CN110177557A - Method for reducing the albuminuria in the people experimenter for suffering from IgANP - Google Patents

Method for reducing the albuminuria in the people experimenter for suffering from IgANP Download PDF

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CN110177557A
CN110177557A CN201780060710.5A CN201780060710A CN110177557A CN 110177557 A CN110177557 A CN 110177557A CN 201780060710 A CN201780060710 A CN 201780060710A CN 110177557 A CN110177557 A CN 110177557A
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masp
antibody
fibrosis
inhibitor
seq
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N.J.布伦斯基尔
G.A.德莫普洛斯
T.杜德勒
H-W.施维布尔
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University of Leicester
Omeros Corp
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Omeros Medical Systems Inc
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Priority claimed from US15/399,524 external-priority patent/US10736960B2/en
Priority claimed from US15/470,647 external-priority patent/US20170253667A1/en
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Priority to CN202310450575.2A priority Critical patent/CN116726163A/en
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Abstract

On the one hand, the present invention provides for reduces suffer from or it is risky generation IgANP (IgAN) people experimenter in albuminuria method.This method includes the steps that the MASP-2 inhibiting antibody for giving the amount that subject in need effectively inhibits MASP-2- dependent complement activation.

Description

For reducing the albuminuria in the people experimenter for suffering from IgANP Method
Statement about sequence table
Sequence table relevant to the application is provided with text formatting instead of paper cover, and this explanation is incorporated herein by reference Book.The entitled MP_1_0269_PCT_Sequence_Listing_20171013_ of text file comprising the sequence table ST25.This article this document is 136 KB, is created on October 10th, 2017；And as the submission of this specification is via EFS- Web is submitted.
Background of invention
Complement system is to start in people and other vertebrates, enhance and coordinate to microorganism infection and other acute injuries Immune response provide early stage mechanism of action (M.K. Liszewski and J.P. Atkinson, 1993, inFundamental Immunology, the third edition, W.E. Paul edits, Raven Press, Ltd., New York).To the greatest extent Pipe complement activation provides the important First Line for potential pathogen and defends, but promotes the complement of protective immune response Activity can also show to the potential threat of host (K.R. Kalli, et al.,Springer Semin. Immunopathol. 15:417-431, 1994; B.P. Morgan, Eur. J. Clinical Investig. 24: 219-228, 1994).For example, C3 and C5 protein hydrolysate is raised and activates neutrophil cell.Although not to host defense It can lack, but the neutrophil leucocyte activated discharges destructive enzyme indiscriminately, may cause organ damage.In addition, complement The complement component that activation may cause lysis deposits on nigh host cell and microorganism target, and host cell is caused to split Solution.
Complement system has also come into the picture into the pathogenesis of many acute and chronic disease conditions, the disease condition packet It includes: capillary vessel leak, cardiopulmonary point after myocardial infarction, apoplexy, ARDS, reperfusion injury, septic shock, thermal burn Flow post-operation inflammatory, graft rejection, rheumatoid arthritis, multiple sclerosis, myasthenia gravis and Alzheimer's disease.Several In all these diseases, complement not instead of causes of disease, one of a number of factors involved in pathogenesis.Nevertheless, Complement activation may be main pathomechanism, and represent available point in the clinic control of many this kind of disease conditions.To each The increased understanding of the importance of the tissue damage of complement-mediated is enhanced to effective Complement inhibition drug in kind disease condition Demand.So far, Yi Kuli monoclonal antibody (Solaris) is the unique benefit for being approved for people and having used for the antibody of C5 Body-targeted drug.However, C5 is one of several effector molecules positioned at complement system " downstream ", and the blocking of C5 does not inhibit The activation of complement system.Therefore, the inhibitor of the initial step of complement activation has significant relative to " downstream " complement inhibitor Advantage.
Currently, generally believing that complement system can be activated by three kinds of different approach: classical pathway, lectin pathway and Alternative route.Classical pathway is usually to combine the compound triggering constituted with foreign particle (i.e. antigen) by host antibodies, Therefore it needs to be exposed to antigen in advance to generate specific antibody response.Because the activation of classical pathway depends on the previous of host Adaptive immune response, so classical pathway is a part of acquired immune system.On the contrary, lectin pathway and substitution way Both diameters are a part of innate immune system independent of adaptive immunity (adaptive immunity).
The activation of complement system results in the sequential activation of serine protease zymogen.Classical pathway activation the first step be Specific recognition molecules C1q is in conjunction with IgG the and IgM molecule for combining antigen.C1q and C1r and C1s serine protease zymogen It is combined into the referred to as compound of C1.After C1q is in conjunction with immune complex, the site Arg-Ile of C1r carries out self albumen water Solution cracking, followed by the activation of the C1r cracking and C1s mediated, to obtain c4 cleavage and the ability of C2.C4 is cracked into two pieces Section, referred to as C4a and C4b, and similarly, C2 is cracked into C2a and C2b.C4b segment can be formed altogether with neighbouring hydroxyl or amino Valence link, and C3 convertase (C4b2a) is generated and carrying out noncovalent interaction with the C2a segment of activation C2.C3 convertase (C4b2a) C3 is activated at C3a and C3b subfraction by proteolytic cleavage, causes to generate C5 convertase (C4b2a3b), institute State C5 convertase (C4b2a3b) by cracking c5 result in membrane attack complex (C5b is combined with C6, C7, C8 and C-9, Referred to as " MAC "), the membrane attack complex can destroy cell membrane, lead to cell cracking.C3 and C4 activated form (C3b and C4b it) is covalently deposited on external source target surface, is identified by the complement receptors on a variety of phagocytes.
Independently, complement system is also the combination of specific recognition molecules by the first step that lectin pathway activates, and is connect Be combined serine protease zymogen activation.However, the identification molecule in lectin pathway includes one group of carbon aquation Close object binding protein (mannan-binding lectin (MBL), H- fiber gelatinized protein (H-ficolin), the fiber gelatinized egg of M- White, L- fiber gelatinized protein and c-type agglutinin CL-11) (being referred to as agglutinin), rather than combined by C1q be immunized it is compound Object.Referring to J. Lu et al.,Biochim. Biophys. Acta 1572:387-400, (2002);Holmskov et al.,Annu. Rev. Immunol. 21:547-578 (2003);Teh et al.,Immunology 101:225-232 (2000)).J. Luet et al. is seen also,Biochim Biophys Acta 1572:387-400 (2002); Holmskov Et al.,Annu Rev Immunol21:547-578 (2003);Teh et al.,Immunology 101:225-232 (2000);Hansen et al.,J. Immunol 185(10):6096-6104 (2010)。
Ikeda et al. is proved first, similar with C1q, MBL energy after with the coated erythrocyte binding of yeast mannans It is enough make in such a way that C4 is relied on complement system activation (Ikeda et al.,J. Biol. Chem. 262:7451-7454, (1987)).MBL is the member of collectin protein family, is Ca-dependent agglutinin, and has that be positioned at pyranose ring red The carbohydrate of 3- hydroxyl and 4- hydroxyl on road face combines.Therefore the important ligand of MBL is D-MANNOSE and N- acetyl-D- Aminoglucose, and do not meet affinity that the carbohydrate of this space requirement then can not detect MBL (Weis et al.,Nature360:127-134, (1992)).MBL and unit price sugar between interaction be it is extremely weak, dissociation constant usually exists In the range of units mM (single-digit millimolar).MBL is by affinity (i.e. by simultaneously and each other Multiple monosaccharide residues of closely adjacent positioning interact) realize with glycan ligand it is close specifically bind (Lee et al.,Archiv. Biochem. Biophys.299:129-136, (1992)).The usual modified microorganism such as bacterium of MBL identification, Yeast, helminth and certain viruses carbohydrate patterns.On the contrary, MBL nonrecognition D- galactolipin and sialic acid, i.e., reciprocal the Two sugar and the sugar of position last, they generally modify present on mammalian plasma and cell surface glycoprotein " at It is ripe " complexity glycoconjugate.Think that this binding specificity promotes the identification on " external source " surface and helps to protect against " itself work Change ".However, MBL, really with high-affinity combination high mannose " precursor " glycan cluster, these clusters, which are located at, is isolated in mammal On the glycoprotein and glycolipid that endocytoplasmic reticulum is connected with the intracorporal N- of Gorky (Maynard et al.,J. Biol. Chem. 257:3788-3794, (1982)).Therefore, damaged cell is the potential target of the lectin pathway activation combined by MBL.
The Lectin domain that there is fiber gelatinized protein (ficolin) type to be different from MBL, referred to as fibrin are as former state Structural domain.Fiber gelatinized protein is not to depend on Ca++Mode combine saccharide residue.In human body, it has been identified that fiber gelatinized The three types (L- fiber gelatinized protein, M- fiber gelatinized protein and H- fiber gelatinized protein) of albumen.L- fiber gelatinized protein Both serum fiber gelatinized proteins have specificity to N- acetyl-d-glucosamine jointly with H- fiber gelatinized protein；However, H- Fiber gelatinized protein is herein in connection with N- acetyl-D-galactosamine.L- fiber gelatinized protein, H- fiber gelatinized protein, CL-11 and MBL Sugar specificity difference mean different agglutinins may be it is complementary, in spite of overlapping, but may target different Glycoconjugate.This viewpoint has obtained the support of latest report, i.e., in the known agglutinin of lectin pathway, only L- fiber Gelled protein and lipoteichoicacid are specifically bound, and lipoteichoicacid is a kind of cell wall sugar being present on all gram-positive bacterias Conjugate (Lynch et al.,J. Immunol. 172:1198-1202, (2004)).Collectin (i.e. MBL) and fiber Gelled protein is on amino acid sequence without significant similitude.However, two histone matter have similar structural domain group structure, with C1q is similar, is assembled into oligomeric structure, allows for a possibility that multidigit point combines maximization in this way.
The serum-concentration of MBL is alterable height in healthy population, this be genetically by mbl gene promoter and Polymorphism/mutation of both code areas is controlled.As acute phase protein, the expression of MBL is dialled further up during inflammation. L- fiber gelatinized protein concentration present in serum is similar with the concentration of MBL.Therefore, the fiber gelatinized egg of the L- of lectin pathway White branch may be suitable with MBL arm in strength.MBL and fiber gelatinized protein may also work as opsonin, this permission Surface that phagocyte is decorated targeted to MBL and fiber gelatinized protein (referring to 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 opsonification needs these protein and phagocytosis Cell receptor interaction (Kuhlman et al.,J. Exp. Med. 169:1733, (1989);Matsushita et al.,J. Biol. Chem. 271: 2448-54, (1996)), identity is not determined also.
People MBL passes through its collagen spline structure domain and unique C1r/C1s sample serine protease (the relevant silk of referred to as MBL Serine protease (MBL-associated serine proteases, MASP)) form specificity, the phase interaction of high-affinity With.It is heretofore described three kinds of MASP.Firstly, single enzyme " MASP " is identified, it is characterized in that being responsible for starting complement Cascade (i.e. cracking C2 and C4) enzyme (Matsushita et al.,J Exp Med 176(6):1497-1502 (1992); Ji Et al.,J. Immunol. 150:571-578, (1993)).It is later determined that MASP is active, actually two kinds of protease MASP-1 and MASP-2 mixture (Thiel et al.,Nature 386:506-510, (1997)).However, demonstrating only MBL-MASP-2 compound be just enough to make complement activation (Vorup-Jensen et al.,J. Immunol.165:2093-2100, (2000)).In addition, only MASP-2 rapid cleavage C2 and C4 (Ambrus et al.,J. Immunol. 170:1374-1382, (2003)).Therefore, MASP-2 is responsible for activation C4 and C2 to generate the protease of C3 convertase C4b2a.This is to be different from classics The significant difference of the C1 compound of approach, two species specificity serine proteases (C1r and C1s) synergistic effect is led in C1 compound The activation of complement system is caused.In addition, isolated the third new protease MASP-3 (Dahl, M.R., et al.,Immunity 15:127-35, 2001).MASP-1 and MASP-3 is the alternative splicing product of same gene.
Those of the enzyme component C1r and C1s of MASP and C1 compound share identical structural domain group structure (Sim et al.,Biochem. Soc. Trans. 28:545,2000).These structural domains include N-terminal C1r/C1s/ sea urchin VEGF/ bon e formation Albumen (CUB) structural domain, epidermal growth factor-like structural domain, the 2nd CUB structural domain, complement regulatory proteins structural domain series connection and Serine protease domain.With in C1 protease, the activation of MASP-2 passes through near serine protease domain Arg-Ile bond cleavage solution and generate, this by enzyme be divided into disulfide bond connection A chain and B chain, the latter is by serine protease domain It constitutes.
MBL can also be with variable sliced form (the MBL GAP-associated protein GAP (MAp19) or small referred to as 19 kDa of MASP-2 MBL GAP-associated protein GAP (sMAP) lacks the catalytic activity of MASP-2) it combines.(Stover,J. Immunol. 162:3481- 90, (1999);Takahashi et al.,Int. Immunol. 11:859-863, (1999)).MAp19 includes MASP-2 The first two structural domain, be followed by the additional sequences of 4 unique amino acids.The function of MAp19 it is indefinite (Degn et al.,J Immunol. Methods, 2011).MASP-1 and MASP-2 gene is located on No. 3 chromosomes of people and No. 1 chromosome (Schwaeble et al.,Immunobiology 205:455-466, (2002))。
Several evidences show that there are different MBL-MASP compounds, and in serum MASP major part it is not compound with MBL (Thiel, et al.,J. Immunol. 165:878-887, (2000)).H- fiber gelatinized protein and L- fiber gelatinized protein All in conjunction with all MASP, and agglutinin complement pathway is activated, the same with MBL (Dahl et al.,Immunity 15: 127-35, (2001);Matsushita et al.,J. Immunol. 168:3502-3506, (2002)).Lectin pathway Common C3 convertase (C4b2a) is all formed with classical pathway, this two approach merge in this step.
It generally believes in natural host, lectin pathway supports in anti-infectious defence in host to play a significant role.MBL Participate in host defense strong evidence from the patient that functional MBL serum levels are reduced analysis (Kilpatrick,Biochim. Biophys. Acta1572:401-413, (2002)).These patients show recurrent bacterial and fungi sense The neurological susceptibility of dye.These symptoms are usually it will be evident that because what is obtained from parent is anti-during the apparent window of rapid wear in life early stage Body potency reduces, but before whole antibody response development.This symptom is often several sites due to MBL collagenous portion Caused by mutation, being properly formed for MBL oligomer is disturbed.However, since MBL can be used as the conditioning independent of complement Element works, so having no knowledge about to the neurological susceptibility increase of infection is to what extent due to caused by impaired complement activation.
Think that all three approach (i.e. classical, agglutinin and alternative route) meet at C5, it is cleaved to be formed with more The product of kind pro-inflammatory effect.Approach after congregation is referred to as terminal complement approach.C5a is most effective anaphylatoxin, causes to put down The change of sliding flesh and vascular tone and vasopermeability.It is also the strong of both neutrophil cell and monocyte Chemotactic factor (CF) and activity factor.The cell activation that C5a is mediated can discharge a variety of other inflammatory mediators by induction to show Amplification inflammatory reaction is write, inflammatory mediator in addition includes cell factor, hydrolase, arachidonic acid metabolite and active oxygen Not.C5 cracks the formation for resulting in C5b-9, it is also referred to as membrane attack complex (MAC).Current strong evidence shows The MAC deposition of Asia cracking is other than plaing a part of to crack pore-forming compound, it is also possible to also play a significant role in inflammation.
Other than the basic role in immune defense, complement system is to cause the original of the tissue damage of many clinical diseases Cause.Although a large amount of evidences show in the pathogenesis of non-infectious human diseases, classic complement approach and substitution complement pathway Both exist, but the evaluation of lectin pathway effect is just started.The evidence that current research provides shows agglutinin The activation of approach may be the reason of causing complement activation and related inflammation in ischemia/reperfusion injury.Collard et al. (2000) it reports the endothelial cell combination MBL of the culture by oxidative stress, and shows that C3 is heavy after being exposed to human serum Product (Collard et al.,Am. J. Pathol. 156:1549-1556, (2000)).In addition, with the anti-MBL Dan Ke of blocking property Grand antibody processing human serum inhibits MBL combination and complement activation.These discoveries are expanded into ischemia-reperfusion rat mould In type, wherein the rat handled with the blocking antibody for rat MBL is coronal dynamic compared with the rat handled with control antibodies After arteries and veins occlusion display myocardial damage significantly relatively it is light (Jordan et al.,Circulation 104:1413-1418, (2001)).Molecular mechanism of the MBL in conjunction with blood vessel endothelium after unclear oxidative stress；It has recently been demonstrated that oxidative stress The activation of lectin pathway may be and be mediated in conjunction with vascular endothelial cell keratin by MBL afterwards, rather than be conjugated with sugar Object in conjunction in conjunction with and mediate (Collard et al.,Am. J. Pathol. 159:1045-1054, (2001)).It is other to grind Study carefully and has shown that classical pathway in ischemia/reperfusion injury pathogenesis and alternative route and lectin pathway in this disease Effect in disease still have dispute (Riedermann, N.C., et al.,Am. J. Pathol. 162:363-367, 2003)。
Fibrosis is that excessive connective tissue is formed in organ or tissue, is normally in response to damage or damage.Fibrosis Mark is that excessive extracellular matrix is generated after local trauma.Connective tissue deposition is caused to the normal physiological reaction of damage, But this initial beneficial repair process may continue and become morbid state, change the structure and function of tissue.In cellular level On, epithelial cell and fibroblast proliferation and it is divided into myofibroblast, causes contracted matrix, intensity to increase, capilary Compressing and anoxic.The inflow of inflammatory cell (including macrophage and lymphocyte) cause cytokine release and expand collagen, The deposition of fibronectin and other molecular markers of fibrosis.Conventional treatments mainly use corticosteroid and immune suppression Property drug processed targets the inflammatory process of fibrosis.Unfortunately, these anti-inflammatory agents have with little to no clinical effectiveness.At present There is no effective therapy or therapeutic agent for fibrosis, but the people of zooscopy and faithful record report shows that fibrosed tissue damages Wound it is reversible (Tampe and Zeisberg,Nat Rev Nephrol, Vol 10:226-237,2014).
The ability of kidney from injury recovery is limited.Various nephrosis reasons lead to local inflammation, cause cicatrization and nephridial tissue Fibrosis.The progressive kidney of perpetuation driving the Tubulointerstitial inflammation and fibrosis and chronic kidney diseases of inflammatory stimulus Function is impaired.It is related with significant morbidity and mortality to end-stage renal failure.Because Tubulointerstitial fibrosis is The common terminal of a variety of nephrosis reasons, which represent crucial therapeutic targets, it is therefore intended that prevention kidney failure.Independent of primary The risk factors (for example, albuminuria) of kidney diaseases promote development and the renal excretion function of kidney fibrosis by driving local inflammation Loss, and then enhance progression of disease.
In view of effect of the fibrosis in many diseases and illness, for example, Tubulointerstitial fibrosis leads to chronic renal disease Disease, the effective medicament for the treatment of of disease and the patient's condition that split hair is caused or aggravated by fibrosis in treatment, there are pressing needs. The new sum for further promoting fibrosis approach in view of the inflammatory in targeting kidney diaseases has the scarcity for the treatment of, to exploitation treatment, suppression System, prevention and/or reversing renal fibrosis, thus the effective medicament of the treatment for preventing progressive chronic kidney diseases, there are demands.
Summary of the invention
This general introduction is provided, to introduce the selection of concept in simplified form, is further described in following detailed description of the invention.This is general The key feature for being both not intended to identify claimed theme is stated, the range for assisting to determine claimed theme is also not intended to.
In one aspect, the present invention is provided to treat, inhibit, mitigate or prevent to suffer from or risky generation is by fibrosis And/or the method for the fibrosis of the mammalian subject of disease or illness that inflammation causes or aggravates, including give subject Effectively inhibit the MASP-2 inhibitor of the amount of fibrosis.In one embodiment, MASP-2 inhibitor be MASP-2 antibody or Its segment.In one embodiment, MASP-2 inhibitor is that the MASP-2 of a part for specifically binding SEQ ID NO:6 is mono- Clonal antibody or its segment.In one embodiment, MASP-2 inhibitor selectively inhibits lectin pathway complement activation, Without significantly inhibiting C1q- dependent complement activation.In one embodiment, subject suffers from is drawn by least one of below The disease or illness for rising or aggravating: (i) fibrosis related with ischemic damage and reperfusion damage and/or inflammation, (ii) kidney fiber Change and/or nephritis disease is (for example, Tubulointerstitial fibrosis, chronic kidney diseases, chronic renal failure, renal glomerular disease are (for example, office Stove segmental glomerulosclerosis), that compound illness (for example, IgA nephrosis, membranous nephropathy), lupus nephritis, nephrosis is immunized is comprehensive Sign, diabetic nephropathy, Tubulointerstitial damage and glomerulonephritis (for example, C3 glomerulopathy), (iii) pulmonary fibrosis and/ Or inflammation is (for example, Chronic Obstructive Pulmonary Disease, cystic fibrosis, pulmonary fibrosis related with chorionitis, bronchiectasis and lung Arterial hypertension), (iv) liver fibrosis and/or inflammation (for example, cirrhosis, non-alcohol fatty liver (steatohepatitis)), Hepatic fibrosis-renal tubular ectasia syndrome secondary to alcohol abuse, the hepatic fibrosis-renal tubular ectasia syndrome secondary to acute or chronic hepatitis, biliary diseases and toxicity liver damage Hurt (for example, hepatotoxicity wind agitation caused by hepatic injury drug-induced as caused by paracetamol or other medicines), (v) heart Fibrosis and/or inflammation are (for example, cardiac fibrosis, myocardial infarction, valvular fibrosis, Atrial fibrosis, endocardium fiber Change, Arrhythmogenic right ventricular cardiomyopathy (ARVC), (vi) vascular fibrosis are (for example, vascular diseases, atherosclerosis Property vascular diseases, hemadostewnosis, restenosis, vasculitis, phlebitis, deep vein thrombosis formed and abdominal aneurvsm), (vii) Skin fibrosis (for example, excessive wound repair, chorionitis, Sjogren's syndrome, cheloid, connective tissue disease, cicatrization and Hypertrophic scar), the fibrosis (for example, joint fibrosis) in the joint (viii), the fibrosis (example of (ix) central nervous system Such as, apoplexy, traumatic brain injury and spinal cord injury), the fibrosis of (x) digestive system is (for example, Crohn disease, pancreas fibrosis And ulcerative colitis), (xi) eye fibrosis is (for example, anterior subcapsular cataract, rear capsule milkiness, macular degeneration and retina and glass Glass body retinopathy), the fibrosis of (xii) muscle skeleton soft tissue structure is (for example, adhesive capsulitis, dupp Yi Telunshi contraction Contracting and myelofibrosis), the fibrosis of (xiii) reproductive organs (for example, mullerianosis and Perun Nie Shi disease), (xiv) Cause the chronic infectious disease of fibrosis and/or inflammation (for example, α is viral, hepatitis A, hepatitis B, hepatitis C, knot Core disease, HIV and influenza), (xv) cause the autoimmune disease of fibrosis and/or inflammation (for example, chorionitis and systematicness Lupus erythematosus (SLE), (xvi) cicatrization related with wound are (for example, wherein cicatrization related with wound is selected from hand (for example, surgical adhesions, wherein cicatricial tissue can be formed art complication between internal organs, led to contracture, pain and be can lead to Infertility), the fibrosis of chemotherapeutic agent induction, radiation-induced fibrosis and with the related cicatrization of burning) or (xvii) organ transplant, Breast fibroadenoma, fibro-muscular, retroperitoneal fibrosis, thyroid gland fibrosis, lymph node fibrosis, Cystic fibrosis and fibrosis of pleura.
On the other hand, the present invention is provided to treat, inhibit, mitigate or prevent to suffer from or risky generation is by kidney fiber Change and/or the method for the kidney fibrosis of the mammalian subject of disease or illness that inflammation causes or aggravates, including give by Examination person effectively inhibits the MASP-2 inhibitor of the amount of kidney fibrosis.In one embodiment, MASP-2 inhibitor is MASP-2 Antibody or its segment.In one embodiment, MASP-2 inhibitor is a part for specifically binding SEQ ID NO:6 MASP-2 monoclonal antibody or its segment.In one embodiment, MASP-2 antibody or its segment are with its conjugated complement system At least 10 times of not synantigen of affinity specific binding include SEQ ID NO:6 polypeptide.In one embodiment, resist Antibody, chimeric antibody, humanized antibody and the people of the effector function of body or its segment selected from recombinant antibodies, with reduction are anti- Body.In one embodiment, MASP-2 inhibitor selectively inhibits lectin pathway complement activation, without significantly inhibiting C1q- dependent complement activation.In one embodiment, MASP-2 inhibitor is subcutaneous, peritonaeum is interior, intramuscular, intra-arterial, vein It is interior or given as inhalant.In one embodiment, MASP-2 inhibitor is effectively to inhibit Tubulointerstitial fibrosis Amount is given.In one embodiment, MASP-2 inhibitor is to effectively reduce, postpone or eliminate the amount that the dialysis of subject needs It gives.In one embodiment, subject suffers from selected from chronic kidney diseases, chronic renal failure, renal glomerular disease (for example, office Stove segmental glomerulosclerosis), that compound illness (for example, IgA nephrosis, membranous nephropathy), lupus nephritis, nephrosis is immunized is comprehensive Sign, diabetic nephropathy, Tubulointerstitial damage and the kidney diaseases or illness of glomerulonephritis (for example, C3 glomerulopathy).? In one embodiment, subject effectively reduced with albuminuria and MASP-2 inhibitor the amount of the albuminuria of subject to It gives.In one embodiment, MASP-2 inhibitor compared with the baseline twenty-four-hour urine protein secretion for treating preceding subject to have It imitates and realizes that twenty-four-hour urine protein secretion at least 20% reduces (for example, at least 30% reduction or at least 40% reduction or at least 50% reduction) Amount and the time give.In one embodiment, subject suffers from kidney diaseases related with albuminuria or illness, is selected from kidney Sick syndrome, pre-eclampsia, eclampsia, the toxic lesion of kidney, amyloidosis, collagen vascular diseases are (for example, systemic red yabbi Sore), dehydration, renal glomerular disease is (for example, membranous glomerulonephritis, focal segmental glomerulonephritis, C3 glomerulopathy, micro- Small lesion disease, lipoid nephrosis), strenuous exercise, stress, benign orthostatic position (position) albuminuria, Focal segmental kidney it is small Ball hardening, IgA nephrosis (that is, Bei Geershi is sick), IgM nephrosis, membranoprolifer ative glomerulonephritis, membranous nephropathy, minute lesion disease Disease, sarcoidosis, Alport's syndrome, diabetes (diabetic nephropathy), drug-induced toxicity are (for example, NSAIDS, Ni Gu Fourth, penicillamine, lithium carbonate, gold and other heavy metals, Vel-Tyr-Pro-Trp-Thr-Gln-Arg-Phe, antibiotic (for example, adriamycin) or opiate (for example, Heroin) or other nephrotoxins)；Fabry disease, infection after HIV, syphilis, first, second or hepatitis C, streptococcus (for example, feel Dye, urinary schistosmiasis)；Amino acid uria, Fanconi syndrome, hypertensive nephrosclerosis, interstitial nephritis, drepanocytosis, Hemoglobinuria, Huppert's disease, myoglobinuria, organ rejection's (for example, renal transplant rejection), Ebola hemorrhagic fever, first Kneecap syndrome, familial Mediterranean fever, HELLP syndrome, systemic loupus erythematosus, Wei Genashi granulomatosis, rheumatoid Arthritis, 1 type of glycogen storage disease, Goodpasture's syndrome, anaphylactoid purpura, the urinary tract infections for having spread to kidney, Si Ye Glomerulonephritis after Glenn Cotard and infection.In one embodiment, subject suffers from IgA nephrosis.In a reality It applies in scheme, subject suffers from membranous nephropathy.
On the other hand, the present invention provides prevention or reduces the subject's with disease related with albuminuria or the patient's condition The method of injury of kidney, the MASP-2 inhibitor of the amount including giving the albuminuria for effectively reducing or preventing subject.In a reality It applies in scheme, MASP-2 inhibitor is MASP-2 antibody or its segment.In one embodiment, MASP-2 inhibitor is special The MASP-2 monoclonal antibody or its segment of a part of property combination SEQ ID NO:6.In one embodiment, MASP-2 presses down Preparation selectively inhibits lectin pathway complement activation, without significantly inhibiting C1q- dependent complement activation.Implement at one In scheme, disease related with albuminuria or the patient's condition are selected from nephrotic syndrome, pre-eclampsia, eclampsia, the toxic lesion of kidney, shallow lake The change of powder sample, collagen vascular diseases (for example, systemic loupus erythematosus), dehydration, renal glomerular disease (for example, membranous glomerulonephritis, Focal segmental glomerulonephritis, C3 glomerulopathy, minimal change disease, lipoid nephrosis), strenuous exercise, stress, it is benign Orthostatic position (position) albuminuria, focal segmental glomerulosclerosis, IgA nephrosis (that is, Bei Geershi is sick), IgM nephrosis, film increase Growing property glomerulonephritis, membranous nephropathy, minimal change disease, sarcoidosis, Alport's syndrome, diabetes (diabetogenous nephrosis Disease), drug-induced toxicity (for example, NSAIDS, nicotine, penicillamine, lithium carbonate, gold and other heavy metals, Vel-Tyr-Pro-Trp-Thr-Gln-Arg-Phe, Antibiotic (for example, adriamycin) or opiate (for example, heroin))；Fabry disease, infection (for example, HIV, syphilis, first, Second or hepatitis C, streptococcus postoperative infection, urinary schistosmiasis)；Amino acid uria, Fanconi syndrome, hypertensive nephrosclerosis, Interstitial nephritis, drepanocytosis, hemoglobinuria, Huppert's disease, myoglobinuria, organ rejection are (for example, kidney transplant Repel), Ebola hemorrhagic fever, chatelain's syndrome, familial Mediterranean fever, HELLP syndrome, systemic loupus erythematosus, Wei Ge Na Shi granulomatosis, 1 type of glycogen storage disease, Goodpasture's syndrome, anaphylactoid purpura, has expanded at rheumatoid arthritis It is scattered to glomerulonephritis after urinary tract infections, siogren's syndrome and the infection of kidney.In one embodiment, MASP-2 Inhibitor is effectively to realize twenty-four-hour urine protein secretion at least compared with the baseline twenty-four-hour urine protein secretion for treating preceding subject The amount of 20% reduction (for example, at least 30% reduction or at least 40% reduction or at least 50% reduction) and time are given.
On the other hand, the present invention provides the method for inhibiting the progress of chronic kidney diseases, including gives and effectively reduce or in advance Prevent the MASP-2 inhibitor of the amount of the kidney fibrosis (for example, Tubulointerstitial fibrosis) of subject in need.In a reality It applies in scheme, MASP-2 inhibitor is MASP-2 antibody or its segment.In one embodiment, MASP-2 inhibitor is special The MASP-2 monoclonal antibody or its segment of a part of property combination SEQ ID NO:6.In one embodiment, MASP-2 presses down Preparation selectively inhibits lectin pathway complement activation, without significantly inhibiting C1q- dependent complement activation.Implement at one In scheme, before giving MASP-2 inhibitor, subject in need shows that albuminuria and giving for MASP-2 inhibitor drop The albuminuria of low subject.In one embodiment, MASP-2 inhibitor is with the baseline twenty-four-hour urine with subject before treatment Protein secretion, which is compared, effectively realizes that twenty-four-hour urine protein secretion at least 20% is reduced (for example, at least 30% reduction or at least 40% reduction Or at least 50% reduce) amount and the time give.In one embodiment, MASP-2 inhibitor is to effectively reduce, postpone or disappear Except the amount that the dialysis of subject needs is given.
On the other hand, the present invention provides method of the kidney from injury of kidney of protection subject, the subject undergone, It is undergoing or will be undergone the treatment of one or more renal toxicity agent, effectively prevents or improve drug-induced kidney including giving The MASP-2 inhibitor of the amount of disease.In one embodiment, MASP-2 inhibitor is MASP-2 antibody or its segment.At one In embodiment, MASP-2 inhibitor be specifically bind SEQ ID NO:6 a part MASP-2 monoclonal antibody or its Segment.In one embodiment, MASP-2 inhibitor selectively inhibits lectin pathway complement activation, without significantly inhibiting C1q- dependent complement activation.
On the other hand, the present invention provides the method for people experimenter of the treatment with IgANP (IgAN), packet Including and giving subject includes the MASP-2 inhibiting antibody or its antigen knot for effectively inhibiting the amount of MASP-2- dependent complement activation Close the composition of segment.In one embodiment, subject suffers from steroid-dependent IgAN.In one embodiment, MASP-2 inhibiting antibody is the monoclonal antibody or its segment for specifically binding people MASP-2.In one embodiment, resist Antibody, chimeric antibody, humanized antibody and the people of the effector function of body or its segment selected from recombinant antibodies, with reduction are anti- Body.In one embodiment, MASP-2 inhibiting antibody does not significantly inhibit classical pathway.In one embodiment, MASP- 2 inhibiting antibodies are with 30 nM or smaller IC50Inhibit C3b deposition in 90% human serum.In one embodiment, the side Method further comprises giving the MASP-2 inhibiting antibody or its antigen binding that subject includes the amount for being effectively improved renal function Before the step of composition of segment, identifying has the people experimenter of steroid-dependent IgAN.In one embodiment, MASP-2 inhibiting antibody or its antigen-binding fragment are given with the amount for being effectively improved renal function.In one embodiment, MASP-2 inhibiting antibody or its antigen-binding fragment are with real compared with the baseline twenty-four-hour urine protein secretion for treating preceding subject The effective quantity and enough time of existing twenty-four-hour urine protein secretion at least 20% reduction are given.In one embodiment, composition with It is enough to improve renal function in the subject and the amount of reduction corticosteroid dosage is given.In one embodiment, MASP-2 inhibiting antibody or its antigen-binding fragment include heavy chain variable region, and it includes amino acid shown in SEQ ID NO:67 CDR-H1, CDR-H2 and CDR-H3 of sequence；And light chain variable region, it includes amino acid sequences shown in SEQ ID NO:70 CDR-L1, CDR-L2 and CDR-L3.
On the other hand, the present invention provide treatment with membranous nephropathy (MN) people experimenter method, including give by Examination person includes the MASP-2 inhibiting antibody or its antigen-binding fragment for the amount for effectively inhibiting MASP-2- dependent complement activation Composition.In one embodiment, subject suffers from steroid-dependent MN.In one embodiment, MASP-2 inhibits Property antibody be specifically bind people MASP-2 monoclonal antibody or its segment.In one embodiment, MASP-2 inhibition Antibody or its antigen-binding fragment are given with the amount for being effectively improved renal function.In one embodiment, MASP-2 inhibition is anti- Body or its antigen-binding fragment are to realize twenty-four-hour urine albumen compared with the baseline twenty-four-hour urine protein secretion for treating preceding subject The effective quantity and enough time of secretion at least 20% reduction are given.In one embodiment, composition is enough described tested The amount for improving renal function and reduction corticosteroid dosage in person is given.In one embodiment, MASP-2 inhibiting antibody Or its antigen-binding fragment include heavy chain variable region, it includes the CDR-H1 of amino acid sequence shown in SEQ ID NO:67, CDR-H2 and CDR-H3；And light chain variable region, it includes CDR-L1, CDR-L2 of amino acid sequence shown in SEQ ID NO:70 And CDR-L3.
On the other hand, the present invention provide treatment with lupus nephritis (LN) people experimenter method, including give by Examination person includes the MASP-2 inhibiting antibody or its antigen-binding fragment for the amount for effectively inhibiting MASP-2- dependent complement activation Composition.In one embodiment, subject suffers from steroid-dependent LN.In one embodiment, MASP-2 inhibits Property antibody be specifically bind people MASP-2 monoclonal antibody or its segment.In one embodiment, MASP-2 inhibition Antibody or its antigen-binding fragment are given with the amount for being effectively improved renal function.In one embodiment, MASP-2 inhibition is anti- Body or its antigen-binding fragment are to realize twenty-four-hour urine albumen compared with the baseline twenty-four-hour urine protein secretion for treating preceding subject The effective quantity and enough time of secretion at least 20% reduction are given.In one embodiment, composition is enough described tested The amount for improving renal function and reduction corticosteroid dosage in person is given.In one embodiment, MASP-2 inhibiting antibody Or its antigen-binding fragment include heavy chain variable region, it includes the CDR-H1 of amino acid sequence shown in SEQ ID NO:67, CDR-H2 and CDR-H3；And light chain variable region, it includes CDR-L1, CDR-L2 of amino acid sequence shown in SEQ ID NO:70 And CDR-L3.
On the other hand, the present invention provides the method for reducing the albuminuria in the people experimenter with IgAN, including basis Following dosage gives subject MASP-2 inhibiting antibody or its antigen-binding fragment, the antibody or its antigen binding fragment Section includes heavy chain variable region, and it includes CDR-H1, CDR-H2 and CDR-H3 of amino acid sequence shown in SEQ ID NO:67；With Light chain variable region, it includes CDR-L1, CDR-L2 and CDR-L3 of amino acid sequence shown in SEQ ID NO:70:
A. (i.e. 3.6mg/kg is described to 4.4 mg/kg's) by subject about 4mg/kg of the intravenous administration with IgAN once a week Antibody continues at least 12 weeks treatment phases；Or
B. (i.e. 162 mg are to 797 mg's) by subject about 180mg to about 725mg of the intravenous administration with IgAN once a week The antibody continues at least 12 weeks treatment phases,
Wherein the method reduces the albuminuria in the people experimenter.
Detailed description of the invention
By reference to following detailed description together with attached drawing, aspect above-mentioned of the invention and many bonus will more It is comprehensible, equally also it is best understood from, in which:
Fig. 1 is the figure for illustrating people's MASP-2 genome structure；
Fig. 2A is the schematic diagram for illustrating the domain constructs of people's MASP-2 albumen；
Fig. 2 B is the schematic diagram for illustrating the domain constructs of people's MAp19 albumen；
Fig. 3 is to illustrate that mouse MASP-2 knocks out the figure of strategy；
Fig. 4 is the figure for illustrating the micro- gene construct of people MASP-2；
Fig. 5 A, which is provided, proves that the C4 that MASP-2 defect causes lectin pathway to mediate activates losing as a result, such as gathering by sweet dew C4b deposits what defect was measured on sugar, as described in Example 2；
Fig. 5 B, which is provided, proves that the C4 that MASP-2 defect causes lectin pathway to mediate activates losing as a result, such as gathering by yeast C4b deposits what defect was measured on sugar, as described in Example 2；
Fig. 5 C provide prove to be obtained from MASP-2 is +/-, MASP-2 -/- and the opposite C4 of blood serum sample of wild-type lines activate It is horizontal as a result, as deposited and measured by the C4b on mannosan and zymosan, as described in Example 2；
Fig. 6 provides extensive in such a way that protein concentration relies in proof addition mouse recombination MASP-2 to MASP-2 -/- blood serum sample Answered the C4 activation of lectin pathway mediation by C4b on mannosan as a result, as deposited and measured, such as embodiment 2 It is described；
Fig. 7, which is provided, proves that classical pathway is functional as a result, as described in Example 8 in MASP-2 -/- strain；
Fig. 8 A provide prove that anti-MASP-2 Fab2 antibody #11 inhibits that C3 convertase formed as a result, as described in Example 10；
Fig. 8 B provide prove anti-MASP-2 Fab2 antibody #11 in conjunction with natural rat MASP-2 as a result, such as 10 institute of embodiment It states；
Fig. 8 C provide prove anti-MASP-2 Fab2 antibody #41 inhibit C4 cracking as a result, as described in Example 10；
Fig. 9, which is provided, proves that all anti-MASP-2 Fab2 antibody for inhibiting C3 convertase to be formed after tested are also found to inhibit C4 Cracking as a result, as described in Example 10；
Figure 10 is to illustrate the recombinant polypeptide derived from the rat MASP-2 to map for MASP-2 blocking property Fab2 antibody epitope Schematic diagram, as described in Example 11；
Figure 11 provide prove anti-MASP-2 Fab2 #40 and #60 with rat MASP-2 polypeptide in conjunction with as a result, such as embodiment 11 It is described；
Figure 12 A is shown under the conditions of lectin pathway specific assay presence or absence of people MASP-2 monoclonal antibody (OMS646) level that MAC is deposited in the case where, it was demonstrated that OMS646 is with the IC of about 1 nM50Value inhibits the MAC of lectin-mediated Deposition, as described in Example 12；
Figure 12 B is shown under the conditions of classical pathway specific assay presence or absence of people MASP-2 monoclonal antibody (OMS646) level that MAC is deposited in the case where, it was demonstrated that the MAC deposition that OMS646 does not inhibit classical pathway to mediate, such as embodiment Described in 12；
Figure 12 C is shown under the conditions of alternative route specific assay presence or absence of people MASP-2 monoclonal antibody (OMS646) level that MAC is deposited in the case where, it was demonstrated that the MAC deposition that OMS646 does not inhibit alternative route to mediate, such as embodiment Described in 12；
Figure 13 is shown in pharmacokinetics (PK) feature of people MASP-2 monoclonal antibody (OMS646) in mouse, shows OMS646 Function of the concentration (n=3 animal/group mean value) as the time after being given with prescribed dose, as described in Example 12；
The pharmacodynamics (PD) that Figure 14 A is shown in after intravenous administration the people MASP-2 monoclonal antibody (OMS646) in mouse is anti- It answers, as the active decline measurement of system lectin pathway, as described in Example 12；
Figure 14 B is shown in after subcutaneous administration pharmacodynamics (PD) reaction of the people MASP-2 monoclonal antibody (OMS646) in mouse, It is as the active decline measurement of system lectin pathway, as described in Example 12；
Figure 15 illustrate the computer based image analysis being sliced with the nephridial tissues of sirius red stains as a result, wherein tissue is cut Piece is obtained from wild type and MASP-2-/- mouse and sham-operation wild type and MASP- of unilateral ureteral obstruction (UUO) after 7 days 2-/- mouse, as described in Example 14；
The computer based image analysis for the nephridial tissue slice that Figure 16 diagram is dyed with F4/80 macrophage specific antibody As a result, wherein histotomy is obtained from wild type and MASP-2-/- mouse of unilateral ureteral obstruction (UUO) after 7 days, and artificial hand Art wild type and MASP-2-/- mouse, as described in Example 14.
Figure 17 is shown in obtained from the wild type and MASP-2-/- mouse and artificial hand after unilateral ureteral obstruction (UUO) 7 days Pass through the opposite mRNA of the collagen -4 of quantitative PCR (qPCR) measurement in art wild type and MASP-2-/- mouse nephridial tissue slice Expression, as described in Example 14.
Figure 18 is shown in obtained from the wild type and MASP-2-/- mouse and artificial hand after unilateral ureteral obstruction (UUO) 7 days Pass through the transforming growth factor-β_1 (TGF β -1) of qPCR measurement in art wild type and MASP-2-/- mouse nephridial tissue slice Relative mRNA expression levels, as described in Example 14.
Figure 19 is shown in obtained from the wild type and MASP-2-/- mouse and artificial hand after unilateral ureteral obstruction (UUO) 7 days Pass through the opposite of the interleukin-6 (IL-6) of qPCR measurement in art wild type and MASP-2-/- mouse nephridial tissue slice MRNA expression, as described in Example 14.
Figure 20 is shown in obtained from the wild type and MASP-2-/- mouse and artificial hand after unilateral ureteral obstruction (UUO) 7 days Water is expressed by the opposite mRNA of the interferon-γ of qPCR measurement in art wild type and MASP-2-/- mouse nephridial tissue slice It is flat, as described in Example 14.
Figure 21 illustrate the computer based image analysis being sliced with the nephridial tissues of sirius red stains as a result, wherein group It knits and is sliced after unilateral ureteral obstruction (UUO) 7 days from the open country handled with MASP-2 inhibiting antibody and Isotype control antibodies Raw type mouse obtains, as described in Example 15.
Figure 22 is illustrated and in the tissue for blocking kidney for being obtained from the wild-type mice handled with IgG4 isotype controls The level of hydroxyproline is compared, from the unilateral ureteral obstruction for being obtained from the wild-type mice handled with MASP-2 inhibiting antibody (UUO) afterwards 7 days harvest kidney hydroxyproline content, as described in Example 15.
Figure 23 be shown in only receive the wild type control mice (n=2) of salt water, receive BSA wild-type mice (n=6) and Receive in MASP-2-/- mouse (n=6) of BSA, in the total amount (mg/ of the serum proteins of albumen overload research measurement in the 15th day Ml), as described in Example 16.
Figure 24 be shown in albumen overload research the 15th day through 24 hours from only receive salt water wild type control mice (n= 2), receive the wild type (n=6) of BSA and receive the albumen of the secretion in the urine that the MASP-2- of BSA/- mouse (n=6) is collected Total amount (mg), as described in Example 16.
Figure 25 is shown in the 15th day representative h and E (H& from following each group mouse of albumen overload research E the nephridial tissue slice) dyed: (figure A) wild type control mice；(figure B) MASP-2-/- control mice；(figure C) is at BSA The wild-type mice of reason；MASP-2-/- mouse that (figure D) is handled with bovine serum albumin(BSA) (BSA), as described in Example 16.
Figure 26 illustrates the computer based image point of the nephridial tissue slice with macrophage specific antibody F4/80 dyeing Analysis as a result, display macrophage be averaged stained area (%), wherein histotomy studies the 15th day from wild type in albumen overload Control mice (n=2) is obtained with the BSA wild-type mice (n=6) handled and the MASP-2- handled with BSA/- mouse (n=5), As described in Example 16.
Figure 27 A diagram is (average by the total secreted proteins and macrophages infiltration measured in the urine of 24 hours samples Stained area %) mapping, macrophage-albuminuria correlation presence in the every wild-type mice (n=6) handled with BSA Analysis, as described in Example 16.
Figure 27 B diagram (is averagely dyed by the total secreted proteins in the urine of 24 hours samples with macrophages infiltration Area %) mapping, macrophage-albuminuria correlation presence point in the every MASP-2- handled with BSA/- mouse (n=5) Analysis, as described in Example 16.
Figure 28 is shown in the MASP-2-/- mouse (n=5) BSA the wild-type mice (n=4) handled and handled with BSA In, use the result of the computer based image analysis of the histotomy of anti-TGF β antibody dyeing (to dye as % TGF β antibody Area measurement), as described in Example 16.
Figure 29 is shown in the MASP-2-/- mouse (n=5) BSA the wild-type mice (n=4) handled and handled with BSA In, the result of the computer based image analysis for the histotomy for using Anti-tnfa antibody to dye (is dyed as % TNF α antibody Area measurement), as described in Example 16.
The wild-type mice (n=7) that Figure 30 is shown in wild type control mice, MASP-2-/- control mice, is handled with BSA With in MASP-2-/- mouse (n=7) for being handled with BSA, with the computer based figure for the histotomy that anti-IL-6 antibody dyes As the result (being measured as % IL-6 antibody stained area) of analysis, as described in Example 16.
Figure 31 be shown in from wild type control mice (n=1), MASP-2-/- control mice (n=1), with BSA handle It wild-type mice (n=6) and is continuously selected with the histotomy of BSA MASP-2-/- mouse (n=7) cortex renis handled The frequency of the TUNEL apoptotic cell counted in 20 high power fields (HPF), as described in Example 16.
Figure 32 is shown in handled with BSA after the 15th day, the representative H&E stained tissue from following each group mouse is cut Piece: (figure A) is used with the wild type control mice of saline treatment, the control mice of (figure B) isotype antibody processing and (figure C) The wild-type mice of MASP-2 inhibiting antibody processing, as described in Example 17.
Figure 33 is shown in come the wild-type mice (n=8) of use by oneself saline control and BSA processing, with Isotype control antibodies and The wild-type mice (n=8) that BSA is handled and the kidney skin with MASP-2 inhibiting antibody and the BSA wild-type mice (n=7) handled The frequency of the TUNEL apoptotic cell counted in 20 continuously selected the high power field (HPF) of the histotomy of matter, such as embodiment Described in 17.
Figure 34 is shown in BSA and the wild-type mice (n=8) of saline treatment, with BSA and Isotype control antibodies and handles Wild-type mice (n=7) and the wild-type mice (n=8) that is handled with BSA and MASP-2 inhibiting antibody in, it is anti-with anti-TGF β The result (being measured as % TGF β antibody stained area) of the computer based image analysis of the histotomy of body dyeing, strictly according to the facts It applies described in example 17.
Figure 35 be shown in BSA and the wild-type mice (n=8) of saline treatment, BSA and Isotype control antibodies (n=7) and In the wild-type mice (n=8) handled with BSA and MASP-2 inhibiting antibody, the histotomy that is dyed with Anti-tnfa antibody The result (being measured as % TNF α antibody stained area) of computer based image analysis, as described in Example 17.
Figure 36 be shown in BSA and the wild-type mice (n=8) of saline treatment, BSA and Isotype control antibodies (n=7) and In the wild-type mice (n=8) handled with BSA and MASP-2 inhibiting antibody, the histotomy that is dyed with anti-IL-6 antibody The result (being measured as % IL-6 antibody stained area) of computer based image analysis, as described in Example 17.
Figure 37 be shown in adriamycin or only salt water (control) processing after the 14th day, the representativeness from following each group mouse H&E staining tissue slides: (figure A-1, A-2, A-3) is with the wild type control mice of only saline treatment；(figure B-1, B-2, B- 3) wild-type mice handled with adriamycin；MASP-2-/- mouse that (figure C-1, C-2, C-3) is handled with adriamycin, such as Described in embodiment 18.
Figure 38 illustrates the computer based image point of the nephridial tissue slice with macrophage specific antibody F4/80 dyeing Analysis as a result, be shown in adriamycin or only salt water (wild type control) processing after the 14th day macrophage from following each group mouse Cell is averaged stained area (%): with the wild type control mice of only saline treatment；The wild-type mice handled with adriamycin；With The only MASP-2- of saline treatment/- mouse and the MASP-2 -/- mouse handled with adriamycin, wherein * *p=0.007, such as implement Described in example 18.
Figure 39 illustrate the computer based image analysis being sliced with the nephridial tissues of sirius red stains as a result, being shown in With adriamycin or only salt water (wild type control) processing after the 14th day collagen deposition stained area from following each group mouse (%): with the wild type control mice of only saline treatment；The wild-type mice handled with adriamycin；With the MASP- of only saline treatment 2-/- mouse and the MASP-2 -/- mouse handled with adriamycin, wherein * *p=0.005, as described in Example 18.
During Figure 40 is shown in 12 weeks research process with MASP-2 inhibiting antibody (OMS646) weekly treatment, at two Urinary albumin/creatinine ratio (uACR) in IgA patient, as described in Example 19.
The uACR (mg/g) of 4 IgAN patients that Figure 41 diagram is treated from baseline by 120 days with OMS646 at any time, such as Described in embodiment 21.
Figure 42 illustrates 24 hours of the 4 IgAN patients treated with OMS646 after treating preceding 1st day baseline to treatment Urine proteins variation, as described in Example 21.
The average change of twenty-four-hour urine albumen after Figure 43 illustrates the 4 IgAN patients treated with OMS646 from baseline to treatment Change, as described in Example 21.
Sequence table description
SEQ ID NO:1 people MAp19 cDNA
SEQ ID NO:2 people MAp19 albumen (has leader sequence)
SEQ ID NO:3 people MAp19 albumen (maturation)
SEQ ID NO:4 people MASP-2 cDNA
SEQ ID NO:5 people MASP-2 albumen (has leader sequence)
SEQ ID NO:6 people MASP-2 albumen (maturation)
SEQ ID NO:7 people MASP-2 gDNA (exons 1-6)
Antigen: (about MASP-2 maturation protein)
SEQ ID NO:8 CUBI sequence (amino acid 1-121)
SEQ ID NO:9 CUBEGF sequence (amino acid 1-166)
SEQ ID NO:10 CUBEGFCUBII (amino acid 1-293)
The area NO:11 EGF ID SEQ (amino acid 1 22-166)
SEQ ID NO:12 serine protease domain (amino acid 429-671)
Serine protease domain (amino acid 610-625, the dashing forward with Ser618 to Ala of SEQ ID NO:13 inactivation Become)
SEQ ID NO:14 TPLGPKWPEPVFGRL (CUBI peptide)
SEQ ID NO:15 TAPPGYRLRLYFTHFDLELSHLCEYDFVKLSSGAKVLATLCGQ (CUBI peptide)
SEQ ID NO:16 TFRSDYSN (combined area MBL core)
SEQ ID NO:17 FYSLGSSLDITFRSDYSNEKPFTGF (combined area MBL)
SEQ ID NO:18 IDECQVAPG (EGF peptide)
SEQ ID NO:19 ANMLCAGLESGGKDSCRGDSGGALV (serine protease combination core) is described in detail
Inhibitor peptides:
SEQ ID NO:20 MBL full-length cDNA
SEQ ID NO:21 MBL full-length proteins
SEQ ID NO:22 OGK-X-GP (shared to combine)
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 (people h- fiber gelatinized protein)
SEQ ID NO:28 GCOGLOGAOGDKGEAGTNGKRGERGPOGPOGKAGPOGPNGAOGEO (the fiber gelatinized egg of people White p35)
SEQ ID NO:29 LQRALEILPNRVTIKANRPFLVFI (C4 cracking site)
Expression inhibiting agent:
The cDNA (the nucleotide 22-680 of SEQ ID NO:4) of SEQ ID NO:30 CUBI-EGF structural domain
SEQ ID NO:31
5' CGGGCACACCATGAGGCTGCTGACCCTCCTGGGC 3'
The nucleotide 12-45 of SEQ ID NO:4, including MASP-2 translation initiation site (sense strand)
SEQ ID NO:32
5'GACATTACCTTCCGCTCCGACTCCAACGAGAAG3'
Encode the nucleotide 361-396 (sense strand) of the SEQ ID NO:4 in the region containing MASP-2 MBL binding site
SEQ ID NO:33
5'AGCAGCCCTGAATACCCACGGCCGTATCCCAAA3'
Encode the nucleotide 610-642 of the SEQ ID NO:4 in the region containing CUBII structural domain
Cloning primer:
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 is the cloning primer of humanized antibody
SEQ ID NO:48 is the peptide bond of 9 amino acid
Expression vector:
SEQ ID NO:49 is MASP-2 mini gene insetion sequence
SEQ ID NO:50 is mouse MASP-2 cDNA
SEQ ID NO:51 is mouse MASP-2 albumen (w/ leader sequence)
SEQ ID NO:52 is mature mouse MASP-2 albumen
SEQ ID NO:53 is rat MASP-2 cDNA
SEQ ID NO:54 is rat MASP-2 albumen (w/ leader sequence)
SEQ ID NO:55 is mature rat MASP-2 albumen
SEQ ID NO:56-59 is the oligonucleotides for the direct mutagenesis of people MASP-2, and the people MASP-2 is used to generate people MASP-2A
SEQ ID NO:60-63 is the oligonucleotides for the direct mutagenesis of mouse MASP-2, and the mouse MASP-2 is used to generate mouse MASP-2A
SEQ ID NO:64-65 is the oligonucleotides for the direct mutagenesis of rat MASP-2, and the rat MASP-2 is used to produce Raw rat MASP-2A
SEQ ID NO:66 encodes 17D20_dc35VH21N11VL (OMS646) heavy chain variable region (VH) (no signal peptide) DNA
SEQ ID NO:67 17D20_dc35VH21N11VL (OMS646) heavy chain variable region (VH) polypeptide
SEQ ID NO:68 17N16mc heavy chain variable region (VH) polypeptide
SEQ ID NO:69 encodes the DNA of 17D20_dc35VH21N11VL (OMS646) light chain variable region (VL)
SEQ ID NO:70 17D20_dc35VH21N11VL (OMS646) light chain variable region (VL) polypeptide
SEQ ID NO:71 17N16_dc17N9 light chain variable region (VL) polypeptide
SEQ ID NO:72:SGMI-2L (overall length)
SEQ ID NO:73:SGMI-2M (medium clipped form)
SEQ ID NO:74:SGMI-2S (cutting back short-form)
SEQ ID NO:75: the mature polypeptide comprising 4 constant region of VH-M2ab6-SGMI-2-N and human IgG has hinge mutation
SEQ ID NO:76: the mature polypeptide comprising 4 constant region of VH-M2ab6-SGMI-2-C and human IgG has hinge mutation
SEQ ID NO:77: the mature polypeptide comprising VL-M2ab6-SGMI-2-N and people's Ig λ constant region
SEQ ID NO:78: the mature polypeptide comprising VL-M2ab6-SGMI-2-C and people's Ig λ constant region
SEQ ID NO:79: peptide linker (10aa)
SEQ ID NO:80: peptide linker (6aa)
SEQ ID NO:81: peptide linker (4aa)
SEQ ID NO:82: coding includes the polypeptide with hinge mutation of 4 constant region of VH-M2ab6-SGMI-2-N and human IgG Polynucleotides
SEQ ID NO:83: coding includes the more with hinge mutation of 4 constant region of VH-M2ab 6-SGMI-2-C and human IgG The polynucleotides of peptide
SEQ ID NO:84: the polynucleotides of polypeptide of the coding comprising VL-M2ab6-SGMI-2-N and people's Ig λ constant region
SEQ ID NO:85: the polynucleotides of polypeptide of the coding comprising VL-M2ab6-SGMI-2-C and people's Ig λ constant region
Detailed description
The present invention is based on the present inventor's it has surprisingly been found that is, the agglutinin Associated Serine for inhibiting mannosan to combine Protease -2 (MASP-2) (key regulator of the lectin pathway of complement system) is in various fibrotic disease animal models (the nephrology mould of unilateral ureteral obstruction (UUO) model, albumen overload model and adriamycin induction including kidney fibrosis Type) in significantly mitigate inflammation and fibrosis.Therefore, inventors have established that, the lectin pathway for inhibiting MASP-2 to mediate activates The effective treatment method for improving, treating or preventing kidney fibrosis, such as Tubulointerstitial inflammation and fibrosis is provided, but regardless of root How is this reason.As further described herein, the use of MASP-2 inhibiting antibody (OMS646) is suffering from immunoglobulin A It is effectively improved renal function in the people experimenter of nephrosis (IgAN) and membranous nephropathy (MN) and reduces corticosteroid demand.
I. it defines
Unless herein clearly stipulate that otherwise all terms used herein all have the those of ordinary skill institute such as field of the present invention The identical meanings of understanding.In order to be specifically used for describing related term of the invention in this specification and the appended claims, mention For following definition.
Terms used herein " MASP-2 dependent complement activation " includes the MASP-2- dependence activation of lectin pathway, It occurs in physiological conditions (that is, in Ca++In the presence of), result in lectin pathway C3 convertase C4b2a, and After C3 pyrolysis product C3b aggregation, subsequent C5 convertase C4b2a (C3b) n is formed, C5 convertase C4b2a (C3b) n is It is confirmed as mainly causing opsonic action.
Terms used herein " alternative route " refers to that the complement activation for example triggered by zymosan, the zymosan come From fungi and yeast cell wall, the lipopolysaccharides (LPS) of Gram-negative outer membrane and rabbit erythrocyte and from a variety of pure polysaccharide, Rabbit erythrocyte, virus, bacterium, animal tumor cell, helminth and damaged cell are traditionally considered always that origin is self-complementary Body factor C3 spontaneous hydrolysis generates caused by C3b.
Terms used herein " lectin pathway " refers to (including sweet by serum and non-serum Carbohydrate binding protein Revealing glycan binding lectin (MBL), CL-11 and fiber gelatinized protein, (H- fiber gelatinized protein, M- fiber gelatinized protein or L- are fine Dimension gelled protein) specific binding and the complement activation that occurs.
Terms used herein " classical pathway " refers to being triggered in conjunction with foreign particle by antibody and needs to combine knowledge The complement activation of other molecule C1q.
Terms used herein " MASP-2 inhibitor " refers in conjunction with MASP-2 or directly interacts and have with MASP-2 Effect inhibits any reagent of MASP-2 dependent complement activation, including anti-MASP-2 antibody and its MASP-2 binding fragment, natural It further include in agglutinin with synthetic peptide, small molecule, soluble M ASP-2 receptor, expression inhibiting agent and isolated natural inhibitor To combine other identification molecules (such as MBL, H- fiber gelatinized protein, M- fiber gelatinized protein or the fiber gelatinized egg of L- in approach It is white) and with MASP-2 competition peptide, but not including that with it is these other identification molecules in conjunction with antibody.For the method for the present invention MASP-2 inhibitor MASP-2 dependent complement activation can be made to be reduced more than 20%, all such as larger than 50%, all such as larger than 90%.? In one embodiment, MASP-2 inhibitor make MASP-2 dependent complement activation be reduced more than 90% (cause only 10% or Lower MASP-2 complement activation).
Terms used herein " fibrosis " refer to the formation or presence of the excessive connective tissue in organ or tissue.Fiber Changing, which can be used as, reacts the reparation or replacement of stimulation such as tissue damage or inflammation and occurs.The mark of fibrosis is excessive cell The generation of epimatrix.Lead to the connective tissue deposition of a part as agglutination, but this to the normal physiological reaction of damage Kind connective tissue deposition is sustainable and becomes morbid state, changes the structure and function of tissue.On a cellular level, epithelial cell and at Fibrocyte proliferation and it is divided into myofibroblast, causes contracted matrix, rigidity to increase, microvascular pressure is forced to make peace anoxic.
" treatment suffers from or the risky disease for occurring to be caused or aggravated by fibrosis and/or inflammation for terms used herein Or the fibrosis of the mammalian subject of illness " refers to reverse, mitigation, improvement or the fibre for inhibiting the mammalian subject Dimensionization.
Terms used herein " albuminuria " refer to the Urine proteins there are abnormal amount, such as in 24 collected from people experimenter More than the amount of 0.3g albumen, or the concentration risen in people experimenter more than 1g/ in hour urine.In some embodiments, suffer from There is the subject of albuminuria to refer in the urine egg that there is the amount more than 1.0g albumen from the twenty-four-hour urine liquid that people experimenter is collected It is white, such as suffer from the subject of immunoglobulin A (IgA) nephrosis.
Terms used herein " improve albuminuria " or " reducing albuminuria " refer to with before MASP-2 inhibitor for treating by The baseline twenty-four-hour urine protein secretion of examination person is compared, and reduces the twenty-four-hour urine protein secretion of the subject with albuminuria up at least 20%, for example, at least 30%, for example, at least 40%, for example, at least 50% or more.In one embodiment, side according to the present invention Method effectively reduces the albuminuria of people experimenter with MASP-2 inhibitor for treating, such as to realize that twenty-four-hour urine protein secretion is more than 20% is reduced such as twenty-four-hour urine protein secretion is more than 30% reduction such as twenty-four-hour urine protein secretion is more than 40% to subtract Few such as twenty-four-hour urine protein secretion is more than 50% reduction.
Terms used herein " antibody " include derived from generate antibody any mammal (such as mouse, rat, rabbit and Primate including people), or (or generated derived from hybridoma, phage selection, recombinant expression or transgenic animals Other methods of antibody or antibody fragment) and it is anti-with target polypeptides such as MASP-2 polypeptide or part thereof specific binding Body and its antibody fragment.It is not intended to about the mode of the source of antibody or its preparation (for example, being selected by hybridoma, bacteriophage Select, recombinantly express, transgenic animals, peptide synthesis etc.) term " antibody " is defined.Illustrative antibody includes Anti-TNF-α Body, monoclonal antibody and recombinant antibodies；Complete specific (pan-specific), multi-specificity antibody (such as bispecific antibody, Three-specific antibody)；Humanized antibody；Mouse antibody；Chimeric, mouse people, mouse primate, primate human monoclonal are anti- Body；And anti-idiotype, and can be any complete antibody or its segment.Terms used herein " antibody " not only included Whole polyclonal or monoclonal antibody, and including its segment (such as dAb, Fab, Fab', F (ab')2, Fv), it is single-stranded (ScFv), variant is synthesized, naturally occurring variant includes antibody moiety and the antigen-binding fragment with required specificity Fusion protein, humanized antibody, chimeric antibody, and comprising with required specificity antigen binding site or segment (epitope know Other site) immunoglobulin molecules any other configuration after modifying.
" monoclonal antibody " refers to homogeneous antibody group, wherein the monoclonal antibody is by participation selective binding epitope Amino acid (naturally occurring amino acid and non-naturally occurring amino acid) constitute.Monoclonal antibody is for target antigen High degree of specificity.Term " monoclonal antibody " not only include intact monoclonal antibodies and full length monoclonal antibodies, but also including Its segment (such as Fab, Fab', F (ab')2, Fv), single-stranded (ScFv), variant, the fusion protein comprising antigen-binding portion thereof, Humanized monoclonal antibodies, chimeric mAb, and include the antigen knot with required specificity and with epitope-binding ability Close any other modified configuration of the immunoglobulin molecules of segment (epitope recognition site).It is not intended to about antibody The mode (for example, passing through hybridoma, phage selection, recombinant expression, transgenic animals etc.) of source or its preparation is defined. The term includes above-mentioned intact immunoglobulins and the segment etc. under " antibody " defines.
Terms used herein " antibody fragment ", which refers to, derives from or is related to full length antibody, such as, such as anti-MASP-2 antibody A part generally comprises antigen binding domain or its variable region.The illustrative example of antibody fragment includes Fab, Fab', F (ab)2、F (ab')2With Fv segment, scFv segment, double antibody, linear antibodies, single-chain antibody molecules and formed by antibody fragment mostly special Property antibody.
" scFv " or " scFv " antibody fragment used herein includes the V of antibodyHAnd VLStructural domain, wherein these structures Domain is present on single polypeptide chain.Fv polypeptide generally further includes VHWith VLPeptide linker between structural domain, this enables scFv Antigen binding structure needed for being formed.
" chimeric antibody " used herein is containing the variable domains for deriving from non-human species (such as rodent) antibody With the recombinant protein of complementary determining region, and the rest part of antibody molecule derive from human antibody.
" humanized antibody " used herein is comprising meeting the specific complementary determining region from non-human immunoglobulin Minmal sequence chimeric antibody, which is implanted in human antibody framework.Humanized antibody is usually it In only complementary antibody determine area be inhuman source recombinant protein.
Terms used herein " mannan-binding lectin " (" MBL ") is equal to maltose-binding protein (" MBP ").
" membrane attack complex " used herein (" MAC ") refers to insertion and destroys 5 kinds of terminal complement component (C5b of film Combine C6, C7, C8 and C-9) compound (also referred to as C5b-9).
" subject " used herein includes all mammals, including but not limited to people, non-human primate, dog, Cat, horse, sheep, goat, ox, rabbit, pig and rodent.
The abbreviation of amino acid residue used herein is as follows: alanine (Ala；A), asparagine (Asn；N), aspartic acid (Asp；D), arginine (Arg；R), cysteine (Cys；C), glutamic acid (Glu；E), glutamine (Gln；Q), glycine (Gly；G), histidine (His；H), isoleucine (Ile；I), leucine (Leu；L), lysine (Lys；K), methionine (Met；M), phenylalanine (Phe；F), proline (Pro；P), serine (Ser；S), threonine (Thr；T), tryptophan (Trp； W), tyrosine (Tyr；) and alanine (Val Y；V).
From most wide meaning, naturally occurring amino acid can be grouped according to the chemical characteristic of each amino acid side chain. " hydrophobic " amino acid refers to Ile, Leu, Met, Phe, Trp, Tyr, Val, Ala, Cys or Pro." hydrophilic " amino acid refer to Gly, Asn, Gln, Ser, Thr, Asp, Glu, Lys, Arg or His.This grouping of amino acid can be further broken down as follows.It is " not charged Lotus it is hydrophilic " amino acid refers to Ser, Thr, Asn or Gln." acidity " amino acid refers to Glu or Asp." alkalinity " amino acid is Refer to Lys, Arg or His.
Terms used herein " conservative amino acid substitution " is illustrated by the substitution between amino acid in following every group: (1) glycine, alanine, valine, leucine and isoleucine；(2) phenylalanine, tyrosine and tryptophan；(3) serine And threonine；(4) aspartic acid and glutamic acid；(5) glutamine and asparagine；(6) lysine, arginine and group ammonia Acid.
Terms used herein " oligonucleotides " refers to ribonucleic acid (RNA) or DNA (DNA) or its analogies Oligomer or polymer.The term is also covered by (skeleton) covalent bond and tool between naturally occurring nucleotide, sugar and nucleosides There are those few nucleobases composed by the oligonucleotides of non-naturally occurring modification.
" epitope " used herein refers to the site on the albumen (for example, people MASP-2 albumen) combined by antibody." overlapping Epitope " includes at least one (for example, two, three, four, five or six) common amino acid residue, including linear and nonlinear table Position.
Terms used herein " polypeptide ", " peptide " and " albumen " may be used interchangeably, and refer to that any peptide of amino acid connects Chain link, no matter length or posttranslational modification.MASP-2 albumen as described herein can contain or can be wild-type protein, or Can be have be no more than 50 (for example, be no more than one, two, three, four, five, six, seven, eight, nine, ten, 12,15,20,25,30, 35,40 or 50) conserved amino acid replace variant.Conservative substitution generally includes the substitution in following group: glycine and the third ammonia Acid；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 amino acid sequence of people MASP-2 albumen can with shown in SEQ ID NO:5 People's MASP-2 albumen of amino acid sequence be or be greater than 70 (for example, 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) % is identical.
In some embodiments, peptide fragment can be at least six (for example, at least 7,8,9,10,11,12,13,14, 15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、 40、41、42、43、44、45、46、47、48、49、50、55、60、65、70、75、80、85、90、95、100、110、120、130、 140,150,160,170,180,190,200,250,300,350,400,450,500 or 600 or more) amino acid residue is long It spends (for example, at least six continuous amino acid residue of SEQ ID NO:5).In some embodiments, people MASP-2 albumen is anti- Former peptide segment be less than 500 (such as less than 450,400,350,325,300,275,250,225,200,190,180,170, 160、150、140、130、120、110、100、95、90、85、80、75、70、65、60、55、50、49、48、47、46、45、44、 43、42、41、40、39、38、37、36、35、34、33、32、31、30、29、28、27、26、25、24、23、22、21、20、19、 18,17,16,15,14,13,12,11,10,9,8,7 or 6) length of amino acid residue is (for example, SEQ ID NOS:5's appoints 500 continuous amino acid residues are less than in one).
Percentage (%) amino acid sequence identity is defined as in aligned sequences and after introducing notch, in candidate sequence, If desired, reaching the percentage of the amino acid identical with the amino acid in reference sequences of maximum percentage sequence identity. For determining that the comparing for purpose of Percent sequence identity can be in a manner of various in art technology, for example, using public Computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software for opening acquisition comes It realizes.For measuring the suitable parameter compared, including realizing the high specific in sequence to be compared to required any algorithm It can be determined by known method.
Terms used herein "about" or "approximately" indicated when before numerical value the numerical value add deduct 10% range.
II. summary of the invention
As described herein, the present inventor has identified center of the lectin pathway in the starting and progression of disease of renal tubule pathology Effect, to imply lectin pathway activation in various kidney diaseases, including IgA nephrosis, C3 glomerulopathy and other glomerulonephritis Key effect in the Pathological Physiology of scorching (glomerulonephritide).As further described herein, human hair of the present invention The agglutinin associated serine protease -2 (MASP-2) for now mannosan being inhibited to combine be (lectin pathway of complement system Important regulator) in various fibrotic disease animal models (unilateral ureteral obstruction (UUO) model, egg including kidney fibrosis White overload model and adriamycin induction nephrology model) in substantially reduce inflammation and fibrosis.Therefore, the present inventor has demonstrate,proved Real, the lectin pathway activation for inhibiting MASP-2 to mediate, which provides, to be improved, it is fine to treat or prevent kidney fibrosis, such as Tubulointerstitial Effective treatment method of dimensionization, but regardless of basic reason how.
Agglutinin (MBP, M- fiber gelatinized protein, H- fiber gelatinized protein and L- fiber gelatinized protein and CL-11) is to draw The specific recognition molecules for sending out complement system congenital, the system include agglutinin starting approach and enlarged distal end complement effect point The associated ends approach of the activation of the agglutinin starting of son amplifies loop.C1q is the specificity knowledge for causing acquired complement system Other molecule, the system include the associated ends of the activation of the C1q starting of classical starting approach and enlarged distal end complement effector molecule Approach amplifies loop.The two main complement activation systems are referred to as Lectin-dependent complement system and C1q by us Dependent complement system.
Other than the basic role in immune defense, complement system is to cause the original of the tissue damage of many clinical diseases Cause.Therefore, there is an urgent need to develop treat upper effective complement inhibitor to inhibit these side effects.If recognizing that it may press down The MASP-2 approach of lectin-mediated processed and keep classical pathway complete, then just it will be appreciated that it is only special that we are in demand Property inhibit cause particular pathologies complement activation system and not exclusively stop complement immune defense ability.For example, wherein For complement activation mainly in the morbid state by the mediation of Lectin-dependent complement system, it will be advantageous that only specificity, which inhibits the system, 's.This will keep the integrality of C1q dependent complement activation system to handle immune complex processing and help host defense Infection.
In the research and development that specificity inhibits the therapeutic agent of Lectin-dependent complement system, preferably as the egg of target Bai Chengfen is MASP-2.Lectin-dependent complement system all known protein ingredients (MBL, H- fiber gelatinized protein, M- fiber gelatinized protein, L- fiber gelatinized protein, MASP-2, C2-C9, factor B, factor D and properdin) in, only MASP-2 It is that Lectin-dependent complement system institute is exclusive and be necessary to this system works.Agglutinin (MBL, H- viscose Coagulated protein, M- fiber gelatinized protein, L- fiber gelatinized protein and CL-11) it is also exclusive in Lectin-dependent complement system Ingredient.However, any forfeiture of these agglutinin ingredients might not inhibit system activation, this is because agglutinin redundancy institute It causes.In order to ensure a suppression of Lectin-dependent complement activation system, inhibit all 5 kinds of agglutinins that may be necessary.In addition, by In it is known that MBL and fiber gelatinized protein have the opsonic activity independent of complement, therefore lectins function is inhibited to will lead to Lose this advantageous anti-infectious host defense mechanism.On the contrary, if MASP-2 is suppression target, it is this independent of The agglutinin opsonic activity of complement can keep complete.MASP-2 is as the therapeutic target for inhibiting Lectin-dependent complement activation system Additional benefit be that the plasma concentration of MASP-2 is (about 500 ng/ml) minimum in all complement proteins；Therefore, to obtain Complete inhibition may enough relatively low intensity of high-affinity MASP-2 inhibitor (Moller-Kristensen, M., etc. People,J. Immunol Methods 282:159-167, 2003)。
As described in embodiment hereof 14, determined in the animal model (unilateral ureteral obstruction UUO) of fibrosis kidney diaseases Compared with wild-type control animals, there is no the mouse of MASP-2 gene (MASP-2-/-) to show significant less kidney diaseases, such as It is shown by the histological markers such as collagen deposition (1/3 is reduced) of inflammatory cell infiltration (75% is reduced) and fibrosis.Such as Embodiment 15 is coagulated it further shows that compared with the wild-type mice that Isotype control antibodies are treated with selectively blocking Collect plain approach and retain the wild-type mice of the complete anti-MASP-2 monoclonal antibody systemic treatment of classical pathway simultaneously and is protected From kidney fibrosis.These results demonstrate that lectin pathway is the key contributing factor of kidney diaseases, and further confirm to block solidifying MASP-2 inhibitor, such as MASP-2 antibody for collecting plain approach, are effective as antifibrotic agents.As embodiment 16 is further Display, proteinuric nephropathies occur with the wild-type mice of bovine serum albumin(BSA) (BSA) processing in albumen overload model, and use MASP-2-/- mouse of the BSA processing of phase same level has reduced injury of kidney.As described in Example 17, it is overloaded in albumen Retained classical pathway simultaneously with selectively blocked lectin element approach in model completely anti-MASP-2 monoclonal antibody is systemic The wild-type mice for the treatment of is protected from injury of kidney.As described in Example 18, it is induced compared with wild-type mice in adriamycin Kidney fibrosis nephrology model in MASP-2-/- mouse show less nephritis disease and Tubulointerstitial damage.As implemented Described in example 19, in the 2 phase open label kidneys test of progress, with the patient with IgA nephrosis of anti-MASP-2 Antybody therapy Be shown in Analysis of urine albumin in entire test: the clinically significant and statistically significant of creatinine ratio (uACR) subtracts Reduction of the few and twenty-four-hour urine protein level from baseline to treatment end.As embodiment 19 further describes, identical 2 In the test of phase kidney, uACR is reduced during being also shown in treatment with the patient with membranous nephropathy of anti-MASP-2 Antybody therapy.Such as Described in embodiment 20, in the 2 phase open label kidneys test of progress, lupus nephritis is suffered from 5 of anti-MASP-2 Antybody therapy (LN) 4 in patient the clinically significant of twenty-four-hour urine protein level when demonstrating from baseline to treatment end subtract It is few.
According to foregoing teachings, the present invention relates to MASP-2 inhibitor, such as MASP-2 inhibiting antibodies as anti-fibrosis The purposes of agent, MASP-2 inhibitor are used to prepare the purposes of the drug of the treatment fibrosis patient's condition, and prevent, treat, mitigate or inverse The method for turning the fibrosis patient's condition of people experimenter in need, the method includes giving the MASP-2 of patient effective amounts suppression Preparation (for example, anti-MASP-2 antibody).
Method of the invention can be used for preventing, treating, mitigate or reverse to suffer from and caused or aggravated by fibrosis and/or inflammation Any disease or illness people experimenter the fibrosis patient's condition, including kidney is (for example, chronic kidney diseases, IgA nephrosis, C3 kidney are small Ball disease and other glomerulonephritis), lung (for example, idiopathic pulmonary fibrosis, cystic fibrosis, bronchiectasis), liver (for example, Cirrhosis, non-alcohol fatty liver), heart is (for example, myocardial infarction, Atrial fibrosis, valvular fibrosis, the internal membrane of heart heart Myofibrosis), brain (for example, apoplexy), skin (for example, excessive wound repair, chorionitis, Sjogren's syndrome, cheloid), blood vessel (for example, atherosclerotic vascular diseases), intestines (for example, Crohn disease), eye are (for example, anterior subcapsular cataract, rear capsule are newborn It is turbid), soft tissue structure's (for example, adhesive capsulitis, dupuytren's contracture, myelofibrosis) of muscle skeleton, genitals The disease of official's (for example, mullerianosis, Perun Nie Shi disease) and some infectious diseases (for example, α is viral, hepatitis C and Hepatitis B).
III. effect of the MASP-2 in the disease and the patient's condition for being caused or being aggravated by fibrosis
Fibrosis is to be formed in organ or tissue or there are excessive connective tissue, be normally in response to damage or damage.Fibrosis Mark be damage after generate excessive cell epimatrix.In kidney, the feature of fibrosis is in the substantial progressive nocuousness knot of kidney Tissue deposition is formed, decreased renal function is inevitably led to, independently of the Primary renal disease for causing initial injury of kidney.It is so-called Epithelium between qualitative change (EMT) (a kind of variation of cell characteristic), wherein Tubular epithelial cell is converted into interstitial into fiber finer Born of the same parents constitute the main mechanism of kidney fibrosis.Fibrosis influences nearly all tissue and tract, and can be used as to stimulation for example Tissue damage or the reparation of inflammation or replacement are reacted and are occurred.Lead to the deposition of connective tissue to the normal physiological reaction of damage, But if the reaction becomes morbid state, cicatrization connective tissue replaces the structure and function that height noble cells changes tissue.? On cellular level, epithelial cell and fibroblast proliferation and it is divided into myofibroblast, contracted matrix, rigidity is caused to increase Add, microvascular pressure is forced to make peace anoxic.There is no an effective therapy or therapeutic agent for fibrosis at present, but zooscopy and the faithful record People's report show fibrosed tissue damage it is reversible (Tampe and Zeisberg,Nat Rev Nephrol, Vol 10:226- 237,2014).
Many diseases lead to fibrosis, cause progressive organ failure, including kidney (for example, chronic kidney diseases, IgA kidney Disease, C3 glomerulopathy and other glomerulonephritis), lung (for example, idiopathic pulmonary fibrosis, cystic fibrosis, bronchiectasis), Liver (for example, cirrhosis, non-alcohol fatty liver), heart (for example, myocardial infarction, Atrial fibrosis, valvular fibrosis, Endomyocardial fibrosis), brain (for example, apoplexy), skin is (for example, excessive wound repair, chorionitis, Sjogren's syndrome, scar Tumor), blood vessel (for example, atherosclerotic vascular diseases), intestines (for example, Crohn disease), eye (for example, anterior subcapsular cataract, Capsule milkiness afterwards), soft tissue structure's (for example, adhesive capsulitis, dupuytren's contracture, myelofibrosis) of muscle skeleton, The disease of reproductive organs (for example, mullerianosis, Perun Nie Shi disease) and some infectious diseases are (for example, α is viral, the third type Hepatitis, hepatitis B etc.).
When fibrosis occurs in many tissues and disease, to its pathology, there are common molecules and cell mechanism.It is logical Cross fibroblastic extrtacellular matrix deposition concomitant immunity cellular infiltration, mainly monocyte (referring to Wynn T.,Nat Rev Immunol4 (8): 583-594,2004 are incorporated herein by reference).Steady inflammatory reaction leads to growth factor (TGF-β, VEGF, hepatocyte growth factor, Connective Tissue Growth Factor), cell factor and hormone (Endothelin, IL-4, IL-6, IL-13, chemotactic factor (CF)), degrading enzyme (pancreatopeptidase E, matrix metalloproteinase, cathepsin) and extracellular matrix protein (glue Original, fibronectin, integrin) expression.
In addition, complement system is activated in many fibrotic diseases.Complement component, including membrane attack complex, exist It is identified in many fibrosed tissue samples.For example, the component of lectin pathway kidney diaseases (Satomura et al.,Nephron. 92(3):702-4 (2002);Sato et al.,Lupus 20(13):1378-86 (2011);Liu et al. people,Clin Exp Immunol,174(1):152-60 (2013))；Liver disease (Rensen et al.,Hepatology 50(6): 1809-17 (2009))；With lung disease (Olesen et al.,Clin Immunol121 (3): 324-31 (2006)) fiber Change and is found in damage.
Have determined that excessive (overshooting) complement activation is that immune complex mediates and antibody dependent/non-dependent kidney The key contributing factor of bead ephritis.However, strong trail of evidence confirms, uncontrolled complement is living in non-renal glomerular disease Change it is in situ inherently participate in TI fibrosis Pathological Physiology progress (Quigg R.J,J Immunol171:3319-3324, 2003, Naik A. et al.,Semin Nephrol33:575-585,2013, Mathern D.R. et al.,Clin J Am Soc Nephrol10:P1636-1650,2015).By the strong pro-inflammatory signals of local complement's activation triggers can by filter into Enter proximal tubule and subsequently enters the complement component of interstitial space, or the benefit by tubule or other residential cells and infiltrating cells The abnormal synthesis of body component, or changed by the expression of Complement Regulatory Protein on nephrocyte, or adjust function in component in complement Mutation lack or lose or obtain and start (Mathern D.R. et al.,Clin J Am Soc Nephrol 10:P1636- 1650,2015, Sheerin N.S. et al.,FASEB J22: 1065-1072,2008).Such as in mouse, complement is adjusted PROTEIN C R1 related gene/albumen y (Crry) defect leads to Tubulointerstitial (TI) complement activation, wherein in people's TI disease See the typical then inflammation of damage and fibrosis (Naik A. et al.,Semin Nephrol33:575-585,2013, Bao L. et al.,J Am Soc Nephrol18:811-822,2007).Tubular epithelial cell, which is exposed to anaphylatoxin C3a, to be caused Skin between qualitative change (Tsang Z. et al.,J Am Soc Nephrol20:593-603,2009).It has recently been shown that, by independent C3a receptor block C3a signal transduction albuminuria and non-protein urine animal in mitigate kidney TI fibrosis (Tsang Z. et al.,J Am Soc Nephrol20:593-603,2009, Bao L. et al.,Kidney Int80:524-534,2011).
As described herein, the present inventor has identified lectin pathway in the starting and progression of disease of renal tubule pathology Central role, so that implying that lectin pathway is activated in various kidney diaseases includes IgA nephrosis, C3 glomerulopathy and other glomerulus Ephritis (Endo M. et al.,Nephrol Dialysis Transplant13:1984-1990,1998;Hisano S. etc. People,Am J Kidney Dis45:295-302,2005;Roos A. et al.,J Am Soc Nephrol 17: 1724- 1734,2006;Liu L.L. et al.,Clin Exp. Immunol174:152-160,2013;Lhotta K. et al.,Nephrol Dialysis Transplant14:881-886,1999;Pickering et al.,Kidney International 84:1079-1089,2013), diabetic nephropathy (Hovind P. et al.,Diabetes 54:1523- 1527,2005), ischemical reperfusion injury (Asgari E. et al.,FASEBJ 28:3996-4003,2014) and graft rejection (Berger S.P. et al.,Am J Transplant5:1361-1366,2005) the key effect in Pathological Physiology.
As further described herein, inventors have established that MASP-2 presses down in the mouse model of Tubulointerstitial disease System reduces inflammation and fibrosis.Therefore, it is contemplated that MASP-2 inhibitor can be used for treating kidney fibrosis, including Tubulointerstitial inflammation With fibrosis, albuminuria, IgA nephrosis, C3 glomerulopathy and other glomerulonephritis and renal ischemic reperfusion injury.
Kidney diaseases and illness
According to National Kidney Foundation, 2.6 thousand ten thousand U.S. adults suffer from chronic kidney diseases (CKD).Mostly Number patient has the progressive disease for leading to kidney failure, needs to be moved with RBC acceptor garland rate stimulating drug, dialysis or kidney to survive Plant treatment.There are several drugs, the cardinal symptom hypertension of CKD can be treated, but currently without the medicine for solving its basic reason Object.
Studies have shown that progressive injury of kidney is caused by the capillary hypertension in the substructure (the referred to as nephron) of kidney (Whitworth J.A.,Annals Acad of Med,vol 34(1):2005).Because the nephron (filter unit of kidney) exists Should during it is impaired or be destroyed, inflammation and organize cicatrization, with the non-functional cicatricial tissue replacement nephron.As a result It is that the ability of kidney filtering blood declines at any time.This is referred to as kidney fibrosis, is the common pathway of progressive kidney diaseases.No How is the property of pipe initial insult, it is believed that kidney fibrosis is common final pathway of the kidney diaseases to end-stage renal failure.Kidney is fine The improvement of dimensionization can pass through following one or more determinations: assessment interstitial volume, collagen iv deposition and/or connective tissue growth MRNA level in-site.Compounds and methods for as described herein can be used for treating kidney fibrosis.
Kidney fibrosis and inflammation are the main features of the end-stage renal disease of the substantially any cause of disease (referring to Boor et al., Boor P. et al.,J of Am Soc of Nephrology18:1508-1515,2007 and Chevalier et al.,Kidney International75:1145-1152,2009).Kidney failure can be caused by one group of heterologous illness.Progressive renal dysfunction Lead to albuminuria and renal insufficiency.As patient health deteriorates, dialysis can only to prevent injury of kidney and prevention multisystem failure It is required.Kidney failure and renal insufficiency can be completely or almost completely at any time to end-stage renal disease (ESRD) Renal function permanent loss.According to the form of kidney diaseases, renal function can be lost within about a couple of days or several weeks, or can be in decades Period is slow and runs down.Once patient progress needs to dialyse (half dialysis or peritoneal dialysis) to ESRD to prevent death.Suffer from Person must keep the Dialysis scheme of a certain form, or must obtain kidney transplant.
In the fibrosis lesion of kidney diaseases it has been found that lectin pathway component (Satomura et al.,Nephron. 92 (3):702-4 (2002);Sato et al.,Lupus 20(13):1378-86 (2011);Liu et al. people,Clin Exp Immunol, 174 (1): 152-60 (2013)).In IgA nephrosis, compared with the patient of no MBL deposition, there is glomerulus The patient of MBL deposition has more serious albuminuria, and kidney function can be reduced, lower level seralbumin, more serious tissue Learn and bigger hypertension (Liu et al. people,Clin Exp Immunol. 2013 Oct;174(1):152-60).With systemic lupus erythematosus kidney Scorching (Sato et al., Lupus, 20 (13): 1378-86,2011) and chronic renal failure (Satomura et al.,Nephron 92 (3): 702-4,2002 patient) also has the MBL and lectin pathway activity of increase level.
Also it has proven convenient that the non-protein in the damage of primary Tubulointerstitial urinates model (i.e. unilateral ureteral obstruction (UUO)) In, C5 deficiency cause the main component of kidney fibrosis significantly improve (Boor P. et al.,J of Am Soc of Nephrology 18:1508-1515,2007).Also it has been reported that C3 gene expression increases in wild-type mice after UUO, and it is small with wild type Mouse is compared, and collagen deposition substantially reduces in C3-/- mouse after UUO, shows effect of the complement activation in kidney fibrosis (Fearn et al.,Mol Immunol48:1666-1733,2011: abstract).However, being described herein by the present inventor Discovery before, the complement component for participating in kidney fibrosis is not able adequately determines also.As described in embodiment hereof 14-17, the present inventor It unexpectedly determines, MASP-2 defect or MASP-2 selectively are blocked lectin pathway while to retain classical pathway complete Inhibiting antibody block, clearly protect mice against kidney fibrosis in the various animal models of kidney diaseases.
Therefore, in certain embodiments, present disclosure provides to inhibit to suffer from and is caused or added by fibrosis and/or inflammation The method of the kidney fibrosis of the subject of the kidney diaseases or illness of weight, including give subject MASP-2 inhibitor in need, Such as anti-MASP-2 antibody.This method includes giving kidney diaseases or illness with being caused or being aggravated by fibrosis and/or inflammation Subject include effectively inhibit kidney fibrosis amount MASP-2 inhibitor composition.
MASP-2 inhibition composition can administer locally to the region of fibrosis, such as during operation or locally injecting, directly It connects or distal end is for example by conduit, pass through topical composition.Alternatively, MASP-2 inhibitor can whole body give subject, example Such as by intra-arterial, intravenous, intramuscular, sucking, intranasal, it is subcutaneous or it is other parenterally give, or for it is non-peptide energy medicament possibility It is given by oral.Giving may be repeated, and be determined by doctor, until the patient's condition has subsided or is controlled.
In certain embodiments, MASP-2 inhibitor (for example, anti-MASP-2 antibody) and it is suitable for potential kidney diaseases Or one or more medicaments or the therapeutic modality combination of the patient's condition are given.In certain embodiments, MASP-2 inhibitor (for example, Anti- MASP-2 antibody) it combines and gives with dialysis or plasmaphoresis scheme.In certain embodiments, MASP-2 inhibitor (example Such as, anti-MASP-2 antibody) for reducing the frequency for needing dialysis or plasmaphoresis.In certain other embodiments, MASP-2 inhibitor (for example, anti-MASP-2 antibody) is applied in combination with kidney transplant.In certain other embodiments, waiting MASP-2 inhibitor (for example, anti-MASP-2 antibody) is for controlling renal insufficiency and prevention renal function in the patient of kidney transplant Further decline.
For example, in certain embodiments, anti-MASP-2 antibody is for inhibiting kidney fibrosis, thus treatment or improvement kidney Bead disease such as focal segmental glomerulosclerosis and nephrotic syndrome (symptom including treating or improving disease).It can control The illustrative symptom treated includes but is not limited to hypertension, albuminuria, hyperlipidemia, blood urine and hypercholesterolemia.Some In embodiment, MASP-2 inhibitor inhibits Tubulointerstitial fibrosis.In certain embodiments, treatment includes improving kidney function Albuminuria can, be reduced, hypertension is improved and/or reduces kidney fibrosis.In certain embodiments, treatment include (i) delay or Progress of preventing is to renal insufficiency, kidney failure or ESRD；(ii) postpone, reduce or prevent the demand to dialysis；Or (iii) prolongs Demand slow or that prevention is to kidney transplant.
The certain specific kidney diaseases and illness for being caused or being aggravated by fibrosis and/or inflammation are described below.
In certain embodiments, the kidney diaseases for being caused or being aggravated by fibrosis and/or inflammation be renal glomerular disease for example Focal segmental glomerulosclerosis (FSGS).Renal glomerular disease damages glomerulus, makes albumen and red blood cell leaks to urine sometimes In.Sometimes, renal glomerular disease also interferes the waste scavenging rate by kidney, therefore they start to accumulate in blood.Glomerulus disease The symptom of disease includes albuminuria, blood urine, glomerular filtration rate reduction, hypoproteinemia and oedema.Many different diseases can Lead to renal glomerular disease.It may be infection or the drug toxic to kidney direct result or it can be by influencing entire body Disease cause, such as hypertension, diabetes or lupus.FSGS is a kind of specific renal glomerular disease, but even this feature There can also be many reasons for the particular condition of cicatrization in kidney.FSGS patient is usually to advanced stage kidney disease in 5-20 Disease, but the patient of the disease with invasion form is interior to ESRD in 2-3.
In certain embodiments, the kidney diaseases for being caused or being aggravated by fibrosis and/or inflammation are diabetic nephropathy (DN), It is significant unsatisfied medical need field.Diabetic nephropathy is the kidney diaseases generated as the complication of diabetes or damage Wound.The patient's condition is aggravated by hypertension, elevated blood glucose levels and high cholesterol and lipid level.The precise reason why of diabetic nephropathy is not Know.However, without being bound by theory thinking that uncontrolled hyperglycemia leads to the fibrosis that injury of kidney, such as tissue occurs And cicatrization.In people, DN is shown as by albuminuria, the glomerular filtration rate being gradually reduced (GFR) and cardiovascular disease Risk increase constitute clinical syndrome.Diabetes albuminuria is related with characteristic Histopathological Characteristics occur, including Glomerular basement membrane (GBM) is thickened to be expanded with glomerular mesangium.As albuminuria progress and renal insufficiency are ensued, kidney Bead sclerosis, arteriole hyalinosis and Tubulointerstitial fibrosis occur.
Therefore, in one embodiment, present disclosure provides the method for treating diabetic nephropathy, including gives A effective amount of MASP-2 inhibitor of subject (for example, MASP-2 inhibiting antibody) in need.In certain embodiments, it controls Treating includes the one or more symptoms for mitigating diabetic nephropathy.In certain embodiments, treatment includes mitigating, postpone or eliminating Dialysis needs.In certain embodiments, treatment includes mitigating, postpone or eliminating kidney transplant to need.In certain embodiments, Treatment includes delay, prevention or reverting diabetes nephrosis to kidney failure or end-stage renal disease.
In certain embodiments, the kidney diaseases for being caused or being aggravated by fibrosis and/or inflammation are lupus nephritis.It is as follows In greater detail, lupus nephritis is the severe complication of systemic loupus erythematosus (SLE) to text, is available MASP-2 suppression Another example of the kidney fibrosis of preparation (for example, anti-MASP-2 antibody) treatment.
Therefore, in one embodiment, present disclosure, which is provided, is caused for inhibiting to suffer from by fibrosis and/or inflammation Or the method for the kidney fibrosis of the subject of the kidney diaseases or illness aggravated, including give a effective amount of MASP-2 inhibitor (example Such as, MASP-2 inhibiting antibody).In some embodiments, it is selected from by the kidney diaseases or illness of fibrosis and/or increased inflammation Chronic renal failure, renal glomerular disease (for example, focal segmental glomerulosclerosis), compound illness (example is immunized in chronic kidney diseases Such as, IgA nephrosis, membranous nephropathy), lupus nephritis, nephrotic syndrome, diabetic nephropathy, Tubulointerstitial damage and C3 kidney it is small Ball disease or other types of glomerulonephritis.
The method of prevention or the treatment injury of kidney as caused by drug-induced toxicity
Another reason for injury of kidney includes drug-induced toxicity.For example, the nephrotoxin can cause Tubular epithelial cell directly Toxicity.As described herein, inventors have established that, MASP-2 deficient mice is protected against the nephrosis of adriamycin induction.
The nephrotoxin includes but is not limited to therapeutic agent (for example, cis-platinum, gentamicin, cefaloridine, cyclosporin, both sexes Mycin, adriamycin), radiocontrast's dyestuff, insecticide (for example, paraquat) and environmental contaminants are (for example, trichloro ethylene and two Chloroacetylene).Other examples include amino nucleoside puromycin (PAN)；Glucosaminide such as gentamicin；Cephalosporin is for example Cefaloridine；Calcineurin inhibitor such as tacrolimus or sirolimus.Drug-induced renal toxicity can also be by non-steroidal Anti-inflammatory agent, antiretroviral agent, anti-cytokine, immunosuppressor, oncologic or Vel-Tyr-Pro-Trp-Thr-Gln-Arg-Phe cause.Drug lures The renal toxicity led can further by fenoprofen abuse, Ciprofloxacin, clopidogrel, cocaine, cox-2 inhibitor, diuretics, Phosphonic acid, gold, ifosfamide, immunoglobulin, Chinese herb, interferon, lithium, mannitol, Mesalazine, mitomycin, Nitroso ureas, penicillamine, penicillin, pentamidine, quinine, rifampin, streptozotocin, sulfanilamide (SN), ticlopidine, triamterene, the third penta Acid, Doxorubicin, glycerol, cidofovir, tobramycin, neomycinsulphate, Sulfomyxin, vancomycin, amikacin, cephalo Thiophene oxime, cis-platinum, aciclovir, lithium, interleukin 2, cyclosporin or indinavir cause.
Therefore, in one embodiment, risky generation or subject suffering from kidney injury may receive a kind of or more Kind has the therapeutic agent of Nephrotoxicity.These subjects can give of the invention before the therapeutic agent or simultaneously MASP-2 inhibitor.Equally, MASP-2 inhibitor can be after therapeutic agent to the possibility for being treated or being reduced generation renal toxicity Property.
Disease relevant to albuminuria and the patient's condition
It has been determined that the impaired progressive loss for leading to albuminuria and accelerating the nephron of the glomerular filtration of albumen, occurs in institute Have in chronic kidney diseases (Remuzzi and Bertani,New Eng. J MedVol 339 (20): 1448-1456,1998). For example, in Eddy et al.,Am J Pathol135:719-33, described in 1989 research in, the glomerular filtration of albumin Chromic fibrous damage and cicatrization always occur later.It such as further describes, has by egg in Eddy et al., 1989 It observes that Complement C_3 deposits on the chamber surface of proximal tubule in the rat of nephrosis caused by white-overload, shows by glomerulus mistake The complement system component of filter can cause chromic fibrous damage.It has been confirmed that complement, which exhausts or lack C6, improves albuminuria animal model In Tubulointerstitial damage, such as Pathology of Mesangial Proliferative Glomerulonephritis, mRNA IN ADRIAMYCIN NEPHROPATHY, 5/6 nephrectomy and aminonucleoside Puromycin aminonucleoside nephrosis (Boor et al.,J of Am Soc of Nephrology: JASN 18:1508-1515,2007).The mankind Studies have shown that albuminuria is the independent prediction object of chronic kidney diseases progress and albuminuria reduction is that kidney is protective (Ruggenenti P. et al.,J Am Soc Nephrol23:1917-1928,2012).
Therefore, in one embodiment, present disclosure is provided with disease related with albuminuria or the patient's condition Prevent in subject or mitigate albuminuria and/or prevention or mitigate the method for injury of kidney, including give effectively reduce or prevent by The MASP-2 inhibitor (for example, MASP-2 inhibiting antibody) of the amount of the albuminuria of examination person.In some embodiments, with albumen It urinates related disease or the patient's condition is selected from nephrotic syndrome, pre-eclampsia, eclampsia, the toxic lesion of kidney, amyloidosis, collagen blood Pipe disease (for example, systemic loupus erythematosus), dehydration, renal glomerular disease are (for example, membranous glomerulonephritis, Focal segmental Glomerulonephritis, minimal change disease, lipoid nephrosis), strenuous exercise, stress, it is benign orthostatic position (position) albuminuria, focal Property segmental glomerulosclerosis, IgA nephrosis (that is, Bei Geershi is sick), IgM nephrosis, membranoprolifer ative glomerulonephritis, film property kidney Disease, minimal change disease, sarcoidosis, Alport's syndrome, diabetes (diabetic nephropathy), drug-induced toxicity (example Such as, NSAIDS, nicotine, penicillamine, lithium carbonate, gold and other heavy metals, Vel-Tyr-Pro-Trp-Thr-Gln-Arg-Phe, antibiotic or opiate (example Such as, heroin))；Fabry disease, infection are (for example, HIV, syphilis, first, second or hepatitis C, streptococcus postoperative infection, urinary tract blood Fluke disease)；Amino acid uria, Fanconi syndrome, hypertensive nephrosclerosis, interstitial nephritis, drepanocytosis, hemoglobin Urine, Huppert's disease, myoglobinuria, organ rejection's (for example, renal transplant rejection), Ebola hemorrhagic fever, chatelain's syndrome, Familial Mediterranean fever, HELLP syndrome, systemic loupus erythematosus, Wei Genashi granulomatosis, rheumatoid arthritis, sugar Former 1 type of thesaurismosis, Goodpasture's syndrome, anaphylactoid purpura, the urinary tract infections for having spread to kidney, Siogren are comprehensive Seek peace infection after glomerulonephritis.
Liver diseases
Hepatic fibrosis-renal tubular ectasia syndrome, also known as liver fibrosis are gathered in liver by cicatricial tissue and are caused, and are most types of livers The feature of dirty disease.Healthy liver tissue is replaced the ability that damage liver correctly functions by cicatricial tissue.If causing scar The patient's condition that trace is formed is not treated, and hepatic fibrosis-renal tubular ectasia syndrome can develop as cirrhosis and complete hepatic failure, a kind of patient's condition of life-threatening. The main reason for hepatic fibrosis-renal tubular ectasia syndrome is alcohol abuse, chronic hcv infection, nonalcoholic fatty liver disease and liver poison Property (for example, hepatic injury drug-induced as caused by paracetamol or other medicines).
In the fibrotic lesions of liver diseases have been found that lectin pathway component (Rensen et al.,Hepatology50(6): 1809-17 (2009)).For example, at nonalcoholic fatty liver disease (also known as fatty liver) In, there are the generalized activations of complement system protein, and its expression it is related with disease severity (Rensen et al.,Hepatology50 (6): 1809-17 (2009), wherein other than C3 and C9 is deposited, it was found that MBL accumulation, it was confirmed that The activation of lectin pathway.
Therefore, in certain embodiments, present disclosure is provided with by fibrosis and/or inflammation being caused or aggravated Liver diseases or illness subject in inhibit the method for liver fibrosis, including give subject MASP-2 in need and inhibit Agent, such as MASP-2 inhibiting antibody.This method includes giving the liver disease with being caused or being aggravated by fibrosis and/or inflammation The subject of disease or illness includes the composition for effectively inhibiting the MASP-2 inhibitor of amount of liver fibrosis.
MASP-2 inhibition composition can administer locally to the region of fibrosis, such as during operation or locally injecting, directly It connects or distal end (such as passing through conduit) passes through topical composition.Alternatively, MASP-2 inhibitor can whole body give subject, example Such as by intra-arterial, intravenous, intramuscular, sucking, intranasal, it is subcutaneous or it is other parenterally give, or for it is non-peptide energy drug possibility It is given by oral.Giving may be repeated, and be determined by doctor, until the patient's condition has been eliminated or controls.
In certain embodiments, MASP-2 inhibitor (for example, MASP-2 inhibiting antibody) and it is suitable for potential liver The combination of the one or more medicaments or form of therapy of dirty disease or the patient's condition is given.
In some embodiments, the liver diseases or illness for being caused or being aggravated by fibrosis and/or inflammation are selected from: liver is hard Change, non-alcohol fatty liver (steatohepatitis), the hepatic fibrosis-renal tubular ectasia syndrome secondary to alcohol abuse, secondary to acute or chronic Hepatic fibrosis-renal tubular ectasia syndrome, biliary diseases and the toxicity hepatic injury of hepatitis are (for example, the drug due to caused by paracetamol or other medicines Hepatotoxicity wind agitation caused by the hepatic injury of induction).
Lung disease
Pulmonary fibrosis is formation or development of the excess fibrous connective tissue in lung, wherein normal lung tissue is by fibrosed tissue Displacement.This cicatrization causes lung stiff and lung structure and function are impaired.In people, pulmonary fibrosis is considered by lung In small air bag (alveole) and between repetition tissue damage cause.Under experimental setup, various animal models have human disease Repetition in terms of.For example, external agent such as bleomycin, fluorescein isothiocynate, silica or asbestos can be inculcated to dynamic In the tracheae of object (Gharaee-Kermani et al.,Animal Models of Pulmonary Fibrosis. Methods Mol. Med., 2005, 117:251-259)。
Therefore, in certain embodiments, present disclosure is provided with by fibrosis and/or inflammation being caused or aggravated Lung disease or illness subject in inhibit the method for pulmonary fibrosis, including give subject MASP-2 in need and inhibit Agent, such as MASP-2 inhibiting antibody.This method include give comprising effectively inhibit pulmonary fibrosis, reduce pulmonary fibrosis and/or Improve the composition of the MASP-2 inhibitor of the amount of lung function.It includes that lung function and/or lung capacity change that the symptom of lung function, which improves, Kind, reduction fatigue and oxygen saturation improve.
In some embodiments, present disclosure provides the treatment in the subject with cystic fibrosis, inhibits, is pre- Method that is anti-or improving pulmonary fibrosis, including to give subject MASP-2 inhibitor in need, such as MASP-2 inhibition anti- Body.
MASP-2 inhibition composition can administer locally to the region of fibrosis, such as during operation or locally injecting, directly It connects or distal end (such as passing through conduit) passes through topical composition.Alternatively, MASP-2 inhibitor can whole body give subject, example Such as by intra-arterial, intravenous, intramuscular, sucking, intranasal, it is subcutaneous or it is other parenterally give, or for it is non-peptide energy drug possibility It is given by oral.Giving may be repeated, and be determined by doctor, until the patient's condition has been eliminated or controls.
In certain embodiments, MASP-2 inhibitor (for example, MASP-2 inhibiting antibody) and it is suitable for potential lung The combination of the one or more medicaments or form of therapy of disease or the patient's condition is given.
The certain specific lung diseases and illness for being caused or being aggravated by fibrosis and/or inflammation are described below.
In certain embodiments, the lung disease for being caused or being aggravated by fibrosis and/or inflammation is Chronic Obstructive Pulmonary Disease Sick (COPD).COPD be a kind of wherein airway walls with the accumulation of myofibroblast and collagen the disease of fibrosis, be disability The main reason for, and in the U.S., it is the fourth-largest cause of death.COPD blocks air flowing and keeps patient respiratory gradually difficult. COPD is caused by the damage to air flue, resultant interference oxygen and carbon dioxide exchange in lung.COPD includes chronic obstructive Bronchitis and pulmonary emphysema, and usually the two has.Lung damaged with lung function damaged COPD patient be in it is thin In the increase risk of bacterium and the related complication of virus infection.
Therefore, in one embodiment, present disclosure provides the method for the treatment of Chronic Obstructive Pulmonary Disease (COPD), Including give subject in need effectively inhibit and/or reduce pulmonary fibrosis amount MASP-2 inhibitor (for example, anti- MASP-2 antibody).In certain embodiments, treatment includes reducing one or more symptoms of COPD.COPD and/or lung fiber The symptom of change include but is not limited to the cough with mucus, gentle activity can become (expiratory dyspnea) short of breath that deteriorates, it is tired, Frequent respiratory tract infection stridulates, feeling of chest tightness, heartbeat irregular (arrhythmia cordis), needs ventilator and oxygen therapy, right side It is heart failure or cor pulmonale (heart swelling and heart failure as caused by chronic lung disease), pneumonia, pneumothorax, serious Weight loss and malnutrition.Symptom further includes lung function reduction, as evaluated using one or more standard pulmonary function tests.
In certain embodiments, the lung disease for being caused or being aggravated by fibrosis and/or inflammation is related with chorionitis Pulmonary fibrosis.As described in more detail below, pulmonary fibrosis related with chorionitis be available MASP-2 inhibitor (for example, MASP-2 inhibiting antibody) treatment pulmonary fibrosis another example.
In some embodiments, the lung disease or illness for being caused or being aggravated by fibrosis and/or inflammation are selected from: chronic resistance Plug property lung disease, cystic fibrosis, pulmonary fibrosis related with chorionitis, bronchiectasis and pulmonary hypertension.
Heart and vascular diseases
Many different hearts and vascular pathological are caused by common fibrotic processes.Fibrosed tissue over-deposit in heart Lead to heart pathology, wherein the excessive of extracellular matrix protein generates the structure for changing heart, construction, shape and influence heart Contractile function (Khan and Sheppard,Immunology118:10-24,2006).
Research shows that fibrosis can significantly promote the barrier of the cardiac function in ischemic, dilatancy and hypertrophic cardiomyopathy Hinder.Such as, it has already been proven that the Finding case with Chronic Atrial Fibrillation has higher levels of Myocardial interstitial compared with the control Fibrosis (Khan and Sheppard,Immunology118:10-24,2006).As another example, it has been determined that in beauty State, most of Arrhythmogenic right ventricular cardiomyopathy (ARVC) cases show fatty infiltration and cicatrization (fibrofatty ARVC) (Burke et al.,Circulation97:1571-1580,1998).In the heart for checking the people experimenter with ARVC In the research of the histopathologic characteristics of room flesh, it has been determined that extensive fibrosis is present in the work from the pediatric patients with ARVC In sample sheet (Nishikawa T. et al.,Cardiovascular PathologyVol 8 (4): 185-189,1999).
Therefore, in certain embodiments, present disclosure is provided with by fibrosis and/or inflammation being caused or aggravated Heart or vascular diseases or illness subject in prevent, treat, reverse, inhibit and/or mitigate fibrosis and/or inflammation Method, including give subject MASP-2 inhibitor, such as MASP-2 inhibiting antibody in need.This method includes giving to wrap The group of MASP-2 inhibitor containing effective amount for inhibiting heart and/or vascular fibrosis and/or improving heart and/or vascular function Close object.
In some embodiments, present disclosure provides the treatment in the subject with valvular fibrosis, inhibits, is pre- Method that is anti-or improving fibrosis, including give subject MASP-2 inhibitor, such as MASP-2 inhibiting antibody in need.
MASP-2 inhibition composition can administer locally to the region of fibrosis, such as during operation or locally injecting, directly It connects or distal end (such as passing through conduit) passes through topical composition.Alternatively, MASP-2 inhibitor can whole body give subject, example Such as by intra-arterial, intravenous, intramuscular, sucking, intranasal, it is subcutaneous or it is other parenterally give, or for it is non-peptide energy drug can It can be given by oral.Giving may be repeated, and be determined by doctor, until the patient's condition has been eliminated or controls.
In certain embodiments, MASP-2 inhibitor (for example, MASP-2 inhibiting antibody) and it is suitable for the potential heart The combination of the one or more medicaments or form of therapy of dirty disease or vascular diseases or the patient's condition is given.
In some embodiments, heart or vascular diseases or the illness choosing for being caused or being aggravated by fibrosis and/or inflammation From: cardiac fibrosis, myocardial infarction, Atrial fibrosis, endomyocardial fibrosis Arrhythmogenic right ventricular cardiomyopathy (ARVC), vascular diseases, atherosclerotic vascular diseases, hemadostewnosis, restenosis, vasculitis, phlebitis, Deep vein Thrombosis and abdominal aneurvsm.
Chronic infectious disease
Chronic infectious disease, such as hepatitis C and hepatitis B cause tissue inflammation and fibrosis, and high agglutinin way Diameter activity may be harmful.In such disease, the inhibitor of MASP-2 may be beneficial.For example, it was discovered that MBL and MASP-1 level be Hepatitis C Virus (HCV) infection hepatic fibrosis-renal tubular ectasia syndrome seriousness important prediction object (Brown et al.,Clin Exp Immunol147 (1): 90-8,2007;Saadanay et al.,Arab J Gastroenterol. 12(2): 68-73,2011;Saeed et al.,Clin Exp Immunol174 (2): 265-73,2013).It is had shown before MASP-1 Be MASP-2 and lectin pathway effective activator (Megyeri et al.,J Biol Chem. 29: 288(13):8922- 34,2013).Alphavirus such as chikungunya virus and ross river virus induce strong host inflammatory to react, cause arthritis and Myositis, and the pathology mediated by MBL and lectin pathway (Gunn et al.,PLoS Pathog8 (3): e1002586, 2012)。
Therefore, in certain embodiments, present disclosure provide suffer from or before suffered from and cause inflammation and/or fibre The side of fibrosis and/or inflammation is prevented, treated, reverses, inhibits and/or mitigated in the subject of the chronic infectious disease of dimensionization Method, including give subject MASP-2 inhibitor, such as MASP-2 inhibiting antibody in need.
MASP-2 inhibition composition can administer locally to the region of fibrosis, such as during operation or locally injecting, directly It connects or distal end (such as passing through conduit) passes through topical composition.Alternatively, MASP-2 inhibitor can whole body give subject, example Such as by intra-arterial, intravenous, intramuscular, sucking, intranasal, it is subcutaneous or it is other parenterally give, or for it is non-peptide energy drug can It can be given by oral.Giving may be repeated, and be determined by doctor, until the patient's condition has been eliminated or controls.
In certain embodiments, MASP-2 inhibitor (for example, MASP-2 inhibiting antibody) and it is suitable for potential slow One or more medicaments or the form of therapy combination of sexy infectious diseases are given.
In some embodiments, the chronic infectious disease of inflammation and/or fibrosis is caused to be selected from: α virus, A type liver Inflammation, hepatitis B, hepatitis C, tuberculosis, HIV and influenza.
Autoimmune disease
Chorionitis is the chronic auto-immune disease that feature is fibrosis, Blood vessel pattern and autoantibody.It is main there are two kinds Form: restricted system chorionitis and dispersivity system chorionitis.The skin symptom of restricted system chorionitis influence hand, arm and Face.The patient of chorionitis with this form usually has one or more following complication: calcinosis, Raynaud's phenomenon, Esophagus Function obstacle, acroscleroderma and capillarectasia.The development of dispersivity system chorionitis quickly, and influences the skin of large area With one or more internal organs, usually kidney, oesophagus, heart and/or lung.
Chorionitis influences the thin vessels for being known as parteriole in all organs.Firstly, the endothelial cell of parteriole and smooth Myocyte gradually decreases through apoptosis.These cells are replaced by collagen and other fibrous materials.Inflammatory cell, especially CD4+ are auxiliary T cell is helped, infiltrates parteriole, and cause further to damage.
The cutaneous manifestations of chorionitis can be pain, can damage involved area use (for example, hand, finger, toe, The use of foot etc.) and can damage appearance.Skin ulcer can occur, and such ulcer can be easy to infection or even gangrenous.It bursts The skin of ulcer is likely difficult to cure or cure slow.The difficult of skin ulcer is cured (such as to have in the patient for having circulation impaired Have those of Raynaud's phenomenon) in may especially aggravate.In certain embodiments, the method for present disclosure is for treating sclerderm Disease, such as the skin symptom of chorionitis.In certain embodiments, treatment chorionitis includes treatment skin ulcer, such as finger Ulcer.Give the chorionitis that MASP-2 inhibitor for example anti-MASP-2 antibody can be used for reducing affected tissue and/or organ Fibrosis and/or inflammatory symptoms.
Other than skin symptom/performance, chorionitis can also influence heart, kidney, lung, joint and alimentary canal.In certain realities It applies in scheme, treatment chorionitis includes treating the symptom of the disease in these any one or more of tissues, such as by subtracting Few fibrosis and/or inflammatory symptoms.Lung problem is one of complication of most serious of chorionitis, and is related with the disease The reason of most of death rates.Two kinds of main tuberculosis conditions related with chorionitis are pulmonary fibrosis and pulmonary hypertension.Involve The patient of lung can have any one or two kinds of patient's condition.Pulmonary fibrosis related with chorionitis is available MASP-2 inhibitor for treating One example of pulmonary fibrosis.The chorionitis for being related to lung leads to cicatrization (pulmonary fibrosis).Such pulmonary fibrosis is about Occur in 70% Patients with scleroderma, although it is in progress, usually slow and symptom is widely varied in terms of seriousness in patients. For with the patient of symptom related with pulmonary fibrosis, symptom includes dry cough, short of breath and locomitivity is reduced really.About Severe pulmonary fibrosis occurs for the patient of 16% pulmonary fibrosis with certain level.Patient experience with severe pulmonary fibrosis Lung function be decreased obviously and pulmonary alveolitis.
In certain embodiments, the method for present disclosure is for treating chorionitis, lung for example related with chorionitis Fibrosis.Give MASP-2 inhibitor, the fibrosis that such as MASP-2 inhibiting antibody can be used for reducing the chorionitis in lung Shape.For example, the method can be used for improving lung function and/or reduce the mortality risk as caused by chorionitis.
Involving kidney is also common in Patients with scleroderma.Kidney fibrosis related with chorionitis is can be by giving MASP-2 inhibitor, for example anti-MASP-2 Antybody therapy kidney fibrosis example.In certain embodiments, in the disclosure The method of appearance is for treating chorionitis, kidney fibrosis for example related with chorionitis.In one embodiment, MASP-2 is given Inhibiting antibody can be used for reducing the fibrotic symptoms of the chorionitis in kidney.For example, the method can be used for improving renal function, subtract Albumen in oliguresis reduces hypertension and/or reduces the risk that can lead to the renal crises of lethal kidney failure.
Systemic loupus erythematosus (SLE) is a kind of chronic inflammatory autoimmune disease disease, feature be spontaneous B and T cell from Body reactivity and multiple organ immunologic mjury, and skin, joint, kidney and other organs can be influenced.Nearly all people's tool with SLE There are arthralgia, and most of generation arthritis.Often impacted joint is finger, hand, wrist and knee.The general symptom of SLE It include: arthritis；Fatigue；General malaise, uneasiness or malice (dispirited)；Arthralgia and swelling；Courbature；Nausea and vomit It spits；And fash.In addition, symptom may also include that abdominal pain；Hematuria；Finger changes colour when being pressurized or is cold；Numb and shouting pain；And skin Erythema.Among the patients, SLE, which has, involves lung or kidney.Without being bound by theory the inflammation and/or fibrosis of lung and kidney are damaged Hurt these organs, and leads to symptom related with lung and/or injury of kidney.In some cases, the patient with SLE is known as The specific nephrosis condition of lupus nephritis.In certain embodiments, present disclosure provides the method for the treatment of SLE, including gives For example anti-MASP-2 antibody of a effective amount of MASP-2 inhibitor.Give one kind that MASP-2 inhibiting antibody can be used for reducing SLE Or a variety of symptoms.In certain embodiments, anti-MASP-2 antibody is given for treating the SLE of Lupus Nephritis Patients.At this In the case where sample, treatment SLE includes treatment lupus nephritis, such as the symptom by reducing lupus nephritis.In certain implementations In scheme, treatment includes the skin symptom for the treatment of SLE.In certain embodiments, treatment includes reduce lupus nephritis one Kind or a variety of symptoms.In certain embodiments, treatment includes reducing, postpone or eliminating dialysis to need.In certain embodiments In, treatment includes reduction, delay or the needs for eliminating kidney transplant.In certain embodiments, treatment includes postponing or preventing wolf Sore ephritis is to kidney failure or end-stage renal disease.
Lupus nephritis is the inflammation of kidney, and is the severe complication of systemic loupus erythematosus (SLE).In kidney, wolf Sore ephritis can lead to the reduction loss of function.Lupus Nephritis Patients can finally develop kidney failure and dialysis or kidney is needed to move It plants.The related complication that the method that present disclosure can also be used is treated includes interstitial nephritis and nephrotic syndrome.Systemic lupus erythematosus The symptom of ephritis includes: hematuria, the foamy appearance of urine, hypertension, Urine proteins, fluid retention and oedema.Other symptoms include kidney The sign and symptom of fibrosis and/or kidney failure.If do not treated, lupus nephritis can lead to kidney failure, and even advanced stage kidney Disease.
Therefore, in certain embodiments, present disclosure is provided with causing or aggravate fibrosis and/or inflammation The method that fibrosis and/or inflammation is prevented, treated, reverses, inhibited and/or mitigated in the subject of autoimmune disease, packet It includes and gives subject MASP-2 inhibitor, such as MASP-2 inhibiting antibody in need.This method includes giving comprising effective Inhibit the composition of the MASP-2 inhibitor of the amount of fibrosis.
MASP-2 inhibition composition can administer locally to the region of fibrosis, such as during operation or locally injecting, directly It connects or distal end (such as passing through conduit) passes through topical composition.Alternatively, MASP-2 inhibitor can whole body give subject, example Such as by intra-arterial, intravenous, intramuscular, sucking, intranasal, it is subcutaneous or it is other parenterally give, or for it is non-peptide energy drug can It can be given by oral.Giving may be repeated, and be determined by doctor, until the patient's condition has been eliminated or controls.
In certain embodiments, MASP-2 inhibitor (for example, MASP-2 inhibiting antibody) and be suitable for it is potential from One or more medicaments or the form of therapy combination of body immunity disease are given.
In some embodiments, the autoimmune disease for causing or aggravating fibrosis and/or inflammation is selected from: chorionitis With systemic loupus erythematosus (SLE).
Central nervous system disease and the patient's condition
In certain embodiments, present disclosure is provided with the maincenter mind for being caused or being aggravated by fibrosis and/or inflammation Disease through system or the side that fibrosis and/or inflammation is prevented, treated, reverses, inhibited and/or mitigated in the subject of illness Method, including give subject MASP-2 inhibitor in need, for example anti-MASP-2 antibody.This method includes giving to include Effect inhibits the composition of the MASP-2 inhibitor of the amount of fibrosis and/or inflammation.
MASP-2 inhibition composition can administer locally to the region of fibrosis, such as during operation or locally injecting, directly It connects or distal end (such as passing through conduit) passes through topical composition.Alternatively, MASP-2 inhibitor can whole body give subject, example Such as by intra-arterial, intravenous, intramuscular, sucking, intranasal, it is subcutaneous or it is other parenterally give, or for it is non-peptide energy drug can It can be given by oral.Giving may be repeated, and be determined by doctor, until the patient's condition has been eliminated or controls.
In certain embodiments, MASP-2 inhibitor (for example, MASP-2 inhibiting antibody) and it is suitable in potential The combination of the one or more medicaments or form of therapy of pivot nervous system disease or illness is given.
In some embodiments, the disease or disease for the central nervous system for being caused or being aggravated by fibrosis and/or inflammation Disease is selected from: apoplexy, traumatic brain injury and spinal cord injury.
Skin disease and the patient's condition
In certain embodiments, present disclosure is provided with the skin disease for being caused or being aggravated by fibrosis and/or inflammation The method of fibrosis and/or inflammation is prevented, treated, reversed, inhibited and/or mitigated in the subject of disease or illness, including is given Subject MASP-2 inhibitor, such as MASP-2 inhibiting antibody in need.This method includes giving comprising effectively inhibiting fine The composition of the MASP-2 inhibitor of the amount of dimensionization and/or inflammation.
MASP-2 inhibition composition can administer locally to the region of fibrosis, such as pass through topical composition to skin Skin, or direct or distal end (such as passing through conduit) local application during operation or locally injecting.Alternatively, MASP-2 inhibitor can Whole body gives subject, for example, by intra-arterial, intravenous, intramuscular, sucking, intranasal, it is subcutaneous or it is other parenterally give, pass through It administers locally to, or non-peptide energy drug may be given by oral.Giving may be repeated, and be determined by doctor, until disease Condition has been eliminated or has controlled.
In certain embodiments, MASP-2 inhibitor (for example, MASP-2 inhibiting antibody) and it is suitable for potential skin The combination of the one or more medicaments or form of therapy of skin disease or illness is given.
In some embodiments, the skin disease or illness for being caused or being aggravated by fibrosis and/or inflammation are selected from: skin Fibrosis, wound healing, chorionitis, Sjogren's syndrome, cheloid, connective tissue disease, cicatrization and hypertrophic scar.
The bone of muscle skeleton and disorder of soft tissue and the patient's condition
In certain embodiments, present disclosure is provided with the bone that is caused or aggravated by fibrosis and/or inflammation or soft The method for preventing, treating in the subject of tissue disease or illness, reversing, inhibit and/or mitigate fibrosis and/or inflammation, packet It includes and gives subject MASP-2 inhibitor, such as MASP-2 inhibiting antibody in need.This method includes giving comprising effective Inhibit the composition of the MASP-2 inhibitor of the amount of fibrosis and/or inflammation.
MASP-2 inhibition composition can administer locally to the region of fibrosis, for example, by topical composition to bone or Soft tissue structure, or direct or distal end (such as passing through conduit) local application during operation or locally injecting.Alternatively, MASP-2 Inhibitor can whole body give subject, such as pass through intra-arterial, intravenous, intramuscular, sucking, intranasal, subcutaneous or other parenteral It gives, may be given by oral by administering locally to, or for non-peptide energy drug.Giving may be repeated, true by doctor It is fixed, until the patient's condition has been eliminated or controls.
In certain embodiments, MASP-2 inhibitor (for example, MASP-2 inhibiting antibody) and it is suitable for potential bone Or soft tissue disease or the combination of the one or more medicaments or form of therapy of illness are given.
In some embodiments, bone or soft tissue disease or the illness choosing for being caused or being aggravated by fibrosis and/or inflammation From: osteoporosis related with such as cystic fibrosis and/or sclerotin are reduced, with the increased osteomyelodysplasia of osteofibrosis The patient's condition, adhesive capsulitis, dupuytren's contracture and myelofibrosis.
Joint disease and the patient's condition
In certain embodiments, present disclosure is provided with the joint disease for being caused or being aggravated by fibrosis and/or inflammation The method of fibrosis and/or inflammation is prevented, treated, reversed, inhibited and/or mitigated in the subject of disease or illness, including is given Subject MASP-2 inhibitor, such as MASP-2 inhibiting antibody in need.This method includes giving comprising effectively inhibiting fine The composition of the MASP-2 inhibitor of the amount of dimensionization and/or inflammation.
MASP-2 inhibition composition can administer locally to the region of fibrosis, such as pass through topical composition to pass Section, or direct or distal end (such as passing through conduit) local application during operation or locally injecting.Alternatively, MASP-2 inhibitor can Whole body gives subject, for example, by intra-arterial, intravenous, intramuscular, sucking, intranasal, it is subcutaneous or it is other parenterally give, pass through It administers locally to, or non-peptide energy drug may be given by oral.Giving may be repeated, and be determined by doctor, until disease Condition has been eliminated or has controlled.
In certain embodiments, MASP-2 inhibitor (for example, MASP-2 inhibiting antibody) be suitable for potentially closing The combination of the one or more medicaments or form of therapy of section disease or illness is given.
In some embodiments, the joint disease or illness for being caused or being aggravated by fibrosis and/or inflammation are joint fibres Dimensionization.
Digestive disease and the patient's condition
In certain embodiments, present disclosure is provided with the digestion disease for being caused or being aggravated by fibrosis and/or inflammation The method of fibrosis and/or inflammation is prevented, treated, reversed, inhibited and/or mitigated in the subject of disease or illness, including is given Subject MASP-2 inhibitor, such as MASP-2 inhibiting antibody in need.This method includes giving comprising effectively inhibiting fine The composition of the MASP-2 inhibitor of the amount of dimensionization and/or inflammation.
MASP-2 inhibition composition can administer locally to the region of fibrosis, such as by during operation or locally injecting Direct or distal end (such as passing through conduit) local application.Alternatively, MASP-2 inhibitor can whole body give subject, such as by dynamic Arteries and veins is interior, intravenous, intramuscular, sucking, intranasal, it is subcutaneous or it is other parenterally give, by administering locally to, or for it is non-peptide can medicine Object may be given by oral.Giving may be repeated, and be determined by doctor, until the patient's condition has been eliminated or controls.
In certain embodiments, MASP-2 inhibitor (for example, MASP-2 inhibiting antibody) be suitable for potentially disappearing One or more medicaments or the form of therapy combination for changing disease or illness are given.
In some embodiments, the digestive disease or illness for being caused or being aggravated by fibrosis and/or inflammation are selected from: Crow Grace disease, ulcerative colitis and pancreas fibrosis.
Eye diseases and the patient's condition
In certain embodiments, present disclosure is provided with the eye diseases for being caused or being aggravated by fibrosis and/or inflammation Or the method that fibrosis and/or inflammation is prevented, treated, reverses, inhibited and/or mitigated in the subject of illness, including given Subject MASP-2 inhibitor, such as MASP-2 inhibiting antibody needed.This method includes giving comprising effectively inhibiting fiber The composition of the MASP-2 inhibitor of the amount of change and/or inflammation.
MASP-2 inhibition composition can administer locally to the region of fibrosis, such as by during operation or locally injecting Direct or distal end (such as passing through conduit) local application.Alternatively, MASP-2 inhibitor can whole body give subject, such as by dynamic Arteries and veins is interior, intravenous, intramuscular, sucking, intranasal, it is subcutaneous or it is other parenterally give, by administering locally to eye (for example, as eye drip Agent), or non-peptide energy drug may be given by oral.Giving may be repeated, and be determined by doctor, until the patient's condition has obtained To elimination or control.
In certain embodiments, MASP-2 inhibitor (for example, MASP-2 inhibiting antibody) and it is suitable for potential eye The combination of the one or more medicaments or form of therapy of disease or illness is given.
In some embodiments, it is selected from by fibrosis and/or inflammation cause or aggravate eye diseases or illness: under preceding capsule Cataract, rear capsule milkiness, macular degeneration and retina and vitreoretinopathy.
The disease and the patient's condition of reproductive organs
In certain embodiments, present disclosure is provided with the reproduction disease for being caused or being aggravated by fibrosis and/or inflammation The method of fibrosis and/or inflammation is prevented, treated, reversed, inhibited and/or mitigated in the subject of disease or illness, including is given Subject MASP-2 inhibitor, such as MASP-2 inhibiting antibody in need.This method includes giving comprising effectively inhibiting fine The composition of the MASP-2 inhibitor of the amount of dimensionization and/or inflammation.
MASP-2 inhibition composition can administer locally to the region of fibrosis, such as by during operation or locally injecting Direct or distal end (such as passing through conduit) local application.Alternatively, MASP-2 inhibitor can whole body give subject, such as by dynamic Arteries and veins is interior, intravenous, intramuscular, sucking, intranasal, it is subcutaneous or it is other parenterally give, by administering locally to, or for it is non-peptide can medicine Object may be given by oral.Giving may be repeated, and be determined by doctor, until the patient's condition has been eliminated or controls.
In certain embodiments, MASP-2 inhibitor (for example, MASP-2 inhibiting antibody) and it is suitable for potential life One or more medicaments or the form of therapy combination for growing disease or illness are given.
In some embodiments, the reproductive disease or illness for being caused or being aggravated by fibrosis and/or inflammation are selected from: uterus Endometriosis and Perun Nie Shi disease.
Cicatrization related with wound
In certain embodiments, present disclosure is provided with the disease or disease generated by cicatrization related with wound The method that fibrosis and/or inflammation is prevented, treated, reverses, inhibited and/or mitigated in the subject of condition, including give in need Subject MASP-2 inhibitor, such as MASP-2 inhibiting antibody.This method include give comprising effectively inhibit fibrosis and/ Or the composition of the MASP-2 inhibitor of the amount of inflammation.
MASP-2 inhibition composition can administer locally to the region of fibrosis, such as by during operation or locally injecting Direct or distal end (such as passing through conduit) local application.Alternatively, MASP-2 inhibitor can whole body give subject, such as by dynamic Arteries and veins is interior, intravenous, intramuscular, sucking, intranasal, it is subcutaneous or it is other parenterally give, by administering locally to, or for it is non-peptide can medicine Object may be given by oral.Giving may be repeated, and be determined by doctor, until the patient's condition has been eliminated or controls.
In certain embodiments, MASP-2 inhibitor (for example, MASP-2 inhibiting antibody) and it is suitable for potential disease The combination of the one or more medicaments or form of therapy of disease or illness is given.
In some embodiments, cicatrization related with wound is selected from: postoperative complication (for example, surgical adhesions, Middle cicatricial tissue can be formed between internal organs, caused contracture, pain and can be caused infertility), chemotherapeutic agent induction Fibrosis, radiation-induced fibrosis and the related cicatrization with burn.
The Other diseases and illness for being caused or being aggravated by fibrosis and/or inflammation
In certain embodiments, present disclosure provide with the disease that is caused or aggravated by fibrosis and/or inflammation or The method that fibrosis and/or inflammation is prevented, treated, reverses, inhibited and/or mitigated in the subject of illness, the disease or disease Disease be selected from organ transplant, Breast fibroadenoma, fibro-muscular, retroperitoneal fibrosis, thyroid gland fibrosis, lymph node fibrosis, Cystic fibrosis and fibrosis of pleura, the method includes giving subject MASP-2 inhibitor in need, such as MASP-2 Inhibiting antibody.This method includes giving the combination comprising the MASP-2 inhibitor for effectively inhibiting the amount of fibrosis and/or inflammation Object.
MASP-2 inhibition composition can administer locally to the region of fibrosis, such as by during operation or locally injecting Direct or distal end (such as passing through conduit) local application.Alternatively, MASP-2 inhibitor can whole body give subject, such as by dynamic Arteries and veins is interior, intravenous, intramuscular, sucking, intranasal, it is subcutaneous or it is other parenterally give, by administering locally to eye (for example, as eye drip Agent), or non-peptide energy drug may be given by oral.Giving may be repeated, and be determined by doctor, until the patient's condition has obtained To elimination or control.
In certain embodiments, MASP-2 inhibitor (for example, MASP-2 inhibiting antibody) and it is suitable for potential disease The combination of the one or more medicaments or form of therapy of disease or illness is given.
In any certain embodiments of various methods as described herein and pharmaceutical composition, MASP-2 inhibition Antibody selectively blocked lectin element approach, while it is complete to retain classical pathway.
IV. MASP-2 inhibitor
In all fields, the present invention provides the method for inhibiting the ill-effect of fibrosis and/or inflammation, including gives in need Subject's MASP-2 inhibitor.MASP-2 inhibitor is effectively to inhibit the MASP-2 dependent complement activation of lived subject Amount give.The present invention this in terms of implementation in, representative MASP-2 inhibitor include: inhibit MASP-2 bioactivity Molecule (such as micromolecular inhibitor, anti-MASP-2 antibody (such as MASP-2 inhibiting antibody) or with MASP-2 phase interaction With or interference Protein-protein interaction blocking peptide), and reduce MASP-2 expression molecule (such as MASP-2 antisense nucleic acid Molecule, MASP-2 specific RNA i molecule and MASP-2 ribozyme), so that MASP-2 be prevented to activate agglutinin complement pathway.MASP- 2 inhibitor can be used alone as main therapy, or be used as complementary therapy to enhance other medicines with the combination of other therapeutic agents The treatment benefit of object treatment.
MASP-2 dependent complement activation inhibits at least one variation below for being characterized in that complement system ingredient, institute State variation be due to giving MASP-2 inhibitor according to the method for the present invention caused by: MASP-2 dependent complement activation system product The inhibition (for example, by embodiment 2 described in measure) that C4b, C3a, C5a and/or C5b-9 (MAC) are formed or generated, C4 cracking and The reduction (for example, measuring by described in embodiment 2) or C3 cracking and the reduction of C3b deposition of C4b deposition are (for example, press embodiment 2 measurements).
According to the present invention, inhibit fibrosis and/or inflammation using effective, and show detectable anti-fibrosis activity and/ Or the MASP-2 inhibitor of inducing fibrosis reduction.In background of the invention, anti-fibrosis activity may include following at least one Item is multinomial: compared with fibrosis activity when lacking MASP-2 inhibitor, (1) inflammation is reduced, such as by activating and raising Macrophage and endothelial cell；By secreting many cell factor/chemotactic factor (CF)s, raise and activated lymphocyte and/or acidophilus Property cell；Discharge cytotoxicity medium and fibrogenic cytokines evaluation；(2) cell Proliferation, ECM synthesis or angiogenesis It reduces；And/or (3) collagen deposition is reduced.
Any technology detection known to technical staff can be used in the assessment of antifibrotic agents, such as MASP-2 inhibitor.Example Such as, the assessment of antifibrotic agents can be assessed (as described in embodiment hereof 12 and 14) in UUO model.If pressed down using MASP-2 Preparation assesses detectable anti-fibrosis activity and/or fibrosis mitigates or reduces, it is believed that such MASP-2 inhibitor is used as For preventing, treating, reversing and/or inhibiting the drug of fibrosis.
The assessment of fibrosis can be carried out periodically, for example, weekly or every month.Increase/reduction of fibrosis and/or anti-fiber Change it is active exist can therefore periodical evaluation, for example, weekly or the moon.It is this assessment preferably for given subject with it is several when Between put and carry out, or for given subject and normal healthy controls with the progress of one or several time points.Assessment can rule when Between interval, for example weekly or every month carry out.Therefore assessment can be carried out regularly, such as weekly or every month.It is assessed when one Result in a finding that fibrosis is reduced or there are when anti-fibrosis activity, it is believed that MASP-2 inhibitor, such as MASP-2 inhibiting antibody Show mitigating or reducing for detectable anti-fibrosis activity and/or inducing fibrosis.
For implement the present invention this in terms of MASP-2 inhibitor include such as MASP-2 antibody and its segment, MASP-2 Peptide for inhibiting, small molecule, MASP-2 soluble recepter and expression inhibiting agent.MASP-2 inhibitor can pass through the biology of blocking MASP-2 It learns function and inhibits MASP-2 dependent complement activation system.For example, inhibitor can effectively block it is mutual between MASP-2 albumen MASP-2 dimerization or assembly are interfered in effect, block Ca2+In conjunction with, interference MASP-2 serine protease site, Huo Zheke Reduce MASP-2 protein expression.
In some embodiments, MASP-2 inhibitor selective depression MASP-2 complement activation maintains C1q dependence Integrality in complement activation system function.
It in one embodiment, is the MASP-2 inhibitor of specificity for the MASP-2 inhibitor of the method for the present invention, Its affinity in conjunction with the polypeptid specificity comprising SEQ ID NO:6 is affine with other antigen bindings in complement system At least 10 times of power.In another embodiment, MASP-2 inhibitor is in conjunction with the polypeptid specificity comprising SEQ ID NO:6 Binding affinity be at least 100 times with the binding affinity of other antigen bindings in complement system.In an embodiment party In case, MASP-2 Inhibitor specificity combination CCP1-CCP2 structural domain (the aa 300-431 of SEQ ID NO:6) or MASP-2's At least one of serine protease domain (the aa 445-682 of SEQ ID NO:6) simultaneously inhibits MASP-2- dependent complement Activation.In one embodiment, MASP-2 inhibitor is the MASP-2 monoclonal antibody or its piece for specifically binding MASP-2 Section.Suitable binding assay can be used to be measured for the binding affinity of MASP-2 inhibitor.
MASP-2 polypeptide has the molecule of protease MASP-1, MASP-3 and C1r and C1s similar to C1 complement system Structure.Representative example (the amino as shown in SEQ ID NO:5 of cDNA molecule encoding MASP-2 shown in SEQ ID NO:4 Acid sequence composition), people's MASP-2 polypeptide with leader sequence (amino acid 1-15) is provided, is cleaved after secretion, is generated mature The people MASP-2 (SEQ ID NO:6) of form.As shown in Figure 2, peopleMASP 2Gene includes 12 exons.People MASP- 2 cDNA are encoded by exon B, C, D, F, G, H, I, J, K and L.Alternative splicing produces 20 kDa albumen, and referred to as MBL is relevant Protein 19 (" MAp19 ", also referred to as " sMAP ") (SEQ ID NO:2), (SEQ that exon B, C, D and E is generated as shown in Figure 2 ID NO:1) coding.The cDNA molecule encoding mouse MASP-2 (amino as shown in SEQ ID NO:51 shown in SEQ ID NO:50 Acid sequence composition), the mouse MASP-2 polypeptide with leader sequence is provided, is cleaved after secretion, the mouse MASP- of mature form is generated 2 (SEQ ID NO:52).CDNA molecule encoding rat MASP-2 is (as shown in SEQ ID NO:54 shown in SEQ ID NO:53 Amino acid sequence composition), the rat MASP-2 polypeptide with leader sequence is provided, is cleaved after secretion, generate mature form Rat MASP-2 (SEQ ID NO:55).
It will be understood by those skilled in the art that being disclosed in SEQ ID NO:4, SEQ ID NO:50 and SEQ ID NO:53 Sequence respectively represent the single allele of people, mouse and rat MASP-2, allelic variation occurs and alternative splicing is Among expectation.The allele of nucleotide sequence shown in SEQ ID NO:4, SEQ ID NO:50 and SEQ ID NO:53 becomes Body, the variant for the mutation for including the variant containing silent mutation and wherein causing amino acid sequence to change, all belongs to of the invention Range.Can according to standard method, by detection cDNA or genomic library cloned from Different Individual MASP-2 sequence etc. Position genetic mutation.
The structural domain of people MASP-2 albumen (SEQ ID NO:6) is shown in Fig. 1 and 2 A, including N-terminal C1r/C1s/ sea urchin Vegf/ bone Form albumen (CUBI) structural domain (amino acid 1-121 of SEQ ID NO:6), epidermal growth factor-like structural domain (amino acid 122-166), the 2nd CUBI structural domain (amino acid 1 67-293) and concatenated complement regulatory proteins structural domain and serine egg White enzyme domains.MASP 2The alternative splicing of gene generates MAp19 shown in Fig. 1.MAp19 is the N-terminal containing MASP-2 The non-zymoprotein in the area CUBI-EGF has 4 additional residues (EQSL) for deriving from exon E shown in Fig. 1.
Some protein have been displayed through the interaction between albumen in conjunction with MASP-2 or interact with MASP-2. For example, as it is known that MASP-2 binding lectin albumen MBL, H- fiber gelatinized protein and L- fiber gelatinized protein, and Ca is formed therewith2+ Dependence compound.Every kind of MASP-2/ agglutinin compound has been displayed to be cracked by the MASP-2 dependence of protein C 4 and C2 Come activating complement (Ikeda, K., et al.,J. Biol. Chem. 262:7451-7454, 1987; Matsushita, M., et al.,J. Exp. Med.176:1497-2284, 2000;Matsushita, M., et al.,J. Immunol. 168:3502-3506, 2002).Studies have shown that the CUBI-EGF structural domain of MASP-2 is must for the combination of MASP-2 and MBL Indispensable (Thielens, N.M., et al.,J. Immunol. 166:5068, 2001).Research is also shown that CUBIEGFCUBII structural domain mediates the dimerization of MASP-2, this be required for forming active MBL compound (Wallis, R., et al., J. Biol. Chem. 275:30962-30969, 2000).It therefore, can be known to MASP-2 dependence to combining The property important target area MASP-2 of complement activation interferes the MASP-2 inhibitor in the region to be identified.
Anti- MASP-2 antibody
The present invention this in terms of some embodiments in, MASP-2 inhibitor include inhibit MASP-2 dependent complement activation The anti-MASP-2 antibody of system.For the present invention this in terms of anti-MASP-2 antibody include derived from it is any generate antibody lactation Polyclonal antibody, monoclonal antibody or the recombinant antibodies of animal, and can be polyspecific, it is chimeric, humanization, anti- Idiotype antibody and antibody fragment.Antibody fragment includes Fab, Fab', F (ab)2、F(ab')2, Fv segment, scFv segment and list Chain antibody is shown in further describing for this paper.
Using measuring method as described herein, MASP-2 antibody can be screened and inhibit MASP-2- dependent complement activation system Ability and anti-fibrosis activity and/or the ability for inhibiting injury of kidney related with the nephrosis that albuminuria or adriamycin induce.It is several MASP-2 antibody has described in the literature and some nearest generations, some of which are listed in the table below in 1.For example, as herein Described in embodiment 10 and 11, the anti-MASP-2 Fab2 antibody for blocking MASP-2- dependent complement activation has been identified.As implemented It described in example 12, and described in WO2012/151481, is incorporated herein by reference, has identified and MASP-2- is blocked to rely on The full people MASP-2 scFv antibody (for example, OMS646) of property complement activation.As described in Example 13, and in WO2014/ It described in 144542, is incorporated herein by reference, the MASP-2 of the carrying SGMI-2 peptide with MASP-2 inhibitory activity is anti- Body and its segment pass through fusion SGMI-2 peptide amino acid sequence (SEQ ID NO:72,73 or 74) to the heavy chain of people's MASP-2 antibody And/or the amino or c-terminus of light chain generate (for example, OMS646-SGMI-2).
It therefore, in one embodiment, include human antibody for the MASP-2 inhibitor of method of the invention, such as OMS646.Therefore, in one embodiment, for the MASP-2 inhibitor of the compositions and methods of the invention include combine by The human antibody of the polypeptide of people MASP-2 (SEQ ID NO:6) composition, wherein the antibody includes: (I) (a) heavy chain variable region, Include: i) heavy chain CDR-H1, the amino acid sequence of the 31-35 comprising SEQ ID NO:67；And ii) heavy chain CDR-H2, include The amino acid sequence of the 50-65 of SEQ ID NO:67；And iii) heavy chain CDR-H3, the 95-107's comprising SEQ ID NO:67 Amino acid sequence and b) light chain variable region include: i) light chain CDR-L1, the amino acid of the 24-34 comprising SEQ ID NO:70 Sequence；And ii) light chain CDR-L2, the amino acid sequence of the 50-56 comprising SEQ ID NO:70；And iii) light chain CDR-L3, The amino acid sequence of 89-97 comprising SEQ ID NO:70, or (II) its variant, comprising having at least with SEQ ID NO:67 90% identity (for example, with SEQ ID NO:67 have 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) heavy chain variable region and have with SEQ ID NO:70 at least 90% same Property (for example, have 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) light chain variable region.
In some embodiments, the method includes give subject include a certain amount of MASP-2 inhibiting antibody or The composition of its antigen-binding fragment, the MASP-2 inhibiting antibody or its antigen-binding fragment include to contain SEQ ID NO: The heavy chain variable region of amino acid sequence shown in 67, and the light chain variable containing amino acid sequence shown in SEQ ID NO:70 Area.
It in some embodiments, include MASP-2 inhibiting antibody or its antigen knot the method includes giving subject The composition of segment is closed, the MASP-2 inhibiting antibody or its antigen-binding fragment specific recognition are by reference antibody OMS646 Know at least part of the epitope on others MASP-2, reference antibody OMS646 includes heavy chain shown in SEQ ID NO:67 can Become light chain variable region shown in area and SEQ ID NO:70.In one embodiment, the MASP-2 for method of the invention Inhibitor includes human antibody OMS646.
Table 1: illustrative MASP-2 specific antibody
The anti-MASP-2 antibody that effector function reduces
The present invention this in terms of some embodiments in, anti-MASP-2 antibody reduces effector function, to mitigate possibility The inflammation generated by classic complement approach activation.The ability that IgG molecule causes classic complement approach has shown to be present in molecule The part Fc (Duncan, A.R., et al.,Nature332:738-740 1988).The portion molecule Fc is wherein removed by enzymatic lysis Point IgG molecule lack this effector function (referring to Harlow,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988).Therefore, effector function can be made to subtract by having The Fc sequence being transformed to minimum genetic engineering, or become human IgG2Or IgG4Thus isotype removes the part Fc of molecule, To generate the antibody of effector function reduction.
Can to IgG heavy chain Fc part carry out standard molecular biology operation come generate effector function reduction antibody, As described herein, it is also described in Jolliffe et al.,Int'l Rev. Immunol. 10: 241-250,1993, and Rodrigues et al.,J. Immunol. 151:6954-6961, 1998.The antibody that effector function reduces further includes activation Complement and/or the human IgG2 reduced with the ability of Fc acceptor interaction and IgG4 isotype (Ravetch, J.V., et al.,Annu. Rev. Immunol. 9:457-492, 1991;Isaacs, J.D., et al.,J. Immunol.148:3062- 3071, 1992;Van de Winkel, J.G., et al.,Immunol. Today 14:215-221, 1993).It can pass through It is special to generate the people MASP-2 being made of IgG2 or IgG4 isotype if one of drying method known to persons of ordinary skill in the art Anisotropic humanized antibody or fully human antibodies, such as Vaughan, T.J., et al.,Nature Biotechnical 16: Described in 535-539,1998.
The generation of anti-MASP-2 antibody
MASP-2 polypeptide (such as overall length MASP-2) can be used or (such as MASP-2 is more using the peptide with antigenicity MASP-2 epitope Peptide moiety) generate anti-MASP-2 antibody.Immunogenic peptide may be as few as five amino acid residue.It is, for example, possible to use include The MASP-2 polypeptide of SEQ ID NO:6 whole amino acid sequence induces the anti-MASP-2 polypeptide for the method for the present invention.It can be with By the known specific MASP-2 structural domain for participating in Protein-protein interaction, such as CUBI and CUBIEGF structural domain and including silk The region of serine protease active site is expressed as recombinant polypeptide described in embodiment 3 and is used as antigen.In addition, comprising The peptide of the part of MASP-2 polypeptide (SEQ ID NO:6) at least six amino acid can also be used to induction MASP-2 antibody.In the following table 2 Provide other examples for inducing the derivative antigen of MASP-2 of MASP-2 antibody.For generating the MASP-2 peptide of antibody and more Peptide can be used as the recombinant polypeptide (such as MASP-2A) of natural polypeptides or recombinant peptide or synthetic peptide and catalytically inactive and by It separates, as further described herein.The present invention this in terms of some embodiments in, use transgenic mouse strain System obtains anti-MASP-2 antibody, as described herein.
Antigen for generating anti-MASP-2 antibody further includes fused polypeptide, such as MASP-2 or part thereof and immune globulin White polypeptide or fusions with maltose-binding protein.Polypeptide immunogen can be full-length molecule or part thereof.If polypeptide Part is haptens sample, then preferably can combine or be connected to macromolecular carrier (such as keyhole limpet hemocyanin for such part (KLH), bovine serum albumin(BSA) (BSA) or tetanus toxoid) on for being immunized.
Antigen derived from table 2:MASP-2
Polyclonal antibody
It can be exempted from by using the well-known method of those of ordinary skill in the art with MASP-2 polypeptide or its immunogenic portion Epidemic disease animal prepares the polyclonal antibody of anti-MASP-2.See, e.g., Green et al., " Production of Polyclonal Antisera, " is loaded inImmunochemical Protocols(Manson chief editor), page 105.It can lead to It crosses using adjuvant and increases the immunogenicity of MASP-2 polypeptide, the adjuvant includes inorganic gel (such as aluminium hydroxide) or Freund Adjuvant (completely or not exclusively), surfactant (such as lysolecithin), Pu Luonike polyalcohol (pluronic Polyol), polyanion, fat liquor, keyholeHemocyanin and dinitrophenol.Polyclonal antibody generally by animal such as Horse, ox, dog, chicken, rat, mouse, rabbit, cavy, goat or sheep generate.Alternatively, being used for anti-MASP-2 antibody of the invention It is available from subhuman primate.Generated in baboon diagnosis with and the general technology for the treatment of antibody can be found in for example The International Patent Publication No. WO 91/11465 and Losman, M.J. of Goldenberg et al., et al.,Int. J. Cancer 46:310, 1990.Then standard method well-known in the art is used, is produced from the blood for the animal that these were immunized The raw serum containing immunoreactivity antibody.
Monoclonal antibody
In some embodiments, MASP-2 inhibitor is anti-MASP-2 monoclonal antibody.Anti- MASP-2 monoclonal antibody is high Degree specificity, for single MASP-2 epitope.Modifier " monoclonal " used herein refers to the antibody obtained from basic homogeneity The antibody characteristic of group, must not be interpreted as needing to generate antibody by any ad hoc approach.It can be used through the company in culture Continuous cell line obtains monoclonal antibody, such as Kohler, G. to provide any technology of antibody molecule generation, et al.,Nature 256: hybridoma method described in 495,1975, or monoclonal antibody can be prepared by recombinant DNA method (see, for example, the U.S. Patent number 4,816,567 of Cabilly).Clackson, T. can also be used, et al.,Nature 352: 624-628,1991, and Marks, J.D., et al.,J. Mol. Biol. 222: 581-597,1991 description Technology separates monoclonal antibody from phage antibody library.Such antibody can be any immunoglobulin class, including IgG, IgM, IgE, IgA, IgD and its any subclass.
For example, can by by include MASP-2 polypeptide or part thereof composition be administered to suitable mammal (such as BALB/c mouse) and obtain monoclonal antibody.After during the scheduled time, splenocyte is taken out from mouse, is allowed to be suspended in In cell culture medium.Then splenocyte is merged to form hybridoma with immortal cell line.By the hybridoma of formation in cell It is cultivated in culture medium, the ability for generating anti-MASP-2 monoclonal antibody to them is screened.It is mono- to further describe anti-MASP-2 Clonal antibody generate example provided herein is (also referring toCurrent Protocols in Immunology, Vol. 1., John Wiley & Sons, pages 2.5.1-2.6.7, 1991.)。
Can obtain human monoclonal antibodies by using transgenic mice, the transgenic mice by it is engineered with Human antibodies specific is generated when response antigen attack.In this technique, by human immunoglobulin heavy chain and light chain gene seat member Part is introduced into the mouse species derived from embryonic stem cell line, and the embryonic stem cell line contains the endogenous immune ball for being directed destruction Ferritin heavy chain and light chain gene seat.The transgenic mice can be synthesized to human antigen's (all MASP-2 antigen as described herein) specificity Human antibody, the mouse can be used generate secretion people MASP-2 antibody hybridoma, by using conventional Kohler- Milstein technology merges the B cell from these animals with suitable myeloma cell line, as further described herein. Transgenic mice with human immunoglobulin gene's group be commercially available (such as purchased from Abgenix, Inc., Fremont, CA, and Medarex, Inc., Annandale, N.J.).Method for obtaining human antibody from transgenic mice describes In such as Green, L.L., et al.,Nature Genet. 7:13, 1994;Lonberg, N., et al.,Nature 368:856, 1994;And Taylor, L.D., et al.,Int. Immun. 6:579, 1994。
It can be separated from Hybridoma culture by the various technologies established and monoclonal antibody purification.These separation skills Art include with A albumen Ago-Gel affinity chromatography, size exclusion chromatography and ion-exchange chromatography (see, for example, Coligan, the 2.7.1-2.7.12 page and the 2.9.1-2.9.3 pages；Baines et al., " Purification of Immunoglobulin G (IgG), it " is loaded inMethods in Molecular Biology, The Humana Press, Inc., volume 10,79- Page 104,1992).
Polyclonal antibody, monoclonal antibody or the antibody derived from bacteriophage just will measure it to MASP- once generating first 2 specificity combined.Various measuring methods well known by persons skilled in the art be can use to detect specific binding MASP-2's Antibody.Illustrative measuring method includes that the western blot method carried out by standard method or immunoprecipitation analysis (are described in Such as Ausubel et al.), immunoelectrophoresis, enzyme linked immunosorbent assay (ELISA), dot blotting, inhibition or competition assay and folder Heart measuring method (is described in Harlow and Land, antibody: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988).Once identifying the antibody of specific binding MASP-2, it will pass through following several measuring methods One of, measure the ability that anti-MASP-2 antibody plays a role as MASP-2 inhibitor: such as agglutinin specific C 4 is split Solution measuring method (being described in embodiment 2), C3b deposition measuring method (being described in embodiment 2) or C4b deposition measuring method (are described in Embodiment 2).
Those of ordinary skill in the art can be easily determined anti-MASP-2 monoclonal antibody affinity (see, for example, Scatchard, A., NY Acad. Sci. 51:660-672, 1949).In one embodiment, it is used for present invention side The anti-MASP-2 monoclonal antibody of method can be in conjunction with MASP-2, binding affinity < 100 nM, preferably < 10 nM, most preferably < 2 nM。
Chimeric/humanized antibody
Monoclonal antibody for the method for the present invention includes chimeric antibody and the segment of these antibody, wherein heavy chain and/or light Chain part with derive from particular species or to belong to the corresponding sequence of antibody of specific antibodies classification or subclass identical or homologous, and chain Rest part with derive from another species or to belong to the corresponding sequence of antibody of another antibody isotype or subclass identical or homologous (the U.S. Patent number 4,816,567 of Cabilly；And Morrison, S.L., et al.,Proc. Nat'l Acad. Sci.USA 81:6851-6855, 1984)。
Form for a kind of chimeric antibody of the invention is the anti-MASP-2 antibody of Humanized monoclonal.Inhuman (such as mouse) The humanization form of antibody is chimeric antibody, contains the minmal sequence derived from non-human immunoglobulin.By by it is inhuman (such as Mouse) complementary determining region (CDR) from the variable heavy chain and variable light of mouse immune globulin is transferred to people variable region, to produce Raw Humanized monoclonal antibodies.Then, typical way is the framework that the rest part of human antibody is substituted into inhuman corresponding part Area.In addition, humanized antibody may include the residue being not present in receptor antibody or donor antibody.These modifications are used to further Improve antibody performance.In general, humanized antibody will include substantially all of at least one, usual two kinds of varistructures Domain, wherein all or substantially all hypermutation rings both corresponds to the hypermutation ring of non-human immunoglobulin, all or substantially institute Fv framework region be all human immunoglobulin sequence Fv framework region.Humanized antibody optionally may include that at least part is immune Immunoglobulin constant area (Fc), the usually constant region of human immunoglobulin(HIg).More details can be found in Jones, P.T., et al.,Nature 321:522-525, 1986;Reichmann, L., et al.,Nature 332:323-329, 1988;With Presta, Curr. Op. Struct. Biol. 2:593-596, 1992。
For the human monoclonal antibodies that humanized antibody of the invention includes at least containing the area MASP-2 combination CDRH3.This Outside, the part Fc can be replaced to generate IgA or IgM and human IgG antibody.These humanized antibodies will have specific clinical effect With because they specifically identify people MASP-2, but human body will not being caused to the immune response of antibody itself.Therefore it Be more suitable for the vivo medicine-feeding of human body, especially required repeated or chronic administration when.
Embodiment hereof 6 is shown in by the example that the anti-MASP-2 monoclonal antibody of mouse obtains the anti-MASP-2 antibody of humanization.Source of people The production technology for changing monoclonal antibody is also recorded in such as Jones, P.T., et al.,Nature 321:522, 1986; Carter, P., et al.,Proc. Nat'l. Acad. Sci. USA 89:4285, 1992; Sandhu, J.S.,Crit. Rev. Biotech. 12:437, 1992;Singer, I.I., et al.,J. Immun. 150:2844, 1993;Sudhir (chief editor),Antibody Engineering Protocols, Humana Press, Inc., 1995;Kelley, " Engineering Therapeutic Antibidies, " is loaded inProtein Engineering: Principles and Practice, Cleland et al. (chief editor), John Wiley & Sons, Inc., the 399- Page 434,1996；And the U.S. Patent number 5 of Queen, 693,762, (1997).In addition, there are also synthesize from specific mouse antibody district The commercial entity of humanized antibody, such as Protein Design Labs (Mountain View, CA).
Recombinant antibodies
Recombination method can also be used to prepare anti-MASP-2 antibody.For example, can be used human immunoglobulin(HIg) expression library (available from example Such as Stratagene, Corp., La Jolla, CA) generate human antibody segment (VH、VL, Fv, Fd, Fab or F (ab')2) make Standby human antibody.Then using the technology for generating chimeric antibody is similar to, by these segments to construct complete human antibody.
Anti-idiotype
Once identify the anti-MASP-2 antibody with required inhibitory activity, can using technology well-known in the art by this A little antibody are used to generate the anti-idiotype of similar portions MASP-2.See, for example, Greenspan, N.S., et al.,FASEB J. 7:437, 1993.For example, the phase of the MASP-2 albumen in conjunction with needed for MASP-2 and Reverse transcriptase complement activation The antibody of interaction can be used to generate the anti-idiotype of the MBL binding site on similar MASP-2 albumen, thus in conjunction with simultaneously The binding partner of MASP-2 is neutralized, such as, such as MBL.
Immunoglobulin fragment
MASP-2 inhibitor for the method for the present invention not only includes complete immunoglobulin molecules, but also including many institute's weeks The segment known, these segments include Fab, Fab', F (ab)2、F(ab')2With Fv segment, scFv segment, double antibody, linear antibodies, Single-chain antibody molecules and the multi-specificity antibody formed by antibody fragment.
It is well known in the art that the antibody molecule of only fraction, that is, paratope participates in the combination of antibody and its epitope (see, for example, Clark, W.R.,The Experimental Foundations of Modern Immunology, Wiley & Sons, Inc., NY, 1986).The area pFc' and the area Fc of antibody are the effectors of classic complement approach, but not Participate in antigen binding.Wherein the area pFc' is referred to as F by the antibody of enzymatic lysis or the generated antibody without the area pFc' (ab')2Segment, it remains the antigen-binding site of complete antibody.Isolated F (ab')2Segment is since there are two antigen bindings Position and referred to as divalent monoclonal segment.Similarly, wherein the area Fc by the antibody of enzymatic lysis or it is generated do not have Fc The antibody in area is referred to as Fab segment, it remains an antigen-binding site of complete antibody molecule.
Antibody fragment can be obtained by proteolysis, such as be disappeared by conventional method through pepsin or papain Change complete antibody.For example, antibody fragment can be generated by carrying out antibody enzymatic lysis with pepsin, it is known as F to provide (ab')25S segment.The segment can reuse the cracking of thiol reduction reagent, obtain 3.5S Fab' monovalent fragment.Can optionally it make Cracking reaction is carried out with the end-capping group of the sulfydryl of disulfide bond cracking generation.Alternatively, using the enzyme of pepsin Cracking directly generates two monovalent Fab fragments and a Fc segment.The U.S. that these methods are recorded in such as Goldenberg is special Benefit number 4,331,647；Nisonoff, A., et al.,Arch. Biochem. Biophys. 89:230, 1960; Porter, R.R., Biochem. J. 73:119, 1959;Edelman, et al., it is loaded inMethods in Enzymology 1:422, Academic Press, 1967；And the 2.8.1-2.8.10 pages and of Coligan 2.10.-2.10.4 page.
In some embodiments it is preferred that being swashed using the antibody fragment for lacking the area Fc to avoid after Fc combination Fc γ receptor Classic complement approach living.There is several methods that can produce the MoAb avoided with Fc γ acceptor interaction.For example, monoclonal antibody The area Fc can (such as ficin enzymic digestion) partial digested by using proteolytic enzyme, to be removed with chemical method, therefore It generates for example in conjunction with the antibody fragment of antigen, such as Fab or F (ab)2Segment (Mariani, M., et al.,Mol. Immunol. 28:69-71, 1991).Fc is not combined alternatively, can use during constructing humanized antibody as described herein The 4 IgG isotype of people γ of γ receptor.Can also be used recombinant technique described herein come the engineered antibody for lacking Fc structural domain, Single-chain antibody and the structural domain for combining antigen.
Single chain antibody fragments
Alternatively, the single peptide chain combination molecule to MASP-2 specificity can be prepared, wherein heavy chain is connected with the area light chain Fv.Fv Segment can be connected by peptide linker, be formed single chain antigen binding protein (scFv).Include coding V by buildingHAnd VLStructural domain The structural gene of DNA sequence dna prepares these single chain antigen binding proteins, passes through oligonucleotides between each structural domain and connects.It will knot Structure gene is inserted into expression vector, is then introduced it into host cell (such as Escherichia coli).Recombinant host cell synthesizes The single polypeptide chain in two V structure domains is bridged by joint peptide.The preparation method of scFv is recorded in such as Whitlow, et al., " Methods: A Companion to Methods in Enzymology" 2:97, 1991;Bird, et al.,Science 242:423, 1988；The U.S. Patent number 4,946,778 of Ladner；Pack, P., et al.,Bio/Technology 11: 1271, 1993。
It for example, can be by the way that lymphocytes in vitro be exposed to MASP-2 polypeptide, and in bacteriophage or similar substrates It selects antibody display libraries (such as by using immobilization or label MASP-2 albumen or peptide), obtains MASP-2 specificity scFv.The random peptide library that can be shown on to bacteriophage or bacterium (such as Escherichia coli) obtains coding tool and screening The gene of the polypeptide of possible MASP-2 polypeptide binding structural domain.These Random peptide display libraries can be used for screening and MASP-2 The peptide of interaction.The technology in these Random peptide display libraries of construction and screening is the (beauty of Lardner well-known in the art State's patent No. 5,223,409；The U.S. Patent number 4,946,778 of Lardner；The U.S. Patent number 5,403,484 of Lardner； The U.S. Patent number 5,571,698 of Lardner；And Kay et al.,Phage Display of Peptides and ProteinsAcademic Press, Inc., 1996), and Random peptide display library and the reagent for screening these libraries Box is commercially available, such as purchased 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 for anti-MASP-2 antibody fragment of the present invention in terms of this is the single complementary determining region of coding (CDR) peptide, in conjunction with MASP-2 antigen epitope and inhibit MASP-2 dependent complement activation.It can be by constructing encoding target The gene of antibody CDR and obtain CDR peptide (" minimum recognition unit ").Such as it can be raw from antibody by using polymerase chain reaction At the synthetic variable region RNA of cell, thus prepare these genes (see, for example, Larrick et al.,Methods: A Companion to Methods in Enzymology 2:106, 1991; Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies, " is loaded inMonoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (chief editor), page 166, Cambridge University Press, 1995;And Ward et al., " Genetic Manipulation and Expression of Antibodies, " is loaded inMonoclonal Antibodies: Principles and Applications, Birch et al. (chief editor), page 137, Wiley-Liss, Inc., 1995)).
MASP-2 antibody described herein, which is given, needs its subject to inhibit MASP-2 dependent complement activation.One In a little embodiments, MASP-2 inhibitor is that the high affinity human that effector function reduces or the anti-MASP-2 of Humanized monoclonal are anti- Body.
Inhibitor peptides
The present invention this in terms of some embodiments in, MASP-2 inhibitor includes isolated MASP-2 inhibitor peptides, Natural inhibitor peptides and synthesis inhibitor peptides including inhibiting the separation of MASP-2 dependent complement activation system.Art used herein Language " isolated MASP-2 inhibitor peptides " refers to by combining MASP-2, competing with MASP-2 with its in binding lectin approach It identify molecule (such as MBL, H- fiber gelatinized protein, M- fiber gelatinized protein or L- fiber gelatinized protein), and/or directly with MASP-2 interacts to inhibit MASP-2 dependent complement activation, thus inhibit the peptide of MASP-2 dependent complement activation, institute Peptide is stated to purify substantially, there is no it is other be present in nature therewith with reach it is practical and be suitable for its set use The substance of way degree.
Successfully disturb the interaction and catalytic site between albumen in vivo using inhibitor peptides.For example, most Closely, in structure the inhibitor peptides of adhesion molecule related with LFA-1 be approved to be clinically used for coagulopathy (Ohman, E.M., etc. People,European Heart J. 16:50-55, 1995).Research discloses short linear peptides (< 30 amino acid) and prevents or do Disturb integrin dependence adherency (Murayama, O., et al.,J. Biochem. 120:445-51, 1996).Also use Length range be 25-200 amino acid residue longer peptide, successfully blocked integrin dependence adhere to (Zhang, L., Et al.,J. Biol. Chem. 271(47):29953-57, 1996).In general, longer inhibitor peptides have than short The high affinity of peptide and/or slower dissociation speed, therefore be more effective inhibitor.Research is it is also shown that cyclic peptide inhibitor is Inhibitor in effective integrin body, for treat people's inflammatory disease (Jackson, D.Y., et al.,J. Med. Chem. 40:3359-68, 1997).A kind of method of generation cyclic peptide includes the conjunction that the end amino acid of wherein peptide is the peptide of cysteine At so that peptide can exist by the disulfide bond between end amino acid with annular form, it has already been proven that made for treating When blood system tumor, affinity and Half-life in vivo (such as U.S. Patent number 6,649,592 of Larson) are improved.
The MASP-2 inhibitor peptides of synthesis
The amino acid sequence of target area is important for MASP-2 function by simulating, come exemplary illustration for the present invention this MASP-2 peptide for inhibiting in aspect method.The magnitude range of peptide for inhibiting for implementing the method for the present invention be about 5 amino acid extremely About 300 amino acid.Table 3 provide for implement the present invention this in terms of exemplary peptide for inhibiting list.It can be by several The ability that one of measuring method works to candidate MASP-2 peptide for inhibiting as MASP-2 inhibitor is measured, these measurements Method includes that C4 cracking measuring method (referring to embodiment 2) of such as agglutinin specificity and C3b deposit measuring method (referring to embodiment 2)。
In some embodiments, MASP-2 peptide for inhibiting derives from MASP-2 polypeptide and is selected from the mature MASP-2 albumen of overall length (SEQ ID NO:6), or the specific domain derived from MASP-2 albumen, such as CUBI structural domain (SEQ ID NO:8), CUBIEGF structural domain (SEQ ID N O:9), EGF structural domain (SEQ ID NO:11) and serine protease domain (SEQ ID NO:12).As previously mentioned, research shows that the area CUBEGFCUBII be dimerization and in conjunction with MBL required for (Thielens Et al., ibid).It is especially confirmed in research of the identification human body with Asp105 to Gly105 homozygous mutation, the CUBI of MASP-2 Peptide sequence TFRSDYN (SEQ ID NO:16) in structural domain participates in combining MBL, and the mutation causes to lose from MBL compound Go MASP-2 (Stengaard-Pedersen, K., et al.,New England J. Med. 349:554-560, 2003)。
In some embodiments, MASP-2 peptide for inhibiting derives from conjunction with MASP-2 and participates in the agglutination of agglutinin complement pathway Fibroin.Several different agglutinins for participating in the approach, including mannan-binding lectin (MBL), L- are identified Fiber gelatinized protein, M- fiber gelatinized protein and H- fiber gelatinized protein (Ikeda, K., et al.,J. Biol. Chem. 262:7451-7454, 1987;Matsushita, M., et al.,J. Exp. Med. 176:1497-2284, 2000; Matsushita, M., et al.,J. Immunol. 168:3502-3506, 2002).These agglutinins are as homologous trimerization The oligomer of body subunit and be present in serum, each subunit have with carbohydrate identification structural domain N-terminal collagen Sample fiber.Have confirmed these different agglutinin combination MASP-2, agglutinin/MASP-2 compound by crack protein C4 and C2 and activating complement.H- fiber gelatinized protein have 24 amino acid amino petiolarea, with 11 Gly-Xaa-Yaa it is duplicate Collagen spline structure domain, the neck structure domain of 12 amino acid and 207 amino acid fibrinogen spline structure domain (Matsushita, M., et al.,J. Immunol. 168:3502-3506, 2002).H- fiber gelatinized protein combines GlcNAc, and make by derive from salmonella typhimurium (S. typhimurium), salmonella minnesota (S. minnesota) and Escherichia coli LPS coated human red blood cells agglutination.Have confirmed H- fiber gelatinized protein combination MASP-2 With MAp19 and activate lectin pathway, ibid.L- fiber gelatinized protein/P35 has been found and people also in relation with GlcNAc MASP-2 in serum and MAp19 is combined, and confirm compound activation lectin pathway (Matsushita, M., et al.,J. Immunol. 164:2281, 2000).It therefore, may include selected from MBL albumen for MASP-2 peptide for inhibiting of the invention (SEQ ID NO:21), H- fiber gelatinized protein (Genbank searching number NM_173452), M- fiber gelatinized protein (Genbank Searching number O00602) and L- fiber gelatinized protein (Genbank searching number NM_015838) at least five amino acid region.
More specifically, it is in 12 Gly-X-Y triplets that scientist, which has identified the MASP-2 binding site on MBL, “GKD GRD GTK GEK GEP GQG LRG LQG POG KLG POGNOG PSG SOG PKG QKG DOG KS”(SEQ ID NO:26) in, the triplet be located between the hinge and neck of MBP collagen spline structure domain C-terminal part (Wallis, R., et al.,J. Biol. Chem. 279:14065, 2004).The MASP-2 binding site area is fine in people H- fiber gelatinized protein and people L- It is also highly conserved for tieing up in gelled protein.Research points out that all three include amino acid sequence " OGK-X-GP " (SEQ ID NO:22 there is shared binding site in agglutinant protein), alphabetical " O " indicates hydroxyproline, letter in the amino acid sequence " X " expression hydrophobic residue (Wallis et al., 2004, ibid).Therefore, in some embodiments, for this side of the invention The MASP-2 peptide for inhibiting length in face is at least six amino acid and includes SEQ ID NO:22.It has been confirmed that including amino acid sequence It arranges " GLR GLQ GPO GKL GPO G " (SEQ ID NO:24) and the peptide derived from MBL combines MASP-2 (Wallis etc. in vitro People, 2004, ibid).For the combination of enhancing and MASP-2, peptide (" the GPO GPO in the adjacent two GPO triplets in every end can be synthesized GLR GLQ GPO GKL GPO GGP OGP O " SEQ ID NO:25), to enhance found in natural MBL albumen three strands Spiral formation (further describe and see Wallis, R., et al.,J. Biol. Chem. 279:14065, 2004)。
MASP-2 peptide for inhibiting is also available from people's H- fiber gelatinized protein comprising shared from H- fiber gelatinized protein The combined area MASP-2 sequence " GAO GSO GEK GAO GPQ GPO GPO GKM GPK GEO GDO " (SEQ ID NO: 27).It further include the peptide derived from people L- fiber gelatinized protein comprising the shared combined area MASP-2 from L- fiber gelatinized protein Sequence " GCO GLO GAO GDK GEA GTN GKR GER GPO GPO GKA GPO GPN GAO GEO " (SEQ ID NO:28)。
MASP-2 peptide for inhibiting is also available from C4 cracking position, is such as connected to the C4 cracking of Antithrombin III C-terminal part Position " LQRALEILPNRVTIKANRPFLVFI " (SEQ ID NO:29) (Glover, G.I., et al.,Mol. Immunol. 25:1261 (1988))。
Table 3: illustrative MASP-2 peptide for inhibiting
Note: alphabetical " O " indicates hydroxyproline.Alphabetical " X " is hydrophobic residue.
To the peptide for deriving from C4 cracking position and other peptides at MASP-2 serine protease position can be inhibited to carry out chemistry Modification, to become irreversible protease inhibitors.For example, suitable modification may include still being not necessarily limited to C-terminal, Asp Or Glu or the halogen methyl ketone (Br, Cl, I, F) being added on function side chain；Halogen second on amino or other function side chains Acyl (or other α-halogen acetyl) group；Epoxy group on aminoterminal or c-terminus or function side chain or containing the group of imines； The either imidoate on aminoterminal or c-terminus or other function side chains.These modifications provide the covalent knot by peptide Close and permanently inhibit this advantage of enzyme.This may cause effective dose is lower and/or inhibitor peptides need administration frequency compared with It is low.
Other than above-mentioned peptide for inhibiting, the MASP-2 peptide for inhibiting for the method for the present invention includes containing by methods described herein The peptide in the area CDRH3 of the combination MASP-2 of the anti-MASP-2 MoAb obtained.The sequence of CDR region for synthetic peptide can pass through Means known in the art measure.Heavy chain variable region is the peptide that normal length range is 100-150 amino acid.Light chain variable region It is the peptide that normal length range is 80-130 amino acid.CDR sequence inside heavy chain and light chain variable region includes only about 3-25 amino acid sequence, those of ordinary skill in the art are easy to that it is sequenced.
It will be understood by those skilled in the art that the substantially homologous variation of above-mentioned MASP-2 peptide for inhibiting can also have MASP-2 inhibitory activity.Illustrative variation including but not necessarily limited to the c-terminus or amino terminal portion of subject peptide have insertion, The peptide and its mixture of missing, displacement and/or the amino acid added.It is therefore believed that these are with MASP-2 inhibitory activity Homeopeptide method for use in the present invention.The peptide may also include duplication motif and other modifications with conservative substitution.This Literary other parts describe conservative variant, including a kind of amino acid with the another of the properties such as same charge, size or hydrophobicity A kind of exchange of amino acid.
MASP-2 peptide for inhibiting can be modified to increase solubility and/or maximize positive or negative charge, to make The section for being more closely similar to intact proteins.Derivative can have or the accurate grade amino acid knot of no peptide disclosed herein Structure, as long as derivative functionally still retains required MASP-2 rejection characteristic.The modification may include amino acid substitution, i.e., By one of 20 kinds of commonly known amino acid or another amino acid substitution, it is attached to required feature (such as enzyme degradation-resistant) Derivatization amino acid or substituted amino acid replace perhaps by D amino acid substitution or by the other one or more ammonia of simulation The molecule or compound (such as carbohydrate) of the native conformation and function of base acid or peptide replace；Amino acid deletions；Amino acid Insertion, that is, be inserted into one of 20 kinds of commonly known amino acid or another amino acid, insertion are attached with required feature (such as enzyme drop Solution resistance) derivatization amino acid or substituted amino acid be perhaps inserted into D amino acid or by the other one or more ammonia of simulation The molecule or compound (such as carbohydrate) of the native conformation and function of base acid or peptide replace；Or it is female by other simulation The native conformation of body peptide, the molecule of distribution of charges and function or compound (such as carbohydrate or nucleic acid monomer) replace.Peptide It can also be modified by acetylation or amidation.
Peptide for inhibiting derived from being synthesized according to known technologies such as peptide biosynthesis, carbohydrate biosynthesis.When beginning, Technical staff can determine the conformation of target peptide according to suitable computer program.Once knowing the structure of peptide disclosed herein As then technical staff just can be determined to carry out taking for what classification in one or more sites in a manner of rational design In generation, so that the derivative of formation remains the basic conformation and distribution of charges of parent peptide, but can possess in parent peptide and not deposit Feature or its feature better than feature existing for parent peptide.Once identifying candidate derivative molecule, so that it may use this paper institute Measuring method is stated to measure derivative, to determine whether they play the effect of MASP-2 inhibitor.
The screening of MASP-2 peptide for inhibiting
Molecule modeling and rational molecular design can be used also to generate and screen the molecular structure of the simulation critical binding domain MASP-2 And inhibit the peptide of the complement activity of MASP-2.Molecular structure for modeling includes the CDR region of anti-MASP-2 monoclonal antibody, with And it is known to the highly important target area of MASP-2 function, these regions include area required for foregoing dimerization Domain is related to combining the region and serine protease position of MBL.For identifying that the method for combining the peptide of particular target is this Field is well-known.For example, molecular imprinting can be used for from the beginning constructing macromolecular structure, such as in conjunction with the peptide of specific molecular.Ginseng See such as Shea, K.J., " Molecular Imprinting of Synthetic Network Polymers:The De Novo synthesis of Macromolecular Binding and Catalytic Sties," TRIP 2(5) 1994。
For example, a kind of method for preparing MASP-2 combination peptide mimics is as follows.Make known MASP-2 binding peptide or tool There is the functional monomer (template) of the combined area of the anti-MASP-2 antibody of MASP-2 inhibiting effect to polymerize.Then removing template is removed, is connect Make the second class monomer polymerization in the gap that template leaves, obtain with properties needed for one or more similar to template Recruit.Other than preparing peptide in this way, other MASP-2 binding molecules as MASP-2 inhibitor can be also prepared, it is all Such as polysaccharide, nucleosides, drug, nucleoprotein, lipoprotein, carbohydrate, glycoprotein, steroids, lipid and other bioactivity materials Material.This method is suitable for designing various biological simulation object more more stable than its natural counterpart, because they are usually Made of free radical polymerization by function monomer, the compound with the biological skeleton that cannot degrade is produced.
Peptide synthesis
Technology preparation MASP-2 peptide for inhibiting well-known in the art, the solid phase such as initially proposed by Merrifield can be used Synthetic technology (Merrifield,J. Amer. Chem. Soc. 85:2149-2154, 1963).For example, can be according to manufacturer The specification of offer is realized automatic using Applied Biosystems 431A peptide synthesizer (Foster City, Calif.) Synthesis.Other technologies see, for example, Bodanszky, M., et al., peptideSynthesis, the second edition, John Wiley & Sons, 1976 and other bibliography well known by persons skilled in the art.
The engineered technology of standard genetic well known by persons skilled in the art also can be used to prepare peptide.For example, can pass through The method of enzyme carries out DNA expression, by DNA in the presence of required amino acid in the nucleic acid insertion expression vector of encoded peptide Peptide is translated into, thus to prepare peptide.Then, using chromatographic technique or electrophoretic techniques by peptide purification, or by by encoded peptide Carrier protein method in sequence insertion expression vector synchronous with the nucleic acid sequence of encoding carrier proteins realizes, the carrier egg It is white to be merged with peptide, then scaled off from peptide again.Can be used chromatographic technique, electrophoretic techniques or immunological technique (such as through The antibody of carrier protein and be integrated on resin) protein isolate-peptide fusions.It can be used chemical method or enzyme method (such as logical Perhydrolase) carry out cleavage of peptide.
The MASP-2 peptide for inhibiting for the method for the present invention can also be produced with recombinant host cell according to routine techniques.For Expression MASP-2 inhibits peptide-coding sequence, it is necessary to by the adjusting of the nucleic acid molecules of encoded peptide and control expression vector transcriptional expression Sequence is operatively connected, and is then introduced into host cell.Other than transcriptional regulatory sequences (such as promoter and enhancer), table It may include translational regulation sequence and marker gene up to carrier, marker gene is suitable for the cell that selection carries expression vector.
It can be with " gene instrument " and with the methods of phosphoramidite method come the nucleic acid molecules of composite coding MASP-2 peptide for inhibiting.If Chemically synthesized double-stranded DNA is needed in such as application of gene or genetic fragment synthesis, then prepares every complementary strand respectively.Short base It, can be by synthesizing complementary strand because the production of (60-80 base-pair) is technically simple and easy, then they anneal, and it is real to come It is existing.For the production of longer gene, to be assembled by the single-chain fragment that length is 20-100 nucleotide by modular form It synthesizes gene (double-strand).The summary of related polynucleotides 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 Climie, S. et al.,Proc. Nat'l Acad. Sci. USA 87:633, 1990。
Micromolecular inhibitor
In some embodiments, MASP-2 inhibitor is micromolecular inhibitor, including the natural and synthesis with low molecular weight Substance, such as peptide, peptide mimics and Nonpeptide inhibitors (including oligonucleotides and organic compound).The small molecule of MASP-2 Inhibitor can be generated according to the molecular structure of the variable region of anti-MASP-2 antibody.
Micromolecular inhibitor can also be designed according to the crystal structure of MASP-2 using computer drug to be designed and produce Raw (Kuntz I.D., et al.,Science 257:1078, 1992).It has been confirmed that the crystal structure of rat MASP-2 (Feinberg, H., et al.,EMBO J. 22:2348-2359, 2003).Using the method for the descriptions such as Kuntz, by MASP- 2 crystal structural coordinates input computer program (such as DOCK), and computer will export a series of expected small points for combining MASP-2 Minor structure.The use of these computer programs is well-known to those skilled in the art.For example, being pressed down using HIV-1 protease The crystal structure of preparation is evaluated by application program DOCK from Cambridge crystallographic data library (Cambridge Crystallographic database) in the compound that finds and enzyme binding site matching, to identify as HIV- 1 protease inhibitors unique non-peptide ligand (Kuntz, I.D., et al.,J. Mol. Biol. 161:269-288, 1982;DesJarlais, R.L., et al.,PNAS 87:6644-6648, 1990)。
It is accredited as potentially to screen with computer approach using all MASP-2 binding assays as described in Example 10 A series of small molecule structures of MASP-2 inhibitor.Then in all functional examination methods as described in Example 2 to being confirmed as It is analyzed in conjunction with the small molecule of MASP-2, to determine whether they inhibit MASP-2 dependent complement activation.
MASP-2 soluble recepter
Think that other suitable MASP-2 inhibitor include MASP-2 soluble recepter, those of ordinary skill in the art can be used Known technology is produced.
The expression inhibiting agent of MASP-2
The present invention this in terms of another embodiment in, MASP-2 inhibitor is to be able to suppress MASP-2 dependent complement The MASP-2 expression inhibiting agent of activation.The present invention this in terms of implementation in, representative MASP-2 expression inhibiting agent includes MASP-2 antisense nucleic acid molecule (such as antisense mRNA, antisense DNA or antisense oligonucleotides), MASP-2 ribozyme and MASP-2 RNAi molecule.
Antisense RNA and anti-sense DNA molecules are played and hybridizing and preventing the translation of MASP-2 albumen with MASP-2 mRNA Directly block the effect of MASP-2 mRNA translation.Antisense nucleic acid molecule can construct in a number of different ways, as long as it can Interfere the expression of MASP-2.For example, can by make MASP-2 cDNA (SEQ ID NO:4) code area (or part thereof) it is opposite It is inverted in its normal transcription direction for the transcription of its complementary series, to construct antisense nucleic acid molecule.
Antisense nucleic acid molecule is usually essentially identical at least part of one or more target genes.However, nucleic acid is not It needs identical to inhibit expression.Higher homology is generally available to mend the use of shorter antisense nucleic acid molecule It repays.The smallest percentage identity normally about 65% or more, but higher percentage identity can send out the expression of endogenous sequence Wave more effective resistance inhibitor action.Generally preferably essentially about 80% or more biggish percentage identity, although usually optimal Choosing about 95% is to identical.
Antisense nucleic acid molecule does not need have introne identical with target gene or exon mode, the non-coding of target gene Section may be equivalent with coding section in terms of the Antisense Suppression for obtaining expression of target gene.At least about 8 or so nucleosides The DNA sequence dna of acid can be used as antisense nucleic acid molecule, although it is preferred that longer sequence.In the present invention, useful MASP-2 inhibits The representative example of agent is antisense MASP-2 nucleic acid molecules, what the molecule and the nucleic acid sequence shown in SEQ ID NO:4 formed The complementary series of MASP-2 cDNA has at least 90% identity.Nucleic acid sequence encoding shown in SEQ ID NO:4 is by SEQ ID NO:5 Shown in amino acid sequence composition MASP-2 albumen.
It is the another kind that can be used for reducing MASP-2 albumen synthesis level that antisense oligonucleotides targeting, which combines MASP-2 mRNA, Mechanism.For example, the synthesis of 2 type acetylcholinergic receptor of polygalacturonase and hydroxycholine is directed to their corresponding mRNA sequences Antisense oligonucleotides inhibit (U.S. Patent number 5,739,119 of Cheng and the U.S. Patent number 5,759 of Shewmaker, 829).In addition, with nucleoprotein cyclin, multidrug resistance gene (MDG1), ICAM-1, e-selectin, STK-1, Corpus straitum GABAAReceptor and people EGF confirm Antisense Suppression example (see, for example, the U.S. Patent number 5 of Baracchini, 801,154；The U.S. Patent number 5,789,573 of Baker；The U.S. Patent number 5,718,709 of Considine；And The U.S. Patent number 5,610,288 of Reubenstein).
Document describes so that those of ordinary skill can determine which oligonucleotides system for use in the present invention, including RNA enzyme H cracking is used to detect the proper site of said target mrna as the mark of sequence accessibility in transcript.Scherr, M., Et al.,Nucleic Acids Res. 26:5079-5085, 1998;Lloyd, et al.,Nucleic Acids Res. 29:3665-3673, 2001.The antisense oligonucleotides acid blend complementary with MASP-2 transcript some regions is added to expression In the cell extract (such as liver cell) of MASP-2, hybridized to generate the position attacked vulnerable to RNA enzyme H.This method can To be combined with area of computer aided sequence selection, the computer assisted sequence selection can according to sequence formed dimer, Hairpin structure or the relative ability of other secondary structures select to predict the optimal sequence for antisense composition, and the structure can drop Specific binding low or that inhibition is to host cell said target mrna.Can be used OLIGO Primer Analysis Software (Rychlik, I., 1997) and BLASTN 2.0.5 algorithm software (Altschul, S.F., et al.,Nucl. Acids Res. 25:3389- 3402,1997) it is considered to carry out these secondary structure analysis and target area selection.For the length of the antisense compounds of target sequence Degree preferably includes about 8 to about 50 nucleotide.It particularly preferably include the antisense oligonucleotides of about 9 to about 35 or so nucleotide Acid.Inventors believe that implementing the present invention is based on the highly preferred range of the method for antisense oligonucleotides to be 9 to 35 nucleotide (i.e. 9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33, The nucleotide of 34 or 35 bases longs or so) all oligonucleotides composition.Highly preferred MASP-2 mRNA target region is position In or neighbouring AUG translation initiation codon region, and with the basic complementary sequence in the 5th ' area of mRNA, such asMASP 2Gene Sequence between -10 and+10 regions of nucleotide sequence (SEQ ID NO:4).Illustrative MASP-2 expression inhibiting agent is shown in Table 4。
The exemplary table of table 4:MASP-2 reaches inhibitor
As described above, terms used herein " oligonucleotides " refers to ribonucleic acid (RNA) or DNA (DNA) or its mould The oligomer or polymer of quasi- object.The term also includes by (skeleton) covalent bond institute between naturally occurring nucleotide, sugar and nucleosides The few nucleobase (oligonucleobase) of those of composition and the oligonucleotides with non-naturally occurring modification.These are repaired It adorns and makes it possible to introduce certain certain required properties that can not be provided by naturally occurring oligonucleotides, such as toxicity reduction, The stability of nuclease-resistant degradation improves and cellular uptake enhancing.In an exemplary embodiment, antisense chemical combination of the invention Object is different from n DNA since the modification of phosphodiester backbone extends the service life of antisense oligonucleotides, and wherein phosphoric acid replaces Base is by thiophosphoric acid ester interchange.Similarly, the one or both ends of oligonucleotides can be inserted in phase in nucleic acid chains between one or more Acridine derivatives between adjacent base-pair replace.
Another substitution of antisense strategies is using " RNA interference " (RNAi).Double-stranded RNA (dsRNA) can be in mammalian body Inside cause gene silencing.The natural function and co-suppression of RNAi seems that Protecting gene group is not (such as anti-by Mobile genetic elements Transcribe transposons) and generated in host cell when its activation aberrant RNAs or dsRNA virus attack (see, for example, Jensen, J., et al.,Nat. Genet. 21:209-12, 1999).Double-stranded RNA point can be capable of forming by synthesizing two The RNA chain of son prepares double stranded rna molecule, and the length of every chain is about 19-25 (such as 19-23) a nucleotide.For example, with In the dsRNA molecule of the method for the present invention may include the RNA for being equivalent to sequence listed by table 4 and its complementary series.Preferably at least one RNA chain has 3 ' jags of 1-5 nucleotide.The RNA chain of synthesis is combined under conditions of forming duplex molecule. RNA sequence may include the part of at least eight nucleotide of SEQ ID NO:4, and total length is 25 nucleotide or following.Particular target The those of ordinary skill in the art that are designed as of siRNA sequence grasped.Can get design siRNA sequence and guarantee expression have to Few 70% strikes the commerce services (Qiagen, Valencia, Calif) subtracted.
DsRNA can be used as pharmaceutical composition and give and carry out by known method, and amplifying nucleic acid is introduced into required target cell In.Usually used gene transfer method includes calcium phosphate, DEAE- glucan, electroporation, microinjection and viral methods.This A little methods in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & It is instructed in Sons, Inc., 1993.
Ribozyme can also be used to reduce the amount of MASP-2 and/or bioactivity, such as target the ribozyme of MASP-2 mRNA. Ribozyme is catalytic RNA molecules, can be cracked with the nucleic acid molecules with the complete or partial homologous sequence of ribozyme sequence.It can be with Ribozyme transgenosis is designed, the ribozyme transgenes encoding and target RNA-specific match and crack phosphodiester backbone in specific position RNA ribozyme, to make target RNA Functional inactivation.When carrying out this cracking, ribozyme itself has no change, therefore can be again It recycles and cracks other molecules.It include that ribozyme sequence imparts the activity of antisense RNA cleaving rna in antisense RNA, to increase The activity of antisense constructs.
Ribozyme for carrying out the present invention generally includes hybridization region and catalytic domain, hybridization region at least about 9 nucleotide, and extremely The nucleotide sequence of small part target MASP-2 mRNA is complementary, and catalytic domain is suitable for cracking target MASP-2 mRNA (referring generally to EPA No. 0 321 201; WO88/04300;Haseloff, J. et al.,Nature 334:585-591, 1988; Fedor, M.J. et al.,Proc. Natl. Acad. Sci. USA 87:1668-1672, 1990;Cech, T.R. et al.,Ann. Rev. Biochem.55:599-629, 1986)。
The form that ribozyme can mix the RNA oligonucleotide of ribozyme sequence is directly targeted cell, or as needed for coding The expression vector for the ribozyme rna wanted and be introduced into cell.It can make with the roughly the same mode for antisense polynucleotides With and using ribozyme.
It can be prepared by any method known in the art synthesized for DNA and RNA molecule for the method for the present invention Antisense RNA and DNA, ribozyme and RNAi molecule.These methods include well-known in the art for few dezyribonucleoside The chemical synthesising technology of acid and oligoribonucleotide, such as solid phase phosphoramidite chemical synthesis.Alternatively, encoding antisense can be passed through The in vitro and in vivo of the DNA sequence dna of RNA molecule is transcribed to generate RNA molecule.This DNA sequence dna can be impregnated in various loads Suitable RNA polymerase promoter (such as T7 or SP6 polymerase promoter) is inserted in body, in carrier.Alternatively, can will depend on In promoter used, the antisense cDNA constructs of composing type or inductive synthesis antisense RNA are steadily introduced into cell line.
Various well-known DNA molecular modifications can be introduced, to increase stability and half-life period.Useful modification includes But it is not limited to for ribonucleotide or deoxyribonucleotide flanking sequence to be added to 5 ' ends of molecule and/or 3 ' ends, or in widow Thiophosphate or 2 ' O- methyl are used in deoxyribonucleotide skeleton rather than phosphodiester bond.
V. pharmaceutical composition and delivering method
Administration
On the other hand, the present invention is provided to inhibit in the subject with disease as described herein or the patient's condition MASP2 according to Rely the composition of the side effect of property complement activation comprising by the MASP-2 inhibitor comprising therapeutically effective amount and pharmaceutically acceptable The composition of carrier give subject.The treatment that can treat or improve MASP-2 dependent complement activation related disease is effective MASP-2 inhibitor is given the subject for needing it by dosage.Treatment effective dose refers to that MASP-2 inhibitor is enough to cause to change The amount of kind symptom relevant to disease or the patient's condition.
Can be by standard pharmaceutical procedures, using experimental animal model, (all expression people MASP-2 as described in example 1 above turn The mouse MASP-2-of gene/- mouse model), to measure the toxicity and therapeutic efficiency of MASP-2 inhibitor.Use these animal moulds Standard method can be used to determine NOAEL (side effect of no observation is horizontal) and MED (minimum effective dose) in type.NOAEL/ Dose ratio between MED effect is treatment ratio, is indicated with NOAEL/MED ratio.It most preferably treats ratio or index is high MASP-2 inhibitor.The data obtained from cell culture analytical and zooscopy can be used to the dosage for being formulated for human body Range.The dosage of MASP-2 inhibitor is preferably within the scope of circulation composition, including almost without toxicity or does not have virose MED.Dosage can change within this range, this depends on used dosage form and used administration route.
In some embodiments, MASP-2 inhibitor is for treating, inhibiting, mitigating or preventing to suffer from or risky generation The therapeutic efficiency of the fibrosis of the mammalian subject of the disease or illness that are caused or aggravated by fibrosis and/or inflammation passes through One or more determinations below: inflammation and one or more markers of cicatrization in nephridial tissue (for example, TGF β -1, CTFF, IL-6, Apoptosis, fibronectin, laminin, collagen, EMT, infiltrating macrophages) reduction；Inflammation and fibrosis kidney The soluble markers of disease, which discharge to urine and blood plasma, reduces (for example, by measurement renal excretion function).
For any compound preparation, animal model can be used to evaluate treatment effective dose.For example, can be in animal Prepared in model up to circulating plasma concentration range including MED dosage.It can also be for example, by high performance liquid chromatography (HPLC) To measure the quantitative level of MASP-2 inhibitor in blood plasma.
Other than toxicity research, the amount of MASP-2 albumen according to present in lived subject and The binding affinity of MASP-2 inhibitor estimates effective dose.It has been confirmed that MASP-2 present in normal human subject's serum MASP-2 quantitative determination process can be used within the scope of 500 ng/ml low-levels to measure the MASP-2 water of specific subject in level Flat (it is described in Moller-Kristensen M., et al.,J. Immunol. Methods 282:159-167, 2003)。
Composition comprising MASP-2 inhibitor give dosage generally according to subject age, weight, height, gender, Overall medical condition and medical history and change.It for example, can be in about 0.010-10.0 mg/kg, preferably 0.010-1.0 mg/ Kg, more preferable 0.010-0.1 mg/kg subject weight dosage range in give MASP-2 inhibitor, such as anti-MASP-2 is anti- Body.In certain embodiments, the composition includes the combination of anti-MASP-2 antibody and MASP-2 peptide for inhibiting.
The MASP- of the present invention of particular subject can be measured according to complement measuring method well known to the skilled person The therapeutic efficiency and suitable dosage of 2 inhibitor combinations and method.Complement generates a variety of specific products.Nearest ten Nian Jian has developed sensitive and specific measuring method, and most of this kind of activation products are all commercially available, including small Activation segment C3a, C4a and C5a and big activation segment iC3b, C4d, Bb and sC5b-9.Most of this kind of measuring method is all Segment is utilized and is exposed to without being an exposure to neoantigen (the new antigen/ that it is formed by native protein Neoantigen the monoclonal antibody) to react, this makes these measuring methods very simple and specific.It is most of to all rely on Elisa technique, in spite of when radioimmunoassay be still used for C3a and C5a.Radioimmunoassay method crude Section and its " desArg " segment, these segments are the principal modes being present in circulation.Crude segment and C5adesArgIt is logical It crosses combination cell surface receptor and is rapidly cleared in humans, thus exist with extremely low concentration, and C3adesArgThen not combination cell, only It is built up in blood plasma.Measurement C3a provides the mark that complement activation is sensitive, does not depend on approach.Can by measurement Bb segment come Evaluate alternative route activation.The liquid product sC5b-9 for detecting film attack pathway activation, provides the card that complement is fully active According to.Because lectin pathway and classical pathway all generate same activation products C4a and C4d, both segments are measured simultaneously It is not provided with which approach in this two approach that closes produces any information of activation products.
MASP-2 dependent complement activation inhibits at least one variation below for being characterized in that complement system ingredient, institute State variation be due to giving MASP-2 inhibitor according to the method for the present invention caused by: MASP-2 dependent complement activation system product Inhibition (such as by described in embodiment 2 measure) that C4b, C3a, C5a and/or C5b-9 (MAC) are formed or generated, C4 cracking and The reduction (such as by described in embodiment 10 measure) or C3 cracking and the reduction of C3b deposition of C4b deposition are (such as by embodiment 10 measurements).
Other medicaments
In certain embodiments, preventing, treating, reverse and/or inhibit the method for fibrosis and/or inflammation includes giving The a part of MASP-2 inhibitor (for example, MASP-2 inhibiting antibody) as therapeutic scheme, and be suitable for inhibiting fibrosis And/or one or more other medicines, biological products or the therapy intervention of inflammation.In certain embodiments, other medicines, life Tetramune or therapy intervention are suitble to specific symptoms related with the disease or illness that are caused or aggravated by fibrosis and/or inflammation. For example, MASP-2 inhibiting antibody can be used as a part of therapeutic scheme together with one or more immunosuppressor such as first ammonia butterfly Purine, cyclophosphamide, imuran and mycophenolate are given.In addition for example, MASP-2 inhibiting antibody can be used as therapeutic scheme A part is together with one or more medicaments through design increase blood flow (for example, nifedipine, Amlodipine, diltiazem, non-Lip river Horizon or nicardipine) it gives.In addition for example, a part that MASP-2 inhibiting antibody can be used as therapeutic scheme subtracts together with expection One or more medicaments of few fibrosis, such as d- penicillamine, colchicin, PUVA, relaxain, cyclosporin, TGF β are blocked Agent and/or p38 MAPK blocking agent are given.In addition for example, MASP-2 inhibiting antibody can be used as a part of therapeutic scheme together with Steroids or bronchodilators are given.
Composition and method comprising MASP-2 inhibitor (for example, MASP-2 inhibiting antibody) can optionally comprising a kind of or A variety of other therapeutic agents, can enhance MASP-2 inhibitor activity or its with addition or cooperative mode provide relevant treatment Function.For example, in the case where treatment suffers from the subject of the disease or illness that are caused or aggravated by fibrosis and/or inflammation, One or more MASP-2 inhibitor can with one or more other antifibrotic agents and/or one or more anti-inflammatories and/ Or immunosuppressive combination gives (including co- give).
MASP-2 inhibitor (for example, MASP-2 inhibiting antibody) can be with for example general immunosupress of other therapeutic agents Property drug such as corticosteroid, inhibitive ability of immunity or cytotoxic agent and/or antifibrotic agents are applied in combination.
Pharmaceutical carrier and delivery vehicle
In general, MASP-2 inhibitor combination of the invention is suitble to when being combined with any other selected therapeutic agent In included in pharmaceutically acceptable carrier.Carrier be it is nontoxic, biocompatible, must not select to MASP-2 inhibitor The bioactivity of (and with its associated with any other therapeutic agent) has adverse effect on.Peptide it is exemplary pharmaceutically acceptable Carrier be described in the U.S. Patent number 5,211,657 of Yamada.It can be with for anti-MASP-2 antibody of the invention and peptide for inhibiting Be configured to solid, semisolid, gel, liquid or gas form preparation, such as tablet, capsule, pulvis, granule, ointment, Solution, suppository (depositories), inhalant and injection, for oral, parenteral or surgical administration.The present invention is also Including administering locally to composition by coating medical device etc..
It include distilled water, phosphorus for the suitable carrier by injection, infusion or flushing and the parenteral delivery of local delivery Acid buffering physiological saline, standard ringer's solution or Lactated Ringer liquid, glucose solution, HankShi solution or propylene glycol.This Outside, sterile expressed oi can be used as solvent or suspension media.For this purpose, any biocompatibility oil can be used, including close At monoglyceride or diglyceride.In addition, fatty acid (such as oleic acid) is in the preparation of injection.It can be by carrier and drug Preparing becomes liquid preparation, suspension, polymerizable or not polymerizable gelling agent, paste or ointment.
Carrier may also comprise delivery vehicle so that drug delivery continues (extend, delay or adjust), or enhancing is controlled Treat delivering, absorption, stability or the Pharmacokinetic Characteristics of drug.This delivery vehicle may include but be not limited to following reality Example: by protein, liposome, carbohydrate, anthropogenics, inorganic compound, polymeric hydrogel or copolymerization water-setting Particle, microballoon, nanosphere or the nanoparticle of glue and polymer micelle composition.Suitable hydrogel and micella delivery system include WO PEO:PHB:PEO copolymer and copolymer/cyclodextrin complexes disclosed in 2004/009664 A2 and U.S. Patent application PEO and PEO/ cyclodextrin complexes disclosed in 2002/0019369 A1 of publication number.These hydrogels can locally injecting to expectation Action position, or subcutaneously or intramuscularly inject to form slow-release depot.
For intra-articular delivering, MASP-2 inhibitor can be loaded on the liquid or gel carrier, above-mentioned of above-mentioned injectable In the sustained release delivery medium or hyaluronic acid or derivatives of hyaluronic acids of injectable.
Oral administration for non-peptide energy drug, MASP-2 inhibitor can be loaded into inert filler or diluent, all In sucrose, cornstarch or cellulose.
For local administration, MASP-2 inhibitor can be loaded into ointment, lotion, cream, gelling agent, drops, suppository, In spray, liquid preparation or pulvis or via in the gel or microcapsules delivery system of transdermal patch.
Various intranasals and transpulmonary delivery system are being researched and developed, including aerosol, metered dose inhaler, Diskus and Nebulizer can be suitably adapted for delivering the drug of the present invention in aerosol, inhalant or atomization delivery vehicle respectively.
(ICV) is delivered in intrathecal (IT) or the ventricles of the brain, suitable aseptic delivery system (such as liquid preparation；Gelling agent is mixed Suspension etc.) it can be used to give drug of the invention.
Composition of the invention may also include biocompatible excipient, such as dispersing agent or wetting agent, suspending agent, dilution Agent, buffer, penetration enhancer, emulsifier, adhesive, thickener, corrigent (for being administered orally).
The pharmaceutical carrier of antibody and peptide
More particularly, as anti-MASP-2 antibody and peptide for inhibiting, can by the compound of injection dosage solution or Suspension parenterally gives exemplary dosage forms, and the compound is included in physiologically acceptable diluent and pharmaceutical carrier Interior, pharmaceutical carrier can be sterile liquid, such as water, oil, salt water, glycerol or ethyl alcohol.In addition, including anti-MASP-2 antibody and suppression Auxiliary substance wetting agent or emulsifier, surfactant, pH buffer substance etc. may be present in the composition of peptide processed.Medicine group The additional component for closing object includes grease (such as grease of animal, plant or synthesis source), such as soybean oil and mineral oil.Generally For, dihydric alcohol such as propylene glycol or polyethylene glycol are the liquid-carriers of preferred injection solution.
Anti- MASP-2 antibody and peptide for inhibiting can also be given in the form of long-acting injection or implantation preparation, these preparations can be pressed Allow the mode of active agent sustained release or pulse release to prepare.
The pharmaceutically acceptable carrier of expression inhibiting agent
More particularly, as the expression inhibiting agent for the method for the present invention, providing can comprising above-mentioned expression inhibiting agent and pharmacy The carrier of receiving or the composition of diluent.Composition also may include colloidal dispersion system.
Pharmaceutical composition including expression inhibiting agent may include but be not limited to solution, emulsion and the system containing liposome Agent.These compositions can be prepared by various components, and the component includes but is not limited to preformed liquid, self-emulsification solid and self-emulsifying Semisolid.The preparation of these compositions generally includes to mix expression inhibiting agent and one or more ingredients below: buffering Agent, antioxidant, low molecular weight polypeptide, protein, amino acid, carbohydrate (including glucose, sucrose or dextrin), chelating Agent (such as EDTA), glutathione and other stabilizers and excipient.Neutral buffered saline is white with nonspecific sera The salt water of albumen mixing is the example of suitable diluents.
In some embodiments, composition can be produced and be configured to emulsion, and emulsion is usually a kind of liquid with drop Form is dispersed in the heterogeneous system in another liquid and (referring to Idson, is loaded inPharmaceutical Dosage Forms, The first volume, Rieger and Banker (chief editor), Marcek Dekker, Inc., N.Y., 1988).For emulsion preparations The example of naturally occurring emulsifier include gum arabic, beeswax, lanolin, lecithin and phosphatide.
In one embodiment, the composition including nucleic acid can be configured to microemulsion.Microemulsion used herein refers to The system of water, oil and amphiphile, it is that single optical isotropy and thermodynamically stable liquid solution (are carried referring to Rosoff InPharmaceutical Dosage Forms, the first volume).Method of the invention can also be used liposome by antisense widow's core Thuja acid shifts and is delivered to required position.
The pharmaceutical composition and preparation of expression inhibiting agent for local administration may include transdermal patch, ointment, lotion, Cream, gelling agent, drops, suppository, spray, liquid preparation and pulvis.Conventional pharmaceutical carriers and aqueous base can be used Matter, powdered substrate or oleaginous base and thickener etc..
Administration mode
The pharmaceutical composition comprising MASP-2 inhibitor can be given in many ways, this depends on being part or systemic applications Mode is most suitable for disease to be treated.In addition, composition of the invention can be by being coated with or mixing implantable doctor by composition Treat device above or the inside and deliver.
Systemic delivery
Terms used herein " systemic delivery " and " systemic applications " refer to including but not limited to oral and parenteral outer approach, packet Include intramuscular (IM), subcutaneous, intravenous (IV), intra-arterial, sucking, sublingual, oral cavity, part, percutaneously, intranasal, rectum, vagina and its Its administration route, the drug delivered is effectively dispersed to one or more positions of expected therapeutic effect by they.For this The optimization approach of the systemic delivery of invention includes intravenous, intramuscular, subcutaneous and sucking.It should be understood that for for this Selected drug, exact systemic applications approach will partly consider drug pair and specific administration in invention concrete composition The sensibility of the relevant metabolic conversion approach of approach is determined.For example, other than peptide energy drug may be most suitable for by taking orally Approach is given.
Antibody and polypeptide delivery can be inhibited into the subject for needing it MASP-2 by any suitable method.Delivering The method of MASP-2 antibody and polypeptide include by oral administration, lung, it is parenteral (such as in intramuscular, peritonaeum, intravenous (IV) or subcutaneous Injection), sucking (such as via fine powder preparation), percutaneous, intranasal, vagina, rectum or sublingual administration routes and give, can will It is configured to the dosage form suitable for respective administration route.
For example, can be by inhibiting antibody and peptide to be applied on the body film that can absorb polypeptide MASP-2, example Such as nose film, gastrointestinal membranes and bung skin, and it is introduced into vivo.Usually polypeptide and penetration enhancer are applied to together absorbable On film (see, for example, Lee, V.H.L.,Crit. Rev. Ther. Drug Carrier Sys. 5:69, 1988; Lee, V.H.L., J. Controlled Release13:213, 1990; Lee, V.H.L., Ed., Peptide and Protein Drug Delivery,Marcel Dekker, New York (1991);DeBoer, A.G. et al.,J. Controlled Release 13:241, 1990).For example, STDHF is the synthesis of derivatives of fusidinic acid, it is class in structure It is similar to the steroid surfactant of cholate, has been used as the penetration enhancer of nasal delivery there.(Lee, W.A.,Biopharm.22, 11/12 month nineteen ninety).
The MASP-2 combined with other molecules (such as lipid) can be introduced and inhibit antibody and polypeptide, with protect polypeptide not by Enzyme degradation.For example, the covalent bond of polymer, especially polyethylene glycol (PEG) has been used to protect certain protein not by body Interior enzyme hydrolysis, thus extend half-life period (Fuertges, F., et al.,J. Controlled Release 11:139, 1990).It has been reported that it is many for protein delivery polymer system (Bae, Y.H. et al.,J. Controlled Release 9:271, 1989;Hori, R. et al.,Pharm. Res. 6:813, 1989;Yamakawa, I. et al.,J. Pharm. Sci. 79:505, 1990;Yoshihiro, I. et al.,J. Controlled Release 10:195, 1989;Asano, M. et al.,J. Controlled Release 9:111, 1989;Rosenblatt, J. et al.,J. Controlled Release 9:195, 1989; Makino, K., J. Controlled Release 12:235, 1990;Takakura, Y. et al.,J. Pharm. Sci. 78:117, 1989;Takakura, Y. et al.,J. Pharm. Sci. 78:219, 1989)。
Recently, liposome that serum stability and circulating half-life are improved is developed (see, for example, the U.S. of Webb The patent No. 5,741,516).Moreover, to liposome and liposome sample prepared product as possible pharmaceutical carrier various methods into Summary gone (see, for example, the U.S. Patent number 5,567,434 of Szoka；The U.S. Patent number 5,552,157 of Yagi； The U.S. Patent number 5,565,213 of Nakamori；The U.S. Patent number 5,738,868 of Shinkarenko and the U.S. of Gao are special Benefit number is 5,795,587).
For percutaneously applying, MASP-2 can be inhibited antibody and polypeptide and other suitable ingredient (such as carriers and/or assistant Agent) combination.To the property of these other ingredients, there is no limit in addition to that must be pharmaceutically acceptable for its expectation administration , and the activity of active constituent in composition cannot be reduced.The example of suitable medium object includes with or without collagen purification Ointment, emulsifiable paste, gel or suspension.MASP-2 inhibits antibody and polypeptide that can also be impregnated into transdermal patch, plaster and bandage, Preferred liquid or semi-liquid form.
Can the level needed for for maintenance therapy effect and determine interval periodic basis on, it is systemic to give this hair Bright composition.For example, composition (such as subcutaneous injection) can be given by every 2-4 weeks or with more low-frequency interval.Agent Amount scheme determines the various factors for considering may to influence the effect of drug combination by doctor.These factors may include to be treated Progress extent, patient age, gender and the weight of disease and other clinical factors.The dosage of each separate medical ingredient will be with combination The presence and property of MASP-2 inhibitor included in object and any drug delivery medium (such as sustained release delivery medium) Matter and change.In addition, can be carried out after considering the variation of Pharmacokinetic Characteristics of administration frequency and delivered drug to dosage Adjustment.
Local delivery
Terms used herein " part " includes drug on scheduled limitation site of action or surrounding application, it may include example Such as it is delivered locally to skin or other affected tissues；Eye delivering；(ICV) in intrathecal (IT), the ventricles of the brain, intra-articular, intracavitary, encephalic or It gives, dispose or rinses in alveolar.The local administration of low dosage can preferably be given to avoid systemic side effects, Yi Jigeng Accurately control the surfactant concentration of delivering opportunity and local delivery site.No matter in the side such as metabolism, blood flow between patient The variation in face, local administration all obtain known concentration in target site.Dosage is controlled by directly delivering mode also to be changed It is kind.
The local delivery of MASP-2 inhibitor can for treat the disease for being caused or being aggravated by fibrosis and/or inflammation or In the case where the operation method of illness, such as realize during program is for example performed the operation.
Therapeutic scheme
In prophylactic use, the pharmaceutical composition comprising MASP-2 inhibitor (for example, MASP-2 inhibiting antibody) is given It is sensitive to the disease or illness that are caused or aggravated by fibrosis and/or inflammation or otherwise it is risky generation by fibrosis and/or inflammation The subject of disease or illness that disease causes or aggravates presents in an amount at least sufficient to and inhibits fibrosis and/or inflammation, to eliminate or reduce hair The risk of the symptom of the raw patient's condition.In some embodiments, pharmaceutical composition with therapeutically effective amount give suspection or Subject with the disease or illness that are caused or aggravated by fibrosis and/or inflammation, the amount are enough to mitigate or at least partly Reduce the symptom of the patient's condition.In preventing simultaneous therapeutic scheme, the composition comprising MASP-2 inhibitor can be given dosage for several times It gives, until realizing enough treatment results in subject.The administration of MASP-2 inhibition composition of the invention can pass through list It is secondary to give composition or limited times sequence is given and carried out, to treat the acute patient's condition with fibrosis and/or inflammation-related.Alternatively, Composition can be given at regular intervals in long term time, to treat the chronic conditions with fibrosis and/or inflammation-related.
In preventing simultaneous therapeutic scheme, the composition comprising MASP-2 inhibitor, Zhi Dao can be given with several dosage Sufficient treatment results are obtained in subject.In one embodiment of the invention, MASP-2 inhibitor is anti-comprising MASP-2 Body suitably can give adult patients (for example, average adult's weight of 70 kg): 0.1 mg to 10 with following dosage, 000 mg, more suitably 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 50.0 mg to 500 mg.For child patient, dosage can be scaled with patient weight. It can be by the single-dose or limited successive administration of composition, to apply MASP-2 inhibitor combination of the invention to treat It suffers from or the risky subject of disease or illness for occurring to be caused or aggravated by fibrosis and/or inflammation.Alternatively, can extend Time during at regular intervals (such as daily, twice a week, weekly, every other week, it is monthly or two months every) give group Object is closed, is suffered from or the subject of disease or illness that risky generation is caused or aggravated by fibrosis and/or inflammation for treat.
Prevent and therapeutic scheme in, the composition comprising MASP-2 inhibitor dosage can be given for several times, until by Enough treatment results are realized in examination person.
In some embodiments, tested by determining that subject has one or more symptoms of impaired renal function Person is identified in the risk occurred by fibrosis or inflammation cause or aggravate disease or illness, such as example by measuring blood Clear creatinine levels, serum creatinine clearance rate, blood urea nitrogen be horizontal, albumen in urine and/or by measurement and kidney diaseases or Damage related one or more biomarker evaluations.
Method for evaluating renal function is well-known in the art, and includes but is not limited to measurement whole body and glomerulus Capillary pressure, albuminuria (for example, albuminuria), microcosmic and macroscopic blood urine, serum creatinine level (for example, One is used to estimate the normal renal function that the creatinine levels of 2.0 mg/dl are equal to 50% by the formula of the renal function of people, and 4.0 mg/dl be equal to 25%), the decline of glomerular filtration rate (for example, creatinine clearance) and pipe damage degree.For example, The assessment of renal function may include using biology and/or physiologic parameters such as serum creatinine level, creatinine clearance, 24 Hour Urine proteins secretion, glomerular filtration rate, urinary albumin creatinine ratio, albumin secreting rate and Renal biospy are (for example, logical The deposition for crossing measurement collagen and/or fibronectin, determines the degree of kidney fibrosis), evaluate at least one renal function.
VI. embodiment
The following examples only illustrate it is presently contemplated that best mode for carrying out the present invention, but should not be construed as limiting The system present invention.All documents cited herein is all incorporated herein by reference.
Embodiment 1
Present embodiment describes the productions for the mouse species for lacking MASP-2 (MASP-2-/-) but MAp19 sufficient (MAp19+ /+) It is raw.
Materials and methods: design targeting vector pKO-NTKV 1901 is shown outside to destroy three of coding mouse MASP-2 C-terminal Son, the exon including encoding serine protease domain, is shown in Fig. 3.Mouse ES cell line is transfected using PKO-NTKV 1901 E14.1a (SV129 Ola).Select the clone of neomycin resistance and thymidine kinase sensitivity.600 ES clones are filtered out, at it In identify 4 different clones, the targeting event (targeting containing expected selection is confirmed by Southern trace Event) and recombination event, see Fig. 3.Chimera is generated by this 4 positive colonies by embryo transfer.Then chimera is being lost It passes and is returned under background C57/BL6 to generate Transgenic male mouse.Transgenic male mouse is hybridized with Female Rats and generates F1,50% Offspring showsMASP-2The heterozygosity that gene is destroyed.After chimeric mice mutually being handed over to generate homozygous MASP-2 deficiency In generation, generates chimeric mice and wild-type mice in the ratio of 1:2:1 respectively.
And phenotype as a result: homozygosis MASP-2-/- deficient mice caused by finding can survive and can educate, and pass through Southern trace confirms to lack MASP-2, to confirmed correct targeting event, confirms to lack through Northern trace MASP-2 mRNA confirms to lack MASP-2 albumen through western blot (data are not shown).Existed using time resolution RT-PCR It is further demonstrated on LightCycler machine there are MAp19 mRNA, lacks MASP-2 mRNA.MASP-2-/- mouse is really such as It is expected that continuing to express MAp19, MASP-1 and MASP-3 mRNA and protein like that (data are not shown).Pass through LightCycler Analyze to properdin in MASP-2-/- mouse, factor B, factor D, C4, C2 and C3 mRNA presence and abundance commented Valence is found and identical (data are not shown) in wild type littermates control.It is complete from homozygous MASP-2-/- mouse blood plasma The full complement activation for lacking lectin pathway and mediating, as embodiment 2 further describes.
MASP-2-/- strain is generated under pure C57BL6 background: MASP-2-/- mouse and pure C57BL6 strain are returned After 9 generations, MASP-2-/- strain is used as experimental animal model.
As mouse MASP-2-/-, MAp19+ /+and expression people MASP-2 transgenosis (mouse MASP-2 knock out and people MASP-2 strikes Enter) transgenic mouse lines also generate it is as follows:
Materials and methods: building includes peopleMASP 2Gene promoter area and encoding human MASP-2 mini gene (SEQ ID NO: 49), referred to as " mini hMASP-2 ", see Fig. 4, it includes preceding 3 exons (exons 1 to exon 3), followed by represent with The cDNA sequence of the coded sequence of 8 exons afterwards, thus the overall length MASP-2 albumen that coding is driven by its endogenesis promoter.It will Mini hMASP-2 construct be injected into MASP-2-/- fertilized eggs in, replaced with will pass through transgene expression people MASP-2 The mouse of defectMASP 2Gene.
Embodiment 2
The present embodiment confirms that MASP-2 is carried out necessary to complement activation via lectin pathway.
Method and material:
Lectin pathway specific C 4 cracks measuring method: C4 cracking measuring method is recorded in Petersen, et al.,J. Immunol. Methods 257: 107 (2001), the measuring method are determined by coming from staphylococcus aureus, in conjunction with L- fiber gelatinized protein Lipoteichoicacid (LTA) caused by lectin pathway activation.To Petersen et al., measuring method described in (2001) is carried out Improve, after being coated with plate with LPS and mannosan or zymosan, is added and derives from MASP-2 -/- mouse serum, come The activation of the lectin pathway as caused by MBL is measured, as described below.Also the measuring method improve to eliminate by classical way A possibility that C4 caused by diameter is cracked.This realizes that this to coagulate by using the sample dilution buffer containing 1 M NaCl Collect plain approach identification component and combine their ligand with high-affinity, but prevent the activation of endogenous C4, thus by making C1 Complex dissociation and the participation for eliminating classical pathway.Briefly, in improved measuring method, blood serum sample (is diluted in In (1 M NaCl) buffer with high salt) it is added on ligand coating plate, it is subsequently added into buffer (salt with physiological concentration) The pure C4 of constant.C4b is caused to deposit in conjunction with recognition complex c4 cleavage containing MASP-2.
Measuring method:
1) be diluted in coating buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6) 1 μ g/ml mannosan (M7504 Sigma) or any other ligand (such as such as following ligand), to be coated with Nunc Maxisorb microtiter plate (Maxisorb®, Nunc, catalog number (Cat.No.) 442404, Fisher Scientific).
Following reagent is in this measuring method:
A. mannosan (1 hole μ g/ mannosan (M7504 Sigma), in 100 μ l coating buffer)；
B. zymosan (1 hole μ g/ zymosan (Sigma), in 100 μ l coating buffer)；
C.LTA (1 hole μ g/, in 100 μ l coating buffer in or 2 holes μ g/, in 20 μ l methanol)；
D.1 μ g H- fiber gelatinized protein specificity Mab 4H5, in coating buffer；
E. from light green aerococcus (Aerococcus viridans) PSA (2 holes μ g/, in 100 μ l be coated with buffer)；
F.100 the fixed staphylococcus aureus DSM20233 (OD of the hole μ l/ formalin550=0.5), in coating buffer In.
2) plate is incubated overnight at 4 DEG C.
3) after being incubated overnight, by by plate and 0.1% HSA-TBS Block buffer (0.1% (weight/volume) HSA's 10 mM Tris-HCl、140 mM NaCl、1.5 mM NaN3Solution (pH 7.4)) after incubation 1-3 hours, then spat with TBS/ Temperature (tween)/Ca2+(contain 0.05% polysorbas20 and 5 mM CaCl2、1 mM MgCl2TBS (pH 7.4)) board-washing 3 times, thus It is saturated residual protein binding site.
4) diluted sample is added to by serum samples diluted to be tested in MBL combination buffer (1 M NaCl) On plate, it is incubated overnight at 4 DEG C.Only plus the hole of buffer is used as negative control.
5) after being incubated overnight at 4 DEG C, by plate TBS/ tween/Ca2+Washing 3 times.Then by people C4 (100 μ The hole l/, 1 μ g/ml are diluted in BBS (4 mM barbitals, 145 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4) in) It is added on plate, is incubated for 90 minutes at 37 DEG C.Plate TBS/ tween/Ca2+It washs again 3 times.
6) (TBS/ tween/Ca is diluted in the anti-human C4c of chicken that alkaline phosphatase is conjugated with 1:10002+In) detect C4b deposition, is added on plate, is incubated at room temperature 90 minutes.Then plate TBS/ tween/Ca2+Washing 3 times.
7) it by the way that 100 μ l p-nitrophenyl phosphate substrate solutions are added come detection of alkaline phosphatase, is incubated at room temperature 20 minutes, OD is read in microtiter plate read plate instrument405。
As a result: Fig. 5 A-B shows from MASP-2+ /+(cross spider), MASP-2+/- (filled circles) and MASP-2-/- The amount that C4b is deposited on mannosan (Fig. 5 A) and zymosan (Fig. 5 B) in the serum dilution of (triangles).Fig. 5 C is aobvious Show from MASP-2- /+mouse (n=5) and MASP-2-/- mouse (n=4) zymosan (white bar shaped column) or mannosan (shade bar shaped column) is coated with the C4 invertase relative activity on plate relative to wild-type mice (n=5), and institute is according to measurement needle To the amount of the C4b deposition of wild type serum homogenization.Error bars represent standard deviation.As shown in figures 5 a-c, from MASP-2-/- The complement activation that the blood plasma of mouse mediates on mannosan and zymosan coating plate complete lack of lectin pathway.These knots Fruit clearly illustrates that MASP-2 is the effect subconstiuent of lectin pathway.
Recombinate the lectin pathway dependence C4 activation in MASP-2 reconstruct MASP-2-/- mice serum
It is MASP-2-/- mouse lectin approach dependence C4 activation forfeiture immediate cause to confirm that MASP-2 lacks, In above-mentioned C4 cracking measurement test effect recombination MASP-2 albumen being added in blood serum sample.According to the following examples in 3 The method is prepared for mouse MASP-2A (the wherein serine protease of functional active mouse MASP-2 and catalytically inactive Serine residue on domain activity position is replaced by alanine residue) it recombinant protein and is purified.4 will be derived from The recombinant murine MASP-2 or inactive recombinant murine MASP-2A that MASP-2 -/- mouse combining anteserum and protein concentration are incremented by Preincubate together measures C4 invertase activity according to the above method.
As a result: being obtained from as shown in fig. 6, functional active mouse recombination MASP-2 albumen (being indicated with open triangles) is added to In MASP-2 -/- mouse serum, restores lectin pathway dependence C4 in such a way that protein concentration relies on and activated, and nothing The mouse MASP-2A albumen (being indicated with asterisk) of catalytic activity does not restore C4 activation.Result shown in Fig. 6 is directed to and uses combined open country C4 activation results homogenization (being indicated by a dotted line) observed by raw type mice serum.
Embodiment 3
Present embodiment describes recombinant full-lenght people, rat and mouse MASP-2, MASP-2 derived peptides and catalytically inactives The recombinant expression and protein production of MASP-2 mutant form.
The expression of overall length people, mouse and rat MASP-2:
People MASP-2 full length cDNA sequence (SEQ ID NO:4) is equally subcloned into mammalian expression vector pCI-Neo (Promega) in, driven under the control of cmv enhancer/promoter region eukaryotic expression (be described in Kaufman R.J. et al.,Nucleic Acids Research19:4485-90, 1991; Kaufman, Methods in Enzymology, 185: 537-66 (1991)).By overall length mouse cDNA (SEQ ID NO:50) and rat MASP-2 cDNA (SEQ ID NO:53) It is subcloned into pED expression vector respectively.Then using standard calcium phosphate transfection method (be described in Maniatis et al., 1989), MASP-2 expression vector is transfected into adherent Chinese hamster ovary line DXB1.It is transfected with these constructs Cell grows very slow, shows that encoded protease has cytotoxicity.
In another approach, by the mini gene construct containing the people MASP-2 cDNA driven by its endogenesis promoter (SEQ ID NO:49) is transiently transfected in Chinese hamster ovary cell (CHO).People's MASP-2 albumen is secreted into culture medium, It is separated Ru following.
The expression of the MASP-2 of overall length catalytically inactive:
Basic principle: in identification subcomponent MBL or fiber gelatinized protein (L- fiber gelatinized protein, H- fiber gelatinized protein or M- Fiber gelatinized protein) in conjunction with after their own carbohydrate patterns, MASP-2 passes through autocatalytic cleavage and activates.Cause The autocatalytic cleavage of MASP-2 activation is frequently occurred in separate MASP-2 from serum during, or it is pure after recombinant expression During change.It is used as antigen to obtain more stable protein prepared product, by being urged with alanine residue replacement protein enzyme domains Change serine residue present in triplet, generates MASP-2, the referred to as MASP-2A of catalytically inactive form, in rats (SEQ ID NO:55 Ser617 becomes Ala617)；In mouse (SEQ ID NO:52 Ser617 becomes Ala617)；Or In people (SEQ ID NO:6 Ser618 becomes Ala618).
For people and the mouse MASP-2A albumen for generating catalytically inactive, direct mutagenesis is carried out using oligonucleotides shown in table 5. In order to which serine codon is become alanine codon, devising the oligonucleotides in table 5 makes coding have enzymatic activity serine People and the annealing of the area mouse cDNA, oligonucleotides contains mispairing.For example, using PCR oligonucleotides SEQ ID NO:56-59 combination people MASP-2 cDNA (SEQ ID NO:4), to be amplified from initiation codon to the region for having enzymatic activity serine and from silk ammonia Acid arrives the region of terminator codon, to generate the saltant type containing Ser618 to Ala618 mutation of Complete Open readable form MASP-2A.PCR product is purified after agarose gel electrophoresis and band preparation, generates simple gland glycosides using standard tailing method Overlapping.Then the MASP-2A for adding adenosine tail is cloned into pGEM-T easy carrier, then be transformed into Escherichia coli.
By by SEQ ID NO:64 and SEQ ID two kinds of oligonucleotides kinases (kinasing) of NO:65, and pass through Both oligonucleotides are combined with equimolar amounts, after being heated 2 minutes at 100 DEG C, is slowly cooled to room temperature and anneals, to produce The rat MASP-2A albumen of raw catalytically inactive.Obtained annealing segment has Pst1 and Xba1 termini compatible, by the segment Insertion is to replace the Pst1-Xba1 segment of wild-type rats MASP-2 cDNA (SEQ ID NO:53) to generate rat MASP-2A。
5 'GAGGTGACGCAGGAGGGGCATTAGTGTTT 3' (SEQ ID NO:64)
5' CTAGAAACACTAATGCCCCTCCTGCGTCACCTCTGCA 3' (SEQ ID NO:65)
As follows, further by people, mouse and rat MASP-2A be subcloned into respectively mammalian expression vector pED or PCI-Neo is simultaneously transfected into Chinese hamster ovary line DXB1.
In another approach, using MASP-2 (Chen etc. of Chen et al. the method building catalytically inactive form People,J. Biol. Chem., 276(28):25894-25902, 2001).Briefly, overall length people MASP-2 will be contained CDNA plasmid (be described in Thiel et al.,Nature 386: 506,1997) it usesXho1 HeEcoR1 digestion, by MASP-2 CDNA (referring to the SEQ ID NO:4 of this paper) is cloned into pFastBac1 baculovirus transfer vector (Life Technologies, NY) in corresponding restriction site.Then by replacing encoded peptide area with natural areas amino acid 610-625 The double chain oligonucleotide of domain amino acid 610-625 (SEQ ID NO:13), by MASP-2 serine protease site Ser618 becomes Ala618, to generate the MASP-2 full-length polypeptide for having inactive protein enzyme domains.
The building of expression plasmid containing the peptide zone derived from people Masp-2
Use MASP-2 signal peptide (the residue 1-15 of SEQ ID NO:5) different to secrete to generate following construct MASP-2 structural domain.(N-terminal CUBI is equivalent to by the region that PCR amplification encodes MASP-2 (SEQ ID NO:6) residue 1-121 Structural domain) come prepare expression people MASP-2 CUBI structural domain (SEQ ID NO:8) construct.It is encoded by PCR amplification People MASP-2 is expressed to prepare in the region (being equivalent to N-terminal CUBIEGF structural domain) of MASP-2 (SEQ ID NO:6) residue 1-166 The construct of CUBIEGF structural domain (SEQ ID NO:9).MASP-2 (SEQ ID NO:6) residue 1- is encoded by PCR amplification People MASP-2 CUBIEGFCUBII structural domain is expressed to prepare in 293 region (being equivalent to N-terminal CUBIEGFCUBII structural domain) The construct of (SEQ ID NO:10).Using VentRPolymerase, pBS-MASP-2 is as template, according to the side PCR established Method, through PCR amplification above structure domain.Sense primer (5'-CGGGATCCATGAGGCTGCTGACCCTC-3' SEQ ID NO: 34) 5 ' primer sequences introduce at 5 ' ends of PCR productBamHI restriction site (underscore).Each MASP-2 shown in the following table 5 The antisense primer of structural domain is designed in various PCR product endsEcoIt is close that termination is introduced before the site RI (underscore) Numeral (runic).DNA fragmentation is once amplified, and is just usedBamHI andEcoRI digestion, and it is corresponding to be cloned into pFastBac1 carrier In site.Gained construct is characterized with restriction mapping, and is confirmed by dsDNA sequencing.
Table 5:MASP-2 PCR primer
The protein production of the mouse of the recombinant eukaryon expression of MASP-2 and non-enzymatic activity, rat and people MASP-2A
Using standard calcium phosphate transfection method (Maniatis et al., 1989), by above-mentioned MASP-2 and MASP-2A expression construct It is transfected into DXB1 cell.MASP-2A is generated, in serum free medium to ensure that prepared product is dirty not by other haemocyanins Dye.Every other day from converge in cell harvest culture medium (totally four times).The recombination MASP-2A level of each in three species is flat It is each about 1.5 mg/ and rises culture medium.
MASP-2A protein purification: make MASP-2A (above-mentioned Ser-Ala on MBP-A agarose column by affinity chromatography Mutant) purifying.This strategy can be rapidly purified without using external label.By MASP-2A (100-200 ml culture With isometric sample loading buffer, (50 mM Tris-Cl, pH 7.5 contain 150 mM NaCl and 25 mM CaCl to base2) dilute Release) it is added on MBP- agarose affinity column (4 ml), column is pre-equilibrated with 10 ml sample loading buffers.Another 10 ml sample loading buffers After washing, eluted in the 1 ml fraction of the 50 mM Tris-Cl (pH 7.5) containing 1.25 M NaCl and 10 mM EDTA Protein.The fraction containing MASP-2A is identified with SDS- polyacrylamide gel electrophoresis.When necessary, MASP-2A passes through ion Displacement chromatography is further purified on MonoQ column (HR 5/5).50 mM Tris-Cls of the protein containing 50 mM NaCl (pH 7.5) dialysis, is added on the column balanced with same buffer.After washing, separately washed with the 0.05-1 M NaCl gradient of 10 ml Deviate from the MASP-2A combined.
As a result: the MASP-2A albumen that yield is 0.25-0.5 mg is obtained from 200 ml culture mediums.Since glycosyl is turned into With so the molecular weight for measuring 77.5 kDa through MALDI-MS is bigger than the calculated value (73.5 kDa) of unmodified polypeptide.Each N- glycosylation site is connected with the reason of glycan is surveyed molecular weight.MASP-2A is as single band in SDS- polyacrylamide It is migrated on gel, it was demonstrated that do not processed by proteolysis during biosynthesis.The weight measured by balancing supercentrifugation Average molecular weight is consistent with the calculated value of glycosylated polypeptides homodimer.
The production of recombined human MASP-2 polypeptide
Another method of production recombination MASP-2 and MASP-2A derived peptides is described in Thielens, N.M., et al.,J. Immunol. 166:5068-5077, 2001.Briefly, make Spodopterafrugiperda (Spodoptera frugiperda) insect Cell (being obtained from Novagen, the Ready-Plaque Sf9 cell of Madison, WI) is in Sf900II serum free medium It grows and maintains in (Life Technologies), which supplements 50 IU/ml penicillin and 50 mg/ml streptomysins (Life Technologies).Make cabbage looper (Trichoplusia ni) (High Five) insect cell is (by Jadwiga Chroboczek, Institut de Biologie Structurale, Grenoble, France are provided) it is trained in TC100 It supports in base (Life Technologies) and maintains, which contains supplemented with 50 IU/ml penicillin and 50 mg/ml strepto-s 10% FCS (Dominique Dutscher, Brumath, France) of element.Using Bac-to-Bac system (Life Technologies) recombinant baculovirus is generated.Rod granule (bacmid) DNA prepares purification system using amount in Qiagen (Qiagen) it purifies, and is used to according to scheme described in manufacturer, in the Sf900 II SFM culture medium (Life of cellfectin Technologies transfection Sf 9 insect cell in).Recombinant virus particle is collected after 4 days, measures and titrates through Virus plaque, according to (King and Possee, are loaded in King and Possee the methodThe Baculovirus Expression System: A Laboratory Guide, 111-114 pages of Ltd., London, the of Chapman and Hall, 1992) and it is expanded.
In Sf900 II SFM culture medium, High is infected at 28 DEG C with the recombinant virus containing MASP-2 polypeptide Five cell (1.75 x 107A cell/175 cm2Tissue culture flasks) 96 hours, infection multiplicity 2.Through supernatant is collected by centrifugation Diisopropyl fluorophosphate (DFP) is added to 1 mM of final concentration in liquid.
MASP-2 polypeptide is secreted into culture medium.By culture supernatants to 50 mM NaCl, 1 mM CaCl2、50 MM Triethanolammonium chloride (pH 8.1) dialysis, it is solidifying with the Q- agarose that 1.5 ml/ minutes speed is added to same buffer balance On the fast flow velocity column of glue (Amersham Pharmacia Biotech) (2.8 x, 12 cm).In same buffer, with 1.2 liters Linear gradient (to 350 mM NaCl) eluted.It is identified by Western blot analysis method more containing recombination MASP-2 The fraction of peptide passes through addition (NH4)2SO4It is precipitated to 60% (weight/volume), 4 DEG C stand overnight.By pellet resuspended in 145 mM NaCl、1 mM CaCl2, in 50 mM Triethanolammonium chlorides (pH 7.4), be added to the TSK of same buffer balance On G3000 SWG column (7.5 x, 600 mm) (Tosohaas, Montgomeryville, PA).It then will be purified more Peptide passes through in Microsep microconcentrator (molecular cut off=10,000) (Filtron, Karlstein, Germany) It is concentrated by ultrafiltration to 0.3 mg/ml.
Embodiment 4
Present embodiment describes the production methods of the polyclonal antibody of anti-MASP-2 polypeptide.
Materials and methods:
MASP-2 antigen: being immunized rabbit with following isolated MASP-2 polypeptide, to produce polyclonal anti-human MASP-2 antiserum: separation From the people MASP-2 (SEQ ID NO:6) of serum；Recombined human MASP-2 (SEQ ID NO:6), contains inactive protease The MASP-2A of structural domain (SEQ ID NO:13), is shown in embodiment 3；Recombinant C UBI (SEQ ID NO:8), the CUBEGFI of expression (SEQ ID NO:9) and CUBEGFCUBII (SEQ ID NO:10), as described in example 3 above.
Polyclonal antibody: used BCG (bacille Calmette-Guerin (bacillus Calmette-Guerin vaccine)) immune Six week old rabbits, injected 100 μ g MASP-2 polypeptides and be immunized, it is molten that MASP-2 polypeptide by 100 μ g/ml is dissolved in sterile salt Liquid.It is injected within every 4 weeks, antibody titer is monitored by ELISA measuring method, is described in embodiment 5.Collect culture supernatants, For carrying out antibody purification by A albumen affinity chromatography.
Embodiment 5
Present embodiment describes anti-rat or the production methods of the mouse monoclonal antibody of people's MASP-2 polypeptide.
Materials and methods:
100 μ g people or rat rMASP-2 or rMASP-2A polypeptide (preparing by described in embodiment 3) are subcutaneously injected into 8-12 The male A/J mouse (Harlan, Houston, Tex.) of week old, the polypeptide are dissolved in 200 μ l phosphate buffered saline solutions (PBS) complete Freund's adjuvant in (pH 7.4) (Difco Laboratories, Detroit, Mich.).Two weeks when Between be spaced under, 50 μ g are dissolved in the people of incomplete Freund's adjuvant or rat rMASP-2 or rMASP-2A polypeptide are subcutaneously injected twice To mouse.In 4th week, 50 μ g people or rat rMASP-2 or the rMASP-2A polypeptide of PBS are dissolved in mouse injection, 4 days laggard Row fusion.
For merging every time, single cell suspension is prepared from by immune mouse spleen, is used for and Sp2/0 myeloma cell Fusion.By 5x108A Sp2/0 and 5x108A splenocyte containing 50% polyethylene glycol (molecular weight 1450) (Kodak, Rochester, N.Y.) and the culture medium of 5% dimethyl sulfoxide (Sigma Chemical Co., St. Louis, Mo.) in carry out Fusion.Then cell is adjusted to Iscove culture medium (Gibco, Grand Island, N.Y.) suspension to every 200 μ l 1.5x105The concentration of splenocyte, supplement in culture medium 10% fetal calf serum, 100 units/ml penicillin, 100 μ g/ml streptomysins, 0.1 mM hypoxanthine, 0.4 μM of aminopterin and 16 μM of thymidines.200 microlitres of cell suspensions are added to about 20 96 holes In each hole of microtest plate.After about 10 days, culture supernatants are taken out, for the screening in ELISA measuring method and purifying The reactivity of factor M ASP-2.
ELISA measuring method: by be added 50 μ l purified 50 ng/ml hMASP-2 or rat rMASP-2 (or RMASP-2A) at room temperature overnight, it is coated with Immulon® 2 (Dynatech Laboratories, Chantilly, Va.) Each hole of micro-scale testing plate.Make it possible to select high-affinity antibody for coated low concentration MASP-2.Gently patting training Support plate remove coating solution after, by the PBS solution of the bovine lacto transfer technique optimizer (skimmed milk power) of 200 μ l be added to each Kong Zhongda 1 hour with Close non-specific sites.After one hour, then each hole is washed with buffer PBST (PBS containing 0.05% polysorbas20). 50 microlitres of culture supernatants are collected from each fusion hole, and are mixed with the bovine lacto transfer technique optimizer of 50 μ l, micro-scale testing plate is then added to Each hole in.After being incubated for 1 hour, each hole is washed with PBST.Then the goat by being conjugated with horseradish peroxidase (HRP) Anti-mouse IgG (to Fc specificity) (Jackson ImmunoResearch Laboratories, West Grove, Pa.) Reaction is diluted in bovine lacto transfer technique optimizer to detect the mouse antibody of combination with 1:2,000.It will join containing 0.1% 3,3,5,5- tetramethyl Aniline (Sigma, St. Louis, Mo.) and the Peroxidase substrate solution of 0.0003% hydrogen peroxide (Sigma) are added to respectively Kong Zhong develops the color 30 minutes.50 μ l 2M H are added in every hole2SO4Terminate reaction.Use BioTek®ELISA plate reader (BioTek® Instruments, Winooski, Vt.) reaction mixture is read in the optical density (OD) of 450 nm.
MASP-2 binding assay:
Test can be measured for positive culture supernatants in binding assay in above-mentioned MASP-2 ELISA measuring method, To measure MASP-2 inhibitor to the binding affinity of MASP-2.Similar measuring method can also be used to determine whether inhibitor combines Other antigens in complement system.
Pass through phosphate buffered saline solution (PBS) (pH 7.4) (20 holes ng/100 μ l/, the Advanced with MASP-2 Research Technology, San Diego, CA) it is stayed overnight at 4 DEG C, to be coated with each hole of polystyrene microtiter plates (96 hole culture medium boards, Corning Costar, Cambridge, MA).After MASP-2 solution is sucked out, each hole is with containing 1% bovine serum albumin(BSA) (BSA；Sigma Chemical) PBS close at room temperature 2 hours.The no coated hole MASP-2 is used as Ground control.Divide the doma supernatant of various concentration in lock solution or purified anti-MASP-2 MoAb etc. to solution It is added in each hole.It is incubated at room temperature after 2 hours, each hole is sufficiently rinsed with PBS.By the way that peroxidating is added in lock solution The goat anti-mouse IgG (Sigma Chemical) of object enzyme conjugation is incubated at room temperature 1 hour, to detect in conjunction with MASP-2's Anti- MASP-2 MoAb.After the plate is sufficiently rinsed again with PBS, 100 μ l, 3,3', 5,5'- tetramethyl benzidine (TMB) is added Substrate (Kirkegaard and Perry Laboratories, Gaithersburg, MD).The 1 M phosphoric acid of 100 μ l is added Come quench TMB reaction, by plate micro plate plate reader (SPECTRA MAX 250, Molecular Devices, Sunnyvale, CA) in read at 450 nm.
Then measuring the culture supernatants from positive hole, (all C4 cracking as described in Example 2 are surveyed in functional examination method Determine method) in inhibit complement activation ability.Then through the cell in limited dilution cloning positive hole.It is measured again in above-mentioned ELISA The reactivity of MoAb and hMASP-2 is measured in method.It grows the hybridoma selected in rolling bottle, collects the supernatant for exhausting culture Liquid passes through A albumen affinity chromatography antibody purification.
Embodiment 6
Present embodiment describes the generations and production of humanized murine MASP-2 antibody and antibody fragment.
According to 5 the method for embodiment, the anti-MASP-2 monoclonal antibody of mouse is generated in male A/J mouse.Then according to Murine constant regions are replaced with its people's counterpart to generate the chimera of IgG and Fab fragments, make mouse antibody human by following methods To reduce its immunogenicity, the intracorporal MASP-2 dependence of people experimenter that the chimera is used to inhibit of the invention is mended in source The side effect of body activation.
1. cloning anti-MASP-2 variable region gene by mouse hybridoma
According to the scheme (Biotech, Houston, Tex.) of manufacturer, using RNAzol by secreting anti-MASP-2 MoAb's Total serum IgE is isolated in hybridoma (obtaining according to method described in embodiment 7).Using oligomerization dT as primer, by total serum IgE Synthesize the first cDNA chain.Using 3 ' primers derived from the constant area C of immunoglobulin and derive from leader peptide or mouse VHOr VKThe of gene The degenerate primer group of one framework region carries out PCR as 5 ' primer pairs.By Chen and Platsucas the method (Chen, P.F., Scand. J. Immunol. 35:539-549,1992) carry out anchor PCR.For cloning VKGene uses Not1- MAK1 primer (5'-TGCGGCCGCTGTAGGTGCTGTCTTT-3'SEQ ID NO:38) prepares double-strand cDNA.Annealing is held in the mouth Meet sub- AD1 (5'-GGAATTCACTCGTTATTCTCGGA-3'SEQ ID NO:39) and AD2 (5'- TCCGAGAATAACGAGTG-3'SEQ ID NO:40) it is connected to the 5 ' ends and 3 ' ends of double-strand cDNA.3 ' are removed through Notl digestion Hold adapter.Then digestion product is used as pcr template, using AD1 oligonucleotides as 5 ' primers, MAK2 (5'- CATTGAAAGCTTTGGGGTAGAAGTTGTTC-3'SEQ ID NO:41) it is used as 3 ' primers.By the DNA piece of about 500 bp Section is cloned into pUC19.It selects several clones and carries out sequence analysis to confirm that cloned sequence includes expected rat immune globulin Constant region.Not1-MAK1 and MAK2 oligonucleotides derives from VKArea, respectively 182 bp and 84 of the first base pairs upstream of C kappa gene bp.Selection includes complete VKWith the clone of leader peptide.
For cloning VHGene uses Not1 MAG1 primer (5'-CGCGGCCGCAGCTGCTCAGAGTGTAGA-3' SEQ ID NO:42) prepare double-strand cDNA.Anneal adapter AD1 and AD2 are connected to the 5 ' ends and 3 ' ends of double-strand cDNA.Through Notl digestion removes 3 ' end adapters.Digestion product is used as pcr template, with AD1 oligonucleotides and MAG2 (5'- CGGTAAGCTTCACTGGCTCAGGGAAATA-3'SEQ ID NO:43) it is used as primer.The DNA for being 500-600 bp by length Segment is cloned into pUC19.Not1-MAG1 and MAG2 oligonucleotides derives from the area mouse C γ .7.1, respectively mouse C γ .7.1 gene 180 bp and 93 bp of first base pairs upstream.Selection includes complete VHWith the clone of leader peptide.
2. the building of the expression vector of chimeric MASP-2 IgG and Fab
By the V of above-mentioned cloneHAnd VKGene is used as the template of PCR reaction, so that Kozak consensus sequence is added to 5 ' ends, will cut Connect the 3 ' ends that donor is added to nucleotide sequence.After carrying out the determining no PCR error of analysis to the sequence, by VHAnd VKGene difference It is inserted into the expression vector box of the γ of C. containing someone 1 and C. κ, obtains pSV2neoVH-huC γ 1 and pSV2neoV-huC γ. Pass through electroporation transfection COS cell using the Plasmid DNA of CsCl gradient-purified heavy chain and light chain vector.After 48 hours, pass through ELISA measures culture supernatants, it was demonstrated that there is the chimeric IgG of about 200 ng/ml.Cell is harvested, total serum IgE is prepared.It uses Oligomerization dT synthesizes the first cDNA chain from total serum IgE as primer.The cDNA is used as pcr template to generate Fd and κ DNA fragmentation.It is right In Fd gene, 5'-AAGAAGCTTGCCGCCACCATGGATTGGCTGTGGAACT-3'(SEQ ID NO:44 is used) as 5 ' 3 ' primers (5'-CGGGATCCTCAAACTTTCTTGTCCACCTTGG-3'SEQ ID NO:45) derived from primer and CH1 into Row PCR.Confirm the V that DNA sequence dna contains complete human IgG1HWith CH1 structural domain.After with suitable enzymic digestion, by Fd DNA HindIII the and BamHI restriction site of fragment inserting expressioning carrier box pSV2dhfr-TUS, obtains pSV2dhfrFd.PSV2 matter Grain is commercially available, and is made of the DNA section of separate sources: pBR322 DNA (filament) contains pBR322 DNA replication dna starting point (pBR ori) and lactamase ampicillin resistance gene (Amp)；SV40 DNA is indicated and is marked by thicker hachure, contained There are SV40 DNA replication dna starting point (SV40 ori), early promoter (5 ' ends of dhfr and neo gene) and polyadenylation letter Number the 3 ' of dhfr and neo gene (end).Polyadenylation signal derived from SV40 (pA) also is located at 3 ' ends of Fd gene.
For kappa gene, 5'- AAGAAAGCTTGCCGCCACCATGTTCTCACTAGCTCT-3'(SEQ ID NO is used: 46) 5 ' primers and C are used asKDerivative 3 ' primer (5'-CGGGATCCTTCTCCCTCTAACACTCT-3'SEQ ID NO:47) Carry out PCR.Validating DNA sequence contains complete VKWith people CKRegion.After with suitable limitation enzymic digestion, κ DNA fragmentation is inserted HindIII the and BamHI restriction site for entering expression vector box pSV2neo-TUS, obtains pSV2neoK.The expression of Fd and kappa gene It is driven by enhancer derived from HCMV and promoter element.Because Fd gene does not include being related to half Guang ammonia of interchain disulfide bond Sour amino acid residue, so the chimeric Fab of the recombination just contains the heavy chain and light chain of non-covalent linking.The chimeric Fab is named For cFab.
In order to obtain the recombinant Fab for having heavy chain and light interchain disulfide bond, above-mentioned Fd gene can be extended to include coming From the coded sequence of additional 9 amino acid (EPKSCDKTH SEQ ID NO:48) of human IgG1's hinge area.Encode Fd gene 3 ' The BstEII-BamHI DNA section of 30 amino acid at end can be encoded the DNA section displacement of extended Fd, generate pSV2dhfrFd/9aa。
3. the expression and purifying of chimeric anti-MASP-2 IgG
To generate the cell line for secreting chimeric anti-MASP-2 IgG, with purified 1 He of pSV2neoVH-huC. γ The Plasmid DNA of pSV2neoV-huC κ passes through electroporation transfection NSO cell.It selects and is transfected in the presence of 0.7 mg/ml G418 Cell.Cell is cultivated in 250 ml rolling bottles with the culture medium containing serum.
By the culture supernatants of 100 ml spinner cultures be added to 10 ml PROSEP-A columns (Bioprocessing, Inc., Princeton, N.J.) on.Column is washed with the PBS of 10 bed volumes.In conjunction with antibody it is slow with 50 mM citric acids Fliud flushing (pH 3.0) elution.1 isometric M Hepes (pH 8.0) is added in the fraction containing antibody purification, pH is adjusted To 7.0.By the ultrafiltration of Millipore film (molecular cut off: 3,000), it is remaining to remove that buffer-exchanged is carried out with PBS Salt.The protein concentration of purified antibody is measured by BCA method (Pierce).
4. the expression and purifying of chimeric anti-MASP-2 Fab
In order to generate the cell line of the chimeric anti-MASP-2 Fab of secretion, with purified pSV2dhfrFd (or PSV2dhfrFd/9aa) and the Plasmid DNA of pSV2neo κ passes through electroporation transfection Chinese hamster ovary celI.Exist in G418 and methotrexate (MTX) Under select the cell being transfected.Selected cell line is expanded in the methotrexate (MTX) of increasing concen-trations.Through limiting dilution to thin Born of the same parents carry out unicellular subclone.Then by the unicellular subcloned cells of high yield tie up in 100 ml rolling bottles with serum free medium into Row culture.
Using the small murine anti-idiotypic MoAb of anti-MASP-2 MoAb, by affinity chromatography to chimeric anti-MASP-2 Fab is purified.It can use and keyholeThe anti-MASP-2 MoAb of mouse of hemocyanin (KLH) conjugation filters out energy to mouse immune Enough in conjunction with the specific MoAb that people MASP-2 is competed, thus to prepare antiidiotype MASP-2 MoAb.As for purifying, in the future It is added to and antiidiotype MASP-2 from 100 ml supernatants of the Chinese hamster ovary celI for the turn culture for generating cFab or cFab/9aa On the affinity column of MoAb coupling.Then after the column is sufficiently washed with PBS, in conjunction with Fab washed with 50 mM diethylamine (pH 11.5) It takes off.Remaining salt is removed as described above by buffer-exchanged.It is measured by BCA method (Pierce) purified The protein concentration of Fab.
Can inhibition measuring method described in Application Example 2 or embodiment 7 come measure chimeric MASP-2 IgG, cFab and The ability of cFAb/9aa inhibition MASP-2 dependent complement approach.
Embodiment 7
Present embodiment describes the external C4 for being used as functionality screening to crack measuring method, so that identification can be blocked via L- fiber Gelled protein/P35, H- fiber gelatinized protein, M- fiber gelatinized protein or mannosan MASP-2 dependent complement activation MASP-2 inhibitor.
C4 crack measuring method: Petersen, S.V. et al. describe C4 cracking measuring method (Petersen, S.V., etc. People,J. Immunol. Methods 257:107,2001), determine the golden yellow grape by combining L- fiber gelatinized protein The activation of lectin pathway caused by the lipoteichoicacid (LTA) of coccus.
Reagent: the fixed staphylococcus aureus (DSM20233) of formalin prepares as follows: by bacterium in tryptone beans peptone In 37 DEG C of overnight incubations in blood culture medium (tryptic soy blood medium), washed three times with PBS, then in room temperature Under 1 hour is fixed in PBS/0.5% formalin, then after being washed three times with PBS, be resuspended to coating buffer (15 mM Na2CO3、35 mM NaHCO3, pH 9.6) in.
Measuring method: Nunc MaxiSorb®Microtiter plate (Nalgene Nunc International, Rochester, NY) each hole be coated with ingredient below: the fixed staphylococcus aureus of 100 μ l formalin DSM20233 (OD550=0.5) the coating buffer of coating buffer and 1 μ g L- fiber gelatinized protein.It is incubated overnight it Afterwards, TBS solution (10 mM Tris-HCl, 140 mM NaCl, the pH 7.4) envelope of each hole 0.1% human serum albumins (HSA) It closes, then uses and contain 0.05% polysorbas20 and 5 mM CaCl2TBS solution (washing buffer) washing.Human serum sample is dilute It releases in 20 mM Tris-HCl, 1 M NaCl, 10 mM CaCl2、0.05% Triton X-100、0.1% HSA (pH 7.4) In, This prevents the activation of endogenous C4, and dissociate C1 compound (being made of C1q, C1r and C1s).By various concentration MASP-2 inhibitor (including anti-MASP-2 MoAb and peptide for inhibiting) is added in blood serum sample.Diluted sample is added in plate, It is incubated overnight at 4 DEG C.After 24 hours, each plate is sufficiently washed with washing buffer, and 0.1 μ g is then added into each hole and is dissolved in 100 μ l, 4 mM barbital, 145 mM NaCl, 2 mM CaCl2、1 mM MgCl2The purified people C4 of (pH 7.4) (is pressed According to Dodds, A.W.,Methods Enzymol. 223:46,1993 the method obtains).37 DEG C after 1.5 hours, each plate The anti-human C4c of chicken (being obtained from Immunsystem, Uppsala, Sweden) that is washed again, being conjugated using alkaline phosphatase, C4b deposition is detected, and is measured using colorimetric substrates p-nitrophenyl phosphate.
C4 about mannosan is measured: modification said determination method is being added with measuring the lectin pathway activation via MBL Enter with before the serum of various MASP-2 inhibitor mixeds, is coated with assay plate with LSP and mannosan.
C4 about H- fiber gelatinized protein (Hakata Ag) is measured: modification said determination method is to measure via H- fiber The lectin pathway of gelled protein activates, before the serum with various MASP-2 inhibitor mixeds is added, with LSP and H- fiber Gelled protein is coated with assay plate.
Embodiment 8
Following measuring method demonstrates in wild type and MASP-2-/- mouse that there are classical pathway activation.
Method: microtiter plate (Maxisorb®, Nunc, catalog number (Cat.No.) 442404, Fisher Scientific) and with 0.1% 10 mM Tris of human serum albumins, 140 mM NaCl (pH 7.4) solution are coated with 1 hour at room temperature, then with by 1: 1000 are diluted in TBS/ tween/Ca2+In the anti-whole serum of sheep antiserum (Scottish Antibody Production Unit, Carluke, Scotland) it is incubated overnight at 4 DEG C, so that in situ generate immune complex.From wild type and Blood serum sample is obtained in MASP-2-/- mouse, is added in coating plate.Prepare control sample, wherein from wild type and MASP-2-/- C1q is exhausted in blood serum sample.According to the specification of supplier, using be coated with rabbit-anti people C1q IgG (Dako, Glostrup, Denmark the Dynabead of A albumen coupling)®(Dynal Biotech, Oslo, Norway) come prepare C1q exhaust it is small Mouse serum.Each plate is incubated for 90 minutes at 37 DEG C.TBS/ tween/Ca is diluted in by 1:1000++In polyclonal anti-human C3c Antibody (Dako A 062) detects the C3b of combination.Secondary antibody is goat anti-rabbit igg.
As a result: Fig. 7 is indicated with IgG and wild type serum, MASP-2-/- serum, is exhausted the wild type of C1q and exhaust C1q MASP-2-/- serum coating plate on C3b relative deposition it is horizontal.These the results show that classical pathway in MASP-2-/- mouse It is complete in strain.
Embodiment 9
Following measuring method is used to by analyzing MASP-2 inhibitor in the case where classical pathway is started by immune complex Effect, to detect whether MASP-2 inhibitor has blocked classical pathway.
Method: in order to detect the complement activation situation that MASP-2 inhibitor is started wherein classical pathway by immune complex Influence, at 37 DEG C, by three parts of 50 μ l sample containing 90% NHS in 10 μ g/ml immune complexs (IC) or PBS In the presence of be incubated for, be incubated at 37 DEG C there are also parallel sample (+/- IC) formula containing the anti-properdin monoclonal antibody of 200 nM Three parts.After 37 DEG C are incubated for two hours, 13 mM EDTA are added in all samples and terminate further complement activation, immediately by sample Product are cooled to 5 DEG C.According to manufacturer's specification, carried out using ELISA kit (Quidel, catalog number (Cat.No.) A015 and A009) Before complement activation products (C3a and sC5b-9) analysis, sample is stored in -70 DEG C.
Embodiment 10
Present embodiment describes the identifications for blocking the anti-MASP-2 Fab2 antibody fragment of the active high-affinity of MASP-2.
Background and basic principle: MASP-2 is the complex proteins with many independent functional domains, comprising: MBL and The binding site of fiber gelatinized protein, serine protease catalytic position, proteolysis substrate C2 binding site, proteolysis The binding site of substrate C4, the cracking position MASP-2 of MASP-2 proenzyme autoactivation and two Ca++Binding site.Identify with The Fab2 antibody fragment of high-affinity combination MASP-2 measures identified Fab2 segment, in functional examination method with determination Whether they can block MASP-2 functional activity.
In order to block MASP-2 functional activity, antibody or Fab2 antibody fragment must combine and hinder MASP-2 functional activity Structure epi-position on required MASP-2.Therefore, many or all of high-affinities combines anti-MASP-2 Fab2 that cannot inhibit MASP-2 functional activity, unless they can be in conjunction with the structure epi-position for the MASP-2 for directly participating in MASP-2 functional activity.
The functional examination method that measurement inhibits lectin pathway C3 convertase to be formed is used to evaluate anti-MASP-2 Fab2's " blocking activity ".Major physiological effect of the known MASP-2 in lectin pathway is the complement pathway for generating lectin-mediated Next functional component, i.e. lectin pathway C3 convertase.Lectin pathway C3 convertase is that proteolytic cleavage C3 becomes C3a With the crucial multienzyme complex (C4bC2a) of C3b.MASP-2 is not the constituent of lectin pathway C3 convertase (C4bC2a)；So And MASP-2 functional activity is needed to generate two protein components (C4b, C2a) for forming lectin pathway C3 convertase.This Outside, in order to make MASP-2 generate lectin pathway C3 convertase, it appears that need all above-mentioned MASP-2 standalone feature activity.Out In these reasons, it is believed that " blocking activity " preferred measuring method for evaluating anti-MASP-2 Fab2 is that measurement inhibits agglutinin The functional examination method that approach C3 convertase is formed.
The generation of high-affinity Fab2: using the phage display library and use of people's variable light and heavy chain antibody sequence In the automatic antibody screening techniques for the Fab2 that identification is reacted with selected target ligand, to generate anti-rat MASP-2 albumen The high-affinity Fab2 of (SEQ ID NO:55).It is carried out using rat MASP-2 (~ 1 mg, the purity > 85%) albumen of known quantity Antibody screening.It is expanded using three-wheel to select the antibody with highest affinity.Select about 250 different expression antibody fragments Target (hits) for ELISA screen.Then high-affinity target is sequenced the uniqueness to determine different antibodies.
By 50 unique anti-MASP-2 antibody purifications, it is used for each purified Fab2 antibody of 250 μ g to characterize MASP-2 Binding affinity and complement pathway functional examination, details are as follows.
Inhibit (blocking) active measuring method for evaluating anti-MASP-2 Fab2
1. the measuring method that measurement inhibits lectin pathway C3 convertase to be formed:
Background: lectin pathway C3 convertase be proteolytic cleavage C3 become two effective proinflammatory segment anaphylatoxin C3a with The multienzyme complex (C4bC2a) of opsonin C3b.The formation of C3 convertase is seemingly in the lectin pathway in terms of transmitting inflammation Committed step.MASP-2 is not the constituent of lectin pathway C3 convertase (C4bC2a)；Therefore, anti-MASP-2 antibody The activity of already present C3 convertase before (or Fab2) will not directly inhibit.However, in order to generate composition lectin pathway C3 Two protein components (C4b, C2a) of invertase, MASP-2 serine protease is required.Therefore, inhibit MASP- From the beginning the anti-MASP-2 Fab2 (i.e. the anti-MASP-2 Fab2 of blocking property) of 2 functional activities can inhibit lectin pathway C3 convertase It is formed.C3 contains unique high response thioester group as its structure division.When C3 convertase cracks C3 in the measuring method When, thioester group on C3b can be with the hydroxyl or amino that are fixed in the macromolecular of plastic eyelet bottom by ester bond or amido bond Covalent bond is formed, to be conducive to detect C3b in ELISA measuring method.
Yeast mannans are the known activator of lectin pathway.In the following method that measurement C3 convertase is formed, 30 minutes will be incubated at 37 DEG C to activate lectin pathway with the coated plastic eyelet of mannosan and diluted rat blood serum.So After wash each hole, and the C3b being fixed in hole is measured using standard ELISA assay.The amount of generated C3b in the measuring method Directly reflect the from the beginning formation of lectin pathway C3 convertase.The anti-MASP-2 of selected concentration is measured in the measuring method Fab2 inhibits the ability that C3 convertase is formed and subsequent C3b is generated.
Method:
By 96 hole Costar culture medium boards and the mannosan of 50 mM carbonate buffer solutions (pH 9.5) is diluted in 1 μ The hole g/50 μ l/ is incubated overnight at 5 DEG C.After being incubated overnight, each hole is washed three times with 200 μ l PBS.Then 100 μ of each hole The PBS solution of 1% bovine serum albumin(BSA) in the hole l/ is closed, and is gently agitated for being incubated for 1 hour at room temperature.Then 200 μ l of each hole PBS is washed three times.At 5 DEG C, anti-MASP-2 Fab2 sample is diluted in containing Ca++And Mg++(4.0 mM bars of GVB buffer Than appropriate, 141 mM NaCl, 1.0 mM MgCl2, 2.0 mM CaCl2, 0.1% gelatin, pH 7.4) extremely select concentration.At 5 DEG C, 0.5% rat blood serum is added in above-mentioned sample, 100 μ l are transferred to each hole.Plate is covered, 30 points are incubated in 37 DEG C of water-baths Clock is so as to complement activation.Plate is transferred in the container equipped with ice-water mixture from 37 DEG C of water-baths makes reaction terminating.Each Kong Yi It is secondary to be washed 5 times with 200 μ l PBS- polysorbas20s (PBS solution of 0.05% polysorbas20), it is washed 2 times with 200 μ l PBS.It is added 1:10 is pressed in 100 holes μ l/, and 000 diluted primary antibody (rabbit-anti people C3c, DAKO A0062), which is dissolved in containing 2.0 mg/ml oxen In sero-abluminous PBS, it is gently agitated for being incubated for 1 hour at room temperature.It is washed 5 times with 200 μ l PBS in each hole.100 μ are added The hole l/ press 1:10,000 diluted secondary antibody (goat anti-rabbit igg of peroxidase conjugated, American Qualex A102PU), The antibody is dissolved in the PBS containing 2.0 mg/ml bovine serum albumin(BSA)s, is gently agitated for being incubated for 1 hour at room temperature in an oscillator. It is washed 5 times with 200 μ l PBS in each hole.100 hole μ l/ peroxidase substrate TMB (Kirkegaard & Perry are added Laboratories), it is incubated at room temperature 10 minutes.By the way that 100 hole μ l/, 1.0 M H is added3PO4It is anti-to terminate peroxidase It answers, measures OD450。
2. measuring the measuring method that MASP-2 dependence C4 cracking inhibits
Background: the serine protease of MASP-2 is high degree of specificity, only identifies two kinds of albumen for MASP-2 Matter substrate: C2 and C4.C4 cracking generates C4a and C4b.Anti- MASP-2 Fab2 is in combination with the MASP-2's for directly participating in C4 cracking Structure epi-position (such as the MASP-2 binding site of C4；MASP-2 serine protease catalytic position), to inhibit MASP-2's C4 cracks functional activity.
Yeast mannans are the known activator of lectin pathway.In the following side of the C4 lytic activity of measurement MASP-2 In method, 30 minutes will be incubated at 37 DEG C with the coated plastic eyelet of mannosan and diluted rat blood serum to activate agglutinin way Diameter.Because the primary antibody in the ELISA measuring method only identifies people C4, diluted rat serum is checked up and return supplemented with people C4 (1.0 μg/ml).It is washed out each hole, the people C4b being fixed in hole is measured using standard ELISA assay.In the measuring method, The amount of generated C4b is to rely on the measurement of the C4 lytic activity of MASP-2.In the measuring method, the anti-of selected concentration is measured MASP-2 Fab2 inhibits the ability of C4 cracking.
Method: by 96 hole Costar culture medium boards and the sweet dew for being diluted in 50 mM carbonate buffer solutions (pH 9.5) Glycan is incubated overnight at 5 DEG C by 1 hole μ g/50 μ l/.It is washed 3 times with 200 μ l PBS in each hole.Then 100 μ l/ of each hole The PBS solution of 1% bovine serum albumin(BSA) of hole is closed, and is gently agitated for being incubated for 1 hour at room temperature.3 are washed with 200 μ l PBS in each hole It is secondary.At 5 DEG C, anti-MASP-2 Fab2 sample is diluted in containing Ca++And Mg++GVB buffer (4.0 mM barbitals, 141 MM NaCl, 1.0 mM MgCl2, 2.0 mM CaCl2, 0.1% gelatin, pH 7.4) extremely select concentration.1.0 μ g/ml people C4 (Quidel) it is also included in these samples.At 5 DEG C, 0.5% rat blood serum is added in above-mentioned sample, 100 μ l is shifted To each hole.Plate is covered, is incubated for 30 minutes in 37 DEG C of water-baths so as to complement activation.Plate is transferred to from 37 DEG C of water-baths and is equipped with Make reaction terminating in the container of ice-water mixture.200 μ l PBS- polysorbas20s (PBS solution of 0.05% polysorbas20) of each hole Washing 5 times, then each hole is washed 2 times with 200 μ l PBS.100 holes μ l/ are added with the chicken of the diluted biotin-conjugated of 1:700 The PBS solution of anti-human C4c (Immunsystem AB, Uppsala, Sweden) (contains 2.0 mg/ml bovine serum albumin(BSA)s (BSA)) it, is gently agitated for being incubated for 1 hour at room temperature.It is washed 5 times with 200 μ l PBS in each hole.0.1 μ in 100 holes μ l/ is added The PBS solution of the streptavidin (Pierce Chemical #21126) of g/ml peroxidase conjugated (contains 2.0 mg/ Ml BSA), it is gently agitated for being incubated for 1 hour at room temperature in an oscillator.It is washed 5 times with 200 μ l PBS in each hole.100 μ are added The hole l/ peroxidase substrate TMB (Kirkegaard & Perry Laboratories), is incubated at room temperature 16 minutes.It is logical It crosses and 100 hole μ l/, 1.0 M H is added3PO4Peroxidase reaction is terminated, OD is measured450。
3. the binding assay of the anti-rat MASP-2 Fab2 of anti-" natural " rat MASP-2
Background: MASP-2 is usually as further including specific agglutination element molecule (mannose-binding protein (MBL) and fiber gelatinized egg It is white) MASP-2 dimer complex be present in blood plasma.Therefore, if the interesting anti-MASP-2 Fab2 of research and physiology phase The MASP-2 of pass form is combined, then it is important that developing binding assay, wherein that utilize is Fab2 and blood plasma " natural " Between MASP-2, rather than the interaction between the recombination MASP-2 of purifying.In the binding assay, will first it come from " natural " MASP-2-MBL compound of 10% rat blood serum is fixed in the coated hole of mannosan.Then standard ELISA is used Method studies the binding affinity of the anti-MASP-2 Fab2 of various anti-immobilization " natural " MASP-2.
Method: by 96 hole Costar high boards and the mannosan for being diluted in 50 mM carbonate buffer solutions (pH 9.5) It is incubated overnight at 5 DEG C by 1 hole μ g/50 μ l/.It is washed 3 times with 200 μ l PBS in each hole.Each hole is with the 0.5% of 100 holes μ l/ The PBST solution (PBS and 0.05% polysorbas20) of skimmed milk power is closed, and is gently agitated for being incubated for 1 hour at room temperature.Each hole is with 200 μ l TBS/ tween/Ca++(Tris buffer salt solution, 0.05% polysorbas20 contain 5.0 mM CaCl to washing buffer2, pH 7.4) Washing 3 times.Combination buffer with high salt (the 20 mM Tris, 1.0 M NaCl, 10 mM of 10% rat blood serum are prepared on ice CaCl2, 0.05% Triton-X100,0.1% (weight/volume) bovine serum albumin(BSA), pH 7.4).100 μ l are added in every hole, It is incubated overnight at 5 DEG C.Each hole 200 μ l TBS/ tweens/Ca++Washing buffer is washed 3 times.Then 200 μ l of each hole PBS is washed 2 times.100 holes μ l/ are added to be diluted in containing Ca++And Mg++GVB buffer (4.0 mM barbitals, 141 mM NaCl, 1.0 mM MgCl2, 2.0 mM CaCl2, 0.1% gelatin, pH 7.4) selected concentration anti-MASP-2 Fab2, in room It is gently agitated for being incubated for 1 hour under temperature.It is washed 5 times with 200 μ l PBS in each hole.100 holes μ l/ are added to be dissolved in by 1:5000 is diluted The anti-Fab2 of goat (Biogenesis catalog number (Cat.No.) 0500-0099) of 2.0 mg/ml bovine serum albumin(BSA)s/PBS HRP conjugation, It is gently agitated for being incubated for 1 hour at room temperature.It is washed 5 times with 200 μ l PBS in each hole.100 hole μ l/ peroxidase substrates are added TMB (Kirkegaard & Perry Laboratories), is incubated at room temperature 70 minutes.By the way that 100 holes μ l/ are added 1.0 M H3PO4Peroxidase reaction is terminated, OD is measured450。
As a result:
About 250 different Fab2 reacted with anti-rat MASP-2 albumen progress high-affinity are picked to screen for ELISA. Sequencing has been carried out to determine the uniquenesses of different antibodies to these high-affinities Fab2, to 50 unique anti-MASP-2 antibody into Row purifying is for further analyzing.Each purified Fab2 antibody of 250 μ g is used to characterize MASP-2 binding affinity and complement way Diameter functional test.The result of the analysis see the table below 6.
Table 6: the anti-MASP-2 FAB2 of blocked lectin element approach complement activation
Shown in table 6 as above, in 50 anti-MASP-2 Fab2 surveyed, 17 Fab2 are accredited as MASP-2 blocking property Fab2, Effectively C3 convertase is inhibited to be formed, IC50 ≤ 10 nM Fab2 (34% positive chooses rate).In 17 Fab2 identified, There are 8 IC50Range is nanomole or less.In addition, all 17 MASP-2 blocking property Fab2 is on agglutinin way shown in table 6 Substantially completely C3 convertase is inhibited to be formed in diameter C3 convertase measuring method.Fig. 8 A illustrates C3 convertase and forms measuring method Fab2 antibody #11's as a result, representative in other measured Fab2 antibody, the result is shown in tables 6.Because even working as When each MASP-2 molecule is combined by Fab2, " blocking property " only possible pole Fab2 faintly inhibits MASP-2 function, this is in theory It is possible, therefore to consider carefully.
Although mannosan is the known activator of lectin pathway, the anti-mannosan existing for rat blood serum is anti- Body is also possible to activation classical pathway, and generates C3b by classical pathway C3 convertase, this is theoretically possible.However, Each of 17 anti-MASP-2 Fab2 of blocking property listed by the present embodiment restrained effectively C3b and generate (> 95%), Hence it is demonstrated that specificity of this measuring method for lectin pathway C3 convertase.
In order to calculate the apparent K of each antibodyd, measurement is all combined to all 17 of blocking property Fab2.6 blockings The result of the binding assay of the anti-rat MASP-2 Fab2 of the anti-natural rat MASP-2 of property Fab2 is also shown in Table 6.Fig. 8 B passes through figure Show and illustrates the result of the binding assay with Fab2 antibody #11.Similar binding assay has also been carried out for other Fab2, It the results are shown in Table 6.Generally, for each combination " natural " MASP-2 apparent K obtained of 6 Fab2dTurn in C3 Change the IC of Fab2 in enzyme functional examination50Correspondence it is rather appropriate.Evidence suggests, after activating its proteinase activity, MASP-2 experienced from " inactive " to " activity " form conformation change (Feinberg et al.,EMBO J 22:2348-59 (2003);Gal et al.,J. Biol. Chem. 280:33435-44 (2005)).Measuring method is being formed for C3 convertase Normal rat plasma in, MASP-2 mainly with " inactive " proenzyme conformation presence.In contrast, in binding assay, MASP-2 exists as the component part for the compound for being integrated to immobilization mannosan together with MBL；Therefore, MASP-2 may For " activity " conformation (Petersen et al.,J. Immunol Methods 257:107-16, 2001).Therefore, for this two Each of 17 measured in a functional examination method blocking property Fab2, it may not be necessary to it is expected that IC50With KdBetween it is exact right Ying Xing, because Fab2 may be in conjunction with the MASP-2 of different conformational forms in each measuring method.Nevertheless, in addition to Fab2 # Other than 88, other 16 Fab2 for being surveyed in two kinds of measuring methods each, IC50With apparent KdBetween seem to have it is quite close Correspondence (referring to table 6).
Several blocking property Fab2 of C4 cracking for inhibiting MASP-2 to mediate is evaluated.Fig. 8 C passes through diagram Illustrate C4 cracking measuring method as a result, show with Fab2 #41 inhibit IC50=0.81 nM (referring to table 6).As shown in figure 9, It was found that the Fab2 of all tests inhibits C4 to crack, IC50Similar to IC obtained in C3 convertase measuring method50(referring to table 6)。
Although mannosan is the known activator of lectin pathway, anti-mannosan antibody is deposited in rat blood serum Classical pathway may also be being activated, so that the C4 cracking mediated by C1s generates C4b, this is theoretically possible.So And, it has been identified that effectively inhibit C4b to generate the several anti-MASP-2 Fab2 of (> 95%), hence it is demonstrated that the measuring method pair In the specificity for the C4 cracking that MASP-2 is mediated.C4 contains unique high response thioester group as its knot as C3 Structure part.In the measuring method after passing through MASP-2 c4 cleavage, thioester group on C4b can with pass through ester bond or amido bond It is fixed on hydroxyl or amino in the macromolecular of plastic eyelet bottom and forms covalent bond, therefore be conducive to detect in ELISA measuring method C4b out.
These researchs clearly show, the generation for the high-affinity Fab2 of rat MASP-2 albumen, functional ground resistance Disconnected C4 and C3 convertase activity, so that lectin pathway be prevented to activate.
Embodiment 11
Present embodiment describes to several anti-rat MASP-2 Fab2 antibody of blocking generated according to 10 the method for embodiment The epitope mapping of progress.
Method:
As shown in Figure 10, using pED4 carrier, the following protein all with 6 His of N-terminal label is expressed in Chinese hamster ovary celI:
Rat MASP-2A, a kind of overall length MASP-2 albumen change into alanine (S613A) by the serine on activated centre And it inactivates；
Rat MASP-2K reduces the overall length MASP-2 albumen (R424K) of autoactivation through changing；
CUBI-II, a kind of N-terminal segment of the rat MASP-2 only containing CUBI, EGF sample and CUBII structural domain；With
CUBI/EGF sample, a kind of N-terminal segment of the rat MASP-2 only containing CUBI and EGF spline structure domain.
These protein are purified (Chen etc. by nickel affinity chromatography from culture supernatant according to preceding method People,J. Biol. Chem. 276:25894-02 (2001))。
Using pTrxFus (Invitrogen), make the C containing CCPII and rat MASP-2 serine protease domain It holds polypeptide (CCPII-SP), becomes thioredoxin fusion protein in expression in escherichia coli.It will with Thiobond affine resin Protein is purified from cell lysate.Thioredoxin fusion object gametophyte is expressed by pTrxFus empty carrier as yin Property control.
All recombinant proteins are carried out dialysis in TBS buffer, by measuring OD (280 nm), measure its concentration.
Dot blotting analysis:
Using 5 recombination MASP-2 polypeptides shown in above-mentioned and Figure 10 of serial dilution (and thioredoxin polypeptide as The negative control of CCPII- serine protease polypeptide) it puts on nitrocellulose filter.The point sample amount of protein is in 5 weight steps Range be 100 ng-6.4 pg.In experiment later, the point sample amount of protein again the range in 5 weight steps by 50 Ng is down to 16 pg.The TBS solution (Block buffer) of 5% skimmed milk power of the film is closed, then with the 1.0 anti-MASP-2 of μ g/ml The Block buffer of Fab2 (contains 5.0 mM Ca2+) be incubated with.Anti-human Fab (the AbD/Serotec being conjugated with HRP；1/ 10,000 dilution) and ECL detection kit (Amersham) detection combination Fab2.One block film and more grams as positive control Grand rabbit-anti people MASP-2 Ab (referring to Stover et al.,J Immunol 163: 6848-59 (1999)) it is incubated with.At this In the case of kind, with the goat anti-rabbit igg (Dako of HRP conjugation；1/2,000 dilution), detect the Ab combined.
MASP-2 binding assay
The carbonate buffer solution (pH 9.0) of the recombination MASP-2A or CUBI-II polypeptide in 1.0 holes μ g/ of elisa plate is at 4 DEG C Coating is overnight.Hole is closed with the TBS solution of 1% BSA, and the TBS solution that the anti-MASP-2 Fab2 of serial dilution is then added (contains There are 5.0 mM Ca2+).The plate is incubated at room temperature 1 hour.With TBS/ tween/Ca2+After washing 3 times, it is added and presses 1/10, 000 is diluted in TBS/Ca2+HRP conjugation anti-human Fab (AbD/Serotec), it is small that the plate is incubated at room temperature 1 again When.The antibody combined is detected with TMB peroxidase substrate kit (Biorad).
As a result:
Show the results are provided in the following table 7 for the reactive dot blotting analysis of Fab2 and various MASP-2 polypeptides.Table 7 The numerical value of offer shows to provide protein point sample amount required for the about half of maximum signal.As indicated, all polypeptides (only thioredoxin fusion object gametophyte makes an exception) by positive control Ab, (polyclonal anti-human MASP-2 serum, produces in rabbit It is raw) identification.
Table 7: in dot blotting with the reactivity of each recombinant rat MASP-2 polypeptide
Fab2 antibody # MASP-2A CUBI-II CUBI/EGF- sample CCPII-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
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=reactionless.Positive control antibodies are the polyclonal anti-human MASP-2 serum generated in rabbit.
All Fab2 react (data are not shown) with MASP-2A and MASP-2K.Most of Fab2 identify CCPII-SP Polypeptide but nonrecognition N-terminal segment.Fab2 #60 and Fab2 #57 are two exceptions.Fab2 #60 identifies MASP-2A and CUBI-II Segment, but nonrecognition CUBI/EGF sample polypeptide or CCPII-SP polypeptide, this prompts it can be in conjunction with the epitope or leap of CUBII CUBII and EGF spline structure domain.Fab2 #57 identifies MASP-2A but any tested MASP-2 segment of nonrecognition, this shows this The epitope of kind Fab2 identification CCP1.Fab2 #40 and #49 is only in conjunction with complete MASP-2A.The ELISA shown in Figure 11, which is combined, to be surveyed Determine in method, Fab2 #60 is herein in connection with CUBI-II polypeptide, although only having slightly lower apparent affinity.
These are observation indicate that unique identification for blocking property Fab2 for MASP-2 albumen multiple regions.
Embodiment 12
Present embodiment describes using phage display identification to combine MASP-2 and inhibiting the complement activation of lectin-mediated, simultaneously Retain the complete full people scFv antibody of classics (C1q- dependence) pathway component of immune system.
It summarizes:
Full people's high-affinity MASP-2 antibody passes through screening phage display library identification.The variable light and heavy chain fragment of antibody It is separated in the form of scFv with overall length IgG form.People's MASP-2 antibody can be used for the complement pathway for inhibiting to mediate with lectin pathway Related cellular damage is activated, while classics (C1q- dependence) pathway component for retaining immune system is complete.In some implementations In scheme, the MASP-2 inhibiting antibody have the feature that (a) to the high-affinity of people MASP-2 (for example, 10 nM or Smaller KD) and (b) in 90% human serum with 30 nM or smaller IC50 inhibition MASP-2- dependent complement activity.
Method:
The expression of the MASP-2 of overall length catalytically inactive:
Full length cDNA sequence (the SEQ ID of the people MASP-2 of encoding human MASP-2 polypeptide and leader sequence (SEQ ID NO:5) NO:4 it) is subcloned to mammalian expression vector pCI-Neo (Promega), in the control of cmv enhancer/promoter region The lower driving eukaryotic expression of system (it is described in Kaufman R.J. et al.,Nucleic Acids Research19: 4485-90, 1991;Kaufman,Methods in Enzymology,185:537-66 (1991))。
In order to generate people's MASP-2A albumen of catalytically inactive, by carrying out direct mutagenesis described in US2007/0172483, It is incorporated herein by reference.PCR product purifies after agarose gel electrophoresis and band preparation, and is produced using standard tailing program Raw simple gland glycosides overlapping.Then the MASP-2A of adenosine tailing is cloned into pGEM-T easy carrier and conversion to Escherichia coli.People MASP-2A is further subcloned into either one or two of mammalian expression vector pED or pCI-Neo.
Above-mentioned MASP-2A expression construct is transfected using standard calcium phosphate transfection procedures (Maniatis et al., 1989) To DXB1 cell.MASP-2A is generated in serum free medium, to ensure that prepared product is not polluted by other serum proteins.Often Culture medium (in total four times) are harvested from the cell converged every two days.About 1.5 mg/ of horizontal average out to for recombinating MASP-2A rises training Support base.MASP-2A (above-mentioned Ser-Ala mutant) is purified on MBP-A- agarose column by affinity chromatography.
To the MASP-2A ELISA by elutriation/scFv conversion and the ScFv candidate clone of filter screening and identification
Antigen elutriation is carried out to the phage display library of human immunoglobulin(HIg) light chain and weight chain variabl area sequence, is then automated Antibody screening and selection are to identify the high-affinity scFv antibody to people's MASP-2 albumen.For HIS- label or biotin-mark The MASP-2A of label carries out the three-wheel elutriation of scFv phage library.Third round elutriation is eluted with MBL first, then uses TEA (alkali Property) elution.In order to which monitoring and displaying is for the specific enrichment of the bacteriophage of the scFv segment of target MASP-2A, for fixed MASP-2A carries out polyclonal Phage-ELISA.By the scFv gene cloning taken turns from elutriation the 3rd to pHOG expression vector and into The small-scale filter screening of row is to find the specificity clone for being directed to MASP-2A.
The bacterial clump for choosing the plasmid comprising scFv segment of the coding from third round elutriation, is transferred to nitrocellulose On film, and grows and stayed overnight to generate motherboard on non-induced culture medium.From third round elutriation, 18,000 bacterium colonies are picked in total And analysis, half are eluted from competitive elution and half from subsequent TEA.For MASP-2A elutriation scFv phasmid text Library then scFv conversion and filter screening, obtains 137 positive colonies.108/ in the ELISA measurement combined for MASP-2 137 clones are positive (data are not shown), block MASP-2 wherein further analyzing 45 and being cloned in normal human serum Active ability.
Measurement inhibits the measuring method of the formation of lectin pathway C3 convertase
The functional examination method that measurement inhibits lectin pathway C3 convertase to be formed is for evaluating MASP-2 scFv candidate clone " Blocking activity ".MASP-2 serine protease is needed to generate two kinds of protein components (C4b, C2a), and it includes agglutinins Approach C3 convertase.Therefore, inhibit the MASP-2 scFv of MASP-2 functional activity (that is, blocking MASP-2 scFv) that will inhibit solidifying Collect re-forming for plain approach C3 convertase.C3 includes one of uncommon and highly reactive thioester group as its structure Part.In this measuring method after C3 is cracked by C3 convertase, thioester group on C3b can be fixed on the big of plastic eyelet bottom Hydroxyl or amino on molecule form covalent bond by ester or amido bond, to be conducive to detect C3b in ELISA measurement.
Yeast mannans are the known activator of lectin pathway.In the following methods of the formation of measurement C3 convertase In, it is incubated for the coated plastic eyelet of mannosan with diluted human serum, to activate lectin pathway.It is washed out hole, and is made The C3b being fixed on hole is measured with standard ELISA assay.The amount of the C3b generated in this measurement is lectin pathway C3 conversion The direct reflection that enzyme re-forms.The MASP-2 scFv clone inhibition C3 convertase of test selection concentration is formed in this measurement With the ability that therefore C3b is generated.
Method:
By 45 candidate clones of above-mentioned identification through expression, purify and be diluted to identical stock concentrations, contain Ca++ and Mg+ + GVB buffer (4.0 mM barbitals, 141 mM NaCl, 1.0 mM MgCl2,2.0 mM CaCl2,0.1% gelatin, pH 7.4) it is diluted again in ensure that all clones have same amount of buffer.ScFv clone is respectively in triplicate with 2 μ g/mL Concentration determination.Positive control is OMS100 Fab2 and is tested with 0.4 μ g/mL.It is cloned in scFv/IgG and exists and be not present In the case where monitor C3c formed.
In 50mM carbonate buffer solution (+1.5 mM NaN3 of 15mM Na2CO3+35mM NaHCO3) pH 9.5 Mannosan is diluted to the concentration (1 hole μ g/) of 20 μ g/mL and is stayed overnight on elisa plate in 4oC coating.Second day, sweet dew The coated plate of glycan-is washed 3 times with 200 μ l PBS.Then by the 1% HSA lock solution adding hole of 100 μ l, and in room Temperature is lower to be incubated for 1 hour.Plate is washed 3 times with 200 μ l PBS, and is stored on ice with 200 μ l PBS, until sample is added.
Normal human serum is diluted to 0.5% in CaMgGVB buffer, and scFv clone or OMS100 Fab2 is positive The buffer and on ice is added with 0.01 μ g/mL, 1 μ g/mL (only OMS100 compare) and 10 μ g/mL in control in triplicate Preincubate 45 minutes, closed elisa plate is then added.Start within 1 hour reaction and by shifting plate by being incubated in 37oC Stop into ice bath.Rabbit α-mouse C3c antibody then goat α-rabbit HRP the detection of C3b deposition.Negative control is free from antibody Buffer (no antibody=maximum C3b deposition), and positive control is the buffer (no C3b deposition) containing EDTA.Pass through progress Same measured method measures background, other than the hole is without mannosan.It will be for the background signal of the plate without mannosan It is subtracted from the signal in the hole containing mannosan.Cut-off standard is set as unrelated scFv clone (VZV) and the only half of buffer Activity.
As a result: according to cut-off standard, discovery clones the activity for blocking MASP-2 for 13 in total.> 50% approach is generated to inhibit All 13 clones selected and be sequenced, obtain 10 Unique clones.It was found that all 10 clones light chain having the same is sub- Class λ 3, but three different heavy chain subclass: VH2, VH3 and VH6.In functional examination method, 0.5% human serum, 5/10 time are used ScFv clone is selected to provide the IC50 nM value less than 25 nM target criterias.
In order to identify the antibody with improved effect, light chain is carried out to the three mother scFv clone identified as described above Reorganization.The process includes generating to be matched by the library of the VH and the natural human lambda light chain (VL) for being originated from 6 healthy donors of each female clone To the combinatorial libraries of composition.Then screening in the library, there is improved binding affinity and/or functional scFv to clone.
Table 8: the function effect of the IC50 (nM) of main son clone and their corresponding female clones's (all scFv forms) Compare
Provided hereinafter heavy chain variable region (VH) sequences of the female clone shown for table 8 above and son clone.
Kabat CDRs (31-35 (H1), 50-65 (H2) and 95-107 (H3)) is indicated with runic；And Chothia CDRs (26-32 (H1), 52-56 (H2) and 95-101 (H3)) is indicated with underscore.
17D20_35VH-21N11VL heavy chain variable region (VH) (SEQ ID NO:67 is encoded by SEQ ID NO:66)
QVTLKESGPVLVKPTETLTLTCTVSGFSLSRGKMGVSWIRQPPGKALEWLAHIFSSDEKSYRTSLKSRLTISK DTSKNQVVLTMTNMDPVDTATYYCARIRRGGIDYWGQGTLVTVSS
D17N9 heavy chain variable region (VH) (SEQ ID NO:68)
QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSTSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITI NPDTSKNQFSLQLNSVTPEDTAVYYCARDPFGVPFDIWGQGTMVTVSS
Provided hereinafter light chain variable region (VL) sequences of the female clone shown for table 8 above and son clone.
Kabat CDRs (24-34 (L1); 50-56 (L2);It is indicated with 89-97 (L3) with runic；And Chothia CDRs (24-34 (L1);50-56 (L2) and 89-97 (L3) are indicated with underscore.These regions be it is identical, either Pass through Kabat or Chothia System Number.
17D20m_d3521N11 light chain variable region (VL) (SEQ ID NO:70 is encoded by SEQ ID NO:69)
QPVLTQPPSLSVSPGQTASITCSGEKLGDKYAYWYQQKPGQSPVLVMYQDKQRPSGIPERFSGSNSGNTATLT ISGTQAMDEADYYCQAWDSSTAVFGGGTKLTVL
17N16m_d17N9 light chain variable region (VL) (SEQ ID NO:71)
SYELIQPPSVSVAPGQTATITCAGDNLGKKRVHWYQQRPGQAPVLVIYDDSDRPSGIPDRFSASNSGNTATLT ITRGEAGDEADYYCQVWDIATDHVVFGGGTKLTVLAAAGSEQKLISE
MASP-2 antibody OMS100 and MoAb_d3521N11VL (include heavy chain variable region and SEQ shown in SEQ ID NO:67 Light chain variable region shown in ID NO:70, also known as " OMS646 " and " mAb6 "), it has proven to high-affinity combination people MASP-2 and ability with block function complement activity, by Dot blot about epitope binding analysis.The result shows that OMS646 and OMS100 antibody is to MASP-2 high degree of specificity, and does not combine MASP-1/3.There is no antibody combination MAp19, does not have yet There is antibody to combine the MASP-2 segment of the CCP1 structural domain not comprising MASP-2, cause to draw a conclusion, binding site includes CCP1.
When compared with C1s, C1r or MASP-1, measurement MASP-2 antibody OMS646 is combined closely with > 5000 times of selectivity It recombinates MASP-2 (Kd 60-250pM) (referring to the following table 9):
Table 9: by the OMS646 MASP-2 antibody-MASP-2 of the solid phase ELISA research evaluation affinity to interact and specifically Property
Antigen KD (pM)
MASP-1 >500,000
MASP-2 62±23*
MASP-3 >500,000
The people C1r of purifying >500,000
The people C1s of purifying ~500,000
* average value ± SD; n=12
The Lectin-dependent of OMS646 specific inhibition terminal complement component activates
Method:
Film is attacked using the approach particular conditions analysis OMS646 for lectin pathway, classical pathway and alternative route multiple Close the influence of object (MAC) deposition.For this purpose, Wieslab Comp300 complement screening reagent box (Wieslab, Lund, Sweden) is pressed It is used according to the specification of manufacturer.
As a result:
Figure 12 A is surveyed in the case where being shown in presence or absence of anti-MASP-2 antibody (OMS646) in lectin pathway specificity The level that MAC is deposited under fixed condition.Figure 12 B be shown in presence or absence of anti-MASP-2 antibody (OMS646) in the case where The level that MAC is deposited under the conditions of classical pathway specific assay.Figure 12 C is shown in presence or absence of anti-MASP-2 antibody (OMS646) level that MAC is deposited under the conditions of alternative route specific assay in the case where.
As illustrated in fig. 12, OMS646 is with the IC of about 1nM50The activation that value blocks the lectin pathway of MAC deposition to mediate. However, what OMS646 generated the activation (Figure 12 B) mediated from classical pathway or the activation (Figure 12 C) mediated from alternative route MAC is deposited without influence.
The pharmacokinetics and pharmacodynamics of OMS646 after intravenous (IV) or subcutaneous (SC) give mouse
The pharmacokinetics (PK) and pharmacodynamics (PD) of OMS646 are evaluated in 28 days single-dose PK/PD research of mouse.It should Research tests the dosage level and intravenous administration (IV) of the OMS646 of subcutaneous administration (SC) 5mg/kg and 15mg/kg The dosage level of 5mg/kg OMS646.
About the PK feature of OMS646, (n=3 are dynamic for OMS646 concentration after Figure 13 illustrates the OMS646 for giving prescribed dose Object/group average value) function as the time.As shown in figure 13, with about 1-2 days after 5mg/kg SC administration, OMS646 reaches The maximal plasma concentration of 5-6 μ g/mL.Bioavilability with 5 mg/kg SC, OMS646 is about 60%.As Figure 13 is further Display, with about 1-2 days after 15 mg/kg SC administration, OMS646 reached the maximal plasma concentration of 10-12 μ g/mL.For All groups, OMS646 is slowly removed from system circulation, and wherein end-stage half-life period is about 8-10 days.OMS646 is characterized in small It is typical for human antibody in mouse.
The PD activity of OMS646 is shown in Figure 14 A and 14B.Figure 14 A and 14B are shown for 5mg/kg IV (Figure 14 A) (decline of system lectin pathway activity) is reacted with the PD of each mouse in 5mg/kg SC (Figure 14 B) group.Dotted line indicates measurement Baseline (maximum suppression；Test the Naive mice serum of the outer excessive addition OMS646 of precursor).As shown in Figure 14 A, it is given in IV After the OMS646 of 5mg/kg, system lectin pathway activity drops to almost undetectable level immediately, and observes through 28 days Phase, lectin pathway activity only show appropriateness recovery.As shown in Figure 14B, in the mouse of OMS646 SC for giving 5mg/kg, Observe the active time-dependent inhibition of lectin pathway.Lectin pathway activity drug give dropped in 24 hours it is several Undetectable level and holding low-level at least 7 days.Lectin pathway activity gradually increases at any time, but observes at 28 days It is horizontal before not restoring in phase to administration.The curve of the lectin pathway observed after giving 15mg/kg SC active reduced time Similar to 5 mg/kg SC dosage (data are not shown), show that PD terminal is saturated.Data further demonstrate that IV or SC give 5mg/ The weekly dosage of the OMS646 of kg is enough to realize the active lasting inhibition of system lectin pathway in mouse.
Embodiment 13
Present embodiment describes the generation for the recombinant antibodies for inhibiting MASP-2, the recombinant antibodies include heavy chain and/or light chain can Become area (one or more CDR comprising specifically binding MASP-2) and at least one SGMI Core peptide sequence (also known as has The MASP-2 antibody or its antigen-binding fragment of SGMI peptide).
Background/basic principle:
The generation of the referred to as specific inhibitor of the MASP-2 of SGMI-2 is described in Heja et al.,J Biol Chem 287: 20290 (2012) and Heja et al.,PNAS109:10498 (2012), each by being incorporated herein by reference.SGMI-2 is The peptide of 36 amino acid, selected from desert locust (Schistocerca gregaria) protease inhibitors 2 variant bacteriophage Library, wherein 6 in the position of the 8 of protease coupling collar are by completely random.Subsequent Evolution in vitro obtains monospecific suppression Preparation, with single digit nM KIValue (Heja et al.,J. Biol. Chem287:20290,2012).Structural research table Bright, the protease coupling collar of optimization forms the principal binding sites for limiting the specificity of two kinds of inhibitor.It is extended secondary and interior The amino acid sequence of portion combined area is that two kinds of inhibitor are shared, and facilitate contact interface (Heja et al., 2012.J. Biol. Chem. 287:20290).In mechanism, SGMI-2 blocks the lectin pathway of complement activation without influencing classical way Diameter (Heja et al., 2012. Proc. Natl. Acad. Sci. 109:10498)。
The amino acid sequence of SGMI-2 inhibitor is as shown below:
SGMI-2- overall length:
LEVTCEPGTTFKDKCNTCRCGSDGKSAVCTKLWCNQ (SEQ ID NO:72)
SGMI-2- is medium:
TCEPGTTFKDKCNTCRCGSDGKSAVCTKLWCNQ (SEQ ID NO:73)
SGMI-2- is short:
………………………………..TCRCGSDGKSAVCTKLWCNQ (SEQ ID NO:74)
As described in the embodiment, and described in WO2014/144542, MASP-2 antibody and its segment with SGMI-2 peptide By fusion SGMI-2 peptide amino acid sequence (for example, SEQ ID NO:72,73 or 74) to people MASP-2 antibody heavy chain and/ Or it is generated on the amino or carboxyl terminal of light chain.When being measured in C3b or C4b deposition measuring method using human serum, and do not wrap The naked MASP-2 scaffold antibody of the peptide sequence containing SGMI-2 is compared, and MASP-2 antibody and segment with SGMI-2 peptide have raising Inhibitory activity, as described in WO2014/144542, and when being measured in mouse in vivo models, with naked MASP-2 bracket Antibody is compared to the inhibitory activity also with raising.The method for generating the MASP-2 antibody with SGMI-2 peptide is described below.
Method:
The expression construct of four kinds of the coding illustrative MASP-2 antibody with SGMI-2 peptide is generated, wherein SGMI-2 peptide merges To the heavy chain of representative MASP-2 inhibiting antibody OMS646 (generating as described in Example 12) or the end N- or C- of light chain.
Table 10:MASP-2 antibody/SGMI-2 fusions
Abbreviation in table 10:
The amino terminal of " H-N "=heavy chain
The carboxyl terminal of " H-C "=heavy chain
The amino terminal of " L-N "=light chain
The carboxyl terminal of " L-C "=light chain
" M2 "=MASP-2 ab bracket (representative OMS646)
For N- terminal fusion shown in table 10, peptide linker (' GTGGGSGSSS ' SEQ ID NO:79) in SGMI-2 peptide and It is added between variable region.
For C- terminal fusion shown in table 10, peptide linker (' AAGGSG ' SEQ ID NO:80) in constant region and It is added between SGMI-2 peptide and the second peptide " GSGA " (SEQ ID NO:81) is added in the end C- of fused polypeptide to protect C- End SGMI-2 peptide is from degradation.
For following representative MASP-2 antibody/SGMI-2 fusions, amino acid sequence is provided below:
H-M2ab6-SGMI-2-N (SEQ ID NO:75 is encoded by SEQ ID NO:82):
LEVTCEPGTTFKDKCNTCRCGSDGKSAVCTKLWCNQ GTGGGSGSSS QVTLKESGPVLVKPTETLTLTCTVSGF SLSRGKMGVSWIRQPPGKALEWLAHIFSSDEKSYRTSLKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCARIRRGG IDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDK SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
[491 aa albumen, aa 1-36=SGMI-2 (underscore), aa37-46=connector (italic), aa47-164=MASP-2 The heavy chain variable region (underscore) of ab#6, the aa165-491=IgG4 constant region with hinge mutation]
H-M2ab6-SGMI-2-C (SEQ ID NO:76 is encoded by SEQ ID NO:83):
QVTLKESGPVLVKPTETLTLTCTVSGFSLSRGKMGVSWIRQPPGKALEWLAHIFSSDEKSYRTSLKSRLTISK DTSKNQVVLTMTNMDPVDTATYYCARIRRGGIDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPC PAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKAAGGSG LEVTCEP GTTFKDKCNTCRCGSDGKSAVCTKLWCNQ GSGA
[491aa albumen, the heavy chain variable region (underscore) of aa1-118=MASP-2 ab#6, aa 119-445=there is hinge The IgG4 constant region of mutation, the connector of aa 446-451=the 1st (italic), aa 452-487=SGMI-2, aa488-491=the 2nd connect Head (italic)]
L-M2ab6-SGMI-2-N (SEQ ID NO:77 is encoded by SEQ ID NO:84):
LEVTCEPGTTFKDKCNTCRCGSDGKSAVCTKLWCNQ GTGGGSGSSS QPVLTQPPSLSVSPGQTASITCSGEKL GDKYAYWYQQKPGQSPVLVMYQDKQRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQAWDSSTAVFGGGTKLT VLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLT PEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
[258aa albumen, aa1-36=SGMI-2 (underscore), aa37-46=connector (italic), aa47-152=MASP-2 ab# 6 light chain variable region (underscore), aa153-258=people Ig λ constant region]
L-M2ab6-SGMI-2-C (SEQ ID NO:78、encoded by SEQ ID NO:85):
QPVLTQPPSLSVSPGQTASITCSGEKLGDKYAYWYQQKPGQSPVLVMYQDKQRPSGIPERFSGSNSGNTATLT ISGTQAMDEADYYCQAWDSSTAVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKA DSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSAAGGSG LEVTCEPGT TFKDKCNTCRCGSDGKSAVCTKLWCNQ GSGA
[258aa albumen, the light chain variable region (underscore) of aa1-106=MASP-2 ab#6, aa 107-212=people Ig λ are constant Area, the connector of aa 213-218=the 1st, the connector of aa219-254=SGMI-2, aa255-258=the 2nd]
Functional examination method:
Four kinds of MASP-2-SGMI-2 fusion antibody constructs transient expression in Expi293F cell (Invitrogen), passes through Protein A affinity chromatography purifying, and tested in 10% normal human serum in the mannosan coating pearl measuring method being described below The inhibition of C3b deposition.
MASP-2-SGMI-2 fusions are tested in the mannosan coating pearl measuring method deposited for C3b
The lectin pathway of MASP-2-SGMI-2 fusion antibody is evaluated on mannosan coating pearl in C3b deposition measuring method Inhibit.The measuring method provides the resolution ratio bigger than Wieslab measuring method by Flow Cytometry Assay level of activity. Lectin pathway pearl measuring method carries out as follows: inhaling mannosan in carbonate-bicarbonate buffer solution (pH 9.6) in 4oC It is attached to 7 μM of diameter polystyrene pearl (Bangs Laboratories;Fishers, IN, USA) overnight.Pearl is washed in PBS Be exposed to 10% human serum, or with antibody or 10% serum of inhibitor preincubate.Serum-pearl mixture is incubated at room temperature It 1 hour, stirs simultaneously.After serum is incubated for, pearl is washed, and pass through with anti-C3c rabbit polyclonal antibody (Dako North America;Carpinteria, CA, USA) and PE-Cy5 conjugation anti-rabbit secondary antibody (the Southern Biotech of goat; Birmingham, AL, USA) detection, measure the C3b deposition on pearl.After dyeing procedure, FACSCalibur flow cytometer is used Analyze pearl.Pearl is gated as uniform group using forward scattering and sidescattering, and it is bright that C3b, which is deposited as FL3- positive particle, Aobvious (FL-3 or " channel FL-3 " indicate the 3rd or red channel on cell instrument).For each experiment condition, the geometry of the group Average fluorescent strength (MFI) is relative to antibody/inhibitor concentration mapping, to evaluate lectin pathway inhibition.
IC50Value is calculated using GraphPad PRISM software.Particularly, IC50Value passes through using variable slope (four ginsengs Number), Nonlinear Quasi be bonded to obtained from cell counting measuring log (antibody) and average fluorescent strength curve obtain.
As the result is shown in table 11.
Table 11: the C3b deposition in 10% human serum (mannosan is coated with pearl measuring method)
Construct IC50 (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
As a result:
Control, MASP-2 " naked " scaffold antibody (mAb#6) without SGMI, is inhibition in this measuring method, have >= The IC50 value of 3.63 nM, it is consistent with the inhibition result that embodiment 12 is observed.It is apparent that as shown in table 11, test is owned SGMI-2-MASP-2 antibody fusions improve the effect of MASP-2 scaffold antibody in the measuring method, show price increase It is advantageously possible for the inhibition of C3b deposition.
MASP-2-SGMI-2 is tested in the mannosan coating pearl measuring method for C4b deposition measurement with 10% human serum Fusions
It is carried out C4b using above-mentioned determination condition identical for C3b deposition measuring method with 10% human serum and deposits measuring method, tool There is following modification.Before flow cytometry, by being dyed with anti-C4b mouse monoclonal antibody (1:500, Quidel) Deposition reactant and be conjugated to PE Cy5 2 secondary antibody of goat anti mouse F (ab ') (1:200, Southern Biotech) contaminate Color carries out C4b detection and flow cytometry.
As a result:
Compared with MASP-2 scaffold antibody (HL-M2:IC50=0.78nM), the MASP-2-N- terminal antibody with SGMI-2 melts Close object (H-M2-SGMI-2-N:IC50=0.34nM), the nM of L-M2-SGMI-2-N:IC50=0.41)) all have increased effect Power.
Similarly, with MASP-2 scaffold antibody (HL-M2:IC50=1.2 nM) it compares, the end C- with single SGMI-2 MASP-2 antibody fusions (H-M2-SGMI-2-C:IC50=0.45nM and L-M2-SGMI-2C:IC50=0.47 nM) it all has Increased effect.
With 10% mice serum, test MASP-2-SGMI-2 melts in the mannosan coating pearl measuring method deposited for C3b Close object
Pearl measuring method is coated with the mannosan that 10% mice serum deposit for C3b as described above.Similar in human serum Middle observation as a result, determining in mice serum compared with MASP-2 scaffold antibody, MASP-2 fusions with SGMI-2 tool There is increased effect.
As a result summarize: the present embodiment as a result, it was confirmed that all SGMI-2-MASP-2 antibody fusions of test improve The effect of MASP-2 scaffold antibody.
Embodiment 14
The present embodiment provides the unilateral urine outputs for using the kidney fibrosis in MASP-2 -/- defect and MASP-2+/+abundance mouse Pipe block (UUO) model generate as a result, to evaluate effect of the lectin pathway in kidney fibrosis.
Background/basic principle:
Kidney fibrosis and inflammation are the main features of end-stage renal disease.Renal tubular interstitium fibrosis is progressive process, is related to P cell damage, abnormal healing, resident and infiltration nephrocyte activation, cytokine release, inflammation and nephrocyte phenotype are living Change to generate extracellular matrix.Renal tubular interstitium (TI) fibrosis is the common terminal of a variety of nephrosis reason and represents purpose and exist The key target of the potential therapy of progressive renal impairment in prevention chronic kidney diseases (CKD).Kidney TI damage is small with kidney Decreased renal function tight association in ball disease (Risdon R.A. et al.,Lancet 1:363-366,1968; Schainuck L.I. et al.,Hum Pathol1:631-640,1970;Nath K.A.,Am J Kid Dis20:1-17,1992), and And be the feature of CKD, wherein there are the accumulations of myofibroblast, and intercapillary potential around renal tubule and pipe Space is occupied by the matrix comprising collagen and other proteoglycans.Still there is extensive arguement in the source of TI myofibroblast, but fine Dimensionization is usually after inflammation, and the inflammation initial characteristics are T lymphocyte, followed by the TI of macrophage gathers (Liu Y. Et al.,Nat Rev Nephrol7:684-696,2011;Duffield J.S.,J Clin Invest 124:2299- 2306、2014)。
The rodent model of UUO generates progressive kidney fibrosis, a kind of progressive kidney diaseases of the substantially any cause of disease Mark (Chevalier et al., Kidney International 75:1145-1152,2009).It has been reported that C3 gene table Up to increasing in the wild-type mice after UUO, and compared with wild-type mice, C3-/- knock-out mice of the collagen deposition after UUO In substantially reduce, show complement activation in kidney fibrosis effect (Fearn et al.,Mol Immunol 48:1666-1733、 2011).Also it has been reported that C5 deficiency causes the main component of the kidney fibrosis in the model that renal tubular interstitium damages to significantly improve (Boor P. et al.,J of Am Soc of Nephrology: 18:1508-1515,2007).However, the present inventor into Before capable research as described herein, the specific complement component for participating in kidney fibrosis is still uncertain.Therefore, following research is carried out To evaluate MASP-2 (-/-) in unilateral ureteral obstruction (UUO) model and MASP-2 (+/+) male mice.
Method:
MASP-2-/- mouse is generated as described in embodiment 1, and is returned for 10 generations with C57BL/6.Male wild-type (WT) C57BL/6 Homozygous MASP-2 defect (MASP-2-/-) mouse of mouse and C57BL/6 background is maintained at the standard bar of 12/12 day night circulation Under part, feeding standard feed granules and food and water are freely obtained.10 week old mouse, are used in 1.5 L/min oxygen by 6/group In 2.5% isoflurane anesthesia.The right ureter of two group of 10 week old male C56/BL6 mouse (wild type and MASP-2-/-) is handled Art ligation.Right kidney passes through the exposure of 1cm lateral incision.Right ureter is hindered completely using 6/0 polyglactin suture in two points Plug.It provides within every 12 hours after buprenorphine analgesic before surgery, at most 5 dosage depend on pain scores.Local Bupivacaine Anesthetic is given once during operation.
7 days execution mouse and collection nephridial tissue, fix and are embedded in paraffin mass after operation.It is pierced under anaesthesia by heart It wears from mouse and collects blood, and mouse is eliminated by avascularization after nephrectomy.Blood is set to solidify 2 hours on ice and pass through It is centrifugated serum and keeps freezing in -80oC by equal part.
The immunohistochemistry of nephridial tissue
In order to measure the degree of the kidney fibrosis indicated by collagen deposition, the bitter Picro-Sirius red of 5 microns of paraffin embedding kidney segments A kind of (collagen specificity stain) dyeing, such as Whittaker P. et al.,Basic Res Cardiol89:397-410,1994 It is described.In short, kidney segment is taken off paraffin, bitter Picro-Sirius red water rehydrated and in 500 mL picric acid saturated aqueous solutions Solution (0.5 gm Picro-Sirius red, Sigma, Dorset UK) was by collagen staining 1 hour.In acidifying water (0.5% glacial acetic acid/distillation Water) in slide is washed twice, it is respective 5 minutes, then rehydrated and fixed.
In order to measure the degree of inflammation indicated by macrophages infiltration, kidney segment macrophage-specific antibody F4/ 80 dyeing as described below.Formalin is fixed, 5 microns of kidney segments of paraffin embedding take off paraffin and rehydrated.In citrate It is carried out antigen recovery 20 minutes in buffer in 95oC, then by 3% H2O2Middle incubation quenching endogenous peroxidating in 10 minutes Object enzymatic activity.By histotomy in Block buffer (10% heat-inactivated Normal Goat Serum and 1% bovine serum albumin(BSA)/phosphorus Hydrochlorate buffered saline (PBS)) in be incubated at room temperature 1 hour, then avidin biotin bonds are closed.By group after each step It knits slice to wash in PBS three times, continues 5 minutes.Apply 1:100 and is diluted in the F4/80 macrophage one in Block buffer Anti- (Santa Cruz, Dallas, TX, USA) continues 1 hour.Then it is anti-big to apply the diluted biotinylated goat of 1:200 Mouse secondary antibody continues 30 minutes, then applies horseradish peroxidase (HRP) conjugated enzyme, continues 30 minutes.Use benzidine Amine (DAB) substrate (Vector Labs, Peterborough UK) carries out dyeing colour developing 10 minutes and washs slide in water, then It is hydrated and fixed, without redying, in favor of computer based analysis.
Image analysis
The percentage of cortex renis dyeing presses Furness P. N. et al.,J Clin Pathol50:118-122,1997 surveys It is fixed.In short, 24 color images from around the entire periphery of kidney segment immediately the cortex renis below the scrotum continuously not Overlapped view capture.After each image capture, NIH image is for extracting red channel as 8 monochrome images.Background illumination Inhomogeneities is reduced using the pre-recorded image of the bright field of microscope in place not being sliced.Image is set to be subjected to fixing Threshold value corresponds to the image-region of stained positive to identify.Then the percentage of black picture element is calculated, and in the institute for surrounding kidney After thering is image to be measured by this method, average percent is recorded, is provided corresponding to the percentage of stained area in kidney segment Value.
Gene expression analysis
The expression of several genes about nephritis disease and fibrosis in Mouse Kidney is measured as follows by quantitative PCT (qPCR).Always RNA uses Trizol from cortex renis®(ThermoFisher Scientific, Paisley, UK) according to the manufacturer's instructions Separation.The RNA of extraction is handled with Turbo DNA-free kit (ThermoFisher Scientific) to eliminate DNA dirt Dye, then using AMV Reverse Transcription System (Promega, Madison, WI, USA) synthesis first Chain cDNA.CDNA integrality is by using TaqMan GAPDH Assay's (Applied Biosystems, Paisley UK) Single qPCR reaction, then uses Custom TaqMan Array 96- orifice plate (Life Technologies, Paisley, UK) QPCR react confirm.
12 genes are had studied in the analysis:
(the col4 α 1 of Collagen type IV α 1;Measure ID:Mm01210125_m1)
Transforming growth factor-β_1 (TGF β -1;Measure ID:Mm01178820_m1);
1 (Cdh1 of cadherin;Measure ID:Mm01247357_m1);
1 (Fn1 of fibronectin;Measure ID:Mm01256744_m1);
Interleukin-6 (IL6;Measure ID Mm00446191_m1);
Interleukin 10 (IL10;Measure ID Mm00439614_m1);
Interleukin 12 a (IL12a;Measure ID Mm00434165_m1);
Vimentin (Vim;Measure ID Mm01333430_m1);
1 (Actn1 of actinine α;Measure ID Mm01304398_m1);
Tumor necrosis factor-alpha (TNF-α;Measure ID Mm00443260_g1)
3 (C3 of complement component;Measure ID Mm00437838_m1);
Interferon gamma (Ifn- γ;Measure ID Mm01168134)
Use following crt gene of running one's home:
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH;Measure ID Mm99999915_g1);
Glucuronidase β (Gus β;Measure ID Mm00446953_m1);
Eukaryon 18S rRNA (18S;Measure ID Hs99999901_s1);
Hypoxanthine guanine phosphoribosyltransferase (HPRT;Measure ID Mm00446968_m1)
12 μ L reactants are expanded using TaqMan Fast Universal Master Mix (Applied Biosystems) 40 circulations.Real time PCR amplification data are analyzed using 7000 SDS v1.4 software of Applied Biosystems.
As a result:
After unilateral ureteral obstruction (UUO), obstruction kidney channel goes through inflammatory cell, particularly macrophage flows into, and then quickly sends out Raw fibrosis, such as by the tubular ectasia and thinning proof of collagenous accumulations and proximal tubule epithelium (referring to Chevalier R. L. et al.,Kidney Int75:1145-1152,2009).
Figure 15 illustrate the computer based image analysis being sliced with the nephridial tissues of sirius red stains as a result, wherein group It knits and is sliced wild type and MASP-2-/- mouse being obtained from ureteral obstruction (UUO) after 7 days or obtained from sham-operation control mice.Such as Shown in Figure 15, the kidney segment of the wild-type mice after ureteral obstruction 7 days is shown than MASP-2-/significantly bigger collagen of-mouse It deposits (p value=0.0096).For the average value ± average value of UUO operation mouse in wild type and MASP-2-/- group Standard error is 24.79 ± 1.908 (n=6) and 16.58 ± 1.3 (n=6) respectively.Such as Figure 15 it further shows that from vacation Operating comparison wild type and sham-operation control MASP-2-/- mouse histotomy show the collagen staining of extremely low level, such as institute It is desired.
The computer based image point for the nephridial tissue slice that Figure 16 diagram is dyed with F4/80 macrophage specific antibody Analysis as a result, wherein histotomy be obtained from ureteral obstruction 7 days after wild type and MASP-2-/- mouse or be obtained from sham-operation pair According to mouse.As shown in figure 16, compared with wild-type mice, after ureteral obstruction 7 days, obtained from MASP-2-/- mouse UUO kidney The significant less macrophages infiltration of tissue display (% macrophage stained area be WT:2.23 ± 0.4 compare MASP-2-/-: 0.53 ± 0.06, p=0.0035).Such as Figure 16 it further shows that coming from sham-operation wild type and sham-operation MASP-2-/- mouse Histotomy show no detectable macrophage dyeing.
With the open country of the gene expression analysis of the associated various genes of nephritis disease and fibrosis after being obtained from ureteral obstruction 7 days It is carried out in raw type and MASP-2-/- mouse and sham-operation wild type and MASP-2-/- mouse nephridial tissue slice.Figure 17-20 The data of middle display are to indicate the Log10 and bar of the relative populations of wild type sham-operation sample the standard error of average value. Gene expression analysis about fibrosis associated genes as a result, the opposite mRNA of Figure 17 diagram Collagen type IV α 1 (collagen -4) Expression, such as the kidney group of the wild type after being obtained from ureteral obstruction 7 days and MASP-2-/- mouse and sham-operation control mice It knits in slice through qPCR measurement.Figure 18 illustrates the relative mRNA expression levels of transforming growth factor-β_1 (TGF β -1), such as In the nephridial tissue slice of the wild type and MASP-2-/- mouse and sham-operation control mice after being obtained from ureteral obstruction 7 days Pass through qPCR measurement.As shown in FIG. 17 and 18, compared with the sham-operation kidney of wild-type mice, the stalk from wild-type mice Resistance kidney confirms that the expression of fibrosis associated genes Collagen type IV (Figure 17) and TGF β -1 (Figure 18) dramatically increase, it was demonstrated that these are fine Dimensionization related gene is induced after the UUO damage of wild-type mice, as expected.In contrast, such as Figure 17 and 18 into one Step display, with by UUO damage wild-type mice compared with, from by UUO damage MASP-2-/- obstruction kidney show The expression of Collagen type IV substantially reduces (Figure 17, p=0.0388) and the expression of TGF β -1 substantially reduces (Figure 18, p=0.0174).
Gene expression analysis about inflammation-related gene as a result, the phase of Figure 19 diagram interleukin-6 (IL-6) To mRNA expression, the wild type and MASP-2-/- mouse and sham-operation control such as after being obtained from ureteral obstruction 7 days are small Pass through qPCR measurement in the nephridial tissue slice of mouse.Figure 20 illustrates the relative mRNA expression levels of interferon-γ, such as obtained from defeated It is surveyed in the nephridial tissue slice of wild type and MASP-2-/- mouse and sham-operation control mice after urinary catheter obstruction 7 days by qPCR Amount.As shown in Figures 19 and 20, compared with the sham-operation kidney of wild-type mice, the obstruction kidney from wild-type mice confirms scorching The expression of disease related gene interleukin-6 (Figure 19) and interferon-γ (Figure 20) dramatically increases, it was demonstrated that these inflammation phases Correlation gene is induced after the UUO damage of wild-type mice.In contrast, such as Figure 19 and 20 it further shows that with UUO is passed through The wild-type mice of damage is compared, from by UUO damage MASP-2-/- obstruction kidney show interleukin-6 (figure 19, p=0.0109) it is substantially reduced with the expression of interferon-γ (Figure 20, p=0.0182).
Note that in the UUO kidney for being obtained from both wild type and MASP-2-/- mouse for Vim, Actn-1, TNF α, C3 and All discovery is significantly raised for the gene expression of IL-10, the table of these specific genes between wild type and MASP-2-/- mouse There is no notable difference up to level (data are not shown).The gene expression dose of Cdh-1 and IL-12a is in the animal from any group Obstruction kidney in have not been changed (data are not shown).
It discusses:
The UUO model of generally acknowledged rodent induction early stage, activity and significantly damage, 1-2 week after obstruction in obstruction kidney It is interior that there are reduced renal hemodynamic, interstitial inflammation and faster fibres, and it is widely used in the inflammation and fibrosis for understanding kidney Common Mechanism and medium (see, for example, Chevalier R.L.,Kidney Int75:1145-1152,2009; Yang H. Et al.,Drug Discov Today Dis Models7:13-19,2010).
Described in the present embodiment as a result, it was confirmed that relative to wild type (+/+) control mice, in MASP-2 (-/-) mouse Substantially reducing there are collagen deposition and macrophages infiltration in UUO operation kidney.It is shown in 2 kinds of levels of histology and gene expression Show that in MASP-2-/- animal injury of kidney substantially reduces this it is unexpected as a result, it was confirmed that complement activation lectin pathway Remarkably promote the generation of inflammation and fibrosis in obstruction kidney.Although being not intended to the constraint by specific theory, it is believed that agglutinin way Diameter is by triggering and keeps proinflammatory sexual stimulus, fatefully promotes the Pathological Physiology of fibrotic disease, the proinflammatory sexual stimulus Vicious circle is maintained when cellular damage drives inflammation, and then causes further cellular damage, cicatrization and tissue damage It loses.Inhibit as a result, being expected with inhibitor according to these or block MASP-2 that will have inhibition or preventing renal fibrosis, and inhibits Or the prevention and/or therapeutic effect of the universal fibrosis of prevention (that is, independent of tissue or organ).
Embodiment 15
Present embodiment describes monoclonal MASP-2 inhibiting antibodies in a kind of unilateral ureteral obstruction (UUO) model (kidney The mouse model of fibrosis) in effect analysis.
Background/basic principle:
The improvement of renal tubular interstitium fibrosis (the common terminals of a variety of nephrosis reasons) represents purpose and is to prevent progressive kidney The key target of the therapeutic strategy of disease.Have in view of the new sum that the inflammatory for lacking targeting kidney diaseases promotees fibrosis approach and controls It treats, to exploitation new treatment, there are urgent needs.Many patients with albuminuria kidney diaseases show renal tubular interstitium inflammation and Progressive fibrosis, closely with decreased renal function.Albuminuria itself induces renal tubular interstitium inflammation and proteinuric nephropathies Generation (Brunskill N.J. et al.,J Am Soc Nephrol15:504-505,2004).No matter Primary renal disease, Renal tubular interstitium inflammation and fibrosis are always seen in the patient with progressive injury of kidney, and close with excretory function decline Cut phase pass (Risdon R.A. et al.,Lancet1:363-366,1968;Schainuck L.I. et al.,Hum Pathol 1:631-640,1970).Leading to the therapy of the potentiality of the crucial common cell approach of fibrosis with blocking has kidney disorders Widely applied prospect.
As described in Example 14, in the UUO model of non-protein urine kidney fibrosis, determination and wild-type control animals phase Show significant less kidney fibrosis and inflammation than, MASP-2-/- mouse, such as by inflammatory cell infiltration (75% is reduced) and The histological markers of fibrosis such as collagen (1/3 is reduced) proves, to establish lectin pathway in kidney fibrosis Key effect.
As described in Example 13, generate monoclonal MASP-2 antibody (OMS646-SGMI-2 fusions, comprising being fused to The SGMI-2 peptide of the C-terminal of the heavy chain of OMS646), the function of specific inhibition people's lectin pathway has also been shown that in mouse Middle blocked lectin element approach.In the present embodiment, UUO mouse mould of the OMS646-SGMI-2 in the kidney fibrosis of wild-type mice It is analyzed in type, to determine whether the specific inhibitor of MASP-2 is able to suppress kidney fibrosis.
Method:
This research evaluation is compared with 4 Isotype control antibodies of human IgG (10 mg/kg ET904) and Vehicle controls, MASP-2 suppression Effect of the property antibody (10 mg/kg OMS646-SGMI-2) processed in male WT C57BL/6 mouse.In UUO operation consent the 7th It, the 4th day and the 1st day and after surgery the 2nd day by (ip) injection in peritonaeum give each group 9 for antibody (10mg/kg) Mouse.Blood sample is obtained at the end of before antibody is given with experiment to evaluate lectin pathway functional activity.
UUO operation, tissue collecting and with Picro-Sirius red and macrophage specific antibody F4/80 dyeing use embodiment 14 The method carries out.
The hydroxyproline content of Mouse Kidney uses specific colorimetric estimation test kit (Sigma) saying according to manufacturer Bright book measurement.
In order to evaluate pharmacodynamic action of the MASP-2 inhibition mAb in mouse, system lectin pathway activity by It is solidifying that MASP-2 mAb or control mAb i.p. gives quantization in the diluted blood serum sample of minimum of the specified time collection after mouse The C3 of collection element induction activates evaluation.In short, 7 μM of diameter polystyrene microspheres (Bangs Laboratories, Fisher IN, USA) it is incubated overnight by 30 μ g/mL mannosans (Sigma) being used in sodium bicarbonate buffer liquid (pH 9.6), use is sweet Reveal glycan coating, be washed out, with 1% fetal calf serum/PBS closing and with 1x108The final concentration of a pearl/mL is resuspended in PBS.It mends Body deposition reaction is by being added mannosan coating pearl (~ 250,000 pearls) of 2.5 μ L to the diluted mouse of minimum of 50 μ L Blood serum sample (90% whole serum-concentration) starts, and is then incubated for 40 minutes in 4oC.It is thin by the ice-cold streaming that 250 μ L are added After born of the same parents' art buffer (FB: PBS containing 0.1% fetal calf serum) terminates deposition reaction, by the way that pearl is collected by centrifugation and with 300 μ L ice Cold FB is washed twice again.
In order to quantify the C3 activation of agglutinin induction, by the rabbit Anti-Human's C3c antibody for being diluted in FB of 50 μ L of pearl (Dako, Carpenteria, CA, USA) is incubated for 1 hour in 4oC.After being washed twice with FB to remove unbonded substance, by pearl 4oC with 50 μ L be diluted in FB be conjugated to PE-Cy5 goat anti-rabbit antibodies (Southern Biotech, Birmingham, AL, USA) it is incubated for 30 minutes.After being washed twice with FB to remove unbonded substance, by pearl be resuspended in FB and Pass through FACS Calibur Cytometric Analysis.Pearl is gated as uniform group using forward scatter and sidescattering, and each sample C3b in product deposits quantification of average fluorescent strength (MFI).
As a result:
The assessment of collagen deposition:
Figure 21 illustrate the computer based image analysis being sliced with the nephridial tissues of sirius red stains as a result, wherein in urine output After pipe blocks 7 days, histotomy is obtained from the wild-type mice handled with MASP-2 inhibiting antibody or Isotype control antibodies. As shown in figure 21, be obtained from IgG4 isotype controls handle wild-type mice obstruction kidney histotomy collagen deposition Amount compare, the obstruction (UUO) obtained from the wild-type mice handled with MASP-2 inhibiting antibody afterwards 7 days harvest kidney tissue Slice display collagen deposition substantially reduces (p=0.0477).
The assessment of Hydroxyproline content:
Hydroxy-proline measures in nephridial tissue as the instruction of collagen content.Hydroxy-proline is that height indicates in the model The parameter of the Pathological Physiology progress of the disease of induction.
Figure 22 diagram is obtained from the obstruction of the wild-type mice handled with MASP-2 inhibiting antibody or Isotype control antibodies (UUO) afterwards 7 days harvest kidney hydroxyproline content.As shown in figure 22, and come IgG4 isotype controls mAb processing of using by oneself The kidney of mouse is compared, and the obstruction nephridial tissue of the mouse of Lai Ziyong MASP-2 inhibiting antibody processing confirms significant less hydroxyl dried meat ammonia Sour (a kind of instruction of collagen content) (p=0.0439).
The assessment of inflammation:
From wild type, the animal of Isotype control antibodies processing and with the wild animal of MASP-2 inhibiting antibody processing Block the active infiltration of kidney confirmation macrophage.Carefully quantization shows the macrophage percentage stained area between the two groups It is not significantly different (data are not shown).However, although the infiltrating macrophages of equivalent, it is dynamic with being injected from isotype controls The obstruction kidney of object is compared, and the obstruction kidney of the animal from the injection of MASP-2 inhibiting antibody shows significant less fibrosis, such as Judged by sirius red stains, which has with come the obstruction nephridial tissue of the mouse for MASP-2 inhibiting antibody processing of using by oneself There is the result of hydroxyproline significantly more less than the kidney handled with IgG4 isotype controls mAb consistent.
It discusses
As a result, it was confirmed that being provided in UUO model using MASP-2 inhibiting antibody for kidney fibrosis described in the present embodiment Protective effect, this is consistent with result described in embodiment 14, and embodiment 14 confirms MASP-2-/- mouse compared with wild-type mice There is the kidney fibrosis substantially reduced and inflammation in UUO model.The present embodiment resists with MASP-2 inhibition as the result is shown Fibrosis is reduced in the mouse of body processing.In the UUO kidney for reducing or blocking the active animal of MASP-2- dependence lectin pathway The new discovery for being the discovery that highly significant that middle fibrosis is reduced.In short, embodiment 14 and result provided in this embodiment confirm MASP-2 inhibits to have renal tubular interstitium inflammation, tubule cells damage, the release of the rush brotic cells factor and cicatrization Benefit effect.The mitigation of kidney fibrosis is still the common-denominator target of kidney therapy.UUO model is the accurate model for accelerating kidney fibrosis, and And the intervention of the fibrosis of the model is reduced, such as using MASP-2 inhibiting antibody, it is possibly used for inhibiting or preventing kidney fiber Change.Result from UUO model may be transferred to the kidney diaseases that feature is glomerulus and/or albuminuria Tubular damage.
Embodiment 16
The present embodiment provides MASP-2-/- and wild-type mice in using kidney fibrosis, inflammation and Tubulointerstitial damage Albumen overload albuminuria model generate as a result, to evaluate effect of the lectin pathway in proteinuric nephropathies.
Background/basic principle:
Albuminuria is the risk factors that kidney fibrosis occurs and loses renal excretion function, regardless of primary kidney diaseases (Tryggvason K. et al.,J Intern Med254:216-224,2003, Williams M.,Am J. Nephrol 25:77-94,2005).The excess protein for the result that the conceptual description of proteinuric nephropathies is damaged as glomerulus differential permeability Into proximal tubule toxic effect (Brunskill N.J.,J Am Soc Nephrol 15:504-505,2004, Baines R.J.,Nature Rev Nephrol7:177-180,2011).This phenomenon common for many renal glomerular diseases causes Proinflammatory cicatrization environment and feature are the signal transduction path imbalance due to the small pipe fluid stimulation of albuminuria in kidney, closely Tubule cells growth, apoptosis, genetic transcription and inflammatory cytokine is held to generate change.Proteinuric nephropathies are known as various primary The key contributing factor of the shared progressive injury of kidney of nephrosis reason.
Chronic kidney diseases are more than 15% adult population in U.S. influence, and are in the world annual about 750, and 000 dead The reason of dying (Lozano R. et al.,LancetVol 380, Issue 9859:2095-2128,2012).Albuminuria is chronic The instruction of kidney diaseases, and promote the factor of progression of disease.Many patients with albuminuria kidney diaseases show renal tubular interstitium Property inflammation and progressive fibrosis, it is closely related with decreased renal function.Albuminuria itself induce renal tubular interstitium inflammation and Proteinuric nephropathies generation (Brunskill N.J. et al.,J Am Soc Nephrol15:504-505,2004).In albumen It urinates in kidney diaseases, excessive albumin and other macromoleculars are filtered through glomerulus, and by proximal tubular epithelial cells reabsorption. This leads to the inflammatory vicious circle mediated by complement activation, leads to cell factor and leukocyte infiltration, causes renal tubular interstitium Property damage and fibrosis, thus aggravate albuminuria and cause lose renal function and finally progress to end-stage renal failure (see, for example, Clark et al.,Canadian Medical Association Journal178:173-175,2008).Adjust inflammation and egg The expected result for improving chronic kidney diseases of the therapy of this vicious cycle of albiduria.
Inhibit beneficial outcomes in the UUO model that renal tubular interstitium damage according to MASP-2, carry out it is following test with Determine that MASP-2 inhibits the injury of kidney in albumen overload model whether can be reduced.The research and utilization albumen, which overloads inducible protein, urinates kidney Disease, such as Ishola et al.,European Renal Association21:591-597, described in 2006.
Method:
MASP-2-/- mouse was returned for 10 generations by generation described in embodiment 1 and with BALB/c.Current research is compared as follows in albumen Overload wild type and MASP-2-/- BALB/c mouse result in albuminuria model.
To see optimum response, the last week is tested, by mouse unilateral nephrectomy before albumen overload attacks.The albumen used Urinating inducer is low endotoxin bovine serum albumin(BSA) (BSA, Sigma), and by following dosage, i.p. gives WT (n in normal salt water =7) and MASP-2 -/- mouse (n=7): respective 2mg BSA/gm, 4mg BSA/gm, 6mg BSA/gm, 8mg BSA/gm, 10mg The 15mg BSA/gm weight of one dosage of BSA/gm and 12mg BSA/gm weight and 9 dosage, in total through 15 day time I.p. 15 dosage are given.Control WT (n=4) and MASP-2-/- (n=4) mouse only receive the salt water that i.p gives.It gives most After the latter dosage, the independent cage of animal in metabolic cage 24 hours to collect urine.It is collected by cardiac puncture under anaesthesia Blood solidifies blood on ice 2 hours and passes through centrifuge separation with serum.Serum and urine are collected at the end of experiment in the 15th day Sample, storage and freezing are for analyzing.
24 hours execution mouse after 15th day last time BSA gives, and various tissues are collected for analyzing.Harvest kidney simultaneously It is handled to carry out H&E and immunostaining.Immunohistochemical staining carries out as follows.Formalin from each mouse fixes, 5 microns of nephridial tissues slice of paraffin embedding is through de- paraffin and rehydrated.Antigen recovery in citrate buffer 95oC into Row 20 minutes, then in 3% H2O2Middle incubation tissue 10 minutes.Then group is woven in the Block buffer containing 10% avidin solution It is incubated at room temperature 1 hour in (10% serum and 1% BSA/PBS of the species generated from secondary antibody).It will be cut after each step Piece washs three times in PBS, and respective 5 minutes.The primary antibody 1 being then applied in the Block buffer containing 10% biotin solution is small When, for antibody F4/80 (Santa Cruz cat# sc-25830), TGF β (Santa Cruz cat# sc-7892), The concentration of IL-6 (Santa Cruz cat# sc-1265) is 1:100 and for TNF α antibody (Santa Cruz cat# sc- 1348) concentration is 1:50.Then apply biotinylation secondary antibody 30 minutes, be 1 for F4/80, TGF β and IL-6 slice concentration: 200, it is 1:100 for TNF α slice concentration, then applies HRP conjugated enzyme other 30 minutes.It uses diaminobenzidine (DAB) Substrate reagent box (Vector labs) develop the color 10 minutes and wash slide in water, dehydration and fixation, without redying in favor of base In the image analysis of computer.Staining tissue slides from cortex renis are by digital image capture, then using automation figure As analysis software quantification is analyzed.
As follows by being commented in histotomy with terminal deoxynucleotidyl transferase dUTP notch end label (TUNEL) dyeing Valence apoptosis.Apoptotic cell in kidney segment is dyed as follows using ApopTag peroxidase conjugation kit (Millipore).It will The fixed kidney segment of paraffin embedding from each mouse, formalin takes off paraffin, rehydrated, then uses Proteinase K (20 μ G/mL) albumen permeates, and is applied to each sample at room temperature 15 minutes.Sample washs in PBS between the steps.Endogenous mistake Peroxidase activity passes through in 3% H2O2It is quenched within middle incubation tissue 10 minutes.Then group is woven in equilibration buffer and is incubated for, connect With TdT enzyme 37oC be incubated for 1 hour.It is washed in termination/washing buffer after ten minutes, anti-foxalin conjugate Apply 30 minutes at room temperature, then washs.It develops the color 4 minutes in DAB substrate reagent box, then washs in water.By tissue It redyes in hematoxylin and is fixed in DBX.The frequency usage Leica DBXM optics of (brown) apoptotic cell of TUNEL dyeing Microscope manual count under 20 continuously selected the high power field from cortex.
As a result:
The assessment of albuminuria
For confirm mouse in albuminuria presence, the 15th day analysis serum in total protein and research the 15th day through 24 hours The total secreted proteins in urine are measured in the urine sample of collection.
Figure 23 be shown in only receive the wild type control mice (n=2) of salt water, receive BSA wild-type mice (n=6) and Receive in MASP-2-/- mouse (n=6) of BSA, the total amount (mg/ml) of the serum proteins of measurement in the 15th day.As shown in figure 23, In wild type and MASP-2-/- group, gives BSA and increase total serum protein level to the control group for only receiving salt water more than twice Concentration, and be not significantly different between processing group.
Figure 24 is shown in the Tot Prot (mg) secreted in the 15th day urine collected through 24 hours of research, from only receiving The wild type control mice (n=2) of salt water, the wild-type mice (n=6) for receiving BSA and the MASP-2-/- mouse for receiving BSA (n= 6).As shown in figure 24, at the 15th day of this research, compared with the false processing control group for only receiving salt water, in BSA processing group In urine there are about 6 times of increases in total secreted proteins.The result of the display of Figure 23 and 24 confirms that albuminuria model is effective, as expected 's.
The histological change of kidney is assessed
Figure 25 shows representative H&E dyeing nephridial tissue slice, harvests at albumen overload research the 15th day from following mouse group: (figure A) wild type control mice；(figure B) MASP-2-/- control mice；The wild-type mice that (figure C) is handled with BSA；(figure D) MASP-2-/- the mouse handled with BSA.As shown in figure 25, it under the albumen overload attacks of phase same level, is overloaded with wild type Group (figure C) is compared, and there are the tissue preserrations of higher degree in MASP-2-/- overload group (figure D).For example, observe with it is wild The Bowman's capsule of type control group (figure A) is compared, and the Bowman's capsule in the wild-type mice (overload) handled with BSA greatly expands (figure C).In contrast, MASP-2-/- mouse (overload) Bowman's capsule (figure D) handled with the BSA of phase same level keeps class It is similar to the morphology of MASP-2-/- control mice (figure B) and wild type control mice (figure A).It is such as Figure 25 it further shows that big Albumen cast structure gathers in the proximally and distally tubule of wild type kidney segment (figure C), with MASP-2-/- mouse (figure D) big and richer compared to more.
It is also noted that the kidney segment from this research by emission electron microscope analysis shows, with BSA processing mouse Overall impairment with the cilium boundary to proximally and distally tubule cells, wherein cellular content and core are burst into tubule Chamber.In contrast, it organizes to be saved in the MASP-2- handled with BSA/- mouse.
The assessment of macrophages infiltration in kidney
In order to measure the degree of inflammation indicated by macrophages infiltration, the histotomy of the kidney of harvest is also special with macrophage Property antibody F4/80 use is described in Boor et al.,J of Am Soc of Nephrology18:1508-1515, in 2007 Method dyeing.
Figure 26 illustrates the computer based image point of the nephridial tissue slice with macrophage specific antibody F4/80 dyeing Analysis as a result, display macrophage be averaged stained area (%), wherein studying the 15th day from wild type control mice in albumen overload (n=2) obtain histotomy with the BSA wild-type mice (n=6) handled and the MASP-2- handled with BSA/- mouse (n=5). As shown in figure 26, it is sliced display with the nephridial tissue of the anti-macrophage antibody dyeing of F4/80, although with wild type vacation control group phase Than, two groups handled with BSA show that kidney macrophages infiltration dramatically increases and (is measured as %F4/80 antibody stained area), but with Macrophages infiltration in the histotomy of wild-type mice from BSA processing is compared, and is observed and is being handled from BSA Macrophages infiltration in MASP-2-/- mouse histotomy substantially reduces (p value=0.0345).
Figure 27 A illustrates the total secreted proteins and macrophages infiltration by measuring in the urine from 24 hours samples (average stained area %) mapping, analysis macrophage-albuminuria correlation in each wild-type mice (n=6) handled with BSA Presence.As shown in fig. 27 a, most of sample from wild type kidney shows existing Proteinuria level and macrophages infiltration Degree is positively correlated.
Figure 27 B diagram (is averagely dyed by the total secreted proteins in the urine of 24 hours samples with macrophages infiltration Area %) mapping, analysis macrophage-albuminuria correlation is deposited in each MASP-2- handled with BSA/- mouse (n=5) ?.As shown in figure 27b, do not observed in MASP-2-/- mouse observed in wild-type mice in Proteinuria level and Positive correlation between macrophages infiltration degree (Figure 27 A is shown).Although there be no wish to be bound by any particular theory, these are tied Fruit can be shown that there are inflammation purge mechanisms under high-caliber albuminuria in MASP-2-/- mouse.
The assessment of cell factor infiltration
Interleukin-6 (IL-6), transforming growth factor β (TGF β) and tumor necrosis factor α (TNF α) are proinflammatory cytokines The factor, it is known raised in the proximal tubule of the wild-type mice of albuminuria model (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 Dialysis and Transplant Association- European Renal Association12:51-56, 1997).The histotomy of kidney is dyed as described above with cytokine-specific antibody.
Figure 28 is shown in in the BSA wild-type mice (n=4) handled and MASP-2-/- mouse (n=5) with BSA processing The result of the computer based image analysis of the histotomy dyed with anti-TGF β antibody (is measured as the dyeing of % TGF β antibody Area).As shown in figure 28, it observes compared with MASP-2-/- BSA processing (overload) group, handles (overload) in wild type BSA What TGF β was dyed in group dramatically increases (p=0.026).
Figure 29 is shown in in the BSA wild-type mice (n=4) handled and MASP-2-/- mouse (n=5) with BSA processing The result of the computer based image analysis of the histotomy dyed with Anti-tnfa antibody (is measured as the dyeing of % TNF α antibody Area).As shown in figure 29, it compared with MASP-2-/- BSA processing (overload) group, is seen in wild type BSA processing (overload) group It observes TNF α dyeing and dramatically increases (p=0.0303).
The wild-type mice (n=7) that Figure 30 is shown in wild type control mice, MASP-2-/- control mice, is handled with BSA With in MASP-2-/- mouse (n=7) for being handled with BSA, with the computer based figure for the histotomy that anti-IL-6 antibody dyes As the result (being measured as % IL-6 antibody stained area) of analysis.As shown in figure 30, compared with MASP-2-/- BSA processing group, Observe that IL-6 dyeing highly significant increases (p=0.0016) in wild type BSA processing group.
Apoptosis assessment
Apoptosis is assessed in histotomy by being dyed with terminal deoxynucleotidyl transferase dUTP notch end label (TUNEL), It is counted in 20 continuously selected the high power field (HPF) from cortex with the frequency of TUNEL stained apoptotic cells.
Figure 31 be shown in from wild type control mice (n=1), MASP-2-/- control mice (n=1), with BSA handle It wild-type mice (n=6) and is continuously selected with the histotomy of BSA MASP-2-/- mouse (n=7) cortex renis handled The frequency of the TUNEL apoptotic cell counted in 20 high power fields (HPF).As shown in figure 31, it is handled with BSA with being obtained from MASP-2-/- mouse kidney is compared, significant higher in cortex with observing in the kidney of the BSA wild-type mice handled being obtained from Apoptosis rate (p=0.0001).
As a result with conclusion summation:
The present embodiment as a result, it was confirmed that in albumen overload model MASP-2-/- mouse have reduced injury of kidney.Therefore, MASP-2 inhibitor, such as MASP-2 inhibiting antibody, it is contemplated that inhibit or prevent the vicious cycle of inflammation and albuminuria, and improve The result of chronic kidney diseases.
Embodiment 17
It is anti-present embodiment describes monoclonal MASP-2 inhibition in albuminuria model is overloaded in the murine protein of wild-type mice Body mitigates and/or prevents the analysis of the effect of nephritis disease and Tubulointerstitial damage.
Background/basic principle:
As described in Example 16, it in the albumen overload model of albuminuria, determines that MASP-2-/- mouse shows and compares wild-type mice Significant preferably result (for example, less Tubulointerstitial damage and less nephritis disease), implies lectin pathway in albuminuria Pathogenic effects in kidney diaseases.
As described in Example 13, monoclonal MASP-2 inhibiting antibody (OMS646-SGMI-2) is generated, specific inhibition The function of people's lectin pathway and also show the blocked lectin element approach in mouse.In the present embodiment, it is overloaded in murine protein MASP-2 inhibiting antibody OMS646-SGMI-2 is analyzed in albuminuria model mitigates and/or prevent ephritis in wild-type mice The effect of disease and renal tubular interstitium damage.
Method:
This research evaluation MASP-2 compared with the 4 Isotype control antibodies ET904 of human IgG (10 mg/kg) and saline control presses down The effect of property antibody (10 mg/kg OMS646-SGMI-2) processed.
Similar to research described in embodiment 16, this research and utilization albumen overload takes inducible protein and urinates kidney diaseases (Ishola Et al.,European Renal Association21:591-597,2006).Albuminuria is small in the Balb/c of unilateral nephrectomy In mouse by daily i.p. inject ascending-dose (the low endotoxin bovine serum albumin(BSA) (BSA) of 2 g/kg to 15 g/kg) induces, 15 days in total, as described in Example 16.
Antybody therapy starts i.p. injection biweekly for first 7 days by albuminuria induction and gives, and continues entirely to study.It should Dosage is selected according to the PK/PD before and pharmaceutical research that confirm to continue lectin pathway inhibition (data are not shown).? 15th day execution mouse harvests kidney and is handled to carry out H&E and immunostaining.Staining tissue slides from cortex renis pass through Digital image capture is then analyzed using Automated Image Analysis software quantification.
Immunohistochemical staining and apoptosis assessment carry out as described in Example 16.
As a result:
Albuminuria assessment
In order to confirm the presence of albuminuria in mouse, measured in the urine sample that the 15th day (experiment terminates) collected through 24 hours Total secreted proteins in urine.It has determined compared with the control group of unused BSA processing, urine sample is shown in BSA processing The average almost 6 times of increases of total protein levels in group (data are not shown), it was confirmed that albuminuria deposits in the mouse handled with BSA ?.Protein level significant difference is not observed between BSA processing group.
Histological change assessment
Figure 32 is shown handled with BSA after the 15th day, from the representative H&E staining tissue slides of the mouse of the following group: (figure A) With the wild type control mice of saline treatment；The control mice of (figure B) isotype antibody processing；(figure C) is pressed down with MASP-2 The wild-type mice of property antibody processing processed.
As shown in figure 32, under the albumen overload attacks of phase same level, and with salt water (figure A) or isotype controls (figure B) The wild type group of processing is compared, and there are the tissue preserrations of higher degree in MASP-2 inhibiting antibody-processing group (figure C).
Apoptosis assessment
By evaluating apoptosis in histotomy with terminal deoxynucleotidyl transferase dUTP notch end label (TUNEL) dyeing, It is counted in 20 continuously selected the high power field (HPF) from cortex with the frequency of TUNEL stained apoptotic cells.Figure 33 figure Show handled come the wild-type mice (n=8) of use by oneself saline control and BSA processing, with Isotype control antibodies and BSA it is wild Type mouse (n=8) and with the histotomy of the cortex renis of MASP-2 inhibiting antibody and the BSA wild-type mice (n=7) handled The frequency of the TUNEL apoptotic cell counted in 20 high power fields (HPF) continuously selected.As shown in figure 33, with salt water and together Kind type control treatment group is compared, and observes the apoptosis rate height in cortex in the kidney for being obtained from MASP-2 inhibiting antibody processing group It substantially reduces (for saline control and MASP-2 inhibiting antibody, p=0.0002；For isotype controls and MASP-2 inhibition Antibody, p=0.0052).
The assessment of cell factor infiltration
Interleukin-6 (IL-6), transforming growth factor β (TGF β) and tumor necrosis factor α (TNF α), they are known The pro-inflammatory cytokine raised in the proximal tubule of the wild-type mice of albuminuria model, in the kidney group that this research obtains It knits and is evaluated in slice.
Figure 34 is shown in BSA and the wild-type mice (n=8) of saline treatment, with BSA and Isotype control antibodies and handles Wild-type mice (n=7) and the wild-type mice (n=8) that is handled with BSA and MASP-2 inhibiting antibody in, it is anti-with anti-TGF β The result (being measured as % TGF β antibody stained area) of the computer based image analysis of the histotomy of body dyeing.Such as Figure 34 Shown, the quantization of TGF β stained area shows to press down compared with the control group that salt water and Isotype control antibodies are handled in MASP-2 TGF β level substantially reduces (respectively, p value=0.0324 and 0.0349) in the mouse of property antibody processed processing.
Figure 35 is shown in BSA and the wild-type mice (n=8) of saline treatment, with BSA and Isotype control antibodies and handles Wild-type mice (n=7) and the wild-type mice (n=8) that is handled with BSA and MASP-2 inhibiting antibody in, it is anti-with anti-TNF alpha The result (being measured as % TNF α antibody stained area) of the computer based image analysis of the histotomy of body dyeing.Such as Figure 35 It is shown, stained slice analysis shows, compared with saline control group (p=0.011) and isotype controls group (p=0.0285), TNF α level substantially reduces in MASP-2 inhibiting antibody processing group.
Figure 36 is shown in BSA and the wild-type mice (n=8) of saline treatment, with BSA and Isotype control antibodies and handles Wild-type mice (n=7) and the wild-type mice (n=8) that is handled with BSA and MASP-2 inhibiting antibody in, it is anti-with anti-IL-6 The result (being measured as % IL-6 antibody stained area) of the computer based image analysis of the histotomy of body dyeing.Such as Figure 36 It is shown, stained slice analysis shows, compared with saline control group (p=0.0269) and isotype controls group (p=0.0445), IL-6 level substantially reduces in MASP-2 inhibiting antibody processing group.
As a result with conclusion summation:
The present embodiment as a result, it was confirmed that the guarantor for injury of kidney is provided in albumen overload model using MASP-2 inhibiting antibody Shield effect, this is consistent with the result for being described in embodiment 16, and embodiment 16 confirms MASP-2-/- mouse tool in albuminuria model The injury of kidney being reduced.
Embodiment 18
The present embodiment provides MASP-2-/- and wild-type mice in damaged using kidney fibrosis, inflammation and renal tubular interstitium The nephrology model of adriamycin induction generate evaluating effect of the lectin pathway in the nephrosis that adriamycin induces As a result.
Background/basic principle:
Adriamycin is the anthracene nucleus of the cancer for treating various cancers, including hematologic malignancies, soft tissue sarcoma and many types Antitumor antibiotics.The nephrosis of adriamycin induction is sufficiently established in the rodent model of chronic kidney diseases, can be more preferable Understand progress (Lee and the Harris of chronic albuminuria, Nephrology,16:30-38,2011).The nephrosis of adriamycin induction Structure and function damage type be very similar to people chronic albuminuria kidney diaseases (Pippin et al.,American Journal of Renal Physiology296:F213-29,2009).
The nephrosis of adriamycin induction is characterized in that Podocytes in Renal Tissue, and then glomerulosclerosis, renal tubular interstitium are scorching Disease and fibrosis.Have shown that the nephrosis of adriamycin induction is adjusted by immune and nonimmune source mechanism in many researchs (Lee and Harris,Nephrology,16:30-38,2011).If the nephrosis of adriamycin induction has as the model of kidney diaseases Dry advantage.Firstly, it is that height reproduce and predictable renal injury model.This is because its feature is several to give in drug Injury of kidney is induced in it, allows to be easy to carry out experimental design since the time of damage is consistent this.Still wherein tissue damages for it The degree of wound is serious, but with the model of the acceptable death rate (< 5%) and disease incidence (weight saving).Therefore, because The seriousness of the injury of kidney of the nephrosis of adriamycin induction and time, it is suitable for the mould that test protects against the intervention of injury of kidney Type.
As described in embodiment 16 and 17, MASP-2-/- mouse and use have been determined in the albumen overload model of albuminuria The mouse of MASP-2 inhibiting antibody processing shows better result more significant than wild-type mice (for example, between less renal tubule Matter damage and less nephritis disease), imply pathogenic effects of the lectin pathway in albuminuria kidney diaseases.
In the present embodiment adriamycin induction nephropathy model (AN) in wild-type mice comparative analysis MASP-2-/- Mouse, to determine whether MASP-2 defect reduces and/or prevent the nephritis disease induced by adriamycin and renal tubular interstitium damage.
Method:
1. dosage and time point optimization
It carries out initial experiment and is suitable for testing the level of therapeutic intervention with the dosage and BALB/c mouse that determine adriamycin Nephritis disease time point.
Adriamycin (10.5 mg/kg) injection of three groups of wild type BALB/c mouse (n=8) IV single doses given.? Three time points eliminate mouse: adriamycin gives latter week, two weeks and surrounding.Control mice only uses salt water injection.
As a result: all mouse of three groups show the sign of glomerulosclerosis and albuminuria, are such as dyed and are determined by H&E , and the tissue inflammation of degree is gradually increased, such as pass through (data are not shown) of the macrophages infiltration measurement in kidney.Tissue The degree of damage is slight in a Zhou Zuzhong, is medium in two Zhou Zuzhong, and is that serious (data are not shown in surrounding group Show).2 week points are selected for remaining research.
2. analyzing the nephrosis of adriamycin induction in wild type and MASP-2-/- mouse
It, will under the adriamycin of same dose in order to illustrate effect of the lectin pathway of complement in the nephrosis that adriamycin induces One group of MASP-2-/- mouse (BALB/c) is compared with wild-type mice (BALB/c).MASP-2-/- mouse and BALB/c mouse return Handed over for 10 generations.
Wild type (n=8) and MASP-2-/- (n=8) are injected with adriamycin (10.5 mg/kg) IV and each strain Three mouse are given only salt water as control.All mouse are eliminated for two weeks after treatment, and collect tissue.Histopathological lesions Degree pass through H&E staining evaluation.
As a result:
Figure 37 be shown in adriamycin or only salt water (control) processing after the 14th day, from the representative H&E of the following group mouse Staining tissue slides: (figure A-1, A-2, A-3) only uses the wild type control mice of saline treatment；(figure B-1, B-2, B-3) use Ah The wild-type mice of mycin processing；MASP-2-/- mouse that (figure C-1, C-2, C-3) is handled with adriamycin.Each figure (example Such as, scheme A-1, A-2, A-3) represent different mouse.
As shown in figure 37, compared with the wild type group handled with the adriamycin of same dose, what is handled with adriamycin There are the tissue preserrations of higher degree in MASP-2-/- group.
Figure 38 illustrates the computer based image point of the nephridial tissue slice with macrophage specific antibody F4/80 dyeing Analysis as a result, be shown in adriamycin or only salt water (wild type control) processing after the 14th day, from the macrophage of the following group mouse Cell is averaged stained area (%): only using the wild type control mice of saline treatment；The wild-type mice handled with adriamycin；Only With MASP-2-/- mouse of saline treatment, and the MASP-2 -/- mouse handled with adriamycin.As shown in figure 38, and with Ah mould The wild-type mice of element processing is compared, and has reduced macrophages infiltration (* * with MASP-2-/- mouse that adriamycin is handledp= 0.007)。
Figure 39 illustrate the computer based image analysis being sliced with the nephridial tissues of sirius red stains as a result, being shown in With adriamycin or only salt water (wild type control) processing after the 14th day, from the collagen deposition stained area of the mouse of the following group (%): the wild type control mice of saline treatment is only used；The wild-type mice handled with adriamycin；Only use the MASP- of saline treatment 2-/- mouse, and the MASP-2 -/- mouse handled with adriamycin.As shown in figure 39, with the wild-type mice that is handled with adriamycin It compares, there is reduced collagen deposition (* * with MASP-2-/- mouse that adriamycin is handledp=0.005)。
Summation and conclusion:
The improvement of renal tubular interstitium inflammation is the common-denominator target for treating kidney diaseases.It is provided herein the result shows that, complement activation Lectin pathway remarkably promote the generation of renal tubular interstitium inflammation.As further confirmed herein, MASP-2 inhibitor, example Such as MASP-2 inhibiting antibody, it can be used as treatment albumen urine nephrosis, mRNA IN ADRIAMYCIN NEPHROPATHY and improve the new of renal tubular interstitium inflammation Treatment method.
Embodiment 19
Present embodiment describes evaluation human monoclonal MASP-2 inhibiting antibodies with steroid-dependent immunoglobulin Safety and clinical efficacy in the adult of A nephrosis (IgAN) and in the adult with steroid-dependent membranous nephropathy (MN) 2 clinical trial phases just carried out initial results.
Background:
Chronic kidney diseases influence the U.S. be more than 2 million peoples (Drawz P. et al.,Ann Intern Med 162(11); ITC1- 16,2015).Glomerulonephropathy (GN), including IgAN and MN be wherein glomerular injury and frequently result in end-stage renal disease and The kidney diaseases of dialysis.If in the presence of the primary GN of dry type, most commonly IgAN.These many patients have lasting ephritis Disease and progressive damage.In general, these patient's corticosteroids or immunosuppressant treatment, with many serious long-term Adverse consequences.Many patients are continuous worsening, or even in these treatments.It does not treat and is approved for treatment IgAN or MN.
IgANephrosis
IgANP (IgAN) is a kind of autoimmune kidney diaseases, leads to inflammation and injury of kidney in kidney.IgAN is complete World's most common form primary glomerulonephritis (Magistroni et al.,Kidney Int. 88(5):974-89, 2015).According to about 2.5/100,000 every Annual occurence rate, in 1/1400 people of the U.S. IgAN will occur for estimation.After diagnosis 20 In year, end-stage renal disease (ESRD) (Coppo R., D ' Amico G., J Nephrol will occur for up to 40% IgAN patient 18(5):503-12, 2005;Xie et al., PLoS One, 7 (6): e38904 (2012)).Patient typically appears as micro- Mirror blood urine and slightly to moderate albuminuria and change level renal insufficiency (Wyatt R.J. et al.,N Engl J Med368 (25): 2402-14,2013).Clinical marker such as impaired renal function, continuous hypertension and weight albuminuria are (more than 1 G/ days) related with poor prognosis (Goto M et al.,Nephrol Dial Transplant24 (10): 3068-74,2009; Berthoux F. et al.,J Am Soc Nephrol22 (4): 752-61,2011).In multiple large-scale observational studies and prediction Property test in, albuminuria is most strong prognostic factor, independent of other risks and assumptions (Coppo R. et al.,J Nephrol 18 (5): 503-12,2005;Reich H. N. et al.,J Am Soc Nephrol18 (12): 3177-83,2007).If no Treatment, estimate disease occur 10 years in 15-20% patient reach ESRD (D ' Amico G.,Am J Kidney Dis 36 (2): 227-37,2000).
The diagnostic markers of IgAN are the significant IgA deposition in glomerulus mesangium, independent or adjoint IgG, IgM or two Person.In IgAN, the kidney that Renal biospy discloses mannan-binding lectin (MBL) (the crucial identification molecule of MASP-2 activation) is small Ball deposition, MASP-2 is the effect enzyme of the lectin pathway of complement system.Glomerulus MBL deposition, usually with IgA common location and table Bright complement activation and high-caliber urine MBL are related with the unfavorable prognosis of IgAN, and wherein these patients confirm more heavy than no MBL It is long-pending or it is high-caliber urine MBL the more serious histological change of patient and Parameters Associated with Mesenteric Proliferation (Matsuda M. et al.,Nephron 80 (4): 408-13,1998;Liu LL et al., Clin Exp Immunol 169 (2): 148-155,2012;Roos A. etc. People,J Am Soc Nephrol17 (6): 1724-34,2006;Liu LL et al.,Clin Exp Immunol 174(1): 152-60,2013).The remission rate of patient with MBL deposition also it is significant it is lower (Liu LL et al.,Clin Exp Immunol 174 (1): 152-60,2013).
The method for the treatment of IgAN all attempts to slow down, stop or postpone the deterioration of renal function at present.The kidney disease of glomerulonephritis Disease improves total result (Kidney Disease Improving Global Outcomes, KDIGO) clinical practice guideline and pushes away IgAN treatment plan is recommended, mainly emphasizes to block by renin-angiotensin system (RAS) to control blood pressure [KDIGO Work Group 2012].For continuing the patient of daily albuminuria with 1 g or more, although the anti-height of maximum tolerated dose Blood pressure medication and the blood pressure well controlled, but the treatment recommended includes corticosteroid and/or other immunosuppressor, such as ring Phosphamide, imuran or mycophenolate.The kidney diaseases of glomerulonephritis improve total result (KDIGO) guide (Int. Soc of Nephrol2 (2): 139-274,2012) suggest that corticosteroid should be given and is greater than or equal to 1 g/ days with albuminuria Patient, general therapeutic duration are 6 months.For being defined as having crescent in > 50% glomerulus with crescent IgAN() The patient that deteriorates rapidly of function is removed with kidney, another kind immunosuppressor (such as ring phosphinylidyne can be added into corticosteroid Amine).However, related with serious long-term sequelae, some patients still have even with the immunosupress sex therapy of invasion The progressive of renal function damages.The IgAN therapy for not having FDA to ratify, and press down even with angiotensin converting enzyme (ACE) Preparation or angiotensin receptor blocker (ARB) control blood pressure, and albuminuria continues to increase among the patients.These treatments None shows to stop or even slow down the progression of disease in the patient for the risk for having IgAN rapid progress.It can reduce or eliminate Unsatisfied medical demand will be solved clearly to the replacement therapy of the needs of chronic corticosteroid and/or immunosuppressive therapy.
Membranous nephropathy
Every Annual occurence rate of membranous nephropathy (MN) is about 10-12/1,000,000.Patient with MN modified can face Bed process, but about 25% will occur end-stage renal disease.
Membranous nephropathy is one of the most common reason of nephrotic syndrome in immune-mediated renal glomerular disease and adult.The disease Sick feature is that Immune deposits, mainly IgG4 are formed on the outside of glomerular basement membrane, it includes sertoli cell antigen and to this The antibody of a little antigentic specificities, leads to complement activation.The initial representation of MN is related to nephrotic syndrome: albuminuria, blood albumin subtract Less, hyperlipidemia and oedema.
Although MN can without treatment spontaneous remission, up to 1/3 patient confirm renal function progressive loss and with 5 years medians are to ESRD after diagnosis.In general, corticosteroid is used to treat MN and exist the need of exploitation alternative medicine It wants.In addition, determining the patient in medium progress risk with prednisone combination cyclophosphamide or mind according to the seriousness of albuminuria Through calcium albumen (calcinuerin) inhibitor for treating, and both treatments usually have with serious systemic ill-effect together It closes.
Method:
Two 1 clinical trial phases carried out in healthy volunteer are it has been shown that intravenous and subcutaneous administration MASP-2 inhibition is anti- Body OMS646 causes lasting lectin pathway to inhibit.
Present embodiment describes from MASP-2 inhibiting antibody OMS646 is being just in the subject with IgAN and MN Interim findings of 2 phases carried out without control multicenter study.All patients in the standard requirements research are included in regardless of kidney diaseases Hypotype is how, research recruit before maintained corticosteroid at least 12 weeks of consistent dose (that is, patient is steroid-dependent ).Research is single group pilot study, has treatment in 12 weeks and 6 weeks follow-up periods.
About 4 subjects are recruited in every kind of disease plan.Whether research is designed to evaluation OMS646 can be with IgAN With improvement renal function (for example, improving albuminuria) and reduction corticosteroid demand in the subject of MN.So far, have 2 patients of IgA nephrosis and 2 patients with membranous nephropathy complete treatment under study for action.
When studying entrance, each subject must have high-caliber albumen in urine, although just with stable cortex Steroids dosage is treated.The selection of these standards can not spontaneous improved patient during research.
Subject age >=18 and only when they diagnose following one including under study for action in screening: Renal biospy is examined It is disconnected to there is IgAN or Renal biospy diagnosis to have primary MN.The patient of recruitment, which must also meet, is included in standard below all:
(1) from 2 times during screening access it is each before three samples that are continuous and collecting daily, have mean urinary white Albumen/kreatinin ratio > 0.6；
(2) screening access 1 before given >=10 mg prednisone or equivalent dose at least 12 weeks；
(3) it if giving immunosupress sex therapy (for example, cyclophosphamide, mycophenolate), has been given before screening access 1 Consistent dose at least two moon, desired amount non-for the research duration change；
(4) there are >=30 mL/min/1.73m2Estimation glomerular filtration rate (eGFR), pass through MDRD formula1It calculates；
(5) doctor of angiotensin converting enzyme inhibitors (ACEI) and/or angiotensin receptor blocker (ARB) is given The diastolic blood pressure of the systolic blood pressure and < 90mmHg when stablizing optimization treatment and rest with < 150 mmHg of guidance；
(6) belimumab, eculizumab or rituzimab is not used in 6 months of screening access 1；With
(7) do not have kidney transplant history.
1MDRD formula: eGFR (mL/min/1.73m2) = 175 x (SCr)-1.154 x (Age)-0.203X (0.742, If women) x (1.212, if African American).Note: SCr=serum creatinine measurement should be mg/dL.
Monoclonal antibody OMS646 used in this research is to combine and inhibit 4 monoclonal of full human IgG of people MASP-2 anti- Body.MASP-2 is the effect enzyme of lectin pathway.As embodiment 12 confirms, OMS646, which combines closely, recombinates MASP-2 (apparently Equilibrium dissociation constant is within the scope of 100 pM) and relative to homologous protein C1s, C1r and MASP-1 show be greater than 5,000 times selection Property.OMS646 (leads to 50% inhibition [IC with nanomole effect in functional examination method50] concentration be about 3 nM) inhibit people solidifying Collect plain approach, but classical pathway is had no significant effect.Mouse, non-human primates are injected by intravenous (IV) or subcutaneous (SC) Animal and people and giving OMS646 leads to high plasma concentration, inhibition of its activation with lectin pathway has in ex vivo assays It closes.
In our current research, OMS646 drug is provided with the concentration of 100 mg/mL, is given by further diluting for IV. Suitable 100 mg/mL of the OMS646 injection solution for calculating volume is extracted out using syringe from bottle, standby for dosimetric system.Infusion Bag is given in preparation 4 hours.
Research is formed by screening (28 days), treatment (12 weeks) and follow-up (6 weeks) phase, institute in researching and designing flow chart as follows Show.
Researching and designing flow chart
In screening and for the first time before OMS646 dosage, the subject of agreement provides in each of two time for three days on end Three urine samples (collecting once a day) are to establish the baseline value of urinary albumin Yu kreatinin ratio.After screening, qualified is tested Person receives 4 mg/kg IV of OMS646 once a week, continues 12 weeks (treatment phase).Have 6 after the OMS646 of the last one dosage All follow-up periods.
During the starting treated with OMS646 4 weeks, the cortex class for the research predose that subject keeps them stable is solid Alcohol.At the end of starting 4 weeks of 12 weekly treatment periods, if tolerance, subject successively decrease through 4 weeks progress corticosteroids (that is, Corticosteroid dosage is reduced), 4 weeks of the corticosteroid dosage then kept.Target is to be decremented to daily≤6 mg Prednisone (or equivalent dose).In this stage, stop successively decreasing in the subject with renal function exacerbation that such as researcher determines. Subject is treated during corticosteroid successively decreases and between entire 12 weekly treatment period with OMS646.Patient then they most Afterwards by other 6 weeks after seance.Successively decreasing for corticosteroid allows to assess whether OMS646 allows to tie up with OMS646 treatment The corticosteroid dosage needed for fixed renal function that keeps steady is reduced.
Key effects measurement in this research be from baseline to 12 weeks, in urine albumin and kreatinin ratio (uACR) and The variation of 24- hours protein levels.The measurement of Urine proteins or albumin is involved conventionally used for evaluation kidney, and continual high levels Urine proteins are related with kidney diaseases progress.UACR is used clinically for evaluation albuminuria.
Efficiency analysis
The assay value of uACR is defined as the average value of all values obtained for time point.The plan numerical value of uACR is each predetermined Time point be 3.The baseline value of uACR is defined as screening the average value of the assay value of access twice.
As a result:
Figure 40 be shown in during 12 weeks research process with 4 mg/kg MASP-2 inhibiting antibody (OMS646) weekly treatments UACR in two IgAN patients.As shown in figure 40, by non-transformed analysis, time point " a " (p=0.003)；Time point “b” (p=0.007) and time point " c " (pIt=0.033) is, statistically significant from the variation of baseline.Table 12 provide for The 24- hour proteinuria data of two IgAN patients of OMS646 treatment.
Table 12: the 24- hour Urine proteins (mg/ days) in the IgAN patient of OMS646 treatment
As shown in Figure 40 and table 12, the patient with IgAN confirms that clinical and statistically significant renal function changes in the course of the research It is kind.There are statistically significant reductions for uACR (see Figure 40) and twenty-four-hour urine protein concentration (being shown in Table 12).Such as the uACR number of Figure 40 Shown in, average baselining uACR is 1264 mg/g and reaches 525 mg/g (p=0.011) in treatment end, in follow-up period At the end of be reduced to 128 mg/g.Such as Figure 40 it further shows that therapeutic effect is maintained in entire follow-up period.24 hours UACR is tracked in the measurement of Urine proteins secretion, is reduced from 3156 mg/24 hourly averages to 1119 mg/24 hours (p=0.017).? Therapeutic effect height is consistent between two patients.The reduction in mg/ days of two patient experiences about 2000 and reach part alleviation It (is defined as the protein secretion that twenty-four-hour urine protein secretion is reduced and/or obtained greater than 50% and is less than 1000 mg/ days；Complete incidence graph It is defined as protein secretion and is less than 300 mg/ days).The amplitude of albuminuria reduction in 24- hours is deposited with kidney in two IgA nephrotics Living significantly improving is related.Two IgA nephrotics can also significantly successively decrease their steroids, respectively reduce daily dosage extremely ≤ 5 mg (60 mg to 0 mg；30 mg to 5 mg).
Two MN patients have also demonstrated the uACR during being treated with OMS646 and reduce.The uACR of one MN patient is from 1003 Mg/g is reduced to 69 mg/g and is kept the low-level in entire follow-up period.The uACR of another MN patient is reduced from 1323 mg/g To 673 mg/g, process after modified treatment.First MN patient shows that 24- hours urine protein levels substantially reduce (base The 10 of line, 325 mg/24 hours of 771 mg/24 hours to the 85th day), it realizes part and almost alleviates, and another Remaining basically unchanged of patient (4502 mg/24 of 4272 mg/24 hours of baseline to the 85th day).Steroids is in two MN 15 mg are decremented to from 30 mg in patient and are decremented to 5 mg from 10 mg.
In short, observing consistent kidney function in IgAN the and MN subject treated with MASP-2 inhibiting antibody OMS646 It can improve.OMS646 treatment is steady and consistent in the patient with IgAN, shows strong effect signal.This A little effects obtain the support of the result in MN patient.The time course and amplitude that uACR changes during treatment with IgAN and It is consistent between all four patients of MN.Apparent safety issue is not observed.Patient in this research, which represents, to be controlled Difficult group is treated, and thinks that MASP-2 inhibiting antibody, such as OMS646 can be predicted in IgAN and MN in the therapeutic effect of these patients Patient, the patient for example with steroid-dependent IgAN and MN are (that is, experience stabilization before being treated with MASP-2 inhibiting antibody Corticosteroid dosage treatment patient), including effect in those of risky rapid progress to end-stage renal disease patient.
According to foregoing teachings, in one embodiment, the present invention provides people experimenter of the treatment with IgAN or MN Method includes the MASP-2 inhibiting antibody for effectively inhibiting the amount of MASP-2- dependent complement activation including giving subject Composition.In one embodiment, method includes giving the people experimenter with IgAN or MN to be enough to improve renal function (example Such as, improve albuminuria) amount MASP-2 inhibiting antibody.In one embodiment, subject suffers from steroid-dependent IgAN.In one embodiment, subject suffers from steroid-dependent MN.In one embodiment, MASP-2 inhibition Antibody be enough in the subject improve renal function and/or reduce corticosteroid dosage amount give with steroids according to Rely the subject of property IgAN or steroid-dependent MN.
In one embodiment, it includes effectively to inhibit MASP-2- dependence that method, which further comprises giving subject, Before the step of composition of the MASP-2 inhibiting antibody of the amount of complement activation, identification with steroid-dependent IgAN people by Examination person.
In one embodiment, it includes effectively to inhibit MASP-2- dependence that method, which further comprises giving subject, Before the step of composition of the MASP-2 inhibiting antibody of the amount of complement activation, people of the identification with steroid-dependent MN is tested Person.
According to any one embodiment disclosed herein, MASP-2 inhibiting antibody shows that at least one or more is following special Sign: table of the antibody with 10 nM or less KD combination people MASP-2, in the CCP1 structural domain of the antibody combination MASP-2 Position, the antibody inhibit C3b deposition, the antibody in 1% human serum in vitro with 10 nM or less IC50 in measuring method Inhibit C3b deposition in 90% human serum with 30 nM or less IC50, wherein antibody is selected from Fv, Fab, Fab', F (ab) 2 With the antibody fragment of F (ab') 2, wherein the antibody is single chain molecule, wherein the antibody is IgG2 molecule, wherein described anti- Body is IgG1 molecule, wherein the antibody is IgG4 molecule, wherein IgG4 molecule is mutated comprising S228P.In an embodiment In, the antibody combination MASP-2 and selective depression lectin pathway and do not significantly inhibit classical pathway (that is, inhibit agglutinin It is complete that approach retains classic complement approach simultaneously).
In one embodiment, MASP-2 inhibiting antibody is related with renal function to be effectively improved at least one or more The amount of clinical parameter give, such as albuminuria improve (for example, uACR is reduced and/or urinary protein concentrations reduction in 24- hour, example It is reduced as twenty-four-hour urine protein secretion is greater than 20%, or such as twenty-four-hour urine protein secretion is greater than 30% and reduces, such as twenty-four-hour urine Protein secretion is greater than 40% and reduces, such as twenty-four-hour urine protein secretion is greater than 50% and reduces).
In some embodiments, the method includes by conduit (for example, intravenous) give with IgAN (such as Steroid-dependent IgAN) subject's MASP-2 inhibiting antibody, continue the first segment time (for example, at least one day to one week Or two weeks or three weeks or surrounding or more long), then subcutaneous administration subject MASP-2 inhibiting antibody, continues the second segment time (for example, at least two weeks or more long chronic phase).
In some embodiments, method includes being given by conduit (for example, intravenous) with MN (such as steroids Dependence MN) subject's MASP-2 inhibitor, continue the first segment time (for example, at least one day to one week or two weeks or three weeks Or surrounding or more long), then subcutaneous administration subject MASP-2 inhibiting antibody, continues the second segment time (for example, at least two weeks Or more long chronic phase).
In some embodiments, method include intravenous, intramuscular or subcutaneous administration with IgAN (such as steroids according to Rely property IgAN) or MN (such as steroid-dependent MN) subject's MASP-2 inhibiting antibody.Treatment can be it is chronic, Daily to monthly giving, but preferably at least every two weeks or at least weekly, such as biweekly or three times a week give.
In one embodiment, method includes that treatment suffers from IgAN (such as steroid-dependent IgAN) or MN (example Such as the subject of steroid-dependent MN), including giving subject includes a certain amount of MASP-2 inhibiting antibody or its antigen The composition of binding fragment, the antibody or its antigen-binding fragment include containing amino acid sequence shown in SEQ ID NO:67 CDR-H1, CDR-H2 and CDR-H3 heavy chain variable region and CDR- containing amino acid sequence shown in SEQ ID NO:70 The light chain variable region of L1, CDR-L2 and CDR-L3.In some embodiments, composition includes MASP-2 inhibiting antibody, Comprising (a) heavy chain variable region, include: i) heavy chain CDR-H1, the amino acid sequence of the 31-35 comprising SEQ ID NO:67；With Ii) heavy chain CDR-H2, the amino acid sequence of the 50-65 comprising SEQ ID NO:67；And iii) heavy chain CDR-H3, it include SEQ The amino acid sequence of the 95-107 of ID NO:67 and b) light chain variable region include: i) light chain CDR-L1, include SEQ ID The amino acid sequence of the 24-34 of NO:70；And ii) light chain CDR-L2, the amino acid sequence of the 50-56 comprising SEQ ID NO:70 Column；And iii) light chain CDR-L3, the amino acid sequence of the 89-97 comprising SEQ ID NO:70, or (II) its variant, include With SEQ ID NO:67 have at least 90% identity (for example, with SEQ ID NO:67 at least 91%, at least 92%, at least 93%, extremely Few 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity) heavy chain variable region and with SEQ ID NO:70 have at least 90% identity (for example, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, At least 97%, at least 98%, at least 99% identity) light chain variable region.
In some embodiments, including giving, subject includes a certain amount of MASP-2 inhibiting antibody to method or it is anti- The composition of former binding fragment, the antibody or its antigen-binding fragment include containing amino acid sequence shown in SEQ ID NO:67 The heavy chain variable region of column and light chain variable region containing amino acid sequence shown in SEQ ID NO:70.
In some embodiments, method includes giving subject to include MASP-2 inhibiting antibody or its antigen binding fragment The composition of section, the antibody or its antigen-binding fragment specific recognition are included weight chain variable shown in SEQ ID NO:67 The reference antibody OMS646 of light chain variable region shown in area and SEQ ID NO:70 knows the epitope on others MASP-2 at least A part.
In some embodiments, method includes giving to suffer from or risky generation IgAN (such as steroid-dependent IgAN) or the subject of MN (such as steroid-dependent MN) includes MASP-2 inhibiting antibody or its antigen-binding fragment Composition, the antibody or its antigen-binding fragment include the weight chain variable containing amino acid sequence shown in SEQ ID NO:67 Area and light chain variable region containing amino acid sequence shown in SEQ ID NO:70, dosage are 1 mg/kg-10 mg/kg (that is, 1 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), one week at least once (such as one week at least twice or one week at least three times), continue at least 3 weeks or at least 4 weeks, Or at least 5 weeks or at least 6 weeks or at least 7 weeks or at least 8 weeks or at least 9 weeks or at least 10 weeks or at least 11 weeks or extremely Few 12 weeks time.
Embodiment 20
Present embodiment describes evaluation human monoclonal MASP-2 inhibiting antibodies with steroid-dependent lupus nephritis (LN) initial results for 2 clinical trial phases of the safety and clinical efficacy in adult just carried out.
Background:
Chronic kidney diseases influence the U.S. be more than 2 million peoples (Drawz P. et al.,Ann Intern Med 162(11); ITC1- 16,2015).Glomerulonephropathy (GN), including IgAN, MN and LN are wherein glomerular injury and to frequently result in end-stage renal disease With the kidney diaseases of dialysis.There is these many patients lasting nephritis disease and progressive to damage.In general, these patients cortex class Sterol or immunosuppressant treatment, with many serious long-term adverse consequences.Many patients are continuous worsening, or even at these When treatment.
Lupus nephritis
The major complications of systemic loupus erythematosus (SLE) are ephritis, also referred to as lupus nephritis, are classified as glomerulonephritis Ondary forms.Up to 60% adult with SLE has some form of kidney accumulation (Koda-Kimble the course of disease later period Et al., Koda-Kimble and Young ' s Applied Therapeutics:the clinical use of drugs, 10thEd, Lippincott Williams & Wilkins:pages 792-9,2012), in the U.S., illness rate is 20-70 people in every 100,000 people.Lupus nephritis is typically found in the other symptoms of activity SLE, including fatigue, fever, fash, In the patient of arthritis, scrositis or central nervous system disease (Pisetsky D.S et al.,Med Clin North Am 81(1):113-28, 1997).Some patients have asymptomatic lupus nephritis;However, laboratory is different during regular follow-up Often as serum creatinine level increases, albumin level is low or Urine proteins or sediment prompt activity lupus nephritis.Autoimmunity It plays a major role in the pathogenesis of lupus nephritis.These autoantibodies form pathogenic immune compound in the blood vessels, It is deposited in glomerulus.Autoantibody can also be formed in situ immune with the antigen binding having been positioned in glomerular basement membrane Compound.Immune complex by complement activation and attract inflammatory cell come promote inflammatory reaction (D ' Agati V.D et al., Lupus nephritis: pathology and pathogenesis: Wallace D.J. Hahn, Dubois’ Lupus Erythematosus, 7th Ed Philadelpha: Lippincott Williams & Wiklins: p1094-111, 2007).Therefore, the complement activation that immune complex mediates plays a crucial role in the pathogenesis of lupus nephritis.C4d deposit It is present in nephridial tissue, and usually related to immune complex deposit object Clq and C3, causes classical pathway.In some cases In, there are C4d deposit in the case where no Clq, show that possible lectin pathway participates in (Kim M.K., et al.Int J Clin Exp Pathol 6(10):2157-67, 2013)。
The further support of significant contribution as lectin pathway, the deposit of MBL appear in the skin damage of SLE patient In wound (Wallim L. R et al.,Hum Immunol75 (7): 629-32,2014).In addition, it has been observed that from having In most of Renal biospies of the patient of lupus nephritis MBL and fiber gelatinized protein strong deposition (Nisihara R. M et al.,Hum Immunol74 (8): 907-10,2013).Kidney MBL is deposited on the most obvious in the patient with high protein urine.This Outside, plasma MBL levels are significantly higher than normal healthy controls in SLE patient, and MBL level is related to disease activity, show MBL water It is flat may represent SLE disease activity biomarker (Panda A.K et al.,Arthritis Res Ther 14(5): R218,2012).Corticosteroid is the main conventional therapy selection for the patient with slight lupus nephritis.For tighter The case of weight, high dose prednisone, methylprednisolone, mycophenolate, cyclophosphamide, imuran and cyclosporin have been used for Clinical practice.The therapeutic choice of SLE and lupus nephritis has high relevant morbidity and mortality.Side effect, especially for a long time The use of corticosteroid limits the compliance of patient, and then influences therapeutic efficiency.It needs to develop better tolerance to control Treatment scheme.
Method:
Such as the description in example 19 above, two 1 clinical trial phases carried out in healthy volunteer it has been shown that it is intravenous and Subcutaneous administration MASP-2 inhibiting antibody OMS646 causes lasting lectin pathway to inhibit.
Present embodiment describes from the MASP-2 inhibiting antibody OMS646 in the subject with lupus nephritis (LN) 2 phases just carried out without control multicenter study interim findings.All patients in the standard requirements research are included in regardless of kidney Disease subtypes are how, maintained before research is recruited consistent dose corticosteroid at least 12 weeks (that is, patient be steroids according to Rely property).Research is single group pilot study, has treatment in 12 weeks and 6 weeks follow-up periods.
Research is designed to whether evaluation OMS646 can improve renal function (for example, improving egg in the subject with LN Albiduria) and reduction corticosteroid demand.So far, 5 patients with lupus nephritis (LN) complete to control under study for action It treats.
When studying entrance, each subject must have high-caliber albumen in urine, although just with stable cortex Steroids dosage is treated.The selection of these standards can not spontaneous improved patient during research.
In screening, subject age >=18 and being only included in when their Renal biospies, which diagnose, the diagnosis of lupus nephritis are ground In studying carefully.The patient of recruitment, which must also meet, is included in standard below all:
(1) from 2 times during screening access it is each before three samples that are continuous and collecting daily, have mean urinary white Albumen/kreatinin ratio > 0.6；
(2) screening access 1 before given >=10 mg prednisone or equivalent dose at least 12 weeks；
(3) it if giving immunosupress sex therapy (for example, cyclophosphamide, mycophenolate), has been given before screening access 1 Consistent dose at least two moon, desired amount non-for the research duration change；
(4) there are >=30 mL/min/1.73m2Estimation glomerular filtration rate (eGFR), pass through MDRD formula1It calculates；
(5) doctor of angiotensin converting enzyme inhibitors (ACEI) and/or angiotensin receptor blocker (ARB) is given The diastolic blood pressure of the systolic blood pressure and < 90mmHg when stablizing optimization treatment and rest with < 150 mmHg of guidance；
(6) belimumab, eculizumab or rituzimab is not used in 6 months of screening access 1；With
(7) do not have kidney transplant history.
1MDRD formula: eGFR (mL/min/1.73m2) = 175 x (SCr)-1.154 x (Age)-0.203X (0.742, If women) x (1.212, if African American).Note: SCr=serum creatinine measurement should be mg/dL.
Monoclonal antibody OMS646 used in this research is to combine and inhibit 4 monoclonal of full human IgG of people MASP-2 anti- Body.MASP-2 is the effect enzyme of lectin pathway.As embodiment 12 confirms, OMS646, which combines closely, recombinates MASP-2 (apparently Equilibrium dissociation constant is within the scope of 100 pM) and relative to homologous protein C1s, C1r and MASP-1 show be greater than 5,000 times selection Property.OMS646 (leads to 50% inhibition [IC with nanomole effect in functional examination method50] concentration be about 3 nM) inhibit people solidifying Collect plain approach, but classical pathway is had no significant effect.Mouse, non-human primates are injected by intravenous (IV) or subcutaneous (SC) Animal and people and giving OMS646 leads to high plasma concentration, inhibition of its activation with lectin pathway has in ex vivo assays It closes.
In our current research, OMS646 drug is provided with the concentration of 100 mg/mL, is given by further diluting for IV. Suitable 100 mg/mL of the OMS646 injection solution for calculating volume is extracted out using syringe from bottle, standby for dosimetric system.Infusion Bag is given in preparation 4 hours.
Research is formed by screening (28 days), treatment (12 weeks) and follow-up (6 weeks) phase, institute in researching and designing flow chart as follows Show.
Researching and designing flow chart
In screening and for the first time before OMS646 dosage, the subject of agreement provides in each of two time for three days on end Three urine samples (collecting once a day) are to establish the baseline value of twenty-four-hour urine albumen and urinary albumin and kreatinin ratio.Screening Afterwards, qualified subject receives 4 mg/kg IV of OMS646 once a week, continues 12 weeks (treatment phase).In the last one dosage OMS646 after have 6 weeks follow-up periods.
During the starting treated with OMS646 4 weeks, the cortex class for the research predose that subject keeps them stable is solid Alcohol.At the end of starting 4 weeks of 12 weekly treatment periods, if tolerance, subject successively decrease through 4 weeks progress corticosteroids (that is, Corticosteroid dosage is reduced), 4 weeks of the corticosteroid dosage then kept.Target is to be decremented to daily≤6 mg Prednisone (or equivalent dose).In this stage, stop successively decreasing in the subject with renal function exacerbation that such as researcher determines. Subject is treated during corticosteroid successively decreases and between entire 12 weekly treatment period with OMS646.Patient then they most Other 6 weeks of follow-up after seance afterwards.Successively decreasing for corticosteroid allows to assess whether OMS646 allows to tie up with OMS646 treatment The corticosteroid dosage needed for fixed renal function that keeps steady is reduced.
Efficiency analysis
Key effects measurement in this research is the variation of 24 hours protein levels from baseline to 12 weeks.Urine proteins or albumin Measurement involve conventionally used for evaluation kidney, and the Urine proteins of continual high levels are related with kidney diaseases progress.Alleviate definition in part It is reduced for twenty-four-hour urine protein secretion and is greater than 50%.
As a result:
Table 13 is provided for twenty-four-hour urine albumen (mg/ days) data of the 5 LN patients treated with OMS646.
Table 13: the twenty-four-hour urine albumen (mg/ days) in the LN patient of OMS646 treatment
Note: patient #1 experienced systemic disease aggravation during research.
As shown in table 13, the patient with LN confirms renal function improvement clinical and statistically significant in the course of the research. As shown in table 13, the albumen secretion of urine in 24 hours in treatment phase of 4 in 5 LN patients shows significant (average value 69%) It reduces.5th patient (patient #1) experienced systemic disease and aggravate and show to dramatically increase.Most of lupus reactors Can significantly be successively decreased their steroids dosage.
In short, observing significant kidney in 4 in the 5 LN patients treated with MASP-2 inhibiting antibody OMS646 Function improves.OMS646 treatment is strong and consistent in the patient with LN, shows strong effect signal.It does not see Observe apparent safety issue.Patient in this research represents the difficult group for the treatment of, and thinks the therapeutic effect of these patients Predictable MASP-2 inhibiting antibody, such as OMS646 in LN patient, for example with steroid-dependent LN patient (that is, with The patient of the stable corticosteroid dosage treatment of experience before the treatment of MASP-2 inhibiting antibody) including it is risky quickly into Open up effect into those of end-stage renal disease patient.
According to foregoing teachings, in one embodiment, the present invention provides the method for people experimenter of the treatment with LN, packet Including and giving subject includes the composition for effectively inhibiting the MASP-2 inhibiting antibody of amount of MASP-2- dependent complement activation. In one embodiment, method includes giving the people experimenter with LN to be enough to improve renal function (for example, improving albuminuria) Amount MASP-2 inhibiting antibody.In one embodiment, subject suffers from steroid-dependent LN.In an embodiment party In case, MASP-2 inhibiting antibody is to be enough to improve renal function in the subject and/or reduce corticosteroid dosage Amount gives the subject with steroid-dependent LN.
In one embodiment, it includes effectively to inhibit MASP-2- dependence that method, which further comprises giving subject, Before the step of composition of the MASP-2 inhibiting antibody of the amount of complement activation, people of the identification with steroid-dependent LN is tested Person.
According to any one embodiment disclosed herein, MASP-2 inhibiting antibody shows that at least one or more is following special Sign: table of the antibody with 10 nM or less KD combination people MASP-2, in the CCP1 structural domain of the antibody combination MASP-2 Position, the antibody inhibit C3b deposition, the antibody in 1% human serum in vitro with 10 nM or less IC50 in measuring method Inhibit C3b deposition in 90% human serum with 30 nM or less IC50, wherein antibody is selected from Fv, Fab, Fab', F (ab) 2 With the antibody fragment of F (ab') 2, wherein the antibody is single chain molecule, wherein the antibody is IgG2 molecule, wherein described anti- Body is IgG1 molecule, wherein the antibody is IgG4 molecule, wherein IgG4 molecule is mutated comprising S228P.In an embodiment In, the antibody combination MASP-2 and selective depression lectin pathway and do not significantly inhibit classical pathway (that is, inhibit agglutinin It is complete that approach retains classic complement approach simultaneously).
In one embodiment, MASP-2 inhibiting antibody is related with renal function to be effectively improved at least one or more Clinical parameter, such as improve albuminuria (for example, uACR is reduced and/or twenty-four-hour urine protein concentration is reduced, such as 24 hours Urine proteins secretion is greater than 20% and reduces, or such as twenty-four-hour urine protein secretion is greater than 30% and reduces, such as twenty-four-hour urine protein secretion Greater than 40% reduce, such as twenty-four-hour urine protein secretion be greater than 50% reduce) amount give the subject with LN.In some implementations In scheme, effectively to cause albuminuria part to be alleviated, (that is, compared with baseline, 50%) the albumen secretion of urine in 24 hours, which is reduced, to be greater than Amount gives subject's MASP-2 inhibiting antibody with LN.
In some embodiments, the method includes being given by conduit (for example, intravenous), with LN, (such as class is solid Alcohol dependence LN) subject's MASP-2 inhibiting antibody, continue the first segment time (for example, at least one day to one week or two weeks Or three weeks or surrounding or more long), then subcutaneous administration subject MASP-2 inhibiting antibody, continues the second segment time (for example, extremely Few two weeks or more long chronic phases).
In some embodiments, method includes intravenous, intramuscular or subcutaneous administration with LN (such as non-steroid dependant Property LN) (such as steroid-dependent LN) subject's MASP-2 inhibiting antibody.Treatment can be it is chronic, daily to every It month gives, but preferably at least every two weeks or at least weekly, such as biweekly or three times a week gives.
In one embodiment, method includes the subject that treatment suffers from LN (such as steroid-dependent LN), including Give the composition that subject includes a certain amount of MASP-2 inhibiting antibody or its antigen-binding fragment, the antibody or it is anti- Former binding fragment includes the heavy chain of CDR-H1, CDR-H2 and CDR-H3 containing amino acid sequence shown in SEQ ID NO:67 can Become the light chain variable region in area and CDR-L1, CDR-L2 and CDR-L3 containing amino acid sequence shown in SEQ ID NO:70.? In some embodiments, composition includes MASP-2 inhibiting antibody, and it includes (a) heavy chain variable regions, include: i) heavy chain CDR-H1, the amino acid sequence of the 31-35 comprising SEQ ID NO:67；And ii) heavy chain CDR-H2, it include SEQ ID NO:67 50-65 amino acid sequence；And iii) heavy chain CDR-H3, the amino acid sequence of the 95-107 comprising SEQ ID NO:67, and B) light chain variable region includes: i) light chain CDR-L1, the amino acid sequence of the 24-34 comprising SEQ ID NO:70；And ii) light Chain CDR-L2, the amino acid sequence of the 50-56 comprising SEQ ID NO:70；And iii) light chain CDR-L3, include SEQ ID NO: The amino acid sequence of 70 89-97, or (II) its variant, comprising there is at least 90% identity (example with SEQ ID NO:67 Such as, with SEQ ID NO:67 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) heavy chain variable region and with SEQ ID NO:70 have at least 90% identity (for example, at least 91%, At least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity) light chain Variable region.
In some embodiments, method include give with LN(such as steroid-dependent LN) subject include one The composition of quantitative MASP-2 inhibiting antibody or its antigen-binding fragment, the antibody or its antigen-binding fragment contain There is the heavy chain variable region of amino acid sequence shown in SEQ ID NO:67 and containing amino acid sequence shown in SEQ ID NO:70 Light chain variable region.
In some embodiments, method include give with LN(such as steroid-dependent LN) subject include The composition of MASP-2 inhibiting antibody or its antigen-binding fragment, the antibody or its antigen-binding fragment specific recognition quilt Reference antibody comprising light chain variable region shown in heavy chain variable region shown in SEQ ID NO:67 and SEQ ID NO:70 OMS646 knows at least part of the epitope on others MASP-2.
In some embodiments, method includes giving to suffer from or risky LN (such as steroid-dependent LN) occurs Subject includes MASP-2 inhibiting antibody or the composition of its antigen-binding fragment, the antibody or its antigen-binding fragment packet Containing the heavy chain variable region containing amino acid sequence shown in SEQ ID NO:67 and contain amino acid shown in SEQ ID NO:70 The light chain variable region of sequence, dosage are 1 mg/kg-10 mg/kg (that is, 1 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), one week at least once (such as one week extremely It is few twice or one week at least three times), continue at least 3 weeks or at least 4 weeks or at least 5 weeks or at least 6 weeks or at least 7 weeks or At least 8 weeks or at least 9 weeks or at least 10 weeks or at least 11 weeks or at least 12 weeks time.
Embodiment 21
This embodiment describes the additional results obtained in ongoing 2 clinical trial phase, to assess human monoclonal MASP-2 inhibiting antibody OMS646 has glomerulus nephrosis, the adult patients including the IgAN as described in embodiment 19 to reduction In albuminuria safety and clinical efficacy.
Method:
As described in example 19 above, the test of 2 phases includes the IgAN patient for receiving corticosteroid when studying and entering, all patients Receive OMS646 in a manner of open label, and obtains positive findings from two IgAN patients.For having completed the total of test Totally 4 IgAN patients have completed the administration of other two IgAN patient using method described in embodiment 19 now.
For being included in this experiment, IgAN patient be have to prove that: (1) IgAN of biopsy diagnosis, (2) uACR > 0.6 g/g, (3) eGFR≥30 mL/min/1.73 m2, the blood pressure that (4) are controlled with stable ACEI/ARB treatment, and (5) >= The stabilization steroids dosage of 10 mg prednisones continues at least 12 weeks.
There is pre-existing injury of kidney with all 4 IgAN adult patients of OMS646 treatment in this experiment, enter When the glomerular filtration rate (eGFR) estimated be 30 to 46 mL/mg/1.73m2, and 24- hours protein measurement values be 2.44 to 4.87 grams/24 hours.All patients study enter when receive stable renin-angiotensin system (RAS) block and The corticosteroid treatment of at least three moon.
All patients receive OMS646 IV once a week, continue 12 weeks.It 4 weeks introduction periods of patient experience, then carries out OMS646 treatment comprising stable steroids dosage 4 weeks, if tolerance, 4 weeks steroid tapers, and the class successively decreased are solid Alcohol dosage 4 weeks.After OMS646 treatment, patient in test additional 6 weeks of follow-up again.After the test, researcher's follow-up Patient.
In test, effect measured value be (1) before the treatment (baseline) measure 6 times and treat and follow-up during each function Measurement 3 times urinary albumin/kreatinin ratios (uACR) when effect assessment;(2) measurement is primary before OMS646 is treated and completes Primary twenty-four-hour urine albumen is measured within 2-4 weeks after OMS646 treatment.According to scheme, if clinically suitable, corticosteroid exists Successively decrease between 4th week and the 8th week.
As a result:
4 IgAN patients complete 6 weeks follow-up periods after treatment.Table 14 provides demography and the baseline spy of these patients Sign.
Table 14: demography and baseline characteristic
The glomerular filtration rate of eGFR- estimation;SSA- standard scale area (1.73 m2);UACR- urinary albumin/kreatinin ratio.
All patients show substantially reducing for albuminuria during OMS646 is treated.As shown in figs. 41 and 42, exist Statistics and clinically significant improvement are observed in uACR and 24 hour protein measurement value.
The uACR (mg/g) of 4 IgAN patients that Figure 41 diagram is treated from baseline by 120 days with OMS646 at any time.Such as Shown in Figure 41, terminate from baseline to research, average uACR, which reduces 1.13g/g ± 0.27(, reduces by 77%, p=0.026).Such as Further shown in Figure 41, after OMS646 treatment, in last time follow-up, relative to baseline, uACR points of each patient Not reducing 94%, 86%, 47% and 89%(respectively is patient 1-4).
Figure 42 illustrates 24 hours of the 4 IgAN patients treated with OMS646 after treating preceding 1st day baseline to treatment Urine proteins variation.As shown in figure 42, it is to suffer from that 24- hours Urine proteins, which reduce 54%, 81%, 63% and 95%(respectively from baseline, Person 1-4).
The average change of twenty-four-hour urine albumen after Figure 43 illustrates the 4 IgAN patients treated with OMS646 from baseline to treatment Change.As shown in figure 43, average twenty-four-hour urine albumen, which reduces 2.87 ± 1.08 g/24 hours, (reduces 73%;P=0.013).
All patients can deactivate corticosteroid during the research phase or after the research phase soon, it was demonstrated that OMS646 pairs The effect of albuminuria is less likely related to corticosteroid.The glomerular filtration rate (eGFR) of estimation (passes through kidney diaseases formula In metatrophia calculate) in entire treatment and follow-up period be stable.
All patients can be resistant to OMS646 well.
In short, being observed in all IgAN patients for treating 12 weeks with OMS646 in the open label 2 phase clinical research The significant and lasting reduction to uACR.The significant reduction of 24 hours albuminuria in all patients.The albuminuria observed reduces journey Spend it is related to significantly improving for kidney prognosis and clinical effectiveness (Inker L. A et al.,Am J Kidney Dis 68(3):392- 401 (2016)).A kind of monoclonal antibody for MASP-2 of the influence for the lectin pathway for eliminating complement of OMS646() it controls Albuminuria largely reduces after treatment, the discovery for allowing steroids to be discontinued, and support uses OMS646 as improvement IgA glomerulopathy result Therapeutic agent.Effect of the OMS646 in IgAN patient is strong and consistent, it was demonstrated that effect in the group.
Embodiment 22
Alleviation after completing OMS646 treatment in IgA nephrosis (IgAN) patient maintains
Background/principle
As described in embodiment 19 and 21, in the 2 phases research of IgAN patient, 4 IgAN patients are treated with OMS646, OMS646 It is a kind of active human monoclonal antibodies of inhibition MASP-2.As described in embodiment 19 and 21, all patients connect once a week By OMS646 IV, continue 12 weeks.As described in example 21 above, after OMS646 treatment, 6 have in test been carried out again to patient All follow-ups, and all IgAN patients by OMS646 treatment realize part alleviation and (are defined as twenty-four-hour urine protein secretion It reduces by more than 50% and/or obtained protein secretion is less than 1000 mg/days).As described in this embodiment, follow-up after experiment This four patients, and assess the alleviation duration after OMS646 treatment.
Method:
Complete described in embodiment 21 after 2 clinical trial phases, with 4 IgAN patients of OMS646 treatment by researcher with It visits.In test, terminal is uACR and 24 hour albuminuria.As described in example 21 above, all 4 IgAN patients tie in test Part alleviation is realized when beam.After experiment in follow-up, measurement Urine proteins and kreatinin ratio (uPCR).By will be every multiplied by 0.64 A uPCR value is converted to uACR(urinary albumin/kreatinin ratio) (referring to Zhao et al.,Clin J Am Soc Nephrol 11: 947-55,2016).
As a result:
All patients realize that part is alleviated after OMS646 treatment.The average age of 3 women and 1 male are 42 years old, 3 It is white people, 1 is Asian.Average eGFR is 41 mL/min/1.73m2, and being averaged into steroids dosage is 55mg. Follow-up range is 2 to 10 months after last time OMS646 dosage.As described in example 21 above, during test, average uACR Reduce 77% (p=0.026).3 patients holding part during obtainable follow-up is alleviated (at 12,12 and 5 months 54%, 93% and 78%uACR is reduced respectively).1 patient had 88% baseline uACR at 7 months.During follow-up, 3 Patient, which also shows eGFR, improves 7,13 and 7 ml/min/1.73 m2.The eGFR of 4th patient stablizes.All patients deactivate Steroids.OMS646 is well tolerable.
In short, as described in example 21 above, with OMS646 treat 12 weeks and including treatment in test after 6 periods Between, albuminuria substantially reduces in IgAN patient.After the completion for the treatment of, the reduction of albuminuria was maintained until 10 months.These data branch It holds and uses OMS646 as the therapeutic agent for improving IgA glomerulopathy result.
From researcher update (after the single 12 weeks courses for the treatment of for using OMS646 to treat, about one year with The situation of 4 patients described in the present embodiment in visit) in, it was recently reported that 3 in 4 patients there is the albuminuria kept to reduce.? In this 3 patients, uACR keeps reducing 14%, 23% and 24% of the baseline value of patient before OMS646 is treated.In addition, trying After testing, changing for glomerular filtration rate (eGFR) (a kind of measurement of renal function) of estimation is observed in 3 in 4 patients It is kind.The patient that renal function reduces most serious shows eGFR from 30 mL/min/1.73 m2Improve to 47 mL/min/1.73 m2, Improve 57%.
In short, the lasting reduction of albuminuria continues to make us printing in follow-up in 1 year after completing the single OMS646 course for the treatment of As deep.The improvement observed in eGFR be it is unexpected, especially in follow-up in 1 year, because it is expected that needing significant Longer time can just show.As described above, two in 4 patients show eGFR and are slightly increased, wherein one of patient is showed 50% improved infusive reaction out.The improvement observed in eGFR show OMS646 can by potentially excluding or It is significant extend to reach need the time dialysed and reduce to mention to the be in progress risk of relevant complication of chronic kidney diseases for patient For further benefit.
According to aforementioned, in one embodiment, the present invention provides the albuminuria reduced in the people experimenter with IgAN Method, including give subject MASP-2 inhibiting antibody or its antigen-binding fragment according to following dosage, it is described anti- Body or its antigen-binding fragment include heavy chain variable region, it includes the CDR-H1 of amino acid sequence shown in SEQ ID NO:67, CDR-H2 and CDR-H3；And light chain variable region, it includes CDR-L1, CDR-L2 of amino acid sequence shown in SEQ ID NO:70 And CDR-L3:
C. (i.e. 3.6 mg/kg are to 4.4 mg/kg's) by subject about 4 mg/kg of the intravenous administration with IgAN once a week The antibody continues at least 12 weeks treatment phases；Or
D. once a week intravenous administration suffer from IgAN about 180 mg of subject to about 725 mg (i.e. 162 mg to 797 mg) The antibody, continue at least 12 weeks treatment phases,
Wherein the method reduces the albuminuria in the people experimenter.
In one embodiment, the dosage of MASP-2 inhibiting antibody is about 4 mg/kg (i.e. 3.6 mg/kg to 4.4 ), mg/kg for example, about 3.6 mg/kg, about 3.7 mg/kg, about 3.8 mg/kg, about 3.9 m/kg, about 4.0 mg/kg, about 4.1 Mg/kg, about 4.2 mg/kg, about 4.3 mg/kg or about 4.4 mg/kg.
In one embodiment, the dosage of MASP-2 inhibiting antibody be about 180mg to about 725mg(that is, 160mg extremely 800mg, or about 300mg to 500mg, for example, about 300mg to the fixed dosage of about 300mg), for example, about 160 mg, about 165 mg, About 170 mg, about 175 mg, about 180 mg, about 185 mg, about 190 mg, about 195 mg, about 200 mg, about 205 mg, about 210 Mg, about 215 mg, about 220 mg, about 225 mg, about 230 mg, about 240 mg, about 245 mg, about 250 mg, about 255 mg, about 260 mg, about 265 mg, about 270 mg, about 275 mg, about 280 mg, about 285 mg, about 290 mg, about 295 mg, about 300 Mg, about 305 mg, about 310 mg, about 315 mg, about 320 mg, about 325 mg, about 330 mg, about 335 mg, about 340 mg, about 345 mg, about 350 mg, about 355 mg, about 360 mg, about 365 mg, about 370 mg, about 375 mg, about 380 mg, about 385 Mg, about 390 mg, about 395 mg, about 400 mg, about 405 mg, about 410 mg, about 415 mg, about 420 mg, about 425 mg, about 430 mg, about 435 mg, about 440 mg, about 445 mg, about 450 mg, about 455 mg, about 460 mg, about 465 mg, about 470 Mg, about 475 mg, about 480 mg, about 485 mg, about 490 mg, about 495 mg, about 500 mg, about 505 mg, about 510 mg, about 515 mg, about 520 mg, about 525 mg, about 530 mg, about 535 mg, about 540 mg, about 545 mg, about 550 mg, about 555 Mg, about 560 mg, about 565 mg, about 570 mg, about 575 mg, about 580 mg, about 585 mg, about 590 mg, about 595 mg, about 600 mg, about 605 mg, about 610 mg, about 615 mg, about 620 mg, about 625 mg, about 630 mg, about 635 mg, about 640 Mg, about 645 mg, about 650 mg, about 655 mg, about 660 mg, about 665 mg, about 670 mg, about 675 mg, about 680 mg, about 685 mg, about 690 mg, about 695 mg, about 700 mg, about 705 mg, about 710 mg, about 715 mg, about 720 mg, about 725 Mg, about 730 mg, about 735 mg, about 740 mg, about 745 mg, about 750 mg, about 755 mg, about 760 mg, about 765 mg, about 770 mg, about 775 mg, about 780 mg, about 785 mg, about 790 mg, about 795 mg or about 800 mg.
In one embodiment, treatment phase is 12 weeks.
It in one embodiment, is the rest period of at least two moon after treatment phase (that is, not giving MASP-2 inhibition Agent) or at least three moon rest period at least four moon rest period or at least five moon rest period or at least six moon or The rest period of longer rest period, for example, at least 7 months rest periods or at least eight moon or the rest period of at least nine moon, or The rest period of at least ten moon, or at least 11 months rest periods, or at least 12 months or longer rest period.
In some embodiments, this method further comprises in treatment phase and/or periodic monitoring subject in the rest period In urine protein level, and optionally find albuminuria recurrence when restore with MASP-2 inhibiting antibody treat.
In some embodiments, as measured at the end for the treatment of phase and/or at the end of the rest period, this method is effectively Albuminuria in subject with IgAN is reduced at least 30%, for example, at least 40% from baseline (before treatment), or at least 50%, or it is greater than 50%.
In some embodiments, this method is effectively low increases the glomerular filtration rate estimated in the subject with IgAN (eGFR).
In some embodiments, the subject with IgAN has greater than 1 gram albumen/twenty-four-hour urine albumen before the treatment The albuminuria of secretion, and as measured at the end for the treatment of phase and/or at the end of the rest period, this method is effectively by subject In albuminuria reduce at least 30%, for example, at least 40%, or at least 50% from baseline (before treatment), or be greater than 50%, and/or As measured at the end for the treatment of phase and/or at the end of the rest period, albuminuria is reduced to less than 1 gram of albumen/twenty-four-hour urine egg White secretion.
In some embodiments, although the subject with IgAN has used the anti-height of maximum tolerated dose before the treatment Blood pressure drug and have and control good blood pressure, but has and be greater than 1 gram of albumen/twenty-four-hour urine protein secretion albuminuria, and As measured at the end for the treatment of phase and/or at the end of the rest period, this method is effectively by the albuminuria in subject from baseline (before treatment) reduces at least 30%, for example, at least 40%, or at least 50%, or is greater than 50%, and/or such as at the end for the treatment of phase And/or measured at the end of the rest period, albuminuria is reduced to less than 1 gram of albumen/twenty-four-hour urine protein secretion.
In some embodiments, subject's unused steroid therapy at least a year with IgAN.In some embodiment party In case, the subject with IgAN carries out steroids at least part time of the treatment in 12 weeks carried out with OMS646 and controls It treats.In some embodiments, at least part time of the subject with IgAN in the treatment in 12 weeks carried out with OMS646 Middle carry out steroid therapy, and albuminuria is efficiently reduced simultaneously at the end of this method to treatment phase and/or at the end of the rest period Reduce or eliminate the needs to steroid therapy.
Other embodiments
All publications, patent applications and patents referred in this specification are incorporated herein by reference.
Without departing from the scope and spirit of the present invention, the various modifications of the method described in the present invention and composition It will be apparent to those skilled in the art with variation.Although the present invention has combined specific required embodiment to carry out Description, it should be understood that claimed invention should not excessively be limited to such specific embodiment.
According to above content, the present invention is characterized in that following embodiments.
1A. is used to suffer from or the risky food in one's mouth of disease or illness for occurring to be caused or aggravated by fibrosis and/or inflammation The method for treating in newborn animal subjects, inhibiting, mitigating or prevent fibrosis, effectively inhibits fibrosis including giving subject The MASP-2 inhibitor of amount.
2A. is according to the method for paragraph 1A, and wherein MASP-2 inhibitor is MASP-2 antibody or its segment.
3A. is according to the method for paragraph 2A, and wherein MASP-2 inhibitor is a part for specifically binding SEQ ID NO:6 MASP-2 monoclonal antibody or its segment.
4A. is according to the method for paragraph 2A, and wherein MASP-2 antibody or its segment are anti-with the difference in its conjugated complement system It include the polypeptide of SEQ ID NO:6 as far as few 10 times of affinity specific binding.
5A. is according to the method for paragraph 2A, and wherein antibody or its segment are selected from recombinant antibodies, the effector function with reduction Antibody, chimeric antibody, humanized antibody and the human antibody of energy.
6A. is according to the method for paragraph 1A, and wherein MASP-2 inhibitor selectively inhibits lectin pathway complement activation, Without significantly inhibiting C1q- dependent complement activation.
For 7A. according to the method for paragraph 1A, wherein MASP-2 inhibitor is subcutaneously, peritonaeum is interior, intramuscular, intra-arterial, intravenous Or it is given as inhalant.
8A. is according to the method for any one in paragraph 1A-7A, wherein the disease for being caused or being aggravated by fibrosis and/or inflammation Disease or illness are related with ischemic damage and reperfusion damage.
9A. is according to the method for any one in paragraph 1A-7A, wherein the disease for being caused or being aggravated by fibrosis and/or inflammation Disease or illness are unrelated with ischemic damage and reperfusion damage.
10A. is according to the method for any one in paragraph 1A-7A, wherein subject is aobvious before giving MASP-2 inhibitor Show the albuminuria for giving reduction subject of albuminuria and MASP-2 inhibitor.
11A. is according to the method for any one in paragraph 1A-7A, and wherein subject suffers from by kidney fibrosis and/or inflammation The disease or illness for causing or aggravating.
12A. is according to the method for paragraph 11A, and wherein MASP-2 inhibitor is effectively to inhibit the amount of Tubulointerstitial fibrosis It gives.
13A. is according to the method for paragraph 11A, and wherein MASP-2 inhibitor is to effectively reduce, postpone or eliminate subject's The amount that dialysis needs is given.
14A. is according to the method for paragraph 11A, wherein the disease or illness are selected from chronic kidney diseases, chronic renal failure, kidney Compound illness (for example, IgA nephrosis, membranous nephropathy), wolf is immunized in bead disease (for example, focal segmental glomerulosclerosis) Sore ephritis, nephrotic syndrome, diabetic nephropathy, Tubulointerstitial damage and glomerulonephritis (for example, C3 glomerulopathy).
15A. is according to the method for any one in paragraph 1A-7A, and wherein subject suffers from by pulmonary fibrosis and/or inflammation The disease or illness for causing or aggravating.
16A. is according to the method for paragraph 15A, wherein to be selected from Chronic Obstructive Pulmonary Disease, capsule fine for the disease or illness Dimensionization, pulmonary fibrosis related with chorionitis, bronchiectasis and pulmonary hypertension.
17A. is according to the method for any one in paragraph 1A-7A, and wherein subject suffers from by liver fibrosis and/or inflammation The disease or illness for causing or aggravating.
18A. is according to the method for paragraph 17A, wherein the disease or illness are selected from cirrhosis, non-alcoholic fatty liver Sick (steatohepatitis), the hepatic fibrosis-renal tubular ectasia syndrome secondary to alcohol abuse, the hepatic fibrosis-renal tubular ectasia syndrome secondary to acute or chronic hepatitis, gallbladder Disease and toxicity hepatic injury are (for example, due to drug-induced as caused by paracetamol or other medicines, such as nephrotoxin Hepatic injury hepatotoxicity wind agitation).
19A. is according to the method for any one in paragraph 1A-7A, and wherein subject suffers from by cardiac fibrosis and/or inflammation The disease or illness that disease causes or aggravates.
20A. is according to the method for paragraph 19A, wherein the disease or the patient's condition are selected from cardiac fibrosis, myocardial infarction, valve Fibrosis, Atrial fibrosis, endomyocardial fibrosis, Arrhythmogenic right ventricular cardiomyopathy (ARVC).
According to the method for any one in paragraph 1A-7A, wherein subject suffers from is caused by vascular fibrosis or is added 21A. The disease or illness of weight.
22A. is according to the method for paragraph 21A, wherein the disease or illness are selected from vascular diseases, atherosclerotic Vascular diseases, hemadostewnosis, restenosis, vasculitis, phlebitis, deep vein thrombosis is formed and abdominal aneurvsm.
23A. according to the method for any one in paragraph 1A-7A, wherein subject suffer from caused by the fibrosis of skin or The disease or illness of exacerbation.
24A. is according to the method for paragraph 23A, wherein the disease or illness are selected from excessive wound repair, chorionitis, whole body Property hardening, cheloid, connective tissue disease, cicatrization and hypertrophic scar.
25A. according to the method for any one in paragraph 1A-7A, wherein subject suffer from caused by the fibrosis in joint or The disease or illness of exacerbation.
26A. is according to the method for paragraph 25A, wherein the disease or illness are joint fibrosis.
27A. is according to the method for any one in paragraph 1A-7A, and wherein subject suffers from the fiber by central nervous system Change the disease or illness for causing or aggravating.
28A. is according to the method for paragraph 27A, wherein the disease or illness are selected from apoplexy, traumatic brain injury and spinal cord Damage.
29A. is according to the method for any one in paragraph 1A-7A, and wherein subject suffers from and drawn by the fibrosis of digestive system The disease or illness for rising or aggravating.
30A. is according to the method for paragraph 29A, wherein the disease or illness are selected from Crohn disease, pancreas fibrosis and burst Ulcer colitis.
31A. is according to the method for any one in paragraph 1A-7A, and wherein subject suffers from and caused or aggravated by eye fibrosis Disease or illness.
32A. is according to the method for paragraph 31A, wherein the disease or illness are selected from anterior subcapsular cataract, rear capsule milkiness, Huang Spot denaturation and retina and vitreoretinopathy.
33A. is according to the method for any one in paragraph 1A-7A, and wherein subject suffers from by the bone of muscle skeleton or soft group The disease or illness that the fibrosis for knitting structure causes or aggravates.
34A. is according to the method for paragraph 33A, wherein the disease or illness are dredged selected from sclerotin related with cystic fibrosis Pine and/or sclerotin reduce, have the increased osteomyelodysplasia patient's condition of osteofibrosis, adhesive capsulitis, dupp Yi Telunshi contraction Contracting and myelofibrosis.
35A. is according to the method for any one in paragraph 1A-7A, and wherein subject suffers from and drawn by the fibrosis of reproductive organs The disease or illness for rising or aggravating.
36A. is according to the method for paragraph 35A, wherein the disease or illness are selected from mullerianosis and Perun Nie Shi Disease.
For 37A. according to the method for any one in paragraph 1A-7A, wherein subject, which suffers from, causes fibrosis and/or inflammation Chronic infectious disease.
38A. according to the method for paragraph 37A, wherein the infectious diseases be selected from α virus, hepatitis A, hepatitis B, Hepatitis C, tuberculosis, HIV and influenza.
For 39A. according to the method for any one in paragraph 1A-7A, wherein subject, which suffers from, causes fibrosis and/or inflammation Autoimmune disease.
40A. is according to the method for paragraph 39A, wherein the autoimmune disease is selected from chorionitis and systemic red yabbi Sore (SLE).
41A. is according to the method for any one in paragraph 1A-7A, and wherein subject suffers from scar shape related with wound At.
42A. is according to the method for paragraph 41A, wherein cicatrization related with wound is selected from postoperative complication (for example, hand Art adhesion, wherein cicatricial tissue can be formed between internal organs, lead to contracture, pain, and can lead to infertility), chemotherapy Drug-induced fibrosis, radiation-induced fibrosis and the related cicatrization with burn.
43A. is according to the method for any one in paragraph 1A-7A, wherein being caused or being aggravated by fibrosis and/or inflammation Disease or illness are selected from organ transplant, Breast fibroadenoma, fibro-muscular, retroperitoneal fibrosis, thyroid gland fibrosis, lymph node Fibrosis, cystic fibrosis and fibrosis of pleura.
The method that 1B. prevents in the subject with disease related with albuminuria or the patient's condition or reduces injury of kidney, packet Include the MASP-2 inhibitor for giving the amount for the albuminuria for effectively reducing or preventing subject.
2B. is according to the method for paragraph 1B, and wherein MASP-2 inhibitor is MASP-2 inhibiting antibody or its segment.
3B. according to the method for paragraph 1B or 2B, wherein MASP-2 inhibitor with the baseline twenty-four-hour urine albumen before treatment Secretion is than effectively realizing that twenty-four-hour urine the protein secretion at least amount of 20% reduction and time are given.
4B. is according to the method for any one in paragraph 1B-3B, wherein disease related with albuminuria or the patient's condition are selected from kidney Sick syndrome, pre-eclampsia, eclampsia, the toxic lesion of kidney, amyloidosis, collagen vascular diseases are (for example, systemic red yabbi Sore), lupus nephritis, dehydration, renal glomerular disease is (for example, membranous glomerulonephritis, focal segmental glomerulonephritis, C3 kidney Bead disease, minimal change disease, lipoid nephrosis), strenuous exercise, stress, benign orthostatic position (position) albuminuria, focal section It is section property glomerulosclerosis, IgA nephrosis (that is, Bei Geershi is sick), IgM nephrosis, membranoprolifer ative glomerulonephritis, membranous nephropathy, micro- Small lesion disease, sarcoidosis, Alport's syndrome, diabetes (diabetic nephropathy), drug-induced toxicity (for example, NSAIDS, nicotine, penicillamine, lithium carbonate, gold and other heavy metals, Vel-Tyr-Pro-Trp-Thr-Gln-Arg-Phe, antibiotic (for example, adriamycin) or Ah Tablet preparation (for example, heroin) or other nephrotoxins)；Fabry disease, infection are (for example, HIV, syphilis, first, second or the third type liver Scorching, streptococcus postoperative infection, urinary schistosmiasis)；Amino acid uria, Fanconi syndrome, hypertensive nephrosclerosis, chromic fibrous kidney Inflammation, drepanocytosis, hemoglobinuria, Huppert's disease, myoglobinuria, organ rejection's (for example, renal transplant rejection), angstrom It is rich to pull out blood-head, chatelain's syndrome, familial Mediterranean fever, HELLP syndrome, systemic loupus erythematosus, Wei Genashi granulation Swollen disease, 1 type of glycogen storage disease, Goodpasture's syndrome, anaphylactoid purpura, has spread to kidney at rheumatoid arthritis Glomerulonephritis after urinary tract infections, siogren's syndrome and infection.
5B. is according to the method for any one in paragraph 1B-3B, wherein disease related with albuminuria or the patient's condition are IgA kidneys Sick (that is, Bei Geershi is sick).
6B. is according to the method for any one in paragraph 1B-3B, wherein disease related with albuminuria or the patient's condition are film properties Nephrosis.
7B. is according to the method for any one in paragraph 1B-3B, wherein disease related with albuminuria or the patient's condition are lupus Ephritis.
The method that 1C. inhibits the progress of chronic kidney diseases effectively reduces including giving or prevents subject's in need The MASP-2 inhibitor of the amount of Tubulointerstitial fibrosis.
2C. is according to the method for paragraph 1C, and wherein MASP-2 inhibitor is MASP-2 inhibiting antibody or its segment.
3C. is according to the method for paragraph 1C, wherein subject in need shows albumen before giving MASP-2 inhibitor Urine, and the albuminuria that MASP-2 inhibitor reduces subject is given, so that the baseline twenty-four-hour urine albumen with subject before treatment Secretion ratio, subject are reduced with twenty-four-hour urine protein secretion at least 20%.
4C. is according to the method for paragraph 1C, and wherein MASP-2 inhibitor is to effectively reduce, postpone or eliminate the saturating of subject The amount that analysis needs is given.
1D. protects kidney from the method for injury of kidney in subject, and the subject undergone, is undergoing or will be through It goes through and is treated with one or more renal toxicity agent, effectively prevent or improve the amount of the incidence of drug-induced nephrosis including giving MASP-2 inhibitor.
2D. is according to the method for paragraph 1D, and wherein MASP-2 inhibitor is MASP-2 inhibiting antibody or its segment.
3D. is according to the method for paragraph 1D, and wherein MASP-2 inhibitor is given before the renal toxicity agent.
4D. according to the method for paragraph 1D, wherein MASP-2 inhibitor with the renal toxicity agent is co- simultaneously gives.
5D. is according to the method for paragraph 1D, and wherein MASP-2 inhibitor is malicious to kidney is treated after the renal toxicity agent Property.
The method of people experimenter of the 1E. treatment with IgANP (IgAN), including give subject and include Effectively inhibit the MASP-2 inhibiting antibody of the amount of MASP-2- dependent complement activation or the composition of its antigen-binding fragment.
2E. is according to the method for 1 E of paragraph, wherein the subject suffers from steroid-dependent IgAN.
3E. is according to the method for paragraph 1E or 2E, and wherein MASP-2 inhibiting antibody is the list for specifically binding people MASP-2 Clonal antibody or its segment.
4E. is according to the method for any one in paragraph 1E-3E, and wherein antibody or its segment are selected from recombinant antibodies, have and subtract Antibody, chimeric antibody, humanized antibody and the human antibody of few effector function.
5E. is according to the method for any one in paragraph 1E-4E, and wherein MASP-2 inhibiting antibody does not significantly inhibit classics Approach.
6E. is according to the method for any one in paragraph 1E-3E, and wherein MASP-2 inhibiting antibody is with 30 nM or smaller IC50Inhibit C3b deposition in 90% human serum.
7E. is according to the method for paragraph 2E, wherein the method further includes including to be effectively improved kidney giving subject Before the step of composition of the MASP-2 inhibiting antibody of the amount of function or its antigen-binding fragment, identification have steroids according to Rely the people experimenter of property IgAN.
8E. is according to the method for any one of paragraph 1E-7E, wherein MASP-2 inhibiting antibody or its antigen-binding fragment It is given with being effectively improved the amount of renal function.
9E. according to the method for paragraph 8E, wherein MASP-2 inhibiting antibody or its antigen-binding fragment with before treatment by The baseline twenty-four-hour urine protein secretion of examination person compared to realize twenty-four-hour urine protein secretion at least the effective quantity of 20% reduction and it is enough when Between give.
10E. is according to the method for paragraph 1E, and wherein composition is to be enough to improve renal function and reduction in the subject The amount of corticosteroid dosage is given.
11E. is according to the method for any one in paragraph 1E-10E, wherein MASP-2 inhibiting antibody or its antigen binding Segment includes heavy chain variable region, and it includes CDR-H1, CDR-H2 and CDR-H3 of amino acid sequence shown in SEQ ID NO:67； And light chain variable region, it includes CDR-L1, CDR-L2 and CDR-L3 of amino acid sequence shown in SEQ ID NO:70.
The method of people experimenter of the 1F. treatment with membranous nephropathy (MN), including giving subject includes effectively to inhibit The MASP-2 inhibiting antibody of the amount of MASP-2- dependent complement activation or the composition of its antigen-binding fragment.
2F. is according to the method for paragraph 1F, wherein the subject suffers from steroid-dependent MN.
3F. is according to the method for paragraph 1F or 2F, and wherein MASP-2 inhibiting antibody is the list for specifically binding people MASP-2 Clonal antibody or its segment.
4F. is according to the method for any one in paragraph 1F-3F, wherein the antibody or its segment are selected from recombinant antibodies, tool Antibody, chimeric antibody, humanized antibody and the human antibody for the effector function being reduced.
5F. is according to the method for any one in paragraph 1F-4F, and wherein MASP-2 inhibiting antibody does not significantly inhibit classics Approach.
6F. is according to the method for any one in paragraph 1F-5F, and wherein MASP-2 inhibiting antibody is with 30 nM or smaller IC50Inhibit C3b deposition in 90% human serum.
7F. is according to the method for paragraph 1F, wherein the method further includes including to be effectively improved kidney giving subject Before the step of composition of the MASP-2 inhibiting antibody of the amount of function or its antigen-binding fragment, identification have steroids according to Rely the people experimenter of property MN.
8F. is according to the method for any one in paragraph 1F-7F, wherein MASP-2 inhibiting antibody or its antigen binding fragment Section is given with the amount for being effectively improved renal function.
9F. according to the method for paragraph 8F, wherein MASP-2 inhibiting antibody or its antigen-binding fragment with before treatment by The baseline twenty-four-hour urine protein secretion of examination person compared to realize twenty-four-hour urine protein secretion at least the effective quantity of 20% reduction and it is enough when Between give.
10F. according to the method for paragraph 1F or 2F, wherein composition be enough to improve in the subject renal function and The amount for reducing corticosteroid dosage is given.
11F. is according to the method for any one in paragraph 1F-10F, wherein MASP-2 inhibiting antibody or its antigen binding Segment includes heavy chain variable region, and it includes CDR-H1, CDR-H2 and CDR-H3 of amino acid sequence shown in SEQ ID NO:67； And light chain variable region, it includes CDR-L1, CDR-L2 and CDR-L3 of amino acid sequence shown in SEQ ID NO:70.
The method of people experimenter of the 1G. treatment with lupus nephritis (LN), including giving subject includes effectively to inhibit The MASP-2 inhibiting antibody of the amount of MASP-2- dependent complement activation or the composition of its antigen-binding fragment.
2G. is according to the method for paragraph 1G, wherein the subject suffers from steroid-dependent MN.
3G. is according to the method for paragraph 1G or 2G, and wherein MASP-2 inhibiting antibody is the list for specifically binding people MASP-2 Clonal antibody or its segment.
4G. is according to the method for any one in paragraph 1G-3G, wherein the antibody or its segment are selected from recombinant antibodies, tool Antibody, chimeric antibody, humanized antibody and the human antibody for the effector function being reduced.
5G. is according to the method for any one in paragraph 1G-4G, and wherein MASP-2 inhibiting antibody does not significantly inhibit classics Approach.
6G. is according to the method for any one in paragraph 1G-5G, and wherein MASP-2 inhibiting antibody is with 30 nM or smaller IC50Inhibit C3b deposition in 90% human serum.
7G. is according to the method for paragraph 1G, wherein the method further includes including to be effectively improved kidney giving subject Before the step of composition of the MASP-2 inhibiting antibody of the amount of function or its antigen-binding fragment, identification have steroids according to Rely the people experimenter of property LN.
8G. is according to the method for any one in paragraph 1G-7G, wherein MASP-2 inhibiting antibody or its antigen binding fragment Section is given with the amount for being effectively improved renal function.
9G. according to the method for paragraph 8G, wherein MASP-2 inhibiting antibody or its antigen-binding fragment with before treatment by The baseline twenty-four-hour urine protein secretion of examination person compared to realize twenty-four-hour urine protein secretion at least the effective quantity of 20% reduction and it is enough when Between give.
10G. according to the method for paragraph 1G or 2G, wherein composition be enough to improve in the subject renal function and The amount for reducing corticosteroid dosage is given.
11G. is according to the method for any one in paragraph 1G-10G, wherein MASP-2 inhibiting antibody or its antigen binding Segment includes heavy chain variable region, and it includes CDR-H1, CDR-H2 and CDR-H3 of amino acid sequence shown in SEQ ID NO:67； And light chain variable region, it includes CDR-L1, CDR-L2 and CDR-L3 of amino acid sequence shown in SEQ ID NO:70.
While there have been illustrated and described what illustrative embodiment, it should be understood that various change can be carried out wherein without de- From the spirit and scope of the present invention.

Claims (20)

1. the method for reducing the albuminuria in the people experimenter with IgAN, including subject is given according to following dosage MASP-2 inhibiting antibody or its antigen-binding fragment, the antibody or its antigen-binding fragment include: heavy chain variable region, packet CDR-H1, CDR-H2 and CDR-H3 of amino acid sequence shown in the NO:67 of ID containing SEQ；And light chain variable region, it includes SEQ CDR-L1, CDR-L2 and CDR-L3 of amino acid sequence shown in ID NO:70:
A. (i.e. 3.6 mg/kg are to 4.4 mg/kg's) by subject about 4 mg/kg of the intravenous administration with IgAN once a week The antibody continues at least 12 weeks treatment phases；Or
B. once a week intravenous administration suffer from IgAN about 180 mg of subject to about 725 mg (i.e. 162 mg to 797 Mg the antibody), continues at least 12 weeks treatment phases,
Wherein the method reduces the albuminuria in the people experimenter.
2. the method for claim 1 wherein the treatment phase is 12 weeks.
3. the method for claims 1 or 2, wherein being the rest period of at least two moon (that is, not giving MASP- after the treatment phase 2 inhibitor).
4. the method for claims 1 or 2, wherein being the rest period of at least three moon (that is, not giving MASP- after the treatment phase 2 inhibitor).
5. the method for claims 1 or 2, wherein being the rest period of at least four moon (that is, not giving MASP- after the treatment phase 2 inhibitor).
6. the method for claims 1 or 2, wherein being the rest period of at least five moon (that is, not giving MASP- after the treatment phase 2 inhibitor).
7. the method for claims 1 or 2, wherein being the rest period of at least six moon (that is, not giving MASP- after the treatment phase 2 inhibitor).
8. the method for any one of claim 1-7, wherein described tested at the end for the treatment of phase and/or at the end of the rest period Albuminuria in person, which reduces at least 20%(, that is, uACR reduction and/or twenty-four-hour urine protein concentration from baseline (before treatment), to be reduced).
9. the method for any one of claim 1-7, wherein described tested at the end for the treatment of phase and/or at the end of the rest period Albuminuria in person, which reduces at least 30%(, that is, uACR reduction and/or twenty-four-hour urine protein concentration from baseline (before treatment), to be reduced).
10. the method for any one of claim 1-7, wherein at the end for the treatment of phase and/or at the end of the rest period it is described by Albuminuria in examination person reduces at least 40%(, that is, uACR reduction and/or twenty-four-hour urine protein concentration drop from baseline (before treatment) It is low).
11. the method for any one of claim 1-7, wherein at the end for the treatment of phase and/or at the end of the rest period it is described by Albuminuria in examination person reduces at least 50%(, that is, uACR reduction and/or twenty-four-hour urine protein concentration drop from baseline (before treatment) It is low).
12. the method for any one of claim 1-7, wherein at the end for the treatment of phase and/or at the end of the rest period it is described by Albuminuria in examination person is reduced from baseline (before treatment) is greater than 50(, that is, uACR reduction and/or the reduction of twenty-four-hour urine protein concentration).
13. the method for any one of claim 1-7, wherein the method further includes in treatment phase and/or in the rest period Urine protein level in periodic monitoring subject.
14. the method for any one of claim 1-7, the glomerular filtration rate wherein estimated in subject (eGFR) increases.
15. the method for any one of claim 1-7, wherein with started with the treatment of MASP-2 inhibiting antibody before the cortex taken Steroids dosage is compared, and the subject stops or significantly reduce skin at the end for the treatment of phase and/or at the end of the rest period Matter steroids dosage.
16. the method for claim 1 wherein the MASP-2 inhibiting antibody or its segments to be selected from recombinant antibodies, has and reduces Effector function antibody, chimeric antibody, humanized antibody and human antibody.
17. the method for claim 1 wherein the MASP-2 inhibiting antibody or its segments to include:
(a) heavy chain variable region, it includes: i) the heavy chain CDR- of the amino acid sequence of the 31-35 comprising SEQ ID NO:67 H1；And ii) comprising SEQ ID NO:67 50-65 amino acid sequence heavy chain CDR-H2；And iii) it include SEQ ID The heavy chain CDR-H3 of the amino acid sequence of the 95-107 of NO:67, and
(b) light chain variable region, it includes: i) the light chain CDR- of the amino acid sequence of the 24-34 comprising SEQ ID NO:70 L1；And ii) comprising SEQ ID NO:70 50-56 amino acid sequence light chain CDR-L2；And iii) it include SEQ ID The light chain CDR-L3 of the amino acid sequence of the 89-97 of NO:70.
18. the method for claim 1 wherein the MASP-2 inhibiting antibody or its segments to include: heavy chain variable region, packet Containing the amino acid sequence with amino acid sequence shown in SEQ ID NO:67 at least 95% identity；And light chain variable region, Comprising having the amino acid sequence of at least 95% identity with amino acid sequence shown in SEQ ID NO:70.
19. the method for claim 1 wherein the MASP-2 inhibiting antibody or its segment include heavy chain variable region, and wrapping Light chain variable region shown in the NO:70 of ID containing SEQ, the heavy chain variable region include amino acid sequence shown in SEQ ID NO:67 Column.
20. the method for claim 1 wherein the subjects with IgAN to have greater than 1 gram albumen/24 hour before the treatment The albuminuria of Urine proteins secretion, and the albuminuria in the subject is effectively reduced at least 30% by the method.
CN201780060710.5A 2016-10-13 2017-10-12 Method for reducing the albuminuria in the people experimenter for suffering from IgANP Pending CN110177557A (en)

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