Compositions and methods for inhibiting MASP-3 for the treatment of various diseases and disorders

Abstract

The present invention relates to compositions and methods for treating various diseases and disorders by inhibiting MASP-3. In particular, the present invention relates to MASP-3 inhibiting antibodies and compositions comprising such antibodies for use in inhibiting adverse effects of MASP-3 dependent complement activation.

Description

[0001] This application is a divisional application of Chinese patent application 201780047995.9, filed on July 31, 2017, entitled "Compositions and methods for inhibiting MASP-3 for treating various diseases and symptoms".

[0002] Cross-references to related applications

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 369,674, filed August 1, 2016; U.S. Provisional Application No. 62 / 419,420, filed November 8, 2016; and U.S. Provisional Application No. 62 / 478,336, filed March 29, 2017, all of which are incorporated herein by reference in their entirety.

[0004] Declaration of sequence list

[0005] A text-formatted sequence list related to this application is provided in lieu of a paper copy and is incorporated herein by reference. The text file containing the sequence list is named MP_1_0254_US_Sequence_Listing_20170628_ST25; the file size is 191 KB; it was created on June 28, 2017 and submitted with this specification via EFS-Web. Technical Field

[0006] This invention relates to the field of antibodies, and more specifically to MASP-3 inhibitory antibodies and compositions comprising such antibodies. Background Technology

[0007] The complement system provides an early mechanism of action for initiating, amplifying, and arranging immune responses against microbial infections and other acute injuries in humans and other vertebrates (MK Liszewski and JP Atkinson, 1993). Fundamental Immunology 3rd Edition, edited by WE Paul, Raven Press, Ltd., New York. While complement activation provides an important first line of defense against potential pathogens, complement activity that promotes protective immune responses can also pose a potential threat to the host (KR Kalli et al.). Springer Semin. Immunopathol.15 :417-431, 1994; BP Morgan, Eur. J. Clinical Investig. 24(219-228, 1994). For example, C3 and C5 proteolytic products recruit and activate neutrophils. Although essential for host defense, activated neutrophils release their destructive enzymes indiscriminately, which can lead to organ damage. Furthermore, complement activation can result in the deposition of cytolytic complement components on nearby host cells and microbial targets, leading to host cell lysis.

[0008] The complement system is also involved in the pathogenesis of many acute and chronic disease states, including myocardial infarction, stroke, ARDS, reperfusion injury, septic shock, capillary leakage after thermal 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 is not the cause, but rather one of several factors involved in the pathogenesis. Nevertheless, complement activation can be an important pathological mechanism and has proven effective in the clinical control of many of these disease states. The growing recognition of the importance of complement-mediated tissue damage in various disease states underscores the need for effective complement inhibitors. To date, eculizumab (Solaris®), an antibody targeting C5, is the only approved complement-targeting drug for human use. However, C5 is one of several effector molecules located "downstream" of the complement system, and blocking C5 does not inhibit complement system activation. Therefore, inhibitors of the initiation step of complement activation will have a significant advantage over "downstream" complement inhibitors.

[0009] Currently, it is generally accepted that the complement system can be activated through three distinct pathways: the classical pathway, the lectin pathway, and the alternative pathway. The classical pathway is typically triggered by a complex of host antibodies binding to exogenous particles (i.e., antigens), and therefore requires prior exposure to the antigen to elicit a specific antibody response. Because activation of the classical pathway depends on the host's prior acquired immune response, it is part of the acquired immune system. In contrast, both the lectin and alternative pathways are independent of acquired immunity and are part of the innate immune system.

[0010] Activation of the complement system leads to the successive activation of serine protease zymogens. The first step in activation via the classical pathway is the binding of the specific recognition molecule C1q to IgG and IgM molecules bound to the antigen. C1q binds to the C1r and C1s serine protease zymogens to form a complex called C1. When C1q binds to the immune complex, the Arg-Ile site of C1r undergoes autoproteolytic cleavage, followed by C1r-mediated Cls cleavage and activation, which then acquires the ability to cleave C4 and C2. C4 is cleaved into two fragments, called C4a and C4b, and similarly, C2 is cleaved into C2a and C2b. The C4b fragment can form covalent bonds with adjacent hydroxyl or amino groups and generates C3 convertase (C4b2b) through non-covalent interactions with the C2a fragment of activated C2. C3 convertase (C4b2b) activates C3 by proteolytically cleaving it into C3a and C3b subcomponents, leading to the generation of C5 convertase (C4b2a3b). C5 convertase, in turn, cleaves C5, resulting in the formation of the membrane attack complex (C5b, also known as "MAC"), which can disrupt the cell membrane and cause cell lysis. The activated forms of C3 and C4 (C3b and C4b) are covalently deposited on exogenous target surfaces and are recognized by complement receptors on various phagocytes.

[0011] Independently, the first step in complement system activation via the lectin pathway is also the binding of a specific recognition molecule, followed by activation of the bound serine protease prozyme. However, the recognition molecules in the lectin pathway include a group of glyco-binding proteins collectively known as lectins (mannan-binding lectin (MBL), H-ficolin, M-ficolin, L-ficolin, and C-type lectin CL-11), rather than binding to immune complexes via Clq. See J. Lu et al., Biochim. Biophys. Acta 1572 :387-400, (2002); Holmskov et al., Annu. Rev. Immunol 21:547-578 (2003); Teh et al., Immunology101 :225-232 (2000)). See also J. Luet et al., Biochim Biophys Acta 1572:387-400 (2002); Holmskov et al., Annu Rev Immunol 21:547-578 (2003); Teh et al., Immunology 101:225-232 (2000); Hansen et al., J. Immunol 185(10):6096-6104 (2010).

[0012] Ikeda et al. first demonstrated that, similar to C1q, MBL can activate the complement system in a C4-dependent manner after binding to yeast mannan-coated erythrocytes (Ikeda et al., J.Biol. Chem. 262 MBL (7451-7454, (1987)) is a member of the collagen lectin family and is a calcium-dependent lectin that binds to carbohydrates with 3- and 4-hydroxyl groups oriented towards the equatorial plane of the pyranose ring. Therefore, the important ligands for MBL are D-mannose and N-acetyl-D-glucosamine, while carbohydrates that do not meet this spatial requirement have no detectable affinity for MBL (Weis et al., 7451-7454, (1987)). Nature 360:127-134, (1992)). The interaction between MBL and monovalent sugars is quite weak, with dissociation constants typically in the single-digit millimole range. MBL achieves its specific tight binding to glycan ligands through affinity, i.e., by simultaneously interacting with multiple monosaccharide residues located close to each other (Lee et al., Archiv. Biochem. Biophys. 299:129-136, (1992)). MBL recognizes carbohydrate patterns that typically modify microorganisms such as bacteria, yeast, parasites, and certain viruses. Conversely, MBL does not recognize D-galactose and sialic acid, the penultimate and penultimate sugars, which generally modify "mature" complex glycoconjugates present on mammalian plasma and cell surface glycoproteins. This binding specificity is thought to facilitate recognition of "exogenous" surfaces and contribute to protection against "self-activation." However, MBL does bind with high affinity to high-mannose "precursor" glycan clusters located on N-linked glycoproteins and glycolipids isolated in the endoplasmic reticulum and Golgi apparatus of mammalian cells (Maynard et al., 2009). J. Biol. Chem. 257 :3788-3794, (1982)). Furthermore, it has been demonstrated that MBL can bind to polynucleotides, DNA, and RNA exposed on necrotic and apoptotic cells (Palaniyar et al., ). Ann. NY Acad. Sci., 1010:467-470 (2003); Nakamura et al., J. Leuk. Biol. 86:737-748 (2009)). Therefore, damaged cells are potential targets for activation via the MBL-bound lectin pathway.

[0013] Fibrin possesses a lectin domain of a different type than that of MBL, called the fibrinogen-like domain. Fibrin generates calcium-independent... ++The lectin binds to sugar residues via a specific pathway. In humans, three types of fibrinogens (L-fibrinogen, M-fibrinogen, and H-fibrinogen) have been identified. Both L-fibrinogen and H-fibrinogen, two serum fibrinogens, are specific for N-acetyl-D-glucosamine; however, H-fibrinogen also binds to N-acetyl-D-galactosamine. The difference in sugar specificity among L-fibrinogen, H-fibrinogen, CL-1I, and MBL suggests that different lectins can be complementary and, despite overlap, can target different glycoconjugates. This view is supported by recent reports that, among the known lectins of the lectin pathway, only L-fibrinogen specifically binds to lipoteichoic acid, a cell wall glycoconjugate found in all Gram-positive bacteria (Lynch et al.). J. Immunol. 172 :1198-1202, (2004)). In addition to the acetylated sugar portion, fibrinogen can also bind acetylated amino acids and peptides (Thomsen et al., Mol. Immunol 48(4):369-81(2011)). Collagen lectins (MBL) and fibrinogens do not share significant similarities in their amino acid sequences. However, these two groups of proteins have similar domain structures and, like C1q, assemble into oligomeric structures, thus maximizing the possibility of multi-site binding.

[0014] Serum concentrations of MBL are highly variable in healthy individuals, and this is genetically controlled by polymorphisms / mutations in both the promoter and coding region of the MBL gene. As an acute-phase protein, MBL expression is further upregulated during inflammation. L-fibrinogen is present in serum at concentrations similar to those of MBL. Therefore, the L-fibrinogen branch of the lectin pathway may be comparable in strength to the MBL branch. MBL and fibrinogen may also function as opsonins, allowing phagocytes to target MBL- and fibrinogen-modified surfaces (see Jack et al.). J Leukoc Biol ., 77(3):328-36 (2004), Matsushita and Fujita, Immunobiology , 205(4-5):490-7(2002), Aoyagi et al., J Immunol, 174(1):418-25(2005)). This opsonin action requires the interaction of these proteins with phagocytic receptors (Kuhlman et al., J. Exp. Med. 169 :1733, (1989); Matsushita et al., J. Biol. Chem. 271 The identities of these phagocytic receptors have not yet been determined (2448-54, (1996)).

[0015] Human MBL forms a specific and high-affinity interaction with a unique C1r / C1s-like serine protease (called MBL-associated serine protease (MASP)) through its collagen-like domain. Three MASPs have been described to date. First, a single enzyme, “MASP,” was identified, characterized as an enzyme responsible for initiating the complement cascade (i.e., cleavage of C2 and C4) (Matsushita et al., J Exp Med 176(6):1497-1502 (1992); Ji et al., J. Immunol. 150 :571-578, (1993)). Subsequently, it was determined that MASP activity was actually a mixture of two proteases, MASP-1 and MASP-2 (Thiel et al., Nature 386 :506-510, (1997)). However, it has been confirmed that the MBL-MASP-2 complex alone is sufficient to activate complement (Vorup-Jensen et al., ). J. Immunol. 165 :2093-2100, (2000)). Furthermore, only MASP-2 cuts C2 and C4 at high speeds (Ambrus et al., J. Immunol. 170 :1374-1382, (2003)). Therefore, MASP-2 is the protease responsible for activating C4 and C2 to produce the C3 convertase C4b2a. This is a significant difference from the C1 complex in the classical pathway, where the synergistic action of two specific serine proteases (C1r and C1s) leads to the activation of the complement system. In addition, a third novel protease, MASP-3, has been isolated (Dahl, MR et al., ). Immunity 15 MASP-1 and MASP-3 are alternative splicing products of the same gene (127-35, 2001).

[0016] The enzymatic components C1r and C1s of the MASP-Cl complex share the same domain structure (Sim et al.). Biochem. Soc. Trans. 28 :545, (2000)). These domains include an N-terminal C1r / C1s / sea urchin VEGF / bone morphogenetic protein (CUB) domain, an epidermal growth factor-like domain, a second CUB domain, a tandem complement regulatory protein domain, and a serine protease domain. As in the C1 protease, MASP-2 activation occurs via the cleavage of the Arg-I1e bond near the serine protease domain, which breaks the enzyme into a disulfide-linked A chain and a B chain, the latter consisting of the serine protease domain.

[0017] MBL also associates with an alternative splicing form of MASP-2, known as 19 kDa MBL-associated protein (MAp19) or small MBL-associated protein (sMAP), which lacks the catalytic activity of MASP-2 (Stover). J. Immunol. 162 :3481-90, (1999); Takahashi et al., Int. Immunol. 11 MAp19 comprises the first two domains of MASP-2, followed by an additional sequence of four unique amino acids. The function of MAp19 is unclear (Degn et al., 859-863, (1999)). J Immunol. Methods MASP-1 and MASP-2 genes are located on human chromosomes 3 and 1, respectively (Schwaeble et al., 2011). Immunobiology 205 :455-466, (2002)).

[0018] Several pieces of evidence suggest the existence of distinct MBL-MASP complexes, and that most MASPs in serum do not complex with MBL (Thiel et al., J. Immunol. 165 :878-887, (2000)). Both H-fibrin and L-fibrin bind to all MASPs and activate the lectin complement pathway, as is the case with MBL (Dahl et al., 878-887, (2000)). Immunity 15 :127-35, (2001); Matsushita et al., J. Immunol. 168 :3502-3506, (2002)). Both the lectin pathway and the classical pathway form a common C3 convertase (C4b2a), and the two pathways converge at this step.

[0019] The lectin pathway is generally considered to play an important role in host defense against infection in naïve hosts. Strong evidence for MBL involvement in host defense comes from an analysis of patients with functionally reduced serum MBL levels (Kilpatrick, Biochim. Biophys. Acta 1572 (401-413, (2002)). These patients exhibit susceptibility to recurrent bacterial and fungal infections. These symptoms are typically seen early in life, during the vulnerable epigenetic window, due to reduced antibody titers acquired from the mother, but prior to the development of a complete antibody response profile. This syndrome is often caused by mutations at several sites in the collagen portion of MBL, which interfere with the proper formation of MBL oligomers. However, because MBL can function as a complement-independent opsonin, the extent to which the increased susceptibility to infection is due to impaired complement activation is unknown.

[0020] Unlike the classical and lectin pathways, no initiator has been previously identified for the recognition function in the alternative pathway, whereas in the other two pathways it is C1q and lectins that perform the recognition function. It is generally accepted that the alternative pathway spontaneously undergoes low-level turnover activation, which can be readily amplified on foreign or other aberrant surfaces (bacterial, yeast, virus-infected cells, or damaged tissues) lacking the appropriate molecular elements to maintain controlled spontaneous complement activation. Four plasma proteins are directly involved in the activation of the alternative pathway: C3, factors B and D, and properdin.

[0021] Despite substantial evidence suggesting that both the classical and alternative complement pathways are involved in the pathogenesis of non-infectious human diseases, the evaluation of the role of the lectin pathway is still in its early stages. Recent studies provide evidence 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 bound to MBL and exhibited C3 deposition upon exposure to human serum (Collard et al., Am. J. Pathol. 156 :1549-1556, (2000)). Furthermore, treatment of human serum with a blocking anti-MBL monoclonal antibody inhibited MBL binding and complement activation. These findings were extended to a rat model of myocardial ischemia-reperfusion, in which rats treated with a blocking antibody against rat MBL showed significantly less myocardial injury in coronary artery occlusion compared to rats treated with a control antibody (Jordan et al., ). Circulation 104 :1413-1418, (2001)). The molecular mechanism by which MBL binds to vascular endothelium after oxidative stress remains unclear; recent studies suggest that activation of the lectin pathway after oxidative stress may be mediated by MBL binding to vascular endothelial cytokeratin, rather than by binding to glycoconjugates (Collard et al., 1413-1418, (2001)). Am. J. Pathol. 159 :1045-1054, (2001)). Other studies have shown classical and alternative pathways in the pathogenesis of ischemia / reperfusion injury, and the role of the lectin pathway in this disease remains controversial (Riedermann, NC et al., ). Am. J. Pathol. 162 :363-367, 2003).

[0022] Recent studies have shown that MASP-1 and MASP-3 convert the alternative pathway activator factor D from its zymogen form to its active enzyme form (see Takahashi M. et al.). J Exp Med207(1):29-37 (2010); Iwaki et al., J. Immunol. 187:3751-58 (2011)). The physiological importance of this process is highlighted by the absence of alternative pathway functional activity in the plasma of MASP-1 / 3-deficient mice. For the alternative pathway, C3b generated from the hydrolysis of native C3 proteins is required to function. Since the alternative pathway C3 convertase (C3bBb) contains C3b as an essential subunit, the question of the first C3b source via the alternative pathway has raised perplexing questions and spurred considerable research.

[0023] C3 belongs to a family of proteins with very few post-translational modifications known as thioester bonds (along with C4 and α-2 macroglobulins). The thioester group consists of glutamine, with its terminal carbonyl group covalently linked to the thiol group of a cysteine ​​residue three amino acids away. This bond is unstable, and the electrophilic glutamyl-thioester can react with nucleophilic moieties such as hydroxyl or amino groups, thereby forming covalent bonds with other molecules. When confined within the hydrophobic pocket of intact C3, the thioester bond is fairly stable. However, C3 is proteolytically cleaved into C3a and C3b, resulting in the exposure of the highly reactive thioester bond on C3b, which then covalently binds to the target via nucleophilic attack through neighboring moieties including hydroxyl or amino groups. In addition to its well-documented role in the covalent binding of C3b to the complement target, the C3 thioester is considered to play a key role in triggering alternative pathways. According to the widely accepted "tick-over theory," the alternative pathway is initiated by the generation of the liquid-phase invertase iC3Bb, which is formed from C3 with hydrolyzed thioesters (iC3; C3(H2O)) and factor B (Lachmann, PJ et al.). Springer Semin. Immunopathol. 7 :143-162, (1984)). C3b-like C3 (H2O) is produced from natural C3 through the slow spontaneous hydrolysis of internal thioesters in proteins (Pangburn, MK, et al.). J. Exp. Med. 154 (856-867, 1981). Through the activity of C3(H2O)Bb convertase, C3b molecules are deposited on the target surface, thereby initiating the alternative pathway.

[0024] Prior to the findings described herein, little was known about the initiators of alternative pathway activation. Activators were thought to include yeast cell walls (yeast polysaccharides), many pure polysaccharides, rabbit erythrocytes, certain immunoglobulins, viruses, fungi, bacteria, animal tumor cells, parasites, and damaged cells. The only common feature among these activators was the presence of carbohydrates; however, the complexity and diversity of carbohydrate structures made it difficult to identify the shared molecular determinants. It is widely accepted that alternative pathway activation is controlled by a fine balance among inhibitory regulatory components of this pathway, such as factor H, factor I, DAF, CR1, and properdin, the latter being the only positive regulator of the alternative pathway (see Schwaeble WJ and Reid K.B.). Immunol Today 20(1):17-21 (1999)).

[0025] In addition to the obvious unregulated activation mechanism described above, the alternative pathway can also provide a powerful amplification loop for the lectin / classical pathway C3 convertase (C4b2a), because any generated C3b can participate with factor B to form additional alternative pathway C3 convertase (C3bBb). The alternative pathway C3 convertase is stabilized by binding properdin. Properdin extends the half-life of the alternative pathway C3 convertase by six to ten times. Adding C3b to the alternative pathway C3 convertase leads to the formation of the alternative pathway C5 convertase.

[0026] It has long been believed that all three pathways (classical, lectin, and alternative pathways) converge at C5, which is cleaved to form products with multiple pro-inflammatory effects. This convergence pathway is known as the terminal complement pathway. C5a is the most potent anaphylatoxin, causing alterations in smooth muscle and vascular tone and permeability. It is also a potent chemokine and activator for both neutrophils and monocytes. C5a-mediated cell activation can significantly amplify the inflammatory response by inducing the release of various other inflammatory mediators, including cytokines, hydrolases, arachidonic acid metabolites, and reactive oxygen species. C5 cleavage leads to the formation of C5b-9, also known as the membrane attack complex (MAC). Strong evidence suggests that subcleaved MAC deposits may play an important role in inflammation, in addition to acting as a pore-forming complex.

[0027] Besides its crucial role in immune defense, the complement system also contributes to tissue damage in many clinical conditions. Therefore, there is an urgent need to develop therapeutically effective complement inhibitors to prevent these adverse effects. Summary of the Invention

[0028] In one aspect, the present invention provides isolated monoclonal antibodies or antigen-binding fragments thereof, which have high affinity (having a K+ of less than 500 pM). DIt specifically binds to the serine protease domain of human MASP-3 (amino acid residues 450 to 728 of SEQ ID NO: 2), wherein the antibody or its antigen-binding fragment inhibits alternative pathway complement activation. In some embodiments, the antibody or antigen-binding fragment is characterized by at least one or more of the following properties: (a) inhibiting prefactor D maturation; (b) not binding to human MASP-1 (SEQ ID NO: 8); (c) inhibiting the alternative pathway in mammalian subjects at a molar ratio of about 1:1 to about 2.5:1 (MASP-3 target: mAb); (d) not inhibiting the classical pathway; (e) inhibiting hemolysis and / or opsonization; (f) inhibiting substrate-specific cleavage of MASP-3 serine protease; (g) reducing hemolysis or C3 cleavage and C3b surface deposition; (h) reducing factor B and / or Bb deposition on activated surfaces; (i) reducing the resting level of active factor D relative to prefactor D (in cycling and without experimental addition of activated surfaces); (j) reducing the level of active factor D relative to prefactor D in response to activated surfaces; (k) reducing the generation of resting and surface-induced levels of Ba, Bb, C3b, or C3a in the fluid phase; and / or (l) reducing factor P deposition. In some embodiments, the isolated antibody or antigen-binding fragment of paragraph 1 or 2 specifically binds to an epitope located within the serine protease domain of human MASP-3, wherein the epitope is located within at least one or more of the following: 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 its antigen-binding fragment binds to at least one of the following epitopes: ECGQPSRSLPSLV (SEQ ID NO: 16), RNAEPGLFPWQ (SEQ ID NO: 17); KWFGSGALLSASWIL (SEQ ID NO: 18); EHVTVYLGLH (SEQ ID NO: 19); PVPLGPHVMP (SEQ ID NO: 20); APHMLGL (SEQ ID NO: 21); SDVLQYVKLP (SEQ ID NO: 22); and / or AFIIFDDLSQRW (SEQ ID NO: 23).

[0029] In another aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof that binds to MASP-3, comprising: (a) a heavy chain variable region comprising HC-CDR1 as shown in SEQ ID NO: 209 (XXDIN, wherein X at position 1 is S or T, and X at position 2 is N or D); HC-CDR2 as shown in SEQ ID NO: 210 (WIYPRDXXXKYNXXFXD, wherein X at position 7 is G or D; X at position 8 is S, T, or R; X at position 9 is I or T; X at position 13 is E or D; X at position 14 is K or E; X at position 16 is T or K); and HC-CDR3 as shown in SEQ ID NO: 211 (XEDXY, wherein X at position 1 is L or V, and X at position 4 is T or S); and (b) a light chain variable region comprising HC-CDR3 as shown in SEQ ID NO: LC-CDR1 as shown in 212(KSSQSLLXXRTRKNYLX, where X at position 8 is N, I, Q or A; X at position 9 is S or T; and X at position 17 is A or S); LC-CDR2 as shown in SEQ ID NO: 144 (WASTRES) and LC-CDR3 as shown in SEQ ID NO: 146 (KQSYNLYT).

[0030] In another aspect, the present invention provides isolated antibodies or antigen-binding fragments thereof that bind to MASP-3, comprising: (a) a heavy chain variable region comprising HC-CDR1 as shown in SEQ ID NO: 213 (SYGXX, wherein X at position 4 is M or I, and X at position 5 is S or T); HC-CDR2 as shown in SEQ ID NO: 74; and HC-CDR3 as shown in SEQ ID NO: 214 (GGXAXDY, wherein X at position 3 is E or D, and X at position 5 is M or L); and (b) a light chain variable region comprising LC-CDR1 as shown in SEQ ID NO: 215 (KSSQSLLDSXXKTYLX, wherein X at position 10 is D, E, or A; X at position 11 is G or A; and X at position 16 is N or S); LC-CDR2 as shown in SEQ ID NO: 155; and HC-CDR3 as shown in SEQ ID NO: 216. (WQGTHFPXT, where X at position 8 is W or Y) as shown in LC-CDR3.

[0031] In another aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof that binds to MASP-3, comprising: (a) a heavy chain variable region comprising HC-CDR1 as shown in SEQ ID NO: 84 (GKWIE); HC-CDR2 as shown in SEQ ID NO: 86 (EILPGTGSTNYNEKFKG) or SEQ ID NO: 275 (EILPGTGSTNYAQKFQG); and HC-CDR3 as shown in SEQ ID NO: 88 (SEDV); and (b) a light chain variable region comprising HC-CDR3 as shown in SEQ ID NO: 142 (KSSQSLL). N SRTRKNYLA), SEQ ID NO: 257 (KSSQSLL QS RTRKNYLA); SEQ ID NO: 258 (KSSQSLL AS RTRKNYLA); or SEQ ID NO: 259 (KSSQSLL NT LC-CDR1 as shown in RTRKNYLA, LC-CDR2 as shown in SEQ ID NO: 144 (WASTRES), and LC-CDR3 as shown in SEQ ID NO: 161 (KQSYNIPT).

[0032] In another aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof that binds to MASP-3, comprising: (a) a heavy chain variable region comprising HC-CDR1 as shown in SEQ ID NO: 91 (GYWIE); HC-CDR2 as shown in SEQ ID NO: 93 (EMLPGSGSTHYNEKFKG); and HC-CDR3 as shown in SEQ ID NO: 95 (SIDY); and (b) a light chain variable region comprising LC-CDR1 as shown in SEQ ID NO: 163 (RSSQSLVQSNGNTYLH), LC-CDR2 as shown in SEQ ID NO: 165 (KVSNRFS), and LC-CDR3 as shown in SEQ ID NO: 167 (SQSTHVPPT).

[0033] In another aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof that binds to MASP-3, comprising:

[0034] (a) A heavy chain variable region comprising HC-CDR1 as shown in SEQ ID NO: 109 (RVHFAIRDTNYWMQ); HC-CDR2 as shown in SEQ ID NO: 110 (AIYPGNGDTSYNQKFKG); HC-CDR3 as shown in SEQ ID NO: 112 (GSHYFDY); and a light chain variable region comprising LC-CDR1 as shown in SEQ ID NO: 182 (RASQSIGTSIH), LC-CDR2 as shown in SEQ ID NO: 184 (YASESIS), and LC-CDR3 as shown in SEQ ID NO: 186 (QQSNSWPYT); or

[0035] (b) A heavy chain variable region comprising HC-CDR1 as shown in SEQ ID NO: 125 (DYYMN), HC-CDR2 as shown in SEQ ID NO: 127 (DVNPNNDGTTYNQKFKG), HC-CDR3 as shown in SEQ ID NO: 129 (CPFYYLGKGTHFDY), and a light chain variable region comprising LC-CDR1 as shown in SEQ ID NO: 196 (RASQDISNFLN), LC-CDR2 as shown in SEQ ID NO: 198 (YTSRLHS), and LC-CDR3 as shown in SEQ ID NO: 200 (QQGFTLPWT); or

[0036] (c) A heavy chain variable region comprising HC-CDR1 as shown in SEQ ID NO: 137, HC-CDR2 as shown in SEQ ID NO: 138, and HC-CDR3 as shown in SEQ ID NO: 140; and a light chain variable region comprising LC-CDR1 as shown in SEQ ID NO: 206, LC-CDR2 as shown in SEQ ID NO: 207, and LC-CDR3 as shown in SEQ ID NO: 208; or

[0037] (d) A heavy chain variable region comprising HC-CDR1 as shown in SEQ ID NO: 98, HC-CDR2 as shown in SEQ ID NO: 99, and HC-CDR3 as shown in SEQ ID NO: 101; and a light chain variable region comprising LC-CDR1 as shown in SEQ ID NO: 169, LC-CDR2 as shown in SEQ ID NO: 171, and LC-CDR3 as shown in SEQ ID NO: 173; or

[0038] (e) a heavy chain variable region comprising HC-CDR1 as shown in SEQ ID NO: 103, HC-CDR2 as shown in SEQ ID NO: 105, and HC-CDR3 as shown in SEQ ID NO: 107; and a light chain variable region comprising LC-CDR1 as shown in SEQ ID NO: 176, LC-CDR2 as shown in SEQ ID NO: 178, and LC-CDR3 as shown in SEQ ID NO: 193; or

[0039] (f) a heavy chain variable region comprising HC-CDR1 as shown in SEQ ID NO: 114, HC-CDR2 as shown in SEQ ID NO: 116, and HC-CDR3 as shown in SEQ ID NO: 118; and a light chain variable region comprising LC-CDR1 as shown in SEQ ID NO: 188, LC-CDR2 as shown in SEQ ID NO: 178, and LC-CDR3 as shown in SEQ ID NO: 190; or

[0040] (g) a heavy chain variable region comprising HC-CDR1 as shown in SEQ ID NO: 114, HC-CDR2 as shown in SEQ ID NO: 121, and HC-CDR3 as shown in SEQ ID NO: 123; and a light chain variable region comprising LC-CDR1 as shown in SEQ ID NO: 191, LC-CDR2 as shown in SEQ ID NO: 178, and LC-CDR3 as shown in SEQ ID NO: 193.

[0041] In another aspect, the present invention provides a method for inhibiting alternative pathway complement activation in mammals, the method comprising administering to a mammalian subject in need an amount sufficient to inhibit alternative pathway complement activation in mammals a composition comprising a high-affinity MASP-3 inhibitory antibody or an antigen-binding fragment thereof. In one embodiment of the method, the antibody or antigen-binding fragment thereof binds to MASP-3 with an affinity of less than 500 pM. In one embodiment of the method, as a result of administration of a composition comprising an antibody or antigen-binding fragment, one or more of the following are present in a mammalian subject: (a) inhibition of factor D maturation; (b) inhibition of alternative pathways when administered to the subject at 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) reduction of hemolysis or reduction of C3 cleavage and C3b surface deposition; (f) reduction of factor B and Bb deposition on activated surfaces; (g) reduction of the resting level of active factor D relative to pre-factor D (in cycling, and without experimental addition of activated surfaces); (h) reduction of the level of active factor D relative to pre-factor D in response to activated surfaces; and / or (i) reduction of the production of resting and surface-induced levels of fluid phase Ba, Bb, C3b, or C3a. In one embodiment of the method, the composition comprises a MASP-3 inhibitory antibody that inhibits the alternative pathway at a molar ratio of about 1:1 to about 2.5:1 (MASP-3 target: mAb).

[0042] In another aspect, the present invention provides a method for inhibiting MASP-3-dependent complement activation in subjects suffering from paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy, asthma, dense deposit disease, microimmune necrotizing crescentic glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis optica, or Bechtel's disease. The method includes the step of administering to the subject a composition comprising an amount of a high-affinity MASP-3 inhibitor that effectively inhibits MASP-3-dependent complement activation. In some embodiments, the method further includes administering to the subject a composition comprising a MASP-2 inhibitor.

[0043] In another aspect, the present invention provides a method for preparing a medicament for inhibiting MASP-3-dependent complement activation in living subjects in which this is desired, comprising combining a therapeutically effective amount of a MASP-3 inhibitor in a drug carrier. In some embodiments, the MASP-3 inhibitor is a high-affinity MASP-3 inhibitory antibody. In some embodiments, the method according to this aspect of the invention comprises preparing a medicament for inhibiting MASP-3-dependent complement activation in subjects suffering from or at risk of developing a disease or condition selected from: paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy, asthma, dense deposit disease, microimmune necrotizing crescentic glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis optica, or Bechtel's disease. In some embodiments, the method further includes combining a therapeutically effective amount of a MASP-2 inhibitor into a medicament containing a MASP-3 inhibitor or combining a therapeutically effective amount of a MASP-2 inhibitor with a medicament containing a MASP-3 inhibitor.

[0044] 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, the high-affinity MASP-3 antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region comprising (i) VHCDR1 comprising SEQ ID NO: 84, (ii) VHCDR2 comprising SEQ ID NO: 86 or SEQ ID NO: 275, and (iii) VHCDR3 comprising SEQ ID NO: 88; and (b) a light chain variable region comprising (i) VLCDR1 comprising SEQ ID NO: 142, SEQ ID NO: 257, SEQ ID NO: 258, or SEQ ID NO: 259, (ii) VLCDR2 comprising SEQ ID NO: 144, and (iii) VLCDR3 comprising SEQ ID NO: 161.

[0045] In another aspect, the present invention provides a method for treating subjects who have or are at risk of developing paroxysmal nocturnal hemoglobinuria (PNH), comprising administering to the subject a pharmaceutical composition comprising an effective amount of a high-affinity monoclonal antibody or an antigen-binding fragment thereof that binds to human MASP-3 and inhibits alternative pathway complement activation to treat PNH in the subject or reduce the risk of PNH in the subject. In one embodiment, the antibody or its antigen-binding fragment comprises (a) a heavy chain variable region comprising (i) VHCDR1 comprising SEQ ID NO: 84, (ii) VHCDR2 comprising SEQ ID NO: 86 or SEQ ID NO: 275, and (iii) VHCDR3 comprising SEQ ID NO: 88; and (b) a light chain variable region comprising (i) VLCDR1 comprising SEQ ID NO: 142, SEQ ID NO: 257, SEQ ID NO: 258, or SEQ ID NO: 259, (ii) VLCDR2 comprising SEQ ID NO: 144, and (iii) VLCDR3 comprising SEQ ID NO: 161. In some embodiments, the pharmaceutical composition increases the survival of red blood cells in subjects suffering from PNH. In some embodiments, a subject with or at risk of developing PNH exhibits one or more of the following: (i) lower than normal hemoglobin levels, (ii) lower than normal platelet counts, (iii) higher than normal reticulocyte counts, and (iv) higher than normal bilirubin levels. In some embodiments, the pharmaceutical composition is administered systemically (e.g., subcutaneously, intramuscularly, intravenously, intra-arterially, or as an inhaler) to a subject with or at risk of developing PNH. In some embodiments, the subject with or at risk of developing PNH has previously received or is receiving treatment with a terminal complement inhibitor that inhibits complement protein C5 cleavage. In some embodiments, the method further includes administering a terminal complement inhibitor that inhibits complement protein C5 cleavage to the subject. 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.

[0046] In another aspect, the present invention provides a method for treating a subject suffering from or at risk of developing arthritis (inflammatory and non-inflammatory arthritis), comprising administering to the subject a pharmaceutical composition comprising an effective amount of a high-affinity monoclonal antibody or an antigen-binding fragment thereof that binds to human MASP-3 and inhibits alternative pathway complement activation to treat arthritis in the subject or reduce the risk of arthritis in the subject. In one embodiment, the antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region comprising (i) VHCDR1 comprising SEQ ID NO: 84, (ii) VHCDR2 comprising SEQ ID NO: 86 or SEQ ID NO: 275, and (iii) VHCDR3 comprising SEQ ID NO: 88; and (b) a light chain variable region comprising (i) VLCDR1 comprising SEQ ID NO: 142, SEQ ID NO: 257, SEQ ID NO: 258, or SEQ ID NO: 259, (ii) VLCDR2 comprising SEQ ID NO: 144, and (iii) VLCDR3 comprising SEQ ID NO: 161. In some embodiments, the subject suffers from arthritis selected from osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, ankylosing spondylitis, Bechtel's disease, infection-related arthritis, and psoriatic arthritis. In some embodiments, the pharmaceutical composition is administered systemically (e.g., subcutaneously, intramuscularly, intravenously, intra-arterially, or as an inhaler). In some embodiments, the pharmaceutical composition is applied topically to the joint.

[0047] As described herein, multiple embodiments of the pharmaceutical compositions of the present invention may use high-affinity MASP-3 inhibitory antibodies, and multiple embodiments optionally combined with MASP-2 inhibitors.

[0048] As described herein, the pharmaceutical compositions of the present invention can be used according to the methods of the present invention.

[0049] These and other aspects and embodiments of the invention described herein will be apparent from the following detailed description and accompanying drawings. All U.S. patents, U.S. patent applications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referenced in this specification are incorporated herein by reference in their entirety as if they were individually incorporated herein. Attached Figure Description

[0050] The foregoing aspects and many incidental advantages of the invention will be more readily understood and better appreciated by referring to the following detailed description of the invention in conjunction with the accompanying drawings, in which:

[0051] Figure 1 This demonstrates a new understanding of the lectin pathway and alternative pathways;

[0052] Figure 2 It is adapted from Schwaeble et al. Immunobiol The schematic diagram in 205:455-466 (2002) is by Yongqing et al. BBA 1824:253 (2012) amended to describe the protein domains of MASP-1, MASP-3 and MAp44 and the exons encoding them;

[0053] Figure 3 The amino acid sequence of human MASP-3 (SEQ ID NO: 2) is depicted, with the leader sequence shown underlined;

[0054] Figure 4 This shows a comparison of full-length MASP-3 proteins from multiple species;

[0055] Figure 5 This shows a comparison of the SP domains of MASP-3 proteins from multiple species;

[0056] Figure 6 This is the Kaplan-Mayer curve, which illustrates the effect of administering an infectious dose of 2.6 x 10⁷ CFU of Neisseria meningitidis (…). N. meningitidis The percentage survival of MASP-2 KO and WT mice after serum group A Z2491 indicates that MASP-2 deficient mice are protected from Neisseria meningitidis-induced death, as described in Example 1;

[0057] Figure 7 The figure shows the percentage survival of MASP-2 KO and WT mice after administration of an infectious dose of 6 x 10⁶ CFU of Neisseria meningitidis sera B strain MC58, indicating that MASP-2 deficient mice are protected from Neisseria meningitidis-induced death, as described in Example 1.

[0058] Figure 8 The figure illustrates the log cfu / mL of Neisseria meningitidis MC58 recovered per mL of blood from MASP-2 KO and WT mice at different time points following infection with 6 x 10⁶ CFU of Neisseria meningitidis MC58 ip (n=3 for both groups of mice at different time points). This indicates that although MASP-2 KO mice were infected with the same dose of Neisseria meningitidis MC58 ip as WT mice, MASP-2 KO mice had a higher bacteremia clearance rate compared to WT mice, as described in Example 1.

[0059] Figure 9The figure illustrates the mean disease scores of MASP-2 KO and WT mice at 3, 6, 12, and 24 hours post-infection with 6x106 CFU Neisseria meningitidis sera strain B MC58, showing that MASP-2-deficient mice exhibited significantly lower disease scores at 6, 12, and 24 hours post-infection compared to WT mice, as described in Example 1.

[0060] Figure 10 The figure shows the percentage survival rate of mice after administration of an infectious dose of 4 x 10⁶ CFU of Neisseria meningitidis serogroup B strain MC58, followed by administration of inhibitory MASP-2 antibody (1 mg / kg) or control isotype antibody 3 hours post-infection. This demonstrates that MASP-2 antibody is effective in treating and improving the survival rate of subjects infected with Neisseria meningitidis, as described in Example 2.

[0061] Figure 11 The illustration shows the log cfu / mL count of Neisseria meningitidis serogroup B strain MC58 recovered at different time points in human serum samples collected at different time points after incubation with Neisseria meningitidis serogroup B strain MC58, as described in Example 3.

[0062] Figure 12 The illustration shows the log cfu / mL of viable counts of Neisseria meningitidis serum group B-MC58 recovered at different time points in the human serum samples shown in Table 8, indicating that complement-dependent killing of Neisseria meningitidis in 20% (v / v) human serum is MASP-3 and MBL-dependent, as described in Example 3.

[0063] Figure 13 The illustration shows the log cfu / mL viable counts of Neisseria meningitidis serum group B-MC58 recovered from mouse serum samples at different time points as shown in Table 10. It shows that compared with WT mouse serum, MASP-2 - / - knockout mouse serum (referred to as "MASP-2 - / -") has a higher level of bactericidal activity against Neisseria meningitidis, while MASP-1 / 3 - / - mouse serum has no bactericidal activity, as described in Example 3;

[0064] Figure 14 The illustration shows the C3 activation kinetics in the serum of WT, C4- / -, MASP-1 / 3- / -, factor B- / -, and MASP-2- / - mice under lectin pathway-specific conditions (1% plasma), as described in Example 4;

[0065] Figure 15The illustration shows the effects of conventional alternative pathway-specific (AP-specific) conditions (i.e., BBS / EGTA / Mg) on ​​serum samples obtained from MASP-3-deficient, C4-deficient, and MBL-deficient human subjects. ++ No Ca ++ Under these conditions, as a function of serum concentration, the alternative pathway-driven (AP-driven) C3b deposition level on a yeast polysaccharide-coated microtiter plate, as described in Example 4;

[0066] Figure 16 The illustration shows the results of testing in 10% human serum samples obtained from MASP-3-deficient, C4-deficient, and MBL-deficient human subjects under "conventional" AP-specific conditions (i.e., BBS / EGTA / Mg). ++ No Ca ++ Under these conditions, as a function of time, the AP-driven C3b deposition level on the yeast polysaccharide-coated microtiter plate, as described in Example 4;

[0067] Figure 17A The illustration shows the effects of conventional AP-specific conditions (i.e., BBS / EGTA / Mg) on ​​serum samples obtained from WT, MASP-2-deficient, and MASP-1 / 3-deficient mice. ++ No Ca ++ Under conditions that allow both the lectin pathway and the alternative pathway (AP) to function (BBS / Mg) ++ / Ca ++ The level of C3b deposition on the mannan-coated microtiter plate as a function of serum concentration, as described in Example 4;

[0068] Figure 17B The illustration shows the effects of conventional AP-specific conditions (i.e., BBS / EGTA / Mg) on ​​serum samples obtained from WT, MASP-2-deficient, and MASP-1 / 3-deficient mice. ++ No Ca ++ Or in physiological conditions that allow both the lectin pathway and the alternative pathway to function (BBS / Mg) ++ / Ca ++ The level of C3b deposition on a yeast polysaccharide-coated microtiter plate as a function of serum concentration, as described in Example 4;

[0069] Figure 17C The illustration shows the effects of conventional AP-specific conditions (i.e., BBS / EGTA / Mg) on ​​serum samples obtained from WT, MASP-2-deficient, and MASP-1 / 3-deficient mice. ++ No Ca ++Under conditions where both the lectin pathway and the alternative pathway are permitted to function (BBS / Mg) or in physiological conditions where both pathways are allowed to operate. ++ / Ca ++ Under these conditions, as a function of serum concentration, in Streptococcus pneumoniae ( S. pneumoniae C3b deposition levels on D39-coated microtiter plates, as described in Example 4;

[0070] Figure 18A The illustration shows the traditional AP-specific conditions (i.e., BBS / EGTA / Mg). ++ No Ca ++ Or in physiological conditions that allow both the lectin pathway and the alternative pathway to function (BBS / Mg) ++ / Ca ++ The results of C3b deposition assays in highly diluted serum were performed on a mannan-coated microtiter plate, using serum concentrations ranging from 0% to 1.25%, as described in Example 4.

[0071] Figure 18B The illustration shows the traditional AP-specific conditions (i.e., BBS / EGTA / Mg). ++ No Ca ++ Or in physiological conditions that allow both the lectin pathway and the alternative pathway to function (BBS / Mg) ++ / Ca ++ The results of C3b deposition assays performed on yeast polysaccharide-coated microtiter plates, using serum concentrations ranging from 0% to 1.25%, as described in Example 4;

[0072] Figure 18C The illustration shows the traditional AP-specific conditions (i.e., BBS / EGTA / Mg). ++ No Ca ++ Or in physiological conditions that allow both the lectin pathway and the alternative pathway to function (BBS / Mg) ++ / Ca ++ The results of C3b deposition assays performed on microtiter plates coated with Streptococcus pneumoniae D39 were obtained using serum concentrations ranging from 0% to 1.25%, as described in Example 4.

[0073] Figure 19 The figure illustrates the effects of a series of serum dilutions on human serum under physiological conditions (i.e., at Ca2+) from MASP-3- / -, heat-inactivated normal human serum (HI NHS), MBL- / -, NHS + MASP-2 monoclonal antibody, and NHS control serum. ++In the presence of mannan-coated mouse erythrocytes, the level of hemolysis is (as measured by the release of hemoglobin from lysed mouse erythrocytes (Crry / C3- / -) into a supernatant, which is measured by photometric method), as described in Example 5;

[0074] Figure 20 The figure illustrates a series of serum concentrations of human serum under physiological conditions (i.e., at Ca2+) from sera derived from MASP-3- / -, heat-inactivated (HI) NHS, MBL- / -, NHS+ MASP-2 monoclonal antibody, and NHS control. ++ In the presence of mannan-coated mouse erythrocytes, the level of hemolysis is (as measured by the release of hemoglobin from lysed mouse erythrocytes (Crry / C3- / -) into a supernatant, which is measured by photometric method), as described in Example 5;

[0075] Figure 21 The illustration shows a series of serum concentrations of human serum under physiological conditions (i.e., at Ca2+) from sera derived from 3MC (MASP-3- / -), heat-inactivated (HI) NHS, MBL- / -, NHS+ MASP-2 monoclonal antibody, and NHS control. ++ The level of hemolysis of non-coated mouse erythrocytes (in the presence of the supernatant) is measured (e.g., by the release of hemoglobin from lysed WT mouse erythrocytes into a supernatant measured by photometric method), as described in Example 5;

[0076] Figure 22 The figure illustrates a series of serum concentrations of human serum under physiological conditions (i.e., at Ca2+) from heat-inactivated (HI) NHS, MBL- / -, NHS+ MASP-2 monoclonal antibody, and NHS control serum. ++ In the presence of the substance, non-coated mouse erythrocytes are hemolyzed (as measured by the release of hemoglobin from lysed mouse erythrocytes (CD55 / 59- / -) into a supernatant, which is measured by photometric method), as described in Example 5;

[0077] Figure 23 The figure illustrates a series of serum concentrations of MASP-1 / 3- / - mouse serum and WT control mouse serum under physiological conditions (i.e., at Ca2+). ++ In the presence of mannan-coated rabbit erythrocytes, hemolysis is achieved (as measured by the release of hemoglobin from lysed rabbit erythrocytes into a supernatant, which is measured by photometric method), as described in Example 6;

[0078] Figure 24A This is a FACS bar chart of MASP-3 antigen / antibody binding for clone M3J5, as described in Example 7;

[0079] Figure 24B This is a FACS bar chart of MASP-3 antigen / antibody binding for clone M3M1, as described in Example 7;

[0080] Figure 25 The illustration shows the saturation binding curve of clone M3J5 (clone 5) for the MASP-3 antigen, as described in Example 7;

[0081] Figure 26A The comparison is between the VH regions of M3J5, M3M1, D14, and 1E10 and the amino acid sequence of the chicken DT40 VH sequence. The dots indicate the amino acid identity with the DT40 sequence, and the horizontal bars indicate the introduction of vacancies to maximize the comparison, as described in Example 7.

[0082] Figure 26B The comparison is between the VL regions of M3J5, M3M1, D14 and 1E10 and the amino acid sequence of chicken DT40 VL sequence, where the dots indicate amino acid identity with the DT40 sequence and the horizontal bars indicate the introduction of vacancies to maximize the comparison, as described in Example 7.

[0083] Figure 27 The bar chart shows the inhibitory activity of monoclonal antibody (mAb) 1E10 in the Wieslab complement system screening MBL pathway compared to the positive serum provided in the assay kit and the isotype control antibody. It indicates that mAb 1E10 partially inhibits LEA-2-dependent activation (by inhibiting MASP-2 MASP-1-dependent activation), while the isotype control antibody does not, as described in Example 7.

[0084] Figure 28A Provide heat-inactivated Staphylococcus aureus ( Staphylococcus aureus Flow cytometry analysis of C3b deposition on [a specific substance] indicated that no C3b deposition was observed in normal human serum in the presence of EDTA (which is known to inactivate lectins and alternative pathways). Figure 1 ), using Mg ++ Alternative pathway-driven C3b deposition (small) was observed in normal human serum treated with EGTA. Figure 2 ), and like small Figure 3 , 4 As shown in Figure 5, no alternative pathway-driven C3b deposition was observed in serum with factor B-depletion, factor D-depletion, and properdin (factor P)-depletion, as described in Example 8.

[0085] Figure 28BFlow cytometry analysis of C3b deposition on heat-inactivated Staphylococcus aureus provides results indicating that, as in EDTA-treated normal serum (small... Figure 1 In 3MC serum, in Mg ++ / EGTA is present but AP-driven C3b deposition is absent (small Figure 3 ), and small Figure 4 and 5 The activity of full-length rMASP-3 (small) was demonstrated. Figure 4 ) and active rMASP-3 (CCP1-CCP2-SP) (small Figure 5 Both restored AP-driven C3b deposition in 3MC serum to the level achieved with Mg ++ The level observed in normal serum treated with EGTA (small) Figure 2 ), while inactive rMASP-3 (S679A) (small Figure 6 ) or wild-type rMASP-1 (small Figure 7 None of these methods could restore AP-driven C3b deposition in 3MC serum, as described in Example 8;

[0086] Figure 29 Western blot analysis of Staphylococcus aureus factor B cleavage in 3MC serum, with or without rMASP-3, showed that the cleavage was in response to Mg2+ cleavage. ++ For normal human serum in the presence of EGTA (as shown in lane 2 (positive control)), normal human serum in the presence of EDTA (negative control, lane 1) showed very low factor B cleavage, as further shown in lane 3. 3MC serum showed significantly lower Mg cleavage. ++ Very little factor B cleavage was observed in the presence of / EGTA. However, as shown in lane 4, factor B cleavage was restored by adding full-length, recombinant MASP-3 protein to 3MC serum and pre-incubating, as described in Example 8;

[0087] Figure 30 Coomassie staining of the protein gel showed that factor B cleavage was optimal in the presence of C3, MASP-3 and pro-factor D (lane 1); and as shown in lanes 4 and 5, factor B cleavage could be mediated by either MASP-3 alone or pro-factor D alone, as long as C3 was present, as described in Example 8.

[0088] Figure 31The figure illustrates the mean fluorescence intensity (MFI) of C3b staining of Staphylococcus aureus derived from mAbD14 (which binds to MASP-3), mAb1A5 (negative control antibody), and isotype control antibody in 3MC serum as a function of mAb concentration, in the presence of rMASP-3. This indicates that mAbD14 inhibits MASP-3-dependent C3b deposition in a concentration-dependent manner, as described in Example 8.

[0089] Figure 32 Western blot analysis of pre-factor D substrate cleavage is shown, wherein full-length wild-type recombinant MASP-3 (lane 2) and MASP-1 (lane 5) completely or partially cleave pre-factor D to produce mature factor D, as described in Example 9, compared to pre-factor D alone (lane 1) or inactive full-length recombinant MASP-3 (S679A; lane 3) or MASP-1 (S646A; lane 4);

[0090] Figure 33 The results are Western blots showing that, compared with the control response containing only MASP-3 and prefactor D (no mAb, lane 1) and the control response containing mAb derived from the DTLacO library (which binds to MASP-1 but not to MASP-3) (lane 4), the mAbs D14 (lane 2) and M3M1 (lane 3) binding to MASP-3 showed inhibitory activity against MASP-3-dependent prefactor D cleavage, as described in Example 9.

[0091] Figure 34 The figure illustrates the AP-driven C3b deposition level as a function of serum concentration on a yeast-polysaccharide-coated microtiter plate in serum samples obtained from MASP-3-deficient (3MC), C4-deficient, and MBL-deficient subjects. It shows that MASP-3-deficient sera from patients 2 and 3 exhibited residual AP activity at high serum concentrations (25%, 12.5%, and 6.25% serum concentrations), but significantly higher AP levels. 50 (That is, 8.2% and 12.3% serum are required to achieve 50% maximum C3 deposition), as described in Example 10;

[0092] Figure 35A The illustration shows the results of testing in 10% human serum samples obtained from MASP-3-deficient, C4-deficient, and MBL-deficient human subjects under "conventional" AP-specific conditions (i.e., BBS / EGTA / Mg). ++ No Ca ++ Under these conditions, as a function of time, the AP-driven C3b deposition level on a yeast polysaccharide-coated microtiter plate, as described in Example 10;

[0093] Figure 35B Western blots are shown for plasma from 3MC patient #2 (MASP-3(- / -), MASP-1(+ / +)), 3MC patient #3 (MASP-3(- / -), MASP-1(- / -)), and serum from normal donors (W), wherein human pre-factor D (25040 Daltons) and / or mature factor D (24405 Daltons) were detected with a human factor D-specific antibody, as described in Example 10;

[0094] Figure 35C The illustration shows the results of Weislab classical, lectin and alternative pathway assays for plasma obtained from 3MC patients #2 and #3 and normal human serum, as described in Example 10;

[0095] Figure 36 The illustration shows the concentration of Ca in serum from two normal human subjects (NHS) and two patients with 3MC (Patient 2 and Patient 3). ++ The percentage of hemolysis of mannan-coated rabbit erythrocytes at a series of serum concentrations in the absence of MASP-3 (measured, for example, by the release of hemoglobin from lysed rabbit erythrocytes into a supernatant measured by photometric method) indicated that MASP-3 deficiency reduced the percentage of complement-mediated mannan-coated erythrocyte lysis compared to normal human serum, as described in Example 10.

[0096] Figure 37 The illustration shows the addition of the drug to a patient with 3MC (MASP-3) from human. - / - The concentration of recombinant full-length MASP-3 protein in serum samples was used as a function of the AP-driven C3b deposition levels on yeast-glycan-coated microtiter plates, indicating that active recombinant MASP-3 protein reconstituted AP-driven C3b deposition on yeast-glycan-coated plates in a concentration-dependent manner compared to the negative control inactive recombinant MASP-3 (MASP-3A; S679A), as described in Example 10;

[0097] Figure 38 The illustration shows that in Ca ++The percentage of hemolysis of mannan-coated rabbit erythrocytes was measured in the following sera at a range of serum concentrations (as measured by the release of hemoglobin from lysed rabbit erythrocytes into a supernatant, which was measured by photometric method): (1) normal human serum (NHS); (2) serum from a 3MC patient; (3) serum from a 3MC patient with active full-length recombinant MASP-3 (20 µg / ml); and (4) heat-inactivated human serum (HIS); indicating that the percentage of rabbit erythrocyte lysis was significantly increased in 3MC serum containing rMASP-3 compared to the percentage of hemolysis in 3MC serum without recombinant MASP-3 (p=0.0006), as described in Example 10;

[0098] Figure 39 The figure illustrates the effect of using active recombinant MASP-3 (in BBS / Mg) at concentrations ranging from 0 to 110 µg / ml. ++ The percentage of rabbit erythrocyte lysis in 7% human serum from patients 2 and 3 of 3MC (in EGTA) showed that the percentage of rabbit erythrocyte lysis increased in a concentration-dependent manner with the amount of recombinant MASP-3, as described in Example 10.

[0099] Figure 40 The illustration shows the LEA-2-driven C3b deposition levels on a mannan-coated ELISA plate as a function of the concentration of human serum diluted in BBS buffer, for serum from normal human subjects (NHS), from two 3MC patients (Patient 2 and Patient 3), from the parents of Patient 3, and from MBL-deficient subjects, as described in Example 10.

[0100] Figure 41 The illustration shows a representative example of a binding assay performed with human MASP-3, where M3-1 Fab (also known as 13B1) shows an apparent binding affinity (EC50) of approximately 0.117 nM for the human protein. 50 As described in Example 11;

[0101] Figure 42 The illustration shows a representative example of a binding assay performed with mouse MASP-3, where M3-1 Fab (also known as 13B1) shows an apparent binding affinity (EC50) of approximately 0.214 nM to the mouse protein. 50 As described in Example 11;

[0102] Figure 43The illustration shows the level of complement factor Bb deposition on yeast glycan particles (as determined by flow cytometry in MFI units) in the presence of different concentrations of mAb M3-1 (also known as 13B1) in the serum of CFD-depleted human patients, as described in Example 11.

[0103] Figure 44 The figure illustrates the level of C3 deposition on yeast polysaccharide particles at various time points following a single dose of mAb M3-1 (13B1) (10 mg / kg iv) in wild-type mice, as described in Example 11;

[0104] Figure 45 The figure illustrates the percentage of donor RBCs (WT or Crry-) survival over a 14-day period in wild-type recipient mice treated with mAb M3-1 (13B1) (at -11, 4, -1, and +6 days, 10 mg / kg), mice treated with mAb BB5.1, or mice treated with the vector, as described in Example 12.

[0105] Figure 46 The figure illustrates the percentage of donor RBCs (WT or Crry-) survival over a 16-day period in wild-type recipient mice or mice treated with the vector after a single dose of mAb M3-1 (13B1) (20 mg / kg on day -6), as described in Example 12.

[0106] Figure 47 The illustration shows the clinical scores of mice treated with mAb M3-1 (13B1) (5 mg / kg or 20 mg / kg) or the mediator over a 14-day period in a collagen-antibody-induced arthritis model, as described in Example 13.

[0107] Figure 48 The illustration shows the percentage incidence of arthritis in mice treated with mAb M3-1 (13B1) (5 mg / kg or 20 mg / kg) or the carboxylant over a 14-day period in a collagen-antibody-induced arthritis model, as described in Example 13.

[0108] Figure 49A The amino acid sequence of the VH region of the high-affinity (≤500pM) anti-human MASP-3 inhibitory mAb is shown as described in Example 15;

[0109] Figure 49B The amino acid sequence of the VL region of the high-affinity (≤500 pM) anti-human MASP-3 inhibitory mAb is shown as described in Example 15;

[0110] Figure 50AIt is a dendrogram of the VH region of a high-affinity anti-human MASP-3 inhibitory mAb, as described in Example 15;

[0111] Figure 50B It is a dendrogram of the VL region of a high-affinity anti-human MASP-3 inhibitory mAb, as described in Example 15;

[0112] Figure 51A The illustrations combine experimental results, showing that the representative purified recombinant anti-human MASP-3 inhibitory antibody exhibits an epigenetic binding affinity of less than 500 pM (e.g., 240 pM to 23 pM) to human MASP-3 protein, as described in Example 16;

[0113] Figure 51B The illustrations combine experimental results, showing that representative purified recombinant anti-human MASP-3 inhibitory antibodies exhibit an apparent binding affinity of less than 500 pM (e.g., 91 pM to 58 pM) to human MASP-3 protein, as described in Example 16.

[0114] Figure 51C The illustration shows the results of the combined experiment, in which the representative purified recombinant high-affinity anti-human MASP-3 inhibitory antibody shows selectivity for binding to MASP-3 and does not bind to human MASP-1, as described in Example 16;

[0115] Figure 51D The illustration shows the results of the combined experiment, which demonstrates that the representative purified recombinant high-affinity anti-human MASP-3 inhibitory antibody is selective for binding to MASP-3 and does not bind to human MASP-2, as described in Example 16;

[0116] Figure 52 The illustrations combine the experimental results, in which the representative purified recombinant anti-human MASP-3 inhibitory antibody also showed a high binding affinity to mouse MASP-3 protein, as described in Example 16;

[0117] Figure 53 The illustration shows the experimental results of measuring the ability of a representative high-affinity MASP-3 antibody to inhibit the cleavage of fluorescent tripeptides, as described in Example 16;

[0118] Figure 54 The Western blot showed that the representative high-affinity MASP-3 inhibitory mAb blocked the ability of recombinant MASP-3-mediated pre-factor D cleavage into factor D in an in vitro assay, as described in Example 16;

[0119] Figure 55AThe illustration shows the level of complement factor Bb deposition on yeast glycan particles (as determined by flow cytometry in MFI units) in human serum with different concentrations of high-affinity MASP-3 mAb1F3, 1G4, 2D7 and 4B6 in factor D-depleted human serum, as described in Example 16.

[0120] Figure 55B The illustration shows the level of complement factor Bb deposition on yeast glycan particles (as determined by flow cytometry in MFI units) in human serum with different concentrations of high-affinity MASP-3 mAb4D5, 10D12 and 13B1 in factor D-depleted human serum, as described in Example 16.

[0121] Figure 56A The figures illustrate the inhibitory levels of rabbit erythrocyte lysis in the presence of different concentrations of high-affinity MASP-3 mAb 1A10, 1F3, 4B6, 4D5 and 2F2, as described in Example 16;

[0122] Figure 56B The figures illustrate the inhibitory levels of rabbit erythrocyte lysis in the presence of different concentrations of high-affinity MASP-3 mAb 1B11, 1E7, 1G4, 2D7 and 2F5, as described in Example 16;

[0123] Figure 57 Western blot analysis was performed on the levels of prefactor D and factor D in the serum of 3MC patients (Patient B) in the presence of active recombinant MASP-3 (rMASP-3), inactive rMASP-3, and active rMASP-3 plus high-affinity MASP-3 mAb 4D5, as described in Example 16.

[0124] Figure 58 The figures illustrate the levels of C3 / C3b / iC3b deposition on yeast polysaccharide particles at various time points following a single dose of high-affinity MASP-3 mAb M3-1 (13B1, 10 mg / kg) or 10D12 (10 mg / kg) in wild-type mice, as described in Example 17.

[0125] Figure 59 Western blot analysis was performed on the state of factor B factor Ba fragment in mice treated with high-affinity MASP-3 mAb 10D12 (10 mg / kg) or in mice treated with a vector control, as described in Example 17.

[0126] Figure 60The figure illustrates the level of hemolysis inhibition from 20% serum of mice treated with high-affinity MASP-3 mAb 10D12 (10 mg / kg or 25 mg / kg), as described in Example 17;

[0127] Figure 61A The illustration shows the results of competitive binding analysis to identify high-affinity MASP-3 mAb that blocks the interaction between high-affinity MASP-3 mAb 4D5 and human MASP-3, as described in Example 18;

[0128] Figure 61B The illustration shows the results of competitive binding analysis to identify high-affinity MASP-3 mAb that blocks the interaction between high-affinity MASP-3 mAb 10D12 and human MASP-3, as described in Example 18;

[0129] Figure 61C The illustration shows the results of competitive binding analysis to identify the high-affinity MASP-3 mAb that blocks the interaction between high-affinity MASP-3 mAb 13B1 and human MASP-3, as described in Example 18;

[0130] Figure 61D The illustration shows the results of competitive binding analysis to identify high-affinity MASP-3 mAb that blocks the interaction between high-affinity MASP-3 mAb 1F3 and human MASP-3, as described in Example 18;

[0131] Figure 61E The illustration shows the results of competitive binding analysis to identify high-affinity MASP-3 mAb that blocks the interaction between high-affinity MASP-3 mAb 1G4 and human MASP-3, as described in Example 18;

[0132] Figure 62 A schematic diagram showing the contact area of ​​high-affinity MASP-3 mAb on human MASP-3, as determined by Pepscan analysis, is provided, as described in Example 18;

[0133] Figure 63A The contact region between human MASP-3 and high-affinity MASP-3 mAb 1F3, 4D5 and 1A10 is shown, including amino acid residues 498-509 (SEQ ID NO: 9), 544-558 (SEQ ID NO: 11), 639-649 (SEQ ID NO: 13) and 704-713 (SEQ ID NO: 14) of MASP-3, as described in Example 18;

[0134] Figure 63B The contact region between human MASP-3 and high-affinity MASP-3 mAb 10D12 is shown, including amino acid residues 498 to 509 of MASP-3 (SEQ ID NO: 9), as described in Example 18;

[0135] Figure 64 The contact region between human MASP-3 and high-affinity MASP-3 mAb 13B1 is shown, including amino acid residues 494 to 508 (SEQ ID NO: 10) and amino acid residues 626 to 638 (SEQ ID NO: 12) of MASP-3, as described in Example 18;

[0136] Figure 65 The contact region between human MASP-3 and high-affinity MASP-3 mAb 1B11 is shown, including amino acid residues 435-447 (SEQ ID NO: 16), 454-464 (SEQ ID NO: 17), 583-589 (SEQ ID NO: 21), and 614-623 (SEQ ID NO: 22) of MASP-3, as described in Example 18;

[0137] Figure 66 The contact region between human MASP-3 and high-affinity MASP-3 mAb 1E7, 1G4 and 2D7 is shown, including amino acid residues 454 to 464 (SEQ ID NO: 17), amino acid residues 514 to 523 (SEQ ID NO: 19) and amino acid residues 667 to 678 (SEQ ID NO: 23) of MASP-3, as described in Example 18;

[0138] Figure 67 The contact region between human MASP-3 and high-affinity MASP-3 mAb 15D9 and 2F5 is shown, including amino acid residues 454-464 (SEQ ID NO: 17), 479-493 (SEQ ID NO: 18), 562-571 (SEQ ID NO: 20), and 667-678 (SEQ ID NO: 23) of MASP-3, as described in Example 18;

[0139] Figure 68The illustration shows the results of an experimental autoimmune encephalomyelitis (EAE) model in mice treated with high-affinity MASP-3 inhibitory mAb 13B1 (10 mg / kg), factor B mAb 1379 (30 mg / kg), or an isotype control mAb (10 mg / kg), as described in Example 20.

[0140] Figure 69 The illustration shows APC activity measured by flow cytometry of the average MFI of complement factor Bb on the surface of yeast polysaccharide particles in serum samples obtained from three groups of cynomolgus monkeys after treatment with high-affinity MASP-3 mAb h13B1X over time, in the presence or absence of anti-factor D antibodies incorporated into the serum sample, as described in Example 21.

[0141] Figure 70 The illustration shows APC activity as determined by Bb deposition on yeast polysaccharides in serum samples obtained from cynomolgus groups (3 animals / group) treated with a single intravenous dose of 5 mg / kg of the high-affinity MASP-3 inhibitory mAbs h4D5X, h10D12X or h13B1X, as described in Example 21.

[0142] Figure 71A The illustration shows APC activity measured in the fluid phase Ba from serum samples obtained over time from a group of cynomolgus monkeys (3 animals / group) following a single intravenous dose of 5 mg / kg of mAb h4D5X, h10D12X and h13B1X, as described in Example 21.

[0143] Figure 71B The illustration shows APC activity as determined by fluid phase Bb in serum samples obtained from cynomolgus monkey groups (3 animals / group) over time following a single intravenous dose of 5 mg / kg of mAb h4D5X, h10D12X and h13B1X, as described in Example 21.

[0144] Figure 71C The illustration shows APC activity as measured by fluid phase C3a in serum samples obtained from cynomolgus monkey groups (3 animals / group) over time after treatment with a single intravenous dose of 5 mg / kg of mAb h4D5X, h10D12X and h13B1X, as described in Example 21.

[0145] Figure 72A The illustration shows the molar ratio of the target (MASP-3) to the high-affinity MASP-3 inhibitory antibody h4D5X at the time point of complete APC inhibition, as measured by fluid phase Ba, as described in Example 21;

[0146] Figure 72B The illustration shows the molar ratio of the target (MASP-3) to the high-affinity MASP-3 inhibitory antibody h10D12X at the time point of complete APC inhibition, as measured by fluid phase Ba, as described in Example 21;

[0147] Figure 72C The illustration shows the molar ratio of the target (MASP-3) to the high-affinity MASP-3 inhibitory antibody h13B1X at the time point of complete APC inhibition, as measured by fluid phase Ba, as described in Example 21; and

[0148] Figure 73 Western blot analysis showed the levels of prefactor D and factor D in serum from cynomolgus monkeys over time (hours) following a single intravenous dose of mAb h13B1X at a dose of 5 mg / kg, as described in Example 21.

[0149] Description of sequence lists

[0150] SEQ ID NO: 1 Human MASP-3 cDNA

[0151] SEQ ID NO: 2 Human MASP-3 protein (with leader region)

[0152] SEQ ID NO: 3 Mouse MASP-3 protein (with leader region)

[0153] SEQ ID NO: 4 Rat MASP-3 protein

[0154] SEQ ID NO: 5 Chicken MASP-3 protein

[0155] SEQ ID NO: 6 Rabbit MASP-3 protein

[0156] SEQ ID NO: 7 cynomolgus monkey MASP-3 protein

[0157] SEQ ID NO: 8 Human MASP-1 protein (with leader region)

[0158] Human MASP-3 SP domain peptide fragment:

[0159] SEQ ID NO: 9 (Human MASP-3 w / leader region aa 498-509)

[0160] SEQ ID NO: 10 (Human MASP-3 w / leader aa 494-508)

[0161] SEQ ID NO: 11 (Human MASP-3 w / leader region aa 544-558)

[0162] SEQ ID NO: 12 (Human MASP-3 w / leader aa 626-638)

[0163] SEQ ID NO: 13 (Human MASP-3 w / leader region aa 639-649)

[0164] SEQ ID NO: 14 (Human MASP-3 w / leader region aa 704-713)

[0165] SEQ ID NO: 15 (Human MASP-3 w / leader region aa 498-508)

[0166] SEQ ID NO: 16 (Human MASP-3 w / leader region aa 435-447)

[0167] SEQ ID NO: 17 (Human MASP-3 w / leader region aa 454-464)

[0168] SEQ ID NO: 18 (Human MASP-3 w / leader region aa 479-493)

[0169] SEQ ID NO: 19 (Human MASP-3 w / leader region aa 514-523)

[0170] SEQ ID NO: 20 (Human MASP-3 w / leader region aa 562-571)

[0171] SEQ ID NO: 21 (Human MASP-3 w / leader region aa 583-589)

[0172] SEQ ID NO: 22 (Human MASP-3 w / leader region aa 614-623)

[0173] SEQ ID NO: 23 (Human MASP-3 w / leader region aa 667-678)

[0174] SEQ ID NO: 24-39: Heavy chain variable region - mouse parent

[0175] SEQ ID NO: 24 4D5_VH

[0176] SEQ ID NO: 25 1F3_VH

[0177] SEQ ID NO: 26 4B6_VH

[0178] SEQ ID NO: 27 1A10_VH

[0179] SEQ ID NO: 28 10D12_VH

[0180] SEQ ID NO: 29 35C1_VH

[0181] SEQ ID NO: 30 13B1_VH

[0182] SEQ ID NO: 31 1G4_VH

[0183] SEQ ID NO: 32 1E7_VH

[0184] SEQ ID NO: 33 2D7_VH [[ID=二十三]]

[0185] SEQ ID NO: 34 49C11_VH

[0186] SEQ ID NO: 35 15D9_VH

[0187] SEQ ID NO: 36 2F5_VH

[0188] SEQ ID NO:

[0189] SEQ ID NO: 38 2F2_VH

[0190] SEQ ID NO: 39

[0191] SEQ ID NO: 40-54: Light chain variable region - mouse parental

[0192] SEQ ID NO: 40 4D5_VL

[0193] SEQ ID NO: 41 1F3_VL

[0194] SEQ ID NO: 42 4B6 / 1A10_VL

[0195] SEQ ID NO: 43 10D12_VL

[0196] SEQ ID NO: 44 35C1_VL

[0197] SEQ ID NO: 45 13B1_VL It should be noted that there seems to be an error in the original text where "SEQ ID NO: 34 1B11_VH" is incomplete in the English translation part. Also, "SEQ ID NO:

[0190] SEQ ID NO: 39 " seems to be incorrect or incomplete in the original. Please check and correct the original text for a more accurate translation.

[0198] SEQ ID NO: 46 1G4_VL

[0199] SEQ ID NO: 47 1E7_VL

[0200] SEQ ID NO: 48 2D7_VL

[0201] SEQ ID NO: 49 49C11_VL

[0202] SEQ ID NO: 50 15D9_VL

[0203] SEQ ID NO: 51 2F5_VL

[0204] SEQ ID NO: 52 1B11_VL

[0205] SEQ ID NO: 53 2F2_VL

[0206] SEQ ID NO: 54 11B6_VL

[0207] SEQ ID NO: 55-140: Heavy chain framework region (FR) and complementarity-determining region (CDR) from mouse parent MASP-3 mAb

[0208] SEQ ID NO: 141-208: Light chain FR and CDR from mouse parent MASP-3 mAb

[0209] SEQ ID NO: 209-216: CDR Common Sequence

[0210] SEQ ID NO: 217-232: DNA encoding the heavy chain variable region (mouse parent)

[0211] SEQ ID NO: 233-247: DNA encoding the light chain variable region (mouse parent)

[0212] SEQ ID NO: 248: Humanized 4D5_VH-14 (h4D5_VH-14) heavy chain variable region

[0213] SEQ ID NO: 249: Humanized 4D5_VH-19 (h4D5_VH-19) heavy chain variable region

[0214] SEQ ID NO: 250: Humanized 4D5_VL-1 (h4D5_VL-1) light chain variable region

[0215] SEQ ID NO: 251: Humanized 10D12_VH-45 (h10D12_VH-45) heavy chain variable region

[0216] SEQ ID NO: 252: Humanized 10D12_VH-49 (h10D12_VH-49) heavy chain variable region

[0217] SEQ ID NO: 253: Humanized 10D12_VL-21 (h10D12-VL-21) light chain variable region

[0218] SEQ ID NO: 254: Humanized 13B1_VH-9 (h13B1-VH-9) heavy chain variable region

[0219] SEQ ID NO: 255: Humanized 13B1_VH-10 (h13B1-VH-10) heavy chain variable region

[0220] SEQ ID NO: 256: Humanized 13B1-VL-1 (h13B1-VL-1) light chain variable region

[0221] SEQ ID NO: 257: 4D5 and 13B1 LC-CDR1-NQ

[0222] SEQ ID NO: 258: 4D5 and 13B1 LC-CDR1-NA

[0223] SEQ ID NO: 259: 4D5 and 13B1 LC-CDR1-ST

[0224] SEQ ID NO: 260: LC-CDR1 shared by the parents 4D5 and 13B1 and variants

[0225] SEQ ID NO: 261:10D12 LC-CDR1-DE

[0226] SEQ ID NO: 262:10D12 LC-CDR1-DA

[0227] SEQ ID NO: 263:10D12 LC-CDR1-GA

[0228] SEQ ID NO: 264-277: HC FR and CDR of humanized 4D5, 10D12 and 13B1

[0229] SEQ ID NO: 278: h4D5_VL-1-NA

[0230] SEQ ID NO: 279:h10D12_VL-21-GA

[0231] SEQ ID NO: 280:h13B1_VL-1-NA

[0232] SEQ ID NO: 281-287 LC FR and CDR of humanized 4D5, 10D12 and 13B1

[0233] SEQ ID NO: 288-293: DNA encoding humanized 4D5, 10D12, 13B1 heavy chain variable regions and variants

[0234] SEQ ID NO: 294-299: DNA encoding humanized 4D5, 10D12, 13B1 light chain variable regions and variants

[0235] SEQ ID NO: 300: Parental DTLacO heavy chain variable region (VH) polypeptide

[0236] SEQ ID NO: 301: MASP-3 specific clone of M3J5 heavy chain variable region (VH) polypeptide

[0237] SEQ ID NO: 302: MASP-3 specific clone M3M1 heavy chain variable region (VH) polypeptide

[0238] SEQ ID NO: 303: Parental DTLacO light chain variable region (VL) polypeptide

[0239] SEQ ID NO: 304: MASP-3 specific clone M3J5 light chain variable region (VL) polypeptide

[0240] SEQ ID NO: 305: MASP-3 specific clone M3M1 light chain variable region (VL) polypeptide

[0241] SEQ ID NO: 306: MASP-3 clone D14 heavy chain variable region (VH) polypeptide

[0242] SEQ ID NO: 307: MASP-3 clone D14 light chain variable region (VL) polypeptide

[0243] SEQ ID NO: 308: MASP-1 clone 1E10 heavy chain variable region (VH) polypeptide

[0244] SEQ ID NO: 309: MASP-1 clone 1E10 light chain variable region (VL) polypeptide

[0245] SEQ ID NO: 310: Human IgG4 constant region

[0246] SEQ ID NO: 311: Human IgG4 constant region with S228P mutation

[0247] SEQ ID NO: 312: Human IgG4 constant region with S228P mutation_X

[0248] SEQ ID NO: 313: Human IgK constant region. Detailed Implementation

[0249] I. Definition

[0250] Unless expressly stated herein, all terms used herein have the same meaning as understood by one of ordinary skill in the art. When these terms are used in the specification and claims to describe the invention, the following definitions are provided to clarify the terms.

[0251] As used in this paper, lectin pathway effector branch (arm) 1 (“LEA-1”) refers to the lectin-dependent activation of factors B and D caused by MASP-3.

[0252] As used in this article, lectin pathway effector branch 2 (“LEA-2”) refers to MASP-2-dependent complement activation.

[0253] As used herein, the term “MASP-3-dependent complement activation” comprises two parts: (i) MASP-3-dependent activation of lectins of factors B and D, which includes LEA-1-mediated complement activation, in Ca ++ This occurs when present, typically leading to the transformation of C3bB to C3bBb and the transformation of pre-factor D to factor D; and (ii) lectin-independent transformations of factor B and factor D, which can occur in Ca ++ In their absence, C3bB typically transforms into C3bBb and pre-factor D into factor D. LEA-1-mediated complement activation and lectin-independent conversion of factor B and factor D have been established to lead to opsonization and / or cell lysis. While not wishing to be bound by any particular theory, it is believed that C3bBb C3 convertases only alter their substrate specificity and cleave C5 to the alternative pathway C5 convertase, i.e., C3bBb(C3b)n, when multiple C3b molecules are closely associated and bound.

[0254] As used herein, the term "MASP-2-dependent complement activation" is also referred to herein as LEA-2-mediated complement activation, including MASP-2 lectin-dependent activation, which in Ca ++It occurs when present, leading to the formation of C3 convertase C4b2a in the lectin pathway and the subsequent formation of C5 convertase C4b2a(C3b)n following the accumulation of the C3 cleavage product C3b, which has been identified as contributing to opsonization and / or cell lysis.

[0255] As used herein, the term “conventional understanding of alternative pathways” and also “conventional alternative pathways” refers to alternative pathways prior to those described herein, namely complement activation triggered, for example, by yeast polysaccharides from fungal and yeast cell walls, lipopolysaccharides (LPS) from Gram-negative outer membranes, and rabbit erythrocytes, as well as various pure polysaccharides, viruses, bacteria, animal tumor cells, parasites, and damaged cells, and has traditionally been considered to be caused by C3b generated from the spontaneous proteolytic hydrolysis of complement factor C3. As used herein, activation of “conventional alternative pathways” (also referred to herein as “alternative pathways”) occurs in Mg… ++ / EGTA buffer (i.e., in Ca) ++ (Measurement when it does not exist)

[0256] As used herein, the term "lectin pathway" refers to complement activation that occurs through the specific binding of serum and non-serum glyco-binding proteins, including mannan-binding lectin (MBL), CL-11, and fibrinogens (H-fibrinogen, M-fibrinogen, or L-fibrinogen). As described herein, the inventors have discovered that the lectin pathway is driven by two effector branches: lectin pathway effector branch 1 (LEA-1), which is now known to be MASP-3-dependent; and lectin pathway effector branch 2 (LEA-2), which is MASP-2-dependent. As used herein, activation of the lectin pathway utilizes Ca2+-containing... ++ The buffer solution is used for evaluation.

[0257] As used in this article, the term "classical pathway" refers to complement activation triggered by the binding of an antibody to an exogenous particle and requiring the binding of the recognition molecule C1q.

[0258] As used in this article, the term "HTRA-1" refers to the high-temperature requirement of serine peptidase for serine protease A1.

[0259] As used herein, the term "MASP-3 inhibitor" refers to any agent that directly inhibits MASP-3-dependent complement activation, including agents that bind to or directly interact with MASP-3, including MASP-3 antibodies and their MASP-3-binding fragments, natural and synthetic peptides, competitive substrates, small molecules, expression inhibitors, and isolated natural inhibitors, and also includes peptides that competitively bind to another recognition molecule (e.g., MBL, CL-11, H-fibrinogen, M-fibrinogen, or L-fibrinogen) in the lectin pathway. In one embodiment, the MASP-3 inhibitor is specific for MASP-3 and does not bind to MASP-1 or MASP-2. Inhibitors that directly inhibit MASP-3 may be referred to as direct MASP-3 inhibitors (e.g., MASP-3 antibodies), while inhibitors that indirectly inhibit MASP-3 may be referred to as indirect MASP-3 inhibitors (e.g., MASP-1 antibodies that inhibit MASP-3 activation). Examples of direct MASP-3 inhibitors are MASP-3-specific inhibitors, such as MASP-3 inhibitors that specifically bind to a portion of human MASP-3 (SEQ ID NO: 2) with a binding affinity at least 10-fold higher than other components of the complement system. Another example of a direct MASP-3 inhibitor is a high-affinity MASP-3 antibody that specifically binds to the serine protease domain of human MASP-3 (SEQ ID NO: 2) with an affinity of less than 500 pM and does not bind to MASP-1 (SEQ ID NO: 8). In one embodiment, the MASP-3 inhibitor indirectly inhibits MASP-3 activity, for example, MASP-3 activation inhibitors, including MASP-1-mediated MASP-3 activation inhibitors (e.g., MASP-1 antibodies or their MASP-1 binding fragments, natural and synthetic peptides, small molecules, expression inhibitors, and isolated natural inhibitors, and also including peptides that competitively bind to MASP-3 with MASP-1). In a preferred embodiment, a MASP-3 inhibitor, such as an antibody or its antigen-binding fragment or peptide, inhibits MASP-3-mediated factor D maturation. In another embodiment, the MASP-3 inhibitor inhibits MASP-3-mediated factor B activation. The MASP-3 inhibitor used in the method of the present invention can reduce MASP-3-dependent complement activation by more than 10%, for example, more than 20%, more than 50%, or more than 90%. In one embodiment, the MASP-3 inhibitor reduces MASP-3-dependent complement activation by more than 90% (i.e., resulting in MASP-3 complement activation of only 10% or less). It is anticipated that MASP-3 inhibition will completely or partially block both LEA-1-related cell lysis and opsonization, and the lectin-independent conversion of factor B and factor D-related cell lysis and opsonization.

[0260] In one embodiment, a high-affinity MASP-3 inhibitory antibody binds to the serine protease domain of MASP-3 (amino acid residues 450 to 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 alternative pathways of complement activation in the blood of mammalian subjects 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).

[0261] An antibody is an immunoglobulin molecule that can specifically bind to a target, such as a polypeptide, through at least one epitope recognition site located in the variable region (also referred to herein as the variable domain) of an immunoglobulin molecule.

[0262] As used herein, the term "antibody" includes antibodies and antibody fragments thereof that are derived from any antibody-producing mammal (such as mice, rats, rabbits, and primates, including humans), or from hybridomas, phage selection, recombinant expression, or transgenic animals (or other methods of producing antibodies or antibody fragments), and that specifically bind to a target polypeptide (such as MASP-1, MASP-2, or MASP-3 polypeptides or portions thereof). The term "antibody" is not intended to be limited by its source or method of preparation (e.g., via hybridomas, phage selection, recombinant expression, transgenic animals, peptide synthesis, etc.). Exemplary antibodies include polyclonal antibodies, monoclonal antibodies, and recombinant antibodies; pan-specific, multispecific antibodies (such as bispecific antibodies, trispecific antibodies); humanized antibodies; mouse antibodies; chimeric mouse-human, mouse-primate, and primate-human monoclonal antibodies; and anti-idiotype antibodies, and may be any complete antibody or fragment thereof. As used herein, the term “antibody” includes not only complete polyclonal or monoclonal antibodies, but also fragments thereof, such as single variable region antibodies (dAb), or other known antibody fragments such as Fab, Fab', F(ab')2, Fv, etc., single chains (ScFv), their synthetic variants, naturally occurring variants, fusion proteins including antibody portions of antigen-binding fragments with desired specificity, humanized antibodies, chimeric antibodies, bispecific antibodies, and any other modified conformation of immunoglobulin molecules containing antigen-binding sites or fragments (epitope recognition sites) with desired specificity.

[0263] "Monoclonal antibody" refers to a group of homogeneous antibodies, wherein the monoclonal antibody is composed of amino acids (naturally occurring and non-naturally occurring) involved in the selection of binding epitopes. Monoclonal antibodies are highly specific to target antigens. The term "monoclonal antibody" includes not only complete and full-length monoclonal antibodies, but also fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), single chains (ScFv), their variants, fusion proteins including antigen-binding portions, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified conformation of immunoglobulin molecules including antigen-binding fragments (epitope recognition sites) with the desired specificity and ability to bind epitopes. It is not intended to limit it based on the antibody's source or its preparation method (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term includes complete immunoglobulins as well as fragments described in the above definition of "antibody".

[0264] As used herein, the term "antibody fragment" refers to a portion of a full-length antibody (e.g., an antibody derived from, MASP-1, MASP-2, or MASP-3) that generally includes its antigen-binding region or its variable region. Illustrative examples of antibody fragments include Fab, Fab', F(ab)2, F(ab')2, and Fv fragments, scFv fragments, biantibodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0265] In some embodiments, the antibody and its antigen-binding fragment, as described herein, include sets of heavy chain (VH) and light chain (VL) complementarity-determining regions (“CDRs”) respectively inserted between sets of heavy and light chain framework regions (FRs), which support the CDRs and define their spatial relationship relative to each other. As used herein, the term “set of CDRs” refers to three hypervariable regions of the heavy or light chain V region. Derived from the N-terminus of the heavy or light chain, these regions are designated as “CDR1”, “CDR2”, and “CDR3”, respectively. Thus, the antigen-binding site comprises six CDRs, which contain a set of CDRs from each of the heavy and light chain V regions.

[0266] As used herein, the term "FR set" refers to the four side amino acid sequences of a CDR framework that forms the CDR set of the heavy or light chain V region. Some FR residues are accessible to the bound antigen; however, the FRs are primarily responsible for folding the V region into an antigen-binding site, especially the FR residues directly adjacent to the CDR. Within the FR, certain amino acid residues and certain structural features are highly conserved. In this respect, all V region sequences contain an internal disulfide ring of approximately 90 amino acid residues. With the V region folded into a binding site, the CDR appears as a prominent ring motif forming the antigen-binding surface. It is generally recognized that conserved structural regions of the FR influence the folding shape of the CDR ring to certain "canonical" structures—regardless of the precise CDR amino acid sequence.

[0267] The structure and location of the immunoglobulin variable region can be determined by referring to Kabat, EA et al., Sequences of Proteins of Immunological Interest, 4th edition, US Department of Health and Human Services, 1987, and its current updates available on the Internet (immuno.bme.nwu.edu.).

[0268] As used herein, a “single-chain Fv” or “scFv” antibody fragment comprises the VH and VL domains of the antibody, wherein these domains are located on a single polypeptide chain. Typically, Fv polypeptides also include a polypeptide linker between the VH and VL domains, which allows scFv to form the desired antigen-binding structure.

[0269] As used herein, a "chimeric antibody" is a recombinant protein containing a variable domain and a complementarity-determining region derived from an antibody from a non-human species (such as rodents), while the remainder 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 operatively linked to or otherwise fused to a heterologous Fc portion of a different antibody. In some embodiments, the heterologous Fc domain may be derived from an Ig type different from the parent antibody, including IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM.

[0270] As used herein, a “humanized antibody” is a chimeric molecule typically prepared using recombinant techniques, having an antigen-binding site derived from an immunoglobulin of a non-human species and the remaining immunoglobulin structure of the molecule based on the structure and / or sequence of a human immunoglobulin. The antigen-binding site may comprise a fully variable region fused to a constant domain or a CDR transplanted only to an appropriate scaffold region within the variable domain. Epitope binding sites may be wild-type or modified by substitution of one or more amino acids. Another approach focuses not only on providing a human-derived constant region but also on modifying the variable regions to remodel them as closely as possible to their human form. In some embodiments, the humanized antibody retains all CDR sequences (e.g., a humanized mouse antibody containing all six CDRs from a mouse antibody). In other embodiments, the humanized antibody has one or more CDRs (one, two, three, four, five, or six) altered relative to the original antibody, also referred to as one or more CDRs “derived” from one or more CDRs of the original antibody.

[0271] An antibody binds with a greater affinity and / or cohesion than it binds to other substances, thus "specifically binding" to its target. In one embodiment, the antibody or its antigen-binding fragment specifically binds to the serine protease domain of human MASP-3 (amino acid residues 450 to 728 of SEQ ID NO: 2). In one embodiment, the antibody or its antigen-binding fragment specifically binds to the substances described in Tables 4 and 28. Figure 62 One or more tabletops are displayed in the table.

[0272] As used herein, the term “mannan-binding lectin” (“MBL”) is equivalent to mannan-binding protein (“MBP”).

[0273] As used herein, the “membrane attack complex” (“MAC”) refers to a complex (also known as C5b-9) that inserts into and disrupts the terminal five complement components (C5b, as well as C6, C7, C8, and C9) of the membrane.

[0274] As used herein, “object” includes all mammals, including but not limited to humans, non-human primates, dogs, cats, horses, sheep, goats, cattle, rabbits, pigs, and rodents.

[0275] As used herein, the abbreviations for amino acid residues are as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0276] In the broadest sense, naturally occurring amino acids can be grouped according to the chemical properties of their side chains. "Hydrophobic" amino acids are any one of Ile, Leu, Met, Phe, Trp, Tyr, Val, Ala, Cys, or Pro. "Hydrophilic" amino acids are any one of Gly, Asn, Gln, Ser, Thr, Asp, Glu, Lys, Arg, or His. This grouping of amino acids can be further subdivided as follows: "Uncharged hydrophilic" amino acids are any one of Ser, Thr, Asn, or Gln. "Acidic" amino acids are any one of Glu or Asp. "Basic" amino acids are any one of Lys, Arg, or His.

[0277] As used herein, the term “conservative amino acid substitution” is described by substitutions between amino acids in each of the following groups: (1) glycine, alanine, valine, leucine, and isoleucine; (2) phenylalanine, tyrosine, and tryptophan; (3) serine and threonine; (4) aspartic acid and glutamic acid; (5) glutamine and asparagine; and (6) lysine, arginine, and histidine.

[0278] As used herein, the term “oligonucleotide” refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or its analogues. The term also includes oligonucleotide bases consisting of naturally occurring nucleotides, sugars, and internucleotide (backbone) covalent bonds, as well as oligonucleotides with non-naturally occurring modifications.

[0279] As used herein, an epitope is a site on a protein (e.g., human MASP-3 protein) that binds to an antibody. An overlapping epitope includes at least one (e.g., 2, 3, 4, 5, or 6) common amino acid residues, including linear and nonlinear epitopes.

[0280] As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably and refer to any peptide-linked chain of amino acids, regardless of length or post-translational modifications. The MASP-3 protein described herein may contain or be a wild-type protein, or may be a variant with no more than 50 (e.g., no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, or 50) conserved amino acid substitutions. Conserved 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.

[0281] In some embodiments, the human MASP-3 protein may have an amino acid sequence that has an identity of 70% or greater (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%) with the human MASP-3 protein having the amino acid sequence shown in SEQ IDNO: 2.

[0282] In some implementations, the peptide fragment can be at least 6 (e.g., at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, or 600 or more) amino acid residue length (e.g., SEQ ID NO: 2 (at least 6 consecutive amino acid residues). In some embodiments, the antigenic peptide fragment of human MASP-3 protein is less than 500. (e.g., less than 450, 400, 350, 325, 300, 275, 250, 225, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6) amino acid residue length (e.g., SEQ ID). NO: 2 contains fewer than 500 consecutive amino acid residues.

[0283] In some embodiments, when generating antibodies that bind to MASP-3, the peptide fragment is antigenic and retains 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 mammals (see “Methods for generating antibodies” below).

[0284] The percentage (%) of amino acid sequence identity is defined as the percentage of amino acids in a candidate sequence that are identical to those in a reference sequence after alignment and the introduction of gaps (if necessary) to achieve maximum percentage sequence identity. Alignments for determining the percentage of sequence identity can be performed in various ways within the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Suitable parameters for determining the alignment, including any algorithm required to achieve maximum alignment across the full length of the sequences to be compared, can be determined by known methods.

[0285] In a representative embodiment, the human MASP-3 protein (SEQ ID NO: 2) is encoded by the cDNA sequence shown in SEQ ID NO: 1. Those skilled in the art will recognize that the cDNA sequence disclosed in SEQ ID NO: 1 represents a single allele of human MASP-3 and is expected to be subject to allelic variations and alternative splicing. Allelic variants of the nucleotide sequence shown in SEQ ID NO: 1, including those containing silent mutations and those in which mutations result in changes to the amino acid sequence, are within the scope of this invention. Allelic variants of the MASP-3 sequence can be cloned according to standard procedures by probing cDNA or genomic libraries from different individuals, or allelic variants of the MASP-1, MASP-2, or MASP-3 sequences can be identified by homology comparison searches (e.g., BLAST searches) using databases containing said information.

[0286] As used herein, a “separated nucleic acid molecule” is a nucleic acid molecule (e.g., a polynucleotide) that is not integrated into the genomic DNA of an organism. For example, a DNA molecule encoding a growth factor that has been separated from the genomic DNA of a cell is a separated DNA molecule. Another example of a separated nucleic acid molecule is a chemically synthesized nucleic acid molecule that is not integrated into the genome of an organism. Nucleic acid molecules that have been separated from a particular species are smaller than the complete DNA molecules of the chromosomes from that species.

[0287] As used in this article, “nucleic acid molecule constructs” are single-stranded or double-stranded nucleic acid molecules that have been modified by human intervention to contain nucleic acid segments in arrangements and parallel arrangements that do not exist in nature.

[0288] As used herein, an "expression vector" is a nucleic acid molecule that encodes a gene expressed in a host cell. Typically, an expression vector contains a transcription promoter, a gene, and a transcription terminator. Gene expression is usually under the control of a promoter, and such a gene is said to be "operably linked" to the promoter. Similarly, if a regulatory element regulates the activity of a core promoter, the regulatory element and the core promoter are operably linked.

[0289] As used herein, the term “about” is intended to specify that a particular value provided may vary to a certain extent, such as within a range of ±10%, preferably ±5%, most preferably ±2%, including the given value.

[0290] Where a range is specified, the endpoints are included within that range, unless otherwise stated or obvious from the context.

[0291] As used herein, the singular forms “a,” “an,” and “the” include the plural aspect unless the context clearly specifies otherwise. Thus, for example, reference to “excipient” includes a variety of such excipients and their equivalents known to those skilled in the art; reference to “pharmaceutical” includes one pharmaceutical and two or more reagents; reference to “antibody” includes a variety of such antibodies; and reference to “architecture region” includes reference to one or more archetype regions and their equivalents known to those skilled in the art, and so on.

[0292] Unless otherwise expressly stated, each embodiment in this specification should be modified as necessary to apply to each other embodiment. It can be understood that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, the compositions of the invention can be used to implement the methods of the invention.

[0293] II. The lectin pathway: A new understanding

[0294] i. Overview: The lectin pathway has been redefined

[0295] As described herein, the inventors have made a surprising discovery: the complement lectin pathway has two effector branches that activate complement, both driven by lectin pathway activation complexes formed by carbohydrate recognition components (MBL, CL-11, and fibrinogen): (i) an effector branch formed by lectin pathway-associated serine proteases MASP-1 and MASP-3, referred herein as "lectin pathway effector branch 1" or "LEA-1"; and (ii) an activation effector branch driven by MASP-2, referred herein as "lectin pathway effector branch 2" or "LEA-2". Both LEA-1 and LEA-2 can have cell lysis and / or opsonization effects.

[0296] It has also been determined that MASP-3-induced lectin-independent transformation of factor B and HTRA-1, MASP-1, and MASP-3-induced lectin-independent transformation of factor D (both of which can be observed in Ca...) ++(When not present) This typically leads to the conversion of C3bB to C3bBb and the conversion of pre-factor D to factor D. Therefore, inhibition of MASP-3 can simultaneously inhibit the lectin-independent activation of LEA-1 and factor B and / or factor D, which can lead to inhibition of cell lysis and / or opsonization.

[0297] Figure 1 This illustrates a new understanding of the complement activation pathway. For example... Figure 1 As shown, LEA-1 is driven by lectin-bound MASP-3, which can activate factor D prozymogen to its active form and / or cleave C3b- or C3b(H2O)-bound factor B, resulting in the conversion of the C3bB prozymogen complex to its enzymatically active form, C3bBb. Activated factor D produced by MASP-3 can also convert the C3bB or C3b(H2O) prozymogen complex to its enzymatically active form. MASP-1 can rapidly self-activate, while MASP-3 cannot. In many cases, MASP-1 is an activator of MASP-3.

[0298] Although in many instances, lectins (i.e., MBL, CL-11, or fibrinogen) can direct activity toward the cell surface, Figure 1 The lectin-independent functions of MASP-3, MASP-1, and HTRA-1 in factor B activation and / or factor D maturation are also outlined. Just as the lectin-related form of MASP-3 in LEA-1, the lectin-independent form of MASP-3 can mediate the conversion of C3bB or C3b(H2O) to C3bBb (see also...). Figure 29 and 30 ) and transforming the former factor D into factor D (see Figure 32 MASP-1 (see also MASP-1) Figure 32 The non-MASP-related protein HTRA-1 can also activate factor D (Stanton et al., Evidence That the HTRA1 Interactome Influences Susceptibility to Age-Related Macular Degeneration (Submitted at The Association for Research in Vision and Ophthalmology 2011 meeting on May 4, 2011), its method does not require lectin components.

[0299] Therefore, MASP-1 (via LEA-1 and lectin-independent form), MASP-3 (via LEA-1 and lectin-independent form), and HTRA-1 (lectin-independent only) can be activated directly or indirectly at one or more points along the MASP-3-factor D-factor B axis. In this case, they produce C3bBb (an alternative pathway C3 convertase) and stimulate the production and deposition of C3b on the microbial surface. C3b deposition plays a key role in opsonization, marking the microbial surface for destruction by host phagocytic cells (e.g., macrophages). As an example in this paper ( Figure 28A and 28B MASP-3 is crucial for the opsonization of Staphylococcus aureus. C3b deposition on Staphylococcus aureus exposed to human serum occurs rapidly in a MASP-3-dependent manner. Figure 28A and 28B ).

[0300] However, the contributions of LEA-1 and MASP-3, or MASP-1 or HTRA-1 to lectin-independent functions are not limited to opsonization. For example... Figure 1 As shown, these three components can also induce cell lysis and the production of C3b through indirect or direct activation of factor B. These components form a complex that produces the alternative pathway C5 convertase, C3bBb (C3b). n As further described in this article, in the cell lysis of Neisseria meningitidis (see...) Figure 11 , 12 The need for MASP-3 and MBL instead of MASP-2 (and, therefore, LEA-2 in this instance) in 13) indicates the role of LEA-1 in cell lysis. In summary, the opsonization results from Staphylococcus aureus studies and the cell lysis results observed in Neisseria meningitidis studies support the role of LEA-1 in both processes (e.g., Figure 1 (As shown). Furthermore, these studies indicate that both opsonization and cell lysis can originate from the conversion of C3bB or C3b(H2O) and / or the conversion of pro-factor D to factor D; therefore, these two processes may be the result of lectin-independent effects of MASP-3, MASP-1, or HTRA-1. Therefore, in Figure 1 The inventors' model supports the use of inhibitors of MASP-3, as well as inhibitors of MASP-1 and / or HTRA-1, to block opsonization and / or cell lysis and treat pathology caused by the dysregulation of these processes.

[0301] 1. Lectin pathway effector branch (LEA-1)

[0302] The first effector branch of the lectin pathway, LEA-1, is formed by the lectin pathway-associated serine proteases MASP-1 and MASP-3. As described herein, the inventors have now demonstrated that the alternative pathway is not effectively activated at the surface structure in the absence of MASP-3 and in the presence of MASP-1. These results indicate that MASP-3 plays a previously undisclosed role in initiating the alternative pathway, and this is confirmed using MASP-3-deficient 3MC serum obtained from patients with a rare 3MC autosomal recessive disorder (Rooryck C, et al., Nat Genet. 43(3):197-203 (2011)), who have mutations that dysregulate the MASP-3 serine protease domain. Based on these new findings, complement activation involving the alternative pathway is expected to be MASP-3-dependent, as conventionally defined. In fact, MASP-3, and its LEA-1 activation, could represent an initiator of the alternative pathway that has not been understood until now.

[0303] As further described in Examples 1-4 of this document, the inventors observed higher activity of the lectin-dependent alternative pathway activation in MASP-2-deficient serum, which resulted in higher bactericidal activity (i.e., cell lysis activity) against Neisseria meningitidis. 。 While not wishing to be bound by any particular theory, it is thought that in the absence of MASP-2, carbohydrate recognition complexes with MASP-1 are more likely to bind tightly to carbohydrate recognition complexes with MASP-3 to activate MASP-3. It is known that in many cases, the activation of MASP-3 depends on the activity of MASP-1 because MASP-3 is not a self-activating enzyme and often requires the activity of MASP-1 to convert from its zymogen form to its enzymatically active form. MASP-1 (like MASP-2) is a self-activating enzyme, while MASP-3 is not, and in many cases, requires the enzymatic activity of MASP-1 to convert to its enzymatically active form. See Zundel S, et al. J Immunol., 172(7):4342-50(2004). In the absence of MASP-2, all lectin pathway recognition complexes are loaded with either MASP-1 or MASP-3. Therefore, the absence of MASP-2 promotes the conversion of MASP-1-mediated MASP-3 to its enzymatically active form. Once MASP-3 is activated, the activated MASP-3 initiates alternative pathway activation (now called “LEA-1” activation) via the MASP-3-mediated conversion of C3bB to C3bBb and / or the conversion of pre-factor D to factor D. C3bBb, also known as the alternative pathway C3 convertase, cleaves additional C3 molecules, resulting in the deposition of opsonin C3b molecules. If several C3b fragments are close together and bind to the C3bBb convertase complex, this leads to the formation of the alternative pathway C5 convertase C3bBb(C3b)n, which promotes MAC formation. Additionally, C3b molecules deposit on the surface, forming new sites for factor B binding, which can now be cleaved by factor D and / or MASP-3 to form additional sites, where alternative pathway C3 and C5 convertase complexes can form. This latter process is required for efficient cell lysis, and once initial C3b deposition has occurred, lectins are not needed. Recent publications (Iwaki D. et al., J Immunol 187(7):3751-8(2011)) and the data obtained by the inventor ( Figure 30 This indicates that the alternative pathway C3 convertase zymogen complex C3bB is converted to its enzymatically active form by activating MASP-3. The inventors have now discovered that MASP-3-mediated cleavage of factor B represents a subcomponent of the newly described LEA-1, which promotes the lectin-dependent formation of the alternative pathway C3 convertase C3bBb.

[0304] 2. Lectin pathway effector branch (LEA-2)

[0305] The second effector branch of the lectin pathway, LEA-2, is formed by the lectin pathway-associated serine protease MASP-2. MASP-2 is activated upon binding of the recognition component to its respective pattern, and can also be activated by MASP-1, subsequently cleaving complement component C4 into C4a and C4b. When the cleavage product C4b binds to plasma C2, the C4b-bound C2 becomes the substrate for the second MASP-2-mediated cleavage step, which converts the C4b-bound C2 into the enzymatically active complex C4bC2a and a small C2b cleavage fragment. C4b2a is a C3-converting enzyme of the lectin pathway, converting abundant plasma component C3 into C3a and C3b. C3b binds to any nearby surface via a thioester bond. If several C3b fragments come close together and bind to the C3 convertase complex C4b2a, the convertase changes its specificity, converting C5 to C5b and C5a, forming the C5 convertase complex C4b2a(C3b)n. Although this C5 convertase can initiate MAC formation, this process is not considered to effectively promote cell lysis on its own. Instead, the initial C3b opsonin generated by LEA-2 forms a nucleus to form new alternative pathway C3 convertase and C5 convertase sites, which ultimately leads to massive MAC formation and cell lysis. This latter event is mediated by factor D activation of factor B associated with C3b formation by LEA-2, and is therefore dependent on LEA-1 due to the essential role of MASP-1 in factor D maturation. There also exists a MASP-2-dependent C4-alternative activation pathway to activate C3 in the absence of C4, which plays an important role in the pathophysiology of ischemia-reperfusion injury because C4-deficient mice cannot protect themselves from ischemia-reperfusion injury, while MASP-2-deficient mice can (Schwaeble et al., PNAS , 2011 supra LEA-2 is also involved in the coagulation pathway, including the cleavage of prothrombin into thrombin (common pathway) and also into factor XII (contact factor) to convert it into its enzymatically active form, XIIa. Factor XIIa, in turn, cleaves factor XI into factor XIa (inherent pathway). Activation of the inherent pathway of the coagulation cascade leads to fibrin formation, which is crucial for thrombosis.

[0306] Figure 1 Based on the results presented in this paper, we have developed a new understanding of the lectin pathway and alternative pathways. Figure 1 The role of LEA-2 in both opsonization and cell lysis was described. Although MASP-2 is an initiator of "downstream" C3b deposition (and the resulting opsonization) in multiple physiological lectin-dependent environments (…), Figure 18A , 18B (18C), but it also plays a role in the cell lysis of serum-sensitive bacteria. For example... Figure 1 As shown, for serosensitive pathogens such as Neisseria meningitidis, the proposed molecular mechanism responsible for increased bactericidal activity in MASP-2-deficient or MASP-2-depleted serum / plasma is that, for bacterial cell lysis, the lectin pathway recognition complex associated with MASP-1 and MASP-3 must bind close to each other to the bacterial surface, thereby allowing MASP-1 to cleave MASP-3. Unlike MASP-1 and MASP-2, MASP-3 is not a self-activating enzyme, but in many cases requires activation / cleavage by MASP-1 to be converted to its enzymatically active form.

[0307] Further as Figure 1 As shown, activated MASP-3 can then cleave C3b-bound factor B on the pathogen surface, initiating an alternative activation cascade by forming C3bBb and C3bBb(C3b)n, respectively, the alternative pathway C3 and C5 convertases. The lectin-pathway activation complex carrying MASP-2 does not participate in MASP-3 activation, and in the absence of MASP-2 or after its depletion, all lectin-pathway activation complexes will be loaded with either MASP-1 or MASP-3. Therefore, in the absence of MASP-2, the likelihood of lectin-pathway activation complexes carrying MASP-1 and MASP-3 approaching each other on the microbial surface is significantly increased, leading to the activation of more MASP-3, resulting in a higher rate of MASP-3-mediated C3b-bound factor B cleavage and the formation of alternative pathway C3 and C5 convertases C3bBb and C3bBb(C3b)n on the microbial surface. This leads to the activation of the terminal activation cascade C5b-C9, forming a membrane attack complex composed of surface-bound C5b-C6 associations, C5bC6-C7 associations, C5bC6C7-C8 associations, and C5bC6C7C8, resulting in C9 polymerization, which inserts into the bacterial surface structure and forms pores in the bacterial wall, leading to complement-targeted osmotic killing of bacteria.

[0308] The core of this new concept is that the data provided in this paper clearly demonstrate that the lectin pathway activation complex drives the following two distinct activation pathways, such as... Figure 1 As shown:

[0309] i) LEA-1: A MASP-3-dependent activation pathway that initiates and drives complement activation by generating the alternative pathway convertase C3bBb through initial cleavage and activation of factor B on the activator surface, subsequently catalyzing C3b deposition and the formation of the alternative pathway convertase C3bBb. The MASP-3-driven activation pathway plays a crucial role in opsonization and microbial cell lysis, driving the alternative pathway on the bacterial surface, leading to optimal activation rates and the generation of the membrane attack complex; and

[0310] ii) LEA-2: The MASP-2-dependent activation pathway leads to the formation of C3 convertase C4b2a in the lectin pathway, and after the accumulation of the C3 cleavage product C3b, C5 convertase C4b2a(C3b)n is subsequently formed. In the absence of complement C4, MASP-2 can form a substitute C3 convertase complex, which includes C2 and coagulation factor XI.

[0311] In addition to its role in cell lysis, the MASP-2-driven activation pathway also plays a crucial role in bacterial opsonization, leading to the coating of microorganisms with covalently bound C3b and its cleavage products (i.e., iC3b and C3dg). This makes them targets for uptake and killing by phagocytes carrying C3 receptors (e.g., granulocytes, macrophages, monocytes, microglia) and the reticuloendothelial system. This is the most effective pathway for clearing bacteria and microorganisms resistant to complement cell lysis. These include most Gram-positive bacteria.

[0312] In addition to LEA-1 and LEA-2, there is a possibility of lectin-independent activation of factor D caused by MASP-3, MASP-1 and / or HTRA-1, and there is also a possibility of lectin-independent activation of factor B caused by MASP-3.

[0313] Although not wishing to be bound by any particular theory, it is believed that each of (i) LEA-1, (ii) LEA-2 and (iii) factor B and / or factor D in lectin-independent activation leads to opsonization and / or MAC formation accompanied by some cell lysis.

[0314] ii. Background of MASP-1, MASP-2 and MASP-3

[0315] Three mannan-binding lectin-associated serine proteases (MASP-1, MASP-2, and MASP-3) are currently known to be associated with human serum containing mannan-binding lectins (MBL). Mannan-binding lectins are also referred to as “mannose-binding proteins” or “mannose-binding lectins” in recent literature. The MBL-MASP complex plays an important role in innate immunity through the binding of MBL to carbohydrate structures present on various microorganisms. The interaction of MBL with specific arrangements of carbohydrate structures leads to the activation of the MASP proenzyme, which in turn activates complement by cleaving complement components C4 and C2 to form the C3 convertase C4b2b (Kawasaki et al.). J. Biochem 106:483-489 (1989); Matsushita & Fujita, J. Exp Med176:1497-1502 (1992); Ji et al. J. Immunol 150:571-578 (1993)).

[0316] The MBL-MASP proenzyme complex was until recently thought to contain only one type of protease (MASP-1), but it is now clear that two other distinct proteases (MASP-2 and MASP-3) are associated with MBL (Thiel et al.). Nature 386:506-510 (1997); Dahl et al., Immunity 15:127-135 (2001), and another serum protein of 19 kDa, called "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)).

[0317] MAp19 is the alternative splicing gene product of the structural genes of MASP-2 and lacks all four C-terminal domains of MASP-2, including the serine endopeptidase domain. Alternative splicing / polyadenylation events in the MASP-2 gene produce a heavily expressed truncated mRNA transcript encoding MAp19. Through a similar mechanism, the MASP-1 / 3 genes result in three major gene products: two serine proteases, MASP-1 and MASP-3, and a truncated 44 kDa gene product called “MAp44” (Degn et al.). J. Immunol 183(11):7371-8 (2009); Skjoedt et al., J Biol Chem 285:8234-43(2010)).

[0318] MASP-1 was first described as a component of the P-100 protease of serum Ra-response factor, and it is now considered to be a complex consisting of MBL and MASP (Matsushita et al.). Collectins and Innate Immunity , (1996); Ji et al., J Immunol150:571-578 (1993). The ability of the MBL-associated endopeptidase within the MBL-MASP complex to act on complement components C4 and C2 in a manner remarkably similar to that of the C1s enzyme within the C1q-(Clr)2-(Cls)2 complex in the classical complement pathway suggests the existence of an MBL-MASP complex functionally similar to the C1q-(C1r)2-(C1s)2 complex. The C1q-(C1r)2-(C1s)2 complex is activated by the interaction of C1q with the Fc region of antibodies IgG or IgM present in the immune complex. This leads to the autoactivation of the C1r proenzyme, which in turn activates the C1s proenzyme, which then acts on complement components C4 and C2.

[0319] The stoichiometry of the MBL-MASP complex differs from that found in the C1q-(C1r)2-(C1s)2 complex in that different MBL oligomers appear to be associated with different ratios of MASP-1 / MAp19 or MASP-2 / MASP-3 (Dahl et al.). Immunity 15:127-135 (2001). Most MASP and MAP19 present in serum do not complex with MBL (Thiel et al., J Immunol 165:878-887 (2000)) and can partially associate with fibrinogen, a group of lectins currently described that possess fibrinogen-like domains and can bind to N-acetylglucosamine residues on the surface of microorganisms (Le et al., FEBS Lett 425:367 (1998); Sugimoto et al., J. Biol Chem 273:20721 (1998)). Among these, human L-fibrin, H-fibrin, and M-fibrin associate with MASP and MAp19, and upon binding to specific carbohydrate structures recognized by fibrin, they can activate the lectin pathway (Matsushita et al., J Immunol 164:2281-2284 (2000); Matsushita et al., J Immunol 168:3502-3506 (2002)). Besides fibrinogen and MBL, MBL-like lectin collagen lectin (referred to as CL-11) has been identified as a lectin pathway recognition molecule (Hansen et al., J Immunol 185:6096-6104 (2010); Schwaeble et al. PNAS108:7523-7528 (2011)). There is very clear evidence of the physiological importance of these alternative carbohydrate recognition molecules, therefore it is important to understand that MBL is not the only recognition component of the lectin activation pathway and that MBL defects are not mistaken for lectin-pathway defects. The possible existence of a set of alternative carbohydrate-recognition complexes associated with MBL structure could broaden the microbial structural profile of direct responses to the innate immune system initiated via complement activation.

[0320] All lectin pathways recognize molecules characterized by a specific MASP-binding motif within their collagen-homogeneous stem region (Wallis et al.). J. Biol Chem 279:14065-14073 (2004)). The MASP-binding site in MBL, CL-11, and fibrin is characterized by a unique motif within this domain: Hyp-Gly-Lys-Xaa-Gly-Pro, where Hyp is a hydroxyproline residue and Xaa is typically an aliphatic residue. Point mutations in this sequence disrupt MASP binding.

[0321] 1. The respective structures, sequences, chromosomal locations, and splicing variants of MASP-1 and MASP-3.

[0322] Figure 2 This is a schematic diagram illustrating the domain structures of human MASP-1 polypeptide (SEQ ID NO: 8), human MASP-3 polypeptide (SEQ ID NO: 2), and human Map44 polypeptide, as well as the exons encoding them. Figure 2 As shown, the serine proteases MASP-1 and MASP-3 consist of six unique domains arranged as seen in C1r and C1s; namely (I) an N-terminal C1r / C1s / sea urchin VEGF / bone morphogenetic protein (or CUBI) domain; (II) an epidermal growth factor (EGF)-like domain; (III) a second CUB domain (CUBII); (IV and V) two complement control protein (CCP1 and CCP2) domains; and (VI) a serine protease (SP) domain.

[0323] cDNA-derived amino acid sequences of human and mouse MASP-1 (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); cDNA-derived amino acid sequences of human, mouse, and rat MASP-2 (Thiel et al., Nature386:506-510 (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 the cDNA-derived amino acid sequence of human MASP-3 (Dahl et al., Immunity References 15:127-135 (2001) indicate that these proteases are serine peptidases with a characteristic triplet of His, Asp, and Ser residues in their putative catalytic domain (Genbank accession numbers: 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, as accessed in Genbank on 2 / 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 monkey (SEQ ID NO: 7)).

[0324] Further as Figure 2 As shown, when the proenzyme is converted to its active form, the heavy chain (α or A chain) and the light chain (β or B chain) split to yield a disulfide-linked A-chain and a smaller B-chain representing the serine protease domain. The single-chain proenzyme MASP-1 is activated by cleaving the Arg-Ile bond located between the second CCP domain (domain V) and the serine protease domain (domain VI) (like proenzymes C1r and C1s). Proenzymes MASP-2 and MASP-3 are thought to be activated in a similar manner to MASP-1. Each MASP protein forms a homodimer and is charged with Ca2+. ++ - It associates with MBL and fibrinogen in a dependent manner, respectively.

[0325] Human MASP-1 polypeptide (SEQ ID NO: 8) and MASP-3 polypeptide (SEQ ID NO: 2) originate from a single structural gene (Dahl et al., Immunity 15:127-135 (2001), which has been mapped to the 3q27-28 region of the long arm of chromosome 3 (Takada et al., Genomics25:757-759 (1995)). The mRNA transcripts of MASP-3 and MASP-1 are produced from the primary transcript via alternative splicing / polyadenylation. The MASP-3 translation product consists of an α-chain (shared by MASP-1 and MASP-3) and a β-chain (serine protease domain) (unique to MASP-3). For example... Figure 2 As shown, the human MASP-1 gene comprises 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. Further details are as follows... Figure 2 As shown, the human MASP3 gene comprises 12 exons. The human MASP-3 cDNA (as described in SEQ ID NO: 1) is encoded by exons 2, 3, 4, 5, 6, 7, 8, 10, 11, and 12. Alternative splicing produces a protein called MBL-associated protein 44 (“MAp44”), derived from exons 2, 3, 4, 5, 6, 7, 8, and 9.

[0326] The human MASP-1 polypeptide (SEQ ID NO: 8 from Genbank BAA04477.1) has 699 amino acid residues, including a 19-residue precursor peptide. When the precursor peptide is omitted, the calculated molecular weight of MASP-1 is 76,976 Da. Figure 2 As shown, the MASP-1 amino acid sequence contains four N-linked glycosylation sites. The human MASP-1 protein domain (see SEQ ID NO: 8) is shown in... Figure 2 It also includes the tandem N-terminal C1r / C1s / sea urchin VEFG / bone morphogenetic protein (CUBI) domain (SEQ ID NO: 8, aa 25-137), epidermal growth factor-like domain (SEQ ID NO: 8, aa 139-181), second CUB domain (CUBII) (SEQ ID NO: 8, aa 185-296), and complement control protein (SEQ ID NO: 8, CCP1aa 301-363 and CCP2aa 367-432) domains, as well as the serine protease domain (SEQ ID NO: 8, aa 449-694).

[0327] Human MASP-3 polypeptide (SEQ ID NO: 2, from Genbank AAK84071) has 728 amino acid residues (e.g., Figure 3 As shown in the figure, it includes a 19-residue precursor peptide (as shown in the figure). Figure 3 (The amino acid sequence is shown as underlined in the image).

[0328] When the leader peptide is omitted, the calculated molecular weight of MASP-3 is 81,873 Da. For example... Figure 2 As shown, MASP-3 has seven N-linked glycosylation sites. The domains of the human MASP-3 protein (see SEQ ID NO: 2) are shown in... Figure 2 It also includes the tandem N-terminal C1r / C1s / sea urchin VEGF / bone morphogenetic protein (CUBI) domain (SEQ ID NO: 2, aa 25-137), epidermal growth factor-like domain (SEQ ID NO: 2, aa 139-181), second CUB domain (CUBII) (SEQ ID NO: 2, aa 185-296), and complement control protein (SEQ ID NO: 2, CCP1, aa 299-363 and CCP2, aa 367-432) domains, as well as the serine protease domain (SEQ ID NO: 2, aa 450-728).

[0329] The MASP-3 translation product consists of an α chain (heavy chain) (α chain: aa 1-448 of SEQ ID NO: 2) and a light chain (β chain: aa 449-728 of SEQ ID NO: 2); the α chain contains a CUB-1-EGF-CUB-2-CCP-1-CCP-2 domain, which is common to both MASP-1 and MASP-3, and the light chain contains a serine protease domain, which is unique to MASP-3.

[0330] 2. Comparison of MASP-3 amino acid sequences from various species

[0331] Figure 4 Multispecies comparisons of MASP-3 are provided, showing comparisons of full-length MASP-3 proteins from humans (SEQ ID NO: 2), cynomolgus monkeys (SEQ ID NO: 7), rats (SEQ ID NO: 4), mice (SEQ ID NO: 3), chickens (SEQ ID NO: 5), and rabbits (SEQ ID NO: 6). Figure 5 Provides multi-species comparisons of serine protease (SP) domains from humans (SEQ ID NO: 2, aa 450-728); rabbits (SEQ ID NO: 6, aa 450-728); mice (SEQ ID NO: 3, aa 455-733); rats (SEQ ID NO: 4, aa 455-733); and chickens (SEQ ID NO: 5, aa aa 448-730).

[0332] like Figure 4As shown, the MASP-3 polypeptide exhibits high levels of amino acid sequence conservation across different species, particularly within the SP domain. Figure 5 ).like Figure 5 As further shown, the catalytic triad (referencing full-length human MASP-3 (SEQ ID NO:2), H at residue 497; D at residue 553 and S at residue 664) is conserved across species. Table 1 summarizes the percentage of identity of the MASP-3 SP domains across species.

[0333] Table 1: Percentage identity of MASP-3 SP domains among species

[0334] Crab-eating macaques rabbit rats mice chicken people 95% 94% 92% 91% 79% Crab-eating macaques 94% 90% 90% 79% rabbit 92% 92% 81% rats 97% 78% mice 78%

[0335] MASP-3 has no proteolytic activity against C4, C2, or C3 substrates. Instead, MASP-3 was initially reported to act as an inhibitor of the lectin pathway (Dahl et al.). Immunity 15:127-135 (2001)). This conclusion may be drawn because, unlike MASP-1 and MASP-2, MASP-3 is not a self-activating enzyme (Zundel S. et al., J Immunol 172:4342-4350 (2004); Megyeri et al., J. Biol. Chem. 288:8922–8934 (2013).

[0336] Recently, using mouse strains deficient in both MASP-1 and MASP-3, evidence has been obtained from transgenic mouse studies regarding the potential physiological functions of MASP-1 and MASP-3. Although MASP-1 / 3 knockout mice possess a functional lectin pathway (Schwaeble et al., PNAS 108:7523-7528 (2011)), but they appear to lack alternative pathway activity (Takahashi et al., JEM 207(1):29-37(2010)). The lack of alternative pathway activity appears to be due to a processing defect of complement factor D, which is essential for alternative pathway activity. In MASP-1 / 3 knockout mice, all factor D circulates in its pro-form form, which is inactive due to proteolysis, while in normal mouse serum, almost all factor D is in its active form. Biochemical analysis suggests that MASP-1 can convert complement factor D from its prozymogeneous form to its enzymatically active form ( ). Figure 32 Takahashi et al. JEM 207(1):29-37(2010)). MASP-3 also cleaves prefactor D zymogen and produces active factor D in vitro. Figure 32Takahashi et al. JEM 207(1):29-37(2010)). Factor D exists in circulation in normal individuals as an active enzyme, and MASP-1 and MASP-3, as well as HTRA-1, are likely responsible for its activation. Furthermore, mice with combined MBL and fibrinogen deficiencies still produce normal levels of factor D and have a fully functional alternative pathway. Therefore, these physiological functions of MASP-1 and MASP-3 are not necessarily involved in lectins and are therefore unrelated to the lectin pathway. Recombinant mouse and human MASP-3 also appear to cleave factor B in vitro and support C3 deposition on Staphylococcus aureus (…). Figure 29 Iwaki D. et al., J Immunol 187(7):3751-8(2011)).

[0337] A recent study of patients with 3MC syndrome (formerly known as Carnevale, Mingarelli, Malpuech, and Michels syndrome; OMIM# 257920) has revealed an unexpected physiological role of MASP-3. These patients exhibit severe developmental abnormalities, including cleft palate, cleft lip, cranial malformations, and intellectual disability. Genetic analysis identified 3MC patients with a dysfunctional MASP-3 gene who were homozygous (Rooryck et al., Ro ... Nat Genet. 43(3):197-203 (2011)). Another group of 3MC patients were found to be homozygous for a mutation in the MASP-1 gene, which resulted in the absence of functional MASP-1 and MASP-3 proteins. Yet another group of 3MC patients lacked the functional CL-11 gene (Rooryck et al., Nat Genet . 43(3):197-203(2011)). Therefore, the CL-11 MASP-3 axis appears to play a role during embryonic development. The molecular mechanism of this developmental pathway is unclear. However, it is unlikely to be mediated by a conventional complement-driven process, as this syndrome does not occur in individuals with deficiencies in the common complement component C3. Therefore, prior to the inventors' discovery, the functional role of MASP-3 in lectin-dependent complement activation, as described herein, had not been previously determined.

[0338] The structures of the MASP-1 and MASP-2 catalytic fragments have been determined by X-ray crystallography. Structural comparisons of the MASP-1 protease domain with other complement proteases reveal the basis for its non-strict substrate specificity (Dobó et al.). J. Immunol 183:1207-1214 (2009)). Although the accessibility of the substrate binding trench of MASP-2 is limited by the surface ring (Harmat et al., J Mol Biol342:1533-1546 (2004)), but MASP-1 has an open substrate-binding pocket, which is similar to trypsin rather than other complement proteases. The thrombin-like nature of the MASP-1 structure is the unusually large 60-amino acid ring (ring B) that can interact with the substrate. Another attractive property of the MASP-1 structure is the internal salt bridge between S1 Asp189 and Arg224. Similar salt bridges can be found in the substrate-binding pocket of factor D, which can regulate its protease activity. C1s and MASP-2 have almost identical substrate specificity. Surprisingly, some of the eight surface rings of MASP-2 that determine substrate specificity have completely different conformations compared to C1s. This means that these two functionally related enzymes interact with the same substrate in different ways. The structure of prozymogen MASP-2 shows an inactive protease domain with a disrupted oxyanion hole and substrate-binding pocket (Gál et al., J Biol Chem 280:33435-33444 (2005)). Surprisingly, prozymogen MASP-2 exhibits considerable activity on the large protein substrate C4. It is likely that the structure of prozymogen MASP-2 is quite flexible, enabling the conversion between its inactive and active forms. This flexibility reflected in its structure may play a role in the self-activation process.

[0339] Northern blot analysis indicated that the liver is the primary source of MASP-1 and MASP-2 mRNA. Using a 5' specific cDNA probe targeting MASP-1, large MASP-1 transcripts at 4.8 kb and small ones at approximately 3.4 kb were observed in both 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 highly expressed in liver tissue. MASP-3 is expressed in the liver and also in many other tissues, including neural tissue (Lynch NJ et al., 4:374-84 (2003)). J Immunol 174:4998-5006 (2005)).

[0340] Patients with a history of infection and chronic inflammatory disease were found to have a mutated form of MASP-2 that was unable to form the active MBL-MASP complex (Stengaard-Pedersen et al., N Engl J Med 349:554-560 (2003). Some researchers have identified MBL deficiency as a predisposition to frequent infections in children (Super et al., Lancet 2:1236-1239 (1989); Garred et al., Lancet346:941-943 (1995) and increased resistance to HIV infection (Nielsen et al., Clin Exp Immunol 100:219-222 (1995); Garred et al., Mol Immunol 33 (Supplement 1):8 (1996)). However, other studies have not shown a significant association between low MBL levels and increased infection (Egli et al., PLoS One. 8(1):e51983 (2013); Ruskamp et al., J Infect Dis. 198(11):1707-13 (2008); Israëls et al., Arch Dis Child Fetal Neonatal Ed. 95(6):F452-61 (2010)). Although the literature is mixed, the deficiency or ineffectiveness of MASP may have adverse effects on an individual's ability to generate direct, non-antibody-dependent defenses against certain pathogens.

[0341] New understanding supports data, emphasizing the lack of Ca ++ Traditional determination conditions and uses include Ca ++ More physiological The result is obtained by setting the conditions.

[0342] This article provides several independent and compelling experimental pieces of evidence that the complement lectin pathway activates complement through two independent effector mechanisms: i) LEA-2:MASP-2-driven pathway, which mediates complement-driven opsonization and 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, namely through the cleavage and activation of factor B on the activator surface to produce the alternative pathway convertase C3bBb, which then catalyzes the deposition of C3b and the formation of the alternative pathway convertase C3bBb, which can lead to cell lysis and opsonization. Additionally, as described herein, individual lectin-independent activation of factor B and / or factor D by MASP-1, MASP-3, or HTRA-1, or any combination of these three, can also lead to complement activation via the alternative pathway.

[0343] Alternative pathways, such as the lectin pathway-dependent MASP-3-driven activation, appear to facilitate well-established factor D-mediated cleavage of C3b-binding factor B to achieve optimal activation rates for complement-dependent cell lysis via a terminal activation cascade, lysing bacterial cells through the formation of the C5b-9 membrane attack complex (MAC) on the cell surface. Figure 12-13This rate-limiting event appears to require optimal coordination because it is defective in the absence of MASP-3 functional activity and in the absence of factor D functional activity. As described in Examples 1-4 of this paper, the inventors discovered the function of this MASP-3-dependent lectin pathway when studying the phenotypes of MASP-2 deficiency and MASP-2 inhibition in an experimental mouse model of Neisseria meningitidis infection. Genetically targeted MASP-2-deficient mice and wild-type mice treated with antibody-based MASP-2 inhibitors exhibited high resistance to experimental Neisseria meningitidis infection (see...). Figure 6-10 When the infection dose was adjusted to achieve a mortality rate of approximately 60% in wild-type littermates, all MASP-2-deficient or MASP-2-depleted mice cleared the infection and survived (see [link]). Figure 6 and Figure 10 The significantly increased bactericidal activity in the serum of MASP-2-deficient or MASP-2-depleted mice reflects this extremely high level of resistance. Further experiments showed that this bactericidal activity depends on an alternative pathway-driven bacterial lysis. Serum from mice lacking factor B, factor D, or C3 showed no bactericidal activity against *Neisseria meningitidis*, indicating that the alternative pathway is necessary to drive the terminal activation cascade. Surprisingly, mouse serum lacking MBL-A and MBL-C (both lectin-pathway recognition molecules for *Neisseria meningitidis*) and mouse serum lacking lectin pathway-associated serine proteases MASP-1 and MASP-3 lost all lytic activity against *Neisseria meningitidis*. Figure 13 Recent papers (Takahashi M. et al., JEM 207: 29-37 (2010) and the work presented in this paper ( Figure 32 This indicates that MASP-1 can convert the proenzyme form of factor D into its enzymatically active form and may partially explain the loss of cell lysis activity due to the absence of the enzymatically active factor D in these serums. This does not explain the lack of bactericidal activity in MBL-deficient mice, as these mice have normal enzymatically active factor D (Banda et al., Mol Imunol 49(1-2):281-9 (2011)). Notably, when human serum from patients with rare 3MC autosomal recessive disease (who have mutations that dysfunction the MASP-3 serine protease domain) was tested (Rooryck C, et al., Nat Genet. 43(3):197-203), no bactericidal activity against Neisseria meningitidis was detected (note: these sera have MASP-1 and factor D, but no MASP-3).

[0344] The hypothesis that human serum requires agglutinin pathway-mediated MASP-3-dependent activity to develop bactericidal activity is further supported by the following observations: MBL-deficient human serum also cannot lyse Neisseria meningitidis. Figure 11-12 MBL is the only human lectin-pathway recognition molecule that binds to this pathogen. Because MASP-3 is not self-activating, the inventors hypothesize that the increased bacterial lysis activity in MASP-2-deficient serum can be explained by the favorable activation of MASP-3 via MASP-1, since in the absence of MASP-2, all lectin-pathway activation complexes bound to the bacterial surface would be loaded with either MASP-1 or MASP-3. This is because activated MASP-3 simultaneously cleaves factor D (…) in vitro. Figure 32 ) and factor B, producing their respective enzymatic active forms ( Figure 30 And Iwaki D., et al. J. Immunol .187(7):3751-3758(2011)), so the most likely function of MASP-3 is to promote the formation of alternative pathway C3 convertase (i.e. C3bBb).

[0345] Although the data on lectin-dependent effects are noteworthy, multiple experiments suggest that MASP-3 and MASP-1 are not necessarily functional in complexes with lectin molecules. For example... Figure 28B The experiments shown demonstrate the ability of MASP-3 to activate alternative pathways in the absence of the complex with lectins (i.e., in the presence of EGTA) (as demonstrated by C3b deposition in Staphylococcus aureus). Figure 28A This indicates that deposition under these conditions depends on factors B, D, and P, all of which are key components of the alternative pathway. Additionally, MASP-3 and MASP-1-induced activation of factor D ( Figure 32 ) and MASP-3-induced factor B activation ( Figure 30 This can occur in vitro in the absence of lectins. Finally, hemolysis of mouse erythrocytes in the presence of human serum demonstrated the clear role of both MBL and MASP-3 in cell lysis. However, MBL deficiency does not fully reproduce the severity of MASP-3 deficiency, contrary to what would be expected if all functional MASP-3 were combined with MBL. Therefore, the inventors do not wish to be limited by the concept that all the effects of MASP-3 (and MASP-1) shown herein can be attributed solely to lectin-related functions.

[0346] The identification of two effector branches of the lectin pathway and the possible lectin-independent functions of MASP-1, MASP-3, and HTRA-1 presents new opportunities for therapeutic interventions to effectively treat specified human pathologies caused by excessive complement activation in the presence of microbial pathogens or altered host cells or metabolic deposits. As described herein, the inventors have now discovered that alternative pathways are not activated at the surface structure in the absence of MASP-3 and in the presence of MASP-1 (see [link to article]). Figure 15-16 28B, 34-35A, B, 38-39). Because alternative pathways are important in driving rate-limiting events leading to bacterial and cell lysis (Mathieson PW et al., J Exp Med 177(6):1827-3 (1993)), therefore our results indicate that activated MASP-3 plays an important role in complement cytolytic activity. Figure 12-13 As shown in 19-21, 36-37 and 39-40, the terminal activation cascade of complement cellular lysis is defective in the serum of 3MC patients who lack MASP-3 but not MASP-1. Figure 12 and 13 The data shown indicate a loss of lysing activity in the absence of MASP-3 and / or MASP-1 / MASP-3 functional activity. Similarly, loss of hemolytic activity was observed in MASP-3-deficient human serum. Figure 19-21 (36-37 and 39-40), and the ability to reconstruct hemolysis by adding recombinant MASP-3 ( Figures 39-40 This strongly supports the conclusion that activation of the alternative pathway on the target surface (which is essential for driving complement-mediated cell 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 the target surface therefore depends on the lectin-independent activation of LEA-1 and / or factor B and / or factor D (which is also mediated by MASP-3), and therefore, agents that block MASP-3-dependent complement activation will prevent activation of the alternative pathway on the target surface.

[0347] The published information regarding the necessary role of MASP-3-dependent initiation for the activation of alternative pathways suggests that alternative pathways are not an independent, singular pathway of complement activation, as is stated in virtually all existing medical textbooks and recent review articles on complement. The existing and widely held scientific concept is that alternative pathways are activated on the surfaces of certain specific targets (microorganisms, yeast glycans, and rabbit erythrocytes) through amplification of spontaneous “tick-over” C3 activation. However, the absence of any alternative pathway activation on yeast glycan-coated plates and two different bacteria (Neisseria meningitidis and Staphylococcus aureus) in the sera of MASP-1 and MASP-3 dual-deficient mice and in the sera of human 3MC patients, and the reduced erythrocyte hemolysis in MASP-3-deficient sera from humans and mice, all suggest that the initiation of alternative pathway activation on these surfaces requires functional MASP-3. The desired function of MASP-3 can be either lectin-dependent or lectin-independent, and results in the formation of the alternative pathway C3 convertase and C5 convertase complexes, namely C3bBb and C3bBb(C3b)n, respectively. Therefore, the inventors here disclose a previously elusive initiation pathway for the alternative pathway. This initiation pathway depends on (i) the activation branch of LEA-1, a newly discovered lectin pathway, and / or (ii) the lectin-independent function of proteins MASP-3, MASP-1, and HTRA-1.

[0348] 3. MASP-3 inhibitors are used to treat diseases and conditions associated with alternative pathways.

[0349] As described herein, high-affinity MASP-3 inhibitory antibodies (e.g., with binding affinity less than 500 pM) have been shown to completely inhibit alternative pathways in mammalian subjects, such as rodents and nonprimates, at molar concentrations less than the concentration of the MASP-3 target (e.g., at 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, a single dose of the high-affinity MASP-3 inhibitory antibody mAb 13B1 in mice resulted in near-complete elimination of systemic alternative pathway complement activity for at least 14 days. As further described in Example 12, studies in well-established animal models associated with PNH have shown that mAb 13B1 significantly improves the survival of PNH-like erythrocytes and provides significantly better protection against PNH-like erythrocytes than C5 inhibition. As described in Example 13, mAb 13B1 further demonstrates a reduction in the incidence and severity of disease in a mouse model of arthritis. The results in this embodiment demonstrate that representative high-affinity MASP-3 inhibitory mAbs 13B1, 10D12, and 4D5 are highly effective in blocking alternative pathways in primates. Single-dose administration of mAb 13B1, 10D12, or 4D5 to cynomolgus monkeys resulted in sustained elimination of systemic alternative pathway activity lasting approximately 16 days. The extent of alternative pathway elimination in cynomolgus monkeys treated with high-affinity MASP-3 inhibitory antibodies was comparable to that achieved through in vitro and in vivo factor D blockade, indicating complete blockade of factor D conversion by MASP-3 inhibitory antibodies. Therefore, high-affinity MASP-3 inhibitory mAbs have therapeutic efficacy in treating patients with diseases associated with excessive alternative pathway activity.

[0350] Therefore, in one aspect, the present invention provides a method for inhibiting alternative pathways in mammalian subjects where such inhibition is desired, comprising administering a composition to the subject in an amount effective in inhibiting complement activation of the alternative pathway in the subject, said composition comprising an isolated monoclonal antibody or an antigen-binding fragment thereof having high affinity (having a K+ of less than 500 pM). DIt specifically binds to the serine protease domain of human MASP-3 (amino acid residues 450 to 728 of SEQ ID NO: 2). In some embodiments, the subject suffers from an alternative pathway-related disease or condition (i.e., a disease or condition associated with excessive activity of the alternative pathway), such as paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD, including wet and dry AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy (including hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), thrombotic thrombocytopenic purpura (TTP), or transplant-associated TMA), asthma, dense deposit disease, microimmune necrotizing crescentic glomerulonephritis, traumatic brain injury, inhalation Pneumonia, endophthalmitis, neuromyelitis optica, Bechtel's disease, multiple sclerosis, Guillain-Barré syndrome, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), lupus nephritis, systemic lupus erythematosus (SLE), diabetic retinopathy, uveitis, chronic obstructive pulmonary disease (COPD), C3 glomerulonephritis, transplant rejection, graft-versus-host disease (GVHD), hemodialysis, sepsis, systemic inflammatory response syndrome (SIRS), acute respiratory distress syndrome (ARDS), ANCA vasculitis, antiphospholipid syndrome, atherosclerosis, IgA nephropathy, and myasthenia gravis, as further described below.

[0351] A. The role of MASP-3 in paroxysmal nocturnal hemoglobinuria and treatment methods using MASP-3 inhibitory antibodies and optional combinations of MASP-2 inhibitory antibodies.

[0352] Overview of PNH

[0353] Paroxysmal nocturnal hemoglobinuria (PNH), sometimes called Marchifava-Micheli syndrome, is an acquired, potentially life-threatening blood disorder. PNH can occur spontaneously, known as "primary PNH," or in the context of other bone marrow disorders such as aplastic anemia, known as "secondary PNH." Most cases are primary PNH. PNH is characterized by complement-induced erythrocyte destruction (hemolysis), low red blood cell count (anemia), thrombosis, and bone marrow failure. Laboratory findings in PNH show changes consistent with intravascular hemolytic anemia: low hemoglobin, elevated lactate dehydrogenase, elevated reticulocyte count (immature red blood cells released from the bone marrow to replace destroyed cells), and elevated bilirubin (a degradation product of hemoglobin) in the absence of autoreactive RBC-binding antibodies, which are a possible contributing factor.

[0354] PNH is characterized by chronic complement-mediated hemolysis resulting from the activation of unregulated terminal complement components, including membrane attack complexes, on the surface of circulating red blood cells (RBCs). PNH RBCs undergo uncontrolled complement activation and hemolysis due to the absence of complement regulators CD55 and CD59 on their surface (Lindorfer, MA, et al.). Blood 115(11):2283-91 (2010), Risitano, et al. Mini-Reviews in Medicinal Chemistry ,11:528-535 (2011)). CD55 and CD59 are highly expressed on normal RBCs and control complement activation. CD55 acts as a negative regulator of the alternative pathway, inhibiting the assembly of the alternative pathway C3 convertase (C3bBb) complex and accelerating the decay of pre-formed convertases, thus blocking the formation of the membrane attack complex (MAC). CD59 inhibits the complement membrane attack complex directly by binding to the C5b678 complex and preventing the binding and polymerization of C9.

[0355] Although hemolysis and anemia are the main clinical features of PNH, the disease is a complex hematological condition that further includes thrombosis and bone marrow failure as part of the clinical findings (Risitano et al., MiniReviews in Med Chem 11:528-535 (2011)). At the molecular level, PNH results from the aberrant clonal expansion of hematopoietic stem cells lacking the functional PIG A gene. PIG A is an X-linked gene encoding glycosylated phosphatidylinositol transferase, an enzyme required for the stable surface expression of GPI-anchored class A glycoproteins (including CD55 and CD59). For reasons currently under investigation, hematopoietic stem cells with a dysfunctional PIG A gene, resulting from spontaneous somatic mutations, can be clonally expanded to the point where their progeny constitute a significant portion of the peripheral hematopoietic pool. Although the progeny of erythrocytes and lymphocytes from the mutant stem cell clone lack CD55 and CD59, only RBCs undergo significant hemolysis upon entering circulation.

[0356] Current treatment for PNH includes blood transfusions to combat anemia, anticoagulation to combat thrombosis, and the use of the monoclonal antibody eculizumab (Soliris®), which protects blood cells from immune destruction caused by the suppression of 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 complement component C5, blocking its cleavage by C5 convertase, thereby preventing the production of C5a and the assembly of MAC. Treatment of PNH patients with eculizumab resulted in reduced intravascular hemolysis (as measured by lactate dehydrogenase (LDH)) in approximately half of the patients, leading to hemoglobin stabilization and transfusion independence (Risitano et al., 350(6):552-559 (2004)). Mini-Reviews in Medicinal Chemistry , 11(6) (2011)). Although almost all patients treated with eculizumab achieved normal or near-normal LDH levels (due to control of intravascular hemolysis), only about one-third of the patients had hemoglobin levels of about 11 gr / dL, and the remaining patients receiving eculizumab continued to exhibit moderate to severe (i.e., transfusion-dependent) anemia in about the same proportion (Risitano AM et al., , Blood 113:4094-100 (2009)). As Risitano et al., Mini-Reviews in Medicinal Chemistry As described in 11:528-535 (2011), it has been shown that PNH patients receiving eculizumab contain a large number of C3 fragments bound to their PNH erythrocytes (unlike untreated patients). This finding leads to the understanding that in PNH patients treated with Soliris, PNH RBCs that are no longer hemolyzed due to C5 blockade can now accumulate large amounts of membrane-bound C3 fragments, which act as opsonins, causing them to be captured by specific C3 receptors into reticuloendothelial cells and subsequently leading to extravascular hemolysis. Therefore, despite preventing intravascular hemolysis and the resulting outcome, eculizumab treatment merely shifts the disposal of these RBCs from intravascular to extravascular hemolysis, resulting in a large amount of residual untreated anemia in many patients (Risitano AM et al., Blood 113:4094-100 (2009)). Therefore, patients experiencing C3-fragment-mediated extravascular hemolysis require treatment strategies other than eculizumab because they continue to require red blood cell transfusions. Such C3-fragment targeting approaches have already demonstrated their usefulness in experimental systems (Lindorfer et al., Blood 115:2283-91, 2010).

[0357] complement-initiation mechanism in PNH

[0358] The causal relationship between the deficient surface expression of the negative complement regulators CD55 and CD59 in PNH, and the effectiveness of eculizumab in preventing intravascular hemolysis, clearly defines PNH as a complement system-mediated condition. Although this paradigm is widely accepted, the nature of the initiating complement activation events and the complement activation pathways involved remain to be resolved. Because CD55 and CD59 negatively regulate the terminal amplification step in the complement cascade common to all complement initiation pathways, deficiencies in these molecules will lead to excessive formation and membrane integration of the membrane attack complex, regardless of whether complement activation is initiated by spontaneous renewal via the lectin pathway, the classical pathway, or the alternative pathway. Therefore, in PNH patients, any complement activation event leading to C3b deposition on the RBC surface can trigger subsequent amplification and pathological hemolysis (intravascular and / or extravascular) and contribute to hemolytic crisis. A clear understanding of the molecular mechanisms triggering hemolytic crisis in PNH subjects remains elusive. Because no complement initiation events are evident in PNH patients experiencing hemolytic crisis, the prevailing view is that complement activation in PNH can occur spontaneously due to low-level “tick-over” activation of alternative pathways, which is subsequently amplified by inappropriate control of terminal complement activation due to the lack of CD55 and CD59.

[0359] However, it is important to note that in its natural history, PNH usually occurs or worsens after certain events such as infection or injury (Risitano, Biologics 2:205-222 (2008) has shown that the events described trigger complement activation. Such complement activation does not depend on prior host immunity against the stimulating pathogen and therefore may not involve the classical pathway. Rather, it appears that such complement activation is initiated by lectin binding to exogenous or “self-altered” carbohydrate patterns expressed on the surface of microbial agents or damaged host tissues. Therefore, events that induce hemolytic crisis in PNH are closely related to complement activation initiated via lectins. This makes it possible that the lectin activation pathway provides the trigger for initiation, which ultimately leads to hemolysis in PNH patients.

[0360] Using a well-defined pathogen that activates complement via lectins as an experimental model to analyze the activation cascade at the molecular level, we demonstrated that complement activation can be initiated by either LEA-2 or LEA-1, leading to opsonization and / or cell lysis, depending on the stimulating microbe. The same principle for the dual response of lectin-initiated events (i.e., opsonization and / or cell lysis) may also apply to other types of infectious agents, or to lectin-induced complement activation following host tissue injury, or to other lectin-driven complement activation events that may contribute to PNH. Based on this duality in the lectin pathway, we infer that in PNH patients, LEA-2- and / or LEA-1-initiated complement activation promotes opsonization and / or RBC lysis via C3b, followed by subsequent extravascular and intravascular hemolysis. Therefore, in the case of PNH, simultaneous inhibition of both LEA-1 and LEA-2 is expected to resolve both intravascular and extravascular hemolysis, providing a significant advantage over the C5 inhibitor eculizumab.

[0361] It has been established that Streptococcus pneumoniae exposure preferentially triggers lectin-dependent activation of LEA-2, leading to opsonization of the microbe via C3b. Because Streptococcus pneumoniae is resistant to MAC-mediated cell lysis, its clearance from circulation occurs through opsonization via C3b. This opsonization and subsequent clearance from circulation are LEA-2-dependent, as demonstrated in MASP-2-deficient mice and in bacterial controls exposed to MASP-2 monoclonal antibodies (PLOS Pathog., 8: e1002793. (2012)).

[0362] In investigating the role of LEA-2 in the innate host response to microbial agents, we tested additional pathogens. When studying Neisseria meningitidis (… Neisseriameningitidis When used as a model organism, very different results were observed. Neisseria meningitidis also activates complement via lectins, and complement activation is essential for Neisseria meningitidis infection in the host first used in the experiment. However, LEA-2 did not play a host-protective role in this response: such as Figure 6 and 7 As shown, LEA-2 blockade via genetic elimination of MASP-2 did not decrease survival rates after Neisseria meningitidis infection. On the contrary, in these studies, LEA-2 blockade via MASP-2 elimination significantly improved survival rates. Figure 6 and 7 ) and disease score ( Figure 9 The same result was obtained by LEA-2 blockade induced by the administration of MASP-2 antibody. Figure 10In knockout mouse strains, secondary or compensatory effects that could be a cause were eliminated. These favorable results in LEA-2-eliminated animals were associated with more rapid clearance of Neisseria meningitidis from the bloodstream. Figure 8 Additionally, as described in this article, incubating Neisseria meningitidis with normal human serum kills Neisseria meningitidis. Figure 11 Adding a human MASP-2-specific functional monoclonal antibody that blocks LEA-2, but without administering an isotype control monoclonal antibody, can enhance its cytotoxic response. However, this process depends on lectins and at least a partially functional complement system, because MBL-deficient human serum or heat-inactivated human serum cannot kill Neisseria meningitidis. Figure 11 In summary, these new findings suggest that Neisseria meningitidis infection is controlled by a complement-activated lectin-dependent but LEA-2-independent pathway in the presence of a functional complement system.

[0363] Using a serum sample from a patient with 3MC, the hypothesis that LEA-1 might be responsible for the complement pathway in the lectin-dependent killing of Neisseria meningitidis was tested. This patient was homozygous for a nonsense mutation in exon 12 of the MASP-1 / 3 gene. As a result, the patient lacked functional MASP-3 protein, but other complement was adequate (exon 12 is specific for MASP-3 transcripts; this mutation had no effect on MASP-1 function or expression levels) (see [link to relevant documentation]). Nat Genet 43(3):197-203(2011)). Normal human serum is effective in killing Neisseria meningitidis, but heat-inactivated serum lacking MBL (a recognition molecule in the lectin pathway) and serum lacking MASP-3- cannot kill Neisseria meningitidis. Figure 12 Therefore, LEA-1 appears to mediate the killing of Neisseria meningitidis. This finding was confirmed using serum samples from knockout mouse strains. Although serum from normal mice containing complement readily killed Neisseria meningitidis, serum from MBL-deficient or MASP-1 / 3-deficient mice was as ineffective as heat-inactivated serum lacking functional complement. Figure 13 Conversely, MASP-2-deficient serum showed effective killing of Neisseria meningitidis.

[0364] These findings provide evidence of a previously unknown duality in the lectin pathway by revealing the existence of separate LEA-2 and LEA-1 pathways for lectin-dependent complement activation. In the examples detailed above, LEA-2 and LEA-1 are non-redundant and mediate distinct functional outcomes. Data suggest that certain types of lectin pathway activators (including, but not limited to, Streptococcus pneumoniae) preferentially initiate complement activation via LEA-2, leading to opsonization, while others (such as Neisseria meningitidis) preferentially initiate complement activation via LEA-1 and promote cell lysis. However, the data do not necessarily limit LEA-2 to opsonization and LEA-1 to cell lysis, as both pathways can mediate opsonization and / or cell lysis in other cases.

[0365] In the case of lectin-dependent complement activation induced by Neisseria meningitidis, the LEA-2 and LEA-1 branches appear to compete with each other, as blockade of LEA-2 in vitro enhances cellular lysis and disruption in LEA-1-dependent organisms. Figure 13 As detailed above, this finding can be explained as follows: In the absence of MASP-2, the likelihood of the lectin MASP-1 complex remaining near the lectin MASP-3 complex increases, which enhances LEA-1 activation and thus promotes more effective cell lysis of Neisseria meningitidis. Since cell lysis of Neisseria meningitidis is the primary protective mechanism in the host in which the experiment was first conducted, the blockade of LEA-2 in vivo increases the clearance of Neisseria meningitidis and leads to increased killing.

[0366] While the examples above illustrate the opposing effects of LEA-2 and LEA-1 on outcomes following Neisseria meningitidis infection, other scenarios exist where LEA-2 and LEA-1 can synergistically produce certain outcomes. As detailed below, in other cases of pathological complement activation via lectins (e.g., those present in PNH), LEA-2- and LEA-1-driven complement activation can synergistically promote the overall pathology of PNH. Additionally, as discussed herein, MASP-3 also promotes lectin-independent conversion of factors B and D, which can occur in Ca... ++ When it is absent, it usually leads to the transformation of C3bB to C3bBb and the transformation of prefactor D to factor D, which can further promote PNH pathology.

[0367] Biological and expected functional activities of PNH

[0368] This section describes the inhibitory effect of LEA-2 and LEA-1 blockade on hemolysis in an in vitro model of PNH. The findings support the use of LEA-2-blockers (including, but not limited to, antibodies that bind to and block the function of MASP-2) and LEA-1-blockers (including, but not limited to, antibodies that bind to and block the MASP-3 function of MASP-3 MASP-1-mediated activation, antibodies that block the function of MASP-3, or antibodies that block both) to treat subjects with one or more aspects of PNH, and also the use of inhibitors of LEA-2 and / or LEA-1, and / or MASP-3-dependent, lectin-independent complement activation inhibitors (including MASP-2 inhibitors, MASP-3 inhibitors, and dual- or bispecific MASP-2 / MASP-3 or MASP-1 / MASP-2 inhibitors, and panspecific MASP-1 / MASP-2 / MASP-3 inhibitors) to improve C3-fragment-mediated extravascular hemolysis in PNH patients who have received C5-inhibition therapy such as eculizumab.

[0369] MASP-2 inhibitors block opsonization and extravascular hemolysis of PNH RBCs through the reticuloendothelial system.

[0370] As detailed above, PNH patients are anemic due to two distinct mechanisms of RBC clearance from circulation: intravascular hemolysis via activation of the membrane attack complex (MAC), and extravascular hemolysis following opsonization at C3b and subsequent clearance via complement receptor binding and uptake through the reticuloendothelial system. When patients are treated with eculizumab, intravascular hemolysis is significantly inhibited. Because eculizumab blocks the terminal cleavage effector mechanism (which occurs downstream of the complement-initiated activation event and subsequent opsonization), it does not block extravascular hemolysis (Risitano AM et al., ...). Blood 113:4094-100 (2009)). Instead, in untreated PNH subjects, RBCs undergoing hemolysis can now accumulate activated C3b protein on their surface, which increases uptake by the reticuloendothelial system and amplifies their extravascular hemolysis. Therefore, eculizumab treatment effectively shifts the management of RBCs from intravascular hemolysis to possible extravascular hemolysis. As a result, some eculizumab-treated PNH patients still suffer from anemia. Therefore, agents that block upstream complement activation and inhibit opsonization of PNH RBCs may be particularly suitable for blocking incidentally observed extravascular hemolysis with eculizumab.

[0371] The microbial data presented in this article indicate that LEA-2 is typically the primary pathway for lectin-dependent opsonization. Furthermore, when activated on surfaces containing three prototype lectins (mannan, ...), ... Figure 17A Yeast polysaccharides, Figure 17B and Streptococcus pneumoniae; Figure 17CWhen evaluating lectin-dependent opsonization (measured as C3b deposition) on a chromogenic basis, LEA-2 appears to exhibit better effects under physiological conditions (i.e., under Ca2+ deposition). ++ When present, all complement pathways are effective, which is the main pathway for lectin-dependent opsonization. Under these experimental conditions, MASP-2-deficient serum (lacking LEA-2) was substantially worse than WT serum in opsonization assays. MASP-1 / 3-deficient serum (lacking LEA-1) was also deficient, although the effect was much less pronounced compared to LEA-2-deficient serum. The relative contributions of LEA-2 and LEA-1 to lectin-driven opsonization were... Figure 18A Further details are shown in -18C. Although alternative complement pathways have been reported to support the opsonization of lectin-activated surfaces in the absence of the lectin pathway or the classical pathway (Selander et al., J Clin Invest 116(5):1425-1434(2006)), but in isolation (in the absence of Ca ++ The alternative pathway (measured under the same conditions) appears to be substantially less efficient than the LEA-2- and LEA-1-initiated processes described herein. Extrapolating these data, they suggest that opsonization of PNH RBCs can also be preferentially initiated by LEA-2, and to a lesser extent by LEA-1 (possibly by the amplification loop of the alternative pathway), rather than as a result of lectin-independent alternative pathway activation. Therefore, LEA-2 inhibitors can be expected to be most effective in limiting opsonization and preventing extravascular hemolysis of PNH. However, it is important to recognize that lectins, rather than MBLs (e.g., fibrinogen), bind to non-carbohydrate structures (e.g., acetylated proteins), and that MASP-3 preferentially associates with H-fibrinogen (Skjoedt et al., ...). Immunobiol The study (215:921-931, 2010) also leaves the possibility of an important role for LEA-1 in the opsonization of PNH-related RBCs unresolved. Therefore, LEA-1 inhibitors are expected to have additional anti-opsonization effects, and the combination of LEA-1 and LEA-2 inhibitors is expected to be optimal and provide the most potent therapeutic benefit in PNH patients in limiting opsonization and mediating extravascular hemolysis. Thus, LEA-2 and LEA-1 act in an additive or synergistic manner to promote opsonization, and cross-reactive or bispecific LEA-1 / LEA-2 inhibitors are expected to be most effective in blocking opsonization and extravascular hemolysis in PNH.

[0372] The role of MASP-3 inhibitors in PNH

[0373] Using an in vitro model of PNH, we demonstrated that complement activation and resulting hemolysis in PNH are indeed initiated by LEA-2 and / or LEA-1 activation, and that it is not a function independent of the alternative pathway. These studies used mannan-sensitized RBCs from different mouse strains, including RBCs from Crry-deficient mice (an important negative regulator of the terminal complement pathway in mice) and RBCs from CD55 / CD59-deficient mice (which lack the aforementioned complement regulator absent in PNH patients). When mannan-sensitized Crry-deficient RBCs were exposed to complement-sufficient human serum, effective hemolysis (…) was achieved at a serum concentration of 3%. Figure 19 and 20 Complement-deficient serum (HI: heat-inactivated) did not hemolyze. Notably, adequate complement-supplemented serum (where LEA-2 is blocked by the addition of MASP-2 antibody) exhibited reduced hemolytic activity, and 6% serum was required for effective hemolysis. Similar observations were obtained when testing CD55 / CD59-deficient RBCs. Figure 22 Supplementing with complement-enhanced human serum (i.e., serum where LEA-2 is inhibited) is approximately 2-fold less effective than untreated serum in supporting hemolysis. Furthermore, higher concentrations of LEA-2-blocking serum (i.e., serum treated with anti-MASP-2 monoclonal antibody) are required to promote effective hemolysis of untreated WT RBCs compared to untreated serum. Figure 21 ).

[0374] Even more surprisingly, serum from patients with 3MC who were homozygous for dysfunctional MASP-3 protein (and therefore lacking LEA-1) completely failed to induce hemolysis of mannan-sensitized Crry-deficient RBCs. Figure 20 and Figure 21 Similar results were observed when using unsensitized normal RBCs: such as Figure 21 As shown, LEA-1-deficient serum isolated from patients with 3MC was completely ineffective in mediating hemolysis. In summary, these data suggest that while LEA-2 significantly promotes intravascular hemolytic responses, LEA-1 is the dominant complement-initiated pathway leading to hemolysis. Therefore, although LEA-2 blockers are expected to significantly reduce intravascular hemolysis of RBCs in PNH patients, LEA-1 blockers are expected to have a more profound effect and significantly eliminate complement-driven hemolysis.

[0375] It should be noted that, when tested under standard alternative pathway assay conditions, the serum from LEA-1-deficient 3MC patients used in this study exhibited reduced but functional alternative pathways (…). Figure 15This finding suggests that LEA-1 contributes significantly to hemolysis compared to alternative pathway activity, as is routinely defined in this experimental setting of PNH. Extrapolating from this, this indicates that LEA-1 blockers are at least as effective as blockers of other aspects of the alternative pathway in preventing or treating intravascular hemolysis in patients with PNH.

[0376] The role of MASP-2 inhibitors in PNH

[0377] The data presented in this article indicate the following pathogenesis of anemia in PNH: intravascular hemolysis due to RBC hemolysis caused by unregulated activation of terminal complement components and MAC formation, primarily (but not exclusively) initiated by LEA-1; and extravascular hemolysis due to opsonization of RBCs via C3b, which appears to be primarily initiated by LEA-2. Although the identifiable role of LEA-2 in initiating complement activation and promoting MAC formation and hemolysis is evident, this process appears to be significantly less effective than LEA-1-initiated complement activation leading to hemolysis. Therefore, LEA-2-blockers are expected to significantly reduce intravascular hemolysis in PNH patients, although this therapeutic activity is expected to be only partial. By comparison, LEA-1-blockers are expected to significantly reduce intravascular hemolysis in PNH patients.

[0378] Extravascular hemolysis (although insignificant, it remains an equally important mechanism contributing to RBC destruction and anemia in PNH), primarily a result of C3b opsonization, appears to be mainly mediated by LEA-2. Therefore, LEA-2-blockers are expected to preferentially block RBC opsonization and subsequent extravascular hemolysis in PNH. This unique therapeutic activity of LEA-2-blockers is anticipated to provide significant therapeutic benefit to all PNH patients, as there is currently no treatment for PNH patients undergoing this pathological process.

[0379] LEA-2 inhibitors as adjuvant therapy for LEA-1 inhibitors or terminal complement blockers

[0380] This article provides data detailing two pathogenesis mechanisms of RBC clearance and anemia in PNH, which can be targeted individually or in combination by different types of therapeutic agents: intravascular hemolysis primarily initiated by (but not exclusively) LEA-1 and therefore expected to be effectively prevented by LEA-1-blockers; and extravascular hemolysis primarily caused by C3b opsonization driven by LEA-2 and therefore effectively prevented by LEA-2-blockers.

[0381] There is ample documentation demonstrating that both intravascular and extravascular hemolytic mechanisms contribute to anemia in PNH patients (Risitano et al., Blood113:4094-4100 (2009)). Therefore, it is expected that the combined use of a LEA-1-blocker for preventing intravascular hemolysis and a LEA-2-blocker for primarily preventing extravascular hemolysis will be more effective than either of the aforementioned agents in preventing anemia in patients with PNH. In fact, it is expected that the combined use of LEA-1- and LEA-2-blockers will prevent all relevant mechanisms of complement initiation in PNH and thus block all anemia symptoms in PNH.

[0382] It is also known that C5-blockers (e.g., eculizumab) effectively block intravascular hemolysis without interfering with opsonization. This leaves some PNH patients who have received anti-C5-therapy with significant residual anemia due to untreated LEA-2-mediated extravascular hemolysis. Therefore, it is anticipated that the combination of a C5-blocker (e.g., eculizumab) for preventing intravascular hemolysis with a LEA-2 blocker for reducing extravascular hemolysis will be more effective than either of these agents alone in preventing anemia in PNH patients.

[0383] Other agents that block the terminal amplification loop of the complement system, leading to C5 activation and MAC deposition (including, but not limited to, agents that block properdin, factor B, or factor D, or enhance the inhibitory activity of factor I, factor H, or other complement inhibitory factors) are also expected to inhibit intravascular hemolysis. However, these agents are not expected to interfere with LEA-2-mediated opsonization in PNH patients. This leaves some PNH patients treated with the aforementioned agents with substantial residual anemia due to untreated LEA-2-mediated extravascular hemolysis. Therefore, treatment with the aforementioned agents that prevent intravascular hemolysis is expected to be more effective in preventing anemia in PNH patients than either of the aforementioned agents alone. In fact, the combination of the aforementioned agents and LEA-2-blockers is expected to prevent all relevant mechanisms of RBC destruction in PNH and thus block all anemia symptoms in PNH.

[0384] Using multiple bispecific or panspecific antibodies of LEA-1 and LEA-2 to treat PNH

[0385] As detailed above, the combined use of pharmaceutical agents that block LEA-1 and LEA-2 separately and thus jointly block all complement activation events mediating intravascular and extravascular hemolysis is expected to provide optimal clinical outcomes for PNH patients. This can be achieved, for example, by co-administering antibodies with LEA-1-blocking activity and antibodies with LEA-2-blocking activity. In some embodiments, LEA-1- and LEA-2-blocking activities are combined into a single molecular entity, and such entities with combined LEA-1- and LEA-2-blocking activities will effectively block intravascular and extravascular hemolysis and prevent anemia in PNH. Such entities may contain or consist of bispecific antibodies in which one antigen-binding site specifically recognizes MASP-1 and blocks LEA-1 and reduces LEA-2, while a second antigen-binding site specifically recognizes MASP-2 and further blocks LEA-2. Alternatively, such entities could be composed of bispecific monoclonal antibodies: one antigen-binding site specifically recognizes MASP-3 and thus blocks LEA-1, and the second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. These entities are preferably composed of bispecific monoclonal antibodies: one antigen-binding site specifically recognizes both MASP-1 and MASP-3 and thus blocks LEA-1 and reduces LEA-2, while the second antigen-binding site specifically recognizes MASP-2 and further blocks LEA-2. Based on the similarity in total protein sequence and structure, it is also expected that conventional antibodies with two identical binding sites can be developed that specifically bind to MASP-1 and MASP-2 and MASP-3 in a functional manner, thus achieving functional blockade of LEA-1 and LEA-2. Such antibodies with pan-MASP inhibitory activity are expected to simultaneously block intravascular and extravascular hemolysis and therefore be effective in treating anemia in PNH patients.

[0386] As described in Examples 11-21 of this document, high-affinity MASP-3 inhibitory antibodies have been produced, which have therapeutic efficacy in inhibiting alternative pathways in AP-related diseases or conditions such as PNH.

[0387] Therefore, in one embodiment, the present invention provides a method for treating subjects who have or are at risk of developing PNH, comprising administering an effective amount of a high-affinity monoclonal antibody or antigen-binding fragment thereof that binds to human MASP-3 and inhibits alternative pathway complement activation to treat PNH in the subject or reduce the risk of PNH in the subject.

[0388] In one embodiment, the present invention provides a method for treating a subject suffering from or at risk of developing paroxysmal nocturnal hemoglobinuria (PNH), comprising administering to the subject a pharmaceutical composition comprising an effective amount of a monoclonal antibody or antigen-binding fragment thereof, as disclosed herein, that binds to human MASP-3 and inhibits alternative pathway complement activation, to treat PNH in the subject or reduce the risk of PNH in the subject, 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) VLCDR2 comprising SEQ ID NO: 144. VLCDR3 of 161. In some embodiments, the pharmaceutical composition increases the survival of red blood cells in subjects with PNH. In some embodiments, subjects with or at risk of developing PNH exhibit one or more of the following: (i) hemoglobin levels below normal, (ii) platelet counts below normal, (iii) reticulocyte counts above normal, and (iv) bilirubin levels above normal. In some embodiments, the pharmaceutical composition is administered systemically (e.g., subcutaneously, intramuscularly, intravenously, intra-arterially, or as an inhaler) to subjects with or at risk of developing PNH. In some embodiments, subjects with or at risk of developing PNH have previously received or are receiving treatment with a terminal complement inhibitor that inhibits the cleavage of complement protein C5. In some embodiments, the method further includes administering a terminal complement inhibitor that inhibits the cleavage of complement protein C5 to the subject. 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.

[0389] B. The role of MASP-3 in age-related macular degeneration and treatment methods using MASP-3 inhibitory antibodies and optional combinations of MASP-2 inhibitors.

[0390] Age-related macular degeneration (AMD) is the leading cause of vision impairment and blindness in older adults, accounting for up to 50% of blindness cases in developed countries. The prevalence of AMD in adults is approximately 3%, and it increases with age, meaning that nearly two-thirds of the population over 80 years of age will have some symptoms. It is estimated that more than 1.75 million individuals in the United States have advanced AMD, and the prevalence is increasing with an aging population, projected to reach nearly 3 million by 2020 (Friedman, DS et al.). Arch. Ophthalmol. AMD (122:564-572, 2004) is an abnormality of the retinal pigment epithelial cells (RPE), which leads to degeneration of the photoreceptors overlying the central retina, or macula, and loss of central vision. Early and intermediate forms of AMD are characterized by progressive deposition of drusen in the subretinal space adjacent to the RPE, accompanied by pigment irregularities in the retina. Drusen are pale yellow substances containing lipids, proteins, lipoproteins, and cellular debris. Late AMD consists of two clinical subtypes: non-neovascular geomorphic atrophy (“dry”) AMD and neovascular exudative AMD (“wet”). Although dry AMD accounts for 80–90% of late AMD cases, most sudden and severe vision loss occurs in patients with wet AMD. It is not yet known whether these two types of AMD represent different phenotypes arising from similar pathology or two different conditions. The U.S. Food and Drug Administration (FDA) has not yet approved any treatments for dry AMD. FDA-approved treatment options for wet AMD include intravitreal injection of anti-angiogenic drugs (ranibizumab, piperacanib sodium, aflibercept), laser therapy, photodynamic laser therapy, and implantable telescopes.

[0391] The etiology and pathophysiology of AMD are complex and not fully understood. Several pieces of evidence support the role of complement system dysregulation in the pathogenesis of AMD. Gene association studies have identified multiple gene loci associated with AMD, including genes encoding a range of complement proteins, factors, and regulators. The strongest association is with the complement factor H (CFH) gene polymorphism, in which homozygotes of the Y402H variant have an approximately 6-fold increased risk of AMD compared to the non-risk genotype, and heterozygotes of the Y402H variant have an approximately 2.5-fold increased risk (Khandhadia, S. et al.). Immunobiol. 217:127-146, 2012). Mutations in other complement pathway-encoding genes have also been associated with increased or decreased risk of AMD, including complement factor B (CFB), C2, C3, factor I, and CFH-related proteins 1 and 3 (Khandhadia et al.). Immunohistochemical and proteomic studies in donor eyes of AMD patients have shown increased complement cascade proteins and localization to drusen (Issa, PC et al., Graefes. Arch. Clin. Exp. Ophthalmol. 249:163-174, 2011). In addition, AMD patients have increased systemic complement activation, as measured in peripheral blood (Issa et al., 2011, ibid.).

[0392] In the pathogenesis of AMD, the complement substitution pathway appears to be more relevant than the classical pathway. Immunohistochemical analysis revealed that C1q, an essential recognition component for activating the classical pathway, was not detected in drusen (Mullins et al.). FASEB J. 14 :835-846, 2000; Johnson et al., Exp. Eye Res. 70 (441-449, 2000). Genetic association studies have involved the CFH and CFB genes. These proteins are involved in the alternative pathway amplification loop, where CFH is a fluid-phase inhibitor and CFB is an activating protease component of the alternative pathway. The Y402H variant of CFH affects ligand-binding interactions, including binding to C-reactive protein, heparin, M protein, and glycosaminoglycans. This alteration in ligand binding reduces binding to the cell surface, which in turn may lead to reduced factor I-mediated degradation of the activated C3b fragment and impaired regulation of the alternative C3 convertase, resulting in overactivation of the alternative pathway (Khandhadia et al., 2012, ibid.). CFB gene variants are associated with protective effects against AMD development. The functional variant fB32Q has a C3b binding affinity that is 1 / 4 that of the risk variant fB32R, resulting in reduced C3 convertase formation (Montes, T. et al., Proc. Natl. Acad. Sci. USA 106:4366-4371, 2009).

[0393] Complement Activation Mechanism in AMD

[0394] The human genetic linkage studies discussed above highlight the important role of the complement system in the pathogenesis of AMD. Furthermore, complement activation products are abundant in drusen (Issa, PC et al.), Graefes. Arch. Clin. Exp. Ophthalmol. (249:163-174, 2011), which is a hallmark pathological lesion in both wet and dry AMD. However, the nature of the events that initiate complement activation and the complement activation pathways involved are still not fully understood.

[0395] It is important to note that drusen deposits consist of cellular debris originating from the retina and oxidative waste products (which accumulate beneath the RPE with age). Furthermore, oxidative stress appears to play a significant role (Cai et al.; Front Biosci ., 17:1976-95, 2012), and has been shown to lead to RPE complement activation ( JBiol Chem., 284(25):16939-47, 2009). It is widely believed that both oxidative stress and cellular or tissue damage activate lectins in the complement system. For example, Collard et al. have shown that endothelial cells exposed to oxidative stress trigger massive complement deposition mediated by lectins (Collard CD et al., 284(25):16939-47, 2009). Mol Immunol ., 36(13-14):941-8, 1999; Collard CD et al., Am J Pathol ., 156(5):1549-56, 2000), and the results in an experimental model of oxidative stress damage improved by blocking lectin binding and lectin-dependent complement activation (Collard CD et al., 156(5):1549-56, 2000). Am J Pathol .,156(5):1549-56,2000). Therefore, it seems possible that oxidative waste products present in drusen also activate complement via lectin. It is inferred that lectin-dependent complement activation may play a key role in the pathogenesis of AMD.

[0396] The role of the complement system has been evaluated in mouse models of AMD. In a photodamage mouse model (an experimental model of oxidative stress-mediated photoreceptor degeneration), knockout mice with classical pathway elimination (C1qα in a C57BL / 6 background) were assessed. - / - Compared to wild-type littermates, they showed the same sensitivity to photodamage, while eliminating the alternative pathway of complement factor D (CFD) - / - This leads to protection against light damage (Rohrer, B. et al., Invest. Ophthalmol. Vis. Sci. 48:5282-5289, 2007). In a mouse model of choroidal neovascularization (CNV) induced by laser photocoagulation of Bruch's membrane, compared with wild-type mice, mice without complement factor B knockout (CFB) - / - Protected from CNV (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 (CR2-fH) targeting complement activation sites reduced the extent of CNV. This protective effect was observed regardless of whether CR2-fH was administered during or after laser injury. A human therapeutic form of CR2-fH (TT30) was also effective in a mouse CNV model (Rohrer, B. et al.). J. Ocul. Pharmacol. Ther., 28: 402-409, 2012Because fB is activated by LEA-1, and because MASP-1 and MASP-3 contribute to the maturation of factor D, these findings suggest that LEA-1 inhibitors may have a therapeutic benefit in patients with AMD. Furthermore, recent results from a phase 2 study have shown that monthly intravitreal injections of lanpazumab (formerly known as FCFD4514S and anti-factor D, which is an antigen-binding fragment of a humanized monoclonal antibody against factor D) reduced the progression of geographic atrophy regions in patients with geographic atrophy secondary to AMD (Yaspan BL et al., Sci Transl. Med . 9, Issue 395, June 21, 2017).

[0397] Initial experimental studies using MBL-deficient mice in an AMD rodent model did not support the crucial 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 lectins other than MBL may induce complement activation in AMD. In fact, our previous work has shown that the rate-limiting serine protease MASP-2, which is extremely needed in the function of the lectin pathway, plays a key role in AMD. As described in U.S. Patent 7,919,094 (assigned to Omeros Corporation), which is incorporated herein by reference, MASP-2 deficient mice and mice treated with MASP-2 antibodies were protected in a laser-induced CNV mouse model (a validated preclinical model of wet AMD) (Ryan et al., Tr Am Opth Soc LXXVII (707-745, 1979). Therefore, inhibitors of LEA-2 are expected to be effective in preventing CNV and improving outcomes in AMD patients.

[0398] Therefore, given the above, LEA-1 and LEA-2 inhibitors are expected to have independent therapeutic benefits in AMD. Furthermore, the combined use of LEA-1 and LEA-2 inhibitors can achieve additional therapeutic benefits compared to either agent alone, or can provide effective treatment for a wider range of patient subgroups. Combined LEA-1 and LEA-2 inhibition can be achieved by co-administration of LEA-1 and LEA-2 blockers. Ideally, the inhibitory function of LEA-1 and LEA-2 can be contained in a single molecular entity, such as a bispecific antibody containing specific binding sites for MASP-1 / 3 and MASP-2, or a bispecific antibody where each binding site can bind to and block either MASP-1 / 3 or MASP-2.

[0399] Based on the foregoing, one aspect of the present invention provides a method for treating age-related macular degeneration (wet and dry forms) by inhibiting LEA-1-dependent complement activation, said method being carried out by administering a composition to a subject suffering from said condition, said composition comprising a therapeutically effective amount of a combination of a MASP-1 inhibitor, a MASP-3 inhibitor, or a combination of MASP-1 / 3 inhibitors in a drug carrier. The MASP-1, MASP-3, or MASP-1 / 3 inhibitory composition can be applied topically to the eye, for example by irrigation, intravitreal administration, or in the form of a gel, ointment, or drops. Alternatively, the MASP-1, MASP-3, or MASP-1 / 3 inhibitor can be administered systemically to the subject, for example by intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous, or other parenteral administration, or possibly by oral administration of a non-peptide agent. Administration can be repeated as determined by a physician until the condition is resolved or controlled.

[0400] In one embodiment, the method according to this aspect of the invention further includes inhibiting LEA-2-dependent complement activation in subjects with age-related macular degeneration, including administering a therapeutically effective amount of a MASP-2 inhibitor and a MASP-1, MASP-3, or MASP 1 / 3 inhibitor to the subject in need. As detailed above, in AMD patients, a combination of pharmacological agents that each block LEA-1 and LEA-2 is expected to provide improved therapeutic outcomes compared to inhibiting LEA-1 alone. This outcome can be achieved, for example, by co-administering an antibody with LEA-1-blocking activity along with an antibody with LEA-2-blocking activity. In some embodiments, LEA-1- and LEA-2-blocking activities are combined into a single molecular entity, and said entity has combined LEA-1- and LEA-2-blocking activities. Such an entity may comprise or consist of a bispecific antibody, wherein 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 an entity could be composed of bispecific monoclonal antibodies, one of which has an antigen-binding site that specifically recognizes MASP-3, thus blocking LEA-1, while the second antigen-binding site specifically recognizes MASP-2, thus blocking LEA-2. Ideally, such an entity could be composed of bispecific monoclonal antibodies, one of which has an antigen-binding site that specifically recognizes both MASP-1 and MASP-3, thus blocking LEA-1, while the second antigen-binding site specifically recognizes MASP-2, thus blocking LEA-2.

[0401] MASP-2 inhibitory compositions can be applied topically to the eye, for example, by irrigation, intravitreal injection, or as a gel, ointment, or drops. Alternatively, MASP-2 inhibitors can be administered systemically, for example, by intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous, or other parenteral administration, or possibly by oral administration of non-peptide agents. Administration may be repeated as determined by a physician until the condition is resolved or controlled.

[0402] The MASP-3 inhibitory composition and optionally the MASP-2 inhibitory composition of the present invention can be administered for the treatment of AMD by a single application of the composition (e.g., a single composition containing both MASP-2 and MASP-3 inhibitors or bispecific or dual inhibitors, or co-administration of separate compositions), or by administration in a limited sequence. Alternatively, the composition can be administered for the treatment of AMD at periodic intervals, such as daily, every two weeks, weekly, every other week, monthly, or every two months over a longer period of time.

[0403] As described in Examples 11-21 of this document, high-affinity MASP-3 inhibitory antibodies have been produced, which have therapeutic efficacy in inhibiting alternative pathways in AP-related diseases or conditions such as AMD.

[0404] Therefore, in one embodiment, the present invention provides a method for treating a subject who has or is at risk of developing AMD, comprising administering an effective amount of a high-affinity monoclonal antibody or an antigen-binding fragment thereof that binds to human MASP-3 and inhibits alternative pathway complement activation to treat AMD in the subject or reduce the risk of AMD in the subject. In one embodiment, the present invention provides a method for treating a subject suffering from or at risk of developing AMD, comprising administering to the subject a pharmaceutical composition comprising an effective amount of a monoclonal antibody or antigen-binding fragment thereof, as disclosed herein, that binds to human MASP-3 and inhibits alternative pathway complement activation, to treat AMD in the subject or reduce the risk of AMD in the subject, 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.

[0405] C. The role of MASP-3 in ischemia-reperfusion injury and treatment methods using MASP-3 inhibitory antibodies and optional combinations of MASP-2 inhibitors.

[0406] Tissue ischemia is the basis of a wide range of clinical conditions. While timely restoration of blood flow is necessary to protect ischemic tissue, it has long been recognized that reperfusion, which can occur spontaneously or through therapeutic intervention, can lead to additional tissue damage; this phenomenon is known as ischemia-reperfusion (I / R) injury (Eltzschig, HK and Tobias, E., Nat. Med. 17:1391-1401, 2011). I / R injury can affect single organs, such as the heart (acute coronary syndrome), kidney (acute kidney injury), intestine (intestinal I / R), and brain (stroke). I / R injury can also affect multiple organs, such as the following major traumas and resuscitations (multiple organ failure), circulatory arrest (hypoxic brain injury, acute kidney injury), peripheral vascular disease, and sickle cell disease (acute chest syndrome, acute kidney injury). Major surgeries can be associated with ischemic / reperfusion injuries, including cardiac surgery (acute heart failure after cardiopulmonary bypass), thoracic surgery (acute lung injury), peripheral vascular surgery (septal syndrome), vascular surgery (acute kidney injury), and solid organ transplantation (acute transplant failure). Currently, there are no specific treatments for ischemic / reperfusion injuries; effective treatments are needed to maximize the salvage of ischemic tissue and improve functional outcomes in these common conditions.

[0407] The pathophysiology of I / R injury is complex, characterized by a robust inflammatory response following reperfusion. Activation of the complement system has been suggested as a crucial component of I / R injury, and inhibition of complement activity has been effective in various animal models (Diepenhorst, GMP et al.). Ann. Surg. (249:889-899, 2009). The relative importance of classical, lectin, and alternative pathways in I / R injury is largely unstable and can vary depending on the organ affected. Recent availability of knockout mice deficient in specific complement proteins and pathway-specific inhibitors has yielded data concerning lectins and alternative pathways in I / R injury.

[0408] The role of alternative pathways in gastrointestinal I / R injury was investigated using factor D-deficient (- / -) mice and heterozygous (+ / -) mice (Stahl, GL et al.). Am. J. Pathol.162:449-455, 2003. Compared with heterozygous mice, factor D-deficient mice reduced, but did not prevent, intestinal and lung injury after transient gastrointestinal ischemia, and the addition of human factor D to factor D(- / -) mice restored I / R injury. The same model was evaluated in C1q-deficient and MBL-A / C-deficient mice, and the results showed that gastrointestinal I / R injury was independent of C1q and classical pathway activation, but MBL and lectin pathway activation were essential for intestinal injury (Hart, ML et al. J. Immunol. 174:6373-6380, 2005). Conversely, C1q recognition molecules of the classical pathway were responsible for lung injury after intestinal I / R (Hart, ML et al. J. Immunol. 174:6373-6380, 2005). One hypothesis is that complement activation during I / R injury occurs via the binding of native IgM to autoantigens (e.g., non-muscle myosin heavy chain type II) presented on the surface of ischemic (but not normal) tissue. In a mouse model of gastrointestinal I / R injury, the presence of initiating factors in immune complexes derived from intestinal tissue was evaluated via the classical (C1q), lectin (MBL), or alternative (factor B) pathways (Lee, H. et al.). Mol. Immunol. 47:972-981, 2010). The results showed that C1q and MBL were detected in these immune complexes, but factor B was not, indicating the involvement of both classical and lectin pathways, but not alternative pathways. In the same model, factor B-deficient mice were not protected from local tissue damage, providing additional support for the lack of involvement of alternative pathways. Direct evaluation of the role of the lectin pathway in gastrointestinal I / R injury in MASP-2-deficient mice showed reduced gastrointestinal damage compared to wild-type controls; treatment with MASP-2 monoclonal antibodies was also protective (Schwaeble, WJ et al., 2010). Proc. Natl. Acad. Sci. (108:7523-7528, 2011). In summary, these results support the involvement of the lectin pathway in gastrointestinal I / R injury, while conflicting data exist regarding the involvement of alternative pathways.

[0409] In a mouse model of myocardial I / R injury, the lectin pathway showed pathogenicity, as MBL-deficient mice were protected from myocardial injury, while C1q-deficient and C2 / fB-deficient mice were not (Walsh, MC et al.). J. Immunol. 175:541-546, 2005). A protective effect against myocardial I / R injury was also observed in MASP-2 deficient mice (Schwaeble, WJ et al.). Proc. Natl. Acad. Sci.108:7523-7528, 2011). Treatment of rats with a monoclonal antibody against myocardial ischemia / reperfusion (MBL) in a myocardial ischemia / reperfusion model resulted in reduced ischemia-reperfusion injury (Jordan, JE et al.). Circulation 104 :1413-18, 2001). In studies of myocardial infarction patients treated with angioplasty, MBL deficiency was associated with reduced 90-day mortality compared to its adequate counterpart (M Trendelenburg et al., Eur Heart J. 31:1181, 2010). Furthermore, MBL levels in myocardial infarction patients who developed heart failure after angioplasty were approximately three times higher than in patients with functional recovery (Haahr-Pedersen S. et al., ). J Inv Cardiology MBL antibodies also reduce complement deposition on endothelial cells in vitro after oxidative stress, indicating the role of the lectin pathway in myocardial I / R injury (Collard, CD et al., 21:13, 2009). Am. J. Pathol. 156:1549-56, 2000). In a mouse model of heterotopic syngeneic heart transplantation with I / R injury, the role of alternative pathways was investigated using the pathway-specific fusion protein CR2-fH (Atkinson, C. et al.). J. Immunol. (185:7007-7013, 2010). Immediate systemic administration of CR2-fH after transplantation resulted in a reduction in myocardial I / R injury to a degree comparable to that of CR2-Crry treatment, which inhibited all complement pathways, suggesting that alternative pathways are extremely important in this model.

[0410] In mouse models of renal I / R injury, alternative pathways are involved because, compared to wild-type mice, factor B-deficient mice are protected from the decline in renal function and renal tubular damage (Thurman, JM et al.). J. Immunol. 170:1517-1523, 2003). Treatment with an inhibitory monoclonal antibody against factor B prevents complement activation and reduces renal I / R damage in mice (Thurman, JM et al., J. Am. Soc. Nephrol. 17:707-715, 2006). In a bilateral renal I / R injury model, MBL-A / C deficient mice were protected from kidney injury compared to wild-type mice, and recombinant human MBL reversed the protective effect of MBL-A / C deficient mice, suggesting 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 renal I / R injury, after I / R, inhibition of MBL with a monoclonal antibody targeting MBL-A preserved renal function (van der Pol, P. et al.). Am. J. Transplant. 12:877-887, 2010). Notably, the role of MBL in this model did not involve the activation of terminal complement components, as treatment with C5 antibodies was ineffective in preventing kidney injury. Instead, MBL appeared to have a direct toxic effect on renal tubular cells, as human proximal tubular cells incubated with MBL internalized MBL in vitro, followed by apoptosis. In a porcine model of kidney I / R, Castellano G. et al., ( Am J Pathol, 176(4):1648-59, 2010) tested C1 inhibitors, which irreversibly inactivate C1r and C1s proteases in the classical pathway and MASP-1 and MASP-2 proteases in the MBL complex of the lectin pathway, and found that C1 inhibitors reduced complement deposition in peritubular capillaries and glomeruli and reduced tubular damage.

[0411] Alternative pathways appear to be involved in experimental traumatic brain injury because, compared to wild-type mice, factor B-deficient mice exhibit reduced systemic complement activation (as measured by serum C5a levels) and reduced post-traumatic neuronal death (Leinhase, I. et al.). BMC Neurosci. 7:55-67, 2006). In human stroke, the detection of complement components C1q, C3c, and C4d by immunohistochemical staining in ischemic injury indicates activation via the classical pathway (Pedersen, ED, et al.). Scand. J. Immunol. 69:555-562, 2009). Targeting the classical pathway in animal models of cerebral ischemia has yielded mixed results, with some studies showing protective effects while others have shown no benefit (Arumugam, TV et al., 2009). Neuroscience 158:1074-1089, 2009). Experimental and clinical studies have provided strong evidence for the involvement of the lectin pathway. In experimental stroke models, the lack 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 with low levels of MBL have better prognosis compared to their MBL-adequate counterparts (Osthoff M. et al., PLoS One, 6(6):e21338, 2011).

[0412] In a baboon model of cardiopulmonary bypass, treatment with factor D monoclonal antibodies suppressed systemic inflammation (as measured by plasma levels of C3a, SC5b-9, and IL-6) and reduced myocardial tissue damage, indicating the involvement of alternative pathways in this model (Undar, A. et al.). Ann. Thorac. Surg. 74:355-362, 2002).

[0413] Therefore, depending on the organ affected by ischemia / reperfusion (I / R), all three complement pathways can contribute to pathogenesis and adverse outcomes. Based on the experimental and clinical findings detailed above, LEA-2 inhibitors are expected to be protective in most I / R cases. At least in some cases, lectin-dependent activation of LEA-1 can lead to complement activation via the alternative pathway. Furthermore, LEA-2-initiated complement activation can be further amplified by the alternative pathway amplification loop, thereby exacerbating I / R-related tissue damage. Therefore, LEA-1 inhibitors are expected to provide additional or complementary therapeutic benefits in patients with ischemia-related conditions.

[0414] In light of the foregoing, LEA-1 and LEA-2 inhibitors are expected to have independent therapeutic benefits in treating, preventing, or reducing the severity of ischemia-reperfusion-related conditions. Furthermore, the combined use of LEA-1 and LEA-2 inhibitors can achieve additional therapeutic benefits compared to either agent alone. Therefore, optimal and effective treatment for I / R-related conditions includes blocking the active pharmaceutical components of both LEA-1 and LEA-2, either alone or in combination. Combined LEA-1 and LEA-2 inhibition can be achieved by co-administration of a LEA-1 blocker and a LEA-2 blocker. Preferably, the inhibitory function of LEA-1 and LEA-2 can be contained in a single molecular entity, such as a bispecific antibody containing specific binding sites for MASP-1 / 3 and MASP-2, or a bispecific antibody wherein each binding site can bind to and block either MASP-1 / 3 or MASP-2.

[0415] Based on the foregoing, one aspect of the present invention therefore provides a method for inhibiting LEA-1-dependent complement activation to treat, prevent, or reduce the severity of ischemia-reperfusion injury, said method being carried out by administering a composition to a subject undergoing ischemia-reperfusion, said composition comprising a therapeutically effective amount of a LEA-1 inhibitor in a drug carrier, said LEA-1 inhibitor comprising a combination of a MASP-1 inhibitor, a MASP-3 inhibitor, or a combination of MASP-1 / 3 inhibitors. The MASP-1, MASP-3, or MASP-1 / 3 inhibitory composition may be administered intra-arterially, intravenously, intracranially, intramuscularly, subcutaneously, or otherwise parenterally, and potentially orally for non-peptidase inhibitors, and most preferably via intra-arterial or intravenous administration. Administration of the LEA-1 inhibitory composition of the present invention is preferably initiated immediately after or as soon as possible following an ischemia-reperfusion event. In cases where reperfusion occurs in a controlled environment (e.g., after aortic aneurysm repair, organ transplantation, or repositioning of a severed or traumatized limb or finger / toe), LEA-1 inhibitors may be administered before and / or during and / or after reperfusion. Administration may be determined by the physician and repeated periodically to achieve optimal therapeutic effect.

[0416] In some implementations, the method is used to treat or prevent ischemia-reperfusion injury associated with at least one of the following: aortic aneurysm repair, cardiopulmonary bypass, vascular reanastomosis related to organ transplantation and / or limb / finger / toe replantation, stroke, myocardial infarction and shock, and / or hemodynamic resuscitation after surgery.

[0417] In some embodiments, the method is used to treat or prevent ischemia-reperfusion injury in a subject who is about to undergo, is undergoing, or has already undergone organ transplantation. In some embodiments, the method is used to treat or prevent ischemia-reperfusion injury in a subject who is about to undergo, is undergoing, or has already undergone organ transplantation, provided that the organ transplantation is not a kidney transplant.

[0418] In one embodiment, the method according to this aspect of the invention further includes inhibiting LEA-2-dependent complement activation in a subject undergoing ischemia-reperfusion injury, including administering to the subject a therapeutically effective amount of a MASP-2 inhibitor and a MASP-1, MASP-3, or MASP-1 / 3 inhibitor. As detailed above, in treating, preventing, or reducing the severity of ischemia-reperfusion injury, a combination of pharmacological agents that individually block LEA-1 and LEA-2 is expected to provide improved therapeutic outcomes compared to inhibiting LEA-1 alone. This outcome can be achieved, for example, by co-administering an antibody having LEA-1 blocking activity along with an antibody having LEA-2 blocking activity. In some embodiments, LEA-1- and LEA-2- blocking activities are combined into a single molecular entity, and said entity has combined LEA-1- and LEA-2- blocking activities. Such an entity may comprise or consist of a bispecific antibody, wherein 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 an entity could be composed of bispecific monoclonal antibodies, one of which has an antigen-binding site that specifically recognizes MASP-3, thus blocking LEA-1, while the second antigen-binding site specifically recognizes MASP-2, thus blocking LEA-2. Ideally, such an entity could be composed of bispecific monoclonal antibodies, one of which has an antigen-binding site that specifically recognizes both MASP-1 and MASP-3, thus blocking LEA-1, while the second antigen-binding site specifically recognizes MASP-2, thus blocking LEA-2.

[0419] MASP-2 inhibitory compositions may be administered intra-arterially, intravenously, intracranially, intramuscularly, subcutaneously, or otherwise parenterically, and potentially orally for non-peptidase inhibitors, but are most suitable for intra-arterial or intravenous administration to patients in need. Administration of the MASP-2 inhibitory compositions of the present invention is suitable to be initiated immediately or as soon as possible after an ischemia-reperfusion event. In cases where reperfusion occurs in a controlled environment (e.g., after aortic aneurysm repair, organ transplantation, or reduction of a severed or traumatized limb or finger / toe), the MASP-2 inhibitor may be administered prior to and / or during and / or after reperfusion. Administration may be determined by a physician and repeated periodically to achieve optimal therapeutic effects.

[0420] The MASP-3 inhibitory composition and optionally the MASP-2 inhibitory composition of the present invention can be administered by a single application of the composition (e.g., a single composition containing both MASP-2 and MASP-3 inhibitors or bispecific or dual inhibitors, or co-administration of separate compositions) or by a limited sequence of administration, for the treatment or prevention of ischemia-reperfusion injury. Alternatively, the composition can be administered at periodic intervals, such as daily, every two weeks, weekly, every other week, monthly, or every two months, over a longer period of time, to treat subjects experiencing ischemia-reperfusion.

[0421] As described in Examples 11-21 of this document, high-affinity MASP-3 inhibitory antibodies have been developed that have therapeutic efficacy in inhibiting alternative pathways in AP-related diseases or conditions (e.g., in subjects undergoing ischemia-reperfusion).

[0422] Therefore, in one embodiment, the present invention provides a method for treating subjects who have or are at risk of developing ischemia-reperfusion, comprising administering an effective amount of a high-affinity monoclonal antibody or antigen-binding fragment thereof that binds to human MASP-3 and inhibits alternative pathway complement activation to treat ischemia-reperfusion-related tissue damage in the subject or reduce the risk of ischemia-reperfusion-related tissue damage in the subject.

[0423] D. The role of MASP-3 in inflammatory and non-inflammatory arthritis and treatment methods using MASP-3 inhibitory antibodies and optional combinations of MASP-2 inhibitors.

[0424] Rheumatoid arthritis (RA) is a chronic inflammatory disease of the synovial joints, which can also present as systemic symptoms. RA affects approximately 1% of the world's population, with women being two to three times more likely to be affected. Joint inflammation manifests as redness, swelling, pain, and stiffness. As the disease progresses, joint erosion and destruction can occur, leading to impaired range of motion and deformities. Treatment goals for RA include preventing or controlling joint damage, preventing joint function loss and disease progression, alleviating symptoms and improving quality of life, and achieving drug-free remission. Pharmacological treatment of RA includes disease-modifying antirheumatic drugs (DMARDs), analgesics, and anti-inflammatory drugs (glucocorticoids and nonsteroidal anti-inflammatory drugs). DMARDs are the most important treatment because they can induce durable remission and delay or prevent the development of irreversible joint destruction. Traditional DMARDs include small molecules such as methotrexate, sulfasalazine, hydroxychloroquine, gold salts, leflunomide, D-penicillamine, cyclosporine, and azathioprine. If traditional DMARDs are insufficient to control the disease, several biologics targeting inflammatory cells or mediators are available treatment options, such as tumor necrosis factor inhibitors (etanercept, infliximab, adalimumab, certolizumab pegol, and golimumab), cytokine antagonists (anaspirin and tocilizumab), rituximab, and abatacept.

[0425] Although adaptive immunity is clearly important for the pathogenesis of RA (as indicated by genetic association with T-cell activation genes and the presence of autoantibodies), innate immune mechanisms are also involved (McInnes, IB and Schett, G). New Engl. J. Med. 365:2205-2219, 2011). In human RA, the synovial fluid level of the alternative pathway cleavage fragment Bb was several times higher than that in samples from patients with crystal-induced arthritis or degenerative joint disease, suggesting preferential activation of the alternative pathway in RA patients (Brodeur, JP et al.). Arthritis Rheum. 34:1531-1537, 1991). In an experimental passive transfer model of type II collagen antibodies in arthritis, factor B-deficient mice exhibited reduced inflammation and joint destruction compared to wild-type mice, while C4-deficient mice showed similar disease activity to wild-type mice, indicating the need for an alternative pathway rather than the classical pathway in this model (Banda, NK et al., 1991). J. Immunol. 177:1904-1912, 2006). In the same experimental model of collagen antibody-induced arthritis (CAIA), mice with activity only in the classical pathway or only in the lectin pathway were unable to develop arthritis (Banda, NK et al., 2006). Clin. Exp. Immunol. 159:100-108, 2010). Data from this study indicate that both the classical and lectin pathways can activate low levels of C3 in vitro. However, without the alternative pathway amplification loop, the level of C3 deposited in the joints is insufficient to produce clinical disease. A key step in the activation of the alternative pathway is the conversion of factor D (pre-factor D) proenzyme to mature factor D, which is generated by MASP-1 and / or MASP-3 (Takahashi, M. et al., 2010). 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 (Provided by The Association for Research in Vision and Ophthalmology 2011 meeting, March 4, 2011). The role of MASP-1 / 3 was evaluated in mouse CAIA, and results showed that MASP-1 / 3-deficient mice were protected from arthritis compared to wild-type mice (Banda, NK et al.). J. Immunol. 185:5598-5606, 2010). In MASP-1 / 3 deficient mice, pre-factor D, but not mature factor D, was detected in serum during CAIA development, and the addition of human factor D to serum from these mice reconstructed C3 activation and C5a production in vitro. In contrast, in mouse models of the effector phase of arthritis, C3-deficient mice developed very mild arthritis compared to WT mice, while factor B-deficient mice still developed arthritis, indicating independent contributions from both the classical / lectin and alternative pathways (Hietala, MA et al., 2010). Eur. J. Immunol. 34:1208-1216, 2004). In a K / BxN T-cell receptor transgenic mouse model of inflammatory arthritis, mice lacking C4 or C1q developed arthritis similar to wild-type mice, while mice lacking factor B did not develop arthritis or had mild arthritis, suggesting that an alternative pathway is needed in this model rather than the classical pathway (Ji H. et al., Immunity 16:157-168, 2002). In the K / BxN model, mice lacking MBL-A are unprotected from serum-induced arthritis, but since the role of MBL-C has not been studied, its potential role in the lectin pathway cannot be ruled out (Ji et al., 2002, ibid.).

[0426] Two research groups have independently proposed that lectin-dependent complement activation promotes inflammation in RA patients through the interaction of MBL with specific IgG glycoform (Malhotra et al., Nat. Med. 1 :237-243, 1995; Cuchacovich et al., J. Rheumatol. 23 (44-51, 1996). It should be noted that rheumatoid condition is associated with a significant increase in IgG glycoform lacking galactose in the Fc region of this molecule (referred to as IgG0 glycoform) (Rudd et al., 1996). Trends Biotechnology 22 The percentage of IgG0 glycoform increases with disease progression in rheumatoid arthritis (RA) and returns to normal when patients are in remission. In vivo, IgG0 is deposited in synovial tissue, and MBL is present in the synovial fluid of RA individuals at increased levels. RA-associated agalactosyl IgG (IgG0) can bind to MBL and thus initiate lectin-dependent complement activation via LEA-1 and / or LEA-2. Moreover, results from observing MBL allele variants in RA patients in clinical studies suggest that MBL may play an exacerbating inflammatory role in the disease (Garred et al., 524-30, 2004). J. Rheumatol. 27 (26-34, 2000). Therefore, lectin-dependent complement activation via LEA-1 and / or LEA-2 may play an important role in the pathogenesis of RA.

[0427] Complement activation also plays an important role in juvenile rheumatoid arthritis (Mollnes, TE et al.). Arthritis Rheum . 29 (1359-64, 1986). Similar to adult RA, in juvenile rheumatoid arthritis, serum and synovial fluid levels of the alternative pathway complement activation product Bb are elevated compared to C4d (a marker of classical or LEA-2 activation), suggesting that complement activation is primarily mediated by LEA-1 (El-Ghobarey, AF et al., 1986). J. Rheumatology 7 :453-460, 1980; Agarwal, A. et al., Rheumatology 39 :189-192, 2000).

[0428] Similarly, complement activation plays an important role in psoriatic arthritis. Patients with this condition have increased complement activation products in their circulation, and their red blood cells appear to have lower levels of the complement regulator CD59 (Triolo). Clin Exp Rheumatol ., 21(2):225-8, 2003). Complement levels are associated with disease activity and have a high predictive value for determining treatment efficacy (Chimenti et al., 21(2):225-8, 2003). Clin Exp Rheumatol ., 30(1):23-30,2012). In fact, recent studies have shown that the effectiveness of anti-TNF therapy for this condition can be attributed to complement regulation (Ballanti et al., 30(1):23-30,2012). Autoimmun Rev ., 10(10):617-23, 2011). Although the precise role of complement in psoriatic arthritis has not been determined, the presence of C4d and Bb complement activation products circulating in these patients suggests they play an important role in pathogenesis. Based on the observed products, LEA-1, and possibly LEA-2, are considered to be responsible for pathological complement activation in these patients.

[0429] Osteoarthritis (OA) is the most common form of arthritis, affecting more than 25 million people in the United States. OA is characterized by the breakdown and eventual loss of articular cartilage, accompanied by new bone formation and synovial hyperplasia, leading to pain, stiffness, loss of joint function, and disability. Joints frequently affected by OA are the hands, neck, lower back, knees, and hips. The disease is progressive, and current treatments are symptomatic pain relief and do not alter the natural history of the disease. The pathogenesis of OA is not fully understood, but the role of complement has been identified. In proteomic and transcriptomic analyses of synovial fluid from OA patients, several components of complement were aberrantly expressed compared to samples from healthy individuals, including classical (C1s and C4A) and alternative (factor B) pathways, as well as C3, C5, C7, and C9 (Wang, Q. et al.). Nat. Med. 17:1674-1679, 2011). Furthermore, in a mouse model of OA induced by medial meniscectomy, C5-deficient mice had less cartilage loss, osteophyte formation, and synovitis than C5-positive mice, and treatment of wild-type mice with CR2-fH (a fusion protein that inhibits the alternative pathway) attenuated the development of OA (Wang et al., 2011, ibid.).

[0430] Ross River virus (RRV) and Chikungunya virus (CHIKV) belong to a group of mosquito-borne viruses that can cause acute and persistent arthritis and myositis in humans. In addition to causing endemicities, these viruses can cause epidemics involving millions of infected individuals. Arthritis is thought to be initiated by viral replication in the joints and a host inflammatory response, with the complement system being invoked as a key component in this process. The synovial fluid of people with RRV-induced polyarthritis contains higher levels of C3a than that of people with osteoarthritis (OA) (Morrison, TE et al., J. Virol. 81:5132-5143, 2007). In a mouse model of RRV infection, C3-deficient mice developed less severe arthritis compared to wild-type mice, suggesting the role of complement (Morrison et al., 2007, ibid.). The specific complement pathways involved were investigated, as well as mice with the inactivated lectin pathway (MBL-A). - / - and MBL-C - / - Compared to wild-type mice, these mice exhibited reduced arthritis. In contrast, mice with inactivated classical pathway (C1q) showed reduced arthritis.- / - ) or alternative pathways (Factor B) - / - Mice developed severe arthritis, indicating that the MBL-induced lectin pathway plays an important role in this model (Gunn, BM et al.). PLoS Pathog. 8:e1002586, 2012). Since arthritis involves joint damage, initial joint injury from various etiologies can trigger a second wave of complement activation via LEA-2. To support this concept, our previous work has shown that MASP-2 KO mice have reduced joint damage compared to WT mice in a collagen-induced RA model.

[0431] Given the substantial evidence detailed above, LEA-1 and LEA-2 inhibitors, alone or in combination, are expected to be therapeutically useful for treating arthritis. Therefore, the optimal and effective treatment for arthritis may comprise an active pharmaceutical ingredient that, alone or in combination, blocks both LEA-1 and LEA-2. Combined LEA-1 and LEA-2 inhibition can be achieved by co-administering a LEA-1 blocker and a LEA-2 blocker. Preferably, the inhibitory function of LEA-1 and LEA-2 can be contained in a single molecular entity, such as a bispecific antibody containing specific binding sites for MASP-1 / 3 and MASP-2, or a bispecific antibody wherein each binding site can bind to and block either MASP-1 / 3 or MASP-2. Based on the foregoing, one aspect of the present invention thus provides a method for inhibiting LEA-1-dependent complement activation to treat, prevent, or reduce the severity of inflammatory or non-inflammatory arthritis (including osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, and psoriatic arthritis), said method being carried out by administering a composition to a subject suffering from or at risk of developing inflammatory or non-inflammatory arthritis, said composition comprising a therapeutically effective amount of a LEA-1 inhibitor in a drug carrier, said LEA-1 inhibitor comprising a combination of a MASP-1 inhibitor, a MASP-3 inhibitor, or a combination of MASP-1 / 3 inhibitors. The MASP-1, MASP-3, or MASP-1 / 3 inhibitory composition may be administered systemically to the subject, for example, via intra-arterial, intravenous, intramuscular, subcutaneous, or other parenteral administration, or via oral administration. Alternatively, administration may be by local delivery, for example, via intra-articular injection. The LEA-1 inhibitor can be administered periodically over an extended period to treat or control chronic conditions, or it can be administered once or repeatedly before, during, and / or after an acute trauma or injury (including surgery on a joint).

[0432] In one embodiment, the method according to this aspect of the invention further includes inhibiting LEA-2-dependent complement activation in subjects suffering from or at risk of developing inflammatory or non-inflammatory arthritis (including osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, and psoriatic arthritis) by administering a therapeutically effective amount of a MASP-2 inhibitor and a MASP-1, MASP-3, or MASP-1 / 3 inhibitor to the subject. As detailed above, the combination of pharmacological agents that respectively block LEA-1 and LEA-2 is expected to provide improved therapeutic or preventative outcomes for arthritis compared to inhibiting LEA-1 alone. This outcome can be achieved, for example, by co-administering an antibody with LEA-1-blocking activity along with an antibody with LEA-2-blocking activity. In some embodiments, LEA-1- and LEA-2-blocking activities are combined into a single molecular entity, and said entity has the combined LEA-1- and LEA-2-blocking activities. Such an entity may comprise or consist of bispecific antibodies, wherein one antigen-binding site specifically recognizes MASP-1 and blocks LEA-1, while the second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Alternatively, such an entity may consist of bispecific monoclonal antibodies, wherein one antigen-binding site specifically recognizes MASP-3, thus blocking LEA-1, while the second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Such an entity is optimally composed of bispecific monoclonal antibodies, wherein one antigen-binding site specifically recognizes both MASP-1 and MASP-3, thus blocking LEA-1, while the second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2.

[0433] MASP-2 inhibitory compositions can be administered systemically to subjects in need, such as via intra-arterial, intravenous, intramuscular, subcutaneous, or other parenteral administration, or potentially orally for non-peptide inhibitors. Alternatively, administration can be via local delivery, such as via intra-articular injection. The MASP-2 inhibitors can be administered periodically over extended periods to treat or control chronic conditions, or via single or repeated administration before, during, and / or after acute trauma or injury (including surgery on joints).

[0434] The MASP-3 inhibitory composition and optionally the MASP-2 inhibitory composition of the present invention can be administered by a single application of the composition (e.g., a single composition containing both MASP-2 and MASP-3 inhibitors or bispecific or dual inhibitors, or co-administration of separate compositions), or by a limited sequential application, to treat, prevent, or reduce the severity of inflammatory or non-inflammatory arthritis. Alternatively, the composition can be administered at periodic intervals, such as daily, every two weeks, weekly, every other week, monthly, or every two months, over a prolonged period to treat subjects with inflammatory or non-inflammatory arthritis.

[0435] As described in Examples 11-21 of this document, high-affinity MASP-3 inhibitory antibodies have been produced, which have therapeutic efficacy in inhibiting alternative pathways in AP-related diseases or conditions such as arthritis.

[0436] Therefore, in one embodiment, the present invention provides a method for treating a subject suffering from or at risk of developing arthritis (inflammatory and non-inflammatory arthritis), comprising administering to the subject a pharmaceutical composition comprising an effective amount of a high-affinity monoclonal antibody or antigen-binding fragment thereof, as disclosed herein, that binds to human MASP-3 and inhibits alternative pathway complement activation, to treat arthritis in the subject or reduce the risk of arthritis in the subject, 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) VLCDR2 comprising SEQ ID NO: 144. VLCDR3 of 161. In some embodiments, the subject suffers from arthritis selected from osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, ankylosing spondylitis, Bechtel's disease, infection-related arthritis, and psoriatic arthritis. In some embodiments, the pharmaceutical composition is administered systemically (e.g., subcutaneously, intramuscularly, intravenously, intra-arterially, or as an inhaler). In some embodiments, the pharmaceutical composition is applied topically to the joint.

[0437] E. The role of MASP-3 in disseminated intravascular coagulation (DIC) and treatment methods using MASP-3 inhibitory antibodies, optionally combined with MASP-2 inhibitors.

[0438] Disseminated intravascular coagulation (DIC) is a syndrome of pathological overstimulation of the coagulation system, which can clinically present as bleeding and / or thrombosis. DIC does not occur as a primary condition but is associated with various disease processes, including tissue damage (trauma, burns, heatstroke, transfusion reactions, acute transplant rejection), tumor formation, infection, obstetric conditions (placenta previa, amniotic fluid embolism, preeclampsia), and other conditions such as cardiogenic shock, near drowning, fat embolism, and aortic aneurysm. Thrombocytopenia is a common abnormality in intensive care unit patients, with an incidence of 35%–44%, and in approximately 25% of these cases, DIC is the cause; that is, DIC occurs in approximately 10% of critically ill patients (Levi, M. and Opal, SM). Crit. Care (10:222-231, 2006). The pathophysiology of DIC lies in the underlying disease process that initiates the physiological coagulation response. However, thrombotic substances suppress normal balancing mechanisms, leading to inappropriate deposition of fibrin and platelets in the microcirculation, resulting in organ ischemia, hypofibrinogenemia, and thrombocytopenia. The diagnosis of DIC is based on clinical presentation within the appropriate underlying disease or process, as well as abnormal laboratory parameters (prothrombin time, partial thromboplastin time, fibrin degradation products, D-dimer, or platelet count). The primary treatment for DIC is to address the underlying condition as the responsible trigger. Support with blood products in the form of red blood cells, platelets, fresh frozen plasma, and cryoprecipitate may be necessary for the treatment or prevention of clinical complications.

[0439] The role of the complement pathway in DIC has been investigated in several studies. Complement activation was evaluated in pediatric patients with meningococcal infection, and clinical course was compared with that of the MBL genotype (Sprong, T. et al.). Clin. Infect. Dis. (49:1380-1386, 2009). At admission, patients with MBL deficiency had lower circulating levels of C3bc, terminal complement complex, C4bc, and C3bBbP than those with adequate MBL, indicating lower levels of activation of cocomplement, terminal complement, and alternative pathways. Furthermore, the degree of systemic complement activation was associated with the severity and parameters of DIC, with patients with MBL deficiency exhibiting a milder clinical course than those with adequate MBL. Therefore, although MBL deficiency is a risk factor for susceptibility to infection, MBL deficiency during septic shock may be associated with lower disease severity.

[0440] As illustrated in Examples 1-4 of this document, experimental studies have highlighted the significant contributions of MBL and MASP-1 / 3 to the innate immune response against Neisseria meningitidis (the pathogen of meningococcal infection). MBL-deficient sera from mice or humans, MASP-3-deficient human sera, or MASP-1 / 3 knockout mice showed poorer efficacy in activating complement and lysing meningococci in vitro compared to wild-type sera. Similarly, MASP-1 / 3 knockout mice used in experiments for the first time were more susceptible to Neisseria infection than their wild-type counterparts. Therefore, in the absence of adaptive immunity, the LEA-1 pathway contributes to innate host resistance to Neisseria infection. Conversely, LEA-1 enhances pathological complement activation, triggering harmful host responses, including disseminated intravascular coagulation (DIC).

[0441] In a mouse model of arterial thrombosis, FeCl3-induced thrombosis was reduced in MBL-free and MASP-1 / -3 knockout mice compared to wild-type or C2 / factor B-free mice, and this deficiency was reconstructed by recombinant human MBL (La Bonte, LR et al.). J. Immunol. (188:885-891, 2012). In vitro, serum from MBL-free or MASP-1 / -3 knockout mice showed reduced thrombin substrate cleavage compared to wild-type or C2 / factor B-free mouse serum; in the serum of MASP-1 / -3 knockout mice, the addition of recombinant human MASP-1 restored thrombin substrate cleavage (La Bonte et al., 2012, ibid.). These results indicate that the MBL / MASP complex, particularly MASP-1, plays a crucial role in thrombosis. Therefore, LEA-1 may play an important role in pathological thrombosis, including DIC.

[0442] Experimental studies have established the equally important role of LEA-2 in pathological thrombosis. In vitro studies have also shown that LEA-2 provides a molecular link between the complement system and the coagulation system. MASP-2 has factor Xa-like activity and activates prothrombin to form thrombin by cleaving it, which in turn can clear fibrinogen and promote fibrin clot formation (see also Krarup et al., PLoS One, 18:2(7):e623, 2007).

[0443] Independent studies have shown that the lectin-MASP complex can promote clot formation, fibrin deposition, and fibrin peptide release in a MASP-2-dependent process (Gulla et al., Immunology , 129(4):482-95, 2010). Therefore, LEA-2 promotes simultaneous lectin-dependent activation of complement and coagulation systems.

[0444] In vitro studies have also shown that MASP-1 has thrombin-like activity (Presanis JS, et al.). Mol Immunol , 40(13):921-9, 2004), and cleave fibrinogen and factor XIII (Gulla KC et al., Immunology (129(4):482-95, 2010), indicating that LEA-1 can activate the coagulation pathway independently or in conjunction with LEA-2.

[0445] The data detailed above demonstrate that LEA-1 and LEA-2 provide an independent link between lectin-dependent complement activation and coagulation. Therefore, given the above, LEA-1 and LEA-2 inhibitors are expected to provide independent therapeutic benefit in treating subjects with disseminated intravascular coagulation (DIC). In some embodiments, the subject has DIC secondary to: sepsis, trauma, infection (bacterial, viral, fungal, parasitic), malignancy, transplant rejection, transfusion reaction, obstetric complications, vascular aneurysm, liver failure, heatstroke, burns, radiation exposure, shock, or severe toxic reactions (such as snake bites, insect bites, transfusion reactions). In some embodiments, the trauma is neurological. In some embodiments, the infection is a bacterial infection, such as Neisseria meningitidis infection.

[0446] Furthermore, the combined use of LEA-1 and LEA-2 inhibitors offers additional therapeutic benefits compared to either agent alone. Since both LEA-1 and LEA-2 are known to be activated through conditions leading to DIC (e.g., infection or trauma), LEA-1 and LEA-2 blockers, alone or in combination, are expected to have therapeutic utility in the treatment of DIC. LEA-1 and LEA-2 blockers prevent different cross-talk mechanisms between complement and coagulation. Therefore, LEA-1 and LEA-2 blockers may have complementary, additive, or synergistic effects in the prevention of DIC and other thrombotic conditions.

[0447] Furthermore, the combined use of LEA-1 and LEA-2 inhibitors can provide additional therapeutic benefits compared to either agent alone, or offer effective treatment for a wider range of patient subgroups. Combined LEA-1 and LEA-2 inhibition can be achieved through the co-administration of LEA-1 and LEA-2 blockers. Ideally, the inhibitory function of LEA-1 and LEA-2 can be contained in a single molecular entity, such as a bispecific antibody containing specific binding sites for MASP-1 / 3 and MASP-2, or a bispecific antibody where each binding site can bind to and block either MASP-1 / 3 or MASP-2.

[0448] Based on the foregoing, one aspect of the present invention therefore provides a method for inhibiting LEA-1-dependent complement activation to treat, prevent, or reduce the severity of disseminated intravascular coagulation (DIC) in subjects with this need. The method comprises administering a composition to a subject suffering from DIC or at risk of developing DIC, the composition comprising a therapeutically effective amount of a LEA-1 inhibitor in a pharmaceutical carrier, comprising a MASP-1 inhibitor, a MASP-3 inhibitor, or a combination of MASP-1 / 3 inhibitors. The MASP-1, MASP-3, or MASP-1 / 3 inhibitory composition may be administered systemically to the subject, for example, via intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous, or other parenteral administration, or possibly via oral administration of a non-peptide agent. Administration may be determined by a physician and repeated until the condition is resolved or controlled. For the treatment or prevention of disseminated intravascular coagulation (DIC) secondary to trauma or other acute events, the LEA-1 inhibitory composition may be administered immediately after the traumatic injury, or prophylactically before, during, or after the trauma-induced injury or condition (such as surgery in a patient considered at risk of DIC), or for a period of 1 to 7 days or longer, such as 24 to 72 hours. In some embodiments, the LEA-1 inhibitory composition may be appropriately administered in a rapid-acting dosage form, such as intravenous or intra-arterial delivery via a bolus solution containing the LEA-1 inhibitory composition.

[0449] In one embodiment, the method according to this aspect of the invention further includes inhibiting LEA-2-dependent complement activation in a subject with this need to treat, prevent, or reduce the severity of disseminated intravascular coagulation (DIC), including administering to the subject a therapeutically effective amount of a MASP-2 inhibitor and a MASP-1, MASP-3, or MASP-1 / 3 inhibitor. As detailed above, a combination of pharmacological agents that each block LEA-1 and LEA-2 is expected to provide improved therapeutic outcomes in the treatment or prevention of DIC compared to inhibiting LEA-1 alone. This outcome can be achieved, for example, by co-administering an antibody having LEA-1 blocking activity along with an antibody having LEA-2 blocking activity. In some embodiments, LEA-1- and LEA-2- blocking activities are combined into a single molecular entity, and said entity has combined LEA-1- and LEA-2- blocking activities. Such an entity may comprise or consist of a bispecific antibody, wherein 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 an entity could be composed of bispecific monoclonal antibodies, one of which has an antigen-binding site that specifically recognizes MASP-3, thus blocking LEA-1, while the second antigen-binding site specifically recognizes MASP-2, thus blocking LEA-2. Ideally, such an entity could be composed of bispecific monoclonal antibodies, one of which has an antigen-binding site that specifically recognizes both MASP-1 and MASP-3, thus blocking LEA-1, while the second antigen-binding site specifically recognizes MASP-2, thus blocking LEA-2.

[0450] MASP-2 inhibitors can be administered systemically to those in need, such as via intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous, or other parenteral administration, and potentially orally for non-peptide agents. Administration may be repeated as determined by a physician until the condition is resolved or controlled. For DIC secondary to trauma or other acute events, MASP-2 inhibitory compositions may be administered immediately after the traumatic injury, or prophylactically before, during, or after the trauma-induced injury or condition (such as surgery in patients considered at risk of DIC), or for a period of 1 to 7 days or longer, such as 24 to 72 hours. In some embodiments, the MASP-2 inhibitory composition may be appropriately administered in a rapid-acting dosage form, such as via intravenous or intra-arterial delivery of a bolus solution containing the MASP-2 inhibitory composition.

[0451] The MASP-3 inhibitory composition and optionally the MASP-2 inhibitory composition of the present invention can be administered by a single application of the composition (e.g., a single composition containing both MASP-2 and MASP-3 inhibitors or bispecific or dual inhibitors, or by co-application of separate compositions) or by limited sequential application, to treat, prevent, or reduce the severity of disseminated intravascular coagulation (DIC) in subjects in need. Alternatively, the composition can be administered at periodic intervals, such as daily, every two weeks, weekly, every other week, monthly, or every two months, over a longer period, to treat subjects with DIC or at risk of developing DIC.

[0452] As described in Examples 11-21 of this document, high-affinity MASP-3 inhibitory antibodies have been generated that have therapeutic efficacy in inhibiting alternative pathways in AP-related diseases or conditions such as disseminated intravascular coagulation.

[0453] Therefore, in one embodiment, the present invention provides a method for treating a subject suffering from or at risk of developing disseminated intravascular coagulation (DIC), comprising administering an effective amount of a high-affinity monoclonal antibody or antigen-binding fragment thereof that binds to human MASP-3 and inhibits alternative pathway complement activation to treat DIC or reduce the risk of developing DIC, 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) VLCDR2 ... and (iii) VLCDR2 comprising SEQ ID NO: 142, SEQ ID NO: 257, SEQ ID NO: 258, or SEQ ID NO: 259, and (iii) VLCDR2 comprising SEQ ID NO: 142, SEQ ID NO: 257, SEQ ID NO: 258, or SEQ ID NO: 259, and (iv) VLCDR2 comprising SEQ ID NO: 142, SEQ ID NO: 257, SEQ ID NO: 258, or SEQ ID VLCDR3 of 161.

[0454] F. The role of MASP-3 in thrombotic microangiopathy (TMA), including hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (AHUS), and thrombotic thrombocytopenic purpura (TTP), and treatment methods using MASP-3 inhibitory antibodies and optional combinations of MASP-2 inhibitors.

[0455] Thrombotic microangiopathy (TMA) refers to a group of conditions clinically characterized by thrombocytopenia, microangiopathic hemolytic anemia, and multiple organ ischemia. The characteristic pathological features of TMA are platelet activation and the formation of microthrombi in arterioles and venules. Classic TMAs include hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). HUS differs from TTP in the presence of acute renal failure. HUS occurs in two forms: diarrhea-associated (D+) or typical HUS and non-diarrheal (D-) or atypical HUS (aHUS).

[0456] HUS

[0457] D+HUS is associated with prodromal diarrhea, typically caused by Escherichia coli O157 or another Shiga toxin-producing strain, accounting for over 90% of HUS cases in children and being the most common cause of acute renal failure in children. Although human infection with Escherichia coli O157 is relatively frequent, the percentage of bloody diarrhea developing into D+HUS ranges from 3% to 7% in sporadic cases and 20% to 30% in some outbreaks (Zheng, XL and Sadler, JE, Annu. Rev. Pathol. (3:249-277, 2008). D+HUS typically occurs 4 to 6 days after the onset of diarrhea, and approximately two-thirds of children require dialysis during the acute phase of the illness. Treatment for D+HUS is supportive, as no specific treatment has been shown to be effective. The prognosis for D+HUS is good, with most patients recovering kidney function.

[0458] The pathogenesis of D+HUS involves Shiga toxin produced by bacteria that binds to the membranes of microvascular endothelial cells, monocytes, and platelets. Renal microvessels are most frequently affected. Upon binding, the toxin is internalized, leading to the release of inflammatory mediators and ultimately cell death. Endothelial cell damage is thought to trigger renal microvascular thrombosis by promoting the activation of the coagulation cascade. There is evidence of complement system activation in D+HUS. Compared to normal controls, children with D+HUS had increased plasma levels of Bb and SC5b-9 at hospitalization, which normalized by day 28 after discharge (Thurman, JM et al.). Clin. J. Am. Soc. Nephrol. 4:1920-1924, 2009). It was found that because activation occurs in the presence of ethylene glycol tetraacetic acid, which blocks the classical pathway, Shiga toxin 2 (Stx2) primarily activates human complement in the fluid phase in vitro via an alternative pathway (Orth, D. et al.). J. Immunol. 182:6394-6400, 2009). Furthermore, Stx2 binds to factor H but not factor I and delays the cofactor activity of factor H on the cell surface (Orth et al., 2009, ibid.). These results suggest that Shiga toxin may cause kidney damage through multiple potential mechanisms, including direct toxicity and indirect effects through complement activation or inhibition of complement regulators. Endothelial toxicity is expected to occur through complement activation by LEA-2, as demonstrated by the effectiveness of MASP-2 blockade in preventing complement-mediated reperfusion injury in various vascular beds, as suggested by Schwaeble, WJ et al. Proc. Natl. Acad. Sci. As stated in 108:7523-7528, 2011.

[0459] In a mouse HUS model induced by co-injection of Shiga toxin and lipopolysaccharide, factor B-deficient mice exhibited less thrombocytopenia and were protected from kidney damage compared to wild-type mice, suggesting that the alternative pathway involves LEA-1-dependent activation in microvascular thrombosis (Morigi, M. et al.). J. Immunol. 187:172-180, 2011). As described in this paper, MASP-2 antibody administration was also effective in the same model and increased survival after STX challenge, suggesting the LEA-2-dependent complement pathway in microvascular thrombosis.

[0460] In light of the foregoing, LEA-1 and LEA-2 inhibitors are expected to offer independent therapeutic benefits in the treatment or prevention of HUS. Furthermore, the combined use of LEA-1 and LEA-2 inhibitors may provide additional therapeutic benefits or offer effective treatment for a wider range of patient subgroups compared to either agent alone. Combined LEA-1 and LEA-2 inhibition can be achieved through the co-administration of LEA-1 and LEA-2 blockers. Ideally, the inhibitory function of LEA-1 and LEA-2 can be contained in a single molecular entity, such as a bispecific antibody containing specific binding sites for MASP-1 / 3 and MASP-2, or a bispecific antibody where each binding site can bind to and block either MASP-1 / 3 or MASP-2.

[0461] aHUS

[0462] Atypical HUS is a rare disease with an estimated incidence of 2 per million in the United States (Loirat, C. and Fremeaux-Bacchi, V.). Orphanet J. Rare Dis. 6:60-90, 2011). Atypical HUS can develop at any age, but most patients experience onset in childhood. Atypical HUS is heterogeneous: some cases are familial, some are recurrent, and some are caused by infectious diseases, usually upper respiratory tract or gastroenteritis. The onset of aHUS is usually sudden, and most patients require dialysis upon admission. Additional renal manifestations are present in approximately 20% of patients and may involve the central nervous system, myocardial infarction, distal ischemic gangrene, or multiple organ failure. Treatment for aHUS includes supportive care for organ dysfunction, plasma transfusion or plasma exchange, and eculizumab (eculizumab), a humanized monoclonal antibody against C5, recently approved for use in the US and EU. The prognosis for aHUS is worse than that for D+HUS, with an approximately 25% mortality rate in the acute phase, and most survivors develop end-stage renal disease.

[0463] Atypical HUS is characterized as a disease of complement regulation disorder, with approximately 50% of patients having mutations in genes encoding complement regulatory proteins (Zheng and Sadler, 2008, ibid.). Most mutations are found in factor H (FH); other mutations include membrane cofactor protein (MCP), factor I (FI), factor B, and C3. Functional studies have shown that FH, MCP, and FI mutations lead to loss of function and thus increased complement activation, while factor B mutations result in gain of function. The effects of these mutations primarily influence alternative pathways. These genetic abnormalities are risk factors, not the sole cause of the disease, as approximately 50% of family members carrying the mutation do not present with the disease by age 45 (Loirat and Fremeaux-Bacchi, 2011, ibid.).

[0464] Factor H (FH) is a complement regulatory protein that protects host tissues from alternative pathway complement attack. FH regulates the alternative pathway amplification loop in three ways: it is a cofactor for FI, which cleaves C3b; it inhibits the formation of C3bBb, an alternative pathway C3 convertase; and it binds to polyanions on the cell surface and tissue matrix, blocking C3b deposition (Atkinson, JP and Goodship, THJ, J. Exp. Med. 6:1245-1248, 2007). Most FH mutations in patients with aHUS occur in the short concordant repeat domain of the protein at the C-terminus, leading to defects in FH binding to heparin, C3b, and endothelium, but without altering plasma C3 regulation residing in the N-terminal domain (Pickering, MC et al.). J. Exp. Med. 204:1249-1256, 2007). FH-deficient mice exhibit uncontrolled plasma C3 activation and spontaneous development of membranoproliferative glomerulonephritis type II, but not aHUS. However, FH-deficient mice transgenic with functionally equivalent expression of the aHUS-related human FH mutant spontaneously develop HUS but not membranoproliferative glomerulonephritis type II, providing in vivo evidence that defective control of alternative pathway activation in the renal endothelium is a key event in the pathogenesis of FH-related aHUS (Pickering et al., 2007, ibid.). Another form of FH-related aHUS occurs in patients with anti-FH autoantibodies that lead to loss of FH functional activity; most of these patients have deletions of genes encoding five FH-related proteins (Loirat and Fremeaux-Bacchi, 2011, ibid.).

[0465] Similar to FH, MCP inhibits complement activation by regulating C3b deposition on target cells. MCP mutations produce proteins with low C3b binding and cofactor activity, thus allowing dysregulated alternative pathway activation. FI is a serine protease that cleaves C3b and C4b in the presence of cofactors such as FH and MCP, thereby preventing the formation of C3 and C5 convertases and inhibiting both alternative and classical complement pathways. Most FI-associated aHUS mutations result in reduced FI activity for C3b and C4b degradation (Zheng and Stadler, 2008, ibid.). FB is a zymogen carrying the catalytic site of the alternative pathway convertase C3bBb. Functional analysis showed that aHUS-associated FB mutations lead to increased alternative pathway activation (Loirat and Fremeaux-Bacchi, 2011, ibid.). Heterozygous mutations in C3 are associated with aHUS. Most C3 mutations result in C3-binding MCP deficiency, leading to increased ability of FB to bind C3b and increased formation of C3 convertase (Loirat and Fremeaux-Bacchi, 2011, ibid.). Therefore, aHUS is a disease closely associated with complement gene mutations leading to inadequate control of the alternative pathway amplification loop. Since the alternative pathway amplification loop depends on factor B proteolytic activity, and since LEA-1 is required for factor B activation (via MASP-3-dependent cleavage or via factor D-type mediated cleavage, where MASP-1 contributes to factor D maturation), LEA-1 blockers are expected to prevent uncontrolled complement activation in susceptible individuals. Therefore, LEA-1 blockers are expected to be effective in treating aHUS.

[0466] Although the central role of the dysregulated alternative pathway amplification loop in aHUS is widely accepted, the triggers for initiating complement activation and the molecular pathways involved remain unresolved. Not all individuals carrying the aforementioned mutations develop aHUS. In fact, familial studies have shown that the penetrance of aHUS is only about 50% (Sullivan M. et al.). Ann Hum Genet 74:17-26 2010). The natural history of the disease suggests that aHUS most commonly develops after initiating events such as infectious onset or injury. Infectious agents are known to activate the complement system. In the absence of pre-existing adaptive immunity, complement activation by infectious agents can be initiated primarily via LEA-1 or LEA-2. Therefore, in aHUS-susceptible individuals, lectin-dependent complement activation induced by infection can represent an initiating trigger for subsequent pathological amplification of complement activation, which may ultimately lead to disease progression. Therefore, another aspect of the invention includes treating patients with aHUS secondary to infection by administering an effective amount of an LEA-1 or LEA-2 inhibitor.

[0467] Other forms of host tissue injury will activate complement via LEA-2, particularly vascular endothelial injury. Human vascular endothelial cells undergo oxidative stress and respond, for example, by expressing surface portions of the LEA-2 complement pathway that bind lectins (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). In this case, activation of the lectin pathway 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, ibid.).

[0468] Therefore, other mechanisms that facilitate the activation of LEA-1 or LEA-2 in aHUS are also known. It is therefore possible that in individuals genetically predisposed to aHUS, the LEA-1 and / or LEA-2 pathways may represent an inappropriately amplified initial complement activation mechanism in a dysregulated manner, thereby initiating aHUS pathogenesis. Consequently, it is inferred that in aHUS-susceptible individuals, agents that block complement activation via LEA-1 and / or LEA-2 are expected to prevent disease progression or reduce disease severity.

[0469] Further supporting this concept is the recent identification of Streptococcus pneumoniae as an important pathogen in pediatric cases of aHUS (Lee, CS et al.). Nephrology , 17(1):48-52 (2012); Banerjee R. et al., Pediatr Infect Dis J ., 30(9):736-9 (2011)). This particular etiology appears to have an unfavorable prognosis, with significant mortality and long-term morbidity. Notably, these cases involve non-enteric infections leading to manifestations of microangiopathy, uremia, and hemolysis, without evidence of known complicating mutations in complement genes that predispose to aHUS. It is important to note that Streptococcus pneumoniae is particularly effective at activating complement and primarily through LEA-2. Therefore, in cases of non-enteric HUS associated with pneumococcal infection, manifestations of microangiopathy, uremia, and hemolysis are expected to be primarily driven by LEA-2 activation, and agents that block LEA-2, including MASP-2 antibodies, are expected to prevent the progression of aHUS or reduce the severity of the disease in these patients. Therefore, another aspect of the invention comprises treating patients with non-enteric aHUS associated with Streptococcus pneumoniae infection by administering an effective amount of a MASP-2 inhibitor.

[0470] TTP

[0471] Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disorder of the blood clotting system caused by autoimmune or hereditary dysfunction of the activated coagulation cascade or complement system (George, JN, N Engl J Med (354:1927-35, 2006). This leads to a large number of microclots or thrombi in the small blood vessels throughout the body, a characteristic feature of TMA. Red blood cells are subjected to shear stress, which damages their membranes, leading to intravascular hemolysis. The resulting reduced blood flow and endothelial damage cause organ damage, including to the brain, heart, and kidneys. The clinical features of TTP are thrombocytopenia, microangiopathic hemolytic anemia, neurological changes, renal failure, and fever. Before plasma exchange, the mortality rate during acute attacks was 90%. Even with plasma exchange, the six-month survival rate is approximately 80%.

[0472] TTP can result from hereditary or acquired inhibition of the enzyme ADAMTS-13, a metalloproteinase responsible for cleaving large multimers of von Willebrand factor (vWF) into smaller units. Inhibition or insufficiency of ADAMTS-13 ultimately leads to increased coagulation (Tsai, H). J Am Soc Nephrol 14: 1072–1081, 2003). ADAMTS-13 regulates vWF activity; in the absence of ADAMTS-13, vWF forms large polymers that are more likely to bind to platelets and make patients susceptible to platelet aggregation and microvascular thrombosis.

[0473] Numerous ADAMTS13 mutations have been identified in individuals with TTP. The disease can also develop due to autoantibodies against ADAMTS-13. Furthermore, TTP can occur in breast cancer, gastrointestinal cancer, or prostate cancer (George JN., Oncology (Williston Park). 25:908-14, 2011), Pregnancy (mid-pregnancy or postpartum) (George JN., Curr Opin Hematol It may develop during the period of 10:339-344, 2003, or be associated with diseases such as HIV or autoimmune diseases such as systemic lupus erythematosus (Hamasaki K et al., 2003). Clin Rheumatol .22:355-8, 2003). TTP can also be caused by certain drug therapies, including heparin, quinine, immune-mediated components, cancer chemotherapy agents (bleomycin, cisplatin, cytarabine, daunorubicin, gemcitabine, mitomycin C and tamoxifen), cyclosporine A, oral contraceptives, penicillin, rifampin and antiplatelet drugs including ticlopidine and clopidogrel (Azarm, T. et al., 22:355-8, 2003). J Res Med Sci ., 16: 353–357, 2011). Other factors or conditions associated with TTP include toxins such as bee venom, septicemia, spleen sequestration, transplantation, vasculitis, vascular surgery, and infections such as Streptococcus pneumoniae and cytomegalovirus infection (Moake JL., 16: 353–357, 2011). N Engl J Med ., 347:589–600, 2002). Transient functional ADAMTS-13 deficiency (TTP) can occur due to endothelial cell damage associated with Streptococcus pneumoniae infection. Pediatr Nephrol , 26:631-5, 2011).

[0474] Plasma exchange is the standard treatment for TTP (Rock GA et al., N Engl J Med 325:393-397, 1991). Plasma exchange replenishes ADAMTS-13 activity in patients with genetic defects and removes ADAMTS-13 autoantibodies in those with acquired autoimmune TTP (Tsai, HM, Hematol Oncol Clin North Am ., 21(4): 609–v,2007). Additional agents such as immunosuppressants are routinely added to the therapy (George, JN, N Engl J Med, 354:1927-35, 2006). However, plasma exchange is unsuccessful in about 20% of patients, relapses occur in more than one-third of patients, and plasma exchange is costly and technically demanding. In addition, many patients cannot tolerate plasma exchange. Therefore, there is still an urgent need for additional and better treatments for TTP.

[0475] Because TTP is a disorder of the blood coagulation cascade, treatment with complement system antagonists can help stabilize and correct the disease. Although pathological activation of the alternative complement pathway is associated with aHUS, the role of complement activation in TTP is less clear. Insufficient ADAMTS13 function is important for TTP susceptibility, but it is insufficient to cause acute attacks. Environmental factors and / or other genetic variations can contribute to TTP presentation. For example, genes encoding proteins involved in the regulation of the coagulation cascade, vWF, platelet function, endothelial vascular surface components, or the complement system can be involved in the development of acute thrombotic microangiopathy (Galbusera, M. et al.). Haematologica (References: 94: 166–170, 2009). Specifically, complement activation has been shown to play a crucial role; serum from thrombotic microangiopathy associated with ADAMTS-13 deficiency has been shown to lead to C3 and MAC deposition and subsequent neutrophil activation, which may be eliminated by complement inactivation (Ruiz-Torres MP et al., 94: 166–170, 2009). Thromb Haemost (93:443-52, 2005). Furthermore, it has recently been shown that levels of C4d, C3bBbP, or C3a increase during acute exacerbations of TTP (M. Réti et al., 93:443-52, 2005). J Thromb Haemost. (10(5):791-798, 2012), which is consistent with the activation of the classical, lectin and alternative pathways. This increased complement activation during acute exacerbations can initiate terminal pathway activation and is responsible for further deterioration of TTP.

[0476] The roles of ADAMTS-13 and vWF in TTP are clearly responsible for platelet activation and aggregation, and their subsequent roles in shear stress and deposition in microangiopathy. Activated platelets interact with and trigger both classical and alternative complement pathways. Platelet-mediated complement activation increases inflammatory mediators C3a and C5a (Peerschke E. et al., Mol Immunol, 47:2170-5 (2010)). Platelets may therefore serve as a target for classical complement activation in genetic or autoimmune TTP.

[0477] As described above, lectin-dependent complement activation, through MASP-1's thrombin-like activity and LEA-2-mediated prothrombin activation, is a major molecular pathway linking endothelial injury with coagulation and microvascular thrombosis occurring in HUS. Similarly, activation of LEA-1 and LEA-2 can directly drive the coagulation system in TTP. Activation of the LEA-1 and LEA-2 pathways can be initiated in response to initial endothelial injury caused by ADAMTS-13 deficiency in TTP. Therefore, LEA-1 and LEA-2 inhibitors, including but not limited to antibodies that block MASP-2, MASP-1, MASP-3, or both MASP-1 and MASP-3 functions, are expected to alleviate microangiopathy associated with intravascular coagulation, thrombosis, and hemolysis in patients with TTP.

[0478] Patients with TTP typically present in the emergency room with one or more of the following: purpura, renal failure, low platelet count, anemia, and / or thrombosis, including stroke. Current standards of care for TTP include intracatheter delivery (e.g., intravenous or other forms of catheter) plasma exchange for up to two weeks or longer, generally three times weekly, but up to daily. If the subject tests positive for an ADAMTS13 inhibitor (i.e., an endogenous antibody against ADAMTS13), plasma exchange may be combined with immunosuppressive therapy (e.g., corticosteroids, rituximab, or cyclosporine). Subjects with refractory TTP (approximately 20% of TTP patients) do not respond to at least two weeks of plasma exchange therapy.

[0479] Based on the foregoing, in one embodiment, in the event of a preliminary diagnosis of TTP, or in subjects exhibiting one or more symptoms consistent with a diagnosis of TTP (e.g., central nervous system involvement, severe thrombocytopenia (platelet count less than or equal to 5000 / μL without aspirin, less than or equal to 20000 / μL with aspirin), severe cardiac involvement, severe pulmonary involvement, gastrointestinal infarction, or gangrene), a method is provided to treat subjects with an effective amount of an LEA-2 inhibitor (e.g., a MASP-2 antibody) or a LEA-1 inhibitor (e.g., a MASP-1 or MASP-3 antibody) as first-line therapy (in the absence of plasma exchange, or in combination with plasma exchange). As first-line therapy, the LEA-1 and / or LEA-2 inhibitors may be administered systemically to the subject, for example, via intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous, or other parenteral administration. In some implementations, LEA-1 and / or LEA-2 inhibitors are administered to subjects as first-line therapy in the absence of plasma exchange to avoid potential complications of plasma exchange, such as bleeding, infection, and exposure to conditions and / or allergies inherent to the plasma donor, or in subjects who are additionally averse to plasma exchange, or when plasma exchange is unavailable. In some implementations, LEA-1 and / or LEA-2 inhibitors are administered in combination with immunosuppressants (e.g., corticosteroids, rituximab, or cyclosporine) (including co-administration) and / or in combination with concentrated ADAMTS-13 to subjects with TTP.

[0480] In some embodiments, the method includes administering LEA-1 and / or LEA-2 inhibitors to a subject with TTP via a catheter (e.g., intravenously) during a first time period (e.g., an acute phase lasting at least one day to one or two weeks), followed by subcutaneous administration of the LEA-1 and / or LEA-2 inhibitors to the subject during a second time period (e.g., a chronic phase lasting at least two weeks or longer). In some embodiments, the administration during the first and / or second time periods is performed without plasma exchange. In some embodiments, the method is used to maintain the subject to prevent the subject from developing one or more TTP-related symptoms.

[0481] In another embodiment, a method is provided for treating subjects with refractory TTP (i.e., subjects who have not responded to at least two weeks of plasma exchange therapy) by administering an amount of LEA-1 and / or LEA-2 inhibitors that effectively reduce one or more symptoms of TTP. In one embodiment, LEA-1 and / or LEA-2 inhibitors are administered subcutaneously or otherwise parenterally for a period of at least two weeks or longer to subjects with chronic refractory TTP. Administration may be repeated as determined by a physician until the condition is resolved or controlled.

[0482] In some embodiments, the method further includes determining the level of at least one complement factor (e.g., C3, C5) in the subject before treatment and optionally during treatment, wherein a determination of a decrease in the level of at least one complement factor compared to a standard value or a healthy control subject indicates the need for continued treatment with LEA-1 and / or LEA-2 inhibitors.

[0483] In some implementations, the method involves administering LEA-1 and / or LEA-2 inhibitors subcutaneously or intravenously to subjects with TTP or at risk of developing TTP. Treatment is preferably performed daily, but can also be less frequent, such as monthly. Treatment continues until the subject's platelet count is greater than 150,000 / ml for at least two consecutive days.

[0484] In summary, LEA-1 and LEA-2 inhibitors are expected to provide independent therapeutic benefits in the treatment of TMA, including HUS, aHUS, and TTP. Furthermore, the combined use of LEA-1 and LEA-2 inhibitors is expected to achieve additional therapeutic benefits compared to either agent alone, or to provide effective treatment for a wider range of patient subgroups with TMA variants. Combined LEA-1 and LEA-2 inhibition can be achieved through the co-administration of LEA-1 and LEA-2 blockers. Ideally, the inhibitory function of LEA-1 and LEA-2 can be contained in a single molecular entity, such as a bispecific antibody containing specific binding sites for MASP-1 / 3 and MASP-2, or a bispecific antibody where each binding site can bind to and block either MASP-1 / 3 or MASP-2.

[0485] Based on the foregoing, one aspect of the present invention provides a method for inhibiting LEA-1-dependent complement activation to treat, prevent, or reduce the severity of thrombotic microangiopathy, such as hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), or thrombotic thrombocytopenic purpura (TTP). The method comprises administering to a subject suffering from or at risk of developing thrombotic microangiopathy a composition comprising a therapeutically effective amount of a LEA-1 inhibitor in a drug carrier, comprising a MASP-1 inhibitor, a MASP-3 inhibitor, or a combination of MASP-1 / 3 inhibitors. The MASP-1, MASP-3, or MASP-1 / 3 inhibitory composition may be administered systemically to the subject, for example, via intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous, or other parenteral administration, or, for non-peptide agents, oral administration. Administration may be determined by a physician and repeated until the condition is resolved or controlled.

[0486] In one embodiment, the method according to this aspect of the invention further includes inhibiting LEA-2-dependent complement activation to treat, prevent, or reduce the severity of thrombotic microangiopathy, such as hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), or thrombotic thrombocytopenic purpura (TTP). The method includes administering a therapeutically effective amount of a MASP-2 inhibitor and a MASP-1, MASP-3, or MASP-1 / 3 inhibitor to a subject with or at risk of developing thrombotic microangiopathy. As detailed above, in treating or preventing or reducing the severity of thrombotic microangiopathy, a combination of pharmacological agents that respectively block LEA-1 and LEA-2 is expected to provide improved therapeutic outcomes compared to inhibiting LEA-1 alone. This outcome can be achieved, for example, by co-administering an antibody with LEA-1-blocking activity along with an antibody with LEA-2-blocking activity. In some embodiments, LEA-1 and LEA-2 blocking activities are combined into a single molecular entity, and said entity has combined LEA-1 and LEA-2 blocking activities. Such an entity may comprise or consist of a bispecific antibody, wherein one antigen-binding site specifically recognizes MASP-1 and blocks LEA-1, while the second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Alternatively, such an entity may consist of a bispecific monoclonal antibody, wherein one antigen-binding site specifically recognizes MASP-3, thus blocking LEA-1, while the second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2. Such an entity is optimally composed of a bispecific monoclonal antibody, wherein one antigen-binding site specifically recognizes both MASP-1 and MASP-3, thus blocking LEA-1, while the second antigen-binding site specifically recognizes MASP-2 and blocks LEA-2.

[0487] MASP-2 inhibitors can be administered systemically, such as via intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous, or other parenteral administration, or, for non-peptide agents, oral administration. Administration may be repeated as determined by a physician until the condition is resolved or controlled.

[0488] The administration of the MASP-3 inhibitory composition and optionally the MASP-2 inhibitory composition of the present invention can be carried out by a single administration of the composition (e.g., a single composition comprising MASP-2 and MASP-3 inhibitors or bispecific or dual inhibitors, or co-administration of separate compositions) or by limited sequential administration, for the treatment, prevention, or reduction of the severity of thrombotic microvascular disease in subjects who have or are at risk of developing thrombotic microvascular disease. Alternatively, the composition can be administered at regular intervals (e.g., daily, every two weeks, weekly, every other week, monthly, or every two months) over an extended period of time for the treatment of subjects in need.

[0489] As described in Examples 11-21 of this document, high-affinity MASP-3 inhibitory antibodies have been generated that have therapeutic efficacy in inhibiting alternative pathways 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)).

[0490] Therefore, in one embodiment, the present invention provides a method for treating a subject suffering from or at risk of developing thrombotic microangiopathy (e.g., hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), or thrombotic thrombocytopenic purpura (TTP)), comprising administering an effective amount of a high-affinity monoclonal antibody or antigen-binding fragment thereof that binds to human MASP-3 and inhibits alternative pathway complement activation to treat thrombotic microangiopathy (e.g., hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), thrombotic thrombocytopenic purpura (TTP), or transplant-associated TMA (TA-TMA)) or reducing the development of thrombotic microangiopathy (e.g., hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), thrombotic thrombocytopenic purpura (TTP), or transplant-associated TMA). The risk of (TA-TMA) is, for example, that the antibody or its antigen-binding fragment 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.

[0491] G. The role of MASP-3 in asthma and treatment methods using MASP-3 inhibitory antibodies and optional combinations of MASP-2 inhibitors.

[0492] Asthma is a common chronic inflammatory disease of the respiratory tract. Approximately 25 million people in the United States have asthma, including 7 million children under the age of 18, more than half of whom experience at least one asthma attack per year, resulting in more than 1.7 million emergency room visits and 450,000 hospitalizations annually (world-wide web accessed May 4, 2012 at gov / health / prof / lung / asthma / naci / asthma-info / index.htm). The disease is atypical, with multiple clinical phenotypes. The most common phenotype is allergic asthma. Other phenotypes include non-allergic asthma, aspirin-exacerbated respiratory disease, post-infectious asthma, occupational asthma, airborne irritant-induced asthma, and exercise-induced asthma. Key characteristics of allergic asthma include airway hyperresponsiveness (AHR) to a variety of specific and non-specific stimuli, excessive airway mucus production, increased pulmonary eosinophils, and elevated serum IgE levels. Symptoms of asthma include cough, wheezing, chest tightness, and shortness of breath. The goal of asthma treatment is to control the disease and minimize its severity, daily symptoms, and physical activity. Current treatment guidelines recommend a step-by-step approach until asthma control is achieved. The first step is to use a rapid-acting inhaled beta-2 agonist as needed, followed by controller medications such as inhaled corticosteroids, long-acting inhaled beta-2 agonists, leukotriene modifiers, theophylline, oral corticosteroids, and anti-IgE monoclonal antibodies.

[0493] Although the origin of asthma is multifactorial, it is generally believed to result from an inappropriate immune response to common environmental antigens in genetically susceptible individuals. Asthma is associated with complement activation, and anaphylactic toxins (AT) C3a and C5a possess pro-inflammatory and immunomodulatory properties, which are relevant to the development and regulation of anaphylactic responses (Zhang, X., and Kohl, ). J. Expert. Rev. Clin. Immunol (6:269-277, 2010). However, the relative involvement of the classical, alternative, and lectin complement pathways in asthma is not well understood. The alternative pathway can be activated on the surface of allergens, while the lectin pathway can be activated by recognizing allergen polysaccharide structures; both processes lead to the production of AT. Depending on the causative allergen involved, complement can be activated via different pathways. For example, highly allergenic grass pollen from the family Parietaria congena very effectively promotes MBL-dependent activation of C4, which involves LEA-2. Conversely, house dust mite allergens do not require MBL for complement activation (Varga et al.). Mol Immunol., 39(14):839-46, 2003).

[0494] Environmental triggers for asthma can activate complement via alternative pathways. For example, in vitro exposure of human serum to cigarette smoke or diesel exhaust particles leads to complement activation, an effect unaffected by the presence of EDTA, indicating activation via alternative pathways rather than the classical pathway (Robbins, RA et al., Am. J. Physiol. 260:L254-L259, 1991;Kanemitsu, H. et al., Biol. Pharm. Bull. 21:129-132, 1998). Evaluation of the role of the complement pathway in allergic respiratory tract inflammation in a mouse ovalbumin sensitization and challenge model. Wild-type mice develop AHR and respiratory tract inflammation in response to airborne allergen challenge. In a mouse ovalbumin model of allergic lung inflammation, the Crry-Ig fusion protein, which inhibits all pathways of complement activation, effectively prevented AHR and lung inflammation when administered systemically or via inhalation (Taube et al., 1998). Am J Respir Crit Care Med ., 168(11):1333-41, 2003).

[0495] Compared to wild-type mice, factor B-deficient mice showed less acute respiratory heart rate (AHR) and respiratory inflammation, while C4-deficient mice had similar effects to wild-type mice (Taube, C. et al.). Proc. Natl. Acad. Sci. USA 103:8084-8089, 2006). These results support the role of alternative pathways, rather than classical pathways, in a mouse model of airborne allergen challenge. In a model using the same mice, studies on factor H (FH) provide further evidence for the importance of alternative pathways (Takeda, K. et al., 2006). J. Immunol. 188:661-667, 2012). FH is a negative regulator of the alternative pathway, which acts to prevent self-damage to tissues. Endogenous FH has been found in the respiratory tract during allergen challenge, and inhibition of FH with recombinant competitive antagonists increased the severity of acute respiratory heart rate (AHR) and respiratory tract inflammation (Takeda et al., 2012, ibid.). Therapeutic delivery of CR2-fH (a chimeric protein linking the iC3b / C3d binding region of CR2 to the complement regulatory region of FH, which targets the complement regulatory activity of fH to existing complement activation sites) protects against the development of AHR and eosinophil infiltration into the respiratory tract after allergen challenge (Takeda et al., 2012, ibid.). This protective effect was demonstrated using ovalbumin and ragweed allergen (a human-associated allergen).

[0496] The role of lectin-dependent complement activation in asthma was evaluated in a mouse model of fungal asthma (Hogaboam et al.). J. Leukocyte Biol. 75(805-814, 2004). These studies used mannan-binding lectin A (MBL-A) genetically deficient mice, which are carbohydrate-binding proteins that act as recognition components for activating the lectin complement pathway. Using Aspergillus fumigatus conidia... it Mice sensitized with MBL-A (+ / +) and MBL-A (- / -) Aspergillus fumigatus were examined on days 4 and 28 post-conidial challenge. Compared to the sensitized MBL-A (+ / +) group, the acute respiratory rate (AHR) of sensitized MBL-A (- / -) mice was significantly reduced at both time points post-conidial challenge. Lung TH2 cytokine levels (IL-4, IL-5, and IL-13) were significantly lower in MBL-A (- / -) mice on day 4 post-conidial challenge compared to the wild-type group. These results suggest that the MBL-A and lectin pathways play major roles in the development and maintenance of AHR during chronic fungal asthma.

[0497] The findings detailed above demonstrate the involvement of lectin-dependent complement activation in the pathogenesis of asthma. Experimental data indicate that factor B activation plays a crucial role. Given the fundamental role of LEA-1 in the lectin-dependent activation of factor B and subsequent activation of the alternative pathway, LEA-1 blockers are expected to be beneficial in treating certain forms of asthma mediated by the alternative pathway. Therefore, such treatment could be particularly effective for asthma induced by house dust mites or environmental triggers such as cigarette smoke or diesel exhaust. On the other hand, asthma responses triggered by grass pollen may induce LEA-2-dependent complement activation. Therefore, LEA-2 blockers are expected to be particularly effective in treating asthma in this subgroup of patients.

[0498] Based on the data detailed above, the inventors believe that LEA-1 and LEA-2 mediate pathological complement activation in asthma. Depending on the sensitizing agent, either LEA-1 or LEA-2 may preferentially participate. Therefore, combinations of LEA-1 and LEA-2 blockers may be effective in treating various forms of asthma, regardless of the underlying etiology. LEA-1 and LEA-2 blockers may have complementary, additive, or synergistic effects in the prevention, treatment, or reversal of lung inflammation and asthma symptoms.

[0499] Combined LEA-1 and LEA-2 inhibition can be achieved by co-administration of a LEA-1 blocker and a LEA-2 blocker. Ideally, the LEA-1 and LEA-2 inhibitory function can be contained in a single molecular entity, such as a bispecific antibody containing specific binding sites for MASP-1 / 3 and MASP-2, or a bispecific antibody where each binding site can bind to and block either MASP-1 / 3 or MASP-2.

[0500] Based on the foregoing, one aspect of the present invention therefore provides a method for inhibiting LEA-1-dependent complement activation to treat, prevent, or reduce the severity of asthma, the method comprising administering to a subject suffering from or at risk of developing asthma a composition comprising a therapeutically effective amount of a LEA-1 inhibitor in a drug carrier, comprising a MASP-1 inhibitor, a MASP-3 inhibitor, or a combination of MASP-1 / 3 inhibitors. The MASP-1, MASP-3, or MASP-1 / 3 inhibitory composition may be administered systemically to the subject, for example, via intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous, or other parenteral administration, or, for non-peptide agents, oral administration. Administration may be determined by a physician and repeated until the condition is resolved or controlled.

[0501] In one embodiment, the method according to this aspect of the invention further includes inhibiting LEA-2-dependent complement activation to treat, prevent, or reduce the severity of asthma, said method comprising administering a therapeutically effective amount of a MASP-2 inhibitor and a MASP-1, MASP-3, or MASP-1 / 3 inhibitor to a subject suffering from or at risk of developing asthma. As detailed above, in treating or preventing or reducing the severity of asthma, a combination of pharmacological agents that each block LEA-1 and LEA-2 is expected to provide improved therapeutic outcomes compared to inhibiting LEA-1 alone. This outcome can be achieved, for example, by co-administering an antibody having LEA-1 blocking activity along with an antibody having LEA-2 blocking activity. In some embodiments, LEA-1- and LEA-2- blocking activities are combined into a single molecular entity, and said entity has combined LEA-1- and LEA-2- blocking activities. Such an entity may comprise or consist of a bispecific antibody, wherein 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 an entity could be composed of bispecific monoclonal antibodies, one of which has an antigen-binding site that specifically recognizes MASP-3, thus blocking LEA-1, while the second antigen-binding site specifically recognizes MASP-2, thus blocking LEA-2. Ideally, such an entity could be composed of bispecific monoclonal antibodies, one of which has an antigen-binding site that specifically recognizes both MASP-1 and MASP-3, thus blocking LEA-1, while the second antigen-binding site specifically recognizes MASP-2, thus blocking LEA-2.

[0502] MASP-2 inhibitors can be administered systemically, such as via intra-arterial, intravenous, intramuscular, inhalation, nasal, subcutaneous, or other parenteral administration, or, for non-peptide agents, oral administration. Administration may be repeated as determined by a physician until the condition is resolved or controlled.

[0503] The MASP-3 inhibitory composition and optionally the MASP-2 inhibitory composition of the present invention can be administered by a single application of the composition (e.g., a single composition containing both MASP-2 and MASP-3 inhibitors or bispecific or dual inhibitors, or by co-application of separate compositions), or by application in a limited sequence, to treat, prevent, or reduce the severity of asthma in subjects who have asthma or are at risk of developing asthma. Alternatively, the composition can be administered at periodic intervals, such as daily, every two weeks, weekly, every other week, monthly, or every two months, over a longer period of time to treat subjects in need.

[0504] As described in Examples 11-21 of this document, high-affinity MASP-3 inhibitory antibodies have been produced, which have therapeutic efficacy in inhibiting alternative pathways in AP-related diseases or conditions such as asthma.

[0505] Therefore, in one embodiment, the present invention provides a method for treating a subject suffering from or at risk of developing asthma, comprising administering an effective amount of a high-affinity monoclonal antibody or antigen-binding fragment thereof that binds to human MASP-3 and inhibits alternative pathway complement activation to treat asthma or reduce the risk of developing asthma, 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.

[0506] The role of H. MASP-3 in dense sedimentary disease and treatment methods using MASP-3 inhibitory antibodies and optional combinations of MASP-2 inhibitors.

[0507] Membranoproliferative glomerulonephritis (MPGN) is a kidney disease morphologically characterized by the proliferation of glomerular mesangial cells and thickening of the glomerular capillary walls due to the subendothelial extension of the glomerular mesangium. MPGN is classified as primary (also known as idiopathic) or secondary, with underlying diseases such as infectious diseases, systemic immune complex disorders, tumors, and chronic liver disease. Idiopathic MPGN includes three morphological types. Type I, or classic MPGN, is characterized by subendothelial deposition of immune complexes and activation of the classical complement pathway. Type II, or dense deposit disease (DDD), is characterized by additional intramembranous dense deposits. Type III is characterized by additional subepithelial deposits. Idiopathic MPGN is rare, accounting for approximately 4% to 7% of primary kidney causes of nephrotic syndrome (Alchi, B. and Jayne, D.). Pediatr. Nephrol. 25:1409-1418, 2010). MPGN primarily affects children and adolescents, and may present as nephrotic syndrome, acute nephritis syndrome, asymptomatic proteinuria and hematuria, or recurrent gross hematuria. Renal dysfunction occurs in most patients, and the disease has a slow, progressive course; approximately 40% of patients develop end-stage renal disease within 10 years of diagnosis (Alchi and Jayne, 2010, ibid.). Current treatment options include glucocorticoids, immunosuppressants, antiplatelet therapy, and plasma exchange.

[0508] DDD is diagnosed by immunofluorescence staining of renal biopsy, based on the absence of immunoglobulins and the presence of C3. Electron microscopy reveals characteristic dense osmophilic deposits along the glomerular basement membrane. DDD is caused by dysregulation of the complement replacement pathway (Sethi et al.). Clin J Am Soc Nephrol . 6(5):1009-17, 2011), which can be caused by many different mechanisms. The most common complement system abnormality in DDD is the presence of C3 nephritis factor, which is an autoantibody against the alternative pathway C3 convertase (C3bBb), which increases its half-life and thus the activation of this pathway (Smith, RJH et al., 6(5):1009-17, 2011). Mol. Immunol. 48:1604-1610, 2011). Other alternative pathway abnormalities include factor H autoantibodies that block factor H function, gain-of-function C3 mutations, and genetic defects in factor H (Smith et al., 2011, ibid.). Recent case reports have shown that eclizumab (anti-C5 monoclonal antibody) treatment was associated with improved ren...

Claims

1. An isolated antibody or antigen-binding fragment thereof that binds to MASP-3, comprising: (a) Heavy chain variable region comprising HC-CDR1 as shown in SEQ ID NO: 72 (SYGMS); HC-CDR2 as shown in SEQ ID NO: 74 (WINTYSGVPTYADDFKG); and HC-CDR3 as shown in SEQ ID NO: 76 (GGEAMDY); and (b) Light chain variable region comprising LC-CDR1 selected from SEQ ID NO: 153 (KSSQSLLDSDGKTYLN), SEQ ID NO: 261 (KSSQSLLDSEGKTYLN), SEQ ID NO: 262 (KSSQSLLDSAGKTYLN) and SEQ ID NO: 263 (KSSQSLLDSDAKTYLN); LC-CDR2 as shown in SEQ ID NO: 155 (LVSKLDS); and LC-CDR3 as shown in SEQ ID NO: 157 (WQGTHFPWT).

2. The antibody of claim 1 or its antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment is selected from humanized antibodies, chimeric antibodies, mouse antibodies and antigen-binding fragments of any of the above.

3. The antibody of claim 1 or its antigen-binding fragment thereof, wherein the antibody or its antigen-binding fragment is selected from single-chain antibodies, Fab fragments, Fab' fragments, F(ab')2 fragments, monovalent antibodies lacking hinge regions, and whole antibodies.

4. The antibody of claim 1 or its antigen-binding fragment, further comprising an immunoglobulin constant region.

5. The antibody of claim 1 or its antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment is humanized.

6. The antibody of claim 1 or its antigen-binding fragment, wherein the antibody binds to the serine protease domain of human MASP-3 with an affinity of less than 500 pM.

7. The isolated antibody or antigen-binding fragment thereof of any one of claims 1-6, wherein the LC-CDR1 is composed of SEQ ID NO:

153.

8. The isolated antibody or antigen-binding fragment thereof of any one of claims 1-6, wherein the LC-CDR1 is composed of SEQ ID NO:261 (KSSQSLLDSEGKTYLN).

9. The isolated antibody or antigen-binding fragment thereof of any one of claims 1-6, wherein the LC-CDR1 is composed of SEQ ID NO:262 (KSSQSLLDSAGKTYLN).

10. The isolated antibody or antigen-binding fragment thereof of any one of claims 1-6, wherein the LC-CDR1 is composed of SEQ ID NO:263 (KSSQSLLDSDAKTYLN).

11. The isolated antibody or antigen-binding fragment thereof of any one of claims 1-6, wherein the HC-CDR1 is composed of SEQ ID NO:72, the HC-CDR2 is composed of SEQ ID NO:74, the HC-CDR3 is composed of SEQ ID NO:76, the LC-CDR1 is composed of SEQ ID NO:153, the LC-CDR2 is composed of SEQ ID NO:155, and the LC-CDR3 is composed of SEQ ID NO:

157.

12. The isolated antibody or antigen-binding fragment thereof of any one of claims 1-6, wherein the HC-CDR1 is composed of SEQ ID NO:72, the HC-CDR2 is composed of SEQ ID NO:74, the HC-CDR3 is composed of SEQ ID NO:76, the LC-CDR1 is composed of SEQ ID NO:263, the LC-CDR2 is composed of SEQ ID NO:155, and the LC-CDR3 is composed of SEQ ID NO:

157.

13. The isolated antibody or antigen-binding fragment thereof of any one of claims 1-6, wherein the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NO:28, SEQ ID NO:251 and SEQ ID NO:

252.

14. The isolated antibody or antigen-binding fragment thereof of any one of claims 1-6, wherein the light chain variable region comprises an amino acid sequence selected from SEQ ID NO:43, SEQ ID NO:253 and SEQ ID NO:

279.

15. The isolated antibody or antigen-binding fragment thereof of any one of claims 1-6, comprising a heavy chain variable region selected from SEQ ID NO:28, SEQ ID NO:251 and SEQ ID NO:252 and a light chain variable region selected from SEQ ID NO:43, SEQ ID NO:253 and SEQ ID NO:

279.

16. The isolated antibody or antigen-binding fragment thereof of claim 15, wherein the heavy chain variable region is composed of SEQ ID NO:28 and the light chain variable region is composed of SEQ ID NO:

43.

17. The isolated antibody or antigen-binding fragment thereof of claim 15, wherein the heavy chain variable region is composed of SEQ ID NO:251 or SEQ ID NO:252, and the light chain variable region is composed of SEQ ID NO:

253.

18. The isolated antibody or antigen-binding fragment thereof of claim 15, wherein the heavy chain variable region is composed of SEQ ID NO:251 or SEQ ID NO:252, and the light chain variable region is composed of SEQ ID NO:

279.

19. An isolated nucleic acid molecule encoding a heavy chain variable region and a light chain variable region of an antibody or antigen-binding fragment of any one of claims 1-18.

20. A cloning or expression vector comprising one or more nucleic acid molecules of claim 19.

21. A host cell comprising one or more of the cloning or expression vectors of claim 20.

22. A method for generating the antibody or antigen-binding fragment of claim 1, comprising culturing the host cell of claim 21 and isolating the antibody or its antigen-binding fragment.

23. A composition comprising an antibody or antigen-binding fragment of any one of claims 1-18, and a pharmaceutically acceptable excipient.

24. Use of the antibody or antigen-binding fragment of any one of claims 1-18, or the composition of claim 20, in the preparation of a medicament for treating a disease or condition associated with MASP-3-dependent complement activation, said disease or condition being selected from paroxysmal nocturnal hemoglobinuria (PNH).

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