Compositions and methods for inhibiting MASP-1, MASP-2 and / or MASP-3 for treatment of paroxysmal nocturnal hemoglobinuria

Abstract

Methods and compositions are provided for inhibiting MASP-3-dependent complement activation in a subject suffering from paroxysmal nocturnal hemoglobinuria by administering to the subject a composition comprising an amount of a MASP-3 inhibitor in an amount effective to inhibit MASP-3-dependent complement activation. Methods and compositions for increasing red blood cell survival in a subject suffering from paroxysmal nocturnal hemoglobinuria are provided by administering to the subject a composition comprising an amount of at least one of a MASP-1 inhibitor and / or a MASP-3 inhibitor, the amount of the inhibitor being effective to increase red blood cell survival. A MASP-2 inhibitor and a MASP-1 inhibitor, a MASP-2 inhibitor and a MASP-3 inhibitor, a MASP-3 inhibitor and a MASP-1 inhibitor, or a MASP-1 inhibitor, a MASP-2 inhibitor and a MASP-3 inhibitor may be administered to the subject.

Description

[0001] This application is a divisional application. The original application was filed on April 5, 2013, with application number 201380029994.3 (PCT / US2013 / 035488), and the invention title was "Composition and method for treating paroxysmal nocturnal hemoglobinuria by inhibiting MASP-1, MASP-2 and / or MASP-3".

[0002] Cross-reference to related applications This application claims the benefit of application number 61 / 621,461, filed on April 6, 2012.

[0003] Declaration of sequence lists 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_0146_PCT_Sequence_20130327_ST25.txt. This text file is 85 KB in size; created on April 1, 2013; and submitted via EFS-Web along with the documents in this specification.

[0004] background 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, published in...). 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.

[0005] 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 complement-targeting drug approved 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.

[0006] 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.

[0007] 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.

[0008] 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).

[0009] 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.

[0010] 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, targeting different glycoconjugates despite overlap. This view is supported by recent reports that among the known lectins via 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 moiety, fibrin 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.

[0011] Serum concentrations of MBL are highly variable in healthy individuals, 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 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)).

[0012] 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 shown 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).

[0013] The enzymatic components C1r and C1s of the MASP-Cl complex share the same domain architecture (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.

[0014] 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)).

[0015] Several pieces of evidence suggest the existence of different 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.

[0016] The lectin pathway is generally considered to play a crucial role in host defense against infection in naïve hosts used in experiments for the first time. 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. During the apparent window of vulnerability, these symptoms are often apparent early in life due to reduced maternal antibody titers, but prior to the development of a complete antibody response profile. This syndrome is frequently 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.

[0017] Unlike the classical and lectin pathways, no initiator was found to perform 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, factor B, factor D, and properdin.

[0018] 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 a cause of 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 showed 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 that the classical and alternative pathways, as well as the role of the lectin pathway, in the pathogenesis of ischemia / reperfusion injury remain controversial (Riedermann, NC et al., ). Am. J. Pathol. 162 :363-367, 2003).

[0019] 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.

[0020] C3 belongs to a family of proteins with a very small number of 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 relatively 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.

[0021] 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)).

[0022] 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.

[0023] 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.

[0024] 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.

[0025] Brief On one hand, the present invention provides a method for inhibiting MASP-3-dependent complement activation in subjects suffering from paroxysmal nocturnal hemoglobinuria (PNH). The method includes administering the subject a composition containing an amount of a MASP-3 inhibitor, said inhibitor effectively inhibiting MASP-3-dependent complement activation. In some embodiments, the method further includes administering the subject a composition containing a MASP-2 inhibitor.

[0026] On the other hand, the present invention provides a pharmaceutical composition comprising at least one inhibitor and a pharmaceutically acceptable carrier, wherein the at least one inhibitor comprises a MASP-2 inhibitor and a MASP-3 inhibitor.

[0027] On the other hand, the present invention provides a pharmaceutical composition comprising a MASP-3 inhibitor that binds to a portion of MASP-1 (SEQ ID NO:10: full length) and also to a portion of MASP-3 (SEQ ID NO:8) and a pharmaceutical carrier.

[0028] On the other hand, the present invention provides a pharmaceutical composition comprising a MASP-3 inhibitor that binds to a portion of MASP-2 (SEQ ID NO:5: full length) and also to a portion of MASP-3 (SEQ ID NO:8) and a pharmaceutical carrier.

[0029] On the other hand, the present invention provides a pharmaceutical composition comprising a MASP-3 inhibitor that binds to a portion of MASP-1 (SEQ ID NO:10: full length) and also to a portion of MASP-2 (SEQ ID NO:5) and a pharmaceutical carrier.

[0030] On the other hand, the present invention provides a pharmaceutical composition comprising a MASP-3 inhibitor that binds to a portion of MASP-1 (SEQ ID NO: 10 full length), a portion of MASP-2 (SEQ ID NO: 5: full length), and a portion of MASP-3 (SEQ ID NO: 8), and a pharmaceutical carrier.

[0031] As described herein, the pharmaceutical compositions of the present invention can be used according to the methods of the present invention.

[0032] These and other aspects and embodiments of the invention described herein will become apparent from the following detailed description and accompanying drawings. All U.S. patents, U.S. patent applications, U.S. patents, 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.

[0033] Attached Figure Description The foregoing aspects and many incidental advantages of the invention will be more readily understood by referring to the following detailed description of the invention in conjunction with the accompanying drawings, in which: Figure 1 This demonstrates a new understanding of the lectin pathway and alternative pathways; Figure 2 It is adapted from Schwaeble et al. Immunobiol The schematic diagram in 205:455-466 (2002) is by Yongqing et al. BBA Modified 1824:253 (2012), indicating the protein domains of MASP-2 and MAp19 and the exons encoding them; Figure 3 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 indicate the protein domains of MASP-1, MASP-3 and MAp44 and the exons encoding them; Figure 4 The amino acid sequences of MASP-1, MASP-2, and MASP-3 proteins are aligned and their common regions are indicated. Figure 5 The amino acid sequences of the MASP-1, MASP-2, and MASP-3 α chains are shown in the comparison. Figure 6 The amino acid sequences of the MASP-1, MASP-2, and MASP-3 β chains are shown in the alignment. Figure 7A The amino acid sequences of the MASP-1 and MASP-2 protease domains (β-chains) are shown in a contrastive pair. Figure 7B The amino acid sequences of the MASP-1 and MASP-3 protease domains (β-chains) are shown in a contrastive pair. Figure 7C The amino acid sequences of the MASP-2 and MASP-3 protease domains (β-chains) are shown in a contrastive pair. Figure 8 This is the Kaplan-Mayer curve, illustrated in the figure, showing 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 demonstrated that MASP-2 deficient mice were protected from Neisseria meningitidis-induced death, as described in Example 1. Figure 9 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 serogroup B strain MC58, demonstrating that MASP-2 deficient mice are protected from Neisseria meningitidis-induced death, as described in Example 1. Figure 10 The figure shows log cfu / mL of Neisseria meningitidis MC58 recovered per mL of blood from MASP-2 KO and WT mice at different time points after infection with 6 x 10⁶ CFU of Neisseria meningitidis MC58 ip (n=3, at different time points for both groups of mice), demonstrating 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; Figure 11 The figure shows the mean disease scores of MASP-2 KO and WT mice at 3, 6, 12 and 24 hours after infection with 6x106 CFU Neisseria meningitidis sera B strain MC58, demonstrating that MASP-2-deficient mice showed much lower disease scores at 6, 12 and 24 hours after infection compared with WT mice, as described in Example 1; Figure 12 The figure shows the percentage survival rate of mice that were given 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. Figure 13 The figure shows the log cfu / mL count of viable Neisseria meningitidis serogroup B strain MC58 collected at different time points after incubation with Neisseria meningitidis serogroup B strain MC58, as described in Example 3. Figure 14 The figure 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 7, 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; Figure 15The figure shows the log cfu / mL viable count of Neisseria meningitidis serum group B-MC58 recovered from mouse serum samples at different time points as shown in Table 9. It shows that MASP-2 - / - knockout mouse serum (referred to as "MASP-2 - / -") has a higher level of bactericidal activity against Neisseria meningitidis compared with WT mouse serum, while MASP-1 / 3 - / - mouse serum has no bactericidal activity as described in Example 3. Figure 16 The figure illustrates 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; Figure 17 The figure illustrates 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, the level of alternative pathway-driven (AP-driven) C3b deposition on a yeast polysaccharide-coated microtiter plate varied with serum concentration as described in Example 4; Figure 18 The figure illustrates the results of testing in 10% of human serum samples from MASP-3-deficient, C4-deficient, and MBL-deficient human subjects under "conventional" AP-specific conditions (i.e., BBS / EGTA / Mg). ++ No Ca ++ The changes in AP-driven C3b deposition levels over time on a yeast polysaccharide-coated microtiter plate are as described in Example 4. Figure 19A The figure illustrates 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 C3b deposition level on the mannan-coated microtiter plate varied with serum concentration as described in Example 4. Figure 19B The figure illustrates 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 C3b deposition level on the yeast polysaccharide-coated microtiter plate varied with serum concentration as described in Example 4. Figure 19C The figure illustrates 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 the influence of Streptococcus pneumoniae ( S. pneumoniae The level of C3b deposition on the D39-coated microtiter plate varies with serum concentration, as described in Example 4; Figure 20A The diagram illustrates 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. Figure 20B The diagram illustrates 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; Figure 20C The diagram illustrates 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. Figure 21 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 was measured (by releasing hemoglobin from the dissolved mouse erythrocytes (Crry / C3- / -) into a supernatant, which was measured by photometric method), as described in Example 5; Figure 22 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 was measured (by releasing hemoglobin from the dissolved mouse erythrocytes (Crry / C3- / -) into a supernatant, which was measured by photometric method), as described in Example 5; Figure 23 The figure illustrates 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) was measured by releasing hemoglobin from dissolved WT mouse erythrocytes into a supernatant measured by photometric method, as described in Example 5; Figure 24 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 virus, non-coated mouse erythrocytes are hemolyzed (measured by releasing hemoglobin from the lysed mouse erythrocytes (CD55 / 59- / -) into a supernatant, which is measured by photometric method), as described in Example 5; Figure 25 The figure shows 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 (measured by releasing the hemoglobin of the dissolved rabbit erythrocytes into a supernatant, which is measured by photometric method), as described in Example 6; Figure 26The figure illustrates the change in C3b deposition level (OD 405 nm) on a yeast polysaccharide-coated microtiter plate with serum concentration in serum samples from D factor- / -, MASP-2- / -, and WT mice under AP-specific conditions, as described in Example 7. Figure 27 The diagram illustrates the physiological conditions (in Ca) ++ In the C3 deposition assay performed (in the presence of ... Figure 28 The diagram illustrates the physiological conditions (in Ca) ++ In the C3b deposition assay performed in the presence of MASP-2 monoclonal antibodies, the C3b deposition level (OD 405 nm) on a yeast polysaccharide-coated microtiter plate in mouse serum samples obtained from factor D- / -; factor B- / -; plus and minus MASP-2 monoclonal antibodies changed with serum incubation time (minutes) as described in Example 7. Figure 29A The figure illustrates the in vitro determination of lectin pathway-specific C4b deposition on a yeast polysaccharide-coated microtiter plate at different time points after subcutaneous administration of 0.3 mg / kg or 1.0 mg / kg mouse MASP-2 MoAb, as described in Example 13; Figure 29B The figure illustrates the time progression of lectin pathway recovery within 3 weeks following a single intraperitoneal administration of 0.6 mg / kg MASP-2 MoAb to mice, as described in Example 13; Figure 30A This is the FACS histogram of MASP-3 antigen / antibody binding for clone M3J5, as described in Example 15; Figure 30B This is the FACS histogram of MASP-3 antigen / antibody binding for clone M3M1, as described in Example 15; Figure 31 The figure shows the saturation binding curve of clone M3J5 (clone 5) for the MASP-3 antigen, as described in Example 15; Figure 32A 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 15. Figure 32B 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 dot indicates the amino acid identity with the DT40 sequence and the horizontal bar indicates the introduction of a vacancy to maximize the comparison, as described in Example 15. Figure 33 The bar chart shows the inhibitory activity of mAb1E10 in the MBL pathway in the Wieslab complement system screening compared to the positive serum provided in the assay kit and the isotype control antibody. It demonstrates that mAb1E10 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 15. Figure 34 The figure shows the C3b deposition levels in 1% normal human serum plus allotype control, SGMI-1Fc, or SGMI-2Fc at concentrations ranging from 0.15 to 1000 nM, demonstrating that both SGMI-1Fc and SGMI-2Fc inhibit C3b deposition from normal serum in mannan-coated ELISA wells, with an IC50 value of [missing value]. 50 The values ​​were approximately 27 nM and 300 nM, respectively, as described in Example 16; Figure 35A Provides heat-inactivated Staphylococcus aureus ( Staphylococcus aureus Flow cytometry analysis of C3b deposition on [a specific cell line] demonstrated 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). [Further details regarding Mg2+ and Mg2+ are needed for accurate translation.] ++ Alternative pathway-driven C3b deposition was observed in normal human serum treated with / EGTA (Figure 2), and as shown in Figures 3, 4 and 5, no alternative pathway-driven C3b deposition was observed in serum depleted of factor B, factor D and factor P, respectively, as described in Example 17. Figure 35B Flow cytometry analysis of C3b deposition on heat-inactivated Staphylococcus aureus provides evidence that, as in EDTA-treated normal serum (Figure 1), C3b deposition in 3MC serum is significantly higher than that in Mg2+. ++ In the presence of EGTA, AP-driven C3b deposition was absent (Figure 3), while Figures 4 and 5 show that AP-driven C3b deposition in both active full-length rMASP-3 (Figure 4) and active rMASP-3 (CCP1-CCP2-SP) (Figure 5) in 3MC serum was restored to normal levels. ++The levels observed in normal serum treated with / EGTA (Figure 2) were not restored by inactive rMASP-3 (S679A) (Figure 6) or wild-type rMASP-1 (Figure 7) in 3MC serum, as described in Example 17; Figure 36 This shows the results of Western blot analysis of factor B cleavage in 3MC serum in response to Staphylococcus aureus, with or without rMASP-3, demonstrating the effect relative to Mg ++ Compared to 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 little factor B cleavage, as further shown in lane 3. 3MC serum showed significantly lower levels of Mg... ++ 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 17; Figure 37 Coomassie staining of the protein gel is shown, in which factor B cleavage was analyzed, demonstrating that factor B cleavage is optimal in the presence of C3, MASP-3, and pro-factor D (lane 1); as shown in lanes 4 and 5, factor B cleavage can be mediated by either MASP-3 alone or pro-factor D alone, as long as C3 is present, as described in Example 17. Figure 38 The figure shows 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 against mAb concentration in 3MC serum in the presence of rMASP-3, demonstrating that mAbD14 inhibits MASP-3-dependent C3b deposition in a concentration-dependent manner, as described in Example 17; Figure 39 Western blot analysis of pre-D factor substrate cleavage is shown, wherein full-length wild-type recombinant MASP-3 (lane 2) and MASP-1 (lane 5) completely or partially cleaved pre-D factor to produce mature D factor, as described in Example 18, compared to pre-D factor alone (lane 1) or inactive full-length recombinant MASP-3 (S679A; lane 3) or MASP-1 (S646A; lane 4); Figure 40The results are Western blots showing that, compared with a control response containing only MASP-3 and pre-D factor (no mAb, lane 1) and a control response containing mAb derived from the DTLacO library (which binds to MASP-1 but not to MASP-3) (lane 4), the MASP-3 binding mAbs D14 (lane 2) and M3M1 (lane 3) exhibited inhibitory activity against MASP-3-dependent pre-D factor cleavage, as described in Example 18. Figure 41 The figure illustrates the variation of AP-driven C3b deposition levels with serum concentration in serum samples from MASP-3-deficient (3MC), C4-deficient, and MBL-deficient subjects on a yeast-polysaccharide-coated microtiter plate. This demonstrates 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 with 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 19; Figure 42 The figure illustrates the results of testing in 10% of human serum samples from MASP-3-deficient, C4-deficient, and MBL-deficient human subjects under "conventional" AP-specific conditions (i.e., BBS / EGTA / Mg). ++ No Ca ++ The changes in AP-driven C3b deposition levels over time on yeast polysaccharide-coated microtiter plates, as described in Example 19; Figure 43 The figure illustrates the concentration of Ca in serum from two normal human subjects (NHS) and two patients with 3MC (Patient 2 and Patient 3). ++ A series of serum concentrations of mannan-coated rabbit erythrocyte hemolysis percentages were measured in the absence of MASP-3 (measured by releasing hemoglobin from lysed rabbit erythrocytes into a supernatant, which was measured photometrically), demonstrating that MASP-3 deficiency reduced the percentage of complement-mediated mannan-coated erythrocyte lysis compared to normal human serum, as described in Example 19; Figure 44 The figure illustrates the AP-driven C3b deposition levels on a yeast polysaccharide-coated microtiter plate as a function of the amount of protein added to a human 3MC patient 2 (MASP-3). - / - The change in the concentration of recombinant full-length MASP-3 protein in serum samples demonstrated that, compared with the negative control inactive recombinant MASP-3 (MASP-3A; S679A), active recombinant MASP-3 protein reconstituted AP-driven C3b deposition on yeast glycan-coated plates in a concentration-dependent manner, as described in Example 19. Figure 45 The diagram illustrates the situation in Ca. ++ The percentage of hemolysis of mannan-coated rabbit erythrocytes in a series of serum concentrations in the absence of recombinant MASP-3 was measured (by releasing the hemoglobin of the dissolved 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); demonstrating that the percentage of hemolysis of rabbit erythrocytes in 3MC serum containing rMASP-3 was significantly increased (p=0.0006) compared to the percentage of hemolysis in 3MC serum without recombinant MASP-3, as described in Example 19; Figure 46 The figure illustrates the effects of 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) demonstrated that the percentage of rabbit erythrocyte lysis increased in a concentration-dependent manner with increasing amounts of recombinant MASP-3, as described in Example 19; and Figure 47 The figure illustrates the variation of LEA-2-driven C3b deposition levels on mannan-coated ELISA plates with human serum concentrations diluted in BBS buffer for serum from normal human subjects (NHS), two 3MC patients (patient 2 and patient 3), the parents of patient 3, and subjects with MBL-deficient ELISA.

[0034] Sequence List Description SEQ ID NO:1 Human MAp19 cDNA SEQ ID NO:2 Human MAp19 protein (with leader sequence) SEQ ID NO:3 Human MAp19 protein (mature) SEQ ID NO:4 human MASP-2 cDNA SEQ ID NO:5 Human MASP-2 protein (with leader sequence) SEQ ID NO:6 Human MASP-2 protein (mature) SEQ ID NO:7 human MASP-3 cDNA SEQ ID NO:8 Human MASP-3 protein (w / leader sequence) SEQ ID NO:9 human MASP-1 cDNA SEQ ID NO:10 Human MASP-1 protein (w / leader sequence) SEQ ID NO:11 Human MAp44 protein (w / leader sequence) SEQ ID NO:12 Rat MASP-2 cDNA SEQ ID NO:13 Rat MASP-2 protein (with leader sequence) SEQ ID NO:14 encodes DNA of the heavy chain variable region (VH) (without signal peptide) of 17D20_dc35VH21N11VL (OMS646). SEQ ID NO:15 17D20_dc35VH21N11VL (OMS646) Heavy Chain Variable Region (VH) Peptide SEQ ID NO:16 17N16mc heavy chain variable region (VH) polypeptide SEQ ID NO:17 17D20_dc21N11VL (OMS644) Light Chain Variable Region (VL) Peptide SEQ ID NO:18 encodes DNA of the 17N16_dc17N9 (OMS641) light chain variable region (VL) (no signal peptide). SEQ ID NO:19 17N16_dc17N9 (OMS641) light chain variable region (VL) polypeptide SEQ ID NO:20: scFv daughter clone 17N16m_d17N9 full-length polypeptide SEQ ID NO:21: scFv subclone 17D20m_d3521N11 full-length polypeptide SEQ ID NO:22: scFv subclone 17N16m_d17N9 DNA encoding a full-length polypeptide (signal peptide-free) SEQ ID NO:23: scFv subclone 17D20m_d3521N11 DNA encoding a full-length polypeptide (signal peptide-free) SEQ ID NO:24: Parental DTLacO heavy chain variable region (VH) polypeptide SEQ ID NO:25: MASP-3 specific clone M3J5 heavy chain variable region (VH) peptide SEQ ID NO:26: MASP-3 specific clone M3M1 heavy chain variable region (VH) polypeptide SEQ ID NO:27: Parental DTLacO light chain variable region (VL) polypeptide SEQ ID NO:28: MASP-3 specific clone M3J5 light chain variable region (VL) polypeptide SEQ ID NO:29: MASP-3 specific clone M3M1 light chain variable region (VL) polypeptide SEQ ID NO:30: MASP-3 clone D14 heavy chain variable region (VH) polypeptide SEQ ID NO:31: MASP-3 clone D14 light chain variable region (VL) polypeptide SEQ ID NO:32: MASP-1 clone 1E10 heavy chain variable region (VH) polypeptide SEQ ID NO:33: MASP-1 clone 1E10 light chain variable region (VL) polypeptide SEQ ID NO:34 SGMI-1 peptide SEQ ID NO:35 SGMI-2 peptide SEQ ID NO:36 Human IgG1-Fc polypeptide; SEQ ID NO:37 Peptide adapter #1 (12aa); SEQ ID NO:38: Peptide linker #2 (10aa); SEQ ID NO:39: Nucleic acid encoding a polypeptide fusion containing human IL-2 signal sequence, SGMI-1, adapter #1 and human IgG1-Fc; SEQ ID NO:40: A mature polypeptide fusion (SGMI-1Fc) containing SGMI-1, linker #1 and human IgG1-Fc; SEQ ID NO:41: Nucleic acid encoding a polypeptide fusion containing human IL-2 signal sequence, SGMI-2, adapter #1 and human IgG1-Fc; SEQ ID NO:42: A mature polypeptide fusion (SGMI-2Fc) containing SGMI-2, linker #1 and human IgG1-Fc. Invention Details I. Definition 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.

[0036] 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.

[0037] As used in this article, lectin pathway effector branch 2 (“LEA-2”) refers to MASP-2-dependent complement activation.

[0038] The term “MASP-3-dependent complement activation” as used in this paper comprises two parts: (i) MASP-3-dependent activation of factor B and factor D lectins, which includes LEA-1-mediated complement activation, in Ca ++ When present, this typically leads to the conversion of C3bB to C3bBb and the conversion of the pre-D factor to the D factor; and (ii) lectin-independent conversions of the B and D factors, which can occur in Ca ++ In their absence, C3bB typically transforms into C3bBb and pre-D factor into D factor. LEA-1-mediated complement activation and lectin-independent conversion of factors B and 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 associate and bind in close proximity.

[0039] The term “MASP-2-dependent complement activation” as used herein 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.

[0040] As used herein, the term "conventional understanding of alternative pathways," also referred to as "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)

[0041] 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.

[0042] The term "classical pathway" as used in this article refers to complement activation triggered by the binding of an antibody to an exogenous particle and requiring the binding of the recognition molecule C1q.

[0043] The term "HTRA-1" used in this article refers to the high-temperature requirement of serine peptidase for serine protease A1.

[0044] As used herein, the term "MASP-3 inhibitor" refers to any agent that directly or indirectly 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 MASP-3 (SEQ ID NO:8) with a binding affinity at least 10 times that of other components in the complement system. In one embodiment, the MASP-3 inhibitor indirectly inhibits MASP-3 activity, for example, as an MASP-3 activation inhibitor, 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 another embodiment, the MASP-3 inhibitor inhibits MASP-3-mediated factor D maturation. In yet another embodiment, the MASP-3 inhibitor inhibits MASP-3-mediated factor B activation. The MASP-3 inhibitor used in the methods 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 implementation, 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). MASP-3 inhibition is expected to 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.

[0045] As used herein, the term "MASP-1 inhibitor" refers to any agent that binds to or directly interacts with MASP-1 and inhibits at least one of the following: (i) MASP-3-dependent complement activation and / or (ii) MASP-2-dependent complement activation and / or (iii) lectin-independent or lectin-dependent MASP-1-mediated maturation of factor D, wherein lectin-dependent MASP-1 maturation of factor D involves direct activation of factor D, including MASP-1 antibodies and 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 another recognition molecule (e.g., MBL, CL-11, H-fibrinogen, M-fibrinogen, or L-fibrinogen) in the lectin pathway. In one embodiment, the MASP-1 inhibitor used in the method of the present invention reduces 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-1 inhibitor reduces MASP-3-dependent complement activation by more than 90% (i.e., resulting in MASP-3 complement activation of only 10% or less). In another embodiment, the MASP-1 inhibitor used in the method of the present invention reduces MASP-2-dependent complement activation by more than 10%, for example, more than 20%, more than 50%, or more than 90%. In one embodiment, the MASP-1 inhibitor reduces MASP-2-dependent complement activation by more than 90% (i.e., resulting in MASP-2 complement activation of only 10% or less).

[0046] In another embodiment, the MASP-1 inhibitor used in the method of the present invention reduces MASP-3-dependent complement activation (LEA-1), lectin-independent conversion of factors B and D, and MASP-2-dependent complement activation (LEA-2) by greater than 10%, for example, greater than 20%, greater than 50%, or greater than 90%. In one embodiment, the MASP-1 inhibitor reduces MASP-3-dependent complement activation (LEA-1), lectin-independent conversion of factors B and D, and MASP-2-dependent complement activation (LEA-2) by greater than 90% (i.e., resulting in MASP-3 complement activation of only 10% or less and MASP-2 complement activation of only 10% or less).

[0047] Examples of direct MASP-1 inhibitors are MASP-1-specific inhibitors, such as MASP-1 inhibitors that specifically bind to a portion of MASP-1 (SEQ ID NO: 10) with a binding affinity at least 10 times greater than other components of the complement system. In many cases, given that MASP-1 can activate MASP-3, and given that MASP-1 can activate MASP-2, inhibition of MASP-1 is expected to effectively inhibit MASP-3 and / or MASP-2. However, in some cases, inhibition of either MASP-1 or MASP-3 or MASP-2 may be a preferred embodiment, relative to inhibition of other MASP targets. For example, in the case of Staphylococcus aureus infection, MASP-3 has been shown to be activated and responsible for opsonization of Staphylococcus aureus in the absence of MASP-1 (see Iwaki D. et al.). J Immunol 187(7):3751-8 (2011)). Therefore, in the treatment of, for example, paroxysmal nocturnal hemoglobinuria (PNH), direct inhibition of MASP-1 instead of MASP-3 may be advantageous, thereby reducing potential susceptibility to Staphylococcus aureus during LEA-1-inhibitory treatment of PNH.

[0048] As used herein, the term "MASP-2 inhibitor" refers to any agent that binds to or directly interacts with MASP-2 and inhibits at least one of the following: (i) MASP-2-dependent complement activation and / or (ii) MASP-1-dependent complement activation, including MASP-2 antibodies and their MASP-2-binding fragments, natural and synthetic peptides, 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. MASP-2 inhibitors used in the methods of this invention can reduce MASP-2-dependent complement activation by more than 10%, for example, more than 20%, more than 50%, or more than 90%. In one embodiment, the MASP-2 inhibitor reduces MASP-2-dependent complement activation by more than 90% (i.e., resulting in MASP-2 complement activation of only 10% or less). Examples of direct MASP-2 inhibitors are MASP-2-specific inhibitors, such as MASP-2 inhibitors that specifically bind to a portion of MASP-2 (SEQ ID NO:5) with a binding affinity at least 10 times that of other components in the complement system.

[0049] 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 and multispecific antibodies (such as bispecific and 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 (e.g., dAb, Fab, Fab', F(ab')2, Fv), single chains (ScFv), synthetic variants thereof, naturally occurring variants thereof, fusion proteins including antibody portions of antigen-binding fragments with desired specificity, humanized antibodies, chimeric antibodies, and any other modified conformations of immunoglobulin molecules containing antigen-binding sites or fragments (epitope recognition sites) with desired specificity.

[0050] "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 according to 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".

[0051] As used herein, the term "antibody fragment" refers to a portion of a full-length antibody (e.g., an antibody derived from, or involving, MASP-1, MASP-2, or MASP-3), generally including 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.

[0052] The “single-chain Fv” or “scFv” antibody fragments used in this article comprise the VH and VL domains of the antibody, which are located on a single polypeptide chain. Typically, Fv polypeptides also include a polypeptide linker between the VH and VL domains, enabling scFv to form the desired antigen-binding structure.

[0053] The “chimeric antibody” used in this article is a recombinant protein containing variable domains and complementarity-determining regions derived from antibodies from non-human species (such as rodents), while the rest of the antibody molecule is derived from human antibodies.

[0054] The term "humanized antibody" as used in this article refers to a chimeric antibody containing a minimal sequence conforming to a specific complementarity-determining region (CDR) derived from a non-human immunoglobulin, which is embedded within a human antibody framework. Humanized antibodies are typically recombinant proteins (including antibodies generated from phage display or yeast) in which only the antibody CDR is of non-human origin.

[0055] The term “mannan-binding lectin” (“MBL”) used in this article is equivalent to mannan-binding protein (“MBP”).

[0056] The “membrane attack complex” (“MAC”) used in this article refers to a complex (also known as C5b-9) that inserts into and disrupts the terminal five complement components (C5b, C6, C7, C8, and C9) of the membrane.

[0057] The “subjects” used in this article include all mammals, including but not limited to humans, non-human primates, dogs, cats, horses, sheep, goats, cattle, rabbits, pigs, and rodents.

[0058] The abbreviations for the amino acid residues used in this article 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).

[0059] 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.

[0060] The term “conservative amino acid substitution” as used in this article is explained 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.

[0061] As used herein, the term "oligonucleotide" refers to oligomers or polymers of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or their analogues. The term also includes oligonucleotide bases composed of naturally occurring nucleotides, sugars, and internucleotide (backbone) covalent bonds, as well as oligonucleotides with non-naturally occurring modifications.

[0062] As used in this article, an "epitope" refers to 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.

[0063] 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 proteins (MASP-1, MASP-2, or MASP-3) described herein may contain or be wild-type proteins, or may be variants 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.

[0064] The human MASP-1 protein (as shown in SEQ ID NO:10), human MASP-2 protein (as shown in SEQ ID NO:5), and human MASP-3 protein (as shown in SEQ ID NO:8) described herein also include “peptide fragments” of the proteins that are shorter than the full-length and / or immature (pre-pro) MASP proteins. The included MASP protein peptide fragments include the protein's terminals and internal deletion variants. Deletion variants may lack (two or more amino acids) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid segments or discontinuous single amino acids. In some embodiments, the human MASP-1 protein may have an amino acid sequence that is equal to or greater than 70% (e.g., 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) of the amino acid sequence given in SEQ ID NO: 10.

[0065] In some embodiments, the human MASP-3 protein may have an amino acid sequence that is equal to or greater than 70% (e.g., 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) of the amino acid sequence given in SEQ ID NO: 8.

[0066] In some embodiments, the human MASP-2 protein may have an amino acid sequence that is equal to or greater than 70% (e.g., 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) of the amino acid sequence given in SEQ ID NO: 5.

[0067] In some implementations, the peptide fragment may be at least 6 [units]. (e.g., at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, or 600 or more) amino acid residues (e.g., SEQ ID NO: (At least 6 consecutive amino acid residues of any one of 5, 8, or 10). In some embodiments, the length of the antigenic peptide fragment of the human MASP protein is less than 500. (e.g., fewer than 450, 400, 350, 325, 300, 275, 250, 225, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6) amino acid residues (e.g., SEQ ID NO) NO: Less than 500 consecutive amino acid residues of any one of 5, 8 or 10.

[0068] In some embodiments, when generating antibodies that bind to MASP-1, MASP-2, and / or 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).

[0069] 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.

[0070] In representative embodiments, the human MASP-1 protein (SEQ ID NO:10) is encoded by the cDNA sequence given in SEQ ID NO:9; the human MASP-2 protein (SEQ ID NO:5) is encoded by the cDNA sequence given in SEQ ID NO:4; and the human MASP-3 protein (SEQ ID NO:8) is encoded by the cDNA sequence given in SEQ ID NO:7. Those skilled in the art will recognize that the cDNA sequences disclosed in SEQ ID NO:9, SEQ ID NO:4, and SEQ ID NO:7 represent individual alleles of human MASP-1, MASP-2, and MASP-3, respectively, and allelic variations and alternative splicing are expected. Allelic variants of the nucleotide sequences shown in SEQ ID NO:9, SEQ ID NO:4, and SEQ ID NO:7, 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-1, MASP-2, or MASP-3 sequences can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures, or can be identified by homology comparison searches (e.g., BLAST searches) using databases containing said information.

[0071] II. The lectin pathway: A new understanding i. Overview: The lectin pathway has been redefined As described herein, the inventors have made a surprising discovery: the complement lectin pathway has two effector branches that activate complement, both driven by a lectin pathway activation complex 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 cytolytic and / or opsonizing effects.

[0072] 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-D factor to D factor. Therefore, inhibition of MASP-3 can simultaneously inhibit the lectin-independent activation of LEA-1 and factors B and / or D, which can lead to inhibition of cell lysis and / or opsonization.

[0073] 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 zymogen to its active form and / or cleave C3b- or C3b(H2O)-bound factor B, resulting in the conversion of the C3bB zymogen complex to its enzymatically active form, C3bBb. The activated factor D produced by MASP-3 can also convert the C3bB or C3b(H2O) zymogen 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.

[0074] 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 36 and 37 ) and transforming the pre-D factor into the D factor (see Figure 39 MASP-1 (see also) Figure 39The 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.

[0075] 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-D-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 crucial role in opsonization, labeling the microbial surface for destruction by host phagocytic cells (e.g., macrophages). As an example in this paper (Figure 35), MASP-3 is essential for opsonization in Staphylococcus aureus. C3b deposition on Staphylococcus aureus exposed to human serum occurs rapidly in a MASP-3-dependent manner (Figure 35).

[0076] 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 generates the alternative pathway C5 convertase, C3bBb (C3b). n As further described in this article, in the cytolysis of Neisseria meningitidis (see... Figure 13 , 14 The need for MASP-3 and MBL instead of MASP-2 (and, therefore, LEA-2 in this instance) in 15) demonstrates 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 demonstrate that both opsonization and cytolysis can originate from the conversion of C3bB or C3b(H2O) and / or the conversion of pre-D factor to D factor; therefore, these two processes may be the result of lectin-independent effects of MASP-3, MASP-1, or HTRA-1. Therefore, in Figure 1The 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.

[0077] 1. Lectin pathway effector branch (LEA-1) 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 demonstrate 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.

[0078] 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., cytolytic 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 conversion of MASP-3-mediated C3bB to C3bBb and / or the conversion of pre-D factor to D factor. 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 be formed. 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 37 It has been demonstrated that the alternative pathway C3 convertase proenzyme 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.

[0079] 2. Lectin pathway effector branch (LEA-2) 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 and C5 convertase sites, which ultimately leads to massive MAC formation and cell lysis. This latter event is mediated by the activation of factor D 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. A MASP-2-dependent C4 bypass activation pathway also exists, activating C3 in the absence of C4. This 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.

[0080] 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 20A , 20B (20°C), but it also plays a role in the 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.

[0081] 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 enzymatically active alternative pathway C3 and C5 convertases C3bBb and C3bBb(C3b)n, respectively. 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.

[0082] 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: i) LEA-1: A MASP-3-dependent activation pathway that initiates and drives complement activation by generating the alternative pathway invertase C3bBb through initial cleavage and activation of factor B on the activator surface, subsequently catalyzing C3b deposition and the formation of the alternative pathway invertase 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 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.

[0083] 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 that resist complement cell lysis. These include most Gram-positive bacteria.

[0084] 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.

[0085] While 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 degree of cell lysis.

[0086] ii. Background of MASP-1, MASP-2 and MASP-3 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 Med 176:1497-1502 (1992); Ji et al. J. Immunol150:571-578 (1993)).

[0087] 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)).

[0088] 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)).

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 1. Their respective structures, sequences, chromosomal locations, and splicing variants. Figure 2 This is a schematic diagram showing the domain structures of the MASP-2 polypeptide (SEQ ID NO:5) and the MAp19 polypeptide (SEQ ID NO:2) and the exons encoding them. Figure 3 This is a schematic diagram showing the domain structures of the MASP-1 polypeptide (SEQ ID NO:10), MASP-3 polypeptide (SEQ ID NO:8), and MAp44 polypeptide (SEQ ID NO:11) and the exons encoding them. Figure 2 and 3 As shown, the serine proteases MASP-1, MASP-2, 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.

[0093] 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. Immunol152:2308-2316 (1994); cDNA-derived amino acid sequences of human, mouse, and rat MASP-2 (Thiel et al., Nature 386:506-510 (1997); Endo et al., J Immunol 161:4924-30 (1998); Stover et al., J. Immunol 162:3481-3490 (1999); Stover et al., J. Immunol 163:6848-6859 (1999)); and the cDNA-derived amino acid sequence of human MASP-3 (Dahl et al., Immunity 15:127-135 (2001) indicates 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; mouse MASP-1: BAA03944; rat MASP-1: AJ457084; human MASP-3: AAK84071; mouse MASP-3: AB049755, as accessed in Genbank on 2 / 15 / 2012, each of which is incorporated herein by reference).

[0094] Further as Figure 2 and 3 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.

[0095] 2. MASP-1 / 3 Human MASP-1 polypeptide (SEQ ID NO:10) and MASP-3 polypeptide (SEQ ID NO:8) 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 generated 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 3 As shown, the human MASP-1 gene comprises 18 exons. The human MASP-1 cDNA (as given in SEQ ID NO:9) is encoded by exons 2, 3, 4, 5, 6, 7, 8, 10, 11, 13, 14, 15, 16, 17, and 18. Further as... Figure 3 As shown, the human MASP3 gene comprises 12 exons. The human MASP-3 cDNA (as given in SEQ ID NO:7) 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”) (as given in SEQ ID NO:11), derived from exons 2, 3, 4, 5, 6, 7, 8, and 9.

[0096] The human MASP-1 polypeptide (SEQ ID NO: 10 from Genbank BAA04477.1) has 699 amino acid residues, including a 19-residue leader peptide. With the leader peptide omitted, the calculated molecular weight of MASP-1 is 76,976 Da. Figure 3 As shown, the MASP-1 amino acid sequence contains four N-linked glycosylation sites. The human MASP-1 protein domain (see SEQ ID NO: 10) is shown below. Figure 3 It also includes an N-terminal C1r / C1s / sea urchin VEFG / bone morphogenetic protein (CUBI) domain (SEQ ID NO:10, aa 25-137), an epidermal growth factor-like domain (SEQ ID NO:10, aa 139-181), a second CUB domain (CUBII) (SEQ ID NO:10, aa 185-296), and tandem complement control protein (SEQ ID NO:10, CCP1aa 301-363 and CCP2aa 367-432) domain and a serine protease domain (SEQ ID NO:10, aa 449-694).

[0097] The human MASP-3 polypeptide (SEQ ID NO:8, from Genbank AAK84071) has 728 amino acid residues, including a 19-residue leader peptide. When the leader peptide is omitted, the calculated molecular weight of MASP-3 is 81,873 Da. Figure 3As shown, MASP-3 has 7 N-linked glycosylation sites. The human MASP-3 protein domain (see SEQ ID NO:8) is shown below. Figure 3 It includes an N-terminal C1r / C1s / sea urchin VEGF / bone morphogenetic protein (CUBI) domain (SEQ ID NO:8, aa 25-137), an epidermal growth factor-like domain (SEQ ID NO:8, aa 139-181), a second CUB domain (CUBII) (SEQ ID NO:8, aa 185-296), and tandem complement control protein (SEQ ID NO:8, CCP1, aa 301-363 and CCP2, aa 367-432) domain and a serine protease domain (SEQ ID NO:8, aa 450-711).

[0098] The MASP-3 translation product consists of an α chain (heavy chain) (α chain: aa 1-448 of SEQ ID NO:8) and a light chain (β chain: aa 449-728 of SEQ ID NO:8); 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 and MASP-1.

[0099] 3. MASP-2 The human MASP-2 gene is located on chromosome 1p36.3-2 (Stover et al., Cytogenet and Cell Genet 84:148-149 (1999) and includes 12 exons, such as Figure 2 As shown. MASP-2 (SEQ ID NO:5) and MAp19 (SEQ ID NO:2) are encoded by transcripts of single structural genes generated through alternative splicing / polyadenylation (Stover et al., Genes and Immunity 2:119-127 (2001)). Human MASP-2 cDNA (SEQ ID NO:4) is encoded by exons 2, 3, 4, 6, 7, 8, 9, 10, 11, and 12. A 20 kDa protein, called MBL-associated protein 19 ("MAp19", also known as "sMAP") (SEQ ID NO:2), is encoded by (SEQ ID NO:1) and is derived from exons 2, 3, 4, and 5. MAp19 is a non-enzymatic protein containing the N-terminal CUB1-EGF region of MASP-2 and four additional residues (EQSL) derived from exon 5, such as... Figure 2 As shown.

[0100] The MASP-2 polypeptide (SEQ ID NO:5) has 686 amino acid residues, including a 15-residue leader peptide that is cleaved after secretion to produce the mature form of human MASP-2 (SEQ ID NO:6). Figure 2 As shown, the MASP-2 amino acid sequence does not contain any N-linked glycosylation sites. The MASP-2 polypeptide has a molecular structure similar to MASP-1, MASP-3, and C1r and C1s (proteases of the C1 complement system). The human MASP-2 protein domains (as per SEQ ID NO:5) are shown below. Figure 2 It includes an N-terminal C1r / C1s / sea urchin VEGF / bone morphogenetic protein (CUBI) domain (SEQ ID NO:5, aa 24-136), an epidermal growth factor-like domain (SEQ ID NO:5, aa 138-180), a second CUB domain (CUBII) (SEQ ID NO:5, aa 184-295), and tandem complement control protein (SEQ ID NO:5, CCP1, aa 300-359 and CCP2, aa 364-431) domain and a serine protease domain (SEQ ID NO:5, aa 445-682).

[0101] like Figure 2 As shown, the MASP-2 peptide has an α-chain (heavy chain) (α-chain: aa 1-443 of SEQ ID NO:5) containing a CUB-1-EGF-CUB-2-CCP-1-CCP-2 domain and a β-chain (light chain) (β-chain: aa444-686) containing a serine protease domain. The CUB-1, EGF, and CUB-2 domains are required for dimerization, and the CUB-1, EGF, CUB-2, and CCP-1 domains contain MBP binding sites. As Wallis et al., J. Biol Chem As described in 279:14065-14073 (2004), each MASP-2 dimer binds to two MBL subunits.

[0102] 4. Comparison of the amino acid sequences of MASP-1, MASP-2, and MASP-3 Figure 4 This is an amino acid alignment of the protein sequences of MASP-1 (SEQ ID NO:10), MASP-2 (SEQ ID NO:6), and MASP-3 (SEQ ID NO:8), showing conserved catalytic triplet residues (H, D, S) in the CUBI, EGF, CUBII, CCP1, CCP2 domains and the serine protease (SP) domain. The symbol "." indicates the same amino acid sequence.

[0103] Figure 5 This is an amino acid alignment of the α-chain sequences of MASP-1 (α-chain: aa1-447 of SEQ ID NO:10), MASP-2 (α-chain: aa1-443 of SEQ ID NO:5), and MASP-3 (α-chain: aa1-448 of SEQ ID NO:8), including CUBI-EGF-CUBII-CCP1-CCP2. Multiple identity patches exist within the CUBI, EGF, and CUBII domains, such as... Figure 5 The dashed boxes in the table are shown. The CCP1 and CCP2 domains are represented by black shaded boxes. The overall % identity between the α chains of human MASP1 / 3 and human MASP-2 is provided in Table 1 below.

[0104] Figure 6 This is an amino acid alignment of the β-chain sequences (including the serine protease domain) of MASP-1 (β-chain: aa 448-699 of SEQ ID NO:10), MASP-2 (β-chain: aa 444-686 of SEQ ID NO:5), and MASP-3 (β-chain: aa 449-728 of SEQ ID NO:8). Figure 7A Paired amino acid alignments between the β chain sequences of MASP-1 (β chain: aa 448-699 of SEQ ID NO:10) and MASP-2 (β chain: aa 444-686 of SEQ ID NO:5) are shown. Figure 7B Paired amino acid alignments between the β chain sequences of MASP-1 (β chain: aa 448-699 of SEQ ID NO:10) and MASP-3 (β chain: aa 449-728 of SEQ ID NO:8) are shown. Figure 7C Paired amino acid alignments between the β chain sequences of MASP-2 (β chain: aa 444-686 of SEQ ID NO:5) and MASP-3 (β chain: aa 449-728 of SEQ ID NO:8) are shown. Figure 5 The identity region in -7 is shown as a dashed box around the same amino acid (displayed as a "." symbol).

[0105] The percentage identity between the α and β chains of human MASP-1, MASP-2, and MASP-3 proteins is provided in Table 1 below.

[0106] Table 1: Percentage Identities Among Human MASP Proteins For the α chain (heavy chain), as shown in Table 1 above, the α chains of MASP-1 and MASP-3 are identical (except for the 15 amino acid sequences at the 3' end). The overall % identity between the α chains of MASP-2 and MASP-3 is 45.4%, with multiple identity complements in the CUBI-EGF-CUBII domain, such as... Figure 5 As shown.

[0107] For the β chain (light chain), the overall percentage identity among the three β chains is low, ranging from 27% to 28%. However, despite the low overall identity among the three β chains, there are several identity complements, such as... Figure 6 As shown. Further as... Figure 7A As shown in -C, the same complements in the sequence are more widely distributed between MASP-2 and 3 than between 1 and 2 or between 1 and 3.

[0108] All cysteine ​​residues present in MASP-2, MASP-3, C1r, and C1s were compared with their counterparts in MASP-1; however, MASP-1 has two cysteine ​​residues (positions 465 and 481 in the L chain) that are absent in MASP-2, MASP-3, C1r, and C1s. These two cysteine ​​residues in MASP-1, at their expected positions, are used to form a histidine-ring disulfide bridge, as seen in trypsin and chymotrypsin. This suggests that MASP-2, MASP-3, C1r, and C1s may have evolved from MASP-1 through gene duplication and divergence (Nonaka & Miyazawa). Genome Biology 3 Reviews1001.1-1001.5 (2001)).

[0109] 5. Their respective biological functions / activities, including relevant human genetic data. The role of the MBL / fibrin-MASP complex in innate immunity is mediated via the following binding pathways: calcium-dependent binding via the C-type lectin domain (present in the MBL molecule) to carbohydrate structures present on yeast, bacteria, viruses, and fungi, or binding via the fibrinogen-like domain (present in the fibrin molecule) to carbohydrate structures present on yeast, bacteria, viruses, and fungi. This recognition phase leads to activation of the proenzyme MASP-2, which subsequently mimics the action of activated C1s within the C1q-(C1r)2-(C1s)2 complex, forming the C3 convertase C4b2b by cleaving C4 and C2. This allows C4b and C3b to deposit on target pathogens and thus promote killing and clearance through phagocytosis.

[0110] Recent literature suggests that the lectin pathway activation complex requires only MASP-2 activity to cleave both C4 and C2: i) Reconstruction of the minimal lectin-pathway activation complex using recombinant MBL and recombinant MASP-2 is clearly sufficient to efficiently cleave both C4 and C2 in vitro (Vorup-Jensen et al., J. Immunol 165:2093-2100 (2000); Rossi et al., J Biol Chem 276:40880-40887 (2001); Ambrus et al., J Immunol 170:1374-1382 (2003); Gál et al., J Biol Chem 280:33435-33444 (2005); ii) mouse serum with MASP-2 gene-targeting deficiency lacks any lectin pathway functional activity (Schwaeble et al., PNAS 108:7523-7528 (2011)). Genetically determined defects in MASP-2 have recently been described (Stengaard-Pedersen et al., New Eng. J. Med. 349:554-560, (2003)). Single nucleotide mutations result in an Asp-Gly exchange in the CUB1 domain and prevent MASP-2 from binding to MBL.

[0111] Furthermore, functional characterization of serum from mice lacking both MASP-1 and MASP-3 showed that, under physiological conditions, comparisons between wild-type and MASP-1 / MASP-3 knockout mice (MASP-1 / 3...) revealed... - / - In mouse serum, the activity of the lectin pathway is slowed, but not eliminated (Takahashi et al., J. Immunol 180:6132-6138 (2008); Schwaeble et al., PNAS (2011)). These studies indicate that, unlike the classical pathway effector endopeptidase C1s, MASP-2 activation does not necessarily involve or require the activity of any other MBL-associated serine endopeptidases (i.e., MASP-1 or MASP-3), and that the proteolytic activity of MASP-2 is sufficient to convert the binding of carbohydrate recognition molecules (i.e., MBL, fibrinogen, or CL-11) in the lectin pathway into complement activation. However, recent studies have demonstrated that although MASP-2 possesses self-activation capabilities, the catalytic rate of MASP-1 activation of the MASP-2 prozymogen exceeds the cleavage rate of its own prozymogen form of MASP-2 by approximately 85,000 times (Héja et al., 2011). PNAS 106:10498-503 (2011); Megyeri et al., J. Biol. Chem. 288(13):8922-34 (2013)). Therefore, in a physiological environment, it is likely that the major activator of MASP-2 is MASP-1. Judging from the size of the resulting C4 fragment and the resulting functional C3 convertase activity, it seems likely that activated MASP-2 cleaves C4 and C2 in the same manner as activated C1s, i.e., at a single argininoyl bond in the α-chain of C4 (Arg76 Ala77) and at a single argininoyl bond in the proenzyme chain of C2 (Arg223 Lys224). It has also been reported that mouse MASP (in the form of the mouse MBL-MASP complex, referred to as Ra-reactive factor), unlike C1s, can cleave the α-chain of complement component C3 to yield the bioactive fragments C3a and C3b (Ogata et al., J. Immunol 154:2351-2357 (1995)). If this were to occur in the human system, it would require cleaving a single argininoyl bond (Arg77 Ser78) within the α-chain of C3. Activation of MASP-2, as with activation of C1s, prevents the cleavage of complement component C5. The proteolytic activity of MASP-1 and MASP-2 is inhibited by C1 inhibitors (Matsushita et al., J Immunol 165:2637-2642 (2000), while C1-inhibitors do not react with MASP-3 (Dahl et al., Immunity 15:127-135 (2001); Zundel et al., J Immunol 172:4342-4350 (2004)).

[0112] The biological functions of MASP-1 and MASP-3 have been slowly uncovered. The substrate specificity and physiological role of MASP-1 have been a subject of debate since its discovery. In recent years, a large number of potential substrates have been identified. It has been proposed that MASP-1 can slowly cleave native C3, and that this direct cleavage of C3 may initiate a complement cascade, perhaps via an alternative pathway (Matsushita et al., J Immunol 165:2637-2642 (2000)). It was subsequently demonstrated that recombinant MASP-1 cleaves the inactive (thioester-hydrolyzed) form of C3, which is futile for initiating the complement cascade (Ambrus et al., J Immunol 170:1374-1382 (2003)). The lack of lectin pathway activity in serum dilutions of MASP-2-deficient mice clearly demonstrates the absence of the MASP-1-driven C3-bypass mechanism (Schwaeble et al., PNAS108:7523-7528 (2011)). The complement component cleaved by MASP-1 with significant efficiency is C2 (Rossi et al., J Biol Chem 276:40880-40887 (2001); Ambrus et al., J Immunol 170:1374-1382 (2003)) and the proenzyme form of factor D (Takahashi et al., J Exp Med 207:29-37 (2010)). Just as with the ability of MASP-1 to cleave C2, it seems possible that MASP-1 could therefore amplify the C3-convertase (C4b2a)-forming ability of MASP-2 via C2 cleavage. This suggestion is supported by the view that the activity of the lectin pathway is reduced in MASP-1-depleted human serum and in MASP-1 / 3-deficient mouse serum (Takahashi et al., J Immunol 180:6132-6138 (2008)), the viewpoint also suggests that MASP-1 plays a role in the activation of MASP-2. Furthermore, although each C4b deposited via the MBL-MASP complex can form a C4b2a convertase, only one of the four C4b deposited via the classical C1 complex can do so (Rawal et al., J Biol Chem 283 (12):7853-63 (2008)).

[0113] MASP-1 also cleaves MASP-2 and MASP-3 (Megyeri M., et al., J Biol Chem. 2013 Mar29; 288(13):8922-34). Recent experiments have shown that although MASP-2 can self-activate, MASP-1 is the major activator of proenzyme MASP-2. In the serum of MASP-1 knockout mice, the activation of MASP-2 is delayed (Takahashi et al., J Immunol 180: 6132-6138 (2008)), and similar results were observed in normal human serum when MASP-1 activity was blocked by specific inhibitors (Kocsis et al., J Immunol 185(7):4169-78 (2010)). Furthermore, Degn et al., ( J. Immunol. 189(8): 3957-69 (2012) found that in human serum, MASP-1 is crucial for the activation of MASP-2 and subsequent C4 cleavage. The catalytic rate of conversion of prozymogen MASP-2 to active MASP-2 is more than 85,000 times greater than the rate at which MASP-2 can be self-activated (Megyeri et al., J. Biol. Chem.288:8922–8934 (2013); Héja et al., J. Biol. Chem. 287(24):20290-300 (2012); Héja et al., PNAS 109:10498-503 (2012)).

[0114] Recent studies have also linked MASP-1 to alternative pathways. MASP-1 can convert prozymogen D into its enzymatically active form (…). Figure 39 Takahashi et al. J Exp Med 207:29-37 (2010)). Furthermore, MASP-1 activates the proenzyme form of MASP-3 (Megyeri et al., J. Biol. Chem. 288:8922–8934 (2013); Degn et al., J. Immunol. 189(8): 3957-69 (2012)), which itself can activate prozymogen D factor ( Figure 39 And cleaving factor B (another essential component of the alternative pathway) into its active form (Iwaki et al., J. Immunol. 187:3751-58(2011)). However, the conversion between pre-D and pre-B factors may be independent of the activation state of LEA-2 and can occur via non-complex-bound MASP-1.

[0115] Several pieces of evidence suggest that MASP-1 is a thrombin-like enzyme and plays an important role in the activation of the coagulation pathway. MASP-1 can cleave several substrates of thrombin, including fibrinogen (Hajela K. et al., Immunobiology 205(4-5):467-75 (2002)), Factor XIII (Krarup et al., Biochim Biophys Acta 1784(9):1294-1300(2008)) and protease-activated receptor 4 (PAR4) (Megyeri et al., J Immunol 183(5):3409-16(2009)). Furthermore, antithrombin in the presence of heparin is a more effective MASP-1 inhibitor than C1-inhibitors (Dobó et al., J Immunol 183:1207-1214 (2009)). The following observations also highlight the link between complement and the coagulation pathway: MASP-2 can activate prothrombin (Krarup A. et al., PLoS One2(7):e623 (2007)). Limited coagulation represents an ancient type of innate immunity when fibrin clots prevent the spread of invading pathogens. Released fibrin peptide B has pro-inflammatory activity. MASP-1-mediated PAR4 cleavage activates endothelial cells-initiated inflammatory responses (Megyeri et al., J Immunol 183(5):3409-16 (2009)).

[0116] MASP-3 has no proteolytic activity against C4, C2, or C3 substrates. Instead, MASP-3 has been 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).

[0117] 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 factors D circulate in their pro-proteolytic, inactive form, while in normal mouse serum, almost all factors D are in their active form. Biochemical analysis suggests that MASP-1 can convert complement factor D from its prozymogeneous form to its enzymatically active form ( ). Figure 39 Takahashi et al. JEM 207(1):29-37(2010)). MASP-3 also cleaves pre-D factor zymogen and produces active D factor in vitro. Figure 39 Takahashi et al. JEM207(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 36 Iwaki D. et al., J Immunol 187(7):3751-8(2011)).

[0118] 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.

[0119] The structures of the catalytic fragments of MASP-1 and MASP-2 have been determined by X-ray crystallography. Comparison of the MASP-1 protease domain with other complement proteases reveals 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, which 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, making the conversion between inactive and active forms possible. This flexibility reflected in the structure may play a role in the self-activation process.

[0120] Western 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, the large MASP-1 transcript was found to be 4.8 kb and the small one to be approximately 3.4 kb, both of which are present in human and mouse livers (Stover et al.). Genes Immunity 4:374-84 (2003)). MASP-2 mRNA (2.6 kb) and MAp19 mRNA (1.0 kb) are 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)).

[0121] 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 demonstrated 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)). Despite the mixed literature, the deficiency or ineffectiveness of MASP may have adverse effects on an individual's ability to develop direct, non-antibody-dependent defenses against certain pathogens.

[0122] iii. Supporting data for the new understanding, emphasizing the lack of Ca ++ Traditional determination conditions and uses include Ca ++ The result is obtained by setting more physiological conditions.

[0123] This article provides several independent and compelling experimental pieces of evidence indicating that the complement lectin pathway is activated through two independent effector mechanisms: i) LEA-2: a MASP-2-driven pathway that 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 surface of an activator 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 caused by MASP-1, MASP-3, or HTRA-1, or any combination of these three, can also lead to complement activation via the alternative pathway.

[0124] Alternative pathways, such as the lectin pathway-dependent MASP-3-driven activation, appear to facilitate well-established C3b-binding factor B-mediated factor D cleavage 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 14-15This 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 document, 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 8-12 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 8 and Figure 12 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 an 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 15 Recent papers (Takahashi M. et al., JEM 207: 29-37 (2010) and the work presented in this paper ( Figure 39 This study demonstrates that MASP-1 can convert the proenzyme form of factor D to its enzymatically active form and can partially explain the loss of cytolytic activity due to the absence of enzymatically active factor D in these serums. This does not explain the lack of bactericidal activity in MBL-deficient mice, which possess 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).

[0125] 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 13-14 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 higher 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 will be loaded with either MASP-1 or MASP-3. This is because activated MASP-3 simultaneously cleaves factor D in vitro (…). Figure 39 Factor B and its respective enzymatic active forms ( Figure 37 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).

[0126] Although the data on lectin-dependent effects are noteworthy, multiple experiments suggest that MASP-3 and MASP-1 are not necessarily functional in their complexes with lectin molecules. For example... Figure 35B 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 35A The study demonstrated that deposition under these conditions depended 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 39 ) and MASP-3-induced factor B activation ( Figure 37 This can occur in vitro in the absence of lectins. Finally, hemolysis studies of mouse erythrocytes in the presence of human serum demonstrated the clear role of both MBL and MASP-3 in cell lysis. However, a deficiency in MBL does not fully reproduce the severity of a deficiency in MASP-3, 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) demonstrated herein can be attributed solely to lectin-related functions.

[0127] 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 17-18 35B, 41-42, 45-46). 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 demonstrate that activated MASP-3 plays an important role in complement cytolytic activity. Figure 14-15 As shown in 21-23, 43-44 and 46-47, the terminal activation cascade of complement cytolysis is defective in the serum of 3MC patients who lack MASP-3 but not MASP-1. Figure 14 and 15 The data shown demonstrate the loss of lysing activity in the absence of MASP-3 and / or MASP-1 / MASP-3 functional activity. Similarly, the loss of hemolytic activity in MASP-3-deficient human serum ( Figure 21-23 (43-44 and 46-47), and the ability to reconstruct hemolysis by adding recombinant MASP-3 ( Figures 46-47 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.

[0128] 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.

[0129] III. The roles of MASP-2 and MASP-3 in paroxysmal nocturnal hemoglobinuria and treatment methods using MASP-2 and MASP-3 inhibitors i. Overview of PNH 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 occur in 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 of 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 precipitating factor.

[0130] 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.

[0131] 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.

[0132] 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. Med350(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 demonstrated that PNH patients receiving eculizumab contain a large number of C3 fragments bound to their PNH red blood cells (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 methods have already demonstrated their usefulness in experimental systems (Lindorfer et al., Blood 115:2283-91, 2010).

[0133] ii. Complement-Initiation Mechanism in PNH 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.

[0134] 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 demonstrated 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 lectins 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.

[0135] 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 governing 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, offering a significant advantage over the C5 inhibitor eculizumab.

[0136] 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 cytolysis, 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)).

[0137] 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 8 and 9 As shown, LEA-2 blockade via genetic ablation of MASP-2 did not decrease survival rates following Neisseria meningitidis infection. On the contrary, in these studies, LEA-2 blockade via MASP-2 ablation significantly improved survival rates. Figure 8 and 9 ) and disease score ( Figure 11 The same result was obtained by LEA-2 blockade induced by MASP-2 antibody administration. Figure 12In knockout mouse strains, secondary or compensatory effects that could be a cause were eliminated. These favorable results in animals subjected to LEA-2 ablation were associated with more rapid clearance of Neisseria meningitidis from the bloodstream. Figure 10 Additionally, as described in this article, incubating Neisseria meningitidis with normal human serum kills Neisseria meningitidis. Figure 13 Adding a human MASP-2-specific functional monoclonal antibody that blocks LEA-2, without administering an isotype control monoclonal antibody, can enhance the 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 13 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.

[0138] 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 the MASP-3 transcript; 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 14 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 15 Conversely, MASP-2-deficient serum showed effective killing of Neisseria meningitidis.

[0139] 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 cytolysis. However, the data do not necessarily limit LEA-2 to opsonization and LEA-1 to cytolysis, as both pathways can mediate opsonization and / or cytolysis in other cases.

[0140] 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 enhances LEA-1-dependent cytolytic destruction in vitro. Figure 15 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 cytolysis of Neisseria meningitidis. Since cytolysis of Neisseria meningitidis is the primary protective mechanism in the host where the experiment was first conducted, blocking LEA-2 in vivo increases the clearance of Neisseria meningitidis and leads to increased killing.

[0141] 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 conversion of C3bB to C3bBb and the conversion of pre-D factor to D factor, which can further promote PNH pathology.

[0142] iii. Biological and expected functional activities in PNH 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 treatment with C5-inhibitors such as eculizumab.

[0143] iv. MASP-2 inhibitors block opsonization and extravascular hemolysis of PNH RBCs through the reticuloendothelial system. 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. Treatment of patients with eculizumab significantly inhibits intravascular hemolysis. Because eculizumab blocks the terminal dissolution 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.

[0144] 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 19A Yeast polysaccharides, Figure 19B and Streptococcus pneumoniae; Figure 19C When 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 20A Further details are shown in -20C. 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 by 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 left 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. This concept is further supported by... Figure 28Support for the opsonization data shown: Compared to WT serum, factor D-deficient mouse serum (which lacks the ability to activate alternative pathways in the liquid phase but possesses functional classical pathways as well as functional LEA-1 and LEA-2 pathways) showed no deficiency in opsonization. Factor B-deficient serum (which lacks LEA-1) showed reduced opsonization, while treatment with the MASP-2 monoclonal antibody to block LEA-2-mediated complement activation in factor D-deficient serum resulted in a more potent inhibition of opsonization. Figure 28 Importantly, adding a MASP-2 monoclonal antibody to factor B-deficient serum is more effective in inhibiting opsonization than MASP-2 blockade or factor D blockade alone. Therefore, LEA-2 and LEA-1 act in an additive or synergistic manner to enhance 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.

[0145] v. The role of MASP-3 inhibitors in PNH 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 21 and 22 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 24 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 23 ).

[0146] 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 22 and Figure 23 Similar results were observed when using unsensitized normal RBCs: such as Figure 23 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.

[0147] 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 17 This 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, it is suggested 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.

[0148] vi. The role of MASP-2 inhibitors in PNH 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.

[0149] 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.

[0150] vii. LEA-2 inhibitors as adjuvant therapy for LEA-1 inhibitors or terminal complement blockers This article provides data detailing two pathogenesis mechanisms of RBC clearance and anemia in PNH, which can be targeted individually or in combination with 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.

[0151] There is ample documentation demonstrating that both intravascular and extravascular hemolytic mechanisms contribute to anemia in PNH patients (Risitano et al., Blood 113:4094-4100 (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.

[0152] 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 undergone 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.

[0153] 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 mechanisms related to RBC destruction in PNH and thus block all anemia symptoms in PNH.

[0154] viii. Use multiple bispecific or panspecific antibodies against LEA-1 and LEA-2 to treat PNH 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 comprise 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. Ideally, such entities could be 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.

[0155] IV. MASP inhibitors With the recognition that the complement lectin pathway consists of two main complement activation branches (LEA-1 and LEA-2) and that a lectin-independent complement activation branch also exists, it has become increasingly clear that there is a strong need to specifically inhibit one or more of these effector branches that lead to PNH-related pathology, without completely shutting down complement's immune defense capabilities (i.e., leaving the complete classical pathway intact). This would leave the complete C1q-dependent complement activation system intact for the processing of immune complexes and to aid in host defense against infection.

[0156] i. Compositions that inhibit LEA-1-mediated complement activation As described herein, the inventors have unexpectedly discovered that the activation of LEA-1, which leads to cell lysis, is MASP-3-dependent. As further described herein, under physiological conditions, MASP-3-dependent LEA-1 activation also promotes opsonization, thereby providing additional effects with LEA-2-mediated complement activation. As shown in Example 7, in Ca... ++ When it exists, a D factor is not needed because MASP-3 has a D factor. - / - LEA-1 activation can be driven in serum. MASP-3, MASP-1, and HTRA-1 can convert pre-D factor into active D factor. Similarly, MASP-3 activation appears to depend on MASP-1 in many cases because MASP-3 (unlike MASP-1 and MASP-2) is not a self-activating enzyme and cannot be converted to its active form without the help of MASP-1 (Zundel, S. et al.). J. Immunol. 172: 4342-4350 (2004); Megyeri et al., J. Biol. Chem 288:8922–8934 (2013). Because MASP-3 is not self-activating and requires MASP-1 activity in many cases to be converted to its enzymatically active form, the MASP-3-mediated activation of the alternative pathway C3 convertase C3Bb can be inhibited by targeting the pro-MASP-3 zymogen or already activated MASP-3, or by targeting the MASP-1-mediated activation of MASP-3, or by both of these, because in many cases, when MASP-1 functional activity is absent, MASP-3 remains in its pro-zymogen form and cannot drive LEA-1 by directly forming the alternative pathway C3 convertase (C3bBb).

[0157] Therefore, in one aspect of the invention, the preferred protein component targeted in the development of a therapeutic that specifically inhibits LEA-1 is an inhibitor of MASP-3 (including an inhibitor of MASP-1-mediated MASP-3 activation (e.g., a MASP-1 inhibitor that inhibits MASP-3 activation)).

[0158] Based on the foregoing, in one aspect, the present invention provides a method for inhibiting the adverse effects (i.e., hemolysis and opsonization) of LEA-1 in subjects suffering from PNH or at risk of developing PNH, comprising administering a pharmaceutical composition to the subject, the pharmaceutical composition comprising a certain amount of a MASP-3 inhibitor that effectively inhibits MASP-3-dependent complement activation and a pharmaceutically acceptable carrier.

[0159] In live subjects with PNH or at risk of developing PNH, a MASP-3 inhibitor is administered in an amount that effectively inhibits MASP-3-dependent complement activation. In practice of this aspect of the invention, representative MASP-3 inhibitors include: molecules that inhibit MASP-3 biological activity, including molecules that inhibit at least one or more of the following: MASP-3-dependent activation of factor B lectins, MASP-3-dependent activation of pre-D lectins, MASP-3-dependent and lectin-independent activation of factor B, and MASP-3-dependent and lectin-independent activation of pre-D (e.g., small molecule inhibitors, MASP-3 antibodies and fragments thereof, or blocking peptides that interact with or interfere with protein-protein interactions with MASP-3); and molecules that reduce MASP-3 expression (e.g., MASP-3 antisense nucleic acid molecules, MASP-3-specific RNAi molecules, and MASP-3 ribozymes). MASP-3 inhibitors can effectively block MASP-3 protein-protein interactions, interfere with MASP-3 dimerization or assembly, and block Ca2+. ++ By binding to or reducing the active site of the MASP-3 serine protease, or by decreasing MASP-3 protein expression, MASP-3 is prevented from activating LEA-1-mediated complement activation or lectin-independent complement activation. MASP-3 inhibitors can be used alone as primary therapy or as adjuvant therapy in combination with other therapeutic agents to enhance the therapeutic benefits of other treatments, as further described herein.

[0160] In one embodiment, the MASP-3 inhibitor specifically binds to a portion of MASP-3 (SEQ ID NO:8) with a binding affinity at least 10 times that of other components in the complement system. In another embodiment, the MASP-3 inhibitor specifically binds to a portion of MASP-3 (SEQ ID NO:8) with a binding affinity at least 100 times that of other components in the complement system. In one embodiment, the MASP-3 inhibitor specifically binds to the serine protease domain of MASP-3 (aa 450-711 of SEQ ID NO:8) and inhibits MASP-3-dependent complement activation, provided that the inhibitor does not bind to the serine protease domain of MASP-1 (SEQ ID NO:10) and it does not bind to the serine protease domain of MASP-2 (SEQ ID NO:5). In one embodiment, the MASP-3 inhibitor is a MASP-3 monoclonal antibody or a fragment thereof that specifically binds to MASP-3.

[0161] In another embodiment, the MASP-3 inhibitor specifically binds to a portion of MASP-1 (SEQ ID NO:10) with a binding affinity at least 10 times that of other components of the complement system and inhibits MASP-1-mediated activation of MASP-3. In another embodiment, the MASP-3 inhibitor specifically binds to a portion of MASP-1 (SEQ ID NO:10) with a binding affinity at least 100 times that of other components of the complement system (i.e., peptides or fragments thereof) and inhibits MASP-1-mediated activation of MASP-3. In some embodiments, the MASP-3 inhibitor specifically binds to the serine protease domain of MASP-1 (aa 449-694 of SEQ ID NO:10) and inhibits MASP-1-mediated activation of MASP-3, provided that the inhibitor does not bind to the serine protease domain of MASP-2 (SEQ ID NO:5) and it does not bind to the serine protease domain of MASP-3 (SEQ ID NO:8). In one embodiment, the MASP-3 inhibitor is a MASP-1 monoclonal antibody or a fragment thereof that specifically binds to MASP-1 and inhibits MASP-1-mediated activation of MASP-3. In some embodiments, the MASP-3 inhibitor bound to MASP-1 inhibits MASP-1-mediated activation of MASP-3 and further inhibits MASP-1-mediated maturation of factor D.

[0162] In another embodiment, the MASP-3 inhibitor binds to a portion of MASP-3 (SEQ ID NO:8) and also to a portion of MASP-1 (SEQ ID NO:10), provided that the inhibitor does not bind to MASP-2 (SEQ ID NO:5) or MAp19 (SEQ ID NO:3). In another embodiment, the MASP-3 inhibitor binds to a portion of MASP-3 (SEQ ID NO:8) and also to a portion of MASP-1 (SEQ ID NO:10), provided that the inhibitor does not bind to MASP-2 (SEQ ID NO:5) or MAp19 (SEQ ID NO:3). In one embodiment, the MASP-3 inhibitor binds to a portion of MASP-3 (SEQ ID NO:8) and also to a portion of MASP-1 (SEQ ID NO:10), provided that the inhibitor does not bind to MASP-2 (SEQ ID NO:5), MAp19 (SEQ ID NO:3), or MAp44 (SEQ ID NO:11), thereby allowing a lower effective dose to inhibit MASP-3-dependent complement activation due to the lack of binding to MAp44, which is present in high concentrations in human serum.

[0163] In one embodiment, the MASP-3 inhibitor is a dual MASP-1 / MASP-3 inhibitor that binds to an epitope within a conserved amino acid region between MASP-1 and MASP-3, such as the CUBI-CCP2 domain (aa 25-432 of SEQ ID NO: 10), as... Figure 3-5 As shown. In one embodiment, the MASP-3 inhibitor is a dual MASP-1 / MASP-3 inhibitor that binds to an epitope within a conserved amino acid region between MASP-1 and MASP-3, provided that the inhibitor does not bind to MAp44, such as the CCP domain (aa 367-432 of SEQ ID NO:10). In another embodiment, the MASP-3 inhibitor is a bispecific inhibitor, such as a bispecific monoclonal antibody, that specifically binds to an epitope on both the MASP-3 protein (SEQ ID NO:8) and the MASP-1 protein (SEQ ID NO:10). In some embodiments, the MASP-3 inhibitor is a bispecific monoclonal antibody that binds to the serine protease domain of MASP-1 (aa 449-694 of SEQ ID NO:10) and also to the serine protease domain of MASP-3 (aa 450-711 of SEQ ID NO:8).

[0164] The binding affinity of the MASP-3 inhibitor can be detected using a suitable binding assay.

[0165] The inhibition of MASP-3-dependent complement activation is characterized by at least one of the following changes in the components of the complement system that occur as a result of administration of a MASP-3 inhibitor according to the method of the present invention: inhibition of LEA-1-mediated complement activation (inhibition of hemolysis and / or opsonization); inhibition of factor B lectin-independent conversion; inhibition of factor D lectin-independent conversion; inhibition of MASP-3 serine protease substrate-specific cleavage; reduction of hemolysis (as determined, for example, as described in Example 5); or reduction of C3 cleavage and C3b deposition (as determined, for example, as described in Examples 4 and 11).

[0166] In some embodiments, the MASP-3 inhibitor selectively inhibits MASP-3-dependent complement activation (i.e., LEA-1-mediated complement activation and / or lectin-independent conversion of factor B and / or lectin-independent conversion of factor D), while leaving the fully functional C1q-dependent complement activation system intact.

[0167] In some embodiments, the MASP-3 inhibitor is an antibody or a fragment thereof, including a MASP-3 antibody and its MASP-3 binding fragment, a MASP-1 antibody and its fragment, a natural or synthetic peptide, or a small molecule. In some embodiments, the MASP-3 inhibitor is a small molecule protease inhibitor that is selective for MASP-1, or selective for MASP-3, or selective for both MASP-1 and MASP-3.

[0168] ii. Compositions that inhibit LEA-2 activation As described herein, LEA-2-mediated complement activation is MASP-2-dependent, leading to opsonization and / or cell lysis. Therefore, MASP-2 is a preferred target protein component in the development of therapeutics that specifically inhibit the LEA-2 lectin-dependent complement system. Several proteins have been shown to bind to or interfere with MASP-2 through protein-protein interactions. For example, MASP-2 is known to bind to lectin proteins MBL, H-fibrinogen and L-fibrinogen, and collagen lectin-11, forming calcium-dependent complexes with them. (Ma Y., et al.) J Innate Immun Epub Dec4 (2012). Each MASP-2 / lectin complex has been shown to activate complement via MASP-2-dependent cleavage of proteins C4 and C2 (Ikeda, K., et al.). J. Biol. Chem .262 :7451-7454, (1987); Matsushita, M., et al. J. Exp. Med. 176 :1497-2284, (2000); Matsushita, M., et al. J. Immunol. 168 :3502-3506, (2002)). Studies have shown that the CUB1-EGF domain of MASP-2 is essential for MASP-2 association with MBL (Thielens, NM, et al., ). J. Immunol. 166 :5068, (2001)). It has also been demonstrated that the CUB1EGFCUBII domain mediates the dimerization of MASP-2, which is essential for the formation of the active MBL complex (Wallis, R., et al., ). J. Biol. Chem.275 (30962-30969, 2000). Therefore, MASP-2 inhibitors that bind to or interfere with MASP-2 target regions known to be important for MASP-2-dependent complement activation can be identified.

[0169] Based on the foregoing, in one aspect, the present invention provides a method for inhibiting the adverse effects of LEA-2-mediated complement activation in subjects with PNH or at risk of developing PNH, comprising administering a pharmaceutical composition to the subject, the pharmaceutical composition comprising a certain amount of a MASP-2 inhibitor that effectively inhibits MASP-2-dependent complement activation and a pharmaceutically acceptable carrier.

[0170] In live subjects with PNH or at risk of developing PNH, a MASP-2 inhibitor is administered in an amount that effectively inhibits MASP-2-dependent LEA-2. In practice of this aspect of the invention, representative MASP-2 inhibitors include molecules that inhibit the biological activity of MASP-2 (e.g., small molecule inhibitors, MASP-2 antibodies, or blocking peptides that interact with or interfere with protein-protein interactions with MASP-2) and molecules that reduce MASP-2 expression (e.g., MASP-2 antisense nucleic acid molecules, MASP-2-specific RNAi molecules, and MASP-2 ribozymes), thereby blocking MASP-2 to prevent activation of LEA-2.

[0171] MASP-2 inhibitors can effectively block MASP-2 protein-protein interactions, interfere with MASP-2 dimerization or assembly, and block Ca2+ protein-protein interactions. ++ By binding and interfering with the active site of the MASP-2 serine protease, MASP-2 protein expression may be reduced, thereby preventing MASP-2 from activating LEA-2. MASP-2 inhibitors can be used alone as primary treatment or as adjunctive therapy in combination with other treatments to enhance the therapeutic benefits of other drugs, as further described herein.

[0172] In one embodiment, the MASP-2 inhibitor specifically binds to a portion of MASP-2 (SEQ ID NO:5) with a binding affinity at least 10 times that of other antigens of the complement system. In another embodiment, the MASP-2 inhibitor specifically binds to a portion of MASP-2 (SEQ ID NO:5) with a binding affinity at least 100 times that of other antigens of the complement system. In one embodiment, the MASP-2 inhibitor specifically binds to at least one of the following: (i) the CCP1-CCP2 domain (aa 300-431 of SEQ ID NO:5) or the serine protease domain of MASP-2 (aa 445-682 of SEQ ID NO:5) and inhibits MASP-2-dependent complement activation, provided that the inhibitor does not bind to the serine protease domain of MASP-1 (SEQ ID NO:10) and it does not bind to the serine protease domain of MASP-3 (SEQ ID NO:8). In one embodiment, the MASP-2 inhibitor is a MASP-2 monoclonal antibody or a fragment thereof that specifically binds to MASP-2.

[0173] The binding affinity of the MASP-2 inhibitor can be detected using a suitable binding assay.

[0174] The inhibition of MASP-2-dependent complement activation is characterized by at least one of the following changes in the components of the complement system that occur as a result of administering a MASP-2 inhibitor according to the method of the present invention: inhibition of the production or generation of MASP-2-dependent complement-activation-system products C4b, C3a, C5a and / or C5b-9 (MAC) (as determined, for example, as described in Example 2 of U.S. Patent No. 7,919,094), reduction of C4 cleavage and C4b deposition (as determined, for example, as described in Example 8 or Example 9), or reduction of C3 cleavage and C3b deposition (as determined, for example, as described in Example 11).

[0175] In some implementations, the MASP-2 inhibitor selectively inhibits MASP-2 complement activation (i.e., LEA-2), while leaving the fully functional C1q-dependent complement activation system intact.

[0176] In some embodiments, the MASP-2 inhibitor is an antibody or a fragment thereof, including a MASP-2 antibody and its MASP-2 binding fragment, a natural or synthetic peptide, or a small molecule. In some embodiments, the MASP-2 inhibitor is a small molecule protease inhibitor that is selective for MASP-2.

[0177] iii. Compositions that inhibit LEA-1-mediated complement activation and LEA-2-mediated complement activation On the other hand, the present invention provides a method for inhibiting the adverse effects of LEA-1 and the adverse effects of LEA-2 in subjects who have one or more aspects of PNH or are at risk of developing PNH.

[0178] In one embodiment, this aspect of the invention relates to a method of increasing red blood cell survival in a subject with PNH, comprising administering a composition to the subject comprising at least one of a MASP-1 inhibitor and / or a MASP-3 inhibitor that effectively increases red blood cell survival.

[0179] In one embodiment, the composition comprises a MASP-1 inhibitor. In one embodiment, the MASP-1 inhibitor inhibits MASP-3-mediated complement activation and also inhibits MASP-2-mediated complement activation.

[0180] In one embodiment, the composition comprises a MASP-3 inhibitor. In one embodiment, the MASP-3 inhibitor inhibits at least one of the following: MASP-3-dependent lectin activation of factor B; MASP-3-dependent lectin activation of factor D; MASP-3-dependent, lectin-independent activation of factor B; and / or MASP-3-dependent, lectin-independent activation of factor D.

[0181] In one embodiment, the composition comprises a MASP-1 inhibitor and a MASP-3 inhibitor.

[0182] In some embodiments, the method further includes administering the subject a composition comprising a MASP-2 inhibitor.

[0183] In another embodiment, this aspect of the invention includes administering a pharmaceutical composition to a subject suffering from PNH, the pharmaceutical composition comprising a certain amount of a MASP-2 inhibitor that effectively inhibits MASP-2-dependent complement activation and a certain amount of a MASP-3 inhibitor that effectively inhibits MASP-3-dependent complement activation and a pharmaceutically acceptable carrier.

[0184] In some embodiments, the composition comprises a single agent that inhibits both LEA-1 and LEA-2 (i.e., a dual MASP-2 / MASP-3 inhibitor, a dual MASP-1 / MASP-2 inhibitor, a bispecific MASP-2 / MASP-3 inhibitor, a bispecific MASP-1 / MASP-2 inhibitor, or a pan-MASP-1 / 2 / 3 inhibitor or a trispecific MASP-1 / 2 / 3 inhibitor). In some embodiments, the composition comprises a combination of LEA-1 and LEA-2 inhibitors, for example, a combination of a dual inhibitor plus a single inhibitor, a combination of a bispecific inhibitor plus a single inhibitor, or any combination of MASP-1, MASP-2, and / or MASP-3 inhibitors described herein that inhibits both LEA-1 and LEA-2, as further described herein.

[0185] In one embodiment, the present invention provides a pharmaceutical composition for inhibiting both LEA-1 and LEA-2, comprising at least one MASP-3 inhibitor and at least one MASP-2 inhibitor and a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition comprises a combination of a first molecule and a second molecule, the first molecule being a MASP-3 inhibitor and the second molecule being a MASP-2 inhibitor. In another embodiment, the pharmaceutical composition comprises a single molecular entity comprising activity as a MASP-3 inhibitor and activity as a MASP-2 inhibitor (i.e., an inhibitor of both MASP-2-mediated LEA-2 activation and MASP-3-mediated LEA-1 activation). In one embodiment, the inhibitor is a dual MASP-2 / MASP-3 inhibitor that binds to an epitope within a conserved amino acid region between MASP-2 (SEQ ID NO:5) and MASP-3 (SEQ ID NO:8), such as a serine protease domain, such as the N-terminal region of the β-chain (e.g., the first 150 aa of the N-terminal region of the β-chain in SEQ ID NO:5 and SEQ ID NO:8), as... Figure 4 , 6As shown in 7C. In one embodiment, the inhibitor is a bispecific inhibitor, such as a bispecific monoclonal antibody, which specifically binds to an epitope on the MASP-2 protein (SEQ ID NO:5) and an epitope on the MASP-3 protein (SEQ ID NO:8). In some embodiments, the inhibitor is a bispecific monoclonal antibody that binds to at least one of the CCP1-CCP2 domains of MASP-2 (aa 300-431 of SEQ ID NO:5) or the serine protease domain of MASP-2 (aa 445-682 of SEQ ID NO:5) and also binds to an epitope in the serine protease of MASP-3 (aa 450-711 of SEQ ID NO:8).

[0186] In another embodiment, the present invention provides a composition that inhibits both LEA-1 and LEA-2, comprising an inhibitor that inhibits both MASP-2-mediated LEA-2 activation and MASP-3 MASP-1-mediated activation, thereby inhibiting MASP-3-mediated LEA-1 activation (and optionally also inhibiting MASP-1-mediated D factor maturation). In one embodiment, the inhibitor is a dual MASP-1 / MASP-2 inhibitor that binds to an epitope within a conserved amino acid region between MASP-1 (SEQ ID NO:10) and MASP-2 (SEQ ID NO:5), such as a serine protease domain, as shown below. Figure 4 , 6 As shown in 7A. In one embodiment, the inhibitor is a bispecific inhibitor, such as a bispecific monoclonal antibody, which specifically binds to the epitopes of the MASP-1 protein (SEQ ID NO: 10) and the MASP-2 protein (SEQ ID NO: 5). In some embodiments, the inhibitor is a bispecific monoclonal antibody that binds to at least one of the serine protease domains of MASP-1 (aa 449-694 of SEQ ID NO: 10) and also to at least one of the CCP1-CCP2 domains of MASP-2 (aa 300-431 of SEQ ID NO: 5) or the serine protease domains of MASP-2 (aa 445-682 of SEQ ID NO: 5).

[0187] In another embodiment, the present invention provides a composition for inhibiting both LEA-1 and LEA-2, comprising an inhibitor that inhibits MASP-2-mediated LEA-2 activation, inhibits MASP-3-mediated LEA-1 activation by direct binding to MASP-3, and also inhibits MASP-3-mediated LEA-1 activation by inhibiting MASP-3 (and optionally also inhibits MASP-1-mediated D factor maturation). In one embodiment, the inhibitor is a pan-MASP inhibitor that binds to a conserved amino acid region between MASP-1 (SEQ ID NO:10), MASP-2 (SEQ ID NO:5), and MASP-3 (SEQ ID NO:8), such as a conserved region in the CUBI-EGF-CUB2 domain, as shown below. Figure 4 and 5 As shown. Figure 4 and 5 As shown, multiple identity complements shared by MASP-1, MASP-2, and MASP-3 exist within the CUBI-EGF-CUBII domain, thereby allowing the generation of pan-specific MASP antibodies. In some embodiments, the pan-specific MASP antibodies can bind to epitopes within the CUB2 domain of MASP-1 (aa 185-296 of SEQ ID NO: 10), the CUB2 domain of MASP-2 (aa 184-295 of SEQ ID NO: 5), and the CUB2 domain of MASP-3 (aa 185-296 of SEQ ID NO: 8). It should be noted that pan-specific MASP inhibitors binding to the CUBI-EGF of MASP-1, MASP-2, and MASP-3 also bind to MAp19 and MAp44; therefore, the effective therapeutic dose of such inhibitors will be adjusted to higher levels to compensate for this binding. It should also be noted that pan-specific MASP inhibitors that bind to the CUBII domains of MASP-1, MASP-2, and MASP-3 also bind to MAp44. Therefore, the effective therapeutic dose of these inhibitors will be adjusted to higher levels to compensate for this binding.

[0188] In one embodiment, the inhibitor is a trispecific MASP-1 / 2 / 3 inhibitor that binds to an epitope on the MASP-1 protein (SEQ ID NO:10), an epitope on the MASP-2 protein (SEQ ID NO:5), and an epitope on the MASP-3 protein (SEQ ID NO:8). In some embodiments, the inhibitor is a trispecific monoclonal antibody that binds to at least one of the serine protease domains of MASP-1 (aa 449-694 of SEQ ID NO:10), to at least one of the CCP1-CCP2 domains of MASP-2 (aa 300-431 of SEQ ID NO:5) or the serine protease domain of MASP-2 (aa 445-682 of SEQ ID NO:5), and also to an epitope on the MASP-3 serine protease (aa 450-711 of SEQ ID NO:8).

[0189] Exemplary inhibitors that inhibit LEA-1, LEA-2, or both LEA-1 and LEA-2 are described in Table 2 below.

[0190] Table 2: MASP Inhibitors *For the cross-reactivity column given in Table 2, the specified MASP inhibitor binds to the inhibitor-binding domain with a binding affinity at least 10 times (e.g., at least 20 times, at least 50 times, or at least 100 times) greater than other complement components (i.e., peptides or fragments thereof) listed as “not” binding.

[0191] In some embodiments, the composition comprises a combination of inhibitors of LEA-1 and LEA-2, such as combinations of single inhibitors as described above and in Table 2. For example, in one embodiment, the composition comprises a combination of MASP-1 and MASP-2 antibodies. In one embodiment, the composition comprises a combination of MASP-1 and MASP-3 antibodies. In one embodiment, the composition comprises a combination of MASP-2 and MASP-3 antibodies. In one embodiment, the composition comprises a combination of MASP-1, MASP-2, and MASP-3 antibodies. In some embodiments, the method of the present invention comprises administering a single composition comprising a combination of inhibitors. In other embodiments, the method of the present invention comprises co-administering separate compositions.

[0192] In some embodiments, the composition comprises a combination of a dual inhibitor plus a single inhibitor (i.e., a dual inhibitor of MASP-2 / 3 plus a MASP-1 inhibitor; a dual inhibitor of MASP-1 / 3 plus a MASP-2 inhibitor; or a dual inhibitor of MASP-1 / 2 plus a MASP-3 inhibitor). In other embodiments, the method of the present invention comprises co-administering separate compositions comprising a dual inhibitor and a single inhibitor.

[0193] In some embodiments, the composition comprises a combination of a bispecific inhibitor and a single inhibitor (i.e., a MASP-2 / 3 bispecific inhibitor plus a MASP-1 inhibitor; a MASP-1 / 3 bispecific inhibitor plus a MASP-2 inhibitor; or a MASP-1 / 2 bispecific inhibitor plus a MASP-3 inhibitor). In other embodiments, the method of the present invention comprises co-administering separate compositions comprising a bispecific inhibitor and a single inhibitor.

[0194] According to different embodiments of the present invention, it should be noted that, compared with C5 antibodies that must be localized to the site of action, MASP-3 inhibitors and / or MASP-2 inhibitors and / or MASP-1 inhibitors will be used to remove the target protein from the plasma.

[0195] V. MASP antibody In certain embodiments of this aspect of the invention, the MASP inhibitor comprises a MASP antibody (e.g., an antibody against MASP-1, MASP-2, or MASP-3) that inhibits at least one of the LEA-1 and / or LEA-2 complement activation pathways. MASP antibodies used in this aspect of the invention include polyclonal antibodies, monoclonal antibodies, or recombinant antibodies derived from any antibody-producing mammal and may be multispecific antibodies (e.g., bispecific or trispecific antibodies), chimeric antibodies, humanized antibodies, fully human antibodies, anti-idiotype antibodies, and antibody fragments. Antibody fragments include Fab, Fab', F(ab)2, F(ab')2, Fv fragments, scFv fragments, and single-chain antibodies, as further described herein.

[0196] The assays described herein can be used to screen for the ability of MASP antibodies to inhibit the LEA-1 or LEA-2-dependent complement activation system. Several MASP-1, MASP-2, and MASP-3 antibodies have been described in the literature, some of which are newly developed; some of these are listed in Table 3 below. The assays described herein can be used to screen for the ability of these exemplary MASP antibodies to inhibit the LEA-1- and / or LEA-2-dependent complement activation system. For example, as described in Examples 11-13 herein, an anti-rat MASP-2 Fab2 antibody has been identified that blocks MASP-2-dependent complement activation. Further, as described in Example 14, a fully human MASP-2 scFv antibody has been identified that blocks MASP-2-dependent complement activation. Further, as described in Example 15, a MASP-3 antibody has been generated. Once a MASP antibody is identified as acting as an inhibitor of LEA-1 or LEA-2, it can be used in the pharmaceutical compositions described herein, and it can also be used to generate bispecific and trispecific inhibitors, as shown in Table 2 and further described herein (see, for example, Example 8).

[0197] Table 3: Specific antibodies against MASP-1, MASP-2, and MASP-3 i. MASP antibodies with reduced effector function In certain embodiments of this aspect of the invention, in order to reduce inflammation that can be caused by activation of the classical complement pathway, the MASP antibody described herein reduces effector function. The ability of IgG molecules to trigger the classical complement pathway has been shown to be within the Fc portion of the molecule (Duncan, AR, et al.). Nature332 IgG molecules whose Fc moiety is removed by enzyme cleavage lack this effector function (see Harlow, 738-740 (1988)). Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory, New York, 1988). Therefore, by having a genetically modified Fc sequence that minimizes the effector's function, or by becoming a human IgG2 or IgG4 isotype, antibodies with reduced effector function can be produced due to the lack of the Fc portion of the molecule.

[0198] Antibodies with reduced effector function can be generated by standard molecular biology manipulation of the Fc region of the IgG heavy chain, as demonstrated by Jolliffe et al. Int'l Rev. Immunol. 10 :241-250, (1993) and Rodrigues et al.,J. Immunol. 151 As described in :6954-6961, (1998). Antibodies with reduced effector function also include human IgG2 and IgG4 isotypes, which have reduced ability to activate complement and / or interact with Fc receptors (Ravetch, JV, et al.). Annu. Rev. Immunol. 9 :457-492, (1991); Isaacs, JD, et al. J. Immunol. 148 :3062-3071, 1992; van de Winkel, JG, et al., Immunol. Today 14 :215-221, (1993)). Humanized or full-length human antibodies (including dual, pan-, bispecific, or trispecific antibodies) containing human MASP-1, MASP-2, or MASP-3 specificity of IgG2 or IgG4 isotypes can be produced by one of several methods known to those skilled in the art, such as Vaughan, TJ, et al. Nature Biotechnical 16 As stated in 535-539, (1998).

[0199] ii. Production of MASP antibodies Antibodies against MASP-1, MASP-2, or MASP-3 can be generated using MASP-1, MASP-2, or MASP-3 peptides (e.g., full-length MASP-1, MASP-2, or MASP-3) or peptides carrying antigenic MASP-1, 2, or 3 epitopes (e.g., a portion of a MASP-2 peptide). Immunogenic peptides can be as few as 5 amino acid residues. For example, a MASP-2 peptide comprising the complete amino acid sequence of SEQ ID NO:5 can be used to induce MASP-2 antibodies for use in the methods of the present invention. Using methods well known in the art, specific MASP domains known to be involved in protein-protein interactions (e.g., CUBI and CUBIEGF domains, and regions including serine protease active sites, as shown in Table 2, for example) can be expressed as recombinant peptides and used as antigens. Additionally, peptides comprising at least six amino acids of MASP-1 peptide (SEQ ID NO:10), MASP-2 peptide (SEQ ID NO:5), or MASP-3 peptide (SEQ ID NO:8) are also used to induce antibodies against MASP-1, MASP-2, or MASP-3, respectively. The MASP peptides and polypeptides used to generate antibodies can be isolated as natural polypeptides, recombinant peptides, synthetic peptides, or non-catalytically active recombinant peptides. Antigens used to generate anti-MASP antibodies also include fusion polypeptides, such as fusions of MASP polypeptides or portions thereof with immunoglobulin polypeptides or with maltose-binding proteins. The polypeptide immunogen can be a full-length molecule or a portion thereof. If the polypeptide portion is hapten-like, the portion can advantageously bind to or be linked to a macromolecular carrier (e.g., a keyhole). It is used for immunization with hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid.

[0200] iii. Polyclonal antibodies Polyclonal antibodies against MASP-1, MASP-2, or MASP-3 can be prepared by immunizing animals with the MASP-1, MASP-2, or MASP-3 peptides or their immunogenic fractions using methods well known to those skilled in the art. See, for example, Green et al., "Production of Polyclonal Anti-sera," published in [Journal Name]. Immunochemical Protocols(Edited by Manson). The immunogenicity of MASP peptides can be increased by using adjuvants, including inorganic gels (e.g., aluminum hydroxide) or Freund's adjuvant (complete or incomplete), surfactants (e.g., lysophosphatidylcholine), pluronic polyols, polyanionic emulsions, oil emulsions, KLH, and dinitrophenol. Polyclonal antibodies are generally produced by animals such as horses, cattle, dogs, chickens, rats, mice, rabbits, guinea pigs, goats, or sheep. Alternatively, the MASP antibodies used in this invention can also be obtained from primates similar to humans. General techniques for producing diagnostic and therapeutic antibodies in baboons can be found, for example, in Goldenbenberg et al., International Patent Publication No. WO 91 / 11465, and Losman, MJ, et al. Int. J. Cancer 46 :310, (1990). Then, using standard methods well-known in the art, serum containing immunologically active antibodies was obtained from the blood of these immunized animals.

[0201] iv. Monoclonal antibodies In some embodiments, the LEA-2 inhibitor is a MASP-2 monoclonal antibody and / or the LEA-1 inhibitor is a MASP-3 monoclonal antibody or a MASP-1 monoclonal antibody. As mentioned above, in some embodiments, the monoclonal antibodies against MASP-1, MASP-2, and MASP-3 are highly specific, targeting a single MASP-1, MASP-2, or MASP-3 epitope. The modifier “monoclonal” as used herein refers to the antibody properties derived from a substantially homologous group of antibodies and is not construed as requiring the production of the antibody by any particular method. Monoclonal antibodies can be obtained using any technique that provides antibody molecule production through continuous cell lines in a culture, such as those employed by Kohler, G., et al. Nature256 The hybridoma method described in :495, (1975) can be used, or monoclonal antibodies can be prepared by recombinant DNA methods (see, for example, US Patent No. 4,816,567 of Cabilly). Clackson, T., et al. can also be used. Nature 352 :624-628, (1991) and Marks, JD, et al., J. Mol. Biol. 222 The technique described in 581-597, (1991) isolates monoclonal antibodies from a phage antibody library. These antibodies may be of any immunoglobulin class, including IgG, IgM, IgE, IgA, IgD and any of their subclasses.

[0202] For example, monoclonal antibodies can be obtained by injecting a composition containing a peptide of MASP-1, a peptide of MASP-2, or a peptide of MASP-3, or a portion thereof, into a suitable mammal (e.g., a BALB / c mouse). After a predetermined time, spleen cells are removed from the mouse and suspended in cell culture medium. The spleen cells are then fused with an indefinitely proliferating cell line to form hybridomas. The resulting hybridomas are cultured in cell culture medium, and their ability to produce monoclonal antibodies against MASP-1, MASP-2, or MASP-3 is screened. (See also...) Current Protocols in Immunology (Volume 1, John Wiley & Sons, pp. 2.5.1-2.6.7, 1991.)

[0203] Human monoclonal antibodies can be obtained using transgenic mice engineered to produce specific human antibodies in response to antigen challenge. In this technique, human immunoglobulin heavy and light chain locus elements are introduced into a mouse strain derived from an embryonic stem cell line containing directionally disrupted endogenous immunoglobulin heavy and light chain loci. This transgenic mouse can synthesize human antibodies specific to human antigens (such as the MASP-2 antigen described herein), and can be used to generate hybridomas that secrete human MASP-2 antibodies, specifically by fusing B cells from the animal with a suitable myeloma cell line using the conventional Kohler-Milstein technique. Methods for obtaining human antibodies from transgenic mice are described, for example, in Green, LL, et al. Nature Genet. 7 :13, 1994; Lonberg, N., et al. Nature 368 :856, 1994; and Taylor, LD, et al. Int. Immun.6 :579,1994.

[0204] Monoclonal antibodies can be isolated and purified from hybridoma cultures using a variety of established techniques. These techniques include affinity chromatography with protein A agarose gel, size exclusion chromatography, and ion exchange chromatography (see, for example, Coligan, pp. 2.7.1–2.7.12 and 2.9.1–2.9.3; Baines et al., "Purification of Immunoglobulin G (IgG)," published in...). Methods in Molecular Biology (The Humana Press, Inc., Vol. 10, pp. 79-104, 1992).

[0205] Once polyclonal antibodies, monoclonal antibodies, or phage-derived antibodies are generated, their specificity for binding to MASP-1, MASP-2, or MASP-3 must first be tested, or, if necessary, for binding to dual MASP-1 / 3, MASP-2 / 3, or MASP-1 / 2. Methods for determining whether an antibody binds to a protein antigen and / or the antibody's affinity for the protein antigen are known in the art. For example, various techniques can be used to determine and / or quantify the binding of antibodies to protein antigens, such as, but not limited to, Western blotting, dot blotting, plasmonic surface resonance methods (e.g., the BIAcore system; Pharmacia Biosensor AB, Uppsala, Sweden, and Piscataway, NJ), or enzyme-linked immunosorbent assay (ELISA). See, for example, Harlow and Lane (1988) "Antibodies: A Laboratory Manual," Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Benny KC Lo (2004) "Antibody Engineering: Methods and Protocols," Humana Press (ISBN: 1588290921); Borrebaek (1992) "Antibody Engineering, A Practical Guide," WH Freeman and Co., NY; Borrebaek (1995) "Antibody Engineering," 2nd ed., OxfordUniversity Press, NY, Oxford; Johne et al. (1993), Immunol. Meth. 160:191-198; Jonsson et al. (1993) Ann. Biol. Clin. 51: 19-26; and Jonsson et al. (1991) Biotechniques 11:620-627. See also U.S. Patent No. 6,355,245.

[0206] Those skilled in the art can readily determine the affinity of MASP monoclonal antibodies (see, for example, Scatchard, A., NY Acad. Sci.51 (660-672, 1949). In one embodiment, the monoclonal antibody of MASP-1, MASP-2 or MASP-3 used in the method of the present invention binds to MASP-1, MASP-2 or MASP-3 with a binding affinity of <100 nM, preferably <10 nM and most preferably <2 nM.

[0207] Once antibodies that specifically bind to MASP-1, MASP-2, or MASP-3 are identified, their ability to act as LEA-1 or LEA-2 inhibitors is tested in one of several functional assays, such as those described in Table 2. For example, the ability of antibodies identified as specifically binding to MASP-2 to act as LEA-2 inhibitors is tested in one of several functional assays, such as those described in Table 2 (e.g., lectin-specific C4 cleavage assay (e.g., the assay described in Example 8 or Example 9), or C3b deposition assay (e.g., the assay described in Example 4 or Example 11)). As another example, the ability of antibodies identified as specifically binding to MASP-1 or MASP-3 to act as LEA-1 inhibitors is tested in one of several functional assays, such as those described in Table 2 (e.g., reduction in hemolysis (e.g., as measured in Example 5) or reduction in C3 cleavage and C3b deposition (e.g., as measured in Examples 4 and 11)).

[0208] v. Chimeric / humanized antibodies Monoclonal antibodies used in the methods of the present invention include chimeric antibodies and fragments of these antibodies, wherein a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence of an antibody derived from a particular species, or belongs to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence of an antibody derived from another species, or belongs to another antibody class or subclass (Cabilly, U.S. Patent No. 4,816,567; and Morrison, SL, et al.). Proc. Nat'l Acad. Sci.USA 81 :6851-6855, (1984)).

[0209] One form of chimeric antibody used in this invention is a humanized monoclonal MASP-1, MASP-2, or MASP-3 antibody. The humanized form of a non-human (e.g., mouse) antibody is a chimeric antibody containing a minimal sequence derived from a non-human immunoglobulin. Humanized monoclonal antibodies are produced by transferring a non-human (e.g., mouse) complementarity-determining region (CDR) from the variable heavy and light chains of a mouse immunoglobulin to a human variable domain. Typically, the remaining portion of the human antibody is then substituted into the corresponding non-human scaffold region. Furthermore, the humanized antibody may include residues not present in the receptor or donor antibody. These modifications are used to further improve antibody performance. Generally, the humanized antibody will contain substantially all of at least one, typically two, variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of the non-human immunoglobulin, and all or substantially all of the Fv scaffold regions are Fv scaffold regions of the human immunoglobulin sequence. The humanized antibody optionally also contains at least a portion of the immunoglobulin constant region (Fc) (typically the constant region of a human immunoglobulin). For more details, please refer to Jones, PT, et al. Nature 321 :522-525, (1986); Reichmann, L., et al. Nature 332 :323-329, (1988); and Presta, Curr. Op. Struct. Biol. 2 :593-596, (1992).

[0210] The humanized antibodies used in this invention comprise human monoclonal antibodies containing at least the MASP-1, MASP-2, or MASP-3 binding CDR3 region. Furthermore, the Fc portion can be replaced to generate IgA or IgM and human IgG antibodies. Such humanized antibodies will have specific clinical utility because they specifically recognize human MASP-1, MASP-2, or MASP-3, but do not elicit an immune response in the human body to the antibody itself. Therefore, they are more suitable for in vivo administration, especially when repeated or long-term administration is necessary.

[0211] Techniques for generating humanized monoclonal antibodies have also been described, for example, by Jones, PT, et al. Nature 321 :522, (1986); Carter, P., et al. Proc. Nat'l. Acad. Sci. USA 89 :4285, (1992); Sandhu, JS, Crit. Rev. Biotech. 12 :437, (1992); Singer, II, et al. J. Immun. 150 :2844, (1993); Sudhir (ed.), Antibody Engineering Protocols, Humana Press, Inc., (1995); Kelley, "Engineering Therapeutic Antibodies," in Protein Engineering: Principles and Practice Cleland et al. (eds.), John Wiley & Sons, Inc., pp. 399-434, (1996); and Queen's U.S. Patent No. 5,693,762, 1997. In addition, there are commercial companies that synthesize humanized antibodies from specific mouse antibody regions, such as Protein Design Labs (Mountain View, CA).

[0212] vi. Recombinant antibodies Antibodies against MASP-1, MASP-2, or MASP-3 can also be prepared using recombinant methods. For example, human antibodies can be prepared using human immunoglobulin expression libraries (available, for example, from Stratagene, Corp., La Jolla, CA) to generate human antibody fragments (VH, VL, Fv, factor D, Fab, or F(ab')2). These fragments are then used to construct complete human antibodies using techniques similar to those used to generate chimeric antibodies.

[0213] vii. Anti-idiotype antibodies Once antibodies against MASP-1, MASP-2, or MASP-3 with the desired inhibitory activity are identified, these antibodies can be used using techniques well-known in the art to generate anti-idiotype antibodies that resemble a portion of MASP-1, MASP-2, or MASP-3. See, for example, Greenspan, NS, etc. FASEB J 7:437, (1993). For example, antibodies that bind to MASP-2 and competitively inhibit the interaction of the MASP-2 protein required for complement activation can be used to generate anti-idiotype antibodies that resemble the MBL binding site on the MASP-2 protein, thereby binding to and neutralizing MASP-2 binding ligands, such as MBL.

[0214] viii. Immunoglobulin fragments Inhibitors of MASP-2 and MASP-3 used in the methods of the present invention include not only complete immunoglobulin molecules, but also well-known fragments, including Fab, Fab', F(ab)2, F(ab')2 and Fv fragments, scFv fragments, biantibodies, linear antibodies, single-chain antibody molecules, and multispecific (e.g., bispecific and trispecific) antibodies formed from antibody fragments.

[0215] It is well known in the art that only a small portion of the antibody molecule, namely the paratope, is involved in the binding of the antibody to its epitope (see, for example, Clark, WR, The Experimental Foundations of Modern Immunology (Wiley & Sons, Inc., NY, 1986). The pFc' and Fc regions of an antibody are effectors of the classical complement pathway but do not participate in antigen binding. An antibody whose pFc' region has been cleaved by an enzyme, or an antibody lacking a pFc' region, is called an F(ab')2 fragment, which retains two of the antigen-binding sites of the intact antibody. The isolated F(ab')2 fragment is called a bivalent monoclonal fragment because it has two antigen-binding sites. Similarly, an antibody whose Fc region has been cleaved by an enzyme, or an antibody lacking a Fc region, is called a Fab fragment, which retains one of the antigen-binding sites of the intact antibody molecule.

[0216] Antibody fragments can be obtained through proteolysis, such as digesting intact antibodies with pepsin or papain using conventional methods. For example, antibody fragments can be generated by enzymatic cleavage of antibodies with pepsin, providing a 5S fragment called F(ab')2. This fragment can be further cleaved with a thiol reducing agent to obtain a 3.5S Fab' monovalent fragment. Optionally, the cleavage reaction can be performed using the thiol blocking group generated by disulfide bond cleavage. As an alternative method, enzymatic cleavage with pepsin directly generates two monovalent Fab fragments and one Fc fragment. These methods are described, for example, in U.S. Patent No. 4,331,647 to Goldenborg; Nisonoff, A., et al. Arch. Biochem. Biophys. 89 :230, (1960); Porter, RR, Biochem. J.73 :119, (1959); Edelman, et al., published in Methods in Enzymology1 :422, Academic Press, (1967); and Coligan, pp. 2.8.1-2.8.10 and 2.10.-2.10.4.

[0217] In some implementations, it is preferred to use antibody fragments lacking the Fc region to avoid activation of the classical complement pathway initiated when the Fc binds to the Fcγ receptor. Several methods exist for generating monoclonal antibodies that avoid interaction with the Fcγ receptor. For example, the Fc region of a monoclonal antibody can be chemically removed by partial digestion with a proteolytic enzyme (e.g., figase digestion), thus producing antibody fragments that bind to antigens, such as Fab or F(ab)2 fragments (Mariani, M., et al.). Mol. Immunol.28 (69-71, (1991)). Alternatively, a human γ4 IgG isotype that does not bind to the Fcγ receptor can be used during the construction of the humanized antibodies described herein. The recombinant techniques described herein can also be used to engineer antibodies, single-chain antibodies, and antigen-binding domains lacking the Fc domain.

[0218] ix. Single-chain antibody fragments Alternatively, single-chain peptide-binding molecules specific to MASP-1, MASP-2, or MASP-3 can be constructed, wherein the heavy and light chain Fv regions are linked. The Fv fragments are linked via peptide linkers to form single-chain antigen-binding proteins (scFv). These single-chain antigen-binding proteins are prepared by constructing structural genes containing DNA sequences encoding VH and VL domains linked by oligonucleotides. These structural genes are inserted into expression vectors, which are then introduced into host cells (e.g., *E. coli*). The recombinant host cells synthesize a single polypeptide chain with the two V domains bridged by linker peptides. Methods for scFv preparation are described, for example, in Whitlow et al., "Methods: A Companion to Methods in Enzymology". 2 :97,(1991); Bird, et al., Science 242 :423, (1988); Ladner's U.S. Patent No. 4,946,778; Pack, P., et al. Bio / Technology11 :1271, (1993).

[0219] For example, MASP-3-specific scFvs can be obtained by exposing lymphocytes to MASP-3 peptides in vitro and selecting antibody display libraries in phages or similar vectors (e.g., by using immobilized or labeled MASP-3 proteins or peptides). Genes encoding peptides with potential MASP-3 peptide-binding domains can be obtained by screening random peptide libraries displayed on phages or bacteria (such as E. coli). These random peptide display libraries can be used to screen peptides that interact with MASP-3. Techniques for constructing and screening these random peptide display libraries are well known in the art (Lardner's U.S. Patent No. 5,223,409; Lardner's U.S. Patent No. 4,946,778; Lardner's U.S. Patent No. 5,403,484; Lardner's U.S. Patent No. 5,571,698; and Kay et al.). Phage Display of Peptides and Proteins Academic Press, Inc. (1996). Random peptide display libraries and kits for screening these libraries are commercially available, for example from CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc. (Beverly, Mass.), and Pharmacia LKB Bio technology Inc. (Piscataway, NJ).

[0220] Another form of the MASP-3 antibody fragment used in this aspect of the invention is a peptide encoding a single complementation-determining region (CDR) that binds to an epitope on the MASP-3 antigen and inhibits MASP-3-dependent complement activation (i.e., LEA-1). Another form of the MASP-1 antibody fragment used in this aspect of the invention is a peptide encoding a single complementation-determining region (CDR) that binds to an epitope on the MASP-1 antigen and inhibits MASP-3-dependent complement activation (i.e., LEA-1). Another form of the MASP-2 antibody fragment used in this aspect of the invention is a peptide encoding a single complementation-determining region (CDR) that binds to an epitope on the MASP-2 antigen and inhibits MASP-2-dependent complement activation (i.e., LEA-2).

[0221] CDR peptides (“minimum recognition units”) can be obtained by constructing genes encoding the target antibody’s CDR. These genes can be prepared, for example, by using a polymerase chain reaction to synthesize variable regions of antibody-generating RNA from cells (see, for example, Larrick et al.). Methods: A Companion to Methods in Enzymology 2 :106, (1991); Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies," in Monoclonal Antibodies: Production, Engineeringand Clinical Application Ritter et al. (eds.), p. 166, Cambridge University Press, (1995); and Ward et al., "Genetic Manipulation and Expression of Antibodies," published in Antibodies: Principles and Applications , Birch et al. (eds.), p. 137, Wiley-Liss, Inc., 1995).

[0222] The MASP antibodies described herein are administered to subjects in need to inhibit complement activation of LEA-1, LEA-2, or a combination of LEA-1 and LEA-2. In some embodiments, the MASP inhibitor is a high-affinity human or humanized monoclonal MASP-1, MASP-2, or MASP-3 antibody with reduced effector function.

[0223] x. Bispecific antibodies Inhibitors of MASP-2 and MASP-3 used in the methods of the present invention include multispecific (i.e., bispecific and trispecific) antibodies. Bispecific antibodies are monoclonal antibodies, preferably human or humanized antibodies, which have binding specificity to at least two different antigens. As described above and as shown in Table 2, in one embodiment, the method includes the use of a bispecific antibody containing binding specificity to MASP-2 (e.g., binding to at least one of the CCP1-CCP2 or serine protease domains of MASP-2) and binding specificity to MASP-3 (e.g., binding to the serine protease domain of MASP-3). In another embodiment, the method includes the use of a bispecific antibody containing binding specificity to MASP-1 (e.g., binding to the serine protease domain of MASP-1) and binding specificity to MASP-2 (e.g., binding to at least one of the CCP1-CCP2 or serine protease domains of MASP-2). In another embodiment, the method includes the use of a bispecific antibody comprising binding specificity to MASP-1 (e.g., binding to a serine protease domain of MASP-1) and binding specificity to MASP-3 (e.g., binding to a serine protease domain of MASP-3). In another embodiment, the method includes the use of a trispecific antibody comprising binding specificity to MASP-1 (e.g., binding to a serine protease domain of MASP-1), binding specificity to MASP-2 (e.g., binding to at least one of the CCP1-CCP2 or serine protease domains of MASP-2), and binding specificity to MASP-3 (e.g., binding to a serine protease domain of MASP-3).

[0224] Methods for preparing bispecific antibodies are within the capabilities of those skilled in the art. Traditionally, the recombinant generation of bispecific antibodies is based on the co-expression of two immunoglobulin heavy / light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, ...). Nature 305:537-539 (1983)). Antibody variable domains with desired binding specificity (antibody-antigen binding site) can be fused to immunoglobulin constant domain sequences. The fusion product preferably has an immunoglobulin heavy chain constant domain, which includes at least a portion of the hinge region, C... H Zone 2 and C H 3. DNA encoding the immunoglobulin heavy chain fusion and (if necessary) the immunoglobulin light chain is inserted into separate expression vectors and then co-transfected into a suitable host organism. For further details on illustrative existing known methods for generating bispecific antibodies, see, for example, Suresh et al. Methods in Enzymology 121:210 (1986); WO96 / 27011; Brennan et al., Science 229:81 (1985); Shalaby et al., J. Exp. Med 175:217-225 (1992); Kostelny et al., J. Immunol 148(5):1547-1553 (1992); Hollinger et al. Proc. Natl. Acad. Sci USA90:6444-6448 (1993); Gruber et al. J. Immunol. 152:5368 (1994); and Tutt et al., J. Immunol. 147:60 (1991). Bispecific antibodies also include cross-linked or heteroconjugate antibodies. Heteroconjugate antibodies can be prepared using any conventional cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Patent No. 4,676,980 and many cross-linking techniques.

[0225] Several techniques for preparing and isolating bispecific antibody fragments directly from recombinant cell cultures have also been described. For example, bispecific antibodies have been prepared using leucine zippers. (See, for example, Kostelny et al.) J.Immunol . 148(5):1547-1553 (1992)). Hollinger et al., Proc. Natl. Acad. Sci The “biantibody” technique described in USA 90:6444-6448 (1993) has provided an alternative mechanism for preparing bispecific antibody fragments. These fragments contain a heavy chain variable domain (VH) linked to a light chain variable domain (VL) via a linker that is too short to allow pairing between the two domains on the same chain. Therefore, the VH and VL domains of one fragment are forced to pair with complementary VL and VH domains of another fragment, resulting in two antigen-binding sites. Bispecific biantibodies can also be particularly useful, as they can be readily constructed and expressed in *E. coli*. Using phage display (WO94 / 13804), biantibodies (and many other peptides, such as antibody fragments) with suitable binding specificity can be readily selected from a library. If one arm of the biantibody remains constant, for example, with specificity against antigen X, a library can be constructed where the other arm differs and antibodies with suitable specificity are selected.

[0226] Another strategy for preparing bispecific antibody fragments by using single-chain Fv (scFv) dimers has also been reported (see, for example, Gruber et al.). J. Immunol ., 152:5368 (1994)). Alternatively, the antibody can be a "linear antibody," as described in Zapata et al., Protein Eng. 8(10):1057-1062 (1995). Briefly, these antibodies contain a pair of tandem D factor segments (V... H -C H IV H -C H I) This forms a pair of antigen-binding regions. Linear antibodies can be bispecific or monospecific. The method of the present invention also includes the use of variant forms of bispecific antibodies, such as those described by Wu et al. Nat Biotechnol The tetravalent dual variable domain immunoglobulin (DVD-Ig) molecule described in 25:1290-1297 (2007) is called a DVD-Ig molecule because two distinct light chain variable regions (VLs) from two different parent antibodies are tandemly linked, either directly or via short linkers, using recombinant DNA technology, followed by a light chain constant region. Methods for generating DVD-Ig molecules from two parent antibodies are further described, for example, in WO08 / 024188 and WO07 / 024715, the disclosures of which are incorporated herein by reference in their entirety.

[0227] VI. Non-peptide inhibitors In some embodiments, the MASP-3 or MASP-2 inhibitor is an inhibitory peptide of MASP-3, MASP-2, or MASP-1, or a non-peptide inhibitor of MASP-3, MASP-2, or MASP-1. Non-peptide MASP inhibitors can be administered to the subject systemically, for example, via intra-arterial, intravenous, intramuscular, subcutaneous, or other parenteral administration, or via oral administration. MASP inhibitors can be administered periodically over an extended period to treat or control chronic conditions, or can be administered once or repeatedly before, during, or after an acute trauma or injury.

[0228] VII. Pharmaceutical Compositions and Delivery Methods dose On the other hand, the present invention provides compositions for inhibiting the adverse effects of MASP-3-dependent complement activation in subjects suffering from hemolytic diseases such as PNH, comprising administering the composition to the subject, the composition comprising a certain amount of a MASP-3 inhibitor that effectively inhibits MASP-3-dependent complement activation and a pharmaceutically acceptable carrier. In some embodiments, the method further comprises administering a composition comprising a MASP-2 inhibitor. Therapeuticly effective doses of inhibitors of MASP-3 and MASP-2 may be administered to subjects in need to treat or improve conditions associated with MASP-3-dependent complement activation (LEA-1) and optionally also associated with MASP-2-dependent complement activation (LEA-2). A therapeutically effective dose refers to an amount of MASP-3 inhibitor or a combination of MASP-3 inhibitor and MASP-2 inhibitor sufficient to result in improvement of symptoms of the condition.

[0229] The toxicity and therapeutic efficacy of MASP-3 and MASP-2 inhibitors can be determined using experimental animal models through standard pharmaceutical methods. Using these animal models, standard methods can be used to determine the NOAEL (no obvious adverse effect level) and MED (minimum effective dose). The dose ratio between the NOAEL and MED effects is the therapeutic ratio, expressed as the NOAEL / MED ratio. MASP-3 and MASP-2 inhibitors with high therapeutic ratios or exponents are preferred. Data obtained from cell culture assays and animal studies can be used to determine the dosage range for human use. The dosages of MASP-3 and MASP-2 inhibitors are preferably within the range of circulating concentrations, including those with virtually no or no toxicity (MED). The dosage can vary within this range depending on the dosage form and route of administration.

[0230] For any compound formulation, animal models can be used to evaluate the therapeutically effective dose. For example, doses reaching the range of circulating plasma concentrations, including MED, can be formulated in animal models. The quantitative levels of MASP-3 inhibitors or MASP-2 inhibitors in plasma can also be measured using, for example, high-performance liquid chromatography.

[0231] In addition to toxicity studies, the effective dose can be estimated based on the amount of target MASP protein present in live subjects and the binding affinity of inhibitors of MASP-3 or MASP-2.

[0232] MASP-1 levels have been reported in the serum of normal human subjects ranging from 1.48 to 12.83 µg / mL (Terai I. et al., Clin Exp Immunol 110:317-323 (1997); Theil et al., Clin. Exp. Immunol . 169:38 (2012)). The mean serum MASP-3 concentrations in normal human subjects have been reported to range from approximately 2.0 to 12.9 µg / mL (Skjoedt M et al., ). Immunobiology 215(11):921-31 (2010); Degn et al., J. Immunol Methods, 361-37 (2010); Csuka et al., Mol. Immunol 54:271 (2013). It has been confirmed that MASP-2 levels in the serum of normal human subjects are in the low range of 500 ng / ml. The MASP-2 quantification method described in the following literature can be used to determine the MASP-2 levels of specific subjects: Moller-Kristensen M., et al. J. Immunol. Methods282 :159-167 (2003) and Csuka et al., Mol. Immunol 54:271(2013).

[0233] Typically, the dosage of a composition containing a MASP-3 inhibitor or a MASP-2 inhibitor varies depending on factors such as the subject's age, weight, height, sex, general medical condition, and medical history. For example, the MASP-3 inhibitor or MASP-2 inhibitor (e.g., MASP-3 antibody, MASP-1 antibody, or MASP-2 antibody) may be administered in a dose range of approximately 0.010 to 100.0 mg / kg, preferably 0.010 to 1.0 mg / kg, more preferably 0.010 to 0.1 mg / kg of the subject's body weight. In some embodiments, the dose range of the MASP-2 inhibitor (e.g., MASP-2 antibody) administered is approximately, preferably, 0.010 to 10 mg / kg, preferably 0.010 to 1.0 mg / kg, more preferably 0.010 to 0.1 mg / kg of the subject's body weight. In some embodiments, the dose range of administering a MASP-1 inhibitor (e.g., a MASP-1 antibody) or a MASP-3 inhibitor (e.g., a MASP-3 antibody) is approximately 0.010 to 100.0 mg / kg, preferably 0.010 to 10 mg / kg, more preferably 0.010 to 1.0 mg / kg, and more preferably 0.010 to 0.1 mg / kg of the subject's body weight.

[0234] According to complement assays well known to those skilled in the art, the therapeutic efficacy and appropriate dosage of a MASP-3 inhibitory composition (optionally combined with a MASP-2 inhibitory composition) or a MASP-1 inhibitory composition (optionally combined with a MASP-2 inhibitory composition) in a specified subject, as well as the method of the present invention, can be determined. Complement produces a variety of specific products. In the last decade, sensitive and specific assays for most of these activated products have been developed and are commercially available, including small activated fragments C3a, C4a, and C5a and large activated fragments iC3b, C4d, Bb, and sC5b-9. Most of these assays utilize monoclonal antibodies that react with neoantigens exposed to the fragments rather than to the native proteins from which they are formed, making these assays very simple and specific. Most rely on ELISA technology, although radioimmunoassays are sometimes used for C3a and C5a. Radioimmunoassays measure both the unprocessed fragments and their “de-Arg” fragments, which are the predominant forms present in circulation. Unprocessed fragments and C5a desArg It is rapidly eliminated by binding to cell surface receptors, and thus exists in extremely low concentrations, while 3a desArgIt does not bind to cells and accumulates in plasma. Measurement of C3a provides a sensitive, pathway-independent biomarker of complement activation. Alternative pathway activation can be evaluated by measuring the Bb fragment and / or factor D activation. Detection of the liquid-phase product sC5b-9, activated by the membrane attack pathway, provides evidence of complete complement activation. Because both the lectin and classical pathways produce the same activation products C4a and C4d, measuring these two fragments does not provide any information about which of the two pathways produced the activation products.

[0235] The inhibition of MASP-3-dependent complement activation is characterized by at least one of the following changes in the components of the complement system that occur as a result of administration of a MASP-3 inhibitor according to the method of the present invention: inhibition of LEA-1-mediated complement activation (inhibition of hemolysis and opsonization); inhibition of MASP-3 serine protease substrate-specific cleavage; reduction of hemolysis (as determined, for example, as described in Example 5); or reduction of C3 cleavage and C3b deposition (as determined, for example, as described in Example 4 or Example 11).

[0236] The inhibition of MASP-2-dependent complement activation is characterized by at least one of the following changes in the components of the complement system that occur as a result of administering a MASP-2 inhibitor according to the method of the present invention: inhibition of the production or generation of MASP-2-dependent complement activation system products C4b, C3a, C5a and / or C5b-9 (MAC) (as determined, for example, as described in Example 2 of U.S. Patent No. 7,919,094); reduction of C4 cleavage and C4b deposition (as determined, for example, as described in Example 8 or Example 9); or reduction of C3 cleavage and C3b deposition (as determined, for example, as described in Example 11).

[0237] i. Pharmaceutical carriers and delivery media Generally, compositions of the present invention, including MASP-3 inhibitor compositions and MASP-2 inhibitor compositions, or compositions comprising inhibitors of MASP-2 and MASP-3, can be combined with any other selected therapeutic agent and suitably contained in a pharmaceutically acceptable carrier. The carrier is non-toxic, biocompatible, and selected so as not to adversely affect the biological activity of the MASP-3 inhibitor or MASP-2 inhibitor (and any other therapeutic agent combined therewith). Exemplary pharmaceutically acceptable carriers for peptides are described in U.S. Patent No. 5,211,657 to Yamada. As described herein, MASP antibodies for use in the present invention can be formulated into preparations in solid, semi-solid, gel, liquid, or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, inhalers, and injections, for oral, parenteral, or surgical administration. The invention also includes topical administration by applying the composition to a medical device.

[0238] Suitable carriers for parenteral and local delivery via injection, infusion, or flushing include distilled water, phosphate-buffered saline, standard Ringer's solution or lactated Ringer's solution, glucose solution, Hank's solution, or propylene glycol. Additionally, sterile, non-volatile oils can be used as solvents or suspension media. For this purpose, any biocompatible oil can be used, including synthetic monoglycerides or diglycerides. Furthermore, fatty acids (e.g., oleic acid) can be used in the preparation of injectable formulations. Carriers and reagents can be formulated as liquid preparations, suspensions, polymerizable or non-polymerizable gels, pastes, or ointments.

[0239] The carrier may also include a delivery medium to sustain (i.e., prolong, delay, or modulate) the delivery of one or more agents, or to enhance the delivery, uptake, stability, or pharmacokinetics of one or more therapeutic agents. Such delivery media may, by way of non-limiting examples, include microparticles, microspheres, nanospheres, or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymeric hydrogels or copolymeric hydrogels, and polymeric micelles. Suitable hydrogel and micelle delivery systems include the PEO:PHB:PEO copolymers and copolymer / cyclodextrin complexes disclosed in WO 2004 / 009664 A2 and the PEO and PEO / cyclodextrin complexes disclosed in U.S. Patent Application Publication No. 2002 / 0019369 A1. These hydrogels may be injected locally to the intended site of action, or injected subcutaneously or intramuscularly to form a sustained-release depot.

[0240] The compositions of the present invention can be formulated for subcutaneous, intramuscular, intravenous, intra-arterial delivery or as an inhalant.

[0241] For intra-articular drug delivery, MASP-3 inhibitors or MASP-2 inhibitors may be loaded into the aforementioned injectable liquid or gel carriers, the aforementioned injectable sustained-release drug delivery media, or hyaluronic acid or hyaluronic acid derivatives.

[0242] For oral administration of non-peptide drugs, MASP-3 inhibitors or MASP-2 inhibitors may be loaded into inert fillers or diluents such as sucrose, corn starch, or cellulose.

[0243] For topical administration, MASP-3 inhibitors or MASP-2 inhibitors may be contained in ointments, lotions, creams, gels, drops, suppositories, sprays, liquid formulations or powders, or delivered via transdermal patches, gels or microcapsules.

[0244] Various nasal and pulmonary delivery systems are under development, including aerosol inhalers, dosing inhalers, dry powder inhalers, and nebulizers, which may be adapted to deliver the present invention in aerosols, inhalers, or nebulized drug delivery solvents.

[0245] For intrathecal (IT) or intraventricular (ICV) delivery, a suitable sterile delivery system (e.g., liquid formulation; gel, suspension, etc.) can be used to administer the present invention.

[0246] The compositions of the present invention may further include biocompatible excipients, such as dispersants or wetting agents, suspending agents, diluents, buffers, penetration enhancers, emulsifiers, binders, thickeners, and flavoring agents (for oral administration).

[0247] ii. Pharmaceutical carriers for antibodies and peptides Regarding the MASP antibodies described herein, more specifically, exemplary formulations may be administered parenterally in solutions or suspensions of the compound at injectable doses, the compound being contained in a physiologically acceptable diluent and a pharmaceutical carrier, which may be a sterile liquid such as water, oil, saline, glycerol, or ethanol. Furthermore, the composition containing the MASP antibody may contain excipients such as wetting agents or emulsifiers, surfactants, pH buffers, etc. Extracellular components of the pharmaceutical composition include petroleum (e.g., petroleum of animal, plant, or synthetic origin), such as soybean oil and mineral oil. Generally, diols such as propylene glycol or polyethylene glycol are preferred liquid carriers for injectable solutions.

[0248] MASP antibodies can also be administered in the form of reservoir-injectable or implantable formulations, which can be formulated to allow for sustained or pulsed release of the active agent.

[0249] VIII. Administration Method Pharmaceutical compositions containing MASP-3 inhibitors or MASP-2 inhibitors can be administered in various ways, depending on whether local or systemic administration is most suitable for the condition to be treated. Furthermore, the compositions of the present invention can be delivered by coating or incorporating the composition onto or into an implantable medical device.

[0250] i. System delivery As used herein, the terms "systemic delivery" and "systemic administration" are intended to include, but are not limited to, oral and parenteral routes, including intramuscular (IM), subcutaneous, intravenous (IV), intra-arterial, inhalation, sublingual, sublingual, topical, percutaneous, nasal, rectal, vaginal, and other routes of administration, which effectively disperse the delivered drug to one or more sites of intended therapeutic action. Preferred routes of systemic delivery for the compositions of the present invention include intravenous, intramuscular, subcutaneous, intra-arterial, and inhalation. It should be understood that, for the drug selected in a particular composition of the present invention, the exact route of systemic administration will be determined in part by taking into account the drug's sensitivity to metabolic pathways associated with that particular route of administration. For example, peptide drugs may be best suited for administration via routes other than oral.

[0251] The MASP inhibitory antibodies described herein can be delivered to subjects in need by any suitable method. Methods of delivering MASP antibodies and peptides include oral, pulmonary, parenteral (e.g., intramuscular, intraperitoneal, intravenous (IV), or subcutaneous), inhalation (e.g., via micropowder formulation), transdermal, nasal, vaginal, rectal, or sublingual administration, and they can be formulated into dosage forms suitable for their respective routes of administration.

[0252] For example, MASP inhibitory antibodies and peptides can be introduced into the body by applying them to body membranes capable of absorbing the peptides, such as nasal membranes, gastrointestinal membranes, and rectal membranes. Typically, peptides are applied together with permeation enhancers to absorbable membranes (see, for example, Lee, VHL). Crit. Rev. Ther. Drug Carrier Sys . 5 :69,(1988); Lee, VHL, J. Controlled Release13 :213, (1990); Lee, editor-in-chief of VHL, Peptide and Protein Drug Delivery, Marcel Dekker, New York (1991); DeBoer, AG, et al., J. Controlled Release13 :241, (1990). For example, STDHF, a synthetic derivative of clostridial acid, is a steroidal surfactant structurally similar to bile salts and has been used as a nasal penetration enhancer (Lee, WA, Biopharm . 22 (November / December 1990)

[0253] MASP inhibitory antibodies, as described herein, can be introduced to associate with other molecules (e.g., lipids) to protect peptides from enzymatic degradation. For example, covalently bound polymers, particularly polyethylene glycol (PEG), have been used to protect certain proteins from enzymatic hydrolysis in vivo, thereby extending their half-life (Fuertges, F., et al.). J. Controlled Release 11 :139,(1990)). Many polymer systems for protein delivery have been reported (Bae, YH, et al., J. Controlled Release9 :271, (1989); Hori, R., et al. Pharm. Res . 6 :813, (1989); Yamakawa, I., et al., J. Pharm. Sci . 79 :505, (1990); Yoshihiro, I., et al. J. Controlled Release 10 :195, (1989); Asano, M., et al. J. Controlled Release9 :111, (1989); Rosenblatt, J., et al. J. Controlled Release9 :195, (1989); Makino, K., J. Controlled Release 12 :235, (1990); Takakura, Y., et al. J. Pharm. Sci . 78 :117, (1989); Takakura, Y., et al. J. Pharm. Sci . 78 :219, (1989)).

[0254] Recently, liposomes with improved serum stability and circulating half-life have been developed (see, for example, Webb's U.S. Patent No. 5,741,516). Furthermore, various methods for using liposomes and liposome-like preparations as potential drug carriers have been reviewed (see, for example, Szoka's U.S. Patent No. 5,567,434; Yagi's U.S. Patent No. 5,552,157; ​​Nakamori's U.S. Patent No. 5,565,213; Shinkarenko's U.S. Patent No. 5,738,868; and Gao's U.S. Patent No. 5,795,587).

[0255] For transdermal applications, the MASP inhibitory antibody described herein can be mixed with other suitable components (e.g., carriers and / or adjuvants). There are no limitations on the properties of these other components, only that they must be pharmaceutically acceptable for the intended administration and must not diminish the activity of the active ingredient in the composition. Examples of suitable solvents include ointments, creams, gels, or suspensions with or without purified collagen. The MASP inhibitory antibody can also be impregnated into transdermal patches, plasters, and bandages, preferably in liquid or semi-liquid form.

[0256] The compositions of the present invention can be administered systematically at periodic intervals determined to maintain the required level of therapeutic effect. For example, the compositions can be administered every 2-4 weeks or at lower frequencies (e.g., subcutaneous injection). The dosing regimen will be determined by the physician considering various factors that may affect the effects of the combined use of the drugs. These factors include the progression of the disease to be treated, the subject's age, sex, weight, and other clinical factors. The dosage of each individual drug will vary depending on the presence and nature of any MASP-3 or MASP-2 inhibitor contained in the composition and any drug delivery solvent (e.g., sustained-release delivery solvent). Furthermore, the dosage may be adjusted after taking into account changes in dosing frequency and the pharmacokinetic behavior of the delivered drugs.

[0257] ii. Local delivery As used herein, the term "local" includes application of a drug at or around the intended site of localized action, which may include, for example, local delivery to the skin or other affected tissues; ocular delivery; intrathecal (IT), intraventricular (ICV), intra-articular, intracavitary, intracranial, or alveolar administration, placement, or irrigation. Local administration is preferred to allow for the delivery of lower doses to avoid systemic adverse effects and to provide more precise control over the timing of delivery and the concentration of the active agent at the local delivery site. Regardless of variations in metabolism, blood flow, etc., between patients, local administration provides a known concentration at the target site. Direct delivery also provides improved dose control.

[0258] Local delivery of MASP-3 or MASP-2 inhibitors can be achieved in conjunction with surgical procedures to treat diseases or conditions, such as during arterial bypass surgery, percutaneous transluminal plaque excision, laser surgery, ultrasound surgery, balloon angioplasty, and stent placement. For example, MASP-3 or MASP-2 inhibitors can be administered to a subject in combination with balloon angioplasty. Balloon angioplasty involves inserting a catheter with a deflated balloon into an artery. The deflated balloon is placed near an atherosclerotic plaque, and inflation of the balloon compresses the plaque against the vessel wall. As a result, the balloon surface comes into contact with the endothelial cell layer of the vessel surface. MASP-3 or MASP-2 inhibitors can be attached to the balloon angioplasty catheter in a manner that allows for drug release at the site of the atherosclerotic plaque. The drug can be attached to the balloon catheter according to standard methods known in the art. For example, the drug can be stored in a compartment of the balloon catheter until the balloon is inflated, at which point the drug is released into the local environment. Alternatively, the drug can be impregnated on the surface of the balloon, so that when the balloon inflates, the drug comes into contact with the cells of the arterial wall. Drug delivery can also be achieved using porous balloon catheters, as described by Flugelman, MY, et al. Circulation85 Those disclosed in :1110-1117, (1992). See also the exemplary method for attaching therapeutic proteins to balloon angioplasty catheters in published PCT application WO 95 / 23161. Similarly, MASP-3 inhibitors or MASP-2 inhibitors may be encapsulated in a gel or polymeric coating material applied to a stent, or may be incorporated into the stent material such that the stent elutes the MASP-3 inhibitors or MASP-2 inhibitors after vascular placement.

[0259] MASP-3 inhibitors or MASP-2 inhibitors used to treat arthritis and other musculoskeletal disorders may be delivered locally via intra-articular injection. Such compositions may suitably include a sustained-release delivery solvent. As another example where local delivery may be necessary, MASP-2 inhibitory compositions for treating urogenital conditions may suitably be instilled into the bladder or other urogenital structures.

[0260] IX. Treatment Plan In prophylactic use, the pharmaceutical composition is given to subjects who are susceptible to PNH or otherwise at risk of developing PNH, in an amount sufficient to eliminate or reduce the risk of disease symptom development. In therapeutic use, the pharmaceutical composition is given to subjects suspected of having or already having PNH in a therapeutically effective amount sufficient to alleviate or at least partially reduce disease symptoms.

[0261] In one embodiment, the subject's red blood cells are conditioning with a C3 fragment in the absence of the composition, and the composition is administered to the subject to increase red blood cell survival in the subject. In one embodiment, the subject exhibits one or more symptoms selected from the following when the composition is absent: (i) hemoglobin below normal levels, (ii) platelets below normal levels, (iii) reticulocytes above normal levels, and (iv) bilirubin above normal levels, and administering the composition to the subject improves at least one or more symptoms, resulting in (i) increased, normal or near-normal levels of hemoglobin, (ii) increased, normal or near-normal levels of platelets, (iii) decreased, normal or near-normal levels of reticulocytes, and / or (iv) decreased, normal or near-normal levels of bilirubin.

[0262] In a prophylactic / treatment regimen, several doses of a composition comprising a MASP-3 inhibitor and optionally a MASP-2 inhibitor may be administered until adequate therapeutic outcomes are achieved in the subject. In one embodiment of the invention, the MASP-3 and / or MASP-2 inhibitor comprises a MASP-1 antibody, a MASP-2 antibody, or a MASP-3 antibody, which may be suitably administered to adult patients (e.g., average adult weight 70 kg) at doses from 0.1 mg to 10,000 mg, more preferably 1.0 mg to 5,000 mg, more preferably 10.0 mg to 2,000 mg, more preferably 10.0 mg to 1,000 mg, and even more preferably 50.0 mg to 500 mg, or 10 to 200 mg. For pediatric patients, the dose may be adjusted proportionally to the patient's body weight.

[0263] The MASP-3 inhibitory composition and optionally the MASP-2 inhibitory composition of the present invention can be administered for the treatment of PNH by a single dose of the composition (e.g., a single composition containing both MASP-2 and MASP-3 inhibitors, or a bispecific or dual inhibitor, or by co-administering separate compositions) or by a limited sequence of administration. Alternatively, the composition can be administered at regular intervals over an extended period of time for the treatment of PNH, such as daily, twice weekly, weekly, every other week, monthly, or twice monthly.

[0264] In some embodiments, a first composition containing at least one MASP-3 inhibitor and a second composition containing at least one MASP-2 inhibitor are administered to the PNH subject. In one embodiment, the first composition containing at least one MASP-3 inhibitor and the second composition containing at least one MASP-2 inhibitor are administered simultaneously (i.e., at an interval not exceeding approximately 15 minutes, such as not exceeding any one of 10, 5, or 1 minute). In one embodiment, the first composition containing at least one MASP-3 inhibitor and the second composition containing at least one MASP-2 inhibitor are administered sequentially (i.e., the first composition is administered before or after the second composition, wherein the interval between administrations is greater than 15 minutes). In some embodiments, the first composition containing at least one MASP-3 inhibitor and the second composition containing at least one MASP-2 inhibitor are administered concurrently (i.e., the administration time of the first composition overlaps with the administration time of the second composition). For example, in some embodiments, the first composition and / or the second composition are administered for a period of at least 1, 2, 3, or 4 weeks or longer. In one embodiment, at least one MASP-3 inhibitor and at least one MASP-2 inhibitor are mixed in a unit dosage form. In one embodiment, a first composition comprising at least one MASP-3 inhibitor and a second composition comprising at least one MASP-2 inhibitor are packaged together in a kit for the treatment of PNH.

[0265] In some embodiments, the PNH subject has previously received, or is currently receiving, treatment with a terminal complement inhibitor that inhibits complement protein C5 cleavage. In some embodiments, the method comprises administering the subject the composition of the present invention, the composition comprising an inhibitor of MASP-3 and optionally MASP-2; and further administering the subject a terminal complement inhibitor that inhibits complement protein C5 cleavage. 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.

[0266] X. Example The following examples are merely illustrative of what is currently considered the best mode for carrying out the invention, and should not be construed as limiting the invention. All references cited herein are expressly incorporated by way of reference.

[0267] Example 1 This example demonstrates that MASP-2 deficient mice infected with Neisseria meningitidis serum group A or Neisseria meningitidis serum group B are protected from Neisseria meningitidis-induced death.

[0268] method: MASP-2 knockout mice (MASP-2 KO mice) were generated as described in Example 1 of US 7,919,094, which is incorporated herein by reference. Ten-week-old MASP-2 KO mice (n=10) and wild-type (WT) C57 / BL6 mice (n=10) were inoculated intraperitoneally (ip) with 2.6 x 10⁷ CFU, 100 µl of Neisseria meningitidis serum A Z2491. The infectious dose, along with iron dextran, was administered to the mice at a final concentration of 400 mg / kg. Survival rates of the infected mice were monitored over a 72-hour period.

[0269] In different experiments, 10-week-old MASP-2 KO mice (n=10) and WT C57 / BL6 mice (n=10) were injected intraperitoneally with 6 x 10⁶ CFU, 100 µl of Neisseria meningitidis serum strain B MC58. The infectious dose, along with iron dextran, was administered to the mice at a final dose of 400 mg / kg. Survival rates of the infected mice were monitored over a 72-hour period. During the 72-hour post-infection period, disease scores were also determined in WT and MASP-2 KO mice according to the disease scoring parameters described in Table 4, which are based on a slightly modified protocol by Fransen et al. (2010).

[0270] Table 4: Disease scores associated with clinical signs in infected mice physical signs score normal 0 Slightly wrinkled fur 1 Wrinkled fur, dull and sticky eyes 2 Wrinkled fur, drowsy and with eyes closed 3 Immobility after severe illness and stimulation 4 die 5 Blood samples were collected from mice hourly after infection and analyzed to determine the serum level of Neisseria meningitidis (log cfu / mL) to confirm infection and measure the clearance rate of bacteria from the serum.

[0271] result: Figure 8 The figure shows the Kaplan-Mayer curves, illustrating the percentage survival of MASP-2 KO and WT mice after administration of an infectious dose of 2.6 x 10⁷ CFU of Neisseria meningitidis serum in group A Z2491. Figure 8 As shown, 100% of MASP-2KO mice survived within 72 hours post-infection. In contrast, only 80% of WT mice ( p MASP-2 deficient mice (=0.012) survived 24 hours post-infection, while only 50% of WT mice survived 72 hours post-infection. These results demonstrate that MASP-2 deficient mice are protected from death induced by Neisseria meningitidis serogroup A Z2491.

[0272] Figure 9The figure shows the Kaplan-Mayer curves, illustrating the percentage survival of MASP-2 KO and WT mice after administration of an infectious dose of 6 x 10⁶ CFU of Neisseria meningitidis serum strain B MC58. Figure 9 As shown, 90% of MASP-2KO mice survived within 72 hours post-infection. In contrast, only 20% of WT mice ( p MASP-2-deficient mice (=0.0022) survived 24 hours after infection. These results demonstrate that MASP-2-deficient mice are protected from death induced by Neisseria meningitidis serogroup B strain MC58-.

[0273] Figure 10 The figure shows the log CFU / mL of Neisseria meningitidis MC58 recovered from blood samples collected at different time points from MASP-2 KO and WT mice after intraperitoneal infection with 6 x 10⁶ CFU of Neisseria meningitidis MC58 serogroup B (n=3, at different time points for both groups of mice). Results are presented as mean ± SEM. Figure 10 As shown, in WT mice, the level of Neisseria meningitidis in the blood peaked at approximately 6.0 log cfu / mL at 24 hours post-infection and decreased to approximately 4.0 log cfu / mL at 36 hours post-infection. In contrast, in MASP-2 KO mice, the level of Neisseria meningitidis peaked at approximately 4.0 log cfu / mL at 12 hours post-infection and decreased to approximately 1.0 log cfu / mL at 36 hours post-infection (* indicates p < 0.05; ** indicates p = 0.0043). These results demonstrate that although MASP-2 KO mice were infected with the same dose of Neisseria meningitidis serogroup B strain MC58 as used in WT mice, MASP-2 KO mice exhibited a higher rate of bacteremia clearance compared to WT mice.

[0274] Figure 11 The figure shows the mean disease scores of MASP-2 KO and WT mice at 3, 6, 12, and 24 hours after infection with 6 x 10⁶ CFU Neisseria meningitidis serogroup B strain MC58. Figure 11 As shown, MASP-2-deficient mice exhibited high resistance to infection, with significantly lower disease scores at 6 hours (*) (p=0.0411), 12 hours (**) (p=0.0049), and 24 hours (***) (p=0.0049) post-infection compared to WT mice. Figure 11 The results are expressed as mean ± SEM.

[0275] In summary, the results of this embodiment demonstrate that MASP-2-deficient mice are protected from Neisseria meningitidis-induced death after infection with either Neisseria meningitidis serogroup A or Neisseria meningitidis serogroup B.

[0276] Example 2 This example demonstrates that administration of MASP-2 antibody after Neisseria meningitidis infection increased the survival rate of Neisseria meningitidis-infected mice.

[0277] Background / Principle: As described in Example 24 of U.S. Patent 7,919,094 (incorporated herein by reference), rat MASP-2 protein was used to pan Fab phage display libraries, from which Fab2 #11 was identified as a functionally active antibody. Full-length antibodies of rat IgG2c and mouse IgG2a isotypes were generated from Fab2 #11. Pharmacokinetic parameters of the full-length MASP-2 antibody of mouse IgG2a isotype were characterized (as described in Example 38 of U.S. Patent 7,919,094).

[0278] In this embodiment, the full-length MASP-2 antibody from mice from Fab2 #11 was analyzed in a mouse model of Neisseria meningitidis infection.

[0279] method: In a mouse model of Neisseria meningitidis infection, the following tests were performed on the mouse IgG2a full-length MASP-2 antibody isotype generated from Fab2 #11 as shown above.

[0280] 1. Administer mouse-MASP-2 monoclonal antibody (MoAb) after infection. Three hours after intraperitoneal injection of a high dose (4 x 10⁶ CFU) of Neisseria meningitidis serum strain B MC58, 9-week-old C57 / BL6 Charles River mice were treated with either an inhibitory mouse MASP-2 antibody (1.0 mg / kg) (n=12) or a control isotype antibody (n=10).

[0281] result: Figure 12 This is a Kaplan-Mayer curve, illustrating the percentage survival of mice treated with an infectious dose of 4 x 10⁶ CFU of Neisseria meningitidis serogroup B strain MC58, followed by administration of an inhibitory MASP-2 antibody (1.0 mg / kg) or a control isotype antibody 3 hours post-infection. Figure 12As shown, 90% of mice treated with the MASP-2 antibody survived within 72 hours post-infection. In contrast, only 50% of mice treated with the isotype control antibody survived within 72 hours post-infection. The symbol "*" indicates p=0.0301, as determined by comparing two survival curves.

[0282] These results demonstrate that administration of MASP-2 antibodies is effective in treating and improving the survival rate of subjects infected with Neisseria meningitidis.

[0283] As demonstrated in this paper, the use of MASP-2 antibodies is effective in treating subjects infected with Neisseria meningitidis when administered within 3 hours of infection, and is expected to be effective within 24 to 48 hours of infection. Meningococcal disease (meningococcal bacteremia or meningitis) is a medical emergency, and treatment is usually initiated immediately if meningococcal disease is suspected (i.e., before Neisseria meningitidis is definitively identified as the pathogen).

[0284] Based on the results of the MASP-2 KO mice given in Example 1, it is believed that administering MASP-2 antibodies before Neisseria meningitidis infection will also effectively prevent or improve the severity of the infection.

[0285] Example 3 This embodiment demonstrates that the complement-dependent killing of Neisseria meningitidis in human serum is MASP-3-dependent.

[0286] principle: Patients with reduced serum levels of functional MBL have increased susceptibility to recurrent bacterial and fungal infections (Kilpatrick et al.). Biochim Biophys Acta 1572:401-413 (2002)). It is known that Neisseria meningitidis is recognized by MBL, and it has been confirmed that serum lacking MBL does not lyse Neisseria meningitidis.

[0287] Based on the results described in Examples 1 and 2, a series of experiments were conducted to determine the efficacy of MASP-2 antibody in treating Neisseria meningitidis infection in complement-deficient and control human serum. Experiments were performed in high-concentration serum (20%) to protect the complement pathway. method: 1. Serum bactericidal activity in human sera with different complement deficiencies and in human sera treated with human MASP-2 antibody. The following complement-deficient human serum and control human serum were used in this experiment: Table 5: Human serum samples tested (e.g.) Figure 13 (As shown) sample Serum type A Normal human serum (NHS) + human MASP-2 Ab B NHS + Allotype Control Ab C MBL - / - Human Serum D NHS E Heat-inactivated (HI) NHS Recombinant antibodies against human MASP-2 were isolated from combinatorial antibody libraries (Knappik, A., et al.). J. Mol. Biol. 296 :57-86 (2000)), using recombinant human MASP-2A as the antigen (Chen, CB and Wallis, J. Biol. Chem. 276 :25894-25902 (2001)). An anti-human scFv fragment that strongly inhibits the C4 and C3 lectin pathway-mediated activation (IC50~20 nM) in human plasma was identified and converted into a full-length human IgG4 antibody.

[0288] Neisseria meningitidis serum group B-MC58 was incubated with different sera (each with a serum concentration of 20%) as shown in Table 5 at 37°C with shaking, with or without inhibitory human MASP-2 antibody (3 µg in 100 µl total volume). Samples were collected at 0, 30, 60, and 90-minute intervals, plated, and viable bacterial counts were determined. Heat-inactivated human serum was used as a negative control.

[0289] result: Figure 13 The figure shows the log cfu / mL viable count of Neisseria meningitidis MC58 serological group B strain recovered from human serum samples at different time points, as shown in Table 5. Table 6 provides... Figure 13 The results of Student's t-test.

[0290] Table 6: Figure 13 Results of Student's t-test (time point 60 minutes) like Figure 13 As shown in Table 6, the addition of human MASP-2 inhibitory antibody significantly enhanced complement-dependent killing of Neisseria meningitidis in 20% human serum.

[0291] 2. Serum bactericidal activity in human sera with different complement deficiencies The following complement-deficient human serum and control human serum were used in this experiment: Table 7: Human serum samples tested (as shown) Figure 14 ) sample Serum type A Normal human serum (NHS) B Heat-inactivated NHS C MBL - / - D MASP-3 - / - (MASP-1 +) Note: The MASP-3 - / - (MASP-1 +) serum in sample D was collected from subjects with 3MC syndrome, a unified term covering Carnevale, Mingarelli, Malpuech, and Michels syndromes. As further described in Example 4, mutations in exon 12 of the MASP-1 / 3 gene cause dysfunction of the serine protease domain of MASP-3 (but not MASP-1). Factor D is also known to be intact in 3MC serum.

[0292] Neisseria meningitidis serological group B-MC58 was incubated with human sera of different complement-deficient groups (each with a serum concentration of 20%) at 37°C with shaking. Samples were collected at intervals of 0, 15, 30, 45, 60, 90, and 120 minutes, plates were poured, and viable bacterial counts were determined. Heat-inactivated human serum was used as a negative control.

[0293] result: Figure 14 The figure shows the log cfu / mL viable count of Neisseria meningitidis serum group B-MC58 recovered from human serum samples at different time points, as shown in Table 7. Figure 14 As shown, WT (NHS) serum exhibited the highest level of bactericidal activity against Neisseria meningitidis. In contrast, MBL- / - and MASP-3- / - (which is MASP-1- sufficient) human serum showed no bactericidal activity. These results indicate that complement-dependent killing of Neisseria meningitidis in 20% (v / v) human serum is MASP-3- and MBL-dependent. Table 8 provides... Figure 14 The results of Student's t-test.

[0294] Table 8: Figure 14 Student's t-test results In short, Figure 14 The results shown in Table 8 indicate that complement-dependent killing of Neisseria meningitidis in 20% human serum is MASP-3- and MBL-dependent.

[0295] 3. Complement-dependent killing of Neisseria meningitidis in 20% (v / v) mouse serum lacking MASP-2, MASP-1 / 3, or MBL A / C.

[0296] The following complement-deficient mouse serum and control mouse serum were used in this experiment: Table 9: Mouse serum samples tested (e.g.) Figure 15 (As shown) sample Serum type A WT B MASP-2 - / - C MASP-1 / 3 - / - D MBL A / C - / - E WT heat-inactivated (HIS) Neisseria meningitidis serum group B-MC58 was incubated with different complement-deficient mouse sera (each with a serum concentration of 20%) at 37°C with shaking. Samples were collected at 0, 15, 30, 60, 90, and 120-minute intervals, plates were poured, and viable bacterial counts were determined. Heat-inactivated human serum was used as a negative control.

[0297] result: Figure 15 The figure shows the log cfu / mL viable count of Neisseria meningitidis serum group B-MC58 recovered from mouse serum samples at different time points, as shown in Table 9. Figure 15 As shown, MASP-2 - / - mouse serum exhibited higher levels of bactericidal activity against Neisseria meningitidis compared to WT mouse serum. In contrast, MASP-1 / 3 - / - mouse serum showed no bactericidal activity. The symbol "**" indicates p=0.0058, and the symbol "***" indicates p=0.001. Table 10 provides... Figure 15 The results of Student's t-test.

[0298] Table 10: Figure 15 Student's t-test results In summary, the results of this embodiment demonstrate that MASP-2 - / - serum has a higher level of bactericidal activity against Neisseria meningitidis compared to WT serum, and that complement-dependent killing of Neisseria meningitidis in 20% serum is MASP-3- and MBL-dependent.

[0299] Example 4 This embodiment describes a series of experiments conducted to determine the mechanism of MASP-3-dependent resistance to Neisseria meningitidis infection observed in MASP-2KO mice as described in Examples 1-3.

[0300] principle: To determine the mechanism of the MASP-3-dependent resistance to Neisseria meningitidis infection (described in Examples 1-3 above) observed in MASP-2 KO mice, a series of experiments were performed as follows.

[0301] 1. MASP-1 / 3-deficient mice do not lack lectin pathway functional activity (also known as "LEA-2"). method: To determine whether MASP-1 / 3-deficient mice lack lectin pathway functional activity (also known as LEA-2), assays were performed to detect the kinetics of C3 convertase activity in serum from different complement-deficient mouse strains, measured under lectin activation pathway-specific assay conditions (1% plasma), as described, for example, in Schwaeble W. et al., PNAS 108(18):7523-7528 (2011), which is incorporated herein by reference.

[0302] The plasma from WT, C4- / -, MASP-1 / 3- / -, Factor B- / -, and MASP-2- / - mice was tested as described below.

[0303] To determine C3 activation, microtiter plates were coated with a coating buffer (15 mM Na₂Co₃, 35 mM NaHCO₃) containing mannan (1 µg / well) and yeast polysaccharide (1 µg / well), or with immune complexes, by coating with a coating buffer containing 1% human serum albumin (HSA). Then, TBS (10 mM Tris, 140 mM NaCl, pH 7.4) containing sheep anti-HAS serum (2 µg / mL) and 0.05% Tween 20 and 5 mM CaO were added. ++ In-situ generation. Each plate was sealed with TBS containing 0.1% HAS and treated with TBS / Tween 20 / Ca. ++ Wash three times. Plasma samples were diluted in 4 mM barbiturate, 145 mM NaCl, 2 mM CaCl2, and 1 mM MgCl2 (pH 7.4), added to each plate, and incubated at 37°C for 1.5 h. After washing, the binding C3b was measured using rabbit anti-human C3c (Dako), followed by alkaline phosphatase-conjugated goat anti-rabbit IgG and p-nitrobenzene phosphate.

[0304] result: C3 activation kinetics under lectin pathway-specific conditions (determined by C3b deposition on mannan-coated plates using 1% serum) are shown in [reference needed]. Figure 16 No C3 cleavage was observed in MASP-2- / - plasma. Factor B- / - plasma cleaved C3 at half the rate of WT plasma, possibly due to a lack of amplification loops. In C4- / - plasma (T... 1 / 2 =33min) and in MASP-1 / 3- / - deficient plasma (T 1 / 2In a study of 49 min, a significant delay was observed in the lectin pathway-dependent conversion of C3 to C3b. This delay in C3 activation in MASP-1 / 3- / - plasma has been shown to be MASP-1-dependent, rather than MASP-3-dependent (see Takahashi M. et al., J Immunol 180:6132-6138 (2008)). These results demonstrate that MASP-1 / 3-deficient mice do not lack lectin pathway functional activity (also known as "LEA-2").

[0305] 2. The effect of hereditary MASP-3 deficiency on the activation of alternative pathways.

[0306] principle: The effect of inherited MASP-3 deficiency on the activation of alternative pathways was determined by measuring serum from patients with 3MC syndromes (caused by frameshift mutations in the exon encoding the MASP-3 serine protease). 3MC syndromes are a unified term encompassing Carnevale, Mingarelli, Malpuech, and Michels syndromes. These rare autosomal recessive disorders exhibit a spectrum of developmental features, including characteristic facial deformities, cleft lip and / or palate, craniosynostosis, learning disabilities, and genital, limb, and bladder / kidney abnormalities. Rooryck et al., Nature Genetics 43:197-203 (2011) investigated 11 families of 3MC syndromes and identified two mutated genes: COLEC11 and MASP-1. Mutations in the MASP-1 gene dysfunction the exon encoding the serine protease domain of MASP-3, but not the exon encoding the serine protease of MASP-1. Therefore, 3MC patients with mutations in the exon encoding the serine protease of MASP-3 lack MASP-3, but have sufficient MASP-1.

[0307] method: MASP-3-deficient serum was obtained from 3MC patients, their parents (both heterozygous individuals carrying a mutated allele that dysfunctions the exon encoding the MASP-3 serine protease domain), and from C4-deficient patients (deficient in two human C4 genes) and MBL-deficient subjects. (For example, Bitter-Suermann et al.) Eur. J. Immunol As described in 11:291-295 (1981), on yeast polysaccharide-coated microtiter plates, within the range of 0.5% to 25% serum concentration, under conventional AP-specific conditions (BBS / Mg). ++ / EGTA, without Ca++ The alternative pathway was determined under the condition that BBS (barbiturate buffered saline containing sucrose) and C3b deposition was measured over time.

[0308] result: Figure 17 The figure illustrates the variation of alternative pathway-driven C3b deposition levels with serum concentration in serum samples obtained from MASP-3-deficient, C4-deficient, and MBL-deficient subjects on a yeast polysaccharide-coated microtiter plate. Figure 17 As shown, serum from MASP-3-deficient patients exhibited residual alternative pathway (AP) activity at high serum concentrations (25%, 12.5%, and 6.25% serum concentrations), but significantly higher AP levels. 50 (That is, 9.8% serum is required to achieve 50% maximum C3 deposition).

[0309] Figure 18 The figure illustrates the results of testing in 10% of human serum samples from MASP-3-deficient, C4-deficient, and MBL-deficient human subjects under “conventional” alternative pathway-specific (AP-specific) conditions (i.e., BBS / EGTA / Mg). ++ No Ca ++ The variation of alternative pathway-driven C3b deposition levels over time on yeast polysaccharide-coated microtiter plates.

[0310] Table 11 below summarizes Figure 17 AP shown 50 Results and Figure 18 The C3b deposition time is shown as half of the time.

[0311] Table 11: Figure 17 and 18 Overview of the results shown Serum type <![CDATA[AP 50 (%)]]> <![CDATA[T 1 / 2 (min)]]> MASP-3 Defect Type (3MC Patients) 9.8 37.4 The mother (heterozygous) of the 3MC subject 4.3 17.2 The father (heterozygous) of the 3MC subject 4.3 20.9 C4 - Defective 4.0 11.6 MBL - Defective 4.8 11.0 Note: In BBS / Mg ++ In EGTA buffer, the lectin pathway-mediated function is lacking because of the absence of Ca in the buffer. ++ .

[0312] While not wishing to be bound by any particular theory, it is believed that the lower alternative pathway activity seen in MASP-3-deficient serum is due to the presence of active factor D in the serum of 3MC patients, and that the conversion of pre-factor D can still occur in the absence of MASP-3, albeit at a lower level, because these patients still express MASP-1 and HTRA1.

[0313] 3. In the serum of mice lacking MASP-2 or MASP-1 / 3, C3b deposition on mannan, yeast polysaccharide, and Streptococcus pneumoniae D39 was measured.

[0314] method: C3b deposition was determined on microtiter plates coated with mannan, yeast polysaccharide, and Streptococcus pneumoniae D39-, using mouse serum at concentrations ranging from 0% to 20% derived from MASP-2- / -, MASP-1 / 3- / -, and WT mice. This was done under “traditional” alternative pathway-specific conditions (i.e., BBS / EGTA / Mg). ++ No Ca ++ ), or under physiological conditions that allow both the lectin pathway and the alternative pathway to function (i.e., BBS / Mg ++ / Ca ++ C3b deposition was measured under these conditions.

[0315] result: Figure 19A The figure illustrates the effects of conventional alternative pathway-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 under physiological conditions that allow both the lectin pathway and the alternative pathway to function (BBS / Mg). ++ / Ca ++ The level of C3b deposition on a mannan-coated microtiter plate varies with serum concentration. Figure 19B The figure illustrates 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 C3b deposition level on a yeast polysaccharide-coated microtiter plate varied with serum concentration. Figure 19C The figure illustrates 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 under physiological conditions that allow both the lectin pathway and the alternative pathway to function (BBS / Mg). ++ / Ca ++The level of C3b deposition on a microtiter plate coated with Streptococcus pneumoniae D39- varies with serum concentration.

[0316] Figure 20A The diagram illustrates 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 mannan-coated microtiter plates, using serum concentrations ranging from 0% to 1.25%. Figure 20B The diagram illustrates 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 / EGTA / Mg) ++ / Ca ++ The results of C3b deposition assays performed on yeast polysaccharide-coated microtiter plates were obtained using serum concentrations ranging from 0% to 1.25%. Figure 20C The diagram illustrates 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 / EGTA / 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%.

[0317] like Figure 20A As shown in -C, it also applies to traditional alternative pathways under 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 ++ C3b deposition was measured using serum at higher dilutions ranging from 0% to 1.25% on mannan-coated plates. Figure 20A ); Yeast polysaccharide-coated plates ( Figure 20B ) and Streptococcus pneumoniae D39-coated plates ( Figure 20C At higher serum dilutions, the alternative pathway gradually disappears, resulting in lower levels of Ca2+. ++ The activity observed in MASP-1 / 3-deficient serum when present is MASP-2-mediated LP activity, and in Ca++ Its activity in MASP-2-deficient serum when present is MASP-1 / 3-mediated residual AP activation.

[0318] discuss: The results described in this embodiment demonstrate that the MASP-2 inhibitor (or MASP-2 KO) provides significant protection against Neisseria meningitidis infection by promoting the activation of the MASP-3-driven alternative pathway. Results from mouse and human serum lysate assays further confirm that, by monitoring serum bactericidal activity against Neisseria meningitidis, bactericidal activity against Neisseria meningitidis is absent in MBL-deficient serum (mouse MBL A and MBL C dual-deficient and human MBL-deficient serum).

[0319] 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 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 20A , 20B (20°C), but it also plays a role in the lysis of serum-sensitive bacteria. For example... Figure 1 As shown, for serologically susceptible pathogens such as Neisseria meningitidis, the proposed molecular mechanism responsible for the increased bactericidal activity of MASP-2-deficient or MASP-2-depleted serum / plasma is that, for bacterial 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, it needs to be activated / cleaved by MASP-1 to be converted into its enzymatically active form.

[0320] Further as Figure 1As 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 load 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 cleavage of C3b-bound factor B, 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. This results in C9 polymerization, which inserts into the bacterial surface structure and forms pores in the bacterial wall, leading to complement-targeted osmotic killing of the bacteria.

[0321] 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.

[0322] Example 5 This example demonstrates the inhibitory effect of MASP-2 deficiency and / or MASP-3 deficiency on erythrocyte lysis in blood samples obtained from a mouse model of paroxysmal nocturnal hemoglobinuria (PNH).

[0323] Background / Principle: Paroxysmal nocturnal hemoglobinuria (PNH), also known as Marchifava-Micheli syndrome, is an acquired, potentially life-threatening blood disorder characterized by complement-induced intravascular hemolytic anemia. The hallmark of PNH is chronic complement-mediated intravascular hemolysis, a consequence of unregulated activation of the alternative complement pathway caused by the lack of complement regulators CD55 and CD59 on PNH erythrocytes, followed by hemoglobinuria and anemia. (Lindorfer, MA, et al.) Blood 115 (11) (2010), Risitano, AM, Mini-Reviews in Medicinal Chemistry, 11:528-535 (2011). Anemia in PNH is caused by the destruction of red blood cells in the bloodstream. Symptoms of PNH include hematuria (due to the presence of hemoglobin in the urine), back pain, fatigue, shortness of breath, and thrombosis. PNH can occur spontaneously, known as "primary PNH," or occur in other bone marrow disorders such as aplastic anemia, known as "secondary PNH." Treatment for PNH includes blood transfusions to treat the anemia, anticoagulation to treat 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., 11:528-535 (2011)). N. Engl. J. Med. 350 (6):552-9 (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 (Hillmen P, et al., Mini-Reviews in Medicinal Chemistry, vol 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 achieved hemoglobin values ​​of approximately 11 gr / dL, and the remaining patients receiving eculizumab continued to exhibit moderate to severe (i.e., transfusion-dependent) anemia in approximately the same proportion (Risitano AM et al., Blood 113:4094-100 (2009)). As Risitano et al., Mini-Reviews Medicinal Chemistry As stated in 11:528-535 (2011), it has been demonstrated that PNH patients receiving eculizumab contain C3 fragments that bind to most of their PNH erythrocytes (unlike untreated patients), leading to the conclusion that the membrane-bound C3 fragments act as opsonins on PNH erythrocytes, causing them to be entrained in reticuloendothelial cells via specific C3 receptors and subsequently leading to extravascular hemolysis. Therefore, for those patients experiencing C3-fragment-mediated extravascular hemolysis, a treatment strategy other than eculizumab is required because they continue to require erythrocyte transfusions.

[0324] This embodiment describes a method for evaluating the effect of MASP-2- and MASP-3-deficient serum on erythrocyte lysis in blood samples obtained from a PNH mouse model, and demonstrates the efficacy of MASP-2 inhibition and / or MASP-3 inhibition in treating PNH subjects. It also supports the use of MASP-2 inhibitors and / or MASP-3 inhibitors (including dual or bispecific MASP-2 / MASP-3 inhibitors) in PNH subjects who have received treatment with C5 inhibitors such as eculizumab to improve C3 fragment-mediated extravascular hemolysis.

[0325] method: PNH animal model : Blood samples were obtained from gene-targeted mice with Crry and C3 deficiencies (Crry / C3- / -) and CD55 / CD59-deficient mice. These mice lost their respective surface complement regulators on their erythrocytes, making these erythrocytes susceptible to spontaneous complement autolysis, similar to PNH human erythrocytes.

[0326] To further sensitize these red blood cells, they were used with or without mannan coating, and then their hemolysis was tested in WTC56 / BL6 plasma, MBL-free plasma, MASP-2 - / - plasma, MASP-1 / 3 - / - plasma, human NHS, human MBL - / - plasma, and NHS treated with human MASP-2 antibody.

[0327] 1. Hemolysis assay of Crry / C3 and CD55 / CD59 dual-deficient mouse erythrocytes in MASP-2-deficient / depleted serum and controls. Day 1. Preparation of mouse RBCs (±mannan coating).

[0328] Materials include: Fresh mouse blood, BBS / Mg ++ / Ca ++ (4.4 mM barbituric acid, 1.8 mM sodium barbital, 145 mM NaCl, pH 7.4, 5 mM Mg) ++ 5mM Ca ++ Chromium chloride, CrCl3·6H2O (0.5 mg / mL in BBS / Mg) ++ / Ca ++ (in Chinese) and mannan, 100 µg / mL in BBS / Mg ++ / Ca ++ middle.

[0329] Whole blood (2 mL) was centrifuged at 2000 x g for 1–2 min in a refrigerated centrifuge at 4 °C. The plasma and erythrocyte sedimentation rate (ESR) layer were aspirated. The RBC precipitate was then resuspended in 2 mL of ice-cold BBS / gelatin / MgSO4 solution. ++ / Ca ++ The centrifugation process was repeated, and the sample was washed three times. After the third wash, the precipitate was resuspended in 4 mL of BBS / MgSO4. ++ / Ca ++ In the middle stage, 2 mL of RBC from an aliquot of the sample was reserved as an uncoated control. 2 mL of CrCl3 and 2 mL of mannan were added to the remaining 2 mL, and the sample was incubated at room temperature with gentle stirring for 5 min. The reaction was mediated by adding 7.5 mL of BBS / gelatin / Mg. ++ / Ca ++ The process was terminated. The sample was centrifuged as described above and resuspended in 2 mL of BBS / gelatin / Mg. ++ / Ca ++ Wash twice more as described above, and then store at 4°C.

[0330] Day 2. Hemolysis test Materials include BBS / gelatin / Mg ++ / Ca ++ (As above), test serum, 96-well round-bottom and flat-bottom plates, and a spectrophotometer, which reads the 96-well plates at 410–414 nm.

[0331] First, the RBC concentration was measured and the cell count was adjusted to 10. 9 / mL, and store at this concentration. Before use, dilute the cells to 10 in assay buffer. 8 / mL, then use 100 μL per well. Hemolysis is measured at 410–414 nm (higher sensitivity than 541 nm is permissible). In ice-cold BBS / gelatin / Mg ++ / Ca ++ Prepare test serum diluents. Transfer 100 µl of each serum diluent into a round-bottom plate. Add 100 µl of appropriately diluted RBC preparation (i.e., 10... 8 Incubate the plates at 37°C for approximately 1 hour, observing for hemolysis. (Photographs of each plate can be taken during this time.) Then centrifuge the plates at maximum speed for 5 minutes. Aspirate 100 µl of the liquid phase, transfer it to a flat-bottomed plate, and record the OD at 410–414 nm. Retain the RBC precipitates (these can be dissolved in water subsequently to obtain the opposite results).

[0332] Experiment #1 Fresh blood was obtained from CD55 / CD59 double-deficient mice, and blood and erythrocytes from Crry / C3 double-deficient mice were prepared as detailed above. The cells were separated, with half coated with mannan and the other half left untreated, and the final concentration was adjusted to 10⁸ / mL, of which 100 µl was used for the hemolysis assay, which was performed as described above.

[0333] Results of Experiment #1: In the PNH animal model, the lectin pathway is involved in erythrocyte lysis. In preliminary experiments, it was determined that uncoated WT mouse erythrocytes did not lyse in any mouse serum. Further analysis showed that mannan-coated Crry- / - mouse erythrocytes lysed slowly in WT mouse serum (over 3 hours at 37°C), but did not lyse in MBL-free serum (data not shown).

[0334] Mannan-coated Crry- / - mouse erythrocytes were found to lyse rapidly in human serum but not in heat-inactivated NHS. Importantly, mannan-coated Crry- / - mouse erythrocytes lysed at NHS dilutions up to 1 / 640 (i.e., lysed at all dilutions of 1 / 40, 1 / 80, 1 / 160, 1 / 320, and 1 / 640) (data not shown). At this dilution, alternative pathways were ineffective (AP functional activity was significantly reduced at serum concentrations below 8%).

[0335] Conclusion of Experiment #1 Mannan-coated Crry- / - mouse erythrocytes dissolved very well in highly diluted human serum with MBL, but not in highly diluted human serum without MBL. The efficient dissolution at each serum concentration measured suggests that alternative pathways are not involved in this dissolution or that such dissolution does not require alternative pathways. The inability of MBL-deficient mouse serum and human serum to dissolve mannan-coated Crry- / - mouse erythrocytes indicates that the classical pathway also does not play a role in the observed dissolution. Because the lectin pathway is required to recognize the molecule (i.e., MBL), this dissolution is mediated by the lectin pathway.

[0336] Experiment #2 Fresh blood was obtained from Crry / C3 and CD55 / CD59 dual-deficient mice, and mannan-coated Crry- / - mouse erythrocytes were analyzed in the presence of the following human serum in the hemolysis assay described above: MASP-3 - / -; MBL-free; WT; NHS pretreated with human MASP-2 antibody; and heat-inactivated NHS as a control.

[0337] Results of Experiment #2: In the PNH animal model, MASP-2 inhibitors and MASP-3 deficiency prevented erythrocyte lysis. Mannan-coated Crry- / - mouse erythrocytes were incubated with the following: NHS diluted to 1 / 640 (i.e., 1 / 40, 1 / 80, 1 / 160, 1 / 320, and 1 / 640), human MBL- / - serum, human MASP-3-deficient serum (from 3MC patients), and NHS pretreated with MASP-2 mAb, and heat-inactivated NHS as controls.

[0338] Centrifuge the ELISA microtiter plate and collect non-lysed red blood cells at the bottom of the round wells. Collect the supernatant from each well and determine the amount of hemoglobin released from the lysed red blood cells by reading OD415 nm in an ELISA plate reader.

[0339] MASP-3- / - serum was observed to be completely insoluble in mannan-coated mouse erythrocytes. As expected, no lysis was observed in the control heat-inactivated NHS (negative control). MBL- / - human serum lysed mannan-coated mouse erythrocytes at 1 / 8 and 1 / 16 dilutions. MASP-2-antibody-pretreated NHS lysed mannan-coated mouse erythrocytes at 1 / 8 and 1 / 16 dilutions, while WT human serum lysed mannan-coated mouse erythrocytes at dilutions as low as 1 / 32.

[0340] Figure 21 The illustration shows how a series of serum dilutions of human serum caused hemolysis of mannan-coated mouse erythrocytes in sera from MASP-3- / -, heat-inactivated (HI) NHS, MBL- / -, NHS pretreated with MASP-2 antibody, and NHS controls (measured by the release of hemoglobin from lysed mouse erythrocytes (Crry / C3- / -) into the supernatant, which was determined by photometric methods).

[0341] Figure 22 The illustration shows how a range of serum concentrations of human serum caused hemolysis of mannan-coated mouse erythrocytes in sera from MASP-3- / -, heat-inactivated (HI) NHS, MBL- / -, NHS pretreated with MASP-2 antibody, and NHS controls (measured by the release of hemoglobin from lysed mouse erythrocytes (Crry / C3- / -) into a supernatant, which was determined by photometric methods).

[0342] according to Figure 21 and 22 The results demonstrate that inhibiting MASP-3 prevents any complement-mediated lysis of sensitized erythrocytes lacking protection from autologous complement activation. Inhibition of MASP-2 by the MASP-2 antibody significantly altered the CH... 50 It is protective to some extent, but MASP-3 inhibition is more effective.

[0343] Experiment #3 In the hemolysis assay described above, uncoated Crry- / - mouse erythrocytes from fresh blood of Crry / C3 and CD55 / CD59 dual-deficient mice were analyzed in the presence of the following sera: MASP-3- / -; MBL- / -; WT; NHS pretreated with human MASP-2 antibody; and heat-inactivated NHS as a control.

[0344] result: Figure 23 The figure illustrates the hemolysis of uncoated mouse erythrocytes in a range of serum concentrations from 3MC (MASP-3- / -) patients, heat-inactivated (HI) NHS, MBL- / -, MASP-2 antibody-pretreated NHS, and NHS controls (measured by the release of hemoglobin from lysed WT mouse erythrocytes into the supernatant, which was determined spectrophotometrically). Figure 23 As shown in the diagram and summarized in Table 12, inhibition of MASP-3 demonstrates that it inhibits complement-mediated lysis of non-sensitized WT mouse erythrocytes.

[0345] Figure 24 The figure illustrates the hemolysis of uncoated mouse erythrocytes by a range of serum concentrations from heat-inactivated (HI) NHS, MBL- / -, MASP-2 antibody-pretreated NHS, and NHS controls (measured by the release of hemoglobin from lysed mouse erythrocytes (CD55 / 59- / -) into a supernatant, which was determined photometrically). Figure 24 As shown in the diagram and summarized in Table 12, it is demonstrated that inhibiting MASP-2 provides protection to a limited extent.

[0346] Table 12: CH4 concentrations expressed as serum concentrations 50 value serum WT CD55 / 59 - / - 3MC patients Insoluble Insoluble Heat-inactivated NHS Insoluble Insoluble MBL AO / XX donor (MBL defective type) 7.2% 2.1% NHS + MASP-2 antibody 5.4% 1.5% NHS 3.1% 0.73% Note: CH 50 "This is the point at which complement-mediated hemolysis reaches 50%."

[0347] In summary, the results of this embodiment demonstrate that inhibition of MASP-3 prevents the lysis of any complement-sensitized and non-sensitized erythrocytes lacking protection from autocomplement activation. MASP-2 inhibition also provides some degree of protection. Therefore, inhibitors of MASP-2 and MASP-3, alone or in combination (i.e., co-administered, sequentially administered) or bispecific or dual inhibitors of MASP-2 / MASP-3 can be used to treat PNH subjects and can also be used to improve (i.e., inhibit, prevent, or reduce the severity of) extravascular hemolysis in PNH patients who have received treatment with C5 inhibitors such as eculizumab (Soliris®).

[0348] Example 6 This example describes a hemolysis assay, which measures the lysis of mannan-coated rabbit erythrocytes in the presence of WT or MASP-1 / 3- / - mouse serum.

[0349] method: 1. Determination of hemolysis of rabbit RBCs (mannan-coated) in mouse MASP-1 / 3-deficient serum and WT control serum Day 1. Preparation of rabbit RBCs.

[0350] Materials include: fresh rabbit blood, BBS / Mg ++ / Ca ++ (4.4 mM barbituric acid, 1.8 mM sodium barbital, 145 mM NaCl, pH 7.4, 5 mM Mg) ++ 5 mM Ca ++ BBS / Mg containing 0.1% gelatin ++ / Ca ++ 1. Buffer solution containing chromium chloride, i.e., CrCl3·6H2O (0.5 mg / mL in BBS / Mg ++ / Ca ++ (in Chinese) and mannan, 100 µg / mL in BBS / Mg ++ / Ca ++ middle.

[0351] 1. Divide rabbit whole blood (2 mL) into two 1.5 mL microcentrifuge tubes and centrifuge at 8000 rpm (approximately 5.9 rcf) for 3 minutes in a refrigerated microcentrifuge at 4°C. Resuspend in ice-cold BBS / Mg ++ / Ca ++ The RBC precipitate was then washed three times. After the third wash, the precipitate was resuspended in 4 mL of BBS / MgSO4. ++ / Ca ++In the first step, 2 mL of the aliquot was added to a 15 mL Falcon tube as an uncoated control. The remaining 2 mL of the RBC aliquot was diluted in 2 mL of CrCl3 buffer, 2 mL of mannan solution was added, and the suspension was incubated at room temperature for 5 minutes with gentle stirring. The solution was then further diluted by adding 7.5 mL of BBS / 0.1% gelatin / Mg... ++ / Ca ++ The reaction was then terminated. The red blood cells were precipitated and treated as described above with BBS / 0.1% gelatin / Mg. ++ / Ca ++ RBCs were washed twice. The RBC suspension was stored at 4°C in BBS / 0.1% gelatin / Mg ++ / Ca ++ middle.

[0352] 2. Dilute 100 µl of suspended RBC with 1.4 mL of water and centrifuge at 8000 rpm (approximately 5.9 rcf) for 3 minutes. Adjust the OD of the supernatant at 541 nm to 0.7 (an OD of 0.7 at 541 nm is equivalent to approximately 10...). 9 Red blood cells / mL).

[0353] 3. Resuspend the RBCs in BBS / 0.1% gelatin / Mg ++ / Ca ++ Dilute to a concentration of 10 8 / mL.

[0354] 4. In cold BBS / gelatin / Mg ++ / Ca ++ Prepare test serum diluents and transfer 100 µl of each serum diluent into the corresponding well of the round-bottom plate. Add 100 µl of appropriately diluted RBC (i.e., 10... 8 / mL) to each well. As a control for complete dissolution, purified water (100 µL) was mixed with diluted RBC (100 µL), resulting in 100% dissolution, while BBS / 0.1% gelatin / Mg ++ / Ca ++ Serum-free (100 µL) was used as a negative control. The plate was then incubated at 37°C for 1 hour.

[0355] 5. Centrifuge the round-bottom plate at 3250 rpm for 5 minutes. Transfer the supernatant (100 µL) from each well to the corresponding well of the flat-bottom plate and read the OD at 415–490 nm using an ELISA reader. Report the results as the ratio of the OD at 415 nm to the OD at 490 nm.

[0356] result: Figure 25 The illustration shows how a range of mouse serum concentrations induced hemolysis of mannan-coated rabbit erythrocytes in sera from MASP-1 / 3- / - and WT controls (measured by the release of hemoglobin from the lysed rabbit erythrocytes into a supernatant, which was analyzed photometrically). Figure 25 As shown, inhibition of MASP-3 demonstrates that complement-mediated lysis of mannan-coated WT rabbit erythrocytes is prevented. These results further support the use of MASP-3 inhibitors to treat one or more aspects of PNH as described in Example 5.

[0357] Example 7 This example illustrates the effect of Ca in factor D-deficient serum. ++ When they are present, alternative pathways are activated.

[0358] Experiment #1: Determination of C3b deposition under alternative pathway-specific conditions method: Determination of C3b deposition on yeast polysaccharide-coated microtiter plates under alternative pathway-specific conditions (BBS / EGTA / Mg) ++ No Ca ++ The following mouse serum was used for incremental dilutions: Factor D - / -; MASP - / -; and WT.

[0359] result: Figure 26 The figure illustrates the variation of C3b deposition levels (OD 405 nm) with serum concentration in serum samples from factor D- / -, MASP-2- / -, and WT mice under alternative pathway-specific conditions. Figure 26 As shown, under these conditions, C3 was completely unactivated in factor D- / - mouse serum and the alternative pathway did not function. MASP-2- / - serum showed activation of the alternative pathway at a rate similar to that of WT serum. These results confirm that, under Ca... ++ In its absence, the D factor is required for C3b deposition. This is consistent with evidence that MASP-3 cannot be converted to its enzymatically active form under these conditions, since the interactions of MASP-1, MASP-3 activating enzymes, and MASP-3 with their respective carbohydrate recognition components are Ca... ++ - Dependency.

[0360] Experiment #2: Determination of C3b deposition under physiological conditions method: Under physiological conditions (BBS / Ca) ++ / Mg++ (Also allowing LP and AP to function) C3b deposition assays were performed using the following incremental dilutions of mouse serum: Factor D - / -; MASP-2 - / -; and WT.

[0361] result: Figure 27 The diagram illustrates the physiological conditions (in Ca) ++ In a C3b deposition assay performed (in the presence of the virus), samples from serum of factor D- / -, MASP-2- / -, and WT mice were used. The C3b deposition level (OD 405 nm) varied with serum concentration. Figure 27 As shown, C3 activation in factor D- / - mouse serum was achieved via both lectin and alternative pathways, with no difference compared to WT serum at specified serum dilutions. MASP- / - serum showed C3 turnover at lower serum dilutions via the alternative pathway only (i.e., MASP-3-driven alternative pathway activation). These results indicate that in Ca... ++ When present, factor D is not needed because MASP-3 can drive alternative pathway activity.

[0362] Experiment #3: Determination of C3b deposition in the presence or absence of MASP-2 mAb using serum from mice deficient in factor B or factor D. method: Under physiological conditions (BBS / Ca) ++ / Mg ++ C3b deposition was determined on a mannan-coated microtiter plate as described below: 1. Microtitering of ELISA plates: Coating with a coating buffer containing mannan (1 µg / mL) (15 mM Na2CO3, 35 mM NaHCo3, 0.02% sodium azide, pH 9.6) overnight at 4°C.

[0363] 2. The following day, the remaining protein binding sites were blocked at room temperature for 2 hours with 250 µl / well of BBS containing 0.1% HSA (4 mM barbiturate, 145 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4) at room temperature.

[0364] 3. Wash each plate three times with washing buffer (TBS containing 0.05% Tween 20 and 5 mM CaCl2).

[0365] 4. At the specified time points, add serum samples diluted 1:10 in BBS to each well. Wells containing only buffer serve as negative controls. Incubate the plate at 37°C for up to 40 minutes.

[0366] 5. Then wash each plate three times with washing buffer.

[0367] 6. Then add 100 µl of rabbit anti-human C3c (Dako) diluted 1:5000 in washing buffer to the wells and incubate each plate at 37°C for 90 minutes.

[0368] 7. After washing three times with washing buffer, add 100 µl of alkaline phosphatase-conjugated anti-rabbit solution diluted 1:5000 in washing buffer to the wells and incubate at room temperature for 90 minutes.

[0369] 8. After washing, alkaline phosphatase was detected by adding 100 µl of substrate solution.

[0370] 9. After incubation for 15 minutes, the optical density was measured at OD 405 nm.

[0371] result: Figure 28 The diagram illustrates the physiological conditions (in Ca) ++ In a C3b deposition assay performed in the presence of MASP-2 mAb, the C3b deposition level (OD405 nm) in mouse serum samples obtained from factor D- / - or factor B- / - mice varied with serum incubation time (minutes) in the presence or absence of MASP-2 mAb. Figure 28 As shown, there was no difference in C3b deposition between WT and factor D- / - serum, strongly supporting the conclusion that MASP-3 can initiate alternative pathway activation, even in the absence of factor D. The observed signal is considered to be due to the simultaneous activation of both the lectin pathway and the alternative pathway. Further... Figure 28 As shown, Factor D- / - plus MASP-2 mAb showed activation of the alternative pathway mediated only by MASP-3-. Factor B- / - plus MASP-2 mAb was only background (data not shown). Heat-inactivated serum was used as a background control, which was the same as that for Factor D- / - and Factor B- / - and MASP-2 (data not shown).

[0372] In summary, the results of this embodiment demonstrate that only under non-physiological conditions (i.e., when in BBS / EGTA / Mg) ++ In Ca ++ Factor D is only needed when the alternative pathway is activated (in the absence of the pathway). I...

Claims

1. A pharmaceutical composition comprising at least one complement pathway inhibitor and a pharmaceutically acceptable carrier, wherein the at least one inhibitor comprises at least one of a MASP-2 inhibitor, a MASP-3 inhibitor, and / or a MASP-1 inhibitor.

2. The pharmaceutical composition of claim 1, wherein the at least one inhibitor comprises a combination of a first molecule and a second molecule, the first molecule being a MASP-3 inhibitor and the second molecule being a MASP-2 inhibitor.

3. The pharmaceutical composition of claim 1, wherein the at least one inhibitor comprises a single molecular entity having activity as a MASP-3 inhibitor and activity as a MASP-2 inhibitor.

4. The pharmaceutical composition of claim 1, wherein the MASP-3 inhibitor is a MASP-3 monoclonal antibody or an antigen-binding fragment thereof.

5. The pharmaceutical composition of claim 1, wherein the MASP-2 inhibitor is a MASP-2 antibody or an antigen-binding fragment thereof.

6. The pharmaceutical composition of claim 1, wherein the MASP-3 inhibitor inhibits MASP-3-mediated factor B cleavage.

7. The pharmaceutical composition of claim 1, wherein the MASP-3 inhibitor inhibits factor D maturation.

8. The pharmaceutical composition of claim 1, wherein the MASP-1 inhibitor is a MASP-1 antibody or a fragment thereof.

9. The pharmaceutical composition of claim 1, wherein the MASP-1 inhibitor specifically binds to a portion of MASP-1, having an affinity at least 10 times greater than that binding to MASP-3 (SEQ ID NO:8).

10. The pharmaceutical composition of claim 1, wherein the MASP-1 inhibitor specifically binds to the serine protease domain of MASP-1 (aa 449-694 of SEQ ID NO: 10).

11. The pharmaceutical composition of claim 1, wherein the MASP-1 inhibitor is further bound to a portion of MASP-3 (SEQ ID NO:8).

12. The pharmaceutical composition of claim 1, wherein the MASP-1 inhibitor is further bound to a portion of MASP-2 (SEQ ID NO:5).

13. The pharmaceutical composition of claim 1, wherein the MASP-3 inhibitor specifically binds to a portion of MASP-3 (SEQ ID NO:8: full length).

14. The pharmaceutical composition of claim 1, wherein the MASP-3 inhibitor specifically binds to a portion of MASP-3, having an affinity at least 10 times greater than that binding to MASP-1 (SEQ ID NO: 10).

15. The pharmaceutical composition of claim 13, wherein the MASP-3 inhibitor specifically binds to the serine protease domain of MASP-3 (aa 450-711 of SEQ ID NO: 8).

16. The pharmaceutical composition of claim 1, wherein the MASP-3 inhibitor is further bound to a portion of MASP-2 (SEQ ID NO:5).

17. The pharmaceutical composition of claim 16, wherein the MASP-3 inhibitor is a dual MASP-2 / MASP-3 inhibitor that binds to conserved regions within the serine protease domains of MASP-2 and MASP-3.

18. The pharmaceutical composition of claim 1, wherein the MASP-3 inhibitor is a MASP-3 antibody, and wherein the composition further comprises a MASP-2 antibody.

19. The pharmaceutical composition of claim 1, wherein the MASP-3 inhibitor is a MASP-3 antibody, and wherein the composition further comprises a MASP-1 antibody.

20. The pharmaceutical composition of claim 1, wherein the composition comprises MASP-1 antibody, MASP-2 antibody, and MASP-3 antibody.

21. The pharmaceutical composition of any one of claims 4, 5 and 8, wherein the antibody or a fragment thereof is selected from recombinant antibodies, antibodies with reduced effector function, chimeric antibodies, and humanized antibodies or human antibodies.

22. The pharmaceutical composition of claim 1, wherein the composition is formulated for systemic delivery.

23. The pharmaceutical composition of claim 22, wherein the composition is formulated for subcutaneous, intramuscular, intravenous, intra-arterial delivery or delivery as an inhaler.

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