OA18820A - Methods for inhibiting fibrosis in a subject in need thereof. - Google Patents

Methods for inhibiting fibrosis in a subject in need thereof. Download PDF

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OA18820A
OA18820A OA1201800254 OA18820A OA 18820 A OA18820 A OA 18820A OA 1201800254 OA1201800254 OA 1201800254 OA 18820 A OA18820 A OA 18820A
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masp
disease
antibody
inhibitory
administration
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OA1201800254
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Gregory A. Demopulos
Thomas Dudler
Hans-Wilhelm Schwaeble
Nigel John BRUNSKILL
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Omeros Corporation
University Of Leicester
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Abstract

In one aspect, the invention provides methods for treating, inhibiting, alleviating or preventing fibrosis in a mammalian subject suffering, or at risk of developing a disease or disorder caused or exacerbated by fibrosis and/or inflammation. In one embodiment, the invention provides methods of treating a subject suffering from rénal fibrosis. In one embodiment, the invention provides methods of reducing proteinuria in a subject suffering from a renal disease or condition associated with proteinuria. The methods comprise the step of administering, to a subject in need thereof, an amount of a MASP-2 inhibitory agent effective to inhibit MASP-2-dependent complément activation.

Description

METHODS FOR INHIBITING FIBROSIS IN A SUBJECT IN NEED THEREOF
CROSS-REFERENCES TO RELATED APPLICATIONS
This application claims the benefit of Provisîonal Application No. 62/275,025, filed January 5, 2016, and Provisîonal Application No. 62/407,979, filed October 13, 2016, both of which are incorporated herein by reference in their entirety.
STATEMENT REGARDING SEQUENCE LISTING
The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the spécification. The name of the text file containing the sequence listing is MP_l_0250_PCT_Sequence_Listing_20l70l04_ST25. The text file is 136 KB; was created on January 4, 2017; and is being submitted via EFS-Web with the filing of the spécification.
BACKGROUND
The complément system provides an early acting mechanism to initiate, amplify and orchestrate the immune response to microbial infection and other acute insults (M.K. Liszewski and J.P. Atkinson, 1993, in Fundamenlal hnmunology, Third Edition, edited by W.E. Paul, Raven Press, Ltd., New York), in humans and other vertebrates. While complément activation provides a valuable first-line defense against potential pathogens, the activities of complément that promote a protective immune response can also represent a potential threat to the host (K.R. Kalli, et al., Springer Semin. Immunopathol. 15'A\7-43\, 1994; B.P. Morgan, Eur. J Clinical Investig. 24:219-228, 1994). For example, C3 and C5 proteolytic products recruit and activate neutrophils. While indispensable for host defense, activated neutrophils are îndiscriminate in their release of destructive enzymes and may cause organ damage. In addition, complément activation may cause the déposition of lytic complément components on nearby host cells as well as on microbial targets, resulting in host cell lysis.
The complément system has also been implicated în the pathogenesis of numerous acute and chronic disease States, including: myocardial infarction, stroke, ARDS, reperfusion injury, septic shock, capillary leakage following thermal burns, postcardiopulmonary bypass inflammation, transplant rejection, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, and Alzheimer’s disease. In almost ail of these conditions, complément is not the cause but is one of several factors involved in pathogenesis. Nevertheless, complément activation may be a major pathological mechanism and represents an effective point for clinical control in many of these disease States. The growing récognition of the importance of complement-mediated tissue injury in a variety of disease States underscores the need for effective complément inhibitory drugs. To date, Eculizumab (Solaris®), an antibody against C5, is the only complementtargeting drug that has been approved for human use. Yet, C5 is one of several effector molécules located “downstream” in the complément system, and blockade ofC5 does not inhibit activation of the complément system. Therefore, an inhibitor of the initiation steps of complément activation would hâve significant advantages over a “downstream” complément inhibitor.
Currently, it is widely accepted that the complément system can be activated through three distinct pathways: the classical pathway, the lectin pathway, and the alternative pathway. The classical pathway is usually triggered by a complex composed of host antibodies bound to a foreign particle (i.e., an antigen) and thus requires prior exposure to an antigen for the génération of a spécifie antibody response. Since activation of the classical pathway dépends on a prior adaptive immune response by the host, the classical pathway is part of the acquired immune system. In contrast, both the lectin and alternative pathways are independent of adaptive immunity and are part of the innate immune system.
The activation of the complément system results in the sequential activation of serine protease zymogens. The first step in activation of the classical pathway is the binding of a spécifie récognition molécule, Clq, to antigen-bound IgG and IgM molécules. Clqis associated with the Clr and Cls serine protease proenzymes as a complex called Cl. Upon binding ofClq to an immune complex, autoproteolytic cleavage of the Arg-lle site of Clr is followed by Clr-mediated cleavage and activation of Cls, which thereby acquires the ability to cleave C4 and C2. C4 is cleaved into two fragments, designated C4a and C4b, and, similarly, C2 is cleaved into C2a and C2b. C4b fragments are able to form covalent bonds with adjacent hydroxyl or amino groups and generate the C3 convertase (C4b2a) through noncovalent interaction with the C2a 5 fragment of activated C2. C3 convertase (C4b2a) activâtes C3 by proteolytic cleavage into C3a and C3b subcomponents leading to génération of the C5 convertase (C4b2a3b), which, by cleaving C5 leads to the formation of the membrane attack complex (C5b combined with C6, C7, C8 and C-9, also referred to as “MAC”) that can disrupt cellular membranes leading to cell lysis. The activated forms ofC3 and C4 (C3b and C4b) are 10 covalentiy deposîted on the foreign target surfaces, which are recognized by complément receptors on multiple phagocytes.
Independently, the first step in activation of the complément system through the lectîn pathway is also the binding of spécifie récognition molécules, which is followed by the activation of associated serine protease proenzymes. However, rather than the 15 binding of immune complexes by Clq, the récognition molécules în the lectin pathway comprise a group of carbohydrate-binding proteins (mannan-binding lectin (MBL), H-ficolin, M-ficolin, L-ficolîn and C-type lectin CL-ll), collectively refened to as lectins. See J. Lu et al., Biochim. Biophys. Acta 7 5 72:387-400, (2002); Holmskov et al., Annu. Rev. Immunol. 21:547-578 (2003); Teh étal., Immunology 101:225-232 (2000)). 20 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. first demonstrated that, like Clq, MBL could activate the complément system upon binding to yeast mannan-coated erythrocytes in a C4-dependent manner 25 (Ikeda étal., J. Biol. Chem. 262:7451-7454, (1987)). MBL, a member of the collectin protein family, is a calcium-dependent lectin that binds carbohydrates with 3- and 4-hydroxy groups oriented in the équatorial plane of the pyranose ring. Prominent ligands for MBL are thus D-mannose and N-acetyl-D-glucosamine, while carbohydrates not fitting this steric requirement hâve undetectable affinity for MBL (Weisetal., 30 Nature 360:127-134, (1992)). The interaction between MBL and monovalent sugars is extremely weak, with dissociation constants typically in the single-digit millimolar range.
MBL achieves tight, spécifie binding to glycan ligands by avidity, i.e., by interacting simultaneously with multiple monosaccharide residues located in close proximity to each other (Lee et al., Archiv. Biochem. Biophys. 299:129-136, (1992)). MBL recognîzes the carbohydrate patterns that commonly decorate microorganisms such as bacteria, yeast, parasites and certain viruses. In contrast, MBL does not recognize D-galactose and sialic acid, the penultimate and ultimate sugars that usually decorate mature complex glycoconjugates présent on mammalian plasma and cell surface glycoproteins. This binding specificîty is thought to promote récognition of “foreign” surfaces and help protect from ‘‘self-activation.” However, MBL does bind with high affinity to clusters of high-mannose precursor glycans on N-linked glycoproteins and glycolipîds sequestered in the endoplasmic réticulum and Golgi of mammalian cells (Maynard et al., J. Biol. Chem. 257-.31^,-3794, (1982)). Therefore, damaged cells are potential targets for lectin pathway activation via MBL binding.
The ficolins possess a different type of lectin domain than MBL, called the fibrinogen-like domain. Ficolins bind sugar residues in a Ca^-independent manner. In humans, three kinds of ficolins (L-ficolin, M-ficolin and H-ficolin) hâve been identified. The two sérum ficolins, L-ficolin and Η-ficolin, hâve in common a specificity for N-acetyl-D-glucosamine; however, H-ficolin also binds N-acetyl-D-galactosamine. The différence in sugar specificity of L-ficolin, H-ficolin, CL-11, and MBL means that the different lectins may be complementary and target different, though overlapping, glycoconjugates. This concept is supported by the recent report that. ofthe known lectins in the lectin pathway, only L-ficolin binds specifically to lipoteichoic acid, a cell wall glycoconjugate found on ail Gram-positive bacteria (Lynch et aL, J. Immunol. /72:1198-1202, (2004)). The collectins (i.e., MBL) and the ficolins bear no significant similarity in amino acid sequence. However, the two groups of proteins hâve similar domain organizations and, like Clq, assemble into oligomerîc structures, which maximize the possibility of multisite binding.
The sérum concentrations of MBL are highly variable in healthy populations and this is genetically controlled by polymorphisms/mutations in both the promoter and coding régions of the MBL gene. As an acute phase protein, the expression of MBL is further upregulated during inflammation. L-ficolin is présent in sérum at concentrations similar to those of MBL. Therefore, the L-ficolin branch of the lectin pathway is potentially comparable to the MBL arm in strength. MBL and ficolîns can also function as opsonins, which allow phagocytes to target MBL- and ficolin-decorated surfaces (see Jack et al., J Leukoc Biol., 77(3):328-36 (2004), Matsushita and Fujita, Immunobiology, 205(4-5):490-7 (2002), Aoyagi et al., J Immunol, 174(1):418-25(2005). This opsonization requires the interaction of these proteins with phagocyte receptors (Kuhlman étal., J. Exp. Med. /69:1733, (1989); Matsushita et al., J. Biol. Chem. 277:2448-54, (1996)), the identity of which has not been established.
Human MBL forms a spécifie and hîgh-affinity interaction through its collagen-like domain with unique C1 r/CIs-like serine proteases, termed MBL-associated serine proteases (MASPs). To date, three MASPs hâve been described. First, a single enzyme MASP was identified and characterized as the enzyme responsible for the initiation of the complément cascade (i.e., cleaving C2 and C4) (Matsushita et al., J Exp Med 176(6):1497-1502 (1992); Ji et al., J. Immunol. 750:571-578, (1993)). It was subsequently determined that the MASP activity was, in fact, a mixture of two proteases: MASP-1 and MASP-2 (Thiel étal., Nature 356:506-510, (1997)). However, it was demonstrated that the MBL-MASP-2 complex alone is sufficient for complément activation (Vorup-Jensen étal., J. Immunol. 765:2093-2100, (2000)). Furthermore, only MASP-2 cleaved C2 and C4 at high rates (Ambrus étal., J. Immunol. 770:1374-1382, (2003)). Therefore, MASP-2 is the protease responsible for activating C4 and C2 to generate the C3 convertase, C4b2a. This is a significant différence from the C1 complex of the classîcal pathway, where the coordinated action of two spécifie serine proteases (Clr and Cl s) leads to the activation of the complément system. In addition, a third novel protease, MASP-3, has been isolated (Dahl, M.R., et aL, Immunity 75:127-35, 2001). MASP-1 and MASP-3 are alternatively spliced products of the same gene.
MASPs share identical domain organîzatîons with those of Clr and Cls, the enzymatic components of the Cl complex (Sim et al., Biochem. Soc. Trans. 25:545, (2000)). These domains include an N-terminal Clr/Cls/sea urchin VEGF/bone morphogenic protein (CUB) domain, an epidermal growth factor-like domain, a second CUB domain, a tandem of complément control protein domains, and a serine protease domain. As in the Cl proteases, activation of MASP-2 occurs through cleavage of an
Arg-Ile bond adjacent to the serine protease domain, which splits the enzyme into disulfide-linked A and B chains, the latter consisting of the serine protease domain.
MBL can also associate with an alternatively sliced form of MASP-2, known as MBL-associated protein of 19 kDa (MApl9) or small MBL-associated protein (sMAP), which lacks the catalytîc activity of MASP-2. (Stover, J. Immunol. /62:3481-90, (1999); Takahashi et al., Int. Immunol. 77:859-863, (1999)). MApl9 comprises the first two domains of MASP-2, followed by an extra sequence of four unique amino acids. The function of MApl9 is unclear (Degn et al., J Immunol. Methods, 2011). The MASP-1 and MASP-2 genes are located on human chromosomes 3 and 1, respectively (Schwaeble et al., Immunobiology 205:455-466, (2002)),
Several lines of evidence suggest that there are different MBL-MASP complexes and a large fraction of the MASPs in sérum is not complexed with MBL (Thiel, et al., J. Immunol. 165:%ΊΖ-ΜΊ, (2000)). Both H- and L-ficolin bind to ail MASPs and activate the lectin complément pathway, as does MBL (Dahl étal., Immunity 75:127-35, (2001); Matsushita et aL, J. Immunol. 765:3502-3506, (2002)). Both the lectin and classical pathways form a common C3 convertase (C4b2a) and the two pathways converge at this step.
The lectin pathway is widely thought to hâve a major rôle in host defense against infection in the naïve host. Strong evidence for the involvement of MBL in host defense cornes from analysis of patients with decreased sérum levels of functional MBL (Kilpatrick, Biochim. Biophys. Acta 75 72:401-413, (2002)). Such patients display susceptibility to récurrent bacterial and fiingal infections. These symptoms are usually évident early in life, during an apparent window of vulnerability as matemally derived antibody titer wanes, but before a full répertoire of antibody responses develops. This syndrome often results from mutations at several sites in the collagenous portion of MBL, which interfère with proper formation of MBL oligomers. However, since MBL can function as an opsonin independent of complément, it is not known to what extent the increased susceptibility to infection is due to impaired complément activation.
Ail three pathways (i.e., the classical, lectin and alternative) hâve been thought to converge at C5, which is cleaved to form products with multiple proinflammatory effects. The converged pathway has been referred to as the terminal complément pathway, C5a is the most potent anaphylatoxin, înducing alterations in smooth muscle and vascular tone, as well as vascular permeability. It is also a powerful chemotaxin and activator of both neutrophils and monocytes. C5a-medîated ceilular activation can significantly amplify inflammatory responses by inducing the release of multiple additional inflammatory medîators, including cytokines, hydrolytic enzymes, arachidonic acid métabolites, and reactive oxygen species. C5 cleavage leads to the formation of C5b-9, also known as the membrane attack complex (MAC). There is now strong evidence that sublytic MAC déposition may play an important rôle in inflammation in addition to its rôle as a lytic pore-forming complex.
In addition to its essentîal rôle in immune defense, the complément system contributes to tissue damage in many clinical conditions. Although there is extensive evidence implicating both the classical and alternative complément pathways in the pathogenesis of non-infectious human diseases, the rôle of the lectin pathway is just beginning to be evaluated. Recent studies provide evidence that activation of the lectin pathway can be responsible for complément activation and related inflammation in ischemia/reperfusion injury. Collard et al. (2000) reported that cultured endothélial cells subjected to oxidative stress bind MBL and show déposition of C3 upon exposure to human sérum (Collard et al., Am. J. Pathol. 156:1549-1556, (2000)). In addition, treatment of human sera with blocking anti-MBL monoclonal antibodies inhibited MBL binding and complément activation. These fmdings were extended to a rat model of myocardial ischemia-reperfusion in which rats treated with a blocking antibody directed against rat MBL showed significantly less myocardial damage upon occlusion of a coronary artery than rats treated with a control antibody (Jordan et al., Circulation /07:1413-1418, (2001)). The molecular mechanism of MBL binding to the vascular endothélium after oxidative stress is unclear; a recent study suggests that activation of the lectin pathway after oxidative stress may be mediated by MBL binding to vascular endothélial cytokeratins, and not to glycoconjugates (Collard étal., Am. J. Pathol. /59:1045-1054, (2001)). Other studies hâve implîcated the classical and alternative pathways in the pathogenesis of ischemia/reperfusion injury and the rôle of the lectin pathway in this disease remains controversial (Riedermann, N.C., et al., Am. J. Pathol. 162:363-361, 2003).
Fibrosis is the formation of excessive connective tissue in an organ or tissue, commonly in response to damage or injury. A hallmark of fibrosis is the production of excessive extracellular matrix following local trauma. The normal physiological response to injury results in the déposition of connective tissue, but this inîtially bénéficiai reparative process may persist and become pathological, altering the architecture and function of the tissue. At the cellular level, épithélial cells and fibroblasts proliferate and differentiate into myofibrobiasts, resulting in matrix contraction, increased rigidity, microvascular compression, and hypoxia. An influx of inflammatory cells, including macrophages and lymphocytes, results in cytokine release and amplifies the déposition of collagen, fibronectin and other molecular markers of fibrosis. Conventional therapeutic approaches hâve largely been targeted towards the inflammatory process of fibrosis, using corticosteroids and immunosuppressive drugs. Unfortunately, these antiinflammatory agents hâve had little to no clinical effect. Currently there are no effective treatments or therapeutics for fibrosis, but both animal studies and anecdotal human reports suggest that fibrotic tissue damage may be reversed (Tampe and Zeîsberg, Nat Rev Nephrol,No} 10:226-237,2014).
The kidney has a limited capacity to recover from injury. Various rénal pathologies resuit in local inflammation that causes scarring and fibrosis of rénal tissue. The perpétuation of inflammatory stimuli drives tubulointerstitial inflammation and fibrosis and progressive rénal functional impairment in chronic kidney disease. Its progression to end-stage rénal failure is associated with significant morbidîty and mortality. Since tubulointerstitial fibrosis is the common end point of multiple rénal pathologies, it represents a key target for thérapies aimed at preventing rénal failure. Risk factors (e.g., proteinuria) independent of the primary rénal disease contribute to the development of rénal fibrosis and loss of rénal excretory function by driving local inflammation, which in tum enhances disease progression.
In view of the rôle of fibrosis in many diseases and disorders, such as, for example, tubulointerstitial fibrosis leading to chronic kidney disease, there is a pressing need to develop therapeutically effective agents for treating diseases and conditions caused or exacerbated by fibrosis. In further view of the paucity of new and existing treatments targeting inflammatory pro-fibrotic pathways in rénal disease, there is a need to develop therapeutically effective agents to treat, inhibit, prevent and/or reverse rénal fîbrosis and thereby prevent progressive chronic kidney disease.
SUMMARY
This summary is provided to introduce a sélection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.
In one aspect, the invention provides a method for treating, inhibiting, alleviatïng or preventing fîbrosis in a mammalian subject suffering, or at risk of developing a disease or disorder caused or exacerbated by fîbrosis and/or inflammation, comprising administering to the subject an amount of a MASP-2 inhibitory agent effective to inhibit fîbrosis. In one embodiment, the MASP-2 inhibitory agent is a MASP-2 antibody or fragment thereof. In one embodiment, the MASP-2 inhibitory agent is a MASP-2 monoclonal antibody, or fragment thereof that specifically binds to a portion of SEQ ID NO:6. In one embodiment, the MASP-2 inhibitory agent selectively inhibits lectin pathway complément activation without substantially inhibiting Clq-dependent complément activation. In one embodiment, the subject is suffering from a disease or disorder caused by or exacerbated by at least one of (i) fîbrosis and/or inflammation associated with an ischemia reperfusion injury, (ii) rénal fîbrosis and/or rénal inflammation (e.g., tubulointerstitial fîbrosis, chronic kidney disease, chronic rénal failure, glomerular disease (e.g., focal segmentai glomerulosclerosis), an immune complex disorder (e.g., IgA nephropathy, membraneous nephropathy), lupus nephritis, nephrotic syndrome, diabetîc nephropathy, tubulointerstitial damage and glomerulonepthritis (e.g., C3 glomerulopathy), (ni) pulmonary fîbrosis and/or inflammation (e.g., chronic obstructive pulmonary disease, cystic fîbrosis, pulmonary fîbrosis associated with scleroderma, bronchiectasis and pulmonary hypertension), (iv) hepatic fîbrosis and/or inflammation (e.g., cirrhosîs, nonalcoholic fatty lîver disease (steatohepatitis)), liver fîbrosis secondary to alcohol abuse, lîver fîbrosis secondary to acute or chronic hepatitis, biliary disease and toxic liver injury (e.g., hepatotoxicity due to drug-induced liver damage induced by acetaminophen or other drug), (v) cardiac fîbrosis
ΙΟ and/or inflammation (e.g., cardiac fibrosis, myocardîal infarction, valvular fibrosis, atrial fibrosis, endomyocardial fibrosis arrhythmogenic right ventricular cardiomyopathy (ARVC), (vi) vascular fibrosis (e.g., vascular disease, an atherosclerotic vascular disease, vascular stenosis, restenosis, vasculitis, phlebitis, deep vein thrombosis and abdominal aortic aneurysm), (vii) fibrosis of the skin (e.g., excessive wound healing, scleroderma, systemic sclerosis, keloids, connective tissue diseases, scarrîng, and hypertrophie scars), (viii) fibrosis of the joints (e.g., arthrofibrosis), (ix) fibrosis of the central nervous system (e.g., stroke, traumatic brain injury and spinal cord injury), (x) fibrosis of the digestive system (e.g., Crohn’s disease, pancreatic fibrosis and ulcerative colitis), (xi) ocular fibrosis (e.g., anterior subcapsular cataract, posterior capsule opacification, macular degeneration, and retinal and vitreal retinopathy), (xiî) fibrosis of musculoskeletal softtîssue structures (e.g., adhesive capsulitis, Dupuytren’s contracture and myelofibrosis), (xiii) fibrosis of the reproductive organs (e.g., endometriosis and Peyronie’s disease), (xiv) a chronic infectious disease that causes fibrosis and/or inflammation (e.g., alpha virus, Hepatitis A, Hepatitis B, Hepatitis C, tuberculosis, HIV and influenza), (xv) an autoimmune disease that causes fibrosis and/or inflammation (e.g., scleroderma and systemic lupus erythematosus (SLE), (xvi) scarring associated with trauma (e.g., wherein the scarring associated with trauma is selected from the group consisting of surgical complications (e.g., surgical adhesions wherein scar tissue can form between 20 internai organs causing contracture, pain and can cause înfertility), chemotherapeutic drug-induced fibrosis, radial ion-induced fibrosis and scarring associated with burns), or (xvii) organ transplant, breast fibrosis, muscle fibrosis, rétropéritonéal fibrosis, thyroid fibrosis, lymph node fibrosis, bladder fibrosis and pleural fibrosis.
In another aspect, the present invention provides a method for treating, inhibiting, 25 alleviating or preventing rénal fibrosis in a mammalian subject suffering, or at risk of developing a disease or disorder caused or exacerbated by rénal fibrosis and/or inflammation, comprising administering to the subject an amount of a MASP-2 inhibitory agent effective to inhibit rénal fibrosis. In one embodiment, the MASP-2 inhibitory agent îs a MASP-2 antibody or fragment thereof. In one embodiment, the MASP-2 inhibitory 30 agent is a MASP-2 monoclonal antibody, or fragment thereof that specifically binds to a portion of SEQ ID NO:6. In one embodiment. the MASP-2 antibody or fragment thereof
H specifically binds to a polypeptide comprising SEQ ID NO:6 with an affinity of at least 10 times greater than it binds to a different antigen in the complément system. 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 MASP-2 inhibitory agent selectively înhibîts lectin pathway complément activation without substantially inhibiting Clq-dependent complément activation. In one embodiment, the MASP-2 inhibitory agent is administered subcutaneously, intraperitoneally, intramuscularly, intra-arterially, intravenously, or as an inhalant. In one embodiment, the MASP-2 inhibitory agent is administered in an amount effective to inhibit tubulointerstitial fibrosis. In one embodiment, the MASP-2 inhibitory agent is administered in an amount effective to reduce, delay or eliminate the need for dialysis in the subject. In one embodiment, the subject is suffering from a rénal disease or disorder selected from the group consisting of chronic kidney disease, chronic rénal failure, glomerular disease (e.g., focal segmentai glomerulosclerosis), an immune complex disorder (e.g., IgA nephropathy, membraneous nephropathy), lupus nephritis, nephrotic syndrome, diabetic nephropathy, tubulointerstitial damage and glomerulonepthritis (e.g., C3 glomerulopathy). In one embodiment, the subject is suffering from proteinuria and the MASP-2 inhibitory agent is administered in an amount effective to reduce proteinuria in the subject. In one embodiment, the MASP-2 inhibitory agent is administered in an amount and for a time effective to achieve at least a 20 percent réduction (e.g., at least a 30 percent réduction, or at least a 40 percent réduction, or at least a 50 percent réduction) in 24-hour urine protein excrétion as compared to baseline 24-hour urine protein excrétion in the subject prior to treatment. In one embodiment, the subject is suffering from a rénal disease or disorder associated with proteinuria selected from the group consisting of nephrotic syndrome, pre-eclampsia, eclampsia, toxic lésions of kidneys, amyloidosis, collagen vascular diseases (e.g., systemic lupus erythematosus), déhydration, glomerular diseases (e.g. membranous glomerulonephritis, focal segmentai glomerulonephritis, C3 glomerulopathy, minimal change disease, lipoid nephrosis), strenuous exercise, stress, benign orthostatis (postural) proteinuria, focal segmentai glomerulosclerosis, IgA nephropathy (i.e., Berger’s disease), IgM nephropathy, membranoproli ferai ive glomerulonephrîtis, membranous nephropathy, minimal change disease, sarcoidosis, Alport’s syndrome, diabètes mellitus (diabetic nephropathy), druginduced toxicity (e.g., NSAIDS, nicotine, penicillamine, lithium carbonate, gold and other heavy metals, ACE inhibitors, antibiotics (e.g., adriamycin) or opiates (e.g. heroin) or other nephrotoxins); Fabry’s disease, infections (e.g., HIV, syphilis, hepatitis A, B or C, poststreptococcal infection, urinary schistosomiasis); aminoaciduria, Fanconi syndrome, hypertensive nephrosclerosis, interstitial nephritis, sickle cell disease, hemoglobinuria, multiple myeloma, myoglobinurta, organ rejection (e.g., kîdney transplant rejection), ebola hémorrhagie fever, Nail pateila syndrome, familial mediterranean fever, HELLP syndrome, systemic lupus erythematosus, Wegener’s granulomatosis, Rheumatoid arthritis, Glycogen storage disease type 1, Goodpasture’s syndrome, Henoch-Schônlein purpura, urinary tract infection which has spread to the kidneys, Sjôgren’s syndrome and post-infections glomerulonepthritis. In one embodiment, the subject is suffering from IgA nephropathy. In one embodiment, the subject is suffering from membranous nephropathy.
In another aspect, the present invention provides a method of preventing or reducing rénal damage in a subject suffering from a disease or condition associated with proteinuria comprising administering an amount of a MASP-2 inhibitory agent effective to reduce or prevent proteinurea in the subject. In one embodiment, the MASP-2 inhibitory agent is a MASP-2 antibody or fragment thereof. In one embodiment, the MASP-2 inhibitory agent is a MASP-2 monoclonal antibody or fragment thereof that specifically binds to a portion of SEQ ID NO:6. In one embodiment, the MASP-2 inhibitory agent selectîvely inhibits lectin pathway complément activation without substantially înhibiting C Iq-dependent complément activation. In one embodiment, the disease or condition associated with proteinuria is selected from the group consisting of nephrotic syndrome, pre-eclampsia, eclampsia, toxic lésions of kidneys, amyloidosis, collagen vascular diseases (e.g., systemic lupus erythematosus), déhydration, glomerular diseases (e.g. membranous glomerulonephrîtis, focal segmentai glomerulonephrîtis, C3 glomerulopathy, minimal change disease, lipoid nephrosis), strenuous exercise, stress, benign orthostatis (postural) proteinuria, focal segmentai glomerulosclerosis, IgA nephropathy (i.e., Berger’s disease), IgM nephropathy, membranoproliferative
I3 glomerulonephritis, membranous nephropathy, minimal change disease, sarcoidosis, Alport’s syndrome, diabètes mellitus (diabetic nephropathy), drug-induced toxicity (e.g., NSAIDS, nicotine, penicillamine, lithium carbonate, gold and other heavy metals, ACE inhibitors, antibiotics (e.g., adriamycin) or opiates (e.g. heroin)); Fabry’s disease, infections (e.g., HIV, syphilis, hepatitis A, B or C, poststreptococcal infection, urinary schistosomiasis); amtnoaciduria, Fanconi syndrome, hypertensive nephrosclerosis, interstitial nephritis, sickle cell disease, hemoglobinuria, multiple myeloma, myoglobînuria, organ rejection (e.g., kidney transplant rejection), ebola hémorrhagie fever, Nail patella syndrome, familial mediterranean fever, HELLP syndrome, systemîc lupus erythematosus, Wegener’s granulomatosis, Rheumatoid arthritis, Glycogen storage disease type l, Goodpasture’s syndrome, Henoch-Schonlein purpura, urinary tract infection which has spread to the kidneys, Sjogren’s syndrome and post-infections glomerulonepthritis. In one embodiment, the MASP-2 inhibîtory agent is administered in an amount and for a time effective to achieve at least a 20 percent réduction (e.g., at least a 30 percent réduction, or at least a 40 percent réduction, or at least a 50 percent réduction) in 24-hour urine protein excrétion as compared to baseline 24-hour urine protein excrétion in the subject prior to treatment.
In another aspect, the present invention provides a method of inhibiting the progression of chronic kidney disease, comprising administering an amount of a MASP-2 inhibîtory agent effective to reduce or prevent rénal fibrosis, e.g., tubulointerstitial fibrosis, in a subject in need thereof. In one embodiment, the MASP-2 inhibîtory agent is a MASP-2 antibody or fragment thereof. In one embodiment, the MASP-2 inhibîtory agent is a MASP-2 monoclonal antibody, or fragment thereof that specifically binds to a portion of SEQ ID NO:6. In one embodiment, the MASP-2 inhibîtory agent selectively inhibits lectin pathway complément activation without substantially inhibiting Clq-dependent complément activation. In one embodiment, the subject in need thereof exhibits proteinuria prior to administration ofthe MASP-2 inhibîtory agent and administration of the MASP-2 inhibîtory agent decreases proteinuria in the subject. In one embodiment, the MASP-2 inhibîtory agent is administered in an amount and for a time effective to achieve at least a 20 percent réduction (e.g., at least a 30 percent réduction, or at least a 40 percent réduction, or at least a 50 percent réduction) in 24-hour urine protein excrétion as compared to baseline 24-hour urine protein excrétion in the subject prior to treatment. In one embodiment, the MASP-2 inhibitory agent is administered in an amount effective to reduce, delay or eliminate the need for dialysis in the subject.
In another aspect, the invention provides a method of protecting a kidney from rénal injury in a subject that has undergone, is undergoing, or will undergo treatment with one or more nephrotoxic agents, comprising administering an amount of a MASP-2 inhibitory agent effective to prevent or ameliorate druginduced nephropathy. In one embodiment, the MASP-2 inhibitory agent is a MASP-2 antibody or fragment thereof. In one embodiment, the MASP-2 inhibitory agent is a MASP-2 monoclonal antibody or fragment thereof that specifically binds to a portion of SEQ ID NO;6. In one embodiment, the MASP-2 inhibitory agent selectively inhibits lectin pathway complément activation without substantially inhibiting Clqdependent complément activation.
In another aspect, the invention provides a method of treating a human subject suffering from Immunoglobulin A Nephropathy (IgAN) comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen-binding fragment thereof, effective to inhibit MASP2-dependent complément activation. In one embodiment, the subject is suffering from steroid-dependent IgAN. In one embodiment, the MASP-2 inhibitory antibody is a monoclonal antibody, or fragment thereof that specifically binds to 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 MASP-2 inhibitory antibody does not substantially inhibit the classical pathway. In one embodiment, the MASP-2 inhibitory antibody inhibits C3b déposition in 90% human sérum with an IC50 of 30 nM or less. In one embodiment, the method further comprises identifying a human subject having steroiddependent IgAN prior to the step of administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen-binding fragment thereof, effective to improve rénal fonction. In one embodiment, the MASP-2 inhibitory antibody or antigen-binding fragment thereof is administered in an amount effective to improve rénal fonction. In one embodiment, the MASP-2 inhibitory antibody or antigen-binding fragment thereof is administered in an amount effective and for a time sufficient to achieve at least a 20 percent réduction in 24-hour urine protein excrétion as compared to baseline 24-hour urine protein excrétion in the subject prior to treatment In one embodiment, the composition is administered in an amount sufficient to improve rénal fonction and decrease the corticosteroid dosage in said subject. In one embodiment, the MASP-2 inhibitory antibody or antigen-binding fragment thereof comprises a heavy chain variable région comprising CDR-H1, CDR-H2 and CDR-H3 of the amino acid sequence set forth as SEQ ID N0:67 and a light chain variable région comprising CDR-L1, CDR-L2 and CDR-L3 of the amino acid sequence set forth as SEQ ID NO:70.
In another aspect, the invention provides a method of treating a human subject suffering from membranous nephropathy (MN) 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-dépendent complément activation. In one embodiment, the subject is suffering from steroiddependent MN. In one embodiment, the MASP-2 inhibitory antibody is a monoclonal antibody, or fragment thereof that specifically binds to human MASP-2. In one embodiment, the MASP-2 inhibitory antibody or antigen-binding fragment thereof is administered in an amount effective to improve rénal fonction. In one embodiment, the MASP-2 inhibitory antibody or antigen-binding fragment thereof is administered in an amount effective and for a time sufficient to achieve at least a 20 percent réduction in 24-hour urine protein excrétion as compared to baseline 24hour urine protein excrétion in the subject prior to treatment. In one embodiment, the composition is administered in an amount sufficient to improve rénal fonction and decrease the corticosteroid dosage in said subject. In one embodiment, the MASP-2 inhibitory antibody or antigen-binding fragment thereof comprises a heavy chain variable région comprising CDR-H1, CDR-H2 and CDR-H3 of the amino acid
I6 sequence set forth as SEQ ID N0:67 and a light chain variable région comprising CDR-L1, CDR-L2 and CDR-L3 of the amino acid sequence set forth as SEQ ID NO:70.
DESCRIPTION OF THE DRAWINGS
The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:
FIGURE l is a diagram illustrating the genomic structure of human MASP-2;
FIGURE 2A is a schematic diagram illustrating the domain structure of human MASP-2 protein;
FIGURE 2B is a schematic diagram illustrating the domain structure of human MApl9 protein;
FIGURE 3 îs a diagram illustrating the murine MASP-2 knockout strategy;
FIGURE 4 is a diagram illustrating the human MASP-2 minigene construct;
FIGURE 5A présents results demonstrating that MASP-2-defîciency leads to the loss of lectin-pathway-mediated C4 activation as measured by iack of C4b déposition on mannan, as described in Example 2;
FIGURE 5B présents results demonstrating that MASP-2-defïciency leads to the loss of lectin-pathway-mediated C4 activation as measured by lack of C4b déposition on zymosan, as described in Example 2;
FIGURE 5C présents results demonstrating the relative C4 activation levels of sérum samples obtained from MASP-2+/-; MASP-2-/- and wild-type strains as measure by C4b déposition on mannan and on zymosan, as described in Example 2;
FIGURE 6 présents results demonstrating that the addition of murine recombinant MASP-2 to MASP-2-/- sérum samples recovers lectin-pathway-mediated C4 activation in a protein concentration dépendent manner, as measured by C4b déposition on mannan, as described in Example 2;
FIGURE 7 présents results demonstrating that the classical pathway is functional in the MASP-2-/- strain, as described in Example 8;
FIGURE 8A présents results demonstrating that anti-MASP-2 Fab2 antibody #l I inhibits C3 convertase formation, as described in Example 10;
FIGURE 8B présents results demonstrating that anti-MASP-2 Fab2 antibody #l l binds to native rat MASP-2, as described in Example I0;
FIGURE 8C présents results demonstrating that anti-MASP-2 Fab2 antibody #4l inhibits C4 cleavage, as described in Example 10;
FIGURE 9 présents results demonstrating that ail of the anti-MASP-2 Fab2 antibodies tested that inhibited C3 convertase formation also were found to inhibit C4 cleavage, as described in Example 10;
FIGURE 10 is a diagram îllustrating the recombinant polypeptides derived from rat MASP-2 that were used for epîtope mapping of the MASP-2 blocking Fab2 antibodies, as described in Example H;
FIGURE 11 présents results demonstrating the binding of anti-MASP-2 Fab2 #40 and #60 to rat MASP-2 polypeptides, as described in Example 11 ;
FIGURE 12A graphically illustrâtes the level of MAC déposition in the presence or absence of human MASP-2 monoclonal antibody (OMS646) under lectin pathwayspecifîc assay conditions, demonstrating that OMS646 inhibits lectin-mediated MAC déposition with an IC50 value of approximately 1 nM, as described ïn Example 12;
FIGURE 12B graphically illustrâtes the level of MAC déposition in the presence or absence of human MASP-2 monoclonal antibody (OMS646) under classical pathwayspecifîc assay conditions, demonstrating that OMS646 does not inhibit classical pathwaymedîated MAC déposition, as described in Example 12;
FIGURE 12C graphically illustrâtes the level of MAC déposition in the presence or absence of human MASP-2 monoclonal antibody (OMS646) under alternative pathway-specific assay conditions, demonstrating that OMS646 does not inhibit alternative pathway-mediated MAC déposition, as described in Example 12;
FIGURE 13 graphically illustrâtes the pharmacokinetic (PK) profile of human MASP-2 monoclonal antibody (OMS646) in mice, showing the OMS646 concentration (mean of n=3 animals/groups) as a function of time after administration at the ïndicated dose, as described in Example 12;
FIGURE 14A graphically illustrâtes the pharmacodynamie (PD) response of human MASP-2 monoclonal antibody (OMS646), measured as a drop in systemic lectin pathway activity, in mice following intravenous administration, as described in Example 12;
FIGURE 14B graphically illustrâtes the pharmacodynamie (PD) response of human MASP-2 monoclonal antibody (OMS646), measured as a drop in systemic lectin pathway activity, in mice following subeutaneous administration, as described in Example 12;
FIGURE 15 graphically illustrâtes the results of computer-based image analysis of kidney tissue sections stained with Sirius red, wherein the tissue sections were obtained from wild-type and MASP-2-/- mice following 7 days of unilatéral ureteric obstruction (UUO) and sham-operated wild-type and MASP-2-/- mice, as described in Example 14;
FIGURE 16 graphically illustrâtes the results of computer-based image analysis of kidney tissue sections stained with the F4/80 macrophage-specific antibody, wherein the tissue sections were obtained from wild-type and MASP-2-/- mice following 7 days of unilatéral ureteric obstruction (UUO) and sham-operated wild-type and MASP-2-/- mice, as described in Example 14.
FIGURE 17 graphically illustrâtes the relative mRNA expression levels of collagen-4, as measured by quantitative PCR (qPCR), in kidney tissue sections obtained from wild-type and MASP-2-/- mice following 7 days of unilatéral ureteric obstruction (UUO) and sham-operated wild-type and MASP-2-/- mice, as described in Example 14.
FIGURE 18 graphically illustrâtes the relative mRNA expression levels of Transforming Growth Factor Beta-1 (TGFp-l), as measured by qPCR, in kidney tissue sections obtained from wild-type and MASP-2-/- mice following 7 days of unilatéral ureteric obstruction (UUO) and sham-operated wild-type and MASP-2-/- mice, as described in Example 14.
FIGURE 19 graphically illustrâtes the relative mRNA expression levels of Interleukin-6 (IL-6), as measured by qPCR, in kidney tissue sections obtained from wildtype and MASP-2-/- mice following 7 days of unilatéral ureteric obstruction (UUO) and sham-operated wild-type and MASP-2-/- mice, as described in Example 14.
FIGURE 20 graphically illustrâtes the relative mRNA expression levels of Interferon-γ, as measured by qPCR, in kidney tissue sections obtained from wild-type and
MASP-2-/- mice following 7 days of unilatéral ureteric obstruction (UUO) and shamoperated wild-type and MASP-2-/- mice, as described in Example 14.
FIGURE 2I graphically illustrâtes the results of computer-based image analysis of kidney tissue sections stained with Siruis red, wherein the tissue sections were obtained following 7 days of unilatéral ureteric obstruction (UUO) from wild-type mice treated with a MASP-2 înhibitory antibody and an isotype control antibody, as described in Example 15.
FIGURE 22 graphically illustrâtes the hydroxyl proline content from kidneys harvested 7 days after unilatéral ureteric obstruction (UUO) obtained from wild-type mice treated with MASP-2 înhibitory antibody as compared with the level of hydroxyl proline in tissue from obstructed kidneys obtained from wild-type mice treated with an IgG4 isotype control, as described in Example 15.
FIGURE 23 graphically illustrâtes the total amount of sérum proteins (mg/ml) measured on day 15 of the protein overload study in wild-type control mice (n=2) that received saline only, wild-type mice that received BSA (n=6) and MASP-2-/- mice that received BSA (n=6), as described in Example 16.
FIGURE 24 graphically illustrâtes the total amount of excreted protein (mg) in urine collected over a 24 hour period on day 15 of the protein overload study from wildtype control mice (n=2) that received saline only, wild-type that received BSA (n=6) and MASP-2-/- mice that received BSA (n=6), as described in Example 16.
FIGURE 25 shows représentative hematoxylin and eosin (H&E) stained rénal tissue sections from the following groups of mice on day 15 of the protein overload study as follows: (panel A) wild-type control mice; (panel B) MASP-2-/- control mice, (panel C) wild-type mice treated with BSA; and (panel D) MASP-2-/- mice treated with bovine sérum albumin (BSA), as described in Example 16.
FIGURE 26 graphically illustrâtes the results of computer-based image analysis of kidney tissue sections stained with macrophage-specific antibody F4/80, showing the macrophage mean stained area (%), wherein the tissue sections were obtained on day 15 of the protein overload study from wild-type control mice (n=2), wild-type mice treated with BSA (n=6), and MASP-2-/- mice treated with BSA (n=5), as described in Example
16.
FIGURE 27A graphîcaliy illustrâtes the analysis for the presence of a macrophage-proteinuria corrélation in each wîld-type mouse (n=6) treated with BSA by plotting the total excreted proteins measured în urine from a 24-hour sample versus the macrophage infiltration (mean stained area %), as described in Example 16.
FIGURE 27B graphîcaliy illustrâtes the analysis for the presence of a macrophage-proteinuria corrélation în each MASP-2-/- mouse (n=5) treated with BSA by plotting the total excreted proteins in urine în a 24-hour sample versus the macrophage infiltration (mean stained area %), as described in Example 16.
FIGURE 28 graphîcaliy illustrâtes the results of computer-based image analysis of stained tissue sections with anti-TGFP antibody (measured as % TGFp antibodystained area) in wild-type mice treated with BSA (n=4) and MASP-2-/- mice treated with BSA (n=5), as described in Example 16.
FIGURE 29 graphîcaliy illustrâtes the results of computer-based image analysis of stained tissue sections with anti-TNFa antibody (measured as % TNFa antibody-staîned area) in wild-type mice treated with BSA (n=4) and MASP-2-/- mice treated with BSA (n=5), as described în Example 16.
FIGURE 30 graphîcaliy illustrâtes the results of computer-based image analysis of stained tissue sections with anti-IL-6 antibody (measured as % IL-6 antibody-stained area) in wild-type control mice, MASP-2-/- control mice, wild-type mice treated with BSA (n=7) and MASP-2-/- mice treated with BSA (n=7), as described in Example 16.
FIGURE 31 graphîcaliy illustrâtes the frequency of TUNEL apoptotic cells counted în serially selected 20 high power fields (HPFs) from tissue sections from the rénal cortex in wild-type control mice (n=l), MASP-2-/- control mice (n=l), wild-type mice treated with BSA (n=6) and MASP-2-/- mice treated with BSA (n=7), as described in Example I6.
FIGURE 32 shows représentative H&E stained tissue sections from the following groups of mice at day 15 after treatment with BSA: (panel A) wild-type control mice treated with saline, (panel B) isotype antibody treated control mice and (panel C) wildtype mice treated with a MASP-2 inhibitory antibody, as described in Example 17.
FIGURE 33 graphîcaliy illustrâtes the frequency of TUNEL apoptotic cells counted in serially selected 20 high power fields (HPFs) from tissue sections from the rénal cortex in wild-type mice treated with saline control and BSA (n=8), wild-type mice treated with the isotype control antibody and BSA (n=8) and wild-type mice treated with a MASP-2 inhibitory antibody and BSA (n=7), as described in Example 17.
FIGURE 34 graphically illustrâtes the results of computer-based image analysis of stained tissue sections with anti-TGFfl antibody (measured as % TGFp antibody-stained area) in wild-type mice treated with BSA and saline (n=8), wild-type mice treated with BSA and isotype control antibody (n=7) and wild-type mice treated with BSA and MASP-2 inhibitory antibody (n=8), as described in Example 17.
FIGURE 35 graphically illustrâtes the results of computer-based image analysis of stained tissue sections with anti-TNFa antibody (measured as % TNFa antibody-stained area) in wild-type mice treated with BSA and saline (n=8), BSA and isotype control antibody (n=7) and wild-type mice treated with BSA and MASP-2 inhibitory antibody (n=8), as described in Example 17.
FIGURE 36 graphically illustrâtes the results of computer-based image analysis of stained tissue sections with anti-lL-6 antibody (measured as % IL-6 antibody-stained area) in in wild-type mice treated with BSA and saline (n=8), BSA and isotype control antibody (n=7) and wild-type mice treated with BSA and MASP-2 inhibitory antibody (n=8), as described in Example 17.
FIGURE 37 shows représentative H&E stained tissue sections from the following groups of mice at day 14 after treatment with Adriamycin or saline only (control): (panels A-I, A-2, A-3) wild-type control mice treated with only saline; (panels B-l, B-2, B-3) wild-type mice treated with Adriamycin; and (panels C-l, C-2, C-3) MASP-2-/- mice treated with Adriamycin, as described in Example 18;
FIGURE 38 graphically illustrâtes the results of computer-based image analysis of kïdney tissue sections stained with macrophage-specific antibody F4/80 showing the macrophage mean stained area (%) from the following groups of mice at day 14 after treatment with Adriamycin or saline only (wild-type control): wild-type control mice treated with only saline; wild-type mice treated with Adriamycin; MASP-2-/- mice treated with saline only, and MASP-2 -/- mice treated with Adriamycin, wherein **/7=0.007, as described in Example 18;
FIGURE 39 graphically illustrâtes the results of computer-based image analysis of kidney tissue sections stained with Sirius Red, showing the collagen déposition stained area (%) from the following groups of mice at day 14 after treatment with Adriamycin or saline only (wild-type control): wild-type control mice treated with only saline; wild-type mice treated with Adriamycin; MASP-2-/- mice treated with saline only, and MASP-2 -/mice treated with Adriamycin, wherein **p=0,005, as described in Example 18; and
FIGURE 40 graphically illustrâtes the urine albumin/creatinine ratio (uACR) in two IgA patients during the course of a twelve week study with weekly treatment with a MASP-2 inhibitory antibody (OMS646), as described in Example 19.
DESCRIPTION OF THE SEQUENCE LISTING
SEQ IDNO:l human MApl9 cDNA
SEQ ID NO:2 human MApl9 protein (with leader)
SEQ ID NO:3 human MApl9 protein (mature)
SEQ ID NO:4 human MASP-2 cDNA
SEQ ID NO:5 human MASP-2 protein (with leader)
SEQ ID NO:6 human MASP-2 protein (mature)
SEQ ID NO:7 human MASP-2 gDNA (exons l-6)
ANTIGENS: (IN REFERENCE TO THE MASP-2 MATURE PROTEIN)
SEQ ID NO:8 CUBI sequence (aa l -121 )
SEQ ID NO:9 CUBEGF sequence (aa l -166)
SEQ ID NO:l0 CUBEGFCUBII (aa 1-293)
SEQ IDNO:l l EGF région (aa 122-166)
SEQ ID NO:l2 serine protease domain (aa 429 - 671)
SEQ ID NO:l3 serine protease domain inactive (aa 610-625 with Ser618 to Ala mutation)
SEQ ID NO: 14 TPLGPKWPEPVFGRL (CUBI peptide)
SEQ ID NO: 15
TAPPGYRLRLYFTHFDLELSHLCEYDFVKLSSGAKVLATLC
GQ (CUBI peptide)
SEQ ID NO: 16 TFRSDYSN (MBL binding région core)
SEQ ID NO:l7 FYSLGSSLDITFRSDYSNEKPFTGF (MBL binding région) SEQ ID NO:I8 IDECQVAPG (EGF PEPTIDE)
SEQ ID NO:l9 ANMLCAGLESGGKDSCRGDSGGALV (serine protease binding core)Detailed Description
PEPTIDE INHIBITORS:
SEQ ID NO:20 MBL full length cDNA
SEQ ID NO:2l MBL full length protein
SEQ ID NO:22 OGK-X-GP (consensus binding)
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 GAOGSOGEKGAOGPQGPOGPOGKMGPKGEOGDO (human h-ficolin)
SEQ ID NO:28
GCOGLOGAOGDKGEAGTNGKRGERGPOGPOGKAGPOGPN
GAOGEO (human ficolin p35)
SEQ ID NO:29 LQRALEILPNRVTIKANRPFLVFI (C4 cleavage site) EXPRESSION INHIBITORS:
SEQ ID NO:30 cDNA of CUBI-EGF domain (nucléotides 22-680 of SEQ ID NO:4)
SEQ IDNO:31
5' CGGGCACACCATGAGGCTGCTGACCCTCCTGGGC 3’ Nucléotides 12-45 of SEQ ID NO:4 including the MASP-2 translation start site (sense)
SEQ ID NO:32
5’GACATTACCTTCCGCTCCGACTCCAACGAGAAG3'
Nucléotides 361-396 of SEQ ID NO:4 encoding a région comprising the MASP-2 MBL binding site (sense)
SEQ ID NO:33
5'AGCAGCCCTGAATACCCACGGCCGTATCCCAAA3' Nucléotides 610-642 of SEQ ID NO:4 encoding a région comprising the CUBII domain
CLONING 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 NOS:38-47 are cloning primers for humanized antibody
SEQ ID NO:48 is 9 aa peptide bond
EXPRESSION VECTOR:
SEQ ID NO:49 is the MASP-2 minigene insert
SEQ ID NO: 50 is the murine MASP-2 cDNA
SEQ ID NO: 51 is the murine MASP-2 protein (w/leader)
SEQ ID NO: 52 is the mature murine MASP-2 protein
SEQ ID NO: 53 the rat MASP-2 cDNA
SEQ ID NO: 54 is the rat MASP-2 protein (w/ leader)
SEQ ID NO: 55 is the mature rat MASP-2 protein
SEQ ID NO: 56-59 are the oligonucleotides for site-directed mutagenesis of human MASP-2 used to generate human MASP-2A
SEQ ID NO: 60-63 are the oligonucleotides for site-directed mutagenesis of murine MASP-2 used to generate murine MASP-2A
SEQ ID NO: 64-65 are the oligonucleotides for site-directed mutagenesis of rat MASP-2 used to generate rat MASP-2A
SEQ ID NO: 66 DNA encoding I7D20_dc35VH21Nl 1VL (OMS646) heavy chain variable région (VH) (without signal peptide)
SEQ ID NO: 67 17D20_dc35VH21Nl 1VL (OMS646) heavy chain variable région (VH) polypeptide
SEQ ID NO: 68 17N16mc heavy chain variable région (VH) polypeptide
SEQ ID NO: 69 DNA encoding l7D20_dc35VH21Nl l VL (OMS646) light chain variable région (VL)
SEQ ID NO: 70 !7D20_dc35VH2lNl IVL (OMS646) light chain variable région (VL) polypeptide
SEQ ID NO: 71 !7N16_dcl7N9 light chain variable région (VL) polypeptide
SEQ ID NO:72: SGMI-2L(full-length)
SEQ ID NO: 73: SGMI-2M (medium truncated version)
SEQ ID NO:74: SGMI-2S (short truncated version)
SEQ ID NO:75: mature polypeptide comprising the VH-M2ab6-SGMI-2N and the human IgG4 constant région with hinge mutation
SEQ ID NO:76: mature polypeptide comprising the VH-M2ab6-SGMI-2C and the human IgG4 constant région with hinge mutation
SEQ ID NO:77: mature polypeptide comprising the VL-M2ab6-SGMI-2N and the human Ig lambda constant région
SEQ ID NO:78: mature polypeptide comprising the VL-M2ab6-SGMI-2C and the human Ig lambda constant région
SEQ ID NO:79: peptide linker (lOaa)
SEQ ID NO:80: peptide linker (6aa)
SEQ ID NO:8l : peptide linker (4aa)
SEQ ID NO:82: polynucleotide encoding the polypeptide comprising the VH-M2ab6-SGMI-2-N and the human lgG4 constant région with hinge mutation
SEQ ID NO:83: polynucleotide encoding the polypeptide comprising the VH-M2ab 6-SGMI-2-C and the human IgG4 constant région with hinge mutation
SEQ ID NO:84: polynucleotide encoding the polypeptide comprising the VL-M2ab6-SGMI-2-N and the human Ig lambda constant région
SEQ ID NO:85: polynucleotide encoding the polypeptide comprising the VL-M2ab6-SGMI-2-C and the human Ig lambda constant région
DETAILED DESCRIPTION
The present invention is based upon the surprising discovery by the present inventors that inhibition of mannan-binding lectin-associated serine protease-2 (MASP-
2), the key reguiator of the lectin pathway of the complément system, significantly reduces inflammation and fîbrosis in various animal models of fibrotic disease including the unilatéral urétéral obstruction (UUO) model, the protein overload model and the adriamycin-induced nephrology model of rénal fîbrosis. Therefore, the inventors hâve demonstrated that inhibition of MASP-2-mediated lectin pathway activation provides an effective therapeutic approach to ameliorate, treat or prevent rénal fîbrosis, e.g., tubulointerstitial inflammation and fîbrosis, regardless of the underlying cause. As further described herein, the use of a MASP-2 inhibitory antibody (OMS646) is effective to improve rénal function and decrease corticosteroid needs in human subjects suffering from Immunoglobulin A Nephropathy (IgAN) and membranous nephropathy (MN).
L DEFINITIONS
Unless specifically defined herein, ail terms used herein hâve the same meaning as would be understood by those of ordinary skill în the art of the present invention. The following définitions are provided in order to provide clarity with respect to the terms as they are used in the spécification and daims to describe the present invention.
As used herein. the term “MASP-2-dependent complément activation” comprises MASP-2- dépendent activation of the lectin pathway, which occurs under physiological conditions (i.e., in the presence of Ca4^) leading to the formation of the lectin pathway C3 convertase C4b2a and upon accumulation of the C3 cleavage product C3b subsequently to the C5 convertase C4b2a(C3b)n, which has been determined to primarily cause opsonization.
As used herein, the term alternative pathway refers to complément activation that is triggered, for example, by zymosan from fungal and yeast cell walls, lipopolysaccharide (LPS) from Gram négative outer membranes, and rabbit érythrocytes, as well as from many pure polysaccharides, rabbit érythrocytes, viruses, bacteria, animal tumor cells, parasites and damaged cells, and which has traditionally been thought to arise from spontaneous proteolytlc génération of C3b from complément factor C3.
As used herein, the term lectin pathway refers to complément activation that occurs via the spécifie binding of sérum and non-serum carbohydrate-binding proteins 5 including mannan-binding lectin (MBL), CL-ll and the ficolins (H-ficolin, M-fîcolin, or L-ficolin).
As used herein, the term classîcal pathway refers to complément activation that is triggered by antibody bound to a foreign particle and requires binding of the récognition molécule Clq.
As used herein, the term MASP-2 inhibitory agent refers to any agent that binds to or directly interacts with MASP-2 and effectively inhibits MASP-2-dependent complément activation, including anti-MASP-2 antibodies and MASP-2 binding fragments thereof, natural and synthetic peptides, small molécules, soluble MASP-2 receptors, expression inhibitors and isolated natural inhibitors, and also encompasses 15 peptides that compete with MASP-2 for binding to another récognition molécule (e.g., MBL, H-ficolin, M-ficolin, or L-ficolin) in the lectin pathway, but does not encompass antibodies that bind to such other récognition molécules. MASP-2 inhibitory agents useful in the method of the invention may reduce MASP-2-dependent complément activation by greater than 20%, such as greater than 50%, such as greater than 90%. In 20 one embodiment, the MASP-2 inhibitory agent reduces MASP-2-dependent complément activation by greater than 90% (i.e., resulting in MASP-2 complément activation of only 10% or less).
As used herein, the term “fibrosis” refers to the formation or presence of excessive connective tissue in an organ or tissue. Fibrosis may occur as a repair or 25 replacement response to a stimulus such as tissue injury or inflammation. A hallmark of fibrosis is the production of excessive extracellular matrix. The normal physiologîcal response to injury results in the déposition of connective tissue as part of the healing process, but this connective tissue déposition may persist and become pathological, altering the architecture and function of the tissue. At the cellular level, épithélial cells 30 and fibroblasts proliferate and differentiate into myofibroblasts, resulting in matrix contraction, increased rigidity, microvascular compression, and hypoxia.
As used herein, the term treating fibrosis in a mammalian subject suffering from or at risk of developing a disease or disorder caused or exacerbated by fibrosis and/or inflammation refers to reversing, alleviating, ameliorating, or inhibiting fibrosis in said mammalian subject.
As used herein, the term “proteinuria” refers to the presence of urinary protein in an abnormal amount, such as in amounts exceedîng 0.3g protein in a 24-hour urine collection from a human subject, or in concentrations of more than l g per liter in a human subject.
As used herein, the term “improving proteinuria” or “reducing proteinuria’ refers to reducing the 24-hour urine protein excrétion in a subject suffering from proteinuria by at least 20%, such as at least 30%, such as at least 40%, such at least 50% or more in comparison to baseline 24-hour urine protein excrétion in the subject prior to treatment with a MASP-2 inhibitory agent. In one embodiment, treatment with a MASP-2 inhibitory agent in accordance with the methods of the invention is effective to reduce proteinuria in a human subject such as to achieve greater than 20 percent réduction in 24hour urine protein excrétion, or such as greater than 30 percent réduction in 24-hour urine protein excrétion, or such as greater than 40 percent réduction in 24-hour urine protein excrétion, or such as greater than 50 percent réduction in 24-hour urine protein excrétion).
As used herein, the terni antibody encompasses antibodies and antibody fragments thereof, derived from any antibody-producing mammal (e.g., mouse, rat, rabbit, and primate including human), or from a hybridoma, phage sélection, recombinant expression or transgenic animais (or other methods of producing antibodies or antibody fragments”), that specifically bind to a target polypeptide, such as, for example, MASP-2, polypeptides or portions thereof. It is not intended that the term “antibody” limited as regards to the source of the antibody or the manner in which it is made (e.g., by hybridoma, phage sélection, recombinant expression, transgenic animal, peptide synthesis, etc). Exemplary antibodies include polyclonal, monoclonal and recombinant antibodies; pan-specific, multispecific antibodies (e.g., bispecifîc antibodies, trispecific antibodies); humanized antibodies; murine antibodies; chimeric, mouse-human, mouse-primate, primate-human monoclonal antibodies; and anti-idiotype antibodies, and may 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 (ScFv), synthetic variants thereof, naturally occurring variants, fusion proteins comprising an antibody portion with an antigen-binding fragment of the required specificity, humanized antibodies, chimeric antibodies, and any other modified configuration of the immunoglobulin molécule that comprises an antigen-binding site or fragment (epitope récognition site) of the required specificity.
A “monoclonal antibody refers to a homogeneous antibody population wherein the monoclonal antibody is comprised of amino acids (naturally occurring and nonnaturally occurring) that are involved in the sélective binding of an epitope. Monoclonal antibodies are highly spécifie for the target antigen. The term monoclonal antibody encompasses not only intact monoclonal antibodies and fuil-length monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab')2, Fv), single chain (ScFv), variants thereof, fusion proteins comprising an antigen-binding portion, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration ofthe immunoglobulin molécule that comprises an antigenbinding fragment (epitope récognition site) of the required specificity and the ability to bind to an epitope. It is not intended to be limited as regards the source of the antibody or the manner in which it is made (e.g., by hybridoma, phage sélection, recombinant expression, transgenic animais, etc.). The term includes whole immunoglobulins as well as the fragments etc. described above under the définition of 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, generally including the antigen binding or variable région thereof. Illustrative examples of antibody fragments include Fab, Fab', F(ab)2, F(ab’)2 and Fv fragments, scFv fragments, diabodies, linear antibodies, single-chain antibody molécules and multispecîfic antibodies formed from antibody fragments.
As used herein, a single-chain Fv or scFv antibody fragment comprises the V[_[ and Vl domains of an antibody, wherein these domains are présent in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the Vpj and Vl domains, which enables the scFv to form the desired structure for antigen binding.
As used herein, a chimeric antibody is a recombinant protein that contains the variable domains and complementarity-determining régions derived from a non-human species (e.g., rodent) antibody, while the remainder of the antibody molécule is derived from a human antibody.
As used herein, a humanized antibody is a chimeric antibody that comprises a minimal sequence that conforms to spécifie complementarity-determining régions derived from non-human immunoglobulin that is transplanted into a human antibody framework. Humanized antibodies are typically recombinant proteins in which only the antibody complementarity-determining régions are of non-human origin.
As used herein, the term mannan-binding lectin (MBL) is équivalent to mannan-binding protein (MBP).
As used herein, the membrane attack complex (MAC) refers to a complex of the terminal five complément components (C5b combined with C6, C7, C8 and C-9) that inserts into and disrupts membranes (also referred to as C5b-9).
As used herein, a subject includes ail mammals, including without limitation humans, non-human primates, dogs, cats, horses, sheep, goats, cows, rabbits, pigs and rodents.
As used herein, the amino acid residues are abbreviated as follows: alanine (Ala;A), asparagine (Asn;N), aspartic acid (Asp;D), arginine (Arg;R), cysteine (Cys;C), glutamic acid (Glu;E), glutamine (Gln;Q), glycine (Gly;G), hîstidine (His;H), isoleucine (Ile;I), leucine (Leu;L), lysine (Lys;K), méthionine (Met;M), phenylalanine (Phe;F), proline (Pro;P), serine (Ser;S), threonine (Thr;T), tryptophan (Trp;W), tyrosine (Tyr;Y), and valîne (Val;V).
In the broadest sense, the naturally occurring amino acids can be divided into groups based upon the Chemical characteristic of the side chain of the respective amino acids. By hydrophobie amino acid is meant either Ile, Leu, Met, Phe, Trp, Tyr, Val, Ala, Cys or Pro. By hydrophilic amino acid is meant either Gly, Asn, Gin, Ser, Thr, Asp, Glu, Lys, Arg or His. This grouping of amino acids can be further subclassed as
3l follows. By uncharged hydrophilic amino acid is meant either Ser, Thr, Asn or Gin. By acidic amino acid is meant either Glu or Asp. By basic amino acid is meant either Lys, Arg or His.
As used herein the term conservative amino acid substitution is illustrated by a substitution among amino acids within each of the following groups: (l) glycine, alanine, valine, leucine, and isoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine, (4) aspartate and glutamate, (5) glutamine and asparagine, and (6) lysine, arginine and histidine.
The term oligonucleotide as used herein refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term also covers those olîgonucleobases composed of naturally-occurring nucléotides, sugars and covalent intemucleoslde (backbone) linkages as well as oligonucleotides having non-naturally-occurring modifications.
As used herein, an epitope refers to the site on a protein (e.g., a human MASP-2 protein) that is bound by an antibody. Overlapping epitopes include at least one (e.g., two, three, four, five, or six) common amino acid resîdue(s), including linear and nonlinear epitopes.
As used herein, the terms polypeptide, peptide, and protein are used interchangeably and mean any peptide-linked chain of amino acids, regardless of length or post-translatïonal modification. The MASP-2 protein described herein can contain or be wild-type proteins or can be variants that hâve not more than 50 (e.g., not more than one, two, three, four, five, six, seven, eîght, 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, the human MASP-2 protein can hâve an amino acid sequence that is, or is greater than, 70 (e.g., 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100) % identical to the human MASP-2 protein having the amino acid sequence set forth in SEQ ID NO: 5.
In some embodiments, peptide fragments can be at least 6 (e.g., at least 7, 8, 9, 10, 11, 12, 13, I4, 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 in length (e.g., at least 6 contiguous amino acid residues of SEQ ID NO: 5). In some embodiments, an antigenic peptide fragment of a human MASP-2 protein is fewer than 500 (e.g., fewer than 450, 400, 350, 325, 300, 275, 250, 225, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22,21,20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6) amino acid residues in length (e.g., fewer than 500 contiguous amino acid residues in any one of SEQ ID NOS: 5).
Percent (%) amino acid sequence identity is defined as the percentage of amino acids in a candidate sequence that are identical to the amino acids in a reference sequence, after aligning the sequences and mtroducîng gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, ustng publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorîthms needed to achieve maximal alignment over the fulllength of the sequences being compared can be determined by known methods.
IL OverView of the Invention
As described herein, the inventors hâve identified the central rôle of the lectin pathway in the initiation and disease progression of tubular rénal pathology, thereby implîcating a key rôle of the lectin pathway activation in the pathophysiology of a diverse range of rénal diseases including IgA nephropathy, C3 glomerulopathy and other glomerulonephritides. As further described herein, the inventors discovered that inhibition of mannan-binding lectin-associated serine protease-2 (MASP-2), the key regulator of the lectin pathway of the complément system, significantly reduces inflammation and fibrosis în various animal models of fibrotic disease Including the unilatéral urétéral obstruction (UDO) model, the protein overload model and the adriamycin-induced nephrology model of rénal fibrosis. Therefore, the inventors hâve demonstrated that inhibition of MASP-2-mediated lectin pathway activation provides an effective therapeutic approach to ameliorate, treat or prevent rénal ftbrosis, e.g., tubulointerstitial fibrosis, regardless ofthe underlying cause.
Lectins (MBL, M-ficolin, H-ficolin, L-ficolin and CL-ll) are the spécifie récognition molécules that trigger the innate complément system and the system includes the lectin initiation pathway and the associated terminal pathway amplification loop that amplifies lectin-înitiated activation of terminal complément effector molécules. Clq is the spécifie récognition molécule that triggers the acquired complément system and the system includes the classical initiation pathway and associated terminal pathway amplification loop that amplifies Clq-initiated activation of terminal complément effector molécules. We refer to these two major complément activation Systems as the lectin-dependent complément system and the Clq-dependent complément system, respectively.
In addition to its essential rôle in immune defense, the complément system contributes to tissue damage in many clinical conditions. Thus, there is a pressing need to develop therapeutically effective complément inhibitors to prevent these adverse effects. With the récognition that it is possible to inhibit the lectin mediated MASP-2 pathway while leaving the classical pathway intact cornes the realization that it would be highly désirable to specifically inhibit only the complément activation system causing a particular pathology without completely shutting down the immune defense capabilities of complément. For example, in disease States in which complément activation is mediated predominantly by the lectin-dependent complément system, it would be advantageous to specifically inhibit only this system. This would leave the Clq-dependent complément activation system intact to handle immune complex processing and to aid in host defense against infection.
The preferred protein component to target in the development of therapeutic agents to specifically inhibit the lectin-dependent complément system is MASP-2. Of ail the known protein components of the lectin-dependent complément system (MBL, H-ficolin, M-ficolin, L-ficolin, MASP-2, C2-C9, Factor B, Factor D, and properdin), only
MASP-2 is both unique to the lectin-dependent complément system and required for the system to function. The iectins (MBL, H-ficolin, M-ficolin, L-ficolin and CL-ll) are also unique components in the lectin-dependent complément system. However, loss of any one of the lectin components would not necessarily inhibit activation of the system due to lectin redundancy. It would be necessary to inhibit ail five Iectins in order to guarantee inhibition of the lectin-dependent complément activation system. Furthermore, since MBL and the ficolîns are also known to hâve opsonic activity independent of complément, inhibition of lectin function would resuit in the loss of this bénéficiai host defense mechanism against infection. In contrast, this complement-independent lectin opsonic activity would remain intact if MASP-2 was the inhibltory target. An added benefit of MASP-2 as the therapeutic target to inhibit the lectin-dependent complément activation system is that the plasma concentration of MASP-2 is among the lowest of any complément protein (= 500 ng/mi); therefore, correspondingly low concentrations of high-affmity inhibitors of MASP-2 may be sufficient to obtain full inhibition (Moller-Kristensen, M., et al., J. Immunol Methods 282:159-167, 2003).
As described herein in Example 14, it was determined in an animal model of fibrotic kîdney disease (unilatéral urétéral obstruction UUO) that mice without the MASP-2 gene (MASP-2-/-) exhibited significantly less kidney disease compared to wild-type control animais, as shown by inflammatory cell infiltrâtes (75% réduction) and histological markers of fibrosis such as collagen déposition (one third réduction). As further shown in Example 15, wild-type mice systemically treated with an anti-MASP-2 monoclonal antibody that selectively blocks the lectin pathway while leaving the classical pathway intact, were protected from rénal fibrosis, as compared to wild-type mice treated with an isotype control antibody. These results demonstrate that the lectin pathway is a key contributor to kidney disease and further demonstrate that a MASP-2 inhibitor that blocks the lectin pathway, such as a MASP-2 antibody, is effective as an antifibrotic agent. As further shown in Example 16, in the protein overload model, wild-type mice treated with bovine-serum album in (BSA) developed protéinurie nephropathy, whereas MASP-2-/mice treated with the same level of BSA had reduced rénal injury. As shown in Example 17, wild-type mice systemically treated with an anti-MASP-2 monoclonal antibody that selectively blocks the lectin pathway while leaving the classical pathway intact, were protected from rénal injury in the protein overload model. As described in Example 18, MASP-2-/- mice exhibited less rénal inflammation and tubulointerstitial injury in an Adriamycin-induced nephrology model of rénal fïbrosis as compared to wild-type mice. As described in Example 19, in an ongoing Phase 2 open-label rénal trial, patients with IgA nephropathy that were treated with an anti-MASP-2 antibody demonstrated a clinically meaningfül and statistically significant decrease in urine albumin-to-creatinine ratios (uACRs) throughout the trial and réduction in 24-hour urine protein levels from baseline to the end of treatment. As further described in Example 19, in the same Phase 2 rénal trial, patients with membranous nephropathy that were treated with an anti-MASP2 antibody also demonstrated réductions in uACR during treatment.
In accordance with the foregoing, the présent invention relates to the use of MASP-2 inhibitory agents, such as MASP-2 inhibitory antibodies, as antifibrotic agents, the use of MASP-2 inhibitory agents for the manufacture of a médicament for the treatment of a fibrotic condition, and methods of preventing, treating, alleviating or reversîng a fibrotic condition in a human subject in need thereof, said method comprising administering to said patient an efficient amount of a MASP-2 inhibitory agent (e.g., an anti-MASP-2 antibody).
The methods of the invention can be used to prevent, treat, alleviate or reverse a fibrotic condition in a human subject suffering from any disease or disorder caused or exacerbated by fïbrosis and/or inflammation, including diseases of the kidney (e.g., chronic kidney disease, IgA nephropathy, C3 glomerulopathy and other glomerulonephritides), lung (e.g., idiopathic pulmonary fïbrosis, cystic fïbrosis, bronchiectasis), liver (e.g., cirrhosis, nonalcoholic fatty liver disease), heart (e.g., myocardial infarction, atrial fïbrosis, valvular fïbrosis, endomyocardial fïbrosis), brain (e.g., stroke), skin (e.g., excessive wound healing, scleroderma, systemic sclerosis, keloids), vasculature (e.g., atherosclerotic vascular disease), intestine (e.g., Crohn’s disease), eye (e.g., anterior subcapsular cataract, posterior capsule opacification), musculoskelétal soft-tissue structures (e.g., adhesive capsulitis, Dupuytren’s contracture, myelofibrosis), reproductive organs (e.g., endometriosis, Peyronie’s disease), and some infectious diseases (e.g., alpha virus, Hepatitis C, and Hepatitis B).
III. THE ROLE OF MASP-2 IN DISEASES AND CONDITIONS CAUSED OR
EXACERBATED BY FIBROSIS
Fibrosis is the formation or presence of excessive connective tissue in an organ or tissue, commonly in response to damage or injury. A hallmark of fibrosis is the production of excessive extracellular matrix following an injury. In the kidney, fibrosis is characterized as a progressive detrimental connective tissue déposition on the kidney parenchyma which inevitably leads to a décliné in rénal function independently of the primary rénal disease which causes the original kidney tnjury. So called épithélial to mesenchymal transition (EMT), a change in cellular characteristics in which tubular épithélial cells are transformed to mesenchymal fibroblasts, constitutes the principal mechanism of rénal fibrosis. Fibrosis affects nearly ail tissues and organ Systems and may occur as a repair or replacement response to a stimulus such as tissue injury or inflammation. The normal physiological response to injury results in the déposition of connective tissue but, if this process becomes pathological, the replacement of highly differentiated cells by scarring connective tissue alters the architecture and function of the tissue. At the cellular level, épithélial cells and fibroblasts proliferate and differentiate into myofibroblasts, resulting in matrix contraction, increased rigidity, microvascular compression, and hypoxia. Currently there are no effective treatments or therapeutics for fibrosis, but both animal studies and anecdotal human reports suggest that fibrotic tissue damage may be reversed (Tampe and Zeisberg, Nat Rev Nephrol, vol 10:226-237, 2014).
Many diseases resuit in fibrosis that causes progressive organ failure, including diseases of the kidney (e.g., chronic kidney disease, IgA nephropathy, C3 glomerulopathy and other glomerulonephritides), lung (e.g., idiopathîc pulmonary fibrosis, cystic fibrosis, bronchiectasis), liver (e.g., cirrhosis, nonalcoholic fatty liver disease), heart (e.g., myocardial infarction, atrial fibrosis, valvular fibrosis, endomyocardial fibrosis), brain (e.g., stroke), skin (e.g., excessive wound healing, scleroderma, systemic sclerosis, keloids), vasculature (e.g., atherosclerotic vascular disease), intestine (e.g., Crohn’s disease), eye (e.g., anterior subcapsular cataract, posterior capsule opacification), musculoskeletal soft-tissue structures (e.g., adhesive capsulitis, Dupuytren's contracture, myelofibrosis), reproductive organs (e.g., endometriosis, Peyronie’s disease), and some infectious diseases (e.g., alpha virus, Hepatitis C, Hepatitis B, etc.).
While fibrosis occurs in many tissues and diseases, there are common molecular and cellular mechanisms to its pathology. The déposition of extracellular matrix by fibroblasts is accompanied by immune cell infiltrâtes, predominately mononuclear cells (see Wynn T., Nat Rev Immunol 4(8):583-594, 2004, hereby incorporated herein by reference). A robust inflammatory response results in the expression of growth factors (TGF-beta, VEGF, Hépatocyte Growth Factor, connective tissue growth factor), cytokines and hormones (endothelin, IL-4, lL-6, IL-13, chemokines), degradatîve enzymes (elastase, matrix métaloprotéinases, cathepsins), and extracellular matrix proteins (collagens, fibronectin, integrins).
In addition, the complément system becomes activated in numerous fibrotic diseases. Complément components, including the membrane attack complex, hâve been identified in numerous fibrotic tissue specimens. For example, components of the lectin pathway hâve been found in fibrotic lésions of kidney disease (Satomura et al., Nephron. 92(3):702-4 (2002); Sato et al., Lupus 20(13):1378-86 (2011); Liu et al., Clin Exp Immunol, 174(1):152-60 (2013)); liver disease (Rensen et al., Hepatology 50(6): 1809-17 (2009)); and lung disease (Olesen et al., Clin Immunol 121(3):324-31 (2006)).
Overshooting complément activation has been established as a key contributor to immune complex-mediated as weil as antibody independent glomerulonephritides. There is, however, a strong line of evidence demonstrating that uncontrolled activation of complément in situ is intrinsically involved in the pathophysiological progression of Ή fibrosis in non-glomerular disease (Quigg RJ, J Immunol 171:3319-3324, 2003, Naik A. et al., Semin Nephrol 33:575-585, 2013, Mathem D.R. et al., Clin J Am Soc Nephrol 10:P 1636-1650, 2015). The strong proînflammatory signais that are triggered by local complément activation may be initiated by complément components filtered into the proximal tubule and subsequently entering the interstitial space, or abnormal synthesis of complément components by tubular or other résident and infiltrating cells, or by altered expression of complément regulatory proteins on kidney cells, or absence or loss or gain for function mutations in complément regulatory components (Mathem D.R. et al., Clin J Am Soc Nephrol 10:P 1636-1650, 2015, Sheerin N.S., et al., FASEB J 22: 1065-1072, 2008). In mice for example, deficiency of the complément regulatory protein CR1related gene/protein y (Crry), results in tubulointerstitial (TI) complément activation with conséquent inflammation and fibrosis typical of the injury seen in human TI diseases (Naik A. et al., Semin Nephrol 33:575-585, 2013, Bao L. et al., J Am Soc Nephrol 18:811822, 2007). Exposure of tubular épithélial cells to the anaphylatoxin C3a results in épithélial to mesenchymal transition (Tsang Z. et ak, J Am Soc Nephrol 20:593-603, 2009). Blocking C3a signaling via the C3a receptor alone has recently been shown to lessen rénal TI fibrosis in protéinurie and non-proteinuric animais (Tsang Z. et al., J Am Soc Nephrol 20:593-603, 2009, Bao L. et al., Kidney Int. 80: 524-534, 2011).
As described herein, the înventors hâve identified the central rôle of the lectin pathway în the initiation and disease progression of tubular rénal pathology, thereby implicating a key rôle ofthe lectin pathway activation in the pathophysiology ofa diverse range of rénal diseases including IgA nephropathy, C3 glomerulopathy and other glomerulonephritides (Endo M. et al., Nephrol Dialysis Transplant 13: 1984-1990, 1998; Hisano S. et al., Am J Kidney Dis 45:295-302, 2005; Roos A. et al., J Am Soc Nephrol 17: 1724-1734, 2006; Liu L.L. et al., Clin Exp. Immunol 174:152-160, 2013; Lhotta K. et al., Nephrol Dialysis Transplant 14:881-886, 1999; Pickering et al., Kidney International 84:1079-1089, 2013), diabetic nephropathy (Hovind P. et al., Diabètes 54:1523-1527, 2005), ischaemic reperfusion injury (Asgari E. et al., FASEB J 28:3996-4003, 2014) and transplant rejection (Berger S.P. et al., Am J Transplant 5:1361-1366, 2005).
As further described herein, the înventors hâve demonstrated that MASP-2 inhibition reduces inflammation and fibrosis in mouse models of tubulointerstitial disease. Therefore, MASP-2 inhibîtory agents are expected to be useful in the treatment of rénal fibrosis, including tubulointerstitial inflammation and fibrosis, proteinuria, IgA nephropathy, C3 glomerulopathy and other glomerulonephritides and rénal ischaemia reperfusion injury.
Kidney Diseases and Disorders
According to the National Kidney Foundation, 26 million American adults suffer from Chronic Kidney Disease (CKD). Most patients hâve progressive disease leading to kidney faîlure, requirîng treatment with erythropoiesis stimulating drugs, dialysis or a kidney transplant for survivaL There are several drugs that can treat the main symptom of CKD, hypertension, but currently there are no drugs that address its root cause.
Studies hâve shown that progressive rénal injury is caused by capillary hypertension in substructures of the kidney known as nephrons (Whitworth J.A., Annals Acad of Med, vol 34(1):2005). As nephrons (the filtration units of the kidney) are injured or destroyed in this process, inflammation and tissue scarrîng occur, replacing nephrons with non-functional scar tissue. As a resuit, the ability of the kidney to filter blood déclinés over time. This is referred to as rénal fibrosis, which is the common pathway of progressive rénal disease. Irrespective of the nature of the initial insult, rénal fibrosis is considered to be the common final pathway by which kidney disease progresses to endstage rénal failure. Amelioration of rénal fibrosis may be determined by one or more of the following: assessment of interstitial volume, coilagen IV déposition, and/or connective tissue growth mRNA levels. The compounds and methods described herein are useful in the treatment of rénal fibrosis.
Rénal fibrosis and inflammation are prominent features of late-stage kidney disease of virtually any etîology (see Boor et al., Boor P. et al., J of Am Soc of Nephrology 18:1508-1515, 2007 and Chevalier et al., Kidney International 75:11451152, 2009). Kidney failure can be caused by a heterogeneous group of disorders. Progressive kidney dysfunction leads to proteinuria and rénal insufficiency. As patient health deteriorates, dîalysis may be necessary simply to forestall the damage to the kidney and to prevent multi-system failure. Over time, kidney failure and rénal insufficiency can progress to end-stage rénal disease (ESRD), which is total, or nearly total, permanent loss of kidney function. Depending on the form of kidney disease, rénal function may be lost in a matter of days or weeks or may deteriorate slowly and graduaily over the course of décades. Once a patient has progressed to ESRD, dîalysis (hemidialysis or peritoneal dîalysis) is requîred to prevent death. Patients must remain on some form of dîalysis regimen or must obtain a kidney transplant.
Components of the lectïn pathway hâve been found in fibrotic lésions of kidney disease (Satomura et ai., Nephron. 92(3):702-4 (2002); Sato et al., Lupus 20(13):1378-86 (2011); Liu et al., Clin Exp Immunol, 174(1):152-60 (2013)). In IgA nephropathy, patients with glomerular MBL déposition had more severe proteinuria, decreased rénal function, lower levels of sérum albumin, more severe histology, and greater hypertension than patients without MBL déposition (Liu et al., Clin Exp Immunol. 2013
Oct; 174( 1):152-60). Patients with lupus nephritis (Sato et al., Lupus, 20(13):1378-86, 2011) and chronic rénal failure (Satomura et al., Nephron 92(3):702-4, 2002) also hâve increased levels of MBL and lectin pathway activity.
It has also been demonstrated that C5 deficiency led to a significant amelioration of major components of rénal fibrosis in a nonproteinuric model of primary tubulointerstitial damage, namely unilatéral urétéral obstruction (UUO) (Boor P. et al., J of Am Soc ofNephrology 18:1508-1515, 2007). It has also been reported that C3 gene expression was increased in wild-type mice following UUO, and that collagen déposition was significantly reduced in C3-/- mice following UUO as compared to wild-type mice, suggesting a rôle of complément activation in rénal fibrosis (Feam et al., Mol Immunol 48:1666-1733, 2011: Abstract). However, prior to the discovery described herein by the present inventors, the complément components involved in rénal fibrosis were not well defined. As described herein in Examples 14-17, the present inventors hâve unexpectedly determined that a deficiency of MASP-2 or blockade of MASP-2 with an inhibitory antibody that selectively blocks the lectin pathway, while leaving intact the classical pathway, clearly protects mice from rénal fibrosis în various animal models of kidney disease.
Accordingly, in certain embodiments, the disclosure provides a method of inhibiting rénal fibrosis in a subject suffering from a kidney disease or disorder caused or exacerbated by fibrosis and/or inflammation comprising administering a MASP-2 inhibitory agent, such as an anti-MASP-2 antibody, to a subject in need thereof. This method includes administering a composition comprising an amount of a MASP-2 inhibitor effective to inhibit rénal fibrosis to a subject suffering from a kidney disease or disorder caused or exacerbated by fibrosis and/or inflammation.
The MASP-2 inhibitory composition may be administered locally to the région of fibrosis, such as by local application of the composition during surgery or local injection, either directly or remotely, for example, by cathéter. Altemately, the MASP-2 inhibitory agent may be administered to the subject systemîcally, such as by intra-arterial, intravenous, intramuscular, inhalational, nasal, subcutaneous or other parentéral administration, or potentially by oral administration for ποη-peptidergic agents.
Administration may be repeated as determined by a physician until the condition has been resolved or is controlled.
In certain embodiments, the MASP-2 inhibitory agents (e.g., anti-MASP-2 antibodies) are administered in combination with one or more agents or treatment modalities appropriate for the underiying kidney disease or condition. In certain embodiments, the MASP-2 inhibitory agents (e.g., anti-MASP-2 antibodies) are administered in combination with a dialysis or plasmapheresis regimen. In certain embodiments, the MASP-2 inhibitory agents (e.g., anti-MASP-2 antibodies) are used to decrease the frequency with which dialysis or plasmapheresis is required. In certain other embodiments, the MASP-2 inhibitory agents (e.g., anti-MASP-2 antibodies) are used in combination with kidney transplantation. In certain other embodiments, the MASP-2 inhibitory agents (e.g., anti-MASP-2 antibodies) are used to control rénal însufficiency and prevent the further décliné in rénal function in patients awaiting kidney transplantation.
By way of example, in certain embodiments, anti-MASP-2 antibodies are used to inhibit rénal fîbrosis and thereby treat or ameliorate (including treating or ameliorating the symptoms of a disease) glomerular diseases such as focal segmentai glomerulosclerosis and nephrotîc syndrome. Exemplary symptoms that can be treated include, but are not limited to, hypertension, proteinuria, hyperlipidemia, hematuria, and hypercholestermia. In some embodiments, the MASP-2 inhibitory agent inhibits tubulointerstitial fîbrosis. In certain embodiments, treating comprises improving rénal function, decreasing proteinuria, improving hypertension, and/or decreasing rénal fîbrosis. In certain embodiments, treating comprises (i) delaying or preventing progression to rénal însufficiency, rénal failure, or ESRD; (ii) delaying, reducing, or preventing need for dialysis; or (iii) delaying or preventing need for kidney transplantation.
Certain spécifie kidney diseases and disorders caused or exacerbated by fîbrosis and/or inflammation are described below.
In certain embodiments, the kidney disease caused or exacerbated by fîbrosis and/or inflammation is a glomerular disease such as focal segmentai glomerulosclerosis (FSGS). Glomerular diseases damage the glomeruli, letting protein and sometimes red blood cells leak into the urine. Sometimes a glomerular disease also interfères with the clearance of waste products by the kidney, so they begin to build up in the blood. Symptoms of glomerular disease include proteinuria, hematuria, reduced glomerular filtration rate, hypoproteinemia, and edema. A number of different diseases can resuit in glomerular disease. It may be the direct resuit of an infection or a drug toxïc to the kidneys, or it may resuit from a disease that affects the entire body, such as hypertension, diabètes or lupus. FSGS is one particular glomerular disease, but even this particular condition characterized by scarring in the kidney can hâve numerous causes. Patients with FSGS typically progress to end stage rénal disease within 5-20 years, although patients with aggressive forms of the disease progress to ESRD in 2 to 3 years.
In certain embodiments, the kidney disease caused or exacerbated by fibrosis and/or inflammation is diabetic nephropathy (DN), which is an area of substantial unmet medical need. Diabetic nephropathy is kidney disease or damage that results as a complication of diabètes. The condition is exacerbated by high blood pressure, high blood sugar levels, and high cholestérol and lipid levels. The exact cause of diabetic nephropathy is unknown. However, without being bound by theory, it is believed that uncontrolled high blood sugar leads to the development of kidney damage, such as fibrosis and scarring of tissue. In humans, DN manifests as a clinical syndrome that is composed of albuminuria, progressively declining glomerular filtration rate (GFR) and increased risk for cardiovascular disease. Diabetic albuminuria is associated with the development of characteristic histo-pathologic features, including ticking of the glomerular basement membrane (GBM) and mesangial expansion. As albuminuria progress and rénal insufficiency ensues, glomerulosclerosis, arteriolar hyalinosis and tubulointerstitial fibrosis develop.
Accordingly, in one embodiment, the present disclosure provides methods for treating diabetic nephropathy comprising administering an effective amount of a MASP-2 inhibitory agent (e.g., a MASP-2 inhibitory antibody) to a subject in need thereof. In certain embodiments, treating comprises reducing one or more symptoms of diabetic nephropathy. In certain embodiments, treating comprises reducing, delaying or eliminating the need for dialysis. In certain embodiments, treating comprises reducing, delaying, or eliminating the need for kidney transplantation. In certain embodiments, treating comprises delaying, preventing or reversing the progression of diabetic nephropathy to rénal failure or end stage rénal disease.
In certain embodiments, the kidney disease caused or exacerbated by fibrosis and/or inflammation is lupus nephritis. As described in more detail below, lupus nephritis, which is a severe complication of systemic lupus erythematosus (SLE), is another example of rénal fibrosis that can be treated with MASP-2 inhibitory agents (e.g., anti-MASP-2 antibodies).
Accordingly, in one embodiment, the present disclosure provides methods for inhibiting rénal fibrosis in a subject suffering from a kidney disease or disorder caused or exacerbated by fibrosis and/or inflammation comprising administering an effective amount of a MASP-2 inhibitory agent (e.g., a MASP-2 inhibitory antibody). In some embodiments, the kidney disease or disorder exacerbated by fibrosis and/or inflammation is selected from the group consisting of chronic kidney disease, chronic rénal failure, glomerular disease (e.g., focal segmentai glomerulosclerosis), an immune complex disorder (e.g., IgA nephropathy, membranous nephropathy), lupus nephritis, nephrotic syndrome, diabetic nephropathy, tubulointerstitiai damage and C3 glomerulopathy or other types of glomerulonepthrîtis.
Methods of Preventing or Treating Rénal Injury caused by drug-induced toxicity
Another cause of rénal injury includes drug-induced toxicity. For example, nephrotoxins can cause direct toxicity on tubular épithélial cells. As described herein, the inventors hâve demonstrated that MASP-2 déficient mice are protected from Adriamycin-induced nephropathy.
Nephrotoxins include, but are not limited to, therapeutic drugs, (e.g., cisplatin, gentamicin, cephaloridine, cyclosporîn, amphotericin, Adriamycin), radiocontrast dye, pesticides (e.g., paraquat), and environmental contaminants (e.g., trichloriethylene and dichloroacetylene). Other examples include puromycin aminonucleosîde (PAN); amînoglycosides, such as gentamicin; cephalosporins, such as cephaloridine; calcineurin inhibitors, such as tacrolimus or sirolimus. Drug-induced nephrotoxicity may also be caused by non-steroidal anti-inflammatories, anti-retrovirals, anti-cytokines, immunosuppressants, oncological drugs or ACE inhibitors. The drug-induced nephrotoxicity may further be caused by nalgesic abuse, ciprofloxacin, clopidogrel, cocaïne, cox-2 inhibitors, diuretics, foscamet, gold, ifosfamide, immunoglobin, Chinese herbs, interferon, lithium, mannitol, mesalamine, mitomycin, nitrosoureas, penicillamine, penicillins, pentamidine, quinine, rifampin, streptozocin, sulfonamides, ticlopidîne, triamterene, valproic acid, doxorubicin, glycerol, cidofovir, tobramycîn, neomycin 5 sulfate, colistimethate, vancomycin, amikacîn, cefotaxime, cisplatin, acyclovir, lithium, interleukin-2, cyclosporin or indinavir.
Accordingly, in one embodiment, a subject at risk for developing or suffering from rénal injury may be receiving one or more therapeutic drugs that hâve a nephrotoxic effect. These subjects may be administered the MASP-2 inhibitors of the invention prior 10 to or simultaneously with such therapeutic agents. Likewise, MASP-2 inhibitors may be administered after the therapeutic agent to treat or reduce the likelihood of developing nephrotoxicîty.
Diseases and Conditions Associated with Proteinuria
It has been established that impaired glomerular filtration of protein results in 15 proteinuria and accelerates the progressive loss of nephrons that occurs in ail chronic rénal diseases (Remuzzi and Bertani, New Eng. J Med vol 339 (20):1448-1456, 1998). For example, in a study described in Eddy et al., Am J Pathol 135:719-33, 1989, glomerular filtration of albumin was consistently followed by the development of interstitial lésions and scarring. As further described in Eddy et al., 1989, déposition of 20 complément C3 on the luminal surface of proximal tubules was observed in the rats with nephropathy induced by protein-overload, indicating that components of the complément system that are filtered by glomeruli can cause interstitial injury. It has been demonstrated that complément déplétion or the lack of C6 ameliorated tubulointerstitial injury in protéinurie animal models such as mesangioprofiferative glomerulonephritis, 25 Adriamycîn nephropathy, five-sixths nephrectomy and puromycin aminonucieoside nephrosis (Boor et al., et al., J of Am Soc of Nephrology: JASN 18:1508-1515, 2007). Human studies hâve shown that proteinuria is an independent predîctor of progression of chronic kidney disease and that réduction in proteinuria is renal-protective (Ruggenentî P. et al., J Am Soc Nephrol 23:1917-1928, 2012).
Accordingly, in one embodiment, the présent disclosure provides methods for preventing or reducing proteînurea and/or preventing or reducing rénal damage in a subject suffering from a disease or condition associated with proteinuria comprising administering an amount of a MASP-2 inhibitory agent (e.g., a MASP-2 inhibitory antibody) effective to reduce or prevent proteinurea in the subject. In some embodiments, the disease or condition associated with proteinuria is selected from the group consisting of nephrotic syndromes, pre-eclampsia, eclampsia, toxic lésions of kidneys, amyloidosis, collagen vascular diseases (e.g., systemic lupus erythematosus), déhydration, glomerular diseases (e.g. membranous glomerulonephritis, focal segmentai glomerulonephritis, minimal change disease, lipoid nephrosis), strenuous exercise, stress, benign orthostatis (postural) proteinuria, focal segmentai glomeruiosclerosis, IgA nephropathy (i.e., Berger’s disease), IgM nephropathy, membranoproliferative glomerulonephritis, membranous nephropathy, minimal change disease, sarcoidosis, Alport’s syndrome, diabètes mellîtus (diabetic nephropathy), drug-induced toxicity (e.g., NSAIDS, nicotine, penicillamine, lithium carbonate, gold and other heavy metals, ACE inhibitors, antîbiotics or opiates (e.g. heroin)); Fabry’s disease, infections (e.g., HIV, syphilis, hepatitis A, B or C, poststreptococcal infection, urinary schistosomiasis); aminoaciduria, Fanconi syndrome, hypertensive nephrosclerosis, interstîtial nephritis, stckle cell disease, hemoglobinuria, multiple myeloma, myoglobinuria, organ rejection (e.g., kidney transplant rejection), ebola hémorrhagie fever, Nail patella syndrome, familial mediterranean fever, HELLP syndrome, systemic lupus erythematosus, Wegener’s granulomatosis, Rheumatoid arthritis, Glycogen storage disease type l, Goodpasture’s syndrome, Henoch-Schônlein purpura, urinary tract infection which has spread to the kidneys, Sjogren’s syndrome and post-infect ion s glomerulonepthrîtis.
Liver Disease
Liver fibrosis, also called hepatic fibrosis, is caused by the accumulation of scar tissue in the liver and is a characteristic of most types of liver disease. The replacement of healthy liver tissue with scar tissue impairs the ability ofthe liver to function properiy. If the condition causing the scarring is not treated, liver fibrosis may progress to liver cirrhosis and complété liver failure, a lîfe-threatening condition. The major causes of liver fibrosis are alcohol abuse, chronic hepatitis C virus infection, nonalcoholic steatohepatitis and hepatotoxicity (e.g., drug-induced liver damage induced by acetaminophen or other drug).
Components of the lectin pathway hâve been found in fibrotic lésions of liver disease (Rensen et al., Hepatology 50(6): 1809-17 (2009)). For example, in nonalcoholic steatohepatitis (also known as fatty liver disease), there is widespread activation of complément System proteins, and their expression is associated with disease severity (Rensen et al., Hepatology 50(6): 1809-17 (2009), where in addition to C3 and C9 déposition, MBL accumulation was found, confîrming activation of the lectin pathway.
Accordingly, in certain embodiments, the disclosure provides a method of inhibiting hepatic fîbrosis in a subject suffering from a lîver disease or disorder caused or exacerbated by fîbrosis and/or inflammation comprising administering a MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody, to a subject in need thereof. This method includes administering a composition comprising an amount of a MASP-2 inhibitor effective to inhibit hepatic fîbrosis to a subject suffering from a liver disease or disorder caused or exacerbated by fîbrosis and/or inflammation.
The MASP-2 inhibitory composition may be administered iocally to the région of fîbrosis, such as by local application of the composition during surgery or local injection, either directly or remotely, for example, by cathéter. Altemately, the MASP-2 inhibitory agent may be administered to the subject systemically, such as by intra-arterial, intravenous, intramuscular, inhalatîonal, nasal, subcutaneous or other parentéral administration, or potentialiy by oral administration for non-peptidergic agents. Administration may be repeated as determined by a physician until the condition has been resolved or is controlled.
In certain embodiments, the MASP-2 inhibitory agents (e.g., MASP-2 inhibitory antibodîes) are administered in combination with one or more agents or treatment modalities appropriate for the underlying liver disease or condition.
In some embodiments, the liver disease or disorder caused or exacerbated by fîbrosis and/or inflammation is selected from the group consisting of: cirrhosis, nonalcoholic fatty liver disease (steatohepatitis), liver fîbrosis secondary to alcohol abuse, liver fîbrosis secondary to acute or chronic hepatitis, biliary disease and toxic liver injury (e.g., hepatotoxicity due to drug-induced liver damage induced by acetaminophen or other drug).
Lung Disease
Pulmonary fibrosîs is the formation or development of excess fibrous connective tissue in the lungs, wherein normal lung tissue is replaced with fibrotic tissue. This scarring leads to stifïhess of the lungs and impaired lung structure and function. In humans, pulmonary fibrosis is thought to resuit from repeated injury to the tissue within and between the tiny air sacs (alveolt) in the lungs. In an experimental setting, a variety of animal models hâve replicated aspects of the human disease. For example, a foreign agent such as bleomycin, fluorescein isothiocyanate, silica, or asbestos may be instilled into the trachea of an animal (Gharaee-Kermani et al., Animal Models of Pulmonary Fibrosîs. Methods Mol. Med., 2005, 117:251-259).
Accordingly, in certain embodiments, the disclosure provides a method of inhibiting pulmonary fibrosîs in a subject suffering from a lung disease or disorder caused or exacerbated by fibrosîs and/or inflammation comprising administering a MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody, to a subject in need thereof. This method includes administering a composition comprising an amount of a MASP-2 inhibitor effective to inhibit pulmonary fibrosîs, decrease lung fibrosîs, and/or improve lung function. Improvements in symptoms of lung function include improvement of lung function and/or capacity, decreased fatigue, and improvement in oxygen saturation.
In some embodiments, the disclosure provides a method of treating, inhibiting, preventing or ameliorating pulmonary fibrosîs in a subject suffering from cystic fibrosîs comprising administering a MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody to a subject in need thereof.
The MASP-2 inhibitory composition may be administered locally to the région of fibrosîs, such as by local application of the composition during surgery or local injection, either directly or remotely, for example, by cathéter. Altemately, the MASP-2 inhibitory agent may be administered to the subject systemically, such as by intra-arterial, intravenous, intramuscular, inhalational, nasal, subcutaneous or other parentéral administration, or potentially by oral administration for non-peptidergic agents. Administration may be repeated as determined by a physician until the condition has been resolved or is controlled.
In certain embodiments, the MASP-2 inhibitory agents (e.g., MASP-2 inhibitory antibodies) are administered in combination with one or more agents or treatment modalities appropriate for the underlying lung disease or condition.
Certain spécifie lung diseases and disorders caused or exacerbated by fibrosis and/or inflammation are described below.
In certain embodiments, the lung disease caused or exacerbated by fibrosis and/or inflammation is chronic obstructive pulmonary disease (COPD). COPD is a disease in which airway walls are fibrotic with the accumulation of myofibroblasts and collagen, is a major cause of disability, and it's the fourth leading cause of death in the United States. COPD blocks airflow and makes it încreasingly difficult for a sufferer to breathe. COPD is caused by damage to the airways that eventually interfères with the exchange of oxygen and carbon dioxide in the lungs. COPD includes chronic obstructive bronchitis and emphysema and often both. COPD patients, whose lungs are aiready damaged and whose lung function is aiready compromised, are at increased risk of complications associated with bacterial and viral infections.
Accordingly, in one embodiment, the présent disclosure provides methods for treating chronic obstructive pulmonary disease (COPD) comprising administering an effective amount of a MASP-2 inhibitory agent (e.g., an anti-MASP-2 antibody) to inhibit and/or decrease lung fibrosis in a subject in need thereof. In certain embodiments, treating comprises reducing one or more symptoms of COPD. Symptoms of COPD and/or lung fibrosis include, but are not limited to, cough with mucus, shortness of breath (dyspnea) that may get worse with mild activity, fatigue, frequent respiratory infections, wheezing, chest tightness, irregular heartbeats (arrhythmias), need for breathing machine and oxygen therapy, right-sîded heart failure or cor pulmonale (heart swelling and heart failure due to chronic lung disease), pneumonia, pneumothorax, severe weight loss and malnutrition. Symptoms also include decrease in lung function, as evaluated using one or more standard tests of lung function.
In certain embodiments, the lung disease caused or exacerbated by fibrosis and/or inflammation is pulmonary fibrosis associated with scleroderma. As described in more detail below, pulmonary fibrosis associated with scleroderma is another example of pulmonary fibrosis that can be treated with MASP-2 inhibitory agents (e.g., MASP-2 inhibitory antibodies).
In some embodiments, the lung disease or disorder caused or exacerbated by fibrosis and/or inflammation is selected from the group consisting of: chronic obstructive pulmonary disease, cystic fibrosis, pulmonary fibrosis associated with scleroderma, bronchiectasis and pulmonary hypertension.
Heart and Vascular Diseases
A number of different cardiac and vascular pathologies are caused by a common fibrotic process. Excessive déposition of fïbrotic tissue in the heart results in cardiac pathology, în which the excess production of extracellular matrix proteins alter the structure, architecture, shape and affect the contractile function of the heart (Khan and Sheppard, Immunology 118: 10-24, 2006).
Studies indicate that fibrosis may contribute significantly to cardiac dysfunction in ischaemic, dilated and hypertrophie cardiomyopathy. For example, it has been demonstrated that patients with chronic atrial fibrillation were found to hâve higher levels of myocardial interstitial fibrosis as compared to Controls (Khan and Sheppard, Immunology 118: 10-24, 2006). As another example, it has been determined that most cases of arrhythmogenic right ventricular cardiomyopathy (ARVC) in the US exhibit fat infiltration and scarring (fibrofatty ARVC) (Burke et al., Circulation 97:1571-1580, 1998). In a study that examined the histopathologie characteristics of the ventricular myocardium in human subjects with ARVC it was determined that extensive fibrosis was present in biopsy specimens from pédiatrie patients with ARVC (Nishikawa T. et al., Cardiovascular Pathology vol 8 (4):185-189, 1999),
Accordingly, in certain embodiments, the disclosure provides a method of preventing, treating, reverting, inhibiting and/or reducing fibrosis and/or inflammation in a subject suffering from a cardiac or vascular disease or disorder caused or exacerbated by fibrosis and/or inflammation comprising administering a MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody, to a subject in need thereof. This method includes administering a composition comprising an amount of a MASP-2 inhibitor effective to inhibit cardiac and/or vascular fibrosis, and/or improve cardiac and/or vascular function.
In some embodiments, the disclosure provides a method of treating, inhibiting, preventing or ameliorating fibrosîs in a subject suffering from valvular fibrosis comprising administering a MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody to a subject in need thereof.
The MASP-2 inhibitory composition may be administered locally to the région of fibrosis, such as by local application of the composition during surgery or local injection, either directly or remotely, for example, by catheter. Aitemately, the MASP-2 inhibitory agent may be administered to the subject systemically, such as by intra-arterial, intravenous, intramuscular, inhalational, nasal, subcutaneous or other parentéral administration, or potentially by oral administration for non-peptidergic agents. Administration may be repeated as determined by a physician until the condition has been resolved or is controlled.
In certain embodiments, the MASP-2 inhibitory agents (e.g., MASP-2 inhibitory antibodies) are administered in combination with one or more agents or treatment modalitîes appropriate for the underlying heart disease, or vascular disease or condition.
In some embodiments, the cardiac or vascular disease or disorder caused or exacerbated by fibrosis and/or inflammation is selected from the group consisting of: cardiac fibrosis, myocardial infarction, atrial fibrosis, endomyocardial fibrosis arrhythmogenic right ventricular cardiomyopathy (ARVC), vascular disease, atherosclerotic vascular disease, vascular stenosis, restenosis, vasculitis, phlebitis, deep vein thrombosis and abdominal aortic aneurysm.
Chronic Infections Diseases
Chronic infectious diseases such as Hepatitis C and Hepatitis B cause tissue inflammation and fibrosis, and high lectin pathway activity may be detrimental. In such diseases, inhibitors of MASP-2 may be bénéficiai. For example, MBL and MASP-i levels are found to be a significant predictor of the severity of liver fibrosis in hepatitis C virus (HCV) infection (Brown et al., Clin Exp Immunol. 147(1):90-8, 2007; Saadanay et al., Arab J Gastroenterol. 12(2):68-73, 2011; Saeed et al., Clin Exp Immunol. 174(2):26573, 2013). MASP-1 has previously been shown to be a potent activator of MASP-2 and the lectin pathway (Megyeri et al., J Biol Chem. 29: 288(13):8922-34, 2013). Alphaviruses such as chikungunya virus and Ross River virus induce a strong host inflammatory response resulting in arthritis and myositis, and this pathology is mediated by MBL and the lectin pathway (Gunn et al., PLoS Pathog. 8(3):e 1002586, 2012).
Accordingly, in certain embodiments, the disclosure provides a method of preventing, treating, reverting, inhibiting and/or reducing fibrosis and/or inflammation în a subject suffering from, or having previously suffered from, a chronic înfectious disease that causes inflammation and/or fibrosis, comprising administering a MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody, to a subject in need thereof.
The MASP-2 inhibitory composition may be administered locally to the région of fibrosis, such as by local application of the composition during surgery or local injection, either dîrectly or remotely, for example, by cathéter. Altemately, the MASP-2 inhibitory agent may be administered to the subject systemically, such as by intra-arterial, intravenous, intramuscular, inhalational, nasal, subcutaneous or other parentéral administration, or potentially by oral administration for non-peptidergic agents. Administration may be repeated as determined by a physician until the condition has been resolved or is controlled.
In certain embodiments, the MASP-2 inhibitory agents (e.g., MASP-2 inhibitory antibodies) are administered in combination with one or more agents or treatment modalities appropriate for the underlying chronic înfectious disease.
In some embodiments, the chronic înfectious disease that causes inflammation and/or fibrosis is selected from the group consisting of: alpha virus, Hepatitis A, Hepatitis B, Hepatitis C, tuberculosis, HIV and influenza.
Autoimmune diseases:
Scleroderma is a chronic autoimmune disease characterized by fibrosis, vascular alterations, and autoantibodies. There are two major forms: limited systemic scleroderma and diffuse systemic scleroderma. The cutaneous symptoms of limited systemic scleroderma affect the hands, arms and face. Patients with this form of scleroderma frequently hâve one or more of the following complications: calcinosis, Raynaud's phenomenon, esophageal dysfonction, sclerodactyl, and telangiectasias. Diffuse systemic scleroderma is rapidly progressing and affects a large area of the skin and one or more internai organs, frequently the kidneys, esophagus, heart and/or lungs.
Scleroderma affects the small blood vessels known as artérioles, In ail organs. First, the endothélial cells of the arteriole die off apoptotically, along with smooth muscle cells. These cells are replaced by collagen and other fibrous material. Inflammatory cells, particularly CD4+ helper T cells, infiltrate the arteriole, and cause further damage.
The skin manifestations of scleroderma can be painful, can impair use of the affected area (e.g., use of the hands, fmgers, toes, feet, etc.) and can be disfiguring. Skin ulcération may occur, and such ulcers may be prone to infection or even gangrené. The ulcerated skin may be difïïcult or slow to heal. Difficulty in healing skin ulcérations may be particularly exacerbated in patients with împaired circulation, such as those with Raynaud's phenomenon. In certain embodiments, the methods of the present disclosure are used to treat scleroderma, for example skin symptoms of scleroderma. In certain embodiments, treating scleroderma comprises treating skin ulcération, such as digital ulcers. Administration of MASP-2 inhibitory agent such as anti-MASP-2 antibodies can be used to reduce the fibrotic and/or inflammatory symptoms of scleroderma in affected tissue and/or organs.
In addition to skin symptoms/manifestations, scleroderma may also affect the heart, kidney, lungs, joints, and digestive tract. In certain embodiments, treating scleroderma includes treating symptoms of the disease in any one or more of these tissues, such as by reducing fibrotic and/or inflammatory symptoms. Lung problems are amongst the most serious complications of scleroderma and are responsible for much of the mortality associated with the disease. The two prédominant lung conditions associated with scleroderma are pulmonary fibrosis and pulmonary hypertension. A patient with lung involvement may hâve either or both conditions. Lung fibrosis associated with scleroderma is one example of pulmonary fibrosis that can be treated with MASP-2 inhibitory agents. Scleroderma involving the lung causes scarring (pulmonary fibrosis). Such pulmonary fibrosis occurs in about 70% of scleroderma patients, although its progression is typically slow and symptoms vary widely across patients in terms of severity. For patients that do hâve symptoms associated with pulmonary fibrosis, the symptoms include a dry cough, shortness of breath, and reduced ability to exercise. About 16% of patients with some level of pulmonary fibrosis develop severe pulmonary fibrosis. Patients with severe pulmonary fibrosis expérience significant décliné in lung function and alveolitis.
In certain embodiments, the methods of the present disclosure are used to treat scleroderma, for example lung fibrosis associated with scleroderma. Administration of MASP-2 înhibitory agents, such as MASP-2 înhibitory antibodies can be used to reduce the fibrotic symptoms of scleroderma tn lung. For example, the methods can be used to improve lung function and/or to reduce the risk of death due to scleroderma.
Kidney involvement is also common in scleroderma patients. Rénal fibrosis associated with scleroderma is an example of rénal fibrosis that can be treated by administration of MASP-2 înhibitory agents, such as anti-MASP-2 antibodies. In certain embodiments, the methods of the present disclosure are used to treat scleroderma, for example kidney fibrosis associated with scleroderma. In one embodiment, administration of MASP-2 înhibitory antibodies can be used to reduce the fibrotic symptoms of scleroderma in kidney. For example, the methods can be used to improve kidney function, to reduce protein in the urine, to reduce hypertension, and/or to reduce the risk of rénal crisis that may lead to fatal rénal failure.
Systemic lupus erythematosus (SLE) is a chronic, inflammatory autoimmune disorder characterized by spontaneous B and T cell autoreactivity and multiorgan immune injury and may affect the skin, joints, kidneys, and other organs. Almost ail people with SLE hâve joint pain and most develop arthritis. Frequently affected joints are the fingers, hands, wrists, and knees. General symptoms of SLE include: arthritis; fatigue; general discomfort, uneasiness or îll feeling (malaise); joint pain and swelling; muscle aches; nausea and vomiting; and skin rash. Additionally symptoms may also include: abdominal pain; blood in the urine; fingers that change color upon pressure or in the cold; numbness and tîngling; and red spots on skin. In some patients, SLE has lung or kidney involvement. Without being bound by theory, inflammation and/or fibrosis in lung and kidney damages those organs and leads to symptoms associated with lung and/or kidney damage. In some cases, patients with SLE develop a particular kidney condition called lupus nephritis. In certain embodiments, the disclosure provides methods of treating SLE comprising admintsterîng an effective amount of a MASP-2 înhibitory agent such as an anti-MASP-2 antibody. Administering MASP-2 Înhibitory antibodies can be used to decrease one or more symptoms of SLE. In certain embodiments, administering antiMASP-2 antibodies is used to treat SLE in a patient with lupus nephritis. In such cases, treating SLE comprises treating lupus nephritis, such as by reducing symptoms of lupus nephritis. In certain embodiments, treating comprises treating the skin symptoms of SLE. In certain embodiments, treating comprises reducing one or more symptoms of lupus nephritis. In certain embodiments, treating comprises reducing, delaying or eliminating the need for dialysis. In certain embodiments, treating comprises reducing, delaying, or eliminating the need for kidney transplantation. In certain embodiments, treating comprises delaying or preventing progression of lupus nephritis to rénal failure or end stage rénal disease.
Lupus nephritis is an inflammation of the kidney, and is a severe complication of systemic lupus erythematosus (SLE). In the kidney, lupus nephritis can lead to debilitating loss of function. Patients with lupus nephritis may eventually develop kidney failure and require dialysis or kidney transplantation. Related complications that can also be treated using the methods of the disclosure include interstitiel nephritis and nephrotic syndrome. Symptoms of lupus nephritis include: blood in the urine, foamy appearance to urine, high blood pressure, protein in the urine, fluid rétention, and edema. Other symptoms include signs and symptoms of rénal fïbrosis and/or kidney failure. If left untreated, lupus nephritis may lead to kidney failure, and even end stage rénal disease.
Accordingly, in certain embodiments, the disclosure provides a method of preventing, treating, reverting, inhibiting and/or reducing fibrosis and/or inflammation in a subject suffering from an autoimmune disease that causes or exacerbâtes fïbrosis and/or inflammation comprising administering a MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody, to a subject in need thereof. This method includes administering a composition comprising an amount of a MASP-2 inhibitor effective to inhibit fïbrosis.
The MASP-2 inhibitory composition may be administered locally to the région of fïbrosis, such as by local application of the composition during surgery or local injection, either directly or remotely, for example, by catheter. Altemately, the MASP-2 inhibitory agent may be administered to the subject systemically, such as by intra-arterial, intravenous, intramuscular, inhalational, nasal, subcutaneous or other parentéral administration, or potentially by oral administration for non-peptidergic agents.
Administration may be repeated as determined by a physician until the condition has been resolved or is controlled.
In certain embodiments, the MASP-2 inhibitory agents (e.g., MASP-2 inhibitory antibodies) are administered in combination with one or more agents or treatment modalities appropriate for the underlying autoimmune disease.
In some embodiments, the autoimmune disease that causes or exacerbâtes fibrosis and/or inflammation is selected from the group consisting of: scleroderma and Systemic lupus erythematosus (SLE).
Central Nervous System Diseases and Conditions:
In certain embodiments, the disclosure provides a method of preventing, treating, reverting, inhibiting and/or reducing fibrosis and/or inflammation in a subject suffering from a disease or disorder of the central nervous system caused or exacerbated by fibrosis and/or inflammation comprising administering a MASP-2 inhibitory agent, such as an anti-MASP-2 antibody, to a subject in need thereof. This method includes administering a composition comprising an amount of a MASP-2 inhibitor effective to inhibit fibrosis and/or inflammation.
The MASP-2 inhibitory composition may be administered locally to the région of fibrosis, such as by local application of the composition during surgery or local injection, either directly or remotely, for example, by cathéter. Altemately, the MASP-2 inhibitory agent may be administered to the subject systemically, such as by întra-arterial, intravenous, intramuscular, inhalational, nasal, subcutaneous or other parentéral administration, or potentially by oral administration for non-peptidergic agents. Administration may be repeated as determined by a physician until the condition has been resolved or is controlled.
In certain embodiments, the MASP-2 inhibitory agents (e.g., MASP-2 inhibitory antibodies) are administered in combination with one or more agents or treatment modalities appropriate for the underlying disease or disorder of the central nervous System.
In some embodiments, the disease or disorder of the central nervous system caused or exacerbated by fibrosis and/or inflammation is selected from the group consisting of: stroke, traumatic brain injury and spinal cord injury.
Skin Diseases and Conditions
In certain embodiments, the disclosure provides a method of preventing. treating, reverting, inhibiting and/or reducing fibrosis and/or inflammation in a subject suffering from a skin disease or disorder caused or exacerbated by fibrosis and/or inflammation comprising administering a MASP-2 inhibîtory agent, such as a MASP-2 inhibîtory antibody, to a subject in need thereof. This method includes administering a composition comprising an amount of a MASP-2 inhibitor effective to inhibit fibrosis and/or inflammation.
The MASP-2 inhibîtory composition may be administered locally to the région of fibrosis, such as by local application of the composition to the skin, or local application during surgery or local injection, either directly or remotely, for example, by cathéter. Altemateiy, the MASP-2 inhibîtory agent may be administered to the subject systemically, such as by intra-arterial, întravenous, intramuscular, înhalational, nasal, subcutaneous or other parentéral administration, by topical administration, or potentially by oral administration for non-peptidergic agents. Administration may be repeated as determined by a physician until the condition has been resolved or is controlled.
In certain embodiments, the MASP-2 inhibitory agents (e.g., MASP-2 inhibitory antibodies) are administered in combination with one or more agents or treatment modalities appropriate for the underlying skin disease or disorder.
In some embodiments, the skin disease or disorder caused or exacerbated by fibrosis and/or inflammation is selected from the group consisting of: skin fibrosis, wound healing, scleroderma, systemic sclerosis, keloids, connective tissue diseases, scarring, and hypertrophie scars.
Musculoskeletal Bone and Soft-Tissue Disorders and Conditions
In certain embodiments, the disclosure provides a method of preventing, treating, reverting, inhibiting and/or reducing fibrosis and/or inflammation in a subject suffering from a bone or soft-tissue disease or disorder caused or exacerbated by fibrosis and/or inflammation comprising administering a MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody, to a subject in need thereof. This method includes administering a composition comprising an amount of a MASP-2 inhibitor effective to inhibit fibrosis and/or inflammation.
The MASP-2 inhibitory composition may be administered locally to the région of fibrosis, such as by local application of the composition to the bone or soft-tissue structure, or local application during surgery or local injection, either directly or remotely, for example, by cathéter. Altemately, the MASP-2 inhibitory agent may be administered to the subject systemically, such as by intra-arterial, intravenous, intramuscular, inhalational, nasal, subcutaneous or other parentéral administration, by topical administration, or potentially by oral administration for non-peptidergic agents. Administration may be repeated as determined by a physician until the condition has been resoived or is controlled.
In certain embodiments, the MASP-2 inhibitory agents (e.g., MASP-2 inhibitory antibodies) are administered in combination with one or more agents or treatment modalities appropriate for the underlying bone or soft-tissue disease or disorder.
In some embodiments, the bone or soft-tissue disease or disorder caused or exacerbated by fibrosis and/or inflammation is selected from the group consisting of: osteoporosis and/or osteopenia associated with, for example, cystic fibrosis, myelodysplastîc conditions with increased bone fibrosis, adhesive capsulitis, Dupuytren’s contracture and myelofibrosis.
Joint Diseases and Conditions
In certain embodiments, the disclosure provides a method of preventing, treating, reverting, inhibiting and/or reducing fibrosis and/or inflammation in a subject suffering from a joint disease or disorder caused or exacerbated by fibrosis and/or inflammation comprising administering a MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody, to a subject în need thereof. This method includes administering a composition comprising an amount of a MASP-2 inhibitor effective to inhibit fibrosis and/or inflammation.
The MASP-2 inhibitory composition may be administered locally to the région of fibrosis, such as by local application of the composition to the joint, or local application during surgery or local injection, either directly or remotely, for example, by cathéter. Altemateiy, the MASP-2 inhibitory agent may be administered to the subject systemically, such as by intra-arterial, întravenous, intramuscular, inhalational, nasal, subcutaneous or other parentéral administration, by topical administration, or potentially by oral administration for non-peptidergic agents. Administration may be repeated as determined by a physician until the condition has been resolved or is controlled.
In certain embodiments, the MASP-2 inhibitory agents (e.g., MASP-2 inhibitory antibodies) are administered in combination with one or more agents or treatment modalities appropriate for the underlying joint disease or disorder.
In some embodiments, the joint disease or disorder caused or exacerbated by fibrosis and/or inflammation is arthrofibrosis.
Digestive Diseases and Conditions
In certain embodiments, the disclosure provides a method of preventing, treating, reverting, inhibiting and/or reducing fibrosis and/or inflammation in a subject suffering from a digestive disease or disorder caused or exacerbated by fibrosis and/or inflammation comprising admînistering a MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody, to a subject in need thereof. This method includes admînistering a composition comprising an amount of a MASP-2 inhibitor effective to inhibit fibrosis and/or inflammation.
The MASP-2 inhibitory composition may be administered locally to the région of fibrosis, such as by local application during surgery or local injection, either directly or remotely, for example, by cathéter. Altemateiy, the MASP-2 inhibitory agent may be administered to the subject systemically, such as by intra-arterial, întravenous, intramuscular, inhalational, nasal, subcutaneous or other parentéral administration, by topical administration, or potentially by oral administration for non-peptidergic agents. Administration may be repeated as determined by a physician until the condition has been resolved or is controlled.
In certain embodiments, the MASP-2 inhibitory agents (e.g., MASP-2 inhibitory antibodies) are administered in combination with one or more agents or treatment modalities appropriate for the underlying digestive disease or disorder.
In some embodiments, the digestive disease or disorder caused or exacerbated by fîbrosis and/or inflammation is selected from the group consisting of: Crohn’s disease, ulcerative colitis and pancreatic fibrosis.
Ocular Diseases and Conditions
In certain embodiments, the disclosure provides a method of preventing, treating, reverting, inhibiting and/or reducing fibrosis and/or inflammation in a subject suffering from an ocular disease or disorder caused or exacerbated by fibrosis and/or inflammation comprising administering a MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody, to a subject in need thereof. This method includes administering a composition comprising an amount of a MASP-2 inhibitor effective to inhibit fibrosis and/or inflammation.
The MASP-2 inhibitory composition may be administered locally to the région of fibrosis, such as by local application during surgery or local injection, either directly or remotely, for example, by cathéter. Altemately, the MASP-2 inhibitory agent may be administered to the subject systemically, such as by intra-arterial, întravenous, intramuscular, inhalational, nasal, subcutaneous or other parentéral administration, by topical administration to the eye (e.g., as eye drops), or potentially by oral administration for non-peptidergic agents. Administration may be repeated as determined by a physician until the condition has been resolved or is controlled.
in certain embodiments, the MASP-2 inhibitory agents (e.g., MASP-2 inhibitory antibodies) are administered in combination with one or more agents or treatment modalities appropriate for the underlying ocular disease or disorder.
In some embodiments, the ocular disease or disorder caused or exacerbated by fîbrosis and/or inflammation is selected from the group consisting of: anterior subcapsular cataract, posterior capsule opacification, macular degeneration, and retinal and vitreal retinopathy.
Diseases and Conditions of the Reproductive Organs
In certain embodiments, the disclosure provides a method of preventing, treating, reverting, inhibiting and/or reducing fibrosis and/or inflammation in a subject suffering from a reproductive disease or disorder caused or exacerbated by fibrosis and/or inflammation comprising administering a MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody, to a subject in need thereof. This method includes administering a composition comprising an amount of a MASP-2 inhibitor effective to inhibit fibrosis and/or inflammation.
The MASP-2 inhibitory composition may be administered locally to the région of fibrosis, such as by local application during surgery or local injection, either directly or remotely, for example, by cathéter. Altemately, the MASP-2 inhibitory agent may be administered to the subject systemically, such as by intra-arterial, intravenous, intramuscular, inhalational, nasal, subcutaneous or other parentéral administration, by topical administration, or potentially by oral administration for non-peptidergic agents. Administration may be repeated as determined by a physician until the condition has been resolved or is controlled.
In certain embodiments, the MASP-2 inhibitory agents (e.g., MASP-2 inhibitory antibodies) are administered in combination with one or more agents or treatment modalities appropriate for the underlying reproductive disease or disorder.
In some embodiments, the reproductive disease or disorder caused or exacerbated by fibrosis and/or inflammation is selected from the group consisting of: endometriosis and Peyronies disease.
Scarring Associated with Trauma
In certain embodiments, the disclosure provides a method of preventing, treating, reverting, inhibiting and/or reducing fibrosis and/or inflammation in a subject suffering from a disease or condition resulting from scarring associated with trauma comprising
6I administering a MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody, to a subject in need thereof. This method includes administering a composition comprising an amount of a MASP-2 înhibitor effective to inhibit fîbrosis and/or inflammation.
The MASP-2 inhibitory composition may be administered locally to the région of fîbrosis, such as by local application during surgery or local injection, either directly or remotely, for example, by cathéter. Altemately, the MASP-2 inhibitory agent may be administered to the subject systemically, such as by intra-arterial, intravenous, intramuscular, înhaiational, nasal, subcutaneous or other parentéral administration, by topical administration, or potentialiy by oral administration for non-peptidergic agents. Administration may be repeated as determined by a physician until the condition has been resolved or is controlled.
In certain embodiments, the MASP-2 inhibitory agents (e.g., MASP-2 inhibitory antibodies) are administered in combination with one or more agents or treatment modalities appropriate for the underlying disease or disorder.
In some embodiments, the scarring associated with trauma is selected from the group consisting of: surgical complications (e.g., surgîcal adhesions wherein scar tissue can form between internai organs causing contracture, pain and can cause infertilîty), chemotherapeutic drug-induced fîbrosis, radiation-induced fîbrosis and scarring associated with bums.
Additional Diseases and Disorders caused or exacerbated by fîbrosis and/or inflammation
In certain embodiments, the disclosure provides a method of preventing, treating, reverting, inhibiting and/or reducing fîbrosis and/or inflammation în a subject suffering from a disease or disorder caused or exacerbated by fîbrosis and/or inflammation selected from the group consisting of organ transplant, breast fîbrosis, muscle fîbrosis, rétropéritonéal fîbrosis, thyroid fîbrosis, lymph node fîbrosis, bladder fîbrosis and pleural fîbrosis, comprising administering a MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody, to a subject in need thereof. This method includes administering a composition comprising an amount of a MASP-2 inhibitor effective to inhibit fibrosis and/or inflammation.
The MASP-2 inhibitory composition may be administered locally to the région of fibrosis, such as by local application during surgery or local injection, either directly or remotely, for example, by cathéter. Altemately, the MASP-2 inhibitory agent may be administered to the subject systemically, such as by intra-arterial, intravenous, intramuscular, inhalational, nasal, subeutaneous or other parentéral administration, by topical administration to the eye (e.g,, as eye drops), or potentially by oral administration for non-peptidergic agents. Administration may be repeated as determined by a physician until the condition has been resolved or is controlled.
In certain embodiments, the MASP-2 inhibitory agents (e.g., MASP-2 inhibitory antibodies) are administered in combination with one or more agents or treatment modalities appropriate for the underlying disease or disorder.
In certain embodiments of any of the various methods and pharmaceutical compositions described herein, the MASP-2 inhibitory antibody selectively blocks the lectin pathway while leaving intact the classical pathway.
IV. MASP-2 INHIBITORY AGENTS
In various aspects, the present invention provides methods of inhibiting the adverse effects of fibrosis and/or inflammation comprising adminîstering a MASP-2 inhibitory agent to a subject in need thereof. MASP-2 inhibitory agents are administered in an amount effective to inhibit MASP-2-dependent complément activation in a living subject. In the practice of this aspect of the invention, représentative MASP-2 inhibitory agents include: molécules that inhibit the biological activity of MASP-2 (such as small molécule inhibitors, anti-MASP-2 antibodies (e.g., MASP-2 inhibitory antibodies) or blocking peptides which interact with MASP-2 or interféré with a protein-proteîn interaction), and molécules that decrease the expression of MASP-2 (such as MASP-2 antisense nucleic acid molécules, MASP-2 spécifie RNAi molécules and MASP-2 ribozymes), thereby preventing MASP-2 from activating the lectin complément pathway. The MASP-2 inhibitory agents can be used alone as a primary therapy or in combination with other therapeutics as an adjuvant therapy to enhance the therapeutic benefits of other medical treatments.
The inhibition of MASP-2-dependent complément activation is characterized by at least one of the following changes in a component of the complément system that occurs as a resuit of administration of a MASP-2 inhibitory agent in accordance with the methods of the invention: the inhibition of the génération or production of MASP-2-dependent complément activation system products C4b, C3a, C5a and/or C5b-9 (MAC) (measured, for example, as described în Example 2), the réduction of C4 cleavage and C4b déposition (measured, for example as described in Example 2), or the réduction of C3 cleavage and C3b déposition (measured, for example, as described in Example 2).
According to the present invention, MASP-2 inhibitory agents are utilized that are effective in inhibiting fibrosis and/or inflammation, and exhibit a détectable antifibrotic activity and/or induce a decrease of fibrosis. Within the context of the invention, an antifibrotic activity may comprise at least one or more of the following: (l) réduction in inflammation, for example, as assessed by activation and recruitment of macrophages and endothélial cells; recruitment and activation of lymphocytes and/or eosinophils via sécrétion of a number of cytokines/chemokines; release of cytotoxic mediators and fibrogenîc cytokines; (2) réduction of cell prolifération, ECM synthesis or angiogenesis, and/or (3) réduction in collagen déposition, as compared to the fibrotic activity in the absence of the MASP-2 inhibitory agent.
Assessment of an antifibrotic agent, such as a MASP-2 inhibitory agent, may be detected using any technique known to the skilled person. For example, assessment of an antifibrotic agent may be assessed in a UUO model (as described in Examples 12 and 14 herein). If a détectable antifibrotic activity and/or a réduction or decrease of fibrosis is assessed using a MASP-2 inhibitory agent, such MASP-2 inhibitory agent is said to be used as a médicament for preventing, treating, reverting, and/or inhibiting fibrosis.
The assessment of fibrosis may be carried out periodically, e.g., each week, or each month. The increase/decrease of fibrosis and/or presence of an antifibrotic activity may therefore be assessed periodically, e.g. each week, or month. This assessment is preferably carried out at several time points for a given subject or at one or several time points for a given subject and a healthy control. The assessment may be carried out at regular time intervals, e.g. each week, or each month. The assessment may therefore be assessed regularly, e.g. each week, or each month. When one assessment has led to the fmding of a decrease of fibrosîs or to the presence of an antifibrotic activity, a MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody, is said is exhibit a détectable antifibrotic activity and/or inducing a réduction or decrease of fibrosîs.
MASP-2 inhibitory agents useful in the practice of this aspect of the invention include, for example, MASP-2 antibodies and fragments thereof, MASP-2 inhibitory peptides, small molécules, MASP-2 soluble receptors and expression inhibitors. MASP-2 inhibitory agents may inhibit the MASP-2-dependent complément activation system by blocking the biological function of MASP-2. For example, an inhibitory agent may effectively block MASP-2 protein-to-protein interactions, interfère with MASP-2 dimerization or assembly, block Ca2+ binding, interfère with the MASP-2 serine protease active site, or may reduce MASP-2 protein expression.
In some embodiments, the MASP-2 inhibitory agents selectively inhibit MASP-2 complément activation, leavîng the Clq-dependent complément activation system functionally intact.
In one embodiment, a MASP-2 inhibitory agent useful in the methods of the invention is a spécifie MASP-2 inhibitory agent that specifically binds to a polypeptide comprising SEQ ID NO:6 with an affinity of at least ten times greater than to other antigens in the complément system. In another embodiment, a MASP-2 inhibitory agent specifically binds to a polypeptide comprising SEQ ID NO:6 with a binding affinity of at least 100 times greater than to other antigens in the complément system. In one embodiment, the MASP-2 inhibitory agent specifically binds to at least one of (i) the CCP1-CCP2 domain (aa 300-431 of SEQ ID NO:6) or the serine protease domain of MASP-2 (aa 445-682 of SEQ ID NO:6) and inhibits MASP-2-dependent complément activation. In one embodiment, the MASP-2 inhibitory agent is a MASP-2 monoclonal antibody, or fragment thereof that specifically binds to MASP-2. The binding affinity of the MASP-2 inhibitory agent can be determined using a suitable binding assay.
The MASP-2 polypeptide exhibits a molecular structure similar to MASP-1, MASP-3, and Clr and Cls, the proteases of the Cl complément system. The cDNA molécule set forth in SEQ ID NO:4 encodes a représentative example of MASP-2 (consisting of the amino acid sequence set forth in SEQ ID NO:5) and provides the human MASP-2 polypeptide with a leader sequence (aa 1-15) that is cleaved after sécrétion, resulting in the mature form of human MASP-2 (SEQ ID NO:6). As shown in FIGURE 2, the human MASP 2 gene encompasses twelve exons. The human MASP-2 cDNA is encoded by exons B, C, D, F, G, H, I, J, K AND L. An alternative splice results in a 20 kDa protein termed MBL-associated protein 19 (MApl9, also referred to as sMAP) (SEQ ID NO:2), encoded by (SEQ ID NO:l) arising from exons B, C, D and E as shown in FIGURE 2. The cDNA molécule set forth in SEQ ID NO:50 encodes the murine MASP-2 (consisting of the amino acid sequence set forth in SEQ ID NO:5l) and provides the murine MASP-2 polypeptide with a leader sequence that is cleaved after sécrétion, resulting in the mature form of murine MASP-2 (SEQ ID NO:52). The cDNA molécule set forth in SEQ ID NO:53 encodes the rat MASP-2 (consisting of the amino acid sequence set forth in SEQ ID NO:54) and provides the rat MASP-2 polypeptide with a leader sequence that ts cleaved after sécrétion, resulting in the mature form of rat MASP-2 (SEQ ID NO:55).
Those skilled in the art will recognize that the sequences disclosed in SEQ ID NO:4, SEQ ID NO;50 and SEQ ID NO:53 represent single alieles of human, murine and rat MASP-2 respectively, and that allelic variation and alternative splicing are expected to occur. Allelic variants of the nucléotide sequences shown in SEQ ID NO:4, SEQ ID NO:50 and SEQ ID NO:53, including those containing silent mutations and those in which mutations resuit in amino acid sequence changes, are within the scope of the présent invention. Allelic variants of the MASP-2 sequence can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures.
The domains of the human MASP-2 protein (SEQ ID NO:6) are shown in FIGURE 1 and 2A and include an N-terminal Clr/Cls/sea urchîn Vegfrbone morphogenic protein (CUBI) domain (aa 1-121 of SEQ ID NO:6). an epidermal growth factor-like domain (aa 122-166), a second CUBI domain (aa 167-293), as well as a tandem of complément control protein domains and a serine protease domain. Alternative splicing of the MASP 2 gene results in MApl9 shown in FIGURE 1. MApl9 is a nonenzymatic protein containing the N-terminal CUBI-EGF région of MASP-2 with four additional residues (EQSL) derived from exon E as shown in FIGURE 1.
Several proteins hâve been shown to bind to, or interact with MASP-2 through proiein-to-protein interactions. For example, MASP-2 is known to bind to, and form Ca-+ dépendent complexes with, the lectin proteins MBL, H-ficolin and L-ficolin. Each MASP-2/lectin complex has been shown to activate complément through the MASP-2-dependent cleavage of proteins C4 and C2 (Ikeda, K., et al., J. Biol. Chem. 262:7451-7454, 1987; Matsushita, M., étal., J. Exp. Med. 776:1497-2284, 2000; Matsushita, M., et al., J. Immunol. 765:3502-3506, 2002). Studies hâve shown that the CUBI-EGF domains of MASP-2 are essentîal for the association of MASP-2 with MBL (Thielens, N.M., étal., J. Immunol. 766:5068, 2001). It has also been shown that the CUBIEGFCUBII domains médiate dimerization of MASP-2, which is required for formation of 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 to or interfère with MASP-2 target régions known to be important for MASP-2-dependent complément activation.
ANTI-MASP-2 ANTIBODIES
In some embodiments of this aspect of the invention, the MASP-2 inhibitory agent comprises an anti-MASP-2 antibody that inhibits the MASP-2-dependent complément activation system. The anti-MASP-2 antibodies useful În this aspect of the invention include polyclonal, monoclonal or recombinant antibodies derived from any antibody producing mammal and may be multispecific, chimeric, humanized, anti-idiotype, and antibody fragments. Antibody fragments include Fab, Fab', F(ab)2, F(ab')2, Fv fragments, scFv fragments and single-chain antibodies as further described herein.
MASP-2 antibodies can be screened for the ability to inhibit MASP-2-dependent complément activation system and for antifibrotic activity and/or the ability to inhibit rénal damage associated with proteinuria or Adriamycin-induced nephropathy using the assays described herein. Several MASP-2 antibodies hâve been described in the literature and some hâve been newly generated, some of which are listed below in TABLE 1. For example, as described in Examples 10 and 11 herein, anti-MASP-2 Fab2 antibodies hâve been identified that block MASP-2-dependent complément activation. As described în Example 12, and also described in WO2012/151481, which is hereby incorporated herein by reference, fully human MASP-2 scFv antibodies (e.g., OMS646) hâve been identified that block MASP-2-dependent complément activation. As described in Example 13, and also described in WO2014/144542, which is hereby incorporated herein by reference, SGMI-2 peptide-bearing MASP-2 antibodies and fragments thereof with MASP-2 inhibitory activity were generated by fusing the SGM1-2 peptide amino acid sequence (SEQ ID NO:72, 73 or 74) onto the amino or carboxy termini of the heavy and/or light chains of a human MASP-2 antibody (e.g., OMS646-SGM1-2).
Accordingly, in one embodiment, the MASP-2 inhibitory agent for use in the methods of the invention comprises a human antibody such as, for example OMS646. Accordingly, in one embodiment, a MASP-2 inhibitory agent for use in the compositions and methods of the claimed invention comprises a human antibody that binds a polypeptide consisting of human MASP-2 (SEQ ID NO:6), wherein the antibody comprises: (I) (a) a heavy-chain variable région comprising: i) a heavy-chain CDR-Hl comprising the amino acid sequence from 31-35 of SEQ ID NO:67; and ii) a heavy-chain CDR-H2 comprising the amino acid sequence from 50-65 of SEQ ID NO:67; and iii) a heavy-chain CDR-H3 comprising the amino acid sequence from 95-I07 of SEQ ID NO:67 and b) a light-chain variable région comprising: i) a light-chain CDR-Ll comprising the amino acid sequence from 24-34 of SEQ ID NO:70; and Π) a light-chain CDR-L2 comprising the amino acid sequence from 50-56 of SEQ ID NO:70; and iii) a light-chain CDR-L3 comprising the amino acid sequence from 89-97 of SEQ ID NO:70, or (II) a variant thereof comprising a heavy-chain variable région 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 to SEQ ID NO:67) and a light-chain variable région with at least 90% identity (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 to 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 antigen binding fragment thereof, comprising a heavy-chain variable région comprising the amino acid sequence set forth as SEQ ID NO:67 and a light-chain variable région comprising the amino acid sequence set forth as SEQ ID NO:70.
In some embodiments, the method comprises administering to the subject a composition comprising a MASP-2 inhibitory antibody, or antigen binding fragment thereof, that specifically recognizes at least part of an epitope on human MASP-2 recognized by reference antibody OMS646 comprising a heavy-chain variable région as 5 set forth in SEQ ID NO:67 and a light-chaîn variable région as set forth in SEQ ID NO:70. In one embodiment, the MASP-2 inhibitory agent for use in the methods of the invention comprises the human antibody OMS646.
TABLE l : EXEMPLARY MASP-2 SPECIFIC AN TIBODIES
ANTIGEN ANTIBODY TYPE REFERENCE
Recombinant MASP-2 Rat Polyclonal Peterson, S.V., et ah, Mol. Immunol. 37:803-811, 2000
Recombinant human CCPl/2-SP fragment (MoAb 8B5) Rat MoAb (subclass IgG l ) Moller-Kristensen, M., et al., J, of Immunol. Methods 282:159-167, 2003
Recombinant human MApl9(MoAb 6G12) (cross reacts with MASP-2) Rat MoAb (subclass IgG l ) Moller-Kristensen, M., et al., J. of Immunol. Methods 282:159-167, 2003
hMASP-2 Mouse MoAb (S/P) Mouse MoAb (N-term) Peterson, S.V., et al., Mol. Immunol. 35:409, April 1998
hMASP-2 (CCP1-CCP2-SP domain rat MoAb: NimoablOl, produced by hybridoma cell line 03050904 (ECACC) WO 2004/106384
hMASP-2 (fuli length-his tagged) murine MoAbs: NimoAbl04, produced by hybridoma cell line M0545YM035 (DSMZ) NimoAbl08, produced by hybridoma cell line M0545YM029 (DSMZ) NimoAbI09 produced by hybridoma cell line' M0545YM046 (DSMZ) NimoAbl 10 produced by hybridoma cell line M0545YM048 (DSMZ) WO 2004/106384
ANTIGEN ANTIBODY TYPE REFERENCE
Rat MASP-2 (fulllength) MASP-2 Fab2 antibody fragments Example 10
hMASP-2 (fulllength) Fully human scFv clones Example 12 and WO2012/151481
hMASP-2 (fulllength) SGMI-2 peptide bearing MASP-2 antibodies Example 13 and WO2014/144542
ANTI-MASP-2 ANTIBODIES WITH REDUCED EFFECTOR FUNCTION
In some embodiments of this aspect of the invention, the anti-MASP-2 antibodies hâve reduced effector function in order to reduce inflammation that may arise from the activation of the classical complément pathway. The ability of IgG molécules to trigger the classical complément pathway has been shown to résidé within the Fc portion of the molécule (Duncan, A.R., étal., Nature 332:138-740 1988). IgG molécules in which the Fc portion of the molécule has been removed by enzymatic cleavage are devoid of this effector function (see Harlow, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988). Accordingly, antibodies with reduced effector function can be generated as the resuit of lacking the Fc portion of the molécule by having a genetically engineered Fc sequence that minimizes effector function, or being of either the human IgGj or IgGq isotype.
Antibodies with reduced effector function can be produced by standard molecular biologîcal manipulation of the Fc portion of the IgG heavy chains as described herein and also described in Jelliffe étal., Int'l Rev. immunol. /0:241-250, 1993, and Rodrigues et al., J. Immunol. /57:6954-6961, 1998. Antibodies with reduced effector function also include human IgG2 and IgG4 isotypes that hâve a reduced ability to activate complément 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. 1 -/5:3062-3071, 1992; van de Winkel, J.G., étal., Immunol. Today /4:215-221, 1993). Humanized or fully human antibodies spécifie to human MASP-2 comprised of IgG2 or IgG4 isotypes can be produced by one of several methods known to one of ordinary skilled in the art, as described în Vaughan, T.J., et al., Nature Biotechnical /6:535-539, 1998.
PRODUCTION OF ANTI-MASP-2 ANTIBODIES
Anti-MASP-2 antibodies can be produced using MASP-2 polypeptides (e.g., full length MASP-2) or using antigenic MASP-2 epitope-bearing peptides (e.g., a portion of the MASP-2 polypeptide). Immunogenic peptides may be as small as five amino acid residues. For example, the MASP-2 polypeptide including the entire amino acid sequence of SEQ ID NO:6 may be used to induce anti-MASP-2 antibodies useful in the method of the invention. Particular MASP-2 domains known to be involved in protein-protein interactions, such as the CUBI, and CUBIEGF domains, as well as the région encompassing the serine-protease active site, may 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 the MASP-2 polypeptide (SEQ ID NO:6) are also useful to induce MASP-2 antibodies. Additional examples of MASP-2 derived antigens useful to induce MASP-2 antibodies are provided below in TABLE 2. The MASP-2 peptides and polypeptides used to raise antibodies may be isolated as natural polypeptides, or recombinant or synthetic peptides and catalytically inactive recombinant polypeptides, such as MASP-2A, as further described herein. In some embodiments of this aspect of the invention, anti-MASP-2 antibodies are obtained using a transgenic mouse strain as described herein.
Antigens useful for producing anti-MASP-2 antibodies also include fusion polypeptides, such as fusions of MASP-2 or a portion thereof with an immunoglobulin polypeptide or with maltose-binding protein. The polypeptide immunogen may be a full-length molécule or a portion thereof. If the polypeptide portion is hapten-like, such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine sérum albumin (BSA) or tetanus toxoid) for immunization.
TABLE 2: MASP-2 DERIVED ANTIGENS
SEQ ID NO: Amino Acid Sequence
SEQ ID NO:6 Human MASP-2 protein
SEQ ID NO:5l Murine MASP-2 protein
SEQ ID NO:8 CUBI domain of human MASP-2 (aa 1-121 ofSEQ IDNO:6)
7l
SEQ ID NO: Amino Acid Sequence
SEQ ID NO:9 CUBIEGF domains of human MASP-2 (aa 1-166 of SEQID NO:6)
SEQ ID NO:lO CUBIEGFCUBII domains of human MASP-2 (aa I-293 ofSEQ IDNO:6)
SEQID NO; H EGF domain of human MASP-2 (aa 122-166 ofSEQ ID NO:6)
SEQ IDNO:l2 Serine-Protease domain of human MASP-2 (aa429-67I ofSEQ IDN0:6)
SEQ ID NO:l3 GKDSCRGDAGGALVFL Serine-Protease inactivated mutant form (aa 610-625 ofSEQ ID NO:6 with mutated Ser 618)
SEQ ID NO:l4 TPLGPKWPEPVFGRL Human CUBI peptide
SEQ ID NO:I5: TAPPGYRLRLYFTHFDLEL SHLCEYDFVKLSSGAKVL ATLCGQ Human CUBI peptide
SEQ ID NO:I6: TFRSDYSN MBL binding région in human CUBI domain
SEQ 1DNO:17: FYSLGSSLDITFRSDYSNEK PFTGF MBL binding région in human CUBI domain
SEQ IDNO:18 IDECQVAPG EGF peptide
SEQ IDN0:I9 ANMLCAGLESGGKDSCRG DSGGALV Peptide from serine-protease active site
POLYCLONAL ANTIBODIES
Polyclonal antibodies against MASP-2 can be prepared by immunizing an animal with MASP-2 polypeptide or an immunogenic portion thereof using methods well known 5 to those of ordinary skill in the art. See, for example, Green étal., Production of Polyclonal Antisera, in Immunochemical Protocols (Manson, ed.), page 105. The immunogenicity of a MASP-2 polypeptide can be increased through the use of an adjuvant, including minerai gels, such as aluminum hydroxide or Freund's adjuvant (complété or incomplète), surface active substances such as lysolecithin, pluronic polyols.
polyanions, oil émulsions, keyhole limpet hemocyanin and dinîtrophenol. Polyclonal antibodies are typically raîsed in animais such as horses, cows, dogs, chicken, rats, mice, rabbits, guinea pigs, goats, or sheep. Altematively, an anti-MASP-2 antibody useful in the present invention may also be derived from a subhuman primate. General techniques for raising diagnostically and therapeutically useful antibodies in baboons may be found, for example, in Goldenberg et al., International Patent Publication No. WO 9l/l 1465, and in Losman, M.J., étal., Int. J. Cancer 46:310, 1990. Sera containing immunologically active antibodies are then produced from the blood of such immunized animais using standard procedures well known in the art.
MONOCLONAL ANTIBODIES
In some embodiments, the MASP-2 inhibitory agent is an anti-MASP-2 monoclonal antibody. Anti-MASP-2 monoclonal antibodies are highly spécifie, being directed against a single MASP-2 epîtope. As used herein, the modifier monoclonal indicates the character of the antibody as being obtained from a substantially homogenous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. Monoclonal antibodies can be obtained using any technique that provides for the production of antibody molécules by continuous cell lines în culture, such as the hybridoma method described by Kohler, G., et al., Nature 256:495, 1975, or they may be rnade by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567 to Cabilly). Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques 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 can 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 (e.g., a BALB/c mouse) with a composition comprising a MASP-2 polypeptide or portion thereof. After a predetermined period of time, splénocytes are removed from the mouse and suspended in a cell culture medium. The splénocytes are then fused with an immortal cell line to form a hybridoma. The formed hybridomas are grown in cell culture and screened for their ability to produce a monoclonal antibody against MASP-2. Examples further describing the production of anti-MASP-2 monoclonal antibodies are provided herein (see also Current Protocols in Immunology, Vol. L, John Wiley & Sons, pages2.5.1-2.6.7, I99l.)
Human monoclonal antibodies may be obtained through the use of transgenic mtce that hâve been engineered to produce spécifie human antibodies in response to antigenic challenge. In this technique, éléments of the human immunoglobulin heavy and light chain locus are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous immunoglobulin heavy chain and light chain loci. The transgenic mice can synthesize human antibodies spécifie for human antigens, such as the MASP-2 antigens described herein, and the mice can be used to produce human MASP-2 antibody-secreting hybridomas by fusing B-cells from such animais to suitable myeloma cell lines using conventîonal Kohier-Milstein technology as further described herein. Transgenic mice with a human immunoglobulin genome are commercially available (e.g., from Abgenix, Inc., Fremont, CA, and Medarex, Inc., Annandale, N.J.). Methods for obtaining human antibodies from transgenic mice are described, for example, by Green, L.L., étal., Nature Genei. 7:13, 1994; Lonberg, N., et al., Nature 365:856, 1994; and Taylor, L.D., et al., Int. Immun. 6:579, 1994.
Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of weil-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion 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; Baînes et al., Purification of Immunoglobulin G (IgG), in Methods in Molecular Biology, The Humana Press, Inc., VoL 10, pages 79-104, 1992).
Once produced, polyclonal, monoclonal or phage-derived antibodies are first tested for spécifie MASP-2 binding. A variety of assays known to those skilled in the art may be utilized to detect antibodies which specifically bind to MASP-2. Exemplary assays include Western blot or immunoprécipitation analysis by standard methods (e.g., as described in Ausubel et al.), immunoelectrophoresis, enzyme-linked immuno-sorbent assays, dot blots, inhibition or compétition assays and sandwich assays (as described in Harlow and Land, Antibodies: A Laboralory Manual, Cold Spring Harbor Laboratory Press, 1988). Once antibodies are identified that specifically bind to MASP-2, the anti-MASP-2 antibodies are tested for the ability to function as a MASP-2 inhibitory agent in one of several assays such as, for example, a lectln-specific C4 cleavage assay (described in Example 2), a C3b déposition assay (described in Example 2) or a C4b déposition assay (described in Example 2).
The affinity of anti-MASP-2 monoclonal antibodies can be readily determined by one of ordinary skill in the art (see, e.g., Scatchard, A., NY Acad. Sci. 51:660-612, 1949). In one embodiment, the anti-MASP-2 monoclonal antibodies useful for the methods of the invention bind to MASP-2 with a binding affinity of <100 nM, preferably <10 nM and most preferably <2 nM.
CHIMERIC/HUMAN1ZED 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 with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies (U.S. Patent No. 4,816,567, to Cabilly; and Morrison, S.L., étal., Proc. Nat'l Acad. Sci. USA 57:6851-6855, 1984).
One form of a chimeric antibody useful in the invention is a humanized monoclonal anti-MASP-2 antibody. Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies, which contain minimal sequence derived from non-human immunoglobulin. Humanized monoclonal antibodies are produced by transferring the non-human (e.g., mouse) complementarity determining régions (CDR), from the heavy and light variable chains of the mouse immunoglobulin into a human variable domain. Typically, residues of human antibodies are then substituted in the Framework régions of the non-human counterparts. Furthermore, humanized antibodies may comprise residues that are not found in the récipient antibody or in the donor antibody. These modifications are made to further refîne antibody performance. In general, the humanized antibody will comprise substantially ail of at least one, and typically two variable domains, in which ail or substantially ail of the hypervariable loops correspond to those of a non-human immunoglobulin and ail or substantially ail of the Fv Framework régions are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulîn constant région (Fc), typically that of a human immunoglobulîn. For further details, see Jones, P.T., et al., Nature 321:522-525, 1986; Reichmann, L., et al., Nature 332:323-329, 1988; and Presta. Curr, Op. Struct. Biol. 2:593-596, 1992.
The humanîzed antibodies useful in the invention include human monoclonal antibodies including at least a MASP-2 binding CDRH3 région. In addition, the Fc portions may be replaced so as to produce IgA or IgM as well as human IgG antibodies. Such humanîzed antibodies will hâve particular clinical utility because they will specifically recognize human MASP-2 but will not evoke an immune response in humans against the antibody îtself. Consequently, they are better suited for in vivo administration in humans, especially when repeated or long-term administration is necessary.
An example of the génération of a humanîzed anti-MASP-2 antibody from a murine anti-MASP-2 monoclonal antibody is provided herein in Example 6. Techniques for producing humanîzed monoclonal antibodies are also described, for example, by Jones, P.T., étal., Nature 321:522, 1986; Carter, P., étal., Proc. Nat'l. Acad. Sci. USA 39:4285, 1992; Sandhu, J.S., Crit. Rev. Biotech. 12:437, 1992; Singer, LL, et al., J. Immun. /5(7:2844, 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 by U.S. Patent No. 5,693,762, to Queen, 1997. In addition, there are commercial entities that will synthesize humanîzed antibodies from spécifie murine antibody régions, such as Protein Design Labs (Mountain View, CA).
RECOMBINANT ANTIBODIES
Anti-MASP-2 antibodies can also be made using recombinant methods. For example, human antibodies can be made using human immunoglobulîn expression libraries (available for example, from Stratagene, Corp., La Jolla, CA) to produce fragments of human antibodies (Vjq, Vl, Fv, Fd, Fab or F(ab’)2). These fragments are then used to construct whole human antibodies using techniques similar to those for producing chimeric antibodies.
ANTI-IDIOTYPE ANTIBODIES
Once anti-MASP-2 antibodies are identified with the desired inhibitory activity, these_antibodies can be used to generate anti-idiotype antibodies that resemble a portion of MASP-2 using techniques that are well known in the art. See, e.g., Greenspan, N.S., étal., FASEBJ. 7:437, 1993. For example, antibodies that bind to MASP-2 and competitively inhibit a MASP-2 protein interaction required for complément activation can be used to generate anti-idiotypes that resemble the MBL binding site on MASP-2 protein and therefore bind and neutralize a binding ligand of MASP-2 such as, for example, MBL.
IMMUNOGLOBULIN FRAGMENTS
The MASP-2 inhibitory agents useful in the method of the invention encompass not only intact immunoglobulîn molécules but also the well known fragments including Fab, Fab', F(ab)2, F(ab')2 and Fv fragments, scFv fragments, diabodies, linear antibodies, single-chaîn antibody molécules and multispecific antibodies formed from antibody fragments.
It is well known in the art that only a small portion of an antibody molécule, the paratope, is involved in the binding of the antibody to its epitope (see, e.g., Clark, W.R., The Experimental Foundations of Modem Immunology, Wiley & Sons, Inc., NY, 1986). The pFc' and Fc régions of the antibody are effectors of the classical complément pathway, but are not involved in antigen binding. An antibody from which the pFc' région has been enzymatically cleaved, or which has been produced without the pFc' région, is designated an F(ab')2 fragment and retains both of the antigen binding sites of an intact antibody. An isolated F(ab’)2 fragment is referred to as a bivalent monoclonal fragment because of its two antigen binding sites. Similarly, an antibody from which the Fc région has been enzymatically cleaved, or which has been produced without the Fc région, is designated a Fab fragment, and retains one of the antigen binding sites of an intact antibody molécule.
Antibody fragments can be obtained by proteolytîc hydrolysis, such as by pepsin or papaîn digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted 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 performed using a blocking group for the sulfhydryl groups that resuit from cleavage of disulfide linkages. As an alternative, an enzymatic cleavage using pepsin produces two monovalent Fab fragments and an Fc fragment directly. These methods are 5 described, for example, U.S. Patent No. 4,331,647 to Goldenberg; Nlsonoff, A., étal., Arch. Biochem. Biophys. 59:230, 1960; Porter, R.R., Biochem. J. 73:119, 1959; Edelman, et al., in Methods in Enzymology 7:422, Academie Press, 1967; and by Coligan at pages 2.8.1-2.8.10 and 2.10.-2.10.4.
In some embodiments, the use of antibody fragments lacking the Fc région are 10 preferred to avoîd activation of the classical complément pathway which is initiated upon binding Fc to the Fcy receptor. There are several methods by which one can produce a MoAb that avoids Fcy receptor interactions. For example, the Fc région of a monoclonal antibody can be removed chemically using partial digestion by proteolytic enzymes (such as ficin digestion), thereby generating, for example, antigen-binding antibody fragments 15 such as Fab or F(ab)2 fragments (Mariani, M., étal., Moi. Immunol. 25:69-71, 1991). Alternatively, the human γ4 IgG isotype, which does not bind Fcy receptors, can be used during construction of a humanîzed antibody as described herein. Antibodies, single chain antibodies and antigen-binding domains that lack the Fc domain can also be engineered using recombinant techniques described herein.
SINGLE-CHAIN ANTIBODY FRAGMENTS
Alternatively, one can create single peptide chain binding molécules spécifie for MASP-2 in which the heavy and light chain Fv régions are connected. The Fv fragments may be connected by a peptide linker to form a single-chain antigen binding protein (scFv). These single-chain antigen binding proteins are prepared by constructing a 25 structural gene comprising DNA sequences encoding the Vjq and domains which are connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell, such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing scFvs are described for example, by 30 Whitlow, étal., Methods: A Companîon to Methods in Enzymology 2:97, 1991; Bird, étal., Science 272:423, 1988; U.S. Patent No. 4,946,778, to Ladner; Pack, P., étal., Bio/Technology 77:1271, 1993.
As an illustrative example, a MASP-2 spécifie scFv can be obtained by exposing lymphocytes to MASP-2 polypeptide in vitro and selecting antibody display libraries in phage or similar vectors (for example, through the use of immobilized or labeled MASP-2 protein or peptide). Genes encoding polypeptides having potential MASP-2 polypeptide binding domains can be obtained by screening random peptide libraries displayed on phage or on bacteria such as E. coli. These random peptide display libraries can be used to screen for peptides which interact with MASP-2. Techniques for creating and screening such random peptide display libraries are well known in the art (U.S. Patent No. 5,223,409, to Lardner; U.S. Patent No. 4,946,778, to Ladner; U.S. Patent No. 5,403,484, to Lardner; U.S. Patent No. 5,571,698, to Lardner; and Kay et al., Phage Display of Peptides and Proteins Academie Press, Inc., 1996) and random peptide display libraries and kits for screening such libraries are available commercially, for instance from CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc. (Ipswich, Mass.), and Pharmacia LKB Biotechnology Inc. (Piscataway, N.J.).
Another form of an anti-MASP-2 antibody fragment useful in this aspect of the invention is a peptide coding for a single complementarity-determtning région (CDR) that binds to an epitope on a MASP-2 antigen and inhibits MASP-2-dependent complément activation. CDR peptides (minimal récognition units) can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared. for example, by using the polymerase chain reaction to synthesize the variable région from RNA of antibody-producing cells (see, for example, Larrick etaL, 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 étal, (eds.), page 166, Cambridge University Press, 1995; and Ward et al., Genetic Manipulation and Expression of Antibodies, în Monoclonal Antibodies: Principles and Applications, Birch étal, (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 complément activation. In some embodiments, the MASP-2 inhibitory agent is a high-affinity human or humanized monoclonal anti-MASP-2 antibody with reduced effector function.
PEPTIDE INHIBITORS
In some embodiments of this aspect of the invention, the MASP-2 inhibitory agent comprises isolated MASP-2 peptide inhibitors, including isolated natural peptide inhibitors and synthetic peptide inhibitors that inhibit the MASP-2-dependent complément activation system. As used herein, the term isolated MASP-2 peptide inhibitors refers to peptides that inhibit MASP-2 dépendent complément activation by binding to, competing with MASP-2 for binding to another récognition molécule (e.g., MBL, H-ficoiin, M-ficoIin, or L-ficolin) in the lectin pathway, and/or directly interacting with MASP-2 to inhibit MASP-2-dependent complément activation that are substantially pure and are essentially free of other substances with which they may be found in nature to an extent practîcal and appropriate for their intended use.
Peptide inhibitors hâve been used successfully in vivo to interfère with protein-protein interactions and catalytic sites. For example, peptide inhibitors to adhesion molécules structurally related to LFA-l hâve recently been approved for clinical use in coagulopathies (Ohman, E.M., et al., European Heart J. /6:50-55, 1995). Short linear peptides (<30 amino acids) hâve been described that prevent or interfère with integrin-dependent adhesion (Murayama, O., étal., J. Biochem. /20:445-51, 1996). Longer peptides, ranging in length from 25 to 200 amino acid residues, hâve also been used successfully to block integrin-dependent adhesion (Zhang, L., étal., J. Biol. Chem. 27/(47):29953-57, 1996). In general, longer peptide inhibitors hâve higher affinîties and/or slower off-rates than short peptides and may therefore be more potent inhibitors. Cyclic peptide inhibitors hâve also been shown to be effective inhibitors of integrins in vivo for the treatment of human inflammatory disease (Jackson, D.Y., et al.,
J. Med. Chem. 70:3359-68, 1997). One method of producïng cyclic peptides involves the synthesis of peptides in which the terminal amino acids of the peptide are cysteines, thereby allowing the peptide to exist in a cyclic form by dîsulfide bonding between the terminal amino acids, which has been shown to improve affinity and half-life in vivo for the treatment of hematopoietic neoplasms (e.g., U.S. Patent No. 6,649,592, to Larson).
SYNTHETIC MASP-2 PEPTIDE INHIBITORS
MASP-2 inhibitory peptides useful in the methods of this aspect of the invention are exemplified by amino acid sequences that mimic the target régions important for MASP-2 function. The inhibitory peptides useful in the practice of the methods of the invention range in size from about 5 amino acids to about 300 amino acids. TABLE 3 provides a list of exemplary inhibitory peptides that may be useful in the practice of this aspect of the present invention. A candidate MASP-2 inhibitory peptide may be tested for the ability to function as a MASP-2 inhibitory agent in one of several assays including, for example, a lectin spécifie C4 cleavage assay (described in Example 2), and a C3b déposition 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 particular domain of the MASP-2 protein such as, for example, the CUBI domain (SEQ ID NO:8), the CUB1EGF domain (SEQ ID NO:9), the EGF domain (SEQ ID NO:lI), and the serine protease domain (SEQ ID NO:l2). As previously described, the CUBEGFCUBII régions hâve been shown to be required for dimerization and binding with MBL (Thielens et al., supra). In particular, the peptide sequence TFRSDYN (SEQ ID NO:l6) in the CUBI domain of MASP-2 has been shown to be involved in binding to MBL in a study that identified a human carrying a homozygous mutation at Aspl05 to Glyl05, resulting in the loss of MASP-2 from the MBL complex (Stengaard-Pedersen, K., et al., New EnglandJ. Med. 349:554-560, 2003).
In some embodiments, MASP-2 inhibitory peptides are derived from the lectin proteins that bind to MASP-2 and are involved in the lectin complément pathway. Several different lectins hâve 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„ étal., J. Exp. Med. /76:1497-2284, 2000; Matsushita, M., et al., J. Immunol. /65:3502-3506, 2002). These lectins are present in sérum as oligomers of homotrimeric subunits, each having N-terminal collagen-like fibers with carbohydrate récognition domains. These different lectins hâve been shown
SI to bind to MASP-2, and the lectin/MASP-2 complex activâtes complément through cleavage of proteins C4 and C2. H-ficolin has an amino-terminal région of 24 amino acids, a collagen-like domain with 11 Gly-Xaa-Yaa repeats, a neck domain of 12 amino acids, and a fibrinogen-like domain of 207 amino acids (Matsushita, M., et al., J. Immunol. 765:3502-3506, 2002). H-ficolin binds to GlcNAc and agglutinâtes human érythrocytes coated with LPS derived from S. typhimurium, S. minnesota and E. coli. Η-ficoÜn has been shown to be associated with MASP-2 and MApl9 and activâtes the lectin pathway. Id L-ficoIin/P35 also binds to GlcNAc and has been shown to be associated with MASP-2 and MApl9 in human sérum and this complex has been shown to activate the lectin pathway (Matsushita, M., étal., J. Immunol. 76-7:2281, 2000). Accordingly, MASP-2 inhibitory peptides useful in the present invention may comprise a région of at least 5 amino acids selected from the MBL protein (SEQ ID NO:21), the H-ficolin protein (Genbank accession number NM_173452), the M-ficolin protein (Genbank accession number 000602) and the L-ficoiin protein (Genbank accession number NM 015838).
More specifically, scientists hâve identified the MASP-2 binding site on MBL to be within the 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 the hinge and the neck in the C-terminal portion of the collagen-like domain of MBP (Wallis, R., et al., J. Biol. Chem. 279:14065, 2004). This MASP-2 binding site région is also highly conserved in human H-ficolin and human L-ficolin. A consensus binding site has been described that is present in ail three lectin proteins comprising the amino acid sequence OGK-X-GP (SEQ ID NO:22) where the letter O represents hydroxyproline and the letter X is a hydrophobie residue (Wallis et al., 2004, supra). Accordingly, in some embodiments, MASP-2 inhibitory peptides useful in this aspect of the invention are at least 6 amino acids in length and comprise SEQ ID NO:22. Peptides derived from MBL that include the amino acid sequence GLR GLQ GPO GKL GPO G (SEQ ID NO:24) hâve been shown to bind MASP-2 in vitro (Wallis, et aL, 2004, supra). 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 the formation of triple helices as found în the native MBL protein (as further described in Wallis, R., et al., J. Biol. Chem. 279:14065, 2004).
MASP-2 inhibitory peptides may also be derived from human H-ficolin that include the sequence GAO GSO GEK GAO GPQ GPO GPO GKM GPK GEO GDO 5 (SEQ ID NO:27) from the consensus MASP-2 binding région in H-ficolin. Also included are 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 consensus MASP-2 binding région in L-ficolin.
MASP-2 inhibitory peptides may also be derived from the C4 cleavage site such 10 as LQRALEILPNRVT1KANRPFLVFI (SEQ ID NO:29) which is the C4 cleavage site linked to the C-terminal portion of antithrombin III (Glover, G.I., et al., Mol. Immunol. 25:1261 (1988)).
TABLE 3: EXEMPLARY MASP-2 INHIBITORY PEPTIDES
SEQ ID NO Source
SEQ ID NO:6 Human MASP-2 protein
SEQ ID NO:8 CUBI domain of MASP-2 (aa 1-121 of SEQ ID NO:6)
SEQ ID NO:9 CUBIEGF domains of MASP-2 (aa 1-166 of SEQ ID NO:6)
SEQ ID NO: 10 CUBIEGFCUBII domains of MASP-2 (aa 1-293 ofSEQ ID NO:6)
SEQ ID NO: 11 EGF domain of MASP-2 (aa 122-166)
SEQ IDNO:12 Serine-protease domain of MASP-2 (aa 429-671)
SEQ ID NO; 16 MBL binding région in MASP-2
SEQ ID NO:3 Human MApl9
SEQ IDNO:21 Human MBL protein
SEQ ID NO Source
SEQ ID NO:22 OGK-X-GP, Where 0 = hydroxyproline and X is a hydrophobie amino acid residue Synthetic peptide Consensus binding site from Human MBL and Human ficolins
SEQ ID NO:23 OGKLG Human MBL core binding site
SEQ ID NO:24 GLR GLQ GPO GKL GPOG Human MBP Triplets 6-10- demonstrated binding to MASP-2
SEQ LD NO:25 GPOGPOGLRGLQGPO GKLGPOGGPOGPO Human MBP Triplets with GPO added to enhance formation of triple helices
SEQ ID NO:26 GKDGRDGTKGEKGEP GQGLRGLQGPOGKLG POGNOGPSGSOGPKG QKGDOGKS Human MBP Triplets 1-17
SEQ ID NO:27 GAOGSOGEKGAOGPQ GPOGPOGKMGPKGEO GDO Human H-Ficoün (Hataka)
SEQ ID NO:28 GCOGLOGAOGDKGE AGTNGKRGERGPOGP OGKAGPOGPNGAOGE O Human L-Ficolin P35
SEQ ID NO:29 LQRALEILPNRVTIKA NRPFLVFI Human C4 cleavage site
SEQ ID NO:72 LEVTCEPGTTFKDKCNT CRCGSDGKSAVCTKLW CNQ SGMI-2L (full-length)
SEQ ID NO Source
SEQ ID NO:73 TCEPGTTFKDKCNTCRC GSDGKSAVCTKLWCNQ SGMI-2M (medium truncated version)
SEQ ID NO:74 TCRCGSDGKSAVCTKL WCNQ SGMI-2S (short truncated version)
Note: The letter O represents hydroxyproline. The letter X is a hydrophobie 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 so that they are irréversible 5 protease inhibitors. For example, appropriate modifications may include, but are not necessarily limited to, halomethyl ketones (Br, Cl, I, F) at the C-terminus, Asp or Glu, or appended to functional side chains; haloacetyl (or other α-haloacetyl) groups on amino groups or other functional side chains; epoxide or imine-containing groups on the amino or carboxy termini or on functional side chains; or imidate esters on the amino or carboxy 10 termini or on functional side chains. Such modifications would afford the advantage of permanently inhibiting the enzyme by covalent attachment of the peptide. This could resuit în lower effective doses and/or the need for less frequent administration of the peptide inhibitor.
In addition to the inhibitory peptides described above, MASP-2 inhibitory 15 peptides useful in the method of the invention include peptides containing the MASP-2-binding CDRH3 région of anti-MASP-2 MoAb obtained as described herein. The sequence of the CDR régions for use in synthesizing the peptides may be determined by methods known in the art. The heavy chain variable région is a peptide that generally ranges from 100 to 150 amino acids in length. The light chain variable région is a peptide 20 that generally ranges from 80 to 130 amino acids in length. The CDR sequences within the heavy and light chain variable régions include only approximately 3-25 amino acid sequences that may be easily sequenced by one of ordinary skîll in the art.
Those skilled in the art will recognize that substantially homologous variations of the MASP-2 inhibitory peptides described above will also exhibit MASP-2 inhibitory 25 activity. Exemplary variations include, but are not necessarily limited to, peptides having insertions, délétions, replacements, and/or additional amino acids on the carboxy-termînus or amino-terminus portions of the subject peptides and mixtures thereof. Accordingly, those homologous peptides having MASP-2 inhibitory activity are considered to be useful in the methods of this invention. The peptides described may also include duplicating motifs and other modifications with conservative substitutions. Conservative variants are described elsewhere herein, and include the exchange of an amino acid for another of like charge, size or hydrophobîcity and the like.
MASP-2 inhibitory peptides may be modified to increase solubility and/or to maximize the positive or négative charge in order to more closely resemble the segment in the intact protein. The dérivative may or may not hâve the exact primary amino acid structure of a peptide disclosed herein so long as the dérivative functionally retains the desired property of MASP-2 inhibition. The modifications can include amino acid substitution with one of the commonly known twenty amino acids or with another amino acid, with a derivatized or substituted amino acid with ancillary désirable characteristics, such as résistance to enzymatic dégradation or with a D-amino acid or substitution with another molécule or compound, such as a carbohydrate, which mimics the natural confirmation and fonction of the amino acid, amino acids or peptide; amino acid délétion; amino acid insertion with one of the commonly known twenty amino acids or with another amino acid, with a derivatized or substituted amino acid with ancillary désirable characteristics, such as résistance to enzymatic dégradation or with a D-amino acid or substitution with another molécule or compound, such as a carbohydrate, which mimics the natural confirmation and fonction of the amino acid, amino acids or peptide; or substitution with another molécule or compound, such as a carbohydrate or nucleic acid monomer, which mimics the natural conformation, charge distribution and fonction of the parent peptide. Peptides may also be modified by acétylation or amidation.
The synthesis of dérivative inhibitory peptides can rely on known techniques of peptide biosynthesis, carbohydrate biosynthesis and the like. As a starting point, the artisan may rely on a suitable computer program to détermine the conformation of a peptide of interest. Once the conformation of peptide disclosed herein is known, then the artisan can détermine in a rational design fashion what sort of substitutions can be made at one or more sites to fashion a dérivative that retains the basic conformation and charge distribution of the parent peptide but which may possess characteristics which are not present or are enhanced over those found in the parent peptide. Once candidate dérivative molécules are identified, the dérivatives can be tested to détermine if they function as MASP-2 înhibitory agents using the assays described herein.
SCREENING FOR MASP-2 INHIBITORY PEPTIDES
One may also use molecular modeling and rational molecular design to generate and screen for peptides that mimic the molecular structures of key binding régions of MASP-2 and inhibit the complément activities of MASP-2. The molecular structures used for modeling include the CDR régions of anti-MASP-2 monoclonal antibodies, as well as the target régions known to be important for MASP-2 function including the région required for dimerization, the région involved in binding to MBL, and the serine protease active site as previously described. Methods for identifying peptides that bind to a particular target are well known in the art. For example, molecular imprinting may be used for the de novo construction of macromolecular structures such as peptides that bind to a particular molécule. 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 of preparîng mimîcs of MASP-2 binding peptides is as follows. Functional monomers of a known MASP-2 binding peptide or the binding région of an anti-MASP-2 antibody that exhibits MASP-2 inhibition (the template) are polymerized. The template is then removed, followed by polymerization of a second class of monomers in the void left by the template, to provide a new molécule that exhibits one or more desired properties that are similar to the template. In addition to preparîng peptides in this manner, other MASP-2 binding molécules that are MASP-2 înhibitory agents such as polysaccharides, nucleosides, drugs, nucleoproteins, lipoproteins, carbohydrates, glycoproteins, steroid, lipids and other bîologically active materials can also be prepared. This method is useful for designing a wide variety of biological mimics that are more stable than their natural counterparts because they are typically prepared by free radical polymerization of function monomers, resulting in a compound with a nonbiodegradable backbone.
PEPTIDE SYNTHESIS
The MASP-2 inhibitory peptides can be prepared using techniques well known in the art, such as the solid-phase synthetic technique initially described by Merrifield, in J. Amer. Chem. Soc. 55:2149-2154, 1963. Automated synthesis may be achieved, for example, using Applied Biosystems 431A Peptide Synthesizer (Foster City, Calif.) in accordance with the instructions provided by the manufacturer. Other techniques may be found, for example, in Bodanszky, M., et al., Peptide Synthesis, second édition, John Wiley & Sons, 1976, as well as in other reference works known to those skîlled in the art.
The peptides can also be prepared using standard genetic engineering techniques known to those skîlled in the art. For example, the peptide can be produced enzymatically by inserting nucleic acid encoding the peptide into an expression vector, expressing the DNA, and translating the DNA into the peptide în the presence of the required amino acids. The peptide is then purified using chromatographie or electrophoretic techniques, or by means of a carrier protein that can be fused to, and subsequently cleaved from, the peptide by inserting into the expression vector in phase with the peptide encoding sequence a nucleic acid sequence encoding the carrier protein. The fusion protein-peptide may be isolated using chromatographie, electrophoretic or immunological techniques (such as binding to a resin via an antibody to the carrier protein). The peptide can be cleaved using Chemical methodology or enzymatically, as by, for example, hydrolases.
The MASP-2 inhibitory peptides that are useful in the method of the invention can also be produced in recombinant host cells following conventional techniques. To express a MASP-2 inhibitory peptide encoding sequence, a nucleic acid molécule 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 regulatory sequences, such as promoters and enhancers, expression vectors can include translational regulatory sequences and a marker gene, which are suitable for sélection of cells that carry the expression vector.
Nucleic acid molécules that encode a MASP-2 inhibitory peptide can be synthesized with gene machines using protocols such as the phosphoramidite method. If chemically synthesized double-stranded DNA is required for an application such as the synthesis of a gene or a gene fragment, then each complementary strand is made separately. The production of short genes (60 to 80 base pairs) is technically straightforward and can be accomplished by synthesizing the complementary strands and then annealing them. For the production of longer genes, synthetic genes 5 (double-stranded) are assembled in modular form from single-stranded fragments that are from 20 to I00 nucléotides in length. For reviews on polynucleotide synthesis, see, for example, Glick and Pasternak, Molecular Biotechnology, Principles and Applications of Recombinant DNA, ASM Press, 1994; Itakura, K., et al., Annu. Rev. Biochem. 53:323, 1984; and Climîe, S., et aL, Proc. Nat'l Acad. Sci. USA 57:633, 1990.
SMALL MOLECULE INHIBITORS
In some embodiments, MASP-2 inhibitory agents are small molécule inhibitors including natural and synthetic substances that hâve a low molecular weight, such as for example, peptides, peptidomimetics and nonpeptide inhibitors (including oligonucleotides and organic compounds). Small molécule inhibitors of MASP-2 can be 15 generated based on the molecular structure of the variable régions of the anti-MASP-2 antibodies.
Small molécule inhibitors may also be designed and generated based on the MASP-2 crystal structure using computational drug design (Kuntz LD., étal., Science 257:1078, 1992). The crystal structure of rat MASP-2 has been described 20 (Feinberg, H., étal., EMBO J. 22:2348-2359, 2003). Using the method described by Kuntz et al., the MASP-2 crystal structure coordinates are used as an input for a computer program such as DOCK, which outputs a Hst of small molécule structures that are expected to bind to MASP-2. Use of such computer programs is well known to one of skill in the art. For example, the crystal structure of the H1V-1 protease inhibitor was 25 used to identify unique nonpeptide ligands that are HIV-1 protease inhibitors by evaluating the fit of compounds found in the Cambridge Crystallographic database to the binding site of the enzyme using the program DOCK (Kuntz, I.D., et al., J. Mol. Biol. /67:269-288, 1982; DesJarlais, R.L., et al., PNAS 57:6644-6648, 1990).
The list of small molécule structures that are identified by a computational method 30 as potential MASP-2 inhibitors are screened using a MASP-2 binding assay such as described in Example 10. The small molécules that are found to bind to MASP-2 are then assayed in a functional assay such as described in Example 2 to détermine if they inhibit MASP-2-dependent complément activation.
MASP-2 SOLUBLE RECEPTORS
Other suitable MASP-2 inhibitory agents are believed to include MASP-2 soluble receptors, which may be produced using techniques known to those of ordinary skill in the art.
EXPRESSION INHIBITORS OF MASP-2
In another embodiment of this aspect of the invention, the MASP-2 inhibitory agent is a MASP-2 expression inhibitor capable of inhibiting MASP-2-dependent complément activation. In the practice of this aspect of the invention, représentative MASP-2 expression inhibitors include MASP-2 antisense nucleic acid molécules (such as antisense mRNA, antisense DNA or antisense oligonucleotides), MASP-2 ribozymes and MASP-2 RNAi molécules.
Anti-sense RNA and DNA molécules act to directly block the translation of MASP-2 mRNA by hybridizing to MASP-2 mRNA and preventing translation of MASP-2 protein. An antisense nucleic acid molécule may be constructed in a number of different ways provided that it is capable of interfering with the expression of MASP-2. For example, an antisense nucleic acid molécule can be constructed by inverting the coding région (or a portion thereof) of MASP-2 cDNA (SEQ ID NO:4) relative to its normal orientation for transcription to allow for the transcription of its complément.
The antisense nucleic acid molécule is usually substantially identical to at least a portion of the target gene or genes. The nucleic acid, however, need not be perfectly identical to inhibit expression. Generally, higher homology can be used to compensate for the use of a shorter antisense nucleic acid molécule. The minimal percent identity is typically greater than about 65%, but a higher percent identity may exert a more effective repression of expression of the endogenous sequence. Substantially greater percent identity of more than about 80% typically is preferred, though about 95% to absolute identity is typically most preferred.
The antisense nucleic acid molécule need not hâve the same întron or exon pattern as the target gene, and non-coding segments of the target gene may be equally effective in achieving antisense suppression of target gene expression as coding segments. A DNA sequence of at least about 8 or so nucléotides may be used as the antîsense nucleic acid molécule, although a longer sequence is préférable. In the présent invention, a représentative example of a useful inhibitory agent of MASP-2 is an antîsense MASP-2 nucleic acid molécule which is at least nînety percent identical to the complément of the MASP-2 cDNA consisting of the nucleic acid sequence set forth in SEQ ID NO:4. The nucleic acid sequence set forth in SEQ ID NO:4 encodes the MASP-2 protein consisting of the amino acid sequence set forth in SEQ ID NO:5,
The targeting of antîsense oligonucleotides to bind MASP-2 mRNA is another mechanism that may be used to reduce the level of MASP-2 protein synthesis. For example, the synthesis of polygalacturonase and the muscarîne type 2 acétylcholine receptor is inhibited by antîsense oligonucleotides directed to their respective mRNA sequences (U.S. Patent No. 5,739,119, to Cheng, and U.S. Patent No. 5,759,829, to Shewmaker). Furthermore, examples of antîsense inhibition hâve been demonstrated with the nuclear protein cyclin, the multiple drug résistance gene (MDG1), ICAM-1, E-selectin, STK-1, striatal GABAa receptor and human EGF (see, e.g., U.S. Patent No. 5,801,154, to Baracchini; U.S. Patent No. 5,789,573, to Baker; U.S. Patent No. 5,718,709, to Consîdine; and U.S. Patent No. 5,610,288, to Reubenstein).
A System has been described that allows one of ordînary skill to détermine which oligonucleotides are useful in the invention, which involves probing for suitable sites in the target mRNA using RNAse H cleavage as an indicator for accessibility of sequences within the transcripts. Scherr, M., étal., Nucleic Acids Res. 26:5079-5085, 1998; Lloyd, et al., Nucleic Acids Res. 29:3665-3673, 2001. A mixture of antîsense oligonucleotides that are complementary to certain régions of the MASP-2 transcript is added to cell extracts expressing MASP-2, such as hépatocytes, and hybrîdized in order to create an RNAse H vulnérable site. This method can be combined with computer-assîsted sequence sélection that can predict optimal sequence sélection for antîsense compositions based upon their relative ability to form dimers, hairpins, or other secondary structures that would reduce or prohibit spécifie binding to the target mRNA in a host cell. These secondary structure analysis and target site sélection considérations may be performed using the OLIGO primer analysis software (Rychlik, L, 1997) and the BLASTN 2.0.5 algorithm software (Altschui, S.F., et al., Nucl. Acids Res. 25:3389-3402,
9l
1997). The antîsense compounds directed towards the target sequence preferably comprise from about 8 to about 50 nucléotides in length. Antisense oligonucleotides comprising from about 9 to about 35 or so nucléotides are particularly preferred. The inventors contemplate ail oligonucleotide compositions in the range of 9 to 35 nucléotides (i.e., those of9, ΙΟ, 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 bases in length) are highly preferred for the practice of antisense oligonucleotide-based methods of the invention. Highly preferred target régions of the MASP-2 mRNA are those that are at or near the AUG translation initiation codon, and those sequences that are substantially complementary to 5' régions of the mRNA, e.g., between the -10 and +10 régions of the MASP-2 gene nucléotide sequence (SEQ ID NO:4). Exemplary MASP-2 expression inhibitors are provided in TABLE 4.
TABLE 4: EXEMPLARY EXPRESSION INHIBITORS OF MASP-2
SEQ ID NO:30 (nucléotides 22-680 of SEQ ID NO:4) Nucleic acid sequence of MASP-2 cDNA (SEQ ID NO:4) encoding CUBIEGF
SEQIDNO:31 5’CGGGCACACCATGAGGCTGCTG ACCCTCCTGGGC3 Nucléotides 12-45 of SEQ ID NO:4 including the MASP-2 translation start site (sense)
SEQ ID NO:32 5’GACATTACCTTCCGCTCCGACTC CAACGAGAAG3' Nucléotides 361-396 of SEQ ID NO:4 encoding a région comprising the MASP-2 MBL binding site (sense)
SEQ ID NO:33 5AGCAGCCCTGAATACCCACGGCC GTATCCCAAA3’ Nucléotides 610-642 of SEQ ID NO:4 encoding a région comprising the CUBII domain
As noted above, the term oligonucleotide as used herein refers to an oiigomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term also covers those oligonucleobases composed of naturally occurring nucléotides, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally occurring modifications. These modifications allow one to introduce certain désirable properties that are not offered through naturally occurring oligonucleotides, such as reduced toxic properties, increased stability against nuclease dégradation and enhanced celiular uptake. In illustrative embodiments, the antisense compounds of the invention differ from native DNA by the modification ofthe phosphodîester backbone to extend the life of the antisense oligonucleotide in which the phosphate substituents are replaced by phosphorothioates. Likewise, one or both ends of the oligonucleotide may be substituted by one or more acridine dérivatives that intercalate between adjacent basepairs within a strand of nucleic acid.
Another alternative to antisense is the use of RNA interférence (RNAi). Double-stranded RNAs (dsRNAs) can provoke gene silencing in mammals in vivo. The natural function of RNAÎ and co-suppression appears to be protection of the genome against invasion by mobile genetic éléments such as retrotransposons and viruses that produce aberrant RNA or dsRNA in the host cell when they become active (see, e.g., Jensen, J., et al., Nat. Genet. 27:209-12, 1999). The double-stranded RNA molécule may be prepared by synthesizing two RNA strands capable of forming a double-stranded RNA molécule, each having a length from about 19 to 25 (e.g., 19-23 nucléotides). For example, a dsRNA molécule useful in the methods of the invention may comprise the RNA corresponding to a sequence and its complément listed in TABLE 4. Preferably, at least one strand of RNA has a 3' overhang from 1-5 nucléotides. The synthesized RNA strands are combined under conditions that form a double-stranded molécule. The RNA sequence may comprise at least an 8 nucléotide portion of SEQ ID NO:4 with a total length of 25 nucléotides or less. The design of siRNA sequences for a given target is within the ordînary skill of one in the art. Commercial services are available that design siRNA sequence and guarantee at least 70% knockdown of expression (Qiagen, Valencia, Calif).
The dsRNA may be administered as a pharmaceutical composition and carried out by known methods, wherein a nucleic acid is introduced into a desired target cell. Commonly used gene transfer methods include calcium phosphate, DEAE-dextran, electroporation, microinjection and viral methods. Such methods are taught in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., 1993.
Ribozymes can also be utilized to decrease the amount and/or biological activity of MASP-2, such as ribozymes that target MASP-2 mRNA. Ribozymes are catalytic RNA molécules that can cleave nucleic acid molécules having a sequence that is completely or partially homologous to the sequence of the ribozyme. It is possible to design ribozyme transgenes that encode RNA ribozymes that specifically pair with a target RNA and cleave the phosphodiester backbone at a spécifie location, thereby functionally inactivating the target RNA. In carrying out this cleavage, the ribozyme is not itself altered, and is thus capable of recycling and cleaving other molécules. The inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the antisense constructs.
Ribozymes useful in the practice of the invention typically comprise a hybridizing région of at least about nine nucléotides, which is complementary in nucieotide sequence to at least part of the target MASP-2 mRNA, and a catalytic région that is adapted to cleave the target MASP-2 mRNA (see generally, EPA No. 0 321 201; W088/04300; Haseloff, J., et al., Nature 337:585-591, 1988; Fedor, M.J., étal., Proc. Natl. Acad. Sci. USA 87:1668-1672, 1990; Cech, T.R., et al., Ann. Rev. Biochem. 55:599-629, 1986).
Ribozymes can either be targeted directly to cells in the form of RNA oligonucleotides incorporating ribozyme sequences, or introduced into the cell as an expression vector encoding the desired ribozyma! RNA. Ribozymes may be used and applied in much the same way as described for antisense polynucleotides.
Anti-sense RNA and DNA, ribozymes and RNAÎ molécules useful in the methods of the invention may be prepared by any method known in the art for the synthesis of DNA and RNA molécules. These include techniques for chemically synthesizing oligodeoxyribonucleotides and oligoribonucleotides well known in the art, such as for example solid phase phosphoramidite Chemical synthesis. Altematively, RNA molécules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molécule. Such DNA sequences may be încorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Altematively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.
Various well known modifications of the DNA molécules may be introduced as a means of increasing stability and half-life. Useful modifications include, but are not limited to, the addition of flankîng sequences of ribonucleotides or deoxyribonucleottdes to the 5' and/or 3’ ends of the molécule or the use of phosphorothioate or 2' O-methyi rather than phosphodiesterase linkages within the oligodeoxyribonucleotide backbone.
V. PHARMACEUTICAL COMPOSITIONS AND DELIVERY METHODS
DOSING
In another aspect, the invention provides compositions for inhibiting the adverse effects of MASP-2-dependent complément activation în a subject suffering from a disease or condition as disclosed herein, comprising administering to the subject a composition comprising a therapeutically effective amount of a MASP-2 inhibitory agent and a pharmaceutically acceptable carrier. The MASP-2 inhibitory agents can be I0 administered to a subject in need thereof, at therapeutically effective doses to treat or ameliorate conditions associated with MASP-2-dependent complément activation. A therapeutically effective dose refers to the amount of the MASP-2 inhibitory agent sufficient to resuit in amelioration of symptoms associated with the disease or condition.
Toxicity and therapeutic efficacy of MASP-2 inhibitory agents can be determined 15 by standard pharmaceutical procedures employing experimental animal models, such as the murine MASP-2 -/- mouse model expressing the human MASP-2 transgene described in Example l. Using such animal models, the NOAEL (no observed adverse effect level) and the MED (the minîmally effective dose) can be determined using standard methods. The dose ratio between NOAEL and MED effects is the therapeutic ratio, which is 20 expressed as the ratio NOAEL/MED. MASP-2 inhibitory agents that exhibit large therapeutic ratios or indices are most preferred. The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosages for use in humans. The dosage of the MASP-2 inhibitory agent preferably lies within a range of circulating concentrations that include the MED with little or no toxicity. The dosage 25 may vary within this range depending upon the dosage form employed and the route of administration utilized.
In some embodiments, therapeutic efficacy of the MASP-2 inhibitory agents for treating, inhibiting, alleviating or preventing fibrosis in a mammalian subject suffering, or at risk of developing a disease or disorder caused or exacerbated by fibrosis and/or 30 inflammation is determined by one or more of the following: a réduction in one of more markers of inflammation and scarring (e.g., TGFp-I, CTFF, IL-6, apoptosis, fibronectin, laminin, collagens, EMT, infiltrating macrophages) in rénal tissue; a réduction in the release of soluble markers of inflammation and fibrotic rénal disease into urine and plasma (e.g., by the measurement of rénal excretory fonctions).
For any compound formulation, the therapeutically effective dose can be estimated using animal models. For example, a dose may be formulated in an animal model to achieve a circulating plasma concentration range that includes the MED. Quantitative levels of the MASP-2 inhibitory agent in plasma may also be measured, for example, by high performance liquid chromatography.
In addition to toxicity studies, effective dosage may also be estimated based on the amount of MASP-2 protein present in a living subject and the binding affinity of the MASP-2 inhibitory agent. It has been shown that MASP-2 levels in normal human subjects is present in sérum in low levels in the range of 500 ng/ml, and MASP-2 levels in a particular subject can be determined using a quantitative assay for MASP-2 described in Moller-Kristensen M., et al., J. Immunol. Methods 252:159-167, 2003.
Generally, the dosage of administered compositions comprising MASP-2 inhibitory agents varies depending on such factors as the subject's âge, weight, height, sex, general medical condition, and previous medical history. As an illustration, MASP-2 inhibitory agents, such as anti-MASP-2 antibodies, can be administered in dosage ranges from about 0.010 to 10.0 mg/kg, preferably 0.010 to 1.0 mg/kg, more preferably 0.010 to 0.1 mg/kg of the subject body weight. In some embodiments the composition comprises a combination of anti-MASP-2 antibodies and MASP-2 inhibitory peptides.
Therapeutic efficacy of MASP-2 inhibitory compositions and methods of the present invention in a given subject, and appropriate dosages, can be determined in accordance with complément assays well known to those of skill in the art. Complément generates numerous spécifie products. During the last decade, sensitive and spécifie assays hâve been developed and are available commercîally for most of these activation products, including the small activation fragments C3a, C4a, and C5a and the large activation fragments iC3b, C4d, Bb, and sC5b-9. Most of these assays utilize monoclonal antibodies that react with new antigens (neoantigens) exposed on the fragment, but not on the native proteins from which they are formed, making these assays very simple and spécifie. Most rely on EL1SA technology, although radioimmunoassay is still sometimes used for C3a and C5a. These latter assays measure both the unprocessed fragments and their 'desArg' fragments, which are the major forms found in the circulation. Unprocessed fragments and CSadesArg are rapidly cleared by binding to cell surface receptors and are hence present in very low concentrations, whereas C3adesArg does not bînd to cells and accumulâtes in plasma. Measurement ofC3a provides a sensitive, pathway-independent indicator of complément activation. Alternative pathway activation can be assessed by measuring the Bb fragment. Détection of the fluid-phase product of membrane attack pathway activation, sC5b-9, provides evidence that complément is being activated to completion. Because both the lectin and classical pathways generate the same activation products, C4a and C4d, measurement of these two fragments does not provide any information about which of these two pathways has generated the activation products.
The inhibition of MASP-2-dependent complément activation is characterized by at least one of the following changes in a component of the complément system that occurs as a resuit of administration of a MASP-2 inhibitory agent in accordance with the methods of the invention: the inhibition of the génération or production of MASP-2-dependent complément activation system products C4b, C3a, C5a and/or C5b-9 (MAC) (measured, for example, as described in measured, for example, as described in Example 2, the réduction of C4 cleavage and C4b déposition (measured, for example as described in Example 10), or the réduction of C3 cleavage and C3b déposition (measured, for example, as described in Example 10).
ADDITIONAL AGENTS
In certain embodiments, methods of preventing, treating, reverting and/or inhibiting fibrosis and/or inflammation include administering an MASP-2 inhibitory agent (e.g., a MASP-2 inhibitory antibody) as part of a therapeutic regimen along with one or more other drugs, biologîcs, or therapeutic interventions appropriate for inhibiting fibrosis and/or inflammation. In certain embodiments, the additional drug, biologie, or therapeutic intervention is appropriate for particular symptoms associated with a disease or disorder caused or exacerbated by fibrosis and/or inflammation. By way of example, MASP-2 inhibitory antibodies may be administered as part of a therapeutic regimen along with one or more immunosuppressive agents, such as methotrexate, cyclophosphamide, azathioprine, and mycophenolate mofetil. By way of further example, MASP-2 inhibitory antibodies may be administered as part of a therapeutic regimen along with one or more agents designed to increase blood flow (e.g., nifedipine, amlodipine, diltiazem, felodipine, or nîcardipine). By way of further example, MASP-2 inhibitory antibodies may be administered as part of a therapeutic regimen along with one or more agents intended to decrease fibrosis, such as d-penicîllamine, colchicine, PUVA, Relaxin, cyclosporine, TGF beta blockers and/or p38 MAPK blockers. By way of further example, MASP-2 inhibitory antibodies may be administered as part of a therapeutic 10 regimen along with steroids or broncho-dilators.
The compositions and methods comprising MASP-2 inhibitory agents (e.g., MASP-2 inhibitory antibodies) may optionally comprise one or more additional therapeutic agents, which may augment the activity of the MASP-2 inhibitory agent or that provide related therapeutic functions in an additive or synergistic fashion. For 15 example, in the context of treating a subject suffering from a disease or disorder caused or exacerbated by fibrosis and/or inflammation one or more MASP-2 inhibitory agents may be administered in combination (including co-administration) with one or more additional antifibrotic agents and/or one or more anti-inflammatory and/or immunosuppressive agents.
MASP-2 inhibitory agents (e.g., MASP-2 inhibitory antibodies) can be used in combination with other therapeutic agents such as general immunosuppressive drugs such as corticosteroids, immunosuppressive or cytotoxic agents, and/or antifibrotic agents.
PHARMACEUTICAL CARRIERS AND DELIVERY VEHICLES
In general, the MASP-2 inhibitory agent compositions of the present invention, combined with any other selected therapeutic agents, are suitably contained in a pharmaceutically acceptable carrier. The carrier is non-toxic, biocompatible and is selected so as not to detrimentally affect the biological activity of the MASP-2 inhibitory agent (and any other therapeutic agents combined therewith). Exemplary pharmaceutically acceptable carriers for peptides are described in U.S. Patent
No. 5,211,657 to Yamada. The anti-MASP-2 antibodies and inhibitory peptides useful in the invention may be formulated into préparations in solid, semi-solid, gel, liquid or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, depositories, inhalants and injections allowing for oral, parentéral or surgical administration. The invention also contemplâtes local administration ofthe compositions by coating medical devices and the like.
Suitable carriers for parentéral delivery via injectable, infusion or irrigation and topical delivery include distilled water, physiological phosphate-buffered saline, normal or lactated Ringer's solutions, dextrose solution, Hank's solution, or propanediol. In addition, stérile, fixed oîÎs may be employed as a solvent or suspending medium. For this purpose any biocompatible oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the préparation of injectables. The carrier and agent may be compounded as a liquid, suspension, polymerizable or non-polymerizable gel, paste or salve.
The carrier may also comprise a delivery vehicle to sustain (i.e., extend, delay or regulate) the delivery of the agent(s) or to enhance the delivery, uptake, stability or pharmacokinetîcs of the therapeutic agent(s). Such a delivery vehicle may include, by way of non-limiting example, microparticies, mïcrospheres, nanospheres or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymeric or copolymeric hydrogels and polymeric micelles. Suitable hydrogel and micelle delivery Systems include the PEO:PHB:PEO copolymers and copolymer/cyclodextrin complexes disclosed in WO 2004/009664 A2 and the PEO and PEO/cyclodextrin complexes disclosed in U.S. Patent Application Publication No. 2002/0019369 Al. Such hydrogels may be injected locally at the site of intended action, or subcutaneously or intramuscularly to form a sustained release depot.
For intra-articular delivery, the MASP-2 inhibitory agent may be carried in above-described liquid or gel carriers that are injectable, above-described sustaîned-release delivery vehtcles that are injectable, or a hyaluronic acid or hyaluronic acid dérivative.
For oral administration of non-peptîdergic agents, the MASP-2 inhibitory agent may be carried in an inert fïller or diluent such as sucrose, comstarch, or cellulose.
For topical administration, the MASP-2 inhibitory agent may be carried in ointment, lotion, cream, gel, drop, suppository, spray, liquid or powder, or in gel or microcapsular delivery Systems via a transdermal patch.
Various nasal and pulmonary delivery Systems, including aérosols, metered-dose inhalers, dry powder inhalers, and nebulizers, are being developed and may suitably be adapted for delivery of the present invention in an aérosol, inhalant, or nebulîzed delivery vehicle, respectively.
For intrathecal (IT) or intracerebroventricular (ICV) delivery, approprîately stérile delivery Systems (e.g., liquids; gels, suspensions, etc.) can be used to administer the present invention.
The compositions of the present invention may also include biocompatible excipients, such as dispersing or wetting agents, suspending agents, diluents, buffers, pénétration enhancers, emulsifiers, binders, thickeners, flavouring agents (for oral administration).
PHARMACEUTICAL CARRIERS FOR ANTIBODIES AND PEPTIDES
More specifically with respect to anti-MASP-2 antibodies and inhibitory peptides, exemplary formulations can be parenterally administered as injectable dosages of a solution or suspension of the compound in a physiologically acceptable diluent with a pharmaceutical carrier that can be a stérile liquid such as water, oils, saline, glycerol or éthanol. Additionally, auxiliary substances such as wetting or emulsifying agents, surfactants, pH buffering substances and the like can be present in compositions comprising anti-MASP-2 antibodies and inhibitory peptides. Additional components of pharmaceutical compositions include petroleum (such as of animal, vegetable or synthetic origin), for example, soybean oil and minerai oil. In general, glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers for injectable solutions.
The anti-MASP-2 antibodies and inhibitory peptides can also be administered in the form of a depot injection or implant préparation that can be formulated in such a manner as to permit a sustained or pulsatile release of the active agents.
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PHARMACEUTICALLY ACCEPTABLE CARRIERS FOR EXPRESSION
INHIBITORS
More specifically with respect to expression inhibitors useful ih the methods of the invention, compositions are provided that comprise an expression inhibîtor as described above and a pharmaceutically acceptable carrier or diluent. The composition may further comprise a colloïdal dispersion system.
Pharmaceutical compositions that include expression inhibitors may include, but are not limited to, solutions, émulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifÿing solids and self-emulsifying semisolids. The préparation of such compositions typically involves combining the expression inhibîtor with one or more of the following: buffers, antioxidants, low molecular weight polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with non-specific sérum albumin are examples of suitable diluents.
in some embodiments, the compositions may be prepared and formulated as émulsions which are typically heterogeneous Systems of one liquid dispersed in another in the form of droplets (see, Idson, in Pharmaceutical Dosage Forms, Vol. I, Rieger and Banker (eds.), Marcek Dekker, Inc., N.Y., 1988). Examples of naturally occurring emulsifiers used in émulsion formulations include acacia, beeswax, lanoiin, lecithin and phosphatides.
In one embodiment, compositions including nucleic acids can be formulated as microemulsions. A microemulsion, as used herein refers to a system of water, oil, and amphiphile, which is a single optically isotropie and thermodynamically stable liquid solution (see Rosoff in Pharmaceutical Dosage Forms, Vol. I). The method of the invention may also use liposomes for the transfer and delivery of antisense oligonucleotides to the desired site.
Pharmaceutical compositions and formulations of expression inhibitors for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops.
ΙΟΙ suppositortes, sprays, liquids and powders. Conventionai pharmaceutical carriers, as well as aqueous, powder or oily bases and thickeners and the like may be used.
MODES OF ADMINISTRATION
The pharmaceutical compositions comprising MASP-2 inhibitory agents may be administered in a number of ways depending on whether a local or systemic mode of administration is most appropriate for the condition being treated. Further, the compositions of the present invention can be delivered by coating or incorporating the compositions on or into an implantable medical device.
SYSTEMIC DELIVERY
As used herein, the terms systemic delivery and systemic administration are intended to include but are not limited to oral and parentéral routes including intramuscular (IM), subeutaneous, intravenous (IV), intra-arterial, inhalational, sublingual, buccal, topical, transdermaï, nasal, rectal, vaginal and other routes of administration that effectively resuit in dispersement of the delivered agent to a single or multiple sites of intended therapeutic action. Preferred routes of systemic delivery for the present compositions include intravenous, intramuscular, subeutaneous and inhalational. It will be appreciated that the exact systemic administration route for selected agents utilized in particular compositions of the present invention will be determined in part to account for the agent's susceptibility to metabolic transformation pathways associated with a given route of administration. For example, peptidergic agents may be most suitably administered by routes other than oral.
MASP-2 inhibitory antibodies and polypeptides can be delivered into a subject in need thereof by any suitable means. Methods of delivery of MASP-2 antibodies and polypeptides include administration by oral, puimonary, parentéral (e.g., intramuscular, intraperitoneal, intravenous (IV) or subeutaneous injection), inhalation (such as via a fine powder formulation), transdermaï, nasal, vaginal, rectal, or sublingual routes of administration, and can be formulated in dosage forms appropriate for each route of administration.
By way of représentative example, MASP-2 inhibitory antibodies and peptides can be introduced into a living body by application to a bodily membrane capable of absorbing the polypeptides, for example the nasal, gastrointestinal and rectal membranes.
102
The polypeptides are typically applied to the absorptive membrane in conjunction with a permeation enhancer. (See, e.g., Lee, V.H.L., Crû. Rev. Ther. Drug Carrier Sys. 5:69, 1988; Lee, V.H.L., J. Controlled Release 75:213, 1990; Lee, V.H.L., Ed., Peptide and Protein Drug Delivery, Marcel Dekker, New York (1991); DeBoer, A.G., étal., J. Controlled Release 75:241, 1990.) For example, STDHF is a synthetic dérivative of fusidic acid, a stéroïdal surfactant that is similar in structure to the bile salts, and has been used as a permeation enhancer for nasal delivery. (Lee, W.A., Biopharm. 22, Nov./Dec. 1990.)
The MASP-2 înhibitory antibodies and polypeptides may be introduced in association with another molécule, such as a lipid, to protect the polypeptides from enzymatic dégradation. For example, the covalent attachment of polymers, especially polyethylene glycol (PEG), has been used to protect certain proteins from enzymatic hydrolysis in the body and thus prolong half-life (Fuertges, F., et al., J. Controlled Release 77:139, 1990). Many polymer Systems hâve been reported for protein delivery (Bae, Y.H., et al., J. Controlled Release 9:271, 1989; Hori, R., et al., Pharm. Res. 6:813, 1989; Yamakawa, L, et al., J. Pharm. Sci. 79:505, 1990; Yoshihiro, L, et al., J. Controlled Release 10Λ95, 1989; Asano, M., etaL, J. Controlled Release 9:111, 1989; Rosenblatt, J., étal., J. Controlled Release 9:195, 1989; Makino, K., J. Controlled Release 72:235, 1990; Takakura, Y., étal., J. Pharm. Sci. 78:\\Ί, 1989; Takakura, Y., et al., J. Pharm. Sci. 75:219, 1989).
Recently, liposomes hâve been developed with împroved sérum stability and circulation half-times (see, e.g., U.S. Patent No. 5,741,516, to Webb). Furthermore, various methods of liposome and liposome-like préparations as potential drug carriers hâve been reviewed (see, e.g., U.S. Patent No. 5,567,434, to Szoka; U.S. Patent No. 5,552,157, to Yagi; U.S. Patent No. 5,565,213, to Nakamorî; U.S. Patent No. 5,738,868, to Shinkarenko; and U.S. Patent No. 5,795,587, to Gao).
For transdermal applications, the MASP-2 înhibitory antibodies and polypeptides may be combined with other suitable ingrédients, such as carriers and/or adjuvants. There are no limitations on the nature of such other ingrédients, except that they must be pharmaceutically acceptable for their intended administration, and cannot dégradé the activity of the active ingrédients of the composition. Examples of suitable vehicles
103 include ointments, creams, gels, or suspensions, with or without purified collagen. The MASP-2 inhibitory antibodies and polypeptides may also be impregnated into transdermal patches, plasters, and bandages, preferably in liquid or semi-liquid form.
The compositions of the present invention may be systemically administered on a periodic basis at intervals determined to maintain a desired level of therapeutic effect. For example, compositions may be administered, such as by subcutaneous injection, every two to four weeks or at less frequent intervals. The dosage regimen will be determined by the physicien considering various factors that may influence the action of the combination of agents. These factors will include the extent of progress of the condition being treated, the patienfs âge, sex and weight, and other clinical factors. The dosage for each individual agent will vary as a function ofthe MASP-2 inhibitory agent that is included in the composition, as well as the presence and nature of any drug delivery vehicle (e.g., a sustained release delîvery vehicle). In addition, the dosage quantity may be adjusted to account for variation in the frequency of administration and the pharmacokinetic behavior of the delivered agent(s).
LOCAL DELIVERY
As used herein. the term local encompasses application of a drug în or around a site of intended localized action, and may include for example topical delivery to the skin or other affected tissues, ophthalmic delivery, intrathecal (IT), intracerebroventricular (ICV), intra-articular, intracavity, intracranial or intravesicular administration, placement or irrigation. Local administration may be preferred to enable administration of a lower dose, to avoid systemic side effects, and for more accurate control of the timing of delivery and concentration of the active agents at the site of local delivery. Local administration provides a known concentration at the target site, regardless of interpatient variabilîty in metabolism, blood flow, etc. Improved dosage control is also provided by the direct mode of delivery.
Local delivery of a MASP-2 inhibitory agent may be achieved in the context of surgical methods for treating disease or disorder caused or exacerbated by fibrosis and/or inflammation such as for example during procedures such as surgery.
104
TREATMENT REGIMENS
In prophylactic applications, the pharmaceutical compositions comprising a MASP-2 inhibitory agent (e.g., a MASP-2 inhibitory antibody) are administered to a subject susceptible to, or otherwise at risk of developing a disease or disorder caused or exacerbated by fibrosis and/or inflammation in an amount sufficient to inhibit fibrosis and/or inflammation and thereby eliminate or reduce the risk of developing symptoms of the condition. In some embodiments, the pharmaceutical compositions are administered to a subject suspected of, or already suffering from, a disease or disorder caused or exacerbated by fibrosis and/or inflammation în a therapeutically effective amount sufficient to relieve, or at least partially reduce, the symptoms of the condition. In both prophylactic and therapeutic regimens, compositions comprising MASP-2 inhibitory agents may be administered in several dosages until a sufficient therapeutic outcome has been achieved în the subject. Application of the MASP-2 inhibitory compositions of the present invention may be carried out by a single administration of the composition, or a limited sequence of administrations, for treatment of an acute condition associated with fibrosis and/or inflammation. Alternatively, the composition may be administered at periodic intervals over an extended period of time for treatment of chronic conditions associated with fibrosis and/or inflammation.
In both prophylactic and therapeutic regimens, compositions comprising MASP-2 inhibitory agents may be administered în several dosages until a sufficient therapeutic outcome has been achieved in the subject. In one embodiment of the invention, the MASP-2 inhibitory agent comprises a MASP-2 antibody, which suitably may be administered to an adult patient (e.g., an average adult weight of 70 kg) in a dosage of from 0.1 mg to 10,000 mg, more suitably from 1.0 mg to 5,000 mg, more suitably 10.0 mg to 2,000 mg, more suitably 10.0 mg to 1,000 mg and still more suitably from 50.0 mg to 500 mg. For pédiatrie patients, dosage can be adjusted în proportion to the patient’s weight. Application of the MASP-2 inhibitory compositions of the present invention may be carried out by a single administration of the composition, or a limited sequence of administrations, for treatment of a subject suffering from or at risk for developing a disease or disorder caused or exacerbated by fibrosis and/or inflammation. Alternatively, the composition may be administered at periodic intervals such as daily, biweekly,
105 weekly, every other week, monthly or bimonthly over an extended period of time for treatment of a subject suffering from or at risk for developing a disease or disorder caused or exacerbated by fibrosis and/or inflammation.
In both prophylactîc and therapeutic regimens, compositions comprising MASP-2 inhibitory agents may be administered in several dosages until a sufïïcient therapeutic outcome has been achieved in the subject.
In some embodiments, a subject is identified to be at risk for developing a disease or disorder caused or exacerbated by fibrosis or inflammation by determining that the subject has one or more symptoms of impaired kidney function. as assessed, for example, by measuring sérum créatinine levels, sérum créatinine clearance, blood urea nitrogen levels, protein in the urine, and/or by measuring one or more biomarkers associated with a rénal disease or injury.
Methods for assessing rénal function are well known in the art and include, but art not limited to, measurements of blood systemic and glomerular capîllary pressure, proteinuria (e.g., albuminuria), microscopie and macroscopie hematuria, sérum creatînine level (e.g., one formula for estimating rénal function in humans equates a creatînine level of 2.0 mg/dl to 50 percent of normal kidney function and 4.0 mg/dl to 25 percent), décliné in the glomerular filtration rate (e.g., rate of creatînine clearance), and degree of tubular damage. For example, assessment of kidney function may include evaluating at least one kidney function using biological and/or physiological parameters such as sérum creatînine level, creatînine clearance rate, 24-hour urinary protein sécrétion, glomerular filtration rate, urinary albumin creatînine ratio, albumin excrétion rate, and rénal bîopsy (e.g., determining the degree of rénal fibrosis by measuring déposition of collagen and/or fibronectin).
VL EXAMPLES
The following examples merely illustrate the best mode now contemplated for practicing the invention, but should not be construed to limit the invention. Ali literature citations herein are expressly incorporated by reference.
106
EXAMPLE 1
This example describes the génération of a mouse strain déficient in MASP-2 (MASP-2-/-) but sufficient of MApl9 (MApl9+/+).
Materials and Methods: The targeting vector pKO-NTKV 1901 was designed 5 to disrupt the three exons coding for the C-terminal end of murine MASP-2, including the exon that encodes the serine protease domain, as shown in FIGURE 3. PKO-NTKV 1901 was used to transfect the murine ES cell lineE14,la (SV129Ola). Neomycin-resistant and Thymidine Kinase-sensitive clones were selected. 600 ES clones were screened and, of these, four different clones were identified and verified by southem 10 blot to contain the expected sélective targeting and recombination event as shown in FIGURE 3. Chimeras were generated from these four positive clones by embryo transfer. The chimeras were then backcrossed in the genetic background C57/BL6 to create transgenic males. The transgenic males were crossed with females to generate Fis with 50% of the offspring showing heterozygosity for the disrupted MASP-2 gene. The 15 heterozygous mice were intercrossed to generate homozygous MASP-2 déficient offspring, resulting in heterozygous and wild-type mice in the ration of 1:2:1, respectively.
Results and Phenotype: The resulting homozygous MASP-2-/- déficient mice were found to be viable and fertile and were verified to be MASP-2 déficient by southem 20 blot to confirm the correct targeting event, by Northern blot to confirm the absence of MASP-2 mRNA, and by Western blot to confirm the absence of MASP-2 protein (data not shown). The presence ofMApl9 mRNA and the absence of MASP-2 mRNA were further confirmed using time-resolved RT-PCR on a LightCycler machine. The MASP-2-/- mice do continue to express MApl9, MASP-1, and MASP-3 mRNA and 25 protein as expected (data not shown). The presence and abundance of mRNA in the MASP-2-/- mice for Properdin, Factor B, Factor D, C4, C2, and C3 was assessed by LightCycler analysis and found to be identical to that of the wild-type littermate Controls (data not shown). The plasma from homozygous MASP-2-/- mice is totally déficient of lectin-pathway-mediated complément activation as further described in Example 2.
107
Génération of a MASP-2-/- strain on a pure C57BL6 Background: The MASP-2-/- mice were back-crossed with a pure C57BL6 line for nine générations prior to use of the MASP-2-/- strain as an experimental animal mode!.
A transgenic mouse strain that is murine MASP-2-/-, MApl9+/+ and that expresses a human MASP-2 transgene (a murine MASP-2 knock-out and a human MASP-2 knock-in) was also generated as follows:
Materials and Methods: A minigene encoding human MASP-2 called mini hMASP-2 (SEQ ID NO:49) as shown in FIGURE 4 was constructed which includes the promoter région of the human MASP 2 gene, including the first 3 exons (exon l to exon 3) followed by the cDNA sequence that represents the coding sequence of the following 8 exons, thereby encoding the full-length MASP-2 protein driven by its endogenous promoter. The mini hMASP-2 construct was injected into fertilized eggs of MASP-2-/- in order to replace the déficient murine MASP 2 gene by transgenically expressed human MASP-2.
EXAMPLE 2
This example demonstrates that MASP-2 is required for complément activation via the lectin pathway.
Methods and Materials:
Lectin pathway spécifie C4 Cleavage Assay: A C4 cleavage assay has been described by Petersen, et ai., J. Immunol. Methods 257:107 (2001) that measures lectin pathway activation resulting from lipoteichoic acid (LTA) from S. auretts, which binds L-ficolin. The assay described by Petersen et al., (2001) was adapted to measure lectin pathway activation via MBL by coating the plate with LPS and mannan or zymosan prior to adding sérum from MASP-2 -/- mice as described below. The assay was also modified to remove the possibility of C4 cleavage due to the classîcal pathway. This was achieved by using a sample dilution buffer containing l M NaCl, which permits high affînity binding of lectin pathway récognition components to their ligands but prevents activation of endogenous C4, thereby excluding the participation of the classîcal pathway by dissociating the C l complex. Briefly described, in the modified assay sérum samples (diluted in high sait (l M NaCl) buffer) are added to ligand-coated plates, followed by the
108 addition of a constant amount of purified C4 in a buffer with a physîological concentration of sait. Bound récognition complexes containing MASP-2 cleave the C4, resultîng in C4b déposition.
Assay Methods:
1) Nunc Maxisorb microtiter plates (MaxiSorb®, Nunc, Cat. No. 442404, Fisher Scientific) were coated with 1 pg/ml mannan (M7504 Sigma) or any other ligand (e.g., such as those listed below) diluted in coating buffer (15 mM Na2CC>3, 35 mM NaHCCty, pH 9.6).
The following reagents were used in the assay:
a. mannan (l pg/well mannan (M7504 Sigma) in 100 pl coating buffer):
b. zymosan (1 pg/well zymosan (Sigma) în 100 pl coating buffer);
c. LTA (1 pg/well in 100 pl coating buffer or 2 pg/well in 20 pl methanol)
d. 1 pg of the H-ficolin spécifie Mab 4H5 in coating buffer
e. PSA from Aerococcus viridans (2 pg/well în 100 pl coating buffer)
f. lOOpl/well of formalin-fixed S. aureus DSM20233 (OD55Q=0.5) in coating buffer.
2) The plates were incubated ovemight at 4°C.
3) After ovemight incubation, the residual protein binding sites were saturated by incubated the plates with 0.1% HSA-TBS biocking buffer (0.1% (w/v) HSA in 10 mM Tris-CL, 140 mM NaCl, 1.5 mM NaNj, pH 7.4) for 1-3 hours, then washing the plates 3X with TBS/tween/Ca^4, (TBS with 0.05% Tween 20 and 5 mM CaCty, 1 mM MgC12, pH 7.4).
4) Sérum samples to be tested were diluted in MBL-binding buffer (1 M NaCl) and the diluted samples were added to the plates and incubated ovemight at 4°C. Wells receiving buffer only were used as négative Controls.
5) Following incubation ovemight at 4°C, the plates were washed 3X with TBS/tween/Ca2+. Human C4 (100 pl/well of 1 pg/ml diluted in BBS (4 mM barbital, 145 mM NaCl, 2 mM CaCfi, 1 mM MgC12, pH 7.4)) was then added to the plates and incubated for 90 minutes at 37°C. The plates were washed again 3X with TBS/tween/Ca2+.
109
6) C4b déposition was detected with an alkaline phosphatase-conjugated chicken anti-human C4c (diluted l : 1000 în TBS/tween/Ca2+), which was added to the plates and incubated for 90 minutes at room température. The plates were then washed agaîn 3X with TBS/tween/Ca^+.
7) Alkaline phosphatase was detected by adding 100 μΐ of p-nitrophenyl phosphate substrate solution, incubating at room température for 20 minutes, and reading the OD4Q5 in a microtiter plate reader.
Results: FIGURES 5A-B show the amount of C4b déposition on mannan (FIGURE 5A) and zymosan (FIGURE 5B) în sérum dilutions from MASP-2+/+ (crosses), MASP-2+/- (closed circles) and MASP-2-/- (closed triangles). FIGURE 5C shows the relative C4 convertase activity on plates coated with zymosan (white bars) or mannan (shaded bars) from MASP-2-/+ mice (n=5) and MASP-2-/- mice (n=4) relative to wild-type mice (n=5) based on measuring the amount of C4b déposition normalized to wîld-type sérum. The error bars represent the standard déviation. As shown în FIGURES 5A-C, plasma from MASP-2-/- mice is totally déficient in lectin-pathway-mediated complément activation on mannan and on zymosan coated plates. These results clearly demonstrate that MASP-2 is an effector component of the lectîn pathway.
Recombinant MASP-2 reconstitutes Lectin Pathway-Dependent C4 Activation in sérum from the MASP-2-/- mice
In order to establish that the absence of MASP-2 was the direct cause of the loss of lectin pathway-dependent C4 activation in the MASP-2-/- mice, the effect of adding recombinant MASP-2 protein to sérum samples was examined in the C4 cleavage assay described above. Functîonally active murine MASP-2 and catalytically inactive murine MASP-2A (in which the active-site serine residue in the serine protease domain was substituted for the alanine residue) recombinant proteins were produced and purified as described below în Example 3. Pooled sérum from 4 MASP-2 -/- mice was pre-incubated with increasing protein concentrations of recombinant murine MASP-2 or inactive recombinant murine MASP-2A and C4 convertase activity was assayed as described above.
I ΙΟ
Results: As shown în FIGURE 6, the addition of ftinctionally active murine recombinant MASP-2 protein (shown as open triangles) to sérum obtained from the MASP-2 -/- mice restored lectin pathway-dependent C4 activation in a protein concentration dépendent manner, whereas the catalytically inactive murine MASP-2A protein (shown as stars) did not restore C4 activation. The results shown în FIGURE 6 are normalized to the C4 activation observed with pooled wîld-type mouse sérum (shown as a dotted line).
EXAMPLE 3
This example describes the recombinant expression and protein production of recombinant full-length human, rat and murine MASP-2, MASP-2 derived polypeptides, and catalytically inactivated mutant forms of MASP-2
Expression of Full-length human, murine and rat MASP-2:
The full length cDNA sequence of human MASP-2 (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 région (described in Kaufman R.J. et al., Nucleic Acids Research /9:4485-90, 1991; Kaufman, Methods in Enzymology, 185:537-66 (1991)). The 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. The MASP-2 expression vectors were then transfected into the adhèrent Chinese hamster ovary cell line DXB1 using the standard calcium phosphate transfection procedure described in Maniatis et al., 1989. Cells transfected with these constructs grew very slowly, implyîng that the encoded protease is cytotoxic.
In another approach, the minigene construct (SEQ ID NO:49) containing the human cDNA of MASP-2 driven by its endogenous promoter is transiently transfected into Chinese hamster ovary cells (CHO). The human MASP-2 protein is secreted into the culture media and isolated as described below.
Expression of Full-length catalytically inactive MASP-2:
Rationale: MASP-2 is activated by autocatalytic cleavage after the récognition subcomponents MBL or ficolins (either L-ficolin, H-fïcolin or M-ficolin) bind to their respective carbohydrate pattern. Autocatalytic cleavage resulting în activation of lll
MASP-2 often occurs during the isolation procedure of MASP-2 from sérum, or during the purification following recombinant expression. In order to obtain a more stable protein préparation for use 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 trîad of the protease domain with an alanine residue in rat (SEQ ID NO:55 Ser6l7 to Ala6l7); in mouse (SEQ ID NO:52 Ser6l7 to Ala6l7); or in human (SEQ ID NO:6 Ser6l8to Ala6i8).
In order to generate catalytically inactive human and murine MASP-2A proteins, site-dîrected mutagenesis was carried out using the oligonucleotides shown in TABLE 5. The oligonucleotides in TABLE 5 were designed to anneal to the région of the human and murine cDNA encoding the enzymatically active serine and oligonucleotide contain a mismatch in order to change the serine codon into an alanine codon. For example, PCR oligonucleotides SEQ ID NOS:56-59 were used in combination with human MASP-2 cDNA (SEQ ID NO:4) to amplify the région from the start codon to the enzymatically active serine and from the serine to the stop codon to generate the complété open reading from of the mutated MASP-2A containing the Ser6l8 to Ala6l8 mutation. The PCR products were purified after agarose gel electrophoresis and band préparation and single adenosine overlaps were generated using a standard tailing procedure. The adenosine tailed MASP-2A was then cloned into the pGEM-T easy vector, transformed into E. coli.
A catalytically inactive rat MASP-2A protein was generated by kinasing and annealing SEQ ID NO:64 and SEQ ID NO:65 by combining these two oligonucleotides in equal molar amounts, heating at l00°C for 2 minutes and slowly cooling to room température. The resulting annealed fragment has Pstl and Xbal compatible ends and was inserted in place of the Pstl-Xbal fragment of the wild-type rat MASP-2 cDNA (SEQ ID NO:53) to generate rat MASP-2A.
’GAGGTGACGCAGGAGGGGCATTAGTGTTT 3’ (SEQ ID NO:64)
5' CTAGAAACACTAATGCCCCTCCTGCGTCACCTCTGCA 3’ (SEQ ID NO:65)
The human, murine and rat MASP-2A were each further subcloned into either of the mammalîan expression vectors pED or pCI-Neo and transfected into the Chinese Hamster ovary cell line DXBl as described below.
1I2
In another approach, a catalytically inactive form of MASP-2 is constructed using the method described in Chen et ai., J. Biol. Chem., 276(28):25894-25902, 2001. Briefly, the plasmid containing the full-length human MASP-2 cDNA (described in Thiel et al., Nature 356:506, 1997) is digested with Xho\ and EcoRA and the MASP-2 cDNA (described herein as SEQ ID NO:4) is cioned into the corresponding restriction sites of the pFastBacl baculovirus transfer vector (Life Technologies, NY). The MASP-2 serine protease active site at Ser6l8 is then altered to Ala6l8 by substituting the double-stranded oligonucleotides encoding the peptide région amino acid 610-625 (SEQ ID NO: 13) with the native région amino acids 610 to 625 to create a MASP-2 full length polypeptide with an inactive protease domain.
Construction of Expression Plasmids Containing Polypeptide Régions Derived from Human Masp-2.
The following constructs are produced using the MASP-2 signal peptide (residues 1-15 of SEQ ID NO:5) to secrete various domains of MASP-2. A construct expressing the human MASP-2 CUBI domain (SEQ ID NO:8) is made by PCR amplifying the région encoding residues 1-121 of MASP-2 (SEQ ID NO:6) (corresponding to the N-terminal CUBI domain). A construct expressing the human MASP-2 CUBIEGF domain (SEQ ID NO:9) is made by PCR amplifying the région encoding residues 1—166 of MASP-2 (SEQ ID NO:6) (corresponding to the N-terminal CUBIEGF domain). A construct expressing the human MASP-2 CUBIEGFCUBII domain (SEQ ID NO: 10) is made by PCR amplifying the région encoding residues 1-293 of MASP-2 (SEQ ID NO:6) (corresponding to the N-terminal CUBIEGFCUBII domain). The above mentioned domains are amplified by PCR using VentR polymerase and pBS-MASP-2 as a template, according to established PCR methods. The 5' primer sequence of the sense primer (5’-CGGGATCCATGAGGCTGCTGACCCTC-3' SEQ ID NO:34) introduces a Bam\A\ restriction site (underlined) at the 5' end of the PCR products. Antîsense primers for each of the MASP-2 domains, shown below in TABLE 5, are designed to introduce a stop codon (boldface) followed by an Eco RI site (underlined) at the end of each PCR product. Once amplified, the DNA fragments are digested with B«mHI and Eco RI and cioned into the corresponding sites of the pFastBacl
II3 vector. The resulting construis are characterized by restriction mapping and confirmed by dsDNA sequencing.
TABLE 5: MASP-2 PCR PRIMERS
MASP-2 domain 5' PCR Primer 3' PCR Primer
SEQ ID NO:8 CUBI (aa 1-121 ofSEQ ID NO;6) 5'CGGGATCCATGAG GCTGCTGACCCTC-3’ (SEQ ID NO:34) 5'GGAATTCCTAGGCTGCA TA (SEQ ID NO:35)
SEQ ID NO:9 CUBIEGF (aa I-I66 of SEQ ID NO:6) 5'CGGGATCCATGAG GCTGCTGACCCTC-3' (SEQ ID NO:34) 5’GGAATTCCTACAGGGCG CT-3' (SEQ ID NO:36)
SEQ ID NO:lO CUBIEGFCUBII (aa 1-293 ofSEQ ID NO:6) 5’CGGGATCCATGAG GCTGCTGACCCTC-3’ (SEQ ID NO:34) 5'GGAATTCCTAGTAGTGG AT 3' (SEQ ID NO:37)
SEQ ID NO:4 human MASP-2 5'ATGAGGCTGCTGA CCCTCCTGGGCCTTC 3’ (SEQ ID NO: 56) hMASP-2 forward 5'TTAAAATCACTAATTAT GTTCTCGATC 3' (SEQ ID NO: 59) hMASP-2_reverse
SEQ ID NO:4 human MASP-2 cDNA 5’CAGAGGTGACGCA GGAGGGGCAC 3' (SEQ ID NO: 58) hMASP-2 ala forward 5'GTGCCCCTCCTGCGTCA CCTCTG 3' (SEQ ID NO: 57) hMASP-2_ala_reverse
SEQ ID NO:50 Murine MASP-2 cDNA 5'ATGAGGCTACTCA TCTTCCTGG3' (SEQ ID NO: 60) mMASP-2 forward 5'TTAGAAATTACTTATTAT GTTCTCAATCC3' (SEQ ID NO: 63) mMASP-2_reverse
SEQ ID NO:50 Murine MASP-2 cDNA 5'CCCCCCCTGCGTC ACCTCTGCAG3' (SEQ ID NO: 62) mMASP-2_aia forward 5'CTGCAGAGGTGACGCAG GGGGGG 3’ (SEQ ID NO: 61) mMASP-2_ala_reverse
Recombinant eukaryotic expression of MASP-2 and protein production of enzymatically inactive mouse, rat, and human MASP-2A.
The MASP-2 and MASP-2A expression construits described above were transfected into DXBl cells using the standard calcium phosphate transfectîon procedure
H4 (Maniatis et al., 1989). MASP-2A was produced in serum-free medium to ensure that préparations were not contaminated with other sérum proteins. Media was harvested from confluent cells every second day (four times in total). The level of recombinant MASP-2A averaged approximately 1.5 mg/liter of culture medium for each of the three 5 species.
MASP-2A protein purification: The MASP-2A (Ser-Ala mutant described above) was purified by affinîty chromatography on MBP-A-agarose columns. This strategy enabled rapid purification without the use of extraneous tags. MASP-2A (100-200 ml of medium diluted with an equal volume of loading buffer (50 mM Tris-Cl, 10 pH 7.5, containing 150 mM NaCI and 25 mM CaCh) was loaded onto an MBP-agarose affinîty column (4 ml) pre-equilibrated with 10 ml of loading buffer. Following washing with a further 10 ml of loading buffer, protein was eluted in 1 ml fractions with 50 mM Tris-Cl, pH 7.5, containing 1.25 M NaCI and 10 mM EDTA. Fractions containing the MASP-2A were identified by SDS-polyacrylamide gel electrophoresis. Where necessary, 15 MASP-2A was purified further by ion-exchange chromatography on a MonoQ column (HR 5/5). Protein was dialyzed with 50 mM Tris-Cl pH 7.5, containing 50 mM NaCI and loaded onto the column equilibrated in the same buffer. Following washing, bound MASP-2A was eluted with a 0.05-1 M NaCI gradient over 10 ml.
Results: Yields of 0.25-0.5 mg of MASP-2A protein were obtained from 200 ml 20 of medium. The molecular mass of 77.5 kDa determined by MALDI-MS is greater than the calculated value of the unmodified polypeptide (73.5 kDa) due to glycosylation. Attachment of glycans at each of the A-glycosylation sites accounts for the observed mass. MASP-2A migrâtes as a single band on SDS-polyacrylamide gels, demonstrating that it is not proteolytically processed during biosynthesis. The weight-average molecular 25 mass determined by equilibrium ultracentrifugation is in agreement with the calculated value for homodimers of the glycosylated polypeptide.
PRODUCTION OF RECOMBINANT HUMAN MASP-2 POLYPEPTIDES
Another method for producing recombinant MASP-2 and MASP2A derived polypeptides is described in Thîelens, N.M., et al., J. Immunol. 166:5068-5077, 2001. 30 Briefly, the Spodoptera frugiperda insect cells (Ready-Plaque Sf9 cells obtained from Novagen, Madison, WI) are grown and maintaîned in SÎ900II serum-free medium (Life
IIS
Technologies) supplemented with 50 ILf/ml penicillin and 50 mg/ml streptomycin (Life Technologies). The Trichophtsia ni (High Five) insect cells (provided by Jadwîga Chroboczek, Institut de Biologie Structurale, Grenoble, France) are maintained in TCI00 medium (Life Technologies) containing 10% FCS (Dominique Dutscher, Brumath, France) supplemented with 50 lU/ml penicillin and 50 mg/ml streptomycin.
(fi) Recombinant baculoviruses are generated using the Bac-to-Bac system (Life Technologies). The bacmid DNA is purified using the Qiagen midiprep purification System (Qiagen) and is used to transfect Sf9 insect cells using cellfectin in SÎ900 II SFM medium (Life Technologies) as described in the manufacturées protocol. Recombinant virus particles are collected 4 days later, titrated by virus plaque assay, and amplified as described by King and Possee, in The Baculovirus Expression System: A Laboratory Guide, Chapman and Hall Ltd., London, pp. 111-114, 1992.
High Five cells (1.75 x IO7 cells/175-cm2 tissue culture flask) are infected with the recombinant viruses containing MASP-2 polypeptides at a multiplicity of infection of 2 in Sf900 II SFM medium at 28°C for 96 h. The supematants are collected by centrifugation and diisopropyl phosphorofiuoridate is added to a final concentration of 1 mM.
The MASP-2 polypeptides are secreted in the culture medium. The culture supematants are dialyzed against 50 mM NaCl, 1 mM CaCh, 50 mM triethanolamine hydrochloride, pH 8.1, and loaded at 1.5 ml/min onto a Q-Sepharose Fast Flow column (Amersham Pharmacia Biotech) (2.8 x 12 cm) equilibrated in the same buffer. Elution is conducted by applying a 1.2 liter linear gradient to 350 mM NaCl in the same buffer. Fractions containing the recombinant MASP-2 polypeptides are identified by Western blot analysis, precîpitated by addition of (NHjJsSCh to 60% (w/v), and left overnight at4°C. The pellets are resuspended in 145 mM NaCl, 1 mM CaCh, 50 mM triethanolamine hydrochloride, pH 7.4, and applied onto a TSK G3000 SWG column (7.5 x 600 mm) (Tosohaas, Montgomeryville, PA) equilibrated in the same buffer. The purified polypeptides are then concentrated to 0.3 mg/ml by ultrafiltration on Microsep microconcentrators (m.w. cut-off= 10,000) (Filtron, Karlstein, Germany).
H6
EXAMPLE 4
This example describes a method of producing polyclonal antibodies against MASP-2 polypeptides.
Materials and Methods:
MASP-2 Antigens: Polyclonal anti-human MASP-2 antiserum is produced by immunizing rabbits with the following isolated MASP-2 polypeptides: human MASP-2 (SEQ ID N0:6) isolated from sérum; recombinant human MASP-2 (SEQ ID NO:6), MASP-2A containing the inactive protease domain (SEQ ID NO:l3), as described în Example 3; and recombinant CUBI (SEQ ID NO:8), CUBEGFI (SEQ ID NO:9), and 10 CUBEGFCUBII (SEQ ID NO: 10) expressed as described above in Example 3.
Polyclonal antibodies: Six-week old Rabbits, primed with BCG (bacillus Calmette-Guerin vaccine) are immunized by injecting 100 pg of MASP-2 polypeptide at 100 gg/ml in stérile saline solution. Injections are done every 4 weeks, with antibody titer monitored by ELISA assay as described in Example 5. Culture supematants are 15 collected for antibody purification by protein A affinity chromatography.
EXAMPLE 5
This example describes a method for producing murine monoclonal antibodies against rat or human MASP-2 polypeptides.
Materials and Methods:
Male A/J mice (Harlan, Houston, Tex.), 8-12 weeks old, are injected subcutaneously with 100 gg human or rat rMASP-2 or rMASP-2A polypeptides (made as described in Example 3) in complété Freund's adjuvant (Difco Laboratories, Detroit, Mich.) în 200 μΐ of phosphate buffered saline (PBS) pH 7.4. At two-week intervals the 25 mice are twice injected subcutaneously with 50 gg of human or rat rMASP-2 or rMASP-2A polypeptide in incomplète Freund's adjuvant. On the fourth week the mice are injected with 50 gg of human or rat rMASP-2 or rMASP-2A polypeptide in PBS and are fused 4 days later.
For each fusion, single cell suspensions are prepared from the spleen of an 30 immunized mouse and used for fusion with Sp2/0 myeloma cells. 5x10^ of the Sp2/0 and 5x10^ spleen cells are fused in a medium containing 50% polyethylene glycol ll7 (M.W. 1450) (Kodak, Rochester, N.Y.) and 5% dlmethylsulfoxide (Sigma Chemical Co., St. Louis, Mo.). The cells are then adjusted to a concentration of l.5xl05 spleen cells per 200 μΐ of the suspension in Iscove medium (Gibco, Grand Island, N.Y.), supplemented with 10% fêtai bovine sérum, lOOunits/ml of penîcillin, 100 pg/ml of streptomycin, 0.1 mM hypoxanthine, 0.4 μΜ aminopterin and 16 μΜ thymidine. Two hundred microliters of the cell suspension are added to each well of about twenty 96-well microculture plates. After about ten days culture supematants are withdrawn for screening for reactivity with purified factor MASP-2 in an ELISA assay.
ELISA Assay: Wells of Immulon® 2 (Dynatech Laboratories, Chantilly, Va.) microtest plates are coated by adding 50 μΐ of purified hMASP-2 at 50 ng/ml or rat rMASP-2 (or rMASP-2A) ovemight at room température. The low concentration of MASP-2 for coating enables the sélection of high-afFinîty antibodies. After the coating solution is removed by flicking the plate, 200 μΐ of BLOTTO (non-fat dry milk) in PBS is added to each well for one hour to block the non-specific sites. An hour later, the wells are then washed with a buffer PBST (PBS containing 0.05% Tween 20). Fifty microliters of culture supematants from each fusion well is collected and mixed with 50 μ! of BLOTTO and then added to the individual wells of the microtest plates. After one hour of incubation, the wells are washed with PBST. The bound murine antibodies are then detected by reaction with horseradish peroxidase (HRP) conjugated goat antî-mouse IgG (Fc spécifie) (Jackson ImmunoResearch Laboratories, West Grave, Pa.) and diluted at 1:2,000 in BLOTTO. Peroxidase substrate solution containing 0.1% 3,3,5,5 tetramethyl benzidine (Sigma, St. Louis, Mo.) and 0.0003% hydrogen peroxide (Sigma) is added to the wells for color development for 30 minutes. The reaction is terminated by addition of 50 μΐ of 2M H2SO4 per well. The Optical Density at 450 nm of the reaction mixture is read with a BioTek® ELISA Reader (BioTek® Instruments, Winooski, Vt.).
MASP-2 Binding Assay:
Culture supematants that test positive in the MASP-2 ELISA assay described above can be tested in a binding assay to détermine the binding affinity the MASP-2 inhibitory agents hâve for MASP-2. A similar assay can also be used to détermine if the inhibitory agents bind to other antigens in the complément system.
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Polystyrène microtiter plate wells (96-well medium binding plates, Corning Costar, Cambridge, MA) are coated with MASP-2 (20 ng/100 μΐ/well, Advanced Research Technology, San Diego, CA) in phosphate-buffered saline (PBS) pH7.4 ovemight at 4°C. After aspirating the MASP-2 solution, wells are blocked with PBS 5 containing l% bovine sérum albumin (BSA; Sigma Chemical) for 2 h at room température. Wells without MASP-2 coating serve as the background Controls. Aliquots of hybridoma supematants or purified anti-MASP-2 MoAbs, at varyÎng concentrations in blocking solution, are added to the wells. Following a 2 h incubation at room température, the wells are extensively rinsed with PBS. MASP-2-bound anti-MASP-2 10 MoAb is detected by the addition of peroxidase-conjugated goat anti-mouse IgG (Sigma Chemical) in blocking solution, which is allowed to incubate for Ih at room température. The plate is rinsed again thoroughly with PBS, and 100 μΐ of 3,3',5,5'-tetramethyl benzidine (TMB) substrate (Kirkegaard and Perry Laboratories, Gaithersburg, MD) is added. The reaction of TMB is quenched by the addition of 100 μΐ of IM phosphoric 15 acid, and the plate is read at 450 nm in a microplate reader (SPECTRA MAX 250, Molecular Devices, Sunnyvale, CA).
The culture supematants from the positive wells are then tested for the ability to inhibit complément activation in a functional assay such as the C4 cleavage assay as described in Example 2. The cells in positive wells are then cloned by limiting dilution. 20 The MoAbs are tested again for reactivity with hMASP-2 in an ELISA assay as described above. The selected hybridomas are grown in spinner flasks and the spent culture supematant collected for antibody purification by protein A affinity chromatography.
EXAMPLE 6
This example describes the génération and production of humanized murine anti-MASP-2 antibodies and antibody fragments.
A murine anti-MASP-2 monoclonal antibody is generated in Male A/J mice as described in Exemple 5. The murine antibody is then humanized as described below to reduce its immunogenicîty by replacîng the murine constant régions with their human 30 counterparts to generate a chimeric IgG and Fab fragment of the antibody, which is useful
119 for inhibiting the adverse effects of MASP-2-dependent complément activation in human subjects in accordance with the present invention.
1. Cloning of anti-MASP-2 variable région genes from murine hybridoma cells. Total RNA is isolated from the hybridoma cells secreting anti-MASP-2 MoAb (obtained as described in Example 7) using RNAzol following the manufacturées protocol (Biotech. Houston, Tex.). First strand cDNA is synthesized from the total RNA using oligo dT as the primer. PCR is performed using the immunoglobulîn constant C region-derived 3' primers and degenerate primer sets derived from the leader peptide or the first framework région of murine Vjq or Vj< genes as the 5' primers. Anchored PCR is carried out as described by Chen and Platsucas (Chen, P.F., Scand. J. Immunol. 35:539-549, 1992). For cloning the Vj< gene, double-stranded cDNA is prepared using a Notl-MAKl primer (5'-TGCGGCCGCTGTAGGTGCTGTCTTT-3' SEQ ID NO:38). Annealed adaptors ADI (5'-GGAATTCACTCGTTATTCTCGGA-3' SEQ ID NO:39) and AD2 (5'-TCCGAGAATAACGAGTG-3' SEQ ID NO:40) are ligated to both 5' and 3' termini of the double-stranded cDNA. Adaptors at the 3' ends are removed by Notl digestion. The digested product is then used as the template in PCR with the ADI oligonucleotide as the 5' primer and MAK2 (5'-CATTGAAAGCTTTGGGGTAGAAGTTGTTC-3' SEQ ID NO:41) as the 3’ primer. DNA fragments of approximately 500 bp are cloned into pUC19. Several clones are selected for sequence analysis to verify that the cloned sequence encompasses the expected murine immunoglobulîn constant région. The Notl-MAKl and MAK2 oligonucleotides are derived from the Vp; région and are 182 and 84 bp, respectively, downstream from the first base pair of the C kappa gene. Clones are chosen that include the complété Vp; and leader peptide.
For cloning the Vpq gene, double-stranded cDNA is prepared using the Notl MAGI primer (5'-CGCGGCCGCAGCTGCTCAGAGTGTAGA-3‘ SEQ ID NO:42). Annealed adaptors ADI and AD2 are ligated to both 5' and 3' termini of the double-stranded cDNA. Adaptors at the 3' ends are removed by Notl digestion. The digested product are used as the template in PCR with the ADI oligonucleotide and MAG2 (5'-CGGTAAGCTTCACTGGCTCAGGGAAATA-3' SEQ ID NO:43) as primers. DNA fragments of 500 to 600 bp in length are cloned into plJC19. The Notl-MAGl and
120
MAG2 oligonucleotides are derived from the murine Cy.7.l région, and are 180 and 93 bp, respectively, downstream from the first bp of the murine Cy.7.l gene. Clones are chosen that encompass the complété Vjq and leader peptide.
2. Construction of Expression Vectors for Chimeric MASP-2 IgG and Fab. The cloned Vjq and genes described above are used as templates in a PCR reaction to add the Kozak consensus sequence to the 5' end and the splice donor to the 3' end of the nucléotide sequence. After the sequences are analyzed to confirm the absence of PCR errors, the Vjq and genes are inserted into expression vector cassettes containing human C.yl and C. kappa respectively, to give pSV2neoVHhuCyl and pSV2neoV-huCy. CsCl gradient-purified plasmid DNAs of the heavy- and light-chain vectors are used to transfect COS cells by electroporation. After 48 hours, the culture supematant is tested by ELISA to confirm the presence of approximately 200 ng/ml of chimeric IgG. The cells are harvested and total RNA is prepared. First strand cDNA is synthesized from the total RNA using oligo dT as the primer. This cDNA is used as the template in PCR to generate the Fd and kappa DNA fragments. For the Fd gene, PCR is carried out using 5'-AAGAAGCTTGCCGCCACCATGGATTGGCTGTGGAACT-3 (SEQ ID NO:44) as the 5' primer and a CH l-derived 3' primer (S'-CGGGATCCTCAAACTTTCTTGTCCACCTTGG-S’ SEQ ID NO:45). The DNA sequence is confirmed to contain the complété Vjq and the CHl domain of human IgGl. After digestion with the proper enzymes, the Fd DNA fragments are inserted at the HindlII and BamHI restriction sites of the expression vector cassette pSV2dhfr-TUS to give pSV2dhfrFd. The pSV2 plasmid is commercially available and consists of DNA segments from various sources: pBR322 DNA (thin line) contains the pBR322 origin of DNA réplication (pBR ori) and the lactamase ampîcillin résistance gene (Amp); SV40 DNA, represented by wider hatching and marked, contains the SV40 origin of DNA réplication (SV40 ori), early promoter (5' to the dhfr and neo genes), and polyadenylation signal (3' to the dhfr and neo genes). The SV40-derived polyadenylation signal (pA) is also placed at the 3' end of the Fd gene.
For the kappa gene, PCR is carried out using 5’AAGAAAGCTTGCCGCCACCATGTTCTCACTAGCTCT-3' (SEQ ID NO:46) as the 5’ primer and a Cj^-derived 3' primer (5'-CGGGATCCTTCTCCCTCTAACACTCT-3' SEQ
I2l
ID NO:47). DNA sequence is confirmed to contain the complété V« and human Cj; régions. After digestion with proper restriction enzymes, the kappa DNA fragments are inserted at the HindiII and BamHI restriction sites of the expression vector cassette pSV2neo-TUS to give pSV2neoK. The expression of both Fd and .kappa genes are driven by the HCMV-derived enhancer and promoter éléments. Since the Fd gene does not include the cysteîne amino acid residue involved in the inter-chain disulfide bond, this recombinant chimeric Fab contains non-covalently linked heavy- and light-chaîns. This chimeric Fab is designated as cFab.
To obtain recombinant Fab with an inter-heavy and light chain disulfide bond, the above Fd gene may be extended to include the coding sequence for additional 9 amino acids (EPKSCDKTH SEQ ID NO:48) from the hinge région of human IgGl. The BstEII-BamHl DNA segment encoding 30 amino acids at the 3’ end of the Fd gene may be replaced with DNA segments encoding the 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 are transfected with purified plasmid DNAs of pSV2neoVjq-huC.yl and pSV2neoV-huC kappa by electroporation. Transfected cells are selected in the presence of 0.7 mg/ml G418. Cells are grown in a 250 ml spinner flask using serum-containing medium.
Culture supematant of lOOml spinner culture is loaded on a ΙΟ-ml PROSEP-A column (Bioprocessing, Inc., Princeton, N.J.). The column is washed with 10 bed volumes of PBS. The bound antibody is eluted with 50 mM citrate buffer, pH 3.0. Equal volume of l M 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 Millîpore membrane ultrafiltration (M.W. cut-off: 3,000). The protein concentration of the purified antibody is determined by the BCA method (Pierce).
4. Expression and purification of chimeric anti-MASP-2 Fab
To generate cell lines secreting chimeric anti-MASP-2 Fab, CHO cells are transfected with purified plasmid DNAs of pSV2dhfrFd (or pSV2dhfrFd/9aa) and pSV2neokappa, by electroporation. Transfected cells are selected in the presence of G418 and methotrexate. Selected cell lines are amplified in increasing concentrations of methotrexate. Cells are single-cell subcloned by limiting dilution. High-producing
122 single-cell subcloned cell Unes are then grown in 100 ml spinner culture using serum-free medium.
Chimeric anti-MASP-2 Fab is purified by affinity chromatography using a mouse anti-idiotypic MoAb to the MASP-2 MoAb. An anti-îdiotypic MASP-2 MoAb can be 5 made by immunizing mice with a murine anti-MASP-2 MoAb conjugated with keyhole limpet hemocyanin (KLH) and screening for spécifie MoAb binding that can be competed with human MASP-2. For purification, 100 ml of supematant from spinner cultures of CHO cells producing cFab or cFab/9aa are loaded onto the affinity column coupled with an anti-idiotype MASP-2 MoAb. The column is then washed thoroughly 10 with PBS before the bound Fab is eluted with 50 mM 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 BCA method (Pierce).
The ability of the chimeric MASP-2 IgG, cFab, and cFAb/9aa to inhibit MASP-2-dépendent complément pathways may be determined by using the inhibitory 15 assays 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 20 complément activation via L-fïcolin/P35, H-ficolin, M-ficoIin or mannan.
C4 Cleavage Assay: A C4 cleavage assay has been described by Petersen,
S.V., et al., J. Immunol. Methods 257:107, 2001, which measures lectin pathway activation resulting from lipoteichoic acid (LTA) from 5. aureus which binds L-ficolin.
Reagents: Formalin-fixed S. aureous (DSM20233) is prepared as follows: 25 bacteria is grown ovemight at 37°C in tryptic soy blood medium, washed three times with PBS, then fixed for 1 h at room température in PBS/0.5% formalin, and washed a further three times with PBS, before being resuspended in coating buffer (15 mM Na2Co3, 35 mM NaHCO3, pH 9.6).
Assay: The wells of a Nunc MaxiSorb® microtiter plate (Nalgene Nunc 30 International, Rochester, NY) are coated with: 100 μΐ of formalin-fixed S. aureus DSM20233 (OD550 = 0-5) in coating buffer with 1 pg of L-ficolin in coating buffer.
123
After ovemight incubation, wells are blocked with 0.1% human sérum albumin (HSA) in TBS (lOmM Tris-HCl, !40mM NaCl, pH 7.4), then are washed with TBS containing 0.05% Tween 20 and 5 mM CaCb (wash buffer). Human sérum samples are diluted in 20 mM Tris-HCl, l M NaCl, 10 mM CaCl2, 0.05% Triton X-lOO, 0.1% HSA, pH 7.4, which prevents activation of endogenous C4 and dissociâtes the Cl complex (composed of Clq, Clr and Cl s). MASP-2 inhibitory agents, including anti-MASP-2 MoAbs and inhibitory peptides are added to the sérum samples in varying concentrations. The diluted samples are added to the plate and incubated ovemight at 4°C. After 24 hours, the plates are washed thoroughly with wash buffer, then 0.1 pg of purified human C4 (obtained as described in Dodds, A.W., Methods Enzymol. 223:46, 1993) in 100 μΐ of 4 mM barbital, 145 mM NaCl, 2 mM CaCl2, l mM MgC^, pH 7.4 is added to each well. After 1.5 h at 37°C, the plates are washed again and C4b déposition is detected using alkaline phosphatase-conjugated chicken anti-human C4c (obtained from Immunsystem, Uppsala, Sweden) and measured using the colorimétrie substrate p-nitrophenyl phosphate.
C4 Assay on mannan: The assay described above is adapted to measure lectin pathway activation via MBL by coating the plate with LSP and mannan prior to adding sérum mixed with various MASP-2 inhibitory agents.
C4 assay on H-ficolin (Hakata Ag): The assay described above is adapted to measure lectin pathway activation via H-ficolîn by coating the plate with LPS and H-ficolin prior to adding sérum mixed with various MASP-2 inhibitory agents.
EXAMPLE 8
The following assay demonstrates the presence of classical pathway activation in wild-type and MASP-2-/- mice.
Methods: Immune complexes were generated in situ by coating microtiter plates (MaxiSorb®, Nunc, cat. No. 442404, Fisher Scientific) with 0.1% human sérum albumin in 10 mM Tris, 140 mM NaCl, pH 7.4 for 1 hours at room température followed by ovemight incubation at 4°C with sheep anti whole sérum antiserum (Scottish Antibody Production Unit, Carluke, Scotland) diluted 1:1000 in TBS/tween/Ca2+. Sérum samples were obtained from wild-type and MASP-2-/- mice and added to the coated plates. Control samples were prepared in which Clq was depicted from wild-type and
I24
MASP-2-/- sérum samples. Clq-depleted mouse sérum was prepared using protein-A-coupled Dynabeads® (Dynal Biotech, Oslo, Norway) coated with rabbît anti-human Clq IgG (Dako, Glostrup, Denmark), according to the supplier's instructions. The plates were incubated for 90 minutes at 37°C. Bound C3b was detected with a 5 polyclonal anti-human-C3c Antibody (Dako A 062) diluted in TBS/tw/ Ca++ at 1:1000.
The secondary antibody is goat anti-rabbit IgG.
Results: FIGURE 7 shows the relative C3b déposition levels on plates coated with IgG in wild-type sérum, MASP-2-/- sérum, Clq-depleted wild-type and Clq-depleted MASP-2-/- sérum. These results demonstrate that the classical pathway is 10 intact in the MASP-2-/- mouse strain.
EXAMPLE 9
The following assay is used to test whether a MASP-2 inhibitory agent blocks the classical pathway by analyzing the effect of a MASP-2 inhibitory agent under conditions 15 in which the classical pathway is initiated by immune complexes.
Methods: To test the effect of a MASP-2 inhibitory agent on conditions of complément activation where the classical pathway is initiated by immune complexes, triplicate 50 μΐ samples containing 90% NHS are incubated at 37°C in the presence of 10 pg/ml immune complex (IC) or PBS, and parallel triplicate samples (+/-IC) are also 20 included which contain 200 nM anti-properdin monoclonal antibody during the 37°C incubation. After a two hour incubation at 37°C, 13 mM EDTA is added to ail samples to stop further complément activation and the samples are immediately cooled to 5°C. The samples are then stored at -70°C prior to being assayed for complément activation products (C3a and sC5b-9) using ELISA kits (Quidel, Catalog Nos. A015 and A009) 25 following the manufacturées instructions.
EXAMPLE 10
This example descrîbes the identification of high affmity anti-MASP-2 Fab2 antibody fragments that block MASP-2 activity.
Background and rationale: MASP-2 is a complex protein with many separate functional domains, including: binding site(s) for MBL and fïcolins, a serine protease
125 catalytic site, a binding site for proteolytic substrate C2, a binding site for proteolytic substrate C4, a MASP-2 cleavage site for autoactivation of MASP-2 zymogen, and two C'a- binding sites. Fab2 antibody fragments were identified that bind with high affinity to MASP-2, and the identified Fab2 fragments were tested in a functional assay to détermine if they were able to block MASP-2 functional activity.
To block MASP-2 functional activity, an antibody or Fab2 antibody fragment must bind and interfère with a structural epitope on MASP-2 that is required for MASP-2 functional activity. Therefore, many or ail of the high affinity binding anti-MASP-2 Fab2s may not inhibit MASP-2 functional activity unless they bind to structural epitopes on MASP-2 that are directly involved in MASP-2 functional activity.
A functional assay that measures inhibition of lectin pathway C3 convertase formation was used to evaluate the blocking activity of anti-MASP-2 Fab2s. It is known that the primary physîological rôle of MASP-2 in the lectin pathway is to generate the next functional component of the lectin-mediated complément pathway, namely the lectin pathway C3 convertase. The lectin pathway C3 convertase is a critical enzymatic complex (C4bC2a) that proteolytically cleaves C3 into C3a and C3b. MASP-2 is not a structural component of the lectin pathway C3 convertase (C4bC2a); however, MASP-2 functional activity is required in order to generate the two protein components (C4b, C2a) that comprise the lectin pathway C3 convertase. Furthermore, ail of the separate functional activities of MASP-2 listed above appear to be required in order for MASP-2 to generate the lectin pathway C3 convertase. For these reasons, a preferred assay to use in evaluating the blocking activity of anti-MASP-2 Fab2s is believed to be a functional assay that measures inhibition of lectin pathway C3 convertase formation.
Génération of High Affinity Fab2s: A phage display library of human variable light and heavy chain antibody sequences and automated antibody sélection technology for identifying Fab2s that react with selected ligands of interest was used to create high affinity Fab2s to rat MASP-2 protein (SEQ ID NO:55). A known amount of rat MASP-2 (~l mg, >85% pure) protein was utilized for antibody screening. Three rounds of amplification were utilized for sélection of the antibodies with the best affinity. Approximately 250 different hits expressing antibody fragments were picked for ELISA
126 screening. High affinity hits were subsequently sequenced to détermine uniqueness of the different antibodies.
Fifty unique anti-MASP-2 antibodies were purified and 250 pg of each purified Fab2 antibody was used for characterization of MASP-2 binding affinity and complément pathway functional testing, as described in more detail below.
Assays used to Evaluate the Inhibitory (blocking) Activity of Anti-MASP-2
Fab2s
1. Assay to Measure Inhibition of Formation of Lectin Pathway C3
Convertase:
Background: The lectin pathway C3 convertase is the enzymatic complex (C4bC2a) that proteolytically cleaves C3 into the two potent proinflammatory fragments, anaphylatoxin C3a and opsonic C3b. Formation of C3 convertase appears to 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); therefore anti-MASP-2 antibodies (or Fab2) will not directly inhibit activity of preexisting C3 convertase. However, MASP-2 serine protease activity is required in order to generate the two protein components (C4b, C2a) that comprise the lectin pathway C3 convertase. Therefore, anti-MASP-2 Fab2 which inhibit MASP-2 functional activity (i.e., blocking anti-MASP-2 Fab2) will inhibit de novo formation of lectin pathway C3 convertase. C3 contains an unusual and highly reactive thioester group as part of its structure. Upon cleavage of C3 by C3 convertase in this assay, the thioester group on C3b can form a covalent bond with hydroxyl or amino groups on macromolecules immobilized on the bottom of the plastic wells via ester or amide linkages, thus faci 1 itatîng détection of C3b in the ELISA assay.
Yeast mannan is a known activator of the lectin pathway. In the following method to measure formation of C3 convertase, plastic wells coated with mannan were incubated for 30 min at 37°C with diluted rat sérum to activate the lectin pathway. The wells were then washed and assayed for C3b immobilized onto the wells using standard ELISA methods. The amount of C3b generated in this assay is a direct reflectîon of the de novo formation of lectin pathway C3 convertase. Anti-MASP-2 Fab2s at selected concentrations were tested in this assay for their ability to inhibit C3 convertase formation and conséquent C3b génération.
I27
Methods:
96-well Costar Medium Binding plates were incubated ovemight at 5°C with mannan diluted in 50 mM carbonate buffer, pH 9.5 at l pg/50 pL/well. After ovemight incubation, each well was washed three times with 200 pL PBS. The wells were then blocked with 100 pL/well of l% bovine sérum albumin in PBS and incubated for one hour at room température with gentle mixîng. Each well was then washed three times with 200 pL of PBS. The anti-MASP-2 Fab2 samples were diluted to selected concentrations in C’a and Mg++ containing GVB buffer (4.0 mM barbital, 141 mM NaCl, i.O mM MgCb, 2.0 mM CaCl2, 0.1% gelatin, pH 7.4) at 5 C. A 0.5% rat sérum was added to the above samples at 5°C and 100 pL was transferred to each well. Plates were covered and incubated for 30 minutes in a 37 C waterbath to allow complément activation. The reaction was stopped by transferring the plates from the 37°C waterbath to a container containing an ice-water mix. Each well was washed five times with 200 pL with PBS-Tween 20 (0.05% Tween 20 in PBS), then washed two times with 200 pL PBS. A 100 pL/well of 1:10,000 dilution of the primary antibody (rabbit anti-human C3c, DAKO A0062) was added in PBS containing 2.0 mg/mi bovine sérum albumin and incubated 1 hr at room température with gentle mixing. Each well was washed 5 x 200 pL PBS. 100 pL/well of 1:10,000 dilution of the secondary antibody (peroxidase-conjugated goat anti-rabbit IgG, American Qualex A102PU) was added in PBS containing 2.0 mg/ml bovine sérum albumin and incubated for one hour at room température on a shaker with gentle mixing. Each well was washed five times with 200 pL with PBS. 100 pL/well of the peroxidase substrate TMB (Kirkegaard & Perry Laboratories) was added and incubated at room température for 10 min. The peroxidase reaction was stopped by adding 100 pL/well of 1.0 Μ H3PO4 and the OD450 was measured.
2. Assay to Measure Inhibition of MASP-2-dependent C4 Cleavage
Background: The serine protease activity of MASP-2 is highly spécifie and only two protein substrates for MASP-2 hâve been identified; C2 and C4. Cleavage of C4 generates C4a and C4b. Anti-MASP-2 Fab2 may bind to structural epitopes on MASP-2 that are directly involved in C4 cleavage (e.g., MASP-2 binding site for C4; MASP-2
128 serine protease catalytic site) and thereby inhibit the C4 cleavage functional activity of MASP-2.
Yeast mannan is a known activator of the lectin pathway. In the following method to measure the C4 cleavage activity of MASP-2, plastic wells coated with mannan were incubated for 30 minutes at 37°C with diluted rat sérum to activate the lectin pathway. Since the primary antibody used in this ELISA assay only recognizes human C4, the diluted rat sérum was also supplemented with human C4 (1.0 gg/ml). The wells were then washed and assayed for human C4b immobilized onto the wells using standard ELISA methods. The amount of C4b generated in this assay is a measure of MASP-2 dépendent C4 cleavage activity. Anti-MASP-2 Fab2 at selected concentrations were tested in this assay for their ability to inhibit C4 cleavage.
Methods: 96-well Costar Medium Binding plates were incubated ovemight at 5°C with mannan diluted in 50 mM carbonate buffer, pH 9.5 at 1.0 gg/50 gL/wetl. Each well was washed 3X with 200 gL PBS. The wells were then blocked with 100 gL/well of 1% bovine sérum albumîn in PBS and incubated for one hour at room température with gentle mixing. Each well was washed 3X with 200 gL of PBS. Anti-MASP-2 Fab2 sampies were diluted to selected concentrations in Ca++ and Mg4-1' containing GVB buffer (4.0 mM barbital, 141 mM NaCl, 1.0 mM MgCh, 2.0 mM CaCh, 0.1% gelatin, pH 7.4) at 5°C. 1.0 gg/ml human C4 (Quidel) was also included in these sampies. 0.5% rat sérum was added to the above sampies at 5°C and 100 gL was transferred to each well. The plates were covered and incubated for 30 minutes in a 37°C waterbath to allow complément activation. The reaction was stopped by transferring the plates from the 37°C waterbath to a container containing an ice-water mix. Each well was washed 5 x 200 gL with PBS-Tween 20 (0.05% Tween 20 in PBS), then each well was washed with 2X with 200 gL PBS. 100 gL/well of 1:700 dilution of bîotin-conjugated chïcken anti-human C4c (Immunsystem AB, Uppsala, Sweden) was added în PBS containing 2.0 mg/ml bovine sérum albumîn (BSA) and incubated one hour at room température with gentle mixing. Each well was washed 5 x 200 gL PBS. 100 gL/well of 0.1 gg/ml of peroxidase-conjugated streptavidin (Pierce Chemical #21126) was added in PBS containing 2.0 mg/ml BSA and incubated for one hour at room température on a shaker with gentle mixing. Each well was washed 5 x 200 gL with PBS. 100 gL/well of the
129 peroxidase substrate TMB (Kirkegaard & Perry Laboratories) was added and incubated at room température for 16 min. The peroxidase reaction was stopped by adding 100 pL/well of l .0 Μ H3PO4 and the OD450 was measured.
3. Binding Assay of anti-rat MASP-2 Fab2 to 'Native' rat MASP-2
Background: MASP-2 is usually present in plasma as a MASP-2 dimer complex that also includes spécifie lectin molécules (mannose-binding protein (MBL) and ficolins). Therefore, if one is interested in studying the binding of anti-MASP-2 Fab2 to the physiologically relevant form of MASP-2, it is important to develop a binding assay in which the interaction between the Fab2 and 'native' MASP-2 in plasma is used, rather than purified recombinant MASP-2. In this binding assay the 'native' MASP-2-MBL complex from 10% rat sérum was first immobilized onto mannan-coated wells. The binding affinity of various anti-MASP-2 Fab2s to the immobilized 'native' MASP-2 was then studied using a standard ELISA methodology.
Methods: 96-well Costar High Binding plates were incubated ovemight at 5°C with mannan diluted in 50 mM carbonate buffer, pH 9.5 at l pg/50 pL/well. Each well was washed 3X with 200 pL PBS. The wells were blocked with 100 pL/well of 0.5% nonfat dry milk in PBST (PBS with 0.05% Tween 20) and incubated for one hour at room température with gentle mixing. Each well was washed 3X with 200 pL of TBS/Tween/Ca^ Wash Buffer (Tris-buffered saline, 0.05% Tween 20, containing 5.0 mM CaCb, pH 7.4. 10% rat sérum in High Sait Binding Buffer (20 mM Tris, LO M NaCl, 10 mM CaCh, 0.05% Triton-Xl00, 0.1% (w/v) bovine sérum albumin, pH 7.4) was prepared on ice. I00 pL/well was added and incubated ovemight at 5°C. Wells were washed 3X with 200 pL of TBS/Tween/Ca++ Wash Buffer. Wells were then washed 2X with 200 pL PBS. 100 pL /well of selected concentration of anti-MASP-2 Fab2 diluted in Ca~ and Mg++ containing GVB Buffer (4.0 mM barbital, I4l mM NaCl, l.OmM MgCh, 2.0 mM CaCb, 0.1% gelatin, pH 7.4) was added and incubated for one hour at room température with gentle mixing. Each well was washed 5 x 200 pL PBS. 100 pL/well of HRP-conjugated goat anti-Fab2 (Biogenesis Cat No 0500-0099) diluted l :5000 in 2.0 mg/ml bovine sérum albumin in PBS was added and incubated for one hour at room température with gentle mixing. Each well was washed 5 x 200 pL PBS. 100 pL/well of the peroxidase substrate TMB (Kirkegaard & Perry Laboratories) was added
I30 and incubated at room température for 70 min. The peroxidase reaction was stopped by adding 100 gL/well of l.O Μ H3PO4 and OD450 was measured.
RESULTS:
Approximately 250 different Fab2s that reacted with high affinîty to the rat
MASP-2 protein were picked for ELISA screening. These high affinîty Fab2s were sequenced to determine the uniqueness of the different antibodies, and 50 unique anti-MASP-2 antibodies were purified for further analysis. 250 pg of each purified Fab2 antibody was used for characterization of MASP-2 binding affinîty and complément pathway functional testing. The resuit of this analysis is shown below in TABLE 6.
TABLE 6: ANTI-MASP-2 FAB2 THAT BLOCK LECTIN PATHWAY
COMPLEMENT ACTIVATION______________________________
Fab2 antibody # C3 Convertase (IC50 (nM) Kd C4 Cleavage IC50 (nM)
88 0.32 4.1 ND
41 0.35 0.30 0.81
H 0.46 0.86 <2 nM
86 0.53 1.4 ND
8l 0.54 2.0 ND
66 0.92 4.5 ND_____
57 0.95 3.6 <2nM
40 l.l 7.2 0.68 _______
58 1.3 2.6 ND
60 1.6 3.1 ND
52 1.6 5.8 <2 nM
63 2.0 6.6 ND
49 2.8 8.5 <2nM ___
89 3.0 2.5 ND
71 3.0 10.5 ND
87 6.0 2.5 ND
67 10.0 7.7 ND
I3l
As shown above in TABLE 6, of the 50 anti-MASP-2 Fab2s tested. seventeen Fab2s were identified as MASP-2 blocking Fab2 that potently inhibit C3 convertase formation with IC50 equal to or less than IO nM Fab2s (a 34% positive hit rate). Eight of the seventeen Fab2s identified hâve IC50S in the subnanomolar range. Furthermore, ail seventeen ofthe MASP-2 blocking Fab2s shown in TABLE 6 gave essentially complété inhibition of C3 convertase formation in the lectin pathway C3 convertase assay. FIGURE 8A graphically illustrâtes the results of the C3 convertase formation assay for Fab2 antibody #l l, which is représentative of the other Fab2 antibodies tested, the results of which are shown in TABLE 6. This is an important considération, since it is theoretically possible that a blocking Fab2 may only fractionally inhibit MASP-2 function even when each MASP-2 molécule is bound by the Fab2.
Although mannan is a known activator of the lectin pathway, it is theoretically possible that the presence of antî-mannan antibodies in the rat sérum might also activate the classîcal pathway and generate C3b via the classical pathway C3 convertase. However, each of the seventeen blocking anti-MASP-2 Fab2s listed in this example potently inhibits C3b génération (>95 %), thus demonstrating the specificity of this assay for lectin pathway C3 convertase.
Binding assays were also performed with ail seventeen of the blocking Fab2s in order to calculate an apparent Kj for each. The results of the binding assays of anti-rat MASP-2 Fab2s to native rat MASP-2 for six of the blocking Fab2s are also shown in TABLE 6. FIGURE 8B graphically illustrâtes the results of a binding assay with the Fab2 antibody #11. Similar binding assays were also carried out for the other Fab2s, the results of which are shown în TABLE 6. In general, the apparent Kjs obtained for binding of each ofthe six Fab2s to 'native' MASP-2 corresponds reasonably well with the IC50 for the Fab2 in the C3 convertase functional assay. There is evidence that MASP-2 undergoes a conformational change from an 'inactive' to an 'active' form upon activation of its protease activity (Feinberg et al., EMBO J 22:2348-59 (2003); Gai et al., J. Biol. Chem. 250:33435-44 (2005)). In the normal rat plasma used in the C3 convertase formation assay, MASP-2 is present primarily in the 'inactive' zymogen conformation. In contrast, in the binding assay, MASP-2 is present as part of a complex with MBL bound to immobilized mannan; therefore, the MASP-2 would be in the 'active' conformation
132 (Petersen étal., J. Immunol Methods 257:107-16, 2001). Consequently, one would not necessarily expect an exact correspondence between the IC50 and Kj for each of the seventeen blocking Fab2 tested in these two functional assays since in each assay the Fab2 would be binding a different conformational form of MASP-2. Never-the-less, with the exception of Fab2 #88, there appears to be a reasonably close correspondence between the IC5Q and apparent Kd for each of the other sixteen Fab2 tested in the two assays (see TABLE 6).
Several of the blocking Fab2s were evaluated for inhibition of MASP-2 mediated cleavage of C4. FIGURE 8C graphically illustrâtes the results of a C4 cleavage assay, showing inhibition with Fab2 #41, with an IC5Q—0.81 nM (see TABLE 6). As shown in FIGURE 9, ail ofthe Fab2s tested were found to inhibit C4 cleavage with IC5QS similar to those obtained in the C3 convertase assay (see TABLE 6).
Although mannan is a known actîvator of the lectin pathway, it is theoretically possible that the presence of anti-mannan antibodies in the rat sérum might also activate the classical pathway and thereby generate C4b by CIs-mediated cleavage of C4. However, several anti-MASP-2 Fab2s hâve been îdentified which potently inhibit C4b génération (>95 %), thus demonstrating the specificity of this assay for MASP-2 mediated C4 cleavage. C4, like C3, contains an unusual and highly reactive thioester group as part of its structure. Upon cleavage of C4 by MASP-2 in this assay, the thioester group on C4b can form a covalent bond with hydroxyl or amino groups on macromolecules immobilized on the bottom of the plastic wells via ester or amide linkages, thus facilitating détection ofC4b in the ELISA assay.
These studies clearly demonstrate the création of high affinity Fab2s to rat MASP-2 protein that functionally block both C4 and C3 convertase activity, thereby preventing lectin pathway activation.
EXAMPLE 11
This Example describes the epitope mapping for several of the blocking anti-rat MASP-2 Fab2 antibodies that were generated as described in Example 10.
Methods:
133
As shown in FIGURE 10, the following proteins, ail with N-terminal 6X His tags were expressed in CHO cells using the pED4 vector:
rat MASP-2A, a full length MASP-2 protein, inactivated by altering the serine at the active center to alanine (S613A);
rat MASP-2K, a full-length MASP-2 protein altered to reduce autoactivation (R424K);
CUBI-II, an N-terminal fragment of rat MASP-2 that contains the CUBI, EGF-lîke and CUBII domains only; and
CUBI/EGF-like, an N-terminal fragment of rat MASP-2 that contains the CUBI 10 and EGF-like domains only.
These proteins were purified from culture supematants by nickel-afïinity chromatography, as previously described (Chen et al., J. Biol. Chem. 276:25894-02 (2001)).
A C-terminal polypeptide (CCPII-SP), containing CCPl! and the serine protease 15 domain of rat MASP-2, was expressed in E. coli as a thioredoxin fusion protein using pTrxFus (Invitrogen). Protein was purified from cell lysâtes using Thiobond afïinity resin. The thioredoxin fusion partner was expressed from empty pTrxFus as a négative control.
Ail recombinant proteins were dialyzed into TBS buffer and their concentrations 20 determined by measuring the OD at 280 nm.
DOT BLOT ANALYSIS:
Serial dilutions of the five recombinant MASP-2 polypeptides described above and shown in FIGURE 10 (and the thioredoxin polypeptide as a négative control for CCPII-serine protease polypeptide) were spotted onto a nitrocellulose membrane. The 25 amount of protein spotted ranged from 100 ng to 6.4 pg, in five-fold steps. In later experiments, the amount of protein spotted ranged from 50 ng down to 16 pg, agaîn in five-fold steps. Membranes were blocked with 5% skimmed milk powder in TBS (blocking buffer) then incubated with 1.0 gg/ml anti-MASP-2 Fab2s in blocking buffer (containing 5.0 mM Ca2+). Bound Fab2s were detected using HRP-conjugated 30 anti-human Fab (AbD/Serotec; diluted 1/10,000) and an ECL détection kit (Amersham). One membrane was incubated with polyclonal rabbit-anti human MASP-2 Ab (described
134 în Stover et al., JImmunol 763:6848-59 ( 1999)) as a positive control. In this case, bound Ab was detected using HRP-conjugated goat anti-rabbit IgG (Dako; diluted i/2,000).
MASP-2 Binding Assay
ELISA plates were coated with 1.0 pg/weil of recombinant MASP-2A or CUBI-II polypeptide in carbonate buffer (pH 9.0) ovemight at 4°C. Wells were blocked with 1% BSA in TBS, then serial dilutions of the anti-MASP-2 Fab2s were added in TBS containing 5.0 mM Ca2+. The plates were incubated for one hour at RT. After washing three times with TBS/tween/Ca2+, HRP-conjugated anti-human Fab (AbD/Serotec) diluted 1/10,000 in TBS/ Ca2+ was added and the plates incubated for a further one hour at RT. Bound antibody was detected using a TMB peroxidase substrate kit (Biorad).
RESULTS:
Results of the dot blot analysis demonstrating the reactivity of the Fab2s with various MASP-2 polypeptides are provided below in TABLE 7. The numerical values provided in TABLE 7 indicate the amount of spotted protein required to give approximately half-maximal signal strength. As shown, ail of the polypeptides (with the exception of the thioredoxin fusion partner alone) were recognized by the positive control Ab (polyclonal anti-human MASP-2 sera, raised in rabbits).
TABLE 7: REACTIVITY WITH VARIOUS RECOMBINANT RAT MASP-2
POLYPEPTIDES ON DOT BLOTS
Fab2 Antibody # MASP-2A CUBI-II CUBI/EGF-like CCPH-SP Thioredoxin
40 0.16 ng NR NR 0.8 ng NR
41 0.16 ng NR NR 0.8 ng NR
11 0.16 ng NR NR 0.8 ng NR
49 0.16 ng NR NR >20 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
135
Fab2 Antibody # MASP-2A CUBI-II CUBIÆGF-like CCPII-SP Thioredoxin
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
NR. - No reaction. The positive control antibody is polyclonal anti-human MASP-2 sera, raised in rabbits.
Ail of the Fab2s reacted with MASP-2A as well as MASP-2K (data not shown). The majority of the Fab2s recognized the CCP1I-SP polypeptide but not the N-terminal fragments. The two exceptions are Fab2 #60 and Fab2 #57. Fab2 #60 recognizes MASP-2A and the CUBI-II fragment, but not the CUBI/EGF-like polypeptide or the CCPIl-SP polypeptide, suggesting it binds to an epitope in CUBII, or spanning the CUBII and the EGF-like domain. Fab2 # 57 recognizes MASP-2A but not any of the MASP-2 fragments tested, indicating that this Fab2 recognizes an epitope in CCPl. Fab2 #40 and #49 bound only to complété MASP-2A. In the ELISA binding assay shown in FIGURE 11, Fab2 #60 also bound to the CUBI-II polypeptide, albeit with a slightly lower apparent affinity.
These finding demonstrate the identification of unique blocking Fab2s to multiple régions of the MASP-2 protein.
EXAMPLE 12
This example describes the identification, using phage display, of fully human scFv antibodies that bind to MASP-2 and inhibit lectin-mediated complément activation while leaving the ciassical (Clq-dependent) pathway component of the immune system intact.
OverView:
Fully human, hîgh-affinity MASP-2 antibodies were identified by screening a phage display library. The variable light and heavy chain fragments of the antibodies
136 were isolated in both a scFv format and in a full-length IgG format. The human MASP-2 antibodies are useful for inhibiting cellular injury associated with lectin pathwaymediated complément pathway activation while leaving the classical (Clq-dependent) pathway component of the immune system intact. In some embodiments, the subject MASP-2 inhibitory antibodies hâve the following characteristics: (a) high affinity for human MASP-2 (e.g., a KD of 10 nM or less), and (b) inhibit MASP-2-dependent complément activity in 90% human sérum with an IC5o of 30 nM or less.
Methods:
Expression offull-length catalytically inactive MASP-2:
The full-length cDNA sequence of human MASP-2 (SEQ ID NO: 4), encoding the human MASP-2 polypeptide with leader sequence (SEQ ID NO:5) was subcloned into the mammalian expression vector pCI-Neo (Promega), which drives eukaryotic expression under the control of the CMV enhancer/promoter région (described in Kaufman R.J. et al., Nucleic Acids Research /9:4485-90, I99l; Kaufman, Methods in Enzymology, /55:537-66 (1991)).
In order to generate catalytically inactive human MASP-2A protein, site-dîrected mutagenesis was carried out as described in US2007/0172483, hereby incorporated herein by reference. The PCR products were purified after agarose gel electrophoresis and band préparation and single adenosine overlaps were generated using a standard tailing procedure. The adenosine-tailed MASP-2A was then cloned into the pGEM-T easy vector and transformed into E. coli. The human MASP-2A was further subcloned into either of the mammalian expression vectors pED or pCI-Neo.
The MASP-2A expression construct described above was transfected into DXB1 cells using the standard calcium phosphate transfection procedure (Maniatis et al., 1989). MASP-2A was produced in serum-free medium to ensure that préparations were not contaminated with other sérum proteins. Media was harvested from confluent cells every second day (four times in total). The level of recombinant MASP-2A averaged approximately 1.5 mg/liter of culture medium. The MASP-2A (Ser-Ala mutant described above) was purified by affinity chromatography on MBP-A-agarose columns
MASP-2A ELISA on ScFv Candidate Clones identified by panning/scFv conversion and filter screening
137
A phage display library of human immunoglobulin light- and heavy-chain variable région sequences was subjected to antigen panning followed by automated antibody screening and sélection to identify high-affinity scFv antibodies to human MASP-2 protein. Three rounds of panning the scFv phage library against HIS-tagged or bîotin-tagged MASP-2A were carried out. The third round of panning was eluted first with MBL and then with TEA (alkaline). To monitor the spécifie enrichment of phages displaying scFv fragments against the target MASP-2A, a polyclonal phage ELISA against immobilized MASP-2A was carried out. The scFv genes from panning round 3 were cloned into a pHOG expression vector and run in a small-scale filter screening to look for spécifie clones against MASP-2A.
Bacterial colonies containing plasmids encoding scFv fragments from the third round of panning were pîcked, gridded onto nitrocellulose membranes and grown ovemight on non-inducing medium to produce master plates. A total of 18,000 colonies were picked and analyzed from the third panning round, half from the compétitive elution and half from the subséquent TEA elution. Panning of the scFv phagemid library against MASP-2A followed by scFv conversion and a filter screen yielded 137 positive clones. 108/137 clones were positive in an ELISA assay for MASP-2 binding (data not shown), of which 45 clones were further analyzed for the ability to block MASP-2 activity in normal human sérum.
Assay to Measure Inhibition of Formation of Lectin Pathway C3 Convertase
A functional assay that measures inhibition of lectin pathway C3 convertase formation was used to evaluate the blocking activity of the MASP-2 scFv candidate clones. MASP-2 serine protease activity is required in order to generate the two protein components (C4b, C2a) that comprise the lectin pathway C3 convertase. Therefore, a MASP-2 scFv that inhibits MASP-2 functional activity (i.e., a blocking MASP-2 scFv), will inhibit de novo formation of lectin pathway C3 convertase. C3 contains an unusual and highly reactive thioester group as part of its structure. Upon cleavage of C3 by C3 convertase in this assay, the thioester group on C3b can form a covalent bond with hydroxyl or amino groups on macromolecules immobilized on the bottom of the plastic wells via ester or amide linkages, thus facilitating détection of C3b in the ELISA assay.
138
Yeast mannan is a known activator of the lectin pathway. In the following method to measure formation of C3 convertase, plastic wells coated with mannan were incubated with diluted human sérum to activate the lectin pathway. The wells were then washed and assayed for C3b immobilized onto the wells using standard ELISA methods. The amount of C3b generated in this assay is a direct reflection of the de novo formation of lectin pathway C3 convertase. MASP-2 scFv clones at selected concentrations were tested in this assay for their ability to inhibit C3 convertase formation and conséquent C3b génération.
Methods:
The 45 candidate clones identified as described above were expressed, purified and diluted to the same stock concentration, which was agaïn diluted in Ca++ and Mg^1' containing GVB buffer (4.0 mM barbital, 141 mM NaCl, 1.0 mM MgC12, 2.0 mM CaC12, 0.1% gelatin, pH 7.4) to assure that ali clones had the same amount of buffer. The scFv clones were each tested in triplicate at the concentration of 2 pg/mL. The positive control was OMS 100 Fab2 and was tested at 0.4 pg/mL. C3c formation was monitored in the presence and absence of the scFv/IgG clones.
Mannan was diluted to a concentration of 20 pg/mL (1 pg/well) in 50mM carbonate buffer (15mM Na2CO3 + 35mM NaHCO3 + 1.5 mM NaN3), pH 9.5 and coated on an ELISA plate ovemight at 4°C. The next day, the mannan-coated plates were washed 3 times with 200 pl PBS. 100 pl of 1% HSA blocking solution was then added to the wells and incubated for 1 hour at room température. The plates were washed 3 times with 200 pl PBS, and stored on ice with 200 pl PBS until addition ofthe samples.
Normal human sérum was diluted to 0.5% in CaMgGVB buffer, and scFv clones or the OMS 100 Fab2 positive control were added in tripltcates at 0.01 pg/mL; 1 pg/mL (only OMS 100 control) and 10 pg/mL to this buffer and preincubated 45 minutes on ice before addition to the blocked ELISA plate. The reaction was initiated by incubation for one hour at 37°C and was stopped by transferring the plates to an Ice bath. C3b déposition was detected with a Rabbit α-Mouse C3c antibody followed by Goat a-Rabbit HRP. The négative control was buffer without antibody (no antibody = maximum C3b déposition), and the positive control was buffer with EDTA (no C3b déposition). The background was determined by carrying out the same assay except that the wells were
139 mannan-free. The background signal against plates without mannan was subtracted from the signais in the mannan-containing wells. A cut-off criterion was set at half of the activity of an irrelevant scFv clone (VZV) and buffer alone.
Results: Based on the cut-off criterion, a total of 13 clones were found to block 5 the activity of MASP-2. Ail 13 clones producing > 50% pathway suppression were selected and sequenced, yielding 10 unique clones. Ail ten clones were found to hâve the same light chain subclass, λ3, but three different heavy chain subclasses: VH2, VH3 and VH6. In the functional assay, five out of the ten candidate scFv clones gave IC50 nM values less than the 25 nM target criteria using 0,5% human sérum.
I0 To identify antibodies with improved potency, the three mother scFv clones, identified as described above, were subjected to light-chain shuffling. This process involved the génération of a combinatorial library consisting of the VH of each of the mother clones paired up with a library of naïve, human lambda light chains (VL) derived from six healthy donors. This library was then screened for scFv clones with improved 15 binding affinity and/or functionality.
TABLE 8: Comparison of functional potency in 1C5O (nM) of the lead daughter clones and their respective mother clones (ail in scFv format)
scFv clone 1% human sérum C3 assay (ICS0 nM) 90% human sérum C3 assay (ICsonM) 90% human sérum C4 assay (IC50 nM)
17D20mc 38 nd nd ____
l7D20m d3521NU 26 >1000 140
17N16mc 68 nd nd
17N16m d!7N9 48 15 _______230_______
Presented below are the heavy-chain variable région (VH) sequences for the mother clones and daughter clones shown above in TABLE 8.
The Kabat CDRs (31-35 (Hl), 50-65 (H2) and 95-107 (H3)) are bolded; and the Chothia CDRs (26-32 (Hl), 52-56 (H2) and 95-101 (H3)) are underlined.
140
17D20 35VH-21NHVL heavy chain variable région (VH) (SEQ ID NO:67, encoded by SEQ ID NO:66)
QVTT KFSGPVT VKPTETLTLTCTVSGFSLSRGKMGVSWIRQPPGKALEW
I .AHIFSSDF-KSYRTSLKSRl TISKDTSKNQWLTMTNMDPVDTATYYCARIRRG GIDYWGQGTLVTVSS dl 7N9 heavy chain variable région (VH) (SEQ ID NQ:68)
OVQÏ QQSGPGT .VKPSQTLSLTCAISGDSVSSTSAAWNWIRQSPSRGLEWLGRTY YRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYÇARDPFGVPF DIWGQGTMVTVSS
Presented below are the lîght-chain variable région (VL) sequences for the mother clones and daughter clones shown above in TABLE 8.
The Kabat CDRs (24-34 (Ll); 50-56 (L2); and 89-97 (L3) are bolded; and the Chothia CDRs (24-34 (Ll); 50-56 (L2) and 89-97 (L3) are underlined. These régions are the same whether numbered by the Kabat or Chothia system.
17D20m d352lNll light chain variable région (VL) (SEQ ID NQ:70. encoded by SEP ID NO:69) qpvltqppslsvspgqtasitcsgeklgdkyaywyqqkpgqspvlvmyq DKQRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQAWDSSTAVFGGGTKL TVL !7Nl6m d!7N9 light chain variable région (VL) (SEP ID NQ:7l)
SYF.I IQPPSVSVAPGQTATITCAGDNLGKKRVHWYQQRPGQAPVLVIYD DSDRPSGIPDRFSASNSGNTATLTITRGEAGDEADYYCPVWDIATDHVVFGGGT KLTVLAAAGSEQKLISE
The MASP-2 antibodies PMSI00 and MoAb_d352lNl IVL, (comprising a heavy chain variable région set forth as SEQ ID NQ:67 and a light chain variable région set
141 forth as SEQ ID NO:70, also referred to as ‘OMS646” and “mAb6” ), which hâve both been demonstrated to bind to human MASP-2 with high affinity and hâve the ability to block functional complément activity, were analyzed with regard to epitope binding by dot blot analysis. The results show that OMS646 and OMS 100 antibodies are highly spécifie for MASP-2 and do not bind to MASP-l/3. Neither antibody bound to MApl9 nor to MASP-2 fragments that did not contain the CCPl domain of MASP-2, leading to the conclusion that the binding sites encompass CCPl.
The MASP-2 antibody OMS646 was determined to avidly bind to recombinant MASP-2 (Kd 60-250pM) with >5000 fold selectivity when compared to Cl s, Clr or MASP-l (see TABLE 9 below):
TABLE 9: Affinity and Specificity of OMS646 MASP-2 antibody-MASP-2 interaction as
assessed by solid phase EL SA studies
Antigen Kd (pM)__________________
MASP-l >500.000
MASP-2 62±23*
MASP-3 >500,000
Purified human Clr >500.000
Purified human Cls -500.000
*Mean±SD; n=l2
OMS646 specifically blocks lectin-dependent activation of terminal complément components
Methods:
The effect of OMS646 on membrane attack complex (MAC) déposition was analyzed using pathway-specific conditions for the lectin pathway, the classîcal pathway and the alternative pathway. For this purpose, the Wieslab Comp300 complément screening kit (Wieslab, Lund, Sweden) was used following the manu facturer’s instructions.
Results:
142
FIGURE 12A graphically illustrâtes the level of MAC déposition in the presence or absence of anti-MASP-2 antibody (OMS646) under lectin pathway-specific assay conditions. FIGURE 12B graphically illustrâtes the level of MAC déposition in the presence or absence of anti-MASP-2 antibody (OMS646) under classical pathwayspecific assay conditions. FIGURE 12C graphically illustrâtes the level of MAC déposition in the presence or absence of anti-MASP-2 antibody (OMS646) under alternative pathway-specific assay conditions.
As shown in FIGURE 12A, OMS646 blocks lectin pathway-mediated activation of MAC déposition with an IC50 value of approximately InM. However, OMS646 had no effect on MAC déposition generated from classical pathway-mediated activation (FIGURE 12B) or from alternative pathway-mediated activation (FIGURE 12C).
Pharmacokinetics and Pharmacodynamies of OMS646 following Intravenous (IV) or Subcutaneous (SC) Administration to Mice
The pharmacokinetics (PK) and pharmacodynamies (PD) of OMS646 were evaluated in a 28 day single dose PK/PD study in mice. The study tested dose levels of 5mg/kg and I5mg/kg of OMS646 administered subcutaneously (SC), as well as a dose level of 5mg/kg OMS646 administered intravenously (IV).
With regard to the PK profile of OMS646, FIGURE 13 graphically illustrâtes the OMS646 concentration (mean of n=3 animals/groups) as a function of time after administration of OMS646 at the indicated dose. As shown in FIGURE 13, at 5mg/kg SC, OMS646 reached the maximal plasma concentration of 5-6 pg/mL approximately l-2 days after dosing. The bioavailability of OMS646 at 5 mg/kg SC was approximately 60%. As further shown in FIGURE 13, at 15 mg/kg SC, OMS646 reached a maximal plasma concentration of 10-I2 pg/mL approximately l to 2 days after dosing. For ail groups, the OMS646 was cleared slowly from systemic circulation with a terminal halflife of approximately 8-10 days. The profile ofOMS646 is typical for human antibodies in mice.
The PD activity of OMS646 is graphically illustrated in FIGURES 14A and 14B. FIGURES 14A and 14B show the PD response (drop in systemic lectin pathway activity) for each mouse in the 5mg/kg IV (FIGURE 14A) and 5mg/kg SC (FIGURE 14B) groups.
143
The dashed line indicates the baseline of the assay (maximal inhibition; naïve mouse sérum spiked in vitro with excess OMS646 prior to assay). As shown in FIGURE 14A, following IV administration of 5mg/kg of OMS646, systemic lectin pathway activity immediately dropped to near undetectable levels, and lectin pathway activity showed only a modest recovery over the 28 day observation period. As shown in FIGURE 14B, in mice dosed with 5mg/kg of OMS646 SC, time-dependent inhibition of lectin pathway activity was observed. Lectin pathway activity dropped to near-undetectable levels within 24 hours of drug administration and remained at low levels for at least 7 days. Lectin pathway activity gradually increased with time, but did not revert to pre-dose levels within the 28 day observation period. The lectin pathway activity versus time profile observed after administration of I5mg/kg SC was similar to the 5 mg/kg SC dose (data not shown), indicating saturation of the PD endpoint. The data further indicated that weekly doses of 5mg/kg of OMS646, administered either IV or SC, is sufficient to achieve continuous suppression of systemic lectin pathway activity in mice.
EXAMPLE 13
This Example describes the génération of recombinant antibodies that înhibit MASP-2 comprising a heavy chain and/or a light chain variable région comprising one or more CDRs that specifically bind to MASP-2 and at least one SGMI core peptide sequence (also referred to as an SGMI-peptide bearing MASP-2 antibody or antigen binding fragment thereof)· Backeround/Rationale:
The génération of spécifie inhibitors of MASP-2, termed SGMI-2, is described in Heja et al., J Biol Chem 287:20290 (2012) and Heja et al., PNAS 109:10498 (2012), each of which is hereby incorporated herein by reference. SGMI-2 is a 36 amino acid peptide which was selected from a phage library of variants of the Schistocerca gregaria protease inhibitor 2 in which six of the eight positions ofthe protease binding loop were fully randomized. Subséquent in vitro évolution yielded mono-specîfic inhibitors with single digit nM Ki values (Heja et al., J. Biol. Chem. 287:20290, 2012). Structural studies revealed that the optimized protease binding loop forms the primary binding site that defines the specificity of the two inhibitors. The amino acid sequences of the extended secondary and internai binding régions are common to the two inhibitors and contribute
144 to the contact interface (Heja et al., 2012. J. Biol. Chem. 287:20290). Mechanistically, SGMI-2 blocks the lectin pathway of complément activation without affecting the classical pathway (Heja et al., 2012. Proc. NatL Acad. Sci. 109:10498).
The amino acid sequences of the SGMI-2 inhibitors are set forth below: SGMI-2-full-length: I FVTCEPGTTFKDKCNTCRCGSDGKSAVCTKLWCNQ (SEQ ID NO:72)
SGMI-2-medium: TCF.PGTTFKDK.CNTCRCGSDGKSAVCTKLWCNQ (SEQ ID
NO:73)
SGMI-2-short: ......................................TCRCGSDGKSAVCTKLWCNO (SEQ ID
NO:74)
As described in this Example, and also described in WO2014/144542, SGMI-2 peptidebearing MASP-2 antibodies and fragments thereof were generated by fusing the SGMI-2 peptide amino acid sequence (e.g., SEQ ID NO: 72, 73 or 74) onto the amino or carboxy termini of the heavy and/or light chains of a human MASP-2 antibody. The SGMI-2 peptide-bearing MASP-2 antibodies and fragments hâve enhanced înhibitory activity, as compared to the naked MASP-2 scaffold antibody that does not contain the SGMI-2 peptide sequence, when measured in a C3b or C4b déposition assay using human sérum, as described in WO2014/144542, and also hâve enhanced înhibitory activity as compared to the naked MASP-2 scaffold antibody when measured in a mouse model in vivo.
Methods of generating SGMI-2 peptide bearing MASP-2 antibodies are described below.
Methods:
Expression constructs were generated to encode four exemplary SGMI-2 peptide bearing MASP-2 antibodies wherein the SGMI-2 peptide was fused either to the N- or Cterminus of the heavy or light chain of a représentative MASP-2 înhibitory antibody OMS646 (generated as described in Example 12).
TABLE 10: MASP-2 antîbody/SGMI-2 fusions
Antibody reference Peptide Location on Antibody SEQID
H-N H-C L-N L-C NO:
HL-M2 - - - * 67 + 70
145
(naked MASP-2 OMS646)
H-M2-SGMI-2-N SGMI-2 - - - 75 + 70
H-M2-SGMI-2-C - SGMI-2 - - 76 +70
L-M2-SGMI-2-N SGMI-2 - 67+ 77
L-M2-SGMI-2-C — - - SGMI-2 67+78
Abbreviations in Table 10:
“H-N”= amino terminus of heavy chain ‘Ή-C’ -carboxyl terminus of heavy chain “L-N”=amino terminus of light chain “L-C”=carboxyl terminus of light chain “M2”=MASP-2 ab scaffold (représentative OMS646)
For the N-termînal fusions shown in TABLE 10, a peptide linker (‘GTGGGSGSSS’ SEQ ID NO: 79) was added between the SGMI-2 peptide and the variable région.
For the C-terminal fusions shown in TABLE 10, a peptide linker (‘AAGGSG’ SEQ LD NO: 80) was added between the constant région and the SGMI-2 peptide, and a second peptide “GSGA” (SEQ ID NO: 81) was added at the C-terminal end of the fusion polypeptide to protect C-terminal SGMI-2 peptides from dégradation.
Amino acid sequences are provided below for the following représentative MASP-2 antibody/SGMI-2 fusions:
H-M2ab6-SGMI-2-N (SEQ ID NO:75. encoded by SEQ ID NO:82):
LEVTCEPGTTFKDKCNTCRCGSDGKSAVCTKLWCNOGTGGGSGSSSQVTLKESG
PVLVKPTETLTLTCTVSGFSLSRGKMGVSWIROPPGKALEWLAHIFSSDEKSYRT
SLKSRLTISKDTSKNOVVLTMTNMDPVDTATYYCARIRRGGIDYWGOGTLVTVS
SASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA
VLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP
APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV
I46
HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQK.SLSLSLG
K
[491 aa protein, aa l-36=SGMl-2 (underlined), aa37-46=linker (italicized); aa47I64=heavy chain variable région of MASP-2 ab#6 (underlined); aa165-49 l=IgG4 constant région with hinge mutation.]
H-M2ab6-SGMI-2-C (SEP ID NO:76, encoded bv SEQ ID NO:83):
OVTLKESGPVLVKPTETLTLTCTVSGFSLSRGKMGVSWIRQPPGKALEWLAHIFS SDEKSYRTSLKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCARIRRGGIDYWG QGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESK YGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNW YVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS SIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQK ST SI SI GK44GG5GLEVTCEPGTTFKDKCNTCRCGSDGKSAVCTKLWCNQGS,G>î
[491aa protein, aal-l I8=heavy chain variable région of MASP-2 ab#6 (underlined); aa l i9-445=igG4 constant région with hinge mutation; aa 446-451= lsl linker (italicized); aa 452-487=SGMI-2; aa488-491=2nd linker (italicized).]
L-M2ab6-SGMI-2-N (SEP ID NO:77. encoded bv SEQ ID NO:84):
LEVTCEPGTTFKDKCNTCRCGSDGKSAVCTKLWCNQGTGGG5G55SPPVLTPPPS lsvspgqtasitcsgeklgdkyaywypqkpgospvlvmyodkqrpsgiperfsg SNSGNTATLTISGTQAMDEADYYCPAWDSSTAVFGGGTKLTVLGQPKAAPSVTL
FPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKY AASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
147
[258aa protein, aaI-36=SGMI-2 (underlined); aa37-46=Iînker (italicized); aa47-l52=light chain variable région of MASP-2 ab#6 (underlined); aal53-258=human Ig lambda constant région]
L-M2ab6-SGMI-2-C (SEQ ID NO:78. encoded by SEQ ID NO:85):
OPVLTOPPSLSVSPGOTASITCSGEKLGDKYAYWYOOKPGOSPVLVMYODKQRP SGIPERFSGSNSGNTATLTISGTOAMDEADYYCQAWDSSTAVFGGGTKLTVLGQ PKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTT PSKQSNNK.YAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSÆ4GGSG LEVTCEPGTTFKDKCNTCRCGSDGKSAVCTKLWCNQGSGzt
[258aa protein, aal-l06=light chain variable région of MASP-2 ab#6 (underlined); aa 107-2l2=human Ig lambda constant région; aa 213-218=lst linker; aa2l9-254~SGMI-2; aa255-258=2nd linker]
Functional Assays:
The four MASP-2-SGMI-2 fusion antibody constructs were transiently expressed in Expi293F cells (Invitrogen), purified by Protein A affînity chromatography, and tested in 10% normal human sérum for inhibition of C3b déposition in a mannan-coated bead assay as described below.
Testing the MASP-2-SGMI-2 fusions in the mannan-coated bead assav for C3b déposition
The MASP-2-SGMI-2 fusion antibodies assessed for lectin pathway inhibition in an assay of C3b déposition on mannan-coated beads. This assay, which détermines degree of activity by flow cytometry, offers greater resolution than the Wieslab® assay. The lectin pathway bead assay was carried out as follows: mannan was adsorbed to 7 μΜ-diameter polystyrène beads (Bangs Laboratories; Fîshers, IN, USA) ovemight at 4°C in carbonate-bicarbonate buffer (pH 9.6). The beads were washed in PBS and exposed to 10% human sérum, or 10% sérum pre-incubated with antibodies or inhibitors. The serum-bead mixture was incubated at room température for one hour while agitating. Following the sérum incubation, the beads were washed, and C3b déposition on the beads was measured by détection with an anti-C3c rabbit polyclonal antibody (Dako North
148
America; Carpinteria, CA, USA) and a PE-Cy5 conjugated goat anti-rabbit secondary antibody (Southern Biotech; Birmingham, AL, USA). Following the staining procedure, the beads were analyzed using a FACSCalibur flow cytometer. The beads were gated as a unifbrm population using forward and side scatter, and C3b déposition was apparent as FL3-positîve particles (FL-3, or “FL-3 channel” indicates the 3rd or red channel on the cytometer). The Géométrie Mean Fluorescence Intensity (MFI) for the population for each experimental condition was plotted relative to the antibody/inhibitor concentration to evaluate lectin pathway inhibition.
The IC50 values were calculated using the GraphPad PRISM software. Specifically, IC50 values were obtained by applying a variable slope (four parameter), nonlinear fit to log (antibody) versus mean fluorescence intensity curves obtained from the cytométrie assay.
The results are shown in TABLE 11.
TABLE 11: C3b déposition (mannan-coated bead assay) in 10% human sérum
Construct ICso (nM)
Naked N2 ab (mAb#6) > 3.63 nM
H-M2-SGMI-2-N 2.11 nM
L-M2-SGMI-2-C 1.99 nM
H-M2-SGMI-2-N 2.24 nM
L-M2-SGMI-2-N 3.71 nM
Results:
The control, non-SGMI-containing MASP-2 naked” scaffold antibody (mAb#6), was inhibitory in this assay, with an IC50 value of > 3.63 nM, which is consistent with the inhibitory results observed in Example 12. Remarkably, as shown in TABLE 11, ail of the SGMI-2-MASP-2 antibody fusions that were tested improved the potency of the MASP-2 scaffold antibody in this assay, suggesting that increased valency may also be bénéficiai in the inhibition of C3b déposition.
149
Testing the MASP-2-SGMI-2 fusions in the mannan-coated bead assay for C4b déposition assay with 10% human sérum
A C4b déposition assay was carried out with 10% human sérum using the same assay conditions as described above for the C3b déposition assay with the following modifications. C4b détection and flow cytométrie analysis was carried out by staining the déposition reaction with an anti-C4b mouse monoclonal antibody (1:500, Quidel) and staining with a secondary goat anti-mouse F(ab’)2 conjugated to PE Cy5 (1:200, Southern Biotech) prior to flow cytométrie analysis.
Results:
The SGMI-2-bearing MASP-2- N-terminal antibody fusions (H-M2-SGMI-2-N: lC5O0.34nM), L-M2-SGMI-2-N: IC5O0.41 nM)), both had increased potency as compared to the MASP-2 scaffold antibody (HL-M2: IC50=0.78nM).
Similarly, the single SGMI-2 bearing C-terminal MASP-2 antibody fusions (HM2-SGM1-2-C: IC5o=O.45nM and L-M2-SGMI-2C: IC5o=O.47 nM) both had increased potency as compared to the MASP-2 scaffold antibody (HL-M2: IC50—1.2 nM).
Testing the MASP-2-SGMI-2 fusions in the mannan-coated bead assay for C3b déposition with 10% mouse sérum.
A mannan-coated bead assay for C3b déposition was carried out as described above with 10% mouse sérum. Similar to the results observed in human sérum, it was determined that the SGMl-2-bearing MASP-2 fusions had increased potency as compared to the MASP-2 scaffold antibody in mouse sérum.
Summary of Results: The results in this Example demonstrate that ail of the SGMI-2-MASP-2 antibody fusions that were tested improved the potency ofthe MASP-2 scaffold antibody.
EXAMPLE 14
This Example provides results that were generated using a Unilatéral Ureteric Obstruction (UUO) model of rénal fibrosis in MASP-2 -/- déficient and MASP-2 +/+ sufficient mice to evaluate the rôle of the lectin pathway in rénal fibrosis.
Background/Rationale:
ISO
Rénal fibrosis and inflammation are prominent features of late stage kidney disease. Rénal tubulointerstîtial fibrosis is progressive process involving sustained cell injury, aberrant healing, activation of résident and infiltrating kidney cells, cytokine release, inflammation and phenotypic activation of kidney cells to produce extracellular matrix. Rénal tubulointerstîtial (Tl) fibrosis is the common end point of multiple rénal pathologies and represents a key target for potential thérapies aimed at preventing progressive rénal functional impairment in chronic kidney disease (CKD). Rénal TI injury is closely linked to declining rénal function in glomerular diseases (Risdon R.A. et al., Lancet l: 363-366, 1968; Schainuck L.I. et al. Hum Pathol l: 631-640, 1970; Nath
K.A., Am J Kid Dis 20:1-17, 1992), and is characteristic of CKD where there is an accumulation of myofibroblasts, and the potential space between tubules and peritubular capillaries becomes occupied by matrix composed of collagens and other proteoglycans. The origin of TI myofibroblasts remains intensely controversial, but fibrosis is generally preceded by inflammation characterized initially by TI accumulation of T lymphocytes and then later by macrophages (Liu Y. et al., Nat Rev Nephrol 7:684-696, 2011; Duffield J.S., J Clin Invest 124:2299-2306, 2014).
The rodent model of UUO generates progressive rénal fibrosis, a hallmark of progressive rénal disease of virtually any etiology (Chevalier et al., Kidney International 75:1145-1152, 2009). It has been reported that C3 gene expression was increased in wild-type mice following UUO, and that collagen déposition was significantly reduced in C3-/- knockout mice following UUO as compared to wild-type mice, suggesting a rôle of complément activation in rénal fibrosis (Feam et al., Mol Immunol 48:1666-1733, 2011). It has also been reported that C5 deficiency led to a significant amelioration of major components of rénal fibrosis in a model of tubulointerstîtial injury (Boor P. et al., J of Am Soc of Nephrology: 18:1508-1515, 2007). However, prior to the study described herein carried out by the present inventors, the particular complément components involved in renal fibrosis were not well defined. Therefore, the following study was carried out to
I5l evaluate MASP-2 (-/-) and MASP-2 (+/+) male mice in a unilatéral urétéral obstruction (UUO) model.
Methods:
A MASP-2-/- mouse was generated as described in Example l and backcrossed for 10 générations with C57BL/6. Male wild-type (WT) C57BL/6 mice, and homozygous MASP-2 déficient (MASP-2-/-) mice on a C57BL/6 background were kept under standardized conditions of 12/12 day/night cycle, fed on standard food pellets and given free access to food and water. Ten-week-old mice, 6 per group, were anesthetized with 2.5% isoflurane in 1.5 L/min oxygen. The right ureters of two groups of ten-weekold male C56/BL6 mice, wild-type and MASP-2-/- were surgically ligated. The right kidney was exposed through a lem flank incision. The right ureter was completely obstructed at two points using a 6/0 polyglactin suture. Buprénorphine analgesia was provided perioperatively every 12 hours for up to 5 doses depending on pain scoring. Local bupivacaine anesthetîc was given once during the surgery.
Mice were sacrificed 7 days after the surgery and kidney tissues were collected, fixed and embedded in paraffin blocks. Blood was collected from the mice by cardiac puncture under anesthésia, and mice were culled by exsanguination after nephrectomy. Blood was allowed to clôt on ice for 2 hours and sérum was separated by centrifugation and kept frozen as aliquots at -80°C.
Immunohistochemistry of Kidney Tissue
To measure the degree of kidney fibrosîs as indicated by collagen déposition, 5 micron paraffin embedded kidney sections were stained with picrosirius red, a collagenspecific stain, as described in Whittaker P. et al., Basic Res Cardial 89:397-410, 1994. Briefly described, kidney sections were de-paraffinized, rehydrated and collagen stained for 1 hour with picrosirius red aqueous solution (0,5 gm Sirius red, Sigma, Dorset UK) in 500 mL saturated aqueous solution of picric acid. Slides were washed twice in acidîfied water (0.5% glacial acetic acid in distilled water) for 5 minutes each, then dehydrated and mounted.
To measure the degree of inflammation as indicated by macrophage infiltration, kidney sections were stained with macrophage-specific antibody F4/80 as follows. Formalin fixed, paraffin embedded, 5 micron kidney sections were deparaffinized and
I52 rehydrated. Antigen retrieval was performed in citrate buffer at 95°C for 20 minutes followed by quenching of endogenous peroxidase activity by incubation in 3% H2O2 for IO minutes. Tissue sections were incubated in blocking buffer (10% heat înactivated normal goat sérum with l% bovine sérum albumin in phosphate buffered saline (PBS)) for l hour at room température followed by avidin/biotin blocking. Tissue sections were washed in PBS three times for 5 minutes after each step. F4/80 macrophage primary antibody (Santa Cruz, Dallas, TX, USA) diluted l : 100 in blocking buffer was applied for l hour. A biotinylated goat anti-rat secondary antibody, diluted l :200, was then applied for 30 minutes followed by horse radish peroxidase (MRP) conjugated enzyme for 30 minutes. Staining color was developed using diaminobenzidine (DAB) substrate (Vector Labs, Peterborough UK) for IO minutes and slides were washed in water, dehydrated and mounted without counter staining to facilitate the computer based analysis.
Image Analysis
The percentage of kidney cortical staining was determined as described in Fumess P. N. et al., J Clin Pathol 50:118-122, 1997. Briefly described, 24 bit color images were captured from sequential non-overlapping fields of rénal cortex just beneath the rénal capsule around the entire periphery of the section of kidney. After each image capture NIH Image was used to extract the red channel as an 8 bit monochrome image. Unevenness in the background illumination was subtracted using a pre-recorded image of the illuminated microscope field with no section in place. The image was subjected to a fixed threshold to identify areas of the image corresponding to the staining positivity. The percentage of black pixels was then calculated, and after ail the images around the kidney had been measured in this way the average percentage was recorded, providing a value corresponding to the percentage of stained area in the kidney section.
Gene Expression Analysis
Expression of several genes relevant to rénal inflammation and fibrosis in mouse kidney were measured by quantitative PCT (qPCR) as follows. Total RNA was isolated from kidney cortex using Trîzol® (ThermoFisher Scientifîc, Paisley, UK) according to the manufacturées instructions. Extracted RNA was treated with the Turbo DNA-free kit (ThermoFisher Scientifîc) to elîminate DNA contamination, and then first strand cDNA was synthesized using AMV Reverse Transcription System (Promega, Madison, WI,
153
USA). The cDNA integrity was confirmée! by a single qPCR reaction using TaqMan GAPDH Assay (Applied Bîosystems, Paisley UK) followed by qPCR reaction using Custom TaqMan Array 96-well Plates (Life Technologies, Paisley, UK).
Twelve genes were studied in this analysis:
Collagen type IV alpha l (co!4al; assay ID: Mm0l2l0l25_ml)
Transforming growth factor beta-l (TGFp-l; assay ID: MmOl l78820_ml);
Cadherin l (Cdhl; Assay ID: Mm0l247357_ml);
Fibronectin l (Fnl; Assay ID:Mm0l256744_ml);
Interleukin 6 (IL6; Assay ID Mm0044619l_m l);
Interleukin 10 (ILl 0; Assay ID Mm00439614_m l);
Interleukin I2a (ILl2a; Assay ID Mm00434l65_ml);
Vimentin (Vim; Assay ID Mm0l333430_ml);
Actinin alpha l (Actnl; Assay ID Mm0l304398_ml);
Tumor necrosis factor-α (TNF-a: Assay ID Mm00443260_gl)
Complément component 3 (C3; Assay ID Mm00437838_ml);
Interferon gamma (Ifn-γ; Assay ID MmOl 168134)
The following housekeeping control genes were used:
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Assay ID Mm99999915_gl );
Glucuronidase beta (Gusp; Assay ID Mm00446953_ml);
Eukaryotic 18S rRNA (I8S; Assay ID Hs99999901_sl);
Hypoxanthine guanine phosphoribosyl transferase (HPRT; Assay ID Mm00446968_ml)
Twenty pL reactions were amplified using TaqMan Fast Universal Master Mix (Applied Bîosystems) for 40 cycles. Real time PCR amplification data were analyzed using Applied Bîosystems 7000 SDS vL4 software.
RESULTS:
Following unilatéral ureteric obstruction (UUO), obstructed kidneys expérience an influx of inflammatory cells, particularly macrophages, followed by the prompt
I54 development of fibrosis as evidenced by the accumulation of collagen alongside tubular dilatation and atténuation of the proximal tubular epithelium (see Chevalier R. L. et al., Kidney Int 75:1145-1152, 2009).
FIGURE 15 graphically illustrâtes the results of computer-based image analysis of kidney tissue sections staïned with Sirius red, wherein the tissue sections were obtained from wild-type and MASP-2-/- mice following 7 days of ureteric obstruction (UUO) or from sham-operated control mice. As shown in FIGURE 15, kidney sections of wild-type mice following 7 days of ureteric obstruction showed significantly greater collagen déposition compared to MASP-2-/- mice (p value = 0.0096). The mean values ± standard error of means for UUO operated mice in wild-type and MASP-2-/- groups were 24.79± 1.908 (n=6) and 16.58±1.3 (n=6), respectively. As further shown in FIGURE 15, the tissue sections from the sham-operated control wild-type and the sham operated control MASP-2-/- mice showed very low levels of collagen staining, as expected.
FIGURE 16 graphically illustrâtes the results of computer-based image analysis of kidney tissue sections stained with the F4/80 macrophage-specific antibody, wherein the tissue sections were obtained from wild-type and MASP-2-/- mice following 7 days of ureteric obstruction or from sham-operated control mice. As shown in FIGURE 16, compared to wild-type mice, the tissue obtained from UUO kidneys from MASP-2-/mice exhibited significantly less macrophage infiltration following 7 days of ureteric obstruction (% macrophage area stained in WT:2.23±0.4 vs MASP-2-/-: 0.53±0.06, p=0.0035). As further shown in FIGURE 16, the tissue sections from the sham-operated wild-type and the sham-operated MASP-2-/- mice showed no détectable macrophage staining.
Gene expression analysis of a variety of genes linked to rénal inflammation and fibrosis was carried out in the kidney tissue sections obtained from wild-type and MASP2-/- mice following 7 days of ureteric obstruction and sham-operated wild-type and MASP-2-/- mice. The data shown in FIGURES 17-20 are the LoglO of relative quantitation to a wild-type sham operated sample and bars represent the standard error of means. With regard to the results of the gene expression analysis of the fibrosis-related genes, FIGURE 17 graphically illustrâtes the relative mRNA expression levels of collagen type IV alpha 1 (collagen-4), as measured by qPCR in kidney tissue sections
155 obtained from wild-type and MASP-2-/- mice following 7 days of ureteric obstruction and sbam-operated control mice. FIGURE 18 graphically illustrâtes the relative mRNA expression levels of Transforming Growth Factor Beta-l (TGFP-l), as measured by qPCR in kidney tissue sections obtained from wild-type and MASP-2-/- mice following Ί days of ureteric obstruction and sham-operated control mice. As shown in FIGURES 17 and 18, the obstructed kidneys from the wild-type mice demonstrated significantly increased expression ofthe fibrosis-related genes Collagen-type IV (FIGURE 17) and TGFp-l (FIGURE 18), as compared to the sham-operated kidneys in wild-type mice, demonstratîng that these fibrosis-related genes are induced after UUO injury in wild-type mice, as expected. In contrast, as further shown in FIGURES 17 and 18, the obstructed kidneys from the MASP-2-/- subjected to the UUO injury exhibited a significant réduction in the expression of Collagen-type IV (FIGURE 17, p=0.0388) and a significant réduction in the expression of TGFP-l (FIGURE 18, p=0.0174), as compared to the wild-type mice subjected to the UUO injury.
With regard to the results of the gene expression analysis of the inflammationrelated genes, FIGURE 19 graphically illustrâtes the relative mRNA expression levels of Interleukin-6 (IL-6), as measured by qPCR in kidney tissue sections obtained from wildtype and MASP-2-/- mice following 7 days of ureteric obstruction and sham-operated control mice. FIGURE 20 graphically illustrâtes the relative mRNA expression levels of Interferon-γ, as measured by qPCR in kidney tissue sections obtained from wild-type and MASP-2-/- mice following 7 days of ureteric obstruction and sham-operated control mice. As shown in FIGURES 19 and 20, the obstructed kidneys from the wild-type mice demonstrated significantly increased expression of the inflammation-related genes Interleukin-6 (FIGURE 19) and Interferon-γ (FIGURE 20), as compared to the shamoperated kidneys in wild-type mice, demonstratîng that these inflammation-related genes are induced after UUO injury in wild-type mice. In contrast, as further shown in FIGURES 19 and 20, the obstructed kidneys from the MASP-2-/- subjected to the UUO injury exhibited a significant réduction in the expression of Interleukin-6 (FIGURE 19, p=0.0109) and Interferon-γ (FIGURE 20, p=0.0182) as compared to the wild-type mice subjected to the UUO injury.
156
It Is noted that gene expression for Vim, Actn-1, TNFa, C3 and IL-10 were ail found to be significantly up-regulated in the UUO kidneys obtained from both the wildtype and the MASP-2-/- mice, with no significant différence în the expression levels of these particular genes between the wild-type and MASP-2-/- mice (data not shown). The gene expression levels of Cdh-1 and IL-I2a did not change in obstructed kidneys from animais in any group (data not shown).
Discussion:
The UUO model in rodents is recognized to induce an early, active and profound injury in the obstructed kidney with reduced rénal blood flow, interstitial inflammation and rapid fibrosis within one to two weeks following obstruction and has been used extensively to understand common mechanisms and mediators of inflammation and fibrosis in the kidney (see e.g., Chevalier R.L., Kidney Int 75:1145-1152, 2009; Yang H. et al., Drug Discov Today Dis Models 7:13-19, 2010).
The results described in this Example demonstrate that there is a significant réduction in collagen déposition and macrophage infiltration in UUO operated kidneys in the MASP-2(-/-) mice versus the wild-type (+/+) control mice. The unexpected results showing a significant réduction of rénal injury at both the histological and gene expression levels in the MASP-2-/- animais demonstrates that the lectin pathway of complément activation contributes significantly to the development of inflammation and fibrosis in the obstructed kidney. While not wishing to be bound by a particular theory, it îs believed that the lectin pathway contributes critically to the pathophysiology of fibrotic disease by triggering and maintaining pro-înflammatory stimuli that perpetuate a vicious cycle where cellular injury drives inflammation which in tum causes further cellular injury, scarring and tissue loss. In view of these results, it is expected that that inhibition or blockade of MASP-2 with an inhibitor would hâve a préventive and/or therapeutic effect in the inhibition or prévention of rénal fibrosis, and for the inhibition or prévention of fibrosis in general (i.e., independent of the tissue or organ).
EXAMPLE 15
157
This Example describes analysis ofa monoclonal MASP-2 inhibitory antibody for efficacy in the Unilatéral Ureteric Obstruction (UUO) model, a murine model of renal fïbrosis.
Backsround/Rationale:
Amelioration of renal tubulointerstitial fïbrosis, the common end point of multiple rénal pathologies, represents a key target for therapeutic strategies aîmed at preventing progressive renal diseases. Given the paucity of new and existing treatments targeting inflammatory pro-fibrotic pathways in renal disease, there is a pressing need to develop new thérapies. Many patients with protéinurie renal disease exhibit tubulointerstitial inflammation and progressive fïbrosis which closely parallels declining renal function. Proteinuria per se induces tubulointerstitial inflammation and the development of protéinurie nephropathy (Brunskill N.J. et al., J Am Soc Nephrol 15:504-505, 2004). Regardless of the primary renal disease, tubulointerstitial inflammation and fïbrosis is invariably seen in patients with progressive renal impairment and is closely correlated with declining excretory function (Risdon R.A. et al., Lancet 1:363-366, 1968; Schainuck
L.L, et al., Hum Pathol 1: 631-640, 1970). Thérapies with the potential to interrupt the key common cellular pathways leading to fïbrosis hold the promise of wide applicability in renal disorders.
As described in Example 14, in the UUO model of non-proteinuric renal fïbrosis it was determined that MASP-2-/- mice exhibited signifîcantly less renal fïbrosis and inflammation compared to wild-type control animais, as shown by inflammatory cell infiltrâtes (75% réduction), and histological markers of fïbrosis such as collagen (one third réduction), thereby establîshing a key rôle of the lectin pathway in renal fïbrosis.
As described in Example 13, a monoclonal MASP-2 antibody (OMS646-SGMI-2 fusion, comprising an SGMI-2 peptide fused to the C-terminus of the heavy chain of OMS646) was generated that specificaily blocks the function of the human lectin pathway has also been shown to block the lectin pathway in mice. In this example, OMS646-SGMI-2 was analyzed in the UUO mouse model of renal fïbrosis in wild-type mice to détermine if a spécifie inhibitor of MASP-2 is able to înhîbit renal fïbrosis.
Methods:
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This study evaluated the effect of a MASP-2 inhibitory antibody (10 mg/kg OMS646-SGMI-2), compared to a human IgG4 isotype control antibody (10 mg/kg ET904), and a vehicle control in male WT C57BL/6 mice. The antibodies (lOmg/kg) were administered to groups of 9 mice by intraperitoneal (ip) injection on day 7, day 4 and day l prior to UUO surgery and again on day 2 post-surgery. Blood samples were taken prior to antibody administration and at the end ofthe experiment to assess lectin pathway functional activity.
The UUO surgery, tissue collection and staining with Sirius red and macrophagespecific antibody F4/80 were carried out using the methods described in Example 14.
Hydroxyproline content of mouse kidneys was measured using a spécifie colorimétrie assay test kit (Sigma) according to manufacturer’s instructions.
To assess the pharmacodynamie effect of the MASP-2 inhibitory mAb in mice, systemic lectin pathway activity was evaluated by quantitating lectin-induced C3 activation in minimally diluted sérum samples collected at the indicated time after MASP-2 mAb or control mAb i.p. administration to mice. Briefly described, 7 μΜ diameter polystyrène microspheres (Bangs Laboratories, Fisher IN, USA) were coated with mannan by ovemight incubation with 30pg/mL mannan (Sigma) in sodium bicarbonate buffer (pH 9.6), then washed, blocked with l% fêtai bovine sérum in PBS and resuspended in PBS at a final concentration of IxlO8 beads/mL. Complément déposition reactions were initiated by the addition of 2.5 pL of mannan-coated beads (-250,000 beads) to 50 pL of minimally diluted mouse sérum samples (90% final serum concentration), followed by incubation for 40 minutes at 4°C. Following termination of the déposition reaction by the addition of 250 pL of ice-cold flow cytometry buffer (FB. PBS containing 0.1% fêtai bovine serum), beads were collected by centrifugation and washed two more times with 300 pL of ice-cold FB.
To quantify lectin-induced C3 activation, beads were incubated for l hour at 4°C with 50 pL of rabbit anti-human C3c antibody (Dako, Carpenteria, CA, USA) diluted in FB. Following two washes with FB to remove unbound material, the beads were incubated for 30 minutes at 4°C with 50 pL of goat anti-rabbit antibody conjugated to PECy5 (Southern Biotech, Birmingham, AL, USA) diluted in FB. Following two washes with FB to remove unbound material, the beads were resuspended in FB and analyzed by
I59 a FACS Calibur cytometer. The beads were gated as a uniform population using forward and side scatter, and C3b déposition in each sample was quantitated as mean fluorescent intensity (MFI).
Results:
Assessment of Collagen Déposition:
FIGURE 21 graphically illustrâtes the results of computer-based image analysis of kidney tissue sections stained with Siruis red, wherein the tissue sections were obtained following 7 days of ureteric obstruction from wild-type mice treated with either a MASP2 inhibitory antibody or an isotype control antibody. As shown in FIGURE 21, tissue sections from kidneys harvested 7 days after obstruction (UUO) obtained from wild-type mice treated with MASP-2 inhibitory antibody showed a significant réduction (p=0.0477) in collagen déposition as compared with the amount of collagen déposition in tissue sections from obstructed kidneys obtained from wild-type mice treated with an IgG4 isotype control.
Assessment of Hydroxy proline content:
Hydroxy proline was measured in kidney tissues as an indicator of collagen content. Hydroxy proline is a parameter which is highly indicative of the pathophysiological progression of disease induced in this model.
FIGURE 22 graphically illustrâtes the hydroxyl proline content from kidneys harvested 7 days after obstruction (UUO) obtained from wild-type mice treated with either a MASP-2 inhibitory antibody or an isotype control antibody. As shown in FIGURE 22, the obstructed kidney tissues from mice treated with MASP-2 inhibitory antibody demonstrated significantly less hydroxyl proline, an indicator of collagen content, than the kidneys from mice treated with the IgG4 isotype control mAb (p=0.0439).
Assessment of inflammation:
Obstructed kidneys from wild-type, isotype control antibody-treated animais, and wild-type animais treated with MASP-2 inhibitory antibody demonstrated a brisk infiltrate of macrophages. Careful quantification revealed no significant différence in macrophage percentage stained area between these two groups (data not shown). However, despite équivalent numbers of infiltrating macrophages, the obstructed kidneys
160 from the MASP-2 inhibitory antibody-injected animais exhibited significantly less fibrosis as judged by Sirius red stainîng, compared to obstructed kidneys from isotype control injected animais, which resuit is consistent with the results that obstructed kîdney tissues from mice treated with MASP-2 inhibitory antibody had significantly less hydroxyl proline than the kidneys treated with the IgG4 isotype control mAb.
Discussion
The results described in this Example demonstrate that the use of a MASP-2 inhibitory antibody provides protection against rénal fibrosis in the UUO model, which is consistent with the results described in Example 14 demonstrating that MASP-2-/- mice hâve significantly reduced rénal fibrosis and inflammation in the UUO model as compared to wild-type mice. The results in this Example showing reduced fibrosis in the mice treated with the MASP-2 inhibitory antibody. The finding of reduced fibrosis in the UUO kidneys in animais with a réduction or blockade of MASP-2-dependent lectin pathway activity is highly significant novel finding. Taken together, the results presented in Example 14 and in this Example demonstrate a bénéficiai effect of MASP-2 inhibition on rénal tubulointerstitial inflammation, tubular cell injury, profibrotic cytokine release and scarring. The relief of rénal fibrosis remains a key goal for rénal therapeutics. The UUO model is a severe model of accelerated rénal fibrosis, and an intervention that reduces fibrosis in this model, such as the use of MASP-2 inhibitory antibodies, is likely to be used to inhibit or prevent rénal fibrosis. The results from the UUO model are likely to be transférable to rénal disease characterized by glomerular and/or protéinurie tubular injury.
EXAMPLE 16
This Example provides results that were generated using a protein overload proteinurea model of rénal fibrosis, inflammation and tubulointerstitial injury in MASP2-/- and wild-type mice to evaluate the rôle of the lectin pathway in protéinurie nephropathy.
Background/Rationale:
Proteinuria is a risk factor for the development of rénal fibrosis and loss of rénal excretory function, regardless of the primary rénal disease (Tryggvason K. et al., J Intern Med 254:216-224, 2003, Williams M., Am J. Nephrol 25:77-94, 2005). The concept of
I6l protéinurie nephropathy describes the toxic effects of excess protein entering the proximal tabule as a resuit ofthe impaired glomerular permselectivity (Brunskill NJ., J Am Soc Nephrol 15:504-505, 2004, Baines RJ., Nature Rev Nephrol 7:177-180, 2011). This phenomenon, common to many glomerular diseases, results in a pro-inflammatory scarring environment in the kidney and is characterized by alterations in proximal tubular cell growth, apoptosis, gene transcription and inflammatory cytokine production as a conséquence of dysregulated signaling pathways stimulated by protéinurie tubular fluid. Protéinurie nephropathy is generally recognized to be a key contributor to progressive rénal injury common to diverse primary rénal pathologies.
Chronic kidney disease affects greater than 15% of the adult population in the United States and accounts for approximately 750,000 deaths each year worldwide (Lozano R. et al., Lancet vol 380, Issue 9859:2095-2128, 2012). Proteinuria is an indicator of chronic kidney disease as well as a factor promoting disease progression. Many patients with protéinurie rénal disease exhîbit tubulointerstitîal inflammation and progressive fibrosis which closely parallels declining rénal function. Proteinuria per se induces tubulointerstitîal inflammation and the development of protéinurie nephropathy (Brunskill N.J. et al., J Am Soc Nephrol 15:504-505, 2004). In protéinurie kidney diseases, excessive amounts of albumin and other macromolecules are filtered through the glomeruli and reabsorbed by proximal tubular épithélial cells. This causes an inflammatory vîcious cycle mediated by complément activation leadîng to cytokine and leukocyte infiltrâtes that cause tubule-interstitial injury and fibrosis, thereby exacerbating proteinuria and leading to loss of rénal function and eventually progression to end-stage rénal failure (see, e.g., Clark et al., Canadian Medical Association Journal 178:173-175, 2008). Thérapies that modulate this detrimental cycle of inflammation and proteinuria are expected to improve outcomes in chronic kidney disease.
In view of the bénéficiai effects of MASP-2 inhibition in the UUO model of tubuloînterstital injury, the following experiment was carried out to détermine if MASP-2 inhibition would reduce rénal injury in a protein overload model. This study employed protein overload to induce protéinurie kidney disease as described in Ishola et al., European Rénal Association 21:591-597, 2006.
Methods:
162
A MASP-2-/- mouse was generated as described in Example l and backcrossed for 10 générations with BALB/c. The current study compared the results of wild-type and MASP-2-/- BALB/c mice in a protein overload proteinuria model as follows.
One week prior to the experiment, mice were unilaterally nephrectomised before protein overload challenge in order to see an optimal response. The proteinuria inducing agent used was a low endotoxin bovine sérum albumin (BSA, Sigma) given i.p. in normal saline to WT (n=7) and MASP-2 -/- mice (n=7) at the following doses: one dose each of 2mg BSA/gm, 4mg BSA/gm, 6mg BSA/gm, 8mg BSA/gm, lOmg BSA/gm and I2mg BSA/gm body weight, and 9 doses of I5mg BSA/gm body weight, for a total of 15 doses administered i.p. over a period of 15 days. The control WT (n=4) and MASP-2-/- (n=4) mice received saline only administered i.p. After administration of the last dose, animais were caged separately in metabolic cages for 24 hours to collect urine. Blood was collected by cardiac puncture under anesthésia, blood was allowed to clôt on ice for 2 hours and sérum was separated by centrifugation. Sérum and urine samples were collected at the end of the experiment on day 15, stored and frozen for analysis.
Mice were sacrificed 24 hours after the last BSA administration on day 15 and various tissues were collected for analysis. Kidneys were harvested and processed for H&E and immunostaining. Immunohistochemistry staining was carried out as follows. Formalîn fixed, parafïin-embedded 5 micron kidney tissue sections from each mouse were deparaffînized and rehydrated. Antigen retrieval was performed in citrate buffer at 95°C for 20 minutes followed by incubating tissues in 3% H2O2 for 10 minutes. Tissues were then incubated in blocking buffer (I0% sérum from the species the secondary antibody was raised in and l% BSA in PBS) with 10% avidin solution for l hour at room température. Sections were washed in PBS three times, 5 minutes each, after each step. Primary antibody was then applied in blocking buffer with 10% bîotin solution for l hour at a concentration of l : 100 for the antibodies F4/80 (Santa Cruz cat# sc-25830), TGFP (Santa Cruz cat# sc-7892), IL-6 (Santa Cruz cat# sc-1265) and at l:50 for the TNFa antibody (Santa Cruz cat# sc-1348). A biotinylated secondary antibody was then applied for 30 minutes at a concentration of l :200 for the F4/80, TGFp and IL-6 sections and l : 100 for the TNFa section followed by HRP conjugale enzyme for another 30 minutes. The color was developed using diaminobenzidine (DAB) substrate kit (Vector labs) for
163
IO minutes and slides were washed in water, dehydrated and mounted without counter staining to faciiitate computer-based image analysis. Stained tissue sections from the rénal cortex were analyzed by digital image capture followed by quantification using automated image analysis software.
Apoptosis was assessed in the tissue sections by staining with terminal deoxynucléotidyl transferase dUTP nick end labeling (TUNEL) as follows. Apoptotic cells in the kidney sections were stained using ApopTag® Peroxidase kit (Millipore) as follows. Parraftn embedded, formalîn fixed kidney sections from each mouse were deparaffmized, rehydrated and then protein permeabilized using protéinase K (20 gg/mL) which was applied to each specîmen for 15 minutes at room température. Specimens were washed in PBS between steps. Endogenous peroxidase activity was quenched by incubating tissues in 3% H2O2 for IO minutes. Tissues were then incubated în équilibration buffer followed by incubation with TdT enzyme for l hour at 37°C. After washing in stop/wash buffer for IO minutes, anti-digoxignenin conjugale was applied for
30 minutes at room température followed by washing. Color was developed in DAB substrate kit for 4 minutes followed by washing în water. Tissues were counter stained in haematoxylin and mounted in DBX. The frequency of TUNEL stained (brown colored) apoptotic cells were manually counted in serially selected 20 high power fields from the cortex using Leica DBXM light microscope.
Results:
Assessment of Proteinuria
To confirm the presence of proteinuria in the mice, the total protein in sérum was analyzed at day 15 and the total excreted proteins in urine was measured în urine samples collected over a 24 hour period on day 15 of the study.
FIGURE 23 graphically illustrâtes the total amount of sérum proteins (mg/ml) measured at day 15 in the wild-type control mice (n=2) that received saline only, the wild-type mice that received BSA (n—6) and the MASP-2-/- mice that received BSA (n=6). As shown in FIGURE 23, administration of BSA increased the sérum total protein level in both wild-type and MASP-2-/- groups to more than double the concentration of 30 the control group that received only saline, with no significant différence between the treated groups.
164
FIGURE 24 graphically illustrâtes the total amount of excreted protein (mg) in urine collected over a 24 hour period on day 15 of the study from the wild-type control mice (n=2) that received saline only, the wild-type mice that received BSA (n=6) and the MASP-2-/- mice that received BSA (n^ô). As shown in FIGURE 24, on day 15 of this study, there was an approximately six-fold increase in total excreted proteins in urine in the BSA treated groups as compared to the sham-treated control group that received saline only. The results shown in FIGURES 23 and 24 demonstrate that the proteinuria model was working as expected.
Assessmentof Histological Changes in the Kidney
FIGURE 25 shows représentative H&E stained rénal tissue sections that were harvested on day 15 of the protein overload study from the following groups of mice: (panel A) wild-type control mice; (panel B) MASP-2-/- control mice; (panel C) wild-type mice treated with BSA; and (panel D) MASP-2-/- mice treated with BSA. As shown in FIGURE 25, there is a much higher degree of tissue préservation in the MASP-2-/overload group (panel D) compared to the wild-type overload group (panel C) at the same level of protein overload challenge. For example, Bowman’s capsules in the wild-type mice treated with BSA (overload) were observed to be greatly expanded (panel C) as compared to Bowman’s capsules in the wild-type control group (panel A). In contrast, Bowman’s capsules in the MASP-2-/- mice (overload) treated with the same level of BSA (panel D) retained morphology similar to the MASP-2-/- control mice (panel B) and wild-type control mice (panel A). As further shown in FIGURE 25, large protein cast structures hâve accumulated in proximal and distal tabules of the wild-type kidney sections (panel C), which are larger and more abundant as compared to MASP-2-/- mice (panel D).
It is also noted that analysis of rénal sections from this study by transmitting électron microscope showed that the mice treated with BSA had overall damage to the ciliary borders of distal and proximal tubular cells, with cellular content and nuclei bursting into the tubule lumen. In contrast, the tissue was preserved in the MASP-2-/mice treated with BSA.
Assessment of Macrophage Infiltration in the Kidney
165
To measure the degree of inflammation, as indicated by macrophage infiltration, the tissue sections of the harvested kidneys were also stained with macrophage-specific antibody F4/80 using methods as described in Boor et al., J of Am Soc of Nephrology 18:1508-1515, 2007.
FIGURE 26 graphîcaliy illustrâtes the results of computer-based image analysis of kidney tissue sections stained with macrophage-specific antibody F4/80, showing the macrophage mean stained area (%), wherein the tissue sections were obtained at day 15 of the protein overload study from wild-type control mice (n=2), wild-type mice treated with BSA (n=6), and MASP-2-/- mice treated with BSA (n=5). As shown in FIGURE 26, kidney tissue sections stained with F4/80 anti-macrophage antibody showed that while both groups treated with BSA showed a significant increase in the kidney macrophage infiltration (measured as %F4/80 antibody-stained area) compared to the wild-type sham control, a significant réduction in macrophage infiltration was observed in tissue sections from BSA-treated MASP-2-/- mice as compared with macrophage infiltration in tissue sections from BSA-treated wild-type mice (p value=0.0345).
FIGURE 27A graphîcaliy illustrâtes the analysis for the presence of a macrophage-proteinuria corrélation in each wild-type mouse (n=6) treated with BSA by plotting the total excreted proteins measured in urine from a 24 hour sample versus the macrophage infiltration (mean stained area %). As shown in FIGURE 27A, most of the samples from the wild-type kidneys showed a positive corrélation between the level of proteinuria present and the degree of macrophage infiltration.
FIGURE 27B graphîcaliy illustrâtes the analysis for the presence of a macrophage-proteinuria corrélation in each MASP-2-/- mouse (n=5) treated with BSA by plotting the total excreted proteins in urine in a 24 hour sample versus the macrophage infiltration (mean stained area %). As shown in FIGURE 27B, the positive corrélation observed in wild-type mice between the level of proteinuria and the degree of macrophage infiltration (shown in FIGURE 27A) was not observed in MASP-2-/- mice. While not wishîng to be bound by any particular theory, these results may indicate the presence of a mechanism of inflammation clearance at high levels of proteinuria in MASP-2-/- mice.
Assessment of Cytokine Infiltration
I66
Interleukin 6 (IL-6), Transforming Growth Factor Beta (TGFP) and Tumor Necrosis Factor Alpha (TNFa) are pro-inflammatory cytokines known to be up-regulated in proximal tubules of wild-type mice in a model of proteinuria (Abbate M. et al., Journal of the American Society of Nephrology: JASN, 17: 2974-2984, 2006; David S. et al., Nephrology, Didalysis, Transplantation, Official Publication of the European Diaiysis and Transplant Association- European Rénal Association 12: 51-56, 1997). The tissue sections of kidneys were stained with cytokine-specific antibodies as described above.
FIGURE 28 graphically illustrâtes the results of computer-based image analysis of stained tissue sections with anti-TGFp antibody (measured as % TGFp antibody-stained area) in wild-type mice treated with BSA (n=4) and MASP-2-/- mice treated with BSA (n=5). As shown in FIGURE 28, a significant increase in the staining of TGFp was observed in the wild-type BSA treated (overload) group as compared to the MASP-2-/BSA treated (overload) group (p=0.026).
FIGURE 29 graphically illustrâtes the results of computer-based image analysis of stained tissue sections with anti-TNFa antibody (measured as % TNFa antibody-stained area) in wild-type mice treated with BSA (n=4) and MASP-2-/- mice treated with BSA (n=5). As shown in FIGURE 29, a significant increase in the staining of TNFa was observed in the wild-type BSA treated (overload) group as compared to the MASP-2-/BSA treated (overload) group (p=0.0303).
FIGURE 30 graphically illustrâtes the results of computer-based image analysis of stained tissue sections with anti-IL-6 antibody (measured as % IL-6 antibody-stained area) in wild-type control mice, MASP-2-/- control mice, wild-type mice treated with BSA (n=7) and MASP-2-/- mice treated with BSA (n=7). As shown in FIGURE 30, a highly significant increase in the staining of IL-6 was observed in the wild-type BSA treated group as compared to the MASP-2-/- BSA treated group (p=0.0016).
Assessment of Apoptosis
Apoptosis was assessed in the tissue sections by staining with terminal deoxynucléotidyl transferase dUTP nick end labeling (TUNEL) and the frequency of TUNEL stained apoptotic cells were counted in serially selected 20 high power fields (HPFs) from the cortex.
167
FIGURE 31 graphically illustrâtes the frequency of TUNEL apoptotic cells counted in serially selected 20 high power fields (HPFs) from tissue sections from the rénal cortex in wild-type control mice (n=I), MASP-2-/- control mice (n=l), wild-type mice treated with BSA (n=6) and MASP-2-/- mice treated with BSA (n=7). As shown in FIGURE 31, a significantly higher rate of apoptosis in the cortex was observed în kldneys obtained from wild-type mice treated with BSA as compared to kidneys obtained from the MASP-2-/- mice treated with BSA (p=0.000l).
Overall Summary of Results and Conclusions:
The results in this Example demonstrate that MASP-2-/- mice hâve reduced rénal injury in a protein overload model. Therefore, MASP-2 inhibitory agents, such as MASP-2 inhibitory antibodies would be expected to inhibit or prevent the detrimental cycle of inflammation and proteinuria and improve outcomes in chronîc kidney disease.
EXAMPLE 17
This Example describes analysis of a monoclonal MASP-2 inhibitory antibody for efficacy in reducing and/or preventing rénal inflammation and tubulointerstitial injury in a mouse protein overload proteinurea model in wild-type mice.
Background/Rationale:
As described în Example 16, in a protein overload model of proteinuria it was determined that MASP-2-/- mice exhibited significantly better outcomes (e.g., less tubulointerstitial injury and less rénal inflammation) than wild-type mice, implicating a pathogenic rôle for the lectin pathway in protéinurie kidney disease.
As described in Example 13, a monocional MASP-2 inhibitory antibody (OMS646-SGMI-2) was generated that specifically blocks the function of the human lectin pathway and has also been shown to block the lectin pathway in mice. In this example, the MASP-2 inhibitory antibody OMS646-SGMI-2 was anaiyzed in a mouse protein overload proteinurea model for efficacy in reducing and/or preventing rénal inflammation and tubulointerstitial injury in wild-type mice.
Methods:
168
This study evaluated the effect of MASP-2 inhibitory antibody (10 mg/kg OMS646-SGMI-2), compared to a human IgG4 isotype control antibody, ET904 (10 mg/kg), and a saline control.
Similar to the study described in Example 16, this study employed protein overload to induce protéinurie kidney disease (Ishola et al., European Rénal Association 21:591-597, 2006). Proteinuria was induced in unilaterally nephrectomîzed Balb/c mice by daily i.p. injections with escalating doses (2 g/kg to 15 g/kg) of low endotoxîn bovine sérum albumin (BSA) for a total of 15 days, as described in Example 16.
Antibody treatments were administered by biweekly i.p. injection starting 7 days before proteinuria induction and continued throughout the study. This dosing scheme was selected based on previous PK/PD and pharmacoclogy studies demonstrating sustained lectin pathway suppression (data not shown). Mice were sacrificed on day 15 and kîdneys were harvested and processed for H&E and immunostaining. Stained tissue sections from the rénal cortex were analyzed by digital image capture followed by quantification using automated image analysis software.
Immunohistochemistry staining and apoptosis assessment were carried out as described in Example 16.
Results:
Assessment of Proteinuria
To confirm the presence of proteinuria in the mice, the total excreted proteins in urine was measured in urine samples collected over a 24 hour period at day 15 (the end of the experiment). It was determined that the urine samples showed a mean of almost a six-fold increase in total protein levels in the groups that were treated with BSA as compared to the control groups not treated with BSA (data not shown), confirming the presence of proteinuria in the mice treated with BSA. No significant différence was observed in the protein levels between the BSA-treated groups.
Assessment of Histological Changes
FIGURE 32 shows représentative H&E stained tissue sections from the following groups of mice at day 15 after treatment with BSA: (panel A) wild-type control mice treated with saline; (panel B) isotype antibody treated control mice; and (panel C) wildtype mice treated with MASP-2 inhibitory antibody.
169
As shown in FIGURE 32, there is a much higher degree of tissue préservation in the MASP-2 inhibitory antibody-treated group (panel C) as compared to the wild-type group treated with saline (panel A) or isotype control (panel B) at the same level of protein overload challenge.
Assessment of Apoptosis
Apoptosis was assessed in the tissue sections by staining with terminal deoxynucléotidyl transferase dUTP nick end labeling (TUNEL) and the frequency of TUNEL stained apoptotic cells were counted in serially selected 20 high power fields (HPFs) from the cortex. FIGURE 33 graphically illustrâtes the frequency of TUNEL apoptotic cells counted in serially selected 20 high power fields (HPFs) from tissue sections from the rénal cortex in wild-type mice treated with saline control and BSA (n=8), wild-type mice treated with the isotype control antibody and BSA (n=8) and wildtype mice treated with the MASP-2 inhibitory antibody and BSA (n=7). As shown in FIGURE 33, a highly significantly decrease in the rate of apoptosis in the cortex was observed in kidneys obtained from the MASP-2 inhibitory antibody treated group as compared to the saline and isotype control treated group (p=0.0002 for saline control v MASP-2 inhibitory antibody; p=0.0052 for isotype control v. MASP-2 inhibitory antibody).
Assessment of Cytokine Infiltration
Interleukin 6 (IL-6), Transforming Growth Factor Beta (TGFP) and Tumor Necrosis Factor Alpha (TNFa), which are pro-inflammatory cytokines known to be upregulated in proximal tubules of wild-type mice in a model of proteinuria, were assessed în the kidney tissue sections obtained in this study.
FIGURE 34 graphically illustrâtes the results of computer-based image analysis of stained tissue sections with anti-TGFp antibody (measured as % TGFp antibody-staîned area) in wild-type mice treated with BSA and saline (n=8), wild-type mice treated with BSA and isotype control antibody (n=7) and wild-type mice treated with BSA and MASP-2 inhibitory antibody (n=8). As shown in FIGURE 34, quantification of the TGFp stained areas showed a significant réduction in the levels of TGFP in the MASP-2 inhibitory antibody-treated mice as compared to the saline and isotype control antibodytreated control groups (p values= 0.0324 and 0.0349, respectively).
170
FIGURE 35 graphically illustrâtes the results of computer-based image analysis of stained tissue sections with anti-TNFa antibody (measured as % TNFa antibody-stained area) in wild-type mice treated with BSA and saline (n=8), BSA and isotype control antibody (n=7) and wild-type mice treated with BSA and MASP-2 inhibitory antibody (n=8). As shown in FIGURE 35, analysis of stained sections showed a significant réduction in the level of TNFa in the MASP-2 inhibitory antibody-treated group as compared to the saline control group (p=0.0l l) as well as the isotype control group (p=0.0285).
FIGURE 36 graphically illustrâtes the results of computer-based image analysis of stained tissue sections with anti-IL-6 antibody (measured as % IL-6 antibody-stained area) in in wild-type mice treated with BSA and saline (n—8), BSA and isotype control antibody (n=7) and wild-type mice treated with BSA and MASP-2 inhibitory antibody (n=8). As shown in FIGURE 36, analysis of stained sections showed a significant réduction in the level of IL-6 in the MASP-2 inhibitory antibody-treated group as compared to the saline control group (p=0.0269) as well as to the isotype control group (p=0.0445).
Overall Summary of Results and Conclusions:
The results in this Example demonstrate that the use of a MASP-2 inhibitory antibody provides protection against rénal injury in a protein overload model, which is consistent with the results described in Example 16 demonstratîng that MASP-2-/- mice hâve reduced rénal injury in the proteinuria model.
EXAMPLE 18
This Example provides results generated using an Adriamycin-induced nephrology model of rénal fibrosis, inflammation and tubulointerstitial injury in MASP2-/- and wild-type mice to evaluate the rôle of the lectin pathway in Adriamycin-induced nephropathy.
Background/Rationale:
Adriamycin is an anthracycline antitumor antibiotic used in the treatment of a wide range of cancers, including hematologîcal malignancies, soft tissue sarcomas and many types of carcinomas. Adriamycin-induced nephropathy is well established rodent
I7l model of chronic kidney disease that has enabled a better understanding of the progression of chronic proteinuria (Lee and Harris, Nephrology, 16:30-38, 2011). The type of structural and functional injury in Adriamycin-induced nephropathy is very similar to that of chronic protéinurie renal disease in humans (Pippin et ai., American Journal of Renal Physiology 296:F213-29, 2009).
Adriamycin-induced nephropathy is characterized by an injury to the podocytes followed by glomerulosclerosis, tubulointerstitial inflammation and fïbrosis. It has been shown in many studies that Adriamycin-induced nephropathy is modulated by both immune and non-immune derived mechanisms (Lee and Harris, Nephrology, 16:30-38, 2011). Adriamycin-induced nephropathy has several strengths as a model of kidney disease. First, ît is a highly reproducible and predicable model of renal injury. This is because it is characterized by the induction of renal injury within a few days of dnjg administration, which allows for ease of experimental design as the tîming of injury is consistent. It is also a model in which the degree of tissue injury is severe while associated with acceptable mortality (<5%) and morbidity (weight loss). Therefore, due to the severîty and timing of renal injury in Adriamycin-induced nephropathy, it îs a model suitable for testing interventions that protect against renal injury.
As described in Examples 16 and 17, in a protein overload model of proteinuria it was determined that MASP-2-/- mice and mice treated with a MASP-2 inhibitory antibody exhibïted significantly better outcomes (e.g., less tubulointerstitial injury, and less renal inflammation) than wild-type mice, implicating a pathogenic rôle for the lectin pathway in protéinurie kidney disease.
In this example, MASP-2-/- mice were analyzed in comparison with wild-type mice in the Adriamycin-induced nephrology model (AN) to détermine if MASP-2 deficiency reduces and/or prevents renal inflammation and tubulointerstitial injury induced by Adriamycin.
Methods:
1. Dosage and Time point optimization
An initial experiment was carried out to détermine the dose of Adriamycin and time point at which BALB/c mice develop a level of renal inflammation suitable for testing therapeutic intervention.
172
Three groups of wild-type BALB/c mice (n=8) were injected with a single dose of Adriamycin (l 0.5 mg/kg) administered IV. Mice were culled at three time points: one week, two weeks and four weeks after Adriamycin administration. Control mice were injected with saline only.
Results: Ail mice in the three groups showed signs of glomerulosclerosis and proteinuria, as determined by H&E staining, with incrementally increasing degree of tissue inflammation as measured by macrophage infiltration in the kidney (data not shown). The degree of tissue injury was mild in the one week group, moderate in the two week group and severe in the four week group (data not shown). The two week time 10 point was selected for the rest of the study.
2. Analysis of Adrïamycin-induced nephrology in wild-type and MASP-2-/mice
In order to elucidate the rôle of the lectin pathway of complément in the Adrîamycininduced nephrology, a group of MASP-2-/- mice (BALB/c) were compared to wild-type 15 mice (BALB/c) at the same dose of Adriamycin. The MASP-2-/- mice were backcrossed with BALB/c mice for 10 générations.
Wild-type (n=8) and MASP-2-/- (n=8) were injected IV with Adriamycin (10.5 mg/kg) and three mice of each strain were give saline only as a control. Ail mice were culled two weeks after the treatment and tissues were collected. The degree of 20 histopatholigical injury was assessed by H&E staining.
Results:
FIGURE 37 shows représentative H&E staîned tissue sections from the following groups of mice at day 14 after treatment with Adriamycin or saline only (control): (panels A-l, A-2, A-3) wild-type control mice treated with only saline; (panels B-l, B-2, B-3) 25 wild-type mice treated with Adriamycin; and (panels C-l, C-2, C-3) MASP-2-/- mice treated with Adriamycin. Each photo (e.g., panel A-l, A-2, A-3) represents a different mouse.
As shown in FIGURE 37, there is a much higher degree of tissue préservation in the MASP-2-/- group treated with Adriamycin as compared to the wild-type group treated 30 with the same dose of Adriamycin.
I73
FIGURE 38 graphically illustrâtes the results of computer-based image analysis of kidney tissue sections stained with macrophage-specific antibody F4/80 showing the macrophage mean stained area (%) from the following groups of mice at day 14 after treatment with Adriamycin or saline only (wild-type control): wild-type control mice treated with only saline; wild-type mice treated with Adriamycin; MASP-2-/- mice treated with saline only, and MASP-2 -/- mice treated with Adriamycin. As shown in FIGURE 38, MASP-2-/- mice treated with Adriamycin hâve reduced macrophage infiltration (**/>=0.007) compared to wild-type mice treated with Adriamycin.
FIGURE 39 graphically illustrâtes the results of computer-based image analysis of kidney tissue sections stained with Sirius Red, showing the collagen déposition stained area (%) from the following groups of mice at day 14 after treatment with Adriamycin or saline only (wild-type control): wild-type control mice treated with only saline; wild-type mice treated with Adriamycin; MASP-2-/- mice treated with saline only, and MASP-2 -/mice treated with Adriamycin. As shown in FIGURE 39, MASP-2-/- mice treated with Adriamycin hâve reduced collagen déposition (**/>=0.005) compared to wild-type mice treated with Adriamycin.
Overall Summary and Conclusions:
The amelioration of rénal tubulointerstitial inflammation is a key target for the treatment of kidney disease. The results presented herein indicate that the lectin pathway of complément activation contributes significantly to the development of rénal tubulointerstitial inflammation. As further demonstrated herein, a MASP-2 inhibitory agent, such as a MASP-2 inhibitory antibody, may be used as a novel therapeutic approach in the treatment of protéinurie nephropathy, Adriamycin nephropathy and amelioration of rénal tubulointerstitial inflammation.
EXAMPLE 19
This Example describes the initial results of an ongoing Phase 2 clinïcal trial to evaluate the safety and clinical effîcacy of a fully human monoclonal MASP-2 inhibitory antibody in adults with steroid-dependent îmmunoglobulin A nephropathy (IgAN) and in adults with steroid-dependent membranous nephropathy (MN).
Backeround:
174
Chronic kidney diseases affect more than 20 million people in the United States (Drawz P. et al., Ann Intern Med I62(l l); ITCl-16, 2015). Glomerulonephropathies (GNs), including IgAN and MN are kidney diseases in which the glomeruli are damaged and frequently lead to end-stage rénal disease and dialysis. Several types of primary GNs exist, the most common being IgAN. Many of these patients hâve persistent rénal inflammation and progressive détérioration. Often these patients are treated with corticosteroids or immunosuppressive agents, which hâve many serious long-term adverse conséquences. Many patients continue to deteriorate even on these treatments. No treatments are approved for the treatment of IgAN or MN.
IgA Nephropathy
Immunoglobulin A nephropathy (IgAN) is an autoimmune kidney disease resulting in intrarenal inflammation and kidney injury. IgAN is the most common primary glomerular disease globally. With an annual incidence of approximately 2.5 per 100,000, ît is estimated that 1 in 1400 persons in the U.S. will develop IgAN. As many as 40% of patients with IgAN will develop end-stage rénal disease (ESRD). Patients typically present with microscopie hematuria with mîld to moderate proteinuria and variable levels of rénal însufficiency (Wyatt R.J., et al., N Engl J Med 368(25):2402-14, 2013). Ciinical markers such as impaired kidney function, sustained hypertension, and heavy proteinuria (over 1 g per day) are associated with poor prognosis (Goto M et al., Nephrol Dial Transplant 24(10):3068-74, 2009; Berthoux F. et al., J Am Soc Nephrol 22(4):752-61, 2011). Proteinuria is the strongest prognostic factor independent of other risk factors in multiple large observational studies and prospective trials (Coppo R. et aL, J Nephrol 18(5):503-12, 2005; Reich H. N., et al., J Am Soc Nephrol 18(12):3177-83, 2007). It is estimated that 15-20% of patients reach ESRD within 10 years of disease onset if left untreated (D’Amico G., Am J Kidney Dis 36(2):227-37, 2000).
The diagnostic hallmark of IgAN is the prédominance of IgA deposits, alone or with IgG, IgM, or both, in the glomerular mesangium. In IgAN, rénal biopsies reveal glomerular déposition of mannan-bindtng lectin (MBL), a key récognition molécule for activation of MASP-2, the effector enzyme of the complément system’s lectin pathway. Glomerular MBL deposits, usually co-localized with IgA and indicating complément activation, and high levels of urinary MBL are associated with an unfavorable prognosis
175 in IgAN, with these patients demonstrating more severe histologîcal changes and mesangial prolifération than patients without MBL déposition or high levels ofurînary MBL (Matsuda M. et al., Nephron 80(4):408-13, 1998; Liu LL et al., Clin Exp Immunol 169(2):148-155, 2012; Roos A. et al., J Am Soc Nephrol 17(6):1724-34, 2006; Liu LL et al., Clin Exp Immunol 174(1):152-60, 2013). Remission rates also are substantially lower for patients with MBL déposition (Liu LL et al., Clin Exp Immunol 174(1):152-60, 2013).
Current therapy for IgAN includes blood pressure control and, frequently, corticosteroids and /or other immunosuppressive agents, such as cyclophosphamide, azathioprine, or mycofenolate mofetil, for severe disease (e.g., crescentic IgAN). The Kidney Disease Improving Global Outcomes (KDIGO) Guîdelines for Glomerulonephritis (Inl. Soc ofNephrol 2(2):139-274, 2012) recommend that corticosteroids should be administered to patients with proteinuria of greater than or equal to 1 g/day, with a usual treatment duration of 6 months. However, even with aggressive immunosuppressive treatment, which is associated with serious long-term sequelae, some patients hâve progressive détérioration of rénal function. There is no approved treatment for IgAN, and even with the use of angiotensin-converting enzyme (ACE) inhibitors or angiotensin receptor blockers (ARBs) to control blood pressure, increased proteinuria persists in some patients. None of these treatments hâve been shown to stop or even slow the progression of IgAN in patients who are at risk for rapid progression of the disease.
Membranous Nephropathy
The annual incidence of membranous nephropathy (MN) is approximately 10-12 per 1,000,000. Patients with MN can hâve a variable clinical course, but approximately 25% will develop end-stage rénal disease.
Membranous nephropathy is an immune-mediated glomerular disease and one of the most common causes of the nephrotic syndrome in adults. The disease is characterized by the formation of immune deposits, primarily IgG4, on the outer aspect of the glomerular basement membrane, which contain podocyte antigens and antibodies spécifie to those antigens, resulting în complément activation. Initial manifestations of MN are related to the nephrotic syndrome: proteinuria, hypoalbuminemia, hyperlipîdemia, and edema.
176
Although MN may spontaneously remit without treatment, as many as one third of patients demonstrate progressive loss of kidney function and progress to ESRD at a médian of 5 years after diagnosis. Often, corticosteroids are used to treat MN and there is a need to develop alternative thérapies. Additionally, patients determined to be at moderate risk for progression, based on severity of proteinuria, are treated with prednisone in conjunction with cyclophosphamide or a calcinuerin inhibitor, and these two treatments together are often associated with severe systemic adverse effects.
Methods:
Two Phase l clinicial trials carried out in healthy volunteers hâve demonstrated that both intravenous and subcutaneous dosing of a MASP-2 inhibitory antibody, OMS646, resulted in sustained lectin pathway inhibition.
This Example describes intérim results from an ongoing Phase 2, uncontrolled, multicenter study of a MASP-2 inhibitory antibody, OMS646, in subjects with IgAN and MN. Inclusion criteria requîre that ail patients in this study, regardless of rénal disease subtype, hâve been maintained on a stable dose of corticosteroids for at least 12 weeks prior to study enrollment (i.e., the patients are steroid-dependent). The study is a singlearm pilot study with 12 weeks of treatment and a 6-week follow-up period.
Approximately four subjects are planned to be enrolled per disease. The study is designed to evaluate whether OMS646 may improve rénal function (e.g., improve proteinuria) and decrease corticosteroid needs in subjects with IgAN and MN. To date, 2 patients with IgA nephropathy and 2 patients with membranous nephropathy hâve completed treatment in the study.
At study entry each subject must hâve high levels of protein in the urine despite ongoing treatment with a stable corticosteroid dose. These criteria select for patients who are unlikely to spontaneously improve during the study period.
The subjects were âge > I8 at screening and were only included in the study if they had a diagnosis of one of the following: IgAN diagnosed on kidney biopsy or primary MN diagnosed on kidney biopsy. The enrolled patients also had to meet ail of the following inclusion criteria:
(l) hâve average urine albumin/creatinine ratio > 0.6 from three samples collected consecutively and daîly prior to each of 2 visits during the screening period;
177 (2) hâve been on > 10 mg of prednisone or équivalent dose for at least 12 weeks prior to screening visit l ;
(3) îf on immunosuppressive treatment (e.g., cyclophosphamide, mycophenolate mofetil), hâve been on a stable dose for at least 2 months prior to Screening Visit l with no expected change in the dose for the study duration;
(4) hâve an estimated glotnerular filtration rate (eGFR) > 30 mL/min/1.73m2 calculated by the MDRD équation1;
(5) are on a physician-directed, stable, optimized treatment with angiotensin converting enzyme inhibitors (ACEI) and/or angiotensin receptor blockers (ARB) and hâve a systolic blood pressure of <150 mmHg and a diastolic blood pressure of <90mmHg at rest;
(6) hâve not used belimumab, eculizumab or rituzimab within 6 months of screening visit 1; and (7) do not hâve a history of rénal transplant.
'MDRD Equation: eGFR (mL/min/1.73m2) = 175 x (SCr)’1154 x (Age)’0203 x (0.742 if female) x (1.212 if African American). Note: SCr=Serum Créatinine measurement should be mg/dL.
The monoclonal antibody used in this study, OMS646, is a fully human IgG4 monoclonal antibody that bînds to and inhibits human MASP-2. MASP-2 is the effector enzyme of the lectîn pathway. As demonstrated in Example 12, OMS646 avîdly bînds to recombinant MASP-2 (apparent equilibrium dissociation constant in the range of 100 pM) and exhibits greater than 5,000-fold selectivity over the homologous proteins Cl s, C Ir, and MASP-L In functional assays, OMS646 inhibits the human lectin pathway with nanomolar potency (concentration leading to 50% inhibition [IC50] of approximately 3 nM) but has no significant effect on the classical pathway. OMS646 administered either by intravenous (IV) or subcutaneous (SC) injection to mice, non-human primates, and
178 humans resulted in high plasma concentrations that were associated with suppression of lectin pathway activation in an ex vivo assay.
In this study, the OMS646 drug substance was provided at a concentration of 100 mg/mL, which was further diluted for IV administration. The appropriate calculated volume of OMS646 100 mg/mL injection solution was withdrawn from the vial using a syringe for dose préparation. The infusion bag was administered within four hours of préparation.
The study consists of screening (28 days), treatment (12 weeks) and follow-up (6 weeks) periods, as shown in the Study Design Schematic below.
Study Design Schematic
Within the screening period and before the first OMS646 dose, consented subjects provided three urine samples (collected once daîly) on each of two three-consecutive-day periods to establish baseline values of the urine albumin-to-creatinlne ratio. Following the screening period, eligible subjects received OMS646 4 mg/kg IV once weekly for 12 weeks (treatment period). There was a 6-week follow-up period after the last dose of OMS646.
During the initial 4 weeks of treatment with OMS646, subjects were maintained on their stable pre-study dose of corticosteroids. At the end of the initial 4-weeks of the 12-week treatment period, subjects underwent corticosteroid taper (i.e., the corticosteroid dose was reduced), if tolerated, over 4 weeks, followed by 4 weeks during which the résultant corticosteroid dose was maintained. The target was a taper to < 6 mg prednisone (or équivalent dose) daily. Over this period, the taper was discontinued in subjects who had détérioration of rénal function, as determined by the investigator.
179
Subjects were treated with OMS646 through the corticosteroid taper and through the futl 12 weeks of treatment. The patients were then followed for an additional 6 weeks after their last treatment. The taper of corticosteroids and OMS646 treatment permitted assessment of whether OMS646 allowed for a decrease in the dose of corticosteroid required to maintain stable rénal function.
The key efficacy measures in this study are the change in urine albumin-tocreatinine ratio (uACR) and 24-hour protein levels from baseline to 12 weeks. Measurement of urinary protein or albumin is routinely used to assess kidney involvement and persistent high levels of urinary protein correlates with rénal disease progression. The uACR is used clinically to assess proteinuria.
Efficacy Analyses
The analysis value for uACR is defined as the average of ail the values obtained for a time point. The planned number of uACRs is three at each scheduled time point. The baseline value of the uACR is defined as the average of the analysis values at the two screening vîsîts.
Results:
FIGURE 40 graphically illustrâtes the uACRin two IgAN patients during the course of a twelve week study with weekly treatment with 4 mg/kg MASP-2 inhibitory antibody (OMS646). As shown in FIGURE 40, the change from baseline is statisticaliy significant at time point “a” (p=0.003); time point “b” (p=0.007) and a time point “c” (/2=0.033) by the untransformed analysis. TABLE 12 provides the 24-hour urine-protein data for the two IgAN patients treated with OMS646.
TABLE 12: 24-hour Urine Protein (mg/day) in QMS646-treated IgAN Patients
Time of Sample Patient # 1 (mg/24 hours) Patient #2 (mg/24 hours) Mean
Baseline 3876 2437 3156
Day 85 1783 455 1119
p= 0.017
ISO
As shown in FIGURE 40 and TABLE 12, the patients with IgAN demonstrated a clinically and statistically significant improvement in kidney function over the course of the study. There were statistically significant decreases in both uACR (see FIGURE 40) and 24-hour urine protein concentration (see TABLE I2). As shown in the uACR data in FIGURE 40, the mean baseline uACR was 1264 mg/g and reached 525 mg/g at the end of treatment (p=0.0ll) decreasing to 128 mg/g at the end of the follow-up period. As further shown in FIGURE 40, the treatment effect was maintained throughout the followup period. Measures of 24-hour urine protein excrétion tracked uACRs, with a mean réduction from 3156 mg/24 hours to 1H9 mg/24 hours (p=0.0l7). Treatment effects across the two patients were highly consistent. Both patients experienced réductions of approximately 2000 mg/day and both achieved a partial remission (defined as greater than 50 percent réduction in 24-hour urine protein excrétion and/or résultant protein exretîon less than 1000 mg/day; complété rémission defined as protein excrétion less than 300 mg/day). The magnitude of the 24-hour proteinuria réductions in both IgA nephropathy patients is associated with a significant improvement in rénal survival. Both IgA nephropathy patients were also able to taper their steroids substantially, each reducing the daily dose to < 5 mg (60 mg to 0 mg; 30 mg to 5 mg).
The two MN patients also demonstrated réductions in uACR during treatment with OMS646. One MN patient had a decrease în uACR from 1003 mg/g to 69 mg/g and maintained this low level throughout the follow-up period. The other MN patient had a decrease in uACR from 1323 mg/g to 673 mg/g, with a variable course after treatment. The first MN patient showed a marked réduction in 24-hour urine protein level (10,771 mg/24 hours at baseline to 325 mg/24 hours on Day 85), achieving partial and nearly complété remission, while the other remained essentially unchanged (4272 mg/24 hours at baseline to 4502 mg/24 on Day 85). Steroids were tapered in the two MN patients from 30 mg to 15 mg and from 10 mg to 5 mg.
In summary, consistent improvements in rénal function were observed in IgAN and MN subjects treated with the MASP-2 inhibitory antibody OMS646. The effects of OMS646 treatment in the patients with IgAN are robust and consistent, suggesting a strong efficacy signal. These effects are supported by the results in MN patients. The time course and magnitude of the uACR changes during treatment were consistent
I8l between ail four patients with IgAN and MN. No significant safety concems hâve been observed. Patients in this study represent a difficult-to-treat group and a therapeutic effect in these patients is believed to be prédictive of efficacy with a MASP-2 inhibitory antibody, such as OMS646, in IgAN and MN patients, such as patients suffering from steroid-dependent IgAN and MN (i.e., patients undergoing treatment with a stable corticosteroid dose prior to treatment with a MASP-2 inhibitory antibody), including those at risk for rapid progression to end-stage rénal disease.
In accordance with the foregoing, in one embodiment, the invention provides a method of treating a human subject suffering from IgAN or MN comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody effective to inhibit MASP-2-dependent complément activation. In one embodiment, the method comprises administering to the human subject suffering from IgAN or MN an amount of a MASP-2 inhibitory antibody sufficient to improve rénal function (e.g., improve proteinuria). In one embodiment, the subject is suffering from steroid-dependent IgAN. In one embodiment, the subject is suffering from steroiddependent MN. In one embodiment, the MASP-2 inhibitory antibody is administered to the subject suffering from steroid-dependent IgAN or steroid-dependent MN in an amount sufficient to improve rénal function and/or decrease corticosteroid dosage in said subject.
In one embodiment. the method further comprises identifying a human subject suffering from steroid-dependent IgAN 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 complément activation.
In one embodiment, the method further comprises identifying a human subject suffering from steroid-dependent MN 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-dependenî complément activation.
In accordance with any of the disclosed embodiments 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 CCPl domain of MASP-2, said antibody inhibits C3b déposition in an in
182 vitro assay in l% human sérum at an IC5Q of 10 nM or less, said antibody inhibits C3b déposition in 90% human sérum with an 1C5O of 30 nM or less, wherein the antibody is an antibody fragment selected from the group consisting of Fv, Fab, Fab', F(ab)? and F(ab')-, wherein the antibody is a single-chain molécule, wherein said antibody is an IgG2 molécule, wherein said antibody is an IgGl molécule, wherein said antibody is an IgG4 molécule, wherein the IgG4 molécule comprises a S228P mutation. In one embodiment, the antibody binds to MASP-2 and selectively inhibits the lectin pathway and does not substantially inhibit the classical pathway (i.e., inhibits the lectin pathway while leaving the classical complément pathway intact).
In one embodiment, the MASP-2 inhibitory antibody is administered in an amount effective to improve at least one or more clinical parameters associated rénal function, such as an improvement in proteinuria (e.g., a decrease in uACR and/or a decrease in 24hour urine protein concentration, such as greater than 20 percent réduction in 24-hour urine protein excrétion, or such as greater than 30 percent réduction in 24-hour urine protein excretion, or such as greater than 40 percent réduction in 24-hour urine protein excrétion, or such as greater than 50 percent réduction in 24-hour urine protein excretion).
In some embodiments, the method comprises adminîstering a MASP-2 inhibitory antibody to a subject suffering from IgAN (such as steroid-dependent IgAN), via a cathéter (e.g., intravenously) for a first time period (e.g., at least one day to a week or two weeks or three weeks or four weeks or longer) followed by adminîstering a MASP-2 inhibitory antibody to the subject subcutaneously for a second time period (e.g., a chronic phase of at least two weeks or longer).
In some embodiments, the method comprises adminîstering a MASP-2 inhibitory agent to a subject suffering from MN (such as steroid-dependent MN), via a cathéter (e.g., intravenously) for a first time period (e.g., at least one day to a week or two weeks or three weeks or four weeks or longer) followed by adminîstering a MASP-2 inhibitory antibody to the subject subcutaneously for a second time period (e.g., a chronic phase of at least two weeks or longer).
In some embodiments, the method comprises adminîstering a MASP-2 inhibitory antibody to a subject suffering from IgAN (such as steroid-dependent IgAN) or MN (such
I83 as steroid-dependent MN) either intravenously, intramuscularly, or subcutaneousiy. Treatment may be chronic and administered daily to monthly, but preferably at least every two weeks, or at least once a week, such as twice a week or three times a week.
In one embodiment, the method comprises treating a subject suffering from IgAN (such as steroid-dependent IgAN) or MN (such as steroid-dependent MN) comprising administering to the subject a composition comprising an amount of a MASP-2 inhibitory antibody, or antigen binding fragment thereof, comprising a heavy chain variable région comprising CDR-Hl, CDR-H2 and CDR-H3 of the amino acid sequence set forth as SEQ ID NO:67 and a light-chain variable région comprising CDR-Ll, CDR-L2 and CDR-L3 of the amino acid sequence set forth as SEQ ID NO:70. In some embodiments, the composition comprises a MASP-2 inhibitory antibody comprising (a) a heavy-chain variable région comprising: i) a heavy-chain CDR-Hl comprising the amino acid sequence from 31-35 of SEQ ID NO:67; and ii) a heavy-chain CDR-H2 comprising the amino acid sequence from 50-65 of SEQ ID NO:67; and iii) a heavy-chain CDR-H3 comprising the amino acid sequence from 95-107 of SEQ ID NO:67 and b) a light-chain variable région comprising: i) a light-chain CDR-Ll comprising the amino acid sequence from 24-34 of SEQ ID NO:70; and ii) a light-chain CDR-L2 comprising the amino acid sequence from 50-56 of SEQ ID NO:70; and iii) a light-chain CDR-L3 comprising the amino acid sequence from 89-97 of SEQ ID NO:70, or (II) a variant thereof comprising a heavy-chain variable région 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 to SEQ ID NO:67) and a light-chain variable région with at least 90% identity (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 to 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 antigen binding fragment thereof, comprising a heavy-chain variable région comprising the amino acid sequence set forth as SEQ ID NO:67 and a light-chain variable région comprising the amino acid sequence set forth as SEQ ID NO:70.
In some embodiments, the method comprises administering to the subject a composition comprising a MASP-2 inhibitory antibody, or antigen binding fragment
184 thereof, that specifically recognizes at least part of an epitope on human MASP-2 recognized by reference antibody OMS646 comprising a heavy-chain variable région as set forth in SEQ ID NO:67 and a light-chain variable région as set forth in SEQ ID NO:70.
In some embodiments, the method comprises administering to a subject suffering from, or at risk for developing IgAN (such as steroid-dependent IgAN) or MN (such as steroid-dependent MN), a composition comprising a MASP-2 inhibitory antibody, or antigen binding fragment thereof comprising a heavy-chain variable région comprising the amino acid sequence set forth as SEQ ID NO:67 and a light-chain variable région comprising the amino acid sequence set forth as SEQ ID NO:70 in a dosage from l mg/kg·to 10 mg/kg (i.e., I mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg. 7 mg/kg, 8 mg/kg, 9 mg/kg or 10 mg/kg) at least once weekly (such as at least twice weekly or at least three times weekly) for a period of at least 3 weeks, or for at least 4 weeks, or for at least 5 weeks, or for at least 6 weeks, or for at least 7 weeks, or for at least 8 weeks, or for at least 9 weeks, or for at least 10 weeks, or for at least 11 weeks, or for at least 12 weeks.
Other embodiments
Ail publications, patent applications, and patents mentioned in this spécification are herein incorporated by reference.
Various modifications and variations of the described methods and compositions of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with spécifie desired embodiments, it should be understood that the invention as claimed should not be unduly limited to such spécifie embodiments.
While illustrative embodiments hâve been îllustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

Claims (36)

  1. Claims
    1. A MASP-2 inhibitory monoclonal antibody or antigen-binding fragment thereof for use in preventing or reducing rénal damage in a subject suffering from a disease or condition associated with proteinuria.
  2. 2. The MASP-2 inhibitory monoclonal antibody or antigen-binding fragment thereof for use of claim l, which is for administration in an amount and for a time effective to achieve at least a 20 percent réduction in 24-hour urine protein excrétion as compared to baseline 24-hour urine protein excrétion prior to treatment.
  3. 3. The MASP-2 inhibitory monoclonal antibody or antigen-binding fragment thereof for use of claim l or 2, wherein the disease or condition associated with proteinuria is selected from the group consisting of nephrotic syndrome, pre-eclampsia, eclampsia, toxic lésions of kidneys, amytoidosis, collagen vascular diseases (e.g., systemic lupus erythematosus), déhydration, glomerular diseases (e.g. membranous glomerulonephritis, focal segmentai glomerulonephritis, C3 glomerulopathy, minimal change disease, lipoid nephrosis), strenuous exercise, stress, benign orthostatis (postural) proteinuria, focal segmentai glomerulosclerosis, IgA nephropathy (i.e., Berger’s disease), IgM nephropathy, membranoproliferative glomerulonephritis, membranous nephropathy, minimal change disease, sarcoidosis, Alport’s syndrome, diabètes mellitus (diabetic nephropathy), drug-induced toxÎcity (e.g., NSAIDS, nicotine, peniciIlamine, lithium carbonate, gold and other heavy metals, ACE inhibitors. antibiotics (e.g., adriamycin) or opiates (e.g. heroin) or other nephrotoxins); Fabry’s disease, infections (e.g., HIV, syphilis, hepatitis A, B or C, poststreptococcal infection, urinary schistosomiasis); aminoaciduria, Fanconi syndrome, hypertensive nephrosclerosis, interstitial nephritis, sickle cell disease, hemoglobinuria, multiple myeloma, myoglobinuria, organ rejection (e.g., kidney transplant rejection), ebola hémorrhagie fever, Nail patella syndrome, familial mediterranean fever, HELLP syndrome, systemic lupus erythematosus, Wegener’s granulomatosis, Rheumatoid arthritis, Glycogen storage disease type l, Goodpasture’s syndrome, Henoch-Schonleîn purpura, urinary tract infection which has spread to the kidneys, Sjôgren’s syndrome and post-infections glomerulonepthritis.
    186
  4. 4. The MASP-2 inhibitory monoclonai antibody or antigen-binding fragment thereof for use of claim l or 2, wherein the disease or condition associated with proteinuria is IgA nepropathy (i.e., Berger’s disease).
  5. 5. The MASP-2 inhibitory monoclonal antibody or antigen-binding fragment thereof for use of claim l or 2, wherein the disease or condition associated with proteinuria is membranous nephropathy.
  6. 6. The MASP-2 inhibitory monoclonal antibody or antigen-binding fragment thereof for use of claim 4, which is for administration to a subject suffering from steroiddependent IgAN.
  7. 7. The MASP-2 inhibitory monoclonal antibody or antigen-binding fragment thereof for use of claim l, 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.
  8. 8. The MASP-2 inhibitory monoclonal antibody or antigen-binding fragment thereof for use of any of daims l to 7, which does not substantially inhibit the classical pathway.
  9. 9. The MASP-2 inhibitory monoclonal antibody or antigen-binding fragment thereof for use of any of daims l to 7, which inhibits C3b déposition in 90% human sérum with an IC50 of 30 nM or less.
  10. 10. The MASP-2 inhibitory monoclonai antibody or antigen-binding fragment thereof for use of daim 4, which is for administration in an amount effective to improve rénal function.
  11. 11. The MASP-2 inhibitory monoclonai antibody or antigen-binding fragment thereof for use daim 4, which is for administration in an amount effective and for a time sufficient to achieve at least a 20 percent réduction in 24-hour urine protein excrétion as compared to baseline 24-hour urine protein excrétion in the subject prior to treatment.
    187
  12. 12. The MASP-2 inhibitory monoclonal antibody or antigen-binding fragment thereof for use of claim 6, which is for administration în an amount sufficient to improve rénal function and decrease the corticosteroid dosage in said subject.
  13. 13. The MASP-2 inhibitory monoclonal antibody or antigen-binding fragment thereof for use of claim 5, which is for administration to a subject suffertng from steroid-dependent MN.
  14. 14. The MASP-2 inhibitory monoclonal antibody or antigen-binding fragment thereof for use of claim 5 which is for administration in an amount effective to improve rénal function.
  15. 15. The MASP-2 inhibitory monoclonal antibody or antigen-binding fragment thereof for use of claim 5, which is for administration in an amount effective and for a time sufficient to achieve at least a 20 percent réduction in 24-hour urine protein excrétion as compared to baseline 24-hour urine protein excrétion in the subject prior to treatment.
  16. 16. The MASP-2 inhibitory monoclonal antibody or antigen-binding fragment thereof for use of claim 13, which is for administration in an amount sufficient to improve rénal function and decrease the corticosteroid dosage in said subject.
  17. 17. The MASP-2 inhibitory monoclonal antibody or antigen-binding fragment thereof for use of any of daims l to 16, which comprises a heavy chain variable région comprising CDR-H1, CDR-H2 and CDR-H3 of the amino acid sequence set forth as SEQ ID NO:67 and a light chain variable région comprising CDR-Ll, CDR-L2 and CDR-L3 of the amino acid sequence set forth as SEQ ID NO:70.
  18. 18. The MASP-2 inhibitory monoclonal antibody or antigen-binding fragment thereof for use of any of daims l to 17, which is for administration subcutaneously, intraperitonealiy, intra-muscularly, intra-arterially, intravenously, or as an inhalant.
  19. 19. Use of a MASP-2 inhibitory monoclonal antibody or antigen-binding fragment thereof in the manufacture of a médicament for preventing or reducing rénal damage in a subject suffering from a disease or condition associated with proteinuria.
    188
  20. 20. The use of claim 19, in which the médicament is for administration in an amount and for a time effective to achieve at least a 20 percent réduction in 24-hour urine protein excrétion as compared to baseline 24-hour urine protein excrétion prior to treatment.
  21. 21. The use of claim 19 or 20, wherein the disease or condition associated with proteinuria is selected from the group consisting of nephrotic syndrome, pre-eclampsia, eclampsia, toxic lésions of kidneys, amyloidosis, collagen vascular diseases (e.g., systemic lupus erythematosus), déhydration, glomerular diseases (e.g. membranous glomerulonephritis, focal segmentai glomerulonephritis, C3 glomerulopathy, minimal change disease, lipoid nephrosis), strenuous exercise, stress, benign orthostatis (postural) proteinuria, focal segmentai glomerulosclerosis, IgA nephropathy (i.e., Berger’s disease), IgM nephropathy, membranoproliferative glomerulonephritis, membranous nephropathy, minimal change disease, sarcoidosis, Alport’s syndrome, diabètes mellitus (diabetic nephropathy), drug-induced toxicity (e.g., NSAIDS, nicotine, penicillamine, lithium carbonate, gold and other heavy metals, ACE inhibitors, antibiotics (e.g., adriamycin) or opiates (e.g. heroîn) or other nephrotoxins); Fabry’s disease, infections (e.g., HIV, syphilis, hepatitis A, B or C, poststreptococcal infection, urinary schistosomiasis); aminoaciduria, Fanconi syndrome, hypertensive nephrosclerosis, interstitial nephritis, sickle cell disease, hemoglobînuria, multiple myeloma, myoglobinuria, organ rejection (e.g., kidney transplant rejection), ebola hémorrhagie fever, Nail patella syndrome, familial mediterranean fever, HELLP syndrome, systemic lupus erythematosus, Wegener s granulomatosis, Rheumatoid arthritis, Glycogen storage disease type 1, Goodpasture’s syndrome, Henoch-Schônlein purpura, urinary tract infection which has spread to the kidneys, Sjôgren’s syndrome and post-infections glomerulonepthritis.
  22. 22. The use of claim 19 or 20, wherein the disease or condition associated with proteinuria is IgA nepropathy (i.e., Berger’s disease).
  23. 23. The use of claim 19 or 20, wherein the disease or condition associated with proteinuria is membranous nephropathy.
  24. 24. The use of claim 22, in which the médicament is for administration to a subject suffering from steroid-dependent IgAN.
    189
  25. 25. The use of claim 19, 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.
  26. 26. The use of any of claims 19 to 25, in which the MASP-2 inhibitory monoclonal antibody or antigen-binding fragment thereof does not substantially inhibit the classical pathway.
  27. 27. The use of any of claims 19 to 25, in which the MASP-2 inhibitory monoclonal antibody or antigen-binding fragment thereof inhibits C3b déposition în 90% human serum with an IC50 of 30 nM or less.
  28. 28. The use of claim 22, in which the médicament is for administration in an amount effective to improve renal function.
  29. 29. The use claim 22, in which the médicament is for administration in an amount effective and for a time sufficient to achieve at least a 20 percent réduction in 24-hour urine protein excrétion as compared to baseline 24-hour urine protein excrétion in the subject prior to treatment.
  30. 30. The use of claim 24, in which the médicament is for administration in an amount sufficient to improve renal function and decrease the corticosteroid dosage in said subject.
  31. 31. The use of claim 23, in which the médicament is for administration to a subject suffering from steroid-dependent MN.
  32. 32. The use of claim 23 in which the médicament is for administration in an amount effective to improve renal function.
  33. 33. The use of claim 23, în which the médicament is for administration in an amount effective and for a time sufficient to achieve at least a 20 percent réduction in 24-hour urine protein excrétion as compared to baseline 24-hour urine protein excrétion in the subject prior to treatment.
    190
  34. 34. The use of claim 31, which is for administration in an amount sufficient to improve rénal fonction and decrease the corticosteroid dosage in said subject.
  35. 35. The use of any of daims 19 to 34, ’m which the MASP-2 inhibitory monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable région
    5 comprising CDR-Hl, CDR-H2 and CDR-H3 of the amino acid sequence set forth as SEQ
    ID NO:67 and a light chain variable région comprising CDR-L1, CDR-L2 and CDR-L3 ofthe amino acid sequence set forth as SEQ ID NQ:70.
  36. 36. The use of any of daims 19 to 35, in which the médicament is for administration subcutaneously, intraperitoneally, intra-muscularly, intra-arterially, intravenously, or as 10 an inhalant.
OA1201800254 2016-01-05 2017-01-05 Methods for inhibiting fibrosis in a subject in need thereof. OA18820A (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US62/275,025 2016-01-05
US62/407,979 2016-10-13

Publications (1)

Publication Number Publication Date
OA18820A true OA18820A (en) 2019-07-19

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