intramuscularly, intravenously, intraarterially, or by inhalation). In some embodiments of the invention, the individual suffering from PNH, or an individual at risk of developing PNH, has previously received or is currently undergoing treatment with a terminal complement inhibitor that inhibits the cleavage of complement C5 protein. In some embodiments, the method also includes administering to the individual a terminal complement inhibitor that inhibits the cleavage of complement C5 protein. In some embodiments, the terminal complement inhibitor is a humanized anti-C5 antibody, or an antigen-binding fragment thereof. In some embodiments, the terminal complement inhibitor is eculizumab.

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

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

As described in this application, pharmaceutical compositions according to the invention can be used in accordance with the methods according to the invention.

These and other aspects and embodiments of the invention described herein will be apparent from the following detailed description and description of the drawings. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications cited herein are incorporated herein by reference in their entirety, as if they were all incorporated in the present description separately.

Description of graphic material

The foregoing aspects of the invention and many of the advantages of the invention described herein will be more apparent and better understood by those skilled in the art from the following detailed description, which is accompanied by the accompanying drawings, in which FIG. 1 illustrates the new understanding of the lectin and alternative pathways;

in fig. 2 is a schematic diagram adapted from Schwaeble et al., Immunobiol. 205:455-466 (2002), and modified by Yongqing et al., BBA 1824:253 (2012), showing the domains of the MASP-1, MASP-3 and MAP44 proteins and the exons encoding these domains;

in fig. 3 illustrates the amino acid sequence of human MASP-3 (SEQ ID

- 8 040888

NO: 2) with a leader sequence that is underlined;

in fig. 4 is an alignment of the full-length MASP-3 protein from various species;

in fig. 5 - alignment of the SP domain of the MASP-3 protein derived from various species;

in fig. 6 is a Kaplan-Meier plot illustrating the percent survival of MASP-2 knockout (KO) and wild-type (WT) mice after they were injected with an infectious dose of 2.6 x 107 k.o.u. N. meningitidis serogroup A Z2491, indicating that MASP-2 deficient mice were protected from N. meningitidis-induced death as described in Example 1;

in fig. 7 is a Kaplan-Meier plot illustrating the percentage survival of MASP-2 knockout (KO) mice and wild-type (WT) mice after they were injected with an infectious dose of 6x106 k.a.u. strain N. meningitidis serogroup B MC58, indicating that MASP-2-deficient mice were protected from N. meningitidis-induced death, as described in example 1;

in fig. 8 graphically illustrates log cfu/ml of N. meningitidis strain serogroup B MC58 per ml of blood drawn from MASP-2 knockout (KO) and wild-type (WT) mice at various times after i. r.-infection 6x106 c.o.u. strain of N. meningitidis serogroup B MC58 (n=3 at different times for mice in both groups), indicating that although MASP-2 knockout mice were infected with the same dose of N. meningitidis strain serogroup B MC58 like wild-type mice, however, MASP-2 knockout mice showed enhanced clearance of bacteremia compared to wild-type mice, as described in Example 1;

in fig. 9 graphically illustrates the mean incidence scores of MASP-2 knockout (KO) and wild-type (WT) mice at 3, 6, 12, and 24 hours after 6x106 kb infection. strain of N. meningitidis serogroup B MC58, indicating that in MASP-2-deficient mice, the incidence rate was much lower at 6 hours, 12 hours and 24 hours after infection compared with wild-type mice, as described in the example 1;

in fig. 10 is a Kaplan-Meier plot illustrating the percent survival of mice after they were given an infectious dose of 4x106 k.o.u. strain of N. meningitidis serogroup B MC58 followed by administration 3 hours after infection with either a MASP-2 inhibitory antibody (1 mg/kg) or an isotype control antibody, indicating that the anti-MASP-2 antibody is effective against treatment and increase in life expectancy of individuals infected with N. meningitidis, as described in example 2;

in fig. 11 graphically illustrates the log c.f.u./ml of viable N. meningitidis serogroup B MC58 strain isolated at various times from the human serum samples shown in Table 1. 6 and taken at various times after incubation with N. meningitidis serogroup B MC58 strain as described in Example 3;

in fig. 12 graphically illustrates the log k.o.e./ml of the number of viable N. meningitidis serogroup B MC58 strain isolated at various time periods from the human serum samples presented in table. 8 indicating that the complement dependent lysis of N. meningitidis in human 20% (v/v) serum is MASP-3 and MBL dependent as described in Example 3;

in fig. 13 graphically illustrates the log k.o.e./ml of the number of viable N. meningitidis serogroup B MC58 strain isolated at various time periods from the mouse serum samples presented in table. 10, indicating that sera from MASP-2 knockout (-/-) mice (designated MASP-2/') have a higher level of bactericidal activity against N. meningitidis than sera from wild-type mice, whereas sera MASP-1/3' ^/ '-mice did not have any bactericidal activity, as described in example 3;

in fig. 14 graphically illustrates the kinetics of C3 activation under conditions specific to the lectin pathway (1% plasma) in the serum of wild-type mice, and in C4'/' mice, MASP-l/3' ^/ ' mice, factor B'/'- mice and MASP-2' ^/ '-mice as described in example 4;

in fig. 15 graphically illustrates the level of C3b precipitation induced by the alternative pathway (AP-induced) on zymosan-coated microtiter plates under conventional alternative pathway-specific (AP-specific) conditions (i.e. BBS/EGTA/Mg ⁺⁺ without Ca ⁺⁺ ) depending on the concentration of serum in serum samples taken from a person with a deficiency of MASP-3, C4 and MBL, as described in example 4;

in fig. 16 graphically illustrates the rate of AP-induced C3b precipitation on zymosan-coated microtiter plates under traditional AP-specific conditions (i.e. BBS/EGTA/Mg ⁺⁺ without SC) as a function of time in 10% human serum samples taken from a human with deficiency of MASP-3, C4 and MBL, as described in example 4;

in fig. 17A graphically illustrates the rate of C3b deposition on mannan-coated microtiter plates as a function of serum concentration in serum samples taken from wild-type mice, MASP-2-deficient mice, and MASP-1/3-deficient mice under conventional AP-specific conditions (i.e. .BBS/EGTA/Mg ⁺⁺ without SJ) or under physiological conditions favorable for lectin and alternative pathway (AP) function (BBS/Mg++/Ca ⁺⁺ ) as described in

- 9 040888 example 4;

in fig. 17B graphically illustrates the level of C3b deposition on zymosan-coated microtiter plates as a function of serum concentration in serum samples from wild-type mice, MASP-2-deficient mice, and MASP-1/3-deficient mice under conventional AP-specific conditions (i.e. .BBS/EGTA/Mg ⁺⁺ without QQ) or under physiological conditions favorable for the function of the lectin pathway and alternative pathway (AP) (BBS/Mg++/Ca ⁺⁺ ) as described in example 4;

in fig. 17C graphically illustrates the rate of C3b deposition on microtiter plates coated with S. pneumoniae D39 as a function of serum concentration in serum samples taken from wild-type mice, MASP-2-deficient mice, and MASP-1/3-deficient mice in conventional AP. -specific conditions (ie BBS/EGTA/Mg ⁺⁺ without QQ) or physiological conditions favorable for the function of the lectin pathway and alternative pathway (AP) (BBS/Mg++/Ca ⁺⁺ ), as described in example 4;

in fig. 18A graphically illustrates the results of a high dilution serum C3b precipitation assay performed on mannan-coated microtiter plates under conventional AP-specific conditions (i.e., BBS/EGTA/Mg++ without QQ) or under physiological conditions favorable for lectin function. pathway and alternative pathway (AP) (BBS/Mg++/Ca ⁺⁺ ), using serum at concentrations from 0 to 1.25%, as described in example 4;

in fig. 18B graphically illustrates the results of a C3b precipitation assay performed on zymosan-coated microtiter plates under conventional AP-specific conditions (i.e., BBS/EGTA/Mg++ without QQ) or under physiological conditions favorable for lectin and alternative pathway function ( BBS/Mg ⁺⁺ /Ca++), using serum at concentrations from 0% to 1.25%, as described in example 4;

in fig. 18C graphically illustrates the results of a C3b precipitation assay performed on microtiter plates coated with S. pneumoniae D39 under traditional AP-specific conditions (i.e. BBS/EGTA/Mg++ without QQ) or under physiological conditions favorable for lectin and alternative pathway function. (BBS/Mg++/Ca ⁺⁺ ), using serum concentrations from 0 to 1.25%, as described in example 4;

in fig. 19 graphically illustrates the level of hemolysis (measured by the release of hemoglobin from lysed mouse erythrocytes (Crry/C3-/-) into the supernatant by photometry) of mannan-coated mouse erythrocytes under physiological conditions (i.e., in the presence of Ca ⁺⁺ ) in dilutions of human MASP- 3' ^/ '-serum, heat-inactivated normal human serum (HI NHS), MBL'/'-serum, NHS+anti-MASP-2 monoclonal antibody and NHS control as described in example 5;

in fig. 20 graphically illustrates the level of hemolysis (measured by the release of hemoglobin by lysed mouse erythrocytes (Crry/C3-/-) into the supernatant by photometry) of mannan-coated mouse erythrocytes under physiological conditions (i.e., in the presence of Ca ⁺⁺ ) in dilutions of human MASP-3 ' ^/ '-serum, heat-inactivated serum (HI NHS), MBL'/'serum, NHS+monoclonal anti-MASP-2 antibody and NHS control as described in example 5;

in fig. 21 graphically illustrates the level of hemolysis (measured by the release of hemoglobin by lysed wild-type mouse erythrocytes into the supernatant by photometry) of uncoated mouse erythrocytes under physiological conditions (i.e., in the presence of Ca++) in dilutions of human 3MC (MASP-3'/'- serum), heat-inactivated serum (HI) NHS, MBL'/'serum, NHS+monoclonal anti-MASP-2 antibody and NHS control as described in example 5;

in fig. 22 graphically illustrates the hemolysis (measured by the release of hemoglobin by lysed mouse erythrocytes (CD55/59-/-) into the supernatant by photometry) of uncoated mouse erythrocytes under physiological conditions (i.e., in the presence of QQ) in dilutions of human heat-inactivated serum (HI NHS) , MBL'/'-serum, NHS+monoclonal anti-MASP-2 antibody and NHS control as described in example 5;

in fig. 23 graphically illustrates the hemolysis (measured by the release of hemoglobin by lysed rabbit erythrocytes into the supernatant by photometry) of mannan-coated rabbit erythrocytes in mouse MASP-3'/'-serum and in the serum of wild-type control mice under physiological conditions (i.e., in the presence of QQ). at the serum concentrations described in Example 6;

in fig. 24A is a FACS histogram of MASP-3 antigen/antibody binding for clone M3J5 as described in Example 7;

in fig. 24B is a FACS histogram of MASP-3 antigen/antibody binding for clone M3M1 as described in Example 7;

in fig. 25 graphically illustrates the saturated binding curve of clone M3J5 (clone 5) to the MASP-3 antigen as described in Example 7;

in fig. 26A shows the amino acid sequence alignment of the M3J5, M3M1, D14, and 1E10 VH regions and the chicken DT40 VH sequence, where the dots indicate amino acids identical to the amino acids of the DT40 sequence, and the dotted lines indicate the regions introduced for

- 10 040888 maximizing the alignment as described in example 7;

in fig. 26B - amino acid sequence alignment of VL regions M3J5, M3M1, D14 and

1E10 and the VL sequence of chicken DT40, where the dots indicate amino acids identical to the amino acids of the DT40 sequence, and the dotted line shows the regions introduced to maximize the alignment, as described in example 7;

in fig. 27 is a bar graph illustrating the inhibitory activity of the mAb 1E10 in the Wieslab Complement System screening for the MBL pathway compared to the positive sera present in the assay kit, and also illustrating the inhibitory activity of the isotype control antibody, indicating that mAb1E10 partially inhibits LEA-2-dependent activation (by inhibiting MASP-1-dependent activation of MASP-2), while the isotype control antibody does not have such activity as described in example 7;

in fig. 28A is a flow cytometric analysis of C3b precipitation in heat-inactivated Staphylococcus aureus, indicating that no C3b precipitation was observed in normal human serum in the presence of EDTA, which is known to inactivate lectin and alternative pathways (panel 1); in normal human serum treated with Mg++/EGTA, C3b precipitation induced by an alternative pathway was observed (panel 2), and as shown in panels 3, 4 and 5, in factor B, factor D and properdin (factor P) deficient sera , respectively, the deposition of C3b, induced by an alternative way, was not observed, as described in example 8;

in fig. 28B - Flow cytometric analysis of C3b precipitation in heat-inactivated S. aureus, indicating that, as in EDTA-treated normal serum (panel 1), in 3MC serum, no AP-induced C3b precipitation was observed in the presence of Mg++/EGTA. (panel 3), and panels 4 and 5 show that active full-length rMASP-3 (panel 4) and active rMASP-3 (CCP1-CCP2-SP) (panel 5) maintained AP-induced C3b serum levels at 3MS, at levels seen in normal serum treated with Mg ⁺⁺ /EGTA (panel 2); however, inactive rMASP-3 (S679A) (panel 6) and wild-type rMASP-1 (panel 7) could not maintain the level of AP-induced C3b serum deposition in 3MC as described in example 8;

in fig. 29 - results of Western blot analysis to determine factor B cleavage in response to S. aureus in 3MC serum in the presence or absence of rMASP-3, indicating that normal human serum in the presence of EDTA (negative control, lane 1) shows very low factor B degradation compared to normal human serum in the presence of Mg++/EGTA as shown in lane 2 (positive control) and lane 3; and 3MC serum shows very low factor B degradation in the presence of Mg++/EGTA. However, as shown in lane 4, factor B cleavage is maintained by adding and pre-incubating full-length recombinant MASP-3 protein with 3MC serum as described in Example 8;

in fig. 30 shows Coomassie staining of a protein gel where factor B cleavage is assayed and this analysis showed that factor B cleavage is most optimal in the presence of C3, MASP-3 and pro factor D (lane 1) and as shown in lanes 4 and 5, MASP-3 or pro-factor D alone are able to mediate factor B cleavage provided that C3 is present as described in Example 8;

in fig. 31 graphically illustrates the mean fluorescence intensities (MFI) of S. aureus C3b staining derived from mAbD14 (which binds to MASP-3), mAb1A5 (negative control antibody), and isotype control antibody as a function of serum mAb concentration 3MC in the presence of rMASP-3 , indicating that mAbD14 inhibits MASP-3 dependent C3b precipitation in a concentration dependent manner as described in Example 8;

in fig. 32 illustrates a Western blot analysis for pro factor D substrate cleavage where compared to pro factor D alone (lane 1) or to inactive full-length recombinant MASP-3 (S679A; lane 3) or MASP-1 (S646A) ; lane 4), wild-type full-length recombinant MASP-3 (lane 2) and MASP-1 (lane 5) completely or partially cleave pro-factor D to form mature factor D as described in example 9;

in fig. 33 is a Western blot analysis illustrating the inhibitory activity of MASP3-binding mAb D14 (lane 2) and M3M1 (lane 3) on MASP-3-dependent pro-factor D cleavage compared to a control reaction mixture containing only MASP-3 and pro-factor D (no mAb, lane 1), as well as a control reaction mixture containing mAb isolated from the DTLacO library and binding to MASP-1 but not to MASP-3 (lane 4), as described in example 9 ;

in fig. 34 graphically illustrates the rate of AP-induced C3b precipitation on zymosan-coated microtiter plates as a function of serum concentration in serum samples from MASP-3 (3MC), C4, and MBL deficient individuals, indicating that MASP-3 is deficient. serum taken from patient 2 and patient 3 has residual AP activity at high serum concentrations (25, 12.5, 6.25% of serum concentration), and a significantly higher AP ₅₀ (i.e. for 8.2 and 12.3% serum required to achieve 50% of maximum C3 precipitation) as described in Example 10;

in fig. 35A graphically illustrates the rate of AP-induced C3b precipitation on zymosan-coated microtiter plates under conventional AP-specific conditions (i.e., BBS/EGTA/Mg ⁺⁺ without Ca++) versus time in 10% human serum samples taken from human with deficiency of MASP-3, C4 and MBL, as described in example 10;

in fig. 35B illustrates Western blot analysis of plasma taken from 3MS patient #2 (MASP-3 (-/-), MASP-1 (+/+)), 3MS patient #3 (MASP-3 (-/-) , MASP-1 (-/-)), and serum taken from normal donors (W), where human pro-factor D (25040 Da) and/or mouse factor D (24405 Da) were detected using an antibody specific for human factor D, as described in example 10;

in fig. 35C graphically illustrates the results of Weislab classical, lectin and alternative pathway assays in plasma from 3MS patient #2, 3MS patient #3, and normal human serum as described in Example 10;

in fig. 36 graphically illustrates the percent hemolysis (measured by the release of hemoglobin by lysed rabbit erythrocytes into the supernatant by photometry) of mannan-coated rabbit erythrocytes at certain concentrations of serum taken from two healthy individuals (NHS) and two 3MC patients (patient 2 and patient 3), in the absence of SLA, indicating that MASP-3 deficiency leads to a decrease in the percentage of complement-mediated lysis of mannan-coated erythrocytes compared to normal human serum, as described in example 10;

in fig. 37 graphically illustrates the level of AP-induced C3b precipitation on zymosan-coated microtiter plates as a function of the concentration of recombinant full-length MASP-3 protein added to a serum sample from 3MC patient 2 (MASP-3'/'), indicating that that, compared to the inactive recombinant MASP-3 (MASP-3A; S679A) used as a negative control, the active recombinant MASP-3 protein restored AP-induced C3b precipitation on zymosan-coated plates in a concentration dependent manner as described in Example 10;

in fig. 38 graphically illustrates the percent hemolysis (measured by the release of hemoglobin from lysed rabbit erythrocytes into the supernatant by photometry) of mannan-coated rabbit erythrocytes at specific serum concentrations: (1) in normal human serum (NHS); (2) in the serum of a 3MS patient; (3) in the serum of a 3MC patient containing active full-length recombinant MASP-3 (20 μg/ml); and (4) in heat-inactivated human serum (HIS) in the absence of SLAs, indicating that the percentage of lysis of rabbit erythrocytes is significantly increased in 3MC serum containing rMASP-3 compared to the percentage of lysis in 3MC serum not containing recombinant MASP- 3 (p=0.0006) as described in example 10;

in fig. 39 graphically illustrates the percentage of lysis of rabbit erythrocytes in 7% human serum taken from 3MC patient 2 and 3MC patient 3 and containing active recombinant MASP-3 at a concentration of 0 to 110 μg/ml (in BBS/Mg++/EGTA), which indicates that the percentage of lysis of rabbit erythrocytes increases as the amount of recombinant MASP-3 increases depending on the concentration, as described in example 10;

in fig. 40 graphically illustrates the level of LEA-2-induced C3b precipitation on mannan-coated ELISA plates as a function of concentration of human serum diluted in BBS buffer from a healthy (NHS) human; in two MMS patients (patient 2 and patient 3); in the parents of Patient 3 and in an MBL-deficient individual as described in Example 10;

in fig. 41 graphically illustrates a representative example of a binding experiment that was carried out using human MASP-3, where Fab M3-1 (also referred to as 13B1) bound to a human protein with an apparent binding affinity (EC ₅₀ ) of approximately 0.117 nM as described in Example 11 ;

in fig. 42 graphically illustrates a representative example of a binding experiment that was carried out using mouse MASP-3, where Fab M3-1 (also referred to as 13B1) bound to a mouse protein with an apparent binding affinity (EC ₅₀ ) of approximately 0.214 nM as described in Example 11 ;

in fig. 43 graphically illustrates the level of deposition of complement factor Bb on zymosan particles (determined by cytometric detection in MFI units) in the presence of various concentrations of mAb M3-1 (also referred to as 13B1) in CFD-deficient human serum as described in Example 11;

in fig. 44 graphically illustrates the level of C3 deposition on zymosan particles at various time points after administration of a single dose of mAb M3-1 (13B1) (10 mg/kg i.v.) to wild-type mice as described in Example 11;

in fig. 45 graphically illustrates the percentage of surviving donor RBCs (wild type or Crry-) at 14 days in recipient mice of wild type treated with mAb M3-1 (13B1) (10 mg/kg on days -11, 04, -1 and +6 ), in mice treated with mAb BB5.1 or mice treated with vehicle as described in Example 12;

- 12 040888 in FIG. 46 graphically illustrates the percentage of surviving donor RBCs (wild-type or

Crry-) for 16 days in wild-type recipient mice treated with a single dose of mAb M3-1 (13B1) (20 mg/kg on day -6) or mice treated with vehicle as described in Example 12;

in fig. 47 graphically illustrates clinical scores for mice treated with mAb M3-1 (13B1) (5 or 20 mg/kg) or mice treated with vehicle for 14 days in an anti-collagen antibody induced arthritis model as described in Example 13;

in fig. 48 graphically illustrates the percentage incidence of arthritis in mice treated with mAb M3-1 (13B1) (5 or 20 mg/kg) or in mice treated with vehicle for 14 days in an anti-collagen antibody induced arthritis model as described in Example 13 ;

in fig. 49A shows the amino acid sequences of the VH regions of the high affinity mAbs inhibiting human MASP-3 (<500 pM) as described in Example 15;

in fig. 49B - Amino acid sequences of the VL regions of high affinity human MASP-3 inhibitory mAbs (<500 pM) as described in Example 15;

in fig. 50A is a dendrogram of the VH regions of high affinity human MASP-3 inhibitory mAbs as described in Example 15;

in fig. 50B is a dendrogram of the VL regions of high affinity human MASP-3 inhibitory mAbs as described in Example 15;

in fig. 51A graphically illustrates the results of a binding experiment where representative purified recombinant human MASP-3 inhibitory antibodies bind to human MASP-3 protein with an apparent binding avidity of less than 500 pM (e.g., 240 to 23 pM) as described in the example. 16;

in fig. 51B graphically illustrates the results of a binding experiment where representative purified recombinant human MASP-3 inhibitory antibodies bind to human MASP-3 protein with an apparent binding avidity of less than 500 pM (e.g., 91 to 58 pM) as described in the example. 16;

in fig. 51C graphically illustrates the results of a binding experiment where representative purified recombinant high affinity human MASP-3 inhibitory antibodies selectively bind to MASP-3 but do not bind to human MASP-1 as described in Example 16;

in fig. 51D graphically illustrates the results of a binding experiment where representative purified recombinant high affinity human MASP-3 inhibitory antibodies selectively bind to MASP-3 but do not bind to human MASP-2 as described in Example 16;

in fig. 52 graphically illustrates the results of a binding experiment where representative purified recombinant human MASP-3 inhibitory antibodies also show high binding avidity to mouse MASP-3 protein as described in Example 16;

in fig. 53 graphically illustrates the results of an experiment evaluating the ability of representative high affinity anti-MASP-3 antibodies to inhibit fluorogenic tripeptide cleavage as described in Example 16;

in fig. 54 illustrates Western blot analysis indicating the ability of representative high affinity MASP-3 inhibitory mAbs to block recombinant MASP-3 mediated pro factor D cleavage to factor D in an in vitro assay as described in Example 16;

in fig. 55A is a graphical illustration of the level of complement factor Bb deposition on zymosan particles (determined by flow cytometric detection in MFI units) in the presence of various concentrations of high affinity anti-MASP-3 mAbs 1F3, 1G4, 2D7 and 4B6 in factor D deficient human serum as described in example 16;

in fig. 55B graphically illustrates the level of complement factor Bb precipitation on zymosan particles (determined by flow cytometric detection in MFI units) in the presence of various concentrations of high affinity anti-MASP-3 mAbs 4D5, 10D12 and 13B1 in factor D deficient human serum as described in Example 16 ;

in fig. 56A graphically illustrates the level of inhibition of rabbit erythrocyte lysis in the presence of various concentrations of high affinity anti-MASP-3 mAbs 1A10, 1F3, 4B6, 4D5 and 2F2 as described in Example 16;

in fig. 56B graphically illustrates the level of inhibition of rabbit erythrocyte lysis in the presence of various concentrations of high affinity anti-MASP-3 mAbs 1B11, 1E7, 1G4, 2D7 and 2F5 as described in Example 16;

in fig. 57 illustrates Western blot analysis for determining the level of pro-factor D and factor D in the serum of a 3MC patient (patient B) in the presence of active recombinant MASP-3 (rMASP-3), inactive rMASP-3 and active rMASP-3 plus high affinity anti-MASP-3 mAb 4D5 as described in Example 16;

in fig. 58 graphically illustrates the rate of C3/C3b/iC3b deposition on zymosan particles at various time points following a single dose of high affinity anti-MASP-3 mAb M3-1.

- 13 040888 (13B1, 10 mg/kg) or 10D12 (10 mg/kg) in wild-type mice as described in example 17;

in fig. 59 illustrates Western blot analysis to determine factor Ba fragment status.

In mice treated with high affinity anti-MASP-3 mAb 10D12 (10 mg/kg) or mice treated with vehicle control as described in Example 17;

in fig. 60 graphically illustrates the level of inhibition of hemolysis by 20% serum from mice treated with high affinity anti-MASP-3 mAb 10D12 (10 mg/kg or 25 mg/kg) as described in Example 17;

in fig. 61A graphically illustrates the results of a competitive binding assay to identify high affinity anti-MASP-3 mAbs that block the interaction between the high affinity anti-MASP-3 mAb 4D5 and human MASP-3 as described in Example 18;

in fig. 61B graphically illustrates the results of a competitive binding assay to identify high affinity anti-MASP-3 mAbs that block the interaction between the high affinity anti-MASP-3 mAb 10D12 and human MASP-3 as described in Example 18;

in fig. 61C graphically illustrates the results of a competitive binding assay to identify high affinity anti-MASP-3 mAbs that block the interaction between the high affinity anti-MASP-3 mAb 13B1 and human MASP-3 as described in Example 18;

in fig. 61D graphically illustrates the results of a competitive binding assay to identify high affinity anti-MASP-3 mAbs that block the interaction between the high affinity anti-MASP-3 mAb 1F3 and human MASP-3 as described in Example 18;

in fig. 61E graphically illustrates the results of a competitive binding assay to identify high affinity anti-MASP-3 mAbs that block the interaction between the high affinity anti-MASP-3 mAb 1G4 and human MASP-3 as described in Example 18;

in fig. 62 is a schematic diagram illustrating the contact areas of human MASP-3 with high affinity anti-MASP-3 mAbs determined using the Pepscan assay as described in Example 18;

in fig. 63A shows the contact regions of human MASP-3 with high affinity anti-MASP-3 mAbs 1F3, 4D5 and 1A10, including amino acid residues 498-509 (SEQ ID NO: 9), amino acid residues 544-558 (SEQ ID NO: 11), amino acid residues 639-649 (SEQ ID NO: 13) and amino acid residues 704-713 (SEQ ID NO: 14) of MASP-3 as described in Example 18;

in fig. 63B shows contact regions of human MASP-3 with high affinity anti-MASP-3 mAb 10D12 comprising amino acid residues 498-509 (SEQ ID NO: 9) of MASP-3 as described in Example 18;

in fig. 64 - contact areas of human MASP-3 with high affinity anti-MASP-3 mAb 13B1, including amino acid residues 494-508 (SEQ ID NO: 10) and amino acid residues 626-638 (SEQ ID NO: 12) of MASP-3, as described in example 18;

in fig. 65 - contact areas of human MASP-3 with high affinity anti-MASP-3 mAb 1B11, including amino acid residues 435-447 (SEQ ID NO: 16), amino acid residues 454-464 (SEQ ID NO: 17), amino acid residues 583-589 (SEQ ID NO: 21) and amino acid residues 614-623 (SEQ ID NO: 22) of MASP-3 as described in Example 18;

in fig. 66 - regions of contact of human MASP-3 with high affinity anti-MASP-3 mAbs 1E7, 1G4 and 2D7, including amino acid residues 454-464 (SEQ ID NO: 17), amino acid residues 514-523 (SEQ ID NO: 19) and amino acids residues 667-678 (SEQ ID NO: 23) of MASP-3 as described in Example 18;

in fig. 67 - contact areas of human MASP-3 with high affinity anti-MASP-3 mAbs 15D9 and 2F5, including amino acid residues 454-464 (SEQ ID NO: 17), amino acid residues 479-493 (SEQ ID NO: 18), amino acid residues 562 -571 (SEQ ID NO: 20) and amino acid residues 667-678 (SEQ ID NO: 23) of MASP-3 as described in Example 18;

in fig. 68 graphically illustrates the results of an assay in a mouse experimental autoimmune encephalomyelitis (EAE) model treated with high-affinity MASP-3 inhibitory mAb 13B1 (10 mg/kg), anti-factor B 1379 mAb (30 mg/kg), or isotype control mAb (10 mg/kg). ) as described in example 20;

in fig. 69 graphically illustrates APC activity as determined by mean MFI in a flow cytometric assay for detection of factor Bb complement on the surface of zymosan particles in serum samples taken from a group of three cynomolgus monkeys over a period of time after treatment with the high affinity anti-MASP-3 mAb h13B1X. in the absence of anti-factor D antibody, or in the presence of anti-factor D antibody introduced into the serum sample, as described in example 21;

in fig. 70 graphically illustrates APC activity, as determined by Bb precipitation on zymosan, in serum samples from a group of cynomolgus monkeys (3 animals per group) treated with a single 5 mg/kg intravenous dose of the high affinity MASP-3 inhibitory mAbs h4D5X, h10D12X, or h13B1X. , as described in example 21;

in fig. 71A is a graphical illustration of APC activity as determined by precipitation of liquid-phase Ba in serum samples from a group of cynomolgus monkeys (3 animals per group) after treatment with a single 5 mg/kg intravenous dose of mAb h4D5X, h10D12X, and h13B1X as described. in example 21;

in fig. 71B graphically illustrates APC activity as determined by precipitation of liquid phase Bb in serum samples taken from groups of cynomolgus monkeys (3 animals per group) over a period of time after treatment with a single intravenous dose of 5 mg/kg mAb h4D5X, h10D12X, and h13B1X, as described in example 21;

in fig. 71C is a graphical illustration of APC activity as determined by precipitation of liquid phase C3a in serum samples taken from groups of cynomolgus monkeys (3 animals per group) over a period of time following treatment with a single 5 mg/kg intravenous dose of mAb h4D5X, h10D12X and h13B1X as described. in example 21;

in fig. 72A graphically illustrates the molar ratio of target (MASP-3) to high affinity MASP-3 inhibitory antibody h4D5X at specific time points after completion of APC inhibition as determined using liquid phase Ba and as described in Example 21;

in fig. 72B graphically illustrates the molar ratio of target (MASP-3) to high affinity MASP-3 inhibitory antibody h10D12X at specific time points after completion of APC inhibition as determined using liquid phase Ba and as described in Example 21;

in fig. 72C graphically illustrates the molar ratio of target (MASP-3) to high affinity MASP-3 inhibitory antibody h13B1X at specific time points after completion of APC inhibition as determined using liquid phase Ba and as described in Example 21;

in fig. 73 illustrates a Western blot analysis to determine the level of pro-factor D and factor D in serum taken from a cynomolgus monkey over a period of time (hours) after treatment with a single intravenous dose of 5 mg/kg mAb h13B1X, as described in example 21.

Sequence list

SEQ ID NO: 1: human MASP-3 cDNA;

SEQ ID NO: 2: human MASP-3 protein (with leader sequence);

SEQ ID NO: 3: mouse MASP-3 protein (with leader sequence);

SEQ ID NO: 4: rat MASP-3 protein;

SEQ ID NO: 5: MASP-3 chicken protein;

SEQ ID NO: 6: rabbit MASP-3 protein;

SEQ ID NO: 7: cynomolgus MASP-3 protein;

SEQ ID NO: 8: human MASP-1 protein (with leader sequence).

Fragments of the human MASP-3 SP peptide domain:

SEQ ID NO: 9 (a.k. 498-509 human MASP-3 leader sequence);

SEQ ID NO: 10 (a.k. 494-508 human MASP-3 leader sequence);

SEQ ID NO: 11 (a.k. 544-558 human MASP-3 leader sequence);

SEQ ID NO: 12 (a.k. 626-638 human MASP-3 leader sequence);

SEQ ID NO: 13 (a.k. 639-649 human MASP-3 leader sequence);

SEQ ID NO: 14 (a.k. 704-713 human MASP-3 leader sequence);

SEQ ID NO: 15 (a.k. 498-508 human MASP-3 leader sequence);

SEQ ID NO: 16 (a.k. 435-447 human MASP-3 leader sequence);

SEQ ID NO: 17 (a.k. 454-464 human MASP-3 leader sequence);

SEQ ID NO: 18 (a.k. 479-493 human MASP-3 leader sequence);

SEQ ID NO: 19 (a.k. 514-523 human MASP-3 leader sequence);

SEQ ID NO: 20 (a.k. 562-571 human MASP-3 leader sequence);

SEQ ID NO: 21 (a.k. 583-589 human MASP-3 leader sequence);

SEQ ID NO: 22 (a.k. 614-623 human MASP-3 leader sequence);

SEQ ID NO: 23 (a.k. 667-678 human MASP-3 leader sequence);

SEQ ID NOs: 24-39: parental mouse heavy chain variable regions:

SEQ ID NO: 24 4D5_VH;

SEQ ID NO: 25 1F3_VH;

SEQ ID NO: 26 4B6_VH;

SEQ ID NO: 27 1A10_VH;

SEQ ID NO: 28 10D12_VH;

SEQ ID NO: 29 35C1_VH;

SEQ ID NO: 30 13B1_VH;

SEQ ID NO: 31 1G4_VH;

SEQ ID NO: 32 1E7_VH;

SEQ ID NO: 33 2D7_VH;

SEQ ID NO: 34 49C11_VH;

SEQ ID NO: 35 15D9_VH;

- 15 040888

SEQ ID NO: 36 2F5_VH;

SEQ ID NO: 37 1B11_VH;

SEQ ID NO: 38 2F2_VH;

SEQ ID NO: 39 11B6_VH;

SEQ ID NOs: 40-54: parental mouse light chain variable regions:

SEQ ID NO: 40 4D5_VL;

SEQ ID NO: 41 1F3_VL;

SEQ ID NO: 42 4B6/1A10_VL;

SEQ ID NO: 43 10D12_VL;

SEQ ID NO: 44 35C1_VL;

SEQ ID NO: 45 13B1_VL;

SEQ ID NO: 46 1G4_VL;

SEQ ID NO: 47 1E7_VL;

SEQ ID NO: 48 2D7_VL;

SEQ ID NO: 49 49C11_VL;

SEQ ID NO: 50 15D9_VL;

SEQ ID NO: 51 2F5_VL;

SEQ ID NO: 52 1B11_VL;

SEQ ID NO: 53 2F2_VL;

SEQ ID NO: 54 11B6_VL;

SEQ ID NOs: 55-140: heavy chain framework regions (FR) and hypervariable regions (CDRs) of mouse parental anti-MASP-3 mAb;

SEQ ID NOs: 141-208: Light chain FR and CDR of mouse parental anti-MASP-3 mAb;

SEQ ID NOs: 209-216: CDR consensus sequences;

SEQ ID NO: 217-232: DNA encoding heavy chain variable regions (mouse parent antibody);

SEQ ID NO: 233-247: DNA encoding light chain variable regions (mouse parent antibody);

SEQ ID NO: 248: humanized 4D5_VH-14 heavy chain variable region (h4D5_VH14);

SEQ ID NO: 249: humanized 4D5_VH-19 heavy chain variable region (h4D5_VH19);

SEQ ID NO: 250: humanized 4D5_VL-1 light chain variable region (h4D5_VL-1);

SEQ ID NO: 251: humanized 10D12_VH-45 heavy chain variable region (h10D12_VH45);

SEQ ID NO: 252: humanized 10D12_VH-4 heavy chain variable region 9 (h10D12_VH-49);

SEQ ID NO: 253: light chain variable region of humanized 10D12_VL-21 (h10D12-VL21);

SEQ ID NO: 254: humanized 13B1_VH-9 heavy chain variable region (h13B1-VH9);

SEQ ID NO: 255: humanized 13B1_VH-10 heavy chain variable region (h13B1-VH10);

SEQ ID NO: 256: humanized 13B1-VL-1 light chain variable region (h13B1-VL-1);

SEQ ID NO: 257: 4D5 and 13B1 LC-CDR1-NQ;

SEQ ID NO: 258: 4D5 and 13B1 LC-CDR1-NA;

SEQ ID NO: 259: 4D5 and 13B1 LC-CDR1-ST;

SEQ ID NO: 260: LC-CDR1 consensus for parental antibodies 4D5, 13B1 and variants thereof;

SEQ ID NO: 261: 10D12 LC-CDR1-DE;

SEQ ID NO: 262: 10D12 LC-CDR1-DA;

SEQ ID NO: 263: 10D12 LC-CDR1-GA;

SEQ ID NO: 264-277: HC FR and CDR for humanized 4D5, 10D12 and 13B1;

SEQ ID NO: 278: h4D5_VL-1-NA;

SEQ ID NO: 279: h10D12_VL-21-GA;

SEQ ID NO: 280: h13B1_VL-1-NA;

SEQ ID NO: 281-287 LC FR and CDR for humanized 4D5, 10D12 and 13B1;

SEQ ID NOs: 288-293: DNA encoding humanized 4D5, 10D12, 13B1 heavy chain variable region and variants thereof;

SEQ ID NOs: 294-299: DNA encoding humanized 4D5, 10D12, 13B1 light chain variable region and variants thereof;

SEQ ID NO: 300: parental DTLacO heavy chain variable region (VH) polypeptide;

SEQ ID NO: 301: MASP-3-specific heavy chain variable region (VH) polypeptide

- 16 040888 clone M3J5;

SEQ ID NO: 302: heavy chain variable region (VH) polypeptide of MASP-3-specific clone M3M1;

SEQ ID NO: 303: parent DTLacO variable light chain (VL) polypeptide;

SEQ ID NO: 304: MASP-3-specific clone M3J5 light chain variable region (VL) polypeptide;

SEQ ID NO: 305: MASP-3-specific clone M3M1 light chain variable region (VL) polypeptide;

SEQ ID NO: 306: heavy chain variable region (VH) polypeptide of MASP-3-specific clone D14;

SEQ ID NO: 307: light chain variable region (VL) polypeptide of MASP-3-specific clone D14;

SEQ ID NO: 308: heavy chain variable region (VH) polypeptide of MASP-1-specific clone 1E10;

SEQ ID NO: 309: light chain variable region (VL) polypeptide of MASP-1-specific clone 1E10;

SEQ ID NO: 310: human IgG4 constant region;

SEQ ID NO: 311: human IgG4 constant region with S228P mutation;

SEQ ID NO: 312: human IgG4 constant region with mutation_X S228P;

SEQ ID NO: 313: human IgK constant region.

Detailed description of the invention

I. Definitions.

Unless otherwise stated, all terms used herein have their generally accepted meanings as understood by one of ordinary skill in the art. The following definitions are provided for a better understanding of the terms used in the description and in the claims.

The term lectin pathway 1 effector chain (LEA-1) as used herein refers to the lectin-dependent activation of factor B and factor D by MASP-3.

As used herein, the term lectin pathway effector chain 2 (LEA-2) means MASP-2 dependent complement activation.

As used herein, the term MASP-3-dependent complement activation includes two components: (i) MASP-3-dependent lectin pathway activation for factor B and factor D, which encompasses LEA-1-mediated complement activation in the presence of Ca~, which typically results in conversion of C3bB to C3bBbC3bBb and pro-factor D to factor D; and (ii) lectin-independent conversion of factor B and factor D, which can occur in the absence of Ca++ and typically results in the conversion of C3bB to C3bBbC3bBb and pro-factor D to factor D. LEA-1-mediated complement activation has been determined to be and lectin-independent conversion of factor B and factor D cause opsonization and/or lysis. Without wishing to be bound by any particular theory, the authors merely note that only when multiple C3b molecules associate and bind to each other in close proximity does the C3bBbC3bBb C3 convertase change its substrate specificity and cleave C5 as an alternative pathway C5 convertase. , called C3bbcbbb(cbb)n.

The term MASP-2-dependent complement activation, as used herein, also referred to here as LEA-2-mediated complement activation, includes the lectin-dependent activation of MASP-2 that occurs in the presence of Ca~, leading to the formation of C3-convertase of the C4b2a lectin pathway and after accumulation of the C3 cleavage product of C3b, C5 convertase C4b2a(C3b)n is formed, which has been determined to cause opsonization and/or lysis.

As used herein, the traditional understanding of the alternative path, also referred to herein as the traditional alternative path, refers to an alternative path described prior to the disclosure of the present invention, i.e. to complement activation, which is triggered by, for example, fungal and yeast cell wall zymosan, lipopolysaccharide (LPS) of the outer membranes of gram-negative bacteria and rabbit erythrocytes, as well as many pure polysaccharides, viruses, bacteria, animal tumor cells, parasites and damaged cells, and which , has traditionally been thought to be caused by spontaneous proteolytic generation of C3b from complement factor C3. As used herein, traditional alternative pathway activation, also referred to herein as alternative pathway, is evaluated in Mg ⁺⁺ /EGTA buffer (ie, in the absence of Ca~).

As used herein, the term lectin pathway refers to complement activation that occurs through the specific binding of serum and non-serum carbohydrate-binding proteins, including mannan-binding lectin (MBL), CL-11, and ficolins (H-ficolin, Mficolin, or L-ficolin). As described herein, the present inventors have found that the lectin pathway is triggered by two effector pathways, such as the lectin pathway 1 (LEA-1) effector chain, which is now known to be MASP-3 dependent, and the lectin pathway effector chain. path 2 (LEA-2), which is MASP-2 dependent. The activation of lectin pathways described here is assessed using Ca ^- containing buffers.

- 17 040888

The term classical pathway as used herein refers to complement activation that is triggered by an antibody bound to a foreign particle and requires binding to a C1q recognition molecule.

As used herein, the term HTRA-1 means serine peptidase, which is converted to serine protease A1 at high temperature.

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

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

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

As used herein, the term antibody encompasses antibodies and antibody fragments derived from any antibody-producing mammal (e.g., mice, rats, rabbits, and primates, including humans) or isolated from hybridomas by phage selection, recombinant expression, or transgenic animals (or other methods). obtaining antibodies or antibody fragments), where these antibodies specifically bind to the target polypeptide, such as, for example, MASP-1, MASP-2 or 3-MASP polypeptides or parts thereof. This does not mean that the term antibody is limited to the source of its origin or the method of its production (for example, by isolation from a hybridoma, phage selection, recombinant expression, isolation from a transgenic animal, peptide synthesis, etc.).

Representative antibodies are polyclonal, monoclonal and recombinant antibodies; panspecific, multispecific antibodies (eg, bispecific antibodies, trispecific antibodies); humanized antibodies; mouse antibodies; chimeric antibodies, antibodies

- 18 040888 mouse-human, mouse-primate antibodies, primate-human monoclonal antibodies; and anti-idiotypic antibodies, and these antibodies can be any intact antibody or fragment thereof. As used herein, the term antibody encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof such as a single variable region antibody (dAb) or other known antibody fragments such as Fab, Fab', F(ab') ₂ , Fv and etc., single-chain fragments (ScFv), their synthetic variants, natural variants, fusion proteins comprising part of an antibody together with an antigen-binding fragment with the desired specificity, humanized antibodies, chimeric antibodies, bispecific antibodies, and any other modified configuration of an immunoglobulin molecule containing an antigen-binding site or its fragment (epitope-recognizing site) with the desired specificity.

The term monoclonal antibody means a population of homogeneous antibodies, where the specified monoclonal antibody consists of amino acids (natural and non-natural) involved in the selective binding of the epitope. Monoclonal antibodies are highly specific for the target antigen. The term monoclonal antibody covers not only intact monoclonal antibodies and full-length monoclonal antibodies, but also their fragments (such as Fab, Fab', F(ab') ₂ , Fv), single chain fragments (ScFv), variants thereof, fusion proteins, including an antigen-binding portion, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of immunoglobulin molecules containing an antigen-binding fragment (epitope recognition site) with the desired specificity and ability to bind to an epitope. This does not mean that the term antibody is limited to the source of its origin or the method of its production (for example, by isolation from a hybridoma, phage selection, recombinant expression, isolation from a transgenic animal, etc.). This term includes whole immunoglobulins as well as their fragments and the like, as described above in the definition of the term antibody.

As used herein, the term antibody fragment means a portion derived from or related to a full-length antibody, such as an anti-MASP 1, MASP-2, or MASP-3 antibody, which typically includes its antigen-binding or variable region. Illustrative examples of antibody fragments are Fab-, Fab'-, F(ab') ₂ - and Fv-fragments; scFv fragments; diantibodies; linear antibodies; single-chain antibody molecules, bispecific and multispecific antibodies derived from antibody fragments.

In some embodiments, the antibodies and antigen-binding fragments thereof described herein comprise a set of heavy chain (VH) and light chain (VL) hypervariable regions (CDRs), respectively, located between a set of heavy chain and light chain framework regions (FRs), which ensure the preservation of the CDRs and define the spatial relationship of the CDRs with respect to each other. The term CDR set as used herein refers to the three hypervariable regions of the heavy or light chain V region. These regions, counted from the N terminus of the heavy or light chain, are designated CDR1, CDR2, and CDR3, respectively. Thus, the antigen-binding site includes six CDRs, representing a set of CDRs from each of the heavy and light chain V regions.

The term FR set as used herein refers to the four flanking amino acid sequences framing a CDR from a heavy or light chain V region CDR set. Some FR residues may contact via antigen binding, however, FRs are primarily responsible for laying the V region at the antigen binding site, and in particular, FR residues are immediately adjacent to the CDR. In FR, some amino acid residues and some structural features are highly conserved. In this regard, all V-region sequences contain an internal disulfide loop of approximately 90 amino acid residues. With regard to the stacking of the V-regions at the binding site, the CDRs appear as raised loop motifs that form an antigen-binding surface. As such, conserved FR structural regions are known to affect the way CDR loops fold into certain canonical structures, regardless of the specific composition of the CDR amino acid sequence.

The structures and arrangement of immunoglobulin variable regions can be determined as described in Rabat EA et al., Sequences of Proteins of Immunological Interest, ^4th Edition, US Department of Health and Human Services, 1987, and later editions currently available. on the Internet (immuno.bme.nwu.edu.).

As used herein, the term single chain Fv or ScFv antibody fragment includes the VH and V _L domains of an antibody, where these domains are present in the same polypeptide chain. Typically, the Fv polypeptide also includes a polypeptide linker between the VH - and V _L domains, which allows ScFv to form the desired structure for binding to the antigen.

As used herein, the term chimeric antibody means a recombinant protein that contains variable domains and hypervariable regions derived from a non-human animal (eg, rodent), while the rest of the antibody molecule is derived from a human antibody. In some embodiments, the chimeric antibody consists of an antigen-binding fragment of a MASP-3 inhibitory antibody operably linked or otherwise linked to a heterologous Fc portion of another antibody. In some embodiments, the heterologous Fc domain may be derived from another class of Ig parent antibody, including IgA (including IgA1 and IgA2 subclasses), IgD, IgE, IgG (including IgG1, IgG2, IgG3, and IgG4 subclasses), and IgM.

As used herein, the term humanized antibody means a chimeric molecule, usually produced by recombinant techniques, and having an antigen-binding site derived from an immunoglobulin of a non-human animal, where the rest of the structure of the immunoglobulin molecule is based on the structure and/or sequence of a human immunoglobulin. The antigen binding site may contain either full length variable regions attached to the constant domains, or only CDRs attached to the appropriate framework regions in the variable domains. Epitope binding sites may be wild-type sites or may be modified with one or more amino acid substitutions. Another approach focuses not only on obtaining human constant regions, but also on the modification of variable regions and their reconstruction in order to obtain a form that is closest to the human form. In some embodiments, humanized antibodies retain all CDR sequences (eg, a humanized mouse antibody contains all six CDRs derived from mouse antibodies). In other embodiments, the humanized antibodies have one or more CDRs (one, two, three, four, five, six) that have been modified from the parent antibody, also referred to herein as one or more CDRs derived from one or more CDRs of the parent antibody.

An antibody specifically binds to a target if it binds to the target with greater affinity and/or avidity than it does to other substances. In one embodiment, the antibody or antigen-binding fragment specifically binds to the human MASP-3 serine protease domain (amino acid residues 450-728 of SEQ ID NO: 2). In one embodiment, the antibody, or antigen-binding fragment thereof, specifically binds to one or more of the epitopes described in Table 1. 4, in the table. 28 or shown in FIG. 62.

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

As used herein, the term membrane attack complex (MAC) refers to a complex of five terminal complement components (C5b combined with C6, C7, C8 and C9) that insert into and destroy membranes (such a complex is also referred to as C5b-9).

As used herein, the term individual includes all mammals, which are, but are not limited to, humans; non-human primates; dogs, cats, horses, sheep, goats, cows, rabbits, pigs and rodents.

The amino acid residues used herein have the following abbreviations, namely: 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), serie (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

In a broader sense, natural amino acids can be classified into groups based on the chemical properties of the side chains of the respective amino acids. Hydrophobic amino acids are Ile, Leu, Met, Phe, Trp, Tyr, Val, Ala, Cys or Pro. Hydrophilic amino acids are Gly, Asn, Gln, Ser, Thr, Asp, Glu, Lys, Arg or His. This group of amino acids can also be subdivided into subclasses. The uncharged hydrophilic amino acids are Ser, Thr, Asn or Gln. The acidic amino acids are Glu or Asp. The main amino acids are Lys, Arg or His.

As used herein, the term conservative amino acid substitution means the replacement of one amino acid with another amino acid, where these amino acids are in one of the following groups: (1) glycine, alanine, valine, leucine and isoleucine, (2) phenylalanine, tyrosine and tryptophan, (3) serine and threonine, (4) aspartate and glutamate, (5) glutamine and asparagine, and (6) lysine, arginine and histidine.

As used herein, the term oligonucleotide means an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. The term also covers oligonucleotide bases consisting of naturally occurring nucleotides, sugars and covalent internucleoside bonds (backbone), as well as oligonucleotides having modifications not found in nature.

As used herein, the term epitope refers to a region on a protein (eg, human MASP-3 protein) that is associated with an antibody. Overlapping epitopes include at least one common amino acid residue (eg, two, three, four, five, or six residues), including linear and non-linear epitopes.

As used herein, the terms polypeptide, peptide, and protein are synonymous and refer to any chain of amino acids linked by a peptide bond, regardless of their length or post-translational modification. The MASP-3 proteins described herein may contain or be wild-type proteins, or may be variants that have no more than 50 (e.g., no more than one, two, three, four, five, six, seven, eight, nine, ten, 12, 15, 20, 25, 30, 35, 40, or 50) conservative amino acid substitutions. Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine and threonine; lysine, histidine and arginine; and phenylalanine and tyrosine.

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

In some embodiments, the peptide fragments may be at least 6 (e.g., at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, or 600 or more) amino acid residues (eg, at least 6 contiguous amino acid residues in SEQ ID NO: 2). In some embodiments, the antigenic peptide fragment of the human MASP-3 protein has a length of less than 500 , 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40 , 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15 , 14, 13, 12, 11, 10, 9, 8, 7, or 6) amino acid residues (eg, less than 500 contiguous amino acid residues in SEQ ID NO: 2).

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

Percent amino acid sequence identity (%) is defined as the percentage of amino acids in a candidate sequence that are identical to the amino acids in the compared sequence after sequence alignment and gap insertion, if necessary, to achieve the maximum percent sequence identity.

Alignment for purposes of determining percent sequence identity can be achieved in a variety of ways that are known to those skilled in the art, for example using publicly available computer programs such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR). Appropriate parameters for alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the compared sequences, can be determined by known methods.

In representative embodiments of the invention, the human MASP-3 protein (SEQ ID NO: 2) is encoded by the cDNA sequence shown as SEQ ID NO: 1. Those skilled in the art will recognize that the cDNA sequence shown as SEQ ID NO: 1 is one allele of human MASP-3 and that allelic variants and alternative splicing variants may be present. Allelic variants of the nucleotide sequences set forth in SEQ ID NO: 1, including those containing silent mutations and those resulting in amino acid sequence modifications, are within the scope of the present invention. Allelic variants of the MASP-3 sequence can be cloned by probing cDNA or genomic libraries taken from different individuals according to standard procedures, or can be identified by homology searches by comparison (e.g., search using the BLAST program) in databases containing such information.

As used herein, an isolated nucleic acid molecule is a nucleic acid molecule (eg, a polynucleotide) that is not integrated into the genomic DNA of a given organism. Thus, for example, a DNA molecule that encodes a growth factor that has been separated from the genomic DNA of a cell is an isolated DNA molecule. Another example of an isolated nucleic acid molecule is a chemically synthesized nucleic acid molecule that has not been integrated into the genome of an organism. A nucleic acid molecule isolated from an organism of a particular species is smaller than the full-length chromosome DNA molecule of that species.

As used herein, a nucleic acid molecule construct is a nucleic acid molecule, either single-stranded or double-stranded, that has been modified by man to contain contiguous nucleic acid segments combined into a corresponding structure that does not exist in nature.

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The expression vector used here is a nucleic acid molecule comprising a gene that is expressed in a host cell. Typically, an expression vector contains a transcription promoter, a gene, and a transcription terminator. The expression of a gene is usually under the control of a promoter, in which case such a gene is said to be operably linked to a promoter. Similarly, a regulatory element and a core promoter are considered to be operably linked to each other if the regulatory element modulates the activity of the core promoter.

In the present description, when using the words approximately when they are used with numbers, it is usually understood that these numbers are in the range of values varying within ±10%, and preferably ±5%, and most preferably ±2% of the specified value. If ranges of values are indicated, then it is understood that this interval also includes final values, unless otherwise indicated and follows from the context of the description.

Singular nouns may refer to plural nouns unless the context of the description implies otherwise. Thus, for example, the term excipient includes a variety of such excipients and their equivalents known to those skilled in the art; the word agent can mean one agent, as well as two or more agents; the word antibody can mean a plurality of such antibodies, and the phrase frame region can mean one or more framework regions and their equivalents known to those skilled in the art, and the like.

Each variant in this description is a variant with the corresponding necessary changes (mutatis mutandis) in comparison with each other option, unless it is expressly stated in the present description. It should be noted that any option discussed in this description can be carried out using any of the methods, kits, reagents or compositions according to the invention and vice versa. In addition, the compositions according to the invention can be used to carry out the methods according to the invention.

II. The lectin pathway: a new insight.

i. Overview: The lectin pathway has been redefined.

As described herein, the inventors have made the surprising discovery that the lectin complement pathway has two complement activation effector circuits that are triggered by lectin pathway activation complexes formed by carbohydrate recognition components (MBL, CL-11 and ficolins): i) an effector circuit formed by the lectin pathway-associated serine proteases MASP-1 and MASP-3 and referred to here as the lectin pathway 1 effector chain or LEA-1; and (ii) an activation effector chain driven by MASP-2 and referred to herein as the lectin pathway 2 effector chain or LEA-2. Both of these LEA-1 and LEA-2 chains can carry out lysis and/or opsonization.

It has also been determined that lectin-independent conversion of factor B by MASP-3 and lectin-independent conversion of factor D by HTRA-1, MASP-1 and MASP-3, which can occur in the absence of Ca~, often result in the conversion of C3bB to C3bBbC3bBb and pro-factor D to factor D. Therefore, inhibition of MASP-3 can inhibit LEA-1 and lectin-independent activation of factor B and/or factor D, which can lead to inhibition of lysis and/or opsonization.

In FIG. 1 illustrates this new understanding of complement activation pathways. As shown in FIG. 1, LEA-1 is triggered by lectin-bound MASP-3, which can activate the factor D zymogen to form its active form and/or cleave C3b- or C3b(H ₂ O)-bound factor B, which will result in conversion of the C3bB complex -zymogen into its enzymatically active form C3bBbC3bBb. Activated Factor D generated by MASP-3 can also convert C3b- or C3b(H ₂ O)-zymogen complexes to their enzymatically active form. MASP-1 is capable of rapid self-activation, while MASP-3 does not. In many cases, MASP-1 is an activator of MASP-3.

Although in many instances lectins (ie, MBL, CL-11, or ficolins) can directly activate cell surfaces, however, in FIG. 1 also outlines the lectin-independent functions of MASP-3, MASP-1, and HTRA-1 for factor B activation and/or factor D maturation. As with the lectin-associated form of MASP-3 in LEA-1, The lectin-independent form of MASP-3 is able to mediate the conversion of C3bB or C3b(H ₂ O) to C3bBbC3bBb (see also FIGS. 29 and 30) and profactor D to factor D (see FIG. 32). MASP-1 (see also Fig. 32) and non-MASP-associated HTRA-1 protein can also activate factor D (Stanton et al., Evidence That the HTRA1 Interactome Influences Susceptibility to Age-Related Macular Degeneration presented by experts Association for the Study of the Organs of Vision and Eye Diseases 2011 at the Conference held on May 4, 2011) on a mechanism that does not require a lectin component.

Thus, MASP-1 (acting on LEA-1 and in lectin-independent form), MASP-3 (acting on LEA-1 and in lectin-independent form), and HTRA-1 (only in lectin-independent form) are capable of either directly or indirectly activated at one or more points in the MASP-3 reaction chain - factor D factor B. In doing so, they generate C3bBbC3bBb, an alternative pathway C3 convertase, and stimulate the production and deposition of C3b on microbial surfaces. precipitation

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C3b plays a critical role in opsonization, marking the surface of microbes for destruction by host phagocytes such as macrophages. As an example (FIGS. 28A and 28B), MASP-3 plays a critical role in S. aureus opsonization. Precipitation of C3b occurs rapidly on S. aureus under the action of human serum in a MASP-3 dependent mechanism (FIGS. 28a and 28b).

However, LEA-1 and the lectin-independent functions of MASP-3, MASP-1, or HTRA-1 are not limited to opsonization. As shown schematically in the diagram in FIG. 1, these three components can also induce cell lysis through indirect or direct factor B activation and C3b production. These components form complexes that generate an alternative pathway C5 convertase, C3bc3bb(C3b) _n . As described below, the need for MASP-3 and MBL, but not MASP-2 (and therefore not LEA-2 in this example), in the lysis of N. meningitidis, (see Fig. 11-13) confirms the role of LEA-1 in lysis. Thus, the opsonization results obtained in the S. aureus. studies and the lysis results obtained in the N. meningitidis studies confirm the role of LEA-1 in both processes (as shown in the diagram in Fig. 1). In addition, these studies demonstrated that opsonization and lysis may result from the conversion of C3bB or C3b(H ₂ O) and/or profactor D to factor D, and therefore these processes may be the result of a lectin-independent role of MASP-3, MASP-1 or HTRA-1. Thus, the model developed by the inventors and shown in FIG. 1 confirms the possibility of using inhibitors, especially MASP-3, but also MASP-1 and/or HTRA-1, to block opsonization and/or lysis and to treat pathologies caused by dysregulation of these processes.

1. Effector chain of the lectin pathway (LEA-1).

The first effector strand of the lectin pathway, LEA-1, is formed by the serine proteases MASP-1 and MASP-3 associated with the lectin pathway. As described in this application, the authors of the present invention found that in the absence of MASP-3 and in the presence of MASP-1, the alternative pathway is not effectively activated on surface structures. These results indicated that MASP-3 plays a previously unknown role in the initiation of the alternative pathway, and this is confirmed using MASP-3-deficient 3MC serum taken from patients with a rare 3MC autosomal recessive disorder (Rooryck C. et al., Nat Genet 43 (3):197-203 (2011)) with mutations that render the MASP-3 serine protease domain non-functional. Based on these new results, it has been suggested that complement activation via the alternative pathway previously identified is MASP-3 dependent. Indeed, MASP-3 and its activation by LEA-1 can also be considered as dubious alternative pathway initiators.

As described in Examples 1-4 below, we observed higher lectin-dependent alternative pathway activation activity in MASP-2-deficient serum, resulting in increased bactericidal activity (i.e., lytic activity) against N. meningitidis . Without wishing to be bound by any particular theory, we only speculate that in the absence of MASP-2, MASP-1-bearing carbohydrate-recognition complexes are more likely to be closely associated with MASP-1-bearing carbohydrate-recognition complexes and activate MASP3. It is known that in many cases, MASP-3 activation depends on MASP-1 activity, since MASP-3 is not an autoactivating enzyme, and therefore, in order to convert its zymogenic form into an enzymatically active form, it in most cases requires MASP-1 activity. MASP-1 (like MASP-2) is an auto-activating enzyme, while MASP-3 is not an auto-activating enzyme and, in many cases, it requires the enzymatic activity of MASP-1 to convert its zymogenic form into an enzymatically active form. See Zundel S. et al., J. Immunol., 172(7):4342-50 (2004). In the absence of MASP-2, all lectin pathway-recognizing complexes are loaded with either MASP-1 or MASP-3. Thus, the absence of MASP-2 facilitates the MASP-1-mediated conversion of MASP-3 to its enzymatically active form. Upon activation of MASP-3, activated MASP-3 initiates the activation of an alternative pathway, now referred to as LEA-Γ activation, via MASP-3-mediated conversion of C3bB to C3bBbC3bBb and/or conversion of profactor D to factor D. C3bBb, also referred to herein as C3- an alternative pathway convertase, cleaves additional C3 molecules, followed by precipitation of C3b opsonin molecules. If several C3b fragments are tightly bound to the C3bBbC3bBb convertase complex, this leads to the formation of the alternative pathway C3bbc3b(C3b)n pathway C5 convertase, which promotes MAC formation. In addition, C3b molecules deposited on the surface form new sites for binding to factor B, which can already be cleaved by factor D and/or MASP-3 to form additional sites where C3 and C5 convertase complexes of the alternative pathway can be formed. This latter process is necessary for efficient lysis and does not require the presence of lectins after the initiation of C3b precipitation. In a recent publication (Iwaki D. et al., J. Immunol. 187(7):3751-8 (2011)), as well as based on the data obtained by the authors of the present invention (Fig. 30), it was shown that the C3 complex -convertase - alternative pathway zymogen C3bB is converted to its enzymatically active form via activated MASP-3. The inventors have found that MASP-3-mediated factor B cleavage is a subcomponent of the recently described LEA-1 that stimulates the lectin-dependent production of the C3bBbC3bBb C3 convertase alternative pathway.

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2. Effector chain of the lectin pathway (LEA-2).

The second effector strand of the lectin pathway, LEA-2, is formed by the lectin pathway-associated serine protease MASP-2. MASP-2 is activated after binding of the recognition components of their respective pattern, and can also be activated by MASP-1, which subsequently leads to the cleavage of the C4 component of complement with the formation of C4a and C4b. After C4B cleavage product binds to plasma C2, C2-bound C4b becomes the substrate for a second MASP2-mediated cleavage step that converts C4b-bound C2 into the enzymatically active C4bC2a complex and a small C2b cleavage fragment. C4b2a is a C3-converting C3-convertase of the lectin pathway that promotes the conversion of excess plasma C3 component to C3a and C3b. C3b binds closely to any surface via a thioether bond. If several fragments of C3b bind closely to the C3-convertase complex C4b2a, then this convertase changes its specificity for the conversion of C5 to C5b and C5a with the formation of the C3-convertase complex C4b2a(C3b)n. Although this C5 convertase can initiate MAC formation, however, it is believed that such a process is not efficient enough to stimulate its own lysis. Most likely, the original C3b opsonins produced by LEA-2 form the nucleus for the formation of a novel alternative pathway C3 convertase and C5 convertase sites that will eventually lead to excess MAC production and lysis. This latter event is mediated by the activation of factor B to factor D associated with C3b produced by LEA-2 and is therefore dependent on LEA-1 due to the important role of MASP-1 in factor D maturation. There is also a MASP-2-dependent C4 bypass. an activation pathway that allows C3 to be activated in the absence of C4, and this pathway plays an important role in the pathophysiology of ischemia-reperfusion injury, since C4-deficient mice are not protected from ischemia-reperfusion injury, while MASP-2-deficient mice are so protected (Schwaeble et al., PNAS, 2011, see above). LEA-2 is also associated with a blood coagulation pathway, including the cleavage of prothrombin to form thrombin (common pathway), as well as the cleavage of factor XII (Hageman factor) to its enzymatically active form XIIa. Factor XIIa in turn cleaves factor XI to form XIa (intrinsic pathway). Activation of the intrinsic pathway of the blood coagulation cascade leads to the formation of fibrin, which is critical for thrombus formation.

In FIG. 1 illustrates the new understanding of the lectin pathway and the alternative pathway based on the results reported herein. In FIG. 1 shows the role of LEA-2 in opsonization and lysis. In addition to initiating subsequent C3b precipitation (and ultimately opsonization) in several lectin-dependent physiological processes (FIGS. 18A, 18B, 18C), MASP-2 also plays a role in the lysis of serum-sensitive bacteria. As shown in FIG. 1, the proposed molecular mechanism responsible for the increased bactericidal activity of MASP-2-deficient or MASP-2-depleted serum/plasma against serum sensitive pathogens such as N. meningitidis is that, in order to lyse bacteria, lectin recognition complexes MASP-1 and MASP-3 associated pathways must bind tightly to each other on the bacterial surface, which will allow MASP-1 to cleave MASP-3. Unlike MASP-1 and MASP-2, MASP-3 is not an autoactivating enzyme, but in many cases requires activation/cleavage by MASP-1 to be converted to an enzymatically active form.

As further shown in FIG. 1, activated MASP-3 can then cleave C3b-bound factor B on the pathogen surface to initiate an alternative cascade of activation reactions due to the formation of the enzymatically active C3 and C5 alternative pathway convertases C3bBbC3bBb and C3bBb(C3b) _n , respectively. MASP-2-bearing lectin pathway activation complexes are not involved in MASP-3 activation and, in the absence of MASP-2 or after MASP-2 depletion, all lectin pathway activation complexes will be loaded with either MASP-1 or MASP-3. Thus, in the absence of MASP-2, the likelihood that on the microbial surface, MASP-1- and MASP-3-bearing lectin pathway activation complexes will be located in close proximity to each other, which will lead to greater activation of MASP-3, is greatly increased. and thereby increasing the rate of MASP-3-mediated cleavage of factor B associated with C3b to form C3 and C5 alternative pathway convertases C3bBbC3bBb and C3bBb(C3b)n on the microbial surface. This leads to the activation of the terminal C5b-C9 activation cascades, which form a membrane attack complex consisting of surface-bound C5b associated with C6; C5bC6 associated with C7; C5bC6C7 associated with C8; and C5bC6C7C8, which will polymerize C9, which is incorporated into the bacterial surface structure and forms pores in the bacterial walls, which will cause osmotic lysis of the bacterium that is the target of this complement.

The essence of this new concept is that the data presented here clearly showed that the activation complexes of the lectin pathway trigger two subsequent distinct activation pathways, as shown in FIG. 1:

i) LEA-1: MASP-3 dependent activation pathway that initiates and triggers complement activation by generating the C3bBbC3bBb alternative pathway convertase due to the initial

- 24 040888 mu cleavage and activation of factor B on the surfaces of activators, which will then catalyze the precipitation of C3b and the formation of the alternative pathway convertase C3bBbC3bBb. The MASP-3 induced activation pathway plays an important role in microbial opsonization and lysis and triggers an alternative pathway on the bacterial surface, resulting in an optimal level of activation for the generation of membrane attack complexes; and ii) LEA-2: MASP-2 dependent activation pathway leading to the formation of the C3 convertase of the C4b2a lectin pathway and, after accumulation of the C3 cleavage product, i.e. C3b, to the formation of C5-convertase C3bc3bb(c3b)p. In the absence of complement C4, MASP-2 can form an alternative C3 convertase complex that includes C2 and factor XI.

The MASP-3-induced activation pathway, in addition to its specific role in lysis, plays an important role in the opsonization of bacteria, whereby the microbes are coated with covalently bound C3b and its cleavage products (i.e., iC3b and C3dg), which will serve as a target for uptake. and lysis by C3 receptor-bearing phagocytes such as granulocytes, macrophages, monocytes, microglial cells, and the reticuloendothelial system. This pathway is the most efficient way to clear bacteria and microorganisms that are resistant to complement lysis. These bacteria include most gram-positive bacteria.

In addition to LEA-1 and LEA-2, there is another possible pathway for lectin-independent activation of factor D by MASP-3, MASP-1 and/or HTRA-1, as well as a possible pathway for lectin-independent activation of factor B by MASP-3. .

Without wishing to be bound by any particular theory, we only believe that each of (i) LEA-1, (ii) LEA-2, and (iii) lectin-independent factor B and/or factor D activation results in opsonization and/or formation MAC followed by lysis.

ii. General description of MASP-1, MASP-2 and MASP-3.

II. General description of MASP-1, MASP-2 and MASP-3.

Three mannan-binding and lectin-associated proteases (MASP-1, MASP-2 and MASP-3) are currently known to bind to mannan-binding lectin (MBL) in human serum. In the current literature, a mannan-binding lectin is also referred to as a mannose-binding protein or mannose-binding lectin. The MBL-MASP complex plays an important role in maintaining innate immunity by binding MBL to carbohydrate structures present on a wide variety of microorganisms. Interaction of MBL with specific arrays of carbohydrate structures results in the activation of MASP proenzymes, which in turn activate complement by cleaving the C4 and C2 components of complement to form the C3 convertase C4b2b (Kawasaki et al., J. Biochem 106:483-489 (1989 ); Matsushita & Fujita, J. Exp. Med. 176:1497-1502 (1992); Ji et al., J. Immunol. 150:571-578 (1993)).

Until recently, the MBL-MASP proenzyme complex was thought to contain only one type of protease (MASP-1), but it has now become clear that there are two other distinct proteases (i.e. MASP-2 and MASP-3) associated with MBL (Thiel et al. Nature 386: 506-510 (1997); Dahl et al. Immunity 15: 127135 (2001)), as well as an additional 19 kDa whey protein referred to as MAP19 or sMAP (Stover et al., J. Immunol 162 :3481-3490 (1999); Stover et al., J. Immunol. 163:6848-6859 (1999); Takahashi et al., Int. Immunol. 11:859-63 (1999)).

MAp19 is an alternative splicing gene product of the MASP2 structural gene and lacks the four C-terminal domains of MASP-2, including the serine endopeptidase domain. An overexpressed truncated mRNA transcript encoding MAP19 results from an alternative splicing/polyadenylation event of the MASP-2 gene. By a similar mechanism, the MASP1/3 gene expresses three major gene products, namely the two serine proteases MASP-1 and MASP-3 and a truncated 44 kD gene product designated MAP44 (Degn et al., J. Immunol. 183(11) :7371-8 (2009); Skjoedt et al., J Biol Chem 285:8234-43 (2010)).

MASP-1 was first described as a component of the P-100 protease of the serum Ra-reactive factor, which is now known as a complex of MBL+MASPs (Matsushita et al., Collectins and Innate Immunity, (1996); Ji et al. , J. Immunol 150:571-578 (1993) The ability of the MBL-associated endopeptidase in the MBL-MASP complex to act on the C4 and C2 components of complement is essentially identical to that of the C1s enzyme in the C1q-(C1r) ₂ -(C1s) complex. ₂ of the classical complement pathway, suggesting that there is an MBL-MASPs complex that is functionally similar to the C1q-(C1r) ₂ -(C1s) ₂ complex. The C1q-(C1r) ₂ -(C1s) ₂ complex is activated when C1q interacts with Fc IgG or IgM antibody regions present in immune complexes This results in auto-activation of the C1r proenzyme, which in turn activates the C1s proenzyme, which subsequently acts on the C4 and C2 components of the complement.

The stoichiometry of the MBL-MASPS complex differs from that of the C1q-(C1r) ₂ -(C1s)2 complex in that different MBL oligomers seem to associate with different ratios of MASP-1/MAp19 or MASP-2/MASP-3 (Dahl et al., Immunity 15:127-135 (2001) Most MASPS and MAP19 found in serum do not form complexes with MBL (Thiel et al., J. Immunol. 165:878-887 (2000)) and may partially associate with ficolins, i.e. a recently described group of lectins,

- 25 040888 having a fibrinogen-like domain capable of binding to N-acetylglucosamine residues on microbial surfaces (Le et al., FEBS Lett 425:367 (1998); Sugimoto et al., J. Biol. Chem. 273:20721 (1998 )). Among them, human L-ficolin, H-ficolin and M-ficolin associate with MASPS as well as MAP19 and can activate the lectin pathway after binding to specific carbohydrate structures recognized by ficolins (Matsushita et al., J. Immunol. 164:2281- 2284 (2000); Matsushita et al., J. Immunol. 168:3502-3506 (2002)). In addition to ficolins and MBL, an MBL-like lectin called CL-11 has been identified as a lectin pathway recognition molecule (Hansen et al. J. Immunol. 185:6096-6104 (2010); Schwaeble et al. PNAS 108:7523- 7528 (2011)). There is strong evidence for the physiological importance of these alternative carbohydrate recognition molecules, and therefore it is important to understand that MBL is not the only component that recognizes the lectin activation pathway, and that MBL deficiency cannot be mistaken for a lectin pathway deficiency. The possibility of an array of alternative carbohydrate-recognition complexes structurally related to MBL could expand the spectrum of microbial structures that initiate a direct response of the innate immune system through complement activation.

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

1. Corresponding structures, sequences, chromosomal locations and splicing variants of MASP-1 and MASP-3.

In FIG. 2 is a schematic diagram illustrating the domain structure of human MASP-1 polypeptide (SEQ ID NO: 8), human MASP-3 polypeptide (SEQ ID NO: 2), and human MAP44 polypeptide, and exons encoding these polypeptides. As shown in FIG. 2, the serine proteases MASP-1 and MASP-3 are composed of six distinct domains found to be located at C1r and C1s; those. (I) Sea urchin C1r/C1s/VEGF/bone morphogenesis protein (or CUBI) N-terminal domain; (II) an epidermal growth factor (EGF) like domain; (III) a second CUB domain (CUBII); (IV and V) two complement regulatory protein domains (CCP1 and CCP2); and (VI) a serine protease (SP) domain.

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

In addition, as shown in FIG. 2, after the conversion of the zymogen into its active form, the heavy chain (alpha or A chain) and light chain (beta or B chain) were cleaved to form a disulfide-linked A chain and a smaller B chain, which is a serine domain. proteases. The single chain MASP-1 proenzyme is activated (like the C1r and C1s proenzymes) by cleaving the Arg-Ile bond located between the second domain of the CCP (domain V) and the serine protease domain (domain VI). It is believed that the proenzymes MASP-2 and MASP-3 are activated in the same way as MASP-1. Each MASP protein forms homodimers and separately associates with MBL and ficolins in a Ca^-dependent manner.

The human MASP-1 polypeptide (SEQ ID NO: 8) and the MASP-3 polypeptide (SEQ ID NO: 2) are derived from the same structural gene (Dahl et al., Immunity 15:127-135 (2001), which was mapped by area 3q27-28 of the long arm of chromosome 3 (Takada et al., Genomics 25:757-759 (1995)).The MASP-3 and MASP-1 mRNA transcripts are generated from the primary transcript by alternative splicing/polyadenylation.The translation product of MASP-3 consists of of the alpha chain, which is common to MASP-1 and MASP-3, and the beta chain (serine protease domain), which is unique to MASP-3.As shown in Fig. 2, the human MASP-1 gene includes 18 exons The human MASP-1 cDNA is encoded by exons 2, 3, 4, 5, 6, 7, 8, 10, 11, 13, 14, 15, 16, 17, and 18. As further shown in Figure 2, the human MASP 3 gene includes twelve exons The human MASP-3 cDNA (represented as SEQ ID NO: 1) is encoded by exons 2, 3, 4, 5, 6, 7, 8, 10, 11, and 12. Alternative splicing results in a protein called MBL-associated protein 44 (MAp44) derived from exons 2, 3, 4, 5, 6, 7, 8, and 9.

- 26 040888

The human MASP-1 polypeptide (SEQ ID NO: 8, Genbank BAA04477.1) has 699 amino acid residues, which includes a leader peptide of 19 residues. If the leader peptide is absent, then the calculated molecular weight of MASP-1 is 76976 Da. As shown in FIG. 2, the amino acid sequence of MASP-1 contains four N-linked glycosylation sites. The domains of the human MASP-1 protein (see SEQ ID NO: 8) are shown in FIG. 2 and include the N-terminal domain of sea urchin C1r/C1s/VEGF/bone morphogenesis protein (CUBI) (a.k. 25-137 in SEQ ID NO: 8); an epidermal growth factor-like domain (a.k. 139-181 in SEQ ID NO: 8); a second CUB domain (CUBII) (a.k. 185-296 in SEQ ID NO: 8), as well as a tandem of complement regulation protein domains (CCP1 a.k. 301-363 and CCP2 a.k. 367-432 in SEQ ID NO: 8), and a serine protease domain (a.k. 449-694 in SEQ ID NO: 8).

The human MASP-3 polypeptide (SEQ ID NO: 2, Genbank AAK84071) has 728 amino acid residues (as shown in FIG. 3), which includes a 19 residue leader peptide (shown by underlined amino acid residues in FIG. 3).

If leader peptides are absent, then the calculated molecular weight of MASP-3 is 81873 Da. As shown in FIG. 2, there are seven N-linked glycosylation sites in MASP-3. The domains of the human MASP-3 protein (see SEQ ID NO: 2) are shown in FIG. 2 and include the N-terminal sea urchin C1r/C1s/VEGF/bone morphogenesis protein (CUBI) domain (a.k. 25-137 in SEQ ID NO: 2), an epidermal growth factor-like domain (a.k. 139- 181 in SEQ ID NO: 2); a second CUB domain (CUBII) (a.k. 185-296 in SEQ ID NO: 2), as well as a tandem of complement regulation protein domains (CCP1, a.k. 299-363 and CCP2, a.k. 367-432 in SEQ ID NO: 2), and a serine protease domain (a.k. 450-728 in SEQ ID NO: 2).

The translation product of MASP-3 consists of an alpha chain (heavy chain) containing the domains CUB-1EGF-CUB-2-CCP-1-CCP-2 (alpha chain: a.k. 1-448 in SEQ ID NO: 2 ) that are common to MASP-1 and MASP-3, and a light chain (beta chains: a.k. 449-728 in SEQ ID NO: 2) containing a serine protease domain that is unique to MASP-3.

2. Comparison of the amino acid sequences of MASP-3 from different species.

In FIG. 4 illustrates a multi-species alignment of MASP-3 showing a comparison of full-length human MASP-3 protein (SEQ ID NO: 2), cynomolgus protein (SEQ ID NO: 7), rat protein (SEQ ID NO: 4), mouse protein (SEQ ID NO: 3), chicken protein (SEQ ID NO: 5) and rabbit protein (SEQ ID NO: 6). In FIG. 5 illustrates the sequence alignment of several species of serine protease (SP) domains, ie. human (a.k. 450-728 in SEQ ID NO: 2); rabbit (a.k. 450-728 in SEQ ID NO: 6); murine (a.k. 455-733 in SEQ ID NO: 3); rat (a.k. 455733 in SEQ ID NO: 4) and chicken (a.k. 448-730 in SEQ ID NO: 5).

As shown in FIG. 4, there is a high level of conservation of the amino acid sequences of the MASP-3 polypeptide among different species, and in particular in the SP domain (FIG. 5). As further shown in FIG. 5, the catalytic triad (H at residue 497, D at residue 553, and S at residue 664 in full-length human MASP-3 (SEQ ID NO: 2) is conserved across species. Table 1 summarizes the percent identity of MASP-3 SP domains. in various species.

Table 1. Percentage of MASP-3 SP Domain Identity in Different Species

dog-like monkey Rabbit Rat Mouse Chicken Human 95% 94% 92% 91% 79% dog-like monkey 94% 90% 90% 79% Rabbit 92% 92% 81% Rat 97% 78% Mouse 78%

MASP-3 has no proteolytic activity on C4, C2, or C3 substrates. Conversely, MASP-3 was originally reported to act as an inhibitor of the lectin pathway (Dahl et al., Immunity 15:127-135 (2001)). This conclusion can be drawn because, unlike MASP-1 and MASP2, MASP-3 is not an autoactivating enzyme (Zundel S. et al., J. Immunol. 172:4342-4350 (2004); Megyeri et al., J Biol Chem 288:8922-8934 (2013).

Recently, confirmation of the possible physiological functions of MASP-1 and MASP-3 has been made based on studies in transgenic mice, namely mice with a combined deficiency of MASP-1 and MASP-3. While MASP-1/3 knockout mice have a functional lectin pathway (Schwaeble et al., PNAS 108:7523-7528 (2011)), however, they do not appear to be active on the alternative pathway (Takahashi et al. al., JEM 207(1):29-37 (2010)). The lack of activity in relation to the alternative pathway is apparently due to a defect in the processing of complement factor D, which is required for activity in relation to the alternative pathway. In MASP-1/3 knockout mice, full-length factor D circulates as a proteolytically inactive pro-form, while in the serum of normal mice almost all factor D is in the active form. Biochemical analysis has shown that MASP-1 can convert complement factor D from its zymogenic form to fer

- 27 040888 mentally active form (Fig. 32; Takahashi et al., JEM 207(1):29-37 (2010)). MASP-3 also cleaves the pro-factor D zymogen and produces active factor D in vitro (Fig. 32; Takahashi et al., JEM 207(1):29-37 (2010)). Factor D is present as an active enzyme in the circulation of normal individuals, and MASP-1 and MASP-3, as well as HTRA-1, may be responsible for this activation. In addition, mice with combined MBL and ficolin deficiency still produce normal levels of factor D and have a fully functional alternative pathway. Thus, these physiological functions of MASP-1 and MASP-3 do not necessarily involve lectins and thus are not associated with the lectin pathway. Recombinant mouse and human MASP-3 also appear to cleave factor B and support C3 precipitation on S. aureus in vitro (Fig. 29; Iwaki D. et al., J. Immunol. 187(7):3751-8 (2011 )).

Recent studies of patients with 3MS syndrome (previously called Carneval, Mingarelli, Malpech and Michaelis syndrome; OMIM # 257920) have revealed a surprising physiological role for MASP-3. These patients presented with severe developmental anomalies, including cleft palate, cleft lip, cranial malformations, and mental retardation. Genetic analysis revealed that 3MC patients were homozygous for the dysfunctional MASP-3 gene (Rooryck et al., Nat Genet. 43(3):197-203 (2011)). It was found that another group of 3MC patients were homozygous for a mutation in the MASP-1 gene, which resulted in the absence of functional MASP-1 and MASP3 proteins. Another group of 3MC patients lacked a functional CL-11 gene (Rooryck et al., Nat. Genet. 43(3):197-203 (2011)). Thus, the complex of CL-11 and MASP-3 obviously plays a certain role in the process of embryonic development. The molecular mechanisms of this developmental pathway are not yet clear. However, it is unlikely that they are mediated by the usual complement-induced mechanism, since individuals with a deficiency of common complement C3 components do not develop this syndrome. Thus, prior to the discovery made by the authors of the present invention and described in this application, the functional role of MASP-3 in lectin-dependent complement activation had not previously been established.

The structures of the catalytic fragment of MASP-1 and MASP-2 were determined using X-ray crystallography. Structural comparison of the MASP-1 protease domain with the domains of other complement proteases revealed the reason for its reduced substrate specificity (Dobo et al., J. Immunol. 183:1207-1214 (2009)). Although the accessibility of the MASP-2 substrate-binding groove is limited to superficial loops (Harmat et al., J. Mol. Biol. 342:1533-1546 (2004)), however, MASP-1 has an open substrate-binding pocket that resembles trypsin, but not other complement proteases. The structure of MASP-1, which resembles thrombin in its properties, is an unusually large loop of 60 amino acids (loop B) that can interact with substrates. Another interesting feature of the MASP-1 structure is the presence of an internal salt bridge between S1 Asp189 and Arg224. A similar salt bridge can be found in the factor D substrate-binding pocket, which may regulate its protease activity. C1s and MASP-2 have nearly identical substrate specificities. Surprisingly, some of the eight surface loops of MASP-2, which determine substrate specificities, have completely different conformations compared to C1s. This means that two functionally related enzymes interact with the same substrates in different ways. The structure of the MASP-2 zymogen has an inactive protease domain with a disrupted oxyanion cavity and a substrate-binding pocket (Gal et al., J. Biol. Chem. 280:33435-33444 (2005)).

Surprisingly, the MASP-2 zymogen has significant activity against the large C4 protein substrate. It is likely that the structure of the MASP-2 zymogen is flexible enough to allow transition between the inactive and active forms. This flexibility, which is reflected in the structure, may play a role in the autoactivation process.

Northern blot analysis showed that the liver is the main source of MASP-1 and MASP-2 mRNA. Using a 5'-specific cDNA probe for MASP-1, a 4.8 kb MASP-1 core transcript was generated. and one small transcript of approximately 3.4 kb, which are both present in human and mouse livers (Stover et al., Genes Immunity 4:374-84 (2003)). MASP-2 mRNA (2.6 kb) and MAP19 mRNA (1.0 kb) are overexpressed in liver tissue. MASP-3 is expressed in the liver as well as in many other tissues, including neural tissues (Lynch N.J. et al., J. Immunol. 174:4998-5006 (2005)).

A patient with a history of infections and chronic inflammatory disease has been found to have a mutated form of MASP-2 that does not form an active MBL-MASP complex (Stengaard-Pedersen et al., N. Engl. J. Med. 349:554-560 (2003)). Some researchers have found that MBL deficiency often leads to infections in children (Super et al., Lancet 2:1236-1239 (1989); Garred et al., Lancet 346:941-943 (1995) and to a decrease in HIV resistance -infections (Nielsen et al., Clin. Exp Immunol. 100:219-222 (1995); Garred et al., Mol. Immunol. 33 (suppl 1):8 (1996)). or a significant correlation between low MBL levels and increased incidence of infections (Egli et al., PLoS One. 8(1):e51983 (2013); Ruskamp et al., J. Infect. Dis. 198(11):1707-13 (2008), Israels et al., Arch Dis Child Fetal Neonatal Ed. 95(6):F452-61 (2010)).

- 28 040888 the literature indicates that a deficiency or lack of utilization of MASP, or both, can have a negative impact on the ability of an individual to develop rapid antibody-independent protection against certain pathogens.

Confirmation of the new insights indicating traditional assay conditions that are performed in the absence of Ca ⁺⁺ and results obtained using a broader physiological set of conditions that include Ca++.

This description provides several independent experimental data that strongly support the conclusion that the lectin complement pathway activates complement through two independent effector mechanisms: i) LEA-2: a MASP-2-induced pathway that mediates complement-driven opsonization, chemotaxis (Schwaeble et al., PNAS 108:7523-7528 (2011)) and cell lysis, and ii) LEA-1: a novel MASP-3-dependent activation pathway that initiates complement activation by generating an alternative pathway C3bBbC3bBb convertase after cleavage, and activation of factor B on the surfaces of the activator, which then catalyze the precipitation of C3b and the formation of the alternative pathway C3bBbC3bBb convertase, which can lead to cell lysis as well as microbial opsonization. In addition, as described in this application, a separate lectin-independent activation of factor B and/or factor D by MASP-1, MASP-3 or HTRA-1, or a combination of any of these three enzymes, can also lead to complement activation through an alternative pathway. .

MASP-3-induced lectin pathway-dependent activation of the alternative pathway appears to promote the well-known factor D-mediated cleavage of C3b-related factor B, resulting in an optimal rate of activation of complement-dependent lysis through the terminal activation pathway for bacterial cell lysis. through the formation of membrane attack complexes C5b-9 (MAC) on the cell surface (Fig. 12-13). For such a rate-limited event, optimal coordination is obviously required, which is due to a deficiency in the absence of functional activity of MASP-3, as well as in the absence of functional activity of factor D. As described here in examples 1-4, the authors of the present invention found this MASP- 3-dependent function of the lectin pathway in the study of the MASP-2 deficiency phenotype and MASP-2 inhibition in experimental mouse models of N. meningitidis infection. Mice deficient in the MASP-2 target gene and wild-type mice treated with antibody-based inhibitors of MASP2 showed high resistance to experimental N. meningitidis infection (see FIGS. 6-10). When the infectious dose was adjusted to give approximately 60% mortality in WT littermates, all MASP-2 deficient or MASP-2 deficient mice were free of infection and survived (see Fig. 6 and Fig. 10). This extremely high degree of resistance was reflected in a significant increase in serum bactericidal activity in the sera of MASP-2 deficient or MASP-2 deficient mice. Further experiments showed that this bactericidal activity was dependent on bacterial lysis induced by the alternative route. Serum from mice deficient in factor B, factor D or C3 had no bactericidal activity against N. meningitidis, indicating that the alternative pathway is important for inducing the terminal cascade of activation reactions. An unexpected result was that in the sera of mice deficient in MBL-A and MBL-C (both molecules are lectin pathway recognition molecules that recognize N. meningitidis), as well as in the sera of mice deficient in the serine proteases MASP-1 and MASP- 3, associated with the lectin pathway, there was a complete loss of bacteriolytic activity against N. meningitidis (Fig. 13). In a recent work (Takahashi M. et al., JEM 207: 29-37 (2010)) and in the work presented here (Fig. 32), it is shown that MASP-1 can convert the zymogenic form of factor D to its enzymatically active form and can partially explain the loss of lytic activity due to the absence of enzymatically active factor D in these sera. This does not explain the lack of bactericidal activity in MBL-deficient mice, since these mice have normal enzymatically active factor D (Banda et al., Mol. Imunol. 49(1-2):281-9 (2011)). Notably, when testing human sera taken from 3MC patients with a rare autosomal recessive disorder (Rooryck C. et al., Nat. Genet. 43(3):197-203) with mutations that tell the MASP serine protease domain -3 dysfunctionality, no bactericidal activity was found against N. meningitidis (NB: these sera have MASP-1 and factor D, but not MASP-3).

The suggestion that human sera require a lectin-mediated MASP-3-dependent activity to generate bactericidal activity was further supported by the observation that MBL-deficient human sera were also unable to lyse N. meningitidis (FIGS. 11-12) . MBL is the only human lectin pathway recognition molecule that binds to this pathogen. Since MASP-3 is not an autoactivating enzyme, the authors of the present invention hypothesized that the higher bacteriolytic activity in MASP-2-deficient serum is due to the good activation of MASP-3 by MASP-1, because in the absence of MASP-2, all lectin pathway activation complexes that bind to the bacterial surface will be loaded either

MASP-1 or MASP-3. Because activated MASP-3 cleaves both factor D (Fig. 32) and factor B to form their respective enzymatically active forms in vitro (Fig. 30 and Iwaki D.

et al., J. Immunol. 187(7):3751-3758 (2011)), the most likely function of MASP-3 is to stimulate the formation of an alternative pathway C3 convertase (i.e., C3bBbC3bBb).

Although evidence for a lectin-dependent role is compelling, however, numerous experiments have shown that MASP-3 and MASP-1 do not necessarily have to function in conjunction with lectin molecules. Experiments such as those illustrated in FIG. 28B demonstrated the ability of MASP-3 to activate an alternative pathway (as shown by C3b precipitation on S. aureus) under conditions (ie, in the presence of EGTA) that lack lectin complexes. In FIG. 28A shows that deposition under these conditions depends on the B factor, the D factor, and the P factor, each of which is a major component of the alternative pathway. In addition, factor D activation by MASP-3 and MASP-1 (FIG. 32) and factor B activation by MASP-3 (FIG. 30) can occur in vitro in the absence of lectin. Finally, studies on hemolysis of murine erythrocytes in the presence of human serum demonstrated a clear role for MBL and MASP-3 in cell lysis. However, MBL deficiency does not fully reproduce the severity of MASP-3 deficiency, as would be expected if all functional MASP-3 complexed with MBL. Thus, the authors of the present invention do not want to be limited to the opinion that all the roles of MASP-3 (and MASP-1) demonstrated in this application can be attributed solely to the function associated with lectin.

The identification of the two effector chains of the lectin pathway, as well as the possible lectin-independent functions of MASP-1, MASP-3 and HTRA-1, represents new avenues for therapy to effectively treat certain human pathologies caused by excessive complement activation in the presence of microbial pathogens or modified host cells. or metabolic deposits. As described in this application, the authors of the present invention found that in the absence of MASP-3 and in the presence of MASP-1, the alternative pathway is not activated on surface structures (see Fig. 15-16, 28B, 34-35A,B, 38- 39). Since the alternative pathway plays an important role in triggering the rate-limiting events leading to bacterial lysis as well as cell lysis (Mathieson P.W. et al., J. Exp. Med. 177(6):1827-3(1993)), then the results obtained by the authors showed that activated MASP-3 plays an important role in the lytic activity of complement. As shown in FIG. 12-13, 19-21, 36-37 and 39-40, in the sera of 3MC patients who lack MASP-3 but not MASP-1, the lytic terminal complement activation cascade is defective. The data shown in FIG. 12 and 13 demonstrated a loss of bacteriolytic activity in the absence of MASP-3 and/or MASP-1/MASP-3 functional activity. In addition, the loss of hemolytic activity in MASP-3-deficient human serum (FIGS. 19-21, 36-37, and 3940) combined with the ability to recreate hemolysis by adding recombinant MASP-3 (FIGS. 39-40) strongly supports the conclusion that that the activation of the alternative pathway on target surfaces (which is very important for triggering complement-mediated lysis) depends on the presence of activated MASP-3. Based on the new understanding of the lectin pathway detailed above, activation of the alternative pathway on target surfaces is dependent on LEA-1 and/or on lectin-independent factor B and/or factor D activation, which is also mediated by MASP-3, and therefore agents that block MASP-3-dependent complement activation, will prevent activation of the alternative pathway on target surfaces.

The disclosure of the essential role of MASP-3-dependent initiation of alternative pathway activation indicates that the alternative pathway is not an independent autonomous complement activation pathway as described in virtually all known medical guidelines and recent complement review articles. The current view, shared by many scientists, is that an alternative pathway is activated on the surface of some target particles (microbes, zymosan, and rabbit erythrocytes) by amplifying spontaneous C3 activation in an idler fashion. However, the absence of any activation of the alternative pathway in the sera of mice deficient in MASP-1 and 3-MASP and in human sera of a 3MC patient on zymosan-coated plates and in two different bacteria (N. meningitidis and S. aureus), as well as a decrease hemolysis of erythrocytes in MASP-3-deficient human and mouse sera showed that functional MASP-3 is required to initiate activation of the alternative pathway on these surfaces. This essential role of MASP-3 can be either lectin-dependent or lectin-independent and leads to the formation of alternative pathway C3 convertase and C5 convertase complexes, i.e. C3bBbC3bBb and C3bBbC3bBb(C3b)n, respectively. Thus, the authors of the present invention disclosed the existence of previously difficult to understand mechanisms for initiating an alternative path. This initiation mechanism depends on (i) LEA1, i.e. the recently discovered activation chain of the lectin pathway; and/or (ii) the lectin-independent role of the MASP-3, MASP-1, and HTRA-1 proteins.

3. The use of MASP-3 inhibitory agents for the treatment of diseases and conditions associated with the alternative pathway.

As described herein, high affinity MASP-3 inhibitory antibodies (e.g., with a binding affinity of less than 500 pM) have been shown to completely inhibit the alternative pathway in mammals, such as rodents, and in non-primate animals. , at molar concentrations lower than that of the MASP-3 target (eg, in a molar ratio of about 1:1 to about 2.5:1 (MASP-3 target:mAb) (see Examples 11-21). As described in Example 11, administration of a high-affinity MASP-3 inhibitory antibody, mAb 13B1, to mice results in almost complete removal of systemic complement pathway activity in at least 14 days.As described in more detail in Example 12, in a study conducted on the well-known In an animal model associated with PNH, mAb 13B1 was shown to significantly increase the viability of erythrocytes in PNH and protected erythrocytes in PNH compared to their viability with C5 inhibition. o in example 13, mAb 13B1 was shown to reduce the incidence and severity of disease in a mouse model of arthritis. The results presented in this example showed that the representative high affinity MASP-3 inhibitory mAbs 13B1, 10D12 and 4D5 are highly effective in blocking the alternative pathway in primates. Administration of a single dose of mAb 13B1, 10D12, or 4D5 to cynomolgus monkeys resulted in sustained elimination of systemic alternative pathway activity for approximately 16 days. The degree of elimination of the alternative pathway in cynomolgus monkeys treated with high-affinity MASP-3 inhibitory antibodies was comparable to that achieved with factor D blockade in vitro and in vivo, indicating that complete blockade of factor D conversion is achieved by MASP-3 inhibitory antibodies. antibodies. Thus, high affinity MASP-3 inhibitory mAbs are of therapeutic value in the treatment of patients suffering from diseases associated with alternative pathway hyperactivity.

Accordingly, in one of its aspects, the present invention relates to methods for inhibiting an alternative pathway in a mammal in need thereof, wherein said methods comprise administering to an individual a composition comprising an isolated monoclonal antibody, or an antigen-binding fragment thereof, that specifically binds to a serine protease domain. a human MASP (amino acid residues 450-728 in SEQ ID NO: 2) with high affinity (having a K _d of less than 500 pM) in an amount effective to inhibit alternative pathway complement activation in an individual. In some embodiments of the invention, the individual suffers from a disease or disorder associated with an alternative pathway (i.e., a disease or disorder associated with hyperactivity of the alternative pathway), such as, for example, paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD, including wet and dry IVAT), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy (including hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (AHUS), thrombotic thrombocytopenic purpura (TTP) or transplant-associated TMA ), asthma, hard deposit disease, oligoimmune necrosing crescentic glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, optic neuromyelitis, Behcet's disease, multiple sclerosis, Guillain-Barré syndrome, Alzheimer's disease, amylotrophic lateral sclerosis (ABS), lupus nephritis, systemic lupus erythematosus (SLE), diabetic retinopathy, uveitis, chronic obstructive pulmonary disease (COPD), C3 glomerulopathy, graft rejection, graft-versus-host disease (GVHD), hemodialysis, sepsis, systemic inflammatory syndrome response (SIRS), acute respiratory distress syndrome (ARDS), ANCA vasculitis, antiphospholipid syndrome, atherosclerosis, IgA nephropathy, and myasthenia gravis, described in more detail below.

A. The role of MASP-3 in the development of paroxysmal nocturnal hemoglobinuria and methods of therapy using MASP-3 inhibitory antibodies, optionally in combination with MASP-2 inhibitory agents.

Overview of PNG.

Paroxysmal nocturnal hemoglobinuria (PNH), sometimes also called Marchiafava-Michael syndrome, is an acquired and life-threatening blood disorder. PNH can develop on its own and is called primary PNH, or if it develops in association with other bone marrow disorders such as aplastic anemia, it is called secondary PNH. In most cases, PNH is the primary disease. PNH is characterized by complement-induced destruction of red blood cells (hemolysis), low red blood cell count (anemia), thrombosis, and bone marrow failure. Laboratory data on PNH show changes consistent with intravascular hemolytic anemia, i.e. low hemoglobin, elevated lactate dehydrogenase, increased reticulocytes (immature red blood cells produced by the bone marrow instead of broken cells), elevated bilirubin (a breakdown product of hemoglobin) in the absence of autoreactive RBC-binding antibodies as a possible cause.

The hallmark of PNH is chronic complement-mediated hemolysis caused by unregulated activation of terminal complement components, including the membrane attack complex on the surface of erythrocytes in the bloodstream. Erythrocytes in PNH undergo uncontrolled complement activation and hemolysis due to the lack of CD55 and CD59 regulators on the surface of erythrocytes (Lindorfer M.A. et al., Blood 115 (11) :2283-91 (2010), Risitano et al., Mini-Reviews in Medicinal Chemistry, 11:528-535 (2011)). CD55 and CD59 are overexpressed on normal erythrocytes and regulate complement activation. CD55 acts as a negative regulator of the alternative pathway by inhibiting the assembly of the alternative pathway C3 convertase complex (C3bBbC3bBb) and accelerating the degradation of previously formed convertase, thus resulting in blocking the formation of the membrane attack complex (MAC). CD59 inhibits the membrane attack complement complex directly by binding to the C5b678 complex and preventing C9 binding and polymerization.

Although hemolysis and anemia are the dominant clinical features of PNH, however, this disease is a complex hematological disorder that additionally includes thrombosis and bone marrow failure as part of the clinical findings (Risitano et al. Mini Reviews in Med. Chem., 11:528- 535 (2011)). At the molecular level, PNH is caused by abnormal clonal expansion of hematopoietic stem cells lacking a functional PIG A gene. PIG A is an X-linked gene that encodes a glycosylphosphatidyl inositol transferase required for stable surface expression of class A GPI-anchored glycoproteins, including CD55 and CD59. For unknown reasons, which are currently being investigated, hematopoietic stem cells with a dysfunctional PIG A gene, which are formed as a result of spontaneous somatic mutations, may undergo clonal expansion to the level at which their progeny constitute a significant part of the peripheral pool of hematopoietic cells. Although the erythrocyte and lymphocytic progeny of the mutant stem cell clone do not contain CD55 and CD59, however, only the erythrocytes undergo complete lysis after they enter the bloodstream.

Current treatment for PNH includes blood transfusion for anemia, the use of anticoagulants for thrombosis, and the use of the monoclonal antibody eculizumab (Soliris®), which protects blood cells from immune destruction by inhibiting the complement system (Hillmen P. et al., N. Engl. J. Med. 350(6):552-559 (2004)). Eculizumab (Soliris®) is a humanized monoclonal antibody that targets the C5 component of complement, blocking its cleavage by C5 convertases and thereby preventing C5a production and MAC assembly. Treatment of patients with PNH with eculizumab results in a reduction in intravascular hemolysis, as measured by lactate dehydrogenase (LDH) levels, and thereby stabilization of hemoglobin, with the result that approximately half of patients do not require a blood transfusion (Risitano et al, Mini Reviews in Med Chem, 11:528-535 (2011)). Although almost all patients treated with eculizumab had normal or near normal LDH levels (due to regulation of intravascular hemolysis), however, only approximately one third of patients had hemoglobin levels of approximately 11 g/dL, while the remaining patients treated eculizumab, moderate or severe (i.e. requiring a blood transfusion) anemia was observed, in almost equal proportions (Risitano A.M. et al., Blood 113:4094-100 (2009)). As described in Risitano et al., Mini-Reviews in Medicinal Chemistry, 11(6) (2011), it was demonstrated that PNH patients treated with eculizumab had a large number of C3 fragments associated with PNH erythrocytes (a this was not observed in untreated patients). This discovery led to the recognition that in PNH patients treated with Soliris, PNH erythrocytes that were no longer hemolyzed due to C5 blockade could accumulate significant amounts of membrane-bound C3 fragments that act as opsonins, resulting in their capture by reticuloendothelial cells through specific C3 receptors and subsequent extravascular hemolysis. Thus, while preventing intravascular hemolysis and its consequences, eculizumab therapy simply redirects the intravascular hemolysis of these red blood cells to extravascular, leading to residual refractory anemia in many patients (Risitano A.M. et al., Blood 113:4094-100 (2009)). Thus, in addition to the use of eculizumab, therapeutic strategies need to be developed for those patients who develop C3-mediated extravascular hemolysis, as these patients still require RBC transfusion. Such approaches targeting the C3 fragment have shown some value in experimental systems (Lindorfer et al., Blood 115:2283-91, 2010).

Complement-initiating mechanisms in PNH.

The causal relationship between defective surface expression of complement negative regulators CD55 and CD59 in PNH, combined with the efficacy of eculizumab in preventing intravascular hemolysis, strongly suggests that PNH is a complement-mediated condition. Although this paradigm has been widely accepted, however, the nature of the events that initiate complement activation and the pathway(s) of complement activation are still unclear. Because CD55 and CD59 negatively regulate the terminal amplification steps in the complement cascade that are common to all complement initiation pathways, deficiency of these molecules will result in increased formation and membrane integration of membrane attack complexes, whether or not complement activation is initiated via the lectin pathway. , by class

- 32 040888 to the logical route or by spontaneous switching to an alternative route. Thus, in patients with PNH, any complement activation events that result in C3b deposition on the erythrocyte surface can cause subsequent amplification and pathological hemolysis (intravascular and/or extravascular) and induce a hemolytic crisis. A clear understanding of the mechanism of molecular events initiating a hemolytic crisis in patients with PNH remains unclear. Since in patients with PNH and with a hemolytic crisis, there is generally no overt complement initiation event, however, the prevailing opinion is that complement activation in PNH may occur spontaneously due to the low level of activation of the alternative pathway by the type of idler, which subsequently increases as a result of inadequate regulation of terminal complement activation due to lack of CD55 and CD59.

However, it is important to note that, by its nature, PNH usually develops or worsens after certain events such as infection or trauma (Risitano, Biologics 2:205-222 (2008)), which have been shown to stimulate complement activation. . This complement activation reaction is independent of the host's immunity to the provoking pathogen and therefore most likely does not follow the classical pathway. Most likely, this complement activation reaction is initiated by lectin binding to foreign or self-modified carbohydrate patterns expressed on the surface of microbial agents or damaged host tissue. Thus, the events initiating the hemolytic crisis in PNH are closely related to complement activation initiated by lectins. This greatly raises the possibility that activation along the lectin pathway provides the initiating stimulus that ultimately leads to hemolysis in patients with PNH.

Through the use of well-defined pathogens that activate complement via lectins as experimental models for recognizing activation cascades at the molecular level, the authors demonstrated that, depending on the provoking microbe, complement activation can be initiated by either LEA-2 or LEA-1, leading to opsonization and/or lysis. This same principle of dual responses (i.e., opsonization and/or lysis) to a lectin initiation event probably also applies to other types of infectious agents, or to complement activation by lectins following host tissue injury, or to other complement activation events by lectin that can cause PNH. Based on this dual action of the lectin pathway, the inventors concluded that LEA-2- and/or LEA-1-initiated complement activation in patients with PNH stimulates opsonization and/or lysis of C3b-containing erythrocytes, followed by their extravascular and intravascular hemolysis. . Thus, once PNH is confirmed, inhibition of LEA-1 and LEA-2 would presumably eliminate both intravascular and extravascular hemolysis, thus providing a significant advantage over the C5 inhibitor eculizumab.

It was found that exposure to S. pneumoniae predominantly triggers lectin-dependent activation of LEA-2, which leads to opsonization of this microbe with C3b. Because S. pneumoniae is resistant to MAC-mediated lysis, it is cleared from the circulation by opsonization with C3b. This opsonization and its subsequent clearance from the circulation is LEA-2 dependent, as indicated by bacterial dysregulation in MASP-2-deficient mice and in mice treated with monoclonal anti-MASP-2 antibodies (PLOS Pathog. 8: E1002793 (2012) ).

While studying the role of LEA-2 in innate host responses to microbial agents, the authors tested additional pathogens. A sharply different result was observed when studying Neisseria meningitidis as a model organism. N. meningitidis also activates complement via lectins, and such complement activation is necessary to attenuate infection with N. meningitidis in a previously uninfected host. However, LEA-2 does not play any protective functional role in this response in the host. As shown in FIG. 6 and 7, blockade of LEA-2 by genetic deletion of MASP-2 did not reduce survival after N. meningitidis infection. In contrast, blockade of LEA-2 via elimination of MASP-2 resulted in a significant increase in survival (FIGS. 6 and 7) as well as improved incidence scores (FIGS. 9) in these studies. Blockade of LEA-2 by administration of an anti-MASP-2 antibody gave the same result (FIG. 10), ruling out secondary or compensatory effects in knockout mice as a possible cause. These favorable results in LEA-2-deficient animals were associated with more rapid elimination of N. meningitidis from the blood (Fig. 8). In addition, as described in this application, the incubation of N. meningitidis with normal human serum resulted in the destruction of N. meningitidis (Fig. 11). The addition of a functional monoclonal antibody specific for human MASP-2 that blocks LEA-2, but not the introduction of an isotype control monoclonal antibody, may enhance such a lytic response. However, this process is dependent on lectins and, at least in part, on the functional complement system, since MBL-deficient human serum or heat-inactivated human serum is unable to kill N. meningitidis (FIG. 11). Overall, these new data suggest that N. meningitidis infections in the presence of a functional complement system are regulated by lectin-dependent but not by LEA-2.

- 33 040888 by complement activation.

The hypothesis that LEA-1 may be the complement pathway responsible for the lectin-dependent lysis of N. meningitidis was tested using a serum sample taken from a 3 M patient. This patient was homozygous for a nonsense mutation in exon 12 of the MASP-1/3 gene. As a result, this patient lacked a functional MASP-3 protein, and vice versa, had a sufficient amount of complement (exon 12 is specific for the MASP-3 transcript, and the mutation does not have any effect on the function or expression levels of MASP-1) ( see Nat Genet 43(3):197203 (2011)). Normal human sera effectively killed N. meningitidis, but heat-inactivated sera deficient in MBL (one of the lectin pathway recognition molecules) and MASP-3-deficient sera were unable to kill N. meningitidis (FIG. 12). Thus, LEA-1 appears to mediate the destruction of N. meningitidis. This conclusion was confirmed using serum samples taken from knockout mice. Although complement-containing normal mouse sera easily killed N. meningitidis, however, MBL-deficient or MASP-1/3-deficient mouse sera were ineffective for use as heat-inactivated sera that did not contain functional complement (Fig. 13). On the other hand, MASP-2-deficient serum effectively killed N. meningitidis.

These data confirmed the previously unknown duality of the lectin pathway by revealing the existence of separate LEA-2 and LEA-1 pathways for lectin-dependent complement activation. In the examples described above, LEA-2 and LEA-1 are not redundant and mediate different functional outcomes. These data suggest that certain types of lectin pathway activators (including but not limited to S. pneumoniae) preferentially initiate complement activation by LEA-2, resulting in opsonization, while other types of activators (for example, N. meningitidis) predominantly initiate complement activation via LEA-1 and stimulate cytolytic processes. However, these data do not indicate that LEA-2 will necessarily lead to opsonization and LEA-1 to cytolytic processes, since opsonization and/or lysis may mediate both pathways under other conditions.

In the context of lectin-dependent complement activation by N. meningitidis, the LEA-2 and LEA-1 chains appear to compete with each other, since blockade of LEA-2 enhances the LEA-1-dependent lytic destruction of the organism in vitro (Fig. 13) . As detailed above, this finding can be explained by an increase in the likelihood that lectin-MASP-1 complexes will be in close proximity to lectin-MASP-3 complexes in the absence of MASP-2, which will enhance LEA-1 activation and thus , stimulate more efficient lysis of N.meningitidis. Since lysis of N. meningitidis is the main defense mechanism in a previously uninfected host, LEA-2 blockade in vivo increases the clearance of N. meningitidis and results in more efficient bacterial lysis.

Although the examples discussed above illustrate the opposite effects of LEA-2 and LEA-1 with respect to outcomes after infection with N. meningitidis, however, there may be other conditions under which both LEA-2 and LEA-1 can act synergistically and give a certain result. As detailed below, in other instances of pathological complement activation by lectins, such as those present in PNH, LEA-2- and LEA-1-induced complement activation may act synergistically and contribute to the overall pathology of PNH. In addition, as described in this application, MASP-3 also contributes to the lectin-independent conversion of factor B and factor D, which can be observed in the absence of Ca ⁺⁺ and which usually leads to the conversion of C3bB to C3bBbC3bBb and pro-factor D to factor D, which may also contribute to the pathology of PNH.

Biology and expected functional activity in PNH.

This section describes the inhibitory effect of LEA-2 and LEA-1 blockade on hemolysis in vitro in a PNH model. These data support the value of LEA-2 blocking agents (including, but not limited to, antibodies that bind to MASP-2 and block MASP-2 function) and LEA-1 blocking agents (including, but not limited to, antibodies that bind with MASP-3 and block the function of MASP-1-mediated activation of MASP-3, MASP-3, or both) for the treatment of patients with PNH of one or more types, and the value of using LEA-2 and/or LEA-1 inhibitors and/or MASP-3-dependent and lectin-independent complement activation (including MASP-2 inhibitors, MASP-3 inhibitors and dual-acting or bispecific MASP-2/MASP-3 or MASP-1/MASP-2 inhibitors and inhibitors, specific to all MASP-1/MASP2/MASP-3) to attenuate the effects of C3-mediated extravascular hemolysis in patients with PNH treated with a C5 inhibitor such as eculizumab.

MASP-2 inhibitors that block opsonization and extravascular hemolysis of PNH erythrocytes through the reticuloendothelial system.

As detailed above, patients with PNH develop anemia by two different mechanisms of RBC clearance from the circulation: intravascular hemolysis via activation of the membrane attack complex (MAC) and extravascular hemolysis after opsonization with C3b and subsequent

- 34 040888 clearance after binding to the complement receptor and uptake by the reticuloendothelial system. Intravascular hemolysis is largely prevented when the patient takes eculizumab. Since eculizumab blocks the terminal lytic effector mechanism that occurs after the complement-initiating activation event as well as after opsonization, eculizumab does not block extravascular hemolysis (Risitano A.M. et al., Blood 113:4094-100 (2009)). Instead, erythrocytes that had been hemolyzed in untreated PNH patients could now accumulate activated C3b proteins on their surface, leading to increased uptake by the reticuloendothelial system and increased their extravascular hemolysis. Thus, treatment with eculizumab effectively redirects intravascular erythrocyte hemolysis to possible extravascular hemolysis. As a result, some patients with PNH treated with eculizumab developed anemia. It follows that agents that block complement pre-activation and prevent RBC opsonization may be particularly useful in blocking the extravascular hemolysis sometimes seen with eculizumab.

The microbiological data presented here suggest that LEA-2 is often the dominant lectin-dependent opsonization pathway. In addition, when assessing lectin-dependent opsonization (determined by C3b precipitation) on three prototypical lectin activation surfaces (mannan, Fig. 17A; zymosan, Fig. 17B and S. pneumonia; Fig. 17C), it was found that LEA-2 appears to be the dominant lectin-dependent opsonization pathway under physiological conditions (ie, in the presence of C', where all complement pathways are active). Under these experimental conditions, MASP-2-deficient sera (which lack LEA-2) are essentially less effective at opsonizing test surfaces than wild-type sera. MASP-1/3-deficient serum (which lacks LEA-1) is also defective, although this effect is much less pronounced compared to serum lacking LEA-2. The relative magnitude of the contribution of LEA-2 and LEA-1 to lectin-induced opsonization is further illustrated in FIG. 18A-18C. Although the alternative complement pathway has been reported to support opsonization of lectin-activating surfaces in the absence of the lectin pathway or the classical pathway (Selander et al., J. Clin. Invest. 116(5):1425-1434 (2006)), however, the alternative pathway itself by itself (as determined under assay conditions without Ca++) is significantly less efficient than the LEA2- and LEA-1-triggered processes described herein. When extrapolated, these data suggest that the opsonization of PNH erythrocytes can also be predominantly initiated by LEA-2 and, to a lesser extent, LEA-1 (possibly strengthening such opsonization in the amplification loop along an alternative pathway), and not as a result of lectin- independent activation of an alternative path. Thus, it can be hypothesized that LEA-2 inhibitors may be most effective in limiting opsonization and preventing extravascular hemolysis in PNH. However, recognition that lectins other than MBL, such as ficolins, bind to non-carbohydrate structures such as acetylated proteins, and that MASP-3 preferentially associates with H-ficolin (Skjoedt et al., Immunobiol. 215:921 -931, 2010), leaves open the possibility of a significant role of LEA-1 in the opsonization of PNH-associated erythrocytes. Therefore, it is expected that LEA-1 inhibitors will produce additional anti-opsonizing effects, and that the combination of LEA-1 and LEA-2 inhibitors will be optimal and provide the most effective treatment by limiting opsonization and extravascular hemolysis in patients with PNH. Thus, LEA-2 and LEA-1 act additively or synergistically in stimulating opsonization, and a cross-reactive or bispecific LEA-1/LEA-2 inhibitor is expected to be most effective in blocking opsonization and extravascular hemolysis in PNH.

The role of MASP-3 inhibitors in PNH.

Using an in vitro PNH model, we have shown that complement activation and subsequent hemolysis in PNH is indeed initiated by LEA-2 and/or LEA-1 activation and that such activation is not independent of the alternative pathway. These studies used mannan-sensitized erythrocytes from mice of various species, including erythrocytes from mice deficient in Crry (an important negative regulator of the terminal complement pathway in mice), as well as erythrocytes from CD55/CD59-deficient mice, which lacked the same complement regulators that absent in patients with PNH). When mannan-sensitized Crry-deficient erythrocytes were exposed to human serum with sufficient complement, these erythrocytes underwent efficient hemolysis in 3% serum (FIGS. 19 and 20), while complement-deficient serum (HI: heat-inactivated) did not had a hemolytic function. Notably, serum with sufficient complement, where LEA-2 was blocked by the addition of an anti-MASP-2 antibody, had reduced hemolytic activity, and therefore 6% serum was needed for effective hemolysis. Similar observations were made when testing CD55/CD59-deficient erythrocytes (Fig. 22). Complement-sufficient human sera spiked with an anti-MASP-2 monoclonal antibody (i.e., sera in which LEA-2 was inhibited) was about half as effective at maintaining hemolysis as untreated sera. In addition, higher concentrations of LEA-2-blocked serum (i.e., treated with aHTu-MASP-2 monoclonal antibody) were required to promote efficient hemolysis of untreated wild-type erythrocytes compared to untreated serum (Fig. 21). .

Even more surprisingly, the sera of a 3MC patient homozygous for the dysfunctional MASP-3 protein (and therefore lacking LEA-1) were completely unable to hemolyze the mannan-sensitized Crry-deficient erythrocytes (FIGS. 20 and 21). A similar result was observed using non-sensitized normal erythrocytes: as shown in FIG. 21, LEA-1-deficient serum isolated from a 3MC patient was completely ineffective in mediating hemolysis. Overall, these data indicate that LEA-2 contributes significantly to the response to intravascular hemolysis, and LEA-1 is the predominant complement-initiating pathway leading to hemolysis. Thus, although LEA-2 blocking agents are expected to significantly reduce the rate of intravascular erythrocyte hemolysis in patients with PNH, however, it is believed that LEA-1 blocking agents should have a more pronounced effect and largely eliminate complement-mediated hemolysis.

Of note, the LEA-1-deficient 3MC patient sera used in this study had a reduced but functional alternative pathway when tested under standard alternative pathway assay conditions (FIG. 15). These data suggest that LEA-1 contributes more to hemolysis than the activity of the alternative pathway, as determined by the PNH experimental study. In conclusion, LEA-1 blocking agents will be at least as effective as agents that block other alternative pathways and help prevent or reverse intravascular hemolysis in patients with PNH.

The role of MASP-2 inhibitors in PNH.

The data presented here suggest the presence of the following mechanisms in the pathogenesis of anemia in PNH: intravascular hemolysis caused by unregulated activation of terminal complement components and erythrocyte lysis as a result of MAC formation, which is initiated predominantly, although not exclusively, by LEA-1, and extravascular hemolysis caused by opsonization erythrocytes under the action of C3b, which is supposed to be initiated predominantly by LEA-2. Although the role of LEA-2 in initiating complement activation and stimulating MAC formation and hemolysis is clear, this process was found to be significantly less efficient than LEA-1-initiated complement activation leading to hemolysis. Thus, it is assumed that LEA-2 blocking agents significantly reduce intravascular hemolysis in patients with PNH, although such therapeutic activity, as expected, is only partial. In comparison, LEA-1 blocking agents are expected to significantly reduce intravascular hemolysis in PNH patients to a much greater extent.

Extravascular hemolysis is a less dramatic but no less important mechanism of erythrocyte destruction leading to anemia in PNH and is primarily the result of C3b opsonization, which is thought to be predominantly mediated by LEA-2. Thus, LEA-2 blocking agents would be expected to preferentially block erythrocyte opsonization and subsequent extravascular hemolysis in PNH. It is expected that this unique therapeutic activity of LEA-2 blocking agents will significantly increase the effectiveness of the treatment of all patients with PNH, since at present there is no way to treat patients with PNH who have this pathogenic process.

LEA-2 inhibitors as an adjunct to treatment in addition to LEA-1 inhibitors or agents that block the terminal complement pathway.

The data presented here detail two pathogenic mechanisms of erythrocyte clearance and anemia in PNH that may be targets, alone or in combination, and to which different classes of therapeutic agents may be targeted: intravascular hemolysis, which is initiated predominantly, though not exclusively, by LEA- 1, and which is thus expected to be effectively prevented by a LEA-1 blocking agent; and extravascular hemolysis, which is caused by C3b opsonization induced predominantly by LEA-2, and which can thus be effectively prevented by a LEA-2 blocking agent.

It is well known that intravascular and extravascular mechanisms of hemolysis lead to anemia in PNH patients (Risitano et al., Blood 113:4094-4100 (2009)). Thus, it is expected that a LEA1 blocking agent that prevents intravascular hemolysis in combination with a LEA-2 blocking agent that primarily prevents extravascular hemolysis will be more effective than either of these agents in preventing anemia that develops in patients with PNH. Indeed, it is expected that the combination of LEA-1 and LEA-2 blocking agents will abolish all relevant complement initiation mechanisms in PNH and thus block all symptoms of anemia in PNH.

C5 blocking agents (such as eculizumab) are also known to effectively block intravascular hemolysis but do not prevent opsonization. As a result, in some patients with PNH,

- 36 040888 who were injected with anti-C5 antibody, remained significant residual anemia due to extravascular hemolysis mediated by LEA-2, which remains unresolved. Thus, a C5 blocking agent (such as eculizumab) that prevents intravascular hemolysis in combination with a LEA-2 blocking agent that reduces extravascular hemolysis is expected to be more effective than either agent alone in preventing anemia. that develops in patients with PNH.

It is also contemplated that other agents that block the terminal amplification loop of the complement system leading to C5 activation and MAC precipitation (including, but not limited to, agents that block properdin, factor B, or factor D or enhance the inhibitory activity of factor I, factor H or other complement-inhibiting factors), inhibit intravascular hemolysis. However, these agents are not expected to interfere with LEA-2-mediated opsonization in patients with PNH. As a result, some patients with PNH treated with such agents have maintained significant residual anemia due to extravascular hemolysis mediated by LEA-2, which remains uncorrected. Thus, treatment with agents that prevent intravascular hemolysis, in combination with an LEA-2 blocking agent that reduces extravascular hemolysis, is expected to be more effective than either agent alone in preventing the anemia that develops in patients with PNH. . Indeed, it is expected that the combination of such agents and a LEA-2 blocking agent will abolish all relevant mechanisms of erythrocyte destruction in PNH and thus block all symptoms of anemia in PNH.

Use of multispecific, bispecific or panspecific antibodies against LEA-1 and LEA-2 for the treatment of PNH.

As detailed above, the use of a combination of pharmacological agents that individually block LEA-1 and LEA-2 and therefore, taken in combination, block all complement activation events that mediate intravascular as well as extravascular hemolysis, is expected to provide the best clinical outcome in the treatment of patients with PNH. This result can be achieved, for example, by co-administering an antibody that has LEA-1 blocking activity with an antibody that has LEA-2 blocking activity. In some embodiments, the LEA-1 and LEA-2 blocking activities are combined into one molecule, and such a molecule, having combined LEA-1 and LEA-2 blocking activity, will effectively block intravascular as well as extravascular hemolysis and prevent anemia in PNG. Such a molecule may include a bispecific antibody or consist of a bispecific antibody, where one antigen-binding site specifically recognizes MASP-1 and blocks LEA-1 and reduces the level of LEA-2, and the second antigen-binding site specifically recognizes MASP-2 and also blocks LEA-2. Alternatively, such a molecule may consist of a bispecific monoclonal antibody, where one antigen-binding site specifically recognizes MASP-3 and thus blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Such a molecule can optimally consist of a bispecific monoclonal antibody, where one antigen-binding site specifically recognizes MASP-1 and MASP-3 and thus blocks LEA-1 and reduces the level of LEA-2, and the second antigen-binding site specifically recognizes MASP-2, a also blocks LEA-2. Based on the general similarity of protein sequences and architecture, one can also conclude that it is possible to obtain a standard antibody with two identical binding sites that will specifically bind to MASP-1, MASP-2 and MASP-3 in a functional form, thereby providing, functional blockade of LEA-1 and LEA-2. Such an antibody with all MASP inhibitory activity is expected to block both intravascular and extravascular hemolysis and thus be effective in the treatment of anemia in patients with PNH.

As described in Examples 11-21 herein, high affinity MASP-3 inhibitory antibodies were generated that are therapeutically effective in inhibiting an alternative pathway in AP-related diseases or conditions such as PNH.

Accordingly, in one of its embodiments, the present invention relates to a method of treating an individual suffering from PNH, or an individual at risk of developing PNH, where this method includes the introduction of an effective amount of the high-affinity monoclonal antibody described here, or its antigen-binding fragment, which binds to a human MASP-3 and inhibit alternative pathway complement activation in order to treat or reduce the risk of developing PNH in an individual.

In one of its embodiments, the present invention relates to a method of treating an individual suffering from paroxysmal nocturnal hemoglobinuria (PNH), or an individual at risk of developing paroxysmal nocturnal hemoglobinuria, which method includes administering to the individual a pharmaceutical composition containing an effective amount of a monoclonal antibody described here, or its antigen-binding fragments that bind to human MASP-3 and inhibit alternative pathway complement activation to treat PNH or reduce the risk of developing

- 37 040888 PNH in an individual, where the specified antibody or its antigen-binding fragment includes: (a) a heavy chain variable region containing (i) VHCDR1, including SEQ ID NO: 84, (ii) VHCDR2, including SEQ ID NO: 86 or SEQ ID NO: 275, and (iii) VHCDR3 comprising SEQ ID NO: 88; and (b) a light chain variable region comprising (i) VLCDR1 comprising SEQ ID NO: 142, SEQ ID NO: 257, SEQ ID NO: 258, or SEQ ID NO: 259, (ii) VLCDR2 comprising SEQ ID NO: 144, and (iii) VLCDR3 comprising SEQ ID NO: 161. In some embodiments, the pharmaceutical composition improves the viability of red blood cells in an individual suffering from PNH. In some embodiments, an individual suffering from PNH, or an individual at risk of developing PNH, exhibits one or more symptoms selected from the group consisting of: (i) decrease in hemoglobin to below normal levels, (ii) decrease in platelet count to levels below the norm; (iii) an increase in the number of reticulocytes to levels above normal; and (iv) increasing bilirubin to above normal levels. In some embodiments of the invention, the pharmaceutical composition is administered to an individual suffering from PNH, or an individual at risk of developing PNH, systemically (eg, subcutaneously, intramuscularly, intravenously, intraarterially, or by inhalation). In some embodiments of the invention, the individual suffering from PNH, or an individual at risk of developing PNH, has previously been treated or is currently being treated with a terminal complement inhibitor that inhibits the cleavage of complement C5 protein. In some embodiments, the method also includes administering to the individual a terminal complement inhibitor that inhibits the cleavage of complement C5 protein. In some embodiments, the terminal complement inhibitor is a humanized anti-C5 antibody, or an antigen-binding fragment thereof. In some embodiments, the terminal complement inhibitor is eculizumab.

B. Role of MASP-3 in the development of age-related macular degeneration and methods of therapy using MASP-3 inhibitory antibodies, optionally in combination with MASP-2 inhibitory agents.

Age-related macular degeneration (AMD) is a major cause of visual impairment and blindness in the elderly, and in developed countries, it leads to blindness in approximately 50% of cases. The incidence of ADHD in adults is approximately 3% and increases with age such that almost two-thirds of the population over the age of 80 have some evidence of the disease. More than 1.75 million people in the United States are estimated to have progressive ADHD, and its prevalence increases with age, with nearly 3 million affected by 2020 (Friedman D.S. et al., Arch. Ophthalmol. 122 :564-572, 2004). Retinal pigment epithelium (RPE) is a pathology of the retinal pigment epithelium (RPE) that results in degeneration of the photoreceptors in the upper layer of the central retina or macula and loss of central vision. Early and intermediate forms of ADJD are characterized by progressive drusen deposits, a yellowish substance containing lipid, protein, lipoprotein, and cellular debris in the subretinal space adjacent to the RPE, and retinal pigmentary lesions. Progressive ADHD consists of two clinical subtypes: non-neovascular regional atrophic (dry) ADHD and neovascular exudative (wet) ADHD. Although dry VAD accounts for 80-90% of all cases of progressive VAD, however, in most cases, sudden and severe visual loss occurs in patients with wet VAD. It is not known whether these two types of ADHD have different phenotypes associated with similar pathologies, or if they represent two different diseases. There is currently no FDA-approved treatment for dry ADJD. FDA-approved treatment for wet VAD includes intravitreal injections of anti-angiogenic drugs (ranibizumab, pegaptanib sodium, aflibercept), laser therapy, photodynamic laser therapy, and telescope implantation.

The etiology and pathophysiology of ADJD are complex and not yet fully understood. There is some evidence supporting the role of complement dysregulation in the pathogenesis of ADJD. Gene association studies have been carried out and many genetic loci associated with ADHD have been identified, including genes encoding a variety of complement proteins, factors and regulators. This disease is most associated with polymorphism in the complement factor H (CFH) gene, and, in the case of the variant with Y402H homozygotes, the risk of developing ADHD increases approximately 6 times, and in the case of heterozygotes, approximately 2.5 times compared with the genotype without the risk of developing ADHD (Khandhadia S. et al., Immunobiol. 217:127-146, 2012). Mutations in other genes encoding complement pathways have also been associated with an increased or decreased risk of developing ADHD, including complement factor B (CFB), C2, C3, factor I, and CFH-related proteins 1 and 3 (Khandhadia et al.). Immunohistochemical and proteomic studies of donor eyes in ADJD patients showed that levels of complement cascade proteins were increased and localized in drusen (Issa PC. et al., Graefes. Arch. Clin. Exp. Ophthalmol. 249:163-174, 2011 ). In addition, patients with ADJD showed increased systemic complement activation as measured by peripheral blood analysis (Issa et al., 2011, supra).

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In the pathogenesis of ADJD, the alternative complement pathway appears to be more relevant than the classical pathway. C1q, a major component of classical pathway activation recognition, was not detected in drusen by immunohistochemical assays (Mullins et al., FASEB J. 14:835 846, 2000; Johnson et al., Exp. Eye Res. 70:441 449, 2000 ). Genetic association studies have been carried out on the CFH and CFB genes. These proteins participate in an alternative pathway amplification loop, with CFH being the liquid phase inhibitor and CFB being the activating protease component of the alternative pathway. The Y402 variant of CFH affects ligand interactions, including binding to C-reactive protein, heparin, M-protein, and glycosaminoglycans. This modified ligand binding may reduce binding to cell surfaces, which in turn may lead to reduced degradation of factor I-mediated C3b activation fragment and dysregulation of the alternative pathway C3 convertase, and thereby overactivation of the alternative pathway. (Khandhadia et al., 2012, see above). Modifications in the CFB gene are associated with a protective effect in the development of ADHD. The functional fB32Q variant had a 4-fold lower binding affinity for C3b than the fB32R variant responsible for the risk of developing the disease, which led to a decrease in the level of formation of C3-convertase (Montes T. et al., Proc. Natl. Acad. Sci. U.S.A. 106 :4366-4371, 2009).

Complement-initiating mechanisms in ADJP.

The studies of genetic linkages in humans described above indicate the important role of the complement system in the pathogenesis of ADJD. In addition, complement activation products are abundant in drusen (Issa P.O. et al., Graefes. Arch. Clin. Exp. Ophthalmol. 249:163-174, 2011), which are characteristic pathological lesions in wet and dry ADJD. However, the nature of the events that initiate complement activation and the complement pathway(s) involved in them are not yet fully understood.

It is important to note that drusen deposits consist of cellular debris and retinal oxidation reaction products that accumulate under RPE in the eyes as we age. In addition, oxidative stress appears to play an important role (Cai et al., Front Biosci., 17:1976-95, 2012) and has been shown to induce complement activation in RPE (J. Biol. Chem, 284 ( 25):16939-47, 2009). It is widely known that oxidative stress and damage to cells or tissues activate the lectins of the complement system. For example, Collard et al. have shown that endothelial cells subjected to oxidative stress trigger excessive lectin-mediated complement deposition (Collard C.D. et al., Mol. Immunol., 36 (13-14): 941-8, 1999; Collard C.D. et al., Am. J. Pathol., 156(5): 154956, 2000), and that blockade of lectin binding and lectin-dependent complement activation improves outcomes in experimental models of oxidative stress injury (Collard C.D. et al., Am. J. Pathol., 156(5):1549-56, 2000). Thus, it is likely that the products of oxidative reactions present in drusen also activate complement via lectins. As a result, it can be concluded that lectin-dependent complement activation may play a key role in the pathogenesis of ADJD.

The role of the complement system has been evaluated in mice with an ADJP model. In a mouse model of radiation injury, an experimental model of photoreceptor degeneration induced by oxidative stress in knockout mice with elimination of the classical pathway (C1qa-/- on C57BL/6 shadow phenotype) had the same sensitivity to radiation injury as wild-type littermates, then how elimination of alternative pathway complement factor D (CFD-/-) contributed to protection against radiation injury (Rohrer B. et al., Invest. Ophthalmol. Vis. Sci. 48:5282-5289, 2007). In a mouse model of choroidal neovascularization (CNV) induced by Bruch's membrane laser photocoagulation, complement factor B (CFB-/-) knockout mice developed protection against CNV compared to wild-type mice (Rohrer B. et al., Invest. Ophthalmol Vis Sci 50:3056-3064, 2009). In the same model, intravenous administration of a recombinant form of complement factor H targeting complement activation sites (CR2-FH) resulted in a reduction in CNV. This protective effect was observed when CR2-FH was administered at the time of laser injury or during therapy (after laser injury). The human therapeutic variant CR2-FH (TT30) was also effective in a CNV mouse model (Rohrer B. et al. J. Ocul. Pharmacol. Ther., 28:402-409, 2012). Since Fb is activated by LEA-1, and since MASP-1 and MASP-3 are involved in factor D maturation, these results indicate that LEA-1 inhibitors may have a therapeutic effect in patients with ADHD. In addition, results from recent phase 2 studies have shown that monthly intravitreal injection of lampalizumab (formerly designated FCFD4514S and referred to as anti-factor D antibody, which is the antigen-binding fragment of a humanized monoclonal antibody directed against factor D) resulted in a reduction in the rate of progression of regional atrophy in a specific area in patients with regional atrophy caused by VAD (Yaspan B.L. et al., Sci. Transl. Med. 9, Issue 395, June 21, 2017).

The first experimental studies in a rodent model of ADJD using MBL-deficient mice did not confirm the important role of the lectin pathway in pathogenic complement activation (Rohrer et al., Mol. Immunol. 48:e1-8, 2011). However, MBL is only one of several lectins, and non-MBL lectins can induce complement activation in ADJD.

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Indeed, the authors, in their previous work, have shown that MASP-2, i.e. the rate-limiting serine protease, which is essential for the functioning of the lectin pathway, plays a critical role in the ADJP. As described in U.S. Patent No. 7,919,094 (assigned to Omeros Corporation), which is incorporated herein by reference, MASP-2 deficient mice and mice treated with anti-MASP-2 antibody were protected in a laser-induced CNV mouse model, i.e. . a validated preclinical model of wet ADJD (Ryan et al., Tr. Am. Opth. Soc. LXXVII:707-745, 1979). Thus, LEA-2 inhibitors are expected to effectively prevent CNV and improve treatment outcomes in patients with ADHD.

Thus, in view of the foregoing, it is assumed that LEA-1 and LEA-2 inhibitors should provide an independent therapeutic effect in ADHD. In addition, LEA-1 and LEA-2 inhibitors used together may provide an additive therapeutic effect compared to the effect of either agent alone, or they may provide effective treatment for a broader subgroup of patients. Combined inhibition of LEA-1 and LEA-2 can be accomplished by co-administering a LEA-1 blocking agent and a LEA-2 blocking agent. Optimally, the LEA-1 and LEA inhibitory function can be a single molecule, such as a bispecific antibody consisting of a MASP-1/3 and a MASP-2 specific binding site, or an antibody with dual specificity where each binding site can bind with MASP-1/3 or MASP-2 and block these proteins.

In accordance with the above, in one of its aspects, the present invention relates to a method of inhibiting LEA-1-dependent complement activation for the treatment of age-related macular degeneration (wet and dry forms) by administering a composition containing a therapeutically effective amount of a MASP-1 inhibitory agent , a MASP-3 inhibitory agent, or a combination of MASPl/3 inhibitory agents in a pharmaceutical carrier, to an individual suffering from such a condition. The MASP-1, MASP-3 or MASP-1/3 inhibitory composition may be administered topically to the eye, for example by irrigation, intravitreal administration, or application of the composition in the form of a gel, ointment or drops. Alternatively, the MASP-1, MASP-3, or MASP-1/3 inhibitory agent may be administered to the individual systemically, for example, intra-arterially, intravenously, intramuscularly, by inhalation, intranasally, subcutaneously, or other parenteral route, or possibly by oral administration of non-peptidergic agents. The introduction can be repeated in accordance with the doctor's prescription until such a condition is eliminated or stopped.

In one embodiment, the method according to this aspect of the invention further comprises inhibiting LEA-2-dependent complement activation in an individual suffering from age-related macular degeneration, wherein said method comprises administering a therapeutically effective amount of a MASP-2 inhibitory agent and MASP-1-, MASP-3 or MASP-1/3 inhibitory agent to an individual in need thereof. As detailed above, it is expected that the use of a combination of pharmacological agents that individually block LEA-1 and LEA-2 will improve therapeutic outcome in patients with ADHD compared to inhibition of LEA-1 alone. This result can be achieved, for example, by co-administering an antibody that has LEA-1 blocking activity with an antibody that has LEA-2 blocking activity. In some embodiments, the LEA-1 and LEA-2 blocking activities are combined into one molecule, and such a molecule will have combined LEA-1 and LEA-2 blocking activity. Such a molecule may include a bispecific antibody or consist of a bispecific antibody where one antigen-binding site specifically recognizes MASP-1 and blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Alternatively, such a molecule may consist of a bispecific monoclonal antibody, where one antigen-binding site specifically recognizes MASP-3 and thus blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Such a molecule may optimally consist of a bispecific monoclonal antibody, where one antigen-binding site specifically recognizes MASP-1 and MASP-3 and thus blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and also blocks LEA-2.

The MASP-2 inhibitory composition may be administered topically to the eye, for example, by irrigation, intravitreal administration, or application of the composition in the form of a gel, ointment or drops. Alternatively, the MASP-2 inhibitory agent may be administered to the individual systemically, eg, intra-arterially, intravenously, intramuscularly, by inhalation, intranasally, subcutaneously, or other parenteral route, or possibly by oral administration of non-peptidergic agents. The introduction can be repeated in accordance with the doctor's prescription until such a condition is eliminated or stopped.

The use of MASP-3 inhibitory compositions and optionally MASP-2 inhibitory compositions according to the invention can be carried out by a single administration of the composition (for example, a single composition containing MASP-2 and MASP-3 inhibitory agents or bispecific agents, or agents of dual inhibitory action, or co-administration of individual compositions), or a limited range of administrations for the treatment of ADHD. Alternatively, the composition may be administered intermittently at intervals, such as daily, twice a week, weekly, biweekly, monthly, or bimonthly over an extended period of time for treatment.

VJP.

As described in Examples 11-21 herein, high affinity MASP-3 inhibitory antibodies were generated that are therapeutically effective in inhibiting an alternative pathway in AP-related diseases or conditions such as ADHD.

Accordingly, in one of its embodiments, the present invention relates to a method of treating an individual suffering from ADHD or an individual at risk of developing ADHD, where this method includes the introduction of an effective amount of the high-affinity monoclonal antibody described here, or its antigen-binding fragment, which binds to human MASP -3 and inhibit alternative pathway complement activation in order to treat or reduce the risk of ADHD in an individual. In one of its embodiments, the present invention relates to a method of treating an individual suffering from ADHD, or an individual at risk of developing ADHD, where this method includes administering to the individual a pharmaceutical composition containing an effective amount of a monoclonal antibody described here, or an antigen-binding fragment that binds to human MASP -3 and inhibit complement activation of an alternative pathway to treat ADHD or reduce the risk of developing ADHD in an individual, wherein said antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region comprising (i) VHCDR1 comprising SEQ ID NO: 84, (ii) VHCDR2 comprising SEQ ID NO: 86 or SEQ ID NO: 275, and (iii) VHCDR3 comprising SEQ ID NO: 88; and (b) a light chain variable region comprising (i) VLCDR1 comprising SEQ ID NO: 142, SEQ ID NO: 257, SEQ ID NO: 258 or SEQ ID NO: 259, (ii) VLCDR2 comprising SEQ ID NO: 144, and (iii) VLCDR3 comprising SEQ ID NO: 161.

C. Role of MASP-3 in the development of ischemia-reperfusion injury and methods of therapy using MASP-3 inhibitory antibodies, optionally in combination with MASP-2 inhibitory agents.

Tissue ischemia is the basis of a wide range of clinical disorders. Although the timely restoration of blood flow is very important for the preservation of tissue damaged by ischemia, however, it has long been recognized that reperfusion, which can occur either spontaneously or through therapeutic treatment, can lead to additional tissue damage, i.e. to a phenomenon that has been termed ischemia-reperfusion (I/R) injury (Eltzschig H.K. and Tobias, E., Nat. Med. 17:1391-1401, 2011). I/R damage can affect specific organs such as the heart (acute coronary syndrome), kidneys (acute kidney injury), intestines (intestinal I/R), and brain (stroke). I/R injury can also cause multiple organ damage such as major injury and subsequent reperfusion (multiple organ failure), circulatory arrest (cerebral hypoxia, acute kidney injury), peripheral vascular disease, and sickle cell anemia (acute chest injury syndrome). cells, acute kidney injury). Major surgeries may be associated with I/R injury, including cardiac surgery (in case of acute heart failure after cardiopulmonary bypass), chest surgery (in case of acute pulmonary insufficiency), peripheral vascular surgery (in case of compartment syndrome), vascular surgery (in case of acute renal failure), and solid organ transplantation (in case of acute graft rejection). Currently, there is no specific treatment for I/R injury, and therefore, effective treatments need to be developed to maximize tissue preservation in the ischemic zone and improve functional outcomes with such general procedures.

The pathophysiology of I/R injury is complex and is characterized by a persistent inflammatory response to reperfusion. Activation of the complement system is involved as an important component of I/R damage, and inhibition of complement activity has been effective in animal models of this pathology (Diepenhorst G.M.P. et al., Ann. Surg. 249:889-899, 2009). The relative importance of the classical, lectin, and alternative pathways in I/R injury remains controversial and may differ depending on the affected organs. Recently, the creation of knockout mice deficient in specific complement proteins and specific inhibitors of a particular pathway has provided data on the involvement of the lectin and alternative pathways in I/R damage.

The role of the alternative pathway in I/R injury of the gastrointestinal tract was investigated using factor D(-/-) deficient and heterozygous (+/-) mice (Stahl G. L. et al. Am. J. Pathol. 162:449-455 , 2003). After transient gastrointestinal ischemia, intestinal and pulmonary injury was reduced but not prevented in factor D deficient mice compared to heterozygous mice, and human factor D supplementation in factor D (-/-) deficient mice resulted in recovery of I/ P-damage. The same model was evaluated in C1q-deficient and MBL-A/C-deficient mice, and the results showed that gastrointestinal I/R damage was independent of C1q and classical pathway activation, but MBL and lectin pathway activation always resulted in 41 040888 led to intestinal damage (Hart M.L. et al. J. Immunol. 174:6373-6380, 2005). On the other hand, the C1q classical pathway recognition molecule has been responsible for lung injury after intestinal I/R injury (Hart M.L. et al. J. Immunol. 174:6373-6380, 2005). One hypothesis is that complement activation during I/R injury occurs due to the binding of native IgM to self-antigens present on the surface of ischemic (but not normal) tissue, such as non-muscle type II myosin heavy chains. In a mouse model of gastrointestinal I/R injury, gut tissue immunocomplexes were evaluated for the presence of classical (C1q), lectin (MBL), or alternative (factor B) pathway initiators (Lee H. et al., Mol. Immunol. 47:972- 981, 2010). The results showed that C1q and MBL were detected, and factor B was not detected in these immunocomplexes, indicating that they are involved in the classical and lectin pathways, but not in the alternative pathway. In the same model, FB-deficient mice were not protected from local tissue damage, further confirming that FB was not involved in the alternative pathway. The role of the lectin pathway in gastrointestinal I/R damage was directly assessed in MASP-2-deficient mice, and the results showed that gastrointestinal I/R damage in these mice was reduced compared to wild type controls, and treatment with anti -MASP-2 antibody produced a similar protective effect (Schwaeble W.J. et al., Proc. Natl. Acad. Sci. 108:7523-7528, 2011). Overall, these results supported the involvement of the lectin pathway in gastrointestinal I/R injury, which is inconsistent with data regarding the involvement of an alternative pathway.

In a mouse model of I/R myocardial injury, a pathogenic role was demonstrated for the lectin pathway, as MBL-deficient mice showed protection against myocardial injury, while C1q-deficient and C2/Fb-deficient mice did not (Walsh M.C. et al. al., J. Immunol. 175:541-546, 2005). Protection against I/R myocardial injury has also been observed in MASP-2 deficient mice (Schwaeble W.J. et al., Proc. Natl. Acad. Sci. 108:7523-7528, 2011). Treatment of rats with a model of I/R myocardial injury with monoclonal antibodies against rat MBL resulted in a reduction in postischemic reperfusion injury (Jordan J.E. et al., Circulation 104:1413 18, 2001). In a study of patients with myocardial infarction who underwent angioplasty, MBL deficiency was associated with a reduction in mortality at 90 days compared with patients who had sufficient MBL (M. Trendelenburg et al., Eur. Heart J. 31:1181, 2010). In addition, patients with myocardial infarction who developed heart failure after angioplasty had MBL levels approximately three times higher than those who underwent functional rehabilitation therapy (Haahr-Pedersen S. et al., J. Inv. Cardiology, 21:13, 2009). AntiMBL antibodies also reduced the level of complement deposition on endothelial cells in vitro after oxidative stress, suggesting a role for the lectin pathway in myocardial I/R injury (Collard, C.D. et al., Am. J. Pathol. 156:1549 56, 2000) . In a heterotopic heart isograft mouse model with I/R injury, the role of the alternative pathway was explored using the pathway-specific CR2-fH fusion protein (Atkinson C. et al., J. Immunol. 185:7007-7013, 2010). Systemic administration of CR2-fH immediately after transplantation resulted in a reduction in myocardial I/R damage to a level comparable to that observed after treatment with CR2-Crry, which inhibits all complement pathways, indicating the key role of the alternative pathway in this model.

An alternative pathway is involved in a mouse model of I/R kidney injury, as factor B-deficient mice were protected from decreased kidney function and tubular injury compared to wild-type mice (Thurman J. M. et al., J. Immunol. 170:1517-1523 , 2003). Treatment with inhibitory anti-factor B monoclonal antibody prevented complement activation and reduced I/R kidney damage in mice (Thurman J. M. et al., J. Am. Soc. Nephrol. 17:707-715, 2006). In a bilateral I/R kidney injury model, MBL-A/C-deficient mice were protected from kidney injury compared to wild-type mice, and recombinant human MBL abolished the protective effect in MBL-A/C-deficient mice, indicating the role of MBL in this model (Moller-Kristensen M. et al., Scand. J. Immunol. 61:426-434, 2005). In a rat model of unilateral I/R kidney injury, inhibition of MBL with an anti-MBL-A monoclonal antibody resulted in preservation of kidney function after I/R (van der Pol P. et al., Am. J. Transplant. 12:877-887, 2010). Interestingly, the role of MBL in this model does not appear to be related to terminal complement activation, as anti-C5 antibody treatment was ineffective in preventing kidney damage. Most likely, MBL has a direct toxic effect on tubular cells, since in vitro incubation of human proximal tubule cells with MBL led to the internalization of MBL into these cells, followed by their apoptosis. In a porcine model of I/R kidney injury, Castellano G. et al. (Am J Pathol, 176(4):1648-59, 2010) tested a C1 inhibitor that irreversibly inactivates C1r and C1s proteases in the classical pathway, as well as proteases MASP-1 and MASP-2 in MBL complexes of the lectin pathway, and it was found that the C1 inhibitor reduced the rate of complement deposition in the peritubular capillaries and glomeruli and reduced the rate of tubular injury.

An alternative pathway appears to be involved in experimental traumatic brain injury, as factor B-deficient mice showed reduced systemic activation

- 42 040888 complement, determined in serum by levels of C5a, and reduced post-traumatic neuronal death compared with wild-type mice (Leinhase I. et al., BMC Neurosci. 7:55-67, 2006). In the case of human stroke, complement components C1q, C3c and C4d were detected by immunohistochemical staining in the area of ischemic lesions, suggesting activation along the classical pathway (Pedersen E.D. et al., Scand. J. Immunol. 69:555-562 , 2009). Targeting the classical pathway in an animal model of cerebral ischemia has had mixed results, with some studies showing protection while others showing no effect (Arumugam T.V. et al., Neuroscience 158:1074-1089, 2009). Experimental and clinical studies have provided strong evidence for the involvement of the lectin pathway. In experimental stroke models, deficiency of MBL or MASP-2 resulted in a reduction in infarct size compared to wild-type mice (Cervera A. et al.; PLoS One 3;5(2): e8433, 2010; Osthoff M. et al., PLoS One, 6(6):e21338, 2011). In addition, stroke patients who were found to have low MBL levels had a better prognosis compared to patients who had normal MBL levels (Osthoff M. et al., PLoS One, 6(6):e21338, 2011) .

In baboons with an extracorporeal circulation model, treatment with an anti-factor D monoclonal antibody resulted in inhibition of systemic inflammation, as measured by plasma levels of C3a, SC5b-9, and IL-6, and reduced myocardial tissue damage, indicating involvement of an alternative pathway in this models (Undar A. et al., Ann. Thorac. Surg. 74:355-362, 2002).

Thus, depending on the I/R-damaged organ, all three complement pathways can contribute to the pathogenesis and adverse outcome. Based on the experimental and clinical data detailed above, it has been suggested that LEA-2 inhibitors will be protective in most cases of I/R injury. Lectin-dependent activation of LEA-1 can cause complement activation via an alternative pathway, at least in some cases. In addition, LEA-2-induced complement activation can be further enhanced by the alternative pathway amplification loop and thus exacerbate I/R-associated tissue damage. Thus, it has been suggested that LEA-1 inhibitors provide results in additive or adjuvant therapy in patients suffering from a condition associated with ischemia.

In view of the foregoing, LEA-1 and LEA-2 inhibitors are expected to have independent therapeutic benefits in the treatment, prevention, or amelioration of conditions associated with ischemia and reperfusion. In addition, LEA-1 and LEA-2 inhibitors used together may provide an additive therapeutic effect compared to the effect of any single agent. Therefore, optimally effective treatment of an I/R-associated condition involves the administration of active pharmaceutical ingredients that, alone or in combination, block LEA-1 and LEA-2. Combined inhibition of LEA-1 and LEA-2 can be achieved by co-administration of a LEA-1 blocking agent and a LEA-2 blocking agent. Preferably, the LEA-1 and LEA2 inhibitory function may have a single molecule, such as a bispecific antibody consisting of a MASP-1/3 and a MASP-2 specific binding site, or an antibody with dual specificity where each binding site can bind with MASP-1/3 or MASP-2 and block these proteins.

In accordance with the foregoing, in one of its aspects, the present invention relates to a method of inhibiting LEA-1-dependent complement activation for the treatment, prevention or amelioration of the severity of ischemia-reperfusion injuries by administering a composition containing a therapeutically effective amount of a LEA-1 inhibitory agent, including a MASP-1 inhibitory agent, a MASP-3 inhibitory agent, or a combination of MASP-1/3 inhibitory agents in a pharmaceutical carrier to an individual suffering from ischemic reperfusion. The MASP-1, MASP3, or MASP-1/3 inhibitory composition may be administered to the individual intra-arterially, intravenously, intracranially, intramuscularly, subcutaneously, or by other parenteral route, and optionally orally, for non-peptidergic inhibitors, and most preferably , intraarterially or intravenously. The administration of the LEA-1 inhibitory compositions of the invention will typically begin immediately after an ischemic reperfusion event or, if possible, shortly after such an event. In cases where reperfusion occurs in a controlled environment (eg, after aortic aneurysm repair, organ transplantation, or replantation of diseased or injured limbs or fingers), the LEA-1 inhibitory agent may be administered before and/or during and/or after reperfusion. The introduction can be periodically repeated in accordance with the appointment of a physician until an optimal therapeutic effect is achieved.

In some embodiments, these methods are used to treat or prevent ischemia-reperfusion injury associated with at least aortic aneurysm repair, cardiopulmonary bypass, vascular reanastomosis in connection with organ transplantation, and/or limb/finger replantation, stroke, myocardial infarction, and hemodynamic resuscitation after a stroke and/or surgery.

In some embodiments, these methods are used to treat

- 43 040888 or prevention of ischemia-reperfusion injury in an individual who needs organ transplantation, in an individual who is undergoing organ transplantation, or in an individual who has already undergone organ transplantation. In some embodiments of the invention, these methods are used to treat or prevent ischemia-reperfusion injury in an individual who is in need of an organ transplant, who is undergoing an organ transplant, or who has already received an organ transplant, provided that the organ to be transplanted is not a kidney transplant.

In one embodiment, the method according to this aspect of the invention further comprises inhibiting LEA-2 dependent complement activation in an individual suffering from ischemic reperfusion, wherein said method comprises administering a therapeutically effective amount of a MASP-2 inhibitory agent and MASP-1-, MASP- 3 or MASP-1/3 inhibitory agent to an individual. As detailed above, the use of a combination of pharmacological agents that individually block LEA-1 and LEA-2 is expected to improve therapeutic outcome in the treatment, prevention, or amelioration of ischemia-reperfusion injury compared to inhibition of LEA-1 alone. This result can be achieved, for example, by co-administering an antibody that has LEA-1 blocking activity with an antibody that has LEA-2 blocking activity. In some embodiments, the LEA-1 and LEA-2 blocking activities are combined into one molecule, and such a molecule will have combined LEA-1 and LEA-2 blocking activity. Such a molecule may include a bispecific antibody or consist of a bispecific antibody where one antigen-binding site specifically recognizes MASP-1 and blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Alternatively, such a molecule may consist of a bispecific monoclonal antibody, where one antigen-binding site specifically recognizes MASP-3 and thus blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Such a molecule may optimally consist of a bispecific monoclonal antibody, where one antigen-binding site specifically recognizes MASP-1 and MASP-3 and thus blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2.

The MASP-2 inhibitory composition may be administered to the individual in need thereof intra-arterially, intravenously, intracranially, intramuscularly, subcutaneously, or by other parenteral route, and optionally orally for non-peptidergic inhibitors, and most preferably intra-arterially or intravenously. The administration of the MASP-2 inhibitory compositions of the invention will typically begin immediately after an ischemic reperfusion event or, if possible, shortly after such an event. In cases where reperfusion occurs in a controlled environment (eg, after repair of an aortic aneurysm, organ transplant, or replantation of diseased or injured limbs or fingers), a MASP-2 inhibitory agent may be administered before and/or during and/or after reperfusion. The introduction can be periodically repeated in accordance with the appointment of a physician until an optimal therapeutic effect is achieved.

The use of MASP-3 inhibitory compositions and, optionally, MASP-2 inhibitory compositions according to the invention can be carried out by a single administration of the composition (for example, one composition containing MASP-2 and MASP-3 inhibitory agents or bispecific agents, or agents dual inhibitory action, or co-administration of separate compositions), or a limited range of administrations for the treatment or prevention of ischemia-reperfusion injury.

Alternatively, the composition may be administered intermittently at intervals, such as daily, twice a week, weekly, every other week, monthly, or twice a month for an extended period of time to treat an individual suffering from ischemic reperfusion.

As described here in Examples 11-21, high affinity MASP-3 inhibitory antibodies were generated that are therapeutically effective in inhibiting an alternative pathway in AP-related diseases or conditions, for example, in an individual suffering from ischemic reperfusion.

Accordingly, in one of its embodiments, the present invention relates to a method of treating an individual suffering from ischemic reperfusion, or an individual at risk of developing ischemic reperfusion, where this method includes the introduction of an effective amount of the high affinity monoclonal antibody described here, or its antigen-binding fragment, which binds to human MASP-3 and inhibit alternative pathway complement activation in order to treat or reduce the risk of tissue damage associated with ischemic reperfusion in an individual.

D. Role of MASP-3 in the development of inflammatory and non-inflammatory arthritis and methods of therapy using MASP-3 inhibitory antibodies, optionally in combination with MASP-2 inhibitory agents.

Rheumatoid arthritis (RA) is a chronic inflammatory joint disease

- 44 040888 which can also have system manifestations. RA affects approximately 1% of the world's population, and women are two to three times more likely to develop the disease. Inflammation of the joints manifests itself in the form of swelling, pain and stiffness. As the disease progresses, erosion and destruction of the joint can occur, leading to limited mobility and deformities. The goal of RA treatment is to prevent or reverse joint damage, prevent loss of joint function and disease progression, and reduce symptoms, improve quality of life, and achieve drug-free remission. Pharmacological treatment of RA includes the administration of disease-modifying antirheumatic drugs (DMARDs), analgesics, and anti-inflammatory drugs (glucocorticoids and non-steroidal anti-inflammatory drugs). DMARDs are the most important treatments because they can produce long-term remissions and delay or stop the progression of joint destruction, which is irreversible. Traditional DMARDs are small molecules such as methotrexate, sulfasalazine, hydroxychloroquine, gold salts, leflunomide, D-penicillamine, cyclosporine, and azathioprine. If traditional DMARDs are not sufficient to control the disease, then some biological agents that target inflammatory cells or inflammatory mediators can be used, such as tumor necrosis factor inhibitors (etanercept, infliximab, adalimumab, certolizumab-pegol, and golimumab), cytokine antagonists (anakinra and tocilizumab), rituximab, and abatacept.

Although it is clear that adaptive immunity plays a central role in the pathogenesis of RA, as evidenced by genetic linkage to T-cell activation genes and the presence of autoantibodies, however, natural immune mechanisms are also involved in such pathogenesis (McInnes I.B. and Schett G. New Engl. J. Med. 365:2205-2219, 2011). In human RA, levels of alternative pathway Bb fragment cleavage in synovial fluid were several times higher than levels in samples from patients with crystal-induced arthritis or degenerative joint disease, indicating preferential activation of the alternative pathway in patients with PA (Brodeur J.P. et al., Arthritis Rheum. 34:1531-1537, 1991). In an experimental arthritis model generated by passive transfer of an anti-collagen type II antibody, factor B-deficient mice showed reduced inflammation and joint damage compared to wild-type mice, while disease activity in C4-deficient mice was similar to disease activity in wild-type mice. type, indicating that the alternative rather than the classical pathway plays an important role in this model (Banda, N.K. et al., J. Immunol. 177:1904-1912, 2006). In the same experimental model of collagen antibody-induced arthritis (CAIA), mice with classical or lectin-only pathway activity did not develop arthritis (Banda N.K. et al., Clin. Exp. Immunol. 159:100-108, 2010) . Data from this study showed that the classical or lectin pathways are able to activate low levels of C3 in vitro. However, in the absence of an alternative pathway amplification loop, the level of C3 deposition in the joint was insufficient for the development of clinical disease. A key step in the activation of the alternative pathway is the conversion of the factor D zymogen (pro-factor D) to mature factor D, which is mediated by MASP-1 and/or MASP-3 (Takahashi M. et al., J. Exp. Med. 207:29 -37, 2010) and/or HTRA1 (Stanton et al., Evidence That the HTRA1 Interactome Influences Susceptibility to Age-Related Macular Degeneration, presented at The Association for Research in Vision and Ophthalmology 2011 conference on May 4, 2011). The role of MASP-1/3 was evaluated in murine CAIA and the results showed that MASP-1/3 deficient mice were protected from arthritis compared to wild-type mice (Banda N.K. et al., J. Immunol. 185:5598-5606, 2010). In MASP-1/3-deficient mice, pro-factor D, but not mature factor D, was detected in serum during the development of CAIA, and in vitro addition of human factor D to the serum of these mice resulted in restoration of C3 activation and C5a generation. . In contrast, in a mouse model of the effector phase of arthritis, C3-deficient mice developed very mild arthritis compared to wild-type mice, and factor B-deficient mice continued to develop arthritis, indicating independent involvement of the classical/lectin pathway. and alternative route (Hietala M.A. et al., Eur. J. Immunol. 34:1208-1216, 2004). Transgenic mice in the K/BxN T-cell receptor-induced inflammatory arthritis model and mice lacking C4 or C1q exhibited arthritis similar to wild-type mice, whereas Factor B-deficient mice either did not develop arthritis, or developed in a very mild manner, indicating an important role for the alternative pathway, but not the classical pathway, in this model (Ji H. et al., Immunity 16:157-168, 2002). In the K/BxN model, mice lacking MBL-A were not protected against serum-induced arthritis, but since the role of MBL-C has not yet been investigated, a possible role for the lectin pathway cannot be ruled out (Ji et al., 2002, see . higher).

Two research groups have independently proposed that lectin-dependent complement activation induces inflammation in RA patients through the interaction of MBL with specific IgG glycoforms (Malhotra et al., Nat. Med. 1:237 243, 1995; Cuchacovich et al., J Rheumatol 23:44 51, 1996). Of note, rheumatoid conditions are associated with a marked increase in levels of IgG glycoforms that do not contain galactose (referred to as glycoforms

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IgG0) in the Fc region of the molecule (Rudd et al., Trends Biotechnology 22:524 30, 2004). The percentage of IgG0 glycoforms increases as the rheumatoid disease progresses and returns to normal levels when patients are in remission. In vivo, IgG0 is deposited on synovial tissue and MBL is present at elevated levels in the synovial fluid of patients with RA. Aggregated agalactosyl-IgG (IgG0), which is associated with RA, can bind to MBL, and therefore can initiate lectin-dependent complement activation via LEA-1 and/or LEA-2. In addition, the results of a clinical study conducted to search for MBL allelic variants in RA patients suggest that MBL may play a role in exacerbating inflammatory disease (Garred et al., J. Rheumatol. 27:26 34, 2000). Therefore, lectin-dependent complement activation by LEA-1 and/or LEA-2 may play an important role in the pathogenesis of RA.

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

Similarly, complement activation plays an important role in the development of psoriatic arthritis. In patients with this disease, there is an increase in the levels of complement activation products in the bloodstream, and red blood cells appear with lower levels of the complement regulator CD59 (Triolo, Clin Exp Rheumatol., 21(2):225-8, 2003). Complement levels are associated with disease activity and have a high predictive value for determining treatment outcomes (Chimenti at al., Clin. Exp. Rheumatol., 30(1):23-30, 2012). Indeed, recent studies have shown that the effect of anti-TNF antibody therapy for this condition is due to complement modulation (Ballanti et al., Autoimmun Rev., 10(10):617-23, 2011). Although the role of complement in the development of psoriatic arthritis has not been clearly defined, however, the presence of complement activation products C4d and Bb in the bloodstream of these patients indicates their important role in pathogenesis. Based on the observed products, it is suggested that LEA-1, and possibly also LEA-2, are responsible for the pathological complement activation in these patients.

Osteoarthritis (OA) is the most common form of arthritis, affecting more than 25 million people in the United States. OA is characterized by the destruction and complete loss of articular cartilage and is accompanied by the formation of new bone tissue and the accumulation of synovial fluid, which causes pain, stiffness and loss of joint function and leads to disability. Joints that are frequently affected by OA are those of the arms, neck, lower back, knees, and hips. The disease is progressive, and current treatments aim to relieve symptomatic pain, but do not address the causes of the disease. The pathogenesis of OA remains unclear, but the role of complement in this pathogenesis cannot be ruled out. When proteomic and transcriptomic analyzes of synovial fluid were performed in patients with OA, several complement components were abnormally expressed compared to samples taken from healthy individuals, including the classical pathway (C1s and C4A) and the alternative pathway (factor B), as well as C3, C5, C7 and C9 (Wang, Q. et al., Nat. Med. 17:1674-1679, 2011). In addition, in a mouse model of medial meniscectomy-induced OA, C5-deficient mice showed less cartilage loss, less osteophyte formation, and less severe synovitis than C5-positive mice, and treatment of wild-type mice with CR2-fH, i.e. a fusion protein that inhibits the alternative pathway led to a reduction in the development of OA (Wang et al., 2011, see above).

Ross River virus (RRV) and Chikungunya virus (CHIKV) belong to a group of mosquito viruses that can cause acute and persistent arthritis and myositis in humans. In addition to being endemic, these viruses can cause epidemics that could involve millions of infected people. Arthritis is believed to be initiated by viral replication, and key components of this process are the induction of an inflammatory response in the host joint and activation of the complement system. The synovial fluid of a person with RRV-induced polyarthritis contains higher levels of C3a than the synovial fluid of a person with OA (Morrison TE et al., J. Virol. 81:51325143, 2007). In a mouse model of RRV infection, C3-deficient mice showed less severe arthritis than wild-type mice, suggesting a role for complement in this disease (Morrison et al., 2007, supra). The involvement of the specific complement pathway was investigated, and it was found that in mice with inactivated lectin pathway (MBL-A-/- and MBL-C-/-), arthritis had a milder form compared to wild-type mice. In contrast, mice with inactivated classical pathway (C1q-/-) or alternative pathway (factor B-/-) developed severe arthritis, indicating that the MBL-initiated lectin pathway played a significant role in this model (Gunn B M. et al., PLoS Pathog 8:e1002586, 2012). Because arthritis is associated with joint damage, initial joint damage of various etiologies may trigger a second wave of complement activation by LEA-2. In support of this concept, the authors note,

- 46 040888 that their previous work showed that MASP-2 knockout mice had reduced joint damage compared to wild-type mice in a collagen-induced RA model.

Based on the data detailed above, LEA-1 and LEA-2 inhibitors, alone or in combination, are expected to be therapeutically effective in the treatment of arthritis.

Therefore, the optimally effective treatment of arthritis may include the administration of active pharmaceutical ingredients that, alone or in combination, block LEA-1 and LEA-2. Combined inhibition of LEA-1 and LEA-2 can be achieved by co-administration of a LEA-1 blocking agent and a LEA-2 blocking agent. Preferably, the LEA-1 and LEA-2 inhibitory function may be a single molecule, such as a bispecific antibody consisting of a MASP-1/3 and a MASP-2 specific binding site, or an antibody with dual specificity where each binding site can bind with MASP-1/3 or MASP-2 and block these proteins. In accordance with the above, in one of its aspects, the present invention relates to a method of inhibiting LEA-1-dependent complement activation for the treatment, prevention or reduction of the severity of inflammatory or non-inflammatory arthritis, including osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis and psoriatic arthritis, by administering a composition containing a therapeutically effective amount of a LEA-1 inhibitory agent, including a MASP-1 inhibitory agent, a MASP-3 inhibitory agent, or a combination of MASP-1/3 inhibitory agents in a pharmaceutical carrier, to an individual suffering from inflammatory or non-inflammatory arthritis, or an individual at risk of developing such arthritis. The MASP-1, MASP-3, or MASP-1/3 inhibitory composition may be administered to the individual systemically, for example, intra-arterially, intravenously, intramuscularly, subcutaneously, or by other parenteral route, or orally. Alternatively, the administration may be carried out locally, for example by intra-articular injection. Administration of the LEA-1 inhibitory agent may be given intermittently over an extended period of time to treat or resolve a chronic condition, or it may be given once or more times before, during, and/or after severe injury or injury, including surgery to joints.

In one embodiment, the method according to this aspect of the invention further comprises inhibiting LEA-2-dependent complement activation in an individual suffering from inflammatory or non-inflammatory arthritis (including osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis and psoriatic arthritis), or in an individual at risk of the development of such arthritis, wherein said method comprises administering a therapeutically effective amount of a MASP2 inhibitory agent and a MASP-1, MASP-3 or MASP-1/3 inhibitory agent to an individual. As detailed above, it is expected that the use of a combination of pharmacological agents that individually block LEA-1 and LEA-2 will improve therapeutic outcome in the treatment or prevention of arthritis compared to inhibition of LEA-1 alone. This result can be achieved, for example, by co-administering an antibody that has LEA-1 blocking activity with an antibody that has LEA-2 blocking activity. In some embodiments, the LEA-1 and LEA-2 blocking activities are combined into one molecule, and such a molecule will have combined LEA-1 and LEA-2 blocking activity. Such a molecule may include a bispecific antibody or consist of a bispecific antibody where one antigen-binding site specifically recognizes MASP-1 and blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Alternatively, such a molecule may consist of a bispecific monoclonal antibody, where one antigen-binding site specifically recognizes MASP-3 and thus blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Such a molecule can optimally consist of a bispecific monoclonal antibody, where one antigen-binding site specifically recognizes MASP-1 and MASP-3 and thus blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2.

The MASP-2 inhibitory composition may be administered to an individual in need thereof systemically, eg, intra-arterially, intravenously, intramuscularly, subcutaneously, or by other parenteral route, or possibly orally for non-peptidergic inhibitors. Alternatively, the administration may be carried out locally, for example by intra-articular injection. Administration of the MASP-2 inhibitory agent may be given intermittently over an extended period of time to treat or resolve a chronic condition, or it may be given once or more times before, during, and/or after severe injury or injury, including surgery to joints.

The use of the MASP-3 inhibitory compositions and optionally the MASP-2 inhibitory compositions of the invention may be accomplished by a single administration of the composition (e.g., a single composition containing MASP-2 and MASP-3 inhibitory agents or bispecific agents, or agents dual inhibitory action, or co-administration of separate compositions), or a limited range of administrations for the treatment, prevention or amelioration of the severity of inflammatory or non-inflammatory arthritis. Alternatively, the composition may be administered intermittently at intervals, such as daily, twice a week, weekly, every other week, monthly, or twice a month for an extended period of time to treat an individual suffering from inflammatory or non-inflammatory arthritis. .

As described in Examples 11-21 herein, high affinity MASP-3 inhibitory antibodies were generated that are therapeutically effective in inhibiting an alternative pathway in AP-related diseases or conditions such as arthritis.

Accordingly, in one of its embodiments, the present invention relates to a method of treating an individual suffering from arthritis, or an individual at risk of developing arthritis (inflammatory and non-inflammatory arthritis), which method includes administering to the individual a pharmaceutical composition containing an effective amount of a high affinity monoclonal described here an antibody, or an antigen-binding fragment thereof, that binds to human MASP-3 and inhibits alternative pathway complement activation, for the purpose of treating or reducing the risk of developing arthritis in an individual, wherein, for example, said antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region containing (i) VHCDR1 comprising SEQ ID NO: 84, (ii) VHCDR2 comprising SEQ ID NO: 86 or SEQ ID NO: 275, and (iii) VHCDR3 comprising SEQ ID NO: 88; and (b) a light chain variable region comprising (i) VLCDR1 comprising SEQ ID NO: 142, SEQ ID NO: 257, SEQ ID NO: 258, or SEQ ID NO: 259, (ii) VLCDR2 comprising SEQ ID NO: 144, and (iii) a VLCDR3 comprising SEQ ID NO: 161. In some embodiments, the subject suffers from arthritis selected from the group consisting of osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, ankylosing spondylitis, Behcet's disease, arthritis caused by infection, and psoriatic arthritis. In some embodiments, the pharmaceutical composition is administered systemically (i.e., subcutaneously, intramuscularly, intravenously, intraarterially, or by inhalation). In some embodiments of the invention, the pharmaceutical composition is administered topically to the joint.

E. Role of MASP-3 in the development of disseminated intravascular coagulation (DIC) and methods of therapy using MASP-3 inhibitory antibodies, optionally in combination with MASP-2 inhibitory agents.

Disseminated intravascular coagulation (DIC) is a syndrome of pathological overstimulation of the blood coagulation system, which can present clinically as hemorrhage and/or thrombosis. DIC does not develop as a primary condition, but is usually associated with various pathological processes, including tissue damage (trauma, burns, heat stroke, transfusion reaction, acute graft rejection), neoplasia, infections, complications during childbirth (placenta previa, amniotic fluid embolism, toxicosis pregnancy), as well as various mixed conditions such as cardiogenic shock, lung injury in water accidents, fat embolism, aortic aneurysm. Thrombocytopenia is a pathology that often occurs in patients in the intensive care unit, with a frequency of 35 to 44%, and in approximately 25% of these cases, it is the etiological factor of DIC, i.e. DIC develops in approximately 10% of critically ill patients (Levi M. and Opal S.M. Crit. Care 10:222-231, 2006). Pathophysiology DIC is a pathological process, which is based on the initiation of a physiological reaction of blood coagulation. However, prothrombotic substances suppress the normal balancing mechanisms in such a way that in this case there is an incorrect deposition of fibrin and platelets in the capillary bloodstream, which leads to organ ischemia, hypofibrinogenemia and thrombocytopenia. Diagnosis of DIC is based on a clinical presentation consistent with the underlying disease or pathological process, along with abnormal laboratory parameters (such as prothrombin time, partial thromboplastin time, fibrin degradation products, presence of D-dimers, or platelet count). First-line treatment for DIC is to prevent the underlying condition that is responsible for the development of DIC. To treat or prevent clinical complications, it may be necessary to maintain blood components in the form of red blood cells, platelets, fresh frozen plasma and cryoprecipitate.

The role of complement pathways in the development of DIC has been explored in several studies. Complement activation was assessed in children with meningococcal infection by comparing the clinical course of the disease by MBL genotype (Sprong T. et al., Clin. Infect. Dis. 49:1380-1386, 2009). On admission to the clinic, MBL-deficient patients had lower circulating levels of C3bc, terminal complement complex, C4bc, and C3bBbC3bBbP than those with adequate MBL levels, indicating a lower degree of total complement, terminal complement, and alternative pathway activation. . In addition, the degree of systemic complement activation correlates with the severity of the disease and with the parameters of DIC, and MBL-deficient patients had a milder course of the disease than those with sufficient MBL levels. Therefore, although MBL deficiency is a risk factor for patients susceptible to infection, however, MBL deficiency during septic shock may be associated with reduced disease severity.

As shown here in examples 1-4, experimental studies have revealed an important contribution of MBL and MASP-1/3 to the innate immune response to Neisseria menigitidis, ie. 48 040888 etiological factor of meningococcal infection. MBL-deficient sera from mice or humans, MASP-3-deficient human sera, or sera from MASP-1/3 knockout mice were less effective in complement activation and lysis of meningococci in vitro compared to wild-type sera. Similarly, MASP-1/3 knockout mice, previously unexperimented, were more susceptible to Neisseria infection than similar wild-type mice. Thus, in the absence of adaptive immunity, the LEA-1 pathway contributes to the host's natural resistance to Neisseria infection. Conversely, LEA-1 enhances pathological complement activation, which triggers an unwanted response in the host, including DIC.

In a mouse model of arterial thrombosis, MBL-deficient and MASP-1/3 knockout mice had reduced FeCl ₃ -induced thrombogenesis compared to wild-type or C2/factor B deficient mice, and this defect was repaired with recombinant human MBL (La Bonte LR et al., J. Immunol. 188:885-891, 2012). In vitro, sera from MBL-deficient or MASP-1/3-knockout mice had reduced levels of thrombin cleavage of the substrate compared to sera from wild-type or C2/Factor B deficient mice, and addition of recombinant human MASP-1 resulted in a recovery of the product. substrate cleavage by thrombin in the serum of MASP-1/-3 knockout mice (La Bonte et al., 2012, see above). These results showed that the MBL/MASP complexes, and in particular MASP-1, play a key role in thrombus formation. Thus, LEA-1 may play an important role in pathological thrombosis, including DIC.

Experimental studies have revealed the same important role of LEA-2 in pathological thrombosis. In vitro studies have also demonstrated that LEA-2 provides a molecular link between the complement system and the blood coagulation system. MASP-2 has activity similar to that of factor Xa and activates prothrombin by cleavage to form thrombin, which can subsequently clear fibrinogen and promote fibrin clot formation (see also Krarup et al., PLoS One, 18:2(7):e623 , 2007).

Separate studies have shown that lectin-MASP complexes can promote clot formation, fibrin deposition, and fibrinopeptide release via a MASP-2-dependent mechanism (Gulla K.C. et al., Immunology, 129(4):482-95, 2010). Thus, LEA-2 simultaneously stimulates lectin-dependent complement activation and the blood coagulation system.

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

The data detailed above suggest that LEA-1 and LEA-2 provide an independent link between lectin-dependent complement activation and blood coagulation. Thus, in view of the above data, it is assumed that LEA-1 and LEA-2 inhibitors provide an independent therapeutic effect in the treatment of an individual suffering from disseminated intravascular coagulation. In some embodiments of the invention, the individual suffers from disseminated intravascular coagulation caused by sepsis, trauma, infection (bacterial, viral, fungal, parasitic), malignancy, transplant rejection, blood transfusion reaction, childbirth complications, vascular aneurysm, liver failure, heat shock, burn, radiation exposure, stroke, or severe toxic reaction (eg, snakebite, insect bite, reaction to a blood transfusion). In some embodiments, the injury is a neurological injury. In some embodiments, the infection is a bacterial infection, such as an infection caused by Neisseria meningitidis.

In addition, LEA-1 and LEA-2 inhibitors used together may provide an additive therapeutic effect compared to the effect of any single agent. Since LEA-1 and LEA-2 are known to be activated under conditions leading to DIC (e.g., infection or injury), it is hypothesized that LEA-1 and LEA-2 blocking agents, either alone or in combination , should give a therapeutic effect in the treatment of DIC. LEA-1 and LEA-2 blocking agents can block various crosstalk mechanisms between complement response and blood coagulation. Thus, LEA-1 and LEA-2 blocking agents may have a complementary, additive, or synergistic effect in preventing DIC and other thrombotic disorders.

In addition, LEA-1 and LEA-2 inhibitors used together may provide an additive therapeutic effect compared to the effect of any single agent, or they may be effective in treating a large number of subgroups of patients. Combined inhibition of LEA-1 and LEA2 can be achieved by co-administration of a LEA-1 blocking agent and a LEA-2 blocking agent. The LEA-1 and LEA-2 inhibitory function may optionally have a single molecule, such as a bispecific antibody consisting of a MASP-1/3 and a MASP-2 specific binding site, or an antibody with dual specificity, where each the binding site can bind to MASP-1/3 or MASP-2 and block these proteins.

- 49 040888

In accordance with the above, in one of its aspects, the present invention relates to a method of inhibiting LEA-1-dependent complement activation for the treatment, prevention or amelioration of the severity of disseminated intravascular coagulation in an individual in need thereof, where this method includes the introduction of a composition containing a therapeutically effective amount of a LEA-1 inhibitory agent, including a MASP-1 inhibitory agent, a MASP-3 inhibitory agent, or a combination of MASP-1/3 inhibitory agents in a pharmaceutical carrier, to an individual suffering from or at risk of developing disseminated intravascular coagulation . The MASP-1, MASP-3, or MASP-1/3 inhibitory composition may be administered to the individual systemically, for example, intra-arterially, intravenously, intramuscularly, by inhalation, intranasally, subcutaneously, or by another parenteral route, or possibly by oral administration. non-peptidergic agents. The introduction can be repeated in accordance with the doctor's prescription until such a condition is eliminated or stopped. For the treatment or prevention of DIC caused by trauma or other injury, the LEA-1 inhibitory composition may be administered immediately after traumatic injury, or prophylactically before, during, immediately after, or 1-7 days or more, such as 2472 hours after injury, causing injury, or another situation such as surgery in patients at risk of developing DIC. In some embodiments, the LEA-1 inhibitory composition may be appropriately administered as a rapid-acting dosage form, for example, by intravenous or intra-arterial bolus delivery of a solution containing the LEA-1 inhibitory agent composition.

In one embodiment, the method according to this aspect of the invention also includes inhibiting LEA-2-dependent complement activation to treat, prevent, or ameliorate disseminated intravascular coagulation in an individual in need thereof, wherein said method comprises administering a therapeutically effective amount of MASP- 2 inhibitory agent and MASP-1, MASP-3 or MASP1/3 inhibitory agent to an individual. As detailed above, it is expected that the use of a combination of pharmacological agents that individually block LEA-1 and LEA-2 will improve therapeutic outcome in the treatment or prevention of disseminated intravascular coagulation compared to inhibition of LEA-1 alone. This result can be achieved, for example, by co-administering an antibody that has LEA-1 blocking activity with an antibody that has LEA-2 blocking activity. In some embodiments, the LEA-1 and LEA-2 blocking activities are combined into one molecule, and such a molecule will have combined LEA-1 and LEA-2 blocking activity. Such a molecule may include a bispecific antibody or consist of a bispecific antibody where one antigen-binding site specifically recognizes MASP-1 and blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Alternatively, such a molecule may consist of a bispecific monoclonal antibody, where one antigen-binding site specifically recognizes MASP-3 and thus blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Such a molecule can optimally consist of a bispecific monoclonal antibody, where one antigen-binding site specifically recognizes MASP-1 and MASP-3 and thus blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2.

The MASP-2 inhibitory agent may be administered to an individual in need thereof systemically, eg intraarterially, intravenously, intramuscularly, by inhalation, intranasally, subcutaneously, or other parenteral route, or possibly by oral administration of non-peptidergic agents. The introduction can be repeated in accordance with the doctor's prescription until such a condition is eliminated or stopped. For the treatment of DIC caused by trauma or other injury, the MASP-2 inhibitory composition may be administered immediately after the traumatic injury, or prophylactically before, during, immediately after, or 17 days or more, e.g., 24-72 hours after the injury causing the injury. , or another event such as surgery in patients at risk for DIC. In some embodiments, the MASP-2 inhibitory composition may be appropriately administered in a fast-acting dosage form, for example, by intravenous or intra-arterial bolus delivery of a solution containing the MASP-2 inhibitory agent composition.

The use of the MASP-3 inhibitory compositions and optionally the MASP-2 inhibitory compositions of the invention may be accomplished by a single administration of the composition (e.g., a single composition containing MASP-2 and MASP-3 inhibitory agents or bispecific agents, or agents dual inhibitory action, or co-administration of separate compositions), or a limited range of administrations for the treatment, prevention or reduction of the severity of disseminated intravascular coagulation in an individual in need of it. Alternatively, the composition may be administered intermittently at intervals, for example, daily, twice a week, weekly, biweekly, monthly, or bimonthly for an extended period of time.

- 50 040888 period of time for the treatment of an individual suffering from disseminated intravascular coagulation, or an individual at risk of developing such a disease.

As described in Examples 11-21 herein, high affinity MASP-3 inhibitory antibodies were generated that are therapeutically effective in inhibiting an alternative pathway in AP-related diseases or conditions such as disseminated intravascular coagulation.

Accordingly, in one of its embodiments, the present invention relates to a method of treating an individual suffering from disseminated intravascular coagulation, or an individual at risk of developing such a disease, where this method includes the introduction of an effective amount of the high-affinity monoclonal antibody described here, or its antigen-binding fragment, which bind to human MASP-3 and inhibit alternative pathway complement activation to treat or reduce the risk of disseminated intravascular coagulation, for example, wherein said antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region comprising (i) VHCDR1 comprising SEQ ID NO: 84, (ii) VhCdR2 comprising SEQ ID NO: 86 or SEQ ID NO: 275, and (iii) VHCDR3 comprising SEQ ID NO: 88; and (b) a light chain variable region comprising (i) VLCDR1 comprising SEQ ID NO: 142, SEQ ID NO: 257, SEQ ID NO: 258, or SEQ ID NO: 259, (ii) VLCDR2 comprising SEQ ID NO: 144, and (iii) VLCDR3 comprising SEQ ID NO: 161.

F. The role of MASP-3 in the development of thrombotic microangiopathy (TMA), including hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (AHUS), and thrombotic thrombocytopenic purpura (TTP) and therapies using MASP-3 inhibitory antibodies, optionally in combination with MASP-2 inhibitory agents.

Thrombotic microangiopathy (TMA) refers to a group of disorders characterized by clinical thrombocytopenia, microangiopathic hemolytic anemia, and ischemia of various organs. The characteristic pathological signs of TMA are platelet activation and the formation of microthrombi in small arterioles and venules. The classic TMAs are hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). HUS differs from TTP by the presence of acute renal failure. HUS occurs in two forms: diarrhea-associated (D+) or typical HUS and non-diarrhea-associated (D-) or atypical HUS (aHUS).

GUS.

D+HUS is associated with a prodromal diarrheal illness, usually caused by Escherichia coli 0157 or another Shiga toxin-producing strain of bacteria, which occurs in more than 90% of cases in children and is the most common cause of acute kidney injury in children. Although infection with Escherichia coli 0157 in humans occurs relatively often, however, in isolated cases, the percentage of bloody diarrhea that progresses to D + HUS is from 3 to 7%, and in some outbreaks of infection, it is from 20 to 30% (Zheng X.L. and Sadler J.E., Annu. Rev. Pathol. 3: 249-277, 2008). HUS usually presents 4 to 6 days after the onset of diarrhea, and approximately two-thirds of children with acute illness require dialysis. Treatment for D+HUS is supportive as there is no specific effective treatment for it. The prognosis of D+HUS is favorable, and in most patients, renal function is restored.

The pathogenesis of D+HUS involves bacterially produced Shiga toxins that bind to membranes on microvascular endothelial cells, monocytes, and platelets. In most cases, the microvascular system of the kidneys is affected. Once bound, the toxin is internalized, leading to the release of pro-inflammatory mediators and ultimately cell death. It is believed that endothelial cell damage causes thrombosis of the microvessels of the kidneys by stimulating the activation of the cascade of blood coagulation reactions. There are data indicating activation of the complement system in D+HUS. In children with D+HUS, plasma levels of Bb and SC5b-9 were increased at the time of hospitalization compared to normal controls, and on the 28th day after discharge from the hospital, plasma levels were normal (Thurman J.M. et al., Clin. J. Am. Soc. Nephrol. 4:1920-1924, 2009). Shiga toxin 2 (Stx2) has been found to activate human complement in the liquid phase in vitro mainly via an alternative pathway as it is activated in the presence of ethylene glycol tetraacetic acid, which blocks the classical pathway (Orth D. et al., J. Immunol 182:6394-6400, 2009). In addition, Stx2 binds to factor H, but not to factor I, and slows down the cofactor activity of factor H on the cell surface (Orth et al, 2009, supra). These results suggest that Shiga toxin may cause kidney damage through a variety of potential mechanisms, including direct toxic effects and mediated complement activation or inhibition of complement regulators. It is suggested that toxic effects on the vascular endothelium activate complement via LEA-2, as indicated by the effectiveness of MASP-2 blockade in preventing complement-mediated reperfusion injury in various vascular beds, as described by Schwaeble W.J. et al., Proc. Natl. Acad. sci. 108:7523-7528, 2011.

In a mouse model of HUS induced by a combined injection of Shiga toxin and lipopolysaccharide, factor B-deficient mice exhibited milder thrombocytopenia and were protected from kidney injury compared to wild-type mice, indicating involvement of LEA. -1-dependent activation of an alternative pathway in the development of microvascular thrombosis (Morigi M. et al., J. Immunol. 87:172-180, 2011). As described herein, in the same model, anti-MASP-2 antibody administration was also effective and increased survival after STX challenge, indicating involvement of the LEA-2-dependent complement pathway in the development of microvascular thrombosis.

Based on the data described above, LEA-1 and LEA-2 inhibitors are expected to be therapeutically effective for the treatment or prevention of HUS. In addition, LEA-1 and LEA-2 inhibitors used together may provide an additive therapeutic effect compared to the effect of any single agent, or they may be effective in treating a large number of subgroups of patients. Combined inhibition of LEA-1 and LEA-2 can be achieved by coadministration of a LEA-1 blocking agent and a LEA-2 blocking agent.

Optimally, the LEA-1 and LEA-2 inhibitory function can be a single molecule, such as a bispecific antibody consisting of a MASP-1/3 and a MASP-2 specific binding site, or an antibody with dual specificity, where each binding site can bind to MASP-1/3 or MASP-2 and block these proteins.

aHUS.

Atypical HUS is a rare disease, with an estimated 2 in a million in the United States (Loirat C. and Fremeaux-Bacchi V. Orphanet J. Rare Dis. 6:60-90, 2011). Atypical HUS can develop at any age, although in most patients it begins in childhood. Atypical HUS is heterogeneous: in some cases it is hereditary, in some cases it is recurrent, and in some cases it is caused by an infectious disease, usually an upper respiratory disease or gastroenteritis. aHUS usually develops suddenly, and most patients require dialysis on admission to the hospital. Approximately 20% of patients have extrarenal manifestations, and such manifestations may include damage to the central nervous system, myocardial infarction, ischemic gangrene of distal organs, or multiple organ failure. Treatment of AGUS consists of maintenance therapy to eliminate organ dysfunction, plasma infusion or plasmapheresis and administration of eculizumab, i.e. a humanized monoclonal antibody that targets C5 and has recently been approved for use in the United States and the European Union. The prognosis of aHUS is not as good as that of D+HUS, with approximately 25% of deaths occurring in the acute phase and most of the survivors developing end-stage kidney disease.

Atypical HUS is characterized as a disease associated with complement dysregulation, and approximately 50% of patients have mutations in the genes encoding complement regulation proteins (Zheng and Sadler, 2008, supra). Most of the mutations are observed in factor H (FH) and other mutations are membrane protein cofactor (MCP), factor I (FI), factor B and C3. Functional studies have shown that mutations in FH, MCP, and FI result in loss of function and hence increased complement activation, while mutations in factor B are responsible for increased function. The effects of these mutations predominantly affect the alternative pathway. These genetic abnormalities are risk factors, and are not the only cause of the disease, as approximately 50% of family members who carry the mutation do not develop the disease until the age of 45 (Loirat and Fremeaux-Bacchi, 2011, supra).

Factor H is a complement regulation protein that protects host tissue from exposure to the alternative complement pathway. FH regulates the alternative pathway amplification loop by three mechanisms, namely: a cofactor for FI, which cleaves C3b, inhibits the formation of the alternative pathway C3 convertase, C3bBbC3bBb; binds to polyanions on cell surfaces and tissue matrices; and blocks C3b precipitation (Atkinson J.P. and Goodship T.H.J., J. Exp. Med. 6:1245-1248, 2007). Most FH mutations in aHUS patients occur in the C-terminal short consensus repeat domains of the protein, resulting in defective binding of FH to heparin, C3b, and endothelium, but do not disrupt the regulation of plasma C3, which is located between the N-terminal domains (Pickering M. C. et al., J. Exp. Med. 204:1249-1256, 2007). FH-deficient mice exhibit unregulated plasma C3 activation and spontaneously develop type II membranoproliferative glomerulonephritis, but not aHUS. However, FH-deficient mice that transgenic express a mouse FH protein that is functionally equivalent to aHUS-associated human FH mutants spontaneously develop HUS but not type II membranoproliferative glomerulonephritis, as supported by in vivo data indicating that dysregulation of alternative pathway activation in the renal endothelium is a key event in the pathogenesis of FH-associated aHUS (Pickering et al., 2007, supra). Another form of FH-associated aHUS occurs in patients who have anti-FH autoantibodies, resulting in loss of functional FH activity; and most of these patients have deletions in the genes encoding five FH

- 52 040888 related proteins (Loirat and Fremeaux-Bacchi, 2011, see above).

Like FH, MCP inhibits complement activation by regulating C3b deposition on target cells. MCP mutations lead to the production of proteins with low C3b-binding and cofactor affinity, which leads to impaired activation of the alternative pathway. FI is a serine protease that cleaves C3b and C4b in the presence of cofactors such as FH and MCP and thereby prevents the formation of C3 and C5 convertases and inhibits both the alternative and classical complement pathways. Most FI-associated mutations in aHUS result in reduced FI activity in degrading C3b and C4b (Zheng and Stadler, 2008, supra). FB is a zymogen that carries the catalytic sites of the alternative pathway C3bBbC3bBb convertase. Functional analyzes have shown that aHUS-associated FB mutations result in increased activation of the alternative pathway (Loirat and Fremeaux-Bacchi, 2011, supra). Heterozygous C3 mutations are associated with aHUS. Most C3 mutations induce a disruption in the binding of C3 to MCP, which leads to an increase in the efficiency of FB binding to C3b and to an increase in the production of C3 convertase (Loirat and Fremeaux-Bacchi, 2011, supra). Thus, aHUS is a disease closely associated with mutations in complement genes, which leads to inadequate regulation of the alternative pathway amplification loop. Since the amplification loop of the alternative pathway depends on factor B proteolytic activity, and because LEA-1 is required for factor B activation (either MASP-3 dependent cleavage or factor D-mediated cleavage, where MASP-1 contributes to factor D maturation), it is expected that LEA-1 blocking agents will prevent unregulated complement activation in sensitive individuals. As a result, LEA-1 blocking agents are expected to be effective in the treatment of aHUS.

Although the central role of dysregulation of the alternative pathway amplification loop in the development of aHUS is well known, however, the stimuli that initiate complement activation and the molecular pathways involved in such activation are not yet known. However, aHUS does not develop in all people who carry the mutations described above. Indeed, family history has shown that aHUS occurs in only ~50% of cases (Sullivan M. et al., Ann. Hum. Genet. 74:17-26 2010). The pathogenesis of the disease indicates that aHUS most often develops after an initiation event such as an infectious outbreak or trauma. It is well known that infectious agents activate the complement system. In the absence of pre-existing adaptive immunity, complement activation by infectious agents may be initiated primarily by LEA-1 or LEA-2. Thus, lectin-dependent complement activation initiated by infection may serve as an initiating stimulus for the subsequent pathological amplification of complement activation in individuals with aHUS predisposition, which may ultimately lead to disease progression. Accordingly, in another aspect, the present invention includes treating a patient suffering from aHUS caused by an infection by administering an effective amount of a LEA-1 or LEA-2 inhibitory agent.

Other forms of host tissue damage will activate complement through LEA-2, in particular, will cause damage to the vascular endothelium. Human vascular endothelial cells are subject to oxidative stress, for example, by responding to the expression of surface molecules that bind to lectins and activate the complement LEA-2 pathway (Collard et al., Am. J. Pathol. 156(5):1549-56, 2000). Vascular injury following ischemia/reperfusion also activates complement via LEA-2 in vivo (Moller-Kristensen et al., Scand. J. Immunol. 61(5):426-34, 2005). Activation of the lectin pathway in this situation has pathological consequences for the host, and as shown in examples 22 and 23, inhibition of LEA-2 by blocking MASP-2 prevents further host tissue damage and adverse outcomes (see also Schwaeble PNAS, 2011, supra) .

Thus, it is also known that other processes that cause aHUS lead to the activation of LEA-1 or LEA-2. Therefore, it is likely that the LEA-1 and/or LEA-2 pathway may represent an initial complement activation mechanism that is inappropriately amplified by a dysregulatory mechanism in individuals with a genetic predisposition to AHUS, and thus initiates the pathogenesis of aHUS. From this it can be concluded that agents that block complement activation by LEA-1 and/or LEA-2 are expected to prevent disease progression or reduce its severity in individuals susceptible to aHUS.

To further support this concept, recent studies have shown that the bacterium Streptococcus pneumoniae is an important etiological agent involved in the development of aHUS in children (Lee C.S. et al. Nephrology, 17(1):48-52 (2012); Banerjee R. et al., Pediatr Infect Dis J., 30(9):736-9 (2011)). A disease of this etiology is considered to have a poor prognosis with high mortality and chronic complications. Notably, these cases are non-intestinal infections leading to manifestations of microangiopathy, uremia, and hemolysis without evidence of parallel mutations in the complement genes known to cause aHUS. It is important to note that S.pneumoniae is particularly effective in complement activation and carries out this

- 53 040888 function via LEA-2. Thus, it is assumed that in the case of non-intestinal HUS associated with pneumococcal infection, manifestations of microangiopathy, uremia, and hemolysis are induced predominantly by activation of LEA-2, and it is assumed that agents that block LEA2, including anti-MASP-2 antibodies , will prevent the progression of aHUS or reduce the severity of this disease in patients. Accordingly, in another aspect, the present invention includes treating a patient suffering from non-intestinal aHUS caused by S. pneumoniae infection by administering an effective amount of a MASP-2 inhibitory agent.

TTP.

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disorder of the blood coagulation system caused by autoimmune or hereditary dysfunctions that activate the coagulation cascade or complement system (George J.N., N. Engl. J. Med.; 354:1927-35, 2006 ). This leads to the formation of numerous microscopic clots or blood clots in small blood vessels throughout the body, which is a hallmark of TMA. RBCs are subjected to shear stress that damages their membranes, leading to intravascular hemolysis. As a result, blood flow is reduced and the endothelium is damaged, leading to organ damage, including the brain, heart, and kidneys. TTP is clinically characterized by thrombocytopenia, microangiopathic hemolytic anemia, neurological changes, renal failure, and fever. At a time when plasmapheresis was not yet known, the mortality rate for acute disease was 90%. Even with plasmapheresis, 80% of patients with this disease have a life expectancy of approximately six months.

TTP may result from genetic or acquired inhibition of the ADAMTS-13 enzyme, i.e. a metalloprotease responsible for the breakdown of large von Willebrand factor (vWF) multimers into smaller fragments. Inhibition or deficiency of ADAMTS-13 ultimately leads to an increase in blood clotting (Tsai H.J. Am. Soc. Nephrol. 14: 1072-1081, 2003). ADAMTS-13 regulates vWF activity; and in the absence of ADAMTS-13, vWF form large multimers that tend to bind to platelets, which will predispose patients to platelet aggregation and microvascular thrombosis.

Numerous mutations in ADAMTS13 have been identified in individuals with TTP. This disease can also develop due to the presence of autoantibodies against ADAMTS-13. In addition, TTP can develop with cancer of the breast, gastrointestinal tract or prostate (George J.N., Oncology (Williston Park). 25:908-14, 2011), during pregnancy (in the second trimester or after childbirth) (George J.N. , Curr. Opin. Hematol. 10:339-344, 2003), or TTP may be associated with diseases such as HIV infections or autoimmune diseases such as systemic lupus erythematosus (Hamasaki K. et al., Clin. Rheumatol. 22 :355-8, 2003). TTP may also be induced by some therapeutic drugs, including heparin, quinine, an immune-mediated ingredient, cancer chemotherapy agents (bleomycin, cisplatin, cytosine-arabinoside, daunomycin, gemcitabine, mitomycin C, and tamoxifen), cyclosporin A, oral contraceptives, penicillin, rifampin and anti-platelet drugs including ticlopidine and clopidogrel (Azarm. T. et al., J. Res. Med. Sci., 16: 353-357, 2011). Other factors or conditions associated with TTP are toxins such as bee venom, sepsis, splenic sequestration, transplantation, vasculitis, vascular surgery, and infections such as Streptococcus pneumoniae and cytomegalovirus (Moake J.L., N. Engl. J. Med. 347:589-600, 2002). TTP due to a temporary functional deficiency of ADAMTS-13 may result from endothelial cell damage associated with S. pneumoniae infection (Pediatr. Nephrol., 26:631-5, 2011).

The standard treatment for TTP is plasmapheresis (Rock G.A. et al., N. Engl. J. Med. 325:393-397, 1991). Plasmapheresis replaces ADAMTS-13 activity in patients with genetic defects and removes anti-ADAMTS-13 autoantibodies in patients with acquired autoimmune TTP (Tsai H.-M. Hematol. Oncol. Clin. North. Am., 21(4): 609-v , 2007). Typically, such therapy uses additional agents such as immunosuppressants (George. J. N., N. Engl. J. Med. 354:1927-35, 2006). However, plasmapheresis fails in about 20% of patients, and more than one third of patients relapse, and besides, plasmapheresis is expensive and requires sophisticated technical equipment. In addition, many patients are unable to withstand the plasmapheresis procedure. Therefore, there is an urgent need to develop additional and more effective treatments for TTP.

Since TTP is a disruption in the coagulation cascade, treatment with complement antagonists will help stabilize and reduce the severity of the disease. Although pathological activation of the alternative complement pathway is associated with aHUS, however, the role of complement activation in TTP is not yet very clear. Functional deficiency of ADAMTS13 plays an important role in susceptibility to TTP, but this is not enough to cause disease in the acute phase. Environmental factors and/or other genetic modifications may contribute to the manifestation of TTP. For example, genes encoding proteins involved in the regulation of the cascade

- 54 040888 coagulation reactions, vWF, platelet function, endothelial vascular surface components or the complement system may also be involved in the development of acute thrombotic microangiopathy (Galbusera M. et al., Haematologica, 94: 166-170, 2009). In particular, complement activation has been shown to play a critical role, and serum from a patient with thrombotic microangiopathy associated with ADAMTS-13 deficiency has been shown to cause C3 and MAC precipitation and subsequent activation of neutrophils that can be eliminated by complement inactivation ( Ruiz-Torres M. P. et al., Thromb Haemost, 93:443-52, 2005). In addition, it has recently been shown that during acute attacks of TTP, the levels of C4d, C3bBbC3bBbP and C3a are increased (M. Reti et al., J. Thromb. Haemost. 10(5):791-798, 2012), which corresponds to the activation classical, lectin and alternative pathways. This leads to an increase in complement activation during acute attacks and can initiate terminal pathway activation as well as further exacerbation of TTP.

It appears that ADAMTS-13 and vWF in TTP play a critical role in platelet activation and aggregation and thus a role in shear stress and deposition in microangiopathies. Activated platelets interact with and stimulate the classical and alternative complement pathways. Platelet-mediated complement activation leads to increased levels of inflammatory mediators C3a and C5a (Peerschke E. et al., Mol. Immunol., 47:2170-5 (2010)). Thus, platelets can serve as targets for activation of the classical complement pathway in the case of hereditary or autoimmune TTP.

As described above, lectin-dependent complement activation, mediated by thrombin-like activity of MASP-1 and LEA-2-mediated prothrombin activation, is the dominant molecular pathway causing endothelial damage after blood clotting and microvascular thrombosis that are seen in HUS. Similarly, activation of LEA-1 and LEA-2 can directly induce the blood coagulation system in TTP. Activation of the LEA-1 and LEA-2 pathways may be initiated in response to initial endothelial injury caused by ADAMTS-13 deficiency in TTP. Thus, LEA-1 and LEA-2 inhibitors, including but not limited to antibodies that block MASP-2 function, MASP-1 function, MASP-3 function, or MASP-1 and MASP-3 function, are expected to will attenuate microangiopathies associated with microvascular coagulation, thrombosis and hemolysis in TTP patients.

TTP patients usually present to the emergency room with one or more conditions such as purpura, kidney failure, low platelet count, anemia, and/or thrombosis, including stroke. Currently, the standard treatment for TTP involves the insertion of a catheter (eg, intravenous line or other form of catheter) for plasmapheresis replacement for two weeks or more, and usually three times a week, up to daily insertion. If an individual is positive for an ADAMTS13 inhibitor (ie, an endogenous antibody against ADAMTS13), then plasmapheresis may be performed in combination with immunosuppressive therapy (eg, by administering corticosteroids, rituxan, or cyclosporine). Patients with recurrent TTP (approximately 20% of patients with TTP) do not experience any response to plasmapheresis therapy for at least two weeks.

In accordance with the above, in one of the embodiments of the invention, when making an initial diagnosis of TTP, or if the individual has one or more symptoms consistent with the diagnosis of TTP (for example, central nervous system involvement, severe thrombocytopenia (platelet count less than or equal to 5000/µl , if no aspirin was administered, or less than or equal to 20,000/μL if aspirin was administered), severe heart disease, severe lung disease, gastrointestinal infarction, or gangrene), the method of treating a subject comprises administering an effective amount of a LEA-2 inhibitory agent ( eg, anti-MASP-2 antibodies) or an LEA-1 inhibitory agent (eg, anti-MASP-1 or anti-MASP-3 antibodies) as first-line therapy without plasmapheresis or in combination with plasmapheresis. As a first line therapy, the LEA-1 and/or LEA-2 inhibitory agent may be administered to the individual systemically, for example, intra-arterially, intravenously, intramuscularly, by inhalation, intranasally, subcutaneously, or by other parenteral route. In some embodiments, the LEA-1 and/or LEA-2 inhibitory agent is administered to the individual as first line therapy in the absence of plasmapheresis to avoid possible complications of plasmapheresis such as bleeding, infection, and complications and/or allergies inherent in the plasma donor, or, in the other case, if the individual has an intolerance to plasmapheresis, or in conditions where plasmapheresis is not available. In some embodiments, the LEA-1 and/or LEA-2 inhibitory agent is administered to an individual suffering from TTP in combination (including co-administration) with an immunosuppressive agent (eg, corticosteroids, rituxan, or cyclosporine) and/or in combination with concentrated ADAMTS13 .

In some embodiments, the method comprises administering a LEA-1 and/or LEA2 inhibitory agent to an individual suffering from TTP via a catheter (e.g., intravenously) during a first period of time (e.g., during an acute phase lasting at least one day up to one week or two weeks) followed by subcutaneous administration of an LEA-1-and/or LEA-2 inhibitory agent for a second period of time (e.g., in the chronic phase

- 55 040888 for at least two weeks or more). In some embodiments of the invention, the introduction during the first and/or second period of time is carried out without plasmapheresis. In some embodiments of the invention, this method is used to maintain the condition of the individual in order to prevent the individual from one or more symptoms associated with TTP.

In another embodiment, the present invention relates to a method of treating an individual suffering from refractory TTP (i.e., when he does not respond to plasmapheresis therapy for at least two weeks), by administering an inhibitor of LEA- 1 and/or LEA-2 in an amount effective to relieve one or more symptoms of TTP. In one embodiment, the LEA-1 and/or LEA-2 inhibitor is administered to an individual with refractory TTP over an extended period of time, such as at least two weeks or more, via subcutaneous administration or other parenteral route. The introduction can be repeated in accordance with the doctor's prescription until such a condition is eliminated or stopped.

In some embodiments of the invention, the method also includes determining the level of at least one complement factor (e.g., C3, C5) in the individual prior to treatment, and possibly during treatment, where the presence of a reduced level of at least one complement factor compared to the standard value or value in a healthy individual indicates the need to continue treatment using the LEA-1 and/or LEA-2 inhibitory agent.

In some embodiments, the method includes administering, either subcutaneously or intravenously, an LEA-1 and/or LEA-2 inhibitory agent to an individual suffering from TTP or an individual at risk of developing TTP. Preferably, the treatment is carried out daily, but it can be carried out at least once a month. Treatment is continued until the individual's platelet count exceeds 150,000/mL for at least two consecutive days.

In general, it is expected that inhibitors of LEA-1 and LEA-2 should provide an independent beneficial therapeutic effect in the treatment of TMA, including HUS, aHUS and TTP. In addition, LEA-1 and LEA-2 inhibitors used together may provide an additive therapeutic effect compared to the effect of either agent alone, or they may provide effective treatment for a broader subgroup of patients suffering from various forms of TMA. Combined inhibition of LEA-1 and LEA-2 can be achieved by co-administration of a LEA-1 blocking agent and a LEA-2 blocking agent. Optimally, the LEA-1 and LEA inhibitory function can be a single molecule, such as a bispecific antibody consisting of a MASP-1/3 and a MASP-2 specific binding site, or an antibody with dual specificity where each binding site can bind with MASP-1/3 or MASP-2 and block these proteins.

In accordance with the above, in one of its aspects, the present invention relates to a method of inhibiting LEA-1-dependent complement activation for the treatment, prevention or amelioration of the severity of thrombotic microangiopathy, such as hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS) or thrombotic thrombocytopenic purpura (TTP), wherein said method comprises administering a composition containing a therapeutically effective amount of a LEA-1 inhibitory agent, including a MASP-1 inhibitory agent, a MASP-3 inhibitory agent, or a combination of MASP1/3 inhibitory agents in a pharmaceutical a carrier, an individual suffering from thrombotic microangiopathy, or an individual at risk of developing such a disease. The MASP-1, MASP-3, or MASP-1/3 inhibitory composition may be administered to the individual systemically, for example, intra-arterially, intravenously, intramuscularly, by inhalation, intranasally, subcutaneously, or by another parenteral route, or possibly by oral route. administration of non-peptidergic agents. The introduction can be repeated in accordance with the doctor's prescription until such a condition is eliminated or stopped.

In one embodiment, the method according to this aspect of the invention further comprises inhibiting LEA-2-dependent complement activation for the treatment, prevention, or amelioration of thrombotic microangiopathy, such as hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), or thrombotic thrombocytopenic purpura (TTP) in an individual suffering from or at risk of developing thrombotic microangiopathy, wherein said method comprises administering to said individual a therapeutically effective amount of a MASP-2 inhibitory agent and MASP1-, MASP-3-, or MASP1/3- inhibitory agent. As detailed above, the use of a combination of pharmacological agents that individually block LEA-1 and LEA-2 is expected to improve patient outcome in the treatment, prevention, or amelioration of thrombotic microangiopathy compared to inhibition of LEA-1 alone. This result can be achieved, for example, by co-administering an antibody that has LEA1 blocking activity with an antibody that has LEA-2 blocking activity. In some embodiments, the LEA-1 and LEA-2 blocking activities are combined into one molecule, and such a molecule will have combined LEA-1 and LEA-2 blocking activity. Such a molecule may include a bispecific antibody or consist of a bispecific antibody, where one antigen-binding site specifically recognizes MASP-1 and blocks LEA-1, and the second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Alternatively, such a molecule may consist of a bispecific monoclonal antibody, where one antigen-binding site specifically recognizes MASP-3 and thus blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Such a molecule can optimally consist of a bispecific monoclonal antibody, where one antigen-binding site specifically recognizes MASP-1 and MASP-3 and thus blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2.

The MASP-2 inhibitory agent may be administered to the individual systemically, for example, intra-arterially, intravenously, intramuscularly, by inhalation, intranasally, subcutaneously, or other parenteral route, or possibly by oral administration of non-peptidergic agents. The introduction can be repeated in accordance with the doctor's prescription until such a condition is eliminated or stopped.

The use of the MASP-3 inhibitory compositions and optionally the MASP-2 inhibitory compositions of the invention can be accomplished by a single administration of the composition (e.g., a single composition containing MASP-2 and MASP-3 inhibitory agents or bispecific or dual inhibitory agents). action, or co-administration of individual compositions), or a limited range of administrations for the treatment, prevention or reduction of the severity of thrombotic microangiopathy in an individual suffering from thrombotic microangiopathy, or in an individual at risk of developing such a disease. Alternatively, the composition may be administered intermittently at intervals, such as daily, twice a week, weekly, every other week, monthly, or twice a month for an extended period of time to treat an individual in need thereof.

As described herein in Examples 11-21, high affinity MASP-3 inhibitory antibodies were generated that are therapeutically effective in inhibiting an alternative pathway in AP-related diseases or conditions such as thrombotic microangiopathy (e.g., hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS) or thrombotic thrombocytopenic purpura (TTP)).

Accordingly, in one of its embodiments, the present invention relates to a method of treating an individual suffering from thrombotic microangiopathy (for example, hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS) or thrombotic thrombocytopenic purpura (TTP)), or an individual at risk of developing such a disease, wherein said method comprises administering an effective amount of a high-affinity monoclonal antibody, or antigen-binding fragment thereof, described herein, that binds to human MASP-3 and inhibits alternative pathway complement activation in order to treat or reduce the risk of developing thrombotic microangiopathy (e.g., hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), thrombotic thrombocytopenic purpura (TTP), or transplant-associated TMA (TA-TMA), wherein said antibody or antigen-binding fragment thereof includes: (a) a heavy chain variable region comprising (i) VHCDR1 comprising SEQ ID NO: 84, (ii) VHCDR2 comprising SEQ ID NO: 86 or SEQ ID NO: 275, and (iii) VHCDR3 comprising SEQ ID NO: 88; and (b) a light chain variable region comprising (i) VLCDR1 comprising SEQ ID NO: 142, SEQ ID NO: 257, SEQ ID NO: 258, or SEQ ID NO: 259, (ii) VLCDR2 comprising SEQ ID NO: 144, and (iii) VLCDR3 comprising SEQ ID NO:161.

G. Role of MASP-3 in the development of asthma and methods of therapy using MASP-3 inhibitory antibodies, optionally in combination with MASP-2 inhibitory agents.

Asthma is a common chronic inflammatory disease of the airways. Approximately 25 million people in the United States suffer from asthma, including 7 million children under the age of 18, with more than half experiencing at least one asthma attack each year, resulting in more than 1.7 million emergency room visits. and 450,000 hospital admissions per year (available at www.gov/health/prof/lung/asthma/naci/asthma-info/index.htm., posted May 4, 2012). This disease is heterogeneous and has many clinical phenotypes. The most common phenotype is allergic asthma. Other phenotypes are non-allergic asthma, respiratory disease that is exacerbated by aspirin, post-infectious asthma, occupational asthma, airborne induced asthma, and exercise-induced asthma. The cardinal features of allergic asthma are airway hypersensitivity (ARH) to various specific and non-specific stimuli, excessive mucus production in the airways, pulmonary eosinophilia, and elevated serum IgE concentrations. Asthma symptoms include coughing, wheezing, chest tightness, and shortness of breath. The goal of asthma treatment is to eliminate the disease and minimize exacerbations, daily symptoms, and keep patients physically active. Currently, treatment recommendations include step-by-step procedures until asthma is completely eliminated. The first stage of treatment includes

- 57 040888 it is necessary, the introduction of a fast-acting β2-agonist by inhalation; and then administration of anti-asthma drugs such as corticosteroids administered by inhalation; long-acting β2 agonists administered by inhalation; leukotriene modifier drugs; theophylline, oral glucocorticosteroids, and an anti-IgE monoclonal antibody.

Although asthma is multifactorial in origin, it is generally accepted that it results from inadequate immunological responses to common natural antigens in genetically susceptible individuals. Asthma is associated with complement activation, and anaphyllotoxins (AT) C3a and C5 have pro-inflammatory and immunoregulatory properties that lead to the development and modulation of the allergic response (Zhang X. and Kohl, J. Expert. Rev. Clin. Immunol., 6:269- 211, 2010). However, the relative involvement of the classical, alternative, and lectin complement pathways in the development of asthma is not well understood. The alternative pathway can be activated on the surface of allergens, and the lectin pathway can be activated through the recognition of allergenic polysaccharide structures, both of which lead to the formation of AT. Complement can be activated in different ways depending on the allergen causing asthma. Highly allergenic pollen from a plant of the Parietaria family, for example, is very effective in stimulating MBL-dependent C4 activation by LEA-2. In contrast, house dust mite allergen does not require MBL for complement activation (Varga et al. Mol. Immunol., 39(14):839-46, 2003).

External stimuli that cause asthma can activate complement through an alternative pathway. For example, in vitro exposure of human serum to cigarette smoke or particulates in exhaust gases leads to complement activation, and this effect does not depend on the presence of EDTA, indicating that activation occurs through an alternative, but not through the classical pathway (Robbins R.A. et al Am J Physiol 260: L254-L259, 1991; Kanemitsu H et al Biol Pharm Bull 21:129-132 1998). The role of complement pathways in allergic airway inflammation was evaluated in a mouse model of sensitization and stimulation with ovalbumin. Wild-type mice developed HDP and airway inflammation in response to aeroallergen stimulation. The Crry-Ig fusion protein, which inhibits all pathways of complement activation, was effective in preventing HDP and lung inflammation when administered systemically or topically by inhalation to mice in an ovalbumin model of allergic lung inflammation (Taube et al., Am. J. Respir. Crit. Care Med., 168(11):1333-41, 2003).

Compared to wild-type mice, factor B-deficient mice exhibited less severe HDP and airway inflammation, while C4-deficient mice exhibited effects similar to wild-type mice (Taube C. et al., Proc. Natl. Acad. Sci USA 103:80848089, 2006). These results support the involvement of an alternative pathway, but not the classical pathway, in a mouse model of aeroallergen stimulation. Further support for the important role of the alternative pathway has been obtained in factor H (FH) studies using the same mouse model (Takeda K. et al., J. Immunol. 188:661-667, 2012). FH is a negative regulator of the alternative pathway and acts as an agent to prevent autologous damage to tissues in which it is present. Endogenous FH was found to be present in the airways during allergen stimulation, and FH inhibition by a recombinant competitive antagonist resulted in exacerbation of HDP and airway inflammation (Takeda et al., 2012, supra). Therapeutic delivery of CR2-fH, i.e. a chimeric protein that links the iC3b/c3d-binding region of CR2 to the complement-regulating FH region, which targets the complement-regulating activity of fH to sites of existing complement activation, conferred protection against the development of HDP and against eosinophil infiltration into the airways after allergen stimulation (Takeda et al. , 2012, see above). A protective effect has been demonstrated using ovalbumin as well as the ragweed allergen, which is a relevant allergen for stimulating human allergy.

The role of lectin-dependent complement activation in asthma was evaluated in a mouse fungal asthma model (Hogaboam et al., J. Leukocyte Biol. 75:805 814, 2004). These studies used mice genetically deficient in mannan-binding lectin A (MBL-A), ie. a carbohydrate-binding protein that functions as a component that recognizes the activation of complement lectin pathways. MBL-A(+/+)- and MBL-A(-/-) mice sensitized with Aspergillus fumigatus were examined on days 4 and 28 after i.t.-sensitization with A. fumigatus conidia. HDP in sensitized MBL-A(-/-) mice was significantly attenuated each time after infection with conidia compared to the group of sensitized MBL-A(+/+) mice. Levels of TH2 cytokines in the lungs (IL-4, IL-5 and IL-13) were significantly lower in A. fumigatus-sensitized MBL-A(-/-) mice compared to the wild-type mice group on day 4 after infection with conidia. These results indicate that MBL-A and the lectin pathway play an important role in the development and maintenance of HDP in chronic fungal asthma.

The results obtained, described in detail above, suggest a role for lectin-dependent complement activation in the pathogenesis of asthma. Experimental data indicate that factor B activation plays a critical role. Based on the fundamental role of LEA-1 in lectin-dependent factor B activation and subsequent activation of the alternative pathway, it has been suggested that LEA-1 blocking agents may be effective in the treatment of certain forms of asthma mediated by the alternative pathway. Thus, said treatment may be particularly effective in eliminating house dust mite-induced asthma or asthma caused by external irritants such as cigarette smoke or exhaust fumes. On the other hand, pollen-induced asthmatic responses are likely to induce LEA-2-dependent complement activation. Therefore, LEA-2 blocking agents are expected to be particularly suitable for the treatment of asthmatic conditions in this subgroup of patients.

Based on the data described above, the authors of the present invention suggested that LEA-1 and LEA-2 mediate pathological complement activation in asthma. Depending on the provoking allergic agents, the development of asthma may be predominantly LEA-1 or LEA-2. Thus, a LEA-1 blocking agent in combination with a LEA-2 blocking agent may be effective in the treatment of various forms of asthma, regardless of their etiology. LEA-1 and LEA-2 blocking agents may have a complementary, additive, or synergistic effect in the prevention, treatment, or elimination of lung inflammation and asthma symptoms.

Combined inhibition of LEA-1 and LEA-2 can be achieved by co-administration of a LEA-1 blocking agent and a LEA-2 blocking agent. Optimally, the LEA-1 and LEA-2 inhibitory function can be a single molecule, such as a bispecific antibody consisting of a MASP-1/3 and a MASP-2 specific binding site, or an antibody with dual specificity where each binding site can bind with MASP-1/3 or MASP-2 and block these proteins.

In accordance with the above, in one of its aspects, the present invention relates to a method of inhibiting LEA-1-dependent complement activation for the treatment, prevention or amelioration of asthma, which method includes the introduction of a composition containing a therapeutically effective amount of a LEA-1 inhibitory agent , including a MASP-1 inhibitory agent, a MASP-3 inhibitory agent, or a combination of MASP1/3 inhibitory agents in a pharmaceutical carrier, to an individual suffering from asthma or an individual at risk of developing asthma. The MASP-1, MASP-3, or MASP-1/3 inhibitory composition may be administered to the individual systemically, for example, intra-arterially, intravenously, intramuscularly, by inhalation, intranasally, subcutaneously, or by another parenteral route, or optionally by oral administration. non-peptidergic agents. The introduction can be repeated in accordance with the doctor's prescription until such a condition is eliminated or stopped.

In one embodiment, the method of this aspect of the invention further comprises inhibiting LEA-2-dependent complement activation to treat, prevent, or ameliorate asthma, wherein said method comprises administering to an individual with or at risk of developing asthma a therapeutically effective amount of MASP -2 inhibitory agent; and MASP-1, MASP-3 or MASP1/3 inhibitory agent. As detailed above, it is expected that the use of a combination of pharmacological agents that individually block LEA-1 and LEA-2 will improve therapeutic outcome in the treatment, prevention, or amelioration of asthma compared to inhibition of LEA-1 alone. This result can be achieved, for example, by co-administering an antibody that has LEA1 blocking activity with an antibody that has LEA-2 blocking activity. In some embodiments, the LEA-1 and LEA-2 blocking activities are combined into one molecule, and such a molecule will have combined LEA-1 and LEA-2 blocking activity. Such a molecule may include a bispecific antibody or consist of a bispecific antibody where one antigen-binding site specifically recognizes MASP-1 and blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Alternatively, such a molecule may consist of a bispecific monoclonal antibody, where one antigen-binding site specifically recognizes MASP-3 and thus blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Such a molecule can optimally consist of a bispecific monoclonal antibody, where one antigen-binding site specifically recognizes MASP-1 and MASP-3 and thus blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2.

The MASP-2 inhibitory agent may be administered to the individual systemically, eg, intra-arterially, intravenously, intramuscularly, by inhalation, intranasally, subcutaneously, or other parenteral route, or possibly by oral administration of non-peptidergic agents. The introduction can be repeated in accordance with the doctor's prescription until such a condition is eliminated or stopped.

The use of the MASP-3 inhibitory compositions and optionally the MASP-2 inhibitory compositions of the invention can be accomplished by a single administration of the composition (e.g., a single composition containing MASP-2 and MASP-3 inhibitory agents or bispecific or dual agents). inhibitory action, or co-administration of separate compositions), or a limited range of administrations for the treatment, prevention or reduction of the severity of asthma in an individual suffering from asthma or in an individual at risk of developing asthma. Alternatively, the composition may be administered intermittently at intervals, such as daily, twice a week, weekly, every other week, monthly, or twice a month for an extended period of time to treat an individual in need thereof.

As described in Examples 11-21 herein, high affinity MASP-3 inhibitory antibodies were generated that are therapeutically effective in inhibiting an alternative pathway in AP-related diseases or conditions such as asthma.

Accordingly, in one of its embodiments, the present invention relates to a method of treating an individual suffering from asthma, or an individual at risk of developing asthma, where this method includes the introduction of an effective amount of the high-affinity monoclonal antibody described here, or its antigen-binding fragment, which binds to a human MASP-3 and inhibit alternative pathway complement activation to treat or reduce the risk of developing asthma, wherein said antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region comprising (i) VHCDR1 comprising SEQ ID NO: 84, (ii ) VHCDR2 comprising SEQ ID NO: 86 or SEQ ID nO: 275, and (iii) VHCDR3 comprising SEQ ID NO: 88; and (b) a light chain variable region comprising (i) VLCDR1 comprising SEQ ID NO: 142, SEQ ID NO: 257, SEQ ID NO: 258 or SEQ ID NO: 259, (ii) VLCDR2 comprising SEQ ID NO: 144, and (iii) VLCDR3 comprising SEQ ID NO: 161.

H. Role of MASP-3 in the development of hard deposit disease and methods of therapy using MASP-3 inhibitory antibodies, optionally in combination with MASP-2 inhibitory agents.

Membranoproliferative glomerulonephritis (MPGN) is a renal disorder morphologically characterized by mesangial cell proliferation and thickening of the glomerular capillary wall due to subendothelial dilation of the mesangium. MPGN are classified as either primary (also called idiopathic) or secondary, associated with underlying diseases such as infectious diseases, systemic immune complex diseases, neoplasms, chronic liver disease, and the like. Idiopathic MPGN includes three morphological types. Type I disease, or classic MPGN, is characterized by subendothelial deposits of immune complexes and activation of the classical complement pathway. Type II disease, or hard deposit disease (DLD), is characterized by extra intramembranous hard deposits. Type III disease is characterized by additional subepithelial deposits. Idiopathic MPGN is a rare disease and occurs in approximately 47% of patients with primary kidney disease causing nephrotic syndrome (Alchi B. and Jayne D. Pediatr. Nephrol. 25:1409-1418, 2010). MPGN mainly occurs in children and young adults and may present as nephrotic syndrome, acute renal syndrome, asymptomatic proteinuria and hematuria, or acute recurrent hematuria. Most patients experience impaired renal function and the disease has a slowly progressive course, with approximately 40% of patients developing end-stage renal failure within 10 years of diagnosis (Alchi and Jayne, 2010, supra). Current treatments include the use of corticosteroids, immunosuppressants, antiplatelet treatments, and plasmapheresis.

BPO is diagnosed by the absence of immunoglobulin and the presence of C3 in the immunofluorescent staining of renal biopsy specimens, and electron microscopy shows characteristic dense osmiophilic deposits along the glomerular basement membranes. BPO is caused by dysregulation of the alternative complement pathway (Sethi et al. Clin. J. Am. Soc. Nephrol. 6(5):1009-17, 2011), which can be caused by a number of different mechanisms. The most common complement system anomaly in RPO is the presence of renal C3 factors, which are autoantibodies against alternative pathway C3 convertase (C3bBbC3bBb) that increase their half-life and therefore enhance pathway activation (Smith R.J.H. et al., Mol. Immunol. 48:16041610, 2011). Other alternative pathway abnormalities are the presence of an anti-factor H autoantibody, which blocks factor H function, enhances C3 mutation function, and creates genetic factor H deficiency (Smith et al., 2011, supra). Recent reports have shown that treatment with eclizumab (an anti-C5 monoclonal antibody) is associated with improved kidney function in two patients with LPO (Daina E. et al., New Engl. J. Med. 366:1161-1163, 2012; Vivarelli M. et al., New Engl. J. Med. 366:1163-1165, 2012), suggesting a causal role for complement activation in the outcome of renal disease.

Considering the above genetic, functional, immunohistochemical and interesting clinical data, it is clear that complement plays a central role in the pathogenesis of BPD. Thus, a therapeutic treatment that blocks disease-causing complement activation mechanisms or subsequent complement activation products is expected to be therapeutically effective in treating this condition.

Although human genetic data presumably indicate that misregulation or overactivation of the alternative pathway amplification loop plays a key role, however, complement initiation events have not yet been identified. Immunohistochemical studies of renal biopsy confirm the deposition of MBL in diseased tissue, which indicates a possible involvement of lectin pathways in the initiation of pathological complement activation in LPO (Lhotta et al, Nephrol Dial Transplant., 14(4):881-6, 1999) . The important role of the alternative pathway was further confirmed in experimental models. Mice deficient in factor H develop progressive proteinuria and renal pathological lesions characteristic of this condition in humans (Pickering et al., Nat. Genet., 31(4):424, 2002). Pickering et al. also showed that deletion of factor B, which mediates LEA-1-dependent activation of the alternative pathway, results in complete protection of factor H-deficient mice from BPO (Pickering et al., Nat. Genet., 31(4): 424, 2002).

Thus, it can be assumed that agents that block LEA-1 will effectively block lectin-dependent activation of the alternative pathway, and thus provide an effective treatment for BPO. Considering that the amplification loop of the alternative dysregulation pathway is dysregulated in patients with BPO, agents that block the amplification loop would also be expected to be effective. Since agents targeting LEA-1 block MASP-1 or MASP-1 and MASP-3 and inhibit factor D maturation, it was predicted that such agents would effectively block the alternative pathway amplification loop.

As detailed above, it was found that the pronounced precipitation of MBL in samples of affected kidneys strongly indicates the possible involvement of lectin-initiated activation events in the pathogenesis of BPO. After initial tissue damage in the glomerular capillaries, it has been suggested that MBLs also bind to damaged glomerular endothelium and mesangial structures are involved. It is well known that such tissue damage leads to the activation of LEA-2, which in turn can lead to additional complement activation. Therefore, it is also expected that LEA-2 blocking agents are also effective in preventing subsequent complement activation on damaged glomerular structures, and thus in preventing subsequent disease progression leading to end-stage renal disease.

The data detailed above suggest that LEA-1 and LEA-2 contribute to the development of certain pathological processes of complement activation in LPO. Thus, a LEA1 blocking agent and a LEA-2 blocking agent, taken alone or in combination, are expected to be effective in the treatment of BPO.

LEA-1 and LEA-2 blocking agents used in combination are expected to be more effective than either of the agents alone or to be effective in treating different stages of the disease. Thus, LEA-1 and LEA-2 blocking agents may have a complementary, additive or synergistic effect in the prevention, treatment or elimination of renal failure associated with RPO.

Combined inhibition of LEA-1 and LEA-2 can be achieved by co-administration of a LEA-1 blocking agent and a LEA-2 blocking agent. Optimally, the LEA-1 and LEA-2 inhibitory agents with an inhibitory function may have a single molecule, such as a bispecific antibody consisting of a MASP-1/3 and MASP-2 specific binding site, or an antibody with dual specificity, where each site binding can bind to MASP-1/3 or MASP-2 and block these proteins.

In accordance with the above, in one of its aspects, the present invention relates to a method of inhibiting LEA-1-dependent complement activation for the treatment, prevention or amelioration of the severity of disease of dense deposits, where this method includes the introduction of a composition containing a therapeutically effective amount of LEA-1- an inhibitory agent, including a MASP-1 inhibitory agent, a MASP-3 inhibitory agent, or a combination of MASP-1/3 inhibitory agents in a pharmaceutical carrier, to an individual suffering from or at risk of developing a hard deposit disease. The MASP-1, MASP-3, or MASP-1/3 inhibitory composition may be administered to the individual systemically, for example, intra-arterially, intravenously, intramuscularly, by inhalation, intranasally, subcutaneously, or other parenteral route, or possibly by oral route. administration of non-peptidergic agents. The introduction can be repeated in accordance with the doctor's prescription until such a condition is eliminated or stopped.

In another aspect, the present invention relates to a method of inhibiting LEA-2 dependent complement activation for the treatment, prevention or amelioration of the severity of disease of dense deposits, which method includes the introduction of a therapeutically effective amount of MASP-2 inhibitory agent to an individual suffering from disease of dense deposits, or an individual at risk such a disease. In another aspect, the present invention provides a method for inhibiting LEA-1 and LEA-2 dependent complement activation for the treatment, prevention, or amelioration of hard deposit disease, the method comprising administering a therapeutically effective amount of a MASP-2 inhibitory agent and MASP -1-, MASP-3 - or MASP-1/3 inhibitory agent to an individual suffering from a disease of dense deposits, or an individual at risk of developing this disease.

- 61 040888

In some embodiments of the invention, this method includes inhibition of LEA-1-dependent complement activation and LEA-2-dependent complement activation. As detailed above, it is expected that the use of a combination of pharmacological agents that individually block LEA-1 and LEA-2 will provide an improved therapeutic outcome in the treatment, prevention, or amelioration of hard deposit disease, compared with inhibition of LEA-1 alone. This result can be achieved, for example, by co-administering an antibody that has LEA-1 blocking activity with an antibody that has LEA-2 blocking activity. In some embodiments, the LEA-1 and LEA-2 blocking activities are combined into one molecule, and such a molecule will have combined LEA-1 and LEA-2 blocking activity. Such a molecule may include a bispecific antibody or consist of a bispecific antibody where one antigen-binding site specifically recognizes MASP-1 and blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Alternatively, such a molecule may consist of a bispecific monoclonal antibody, where one antigen-binding site specifically recognizes MASP-3 and thus blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Such a molecule can optimally consist of a bispecific monoclonal antibody, where one antigen-binding site specifically recognizes MASP-1 and MASP-3 and thus blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2.

The LEA-1 and/or LEA-2 inhibitory agents may be administered to the individual systemically, for example intra-arterially, intravenously, intramuscularly, by inhalation, intranasally, subcutaneously, or other parenteral route, or possibly by oral administration of non-peptidergic agents. The introduction can be repeated in accordance with the doctor's prescription until such a condition is eliminated or stopped.

The use of MASP-3 inhibitory compositions and/or MASP-2 inhibitory compositions according to the invention can be carried out by a single administration of the composition (for example, a single composition containing MASP-2 and MASP-3 inhibitory agents or bispecific or dual inhibitory agents). action, or co-administration of individual compositions), or a limited range of administrations for the treatment, prevention or amelioration of the severity of disease of dense deposits in an individual in need of it. Alternatively, the composition may be administered intermittently at intervals, such as daily, twice a week, weekly, every other week, monthly, or twice a month for an extended period of time to treat an individual in need thereof.

As described in Examples 11-21 herein, high affinity MASP-3 inhibitory antibodies have been generated that are therapeutically effective in inhibiting an alternative pathway in AP-related diseases or conditions such as hard deposit disease.

Accordingly, in one of its embodiments, the present invention relates to a method of treating an individual suffering from a disease of dense deposits, or an individual at risk of developing such a disease, which method includes the introduction of an effective amount of the high-affinity monoclonal antibody described here, or its antigen-binding fragment, which bind to human MASP-3 and inhibit alternative pathway complement activation to treat or reduce the risk of developing hard deposit disease, wherein said antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region comprising (i) VHCDR1 comprising SEQ ID NO : 84, (ii) VHCDR2 comprising SEQ ID NO: 86 or SEQ ID NO: 275, and (iii) VHCDR3 comprising SEQ ID NO: 88; and (b) a light chain variable region comprising (i) VLCDR1 comprising SEQ ID NO: 142, SEQ ID NO: 257, SEQ ID NO: 258, or SEQ ID NO: 259, (ii) VLCDR2 comprising SEQ ID NO: 144, and (iii) VLCDR3 comprising SEQ ID NO:161.

I. The role of MASP-3 in the development of oligoimmune necrosing sickle-shaped glomerulonephritis and methods of therapy using MASP-3 inhibitory antibodies, optionally in combination with MASP-2 inhibitory agents.

Oligoimmune necrotizing crescentic glomerulonephritis (ONSH) is a form of rapidly progressive glomerulonephritis in which the glomerular capillary wall has signs of inflammation with poorly detectable deposition of immune complexes or antibodies against the glomerular basement membrane. This condition is associated with a rapid decline in kidney function. Autoantibodies against cytoplasmic neutrophils (ANCA) have been found to be present in the majority of patients with ONSH, and therefore this disease belongs to a group of diseases called ANCA-associated vasculitis. Vasculitis is a blood vessel disorder characterized by inflammation and fibrinoid necrosis of the vessel wall. Systemic vasculitis is classified according to the size of the vessels: large, medium, and small. Some forms of small vessel vasculitis are associated with the presence of ANCA, and such forms are Wegener's granulomatosis, microscopic polyangiitis, Churg-Strauss syndrome, and renal limited vasculitis (ONSH). They may also be manifestations of underlying conditions such as systemic lupus erythematosus. An

- 62 040888 target tigens for ANCA are proteinase-3 (PR3) and myeloperoxidase (MPO). Oligoimmune ONSH is rare; it has been reported to occur in about 4 people per million in Wessex, UK (Hedger N. et al., Nephrol. Dial. Transplant. 15:1593-1599, 2000). In the Wessex cohort of 128 patients with oligoimmune ONSH, 73% were ANCA positive and 59% of patients required initial dialysis and 36% of patients required dialysis for an extended period of time. Treatment of oligoimmune ONSH includes the administration of corticosteroids and immunosuppressive agents such as cyclophosphamide and azathioprine. Additional treatment options for ANCA-associated vasculitis include rituximab and plasmapheresis (Chen M. and Kallenberg, C.G.M. Nat. Rev. Rheumatol. 6:653-664, 2010).

Although ONSH is characterized by insufficient complement deposition, however, an alternative complement pathway is involved in the pathogenesis of ONSH. Analysis of renal biopsy specimens from 7 patients with MPOANCA-associated oligoimmune ONSH revealed the presence of the membrane attack complex, C3d, factor B, and factor P (which were not detected in biopsy specimens from normal controls or patients with minimal pathological abnormalities), while C4d and lectin binding with mannose were not detected, indicating selective activation of the alternative pathway (Xing, GQ et al. J. Clin. Immunol. 29:282-291, 2009). Experimental ONSH can also be initiated by transferring anti-MPO IgG to wild-type mice or anti-MPO splenocyte antibodies to immunodeficient mice (Xiao H. et al. J. Clin. Invest. 110:955-963, 2002). In this mouse model of ONSH, the role of specific complement activation pathways was investigated using knockout mice. After injection of anti-MPO IgG, C4 ^-/ '-mice developed renal disease compared with wild-type mice, while C5 ^-/ '-mice and factor B-deficient mice did not develop renal disease, indicating that that this model involves an alternative pathway, but not the classical and lectin pathway (Xiao H. et al. Am. J. Pathol. 170:52-64, 2007). In addition, incubation of MPO-ANCA or PR3-ANCA IgG from patients with TNF-primed human neutrophils resulted in the release of factors that activated complement in normal human serum, as indicated by C3a production, but this effect was not seen with IgG. healthy individuals, suggesting a possible pathogenic role for ANCA in neutrophil and complement activation (Xiao et al., 2007, supra).

Based on the role described above for the alternative pathway in this condition, blocking the activation of the alternative pathway would be effective in the treatment of ANCA-positive ONSH. Given the fact that fB activation is required for pathogenesis, LEA-1 inhibitors are expected to be particularly effective in treating this condition as well as preventing further decline in kidney function in these patients.

Another subgroup of patients develop progressive renal sickle cell vasculitis accompanied by a rapid decline in renal function in the absence of ANCA. This form of the disease is called ANCA-negative ONSH and occurs in approximately one third of all patients with oligoimmune ONSH (Chen et al., JASN 18(2): 599-605, 2007). Typically, this disease develops in younger patients and this renal disease tends to have a poor outcome (Chen et al., Nat. Rev. Nephrol., 5(6):313-8, 2009). A characteristic pathological feature of the disease in these patients is the deposition of MBL and C4d in renal lesions (Xing et al., J. Clin. Immunol. 30(1):144-56, 2010). The intensity of MBL and C4d staining in renal biopsy specimens is negatively correlated with renal function (Xing et al., 2010, supra). These data indicate the important role of lectin-dependent complement activations in pathogenesis. The fact that C4d, but not factor B, is usually detected in affected tissue samples indicates the involvement of LEA-2.

Based on the role of lectin-dependent complement activation in ANCA-negative ONSH described above, it has been suggested that blocking the activation of the LEA-2 pathway would be effective in the treatment of ANCA-negative ONSH.

The data detailed above suggest that LEA-1 and LEA-2 mediate pathological complement activation in ANCA-positive and ANCA-negative ONSH, respectively. Thus, it is expected that a LEA-1 blocking agent in combination with a LEA-2 blocking agent will be effective in the treatment of all forms of oligoimmune ONSH, regardless of their etiology. Thus, LEA-1 and LEA-2 blocking agents may have a complementary, additive, or synergistic effect in the prevention, treatment, or elimination of ONSH-associated renal failure.

LEA-1 and LEA-2 inhibitors used in combination with each other can provide an additive therapeutic effect compared to any single agent and can provide effective treatment for a wide range of subgroups of patients. Combined inhibition of LEA-1 and LEA-2 can be accomplished by co-administration of a LEA-1 blocking agent and a LEA-2 blocking agent. Optimally, the LEA-1 and LEA-2 inhibitory function can be a single molecule, such as a bispecific antibody consisting of a MASP-1/3 and a MASP-2 specific binding site, or an antibody with dual specificity, where each binding site can bind to MASP-1/3 or MASP-2 and block these proteins.

- 63 040888

In accordance with the above, in one of its aspects, the present invention relates to a method of inhibiting LEA-1-dependent complement activation for the treatment, prevention or reduction of the severity of oligoimmune necrosing crescentic glomerulonephritis, where this method includes the introduction of a composition containing a therapeutically effective amount of LEA-1 inhibitory an agent, including a MASP-1 inhibitory agent, a MASP-3 inhibitory agent, or a combination of MASP-1/3 inhibitory agents in a pharmaceutical carrier, to an individual suffering from or at risk of developing oligoimmune necrotizing crescentic glomerulonephritis. The MASP-1, MASP-3, or MASP-1/3 inhibitory composition may be administered to the individual systemically, for example, intra-arterially, intravenously, intramuscularly, by inhalation, intranasally, subcutaneously, or by another parenteral route, or possibly by oral administration. non-peptidergic agents. The introduction can be repeated in accordance with the doctor's prescription until such a condition is eliminated or stopped.

In another aspect, the present invention provides a method for inhibiting LEA-2 dependent complement activation for the treatment, prevention, or amelioration of oligoimmune necrotizing crescentic glomerulonephritis, which method comprises administering a therapeutically effective amount of a MASP-2 inhibitory agent to an individual suffering from oligoimmune necrotizing crescentic glomerulonephritis, or an individual at risk for such a disease. In another aspect, the present invention provides a method for inhibiting LEA-1 and LEA-2 dependent complement activation for the treatment, prevention, or amelioration of oligoimmune necrotizing crescentic glomerulonephritis, the method comprising administering a therapeutically effective amount of a MASP-2 inhibitory agent, and MASP-1, MASP-3 or MASP-1/3 inhibitory agent to an individual in need thereof.

In some embodiments of the invention, this method includes inhibition of LEA-1-dependent complement activation and LEA-2-dependent complement activation. As detailed above, it is expected that the use of a combination of pharmacological agents that separately block LEA-1 and LEA-2 will provide an improved therapeutic outcome in the treatment, prevention or reduction of the severity of oligoimmune necrotizing crescentic glomerulonephritis compared with inhibition of LEA-1 alone. This result can be achieved, for example, by co-administering an antibody that has LEA-1 blocking activity with an antibody that has LEA-2 blocking activity. In some embodiments, the LEA-1 and LEA-2 blocking activities are combined into one molecule, and such a molecule will have combined LEA-1 and LEA-2 blocking activity. Such a molecule may include a bispecific antibody or consist of a bispecific antibody where one antigen-binding site specifically recognizes MASP-1 and blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Alternatively, such a molecule may consist of a bispecific monoclonal antibody, where one antigen-binding site specifically recognizes MASP-3 and thus blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Such a molecule may optimally consist of a bispecific monoclonal antibody, where one antigen-binding site specifically recognizes MASP-1 and MASP-3 and thus blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2.

The MASP-2 inhibitory agent may be administered to the individual systemically, for example, intra-arterially, intravenously, intramuscularly, by inhalation, intranasally, subcutaneously, or other parenteral route, or possibly by oral administration of non-peptidergic agents. The introduction can be repeated in accordance with the doctor's prescription until such a condition is eliminated or stopped.

The use of MASP-3 inhibitory compositions and/or MASP-2 inhibitory compositions according to the invention can be carried out by a single administration of the composition (for example, a single composition containing MASP-2 and MASP-3 inhibitory agents or bispecific or dual inhibitory agents). action, or co-administration of individual compositions), or a limited range of administrations for the treatment, prevention or amelioration of the severity of oligoimmune necrosing crescentic glomerulonephritis. Alternatively, the composition may be administered intermittently at intervals, such as daily, twice a week, weekly, every other week, monthly, or twice a month for an extended period of time to treat an individual in need thereof.

As described herein in Examples 11-21, high affinity MASP-3 inhibitory antibodies were generated that are therapeutically effective in inhibiting an alternative pathway in AP-related diseases or conditions such as oligoimmune necrotizing crescentic glomerulonephritis (ONSH).

Accordingly, in one of its embodiments, the present invention relates to a method of treating an individual suffering from oligoimmune necrosing crescentic glomerulonephritis (ONSH), or an individual at risk of developing such a disease, where this method includes the introduction

- 64 040888 an effective amount of a high-affinity monoclonal antibody or an antigen-binding fragment thereof described here that binds to human MASP-3 and inhibits complement activation of an alternative pathway, in order to treat or reduce the risk of developing oligoimmune necrosing sickle-shaped glomerulonephritis (ONSH), where the specified antibody or its antigen-binding the fragment includes: (a) a heavy chain variable region comprising (i) VHCDR1 comprising SEQ ID NO: 84, (ii) VHCDR2 comprising SEQ ID NO: 86 or SEQ ID NO: 275, and (iii) VHCDR3 comprising SEQ ID NO: 88; and (b) a light chain variable region comprising (i) VLCDR1 comprising SEQ ID NO: 142, SEQ ID NO: 257, SEQ ID NO: 258, or SEQ ID NO: 259, (ii) VLCDR2 comprising SEQ ID NO: 144, and (iii) VLCDR3 comprising SEQ ID NO:161.

I. The role of MASP-3 in traumatic brain injury and methods of therapy using MASP-3 inhibitory antibodies, optionally in combination with MASP-2 inhibitory agents.

Traumatic brain injury (TBI) is a major global health problem and is responsible for at least 10 million deaths or hospitalizations per year (Langlois J.A. et al., J. Head Trauma Rehabil. 21:375-378, 2006) . In 2003, an estimated 1.6 million TBIs were reported in the United States, including 1.2 million emergency room visits, 290,000 hospitalizations, and 51,000 deaths (Rutland-Brown W. et al., J. Head Trauma Rehabil 21:544-548, 2006). Most TBIs in the United States are caused by falls and traffic accidents. TBI can lead to long-term or lifelong physical, cognitive, behavioral and emotional consequences. More than 5 million Americans have a permanent or life-long disability associated with TBI (Langlois et al., 2006, supra).

TBI can involve penetrating damage to the brain (penetrating injury) or injuries that do not penetrate the brain (closed injuries). Injury profiles and associated neurobehavioral consequences can differ significantly between penetrating and occluded TBIs. Although each injury is unique, however, certain regions of the brain are particularly vulnerable to injury-induced injury, including the frontal cortex and subfrontal white matter, the basal ganglia and diencephalon, the rostral brainstem, and the temporal lobes, including the hippocampus ( McAllister T. W. Dialogues Clin Neurosci 13:287300, 2011). TBI can lead to changes in several neurotransmitter systems, including the release of glutamate and other excitatory amino acids during the acute phase, and to chronic changes in the catecholaminergic and cholinergic systems, which may be associated with neurobehavioral disturbances (McAllister, 2011, supra). Serious TBI survivors often suffer from cognitive impairment, personality disorders, and severe psychiatric disorders, such as depression, anxiety, and post-traumatic stress disorder. Despite intensive research, there is still no clinically effective treatment for TBI that could reduce mortality and morbidity and improve functional outcomes.

Complement factors and TBI.

Numerous studies have identified an association between complement proteins and neurological disorders, including Alzheimer's disease, multiple sclerosis, myasthenia gravis, Guillain-Barré syndrome, cerebral lupus, and stroke (for a review, see Wagner E. et al., Nature Rev. Drug Disc. 9:43 -56, 2010). Recently, the role of C1q and C3 in synapse depletion has been demonstrated, and therefore it is believed that complement factors are likely involved in both normal CNS functions and the development of neurodegenerative disease (Stevens B. et al., Cell 131: 1164-1178 , 2007). The MASP-1 and MASP-3 genes are widely expressed in the brain as well as in the glioma cell line, T98G (Kuraya M. et al., Int. Immunol., 15:109-17, 2003), consistent with the role of the lectin pathway in CNS.

MASP-1 and MASP-3 are a key factor in rapid defense against pathogens and modified cells themselves, but the lectin pathway is also responsible for severe tissue damage after stroke, infarction, and other ischemia-reperfusion injuries. Similarly, MASP-1 and MASP-3 are possible mediators of tissue damage caused by TBI. Inhibition of factor B in the alternative pathway has been shown to attenuate the effects of TBI in two mouse models. Factor B knockout mice were protected from complement-mediated neuroinflammation and neuropathology after TBI (Leinhase I. et al., BMC Neurosci. 7:55, 2006). In addition, anti-factor B antibody attenuated brain tissue damage and prevented neuronal death in TBI-induced mice (Leinhase I. et al., J. Neuroinflammation 4:13, 2007). MASP-3 directly activates factor B (Iwaki. D. et al., J. Immunol. 187:3751-8, 2011), and therefore, it is a possible mediator in TBI.

Similar to the factor B inhibitor, LEA-1 inhibitors, such as anti-MASP-3 antibodies, are expected to represent a promising strategy for the treatment of tissue damage and subsequent complications after TBI.

Thus, LEA-1 and LEA-2 inhibitors may have an independent therapeutic effect in TBI. In addition, LEA-1 and LEA-2 inhibitors used in combination with each other give

- 65 040888 additive therapeutic effect compared to any single agent and can provide effective treatment for subgroups of patients of a wide range. Combined inhibition of LEA-1 and LEA-2 can be accomplished by co-administering a LEA-1 blocking agent and a LEA-2 blocking agent. Optimally, the LEA-1 and LEA-2 inhibitory function may have a single molecule, such as a bispecific antibody consisting of a MASP-1/3 and a MASP-2 specific binding site, or an antibody with dual specificity, where each binding site can bind to MASP-1/3 or MASP-2 and block these proteins.

In accordance with the foregoing, in one of its aspects, the present invention relates to a method of inhibiting LEA-1-dependent complement activation for the treatment or amelioration of the severity of traumatic brain injury, where this method includes the introduction of a composition containing a therapeutically effective amount of LEA-1-inhibitory agent, including a MASP-1 inhibitory agent, a MASP-3 inhibitory agent, or a combination of MASP-1/3 inhibitory agents in a pharmaceutical carrier, to an individual with a traumatic brain injury. The MASP-1, MASP-3, or MASP-1/3 inhibitory composition may be administered to the individual systemically, for example, intra-arterially, intravenously, intramuscularly, by inhalation, intranasally, intracranially, subcutaneously, or by other parenteral route, or optionally by oral administration of non-peptidergic agents. The introduction can be repeated in accordance with the doctor's prescription until such a condition is eliminated or stopped.

In another aspect, the present invention relates to a method of inhibiting LEA-2 dependent complement activation for the treatment or amelioration of a traumatic brain injury, which method comprises administering a therapeutically effective amount of a MASP-2 inhibitory agent to an individual with a traumatic brain injury. In another aspect, the present invention provides a method for inhibiting LEA-1 and LEA-2 dependent complement activation for the treatment or amelioration of traumatic brain injury, which method comprises administering a therapeutically effective amount of a MASP-2 inhibitory agent and MASP- 1-, MASP-3 - or MASP1/3 inhibitory agent to an individual with a traumatic brain injury.

In some embodiments of the invention, this method includes inhibition of LEA-1-dependent complement activation and LEA-2-dependent complement activation. As detailed above, it is expected that the use of a combination of pharmacological agents that individually block LEA-1 and LEA-2 will provide an improved therapeutic outcome in the treatment or reduction of the severity of traumatic brain injury compared with the inhibition of LEA-1 alone. This result can be achieved, for example, by co-administering an antibody that has LEA-1 blocking activity with an antibody that has LEA-2 blocking activity. In some embodiments, the LEA-1 and LEA-2 blocking activities are combined into one molecule, and such a molecule will have combined LEA-1 and LEA-2 blocking activity. Such a molecule may include a bispecific antibody or consist of a bispecific antibody where one antigen-binding site specifically recognizes MASP-1 and blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Alternatively, such a molecule may consist of a bispecific monoclonal antibody, where one antigen-binding site specifically recognizes MASP-3 and thus blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Such a molecule may optimally consist of a bispecific monoclonal antibody, where one antigen-binding site specifically recognizes MASP-1 and MASP-3 and thus blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2.

The MASP-2 inhibitory agent may be administered to the individual systemically, for example, intra-arterially, intravenously, intramuscularly, by inhalation, intranasally, subcutaneously, intracranially, or other parenteral route, or possibly by oral administration of non-peptidergic agents. The introduction can be repeated in accordance with the doctor's prescription until such a condition is eliminated or stopped.

The use of MASP-3 inhibitory compositions and/or MASP-2 inhibitory compositions according to the invention can be carried out by a single administration of the composition (for example, a single composition containing MASP-2 and/or MASP-3 inhibitory agents or bispecific agents or agents dual inhibitory action, or co-administration of separate compositions), or a limited range of administrations for the treatment or amelioration of the severity of traumatic brain injury. Alternatively, the composition may be administered intermittently at intervals, such as daily, twice a week, weekly, every other week, monthly, or twice a month for an extended period of time to treat an individual in need thereof.

As described in Examples 11-21 herein, high affinity MASP-3 inhibitory antibodies were generated that are therapeutically effective in inhibiting an alternative pathway in AP-related diseases or conditions such as traumatic brain injury.

Accordingly, in one of its embodiments, the present invention relates to a method of treating an individual with a traumatic brain injury or an individual at risk of receiving a traumatic

- 66 040888 brain injury, where the specified method includes the introduction of an effective amount described here, a high affinity monoclonal antibody or antigen-binding fragment that binds to human MASP-3 and inhibits complement activation of the alternative pathway in order to treat or reduce the risk of traumatic brain injury, where specified the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region comprising (i) VHCDR1 comprising SEQ ID NO: 84, (ii) VHCDR2 comprising SEQ ID NO: 86 or SEQ ID NO: 275, and (iii) VHCDR3 including SEQ ID N0:88; and (b) a light chain variable region comprising (i) VLCDR1 comprising SEQ ID NO: 142, SEQ ID NO: 257, SEQ ID NO: 258 or SEQ ID NO: 259, (ii) VLCDR2 comprising SEQ ID NO: 144, and (iii) VLCDR3 comprising SEQ ID NO: 161.

K. Role of MASP-3 in the development of aspiration pneumonia and methods of therapy using MASP-3 inhibitory antibodies, optionally in combination with MASP-2 inhibitory agents.

Aspiration is defined as the penetration of the contents of the oropharynx or stomach into the lower respiratory tract. Aspiration can lead to complications of aspiration (chemical) pneumonitis, primary bacterial aspiration pneumonia, or secondary bacterial infection of chemical pneumonitis. Risk factors for aspiration include decreased intelligence (eg, head injury or sensory changes caused by alcohol or drug use, stroke), various gastrointestinal and esophageal disorders, and neuromuscular disorders. It is estimated that 5-15% of the 4.5 million cases of community-acquired pneumonia are caused by aspiration pneumonia (Marik P.E. New Engl. J. Med. 344:665-671, 2001). Treatment for chemical pneumonitis is mainly supportive, and the use of empiric antibiotics is controversial. Bacterial aspiration pneumonia is treated with appropriate antibiotics, and such treatment depends on the site of the aspiration, ie. on whether such aspiration occurred outside the hospital or in the hospital, since the etiological factors of these diseases differ from each other. At the same time, measures should be taken to prevent aspiration in high-risk patients, such as elderly patients in nursing homes who have impaired gag reflexes. Measures that have been shown to promote effective prevention include head elevation in bed during feeding, dental prophylaxis, and oral hygiene. Prophylactic antibiotics have been shown to be ineffective and are not recommended because they may lead to the emergence of resistant organisms.

Complement modulation has been proposed for the treatment of a variety of clinical conditions, including infectious disease such as sepsis, viral, bacterial, and fungal infections, and lung diseases such as respiratory distress syndrome, chronic obstructive pulmonary disease, and cystic fibrosis (for a review, see Wagner E. et al., Nature Rev. Drug Disc 9: 43-56, 2010). This assumption has been confirmed by numerous clinical and genetic studies. For example, there has been a significant decrease in the number of patients with low MBL levels in clinical tuberculosis (Soborg et al., Journal of Infectious Diseases 188:777-82, 2003), suggesting that low MBL levels indicate protection against this disease. .

In a mouse model of acid aspiration injury, Weiser M.R. et al., J. Appl. physiol. 83(4): 1090-1095, 1997, it was demonstrated that C3 knockout mice were protected from severe damage while C4 knockout mice were not, indicating that complement activation is mediated by an alternative pathway. Therefore, blocking the alternative pathway with LEA-1 inhibitors is expected to have a therapeutic effect in the treatment of aspiration pneumonia.

Thus, LEA-1 and LEA-2 inhibitors may have an independent therapeutic effect in aspiration pneumonia. In addition, LEA-1 and LEA-2 inhibitors used in combination with each other may provide an additive therapeutic effect compared to any single agent or may provide effective treatment for a wide range of subgroups of patients. Combined inhibition of LEA-1 and LEA-2 can be achieved by co-administration of a LEA1 blocking agent and a LEA-2 blocking agent. Optimally, the LEA-1 and LEA-2 inhibitory function can be a single molecule, such as a bispecific antibody consisting of MASP-1/3 and a MASP-2 specific binding site, or an antibody with dual specificity where each binding site can bind with MASP-1/3 or MASP-2 and block these proteins.

In one of its aspects, the present invention relates to a method of inhibiting LEA-1 dependent complement activation for the treatment of aspiration pneumonia by administering a composition containing a therapeutically effective amount of a MASP-1 inhibitory agent, a MASP-3 inhibitory agent, or a combination of MASP-1/3 inhibitory agents in a pharmaceutical carrier, to an individual suffering from such a condition or other complement-mediated pneumonia. The MASP-1, MASP-3 or MASP-1/3 inhibitory composition may be administered topically to the lungs, for example by inhalation. Alternatively, the MASP-1, MASP-3, or MASP-1/3 inhibitory agent may be administered to the individual systemically, for example, intra-arterially, intravenously, intramuscularly, by inhalation, intranasally, subcutaneously, or other parenteral route, or optionally 67 040888 but, by oral administration. The introduction can be repeated in accordance with the doctor's prescription until such a condition is eliminated or stopped.

In accordance with the above, in one of its aspects, the present invention relates to a method of inhibiting LEA-1-dependent complement activation for the treatment, prevention or amelioration of the severity of aspiration pneumonia, where this method includes the introduction of a composition containing a therapeutically effective amount of LEA-1-inhibitory agent, including a MASP-1 inhibitory agent, a MASP-3 inhibitory agent, or a combination of MASP-1/3 inhibitory agents in a pharmaceutical carrier, to an individual suffering from aspiration pneumonia, or an individual at risk of developing such a disease. The MASP-1, MASP-3, or MASP-1/3 inhibitory composition may be administered to the individual systemically, for example, intra-arterially, intravenously, intramuscularly, by inhalation, intranasally, subcutaneously, or by another parenteral route, or possibly by oral route. administration of non-peptidergic agents. The introduction can be repeated in accordance with the doctor's prescription until such a condition is eliminated or stopped.

In another aspect, the present invention relates to a method of inhibiting LEA-2 dependent complement activation for the treatment, prevention or amelioration of aspiration pneumonia, which method comprises administering a therapeutically effective amount of a MASP-2 inhibitory agent to an individual suffering from aspiration pneumonia, or an individual at risk of developing such diseases. In another aspect, the present invention provides a method comprising inhibition of LEA-1 and LEA-2 dependent complement activation for the treatment or amelioration of aspiration pneumonia, said method comprising administering a therapeutically effective amount of a MASP-2 inhibitory agent and MASP- 1-, MASP-3 - or MASP-1/3 inhibitory agent to an individual suffering from aspiration pneumonia. In some embodiments of the invention, this method includes inhibition of LEA-1-dependent complement activation and LEA-2-dependent complement activation. As detailed above, it is expected that the use of a combination of pharmacological agents that individually block LEA-1 and LEA-2 will provide an improved therapeutic outcome in the treatment or reduction of aspiration pneumonia compared to inhibition of LEA-1 alone. This result can be achieved, for example, by co-administering an antibody that has LEA-1 blocking activity with an antibody that has LEA-2 blocking activity. In some embodiments, the LEA-1 and LEA-2 blocking activities are combined into one molecule, and such a molecule will have combined LEA-1 and LEA-2 blocking activity. Such a molecule may include a bispecific antibody or consist of a bispecific antibody where one antigen-binding site specifically recognizes MASP-1 and blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Alternatively, such a molecule may consist of a bispecific monoclonal antibody, where one antigen-binding site specifically recognizes MASP-3 and thus blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Such a molecule may optimally consist of a bispecific monoclonal antibody, where one antigen-binding site specifically recognizes MASP-1 and MASP-3 and thus blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2.

The MASP-2 inhibitory agent may be administered to the individual systemically, eg, intra-arterially, intravenously, intramuscularly, by inhalation, intranasally, subcutaneously, or other parenteral route, or possibly by oral administration of non-peptidergic agents. The introduction can be repeated in accordance with the doctor's prescription until such a condition is eliminated or stopped.

The use of MASP-3 inhibitory compositions and/or MASP-2 inhibitory compositions according to the invention can be carried out by a single administration of the composition (for example, a single composition containing MASP-2 and/or MASP-3 inhibitory agents or bispecific agents or agents dual inhibitory action, or co-administration of separate compositions), or a limited range of administrations for the treatment, prevention or reduction of the severity of aspiration pneumonia in an individual in need of it. Alternatively, the composition may be administered intermittently at intervals, such as daily, twice a week, weekly, biweekly, monthly, or bimonthly for an extended period of time to treat an individual in need thereof.

As described in Examples 11-21 herein, high affinity MASP-3 inhibitory antibodies were generated that are therapeutically effective in inhibiting an alternative pathway in AP-related diseases or conditions such as aspiration pneumonia.

Accordingly, in one of its embodiments, the present invention relates to a method of treating an individual suffering from aspiration pneumonia or an individual at risk of developing such a disease, which method includes the introduction of an effective amount of a high-affinity monoclonal antibody described here, or its antigen-binding fragment, which

- 68 040888 bind to human MASP-3 and inhibit alternative pathway complement activation to treat or reduce the risk of developing aspiration pneumonia, wherein said antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region containing (i) VHCDR1 comprising SEQ ID NO: 84, (ii) VHCDR2 comprising SEQ ID NO: 86 or SEQ ID NO: 275, and (iii) VHCDR3 comprising SEQ ID NO: 88; and (b) a light chain variable region comprising (i) VLCDR1 comprising SEQ ID NO: 142, SEQ ID NO: 257, SEQ ID NO: 258, or SEQ ID NO: 259, (ii) VLCDR2 comprising SEQ ID NO: 144, and (iii) VLCDR3 comprising SEQ ID NO:161.

L. Role of MASP-3 in the development of endophthalmitis and methods of therapy using MASP-3 inhibitory antibodies, optionally in combination with MASP-2 inhibitory agents.

Endophthalmitis is an inflammatory disease of the intraocular cavities and is usually caused by an infection. Endophthalmitis can be endogenous and result from hematogenous spread of microorganisms from a distant source of infection (eg, endocarditis), or exogenous from direct inoculation of the organism from outside as a complication of ocular surgery, foreign bodies, and/or blunt or penetrating ocular trauma. Exogenous endophthalmitis is much more common than endogenous endophthalmitis, and in most cases it is caused by eye surgery. In the United States, cataract surgery is the leading cause of endophthalmitis, with 0.1-0.3% of patients developing cataract surgery after cataract surgery, with an increasing number of cases over the past decade (Taban M. et al., Arch. Ophthalmol 123:613-620, 2005). Postoperative endophthalmitis can occur either in an acute form, within 2 weeks after surgery, or with a delay of several months after surgery. Acute endophthalmitis usually presents with pain, redness, swelling of the eyelid, and reduced visual acuity. Delayed onset endophthalmitis is less common than the acute form, and patients with this disease may report only moderate pain and photosensitivity. Treatment of endophthalmitis depends on the cause and may include systemic and/or intravitreal antibiotics. Endophthalmitis can lead to reduction or loss of vision.

As previously described for ADJP, many complement pathway genes are associated with ocular diseases, and such genes are, in particular, those of the lectin pathway. Thus, for example, MBL2 has been identified in the case of ADJD of certain subtypes (Dinu V. et al., Genet. Epidemiol. 31: 22437, 2007). The LEA-1 and LEA-2 pathways are likely involved in the development of inflammatory eye diseases such as endophthalmitis (Chow S.P. et al., Clin. Experiment. Ophthalmol. 39:871-7, 2011). Chow et al. examined MBL levels in patients with endophthalmitis and showed that MBL levels and functional activity of the lectin pathway are significantly increased in human ocular inflammations, but virtually undetectable in non-inflamed eyes in controls. This indicates a certain role of MBL and the lectin pathway in the development of inflammatory eye diseases, which can lead to loss of vision, and in particular to endophthalmitis. In addition, in a mouse model of fungal corneal keratitis, the MBL-A gene is one of five genes responsible for activating the inflammatory pathway (Wang Y. et al., Mol. Vis. 13: 1226-33, 2007).

Thus, it is hypothesized that LEA-1 and LEA-2 inhibitors may have an independent therapeutic effect in the treatment of endophthalmitis. In addition, LEA-1 and LEA-2 inhibitors used in combination with each other may provide an additive therapeutic effect compared to any single agent or may provide effective treatment for a wide range of subgroups of patients. Combined inhibition of LEA-1 and LEA-2 can be achieved by co-administration of a LEA-1 blocking agent and a LEA-2 blocking agent. Optimally, the LEA-1 and LEA2 inhibitory function can be a single molecule, such as a bispecific antibody consisting of a MASP-1/3 and a MASP-2 specific binding site, or an antibody with dual specificity where each binding site can bind with MASP-1/3 or MASP-2 and block these proteins.

In accordance with the above, in one of its aspects, the present invention relates to a method of inhibiting LEA-1-dependent complement activation for the treatment, prevention or amelioration of the severity of endophthalmitis, which method includes the introduction of a composition containing a therapeutically effective amount of a LEA-1 inhibitory agent including a MASP-1 inhibitory agent, a MASP-3 inhibitory agent, or a combination of MASP1/3 inhibitory agents in a pharmaceutical carrier, to an individual suffering from endophthalmitis or an individual at risk of developing endophthalmitis. The MASP-1, MASP-3 or MASP-1/3 inhibitory composition may be administered topically to the eye, for example by irrigation or topical administration of the composition in the form of a gel, ointment or drops, or by administration into the vitreous body. Alternatively, the MASP-1-, MASP-3-, or MASP-1/3 inhibitory agent may be administered to the individual systemically, for example, intra-arterially, intravenously, intramuscularly, by inhalation, intranasally, subcutaneously, or other parenteral route, or possibly by oral administration. non-peptidergic agents. The introduction can be repeated in accordance with the doctor's prescription until such a condition is eliminated or stopped.

In another aspect, the present invention relates to a method for inhibiting LEA-2

- 69 040888 dependent complement activation for the treatment, prevention or amelioration of the severity of endophthalmitis, where the specified method includes the introduction of a therapeutically effective amount of MASP-2 inhibitory agent to an individual suffering from endophthalmitis, or an individual at risk of developing endophthalmitis. In another aspect, the present invention provides a method comprising inhibition of LEA-1 and LEA-2 dependent complement activation for the treatment or amelioration of endophthalmitis, said method comprising administering a therapeutically effective amount of a MASP-2 inhibitory agent and MASP-1 -, MASP-3 - or MASP-1/3 inhibitory agent to an individual suffering from endophthalmitis.

In some embodiments of the invention, this method includes inhibition of LEA-1-dependent complement activation and LEA-2-dependent complement activation. As detailed above, it is expected that the use of a combination of pharmacological agents that individually block LEA-1 and LEA-2 will provide an improved therapeutic outcome in the treatment, prevention, or amelioration of endophthalmitis compared to inhibition of LEA-1 alone. This result can be achieved, for example, by co-administering an antibody that has LEA-1 blocking activity with an antibody that has LEA-2 blocking activity. In some embodiments, the LEA-1 and LEA-2 blocking activities are combined into one molecule, and such a molecule will have combined LEA-1 and LEA-2 blocking activity. Such a molecule may include a bispecific antibody or consist of a bispecific antibody where one antigen-binding site specifically recognizes MASP-1 and blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Alternatively, such a molecule may consist of a bispecific monoclonal antibody, where one antigen-binding site specifically recognizes MASP-3 and thus blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Such a molecule may optimally consist of a bispecific monoclonal antibody, where one antigen-binding site specifically recognizes MASP-1 and MASP-3 and thus blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2.

The MASP-2 inhibitory agent may be administered topically to the eye of an individual, for example, by irrigation or topical administration of a gel, ointment, or drop composition, or by intravitreal injection. Alternatively, the MASP-2 inhibitory agent may be administered to the individual systemically, for example, intra-arterially, intravenously, intramuscularly, by inhalation, intranasally, subcutaneously, or other parenteral route, or possibly by oral administration of non-peptidergic agents. The introduction can be repeated in accordance with the doctor's prescription until such a condition is eliminated or stopped.

The use of MASP-3 inhibitory compositions and/or MASP-2 inhibitory compositions according to the invention can be carried out by a single administration of the composition (for example, a single composition containing MASP-2 and/or MASP-3 inhibitory agents or bispecific agents or agents dual inhibitory action, or co-administration of separate compositions), or a limited range of administrations for the treatment, prevention or amelioration of the severity of endophthalmitis in an individual in need of it. Alternatively, the composition may be administered intermittently at intervals, such as daily, twice a week, weekly, every other week, monthly, or twice a month for an extended period of time to treat an individual in need thereof.

As described in Examples 11-21 herein, high affinity MASP-3 inhibitory antibodies were generated that are therapeutically effective in inhibiting an alternative pathway in AP-related diseases or conditions such as endophthalmitis.

Accordingly, in one of its embodiments, the present invention relates to a method of treating an individual suffering from endophthalmitis, or an individual at risk of developing endophthalmitis, which method includes administering an effective amount of a high affinity monoclonal antibody described here, or an antigen-binding fragment that binds to a human MASP-3 and inhibit alternative pathway complement activation to treat or reduce the risk of endophthalmitis, wherein said antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region comprising (i) VHCDR1 comprising SEQ ID NO: 84, (ii ) VHCDR2 comprising SEQ ID NO: 86 or SEQ ID NO: 275, and (iii) VHCDR3 comprising SEQ ID NO: 88; and (b) a light chain variable region comprising (i) VLCDR1 comprising SEQ ID NO: 142, SEQ ID NO: 257, SEQ ID NO: 258, or SEQ ID NO: 259, (ii) VLCDR2 comprising SEQ ID NO: 144, and (iii) VLCDR3 comprising SEQ ID NO: 161.

M. Role of MASP-3 in the development of neuromyelitis of the optic nerve and methods of therapy using MASP-3 inhibitory antibodies, optionally in combination with MASP-2 inhibitory agents.

Optic neuromyelitis (ONN) is an autoimmune disease that affects the optic nerves and spinal cord. This results in inflammation of the optic nerve, known as optic neuritis, and of the spinal cord, known as myelitis. Spinal cord injury in NCI can cause weakness or paralysis of the legs or arms, blindness, bladder dysfunction, and

- 70 040888 bowel and sensory dysfunction.

NZN shares some similarities with multiple sclerosis (MS) in that both are caused by immune attack on CNS targets and lead to demyelination (Papadopoulos and Verkman, Lancet Neurol., 11(6)535-44, 2013). However, in NZN, molecular targets, treatments, and lesions are different from those in MS. PC is mediated primarily by T cells, and patients with NC usually have antibodies targeting the water-associated channel protein aquaporin 4 (AQP4), i.e. a protein present in astrocytes that surround the blood-brain barrier. Treatment with interferon-beta is one of the most widely used treatments for MS, however, it can often have side effects in the treatment of NZN. Inflammatory lesions in NCI occur in the spinal cord and optic nerve, but may also spread to the brain, including the white and gray matter. The demyelination that is observed in the case of NCI lesions is mediated by complement (Papadopoulos and Verkman, Lancet Neurol., 11(6):535-44, 2013).

Complement-dependent cytotoxicity appears to be the main mechanism causing NZN. More than 90% of patients with ND have anti-AQP4 IgG antibodies (Jarius and Wildemann, Jarius S., Wildemann B., Nat. Rev. Neurol. 2010 Jul; 6 (7): 383-92). These antibodies initiate damage in the blood-brain barrier. The initial antigen-antibody-AQ2P4/AQP4-IgG complex on the surface of astrocytes activates the classical complement pathway. This leads to the formation of a membrane attack complex on the surface of astrocytes, which leads to granulocyte infiltration, demyelination and, ultimately, necrosis of astrocytes, oligodendrocytes and neurons (Misu et al., Acta Neuropathol. 125(6):815-27, 2013 ). These cellular events are reflected in tissue destruction and the formation of cysts and necrotic lesions.

The classical complement pathway certainly plays a critical role in the pathogenesis of ND. NZN lesions indicate deposition of immunoglobulin in the vasomotor system and activation of complement components (Jarius et al., Nat. Clin. Pract. Neurol. 4(4):202-14, 2008). In addition, complement proteins such as C5a have been isolated from the cerebrospinal fluid of patients with NCN (Kuroda et al., J. Neuroimmunol., 254(1-2):178-82, 2013). In addition, serum IgG obtained from patients with ND can cause complement-dependent cytotoxicity in mice with NDN (Saadoun et al., Brain, 133 (Pt 2):349-61, 2010). An anti-C1q monoclonal antibody prevents complement-mediated astrocyte destruction and lesions in the HNV mouse model (Phuan et al., Acta Neuropathol., 125(6):829-40, 2013).

The alternative complement pathway serves to enhance overall complement activity. Harboe et al. (2004) showed that selective blocking of the alternative pathway by more than 80% inhibits the formation of the membrane attack complex induced by the classical pathway (Harboe et al., Clin. Exp. Immunol. 138 (3): 439-46, 2004 ). Tuzun et al. (2013) investigated classical and alternative pathway products in patients with NCN (Tuzun E. et al., J. Neuroimmunol. 233(1-2): 211-5, 2011). C4d, i.e. the degradation product of C4 was evaluated for classical pathway activity, and it was determined that the level of this product in the serum of patients with ONH was higher than in the control (2.14 times). In addition, patients with ONH showed an increase in the level of factor Bb, i.e. alternative pathway factor B cleavage product compared to MS patients or normal controls (i.e., 1.33-fold increase). This suggests that the function of the alternative pathway is also enhanced in NCI. It was assumed that such activation would increase the total complement activation, and indeed, the level of sC5b-9, i.e. the level of the end product of the complement reaction cascade was significantly increased (by 4.14 times).

It is expected that specific inhibitors of MASP-3 will be effective in the treatment of patients suffering from ND. As shown in the present description, serum without MASP-3 cannot activate factor B, i.e. the main component of C5 convertase, or factor D, the central activator of the alternative pathway. Therefore, blocking MASP-3 activity with an inhibitory agent, such as an antibody or small molecule, would also presumably inhibit factor B and factor D activation. Inhibition of these two factors would stop amplification of the alternative pathway, resulting in a decrease in total complement activity. Thus, inhibition of MASP-3 will significantly improve the results of the treatment of ND.

Thus, it is hypothesized that LEA-1 and LEA-2 inhibitors may provide an independent therapeutic effect in the treatment of ND. In addition, LEA-1 and LEA-2 inhibitors used in combination with each other can provide an additive therapeutic effect compared to any single agent and can provide effective treatment for a wide range of subgroups of patients. Combined inhibition of LEA-1 and LEA-2 can be achieved by co-administration of a LEA1 blocking agent and a LEA-2 blocking agent. Optimally, the LEA-1 and LEA-2 inhibitory function can be a single molecule, such as a bispecific antibody consisting of MASP-1/3 and a MASP-2 specific binding site, or an antibody with dual specificity where each binding site can bind with MASP-1/3 or MASP-2 and block these proteins.

In accordance with the above, in one of its aspects, the present invention relates to a method of inhibiting LEA-1-dependent complement activation for the treatment, prevention or treatment of

- 71 040888 easing the severity of NZN, where the specified method includes the introduction of a composition containing a therapeutically effective amount of a LEA-1 inhibitory agent, including a MASP-1 inhibitory agent, a MASP-3 inhibitory agent, or a combination of MASP-1/3 inhibitory agents in a pharmaceutical carrier, an individual suffering from NZN, or an individual at risk of developing NZN. The MASP-1, MASP3 or MASP-1/3 inhibitory composition may be administered topically to the eye, for example by irrigation or topical administration of the composition in the form of a gel, ointment or drops, or by intravitreal injection. Alternatively, the MASP-1, MASP-3, or MASP-1/3 inhibitory agent may be administered to the individual systemically, for example, intra-arterially, intravenously, intramuscularly, by inhalation, intranasally, subcutaneously, or other parenteral route, or possibly by oral administration of non-peptidergic agents. The introduction can be repeated in accordance with the doctor's prescription until such a condition is eliminated or stopped.

In another aspect, the present invention relates to a method of inhibiting LEA-2-dependent complement activation for the treatment, prevention, or amelioration of CMN, which method comprises administering a therapeutically effective amount of a MASP-2 inhibitory agent to an individual suffering from CMN or an individual at risk of development of NZN. In another aspect, the present invention relates to a method comprising the inhibition of LEA-1 and LEA-2 dependent complement activation for the treatment or amelioration of congestive heart failure, which method comprises administering a therapeutically effective amount of a MASP-2 inhibitory agent and MASP-1 -, MASP-3 or MASP-1/3 inhibitory agent to an individual suffering from NZN.

In some embodiments of the invention, this method includes inhibition of LEA-1-dependent complement activation and LEA-2-dependent complement activation. As detailed above, it is expected that the use of a combination of pharmacological agents that individually block LEA-1 and LEA-2 will provide an improved therapeutic outcome in the treatment, prevention or reduction of the severity of ND compared to the inhibition of LEA-1 alone. This result can be achieved, for example, by co-administering an antibody that has LEA-1 blocking activity with an antibody that has LEA-2 blocking activity. In some embodiments, the LEA-1 and LEA-2 blocking activities are combined into one molecule, and such a molecule will have combined LEA-1 and LEA-2 blocking activity. Such a molecule may include a bispecific antibody or consist of a bispecific antibody where one antigen-binding site specifically recognizes MASP-1 and blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Alternatively, such a molecule may consist of a bispecific monoclonal antibody, where one antigen-binding site specifically recognizes MASP-3 and thus blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Such a molecule may optimally consist of a bispecific monoclonal antibody, where one antigen-binding site specifically recognizes MASP-1 and MASP-3 and thus blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2.

The MASP-2 inhibitory agent may be administered topically to the eye of an individual, for example by irrigation or topical administration of a gel, ointment or drop formulation, or by intravitreal injection. Alternatively, the MASP-2 inhibitory agent may be administered to the individual systemically, eg, intra-arterially, intravenously, intramuscularly, by inhalation, intranasally, subcutaneously, or other parenteral route, or possibly by oral administration of non-peptidergic agents. The introduction can be repeated in accordance with the doctor's prescription until such a condition is eliminated or stopped.

The use of MASP-3 inhibitory compositions and/or MASP-2 inhibitory compositions according to the invention can be carried out by a single administration of the composition (for example, a single composition containing MASP-2 and/or MASP-3 inhibitory agents or bispecific agents or agents dual inhibitory action, or co-administration of separate compositions), or a limited range of administrations for the treatment, prevention or amelioration of the severity of ONH in an individual in need of it. Alternatively, the composition may be administered intermittently at intervals, such as daily, twice a week, weekly, every other week, monthly, or twice a month for an extended period of time to treat an individual in need thereof.

As described here in Examples 11-21, high affinity MASP-3 inhibitory antibodies were generated that are therapeutically effective in inhibiting an alternative pathway in AP-related diseases or conditions such as optic neuromyelitis (ONN).

Accordingly, in one of its embodiments, the present invention relates to a method of treating an individual suffering from neuromyelitis of the optic nerve (ONN), or an individual at risk of developing such a disease, where this method includes the introduction of an effective amount of the high-affinity monoclonal antibody described herein or its antigen-binding fragment that binds to human MASP-3 and inhibits alternative pathway complement activation to treat or reduce the risk of developing optic neuromyelitis (ONN), where specified

- 72 040888 the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region comprising (i) VHCDR1 comprising SEQ ID NO: 84, (ii) VhCdR2 comprising SEQ ID NO: 86 or SeQ ID No: 275, and (iii) VHCDR3 comprising SEQ ID nO: 88; and (b) a light chain variable region comprising (i) VLCDR1 comprising SeQ ID NO: 142, SEQ ID NO: 257, SEQ ID NO: 258 or SEQ ID NO: 259, (ii) VLCDR2 comprising SEQ ID NO: 144, and (iii) VLCDR3 comprising SEQ ID NO: 161.

N. Role of MASP-3 in the development of Behcet's disease and methods of therapy using MASP-3 inhibitory antibodies, optionally in combination with MASP-2 inhibitory agents.

Behcet's disease or Behcet's syndrome is a rare immune-mediated systemic small vessel vasculitis that is often associated with mucosal ulceration and eye disease. Behcet's disease (BD) was named in 1937 after the Turkish dermatologist Hulusi Behcet, who first described the triple symptom complex of recurrent oral ulcers, genital ulcers, and uveitis. BD is a systemic relapsing inflammatory disorder of unknown etiology. Inflammatory perivasculitis in BD can affect the gastrointestinal tract, lungs, musculoskeletal system, cardiovascular system, and nervous system. BB can be fatal due to vascular aneurysm rupture or severe neurological complications. Neuropathy and atrophy of the optic nerve can result from vasculitis and blockage of the vessels that feed the optic nerve. See Al-Araji A. et al., Lancet Neurol. , 8(2):192-204, 2009.

BB is most common in the Middle East and Far East regions, but is rare in Europe and North America. Initially, BD often responds to treatment with corticosteroids and immunosuppressants, but many cases of relapse leading to severe illness and death are known. Biological agents including interferon-alpha, IVIG, anti-TNF antibody, anti-IL-6 antibody and anti-CD20 antibody have been shown to be effective in some cases, however, there is no consensus on a more effective therapy .

Although BD is known to be an inflammatory disease, however, its pathobiology is not yet clear. Genetic association studies with HLA antigens and extensive genomic association studies have revealed the involvement of multiple cytokine genes (Kirino et al., Nat Genet, 45(2):202-7, 2013). An overactive immune system appears to be regulated by the complement system. Elevated levels of C3 have been observed in the serum of patients with BD (Bardak and Aridogan, Ocul Immunol. Inflamm 12(1):53-8, 2004), and elevated levels of C3 and C4 in the cerebrospinal fluid have been correlated with this disease (Jongen et al., Arch Neurol, 49(10):1075-8, 1992).

Tuzun et al. (2013) investigated classical and alternative pathway products in the sera of BD patients (Tuzun E. et al., J. Neuroimmunol. 233(1-2): 211-5, 2011). 4d, i.e. the degradation product of C4, is produced in the reaction cascade upstream of the alternative pathway, and this product was evaluated for the original activity of the classical pathway. The level of C4d in the serum of patients with BD was higher than in the control (by 2.18 times). Factor Bb is a cleavage product of factor B and this product was evaluated for alternative pathway activity. In patients with BD, there was an increase in the level of factor Bb compared with normal controls (i.e., an increase of 2.19 times), which corresponded to an increase in the function of the alternative pathway in BD. Since the alternative complement pathway serves to amplify total complement activity, such activation is expected to enhance total complement activity. Harboe et al. (2004) have shown that selective blockade of the alternative pathway inhibits more than 80% the formation of the classical pathway-induced membrane attack complex (Harboe M. et al., Clin. Exp. Immunol., 138 (3): 439- 46, 2004). Indeed, sC5b-9, i.e. the end product of the complement cascade was significantly increased (5.46-fold) in BD patients. Specific inhibitors of MASP-3 will be effective in the treatment of BB. Blocking MASP-3 would inhibit factor B and factor D activation. This would stop the amplification of the alternative pathway, resulting in a decrease in total complement activity. Thus, inhibition of MASP-3 will significantly improve the results of BB treatment. Therefore, it is assumed that inhibitors of LEA-1 and/or LEA-2 can provide an independent therapeutic effect in the treatment of BB. In addition, LEA-1 and LEA-2 inhibitors used in combination with each other may provide an additive therapeutic effect compared to any single agent or may provide effective treatment for a wide range of subgroups of patients. Combined inhibition of LEA-1 and LEA-2 can be accomplished by co-administering a LEA-1 blocking agent and a LEA-2 blocking agent. Optimally, the LEA-1 and LEA-2 inhibitory function may have a single molecule, such as a bispecific antibody consisting of a MASP-1/3 and a MASP-2 specific binding site, or an antibody with dual specificity, where each binding site can bind to MASP-1/3 or MASP-2 and block these proteins.

In accordance with the foregoing, in one of its aspects, the present invention relates to a method of inhibiting LEA-1-dependent complement activation for the treatment, prevention or reduction of the severity of BD, where this method includes the introduction of a composition containing a therapeutically effective amount of a LEA-1 inhibitory agent including a MASP-1 inhibitory agent, a MASP-3 inhibitory agent, or a combination of MASP-1/3 inhibitory agents in a pharmaceutical

- 73 040888 a carrier, an individual suffering from BD, or an individual at risk of developing BD. The MASP-1, MASP-3, or MASP-1/3 inhibitory composition may be administered topically to the eye, for example by irrigation or topical administration of the composition in the form of a gel, ointment or drops, or by intravitreal injection. Alternatively, the MASP-1, MASP-3, or MASP-1/3 inhibitory agent may be administered to the individual systemically, for example, intra-arterially, intravenously, intramuscularly, by inhalation, intranasally, subcutaneously, or other parenteral route, or possibly by oral administration of non-peptidergic agents. The introduction can be repeated in accordance with the doctor's prescription until such a condition is eliminated or stopped.

In another aspect, the present invention relates to a method of inhibiting LEA-2-dependent complement activation for the treatment, prevention, or amelioration of BD, which method comprises administering a therapeutically effective amount of a MASP-2 inhibitory agent to an individual suffering from BD, or an individual at risk of BB development. In another aspect, the present invention relates to a method comprising inhibition of LEA-1 and LEA-2 dependent complement activation for the treatment or amelioration of BD, said method comprising administering a therapeutically effective amount of a MASP-2 inhibitory agent and MASP-1 -, MASP-3 - or MASP-1/3 inhibitory agent to an individual suffering from BD.

In some embodiments of the invention, this method includes inhibition of LEA-1-dependent complement activation and LEA-2-dependent complement activation. As detailed above, it is expected that the use of a combination of pharmacological agents that separately block LEA-1 and LEA-2 will provide an improved therapeutic outcome in the treatment, prevention or reduction of the severity of BD compared with the inhibition of LEA-1 alone. This result can be achieved, for example, by co-administering an antibody that has LEA1 blocking activity with an antibody that has LEA-2 blocking activity. In some embodiments, the LEA-1 and LEA-2 blocking activities are combined into one molecule, and such a molecule will have combined LEA-1 and LEA-2 blocking activity. Such a molecule may include a bispecific antibody or consist of a bispecific antibody where one antigen-binding site specifically recognizes MASP-1 and blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Alternatively, such a molecule may consist of a bispecific monoclonal antibody, where one antigen-binding site specifically recognizes MASP-3 and thus blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Such a molecule may optimally consist of a bispecific monoclonal antibody, where one antigen-binding site specifically recognizes MASP-1 and MASP-3 and thus blocks LEA-1, and a second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2.

The MASP-2 inhibitory agent may be administered topically to the eye of an individual, for example by irrigation or topical administration of a gel, ointment or drop formulation, or by intravitreal injection. Alternatively, the MASP-2 inhibitory agent may be administered to the individual systemically, for example, intra-arterially, intravenously, intramuscularly, by inhalation, intranasally, subcutaneously, or other parenteral route, or possibly by oral administration of non-peptidergic agents. The introduction can be repeated in accordance with the doctor's prescription until such a condition is eliminated or stopped.

The use of MASP-3 inhibitory compositions and/or MASP-2 inhibitory compositions according to the invention can be carried out by a single administration of the composition (for example, a single composition containing MASP-2 and/or MASP-3 inhibitory agents or bispecific agents or agents dual inhibitory action, or co-administration of separate compositions), or a limited range of administrations for the treatment, prevention, or alleviation of the severity of BD in an individual in need of it. Alternatively, the composition may be administered intermittently at intervals, such as daily, twice a week, weekly, every other week, monthly, or twice a month for an extended period of time to treat an individual in need thereof.

As described in Examples 11-21 herein, high affinity MASP-3 inhibitory antibodies were generated that are therapeutically effective in inhibiting an alternative pathway in AP-related diseases or conditions such as Behcet's disease (BD).

Accordingly, in one of its embodiments, the present invention relates to a method of treating an individual suffering from Behçet's disease (BD), or an individual at risk of developing such a disease, which method includes the introduction of an effective amount of the high-affinity monoclonal antibody described here, or its antigen-binding fragment , which bind to human MASP-3 and inhibit alternative pathway complement activation, for the treatment or reduction of the risk of developing Behcet's disease (BD), wherein said antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region containing (i) VHCDR1 , including SEQ ID NO: 84, (ii) VHCDR2, including SEQ ID NO: 86 or SEQ ID NO: 275, and (iii) VHCDR3, including SEQ ID NO: 88; and (b) a light chain variable region comprising (i) VLCDR1 comprising SEQ ID NO: 142, SEQ ID NO: 257, SEQ ID NO: 258, or SEQ ID NO: 259, (ii) VLCDR2 including

- 74 040888 with SEQ ID NO: 144, and (iii) VLCDR3 comprising SEQ ID NO: 161.

MASP-3 Inhibitory agents.

The discovery that the lectin complement pathway consists of two major complement activation chains, LEA-1 and LEA-2, and that this pathway is a lectin-independent complement pathway indicates that it would be highly desirable to specifically inhibit one or more than these effector chains, which cause a disease associated with activation of the alternative complement pathway, for example, at least one of the diseases such as paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD, including wet and dry VAD), ischemic reperfusion injury, arthritis, disseminated intravascular coagulation syndrome, thrombotic microangiopathy (including hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), thrombotic thrombocytopenic purpura (TTP) or graft-associated TMA), asthma, hard deposit disease, oligoimmune necrosing falciform glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis of the optic nerve, Behçet's disease, multiple sclerosis (PC), Guillain-Barré syndrome, Alzheimer's disease, amylotrophic lateral sclerosis (ABS), lupus nephritis, systemic lupus erythematosus (SLE), diabetic retinopathy, uveitis, chronic obstructive pulmonary disease (COPD), C3 glomerulopathy, graft rejection, graft versus host disease (GVHD), hemodialysis, sepsis, systemic inflammatory response syndrome (SIR), acute respiratory distress syndrome (ARDS), ANCA- vasculitis, antiphospholipid syndrome, atherosclerosis, IgA nephropathy, and myasthenia gravis, without complete elimination of complement's immune defense capability (ie. preserving the intact classical path). This will keep the C1q-dependent complement activation system intact to improve the processing of immune complexes and to enhance the host's defense against infection.

Compositions for inhibiting LEA-1-mediated complement activation.

As described in this application, the authors of the present invention was unexpectedly found that the activation of LEA-1, leading to lysis, is MASP-3-dependent. As further described herein, under physiological conditions, MASP-3-dependent activation of LEA-1 also promotes opsonization, thus providing an additive effect along with LEA-2-mediated complement activation. As demonstrated herein, in the presence of Ca ⁺⁺ , factor D is not required because MASP-3 can induce LEA-1 activation in serum without factor D. MASP-3, MASP-1 and HTRA-1 are able to convert pro-factor D into active factor D. Similarly, activation of MASP-3 is in many cases dependent on MASP-1, since MASP-3 (unlike MASP-1 and MASP-2) is not an autoactivating enzyme and cannot be converted to its active form. without MASP-1 assistance (Zundel S. et al., J. Immunol. 172: 4342-4350 (2004); Megyeri et al., J. Biol. Chem. 288:8922-8934 (2013). Since MASP-3 is not an autoactivating enzyme and in many cases it requires MASP-1 activity to be converted to an enzymatically active form, then the MASP-3-mediated activation of the alternative pathway C3-convertase C3Bb can either be inhibited by targeting the MASP-3 zymogen or already activated MASP- 3, either by targeting MASP-1-mediated activation of MASP-3, or both, since during in many cases, in the absence of MASP-1 functional activity, MASP-3 remains in zymogen form and is unable to initiate LEA-1 via direct production of C3 convertase (C3bBbC3bBb) alternative pathway.

Thus, in one aspect of the present invention, a preferred protein component for the development of therapeutics specifically inhibiting LEA-1 is a MASP-3 inhibitor (including inhibitors of MASP-1-mediated activation of MASP-3 (e.g., a MASP-1 inhibitor that inhibits MASP-3 activation)).

In accordance with the foregoing, in one aspect, the present invention relates to methods for inhibiting the side effects of LEA-1 (i.e., hemolysis and opsonization) by introducing a MASP-3 inhibitory agent, such as a MASP-3 inhibitory antibody in an individual, suffering from a disease or disorder, or an individual at risk of developing a disease or disorder selected from the group consisting of: paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathies (including hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS) and thrombotic thrombocytopenic purpura (TTP)), asthma, hard deposit disease, oligoimmune necrosing crescentic glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis of the optic nerve , Beh's disease multiple sclerosis, Guillain-Barré syndrome, Alzheimer's disease, amylotrophic lateral sclerosis (ABS), lupus nephritis, systemic lupus erythematosus (SLE), diabetic retinopathy, uveitis, chronic obstructive pulmonary disease (COPD), C3 glomerulopathy, transplant rejection, graft versus host disease (GVHD), hemodialysis, sepsis, systemic inflammatory response syndrome (SIR), acute respiratory distress syndrome (ARDS), ANCA vasculitis, antiphospholipid syndrome, atherosclerosis, IgA nephropathy, and myasthenia gravis, wherein said method comprises administering - 75 040888 providing an individual with a pharmaceutical composition containing an amount of a MASP-3 inhibitory agent effective to inhibit MASP-3-dependent complement activation and a pharmaceutically acceptable carrier.

MASP-3 inhibitory agents are administered in an amount effective to inhibit MASP-3 dependent complement activation in a living subject suffering from paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy (including hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), and thrombotic thrombocytopenic purpura (TTP)), asthma, hard deposit disease, oligoimmune necrotizing crescentic glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis of the optic nerve, Behcet's disease, multiple sclerosis, Guillain-Barré syndrome, Alzheimer's disease, amylotrophic lateral sclerosis (ABS), lupus nephritis, systemic lupus erythematosus (SLE), diabetic retinopathy, uveitis, chronic obstructive pulmonary disease (X OPD), C3 glomerulopathy, graft rejection, graft-versus-host disease (GVHD), hemodialysis, sepsis, systemic inflammatory response syndrome (SIR), acute respiratory distress syndrome (ARDS), ANCA vasculitis, antiphospholipid syndrome, atherosclerosis, IgA nephropathy, and myasthenia gravis , or an individual at risk of developing these diseases. In accordance with this aspect of the invention, representative MASP-3 inhibitory agents are molecules that inhibit the biological activity of MASP-3, including molecules that inhibit at least one or more of the following functions: MASP-3-dependent activation of factor B lectin pathway, MASP-3-dependent activation of pro-factor D of the lectin pathway, MASP-3-dependent and lectin-independent activation of factor B, and MASP-3-dependent and lectin-independent activation of pro-factor D (such as small molecule inhibitors, anti-MASP- 3 antibodies and fragments thereof, or blocking peptides that interact with MASP-3 or inhibit protein-protein interaction), and molecules that reduce the expression level of MASP-3 (such as MASP-3 antisense nucleic acid molecules, MASP-3-specific RNAi molecules and MASP-3 ribozymes). The MASP-3 inhibitory agent can effectively block MASP-3 protein-protein interactions, interfere with MASP-3 dimerization or assembly, block Ca ⁺⁺ binding, block the active site of MASP-3 serine protease, or reduce the expression level of MASP-3 protein, and thereby preventing LEA-1-mediated or lectin-independent complement activation of MASP-3. MASP-3 inhibitory agents can be used alone as primary therapy or in combination with other therapeutic agents as adjuvant therapy to enhance the therapeutic effect of treatment with other drugs, as described in detail in this application.

High affinity monoclonal MASP-3 inhibitory antibodies.

As described here in examples 11-21, and systematized below in table. 2A, 2B and 3, the present inventors unexpectedly produced high affinity (ie <500 pM) MASP-3 inhibitory antibodies that bind to an epitope in the human MASP-3 serine protease domain. As described herein, the present inventors have demonstrated that these high affinity anti-MASP-3 antibodies are capable of inhibiting the activation of the alternative complement pathway in the serum of humans, rodents and non-human primates. The light and heavy chain variable regions of these antibodies were sequenced, isolated and analyzed in Fab and full length IgG format. As described in Example 15, and as shown in the dendrograms shown in FIG. 50A and 50B, antibodies can be grouped according to their sequence similarity. A brief description of the heavy chain variable regions and the light chain variable regions of these antibodies is provided in FIG. 49A and 49B and are presented below in table. 2A and 2B. Were obtained humanized variants of representative high-affinity MASP-3 inhibitory antibodies, as described in example 19 and systematized in table. 3.

Table 2A. High-affinity MASP-3 inhibitory antibody sequences: mouse parental

Reference anti-MASP-3 antibody Group Variable area heavy chain Light chain variable region (amino acids Heavy chain variable region (DNA) Variable region of the lung

- 76 040888

no (amino acid you) ) chains (DNA) 4D5 IA SIN:24 SIN:40 SIN:217 SIN:233 1F3 IA SIN:25 SIN:41 SIN:218 SIN:234 4B6 IA SIN:26 SIN:42 SIN:219 SIN:235 1А10 IA SIN:27 SIN:42 SIN:220 SIN:235 10D12 IB SIN:28 SIN:43 SIN:221 SIN:236 35C1 IB SIN:29 SIN:44 SIN:222 SIN:237 13B1 IC SIN:30 SIN:45 SIN:223 SIN:238 1G4 II SIN:31 SIN:46 SIN:224 SIN:239 1E7 I IIA SIN:32 SIN:47 SIN:225 SIN:240 2D7 I IIA SIN:33 SIN:48 SIN:226 SIN:241 49C11 I IIA SIN:34 SIN:49 SIN:227 SIN:242 15D9 IIIB SIN:35 SIN:50 SIN:228 SIN:243 2F5 IIIB SIN:36 SIN:51 SIN:229 SIN:244 1B11 IIIC SIN:37 SIN:52 SIN:230 SIN:245 2F2 HID SIN:38 SIN:53 SIN:231 SIN:246 11B6 HID SIN:39 SIN:54 SIN:232 SIN:247

Notes: SIN means SEQ ID NO:

Table 2B. High affinity MASP-3 inhibitory antibodies: CDR

Reference Variable Variable Heavy chain : Light chain : anti- severe area lung area CDR1; CDR2; CDR1; CDR2; MASP-3 chains chains CDR3 (SEQ ID CDR3 (SEQ ID antibody (amino acids) (amino acids) NO) NO) no 4D5 SIN:24 SIN:40 56;58;60 142;144;146 1F3 SIN:25 SIN:41 62;63;65 149;144;146 4B6 SIN:26 SIN:42 62;67;65 149;144;146 1A10 SIN:27 SIN:42 62;69;65 149;144;146 10D12 SIN:28 SIN:43 72;74;76 153;155;157 35C1 SIN:29 SIN:44 79;74;82 159;155;160 13B1 SIN:30 SIN:45 84;86;88 142;144;161 1G4 SIN:31 SIN:46 91;93;95 163;165;167 1E7 SIN:32 SIN:47 109;110;112 182;184;186 2D7 SIN:33 SIN:48 125;127;129 196;198;200 49CH SIN:34 SIN:49 132;133;135 203;165;204 15D9 SIN:35 SIN:50 137;138;140 206;207;208 2F5 SIN:36 SIN:51 98;99;101 169;171;173 1B11 SIN:37 SIN:52 103;105;107 176;178;180 2F2 SIN:38 SIN:53 H4;H6;118 188;178;190 11B6 SIN:39 SIN:54 114;121;123 191;178;193

- 77 040888

Table 3. Representative high affinity MASP-3 inhibitory antibodies: humanized and modified antibodies to remove post-translational modification sites

Reference anti-MAZR-Z antibody No Variable region of the heavy chain, a. to. (SEQ ID NO) Variable region of the lung a.k. chains (SEQID NO) Heavy chain:: CDR1; CDR2; CDR3 (SEQ ID NO) Light chain : CDR1; CDR2; CDR3 (SEQID NO) Parent 4D5 24 40 56;58;60 142;144;146 h4D5-14-l 248 250 56;58;60 142;144;146 h4D5-19-l 249 250 56;58;60 142;144;146 h4D5-14-l-NA 248 278 56;58;60 258;144;146 h4D5-19-l-NA 249 278 56;58;60 258;144;146 Parental 10D12 28 43 72;74;76 153;155;157 hlOD12-45-21 251 253 72;74;76 153;155;157 hlOD12-49-21 252 253 72;74;76 153;155;157 hlOD12-45-21- GA 251 279 72;74;76 263;155;157 hlOD12-49-21- GA 252 279 72;74;76 263;155;157 Parental 13B1 thirty 45 84;86;88 142;144;161 hl3Bl-9-l 254 256 84;275;88 142;144;161 hl3Bl-10-l 255 256 84;86;8 8 142;144;161 hl3Bl-9-l-NA 254 280 84;275;88 258;144;161 hl3Bl-10-l-NA 255 280 84;86;8 8 258;144;161

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

Inhibition of alternative pathway complement activation is characterized by at least one or more of the following modifications in a component of the complement system that result from administration of a high affinity MASP-3 inhibitory antibody in accordance with various embodiments of the invention: inhibition of hemolysis and/or opsonization; inhibition of lectin-independent conversion of factor B; inhibition of lectin-independent factor D conversion, inhibition of substrate-specific cleavage by the MASP-3 serine protease; reducing the degree of hemolysis or reducing the degree of C3 cleavage and deposition of C3b on the surface; reducing the degree of deposition of factors B and Bb on the activating surface; reduction of residual levels (in the bloodstream and without experimental addition of an activating surface) of active factor D compared with pro-factor D; lower levels of active factor D relative to pro-factor D in response to an activating surface; and/or producing residual and surface-induced levels of liquid phase Ba, Bb, C3b or C3a.

Thus, for example, as described herein, high affinity MASP-3 inhibitory antibodies are antibodies or antigen-binding fragments thereof capable of inhibiting factor D maturation (i.e., cleavage of pro-factor D to form factor D) in a mammal. In some embodiments, the high affinity MASP-3 inhibitory antibodies are capable of inhibiting factor D maturation in total serum to less than 50% of that observed in untreated control serum (e.g., less than 40%, e.g., less than

- 78 040888

30%, e.g., less than 25%, e.g., less than 20%, e.g., less than 15%, e.g., less than 10%, e.g., less than 5% compared to untreated control sera that were not exposed to MASP- 3 inhibitory antibody).

In preferred embodiments, the high affinity MASP-3 inhibitory antibodies selectively inhibit the alternative pathway so that the C1q-dependent complement activation system remains functionally intact.

In another aspect, the present invention provides a nucleic acid molecule that encodes one or both of the heavy and light chain polypeptides of any of the MASP-3 inhibitory antibodies described herein, or antigen-binding fragments thereof. The present invention also relates to a vector (eg, a cloning or expression vector) containing a nucleic acid, and a cell (eg, an insect, bacterial, fungal, or mammalian cell) containing the vector. The present invention also relates to a method for producing any of the MASP-3 inhibitory antibodies described herein, or antigen-binding fragments thereof. These methods include obtaining a cell containing an expression vector that contains a nucleic acid encoding one or both of the heavy and light chain polypeptides of any of the antibodies described herein or antigen-binding fragments thereof. The cell or cell culture is cultured under conditions and for a time sufficient for expression in the cell (or cell culture) of the antibody or its antigen-binding fragment encoded by the nucleic acid. Said method may also include isolating the antibody or antigen-binding fragment thereof from the cell (or cell culture) or from the culture medium of the cell or cells.

Epitopes and peptides of MASP-3.

As described in Example 18, shown in FIG. 62 and systematized below in table. 4, the high affinity MASP-3 inhibitory antibodies and antigen-binding fragments thereof according to the invention were found to specifically recognize one or more epitopes in the human MASP-3 serine protease domain (amino acid residues 450-728 of SEQ ID NO: 2). The term specifically recognizes means that the antibody binds to the specified epitope with a significantly higher affinity than to any other molecule or part thereof.

Table 4 Representative High Affinity MASP-3 Inhibitory Antibodies: MASP-3 Epitope Binding Regions (See also FIG. 62)

Peptide-binding fragments (epitopes) in reference human MASP-3 (with leader sequence) Ahth-MASP-3mAb Per. no. 49 ₈ VLRSQRRDTTVI ₅ o ₉ (SIN: 9) 1F3, 4B6, 4D5, 1A10, 10D12, 494TAAHVLRSQRRDTTV508 (SIN: 10) 13B1 544DFNIQNYNHDIALVQ ₅₅₈ (SIN: 11) 1F3, 4B6, 4D5, 1A10 ₆₂₆ PHAECKTSYESRS ₆₃₈ (SIN: 12) 13B1 ₆₃₉ GNYSVTENMFC ₆ 49 (SIN: 13) 1F3, 4B6, 4D5, 1A10 ₇ 04VSNYVDWVWE ₇₁₃ (SIN: 14) 1F3, 4B6, 4D5, 1A10 49 ₈ VLRSQRRDTTV ₅₀₈ (SIN: 15) Group I core sequence 1F3, 4B6, 4D5, 1A10, 10D12, 13B1 4 ₃₅ ECGQPSRSLPSLV447 (SIN: 16) 1B11 454RNAEPGLFPWQ ₄₆ 4 (SIN: 17) Core sequence of Groups II and III 1G4, 1E7, 2D7, 15D9, 2F5, 1B11 47 ₉ KWFGSGALLSASWIL49 ₃ (SIN 18) 15D9, 2F5 ₅₁ 4EHVTVYLGLH ₅₂₃ (SIN: 19) 1E7, 2D7, 1G4 5 ₆₂ PVPLGPPHVMP5 ₇₁ (SIN: 20) 15D9, 2F5 ₅₈₃ APHMLGL ₅₈₉ (SIN: 21) 1B11 ₆₁ 4SDVLQYVKLP ₆₂₃ (SIN :22) 1B11 ₆₆₇ AFVIFDLSQRW ₆ 7 ₈ (SIN: 23) 1G4, 1E7, 2D7, 15D9, 2F5

Accordingly, in some embodiments, the high affinity MASP3 inhibitory antibody, or antigen-binding fragment thereof, specifically binds to an epitope located in the human MASP-3 serine protease domain, wherein said epitope is located in at least one or more of the following sequences: VLRSQRRDTTVI

- 79 040888 (SEQ ID NO: 9), TAAHVLRSQRRDTTV (SEQ ID NO: 10), DFNIQNYNHDIALVQ (SEQ ID NO: 11), PHAECKTSYESRS (SEQ ID NO: 12), GNYSVTENMFC (SEQ ID NO: 13), VSNYVDWVWE (SEQ ID NO: 14) and/or VLRSQRRDTTV (SEQ ID NO: 15). In some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope in SEQ ID NO: 15. In some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope in SEQ ID NO: 9. In some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope in SEQ ID NO: 10. In some embodiments, an antibody or antigen-binding fragment thereof binds to an epitope in SEQ ID NO: 12. In some embodiments, an antibody or antigen-binding fragment thereof binds to an epitope in SEQ ID NO: 10 and SEQ ID NO: 12. In some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope in at least one of SEQ ID NO: 11, SEQ ID NO: 13, and/or SEQ ID NO: 14.

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

CDR areas.

In one aspect of the present invention, the antibody or its functional equivalent contains specific hypervariable regions, designated CDR. Preferably, the CDRs are CDRs as defined by Kabat. CDRs, or hypervariable regions, for example, can be identified by sequence alignment with other antibodies. The CDR regions of the high affinity MASP-3 inhibitory antibodies are shown in Table 1. 18-23.

mAb Group IA.

In one of its aspects, the present invention provides an isolated antibody, or antigen-binding fragment thereof, that binds to MASP-3 and contains: (a) a heavy chain variable region comprising HC-CDR1 represented as SEQ ID NO: 209 (XXDIN, where X in position 1 is S or T and where X in position 2 is N or D); HC-CDR2 shown as SEQ ID NO: 210 (WIYPRDXXXKYNXXFXD where X at position 7 is G or D; X at position 8 is S, T or R; X at position 9 is I or T; X at position 13 is E or D, X at position 14 is K or E, and X at position 16 is T or K); and HC-CDR3 shown as SEQ ID NO: 211 (XEDXY where X at position 1 is L or V and where X at position 4 is T or S); and (b) a light chain variable region comprising LC-CDR1 shown as SEQ ID NO: 212 (KSSQSLLXXRTRKNYLX, where X at position 8 is N, I, Q, or A; where X at position 9 is S or T; and where X at position 17 is A or S); LC-CDR2 shown as SEQ ID NO: 144 (WASTRES) and LC-CDR3 shown as SEQ ID NO: 146 (KQSYNLYT). In one embodiment, the heavy chain variable region HC-CDRI according to (a) comprises SEQ ID NO: 56 (TDDIN). In one embodiment, the heavy chain variable region HC-CDR2 according to (a) comprises SEQ ID NO: 62 (SNDIN). In one embodiment, the heavy chain variable region HC-CDR2 according to (a) comprises SEQ ID NO: 58 (WIYPRDDRTKYNDKFKD). In one embodiment, the heavy chain variable region HC-CDR2 according to (a) comprises SEQ ID NO: 63 (WIYPRDGSIKYNEKFTD). In one embodiment, the heavy chain variable region HCCDR2 according to (a) comprises SEQ ID NO: 67 (WIYPRDGTTKYNEEFTD). In one embodiment, the heavy chain variable region HC-CDR2 according to (a) comprises SEQ ID NO: 69 (WIYPRDGTTKYNEKFTD). In one embodiment, the heavy chain variable region HC-CDR3 according to (a) comprises SEQ ID NO: 60 (LEDTY). In one embodiment, the heavy chain variable region HC-CDR3 according to (a) comprises SEQ ID NO: 65 (VEDSY). In one embodiment, the light chain variable region LC-CDR1 comprises SEQ ID NO: 142 (KSSQSLLNSRTRKNYLA); SEQ ID NO: 257 (KSSQSLLQSRTRKNYLA), SEQ ID NO: 258 (KSSQSLLASRTRKNYLA); or SEQ ID NO: 259 (KSSQSLLNTRTRKNYLA). In one embodiment, the LC-CDR1 contains SEQ ID NO: 258 (KSSQSLLASRTRKNYLA). In one embodiment, the LC-CDR1 contains SEQ ID NO: 149 (KSSQSLLISRTRKNYLS).

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

In one embodiment, HC-CDR1 contains SEQ ID NO: 62, HC-CDR2 contains SEQ ID NO: 63, SEQ ID NO: 67 or SEQ ID NO: 69, HC-CDR3 contains SEQ ID NO: 65 and LCCDR1 contains SEQ ID NO: 149, LC-CDR2 contains SEQ ID NO: 144, and LC-CDR3 contains SEQ ID NO: 146.

mAb Group IB.

In another aspect, the present invention provides an isolated antibody, or antigen-binding fragment thereof, that binds to MASP-3 and contains: (a) a heavy chain variable region comprising HC-CDR1 represented as SEQ ID NO: 213 (SYGXX where X in position 4 is M or I and where X in position 5 is S or T); HC-CDR2 shown as SEQ ID NO: 74; and HC-CDR3 shown as SEQ ID NO: 214 (GGXAXDY where X at position 3 is E or D and where X at position 5 is M or L); and (b) a light chain variable region comprising LC-CDR1 shown as SEQ ID NO: 215 (KSSQSLLDSXXKTYLX, where X at position 10 is D, E, or A; where X at position 11 is G or A; and where X at position 16 is N or S); LC-CDR2 shown as SEQ ID NO: 155; and LC-CDR3 shown as SEQ ID NO: 216 (WQGTHFPXT where X at position 8 is W or Y).

In one embodiment, the heavy chain variable region HC-CDR1 according to (a) comprises SEQ ID NO: 72 (SYGMS). In one embodiment, the HC-CDR1 contains SEQ ID NO: 79 (SYGIT). In one embodiment, the HCCDR3 contains SEQ ID NO: 76 (GGEAMDY). In one embodiment, the HCCDR3 contains SEQ ID NO: 82 (GGDALDY). In one of the embodiments of the invention LCCDR1 contains SEQ ID NO: 153 (KSSQSLLDSDGKTYLN); SEQ ID NO: 261 (KSSQSLLDDSEGKTYLN), SEQ ID NO: 262 (KSSQSLLDSAGKTYLN), or SEQ ID NO: 263 (KSSQSLLDSDAKTYLN). In one embodiment, the LC-CDR1 contains SEQ ID NO: 263 (KSSQSLLDSDAKTYLN). In one embodiment, the LC-CDR1 contains SEQ ID NO: 152. In one embodiment, the LC-CDR3 contains SEQ ID NO: 159 (KSSQSLLDSDGKTYLS).

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

In one embodiment, HC-CDR contains SEQ ID NO: 72, HC-CDR2 contains SEQ ID NO: 74, HC-CDR3 contains SEQ ID NO: 76, and LC-CDR1 contains SEQ ID NO: 153 or SEQ ID NO : 263, LC-CDR2 contains SEQ ID NO: 155, and LC-CDR3 contains SEQ ID NO: 157.

In one embodiment, HC-CDR1 contains SEQ ID NO: 79, HC-CDR2 contains SEQ ID NO: 74, HC-CDR3 contains SEQ ID NO: 82, and LC-CDR1 contains SEQ ID NO: 159, LC-CDR2 contains SEQ ID NO: 155 and LC-CDR3 contains SEQ ID NO: 160.

mAb Group IC.

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

Group II mAbs.

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

- 81 040888 mAb Group III.

In another aspect, the present invention provides an isolated antibody, or antigen-binding fragment thereof, that binds to MASP-3 and contains:

(a) a heavy chain variable region comprising HC-CDR1 shown as SEQ ID NO: 109 (RVHFAIRDTNYWMQ), HC-CDR2 shown as SEQ ID NO: 110 (AIYPGNGDTSYNQKFKG), HC-CDR3 shown as SEQ ID NO: 112 (GSHYFDY); and a light chain variable region comprising LC-CDR1 shown as SEQ ID NO: 182 (RASQSIGTSIH), LC-CDR2 shown as SEQ ID NO: 184 (YASESIS) and LC-CDR3 shown as SEQ ID NO: 186 (QQSNSWPYT ); or (b) a heavy chain variable region comprising HC-CDR1 given as SEQ ID NO: 125 (DYYMN), HC-CDR2 given as SEQ ID NO: 127 (DVNPNNDGTTYNQKFKG), HC-CDR3 given as SEQ ID NO: 129(CPFYYLGKGTHFDY); and a light chain variable region comprising LC-CDR1 given as SEQ ID NO: 196 (RASQDISNFLN), LC-CDR2 given as SEQ ID NO: 198 (YTSRLHS) and LC-CDR3 given as SEQ ID NO: 200 (QQGFTLPWT ); or (c) a heavy chain variable region comprising HC-CDR1, shown as SEQ ID NO: 137, HC-CDR2, shown as SEQ ID NO: 138, HC-CDR3, shown as SEQ ID NO: 140; and a light chain variable region comprising LC-CDR1, shown as SEQ ID NO: 206, LCCDR2, shown as SEQ ID NO: 207, and LC-CDR3, shown as SEQ ID NO: 208; or (d) a heavy chain variable region comprising HC-CDR1, shown as SEQ ID NO: 98, HC-CDR2, shown as SEQ ID NO: 99, HC-CDR3, shown as SEQ ID NO: 101; and a light chain variable region comprising LC-CDR1, shown as SEQ ID NO: 169, LCCDR2, shown as SEQ ID NO: 171, and LC-CDR3, shown as SEQ ID NO: 173; or (e) a heavy chain variable region comprising HC-CDR1, shown as SEQ ID NO: 103, HC-CDR2, shown as SEQ ID NO: 105, HC-CDR3, shown as SEQ ID NO: 107; and a light chain variable region comprising LC-CDR1, shown as SEQ ID NO: 176, LCCDR2, shown as SEQ ID NO: 178, and LC-CDR3, shown as SEQ ID NO: 193; or (f) a heavy chain variable region comprising HC-CDR1, shown as SEQ ID NO: 114, HC-CDR2, shown as SEQ ID NO: 116, HC-CDR3, shown as SEQ ID NO: 118; and a light chain variable region comprising LC-CDR1, shown as SEQ ID NO: 188, LCCDR2, shown as SEQ ID NO: 178, and a LC-CDR3, shown as SEQ ID NO: 190; or (g) a heavy chain variable region comprising HC-CDR1, shown as SEQ ID NO: 114, HC-CDR2, shown as SEQ ID NO: 121, HC-CDR3, shown as SEQ ID NO: 123; and a light chain variable region comprising LC-CDR1, shown as SEQ ID NO: 191, LCCDR2, shown as SEQ ID NO: 178, and LC-CDR3, shown as SEQ ID NO: 193; or (h) a heavy chain variable region comprising HC-CDR1, shown as SEQ ID NO: 132, HC-CDR2, shown as SEQ ID NO: 133, HC-CDR3, shown as SEQ ID NO: 135; and a light chain variable region comprising LC-CDR1, shown as SEQ ID NO: 203, LCCDR2, shown as SEQ ID NO: 165, and LC-CDR3, shown as SEQ ID NO: 204. Heavy chain and light chain variable regions In one of its variants, the present invention provides a high affinity MASP-3 inhibitory antibody comprising a heavy chain variable region comprising or consisting of a sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% identical to any of the SEQs. ID NO: 24-39, 248-249, 251-252, 254-255, or to an antibody containing a heavy chain variable region comprising SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO: 252, SEQ ID NO: 254 or SEQ ID NO: 255.

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

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

In one embodiment, the anti-MASP-3 monoclonal antibody comprises a heavy chain containing a sequence that is at least 80, 85, 90, 95, 98, 99 or

100% identical to SEQ ID NO: 25, and a light chain containing a sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 41.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

- 83 040888

Cross-competitive binding of high-affinity aHTu-MASP-3 antibodies.

As described herein, the high affinity MASP-3 inhibitory antibodies described herein recognize overlapping epitopes in the MASP-3 serine protease domain. As described in Example 18, shown in FIG. 61A-E and 62-67 and systematized in the table. 4 and 28, the cross-competitive binding assay and the Pepscan binding assay showed that high affinity MASP-3 inhibitory antibodies cross-compete for binding to common epitopes located in the MASP-3 serine protease domain. Thus, in one embodiment, the present invention provides high affinity MASP-3 inhibitory antibodies that specifically recognize an epitope or portion thereof in the human MASP-3 serine protease domain recognized by one or more antibodies selected from the group consisting of a monoclonal an antibody comprising a heavy chain variable region shown as SEQ ID NO: 24 and a light chain variable region shown as SEQ ID NO: 40;

a monoclonal antibody comprising a heavy chain variable region shown as SEQ ID NO: 25 and a light chain variable region shown as SEQ ID NO: 41;

a monoclonal antibody comprising a heavy chain variable region shown as SEQ ID NO: 26 and a light chain variable region shown as SEQ ID NO: 42;

a monoclonal antibody comprising a heavy chain variable region shown as SEQ ID NO: 27 and a light chain variable region shown as SEQ ID NO: 42;

a monoclonal antibody comprising a heavy chain variable region shown as SEQ ID NO: 28 and a light chain variable region shown as SEQ ID NO: 43;

a monoclonal antibody comprising a heavy chain variable region shown as SEQ ID NO: 29 and a light chain variable region shown as SEQ ID NO: 44;

a monoclonal antibody comprising a heavy chain variable region shown as SEQ ID NO: 30 and a light chain variable region shown as SEQ ID NO: 45;

a monoclonal antibody comprising a heavy chain variable region shown as SEQ ID NO: 31 and a light chain variable region shown as SEQ ID NO: 46;

a monoclonal antibody comprising a heavy chain variable region shown as SEQ ID NO: 32 and a light chain variable region shown as SEQ ID NO: 47;

a monoclonal antibody comprising a heavy chain variable region shown as SEQ ID NO: 33 and a light chain variable region shown as SEQ ID NO: 48;

a monoclonal antibody comprising a heavy chain variable region shown as SEQ ID NO: 34 and a light chain variable region shown as SEQ ID NO: 49;

a monoclonal antibody comprising a heavy chain variable region shown as SEQ ID NO: 35 and a light chain variable region shown as SEQ ID NO: 50;

a monoclonal antibody comprising a heavy chain variable region shown as SEQ ID NO: 36 and a light chain variable region shown as SEQ ID NO: 51;

a monoclonal antibody comprising a heavy chain variable region shown as SEQ ID NO: 37 and a light chain variable region shown as SEQ ID NO: 52;

a monoclonal antibody comprising a heavy chain variable region shown as SEQ ID NO: 38 and a light chain variable region shown as SEQ ID NO: 53; and a monoclonal antibody comprising the heavy chain variable region shown as SEQ ID NO: 39 and the light chain variable region shown as SEQ ID NO: 54.

In accordance with the present invention, if a given antibody recognizes at least a portion of an epitope recognized by another given antibody, then the two antibodies are said to recognize the same or overlapping epitopes.

In order to determine whether an antibody (also referred to as test antibody) recognizes the same or overlapping epitopes for a particular monoclonal antibody (also referred to as reference antibody), various tests known to those skilled in the art can be performed. Preferably, such analysis includes the steps:

obtaining MASP-3 or its fragment containing an epitope recognized by the reference antibody;

adding a test antibody and a reference antibody to said MASP-3, wherein the test antibody or reference antibody has been labeled with a detectable label. Alternatively, both antibodies can be labeled with different detectable labels;

detecting the presence of a detectable label in MASP-3;

detection to determine if the antibody being tested can displace the reference antibody.

In the case of displacement of the reference antibody, the test antibody recognizes the same or overlapping epitopes for the reference antibody. Thus, if the reference antibody has been labeled with a detectable label, then a weak detectable signal from MASP-3 indicates displacement of the reference antibody. If the test antibody has been labeled with a detectable label, then a strong detectable signal from MASP-3 indicates displacement of the reference antibody. The MASP-3 fragment may preferably be immobilized on a solid support to facilitate its handling. detecting

- 84 040888 May the label can be any directly or indirectly detectable label, such as an enzyme, a radioactive isotope, a heavy metal, a colored compound, or a fluorescent compound. Example 18, in the Competitive Binding Assay section below, describes a representative method for determining the ability of a test antibody to recognize the same epitope or an overlapping epitope recognized by a reference antibody. A person skilled in the art can easily adapt this method to the specific antibodies in question.

Ahtu-MASP-3 antibodies used in this aspect of the invention are monoclonal or recombinant antibodies derived from any antibody-producing mammal, and such antibodies can be multispecific (i.e., bispecific or trispecific), chimeric, humanized, fully human , anti-idiotypic antibodies and their fragments. Antibody fragments are Fab-, Fab'-, F(ab)2-, F(ab') ₂ -, Fv-fragments, scFv-fragments and single-chain antibodies, described in detail in this application.

Ahtu-MASP-3 antibodies can be screened for the ability to inhibit the alternative pathway complement activation system using the assays described here. Inhibition of alternative pathway complement activation is characterized by at least one or more of the following modifications in a component of the complement system that result from administration of a high affinity MASP-3 inhibitory antibody in accordance with various embodiments of the invention: inhibition of hemolysis and/or opsonization; inhibition of lectin-independent conversion of factor B; inhibition of lectin-independent factor D conversion, inhibition of substrate-specific cleavage by the MASP-3 serine protease; reducing the degree of hemolysis or reducing the degree of C3 cleavage and deposition of C3b on the surface; reducing the degree of deposition of factors B and Bb on the activating surface; reduction of residual levels (in the bloodstream and without experimental addition of an activating surface) of active factor D compared to profactor D; lower levels of active factor D relative to pro-factor D in response to an activating surface; and/or producing residual and surface-induced levels of liquid phase Ba, Bb, C3b or C3a.

Ahtu-MASP-3 antibodies with reduced effector function.

In some embodiments of this aspect of the present invention, the high affinity MASP-3 inhibitory antibodies described herein have reduced effector function to reduce inflammation that may result from classical pathway complement activation. The ability of IgG molecules to trigger classical pathway complement has been shown to be retained in the Fc portion of the molecule (Duncan AR et al., Nature 332:138 740 (1988)). IgG molecules in which the Fc portion of the molecule has been removed by enzymatic cleavage do not have this effector function (see Harlow, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988). Accordingly, antibodies with reduced effector function can be generated by removing the Fc portion of the molecule by genetically engineering an Fc sequence that has minimal effector function or is of either the human IgG2 or IgG ₄ isotype.

Antibodies with reduced effector function can be generated using standard molecular biological manipulation using the Fc portion of IgG heavy chains, as described in Jolliffe et al., Int'l Rev. Immunol. 10:241 250, (1993) and Rodrigues et al., J. Immunol. 151:6954 6961, (1998). Antibodies with reduced effector function are also human antibodies of the IgG2 and IgG4 isotypes, which have a reduced ability to activate complement and/or interact with Fc receptors (Ravetch J.V. et al., Annu. Rev. Immunol. 9:451 492, (1991); Isaacs J. D. et al., J. Immunol. 148:3062 3071, 1992; van de Winkel J. G. et al., Immunol. Today 14:215 221 (1993)). Humanized or fully human antibodies specific for human MASP-1, MASP-2, or MASP-3 (including dual-specific, pan-specific, bispecific, or trispecific antibodies) and consisting of IgG2 or IgG4 isotypes can be generated by one of several methods. known to those skilled in the art as described by Vaughan T.J. et al., Nature Biotechnical. 16:535 539, (1998).

Production of high-affinity MASP-3 inhibitory antibodies.

Ahtu-MASP-3 antibodies can be generated using MASP-3 polypeptides (eg, full-length MASP-3) or using antigenic peptides bearing a MASP-3 epitope (eg, a portion of a MASP-3 polypeptide), for example, as described below in Example 14 Immunogenic peptides may have at least five amino acid residues. The MASP-3 peptides and polypeptides used to generate antibodies can be isolated as natural polypeptides or recombinant or synthetic peptides and catalytically inactive recombinant polypeptides. Antigens used to produce anti-MASP-3 antibodies also include fusion polypeptides, such as fusions of a MASP-3 polypeptide or portion thereof with an immunoglobulin polypeptide or maltose binding protein. The polypeptide immunogen may be a full-length molecule or a portion thereof. If a portion of the polypeptide is hapten-like, then such portion may advantageously be attached to or coupled to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), or tetanus toxoid) to

- 85 040888 immunization.

monoclonal antibodies.

As used herein, the term monoclonal indicates the type of antibody obtained from a substantially homogeneous population of antibodies, but this term does not mean that the antibody must be obtained by any particular method. Monoclonal antibodies can be obtained by any method of producing antibody molecules using stable cell lines in culture, such as the hybridoma method described by Kohler G. et al., Nature 256:495 (1975), or recombinant DNA methods (see, for example, US patent No. 4816567, Cabilly). Monoclonal antibodies can also be isolated from antibody phage libraries by the methods described by Clackson T. et al., Nature 352:624-628, (1991), and Marks J.D. et al., J. Mol. Biol. 222:581-597 (1991). Such antibodies can be any class of immunoglobulins, including IgG, IgM, IgE, IgA, IgD, and any subclass.

For example, monoclonal antibodies can be obtained by administering to a suitable mammal (eg, BALB/c mice) a composition containing a MASP-3 polypeptide or a portion thereof. After a predetermined period of time, splenocytes are taken from mice and suspended in cell culture medium. The splenocytes are then fused with an immortalized cell line to form a hybridoma. The resulting hybridomas are cultured in cell culture and screened for their ability to produce anti-MASP-3 monoclonal antibody. (See also Current Protocols in Immunology, Vol. 1., John Wiley & Sons, pages 2.5.1-2.6.7, 1991).

Human monoclonal antibodies can be produced using transgenic mice that have been designed to produce specific human antibodies in response to antigen challenge. In this method, human immunoglobulin heavy and light chain locus elements are administered to mice derived from embryonic stem cell lines that contain targeted disruptions of endogenous immunoglobulin heavy chain and light chain loci. Transgenic mice can synthesize human antibodies specific for human antigens, such as the MASP-2 antigens described here, and these mice can be used to produce anti-human MASP-2 antibody-secreting hybridomas by fusing B cells from these animals, obtaining suitable myeloma cell lines using standard Kohler-Milstein technology. Methods for obtaining human antibodies from transgenic mice are described, for example, by Green L.L. et al., Nature Genet. 7:13, 1994; Lonberg N. et al., Nature 368:856, 1994; and Taylor L.D. et al., Int. Immun. 6:579, 1994.

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

Monoclonal antibodies, once obtained, are first tested for specific binding to MASP-3 or, if necessary, double binding to MASP-1/3, MASP-2/3 or MASP-1/2. Methods for determining the binding of an antibody to a protein antigen and/or determining the affinity of an antibody for a protein antigen are known to those skilled in the art. For example, the binding of an antibody to a protein antigen can be detected and/or quantified by various methods such as, but not limited to, Western blot analysis, dot blot analysis, surface plasmon resonance (e.g., using a system BIAcore; Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ), or ELISA. See, for example, Harlow and Lane (1988) Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Benny KC Lo (2004) Antibody Engineering: Methods and Protocols, Humana Press (ISBN: 1588290921); Borrebaek (1992) Antibody Engineering, A Practical Guide, W.H. Freeman and Co., NY; Borrebaek (1995) Antibody Engineering, ^2nd Edition, Oxford University Press, NY, Oxford; Johne et al. (1993), Immunol. Meth. 160:191-198; Jonsson et al. (1993) Ann. Biol. Clin. 51:19-26; and Jonsson et al. (1991) Biotechniques 11:620627. See also U.S. Patent No. 6,355,245.

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

Once antibodies that specifically bind to MASP-3 are identified, anti-MASP-3 antibodies are tested for their ability to function as an alternative pathway inhibitor in one of several functional assays, such as, for example, the alternative pathway complement activation inhibition assay, which is characterized by at least one or more of the following modifications in a component of the complement system resulting from administration of a high affinity MASP-3 inhibitory antibody in accordance with various embodiments of the invention: inhibition of hemolysis and/or opsonization; inhibition of lectin-independent conversion of factor B; inhibition of lectin-independent factor D conversion; inhibition

- 86 040888 substrate-specific cleavage by serine protease MASP-3; reducing the degree of hemolysis or reducing the degree of C3 cleavage and deposition of C3b on the surface; reducing the degree of deposition of factors B and Bb on the activating surface; reducing residual levels (in the circulation and without experimental addition of an activating surface) of active factor D compared to profactor D; lower levels of active factor D relative to pro-factor D in response to an activating surface; reducing the level of production of residual and surface-induced levels of liquid-phase Ba, Bb, C3b or C3a; and/or reducing factor P precipitation.

Chimeric/humanized antibodies.

The monoclonal antibodies used in the method of the invention include chimeric antibodies in which a portion of the heavy and/or light chain is identical or homologous to the respective sequences of antibodies derived from a particular species or belonging to a particular class or subclass of antibodies, while the remainder of the chain(s) identical or homologous to the corresponding sequences of antibodies derived from other species or belonging to another class or subclass of antibodies, as well as fragments of such antibodies (US patent No. 4816567, Cabilly; and Morrison S.L. et al., Proc. Nat'l Acad. Sci. USA 81:6851 6855 (1984)).

One form of chimeric antibody used in the present invention is a humanized monoclonal anti-MASP-3 antibody. Humanized forms of non-human (eg, murine) antibodies are chimeric antibodies that contain a minimal sequence derived from a non-human immunoglobulin.

Humanized monoclonal antibodies are produced by transferring the non-human (eg, murine) hypervariable regions (CDRs) of the murine immunoglobulin heavy and light chain variable regions into the human variable domain. Then, human antibody residues are usually replaced in the framework regions of the non-human counterparts. In addition, humanized antibodies may contain residues that are not present in the recipient antibody or in the donor antibody. These modifications are introduced to improve the properties of the antibody. In general, a humanized antibody will contain substantially all or at least one, and usually two, variable domains in which all or almost all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or almost all of the Fv framework regions are regions of the human sequence. immunoglobulin.

The humanized antibody also contains, but not necessarily, at least a portion of an immunoglobulin constant region (Fc), and usually a human immunoglobulin. For a more detailed description, see Jones R.T. et al., Nature 321:522 525 (1986); Reichmann L. et al., Nature 332:323 329, (1988); and Presta, Curr. Op. Struct. Biol. 2:593 596, (1992).

Humanized antibodies used in the present invention include human monoclonal antibodies containing at least a CDR3 region that binds to MASP-3. In addition, the Fc portions can be changed so that they produce IgA or IgM as well as human IgG antibodies. Such humanized antibodies will be of particular clinical use as they will specifically recognize human MASP-3 but will not elicit an immune response in humans against the antibody itself. Therefore, they are better suited for administration to the human body in vivo, especially for repeated or prolonged administration.

Methods for preparing humanized monoclonal antibodies are also described, for example, in Jones P.T. et al., Nature 321:522, (1986); Carter P. et al., Proc. Nat.'l. Acad. sci. USA 89:4285, (1992); Sandhu J.S., Crit. Rev. Biotech. 12:431, (1992); Singer I.I. et al., J. Immun. 150:2844, (1993); Sudhir (ed.), Antibody Engineering Protocols, Humana Press, Inc., (1995); Kelley, Engineering Therapeutic Antibodies, in Protein Engineering: Principles and Practice, Cleland et al. (eds.), John Wiley & Sons, Inc., pages 399 434, (1996); and US Pat. No. 5,693,762, Queen, 1997. In addition, there are commercial facilities that synthesize humanized antibodies from specific mouse antibody regions, such as Protein Design Labs (Mountain View, CA).

recombinant antibodies.

Ahtu-MASP-3 antibodies can also be obtained using recombinant techniques. For example, human antibodies can be generated using human immunoglobulin-expressing gene libraries (available from, for example, Stratagene, Corp., La Jolla, CA) to generate human antibody fragments (VH, VL, F _V , factor D, Fab or F(ab') ₂ ). These fragments are then used to construct full-length human antibodies using methods similar to those for making chimeric antibodies.

Fragments of immunoglobulin.

The MASP-3 inhibitory agents used in the method of the invention include not only intact immunoglobulin molecules, but also well known fragments including Fab-, Fab'-, F(ab) ₂ -, F(ab') ₂ - and Fv fragments, ScFv fragments, diabodies, linear antibodies, single chain antibody molecules, and multispecific (eg, bispecific and trispecific) antibodies formed from antibody fragments.

It is well known to those skilled in the art that only a small portion of an antibody molecule,

- 87 040888

those. paratope, is involved in the binding of an antibody to its epitope (see, for example, Clark WR, The Experimental Foundations of Modern Immunology, Wiley & Sons, Inc., NY, 1986). The pFc' and Fc regions of an antibody are complement effectors of the classical pathway but are not involved in antigen binding. An antibody from which a pFc' region is enzymatically cleaved, or which has been produced without a pFc' region, is referred to as a F(ab') ₂ fragment and retains both antigen-binding sites of the intact antibody. The isolated F(ab') ₂ fragment is called a divalent monoclonal fragment because of the two antigen-binding sites. Similarly, an antibody from which an Fc region is enzymatically cleaved, or which has been produced without an Fc region, is referred to as a Fab fragment, and retains one of the antigen-binding sites of the intact antibody molecule.

Antibody fragments can be obtained by proteolytic hydrolysis, for example by hydrolysis of full length antibodies with pepsin or papain by standard methods. Thus, for example, antibody fragments can be obtained by enzymatic cleavage of antibodies with pepsin to form a 5S fragment, designated F(ab') ₂ . This fragment can be further cleaved using a thiol reducing agent to give monovalent 3.5S Fab' fragments. The cleavage reaction may optionally be carried out using a blocking group for the sulfhydryl groups that result from the cleavage of disulfide bonds. Alternatively, enzymatic cleavage with pepsin results in the formation of two monovalent Fab fragments and an Fc fragment itself. These methods are described, for example, in the US patent. No. 4331647, Goldenberg; Nisonoff A. et al., Arch. Biochem. Biophys. 89:230, (1960); Porter RR, Biochem. J. 73:119, (1959); Edelman et al., in Methods in Enzymology 1:422, Academic Press, (1967); and in the Coligan publication, at pp. 2.8.1 2.8.10 and 2.10 2.10.4.

In some embodiments of the invention, the use of antibody fragments that do not contain an Fc region is preferred to prevent complement activation of the classical pathway that is initiated after binding of Fc to the Fcy receptor. There are several ways in which a monoclonal antibody can be produced while avoiding interactions with the Fcy receptor. For example, the Fc region of a monoclonal antibody can be chemically removed by partial cleavage with proteolytic enzymes (eg, such as ficin cleavage), which allows, for example, antigen-binding fragments of the antibody, such as Fab- or F(ab) ₂ -Fragments ( Mariani M. et al., Mol. Immunol. 28:69 71 (1991)). Alternatively, a human IgG y4 isotype antibody that does not bind to Fcy receptors can be used in the construction of a humanized antibody as described herein. Antibodies, single chain antibodies, and antigen binding domains lacking an Fc domain can also be constructed by the recombinant methods described herein.

Fragments of single-chain antibodies.

Alternatively, single peptide chain-binding, MASP-3 specific molecules can be designed in which the heavy and light chain Fv regions are linked. Fv fragments can be joined by a peptide linker to form a single chain antigen binding protein (scFv). These single-stranded antigen-binding proteins are produced by constructing a structural gene containing DNA sequences encoding V _H and VL domains that are linked by an oligonucleotide. The structural gene is inserted into an expression vector which is then introduced into a host cell such as E. coli. Recombinant host cells synthesize a single polypeptide chain with a linker peptide that is bridged to two V domains. Methods for preparing scFv are described, for example, in Whitlow et al., Methods: A Companion to Methods in Enzymology 2:97, (1991); Bird et al., Science 242:423, (1988); US patent No. 4946778, Ladner; Pack P. et al., Bio/Technology 11:1271 (1993).

As an illustrative example, a MASP-3-specific ScFv can be generated by treating lymphocytes with a MASP-3 polypeptide in vitro and selecting libraries to present antibodies in phage or similar vectors (e.g., by using an immobilized or labeled MASP-3 protein or peptide) . Genes encoding polypeptides having potential MASP-3 polypeptide binding domains can be obtained by randomized screening of peptide libraries presented on phage or bacteria such as E. coli. These randomized peptide presentation libraries can be used to screen for peptides that interact with MASP-3. Methods for generating and screening such randomized peptide presentation libraries are well known to those skilled in the art (U.S. Patent No. 5,223,409 to Lardner; U.S. Patent No. 4,946,778 to Ladner; U.S. Patent No. 5,403,484 to Lardner; U.S. Patent No. 5,571,698 to Lardner; and Kay et al. , Phage Display of Peptides and Proteins Academic Press, Inc., 1996), and randomized peptide display libraries and screening kits for such libraries are commercially available from, for example, CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc. (Beverly, Mass.), and Pharmacia LKB Biotechnology Inc. (Piscataway, N.J.).

Another form of the anti-MASP-3 antibody fragment used in this aspect of the invention is

- 88 040888 is a peptide encoding a single hypervariable region (CDR) that binds to an epitope on the MASP-3 antigen and inhibits alternative pathway complement activation.

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

The high affinity MASP-3 inhibitory antibodies described herein are administered to an individual in need thereof to inhibit the activation of an alternative pathway. In some embodiments, the high affinity MASP-3 inhibitory antibody is a humanized anti-MASP-3 monoclonal antibody that has, but not necessarily reduced effector function.

bispecific antibodies.

High affinity MASP-3 inhibitory antibodies useful in the method of the invention include multispecific (ie, bispecific and trispecific) antibodies.

Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificity for at least two different antigens. In one embodiment, the compositions and methods comprise the use of a bispecific antibody having a binding specificity for a MASP-3 serine protease domain and a binding specificity for MASP-2 (e.g., binding to at least one of CCP1-CCP2 or a MASP-3 serine protease domain). 2). In another embodiment, the method comprises using a bispecific antibody having a binding specificity for a MASP-3 serine protease domain and a binding specificity for MASP-1 (eg, a binding specificity for a MASP-1 serine protease domain). In another embodiment, the method comprises using a trispecific antibody having a binding specificity for MASP-3 (e.g., binding specificity for a MASP-3 serine protease domain), a specificity for binding to MASP-2 (e.g., binding specificity for at least one CCP1 -CCP2 or MASP-2 serine protease domain) and binding specificity to MASP-1 (eg, binding specificity to the MASP-1 serine protease domain).

Methods for producing bispecific antibodies are within the skill of the art. Traditionally, the recombinant method for producing bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain/light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature 305:537-539 (1983)). Antibody variable domains with the desired binding specificities (antibody-antigen binding sites) can be fused to immunoglobulin constant domain sequences. Such attachment is preferably carried out using an immunoglobulin heavy chain constant domain, including at least a portion of the hinge, CH2 and CH3 regions. DNA encoding immunoglobulin heavy chain and, if appropriate, immunoglobulin light chain fusions are inserted into separate expression vectors and co-transfected into a suitable host organism. For a more detailed description of illustrative and currently known methods for producing bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology 121:210 (1986); WO96/27011; Brennan et al. Science 229:81 (1985); Shalaby et al., J. Exp. Med. 175:217-225 (1992); Kostelny et al., J. Immunol. 148(5):1547-1553 (1992); Hollinger et al. Proc. Natl. Acad. Sci USA 90:6444-6448 (1993); Gruber et al., J. Immunol. 152:5368 (1994); and Tutt et al., J. Immunol. 147:60 (1991). Bispecific antibodies also include cross-linked or heteroconjugated antibodies. Heteroconjugated antibodies can be made by any of the standard cross-linking methods. Suitable crosslinking agents are well known to those skilled in the art and are described in US Pat. No. 4,676,980, along with various crosslinking techniques.

Various methods have also been described for obtaining and isolating bispecific antibody fragments directly from recombinant cell culture. For example, bispecific antibodies have been generated using leucine zippers (see, eg, Kostelny et al. J. Immunol. 148(5):1547-1553 (1992)). Diabody technologies described by Hollinger et al. Proc. Natl. Acad. Sci USA 90:6444-6448 (1993) are based on an alternative mechanism for generating bispecific antibody fragments. These fragments contain a heavy chain variable domain (VH) linked to a light chain variable domain (VL) via a linker that is too short to pair the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Bispecific diabodies, as opposed to bispecific full length antibodies, may also be particularly preferred because they can be easily constructed and expressed in E. coli. Diabodies (and many other polypeptides such as antibody fragments) having appropriate binding specificities can be readily selected from phage display libraries (WO 94/13804). If one strand of the diantibody remains constant, eg, has a specificity directed against antigen X, then a library can be generated in which the other strand differs from the first strand, and then an antibody with the appropriate specificity can be selected.

The literature also describes another strategy for obtaining bispecific antibody fragments using single-chain Fv dimers (ScFv). (See, for example, Gruber et al. J. Immunol., 152:5368 (1994)). Alternatively, the antibodies may be linear antibodies as described, for example, by Zapata et al., Protein Eng. 8(10):1057-1062 (1995). Briefly, these antibodies contain a pair of factor D tandem segments (VH-CHI-VH-CHI) that form a pair of antigen-binding regions. Linear antibodies can be bispecific or monospecific. The methods of the invention also include the use of various forms of bispecific antibodies, such as the tetravalent dual variable domain immunoglobulin molecules (DVD-Ig) described by Wu et al., Nat. Biotechnol. 25:1290-1297 (2007). DVD-Ig molecules are designed so that two different light chain variable domains (VL) derived from two different parental antibodies are linked in tandem directly or via a short linker using recombinant DNA techniques, followed by the addition of a light chain constant domain. Methods for obtaining DVD-Ig molecules from two parental antibodies are further described, for example, in applications WO 08/024188 and WO 07/024715, each of which is incorporated herein by reference in its entirety.

XVIII. Pharmaceutical Compositions and Dosing Methods.

In another aspect, the present invention relates to compositions containing high affinity MASP-3 inhibitory antibodies for inhibiting the side effects of alternative pathway complement activation in an individual in need of it, for example, an individual suffering from a disease or condition associated with an alternative pathway, such as a hemolytic disease such as PNH, or a disease or disorder selected from the group consisting of: age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy (including hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS) and thrombotic thrombocytopenic purpura (TTP)), asthma, hard deposit disease, oligoimmune necrosing crescentic glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, optic neuromyelitis a, Behcet's disease, multiple sclerosis (PC), Guillain-Barré syndrome, Alzheimer's disease, amylotrophic lateral sclerosis (ABS), lupus nephritis, systemic lupus erythematosus (SLE), diabetic retinopathy, uveitis, chronic obstructive pulmonary disease (COPD), C3 glomerulopathy, graft rejection, graft-versus-host disease (GVHD), hemodialysis, sepsis, systemic inflammatory response syndrome (SIR), acute respiratory distress syndrome (ARDS), ANCA vasculitis, antiphospholipid syndrome, atherosclerosis, IgA nephropathy, and myasthenia gravis .

The methods of this aspect of the present invention comprise administering to an individual a composition comprising an amount of a high affinity MASP-3 inhibitory antibody effective to inhibit alternative pathway complement activation and a pharmaceutically acceptable carrier. In some embodiments, said method also includes administering a composition containing a MASP-2 inhibitory agent. High affinity MASP-3 inhibitory antibodies and MASP-2 inhibitory agents may be administered to an individual in need thereof at therapeutically effective doses to treat or alleviate conditions associated with alternative pathway complement activation, and optionally also MASP-2- dependent complement activation. A therapeutically effective dose corresponds to an amount of a MASP-2 inhibitory antibody or a combination of a MASP-3 inhibitory antibody and a MASP-2 inhibitory agent sufficient to relieve the symptoms of the condition. Inhibition of alternative pathway complement activation is characterized by at least one or more of the following modifications in a component of the complement system that result from administration of a high affinity MASP-3 inhibitory antibody in accordance with various embodiments of the invention: inhibition of hemolysis and/or opsonization; inhibition of lectin-independent conversion of factor B; inhibition of lectin-independent factor D conversion, inhibition of substrate-specific cleavage by the MASP-3 serine protease; reducing the degree of hemolysis or reducing the degree of C3 cleavage and deposition of C3b on the surface; reducing the degree of deposition of factors B and Bb on the activating surface; reduction of residual levels (in the bloodstream and without experimental addition of an activating surface) of active factor D compared with pro-factor D; lower levels of active factor D relative to pro-factor D in response to an activating surface; and/or producing residual and surface-induced levels of liquid phase Ba, Bb, C3b or C3a.

The toxicity and therapeutic efficacy of MASP-3 and MASP-2 inhibitory agents may

- 90 040888 be determined by standard pharmaceutical methods using experimental animal models. With these animal models, NOAEL (no level of side effects) and MED (minimum effective dose) can be determined by standard methods. The dose ratio producing NOAEL and MED is the therapeutic index, which is expressed as the NOAEL/MED ratio. MASP-3 inhibitory agents and MASP-2 inhibitory agents that have high therapeutic ratios or indices are particularly preferred. Data obtained from cell culture assays and from animal studies can be used to calculate doses administered to humans. The dose of the MASP-3 inhibitory agent and the MASP-2 inhibitory agent is preferably in the range of circulating concentrations that include MED with minimal or no toxicity. The dose may vary within this range depending on the dosage form used and the route of administration.

For any composition of compounds, a therapeutically effective dose can be determined using animal models. Thus, for example, a dose may be determined in a model animal in order to calculate a plasma concentration range that includes the MED. Plasma levels of the MASP-3 inhibitory agent or MASP-2 inhibitory agent can also be determined, for example, by high performance liquid chromatography.

In addition to toxicity studies, an effective dose can be determined based on the amount of MASP target protein present in a living individual and the binding affinity of the MASP-3 or MASP-2 inhibitory agent.

Healthy human serum levels of MASP-1 have been reported to range from 1.48 to 12.83 μg/mL (Terai I. et al. Clin. Exp. Immunol. 110:317-323 (1997); Theil et al. ., Clin Exp Immunol 169:38 (2012)). The average concentration of MASP-3 in healthy human serum has been reported to be in the range of approximately 2.0 to 12.9 µg/ml (Skjoedt M. et al., Immunobiology 215(11):921-31 (2010); Degn et al., J. Immunol. Methods, 361-37 (2010) Csuka et al., Mol. Immunol. 54:271 (2013) Healthy human serum levels of MASP-2 have been shown to be low and in the range 500 ng/mL and MASP-2 levels in a given individual can be determined by quantifying MASP-2 as described by Moller Kristensen M. et al., J. Immunol Methods 282:159 167 (2003) and Csuka et al., Mol Immunol 54:271 (2013).

Typically, the dosage of compositions containing MASP-3 inhibitory agents or MASP-2 inhibitory agents administered will vary depending on factors such as the individual's age, weight, height, sex, general health, and medical history. By way of illustration, MASP-3 inhibitory agents or MASP-2 inhibitory agents (such as anti-MASP-3 antibodies, anti-MASP-1 antibodies, or anti-MASP-2 antibodies) can be administered in a dosage range of about 0.010 to 100.0 mg/kg, preferably 0.010 to 10 mg/kg, preferably 0.010 to 1.0 mg/kg, and more preferably 0.010 to 0.1 mg/kg of the individual's body weight. In some embodiments of the invention, MASP-2 inhibitory agents (such as anti-MASP-2 antibodies) are administered in a dosage range, preferably from about 0.010 to 10 mg/kg, preferably from 0.010 to 1.0 mg/kg, and more preferably, from 0.010 to 0.1 mg/kg body weight of the individual. In some embodiments, MASP-1 inhibitory agents (such as anti-MASP-l antibodies) or MASP-3 inhibitory agents (such as anti-MASP-3 antibodies) are administered at a dosage range of about 0.010 to 100.0 mg/kg , preferably from 0.010 to 10 mg/kg, for example from about 1 mg/kg to about 10 mg/kg, preferably from 0.010 to 1.0 mg/kg, and most preferably from 0.010 to 0.1 mg/kg of the individual's body weight.

Therapeutic efficacy of MASP-3 inhibitory compositions, optionally in combination with MASP-2 inhibitory compositions or with MASP-1 inhibitory compositions, optionally in combination with MASP-2 inhibitory compositions, and the methods of the invention applied to a given individual, and appropriate doses can be determined using complement assays well known to those skilled in the art. Complement produces a large number of specific products. In recent decades, sensitive, specific and commercially available assays have been developed for most of these activation products, including small activation fragments C3a, C4a and C5a and large activation fragments iC3b, C4D, Bb and SC5b-9. Most of these assays use monoclonal antibodies that react with novel antigens (neoantigens) present on the fragment but not on the native proteins from which they are derived, making these assays very simple and specific. Although radioimmunoassay is sometimes used to determine C3a and C5a, ELISA technology is used in most cases. These latter analyzes allow to determine the levels of unprocessed fragments and their desArg fragments, which are the main forms present in the bloodstream. The unprocessed fragments and C5a _des Ar _g are rapidly cleared by binding to receptors on the cell surface and are therefore present at very low concentrations, while C3a _des Ar _g does not bind to cells and accumulates in plasma. Measurement of C3a is a sensitive indicator, independent of the complement activation pathway. Alternative pathway activation can be assessed by measuring the level of the Bb fragment and/or measuring the degree of factor D activation. Detection of the liquid phase activation product

- 91 040888 membrane attack pathway, SC5b-9, indicates that complement is activated until the completion of the reaction. Since the lectin pathway and the classical pathway generate the same activation products, C4a and C4d, the assessment of the levels of these two fragments does not provide any information about which of the two pathways generates activation products.

Inhibition of alternative pathway complement activation is characterized by at least one or more of the following modifications in a component of the complement system that result from administration of the high affinity MASP-3 inhibitory antibody described herein: inhibition of factor D maturation; inhibition of an alternative pathway when the antibody is administered to an individual in a molar ratio of from about 1:1 to about 2.5:1 (MASP-3 target: mAb), but not the classical route; inhibition of hemolysis and/or opsonization; reducing the degree of hemolysis or reducing the level of C3 cleavage and deposition of C3b on the surface; reducing the degree of deposition of factors B and Bb on the activating surface; reduction of residual levels (in the bloodstream and without experimental addition of an activating surface) of active factor D compared to profactor D; lower levels of active factor D relative to pro-factor D in response to an activating surface; and/or reducing the level of production of residual and surface-induced liquid phase Ba, Bb, C3b or C3a.

Inhibition of MASP-2 dependent complement activation is characterized by at least one or more of the following modifications in a component of the complement system that result from the administration of a MASP-3 inhibitory agent, in accordance with the methods of the present invention: inhibition of the generation or formation of MASP-2 products β-dependent complement activation system C4b, C3a, C5a and/or C5b-9 (MAC) (as measured and, for example, as described in Example 2 of US Pat. C3 and precipitation of C3b.

Pharmaceutical and delivery vehicles.

Generally speaking, MASP-3 inhibitory antibody compositions or compositions containing a combination of MASP-2 and MASP-3 inhibitory agents may be combined with any other selected therapeutic agents, and are usually contained in a pharmaceutically acceptable carrier. Such a carrier is non-toxic and biocompatible, and is chosen so that it does not adversely affect the biological activity of the MASP-3 inhibitory antibody or the MASP-2 inhibitory antibody (and any other therapeutic agents present in combination with this antibody). Representative pharmaceutically acceptable carriers for polypeptides are described by Yamada in US Pat. No. 5,211,657. powders, granules, ointments, solutions, suppositories, inhalers and injections suitable for oral administration, parenteral administration or surgery. The present invention also contemplates topical administration of compositions using coated medical devices and the like.

Suitable vehicles for parenteral delivery by injection, infusion, or irrigation, and for topical delivery, are distilled water, phosphate buffered saline, normal or lactated Ringer's solutions, dextrose solution, Hanks' solution, or propanediol. In addition, sterile fatty oils can be used as a solvent or suspending medium. For this purpose, any biocompatible oil can be used, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can be used in the preparation of injections. The carrier and agent may be formulated as a liquid, suspension, polymerizable or non-polymerizable gel, paste or ointment.

The carrier may also contain a delivery agent used to delay (i.e., prolong, delay, or regulate) the delivery of the agent(s) or to improve delivery and absorption, to increase the stability, or to improve the pharmacokinetic properties of the therapeutic agent(s). ). Such delivery vehicles may include, but are not limited to, for example, microparticles, microspheres, nanospheres, or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymeric and copolymeric hydrogels, and polymeric micelles. Suitable delivery systems based on hydrogels and micelles are the PEO:PHB:PEO copolymers and copolymer/cyclodextrin complexes described in WO 2004/009664 A2, and the PEO and PEO/cyclodextrin complexes described in US Patent Application Publication No. 2002/0019369 A1 . Such hydrogels can be administered topically to the area intended for their action, or subcutaneously or intramuscularly to form a sustained release depot.

Compositions according to the invention can be prepared for subcutaneous, intramuscular, intravenous, intra-arterial administration or administration by inhalation.

For intra-articular delivery, the MASP-3 inhibitory antibody, optionally in combination with a MASP-3 inhibitory agent, may be administered in the above liquid or in the injectable gel vehicles, in the above injectable sustained delivery vehicles, or in hyaluronic acid or into a hyaluronic acid derivative.

For oral administration of non-peptidergic agents, the MASP-3 inhibitory antibody, optionally in combination with a MASP-3 inhibitory agent, may be administered in an excipient or diluent such as sucrose, corn starch, or cellulose.

For topical administration, the MASP-3 inhibitory antibody, optionally in combination with a MASP-3 inhibitory agent, may be administered as an ointment, lotion, cream, gel, drops, suppositories, spray, liquid, or powder, or as gel or microcapsule delivery systems. using a transdermal patch.

Various systems have been developed for intranasal and pulmonary delivery, including aerosols, metered valve inhalers, insufflators, and aerosol inhalers, and these systems can be adapted to deliver the invention in an aerosol, inhaler, or aerosol inhaler vehicle, respectively.

For intrathecal (IT) or intracerebroventricular (ICV) delivery, appropriate sterile delivery systems (eg liquids, gels, suspensions, etc.) administered by the method of the invention may be used.

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

Pharmaceutical carriers for antibodies and peptides.

More specifically, with respect to the high affinity MASP-3 inhibitory antibodies described herein, representative formulations may be parenterally administered as injectable doses of a solution or suspension of the compound in a physiologically acceptable diluent, together with a pharmaceutical carrier, which may be a sterile liquid such as water, oil, saline, glycerin or ethanol. In addition, adjuvants such as wetting or emulsifying agents, surfactants, pH buffering agents, and the like may also be present in compositions containing anti-MASP-3 antibodies. Additional components of pharmaceutical compositions are petroleum jelly (such as animal, vegetable or synthetic petroleum jelly), for example, soybean oil and mineral oil. In general terms, the preferred liquid carriers for injectable solutions are glycols such as propylene glycol or polyethylene glycol.

Ahtu-MASP-2 antibodies can also be administered in the form of a depot injection or implant, which can be formulated to allow sustained or intermittent release of the active agents.

XVIX. Methods of administration.

Pharmaceutical compositions containing MASP-3 inhibitory antibodies, optionally in combination with MASP-2 inhibitory agents, can be administered in various ways depending on which of the methods of administration, namely, local or systemic, is most suitable for treatment. this state. In addition, compositions according to the invention can be delivered by coating an implantable medical device with said compositions or by incorporating these compositions into the device.

System delivery.

As used herein, systemic delivery and systemic administration means, but are not limited to, oral and parenteral administration, including intramuscular administration (i.m.), subcutaneous administration, intravenous administration (i.v.), intra-arterial administration, administration by inhalation, sublingual administration, buccal administration, topical administration, transdermal administration, intranasal administration, rectal administration, vaginal administration, and other routes of administration that provide effective delivery of the desired antibody to one or more sites of intended therapeutic action. Preferred methods of systemic delivery of compositions according to the invention are intravenous administration, intramuscular administration, subcutaneous administration, intra-arterial administration and administration by inhalation. It should be noted that the specific method of systemic delivery of selected agents used in particular compositions according to the invention is determined in part by taking into account the susceptibility of these agents to the metabolic pathways of transformation associated with a given route of administration. Thus, for example, peptidergic agents may be administered by any route other than oral.

The MASP-3 inhibitory antibodies described herein may be administered to an individual in need thereof by any suitable route. Delivery methods for anti-MASP-3 antibodies and polypeptides include oral administration, pulmonary administration, parenteral administration (e.g., intramuscular, intraperitoneal, intravenous (i.v.) or subcutaneous injection), administration by inhalation (drug in the form of a fine powder), transdermal administration, intranasal administration, vaginal administration, rectal administration, or sublingual administration, and these antibodies and polypeptides can be formulated into dosage forms suitable for each route of administration.

In a representative example, MASP-3 inhibitory antibodies and peptides can be introduced into

- 93 040888 a living organism by introducing them into a biological membrane capable of absorbing these polypeptides, for example, into the nasal mucosa, the mucosa of the gastrointestinal tract and the mucosa of the rectum. The polypeptides are typically incorporated into the absorbent shell along with the shell penetration enhancer. (See, for example, Lee V.H.L., Crit. Rev. Ther. Drug Carrier Sys. 5:69, 1988; Lee, V.H.L., J. Controlled Release 13:213 (1990); Lee V.H.L., Ed., Peptide and Protein Drug Delivery, Marcel Dekker, New York (1991); DeBoer, A. G. et al., J. Controlled Release 13:241 (1990)). For example, STDHF can be used as a permeation enhancer for intranasal delivery; a synthetic fusidic acid derivative, a steroidal surfactant that is similar in structure to bile salts (Lee W.A., Biopharm. 22, Nov./Dec. 1990).

The MASP-3 inhibitory antibodies described herein may be administered in combination with another molecule, such as a lipid, to protect the polypeptides from enzymatic degradation. For example, the covalent binding of polymers, and in particular, polyethylene glycol (PEG), has been used to protect certain proteins from enzymatic hydrolysis in the body and, thereby, to increase their half-life (Fuertges F. et al., J. Controlled Release 11:139 (1990)). Many polymeric protein delivery systems have been described in the literature (Bae Y. H. et al., J. Controlled Release 9:271 (1989); Hori R. et al., Pharm. Res. 6:813, 1989; Yamakawa I. et al. ., J. Pharm. Sci. 79:505 (1990), Yoshihiro, I. et al., J. Controlled Release 10:195 (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)).

Recently, liposomes have been developed with improved serum stability and increased circulating half-life (see, for example, US Pat. No. 5,741,516 to Webb). In addition, various methods have been described for obtaining liposomes and liposome-like preparations as potential drug carriers (see, for example, US patent No. 5567434, Szoka; US patent No. 5552157, Yagi; US patent No. 5565213, Nakamori; US patent No. 5738868, Shinkarenko; and US patent No. 5795587, Gao).

For transdermal administration, the MASP-3 inhibitory antibodies described herein may be combined with other suitable ingredients such as carriers and/or adjuvants. However, any other ingredients may be used without any limitation, except that they must be pharmaceutically acceptable for the intended route of administration and must not adversely affect the activity of the active ingredients of this composition. Examples of suitable carriers are ointments, creams, gels or suspensions, with or without purified collagen. MASP-3 inhibitory antibodies can also be administered by soaking transdermal patches, plaster casts and bandages, preferably with liquid or semi-solid formulations.

Systemic administration of the compositions according to the invention can be carried out periodically at certain intervals to maintain the therapeutic effect at the desired level. Thus, for example, the compositions may be administered by subcutaneous injection every 2-4 weeks or at shorter intervals. Dosing schedule can be determined by the attending physician, taking into account various factors that may affect the effect of this combination of agents. Such factors include the degree of progression of the condition being treated, the age, sex and weight of the patient, and other clinical factors. Doses of each individual agent may vary depending on the function of the MASP-3 inhibitory antibody or MASP-2 inhibitory agent included in the composition, as well as the presence and nature of any drug delivery vehicle (e.g., sustained release delivery vehicle). ). In addition, the amount of dose can be adjusted taking into account changes in the frequency of administration and the pharmacokinetic properties of the delivered(s) agent(s).

local introduction.

As used herein, the term topical administration encompasses administration of a drug to and near the intended localized site of action and may include, for example, topical application to the skin or other affected tissues, ophthalmic administration, intrathecal administration (IT), intracerebroventricular (ICV), intra-articular, intracavitary, intracranial or intravesicular, stent or lavage. Topical administration may be preferred for lower dose administration, to prevent systemic side effects, and to fine-tune the timing of delivery and the concentration of active agents at the site of local administration. Topical administration involves administering a known concentration to the desired site, regardless of differences in metabolic pathways, circulatory system, and the like. The direct delivery method also allows for better dosage control.

Local delivery of a MASP-3 inhibitory antibody or MASP-2 inhibitory agent can be achieved by surgical treatments for a disease or condition, such as during arterial bypass surgery, atherectomy, laser surgery, ultrasound surgery, balloon angioplasty, and stent placement surgery. For example, MASP-3 inhibitory

- 94 040888 an antibody or MASP-2 inhibitory agent may be administered to an individual in combination with balloon angioplasty. Balloon angioplasty involves inserting a catheter containing an deflated balloon into an artery. A balloon with evacuated air is placed near the atherosclerotic plaque and inflated so that the plaque is pressed into the vessel wall. As a result, the surface of the balloon comes into contact with the layer of vascular endothelial cells on the surface of the blood vessels. A MASP-3 inhibitory antibody or a MASP-2 inhibitory agent can be attached to a balloon angioplasty catheter such that the agent is released into the site of an atherosclerotic plaque. This agent can be attached to the balloon catheter by standard methods known to those skilled in the art. For example, such an agent can be stored in the compartment of a balloon catheter until the balloon is inflated with air, and then begins to be released into the environment. Alternatively, the surface of the balloon may be impregnated with the agent so that it contacts the cells of the arterial wall as the balloon is inflated with air. Such an agent may also be delivered to a perforated balloon catheter as described by Flugelman M.Y. et al., Circulation 85: 1110-1117 (1992). See also PCT Published Application WO 95/23161 which describes a representative procedure for coupling a therapeutic protein to a balloon angioplasty catheter. Similarly, the MASP-3 inhibitory agent or MASP-2 inhibitory agent may be included in the gel or polymer coating applied to the stent, or it may be included in the material from which the stent is made so that the stent can release MASP. -3 inhibitory agent or MASP-2 inhibitory agent after it has been placed in the blood vessels.

MASP-3 inhibitory antibodies used to treat arthritis and other musculoskeletal disorders can be administered topically by intra-articular injection. Such compositions may include an appropriate carrier for sustained release delivery. As in another example where local delivery may be desirable, MASP-3 inhibitory compositions used in the treatment of urogenital disorders may be appropriately administered intravesicularly or into another urogenital structure.

XX. Treatment regimens.

Pharmaceutical compositions used prophylactically are administered to an individual susceptible to developing a disease or disorder associated with an alternative pathway, or an individual at risk of developing such a disease or disorder, for example a disease or disorder associated with an alternative pathway and selected from the group consisting of: paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy (including hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS) and thrombotic thrombocytopenic purpura ( TTP)), asthma, disease of dense deposits, oligoimmune necrosing crescentic glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis of the optic nerve, Behcet's disease, multiple sclerosis, syndrome G Yien-Barré, Alzheimer's disease, amylotrophic lateral sclerosis (ABS), lupus nephritis, systemic lupus erythematosus (SLE), diabetic retinopathy, uveitis, chronic obstructive pulmonary disease (COPD), C3 glomerulopathy, graft rejection, graft versus host disease (GVHD), hemodialysis, sepsis, systemic inflammatory response syndrome (SIR), acute respiratory distress syndrome (ARDS), ANCA vasculitis, antiphospholipid syndrome, atherosclerosis, IgA nephropathy and myasthenia gravis, in an amount sufficient to eliminate or reduce the risk of developing symptoms of the disease. Pharmaceutical compositions used for therapeutic purposes are administered to an individual suspected of having a disease or disorder associated with an alternative pathway, or to an individual already suffering from such a disease or disorder, for example, a disease or disorder associated with an alternative pathway and selected from the group consisting of: paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy (including hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS) and thrombotic thrombocytopenic purpura ( TTP)), asthma, disease of dense deposits, oligoimmune necrosing crescentic glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis of the optic nerve, Behçet's disease, multiple sclerosis, Guillain-Barré syndrome, Alzheimer's disease, amylotrophic lateral sclerosis (ABS), lupus nephritis, systemic lupus erythematosus (SLE), diabetic retinopathy, uveitis, chronic obstructive pulmonary disease (COPD), C3 glomerulopathy, graft rejection, graft versus host disease (GVHD), hemodialysis, sepsis, systemic inflammatory response syndrome (SIR), acute respiratory distress syndrome (ARDS), ANCA vasculitis, antiphospholipid syndrome, atherosclerosis, IgA nephropathy, and myasthenia gravis, in a therapeutically effective amount sufficient to relieve or at least partially alleviate the symptoms of the disease.

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In one embodiment, a pharmaceutical composition comprising a high affinity MASP-3 inhibitory antibody is administered to an individual suffering from PNH or at risk of developing PNH. Accordingly, the individual's erythrocytes are opsonized with C3 fragments in the absence of the composition, wherein administering the composition to the individual enhances the viability of the individual's erythrocytes. In one embodiment, the subject, in the absence of the composition, exhibits one or more symptoms selected from the group consisting of: (i) decrease in hemoglobin to below normal levels, (ii) decrease in platelet count to below normal levels; (iii) an increase in the number of reticulocytes to levels above normal; and (iv) increasing bilirubin to levels above normal, and administering the composition to the individual allows at least one or more symptoms to be eliminated, and thereby, (i) increase hemoglobin to normal or to a level close to normal; (ii) increase the number of platelets to normal or to a level close to normal; (iii) reduce the number of reticulocytes to normal or to a level close to normal; and (iv) reduce bilirubin levels to normal or close to normal.

In accordance with prophylaxis and therapy regimens used to treat, prevent or reduce the severity of a disease or condition selected from the group consisting of: paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation blood, thrombotic microangiopathy (including hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS) and thrombotic thrombocytopenic purpura (TTP)), asthma, hard deposit disease, oligoimmune necrosing crescentic glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis of the optic nerve and Behçet's disease, compositions containing high affinity MASP-3 inhibitory antibodies and optionally MASP-2 inhibitory agents may be administered in multiple doses until the desired therapeutic result is observed in the individual. tat. In one embodiment, the high affinity MASP-3 inhibitory antibody and/or MASP-2 inhibitory agent may be administered to an adult patient (e.g., with an average weight of 70 kg) at a dose of 0.1 to 10,000 mg, more preferably 1.0 up to 5000 mg, more preferably 10.0 to 2000 mg, more preferably 10.0 to 1000 mg, and most preferably 50.0 to 500 mg or 10 to 200 mg. For administration to children, the dose may be adjusted according to the body weight of the patient.

The use of high affinity MASP-3 inhibitory antibodies and optionally MASP-2 inhibitory compositions of the invention can be accomplished by a single administration of the composition (e.g., a single composition containing MASP-3 and optionally MASP-2 inhibitory agents or bispecific or dual inhibitory agents, or co-administration of separate compositions), or a limited range of administrations for the treatment of a disease or disorder associated with an alternative route, such as a disease or disorder selected from the group consisting of: paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), ischemic reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy (including hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS) and thrombotic thrombocytopenic purpura (TTP)), asthma, disease of dense deposits th, oligoimmune necrosing sickle-shaped glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis of the optic nerve, Behcet's disease, multiple sclerosis, Guillain-Barré syndrome, Alzheimer's disease, amylotrophic lateral sclerosis (ABS), lupus nephritis, systemic lupus erythematosus (SLE) ), diabetic retinopathy, uveitis, chronic obstructive pulmonary disease (COPD), C3 glomerulopathy, graft rejection, graft versus host disease (GVHD), hemodialysis, sepsis, systemic inflammatory response syndrome (SIR), acute respiratory distress syndrome (ARDS) , ANCA vasculitis, antiphospholipid syndrome, atherosclerosis, IgA nephropathy and myasthenia gravis.

Alternatively, the composition may be administered intermittently at intervals, for example, daily, twice a week, weekly, every other week, monthly, or twice a month for an extended period of time, as directed by a physician, until an optimal therapeutic effect is achieved.

In some embodiments, a first composition containing at least one high affinity MASP-3 inhibitory antibody and a second composition containing at least one MASP-2 inhibitory agent are administered to an individual suffering from a disease or condition, or an individual at risk of developing a disease or condition selected from the group consisting of: paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy (including hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS) and thrombotic thrombocytopenic purpura (TTP)), asthma, hard deposit disease, oligoimmune necrosing crescentic glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, neuritis

- 96 040888 optic nerve romyelitis, Behçet's disease, multiple sclerosis, Guillain-Barré syndrome, Alzheimer's disease, amylotrophic lateral sclerosis (ABS), lupus nephritis, systemic lupus erythematosus (SLE), diabetic retinopathy, uveitis, chronic obstructive pulmonary disease (COPD) , C3 glomerulopathy, graft rejection, graft versus host disease (GVHD), hemodialysis, sepsis, systemic inflammatory response syndrome (SIR), acute respiratory distress syndrome (ARDS), ANCA vasculitis, antiphospholipid syndrome, atherosclerosis, IgA nephropathy, and severe myasthenia gravis.

In one embodiment of the invention, the first composition containing at least one high affinity MASP-3 inhibitory antibody and the second composition containing at least one MASP-2 inhibitory agent are administered simultaneously (i.e., at intervals of approximately no more than than about 15 minutes or less, such as no more than 10, 5, or 1 minutes). In one embodiment of the invention, the first composition containing at least one high affinity MASP-3 inhibitory antibody and the second composition containing at least one MASP-2 inhibitory agent are administered sequentially (i.e., the first composition is administered before or after the introduction of the second composition, where the intervals between the introduction of more than 15 minutes). In some embodiments of the invention, the first composition containing at least one high-affinity MASP-3 inhibitory antibody and the second composition containing at least one MASP-2 inhibitory agent are administered simultaneously (i.e., the time of administration of the first composition overlaps with time of introduction of the second composition). Thus, for example, in some embodiments of the invention, the first composition and/or the second composition is administered over a period of time of at least one, two, three or four weeks or more. In one embodiment of the invention, at least one high affinity MASP-3 inhibitory antibody and at least one MASP-2 inhibitory agent are combined into a unit dosage form. In one embodiment of the invention, a first composition containing at least one high affinity MASP-3 inhibitory antibody and a second composition containing at least one MASP-2 inhibitory agent are packaged together in a kit for use in the treatment of a disease or alternative pathway associated disorder such as paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy (including hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), thrombotic thrombocytopenic purpura (TTP)), asthma, hard deposit disease, oligoimmune necrotizing crescentic glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, optic neuromyelitis, Behcet's disease, multiple sclerosis, Guillain-Barré syndrome, Al Uveitis, chronic obstructive pulmonary disease (COPD), C3 glomerulopathy, graft rejection, graft versus host disease (GVHD), hemodialysis, sepsis , systemic inflammatory response syndrome (SIRS), acute respiratory distress syndrome (ARDS), ANCAvasculitis, antiphospholipid syndrome, atherosclerosis, IgA nephropathy, or myasthenia gravis.

In some embodiments of the invention, an individual suffering from PNH, age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy (including hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), thrombotic thrombocytopenic purpura (TTP)), asthma, hard deposit disease, oligoimmune necrosing crescentic glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, optic neuromyelitis, Behcet's disease, multiple sclerosis, Guillain-Barré syndrome, Alzheimer's disease, amylotrophic lateral sclerosis (ABS), lupus nephritis, systemic lupus erythematosus (SLE), diabetic retinopathy, uveitis, chronic obstructive pulmonary disease (COPD), C3 glomerulopathy, graft rejection, graft versus host disease (GVHD), heme dialysis, sepsis, systemic inflammatory response syndrome (SIRS), acute respiratory distress syndrome (ARDS), ANCA vasculitis, antiphospholipid syndrome, atherosclerosis, IgA nephropathy, or myasthenia gravis, has been treated or is currently being treated with a terminal complement inhibitor, which inhibits the breakdown of complement C5 protein. In some embodiments, the method includes administering to the individual a composition of the invention comprising a high affinity MASP-3 inhibitory antibody and optionally a MASP-2 inhibitor, and administering to the individual a terminal complement inhibitor that inhibits the cleavage of complement C5 protein. In some embodiments, the terminal complement inhibitor is a humanized anti-C5 antibody, or an antigen-binding fragment thereof. In some embodiments, the terminal complement inhibitor is eculizumab.

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XXI. Examples.

The following examples are provided merely to illustrate the best mode for carrying out the invention and should not be construed as limiting the scope of the invention. All documents cited herein are expressly incorporated herein by reference.

Example 1

This example demonstrates that MASP-2-deficient mice were protected from Neisseria meningitidis-induced death after infection with N. meningitidis serogroup A or N. meningitidis serogroup B.

Methods.

MASP-2 knockout mice (MASP-2-HK mice) were prepared as described in Example 1 of US Pat. No. 7,919,094, which is incorporated herein by reference. 10-week-old MASP-2-HK mice (n=10) and wild-type (WT) C57/BL6 mice (n=10) were inoculated by intraperitoneal (IP) injection at a dose of 2.6 χ 10 ⁷ k.o.e. N.meningitidis serogroup A Z2491 in a volume of 100 µl. Mice were administered an infectious dose in combination with iron-containing dextran at a final concentration of 400 mg/kg. The survival of mice after infection was monitored for 72 h.

In a separate experiment, 10-week-old MASP-2-HK mice (n=10) and wild-type (WT) C57/BL6 mice (n=10) were inoculated by intraperitoneal injection at a dose of 6 χ 10 ⁶ k.o.e. strain N. meningitidis serogroup B MC58 in a volume of 100 μl. Mice were administered an infectious dose in combination with iron-containing dextran at a final concentration of 400 mg/kg. The survival of mice after infection was monitored for 72 hours. Disease assessment was also performed for wild-type mice and for MASP-2-HK mice during the 72-hour period after infection, based on the assessment of disease parameters presented in Table 1 below. 5, which was carried out according to the scheme of Fransen et al. (2010) with minor modifications.

Table 5. Evaluation of disease associated with clinical signs in infected mice

signs Grade Normal 0 Slightly bumpy coating 1 Lumpy coating, slow motion and stickiness of the eyes 2 Lumpy plaque, sluggish eye movement, and eyelid closure 3 Very painful condition and lack of movement after stimulation 4 Doom 5

Blood samples were taken from mice at hourly intervals post-infection and analyzed to determine serum levels of N. meningitidis (log cfu/ml) to confirm infection and to determine the rate of clearance of bacteria from serum.

Results.

In FIG. 6 is a Kaplan-Meier plot illustrating the percent survival of MASP-2HK mice and wild-type mice following an infectious dose of 2.6 x 107 k.o.u. N.meningitidis serogroup A Z2491. As shown in FIG. 6, 100% of MASP-2-NK mice survived for 72 h after infection. In contrast, only 80% of wild-type mice (p=0.012) were still alive 24 hours after infection, and only 50% of wild-type mice were still alive 72 hours after infection. These results showed that MASP-2-deficient mice were protected from death caused by N. meningitidis serogroup A Z2491.

In FIG. 7 is a Kaplan-Meier plot illustrating the percent survival of MASP-2HK mice and wild-type mice following an infectious dose of 6 x 106 k.p.u. strain N.meningitidis serogroup B MC58. As shown in FIG. 7, 90% of MASP-2-NK mice survived for 72 h after infection. In contrast, only 20% of wild-type mice (p=0.0022) were still alive 24 hours after infection. These results showed that MASP-2-deficient mice were protected from death caused by N. meningitidis serogroup B MC58.

In FIG. 8 graphically illustrates the log c.f.u./ml of N. meningitidis strain serogroup B MC58 isolated at various time points from blood samples taken from MASP-2-HK mice and wild-type mice after i.p.-infection with 6 χ 106 k.o.u. strain N. meningitidis serogroup B MC58 (n=3 at different times for mice in both groups). The results are expressed as means ± cf. sq. off As shown in FIG. 8, in wild-type mice, blood levels of N. meningitidis peaked at approximately 6.0 log c.f.u./mL at 24 h post-infection and decreased to approximately 4.0 log c.f.u. ./ml 36 hours after infection. In contrast, in MASP-2HK mice, blood levels of N. meningitidis peaked at approximately 4.0 log c.u./mL at 12 h post-infection and decreased to approximately 1.0 log c.o.e. /ml 36 hours after infection (symbol * means p<0.05; symbol ** means p=0.0043). These results showed that although MASP2-HK mice were infected with the same dose of N. meningitidis serogroup B MC58 as wild-type mice, MASP-2-HK mice showed an increased clearance of bacteremia compared to mice. wild type.

In FIG. 9 graphically illustrates the mean incidence score of MASP-2-NK mice and wild-type mice at 3, 6, 12, and 24 hours after infection with 6 x 106 k.p.u. strain N.meningitidis serogroup B MC58. As shown in FIG. 9, MASP-2-deficient mice were highly resistant to infection with much lower disease scores at 6 hours (* means p=0.0411), 12 hours (** means p=0.0049), and 24 hours. (symbol *** means p=0.0049) after infection compared to wild-type mice. The results shown in FIG. 9 are expressed as means ± cf. sq. off

Overall, the results of this example showed that MASP-2-deficient mice were protected from Neisseria meningitidis-induced death after infection with N. meningitidis serogroup A or N. meningitidis serogroup B.

Example 2

This example shows that administration of an anti-MASP-2 antibody after infection with N. meningitidis increased the survival of mice infected with N. meningitidis.

General Description/Rationale.

As described in Example 24 of US Pat. No. 7,919,094, which is incorporated herein by reference, rat MASP-2 protein was used to screen a Fab phage display library, where Fab2 #11 was identified as a functional antibody. A full-length rat IgG2c isotype antibody and a mouse IgG2a isotype antibody were obtained from Fab2 #11. A full-length mouse anti-MASP-2 antibody of the IgG2a isotype was characterized for pharmacodynamic parameters (as described in Example 38 of US Pat. No. 7,919,094).

In this example, a full-length mouse anti-MASP-2 antibody derived from Fab2 #11 was analyzed in a mouse model of N. meningitidis infection.

Methods.

A full-length mouse anti-MASP-2 isotype IgG2a antibody derived from Fab2 #11 was prepared as described above and tested in a mouse model of N. meningitidis infection as follows.

1. Administration of mouse monoclonal anti-MASP-2 antibodies (MoAb) after infection, 9-week-old Charles River C57/BL6 mice were treated with mouse MASP-3 inhibitory antibody (1.0 mg/kg) (n=12) or isotype control antibody (n =10) 3 hours after i.p. injections of a high dose (4 x 106 k.o.e.) of N. meningitidis strain of serogroup B MC58.

Results.

In FIG. 10 is a Kaplan-Meier plot illustrating the percent survival of mice after they were given an infectious dose of 4 x 106 k.o.u. strain N. meningitidis serogroup B MC58, followed by the introduction 3 hours after infection with either a MASP-2 inhibitory antibody (1.0 mg/kg) or an isotype control antibody. As shown in FIG. 10.90% of mice treated with anti-MASP-2 antibody survived for 72 h after infection. In contrast, only 50% of mice treated with the isotype control antibody survived 72 hours after infection. The symbol * means p=0.0301, as determined by comparing two survival curves.

These results indicate that administration of an anti-MASP-2 antibody is effective in treating and prolonging life in individuals infected with N. meningitidis.

As demonstrated herein, the use of an anti-MASP-2 antibody to treat an individual infected with N. meningitidis is effective when administered within 3 hours of infection and is expected to be effective 24-48 hours after infection.

Meningococcal disease (either meningococcal or meningitis) is a medical emergency and treatment is usually started as soon as meningococcal disease is suspected (ie, before N. meningitidis is positively identified as an etiological factor).

In view of the results obtained in MASP-2-HK mice and presented in Example 1, it has been suggested that administration of an anti-MASP-2 antibody prior to N. meningitidis infection may also be effective in preventing infection or attenuating its severity.

Example 3

This example illustrates that complement dependent lysis of N. meningitidis in human serum is MASP-3 dependent.

Rationale.

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Patients with decreased serum functional MBL levels had an increased susceptibility to recurrent bacterial and fungal infections (Kilpatrick et al., Biochim

Biophys Acta 1572:401-413 (2002)). N. meningitidis is known to be recognized by MBL and has been shown to

MBL-deficient serum does not lyse N. meningitidis.

Based on the results described in Examples 1 and 2, a series of experiments were conducted to determine the effectiveness of administering an anti-MASP-2 antibody to eradicate N. meningitidis infection in complement-deficient sera and in control human sera. Experiments were performed in high concentration serum (20%) to assess the preservation of the complement pathway.

Methods.

2. Bactericidal activity in various complement-deficient human sera and in human sera treated with human anti-MASP-2 antibody.

The following complement-deficient human sera and control human sera were used in this experiment.

Table 6: Tested human serum samples (as shown in Fig. 11)

Try Serum type A Normal human serum (NHS) + human anti-MAP-2 AB IN NHS+Ab isotype control WITH Human MBV-serum D NHS E Heat inactivated (HI) NHS

Recombinant anti-human MASP-2 antibody was isolated from a combinatorial antibody library (Knappik A. et al., J. Mol. Biol. 296:57-86 (2000)) using recombinant human MASP-2A as antigen (Chen C.B and Wallis, J Biol Chem 275:25894-25902 (2001)). An anti-human antibody scFv fragment that potentially inhibited lectin-mediated C4 and C3 activation in human plasma (IC ₅₀ ~ 20 nM) was identified and converted into a full length human IgG4 antibody.

Strain N. meningitidis serogroup B-MC58 was incubated with different sera, presented in table. 6 at 20% concentrations, with or without addition of human MASP-2 inhibitory antibody (3 μg in a total volume of 100 μl) at 37° C. with shaking. Then samples were taken at certain time intervals, namely, after 0, 30, 60 and 90 minutes, seeded and the number of viable cells was determined. Heat-inactivated human serum was used as a negative control.

Results.

In FIG. 11 graphically illustrates the log c.f.u./ml of viable N. meningitidis serogroup B MC58 strain isolated at various times from the human serum samples shown in Table 1. 6. In the table. 7 shows the results of Student's t-test for FIG. eleven.

Table 7 Student's t-test results for FIG. 11 (at time 60 min)

Mean Difference (Log) Meaningful? P<0.05? Total value R A compared to B -0.3678 Yes ***(0.0002) And compared to C -1.1053 Yes *** (p<0.0001) A compared to D -0.2111 Yes **(0.0012) Compared to with D 1.9 Yes ***(p<0.0001)

As shown in FIG. 11 and in table. 7, complement-dependent lysis of N. meningitidis in 20% human serum was significantly increased after the addition of human MASP-2 inhibitory antibody.

2. Bactericidal activity in various complement-deficient human sera.

The following complement-deficient human sera and control human sera were used in this experiment.

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Table 8 Human serum samples tested (as shown in Fig. 12)

Try Serum type A Normal human serum (NHS) IN Heat inactivated NHS WITH MBL-/- D MASP-3 -/- (MASP-1+)

Note: MASP-3 ^- / ^- (MASP-1 ⁺ )-serum in sample D was taken from an individual with 3MS syndrome, which is a general term covering Carneval, Mingarelli, Malpech and Michaelis syndromes. As further described in Example 4, mutations in exon 12 of the MASP-1/3 gene confer dysfunction on the serine protease domain of MASP-3, but not MASP-1. As described in Example 10, profactor D is predominantly present in 3MC serum, while activated factor D is predominantly present in normal human serum.

The N.meningitidis strain of serogroup B-MC58 was incubated with various complement-deficient human sera, each at a concentration of 20%, at 37° C. with shaking. Then samples were taken at certain time intervals, namely, after 0, 15, 30, 45, 60, 90 and 120 minutes, seeded and the number of viable cells was determined.

Heat-inactivated human serum was used as a negative control.

Results.

In FIG. 12 graphically illustrates the log c.f.u./ml of viable strain of N. meningitidis serogroup B-MC58 isolated at various times from the human serum samples shown in Table. 8. As shown in FIG. 12, wild-type serum (NHS) has the highest level of bactericidal activity for N. meningitidis. In contrast, MBL ^-/- - and MASP-3 ^{-/- -} human serum (which contains a sufficient amount of MASP-1) does not have any bactericidal activity. These results indicate that the complement dependent lysis of N. meningitidis in 20% human serum (v/v) is MASP-3 and MBL dependent. In table. 9 shows the results of Student's t-test for FIG. 12.

Table 9 Student's t-test results for FIG. 12

Comparison Point of time (min) Mean Difference (Log) Meaningful? P<0.05? The total value of P A compared to B 60 -0.8325 Yes *** (p<0.000 1) A compared to B 90 -1,600 Yes *** (p<0.000 1) And compared to C 60 -1.1489 Yes *** (p<0.000 1) And compared to C 90 -1.822 Yes ***(p<0.000 1) A compared to D 60 -1.323 Yes ***(0.0005) A compared to D 90 -2.185 Yes ***(p<0.000 1)

In general, the results presented in Fig. 12 and in table. 9 showed that complement dependent lysis of N. meningitidis in 20% human serum is MASP-3 and MBL dependent.

3. Complement dependent lysis of N. meningitidis in 20% (v/v) mouse serum deficient in MASP-2, MASP-1/3 or MBL A/C.

The following complement-deficient mouse sera and control mouse sera were used in this experiment.

Table 10 Mouse serum samples tested (as shown in FIG. 13)

Try Serum type A wild type IN MASP-2-/- WITH MASP-1/3-/- D MBL A/C-/- E Heat-inactivated wild-type serum (HIS)

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The N. meningitidis strain of serogroup B-MC58 was incubated with various complement-deficient mouse sera, each at a concentration of 20%, at 37° C. with shaking. Then, samples were taken at certain time intervals, namely, after 0, 15, 30, 60, and 120 minutes, seeded and the number of viable cells was determined. Heat-inactivated human serum was used as a negative control.

Results.

In FIG. 13 graphically illustrates the log c.f.u./ml of viable N. meningitidis serogroup B-MC58 cells isolated at various times from mouse serum samples as shown in Table 13. 10. As shown in FIG. 13, mouse MASP-2' ^/ '-serum has a higher level of bactericidal activity for N. meningitidis than wild-type mouse serum. In contrast, mouse MASP-1/3' ^/ '-serum did not have any bactericidal activity. The symbol ** means p=0.0058 and the symbol *** means p=0.001. In table. 11 shows the results of Student's t-test for FIG. 13.

Table 11 Student's t-test results for FIG. 13

Comparison Moment time Mean Difference (LOG) Meaningful? (p<0.05)? The total value of P A compared to B 6 0 min. 0.39 Yes ** (0.0058) A compared to B 9 0 min. 0.6741 Yes *** (0.001)

Overall, the results of this example showed that MASP-2' ^/ '-serum has a higher level of bactericidal activity for N. meningitidis than wild-type serum, and that complement-dependent lysis of N. meningitidis in 20% human serum is MASP-3- and MBL dependent.

Example 4

This example describes a series of experiments that were carried out to determine the mechanism of MASP-3-dependent resistance to N. meningitidis infection observed in MASP-2-HK mice, as described in examples 1-3.

Rationale.

To determine the mechanism of MASP-3-dependent resistance to N. meningitidis infection observed in MASP-2-HK mice (as described above in examples 1-3), a series of the following experiments was carried out.

1. MASP-1/3-deficient mice are not deficient in the functional activity of the lectin pathway (also called LEA-2).

Methods.

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

Plasma was tested in wild-type mice and mice deficient in C4, MASP-1/3; factor B and MASP-2.

To assess C3 activation, microtiter plates were coated with mannan (1 μg/well), zymosan (1 μg/well) in coating buffer (15 mM Na ₂ CO ₃ , 35 mM NaHCO ₃ ), or immune complexes produced in situ by coating with 1% human serum albumin (HSA) in coating buffer, and then adding sheep anti-HAS serum (2 μg/ml) in TBS (10 mM Tris, 140 mM NaCl, pH 7.4) along with 0.05 % Tween 20 and 5 mM Ca ⁺⁺ . The plates were blocked with 0.1% HSA in TBS and washed three times with TBS/Tween 20/Ca ⁺⁺ . Plasma samples were diluted in 4 mm barbital, 145 mm NaCl, 2 mm CaCl ₂ , 1 mm MgCl ₂ , pH 7.4, added to the plates and incubated for 1.5 h at 37°C. After washing, bound C3b was detected using rabbit anti-human C3c (Dako) followed by alkaline phosphatase-conjugated goat anti-rabbit IgG and p-nitrophenyl phosphate.

Results.

The kinetics of C3 activation (assessed by C3b precipitation on mannan-coated plates with 1% serum) under lectin pathway-specific conditions is illustrated in FIG. 14. In MASP-2' ^/ 'plasma, C3 cleavage was not observed. Factor B (factor B'/') deficient plasma cleaved C3 at half the rate of wild-type plasma, probably due to the absence of an amplification loop. A significant slowdown in the conversion of C3 to C3b, dependent on the lectin pathway, was observed in C4' ^/ '-plasma (T _1/2 \u003d 33 min), and also in MASP-1/3-deficient plasma (T _1/2 \u003d 49 minutes) . This slowing down of C3 activation in MASP-1/3' ^/ '-plasma was observed depending on MASP-1, but not MASP-3 (see.

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Takahashi M. et al., J Immunol. 180:6132-6138 (2008)). These results demonstrate that MASP1/3-deficient mice are not deficient in the functional activity of the lectin pathway (also referred to as LEA-2).

2. Effect of hereditary MASP-3 deficiency on the activation of the alternative pathway.

Rationale.

The effect of inherited MASP-3 deficiency on alternative pathway activation was determined by testing the sera of a MASP-3-deficient patient with 3MC syndrome caused by a frameshift mutation in the exon encoding the MASP-3 serine protease. 3MS syndrome is named as a general term covering Carneval, Mingarelli, Malpech and Michaelis syndromes. These rare autosomal recessive disorders have a range of malformations, including characteristic facial dysmorphism, cleft lip and/or cleft palate, craniosynostosis, mental retardation, and pathologies of the genitals, extremities, and bladder. Rooryck et al. (Rooryck et al. Nature Genetics 43:197-203 (2011)) examined 11 families with 3MC syndrome and identified two mutated genes, COLEC11 and MASP-1. Mutations in the MASP-1 gene conferred dysfunction on the exon encoding the MASP-3 serine protease domain, but not on the exons encoding the MASP-1 serine protease domain. Therefore, 3MC patients with mutations in the exon encoding the serine protease MASP-3 were deficient in MASP-3 but had sufficient amounts of MASP-1.

Methods.

MASP-3-deficient serum was taken from a 3MC patient, from the mother and father of a 3MC patient (heterozygous for an allele carrying a mutation that imparts dysfunction to the exon encoding the MASP-3 serine protease domain), and from a C4-deficient patient (deficient for human C4 genes) and in an MBL-deficient individual. Alternative pathway analysis was performed under traditional AP-specific conditions (BBS/Mg ⁺⁺ /EGTA, no Ca ⁺⁺ , where BBS=barbital buffered saline containing sucrose) as described by Bitter-Suermann et al., Eur. J. Immunol. 11:291-295 (1981)), on zymosan-coated microtiter plates at a serum concentration of 0.5 to 25%, and assessed for C3b precipitation over a period of time.

Results.

In FIG. 15 graphically illustrates the level of C3b precipitation induced by the alternative route on zymosan-coated microtiter plates as a function of concentration in serum samples taken from MASP-3, C4 and MBL deficient individuals. As shown in FIG. 15, sera from MASP-3-deficient patients showed residual alternative pathway (AP) activity at high serum concentrations (serum concentrations 25, 12.5, 6.25%), and a significantly higher AP ₅₀ (i.e. 9, 8% serum required to achieve 50% of maximum C3 precipitation).

In FIG. 16 graphically illustrates the rate of AP-induced C3b precipitation on zymosan-coated microtiter plates under traditional alternative pathway-specific (AP-specific) conditions (i.e., BBS/EGTA/Mg ⁺⁺ without Ca ⁺⁺ ) as a function of time, in samples of 10% human serum taken from a person with a deficiency of MASP-3, C4 and MBL.

In the table below. 12 summarizes the results of the AP ₅₀ shown in FIG. 15 and the semi-deposition time C3b as shown in FIG. 16.

Table 12. Systematized results presented in FIG. 15 and 16

Serum type AR ₅₀ (%) Τι/g (min) MASP-3-deficient (ZMS patient) 9.8 37.4 Mother of an HMS patient (heterozygous) 4.3 17.2 Father of an HMS patient (heterozygous) 4.3 20, 9 C4-deficient 4.0 11, 6 MBL-deficient 4.8 11.0

Note: In BBS/Mg ⁺⁺ /EGTA buffer, lectin-mediated effects were not observed due to the absence in this buffer.

Overall, under the conditions of these assays, the alternative pathway in the 3MS patient was significantly impaired.

3. Evaluation of the level of precipitation of C3b on mannan, zymosan and S. pneumonia D39 in mouse serum deficient in MASP-2 or MASP-1/3.

Methods.

Precipitation of C3b was assessed on microtiter plates coated with mannan, zymosan and S.pneumonia D39 using mouse serum at a concentration ranging from 0 to 20%, taken from MASP-2 ^- / ^- -, MASP-1/3 ^-/- - mice and wild-type mice. C3b precipitation assays were performed either under conventional conditions specific to the alternative pathway (i.e. BBS/EGTA/Mg ⁺⁺ without Ca ⁺⁺ ), or under physiological conditions that ensure the functioning of the lectin pathway and the alternative pathway (i.e. BBS /Mg ⁺⁺ /Ca ⁺⁺ ).

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Results.

In FIG. 17A graphically illustrates the rate of C3b deposition on mannan-coated microtiter plates as a function of serum concentration in serum samples from wild-type mice, MASP-2-deficient mice, and MASP-1/3-deficient mice under traditional alternative pathway-specific conditions ( ie BBS/EGTA/Mg^ without Ca ⁺⁺ ), or under physiological conditions that ensure the functioning of the lectin pathway and an alternative pathway (ie BBS/Mg ⁺⁺ /Ca ++). In FIG. 17B graphically illustrates the rate of C3b deposition on zymosan-coated microtiter plates as a function of serum concentration in serum samples from wild-type mice, MASP-2-deficient mice, and MASP-1/3-deficient mice, or under conventional AP-specific conditions ( ie BBS/EGTA/Mg++ without Ca~), or under physiological conditions that support the functioning of the lectin pathway and an alternative pathway (ie BBS/Mg ⁺⁺ /Ca++). In FIG. 17C graphically illustrates the level of C3b deposition on S.pneumoniae D39 coated microtiter plates as a function of serum concentration in serum samples from wild-type mice, MASP-2-deficient mice, and MASP-1/3-deficient mice, or conventional AP- specific conditions (i.e. BBS/EGTA/Mg++ without Ca~), or under physiological conditions that support the functioning of the lectin pathway and the alternative pathway (i.e. BBS Mg Ca l·

In FIG. 18A graphically illustrates the results of a C3b precipitation assay in highly diluted serum performed on mannan-coated microtiter plates under conventional AP-specific conditions (i.e., BBS/EGTA/Mg ⁺⁺ without Ca~), or under physiological conditions that allow functioning lectin pathway and alternative pathway (i.e. BBS/Mg ⁺⁺ /Ca ++), using serum at concentrations from 0% to 1.25%. In FIG. 18B graphically illustrates the results of a C3b precipitation assay performed on zymosan-coated microtiter plates under conventional AP-specific conditions (i.e., BBS/EGTA/Mg++ without Ca ⁺⁺ ), or under physiological conditions that maintain the lectin pathway and alternative pathway ( i.e. BBS/EGTA/Mg ⁺⁺ /Ca++), using serum concentrations from 0 to 1.25%. In FIG. 18C graphically illustrates the results of a C3b precipitation assay performed on S. pneumoniae D39 coated microtiter plates under conventional AP-specific conditions (i.e., BBS/EGTA/Mg ⁺⁺ without Ca ⁺⁺ ), or under physiological conditions to support lectin function. route and alternative route (ie BBS/EGTA/Mg ⁺⁺ /Ca ⁺⁺ ), using serum concentrations from 0 to 1.25%.

As shown in FIG. 18A-C, C3b precipitation assays were also performed under traditional alternative pathway specific conditions (i.e., BBS/EGTA/Mg ⁺⁺ without Ca~), or under physiological conditions enabling the lectin pathway and the alternative pathway to function (i.e., .e. BBS/Mg ⁺⁺ /Ca++) using higher serum dilutions of 0 to 1.25% on mannan-coated plates (FIG. 18A); on zymosan coated plates (FIG. 18B) and on S. pneumoniae D39 coated plates (FIG. 18C). The alternative pathway was blocked at higher serum dilutions, and therefore the activity observed in MASP-1/3-deficient serum in the presence of Ca~ is MASP-2-mediated LP activity, and the activity in MASP-2-deficient serum in the presence of Ca ~ is MASP-1/3-mediated residual AP activation.

Discussion.

The results described in this example showed that a MASP-2 (or MASP-2-HK) inhibitor provided significant protection against N. meningitidis infection by promoting MASP-3 induced alternative pathway activation. The results of mouse serum bacteriolysis assays and human serum bacteriolysis assays also showed, by monitoring the bactericidal activity of serum against N. meningitidis, that bactericidal activity against N. meningitidis is absent in MBL-deficient mice (in mouse serum deficient in MBL A and MBL C and in human MBL-deficient serum).

In FIG. 1 illustrates the new understanding of the lectin pathway and alternative pathway based on the results reported herein. In FIG. 1 shows the role of LEA-2 in opsonization and lysis. In addition to initiating subsequent C3b precipitation (and ultimately opsonization) in several lectin-dependent physiological processes (FIGS. 18A, 18B, 18C), MASP-2 also plays a role in the lysis of serum-sensitive bacteria. As shown in FIG. 1, the proposed molecular mechanism responsible for the increased bactericidal activity of MASP-2-deficient or MASP-2-depleted serum/plasma against serum sensitive pathogens such as N. meningitidis is that, in order to lyse bacteria, lectin recognition complexes MASP-1 and MASP-3 associated pathways must bind tightly to each other on the bacterial surface, which will allow MASP-1 to cleave MASP-3. Unlike MASP-1 and MASP-2, MASP-3 is not an auto-activating enzyme and in many cases requires activation/cleavage by MASP-1 to be converted to an enzymatically active form.

As further shown in FIG. 1, activated MASP-3 can then cleave bound

- 104 040888 factor B with C3b on the surface of the pathogen with the initiation of an alternative cascade of activation reactions due to the formation of enzymatically active C3- and C5-convertases of the alternative pathway C3bBbC3bBb and C3bBb(C3b)n, respectively. MASP-2-bearing lectin pathway activation complexes are not involved in MASP-3 activation and, in the absence of MASP-2 or after MASP-2 depletion, all lectin pathway activation complexes will be loaded with either MASP-1 or MASP-3. Thus, in the absence of MASP-2, the likelihood that on the microbial surface, MASP-1- and MASP-3-bearing lectin pathway activation complexes will be located in close proximity to each other, which will lead to greater activation of MASP-3, is greatly increased. and thereby increasing the rate of MASP-3-mediated cleavage of factor B associated with C3b to form C3 and C5 alternative pathway convertases C3bBbC3bBb and C3bbc3bb(C3b)n on the microbial surface. This leads to the activation of the terminal C5b-C9 activation cascades, which form a membrane attack complex consisting of surface-bound C5b associated with C6; C5bC6 associated with C7; C5bC6C7 associated with C8; and C5bC6C7C8, which will lead to the polymerization of C9, which is incorporated into the structure of the bacterial surface and forms pores in the bacterial walls, which will lead to osmotic lysis of the bacterium that is the target of this complement.

The essence of this new concept is that the data presented here clearly showed that the activation complexes of the lectin pathway trigger two subsequent distinct activation pathways, as shown in FIG. 1.

Example 5

This example demonstrates the inhibitory effect of MASP-2 deficiency and/or MASP-3 deficiency on the lysis of erythrocytes isolated from blood samples taken from mice with a model of paroxysmal nocturnal hemoglobinuria (PNH).

Background/Rationale.

Paroxysmal nocturnal hemoglobinuria (PNH), sometimes also called Marchiafava-Michael syndrome, is an acquired and life-threatening blood disorder characterized by complement-induced intravascular hemolytic anemia. The hallmark of PNH is chronic complement-mediated intravascular hemolysis caused by unregulated alternative pathway complement activation due to the absence of CD55 and CD59 complement regulators on erythrocytes in PNH, followed by hemoglobinuria and anemia. Lindorfer M.A. et al., Blood 115(11) (2010), Risitano A.M., Mini-Reviews in Medicinal Chemistry, 11:528-535 (2011). Anemia in PNH is caused by the destruction of red blood cells in the bloodstream. Symptoms of PNH are redness of the urine caused by the presence of hemoglobin in the urine, back pain, fatigue, shortness of breath, and thrombosis. PNH can develop on its own, in which case it is called primary PNH, or if it develops in association with other bone marrow disorders such as aplastic anemia, it is called secondary PNH. Treatment of PNH includes blood transfusion for anemia, the use of anticoagulants for thrombosis, and the use of the monoclonal antibody eculizumab (Soliris®), which protects blood cells from immune destruction by inhibiting the complement system (Hillmen P. et al., N. Engl. J. Med. 350 (6):552-559 (2004)). Eculizumab (Soliris®) is a humanized monoclonal antibody that targets the C5 component of complement, blocking its cleavage by C5 convertases and thereby preventing C5a production and MAC assembly. Treatment of PNH patients with eculizumab results in a reduction in intravascular hemolysis, as measured by lactate dehydrogenase (LDH) levels, and thus in stabilization of hemoglobin and elimination of the need for blood transfusion in approximately half of patients (Risitano et al., Mini Reviews in Med Chem., 11:528-535 (2011)). Although almost all patients treated with eculizumab had normal or near normal LDH levels (due to regulation of intravascular hemolysis), however, only approximately one third of patients had hemoglobin levels of approximately 11 g/dL, while the remaining patients treated eculizumab, moderate or severe (i.e. requiring a blood transfusion) anemia was observed, in almost equal proportions (Risitano A.M. et al., Blood 113:4094-100 (2009)). As described in the publication (Risitano et al., Mini-Reviews in Medicinal Chemistry, 11(6) (2011)), it was demonstrated that in patients with PNH treated with eculizumab, there was a large number of C3 fragments associated with PNH erythrocytes (and this was not observed in untreated patients), which led to the conclusion that membrane-bound C3 fragments act as opsonins, resulting in their capture by reticuloendothelial cells through specific C3 receptors and subsequent extravascular hemolysis.

Therefore, in addition to the use of eculizumab, it is necessary to develop therapeutic strategies for those patients who develop extravascular C3-mediated hemolysis, since these patients still require red blood cell transfusion.

This example describes methods for evaluating the effect of MASP-2- and MASP-3-deficient serum on erythrocyte lysis of blood samples taken from mice with a PNH model, and demonstrated the effectiveness of MASP-2 inhibition and/or MASP-3 inhibition for the treatment of individuals, suffering from PNH, as well as the possibility of using MASP-2 inhibitors and / or inhibitors

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MASP-3 (including dual-acting MASP-2/MASP-3 inhibitors or bispecific inhibitors

MASP-2/MASP-3) to attenuate the effects of C3 fragment-mediated extravascular hemolysis in individuals with PNH treated with a C5 inhibitor such as eculizumab.

Methods.

Animals with PNG model.

Blood samples were taken from gene-targeted Crry and C3 deficient (Crry/C3—/—) mice and from CD55/CD59 deficient mice. These mice lacked the appropriate complement regulators on the surface of the erythrocytes and therefore these erythrocytes were susceptible to spontaneous complement autolysis as they are human blood cells with PNH.

To further sensitize these erythrocytes, these cells were used with and without mannan coating, and then they were tested for hemolysis in wild-type C56/BL6 mouse plasma, in non-MBL mouse plasma, in MASP-2 ^-/- -plasma, in MASP-1/3 ^-/- -plasma, in human NHS, in human MBL ^-/- -plasma, and in NHS treated with human anti-MASP-2 antibody.

1. Hemolysis analysis of mouse erythrocytes deficient in two Crry/C3 and CD55/CD59 in MASP2-deficient/depleted serum and controls.

Day 1. Obtaining mouse erythrocytes (± mannan coating).

Reagents included: fresh mouse blood, BBS/Mg ^++/ Ca ⁺⁺ (4.4 mM barbituric acid, 1.8 mM sodium barbitone, 145 mM NaCl, pH 7.4, 5 mM Mg~, 5 mM Ca ⁺⁺ ), chromium chloride, CrCl ₃ -6H ₂ O (0.5 mg/ml in BBS/Mg ⁺⁺ /Ca++) and mannan, 100 µg/ml in BBS/Mg++/Ca ⁺⁺ .

Whole blood (2 ml) was centrifuged for 1-2 min at 2000xg in a refrigerated centrifuge at 4°C. Plasma and buffy coat were aspirated. The sample was then washed three times by resuspending the erythrocyte pellet in 2 ml ice-cold BBS/gelatin/Mg ⁺⁺ /Ca^ and repeating the centrifugation step. After the third wash, the pellet was resuspended in 4 ml BBS/Mg ⁺⁺ /Ca++. A 2 ml aliquot of erythrocytes was set aside as an uncoated control. To the remaining 2 ml were added 2 ml of CrCl ₃ and 2 ml of mannan, and then the sample was incubated with gentle stirring at room temperature for 5 minutes. The reaction was terminated by adding 7.5 ml of BBS/gelatin/Mg ⁺⁺ /Ca ⁺⁺ . The sample was centrifuged as described above, resuspended in 2 ml BBS/gelatin/Mg ⁺⁺ /Ca ⁺⁺ and washed two more times as described above, and then stored at 4°C.

Day 2. Analysis for hemolysis.

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

First, the concentration of erythrocytes was determined, and the cells were adjusted to 10 ⁹ /ml, and then stored at this concentration. Cells were diluted in assay buffer to 10 ⁸ /ml prior to use and then used at a concentration of 100 μl per well. The level of hemolysis was determined at 410-414 nm (which gives greater sensitivity than at 541 nm). Test serum dilutions were prepared in ice-cold BBS/gelatin/Mg ⁺⁺ /Ca ⁺⁺ . 100 μl of each serum dilution was pipetted into a round bottom plate. Then, 100 μl of an appropriately diluted erythrocyte preparation (ie 10 ⁸ /ml) was added, incubated at 37° C. for approximately 1 hour, and lysis was observed. (Plates may be photographed at the time of lysis). The plate was then centrifuged at maximum speed for 5 minutes. 100 μl of the liquid phase was aspirated, transferred to flat-bottomed plates, and OD was recorded at 410-414 nm. The erythrocyte sediment was kept (these erythrocytes can then be subjected to lysis under the action of water to obtain the opposite result).

Experiment #1

Fresh blood was taken from mice deficient in two CD55/CD59, and blood from mice deficient in two Crry/C3 and erythrocytes were obtained as detailed in the protocol above. The cells were separated and half of the cells were coated with mannan and the other half left untreated and brought to a final concentration of 10 ⁸ /ml, of which 100 μl was used in the analysis for hemolysis, which was carried out as described above.

Experiment #1 results. The lectin pathway is involved in erythrocyte lysis in PNH animal models.

In the original experiment, it was found that in wild-type mice, uncoated erythrocytes were not lysed in any of the mouse sera. In addition, it was determined that mannan-coated Crry'/'-mouse erythrocytes were slowly lysed (over 3 h at 37°C) in wild-type mouse sera, but they were not lysed in sera without MBL. (Data not shown).

It was found that mannan-coated Crry'/'-mice erythrocytes were rapidly lysed in human serum, but not in heat-inactivated NHS. Importantly, mannan-coated Crry'/'-mice erythrocytes were lysed in NHS diluted to 1/640 (i.e., all 1/40, 1/80, 1/160, 1/320 and 1/640 dilutions were lysis). (Data not shown). In this dilution, the alternative pathway does not work (the functional activity of AP is significantly reduced below 8%

- 106 040888 serum concentrations).

Conclusions from experiment #1.

Mannan-coated Crgy'/' mouse erythrocytes lysed very well in highly diluted human serum with MBL, but not in serum without MBL. Efficient serum lysis at each concentration tested showed that the alternative pathway does not participate in this lysis or is not necessary for such lysis. The inability of MBL-deficient mouse sera and human sera to lyse mannan-coated erythrocytes from Otu ^−/− mice indicates that the classical pathway is also not associated with observed lysis. Since certain molecules (ie, MBL) are required for recognition of the lectin pathway, this lysis is mediated by the lectin pathway.

Experiment #2

Fresh blood was taken from mice deficient in two Sppu/C3 and CD55/CD59, and mannan-coated erythrocytes from Oty ^{-/- -} mice were analyzed in the hemolysis assay as described above in the presence of the following human sera: MASP-3 ^-/- - sera, MBL-free sera, wild-type sera; NHS pretreated with human anti-MASP-2 antibody and heat-inactivated NHS as control.

Results of experiment #2: MASP-2 inhibitors and MASP-3 deficiency prevent erythrocyte lysis in PNH model animals.

In the case of mannan-coated erythrocytes, Otu ^{-/- -} NHS mice were incubated at dilutions up to 1/640 (i.e. 1/40, 1/80, 1/160, 1/320 and 1/640) for human MBL ^-/- β-serum, human MASP-3 deficient sera (from an MMS patient), NHS pre-treated with anti-MASP-2 mAb, and heat-inactivated NHS as controls.

The microtiter ELISA plate was centrifuged and unlysed erythrocytes were collected at the bottom of the round well plate. The supernatant of each well was collected and the amount of hemoglobin released from lysed erythrocytes was determined by reading the OD at 415 nm in an ELISA reader.

It was noted that MASP-3 ^{-/- -} serum did not lyse mannan-coated mouse erythrocytes at all. In the heat-inactivated control NHS (negative control), as expected, no lysis was observed. Human MBL ^-/- -serum lysed mannan-coated mouse erythrocytes at 1/8 and 1/16 dilutions. NHS pretreated with anti-MASP-2 antibody lysed mannan-coated mouse erythrocytes at 1/8 and 1/16 dilutions, while wild-type human serum lysed mannan-coated mouse erythrocytes at dilutions up to 1/32.

In FIG. 19 graphically illustrates the hemolysis (measured by the release of hemoglobin by lysed mouse erythrocytes (Crgy/C3-/-) into the supernatant by photometry) of mannan-coated mouse erythrocytes with human serum at dilutions of MASP-3 ^-/- -serum, heat inactivated (HI) NHS, MBL ^{- /--} sera, NHS pre-treated with anti-MASP-2 antibody, and NHS controls.

In FIG. 20 graphically illustrates the hemolysis (measured by the release of hemoglobin by lysed mouse erythrocytes (Crgy/C3-/-) into the supernatant by photometry) of mannan-coated mouse erythrocytes with human serum at specific concentrations of MASP-3 ^-/-- serum, heat-inactivated (HI) NHS, MBL ^-/-- sera, NHS pre-treated with anti-MASP-2 antibody, and NHS controls.

Based on the results presented in Fig. 19 and 20, inhibition of MASP-3 has been demonstrated to prevent any complement-mediated lysis of sensitized erythrocytes with insufficient protection against autologous complement activation. Inhibition of MASP-2 by an anti-MASP-2 antibody significantly alters _CH50 and is somewhat protective, but inhibition of MASP-3 was more effective.

Experiment #3

Uncoated mouse Spy ^{-/- -} erythrocytes taken from fresh blood of mice deficient in two Spy/S3 and CD55/CD59 were analyzed in the hemolysis assay as described above in the presence of the following sera: MASP-3 ^-/- -sera , MBL-free sera, wild-type sera; NHS pretreated with human anti-MASP-2 antibody and heat-inactivated NHS as control.

Results.

In FIG. 21 graphically illustrates hemolysis (measured by the release of hemoglobin lysed wild-type mouse erythrocytes into the supernatant by photometry) of uncoated murine erythrocytes at certain concentrations of human serum, namely, the serum of an MMS patient (MASP-3 ^-/- -serum); heat-inactivated (HI) NHS, MBL ^-/- -serum, NHS pre-treated with anti-MASP-2 antibody, and NHS controls. As shown in FIG. 21 and systematized in table. 13, inhibition of MASP-3 was shown to inhibit complement-mediated lysis of unsensitized wild-type mouse erythrocytes.

In FIG. 22 graphically illustrates hemolysis (measured by the release of hemoglobin

- 107 040888 lysed mouse erythrocytes (CD55/59-/-) in the supernatant by photometry) uncoated mouse erythrocytes at certain concentrations of human serum, namely heat-inactivated (HI) NHS, MBL ^-/- -serum, NHS pre-treated with anti -MASP-2 antibody, and NHS control. As shown in FIG. 22 and systematized in table. 13, inhibition of MASP-2 has been shown to be protective to a limited extent.

Table 13 CH ₅₀ values expressed as serum concentration

Serum wild type CD55/59 -/- HMS patient Lysis absent Lysis absent Heat inactivated NHS Lysis absent Lysis absent Donor MBL AO/XX (MBL-deficient) 7.2% 2.1% NH3+anti-MAP-2 antibody 5.4% 1.5% NHS 3.1% 0.73%

Notes: CH ₅₀ represents the point in time when complement-mediated hemolysis reaches 50%.

Overall, the results of this example showed that MASP-3 inhibition prevents any complement lysis of sensitized and non-sensitized erythrocytes with insufficient protection against autologous complement activation. Inhibition of MASP-2 is also protective, but to a certain extent. Thus, MASP-2 and MASP-3 inhibitors alone or in combination (i.e. administered simultaneously or sequentially) or bispecific or dual MASP-2/MASP-3 inhibitors can be used to treat patients suffering from PNH, and they can also be used to attenuate (ie, inhibit, prevent, or lessen the severity of) extravascular hemolysis in PNH patients treated with a C5 inhibitor such as eculizumab (Soliris®).

Example 6

This example describes a hemolysis assay of mannan-coated rabbit erythrocytes to assess lysis in the presence of wild-type or MASP-1/3 ^{-/- -} mouse sera.

Methods.

1. Hemolysis assay of rabbit erythrocytes (mannan-coated) in MASP-1/3 mouse deficient serum and wild-type control serum

Day 1. Obtaining rabbit erythrocytes.

Reagents included: fresh rabbit blood, BBS/Mg ^++/ Ca ⁺⁺ (4.4 mM barbituric acid, 1.8 mM sodium barbitone, 145 mM NaCl, pH 7.4, 5 mM Mg ⁺⁺ , 5 mM Ca ⁺⁺ ), BBS/ Mg ⁺⁺ /Ca ⁺⁺ with 0.1% gelatin, chromium chloride contained in the buffer, i.e. CrCl ₃ -6H ₂ O (0.5 mg/ml in BBS/Mg ⁺⁺ /Ca ⁺⁺ ) and mannan, 100 µg/ml in BBS/Mg ⁺⁺ /Ca ⁺⁺ .

1) Whole rabbit blood (2 ml) was dispensed into two 1.5 ml Eppendorf tubes and centrifuged for 3 minutes at 8000 rpm (approximately 5.9 RCF) in a refrigerated centrifuge at 4°C. The erythrocyte pellet was washed three times after resuspension in ice-cold BBS/Mg ⁺⁺ /Ca ⁺⁺ . After the third wash, the pellet was resuspended in 4 ml BBS/Mg ⁺⁺ /Ca ⁺⁺ . 2 ml of this aliquot was added to a 15 ml Falcon tube and these aliquots were used as an uncoated control. The remaining 2 ml aliquots of erythrocytes were diluted in 2 ml of CrCl ₃ buffer, 2 ml of mannan solution was added and the suspension was incubated at room temperature for 5 min with slight stirring. The reaction was terminated by adding 7.5 ml BBS/0.1% gelatin/Mg ⁺⁺ /Ca ⁺⁺ to the mixture. The erythrocytes were pelleted and washed twice with BBS/0.1% gelatin/Mg ⁺⁺ /Ca ⁺⁺ as described above. The erythrocyte suspension was stored in BBS/0.1% gelatin/Mg ⁺⁺ /Ca ⁺⁺ at 4°C.

2) 100 µl of suspended erythrocytes were diluted with 1.4 ml of water and centrifuged at 8000 rpm (approximately 5.9 RCF) for 3 min and the OD of the supernatant was adjusted to 0.7 at 541 nm (OD equal to 0.7 at 541 nm, corresponds to approximately 10 ⁹ erythrocytes/ml).

3) Resuspended erythrocytes were diluted with BBS/0.1% gelatin/Mg ⁺⁺ /Ca ⁺⁺ to a concentration of 10 ⁸ /ml.

4) A test serum dilution was prepared in ice-cold BBS/gelatin/Mg ⁺⁺ /Ca ⁺⁺ and 100 μl of each serum dilution was pipetted into the appropriate well of a round bottom plate. 100 μl of appropriately diluted erythrocytes (10 ⁸ /ml) was added to each well. Purified water (100 µl) was used as a control to evaluate complete lysis, which was mixed with diluted erythrocytes (100 µl) to initiate 100% lysis, and BBS/0.1% gelatin/Mg ⁺⁺ /Ca ⁺ was used as a negative control. ⁺ without serum (100 µl). The plate was then incubated for 1 hour at 37°C.

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

- 108 040888

Results.

In FIG. 23 graphically illustrates the hemolysis (measured by the release of hemoglobin lysed rabbit erythrocytes into the supernatant by photometry) of mannan-coated rabbit erythrocytes with mouse MASP-3 ^{-/- -} serum at specific concentrations of serum taken from MASP-1/3 ^- / ^- - mice and controls. wild type mice. As shown in FIG. 23, inhibition of MASP-3 was demonstrated to prevent complement-mediated lysis of mannan-coated wild-type rabbit erythrocytes. These results also support the use of MASP-3 inhibitors for the treatment of PNH of one or more of the species described in Example 5.

Example 7

This example describes the production of anti-MASP-1 and anti-MASP-3 monoclonal antibodies using an in vitro system containing a modified DT40 cell line, DTLacO.

Background/Rationale.

Anti-human MASP-1 and MASP-3 antibodies were generated using an in vitro system containing a modified DT40 cell line, DTLacO, which provides a reversible induction of specific polypeptide diversification, as further described in WO 2009029315 and US 2010093033. DT40 is a chicken B cell a line in which the immunoglobulin (Ig) heavy and light chain genes are known to be constitutively mutated. Like other B cells, this constitutive mutagenesis results in mutations in the V region of the Ig genes, and thus in the CDRs of the expressed antibody molecules. Constitutive mutagenesis in DT40 cells occurs through gene conversion, where the donor sequences are an array of non-functional segments of the V gene (pseudo-V; ψV genes) located above each functional V region. It is already known that deletion of the ψV region causes a switch in the diversification mechanism from gene conversion to somatic hypermutation, i.e. a mechanism commonly seen in human B cells. The DT40 chicken B cell lymphoma has been shown to be a promising starting point for ex vivo antibody production (Cumbers S. J. et al. Nat. Biotechnol. 20, 11291134 (2002); Seo H. et al. Nat. Biotechnol. 23, 731-735 (2005)). DT40 cells proliferate only in culture, with a doubling time of 8-10 hours (compared to 20-24 hours for human B cell lines), and they support very efficient homologous gene targeting (Buerstedde J.M. et al. Embo J. 9, 921 -927 (1990)). DT40 cells regulate the pathological diversification of potential V region sequences under the condition that they can communicate access to two different physiological pathways for diversification, gene conversion and somatic hypermutations that create template and non-template mutations, respectively (Maizels N. Annu. Rev. Genet. 39, 23-46 (2005)).

Diversified heavy and light chain immunoglobulins (Ig) are expressed as IgM on the cell surface. Surface IgM has a bivalent form, similar in structure to the IgG molecule. Cells that present IgM with specificity for a particular antigen can be isolated by binding immobilized soluble or membrane variants of the antigen. However, the use of DT40 cells for the production of antibodies in practice is still limited, since, as in other transformed B cell lines, diversification is observed at a level of less than 1% of the physiological level.

In the system used in this example, as described in WO 2009029315 and US 2010093033, DT40 cells were engineered to accelerate the degree of Ig gene diversification without reducing the ability for further genetic modification or potential gene conversion and somatic hypermutation to promote mutagenesis. Two key modifications of DT40 were introduced to increase the degree of diversification and therefore to increase the diversity of binding specificities in the cell library created by the authors. First, Ig gene diversification is under the control of an active E. coli lactose operator/repressor regulatory network. Multimers consisting of approximately 100 polymerized repeats of the E. coli lactose active operator (PolyLacO) were inserted upstream of rearranged and expressed Igλ and IgH genes by targeting homologous genes. Regulatory factors attached to a lactose repressor protein (LacI) can then be attached to LacO regulatory elements to regulate diversification given the high affinity (K _D =10 ^-14 M) of the lactose repressor for operator DNA. DT40 PolyLacO-λκ cells, in which PolyLacO was integrated into Igλ only, had an Ig gene diversification rate 5 times that of parental DT40 cells before any of their construction (Cummings WJ et al. PLoS Biol. 5, e246 (2007 )). Diversification also increased in cells engineered to target the Igλ and IgH (DTLacO) genes with PolyLacO. DTLacO cells have been shown to have a diversification rate that is 2.5 to 9.2 times the diversification rate of 2.8% of the characteristic DT40 PolyLacO^R LacI-HP1 parental lines. Thus, targeting PolyLacO elements in the heavy and light chain genes accelerated diversification by a factor of 21.7 compared to the parental DT40 cell line. Attachment of regulatory factors to Ig loci leads not only to a change in the frequency of mutagenesis, but also to a change in the mutagenesis pathway and to the creation of more

- 109 040888 a large collection of unique sequences with modifications (Cummings et al. 2007; Cummings et al. 2008). Second, a diversified set of baseline sequences was created for Ig gene diversification accelerated by an attached factor. These different sequence starting points were added to DTLacO by targeting rearranged Ig heavy chain variable regions isolated from 2 month old hens at the heavy chain locus. The addition of heavy chain variable regions provides a repertoire of 10 ⁷ new starting points for antibody diversification. The establishment of these new starting points in the DTLacO cell line allows the identification of clones that bind to a particular target, followed by rapid maturation of the binding affinity to the attached factors. After affinity maturation, full-length recombinant chimeric IgG is generated by cloning mature rearranged heavy and light chain variable region sequences (VH and Vλ, consisting of chicken framework regions and hypervariable regions or CDRs) into expression vectors containing human IgG1 constant regions and lambda constant regions. . These recombinant mAbs are suitable for their use in vitro and in vivo and serve as a starting point for humanization.

Methods.

Selection for binding to MASP-1 and MASP-3 antigens Initial selection was made by binding DTLacO populations diversified by gene targeting to spheres complexed with human MASP-1 antigens (SEQ ID NO: 8) and MASP-3 (SEQ ID NO: 2); and subsequent selection was performed by FACS using a fluorescently labeled soluble antigen (Cumbers S. J. et al. Nat. Biotechnol. 20, 1129-1134 (2002); Seo H. et al. Nat. Biotechnol. 23, 731-735 (2005 Since a conserved amino acid sequence is present in the alpha chain, which is common to MASP-1 and MASP-3 (as shown in Figure 2) but differs from the beta chain sequences (as shown in Figure 2), there were separate parallel screens for binding to MASP-1 and MASP-3 were performed to identify MASP-1 specific mAbs, MASP-3 specific mAbs, and mAbs capable of binding to MASP-1 and MASP-3 (bispecific). Two forms of antigens were used for binding: First, recombinant MASP-1 or MASP-3, either in full-length form or as an Fc domain-bound fragment, was attached to Dynal G-protein magnetic spheres, or used in selection methods for based on FACS using PECy5-labeled auto horn antibodies against human IgG (Fc). Alternatively, recombinant variants of MASP-1 or MASP-3 proteins were directly labeled with DyLight Flours reagent and used for selection and screening.

Binding and affinity.

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

Results.

Numerous MASP-1 and MASP-3 binding antibodies have been generated using the methods described above. Binding, as demonstrated by FACS analysis, is described for representative clones of M3J5 and M3M1, which were isolated by screening for binding to MASP-3.

In FIG. 24A is a FACS histogram of MASP-3 antigen/anti-MASP-3 antibody binding for clone DTLacO M3J5. In FIG. 24B is a FACS histogram of MASP3 antigen/anti-MASP-3 antibody binding for clone DTLacO M3M1. In FIG. 24A and 24B, the gray-hatched curves correspond to IgG1-stained negative control, and the bold black curves correspond to MASP-3 staining.

In FIG. 25 graphically illustrates the saturation binding curve of clone M3J5 (clone 5) to MASP-3 antigen. As shown in FIG. 25, the apparent binding affinity of the M3J5 antibody to MASP-3 is approximately 31 nM.

Sequence analysis of identified clones was performed by standard methods. All clones were compared to common VH and VL (DT40) sequences and to each other. The sequences of the above two clones M3J5 and M3M1 were aligned with two additional representative clones D14 and 1E10, which were identified by screening for CCP1CCP2-SP MASP-1 and MASP-3 fragments, respectively. D14 and 1E10 bind to regions that are common to both MASP-1 and MASP-3.

In FIG. 26A illustrates the amino acid sequence alignment of the M3J5, M3M1, D14, and 1E10 VH regions with the chicken DT40 VH sequence.

- 110 040888

In FIG. 26B illustrates the amino acid sequence alignment of the VL regions.

M3J5, M3M1, D14 and 1E10 with the VL sequence of chicken DT40.

The VH and VL amino acid sequences of each clone are shown below.

Heavy chain variable region (VH) sequences.

In FIG. 26A illustrates the amino acid sequence alignment of the heavy chain variable region (VH) for the parental DTLacO (SEQ ID NO: 300), MASP-3 binding clones M3J5 (SEQ ID NO: 301) and M3M1 (SEQ ID NO: 302), and clones binding to two MASP1/MASP-3, D14 (SEQ ID NO: 306) and 1E10 (SEQ ID NO: 308).

The CDRs in the VH sequences, numbered according to Kabat and shown below, are located at the following amino acid positions: H1: a.k. 31-35; H2: a.k. 50-62; and H3: a.k. 95-102.

The CDRs in the VH sequences, numbered according to Chotia and shown below, are located at the following amino acid positions: H1: a.k. 26-32; H2: a.k. 52-56; and H3: a.k. 95-101.

VH parent DTLacO: (SEQ ID NO: 300)

AVTLDESGGGLQTPGGALSLVCKASGFTFSSNAMGWVRQAPGKGLEWVAGIDDDGSGTRYAPA VKGRATISRDNGQSTLRLQLNNLRAEDTGTYYCTKCAYSSGCDYEGGYIDAWGHGTEVIVSS

VH clone M3J5: (SEQ ID NO: 301)

AVTLDESGGGLQTPGGGLSLVCKASGFTFSSYAMGWMRQAPGKGLEYVAGIRSDGSFTLYATA VKGRATISRDNGQSTVRLQLNNLRAEDTATYFCTRSGNVGDIDAWGHGTEVIVSS

VH clone M3M1: (SEQ ID NO: 302)

AVTLDESGGGLQTPGGGLSLVCKASGFDFSSYQMNWIRQAPGKGLEFVAAINRFGNSTGHGAAV KGRVTISRDDGQSTVRLQLSNLRAEDTATYYCAKGVYGYCGSYSCCGVDTIDAWGHGTE VIVSS

VH clone D14: (SEQ ID NO: 306)

VKGRATISRDNGQSTVRLQLNNLRAEDTGTYYCAKTTGSGCSSGYRAEYIDAWGHGTEVI VSS

VH clone 1E10: (SEQ ID NO: 308)

AVTLDESGGGLQTPGGALSLVCKASGFTFSSYDMVWVRQAPGKGLEFVAGISRNDGRYTEYGSA VKGRATISRDNGQSTVRLQLNNLRAEDTATYYCARDAGGSAYWFDAGQIDAWGHGTEVIVSS

Light chain variable region (VL) sequences.

In FIG. 26B illustrates the amino acid sequence alignment of the light chain variable region (VL) for parental DTLacO (SEQ ID NO: 303), MASP-3 binding clones M3J5 (SEQ ID NO: 304) and M3M1 (SEQ ID NO: 305), and clones binding to two MASP1/MASP-3, D14 (SEQ ID NO: 307) and 1E10 (SEQ ID NO: 309).

VL of parent DTLacO: (SEQ ID NO: 303)

ALTQPASVSANLGGTVKITCSGGGSYAGSYYYGWYQQKSPGSAPVTVIYDNDKRPSDIP SRFSGSLSGSTNTLTITGVRADDEAVYFCGSADNSGAAFGAGTTLTVL

VL clone M3J5: (SEQ ID NO: 304)

ALTQPASVSANPGETVKITCSGGYSGYAGSYYYGWYQQKAPGSAPVTLIYYNNKRPSDI PSRFSGSLSGSTNTLTITGVRADDEAVYFCGSADNSGAAFGAGTTLTVL

VL clone M3M1: (SEQ ID NO: 305)

ALTQPASVSANPGETVKITCSGGGSYAGSYYYGWYQQKAPGSAPVTLIYYNNKRPSDIP SRFSGSLSGSTNTLTITGVRADDEAVYFCGSADNSGAAFGAGTTLTVL

VL clone D14: (SEQ ID NO: 307)

ALTQPASVSANPGETVKITCSGGGSYAGSYYYGWYQQKAPGSAPVTLIYYNNKRPSDIP SRFSGSLSGSTNTLTITGVRADDEAVYFCGSADNSGAAFGAGTTLTVL

VL clone 1E10: (SEQ ID NO: 309)

ALTQPASVSANPGETVKITCSGGGSYAGSYYYGWYQQKAPGSAPVTLIYYNNKRPSDIP SRFSGSLSGSTNTLTITGVRADDEAVYFCGSADNSGAAFGAGTTLTVL

Functional analysis for LEA-2 (MASP-2-dependent).

MASP-1 contributes to LEA-2 through its ability to activate MASP-2 (see FIG. 1). The MBL screening assay of the Wieslab® Complement System (Euro Diagnostica, Malmo, Sweden) measures the extent of C5b-C9 deposition under conditions of inhibition of LEA-2-dependent activation (ie, traditional lectin pathway activity). This assay was performed according to the manufacturer's instructions using a representative 1E10 clone tested at a final concentration of 400 nM.

In FIG. 27 is a bar graph showing the inhibitory activity of mAb 1E10 compared to the positive sera present in the assay kit, as well as the inhibitory activity of the isotype control antibody. As shown in FIG. 27, mAb 1E10 partially inhibits LEA-2 dependent activation (by inhibiting MASP-1 dependent MASP-2 activation), while the isotype control antibody does not. Stronger inhibition should be achieved by continuous maturation of the binding affinity of the antibody to MASP-1 using attached factors in the DTLacO system.

Functional analysis for LEA-1 (MASP-3 dependent) for representative mAbs is described below in

- 111 040888 examples 8 and 9.

Brief summary of the results.

The above results showed that DTLacO-based antibodies against MASP-1 and MASP-3 with inhibitory properties against LEA-1 (as shown in Examples 8 and 9 below) and LEA-2 (as shown in this figure) can be rapidly detected ex vivo. example).

Example 8

Complement pathway analysis in 3MS serum with S. aureus.

Background/Rationale.

It was found that MASP-3 is not activated by non-immobilized liquid phase mannan, zymosan A or N-acetylcysteine in the presence or absence of normal human serum. However, recombinant and native MASP-3 were found to be activated on the surface of heat inactivated S. aureus in the presence and absence of normal human serum (NHS) or heat inactivated human serum (HIS) (data not shown). It was also found that C3b precipitation occurs on the surface of S. aureus in the presence of normal human serum and that the precipitation can be monitored using a flow cytometer. Thus, the alternative pathway (AP) response to S. aureus was assessed as described as a means to assess the contribution of MASP-3 to LEA-1.

Methods.

Recombinant MASP-3.

Polynucleotide sequences encoding full-length recombinant human MASP-3, truncated active variant of MASP-3 serine protease (SP) (CCP1-CCP2-SP), and SP-inactivated form of MASP-3 (S679A) were cloned into the mammalian expression vector pTriEx7 (Invivogen). The resulting expression constructs encode full-length MASP-3 or its CCP1-CCP2-SP fragment with an amino-terminal Streptag tag and a carboxy-terminal His ₆ tag. The expression constructs were transfected into Freestyle 293-F cells or Expi293F cells (Invitrogen) according to protocols provided by the manufacturer. After 3-4 days of cultivation in 5% CO ₂ at 37°C, the recombinant proteins were purified using streptactin affinity chromatography.

Recombinant MASP-1.

Full-length or truncated forms of CCP1-CCP2-SP recombinant MASP-1 with or without the addition of a stabilizing R504Q (Dobo et al., J. Immunol. 183:1207, 2009) or SP-inactivating (S646A) mutations along with the amino-terminal tag Steptag and The carboxy-terminal His ₆ tag was generated as described above for recombinant MASP-3.

1. Precipitation of C3b and cleavage of factor B by S. aureus in 3MC serum (human).

A first experiment was performed to demonstrate that flow cytometric analysis can detect the presence or absence of AP-induced C3b (Απ-C3b) precipitation as follows. Five percent of the following sera: normal human serum; factor B-free human serum; factor D-free human serum; and properdin-free human serum (obtained from Complement Technology, Tyler, Texas, USA) were mixed with test antibody in either Mg ⁺⁺ /EGTA buffer or EDTA at 4°C overnight. Heat-inactivated S. aureus (108/reaction mixture) was added to each mixture to a total volume of 100 μl and the mixture was rotated at 37° C. for 40 minutes. The bacteria were washed in wash buffer, the bacterial pellet was resuspended in wash buffer, and 80 µl aliquots of each sample were analyzed for C3b precipitation on the bacterial surface, which was detected using an anti-human C3c antibody (Dako, UK) by flow cytometry.

The results of C3b detection by flow cytometry are shown in FIG. 28A. As shown in FIG. 28A, panel 1, in normal human serum in the presence of EDTA, in which AP is known to be inactive, no precipitation of C3b was observed (negative control). In normal human serum treated with Mg++/EGTA, only lectin-independent complement pathways functioned. In panel 2, using Mg ⁺⁺ /EGTA buffer, where AP is active, AP-induced C3b precipitation was observed (positive control). As shown in panels 3, 4 and 5, in factor B, factor D and properdin free serum, respectively, no alternatively induced C3b precipitation was observed, as expected. These results demonstrated that such an assay is capable of detecting AP-dependent C3b deposition.

C3b precipitation assay on S. aureus was performed as described above to assess the ability of recombinant MASP-3 to restore the AP pathway (LEA-1) in MASP-3 deficient human ZMS serum (Rooryck C et al., Nat. Genet. 43 (3): 197-203 (2011)). The following combinations of reagents were tested.

1) 5% normal human serum + EDTA.

2) 5% normal human serum+Mg/EGTA.

- 112 040888

3) 5% human ZMS serum (MASP-3 ^-/- )+Mg++/EGTA.

4) 5% human ZMS serum (MASP-3'/') + Mg ⁺⁺ /EGTA + active full length rMASP-3.

5) 5% human 3MS serum (MASP-3' ^/ ')+Mg ⁺⁺ /EGTA+active truncated rMASP-3 (CCP1/CCP2/SP).

6) 5% human 3MS serum (MASP-3' ^/ ')+Mg ⁺⁺ /EGTA+inactive rMASP-3 (S679A).

7) 5% human 3MS serum (MASP-3' ^/ ')+Mg ⁺⁺ /EGTA+active full length rMASP-1.

Various mixtures of 5% serum and recombinant proteins (5 μg each) as shown above were incubated under the indicated buffer conditions (either Mg ⁺⁺ /EGTA buffer or EDTA) at 4°C overnight. After overnight incubation, 10 ⁸ heat-inactivated S. aureus were added to each mixture to a total volume of 100 μl and the mixture was subjected to circular rotation at 37° C. for 40 minutes. The bacteria were washed and resuspended in wash buffer, and then an 80 μl aliquot of each sample was analyzed for C3b precipitation by FACS. The remaining 20 µl aliquots of each sample were used to evaluate factor B digestion by Western blot analysis using an anti-factor B antibody, as described below.

The results of C3b detection by flow cytometry are shown in FIG. 28V. The panel numbers correspond to the numbers given for each reagent combination described above. The negative control (panel 1) and the positive control (panel 2), as expected, indicate the absence and presence of C3b precipitation. Panel 3 shows that AP-induced C3b precipitation was absent in 3MC serum. Panels 4 and 5 show that active full-length rMASP-3 (panel 4) and active rMASP-3 (CCP1-CCP2-SP) (panel 5) restore AP-induced C3b precipitation in 3MC serum. Panel 6 shows that inactive rMASP-3 (S679A) did not restore AP-induced C3b precipitation in 3MC serum. Panel 7 shows that rMASP-1 did not restore AP-induced C3b precipitation in 3MC serum.

Overall, these results indicate that MASP-3 is required for AP-induced C3b deposition on S. aureus in human serum.

MASP-3-dependent factor B activation.

For analysis of MASP-3-dependent factor B activation, various mixtures of 5% serum (either normal human serum or 3MC patient serum) and the recombinant proteins described above were analyzed as described above. From each reaction mixture, 20 μl was taken and added to the protein sample loading buffer. Samples were heated at 70°C for 10 min and loaded onto SDSPAAG. Western blot analysis was performed using a polyclonal antibody against factor B (R&D Systems). Factor B activation was apparently due to the formation of two lower molecular weight cleavage products (Bb and Ba) derived from the higher molecular weight pro-factor B protein.

In FIG. 29 shows the results of Western blot analysis to determine factor B cleavage in response to S. aureus in 3MC serum in the presence or absence of rMASP-3. As shown in lane 1, normal human serum in the presence of EDTA (negative control) shows a very low level of factor B cleavage compared to normal human serum in the presence of Mg++/EGTA, as shown in lane 2 (positive control). As shown in lane 3, 3 MC serum shows very low factor B cleavage in the presence of Mg++/EGTA. However, as shown in lane 4, factor B digestion is maintained by the addition and pre-incubation of full-length recombinant MASP-3 protein (5 μg) with 3MC serum.

Assay to determine the effect of rMASP-3 on pro-factor D by factor B/C3 (H ₂ O) digestion.

In order to determine the minimum requirement for MASP-3-dependent factor B activation/cleavage, the following analysis was performed.

G3(H ₂ O) (200 ng), purified plasma factor B (20 μg), recombinant pro-factor D (200 ng), and recombinant human MASP-3 (200 ng) were mixed together in various combinations (as shown in FIG. 30) in a total volume of 100 µl in BBS/Ca ⁺⁺ /Mg ⁺⁺ and incubated at 30°C for 30 min. The reaction was terminated by adding 25 μl of SDS loading dye containing 5% 2-mercaptoethanol. After boiling at 95°C for 10 min with shaking (300 rpm), the mixture was centrifuged at 1400 rpm for 5 min, and then loaded with 20 μl of the supernatant, and separated on a 10% SDS gel. The gel was stained with Coomassie brilliant blue.

Results.

In FIG. 30 shows Coomassie staining of an SDS-PAGE gel for factor B digestion assay. As shown in lane 1, factor B digestion is most optimal in the presence of C3, MASP-3, and pro-factor D. As shown in lane 2, C3 is absolutely essential, however, as shown in lanes 4 and 5, MASP-3 or pro-factor D are able to mediate factor B cleavage provided that C3 is present.

Assay for the ability of an anti-MASP-3 mAb to inhibit MASP-3-dependent AP-induced C3b precipitation.

As described in this example, MASP-3 was demonstrated to be required for AP-induced C3b deposition on S. aureus in human serum. Therefore, an analysis was made

- 113 040888 in order to determine whether a representative aHTu-MASP-3 mAb, identified as described in example 7, can inhibit the activity of MASP-3. An active recombinant MASP-3 protein fragment (CCP1-CCP2-SP) (250 ng) was preincubated with an isotype control mAb, with mAb1A5 (a control antibody isolated from the DTLacO cell line and not binding to MASP-3 or MASP-1), or with mAbD14 (which binds to MASP-3) at three different concentrations (0.5, 2 and 4 μM) for 1 hour on ice. The enzyme-mAb mixture was treated with 5% 3MC serum (MASP-3 deficient) and 5 x 10 ⁷ heat-inactivated S. aureus in a final reaction volume of 50 μl. The reaction mixtures were incubated at 37°C for 30 min and then stained to detect C3b precipitation. Stained bacterial cells were analyzed on a flow cytometer.

In FIG. 31 graphically illustrates the mean fluorescence intensities (MFI) of C3b staining obtained for three antibodies and plotted as a function of mAb concentration in 3MS serum in the presence of rMASP-3. As shown in FIG. 31, mAbD14 shows concentration dependent inhibition of C3b precipitation. In contrast, none of the control mAbs inhibited C3b precipitation. These results indicated that mAbD14 was able to inhibit MASP-3 dependent C3b precipitation. As expected, the increase in inhibitory activity for mAbD14 is a consequence of the prolonged affinity maturation of this antibody to bind to MASP-3 via attached factors in the DTLacO system.

Brief summary of the results.

Overall, the results of this example demonstrated a clear absence of AP in MASP-3 deficient serum. Thus, MASP-3 was shown to be a significant contributor to AP, as indicated by factor B activation and C3b precipitation as functional endpoints. In addition, the addition of functional recombinant MASP-3, including the catalytically active C-terminal portion of MASP-3, abolished the defect in factor B activation and C3b precipitation in the patient's 3M serum. On the other hand, as was also demonstrated in this example, the addition of an anti-MASP-3 antibody (eg, mAbD14) to rMASP-3 3MS serum inhibits AP-induced C3b precipitation. The direct role of MASP-3 in the activation of factor B, and hence AP, was demonstrated by the observation that recombinant MASP-3, along with C3, is sufficient to activate recombinant factor B.

Example 9

This example demonstrates that MASP-1 and MASP-3 activate factor D.

Methods.

Recombinant MASP-1 and MASP-3 were tested for their ability to cleave two different recombinant pro-factor D variants. The first variant (pro-factor D-His) lacks an N-terminal tag but has a C-terminal His tag. Thus, this pro-factor D variant contains a 5 amino acid propeptide that is removed by cleavage during activation. The second variant (ST-pro-factor D-His) has a Strep-TagII sequence at the N-terminus, which increases the cleaved N-terminal fragment to 15 amino acids. ST pro factor D also contains a His ₆ tag at the C terminus. Increasing the propeptide length of the ST pro-factor D-His improves the separation of the cleaved and non-cleaved form by SDS-PAGE compared to the separation that may be possible with pro-factor D-His.

Recombinant MASP-1 or MASP-3 proteins (2 μg) were added to pro-factor D-His or ST-pro-factor D-His substrates (100 ng) and incubated for 1 h at 37°C. Reaction mixtures were electrophoresed on a 12% bis-Tris gel to separate pro-factor D and active factor D cleavage product. The separated proteins were transferred to a PVDF membrane and analyzed by Western blot analysis for detection using a biotinylated anti-factor D antibody. (R&D Systems).

Results.

In FIG. 32 illustrates a Western blot analysis for pro-factor D substrate cleavage.

Table 14. Description of lanes for Western blot analysis illustrated in FIG. 32

Experimental conditions Track 1 Track 2 Track 3 Track 4 Track 5 Pro Factor D + + + + + rMASP-3 (full size) — + - - - rMASP-3a (S679A) - - + - - rMASP-lA (S646A) - - - + - rMASP-l (SSR-1- CCP2-SP) — — — — +

As shown in FIG. 32, full size MASP-3 only (track 2) and MASP-1 fragment CCP1- 114 040888

CCP2-SP (lane 5) cleave ST-pro-factor D-His ₆ . Catalytically inactive full length MASP-3 (S679A; lane 3) and MASP-1 (S646A; lane 3) were unable to cleave any of these substrates. Identical results were obtained for the pro-factor D-His ₆ polypeptide (not shown). Comparison of the molar excess of MASP-1 (CCP1-CCP2-SP) and MASP-3 suggests that MASP-3 is a more efficient pro-factor D cleavage catalyst than MASP-1, at least under the conditions described here.

Conclusions. Both MASP-1 and MASP-3 had the ability to cleave and activate factor D. This activity establishes a direct relationship between LEA-1 and AP activation. More specifically, factor D activation by MASP-1 or MASP-3 results in factor B activation, C3b precipitation, and likely opsonization and/or lysis.

Assay for inhibition of MASP-3-dependent pro-factor D cleavage by anti-MASP-3 antibodies.

An assay was performed to determine the inhibitory effect of representative anti-MASP-3 and anti-MASP-1 mAbs, identified as described in Example 7, on MASP-3-dependent factor D cleavage. Active recombinant MASP-3 protein (80 ng) was preincubated with 1 µg of representative mAb D14, M3M1 and a control antibody (which specifically binds to MASP1 but not to MASP-3) at room temperature for 15 minutes. Pro-factor D with an N-terminal Strep tag (ST-pro-factor D-His, 70 ng) was then added and the mixture was incubated at 37°C for 75 min. The reaction mixtures were then subjected to electrophoresis, blot analysis and stained with anti-factor D antibody as described above.

In FIG. 33 illustrates a Western blot analysis indicating partial inhibitory activity of mAb D14 and M3M1 compared to a control reaction mixture containing only MASP-3 and ST-pro-factor D-His (no mAb; lane 1), as well as control a reaction mixture containing mAb isolated from the DTLacO library and binding to MASP-1 but not to MASP-3 (lane 4). As shown in FIG. 33, in the absence of inhibitory antibody, MASP-3 cleaves approximately 50% of pro-factor D to form factor D (lane 1). The control MASP-1 specific antibody (lane 4) does not alter the ratio of pro-factor D to factor D. In contrast, as shown in lanes 2 and 3, both mAb D14 and mAb M3M1 inhibit MASP-3-dependent cleavage of pro-factor D to form factor D, which leads to a decrease in the level of production of factor D.

Conclusions. These results indicated that anti-MASP-3 mAb D14 and M3M1 were able to inhibit MASP-3-dependent factor D cleavage. by means of attached factors in the DTLacO system.

Example 10

This example demonstrates that MASP-3 deficiency prevents complement-mediated lysis of mannan-coated wild-type rabbit erythrocytes.

Background/Rationale.

As described here in Examples 5 and 6, the effect of MASP-2- and MASP-3-deficient serum on erythrocyte lysis of blood samples taken from PNH mice indicates the efficacy of MASP-2 inhibition and/or MASP-3 inhibition for treatment individuals suffering from PNH, as well as the possibility of using MASP-2 inhibitors and / or MASP-3 inhibitors (including dual-acting MASP-2 / MASP-3 inhibitors or bispecific MASP-2 / MASP-3 inhibitors) to reduce the effects of extravascular hemolysis, mediated by the C3 fragment in individuals with PNH treated with a C5 inhibitor such as eculizumab.

As described in this example, C3b precipitation and hemolysis experiments were performed on MASP3-deficient sera from other 3MC patients, and these experiments confirmed the results obtained in examples 5 and 6. In addition, experiments were performed that showed that the addition of rMASP-3 to 3MC serum helped restore C3b precipitation and hemolytic activity.

Methods.

MASP-3-deficient sera were taken from the following three different 3MC patients:

3MC patient 1. This patient contains an allele carrying a mutation that renders the exon encoding the MASP-3 serine protease domain dysfunctional, and this dysfunction is also seen in the mother and father of the 3MC patient (both of whom are heterozygous for the allele carrying the mutation, which informs the exon encoding the MASP-3 serine protease domain of dysfunction).

3MC patient 2. This patient has a C1489T (H497Y) mutation in exon 12 of MASP-1; in the exon that codes for the MASP-3 serine protease domain, causing MASP-3 to become non-functional and MASP-1 proteins to become functional.

3MC Patient 3 This patient has a confirmed defect in the MASP-1 gene, causing MASP-3 to become non-functional and MASP-1 proteins also to become non-functional.

- 115 040888

Experiment #1 C3b precipitation analysis.

AP analysis was performed under conventional AP-specific conditions (BBS/Mg ⁺⁺ /EGTA, no Ca ⁺⁺ , where BB3=barbital buffered saline containing sucrose) as described by Bitter-Suermann et al., Eur. J. Immunol. 11:291-295 (1981)), on zymosan-coated microtiter plates at a serum concentration of 0.5 to 25%, and assessed for C3b precipitation over a period of time.

Results.

In FIG. 34 graphically illustrates the rate of AP-induced C3b precipitation on zymosan-coated microtiter plates as a function of serum concentration in serum samples taken from MASP-3 (3MC), C4 and MBL deficient individuals. As shown in FIG. 34 and systematized below in table. 15, MASP-3-deficient sera from patient 2 and patient 3 have residual AP activity at high serum concentrations (serum concentrations 25, 12.5, 6.25%), and a significantly higher AP ₅₀ (t i.e. 8.2% and 12.3% serum is needed to achieve 50% of the maximum C3 precipitation).

In FIG. 35A graphically illustrates the rate of AP-induced C3b precipitation on zymosan-coated microtiter plates under traditional AP-specific conditions (i.e., BBS/EGTA/Mg ⁺⁺ without Ca ⁺⁺ ) versus time in 10% human serum samples taken from a person with a deficiency of MASP-3, C4 and MBL.

Below in table. 15 summarizes the results of the AP ₅₀ shown in FIG. 34 and the half-life C3b as shown in FIG. 35A.

Table 15 Summary of the results shown in FIG. 34 and 35A

Serum type ar ₅₀ (%) T _1/2 (min.) Normal 4.5 26, 3 MBL-deficient (MBL-/-) 5, 7 27.5 C4-deficient (C4-/-) 5, 1 28.6 HMS (Patient 3) 8.2 58.2 HMS (Patient 2) 12.3 72.4

Note: In BBS/Mg ⁺⁺ /EGTA buffer, the effects mediated by the lectin pathway are insufficient due to the lack of Ca ⁺⁺ in this buffer.

Experiment #2 Analysis of pro-factor D degradation in the serum of a MMS patient using Western blot analysis.

Methods. Serum was taken from ZMS patient #2 (MASP-3 (-/-), MASP-1 (+/+)) and from ZMS patient #3 (MASP-3 (-/-), MASP-1 (-/+). -)). Patient sera, together with sera from healthy donors (W), were separated on an SDS-polyacrylamide gel and the separated proteins were blotted onto a PVDF membrane. Human pro-factor D (25040 Da) and/or mature factor D (24405 Da) were detected using an antibody specific for human factor D.

Results. The results of Western blot analysis are shown in FIG. 35V. As shown in FIG. 35B, in sera from healthy donors (W), the anti-factor D antibody detected a protein the size of mature factor D (24405 Da). As shown in FIG. 35B, the anti-factor D antibody detected a slightly larger protein in the sera of MMS patient #2 (P2) and MMS patient #3 (P3), consistent with the presence of pro factor D (25040 Da) in these MMS patients.

Experiment #3 Wieslab complement assays in the serum of an MMS patient.

Methods. Serum taken from MMS patient #2 (MASP-3 (-/-), MASP-1 (+/+)) and from MMS patient #3 (MASP-3 (-/-), MASP-1 (- /-)), were also tested for classical, lectin and alternative pathway activity by the Wieslab complement system screening method (Euro-Diagnostica, Malmo, Sweden) according to the manufacturer's instructions. Normal human serum was tested in parallel as a control.

Results. In FIG. 35C graphically illustrates the results of Weislab classical, lectin, and alternative pathway assays using plasma from MMS patient #2, MMS patient #3, and normal human serum. As shown in FIG. 35C, under Wieslab assay conditions, the classical, alternative, and MBL (lectin pathway) pathways are functional in normal human serum. In the serum of 3MC patient #2 (MASP-3 (-/-), MASP-1 (+/+)), the classical pathway and the lectin pathway are functional, however, no activity of the alternative pathway was detected. In the serum of MMS patient #3 (MASP-3 (-/-), MASP-1 (-/-)), the classical pathway is functional, and the activity of the lectin pathway and the alternative pathway was not detected.

The result shown in Fig. 35B and 35C also confirmed the authors' understanding of the role of MASP-1 and MASP-3 in the LEA-1 and LEA-2 pathways. In particular, the absence of an alternative pathway and yet an almost fully functional lectin pathway in the serum of patient 2, who lacked only MASP-3, confirmed that MASP-3 plays an important role in the activation of the alternative pathway. Serum

- 116 040888 patient 3, not containing MASP-1 and MASP-3, lost its ability to activate the lectin pathway, as well as the alternative pathway. This analysis confirmed the mandatory involvement of MASP-1 in the LEA-2 functional pathway and was consistent with the results of Example 7 and data available in the literature, which indicate that MASP-1 activates MASP-2. This apparent inability of sera to activate pro-factor D is also consistent with the data described in Example 9, where MASP-3 was shown to cleave pro-factor D. These observations are consistent with those of the LEA-1 and LEA-2 pathways plotted in FIG. 1.

Experiment #4 Hemolysis assay of mannan-coated rabbit erythrocytes assessed for lysis in the presence of human normal serum or 3MC serum (in the absence of Ca ⁺⁺ ).

Methods.

Obtaining rabbit erythrocytes in the absence of Ca ⁺⁺ (ie using EGTA).

Whole rabbit blood (2 ml) was dispensed into two 1.5 ml Eppendorf tubes and centrifuged for 3 minutes at 8000 rpm (approximately 5.9 RCF) in a refrigerated centrifuge at 4°C. The erythrocyte pellet was washed three times after resuspension in ice-cold BBS/Mg ⁺⁺ /Ca ⁺⁺ (4.4 mM barbituric acid, 1.8 mM sodium barbitone, 145 mM NaCl, pH 7.4, 5 mM Mg ⁺⁺ , 5 mM Ca ⁺⁺ ). After the third wash, the pellet was resuspended in 4 ml BBS/Mg ⁺⁺ /Ca ⁺⁺ . The erythrocytes were pelleted and washed with BBS/0.1% gelatin/Mg ⁺⁺ /Ca ⁺⁺ as described above. The erythrocyte suspension was stored in BBS/0.1% gelatin/Mg ⁺⁺ /Ca ⁺⁺ at 4°C. Then, 100 µl of suspended erythrocytes were diluted with 1.4 ml of water and centrifuged at 8000 rpm (approximately 5.9 RCF) for 3 min and the OD of the supernatant was adjusted to 0.7 at 541 nm (OD equal to 0.7 at 541 nm , corresponds to approximately 10 ⁹ erythrocytes/ml). Thereafter, 1 ml of resuspended erythrocytes at an OD of 0.7 was added to 9 ml of BBS/Mg ⁺⁺ /EGTA to a concentration of 10 ⁸ erythrocytes/ml. Test serum or plasma dilutions were prepared in ice-cold BBS, Mg ⁺⁺ , EGTA, and 100 μl of each serum or plasma dilution was pipetted into the appropriate well of a round bottom plate. 100 μl of appropriately diluted erythrocytes (10 ⁸ erythrocytes/ml) was added to each well. Nano water was used to make a positive control (100% lysis) and a BBS/Mg ⁺⁺ /EGTA dilution without serum or plasma was used as a negative control. The plate was then incubated for 1 hour at 37°C. The round bottom plate was centrifuged at 3750 rpm for 5 minutes. Thereafter, the supernatant from each well was transferred to the corresponding wells of a flat-bottomed plate and the OD was read at 415-490 nm.

Results. In FIG. 36 graphically illustrates the percent hemolysis (measured by the release of hemoglobin from lysed rabbit erythrocytes into the supernatant by photometry) of mannan-coated rabbit erythrocytes at concentrations in serum taken from healthy individuals and two 3MC patients (patient 2 and patient 3) in the absence of Ca ⁺⁺ . As shown in FIG. 36, it has been demonstrated that MASP-3 deficiency leads to a decrease in the percentage of complement-mediated lysis of mannan-coated erythrocytes compared to normal human serum. The differences between the two curves plotted from normal human serum data and the two curves plotted from 3MS data are significant (p=0.013, Friedman's test).

Below in table. 16 summarizes the AP ₅₀ results shown in FIG. 36.

Table 16. Summary of the results shown in FIG. 36

Serum type ar ₅₀ (%) Normal Human Serum #1 7.1 Normal human serum #2 8, 6 HMS patient #2 11.9 HMS patient #3 14.3

It is noteworthy that when combining the serum samples presented in Table. 16, the AP ₅₀ value for normal human serum is 7.9, and the AP ₅₀ value for 3MC serum is 12.8 (p=0.031, Wilcoxon's rank significance test of paired matches).

Experiment #5 Reconstitution of human 3MC serum with recombinant MASP-3 promotes the maintenance of AP-induced C3b precipitation on zymosan-coated plates.

Methods.

AP analysis was performed under conventional AP-specific conditions (BBS/Mg ⁺⁺ /EGTA, no Ca ⁺⁺ , where BBS=barbital buffered saline containing sucrose) as described by Bitter-Suermann et al., Eur. J. Immunol. 11:291-295 (1981)) on zymosan-coated microtiter plates in the following serum samples: (1) 5% human serum from 3MC patient #2 with full-length active rMASP-3 added at 0 to 20 mcg/ml; (2) in 10% human serum taken from 3MS patient #2 with full-length active rMASP-3 added at 0 to 20 μg/ml; and (3) in 5% human serum taken from MMS patient #2 with inactive

- 117 040888 rMASP-3A (S679A) added at 0 to 20 µg/ml.

Results.

In FIG. 37 graphically illustrates the rate of AP-induced C3b precipitation on zymosan-coated microtiter plates as a function of the concentration of rMASP-3 protein added to a serum sample from 3MC patient #2 (MASP-3-deficient). As shown in FIG. 37, active recombinant MASP-3 protein restores AP-induced C3b precipitation on zymosan-coated plates in a concentration dependent manner. As further shown in FIG. 37, no C3b precipitation was observed in 3MC serum containing inactive rMASP-3 (S679A).

Experiment #6 Restoration of human 3MC serum by recombinant MASP-3 promotes the maintenance of hemolytic activity in the serum of a 3MC patient.

Methods.

Hemolytic analysis was performed using rabbit erythrocytes by the methods described above in Experiment #2, in the following sera tested at a concentration of 0 to 12%: (1) in normal human serum; (2) in the serum of a 3MS patient; (3) in the serum of a 3MS patient plus active full-length rMASP-3 (20 μg/ml); and (4) in heat-inactivated human serum.

Results.

In FIG. 38 graphically illustrates the percent hemolysis (measured by the release of hemoglobin from lysed rabbit erythrocytes into the supernatant by photometry) of mannan-coated rabbit erythrocytes at specific serum concentrations: (1) normal human serum; (2) sera from a 3MS patient; (3) sera from a 3MC patient containing active full-length rMASP-3 (20 μg/ml); and (4) heat-inactivated human serum in the absence of Ca ⁺⁺ . As shown in FIG. 38, the percentage of lysis of rabbit erythrocytes is significantly increased in 3MS serum containing rMASP-3 compared to the percentage of lysis in 3MS serum without rMASP-3 (p=0.0006).

In FIG. 39 graphically illustrates the percent lysis of rabbit erythrocytes in 7% human serum taken from 3MC patient 2 and 3MC patient 3 containing active rMASP-3 at a concentration of 0 to 110 μg/ml in BBS/Mg ⁺⁺ /EGTA. As shown in FIG. 39, the percentage of lysis of rabbit erythrocytes is maintained if the activity of rMASP-3 as a function of concentration is 100%.

Experiment #7 Serum of a MASP-3-deficient (3MC) patient has functional MASP2 if MBL is present.

Methods.

C3b precipitation assay was performed on mannan-coated ELISA plates to determine if the 3MS serum was LEA-2 deficient. Citrate-containing plasma was diluted in BBS buffer at several dilutions (1:80, 1:160, 1:320, 1:640, 1:1280, 1:2560) and plated on mannan-coated plates. Precipitated C3b was detected in an assay using chicken anti-human C3b. LEA-2-induced C3b precipitation (plasma dilutions increased to initiate AP and LEA-1 function) on mannan-coated ELISA plates was assessed as a function of normal human serum (NHS) concentration in two 3M patients (patient 2 and patient 3), in the parents of patient 3 and in an MBL-deficient individual.

Results.

In FIG. 40 graphically illustrates the level of LEA-2-induced (ie, MASP-2-induced) C3b precipitation on mannan-coated ELISA plates as a function of concentration of human serum diluted in BBS buffer from a healthy human (NHS); in two 3MS patients (patient 2 and patient 3); in the parents of patient 3 and in an individual with MBL deficiency. These data showed that in patient 2, the level of MBL is sufficient. However, Patient 3 and Patient 3's mother are deficient in MBL and therefore their serum does not precipitate C3b on mannan via LEA-2. Replacement of MBL in these sera resulted in restoration of LEA-2-mediated C3b precipitation in the sera of patient 3 (who is homozygous for SNP, resulting in MASP-3 deficiency) and his mother (who is homozygous for the MASP-3 mutant allele) (data not given). These results indicated that the ZMS serum was not deficient in LEA-2 but had a functional MASP-2.

General conclusions and conclusions.

These results demonstrated that deficiency of MASP-3 in human serum results in loss of AP activity, as indicated by reduced C3b precipitation on zymosan-coated wells and reduced rabbit erythrocyte lysis. AP can be restored in both assays by adding serum containing functional recombinant human MASP-3.

Example 11.

This example demonstrates that an anti-human MASP-3 monoclonal antibody containing a chimeric mouse V region/human IgG4 constant region (mAb M3-1, also referred to as mAb 13B1) is a potent inhibitor of MASP-3 mediated complement activation of the alternative pathway. (ARS).

- 118 040888

Methods.

Obtaining a monoclonal antibody against human MASP-3 containing a chimeric mouse V-region/human IgG4 constant region (mAb M3-1).

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

The MASP-3 inhibitory monoclonal antibody M3-1 (13B1) comprises a heavy chain variable region (VH) shown as SEQ ID NO: 30 and a light chain variable region (VL) shown as SEQ ID NO: 45. Variable region sequences monoclonal antibody M3-1 are presented below.

Heavy chain variable region.

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

mAb M3-1 heavy chain variable region (VH) (SEQ ID NO: 30)

QVQLKQSGAELMKPGASVKLSCKATGYTFTGKWIEWVKQRPGHGLEWIGEILPGTGSTNYNEK FKG KATFTADSSSNTAYMQLSSLTTEDSAMYYCLRSEDVWGTGTTVTVSS

Light chain variable region.

Below is the sequence of the light chain variable region (VL) for mAb M3-1. Kabat CDRs (24-34 (H1), 50-56 (H2) and 89-97 (H3) are underlined and correspond to amino acid residues 24-40 (L1); 56-62 (L2) and 95-102 (L3) SEQ ID NO: 45. These areas are the same regardless of whether they are numbered according to the Kabat or Chotiya system.

Light chain variable region (VL) mAb M3-1 (SEQ ID NO: 45)

DIVMTQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVP DRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNIPTFGGGTKLEIKR mAb М3-1 VH CDR

VHCDR1: GKWIE (SEQ ID NO: 84)

VHCDR2: EILPGTGSTNYNEKFKG (SEQ ID NO: 86)

VHCDR3: SEDV (SEQ ID NO: 88) mAb M3-1 VL CDR

VLCDR1: KSSQSLLNSRTRKNYLA (SEQ ID NO: 142)

VLCDR2: WASTRES (SEQ ID NO: 144)

VLCDR3: KQSYNIPT (SEQ ID NO: 161)

As shown above, the anti-MASP-3 monoclonal antibody M3-1 comprises (a) a heavy chain variable region comprising (i) VHCDR1 comprising SEQ ID NO: 84, (ii) VHCDR2 comprising SEQ ID NO: 86, and ( iii) VHCDR3 comprising SEQ ID NO: 88; and (b) a light chain variable region comprising (i) VLCDR1 comprising SEQ ID NO: 142, (ii) VlCdR2 comprising SEQ ID NO: 144, and (iii) VLCDR3 comprising SEQ ID NO: 161.

Binding of mAb M3-1 to recombinant forms of human and mouse MASP-3.

The monovalent Fab M3-1 variant was tested for binding to recombinant full-length human and mouse MASP-3 protein in an ELISA experiment. Binding affinity determinations were made by coating 96-well plates with an anti-MASP-3 capture antibody that binds to a protein derived from a variety of species. The capture antibody has been shown to bind to the CCP1-CCP2 region of MASP-1 and MASP-3. Full length human and mouse protein variants were immobilized on capture antibody coated ELISA plates and M3-1 Fab at various concentrations were bound to the target protein in separate wells. Bound M3-1 was detected using an anti-kappa chain light chain that was conjugated to HRP (Novus Biologicals NBP1-75064) and visualized using a TMB substrate reagent kit (BD Biosciences 555214).

In FIG. 41 graphically illustrates a representative example of a binding experiment that was carried out using human MASP-3, where Fab M3-1 (also referred to as 13B1) bound to a human protein with an apparent binding affinity (EC ₅₀ ) of approximately 0.117 nM.

In FIG. 42 graphically illustrates a representative example of a binding experiment,

- 119 040888 which was carried out using mouse MASP-3, where Fab M3-1 associated with mouse protein with an apparent binding affinity (EC ₅₀ ) of approximately 0.214 nm.

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

Determination of the ability of mAb M3-1 to inhibit alternative pathway complement activation (APC) and measurement of in vitro activity of mAb M3-1.

As described herein, MASP-3 has been determined to be a key regulator of APC, at least in part because it is required for CFD activation, i. the central enzyme APC. In addition, as described in this application, MASP-3 circulates in the body at a relatively low concentration and has a low rate of catabolism, which allows long-term inhibition of the pro-inflammatory pathway after intravenous, subcutaneous and oral administration of anti-MASP-3 antibodies. The following experiment was performed to determine the effectiveness of mAb M3-1 in inhibiting MASP-3-mediated CFD maturation and inhibition of APC in human serum. Normal human serum contains predominantly active or processed (i.e., mature) CFD, and therefore experiments were performed in which CFD-deficient human serum (Complement Technology A336) was diluted with a recombinant unprocessed form of CFD (pro-CFD). Thus, in this experimental system, APC activation requires processing of pro-CFD into active CFD.

APC was induced by adding zymosan particles which function as an activating surface for complement precipitation. Serum, before adding recombinant pro-CFD and zymosan, was added mAb M3-1 at various concentrations. The mixtures were incubated at 37° C. for 75 min and APC activity was measured by detection of complement factor Bb (Quidel A252) on the surface of zymosan particles by flow cytometry.

In FIG. 43 graphically illustrates the level of deposition of complement factor Bb on zymosan particles (determined by flow cytometric detection in MFI units) in the presence of various concentrations of M3-1 mAb in CFD-deficient human serum. As shown in FIG. 43, mAb M3-1 shows strong APC inhibition capacity in 10% human serum, where the IC ₅₀ is 0.311 nM in this experimental example.

These results demonstrated that MASP-3 plays a key role in the in vitro activation of APC in a human serum model and further showed that mAb M3-1 is a potent inhibitor of APC.

Inhibition of APC by mAb M3-1 in vivo.

To determine the effectiveness of mAb M3-1 in inhibiting APC in vivo, a group of mice (n=4) were injected into the tail vein with a single intravenous injection of 10 mg/kg mAb M3-1. Animal blood was used to obtain serum and to create a matrix for assessing APC activity by flow cytometry in an ex vivo assay that measures C3 (also C3b and iC3b) precipitation on zymosan particles. Serum obtained from pre-dose and some time post-dose blood (at 96 hours, 1 and 2 weeks) was diluted to 7.5% and zymosan particles were added to induce APC. Mice treated with the antibody were compared with a group of control mice (n=4) that received a single intravenous dose of vehicle.

In FIG. 44 graphically illustrates the level of C3 deposition on zymosan particles at various time points after administration of a single dose of M3-1 mAb (10 mg/kg i.v.) to wild-type mice. As shown in FIG. 44, under pre-dose conditions, APC activity levels were comparable. At 96 hours and two subsequent times, the M3-1 mAb-treated group showed almost complete inhibition of APC, while the APC activity of the vehicle-treated group remained at the same level. As shown in FIG. 44, a single dose of mAb M3-1 administered intravenously to mice resulted in almost complete elimination of systemic APC activity after at least 14 days.

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

Example 12.

This example demonstrates that an anti-human MASP-3 monoclonal antibody containing a chimeric mouse V region/human IgG4 constant region (mAb M3-1, also referred to as mAb 13B1) has a clear advantage in terms of survival of Crry-free erythrocytes, in mice with a model of paroxysmal nocturnal hemoglobinuria (PNH).

Methods.

An anti-human MASP-3 monoclonal antibody containing a chimeric mouse V region/human IgG4 constant region (mAb M3-1) was prepared as described in Example 11 and in Example 14. In addition, as described in Example 11, it was determined that the mAb M3-1 is a potent inhibitor of APC in an in vivo mouse model. This example describes the analysis of mAb M3-1 for its effectiveness in a mouse model associated with PNH.

Analysis of mAb M3-1 for its efficacy in a mouse model associated with PNH.

In a mouse model with PNH, erythrocytes (RBC) from Crry-deficient mice that do not contain essential

- 120 040888 cell surface APC repressor were obtained for use as donor cells. In parallel, erythrocytes from wild-type (WT) donor mice were prepared. These donor erythrocytes were separately labeled with fluorescent lipophilic dyes (Sigma): WT (red) and Cny mice (green). In two different experiments, labeled donor WT cells and Crry cells were mixed 1:1 and injected intravenously into recipient mice of wild type, and then the percentage viability of WT erythrocytes and Crry-deficient erythrocytes (compared to viability at an early time period) in recipient mice was determined by analyzing 20,000 live cells using flow cytometry. In the first experiment, a plurality of treatments were performed by pre-dosing mAb M3-1, and the effect of mAb M3-1 was compared with that of another complement-inhibiting antibody, mAb BB5.1 (supplied by Hycult Biotech), which is a C5 inhibitory antibody having efficacy in many studies in mice (Wang et al., PNAS vol 92:8955-8959, 1995; Hugen et al., Kidney Int. 71(7):646-54, 2007). The introduction of a C5 inhibitor is the standard modern course of treatment for a patient suffering from PNH. In a second experiment, one pre-dose of mAb M3-1 was evaluated.

In the first experiment, three different groups of mice (n=4) were evaluated for each condition: vehicle condition, mAb M3-1 condition, and mAb BB5.1 (murine C5 blocking mAb) condition. Labeled cells were injected into mice on day 0 and multiple doses of M3-1 and BB5.1 were administered as follows: M3-1 mAb was administered intravenously (10 mg/kg) on days -11, -4, -1 and +6. mAb BB5 .1 administered by intraperitoneal injection (40 mg/kg) on days -1, +3, +6 and +10. Vehicle treatment was carried out according to the dosing schedule for mAb M3-1.

In FIG. 45 graphically illustrates the percentage of surviving donor RBCs (wild type or Crry-) at 14 days in recipient mice of wild type treated with mAb M3-1 (13B1) (10 mg/kg on days -11, 04, -1 and +6 ), in mice treated with mAb BB5.1 or mice treated with vehicle. As shown in FIG. 45, in contrast to wild-type erythrocytes, whose viability was typical of vehicle-treated mice, Crry-deficient erythrocytes underwent rapid clearance (greater than 75% in 24 h). Treatment of mice with mAb BB5.1 did not improve the viability of Crry-deficient erythrocytes compared to vehicle treatment. In contrast, treatment of animals with mAb M3-1 resulted in a dramatic increase in the viability of Crry-deficient erythrocytes compared to treatment with mAb BB5.1 and vehicle. The protective effect of mAb M3-1 was observed throughout the experiment.

In a second study, separately labeled WT (red) and Crry (green) erythrocytes were evaluated in two different groups of wild-type mice (n=4 per condition): vehicle-treated mice and M3-1 mAb-treated mice. One dose of vehicle or antibody (20 mg/kg) was administered to recipient mice by intravenous injection for six days (day -6) and then labeled donor cells were administered to recipient mice. After that, the labeled donor erythrocytes were analyzed for percentage of viability in the recipient mice at certain intervals after injection for 16 days.

In FIG. 46 graphically illustrates the percentage of surviving donor RBCs (wild-type or Crry-) for 16 days in wild-type recipient mice treated with a single dose of mAb M3-1 (13B1) (20 mg/kg on day -6), or mice treated with carrier. As shown in FIG. 46, a single pre-treatment with a dose of M3-1 mAb resulted in an increase in Crry-RBC viability compared to Crry-RBC viability in vehicle-treated mice. At 96 hours post-injection, approximately 90% of vehicle-treated wild-type erythrocytes survived under control conditions, while only 5% of Crry-RBC survived in vehicle-treated wild-type mice. In contrast to vehicle-treated mice, 40% of CrryRBC survived in M3-1 mAb-treated mice.

Overall, these results indicated that the MASP-3 inhibitory antibody mAb M3-1 conferred a clear advantage in terms of viability of Crry-free erythrocytes, as this antibody is a key complement inhibitor on the cell surface in the PNH mouse model.

Example 13

This example describes a study that demonstrated that a chimeric monoclonal MASP-3 inhibitory antibody (mAb M3-1, also referred to as mAb 13B1) resulted in a reduction in clinical scores of anti-collagen antibody (CAIA)-induced arthritis in a mouse model of rheumatoid arthritis. (RA).

Background/Rationale.

CAIA is a well-established animal model of arthritis. In addition to information on RA, the pathology of the disease in the CAIA model was found to be associated with APC. Banda et al. demonstrated improved treatment outcomes in the CAIA model in mice deficient in APC components such as factor B and factor D (Banda et al., J. Immunol. vol 177:1904-1912, 2006 and Banda et al., Clinical & Exp Imunol Vol 159:100-108, 2009). APC knockout mice had lower scores for arthritis (disease), reduced incidence of disease, and less C3 and factor H precipitation in synovial tissue and surrounding tissues compared to wild-type controls. In addition, scores of disease severity, complement C3 deposition in joint tissue, and histopathological lesions were significantly reduced in MASP1/3 knockout mice (Banda et al.,

J. Immunol. Vol. 185:5598-5606, 2010). Therefore, the MASP-3 inhibitory antibody mAb M3-1 was analyzed for efficacy in the CAIA model.

Methods.

A chimeric anti-MASP-3 monoclonal antibody (mAb M3-1) was prepared as described in Example 11 and in Example 14. In addition, as described in Example 11, mAb M3-1 was determined to be a potent inhibitor of APC in a mouse model in vivo.

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

Clinical scores were given for all four paws of each mouse in studies on days 0-14 according to the following standard scores:

- norm;

- damage to the joint 1 of the hind and / or front paw or minimal diffuse erythema and swelling;

- damage to the joints of 2 hind and / or front legs or mild diffuse erythema and swelling;

- damage to the joints of the 3 hind and / or front paws or diffuse moderate erythema and swelling;

- noticeable diffuse erythema and swelling or damage to the joints of 4 fingers;

- severe diffuse erythema and severe swelling of all paws, and inability to flex the toes.

Incidence was also defined as the % of mice in the treatment group that had symptoms of arthritis.

The results are shown in FIG. 47 (clinical evaluations) and FIG. 48 (incidence of arthritis). In FIG. 47 graphically illustrates clinical scores for mice treated with mAb M3-1 (13B1) (5 or 20 mg/kg) or mice treated with vehicle for 14 days. In FIG. 48 graphically illustrates the percentage incidence of arthritis in mice treated with mAb M3-1 (13B1) (5 or 20 mg/kg) or in mice treated with vehicle for 14 days. As shown in FIG. 47, mAb M3-1 showed a clear therapeutic effect for both endpoints from day 5 until the end of the study. As shown in FIG. 48, although the incidence rate reached 100% in vehicle-treated animals, however, two-thirds of the animals treated with 5 mg/kg mAb M3-1 did not develop disease. In addition, only one animal (ie, only one of all animals, n=7) showed any symptoms of arthritis after administration of 20 mg/kg mAb M3-1.

The results of this study demonstrated that the MASP-3 inhibitory antibody mAb M3-1 has a clear therapeutic effect in the CAIA model, ie. a well-established mouse model of rheumatoid arthritis (RA) and a model closely associated with APC activation. As shown in Example 11, a single dose of M3-1 mAb administered intravenously to mice resulted in almost complete elimination of systemic APC activity after at least 14 days. As indicated in this example, in an animal model induced by administration of anti-mouse connective tissue autoantibodies, mAb M3-1 reduced the incidence and severity of arthritis in accordance with clinical assessments in a dose-dependent manner. Compared to control animals, animals treated with M3-1 mAb experienced a reduction in incidence and disease severity of approximately 80% at the highest dose tested. Therefore, administration of a MASP-3 inhibitory antibody such as mAb M3-1 will, as expected, provide a therapeutic effect in patients suffering from arthritis such as rheumatoid arthritis, osteoarthritis, juvenile rheumatoid arthritis, infection-induced arthritis, psoriatic arthritis, as well as ankylosing spondylitis and Behçet's disease.

Example 14

This example describes the production of high affinity inhibitory antibodies against human MASP-3.

Background/Rationale.

A limited number of antibodies specific to MASP-3 have been described (Thiel et al., Mol. Immunol. 43:122, 2006; Moller-Kristensen et al., Int. Immunol. 19:141, 2006; Skjoedt et al., Immunobiol 215:921, 2010). These antibodies can be used in detection assays such as Western blot, immunoprecipitation, and ELISA assays using capture or detection reagents. However, the antibodies described by Thiel et al., 2006, Moller-Kristensen et al., 2006 and Skjoedt et al., 2010 were found not to inhibit the catalytic activity of MASP-3.

Anti-MASP-3 antibodies were also prepared previously, as described here in Example 7 (and also in Example 15 of WO 2013/192240), by screening a library of chicken antibodies in

- 122 040888 modified DT40 cell line, DTLacO, for MASP-3 binding antibodies. These antibodies bind to human MASP-3 in the nanomolar range at EC ₅ o from 10 to 100 nM and partially inhibit pro-CFD cleavage by MASP-3.

This example describes the production of inhibitory antibodies against human MASP-3 with unusually high binding affinity (ie subnanomolar binding affinity ranging from <500 to 20 pM). The antibodies described in this example bind specifically to human MASP-3 with high affinity (eg, <500 pM), inhibit factor D maturation, and do not bind to human MASP-1 (SEQ ID NO: 8).

Methods.

1. Obtaining a monoclonal antibody against human MASP-3 containing a chimeric mouse V-region/human IgG4 constant region.

7-14-week-old MASP-1/3 knockout C57BL/6 mice were immunized with the human MASP-3 CCP1/CCP2/SP polypeptide (amino acid residues 299-728 of SEQ ID NO: 2) containing the StrepTag II epitope tag at the N-terminus ; or the human MASP-3 SP domain (amino acid residues 450-728 of SEQ ID NO: 2) including StrepTagII at the N-terminus using the Sigma Adjuvant System (Sigma-Aldrich, St. Louis, MO). Mice were intraperitoneally injected with 50 μg of immunogen per mouse. Immunized mice were given a booster dose of additional immunogen in adjuvant 14 days later. The mice were then given a booster dose of the immunogen in PBS every 14-21 days for several weeks. Serum samples were periodically taken from tail blood and tested by ELISA for the presence of antigen-specific antibodies. Mice with high antibody titers were boosted with immunogen in PBS four days prior to splenocyte fusion. Three days prior to fusion, mice were injected subcutaneously at the base of the tail with 50 μg of an anti-CD40 mAb agonist in PBS (R&D Systems, Minneapolis, MN) to increase the number of B cells (see Rycyzyn et al., Hybridoma 27:25-30, 2008 ). Mice were then sacrificed, after which spleen cells were harvested and fused with the selected mouse myeloma cell line P3/NSI/1-AG4-1 (NS-1) (ATCC No. TIB18) using 50% polyethylene glycol or 50% polyethylene glycol plus 10% DMSO . This fusion resulted in the formation of hybridoma cells, which were cultured in 96-well tissue culture plates containing HAT medium (hypoxanthine, aminopterin, and thymidine) to inhibit the proliferation of unfused cells, myeloma hybrids, and spleen cell hybrids. After selection of hybridomas, culture supernatants were analyzed for MASP-3 binding (ELISA) and inhibition of pro-factor D activation. Positive hybridomas were identified and subcloned by serial dilution methods.

Table 17. Summary of fusion experiments

merger Immunogen: Human MASP-3 All hybridomas MASP-3- binding hybridomas Functional MASP-3 hybridomas 1 SP 434 38 10 2 SP 279 13 0 3 CCP1/CCP2/SP 348 40 2 4 CCP1/CCP2/SP 319 60 2 5 CCP1/CCP2/SP 651 152 1 6 CCP1/CCP2/SP 1297 ND 1

Note: ND means that the fusion was only screened for functional inhibition of pro-CFD activation.

Results.

As shown in Table. 17, a total of 3328 hybridomas were screened from induced MASP-1/3NK mice, of which >303 were found to bind to MASP-3 and 16 to bind to MASP-3 and inhibit pro-CFD activation. mAb M3-1 (13V1), described in example 11, is one of the 16 functional MASP-3 inhibitory antibodies described in table. 17. As described in Example 15, all 16 functional MASP-3 inhibitory antibodies were determined to bind to human MASP-3 with an unusually high binding affinity (<500 pM).

Discussion.

This example describes the generation of antibodies that inhibit human MASP-3 with unusually high binding affinity (ie, subnanomolar binding affinity ranging from <500 to 20 pM) by immunizing MASP-1/3 knockout mice. The antibodies described in this example bind specifically to human MASP-3 with high affinity (eg, <500 pM), inhibit factor D maturation, and do not bind to human MASP-1. As described herein, the amino acid sequences of human, mouse and chicken MASP-3 showed that the MASP-3 SP domain is highly conserved, especially in the active site (see FIGS. 4 and 5). It is likely that the ability to produce MASP-3 inhibitory antibodies with an unusual

- 123 040888 The high binding affinity in MASP-1/3-NK mice as described in this example is due in part to a lack of immunological tolerance that can prevent the production of highly active antibodies specific for the MASP-3 catalytic site in wild animals. type.

Example 15

This example describes an assay for cloning and sequencing of high affinity inhibitory mAbs against human MASP-3.

Methods.

Cloning and purification of recombinant antibodies.

The heavy and light chain variable regions were cloned from the hybridomas described in Examples 11 and 14 by RT-PCR and sequenced. Chimeric mouse-human mAbs consisting of mouse mAb variable regions fused to human IgG4 heavy chain (SEQ ID NO: 311) and kappa light chain (SEQ ID NO: 313) constant regions were generated as recombinant proteins in Expi2 93F cells. The IgG4 constant hinge region (SEQ ID NO: 311) contains the stabilizing amino acid substitution S228P. In one embodiment, the chimeric mAbs are attached to a human IgG4 constant hinge region (SEQ ID NO: 312) that contains the S228P amino acid substitution as well as a mutation that promotes interactions with FcRn at low pH.

The sequences of the heavy chain variable regions and the light chain variable regions are shown in FIG. 49A and 49B, respectively (SIN=SEQ ID NO: in FIGS. 49A and 49B), and are described below. The hypervariable regions (CDRs) and framework regions (FRs) of each of these antibodies are shown in Table 1 below. 18-22.

In FIG. 50A is a dendrogram of the VH regions of high affinity human MASP-3 inhibitory mAbs produced in MASP-1/3-HK mice. In FIG. 50B is a dendrogram of the VL regions of high affinity human MASP-3 inhibitory mAbs produced in MASP-1/3-HK mice. As shown in FIG. 50A and 50B, several groups of related antibodies have been identified.

The heavy chain variable region (VH) sequences for each high affinity MASP-3 inhibitory antibody are shown below. CDRs numbered according to Kabat are underlined.

Heavy chain variable regions.

- 124 040888

4D5 VH: SEQ ID NO:24

OVOLKOSGPELVKPGASVKLSCKASGYTFTTDDINWVKQRPGOGLEWIGWIYPRDDRTK YNDKFKDKATLTVDTSSNTAYMDLHSLTSEDSAVYFCSSLEDTYWGOGTLVAVSS

1F3 VH: SEQ ID NO:25

OVOLKOSGPELVKPGASVKLSCKASGYTETSNDINWVKQRPGOGLEWIGWIYPRDGSIK YNEKFTDKATL TVDVS S S TAYME LHS L T S E DSAVY FC S GVEDSYWGOGTLVTVSS

4B6 VH: SEQ ID NO :26

OVOLKOSGPELVKPGASVKLSCKASGYTETSNDINWVKQRPGOGLEWIGWIYPRDGTTK YNEEFTDKATLTVDVSSSTAFMELHSLTSEDSAVYFCSSVEDSYWGOGTLVTVSS

1A1O VH: SEQ ID NO:27

OVOLKOSGPELVKPGASVKLSCKASGYTFTSNDINWVKQRPGOGLEWIGWIYPRDGTTK YNEKFTDKATLTVDVSSSTAFMELHRLTSEDSAVYFCSSVEDSYWGOGTLVTVSS

10D12 VH: SEQ ID NO:28

QIQLVQSGPELKKPGETVKISCKASGYIFTSYGMSWVRQAPGKGLKWMGWINTYSGVPT

YADDFKGRFAFSLETSARTPYLOINNLKNEDTATYFCARGGEAMDYWGOGTSVTVSS

35C1 VH: SEQ ID NO:29

OIOLVOSGPELKTPGETVKISCKASGYIFTSYGITWVKQAPGKGLKWMGWINTYSGVPT

YADDFKGRFAFSLETSASTAYLOINNLKNEDTTTYFCTRGGDALDYWGOGTSVTVSS

13B1 VH: SEQ ID NO:30

OVOLKOSGAELMKPGASVKLSCKATGYTFTGKWIEWVKORPGHGLEWIGEILPGTGSTN YNEKFKGKATFTADSSSNTAYMOLSSLTTEDSAMYYCLRSEDVWGTGTTVTVSS

1G4 VH: SEQ ID NO:31

OVOLKOSGAELMKPGASVKLACKATGYTFTGYWIEWIKORPGOGLEWIGEMLPGSGSTH YNEKFKGKATFTADTSSNTAYMOLSGLTTEDSAIYYCVRSIDYWGOGTTLTVSS

1E7 VH: SEQ ID NO:32

OVOLKOSGPELARPWASVKISCOAFYTFSRRVHFAIRDTNYWMQWVKORPGOGLEWIGA IYPGNGDTSYNQKFKGKATL TADKS S S TAYMQL S S L T S EDSAVYYCASGSHYFDYWGOGTTL TV SS

2D7 VH: SEQ ID NO:33

EVOLOQSGPELVKPGASVKVSCKASGYTLTDYYMNWVKOSHGKSLEWIGDVNPNNDGTT

- 125 040888

YNQKFKGRATLTVDKSSNTASMELRSLTSEDSAVYYCAICPFYYLGKGTHFDYWGOGTSLTVSS 49C11 VH: SEQ ID NO:34

EVQLQQSGPVLVKPGASGKMSCKASGYKFTDYYMIWVKQSHGKSLEWIGVIKIYNGGTS

YNQKFKGRATL TVDKS S S TAYME LNS L T S EDSAVYYCARGPSLYDYDPYWYFDVWGTGTTVTVS

S

15D9 VH: SEQ ID NO:35

QVQLKQSGTELMKPGASVNLSCKASGYTFTAYWIEWVKQRPGHGLEWIGEILPGSGTTN

YNENFKDRATFTADTSSNTAYMOLSSLTSEDSAIYYCARSYYYASRWFAFWGOGTLVTVSS 2F5 VH: SEQ ID NO:36

EVQLQQPGAELVKPGASVKMSCKASGYTFTSYWITWVKQRPGQGLEWIGDIYPGGSTN

YNEKFKSKAT L TVDT S S S TAYMQL S S L T S EDSAVYYCARRRYYATAWFAYWGOGTLVTVSS

1B11 VH: SEQ ID NO:37

QVQLKQSGAELVRPGASVKLSCKASGYTFTDYYINWVKQRPGQGLEWIARIYPGSGNTY

YNEKFKGKATL TAEKS S S TAYMQL S S L T S E DSAVY FCARNYYISSPWFAYWGOGTLVTVSS 2F2 VH: SEQ ID NO:38

QVQLKQSGAELVTPGASVKMSCKASGYTFTTYPIEWMKQNHGKSLEWIGNFHPYNDDTK

YNEKFKGKATLVEKSSNTVYLELSRLTSDDSAVYFCARRVYYSYFWFGYWGHGTLVTVSS

11B6 VH: SEQ ID NO:39

QVQLKQSGELVKPGASVKMSCKASGYTFTTYPIEWMKQNHGKSLEWIGNFHPYNGDSK

YNEKFKGKATLVEKSSSTVYLELSRLPSADSAIYYCARRHYAASPWFAHWGOGTLVTVSS

Table 18. VH sequences of aHTu-MASP-3 antibody (CDR and FR regions, according to Kabat)

Antibody NS FR1 HC CDR1 4D5 QVQLKQSGPELVKPGASVKLSCKASGYTFT (SEQ ID NO:55) TDDIN (SEQ ID NO:56) 1F3 QVQLKQSGPELVKPGASVKLSCKASGYTFT (SEQ ID NO:55) SNDIN (SEQ ID NO:62) 4B6 QVQLKQSGPELVKPGASVKLSCKASGYTFT (SEQ ID NO:55) SNDIN (SEQ ID NO:62) 1A1O QVQLKQSGPELVKPGASVKLSCKASGYTFT (SEQ ID NO:55) SNDIN (SEQ ID NO:62) 10D12 QIQLVQSGPELKKPGETVKISCKASGYFT (SEQ ID NO:71) SYGMS (SEQ ID NO:72) 35С1 QIQLVQSGPELKTPGETVKISCKASGYFT (SEQ ID NO:78) SYGIT (SEQ ID NO:79) 13B1 QVQLKQSGAELMKPGASVKLSCKATGYTFT (SEQ ID NO:83) GKWIE (SEQ ID NO:84)

- 126 040888

1G4 QVQLKQSGAELMKPGASVKLACKATGYTFT (SEQ ID NO:90) GYWIE (SEQ ID NO:91) 2F5 EVQLQQPGAELVKPGASVKMSCKASGYTFT (SEQ ID NO:97) SYWIT (SEQ ID NO:98) 1B11 QVQLKQSGAELVRPGASVKLSCKASGYTFT (SEQ ID NO:102) DYYIN (SEQ ID NO:103) IE7 QVQLKQSGPELARPWASVKISCQAFYTFSR (SEQ ID NO:108) RVHFAIRDTNYWMQ (SEQ ID NO:109) 2F2 QVQLKQSGAELVTPGASVKMSCKASGYTFT (SEQ ID NO:113) TYPE (SEQ ID NO:114) 11B6 QVQLKQSGAELVKPGASVKMSCKASGYTFT (SEQ ID NO:120) TYPE (SEQ ID NO:114) 2D7 EVQLQQSGPELVKPGASVKVSCKASGYTLT (SEQ ID NO:124) DYYMN (SEQ ID NO:125) 49C11 EVQLQQSGPVLVKPGASGKMSCKASGYKFT (SEQ ID NO:131) DYYMI (SEQID NO:132) 15D9 QVQLKQSGTELMKPGASVNLSCKASGYTFT (SEQ ID NO:136) AYWIE (SEQ ID NO:137) Antibody HC-FR2 HC CDR2 4D5 WVKQRPGQGLEWIG (SEQ ID NO:57) WIYPRDDRTKYNDKFKD (SEQ ID NO:58) 1F3 WVKQRPGQGLEWIG (SEQ ID NO:57) WIYPRDGSIKYNEKFTD (SEQ ID NO:63) 4B6 WVKQRPGQGLEWIG (SEQ ID NO:57) WIYPRDGTTKYNEEFTD (SEQ ID NO:67) 1A10 WVKQRPGQGLEWIG (SEQ ID NO:57) WIYPRDGTTKYNEKFTD (SEQ ID NO:69) 10D12 WVRQAPGKGLKWMG (SEQ ID NO:73) WINTYSGVPTYADDFKG (SEQ ID NO:74) 35C1 WVRQAPGKGLKWMG (SEQ ID NO:80) WINTYSGVPTYADDFKG (SEQ ID NO:74) 13B1 WVKQRPGHGLEWIG (SEQ ID NO:85) EILPGTGSTNYNEKFKG (SEQ ID NO:86) 1G4 WIKQRPGQGLEWIG (SEQ ID NO:92) EMLPGSGSTHYNEKFKG (SEQ ID NO:93) 2F5 WVKQRPGQGLEWIG (SEQ ID NO:57) DIYPGSGSTNYNEKFKS (SEQ ID NO:99) 1B11 WVKQRPGQGLEWIA (SEQ ID NQ:104) RIYPGSGNTYYNEKFKG (SEQ ID NQ:105)

- 127 040888

IE7 WVKQRPGQGLEWIG (SEQ ID NO:57) AIYPGNGDTSYNQKFKG (SEQ ID NO:110) 2F2 WMKQNHGKSLEWIG (SEQ ID NO:115) NFHPYNDDTKYNEKFKG (SEQ ID NO:116) 11B6 WMKQNHGKSLEWIG (SEQ ID NO:115) NFHPYNGDSKYNEKFKG (SEQ ID NO:121) 2D7 WVKQSHGKSLEWIG (SEQ ID NO:126) DVNPNNDGTTYNQKFKG (SEQ ID NO:127) 49C11 WVKQSHGKSLEWIG (SEQ ID NO:126) VIKIYNGGTSYNQKFKG (SEQ ID NO:133) 15D9 WVKQRPGHGLEWIG (SEQ ID NO:85) EILPGSGTTNYNENFKD (SEQ ID NO:138) Antibody HC-FR3 HC CDR3 4D5 KATLTVDTSSNTAYMDLHSLTSEDSAVYFCSS (SEQ ID NO:59) LEDTY (SEQ ID NO:60) 1F3 KATLTVDVSSSTAYMELHSLTSEDSAVYFCSG (SEQ ID NO :64) VEDSY (SEQ ID NO:65) 4B6 KATLTVDVSSSTAFMELHSLTSEDSAVYFCSS (SEQ ID NO:68) VEDSY (SEQ ID NO:65) 1A10 KATLTVDVSSSTAFMELHRLTSEDSAVYFCSS (SEQ ID NO:70) VEDSY (SEQ ID NO:65) 10D12 RFAFSLETSARTPYLQINNLKNEDTATYFCAR (SEQ ID NO:75) GGEAMDY (SEQ ID NO:76) 35C1 RFAFSLETSASTAYLQINNLKNEDTTTYFCTR (SEQ ID NO:81) GGDALDY (SEQ ID NO:82) 13B1 KATFTADSSSNTAYMQLSSLTTEDSAMYYCLR (SEQ ID NO:87) SEDV (SEQ ID NO:88) 1G4 KATFTADTSSNTAYMQLSGLTTEDSAIYYCVR (SEQ ID NO:94) SIDY (SEQ ID NO:95) 2F5 KATLTVDTSSSTAYMQLSSLTSEDSAVYYCAR (SEQ ID N0:100) RRYYATAWFAY (SEQ ID NO:101) 1B11 KATLTAEKSSSTAYMQLSSLTSEDSAVYFCAR (SEQ ID NO:106) NYYISSPWFAY (SEQ ID NO:107) IE7 KATLTADKSSSTAYMQLSSLTSEDSAVYYCAS (SEQ ID NO:111) GSHYFDY (SEQ ID NO:112) 2F2 KATLVEKSSNTVYLELSRLTSDDSAVYFCAR (SEQ ID NO:117) RVYYSYFWFGY (SEQ ID NO:118) 11B6 KATLVEKSSSTVYLELSRLPSADSAIYYCAR (SEQ ID NO:122) RHYAASPWFAH (SEQ ID NO:123)

- 128 040888

2D7 RATLTVDKSSNTASMELRSLTSEDSAVYYCAI (SEQ ID NO:128 CPFYYLGKGTHFDY (SEQ ID NO:129) 49C11 KATLTVDKSSSTAYMELNSLTSEDSAVYYCAR (SEQ ID NO:134) GPSLYDYDPYWYFDV (SEQ ID NO:135) 15D9 RATFTADTSSNTAYMQLSSLTSEDSAIYYCAR (SEQ ID NO:139) SYYYASRWFAF (SEQ ID NO:140) Antibody HC FR4 4D5 WGQGTLVAVS (SEQ ID NO:61) 1F3 WGQGTLVTVSS (SEQ ID NO :66) 4B6 WGQGTLVTVSS (SEQ ID NO :66) 1А10 WGQGTLVTVSS (SEQ ID NO :66) 10D12 WGQGTSVTVSS (SEQ ID NO:77) 35С1 WGQGTSVTVSS (SEQ ID NO:77) 13B1 WGTGTTVTVSS (SEQ ID NO:89) 1G4 WGQGTTTLTVSS (SEQ ID NO:96) 2F5 WGQGTLVTVSS (SEQ ID NO :66) 1B11 WGQGTLVTVSS (SEQ ID NO :66) 1E7 WGQGTTTLTVSS (SEQ ID NO:96) 2F2 WGHGTLVTVSS (SEQ ID NO:119) 11B6 WGQGTLVTVSS (SEQ ID NO :66) 2D7 WGQGTSLTVSS (SEQ ID NO:130) 49C11 WGTGTTVTVSS (SEQ ID NO:89) 15D9 WGQGTLVTVSS (SEQ ID NO :66)

The light chain variable region (VL) sequences for each high affinity MASP-3 inhibitory antibody are shown below. CDRs numbered according to Kabat are underlined. These areas are the same regardless of whether they are numbered according to the Kabat or Chotiya system.

Light chain variable regions

- 129 040888

4D5 VL: SEQ ID NO:40

DIVMTQS PS S LAVSAGEKVTMTCKSSOSLLNSRTRKNYLAWYOQKPGOSPKLLIYWAST

RESGVPDRFTGSGSGTDFSLTISSVOAEDLAVYYCKQSYNLYTFGGGTKLEIKR

1F3 VL: SEQ ID NO:41

DIVMTQSPSSLAVSAGERVTMSCKSSQSLLISRTRKNYLSWYQQKPGQSPKLLIYWAST

RESGVPDRFTGSGSGTDFTLTISSVOAEDLAVYYCKQSYNLYTFGGGTKLEIKR

4B6 VL: SEQ ID NO:42 (SAME for 1A1O VL)

DIVMTOSPSSLAVSAGEKVTMSCKSSQSLLISRTRKNYLSWYOQKPGOSPKLLIYWAST

RESGVPDRFTGSGSGTDFTLTISSVOAEDLAVYYCKQSYNLYTFGGGTKLEIKR

10D12 VL: SEQ ID NO:43

DVLMTOTPLTLSVTIGOPASISCKSSQSLLDSDGKTYLNWLLQRPGOSPKRLIYLVSKL

DSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPWTFGGGTKLEIKR

35C1 VL: SEQ ID NO:44

DIVMTOAPLTLSVTIGOPASISCKSSQSLLDSDGKTYLSWLLQRPGOSPKRLIYLVSKL

DSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKLEIKR

13B1 VL: SEQ ID NO:45

DIVMTOSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYOQKPGOSPKLLIYWAST

RESGVPDRFTGSGSGTDFTLTISSVOAEDLAVYYCKQSYNIPTFGGGTKLEIKR

1G4 VL: SEP ID NO :46

DVLMTQT PLS LPVS LGEQASIS CRSSQSLVQSNGNTYLHWYLOKPGOSPKLLIYKVSNR

FSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPTFGGGTKLEIKR

1E7 VL: SEP ID NO:47

DIOLTOSPAILSVSPGERVSFSCRASOSIGTSIHWYOQRTNGSPRLLIKYASESISGIP

SRFSGSGSGTDFTLSINSVESEDIADYYCOQSNSWPYTFGGGTKLEIKR

2D7 VL: SEQ ID NO:48

DIQMT QT PAS L SAS LGDRVTIS CRASQDISNFLNWYOOKPNGTVKLLVFYTSRLHSGVP

SRFSGSGSGAEHSLTISNLEOEDVATYFCOQGFTLPWTFGGGTKVEIKR

49C11 VL:SEQ ID NO:49

DVLMTOTPLSLPVSLGDOASFSCRSSQSLIHSNGNTYLHWYLOKPGOSPKLLIYKVSNR

FSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIKR 15D9 VL: SEQ ID NO:50

DIVMTOSOKFMSTSIGDRVSVTCRASQNVGPNLAWYOOKPGOSPKALIYSASYRFSGVP

DRFTGSGSGTDFTLTISNVOSEDLAEYFCOQYNRYPFTFGSGTKLEIKR

2F5 VL: SEQ ID NO:51

DIVMTQSQKFMSTSVGDRVSITCKASQNVGTAVAWYOQKPGOSPKLLISSASNRYTGVP

DRFTGSGSGTDFTLTISNMOSEDVADYFCOQYNSYPLTFGAGTKLELKR

1B11 VL: SEQ ID NO:52

DIVMTOSOKFMSTSVGDRVSVTCKASQNVGPNVAWYOOKPGOSPKALIYSASYRYSGVP

DRFTGSGSGTDFTLTISNVOSEDLADYFCOQYNRYPLTFGAGTKLELKR

2F2 VL: SEQ ID NO:53

DIVMTOSOKFMSTSVGDRVNVTCKASQNVGTHVAWYOOKPGOSPKALIYSASYRYSGVP

DRFTGSGSGTDFTLTISNVOSEDLAEYFCOQYNSYPRALTFGAGTKLELKR

11B6 VL: SEP ID NO:54

DIVMTQSQKFMSTSVGDRVNVTCKASQNVGPTVAWYOQKPGOSPKALIYSASYRYSGVP

DRFTGSGSGTDFTLTISNVHSEDLAEYFCQQYNSYPFTFGSGTKLEIKR

- 130 040888

Table 19. VL sequences of aHTu-MASP-3 antibody (CDR and FR regions, according to Kabat and Chotiy)

Antibody LC-FR1 LC CDR1 4D5 DIVMTQ S P S S LAVSAGEKVMTTC (SEQ ID NO:141) KSSQSLLNSRTRKNYLA (SEQ ID NO:142) 1F3 DIVMTQSPSSLAVSAGERVTMSC (SEQ ID NO:148) KSSQSLLISRTRKNYLS (SEQ ID NO:149) 4B6 DIVMTQSPSSLAVSAGEKVTMSC (SEQ ID NO:151) KSSQSLLISRTRKNYLS (SEQ ID NO:149) 1А10* [used 4B6 LC: SEQ ID NO:151] [used 4B6 LC: SEQ ID NO:149] 10D12 DVLMTQT P LT L S VTIGQ PAS ISC (SEQ ID NO:152) KSSQSLLDSDGKTYLN (SEQ ID NO:153) 35С1 DIVMTQAPLTLSVTIGQPASISC (SEQ ID NO:158) KSSQSLLDSDGKTYLS (SEQ ID NO:159) 13B1 DIVMTQSPSSLAVSAGEKVTMSC (SEQ ID NO:151) KSSQSLLNSRTRKNYLA (SEQ ID NO:142) 1G4 DVLMTQTPLSLPVSLGEQASISC (SEQ ID NO:162) RSSQSLVQSNGNTYLH (SEQ ID NO:163) 2F5 DIVMTQSQKFMSTSVGDRVSITC (SEQ ID NO:168) KASQNVGTAVA (SEQ ID NO:169) 1B11 DIVMTQSQKFMSTSVGDRVSVTC (SEQ ID NO:175) KASQNVGPNVA (SEQ ID NO:176)

- 131 040888

1E7 DIQLTQSPAILSVSPGERVSFSC (SEQ ID NO:181) RASQSIGTSIH (SEQ ID NO:182) 2F2 DIVMTQSQKFMSTSVGDRVNVTC (SEQ ID NO:187) KASQNVGTHVA (SEQ ID NO:188) 11B6 DIVMTQSQKFMSTSVGDRVNVTC (SEQ ID NO:187) KASQNVGPTVA (SEQ ID NO:191) 2D7 DIQMTQTPASLSASLGDRVTISC (SEQ ID NO:195) RASQDISNFLN (SEQ ID NO:196) 49C11 DVLMTQTPLSLPVSLGDQASFSC (SEQ ID NO:202) RSSQSLIHSNGNTYLH (SEQ ID NO:203) 15D9 DIVMTQSQKFMSTSIGDRVSVTC (SEQ ID NO:205) RASQNVGPNLA (SEQ ID NO:206) Antibody LC-FR2 LC CDR2 4D5 WYQQKPGQSPKLLIY (SEQ ID NO:143) WASTRES (SEQ ID NO:144) 1F3 WYQQKPGQSPKLLIY (SEQ ID NO:143) WASTRES (SEQ ID NO:144) 4B6 WYQQKPGQSPKLLIY (SEQ ID NO:143) WASTRES (SEQ ID NO:144) 1A10 [used 4B6 LC: SEQ ID NO:143] [used 4B6 LC: SEQ ID NO:144] 10D12 WLLQRPGQSPKRLIY (SEQ ID NO:154) LVSKLDS (SEQ ID NO:155) 35С1 WLLQRPGQSPKRLIY (SEQ ID NO:154) LVSKLDS (SEQ ID NO:155) 13B1 WYQQKPGQSPKLLIY (SEQ ID NO:143) WASTRES (SEQ ID NO:144) 1G4 WYLQKPGQSPKLLIY (SEQ ID NO:164) KVSNRFS (SEQ ID NO:165) 2F5 WYQQKPGQSPKLLIS (SEQ ID NO:170) SASNRYT (SEQ ID NO:171) 1B11 WYQQKPGQSPKALIY (SEQ ID NO:177) SASYRYS (SEQ ID NO:178) 1E7 WYQQRTNGSPRLLIK (SEQ ID NO:183) YASESIS (SEQ ID NO:184) 2F2 WYQQKPGQSPKALIY (SEQ ID NO:177) SASYRYS (SEQ ID NO:178) 11B6 WYQQKPGQSPKALIY (SEQ ID NO:177) SASYRYS (SEQ ID NO:178) 2D7 WYQQKPNGTVKLLVF YTSRLHS

- 132 040888

(SEQ ID NO:197) (SEQ ID NO:198) 49C11 WYLQKPGQSPKLLIY (SEQ ID NO:164) KVSNRFS (SEQ ID NO:165) 15D9 WYQQKPGQSPKALIY (SEQ ID NO:177) SASYRFS (SEQ ID NO:207) Antibody LC-FR3 LC CDR3 4D5 GVPDRFTGSGSGTDFSLTISSVQAEDLAVYYC (SEQ ID NO:145) KQSYNLYT (SEQ ID NO:146) 1F3 GVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC (SEQ ID NO:150) KQSYNLYT (SEQ ID NO:146) 4B6 GVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC (SEQ ID NO:150) KQSYNLYT (SEQ ID NO:146) 1A10 [used 4B6 LC: SEQ ID NO:150] [used 4B6 LC: SEQ ID NO:146] 10D12 GVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC (SEQ ID NO:156) WQGTHFPWT (SEQ ID NO:157) 35С1 GVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC (SEQ ID NO:156) WQGTHFPYT (SEQ ID NO:160) 13B1 GVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC (SEQ ID NO:150) KQSYNIPT (SEQ ID NO:161) 1G4 GVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC (SEQ ID NO:166) SQSTHVPPT (SEQ ID NO:167) 2F5 GVPDRFTGSGSGTDFTLTISNMQSEDVADYFC (SEQ ID NO:172) QQYNSYPLT (SEQ ID NO:173) 1B11 GVPDRFTGSGSGTDFTLTISNVQSEDLADYFC (SEQ ID NO:179) QQYNRYPLT (SEQ ID NO:180) 1E7 GIPSRFSGSGSGTDFTLSINSVESEDIADYC (SEQ ID NO:185) QQSNSWPYT (SEQ ID NO:186) 2F2 GVPDRFTGSGSGTDFTLTISNVQSEDLAEYFC (SEQ ID NO:189) QQYNSYPRALT (SEQ ID NO:190) 11B6 GVPDRFTGSGSGTDFTLTISNVHSEDLAEYFC (SEQ ID NO:192) QQYNSYPFT (SEQ ID NO:193) 2D7 GVPSRFSGSGSGAEHSLTISNLEQEDVATYFC (SEQ ID NO:199) QQGFTLPWT (SEQ ID NO:200) 49C11 GVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC (SEQ ID NO:166) SQSTHVPWT (SEQ ID NO:204) 15D9 GVPDRFTGSGSGTDFTLTISNVQSEDLAEYFC (SEQ ID NO:189) QQYNRYPFT (SEQ ID NO:208) Antibody LC-FR4

- 133 040888

4D5 FGGGTKLEIKR (SEQ ID NO:147) 1F3 FGGGTKLEIKR (SEQ ID NO:147) 4B6 FGGGTKLEIKR (SEQ ID NO:147) 1А10 [used 4B6 LC: SEQ ID NO:147] 10D12 FGGGTKLEIKR (SEQ ID NO:147) 35С1 FGGGTKLEIKR (SEQ ID NO:147) 13B1 FGGGTKLEIKR (SEQ ID NO:147) 1G4 FGGGTKLEIKR (SEQ ID NO:147) 2F5 FGAGTKLELKR (SEQ ID NO:174) 1B11 FGAGTKLELKR (SEQ ID NO:174) 1E7 FGGGTKLEIKR (SEQ ID NO:147) 2F2 FGAGTKLELKR (SEQ ID NO:174) 11B6 FGSGTKLEIKR (SEQ ID NO:194) 2D7 FGGGTKVEIKR (SEQ ID NO:201) 49C11 FGGGTKLEIKR (SEQ ID NO:147) 15D9 FGSGTKLEIKR (SEQ ID NO:194)

*Note: The light chain for mAb 1A10 was not identified and therefore the 4B6 light chain was used together with 1A10 HC.

Table 20. Consensus sequences for Group IA HC CDRs

Antibody Region Subsequence 4D5 HC-CDR1 TDDIN (SEQ ID NO:56) 1F3 HC-CDR1 SNDIN (SEQ ID NO:62) 4B6 HC-CDR1 SNDIN (SEQ ID NO:62)

- 134 040888

1A10 HC-CDR1 SNDIN (SEQ ID NO:62) Consensus HC-CDR1 XXDIN (SEQ ID NO:209) Where : X at position 1 is S or T; And X in position 2 is N or D 4D5 HC-CDR2 WIYPRDDRTKYNDKFKD (SEQ ID NO:58) 1F3 HC-CDR2 WIYPRDGSIKYNEKFTD (SEQ ID NO:63) 4B6 HC-CDR2 WIYPRDGTTKYNEEFTD (SEQ ID NO:67) 1А10 HC-CDR2 WIYPRDGTTKYNEKFTD (SEQ ID NO:69) Consensus HC-CDR2 WIYPRDXXXKYNXXFXD (SEQ ID NO:210) where X at position 7 is G or D; X at position 8 is S, T or R; X at position 9 is I or T; X at position 13 is E or D; X at position 14 is K or E; X at position 16 is T or TO 4D5 HC-CDR3 LEDTY (SEQ ID NO:60) 1F3 HC-CDR3 VEDSY (SEQ ID NO:65) 4B6 HC-CDR3 VEDSY (SEQ ID NO:65) 1А10 HC-CDR3 VEDSY (SEQ ID NO:65) Consensus HC-CDR3 XEDXY (SEQ ID NO:211) where X at position 1 is L or V, and where X at position 4 is T or S

Table 21. Consensus sequences for Group IA LC CDRs

Antibody Region Subsequence 4D5 LC-CDR1 KSSQSLLNSRTRKNYLA (SEQ ID NO:142) 4D5-NQ LC-CDR1 KSSQSLLQSRTRKNYLA (SEQ ID NO:257) 4D5-NA LC-CDR1 KSSQSLLASRTRKNYLA (SEQ ID NO:258) 4D5-ST LC-CDR1 KSSQSLLNTRTRKNYLA (SEQ ID NO:259) 1F3 LC-CDR1 KSSQSLLISRTRKNYLS (SEQ ID NO:149)

- 135 040888

4B6 LC-CDR1 KSSQSLLISRTRKNYLS (SEQ ID NO:149) Consensual e* LC-CDR1 KSSQSLLXXRTRKNYLX (SEQ ID NO :212) where X at position 8 is N, I, Q or A; where X at position 9 is S or T; and where X at position 17 is A or S 4D5 LC-CDR2 WASTRES (SEQ ID NO:144) 1F3 LC-CDR2 WASTRES (SEQ ID NO:144) 4B6 LC-CDR2 WASTRES (SEQ ID NO:144) Consensus e LC-CDR2 WASTRES (SEQ ID NO:144) 4D5 LC-CDR3 KQSYNLYT (SEQ ID NO:146) 1F3 LC-CDR3 KQSYNLYT (SEQ ID NO:146) 4B6 LC-CDR3 KQSYNLYT (SEQ ID NO:146) Consensus e LC-CDR3 KQSYNLYT (SEQ ID NO:146)

^Note: The consensus CDR-L1 includes variants prepared as described in Example 19.

Table 22. Group IB HC Consensus CDR Sequences

Antibody Region Subsequence 10D12 HC-CDR1 SYGMS (SEQ ID NO:72) 3501 HC-CDR1 SYGIT (SEQ ID NO:79) Consensus oe HC-CDR1 SYGXX (SEQ ID NO:213) where X at position 4 is M or I; And where X at position 5 is S or T 10D12 HC-CDR2 WINTYSGVPTYADDFKG (SEQ ID NO:74) 3501 HC-CDR2 WINTYSGVPTYADDFKG (SEQ ID NO:74) Consensus oe HC-CDR2 WINTYSGVPTYADDFKG (SEQ ID NO:74) 10D12 HC-CDR3 GGEAMDY (SEQ ID NO:76) 3501 HC-CDR3 GGDALDY (SEQ ID NO:82) Consensus oe HC-CDR3 GGXAXDY (SEQ ID NO:214) where X at position 3 is E or D; And where X at position 5 is M or L

- 136 040888

Table 23. Group IB LC CDR Consensus Sequences

Antibody Region Subsequence 10D12 LC-CDR1 KSSQSLLDSDGKTYLN (SEQ ID NO:153) 10D12-DE LC-CDR1 KSSOSLLDDSEGKTYLN (SEQ ID NO :261) 10D12-DA LC-CDR1 KSSQSLLDSAGKTYLN (SEQ ID NO:262) 10D12-GA LC-CDR1 KSSQSLLDSDAKTYLN (SEQ ID NO:263) 35С1 LC-CDR1 KSSQSLLDSDGKTYLS (SEQ ID NO:159) Consensual e* LC-CDR1 KSSQSLLDSXXKTYLX (SEQ ID NO:215) where X at position 10 is D, E or A; where X at position 11 is G or A; And where X at position 16 is N or S 10D12 LC-CDR2 LVSKLDS (SEQ ID NO:155) 35С1 LC-CDR2 LVSKLDS (SEQ ID NO:155) Consensus e LC-CDR2 LVSKLDS (SEQ ID NO:155) 10D12 LC-CDR3 WQGTHFPWT (SEQ ID NO:157) 35С1 LC-CDR3 WQGTHFPYT (SEQ ID NO:160) Consensus e LC-CDR3 WQGTHFPXT (SEQ ID NO: 216) where X at position 8 is W or Y

*Note: The consensus CDR-L1 includes variants prepared as described in Example 19. DNA encoding heavy and light chains of the mouse mAb.

SEQ ID NO: 217: DNA encoding 4D5 heavy chain variable region (parent)

CAGGTGCAGCTGAAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGTT

GTCCTGCAAGGCTTTCTGGCTACACCTTCACAACCGACGATATAAACTGGTGAAGCAGAGGCCT GGACAGGGAC T T GAGT GGAT T GGAT GGAT T TAT CC TAGAGAT GATAGAAC TAAGTACAAT GACA AGTTCAAGGACAAGGCCACATTGACTGTAGACACATCTTCCAACACAGCGTACATGGACCTCCA

- 137 040888

CAGCCTGACATCTGAGGACTCTGCGGTCTATTTCTGTTCAAGCCTCGAGGATACTTACTGGGGC CAAGGGACTCTGGTCGCTGTCTCTTCA

SEQ ID NO:218: DNA encoding 1F3 heavy chain variable region (parent)

CAGGTGCAGCTGAAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGTT GTCCTGCAAGGCTTCTGGCTACACCTTCACAAGTAACGATATAAACTGGGTGAAGCAGAGGCCT GGACAGGGACTTGAGTGGATTGGATGGATTTATCCTAGAGATGGGAGTATTAAATATAATGAGA AATTCACGGACAAGGCCACATTGACAGTTGACGTATCCTCCAGCACAGCGTACATGGAGCTCCA CAGCCTGACATCTGAGGACTCTGCGGTCTATTTCTGTTCAGGTGTCGAGGATTCTTACTGGGGC CAAGGGACTCTGGTCACTGTCTCTTCA

SEQ ID NO:219: DNA encoding heavy chain variable region 4B6 (parental)

CAGGTGCAGCTGAAGCAGTCTGGACCTGAACTGGTGAAGCCTGGGGCTTCAGTGAAATT GTCCTGCAAGGCTTCTGGCTACACCTTCACAAGTAACGATATAAACTGGGTGAAACAGAGGCCT GGACAGGGACTTGAGTGGATTGGATGGATTTATCCTAGAGATGGTACTACTAAGTACAATGAGG AGTTCACGGACAAGGCCACATTGACTGTTGACGTATCCTCCAGCACAGCGTTCATGGAGCTCCA CAGCCTGACATCTGAGGACTCTGCTGTCTATTTCTGTTCAAGTGTCGAGGATTCTTACTGGGGC CAAGGGACTCTGGTCACTGTCTCTTCA

SEQ ID NO:22Q: DNA encoding heavy chain variable region 1A10 (parent)

CAGGTGCAGCTGAAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGTT GTCCTGCAAGGCTTCTGGCTACACCTTCACAAGTAACGATATAAACTGGGTGAAGCAGAGGCCT GGACAGGGACTTGAGTGGATTGGATGGATTTATCCTAGAGATGGTACTACTAAGTACAATGAGA AGTTCACGGACAAGGCCACATTGACTGTTGACGTATCCTCCAGCACAGCGTTCATGGAGCTCCA CAGGCTGACATCTGAGGACTCTGCGGTCTATTTCTGTTCAAGTGTCGAGGATTCTTACTGGGGC CAAGGGACTCTGGTCACTGTCTCTTCA

SEQ ID NO:221: DNA encoding 10D12 heavy chain variable region (parent)

CAGATCCAGTTGGTACAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGAT CTCCTGCAAGGCTTCTGGGTATATTTTCACAAGCTATGGAATGAGCTGGGTGAGACAGGCTCCA GGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACTCTGGAGTGCCAACATATGCTGATG ACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGAACTCCCTATTTGCAGATCAA CAACCTCAAAAATGAGGACACGGCTACATATTTCTGCGCAAGAGGGGGCGAAGCTATGGACTAC TGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA

SEQ ID NO:222: DNA encoding 35C1 heavy chain variable region (parent)

- 138 040888

CAGATCCAGTTGGTACAGTCTGGACCTGAGCTGAAGACGCCAGGAGAGACAGTCAAGAT CTCCTGCAAGGCTTCTGGGTATATCTTCACATCCTATGGAATTACCTGGGTGAAACAGGCTCCA GGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACTCTGGAGTGCCAACATATGCTGATG ACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACGTCTGCCAGCACTGCCTATTTGCAGATCAA CAACCTCAAAAATGAGGACACGACTACATATTTСTGTACAAGAGGGGGTGATGCTTTGGACTAG TGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA

SEQ ID NO:223: DNA encoding 13B1 heavy chain variable region (parent)

CAGGTGCAGCTGAAGCAGTCTGGAGCTGAGCTGATGAAGCCTGGGGCCTCAGTGAAGCT TTCCTGCAAGGCTACTGGCTACACATTCACTGGCAAGTGGATAGAGTGGGTAAAACAGAGGCCT GGACATGGCCTAGAGTGGATTGGAGAGATTTTACCTGGAACTGGTAGTACTAACTACAATGAGA AGTTCAAGGGCAAGGCCACATTCACTGCAGACTCATCCTCCAACACAGCCTACATGCAACTCAG CAGCCTGACAACTGAAGACTCTGCTATGTATTATTGTTTAAGATCCGAGGATGTCTGGGGCACA GGGACCACGGTCACCGTCTCCTCA

SEQ ID NO:224: DNA encoding 1G4 heavy chain variable region (parent)

CAGGTGCAGCTGAAGCAGTCTGGAGCTGAGCTGATGAAGCCTGGGGCCTCAGTGAAGCT TGCCTGCAAGGCTACTGGCTACACATTCACTGGCTACTGGATAGAGTGGATAAAGCAGAGGCCT GGACAAGGCCTTGAGTGGATTGGAGAGATGTTACCTGGAAGTGGTAGTACTCACTACAATGAGA AGTTCAAGGGTAAGGCCACATTCACTGCAGATACATCCTCCAACACAGCCTACATGCAACTCAG CGGCCTGACAACTGAGGACTCTGCCATCTATTACTGTGTAAGAAGCATAGACTACTGGGGCCAA G G САС САС Т С Т СACAG Т С Т С С Т СА

SEQ ID NO:225: DNA encoding 1E7 heavy chain variable region (parent)

CAGGTGCAGCTGAAGCAGTCTGGGCCTGAGCTGGCAAGGCCTTGGGCTTCAGTGAAGAT ATCCTGCCAGGCTTTCTACACCTTTTCCAGAAGGGTGCACTTTGCCATTAGGGATACCAACTAC TGGATGCAGTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATCGGGGCTATTTATCCTG GAAATGGTGATACTAGTТАСААТCAGAAGTТCAAGGGCAAGGCCACATТGACТGCAGACAAATС CTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGT GCATCCGGTAGCCACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

SEQ ID NO:226: DNA encoding heavy chain variable region 2D7 (parent)

GAGGTCCAGCTGCAACAATCTGGGCCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGGT ATCCTGTAAGGCTTCTGGATACAACGCTCACTGACTACATGAACTGGGTGAAGCAGAGCCAT GGAAAGAGCCTTGAGTGGATTGGAGATGTTAATCCTAACAATGATGGTACTACCTACAACCAGA AATTCAAGGGCAGGGCCACATTGACTGTAGACAAGCCTCTCTCAT

- 139 040888

CAGCCTGACATCTGAGGACTCTGCAGTCTACTACTGTGCAATATGCCCCTTTTATTACCTCGGT AAAGGGACCCACTTTGACTACTGGGGCCAAGGCACCTCTCTCACAGTCTCCTCA

SEQ ID NO:227: DNA encoding 49C11 heavy chain variable region (parent)

GAGGTCCAGCTGCAACAATCTGGACCTGTGCTGGTGAAGCCTGGGGCTTCAGGGAAGAT GTCCTGTAAGGCTTCTGGATACAAATTCACTGACTACTATATGATCTGGGTGAAGCAGAGCCAT GGAAAGAGCCTTGAGTGGATTGGAGTTATTAAAATTTATAACGGTGGTACGAGCTACAACCAGA AGTTCAAGGGCAAGGCCACATTGACTGTTGACAAGTCCTCCAGCACAGCCTACATGGAGCТСАА CAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAGGGCCATCTCTCTATGATTAC GACCCTTACTGGTACTTCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTCA

SEQ ID NO:228: DNA encoding 15D9 heavy chain variable region (parent)

CAGGTGCAGCTGAAGCAGTCTGGAACTGAGCTGATGAAGCCTGGGGCCTCAGTGAACCT TTCCTGCAAGGCTTCTGGCTACACATTCACTGCCTACTGGATAGAGTGGGTAAAGCAGAGGCCT GGACATGGCCTTGAGTGGATTGGAGAGATTTTACCTGGAAGTGGTACTACTAACTACAATGAGA АС Т Т CAAGGACAGGGCCACAT Т САС Т GCAGATACAT CC Т CCAACACAGCC ТАСАТ GCAACТ CAG CAGCCTGACAAGTGAGGACTCTGCCATCTATTACTGTGCAAGATCCTATTACTACGCTAGTAGA TGGTTTGCTTTCTGGGGCCAAGGGACTCTGGTCACTGTCTCTTCA

SEQ ID NO:229: DNA encoding 2F5 heavy chain variable region (parent)

GAGGTCCAGCTGCAGCAGCCTGGGGCTGAGCTTGTGAAGCCTGGGGCTTCAGTGAAGAT GTCCTGTAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATAACCTGGGTGAAGCAGAGGCCT GGACAAGGCCTTGAGTGGATTGGAGATATTТАТCCTGGTAGTGGTAGTACTAACTACAATGAGA AGTTCAAGAGCAAGGCCACACTGACTGTAGACACATCCTCCAGCACAGCCТАСАТGCAGCTCAG CAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGAAGGAGATACTACGCTACGGCC TGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTTCA

SEQ ID NO:23Q: DNA encoding 1B11 heavy chain variable region (parent)

CAGGTGCAGCTGAAGCAGTCTGGGGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGAAGCT GTCCTGCAAGGCTTCTGGCTACACTTTCACTGACTACTATATAAACTGGGTGAAGCAGAGGCCT GGACAGGGACТТGAGTGGATТGCAAGGATТТАТОСТGGAAGTGGTAATACТТАСТАСААТGAGA AGTTCAAGGGCAAGGCCACACTGACTGCAGAAAAATCCTCCAGCACTGCCТАСАТGCAGCTCAG CAGCCTGACATCTGAGGACTCTGCTGTCTATTTCTGTGCAAGAAATTACTACATTAGTAGTCCC TGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTTCA

SEQ ID NO:231: DNA encoding 2F2 heavy chain variable region (parent)

- 140 040888

CAGGTGCAGCTGAAGCAGTCTGGGGCTGAGCTAGTGACGCCTGGAGCCTCAGTGAAGAT GTCCTGCAAGGCTTCTGGCTACACCTTCACTACCTATCCTATAGAGTGGATGAAACAGAATCAT GGAAAGAGC С TAGAG T GGAT T GGAAAT Τ Τ T CAT С C Τ TACAAT GAT GATAC TAAG TACAAT GAAA AGTTCAAGGGCAAGGCCACATTGACTGTAGAAAAATCCTCTAACACAGTCTACTTGGAGCTCAG CCGATTAACATCTGATGACTCTGCTGTTTATTTCTGTGCAAGGAGGGTCTACTATAGTTACTTC TGGTTTGGTTACTGGGGCCACGGGACTCTGGTCACTGTCTCTTCA

SEQ ID NO:232: DNA encoding 11B6 heavy chain variable region (parent)

CAGGTGCAGCTGAAGCAGTCTGGGGCTGAGCTAGTGAAACCTGGAGCCTCAGTGAAGAT GTCCTGCAAGGCTTCTGGCTACACCTTCACTACCTATCCTATAGAGTGGATGAAGCAGAATCAT GGGAAGAGCCTAGAGTGGATTGGAAATTTTCATCCTTACAATGGTGATTCTAAGTACAATGAAA AGTTCAAGGGCAAGGCCACCTTGACTGTAGAAAAATCCTCTAGCACAGTCTACTTAGAACTCAG CCGATTACCATCTGCTGACTCTGCTATTTATTACTGTGCAAGGAGGCACTACGCTGCTAGTCCC TGGTTTGCTCACTGGGGCCAAGGGACTCTGGTCACTGTCTCTTCA

DNA encoding light chain variable region (mouse mAb):

SEQ ID NO:233: DNA encoding 4D5 light chain variable region (parent)

GACATTGTGATGACCCAGTCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGAAGGTCAC TATGACCTGCAAATCCAGTCAGAGTCTGCTCAACAGTAGAACCCGAAAGAACTACTTGGCTTGG TACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTACTGGGCATCCACTAGGGAATCTG GGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCTCTCTCACCATCAGCAGTGT GCAGGCTGAAGACCTGGCAGTTTATTACTGCAAGCAATCTTATAATCTGTACACGTTCGGAGGG GGGACCAAGCTGGAAATAAAACGG

SEQ ID NO:234: DNA encoding 1F3 light chain variable region (parent)

GACATTGTGATGACCCAGTCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGAGGGTCAC ТATGAGСТGСАААТССAGТСAGAGТСТGСТСАТСAGТAGAACССGAAAGAACТАТΤТGТСΤТGG TACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTACTGGGCATCCACTAGGGAATCTG GGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGT ACAGGCTGAAGACCTGGCAGTTTATTACTGCAAGCAATCTTATAATCTGTACACGTTCGGCGGG GGGACCAAGCTGGAAATAAAACGG

SEQ ID NO:235: DNA encoding light chain variable region 4B6/1A10 (parental)

GACATTGTGATGACCCAGTCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGAAGGTCAC

- 141 040888

TACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTATTGGGCATCCACTAGGGAATCTG GGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGT ACAGGCTGAAGACCTGGCAGTTTATTACTGCAAACAATCTTATAATCTGTACACGTTCGGCGG GGGACCAAGCTGGAAATCAAACGG

SEQ ID NO:236: DNA encoding 10D12 light chain variable region (parent)

GATGTTTTGATGACCCAAACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTC CATCTCTTG СAAG Т СAAG Т СAGAG С С Т С Т ТAGATAG Т GAT G GAAAGACАТАТ Т Т GAAT Т G G Т Т G TTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAG TCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGA GGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCGTGGACGTTCGGTGGA GGGACCAAGCТGGAAATCAAACGG

SEQ ID NO:237: DNA encoding 35C1 light chain variable region (parent)

GATATTGTGATGACGCAGGCTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTC CATCTCTTG GAAG Т GAAG Т СAGAG С С Т С Т ТAGATAG Т GAT G GAAAGACАТАТ Т Т GAG Т Т G G Т Т G TTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAG TCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGA GGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCGTACACGTTCGGAGGG GGGACCAAGCTGGAAATAAAACGG

SEQ ID NO:238: DNA encoding 13B1 light chain variable region (parent)

GACATTGTGATGACCCAGTCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGAAGGTCAC TATGAGCTGCAAATCCAGTCAGAGTCTGCTCAACAGTAGAACCCGAAAGAACTACTTGGCTTGG TACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTACTGGGCATCCACTAGGGAATCTG GGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAACAGATTTCACTCTCACCATCAGCAGTGT GCAGGCTGAAGACCTGGCAGTTTATTACTGCAAGCAATCTTATAATATTCCGACGTTCGGTGGA GGGACCAAGCТGGAAATCAAACGG

SEQ ID NO:239: DNA encoding 1G4 light chain variable region (parent)

GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGAACAAGCCTC CATCTCTTG СAGAT СAAG Т СAGAG С С Т Т G ТACAAAG ТAAT G GAAACАС С ТАТ Т ТАСАТ Т G G ТАС CTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGG TCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGA GGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCTCCGACGTTCGGTGGA GGGACCAAGCТGGAAATCAAACGG

- 142 040888

SEQ ID NO:24Q: DNA encoding light chain variable region 1E7 DNA encoding light chain variable region (parent)

GACATCCAGCTGACTCAGTCTCCAGCCATCCTGTCTGTGAGTCCAGGAGAAAGAGTCAG TTTCTCCTGCAGGGCCAGTCAGAGCATTGGCACAAGCATACACTGGTATCAGCAAAGAACAAAT GGTTCTCCAAGGCTTCTCATAAAGTATGCTTCTGAGTCTATCTCTGGGATCCCTTCCAGGTTTA GTGGCAGTGGATCAGGGACAGATTTTACTCTTAGCATCAACAGTGTGGAGTCTGAAGATATTGC AGATTATTACTGTCAACAAAGTAATAGCTGGCCGTACACGTTCGGAGGGGGGACCAAGCTGGAA ATAAAACGG

SEQ ID NO:241: DNA encoding light chain variable region 2D7 (parent)

GATATCCAGATGACACAGACTCCAGCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCAC CATCAGTTGTAGGGCAAGTCAGGACATTAGCAATTTTTTAAACTGGTATCAACAGAAACCGAAT G GAAC Т G Т ТАЛАС TCCTAGTCTTC ТАСАСАТ СAAGATТАСАС Т СAGGAGТ СССАТ СAAGGТ Т СА GTGGCAGTGGGTCTGGAGCAGAGCATTCTCTCACCATTAGCAACCTGGAGCAGGAAGATGTTGC CACTTACTTTTGCCAACAGGGTTTTACGCTTCCGTGGACGTTCGGTGGGGGCACCAAGGTGGAA ATCAAACGG

SEQ ID NO:242: DNA encoding 49C11 light chain variable region (parental)

GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTC CTTCTCTTG СAGAT С ТAG Т СAGAG СОТ ТАТACACAG ТЛАТ G GAAACАС С ТАТ Т ТАСАТ Т G G ТАС CTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGG ТCCCAGACAGGTТCAGTGGCAGTGGATCAGGGACAGATТТCACAOTCAAGATCAGCAGAGTGGA GGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCGTGGACGTTCGGTGGA GGCACCAAGCТGGAAATCAAACGG

SEQ ID NO:243: DNA encoding 15D9 light chain variable region (parent)

GACАТ Т G Т GAT GAC С СAG ТОТ СΑΑΑΑΆΤ Т СAT G Т С САСАТ СЛАТAG GAGACAG G G Т СAG CGTCACCTGCAGGGCCAGTCAGAATGTGGGTCCCAATTTAGCCTGGTATCAACAGAAACCAGGG CAATCTCCTAAAGCACTGATTTACTCGGCATCCTACCGATTCAGTGGAGTCCCTGATCGCTTCA CAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACTTGGC AGAGTATTTCTGTCAGCAATATAACAGGTATCCATTCACGTTCGGCTCGGGGACAAAGTTGGAA ATAAAACGG

SEQ ID NO:244: DNA encoding 2F5 light chain variable region (parent)

GACAT T G T GAT GAC C CAG TOT CΑΑΑΑΑΤ T CAT G T C CACAT CAG TAG GAGACAG G G T CAG

- 143 040888

CATCACCTGCAAGGCCAGTCAGAATGTGGGTACTGCTGTAGCCTGGTATCAACAGAAACCAGGA CAATCTCCTAAACTACTGATTTCCTCGGCATCCAATCGGTACACTGGAGTCCCCTGATCGCTTCA CAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGTAATATGCAGTCTGAAGACGTGGC AGATTTTCTGGACCAGCAATATAGCTGTTCGGACTGACCG

SEQ ID NO:245: DNA encoding light chain variable region 1B11 (parental)

GAG АТ Т G Т GAT GAG С С AG Т С Т С АААААТ Т С AT G Т С САС Т Т С AG TAG GAGAC AG G G Т С AG CGTCACCTGCAAGGCCAGTCAGAATGTGGGTCCTAATGTAGCCTGGTATCAACAGAAACCAGGG CAATCTCCTAAAGCACTGATTTACTCGGCATCCTACCGGTACAGTGGAGTCCCTGATCGCTTCA CAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACTTGGC AGACTATTTCTGTCAGCAATATAACCGCTATCCTCTCACGTTCGGTGCTGGGACCAAACTGGAG CTGAAACGG

SEQ ID NO:246: DNA encoding 2F2 light chain variable region (parent)

GACАТ Т G Т GAT GAC С СAG Т С Т САААААТ Т СAT G Т С САСАТ СAG TAG GAGACAG G G Т СAA CGTCACCTGCAAGGCCAGTCAGAATGTGGGTACTCATGTAGCCTGGTATCAACAGAAACCAGGG CAATCTCCTAAAGCACTGATTTACTCGGCATCCTACCGGTACAGTGGCGTCCCTGATCGCTTCA CAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACCTGGC AGAGTATTTCTGTCAGCAATATAACAGCTATCCTCGAGCGCTCACGTTCGGTGCTGGGACCAAG CTGGAGCTGAAACGG

SEQ ID NO:247: DNA encoding 11B6 light chain variable region (parental)

GACАТ Т G Т GAT GAC С СAG Т С Т САААААТ Т СAT G Т С САСАТ СAG TAG GAGACAG G G Т СAA CGTCACCTGCAAGGCCAGTCAGAATGTGGGTCCTACTGTAGCCTGGTATCAACAGAAACCAGGG CAATCTCCTAAAGCACTAATTTACTCGGCATCCTACCGGTACAGTGGAGTCCCTGATCGCTTCA CAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCACTCTGAAGACTTGGC AGAGTATTTCTGTCAGCAATATAACAGCTATCCATTCACGTTCGGCTCGGGGACAAAGTTGGAA ATAAAACGG

SEQ ID NO:310: Human IgG4 constant region

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLF PPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLT VLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNH YTQKSLSLSLGK

- 144 040888

SEQ ID NO:311: Human IgG4 constant region with S228P mutation

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLF PPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLT VLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNH YTQKSLSLSLGK

SEQ ID NO:312: Human IgG4 constant region with the S228P mutation, as well as a mutation (Xtend) that promotes interactions with FcRn at low pH

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLF PPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLT VLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHSH YTQKSLSLSLGK

SEQ ID NO:313: Human IgK constant region

TVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD

SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Example 16

This example describes the functional characterization of recombinant purified high affinity MASP-3 inhibitory antibodies in several in vitro assays.

Methods.

Recombinant anti-MASP-3 mAbs prepared as described in Examples 11 and 14 were characterized for (i) binding to human MASP-3 and MASP-3 of other species; (ii) the ability to inhibit degradation of the artificial substrate; (iii) the ability to inhibit the cleavage of pro-factor D with the formation of factor D; (iv) inhibition of complement precipitation in human serum; and (v) inhibition of rabbit erythrocyte lysis in human serum, as described below.

1. Human and mouse MASP-3 binding assays.

ELISA assays.

MASP-3 binding assay with purified recombinant anti-MASP-3 mAbs.

Human MASP-3.

A sandwich ELISA was performed to evaluate the binding of 16 purified recombinant anti-MASP-3 antibodies to human MASP-3 (CCP1-CCP2-SP fragment) as follows. The ELISA plate was coated with aM3-259 antibody at 4 μg/ml in carbonate/bicarbonate buffer overnight at 4°C. aM3-259 is a highly avid recombinant chimeric chick-human anti-MASP-3 mAb produced in chickens immunized with the CCP1-CCP2-SP region of human MASP-3. Domain mapping studies have shown that aM3-259 binds to the CCP1-CCP2 region of MASP-3 from a variety of species including humans, cynomolgus monkeys, mice, rats and dogs. As shown in FIG. 51C, aM3-259 also binds to MASP-1.

The plate was then blocked with 1% BSA/PBS, washed in PBS and incubated for one hour at room temperature with MASP-3 CCP1-CCP2-SP (2 μg/ml). The plate was then washed (PBS-T, 0.05%) and anti-MASP-3 candidate antibodies were added, followed by incubation for one hour at room temperature. The plate was washed (PBS-T, 0.05%) and detection antibody (mouse anti-human kappa chain HRP conjugated antibody, SouthernBiotech #9230-05) was added for one hour at room temperature. After another wash (PBS-T, 0.05%), the plate was developed (5 min) with the OPT EIA TMB reagent (BD Biosciences #555214). Optical density readings on the A450 were performed on a Spectramax M5e plate reader.

Results.

In FIG. 51A and 51B graphically illustrate the binding avidity of anti-MASP-3 mAbs (purified recombinant) to human MASP-3 (CCP1-CCP2-SP). As shown in FIG. 51A, in FIG. 51B and in table. 24, anti-MASP-3 mAbs have high binding avidity to human MASP-3, ie. ranging from 0.241 nM to 0.023 nM. These values are 10-100 times lower than those reported for previously described anti-MASP-3 mAbs (see Example 7 here, as well as Example 15 in WO 2013/192240).

- 145 040888

Binding specificity of anti-MASP-3 mAb.

To determine the binding specificity of high affinity anti-MASP-3 mAbs to MASP-3, binding experiments were performed to evaluate the binding of 16 purified recombinant anti-MASP-3 antibodies to human MASP-1 and to human MASP-2. Binding was determined as described in the MASP-3 binding ELISA, except that recombinant MASP-1A (S646A, CCP1-CCP2-SP fragment) and MASP-2 (CCP1-CCP2-SP fragment) were immobilized directly onto tablet.

Results.

In FIG. 51C graphically illustrates the results of a binding experiment where representative purified recombinant high affinity human MASP-3 inhibitory antibodies selectively bind to MASP-3 but do not bind to human MASP-1.

In FIG. 51D graphically illustrates the results of a binding experiment where representative purified recombinant high affinity human MASP-3 inhibitory antibodies selectively bind to MASP-3 but do not bind to human MASP-2.

Mouse MAS P-3.

Binding of the αanti-MASP-3 mAb to mouse MASP-3 was evaluated as described above for human MASP-3, except that recombinant full-length mouse MASP-3 (SEQ ID NO: 3) was captured on aM3-259 plate. The mAb that served as the negative control used in both experiments is mAb77, namely a recombinant chimeric chicken-human mAb produced in the same immunized chickens that produced aM3-259, however, mAb77 does not bind to mouse MASP -3.

Results.

In FIG. 52 graphically illustrates the avidity of representative anti-MASP-3 mAbs (purified recombinant) relative to mouse full-length MASP-3. As shown in FIG. 52, most of the anti-MASP-3 mAbs tested also had high binding avidity to mouse MASP-3.

Avidity values (EC ₅₀ ) of binding of 16 recombinant chimeric anti-MASP-3 mAbs to human and mouse MASP-3 are summarized in Table 1. 24.

Table 24 Avidity of binding of antu-MASP-3 mAb to human and mouse MASP-3 (FIGS. 51A, 51B and 52)

Antibody clone Antigen used For mAb Avidity of binding to human MASP-3 (CCP1-CCP2-SP) (EC ₅₀ nM) Binding avidity to mouse MASP-3 (full-length) (EC ₅₀ nM) 1А10* SP 0.241 0.15 1B11 SP 0.059 1, 10 1E7 SP 0.112 117.00 1F3 SP 0.236 0.111 1G4 SP 0.177 3.70 2D7 SP 0.122 NA 2F2 SP 0.057 0.105 2F5 SP 0.073 0.102 4B6 SP 0.211 0.188 4D5 SP 0.058 0.098 10D12 CCP1-CCP2-SP 0.089 0.081 11B6 CCP1-CCP2-SP 0.060 0.066 13B1 CCP1-CCP2-SP 0.059 0.035 15D9 CCP1-CCP2-SP 0.074 0.092 35С1 CCP1-CCP2-SP 0.091 0.209 49C11 CCP1-CCP2-SP 0.069 0.064

Three anti-MASP-3 mAbs, 13B1, 10D12 and 4D5, were also tested for binding to recombinant MASP-3 cynomolgus monkeys, dogs and rats. These results are summarized in Table 1 below. 25.

- 146 040888

Table 25 Systematized description of anti-MASP-3 mAb cross-linking experiments

Origin of MASP-3 Fab Link Ranking Human 13B1 (pM) « 10D12 (pM) « 4D5 (pM) dog-like monkey 13B1 (pM) " 4D5 (pM) > 10D12 (pM) Dog 13B1 (pm) > 10D12 (pM) » 4D5 (nM) Rat 13B1 (pM) " 10D12 (pM) » 4D5 (nM) Mouse 10D12 (pM) 2 13B1 (pM) » 4D5 (nM)

As shown in Table. 25, anti-MASP-3 mAbs 13B1, 10D12, and 4D5 bind to test MASP-3s from all five species (human, mice, rats, dogs, and cynomolgus monkeys). These mAbs bind to human MASP-3 with high avidity (<500 pM), but they bind to other MASP-3 species with different avidities.

2. Fluorogenic analysis for cleavage of tripeptides.

Background/Rationale.

MASP-3, in addition to its known natural substrates (Iwaki et al., J. Immunol. 187:3751, 2011; Cortesio and Jiang, Arch. Biochem. Biophys. tripeptide substrates (Cortesio and Jiang, ibid.). These molecules, as well as very small substrates, can be used to map the protease catalytic site. Inhibition of tripeptide cleavage is an indication that an inhibitory agent, such as an antibody, either directly blocks access of a small substrate to the catalytic site, or initiates a conformational shift in the SP domain that similarly blocks such access. For example, it is assumed that such an antibody can also block the catalysis of large natural substrates due to a negative effect on the active site of the enzyme. In its function, it most closely matches the antibody in mice lacking MASP-3 or in 3MC patients (deficient in MASP-3).

Methods.

Titrated recombinant mAbs (3-fold dilutions from 666 nM to 0.91 nM) were incubated with MASP-3 CCP1-CCP2-SP (197 nM) for 15 min at room temperature. Tripeptide substrate BOC-V-P-R-AMC (tert-butyloxycarbonyl-Val-Pro-Arg-7-amino-4-methylcoumarin) (R&D Systems, Cat. No. ES011) was added at a final concentration of 0.2 mM. Hydrolysis of the Arg-AMC amide bond promoted the release of AMS, i.e. in a highly fluorescent group. Kinetic values for excitation at 380 nm/emission at 460 nm were recorded every 5 min at 37° C. for 70 min on a Spectramax M5e fluorescent plate reader.

Results.

In FIG. 53 graphically illustrates the results of an assay for inhibition of MASP-3 dependent cleavage of a fluorogenic tripeptide by anti-MASP-3 monoclonal antibodies. As shown in FIG. 53, the anti-MASP-3 mAbs tested were assigned to three different groups.

1. Ahtu-MASP-3 mAb, which are strong inhibitors of peptide cleavage by MASP-3: 1A10 (29.77 nM), 1G4 (29.64 nM), 1F3 (32.99 nM), 4B6 (26.03 nM), 4D5 (27.54 nM), 10D12 (30.94 nM) and 13B1 (30.13 nM).

2. Ahtu-MASP-3 mAbs that are weak or very weak inhibitors of peptide cleavage by MASP-3: 15D9, 11B6, 2F5, 1E7 and 2D7.

3. Ahtu-MASP-3 mAbs that are neutral or can stimulate peptide cleavage by MASP-3: 1B11; 2F2; 77 (control mAb).

3. Inhibition of pro-factor D cleavage to form factor D.

Methods.

Active recombinant human MASP-3 protein (240 ng per reaction) was pre-incubated with representative anti-MASP-3 mAbs and control mAbs (which bind to MASP-1 but not to MASP-3) in GVB ⁺⁺ buffer at full volume of 9 μl at room temperature for 15 minutes. Then, 70 ng of Streptag II N-terminal epitope-tagged pro-factor D (ST-pro-factor D-His) was added to each tube to a final volume of 10 μl per reaction. The reaction mixtures were incubated in a thermowell at 37°C for 6 hours. After that, one tenth of each reaction mixture was subjected to electrophoresis in 12% bis-Tris gel to release pro-factor D and obtain an active factor D cleavage product. The obtained proteins were transferred to The PVDF membrane was analyzed by western blot analysis by detection using a biotinylated anti-factor D antibody (R&D Systems).

Results.

In FIG. 54 illustrates Western blot analysis indicating the ability of representative anti-MASP-3 mAbs to block pro-CFD cleavage mediated by recombinant MASP-3 with

- 147 040888 CFD formation in an in vitro assay. As shown in FIG. 54, representative high affinity MASP-3 inhibitory mAbs 13B1, 4B6, 1G4, 2D7, 10D12, 1A10, 4D5, 1E7, 1F3 and mouse-human chimeric mAbs were found to be unable to completely inhibit pro-CFD cleavage in this assay.

4. Factor Bb precipitation analysis on zymosan.

Methods.

Ahtu-MASP-3 mAb at various concentrations was added to 10% CFD-deficient human serum (Complement Technology A336) and GVB+Mg/EGTA (20 nM) and incubated for 30 min on ice, and then recombinant ST-pro- factor D-His (final volume 2 μg/ml) and zymosan (final volume 0.1 mg/ml). The zymosan particles function as an activating surface for complement deposition. The mixture was incubated at 37° C. and then APC activity was measured by detection of complement factor Bb on a flow cytometer (using Quidel A252 antibody) on the surface of the zymosan particles.

Results.

In FIG. 55A graphically illustrates the level of factor Bb precipitation on zymosan particles (determined by flow cytometric detection in MFI units) in the presence of various concentrations of anti-MASP-3 mAb 1F3, 1G4, 2D7, and 4B6 in factor D deficient human serum at 37° C. for 70 min.

In FIG. 55B graphically illustrates the level of factor Bb precipitation on zymosan particles (determined by flow cytometric detection in MFI units) in the presence of various concentrations of anti-MASP-3 mAbs 4D5, 10D12 and 13B1 in CFD-deficient human serum at 37°C for 70 minutes.

The results shown in FIG. 55A and 55B are summarized in Table 1 below. 26.

Table 26 Inhibition of factor Bb precipitation on zymosan by anti-MASP-3 mAb (FIGS. 55A and 55B)

As shown in FIG. 55A, in FIG. 55B and in table. 26, anti-MASP-3 mAbs show strong inhibition of APC in human serum with IC ₅₀ values from 0.1 to 3.5 nM. These results demonstrated that MASP-3 plays a key role in the activation of APC in an in vitro model in human serum and also demonstrated that MASP-3 inhibitory antibodies are potent inhibitors of APC.

5. Assay for the ability of representative anti-MASP-3 mAbs to inhibit rabbit erythrocyte lysis.

Methods.

To monitor APC inhibition in another experiment, we evaluated the ability of representative anti-MASP-3 mAbs to block rabbit erythrocyte lysis in human serum. Anti-MASP-3 mAb at various concentrations was added to 10% factor D-deficient human serum and GVB+Mg/EGTA (20 nM) and then incubated for 30 min on ice, after which recombinant ST-pro was added. -factor B-His (final volume 2 μg/ml) and erythrocytes (final volume 2.5 x 10 ⁸ cells/ml). The mixtures were incubated at 37°C for 70 min and the degree of APC-mediated hemolysis was assessed by diluting the reaction mixtures and measuring the optical density (A405), which indicates the levels of free hemoglobin.

Results.

In FIG. 56A graphically illustrates the level of inhibition of rabbit erythrocyte lysis in the presence of various concentrations of anti-MASP-3 mAb 1A10, 1F3, 4B6, 4D5, 1G4, and 2F2 in CFD-deficient human serum. In FIG. 56B graphically illustrates the level of inhibition of rabbit erythrocyte lysis in the presence of various concentrations of anti-MASP-3 mAb 1B11, 1E7, 1G4, 2D7, and 2F5 in CFD-deficient human serum. The results are systematized in table. 27.

- 148 040888

Table 27 Inhibition of rabbit erythrocyte lysis by anti-MASP-3 mAb (FIGS. 56A and 56B)

As shown in FIG. 56A, 56B and in the table. 27, anti-MASP-3 mAbs show inhibition of APC-induced hemolysis of rabbit erythrocytes with IC ₅₀ values from 0.1 to 5.4 nM. These results are consistent with the observations of anti-MASP-3 antibodies in the zymosan assay and also indicate that MASP-3 inhibitory antibodies are potent inhibitors of APC.

6. Inhibition of pro-factor D cleavage in the serum of a 3MS patient.

Methods.

A representative recombinant anti-MASP-3 mAb (4D5) was tested for its ability to block pro-factor D cleavage (and activation) by MASP-3 in normal human serum and in the serum of 3MC patient B (Pat B), i.e. . an individual in which MASP-3 is not detected in serum and who has an APC deficiency.

Healthy human serum and Patient B serum (final volume 10%) and GVB+Mg/EGTA (30 nM) were incubated in the absence of the enzyme or in the presence of active recombinant MASP-3 (rMASP-3; 0.5 μg/ml), inactive rMASP-3 or active rMASP-3 plus anti-MASP-3 mAb 4D5 (final concentration 500 nM) on ice for 1 hour. Then added zymosan (final concentration of 0.1 mg/ml) and the mixture was incubated at 37°C. After 2 h, the samples were centrifuged and the supernatants were collected. Samples were immunoprecipitated with goat anti-human factor D (R&D Systems AF1824), thermodenaturated, and treated with peptide-N-glycosidase (New England Biolabs P0704L). Captured and deglycosylated proteins were separated by SDS-PAGE electrophoresis and gels electroblotted in Western blot analysis using a biotinylated anti-CFD antibody (R&D Systems BAF1824) and highly sensitive streptavidin-HRP (Thermo Fischer Scientific 21130).

Results.

In FIG. 57 illustrates Western blot analysis to determine the level of pro-factor D and factor D in the serum of 3MC patient B in the presence of active rMASP-3, inactive rMASP-3 and active rMASP-3 plus mAb 4D5. As shown in FIG. 57, normal human serum contains predominantly the mature form, while Patient B's serum contains mainly factor D in the zymogen form. As shown also in FIG. 57, active rMASP-3 in the presence of zymosan cleaves profactor D in the serum of Patient 3, while the inactive (zymogenic) form of MASP-3 does not have this function. Finally, as shown in FIG. 57, anti-MASP-3 mAb 4D5 blocks pro-factor D cleavage in the serum of Patient 3 in the presence of active rMASP-3. These results also indicate a role for MASP-3 in pro factor D cleavage and APC activation and that the MASP-3 inhibitory mAb is able to block MASP-3 mediated pro factor D cleavage and block APC.

Example 17 Analysis of representative MASP-3 inhibitory mAbs 10D12 and 13B1 for their ability to inhibit APC in vivo.

1. Inhibition of APC by mAb M3-1 (13B1) and 10D12 in vivo.

Methods.

To determine the effectiveness of anti-MASP-3 mAb (M3-1) and 10D12 in inhibiting APC in vivo, a group of mice (n=4) received a single intravenous injection of 10 mg/kg mAb 13B1 into the tail vein, and a second group of mice (n= 4) injected into the tail vein one intravenous injection of 10 mg/kg mAb 10D12. Animal blood was used to obtain serum and to create a matrix for assessing APC activity by flow cytometry in an ex vivo assay that measures the level of C3 (also C3b and iC3b, Dako F020102-2) precipitation on zymosan particles. Serum obtained from blood taken before dosing and some time after dosing (at 96 h, 1 and 2 weeks) was diluted to 7.5% and zymosan particles (at a final concentration of 0.1 mg/ml) were added to in - 149 040888 APC induction. Mice treated with the antibody were compared with a group of control mice (n=4) that received a single intravenous dose of vehicle.

Results.

In FIG. 58 graphically illustrates the level of C3 deposition on zymosan particles at various time points following a single dose of M3-1 (13B1) mAb, 10D12 mAb, or vehicle in wild-type mice. As shown in FIG. 58, prior to dosing, comparable levels of APC activity were observed in the three cases.

At 96 hours and two further time intervals, the mAb-treated group showed almost complete inhibition of systemic APC activity, while the APC activity of the vehicle-treated group remained at the same level.

These results demonstrate that anti-MASP-3 mAb M3-1 (13B1) and mAb 10D12 are potent inhibitors of APC in mice in vivo.

2. Factor B status in mice treated with anti-MASP-3 mAb 10D12.

Methods.

During the conversion of the factor B zymogen into an active proteolytic enzyme, factor B was cleaved by factor D to form Ba (~30 kDa) and Bb (~60 kDa) fragments. The status of the Ba fragment in mouse sera taken from mice treated with anti-MASP-3 mAb 10D12 was determined as follows.

Mice (n=4) received two injections of 10 mg/kg mAb 10D12 via the tail vein. The treatment was carried out after 7 days, and then three days after the second injection, the animals were bled. The second group of four mice received a single intravenous dose of vehicle (PBS). Blood taken from both groups was used to obtain serum as a matrix for complement activation. Zymosan particles (final concentration 0.1 mg/ml) were added to diluted serum (final dilution 7.5%) and incubated for 35 min at 37°C.

Results.

To evaluate APC activation in FIG. 59 illustrates Western blot analysis for Ba fragment status in mouse sera from mice treated with mAb 10D12 or PBS and stimulated with zymosan. In FIG. 59, each lane corresponds to each individual mouse, and the other lanes correspond to sera from a representative vehicle-treated mouse and an anti-MASP-3 mAb-treated mouse used for comparison. Tracks 1 and 2 show two control cases, i.e. mice treated with vehicle or mAb 10D12, respectively (when viewed from the left side of the blot), as examples of representative basal levels of Ba present in serum samples in the absence of zymosan. All lanes 3-10 correspond to the level of the Ba fragment present after incubation with zymosan. In all cases, mice treated with anti-MASP-3 mAb showed reduced levels of the Ba fragment compared to levels in vehicle-treated animals.

3. Serum from mice treated with mAb 10D12 inhibits hemolysis.

Methods.

To further assess the inhibition of APC by MASP-3 inhibitory antibodies, the authors of the present invention analyzed the ability of anti-MASP-3 antibodies to block the lysis of rabbit erythrocytes in the sera of mice treated with a representative anti-MASP-3 mAb 10D12 compared with the sera of control mice, processed by the carrier.

Mice (n=4/group) received three injections of vehicle control (PBS), 10 mg/kg anti-MASP-3 mAb 10D12 or 25 mg/kg anti-MASP-3 mAb 10D12, via the tail vein. The treatment was carried out at intervals of seven days, and three days after the third injection, the animals were bled. Blood was used to obtain serum as a matrix for hemolysis reactions. To a 20% serum pool taken from four mice, erythrocytes (in a final volume of 2.5 x 10 ⁸ cells/ml) were added in GVB+Mg/EGTA (20 nM). The mixtures were incubated at 37°C and the degree of APC-mediated hemolysis was assessed by diluting the reaction mixtures and measuring the optical density (A405).

Results.

In FIG. 60 graphically illustrates the level of inhibition of hemolysis by 20% sera from mice treated with anti-MASP-3 mAb 10D12 (10 mg/kg or 25 mg/kg) or vehicle control. As shown in FIG. 60, sera from mice treated with anti-MASP-3 mAb 10D12 at 10 mg/kg and 25 mg/kg showed less total hemolysis during 1 hour testing compared to vehicle-treated mice.

General conclusions based on the results.

As described in this example, representative high affinity MASP-3 inhibitory mAbs 13B1 and 10D12 inhibit APC in vivo. As described in Example 12, the anti-MASP-3 monoclonal antibody 13B1 (also referred to as mAb M3-1) was determined to provide a clear advantage in terms of viability of Crry-free erythrocytes in a mouse model of paroxysmal nocturnal hemoglobinuria (PNH). As described in Example 13, the anti-MASP-3 mAb M3-1 was determined to reduce arthritis incidence and severity scores in a dose-dependent manner.

- 150 040888

Example 18.

This example describes the results of an epitope binding assay with high potency MASP3 inhibitory mAbs.

1. Analysis for competitive binding.

Methods.

96-well ELISA plates were coated with the capture antibody, aM3-259, an IgG4 isotype mAb that has been shown to bind to the CCP1-CCP2 region of MASP-1 and MASP-3. Full length human MASP-3 protein was immobilized on the plate with the aM3-259 capture antibody. In uncoated single wells, a 2-fold dilution of a batch of test anti-MASP-3 mAb isotype IgG4 was mixed with another test anti-MASP-3 antibody isotype IgG1 at the same concentration. This mixture was added to the coated wells and allowed to bind to the captured MASP-3. Potential competitive binding of the two antibodies was determined by detecting the IgG1 isoform using an anti-human IgG1 hinge-HP-conjugated antibody (Southern Biotech 9052-05) and using a series of TMB substrate reagents (BD Biosciences 555214).

Results.

In FIG. 61A-61E are graphical illustrations of competition binding assay results.

In FIG. 61A graphically illustrates the results of a competitive binding assay to identify anti-MASP-3 mAbs (IgG4) that block the interaction between mAb 4D5 (IgG1) and human MASP-3.

In FIG. 61B graphically illustrates the results of a competitive binding assay to identify anti-MASP-3 mAbs (IgG4) that block the interaction between mAb 10D12 (IgG1) and human MASP-3.

In FIG. 61C graphically illustrates the results of a competitive binding assay to identify anti-MASP-3 mAbs (IgG4) that block the interaction between 13B1 mAb (IgG1) and human MASP-3.

In FIG. 61D graphically illustrates the results of a competitive binding assay to identify anti-MASP-3 mAbs (IgG4) that block the interaction between 1F3 mAb (IgG1) and human MASP-3.

In FIG. 61E graphically illustrates the results of a competitive binding assay to identify anti-MASP-3 mAbs (IgG4) that block the interaction between 1G4 mAb (IgG1) and human MASP-3.

The data in FIG. 61A-61E are summarized in Table 1 below. 28.

These data indicated that the anti-MASP-3 mAbs 4D5, 10D12, 13B1, 1A10, 1F3 and 1G4 share a common epitope or an overlapping epitope on human MASP-3. Surprisingly, it was found that 1G4 has a very limited ability to block the binding of the other five mAbs to MASP-3, but these mAbs almost completely block the binding of 1G4 to MASP-3.

. Assay for mAb binding to peptides representing linear and discontinuous MASP-3 epitopes.

Methods.

of 16 anti-MASP-3 mAbs were evaluated using the Pepscan assay to identify the MASP-3 regions they bind to. To reconstruct the linear and potential discontinuous epitopes of the target molecule, a library of peptides corresponding to amino acid residues 299-728 of SEQ ID NO: 2 (human MASP-3) was synthesized. Amino acid residues 1-298 of MASP-3 were not present in the immunogen and were not included in this analysis.

Pepscan epitope analysis involves the use of CLIPS technology, which locks peptides into defined three-dimensional structures (see Timmerman et al., J. Mol. Recog. 20:283-299, 2007 and Langedijk et al., Analytical Biochemistry 417:149-155, 2011). The binding of each antibody to each synthesized peptide was tested in a Pepscan-based ELISA.

Results.

The results of binding of peptides in the analysis of Pepscan with each analyzed antibody are described below and systematized in table. 4, tab. 28 and in FIG. 62-67.

Antibodies 1F3, 4B6, 4D5 and 1A10 (Groups IA).

When tested under conditions of moderate stringency, 1F3, 4B6, 4D5, and 1A10 antibodies bound to discontinuous epitope mimetics as well as simple conformational and linear mimetics. Analysis of the data showed that all antibodies 1F3, 4B6, 4D5 and 1A10 preferentially recognized the peptide fragment ₄₉₈ VLRSQRRDTTVI ₅₀₉ (SEQ ID NO: 9) of MASP-3. This peptide is in close proximity to the histidine active site of H497. The data obtained for these antibodies that bind to discontinuous mimetics suggest that peptide fragments ₅₄₄ DFNIQNYNHDIALVQ ₅ 5 ₈ (SEQ ID NO: 1 1), ₆₃₉ GNYSVTENMFC ₆₄₉ (SEQ ID NO: 13) and ₇₀₄ VSNYVDWVWE ₇₁₃ (SEQ ID NO : 14) MASP-3 is also involved in binding. Peptide

- 151 040888 ₅₄₄ DFNIQNYNHDIALVQ ₅₅₈ (SEQ ID NO: 11) contains aspartate in the active site (D553).

Antibody 10D12 (Group IB).

When tested under conditions of moderate stringency, the 10D12 antibody bound to peptides having the core sequence ₄₉₈ VLRSQRRDTTVI ₅₀₉ (SEQ ID NO: 9) of MASP-3, where said sequence is located in the vicinity of the active site histidine, H497.

Antibody 13B1 (Group IC).

When tested under conditions of moderate stringency, the 13B1 antibody recognized a discontinuous epitope containing peptide fragments ₄₉₄ TAAHVLRSQRRDTTV ₅₀₈ (SEQ ID NO: 10) and 626PHAECKTSYESRS638 (SEQ ID NO: 12) of MASP-3, where the peptide fragment 626PHAECKTSYESRS638 (SEQ ID NO: 12), is obviously the dominant part of the epitope since it can also bind in a simple conformational form. Peptide ₄₉₄ TAAHVLRSQRRDTTV ₅₀₈ (SEQ ID NO: 10) includes the active site histidine, H497.

Antibody 1G4 (Group II).

When tested under low stringency conditions, the 1G4 antibody recognized a discontinuous epitope containing peptide fragments ₄₅₄ RNAEPGLFPWQ ₄₆₄ (SEQ ID nO: 17), ₅₁₄ EHVTVYLGLH ₅₂₃ (SEQ ID NO: 19) and ₆₆₇ AFVIFDDLSQRW ₆₇₈ (SEQ ID NO: 23) MASP- 3, where the peptide fragment ₆₆₇ AFVIFDLSQRW ₆₇₈ (SEQ ID NO: 23) is the dominant part of the epitope. This dominant peptide is in the three amino acid region of the active site with serine, S664.

Antibodies 1E7 and 2D7 (Group IIIA).

When tested under conditions of high and low stringency, respectively, antibodies 1E7 and 2D7 recognized a discontinuous epitope containing the peptide fragments 454RNAEPGLFPWQ464 (SEQ ID NO: 17), 514EHVTVYLGLH523 (SEQ ID NO: 19) and 667AFVIFDDLSQRW678 (SEQ ID NO: 23) MASP- 3, where the peptide fragment ₆₆₇ AFVIFDLSQRW ₆₇₈ (SEQ ID NO: 23) is the dominant part of the epitope and is in the three amino acid region of the active site with serine, S664.

Antibodies 2F5 and 15D9 (Group IIIB).

When tested under low stringency conditions, 2F5 and 15D9 antibodies dominantly recognized a discontinuous epitope containing peptide fragments ₄₅₄ RNAEPGLFPWQ ₄₆₄ (SEQ ID NO: 17), ₄₇₉ KWFGSGALLSASWIL ₄₉₃ (SEQ ID NO: 18), ₅₆₂ PVPLGPHVMP ₅₇₁ (SEQ ID NO: 20) and ₆₆₇ AFVIFDDLSQRW ₆₇₈ (SEQ ID NO: 23) MASP-3. Peptides ₄₇₉ KWFGSGALLSASWIL ₄₉₃ (SEQ ID NO: 18) and ₆₆₇ AFVIFDLSQRW ₆₇₈ (SEQ ID NO: 23) are localized to four or three amino acid residues of the active site of H497 and S664, respectively.

Antibody 1B11 (Group IIIC).

When tested under conditions of moderate stringency, antibody 1B11 recognized a discontinuous epitope containing the peptide fragments 435ECGQPSRSLPSLV447 (SEQ ID NO: 16), 454RNAEPGLFPWQ464 (SEQ ID NO: 17), 583APHMLGL589 (SEQ ID NO: 21), and 6 _M SDVLQYVKLP623 (SEQ ID NO: 21). : 22) MASP-3.

- 152 040888

Table 28. Organized epitope binding assay data

Ahth-MASP-3mAh per. No./Group Peptide-binding fragments (epitopes) on human MASP-3 (with leader sequence) Compete with antibodies Peptide digestion assay 4D5 Groups ΙΑ 49 ₈ VLRSQRRDTTVI ₅ o ₉ (SIN: 9) 54 ₄ DFNIQNYNHDIALVQ55 ₈ (SIN:11) ₆₃₉ GNYSVTENMFC ₆ 4 ₉ ( SIN : 13 ) ₇ o4VSNYVDWVWE ₇₁₃ (SIN: 14) 1F3, 1G4, 4D5, 10D12, 13B1 inhibits 1F3 Groups ΙΑ 49 ₈ VLRSQRRDTTVI ₅₀ 9 (SIN: 9) 54 ₄ DFNIQNYNHDIALVQ55 ₈ (SIN:11) ₆₃₉ GNYSVTENMFC ₆ 49 ( SIN : 13 ) ₇ 04VSNYVDWVWE ₇₁₃ (SIN: 14) 1F3, 1G4, 4D5, 10D12, 13B1 inhibits 4B6 Groups ΙΑ 49 ₈ VLRSQRRDTTVI ₅₀ 9 (SIN: 9) 544DFNIQNYNHDIALVQ ₅₅₈ (SIN:11) ₆₃₉ GNYSVTENMFC ₆ 49 ( SIN : 13 ) ₇ 04VSNYVDWVWE ₇₁₃ (SIN: 14) 1F3, 1G4, 4D5, 10D12, 13B1 inhibits 1A10 Groups ΙΑ 49 ₈ VLRSQRRDTTVI ₅₀ 9 (SIN: 9) 544DFNIQNYNHDIALVQ ₅₅₈ (SIN:11) ₆₃₉ GNYSVTENMFC ₆ 49 ( SIN : 13 ) ₇ 04VSNYVDWVWE ₇₁₃ (SIN: 14) 1F3, 1G4, 4D5, 10D12, 13B1 inhibits 10D12 IB groups 49 ₈ VLRSQRRDTTVI ₅₀ 9 (SIN: 9) 1F3, 1G4, 4D5, 10D12, 13B1 inhibits 13B1 IC groups 494TAAHVLRSQRRDTTV ₅₀₈ (SIN:10) ₆₂₆ PHAECKTSYESRS ₆₃₈ (SIN:12) 1F3, 1G4, 4D5, 10D12, 13B1 inhibits Cow sequence b Group I ₄₉₈ VLRSQRRDTTV ₅₀₈ (SIN: 15) 1G4 Group II competes for cross-linking with 454RNAEPGLFPWQ464 (SIN: 17) ₅₁ 4EHVTVYLGLH ₅₂₃ (SIN: 19) ₆₆₇ AFVIFDDLSQRW ₆₇₈ (SIN: 23) 1F3, 1G4, 4D5, 10D12, 13B1 inhibits

- 153 040888

Groups I and III 1E7 Groups IIΙΑ 454RNAEPGLFPWQ ₄₆ 4 (SIN: 17) ₅₁ 4EHVTVYLGLH ₅₂ 3 (SIN: 19) ₆₆₇ AFVIFDDLSQRW ₆₇₈ (SIN: 23) 1G4 Weakly inhibits 2D7 Groups IIΙΑ 454RNAEPGLFPWQ464 (SIN: 17) ₅₁₄ EHVTVYLGLH523 (SIN: 19) ₆₆₇ AFVIFDDLSQRW ₆₇₈ (SIN: 23) Weakly inhibits 2F5 Group IIIB 454RNAEPGLFPWQ ₄₆ 4 (SIN: 17) 4 ₇₉ KWFGSGALLSASWIL493 (SIN 18) 5 ₆₂ PVPLGPHVMP ₅₇₁ (SIN: 20) ₆₆₇ AFVIFDDLSQRW ₆₇₈ (SIN :23) No effect 15D9 Group IIIB 454RNAEPGLFPWQ ₄₆ 4 (SIN: 17) 4 ₇₉ KWFGSGALLSASWIL493 (SIN 18) 5 ₆₂ PVPLGPHVMP ₅₇₁ (SIN: 20) ₆₆₇ AFVIFDDLSQRW ₆₇₈ (SIN :23) No effect 1B11 IIIC groups 43 ₅ ECGQPSRSLPSLV44 ₇ (SIN: 16) 454RNAEPGLFPWQ ₄₆ 4 (SIN: 17) 5 ₈₃ APHMLGL ₅₈ 9 (SIN: 21) ₆₁ 4SDVLQYVKLP ₆₂ 3 (SIN: 22) stimulates Cow sequence b Group II and Group III 454RNAEPGLFPWQ464 (SIN: 17) 2F2 Group IV Binding to an epitope determined 1F3, 4D5, 11В6, 2F2 stimulates 11B6 Group IV Binding to an epitope determined 1F3, 4D5, 11В6, 2F2 No effect

In FIG. 62 is a schematic diagram illustrating the contact areas of human MASP-3 with anti-MASP-3 mAb determined using the Pepscan assay. As shown in FIG. 62, all anti-MASP-3 mAbs have contact regions in the beta chain containing the MASP-3 SP domain. One mAb, 1B11, also has a contact region between CCP2 and SP domains in the MASP-3 alpha chain.

In FIG. 63A-67 show 3-E models illustrating contact regions of high-affinity anti-MASP-3 mAbs on the CCP1/2/SP domains of human MASP-3, where the active site of the MASP-3 SP domain is in front and the catalytic triad is shown as side chains. .

In FIG. 63A shows the contact regions of human MASP-3 with high affinity anti-MASP-3 mAbs 1F3, 4D5 and 1A10, including amino acid residues 498-509 (SEQ ID NO: 9), amino acid residues 544-558 (SEQ ID NO: 11), amino acid residues 639-649 (SEQ ID NO: 13) and amino acid residues 704-713 (SEQ ID NO: 14).

In FIG. 63B shows the contact regions of human MASP-3 with the high affinity antu-MASP-3 mAb 10D12 comprising amino acid residues 498-509 (SEQ ID NO: 9).

In FIG. 64 shows contact regions of human MASP-3 with high affinity anti-MASP-3 mAb 13B1 comprising amino acid residues 494-508 (SEQ ID NO: 10) and amino acid residues 626-638 (SEQ ID NO: 12).

In FIG. 65 shows contact regions of human MASP-3 with high affinity antu-MASP-3 mAb 1B11 comprising amino acid residues 435-447 (SEQ ID NO: 16), amino acid residues 454-464 (SEQ ID NO: 17), amino acid residues 583-589 (SEQ ID NO: 21) and amino acid residues 614-623 (SEQ ID NO: 22).

In FIG. 66 shows the contact regions of human MASP-3 with high affinity antu-MASP-3 mAbs 1E7, 1G4 and 2D7, including amino acid residues 454-464 (SEQ ID NO: 17), amino acid residues 514-523 (SEQ ID NO: 19) and amino acid residues 667-678 (SEQ ID NO: 23).

In FIG. 67 shows the contact regions of human MASP-3 with high affinity an-154 040888 mu-MASP-3 mAbs 15D9 and 2F5, including amino acid residues 454-464 (SEQ ID NO: 17), amino acid residues 479-493 (SEQ ID NO: 18) , amino acid residues 562-571 (SEQ ID NO: 20) and amino acid residues 667-678 (SEQ ID NO: 23).

Overall, final binding profiles were evaluated for 12 out of 14 antibodies. All 12 antibodies mapped recognized solvent-accessible epitopes in the peptidase S1 domain. The close proximity of a number of determinant epitopes to residues of the catalytic active site triad (H497, D553, S664) is consistent with a model in which high-affinity MASP-3 inhibitory mAbs block enzymatic activity by inhibiting enzyme-substrate interaction.

Example 19.

This example describes the humanization of representative anti-MASP-3 mAbs and the construction of potential sites for post-translational modification.

Methods.

1. Humanization of representative high affinity anti-MASP-3 mAbs.

Methods.

To reduce the risk of immunogenicity, representative high affinity MASP-3 inhibitory antibodies 4D5, 10D12 and 13B1 were humanized by CDR attachment. The CDRs of each antu-MASP-3 antibody were fused to the most consensus human framework sequences. Some remnants of the Vernier zone were modified by Quickchange site-directed mutagenesis (Agilent Technologies). The resulting humanized VH and VL regions were transferred to pcDNA3.1 based human IgG1 or IgG4 and IgK expression constructs and the recombinant antibodies were expressed and purified as described above. The affinity of the humanized antibodies was determined by ELISA using monovalent Fab fragments, and activity was assessed by C3 precipitation assay using intact IgG4.

Results.

Amino acid sequences of representative humanized variants of the heavy chain and light chain variable regions of mAbs 4D5, 10D12 and 13B1 are shown below. CDR (by Kabat) underlined

4D5:

h4D5 VH-14 (SEQ ID NO:248)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTTDDINWVRQAPGQGLEWIGWIYPRDDRTK

YNDKFKDKATLTVDTSSNTAYMELSSLRSEDTAVYYCSSLEDTYWGOGTLVTVSS h4D5 VH-19 (SEQ ID NO:249)

OVOLVOSGAEVKKPGASVKVSCKASGYTFTTDDINWVROAPGOGLEWIGWIYPRDDRTK

- 155 040888

YNDKFKDRATLTVDTSSNTAYMELSSLRSEDTAVYYCSSLEDTYWGOGTLVTVSS h4D5 VL-l (SEQ ID NO:250) DIVMTOSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYOQKPGOPPKLLIYWAST

RESGVPDRFSGSGSGTDFTLTISSLOAEDVAVYYCKQSYNLYTFGOGTKVEIKR 10D12: hlOD12 VH-45 (SEQ ID NO:251)

QIQLVQSGSELKKPGASVKVSCKASGYIFTSYGMSWVRQAPGKGLKWMGWINTYSGVPT

YADDFKGRFVFSLDTSVRTPYLOISSLKAEDTAVYFCARGGEAMDYWGOGTLVTVSS hlOD12 VH-49 (SEQ ID NO:252)

QIQLVQSGSELKKPGASVKVSCKASGYIFTSYGMSWVRQAPGKGLKWMGWINTYSGVPT

YADDFKGRFVFSLDTSVRTPYLOISSLKAEDTATYFCARGGEAMDYWGOGTLVTVSS hlOD12 VL-21 (SEQ ID NO:253)

DVLMTQTPLSLSVTPGQPASISCKSSQSLLDSDGKTYLNWLLORPGOSPKRLIYLVSKL

DSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPWTFGOGTKVEIKR 13B1 h!3Bl VH-9 (SEQ ID NO:254) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGKWIEWVRQAPGQGLEWIGEILPGTGSTN

YAQKFQGRATFTADSSTSTAYMELSSLRSEDTAVYYCLRSEDVWGOGTLVTVSS hl3Bl VH-10 (SEQ ID NO:255) OVOLVOSGAEVKKPGASVKVSCKASGYTFTGKWIEWVRQAPGOGLEWIGEILPGTGSTN

YNEKFKGRATFTADSSTSTAYMELSSLRSEDTAVYYCLRSEDVWGOGTLVTVSS h!3Bl VL-l (SEQ ID NO:256) DIVMTOSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYOQKPGOPPKLLIYWAST

RESGVPDRFSGSGSGTDFTLTISSLOAEDVAVYYCKQSYNIPTFGOGTKVEIKR

The affinity of representative humanized antibodies 4D5, 10D12 and 13B1 against human MASP-3 is presented in table below. 29.

Table 29 Binding of representative humanized anti-MASP-3 mAbs to MASP-3

Anti-MABR-3 antibody clone (Fab format) Binding to human MASP-3, EC ₅₀ (hM) 4D5, parent Fab 0.107 h4D5_14-l Fab (VH-14 and VL-1) 0.085 h4D5_19-l Fab 0.079 (VH-19 and VL-1) 10D12, parent Fab 0.108 hlOD12_45-21 Fab (VH-45 and VL-21) 0.108 hlOD12_49-21 Fab (VH-49 and VL-21) 0.115 13B1 parent Fab 0.123 hl3Bl_9-l Fab (VH-9 and VL-1) 0.101 hl3Bl_10-l Fab (VH-10 and VL-1) 0.097

Percent Identity between Humanized Framework Sequences and Human Germline Framework Sequences:

h4D5_VH-14=90%; h4D5_VH-19=91%; h4D5_VL-l=100%;

h10D12_VH-45=92%; h10D12_VH-49=91%; h10D12_VL-21=93%;

- 156 040888 h13B1_VH-9=95%; h13B1_VH-10=94%; h13B1_VL-1=100%.

2. Mutagenesis of representative aHTU-MASP-3 mAbs to remove Asn/Asp modification sites in

Light chain variable region CDR-1 4D5, 10D12 and 13B1.

Representative high affinity MASP-3 inhibitory mAbs 4D5, 10D12 and 13B1 were analyzed for post-translational modification. Aspartic residues followed by glycine, serine, histidine, alanine or asparagine (NG, NS, NH, NA or NN motif) are often susceptible to hydrolysis of the amide group of the asparagine side chain or to deamidation. Aspartic acid residues followed by glycine or proline (DG or DP motif) are often susceptible to interconversion or isomerization. Such modifications change the charge and may affect the function of the antibody if they are present in the border binding region. They may also increase the risk of fragmentation, immunogenicity and aggregation.

Potential post-translational modification motifs have been identified in the CDR-1 light chain variable regions 4D5, 10D12 and 13B1.

4D5 and 13B1 contained one possible Asn deamidation site in the light chain CDR1 (shown as NS at positions 8 and 9 of SEQ ID NO: 142, which is underlined in Table 30 below). As also shown in Table 30 below, 10D12 contained one possible Asp isomerization site in the light chain CDR1.

Variants of these humanized anti-MASP-3 mAbs were obtained by site-directed mutagenesis, as shown in table. 30. These variants were expressed and purified as described above. Affinity was determined by ELISA using monovalent Fab fragments and activity was assessed using the C3 precipitation assay using intact IgG4 described above.

Table 30. CDR-L1 option for 4D5, 10D12 and 13B1

Antibody Region Subsequence Parent 4D5 LC-CDR1 KSSQSLLNSRTRKNYLA (SEQ ID NO:142) Mutant 4D5-NQ LC-CDR1 KSSQSLLASRTRKNYLA (SEQ ID NO:257) Mutant 4D5-NA LC-CDR1 KSSQSLLASRTRKNYLA (SEQ ID NO :258) Mutant 4D5-ST LC-CDR1 KSSQSLLNTRTRKNYLA (SEQ ID NO:259) Parental 13B1 LC-CDR1 KSSQSLLNSRTRKNYLA (SEQ ID NO:142) 13B1-NQ LC-CDR1 KSSOSLLQSRTRKNYLA (SEQ ID NO:257) 13B1-NA LC-CDR1 KSSQSLLASRTRKNYLA (SEQ ID NO :258) 13B1-ST LC-CDR1 KSSQSLLNTRTRKNYLA (SEQ ID NO:259) Consensus Sequence for 4D5, 13V1 and options LC-CDR1 KSSQSLLXXRTRKNYLA (SEQ ID NO:260) where X at position 8 is N, Q or A; and where X in position 9 is S or T Parent 10D12 LC-CDR1 KSSQSLLDSDGKTYLN (SEQ ID NO:153) Mutant 10D12-DE LC-CDR1 KSSQSLLDDSEGKTYLN (SEQ ID NO:261) Mutant 10D12-DA LC-CDR1 KSSQSLLDSAGKTYLN (SEQ ID NO:262) Mutant 10D12-GA LC-CDR1 KSSOSLLDSDAKTYLN (SEQ ID NO:263) 35С1 LC-CDR1 KSSQSLLDSDGKTYLS (SEQ ID NO:159) Consensus subsequence LC-CDR1 KSSQSLLDSXXKTYLX (SEQ ID NO:215), where X at position 10 represents for 10D12, 35C1 and variants is D, E or A; where X at position 11 is G or A; And where X at position 16 is N or S

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Table 31 Binding of mutagenic candidate humanized mAbs 4D5, 10D12 and 13B1 to human MASP-3

Anti-MAER-3 antibody clone (Fab format) Binding to human MASP-3, EC ₅₀ (nM) h4D5 19-1, parent Fab (VH-19 and VL-1) 102 h4D5-19-l-NQ Fab (VH-19 and VL-l-NQ) 732 h4D5-19-l-NA Fab (VH-19 and VL-l-NA) 122 h4D5-19-l-ST Fab (VH-19 and VL-l-ST) 151 hlOD12_45-21, parent Fab (VH-45 and VL-21) 108 hlOD12-45-21-DE Fab (VH-45 and VL-21-DE) 326 hlOD12-45-21-DA Fab (VH-45 and VL-21-DA) 294 hlOD12-45-21-GA Fab (VH-45 and VL-21-GA) 181 hl3Bl_10-l, parent Fab (VH-10 and VL-1) 100 hl3Bl_10-l-NQ Fab (VH-10 and VL-l-NQ) 138 hl3Bl_10-l-NA Fab (VH-10 and VL-l-NA) 105 hl3Bl_10-l-ST Fab (VH-10 and VL-l-ST) 120

Table 32 Humanized Anti-MASP-3 Antibody VH Sequences (CDR and FR Regions, by Kabat)

Antibody NS FR1 HC CDR1 Parental QVQ LKQ S GΡELVKP GASVKLSCKAS GYT FT TDDIN

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4D5 (SIN:24) (SEQ ID NO:55) (SEQ ID NO:56) h4D5_VH-14 (SIN:248) QVQ LVQ S GAEVKK P GASVKVSCKAS GYT FT (SEQ ID NO:264) TDDIN (SEQ ID NO:56) h4D5_VH-19 (SIN:249) QVQ LVQ S GAEVKK P GASVKVS C KAS GYT FT (SEQ ID NO:264) TDDIN (SEQ ID NO:56) Parental 10D12 (SIN:28) QIQLVQSGPELKKPGETVKISCKASGYFT (SEQ ID NO:71) SYGMS (SEQ ID NO:72) hlOD12_VH-45 (SIN:251) QIQLVQSGSELKKPGASVKVSCKASGYFT (SEQ ID NO:269) SYGMS (SEQ ID NO:72) hlOD12-VH-49 (SIN:252) QIQLVQSGSELKKPGASVKVSCKASGYFT (SEQ ID NO:269) SYGMS (SEQ ID NO:72) Parental 13B1 (SIN:30) QVQ LKQ S GAELMKP GASVKLS C KATGYT FT (SEQ ID NO:83) GKWIE (SEQ ID NO:84) hl3Bl_VH-9 (SIN:254) QVQ LVQ S GAEVKK P GASVKVS C KAS GYT FT (SEQ ID NO:273) GKWIE (SEQ ID NO:84) hl3Bl_VH-10 (SIN:255) QVQ LVQ S GAEVKK P GASVKVS C KAS GYT FT (SEQ ID NO:273) GKWIE (SEQ ID NO:84) Antibody HC-FR2 HC CDR2 Parent 4D5 WVKQRPGQGLEWIG (SEQ ID NO:57) WIYPRDDRTKYNDKFKD (SEQ ID NO:58) h4D5_VH-14 WVRQAPGQGLEWIG (SEQ ID NO:265) WIYPRDDRTKYNDKFKD (SEQ ID NO:58) h4D5_VH-19 WVRQAPGQGLEWIG (SEQ ID NO:265) WIYPRDDRTKYNDKFKD (SEQ ID NO:58) Parental 10D12 WVRQAPGKGLKWMG (SEQ ID NO:73) WINTYSGVPTYADDFKG (SEQ ID NO:74) hlOD12_VH-45 WVRQAPGKGLKWMG (SEQ ID NO:73) WINTYSGVPTYADDFKG (SEQ ID NO:74) h!0D12-VH-49 WVRQAPGKGLKWMG (SEQ ID NO:73) WINTYSGVPTYADDFKG (SEQ ID NO:74) Parental 13B1 WVKQRPGHGLEWIG (SEQ ID NO:85) EILPGTGSTNYNEKFKG (SEQ ID NO:86) h!3Bl_VH-9 WVRQAPGQGLEWIG (SEQ ID NO:274) EILPGTGSTNYAQKFQG (SEQ ID NO:275)

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h!3Bl_VH-10 WVRQAPGQGLEWIG (SEQ ID NO:274) EILPGTGSTNYNEKFKG (SEQ ID NO:86) Antibody HC-FR3 HC CDR3 Parental 4D5 KAT LTVDT S SNTAYMDLHS LTS EDSAVYFC S S (SEQ ID NO:59) LEDTY (SEQ ID NO:60) h4D5_VH-14 KATLTVDTSSNTAYMELSSLRSEDTAVYYCSS (SEQ ID NO:266) LEDTY (SEQ ID NO:60) h4D5_VH-19 RATLTVDTSSNTAYMELSSLRSEDTAVYYCSS (SEQ ID NO:267) LEDTY (SEQ ID NO:60) Parental 10D12 RFAFSLETSARTPYLQINNLKNEDTATYFCAR (SEQ ID NO:75) GGEAMDY (SEQ ID NO:76) h!0D12_VH-45 RFVFSLDTSVRTPYLQISSLKAEDTAVYFCAR (SEQ ID NO:270) GGEAMDY (SEQ ID NO:76) h!0D12-VH-49 RFVFSLDTSVRTPYLQISSLKAEDTATYFCAR (SEQ ID NO:271) GGEAMDY (SEQ ID NO:76) Parental 13B1 KAT FTAD S S SNTAYMQLSSLTTEDSAMYYCLR (SEQ ID NO:87) SEDV (SEQ ID NO:88) h!3Bl_VH-9 RATFTADSSTSTAYMELSSLRSEDTAVYYCLR (SEQ ID NO:276) SEDV (SEQ ID NO:88) h!3Bl_VH-10 RATFTADSSTSTAYMELSSLRSEDTAVYYCLR (SEQ ID NO:276) SEDV (SEQ ID NO:88) Antibody HC-FR4 Parental 4D5 WGQGTLVAVSS (SEQ ID NO:61) h4D5_VH-14 WGQGTLVTVSS (SEQ ID NO:268) h4D5_VH-19 WGQGTLVTVSS (SEQ ID NO:268) Parental 10D12 WGQGTSVTVSS (SEQ ID NO:77) hlOD12_VH-45 WGQGTLVTVSS (SEQ ID NO:272) h!0D12-VH-49 WGQGTLVTVSS (SEQ ID NO:272) Parental WGTGTTVTVSS 13B1 (SEQ ID NO:89) h!3Bl_VH-9 WGQGTLVTVSS (SEQ ID NO:277) h!3Bl_VH-10 WGQGTLVTVSS (SEQ ID NO:277)

Representative humanized light chain variable regions with variants

- 160 040888 h4D5 VL-l-NA (SEQ ID NO:278)

DIVMTOSPDSLAVSLGERATINCKSSQSLLASRTRKNYLAWYOOKPGOPPKLLIYWAST

RESGVPDRFSGSGSGTDFTLTISSLOAEDVAVYYCKQSYNLYTFGOGTKVEIKR hl0D12 VL-21-GA (SEQ ID NO:279)

DVLMTOTPLSLSVTPGQPASISCKSSOSLLDSDAKTYLNWLLORPGOSPKRLIYLVSKL

DSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPWTFGOGTKVEIKR hl3Bl VL-l-NA (SEQ ID NO:280)

DIVMTOSPDSLAVSLGERATINCKSSQSLLASRTRKNYLAWYOOKPGOPPKLLIYWAST

RESGVPDRFSGSGSGTDFTLTISSLOAEDVAVYYCKQSYNIPTFGOGTKVEIKR

Table 33 Humanized aHTu-MASP-3 antibody VL sequences (CDR and FR regions, according to Kabat) [plus variants in LC-CDR1]

Antibody LC-FR1 LC CDR1 Parental 4D5 (SIN:40) DIVMT Q S P S S LAVSAGEKVTMTC (SEQ ID NO:141) KSSQSLLNSRTRKNYLA (SEQ ID NO:142) h4D5_VL-l (SIN:250) DIVMTQSPDSLAVSLGERATINC (SEQ ID NO:281) KSSQSLLNSRTRKNYLA (SEQ ID NO:142) h4D5_VL-l-NA (SIN:278) DIVMTQSPDSLAVSLGERATINC (SEQ ID NO:281) KSSQSLLASRTRKNYLA (SEQ ID NO:258) Parental 10D12 (SIN:43) DVLMT QT P LTL SVT1GQ PASISC (SEQ ID NO:152) KSSQSLLDSDGKTYLN (SEQ ID NO:153) hlOD12_VL-21 (SIN:253) DVLMTQTPLSLSVTPGQPASISC (SEQ ID NO:285) KSSQSLLDSDGKTYLN (SEQ ID NO:153) hl0D12_VL-21- GA (SIN:279) DVLMTQTPLSLSVTPGQPASISC (SEQ ID NO:285) KSSQSLLDSDAKTYLN (SEQ ID NO:263) Parental 13B1 DIVMT Q S PSS LAVSAGEKVTMSC (SEQ ID NO:151) KSSQSLLNSRTRKNYLA (SEQ ID NO:142) hl3Bl_VL-l DIVMTQSPDSLAVSLGERATINC (SEQ ID NO:281) KSSQSLLNSRTRKNYLA (SEQ ID NO:142)

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h!3Bl_VL-l-NA DIVMTQSPDSLAVSLGERATINC (SEQ ID NO:281) KSSQSLLASRTRKNYLA (SEQ ID NO:258) Antibody LC-FR2 LC CDR2 Parent 4D5 WYQQKPGQSPKLLIY (SEQ ID NO:143) WASTRES (SEQ ID NO:144) h4D5_VL-l WYQQKPGQPPKLLIY (SEQ ID NO:282) WASTRES (SEQ ID NO:144) h4D5_VL-l-NA WYQQKPGQPPKLLIY (SEQ ID NO:282) WASTRES (SEQ ID NO:144) Parental 10D12 WLLQRPGQSPKRLIY (SEQ ID NO:154) LVSKLDS (SEQ ID NO:155) hlOD12_VL-21 WLLQRPGQSPKRLIY (SEQ ID NO:154) LVSKLDS (SEQ ID NO:155) hlOD12_VL-21- GA WLLQRPGQSPKRLIY (SEQ ID NO:154) LVSKLDS (SEQ ID NO:155) Parental 13B1 WYQQKPGQSPKLLIY (SEQ ID NO:143) WASTRES (SEQ ID NO:144) hl3Bl_VL-l WYQQKPGQPPKLLIY (SEQ ID NO:282) WASTRES (SEQ ID NO:144) h!3Bl_VL-l-NA WYQQKPGQPPKLLIY (SEQ ID NO:282) WASTRES (SEQ ID NO:144) Antibody LC-FR3 LC CDR3 Parent 4D5 GVPDRFTGSGSGTDFSLTISSVQAEDLAVYYC (SEQ ID NO:145) KQSYNLYT (SEQ ID NO:146) h4D5_VL-l GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO:283) KQSYNLYT (SEQ ID NO:146) h4D5_VL-l-NA GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO:283) KQSYNLYT (SEQ ID NO:146) Parental 10D12 GVP D RFTGSGSGTDFTLKIS RVEAEDLGVYYC (SEQ ID NO:156) WQGTHFPWT (SEQ ID NO:157) hlOD12_VL-21 GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC (SEQ ID NO:286) WQGTHFPWT (SEQ ID NO:157) hlOD12_VL-21- GA GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC (SEQ ID NO:286) WQGTHFPWT (SEQ ID NO:157) Parental GVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC KQSYNIPT

- 162 040888

13B1 (SEQ ID NO:150) (SEQ ID NO:161) hl3Bl_VL-l GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO:283) KQSYNIPT (SEQ ID NO:161) hl3Bl_VL-l-NA GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO:283) KQSYNIPT (SEQ ID NO:161) Antibody LC-FR4 Parent 4D5 FGGGTKLEIKR (SEQ ID NO:147) h4D5_VL-l FGQGTKVEIKR (SEQ ID NO:284) h4D5_VL-l-NA FGQGTKVEIKR (SEQ ID NO:284) Parental 10D12 FGGGTKLEIKR (SEQ ID NO:147) hlOD12_VL-21 FGQGTKVEIKR (SEQ ID NO:287) hlOD12_VL-21- GA FGQGTKVEIKR (SEQ ID NO:287) Parental 13B1 FGGGTKLEIKR (SEQ ID NO:147) hl3Bl_VL-l FGQGTKVEIKR (SEQ ID NO:284) h!3Bl_VL-l-NA FGQGTKVEIKR (SEQ ID NO:284)

Example 20.

Analysis of a representative MASP-3 inhibitory mAb 13B1 in a mouse model of multiple sclerosis.

Background/Rationale. Experimental autoimmune encephalomyelitis (EAE), i.e. acquired inflammatory and demyelinating autoimmune disorder is an animal model of multiple sclerosis (MS). There is evidence to suggest that APC plays a significant role in the development/progression of EAE and that the disease is attenuated in mice treated with factor B neutralizing antibody (Hu et al., Mol. Immunol. 54:302, 2013). This example describes the assay of a representative high affinity MASP-3 inhibitory antibody, 13B1, in an EAE model.

Methods.

EAE induction.

A kit purchased from Hooke Laboratories (Lawrence, MA) was used to induce EAE in this study. This kit contained the neuroantigen MOG _35-55 in complete Freund's adjuvant (CFA) as well as pertussis toxin.

In this study, 30 female C57 B1/6J wild-type mice were used and these mice were allowed to acclimate to appropriate housing conditions for at least one week prior to induction of EAE. Mice were approximately 10 weeks old at the time of induction. As shown below in Table. 34, at induction, each mouse was given two 100 µl injections of MOG35-55 subcutaneously (sc) and one intraperitoneal (ip) injection of 100 µl (400 ng) pertussis toxin. The second injection of pertussis toxin was administered 24 hours after the first injection.

Treatment: 30 mice were divided into three groups of 10 animals and treated with an irrelevant isotype control mAb (10 mg/kg i.v.); mAb 13B1 (anti-MASP-3 antibody, 10 mg/kg i.v.) or mAb 1379 (anti-factor B antibody (Hi et al., Mol. Immunol. 54:302, 2013) 40 mg/kg i.p.). As shown in Table. 34, the isotype control mAb and α anti-MASP-3 mAb 13B1 were dosed weekly from day -16 until day +12. The dose of mAb 1379 was administered every other day from day +3 to day +11, in accordance with the dosing schedule described by Hu et al., Mol. Immunol. 54:302-308, (2013).

- 163 040888

Table 34. Experimental methods for conducting an EAE study using anti-MASP-3 mAb 13B1

Day entered AND I pertussis toxin 400 ng i ·Ρ· Peptide MOG 35-55 250 mcg mAb 1379 (against factor B) 4 0 mg i.p. mAb control isotype 10 mg/kg i.v. mAb 13B1 (against MASP-3) 10 mg/kg -16 + + -9 + + -2 + + 0 + + +1 + +3 + +5 + + + +7 + +9 + + 11 + + 12 + +

Grade. Mice were assessed the day before symptom onset, after which they were assessed daily. The first signs of the disease, as expected, were observed 7-12 days after immunization. Mice were evaluated according to the scores of the rating scale presented below in table. 35.

Table 35. Evaluation criteria for the EAE model score Clinical Observations 0.0 The mice did not show any change in motor function compared to non-immunized mice. When lifting the tail at the base, there is tension and stretching. The hind limbs are usually spread apart. When moving the mouse, there is no change in gait or head tilt. 0.5 Dangling tip of the tail. When lifting the tail, there is tension at the base, except for the tip of the tail. There is tension in the muscles of the tail, but at the same time, the tail continues to move. 1.0 Tail hanging. When lifting the tail at the base, instead of stretching, the entire tail hangs on the fingers. The hind limbs are usually spread apart. There were no signs of tail movement. 1.5 Hanging of the tail and inhibition of the hind limbs. When lifting the tail at the base, the entire tail hangs on the fingers. When the mouse is lowered onto the hanging rack, at least one hind leg is fully lowered. The gait becomes very unsteady. 2.0 Hanging tail and weakness of the hind limbs. When lifting the tail at the base, the hind legs do not move apart and are located next to each other. When observing the movement of mice, the gait was clearly unsteady. The fingers of one paw moved with difficulty, and the fingers of the other paw hardly moved;

- 164 040888

OR The mice were scored 0.0 but showed clear signs of head tilt when walking. The balance was broken. 2.5 The tail hung down and the hind legs moved with difficulty. Both hind legs moved a little, but still they moved with difficulty (the mouse moved on its hind legs). - OR- movement of one paw was observed / one paw moved with difficulty, but the other paw moved normally - OR - The severity of EAE was assessed as weak when lifting the tail (scored 0.0-1.5), but there was a strong tilt of the head, and therefore the mouse sometimes fell. 3.0 Hanging of the tail and complete paralysis of the hind legs were observed (the most common symptoms). - OR - Hanging tail and almost complete paralysis of the hind legs. One or both hind legs could hobble, but neither hind leg could come forward of the hind thigh - OR - Tail drooping and paralysis of one front and one hind leg were observed - OR - ALL signs: strong head tilt, movement only along the edges of the cage , pushing away from the walls of the cage and turning over when the tail base is raised. 3.5 Hanging of the tail and complete paralysis of the hind legs were observed. In addition: the mouse moved around the cage, but when it was placed on its side, it could not stand up on its own. The hind legs were together but on the same side of the torso - OR the mouse was moving around the cage, but the back of the torso was flat like a pancake, giving the impression that the mouse had a hump in the front of the torso. 4.0 Hanging of the tail, complete paralysis of the hind legs and partial paralysis of the front legs were observed. The mouse made minimal movements in the cage, but in case of alarm and food supply. If the mice were given a score of 4.0 within 2 days, euthanasia is most commonly recommended. However, with daily subcutaneous fluid administration, some mice could score as high as 3.5 or 3.0. If the mouse was euthanized due to severe paralysis, then for the rest of the experiment, the mice were assigned a score of 5.0. 4.5 There was a complete paralysis of the hind legs and partial paralysis of the front legs, and no movement around the cage was observed. The mouse showed no signs of anxiety. The mouse showed minimal movements of the forepaws. The mouse barely responded to touch. In this case, euthanasia is recommended. If the mouse was euthanized due to severe paralysis, then for the rest of the experiment, the mice were assigned a score of 5.0. 5.0 The mouse spontaneously rolled over the cage.

Results.

In FIG. 68 graphically illustrates the results of an EAE model assay in mice treated with either MASP-3 inhibitory mAb 13B1 (10 mg/kg), anti-factor B mAb 1379 (40 mg/kg), or isotype control mAb (10 mg/kg). , where the position of the down arrows indicates the dose of anti-factor B antibody and the position of the up arrows indicates the last dose of mAb 13B1. As shown in FIG. 68, mice treated with MASP-3 inhibitory mAb 13B1 and anti-factor B mAb 1379 showed improvement in clinical symptoms as measured by the parameters shown in Table 1. 35 compared to isotype control antibody treatment.

In accordance with the foregoing, it is expected that MASP-3 inhibitory antibodies, such as the high affinity MASP-3 inhibitory antibodies described herein, will provide a beneficial effect (neuroprotective or neuroregenerative) in the treatment and/or rehabilitation of an individual. suffering from multiple sclerosis, Balo's concentric sclerosis, neuromyelitis of the optic nerve, Marburg's multiple sclerosis, Schilder's disease, multiple sclerosis causing edema and acute disseminated encephalomyelitis (ADE).

Example 21 Pharmacodynamic study using representative high affinity anti-MASP-3 mAbs in cynomolgus monkeys.

Background/Rationale. As demonstrated in rodent studies (FIG. 44), the high affinity MASP-3 inhibitory antibody was capable of sustainably inhibiting (residual) pro-factor D maturation in vivo. This example describes a study that was conducted in cynomolgus monkeys to determine if representative high affinity MASP-3 inhibitory mAbs could inhibit APC activity in a non-human primate.

Methods. To confirm that MASP-3 functions in APC in a non-human primate and that high-affinity anti-MASP-3 antibodies are capable of inhibiting APC in a non-human primate, 9 cynomolgus monkeys (3 animals per mAb status) were injected with one an intravenous 5 mg/kg dose of one of three representative high-affinity MASP-3 inhibitory antibodies: h4D5X, h10D12X, or h13B1X. (h means humanized, IgG4 X constant hinge region (SEQ ID NO: 312) containing a stabilizing amino acid substitution S228P and a human IgG4 constant region with the S228P mutation, as well as a mutation that promotes FcRn interactions at low pH). Plasma (EDTA) and serum samples were collected at regular intervals for three weeks or more.

Two analyzes were performed to evaluate APC activity in sera from treated monkeys. The first assay assessed levels of complement factor Bb deposited on zymosan spheres added to diluted serum. In the second assay, levels of liquid phase products of zymosan-activated APC were assessed, ie. complement factors Ba and Bb, as well as C3a.

For detection of factor Bb precipitated on zymosan, flow cytometric analysis was performed using anti-factor Bb antibody A252 (Quidel). To determine the background signal in the assay after complete APC inhibition, an aliquot of serum (final volume 5% diluted in GVB+Mg/EGTA) from cynomolgus monkeys treated with anti-MASP-3 mAb was supplemented with 300 nM inhibitory factor antibody. D. To determine the degree of APC inhibition by the anti-MASP-3 mAb intravenously administered to monkeys, 300 nM neutral isotype control antibody (not having APC inhibitory activity) was added to a second aliquot of diluted serum, and then the degree of precipitation of factor Bb on zymosan was assessed. The antibody-serum mixtures were incubated for 30 min on ice and then zymosan was added (at a final concentration of 0.1 mg/ml). The mixtures were incubated at 37° C. for 65 min and APC activity was measured on a flow cytometer by detecting complement factor Bb (A252 antibody, Quidel) on the surface of the zymosan particles.

To evaluate the production of the liquid phase markers Ba, Bb and C3a, APC was induced in ex vivo assays by incubating zymosan (at a final concentration of 1 mg/ml) in serum (at a final volume of 5% diluted in GVB+Mg/EGTA) obtained from canine monkeys treated with anti-MASP3 mAb. The mixtures were incubated at 37° C. for 40 minutes and APC activity was measured by detection of complement endpoints by ELISA. Ba, Bb and C3a were detected in the supernatants of the reaction mixture using commercially available ELISA kits (Quidel). The absorbance values in all assays were normalized by taking pre-treatment values as 100% activity, and the pre-treatment value for a sample that had been incubated but not treated with zymosan was taken as 0%.

Serum levels of MASP-3 and MASP-3 inhibitory mAb were determined in monkeys treated with anti-MASP-3 mAb to establish the relationship between the degree of APC inhibition and the antibody-to-target ratio. Serum levels of MASP-3 were assessed using a sandwich ELISA assay. The MASP-3 protein was captured on the plate with aM3-259 (as described in Example 16). Serum samples (diluted 1:40) were first incubated with an unlabeled (non-biotinylated) anti-MASP-3 mAb matched to the treatment mAb at 37°C for 1 hour and then diluted 1:250 (final volume 1:10,000), was added to the tablet and incubated at 37°C for another hour. The plate was washed and a biotinylated version of mAb 10D12 was used as the detection antibody. Prior to the detection step, a large serum dilution was used to separate the target and therapeutic mAb, and to prevent competitive binding of therapeutic antibody and detection antibody. After multiple washes of the plate, streptavidin HRP was used to carry out the final detection step. Optical density values were obtained on a tablet reader at a wavelength of 450 nm. Serum MASP-3 concentrations were extrapolated from a standard curve generated from recombinant full-length MASP-3 cynomolgus protein assay. The amount of anti-MASP-3 antibody present in serum was detected using a human therapeutic IgG4 ELISA kit (Cayman

- 166 040888

Chemicals) in accordance with the manufacturer's instructions.

Western blot analysis was performed to assess the level of pro-factor D and factor D in the serum of cynomolgus monkeys for a certain period of time (hours) after treatment with a single intravenous 5 mg/kg dose of mAb h13B1X. Briefly, Western blot analysis was performed by mixing 20 μl of cynomolgus monkey plasma obtained at various times before treatment (-120 hours, 24 hours) and after treatment (72, 168, 336, 504, 672 and 840 hours) with PBS, and 11.2 µl of anti-CFD antibody (0.5 µg/µl) in a total volume of 400 µl at 4°C for 1 hour. Then 12 µl of protein A/G plus agarose (Santa Cruz Biotech) was added and the mixture was incubated overnight at 4°C. Immunoprecipitates were collected by centrifugation at 1000 x g for 5 min at 4°C. The precipitate was washed five times with PBS. After a final wash, the pellet was resuspended in 30 µl of 1 x glycoprotein denaturation buffer and the glycoprotein was denatured by heating the reaction mixture at 100°C for 10 min. 10x reaction buffer G2, 10% NP-40, and 2.5 μl peptide-K-glycosidase (New England Biolabs, P0704L) were added to each tube and the reaction mixture was incubated at 37°C for 2 h. Agarose spheres were pelleted by centrifugation at 1000 x g for 5 min and 20 μl of the supernatant were collected in new tubes. Captured and deglycosylated proteins were separated by SDS-PAGE electrophoresis (in NuPAGE 12% bis-tris-minigel) and these gels were electroblotted for Western blot analysis using a biotinylated anti-CFD antibody (R&D Systems BAF1824) and streptavidin-HRP, high sensitivity Pierce™ (Thermo Fischer Scientific 21130).

Results.

In FIG. 69 graphically illustrates APC activity in sera taken from a group of three cynomolgus monkeys over a period of time after one time 0 treatment with the high affinity anti-MASP-3 mAb h13B1X. This figure shows the mean MFI in a flow cytometric assay for factor Bb detection of complement on the surface of zymosan particles in 5% serum supplemented with either an anti-factor D APC inhibitory mAb or a neutral isotype control mAb. As shown in FIG. 69, a decrease in APC activity was observed in these animals as early as 4 h. If treatment with anti-MASP-3 antibody blocked APC as effectively as factor D inhibition, identical levels of inhibition of Bb precipitation in post-dose samples were observed under the two antibody dilution conditions. , but not before dosing (or time=0; FIG. 69). As shown in FIG. 69, 72 hours post-treatment, APC activity was reduced to approximately the level achieved by adding the anti-factor D mAb to serum samples. Almost complete inhibition due to treatment with h13B1X antibody, as experimentally determined by comparing the inhibition achieved by treatment with antibody against factor D, lasted up to 336 hours (14 days) after dosing. Thus, these results demonstrate that treatment with a high affinity MASP-3 inhibitory mAb results in complete long-term inhibition of APC in a non-human primate.

In FIG. 70 graphically illustrates APC activity as determined by Bb precipitation on zymosan in serum samples from groups of cynomolgus monkeys (3 animals per group) treated with a single 5 mg/kg intravenous dose of the high affinity MASP-3 inhibitory mAbs h4D5X, h10D12X, or h13B1X. Bb deposition data were collected as described above. APC activity at the time of treatment was normalized by taking the pre-treatment MFI values with non-inhibitory isotype control antibody as 100% activity, and the pre-treatment value for the sample incubated with 50 mM EDTA (to inhibit all complement activity) as 0%. The processing data h13BX used as shown in FIG. 70 are also illustrated in FIG. 69. As shown in FIG. 70, treatment with all three high affinity MASP-3 inhibitory antibodies resulted in greater than 95% inhibition of APC. h4D5X-, h10D12X- and h13B1X-treated animals retained at least 90% APC inhibition for 6.7, 11.7 and 16 days, respectively. Thus, these results demonstrate that treatment with these representative high affinity MASP-3 inhibitory mAbs results in long-term inhibition of APC in a non-human primate at a single dose of 5 mg/kg.

In FIG. 71A-C are graphical illustrations of additional methods for assessing APC activity. Fluid phase Ba (FIG. 71A), Bb (FIG. 71B), and C3a (FIG. 71C) were evaluated in zymosan-treated and diluted sera from groups of cynomolgus monkeys (3 animals per group) over a period of time after treatment with one intravenous dose of 5 mg/kg h4D5X, h10D12X and h13B1X as described above.

As shown in FIG. 71A-C, a single administration of all three high affinity MASP-3 inhibitory antibodies resulted in APC inhibition as determined by the three endpoints for various fluid phase factors. These data are consistent with the level of APC inhibition demonstrated in the Bb precipitation assay in FIG. 70 and also indicate the effectiveness of these mAbs in inhibiting this pathway for several weeks.

In FIG. 72A-C are graphical illustrations of the relationship between APC activity as determined by liquid phase Ba production and the molar ratio of monomeric MASP-3 and anti- 167 040888

MASP-3 antibody detectable in sera of monkeys treated with either h4D5X (FIG. 72A) or h10D12X (FIG. 72B) or h13B1X (FIG. 72C). Each panel in Fig. 72A-C correspond to data for one monkey. The monkeys and serum (or plasma) used in this study are the same as described above (FIGS. 69, 70 and 71).

In FIG. 72A-C graphically illustrate the molar ratio of target (MASP-3) to high affinity MASP-3 inhibitory antibodies h4D5X (FIG. 72A), h10D12X (FIG. 72B), and h13B1X (FIG. 72C) at specific times of complete APC inhibition as was determined from liquid-phase Ba. For comparison, the molar ratio target:antibody=1:1 is shown in each graph with a dotted line. As shown in FIG. 72A-C, a molar ratio of target (MASP-3):high affinity MASP-3 inhibitory mAbs h4D5X, h10D12X, and h13B1X of approximately 2:1 to 2.5:1 (target:antibody) is sufficient for complete inhibition of APC. . These data demonstrated that these three representative MASP-3 inhibitory mAbs are active, high affinity MASP-3 inhibitory antibodies that are capable of inhibiting APC when their molar concentrations are less than the target concentration. These levels of activity clearly indicate that such mAbs can be used in clinical applications for the treatment of diseases or disorders caused by ARS.

In FIG. 73 illustrates a Western blot analysis to determine the level of pro-factor D and factor D in serum taken from a cynomolgus monkey over a period of time (hours) before and after treatment with a single intravenous dose of 5 mg/kg mAb h13B1X. As shown in FIG. 73, factor D is present in plasma as pro-factor D for at least 336 hours (14 days) after administration of a single dose of h13B1X mAb.

Brief description of the results.

As described in Example 11, administration of a single dose of the high affinity MASP-3 inhibitory antibody mAb 13B1 to mice resulted in almost complete elimination of systemic alternative pathway complement activity after at least 14 days. As further described in Example 12, in a study conducted in a well-established animal model associated with PNH, mAb 13B1 was shown to significantly increase the viability of PNH-like erythrocytes and protect PNH-like erythrocytes significantly better than inhibition of C5. As described in Example 13, mAb 13B1 was also shown to reduce the incidence and severity of disease in a mouse model of arthritis. The results described in this example showed that the representative high affinity MASP-3 inhibitory mAbs 13B1, 10D12 and 4D5 are highly effective in blocking the alternative pathway in primates. Administration of a single dose of mAb 13B1, 10D12 or 4D5 to cynomolgus monkeys resulted in prolonged elimination of systemic activity of the alternative pathway for approximately 16 days. The rate of elimination of the alternative pathway in cynomolgus monkeys treated with high affinity MASP-3 inhibitory antibodies was comparable to that achieved with factor D blocking in vitro, indicating complete blockage of factor D conversion by MASP-3 inhibitory antibodies. Therefore, high-affinity MASP-3 inhibitory mAbs have therapeutic efficacy in the treatment of patients suffering from diseases associated with alternative pathway hyperactivity, such as, for example, paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD, including wet and dry AHP) , ischemic-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy (including hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (AHUS), thrombotic thrombocytopenic purpura (TTP) or transplant-associated TMA), asthma, hard deposit disease, oligoimmune necrosing sickle-shaped glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis of the optic nerve, Behcet's disease, multiple sclerosis, Guillain-Barré syndrome, Alzheimer's disease, amylotrophic lateral sclerosis (ABS), lupus nephritis, systemic lupus erythematosus (SLE), diabetic retinopathy, uveitis, chronic obstructive pulmonary disease (COPD), C3 glomerulopathy, graft rejection, graft versus host disease (GVHD), hemodialysis, sepsis, systemic inflammatory response syndrome (SIRS), acute respiratory distress -syndrome (ARDS), ANCA-vasculitis, antiphospholipid syndrome, atherosclerosis, IgA nephropathy and myasthenia gravis.

VII. Other embodiments of the invention.

All publications, patent applications and patents described in this application are hereby incorporated by reference.

Various modifications and variations of the described methods, compositions and compounds according to the invention will be obvious to those skilled in the art and do not go beyond the essence and scope of the invention. While the present invention has been described in terms of specific preferred embodiments, it should be noted that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described methods of carrying out the invention, which are obvious to those skilled in medicine, immunology, pharmacology, oncology, or related fields, are within the scope of the present invention.

In accordance with the foregoing, the present invention is represented by the following variants of its implementation.

High affinity MASP-3 inhibitory antibodies that bind to one or more epitopes in the SP domain.

1A. An isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to the human MASP-3 serine protease domain (amino acid residues 450-728 of SEQ ID NO: 2) with high affinity (having a KD of less than 500 pM), wherein the antibody or antigen-binding fragment thereof inhibit complement activation of the alternative pathway.

2A. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof has at least one or more of the following properties:

(a) inhibit pro-factor D maturation;

(b) do not bind to human MASP-1 (SEQ ID NO: 8);

(c) inhibit the alternative pathway in a molar ratio of about 1:1 to about 2.5:1 (MASP-3 target: mAb) in a mammal;

(d) do not inhibit the classical pathway;

(e) inhibit hemolysis and/or opsonization;

(f) inhibit substrate-specific cleavage of the MASP-3 serine prosthesis;

(g) reduce the degree of hemolysis or degree of C3 cleavage and deposition of surface C3b;

(h) reduce the amount of factor B and Bb deposition on the activating surface;

(i) reduce residual levels (in the circulation and without experimental additional surface activation) of active factor D compared to pro-factor D;

(j) reduce levels of active factor D relative to pro factor D in response to surface activation;

(k) reduce production levels of residual and/or surface-induced liquid phase Ba, Bb, C3b or C3a; and/or (l) reduce factor P deposition.

3A. An isolated antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein said antibody or antigen-binding fragment thereof specifically binds to an epitope located in the human MASP-3 serine prosthesis domain, wherein said epitope is located in at least one or more of: VLRSQRRDTTVI ( SEQ ID NO: 9), TAAHVLRSQRRDTTV (SEQ ID NO: 10), DFNIQNYNHDIALVQ (SEQ ID NO: 11), PHAECKTSYESRS (SEQ ID NO: 12), GNYSVTENMFC (SEQ ID NO: 13), VSNYVDWVWE (SEQ ID NO: 14 ) and/or VLRSQRRDTTV (SEQ ID NO: 15) [Group I].

4A. An antibody or antigen-binding fragment thereof according to claim 3, wherein said antibody or antigen-binding fragment thereof binds to an epitope in SEQ ID NO: 15 [including all Group I Abs].

5A. An antibody or antigen-binding fragment thereof according to claim 3, wherein said antibody or antigen-binding fragment thereof binds to an epitope in SEQ ID NO: 9. [10D12]

6A. An antibody or antigen-binding fragment thereof according to claim 3, wherein said antibody or antigen-binding fragment binds to an epitope in SEQ ID NO: 10. [13B1].

7A. An antibody or antigen-binding fragment thereof according to claim 6, wherein said antibody or antigen-binding fragment thereof also binds to an epitope in SEQ ID NO: 12. [13B1].

8A. An antibody or antigen-binding fragment thereof according to claim 3, wherein said antibody or antigen-binding fragment thereof also binds to an epitope in SEQ ID NO: 10 and/or SEQ ID NO: 12. [13B1].

9A. An antibody or antigen-binding fragment thereof according to claim 3, wherein said antibody or antigen-binding fragment thereof binds to an epitope in SEQ ID NO: 9. [1F3, 4B6, 4D5, 1A10].

10A. An antibody or antigen-binding fragment thereof according to claim 7, wherein said antibody or antigen-binding fragment thereof also binds to an epitope in at least one of SEQ ID NO: 11, SEQ ID NO: 13, and/or SEQ ID NO: 14. [1F3, 4B6, 4D5, 1A10].

11A. An antibody or antigen-binding fragment thereof according to claim 7, wherein said antibody or antigen-binding fragment thereof also binds to an epitope in at least one of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13 and/or SEQ ID NO : 14. [1F3, 4B6, 4D5, 1A10].

12A. An antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein said antibody binds to an epitope in at least one of ECGQPSRSLPSLV (SEQ ID NO: 16), RNAEPGLFPWQ (SEQ ID NO: 17); KWFGSGALLSASWIL(SEQ ID NO: 18); EHVTVYLGLH (SEQ ID NO: 19); PVPLGPPHVMP (SEQ ID NO: 20); APHMLGL (SEQ ID NO: 21); SDVLQYVKLP (SEQ ID NO: 22); and/or AFVIFDLSQRW (SEQ ID NO: 23). [Groups II and III].

13A. An antibody or antigen-binding fragment thereof according to claim 12, wherein said antibody or antigen-binding fragment thereof binds to an epitope in SEQ ID NO: 17 [all Abs of groups II and III].

14A. An antibody or antigen-binding fragment thereof according to claim 13, wherein said antibody or an-

- 169 040888 the tigen-binding fragment also binds to an epitope in EHVTVYLGLH (SEQ ID NO: 19) and/or

AFVIFDDLSQRW (SEQ ID NO: 23). [1G4, 1E7, 2D7 15D9].

15A. An antibody or antigen-binding fragment thereof according to claim 13, wherein said antibody or antigen-binding fragment thereof also binds to an epitope in SEQ ID NO: 23. [1G4, 1E7, 2D7, 15D9,

2F5].

16A. An antibody or antigen-binding fragment thereof according to claim 14, wherein said antibody or antigen-binding fragment thereof also binds to an epitope in SEQ ID NO: 19 and/or SEQ ID NO: 23. [Ig4, 1E7, 2D7].

17A. An antibody or antigen-binding fragment thereof according to claim 14, wherein said antibody or antigen-binding fragment thereof also binds to an epitope in SEQ ID NO: 18, SEQ ID NO: 20 and/or SEQ ID NO: 23. [15D9, 2F5].

18A. An antibody or antigen-binding fragment thereof according to claim 14, wherein said antibody or antigen-binding fragment thereof also binds to an epitope in at least one of SEQ ID NO: 18, SEQ ID NO: 20 and/or SEQ ID NO: 23 [15D9, 2F5 ].

19A. An antibody or antigen-binding fragment thereof according to claim 14, wherein said antibody or antigen-binding fragment thereof also binds to an epitope in at least one of SEQ ID NO: 16, SEQ ID NO: 21, and/or SEQ ID NO: 22. [1B11] .

0A. An antibody or antigen-binding fragment thereof according to claim 14, wherein said antibody or antigen-binding fragment thereof also binds to an epitope in at least one of SEQ ID NO: 16, SEQ ID NO: 21 and/or SEQ ID NO: 22 [1B11].

21A. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 20, wherein said antibody or antigen-binding fragment thereof is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a mouse antibody, and an antigen-binding fragment of any of the above antibodies.

22A. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 21, wherein said antibody or antigen-binding fragment thereof is selected from the group consisting of single chain antibody, ScFv, Fab fragment, Fab' fragment, F(ab') 2 fragment, a monovalent antibody that does not contain a hinge region and a whole antibody.

23A. An antibody or antigen-binding fragment thereof according to any one of paragraphs. 1-22, also including the immunoglobulin constant region.

24A. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 23, wherein the antibody or antigen-binding fragment thereof is humanized.

25A. An antibody or antigen-binding fragment thereof according to any one of paragraphs. 1-24, wherein said antibody binds to the human MASP-3 serine protease domain with an affinity of less than 500 pM.

26A. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 25, wherein said antibody inhibits the activation of an alternative pathway in the blood of a mammal.

27A. A composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1A-26A and a pharmaceutically acceptable excipient.

A. High affinity group IA MASP-3 inhibitory antibodies that bind to one or more epitopes in the SP domain (4D5, 4B6, 1A10 plus 4D5 variants) 1B. An isolated antibody or antigen-binding fragment thereof that binds to MASP-3 and contains:

(a) a heavy chain variable region comprising HC-CDR1 shown as SEQ ID NO: 209 (XXDIN where X at position 1 is S or T and where X at position 2 is N or D); HC-CDR2 shown as SEQ ID NO: 210 (WIYPRDXXXKYNXXFXD where X at position 7 is G or D; X at position 8 is S, T or R; X at position 9 is I or T; X at position 13 is E or D, X at position 14 is K or E, and X at position 16 is T or K); and HC-CDR3 shown as SEQ ID NO: 211 (XEDXY where X at position 1 is L or V and where X at position 4 is T or S); and (b) a light chain variable region comprising LC-CDR1 shown as SEQ ID NO: 212 (KSSQSLLXXRTRKNYLX, where X at position 8 is N, I, Q, or A; where X at position 9 is S or T; and where X at position 17 is A or S); LC-CDR2 shown as SEQ ID NO: 144 (WASTRES) and LC-CDR3 shown as SEQ ID NO: 146 (KQSYNLYT).

2b. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region HC-CDR1 according to (a) comprises SEQ ID NO: 56 (TDDIN). [4D5 and variants].

3B. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region HC-CDR1 according to (a) comprises SEQ ID NO: 62 (SNDIN). [1F3, 4B6 and 1A10].

4B. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region HC-CDR2 according to (a) comprises SEQ ID NO: 58 (WIYPRDDRTKYNDKFKD) [4D5 and variants].

5V. The isolated antibody or antigen-binding fragment thereof according to claim 1, where HC-CDR2 is variable

- 170 040888 heavy chain region according to (a) contains SEQ ID NO: 63 (WIYPRDGSIKYNEKFTD).

[1F3]

6B. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region HC-CDR2 according to (a) comprises SEQ ID NO: 67 (WIYPRDGTTKYNEEFTD). [4B6].

7B. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region HC-CDR2 according to (a) comprises SEQ ID NO: 69 (WIYPRDGTTKYNEKFTD). [1A10]

8B. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region HC-CDR3 according to (a) comprises SEQ ID NO: 60 (LEDTY) [4D5 and variants].

9B. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region HC-CDR3 according to (a) comprises SEQ ID NO: 65 (VEDSY). [1F3, 4B6 and 1A10].

10V. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain variable region LC-CDR1 according to (b) contains SEQ ID NO: 142 (KSSQSLLNSRTRKNYLA); SEQ ID NO: 257 (KSSQSLLQSRTRKNYLA), SEQ ID NO: 258 (KSSQSLLASRTRKNYLA); or SEQ ID NO: 259 (KSSQSLLNTRTRKNYLA). [4D5 and variants].

11B. The isolated antibody or antigen-binding fragment thereof according to claim 10, wherein the light chain variable region LC-CDR1 according to (b) comprises SEQ ID NO: 258 (KSSQSLLASRTRKNYLA). [mutant 4D5 NA].

12V. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain variable region LC-CDR1 according to (b) comprises SEQ ID NO: 149 (KSSQSLLISRTRKNYLS). [1F3 and 4B6].

13b. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein HC-CDR1 contains SEQ ID NO: 56, HC-CDR2 contains SEQ ID NO: 58, HC-CDR3 contains SEQ ID NO: 60, and wherein LC-CDR1 contains SEQ ID NO: : 142, SEQ ID NO: 257, SEQ ID NO: 258 or SEQ ID NO: 259; where LC-CDR2 contains SEQ ID NO: 144 and where LC-CDR3 contains SEQ ID NO: 146. [all 6 CDR 4D5 with variants in lCCDR1].

14V. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein HC-CDR1 contains SEQ ID NO: 62, HC-CDR2 contains SEQ ID NO: 63, SEQ ID NO: 67 or SEQ ID NO: 69, HC-CDR3 contains SEQ ID NO: 65 and where LC-CDR1 contains SEQ ID NO: 149, LC-CDR2 contains SEQ ID NO: 144 and LC-CDR3 contains SEQ ID NO: 146 [all 6 CDRs 1F3, 4B6 and 1A10].

15V. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, wherein said antibody or antigen-binding fragment thereof is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a mouse antibody, and an antigen-binding fragment of any of the above antibodies.

16V. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, wherein said antibody or antigen-binding fragment thereof is selected from the group consisting of single chain antibody, ScFv, Fab fragment, Fab' fragment, F(ab') ₂ fragment, a monovalent antibody that does not contain a hinge region and a whole antibody.

17V. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, also comprising an immunoglobulin constant region.

18V. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, wherein the antibody or antigen-binding fragment thereof is humanized.

19V. An antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising a sequence that is at least 80%, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 24, SEQ ID NO: 248 or SEQ ID NO: 249 and a light chain comprising a sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:40, SEQ ID NO:250 or SEQ ID NO: 278 [parental, humanized and modified versions of 4D5].

20V. An antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising a sequence that is at least 80%, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 25 and a light a chain comprising a sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 41 [1F3].

21B. An antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising a sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 26 and a light a chain comprising a sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 42 [4B6].

22V. An antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising a sequence that is at least 80%, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 27 and a light a chain comprising a sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 42 [1A10].

23b. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 22, wherein said antibody is

- 171 040888 lo binds to human MASP-3 with an affinity of less than 500 pM.

2B. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 23, wherein said antibody inhibits the activation of an alternative pathway in the blood of a mammal.

25V. A composition comprising an antibody or antigen-binding fragment thereof according to any one of

1B-24B and a pharmaceutically acceptable excipient.

B. High affinity group IB MASP-3 inhibitory antibodies that bind to one or more epitopes in the SP domain (options 10D12, 35C1 and 10D12) 1C. An isolated antibody or antigen-binding fragment thereof that binds to MASP-3 and contains:

(a) a heavy chain variable region comprising HC-CDR1 shown as SEQ ID NO: 213 (SYGXX where X at position 4 is M or I and where X at position 5 is S or T); HC-CDR2 shown as SEQ ID NO: 74; and HC-CDR3 shown as SEQ ID NO: 214 (GGXAXDY where X at position 3 is E or D and where X at position 5 is M or L); and (b) a light chain variable region comprising LC-CDR1 represented as SEQ ID NO: 215 (KSSQSLLDSXXKTYLX where X at position 10 is D, E or A; where X at position 11 is G or A; and where X at position 16 is N or S); LC-CDR2 shown as SEQ ID NO: 155; and LC-CDR3 shown as SEQ ID NO: 216 (WQGTHFPXT where X at position 8 is W or Y).

2C. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region HC-CDR1 according to (a) comprises SEQ ID NO: 72 (SYGMS). [10D12 and variants].

3C. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region HC-CDR1 according to (a) comprises SEQ ID NO: 79 (SYGIT). [35C1].

4C. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region HC-CDR3 according to (a) comprises SEQ ID NO: 76 (GGEAMDY). [10D12 and variants].

5C. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region HC-CDR3 according to (a) comprises SEQ ID NO: 82 (GGDALDY). [35C1].

6C. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain variable region LC-CDR1 according to (b) contains SEQ ID NO: 153 (KSSQSLLDSDGKTYLN); SEQ ID NO: 261 (KSSQSLLDDSEGKTYLN), SEQ ID NO: 262 (KSSQSLLDSAGKTYLN), or SEQ ID NO: 263 (KSSQSLLDSDAKTYLN). [10D12 and variants].

7C. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain variable region LC-CDR1 contains SEQ ID NO: 263 (KSSQSLLDSDAKTYLN) [option 10D12 GA].

8C. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain variable region LC-CDR1 contains SEQ ID NO: 152. [35C1].

9C. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain variable region LC-CDR3 according to (b) comprises SEQ ID NO: 159 (KSSQSLLDSDGKTYLS). [10D12].

10C. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain variable region LC-CDR3 according to (b) contains SEQ ID NO: 160 (WQGTHFPYT) [35C1].

11c. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein HC-CDR1 contains SEQ ID NO: 72, HC-CDR2 contains SEQ ID NO: 74, HC-CDR3 contains SEQ ID NO: 76, LC-CDR1 contains SEQ ID NO: 153, SEQ ID NO: 261, SEQ ID NO: 262 or SEQ ID NO: 263; LC-CDR2 contains SEQ ID NO: 155 and LC-CDR3 contains SEQ ID NO: 157 [all 6 CDRs of 10D12 with variants in LC-CDR1].

12C. The isolated antibody or antigen-binding fragment thereof according to claim 1, where HC-CDR1 contains SEQ ID NO: 79, HC-CDR2 contains SEQ ID NO: 74, HC-CDR3 contains SEQ ID NO: 82, LC-CDR1 contains SEQ ID NO: 159, LC-CDR2 contains SEQ ID NO: 155 and LC-CDR3 contains SEQ ID NO: 160 [all 6 CDRs of 35C1].

13C. An isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a mouse antibody, and an antigen-binding fragment of any of the above antibodies.

14C. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, wherein said antibody or antigen-binding fragment thereof is selected from the group consisting of single chain antibody, ScFv, Fab fragment, Fab' fragment, F(ab') 2 fragment, a monovalent antibody that does not contain a hinge region and a whole antibody.

15C. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, also comprising an immunoglobulin constant region.

16C. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, wherein the antibody or antigen-binding fragment thereof is humanized.

17C. An antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising a sequence that is at least

- 172 040888 is 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 28, SEQ ID NO: 251 or SEQ ID NO: 252 and a light chain comprising a sequence that is at least 80% 85, 90, 95, 98, 99 or 100% identical to SEQ ID NO: 43, SEQ ID NO: 253, or SEQ ID NO: 279 [parental, humanized and modified versions of 10D12].

18C. An antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising a sequence that is at least 80%, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 29 and a light a chain comprising a sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 44 [35C1].

19c. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, wherein said antibody binds to human MASP-3 with an affinity of less than 500 pM.

20C. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 19, wherein said antibody inhibits the activation of an alternative pathway in the blood of a mammal.

21c. A composition comprising an antibody or antigen-binding fragment thereof according to any one of paragraphs 1C-20C and a pharmaceutically acceptable excipient.

C. High affinity MASP-3 inhibitory antibodies of the IC group that bind to one or more epitopes in the SP domain (13B1 and variants).

1D. An isolated antibody or antigen-binding fragment thereof that binds to MASP-3 and contains:

(a) a heavy chain variable region comprising HC-CDR1 shown as SEQ ID NO: 84 (GKWIE); HC-CDR2 shown as SEQ ID NO: 86 (EILPGTGSTNYNEKFKG) or SEQ ID NO: 275 (EILPGTGSTNYAQKFQG); and HC-CDR3 shown as SEQ ID NO: 88 (SEDV); and (b) a light chain variable region comprising LC-CDR1 represented as SEQ ID NO: 142 (KSSQSLLNSRTRKNYLA), SEQ ID NO: 257 (KSSQSLLQSRTRKNYLA); SEQ ID NO: 258 (KSSQSLLASRTRKNYLA); or SEQ ID NO: 259 (KSSQSLLNTRTRKNYLA), LC-CDR2 shown as SEQ ID NO: 144 (WASTRES); and LC-CDR3 shown as SEQ ID NO: 161 (KQSYNIPT) [all 6 CDRs of 13B1 and variants in LC-CDR1].

2D. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the LC-CDR1 contains SEQ ID NO: 258. [Option 13B1 LC-CDR1 NA].

3D. An isolated antibody or antigen-binding fragment thereof according to claims 1, 2, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a mouse antibody, and an antigen-binding fragment of any of the above antibodies.

4D. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein said antibody or antigen-binding fragment thereof is selected from the group consisting of single chain antibody, ScFv, Fab fragment, Fab' fragment, F(ab') 2 fragment, a monovalent antibody that does not contain a hinge region and a whole antibody.

5D. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, also comprising an immunoglobulin constant region.

6d. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof is humanized.

7D. An antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising a sequence that is at least 80%, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 30, SEQ ID NO: 254 or SEQ ID NO: 255 and a light chain comprising a sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:45, SEQ ID NO:256 or SEQ ID NO: 280 [parental, humanized and modified versions of 13B1].

8D. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein said antibody binds to human MASP-3 with an affinity of less than 500 pM.

9D. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein said antibody inhibits the activation of an alternative pathway in the blood of a mammal.

10D. A composition comprising an antibody or antigen-binding fragment thereof according to any one of paragraphs 1D-9D and a pharmaceutically acceptable excipient.

D. High affinity group II MASP-3 inhibitory antibodies that bind to one or more epitopes in the SP (1G4) domain.

1E. An isolated antibody or antigen-binding fragment thereof that binds to MASP-3 and contains:

(a) heavy chain variable region comprising HC-CDR1 shown as SEQ ID NO: 91 (GYWIE); HC-CDR2 shown as SEQ ID NO: 93 (EMLPGSGSTHYNEKFKG) and HC-CDR3 shown as SEQ ID NO: 95 (SIDY); and (b) a light chain variable region comprising LC-CDR1 given as SEQ ID NO: 163 (RSSQSLVQSNGNTYLH), LC-CDR2 given as SEQ ID NO: 165 (KVSNRFS) and LC-CDR3 given as SEQ ID NO: 167 (SQSTHVPPT).

- 173 040888

2E. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a mouse antibody, and an antigen-binding fragment of any of the above antibodies.

3E. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, wherein said antibody or antigen-binding fragment thereof is selected from the group consisting of single chain antibody, ScFv, Fab fragment, Fab' fragment, F(ab') ₂ fragment, a monovalent antibody that does not contain a hinge region and a whole antibody.

4E. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, also comprising an immunoglobulin constant region.

5E. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof is humanized.

6E. An antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising a sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 31 and a light chain a chain comprising a sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 46 [1G4].

7E. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein said antibody binds to human MASP-3 with an affinity of less than 500 pM.

8E. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein said antibody inhibits the activation of an alternative pathway in the blood of a mammal.

9E. A composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1E8E and a pharmaceutically acceptable excipient.

F. High affinity group III MASP-3 inhibitory antibodies that bind to one or more epitopes in the SP domain (1E7, 2D7, 15D9, 2F5, 1B11, 2F2, 11B6).

1F. An isolated antibody or antigen-binding fragment thereof that binds to MASP-3 and contains:

(a) a heavy chain variable region comprising HC-CDR1 shown as SEQ ID NO: 109 (RVHFAIRDTNYWMQ), HC-CDR2 shown as SEQ ID NO: 110 (AIYPGNGDTSYNQKFKG), HC-CDR3 shown as SEQ ID NO: 112 (GSHYFDY); and a light chain variable region comprising LC-CDR1 shown as SEQ ID NO: 182 (rASQSIGTSIH), LC-CDR2 shown as SEQ ID NO: 184 (YASESIS) and LC-CDR3 shown as SEQ ID NO: 186 (QQSNSWPYT ) [1E7]; or (b) a heavy chain variable region comprising HC-CDR1 given as SEQ ID NO: 125 (DYYMN), HC-CDR2 given as SEQ ID NO: 127 (DVNPNNDGTTYNQKFKG), HC-CDR3 given as SEQ ID NO: 129(CPFYYLGKGTHFDY); and a light chain variable region comprising LC-CDR1 given as SEQ ID NO: 196 (RASQDISNFLN), LC-CDR2 given as SEQ ID NO: 198 (YTSRLHS) and LC-CDR3 given as SEQ ID NO: 200 (QQGFTLPWT ) [2D7]; or (c) a heavy chain variable region comprising HC-CDR1, shown as SEQ ID NO: 132, HC-CDR2, shown as SEQ ID NO: 133, HC-CDR3, shown as SEQ ID NO: 135; and a light chain variable region comprising LC-CDR1, shown as SEQ ID NO: 203, LCCDR2, shown as SEQ ID NO: 165, and LC-CDR3, shown as SEQ ID NO: 204 [49C11]; or (d) a heavy chain variable region comprising HC-CDR1, shown as SEQ ID NO: 137, HC-CDR2, shown as SEQ ID NO: 138, HC-CDR3, shown as SEQ ID NO: 140; and a light chain variable region comprising LC-CDR1 shown as SEQ ID NO: 206, LCCDR2 shown as SEQ ID NO: 207 and LC-CDR3 shown as SEQ ID NO: 208 [15D9]; or (e) a heavy chain variable region comprising HC-CDR1, shown as SEQ ID NO: 98, HC-CDR2, shown as SEQ ID NO: 99, HC-CDR3, shown as SEQ ID NO: 101; and a light chain variable region comprising LC-CDR1, shown as SEQ ID NO: 169, LCCDR2, shown as SEQ ID NO: 171, and LC-CDR3, shown as SEQ ID NO: 173 [2F5]; or (f) a heavy chain variable region comprising HC-CDR1, shown as SEQ ID NO: 103, HC-CDR2, shown as SEQ ID NO: 105, HC-CDR3, shown as SEQ ID NO: 107; and a light chain variable region comprising LC-CDR1 shown as SEQ ID NO: 176, LCCDR2 shown as SEQ ID NO: 178 and LC-CDR3 shown as SEQ ID NO: 193 [1B11]; or (g) a heavy chain variable region comprising HC-CDR1, shown as SEQ ID NO: 114, HC-CDR2, shown as SEQ ID NO: 116, HC-CDR3, shown as SEQ ID NO: 118; and a light chain variable region comprising LC-CDR1, shown as SEQ ID NO: 188, LCCDR2, shown as SEQ ID NO: 178, and LC-CDR3, shown as SEQ ID NO: 190 [2F2]; or (h) a heavy chain variable region comprising HC-CDR1 shown as SEQ ID NO:

- 174 040888

114, HC-CDR2 given as SEQ ID NO: 121, HC-CDR3 given as SEQ ID NO: 123; and a light chain variable region comprising LC-CDR1, shown as SEQ ID NO: 191, LCcDr2, shown as SEQ ID NO: 178, and LC-CDR3, shown as SEQ ID NO: 193 [11B6].

2F. An isolated antibody or antigen-binding fragment thereof according to claim 1(a)-(g), wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a mouse antibody, and an antigen-binding fragment of any of the above antibodies.

3F. An antibody or antigen-binding fragment thereof according to any one of claims 1, 2, wherein said antibody or antigen-binding fragment thereof is selected from the group consisting of single chain antibody, ScFv, Fab fragment, Fab' fragment, F(ab') ₂ fragment, a monovalent antibody that does not contain a hinge region and a whole antibody.

4F. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, also comprising an immunoglobulin constant region.

5F. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof is humanized.

6F. An antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising a sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 32 and a light a chain comprising a sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 47 [1E7].

7F. An antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising a sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 33 and a light chain a chain comprising a sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 48 [2D7].

8F. An antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising a sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 34 and a light a chain comprising a sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 49 [49C11].

9F. An antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising a sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 35 and a light chain a chain comprising a sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 50 [15D9].

10F. An antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising a sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 36 and a light a chain comprising a sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 51 [2F5].

11F. An antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising a sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 37 and a light a chain comprising a sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 52 [1B11].

12F. An antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising a sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 38 and a light a chain comprising a sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 53 [2F2].

13F. An antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising a sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 39 and a light a chain comprising a sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 54 [11B6].

14F. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, wherein said antibody binds to human MASP-3 with an affinity of less than 500 pM.

15F. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, wherein said antibody inhibits the activation of an alternative pathway in the blood of a mammal.

16F. A composition comprising an antibody or antigen-binding fragment thereof according to any one of paragraph 1F15F and a pharmaceutically acceptable excipient.

E. Use of MASP-3 inhibitory antibodies for the treatment of AP-diseases.

1. A method of inhibiting alternative pathway complement activation in a mammal, wherein the method comprises administering to a mammal in need thereof a composition comprising a high affinity MASP-3 inhibitory antibody or antigen-binding fragment thereof in an amount sufficient to inhibit alternative pathway complement activation in the mammal.

2. The method of claim 1, wherein the antibody, or antigen-binding fragment thereof, binds to MASP-3 with an affinity of less than 500 pM.

3. The method according to claim 1, where as a result of the introduction of a composition containing an antibody or antigen-binding fragment, one or more of the following signs are observed in mammals:

- 175 040888 (a) inhibition of factor D maturation;

(b) inhibition of the alternative pathway when administered to an individual in a molar ratio of about 1:1 to about 2.5:1 (MASP-3 target: mAb);

(c) no classical pathway inhibition;

(d) inhibition of hemolysis and/or opsonization;

(e) reducing the degree of hemolysis or degree of C3 cleavage and deposition of superficial C3b;

(f) reducing the deposition of factor B and Bb on the activating surface;

(g) reduction of residual levels (in the circulation and without experimental additional surface activation) of active factor D compared to pro-factor D;

(h) a decrease in active factor D levels relative to pro factor D in response to surface activation; and/or (i) reducing the level of production of residual and surface-induced levels of liquid phase Ba, Bb, C3b or C3a.

4. The method of claim 1 wherein the antibody inhibits the alternative pathway in a molar ratio of about 1:1 to about 2.5:1 (MASP-3 target: mAb).

5. The method according to any one of claims 1 to 3, wherein the high affinity anti-MASP-3 antibody is characterized according to any one of claims 27A, 25B, 21C, 10D, 9E, or 16F.

6. The method according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment selectively inhibits the alternative pathway but does not affect the activation of the classical pathway.

7. The method according to any one of claims 1 to 4, wherein the mammal suffers from or is at risk of developing a disease or disorder associated with the alternative pathway and is selected from the group consisting of paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD, including wet and dry VAT), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy (including hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), and thrombotic thrombocytopenic purpura (TTP) or transplant-associated TMA), asthma, disease of dense deposits, oligoimmune necrosing crescentic glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis of the optic nerve, Behcet's disease, multiple sclerosis, Guillain-Barré syndrome, Alzheimer's disease, amylotrophic lateral sclerosis (ABS), lupus nephritis, systemic beautiful lupus erythematosus (SLE), diabetic retinopathy, uveitis, chronic obstructive pulmonary disease (COPD), C3 glomerulopathy, graft rejection, graft-versus-host disease (GVHD), hemodialysis, sepsis, systemic inflammatory response syndrome (SIR), acute respiratory distress syndrome (ARDS), ANCA vasculitis, antiphospholipid syndrome, atherosclerosis, IgA nephropathy and myasthenia gravis.

Although the invention describes and illustrates the preferred embodiments of its implementation, however, it is obvious that various changes can be made to them without going beyond the spirit and scope of the invention.

Claims (35)

1. An isolated antibody or antigen-binding fragment thereof that binds to MASP-3 and includes: (a) a heavy chain variable region comprising HC-CDR1 shown as SEQ ID NO: 84 (GKWIE); HC-CDR2 shown as SEQ ID NO: 86 (EILPGTGSTNYNEKFKG); and HC-CDR3 shown as SEQ ID NO: 88 (SEDV); and (b) a light chain variable region comprising LCCDR1 shown as SEQ ID NO: 142 (KSSQSLLNSRTRKNYLA), LC-CDR2 shown as SEQ ID NO: 144 (WASTRES); and LC-CDR3 shown as SEQ ID NO: 161 (KQSYNIPT); or (b) a heavy chain variable region comprising HC-CDR1 shown as SEQ ID NO: 84 (GKWIE); HC-CDR2 shown as SEQ ID NO: 275 (EILPGTGSTNYAQKFQG); and HC-CDR3 shown as SEQ ID NO: 88 (SEDV); and a light chain variable region containing LCCDR1 shown as SEQ ID NO: 142 (KSSQSLLNSRTRKNYLA); LC-CDR2 shown as SEQ ID NO: 144 (WASTRES); and LC-CDR3 shown as SEQ ID NO: 161 (KQSYNIPT); or (c) a heavy chain variable region comprising HC-CDR1 shown as SEQ ID NO: 84 (GKWIE); HC-CDR2 shown as SEQ ID NO: 275 (EILPGTGSTNYAQKFQG); and HC-CDR3 shown as SEQ ID NO: 88 (SEDV); and a light chain variable region comprising LCCDR1 shown as SEQ ID NO: 258 (KSSQSLLASRTRKNYLA); LC-CDR2 shown as SEQ ID NO: 144 (WASTRES) and LC-CDR3 shown as SEQ ID NO: 161 (KQSYNIPT); or (d) a heavy chain variable region comprising HC-CDR1 shown as SEQ ID NO: 84 (GKWIE); HC-CDR2 shown as SEQ ID NO: 86 (EILPGTGSTNYNEKFKG); and HC-CDR3 shown as SEQ ID NO: 88 (SEDV); and a light chain variable region comprising LCCDR1 shown as SEQ ID NO: 258 (KSSQSLLASRTRKNYLA); LC-CDR2 shown as SEQ ID NO: 144 (WASTRES) and LC-CDR3 shown as SEQ ID NO: 161 (KQSYNIPT). - 176 040888 2. An isolated antibody or antigen-binding fragment thereof that binds to MASP-3 and includes: (a) a heavy chain variable region comprising HC-CDR1 shown as SEQ ID NO: 72 (SYGMS); HC-CDR2 shown as SEQ ID NO: 74 (WINTYSGVPTYADDFKG); and HC-CDR3 shown as SEQ ID NO: 76 (GGEAMDY); and a light chain variable region comprising LC-CDR1 shown as SEQ ID NO: 153 (KSSQSLLDSDGKTYLN); LC-CDR2 shown as SEQ ID NO: 155 (LVSKLDS); and LC-CDR3 shown as SEQ ID NO: 157 (WQGTHFPWT); (b) a heavy chain variable region comprising HC-CDR1 shown as SEQ ID NO: 72 (SYGMS); HC-CDR2 shown as SEQ ID NO: 74 (WINTYSGVPTYADDFKG); and HC-CDR3 shown as SEQ ID NO: 76 (GGEAMDY); and a light chain variable region comprising LC-CDR1 shown as SEQ ID NO: 263 (KSSQSLLDSDAKTYLN); LC-CDR2 shown as SEQ ID NO: 155 (LVSKLDS); and LC-CDR3 shown as SEQ ID NO: 157 (WQGTHFPWT); or (c) a heavy chain variable region comprising HC-CDR1 shown as SEQ ID NO: 79 (SYGIT); HC-CDR2 shown as SEQ ID NO: 74 (WINTYSGVPTYADDFKG); and HC-CDR3 shown as SEQ ID NO: 82 (GGDALDY); and a light chain variable region comprising LC-CDR1 shown as SEQ ID NO: 159 (KSSQSLLDSDGKTYLS); LC-CDR2 shown as SEQ ID NO: 155 (LVSKLDS); and LC-CDR3 shown as SEQ ID NO: 160 (WQGTHFPYT). 3. An isolated antibody or antigen-binding fragment thereof that binds to MASP-3 and includes: (a) a heavy chain variable region comprising HC-CDR1 shown as SEQ ID NO: 56 (TDDIN), HC-CDR2 shown as SEQ ID NO: 58 (WIYPRDDRTKYNDKFKD); and HC-CDR3 shown as SEQ ID NO: 60 (LEDTY); and a light chain variable region comprising LCCDR1 shown as SEQ ID NO: 142 (KSSQSLLNSRTRKNYLA); LC-CDR2 shown as SEQ ID NO: 144 (WASTRES); and LC-CDR3 shown as SEQ ID NO: 146 (KQSYNLYT); (b) a heavy chain variable region comprising HC-CDR1 shown as SEQ ID NO: 56 (TDDIN), HC-CDR2 shown as SEQ ID NO: 58 (WIYPRDDRTKYNDKFKD); and HC-CDR3 shown as SEQ ID NO: 60 (LEDTY); and a light chain variable region comprising LCCDR1 shown as SEQ ID NO: 258 (KSSQSLLASRTRKNYLA); LC-CDR2 shown as SEQ ID NO: 144 (WASTRES); and LC-CDR3 shown as SEQ ID NO: 146 (KQSYNLYT); (c) a heavy chain variable region comprising HC-CDR1 shown as SEQ ID NO: 62 (SNDIN), HC-CDR2 shown as SEQ ID NO: 63 (WIYPRDGSIKYNEKFTD); and HC-CDR3 shown as SEQ ID NO: 65 (VEDSY); and a light chain variable region comprising LCCDR1 shown as SEQ ID NO: 149 (KSSQSLLISRTRKNYLS); LC-CDR2 shown as SEQ ID NO: 144 (WASTRES); and LC-CDR3 shown as SEQ ID NO: 146 (KQSYNLYT); (d) a heavy chain variable region comprising HC-CDR1 shown as SEQ ID NO: 62 (SNDIN), HC-CDR2 shown as SEQ ID NO: 67 (WIYPRDGTTKYNEEFTD); and HC-CDR3 shown as SEQ ID NO: 65 (VEDSY); and a light chain variable region comprising LCCDR1 shown as SEQ ID NO: 149 (KSSQSLLISRTRKNYLS); LC-CDR2 shown as SEQ ID NO: 144 (WASTRES); and LC-CDR3 shown as SEQ ID NO: 146 (KQSYNLYT); or (e) a heavy chain variable region comprising HC-CDR1 shown as SEQ ID NO: 62 (SNDIN), HC-CDR2 shown as SEQ ID NO: 69 (WIYPRDGTTKYNEKFTD); and HC-CDR3 shown as SEQ ID NO: 65 (VEDSY); and a light chain variable region comprising LCCDR1 shown as SEQ ID NO: 149 (KSSQSLLISRTRKNYLS); LC-CDR2 shown as SEQ ID NO: 144 (WASTRES); and LC-CDR3 shown as SEQ ID NO: 146 (KQSYNLYT). 4. An antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region comprises SEQ ID NO: 30 and the light chain variable region comprises SEQ ID NO: 45. 5. An antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region comprises SEQ ID NO: 255 and the light chain variable region comprises SEQ ID NO: 256. 6. An antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region comprises SEQ ID NO: 254 and the light chain variable region comprises SEQ ID NO: 256. 7. An antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region comprises SEQ ID NO: 254 and the light chain variable region comprises SEQ ID NO: 280. 8. An antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region comprises SEQ ID NO: 255 and the light chain variable region comprises SEQ ID NO: 280. 9. An antibody or antigen-binding fragment thereof according to claim 2, wherein the heavy chain variable region comprises SEQ ID NO: 28 and the light chain variable region comprises SEQ ID NO: 43. 10. An antibody or antigen-binding fragment thereof according to claim 2, wherein the heavy chain variable region comprises SEQ ID NO: 251 and the light chain variable region comprises SEQ ID NO: 253. 11. An antibody or antigen-binding fragment thereof according to claim 2, wherein the heavy chain variable region comprises SEQ ID NO: 252 and the light chain variable region comprises SEQ ID NO: 253. 12. An antibody or antigen-binding fragment thereof according to claim 2, wherein the heavy chain variable region comprises SEQ ID NO: 251 and the light chain variable region comprises SEQ ID NO: 279. - 177 040888 13. An antibody or antigen-binding fragment thereof according to claim 2, wherein the heavy chain variable region comprises SEQ ID NO: 252 and the light chain variable region comprises SEQ ID NO: 279. 14. An antibody or antigen-binding fragment thereof according to claim 2, wherein the heavy chain variable region comprises SEQ ID NO: 29 and the light chain variable region comprises SEQ ID NO: 44. 15. An antibody or antigen-binding fragment thereof according to claim 3, wherein the heavy chain variable region comprises SEQ ID NO: 24 and the light chain variable region comprises SEQ ID NO: 40. 16. An antibody or antigen-binding fragment thereof according to claim 3, wherein the heavy chain variable region comprises SEQ ID NO: 248 and the light chain variable region comprises SEQ ID NO: 250. 17. An antibody or antigen-binding fragment thereof according to claim 3, wherein the heavy chain variable region comprises SEQ ID NO: 249 and the light chain variable region comprises SEQ ID NO: 250. 18. An antibody or antigen-binding fragment thereof according to claim 3, wherein the heavy chain variable region comprises SEQ ID NO: 248 and the light chain variable region comprises SEQ ID NO: 278. 19. An antibody or antigen-binding fragment thereof according to claim 3, wherein the heavy chain variable region comprises SEQ ID NO: 249 and the light chain variable region comprises SEQ ID NO: 278. 20. An antibody or antigen-binding fragment thereof according to claim 3, wherein the heavy chain variable region comprises SEQ ID NO: 25 and the light chain variable region comprises SEQ ID NO: 41. 21. An antibody or antigen-binding fragment thereof according to claim 3, wherein the heavy chain variable region contains SEQ ID NO: 26 and the light chain variable region contains SEQ ID NO: 42. 22. An antibody or antigen-binding fragment thereof according to claim 2, wherein the heavy chain variable region comprises SEQ ID NO: 27 and the light chain variable region comprises SEQ ID NO: 42. 23. A combination of an isolated DNA sequence encoding a heavy chain variable region and an isolated DNA sequence encoding a light chain variable region of an antibody or antigen-binding fragment thereof according to any one of claims 1-22. 24. An antibody or antigen-binding fragment thereof according to any one of claims 1-22, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a mouse antibody, and an antigen-binding fragment of any of the above antibodies. 25. An antibody or antigen-binding fragment thereof according to any one of claims 1-22, wherein said antibody or antigen-binding fragment thereof is selected from the group consisting of single chain antibody, ScFv, Fab fragment, Fab' fragment, F(ab')2- a fragment, a monovalent antibody that does not contain a hinge region and a whole antibody. 26. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 22, also comprising an immunoglobulin constant region. 27. An antibody or antigen-binding fragment thereof according to any one of claims 1-22, wherein the antibody or antigen-binding fragment thereof is humanized. 28. An antibody or antigen-binding fragment thereof according to any one of claims 1-22, wherein said antibody binds to the human MASP-3 serine protease domain with an affinity of less than 500 pM. 29. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 22, wherein said antibody inhibits the activation of an alternative pathway in the blood of a mammal. 30. An isolated DNA sequence encoding the heavy and light chain variable region of an antibody or antigen-binding fragment thereof according to any one of claims 1-22. 31. A cloning or expression vector containing one or more DNA sequences according to claim 30 or a combination of two or more cloning or expression vectors that contain a combination of DNA sequences according to claim 23. 32. A host cell containing one or more cloning or expression vectors according to claim 31. 33. A method for producing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 22, comprising culturing a host cell according to claim 32 and isolating the antibody or antigen-binding fragment thereof. 34. A composition for inhibiting alternative pathway complement activation, comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-22 and a pharmaceutically acceptable excipient. 35. A method of inhibiting alternative pathway complement activation in a mammal, wherein the method comprises administering to a mammal in need thereof a composition according to claim 34 comprising a high affinity MASP-3 inhibitory antibody or antigen-binding fragment thereof in an amount sufficient to inhibit alternative pathway complement activation in a mammal.