EA042534B1 - METHODS FOR THE TREATMENT OF CONDITIONS ASSOCIATED WITH MASP-2 DEPENDENT COMPLEMENT ACTIVATION - Google Patents

Description

Cross-reference to related applications

This patent application claims the priority of U.S. Provisional Application No. 62/406,726, filed October 11, 2016, and U.S. Provisional Application No. 62/252,814, filed November 9, 2015, the contents of which are incorporated into the present invention by reference to them.

Sequence listing statement

The sequence listing related to this patent is not provided in the form of a paper copy, but as a file in text format, and, therefore, the contents of this file are incorporated into the present invention by reference to it. The text file containing the sequence listing has the following name: MP_1_0248_PCT_Sequence_Listing_as_Filed_20161109.txt. A 115 kilobyte text file, created on November 8, 2016, is provided to the EFS-Web site for electronic registration of applications along with a description of the patent application.

State of the art

The complement system provides an initial mechanism for initiating, amplifying, and directing the immune response to microbial infection and other acute lesions (M.K. Liszewski and J.P. Atkinson, 1993, in Fundamental Immunology, Third Edition, edited by W.E. Paul, Raven Press, Ltd. , New York) in humans and other vertebrates. Complement activation provides the first important protective barrier against potential pathogens, but complement activity that promotes a protective immune response may also pose a potential threat to the host (K. R. Kalli, et al., Springer Semin. Immunopathol. 15:417 431, 1994; B. P. Morgan, Eur. J. Clinical Investig. 24:219 228, 1994). For example, proteolysis products C3 and C5 cause recruitment and activation of neutrophils. Neutrophils are essential for host defense, but activated neutrophils act indiscriminately by releasing destructive enzymes and can cause organ damage. In addition, complement activation can lead to the deposition of lytic complement components on adjacent host cells as well as on target microbes, resulting in lysis of the host cells.

The complement system has also been implicated in the pathogenesis of various acute and chronic disease states, including myocardial infarction, stroke, acute respiratory distress syndrome (ARDS), reperfusion injury, septic shock, increased capillary permeability after thermal burns, inflammation after cardiopulmonary bypass surgery, rejection transplant, rheumatoid arthritis, multiple sclerosis, myasthenia gravis and Alzheimer's disease. In almost all of these conditions, complement itself is not the cause, but it is one of several factors involved in pathogenesis. However, complement activation may be the underlying pathophysiological mechanism and serve as an effective point of clinical control in many of these disease states. Increasing recognition of the important role that complement plays in tissue damage in various disease states points to the need for drugs that effectively inhibit complement. Currently, the only drug that targets complement and is approved for use in humans is eculizumab (Soliris®), which is an anti-C5 antibody. In addition, C5 is one of several effector molecules that act in the subsequent stages of the reaction of the complement system, and blockade of C5 does not lead to inhibition of the activation of the complement system. Therefore, an inhibitor of the stages of initiation of complement activation can provide significant advantages over an inhibitor of the subsequent stages of the reaction of the complement system.

It is now generally accepted that the complement system can be activated in three different ways: the classical pathway, the lectin pathway, and the alternative pathway. The classical pathway is usually triggered by a complex consisting of host antibodies bound to a foreign particle (i.e., an antigen) and, accordingly, prior exposure to the antigen is required to generate a specific humoral immune response. Since the activation of the classical pathway depends on the host's pre-adaptive immune response, the classical pathway is part of the acquired immune system. In contrast, the lectin and alternative pathways are independent of the adaptive immune response and are part of the innate immune system.

Activation of the complement system leads to the subsequent activation of zymogens of serine-protease factors. The first step in activation of the classical pathway is the binding of the C1q specific recognition molecule to an antigen bound to IgG and IgM molecules. C1q is associated with the serine proenzymes C1r and C1s in the form of a complex, designated C1. After binding of C1q to the immune complex, autoproteolytic cleavage of the Arg-Ile site of C1r is followed by C1-mediated cleavage and activation of C1s, giving it the ability to cleave C4 and C2. C4 is cleaved into two fragments, designated C4a and C4b, and similarly, C2 is cleaved into components C2a and C2b. C4b fragments can form covalent bonds with nearby hydroxyl groups or amino groups and generate C3-convertase (C4b2a) as a result of non-covalent interaction with the C2a fragment of activated C2. C3 convertase (C4b2a) activates C3 by proteolytic cleavage into subcomponents C3a and C3b, which leads to the formation of C5 convertase (C4b2a3b), which, as a result of C5 cleavage, forms a membrane attack complex (C5b in combination with C6, C7, C8 and C9 is also called MAC) , which can destroy cell membranes, causing cell lysis. Activated forms of C3 and C4 (C3b and C4b) are covalently deposited on the surfaces of foreign targets, which are recognized by complement receptors on a variety of phagocytes.

Regardless of this, the first step in the activation of the complement system via the lectin pathway is also the binding of specific recognition molecules, after which the associated serine-protease proenzymes are activated. However, instead of immune complex binding by C1q, recognition molecules in the lectin pathway include a group of carbohydrate-binding proteins (mannan-binding lectin (MBL), H-ficolin, M-ficolin, L-ficolin, and C-type lectin CL-11) that collectively referred to as lectins. 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., Immunology 101:225 232 (2000)). See also J. Luet et al., Biochim Biophys Acta 1572:387-400 (2002); Holmskov et al., Annu Rev Immunol 21:547-578 (2003); Teh et al., Immunology 101:225-232 (2000); Hansen et al., J. Immunol 185(10):6096-6104 (2010).

Ikeda et al., J. Biol. Chem. 262:7451 7454, (1987) was the first to demonstrate that MBL, like C1q, can activate the complement system in a C4-dependent manner upon binding to yeast mannan-coated erythrocytes. MBL, a member of the collectin protein family, is a calcium-dependent lectin that binds to carbohydrates at 3- and 4-hydroxy groups oriented in the equatorial plane of the pyranose ring. Therefore, the main ligands for MBL are D-mannose and N-acetyl-O-glucosamine, and carbohydrates that do not meet this steric requirement show little or no affinity for MBL (Weis, W.I., et al., Nature 360:127- 134, 1992). The interaction between MBL and monovalent sugars is very weak and their dissociation constants are usually in the single digit millimolar range. MBL achieves strong specific binding to glycan ligands due to avidity, that is, due to simultaneous interaction with multiple monosaccharide residues located in close proximity to each other (Lee, R.T., et al., Archiv. Biochem. Biophys. 299: 129-136, 1992 ). MBL recognizes carbohydrate moieties that normally decorate the surface of microorganisms such as bacteria, yeasts, parasites and some viruses. In contrast, MBL does not recognize D-galactose and sialic acid, the pre-terminal and terminal sugars that normally decorate the mature complex of glycoconjugates present on mammalian plasma and cell surface glycoproteins. This binding specificity is believed to promote recognition of foreign surfaces and contribute to protection against self-activation. However, MBL does not bind with high affinity to high-mannose glycan precursor clusters on N-linked glycoproteins and glycolipids sequestered in the endoplasmic reticulum and Golgi apparatus of mammalian cells (Maynard, Y., et al., J. Biol. Chem. 257 :3788-37 94, 1982). Therefore, damaged cells are potential targets for activation along the lectin pathway through binding to MBL.

Ficolins contain a lectin domain that is different from the MBL lectin domain, which is called the fibrinogen-like domain. Ficolins bind sugar residues Ca ^++- independently. Three types of ficolin have been identified in humans (L-ficolin, M-ficolin, and H-ficolin). Two serum ficolins, L-ficolin and H-ficolin, both have specificity for N-acetyl-O-glucosamine, but N-ficolin also binds N-acetyl-O-galactosamine. Differences in the sugar specificity of L-ficolin, H-ficolin, CL-11, and MBL mean that different lectins can be complementary and differently targeted, albeit partially overlapping, glycoconjugates. This concept is supported by recently published scientific evidence that of all known lectins involved in the lectin pathway, only L-ficolin specifically binds to lipoteichoic acid, a cell wall glycoconjugate found on all Gram-positive bacteria (Lynch, NJ, et al. , J. Immunol. 172:1198-1202, 2004). The amino acid sequences of collectins (ie MBL) and ficolins do not share any significant similarity. However, these two groups of proteins have similar domain organizations and, like C1q, form into oligomeric structures that maximize their multisite binding capacity.

MBL concentrations in the blood serum of healthy individuals are characterized by high variability and are genetically regulated by polymorphisms/mutations in both the promoter and coding regions of the MBL gene. The expression level of MBL as an acute phase protein is further upregulated during inflammation. L-ficolin is present in serum at concentrations similar to those of MBL. Therefore, the efficiency of the L-ficolin branch of the lectin pathway is, in principle, comparable to that of the MBL branch. MBL and ficolins can also act as opsonins, allowing phagocytes to target MBL- and ficolin-decorated surfaces (see Jack et al., J.

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Leukoc Biol., 77(3):328-36 (2004); Matsushita and Fujita, Immunobiology, 205(4-5):490-7 (2002); Aoyagi et al., J. Immunol. 174(1):418-25 (2005)). Such opsonization requires interaction of these proteins with phagocyte receptors (Kuhlman, M, et al., J. Exp. Med. 169:1733, 1989; Matsushita, M., et al., J. Biol. Chem.

271:2448-54, 1996), whose identity has not yet been established.

Human MBL forms a specific and high affinity interaction through its collagen-like domain with unique C1r/C1s-like serine proteases called MBL-associated serine proteases (MASPs). Three MASPs have been described so far. One MASP enzyme was first identified and characterized as the enzyme responsible for initiating the complement cascade (i.e., C2 and C4 cleavage reactions) (Matsushita M & Fujita T., J. Exp Med 176(6):1497-1502 (1992 ), Ji, Y. H., et al., J. Immunol. 150:571-578, 1993). Then it was found that the activity of MASP is actually the activity of a mixture of two proteases MASP-1 and MASP-2 (Thiel, S., et al., Nature 386:506-510, 1997). However, it has been shown that one MBL-MASP-2 complex is sufficient for complement activation (Vorup-Jensen, T., et al., J. Immunol. 165:2093-2100, 2000). In addition, only MASP-2 cleaved C2 and C4 with high conversion (Ambrus, G., et al., J. Immunol. 170: 1374-1382, 2003). Therefore, MASP-2 is the protease responsible for activating C4 and C2 to generate a C3 convertase, C4b2a. This differs significantly from the C1 complex of the classical pathway, where the coordinated action of two specific serine proteases (C1r and C1s) leads to the activation of the complement system. In addition, a third new MASP-3 protease has been isolated (Dahl, M.R., et al., Immunity 15: 127-35, 2001). MASP-1 and MASP-3 are alternatively spliced products of the same gene.

MASPs have the same domain organizations as those of C1r C1s, the enzymatic components of the C1 complex (Sim, R. B., et al., Biochem. Soc. Trans. 25:545, 2000). These domains include the N-terminal domain of C1r/C1s/vascular endothelial growth factor (VEGF) sea urchin/bone morphogenetic protein (CUB); an epidermal growth factor-like domain; second CUB domain; a tandem of complement regulatory protein domains and a serine protease domain. As in C1 proteases, MASP-2 activation is mediated by cleavage of the Arg-Ile bond adjacent to the serine protease domain, leading to cleavage of the enzyme into disulfide-linked A and B chains, where the last of the two chains consists of the serine protease domain. .

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

A number of findings suggest that various MBLMASP complexes exist and that a large fraction of serum MASP does not complex with MBL (Thiel, S., et al., J. Immunol. 165:878-887, 2000). Both H-ficolin and L-ficolin bind to all MASPs and activate the complement lectin pathway in the same manner as MBL does (Dahl, M.R., et al., Immunity 15: 127-35, 2001; Matsushita, M. , et al., J. Immunol 165:3502-3506, 2002). Both pathways, the lectin pathway and the classical pathway, form a common C3 convertase (C4b2a), and at this stage the two pathways converge.

It is generally accepted that the lectin pathway plays an important role in protecting the uninfected host from infection. Strong evidence for the involvement of MBL in host defense comes from studies of patients with reduced serum levels of functional MBL (Kilpatrick, D.C., Biochim. Biophys. Acta 1572:401-413, 2002). These patients are predisposed to recurrent bacterial and fungal infections. Such symptoms usually appear at a young age during a well-defined time interval of reduced body resistance as maternal antibody titers decline, but before the development of a humoral response with the formation of a full antibody repertoire. This syndrome is often the result of mutations at several sites in the MBL collagen portion that interfere with the normal formation of MBL oligomers. However, since MBL can function as an opsonin independent of complement, it is not known to what extent the increased susceptibility to infection is due to impaired complement activation.

In contrast to the classical and lectin pathways, no alternative pathway initiators have been found that provide the recognition functions found in C1q and lectins in the other two pathways. It is now generally accepted that the alternative pathway spontaneously undergoes low-level cyclic activation, which can easily be amplified on foreign or other abnormal surfaces (bacteria, yeast, virus-infected cells, or damaged tissue) that do not have their own molecular elements that control spontaneous activation. complement. There are four plasma proteins directly involved in the activation of the alternative pathway, namely C3, factors B and D, and properdin.

There is already a large amount of evidence for the involvement of the classical and alternative pathways.

- 3 042534 complement activation in the pathogenesis of non-communicable diseases in humans, but the role of the lectin pathway has yet to be elucidated. Recent studies have shown that activation of the lectin pathway can induce complement activation and associated inflammation in ischemia/reperfusion injury. Collard, CD., et al., Am. J. Pathol. 156:1549-1556, 2000 cultured endothelial cells subjected to oxidative stress have been shown to bind MBL and exhibit C3 precipitation upon exposure to human serum. In addition, treatment of human sera with blocking anti-MBL monoclonal antibodies resulted in inhibition of MBL binding and complement activation. These findings were refined in a rat myocardial ischemia reperfusion model where rats treated with a blocking anti-rat MBL antibody showed a significant reduction in myocardial injury after coronary artery occlusion compared to rats treated with a control antibody (Jordan, J.E., et al., Circulation 104:1413-1418, 2001). The molecular mechanism of MBL binding to vascular endothelium after oxidative stress remains unclear, but recent studies suggest that activation of the lectin pathway after oxidative stress may be mediated by MBL binding to vascular endothelial cytokeratins rather than glycoconjugates (Collard, CD., et al. Am J Pathol 159:1045-1054, 2001). Other studies have identified the role of the classical and alternative pathways in the pathogenesis of ischemia/reperfusion injury, and the role of the lectin pathway in this disease is still a matter of debate (Riedermann, N.C, et al., Am. J. Pathol. 162:363-367, 2003 ).

Recent studies have shown that MASP-1 (and possibly also MASP-3) is required to convert factor D, i.e., the alternative pathway activation enzyme, from its zymogenic form to its enzymatically active form (see Takahashi M. et al. ., J. Exp Med 207(1):29-37 (2010)). The physiological importance of this process is indicated by the absence of functional activity of the alternative pathway in the plasma of MASP-1/3-deficient mice. The alternative pathway requires proteolytic production of C3b from native C3. Since the alternative pathway C3 convertase (C3bBb) contains C3b as the main subunit, the question of the origin of the first C3b through the alternative pathway is a confusing problem and requires in-depth research.

C3 belongs to a family of proteins (along with C4 and macroglobulin α-2) that contain a rare post-translational modification known as a thioether bond. The thioether group consists of glutamine, in which the terminal carbonyl group forms a covalent thioether bond with the sulfhydryl group of cysteine without three amino acids. This bond is unstable, and the electrophilic glutamyl thioether can react with nucleophilic groups such as hydroxyl groups or amino groups, thereby forming a covalent bond with other molecules. The thioether bond is quite stable when sequestered in the hydrophobic pocket of intact C3. However, the proteolytic cleavage of C3 into C3a and C3b leads to the action of a highly reactive thioether bond on C3b, and after the nucleophilic attack of nearby fragments containing hydroxyl groups or amino groups, C3b becomes covalently associated with the target. In addition to the well documented role of the C3 thioester in the covalent binding of C3b to complement targets, the C3 thioester is also believed to play a major role in triggering the alternative pathway. According to the generally accepted blank activation theory, the alternative pathway is initiated by the production of a liquid phase convertase, iC3Bb, which is formed from C3 together with a hydrolyzed thioester (iC3; C3(H ₂ O)) and factor B (Lachmann, PJ, et al., Springer Semin Immunopathol 7:143162, 1984). C3b-sub6 C3(H ₂ O) is produced from native C3 by slow spontaneous hydrolysis of an internal thioester present in the protein (Pangburn, MK, et al., J. Exp. Med. 154:856-867, 1981). Due to the activity of C3(H ₂ O)Bb-convertase, C3b molecules are deposited on the target surface and thus initiate an alternative pathway.

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

In addition to the apparently unregulated activation mechanism described above, the alternative pathway may also provide powerful loop amplification for the C3 convertase (C4b2a) of the lectin/classical pathway, since any C3b produced can co-participate with factor B in the formation of an additional C3 convertase (C3bBb) of the alternative pathway. . The alternative pathway C3 convertase is stabilized by properdin binding. Properdin increases the half-life of the C3-convertase of the alternative pathway by 6-10 times. The addition of C3b to the alternative pathway C3 convertase results in the formation of the alternative pathway C5 convertase.

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

The complement system, in addition to its important role in immune defense, contributes to tissue damage in many clinical conditions. Therefore, the development of therapeutically effective complement inhibitors to prevent these side effects is an urgent need.

The essence of the invention

The Summary of the Invention is intended to introduce a selection of concepts in a simplified form, which are further described below in the Detailed Description of the Invention. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

In one aspect, the present invention provides a method of inhibiting capillary endothelial cell injury and/or thrombus formation in a subject suffering from thrombotic microangiopathy (TMA), comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody effective to inhibit MASP-2 dependent complement activation. . In some embodiments, the subject suffers from or is at risk of developing TMA selected from the group consisting of hemolytic uremic syndrome (HUS), thrombotic thrombocytopenic purpura (TTP), and atypical hemolytic uremic syndrome (aHUS). In some embodiments, prior to administration of the composition, the subject is screened for one or more symptoms selected from the group consisting of (i) anemia, (ii) thrombocytopenia (iii) renal failure, and (iv) creatinine elevation, and the composition is administered at an effective amount and for a period of time sufficient to improve one or more of these symptoms. In some embodiments, the MASP-2 inhibitory agent is an anti-MASP-2 antibody or a fragment thereof. In some embodiments, the MASP-2 inhibitory agent is an anti-MASP-2 monoclonal antibody, or fragment thereof, that specifically binds to a portion of SEQ ID NO: 6. In some embodiments, the MASP-2 inhibitory agent inhibits damage to capillary endothelial cells.

In another aspect, the invention provides a method for inhibiting MASP-2 dependent complement activation in a subject suffering from or at risk of developing atypical hemolytic uremic syndrome (aHUS), comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory agent effective to inhibit MASP-2 dependent complement activation. In one embodiment, prior to administration of the composition, the subject is screened for one or more symptoms selected from the group consisting of (i) anemia, (ii) thrombocytopenia (iii) renal failure, and (iv) creatinine elevation, and the composition is administered at an effective amount and for a period of time sufficient to improve one or more of these symptoms. In one embodiment, the subject suffers from factor H independent aHUS or is at risk of developing factor H independent aHUS. In one embodiment, the subject suffers from aHUS associated with factor I, factor B, or membrane cofactor protein CD46. In one embodiment, the MASP-2 inhibitory agent is an anti-MASP-2 antibody or fragment thereof, such as an anti-MASP-2 monoclonal antibody or fragment thereof, that specifically binds to a portion of the sequence of SEQ ID NO: 6. In one embodiment, MASP- 2 inhibitory agent inhibits damage to capillary endothelial cells. In one embodiment, the MASP-2 inhibitory agent inhibits thrombus formation.

In another aspect, the invention provides a method for reducing the likelihood that a subject at risk for developing atypical hemolytic uremic syndrome (aHUS) will experience clinical symptoms associated with aHUS. The method of this aspect of the invention includes (a) determining the presence in a subject of a genetic marker known to be associated with aHUS; (b) periodically examining the subject to determine the presence or absence of at least one symptom selected from the group consisting of anemia, thrombocytopenia, renal

- 5 042534 insufficiency and increase in creatinine; and (c) administering to the subject a composition comprising an amount of a MASP-2 inhibitory agent effective to inhibit MASP-2 dependent complement activation after determining the presence of at least one of the symptoms, such as anemia, thrombocytopenia, renal failure, or an increase in creatinine, wherein the composition is administered in an effective amount and for a sufficient period of time to improve one or more of these symptoms. In one embodiment, the MASP-2 inhibitory agent is an anti-MASP-2 antibody or fragment thereof, such as an anti-MASP-2 monoclonal antibody or fragment thereof, that specifically binds to a portion of the sequence of SEQ ID NO: 6. In one embodiment of the method, step (a) comprises performing a genetic screening test on a patient sample and detecting the presence of at least one aHUS-related genetic marker in a gene selected from the group consisting of complement factor H (CFH), complement factor I (CFI), complement factor B (CFB), membrane cofactor protein CD46, C3, complement-like factor H protein (CFHR1), thrombodulin anticoagulant protein (THBD), complement-like factor H protein 3 (CFHR3), and protein 4 , similar to complement factor H (CFHR4). In one embodiment, the method further comprises continuously screening the subject for the occurrence of an event known to be associated with the initiation of clinical symptoms of aHUS, and administering to the subject a composition comprising a MASP-2 inhibitory agent before, during, or after the occurrence of the initiating event. . In one embodiment, the event associated with the initiation of the clinical symptoms of aHUS is selected from the group consisting of prolonged drug exposure, infection, malignancy, injury, organ or tissue transplantation, and pregnancy. In one embodiment, the infection is a bacterial infection. In one embodiment, the composition is administered subcutaneously. In one embodiment, the MASP-2 inhibitory agent inhibits damage to capillary endothelial cells. In one embodiment, the MASP-2 inhibitory agent inhibits thrombus formation.

In another aspect, the invention provides a method for inhibiting MASP-2 dependent complement activation in a subject suffering from an atypical hemolytic uremic syndrome (aHUS) infection or at risk of developing an atypical hemolytic uremic syndrome (aHUS) from an infection, comprising administering to the subject a composition containing an amount MASP-2 inhibitory agent effective for inhibiting MASP-2 dependent complement activation. In one embodiment, the subject suffers from non-intestinal aHUS associated with pneumococcal infection or is at risk of developing non-intestinal aHUS associated with pneumococcal infection. In one embodiment, the MASP-2 inhibitory agent is an anti-MASP-2 antibody or fragment thereof, such as an anti-MASP-2 monoclonal antibody or fragment thereof, that specifically binds to a portion of the sequence of SEQ ID NO: 6. In one embodiment, MASP- 2 inhibitory agent inhibits damage to capillary endothelial cells. In one embodiment, the MASP-2 inhibitory agent inhibits thrombus formation.

In another aspect, the invention provides a method of treating a subject suffering from atypical hemolytic uremic syndrome (aHUS) comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory agent effective to inhibit MASP-2 dependent complement activation, wherein administration of the MASP-2 inhibitory agent is through an intravenous catheter or other method of delivery through the catheter. In one embodiment, the method further comprises treating the patient with plasmapheresis. In one embodiment, a composition comprising a MASP-2 inhibitory agent is administered without using a plasmapheresis technique. In one embodiment, the composition comprising the MASP-2 inhibitory agent is administered via a catheter over a first period of time, followed by administration of the composition comprising the MASP-2 inhibitory agent during a second period of time, where the composition is administered subcutaneously during the second period of time. In one embodiment, the method further includes periodically determining the level of at least one complement factor, where the detection of a reduced level of at least one complement factor relative to the normal value or level in a healthy subject is an indication of continued treatment with the composition. In one embodiment, the MASP-2 inhibitory agent is an anti-MASP-2 antibody or fragment thereof, such as an anti-MASP-2 monoclonal antibody or fragment thereof, that specifically binds to a portion of the sequence of SEQ ID NO: 6. In one embodiment, MASP- 2 inhibitory agent inhibits damage to capillary endothelial cells. In one embodiment, the MASP-2 inhibitory agent inhibits thrombus formation.

In another aspect, the invention provides a method of treating a subject suffering from thrombotic thrombocytopenic purpura (TTP) or exhibiting symptoms indicative of a diagnosis of TTP comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory agent effective to inhibit MASP-2 dependent complement activation, wherein introduction

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The MASP-2 inhibitory agent is delivered via an intravenous catheter or other catheter delivery method. In one embodiment, the subject exhibits at least one or more symptoms selected from the group consisting of central nervous system involvement, thrombocytopenia, severe cardiac disorder, severe pulmonary disorder, gastrointestinal infarction, and gangrene. In one embodiment, the subject is tested positive for the presence of an ADAMTS13 inhibitor, and the method further comprises administering an immunosuppressant to the subject. In one embodiment, a composition comprising a MASP-2 inhibitory agent is administered over a first period of time without using a plasmapheresis technique. In one embodiment, the subject is tested positive for the presence of an ADAMTS13 inhibitor and the method further comprises administering ADAMTS-13. In one embodiment, the method further comprises treating the patient using a plasmapheresis technique. In one embodiment, a composition comprising a MASP-2 inhibitory agent is administered using a plasmapheresis technique. In one embodiment, the composition comprising the MASP-2 inhibitory agent is administered via a catheter over a first period of time, followed by administration of the composition comprising the MASP-2 inhibitory agent during a second period of time, where the composition is administered subcutaneously during the second period of time. In one embodiment, the method further includes periodically determining the level of at least one complement factor, where the detection of a reduced level of at least one complement factor relative to the normal value or level in a healthy subject is an indication of continued treatment with the composition. In one embodiment, the MASP-2 inhibitory agent is an anti-MASP-2 antibody or fragment thereof, such as an anti-MASP-2 monoclonal antibody or fragment thereof, that specifically binds to a portion of the sequence of SEQ ID NO: 6. In one embodiment, MASP- 2 inhibitory agent inhibits damage to capillary endothelial cells. In one embodiment, the MASP-2 inhibitory agent inhibits thrombus formation.

In another aspect, the invention provides a method of treating a subject suffering from refractory thrombotic thrombocytopenic purpura (TTP), comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory agent effective to inhibit MASP-2 dependent complement activation. In one embodiment, the composition is administered subcutaneously. In one embodiment, the method further includes periodically determining the level of at least one complement factor, where the detection of a reduced level of at least one complement factor relative to the normal value or level in a healthy subject is an indication of continued treatment with the composition. In one embodiment, the MASP-2 inhibitory agent is an anti-MASP-2 antibody or fragment thereof, such as an anti-MASP-2 monoclonal antibody or fragment thereof, that specifically binds to a portion of the sequence of SEQ ID NO: 6. In one embodiment, MASP- 2 inhibitory agent inhibits damage to capillary endothelial cells. In one embodiment, the MASP-2 inhibitory agent inhibits thrombus formation.

In another aspect, the present invention provides a method for inhibiting MASP-2 dependent complement activation in a subject suffering from thrombotic microangiopathy (TMA) or at risk of developing thrombotic microangiopathy (TMA), wherein TMA is at least one of (i) cancer-related TMAs ; (ii) TMA in chemotherapy, or (iii) TMA in transplant, comprising administering to the subject a composition containing an amount of a MASP-2 inhibitory agent effective to inhibit MASP-2 dependent complement activation. In some embodiments, the subject suffers from or is at risk of developing TMA due to cancer, and the MASP-2 inhibitory agent is administered systemically to the subject in an amount effective to reduce the risk of developing TMA or reduce the severity of TMA. In some embodiments, the subject suffers from TMA during chemotherapy or is at risk of developing TMA during chemotherapy, and the MASP-2 inhibitory agent is administered systemically to the subject before, during, or after chemotherapy in an amount effective to reduce the risk of developing TMA or reduce the severity of TMA. In some embodiments, the subject suffers from or is at risk of developing TMA during transplantation, and the MASP-2 inhibitory agent is administered systemically to the subject before, during, or after the transplant procedure in an amount effective to reduce the risk of developing TMA or reduce the severity of TMA . In some embodiments, the transplant procedure is an allogeneic hematopoietic stem cell transplant. In some embodiments, the subject has previously been or is currently being treated with an inhibitor of terminal complement activation that inhibits the cleavage of complement C5 protein. In some embodiments, the method further comprises administering to the subject an inhibitor of terminal complement activation that inhibits cleavage of the complement C5 protein, such as a humanized anti-C5 antibody or antigen-binding fragment thereof, such as eculizumab.

In another aspect, the invention provides a method for inhibiting MASP-2 dependent activation

- 7 042534 complement in a subject suffering from Upshaw-Schulman syndrome or at risk of developing Upshaw-Schulman syndrome (USS), comprising administering to the subject a composition containing an amount of a MASP-2 inhibitory agent effective to inhibit MASP-2 dependent complement activation. In some embodiments, the method includes treating a subject at risk for developing USS, wherein the method includes administering an amount of a MASP-2 inhibitory agent for a period of time effective to alleviate or prevent one or more clinical symptoms associated with TTP. In some embodiments, the method further comprises periodically examining the subject and administering a MASP-2 inhibitory agent after an event is identified that is known to be associated with the initiation of clinical symptoms of TTP. In some embodiments, the method further comprises periodically examining the subject and administering a MASP-2 inhibitory agent after detecting the presence of anemia, thrombocytopenia, or elevated creatine. In some embodiments, the subject has previously been or is currently being treated with an inhibitor of terminal complement activation that inhibits the cleavage of complement C5 protein. In some embodiments, the method further comprises administering to the subject an inhibitor of terminal complement activation that inhibits cleavage of the complement C5 protein, such as a humanized anti-C5 antibody or antigen-binding fragment thereof, such as eculizumab.

In another aspect, the invention provides a method for inhibiting MASP-2 dependent complement activation in a subject suffering from Degos disease, comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory agent effective to inhibit MASP-2 dependent complement activation. In some embodiments, the subject has previously been or is currently being treated with an inhibitor of terminal complement activation that inhibits the cleavage of complement C5 protein. In some embodiments, the method further comprises administering to the subject an inhibitor of terminal complement activation that inhibits cleavage of complement C5 protein, such as a humanized anti-C5 antibody or antigen-binding fragment thereof, such as eculizumab.

In another aspect, the invention provides a method for inhibiting MASP-2 dependent complement activation in a subject suffering from catastrophic antiphospholipid syndrome (CAPS), comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory agent effective to inhibit MASP-2 dependent complement activation. In some embodiments, the subject has previously been or is currently being treated with an inhibitor of terminal complement activation that inhibits the cleavage of complement C5 protein. In some embodiments, the method further comprises administering to the subject an inhibitor of terminal complement activation that inhibits cleavage of complement C5 protein, such as a humanized anti-C5 antibody or antigen-binding fragment thereof, such as eculizumab.

In some embodiments of any of the disclosed methods of the invention, the MASP-2 inhibitory agent is a MASP-2 inhibitory antibody or fragment thereof. In some embodiments, the MASP-2 inhibitory antibody has reduced effector function. In some embodiments, the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway. In some embodiments, the MASP-2 inhibitory agent is an anti-MASP-2 monoclonal antibody, or fragment thereof, that specifically binds to a portion of the sequence of SEQ ID NO: 6. In some embodiments, the anti-MASP-2 antibody, or fragment thereof, is selected from the group, consisting of a recombinant antibody, an antibody having a reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody. In some embodiments, the MASP-2 inhibitory antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab) ₂ and F(ab') ₂ . In some embodiments, the MASP-2 inhibitory antibody is a single chain molecule. In some embodiments, the MASP-2 inhibitory antibody is selected from the group consisting of an IgG1 molecule, an IgG2 molecule, and an IgG4 molecule. In some embodiments, the MASP-2 inhibitory antibody is an IgG4 molecule comprising an S228P mutation. In some embodiments, the MASP-2 inhibitory antibody binds human MASP-2 with a KD dissociation constant of 10 nM or less. In some embodiments, the MASP-2 inhibitory antibody binds an epitope in the CCP1 domain of MASP-2. In some embodiments, the MASP-2 inhibitory antibody inhibits C3b deposition when assayed in vitro in 1% human serum with an IC ₅₀ value of 10 nM or less. In some embodiments, the MASP-2 inhibitory antibody inhibits C3b deposition in 90% human serum with an IC ₅₀ value of 30 nM or less.

In some embodiments of any of the disclosed methods of the invention, the MASP-2 inhibitory monoclonal antibody, or antigen-binding fragment thereof, comprises: (a) a heavy chain variable region comprising: i) a CDR-H1 heavy chain comprising an amino acid sequence from 31-35 of SEQ ID NO: 67; and ii) a CDR-H2 heavy chain, including

- 8 042534 amino acid sequence from 50-65 of SEQ ID NO: 67; and iii) a CDR-H3 heavy chain comprising the amino acid sequence from 95-102 of SEQ ID NO: 67; and (b) a light chain variable region comprising: i) a CDR-L1 light chain comprising an amino acid sequence from 24-34 of SEQ ID NO: 70; and ii) a CDR-L2 light chain comprising the amino acid sequence from 50-56 of SEQ ID NO: 70; and iii) a CDR-L3 light chain comprising the amino acid sequence from 89-97 of SEQ ID NO: 70. In some embodiments, the MASP-2 inhibitory monoclonal body comprises a heavy chain variable region, referred to as the sequence of SEQ ID NO: light chain region, referred to as the sequence of SEQ ID NO: 70. In some embodiments, the MASP-2 inhibitory antibody, or antigen-binding fragment thereof, specifically recognizes at least a portion of an epitope recognized by a control antibody comprising the heavy chain variable region, referred to as in the sequence SEQ ID NO: 67, and the light chain variable region referred to as in SEQ ID NO: 70.

In another aspect of the invention, methods are provided for inhibiting thrombus formation in a subject suffering from atypical hemolytic uremic syndrome (aHUS), comprising administering to the subject an amount of a MASP-2 inhibitory antibody, or antigen-binding fragment thereof, effective to inhibit MASP-2 dependent complement activation. In some embodiments, the MASP-2 inhibitory antibody inhibits clotting in the serum of a subject suffering from aHUS by at least 40% compared to the serum of an untreated subject. In some embodiments, the MASP-2 inhibitory antibody inhibits blood clotting in the serum of a subject suffering from aHUS at a level of at least 20% greater (e.g., at least 30% greater, at least 40% greater, or at least 50% greater) than its inhibitory effect on the deposition of C5b-9 in the blood serum of the same subject. In some embodiments, the subject is in the acute phase of aHUS. In some embodiments, the subject is in remission of aHUS. In some embodiments, the MASP-2 inhibitory antibody is a monoclonal antibody or fragment thereof that specifically binds to a portion of the sequence of SEQ ID NO: 6. In some embodiments, the MASP-2 inhibitory antibody or fragment is selected from the group consisting of a recombinant antibody, an antibody having a reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody. In some embodiments, the MASP-2 inhibitory antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab) ₂ and F(ab') ₂ . In some embodiments, the MASP-2 inhibitory antibody is a single chain molecule. In some embodiments, the MASP-2 inhibitory antibody is selected from the group consisting of an IgG1 molecule, an IgG2 molecule, and an IgG4 molecule. In some embodiments, the MASP-2 inhibitory antibody is an IgG4 molecule comprising an S228P mutation. In some embodiments, the MASP-2 inhibitory antibody binds human MASP-2 with a KD dissociation constant of 10 nM or less. In some embodiments, the MASP-2 inhibitory antibody binds an epitope in the CCP1 domain of MASP-2. In some embodiments, the MASP-2 inhibitory antibody inhibits C3b deposition when assayed in vitro in 1% human serum with an IC ₅₀ value of 10 nM or less. In some embodiments, the MASP-2 inhibitory antibody inhibits C3b deposition in 90% human serum with an IC ₅₀ value of 3 0 nM or less. In some embodiments, the MASP-2 inhibitory monoclonal body, or antigen-binding fragment thereof, comprises: (a) a heavy chain variable region comprising: i) a CDR-H1 heavy chain comprising the amino acid sequence from 31-35 of SEQ ID NO: 67; and ii) a CDR-H2 heavy chain comprising the amino acid sequence from 50-65 of SEQ ID NO: 67; and iii) a CDR-H3 heavy chain comprising the amino acid sequence from 95-102 of SEQ ID NO: 67; and (b) a light chain variable region comprising: i) a CDR-L1 light chain comprising an amino acid sequence from 24-34 of SEQ ID NO: 70; and ii) a CDR-L2 light chain comprising the amino acid sequence from 50-56 of SEQ ID NO: 70; and iii) a CDR-L3 light chain comprising the amino acid sequence from 89-97 of SEQ ID NO: 70. In some embodiments, the MASP-2 inhibitory monoclonal body comprises a heavy chain variable region, referred to as the sequence of SEQ ID NO: light chain region, referred to as the sequence of SEQ ID NO: 70. In some embodiments, the MASP-2 inhibitory antibody, or antigen-binding fragment thereof, specifically recognizes at least a portion of an epitope recognized by a control antibody comprising the heavy chain variable region, referred to as in the sequence SEQ ID NO: 67, and the light chain variable region referred to as in SEQ ID NO: 70.

In another aspect, the present invention provides a method of treating a subject suffering from plasma therapy resistant aHUS, comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody effective to inhibit MASP-2 dependent complement activation. In one embodiment, the MASP-2 inhibitory antibody is mono

- 9 042534 an anti-MASP-2 clonal antibody or fragment thereof that specifically binds human MASP-2. In one embodiment, the antibody or fragment thereof is selected from the group consisting of a recombinant antibody, an antibody having reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody. In one embodiment, the subject has previously been or is currently being treated with an inhibitor of terminal complement activation that inhibits cleavage of C5 complement protein, wherein the inhibitor of terminal component activation is a humanized anti-C5 antibody or an antigen-binding fragment thereof. In one embodiment, the method further comprises treating the patient with plasmapheresis. In one embodiment, a composition comprising a MASP-2 inhibitory antibody is administered without using a plasmapheresis technique.

In another aspect, the present invention provides a method of treating a subject suffering from TMA associated with hematopoietic stem cell transplantation, comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody effective to inhibit MASP-2 dependent complement activation. In one embodiment, the MASP-2 inhibitory antibody is an anti-MASP-2 monoclonal antibody, or fragment thereof, that specifically binds human MASP-2. In one embodiment, the antibody or fragment thereof is selected from the group consisting of a recombinant antibody, an antibody having reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody. In one embodiment, the subject has been or is currently being treated with an inhibitor of terminal complement activation that inhibits the cleavage of complement C5 protein. In one embodiment, the subject suffers from a hematopoietic stem cell transplant-associated TMA that is refractory to platelet transfusion treatment and/or is refractory to plasmapheresis treatment. In one embodiment, the MASP-2 inhibitory antibody is administered in an amount effective to improve at least one or more clinical parameters associated with TMA associated with hematopoietic stem cell transplantation, such as an increase in platelet count (e.g., at least two-fold, at least three times, at least four times compared with the number of platelets before treatment), an increase in haptoglobin and / or a decrease in lactate dehydrogenase.

In another aspect, the present invention provides a method of treating a human suffering from persistent TMA associated with hematopoietic stem cell transplantation (HSCT-TMA), comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen-binding fragment thereof, effective to inhibit MASP-2 dependent complement activation. In one embodiment, the method further comprises identifying a person having persistent TMA associated with hematopoietic stem cell transplantation prior to administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen-binding fragment thereof, effective to inhibit MASP-2 dependent complement activation. In one embodiment, the MASP-2 inhibitory antibody is a monoclonal antibody, or fragment thereof, that specifically binds human MASP-2. In one embodiment, the antibody or antigen-binding fragment thereof is selected from the group consisting of a recombinant antibody, an antibody having reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody. In one embodiment, the MASP-2 inhibitory antibody does not substantially inhibit the classical pathway. In one embodiment, the MASP-2 inhibitory antibody inhibits C3b deposition in 90% of human serum with an IC ₅₀ value of 3 0 nM or less. In one embodiment, the MASP-2 inhibitory antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3 amino acid sequences, referred to as the sequence of SEQ ID NO: 67, and a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 amino acid sequences, referred to as SEQ ID NO: 70. In one embodiment, the MASP-2 inhibitory antibody is administered to a patient without using a plasmapheresis technique. In one embodiment, the subject has previously been or is currently being treated with a humanized anti-C5 antibody or antigen-binding fragment thereof, such that in this case the humanized anti-C5 antibody or antigen-binding fragment thereof is an inhibitor of terminal complement activation. In one embodiment, the MASP-2 inhibitory antibody is administered systemically to the subject. In one embodiment, the MASP-2 inhibitory antibody, or antigen-binding fragment thereof, is administered in an amount effective to improve at least one or more of the following clinical parameters associated with persistent TMA associated with hematopoietic stem cell transplantation: (i) an increase in platelet count (e.g. at least two times, at least three times, at least four times compared to the platelet count before treatment); (ii) an increase in haptoglobin; (iii) decreased lactate dehydrogenase (LDH); and/or (iv) a decrease in creatinine.

In another aspect, the present invention provides compositions for inhibiting the negative effects of MASP-2 dependent complement activation, comprising a therapeutically effective co

- 10 042534 an amount of a MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody, and a pharmaceutically acceptable carrier. Also provided are methods for the manufacture of a medicament for use in inhibiting the adverse effects of MASP-2 dependent complement activation in living subjects in need thereof, comprising a therapeutically effective amount of a MASP-2 inhibitory agent in a pharmaceutical carrier. Also provided are methods for the manufacture of a medicament for use in inhibiting MASP-2 dependent complement activation for the treatment of each of the conditions, diseases and disorders described herein below.

The methods, compositions, and medicaments of the invention may be used to inhibit the adverse effects of MASP-2 dependent complement activation in vivo in mammals, including humans, suffering from thrombotic microangiopathy (TMA) or at risk of developing thrombotic microangiopathy (TMA), as described. later in the invention.

Description of drawings

The foregoing aspects and many of the advantages of this invention may be more readily appreciated and understood upon reading the following detailed description in conjunction with the accompanying drawings, in which:

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

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

in fig. 2B is a schematic diagram illustrating the domain structure of the human MAP19 protein;

in fig. 3 is a diagram illustrating the MASP-2 knockout strategy in mice;

in fig. 4 is a diagram illustrating the human MASP-2 minigene construct;

in fig. 5A shows the results indicating that MASP-2 deficiency leads to the disappearance of lectin-mediated C4 activation, as measured by the absence of C4b deposition on mannan, as described in Example 2;

in fig. 5B presents the results that demonstrate that MASP-2 deficiency leads to the disappearance of lectin-mediated C4 activation, as measured by the absence of C4b deposition on zymosan, as described in example 2;

in fig. 5C shows results showing relative levels of C4 activation in blood serum samples derived from MASP-2+/-; MASP-2-/- and wild-type strains, measured by C4b deposition on mannan and zymosan as described in Example 2;

in fig. 6 shows results demonstrating that addition of mouse recombinant MASP-2 to MASP-2-/- serum samples restored lectin-mediated C4 activation in a protein concentration dependent manner as measured by C4b deposition on mannan as described in Example 2;

in fig. 7 shows the results showing that the classical pathway is involved in the MASP-2 -/- strain as described in Example 8;

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

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

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

in fig. 9 presents the results which demonstrate that all tested anti-MASP-2 antibody Fab2 fragments that inhibited C3 convertase formation were also found to inhibit C4 cleavage as described in Example 10;

in fig. 10 is a diagram illustrating recombinant rat MASP-2 derived polypeptides used to map the epitope of anti-MASP-2 antibodies Fab2 blocking fragments as described in Example 11;

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

in fig. 12 shows results showing blood urea nitrogen clearance for wild (+/+) and MASP-2 (-/-) mice 24 and 48 hours post-reperfusion in the renal ischemia/reperfusion injury model as described in Example 12;

in fig. 13A shows results showing baseline VEGF protein levels in retinal pigment epithelium (RPE) and choroid complex isolated from wild-type (+/+) and MASP-2 (-/-) mice as described in Example 13;

in fig. 13B shows results showing baseline VEGF protein levels in the retinal pigment epithelium (RPE) and choroid complex in wild (+/+) and MASP-2 (-/-) mice 3 days after laser-induced injury in an experimental yellow degeneration model. spots, as described in example 13;

in fig. 14 presents the results showing the average volume of neovascularization of the vascular

- 11 042534 ocular membranes (CNV) seven days after laser-induced damage in wild-type (+/+) and

MASP-2 (-/-) mice as described in Example 13;

in fig. 15A and 15B are dose-response curves for deposition inhibition.

C4b (FIG. 15A) and inhibition of thrombin activation (FIG. 15B) after administration of the Fab2 fragment of an anti-MASP-2 antibody to healthy rat sera as described in Example 14;

in fig. 16A and 16B depict measured platelet aggregation (expressed as area of aggregation) in MASP-2 (-/-) mice (FIG. 16B) compared to platelet aggregation in untreated wild mice and wild mice in which the complement activation pathway is inhibited by cobra venom factor (CVF)-based phlebotomy and an inhibitor of terminal complement activation (C5aR antagonist) (FIG. 16A) in an experimental model of localized generalized Schwartzmann phenomenon of disseminated intravascular coagulation as described in Example 15;

in fig. 17 is a graphical representation of blood urea nitrogen (BUN) levels measured in either WT (+/+) mice (B6) or MASP-2 (-/-) mice transplanted with donor kidneys from WT (+/+) mice, as shown below. described in example 16;

in fig. 18 is a graphical representation of the percentage of surviving WT (+/+) mice and MASP-2 (-/-) mice as a function of the number of days since microbial infection in the cecal ligation and subsequent puncture (CLP) experimental model as described in the example. 17;

in fig. 19 is a graphical representation of bacterial counts measured in WT (+/+) mice and MASP-2 (-/-) mice after microbial infection in an experimental caecal ligation and subsequent puncture model as described in Example 17;

in fig. 20 is a Kaplan-Meier curve showing the percent survival of WT (+/+) mice, MASP-2 (-/-) mice, and C3 (-/-) mice six days after their intranasal administration of Pseudomonas aeruginosa bacteria as described in example 18;

in fig. 21 is a graphical representation of the level of C4b deposition, measured as % control, in samples taken at various time points after WT mice were dosed subcutaneously with either 0.3 mg/kg or 1.0 mg/kg of mouse anti-MASP-2 monoclonal antibody as this is described in example 19;

in fig. 22 is a graphical representation of the level of C4b deposition, measured as % control, in samples taken at various time points after intraperitoneal administration of WT mice with a dose of 0.6 mg/kg mouse anti-MASP-2 monoclonal antibody as described in Example 19;

in fig. 23 is a graphical representation of the mean choroidal neovascularization volume (CNV) seven days after laser-induced injury in WT (+/+) mice pretreated with a single intraperitoneal injection of 0.3 mg/kg or 1.0 mg/kg murine a monoclonal antibody against MASP-2, as described in example 20;

in fig. 24A is a graphical representation of the percentage survival of MASP-2 (-/-) mice and WT (+/+) mice after infection with 5x108 cfu/100 μl of meningococcus, as described in example 21;

in fig. 24B is a graphical representation of log cfu/ml meningococcus obtained at various time points for blood samples from MASP-2 KO (-/-) mice and WT (+/+) mice infected with 5x108 cfu/100 µl meningococcus. , as described in example 21;

in fig. 25A is a graphical representation of the percentage survival of MASP-2 KO (-/-) mice and WT (+/+) mice after infection with 2x108 cfu/100 μl of meningococcus, as described in example 21;

in fig. 25B is a graphical representation of log cfu/ml meningococcus obtained at various time points from blood samples taken from WT (+/+) mice infected with 2x108 cfu 100 μl meningococcus as described in Example 21;

in fig. 25C is a graphical representation of log cfu/ml meningococcus obtained at various time points from blood samples taken from MASP-2 (-/-) mice infected with 2x108 cfu/100 μl meningococcus as described in Example 21;

in fig. 26A is a graphical representation of the results of a C3b deposition study demonstrating that MASP-2 (-/-) mice retain the functional classical pathway as described in Example 22;

in fig. 26B is a graphical representation of the results of a C3b deposition study on zymosan-coated plates demonstrating that MASP-2 (-/-) mice retain a functional alternative pathway as described in Example 22;

in fig. 27A is a graphical representation of tissue loss caused by myocardial ischemia/reperfusion injury (MIRI) after ligation of the left anterior descending branch of the coronary artery (LAD) and reperfusion in C4 (-/-) mice (n=6) and for controls in WT littermates (n =7), characterized by area at risk (AAR) and infarct size (INF), as described in example 22;

in fig. 27B is a graphical representation of infarct size (INF) versus area at risk (AAR) in C4 (-/-) mice and WT mice treated as described in FIG. 42A, which demonstrates that C4 (-/-) mice are as sensitive to MIRI as those used to control WT mice (dashed line) as described in Example 22;

- 12 042534 in FIG. 28A is a graphical representation of the results of a C3b deposition study using sera from WT mice, C4 (-/-) mice, and sera from C4 (-/-) mice preincubated with mannan as described in Example 22;

in fig. 28B is a graphical representation of C3b deposition on sera from WT mice, C4 (-/-) mice, and MASP-2 (-/-) mice mixed with different concentrations of mouse anti-MASP-2 mAb fragment (mAbM11) as described. in example 22;

in fig. 28C is a graphical representation of the results of a study of C3b deposition on human sera from WT (C4 sufficient) and C4 deficient subjects, and on sera from C4 deficient subjects preincubated with mannan as described in Example 22;

in fig. 28D is a graphical representation of the results of a study of C3b deposition on human sera from WT (C4 sufficient) and C4 deficient subjects mixed with a human anti-MASP-2 mAb (mAbH3) fragment as described in Example 22;

in fig. 29A is a graphical comparison of C3 convertase activity in plasma of various strains of decomplemented mice tested under either the specific conditions of the lectin activation pathway assay or the specific conditions of the classical activation pathway as described in Example 22;

in fig. 29B is a graphical representation of the real-time kinetics of C3 convertase activity in plasma of various strains of decomplemented mice tested under the specific conditions of the lectin activation pathway study as described in Example 22;

in fig. 30 shows Western blot results showing human C3 activation as evidenced by the presence of the a' chain, thrombin substrates FXIa and FXa as described in Example 23;

in fig. 31 shows the results of a study of C3 deposition on serum samples obtained from WT, MASP-2 (-/-), F11 (-/-), F11 (-/-)/C4 (-/-) and C4 (-/-) mice, as described in example 23;

in fig. 32A is a Kaplan-Meier curve illustrating the percent survival over time following 7.0 Gy radiation exposure in control mice and mice treated with mouse anti-MASP-2 antibody (mAbM11) or human anti-MASP-2 antibody (mAbH6) as this is described in example 29;

in fig. 32B is a Kaplan-Meier curve plotting percentage survival over time after 6.5 Gy radiation exposure in control mice and mice treated with mouse anti-MASP-2 antibody (mAbM11) or human anti-MASP-2 antibody (mAbH6), as described in example 29;

in fig. 33 is a Kaplan-Meier curve graphically illustrating the percent survival of MASP-2 KO mice and WT mice following an infective dose of 2.6x107 CFU of serogroup A meningococcus Z2491, demonstrating that MASP-2 deficient mice are protected from meningococcal lethality as this is described in example 30;

in fig. 34 is a Kaplan-Meier curve graphically illustrating the percent survival of MASP-2 KO mice and WT mice following an infective dose of 6x106 CFU of serogroup B meningococcal strain MC58, demonstrating that MASP-2 deficient mice are protected from lethality caused by serogroup B meningococcal strain MC58. group B, as described in example 30;

in fig. 35 is a graphical representation of the log CFU/ml of serogroup B meningococcus strain MC58 obtained at various time points for blood samples taken from MASP-2 KO mice and WT mice after intraperitoneal infection with 6x106 CFU of serogroup B meningococcus strain MC58 (n=3 in different time points for both groups of mice, the results are presented as means ± standard error of the mean), which demonstrate that despite the fact that MASP-2 KO mice were infected with the same dose of serogroup B meningococcus strain MC58 as WT mice, however, MASP-2 KO mice had increased clearance for bacteremia compared to WT mice, as described in Example 30;

in fig. 36 is a graphical representation of the mean disease state scores of MASP-2 WT mice at 3, 6, 12, and 24 h after infection with 6x106 cfu/100 µl of serogroup B meningococcus strain MC58, demonstrating that MASP-2 deficient mice were highly resistant. to infection with significantly lower scores of the disease state after 6 hours, as described in example 30;

in fig. 37 is a Kaplan-Meier curve graphically illustrating the percent survival of mice after an infective dose of 4x106/100 µl CFU of serogroup B meningococcal strain MC58, followed by administration 3 hours after infection with either inhibitory antibody against MASP-2 (1 mg/kg), or a control isotype antibody that demonstrates that the anti-MASP-2 antibody is effective in treating and improving the survival of subjects infected with meningococcus, as described in example 31;

in fig. 38 is a graphical representation of the log CFU/mL viable numbers of serogroup B meningococcus strain MC58 obtained at various time points in 20% human serum following intraperitoneal infection with 6.5x106

cfu/100 µl of serogroup B meningococcal strain MC58 at 0, 30, 60 and 90 minutes after incubation in the presence of: (A) healthy human serum (NHS) plus human anti-

MASP-2; (B) healthy human sera (NHS) plus isotype control antibody;

(C) MBL-/- human sera; (D) healthy human sera (NHS) and (E) heat-inactivated healthy human sera (NHS), which show that complement-dependent killing of meningococcus in human serum was significantly enhanced by the addition of human anti-MASP-2 antibody as described in Example 32;

in fig. 39 is a graphical representation of the log CFU/mL viable counts of serogroup B meningococcus strain MC58 obtained at various time points in mouse sera, demonstrating that MASP-2-/- mouse sera have a higher level of bactericidal activity against meningococcus, chebm serum WT mice, as described in example 32;

in fig. 40 is a graphical representation of hemolysis (determined by the release of hemoglobin supernatant of lysed murine erythrocytes (Crry/C3-/-), which is measured photometrically) of mannan-coated murine erythrocytes exposed to human serum over a range of serum concentrations. Serums tested included heat-inactivated (HI) NHS, MBL-/-, NHS+anti-MASP-2 antibody and NHS control as described in Example 33;

in fig. 41 is a graphical representation of hemolysis (determined by the release of hemoglobin into the supernatant of lysed murine WT erythrocytes, measured photometrically) of mannan-free murine erythrocytes exposed to human serum over a range of serum concentrations. Serums tested included heat-inactivated (HI) NHS, MBL-/-, NHS+anti-MASP-2 and NHS control, which demonstrates that MASP2 inhibition inhibits complement-mediated lysis of unsensitized WT mouse erythrocytes as described in Example 33;

in fig. 42 is a graphical representation of hemolysis (determined by the release of hemoglobin supernatant of lysed murine erythrocytes (CD55/59 -/-), which is measured photometrically) of uncoated murine erythrocytes under the influence of human serum over a range of serum concentrations. Serums tested included heat-inactivated (HI) NHS, MBL-/-, NHS+anti-MASP-2 antibody and NHS control as described in Example 33;

in fig. 43 is a graphical representation of percent survival over time (days) following 8.0 Gy radiation exposure in control mice and in mice treated with human anti-MASP-2 antibody (mAbH6) as described in Example 34;

in fig. 44 is a graphical representation of the time to onset of capillary occlusion after lipopolysaccharide (LPS) injection in MASP-2 −/− mice and WT mice, indicating the percentage of mice with thrombosis measured within 60 minutes, which indicates that thrombus formation in WT mice is detectable at 15 minutes, and after 60 minutes thrombus formation was observed in 80% of WT mice; in contrast, none of the MASP-2 −/− mice showed any signs of thrombus formation at 60 (log-rank test: p=0.0005) as described in Example 35;

in fig. 45 is a graphical representation of the percentage survival of saline-treated control mice (n=5) and anti-MASP-2 antibody-treated mice (n=5) in a Shiga-like toxin/lipopolysaccharide-induced hemolytic uremic syndrome (HUS) model ( STX/LPS) over time (hours), which shows that all control mice died within 42 hours, while, in contrast, 100% of mice injected with anti-MASP-2 antibody remained alive throughout the time of the experiment, as described in example 36;

in fig. 46 is a graphical representation of the percentage of mice with capillary occlusion in the fluorescein isothiocyanate/dextran UV (FITC/Dextran UV) experimental model as a function of time after injury induction, following administration of isotype control antibody or human anti-MASP-2 mAbH6 antibody (10 mg/kg ) 16 hours and 1 hour before the injection of FITC/Dextran, as described in example 37;

in fig. 47 is a graphical representation of occlusion time in minutes for mice treated with human anti-MASP-2 antibody (mAbH6) and isotype control antibody, with data plotted as a scatter of mean points (horizontal lines) and standard error lines (vertical lines). The statistical test used for the analysis was Student's t-test for one sample; where the symbol * denotes p=0,0129, as described in example 37; and in FIG. 48 is a graphical representation of time to occlusion in minutes for wild mice, MASP-2 KO mice, and wild mice that were pre-treated with 10 mg/kg human anti-MASP-2 antibody (mAbH6) intraperitoneally 16 hours before and again 1 hour before. inducing thrombosis in an experimental model of thrombosis as a result of damage to endothelial cells caused by FITC-dextran/low intensity light radiation (800-1500) as described in example 37;

- 14 042534 in FIG. 49 is a Kaplan-Meier curve showing the percentage of mice with thrombi versus time in FITC-dextran-induced thrombotic microangiopathy in mice treated with increasing doses of human MASP-2 inhibitory antibody (mAbH6) or isotype control antibody as described in example 39;

in fig. 50 is a graphical representation of the mean time (in minutes) to thrombus formation versus mAbH6 dose (*p < 0.01 vs control) as described in Example 39;

in fig. 51 is a Kaplan-Meier curve showing the percentage of mice with capillary occlusion versus time in FITC-dextran-induced thrombotic microangiopathy in mice treated with increasing doses of an inhibitory human antibody (mAbH6) against MASP-2 or an isotype control antibody as described in example 39;

in fig. 52 is a graphical representation of the mean time to capillary occlusion versus mAbH6 dose (*p<0.05 vs control) as described in Example 39;

in fig. 53A is a graphical representation of the level of MAC deposition in the presence or absence of a human monoclonal body (OMS646) against MASP-2 under the specific conditions of the lectin pathway study, which shows that OMS646 inhibits lectin pathway mediated MAC deposition with an IC ₅₀ value of approximately 1 nM, as follows: described in example 40;

in fig. 53B is a graphical representation of the level of MAC deposition in the presence or absence of a human monoclonal body (OMS646) against MASP-2 under the specific conditions of a classical pathway study, showing that OMS646 does not inhibit classically mediated MAC deposition as described in Example 40;

in fig. 53C is a graphical representation of the level of MAC deposition in the presence or absence of a human monoclonal body (OMS646) against MASP-2 under the specific conditions of an alternative pathway study, showing that OMS646 does not inhibit alternative pathway mediated MAC deposition as described in Example 40;

in fig. 54 is a graphical pharmacokinetic (PK) profile of human monoclonal body (OMS646) against MASP-2 in mice showing the concentration of OMS646 (mean n=3 animals/group) versus time after the indicated dose as described in Example 40;

in fig. 55A is a graphical representation of the pharmacodynamic (PD) response to a human monoclonal body (OMS646) against MASP-2, measured as a decrease in systemic lectin pathway activity in mice following intravenous administration as described in Example 40;

in fig. 55B is a graphical representation of the pharmacodynamic (PD) response to a human monoclonal body (OMS646) against MASP-2, measured as a reduction in systemic lectin pathway activity in mice after subcutaneous administration, as described in Example 40;

in fig. 56 is a graphical representation of the inhibitory effect of anti-MASP-2 antibody (OMS646) versus sCR1 on aHUS serum-induced deposition of C5b-9 on ADP-activated HMEC-1 cells as described in Example 41;

in fig. 57 is a graphical representation of the inhibitory effect of anti-MASP-2 antibody (OMS646) versus sCR1 on aHUS serum-induced thrombosis on ADP-activated HMEC-1 cells as described in Example 42;

in fig. 58 is a graphical representation of the mean change in platelet count from baseline over time (week) in subjects suffering from persistent thrombotic microangiopathy associated with hematopoietic stem cell transplantation (HSCT-TMA) following administration of an inhibitory antibody (OMS646) against MASP-2 as this is described in example 46;

in fig. 59 is a graphical representation of the mean change in lactate dehydrogenase (LDH) from baseline over time (week) in subjects suffering from persistent thrombotic microangiopathy associated with hematopoietic stem cell transplantation (HSCT-TMA) after administration of an inhibitory antibody (OMS646) against MASP-2 , as described in example 46; and in FIG. 60 is a graphical representation of the mean change in haptoglobin from baseline over time (week) in subjects suffering from persistent thrombotic microangiopathy associated with hematopoietic stem cell transplantation (HSCT-TMA) following administration of an inhibitory antibody (OMS646) against MASP-2, as shown below. described in example 46.

- 15 042534

Description of the sequence listing

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-2 genomic DNA (exons 1-6)

ANTIGENS: (RELATED TO MATURE MASP-2 PROTEIN)

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

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

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

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

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

SEQ ID NO:13 inactivated serine protease domain (aa 610625 with Ser618 mutation in Ala)

SEQ ID NO:14 TPLGPKWPEPVFGRL (CUBI domain peptide)

SEQ ID NO:15 TAPPGYRLRLYFTHFDLELSHLCEYDFVKLSSGAKVLATLCGQ (CUBI domain peptide)

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

SEQ ID NO:17 FYSLGSSLDITFRSDYSNEKPFTGF (core MBL binding region)

SEQ ID NO:18 IDECQVAPG (EGF PEPTIDE)

SEQ ID NO:19 ANMLCAGLESGGKDSCRGDSGGALV (Serine Protease Binding Core)

PEPTIDE INHIBITORS:

SEQ ID NO:20 full length MBL cDNA SEQ ID NO:21 full-length MBL protein SEQ ID NO:22 OGK X GP (binding consensus) SEQ ID NO:23 OGKLG SEQ ID NO:24 GLR GLQ GPO GKL GPO G SEQ ID NO:25 GPO GPO GLR GLQ GPO GKL GPO GPO GPO SEQ ID NO:26 GKDGRDGTKGEKGEPGQGLRGLQGPOGKLGPOG SEQ ID NO : 27 GAOGSOGEKGAOGDO (h-ficolin person) SEQ ID NO : 2 8

- 16 042534 (human ficolin p35)

SEQ ID NO:29 LQRALEILPNRVTIKANRPFLVFI (cleavage site C4) EXPRESSION INHIBITORS:

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

SEQ ID NO:31

5' CGGGCACACCATGAGGCTGCTGACCCTCCTGGGC 3' Nucleotides 12-45 in

SEQ ID NO:4 including MASP-2 translation initiation site (sense)

SEQ ID NO:32

5-GACATTACCTTCCGCTCCGACTCCAACGAGAAG3' Nucleotides 361-396 in

SEQ ID NO:4 encoding the region containing the MASP-2 MBL binding site (sense)

SEQ ID NO:33

5' AGCAGCCCTGAATACCCACGGCCGTATCCCAAA3' Nucleotides 610-642 in

SEQ ID NO:4 encoding region containing CUBII domain CLONE PRIMERS:

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

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

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

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

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

SEQ ID NO:48 is a peptide bond of 9 amino acids

EXPRESSION VECTOR:

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

SEQ ID NO:50 is mouse MASP-2 cDNA

SEQ ID NO:51 is mouse MASP-2 protein (leader)

SEQ ID NO:52 is mouse MASP-2 protein (mature)

SEQ ID NO:53 is the rat MASP-2 cDNA

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

SEQ ID NO:55 is rat MASP-2 protein

- 17 042534 (mature)

SEQ ID NO:56-59 are human MASP-2 site-directed mutagenesis oligonucleotides that are used to generate human MASP-2A

SEQ ID NO:60-63 are mouse MASP-2 site-directed mutagenesis oligonucleotides that are used to generate mouse MASP-2A

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

SEQ ID NO: 66 DNA encoding heavy chain variable region (VH) 17D20_dc35VH2INI1VL (OMS646) (no signal peptide)

SEQ ID NO: 67 17D20_dc35VH21NllVL heavy chain variable region (VH) polypeptide (OMS646)

SEQ ID NO: 68 variable region (VH) severe polypeptide

17N1bshs

SEQ ID NO: 69: DNA encoding light chain variable region (VL) 17D20_dc35VH2INI1VL (OMS646)

SEQ ID NO: 70: 17D20_dc35VH21NllVL variable light chain (VL) polypeptide (OMS646)

SEQ ID NO: 71: 17N16_dcl7N9 variable light chain (VL) polypeptide

Detailed description of the invention

The present invention is based on the inventors' striking discovery that it is possible to inhibit the MASP-2 mediated lectin pathway without affecting the classical pathway. The present invention also describes the use of MASP-2 as a therapeutic target to inhibit lectin-mediated cell damage by complement activation without affecting a component of the classical (Clq dependent) immune system pathway.

I. Definitions.

Unless otherwise defined in the invention, all terms used in the invention have the meanings that are generally accepted by those of ordinary skill in the field to which the present invention pertains. The following definitions are provided in order to clarify the meanings of terms that are used in the description and in the claims to disclose the present invention.

As used herein, the term MASP-2-dependent complement activation includes MASP-2-dependent activation of the lectin pathway that occurs under physiological conditions (i.e., in the presence of Ca ⁺⁺ ) leading to the formation of the C3-convertase C4b2a of the lectin pathway and, after accumulation C3b, a cleavage product of C3, to the formation of C5-convertase C4b2a(C3b)n, which has been found to be the main cause of opsonization.

As used herein, the term alternative pathway refers to complement activation that is initiated by, for example, fungal and yeast cell wall zymosan, gram-negative bacteria outer membrane lipopolysaccharide (LIS), and rabbit erythrocytes, as well as many pure polysaccharides, rabbit erythrocytes, viruses, bacteria. , animal tumor cells, parasites and damaged cells, and which, as is commonly believed, is the result of spontaneous proteolytic generation of C3b from complement factor C3.

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

As used herein, the term classical pathway refers to complement activation that is initiated by an antibody bound to a foreign particle and that requires binding of the Clq recognition molecule.

As used herein, the term MASP-2 inhibitory agent refers to an agent that binds or interacts directly with MASP-2 and effectively inhibits MASP-2 dependent complement activation, and which includes anti-MASP-2 antibodies and MASP-2 binding fragments thereof, natural and synthetic peptides, low molecular weight compounds, soluble

-18042534

MASP-2 receptors, expression inhibitors, and isolated natural inhibitors, and also includes peptides that compete with MASP-2 for binding to another recognition molecule (e.g., MBL, H ficolin, M ficolin, or L ficolin) in the lectin pathway, but do not include antibodies that bind to other such recognition molecules. MASP-2 inhibitory agents used in the method of the invention can reduce MASP-2 dependent complement activation by more than 20%, eg more than 50%, eg more than 90%. In one embodiment, the MASP-2 inhibitory agent reduces MASP-2 dependent complement activation by more than 90% (ie, MASP-2 results in only 10% or less dependent complement activation).

As used herein, the term antibody encompasses antibodies and fragments thereof derived from any antibody-producing mammal (e.g., mice, rats, rabbits, and primates, including humans), or from hybridomas, phage selection, recombinant expression, or transgenic animals (or other methods for producing antibodies or fragments thereof) that specifically bind to the target polypeptide, such as, for example, MASP-2, polypeptides, or portions thereof. It is assumed that the term antibody does not impose restrictions on the sources of origin of the antibody or on the method of its production (for example, using hybridoma, phage selection, recombinant expression, transgenic animals, peptide synthesis, and so on). Examples of antibodies include polyclonal, monoclonal and recombinant antibodies; polyspecific, multispecific antibodies (eg, bispecific antibodies, trispecific antibodies); humanized antibodies; mouse antibodies; chimeric, murine-human, murine-primate, primate-human monoclonal antibodies and anti-idiotypic antibodies, and can be any intact antibody or fragment thereof. As used herein, the term antibody encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as dAb, Fab, Fab', F(ab') ₂ , Fv), single chain fragments (ScFv), synthetic variants thereof, natural variants , fusion proteins comprising an antibody portion with an antigen-binding fragment with the required specificity, humanized antibodies, chimeric antibodies, and any other modified configuration of the immunoglobulin molecule that includes an antigen-binding site or fragment (epitope recognition site) with the required specificity.

Monoclonal antibody refers to a homogeneous population of antibodies in which the monoclonal antibody consists of amino acids (natural and non-natural) involved in the selective binding of an epitope. Monoclonal antibodies are highly specific for the target antigen. The term monoclonal antibody covers not only intact monoclonal antibodies and full-length monoclonal antibodies, but also their fragments (such as Fab, Fab', F(ab')2, Fv), single-chain fragments (ScFv), their variants, hybrid proteins, including antigen-binding a portion, a humanized monoclonal antibody, a chimeric monoclonal antibody, and any other modified configuration of an immunoglobulin molecule that includes an antigen-binding site or fragment (epitope-recognition site) with the desired epitope-binding specificity and ability. It is assumed that the term monoclonal antibody does not impose restrictions on the sources of origin of the antibody or on the method of its production (for example, using hybridoma, phage selection, recombinant expression, transgenic animals, and so on). The term includes all immunoglobulins, as well as their fragments and so on, described above for the term antibody.

As used herein, the term antibody fragment refers to a portion derived from or related to a full length antibody, such as, for example, an anti-MASP-2 antibody, and this portion typically includes the antigen-binding or variable region of the antibody.

Illustrative examples of antibody fragments are Fab, Fab', F(ab)2, F(ab')2 and Fv fragments, scFv fragments, diabodies, linear antibodies, single chain antibody molecules, and multispecific antibodies formed from antibody fragments.

As used herein, the term single chain Fv or scFv antibody fragment includes the VH and VL domains of an antibody, where these domains are present in the same polypeptide chain. Typically, the Fv polypeptide further includes a polypeptide linker between the VH and VL domains that allows the scFv to form the desired structure for antigen binding.

The chimeric antibody used in the invention is a recombinant protein that contains variable domains and complementarity determining regions derived from an antibody of a non-human animal species (for example, rodents), while the rest of the antibody molecule is derived from a human antibody.

The humanized antibody used in the invention is a chimeric antibody that includes a minimum sequence corresponding to specific complementarity determining regions derived from a non-human immunoglobulin that is transplanted into a human antibody scaffold. Humanized antibodies are typically recombinant proteins in which only the complementarity-determining portions of the antibody are of non-human origin.

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

- 19 042534

As used herein, the term membrane attack complex (MAC) refers to the complex of the five final complement components (C5b along with C6, C7, C8 and C9) that invades and destroys membranes (also referred to as C5b-9).

As used herein, the term subject includes all mammals, including but not limited to humans, primates, dogs, cats, horses, sheep, goats, cows, rabbits, pigs, and rodents.

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

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

As used herein, the term conservative amino acid substitution can be illustrated by substitution among amino acids within each of the following groups: (1) glycine, alanine, valine, leucine and isoleucine, (2) phenylalanine, tyrosine and tryptophan, (3) serine and threonine, (4 ) aspartate and glutamate, (5) glutamine and asparagine, (6) lysine, arginine and histidine.

As used herein, the term oligonucleotide refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. The term also covers oligonucleotide bases consisting of natural nucleotides, sugars and covalent internucleoside bonds (between the main chains), as well as oligonucleotides having synthetic modifications.

As used herein, the term epitope refers to a site on a protein (eg, human MASP-2 protein) that is bound by an antibody. Overlapping epitopes include at least one (eg, two, three, four, five, or six) common amino acid residue(s) including linear and non-linear epitopes.

As used herein, the terms polypeptide, peptide, and protein are used interchangeably and refer to any amino acid chain linked to a peptide, regardless of length or post-translational modification. The MASP-2 protein described herein may comprise wild-type proteins, or may be wild-type proteins, or may be variants that have no more than 50 (e.g., no more than one, two, three, four, five, six, seven, eight, nine, ten, 12, 15, 20, 25, 30, 35, 40, or 50) conservative amino acid substitutions. Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine and threonine; lysine, histidine and arginine; and phenylalanine and tyrosine.

In some embodiments, a human MASP-2 protein may have an amino acid sequence that is 70% or greater than 70% identical to a human MASP-2 protein having the amino acid sequence described in SEQ ID NO: 5 (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%).

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

Percent (%) amino acid sequence identity is defined as the percentage of amino acids in the sequence of interest that are identical to the amino acids in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment to determine percent sequence identity can be achieved by various methods known to those skilled in the art, for example, by using publicly available computer software such as BLAST, BLAST2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. . Appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment over the full length of the compared sequences, can be determined by well-known methods.

II. General description of the invention.

Lectins (MBL, M-ficolin, H-ficolin, L-ficolin, and CL-11) are specific recognition molecules that initiate the innate complement system, and this system includes the lectin initiation pathway and its associated final pathway amplification loop, which amplifies lectin-initiated activation of final effector molecules. C1q is a specific recognition molecule that initiates the acquired complement system, and this system includes the classical initiation pathway and its associated final pathway amplification loop that amplifies C1q-initiated activation of final effector molecules. The present inventors refer to these two major complement activation systems as the lectin-dependent complement system and the C1q-dependent complement system, respectively.

The complement system, in addition to its major role in immune defense, contributes to tissue damage in many clinical conditions. Therefore, there is an urgent need to develop therapeutically effective complement inhibitors to prevent these negative consequences. The finding that it is possible to inhibit the lectin pathway mediated by MASP-2 without affecting the classical pathway suggests that it would be highly desirable to provide specific inhibition of only the complement activation system that causes a particular pathology, but does not completely suppress complement's immune defense capabilities. For example, in pathological conditions in which complement activation is predominantly mediated by the lectin-dependent complement system, it would be preferable that only that system be specifically inhibited. This would allow not to affect the system of C1q-dependent complement activation, which regulates the processing of the immune complex and contributes to the protection of the host organism from infection.

The preferred target protein component in the development of therapeutic agents for the specific inhibition of the lectin-dependent complement system is MASP-2. Of all the known protein components of the lectin-dependent complement system (MBL, H-ficolin, Mficolin, L-ficolin, MASP-2, C2-C9, factor B, factor D, and properdin), only MASP-2 is both unique and necessary for the functioning of the lectin-dependent complement system. Lectins (MBL, H-ficolin, M-ficolin, L-ficolin, and CL-11) are also unique components of the lectin-dependent complement system. However, the loss of any one of the lectin components will not necessarily lead to inhibition of the activation of the system, which is due to an excess amount of lectin. All five lectins must be inhibited to ensure that activation of the lectin-dependent complement system is inhibited. In addition, since MBL and ficolins are also known to have complement-independent opsonin activity, inhibition of lectin function would result in the loss of this valuable host defense mechanism against infection. In contrast, the complement-dependent opsonin activity of the lectin would remain unaffected if MASP-2 was the target for inhibition of lectin pathway activation. An additional advantage of MASP-2 as a therapeutic target for inhibiting activation of the lectin-dependent complement system is that the plasma concentration of MASP-2 is the lowest (~500 ng/mL) of any other complement protein, and therefore, for to achieve complete inhibition, correspondingly low concentrations of high affinity MASP-2 inhibitors will be sufficient (Moller Kristensen, M., et al., J. Immunol Methods 282:159 167, 2003).

III. The role of masp-2 in thrombotic microangiopathies and therapeutic methods for the use of masp-2 inhibitory agents.

General description.

Thrombotic microangiopathy (TMA) is a pathology characterized by the presence of blood clots in small blood vessels (Benz, K.; et al., Curr Opin Nephrol Hypertens 19(3):242-7 (2010)). It is believed that stress or damage to the underlying vascular endothelium is the main cause of this pathology. The clinical picture and laboratory findings of TMA include thrombocytopenia, anemia, purpura, and renal failure. Classical examples of TMA are hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). The underlying pathological feature of TMA is platelet activation and the formation of microthrombi in small arterioles and venules. Complement activation, initiated at least in part by microvascular endothelial injury or stress, is also implicated in other types of TMA, including catastrophic antiphospholipid syndrome (CAPS), systemic Degos disease, and TMA in cancer, cancer therapy, and transplants.

Direct evidence of the pathological role of complement in many cases of nephritis is provided by studies of patients with a genetically determined deficiency of specific complement components. A number of studies have shown an association of kidney damage with complement regulatory factor H deficiency (Ault, B.H., Nephrol. 14:1045 1053, 2000; Levy, M., et al., Kidney Int. 30:949 56, 1986; Pickering, M.C. , et al., Nat Genet 31:424 8, 2002). Factor H deficiency results in low plasma levels of factor B and C3 due to activation-related depletion of these components. Circulating C5b-9 levels are also elevated in the serum of these patients, indirectly indicating complement activation. Membranoproliferative glomerulonephritis (MPGN) and idiopathic hemolytic uremic syndrome (HUS) are associated with factor H deficiency or factor H mutations. Y factor H-deficient pigs (Jansen, J.H., et al., Kidney Int. 53:331 49, 1998) and factor H knockout mice (Pickering, M.C., 2002) show MPGN-like symptoms, suggesting the importance of factor H in complement regulation. Deficiency of other complement components is associated with kidney disease associated with systemic lupus erythematosus (SLE) (Walport, M.J., Davies, et al., Ann. N.Y. Acad. Sci. 815:267 81, 1997). Deficiency of C1q, C4 and C2 dramatically provokes the development of SLE through mechanisms related to impaired clearance of immune complexes and apoptotic material. Many of these SLE patients develop lupus nephritis, characterized by the deposition of immune complexes over the entire surface of the glomerulus.

aHUS.

Atypical hemolytic uremic syndrome (aHUS) is part of a group of conditions called thrombotic microangiopathies. In atypical HUS (aHUS), the disease is associated with dysregulation of complement and may be either sporadic or hereditary. Inherited cases of aHUS are associated with mutations in genes encoding complement activation or complement regulatory proteins, including complement factor H, factor I, factor B, membrane cofactor protein CD46, and complement-like factor H protein 1 (CFHR1) and protein 3, complement-like factor H (CFHR3) (Zipfel, P.F., et al., PloS Genetics 3(3):e41 (2007)). One characteristic feature of this heterogeneous array of aHUS-associated genetic mutations is the tendency to increased complement activation on the surface of cells or tissues. Therefore, one aspect of the present invention includes treating a patient suffering from aHUS that is associated with factor H deficiency by administering an effective amount of a MASP-2 inhibitory agent. Another aspect of the present invention includes the treatment of a patient suffering from aHUS that is associated with deficiency of factor I, factor B, membrane cofactor protein CD46, CFHR1 or CFHR3 by administering an effective amount of a MASP-2 inhibitory agent.

Recently, significant progress has been made in understanding the molecular pathophysiology underlying the increased complement activation in aHUS caused by a heterogeneous set of mutant complement factors. This mechanism is best understood for factor H mutations. Factor H is an abundant serum protein comprising 20 short consensus repeat (SCR) domains that act as a negative regulator of complement activation both in solution and on the host cell surface. . It targets the activated form of C3 and, together with factor I and other cofactors, promotes its inactivation, anticipating subsequent complement activation. Effective control of complement activation on the surface of host cells requires interaction of factor H with host cells, which is mediated by SCR domains 16-20. All aHUS-associated factor H mutations described to date cluster in the C-terminal region spanning the 1620 SCR domains. These factor H mutant proteins are fully functional in controlling C3 activation in solution, but are unable to interact with the host cell surface and therefore cannot control C3 activation on the cell surface (Exp Med 204(6):1249-56 (2007)). Thus, specific factor H mutations are associated with aHUS because the mutated factor H protein is unable to interact with host cell surfaces and therefore cannot effectively down-modulate complement activation on host cell surfaces, including microvascular endothelium. . As a result, after initial C3 activation has occurred, subsequent complement activation at the surface of the capillary endothelium is not attenuated in patients with factor H mutations. This uncontrolled complement activation eventually leads to progressive damage to the vascular endothelium, then to platelet aggregation and microvascular coagulation, and hemolysis caused by direct pressure during the passage of red blood cells through partially occluded microvessels. Thus, the painful manifestations of aHUS and its clinical picture and laboratory data are directly related to the violation of the negative regulation of complement on the surface of the microvascular endothelium.

Similarly, factor H mutations, loss-of-function mutations in the negative complement modulator factor I and membrane cofactor protein (CD46) are also associated with aHUS. The opposite was observed for factor B, the C3 protein, in which aHUS was found to be associated with gain-of-function mutations in these proteins (Pediatr Nephrol 25(12):2431-42 (2010)). Thus, a large body of pooled data directly links complement activation to the pathogenesis of aHUS. This point of view is most convincingly supported by the therapeutic efficacy of eculizumab, a monoclonal antibody that blocks final complement C5 protein in the treatment of aHUS.

While the important role of complement as an effector mechanism in aHUS is generally recognized, the triggers that initiate complement activation and the molecular signaling pathways involved in the activation remain unexplored. Not all individuals with the mutations described above develop aHUS. In fact, studies of hereditary factors have concluded that the incidence of the aHUS gene is only ~50% (Ann Hum Genet 74(1):17-26 (2010)). The course of the disease indicates that aHUS more frequently develops after an initiating event such as an outbreak or injury. It is well known that infectious agents activate the complement system. In the absence of pre-existing acquired immunity, complement activation by infectious agents can be initiated primarily via the lectin pathway. Thus, the lectin activation pathway triggered by infection may represent the initiation of a trigger for subsequent pathological amplification of complement activation in aHUS-predisposed individuals, which may eventually lead to the development of the disease. Accordingly, another aspect of the present invention comprises the treatment of a patient suffering from aHUS in the presence of an infection by administering an effective amount of a MASP-2 inhibitory agent.

Other forms of damage to the host tissue, in particular, damage to the vascular endothelium, will also activate complement along the lectin pathway. Human vascular endothelial cells are subjected to oxidative stress, for example, in response to the expression of surface fragments that bind lectins and activate the lectin complement pathway (Am J. Pathol 156(6):1549-56 (2000)). Vascular injury following ischemia/reperfusion also activates complement via the lectin pathway in vivo (Scand J Immunol 61(5):426-34 (2005)). Activation of the lectin pathway under these conditions leads to pathological consequences for the host, and inhibition of the lectin pathway by blocking MASP-2 prevents subsequent host tissue damage and poor outcome (Schwaeble PNAS 2011).

Thus, it is well known that other processes that provoke aHUS also activate the lectin complement pathway. Therefore, the lectin pathway may appear to represent an initial complement activation mechanism that is inappropriately and unregulatedly upregulated in individuals genetically predisposed to aHUS, thus initiating the pathogenesis of aHUS. Based on this, agents that block complement activation via the lectin pathway, including anti-MASP-2 antibodies, would be expected to prevent disease progression or reduce exacerbations in individuals predisposed to developing aHUS.

In support of this concept, recent studies have identified pneumococcus as an important etiological factor in pediatric cases of aHUS (Nephrology (Carlton), 17:48-52 (2012); Pediatr Infect Dis J. 30(9):736-9 (2011 )). Apparently, this particular etiology has a poor prognosis with high mortality and a long course of the disease. Moreover, these cases included non-intestinal infections leading to manifestations of microangiopathy, uremia, and hemolysis without evidence of concomitant mutations in complement genes known to provoke aHUS. It is important to note that pneumococcus is particularly effective in complement activation, and thus acts predominantly via the lectin pathway. Therefore, it is expected that in cases of non-intestinal HUS associated with pneumococcal infection, manifestations of microangiopathy, uremia, and hemolysis will be predominantly due to activation of the lectin pathway, and agents that block the lectin pathway, including antibodies against MASP-2, would be expected to prevent the development of aHUS and reduce the severity of the disease in these patients. Accordingly, another aspect of the present invention includes the treatment of a patient suffering from non-intestinal aHUS that is associated with pneumococcal infection by administering an effective amount of a MASP-2 inhibitory agent.

In accordance with the foregoing, in some embodiments, in the case of a subject at risk of developing aHUS-related renal failure, a method is provided for reducing the likelihood of developing aHUS or developing aHUS-related renal failure, comprising administering an amount of a MASP-2 inhibitory agent for a period time effective to alleviate or prevent renal failure in a subject. In some embodiments, the method further comprises the step of determining if the subject is at risk of developing aHUS prior to the onset of any symptoms associated with aHUS. In other embodiments, the method includes determining if a subject is at risk of developing aHUS after exhibiting at least one or more symptoms indicative of aHUS (e.g., the presence of anemia, thrombocytopenia, and/or renal insufficiency) and/or the presence of thrombotic microangiopathy in a biopsy, taken from the subject. Determining whether a subject is at risk of developing aHUS includes determining whether a subject has a genetic predisposition to develop aHUS, which can be done by evaluating genetic information (eg, from a database containing the subject's genotype) or by conducting at least one genetic screening of the subject for determine the presence or

- 23 042534 absence of a genetic marker associated with aHUS (i.e. determining the presence or absence of a genetic mutation associated with aHUS in genes encoding complement factor H (CFH), factor I (CFI), factor B (CFB), membrane cofactor protein CD46, C3, factor H complement-like protein 1 (CFHR1) or THBD (coding for the anticoagulant thrombodulin protein) or factor H complement-like protein 3 (CFHR3) or factor H complement-like protein 4 (CFHR4)) , or by genome sequencing or gene-specific analysis (eg, polymerase chain reaction (PCR) analysis), and/or by determining whether the subject has aHUS in the family. Methods for genetic screening for the presence or absence of a genetic mutation associated with aHUS are well known and are described, for example, in Noris M et al. Atypical Hemolytic-Uremic Syndrome, 2007 Nov 16 [Updated 2011 Mar 10]. In: Pagon RA, Bird TD, Dolan CR, et al., editors. GeneReviews™, Seattle (WA): University of Washington, Seattle.

For example, the overall frequency of gene expression in a disease in which complement factor H (CFH) mutations occur is 48%, and gene expression for mutations in CD46 is 53%, for mutations in CFI is 50%, for mutations in C3 is 56% and for mutations in THBD is 64% (Caprioli J. et al., Blood, 108:1267-79 (2006); Noris et al., Clin J Am Soc Nephrol 5:1844-59 (2010)). As described in Caprioli et al., cited above, (2006), a significant number of individuals with a mutation in complement factor H (CFH) never develop aHUS, and it has been suggested that suboptimal CFH activity in these individuals is sufficient to protect the host from effects of complement activation under physiological conditions, however, suboptimal CFH activity is insufficient to prevent C3b deposition on vascular endothelial cells when exposed to an agent that activates complement, producing higher than normal amounts of C3b.

Accordingly, in one embodiment, a method is provided for inhibiting MASP-2 dependent complement activation in a subject suffering from factor H-independent atypical hemolytic uremic syndrome or at risk of developing factor H-independent atypical hemolytic uremic syndrome, comprising administering to the subject a composition comprising an amount MASP-2 inhibitory agent effective for inhibiting MASP-2 dependent complement activation. In another embodiment, a method is provided for inhibiting MASP-2 dependent complement activation in a subject at risk for developing factor H dependent atypical hemolytic uremic syndrome, comprising periodically examining the subject for the presence or absence of anemia, thrombocytopenia, or elevated creatinine, and treating with MASP-2 inhibitory agent after determining the presence of anemia, thrombocytopenia, or an increase in creatinine. In another embodiment, a method is provided for reducing the likelihood that a subject at risk for developing Factor H-Dependent aHUS will suffer clinical symptoms associated with aHUS, comprising administering a MASP-2 inhibitory agent prior to, or during, or after an event about which is known to be associated with the initiation of aHUS clinical symptoms, eg, prolonged drug exposure (eg, chemotherapy), infection (eg, bacterial infection), malignancy, injury, organ or tissue transplantation, and pregnancy.

In one embodiment, a method is provided to reduce the likelihood that a subject at risk of developing aHUS will suffer from clinical symptoms associated with aHUS, comprising periodically examining the subject to determine whether or not they have anemia, thrombocytopenia, or elevated creatinine, and treating with using a MASP-2 inhibitory agent after determining the presence of anemia, thrombocytopenia, or an increase in creatinine.

In another embodiment, a method is provided for reducing the likelihood that a subject at risk of developing aHUS will suffer clinical symptoms associated with aHUS, comprising administering a MASP-2 inhibitory agent prior to, or during, or after a known event. that it is associated with the initiation of clinical symptoms of aHUS, such as prolonged exposure to a drug (eg, chemotherapy), infection (eg, bacterial infection), malignancy, injury, organ or tissue transplantation, or pregnancy.

In some embodiments, the MASP-2 inhibitory agent is administered for at least one, two, three, four days or more before, during, or after the event associated with the initiation of the clinical symptoms of aHUS, and the administration may be repeated at the discretion of the attending physician until the disease state is eliminated or under control. In conditions prior to the onset of aHUS, the MASP-2 inhibitory agent may be administered to the subject systemically, for example, intra-arterially, intravenously, intramuscularly, by inhalation, nasally, subcutaneously, or by other parenteral route.

In some embodiments, against the background of a primary diagnosis of aHUS, or in the case of a subject exhibiting one or more symptoms consistent with an aHUS diagnosis (e.g., the presence of anemia, thrombocytopenia, and/or renal failure), the subject is treated with an effect

- 24 042534 active amount of MASP-2 inhibitory agent (for example, antibodies against MASP-2) as first-line therapy without the use of the plasmapheresis method or in combination with plasmapheresis. As a first line therapy, the MASP-2 inhibitory agent may be administered systemically to a subject, for example, intra-arterially, intravenously, intramuscularly, inhaled, nasally, subcutaneously, or by other parenteral route. In some embodiments, the MASP-2 inhibitory agent is administered to the subject as first-line therapy without the use of a plasmapheresis method, in order to avoid possible complications that occur with plasmapheresis, including hemorrhage, infection, and exposure to disorders and/or allergies inherent in the plasma donor, or or when the subject has contraindications to the use of plasmapheresis, or in conditions where plasmapheresis is not available.

In some embodiments, the method comprises administering a MASP-2 inhibitory agent to a subject suffering from aHUS via a catheter (e.g., intravenously) for a first period of time (e.g., at least one day to one week or two weeks), then administering the MASP-2 inhibitory agent subcutaneously to the subject for a second period of time (eg, for an extended period of time, at least two weeks or more). In some embodiments, the implementation of the introduction in the first and/or second period of time is carried out without the use of the method of plasmapheresis. In some embodiments, the method further comprises determining a level of at least one complement factor (e.g., C3, C5) in a subject prior to treatment, and optionally during treatment, wherein detecting a reduced level of at least one complement factor compared to a standard value or compared to a healthy control subject is an indication of the need to continue treatment with a MASP-2 inhibitory agent.

In some embodiments, the method includes administering a MASP-2 inhibitory agent, such as an anti-MASP-2 antibody, to a subject suffering from or at risk of developing aHUS, either intravenously, intramuscularly, or preferably subcutaneously. The treatment may be prolonged and the administration may be from once a day to once a month, but preferably once every two weeks. The anti-MASP-2 antibody can be administered as monotherapy or in combination with a C5 inhibitor such as eculizumab.

HUS.

As in the case of atypical HUS, the typical form of HUS manifests itself in the form of a clinical picture and laboratory findings characteristic of thrombotic microangiopathy (TMA). However, the typical form of HUS is often a pediatric disease and usually has no hereditary component or direct association with mutations in complement genes. The etiology of the typical form of HUS is closely related to infection with specific intestinal pathogens. Patients usually present with acute renal failure, hemoglobinuria, and thrombocytopenia, which usually accompanies an episode of hemorrhagic diarrhea. This condition is caused by intestinal infection with dysentery coli, salmonella, or Shiga toxin, which produces enterohaemorrhagic E. coli stamps such as E. coli 0157:H7. Pathogens come from contaminated food or water sources. HUS is a life-threatening condition with a mortality rate of 5-10%. A significant proportion of surviving patients develop chronic kidney disease (Corrigan and Boineau, Pediatr Rev 22 (11):365-9 (2011)), and a kidney transplant may be necessary.

Microvascular coagulation in typical HUS occurs predominantly, though not exclusively, in the microvasculature of the kidneys. The underlying pathophysiology is mediated by Shiga toxin (STX). Released by enteropathic microbes into the intestinal lumen, STX crosses the intestinal barrier, enters the bloodstream, and binds to vascular endothelial cells via the globotriosyl ceramide receptor CD77 (Boyd and Lingwood Nephron 51:207 (1989)), which is predominantly expressed on glomerular endothelium and mediates the toxic effects of STX.

After binding to the endothelium, STX induces a series of events that damage the vascular endothelium, activate leukocytes, and cause vWF-dependent thrombosis (Forsyth et al., Lancet 2: 411-414 (1989); Zoja et al., Kidney Int. 62: 846 -856 (2002), Zanchi et al., J. Immunol.181:1460-1469 (2008), Morigi et al., Blood 98:1828-1835 (2001), Guessou et al., Infect. Immun., 73 : 8306-8316 (2005)). These microthrombi lead to obstruction or occlusion of the arterioles and capillaries of the kidneys and other organs. The obstruction of blood flow in arterioles and capillaries by microthrombi increases the shear force acting on red blood cells as they pass through constricted blood vessels. This can lead to the destruction of red blood cells by shear force and the formation of fragments of red blood cells, called schistocytes. The presence of schizocytes is a characteristic finding in HUS. This mechanism is known as microangiopathic hemolysis. In addition, obstruction of blood flow leads to ischemia, initiating a complement-mediated inflammatory response, which causes additional damage to the affected organ.

The lectin complement pathway contributes to the pathogenesis of HUS through two main mechanisms: 1) MASP-2-mediated direct activation of the coagulation system induced by damage to the endothelium, and 2) lectin-mediated subsequent complement activation induced by ischemia resulting from initial microvascular occlusion. blood flow.

STX damages vascular endothelial microcells, and damaged endothelial cells are known to activate the complement system. As discussed in detail above, complement activation after damage to endothelial cells is predominantly mediated by the lectin pathway. Human vascular endothelial cells subjected to oxidative stress respond by expressing surface fragments that bind lectins and activate the lectin complement pathway (Collard et al., Am J Pathol. 156(5):1549-56 (2000)). Vascular injury resulting from ischemia/reperfusion also activates complement via the lectin pathway in vivo (Scand J Immunol 61(5): 42634 (2005)). Activation of the lectin pathway under these conditions has pathological consequences for the host, and inhibition of the lectin pathway by blocking MASP-2 prevents subsequent host tissue damage and adverse outcomes (Schwaeble et al., PNAS (2011)). In addition to complement activation, lectin-dependent activation of MASP-2 has been shown to cleave prothrombin to form thrombin and promote coagulation. Thus, activation of the complement lectin pathway by damaged endothelial cells can directly activate the coagulation system. Therefore, the lectin complement pathway, via MASP-2-mediated prothrombin activation, appears to be the dominant molecular pathway linking STX-induced initial endothelial injury to the coagulation and microvascular thrombosis that occurs in HUS. Therefore, lectin pathway inhibitors, including but not limited to antibodies that block MASP-2 function, would be expected to prevent or reduce microvascular coagulation, thrombosis, and hemolysis in patients suffering from HUS. Indeed, administration of an anti-MASP-2 antibody effectively protected mice in a typical HUS experimental model. As described in Example 36 and shown in FIG. 45, all control mice exposed to STX and LPS developed severe HUS and the mice agonized or died within 48 hours. On the other hand, as further shown in FIG. 45, all mice that were treated with anti-MASP-2 antibody and then exposed to STX and LPS survived (Fischer's exact test p<0.01; N=5). Thus, anti-MASP-2 therapy provides effective protection to mice in this HUS experimental model. Administration of a MASP-2 inhibitory agent, such as an anti-MASP-2 antibody, is expected to be effective in treating HUS patients and provide protection against microvascular coagulation, thrombosis, and hemolysis caused by infection with enteropathic Escherichia coli or other STX-producing pathogens.

While illustrated in the invention with respect to HUS caused by STX, it is expected that anti-MASP-2 therapy will also give positive results in the case of HUS-like syndromes due to endothelial damage caused by other toxic agents. These include agents such as mitomycin, ticlopidine, cisplatin, quinine, cyclosporine, bleomycin, as well as other chemotherapeutic drugs and immunosuppressive drugs. Thus, therapy with an anti-MASP-2 antibody or other modes of action that inhibit MASP-2 activity is expected to be effective in preventing or limiting erythrocyte coagulation, thrombosis, and destruction and preventing renal failure in HUS and other TMA-related diseases. (i.e. aHUS and TTP).

Patients suffering from HUS often have diarrhea and vomiting, usually have a low platelet count (thrombocytopenia), and a low red blood cell count (anemia). The pre-HUS phase of diarrhea typically lasts for approximately four days, during which subjects at risk for developing HUS typically present with one or more of the following symptoms in addition to severe diarrhea: a hematocrit level below 30% with evidence of intravascular RBC destruction on smear examination, thrombocytopenia (platelet count < 150x10 ³ /mm ³ ) and / or the presence of impaired renal function (serum creatinine concentration above the upper limit of the reference range for this age). The presence of oligoanuria (urine output <0.5 ml/kg/hour for > 1 day) can be used as a measure of the progress of HUS (see C. Hickey et al., Arch Pediatr Adolesc Med 165(10):884- 889 (2011)). Usually, a study is carried out for the presence of infection with Escherichia coli bacteria (E. coli 0157: H7) or representatives of Shigella or Salmonella. Antibiotics are contraindicated in a subject who tests positive for enterogenic Escherichia coli (eg, E. coli 0157:H7), as antibiotic use may increase the risk of developing HUS through increased production of STX (see Wong C. et al., N Engl J. Med 342:1930-1936 (2000) For subjects who test positive for Shigella or Salmonella, antibiotics are usually given to clear the infection.Other conventional first line therapy for HUS includes volume expansion, dialysis, and plasmapheresis.

- 26 042534

In accordance with the foregoing, in some embodiments, in the case of a subject suffering from one or more symptoms associated with the preceding HUS phase and at risk of developing HUS (i.e., the subject exhibits one or more of the following symptoms: diarrhea, hematocrit level below 30% with evidence of intravascular RBC destruction on smear examination, thrombocytopenia (platelet count < 150x10 ³ /mm ³ ) and/or impaired renal function (serum creatinine concentration above the upper limit of the reference range for this age)), a method is proposed to reduce the risk of developing HUS, or reducing the likelihood of developing renal failure in a subject, comprising administering an amount of a MASP-2 inhibitory agent for a period of time effective to improve or prevent impaired renal function. In some embodiments, the MASP-2 inhibitory agent is administered over a period of time of at least one, two, three, four, or more days, and the administration may be repeated at the discretion of the attending physician until the disease state is resolved or be under control. In the case of a preceding HUS phase, the MASP-2 inhibitory agent may be administered to the subject systemically, for example, intra-arterially, intravenously, intramuscularly, by inhalation, nasally, subcutaneously, or by other parenteral route.

Treatment of E. coli 0157:H7 infection with bactericidal antibiotics, particularly β-lactams, has been associated with an increased risk of developing HUS (Smith et al., Pediatr Infect Dis J 31(1):37-41 (2012). In some embodiments implementation in the case of a subject suffering from symptoms associated with the previous HUS phase, when the subject is known to be infected with enterogenic E. coli and the use of antibiotics is contraindicated (for example, E. coli 0157:H7), a method is provided to reduce the risk of developing HUS or reduce the likelihood of developing renal failure in a subject, comprising administering an amount of a MASP-2 inhibitory agent for a first period of time effective to inhibit or prevent the occurrence of oligoanuria in a subject (for example, for at least one, two, three, or four days), where the administration of the MASP-2 inhibitory agent for the first time period is in the absence of the antibiotic. The method further comprises administering the MASP-2 inhibitory agent to the subject in combination with the antibiotic for a second period of time (eg, at least one to two weeks).

In other embodiments, in the case of a subject suffering from symptoms associated with a pre-HUS phase, where the subject is known to be infected with shigella or salmonella, a method is provided for reducing the risk of developing HUS or reducing the likelihood of developing kidney failure in the subject, comprising administering an amount MASP-2 inhibitory agent for a period of time effective to inhibit or prevent the occurrence of oligoanuria in the subject, where the administration of the MASP-2 inhibitory agent is carried out either in the presence or absence of a suitable antibiotic.

In some embodiments, upon a primary diagnosis of HUS, or if the subject exhibits one or more symptoms indicative of a diagnosis of HUS (e.g., presence of renal failure or microangiopathic hemolytic anemia in the absence of low fibrinogen or thrombocytopenia), the subject is treated with an effective amount of MASP-2 an inhibitory agent (eg, an anti-MASP-2 antibody) as first-line therapy without plasmapheresis or in combination with plasmapheresis. As a first line therapy, the MASP-2 inhibitory agent may be administered systemically to a subject, for example, intra-arterially, intravenously, intramuscularly, inhaled, nasally, subcutaneously, or by other parenteral route. In some embodiments, the MASP-2 inhibitory agent is administered to the subject as first-line therapy without the use of a plasmapheresis method, in order to avoid possible complications arising from plasmapheresis, including hemorrhage, infection, and exposure to disorders and/or allergies inherent in the plasma donor, or or when the subject has contraindications to the use of plasmapheresis, or in conditions where plasmapheresis is not available.

In some embodiments, the method comprises administering a MASP-2 inhibitory agent to a subject suffering from HUS via a catheter (e.g., intravenously) during a first period of time (e.g., during an acute phase lasting from at least one day to a week or two weeks ) then administering the MASP-2 inhibitory agent subcutaneously to the subject for a second period of time (eg, for an extended period of time, at least two weeks or more). In some embodiments, the implementation of the introduction in the first and/or second period of time is carried out without the use of the method of plasmapheresis. In some embodiments, the method further comprises determining a level of at least one complement factor (e.g., C3, C5) in a subject prior to treatment, and optionally during treatment, wherein detecting a reduced level of at least one complement factor compared to a standard value or compared with a healthy control subject is an indication of the need for treatment, and where the detection of a normal level indicates improvement.

In some embodiments, the method comprises administering a MASP-2 inhibitory agent, such as an anti-MASP-2 antibody, to a subject suffering from or at risk of developing HUS, either subcutaneously or intravenously. The treatment is preferably carried out daily, but it can also be carried out with a frequency of once a week or once a month. Treatment may be continued for at least one week to 3 months. The anti-MASP-2 antibody can be administered as monotherapy or in combination with a C5 inhibitor such as eculizumab.

TTR.

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disease of the blood coagulation system caused by autoimmune or inherited disorders that activate the coagulation or complement system (George, JN, N Engl J Med; 354:1927-35 (2006)). This leads to the formation of numerous microscopic clots or to thromboses in small blood vessels throughout the body. RBCs are subjected to shear stress, which disrupts their membranes, leading to intravascular hemolysis. The resulting reduction in blood flow and endothelial damage cause organ damage, including the brain, heart, and kidneys. TTP is clinically characterized by thrombocytopenia, microangiopathic hemolytic anemia, neurological changes, renal failure, and fever. In times before plasma replacement was used, the fatality rate for acute attacks was 90%. Even with plasma replacement, survival in the first six months is approximately 80%.

TTP results from genetic and acquired inhibition of the enzyme ADAMTS13, a metalloprotease responsible for the breakdown of large von Willebrand factor (vWF) multimers into smaller fragments. Inhibition or deficiency of ADAMTS-13 ultimately leads to increased coagulation (Tsai, H. J Am Soc Nephrol 14: 1072-1081, (2003)). ADAMTS-13 regulates vWF activity; in its absence, vWF forms large multimers that most likely bind platelets and induce platelet aggregation and microcirculatory thrombosis in patients.

Upshaw-Schulman syndrome (USS, also described as congenital TTP) is a congenital deficiency in ADAMTS13 activity due to ADAMTS13 gene mutations (Schulman et al., Blood, 16 (1): 943-57, 1960; Upshaw et al., New Engl. J. Med, 298(24):1350-2, 1978). Numerous mutations in ADAMTS13 have been identified in individuals with congenital TTP (Kinoshita et al., International Journal of Hematology, 74:101-108 (2001); Levy et al., Nature, 413 (6855):488-494 (2001); Kokame et al., PNAS 99 (18):11902-11907 (2002); Savasan et al., Blood, 101:4449-4451 (2003); Matsumoto et al., Blood, 103:1305-1310 (2004) and Fujimura et al., Brit J Haemat 144:742-754 (2008)). Subjects with USS typically have 5-10% of normal ADAMTS13 activity (Kokame et al., PNAS 99(18):1190211907, 2002). Although acquired TTP and USS share some similarities, USS has some important differences in clinical features. USS usually presents in infancy or childhood and is characterized by severe hyperbilirubinemia with a negative Coombs test shortly after birth, clinical improvement with fresh plasma infusion, and frequent relapses (Savasan et al., Blood, 101:4449-4451, 2003). In some cases, patients with this congenital ADAMTS13 deficiency have a mild phenotype at birth and only develop TTP-related symptoms in clinical situations with elevated von Willebrand factor levels, such as infection or pregnancy. For example, in Fujimura et al., Brit. J. Haemat 144:742-754, 2008 reported 9 Japanese women from 6 families with genetically confirmed USS who were diagnosed with this disorder during their first pregnancy. Thrombocytopenia occurred during the second to third trimesters in each of these 15 pregnancies, often after TTP. All of these women were found to be significantly deficient in ADAMTS13 activity.

As described above, in some embodiments, in the case of a subject with Upshaw-Schulman Syndrome (USS) (i.e., a subject known to be deficient in ADAMTS13 activity and/or a subject known to be that it has one or more ADAMTS13 gene mutations), a method for reducing the likelihood of developing clinical symptoms associated with congenital TTR (eg, thrombocytopenia, anemia, fever, and/or renal failure) is provided, comprising administering an amount of a MASP-2 inhibitory agent ( for example, anti-MASP-2 antibodies) for a period of time effective to alleviate or prevent one or more clinical symptoms associated with TTP. In some embodiments, the method further comprises the step of determining if the subject is at risk of developing symptoms associated with congenital TTR before the onset of any symptoms associated with TTP, or after the onset of at least one or more symptoms indicative of the presence of TTP (e.g. , the presence of anemia, thrombocytopenia and / or renal failure). Determining whether a subject is at risk of developing symptoms associated with congenital TTP (i.e., whether the subject has USS) includes determining whether the subject has a mutation in the gene encoding ADAMTS13 and/or determining whether the subject has a deficiency in ADAMTS13 activity and/or determining whether whether the subject has a family history of USS. Methods for genetic screening for the presence or absence of a USS-associated genetic mutation are well known, for example, see Kinoshita et al., International Journal of Hematology, 74:101-108 (2001); Levy et al.

Nature, 413 (6855): 488-494 (2001); Kokame et al., PNAS 99(18):11902-11907 (2002); Savasan et al., Blood,

101:4449-4451 (2003); Matsumoto et al., Blood, 103:1305-1310 (2004) and Fujimura et al., Brit. J. Haemat

144:742-754 (2008).

In one embodiment, a method is provided to reduce the likelihood that a subject diagnosed with USS will suffer from clinical symptoms associated with TTR, comprising periodically examining the subject to determine whether or not he has anemia, thrombocytopenia, or elevated creatinine, and treating with using a MASP-2 inhibitory agent (eg, an anti-MASP-2 antibody) after detection of anemia, thrombocytopenia, or elevated creatinine, or after an event known to be associated with the initiation of clinical symptoms of TTP, such as prolonged drug exposure ( eg chemotherapy), infection (eg bacterial infection), malignancy, injury, transplant or pregnancy.

In another embodiment, a method is provided for treating a subject with USS and suffering from clinical symptoms associated with TTP, comprising administering an amount of a MASP-2 inhibitory agent (e.g., an anti-MASP-2 antibody) for a period of time effective to alleviate or prevent one or more clinical symptoms associated with TTR.

The development of TTP can also be caused by an autoantibody against ADAMTS-13. In addition, TTP can occur in breast cancer, gastrointestinal cancer or prostate cancer (George JN., Oncology (Williston Park). 25:908-14 (2011)), pregnancy (second trimester or postpartum ), George JN., Curr Opin Hematol 10:339-344 (2003)), or may be associated with diseases such as AIDS or autoimmune diseases such as systemic lupus erythematosus (Hamasaki K, et al., Clin Rheumatol. 22:355-8 (2003)). TTP can also be caused by the use of specific drugs, including heparin, quinine, an immune-mediated ingredient, chemotherapeutic anti-cancer agents (bleomycin, cisplatin, cytosine arabinoside, daunomycin, gemcitabine, mitomycin C and tamoxifen), cyclosporine A, oral contraceptives, penicillin , rifampin, and antiplatelet drugs, including ticlopidine and clopidogrel (Azarm, T. et al., J Res Med Sci., 16: 353-357 (2011)). Other factors or conditions associated with TTP are toxins such as bee venom, sepsis, splenic sequestration, transplantation, vasculitis, vascular surgery, and infections such as streptococcal pneumonias and generalized cytomegaly (Moake JL., N Engl J Med., 347:589-600 (2002)). TTP due to transient functional deficiency of ADAMTS-13 may result from endothelial cell damage associated with pneumococcal infection (Pediatr Nephrol., 26:631-5 (2011)).

Plasma replacement is the standard approach in the treatment of TTP (Rock GA, et al., N Engl J Med 325:393-397 (1991)). Plasma replacement restores ADAMTS-13 activity in patients with genetic defects and removes ADAMTS-13 autoantibodies in patients with acquired autoimmune TTP (Tsai, H-M, Hematol Oncol Clin North Am., 21(4): 609-v (2007)) . Typically, this therapy uses additional drugs such as immunosuppressants (George, JN, N Engl J Med, 354:1927-35 (2006)). However, plasma exchange fails in 20% of patients, recurrence occurs in more than a third of patients, and plasmapheresis is an expensive treatment and requires special equipment. In addition, many patients cannot tolerate plasma replacement. Therefore, there remains a strong need for additional and improved treatments for TTP.

Since TTP is a disorder of the blood coagulation system, treatment with complement antagonists may help stabilize and correct the disease. While pathological activation of the alternative complement pathway is associated with aHUS, the role of complement activation in TTP is less clear. Functional deficiency of ADAMTS13 is of great importance in terms of susceptibility to TTP, however, but it does not explain the cause of acute attacks. Environmental factors and/or other genetic changes may contribute to the manifestation of TTR. For example, genes encoding proteins involved in the regulation of the coagulation system, vWF, platelet function, vascular endothelial surface components, or the complement system may be directly associated with the development of acute thrombotic microangiopathy (Galbusera, M. et al., Haematologica, 94: 166- 170 (2009)). In particular, complement activation has been shown to play an important role; serum thrombotic microangiopathy associated with ADAMTS-13 deficiency has been shown to cause C3 and MAC deposition and subsequent neutrophil activation that could be abolished by complement inactivation (Ruiz-Torres MP, et al., Thromb Haemost, 93:443 -52 (2005)). In addition, C4d, C3bBbP and C3a levels have recently been shown to increase during acute TTP attacks (M. Reti et al., J Thromb Haemost. Feb 28. (2012) doi: 10.1111/j.1538-7836.2012.04674. x. [electronic publication coming soon]), which corresponds to the activation of the classical/lectin and alternative pathways. This increased amount of complement activation in acute attacks may initiate the final activation pathway and may be responsible for the subsequent exacerbation of TTP.

- 29 042534

Clearly, the role of ADAMTS-13 and vWF in TTP is platelet activation and aggregation and their subsequent involvement in shear stress and deposition in microangiopathies. Activated platelets interact with and initiate both the classical and alternative complement pathways. Platelet-mediated complement activation increases inflammatory mediators C3a and C5a (Peerschke E et al., Mol Immunol, 47:2170-5 (2010)). Thus, platelets can serve as targets for classical complement activation in hereditary or autoimmune TTR.

As described above, the lectin complement pathway via MASP-2 mediated prothrombin activation is the dominant molecular pathway linking endothelial injury to the coagulation and microvascular thrombosis that occurs in HUS. Similarly, activation of the lectin complement pathway can directly trigger the coagulation system in TTP. Activation of the lectin pathway may be initiated in response to initial endothelial injury caused by ADAMTS-13 deficiency in TTP. Therefore, lectin pathway inhibitors, including but not limited to antibodies that block MASP-2 function, would be expected to alleviate microangiopathy conditions associated with microvascular coagulation, thrombosis, and hemolysis in patients suffering from TTP.

Patients suffering from TTP are usually in the emergency department with one or more of the following diagnoses: purpura, kidney failure, low platelets, anemia, and/or thrombosis, including stroke. The current standard treatment for TTP involves catheter (eg, intravenous or other forms of catheter) delivery of plasmapheresis plasma for two weeks or more, typically three times a week, but up to once a day. If the subject's test results indicate the presence of an ADAMTS13 inhibitor (ie, an endogenous anti-ADAMTS13 antibody), then plasmapheresis may be performed in combination with immunosuppressive therapy (eg, corticosteroids, rituxan, or cyclosporine). In the case of subjects with refractory TTR (approximately 20% of patients with TTR), treatment for at least two weeks with plasmapheresis does not give positive results.

As described above, in one embodiment, if a subject is initially diagnosed with TTP or exhibits one or more symptoms indicative of TTP (e.g., central nervous system involvement, severe thrombocytopenia (platelet count less than or equal to 5000/μL without taking aspirin, less than or equal to 200,000/µl when taking aspirin), severe heart disease, severe lung disease, gastrointestinal infarction, or gangrene), a method of treating a subject with an effective amount of a MASP-2 inhibitory agent (e.g., an antibody against MASP-2) as first-line therapy without the use of plasmapheresis or in combination with plasmapheresis. As a first line therapy, the MASP-2 inhibitory agent may be administered systemically to a subject, for example, intra-arterially, intravenously, intramuscularly, inhaled, nasally, subcutaneously, or by other parenteral route. In some embodiments, the MASP-2 inhibitory agent is administered to the subject as first-line therapy without the use of a plasmapheresis method, in order to avoid possible complications arising from plasmapheresis, including hemorrhage, infection, and exposure to disorders and/or allergies inherent in the plasma donor, or or when the subject has contraindications to the use of plasmapheresis, or in conditions where plasmapheresis is not available. In some embodiments, the MASP-2 inhibitory agent is administered to a subject suffering from TTP in combination (including co-administration) with an immunosuppressant (e.g., e.g., corticosteroids, rituxan, or cyclosporine) and/or in combination with concentrated ADAMTS-13 .

In some embodiments, the method comprises administering a MASP-2 inhibitory agent to a subject suffering from TTP through a catheter (e.g., intravenously) during a first period of time (e.g., during the acute phase lasting from at least one day to a week or two weeks ), then administering the MASP-2 inhibitory agent subcutaneously to the subject for a second period of time (eg, during the chronic phase period of at least two weeks or more). In some embodiments, the implementation of the introduction in the first and/or second period of time is carried out without the use of the method of plasmapheresis. In some embodiments, the method is used to maintain the subject in order to prevent the subject from suffering from one or more of the symptoms associated with TTR.

In another embodiment, a method is provided for treating a subject suffering from refractory TTR (i.e., a subject who has not been successfully treated with plasmapheresis for at least two weeks) by administering an amount of a MASP-2 inhibitor effective to alleviate one or more symptoms of TTR. In one embodiment, a MASP-2 inhibitor (eg, an anti-MASP-2 antibody) is administered to a subject with refractory TTR on a long-term basis over a period of time of at least two weeks or more, subcutaneously or by other parenteral route. The introduction may be repeated at the discretion of the attending physician until the disease state is eliminated or is under control.

- 30 042534

In some embodiments, the method further comprises determining a level of at least one complement factor (e.g., C3, C5) in a subject prior to treatment and optionally during treatment, wherein detecting a reduced level of at least one complement factor compared to a standard value, or compared with a healthy control subject, is an indication of the need to continue treatment with a MASP-2 inhibitory agent.

In some embodiments, the method includes administering a MASP-2 inhibitory agent, such as an anti-MASP-2 antibody, to a subject suffering from or at risk of developing TTP, either subcutaneously or intravenously. Treatment is preferably carried out daily, but it can also be carried out with a frequency of once every two weeks. Treatment is continued until the subject's platelet count reaches more than 150,000/ml for at least two consecutive days. The anti-MASP-2 antibody can be administered as monotherapy or in combination with a C5 inhibitor such as eculizumab.

In one embodiment, the MASP-2 inhibitory antibody exhibits at least one or more of the following: said antibody binds human MASP-2 with a KD value of 10 nM or less, said antibody binds a CCP1 epitope in the MASP-2 domain, said antibody inhibits deposition C3b in an in vitro study in 1% human serum with an IC ₅₀ value of 10 nM or less, said antibody inhibits the deposition of C3b in 90% of human serum with an IC ₅₀ value of 30 nM or less, wherein the antibody is an antibody fragment selected from the group consisting of of Fv, Fab, Fab', F(ab)2 and F(ab') ₂ , where the antibody is a single chain molecule, where said antibody is an IgG2 molecule, where said antibody is an IgG1 molecule, where said antibody is an IgG4 molecule, where the IgG4 molecule includes the S228P mutation, and/or where the antibody does not substantially inhibit the classical pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and substantially does not inhibit the alternative pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and does not substantially inhibit the classical or alternative pathway (ie, it inhibits the lectin pathway but does not affect the classical and alternative complement pathways).

In one embodiment, the MASP-2 inhibitory antibody inhibits clotting in serum from a subject suffering from TTP by at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% to 99%, compared to untreated serum. In some embodiments, the MASP-2 inhibitory antibody inhibits clotting in serum from a subject suffering from TTP by at least 20% or more (e.g., at least 30%, at least 40%, at least 50%) more than the inhibitory effect on the deposition of C5b-9 in the blood serum.

In one embodiment, the MASP-2 inhibitory antibody inhibits clotting in serum from a patient with TTP by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95 to 99%, compared to untreated serum.

In one embodiment, the MASP-2 inhibitory antibody is administered to the subject via an intravenous catheter or other catheter delivery method.

In one embodiment, the invention provides a method for inhibiting thrombus formation in a subject suffering from TTP, comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or an antigen-binding fragment thereof, comprising (I) (a) a heavy chain variable region comprising: i) a heavy a CDR-H1 chain comprising the amino acid sequence from 31-35 of SEQ ID NO: 67; and ii) a CDR-H2 heavy chain comprising the amino acid sequence from 50-65 of SEQ ID NO: 67; and iii) a CDR-H3 heavy chain comprising the amino acid sequence from 95-102 of SEQ ID NO: 67 and b) a light chain variable region comprising: i) a CDR-L1 light chain comprising the amino acid sequence from 24-34 of SEQ ID NO: 70; and ii) a CDRL2 light chain comprising the amino acid sequence from 50-56 of SEQ ID NO: 70; and iii) a CDR-L3 light chain comprising an amino acid sequence from 89-97 of SEQ ID NO: 70, or (ii) a variant thereof comprising a heavy chain variable region with at least 90% identity to SEQ ID NO: 67 (e.g. , at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, with at least 98%, at least 99% identity with SEQ ID NO: 67), and a light chain variable region with at least 90% identity (for example, at least 91%, at least 92 %, at least 93%, at least 94%, by

- 31 042534 with at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with SEQ ID NO: 70.

In some embodiments, the method comprises administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising an amino acid sequence referred to as the sequence of SEQ ID NO: 67. In some embodiments, the method comprises administering to the subject the composition , comprising the amount of MASP-2 inhibitory antibody or its antigen-binding fragment, including the variable region of the light chain, including the amino acid sequence, referred to as the sequence of SEQ ID NO: 70.

In some embodiments, the method includes administering to a subject a composition comprising a MASP-2 inhibitory antibody, or an antigen-binding fragment thereof, that specifically recognizes at least a portion of an epitope on human MASP-2 recognized by an OMS646 control antibody, comprising the heavy chain variable region described in sequence of SEQ ID NO: 67, and the variable region of the light chain described in the sequence of SEQ ID NO: 70.

Degos disease.

Degos disease, also known as malignant atrophic papulosis, is a very rare TMA that affects the endothelium of small vessels in the skin, gastrointestinal tract, and central nervous system. This vasculopathy causes venule and arteriole occlusion leading to skin damage, intestinal ischemia, and central nervous system disorders including stroke, epilepsy, and cognitive impairment. In the case of the skin, connective tissue necrosis is due to thrombotic occlusion of small arteries. However, the cause of Degos disease is unknown. It has been associated with vasculitis, coagulopathy, or primary endothelial cell dysfunction. Degos disease has a 50% survival rate over two to three years. There is no effective treatment for Degos disease, but antiplatelet drugs, anticoagulants, and immunosuppressants are used to relieve symptoms.

Although the mechanism of Degos disease is unknown, it has nevertheless been associated with the complement activation pathway. Margo et al., Am J Clin Pathol 135(4):599-610, 2011) reported the identification of prominent C5b-9 deposits in the skin, gastrointestinal tract, and cerebral vessels in four patients with end-stage Degos disease. . Experimental treatment with eculizumab was effective initially in treating skin and intestinal lesions but did not prevent systemic disease (see Garrett-Bakelman F. et al., C5b-9 is a potential effector in the pathophysiology of Degos disease; a case report of treatment with eculizumab (Abstract), Jerusalem: International Society of Hematology;2010, Poster #156;and Polito J. et al., Early detection of systemic Degos disease (DD) or malignant atrophic papulosis (MAP) may increase survival (Abstract), San Antonio, TX: American College of Gastroenterology; 2010, Poster #1205).

Many patients suffering from Degos disease have blood coagulation disorders. This disease is characterized by thrombotic occlusion of small arteries of the skin. Because the complement pathway is involved in this disease, as described for other TMAs, it is expected that inhibitors of the lectin pathway, including, but not limited to, antibodies that block MASP-2 function, may be beneficial in treating patients. suffering from Degos disease.

Accordingly, in another embodiment, the invention provides methods of treating Degos disease by administering a composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent, such as anti-MASP-2 antibodies, in a pharmaceutical carrier, to a subject suffering from Degos disease or a condition arising from due to Degos disease. The MASP-2 inhibitory agent is administered systemically to a subject suffering from Degos' disease or a condition resulting from Degos' disease, for example, intra-arterially, intravenously, intramuscularly, inhaled, subcutaneously, or by another parenteral route, or possibly by oral administration in the case of non-peptidergic drugs. . The anti-MASP-2 antibody can be administered as monotherapy or in combination with a C5 inhibitor such as eculizumab.

In one embodiment, the MASP-2 inhibitory antibody exhibits at least one or more of the following: said antibody binds human MASP-2 with a KD value of 10 nM or less, said antibody binds a CCP1 epitope in the MASP-2 domain, said antibody inhibits deposition C3b in an in vitro study in 1% human serum with an IC ₅₀ value of 10 nM or less, said antibody inhibits the deposition of C3b in 90% of human serum with an IC ₅₀ value of 30 nM or less, wherein the antibody is an antibody fragment selected from the group consisting of of Fv, Fab, Fab', F(ab) ₂ and F(ab') ₂ , where the antibody is a single chain molecule, where the specified antibody is an IgG2 molecule, where the specified antibody is an IgG1 molecule, where the specified antibody is an IgG4 molecule, where the IgG4 molecule includes the S228P mutation, and/or where the antibody does not substantially inhibit the classical pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and does not substantially inhibit

- 32 042534 alternative route. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and does not substantially inhibit the classical or alternative pathway (ie, it inhibits the lectin pathway but does not affect the classical and alternative complement pathways).

In one embodiment, the MASP-2 inhibitory antibody inhibits clotting in serum from a subject suffering from Degos disease by at least 30%, such as at least 40%, such as at least 50%, such as at least at least 60%, such as at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% to 99%, compared to untreated serum. In some embodiments, the MASP-2 inhibitory antibody inhibits blood clotting in serum from a subject suffering from Degos disease by at least 20% or more (e.g., at least 30%, at least 40%, at least 50%) more than the inhibitory effect on the deposition of C5b-9 in the blood serum.

In one embodiment, the MASP-2 inhibitory antibody inhibits clotting in serum from a patient with Degos disease by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95 to 99%, compared to untreated serum.

In one embodiment, the MASP-2 inhibitory antibody is administered to the subject via an intravenous catheter or other catheter delivery method.

In one embodiment, the invention provides a method of inhibiting thrombus formation in a subject suffering from Degos disease, comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or an antigen-binding fragment thereof, comprising (I)(a) a heavy chain variable region comprising: i) heavy chain CDR-H1, including the sequence of amino acids from 31-35 of the sequence of SEQ ID NO: 67; and ii) a CDRH2 heavy chain comprising an amino acid sequence from 50-65 of SEQ ID NO: 67; and iii) a CDR-H3 heavy chain comprising the amino acid sequence from 95-102 of SEQ ID NO: 67 and b) a light chain variable region comprising: i) a CDR-L1 light chain comprising the amino acid sequence from 24-34 of SEQ ID NO: 70; and ii) a CDR-L2 light chain comprising the amino acid sequence from 50-56 of SEQ ID NO: 70; and iii) a CDR-L3 light chain comprising an amino acid sequence from 89-97 of SEQ ID NO: 70, or (ii) a variant thereof comprising a heavy chain variable region with at least 90% identity to SEQ ID NO: 67 ( e.g. at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , with at least 98%, at least 99% identity with SEQ ID NO: 67), and a light chain variable region with at least 90% identity (for example, at least 91%, at least with 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99 % identity with SEQ ID NO: 70.

In some embodiments, the method comprises administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising an amino acid sequence referred to as the sequence of SEQ ID NO: 67. In some embodiments, the method comprises administering to the subject the composition , including the amount of MASP-2 inhibitory antibody or its antigen-binding fragment, including the variable region of the light chain, including the amino acid sequence, referred to as the sequence of SEQ ID NO: 70.

In some embodiments, the method includes administering to a subject a composition comprising a MASP-2 inhibitory antibody, or an antigen-binding fragment thereof, that specifically recognizes at least a portion of an epitope on human MASP-2 recognized by an OMS646 control antibody, comprising the heavy chain variable region described in sequence of SEQ ID NO: 67, and the variable region of the light chain described in the sequence of SEQ ID NO: 70.

Catastrophic antiphospholipid syndrome (CAPS).

Catastrophic antiphospholipid syndrome (CAPS) is an extreme variant of the antiphospholipid antibody syndrome (APLA). CAPS is characterized by venous and arterial thrombosis caused by pathogenic antibodies. CAPS is a TMA with multiple thrombosis, organ ischemia, and organ failure. As in the case of other TMAs, this disease is characterized by occlusion of small vessels in various organs. Mortality in CAPS reaches a high level of about 50%, and is often associated with infection or trauma. Patients have antiphospholipid antibodies, usually IgG.

Clinically, CAPS involves at least three organs or tissues with

- 33 042534 histopathological evidence of small vessel occlusion. Peripheral thrombosis may involve veins and arteries in the central nervous system, cardiovascular, renal, or pulmonary systems. Patients are treated with antibiotics, anticoagulants, corticosteroids, plasma exchange, and intravenous immunoglobulins. However, death can occur as a result of multiple organ failure.

In CAPS, the complement pathway is involved. For example, studies in animal models indicate that complement inhibition may be an effective way to prevent CAPS-associated thrombosis (Shapira L. et al., Arthritis Rheum 64(8):2719-23, 2012). Moreover, as was further reported in the publication cited above, administration of eculizumab to a subject suffering from CAPS at doses that block the complement pathway prevented acute progressive thrombotic complications and reversed thrombocytopenia (see also Lim W., Curr Opin Hematol 18 (5):361-5, 2011). Therefore, as described in the invention for other TMAs, it is expected that inhibitors of the lectin pathway, including, but not limited to, antibodies that block MASP-2 function, can lead to positive results in the treatment of patients suffering from CAPS.

Accordingly, in another embodiment, the invention provides methods of treating CAPS by administering a composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent, such as an anti-MASP-2 antibody, in a pharmaceutical carrier, to a subject suffering from CAPS or a condition resulting from CAPS. . The MASP-2 inhibitory agent is administered systemically to a subject suffering from CAPS or a condition resulting from CAPS, for example, intra-arterially, intravenously, intramuscularly, by inhalation, subcutaneously, or other parenteral route, or possibly by oral administration in the case of non-peptidergic drugs. The anti-MASP-2 antibody can be administered as monotherapy or in combination with a C5 inhibitor such as eculizumab.

In one embodiment, the MASP-2 inhibitory antibody exhibits at least one or more of the following: said antibody binds human MASP-2 with a KD value of 10 nM or less, said antibody binds a CCP1 epitope in the MASP-2 domain, said antibody inhibits deposition C3b in an in vitro study in 1% human serum with an IC ₅₀ value of 10 nM or less, said antibody inhibits the deposition of C3b in 90% of human serum with an IC ₅₀ value of 30 nM or less, wherein the antibody is an antibody fragment selected from the group consisting of of Fv, Fab, Fab', F(ab) ₂ and F(ab') ₂ , where the antibody is a single chain molecule, where the specified antibody is an IgG2 molecule, where the specified antibody is an IgG1 molecule, where the specified antibody is an IgG4 molecule, where the IgG4 molecule contains the S228P mutation, and/or where the antibody does not substantially inhibit the classical pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and substantially does not inhibit the alternative pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and does not substantially inhibit the classical or alternative pathway (ie, it inhibits the lectin pathway but does not affect the classical and alternative complement pathways).

In one embodiment, the MASP-2 inhibitory antibody inhibits clotting in serum from a subject suffering from CAPS by at least 30%, such as at least 40%, such as at least 50%, such as at least at least 60%, such as at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% to 99%, compared to untreated serum. In some embodiments, the MASP-2 inhibitory antibody inhibits blood clotting in serum from a subject suffering from CAPS by at least 20% or more (e.g., at least 30%, at least 40%, at least 50%) more than the inhibitory effect on the deposition of C5b-9 in the blood serum.

In one embodiment, the MASP-2 inhibitory antibody inhibits clotting in serum from a CAPS patient by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% to 99%, compared to untreated serum.

In one embodiment, the MASP-2 inhibitory antibody is administered to the subject via an intravenous catheter or other catheter delivery method.

In one embodiment, the invention provides a method for inhibiting thrombus formation in a subject suffering from CAPS, comprising administering to the subject a composition containing an amount of a MASP-2 inhibitory antibody, or an antigen-binding fragment thereof, comprising (I) (a) a heavy chain variable region comprising: i) a heavy a CDR-H1 chain comprising the amino acid sequence from 31-35 of SEQ ID NO: 67; and ii) a CDR-H2 heavy chain comprising the amino acid sequence from 50-65 of SEQ ID NO: 67; and iii) severe

- 34 042534 a CDR-H3 chain comprising the amino acid sequence from 95-102 of SEQ ID NO: 67 and b) a light chain variable region comprising: i) a CDR-L1 light chain comprising an amino acid sequence from 24-34 of SEQ ID NO : 70; and ii) a CDRL2 light chain comprising the amino acid sequence from 50-56 of SEQ ID NO: 70; and iii) a CDR-L3 light chain comprising the amino acid sequence from 89-97 of SEQ ID NO: 70, or (ii) a variant thereof comprising a heavy chain variable region with at least 90% identity to SEQ ID NO: 67 ( e.g. at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , with at least 98%, at least 99% identity with SEQ ID NO: 67), and a light chain variable region with at least 90% identity (for example, at least 91%, at least with 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99 % identity with SEQ ID NO: 70.

In some embodiments, the method comprises administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising an amino acid sequence referred to as the sequence of SEQ ID NO: 67. In some embodiments, the method comprises administering to the subject the composition , including the amount of MASP-2 inhibitory antibody or its antigen-binding fragment, including the variable region of the light chain, including the amino acid sequence, referred to as the sequence of SEQ ID NO: 70.

In some embodiments, the method includes administering to a subject a composition comprising a MASP-2 inhibitory antibody, or an antigen-binding fragment thereof, that specifically recognizes at least a portion of an epitope on human MASP-2 recognized by an OMS646 control antibody, comprising the heavy chain variable region described in sequence of SEQ ID NO: 67, and the variable region of the light chain described in the sequence of SEQ ID NO: 70.

TMA against the background of cancer.

Systemic malignancies of any type can lead to clinical and pathological manifestations of TMA (see, for example, Batts and Lazarus, Bone Marrow Transplantation 40:709-719, 2007). TMA in cancer is often found in the lungs and appears to be associated with tumor emboli (Francis KK et al., Commun Oncol 2:339-43, 2005). Tumor emboli can reduce blood flow and therefore lead to a state of hypoperfusion in the affected arterioles and venules. It is assumed that the resulting stress and tissue damage locally activates the complement lectin pathway. The activated lectin pathway, in turn, can activate the coagulation system through MASP-2 dependent cleavage of prothrombin to thrombin, leading to the prothrombic state characteristic of TMA.

Inhibition of MASP-2 under these conditions is expected to reduce localized thrombin activation and thereby alleviate the prothrombic state.

Therefore, as described herein for other TMAs, it is contemplated that inhibitors of the lectin pathway, including but not limited to antibodies that block MASP-2 function, may be beneficial in the treatment of patients suffering from TMA in cancer.

Accordingly, in another embodiment, the invention provides methods for treating or preventing TMA in cancer by administering a composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent, such as an anti-MASP-2 antibody, in a pharmaceutical carrier, to a subject suffering from TMA on background of cancer or at risk of developing cancer-related TMA. The MASP-2 inhibitory agent is administered systemically to a subject suffering from cancer-related TMA or at risk of developing cancer-related TMA, for example, intra-arterially, intravenously, intramuscularly, inhaled, subcutaneously, or by other parenteral route, or possibly by oral administration in the case of non-peptidergic drugs. The anti-MASP-2 antibody can be administered as monotherapy or in combination with a C5 inhibitor such as eculizumab.

In one embodiment, the MASP-2 inhibitory antibody exhibits at least one or more of the following: said antibody binds human MASP-2 with a KD value of 10 nM or less, said antibody binds a CCP1 epitope in the MASP-2 domain, said antibody inhibits deposition C3b in an in vitro study in 1% human serum with an IC ₅₀ value of 10 nM or less, said antibody inhibits the deposition of C3b in 90% of human serum with an IC ₅₀ value of 30 nM or less, wherein the antibody is an antibody fragment selected from the group consisting of of Fv, Fab, Fab', F(ab) ₂ and F(ab') ₂ , where the antibody is a single chain molecule, where the specified antibody is an IgG2 molecule, where the specified antibody is an IgG1 molecule, where the specified antibody is an IgG4 molecule, where the IgG4 molecule contains the S228P mutation, and/or where the antibody does not substantially inhibit the classical pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and does not substantially inhibit

- 35 042534 alternative route. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and does not substantially inhibit the classical or alternative pathway (ie, it inhibits the lectin pathway but does not affect the classical and alternative complement pathways).

In one embodiment, the MASP-2 inhibitory antibody inhibits blood clotting in serum taken from a subject suffering from cancer-related TMA by at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95 to 99% compared to untreated serum.

In one embodiment, the MASP-2 inhibitory antibody inhibits blood clotting in serum taken from a patient suffering from cancer-related TMA by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% to 99%, compared to serum, not subjected to treatment.

In one embodiment, the MASP-2 inhibitory antibody is administered to the subject via an intravenous catheter or other catheter delivery method.

In one embodiment, the invention provides a method for inhibiting thrombus formation in a subject suffering from TMA in cancer, comprising administering to the subject a composition containing an amount of a MASP-2 inhibitory antibody, or an antigen-binding fragment thereof, comprising (I)(a) a heavy chain variable region comprising: i) a CDR-H1 heavy chain comprising the amino acid sequence from 31-35 of SEQ ID NO: 67; and ii) a CDR-H2 heavy chain comprising the amino acid sequence from 50-65 of SEQ ID NO: 67; and iii) a CDR-H3 heavy chain comprising the amino acid sequence from 95-102 of SEQ ID NO: 67 and b) a light chain variable region comprising: i) a CDR-L1 light chain comprising the amino acid sequence from 24-34 of SEQ ID NO: 70; and ii) a CDR-L2 light chain comprising the amino acid sequence from 50-56 of SEQ ID NO: 70; and iii) a CDR-L3 light chain comprising an amino acid sequence from 89-97 of SEQ ID NO: 70, or (ii) a variant thereof comprising a heavy chain variable region with at least 90% identity to SEQ ID NO: 67 ( e.g. at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , with at least 98%, at least 99% identity with SEQ ID NO: 67), and a light chain variable region with at least 90% identity (for example, at least 91%, at least with 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99 % identity with SEQ ID NO: 70.

In some embodiments, the method comprises administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising an amino acid sequence referred to as the sequence of SEQ ID NO: 67. In some embodiments, the method comprises administering to the subject the composition , including the amount of MASP-2 inhibitory antibody or its antigen-binding fragment, including the variable region of the light chain, including the amino acid sequence, referred to as the sequence of SEQ ID NO: 70.

In some embodiments, the method includes administering to a subject a composition comprising a MASP-2 inhibitory antibody, or an antigen-binding fragment thereof, that specifically recognizes at least a portion of an epitope on human MASP-2 recognized by an OMS646 control antibody, comprising the heavy chain variable region described in sequence of SEQ ID NO: 67, and the variable region of the light chain described in the sequence of SEQ ID NO: 70.

TMA against the background of anticancer chemotherapy.

Chemotherapy-associated TMA is a condition including thrombocytopenia, microangiopathic hemolytic anemia, and renal dysfunction that develops in 2-10% of patients with a history of malignancy treated with chemotherapeutic agents such as gemcitabine, mitomycin, oxaliplatin, and the like. Chemotherapy-associated TMA is characterized by poor clinical outcomes with high mortality (see, for example, Blake-Haskins et al., Clin Cancer Res 17(18):5858-5866, 2011).

The etiology of chemotherapy-associated TMA is believed to involve non-specific toxic damage to the microvascular endothelium. In an animal model of mitomycin-induced TMA, direct damage to endothelial cells has been demonstrated (Dlott J. et al., Ther Apher Dial 8:102-11, 2004). Damage to endothelial cells has been shown to activate the lectin complement pathway through a number of mechanisms. For example, in Stahl et al. It has been shown that endothelial cells exposed to oxidative stress activate

- 36 042534 lectin complement pathway both in vitro and in vivo (Collard et al., Am J Pathol. 156(5):1549-56, 2000; La Bonte et al., J Immunol. 15; 188(2) :885-91, 2012). In vivo, this process leads to thrombosis, and inhibition of the lectin pathway has been shown to prevent thrombosis (La Bonte et al. J Immunol. 15; 188(2):885-91, 2012). In addition, as illustrated in the invention in examples 37-39, in an experimental model of TMA in mice, in which localized FITCDex photoexcitation was used to induce localized damage to the microcirculatory bed, followed by the development of a response in the form of TMA, the authors of the present invention showed that inhibition of MASP- 2 can prevent TMA. Thus, damage to the microvascular endothelium by chemotherapeutic drugs can activate the lectin complement pathway, which then leads to a localized prothrombotic state and promotes the development of a response in the form of TMA. Since activation of the lectin pathway and the onset of a prothrombotic state depend on MASP-2, it is hypothesized that MASP-2 inhibitors, including, but not limited to, antibodies that block MASP-2 function, may reduce the development of the TMA response and reduce the risk of TMA on the background of anticancer chemotherapy.

Accordingly, in another embodiment, the invention provides methods for treating or preventing TMA during cancer chemotherapy by administering a composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent, such as an anti-MASP-2 antibody, in a pharmaceutical carrier, to a subject suffering from TMA. on the background of anti-cancer chemotherapy or at risk of developing TMA on the background of anti-cancer chemotherapy. The MASP2 inhibitory agent is administered systemically to a subject who has been, is currently undergoing, or will be undergoing chemotherapy, for example, intra-arterially, intravenously, intramuscularly, by inhalation, subcutaneously, or other parenteral route, or possibly by oral administration in the case of non-peptidergic drugs. The anti-MASP-2 antibody can be administered as monotherapy or in combination with a C5 inhibitor such as eculizumab.

In one embodiment, the MASP-2 inhibitory antibody exhibits at least one or more of the following: said antibody binds human MASP-2 with a KD value of 10 nM or less, said antibody binds a CCP1 epitope in the MASP-2 domain, said antibody inhibits deposition C3b in an in vitro study in 1% human serum with an IC ₅₀ value of 10 nM or less, said antibody inhibits the deposition of C3b in 90% of human serum with an IC ₅₀ value of 30 nM or less, wherein the antibody is an antibody fragment selected from the group consisting of of Fv, Fab, Fab', F(ab) ₂ and F(ab') ₂ , where the antibody is a single chain molecule, where the specified antibody is an IgG2 molecule, where the specified antibody is an IgG1 molecule, where the specified antibody is an IgG4 molecule, where the IgG4 molecule contains the S228P mutation, and/or where the antibody does not substantially inhibit the classical pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and substantially does not inhibit the alternative pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and does not substantially inhibit the classical or alternative pathway (ie, it inhibits the lectin pathway but does not affect the classical and alternative complement pathways).

In one embodiment, the MASP-2 inhibitory antibody inhibits clotting in serum taken from a subject suffering from TMA on cancer chemotherapy by at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% to 99% compared to untreated serum.

In one embodiment, the MASP-2 inhibitory antibody inhibits clotting in serum taken from a patient suffering from TMA on cancer chemotherapy by at least 30%, at least 40%, at least 50%, at least at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% to 99%, compared to serum, untreated.

In one embodiment, the MASP-2 inhibitory antibody is administered to the subject via an intravenous catheter or other catheter delivery method.

In one embodiment, the invention provides a method for inhibiting thrombus formation in a subject suffering from TMA during cancer chemotherapy, comprising administering to the subject a composition containing an amount of a MASP-2 inhibitory antibody, or an antigen-binding fragment thereof, comprising (I)(a) a heavy chain variable region comprising : i) heavy chain CDR-H1, including the amino acid sequence from 31-35 of the sequence of SEQ ID NO: 67; and ii) a CDR-H2 heavy chain comprising the amino acid sequence from 50-65 of SEQ ID NO: 67; and iii) a CDR-H3 heavy chain comprising the amino acid sequence from 95-102 of SEQ ID NO: 67 and b) a light chain variable region comprising: i) a CDR-L1 light chain comprising the amino acid sequence from 24-34 of SEQ ID NO: 70; and ii) a CDR-L2 light chain comprising an amino acid sequence of 50-56

- 37 042534 SEQ ID NO: 70; and iii) a CDR-L3 light chain comprising the amino acid sequence from 89-97 of SEQ ID NO: 70, or (ii) a variant thereof comprising a heavy chain variable region with at least 90% identity to SEQ ID NO: 67 ( e.g. at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , with at least 98%, at least 99% identity with SEQ ID NO: 67), and a light chain variable region with at least 90% identity (for example, at least 91%, at least with 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99 % identity with SEQ ID NO: 70.

In some embodiments, the method comprises administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising an amino acid sequence referred to as the sequence of SEQ ID NO: 67. In some embodiments, the method comprises administering to the subject the composition , comprising the amount of MASP-2 inhibitory antibody or its antigen-binding fragment, including the variable region of the light chain, including the amino acid sequence, referred to as the sequence of SEQ ID NO: 70.

In some embodiments, the method includes administering to a subject a composition comprising a MASP-2 inhibitory antibody, or an antigen-binding fragment thereof, that specifically recognizes at least a portion of an epitope on human MASP-2 recognized by an OMS646 control antibody, comprising the heavy chain variable region described in sequence of SEQ ID NO: 67, and the variable region of the light chain described in the sequence of SEQ ID NO: 70.

TMA on the background of transplantation.

Transplant-associated TMA (TA-TMA) is a debilitating symptom that can occur in transplant patients, such as patients undergoing allogeneic hematopoietic stem cell transplantation (see, for example, Batts and Lazarus, Bone Marrow Transplantation 40:709-719, 2007). The pathogenesis of this condition is practically not studied, but, apparently, it involves a fusion of responses that culminate in damage to endothelial cells (Laskin B.L. et al., Blood 118(6):1452-62, 2011). As described above, damage to endothelial cells is the prototypical stimulus for activation of the lectin pathway and generation of prothrombotic environments.

Recent data further support the role of complement activation via the lectin pathway in the pathogenesis of TA-TMA. In Laskin B.L., et al., Transplantation, 27; 96(2):217-23, 2013 showed that C4d deposition in renal arterioles was significantly higher in subjects with histological TA-TMA (75%) than in controls (8%). Therefore, C4d may be a marker of TA-TMA pathology, indicating localized fixation of complement activation along the lectin or classical pathway.

Since activation of the lectin pathway and the onset of a prothrombotic state depend on MASP-2, it is hypothesized that MASP-2 inhibitors, including, but not limited to, antibodies that block MASP-2 function, may reduce the development of the TMA response and reduce the risk of transplantation-associated TMA (TA-TMA).

Accordingly, in another embodiment, the invention provides methods of treating or preventing transplant-related TMA by administering a composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent, such as an anti-MASP2 antibody, in a pharmaceutical carrier, to a subject suffering from cancer-related TMA. chemotherapy or at risk of developing TMA during transplantation. The MASP-2 inhibitory agent is administered systemically to a subject who has been, is currently undergoing, or will be undergoing a transplant procedure, e.g., intra-arterially, intravenously, intramuscularly, inhaled, subcutaneously, or by another parenteral route, or possibly by oral administration in the case of non-peptidergic medicines. The anti-MASP-2 antibody can be administered as monotherapy or in combination with a C5 inhibitor such as eculizumab. In some embodiments, the invention provides methods for treating or preventing TMA in an allogeneic stem cell transplant, comprising administering a composition comprising an amount of a MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody, to a subject prior to, during, or after receiving an allogeneic stem cell transplant. cells.

In one embodiment, the MASP-2 inhibitory antibody exhibits at least one or more of the following: said antibody binds human MASP-2 with a KD value of 10 nM or less, said antibody binds a CCP1 epitope in the MASP-2 domain, said antibody inhibits deposition C3b in an in vitro study in 1% human serum with an IC ₅₀ value of 10 nM or less, said antibody inhibits the deposition of C3b in 90% of human serum with an IC ₅₀ value of 30 nM or less, wherein the antibody is an antibody fragment selected from the group consisting of of Fv, Fab, Fab', F(ab) ₂ and F(ab') ₂ where the antibody is a single chain

- 38 042534 a molecule, where the specified antibody is an IgG2 molecule, where the specified antibody is an IgG1 molecule, where the specified antibody is an IgG4 molecule, where the IgG4 molecule includes the S228P mutation, and/or where the antibody practically does not inhibit the classical pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and substantially does not inhibit the alternative pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and does not substantially inhibit the classical or alternative pathway (ie, it inhibits the lectin pathway but does not affect the classical and alternative complement pathways).

In one embodiment, the MASP-2 inhibitory antibody inhibits clotting in serum taken from a subject suffering from transplant TMA by at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% to 99 % compared to untreated serum.

In one embodiment, the MASP-2 inhibitory antibody inhibits blood clotting in serum taken from a patient suffering from transplant TMA by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% to 99%, compared to serum, not subjected to treatment.

In one embodiment, the MASP-2 inhibitory antibody is administered to the subject via an intravenous catheter or other catheter delivery method.

In one embodiment, the invention provides a method of inhibiting thrombus formation in a subject suffering from TMA on a transplant background, comprising administering to the subject a composition containing an amount of a MASP-2 inhibitory antibody, or an antigen-binding fragment thereof, comprising (I)(a) a heavy chain variable region, comprising: i) a CDR-H1 heavy chain comprising the amino acid sequence from 31-35 of SEQ ID NO: 67; and ii) a CDR-H2 heavy chain comprising the amino acid sequence from 50-65 of SEQ ID NO: 67; and iii) a CDR-H3 heavy chain comprising the amino acid sequence from 95-102 of SEQ ID NO: 67; and b) a light chain variable region comprising: i) a CDRL1 light chain comprising the amino acid sequence from 24-34 of SEQ ID NO: 70; and ii) a CDR-L2 light chain comprising the amino acid sequence from 50-56 of SEQ ID NO: 70; and iii) a CDR-L3 light chain comprising an amino acid sequence from 89-97 of SEQ ID NO: 70, or (ii) a variant thereof comprising a heavy chain variable region with at least 90% identity to SEQ ID NO: 67 ( e.g. at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , with at least 98%, at least 99% identity with SEQ ID NO: 67), and a light chain variable region with at least 90% identity (for example, at least 91%, at least with 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99 % identity with SEQ ID NO: 70.

In some embodiments, the method comprises administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising an amino acid sequence referred to as the sequence of SEQ ID NO: 67. In some embodiments, the method comprises administering to the subject the composition , comprising the amount of MASP-2 inhibitory antibody or its antigen-binding fragment, including the variable region of the light chain, including the amino acid sequence, referred to as the sequence of SEQ ID NO: 70.

In some embodiments, the method includes administering to a subject a composition comprising a MASP-2 inhibitory antibody, or an antigen-binding fragment thereof, that specifically recognizes at least a portion of an epitope on human MASP-2 recognized by an OMS646 control antibody, comprising the heavy chain variable region described in sequence of SEQ ID NO: 67, and the variable region of the light chain described in the sequence of SEQ ID NO: 70.

IV. The role of masp-2 in other diseases and conditions, and therapeutic methods that use MASP-2.

inhibitory agents.

Kidney diseases.

The complement activation system is involved in the pathogenesis of a wide range of kidney diseases, including membranous proliferative glomerulonephritis (IgA nephropathy, Berger's disease) (Endo, M., et al., Clin. Nephrology 55:185-191, 2001), membranous glomerulonephritis (Kerjashki , D., Arch B Cell Pathol 58:253-71, 1990; Brenchley, RE., et al., Kidney Int., 41:933-7, 1992; Salant, D. J., et al., Kidney Int. 35 :976-84, 1989), membranous proliferative glomerulonephritis (mesangiocapillary glomerulo

- 39 042534 nephritis) (Bartlow, B.G., et al. Kidney Int. 15:294-300, 1979; Meri, S., et al., J. Exp. Med. 175:939-50, 1992), acute postinfectious glomerulonephritis (poststreptococcal glomerulonephritis), cryoglobulinemic glomerulonephritis (Ohsawa, I, et al., Clin Immunol. 101:59-66, 2001), lupus nephritis (Gatenby, P.A, Autoimmunity 11:61-6, 1991) and nephritis with purpura Schonlein-Genoha (Endo, M., et al., Am. J. Kidney Dis. 35:401-407, 2000). Complement involvement in kidney disease has been recognized for several decades, however, to date, its precise role in the onset, progression, and end phase of kidney disease is still the subject of much debate. Under normal conditions, complement participation has a beneficial effect on the host, but inappropriate activation and deposition of complement can lead to tissue damage.

There is substantial evidence that glomerulonephritis, an inflammation of the glomerulus, is often initiated by the deposition of immune complexes in glomerular or tubular structures, which then initiates complement activation, inflammation, and tissue damage. In Kahn, T.N, et al., Histopath. 26:351-6, 1995 demonstrated increased deposition of C5b-9 in tubular basement membranes in biopsies taken from patients suffering from various forms of glomerulonephritis. In a study of patients with IgA nephrology (Alexopoulos, A., et al., Nephrol. Dial. Transplant 10:11661172, 1995), deposition of C5b-9 in tubular epithelial/basal membrane structures correlated with plasma creatinine levels. Another study on membranous nephropathy demonstrated an association between clinical outcomes and urinary sC5b-9 levels (Kon, S.P, et al., Kidney Int. 48:1953-58, 1995). Elevated sC5b-9 levels were positively correlated with poor prognosis. In Lento, T., et al., Kidney Int. 47:1403-11, 1995 reports measuring elevated levels of CD59, the complement regulatory factor that inhibits the membrane attack complex in cytoplasmic membranes, and C5b-9 in the urine of patients suffering from membranous glomerulonephritis. Histopathological analysis of biopsy samples taken from the same patients showed the deposition of C3 and C9 proteins in the glomeruli, while the expression of CD59 in these tissues was reduced compared to the expression in normal kidney tissues. These various studies suggest that long-term complement-mediated glomerulonephritis leads to the excretion of complement proteins in the urine, which correlates with the degree of tissue damage and with the prognosis of the disease.

Inhibition of complement activation in various animal models of glomerulonephritis has also shown the importance of complement activation in the etiology of the disease. In a model of membranous proliferative glomerulonephritis (MPGN), infusion of anti-Thy1 antiserum into C6-deficient rats (which are unable to synthesize C5b-9) resulted in a 90% decrease in glomerular cell proliferation, an 80% decrease in platelet and macrophage infiltration, and a decrease in collagen synthesis. type IV (a marker of mesangial matrix expansion) and a 50% reduction in proteinuria compared to wild animals (Brandt, J., et al., Kidney Int. 49:335-343, 1996). These results indicate that C5b-9 acts as the primary mediator of complement tissue damage in this antithymocyte serum model in rats. In another model of glomerulonephritis, infusion of increasing doses of rabbit antiserum to rat glomerular basement membrane produced a dose-dependent migration of polymorphonuclear leukocytes (PMN) that was pre-attenuated by treatment with cobra venom factor (for complement depletion) (Scandrett, A.L, et al., Am. J Physiol 268:F256-F265, 1995). Rats treated with cobra venom factor also showed reduced histopathology, reduced long-term proteinuria, and lower creatinine levels compared to control animals. In Couser, W.G., et al., J. Am. soc. Nephrol. 5:1888-94, 1995, using three models of glomerulonephritis (GN) in rats (antithymocyte serum, concavalin A, anti-concavalin A, and passive Heymann's nephritis), the potential therapeutic efficacy of complement inhibition approaches using the recombinant sCRI protein was demonstrated. Rats treated with sCRI were characterized by a significant decrease in the migration of polymorphonuclear leukocytes, platelets and macrophages, a decrease in mesangiolysis and proteinuria compared to control animals. Additional evidence for the importance of complement activation in glomerulonephritis was obtained using MoAb against C5 in the NZB/W F1 mouse model. Antibodies MoAb against C5 inhibit the cleavage of C5, thereby blocking the generation of C5a and C5b-9. Long-term therapy with MoAb against C5 for 6 months resulted in a significant improvement in the course of glomerulonephritis.

A humanized anti-C5 monoclonal antibody MoAb (5G1.1), which prevents the breakdown of human complement component C5 into its pro-inflammatory components, is currently under development by Alexion Pharmaceuticals, Inc., (New Haven, Connecticut) as a promising drug for the treatment of glomerulonephritis .

Direct evidence of the pathological role of complement in kidney injury has been obtained from a study of patients with a genetically determined deficiency of specific complement components. A number of publications have documented the association of kidney disease with complement regulatory factor H deficiency (Ault, B.H., Nephrol. 14:1045-1053, 2000; Levy, M., et al.,

- 40 042534

Kidney Int. 30:949-56, 1986; Pickering, M.C., et al., Nat. Genet. 31:424-8, 2002). Factor H deficiency results in low plasma levels of factor B and C3 and depletion of C5b-9. Atypical membranous proliferative glomerulonephritis (MPGN) and idiopathic hemolytic uremic syndrome (HUS) are associated with factor H deficiency. Factor H deficient pigs (Jansen, J.H., et al., Kidney Int. 53:331-49, 1998) and factor H knockout mice (Pickering, M.C., 2002) show MPGN-like symptoms, suggesting the importance of factor H in complement regulation. Deficiency of other complement components is associated with kidney disease associated with systemic lupus erythematosus (SLE) (Walport, M.J., Davies, et al., Ann. N.Y. Acad. Sci 815:261-81, 1997). Deficiency in C1q, C4 and C2 strongly provokes the development of SLE through mechanisms related to impaired clearance of immune complexes and apoptotic material. Many of the patients with SLE develop lupus nephritis, characterized by the deposition of immune complexes throughout the glomerulus.

Additional evidence linking complement activation to kidney disease has come from the identification in patients of autoantibodies directed against complement components, some of which have been directly linked to kidney disease (Trouw, LA, et al., Mol. Immunol. 38:199 -206, 2001). The number of these autoantibodies correlates with kidney disease to such a high degree that the term nephritogenic factor (NeF) has been introduced to refer to this activity. In clinical studies, approximately 50% of patients with positive nephritogenic factors developed MPGN (Spitzer, R. E., et al., Clin. Immunol. Immunopathol. 64:177-83, 1992). C3NeF is an autoantibody directed against the alternative pathway C3 convertase (C3bBb) and it stabilizes this convertase, thereby promoting the activation of the alternative pathway (Dana, M.R., et al., J. Immunol. 116:1-7, 1976). Similarly, an autoantibody with specificity for the C3 convertase (C4b2a) of the classical pathway, called C4NeF, stabilizes this convertase and as a result promotes activation of the classical pathway (Dana, M.R. et al., J. Immunol. 125:2051-2054, 1980; Halbwachs, L, et al., J. Clin. Invest. 65:1249-56, 1980). Anti-C1q autoantibodies have been described to be associated with nephritis in patients suffering from SLE (Hovath, L., et al., Clin. Exp. Rheumatol. 19:667-72, 2001; Siegert, C, et al., J. Rheumatol 18:230-34, 1991; Siegert, C, et al., Clin. Exp. Rheumatol. 10:19-23, 1992), and it has been reported that an increase in the titer of these anti-C1q autoantibodies can be used as a predictor of flare-ups. jade (Coremans, I.E., et al., Am. J. Kidney Dis. 26:595601, 1995). Immune deposits extracted from the kidneys of deceased SLE patients showed accumulation of these anti-C1q autoantibodies (Mannick, M., et al., Arthritis Rheumatol. 40:1504-11, 1997). All these facts point to the pathological role of these autoantibodies. However, not all patients with anti-C1q autoantibodies develop kidney disease and, in addition, some healthy individuals also have a low titer of anti-C1q autoantibodies (Siegert, C.E., et al., Clin. Immunol. Immunopathol. 67:204-9, 1993 ).

In addition to the alternative and classical pathways of complement activation, the lectin pathway of complement activation may also play an important pathological role in kidney disease. Elevated levels of MBL, MBL-associated serine protease, and complement activation products have been detected by immunohistochemistry in kidney biopsies taken from patients suffering from a number of different kidney diseases, including Henoch-Schönlein purpura nephritis (Endo, M., et al., Am J. Kidney Dis. 35:401-407, 2000), cryoglobulemic glomerulonephritis (Ohsawa, I, et al., Clin. Immunol. 101:59-66, 2001), and IgA neuropathy (Endo, M., et al. , Clin Nephrology 55:185-191, 2001). Therefore, despite the fact that the link between complement and kidney disease has been known for several decades, research into exactly how complement affects these kidney diseases is far from complete.

Blood diseases.

Sepsis is due to the patient's generalized response to microbial invasion. The main function of the complement system is to control the inflammatory response to invasive bacteria and other pathogens. Numerous studies have shown that complement activation is central to the pathogenesis of sepsis, consistent with the physiological role of complement (Bone, R.C., Annals. Internal. Med. 115:457-469, 1991). The definition of the clinical manifestations of sepsis is still under development. Sepsis is usually described as a systemic host response to infection. However, in many cases, patients with symptoms of sepsis do not show obvious clinical signs of infection (eg, positive bacterial blood cultures). This controversy was first brought to the fore at the 1992 Consensus Conference, which coined the term systemic inflammatory response syndrome (SIRS), which did not require detection of bacterial infection (Bone, R.C., et al., Crit Care Med 20:724-726, 1992). Currently, there is general agreement that sepsis and SIRS are accompanied by a failure to regulate the inflammatory response. For the purposes of this brief review, the inventors believe that the clinical definition of sepsis also includes severe sepsis, septic shock, and SIRS.

Until the late 1980s, the dominant source of infection in patients suffering from sepsis was

- 41 042534 were gram-negative bacteria. Injection of lipopolysaccharide (LPS), a major component of the cell wall of Gram-negative bacteria, into animals has been known to stimulate the release of inflammatory mediators from various cell types and induce acute infectious disease symptoms (Haeney, M.R., et al., Antimicrobial Chemotherapy 41 (Suppl. A): 41-6, 1998). It is interesting to note that the spectrum of microorganisms responsible for the development of the disease, for unknown reasons, clearly shifted from predominantly gram-negative bacteria in the late 1970s and 1980s to predominantly gram-positive bacteria today (Martin, G.S., et al., N. Eng. J. Med. 348:1546-54, 2003).

Numerous studies have demonstrated the important role of complement activation in mediating inflammation and in contributing to the characteristics of shock, in particular septic and hemorrhagic shock. Septic shock is usually caused by both Gram-negative and Gram-positive organisms. LPS is a potent complement activator, predominantly via the alternative pathway, although the classic antibody-mediated activation pathway also occurs (Fearon, D.T., et al., N. Engl. J. Med. 292:937-400, 1975). Peptidoglycan and lipoteichoic acid are the main components of the cell walls of Gram-positive bacteria, and both components are potential activators of the alternative complement pathway, although in the presence of specific antibodies they can also activate the classical complement pathway (Joiner, K.A., et al., Ann. Rev. Immunol. 2:461-2, 1984).

At first, the pathogenesis of sepsis was associated with the complement system, when researchers noted that anaphylatoxins C3a and C5a mediate a number of inflammatory reactions that can also occur in sepsis. These anaphylatoxins induce vasodilation and increased microvascular permeability, complications that play a central role in septic shock (Schumacher, W.A., et al., Agents Actions 34:345-349, 1991).

In addition, anaphylatoxins induce bronchospasm, histamine release from mast cells, and platelet aggregation. Moreover, they have numerous effects on granulocytes such as chemotaxis, aggregation, adhesion, release of lysosomal enzymes, generation of toxic superoxide anion, and formation of leukotrienes (Shin, H.S., et al., Science 162:361-363, 1968; Vogt, W ., Complement 3:177-86, 1986). These biological effects are believed to play a role in the development of complications of sepsis such as shock or acute respiratory distress syndrome (ARDS) (Hammerschmidt, D.E., et al., Lancet 1:947-949, 1980; Slotman, G.T, et al. ., Surgery 99:744-50, 1986). Moreover, elevated levels of anaphylatoxin C3a are associated with death in sepsis (Hack, C.E., et al., Am. J. Med. 86:20-26, 1989). In some experimental animal models of shock, certain complement-deficient strains (eg, C5-deficient strains) are more resistant to the effects of LPS infusion (Hseuh, W., et al., Immunol. 70:30914, 1990).

It has been shown that blocking C5a generation with antibodies early in the development of sepsis in rodents leads to a significant increase in survival (Czermak, B. J., et al., Nat. Med. 5:788-7 92, 1999). Similar conclusions were drawn when blocking the C5a receptor (C5aR) with both antibodies and a small molecule inhibitor with an inhibitor (Huber-Lang, M.S., et al., FASEB J. 16:1567-74, 2002; Riedemann, N.C. et al. ., J. Clin. Invest, 110:101-8, 2002). Previous experimental studies in monkeys suggested that antibody blockade of C5a alleviated E. coli septic shock and adult respiratory distress syndrome (Hangen, D. H., et al., J. Surg. Res. 46:195 -9, 1989; Stevens, J.H., et al., J. Clin. Invest. 77:1812-16, 1986). People with sepsis had elevated C5a levels, which was associated with a significantly reduced survival rate along with multi-organ failure compared to those in patients with less severe sepsis and survivors (Nakae, H., et al., Res. Commun Pathol Pharmacol 84:189-95, 1994; Nakae, et al., Surg. Today 26:225-29, 1996; Bengtson, A., et al., Arch. Surg. 123:645-649 , 1988). The mechanisms by which C5a exerts its detrimental effects in sepsis require more detailed investigation, but recent evidence suggests that C5a generation in sepsis significantly impairs innate immune functions of blood neutrophils (Huber-Lang, M.S., et al., J. Immunol. 169:3223-31, 2002), their ability to increase respiratory activity and generate cytokines (Riedemann, N.C., et al., Immunity 19:193-202, 2003). In addition, C5a generation in sepsis appears to have a procoagulant effect (Laudes, LJ, et al., Am. J. Pathol. 160:1867-75, 2002). The complement-modulating protein Cl INH has also been shown to be effective in experimental animal models of sepsis and ARDS (Dickneite, G, Behring Ins. Mitt. 93:299-305, 1993).

The pectin pathway may also play a role in the pathogenesis of sepsis. It has been shown that MBL can bind to a range of clinically important microorganisms, including both gram-negative and gram-positive bacteria, and activate the lectin pathway (Neth, O., et al., Infect. Immun (68:688, 2000). Lipoteichoic acid (LTA) has recently been increasingly considered as a Gram-positive analog of LPS and is a potential immunostimulant that induces cytokine release from mononuclear phagocytes and whole blood (Morath, S. et al.,

- 42 042534

J. Exp. Med. 195:1635, 2002; Morath, S. et al., Infect. Immun. 70:938, 2002). Lficolin has recently been shown to specifically bind to LTA isolated from numerous Gram-positive bacterial species, including Staphylococcus aureus, and activate the lectin pathway (Lynch, N.J., et al., J. Immunol. 172:1198-02, 2004). , MBL has been shown to bind to LTA of fecal streptococcus, in which the polyglycerophosphate chain is substituted with glycosyl groups, but not to LTA of nine other species, including Staphylococcus aureus (Polotsky, V.Y, et al. Infect. Immun. 64:380, 1996).

Thus, one aspect of the present invention provides a method of treating sepsis or a condition resulting from sepsis by administering a composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent in a pharmaceutically acceptable carrier to a subject suffering from sepsis or a condition resulting from sepsis, including, without limitation, severe sepsis, septic shock, sepsis-associated acute respiratory distress syndrome, and systemic inflammatory response syndrome. Related methods are provided for the treatment of other blood disorders, including hemorrhagic shock, hemolytic anemia, autoimmune thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), or other destructive bone marrow/blood diseases, by administering a composition comprising a therapeutically effective amount of MASP-2 an inhibitory agent in a pharmaceutically acceptable carrier, to a subject suffering from such a condition. The MASP-2 inhibitory agent is administered to the subject systemically, for example, intra-arterially, intravenously, intramuscularly, by inhalation (particularly in the case of ARDS), subcutaneously, or by another parenteral route, or possibly by oral administration in the case of non-peptidergic drugs. The MASP-2 inhibitory agent composition may be combined with one or more additional therapeutic agents to combat the complications of sepsis and/or shock. In the case of an advanced stage of sepsis or shock, or a disorder resulting from these diseases, the MASP-2 inhibitory composition may be appropriately administered as a rapid-acting dosage form, for example, by intravenous or intra-arterial delivery of a bolus of a solution containing the MASP-2 inhibitory composition. facilities. The introduction can be repeated at the discretion of the attending physician until the disease state is eliminated.

Coagulopathy.

Evidence has been provided for the role of the complement system in disseminated intravascular coagulation (DIC), such as DIC, in the setting of severe injury.

Previous studies have shown that C4-/- mice are not protected from renal reperfusion injury (Zhou, W., et al., Predominant role for C5b-9 in renal ischemia/reperfusion injury, J Clin Invest 105:1363-1371 (2000) ). In order to study the ability of C4 −/− mice to activate complement via the classical or lectin pathway, C3 turnover in the plasma of C4 −/− mice was measured by assay methods specific to the direction of activation via the classical or lectin pathway. Although no C3 cleavage was observed upon initiation of classical pathway activation, highly effective lectin pathway dependent C3 activation was observed in C4-deficient serum (FIG. 30). Clearly, in MASP-2 −/− mice, C3b deposition on mannan and zymosan is seriously impaired even under experimental conditions, which, according to many previously published data on the activation of the alternative pathway, should be observed for all three pathways. When using the same sera in wells coated with immunoglobulin complexes instead of mannan or zymosan, C3b deposition and factor B cleavage are observed in the sera of MASP-2+/+ mice and in the sera of MASP-2 -/- mice, but not in sera depleted in C1q . This indicates that activation of the alternative pathway is facilitated in MASP-2-/-serum when the original C3b is provided through classical activity. In FIG. 3° C. graphically illustrates unexpected findings that C3 can be efficiently activated in a lectin pathway dependent manner in C4 deficient plasma.

This bypass C4 activation is abolished by inhibition of lectin pathway activation by preincubation of the plasma with soluble mannan or mannose.

Aberrant non-immune activation of the complement system poses a potential hazard to humans and may also play an important role in the activation of the hematologic pathway, especially in cases of severe injury in which the inflammatory and hematologic pathways are activated. In healthy individuals, C3 conversion is <5% of total plasma C3 protein. In a highly advanced infection involving blood poisoning and immune complex disease, C3 conversion is restored by itself to about 30% with complement levels usually lower than normal due to increased utilization and changes in pool distribution. Immediate activation of the C3 pathway greater than 30% usually provides unequivocal clinical evidence of vasodilation and tissue fluid loss. Above 30% C3 conversion, the initiating mechanisms are predominantly non-immune, and the resulting clinical manifestations of diseases are dangerous for the patient. Complement levels C5 in healthy individuals and in controlled disease are more stable than C3. Significant reductions and or conversions in C5 levels are associated with the patient's response to atypical polytrauma (eg, traffic accidents) and

- 43 042534 probable development of shock lung syndromes. Thus, any evidence of C3 complement activation above 30% of the vascular pool, or any involvement of C5, or both, can be considered as a likely precursor to a dangerous pathological change in the patient.

C3 and C5 release anaphylatoxins (C3a and C5a) that act on mast cells and basophils to release vasodilatory chemicals. They establish chemotoxic gradients to guide polymorphonuclear cells (PMN) to the center of immunological disorders (treatment response), but they differ here as C5a has a specific clumping (aggregation) effect on these phagocytes, preventing them from being inadvertently removed from the site of the reaction. In normal infection control, C3 activates C5. However, in polytrauma, C5 appears to be largely activated, systemically generating C5a anaphylatoxins. This uncontrolled activity causes the polymorphs to form clumps in the vascular system, these clumps are then carried away into the capillaries of the lungs, where they clog and cause local damaging effects as a result of the release of superoxide. Without resorting to any theory as evidence, however, it can be assumed that this mechanism is important in the pathogenesis of acute respiratory failure syndrome (ARDS), although this point of view has recently been questioned. It can be demonstrated that C3a anaphylatoxins are potent platelet aggregators in vitro, but their involvement in vivo is less clear, and release of platelet and plasmin during wound healing may only re-engage complement C3. It is possible that a long rise in C3 activation is needed to generate DIC.

In addition to the cellular and vascular effects of the activated complement component described above, which may explain the association between trauma and DIC, new scientific discoveries have revealed direct molecular links and a dramatic functional response between complement and coagulation systems. Supporting data came from studies of C3-deficient mice. Since C3 is a common component for each of the complement pathways, one would expect C3 deficient mice to lack complete complement function. Surprisingly, however, C3-deficient mice are able to fully activate terminal complement components (Huber-Lang, M., et al., Generation of C5a in the absence of C3: a new complement activation pathway, Nat. Meet 12:682-687 (2006)). In-depth studies have shown that C3-independent activation of terminal complement components is mediated by thrombin, an enzyme that limits the rate of the coagulation cascade (Huber et al., 2006). The molecular components mediating thrombin activation after initial complement activation remain unclear.

The present inventors have explained what is to be considered the molecular basis for the abrupt reaction between complement and coagulation cascades and identified MASP-2 as the central checkpoint linking the two systems. Biochemical studies of the substrate specificity of MASP-2 have identified prothrombin as a possible substrate in addition to the widely known complement proteins C2 and C4. MASP-2 specifically cleaves prothrombin at functionally relevant sites, generating thrombin, the rate-limiting enzyme of the coagulation cascade (Krarup, A., et al., Simultaneous Activation of Complement and Coagulation by MBL-Associated Serine Protease 2, PLoS. ONE. 2: e623 (2007)). Thrombin generated by MASP-2 is able to promote fibrin deposition in a specific repair system in vitro, demonstrating the functional relevance of MASP-2 cleavage (Krarup et al., 2007). As discussed in the examples below in this invention, the authors of the present invention further confirmed the physiological significance of this discovery by documenting the activation of thrombin in the serum of healthy rodents after activation of the lectin pathway, and also demonstrated that this process is blocked by monoclonal antibodies that neutralize MASP- 2.

MASP-2 may represent a central branching point in the lectin pathway capable of promoting activation of both the complement and coagulation systems. Since activation of the lectin pathway is a physiological response to many types of traumatic injury, the present inventors believe that concomitant systemic inflammation (mediated by complement components) and disseminated coagulation (mediated through the coagulation pathway) can be explained by the ability of MASP-2 to activate both pathways. These findings clearly point to a role for MASP-2 in causing DIC and to a beneficial therapeutic effect of MASP2 inhibition in treating or preventing DIC. MASP-2 can provide a molecular link between complement and the coagulation system, and activation of the lectin pathway that occurs in various injuries can directly initiate activation of the coagulation system through the MASP-2-thrombin axis, providing a mechanism for linking injury to DIC. In accordance with an aspect of the present invention, inhibition of MASP-2 would inhibit the activation of the lectin pathway and reduce the generation of both C3a and C5a anaphylatoxins. It is believed that a long rise in C3 activation is required to generate DIC.

Microcirculatory coagulation (clots in capillaries and small blood vessels) occurs in conditions of septic shock.

- 44 042534

The role of the lectin pathway in septic shock has been established and validated in experimental mouse models of sepsis with the protected MASP-2 (-/-) phenotype described in Example 17 and FIG. 18 and 19. Moreover, as shown in Example 15 and FIG. 16A and 16B, MASP-2 (-/-) mice are protected in an experimental localized Schwartzmann reaction model of disseminated intravascular coagulation (DIC), in a microvessel localized coagulation model.

V. MASP-2.

inhibitory agents.

In one aspect, the present invention provides methods for inhibiting MASP-2 dependent complement activation in a subject suffering from or at risk of developing thrombotic microangiopathy. MASP-2 inhibitory agents are administered in an amount effective to inhibit MASP-2 dependent complement activation in living organisms. In the practice of this aspect of the invention, exemplary MASP-2 inhibitory agents include molecules that inhibit the biological activity of MASP-2 (such as small molecule inhibitors, anti-MASP-2 antibodies, or blocking peptides that interact with MASP-2 or interfere with protein-to-protein interaction), and molecules that reduce MASP-2 expression (such as MASP-2 antisense nucleic acid molecules, MASP-2 specific RNAi molecules, and MASP-2 ribozymes) by which MASP-2 prevents activation of the complement lectin pathway. MASP-2 inhibitory agents can be used as primary monotherapy or in combination with other therapeutic agents as an adjuvant therapy to enhance the beneficial therapeutic effects of other therapeutic treatments.

Inhibition of MASP-2 dependent complement activation is characterized by at least one of the following changes in a component of the complement system that results from the administration of a MASP-2 inhibitory agent in accordance with the methods of the invention: inhibition of generation or production of C4b, C3a, C5a and/or C5b products -9 (MAC) MASP-2 dependent activation of the complement system (measured, for example, as described in example 2), a decrease in complement activation, determined in a hemolytic study using non-sensitized rabbit or guinea pig erythrocytes (measured, for example, as described in example 33) , a decrease in C4 splitting and C4b deposition (measured, for example, as described in example 2) or a decrease in C3 splitting and C3b deposition (measured, for example, as described in example 2).

In accordance with the present invention, those MASP-2 inhibitory agents are used which effectively inhibit MASP-2 dependent activation of the complement system. MASP-2 inhibitory agents useful in the practice of this aspect of the invention include anti-MASP-2 antibodies and fragments thereof, MASP-2 inhibitory peptides, small molecules, MASP-2 soluble receptors, and expression inhibitors. MASP-2 inhibitory agents can inhibit MASP-2 dependent activation of the complement system by blocking the biological function of MASP-2. For example, an inhibitory agent can effectively block MASP-2 protein-protein interactions, interfere with MASP-2 dimerization or assembly, block Ca ²⁺ binding, affect the MASP-2 serine protease active site, or reduce MASP-2 protein expression.

In some embodiments, MASP-2 inhibitory agents selectively inhibit MASP-2 complement activation without affecting C1q dependent complement system activation.

In one embodiment, the MASP-2 inhibitory agent used in the methods of the invention is a specific MASP-2 inhibitory agent that specifically binds to a polypeptide comprising the sequence of SEQ ID NO: 6 with an affinity at least ten times higher than with other antigens in the complement system. In another embodiment, the MASP-2 inhibitory agent specifically binds to a polypeptide comprising the sequence of SEQ ID NO: 6 with a binding affinity at least 100 times greater than other antigens in the complement system. The binding affinity of the MASP-2 inhibitory agent can be determined using an appropriate binding assay method.

The MASP-2 polypeptide is characterized by a molecular structure similar to that of MASP 1, MASP 3, and the C1r and C1s proteases of the C1 complement system. The cDNA molecule described in the sequence of SEQ ID NO: 4 encodes a typical example of MASP-2 (consisting of the amino acid sequence described in the sequence of SEQ ID NO: 5) and produces a human MASP-2 polypeptide with a leader sequence (aa 1-15), which is cleaved after secretion, resulting in the mature form of human MASP-2 (SEQ ID NO: 6). As shown in FIG. 2, the human MASP-2 gene spans twelve exons. Human MASP-2 cDNA is encoded by exons B, C, D, F, G, H, I, J, K, and L. Alternative splicing results in a 20 kDa protein called MBL-associated protein 19 (MAp19, also called sMAP) (SEQ ID NO: 2) encoded by SEQ ID NO: 1 arising from exons B, C, D, and E as shown in FIG. 2. The cDNA molecule described in SEQ ID NO: 50 encodes mouse MASP-2 (consisting of the amino acid sequence described in SEQ ID NO: 51) and produces a mouse MASP-2 polypeptide with a leader sequence that is cleaved after secretion, resulting in as a result to

- 45 042534 mature mouse MASP-2 (SEQ ID NO: 52). cDNA molecule described in sequence

SEQ ID NO: 53 encodes a rat MASP-2 consisting of the amino acid sequence described in SEQ ID NO: 54) and produces a rat MASP-2 polypeptide with a leader sequence that is cleaved after secretion, resulting in the mature form of rat MASP -2 (SEQ ID NO:

55).

Those skilled in the art will appreciate that the sequences described in SEQ ID NO: 4, SEQ ID NO: 50, and SEQ ID NO: 53 represent single alleles of human, mouse, and rat MASP-2, respectively, and that allelic variation and alternative splicing. Allelic variations of the nucleotide sequences described in SEQ ID NO: 4, SEQ ID NO: 50, and SEQ ID NO: 53, including those containing silent mutations and those in which mutations result in amino acid sequence changes, are included within the scope of the present invention. Allelic variations of the MASP-2 sequence can be cloned with probe cDNA or gene banks from different individuals according to standard techniques.

The protein domains of human MASP-2 (SEQ ID NO: 6) are shown in FIG. 1 and 2A and include the N terminal domain of C1r/C1s/vascular endothelial growth factor (Vegf) sea urchin/bone morphogenic protein (CUBI) (aa 1-121 of SEQ ID NO: 6), epidermal growth factor like domain (aa 122 -166), the second CUBI domain (aa 167-293), as well as the tandem domains of complement control proteins and the serine protease domain. Alternative splicing of the MASP-2 gene results in MAP19 shown in FIG. 1. MAP19 is a non-enzymatic protein containing the N-terminal CUBI EGF region of MASP-2 with four additional residues (EQSL) derived from the E exon shown in FIG. 1.

Various proteins have been shown to bind or interact with MASP-2 through protein-protein interactions. For example, MASP-2 is known to bind and form Ca ²⁺ dependent complexes with the lectin proteins MBL, H ficolin and L ficolin. Each MASP-2/lectin complex has been shown to activate complement via MASP-2 dependent cleavage of C4 and C2 proteins (Ikeda, K., et al., J. Biol. Chem. 262:7451 7454, 1987; Matsushita, M. , et al., J. Exp. Med. 176:1497 2284, 2000; Matsushita, M., et al., J. Immunol. 168:3502 3506, 2002). Studies have shown that the CUB1-EGF domains of MASP-2 are of great importance in the association of MASP-2 with MBL (Thielens, NM, et al., J. Immunol. 166:5068, 2001). The CUB1EGFCUBII domains have also been shown to mediate MASP-2 dimerization, which is required to form an active MBL complex (Wallis, R., et al., J. Biol. Chem. 275:30962 30969, 2000). Therefore, MASP-2 inhibitory agents can be identified that bind or interact with MASP-2 target regions known to be important for MASP-2 dependent complement activation.

Antibodies against MASP-2.

In some embodiments of this aspect of the invention, the MASP-2 inhibitory agent comprises an anti-MASP-2 antibody that inhibits MASP-2 dependent activation of the complement system. Anti-MASP-2 antibodies useful in this aspect of the invention include polyclonal, monoclonal or recombinant antibodies derived from any antibody produced by a mammal and may be polyspecific, chimeric, humanized, anti-idiotypic antibodies and antibody fragments. Antibody fragments include Fab, Fab', F(ab) ₂ , F(ab') ₂ , Fv fragments, scFv fragments, and single chain antibodies described hereinafter.

The literature describes various antibodies against MASP-2, some of which are listed below in table. 1. These previously described anti-MASP-2 antibodies can be screened for their ability to inhibit MASP-2 dependent activation of the complement system using the assay methods described herein. For example, Fab2 rat anti-MASP-2 antibodies have been identified that block MASP-2 dependent complement activation, as described in more detail in the invention in Examples 10 and 11. Once an anti-MASP-2 antibody that acts as MASP-2 has been identified, 2 inhibitory agent, it can be used to produce anti-idiotypic antibodies and used to identify other MASP-2 binding molecules described below.

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Table 1

MASP-2 specific antibodies described in the scientific literature

ANTIGEN TYPE OF ANTIBODY LINK Recombinant MASP-2 Rat polyclonal Peterson, S.V., et al., Mol. Immunol. 37:803 811, 2000 Recombinant human CCP1/2 SP fragment (MoAb 8B5) Rat MoAb (IgGl subclass) Moller-Kristensen, M., et al., J. of Immunol. Methods 282:159 167, 2003 Recombinant human MAP19 (MoAb 6G12) (cross-reactions with MASP-2) Rat MoAb (IgGl subclass) Moller-Kristensen, M., et al., J. of Immunol. Methods 282:159 167, 2003 hMASP-2 Mouse MoAb (S/P) Mouse MoAb (N-terminal) Peterson, S.V., et al., Mol. Immunol. 35:409 April 1998 hMASP-2 (CCP1- CCP2-SP domain) Rat MoAb: Nimoab 101 produced by hybridoma cell line 03050904 (ECACC) WO2004/106384 hMASP-2 (full exchange labeled with histidine) Mouse MoAbs: NimoAb 104 produced by hybridoma cell line M0545YM035 (DSMZ) NimoAb 108 produced by M0545YM029 hybridoma cell line (DSMZ) NimoAb 109 produced by M0545YM046 hybridoma cell line (DSMZ) NimoAb 110 produced by hybridoma cell line M0545YM048 (DSMZ) WO2004/106384

Anti-MASP-2 antibodies with reduced effector function.

In some embodiments of this aspect of the invention, the anti-MASP-2 antibodies have reduced effector function in order to reduce inflammation that may result from activation of the classical complement pathway. It has been shown that the ability of IgG molecules to initiate the classical complement pathway is inherent in the internal Fc portion of the molecule (Duncan, A.R., et al., Nature 332:738 740 1988). IgG molecules in which the Fc portion of the molecule has been removed by enzymatic cleavage lack this effector function (see Harlow, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988). Accordingly, antibodies with reduced effector function can be formed as a result of a deficiency of the Fc portion of the molecule due to the presence of a genetically engineered Fc sequence that minimizes effector function or is an isotype of human IgG2 or IgG4.

Antibodies with reduced effector function can be produced by standard molecular biological manipulation of the Fc portion of IgG heavy chains as described in the invention in Example 9 and also described in Jolliffe et al., Int'l Rev. Immunol. 10:241 250, 1993, and Rodrigues et al., J. Immunol. 151:6954 6961, 1998. Antibodies with reduced effector function also include human IgG2 and IgG4 isotypes that have reduced ability to activate complement and/or interact with Fc receptors (Ravetch, J.V., et al., Annu. Rev. Immunol. 9 :457 492, 1991; Isaacs, J. D., et al., J. Immunol. 148:3062 3071, 1992; van de Winkel, J. G., et al., Immunol. Today 14:215 221, 1993). Humanized or fully human antibodies specific for human MASP2, consisting of the IgG2 or IgG4 isotypes, can be produced by one of several methods known to any one of ordinary skill in the art, as described in Vaughan, T.J., et al., Nature Biotechnical 16: 535 539, 1998.

Production of antibodies against MASP-2.

Anti-MASP-2 antibodies can be produced by using MASP-2 polypeptides (eg, full-length MASP-2) or by using antigenic peptides bearing a MASP-2 epitope (eg, a portion of a MASP-2 polypeptide). Immunogenic peptides can be up to five amino acid residues in size. For example, a MASP-2 polypeptide comprising the complete amino acid sequence of SEQ ID NO: 6 can be used to induce an antibody against

- 47 042534

MASP-2 used in the method of the invention. Specific MASP-2 domains known to be involved in protein-protein interactions, such as the CUBI and CUBIEGF domains, as well as the serine protease active site spanning region, can be expressed as recombinant polypeptides, as described in example 3, and used as antigens. In addition, peptides comprising a portion of at least 6 amino acids of a MASP-2 polypeptide (SEQ ID NO: 6) are also used to induce MASP-2 antibodies. Additional examples of MASP-2 derived antigens used to induce MASP-2 antibodies are shown in Table 1 below. 2. MASP-2 peptides and polypeptides used to induce antibodies can be isolated in the form of natural polypeptides or recombinant or synthetic peptides and catalytically inactive recombinant polypeptides such as MASP-2A, described further in examples 5-7. In some embodiments of this aspect of the invention, anti-MASP-2 antibodies are generated using a transgenic mouse strain as described in Examples 8 and 9 and further described below.

Antigens used to produce anti-MASP-2 antibodies also include recombinant polypeptides such as recombinants of MASP-2 or portions thereof with an immunoglobulin polypeptide or maltose binding protein. The polypeptide immunogen may be a full length molecule or a portion thereof. If the polypeptide portion is hapten-like, then such a portion can be successfully combined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), or tetanus toxin) for immunization.

table 2

MASP-2 derived antigens

SEQID NO: Amino acid sequence SEQ ID NO:6 Human MASP-2 protein SEQ ID NO:51 Mouse MASP-2 protein SEQ ID NO:8 CUBI domain of human MASP-2 (aa 1-121 of SEQ ID NO:6) SEQ ID NO:9 CUBIEGF human domains MASP-2 (aa 1-166 of SEQ ID NO:6) SEQ ID NO:10 CUBIEGFCUBII domains of human MASP-2 (aa 1-293 of SEQ ID NO:6) SEQ ID NO:11 EGF domains of human MASP-2 (aa 122-166 of SEQ ID NO:6) SEQ ID NO:12 Human MASP-2 serine protease domain (aa 429-671 of SEQ ID NO:6) SEQ ID NO:13 GKDSCRGDAGGALVFL serine protease inactivated mutant form (aa 610-625 of SEQ ID NO:6 with mutated Ser 618) SEQ ID NO:14 TPLGPKWPEPVFGRL human CUBI peptide SEQ ID NO:15: TAPPGYRLRLYFTHFDLELSHLCEYDFVKLSS GAKVLATLCGQ human CUBI peptide SEQ ID NO:16 TFRSDYSN MBL binding region in the human CUBI domain SEQ ID NO:17 FYSLGSSLDITFRSDYSNEKPFTGF MBL binding region in the human CUBI domain SEQ ID NO:18 IDECQVAPG EGF peptide SEQ ID NO:19 ANMLCAGLESGGKDSCRGDSGGALV peptide from the active site of serine protease

polyclonal antibodies.

Anti-MASP-2 polyclonal antibodies can be prepared by immunizing an animal with a MASP-2 polypeptide, or an immunogenic portion thereof, using methods that are well known to those of ordinary skill in the art. See, for example, Green et al., Production of Polyclonal Antisera, in Immunochemical Protocols (Manson, ed.), page 105, and the methods described in Example b below. The immunogenicity of a MASP-2 polypeptide can be enhanced by the use of an adjuvant including mineral gels such as aluminum hydroxide or Freund's adjuvant (complete or incomplete), surfactants such as lysolecithin, polyethylene polypropylene oxide polyols, polyanions, oil emulsions, keyhole hemocyanin, and dinitrophenol. Polyclonal antibodies are commonly produced in animals such as horses, cows, dogs, chickens, rats, mice,

- 48 042534 rabbits, guinea pigs, goats or sheep. Alternatively, the anti-MASP-2 antibody used in the present invention can also be obtained from great apes. General methods for the production of diagnostic and therapeutic antibodies in baboons can be found, for example, in Goldenberg et al., International Patent Publication No. WO 91/11465 and Losman, M.J., et al., Int. J. Cancer 46:310, 1990. Sera containing immunologically active antibodies are then produced from the blood of such immunized animals using well known standard methods.

monoclonal antibodies.

In some embodiments, the MASP-2 inhibitory agent is an anti-MASP-2 monoclonal antibody. Monoclonal antibodies against MASP-2 are highly specific, directed against a single MASP-2 epitope. Used in the invention, the definition of monoclonal indicates that the characteristic feature of the antibody is its production from a substantially homogeneous population of antibodies, but this definition does not imply the need to produce antibodies by any particular method. Monoclonal antibodies can be made by any method that produces stable cell lines of antibody molecules upon culture, such as the hybridoma method described in Kohler, G., et al., Nature 256:495, 1975, or they can be made by recombinant techniques. DNA (see, for example, U.S. Patent No. 4816567 to Cabilly). Monoclonal antibodies can also be isolated from phage antibody libraries using the methods described in Clackson, T., et al., Nature 352:624 628, 1991, and Marks, J.D., et al., J. Mol. Biol. 222:581-597, 1991. Such antibodies may be of any immunoglobulin class, including IgG, IgM, IgE, IgA, IgD, and any subclass thereof.

For example, monoclonal antibodies can be obtained by injecting a suitable mammal (eg, BALB/c mice) with a composition comprising a MASP-2 polypeptide or a portion thereof. After a predetermined period of time, splenocytes are removed from mice and suspended in cell culture medium. The splenocytes then hybridize with an immortalized cell line to form a hybridoma. The resulting hybridomas are grown in cell culture and screened for their ability to produce anti-MASP-2 monoclonal antibody. An example detailing the production of anti-MASP-2 monoclonal antibodies is provided in Example 7. See also Current Protocols in Immunology, Vol. 1., John Wiley & Sons, pages 2.5.1 2.6.7, 1991.).

Human monoclonal antibodies can be obtained by using transgenic mice that have been designed to produce specific human antibodies in response to an antigenic stimulus. In this method, human immunoglobulin heavy and light chain locus elements are introduced into mouse strains derived from embryonic stem cell lines that contain targeted damaged endogenous immunoglobulin heavy and light chain loci. Transgenic mice can synthesize human antibodies specific for human antigens, such as the MASP-2 antigens described herein, and mice can be used to produce human MASP-2 antibodies by isolating hybridomas by hybridizing B cells from such animals with appropriate strains. myeloma cells using the traditional Kohler-Milstein hybridoma technology described in Example 7 below. , Annandale, N.J.). Methods for obtaining human antibodies from transgenic mice are described, for example, in Green, L.L., et al., Nature Genet. 7:13, 1994; Lonberg, N., et al., Nature 368:856, 1994; and Taylor, L.D., et al., Int. Immun. 6:579, 1994.

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

After production, polyclonal, monoclonal, or phage-derived antibodies are first tested for binding specificity to MASP-2. A number of assays, which are well known to those skilled in the art, can be used to detect antibodies that specifically bind to MASP-2. Examples of assays include Western blot or immunoprecipitation assays by standard methods (e.g., described in Ausubel et al.), immunoelectrophoresis, enzyme-labeled immunosorbent assays, dot blot, inhibition or competition assays, and sandwich assays (described in Harlow and Land, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988). Once antibodies have been identified that specifically bind to MASP-2, anti-MASP-2 antibodies are tested for their ability to act as a MASP-2 inhibitory agent in one of several assays, such as, for example, the C4 lectin specific cleavage assay (described in Example 2 ), a C3b deposition study (described in Example 2), or a C4b deposition study (described in Example 2).

The affinity of monoclonal antibodies against MASP-2 can be easily determined by conventional

- 49 042534 a socialist in this field (see, for example, Scatchard, A., NY Acad. Sci. 51:660 672, 1949). In one embodiment, the anti-MASP-2 monoclonal antibodies used in the methods of the invention bind to MASP-2 with a binding affinity value of <100 nM, preferably < 10 nM, and most preferably < 2 nM. In some embodiments, the MASP-2 inhibitory monoclonal antibody used in the methods of the invention is a MASP-2 inhibitory monoclonal antibody, or an antigen-binding fragment thereof, comprising (I) (a) a heavy chain variable region comprising: i) a CDR- heavy chain H1, including the amino acid sequence from 31-35 of the sequence of SEQ ID NO: 67; and ii) a CDR-H2 heavy chain comprising the amino acid sequence from 50-65 of SEQ ID NO: 67; and iii) a CDR-H3 heavy chain comprising the amino acid sequence from 95-102 of SEQ ID NO: 67 and b) a light chain variable region comprising: i) a CDR-L1 light chain comprising the amino acid sequence from 24-34 of SEQ ID NO: 70; and ii) a CDR-L2 light chain comprising the amino acid sequence from 50-56 of SEQ ID NO: 70; and iii) a CDR-L3 light chain comprising an amino acid sequence from 89-97 of SEQ ID NO: 70, or (ii) a variant thereof comprising a heavy chain variable region with at least 90% identity to SEQ ID NO: 67 ( e.g. at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , at least 98%, at least 99% identity with SEQ ID NO: 67) and a light chain variable region with at least 90% identity (for example, at least 91%, at least 92% , at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical with SEQ ID NO: 70.

Chimeric/humanized antibodies.

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

One form of chimeric antibody useful in the invention is a humanized anti-MASP-2 monoclonal antibody. Humanized forms of non-human (eg, murine) antibodies are chimeric antibodies that contain a minimal sequence derived from a non-human immunoglobulin. Humanized monoclonal antibodies are produced by transferring non-human (eg, murine) complementarity-determining regions (CDRs) from murine immunoglobulin heavy and light variable chains into the human variable domain. Typically, human antibody residues are then replaced in the framework regions of the non-human counterparts. In addition, humanized antibodies may include residues that are not found in the recipient antibody or in the donor antibody. These modifications are obtained for the subsequent purification of antibodies. Typically, a humanized antibody may comprise substantially all of at least one, and typically two, variable domains wherein all or substantially all of the hypervariable loops correspond to hypervariable loops of a non-human immunoglobulin and all or substantially all of the Fv frameworks are Fv frameworks. human immunoglobulin sequences. The humanized antibody may optionally also include at least a portion of an immunoglobulin constant region (Fc), which is typically part of a human immunoglobulin. More information can be found in 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.

Humanized antibodies useful in the invention include human monoclonal antibodies containing at least a MASP-2 CDR3 binding site. In addition, Fc portions can be substituted to produce IgA or IgM as well as human IgG antibodies. Such humanized antibodies may have particular clinical utility as they can specifically recognize human MASP-2 but not elicit an immune response in humans against the antibody itself. Therefore, they are better suited for in vivo administration to humans, especially when repeated or prolonged administration is required.

An example of generating a humanized anti-MASP-2 antibody from a mouse anti-MASP-2 monoclonal antibody is given in Example 6. Methods for producing humanized monoclonal antibodies are also described, for example, in Jones, P.T., et al., Nature 321:522, 1986 ; Carter, P., et al., Proc. Nat'l. Acad. sci. USA 89:4285, 1992; Sandhu, J.S., Crit. Rev. Biotech. 12:437, 1992; Singer, 1.1., et al., J. Immun. 150:2844, 1993; Sudhir (ed.), Antibody Engineering Protocols, Humana Press, Inc., 1995; Kelley, Engineering Therapeutic Antibodies, in Protein Engineering: Principles and Practice, Cleland et al. (eds.), John Wiley & Sons, Inc., pages 399-434, 1996; and in the U.S. patent document. Patent no.

- 50 042534

5693762, to Queen, 1997. In addition, there are commercial organizations that can synthesize humanized antibodies from specific regions of mouse antibodies, such as Protein Design

Labs (Mountain View, CA).

recombinant antibodies.

Anti-MASP-2 antibodies can also be obtained using recombinant methods. For example, human antibodies can be made by using a library of human immunoglobulin expressed sequences (available from, for example, Stratagene, Corp., La Jolla, CA) to produce human antibody fragments (VH, VL, Fv, Fd, Fab, or F(ab' ) ₂ ). These fragments are then used to construct the entire human antibody using methods similar to those used to produce chimeric antibodies.

Anti-idiotypic antibodies.

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

Fragments of immunoglobulin.

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

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

Antibody fragments can be prepared by conventional proteolytic hydrolysis methods, such as hydrolysis of total antibodies with pepsin or papain. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to produce a 5S fragment, referred to as F(ab') ₂ . This fragment can be further cleaved using a thiol reducing agent to produce 3.5S Fab' monovalent fragments.

Optionally, the cleavage reaction can be carried out using a group that blocks sulfhydryl groups that result from cleavage of disulfide bonds. Alternatively, enzymatic digestion using pepsin directly produces two monovalent Fab fragments and an Fc fragment directly. These methods are described, for example, in U.S. Patent No. 4331647 to Goldenberg; in Nisonoff, A., et al., Arch. Biochem. Biophys. 89:230, 1960; Porter, R.R., Biochem. J. 73:119, 1959; Edelman, et al., in Methods in Enzymology 1:422, Academic Press, 1967; and by Coligan at pages 2.8.1 2.8.10 and 2.10. 2.10.4.

In some embodiments, it is preferable to use antibody fragments lacking an Fc region in order to prevent activation of the classical complement pathway that is initiated after Fc binds to the Fcy receptor. Several methods of MoAb production are known that prevent Fcy receptor interactions. For example, the Fc region of a monoclonal antibody can be chemically removed using incomplete proteolytic enzyme cleavage (such as ficin enzymatic cleavage), thereby generating, for example, antigen-binding fragments of the antibody, such as Fab or F(ab) ₂ fragments. (Mariani, M., et al., Mol. Immunol. 28:69 71, 1991). Alternatively, a human y4 IgG isotype that does not bind Fcy receptors can be used in the construction of the humanized antibody described herein. Antibodies, single chain antibodies and antigen binding domains that do not have an Fc domain can also be constructed using the recombinant techniques described herein.

Single chain antibody fragments.

Alternatively, you can create a single-chain peptide that binds molecules specific for MASP-2, in which the Fv regions of the heavy and light chain are connected. Fv fragments can

- 51 042534 be connected using a peptide linker to form a single-stranded antigen-binding protein (scFv). These single-stranded antigen-binding proteins are produced by constructing a structural gene that includes DNA sequences encoding VH and VL domains that are joined by an oligonucleotide. The structural gene is inserted into an expression vector, which is then introduced into a host cell such as E. coli. Recombinant host cells synthesize a single chain polypeptide with a peptide linker bridging the two V domains. Methods for the production of scFvs 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; in a U.S. patent document. Patent No. 4946778, to Ladner; in Pack, P., et al., Bio/Technology 11:1271, 1993.

As an illustrative example, a MASP-2 specific scFv can be generated by exposing lymphocytes to a MASP-2 polypeptide in vitro and selecting to display the antibody library in phage or similar vectors (e.g., by using an immobilized or labeled MASP-2 protein or peptide). Genes encoding polypeptides having potential MASP-2 polypeptide binding domains can be obtained by screening randomized peptide libraries for phage displays or bacterial displays, such as E. coli. These randomized display peptide libraries can be used to screen for peptides that interact with MASP-2. Methods for generating and screening such randomized display peptide libraries are well known (U.S. Patent No. 5223409 to Lardner; U.S. Patent No. 4946778 to Ladner; U.S. Patent No. 5403484 to Lardner; U.S. Patent No. 5571698 to Lardner; and Kay et al., Phage Display of Peptides and Proteins, Academic Press, Inc., 1996), and randomized peptide display libraries and screening kits for such libraries can be purchased from appropriate companies such as CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc. (Beverly, Mass.) and Pharmacia LKB Biotechnology Inc. (Piscataway, N.J.).

Another form of anti-MASP-2 antibody fragment useful in this aspect of the invention is a peptide encoding a single hypervariable region (CDR) that binds to an epitope on the MASP-2 antigen and inhibits MASP-2 dependent complement activation. CDR peptides (minimum recognition components) can be obtained by constructing genes encoding the CDRs of the corresponding antibody. Such genes are obtained, for example, by using the polymerase chain reaction to synthesize the variable region from the RNA of the cell-producing antibody (see, for example, Larrick et al., Methods. A Companion to Methods in Enzymology 2:106, 1991; Courtenay Luck, Genetic Manipulation of Monoclonal Antibodies, in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al (eds.), page 166, Cambridge University Press, 1995; and Ward et al., Genetic Manipulation and Expression of Antibodies, in Monoclonal Antibodies: Principles and Applications, Birch et al (eds.), page 137, Wiley Liss, Inc., 1995).

The MASP-2 antibodies described herein are administered to a subject in need thereof to inhibit MASP-2 dependent complement activation. In some embodiments, the MASP-2 inhibitory agent is a high affinity human or humanized anti-MASP-2 monoclonal antibody with reduced effector function.

peptide inhibitors.

In some embodiments of this aspect of the invention, the MASP-2 inhibitory agent includes isolated peptide inhibitors of MASP-2, including isolated natural peptide inhibitors and synthetic peptide inhibitors that inhibit MASP-2 dependent activation of the complement system. As used herein, isolated MASP-2 peptide inhibitors refers to peptides that inhibit MASP-2 dependent complement activation by binding to MASP-2 by competing with MASP-2 for binding to another recognition molecule (e.g., MBL, H-ficolin, M-ficolin or L-ficolin), via the lectin pathway, and/or by interacting directly with MASP-2 to inhibit MASP-2 dependent complement activation that are substantially pure and contain virtually no other substances with which they can be found in nature , to the extent that ensures in practice their intended use and is consistent with their intended use.

Peptide inhibitors have been successfully used in vivo to prevent protein-protein interactions and negative effects on catalytic sites. For example, peptides that inhibit adhesion molecules structurally related to LFA 1 have recently been approved for clinical use in the treatment of coagulopathy (Ohman, E.M., et al., European Heart J. 16:50 55, 1995). Short linear peptides (<30 amino acids) have been described to prevent or adversely affect integrin dependent adhesion (Murayama, O., et al., J. Biochem. 120:445 51, 1996). Longer peptides, ranging in length from 25 to 200 amino acid residues, have been successfully used to block integrin dependent adhesion (Zhang, L., et al., J. Biol. Chem. 271(47):29953 57, 1996). In general, longer peptide inhibitors have higher affinity values and/or lower dissociation rates than shorter peptides and therefore may be more potent inhibitors. Cyclic peptide inhibitors have also been shown to be effective inhibitors of integrins in vivo in the treatment of inflammatory diseases in humans.

- 52 042534 (Jackson, D.Y., et al., J. Med. Chem. 40:3359 68, 1997). One method of producing cyclic peptides is by synthesizing peptides in which the terminal amino acids of the peptide are cysteines, which allows the peptide to exist in a cyclic form as a result of the formation of a disulfide bond between the terminal amino acids, which has been shown to increase affinity and in vivo half-life in the treatment of hematopoietic neoplasia (see, for example, U.S. Patent No. 6649592, to Larson).

Synthetic peptide inhibitors of SMASP-2.

The MASP-2 inhibitory peptides used in the methods of this aspect of the invention are characterized by amino acid sequences that mimic target sites important for MASP-2 function. Inhibitory peptides useful in practicing the methods of the invention range in size from about 5 amino acids to about 300 amino acids. In table. 3 is a list of exemplary inhibitory peptides that may be used in the practice of this aspect of the present invention. A MASP-2 inhibitory peptide of interest can be tested for its ability to act as a MASP-2 inhibitory agent in one of several assays, including, for example, a specific C4 lectin cleavage assay (described in Example 2), and a C3b deposition assay (described in example 2).

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

In some embodiments, the MASP-2 inhibitory peptides are derived from lectin proteins that bind to MASP-2 and are involved in the lectin complement pathway. Several different lectins have been identified that are involved in this pathway, including mannan-binding lectin (MBL), L-ficolin, M-ficolin, and H-ficolin (Ikeda, K., et al., J. Biol. Chem. 262: 7451 7454, 1987; Matsushita, M., et al., J. Exp. Med. 176:1497 2284, 2000; Matsushita, M., et al., J. Immunol. 168:3502 3506, 2002). These lectins are present in serum as oligomers of homotrimeric subunits, each of which has N-terminal collagen-like fibers with carbohydrate recognition domains. These various lectins have been shown to bind to MASP-2 and the lectuh/MASP-2 complex activates complement via cleavage of C4 and C2 proteins. H-ficolin has a 24 amino acid amino-terminal region, a collagen-like domain with 11 Gly-Xaa-Yaa repeats, a 12 amino acid neck domain, and a 207 amino acid fibrinogen-like domain (Matsushita, M., et al., J. Immunol. 168:3502 3506, 2002). N-ficolin binds to GlcNAc and agglutinates human erythrocytes coated with LPS derived from Salmonella subspecies S. typhimurium, S. Minnesota, and E. coli. H-ficolin has been shown to bind to MASP-2 and MAP19 and activate the lectin pathway (in the same publication). L-ficolin/P35 also binds to GlcNAc and has been shown to associate with MASP-2 and MAP19 in human serum, and this complex has been shown to activate the lectin pathway (Matsushita, M., et al., J. Immunol 164:2281, 2000). Accordingly, MASP-2 inhibitory peptides useful in the present invention may include a stretch of at least 5 amino acids selected from MBL protein (SEQ ID NO: 21), H-ficolin protein (Genbank accession number NM 173452), M-ficolin (Genbank accession number O00602) and Lficolin protein (Genbank accession number NM_015838).

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

MASP-2 inhibitory peptides can also be derived from human H-ficolin, which include the sequence GAO GSO GEK GAO GPQ GPO GPO GKM GPK GEO GDO (SEQ ID NO: 27) from the MASP-2 consensus binding site in H-ficolin. In addition, these include peptides derived from human L-ficolin that include the sequence GCO GLO GAO GDK GEA GTN GKR GER GPO GPO GKA GPO GPN GAO GEO (SEQ ID NO: 28) from the MASP-2 consensus binding site in L- ficoline.

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

Table 3

Examples of peptides that inhibit MASP-2

SEQID NO Source SEQ ID NO:6 Human MASP-2 protein SEQ ID NO:8 CUBI domain of MASP-2 (aa 1-121 sequence SEQ ID NO: 6) SEQ ID NO:9 CUBIEGF MASP-2 domains (aa 1166 sequences SEQ ID NO: 6) SEQ ID NO:10 CUBIEGFCUBII MASP-2 domains (aa 1-293 of SEQ ID NO:6) SEQ ID NO:11 EGF domain of MASP-2 (aa 122-166) SEQ ID NO:12 MASP-2 serine protease domain (aa 429-671) SEQ ID NO:16 MBL binding site in MASP2 SEQ ID NO:3 Human MApl9 SEQ ID NO:21 Human MBL protein SEQ ID NO:22 OGK-X-GP where O=hydroxyproline and X is a hydrophobic amino acid residue A consensus binding site for a synthetic peptide from human MBL and human ficolins SEQ ID NO:23 OGKLG Major human MBL binding site SEQ ID NO:24 GLR GLQ GPO GKL GPO G Triplets 6-10 of human MBP showing binding to MASP-2 SEQ ID NO:25 GPOGPOGLRGLQGPOGKLGPOGGPOGPO Triplets with human MBP GPO added to enhance triple helix formation SEQ ID NO:26 GKDGRDGTKGEKGEPGQGLRGLQGPOGKLG POGNOGPSGSOGPKGQKGDOGKS Triplets 1-17 human MVR SEQ ID NO:27 GAOGSOGEKGAOGPQGPOGPOGKMGPKGEO GDO Human N-ficolin (Hataka) SEQ ID NO:28 GCOGLOGAOGDKGEAGTNGKRGERGPOGPO GKAGPOGPNGAOGEO Human L-ficolin P35 SEQ ID NO:29 LQRALEILPNRVTIKANRPFLVFI Human C4 cleavage site

Note: the letter O stands for hydroxyproline, the letter X stands for a hydrophobic residue. Peptides derived from the C4 cleavage site, as well as other peptides that inhibit the MASP-2 serine protease site, can be chemically modified to become irreversibly active protease inhibitors. For example, appropriate modifications may include, but are not limited to, halomethyl ketones (Br, Cl, I, F) at the C-terminus, Asp or Glu, or attached to functional side chains; haloacetyl (or other α-haloacetyl) groups on amino groups or other functional side chains; epoxy or imine groups on the amino or carboxyl terminus, or on functional side chains; or imidate esters at the amino or carboxyl terminus, or at functional side chains. Such modifications may offer the advantage of permanently inhibiting the enzyme by covalently attaching the peptide. This makes it possible to reduce effective doses and/or reduce the frequency of administration of the peptide inhibitor.

In addition to the inhibitory peptides described above, MASP-2 inhibitory peptides useful in the method of the invention include peptides containing the MASP-2 CDR3 binding site of an anti-MASP-2 MoAb, prepared as described in the invention. The sequence of CDRs for use in peptide synthesis can be determined using well-known methods. The heavy chain variable region is a peptide that is typically in the range of 100 to 150 amino acids in length. The light chain variable region is a peptide that is typically in the range of 80 to 130 amino acids in length. The CDR sequences in the heavy and light chain variable regions comprise only about 3-25 amino acid sequences, which can be easily sequenced by one of skill in the art.

Those skilled in the art will appreciate that substantially homologous variations of the MASP-2 inhibitory peptides described above should also exhibit MASP-2 inhibitory activity. Examples of variations include, but are not limited to, peptides having insertions, deletions, substitutions and/or additional amino acids at the carboxyl or amine termini of said peptides or mixtures thereof. Accordingly, it is believed that homologous peptides that have MASP-2 inhibitory activity can be used in the methods of this invention. The described peptides may also include duplicate motifs and other modifications with conservative substitutions. Conservative variants are described elsewhere in the invention and include the exchange of an amino acid for another amino acid with a similar charge, size or hydrophobicity and other similar properties.

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

Synthesis of inhibitory peptide derivatives may be based on known methods for peptide biosynthesis, carbohydrate biosynthesis, and the like. As a starting point, one skilled in the art can use a suitable computer program to determine the conformation of the peptide of interest. Once the conformation of a peptide disclosed in the invention is known, one skilled in the art can determine, using a rational design method, what kind of substitutions can be made at one or more sites in order to obtain a derivative that retains the basic conformation and charge distribution of the parent peptide, but which may have characteristics that are not are present or which are improved over the characteristics found in the parent peptide. Once molecules of promising derivatives have been identified, these derivatives can be tested to determine their suitability as MASP-2 inhibitory agents using the assay methods described herein.

Screening for MASP-2 inhibitory peptides.

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

As an illustrative example, one method for making MASP-2 binding peptide mimics is as follows. Functional monomers of a known MASP-2 binding peptide or binding site of an anti-MASP-2 antibody that inhibits MASP2 (template) are polymerized. Then the template is removed and the polymerization of the second class of monomers is carried out in the empty space remaining after the removal of the heat to obtain a new molecule that has one

- 55 042534 or more desirable properties that are similar to those of heat. In addition to producing peptides, other MASP-2 binding molecules that are MASP-2 inhibitory agents, such as polysaccharides, nucleosides, drugs, nucleoproteins, lipoproteins, carbohydrates, glycoproteins, steroids, lipids and other biologically active materials can also be obtained by this method. . This method is used to construct a large number of biological mimics that are more stable than their natural counterparts, as they are usually prepared by free radical polymerization of functional monomers resulting in a compound with a non-biodegradable backbone.

Synthesis of peptides.

MASP-2 inhibitory peptides can be prepared by well known methods such as the solid phase synthesis method first described in Merrifield, J. Amer. Chem. soc. 85:2149-2154, 1963. Automated synthesis can be performed using, for example, an Applied Biosystems 431A Peptide Synthesizer (Foster City, Calif.) according to the manufacturer's instructions. Description of other methods can be found, for example, in the monograph Bodanszky, M., et al., Peptide Synthesis, second edition, John Wiley & Sons, 1976, as well as in other reference publications known to specialists in this field.

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

The MASP-2 inhibitory peptides that are used in the method of the invention can also be produced in recombinant host cells according to conventional methods. In order to express a sequence encoding a MASP-2 inhibitory peptide, the nucleic acid molecule encoding the peptide must be operably linked to regulatory sequences that control transcriptional expression in an expression vector and then introduced into a host cell. In addition to transcriptional control sequences such as promoters and enhancers, expression vectors may include translational control sequences and a marker gene that are used to select cells capable of carrying the expression vectors.

Nucleic acid molecules that encode a MASP-2 inhibitory peptide can be synthesized by gene machines using protocols such as the phosphoramidite method. If the use of chemically synthesized double-stranded DNA is required, for example, in the synthesis of a gene or its fragment, then each complementary strand is obtained separately. The production of short genes (from 60 to 80 base pairs) is technically rapidly developing and can be carried out by synthesizing complementary chains and then renaturating them. To obtain longer genes, synthetic genes (double-stranded) are assembled into a modular form from single-stranded fragments that are 20 to 100 nucleotides in length. Reviews of polynucleotide synthesis can be found, for example, in Glick and Pasternak, Molecular Biotechnology, Principles and Applications of Recombinant DNA, ASM Press, 1994; in Itakura, K., et al., Annu. Rev. Biochem. 53:323, 1984; and Climie, S., et al., Proc. Nat'l Acad. Sci USA 87:633, 1990.

low molecular weight inhibitors.

In some embodiments, the MASP-2 inhibitory agents are small molecule inhibitors, including natural and synthetic substances that have a low molecular weight, such as, for example, peptides, peptidomimetics, and non-peptide inhibitors (including oligonucleotides and organics). Small molecular weight inhibitors of MASP-2 can be obtained based on the molecular structure of the variable regions of antibodies against MASP-2.

Small molecule inhibitors can be designed and produced based on the crystal structure of MASP-2 using computerized drug design and development (Kuntz I.D., et al., Science 257:1078, 1992). The crystal structure of rat MASP-2 has been described (Feinberg, H., et al., EMBO J. 22:2348-2359, 2003). Using the method described in Kuntz et al., MASP-2 crystal structure coordinates are entered into a computer program such as DOCK, which outputs a list of small molecular weight structures with expected MASP-2 binding ability. The use of such computer programs is well known to those skilled in the art. For example, the crystal structure of an HIV-1 protease inhibitor has been used to identify unique non-peptide ligands that are protease inhibitors.

- 56 042534

HIV-1 by evaluating the correspondence of compounds from the Cambridge Crystallographic database to the binding site of the enzyme using the DOCK program (Kuntz, L.D., et al., J. Mol. Biol. W:269-288, 1982; DesJarlais, R.L, et al., PNAS 87:6644-6648, 1990).

A list of small molecule structures identified by a computerized method as potential MASP-2 inhibitors is screened using the MASP-2 binding assay as described in Example 10. Small molecule compounds found to bind to MASP-2 are then subjected to the test in a functional study, as described in example 2, to determine their ability to inhibit MASP-2 dependent complement activation.

MASP-2 soluble receptors.

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

MASP-2 expression inhibitors.

In another embodiment of this aspect of the invention, the MASP-2 inhibitory agent is an inhibitor of MASP-2 expression capable of inhibiting MASP-2 dependent complement activation. In the practice of this aspect of the invention, exemplary inhibitors of MASP-2 expression include MASP-2 antisense nucleic acid molecules (such as antisense mRNA, antisense DNA, or antisense oligonucleotides), MASP-2 ribozymes, and MASP-2 RNA interference molecules.

Antisense RNA and DNA molecules directly block translation of MASP-2 mRNA by hybridizing to MASP-2 mRNA and preventing translation of the MASP-2 protein. The antisense nucleic acid molecule can be constructed using a variety of methods, provided that it must be able to interfere with the expression of MASP-2. For example, an antisense nucleic acid molecule can be constructed by inverting the coding region (or portion thereof) of the MASP-2 cDNA (SEQ ID NO: 4) from its normal transcriptional orientation to allow transcription of its complement.

Typically, the antisense nucleic acid molecule is substantially identical to at least a portion of the target gene or target genes. However, in order to inhibit expression, the nucleic acid need not be exactly the same. Generally, a higher homology may be used to compensate for the use of a shorter sequence antisense nucleic acid molecule. The minimum percentage of identity is usually at least 65%, but a higher percentage of identity may be characterized by more effective suppression of expression of the endogenous sequence.

A percentage of identity substantially greater than 80% is preferred, and yet an identity of 95% to absolute identity is most preferred.

An antisense nucleic acid molecule may not have the same intron or exon pattern as the target gene, and non-coding segments of the target gene may be as effective in achieving antisense suppression of the target gene as the coding segments. A DNA sequence of at least 8 nucleotides or more can be used as the antisense nucleic acid molecule, although a longer sequence is preferred. In the present invention, a typical example of a MASP-2 inhibitory agent used is a MASP-2 antisense nucleic acid molecule that is at least ninety percent identical to a complementary MASP-2 cDNA consisting of the nucleic acid sequence described in SEQ ID NO: 4. Sequence the nucleic acid described in SEQ ID NO: 4 encodes a MASP-2 protein consisting of the amino acid sequence described in SEQ ID NO: 5.

Targeting antisense oligonucleotides to bind to MASP-2 mRNA is another mechanism that can be used to decrease the level of MASP-2 protein synthesis. For example, the synthesis of polygalacturonase and type II muscarinic acetylcholine receptor is inhibited by antisense oligonucleotides directed to their respective mRNA sequences (U.S. Patent No. 5739119, to Cheng, and U.S. Patent No. 5759829, to Shewmaker). In addition, examples of antisense inhibition by cyclin core protein, multidrug resistance gene (MDG1), ICAM-1, Eselectin, STK-1, striatal GABAA receptor and human EGF have been demonstrated (see, for example, U.S. Patent No. 5801154 , to Baracchini; U.S. Patent No. 5789573, to Baker; U.S. Patent No. 5718709, to Considine; and U.S. Patent No. 5610288, to Reubenstein).

A system is described that allows one of ordinary skill in the art to determine which oligonucleotides may be used in the invention, including probing suitable sites in the target mRNA using ribonuclease H cleavages as an indicator of the availability of sequences within transcripts. Scherr, M., et al., Nucleic Acids Res. 26:5079-5085, 1998; Lloyd, et al., Nucleic Acids Res. 29:3665-3673, 2001. A mixture of antisense oligonucleotides that are complementary in

- 57 042534 to specific regions of the MASP-2 transcript are added to cell extracts expressing MASP-2, such as hepatocytes, and subjected to hybridization to create ribonuclease H susceptible sites. This method can be combined with sequence selection using a computer program that can predict the optimal sequence selection for antisense compositions based on their relative ability to form dimers, hairpins, or other secondary structures that can reduce or prevent specific binding to the target mRNA in the host cell. . Analysis of these secondary structures and recommendations for target site selection can be performed using the OLIGO primer analysis software (Rychlik, I., 1997) and the BLASTN 2.0.5 computer algorithm (Altschul, S.F, et al., Nucl. Acids Res. 25:3389-3402, 1997). Preferably, antisense compounds directed to a target sequence are about 8 to 50 nucleotides in length. Antisense oligonucleotides comprising about 9 to 35 nucleotides or so are particularly preferred. The inventors contemplate that all oligonucleotide compositions in the range of 9 to 35 nucleotides (i.e. those that include 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 or so) are most preferred for the practice of the methods of the invention based on antisense oligonucleotides. The most preferred target regions of the MASP-2 mRNA are those located in close proximity to the AUG translation initiation codon and those sequences that are effectively complementary to the 5' regions of the mRNA, e.g. between the -10 and +10 regions of the nucleotide sequence of the MASP gene. -2 (SEQ ID NO: 4). Examples of inhibitors of MASP-2 expression are presented in table. 4.

Table 4

Examples of Inhibitors of MASP-2 Expression

SEQ ID NO:30 (nucleotides 22-680 of SEQ ID NO:4) MASP-2 cDNA nucleic acid sequence (SEQ ID NO:4) encoding CUBIEGF SEQ ID NO:31 5'CGGGCACACCATGAGGCTGCTGAC CCTCCTGGGC3 Nucleotides 12-45 of SEQ ID NO:4, including MASP-2 translation start site (sense) SEQ ID NO:32 5’GACATTACCTTCCGCTCCGACTCC AACGAGAAG3’ Nucleotides 361-396 of SEQ ID NO:4 encoding the region that contains the MASP2 MBL binding site (sense) SEQ ID NO:33 5'AGCAGCCCTGAATACCCACGGCCG TATCCCAAA3' Nucleotides 610-642 of SEQ ID NO:4 encoding the region that contains the CUBII domain

As noted above, as used in the invention, the term oligonucleotide refers to any oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or their mimics. The term also covers oligonucleotide bases consisting of natural nucleotides, sugars and covalent internucleoside (framework) bonds, as well as oligonucleotides with non-natural modifications. These modifications allow the oligonucleotides to be endowed with certain desirable properties that natural oligonucleotides do not have, such as reduced toxicity, increased resistance to nuclease degradation, and increased cellular uptake. In illustrative embodiments, the antisense compounds of the invention differ from native DNA by modifying the phosphodiester backbone to increase the lifetime of the antisense oligonucleotide in which the phosphate substituents are replaced by phosphorothioate. Similarly, one or both ends of the oligonucleotide may be substituted with one or more acridine derivatives that intercalate between adjacent base pairs in the nucleic acid strand.

Another alternative to antisense is the use of RNA interference (RNAi). Double-stranded RNAs (dsRNAs) can induce gene silencing in mammals in vivo. It seems that the natural function of RNAi and cosuppression is to protect the genome from invasion by mobile genetic elements such as retrotransposons and viruses that produce aberrant RNA or dsRNA in the host cell when they become active (see, for example, Jensen, J., et al., Nat Genet 21:20912, 1999). A double-stranded RNA molecule can be prepared by synthesizing two strands of RNA capable of forming a double-stranded RNA molecule, each about 19 to 25 nucleotides in length (eg, 19-23 nucleotides). For example, the dsRNA molecule used in the methods of the invention may include RNA corresponding to the sequence and its complement shown in table. 4. Preferably, at least one RNA strand has a 3' sticky end of 1-5 nucleotides. The synthesized RNA chains are combined under conditions that form a double-stranded molecule. The RNA sequence may include at least an eight nucleotide portion of SEQ ID NO: 4 with a total length of 25 nucleotides or less. The design of siRNA sequences for a given target is well known to one of ordinary skill in the art. There are commercial organizations that construct siRNA sequences and guarantee at least 70% gene expression knockdown (Qiagen, Valencia, Calif).

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

Ribozymes, such as ribozymes that target MASP-2 mRNA, can also be used to reduce the amount and/or biological activity of MASP-2. Ribozymes are catalytic RNA molecules capable of cleaving nucleic acid molecules with a sequence that is wholly or partly homologous to that of the ribozyme. It is possible to construct ribozyme transgenes encoding RNA ribozymes that specifically pair with the target RNA and cleave the phosphodiester backbone in a specific site, resulting in functional inactivation of the target RNA. When this cleavage is carried out, the ribozyme itself does not change, as a result of which it retains the ability to recycle and cleave other molecules. The incorporation of ribozyme sequences into antisense RNAs provides them with RNA cleavage activity, resulting in increased activity of the antisense constructs.

Ribozymes useful in the practice of the invention typically comprise a hybridization region of at least nine nucleotides that is nucleotide sequence complementary to at least a portion of the target MASP-2 mRNA and a catalytic region that is designed to cleave the target MASP-2 mRNA. 2, (See in general, EPA Patent Documents No. 0321201; WO88/04300; Haseloff, J., et al., Nature 334:585 591, 1988; Fedor, M.J., et al., Proc. Natl. Acad. Sci USA 87:1668 1672, 1990; Cech, T. R., et al., Ann Rev. Biochem 55:599 629, 1986).

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

The antisense RNA and DNA, ribozymes and RNAi molecules used in the methods of the invention can be prepared by any known method for synthesizing DNA and RNA molecules. These include well-known methods for the chemical synthesis of oligodeoxyribonucleotides and oligoribonucleotides, such as, for example, solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules can be generated by in vitro and in vivo transcription of DNA sequences encoding an antisense RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesize antisense RNA either continuously or inducibly, depending on the promoter used, can be stably introduced into cell lines.

Well-known various modifications of DNA molecules can be introduced to improve stability and half-life. Useful modifications include, but are not limited to, adding flanking ribonucleotide or deoxyribonucleotide sequences to the 5' and/or 3' ends of the molecule, or using phosphothioate or 2'O-methyl instead of phosphodiester bonds to the oligodeoxyribonucleotide backbone.

VI. Pharmaceutical compositions and methods of delivery. Dosing.

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

The toxicity and therapeutic efficacy of MASP-2 inhibitory agents can be determined by standard pharmaceutical methods using experimental animal models such as the MASP-2 -/- mouse model expressing the human MASP-2 transgene as described in Example 1. Using such experimental animal models, the no observed adverse effect level (NOAEL) and the minimum effective dose (MED) can be determined by standard methods. The dose ratio that characterizes NOAEL and MED is the therapeutic index, which is expressed as the NOAEL/MED ratio. MASP-2 inhibitory agents that have high therapeutic indices are most preferred. Data obtained from the study of cell cultures and

- 59 042534 animal studies can be used to determine the range of doses for humans.

Preferably, doses of the MASP-2 inhibitory agent are in a range of circulating concentrations that include MED with minimal or no toxicity. The dose may vary within this range depending on the dosage form or the route of administration used.

For any dosage form of a compound, a therapeutically effective dose can be determined in animal models. For example, in an animal model, the dose that achieves the circulating plasma concentration range that includes the MED can be determined. Plasma levels of the MASP-2 inhibitory agent can also be determined, for example, by high performance liquid chromatography.

In addition to toxicological studies, an effective dose can also be estimated by the amount of MASP-2 protein present in a living organism and by the binding affinity of the MASP-2 inhibitory agent. MASP-2 levels have been shown to be present in the serum of healthy individuals in small amounts in the range of 500 ng/mL, and MASP-2 levels in specific subjects can be determined by the MASP-2 quantification method described in MollerKristensen M., et al. ., J. Immunol. Methods 282:159-167, 2003.

Typically, the dose of compositions administered, including MASP-2 inhibitory agents, will vary depending on factors such as age, body weight, height, sex of the subject, his general health and medical history. By way of illustration, MASP-2 inhibitory agents such as anti-MASP-2 antibodies can be administered at a dose of about 0.010 to 10.0 mg/kg, preferably 0.010 to 1.0 mg/kg, more preferably from 0.010 to 0.1 mg/kg subject's body weight. In some embodiments, the composition comprises a combination of an anti-MASP-2 antibody and MASP-2 inhibitory peptides.

The therapeutic efficacy of the MASP-2 inhibitory compositions and methods of the present invention in a given subject and the appropriate doses can be determined by complement studies, which are well known to those skilled in the art. Complement generates many specific products. During the past ten years, sensitive and specific assays have been developed that are commercially available for most of these activation products, including small activation fragments C3a, C4a and C5a and large activation fragments iC3b, C4d, Bb and sC5b-9. Most of these studies use monoclonal antibodies that react with novel antigens (neoantigens) available on the fragment but not on the native proteins from which they are derived, making these assays very simple and specific. The most commonly used enzyme-linked immunosorbent assay (ELISA), although radioimmunoassay for C3a and C5a is sometimes still used. In this latter method, both the unprocessed fragments and their 'desArg' fragments, which are the main forms found in the bloodstream, are measured. Full length fragments and C5adesArg are rapidly cleared by binding to cell surface receptors and therefore present at very low concentrations, while C3adesArg does not bind to cells and accumulates in plasma. The measurement of C3a is a sensitive, pathway-independent indicator of complement activation. An alternative pathway for complement activation can be assessed by measuring the Bb fragment. The detection of the liquid-phase product of the membrane attack pathway, sC5b-9, is evidence that complement is fully activated. Since both lectin and classical activation pathways generate the same activation products, C4a and C4d, measurement of these two fragments does not provide any information as to which of the two complement pathways resulted in the formation of activation products.

Inhibition of MASP-2 dependent complement activation is characterized by at least one of the following changes in a component of the complement system that results from administration of the MASP-2 inhibitory agent by the methods of the invention: and/or C5b-9 (MAC) (measured, for example, as described in Example 2), reduction in C4 splitting and C4b deposition (measured, for example, as described in Example 10), or reduction in C3 splitting and C3b deposition (measured, for example, , as described in example 10).

Complementary medicines.

Compositions and methods comprising MASP-2 inhibitory agents may optionally include one or more additional therapeutic agents that can enhance the activity of the MASP-2 inhibitory agent or that provide similar therapeutic effects additively or synergistically. For example, in the case of treating a subject suffering from TTP where the subject achieves a positive response to treatment with an ADAM-TS13 inhibitor, one or more MASP-2 inhibitory agents may be administered in combination (including concurrently) with one or more immunosuppressive agents. Suitable immunosuppressive agents include corticosteroids, rituxan, cyclosporine, and other similar immunosuppressants. In the case of treating a subject suffering from HUS or aHUS, or at risk of developing HUS or aHUS, one or more MASP-2 inhibitory agents may be administered in combination (including together) with a suitable antibiotic. In the case of treating a subject suffering from aHUS or at risk of developing aHUS, one or more MASP-2 inhibitory agents may be administered in combination (including together) with other complement inhibitors such as eculizumab (Soliris), TT-30 , an antibody to factor B, or other agents that inhibit terminal complement components or alternative pathway amplification.

To achieve the desired therapeutic result, it is necessary to decide on the inclusion and choice of an additional agent (additional agents). In some embodiments, the MASP-2 inhibitory agent may be administered in combination with one or more anti-inflammatory and/or analgesic agents. Suitable anti-inflammatory and/or analgesic agents include serotonin receptor antagonists; serotonin receptor agonists; histamine receptor antagonists; bradykinin receptor antagonists; kallikrein inhibitors; tachykinin receptor antagonists, including neurokinin 1 and neurokinin-2 receptor subtype antagonists; calcitonin gene-related peptide (CGRP) receptor antagonists; interleukin receptor antagonists; inhibitors of enzymes active in the synthetic activation pathway for arachidonic acid metabolites, including phospholipase inhibitors, including PLA2 isoform inhibitors and PLCy isoform inhibitors, cyclooxygenase (COX) inhibitors (which may be either COX-1, COX-2, or non-selective COX- 1 and COX-2), lipoxygenase inhibitors; prostanoid receptor antagonists, including eicosanoid EP-1 and EP-4 receptor subtype antagonists and thromboxane receptor subtype antagonists; leukotriene receptor antagonists, including leukotriene B4 and leukotriene D4 receptor subtype antagonists; opioid receptor agonists, including μ-opioid, δ-opioid and κ-opioid receptor subtype agonists; purinergic receptor agonists and antagonists, including P2X receptor antagonists and P2Y receptor agonists; adenosine triphosphate (ATP) sensitive substances that open potassium channels; MAP kinase inhibitors; cholinomimetic acetylcholine inhibitors; and alpha-adrenergic receptor agonists (including alpha-1, alpha-2 and non-selective alpha-1 and alpha-2 agonists).

The MASP-2 inhibitory agents of the present invention may be administered in combination with one or more other complement inhibitors, such as a C5 inhibitor. Currently, eculizumab (Solaris®), an anti-C5 antibody, is the only complement-targeted drug that has been approved for use in humans. However, pharmacological agents have been shown to block complement in vivo. K76COOH and nafamstat mesylate are two drugs that have shown some efficacy in animal transplant models (Miyagawa, S., et al., Transplant Proc. 24:483 484, 1992). Small molecule heparins have also been shown to be effective in regulating complement activation (Edens, R.E., et al., Complement Today, pp. 96-120, Basel: Karger, 1993). It is believed that these small molecule inhibitors can be used as drugs when used in combination with the MASP-2 inhibitory agents of the present invention.

Other natural complement inhibitors may be used in combination with the MASP-2 inhibitory agents of the present invention. Biological complement inhibitors include soluble complement factor 1 (sCR1). It is a natural inhibitor found in the outer membrane of human cells. Other membrane inhibitors include the membrane inhibitors DAF, MCP and CD59. The recombinant forms were tested for their anti-complement activity in vitro and in vivo. sCRl has been shown to be effective in xenotransplantation when the complement system (both alternative and classical) initiates hyperactive rejection syndrome within minutes of blood perfusion in a newly transplanted organ (Platt J.L, et al., Immunol. Today 11:450 -6, 1990; Marino I.R., et al., Transplant Proc. 1071:6, 1990; Johnstone, P.S., et al., Transplantation 54:573-6, 1992). The use of sCRl protects and prolongs the survival time of the transplanted organ by involving the complement activation pathway in the pathogenesis of organ survival (Leventhal, J. R. et al., Transplantation 55:857-66, 1993; Pruitt, S.K., et al., Transplantation 57:363-70, 1994).

Suitable additional complement inhibitors for use in combination with the compositions of the present invention also include, for example, MoAbs, such as an anti-C5 antibody (eg, eculizumab) developed by Alexion Pharmaceuticals, Inc., New Haven, Connecticut, and MoAbs against properdin.

Pharmaceutical carriers and delivery systems.

Typically, the MASP-2 inhibitory compositions of the present invention, when combined with any other selected therapeutic agents, are suitably enclosed in a pharmaceutically acceptable carrier. The carrier is non-toxic, biocompatible, and is chosen such that it does not adversely affect the biological activity of the MASP-2 inhibitory agent (and any other therapeutic agents combined with it). Examples of pharmaceutically acceptable

- 61 042534 carriers for peptides are described in the patent document U.S. Patent No. 5211657 to Yamada. The anti-MASP-2 antibodies and inhibitory peptides used in the invention can be formulated into solid, semi-solid, gel, liquid and gaseous dosage forms such as tablets, capsules, powders, granules, ointments, solutions, suppositories, inhalation forms and injections for oral, parenteral or surgical administration. The invention also includes topical administration of the compositions by drug coating of medical devices and other similar instruments.

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

The carrier may also include a delivery device to maintain a steady state (i.e., prolonged, delayed, or controlled) delivery of the drug(s) or to enhance delivery, uptake, stability, or pharmacokinetics of the therapeutic(s). Such a delivery system may include, but is not limited to, microparticles, microspheres, nanospheres, or nanoparticles formed from proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymeric or copolymeric hydrogels, and polymeric micelles. Suitable hydrogel and micellar delivery systems include PEO:PHB:PEO (polyethylene oxide: polyhydroxybutyrate:polyethylene oxide) copolymers and copolymer/cyclodextrin complexes disclosed in WO 2004/009664 A2, and PEO and PEO/cyclodextrin complexes disclosed in U.S. Patent Application Publication No. 2002/0019369 A1. Such hydrogels may be injected topically at the site of intended action, or subcutaneously or intramuscularly to form a sustained release drug depot.

In the case of intra-articular delivery, the MASP-2 inhibitory agent can be carried in the injectable liquid or gel carriers described above, in the sustained release drug injectable delivery systems described above, or in hyaluronic acid or a hyaluronic acid derivative.

In the case of oral administration of non-peptidergic agents, the MASP-2 inhibitory agent may be carried in an inert excipient or diluent such as sucrose, corn starch or cellulose.

In the case of topical administration, the MASP-2 inhibitory agent can be transferred in an ointment, lotion, cream, gel, drops, suppository, spray, liquid or powder, or in gel or microcapsular delivery systems via a transdermal patch.

Various nasal and inhalation delivery systems, including aerosols, metered dose inhalers, dry powder inhalers, and nebulizers, are currently being developed and can be suitably adapted to deliver the present invention by aerosol, inhalation, or spray delivery, respectively.

In the case of intrathecal (IT) or intracerebroventricular (ICV) delivery, suitably sterilized delivery systems (eg, liquids, gels, suspensions, etc.) may be used to administer the composition of the present invention.

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

Pharmaceutical carriers for antibodies and peptides.

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

Anti-MASP-2 antibodies and inhibitory peptides may also be administered in the form of an injectable depot or implantable preparation, which may be formulated to provide sustained or pulsatile release of the active agents.

Pharmaceutically acceptable carriers for expression inhibitors.

More specifically, with respect to expression inhibitors used in the methods of the invention, compositions are provided that include an expression inhibitor as described above and a pharmaceutically acceptable carrier or diluent. The composition may further include a colloidal disperse system.

Pharmaceutical compositions that include expression inhibitors may include, but are not limited to, solutions, emulsions, and formulations containing liposomes. These compositions can be prepared from a variety of ingredients which include, but are not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semi-solids. The preparation of such compositions typically involves mixing an expression inhibitor with one or more of the following: buffers, antioxidants, low molecular weight polypeptides, proteins, amino acids, carbohydrates including glucose, sucrase or dextrins, chelating agents such as EDTA, glutathione, and other stabilizers and excipients. . Examples of suitable diluents are neutral buffered saline or saline mixed with non-specific serum albumin.

In some embodiments, the compositions may be formulated and prepared in the form of emulsions, which are usually heterogeneous systems of one liquid dispersed in another liquid in the form of droplets (see Idson, in Pharmaceutical Dosage Forms, Vol. 1, Rieger and Banker (eds. ), Marcek Dekker, Inc., N.Y., 1988). Examples of natural emulsifiers used in emulsion dosage forms include gum arabic, beeswax, lanolin, lecithin, and phosphatides.

In one embodiment, compositions comprising nucleic acids may be prepared in the form of microemulsions. The microemulsion used in the invention refers to a system of water, oil and amphiphilic substance, which is a simple optically isotropic and thermodynamically stable liquid solution (see Rosoff in Pharmaceutical Dosage Forms, Vol. 1). Liposomes can also be used in the method of the invention to carry and deliver antisense oligonucleotides to the desired site.

Pharmaceutical compositions and formulations of topical expression inhibitors may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers may be used, as well as aqueous, powdered or oily bases and thickeners and the like.

Methods of administration.

Pharmaceutical compositions comprising MASP-2 inhibitory agents may be administered in a variety of ways, depending on which route of administration, topical or systemic, is most appropriate for the condition being treated. In addition, as described in the invention above with respect to methods of extracorporeal reperfusion, MASP-2 inhibitory agents can be administered by introducing the compositions of the present invention into circulating blood or plasma. In addition, the compositions of the present invention can be delivered by coating an implantable medical device or by injecting the compositions into an implantable medical device.

System delivery.

The terms systemic delivery and systemic administration as used herein are intended to include, but are not limited to, oral and parenteral routes, including intramuscular (IM), subcutaneous, intravenous (IV), intra-arterial, inhalation, sublingual, buccal, topical, transdermal. , nasal, rectal, vaginal and other routes of administration that allow effective dispersion of the delivered agent at one or more sites of intended therapeutic action. Preferred methods of systemic delivery of the compositions of the present invention include intravenous, intramuscular, subcutaneous and inhalation. It should be borne in mind that the appropriate systemic route of administration for the selected agents used in particular compositions of the present invention should be determined, including taking into account the predisposition of the drug to the metabolic transformation pathways associated with this route of administration. For example, peptidergic agents are most conveniently administered by non-oral routes.

MASP-2 inhibitory antibodies and polypeptides can be delivered to a subject in need thereof by any suitable means. Methods for delivering MASP-2 antibodies and polypeptides include oral, pulmonary, parenteral (eg, intramuscular, intraperitoneal, intravenous (IV) or subcutaneous) injection, inhalation (eg, in fine powder form), transdermal, nasal, vaginal, rectal, or sublingual routes of administration and can be formulated into dosage forms corresponding to each of the routes of administration.

- 63 042534

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

MASP-2 inhibitory antibodies and polypeptides can be co-administered with another molecule, such as a lipid, to protect the polypeptides from enzymatic degradation. For example, covalent attachment of polymers, in particular polyethylene glycol (PEG), has been used to protect specific proteins from enzymatic hydrolysis in the body and thus increase the half-life (Fuertges, F., et al., J. Controlled Release 11:139, 1990 ). Many polymeric protein delivery systems have been reported (Bae, Y.H., et al., J. Controlled Release 9:271, 1989; Hori, R., et al., Pharm. Res. 6:813, 1989; Yamakawa, I. , et al., J. Pharm. Sci. 79:505, 1990; Yoshihiro, I., et al., J. Controlled Release 10:195, 1989; Asano, M., et al., J. Controlled Release 9 :111, 1989; Rosenblatt, J., et al., J. Controlled Release 9:195, 1989; Makino, K., J. Controlled Release 12:235, 1990; Takakura, Y., et al., J. Pharm. Sci. 78:117, 1989; Takakura, Y., et al., J. Pharm. Sci. 78:219, 1989).

Recently, liposomes with increased serum stability and increased circulating half-lives have been developed (see, for example, U.S. Patent No. 5741516, to Webb). In addition, reviews of various methods for the preparation of liposome and liposome-like preparations used as promising drug carriers have been published (see, for example, U.S. Patent No. 5567434, to Szoka; U.S. Patent No. 5552157, to Yagi; U.S. Patent No. 5565213, to Nakamori; U.S. Patent No. 5738868, to Shinkarenko; and U.S. Patent No. 5795587, to Gao).

In the case of transdermal application, MASP-2 inhibitory antibodies and polypeptides may be combined with other suitable ingredients such as carriers and/or excipients. There are no restrictions on the properties of such other ingredients, except that they must be pharmaceutically acceptable for their intended administration, and they are not capable of impairing the effect of the active ingredients of the composition. Examples of suitable media include ointments, creams, gels or suspensions in the presence or absence of purified collagen. MASP-2 inhibitory antibodies and polypeptides, preferably in liquid or semi-liquid form, can also be impregnated into transdermal patches, patches and dressings.

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

Local delivery.

As used herein, the term topical includes the application of the drug at or near the site of the intended localized effect, and this term may include, for example, topical delivery to the skin or other affected tissues, ophthalmic delivery, intrathecal (IT), intracerebroventricular (ICV) , intra-articular, intracavitary, intracranial or intravesical administration, application or lavage. Topical administration may be preferred in cases where low dose administration, prevention of systemic side effects, and more precise control of dose intervals and concentrations of active drugs at the delivery site are required. Local administration ensures the creation of a given concentration at the target site, regardless of interindividual fluctuations in metabolism, blood flow, and so on. The direct delivery method also allows for better dosing control.

Topical delivery of a MASP-2 inhibitory agent may be performed during surgical treatments for a disease or condition, such as during procedures such as arterial bypass, atherectomy, laser procedures, ultrasound procedures, balloon angioplasty, and stent placement. For example, a MASP-2 inhibitor may be administered to a subject concurrently with balloon angioplasty. Balloon angioplasty

- 64 042534 involves inserting a catheter with a deflated balloon into an artery. The deflated balloon is placed in close proximity to the atherosclerotic plaque and inflated to press the plaque against the vessel wall. As a result, the surface of the balloon comes into contact with the layer of vascular endothelial cells on the surface of the blood vessel. The MASP-2 inhibitory agent can be attached to the balloon angioplasty catheter in a manner that releases the drug at the site of the atherosclerotic plaque. The drug may be attached to the balloon catheter by well known standard techniques. For example, the agent may be stored within the interior of a balloon catheter until the balloon is inflated, whereby the agent is released into the local environment. Alternatively, the surface of the balloon may be impregnated with drug, whereby the drug contacts the cells of the arterial wall when the balloon is inflated. The agent may also be delivered in a perforated balloon catheter such as that described in Flugelman, M.Y., et al., Circulation 85:1110 1117, 1992. See also PCT Application WO 95/23161 for an example of therapeutic protein attachment. to the balloon angioplasty catheter. Similarly, the MASP-2 inhibitory agent may be incorporated into the gel or polymer coating applied to the stent, or may be incorporated into the stent material, whereby the stent releases the MASP-2 inhibitory agent upon placement in the vessels.

MASP-2 inhibitory compositions used in the treatment of arthritis and other musculoskeletal disorders can be topically delivered by intra-articular injection. Conveniently, such compositions may include a sustained release delivery vehicle. As a further example of cases in which topical delivery may be required are cases where MASP-2 inhibitory compositions used in the treatment of urogenital conditions may be appropriately instilled into the bladder or into another urogenital structure.

Coating of a medical device.

MASP-2 inhibitory agents, such as antibodies and inhibitory peptides, may be immobilized on (or within) the surface of an implantable or attachable medical device. The modified surface is usually in contact with living tissue after implantation in the animal body. By implantable or attachable medical device is meant any device that is implanted within or attached to tissue in an animal body during normal operation of the device (eg, stents and implantable drug delivery devices). Such implantable or attachable medical devices can be made from, for example, nitrocellulose, diazocellulose, glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, dextran, sepharose, agar, starch, nylon, stainless steel, titanium, and biodegradable and/or biocompatible polymers. The association of the protein with the device may be by any method that does not interfere with the biological activity of the associated protein, such as by attaching one or both of the N-terminal and C-terminal residues of the protein to the device. Attachment may also be made at one or more internal sites in the protein. Multiple attachments (both internal and at the ends of the protein) can also be used. The surface of an implantable or attached medical device may be modified to introduce functional groups (e.g., carboxyl, amide, amine, ether, hydroxyl, cyanide, nitride, sulfanilamide, acetyline, epoxy, silane, anhydride, succinimide, azide) to immobilize the protein on it. . Methods of chemical interaction include, but are not limited to, the formation of derivatives of esters, ethers, amides, azide and sulfanilamide derivatives, cyanate and other bonds with functional groups available on MASP-2 antibodies or inhibitory peptides. MASP-2 antibodies or inhibitory fragments can also be non-covalently attached by adding an affinity tag sequence to the protein, such as GST (D.B. Smith and K.S. Johnson, Gene 67:31, 1988), polyhistidines (E. Hochuli et al., J. Chromatog 411:77, 1987), or biotin. Such affinity tags can be used to reversibly attach a protein to a device.

Proteins can also be covalently attached to the surface of the device body, for example by covalently activating the surface of the medical device. As a typical example, the matrix cell protein(s) can be attached to the device body using any of the following pairs of reactive groups (wherein one member of the pair is present on the surface of the device body and the second member of the pair is present on the matrix cell protein (proteins)): hydroxyl group/carboxylic acid group to form an ester bond; hydroxyl group/anhydride group to form an ester bond; hydroxyl group/isocyanate group to form a urethane bond. The surface of the device body, which does not have suitable reactive groups, can be treated by RFGD plasma etching to form reactive groups in order to allow the deposition of matrix cell protein(s) (e.g., treatment with oxygen plasma to introduce oxygen-containing groups; processing with propylamino

- 65 042534 howling plasma for the introduction of amino groups).

MASP-2 inhibitory agents, including nucleic acid molecules, such as antisense RNAi or DNA-encoding peptide inhibitors, can be enclosed in porous matrices attached to the body of the device. Typical porous matrices used to create the surface layer are matrices prepared from tendon or skin collagen, manufactured and supplied by a number of companies (for example, Sigma and Collagen Corporation), or collagen matrices prepared as described in U.S. patent documents. Patent Nos. 4394370, to Jefferies, and 4975527, to Koezuka. One collagen material is called UltraFiber™ and is manufactured by Norian Corp. (Mountain View, California).

If desired, specific polymeric matrices may also be used, which include acrylic ester polymers and lactic acid polymers, as described, for example, in U.S. Patent Nos. 4526909 and 4563489, to Urist. Specific examples of suitable polymers are polymers of orthoesters, anhydrides, propylene-fumarate copolymers, or a polymer of one or more α-hydroxycarboxylic acid monomers (eg, α-hydroxyacetic acid (glycolic acid) and/or α-hydroxypropionic acid (lactic acid)).

treatment regimens.

In prophylactic use, the pharmaceutical compositions are administered to a subject at risk for a condition associated with MASP-2 dependent complement activation, or at risk for a condition associated with MASP-2 dependent complement activation, in an amount sufficient to eliminate or reduce the risk of developing symptoms of the condition. In therapeutic use, the pharmaceutical compositions are administered to a subject who is suspected of having a condition associated with MASP-2 dependent complement activation, or who already suffers from a condition associated with MASP-2 dependent complement activation, in a therapeutically effective amount, sufficient to alleviate or at least partially alleviate the symptoms of the condition. For both prophylactic and therapeutic regimens, compositions comprising MASP-2 inhibitory agents may be administered in multiple doses until an appropriate therapeutic result is achieved in the subject. The use of the MASP-2 inhibitory compositions of the present invention can be carried out by a single administration of the composition or a limited number of consecutive administrations for the treatment of an acute condition, for example, reperfusion injury or other traumatic injury. Alternatively, the composition may be administered at specific times over an extended period to treat chronic conditions such as arthritis or psoriasis.

The methods and compositions of the present invention can be used to inhibit inflammation and related processes that typically result from diagnostic and therapeutic medical and surgical procedures. To inhibit such processes, the MASP-2 inhibitory composition of the present invention can be applied periprocedurally. As used herein, the term periprocedural refers to the administration of an inhibitory composition before and/or during and/or after a procedure, i.e. before a procedure, before and during a procedure, before and after a procedure, before, during and after a procedure, or after procedures. Periprocedural administration may be by topical administration of the composition to the surgical or procedure site, for example by injection or by continuous or intermittent lavage of the site, or by systemic administration. Suitable methods for local perioperative delivery of MASP-2 inhibitory agent solutions are described in US Patent Nos. 6420432 to Demopulos and 6645168 to Demopulos. Suitable methods for local delivery of chondroprotective compositions comprising MASP-2 inhibitory agent(s) are described in International PCT Patent Application WO 01/07067 A2. Suitable methods and compositions for targeted systemic delivery of chondroprotective compositions comprising MASP-2 inhibitory agent(s) are described in International PCT Patent Application WO 03/063799 A2.

In one aspect of the invention, the pharmaceutical compositions are administered to a subject suffering from thrombotic microangiopathy (TMA) or at risk of developing thrombotic microangiopathy (TMA). In one embodiment, the TMA is selected from the group consisting of hemolytic uremic syndrome (HUS), thrombotic thrombocytopenic purpura (TTP), and atypical hemolytic uremic syndrome (aHUS). In one embodiment, the TMA is aHUS. In one embodiment, the composition is administered to a patient with aHUS during the acute phase of the disease. In one embodiment, the composition is administered to a patient with aHUS during a phase of remission (i.e., in a subject who has recovered or partially recovered from an episode of the acute phase of aHUS, such remission is confirmed, for example, by an increase in platelet count and/or a decrease in serum LDH concentrations, for example, as described in Loirat C et al., Orphanet Journal of Rare Diseases 6:60, 2011, the contents of which are incorporated herein by reference). In one embodiment, the subject suffers from TMA or is susceptible to

- 66 042534 the risk of developing TMA, which is (i) TMA on the background of cancer; (ii) TMA during chemotherapy; or (iii) TMA in the setting of transplantation (eg, organ transplantation such as kidney transplantation or allogeneic hematopoietic stem cell transplantation). In one embodiment, the subject suffers from Upshaw-Schulman syndrome (USS) or is at risk for developing Upshaw-Schulman syndrome (USS). In one embodiment, the subject suffers from Degos disease or is at risk of developing Degos disease. In one embodiment, the subject suffers from catastrophic antiphospholipid syndrome (CAPS) or is at risk of developing catastrophic antiphospholipid syndrome (CAPS). In therapeutic use, the pharmaceutical compositions are administered to a subject suffering from or at risk of developing TMA in a therapeutically effective amount sufficient to inhibit thrombus formation, alleviate, or at least partially alleviate the symptoms of the condition.

In both prophylactic and therapeutic regimens, compositions comprising MASP-2 inhibitory agents may be administered in multiple doses until an appropriate therapeutic result is achieved in the subject. In one embodiment of the invention, the MASP-2 inhibitory agent comprises an anti-MASP-2 antibody that can be administered in an appropriate manner to an adult patient (e.g., an adult patient with an average body weight of 70 kg) at a dose of 0.1 mg to 10,000 mg, more conveniently. , 1.0 mg to 5000 mg, more conveniently, 10.0 mg to 2000 mg, more conveniently, 10.0 mg to 1000 mg, and more conveniently, 50.0 mg to 500 mg. In the case of pediatric patients, the dose may be adjusted in proportion to the patient's body weight. The use of the MASP-2 inhibitory compositions of the present invention may be by single administration of the composition or a limited number of consecutive administrations for the treatment of TMA. Alternatively, the composition can be administered at specific times, once a day, twice a week, once a week, once every two weeks, once a month, twice a month for an extended period of time to treat TMA.

In some embodiments, the subject suffering from or at risk of developing TMA has previously been or is currently being treated with an inhibitor of terminal complement components that inhibits the cleavage of complement C5 protein. In some embodiments, the method includes administering to the subject a composition of the invention comprising a MASP-2 inhibitor and further administering to the subject an inhibitor of terminal complement components that inhibits the cleavage of complement C5 protein. In some embodiments, the terminal complement inhibitor is a humanized anti-C5 antibody or an antigen-binding fragment thereof. In some embodiments, the terminal complement inhibitor is eculizumab.

In one aspect of the invention, the pharmaceutical compositions are administered to a subject susceptible to aHUS or at risk of developing aHUS in an amount sufficient to eliminate or reduce the risk of developing symptoms of the condition. In therapeutic use, the pharmaceutical compositions are administered to a subject suspected of having aHUS or already suffering from aHUS in a therapeutically effective amount sufficient to alleviate or at least partially alleviate the symptoms of the condition. In one aspect of the invention, prior to administration, the subject may be screened to determine if they are exhibiting one or more symptoms of aHUS, including (i) anemia, (ii) thrombocytopenia (iii) renal failure, and (iv) creatinine elevation, and then the composition is administered. of the present invention in an effective amount and for a sufficient period of time to relieve these symptoms.

In another aspect of the invention, the MASP-2 inhibitory compositions of the present invention may be used to prophylactically treat a subject at increased risk of developing aHUS and thereby reduce the likelihood that the subject may develop aHUS. First, the presence in a subject of a genetic marker known to be associated with aHUS is determined by performing a genetic screening test on a sample taken from the patient and identifying the presence of at least one genetic marker associated with aHUS, complement factor H ( CFH), Factor I (CFI), Factor B (CFB), Membrane cofactor protein CD46, C3, Complement-like factor H protein (CFHR1), Thrombodulin anticoagulant protein (THBD), Complement-like factor H protein 3 (CFHR3 ) or complement-like factor H protein 4 (CFHR4). The subject is then periodically monitored (e.g., once a month, once every three months, twice a year, or once a year) to determine the presence or absence of at least one symptom of aHUS, such as anemia, thrombocytopenia, renal failure, and an increase in creatinine. After determining the presence of at least one of these symptoms, the subject may be administered an amount of a MASP-2 inhibitory agent effective to inhibit MASP-2 dependent complement activation in an effective amount and for a sufficient period of time to improve one or more of these symptoms. In yet another aspect of the present invention, a subject at increased risk of developing aHUS due to the presence of one of the genetic markers associated with aHUS during the screening process can be monitored for the occurrence of an event associated with aHUS.

- 67 042534 initiation of clinical symptoms of aHUS, including prolonged drug exposure, infection (eg, bacterial infection), malignancy, injury, organ or tissue transplantation, and pregnancy.

In yet another aspect of the present invention, a composition comprising an amount of a MASP-2 inhibitory agent effective to inhibit MASP-2 dependent complement activation may be administered to a subject suffering from atypical hemolytic uremic syndrome (aHUS) or at risk of developing atypical hemolytic uremic syndrome (aHUS ) due to infection. For example, a patient suffering from non-intestinal aHUS or at risk of developing non-intestinal aHUS associated with pneumococcal infection may be treated with the compositions of the present invention.

In yet another aspect of the present invention, a subject suffering from aHUS may first be treated with a MASP-2 inhibitory composition of the present invention administered through a catheter line, such as an intravenous catheter line or a subcutaneous catheter line, for a first period of time, for example, within 1, 12 hours, 1 day, 2 or 3 days. The subject may then be treated for a second period of time with the MASP-2 inhibitory composition administered by regular subcutaneous injections, e.g., injections once a day, twice a week, once a week, once every two weeks, once once a month or twice a month.

In yet another aspect of the present invention, the MASP-2 inhibitory composition of the present invention may be administered to a subject suffering from aHUS without the use of a plasmapheresis method (i.e., a subject whose aHUS symptoms have not been treated with plasmapheresis and are not being treated with plasmapheresis in period of treatment with a MASP-2 inhibitory composition), in order to prevent potential complications arising from plasmapheresis, including hemorrhage, infection, and susceptibility to disorders and/or allergies present in a plasma donor, or in a subject who rejects the use of plasmapheresis, or in conditions when the use of plasmapheresis is not available.

In yet another aspect of the present invention, the MASP-2 inhibitory composition of the present invention may be administered to a subject suffering from aHUS at the same time that the patient is being treated with plasmapheresis. For example, a subject being treated with plasmapheresis may then be administered a MASP-2 inhibitory composition after plasma replacement or in alternation with plasma replacement.

In another aspect of the present invention, a subject suffering from aHUS or at risk of developing aHUS and currently being treated with a MASP-2 inhibitory composition of the present invention may be monitored by periodic determination, for example, every 12 hours or once every day, the level of at least one complement factor, where the determination of a reduced level of at least one complement factor compared to the standard value with a healthy subject indicates the need to continue treatment with the composition.

In both prophylactic and therapeutic regimens, compositions comprising MASP-2 inhibitory agents may be administered in multiple doses until an appropriate therapeutic result is achieved in the subject. In one embodiment of the invention, the MASP-2 inhibitory agent comprises an anti-MASP-2 antibody that can be administered in an appropriate manner to an adult patient (e.g., an adult patient with an average body weight of 70 kg) at a dose of 0.1 mg to 10,000 mg, more conveniently. , 1.0 mg to 5000 mg, more conveniently, 10.0 mg to 2000 mg, more conveniently, 10.0 mg to 1000 mg, and more conveniently, 50.0 mg to 500 mg. In the case of pediatric patients, the dose may be adjusted in proportion to the patient's body weight. The use of the MASP-2 inhibitory compositions of the present invention may be by single administration of the composition or by a limited number of consecutive administrations for the treatment of aHUS. Alternatively, the composition may be administered at specific time periods, such as once a day, once every two weeks, once a week, once every two weeks, once a month, or twice a month, for an extended time period for aHUS treatment.

In some embodiments, the subject suffering from aHUS has been or is currently being treated with an inhibitor of terminal complement components that inhibits the cleavage of complement C5 protein. In some embodiments, the method includes administering to the subject a composition of the invention comprising a MASP-2 inhibitor, and further administering to the subject an inhibitor of terminal complement components that inhibits cleavage of complement C5 protein. In some embodiments, the terminal complement inhibitor is a humanized anti-C5 antibody or an antigen-binding fragment thereof. In some embodiments, the terminal complement inhibitor is eculizumab.

In one aspect of the invention, the pharmaceutical compositions are administered to a subject susceptible to or at risk of developing HUS in an amount sufficient to eliminate or reduce the risk of developing symptoms of the condition. In therapeutic use, the pharmaceutical compositions are administered to a subject suspected of having HUS or already suffering from HUS in a therapeutically effective amount sufficient to alleviate, or at least partially alleviate, the symptoms of the condition.

In another aspect of the present invention, a subject at risk of developing HUS may be less likely to develop renal dysfunction by administering to the subject a MASP2 inhibitory composition of the present invention in an amount effective to inhibit MASP-2 dependent complement activation. For example, a subject at risk of developing HUS and being treated with a MASP-2 inhibitory composition of the present invention may exhibit one or more of the symptoms associated with HUS, including diarrhea, a hematocrit level below 30% with intravascular destruction of red blood cells detected on a smear, thrombocytopenia and an increase in creatinine levels. As a further example, a subject at risk of developing HUS and being treated with a MASP-2 inhibitory composition of the present invention may be infected with E. coli, Shigella, or Salmonella. Such subjects infected with E. coli, Shigella or Salmonella may be treated with a MASP-2 inhibitory composition of the present invention concurrently with antibiotic treatment, or alternatively may be treated with a MASP-2 inhibitory composition without concomitant treatment. with an antibiotic, in particular in the case of enterogenic Escherichia coli, for which antibiotic treatment is contraindicated. A subject infected with enterogenic Escherichia coli who has been treated with an antibiotic may be at an increased risk of developing HUS, and may be appropriately treated with the MASP-2 inhibitory composition of the present invention to reduce the risk. A subject infected with enterogenic Escherichia coli may be treated for a first period of time with the MASP-2 inhibitory composition of the present invention in the absence of an antibiotic and then, for a second period of time, be treated with both the MASP-2 inhibitory composition of the present invention. of the present invention, and with the help of an antibiotic.

In yet another aspect of the present invention, a subject suffering from HUS may be first treated with a MASP-2 inhibitory composition of the present invention administered through a catheter line, such as an intravenous catheter line or a subcutaneous catheter line, for a first period of time, for example, within 1, 12 hours, 1, 2 or 3 days. The subject may then be treated for a second period of time with the MASP-2 inhibitory composition administered by regular subcutaneous injections, e.g., injections once a day, once every two weeks, once a week, once every two weeks, once a month or twice a month.

In yet another aspect of the present invention, the MASP-2 inhibitory composition of the present invention may be administered to a subject suffering from HUS without the use of a plasmapheresis method (i.e., a subject whose symptoms of HUS have not been treated with plasmapheresis and who is not treated with plasmapheresis during treatment with a MASP-2 inhibitory composition), to prevent the occurrence of potential complications of plasmapheresis, including hemorrhage, infection, and susceptibility to disorders and/or allergies inherent in the plasma donor, or when the subject has contraindications to the use of plasmapheresis, or in conditions where plasmapheresis is not available.

In yet another aspect of the present invention, the MASP-2 inhibitory composition of the present invention may be administered to a subject suffering from HUS concurrently with the patient being treated with plasmapheresis. For example, a subject being treated with plasmapheresis may then be administered a MASP-2 inhibitory composition after plasma replacement or alternating with plasma replacement.

In yet another aspect of the present invention, a subject suffering from or at risk of developing HUS and being treated with a MASP-2 inhibitory composition of the present invention may be monitored by periodically determining, for example, every 12 hours or once a day, the level of at least one complement factor, where the determination of a reduced level of at least one complement factor in comparison with the standard value or with a healthy subject indicates the need to continue treatment with the composition.

In both prophylactic and therapeutic regimens, compositions comprising MASP-2 inhibitory agents may be administered in multiple doses until an appropriate therapeutic result is achieved in the subject. In one embodiment of the invention, the MASP-2 inhibitory agent comprises an anti-MASP-2 antibody that can be administered in an appropriate manner to an adult patient (e.g., an adult patient with an average body weight of 70 kg) at a dose of 0.1 mg to 10,000 mg, more conveniently. , 1.0 mg to 5000 mg, more conveniently, 10.0 mg to 2000 mg, more conveniently, 10.0 mg to 1000 mg, and more conveniently, 50.0 mg to 500 mg. In the case of pediatric patients, the dose may be adjusted in proportion to the patient's body weight. The use of the MASP-2 inhibitory compositions of the present invention may be by single administration of the composition or by a limited number of consecutive administrations for the treatment of HUS. Alternatively, the composition may be administered at specific time periods, such as once a day, once every two weeks, once a week, once every two weeks, once a month, or twice a month, for an extended time period for HUS treatment.

In some embodiments, the subject suffering from HUS has been or is currently being treated with an inhibitor of terminal complement components that inhibits the cleavage of complement C5 protein. In some embodiments, the method includes administering to the subject a composition of the invention comprising a MASP-2 inhibitor, and further administering to the subject an inhibitor of terminal complement components that inhibits cleavage of complement C5 protein. In some embodiments, the terminal complement inhibitor is a humanized anti-C5 antibody or an antigen-binding fragment thereof. In some embodiments, the terminal complement inhibitor is eculizumab.

In one aspect of the invention, the pharmaceutical compositions are administered to a subject susceptible to or at risk of developing TTP in an amount sufficient to eliminate or reduce the risk of developing symptoms of the condition. In therapeutic use, the pharmaceutical compositions are administered to a subject suspected of having TTP or already suffering from TTP in a therapeutically effective amount sufficient to alleviate, or at least partially alleviate, the symptoms of the condition.

In another aspect of the present invention, a subject exhibiting one or more symptoms of TTP, including central nervous system involvement, thrombocytopenia, severe heart disease, severe lung disease, gastrointestinal infarction, and gangrene, may be treated with MASP. -2 inhibitory composition of the present invention. In another aspect of the present invention, a subject found to be downregulated in ADAMTS13 levels and also tested positive for the presence of an ADAMTS13 inhibitor (i.e., antibody) may be treated with a MASP-2 inhibitory composition of the present invention. In another aspect of the present invention, a subject who tests positive for the presence of an ADAMTS13 inhibitor may be treated with an immunosuppressant (e.g., corticosteroids, rituxan, or cyclosporine) concomitantly with treatment with a MASP-2 inhibitory composition of the present invention. In yet another aspect of the present invention, a subject found to have decreased levels of ADAMTS13 and tested positive for the presence of an ADAMTS13 inhibitor may be treated with ADAMTS13 concurrently with treatment with a MASP-2 inhibitory composition of the present invention.

In yet another aspect of the present invention, a subject suffering from TTP may first be treated with a MASP-2 inhibitory composition of the present invention administered through a catheter line, such as an intravenous catheter line or a subcutaneous catheter line, for a first period of time, for example, within 1, 12 hours, 1, 2 or 3 days. The subject may then be treated for a second period of time with the MASP-2 inhibitory composition administered by regular subcutaneous injections, e.g., injections once a day, once every two weeks, once a week, once every two weeks, once a month or twice a month.

The MASP-2 inhibitory composition of the present invention can be administered to a subject suffering from TTP without using a plasmapheresis method (i.e., a subject whose symptoms of TTP have not been treated with plasmapheresis and who is not treated with plasmapheresis during the period of treatment with MASP -2 inhibitory composition), to prevent the occurrence of potential complications of plasmapheresis, including hemorrhage, infection and exposure to disorders and/or allergies inherent in the plasma donor, or when the subject has contraindications to the use of plasmapheresis, or in conditions where plasmapheresis is not available.

In yet another aspect of the present invention, the MASP-2 inhibitory composition of the present invention may be administered to a subject suffering from TTP while the patient is being treated with plasmapheresis. For example, a subject being treated with plasmapheresis may then be administered a MASP-2 inhibitory composition after plasma replacement or alternating with plasma replacement.

In yet another aspect of the present invention, a subject suffering from refractory TTR, i.e. symptoms of TTR that do not respond adequately to another treatment, such as plasmapheresis, may be treated with a MASP-2 inhibitory composition of the present invention, with or without the use of additional plasmapheresis.

In yet another aspect of the present invention, a subject suffering from TTP or exposed to rice

- 70 042534 the development of TTP, and being treated with a MASP-2 inhibitory composition of the present invention, can be monitored by periodically determining, for example, every 12 hours or once a day, the level of at least one complement factor, where the determination a reduced level of at least one complement factor compared to the standard value or with a healthy subject indicates the need to continue treatment with the composition.

In both prophylactic and therapeutic regimens, compositions comprising MASP-2 inhibitory agents may be administered in multiple doses until an appropriate therapeutic result is achieved in the subject. In one embodiment of the invention, the MASP-2 inhibitory agent comprises an anti-MASP-2 antibody that can be administered in an appropriate manner to an adult patient (e.g., an adult patient with an average body weight of 70 kg) at a dose of 0.1 mg to 10,000 mg, more conveniently. , 1.0 mg to 5000 mg, more conveniently, 10.0 mg to 2000 mg, more conveniently, 10.0 mg to 1000 mg, and more conveniently, 50.0 mg to 500 mg. In the case of pediatric patients, the dose may be adjusted in proportion to the patient's body weight. The use of the MASP-2 inhibitory compositions of the present invention may be by single administration of the composition or by a limited number of consecutive administrations for the treatment of TTP. Alternatively, the composition may be administered at specific time periods, such as once a day, once every two weeks, once a week, once every two weeks, once a month, or twice a month, for an extended time period for TTR treatment.

In some embodiments, the subject suffering from TTP has been or is currently being treated with an inhibitor of terminal complement components that inhibits the cleavage of complement C5 protein. In some embodiments, the method includes administering to the subject a composition of the invention comprising a MASP-2 inhibitor, and further administering to the subject an inhibitor of terminal complement components that inhibits cleavage of complement C5 protein. In some embodiments, the terminal complement inhibitor is a humanized anti-C5 antibody, or an antigen-binding fragment thereof. In some embodiments, the terminal complement inhibitor is a humanized anti-C5 antibody or an antigen-binding fragment thereof. In some embodiments, the terminal complement inhibitor is eculizumab.

VI. Examples.

The following examples are only illustrative of the presently proposed best practice of the invention, but should not be construed as limitations on the invention in any way. The content of all cited literary sources is given in the invention by reference to these sources.

Example 1

This example describes the generation of a strain of MASP-2 deficient mice (MASP-2-/-) but sufficient for MAP19 (MAp19+/+).

Materials and methods. The NTKV 1901 pKO targeting vector was constructed to destroy three exons encoding the C-terminal end of mouse MASP-2, including the exon that encodes the serine protease domain shown in FIG. 3. RKO NTKV 1901 was used to transfect mouse embryonic stem (ES) cells of the E14.1a (SV129 Ola) line. Neomycin resistant and thymidine kinase sensitive clones were selected. 600 ES clones were screened, and among them, four different clones were identified and verified by Southern blotting, containing the desired selective targeting and recombination phenomenon, as shown in FIG. 3. From these four positive clones, chimeras were generated by embryo transfer. The chimeras were then backcrossed in the C57/BL6 genetic environment to create transgenic males. The transgenic males were then bred to females to generate F1s with 50% of the offspring showing heterozygosity for the disrupted MASP-2 gene. Heterozygous mice were intercrossed to generate homozygous MASP-2 deficient progeny, resulting in heterozygous and wild-type mice, at a ratio of 1:2:1, respectively.

Results and phenotype. The resulting homozygous MASP-2-/- deficient mice were found to be viable and fertile and were tested for MASP-2 deficiency by Southern blot to confirm the correct targeting event, Northern blot to confirm the absence of MASP-2 mRNA, and Western blotting to confirm absence of MASP-2 protein (data not shown). Then, the presence of MAP19 mRNA and the absence of MASP-2 mRNA were confirmed by real-time reverse transcription polymerase chain reaction (RT PCR) using a LightCycler instrument. As expected, MASP-2 -/- mice did continue to express MAP19, MASP 1 and MASP-3 mRNA and protein (data not shown). The presence and excess of mRNA for properdin, factor B, factor D, C4, C2, and C3 in MASP-2 −/− mice was assessed by the LightCycler assay and found to be identical to those obtained from control littermate wildcats. mice (data not shown). The plasma of homozygous MASP-2-/- mice is completely deficient in lectin-mediated complement activation, as described further in Example 2.

Generation of MASP-2-/- strain in a pure C57/BL6 genetic environment. MASP-2-/- mice were backcrossed to pure C57BL6 for nine generations before using the MASP-2-/- strain as an animal model.

A transgenic mouse strain that is mouse MASP-2-/-, MAP19+/+ and which expresses the human MASP-2 transgene (mouse MASP-2 knockout and human MASP2 knockout) was also generated as follows.

Materials and methods.

A minigene encoding human MASP-2, called hMASP-2 mini (SEQ ID NO: 49), shown in FIG. 4, which includes the promoter region of the human MASP-2 gene, including the first 3 exons (exon 1 to exon 3), followed by a cDNA sequence that represents the coding sequence of the next 8 exons, whereby a full-length MASP-2 protein is encoded under the influence of its endogenous promoter. The mini hMASP-2 construct was injected into fertilized MASP-2 -/- mouse eggs in order to replace the deficient mouse MASP-2 gene with transgenic human MASP-2.

Example 2

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

Methods and materials.

Study of specific C4 cleavage in the lectin pathway of complement activation. A C4 cleavage study has been described in Petersen, et al., J. Immunol. Methods 257:107 (2001), which measured lectin pathway activation mediated by Staphylococcus aureus lipoteichoic acid (LTA), which binds L-ficolin. The study described in Petersen et al., (2001) was adapted to measure lectin pathway activation by MBL by coating the plate with LPS and mannan or zymosan prior to addition of serum from MASP-2 -/- mice, as described below. The study was also modified to exclude the possibility of C4 cleavage along the classical pathway. This was achieved by using a sample dilution buffer containing 1 M NaCl, which allows high affinity binding of lectin pathway recognition components to their ligands, but prevents endogenous C4 activation, thereby precluding the involvement of the classical pathway by dissociation of the C1 complex. Briefly, in a modified assay, serum samples (diluted in high salt buffer (1 M NaCl)) are added to ligand coated plates, then a constant amount of purified C4 in physiological salt buffer is added. Bound recognition complexes containing MASP-2 cleave C4, resulting in the deposition of C4b.

Research methods.

1) Nunc Maxisorb microtiter plates (Maxisorb, Nunc, Cat # 442404, Fisher Scientific) were coated with 1 μg/mL mannan (M7504 Sigma) or any other ligand (such as the ligands listed below) diluted in coating buffer (15 mM Na ₂ CO ₃ , 35 mM NaHCO ₃ , pH 9.6).

The following reagents were used in the study:

a. mannan (1 μg per well of mannan (M7 504 Sigma) in 100 μl coating buffer);

b. zymosan (1 μg per well of zymosan (Sigma) in 100 μl coating buffer);

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

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

e. Aerococcus viridans PSA (2 µg per well in 100 µl coating buffer);

f. 100 µl per well of formalin-fixed Staphylococcus aureus DSM20233 (OD550=0.5) in coating buffer;

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

3) After overnight incubation, saturate residual protein binding sites by incubating plates with 0.1% HSA-TBS blocking buffer (0.1% (w/v) HSA in 10 mM Tris CL, 140 mM NaCl, 1, 5 mM NaN ₃ , pH 7.4) for 1-3 h, then washing the plates 3 times with TBS/tween/Ca ²⁺ (TBS with 0.05% Tween 20 and 5 mM CaCl ₂ , 1 mM MgCl ₂ , pH 7.4).

4) Serum samples to be tested were diluted in MBL binding buffer (1 M NaCl) and the diluted samples were added to the plates and incubated overnight at 4°C. Wells to which only buffer was added were used as negative controls.

5) After overnight incubation at 4°C, the plates were washed three times with TBS/tween/Ca ²⁺ . Then human C4 (100 µl/well 1 µg/ml diluted in BBS (4 mM barbital, 145 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4)) was added to the plates and incubated for 90 minutes at 37 °C. The plates were washed again 3 times with TBS/tween/Ca ²⁺ .

6) C4b deposition was detected using alkaline phosphatase-conjugated chicken

- 72 042534 anti-human c4c antibody (diluted 1:1000 in TBS/tween/Ca ²⁺ ), which was added to the plates and incubated for 90 minutes at room temperature. The plates were then washed again three times with TBS/tween/Ca ²⁺ .

7) Alkaline phosphatase was determined by adding 100 µl of pnitrophenyl phosphate substrate solution, incubating at room temperature for 20 minutes and reading OD405 on a spectrophotometer to read microtiter plates.

Results. In FIG. 5A-B show the amount of C4b deposition on mannan (FIG. 5A) and zymosan (FIG. 5B) in serum dilutions MASP-2+/+ (crosses), MASP-2+/- (solid circles) and MASP-2- /- (shaded triangles) mice. In FIG. 5C shows the relative activity of C4 convertase on zymosan (white bars) or mannan (solid bars) coated plates of MASP-2-/+ mice (n=5) and MASP-2-/- mice (n=4) relative to wild mice ( n=5) obtained by measuring the amount of C4b deposition normalized to the sera of wild type mice. Error bars represent the standard deviation. As shown in FIG. 5AC, MASP-2 −/− mouse plasma is absolutely deficient in lectin pathway-mediated complement activation on zymosan mannan-coated plates. These results clearly show that MASP-2 is an effector component of the lectin pathway.

Recombinant MASP-2 restores lectin pathway-dependent C4 activation in the serum of MASP-2 -/- mice.

In order to establish that the absence of MASP-2 was the direct cause of the reduction in lectin pathway dependent C4 activation in MASP-2 −/− mice, the effect of adding recombinant MASP-2 protein to serum samples in the C4 cleavage study described above was examined. Functional recombinant mouse MASP-2 and catalytically inactive mouse MASP-2A proteins (in which the active site of a serine residue in the serine protease domain was replaced by an alanine residue) were produced and purified as described in Example 3. Mixed serum of four MASP-2 -/- mice incubated with increasing concentrations of recombinant mouse MASP-2 protein or inactive mouse MASP-2A, and C4 convertase activity was determined as described above.

Results. As shown in FIG. 6, addition of functionally active recombinant mouse MASP-2 protein (shown by open triangles) to sera of MASP-2-/- mice restored lectin-pathway dependent C4 activation in a protein concentration-dependent manner, while catalytically inactive mouse MASP-2A protein ( shown by asterisks) did not restore C4 activation. The results shown in FIG. b, normalized to C4 activation observed in wild-type mice mixed sera (shown by dotted line).

Example 3

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

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

The full-length human MASP-2 cDNA sequence (SEQ ID NO: 4) was also subcloned into the mammalian expression vector pCI-Neo (Promega), which drives eukaryotic expression under the control of the CMV enhancer/promoter region (as described in Kaufman RJ. et al., Nucleic Acids Research 19:4485-90, 1991; Kaufman, Methods in Enzymology, 185:531-66 (1991)). Full-length mouse cDNA (SEQ ID NO: 50) and rat MASP-2 cDNA (SEQ ID NO: 53) were each subcloned into the pED expression vector. MASP-2 expression vectors were then transfected into the Chinese Hamster Ovary DXB1 adherent cell line using the standard calcium phosphate transfection method described in Maniatis et al. proteases.

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

Expression of full length catalytically inactive MASP-2.

Relevance. MASP-2 is activated by autocatalytic cleavage after MBL recognition subcomponents or ficolins (either L-ficolin, H-ficolin, or M-ficolin) bind to their respective carbohydrate pattern. Autocatalytic cleavage caused by MASP-2 activation often occurs during the procedure for isolating MASP-2 from serum or during purification after recombinant expression. In order to obtain a more stable preparation of the protein used as an antigen, a catalytically inactive form of MASP-2, designed as MASP-2A, was created by replacing the serine residue that is present in the catalytic triad of the protease domain with an alanine residue in rats (SEQ ID NO: 55 Ser617 on A1a617), in mice (SEQ ID NO: 52 Ser617 on A1a617) or in humans (SEQ ID NO: 3 Ser618 on A1a618).

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To generate catalytically inactive human and mouse MASP-2A proteins, site-directed mutagenesis was performed using the oligonucleotides shown in Table 1. 5. Oligonucleotides in table. 5 were designed to be coupled to a region of human and mouse cDNA encoding an enzymatically active serine, and the oligonucleotides contained a mismatch to convert the serine codon to an alanine codon. For example, the polymerase chain reaction (PCR) of oligonucleotides SEQ ID NOS:56-59 was used in combination with human MASP-2 cDNA (SEQ ID NO:4) to amplify the region from start codon to enzymatically active serine and from serine to stop codon with the purpose of generating a full open read from a mutated MASP-2A containing the Ser618 mutation in A1a618. After agarose gel electrophoresis, the PCR products were purified and a chromosome disk preparation and single adenosine overlaps were generated using a standard extension method. The adenosine-terminated MASP-2A was then cloned into a simple pGEM-T vector transformed into E. coli.

Catalytically inactive rat MASP-2A protein was generated by kinasing and renaturating SEQ ID NO: 64 and SEQ ID NO: 65, combining the two oligonucleotides in equal molar amounts, heating at 100°C for 2 minutes and slowly cooling to room temperature . The resulting annealed fragment contained Pst1 and Xba1 compatible ends and was inserted in place of the Pst1-Xba1 wild rat MASP-2 cDNA fragment (SEQ ID NO: 53) to obtain rat MASP-2A.

'GAGGTGACGCAGGAGGGGCATTAGTGTTT 3' (SEQ ID NO: 64).

5'CTAGAAACACTAATGCCCCTCCTGCGTCACCTCTGCA 3' (SEQ ID NO: 65).

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

In another approach, a catalytically inactive form of MASP-2 was constructed using the method described in Chen et al., J. Biol. Chem., 276(28);25894-25902, 2001. Briefly, a plasmid containing the full-strand human MASP-2 cDNA (described in Thiel et al. Nature 386:506, 1997) was cut with Xho1 and EcoR1, and the MASP cDNA -2 (described in the invention as SEQ ID NO: 4) was cloned into the appropriate restriction sites of the baculovirus transfer vector pFastBad (Life Technologies, NY). The active site of the MASP-2 serine protease in Ser618 was then replaced with Ala618 by replacing the double-stranded oligonucleotides encoding the peptide region from amino acids 610 to 625 (SEQ ID NO: 13) with the natural region from amino acids 610 to 625 to create a full-length MASP polypeptide -2 with an inactive protease domain.

Construction of expression plasmids containing polypeptide regions derived from human MASP-2.

To secrete various MASP-2 domains, the following constructs were produced using the MASP-2 signal peptide (residues 1-15 of SEQ ID NO: 5). A construct expressing the CUBI domain of human MASP-2 (SEQ ID NO: 8) was obtained by PCR amplification of the region encoding residues 1-121 of MASP-2 (SEQ ID NO: 6) (corresponding to the N-terminal domain of CUBI). A construct expressing the CUBIEGF domain of human MASP-2 (SEQ ID NO: 9) was obtained by PCR amplification of the region encoding residues 1-166 of MASP-2 (SEQ ID NO: 6) (corresponding to the N-terminal domain of CUBIEGF). A construct expressing the CUBIEGFCUBII domain of human MASP-2 (SEQ ID NO: 10) was obtained by PCR amplification of the region encoding residues 1-293 of MASP-2 (SEQ ID NO: 6) (corresponding to the N-terminal domain of CUBIEGFCUBII). The above domains are amplified by PCR using VentR polymerase and pBS-MASP-2 as a template, according to conventional PCR methods. The primer sequence of the 5' sense primer (5'CGGGATCCATGAGGCTGCTGACCCTC-3' SEQ ID NO: 34) introduces a BamHI restriction site (underlined) at the 5' end of the PCR product. Antisense primers for each MASP-2 domain, shown below in table. 5 are designed to introduce a stop codon (in bold) after the EcoRI site (underlined) at the end of each PCR product. After amplification, the DNA fragments were cut with BamHI and EcoRI and cloned into the corresponding sites of the pFastBac1 vector. The resulting constructs are characterized by restriction mapping and confirmed by dsDNA sequencing.

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Table 5

MASP-2 PCR primers

MASP-2 domain 5' PCR primer 3' PCR primer SEQ ID NO:8 CUBI (aa 1-121 sequences of SEQ ID NO:6) 5'CGGGATCCATGAGGCTG CTGACCCTC3' (SEQ ID NO:34) 5'GGAATTCCTAGGCTGCA TA (SEQ ID NO:35) SEQ ID NO:9 CUBIEGF (aa 1-166 sequences of SEQ ID NO:6) 5'CGGGATCCATGAGGCTG CTGACCCTC3' (SEQ ID NO:34) 5'GGAATTCCTACAGGGCG CT3' (SEQ ID NO:36) SEQ ID NO:10 CUBIEGFCUBII (aa 1293 sequences of SEQ ID NO:6) 5'CGGGATCCATGAGGCTG CTGACCCTC3' (SEQ ID NO:34) 5'GGAATTCCTAGTAGTGG AT3' (SEQ ID NO:37) SEQ ID NO:4 human MASP-2 5'ATGAGGCTGCTGACCCT CCTGGGCCTTC3' (SEQ ID NO: 56) hMAS P-2_straight hole 5'TTAAAATCACTAATTAT GTTCTCGATC3' (SEQ ID NO: 59) LMAE-2_reverse SEQ ID NO:4 human cDNA MASP-2 5'CAGAGGTGACGCAGGAG GGGCAC3' (SEQ ID NO:58) hMAS P-2_ala_straight pit 5'GTGCCCCTCCTGCGTCA CCTTCTG3' (SEQ ID NO: 57) hMASP2 ala reverse SEQ ID NO:50 mouse MASP-2 cDNA 5'ATGAGGCTACTCATCTT CCTGG3' (SEQ ID NO: 60) mMAS P-2_straight 5'TTAGAAATTACTTATTA TGTTCTCAATCC3' (SEQ ID NO: 63) tMAP-2_reverse SEQ ID NO:50 mouse MASP-2 cDNA 5' CCCCCCCTGCGTCACCT CTGCAG3' (SEQ ID NO: 62) mMASP-2_ala_straight 5'CTGCAGAGGTGACGCAG GGGGGG3' (SEQ ID NO: 61) mMASP- 2 ala reverse

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

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

Purification of the MASP-2A protein. MASP-2A (the mutant form of Ser-Ala described above) was purified by affinity chromatography on MBP-A-agarose columns. This approach allowed fast cleaning without the use of foreign markers. MASP-2A (100-200 ml of culture medium diluted with an equal volume of loading buffer (50 mmol Tris-Cl, pH 7.5, containing 150 mmol NaCl and 25 mmol CaCl ₂ ) was loaded onto an MBP-agarose affinity chromatography column ( 4 ml) previously equilibrated with 10 ml of loading buffer After washing with an additional 10 ml of loading buffer, the protein was eluted into a 1 ml fraction with 50 mmol Tris-Cl, pH 7.5, containing 1.25 mol NaCl and 10 mmol EDTA.Fractions containing MASP-2A were identified by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.If necessary, MASP-2A was further purified by ion exchange chromatography on a MonoQ column (HR 5/5).Protein was dialyzed with 50 mmol Tris-Cl pH 7.5 containing 50 mmol NaCl and loaded onto a column equilibrated with the same buffer After washing, the bound MASP-2A was eluted with a gradient solution of 0.05-1 NaCl in 10 ml.

Results. The yield of MASP-2A protein was 0.25-0.5 mg per 200 ml of culture medium. The molecular weight determined by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) was 77.5 kDa, which is greater than the calculated value for the unmodified polypeptide (73.5 kDa) due to glycosylation. The observed increase in mass is explained by the attachment of glycans to each of the N-glycosylation sites. MASP2A migrates as a single band in a polyacrylamide gel in the presence of sodium dodecyl sulfate, indicating that it is not proteolytically degraded during biosynthesis. The weight average molecular weight determined by the equilibrium ultracentrifugation method is consistent with the calculated value for glycosylated polypeptide homodimers.

Production of recombinant human MASP-2 polypeptides.

Another method for producing polypeptides from recombinant MASP-2 and MASP2A is described in Thielens, N.M., et al., J. Immunol. 166:5068-5077, 2001. Briefly, Spodoptera frugiperda insect cells (Ready-Plaque Sf9 cells obtained from Novagen, Madison, WI) were grown and

- 75 042534 are maintained in Sf90011 serum-free culture medium (Life Technologies) supplemented with 50 IU/ml penicillin and 50 mg/ml streptomycin (Life Technologies). Insect Trichoplusia ni (High Five) cells provided by Jadwiga Chroboczek, Institut de Biologie Structurale, Grenoble, France) are maintained in TC100 culture medium (Life Technologies) containing 10% FCS (Dominique Dutscher, Brumath, France) supplemented with 50 IU/mL penicillin and 50 mg/ml streptomycin. Recombinant baculoviruses are generated using the Bac-to-Bac system (Life Technologies). Bacmid DNA was purified using the Qiagen midi preparative purification system (Qiagen) and used to transfect Sf9 insect cells using Selfectin in Sf900 II SFM culture medium (Life Technologies) as described in the manufacturer's protocol. Recombinant viral particles are harvested after 4 days, titrated with a virological plaque test and amplified as described in King and Possee, The Baculovirus Expression System: A Laboratory Guide, Chapman and Hall Ltd., London, pp.111-114, 1992 .

High Five cells (1.75x107 cells in a 175 cm ² tissue culture flask) were infected with recombinant viruses containing MASP-2 polypeptides at an MOI of 2 in Sf900 II SFM medium at 28°C for 96 hours. Supernatants by centrifugation and diisopropyl phosphorofluoridate was added to a final concentration of 1 mmol.

The MASP-2 polypeptides are secreted into the culture medium. Culture supernatants were dialyzed against 50 mmol NaCl, 1 mmol CaCl ₂ , 50 mmol triethanolamine hydrochloride, at pH 8.1, and loaded at a flow rate of 1.5 ml/min onto a Fast Flow Q-Sepharose column (Amersham Pharmacia Biotech) (2 .8x12 cm) balanced with the same buffer. Elution is carried out with 1.2 liters of the same buffer with a linear gradient to 350 mmol NaCl. Fractions containing recombinant MASP-2 polypeptides are identified by Western blotting, precipitated by adding (NH ₄ ) ₂ SO ₄ to 60% (w/v), and incubated overnight at 4°C. The pellets are resuspended in 145 mmol NaCl, 1 mmol CaCl2, 50 mmol triethanolamine hydrochloride, at pH 7.4, and loaded onto a TSK G3000 SWG (7.5x600 mm) column Tosohaas, Montgomeryville, PA) equilibrated with the same buffer. The purified polypeptides are then concentrated to 0.3 mg/ml by ultrafiltration in Microsep microconcentrators (cut-off molecular weight=10,000) (Filtron, Karlstein, Germany).

Example 4

This example describes a method for producing a polyclonal antibody against MASP-2 polypeptides.

Materials and methods.

MASP-2 antigens. A polyclonal anti-human MASP-2 antiserum is produced by immunizing rabbits with the following isolated MASP-2 polypeptides: serum isolated human MASP-2 (SEQ ID NO: 6), recombinant human MASP-2 (SEQ ID NO: 6), MASP- 2A containing the inactive protease domain (SEQ ID NO: 13) described in Example 3 and recombinant CUBI (SEQ ID NO: 8), CUBEGFI (SEQ ID NO: 9) and CUBEGFCUBII (SEQ ID NO: 10) expressed, as described in example 3 above.

polyclonal antibodies. Six week old rabbits primed with Bacillus Calmette-Guerin (BCG) are immunized by injection of 100 μg MASP-2 polypeptide in 100 μg/ml sterile saline. Injections are made every 4 weeks with antibody titer monitoring by ELISA as described in Example 7. Culture supernatants are collected for antibody purification by protein A binding affinity chromatography.

Example 5

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

Materials and methods.

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

For each pooling event, single cell suspensions are prepared from immunized mouse spleens and used to pool with Sp2/0 myeloma cells. 5x108 Sp2/0 cells and 5x108 spleen cells are pooled in a medium containing 50% polyethylene glycol (molecular weight 1450) (Kodak, Rochester, N.Y.) and 5% dimethyl sulfoxide (Sigma Chemical Co., St. Louis, Mo.). The cell concentration is then adjusted to 1.5x105 spleen cells in 200 µl suspension in Iscove's medium (Gibco, Grand Island, N.Y.) supplemented with 10% fetal serum, 100 units/ml penicillin, 100 µg/ml

- 76 042534 streptomycin, 0.1 mmol hypoxanthine, 0.4 μmol aminopterin and 16 μmol thymidine. Two hundred microliters of cell suspension is added to each well of approximately twenty wells of 96-well microculture plates. Approximately ten days later, culture supernatants are screened for their reactivity with purified MASP-2 factor by ELISA assay.

Enzyme-linked immunosorbent assay (ELISA). Wells of Immunol 2 microassay plates (Dynatech Laboratories, Chantilly, Va.) are coated by adding 50 μl of purified hMASP-2 at 50 ng/ml or rat rMASP-2 (or rMASP-2A) overnight at room temperature. The low concentration of MASP-2 for coating allows the selection of high affinity antibodies. After coating, remove the solution by shaking the plate, and add 200 µl of BLOTTO (skimmed milk powder) in PBS to each well over 1 hour to block non-specific sites. After one hour, the wells are washed with PBST buffer (PBS containing 0.05% Tween 20). Fifty microliters of culture supernatants are removed from each well of the pool and mixed with 50 µl of BLOTTO preparation and then added to individual wells of microassay plates. After 1 hour incubation, the wells are washed with PBST. Bound mouse antibodies are then determined by reaction with horseradish peroxidase (HRP) conjugated goat anti-mouse IgG (Fc specific) (Jackson ImmunoResearch Laboratories, West Grove, Pa.) and diluted 1:2000 in BLOTTO. Peroxidase substrate solution containing 0.1% 3,3,5,5 tetramethylbenzidine (Sigma, St. Louis, Mo.) and 0.0003% hydrogen peroxide (Sigma) is added to the wells to develop color within 30 minutes. The reaction is terminated by adding 50 μl of 2M H ₂ SO ₄ solution to each well. Read the absorbance of the reaction mixture at 450 nm on a BioTek ELISA Reader (BioTek Instruments, Winooski, Vt.).

MASP-2 binding analysis.

Culture supernatants that test positive for MASP-2 in the ELISA assay described above can be assayed by binding assay to determine the binding affinity they have for MASP-2 inhibitory agents for MASP-2. A similar assay can also be used to determine if inhibitory agents can bind to other antigens in the complement system.

Polystyrene microplate wells (96-well medium binding plates, Corning Costar, Cambridge, MA) are coated with MASP-2 (20 ng/100 μl/well, Advanced Research Technology, San Diego, CA) in phosphate buffered saline (PBS ) with pH 7.4 overnight at 4°C. After removing the MASP-2 solution, the wells are blocked with PBS containing 1% bovine serum albumin (BSA, Sigma Chemical) for 2 hours at room temperature. Wells uncoated with MASP-2 are used as controls. Aliquots of hybridoma supernatants or purified anti-MASP-2 MoAbs at varying concentrations in blocking solution are added to the wells. After a two hour incubation at room temperature, the wells are washed thoroughly with PBS. MASP-2-bound anti-MASP-2 MoAb was detected by adding peroxidase-conjugated goat anti-mouse IgG (Sigma Chemical) in blocking solution and incubated for 1 hour at room temperature. The plate is again thoroughly washed with PBS and 100 μl of 3,3',5,5'-tetramethylbenzidine (TMB) substrate (Kirkegaard and Perry Laboratories, Gaithersburg, MD) is added. The TMB reaction is terminated by the addition of 100 μl of a 1M phosphoric acid solution and the plate is read at 450 nm using a SPECTRA MAX 250 plate reader (SPECTRA MAX 250, Molecular Devices, Sunnyvale, CA).

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

Example 6

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

An anti-MASP-2 mouse monoclonal antibody is generated in male A/J mice as described in Example 5. The mouse antibody is then humanized as described below to reduce its immunogenicity by replacing mouse constant regions with their human counterparts to generate chimeric IgG and fragment Fab antibodies that can be used according to the present invention in humans to inhibit the negative effects of MASP-2 dependent complement activation.

1. Cloning of anti-MASP-2 variable region genes from mouse hybridoma cells. Total RNA was isolated from hybridoma cells secreting anti-MASP-2 MoAb (prepared as described in Example 7) using RNAzol according to the manufacturer's protocol (Biotech,

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Houston, Tech.). First strand cDNA is synthesized from total RNA using a synthetic homopolymer oligodeoxyribonucleotide (oligo-dT) as a primer. PCR was performed using 3' primers derived from the immunoglobulin constant C region and degenerate primer sets derived from the leader peptide or the first framework region of the mouse VH or VK genes as 5' primers. Anchor PCR is performed as described in Chen, P.F, Scand. J. Immunol. 35:539-549, 1992. To clone the VK gene, a double-stranded cDNA was prepared using the Not1-MAK1 primer (5'-TGCGGCCGCTGTAGGTGCTGTCTTT-3' SEQ ID NO: 38).

Renatured adapters AD1 (5'-GGAATTCACTCGTTATTCTCGGA-3' SEQ ID NO: 39) and AD2 (5'-TCCGAGAATAACGAGTG-3' SEQ ID NO: 40) are linked to ligands at both the 5' and 3' ends of the double-stranded cDNA. Adapters at the 3' ends are removed by Not1 cleavage. The cleavage product is then used as template in PCR with AD1 oligonucleotide as 5' primer and MAK2 (5'-CATTGAAAGCTTTGGGGTAGAAGTTGTTC-3' SEQ ID NO: 41) as 3' primer. The DNA fragments, with approximately 500 base pairs, are cloned into the pUC19 vector. Several clones are selected for sequence analysis to confirm that the cloned sequence covers the expected murine immunoglobulin constant region. Oligonucleotides Not1MAK1 and MAK2 are derived from the VK region and are 182 and 84 base pairs, respectively, after the first base pair of the kappa C gene. Clones are selected that include the completed VK and the leader peptide.

To clone the VH gene, a double-stranded cDNA was prepared using primer Not1 MAG1 (5'CGCGGCCGCAGCTGCTCAGAGTGTAGA-3' SEQ ID NO: 42). Renatured adapters AD1 and AD2 bind to ligands at both the 5' and 3' ends of the double-stranded cDNA. Adapters at the 3' ends are removed by Not1 cleavage. The cleavage product is then used as template in PCR with ADI oligonucleotide and MAG2 (5'-CGGTAAGCTTCACTGGCTCAGGGAAATA-3' SEQ ID NO: 43) as primers. DNA fragments from 500 to 600 bp in length are cloned into pUC19. The Notl-MAG1 and MAG2 oligonucleotides are derived from the mouse Cy.7.1 region and are 180 and 93 base pairs, respectively, downstream of the first base pair in the mouse Cy.7.1 gene. Clones are selected that include the completed VH and the leader peptide.

2. Construction of Expression Vectors for Chimeric MASP-2 IgG and Fab. The cloned VH and VK genes described above are used as templates in a PCR reaction to add a Kozak consensus sequence to the 5' end and a spliced fragment donor to the 3' end of the nucleotide sequence. The sequences are then analyzed for PCR errors, and the VH and VK genes are inserted into expression vector cassettes containing human C.yI and C. kappa, respectively, to generate pSV2neoVH-huCyI and pSV2neoV-huCy. CsCl gradient-purified heavy and light chain vector DNA plasmids were used to transfect COS cells by electroporation. After 48 hours of incubation, the culture supernatant is analyzed by ELISA to confirm the presence of approximately 200 ng/ml of chimeric IgG. Cells are harvested and total RNA is obtained. The first strand cDNA is synthesized from total RNA using oligo dT as a primer. This cDNA is used as a template in PCR to generate Fd and kappa DNA fragments. For the Fd gene, PCR was performed using 5'-AAGAAGCTTGCCGCCACCATGGATTGGCTGTGGAACT-3' (SEQ ID NO: 44) as 5' primer and 3' primer (5'-CGGGATCCTCAAACTTTCTTGTCCACCTTGG3' SEQ ID NO: 45) derived from CH1. The DNA sequence was confirmed to contain the complete VH and CH1 domain of human IgG1. After digestion with the appropriate enzymes, the Fd DNA fragments are inserted into the HindIII and BamHI restriction sites in the pSV2dhfr-TUS expression vector cassette to generate pSV2dhfrFd. Plasmid pSV2 is commercially produced and consists of DNA segments taken from various sources: pBR322 DNA (depicted by thin line) contains plasmid pBR322 origin of DNA replication (pBR ori) and lactamase resistance gene to ampicillin (Amp); SV40 DNA, which is depicted with broad strokes and labeling, contains the SV4 0 origin of DNA replication plasmid (SV40 ori), an early promoter (from 5' to dhfr and neogenes) and a polyadenylation signal (from 3' to dhfr and neogenes). The polyadenylation signal (pA) derived from SV40 is also placed at the 3' end of the Fd gene.

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

To produce a recombinant Fab with a disulfide bond between the heavy and light chains, the above Fd gene can be extended to include a sequence encoding 9 additional amino acids (EPKSCDKTH SEQ ID NO: 48) from the human IgG1 hinge region.

The BstEII-BamHI DNA segment encoding the 30 amino acids at the 3' end of the Fd gene can be replaced with DNA segments encoding extended Fd, resulting in pSV2dhfrFd/9aa.

3. Expression and purification of chimeric anti-MASP-2 IgG.

To generate cell lines secreting chimeric anti-MASP-2 IgG, NSO cells were transfected with purified pSV2neoVH-huC.y1 and pSV2neoV-huC kappa plasmid DNA by electroporation. Transfected cells are selected in the presence of 0.7 mg/ml G418. Cells are grown in a 250 ml spinner flask in culture medium containing serum.

The culture supernatant from a 100 ml culture spin tube was loaded onto a 10 ml PROSEP-A column (Bioprocessing, Inc., Princeton, N.J.). The column is washed with a volume of PBS equal to 10 column bed volumes. The bound antibody is eluted with 50 mmol citrate buffer pH 3.0. An equal volume of 1 mol Hepes pH 8.0 is added to the fraction containing the purified antibody to adjust the pH to 7.0. Residual salts are removed by buffer exchange with PBS by ultrafiltration through a Millipore membrane (cut-off molecular weight 3000). The protein concentration of the purified antibody is determined by the bicinchoninic acid (BCA) method (Pierce).

4. Expression and purification of the chimeric anti-MASP-2 Fab.

To generate cell lines secreting chimeric anti-MASP-2 Fabs, CHO cells are transfected with purified plasmid DNA pSV2dhfrFd (or pSV2dhfrFd/9aa) and pSV2neokappa by electroporation. Transfected cells are selected in the presence of G418 and methotrexate. Selected cell lines are amplified at increasing concentrations of methotrexate. The cells are a single cell, subcloned by the limiting dilution method. High-yielding cell lines subcloned from single cells are then grown in a 100 ml spin tube in serum-free culture medium.

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

The ability of chimeric MASP-2 IgG, cFab and cFAb/9aa to inhibit MASP-2 dependent complement activation pathways can be determined by performing the inhibition assay described in Example 2 or Example 7.

Example 7

This example describes an in vitro C4 cleavage assay used as a functional screen to identify MASP-2 inhibitory agents capable of blocking MASP-2 dependent complement activation via L-ficolin/P35, H-ficolin, M-ficolin, or mannan.

C4 splitting study. A C4 cleavage study is described in Petersen, S.V., et al., J. Immunol. Methods 257:107, 2001, which measures lectin pathway activation induced by Staphylococcus aureus lipoteichoic acid (LTA), which binds L-ficolin.

Reagents. Formalin-fixed preparation of Staphylococcus aureus (DSM20233) is prepared as follows: cultured bacteria grown overnight at 37°C in trypton-soy blood agar medium, then washed three times with PBS, then fixed for 1 h at room temperature. temperature in PBS/0.5% formalin, and additionally washed three times with PBS, and then resuspended in coating buffer (15 mmol Na ₂ CO ₃ , 35 mmol NaHCO ₃ , pH 9.6.

Study. Wells of a Nunc MaxiSorb microtiter plate (Nalgene Nunc International, Rochester, NY) are coated with 100 µl of formalin-fixed Staphylococcus aureus preparation DSM20233 (OD550=0.5) in coating buffer with 1 µg of L-ficolin. After overnight incubation, wells are blocked with 0.1% human serum albumin (HSA) in TBS (10 mM Tris-HCl, 140 mM NaCl, pH 7.4), then washed with TBS containing 0.05 % Tween 20 and 5 mmol CaCl2 (wash buffer). Human serum samples are diluted in 20 mM Tris-HCl, 1 M NaCl, 10 mM CaCl ₂ , 0.05% Triton X-100, 0.1% HSA, pH 7.4, which prevents activation of endogenous C4 and leads to dissociation of the complex C1 (consisting of C1q, C1r and C1s). Added to the serum samples at various concentrations of MASP-2 inhibitory agents, including MoAb against MASP-2 and inhibitory peptides. The diluted samples are added to the plate and incubated overnight at 4°C. After 24 hours, the plates are thoroughly washed with wash buffer, then 0.1 µg of purified human C4 (prepared as described in Dodds, AW, Methods Enzymol. 223:46, 1993) in 100 µl is added to each well. 4 mmol barbital, 145 mmol NaCl, 2 mmol CaCl ₂ , 1 mmol MgCl ₂ , pH 7.4. After incubation for 1.5 h at 37°C, the plates are washed again and C4b deposition is detected using alkaline phosphatase-conjugated chicken anti-human C4c (obtained from Immunsystem, Uppsala, Sweden) and measured using substrate for colorimetric determination. p-nitrophenyl phosphate.

C4 study on mannan. The assay described above is adapted to measure lectin pathway activation by MBL by coating the plate with a layer of mannan and LSP prior to the addition of serum mixed with various MASP-2 inhibitory agents.

C4 study on H-ficolin (Hakata antigen). The assay described above is adapted to measure lectin pathway activation by N-ficolin by coating the plate with Nficolin and LPS prior to the addition of serum mixed with various MASP-2 inhibitory agents.

Example 8

The study described below demonstrates the presence of the classical activation pathway in wild-type mice and MASP-2-/- mice.

Methods. Immune complexes were generated in situ by coating microtiter plates (Maxisorb, Nunc, Cat # 442404, Fisher Scientific) with a layer of 0.1% human serum albumin in 10 mM Tris, 140 mM NaCl, pH 7.4 for 1 h at room temperature followed by overnight incubation at 4°C with sheep antiserum against whole serum (Scottish Antibody Production Unit, Carluke, Scotland) diluted 1:1000 in TBS/tween/Ca ²⁺ . Serum samples were obtained from wild mice and MASP-2 −/− mice and added to coated plates. Prepared control samples, which contained serum samples of wild mice and MASP-2 -/- mice with a sharply reduced level of C1q. C1q drastically reduced mouse serum was prepared using protein A coupled to Dynabeads Dynal Biotech, Oslo, Norway magnetic beads coated with rabbit anti-human C1q IgG (Dako, Glostrup, Denmark) according to the manufacturer's instructions. The plates were incubated for 90 minutes at 37°C. Bound C3b was detected with an anti-human C3c polyclonal antibody (Dako A 062) diluted 1:1000 in TBS/tw/Ca++. The secondary antibody is goat anti-rabbit IgG.

Results. In FIG. 7 shows the relative levels of C3b deposition on IgG coated plates in wild mouse sera, MASP-2 −/− mouse sera, C1q severely reduced wild mouse sera, and C1q severely reduced MASP-2 −/− mice sera. These results demonstrate that the classical pathway remains unaffected in MASP-2 -/- mice.

Example 9

The following study analyzes the ability of a MASP-2 inhibitory agent to block the classical activation pathway by examining the effect of the MASP-2 inhibitory agent under conditions where the classical pathway is initiated by immune complexes.

Methods. To test the effect of a MASP-2 inhibitory agent under conditions where the classical pathway is initiated by immune complexes, three 50 µl samples of sera containing 90% NHS are incubated at 37° C. in the presence of 10 µg/ml immune complex (IC) or PBS, and, in parallel, incubated at 37° C. three more copies of the sample (+/-IC), which contain 200 nmol of monoclonal antibodies against properdin. After incubation for 2 hours at 37°C, 13 mmol EDTA is added to all samples to stop further complement activation, after which all samples are immediately cooled to 5°C. The samples are then stored at -70°C until used in the assay of complement activation products (C3a and sC5b-9) using ELISA kits (Quidel, catalog numbers A015 and A009) according to the manufacturer's instructions.

Example 10

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

The level of knowledge of the problem and relevance. MASP-2 is a complex protein with many distinct functional domains, including the binding site(s) for MBL and ficolins, the serine protease catalytic site, the C2 proteolytic substrate binding site, the C4 proteolytic substrate binding site, the MASP-2 cleavage site for MASP-2 Zymogen Self-Activation and Two Ca~ Binding Sites Fab2 antibody fragments that bind with high affinity to MASP-2 were identified and the identified Fab2 fragments were tested in a functional assay to determine their ability to block MASP-2 functional activity.

To block functional activity of MASP-2, an antibody or Fab2 fragment of an antibody must bind and negatively affect a structural epitope on MASP-2 that is required for functional activity of MASP-2. Therefore, many or all of the high affinity Fab2 binding fragments of anti-MASP-2 antibodies may not inhibit the functional activity of MASP-2,

- 80 042534 if they do not bind to structural epitopes on MASP-2 that directly confer the functional activity of MASP-2.

A functional assay measuring inhibition of the lectin pathway C3 convertase was used to evaluate the blocking activity of the Fab2 fragment of an anti-MASP-2 antibody. It is known that the main physiological function of MASP-2 in the lectin pathway is the generation of the next functional component of the lectin-mediated complement pathway, namely the lectin pathway C3 convertase. The C3 lectin pathway convertase is a particularly important enzyme complex (C4bC2a) that proteolytically cleaves C3 into C3a and C3b. MASP-2 is not a structural component of the lectin pathway C3 convertase (C4bC2a); however, the functional activity of MASP-2 is required for the generation of two protein components (C4b, C2a) that include the C3-convertase of the lectin pathway. In addition, all of the individual functional activities of MASP-2 listed above appear to be required for MASP-2 to generate the lectin pathway C3 convertase. For these reasons, it is believed that the preferred assay to evaluate the blocking activity of the Fab2 fragment of an anti-MASP-2 antibody is a functional assay that measures inhibition of the formation of lectin pathway C3 convertase.

Generation of high affinity Fab2 fragments. To generate high affinity Fab2 fragments for the rat MASP-2 protein (SEQ ID NO: 55), human antibody light and heavy chain sequence library phage display and automated antibody selection were used to identify Fab2s that interact with selected ligands of interest. A known amount of rat MASP-2 protein (˜1 mg, >85% purity) was used for antibody screening. Three rounds of amplification were performed to select antibodies with the best affinity. Approximately 250 different hits expressing antibody fragments were selected for ELISA screening. The high affinity hits were then sequenced to determine the uniqueness of the different antibodies.

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

Studies conducted to evaluate the inhibitory (blocking) activity of Fab2 fragments of antibodies against MASP-2.

1. Study to measure the inhibition of the formation of C3-convertase of the lectin pathway.

The level of knowledge of the problem. Lectin pathway C3 convertase is an enzyme complex (C4bC2a) that proteolytically cleaves C3 into two potential pro-inflammatory fragments, anaphylotoxin C3a and opsonin C3b. Apparently, the formation of C3 convertase is a key step in the lectin pathway in terms of mediating inflammation. MASP-2 is not a structural component of the lectin pathway C3 convertase (C4bC2a), so antibodies against MASP-2 (or Fab2) will not directly inhibit the activity of an already existing C3 convertase. However, the activity of the MASP-2 serine protease is required for the generation of two protein components (C4b, C2a), which include the C3-convertase of the lectin pathway. Therefore, a Fab2 fragment of an anti-MASP-2 antibody that inhibits MASP-2 functional activity (ie, blocks anti-MASP-2 Fab2) will initially inhibit the formation of lectin pathway C3 convertase. C3 contains as part of its structure an unusual and highly reactive thioether group. After cleaving C3 with C3 convertase, the thioether group in C3b can form a covalent bond with hydroxyl groups or amino groups of macromolecules immobilized at the bottom of plastic wells using ether or amide bonds, which facilitates the determination of C3b using the ELISA assay method.

Yeast mannan is a known activator of the lectin pathway. In the method described below for measuring C3 convertase production, mannan-coated plastic wells were incubated for 30 minutes at 37° C. with diluted rat serum to activate the lectin pathway. The wells were then washed and the C3b immobilized in the wells was determined using standard ELISA methods. The amount of C3b generated in this study is a direct reflection of the C3-convertase of the lectin pathway formed from the very beginning. In this study, Fab2 fragments of anti-MASP-2 antibodies were tested at selected concentrations for their ability to inhibit C3 convertase formation and subsequent C3b generation.

Methods.

Costar medium bound 96-well plates were incubated overnight at 5° C. with mannan diluted in 50 mM pH 9.5 carbonate buffer at 1 μg of mannan in 50 μl of buffer per well. After incubation overnight, each well was washed three times with 200 μl PBS. The wells were then blocked with 1% bovine serum albumin in PBS at 100 μl per well and incubated for 1 hour at room temperature with gentle agitation. Each well was then washed three times with 200 μl PBS. Anti-MASP-2 Fab2 samples were diluted to selected concentrations in Ca++ and Mg ⁺⁺ containing GVB buffer (4.0 mmol barbital, 141 mmol NaCl, 1.0 mmol MgCl, 2.0 mmol CaCl ₂ , 0.1% gelatin , pH 7.4) at 5°C. 0.5% rat blue serum was added to each sample at 5°C and 100 µl was transferred to each well. The plates were covered with lids and incubated for 30 minutes in a water bath at 37°C to initiate complement activation. The reaction was stopped by transferring the plates from the 37° C. water bath to a container containing a mixture of ice and water. Each well was washed five times with 200 µl of PBS-Tween 20 solution (0.05% Tween 20 in PBS), then washed twice with 200 µl of PBS. A 1:10,000 dilution of primary antibody solution (rabbit versus human C3, DAKO A0062) at a volume of 100 µl per well was added to PBS containing 2.0 mg/ml bovine serum albumin and incubated for 1 hour at room temperature with gentle agitation . Each well was washed 5 times with 200 μl PBS. A 1:10,000 dilution of a secondary antibody solution (goat anti-rabbit IgG peroxidase conjugate, American Qualex A102PU) at a volume of 100 µl per well was added to PBS containing 2.0 mg/ml bovine serum albumin and incubated for 1 h at room temperature. temperature with gentle stirring on a shaker. Each well was washed five times with 200 µl PBS. 100 μl per well of TMB peroxidase substrate (Kirkegaard & Perry Laboratories) was added and incubated at room temperature for 10 minutes. The peroxidase reaction was stopped by adding 1.0 M H ₃ PO ₄ solution at 100 μl per well and the OD ₄₅₀ was measured.

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

The level of knowledge of the problem. MASP-2 serine protease activity is highly specific and only two MASP-2 protein substrates have been identified, namely C2 and C4. Cleavage of C4 leads to the formation of C4a and C4b. Anti-MASP-2 Fab2 can bind to structural epitopes on MASP-2 that are directly involved in C4 cleavage (eg, MASP-2 binding site for C4; MASP-2 serine protease catalytic site) and thereby inhibit the functional activity of MASP- 2 for C4 cleavage.

Yeast mannan is a known activator of the lectin pathway. In the method described below for measuring MASP-2 C4 cleavage activity, mannan-coated plastic wells were incubated for 30 minutes at 37° C. with diluted rat serum to activate the lectin pathway. Since the primary antibodies used in this ELISA method only recognize human C4, the diluted rat serum was also supplemented with human C4 (1.0 μg/ml). The wells were then washed and analyzed for human C4b immobilized in the wells using standard ELISA methods. The amount of C4b generated in this assay is a measure of MASP2 dependent C4 cleavage activity. In this study, Fab2 fragments of anti-MASP-2 antibodies were tested at selected concentrations for their ability to inhibit C4 cleavage.

Methods. Costar medium bound 96-well plates were incubated overnight at 5° C. with mannan diluted in 50 mM pH 9.5 carbonate buffer at 1 μg of mannan in 50 μl of buffer per well. Each well was washed three times with 200 μl PBS. The wells were then blocked with 1% bovine serum albumin in PBS at 100 μl per well and incubated for 1 hour at room temperature with gentle agitation. Each well was washed three times with 200 μl PBS. Anti-MASP-2 Fab2 samples were diluted to selected concentrations in Ca ⁺⁺ and Mg ⁺⁺ containing GVB buffer (4.0 mmol barbital, 141 mmol NaCl, 1.0 mmol MgCl, 2.0 mmol CaCl ₂ , 0.1% gelatin , pH 7.4) at 5°C. These samples were also injected with 1.0 μg/ml human C4 (Quidel). In the samples described above, 0.5% rat serum was added at 5° C. and 100 μl was transferred to each well. The plates were covered with lids and incubated for 30 minutes in a water bath at 37°C to initiate complement activation. The reaction was stopped by transferring the plates from the 37° C. water bath to a container containing a mixture of ice and water. Each well was washed five times with 200 µl of PBS-Tween 20 solution (0.05% Tween 20 in PBS), then washed twice with 200 µl of PBS. Biotin-conjugated chick versus human C4 solution (Immunsystem AB, Uppsala, Sweden) diluted 1:700 in a volume of 100 µl per well was added to PBS containing 2.0 mg/ml bovine serum albumin and incubated for 1 h at room temperature. temperature with gentle stirring. Each well was then washed five times with 200 μl PBS. 100 µl per well of peroxidase-conjugated streptavidin at 0.1 µg/ml (Pierce Chemical #21126) in PBS containing 2.0 mg/ml BSA was added and incubated for 1 hour at room temperature with gentle agitation on the apparatus for shaking. Each well was washed five times with 200 μl PBS. 100 μl per well of TMB peroxidase substrate (Kirkegaard & Perry Laboratories) was added and incubated at room temperature for 16 minutes. The peroxidase reaction was stopped by adding 1.0 M H ₃ PO ₄ solution at 100 μl per well and the OD ₄₅₀ was measured.

3. Binding analysis of the Fab2 fragment of the rat anti-MASP-2 antibody to native rat MASP-2.

The level of knowledge of the problem. MASP-2 is normally present in plasma in the form of a dimeric MASP-2 complex, which also includes specific lectin molecules (mannose-binding protein (MBL) and ficolins). Therefore, if there is interest in studying the binding of the Fab2 fragment of an anti-MASP-2 antibody to a physiologically relevant form of MASP-2, it is important to develop a binding assay that uses the plasma interaction between Fab2 and native MASP-2 rather than purified recombinant MASP-2. In this binding assay, native MASP-2 complexed with MBL from 10% rat serum was first immobilized on mannan-coated wells. The binding affinity of various anti-MASP-2 antibody Fab2 fragments to immobilized native MASP-2 was then examined using a standard ELISA assay.

Methods. Costar 96-well high binding plates were incubated overnight at 5° C. with mannan diluted in 50 mM carbonate buffer pH 9.5 at 1 μg/50 μl per well. Each well was washed three times with 200 μl PBS. The wells were then blocked with 0.5% skimmed milk powder in PBST (PBS with 0.05% Tween 20) at 100 μl per well and incubated for 1 hour at room temperature with gentle agitation. Each well was washed three times with 200 µl TBS/Tween/Ca ⁺⁺ wash buffer (Tris buffered saline, 0.5% Tween 20 containing 5.0 mmol CaCl2, pH 7.4. Prepared on ice 10% rat serum in high salt binding buffer (20 mM Tris, 1.0 M NaCl, 10 mM CaCl2, 0.05% Triton-X100, 0.1% (w/v) bovine serum albumin, pH 7.4). 100 µl per well was added and incubated overnight at 5° C. The wells were then washed three times with 200 µl TBS/Tween/Ca++ wash buffer.Then the wells were washed twice with 200 µl PBS. selected concentration of Fab2 fragment of anti-MASP-2 antibody diluted in GVB buffer containing Ca ⁺⁺ and Mg ⁺⁺ (4.0 mmol barbital, 141 mmol NaCl, 1.0 mmol MgCl, 2.0 mmol CaCl2, 0.1 % gelatin, pH 7.4) and incubated for 1 h at room temperature with gentle agitation. Each well was washed five times with 200 µl of PB S. 100 μl per well of an HRP-conjugated goat antibody Fab2 fragment (Biogenesis catalog number 0500-0099) diluted 1:5000 in 2.0 mg/ml bovine serum albumin in PBS was added and incubated for one hour at room temperature at careful mixing. Each well was washed five times with 200 µl PBS. 100 μl per well of TMB peroxidase substrate (Kirkegaard & Perry Laboratories) was added and incubated at room temperature for 70 minutes. The peroxidase reaction was stopped by adding 1.0 mol H3PO ₄ at 100 μl per well and then the OD ₄₅₀ was measured.

Results.

Approximately 250 different Fab2 fragments were selected for screening by ELISA assay, which react with high affinity with the rat MASP-2 protein. These high affinity Fab2s were sequenced to determine the uniqueness of the various antibodies, and 50 unique anti-MASP-2 antibodies were purified for further study. 250 μg of each purified Fab2 antibody fragment was used for MASP-2 binding affinity characterization and complement activation pathway functional testing. The results of this study are shown in table. 6.

Table 6 Fab2 fragments of anti-masp-2 antibodies that block the lectin pathway of complement activation

Fab2 fragment of antibody no. C3-convertase 1C ₅₀ (nM) _Kd Cleavage C4 1C ₅₀ (nM) 88 0.32 4.1 did not define 41 0.35 0.30 0.81 eleven 0.46 0.86 <2 nM 86 0.53 1.4 did not define 81 0.54 2.0 did not define 66 0.92 4.5 did not define 57 0.95 3, 6 <2 nM 40 eleven 7.2 0.68 58 1.3 2, 6 did not define 60 16 3, 1 did not define 52 16 5, 8 <2 nM 63 2.0 6, 6 did not define 49 2.8 8.5 <2 nM 89 thirty 2.5 did not define 71 thirty 10.5 did not define 87 6.0 2.5 did not define 67 100 7.7 did not define

As shown above in Table. 6, of 50 anti-MASP-2 antibody Fab2 fragments tested, seventeen Fab2s were identified as MASP-2 blocking Fab2s that potentially inhibit C3 convertase formation with an IC ₅₀ value equal to or less than 10 nmol Fab2 (34% positive). search). Eight of the seventeen identified Fab2 have an IC ₅₀ value in the subnanomolar range. In addition, all given in table. 6, seventeen Fab2s blocking MASP-2 provide virtually complete inhibition of C3 convertase formation in the lectin pathway C3 convertase assay. In FIG. 8A is a graphical representation of the results of a C3 convertase production study for the Fab2 fragment of antibody #11, which is representative of other antibody Fab2 fragments tested, the results of which are shown in Table 83.042534. 6. This is an important factor because it is theoretically possible that a blocking fragment of Fab2 can only partially inhibit MASP-2 function even when every MASP-2 molecule binds to Fab2.

Although mannan is a well-studied activator of the lectin pathway, there is nevertheless a theoretical possibility that the presence of anti-mannan antibodies in rat serum may also activate the classical pathway and generate C3b via classical pathway C3 convertase. However, each of the seventeen Fab2 blocking fragments of anti-MASP-2 antibodies listed in this example potentially inhibited C3b generation (>95%), thus demonstrating the specificity of this assay for lectin pathway C3 convertase.

All seventeen blocking Fab2s were also analyzed for binding to calculate the apparent Kd for each. The results of studies on the binding of Fab2 fragments of rat antibodies against MASP-2 with native rat MASP-2 for six of the blocking Fab2 are also shown in table. 6. In FIG. 8B is a graphical representation of the results of a binding study with the Fab2 fragment of antibody #11. Similar binding studies were also performed for other Fab2s and the results are shown in Table 8B 6. Overall, the Kd values obtained for the binding of each of the six Fab2s to native MASP-2 closely matched the IC ₅₀ values for Fab2 in the C3 convertase functional assay. There is evidence that MASP-2 undergoes a conformational change from an inactive form to an active form upon initiation of protease activity (Feinberg et al., EMBO J 22:2348-59 (2003); Gal et al., J. Biol. Chem. 280 :33435-44 (2005)). In normal rat plasma used in the study of C3 convertase formation, MASP-2 is present initially in the form of an inactive zymogen conformation. In contrast, in a binding assay, MASP-2 is present as part of a complex with MBL bound to immobilized mannan; therefore, MASP-2 is likely to be in an active conformation (Petersen et al., J. Immunol Methods 257:107-16, 2001). Therefore, one should not expect an exact match between the IC ₅₀ and Kd values for each of the seventeen blocking Fab2 tested in these two functional studies, since in each study, Fab2 could bind different conformational forms of MASP-2. However, it appears that, with the exception of Fab2 #88, there is a fairly good match between _IC50 and apparent Kd for each of the other sixteen Fab2s tested in the two studies (see Table 6).

Some of the blocking Fab2s were evaluated for their ability to inhibit MASP-2 dependent C4 cleavage. In FIG. 8C is a graphical representation of the results of a C4 cleavage assay showing inhibition by Fab2 #41 with an IC ₅₀ value of 0.81 nmol (see Table 6). As shown in FIG. 9, all tested Fab2s were found to inhibit C4 cleavage with similar IC ₅₀ values to those obtained in the C3 convertase assay (see Table 6).

Although mannan is a well-studied activator of the lectin pathway, there is nevertheless a theoretical possibility that the presence of anti-mannan antibodies in rat serum may also activate the classical pathway and generate C4b through C1s-mediated C4 cleavage. However, several Fab2 fragments of anti-MASP-2 antibodies have been identified that potentially inhibit C4b generation (>95%), thus demonstrating the specificity of this MASP-2 mediated C4 cleavage assay. C4, like C3, contains an unusual and highly reactive thioether group as part of the structure. After C4 is cleaved by MASP-2 in this assay, the thioether group on C4b can form a covalent bond with hydroxyl or amino groups on macromolecules immobilized at the bottom of the plastic wells via ester or amide bonds, thus facilitating the determination of C4b in an ELISA assay.

These studies unequivocally demonstrate that FAB2s have been created that have high affinity for the MASP-2 protein and that block the functional activity of C4 and C3 convertase, thereby preventing activation of the lectin pathway.

Example 11.

This example describes epitope mapping of some of the Fab2 blocking fragments of rat anti-MASP-2 antibodies that were generated as described in Example 10.

Methods.

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

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

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

CUBI-II, an N-terminal fragment of rat MASP-2 that contains only CUBI, EGF-like, and CUBII domains; And

A CUBI/EGF-like, N-terminal fragment of rat MASP-2 that contains only

- 84 042534

CUBI and EGF-like domains.

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

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

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

Dot blot analysis.

Serial dilutions of the five recombinant MASP-2 polypeptides described above and shown in FIG. 1 were spotted onto the nitrocellulose membrane. 10 (and thioredoxin polypeptide as negative control for CCPII serine protease polypeptide). The amount of protein applied in the form of spots varied in the range from 100 ng to 6.4 pg at each successive five-fold dilution. In subsequent experiments, the amount of protein applied in the form of spots varied in the range from 50 ng to 16 pg, also at each successive five-fold dilution. The membranes were blocked with 5% skimmed milk powder in TBS (blocking buffer), then incubated with 1.0 μg/ml of anti-MASP-2 Fab2 fragment in blocking buffer (containing 5.0 nmol Ca ²⁺ ). Bound Fab2 fragments were detected using an HRP-conjugated human antibody Fab fragment (AbD/Serotec; diluted 1/10,000) and an ECL detection kit (Amersham). One membrane was incubated with a polyclonal rabbit antibody against human MASP-2 (described in Stover et al., J Immunol 163:6848-59 (1999)) as a positive control. In this case, bound antibody was determined using HRP-conjugated goat anti-rabbit IgG (Dako; diluted 1/2000).

MASP-2 binding analysis.

ELISA plates were coated with recombinant MASP-2A or CUBI-II polypeptide in carbonate buffer (pH 9.0) at 1.0 μg per well overnight at 4°C. The wells were blocked with 1% BSA in TBS, then serial dilutions of anti-MASP-2 antibody Fab2 fragments were added in TBS containing 5.0 nmol Ca ²⁺ . The plates were incubated for 1 h at room temperature. After a triple wash in TBS/tween/Ca ²⁺ , an HRP-conjugated human antibody Fab fragment (AbD/Serotec) diluted 1/10,000 in TBS/Ca ²⁺ was added and the plates were again incubated for 1 hour at room temperature. Bound antibody was detected using the TMB Peroxidase Substrate Kit (Biorad).

Results.

The results of the dot-blot analysis, showing the reactivity of Fab2 when interacting with various MASP-2 polypeptides, are presented below in table. 7. Numerical values given in Table. 7 denote the amount of protein deposited in the form of patches required to deliver a signal at about half its maximum intensity. As shown, all polypeptides (with the exception of only confluent thioredoxin cells) were recognized by a positive control antibody (anti-MASP-2 polyclonal human serum produced in rabbits).

Table 7 Reactivity with various recombinant rat masp-2 polypeptides in dot blot analysis

Fab2 fragment of antibody no. MASP-2A CUBI II CUBI/EGF-like CCP II SP Thioredoxy n 40 0.16 ng NR NR 0.8 ng NR 41 0.16 ng NR NR 0.8 ng NR eleven 0.16 ng NR NR 0.8 ng NR 49 0.16 ng NR NR >2 0 ng NR 52 0.16 ng NR NR 0.8 ng NR 57 0.032 ng NR NR NR NR 58 0.4 ng NR NR 2.0 ng NR 60 0.4 ng 0.4 ng NR NR NR 63 0.4 ng NR NR 2.0 ng NR 66 0.4 ng NR NR 2.0 ng NR 67 0.4 ng NR NR 2.0 ng NR 71 0.4 ng NR NR 2.0 ng NR 81 0.4 ng NR NR 2.0 ng NR 86 0.4 ng NR NR 10 ng NR 87 0.4 ng NR NR 2.0 ng NR Positive control <0.032 ng 0.16 ng 0.16 ng <0.032 ng NR

The activity of the lectin pathway was recorded during the second and third weeks, complete recovery of the lectin pathway in mice occurred on day 17 after administration of the monoclonal antibody against MASP-2. NK=no response. The positive control antibody is a serum anti-human MASP-2 polyclonal antibody produced in rabbits.

All Fab2 antibody fragments interacted with MASP-2A as well as MASP-2K (data not shown). Most of the Fab2 fragments recognized the CCPII-SP polypeptide, but not the N-terminal fragments. Two exceptions are Fab2 #60 and Fab2 #57. Fab2 #60 recognizes MASP-2A and a CUBI-II fragment, but not a CUBI/EGF-like polypeptide or a CCPII-SP polypeptide, suggesting that it binds to an epitope in CUBII, or overlaps the CUBII domain and the EGF-like domain. Fab2 #57 recognizes MASP-2A but none of the MASP-2 fragments tested, indicating that this antibody Fab2 fragment recognizes an epitope in CCPI. Fab2 #40 and #49 only bind to complete MASP-2A. In the ELISA binding assay depicted in FIG. 11, Fab2 #60 also bound to the CUBI-II polypeptide, although the level of apparent affinity was slightly lower.

These results demonstrate the identification of unique Fab2 blocking antibody fragments that act on multiple regions of the MASP-2 protein.

Example 12.

This example describes the study of MASP-2 -/- mice in an experimental model of renal ischemia/reperfusion in mice.

The level of knowledge of the problem and relevance. Kidney injury resulting from ischemia/reperfusion (I/R) at body temperature is related to a number of clinical conditions including hypovolemic shock, renal artery occlusion, and clamping procedures.

Renal ischemia-reperfusion (I/R) is an important cause of acute renal failure, resulting in 50% mortality (Levy et al., JAMA 275:1489-94, 1996; Thadhani et al., N. Engl. J. Med 334:1448-60, 1996). Post-transplant renal failure is a common and life-threatening complication after kidney transplantation (Nicholson et al., Kidney Int. 58:2585-91, 2000). Currently, there is no effective method for the treatment of I/R kidney damage, the only form of treatment is hemodialysis. The pathophysiology of I/R renal disease is highly complex. Recent studies have shown that the lectin complement pathway may play an important role in the pathogenesis of I/R kidney disease (deVries et al., Am. J. Path. 165:1677-88, 2004).

Methods.

A MASP-2(-/-) mouse was created as described in Example 1 and backcrossed for at least 10 generations with C57B1/6 mice. Six male MASP-2(-/-) mice and six wild-type mice (+/+) weighing 22-25 g were injected intraperitoneally with Hypnovel (6.64 mg/kg; Roche products Ltd. Welwyn Garden City, UK) and then anesthetized by isoflurane inhalation (Abbott Laboratories Ltd., Kent, UK). Isoflurane was chosen because it is a mild inhalation anesthetic with minimal hepatotoxicity; its concentrations are produced with high precision and animals recover quickly even after prolonged anesthesia. Hypnovel was administered due to the fact that it causes a state of neuroleptanalgesia in animals, which implies the possibility of administering a lower dose of isoflurane. Animals were placed on warm bedding in order to maintain a constant body temperature. An incision was then made along the midline of the abdominal wall, and the abdominal cavity was left open using a pair of retractors. Connective tissue was trimmed above and below the renal vein and artery of the right and left kidneys, and the renal pedicle was occluded with microaneurysm clamps for 55 minutes. This period of ischemia was originally substantiated as a result of a previous study conducted in this laboratory (Zhou et al., J. Clin. Invest. 105:1363-71 (2000)). In addition, a standard ischemia duration time of 55 minutes was chosen after performing an ischemia titration study, and a time of 55 minutes was found to produce a persistent lesion that is also reversible, with a low mortality rate of less than 5%. After occlusion, 0.4 ml of warm saline (37°C) was injected into the abdominal cavity, and then the abdominal cavity was closed for the period of ischemia. After removal of the clips used in microaneurysm, the kidneys were observed until there was a change in their color, which indicated the resumption of blood circulation in the kidneys. An additional 0.4 ml of warm saline was injected into the abdominal cavity and the incision was sutured, after which the animals were returned to their cages. Blood samples were taken from the tails of the animals 24 hours after removal of the clamps, and 48 hours later the mice were sacrificed and additional blood samples were collected.

Assessment of kidney damage. Kidney function was assessed in six male MASP-2(-/-) mice and six wild mice (+/+) 24 and 48 hours after reperfusion. Determination of creatinine in the blood was carried out by mass spectrometry, which provides reproducible results in determining the index of kidney function (sensitivity <1.0 µmol/l). In FIG. 12 is a graphical representation of blood urea nitrogen clearance data for a control group of C57B1/6 wildcat mice and a group of MASP-2(-/-) mice at 24 hours and 48 hours after reperfusion. As shown in FIG. 12, MASP-2(-/-) mice showed a significant decrease in blood urea through

- 86 042534 and after 48 hours in comparison with control wild mice, which indicates a protective effect of a functional nature against kidney damage in the model of ischemic reperfusion injury.

In general, elevated blood urea levels were observed in both wild-type (+/+) and MASP-2(-/-) mice at 24 and 48 hours after the surgical procedure and ischemic stroke. The blood urea level in the non-ischemic operated wild animal (+/+) was determined separately and was 5.8 mmol/l. In addition to the data presented in Fig. 12, one MASP-2(-/-) animal showed almost complete protection against ischemic stroke, with rates of 6.8 and 9.6 mmol/L at 24 h and 48 h, respectively. This animal was excluded from the study group as an animal, sharply different from the others, which can not experience ischemic damage. Therefore, the final results of the study presented in FIG. 12 included results for 5 MASP-2(-/-) mice and 6 wild-type mice (+/+), and a statistically significant decrease in blood urea was found at 24 h and at 48 h in MASP-2(-/- ) mice (Student's t-test p<0.05). It can be hypothesized that these results indicate that inhibition of MASP-2 activity has a protective or therapeutic effect against ischemia-induced renal injury.

Example 13

This example describes the results obtained in a study conducted on an experimental model of macular degeneration in MASP-2 -/- mice.

The level of knowledge of the problem and relevance. Age-related macular degeneration (AMD) is the leading cause of blindness after the age of 55 in industrialized countries. AMD occurs in two main forms: neovascular (wet) AMD and atrophic (dry) AMD. The neovascular (wet) form accounts for 90% of severe AMD-related vision loss, although only about 20% of individuals develop wet AMD. Clinical features of AMD include multiple drusen, geographic atrophy, and choroidal neovascularization (CNV). In December 2004, the US Food and Drug Administration (FDA) approved the drug makugen (pegaptanib), a new class of ophthalmic drugs to specifically target and block the effects of vascular endothelial growth factor (VEGF), for the treatment of wet (neovascular) AMD forms (Ng et al., Nat Rev. Drug Discov 5:123-32 (2006)). Although makugen represents a promising new therapeutic agent for the treatment of a subgroup of patients with AMD, there remains an urgent need to develop additional therapies for this complex disease. Numerous independent studies point to the central role of complement activation in the pathogenesis of AMD. The pathogenesis of choroidal neovascularization (CNV), the most serious form of AMD, may involve activation of complement pathways.

More than twenty-five years ago, in a publication by Ryan, S.J., Tr. Am. Opth. soc. LXXVII:707 745, 1979, an experimental animal model of laser-induced choroidal neovascularization (CNV) was described (Ryan, S.J., Tr. Am. Opth. Soc. LXXVII: 707-745, 1979). The model was originally developed for use in rhesus monkeys, but the same technology has been used for a long time to develop similar models of CNV in a range of laboratory animals, including mice (Tobe et al., Am. J. Pathol. 153:1641-46, 1998). In this model, laser photocoagulation is used to destroy Bruch's membrane, resulting in the formation of CNV-like membranes. The laser-induced model captures many important manifestations of this human disease (see recently published review by Ambati et al., Survey Ophthalmology 48:257293, 2003). At present, the laser-induced mouse model is well established and is widely used as an experimental basis in a large and growing body of scientific research. There is general agreement that the laser-induced model bears sufficient biological similarity to human choroidal neovascularization (CNV) and that preclinical pathogenesis and drug inhibition studies using this model are relevant to human CNV.

Methods.

A MASP-2(-/-) mouse was created as described in Example 1 and backcrossed for at least 10 generations with C57B1/6 mice. This study compared the results obtained in male MASP-2(-/-) mice and MASP-2(+/+) mice after laser injury in a laser-induced CNV, accelerated model of neovascular AMD, focusing on laser-induced volume CNV, by confocal laser scanning microscopy as a method for measuring tissue damage, and by ELISA assay to determine the levels of VEGF, a potential angiogenic factor involved in CNV, in the retinal pigment epithelium (RPE)/b choroid.

Induction of choroidal neovascularization (CNV).

Laser photocoagulation (532 nm, 200 mW, 100 ms, 75 µm; Oculight GL, Iridex, Mountain View, CA) was performed on both eyes of each animal on day zero by one staff member who did not know which group of animals was administered the drug. laser spots

- 87 042534 was applied in a standardized manner around the optic nerve using a slit lamp and cover slip system as a contact lens. The morphological endpoint of laser damage was considered to be the appearance of a cavitation bubble, a sign that was associated with damage to the Bruch's membrane. Detailing of the methods and evaluation of the obtained results are as follows.

Fluorescent angiography. Fluorescein angiography was performed with a camera and imaging system (TRC 50 1A camera; ImageNet 2.01 system; Topcon, Paramus, NJ) 1 week after laser photocoagulation. Photographs were taken using 20-D lenses in contact with fundus camera lenses after intraperitoneal injection of 0.1 ml of 2.5% sodium fluorescein. A retinal specialist with no involvement in laser photocoagulation or angiography evaluated fluorescein angiograms in a single session without knowledge of prior animal manipulation.

Choroidal neovascularization volume (CNV). One week after laser injury, the eyes were enucleated and fixed with 4% paraformaldehyde for 30 minutes at 4°C. Eyecups were prepared by removing the anterior segments and washed three times in PBS, then dehydrated and rehydrated using methanol. After blocking twice with buffer (PBS containing 1% bovine serum albumin and 0.5% Triton X-100) for 30 minutes at room temperature, the eyecups were incubated overnight at 4°C with 0.5% fluorescein isothiocyanate isolectin B4 (Vector laboratories, Burlingame, CA) diluted with PBS containing 0.2% BSA and 0.1% Triton X-100, which binds terminal e-D-galactose residues on the surface of endothelial cells and selectively labels the murine vasculature. After two washes with PBS containing 0.1% Triton X-100, the neurosensory retina was carefully peeled off and separated from the optic nerve. Four relaxing radial incisions were made and the remaining RPE-choroidal sclera complex was embedded in an anti-discoloration medium (Immu Mount Vectashield Mounting Medium; Vector Laboratories) and the coverslip removed.

Enclosed samples were examined using a laser confocal scanning microscope (TCS SP; Leica, Heidelberg, Germany). Vessels were visualized by excitation at 488 nm in the blue visible spectrum of argon and recording emissions between 515 and 545 nm. All imaging studies used a 40x oil immersion objective. Received horizontal optical sections (with a step of 1 μm) from the surface of the complex RPE-choroidal sclera. It was found that the deepest focal plane in which the surrounding choroidal vascular network associated with the lesion could be identified was the floor of the lesion. Any vessel located in the area that was targeted by the laser, and which is located on the surface of the above area, was evaluated as CNV. Images of each slice were stored digitally. The area of CNV-associated fluorescence was measured using computerized image analysis with microscope software (TCS SP; Leica). The summation of the entire area of fluorescence in each horizontal section was used as an indicator characterizing the volume of CNV. The imaging was performed by the operator, who was not aware of which group of animals was assigned to the administration of drugs.

Since the possibility of each laser injury leading to the development of CNV is affected by its belonging to one or another group (mouse, eye and laser spot), the average lesions volumes were compared using a linear mixed model with split areas with repeated measurements. The full site factor was the genetic group to which the animals belonged, while the split site factor was the eye. Statistical significance was determined at the 0.05 level. Post hoc comparisons of mean values were made using the Bonferroni correction for multiple comparisons.

Analysis of vascular endothelial growth factor (VEGF) by ELISA. Three days after injury with 12 laser spots, the RPE-choroid complex was sonicated in lysis buffer (20 mmol imidazole-HCl, 10 mmol KCl, 1 mmol MgCl ₂ , 10 mmol EGTA, 1% Triton X-100, 10 mmol NaF , 1 mmol sodium molybdate and 1 mmol EDTA with protease inhibitor) on ice for 15 minutes. Supernatant VEGF protein levels were determined using an ELISA assay kit (R&D Systems, Minneapolis, MN) that recognizes all splice variants at 450 to 570 nm (Emax; Molecular Devices, Sunnyvale, CA), and normalized to total protein. Duplicate measurements were performed by an operator who was not involved in photocoagulation, imaging, or angiography. VEGF amounts were presented as the mean +/- standard error of the mean in at least three independent experiments and compared using the Mann-Whitney U-test. The null hypothesis was rejected at P<0.05.

- 88 042534

Results.

Assessment of VEGF levels.

In FIG. 13A is a graphical representation of the levels of VEGF proteins in the RPE-choroid complex isolated from wild type C57B16 mice and MASP-2(-/-) mice on day zero. As shown in FIG. 13A, assessment of VEGF levels indicates a decrease in baseline VEGF levels in MASP-2(-/-) mice compared to C57b1 wild-type control mice. In FIG. 13B is a graphical representation of VEGF protein levels measured on the third day after laser-induced injury. As shown in FIG. 13B, VEGF levels were significantly elevated in wild-type (+/+) mice three days after laser-induced injury, consistent with published studies (Nozaki et al., Proc. Natl. Acad. Sci USA 103:2328-33 (2006 )). However, MASP-2(-/-) mice showed surprisingly very low levels of VEGF.

Assessment of choroidal neovascularization (CNV).

In addition to the reduction in VEGF levels after laser-induced macular degeneration, the area of CNV was determined before and after laser lesion. In FIG. 14 is a graphical representation of CNV volume measured in C57b1 wildcat mice and MASP-2(-/-) mice on day seven after laser-induced injury. As shown in FIG. 14, on the seventh day after laser-induced injury, MASP-2(-/-) mice showed an approximately 30% reduction in CNV area compared to a control group of wild mice.

These data indicate a reduction in VEGF levels and CNV area observed in MASP(-/-) mice compared to wild-type controls (+/+), and also that blocking MASP-2 with an inhibitor may have a prophylactic or therapeutic effect. action in the treatment of macular degeneration.

Example 14

This example shows that thrombin activation can occur after activation of the lectin pathway under physiological conditions, and characterizes the degree of involvement in the pathological process of MASP-2. In healthy rat serum, activation of the lectin pathway results in thrombin activation (as measured by thrombin deposition) simultaneously with complement activation (as measured by C4 deposition). As can be seen in FIG. 15A and 15B, thrombin activation in this system is inhibited by a blocking MASP-2 antibody (in Fab2 format) characterized by a thrombin activation concentration-effect curve (FIG. 15B) that runs parallel to the complement activation concentration-effect curve (FIG. 28A). These data suggest that the activation of the lectin pathway that occurs during injury should lead to the activation of both the complement system and the coagulation system, in a process that is completely dependent on MASP-2. In this regard, it can be assumed that MASP2 blocking antibodies may be effective in reducing the occurrence of excessive systemic coagulation, such as disseminated intravascular coagulation, which is one of the signs leading to mortality in most cases of injury.

Example 15

This example reports the results obtained using the local Schwartzman response model of disseminated intravascular coagulation (DIC) in MASP-2-/- deficient mice and MASP-2 (+/+) deficient mice to evaluate the role of lectin in DIC.

The level of knowledge of the problem and relevance.

As described above, blockade of MASP-2 inhibits the activation of the lectin pathway and reduces the generation of C3a and C5a anaphylatoxins. C3a anaphylatoxins can be shown to be potent platelet aggregators in vitro, but their involvement in vivo is less well understood, and release of platelets and plasmin during wound healing may involve complement C3 only secondarily. In this example, the role of the lectin pathway was examined in MASP-2 (-/-) mice and wild (+/+) mice in order to determine whether a sustained increase in C3 activation is necessary to generate disseminated intravascular coagulation.

Methods.

The MASP-2(-/-) mice used in this study were created as described in Example 1 and backcrossed for at least 10 generations to C57B1/6 mice. In this experiment, the local Schwartzmann reaction model was used. The local Schwartzmann reaction (LSR) is a lipopolysaccharide (LPS) induced reaction with a well-studied involvement of cellular and humoral elements of the innate immune system. The dependence of LSR on complement is well established (Polak, L, et al., Nature 223:738-739 (1969); Fong J.S. et al., J Exp Med 134:642-655 (1971)). In the LSR model, mice were vaccinated for 4 hours with tumor necrosis factor alpha (500 ng, intrascrotum), then the mice were anesthetized and prepared for live microscopy of the levator testis muscle. Networks of postcapillary venules (diameter 15–60 μm) with good blood flow (1–4 mm/s) were selected for observation. Animals were treated with fluorescent antibodies in order to selectively label neutrophils or platelets. The vascular network was sequentially scanned, and the images of all vessels at the last analysis were digitally recorded.

- 89 042534

After recording the basal state of the microcirculation, mice were administered LPS by a single intravenous injection (100 μg) either alone or with the following agents. The same vascular network was then scanned every 10 minutes for 1 hour. Specific fluorophore accumulation was identified by subtracting background fluorescence and amplifying by thresholding image brightness. The intensity of the reactions was measured from the recorded images. Summary data were the primary quantitative assessment of the Schwartzman reactions.

The studies compared MASP-2(+/+) sufficient mice or wild mice exposed to either a known compound that impairs complement pathway activation, cobra venom factor (CVF), or an inhibitor of terminal complement component activation (C5aR antagonist). The results (FIG. 16A) show that CVF, as well as the C5aR antagonist, prevented the appearance of aggregates in the vasculature. In addition, MASP-2(-/-) deficient mice (FIG. 16B) also showed complete inhibition of the local Schwartzman reaction, suggesting involvement of the lectin pathway. These results clearly show the role of MASP-2 in DIC generation and support the use of MASP-2 inhibitors to treat and prevent DIC.

Example 16

This example describes a study of MASP-2(-/-) mice in an experimental mouse kidney transplant model.

The level of knowledge of the problem and relevance.

The role of MASP-2 in the functional outcome of transplantation was evaluated using an experimental mouse model.

Methods.

The functional outcome of kidney transplantation was evaluated using a single kidney isograft to unilaterally nephrectomized recipient mice, among which the transplant recipients were six wild-type mice (+/+) (B6) and six MASP-2(-/-) mice. To assess the function of the transplanted kidney, the remaining native kidney was removed from the recipient 5 days after transplantation, and 24 hours later the kidney function was assessed by measuring blood urea nitrogen (BUN) levels.

Results.

In FIG. 17 is a graphical representation of kidney blood urea nitrogen (BUN) levels in wild-type recipients (+/+) and MASP-2(-/-) recipients on day 6 post-kidney transplantation. As shown in FIG. 17, strongly elevated BUN levels were observed in wild-type (+/+) (B6) transplant recipient mice (normal BUN levels in mice are < 5 mmol), indicating renal failure. In contrast, MASP-2(-/-) isograft recipient mice had significantly lower levels of BUN, indicating improved kidney function. It should be noted that these results were obtained using grafts from wild (+/+) kidney donor mice, suggesting that the absence of only a functional lectin pathway in the transplant recipient is sufficient to achieve a positive therapeutic effect.

Taken together, these results indicate that transient inhibition of the lectin pathway through MASP-2 inhibition provides a way to reduce disease incidence and delayed graft function in kidney transplantation, and that this approach appears to be applicable to other transplant cases.

Example 17.

This example shows that MASP-2(-/-) mice are resistant to septic shock in an experimental mouse model of septic polymicrobial peritonitis.

The level of knowledge of the problem and relevance.

To evaluate the potential effects of MASP-2(-/-) on infection, a cecal ligation and puncture (CLP) model study was performed, a polymicrobial septic peritonitis model. It is believed that this model most accurately mimics the course of septic peritonitis in humans. The cecal ligation and puncture (CLP) model is a model in which the caecum is ligated and punctured with a needle, which leads to the constant penetration of bacteria into the abdominal cavity, which through the lymphatic drainage enter the bloodstream and then spread to all organs of the abdominal cavity, causing multi-organ failure. and septic shock (Eskandari et al., J Immunol 148(9):2724-2730 (1992)). The CLP model mimics the course of sepsis observed in patients and induces an early hyperinflammatory response followed by a pronounced hypoinflammatory phase. During this phase, the animals show a high sensitivity to bacterial infections (Wichterman et al., J. Surg. Res. 29 (2); 189-201 (1980)).

Methods.

Mortality from polymicrobial infection was measured using a caecal ligation and puncture (CLP) model in wild-type (+/+) mice (n=18) and MASP-2 (-/-) mice (n=16). Briefly, MASP-2-deficient mice and littermate wild-type mice were anesthetized, and the caecum was temporarily brought to the surface of the body and ligated 30% above the distal tip. After that, the caecum was punctured once with a needle with a diameter of 0.4 mm. The caecum was then returned to

- 90 042534 the abdominal cavity, and the skin was pulled together with clamps. Within 14 days after CLP, conducted continuous monitoring of the survival of mice subjected to CLP. Sixteen hours after CLP, peritoneal washings were collected from mice to measure bacterial load. Serial dilutions of the peritoneal wash were prepared in PBS and inoculated into Mueller-Hinton plates and then incubated at 37°C under anaerobic conditions for 24 h, after which the bacterial load was determined.

16 hours after CLP, the cytokine response of tumor necrosis factor alpha to bacterial infection was also measured in the lungs and spleens of wild-type mice (+/+) and MASP-2(-/-) mice by quantitative real-time polymerase chain reaction (qRT- PCR). In addition, 16 hours after CLP, the level of tumor necrosis factor alpha in the serum of wildcat mice (+/+) and MASP-2(-/-) mice was quantified by sandwich ELISA analysis.

Results.

In FIG. 18 is a graphical representation of the percentage survival of animals subjected to CLP as a function of the number of days elapsed after the CLP procedure. As shown in FIG. 18, lectin pathway deficiency in MASP-2(−/−) mice does not increase mouse mortality after polymicrobial infection using a caecal ligation and puncture model compared to wild (+/+) mice. However, as shown in FIG. 19, after CLP, peritoneal washings of MASP-2(−/−) mice showed a significantly higher bacterial load (bacteria count was approximately 1000 times greater) than littermate wild (+/+) mice. These results indicate that MASP-2 deficient (-/-) mice exhibit resistance to septic shock. The reduced bacterial clearance in MASP-2 deficient mice in this model may be due to impaired C3b-mediated phagocytosis, as demonstrated by the fact that C3 deposition is MASP-2 dependent.

It was found that the cytokine response of tumor necrosis factor alpha to bacterial infection was not increased in MASP-2(-/-) mice compared to control wild mice (+/+) (data not shown). It was also found that the serum concentration of tumor necrosis factor alpha in wild mice (+/+) 16 h after CLP is significantly higher compared to MASP-2(-/-) mice, in which the level of tumor necrosis factor alpha in serum remained virtually unchanged. These results confirm that the intense inflammatory response to a bacterial infection condition was attenuated in MASP-2(-/-) mice, allowing the animals to survive in the presence of higher numbers of bacteria.

Taken together, these results demonstrate the potential negative effects of the lectin complement pathway in generalized infections and increased mortality in patients with generalized sepsis. In addition, these results indicate that MASP-2 deficiency modulates the inflammatory immune response and reduces expression levels of inflammatory mediators in sepsis. Therefore, inhibition of MASP-2(-/-) by administering inhibitory anti-MASP-2 monoclonal antibodies would be expected to be an effective method of attenuating the inflammatory response in a subject suffering from septic shock.

Example 18.

This example describes the study of MASP-2(-/-) mice in an experimental model of intranasal infection in mice.

The level of knowledge of the problem and relevance.

Pseudomonas aeruginosa (Pseudomonas aeruginosa) is a Gram-negative bacterial pathogen of human opportunistic infection that causes a wide range of infections, especially in immunocompromised individuals. Pseudomonas aeruginosa is the main source of acquired nosocomial infections, in particular, nosocomial pneumonia. It also causes significant morbidity and mortality in patients with cystic fibrosis (CF). Pseudomonas aeruginosa pulmonary infection is characterized by intense neutrophil recruitment and significant lung inflammation leading to extensive tissue damage (Palanki M.S. et al., J. Med. Chem 51:1546-1559 (2008)).

In this example, a study was conducted to determine if the removal of the lectin pathway in MASP-2(-/-) mice increases susceptibility to bacterial infections.

Methods.

Twenty-two wild-type mice (+/+), twenty-two MASP-2(-/-) mice and eleven C3(-/) mice were intranasally injected with a bacterial strain of Pseudomonas aeruginosa. Mice were monitored continuously for six days post-infection and Kaplan-Meier curves were plotted showing percentage survival.

Results.

In FIG. 20 is a Kaplan-Meier Percent Survival curve for non-mouse (+/+), MASP-2(-/-) mice, or C3(-/-) mice six days post infection. As shown in FIG. 20, no differences were found when MASP-2(-/-) mice were compared with wild-type (+/+) mice. However, deletion of the classical pathway (C1q) in C3(-/-) mice resulted in severe

- 91 042534 susceptibility to bacterial infection. These results indicate that inhibition of MASP-2 does not increase susceptibility to bacterial infection, suggesting that it is possible to reduce the incidence of unwanted inflammatory complications in trauma patients by inhibiting MASP-2 without impairing the patient's ability to resist infections using the classical complement pathway.

Example 19.

This example describes a pharmacodynamic study for typical high affinity Fab2 fragments of anti-MASP-2 antibodies that were identified as described in Example 10.

The level of knowledge of the problem and relevance.

As described in Example 10, to identify high affinity antibodies that block the lectin pathway in rats, the rat MASP-2 protein was used to search the phage display for antibody libraries. This library was designed to provide great immunological diversity, and was created using only human immunoglobulin gene sequences. As shown in Example 10, ELISA screening identified approximately 250 individual phage clones that bind to the rat MASP2 protein with high affinity. Sequencing of these clones identified 50 unique phages encoding the MAPS-2 antibody. From these clones, the Fab2 protein was expressed, purified and examined for MASP-2 binding affinity and functional inhibition of the complement lectin pathway.

As shown in Table. 6 of Example 10, this analysis identified 17 Fab2 fragments of anti-MASP-2 antibodies with functional blocking activity (34% blocking antibody search efficiency). Functional inhibition of the lectin complement pathway by Fab2 was detected by the level of C4 deposition, which is a direct indicator of C4 cleavage upon exposure to MASP-2. It is important to note that inhibition was equally confirmed when assessing the activity of C3-convertase, which substantiated the functional blockade of the complement lectin pathway. The 17 MASP-2 blocking Fab2s identified as described in Example 10 effectively inhibit the formation of C3 convertase with IC ₅₀ values equal to or less than 10 nmol. Eight of the 17 identified Fab2 are characterized by the value of the IC ₅₀ in the subnanomolar range of values. In addition, all 17 MASP-2 blocking Fab2s showed virtually complete inhibition of C3 convertase formation when assaying the C3 convertase lectin pathway, as shown in FIG. 8A-C and presented in table. 6 of example 10. Moreover, each of the 17 Fab2, blocking anti-MASP-2, are shown in table. 6 effectively inhibits C3b generation (>95%), thus demonstrating the specificity of this assay for the C3 convertase lectin pathway.

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

Methods.

As described in Example 10, the rat MASP-2 protein was used to search for Fab in the phage display of antibody libraries from which Fab2 #11 was identified. Rat IgG2c and mouse IgG2a full-length antibody isotype variants were generated from Fab2 #11. Rat IgG2c and mouse IgG2a full-length antibody isotypes were characterized in vivo for pharmacodynamic parameters as described below.

An in vivo study in mice.

To study the effect of anti-MASP-2 antibody dosing on plasma lectin pathway activity in vivo, a pharmacodynamic study was performed in mice. In this study, ex vivo C4 deposition was measured in the lectin pathway assay at different time points after subcutaneous (sc) and intraperitoneal (ip) administration of 0.3 mg/kg or 1.0 mg/kg of a mouse monoclonal antibody (MoAb) against MASP- 2 (mouse IgG2a full length antibody isotype derived from Fab2 #11).

In FIG. 21 is a graphical representation of the C4 specific deposition lectin pathway measured ex vivo in undiluted serum samples taken from mice (n=3 mice/group) at various time points after subcutaneous dosing of 0.3 mg/kg or 1.0 mg/kg mouse monoclonal antibodies (MoAb) against MASP-2. Mouse serum samples collected prior to antibody dosing were used as a negative control (100% activity) and serum supplemented in vitro with 100 nmol of the same anti-MASP-2 blocking antibody was used as a positive control (0% activity).

The results shown in FIG. 21 demonstrate rapid and complete inhibition of C4b deposition following a 1.0 mg/kg subcutaneous dose of anti-MASP-2 mouse monoclonal antibody (Mo Ab). Partial inhibition of C4 deposition was observed after a subcutaneous dose of 0.3 mg/kg mouse anti-MASP-2 monoclonal antibody (MoAb).

Restoration of the lectin pathway occurred within three weeks after a single intraperitoneal injection of a mouse monoclonal antibody (MoAb) against MASP-2 to mice at a dose of 0.6

- 92042534 mg/kg. As shown in FIG. 22, a dramatic decrease in lectin pathway activity occurred after antibody dosing, followed by complete inhibition of the lectin pathway, which lasted for approximately 7 days after intraperitoneal administration. Slow recovery of lectin pathway activity was observed during the second and third weeks with complete recovery of the lectin pathway in mice 17 days after administration of a mouse monoclonal antibody (MoAb) against MASP-2.

These results indicate that the mouse anti-MASP-2 monoclonal antibody (MoAb) derived from Fab2 #11 inhibits the mouse lectin pathway in a dose dependent manner when administered systemically.

Example 20.

This example describes the efficacy analysis of a mouse anti-MASP-2 monoclonal antibody (MoAb) derived from Fab2 #11 in a mouse model of age-related macular degeneration.

The level of knowledge of the problem and relevance.

As described in Example 10, the rat MASP-2 protein was used to search for Fab in the phage display of antibody libraries from which Fab2 #11 was identified as a functional antibody. Generated from Fab2 No. 11 isotypes of full-length antibodies of rat IgG2c and mouse IgG2a. Full-length anti-MASP-2 mouse IgG2a isotype was determined for pharmacodynamic parameters as described in Example 19. In this example, full-length anti-mouse MASP-2 antibody derived from Fab2 #11 was tested in an experimental model of age-related macular degeneration (AMD) on mice, which is described in the publication Bora P.S. et al., J Immunol 174:491-497 (2005).

Methods.

The mouse IgG2a full-length antibody isotype derived from Fab2 #11 as described in Example 19 was tested in a mouse age-related macular degeneration (AMD) model as described in Example 13 with the following modifications.

Introduction of monoclonal antibodies (MoAb) against mouse MASP-2.

Two different doses (0.3 mg/kg and 1.0 mg/kg) of anti-mouse MASP-2 monoclonal antibodies (MoAb), along with isotype control monoclonal antibody (MoAb) treatment, were administered intraperitoneally to wild-type mice (+/+) (n=8 mice per group) 16 hours before CNV induction.

Induction of choroidal neovascularization (CNV).

Choroid neovascularization (CNV) induction and CNV volume measurement were performed using laser photocoagulation as described in Example 13.

Results.

In FIG. 23 is a graphical representation of CNV area measured 7 days after laser injury in mice treated with monoclonal antibody (MoAb) for isotype control or monoclonal antibody (MoAb) against mouse MASP-2 (0.3 mg/kg and 1.0 mg/kg ). As shown in FIG. 23, mice pretreated with anti-MASP-2 monoclonal antibody (MoAb) at 1.0 mg/kg showed a statistically significant (p<0.01) approximately 50% reduction in CNV seven days after laser exposure. As further shown in FIG. 23, it was found that a dose of 0.3 mg/kg of anti-MASP-2 monoclonal antibody (MoAb) was not effective in reducing CNV. It should be noted that a dose of 0.3 mg/kg of anti-MASP-2 monoclonal antibody (MoAb) was found to have a partial and transient inhibitory effect on C4b deposition after subcutaneous administration, as described in Example 19 and shown in FIG. 21.

The results described in this example demonstrate that blockade of MASP-2 with an inhibitor such as an anti-MASP-2 monoclonal antibody (MoAb) has a prophylactic and/or therapeutic effect in the treatment of macular degeneration. It should be noted that these results are consistent with those obtained in the MASP-2(-/-) mouse study described in Example 13, in which a 30% reduction in CNV was observed in MASP-2(-/-) 7 days after laser treatment. ) mice compared to control wild mice. Moreover, the results in this example further demonstrate that systemic delivery of anti-MASP-2 antibody provides a local beneficial therapeutic effect in the eye, thus highlighting the efficacy of the systemic route of administration in treating patients with AMD. Thus, these results provide evidence for the use of monoclonal antibodies (MoAb) against MASP-2 in the treatment of AMD.

Example 21.

This example shows that MASP-2 deficient mice after meningococcal infection are protected from meningococcal-induced mortality and have increased clearance when bacteremic compared to wild-type control mice.

Relevance. Meningococcus is a heterotrophic gram-negative diplococcal bacterium known for its role in causing meningitis and other forms of meningococcal disease such as meningococcemia. Meningococcus is the leading cause of morbidity and mortality in childhood. Serious complications include septicemia, Waterhouse syndrome - 93 042534

Friederiksen, adrenal insufficiency and disseminated intravascular coagulation (DIC). See, for example, Rintala E. et at., Critical Care Medicine 28(7):2373-2378 (2000). In this example, the role of the lectin pathway was examined in MASP-2 (-/-) mice and wild (+/+) mice to see if MASP-2 deficient mice were susceptible to meningococcal death.

Methods.

MASP-2 knockout mice were created as described in Example 1 and backcrossed for at least 10 generations to C57B1/6 mice. MASP-2 KO mice (n=10) and wild-type C57/B6 mice (n=10) at 10 weeks of age were inoculated by intravenous injection at a dose of 5 x ^{10 8} cfu/100 µl, 2 x ^{10 8} cfu/100 µl, or 3 x 10 ⁷ cfu/100 µl meningococcus serogroup A Z2491 in 400 mg/kg iron dextran. The survival of mice after infection was continuously monitored for 72 hours. Blood samples were taken from mice one hour after infection and analyzed to determine the level of meningococcus in serum (log CFU / ml) to confirm infection and determine the rate of clearance of bacteria from serum.

Results.

In FIG. 24A is a graphical representation of the percentage survival of MASP-2 KO mice and wild mice following an infective dose of 5 x 10 ⁸ cfu/100 μl of meningococcus. As shown in FIG. 24A, after infection with the highest dose of meningococcus 5 x 10 ⁸ cfu/100 μl, 100% of MASP-2 KO mice survived 72 hours after infection. In contrast, only 20% of the wild mice were still alive 24 hours after infection. These results indicate that MASP-2 deficient mice are protected from death associated with meningococcal infection.

In FIG. 24B is a graphical representation of log cfu/ml of meningococcus measured at various time points in blood samples taken from MASP-2 KO mice and wild mice infected with meningococcus 5 x 10 ⁸ cfu/100 μl. As shown in FIG. 24B, blood levels of meningococcus in wild mice peaked at approximately log CFU/mL 6.5 at 24 hours post-infection and dropped to zero at 48 hours post-infection. In contrast, in MASP-2 KO mice, meningococcal levels peaked at approximately log CFU/mL 3.5 at 6 hours post-infection and dropped to zero at 36 hours post-infection.

In FIG. 25A is a graphical representation of the percentage survival of MASP-2 KO mice and wild mice after infection with a dose of 2 x 10 ⁸ cfu/100 μl of meningococcus. As shown in FIG. 25A, after infection with a dose of 2 x 10 ⁸ cfu/100 μl of meningococcus, 100% of MASP-2 KO mice survived during the 72 hour post-infection period.

In contrast, only 80% of the wild mice remained alive 24 hours after infection. According to the results shown in FIG. 24A, these results further show that MASP-2 deficient mice are protected from meningococcal death.

In FIG. 25B is a graphical representation of log cfu/ml of meningococcus measured at various time points in blood samples taken from wild mice infected with meningococcus 2 x 10 ⁸ cfu/100 μl. As shown in FIG. 25B, blood levels of meningococcus in wild mice infected with 2×10 ⁸ cfu/100 μl reached a maximum value of approximately log cfu/ml 4 12 hours post-infection and decreased to zero 24 hours post-infection. In FIG. 25C is a graphical representation of log cfu/ml meningococcus measured at various time points in blood samples taken from MASP-2 KO mice infected with meningococcus 2 x 10 ⁸ cfu/100 μl. As shown in FIG. 25C, the level of meningococcus in the blood of MASP-2 KO mice infected with 2x10 ⁸ cfu/100 μl, reached a maximum value of approximately log cfu/ml 3.5 at 2 h after infection, and decreased to zero after 3 h after infection. According to the results shown in FIG. 24B, these results show that, despite the fact that MASP-2 KO mice were infected with the same dose of meningococcus as wild-type mice, MASP-2 KO mice have increased bacteremic clearance compared to wild-type mice.

The percentage survival of MASP-2 KO mice and wild mice after infection with the lowest dose of meningococcus 3x10 ⁷ cfu/100 μl was 100% within 72 hours after infection (data not shown).

Discussion.

These results show that with MASP-2 deficient mice are protected from meningococcal death and have increased clearance in bacteremia compared to wild mice. Therefore, in view of these results, the therapeutic use of MASP-2 inhibitors, such as the MASP-2 monoclonal antibody, would be expected to be effective in treating, preventing, or attenuating meningococcal infection (ie, sepsis and DIC). In addition, these results indicate that the therapeutic use of MASP-2 inhibitors, such as the MASP-2 monoclonal antibody, does not increase the risk of meningococcal infection in the subject.

- 94 042534

Example 22.

This example describes the discovery of a new lectin pathway mediated and MASP-2 dependent alternative C4 activation pathway of complement C3.

Relevance.

The main beneficial therapeutic effect of using complement activation inhibitors to limit myocardial injury due to ischemia/reperfusion (MIRI) was convincingly demonstrated in an experimental model of myocardial infarction in rats twenty years ago. Recombinant sCR1, a soluble truncated derivative of the cell surface complement receptor type-1 (CR1), was intravenously administered and evaluated in a rat MIRI model. Treatment with sCRl reduced infarct volume by more than 40% (Weisman, H.F., et al., Science 249:146-151 (1990)). The therapeutic potential of this recombinant inhibitor was then demonstrated in a clinical trial which showed that administration of sCR1 to patients with MI prevented heart failure after ischemia (Shandelya, S., et al., Circulation 87:536-546 (1993)). However, the primary mechanism leading to complement activation in ischemic tissue has not been fully elucidated, mainly due to the lack of appropriate experimental models, insufficient understanding of the molecular processes that lead to complement activation of oxygen-depleted cells, and the mutual influence and synergistic effects between different pathways. complement activation.

As a major component of the immune response, the complement system provides protection against invading microorganisms through both antibody-dependent and independent mechanisms. It governs many cellular and humoral interactions in the immune response, including chemotaxis, phagocytosis, cell adhesion, and B cell differentiation. Three distinct pathways initiate the cascade of complement reactions: the classical pathway, the alternative pathway, and the lectin pathway. The C1q recognition subcomponent of the classical pathway binds to a variety of targets—the most important immune complexes—to initiate the stepwise activation of associated serine proteases, C1r and C1s, providing a major pathogen and immune complex clearing mechanism once the adaptive immune system has initiated. Binding of C1q to immune complexes converts the C1r zymogen dimer to its active form for cleavage and therefore activation of C1s. C1s translates C1q binding into complement activation via two cleavage steps: first, C4 is converted to C4a and C4b, and then C4b-bound C2 is cleaved to form C3 convert C4b2a. This complex converts the C3 component present in abundance in plasma into C3a and C3b. Accumulation of C3b in the immediate vicinity of the C4b2a complex changes the substrate specificity for C3 to C5 with the formation of C5-convertase C4b2a(C3b)n. The C3- and C5-convertase complexes generated as a result of activation of the classical pathway are identical to those generated as a result of activation of the lectin pathway. In an alternative pathway, spontaneous minor hydrolysis of the C3 component leads to the deposition of protein fragments on cell surfaces, initiating complement activation on foreign cells, while cell-bound regulatory proteins on host tissues prevent activation, thereby preventing self-injury. As with the alternative pathway, the lectin pathway can be activated in the absence of immune complexes. Activation is initiated by binding of the lectin pathway's multimolecular activation complex to pathogen-associated molecular patterns (PAMPs), primarily carbohydrate structures present on bacterial, fungal, or viral pathogens, or to aberrant glycosylation patterns on apoptotic, necrotic, malignant, or oxygen-depleted cells. (Collard, C.D., et al., Am. J. Pathol. 156:1549-1556 (2000); Walport, M.J., N. Engl. J. Med. 344:1058-1066 (2001); Schwaeble, W., et al., Immunobiology 205:455466 (2002) and Fujita, T., Nat Rev. Immunol 2:346-353 (2002)).

Mannan-binding lectin (MBL) was the first carbohydrate recognition subcomponent shown to form complexes with a group of new serine proteases called MBL-associated serine proteases (MASPs) and numbered according to the order in which they were discovered (i.e., MASP- 1, MASP-2 and MASP-3). In humans, lectin pathway activation complexes can form with four alternative carbohydrate recognition subcomponents with different carbohydrate binding specificities, i.e. MBL 2, and with three different members of the ficolin family, namely L-ficolin, H-ficolin and M-ficolin , and MASP. The two forms of MBL, MBL A and MBL C, and ficolin-A form lectin pathway activation complexes with MASP in mouse and rat plasma. The inventors have previously cloned and characterized MASP-2 and an additional 19 kDa truncated MASP-2 gene product, referred to as MAP19 or sMAP, in humans, mice and rats (Thiel, S., et al., Nature 386:506- 510 (1997); Stover, C. M., et al., J. Immunol. 162:34813490 (1999); Takahashi, M., et al., Int. Immunol. 11:859-863 (1999); and Stover, C M, et al., J. Immunol 163:6848-6859 (1999)). MAp19/sMAP lacks protease activity but can regulate lectin pathway activation by competing with carbohydrate recognition complexes for MASP binding (Iwaki, D. et al., J. Immunol. 177:8626-8632 (2006)).

There is evidence that confirms that of the three MASPs, only MASP-2 is needed

- 95 042534 to translate binding of lectin pathway recognition complexes into complement activation (Thiel, S., et al. (1997); Vorup-Jensen, T., et al., J. Immunol. 165:2093-2100 (2000); Thiel, S., et al., J. Immunol. 165:878-887 (2000); Rossi, V., et al., J. Biol. Chem. 276:40880-40887 (2001)). This conclusion is supported by the phenotype of a very recently described MASP-1 and MASP-3 deficient mouse strain. Apart from the delay in onset of the lectin pathway mediated by complement activation in vitro, -MASP-1/3 deficient mice retain functional lectin pathway activity. Reconstruction of MASP-1 and MASP-3 deficient serum with recombinant MASP-1 overcomes this delay in lectin pathway activation, indirectly indicating that MASP-1 may promote MASP-2 activation (Takahashi, M., et al., J Immunol 180:6132-6138 (2008)). In the most recent study, MASP-1 (and possibly also MASP-3) has been shown to be required for the conversion of the alternative pathway enzyme activation factor D from its zymogenic form to its enzymatically active form (Takahashi, M., et al., J. Exp Med 207:29-37 (2010)). The physiological significance of this process is confirmed by the absence of functional activity of the alternative pathway in the plasma of MASP-1/3 deficient mice.

Recently generated mouse strains with combined targeted deficiencies in the MBL A and MBL C lectin pathway carbohydrate recognition subcomponents are still able to initiate lectin pathway activation by the remaining mouse ficolin A recognition subcomponent of the lectin pathway (Takahashi, K., et al., Microbes Infect. 4: 773-784 (2002)). The absence of any residual functional activity of the lectin pathway in MASP-2 deficient mice makes it possible to create a valid model for studying the role of this effector group of innate humoral immunity in normal and pathological conditions.

The availability of C4 and MASP-2 deficient mouse strains allows the identification of a novel lectin pathway, a specific but MASP-2 dependent alternative pathway for activation of C4 complement C3. The critical contribution of this new lectin pathway, mediated by an alternative C4 activation pathway, to tissue loss after ischemia is supported by the pronounced protective phenotype of MASP-2 deficiency in myocardial injury due to ischemia/reperfusion (MIRI), while in C4-deficient mice tested in the same model, did not detect this protection.

In this example, a new lectin pathway, mediated and MASP-2 dependent alternative C4 activation pathway of C3 complement is described. The physiological significance of this novel activation pathway has been established based on the protective phenotype of MASP-2 deficiency in an experimental model of myocardial injury due to ischemia/reperfusion (MIRI), in which C4-deficient animals were not protected.

Methods.

MASP-2 deficient mice do not show severe abnormalities. MASP-2 deficient mice were created as described in Example 1. Both heterozygous (+/-) and homozygous (-/-) MASP-2 deficient mice are healthy and fertile, and they do not show severe forms of disorders. Their average lifespan is similar to that of their wildcat littermates (>18 months). Before studying the phenotype of these mice in experimental disease models, MASP-2-/- mice were backcrossed for eleven generations with C57BL/6 mice. The complete absence of MASP-2 mRNA was confirmed by Northern blotting of poly (A+) preparations of selected liver RNAs, while 1.2 kb (thousand nucleotide pairs) of mRNA encoding MAP19 or sMAP (truncated alternative splicing product of the MASP-2 gene) is expressed. in excess.

Real-time quantitative polymerase chain reaction (qRT-PCR) analysis using a pair of primers specific for either the coding sequence for the MASP-2 serine protease domain (B chain) or the coding sequence residue for the A chain showed that the mRNA-coding B strand was not detected in MASP-2-/- mice, while the excess of the broken A-strand mRNA transcript was significantly increased. Similarly, the excess of MAp19/sMAP encoding mRNA is increased in MASP-2+/- mice and MASP-2 -/- mice. Plasma MASP-2 levels determined by ELISA for 5 animals of each genotype were 300 ng/mL for control wild mice (range 260-330 ng/mL), 360 ng/mL for heterozygous mice (range 330-395 ng/mL). ) and were not detected in MASP-2 -/- mice. Using qRT-PCR, mRNA expression profiles were determined that showed that MASP-2 -/- mice express mRNA for MBL A, MBL C, ficolin A, MASP-1, MASP-3, C1q, C1rA, C1sA, factor B, factor D, C4 and C3 in excess, similar to the excess in the case of MASP-2 sufficient littermates (data not shown).

Plasma C3 levels of MASP-2-/- (n=8) and MASP-2+/+ (n=7) litter mice were measured using a commercially available mouse C3 ELISA kit (Kamiya, Biomedical, Seattle, WA). C3 levels of MASP2 deficient mice (mean 0.84 mg/ml + 0.34) were similar to wild control mice (mean 0.92 ± 0.37).

Results.

MASP-2 is essential for the functional activity of the lectin pathway.

- 96 042534

As described in Example 2 and shown in FIG. 5, in vitro assays of MASP-2 -/- in plasma showed a complete lack of functional activity of the lectin pathway on activated mannan- and zymosan-coated surfaces for C4 activation. Similarly, neither lectin pathway dependent C4 nor C3 cleavage was detected in plasma MASP-2-/- on surfaces coated with Nacetylglucosamine, which binds and initiates activation via MBL A, MBL C and ficolin A (data not shown) .

Serum and plasma analyzes of MASP-2 −/− mice clearly showed that MASP-2 is essential for complement activation via the lectin pathway. However, a general deficiency in the functional activity of the lectin pathway does not affect complement activation in other pathways. MASP-2-/- plasma can still activate complement via the classical (FIG. 26A) and alternative pathways (FIG. 26B). In FIG. 26A and 26B, * indicates wild mouse sera (MASP-2 (+/+)); symbol denotes the serum of wild mice (depleted in C1q); the symbol □ denotes the serum of MASP-2 (-/-) mice; and the symbol A denotes the serum of MASP-2 (-/-) mice (depleted in C1q).

In FIG. 26A graphically shows that the MASP-2 -/- mouse retains the functional classical pathway. C3b deposition was studied on microtiter plates coated with immune complexes (generated in situ by applying BSA, then adding goat anti-BSA IgG). In FIG. 26B graphically shows that MASP-2 deficient mice retain a functional alternative pathway. C3b deposition was studied on zymosan-coated microtiter plates under conditions that only allow activation of the alternative pathway (buffer containing Mg2 ⁺ and EGTA). The results shown in FIG. 26A and FIG. 26B are averages of two measurements and are typically obtained from three independent experiments. All figures used the same symbols for plasma data. These results indicate that a functional alternative pathway is present in MASP-2 deficient mice, as evidenced by the results shown in FIG. 26B, under experimental conditions designed to directly initiate an alternative pathway, while inactivating both the classical pathway and the lectin pathway.

The lectin pathway of complement activation highly promotes inflammatory tissue loss in ischemia/reperfusion myocardial injury (MIRI).

To investigate the contribution of functional activity of the lectin pathway to myocardial injury due to ischemia/reperfusion (MIRI), we compared MASP-2-/- mice and control littermate wild mice in an experimental MIRI model after transient ligation and reperfusion of the left anterior descending branch of the coronary artery (LAD ). The presence or absence of complement C4 has no effect on the extent of ischemic tissue loss in MIRI. The effect of C4 deficiency on infarct size after experimental myocardial injury due to ischemia/reperfusion (MIRI) was evaluated. As shown in FIG. 27A and FIG. 27B, identical infarct sizes were found in both C4 deficient mice and wildcat littermates. In FIG. 27A is a graphical representation of MIRI-induced tissue loss after LAD ligation and reperfusion in C4 −/− mice (n=6) and their respective control litter wild mice (n=7). In FIG. 27B is a plot of infarct area (INF) versus area at risk (AAR), clearly demonstrating that C4 −/− mice are also susceptible to MIRI as are their wild-type controls (dashed line).

These results indicate that C4 deficient mice are not protected from MIRI. This result was unexpected, as it contradicts the widely accepted view that the major C4 activation fragment, C4b, is a necessary component of the classical and lectin pathway C3-convertase C4b2a. Therefore, the possibility of detecting residual specific activation of complement C3 along the lectin pathway in the plasma of C4-deficient human mice was investigated.

The lectin pathway can activate complement C3 in the absence of C4 via a novel MASP-2 dependent alternative C4 activation pathway.

Based on the results of scientific studies of the past years, which indicate the existence of alternative activation of C4 in the serum of C4-deficient guinea pigs (May, J. E., and M. Frank, J. Immunol. 111:1671-1677 (1973)), a study was made of the presence in C4-deficient mice of residual functional activity of the classical or lectin pathway and consistently recorded C3 activation under pathway-specific study conditions that exclude the contribution of the alternative pathway.

C3b deposition was tested on mannan-coated microtiter plates using decalcified plasma at plasma concentrations preventing the alternative pathway of activation (1.25% and below). Although C3 cleavage was not detected in C4-deficient plasma tested for classical pathway activation (data not shown), strong residual C3 cleavage activity was nevertheless observed in the plasma of C4-deficient mice upon initiation of complement activation via the lectin pathway. Lectin pathway dependence is demonstrated by competitive inhibition of C3 cleavage after preincubation of dilutions of C4 deficient plasma with soluble mannan (see FIG. 28A). As shown in FIG. 28A-D,

- 97 042534 found MASP-2 dependent activation of C3 in the absence of C4. In FIG. 28A is a graphical representation of C3b deposition with C4+/+ (crosses) and C4-/- (open circles) mouse plasma. Pre-incubation of C4 -/- plasma with an excess (1 μg/ml) of mannan in the liquid phase before the assay resulted in complete inhibition of C3 deposition (solid circles). The results were obtained in three independent experiments. In FIG. 28B is a graphical representation of the results of an experiment in which plasma (1%) of wild-type, MASP-2 deficient (open squares) and C4-/ mice were mixed with various concentrations of anti-rat MASP-2 mAbMll (abscissa) and C3b deposition on plates with applied with mannan. Results are means (± SD) of 4 studies (two replicates for 2 of each plasma type). In FIG. 28C is a graphical representation of the results of an experiment in which human plasma combined with NHS (crosses), C4-/- plasma (open circles) and C4-/- plasma were pre-incubated with 1 μg/ml mannan (solid circles). The results represent three independent experiments. In FIG. 28D graphically shows inhibition of C3b deposition in C4 deficient and C4 deficient human plasma (1%) by anti-human MASP-2 mAbH3 (mean ± SD of three replicates). As shown in FIG. 28B did not detect lectin pathway dependent C3 activation in MASP-2-/- plasma assayed in parallel, indicating that this C4 alternative C3 activation is MASP-2 dependent.

To further confirm these findings, a number of recombinant inhibitory monoclonal bodies (mAbs) were generated, isolated from phage display antibody libraries by affinity screening for recombinant human and rat MASP-2A (where the serine residue of the protease active domain was replaced with an alanine residue by site-directed mutagenesis to prevent autolytic degradation of the antigen). Recombinant anti-MASP-2 antibodies (AbH3 and AbM11) were isolated from combinatorial antibody libraries (Knappik, A., et al., J. Mol. Biol. 296:5786 (2000)), using recombinant human and rat MASP-2A as antigens (Chen, CB and Wallis, J. Biol. Chem. 276:25894-25902 (2001)). An anti-rat Fab2 fragment that effectively inhibited lectin-mediated activation of C4 and C3 in mouse plasma (IC ₅₀ ~ 1 nM) was converted into a full length IgG2a antibody. Polyclonal antiserum against mouse MASP-2A was induced in rats. These tools allowed confirmation of the MASP-2 dependence of this new lectin pathway specific to the C4 alternative pathway of C3 activation, further described below.

As shown in FIG. 28B, M211, an inhibitory monoclonal antibody that selectively binds to mouse and rat MASP-2, inhibited C4-alternative C3 activation in C4-deficient mice, as well as C3 activation in wild-type mice via the lectin pathway, in a concentration-dependent manner with similar _IC50 values. . All studies were performed at high plasma dilutions, rendering the alternative activation pathway dysfunctional (with the highest plasma concentration being 1.25%).

To investigate the presence of a similar lectin pathway specific to human C4 alternative C3 activation, plasma from a donor with congenital deficiency of both human C4 genes (i.e., C4A and C4B) resulting in complete absence of C4 was analyzed (Yang, Y., et al., J Immunol 173:2803-2814 (2004)). In FIG. 28C shows that this patient's plasma effectively activates C3 at high plasma dilutions (making the alternative activation pathway dysfunctional). A specific method for activating C3 via the lectin pathway on mannan-coated plates was demonstrated in C4-deficient mouse plasma (FIG. 28A) and C4-deficient human plasma (FIG. 28C) by adding excess concentrations of mannan in the liquid phase. The MASP-2 mechanism dependence of this C3 activation in C4-deficient human plasma was assessed using AbH3, a monoclonal antibody that specifically binds human MASP-2 and suppresses MASP-2 functional activity. As shown in FIG. 28D, AbH3 inhibited C3b (and C3dg) deposition in both C4 deficient and C4 deficient human plasma with comparable activity.

To assess the possible role of other complement components in C4-alternative C3 activation, plasma testing of MASP-1/3-/- and Bf/C2-/- mice was performed, along with MASP-2-/-, C4-/- and C1q- /-plasma (as a control) under specific study conditions for both the lectin pathway and the classical pathway. The relative magnitude of C3 cleavage was plotted against the amount of C3 deposition using wild mouse plasma.

In FIG. 29A is a graphical comparison of plasma C3 convertase activity in various strains of complement deficient mice tested under specific study conditions for both the lectin pathway and the classical pathway. Diluted plasma samples (1%) wild mice (n=6), MASP-2-/- mice (n=4), MASP-1/3-/- mice (n=2), C4-/- mice (n =8), C4/MASP-1/3-/- mice (n=8), Bf/C2-/- (n=2) and C1q-/- mice (n=2) were tested in parallel. Reconstruction of Bf/C2-/- plasma with 2.5 μg/ml recombinant rat C2 (Bf/C2-/+C2) restored C3b deposition. Results are means (±SD), **p<0.01 (compared to wild mouse plasma). As shown in FIG. 29A, there is a

- 98 042534 venous deposition of C3 in C4-/-plasma tested under specific study conditions for the lectin pathway, but under specific study conditions for the classical pathway. And in this case, C3 deposition was not observed in MASP-2 deficient plasma as a result of activation of the lectin pathway, while in the same plasma C3 deposition occurred along the classical pathway. In MASP-1/3-/- plasma, C3 deposition occurs under both the specific conditions of the lectin pathway study and the specific conditions of the classical pathway study. C3 deposition was not detected in plasma deficient in C4 and MASP-1/3, either using the specific conditions of the lectin pathway assay or using the specific conditions of the classical pathway assay. There is no deposition of C3 in C2/Bf-/- plasma, either via the lectin pathway or the classical pathway. However, plasma reconstruction of C2/Bf −/− mice with recombinant C2 restored C3 cleavage mediated by both the lectin and classical pathways. The study conditions were validated using C1q -/- plasma.

In FIG. 29B is a graphical representation of data from a time-resolved kinetic study of C3 convertase activity in plasma of various strains of complement-deficient wild-type, fB-/-, C4-/-, MASP-1/3-/-, and MASP-2-/- mice tested. under specific conditions of the study of activation of the lectin pathway (1% plasma, the results are usually obtained in three independent experiments). As shown in FIG. 29B, while C3 cleavage was not detected in MASP-2-/- plasma, fB-/- plasma cleaved C3 with similar kinetics to wild mouse plasma. A significant delay in the lectin pathway-dependent conversion of C3 to C3b (and C3dg) was observed in C4-/-plasma as well as in MASP-1/3 deficient plasma. It has recently been shown that this delay in C3 activation in MASP-1/3-/- plasma should be MASP-1 dependent and not MASP-3 dependent (Takahashi, M., et al., J. Immunol. 180:6132- 6138 (2008)).

Discussion.

The results described in this example strongly suggest that MASP-2 functional activity is required for C3 activation via the lectin pathway in both the presence and absence of C4. In addition, C2 and MASP-1 are required for this new lectin-specific C4-alternative C3 activation pathway to function. A comparative analysis of the functional activity of the lectin pathway in MASP-2-/- plasma, as well as in C4-/- plasma, revealed the existence of a previously unrecognized C4-independent, but MASP-2-dependent C3 complement activation pathway, and showed that C3 may be activated in a lectin pathway dependent manner in the complete absence of C4. Although the detailed molecular compositions and sequence of events of activation of this novel MASP-2 dependent C3 convertase remain unclear, the results presented imply that this C4 alternative activation requires the presence of complement C2 as well as MASP-1. The loss of activation of lectin-mediated C3 cleavage in the plasma of mice with combined C4 and MASP-1/3 deficiency may be explained by the more recently described role of MASP-1 in enhancing MASP-2 dependent complement activation by direct cleavage and activation of MASP-2 (Takahashi , M., et al., J. Immunol 180:6132-6138 (2008)). Similarly, MASP1 can enhance the functional activity of MASP-2 due to its ability to cleave C2 (Moller-Kristensen, et al., Int. Immunol. 19:141-149 (2007)). Both of these activities may be explained by the reduced rate at which MASP-1/3 deficient plasma cleaves C3 via activation via the lectin pathway, and that activation of MASP-1 may be required to maintain C3 conversion via the C4 alternative pathway.

The inability of C2/fB-/- plasma to activate C3 via the lectin pathway was shown to be C2-dependent, as the addition of recombinant rat C2 to C2/fB-/- plasma restored the ability of reconstituted plasma to activate C3 on mannan-coated plates.

The finding that C4 deficiency specifically disrupts the classical complement pathway, while the lectin pathway maintains a physiologically very important level of C3 convertase activity through a MASP-2 dependent C4 alternative pathway, calls for a reassessment of the role of the lectin pathway in various experimental disease models, including experimental model of pneumococcal infection (Brown, J.S., et al., Proc. Natl. Acad. Sci. U.S.A. 99:16969-16974 (2002); Experimental Allergic Encephalomyelitis (Boos, L.A., et al., Glia 49:158-160 ( 2005); and a mouse model of C3 dependent liver regeneration (Clark, A., et al., Mol. Immunol. 45:3125-3132 (2008)). The latter group showed that C4-deficient mice can independently activate C3 alternative pathway, since in vivo inhibition of the alternative pathway as a result of antibody-mediated depletion of factor B functional activity had no effect on C3 cleavage-dependent regeneration effects in C4 −/− mice (Clark, A., et al. (2008)). This lectin-mediated C4-alternative C3 activation pathway may also explain the loss of the protective C4 deficiency phenotype in the MIRI model as well as in the previously described kidney allograft rejection model (Lin, T., et al., Am. J. Pathol. 168 :1241-1248 (2006)). In contrast, recent results have independently demonstrated an important protective phenotype in MASP-2 −/− mice in kidney transplant models (Farrar, C.A., et al., Mol. Immunol. 46:2832 (2009)).

Thus, the results of this example support the view that MASP-2 dependent C4-99 042534 alternative C3 activation is a physiologically relevant mechanism that may play an important role in conditions where C4 availability is limited by C3 activation.

Example 23.

This example describes C3 activation by thrombin substrates and C3 deposition on mannan in wild-type (+/+), MASP-2 (-/-), F11 (-/-), F11/C4 (-/-) and C4 (-/-) -) mice.

Relevance.

As described in Example 14, it was found that thrombin activation can occur after activation of the lectin pathway under physiological conditions, and it characterizes the degree of involvement of MASP-2 in the pathological process. C3 plays a central role in the activation of the complement system. C3 activation is required for complement activation via both the classical and alternative pathways. An experiment was performed to find out if C3 is activated by thrombin substrates.

Methods.

C3 activation by thrombin substrates.

C3 activation was measured in the presence of the following activated forms of thrombin substrates: human FCXIa, human FVIIa, bovine FXa, human FXa, human activated protein C, and human thrombin. C3 was incubated with various thrombin substrates, then separated under reducing conditions on polyacrylamide gels in the presence of 10% sodium dodecyl sulfate. After electrophoretic transfer using a cellulose membrane, the membrane was incubated with rat anti-mouse C3 monoclonal biotin-linked antibody detected with a streptavidin-horseradish peroxidase kit and developed using enterochromaffin (ECL) reagent.

Results.

C3 activation involves cleavage of the intact a chain into a truncated a' chain and soluble C3a (not shown in FIG. 30). In FIG. 30 shows the results of a Western blot analysis of human C3 activation by thrombin substrates, where the uncleaved C3 alpha chain and the a' chain activation product are depicted by arrows. As shown in FIG. 30, incubation of C3 with activated forms of human coagulation factor XI and factor X, as well as activated bovine factor X, can cleave C3 in vitro in the absence of any complement proteases.

Deposition of C3 on mannan.

C3 deposition studies were performed on sera obtained from wild-type, MASP-2 (-/-), F11(-/-), F11(-/-)/C4(-/-) and C4(-/-) mice. F11 is the gene encoding factor XI coagulation. To measure C3 activation, a layer of mannan (1 μg/well) was applied to microtiter plates, then sheep antiserum against HSA (2 μg/ml) in TBS/tween/Ca ²⁺ was added. The plates were blocked with 0.1% HSA in TBS and washed as described above. Plasma samples were diluted in 4 mm barbital, 145 mm NaCl, 2 mm CaCl ₂ , 1 mm MgCl ₂ , pH 7.4, added to the plates and incubated for 1.5 h at 37°C. After washing, bound C3b was determined using rabbit anti-human C3c (Dako) followed by alkaline phosphatase-conjugated goat anti-rabbit IgG and pNPP.

Results.

In FIG. 31 shows the results of a study of C3 deposition on serum samples obtained from wild-type, MASP-2 (-/-), F11 (-/-), Fll (-/-)/C4 (-/-) and C4 (-/-) mice. As shown in FIG. 31, a functional lectin pathway exists even in the complete absence of C4. As further shown in FIG. 31, this novel lectin pathway dependent complement activation requires coagulation factor XI.

Discussion.

Prior to the results of this experiment, it was believed by those skilled in the art that C4 activation required the lectin pathway of complement activation. As a consequence, data from C4 knockout mice (and C4 deficient humans) have been interpreted with the assumption that such organisms are deficient in the lectin pathway (in addition to being deficient in the classical pathway). The present results show that this opinion is erroneous. Thus, for example, the conclusions of past studies suggesting that the lectin pathway is not important in cases of specific diseases based on the C4 phenotype of deficient animals may be erroneous. The data described in this example also show that in the physiological context of whole serum, the lectin pathway can activate components of the coagulation system. Thus, it has been shown that there is a relationship between complement and coagulation involving MASP-2.

Example 24.

This example describes methods for evaluating the effect of an anti-MASP-2 antibody on erythrocyte lysis in blood samples from patients with paroxysmal nocturnal hemoglobinuria (PNH).

The level of knowledge of the problem/relevance.

Paroxysmal nocturnal hemoglobinuria (PNH), also called Marchiafave-Micheli syndrome, is an acquired, potentially life-threatening blood disorder characterized by complement-induced intravascular hemolytic anemia. The hallmark of PNH is chronic intravascular hemolysis resulting from uncontrolled complement activation via the alternative pathway (Lindorfer, M.A., et al., Blood 115 (11) (2010)). The anemia in PNH is due to the destruction of red blood cells in the bloodstream. Symptoms of PNH include red urine due to hemoglobin in the urine and thrombosis. PNH can develop on its own, in which case it is called primary PNH, or it can be associated with other bone marrow disorders such as aplastic anemia, in which case it is called secondary PNH. Treatment for PNH includes blood transfusion for anemia, anticoagulation for thrombosis, and the monoclonal antibody eculizumab (Soliris), which protects cells from immune damage by inhibiting the complement system (Hillman P. et al., N. Engl. J. Med. 350( 6):552-9 (2004)). However, a significant proportion of patients with PNH treated with eculizumab remain clinically immune-mediated hemolytic anemia because the antibody does not block complement activation via the alternative pathway.

This example describes methods for evaluating the effect of an anti-MASP-2 antibody on erythrocyte lysis in blood samples from PNH patients (not treated with Soliris), where the erythrocytes are incubated with blood type-matched acidified healthy human serum. Methods. Reagents. Red blood cells from healthy donors and patients suffering from PNH (not treated with Soliris) are obtained by venipuncture and prepared as described in Wilcox, L.A., et al., Blood 78:820-829 (1991), the content of which is included in the present invention by reference to it. Anti-MASP-2 antibodies with functional blocking activity on the lectin pathway can be produced as described in Example 10.

Hemolysis analysis.

Determinations of the ability of anti-MASP-2 antibodies to block erythrocyte hemolysis in patients with PNH are performed using the methods described in Lindorfer, MA, et al., Blood 15 (11):2283-91 (2010) and Wilcox, LA, et al. , Blood 78:820-829 (1991), the contents of which are incorporated herein by reference. As described in Lindorfer et al., erythrocytes from PNH patients are centrifuged, the buffy coat is aspirated, and the cells are washed in gelatin-veronal buffer (GVB) before each experiment. Erythrocytes are tested for susceptibility to APC-mediated lysis as follows. Matched healthy human serum is diluted with GVB containing 0.15 mM CaCl ₂ and 0.5 mM MgCl ₂ (GVB+ ² ) and acidified to pH 6.4 (acidified NHS, aNHS) and used to restore erythrocytes to hematocrit 1.6% in 50% aNHS. The mixtures are then incubated at 37° C. and after 1 hour, the erythrocytes are pelleted by centrifugation. Measure at 405 nm the optical density of an aliquot of the extracted supernatant and use this data to calculate the percentage of lysis. Samples reconstituted in an acidified serum-EDTA mixture are subjected to a similar treatment and used to determine the concomitant non-complement-mediated lysis (usually less than 3%). Complete lysis (100%) is determined after incubation of erythrocytes in distilled water.

To determine the effect of anti-MASP-2 antibodies on erythrocyte hemolysis in PNH, erythrocytes from PNH patients are incubated in aNHS in the presence of gradually increasing concentrations of anti-MASP-2 antibodies, and then the presence/amount of hemolysis is quantified.

Given that anti-MASP-2 antibodies have been found to block subsequent complement activation via an alternative pathway, it is expected that anti-MASP-2 antibodies can effectively block alternative pathway-mediated hemolysis of erythrocytes in PNH and can be used as a therapeutic agent. agents for the treatment of patients suffering from PNH.

Example 25.

This example describes methods for evaluating the effect of a blocking antibody against MASP-2 on complement activation by cryoglobulins in blood samples from patients suffering from cryoglobulinemia.

The level of knowledge of the problem/relevance.

Cryoglobulinemia is characterized by the presence of cryoglobulins in serum. Cryoglobulins are individual or mixed immunoglobulins (usually IgM antibodies) that are susceptible to reversible aggregation at low temperatures. Aggregation leads to activation of the classical complement pathway and inflammation in the vascular network, in particular, in the periphery. Clinical manifestations of cryoglobulinemia include vasculitis and glomerulonephritis.

Cryoglobulinemia can be classified based on the composition of cryoglobulin as follows:

type I cryoglobulinemia, or simple cryoglobulinemia, is caused by a monoclonal immunoglobulin, usually immunoglobulin M (IgM);

cryoglobulinemia type II and type III (mixed cryoglobulinemia) contains rheumatoid factors (RF), which are usually IgM in complexes with the Fc portion of polyclonal IgG.

Conditions associated with cryoglobulinemia include hepatitis C virus infection, lymphoproliferative disorders, and other autoimmune diseases. Cryoglobulin-containing immune complexes cause a clinical syndrome of systemic inflammation, apparently due to their ability to activate complement. Although normally IgG immune complexes activate the classical complement pathway, however, IgM containing complexes can also activate complement via the lectin pathway (Zhang, M., et al., Mol Immunol 44(1-3):103-110 (2007) and

Zhang. M., et al., J. Immunol. 177(7):4727-34 (2006)).

Immunohistochemical studies have further demonstrated that cryoglobulin immune complexes contain components of the lectin pathway, and biopsies from patients with cryoglobulinemic glomerulonephritis have provided immunohistochemical evidence for in situ activation of the lectin pathway (Ohsawa, I., et al., Clin Immunol 101(1):59-66 (2001 )). These results suggest that the lectin pathway may contribute to inflammation and poor outcomes in cryoglobulinemic diseases.

Methods.

Determination of the ability of anti-MASP-2 antibodies to block the negative effects of cryoglobulinemia is carried out using a liquid phase C3 conversion assay as described in Ng YC et al., Arthritis and Rheumatism 31(1):99-107 (1988), the content of which is included in the present invention by reference to it. As described in Ng et al., in essential mixed cryoglobulinemia (EMC), monoclonal rheumatoid factor (mRF), usually IgM, complexes polyclonal IgG to form a characteristic immune complex (IC) cryoprecipitate (type II cryoglobulin). Immunoglobulins and C3 have been found on vessel walls in affected tissues such as skin, nerve fiber, and kidney tissue. As described in Ng et al., ¹²⁵ I-labeled mRF is added to serum (healthy human serum and cryoglobulinemia patient serum), incubated at 37° C., and erythrocyte binding measured.

Determine the conversion of C3 in the liquid phase in serum (to the serum of a healthy person and to the serum of patients suffering from cryoglobulinemia) in the presence or absence of the following immune complex cryoprecipitates (IC): BSA-anti-BSA, mRF, mRF plus IgG or cryoglobulins, in the presence of or the absence of antibodies against MASP-2. C3 and C4 fixation with immune complex cryoprecipitate was measured using a co-precipitation assay with F(ab')2 anti-C3 and F(ab')2 anti-C4.

In view of the fact that anti-MASP-2 antibodies have been shown to block lectin pathway activation, it is expected that anti-MASP-2 antibodies can effectively block the complement-mediated negative effects associated with cryoglobulinemia and can be used as a therapeutic agent to treat patients suffering from cryoglobulinemia.

Example 26.

This example describes methods for evaluating the effect of an anti-MASP-2 antibody on blood samples from patients with Cold Agglutinin Disease, which manifests itself in the form of anemia.

The level of knowledge of the problem/relevance.

Cold agglutinin disease (CAD) is a type of autoimmune hemolytic anemia. Cold agglutinin antibodies (usually IgM) are activated at low temperatures and bind and aggregate with red blood cells. Cold agglutinin antibodies bind to complement and act on the antigen on the surface of red blood cells. This leads to opsonization of the RBCs (hemolysis), which initiates their elimination by the reticuloendothelial system. The temperature at which agglutination occurs varies from patient to patient.

CAD manifests itself in the form of anemia. Anemia occurs when the rate of destruction of red blood cells exceeds the ability of the bone marrow to produce an adequate number of oxygen-carrying cells. CAD can be caused by an underlying disease or disorder (in which case it is called secondary CAD), such as an infectious disease (Mycoplasma pneumoniae, mumps, mononucleosis), a lymphoproliferative disease (lymphoma, chronic lymphocytic leukemia), or a connective tissue disorder. Patients with primary CAD are thought to have a mild lymphoproliferative lesion in the bone marrow. Both primary and secondary CAD are acquired conditions.

Methods.

Reagents.

Erythrocytes from healthy donors of patients suffering from CAD are obtained by venipuncture. Anti-MASP-2 antibodies that block the functional activity of the lectin pathway can be produced as described in Example 10.

The ability of an anti-MASP-2 antibody to block cold agglutinin-mediated lectin pathway activation can be determined as follows. The erythrocytes of Rh positive blood group I patients are sensitized with cold agglutinins (ie, IgM antibodies) in the presence or absence of anti-MASP-2 antibodies. The erythrocytes are then tested for their ability to activate the lectin pathway by measuring C3 binding.

Given the fact that anti-MASP-2 antibodies have been shown to block lectin pathway activation, it is hypothesized that anti-MASP-2 antibodies may be effective in

- 102 042534 blocking complement-mediated adverse effects associated with cold agglutinin disease and can be used as a therapeutic agent for the treatment of patients suffering from cold agglutinin disease.

Example 27.

This example describes methods for evaluating the effect of an anti-MASP-2 antibody on erythrocyte lysis in blood samples from mice with atypical hemolytic uremic syndrome (aHUS).

The level of knowledge of the problem/relevance.

Atypical hemolytic uremic syndrome (aHUS) is characterized by hemolytic anemia, thrombocytopenia, and renal failure caused by platelet clots in the capillaries of the kidney and other organs. aHUS is associated with complement dysregulation and may be either sporadic or hereditary. aHUS is associated with mutations in genes encoding complement activation, including complement factor H, membrane cofactor protein B, and factor I, as well as complement factor H-linked protein 1 (CFHR1) and complement factor H-linked protein 3 (CFHR3) (Zipfel, P.F., et al., PloS Genetics 3(3):e41 (2007)). This example describes methods for evaluating the effect of anti-MASP-2 antibody on erythrocyte lysis in blood samples from aHUS mice.

Methods.

The effect of anti-MASP-2 antibodies in the treatment of aHUS can be determined in a mouse model of the disease in which the endogenous mouse fH gene has been replaced with a human homologue encoding a mutant form of fH often found in aHUS patients. See publication Pickering M.S. et al., J. Exp. Med. 204 (6): 1249-1256 (2007), the contents of which are incorporated into the present invention by reference. As described by Pickering et al., these mice develop aHUS-like pathology. To evaluate the effect of anti-MASP-2 antibodies in the treatment of aHUS, anti-MASP-2 antibodies are administered to mutant aHUS mice and the erythrocyte lysis that occurs in mice treated with anti-MASP-2 antibodies is compared with the lysis of erythrocytes in control untreated mice. In view of the fact that anti-MASP-2 antibodies have been shown to block lectin pathway activation, it is expected that anti-MASP-2 antibodies may be effective in blocking erythrocyte lysis in mammals suffering from aHUS.

Example 28.

This example describes methods for evaluating the effect of an anti-MASP-2 antibody in the treatment of glaucoma.

Relevance/level of knowledge of the problem.

It has been shown that uncontrolled complement activation contributes to the development of degenerative damage to retinal ganglion cells (RGCs), their synapses and axons in glaucoma (see Tezel G. et al., Invest Ophthalmol Vis Sci 51:5071-5082 (2010)). For example, histopathological studies of human tissues and in vivo studies using various animal models have shown that complement components, including C1q and C3, are synthesized and terminal complement complex is formed in the glaucomatous retina (see Stasi K. et al., Invest Ophthalmol Vis Sci 47:1024-1029 (2006), Kuehn M.H. et al., Exp Eye Res 83:620-628 (2006)). As later described in Kuehn M.H. et al., Experimental Eye Research 87:89-95 (2008), complement synthesis and deposition are induced by retinal ischemia/reperfusion, and interruption of the complement cascade arrests degenerative damage to retinal ganglion cells. In this study, mice with targeted C3 complement disorder were found to delay degenerative retinal ganglion cell (RGC) damage after transient retinal ischemia/reperfusion compared to normal animals.

Methods.

The effect of anti-MASP-2 antibodies on retinal ganglion cell (RGC) degenerative injury in an experimental retinal ischemia/reperfusion animal model was determined as described in Kuehn M.H. et al., Experimental Eye Research 87:89-95 (2008 ), the content of which is incorporated herein by reference.As described in Kuehn et al., retinal ischemia is induced by anesthetizing the animals, then inserting a 30-gauge needle connected to a reservoir containing phosphate buffered saline through the cornea into the anterior chamber of the eye.The reservoir of saline is then lifted upward to create an intraocular pressure of 104 mm Hg, sufficient to completely stop blood circulation through the retinal vasculature.Increased intraocular ischemia is confirmed by blanching of the iris, and retinal ischemia is maintained for 45 minutes left eye only; right eye serves as control and he is not catheterized. Mice are then sacrificed either 1 week or 3 weeks after the ischemic stroke. To evaluate the effect of an anti-MASP antibody administered prior to ischemic stroke, anti-MASP-2 antibodies are administered to mice either topically in the eye or systemically.

Conduct immunohistochemical studies of the eyes, using antibodies against C1q and C3, to detect complement deposition. Damage to the optic nerve can also be assessed using standard electron microscopy techniques.

Surviving retinal ganglion cells (RGCs) are quantitated using a gamma-synuclein label.

Results.

As described in Kuehn et al., in control normal mice, transient retinal ischemia leads to degenerative changes in the optic nerve and deposits of C1q and C3 on the retina, which are determined by immunohistochemistry. In contrast, C3 deficient mice showed a marked reduction in axonal degeneration, characterized by only non-significant levels of optic nerve damage 1 week after induction. Based on these results, it is expected that similar results could be obtained by conducting this study in MASP-2 knockout mice, and by administering anti-MASP-2 antibodies to normal mice prior to ischemic stroke induction.

Example 29.

In this example, a MASP-2 inhibitor, such as an anti-MASP-2 antibody, has been shown to be effective for treating post-radiation conditions and/or treating, alleviating, or preventing acute radiation syndrome.

Relevance.

Irradiation with high doses of ionizing radiation leads to death by the following two main mechanisms: toxic effect on the bone marrow and the development of a gastrointestinal syndrome. The toxic effect on the bone marrow leads to a decrease in the number of all hematological cells, provoking a lethal outcome as a result of infection and hemorrhage. Gastrointestinal syndrome is a more severe form and is caused by loss of intestinal barrier function due to disintegration of the intestinal epithelial layer and loss of intestinal endocrine functions. This leads to sepsis and the associated systemic inflammatory response syndrome, which can lead to death.

The complement lectin pathway is an innate immune mechanism that initiates inflammation in response to tissue damage and exposure to foreign surfaces (i.e., bacteria). Blockade of this pathway leads to improved treatment outcomes in mice in a model of ischemic injury to intestinal tissue or septic shock. It has been suggested that the lectin pathway may initiate excessive and dangerous inflammation in response to radiation-induced tissue damage. Therefore, blockade of the lectin pathway may attenuate secondary injury and increase survival after acute radiation exposure.

The aim of the ongoing study described in this example is to evaluate the effect of blockade of the lectin pathway on survival in mice in an experimental model of radiation injury in mice by administration of anti-mouse MASP-2 antibodies.

Methods and materials.

Materials. The following test materials were used in this study: (i) high affinity anti-mouse MASP-2 antibody (mAbMll) and (ii) high affinity anti-human MASP-2 antibody (mAbH6), which block the MASP-2 protein component of the complement lectin pathway produced in transfected mammalian cells. Dosing concentrations were 1 mg/kg anti-mouse MASP-2 antibody (mAbM11), 5 mg/kg anti-human MASP-2 antibody (mAbH6), or sterile saline. For each dosing procedure, an appropriate volume of fresh dosing solutions was prepared.

Animals. Young adult male Swiss-Webster mice were obtained from Harlan Laboratories (Houston, TX). Animals were housed in Alpha-Dri bedded hard bottom cages and provided with PMI 5002 certified rodent chow (Animal Specialties, Inc., Hubbard OR) and ad libitum water. The animal cages were constantly temperature controlled and set to a 12 hour day/12 hour lighting cycle.

Irradiation. After acclimatization for 2 weeks in a special room, the whole body surface area of the mice was irradiated at doses of 6.5 and 7.0 Gy in groups of 10 animals at a dose rate of 0.78 Gy/minute using the Therapax X-RAD 320 system, equipped with a high stability x-ray generator, sintered x-ray tube operating at 320 kV, variable x-ray collimator and filter (Precision X-ray Incorporated, East Haven, CT).

Preparation and administration of medicines. According to the protocol, a certain volume of concentrated stock solutions were diluted with ice-cold saline to obtain solutions with doses of 0.2 mg/ml of anti-mouse MASP-2 antibody (mAbM11) or 0.5 mg/ml of anti-human MASP-2 antibody ( mAbH6). Anti-MASP-2 antibodies mAbM11 and mAbH6 were administered by intraperitoneal injection with a 25 gauge needle based on animal weight to deliver 1 mg/kg mAbM11, 5 mg/kg mAbH6 or saline as placebo.

- 104 042534

Study protocol. Mice were randomly distributed into groups, as indicated in table. 8. Body weight and temperature were measured and recorded once a day. Mice in groups 7, 11 and 13 were sacrificed on day 7 post-irradiation and blood was collected by cardiac puncture under deep anesthesia. Animals surviving 30 days after irradiation were sacrificed in the same way and blood was collected. Plasma was prepared from the collected blood samples in accordance with the protocol and returned to the customer of the clinical trial for analysis.

Table 8 Study Groups

Group number Number of mice in group N Irradiation Level (Gray) Treatment Dosing regimen 1 20 6, 5 placebo 18 hours before exposure, 2 hours after exposure, weekly booster dose 2 20 6, 5 Anti-mouse MASP-2 antibody (mAbMll) only 18 hours before exposure 3 20 6, 5 Anti-mouse MASP-2 antibody (mAbMll) 18 hours before exposure, 2 hours after exposure, weekly booster dose 4 5 6 7 8 9 10 eleven 20 20 20 5 20 20 20 5 6, 5 6, 5 7.0 7.0 7.0 7.0 7.0 7.0 Anti-mouse MASP-2 antibody (mAbMll) Anti-mouse MASP-2 antibody (mAbHb) placebo placebo Anti-mouse MASP-2 antibody (mAbMll) Anti-mouse MASP-2 antibody (mAbMll) Anti-mouse MASP-2 antibody (mAbMll) Anti-mouse MASP-2 antibody (mAbMll) 2 hours after exposure, weekly booster dose 18 hours before exposure, 2 hours after exposure, weekly booster dose 18 hours before exposure, 2 hours after exposure, weekly booster dose only 2 hours after exposure only 18 hours before exposure 18 hours before exposure, 2 hours after exposure, weekly booster dose 2 hours after irradiation only 2 hours after irradiation 12 20 7.0 Anti-human MASP-2 antibody (mAbHb) 18 hours before exposure, 2 hours after exposure, weekly booster dose 13 5 No No No

Statistical analysis.

Kaplan-Meier survival curves were constructed and used to compare mean survival times for treated groups using log-rank and Wilcoxon tests. Averages with standard deviations or averages with standard error of the mean are given.

Conducted statistical comparisons using a two-tailed t-test for one sample between irradiated animals of the control group and individual groups treated.

- 105 042534

Results.

The Kaplan-Meier survival curves for the 7.0 and 6.5 Gray groups are shown in FIG. 32A and 32B, respectively, and summarized in Table. 9 below. Overall, pre-irradiation treatment with anti-mouse MASP-2 antibodies (mAbM11) resulted in improved survival of irradiated mice compared to placebo-treated irradiated controls at radiation doses of 6.5 Gy (20% increase) and 7 .0 Gray (30% increase). At an irradiation dose of 6.5 Gy, post-irradiation administration of anti-mouse MASP-2 antibody resulted in a modest increase in survival (15%) compared to placebo-treated control irradiated animals.

In comparison, all treated animals at a radiation dose of 7.0 Gy showed an increase in survival compared to the survival of irradiated control animals that were injected with placebo. The greatest change in survival was observed in animals treated with mAbH6, namely, a 45% increase in survival compared to control animals. In addition, at an irradiation dose of 7.0 Gy, the first cases of mortality were observed on the 15th day after irradiation in the groups that were administered mAbH6, and on the 8th day after irradiation in the groups of irradiated control animals that were injected with placebo, that is, survival in the treated group, the survival rate of control animals was 7 days higher. The mean time to death in mAbH6-treated mice (27.3±1.3 days) was significantly longer (p=0.0087) compared to control animals (20.7±2.0 days) at a dose of 7. 0 Grey.

Throughout the study, the percentage change in body weight compared to body weight on the day before exposure (day -1) was recorded. A transient decrease in body weight was observed in all irradiated animals, with no difference in body weight changes between the mAbM11 or mAbH6 treated groups compared to controls (data not shown). At the end of the study, all surviving animals showed an increase in body weight compared to the body weight before the study (day -1).

Table 9

Survival rates of test animals exposed to irradiation

Test group Exposure level Survival (%) Time to death (mean ± SEM) days Number of days until first case/last death Control exposure 6.5 Gray 65% 24.0±2.0 9/16 mAbMll administration before irradiation 6.5 Gray 85% 27.7+1.5 13/17 mAbMll administration before and after irradiation 6.5 Gray 65% 24.0+2.0 9/15 mAbMll administration after irradiation 6.5 Gray 80% 26, 3+1.9 9/13 Administration of maHNb before and after irradiation 6.5 Gray 65% 24.6+1.9 9/19 Control exposure 7.0 Gray 35% 20.7+2.0 8/17 mAbMll administration before irradiation 7.0 Gray 65% 23.0+2.3 7/13 mAbMll administration before and after irradiation 7.0 Gray 55% 21.6+2.2 7/16 mAbMll administration after irradiation 7.0 Gray 70% 24.3+2.1 9/14 Introduction of taNb before and after 7.0 Gray 80% 27.3+1.3* 15/20 | exposure

* p=0.0087 two-tailed Student's t-test for one sample of irradiated control animals and a group of irradiated treated animals at the same radiation dose.

- 106 042534

Discussion.

Acute radiation syndrome consists of three distinct subsyndromes: hematopoietic, gastrointestinal, and cerebrovascular. The observed syndrome is dose-dependent, with hematopoietic effects observed in humans with significant partial or total body surface irradiation in excess of 1 Gy. Hematopoietic syndrome is characterized by severe suppression of bone marrow function, leading to pancytopenia with changes in the blood count, the number of erythrocytes, leukocytes, and platelets, accompanied by damage to the immune system. In this case, at a minimum, there are low numbers of neutrophils and platelets in the peripheral blood, neutropenia, high temperature, and, as a complication, sepsis and uncontrolled bleeding, leading to death.

In the present study, mAbH6 administration was found to improve survival in male Swiss-Webster mice irradiated over the entire body surface area with X-rays at a dose of 7.0 Gy. It should be noted that at a radiation dose of 7.0 Gy, 80% of the mAbH6-treated animals lived up to 30 days, while only 35% of placebo-treated control exposed animals experienced such survival. It is important to note that the first case of death of animals in this treated group occurred only on the 15th day after irradiation, that is, 7 days later than that observed in the placebo-treated control animals. Curiously, at a lower dose of x-ray exposure (6.5 Gy), mAbH6 administration did not have any effect on survival or any delay in mortality compared to placebo-treated control irradiated animals. This difference in responses to different doses of radiation can be due to many reasons, although additional studies may be required to test any hypothesis, including preliminary sampling for analysis of microbiological cultures and for the evaluation of hematological parameters. One simple explanation could be that the number of animals belonging to the indicated groups does not allow any minor treatment-related differences to be detected. For example, in a group of twenty animals (n=20), 3 animals showed survival differences ranging from 65% (mAbH6, at 6.5 Gy radiation dose) to 80% (mAbH6, at 7.5 Gy radiation dose). 0 Grey). On the other hand, 9 animals showed differences ranging from 35% (placebo control at 7.0 Gy radiation dose) to 80% (mAbH6, at 7.0 Gy radiation dose), which clearly indicates a difference in results related to to groups of treated animals.

The results indicate that anti-MASP-2 antibodies are effective in treating a mammal at risk for the adverse effects of acute radiation syndrome or suffering from the harmful effects of acute radiation syndrome.

Example 30.

This example shows that MASP-2 deficient mice are protected from death caused by meningococcus (Neisseria meningitidis) after infection with either serogroup A meningococcus or serogroup B meningococcus.

Methods.

MASP-2 knockout mice (MASP-2 KO mice) were generated as described in Example 1. MASP-2 KO mice at 10 weeks of age (n=10) and wild-type C57/BL6 mice (n=10) were inoculated by intraperitoneal injection doses of 2.6x10 ⁷ CFU of meningococcus serogroup A Z2491 in a volume of 100 μl. The infectious dose was administered to mice in combination with iron dextran at a final concentration of 400 mg/kg. Survival of mice after infection was monitored for 72 h.

In a separate experiment, MASP-2 KO mice aged 10 weeks (n=10) and wild-type C57/BL6 mice (n=10) were inoculated by intraperitoneal injection of a dose of 6x106 cfu of serogroup B meningococcus strain MC58 in a volume of 100 μl. The infectious dose was administered to mice in combination with iron dextran at a final concentration of 400 mg/kg. Survival of mice after infection was monitored for 72 hours. For wild mice and MASP-2 KO mice, disease progression was also assessed in scores within 72 hours after infection using the disease scoring parameters described in Table 1 below. 10, which are based on the scheme described in Fransen et al. (2010), but with minor changes.

- 107 042534

Table 10

Scoring of disease progression associated with clinical signs in infected mice

Clinical signs Score in points Normal 0 Slightly ruffled coat 1 Tousled fur, slow gaze and wet eyes 2 Tousled fur, lethargy and closed eyes 3 Depressed state and lack of movement after stimulation 4 Death 5

Blood samples were taken from mice at intervals of 1 hour after infection and their serum meningococcal levels (log cfu/ml) were determined to confirm the presence of infection and to determine the rate of clearance of bacteria from the serum.

Results.

In FIG. 33 is a Kaplan-Meier curve graphically illustrating the percent survival of MASP-2 KO mice and wild mice following an infective dose of 2.6 x 10 ⁷ cfu of serogroup A meningococcus Z2491. As shown in FIG. 33, 100% MASP-2 KO mice survived for 72 hours after infection. In contrast, only 80% of wild mice (p=0.012) were still alive 24 hours after infection, and only 50% of wild mice were still alive 72 hours after infection. These results indicate that MASP-2 deficient mice are protected from death caused by infection with serogroup A meningococcus Z2491.

In FIG. 34 is a Kaplan-Meier curve graphically illustrating the percent survival of MASP-2 KO mice and wild mice following an infective dose of 6 x 10 ⁶ cfu of serogroup B meningococcus strain MC58. As shown in FIG. 34.90% of MASP-2 KO mice survived for 72 h after infection. In contrast, only 20% of wild mice (p=0.0022) were still alive 24 hours after infection. These results indicate that MASP-2 deficient mice are protected from death caused by infection with serogroup B meningococcus strain MC58.

In FIG. 35 is a graphical representation of log cfu/ml of serogroup B meningococcus strain MC58 determined at different time points in blood samples taken from MASP-2 KO mice and wild mice after intraperitoneal infection with 6 x 10 ⁶ cfu of serogroup B meningococcus strain MC58 (n=3 at different time points). time points for both groups of mice). Results are presented as means ± SEM. As shown in FIG. 35, in wild mice blood levels of meningococcus peaked at approximately log CFU/mL 6.0 at 24 hours post-infection and decreased to log CFU/mL 4.0 at 36 hours post-infection. In contrast, in MASP-2 KO mice, blood levels of meningococcus peaked at approximately log CFU/mL 4.0 at 12 h post-infection and decreased to log CFU/mL 1.0 at 36 h post-infection (symbol * denotes p<0.05, symbol ** denotes p=0.0043). These results show that although mouse MASP-2 KOs were infected with the same dose of serogroup B MC58 meningococcus as wildcat mice, mouse MASP-2 KOs nevertheless show improved bacteremia clearance compared to wild mice.

In FIG. 36 is a graphical representation of the mean disease progression scores for MASP-2 KO mice and wild mice at 3, 6, 12, and 24 hours after infection with 6 x 10 ⁶ cfu of serogroup B meningococcus strain MC58. As shown in FIG. 36, MASP-2 deficient mice were highly resistant to infection with much lower disease progression scores at 6 h (* denotes p=0.0411), 12 h (** denote p=0.0049), and 24 h. (symbol *** denotes p=0.0049) after infection compared to wild mice. The results in FIG. 36 are shown as means±SEM.

Thus, the results of this example demonstrate that MASP-2 deficient mice are protected from meningococcal death after infection with either serogroup A meningococcus or serogroup B meningococcus.

Example 31.

This example shows that administration of an anti-MASP-2 antibody after meningococcal infection increases the survival of mice infected with meningococcus.

The level of knowledge of the problem/relevance.

As described in Example 10, rat MASP-2 protein was used for selection from phage display

- 108 042534 Fab library, from which Fab2 No. 11 was identified as a functionally active antibody. From Fab2 #11, full-length antibodies against rat IgG2c and mouse IgG2a isotypes were generated. A full-length antibody against mouse IgG2a isotype MASP-2 was characterized by pharmacodynamic parameters (as described in Example 19).

In this example, a full-length anti-mouse MASP-2 antibody derived from Fab2 #11 was analyzed in an experimental murine meningococcal model.

Methods.

The IgG2a isotype of the full-length anti-mouse MASP-2 antibody derived from Fab2 No. 11, generated as described above, was tested in an experimental mouse meningococcal model as follows.

Anti-mouse MASP-2 Monoclonal Antibody (MoAb) Administration Post-Infection C57/BL6 Charles River mice at 9 weeks of age were injected with an inhibitory anti-mouse MASP-2 antibody (1.0 mg/kg) (n=12) or an isotype control antibody ( n=10) 3 hours after intraperitoneal injection of a high dose (4x106 CFU) of serogroup B meningococcus strain MC58.

Results.

In FIG. 37 is a Kaplan-Meier curve graphically illustrating the percent survival of mice after an infective dose of 4x106 cfu of serogroup B meningococcus strain MC58 followed by 3 hours post-infection with either an anti-MASP2 inhibitory antibody (1.0 mg/kg) or an isotype control antibody. As shown in FIG. 37.90% of mice injected with anti-MASP-2 antibody survived 72 hours after infection. In contrast, only 50% of mice injected with the isotype control antibody survived 72 hours after infection. The symbol * denotes a p=0.0301 value, which will be determined by comparing two survival curves.

These results show that the administration of the anti-MASP-2 antibody allows effective treatment and improves the survival of subjects infected with meningococcus.

As shown in the invention, the use of an anti-MASP-2 antibody in the treatment of a subject infected with meningococcus is effective when administered 3 hours after infection and is expected to be effective for 24 to 48 hours after infection. Meningococcal disease (meningococcemia or meningitis) is a life-threatening condition requiring urgent medical attention, and therapy is usually started immediately if a meningococcal infection is suspected (that is, before meningococcus is reliably identified as an etiological factor).

Based on the results for MASP-2 KO mice given in Example 30, it is believed that administration of an anti-MASP-2 antibody prior to meningococcal infection may also be effective in preventing or reducing the adverse effects of the infection.

Example 32.

This example shows that administration of an anti-MASP-2 antibody is effective in treating meningococcal infection in human serum.

Relevance.

Patients with a reduced level of functional MBL in serum are characterized by increased susceptibility to recurrent bacterial and fungal infections (Kilpatrick et al., Biochim Biophys Acta 1572: 401-413 (2002)). MBL is known to recognize meningococcus, and MBL-deficient sera have been shown not to cause lysis of meningococcus.

Based on the results described in Examples 30 and 31, a series of experiments were performed to determine the efficacy of administering an anti-MASP-2 antibody in the treatment of meningococcal infection in complement-deficient and control human sera. Experiments were performed at high serum concentration (20%) in order to preserve the complement activation pathway.

Methods.

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

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

- 109 042534

Table 11 Human Serum Samples Tested (shown in FIG. 38)

Sample Serum type A Healthy human serum (NHS) + antibody (Ab) against human MASP-2 IN NHS + isotype control AB WITH MBL-/- human serum D NHS E Heat inactivated (HI) NHS

A recombinant anti-human MASP-2 antibody (Knappik, A., et al., J. Mol. Biol., 296:57-86 (2000)) was isolated from a combinatorial antibody library using recombinant human MASP-2A as antigen (Chen , CB and Wallis, J Biol Chem 276: 25894-25902 (2001)). A single chain human scFv antibody fragment was identified that effectively inhibited lectin-mediated C4 and C3 activation in human plasma (IC ₅₀ ~ 20 nM) and converted into a full-length anti-human IgG4 antibody.

Meningococcus serogroup B-MC58 was incubated with the various sera shown in Table 11, each at 20% concentration, with or without the addition of an anti-human MASP-2 inhibitory antibody (3 μg in 100 μl of total volume) at 37°C at mixing. Samples were taken at time points at 0, 30, 60 and 90 minutes, isolated and then the number of viable microorganisms was determined. Heat-inactivated human serum was used as a negative control.

Results.

Figure 38 is a graphical representation of the log CFU/ml number of viable serogroup B-MC58 meningococci determined at different time points in the human serum samples shown in Table 1. 11. In the table. 12 shows the results of applying the parametric Student's t-test to the data shown in FIG. 38.

Table 12 Results of applying the parametric Student's t-test to the data shown in FIG. 38 (time 60 minutes)

Medium Statistically R summary difference (Log) meaningful? P<0.05? values A compared to B -0.3678 Yes *** (0, 0002) And compared to C -1.1053 Yes *** (p<0.0001) And compared to D -0.2111 Yes “(0.0012) C versus D 19 Yes ***(p<0.0001)

As shown in FIG. 38 and in table. 12, complement-dependent death of meningococcus in 20% human serum was significantly increased by the addition of inhibitory antibody against human MASP-2.

2. Complement-dependent death of meningococcus in 20% (v/v) MASP-2 deficient mouse sera.

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

Table 13

Mouse serum samples (shown in Fig. 39)

Sample Serum type A wild-type IN MASP-2-/- WITH MBL A/C-/- D Wild Heat Inactivated (HIS)

Meningococcus serogroup B-MC58 was incubated with various complement-deficient mouse sera, the concentration of each of which was 20%, at 37°C with stirring. Samples were taken at time points at 0, 15, 30, 60, 90 and 120 minutes, isolated and then the number of viable microorganisms was determined. Heat-inactivated human serum was used as a negative control.

Results.

In FIG. 39 is a graphical representation of the log cfu/ml number of viable meningo-110 042534 cocci of serogroup B-MC58 determined at various time points in the mouse serum samples shown in Table 39. 13. As shown in FIG. 39, sera from MASP-2 -/- mice have a higher level of bactericidal activity against meningococcus than sera from wild mice. Symbol ** denotes p value=0.0058, symbol *** denotes p value=0.001. In table. 14 shows the results of applying the parametric Student's t-test to the data shown in FIG. 39.

Table 14

The results of applying the parametric Student's t-test to the data shown in FIG. 39

Comparison Moment time Average Difference (Log) Statistically and significant? P<0.05? Summary of P values A compared to B 60 min 0.39 Yes ** (0.0058) A compared to B 90 min 0.6741 Yes *** (0.001)

Thus, the results of this example show that MASP-2 -/- sera have a higher level of bactericidal activity against meningococcus than wild mouse sera.

Example 33.

This example illustrates the inhibitory effect of MASP-2 deficiency on erythrocyte lysis of blood samples obtained in an experimental model of paroxysmal nocturnal hemoglobinuria (PNH) in mice.

The level of knowledge of the problem/relevance.

Paroxysmal nocturnal hemoglobinuria (PNH), also called Marchiafava 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, which occurs due to unregulated complement activation via an alternative pathway due to the absence of complement regulators CD55 and CD59 on PNH erythrocytes, followed by hemoglobinuria and anemia (Lindorfer, M.A., et al., Blood 115 (11 ) (2010), Risitano, A.M, Mini-Reviews in Medicinal Chemistry, 11: 528-535 (2011)). The anemia in PNH is caused by the destruction of red blood cells in the bloodstream. Symptoms of PNH include red urine due to hemoglobin in the urine, back pain, fatigue, shortness of breath, and thrombosis. PNH can develop on its own, in which case it is called primary PNH, or it can be associated with other bone marrow disorders such as aplastic anemia, in which case it is called secondary PNH. Treatment for PNH includes blood transfusion for anemia, anticoagulation for thrombosis, and the use of the monoclonal antibody eculizumab (Soliris), which protects cells from immune damage by inhibiting the complement system (Hillmen P. et al., N. Engl. J Med. 350 (6 ): 552-9 (2004)). Eculizumab (Soliris®) is a humanized monoclonal antibody that targets complement component C5 and blocks its cleavage by C5 convertases, thereby preventing C5a formation and MAC assembly. Treatment of patients with PNH with eculizumab results in a reduction in intravascular hemolysis, as measured by lactate dehydrogenase (LDH), and consequently in hemoglobin stabilization and transfusion independence in about half of patients (Hillmen P, et al., Mini-Reviews in Medicinal Chemistry, vol. 11 (6) (2011) ). Although almost all patients treated with eculizumab achieve normal or near-normal LDH levels (due to regulation of intravascular hemolysis), however, only about one-third of patients achieve hemoglobin levels above 11 g/dL, and the remainder patients treated with eculizumab continue to present with moderate to severe (i.e., transfusion-dependent) anemia, in roughly equal proportions (Risitano AM et al., Blood 113: 4094-100 (2009)). As described in Risitano et al., Mini-Reviews in Medicinal Chemistry 11: 528-535 (2011), PNH patients treated with eculizumab were found to contain C3 fragments associated with a significant proportion of their PNH erythrocytes (at that time as untreated patients did not contain them), which allows us to conclude that membrane-bound C3 fragments function as opsonins on PNH erythrocytes, which leads to their capture in reticuloendothelial cells through specific C3 receptors and subsequent extravascular hemolysis. Therefore, for those patients who develop C3-mediated extravascular hemolysis, other therapeutic strategies besides eculizumab are needed because they continue to require red blood cell transfusions. 2 inhibitory agent on erythrocyte lysis of blood samples obtained in an experimental model of PNH in mice, and demonstrated the effectiveness of MASP-2 inhibition for the treatment of subjects suffering from PNH, and also confirms the possibility of using MASP-2 inhibitors to improve the condition associated with negative consequences extravascular hemolysis mediated by C3 fragment in subjects with PNH undergoing inhibitor therapy

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C5, such as eculizumab.

Methods.

Experimental animal model of PNH.

Blood samples were taken from gene-targeted mice deficient in Crry and C3 (Crry/C3-/-) and from CD55/CD59-deficient mice. These mice lack the appropriate complement surface regulators and, therefore, their erythrocytes are susceptible to spontaneous complement autolysis, as are human blood cells with PNH.

To further sensitize these RBCs, these cells were used with and without mannan coating and then tested for hemolysis in WT C56/BL6 plasma, MBL null plasma, MASP-2-/-plasma, serum healthy human, MBL-/human plasma, and healthy human serum treated with anti-human MASP-2 antibody.

1. Study of hemolysis of erythrocytes from Crgy/C3 and CD55/CD59 doubly deficient mice in MASP-2-deficient/depleted sera and control sera.

Day 1. Preparation of mouse erythrocytes (± mannan coating).

Materials included: fresh mouse blood, BBS/Mg ²⁺ /Ca ²⁺ (4.4 mM barbituric acid, 1.8 mM sodium barbitol, 145 mM NaCl, pH 7.4, 5 mM Mg ²⁺ , 5 mM Ca ^{2 +} ), chromium chloride, CrCl ₃ -6H ₂ O (0.5 mg/ml in BBS/Mg ²⁺ /Ca ²⁺ ) and mannan, 100 μg/ml in BBS/Mg ²⁺ /Ca ²⁺ .

Whole blood (2 ml) was centrifuged for 1-2 min at 2,000 x g in a refrigerated centrifuge at 4°C. Plasma and buffy coat were aspirated. The sample was then washed three times by resuspending the erythrocyte pellet in 2 ml ice-cold BBS/gelatin/Mg ²⁺ /Ca ²⁺ and repeating the centrifugation step. After the third wash, the pellet was resuspended in 4 ml BBS/Mg ²⁺ /Ca ²⁺ . A 2 ml aliquot of erythrocytes was reserved as an uncoated control. To the remaining 2 ml were added 2 ml of CrCl ₃ and 2 ml of mannan, and the sample was incubated with gentle stirring at room temperature for 5 minutes. The reaction was terminated by adding 7.5 ml of BBS/gelatin/Mg ²⁺ /Ca ²⁺ . The sample was centrifuged as above, resuspended in 2 ml BBS/gelatin/Mg ²⁺ /Ca ²⁺ and washed two more times as above, then stored at 4°C.

Day 2. Study of hemolysis.

Materials included BBS/gelatin/Mg ²⁺ /Ca ²⁺ (as above) tested sera, 96-well round bottom and flat bottom plates, and a spectrophotometer that reads 96-well plates at 410-414 nm.

First, the concentration of erythrocytes was determined, and the concentration of cells was adjusted to 10 ⁹ /ml and stored at this concentration. Before use, the assay buffer was diluted to 10 ⁸ /ml, and then used at 100 μl per well. Hemolysis was measured at 410-414 nm (which allowed for greater sensitivity than at 541 nm). Dilutions of the sera to be tested were prepared in ice-cold BBS/gelatin/Mg ²⁺ /Ca ²⁺ . 100 μl of each serum dilution was dropped into the wells of a round bottom plate (see plate layout). 100 μl of an appropriately diluted erythrocyte preparation (ie 10 ⁸ /ml) was added (see plate layout), incubated at 37° C. for approximately 1 hour and observed for lysis. At this point, the tablets can be photographed. The plate was then centrifuged at maximum speed for 5 minutes. 100 μl was aspirated from the liquid phase, transferred to flat bottom plates, and the optical density (OD) was recorded at 410-414 nm. The erythrocyte pellets were kept (then they can be lysed with water to reverse the result).

Experiment #1

Fresh blood was obtained from CD55/CD59 doubly deficient mice, and blood of doubly deficient Crgy/C3 mice and erythrocytes were obtained as detailed in the above protocol. The cells were separated and half of the cells were coated with mannan, the other half left untreated, bringing the final concentration to 1x10 ⁸ per ml, 100 μl of which was used in the hemolysis study, which was carried out as described above.

Results of Experiment #1 The lectin pathway is involved in erythrocyte lysis in an experimental animal model of PNH.

In the initial experiment, it was found that non-mannan coated wild mouse erythrocytes were not lysed in any of the mouse sera. It was further found that mannan-coated mouse erythrocytes were slowly lysed (over 3 h at 37° C.) in wild mouse sera, but were not lysed in MBL null serum (data not shown).

Mannan-coated Crgy-/-mice erythrocytes were found to rapidly lyse in human serum, but not in heat-inactivated healthy human serum (NHS). Importantly, mannan-coated Crry-/- mouse erythrocytes lysed in NHS diluted to 1/640 (i.e., lysis occurred at all dilutions of 1/40, 1/80, 1/160, 1/320, and 1/640) ( data not shown). At this dilution, the alternative pathway is not functional (the functional activity of the alternative pathway is significantly reduced at serum concentrations below 8%).

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Conclusions from experiment No. 1.

Mannan-coated Crry-/- mouse erythrocytes lyse very well in highly diluted human serum with MBL, but not in serum without MBL. Efficient lysis at each serum concentration tested means that no alternative route is involved or required for that lysis. The inability of MBL-deficient mouse sera and human sera to lyse mannan-coated Crry -/- mouse erythrocytes indicates that the classical pathway also has nothing to do with the observed lysis. Since a lectin pathway is required to recognize molecules (such as MBL), this lysis is mediated by the lectin pathway.

Experiment #2

Fresh blood was obtained from Crry/C3 and CD55/CD59 doubly deficient mice, and mannan-coated Crry-/- mice erythrocytes were analyzed in a hemolysis assay as described above in the presence of the following human serum: MBL null; wild-type; NHS pretreated with anti-human MASP-2 antibody; and heat-inactivated NHS as a control.

Results of experiment #2 MASP-2 inhibitor prevents erythrocyte lysis in an animal model of PNH.

Healthy Human Serum (NHS) with mannan-coated Crry-/- mouse erythrocytes were incubated at dilutions adjusted to 1/640 (i.e. 1/40, 1/80, 1/160, 1/320 and 1/640), MBL- /- human sera, healthy human serum (NHS) pre-treated with monoclonal antibody against human MASP-2, and heat-inactivated healthy human serum as a control.

The ELISA microtiter plate was subjected to centrifugation and unlysed erythrocytes were collected from the bottom of the round bottom plate. The supernatant was collected from each well and the amount of hemoglobin released from the lysed erythrocytes was measured by reading the optical density at 415 nm (OD415) in an ELISA plate reader.

In the heat-inactivated control NHS (negative control), as expected, no lysis was observed. MBL-/- human serum lysed mannan-coated mouse erythrocytes at 1/8 and 1/16 dilutions. Healthy human sera (NHS) pretreated with anti-MASP-2 antibody lysed mannan-coated mouse erythrocytes at 1/8 and 1/16 dilutions, while wild-type human sera lysed mannan-coated mouse erythrocytes to a 1/32 dilution.

In FIG. 40 is a graphical representation of hemolysis (photometrically measured by the release of hemoglobin from lysed murine erythrocytes (Cryy/C3-/-) into the supernatant) of mannan-coated murine erythrocytes when exposed to human serum over a range of concentrations in the case of heat-inactivated (HI) healthy human serum (NHS) , MBL-/- sera, healthy human serum (NHS) pretreated with anti-MASP-2 antibody, and healthy human control serum (NHS).

The results shown in FIG. 40 show that inhibition of MASP-2 with an anti-MASP-2 antibody significantly altered _CH50 and inhibited complement-mediated lysis of sensitized erythrocytes with insufficient protection against autologous complement activation.

Experiment #3

Fresh blood was obtained from Crry/C3 and CD55/CD59 doubly deficient mice, and the erythrocytes of Crry-/- mice not coated with mannan were analyzed in a hemolysis assay as described above in the presence of the following human sera: MBL-/- serum, wild-type serum , healthy human sera pretreated with anti-MASP-2 antibody, and heat-inactivated control healthy human sera.

Results.

In FIG. 41 is a graphical representation of hemolysis (photometrically measured by the release of hemoglobin from lysed wild mouse erythrocytes into the supernatant) of non-mannan-coated mouse erythrocytes when exposed to human serum over a range of concentrations in the case of heat-inactivated (HI) healthy human serum (NHS), MBL-/- serum , healthy human serum (NHS) pretreated with anti-MASP-2 antibody, and healthy human control serum (NHS). The results shown in FIG. 41 show that inhibition of MASP-2 results in inhibition of complement-mediated lysis of erythrocytes in non-sensitized wildcat mice.

In FIG. 42 is a graphical representation of the hemolysis (photometrically measured by the release of hemoglobin from lysed murine erythrocytes (CD55/59-/-) into the supernatant) of mannan-free murine erythrocytes when exposed to human serum over a range of concentrations in the case of heat-inactivated (HI) healthy human serum (NHS). ), MBL-/- sera, healthy human serum (NHS) pretreated with anti-MASP-2 antibody, and healthy human control serum (NHS).

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Table 12

CH ₅₀ values depending on serum concentrations

Serum wild-type CD55/59-/- Heat inactivated serum Lysis Lysis healthy person (NHS) absent absent MBL AO/XX donor (MBL deficient) 7.2% 2.1% NHS+anti-MASP-2 antibody 5.4% 1.5% Healthy Human Serum (NHS) 3.1% 0.73%

Note: CH ₅ o is the value at which complement-mediated hemolysis reaches 50%.

Thus, the results of this example show that inhibition of MASP-2 leads to inhibition of complement-mediated lysis of sensitized and non-sensitized erythrocytes with insufficient protection against autologous complement activation. Therefore, a MASP-2 inhibitor can be used to treat subjects suffering from PNH and can also be used to alleviate (i.e., inhibit, prevent, or lessen the severity of) extravascular hemolysis in PNH patients treated with a C5 inhibitor such as eculizumab ( Soliris®).

Example 34.

This example describes an additional clinical study to that described in Example 29 above, providing additional evidence that a MASP-2 inhibitor, such as an anti-MASP-2 antibody, is effective in treating radiation exposure and/or in treating, alleviating, or preventing acute radiation syndrome.

Relevance. In the original study described in Example 29, administration of anti-MASP-2 antibody to mice prior to irradiation was shown to increase the survival of irradiated mice compared to placebo-treated irradiated controls at irradiation levels of 6.5 Gy and 7.0 Gy. In addition, in Example 29, it was demonstrated that at an irradiation level of 6.5 Gy, administration of an anti-MASP-2 antibody after irradiation resulted in a modest increase in survival compared to irradiated control animals treated with placebo. This example describes a second radiation effects study that was conducted to confirm the results of the first study.

Methods.

Study design A.

Swiss Webster mice (n=50) were exposed to radiation (8.0 Gy).

The effect on mortality of the therapeutic effect of an anti-MASP-2 antibody (mAbH6 5 mg/kg) administered 18 hours before irradiation and 2 hours after irradiation, and once a week thereafter, was assessed.

Research results A.

As shown in FIG. 43, it was found that administration of the anti-MASP-2 antibody mAbH6 increased survival in mice exposed to 8.0 Gy radiation, with adjusted mean survival increasing from 4 to 6 days compared to placebo-treated control mice, and mortality was reduced. by 12% compared to placebo-treated control mice (log-rank test, p=0.040).

Study design B.

Swiss Webster mice (n=50) were exposed to radiation (8.0 Gy) in the following control groups (I: with placebo) saline as control; (II: low dose) anti-MASP-2 mAbH6 (5 mg/kg) administered 18 hours before irradiation and 2 hours after irradiation; (III: high dose) mAbH6 (10 mg/kg) given 18 hours before irradiation and 2 hours after irradiation; and (IV: high-dose post-irradiation) mAbH6 (10 mg/kg) administered only 2 hours post-irradiation.

The results of the study B.

Administration of anti-MASP-2 antibody before and after irradiation resulted in a median survival of 4 to 5 days compared to placebo-treated control animals. Mortality in mice injected with antibodies against MASP-2 was reduced by 6-12% compared to control mice injected with placebo. In addition, it was noted that no significant adverse effects were observed with treatment (data not shown).

Thus, the results reported in this example are consistent with those reported in Example 29, and further demonstrate that anti-MASP-2 antibodies are effective in treating a mammal at risk for or suffering from the adverse effects of acute radiation syndrome.

Example 35.

This study investigates the effect of MASP-2 deficiency in an experimental mouse model of lipopolysaccharide (LPS)-induced thrombosis.

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Relevance.

Hemolytic uremic syndrome (HUS), which is caused by E. coli that produces Shiga toxin, is the leading cause of acute kidney injury in children. In this example, an experimental Schwartzman model of LPS-induced thrombosis (microvascular coagulation) in MASP-2-/- (KO) mice was used to determine the effectiveness of MASP-2 inhibition to inhibit or prevent intravascular thrombus formation.

Methods.

MASP-2-/- mice (n=9) and wild mice (n=10) were studied in an experimental Schwartzman model of LPS-induced thrombosis (microvascular coagulation). Mice were injected with Serratia liposaccharide (LPS) and continuously monitored for clot formation over time. The frequency of occurrence of microthrombi and microvascular coagulation induced by LPS was compared.

Results.

Notably, all MASP-2 −/− mice tested (9/9) did not form intravascular clots after administration of Serratia bacteria liposaccharide (LPS). In contrast, microthrombi were found in 7 out of 10 wild mice tested (p=0.0031, Fisher's exact test). As shown in FIG. 44, after infection with LPS, the time to onset of microvascular occlusion in MASP-2 −/− mice and wild mice was measured, expressing the results as the percentage of wild mice with thrombosis measured within 60 minutes, with thrombus being detected as early as approximately 15 minutes. After 60 minutes, thrombus formation was detected in up to 80% of wild mice. In contrast, as shown in FIG. 44, after 60 minutes, none of the MASP-2 -/- mice were found to have thrombosis (log-rank test: p=0.0005).

These results indicate that inhibition of MASP-2 protects against the development of intravascular thrombi in the HUS model.

Example 36.

This example describes the effect of an anti-MASP-2 antibody in an experimental mouse model of HUS using intraperitoneal injection of purified Shiga toxin 2 (STX2) and LPS.

The level of knowledge of the problem.

An experimental mouse model of HUS was developed using intraperitoneal injection of purified Shiga toxin 2 (STX2) and LPS. Biochemical and microarray analysis of mouse kidneys showed that the effects of STX2 plus LPS differ from those of either agent alone. Analysis of the blood and serum of these mice revealed neutrophilia, thrombocytopenia, erythrocyte hemolysis, and elevated serum creatinine and blood urea nitrogen. In addition, histological analysis and electron microscopy of mouse kidneys revealed glomerular fibrin deposition, erythrocyte congestion, microthrombi formation, and changes in glomerular ultrastructure. This animal model of HUS has been found to induce in C57BL/6 mice all the clinical symptoms of human HUS pathology, including thrombocytopenia, hemolytic anemia, and renal failure, which define human disease (J. Immunol 187 (1): 172-80 (2011)).

Methods.

Female C57BL/6 mice weighing 18 to 20 g were purchased from Charles River Laboratories and divided into 2 groups (5 mice per group). One group of mice was previously intraperitoneally injected with recombinant anti-MASP-2 antibody mAbM11 (100 μg per mouse, corresponding to a final concentration of 5 mg/kg body weight) diluted in a total volume of 150 μl of saline. The control group was injected with saline without any antibody. Six hours after injection of the anti-MASP-2 antibody mAbM11, all mice were combined intraperitoneally injected with a sublethal dose (3 μg/animal, corresponding to 150 μg/kg body weight) of LPS Serratia marcescens (L6136, Sigma-Aldrich, St. Louis, MO) and a dose of 4.5 ng per animal (corresponding to 225 ng/kg) of STX2 (twice the LD ₅₀ ) in a total volume of 150 μl. Saline injections were used as controls.

After dosing, the survival of mice was monitored every 6 hours. Mice were culled as soon as they reached the lethargic stage of HUS pathology. After 36 hours, all mice were culled and both kidneys were removed for immunohistochemical analysis and scanning electron microscopy. Blood samples were taken at the end of the experiment by heart puncture. Serum was separated and stored frozen at -80° C. to measure blood urea nitrogen and serum creatinine levels in both treated and control groups.

Immunohistochemical analysis.

One third of each mouse kidney was fixed in 4% paraformaldehyde for 24 h, processed and embedded in paraffin. Prepared histological sections with a thickness of three microns and placed them on prepared slides for subsequent staining with hematoxylin and eosin.

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Electron microscopy.

Kidney midsections were cut into approximately 1 to 2 mm ³ blocks and fixed overnight at 4°C in 2.5% glutaraldehyde in 1x PBS. The fixed tissue was then examined using the University of Leicester Electron Microscopy Facility.

Cryostat sections.

The other one third of the kidneys were cut into blocks with a size of approximately 1-2 mm ³ and frozen in liquid nitrogen and stored at -80°C for cryostat sections and mRNA analysis.

Results.

In FIG. 45 is a graphical representation of the survival percentage of saline treated mice (n=5) and anti-MASP-2 treated mice (n=5) in the STX/LPS induced model over time (hours). Notably, as shown in FIG. 45, all control mice died after 42 hours. In contrast, 100% of mice injected with anti-MASP-2 antibody were alive throughout the duration of the experiment. According to the results shown in FIG. 45, it was found that all mice not injected with anti-MASP-2 antibody either died or were culled with signs of severe disease, had significant glomerular damage, while the glomeruli of all mice that were injected with anti-MASP-2 antibody appeared normal. (data not shown). These results indicate that an inhibitor of MASP-2s, such as anti-MASP-2 antibodies, can be used to treat subjects suffering from thrombotic microangiopathy (TMA) or at risk of developing thrombotic microangiopathy (TMA) such as hemolytic uremic syndrome (HUS), atypical HUS (aHUS), or thrombotic thrombocytopenic purpura (TTP).

Example 37.

This example describes the effect of MASP-2 deficiency and MASP-2 inhibition in a mouse model of thrombosis resulting from FIT C-dextran/light-induced endothelial cell injury.

The level of knowledge of the problem/relevance. As shown in Examples 35 and 36, MASP-2 deficiency (MASP-2 KO) and MASP-2 inhibition (by administration of a MASP-2 inhibitory antibody) protected mice in a typical HUS model in which all control mice exposed to STX and LPS, severe HUS developed and the mice began to agonize or die within 48 hours. For example, as shown in FIG. 54, all mice treated with MASP-2 inhibitory antibody and then exposed to STX and LPS survived (Fisher's exact test p<0.01; N=5). Thus, anti-MASP-2 therapy protects mice in this HUS model.

The following experiments were performed to analyze the effect of MASP-2 deficiency and MASP-2 inhibition in an experimental mouse model of thrombotic microangiopathy (TMA) resulting from fluorescein isothiocyanate (FITC) dextran-induced endothelial cell damage to further demonstrate the benefit of MASP-2 inhibitors in treatment of HUS, aHUS, TTP, and TMA with other etiologies.

Methods.

Live microscopy.

Mice were prepared for in vivo microscopy as described in Frommhold et al., BMC Immunology 12:56-68, 2011. Briefly, mice were anesthetized by intraperitoneal injection of ketamine (125 mg/kg bw, Ketanest, Pfitzer GmbH, Karlsruhe, Germany ) and xylazine (12.5 mg/kg body weight, Rompun, Bayer, Leverkusen, Germany) and placed on a heated bed to maintain body temperature at 37°C. Intravital microscopy was performed on an upright microscope (Leica, Wetzlar, Germany) with an immersion (saline) objective (magnification 40/0.75 numerical aperture, Zeiss, Jena, Germany). To facilitate breathing, mice were intubated using PE 90 polyethylene tubing (Becton Dickson and Company, Sparks, MD, USA). The left carotid artery was cannulated with a PE 10 tube (Becton Dickson and Company, Sparks, MD, USA) for blood sampling and systemic administration of a monoclonal antibody (mAb).

Preparation of the preparation of the muscle that lifts the testicle.

Surgical preparation of the levator testis muscle for intravital microscopy was performed as described in Sperandio et al., Blood, 97:3812-3819, 2001. Briefly, the scrotum was opened and the levator testicular muscle was made movable. After a longitudinal incision and distribution of the muscles on the coverslip, the epididymis and testicles were moved to the side and fixed for full access of the microscope to the capillary circulation in the muscle that lifts the testis. Elevator testicular venules were recorded with a charge-coupled video camera (CF8/1; Kappa, Gleichen, Germany) on a Panasonic S-VHS recorder. The levator testis muscle was sprayed with temperature controlled (35° C.) bicarbonate buffered saline as previously described in Frommhold et al., BMC Immunology 12:56-68, 2011.

Photoexcitation of a model of damage caused by FITC-dextran.

Controlled light dose-dependent vascular endothelial injury of venules and levator testicular arterioles was induced by phototoxic induction (FITC)-116 042534 dextran (cat# FD150S, Sigma Aldrich, Poole, U.K.). This procedure initiates localized thrombosis. As a phototoxic reagent, 60 μl of 10% (w/v) FITC-dextran solution was injected through the left carotid artery access and allowed to diffuse homogeneously in the circulating blood for 10 minutes. After a well-perfused venule was selected, a low to moderate (800-1500) halogen lamp light was focused on the test vessel to induce FITC-dextran fluorescence and low to moderate phototoxicity to the endothelial surface to induce reproducible controlled thrombosis. The necessary phototoxic light intensity for excitation of FITC-dextran was generated using a halogen lamp (12 V, 100 W, Zeiss, Oberkochen, Germany). The phototoxicity resulting from light-induced excitation of the fluorochrome is dependent on the light intensity threshold and/or illumination duration and is due either to direct heating of the endothelial surface or to the formation of reactive oxygen radicals, as described in Steinbauer et al., Langenbecks Arch Surg 385: 290 -298, 2000.

The intensity of light focused on each vessel was measured for correction using a wavelength changing diode detector for low power measurements (Labmaster LM-2, Coherent, Auburn, USA). Offline analysis of video images was performed using a computerized capillary blood circulation system (CAMAS, Dr. Zeintl, Heidelberg), and erythrocyte velocities were measured as described in Zeintl et al., Int J Microcirc Clin Exp, 8(3): 293302 , 2000.

Use of an inhibitory monoclonal antibody (mAbH6) against human MASP-2 and placebo control before thrombosis induction.

Using a blind protocol, male C57BL/6 wild litter mice at 9 weeks of age were injected intraperitoneally or with a recombinant anti-human MASP-2 monoclonal antibody (mAbH6), an inhibitor of MASP-2 functional activity (administered at a final concentration of 10 mg/kg body weight) , or the same amount of isotype antibody as a control (without inhibitory activity against MASP-2) 16 hours before phototoxic induction of thrombosis in an experimental model of intravital microscopy of the levator testis muscle. One hour prior to thrombosis induction, a second dose of either mAbH6 or control antibody was administered. MASP-2 knockout (KO) mice were also evaluated in this model.

The mAbH6 monoclonal antibody (generated against recombinant human MASP-2) is a potent inhibitor of human MASP-2 functional activity that cross-reacts, binds to MASP-2, and inhibits murine MASP-2, but with lower affinity due to its species specificity ( data not shown). To compensate for the low affinity of mAbH6 for mouse MASP-2, mAbH6 was administered at a high concentration (10 mg/kg body weight) to overcome the species-specific difference and lower affinity for mouse MASP-2 to effectively block the functional activity of mouse MASP-2. under in vivo conditions.

In this blind study, the time required for complete occlusion of each individually tested venule was recorded (selection criteria were comparable diameters and blood flow velocities).

The percentage of mice with microvascular occlusion, the time to onset of occlusion, and the time elapsed to onset of occlusion were assessed when observed for 60 minutes using video recording of the results of the study by intravital microscopy.

Results.

In FIG. 46 is plotted as a function of time elapsed after injury induction, the percentage of mice with microvascular occlusion in the FITC/dextran UV model after isotype control antibody or anti-human MASP-2 antibody mAbH6 (10 mg/kg) dosed per 16 h and 1 h before FITC/dextran injection. As shown in FIG. 46, 85% of wild-type mice treated with isotype control antibody developed occlusion within 30 minutes or less, while only 19% of wild-type mice pretreated with anti-human MASP-2 antibody (mAbH6) developed occlusion within the same period of time, and there was a delay in the time of occurrence of occlusion in mice, which did develop occlusion over time, in the group in which the anti-human MASP-2 antibody was administered. It should also be noted that the three mice treated with anti-MASP-2 mAbH6 did not develop occlusion at all during the 60 minute observation period (ie they were protected from thrombotic occlusion).

In FIG. 47 is a graphical representation of occlusion time in minutes for mice treated with anti-human MASP-2 antibody (mAbH6) and isotype control antibody. Data are presented as a spread of points with mean values (horizontal lines) and standard error lines (vertical lines). This figure shows the time of occlusion in mice, in case occlusion was detected. Therefore, three mice injected with MASP-2 that did not develop occlusion within 60 minutes of observation were excluded from this analysis (there were no control mice that did not develop occlusion). Student's t-test for one sample was used as a statistical criterion in the analysis; where * denotes the p value=0.0129. As shown in

- 117 042534 fig. 47, in four mice injected with anti-MASP-2 antibody (mAbH6) that developed occlusion, administration of anti-MASP-2 antibody significantly increased venous occlusion time in a thrombosis model due to FITC-dextran/light-induced damage to endothelial cells. at low light intensity (800-1500) compared to mice injected with control isotype antibody. The mean time to complete occlusion for the isotype control antibody was 19.75 minutes, while the mean time to complete occlusion for the anti-MASP-2 antibody treated group was 32.5 minutes.

In FIG. 48 is a graphical representation of time to occlusion in minutes for wild mice, MASP-2 KO mice, and wild mice pretreated with anti-human MASP-2 antibody (mAbH6) intraperitoneally at 10 mg/kg over 16 hours and then again at 1 hour. before induction of thrombosis in a model of thrombosis induced by FITC-dextran/light damage to endothelial cells at low light intensity (800-1500). In FIG. 48 shows data only for animals that experienced occlusion; n=2 for wild mice treated with isotype control antibody; n=2 for MASP-2 KO mice; and n=4 for wild mice treated with anti-human MASP-2 antibody (mAbH6). The symbol * denotes a p value <0.01. As shown in FIG. 48, MASP-2 deficiency and inhibition of MASP-2 (mAbH6 at 10 mg/kg) increased venous occlusion time in an experimental model of thrombosis induced by FITC-dextran/light endothelial cell injury at low light intensity (800-1500).

Conclusions.

The results of this example further demonstrate that a MASP-2 inhibitory agent that blocks the lectin pathway (e.g., antibodies that block MASP-2 function) inhibits microvascular coagulation and thrombosis, which are hallmarks of many microangiopathic disorders, in a TMA mouse model. Therefore, administration of a MASP-2 inhibitory agent, such as an anti-MASP-2 inhibitory antibody, is expected to be an effective therapy for patients suffering from HUS, aHUS, TTP or other microangiopathic disorders and may provide protection against microvascular coagulation and thrombosis.

Example 38.

This example describes a study that demonstrates that an antibody (mAbH6) that inhibits human MASP-2 does not affect platelet function in human platelet-rich plasma.

The level of knowledge of the problem/relevance. As shown in Example 37, inhibition of MASP-2 with an anti-human MASP-2 inhibitory antibody (mAbH6) was found to increase venous occlusion time in an experimental model of FITC-dextran/light-induced endothelial cell injury thrombosis. Conducted the following experiment to determine whether the inhibitory antibody MASP-2 mAbH6) affects the function of platelets.

Methods. The effect of anti-human MASP-2 antibody mAbH6 on ADP-induced platelet aggregation was studied as follows. 40 μl of a solution of mAbH6 antibody against human MASP-2 at a concentration of either 1 μg/ml or 0.1 μg/ml was added to 360 μl of freshly prepared platelet-rich plasma. An isotype control antibody was used as a negative control. After adding the antibody to plasma, platelet activation was induced by adding ADP (adenosine diphosphate) at a final concentration of 2 μM. The study was started by stirring the solutions with a small magnet in a 1 ml cuvette. Platelet aggregation was measured on a two-channel optical luminescent aggregometer for whole blood platelets model 700 from Chrono-log.

Results.

The percentage of aggregation in solutions was measured for 5 min. The results are shown below in table. 13.

Table 13

Platelet aggregation within 5 min

Antibody Completeness of aggregation (percentage of aggregation) Curve slope (percentage of aggregation versus time) Anti-MASP-2 antibody (mAbHb) (1 µg/ml) 46% 59 Isotype control antibody (1 µg/ml) 49% 64 Anti-MASP-2 antibody (mAbHb) (0.1 µg/ml) 52% 63 Isotype control antibody (0.1 µg/mL) 46% 59

- 118 042534

As shown above in Table. 13, no significant difference was observed between ADP-induced platelet aggregation treated with control antibody or anti-MASP-2 mAbH6. These results indicate that anti-human MASP-2 antibody (mAbH6) does not affect platelet function. Therefore, the results described in Example 37, which showed that inhibition of MASP-2 by an anti-human MASP-2 inhibitory antibody (mAbH6) increases venous occlusion time in a thrombosis model due to FITCdextran/light-induced endothelial cell damage, were not caused by exposure to mAbH6. on platelet function. Thus, inhibition of MASP-2 prevents thrombosis without directly affecting platelet function, revealing a therapeutic mechanism that is different from existing antithrombotic agents.

Example 39.

This example describes the effect of MASP-2 inhibition on thrombosis and vascular occlusion in an experimental mouse model of TMA.

The level of knowledge of the problem/relevance. The lectin pathway plays a dominant role in the activation of the complement system under conditions of stress or damage to endothelial cells. This activation rapidly increases in an alternative pathway that is dysregulated in many patients presenting with aHUS. On this basis, it is hypothesized that preventing the activation of MASP-2 and the lectin pathway may interrupt the sequence of enzymatic reactions that lead to the formation of the membrane attack complex, platelet activation, and leukocyte recruitment. This effect limits tissue damage.

In addition, MASP-2 has factor Xa-like activity and cleaves prothrombin to form thrombin. This activation of the coagulation system, driven by MASP-2, can lead to hemostasis imbalance and TMA pathology. Thus, inhibition of MASP-2 using a MASP-2 inhibitor, such as a MASP-2 inhibitory antibody that blocks complement activation and coagulation systems, is expected to improve clinical outcomes in aHUS and other TMA-related conditions.

As shown in Example 37, inhibition of MASP-2 with an anti-human MASP-2 inhibitory antibody (mAbH6) was found to increase venous occlusion time in a thrombosis model due to FITC-dextran/light-induced damage to endothelial cells. In this TMA model, mice were sensitized by intravenous administration of FITC-dextran followed by localized photoactivation of FITC-dextran in the microvasculature of the levator testis muscle (Thorlacius H et al., Eur J Clin. Invest 30 (9): 804-10, 2000, Agero et al., Toxicon 50 (5): 698706, 2007).

In order to determine whether the MASP-2 inhibitory antibody (mAbH6) has a dose-dependent effect on thrombus formation and vessel occlusion in an experimental model of TMA in mice, the following experiment was performed.

Methods.

Localized thrombosis was induced by photoactivation with dextran labeled with fluorescein isothiocyanate (FITC-dextran) in the microcirculatory bed of the levator testis muscle of C57 Bl/6 mice, and an intravital microscopy method was used to determine the onset of thrombus formation and vessel occlusion using the methods described in example 37 , with the following changes. Groups of mice were intravenously injected with mAbH6 (2 mg/kg, 10 mg/kg or 20 mg/kg) or isotype control antibody (20 mg/kg) one hour prior to TMA induction. The time of onset of thrombus formation and the time of completion of vascular occlusion were recorded. An analysis of replayed video recordings of intravital microscopy images recorded over a period of 30 to 60 minutes was used to evaluate vessel size, blood flow velocity, light intensity, rate of onset of thrombus formation as equivalent to platelet adhesion, time to onset of thrombus formation, rate of complete vessel occlusion, and time to complete vessel occlusion. . SigmaPlot v12.0 software was used for statistical analysis.

Results.

thrombus initiation.

In FIG. 49 is a Kaplan-Meier curve showing the percentage of mice with thrombi as a function of time in FITC-dextran-induced thrombotic microangiopathy in mice treated with increasing doses of inhibitory antibody against human MASP-2 (mAbH6 at 2 mg/kg, 10 mg/kg). kg or 20 mg/kg) or control isotype antibody. As shown in FIG. 49, a dose-dependent delay in thrombus initiation was observed in mAbH6-treated mice compared to control antibody-treated mice.

In FIG. 50 is a graphical representation of the mean time (in minutes) to onset of thrombus formation as a function of mAbH6 dose (*p<0.01 vs. control). As shown in FIG. 50, the mean time to onset of thrombosis increased with increasing mAbH6 dose from 6.8 minutes in the control group to 17.7 minutes in the 20 mg/kg mAbH6 group (p<0.01). The main experimental data and statistical analysis are given in Table. 14 and 15.

- 119 042534

The time of onset of thrombus formation in individual mice, recorded based on video evaluation, is detailed in Table 1 below. 14.

Table 14

Time of onset of thrombus formation after dye- and light-induced injury

Introduction of control antibodies Injection of the antibody mAHHb Time of onset of thrombus formation (minutes) Control 2 mg/kg 10 mg/kg 20 mg/kg 6, 07 5, 93 12.75 10.00 1.07 6.95 2.53 10.33 8.00 8.92 14.00 21.00 2.40 11.92 3, 05 11.50 8.48 12.75 8.00 19.00 4.00 12.53 8.17 10.37 4.00 15, 83 22, 65 7.83 11.70 16, 37 6, 83 50, 67 21.75* 15.00 32.25* 15, 67

* the vessels did not show the onset of thrombus formation during the indicated period of observation.

Statistical analysis of the comparison of the time of onset of occlusion among animals that were injected with the control antibody and antibody shALHb, is shown below in table. 15.

Table 15

Time to occlusion. Data from a dose-response study on induction with FITC-dextrose

Statistics Control nbNb (2 mg/kg) shashnb (10 mg/kg) shashnb (20 mg/kg) Number of events/ 11/11 6/6 9/9 8/10 number of animals (%) (100%) (100%) (100%) (80.0%) Average time 6, 8 10.4 11.7 17.7 (minutes) (95% CI) (2,4, (5, 9, (2.5, (Yu, 0, 8.5) 12.8) 15, 8) 22.7) Criterion Wilcoxon p-value* 0.2364 0.1963 0.0016

Event = observed time to start.

The mean time (minutes) and its 95% CI were based on a Kaplan-Meier estimate.

NE = not evaluated.

*p-values were calculated using the DunnettHsu comparison test.

microvascular occlusion.

In FIG. 51 is a Kaplan-Meier curve showing the percentage of mice with microvascular occlusion versus time in FITC-dextran-induced thrombotic microangiopathy in mice treated with increasing doses of inhibitory antibody against human MASP-2 (mHHb at 2 mg/kg, 10 mg/kg or 20 mg/kg) or control isotype antibody. As shown in FIG. 51, a delay in the onset of complete microvascular occlusion was observed in the groups of mice administered with nHALHb compared to control mice.

In FIG. 52 is a graphical representation of the mean time to microvascular occlusion as a function of dose of nAbHb (*p<0.05 vs. control). As shown in FIG. 52, the mean time to complete microvascular occlusion increased from 23.3 minutes in the control group to 38.6 minutes in the 2 mg/kg wALH6 group (p<0.05). Doses of 10 mg/kg or 20 mg/kg wALHb had the same effect (mean time to complete microvascular occlusion was 40.3 and 38 minutes, respectively) as in the 2 mg/kg wLHb group. The main experimental data and their statistical analysis are presented in Table. 16 and 17.

The time to complete vessel occlusion in individual mice recorded based on

- 120042534 primary evaluation of videographic records, described in detail below in table. 16.

Table 16

Time of occurrence of occlusion after damage induced by exposure to light and dye

Introduction of control antibodies Injection of the antibody mAHHb Occlusion start time (minutes) Control 2 mg/kg 10 mg/kg 20 mg/kg 37.50 42.3 30, 92 38.00 29, 07 21.91 17.53 28.00 27.12 24.4 51.38 40, 58 19, 38 31.38 36, 88 33.00 19.55 61.17* 26, 83 39, 10 18.00 61.55* 40.28 32, 03 16.50 55, 83 38.53 23.33 71.93* 21.75* 14.83 98.22* 32.25* thirty* 33.17* 61.8*

* Vessels did not experience complete occlusion during the indicated observation time.

Statistical analysis comparing the time to complete occlusion among animals treated with control antibody and mAbH6 antibody is shown in Table 1 below. 17.

Table 17

Time to microvascular occlusion

Data from a dose-response study on induction with FITC-dextrose

Statistics Control nbNb (2 mg/kg) shashnb (10 mg/kg) shashnb (20 mg/kg) Number of events/ 9/11 4/6 7/9 7/10 number of animals (%) (81.8%) (66.7%) (77.8%) (70.0%) Average time 23.3 36, 8 40.3 38.0 (minutes) (95% CI) (16.5, (21.9, (17.5, (28.0, 37.5) NE) NE) 40, 6) Criterion Wilcoxon p-value* 0.0456 0.0285 0.0260

Event=time to start of occlusion.

The mean time (minutes) and its 95% CI were based on a Kaplan-Meier estimate.

NE=not evaluated.

*p-values were calculated using the DunnettHsu comparison test.

Brief conclusions.

As summarized in Table. 18, there was a dose-dependent delay in thrombus initiation in mAbH6 treated mice compared to control antibody treated mice (mean time to thrombosis 10.4 to 17.7 minutes versus 6.8 minutes). The mean time to complete occlusion was significantly delayed in all mAbH6 treated groups compared to control antibody treated groups (Table 18).

- 121 042534

Table 18

Mean time to thrombosis and complete occlusion

Control taNb (2 mg/kg) taNb (10 mg/kg) taNb (20 mg/kg) Mean# time to clot onset (minutes) 6, 8 10.4 11.7 17.7* Mean# time to complete microvascular occlusion (minutes) 23.3 36.8* 40.3* 38.0*

# Means are based on Kaplan-Meier estimation.

*p<0.05 compared to control (Wilcoxon test adjusted with Dunnett-Hsu multiple comparison test.

These results indicate that mAbH6, a human monoclonal antibody that binds to MASP-2 and blocks the lectin pathway of the complement system, reduces microvascular thrombosis in a dose dependent manner in an experimental mouse model of TMA. Therefore, administration of a MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody, is expected to be an effective therapy for patients suffering from HUS, aHUS, TTP, or other microangiopathic disorders such as other forms of TMA, including catastrophic antiphospholipid syndrome (CAPS). ), systemic Degos disease and TMA in cancer, cancer chemotherapy, and transplantation, and may provide protection against microvascular coagulation and thrombosis.

Example 40.

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

General description.

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

Methods.

Expression of full length catalytically inactive MASP-2.

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

To generate a catalytically inactive human MASP-2A protein, site-directed mutagenesis was performed as described in US2007/0172483, the contents of which are hereby incorporated by reference. After agarose gel electrophoresis, the PCR products were purified and a chromosome disk preparation and single adenosine overlaps were generated using a standard extension method. The adenosine-terminated MASP-2A was then cloned into a simple pGEM-T vector transformed into E. coli. Human MASP-2A was further subcloned into either pED or pCINeo mammalian expression vectors.

The MASP-2A expression construct described above was transfected into DXB1 cells using a standard calcium phosphate transfection procedure (Maniatis et al., 1989). MASP-2A was produced in a serum-free medium to ensure that the preparations were not contaminated with other serum proteins. Collected medium from confluent cells every other day (a total of four times). The level of recombinant MASP-2A averaged about 1.5 mg/l of culture medium.

- 122 042534

MASP-2A (Ser-Ala mutant described above) was purified by affinity chromatography on columns with

MBP-A-agarose.

MASP-2A ELISA analysis on ScFv clones of interest identified by panning/scFv conversion and filter screening.

A phage display library of the variable region of human immunoglobulin light and heavy chain sequences was subjected to antigenic panning followed by automated screening and antibody selection to detect high affinity scFv antibodies to human human MASP-2 protein. Three rounds of panning the scFv phage library against HIS-labeled or biotin-labeled MATP-2A were performed. The third round of panning was first eluted with MBL and then with TEA (alkaline). To monitor the specific enrichment of phages displaying scFv fragments against targeted MASP-2A, a polyclonal phage was analyzed against immobilized MASP-2A by ELISA. The scFv genes from the third round of panning were cloned into the pHOG expression vector and small scale filter screening was performed to look for specific clones against MASP-2A.

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

A study to measure the inhibition of the formation of C3-convertase of the lectin pathway.

A functional assay that measures inhibition of lectin pathway C3 convertase formation was used to evaluate the blocking activity of MASP-2 scFv clones of interest. MASP-2 serine protease is required for the generation of two protein components (C4b, C2a) that include the lectin pathway C3 convertase. Therefore, a Fab2 fragment of an anti-MASP-2 antibody that inhibits the functional activity of MASP-2 (ie, MASP-2 blocking scFv) will initially inhibit the formation of lectin pathway C3 convertase. C3 contains as part of its structure an unusual and highly reactive thioether group. After cleaving C3 with C3 convertase, the thioether group in C3b can form a covalent bond with hydroxyl groups or amino groups of macromolecules immobilized at the bottom of plastic wells using ether or amide bonds, which facilitates the determination of C3b using the ELISA assay method.

Yeast mannan is a known activator of the lectin pathway. In the method described below for measuring C3 convertase production, mannan-coated plastic wells were incubated with diluted human serum to activate the lectin pathway. The wells were then washed and the C3b immobilized in the wells was determined using standard ELISA methods. The amount of C3b generated in this assay is a direct reflection of the formation from the very beginning of the C3-convertase of the lectin pathway. In this study, clones of anti-MASP-2 scFv fragments were tested at selected concentrations for their ability to inhibit C3 convertase formation and subsequent C3b generation.

Methods.

clones of interest identified as described above were expressed, purified and diluted to the same initial concentration, which was diluted again in Ca++ and Mg ⁺⁺ containing GVB buffer (4.0 mM barbital, 141 mM NaCl, 1, 0 mM MgCl ₂ , 2.0 mM CaCl ₂ , 0.1% gelatin, pH 7.4) to ensure that all clones have the same amount of buffer. Each of the scFv clones was tested three times at a concentration of 2 μg/ml. OMS100 Fab2 was used as a positive control and tested at a concentration of 0.4 μg/ml. C3c formation was constantly monitored in the presence and absence of scFv/IgG clones.

Mannan was diluted to a concentration of 20 μg/ml (1 μg/well) in 50 mM carbonate buffer (15 mM Na ₂ CO ₃ +35 mM NaHCO ₃ +l.5 mM NaN ₃ ), pH 9.5, and applied to the ELISA plate overnight at 4°C. The next day, the mannan-coated plates were washed 3 times with 200 μl PBS. Then 100 μl of 1% HSA inhibiting solution was added to the wells and incubated for 1 hour at room temperature. The plates were washed three times with 200 µl PBS and kept on ice with 200 µl PBS until samples were added.

Healthy human serum was diluted to 0.5% in CaMgGVB buffer and scFv clones or OMS100 Fab2 positive control were added in triplicate to this buffer at a concentration of 0.01 μg/mL; 1 μg/ml (OMS100 control only) and 10 μg/ml, and pre-incubated 45 minutes on ice before adding to the blocked ELISA plate. The reaction was initiated by incubation for 1 hour at 37° C. and terminated by transferring the plates to an ice bath. The deposition of C3b was determined with

- 123 042534 rabbit antibody against α-mouse C3c, followed by the addition of goat α-rabbit HRP. The negative control was antibody-free buffer (no antibodies=maximum C3b precipitation) and the positive control was EDTA buffer (no C3b precipitation). The background was determined by carrying out the same study, except that the wells did not contain mannan. The background signal of the plates without mannan was subtracted from the signals of the wells containing mannan. Half the activity of an irrelevant scFv clone (VZV) and one buffer was taken as a threshold criterion.

Results. Based on the threshold criterion, a total of 13 clones were found to block MASP-2 activity. All 13 clones producing suppression >50% of the path were selected and sequenced to obtain 10 unique clones. All ten clones were found to have the same λ3 light chain subclass, but three different heavy chain subclasses: VH2, VH3 and VH6. In a functional study, five out of ten scFv clones of interest had lower IC ₅₀ values than the target criteria of 25 nM when using 0.5% human serum.

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

Table 19

Comparison of functional activity in terms of IC ₅₀ (nM) of leading daughter clones and their respective parent clones (all in scFv format)

scFv clone Study of S3 in 1% human serum (1C ₅ 0 NM) Study of SZ in 90% human serum (1C ₅ 0 NM) C4 study in 90% human serum (1C ₅₀ nM) 17D20mc 38 nd nd 17D20m_d3521Nl 1 26 >1000 140 17N16mc 68 nd nd 17N16m_dl7N9 48 15 230

Below are the sequences of the variable regions of the heavy chain (VH) for maternal clones and child clones, above in table. 19.

The hypervariable regions (CDRs) of Rabat (31-35 (H1), 50-65 (H2) and 95-107 (H3)) are in bold, and the hypervariable regions (CDRs) of Chothia (26-32 (H1), 52-56 (H2) and 95-101 (H3) are underlined.

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

QVT L KE S G PVLVKP T E T L T L T C TVSGFSLSRGKMGVSWIRQP P GKALEWLAHIFSSDEK

SYRTSLKS RL TIS KDT S KNQWL TMTNMDPVDTATYYCARIRRGGIDYWGOGTLVTVS S d17N9 heavy chain variable region (VH) (SEQ ID NO: 68)

QVQL QQS G P GLVKP S QT L S L T CAISGDSVSSTSAAWNWIRQ S P S RGLEWLGRTYYRSKW

YNDYAVSVKSRITINPDTSKNOFSLQLNSVTPEDTAVYYCARDPFGVPFDIWGOGTMVTVSS

The sequences of the light chain variable regions (VL) for maternal clones and daughter clones are shown below.

The hypervariable regions (CDRs) of Rabat (24-34 (L1), 50-56 (L2) and 89-97 (L3) are shown in bold, while the hypervariable regions (CDRs) of Chothia (24-34 (L1), 50-56 ( L2) and 8 9-97 (L3) are underlined These sections are the same whether they are numbered Rabat or Chothia.

17D20m_d3521N11 light chain variable region (VL) (SEQ ID NO: 70, coded with SEQ ID NO: 69)

QPVLTQPRSLSVSPGQTASITCSGEKLGDKYAYWYQQKPGQSPVLVMYQDKQRPSGIPE

RFSGSNSGNTATLTISGTQAMDEADYYCQAWDSSTAVFGGGTKLTVL

17N16m_d17N9 light chain variable region (VL) (SEQ ID NO: 71)

SYELIQPPSVSVAPGQTATITCAGDNLGKKRVHWYOQRPGOAPVLVIYDDSDRPSGIPD

RFSASNSGNTATLTI TRGEAGDEADYYCQyWDIATDHWFGGGTKLTVLAAAGSEQKL ISE

Anti-MASP-2 antibodies OMS100 and MoAb_d3521N11VL (comprising the heavy chain variable region referred to as SEQ ID NO: 67 and the light chain variable region referred to as SEQ ID NO: 70, also referred to as OMS646 and mAbH6), according to

- 124 042534 about which they were found to bind to human MASP-2 with high affinity and have the ability to block the functional activity of complement, were analyzed for epitope binding using dot-blot analysis. The results show that the antibodies OMS646 and OMS100 are highly specific for MASP-2 and do not bind to MASP-1/3. The antibody did not bind to either MAP19 or MASP-2 fragments that did not contain the MASP-2 CCP1 domain, suggesting that the binding sites include CCP1.

The anti-MASP-2 antibody OMS646 was found to actively bind to recombinant MASP-2 (Kd 60-250 μM) with a selectivity of 5000 times higher than C1s, C1r or MASP-1 (see Table 20 below).

Table 20

Affinity and Specificity of Anti-MASP-2 Antibody OMS646 in Interaction with MASP-2 Evaluated by Solid-Phase ELISA

Antigen K _D (pM) MASP-1 > 500000 MASP-2 62 ± 23* MASP-3 > 500000 Purified human C1r > 500000 Purified Human Cis ~ 500000

* Mean value ± standard deviation; n=12.

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

Methods.

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

Results.

In FIG. 53A is a graphical representation of the level of MAC precipitation in the presence or absence of anti-MASP-2 antibody (OMS646) under lectin pathway specific assay conditions. In FIG. 53B is a graphical representation of the level of MAC precipitation in the presence or absence of anti-MASP-2 antibody (OMS646) under classical pathway specific assay conditions. In FIG. 53C is a graphical representation of the level of MAC precipitation in the presence or absence of anti-MASP-2 antibody (OMS646) under alternative pathway specific assay conditions.

As shown in FIG. 53A, the OMS646 antibody blocks lectin-mediated activation of MAC precipitation with an IC ₅₀ value of approximately 1 nM. However, the OMS646 antibody had no effect on MAC deposition generated by classical pathway mediated activation (FIG. 53B) or by alternative pathway mediated activation (FIG. 53C).

Study of the pharmacokinetics and pharmacodynamics for the antibody OMS646 after its intravenous or subcutaneous injection in mice.

The pharmacokinetics and pharmacodynamics for the OMS646 antibody were studied in mice after a single dosing of the antibody for 28 days. The study used doses of 5 mg/kg and 15 mg/kg OMS646 subcutaneously, as well as doses of 5 mg/kg OMS646 intravenously.

With regard to the pharmacokinetic profile of OMS646, FIG. 54 is a graphical representation of OMS646 concentration (mean n=3 animals per group) versus time following administration of OMS646 at the indicated dose. As shown in FIG. 54, at a dose of 5 mg/kg s.c., the maximum plasma concentration of OMS646 reached 5-6 μg/ml approximately 1-2 days after dosing. The bioavailability of OMS646 at a dose of 5 mg/kg subcutaneously was approximately 60%. As further shown in FIG. 54, at a dose of 15 mg/kg s.c., the maximum plasma concentration of OMS646 reached 1012 μg/ml approximately 1-2 days after dosing. For all groups, OMS646 was slowly cleared from the systemic circulation with a terminal half-life of approximately 8-10 days. The profile of OMS646 is typical when a human antibody is administered to mice.

The pharmacodynamic activity of OMS64 6 is graphically illustrated in FIG. 55A and 55B. In FIG. 55A and 55B show the pharmacodynamic response (decrease in systemic lectin pathway activity) for each mouse in the 5 mg/kg IV (FIG. 55A) and 5 mg/kg SC (FIG. 55B) dosing groups. The dotted line indicates the initial level in the study (maximum inhibition, serum of intact mice, which were injected with an excess of OMS646 in vitro before the study). As shown in FIG. 55A, after intravenous administration of 5 mg/kg OMS646, the systemic lectin pathway activity immediately decreased to almost undetectable levels, and the lectin pathway activities showed only modest recovery during 28 days of observation. As shown in FIG. 55B, mice dosed with 5 mg/kg OMS646 subcutaneously exhibited time-dependent inhibition.

- 125 042534 activity of the lectin pathway. The activity of the lectin pathway decreased to almost undetectable levels within 24 hours after drug administration and remained at a low level for at least 7 days. The activity of the lectin pathway gradually increased over time, but during the 28 days of observation did not reach the level that was before dosing. The time profile of lectin pathway activity observed after a dose of 15 mg/kg s.c. was similar to the profile at a dose of 5 mg/kg s.c. (data not shown), indicating that the maximum possible pharmacodynamic effect was achieved. In addition, these data indicated that once weekly intravenous or subcutaneous administration of a dose of 5 mg/kg OMS646 is sufficient to provide continuous suppression of systemic lectin pathway activity in mice.

Example 41.

This example shows that a MASP-2 inhibitory antibody (OMS646) inhibits serum-induced aHUS deposition of C5b-9 on the surface of activated human vascular endothelial microcells (HMEC-1) after exposure to serum obtained from patients with atypical hemolytic uremic syndrome (aHUS), taken during the acute and remission phases of the disease.

The level of knowledge of the problem/relevance. The following study was performed to investigate serum aSUS-induced C5b-9 deposition on the surface of activated HMEC-1 cells after exposure to HUS patient sera obtained (1) during the acute phase and (2) during the remission phase of the disease, in the presence or absence of OMS646, an anti-MASP-2 antibody that specifically binds to MASP-2 and inhibits lectin pathway activation.

Methods.

Patients. For this study, four patients with aHUS were selected, who were examined both during the acute phase of the disease and in remission, among those included in the International Registry of HUS/TTP (International Registry of HUS/TTP) and genotyped in the Laboratory of Immunology and Genetics Transplantation and Rare Diseases of the Mario Negri Institute (Laboratory of Immunology and Genetics of Transplantation and Rare Diseases of the Mario Negri Institute). One aHUS patient had a heterozygous complement factor H (CFH) p.R121°C mutation and one patient had anti-CFH autoantibodies, while two other patients had no mutations or anti-CFH antibodies.

In table. Figures 21 and 22 summarize the results of screening for complement gene mutations and anti-CFH autoantibodies in the four patients examined in this study, along with clinical and biochemical data obtained either during the acute phase or in remission.

Table 21

Clinical parameters of four aHUS patients in this study

Case No. Mutation or AB against CFH Illness phase NIA Platelets (15 0400x10 ³ / µl) LDH (2 66- 500 Chalk) Hemoglo bin (14-18 g/dl) s-creatinine (0.55-1.25 mg/dl) #1 No mutations, no AB against CFH acute 31000 1396 12.9 2.37 remission 267000 n.a. 11.5 3.76 #2 CFH- R1210C acute 46000 1962 7 5.7 remission 268000 440 13.4 7.24 #3 Ab vs. CFH acute 40000 3362 9.5 1.77 remission 271000 338 8.8 0.84 #4 No mutations, no AB against CFH acute 83000 1219 7.8 6, 8 remission 222000 495 12.2 13

Note: n.a. = data not available.

- 126 042534

Table 22

Complement parameters of four aHUS patients in this study

Case No. Mutation or AB against CFH Disease phase Serum S3 (83-180 mg/dL) Plasma SC5b- 9 (127-400 ng/ml) #1 No mutations, no AB against CFH acute 51 69 remission η. A. 117 #2 CFH-R1210C acute 79 421 remission 119 233 #3 AB vs CFH acute 58 653 remission 149 591 #4 No mutations, no AB against CFH acute 108 η. A. remission η. A. η. A.

Experimental methods.

Human dermal-derived human vascular endothelial microcell line (HMEC-1) cells were seeded on glass slides and used after confluence. HMEC-1 confluent cells were activated with 10 μM ACP (adenosine diphosphate) for 10 minutes and then incubated for 4 hours with sera from the four aHUS patients described in Table 1 above. 23 and 24 collected either during the acute phase of the disease or in remission, or with sera from 4 healthy subjects used as controls. Serum was diluted 1:2 with control medium (HBSS with 0.5% BSA) in the presence or absence of MASP-2 inhibitory antibody OMS646 (100 μg/ml) generated as described in Example 40 above, or in the presence of soluble complement receptor 1 (sCR1) (150 µg/ml), as a positive control for complement inhibition. At the end of the incubation step, HMEC-1 cells were treated with rabbit anti-human complement C5b-9 followed by FITC-conjugated secondary antibody. In each experiment, sera from one healthy control subject were tested in parallel with sera from an aHUS patient (acute and in remission). A confocal inverted laser microscope was used to register fluorescent staining on the surface of endothelial cells. Registered 15 fields in each sample, and estimated the area of fluorescent staining with automatic edge detection, using built-in specific functions of the software Image J, and the results are expressed as pixel ² per analyzed field. The fields with the lowest and highest values were not used for calculations.

For statistical analysis (one-way ANOVA followed by Tukey's test for multiple comparisons), pixel ² results for 13 fields obtained for each patient and control subject under each experimental condition were used.

Results.

The results of the complement deposition assay using sera from four aHUS patients are shown in Table 23A below, and the results from sera from four healthy subjects are shown in Table 23B below.

- 127 042534

Table 23A

Effect of complement inhibitors on serum-induced C5b-9 precipitation on ADP-activated HMEC-1 cells from patients with aHUS

Patient with aHUS # Acute phase of aHUS aHUS remission phase Without treatment + SCR1 +OMS646 Without treatment + SCR1 +OMS646 Patient #1 (no mutation, no ab against CFH) 5076± 562° 551 ± 80* 3312 ± 422** 4507 ± 533° 598± 101 1650 ± 223 Patient #2 (CFH-R1210C) 5103+ 648° 497+ 67* 2435+ 394* 3705+ 570° 420+ 65 2151+ 250 Patient #3 (ab vs. CFH) 3322± 421° 353± 64* 2582± 479 6790 ± 901° 660 ± 83 2077± 353 Patient #4 (no mutation, 4267 ± 488° 205± 34* 2369 ± 265** 5032 ± 594° 182 ± 29 3290 ± 552 no ab vs CFH)

Table 23B

Effect of complement inhibitors on serum-induced C5b-9 deposition on ADP-activated HMEC-1 cells from four control healthy subjects (not suffering from aHUS)

Control healthy subject # Without treatment + sCRl +OMS646 Control subject #1 (study in parallel with aHUS patient #1) 481 ± 66 375 ± 43 213+57 Control subject #2 (tested in parallel with aHUS patient #2) 651 ± 61 240 ± 33 490+69 Control subject #3 (study in parallel with aHUS patient #3) 602 ± 83 234 ± 35 717 ± 109 Control subject #4 (tested in parallel with aHUS patient #4) 370±53 144+20 313+36

For table. 23A and 23B: data are means ± SE. °P<0.001 relative to control; *P<0.001, **P<0.01 relative to the acute phase of aHUS without treatment; P<0.001, P<0.01, P<0.05 relative to aHUS remission phase without treatment.

In FIG. 56 is a graphical representation of the inhibitory effect of an anti-MASP-2 antibody (OMS646) and sCR1 on serum-induced C5b-9 precipitation on ADP-activated HMEC-1 cells. In FIG. 56, data are means ± SE. °P<0.0001 relative to control; *P<0.0001 relative to the acute phase of aHUS; ^L P<0.0001 relative to the acute phase aHUS+sCR1; P<0.0001 relative to remission phase of aHUS without treatment and #P<0.0001 relative to remission phase of aHUS+sCR1.

- 128 042534

As shown in Table. 23A, 23B and in FIG. 56, ADP-activated HMEC-1 cells exposed to sera from HUS patients (collected either in the acute phase or in remission) for 4 hours under static conditions showed intense C5b-9 deposition on the cell surface, detectable by confocal microscopy. When measuring the area covered by C5b-9, significantly more precipitated C5b-9 was found on cells exposed to sera from HUS patients than on cells exposed to sera from healthy control subjects, regardless of whether the aHUS sera was collected in acute phase or remission phase. There was no difference in endothelial C5b-9 deposits induced by serum collected during the acute and remission phases of aHUS.

In addition, as shown in Table. 23A, 23B and in FIG. 56, the addition of anti-MMSP-2 antibody OMS646 to aHUS sera (obtained from a patient either during the acute phase or in remission) resulted in a significant reduction in C5b-9 deposition on the surface of endothelial cells compared to untreated aHUS sera. However, the inhibitory effect of OMS646 on C5b-9 deposition was less profound than that of the broad complement inhibitor sCR1. Indeed, there was a statistically significant difference between C5b-9 deposits exposed to serum aHUS in the presence of OMS646 compared to the presence of sCRl (FIG. 56 and Tables 23A and 23B).

When calculating the mean value for four patients with aHUS, the percentage reductions in C5b-9 deposits observed in the presence of complement inhibitors (compared to C5b-9 deposits induced by untreated serum of the same patients, taken as 100%) were as follows:

Acute phase:

sCR1 (150 µg/ml): 91% reduction in C5b-9 deposits.

OMS646 (100 µg/mL): 40% reduction in C5b-9 deposits.

Remission phase:

sCR1 (150 µg/ml): 91% reduction in C5b-9 deposits.

OMS646 (100 µg/mL): 54% reduction in C5b-9 deposits.

Conclusions.

The results presented in this example show that the complement lectin pathway is stimulated by activated vascular endothelial microcells, and that this stimulation is an important signal for the complement overactivation response characteristic of aHUS. It has also been shown that this stimulation of the lectin pathway and, as a result, the reciprocal complement overactivation, occurs both during the acute phase and in the phase of clinical remission of aHUS. Moreover, it appears that this finding is not limited to any particular complement defect associated with aHUS. In addition, as shown in this example, selective inhibition of the lectin pathway with a MASP-2 inhibitory antibody such as OMS646 reduces complement deposition in HUS patients of various etiologies.

Example 42.

This example shows that a MASP-2 inhibitory antibody (OMS64 6) inhibits serum-induced platelet aggregation and thrombus formation on the surface of activated human vascular endothelial microcells (HMEC-1) after exposure to HUS patient serum obtained (1) during the acute phase and ( 2) during the remission phase of aHUS.

Methods.

Patients. Three patients (patients #1, #2 and #4 described in Tables 21, 22, 23A and 23B in Example 41) suffering from aHUS were examined (one patient had a heterozygous p.R121°C CFH mutation, two others patients were found to have a mutation or antibodies against CFH) both during the acute phase of the disease and in remission. Four patients were selected for this study among those included in the International Registry of HUS / TTP (International Registry of HUS / TTP) and genotyped in the Laboratory of Immunology and Genetics of Transplantation and Rare Diseases of the Mario Negri Institute (Laboratory of Immunology and Genetics of Transplantation and Rare Diseases of the Mario Negri Institute). Five healthy subjects were also selected as blood donors for perfusion experiments.

Methods. HMEC-1 confluent cells were activated with 10 μM ADP for 10 minutes, and then incubated for 3 hours with sera from three aHUS patients (patients #1, #2, and #4 described in Example 41) collected either during acute phase of the disease, either in remission, or with control sera from healthy subjects. Serum was diluted 1:2 with control medium (HBSS with 0.5% BSA) in the presence or absence of MASP-2 inhibitory antibody OMS646 (100 μg/ml) generated as described in Example 40 above, or in the presence of sCR1 (150 μg /ml), as a positive control for complement inhibition. For patients #1 and #2, additional wells were incubated with sera (acute phase and remission) diluted 1:2 with control medium containing 100 µg/mL of the irrelevant isotype control antibody or 20 µg/mL of OMS646 (for the latter antibody, in case Patient #1 was tested with sera in remission only, and

- 129 042534 in case # 2 - both during the acute phase and in the remission phase).

At the end of the incubation step, HMEC-1 cells were perfused in the flow chamber with heparinized whole blood (10 IU/mL) obtained from healthy subjects (containing the fluorescent dye mecacrin, which labels platelets) at a shear stress characteristic of microcirculation (60 dynes). /cm ² , three minutes). After three minutes of perfusion, monolayers of endothelial cells were fixed in acetone. Fifteen images were recorded for each sample of platelet thrombus on the surface of endothelial cells using a confocal inverted laser microscope, and the areas occupied by thrombi were evaluated using Image J software. The fields with the lowest and highest values were not used for calculations.

For statistical analysis (one-way ANOVA followed by Tukey's test for multiple comparisons), pixel ² results for 13 fields obtained for each patient and control subject under each experimental condition were used.

Results.

The results of clotting experiments with sera from three aHUS patients are shown in Table 1 below. 24A, and the results with the sera of five healthy subjects are shown below in table. 24V.

Table 24A

Effects of Complement Inhibitors on Serum aHUS-Induced Thrombogenesis (pixel ² ±SE) in ADP-activated HMEC-1 Cells

Experiment conditions Stage of the disease Case #1 aHUS thrombosis (pixel ² +SE) (no mutation, no ab against CFH) Case #2 aHUS thrombosis (pixel ² 1SE) (CFH-R1210C) Case #3 aHUS thrombosis (pixel ² 1SE) (no mutation, no ab against CFH) without processing acute 54991600 2232011273° 1029111362° remission 646811012° 33871443° 1767611106° IsCRl (150 µg/ml) acute 43111676 55391578* 533611214*** remission 5731316 9771102 25441498 +OMS646 (20 µg/ml) acute did not define 69741556* did not define remission 8321150 12241252 did not define +OMS646 (100 µg/ml) acute 37051777 99131984* 28361509* remission 33211945 7331102 17001321 + irrelevant isotypic control antibody (100 µg/ml) acute 59951725 1865511699 did not define remission 1088511380 27111371 did not define

- 130 042534

Table 24V

Effects of complement inhibitors on the serum of five control healthy subjects (not suffering from HUS) in the study of thrombosis (pixel ² ± SE) on ADP-activated HMEC-1 cells

Experiment conditions Control #1 Thrombus (Pixel ² 1SE) Control #2 Thrombus (Pixel ² 1SE) Control #3 Thrombus (Pixel ² 1SE) Control #4 Thrombus (Pixel ² 1SE) Control #5 Thrombus (Pixel ² 1SE) without processing 28801510 10461172 11441193 7351124 28111609 + SCR1 (150 µg/ml) 51921637 15271153 11981138 22391243 23841410 +OMS646 (100 µg/ml) 76371888 10361175 7311203 20001356 717711477 + irrelevant isotype control antibody (100 µg/ml) 63251697 10241235 399182 45269 Not determined Investigated in parallel with the serum of a subject with aHUS #1 (serum- acute phase mouth) #1 (serum- phase of remission) #2 (serum- sharp mouth phase) #2 (serum- phase of remission) #5 (serum- rotka acute phases and phases of remission)

For table. 24A and 24B: data are mean ± SE. °P<0.001 relative to control; *P<0.001, ***P<0.05 relative to the acute phase of aHUS without treatment; P<0.001, P<0.05 relative to aHUS remission phase without treatment.

In FIG. 57 is a graphical representation of the effect of anti-MASP-2 antibody (OMS646) and sCR1 on serum-induced thrombosis on ADP-activated HMEC-1 cells. In FIG. 57, data shown are mean ± SE. °P 0.0001, ^oo P<0.01 against control; *P<0.0001, **P<0.01 vs acute phase HUS without treatment; P<0.0001 vs remission phase of aHUS without treatment.

As shown in Table. 24A and in FIG. 57, a marked increase in the area covered by thrombi was observed in HMEC-1 cells treated with aHUS serum collected either during the acute phase or in remission compared to cells exposed to serum from control healthy subjects (Table 24B and FIG. 57 ). As shown in FIG. 57 and in table. 24A, OMS646 antibody (at 100 μg/mL and 20 μg/mL) exerted partial inhibition of thrombus formation on cells pre-exposed to aHUS serum taken during the acute phase. The antithrombogenic effect was comparable for two different doses of OMS646 and did not differ from the effect of sCRl (Figure 57 and Table 24A). The addition of an irrelevant isotype control antibody had no inhibitory effect on aHUS-induced thrombosis.

In addition, as shown in FIG. 57 and in table. 24A, the inhibitory effect of OMS646 was even more evident in the case of aHUS serum collected during the remission phase. Indeed, the addition of OMS646 at doses of 100 μg/mL and 20 μg/mL to aHUS patient's serum collected in remission resulted in almost complete inhibition of thrombus formation, similar to the inhibition observed with the addition of sCR1. The irrelevant isotype control antibody showed no significant inhibitory effect.

When calculating the mean value for three patients with aHUS, observed in the presence of complement inhibitors, the percent reduction in the surface area of HMEC-1 cells covered with thrombi deposits (compared to the area covered with thrombi deposits caused by untreated serum of the same patients, taken as 100%), were next.

- 131 042534

Acute phase:

sCR1 (150 µg/ml): 60% reduction.

OMS646 (100 µg/mL): 57% reduction.

OMS646 (20 µg/mL): 45% reduction.

Remission stage:

sCR1 (150 µg/ml): 85% reduction.

OMS646 (100 µg/mL): 79% reduction.

OMS646 (20 µg/mL): 89% reduction.

The discussion of the results.

The results obtained in this example show that a MASP-2 inhibitory antibody such as OMS646 (generated as described in example 40) has a strong inhibitory effect on serum-induced thrombosis on HMEC-1. Surprisingly, the inhibitory effect of the OMS646 antibody on thrombus formation was greater than its effect on C5b-9 deposits induced on HMEC-1 (as described in Example 41). It is also surprising that the addition of OMS646 at doses of 100 μg/mL and 20 μg/mL to aHUS patient's serum collected at remission resulted in almost complete inhibition of thrombus formation. Another surprising result is the observation that OMS646, both in the acute phase and in remission, was as effective as a positive control for sCR1, which is a broad-acting and near-complete inhibitor of the complement system (Weisman H. et al., Science 249: 146-151, 1990; Lazar H. et al., Circulation 100: 1438-1442, 1999).

Of note, control sera from healthy subjects also induced moderate thrombosis on HMEC-1 cells. No sustained inhibitory effect on control serum-induced thrombosis was found for either OMS646 or sCR1. Without advocating any particular theory, however, it can be assumed that control serum-induced thrombi are complement-independent, as evidenced by the very low levels of C5b-9 deposits found on HMEC-1 cells incubated with control serum (see example 41).

Conclusions.

In conclusion, the observed antithrombotic effect of a MASP-2 inhibitory antibody, such as OMS646, is significantly higher than would be expected based on the inhibitory effect of OMS646 on C5b-9 deposition observed in this experimental system (as described in Example 41 and shown in Fig. 56). For example, in Gastoldi et al., Immunobiology 217: 1129-1222 Abstract 48 (2012), entitled C5a/C5aR interaction mediates complement activation and thrombosis on endothelial cells in atypical hemolytic uremic syndrome (aHUS), it was found that the addition of an anti-C5 antibody , inhibiting C5b-9 deposits (60% reduction), limited thrombus formation on HMEC-1 cells to a comparable extent (60% reduction). In contrast, the MASP-2 inhibitory antibody (OMS646 at 100 μg/mL) inhibited C5b-9 deposition with fairly moderate values (acute phase=40% reduction, remission phase=54% reduction) and inhibited thrombus formation at significantly more a high percentage (acute phase=57% decrease, remission phase=79% decrease). In comparison, the OMS646 antibody inhibited complement precipitation at a lower percentage than the positive control complement inhibitor (sCR1 at 150 μg/mL, inhibition of C5b-9 precipitation in acute phase=91%, in remission phase=91%), but it was as effective as sCR1-positive control in inhibiting thrombus formation (sCR1 at 150 μg/ml, acute phase=60% reduction, remission phase=85% reduction). These results indicate that a MASP-2 inhibitory antibody (eg, OMS646) exhibits unexpected efficacy in inhibiting clotting in sera from aHUS subjects in both the acute and remission phases.

In accordance with the foregoing, in one embodiment, the invention provides a method for inhibiting thrombosis in a subject suffering from thrombotic microangiopathy (TMA) or at risk of developing thrombotic microangiopathy (TMA), comprising administering to the subject a composition containing an amount of a MASP-2 inhibitory antibody effective to inhibit MASP-2 dependent complement activation. In one embodiment, the TMA is selected from the group consisting of hemolytic uremic syndrome (HUS), thrombotic thrombocytopenic purpura (TTP), and atypical hemolytic uremic syndrome (aHUS). In one embodiment, the TMA is aHUS. In one embodiment, the composition is administered to a patient with aHUS during the acute phase of the disease. In one embodiment, the composition is administered to a patient with aHUS during a remission phase (i.e., in a subject who has recovered or partially recovered from an episode of the acute phase of aHUS, such remission is confirmed, for example, by an increase in platelet count and/or reduced serum lactate dehydrogenase (LDH) concentrations , for example, as described in Loirat C et al., Orphanet Journal of Rare Diseases 6:60, 2011, the contents of which are incorporated herein by reference).

- 132 042534

In one embodiment, the MASP-2 inhibitory antibody has at least one or more of the following: said antibody binds human MASP2 with a K _D value of 10 nM or less, said antibody binds an epitope in the CCP1 domain of MASP-2, said antibody inhibits C3b precipitation in an in vitro study in 1% human serum with an IC ₅₀ value of 10 nM or less, said antibody inhibits C3b precipitation in 90% human serum with an IC ₅₀ value of 30 nM or less, where the antibody is an antibody fragment selected from the group, consisting of Fv, Fab, Fab', F(ab) ₂ and F(ab') ₂ where the antibody is a single chain molecule, where the specified antibody is an IgG2 molecule, where the specified antibody is an IgG1 molecule, where the specified antibody is an IgG4 molecule, where the IgG4 molecule contains the S228P mutation, and/or where the antibody does not substantially inhibit the classical pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and substantially does not inhibit the alternative pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and substantially does not inhibit the classical or alternative pathway (ie, inhibits the lectin pathway while leaving the classical and alternative complement pathways unaffected).

In one embodiment, the MASP-2 inhibitory antibody inhibits blood clotting in the serum of a subject suffering from TMA, such as aHUS (acute or remission) by at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, and up to 99% compared to untreated serum. In some embodiments, the MASP-2 inhibitory antibody inhibits blood clotting in the blood serum of a subject suffering from aHUS by at least 20 percent or more (e.g., at least 30%, at least 40%, at least least 50%) exceeding the inhibitory effect on the deposition of C5b-9 in the blood serum.

In one embodiment, the MASP-2 inhibitory antibody inhibits clotting in the serum of an aHUS patient in remission by at least 30%, such as at least 40%, such as at least 50%, such as at least 60%. , such as at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% and up to 99%, compared to serum , untreated with antibody. In some embodiments, the MASP-2 inhibitory antibody inhibits clotting in the serum of an aHUS patient in remission by at least 20 percent or more (e.g., at least 30%, at least 40%, at least least 50%) more than the inhibitory effect on the precipitation of C5b-9 in the blood serum.

In one embodiment, the MASP-2 inhibitory antibody is administered to the subject via an intravenous catheter or other catheter delivery method.

In one embodiment, the invention provides a method for inhibiting thrombus formation in a subject suffering from TMA, comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or an antigen-binding fragment thereof, comprising (I) (a) a heavy chain variable region comprising: i) a heavy a CDR-H1 chain comprising the amino acid sequence from 31-35 of SEQ ID NO: 67; and ii) a CDR-H2 heavy chain comprising the amino acid sequence from 50-65 of SEQ ID NO: 67; and iii) a CDR-H3 heavy chain comprising the amino acid sequence from 95-102 of SEQ ID NO: 67 and b) a light chain variable region comprising: i) a CDR-L1 light chain comprising the amino acid sequence from 24-34 of SEQ ID NO: 70; and ii) a CDRL2 light chain comprising the amino acid sequence from 50-56 of SEQ ID NO: 70; and iii) a CDR-L3 light chain comprising the amino acid sequence from 89-97 of SEQ ID NO: 70, or (ii) a variant thereof comprising a heavy chain variable region with at least 90% identity to SEQ ID NO: 67 ( e.g. at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , at least 98%, at least 99% identity with SEQ ID NO: 67) and a light chain variable region with at least 90% identity (for example, at least 91%, at least 92 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with SEQ ID NO: 70.

In one embodiment, the TMA is selected from the group consisting of atypical hemolytic uremic syndrome (aHUS) (either in the acute phase or in remission), HUS, and TTP. In one embodiment, the subject is in the acute phase of aHUS. In one embodiment, the subject is in remission aHUS.

In some embodiments, the method includes administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising an amino acid sequence,

- 133 042534, referred to as the sequence of SEQ ID NO: 67. In some embodiments, the method includes administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or an antigen-binding fragment thereof, comprising a light chain variable region, comprising an amino acid sequence, referred to as the sequence of SEQ ID NO : 70.

In some embodiments, the method includes administering to the subject a composition comprising a MASP-2 inhibitory antibody, or an antigen-binding fragment thereof, that specifically recognizes at least a portion of an epitope on human MASP-2 recognized by a control antibody 0MS646 comprising a heavy chain variable region, referred to as the sequence of SEQ ID NO: 67, and the light chain variable region, referred to as the sequence of SEQ ID NO: 70. Competition between binding members can be easily investigated in vitro, for example, using an ELISA assay method and/or by labeling a specific reporter group in one of a binding member that can be detected in the presence of other unlabeled binding member(s) to allow identification of specific binding members that bind the same epitope or an overlapping epitope. Thus, the present invention provides a specific antibody, or antigen-binding fragment thereof, comprising the antigen-binding site of a human antibody that competes with the control antibody OMS646 when bound to human MASP-2.

Example 43.

This example shows that human MASP-2 inhibitory antibody (OMS646) is able to inhibit, in TMA patients, plasma-mediated induction of apoptosis in primary human dermal-derived vascular endothelial microcells (MVEC).

The level of knowledge of the problem/relevance.

The pathophysiology of TMA is known to include damage to endothelial cells caused by various factors, followed by occlusion of small vessels (eg, small arterioles and capillaries) by platelet plugs and/or fibrin thrombi (Hirt-Minkowsk P. et al., Nephron Clin Practice 114: c219-c235, 2010; Goldberg RJ et al., Am J Kidney Dis 56 (6): 1168-1174, 2010). Vascular endothelial microcells (MVEC) have been shown to undergo apoptotic damage when exposed in vitro to plasma from patients with TMA-related disorders (see Stefanescu et al., Blood Vol 112 (2): 340-349, 2008; Mitra D. et al., Blood 89: 1224-1234, 1997). TMA-associated apoptotic injury has been found in vascular endothelial microcells (MVEC) obtained from tissue biopsies (skin, bone, bone marrow, spleen, kidney, ileum) of these patients. Apoptotic MVEC lesions have also been shown to reduce the levels of membrane-bound regulatory complement proteins in vascular endothelial microcells (MVEC) (see, for example, Mold & Morris, Immunology 102: 359-364, 2001; Christmas et al., Immunology 119: 522 , 2006).

A positive feedback loop involving terminal complement components is thought to be involved in the pathophysiology of TMA including atypical hemolytic uremic syndrome (aHUS) and TMA associated with catastrophic antiphospholipic syndrome (CAPS), Degos disease and TMA in cancer, cancer chemotherapy , autoimmune response, and transplantation, each of which is known or believed to be susceptible to anti-C5 therapy using the monoclonal antibody (mAb) eculizumab (Chapin J. et al., Brit. J. Hematol 157: 772774, 2012; Tsai et al., Br J Haematol 162 (4): 558-559, 2013); Magro C. M. et al., Journal of Rare Diseases 8: 185, 2013).

The following experiment was conducted to investigate the ability of an inhibitory antibody against human MASP-2 (OMS646) to block plasma-induced apoptosis in primary human dermal vascular endothelial microcells (MVEC) in patients suffering from TMA in plasma samples obtained from patients suffering from HUS, from ADAMTS13 deficiency-associated thrombotic thrombocytopenic purpura (TTP), from catastrophic antiphospholipic syndrome (CAPS) and systemic Degos disease, as well as from TMA due to cancer, transplantation, autoimmune disease and chemotherapy.

Methods.

An in vitro study was made of the efficacy of MASP-2 inhibitory antibody (OMS646) in blocking plasma-mediated apoptosis induction in primary human dermal-derived vascular endothelial microcells (MVEC) in TMA patients as described in Stefanescu R. et al., Blood Vol 112 ( 2): 340-349, 2008, the contents of which are incorporated into the present invention by reference. The plasma samples used in this study were obtained from a collection of plasma from control healthy subjects and patients with thrombotic microangiopathy, either in the acute phase or in the convalescent phase. The presence of microangiopathy in patients with TMA was assessed by detecting schistocytes in a peripheral blood smear. In addition, thrombotic thrombocytopenic purpura (TTP) was diagnosed as described in Stefanescu R. et al., Blood Vol 112 (2): 340-349, 2008.

Culture of endothelial cells (EC).

Described in Stefanescu et al. primary human vascular endothelial microcells

- 134 042534 (MVEC) of dermal origin were supplied by ScienCell Research Labs (San Diego, CA). MVEC expressed CD34 using passages 5 and 6 (Blood 89:1224-1234, 1997). MVEC were maintained in polystyrene mattresses coated with 0.1% gelatin in water in ECM1001 medium (ScienCell Research Labs) containing endothelial cell growth supplement, penicillin, streptomycin and 15% fetal bovine serum. All vascular endothelial microcells (MVEC) were used in passages 2 to 6. Passages included exposure to 0.25% trypsin-EDTA for 5 to 10 minutes.

Apoptosis.

Representative primary human cutaneous vascular endothelial microcells (MVECs) known to be susceptible to TTP/HUS plasma-induced apoptosis were washed with phosphate buffered saline (PBS) and plated in 12-well plates coated with 0.1% gelatin in water at 0.15x10 ⁶ viable cells/ml. Seeded MVEC cells were maintained in complete medium for 24 h and then exposed to various concentrations (2% to 20% v/v) of TMA patient plasma or healthy donor plasma for 18 h in the presence or absence of a monoclonal antibody (mAb) against MASP-2 OMS646 (150 μg/ml), and the cells were then harvested by trypsinization. Each plasma sample from a patient with TMA was examined in two parallel experiments. The extent of plasma-mediated apoptosis was assessed by propidium iodide (PI) staining, analysis of >5×10 ³ cells in a cytofluorograph, and AO peaks determined by software (MCycle Av, Phoenix Flow Systems, San Diego, CA). DNA fragments associated with cytoplasmic histone were also quantified in the cell lysate using an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (Roche Diagnostics, Mannheim, Germany).

Results.

The results of a vascular endothelial microcell apoptosis (MVEC) study induced by TMA patient plasma in the presence of an anti-MASP-2 monoclonal antibody (mAb) (OMS646) are shown in Table 1 below. 25.

Table 25

TMA patient plasma tested on primary human vascular endothelial microcells (MVEC) of dermal origin in the presence of a monoclonal antibody (mAb) against

MASP-2 (OMS646)__________________

Subject # Age / Gender Clinical diagnosis (TMA) and other conditions MASP-2 ng/ml Cre/LDH based diagnosis C5a sC5-b9 ADAMS Activity Diagnosis based on ADAMS activity Protection with OMS646 #2 41/f TTR 174 TTR 34.4 2 772 thirty% aHUS Effect #3 52/f TTR 150 TTR 48.3 2 1399 70% aHUS No effect #4 20/m TTR 224 TTR 36.9 1187 <10% TTR Effect #10 60/f TTR 175.4 TTR 49.5 4406 64% aHUS No effect #eleven 59/f TTR 144.9 TTR 40.3 1352 <10% TTR No effect #13 49/f HUS, cancer, TTP 142.8 TTR 48.6 3843 86% aHUS No effect

- 135 042534

#42 27/t TTP 341.5 TTP 100.0 5332 <5% TTP No effect #46 25/f TTP, Degos disease, SLE 225.1 TTP 53, 9 3426 ND ND Effect #48 53/f TTP, SLE, nephritis after kidney transplantation 788.5 aHUS 31.2 1066 66% aHUS Effect #49 64/f TTR, APLAS, CVA 494.5 35, 4 2100 ND ND Effect #51 25/f ahus, APLAs 313, 1 TTP 26, 8 1595 23% aHUS Effect #52 56/f aHUS, SLE 333, 1 TTP 18.9 1103 97% aHUS No effect #53 56/f aHUS remission 189.9 TTP remy SSIA 28.6 9 344 74% aHUS No effect

Conventional abbreviations used in Table. 25:

APLAs=antiphospholipid antibodies associated with catastrophic antiphospholipid syndrome (CAPS).

SLE=systemic lupus erythematosus.

CVA=acute cerebrovascular accident (stroke).

According to the results published in Stefanescu R. et al., Blood Vol 112 (2): 340-349, 2008, there was significant apoptosis of dermal-derived primary vascular endothelial microcells (MVEC) in the presence of thirteen plasma samples from TMA patients in the absence of antibodies against MASP-2. Control samples of healthy human plasma were tested in parallel and did not induce primary vascular endothelial microcell (MVEC) apoptosis (data not shown). As shown in Table. 25, MASP-2 inhibitory mAb (OMS646) inhibited TMA patient plasma-mediated primary vascular endothelial microcell (MVEC) apoptosis (effect in Table 25) in 6 of 13 patient plasma samples tested (46%). In particular, it should be noted that MASP-2 inhibitory monoclonal antibody mAb (OMS646) inhibited apoptosis in plasma obtained from patients suffering from HUS, TTP, Degos disease, SLE, graft and APLAs (CAPS). Considering that in the seven plasma samples of patients subjected to this study, the anti-MASP-2 monoclonal antibody (mAb) did not block apoptosis (no effect in Table 25), it should be noted that apoptosis can be induced through several pathways, not all of which are complement dependent. For example, as noted in Stefanescu R. et al., Blood Vol 112 (2): 340-349, 2008, apoptosis in endothelial cell (EC) studies is dependent on the basal state of EC activation, which is influenced by plasma factors that can play some role in determining the level of damage required to induce apoptosis. In addition, as noted in the publication of Stefanescu R. et al., additional factors that can modulate apoptosis, such as cytokines and various components of the complement system, may be present in the plasma of a patient with TMA. Therefore, due to these complicating factors, it is not surprising that the anti-MASP-2 antibody was not blocking in all plasma samples that exhibited plasma-induced apoptosis from TMA patients.

In addition, in this regard, it should be noted that a similar study of apoptosis induced by the plasma of patients with TMA was performed in the presence of the anti-C5 antibody eculizumab, and very similar results were obtained (see Chapin et al., Blood (ASH Annual Meeting Abstracts) : Abstract #3342, 120: 2012). The clinical efficacy of eculizumab, a highly successful commercial product, appears to be superior to that demonstrated in this model, suggesting that this in vitro model may underestimate the clinical efficacy of complement inhibitory drugs.

These results show that a MASP-2 inhibitory antibody such as OMS646 is effective

- 136 042534 inhibits plasma-induced apoptosis obtained from patients suffering from TMA, such as aHUS, TTP, Degos disease, SLE, graft and APLAs (CAPS). Endothelial injury and apoptosis are known to play a key role in TMA pathology such as idiopathic TTP and sporadic HUS (Kim et al., Microvascular Research vol 62 (2): 83-93, 2001). As described in Dang et al., Blood 93 (4): 1264-1270, 1999, apoptosis was found in the red pulp of the spleen of patients with TTP, but not in healthy controls. Evidence of apoptosis has also been found in glomerular cells of MVEC origin in a HUS patient (Arends M. J. et al., Hum Pathol 20:89, 1989). Therefore, it can be expected that the administration of a MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody (eg, OMS646), may be an effective therapy for patients suffering from TMA, such as aHUS, TTP, or another microangiopathic disorder, such as another a form of TMA that includes CAPS, systemic Degos disease, and TMA due to cancer, TMA due to chemotherapy, or TMA due to transplantation.

In accordance with the foregoing, in one embodiment, the invention provides a method for inhibiting endothelial cell damage and/or endothelial cell apoptosis and/or thrombosis in a subject suffering from thrombotic microangiopathy (TMA) or at risk of developing thrombotic microangiopathy (TMA), comprising administering the subject of a composition comprising an amount of a MASP-2 inhibitory antibody effective to inhibit MASP-2 dependent complement activation. In one embodiment, the TMA is selected from the group consisting of atypical hemolytic uremic syndrome (aHUS), thrombotic thrombocytopenic purpura (TTP), and hemolytic uremic syndrome (HUS). In one embodiment, the TMA is aHUS. In one embodiment, the composition is administered to a patient with aHUS during the acute phase of the disease. In one embodiment, the composition is administered to a patient with aHUS during a remission phase (i.e., a subject who has recovered or partially recovered from an episode of the acute phase of aHUS and such remission is confirmed, for example, by an increase in platelet count and/or a decrease in serum LDH, for example, as described in Loirat C et al., Orphanet Journal of Rare Diseases 6:60, 2011, the contents of which are incorporated herein by reference).

In one embodiment, the subject suffers from or is at risk of developing TMA, which is (i) TMA in cancer; (ii) TMA during chemotherapy; or (iii) TMA in the setting of transplantation (eg, organ transplantation such as kidney transplantation or allogeneic hematopoietic stem cell transplantation). In one embodiment, the subject suffers from Upshaw-Schulman syndrome (USS) or is at risk for developing Upshaw-Schulman syndrome (USS). In one embodiment, the subject suffers from Degos disease or is at risk of developing Degos disease. In one embodiment, the subject suffers from catastrophic antiphospholipid syndrome (CAPS) or is at risk of developing catastrophic antiphospholipid syndrome (CAPS).

According to any of the embodiments disclosed herein, the MASP-2 inhibitory antibody is characterized by at least one or more of the following: said antibody binds human MASP-2 with a K _D of 10 nM or less, said antibody binds an epitope in the CCP1 domain MASP-2, said antibody inhibits C3b precipitation in an in vitro assay in 1% human serum with an IC ₅₀ value of 10 nM or less, said antibody inhibits C3b precipitation in 90% human serum with an IC ₅₀ value of 30 nM or less, wherein the antibody is a fragment antibodies selected from the group consisting of Fv, Fab, Fab', F(ab)2 and F(ab')2, where the antibody is a single chain molecule, where said antibody is an IgG2 molecule, where said antibody is an IgG1 molecule, where the specified antibody is an IgG4 molecule, where the IgG4 molecule contains the S228P mutation, and/or where the antibody does not substantially inhibit the classical pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and substantially does not inhibit the alternative pathway. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and substantially does not inhibit the classical pathway (ie, it inhibits the lectin pathway while leaving the classical complement pathway unaffected).

In one embodiment, the MASP-2 inhibitory antibody inhibits plasma-induced vascular endothelial microcell (MVEC) apoptosis in the serum of a subject suffering from TMA, such as aHUS (acute or remission), hemolytic uremic syndrome (HUS), thrombotic thrombocytopenic purpura (TTP), TMA on the background of cancer, TMA on the background of chemotherapy; TMA in the background of transplantation (e.g. organ transplantation, e.g. kidney transplantation or hematopoietic stem cell transplantation), or in the blood serum of a subject suffering from Upshaw-Schulman syndrome (USS), or in the blood serum of a subject suffering from Degos disease, or a subject suffering from catastrophic antiphospholipid syndrome (CAPS), wherein plasma-induced vascular endothelial microcell (MVEC) apoptosis is inhibited by at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 60%, such as

- 137 042534 at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% to 99%, compared to serum , untreated with antibody. In some embodiments, the MASP-2 inhibitory antibody inhibits blood clotting in the serum of a subject suffering from TMA (e.g., such as aHUS (acute or remission), hemolytic uremic syndrome (HUS), thrombotic thrombocytopenic purpura (TTP), TMA on cancer background, chemotherapy background TMA, transplant background TMA (for example, organ transplantation such as kidney transplantation or allogeneic hematopoietic stem cell transplantation), or in the blood serum of a subject suffering from Upshaw-Schulman syndrome (USS), or in the blood serum a subject suffering from Degos disease or a subject suffering from catastrophic antiphospholipid syndrome (CAPS)), at a level of at least 20 percent or more (e.g., at least 30%, at least 40%, at least 50%) higher than the inhibitory effect on serum C5b-9 precipitation.

In one embodiment, the MASP-2 inhibitory antibody is administered to the subject via an intravenous catheter or other catheter delivery method.

In one embodiment, the invention provides a method for inhibiting thrombus formation in a subject suffering from TMA (e.g., aHUS (acute or remission), hemolytic uremic syndrome (HUS), thrombotic thrombocytopenic purpura (TTP), TMA in cancer, TMA in in the background of chemotherapy, TMA in the background of transplantation (for example, organ transplantation, such as kidney transplantation or hematopoietic stem cell transplantation), or in the blood serum of a subject suffering from Upshaw-Schulman syndrome (USS), or in the blood serum of a subject suffering from Degos disease , or in a subject suffering from catastrophic antiphospholipid syndrome (CAPS)), comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or antigen-binding fragment thereof, containing (I) (a) a heavy chain variable region comprising: i) a heavy chain CDR-H1 containing the amino acid sequence from 31-35 sequences of SEQ ID NO: 67; and ii) a CDR-H2 heavy chain containing the amino acid sequence from 50-65 of SEQ ID NO: 67; and iii) a CDR-H3 heavy chain comprising the amino acid sequence from 95-102 of SEQ ID NO: 67 and b) a light chain variable region comprising: i) a CDR-L1 light chain comprising an amino acid sequence from 24-34 of SEQ ID NO: 70; and ii) a CDR-L2 light chain containing the amino acid sequence from 50-56 of SEQ ID NO: 70; and iii) a CDR-L3 light chain comprising the amino acid sequence from 89-97 of SEQ ID NO: 70 or (ii) a variant thereof comprising a heavy chain variable region with at least 90% identity to SEQ ID NO: 67 (e.g. , at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, with at least 98%, at least 99% identity with SEQ ID NO: 67) and a light chain variable region with at least 90% identity (for example, at least 91%, at least 92% , at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical with SEQ ID NO: 70.

In one embodiment, the subject suffers from TMA selected from the group consisting of TMA due to cancer, TMA due to chemotherapy, TMA due to transplantation (e.g., organ transplantation such as kidney transplantation or allogeneic hematopoietic stem cell transplantation), Upshaw's syndrome -Schulman (USS), Degos disease and catastrophic antiphospholipid syndrome (CAPS).

In some embodiments, the method includes administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising an amino acid sequence referred to as the sequence of SEQ ID NO: 67. In some embodiments, the method comprises administering the subject of a composition comprising an amount of a MASP-2 inhibitory antibody or an antigen-binding fragment thereof, comprising a light chain variable region comprising an amino acid sequence referred to as the sequence of SEQ ID NO: 70.

In some embodiments, the method comprises administering to a subject a composition comprising a MASP-2 inhibitory antibody or antigen-binding fragment thereof that specifically recognizes at least a portion of an epitope on human MASP-2 recognized by an OMS646 control antibody comprising a heavy chain variable region, referred to as the sequence of SEQ ID NO: 67 and the variable region of the light chain, referred to as the sequence of SEQ ID NO: 70.

Example 44.

This example describes the initial results of an ongoing phase 2 clinical trial to evaluate the safety and clinical efficacy of a fully human anti-MASP-2 monoclonal inhibitory antibody in adult patients with thrombotic microangiopathy (TMA).

- 138 042534

The level of knowledge of the problem.

Thrombotic microangiopathies (TMAs) are a family of rare, debilitating and life-threatening disorders characterized by the formation of an excessive number of thrombi (clots) of platelet aggregates in the capillary circulation of body organs, usually in the kidneys and brain.

Methods.

Phase 2 open-label clinical trials were conducted in patients with primary atypical hemolytic uremic syndrome (aHUS), plasma-resistant aHUS, and thrombotic thrombocytopenic purpura (TTP). This Phase II clinical trial does not use placebo due to the life-threatening nature of the disease.

Subjects aged >18 years were screened and only subjects diagnosed with one of the following TMAs were included in this study:

1) Primary aHUS diagnosed clinically and having ADAMTS13 activity > 10% in plasma. Eligible patients are those with or without a documented mutation in complement, or with an anti-CFH antibody. Patients are classified according to their response to plasma therapy (plasma replacement or plasma infusion):

Plasma-resistant patients with aHUS, for the purposes of this study, fulfill all of the following requirements:

(a) screening for a platelet count < 150,000/µl, despite the fact that four sessions of plasma therapy were performed before screening;

(b) evidence of microangiopathic hemolysis (presence of schizocytes, serum lactate dehydrogenase (LDH) above the upper limit of normal (ULN), haptoglobin < LLN);

and (c) serum creatinine > ULN.

patients with aHUS susceptible to long-term plasma therapy (plasma therapy sensitive) who may require at least plasma therapy once a week for four weeks prior to the first dose of OMS646 when serum creatine > ULN.

2) TTR is determined by all of the following indicators:

platelet count < 150,000/µl;

evidence of microangiopathic hemolysis (presence of schistocytes, serum lactate dehydrogenase (LDH) > ULN, haptoglobin < LLN)); and ADAMTS13 activity < 10% during the current episode of TTP or continuously.

Note: Subjects were excluded from the study if they were treated with eculizumab within three months prior to screening. Criteria for a patient classified as refractory to plasma therapy were used to determine eligibility for participation in this study. In additional aspects of the invention, a plasma therapy resistant patient to be treated with a MASP-2 inhibitor has been previously treated with plasma therapy at least once, and after such treatment with plasma therapy, one or more clinical markers of aHUS are detected, that are not adequately reduced or eliminated as a result of such plasma therapy.

The monoclonal antibody OMS646 used in this study is a fully human IgG4 monoclonal antibody against human MASP-2. As shown in Example 40, OMS646 binds strongly to recombinant MASP-2 (with an apparent equilibrium dissociation constant in the 100 pM range) and exhibits over 5,000-fold selectivity over binding to homologous protein C1s, C1r and MASP-1. In functional studies, OMS646 inhibits the human lectin pathway with activity in the nanomolar concentration range (the concentration resulting in 50% inhibition [IC ₅₀ ] is approximately 3 nM), but does not significantly affect the classical pathway. Intravenous or subcutaneous administration of OMS646 to mice, primates, and humans resulted in high plasma concentrations that were associated with downregulation of lectin pathway activation in ex vivo studies. As further described in Example 42, administration of OMS646 reduced C5b-9 deposition and thrombus formation in experimental mouse models of TMA and thrombus formation in vitro, thereby demonstrating the potential for therapeutic applications of OMS646.

In this study, OMS646 drug was formulated at 100 mg/mL concentrations, which was then diluted for intravenous administration. The corresponding calculated volume of OMS646 100 mg/mL injection solution was withdrawn from the vial with a dose preparation syringe. The infusion bag was used within 4 hours of preparation.

In the first phase of the study, OMS646 was administered at increasing doses in groups of three patients each. Each subject in stage 1 was dosed with OMS646 once a week for four weeks, as shown in Table 1 below. 26.

- 139 042534

Table 26

Dosing schedule at stage 1

Stage, group Number of subjects Dose of OMS646 (mg/kg) 1, group 1 3 0.675 once a week x 4 1, group 2 3 2.0, once a week x 4 1, group 3 3 4.0, once a week x 4

Stage 1.

Study Design Outline

The diluted study drug was administered intravenously over 30 minutes.

primary endpoints.

Concomitant primary endpoints were:

safety, which is assessed by side effects (AE), vital signs, ECG and clinical laboratory tests;

clinical activity, determined by the change in the number of platelets.

secondary endpoints.

Secondary endpoints were:

clinical activity of TMA;

serum LDH;

serum haptoglobin;

hemoglobin;

serum creatinine;

symptoms associated with TMA;

the need for plasma therapy (plasma replacement or plasma infusion);

need for dialysis.

Approved concomitant therapies.

plasma-resistant aHUS - plasma therapy during treatment with OMS646 was allowed if the investigator considered it to be medically indicated.

aHUS susceptible to long-term plasma therapy, the investigators were advised to continue plasma therapy until TMA improved, eg, increased platelet count, decreased LDH, increased haptoglobin, increased hemoglobin, decreased creatinine, after which the investigator was advised to consider discontinuing plasma therapy and conduct continuous monitoring of TMA parameters to assess the possibility of refusing the use of plasma therapy.

TTR - Plasma therapy was allowed if the investigator considered it to be medically indicated.

The investigators were advised that hemodialysis be carried out in accordance with the standard of care.

Eculizumab was not administered during the study.

Results.

The first group of subjects consisted of three aHUS patients receiving the lowest dose of OMS646. There were improvements in TMA markers in all patients in this study group. As markers of disease activity, platelet count, serum lactate dehydrogenase (LDH) content, and serum haptoglobin content were determined. Compared to baseline, platelet counts improved in all patients. Serum LDH levels remained normal in one patient, decreased substantially to near normal range in another patient, and remained elevated in a third patient. Haptoglobin improved in two patients and returned to normal in one patient. One patient with independent kidney function also improved creatinine levels.

According to the study design, three patients were treated with the average dose in the second arm of this clinical trial. Two patients with aHUS are resistant to plasma therapy and one patient has thrombotic thrombocytopenic purpura (TTP). Both patients with aHUS underwent hemodialysis before and during the study. In the second or intermediate dose group, plasma-resistant aHUS patients were characterized by:

47% increase in mean platelet count resulting in both patients having platelet counts in the normal range.

86% reduction in the mean number of schistocytes, with schistocytes disappearing in one patient.

71% increase in mean haptoglobin, with two patients reaching the normal range during treatment, one patient falling slightly below normal during treatment

- 140 042534 one week after the last dosing.

5% reduction in mean LDH levels, with two patients remaining slightly elevated over the normal range.

The patient with TTR in the middle dose group required a repeat plasma infusion before participating in the study. Laboratory parameters showed no consistent improvement, but during treatment with OMS646 and at the end of treatment, the patient did not require plasma therapy.

The drug was well tolerated by all patients throughout the treatment period. Based on the positive results for the second group or the medium dose group, studies were started in the third group or the high dose group, and the study of the patient with aHUS has already been completed. Data for all patients includes measurements within one week of the last dose.

Treatment with OMS646 was also completed for the first patient in the third group (in the high dose group), an aHUS patient who was refractory to plasma therapy and had additional complications including hepatitis C, cryoglobulinemia, and lymphoma. Prior to treatment with OMS646, the patient required repeat dialysis. During and after the completion of the course with OMS646 and to date, the patient has not required dialysis. Hematological and renal parameters showed:

63% improvement in platelet count, return to normal levels, 100% decrease in schistocytes, haptoglobin increased from undetectable levels and returned to normal, 43% decrease in LDH resulting in slightly above normal levels, 24% decrease in creatinine.

The drug was well tolerated in both the first group and the second group.

The primary endpoint in this phase 2 open-label clinical trial is platelet count change. Platelet counts in all three patients with aHUS in the medium and high dose groups (two patients in the medium dose group and one patient in the high dose group) returned to normal, with a statistically significant mean increase from baseline of approximately 68,000 platelets/mL ( p=0.0055).

In summary, pre-Phase 2 data of this clinical trial demonstrate the efficacy of OMS646 in patients with primary aHUS, plasma-resistant aHUS patients, and patients with TTP.

According to the foregoing, in one embodiment, the invention provides a method of treating a subject suffering from plasma therapy resistant aHUS, comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody effective to inhibit MASP-2 dependent complement activation. In some embodiments, the method further comprises the step of identifying a subject suffering from plasma therapy resistant aHUS prior to administering to the subject a composition comprising a MASP-2 inhibitory antibody.

In accordance with any of the embodiments disclosed herein, the MASP-2 inhibitory antibody exhibits at least one or more of the following: said antibody binds human MASP-2 with a KD of 10 nM or less, said antibody binds an epitope in the CCP1 domain of MASP- 2, said antibody inhibits C3b precipitation in an in vitro assay in 1% human serum with an IC ₅₀ value of 10 nM or less, said antibody inhibits C3b precipitation in 90% human serum with an IC ₅₀ value of 3 0 nM or less, wherein the antibody is an antibody fragment , selected from the group consisting of Fv, Fab, Fab', F(ab)2 and F(ab')2, where the antibody is a single chain molecule, where the specified antibody is an IgG2 molecule, where the specified antibody is an IgG1 molecule, where said antibody is an IgG4 molecule, where the IgG4 molecule contains the S228P mutation. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and substantially does not inhibit the classical pathway (ie, inhibits the lectin pathway without affecting the classical complement pathway).

In one embodiment, the MASP-2 inhibitory antibody is administered in an amount effective to improve at least one or more clinical parameters associated with aHUS, such as an increase in platelet count, a decrease in LDH, an increase in haptoglobin, an increase in hemoglobin, and/or a decrease in creatinine.

In some embodiments, the method comprises administering a MASP-2 inhibitory antibody to a subject suffering from plasma therapy resistant aHUS via a catheter (e.g., intravenously) for a first period of time (e.g., for at least one day to a week or two weeks) , and then administering the MASP-2 inhibitory antibody to the subject subcutaneously for a second period of time (eg, in the long-term treatment phase, for at least two weeks or more). In some embodiments, the implementation of the introduction in the first and/or second period of time is carried out without the use of plasma therapy. In some embodiments, the implementation of the introduction in the first and/or second period of time is carried out using plasma therapy.

- 141 042534

In some embodiments, the method includes administering a MASP-2 inhibitory antibody to a subject suffering from plasma therapy resistant aHUS, either intravenously, intramuscularly, or preferably subcutaneously. The treatment can be prolonged and applied from once a day to once a month, but preferably once every two weeks. The MASP-2 inhibitory antibody can be administered alone or in combination with a C5 inhibitor such as eculizumab.

In one embodiment, the method comprises treating a subject suffering from plasma therapy resistant aHUS comprising administering to the subject a composition comprising an amount of a MASP2 inhibitory antibody or antigen-binding fragment thereof containing (I) (a) a heavy chain variable region comprising: i) a CDR heavy chain -H1 containing the amino acid sequence from 31-35 sequences of SEQ ID NO: 67; and ii) a CDR-H2 heavy chain containing the amino acid sequence from 50-65 of SEQ ID NO: 67; and iii) a CDR-H3 heavy chain comprising the amino acid sequence from 95-102 of SEQ ID NO: 67 and b) a light chain variable region comprising: i) a CDR-L1 light chain comprising an amino acid sequence from 24-34 of SEQ ID NO: 70; and ii) a CDR-L2 light chain containing the amino acid sequence from 50-56 of SEQ ID NO: 70; and iii) a CDR-L3 light chain comprising the amino acid sequence from 89-97 of SEQ ID NO: 70 or (ii) a variant thereof comprising a heavy chain variable region with at least 90% identity to SEQ ID NO: 67 (e.g. , at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, with at least 98%, at least 99% identity with SEQ ID NO: 67) and a light chain variable region with at least 90% identity (for example, at least 91%, at least 92% , at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical with SEQ ID NO: 70.

In some embodiments, the method includes administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising the amino acid sequence referred to as the sequence of SEQ ID NO: 67 and a light chain variable region comprising the amino acid sequence , referred to as the sequence SEQ ID NO: 70.

In some embodiments, the method includes administering to a subject a composition comprising a MASP-2 inhibitory antibody, or an antigen-binding fragment thereof, that specifically recognizes at least a portion of an epitope on human MASP-2 recognized by an OMS646 control antibody comprising a heavy chain variable region, referred to as SEQ ID NO: 67, and a light chain variable region referred to as SEQ ID NO: 70.

Example 45.

This example describes additional results from an ongoing phase 2 clinical trial to evaluate the safety and clinical efficacy of a fully human anti-MASP-2 monoclonal inhibitory antibody in adult patients with thrombotic microangiopathy (TMA) described in Example 44.

The level of knowledge of the problem. As described in Example 44, thrombotic microangiopathies (TMAs) are a family of rare, debilitating, and life-threatening disorders characterized by the formation of an excessive amount of blood clots (clots) - platelet aggregates - in the capillary circulation of body organs, usually in the kidneys and brain. As described in the invention, transplant-related TMA (TA-TMA) is a very dangerous syndrome that can occur in transplant patients such as hematopoietic stem cell transplant (HSCT).

This example describes the initial results of a phase 2 clinical trial to evaluate the safety and clinical efficacy of a fully human monoclonal MASP-2 inhibitory antibody in a patient suffering from TMA associated with hematopoietic stem cell transplantation.

Methods.

As described in Example 44, the phase 2 TMA assay consists of a three-level dosing step followed by a fixed dosing step for the MASP-2 inhibitory antibody OMS646. As further described in example 44, consistent and reliable positive results were obtained in the case of patients with aHUS and one patient with TTP. As with the low dose group, OMS646 was well tolerated by all patients in the medium and high dose groups throughout the treatment period. Preclinical toxicity studies have been completed and no safety concerns have been identified with this antibody, allowing long-term dosing in clinical trials.

As described in example 44, data were obtained from a phase 2 clinical study of OMS646 in the case of TMA for patients with aHUS and TTP. Dosing of one additional patient with TMMA associated with stem cell transplantation in the high dose group was completed using the methods described in Example 44. This patient had a history of lymphoma for which he had undergone hematopoietic stem cell transplantation. . The course of his disease after transplantation was complicated by a number of life-threatening disorders, including TMA requiring platelet infusion. Despite the infusion, he retained TMA associated with stem cell transplantation and participated in a phase 2 study of the OMS646 antibody.

Results.

After dosing for four weeks (high dose group) as described in Example 44, a patient with TMA associated with stem cell transplantation was characterized by:

a fourfold increase in the number of platelets, as a result of which the number of platelets exceeded 100,000;

the level of haptoglobin more than doubled and reached the norm;

plasma lactate dehydrogenase level, indicator;

damage to blood vessels decreased by 35%, but was still above normal; single schistocytes were found.

During the entire time of OMS646 dosing and after completion of OMS646 treatment, the patient did not require either platelet infusion or plasmapheresis.

Therefore, the phase 2 data of this clinical trial (as described in Example 44 and in this Example) demonstrates the efficacy of OMS646 in patients with primary aHUS, in plasma-resistant aHUS patients, in TTR patients, and in the patient with TMA associated with hematopoietic stem cell transplantation.

In accordance with the foregoing, in one embodiment, the invention provides a method of treating a human suffering from TMA associated with hematopoietic stem cell transplantation, comprising administering to the subject a composition containing an amount of a MASP-2 inhibitory antibody effective to inhibit MASP-2 dependent complement activation. In one embodiment, the subject suffers from TMA associated with hematopoietic stem cell transplantation, which is resistant to treatment by platelet infusion and/or plasmapheresis. In one embodiment, the method includes identifying a human suffering from a hematopoietic stem cell transplant-associated TMA that is refractory to treatment by platelet infusion and/or plasmapheresis prior to the step of administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody effective to inhibit MASP -2 dependent complement activation.

According to any of the embodiments disclosed herein, the MASP-2 inhibitory antibody exhibits at least one or more of the following: said antibody binds human MASP-2 with a K _D of 10 nM or less, said antibody binds an epitope in the CCP1 domain of MASP -2, said antibody inhibits C3b precipitation in an in vitro assay in 1% human serum with an IC ₅₀ value of 10 nM or less, said antibody inhibits C3b precipitation in 90% human serum with an IC ₅₀ value of 30 nM or less, wherein the antibody is an antibody fragment , selected from the group consisting of Fv, Fab, Fab', F(ab)2 and F(ab')2, where the antibody is a single chain molecule, where the specified antibody is an IgG2 molecule, where the specified antibody is an IgG1 molecule, where said antibody is an IgG4 molecule, where the IgG4 molecule contains the S228P mutation. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and substantially does not inhibit the classical pathway (ie, it inhibits the lectin pathway while leaving the classical complement pathway unaffected).

In one embodiment, the MASP-2 inhibitory antibody is administered in an amount effective to improve at least one or more clinical parameters related to TMA associated with hematopoietic stem cell transplantation, such as an increase in platelet count (e.g., at least two times, at least three times, at least four times, compared with the number of platelets before treatment), an increase in haptoglobin and/or a decrease in lactate dehydrogenase.

In some embodiments, the method comprises administering a MASP-2 inhibitory antibody to a subject suffering from hematopoietic stem cell transplant-associated TMA via a catheter (e.g., intravenously) over a first period of time (e.g., at least one day to weeks or two weeks) followed by subcutaneous administration of the MASP-2 inhibitory antibody to the subject for a second period of time (eg, for an extended period of at least two weeks or more). In some embodiments, the implementation of the introduction in the first and/or second period of time is carried out without the use of plasma therapy. In some embodiments, the implementation of the introduction in the first and/or second period of time is carried out with the use of plasma therapy.

In some embodiments, the method includes administering a MASP-2 inhibitory antibody to a subject suffering from TMA associated with hematopoietic stem cell transplantation, either intravenously, intramuscularly, or preferably subcutaneously. Treatment can be long-term and can be from once a day to once a month, but preferably once every two

- 143 042534 weeks. The MASP-2 inhibitory antibody can be administered alone or in combination with a C5 inhibitor such as eculizumab.

In one embodiment, the method comprises treating a subject suffering from TMA associated with hematopoietic stem cell transplantation, comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or an antigen-binding fragment thereof, comprising (I) (a) a heavy chain variable region comprising : i) heavy chain CDR-H1 containing the amino acid sequence from 31-35 sequences of SEQ ID NO: 67; and ii) a CDR-H2 heavy chain containing the amino acid sequence from 50-65 of SEQ ID NO: 67; and iii) a CDR-H3 heavy chain comprising the amino acid sequence from 95-102 of SEQ ID NO: 67; and b) a light chain variable region comprising: i) a CDRL1 light chain comprising the amino acid sequence from 24-34 of SEQ ID NO: 70; and ii) a CDR-L2 light chain containing the amino acid sequence from 50-56 of SEQ ID NO: 70; and iii) a CDR-L3 light chain comprising the amino acid sequence from 89-97 of SEQ ID NO: 70 or (ii) a variant thereof comprising a heavy chain variable region with at least 90% identity to SEQ ID NO: 67 (e.g. , at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, with at least 98%, at least 99% identity with SEQ ID NO: 67) and a light chain variable region with at least 90% identity (for example, at least 91%, at least 92% , at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical with SEQ ID NO: 70.

In some embodiments, the method includes administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising an amino acid sequence referred to as the sequence of SEQ ID NO: 67, and a light chain variable region comprising amino acid sequence, referred to as SEQ ID NO: 70.

In some embodiments, the method comprises administering to a subject a composition comprising a MASP-2 inhibitory antibody or antigen-binding fragment thereof that specifically recognizes at least a portion of an epitope on human MASP-2 recognized by an OMS646 control antibody comprising a heavy chain variable region, referred to as the sequence of SEQ ID NO: 67, and the variable region of the light chain, referred to as the sequence of SEQ ID NO: 70.

Example 46.

This example describes additional results obtained in an ongoing phase 2 clinical study to evaluate the safety and clinical efficacy of a fully human monoclonal MASP-2 inhibitory antibody in adult patients with thrombotic microangiopathies (TMA) described in examples 44 and 45.

The level of knowledge of the problem.

As described in Example 45, transplant-associated TMA (TA-TMA) is a very dangerous syndrome that can occur in transplant patients such as hematopoietic stem cell transplant (HSCT) recipients. TMAs associated with hematopoietic stem cell transplantation (HSCT-TMA) are a life-threatening complication caused by endothelial damage. The kidneys are the most commonly affected organ, although HSCTTMA can be a systemic disease that also affects the lung, intestines, heart, and brain. The occurrence of even mild TMA is associated with long-term renal failure. The development of post-allogenic HSCT-associated TMA varies in frequency depending on various diagnostic criteria and patterns of prevention of graft rejection in the host, with calcineurin inhibitors most commonly causing HSCT-associated TMA (Ho VT et al., Biol Blood Marrow Transplant , 11(8):571-5, 2005). Modification of the dosing regimen of an immunosuppressive calcineurin inhibitor may lead to improvement in TMA in some patients within a few weeks after discontinuation or discontinuation of the calcineurin inhibitor.

TMA is a potentially life-threatening complication of HSCT, and current management is based largely on reducing exposure to precipitating factors, including avoidance of immunosuppressant drugs (eg, calcineurin inhibitors) and treatment of current infections, as well as supportive therapies such as like hemodialysis. While many HSCT-TMA patients respond well to reductions or interruptions in immunosuppressant medication, there is a subset of patients who develop persistent HSCTTMA despite conservative treatments (i.e., TMA does not respond to immunosuppressant dose reduction or interruption). their acceptance). Patients who do not respond to these conservative treatments have a poor prognosis. Plasma replacement was ineffective and no other therapy was approved.

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Example 45 describes the initial positive results of a phase 2 clinical trial to evaluate the safety and clinical efficacy of a fully human monoclonal MASP-2 inhibitory antibody (OMS646) in a patient suffering from permanent THM associated with hematopoietic stem cell transplantation (HSCT-TMA). This example describes additional results from an ongoing phase 2 clinical trial to evaluate the safety and clinical efficacy of OMS646 in three additional subjects with persistent HSCT-TMA refractory to conservative treatments.

Methods.

As described in Example 44, the phase 2 TMA study consists of a three-level dosing step followed by a fixed dose of the MASP-2 inhibitory antibody OMS646. OMS646 was well tolerated by all patients in the low, medium and high dose groups throughout the treatment period. Preclinical toxicity studies have been completed and no safety concerns have been identified with this antibody, allowing long-term dosing in clinical trials.

The Phase 2 TMA study included subjects suffering from persistent HSCT-related TMA refractory to conservative therapies, which was defined for the purposes of this study as having all of the following, at least two weeks after withdrawal or discontinuation of an immunosuppressive drug. agent (for example, a calcineurin inhibitor) or at least 30 days after transplantation:

thrombocytopenia (platelet count < 150,000/mcL) and evidence of microangiopathic hemolytic anemia (presence of schistocytes, serum lactate dehydrogenase (LDH) > upper limit of normal (ULN) or haptoglobin < lower limit of normal (LLN)).

Approved concomitant therapy for HSCT-related TMA.

The use of plasma therapy during treatment with OMS646 was allowed if the investigator considered it to be medically indicated. Patients treated with plasma therapy could receive additional half doses of OMS646.

The investigators were advised that hemodialysis be carried out in accordance with the standard of care.

Eculizumab was not administered during the study.

This TMA study included subjects suffering from persistent HSCT-TMA that is resistant to standard therapies (i.e., persistent TMA at least two weeks after discontinuation or interruption of calcineurin inhibitor treatment).

As described in Example 45, dosing was completed for a patient suffering from persistent HSCTTMA (Patient #1) in the high dose group (4 mg/kg OMS646 administered intravenously once a week for four weeks) using the methods described in Example 44.

Dosing of an additional 4 patients suffering from persistent HSCT-TMA was completed as described below.

Patient #1 (described in Example 44) was administered OMS646 for four weeks (4 mg/kg IV once a week).

Patients #2 and #3 were given OMS646 for eight weeks (4mg/kg IV once a week).

Patients #4 and #5 were given OMS646 (4 mg/kg IV once a week) for two and three weeks, respectively. Patients #4 and #5, withdrawn from the study after two and three weeks, respectively, did not respond to treatment and worsened. Of note, one of these patients was found to have elevated creatinine at the time of entry into the study.

Results.

This study included five patients with permanent HSCT-TMA. All patients were adults and underwent hematopoietic stem cell transplantation (HSCT) due to the presence of hematological malignancies. Figures 58, 59 and 60 show changes from baseline variables for TMA after treatment with MASP-2 inhibitory antibody OMS646. These figures show data for all five patients, two of whom discontinued the study after 2-3 weeks, after which one patient relapsed and the other received palliative care. As noted in FIG. 58, 59 and 60, the last administration of the antibody could be at week 7.

In FIG. 58 is a graphical representation of the mean change in platelet count from baseline over time (week) in subjects suffering from persistent thrombotic microangiopathy associated with hematopoietic stem cell transplantation (HSCT-TMA) following administration of a MASP-2 inhibitory antibody (OMS646).

In FIG. 59 is a graphical representation of the mean change in LDH from baseline over time (week) in subjects suffering from persistent (HSCT-TMA) after administration of a MASP-2 inhibitory antibody (OMS646).

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In FIG. 60 is a graphical representation of the mean change in haptoglobin from baseline over time (week) in subjects suffering from persistent (HSCT-TMA) after administration of a MASP-2 inhibitory antibody (OMS646).

As shown in FIG. 59 and 60, there was a statistically significant improvement in LDH and haptoglobin during treatment. As shown in FIG. 58, the platelet count improved but was not statistically significant due to the small number of patients. Of the three patients for whom treatment was completed, one patient did not improve in creatinine but received concomitant nephrotoxic therapies. Creatinine improved or remained normal in two other patients. At long-term follow-up, one patient had a transplant failure and was awaiting a second transplant. The condition of the other two patients remained stable.

Despite the possibility of overlapping drug effects associated with bone marrow suppression and nephrotoxicity in two subjects, positive effects of treatment with OMS646 were observed in three subjects suffering from persistent HSCT-TMA (one of which is described in example 45), and which are further described below. Notably, the first positive treatment results in each of the three patients were observed after approximately three weeks of treatment with OMS646.

Patient # 1 (treatment for 4 weeks as described in example 45). To summarize, the platelet count quadrupled, resulting in a platelet count of more than 100,000/µL, haptoglobin more than doubled and was normal, plasma lactate dehydrogenase decreased by 35% but was still above normal, and only one schistocyte was found.

Patient #2 (treatment for 8 weeks). Treatment had no effect on platelet count, although the patient also suffered from bone marrow suppression during concomitant treatment with valganciclovir and later had graft rejection, plasma lactate dehydrogenase decreased from 712 U/L to 256 U/L, and haptoglobin increased from undetectable levels up to 250 mg/dl.

Patient # 3 (treatment for 8 weeks). The number of platelets increased from 1350 0/µl to 133000/µl; the plasma lactate dehydrogenase level decreased from 537 to 225 U/l, the haptoglobin level increased from undetectable levels to 181 mg/dl.

Brief summary of the results.

In the case of three patients suffering from persistent HSCT-related TMA for whom OMS646 dosing was completed, the data demonstrate a strong and sustained effect. During treatment, statistically significant improvements in LDH and haptoglobin were observed. The platelet count improved, but it was not statistically significant in this small number of patients. Of the three patients who completed treatment, one patient had no improvement in creatinine but received concurrent nephrotoxic agents. Creatinine improved or remained normal in two other patients.

Conclusions.

The OMS646 antibody improved TMA markers in patients with persistent HSCT-TMA who did not respond to conservative treatments. HSCT-TMA patients treated with OMS646 are among the most challenging patients to treat, and the OMS646 antibody has demonstrated positive therapeutic effects in patients at high risk for persistent HSCT-TMA despite conservative treatments.

In accordance with the foregoing, in one embodiment, the invention provides a method of treating a human suffering from TMA associated with hematopoietic stem cell transplantation, comprising administering to the subject a composition containing an amount of a MASP-2 inhibitory antibody effective to inhibit MASP-2 dependent complement activation. In one embodiment, the subject suffers from persistent TMA associated with hematopoietic stem cell transplantation that is resistant to conservative treatments. In one embodiment, the method further comprises identifying a human suffering from persistent hematopoietic stem cell transplant-associated TMA that is resistant to conservative therapies, prior to the step of administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody effective to inhibit MASP-2 dependent activation. complement.

In accordance with any of the embodiments disclosed herein, the MASP-2 inhibitory antibody exhibits at least one or more of the following characteristics: said antibody binds human MASP-2 with a KD of 10 nM or less, said antibody binds an epitope in the CCP1 domain of MASP- 2, said antibody inhibits C3b precipitation in an in vitro assay in 1% human serum with an IC ₅₀ value of 10 nM or less, said antibody inhibits C3b precipitation in 90% human serum with an IC ₅₀ value of 30 nM or less, wherein the antibody is a fragment of an 146 042534 titel selected from the group consisting of Fv, Fab, Fab', F(ab)2 and F(ab')2, where the antibody is a single chain molecule, where the specified antibody is an IgG2 molecule, where the specified antibody is a molecule IgG1, where the specified antibody is an IgG4 molecule, where the IgG4 molecule contains the S228P mutation. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and substantially does not inhibit the classical pathway (ie, it inhibits the lectin pathway while leaving the classical complement pathway unaffected).

In one embodiment, the MASP-2 inhibitory antibody is administered in an amount effective to improve at least one or more clinical parameters related to TMA associated with hematopoietic stem cell transplantation, such as an increase in platelet count (e.g., at least a doubling of , at least three times, at least four times more than the number of platelets before treatment), an increase in haptoglobin and / or a decrease in lactate dehydrogenase.

In some embodiments, the method comprises administering a MASP-2 inhibitory antibody to a subject suffering from hematopoietic stem cell transplant-associated TMA via a catheter (e.g., intravenously) over a first period of time (e.g., at least one day to weeks, or two weeks, or three weeks, or four weeks or more), followed by subcutaneous administration of the MASP-2 inhibitory antibody to the subject for a second period of time (e.g., for an extended period of time, at least two weeks or more). In some embodiments, the implementation of the introduction in the first and/or second period of time is carried out without the use of plasma therapy. In some embodiments, the implementation of the introduction in the first and/or second period of time is carried out using plasma therapy.

In some embodiments, the method includes administering a MASP-2 inhibitory antibody to a subject suffering from TMA associated with hematopoietic stem cell transplantation, either intravenously, intramuscularly, or subcutaneously. The treatment can be prolonged and applied from once a day to once a month, but preferably at least once every two weeks, or at least once a week, for example, twice a week or three times a week. The MASP2 inhibitory antibody can be administered alone or in combination with a C5 inhibitor such as eculizumab.

In one embodiment, the method comprises treating a subject suffering from persistent TMA associated with hematopoietic stem cell transplantation, comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or an antigen-binding fragment thereof, containing a heavy chain variable region comprising CDR-H1, CDR -H2 and CDR-H3 amino acid sequence, referred to as the sequence of SEQ ID NO: 67, and the variable region of the light chain, including CDR-L1, CDR-L2 and CDR-L3 amino acid sequence, referred to as the sequence of SEQ ID NO: 70. In some embodiments, the composition comprises a MASP-2 inhibitory antibody comprising (a) a heavy chain variable region comprising: i) a CDR-H1 heavy chain comprising the amino acid sequence from 31-35 of SEQ ID NO: 67; and ii) a CDR-H2 heavy chain containing the amino acid sequence from 50-65 of SEQ ID NO: 67; and iii) a CDR-H3 heavy chain comprising the amino acid sequence from 95-107 of SEQ ID NO: 67 and b) a light chain variable region comprising: i) a CDR-L1 light chain comprising the amino acid sequence from 24-34 of SEQ ID NO: 70; and ii) a CDR-L2 light chain containing the amino acid sequence from 50-56 of SEQ ID NO: 70; and iii) a CDR-L3 light chain comprising an amino acid sequence from 89-97 of SEQ ID NO: 70 or (II) a variant which includes the heavy chain variable region with at least 90% identity with SEQ ID NO: 67 (e.g. at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97 %, at least 98%, at least 100% identity with SEQ ID NO: 67), and the variable region of the light chain, at least 90% identity (for example, at least 91%, at least at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least with 99% identity with SEQ ID NO: 70.

In some embodiments, the method includes administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising the amino acid sequence referred to as the sequence of SEQ ID NO: 67 and a light chain variable region comprising the amino acid sequence , referred to as the sequence SEQ ID NO: 70.

In some embodiments, the method includes administering to the subject a composition comprising a MASP-2 inhibitory antibody or antigen-binding fragment thereof that specifically recognizes at least a portion of an epitope on human MASP-2 recognized by an OMS646 control antibody comprising a heavy chain variable region, referred to as the sequence of SEQ ID NO: 67, and the variable region of the light chain, referred to as the sequence

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SEQ ID NO: 70.

In some embodiments, the method comprises administering to a subject suffering from HSCT-related TMA or at risk of developing HSCT-related TMA, including a subject suffering from persistent hematopoietic stem cell transplant-associated TMA that is resistant to conservative therapies, the compositions comprising a MASP-2 inhibitory antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising an amino acid sequence referred to as the sequence of SEQ ID NO: 67, and a variable region comprising an amino acid sequence referred to as the sequence of SEQ ID NO: 70, at a dose of 1 to 10 mg/kg (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/kg), at least once a week (for example, at least twice a week or at least three times a week) for at least 3 weeks, or for at least 4 weeks, or for at least 5 weeks, and or for at least 6 weeks, or for at least 7 weeks, or for at least 8 weeks.

Although the invention has been described and illustrated non-limiting embodiments, however, it is obvious that various changes can be made to the invention without deviating from the essence and scope of the invention.

Claims (10)

CLAIM 1. A method of treating a human suffering from persistent thrombotic microangiopathy (TMA) associated with hematopoietic stem cell transplantation (HSCT-TMA), comprising administering to the subject a composition containing a MASP-2 inhibitory monoclonal antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region, referred to as the sequence of SEQ ID NO: 67, and the variable region of the light chain, referred to as the sequence of SEQ ID NO: 70, at a dose of at least 4 mg/kg, when administered at least weekly for a period of at least three weeks, where said subject has a TMA that persists despite reduction or discontinuation of an immunosuppressive agent, or where said subject has a TMA that persists at least 30 days after transplantation, wherein said administration improves at least one or more of the following clinical parameters due to persistent TMA, St. associated with hematopoietic stem cell transplantation: (i) increased platelet count; (ii) an increase in haptoglobin; (iii) decreased lactate dehydrogenase (LDH); and/or (iv) a decrease in creatinine. 2. The method of claim 1, wherein the antibody or fragment thereof is selected from the group consisting of a recombinant antibody, an antibody having reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody. 3. The method of claim 1, wherein the MASP-2 inhibitory antibody does not inhibit the classical pathway. 4. The method of claim 1, wherein the MASP-2 inhibitory antibody inhibits C3b deposition in 90% human serum with an IC ₅₀ value of 30 nM or less. 5. The method of claim 1, wherein the MASP-2 inhibitory antibody is administered systemically to the subject. 6. The method of claim 1, wherein the subject has been or is currently being treated with a humanized anti-C5 antibody or antigen-binding fragment thereof. 7. The method of claim 1, wherein the platelet count is increased by at least two times the platelet count prior to said administration. 8. The method of claim 1, wherein the subject has a TMA that persists for at least two weeks after the immunosuppressive agent is reduced or discontinued. 9. The method of claim 1, wherein said subject has a TMA that persists for at least 30 days after transplantation. 10. The method of claim 1 wherein the immunosuppressive agent is a calcineurin inhibitor.