UA126908C2 - Methods for reducing proteinuria in a human subject suffering from immunoglobulin a nephropathy - Google Patents

Abstract

In one aspect, the invention provides methods for reducing proteinuria in a human subject suffering, or at risk of developing Immunoglobulin A Nephropathy (IgAN). The methods comprise the step of administering, to a subject in need thereof, an amount of a MASP-2 inhibitory antibody effective to inhibit MASP-2-dependent complement activation.

Description

STATEMENT REGARDING SEQUENCE LIST

The sequence listing associated with this application is provided in text format in lieu of hard copy and is incorporated into this application by reference. The text file containing the sequence list is named MR 1 0269 PCT Zedyepse I ibzipd 20171013 5725.

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TECHNICAL LEVEL

In humans and other vertebrates, the complement system provides a mechanism of early action for the initiation, amplification and regulation of the immune response to microbial infection and other acute infections (I ie27em/5Ki

M.K. apa AikKipzop UR, 1993, ip Eipdatepai! Ittypo!odu, Tpika Edyiop, edited by ME. Ratsi, Namep

Rgez55, Tsa., Mem/ HogKk). Although complement activation provides an important first line of defense against potential pathogens, complement activity, which stimulates the formation of a protective immune response, can also be a potential threat to the host (Kaiyi K.M. ei ai., 5rhipdeg bZetip.

Ittiporaina. 15:41-431, 1994; Moghdap V.R., Ek. IN). Siipisa! Ipmevzid. 24:219-228, 1994).

For example, proteolytic products of C3 and C5 stimulate the recruitment and activation of neutrophils.

Although neutrophils are necessary for host defense, activated neutrophils are not selective in their release of destructive enzymes and can cause organ damage. In addition, complement activation can cause the deposition of lytic components of complement on neighboring host cells as well as on microbial targets, leading to host cell lysis.

The complement system is also involved in the pathogenesis of numerous acute and chronic diseases, including myocardial infarction, stroke, RDDM, reperfusion injury, septic shock, capillary leak after thermal burns, inflammation after cardiopulmonary bypass surgery, transplant rejection, rheumatoid arthritis, multiple sclerosis, myasthenia gravis and Alzheimer's disease. In almost all of these conditions, complement itself does not cause these conditions, but is one of several factors involved in pathogenesis. However, complement activation may be the underlying pathological mechanism and represents an effective link for clinical control in many of the indicated disease states.

The growing recognition of the importance of understanding the mechanisms of complement-mediated tissue damage in various disease states once again points to the need for

From the development of effective medicinal substances that inhibit complement. For today

Eculizumab (ZoiagieFf (Solaris)), an anti-C5 antibody, is the only complement-targeting drug approved for human use. However, C5 is one of several effector molecules that act at the "late" stages of the complement system, and blockade of C5 does not lead to suppression of activation of the complement system. Therefore, an inhibitor of the initiation stages of complement activation would have a significant advantage over an inhibitor of the complement component acting at later stages.

Currently, it is generally accepted that the complement system can be activated in three different ways: classical, lectin and alternative. The classical pathway is usually initiated by a complex consisting of host antibodies bound to a foreign particle (ie, an antigen) and therefore requires prior exposure to an antigen to generate a specific antibody response. Since the activation of the classical pathway depends on the host's prior adaptive immune response, the classical pathway is part of the acquired immunity system. In contrast, both lectin and alternative pathways are independent of adaptive immunity and are part of the innate immune system.

Activation of the complement system leads to sequential activation of serine protease zymogens. The first stage of activation of the classical pathway is the binding of the specific recognition molecule C1d 3 by antigen-bound molecules /dsC and IM. C14d is associated with serine protease proenzymes Sig and C15 in the form of a complex called C1. When C1d binds to the immune complex, autoproteolytic cleavage of the Ago-Pe site of Cg is accompanied by Sig-mediated cleavage and activation of C15, thus providing the ability to cleave C4 and C2. C4 is cleaved into two fragments, marked C4a and C4b, and C2, similarly, is cleaved into Cga and C2p6. C4B6 fragments can form covalent bonds with neighboring hydroxyl groups or amino groups and generate C3-convertase (C4p2a) through non-covalent interaction with the C2a fragment of activated C2. C3-convertase (C4p2a) activates C3 with the help of proteolytic cleavage into its subcomponents C3 and C3, leading to the formation of C5-convertase (C4rgas), which through the cleavage of C5 leads to the formation of a membrane attacking complex (C5B together with Cb, C7, C8 and C9, as well called MAC), which can destroy cell membranes, leading to cell lysis. Activated forms of C3 and C4 (C3Br and C4b) as a result of covalent binding are deposited on the surfaces of foreign targets, which are recognized by complement receptors on many phagocytes.

Regardless of this, the first stage of activation of the complement system by the lectin pathway is also the binding of specific recognition molecules, after which the activation of proenzymes associated with serine protease occurs. However, instead of binding immune complexes using C14, the recognition molecules in the lectin pathway contain a group of carbohydrate-binding proteins (mannan-binding lectin (MB), H-ficolin, M-ficolin, I-ficolin and C-type SI lectin - 11), known under the common name of lectins. See Gee.). ей ай., Viospit. Viorpuz.

Asia, 1572:387-400, (2002); NoIt5Kom ei ai., Appy. German And so on. 21:547-578 (2003); Tep ey ai.,

Ittypoiodu, 101:225-232 (2000). See also And! tsei U. ei ai., Viospit. Viorpu5. Asia, 1572:387-400 (2002); NoIt5Kom vy aiY, Appy. Nehm. And so on. 21:547-578 (2003); Tep ei ai!., Ittypoiodu, 101:225- 232 (2000); Napsep ei ai, 9. Ittipoi. 185(10):6096-6104 (2010).

Ikeda her amI. demonstrated for the first time that MVI, like C1d, can activate the complement system after binding to erythrocytes covered with yeast mannan by a C4-dependent mechanism (IKeda ei ai., 9. VioI. Speth. 2(52):7451-7454, (1987)). MVI, a member of the collectin protein family, is a calcium-dependent lectin that binds to carbohydrates by 3- and 4-hydroxy groups oriented in the horizontal plane of the pyranose ring. Thus, the main ligands for MV are UO-mannose and M-acetyl-O-glucosamine, and carbohydrates that do not satisfy this steric requirement have undetectable affinity for MV. (Umeiv5 ei ai.,

Maishge, 360:127-134, (1992)). The interaction between MVI and monovalent sugars is very weak with dissociation constants usually in the single-digit millimolar range.

Strong, specific binding of MVI. with glycan ligands is achieved due to avidity, that is, interaction simultaneously with a set of monosaccharide residues localized in close proximity to each other (ee ei ai., Agspim. Viospet. Viorpub. 5. 299:129-136, (1992)). MVI recognizes carbohydrate patterns, usually formed by microorganisms such as bacteria, yeast, parasites and certain viruses. In contrast, MVI does not recognize O-galactose and sialic acid, the penultimate and last sugars, which, as a rule, are contained in "mature" complex glycoconjugates present on the cell surface and in the plasma of mammals. This binding specificity is believed to facilitate the recognition of "foreign" surfaces and help protect against "autoactivation". However, MVI does not bind with high affinity to clusters of glycan "precursors" with a high mannose content on M-linked glycoproteins and glycolipids sequestered in the endoplasmic reticulum and in the Golgi apparatus of mammalian cells (Maupaga et al.,).

VioI. Snet 257:3788-3794, (1982)). Thus, damaged cells are potential targets for activation of the lectin pathway by binding to MVI.

Ficolins contain a lectin domain that differs from the lectin domain of MVI, which is called a fibrinogen-like domain. Ficolins bind to sugar residues in a Ca--independent manner. Three types of ficolins (I-ficolin, M-ficolin and H-ficolin) were identified in humans. Two serum ficolins, I-ficolin and H-ficolin, have a common specificity for M-acetyl-O-glucosamine, but H-ficolin also binds to M-acetyl-O-galactosamine.

The difference in specificity for sugars I-ficolin, H-ficolin, SI-11 and MVI. means that different lectins can be complementary and affect different, even overlapping, glycoconjugates. This concept is confirmed by recent reports in the literature that of the known lectins in the lectin pathway, only I-ficolin specifically binds to lipoteichoic acid, i.e. the glycoconjugate of the cell wall, which is found in all gram-positive bacteria (Gupsp M.. ei ai. , 9U. Ittipoi. 172:1198-1202, 2004). Collectins (ie

MVI) and ficolin have no significant similarity in amino acid sequences. However, these two groups of proteins have the same domain organization and, like C1d, assemble into oligomeric structures, which maximizes the possibility of multisite binding.

Concentrations of MB in serum vary greatly in populations of healthy individuals and are genetically controlled by polymorphisms/mutations in the promoter and coding regions of the gene

MVI.. Expression of MVI, as an acute phase protein, is additionally enhanced during inflammation. I-ficolin is present in the serum in concentrations similar to MV. Therefore, the I-ficolin branch of the lectin pathway is potentially comparable in its strength to the hilua of MV. MV. ficolins also function as opsonins, which allow phagocytes to affect MVI- and ficolin-decorated surfaces (see. Ittipobiiodu, 205(4-5):490-7 (2002), Asuadi et al., U. Ittipoi. 174(1):418-25(2005)). Such opsonization requires the interaction of these proteins with phagocyte receptors (KipItap ei ai., U. Ehr. Med. 169:1733, (1989); Maizivznya ei ai., ».

VioI. Spent 277:2448-54, (1996)), the identity of which has not yet been established.

The specific and high-affinity interaction of human MVI with unique C1g/C15-like serine proteases, called MVI-associated serine proteases (MVI), is carried out through its collagen-like domain. At the moment, three MA5BRs are described. Initially, one enzyme "MABR" was identified, which was characterized as the enzyme responsible for the initiation of the complement cascade (i.e. cleavage of C2 and C4) (Maiziv5nya ei ai.,

U. Ehr. May 176(6):1497-1502 (1992); Phi eyi ai., U. Ittipoi. 750:571-578, (1993)). Then it was determined that the activity of MABR is actually represented by a mixture of two proteases: MA5RA-Yi and

MABR-2 (Tiyei eyi aiYi., Maishte, 556:506-510, (1997)). However, it was shown that one MVI-MABR-2 complex is sufficient for complement activation (Mohyr-Uepvep ei ai., 9. Ittipoi. 165:2093-2100, (2000)). In addition, only MABR-2 cleaved C2 and C4 at a high rate (Atbrgyb ei ai. U. Ittipoi. 770:1374-1382, (2003)) Thus, MABR-2 is the protease responsible for the activation of C4 and Сб2 with the formation SZ-convertases, C4r2a. This is a significant difference from the C1 complex of the classical pathway, where the coordinated action of two specific serine proteases (Sig and C15) leads to the activation of the complement system. In addition, a third new protease, MABR-Z, was isolated (Bani MV et al., Ittipyu, 15:127-35, 2001). MA5R-1 and

MA5P-3 are products of alternative splicing of the same gene.

The organization of MABR domains is identical to that of Stig and C15, which are enzymatic components of the St complex (Zit eyi aiYi., Viospet. os. Tgap5. 28:545, (2000)). These domains include the sea urchin M-terminal C1g/C15/ESE/bone morphogenetic protein (BMP) domain, an epidermal growth factor-like domain, a second SIOV domain, a complement regulatory protein domain tandem, and a serine protease domain. As in C1 proteases, activation

MABR-2 is carried out through the cleavage of the Ago-IPe bond adjacent to the serine protease domain, leading to the separation of the enzyme into A and B chains connected by a disulfide bond, where the latter consists of the serine protease domain.

MVI can also be associated with an alternatively spliced form of MAZBR-2, known as MVI-associated protein 19 kDa (MAp19) or small MVI-associated protein (5MAP), which lacks the catalytic activity of MAZR-2 (5iomega, y). And so on. 162:3481-90 (1999); TaKkanavni ei ai., Iyi. And so on. 11:859-863, (1999)). MAp19 contains the first two domains

MABR-2, followed by an additional sequence of four unique amino acids. Function

MAr19 remains unclear (Oeodp ei ai.,". Ittipoi. Meioa5, 2011). MA5BR-1 and MA5BR-2 genes are localized on human chromosomes 3 and 1, respectively (Z5spugaebie ei ai., Ittypobioiodu, 205:455-466 (2002)).

There are a number of data that suggest that different MVI-MA5R complexes and a large fraction

MABRs in serum do not form complexes with CF. (TpPiei 5. ei ai., U. Ittipoi. 165:878-887, 2000).

Both H- and I-ficolin bind to all MARs and, like MVI, activate the lectin pathway of complement (ShBai!i M.K. ei ai., Ittipyu, 15:127-35, 2001; Maizivnia M. ei ai., 9. Ittypo). 165:3502-3506, 2002). The lectin and classical pathways form a common S3-convertase (C4rga), and at this stage these two pathways converge.

It is widely believed that the lectin pathway plays an important role in the defense of the uninfected host against infection. Convincing evidence for the involvement of MVHI in host defense was obtained from analysis of patients with reduced levels of functional MVHI. in serum (Kiiraigisk

O.b., Viosnit. Viorpuz. Asia, 1572:401-413, 2002). These patients are susceptible to recurrent bacterial and fungal infections. Such symptoms usually appear at an early age within an apparent "window of vulnerability" when maternal antibody titers decline until the full repertoire of antibodies is formed.

This syndrome is often the result of mutations at multiple sites in the collagen portion of MV that prevent proper formation of MV oligomers. However, since MV can function as an opsonin independently of complement, it is not known to what extent the increased susceptibility to infection is due to reduced complement activation.

All three pathways (i.e., classical, lectin, and alternative) are believed to converge in

C5, which is broken down to form products with numerous pro-inflammatory effects.

The pathway in which all three pathways converge was defined as the terminal complement pathway.

Sba is the strongest anaphylotoxin, inducing a change in the tone of smooth muscles and blood vessels, as well as the permeability of blood vessels. It is also a potent chemotaxin and activator of neutrophils and monocytes. Sba-mediated cell activation can significantly enhance inflammatory responses by inducing the release of a variety of additional inflammatory mediators, including cytokines, hydrolytic enzymes, arachidonic acid metabolites, and reactive oxygen species. Cleavage of C5 leads to the formation of C5p-9, also known as membrane attack complex (MAC). At present, there is convincing evidence that sublytic deposition of MAC, in addition to its role as a lytic pore-forming complex, may play an important role in the inflammatory process.

In addition to its important role in immune protection, the complement system is involved in tissue damage in many clinical conditions. Despite the availability of numerous data indicating the involvement of classical and alternative pathways of complement in the pathogenesis of non-infectious diseases in humans, the role of the lectin pathway in such pathogenesis began to be assessed only now. Recent studies have shown that activation of the lectin pathway may be responsible for complement activation and associated inflammation in ischemia/reperfusion injury. SoPaga her a). (2000) showed that cultured endothelial cells exposed to oxidative stress bind to MVI. and show deposition

SZ in response to the influence of human serum (Soyaga ei ai., At. 9. Raiyoi. 156:1549-1556, (2000)).

In addition, treatment of human serum with blocking monoclonal anti-MVI. antibodies suppressed MVI binding and complement activation. These data were confirmed in a rat model of myocardial ischemia/reperfusion, in which rats receiving blocking antibodies to rat MVI showed a significant reduction in myocardial damage against the background of coronary artery occlusion, compared to rats receiving a control antibody (Chogdap "U. E. ei a!., Sigsshayop, 104:1413-1418, 2001). The molecular mechanism of binding of MVI to the vascular endothelium after oxidative stress remains unclear, but recent studies suggest that the activation of the lectin pathway after oxidative stress may be mediated by binding of MVI to vascular endothelial cytokeratins rather than to glycoconjugates (SoPaga C .O. ei ai,, At. U. Raioiyi. 159:1045-1054, 2001). Other studies pointed to the role of classical and alternative pathways in the pathogenesis of ischemic/reperfusion injury, and the role of the lectin pathway in the pathogenesis of this disease still remains a subject of debate (Kivdeptapp M.S. ei ai!., At. y). Raipoi 162:363-367, 2003).

Fibrosis is the formation of an excess amount of connective tissue in an organ or tissue, usually formed in response to injury or damage. A distinctive feature of fibrosis is the production of excess extracellular matrix after a local injury.

The normal physiological response to injury results in the formation of connective tissue, but this initially healing repair process can become protracted and pathological, altering tissue architecture and function. At the cellular level, epithelial cells and fibroblasts proliferate and differentiate into myofibroblasts, leading to matrix contraction, increased stiffness, microvascular compression, and hypoxia. The influx of inflammatory cells, including macrophages and lymphocytes, leads to the release of cytokines and enhances deposition

From collagen, fibronectin and other molecular markers of fibrosis. Traditional therapeutic approaches were mainly aimed at the inflammatory process in fibrosis with the use of corticosteroids and immunosuppressants. Unfortunately, all these anti-inflammatory agents have little clinical effect. There is currently no effective treatment or therapy for fibrosis, but both animal studies and anecdotal reports of human outcomes suggest that fibrotic tissue lesions can be reversed (Tatre apa 7eizrego, Mai. Keu. Merpgoi!., Moi. 10:226-237, 2014).

The kidney has a limited ability to recover from injury. Various pathologies of the kidneys lead to local inflammation, which causes scarring and fibrosis of the kidneys. The influence of inflammatory stimuli, which does not stop, leads to tubulointerstitial inflammation and fibrosis and progressive functional impairment of the kidneys in chronic kidney disease. Its progression to end-stage renal disease is associated with significant morbidity and mortality. Since tubulointerstitial fibrosis is a common endpoint of many renal pathologies, it represents an important target for treatment aimed at preventing renal failure. Risk factors (eg, proteinuria) independent of the primary kidney disease contribute to the development of renal fibrosis and loss of excretory function of the kidneys, stimulating the development of local inflammation, which, in turn, enhances the progression of the disease.

Given the role of fibrosis in many diseases and disorders, such as, for example, tubulointerstitial fibrosis leading to chronic kidney disease, there is an urgent need to develop therapeutically effective agents for the treatment of diseases and conditions caused or complicated by fibrosis. Also, given the lack of new and existing treatments that affect inflammatory profibrotic factors in kidney diseases, it is necessary to develop therapeutically effective means to treat, suppress, prevent and/or stop kidney fibrosis, thereby preventing the progression of chronic kidney disease.

ESSENCE OF THE INVENTION

This section of the description is intended to introduce a set of concepts, presented in a simplified form, which are described in more detail below. This section of the description is not intended to define the key features of the claimed object of the invention and is not intended to define the scope 60 of the claimed object of the invention.

In one aspect, the invention relates to a method of treating, inhibiting, alleviating or preventing fibrosis in a mammal suffering from or at risk of developing a disease or disorder caused or complicated by fibrosis and/or inflammation, which comprises administering to the subject an amount of an MA5BR-2 inhibitory agent, effective for suppressing fibrosis. In one embodiment, the MABR-2 inhibitory agent is a MABR-2 antibody or its fragment. In one of the variants, the MABR-2 inhibitory agent is a monoclonal MA5ZR-2 antibody or its fragment that specifically binds to a part of

ZEO IU MO:6. In one embodiment, the MA5BR-2 inhibitory agent selectively inhibits activation of the complement lectin pathway without substantially inhibiting C1d-dependent complement activation. In one embodiment, the subject suffers from a disease or disorder caused or complicated by at least one of the following: (i) fibrosis and/or inflammation associated with ischemia reperfusion injury, (ii) renal fibrosis and/or inflammation (eg, tubulointerstitial fibrosis, chronic kidney disease, chronic renal failure, glomerular disease (for example, focal segmental glomerulosclerosis)), disorders of the immune complex (for example, Ida nephropathy, membranous nephropathy), lupus nephritis, nephrotic syndrome, diabetic nephropathy, tubulointerstitial damage and glomerulonephritis ( eg, SZ glomerulopathy), (iii) fibrosis and/or inflammation of the lungs (eg, chronic obstructive pulmonary disease, cystic fibrosis, pulmonary fibrosis associated with scleroderma, bronchiectasis and pulmonary hypertension), (im) fibrosis and/or inflammation liver (eg, cirrhosis, nonalcoholic fatty liver disease (steatohepatitis)), liver fibrosis secondary to alcohol abuse, liver fibrosis secondary to acute or chronic hepatitis (drug-induced hepatotoxicity caused by acetaminophen or another drug), (m) fibrosis and/or inflammation of the heart (eg, cardiac fibrosis, myocardial infarction, valvular fibrosis, atrial fibrosis, endomyocardial fibrosis, arrhythmic right ventricular cardiomyopathy (ART), (mi) vascular fibrosis (eg, vascular disease, atherosclerotic vascular disease, vascular stenosis, restenosis , vasculitis, phlebitis, deep vein thrombosis and abdominal aortic aneurysm), (mi) skin fibrosis (eg, excessive wound healing, scleroderma, systemic sclerosis, keloids, connective tissue disease, scarring and hypertrophic

Of scars), (my) fibrosis of joints (eg, arthrofibrosis), (their) fibrosis of the central nervous system (eg, stroke, traumatic brain injury and spinal cord injury), 09 fibrosis of the digestive system (eg, Crohn's disease, pancreatic fibrosis and ulcerative colitis), (xi) ocular fibrosis (for example, anterior subcapsular cataract, opacification of the posterior capsule, macular degeneration and retinal and vitreous retinopathy), (xii) fibrosis of soft tissue structures of the musculoskeletal system (for example, cellular capsulitis, Dupuytren's contracture and myelofibrosis), (chii) fibrosis of the reproductive organs (eg, endometriosis and Peyronie's disease), (chem) chronic infectious disease that causes fibrosis and/or inflammation (eg, alpha virus, hepatitis A, hepatitis B, hepatitis C, tuberculosis, HIV, and influenza), (hm) autoimmune disease that causes fibrosis and/or inflammation (eg, scleroderma and systemic lupus erythematosus (SLE), (hm) trauma-related scarring (eg, where Trauma-related scarring is selected from the group consisting of surgical complications (eg, postoperative adhesions, in which scar tissue can form between internal organs, causing contracture, pain, and can lead to infertility), chemotherapy-induced fibrosis, radiation-induced fibrosis and scarring associated with burns), or (chemically) organ transplants, breast fibrosis, muscle fibrosis, retroperitoneal fibrosis, thyroid fibrosis, lymph node fibrosis, bladder fibrosis, and pleural fibrosis.

In another aspect, the present invention relates to a method of treating, inhibiting, alleviating, or preventing renal fibrosis in a mammal suffering from or at risk of developing a disease or disorder caused or complicated by renal fibrosis and/or inflammation, which comprises administering to the subject an amount of an inhibitory MABR-2 an agent effective in suppressing renal fibrosis. In one embodiment, the MA5P-2 inhibitory agent is a MA5P-2 antibody or its fragment. In one of the variants, the MABR-2 inhibitory agent is a monoclonal MA5ZR-2 antibody or its fragment that specifically binds to a part of

ZEO IU MO:6. In one of the variants of MA5P-2, the antibody or its fragment specifically binds to the polypeptide containing 5EO IUO MO:6, with an affinity that is at least 10 times greater than the affinity of binding to another antigen in the complement system. In one embodiment, the antibody or its fragment is selected from the group consisting of a recombinant antibody, an antibody with reduced effector function, a chimeric antibody, or a humanized antibody, and a human antibody. In one of the variants, the inhibitory MA5P-

2 agent selectively inhibits activation of the lectin pathway of complement without substantially inhibiting the activation of Sid-dependent complement. In one embodiment, the MA5ZR-2 inhibitory agent is administered subcutaneously, intraperitoneally, intramuscularly, intraarterially, intravenously, or by inhalation. In one embodiment, the MA5BR-2 inhibitory agent is administered in an amount effective to inhibit tubulointerstitial fibrosis. In one embodiment, the MA5P-2 inhibitory agent is administered in an amount effective to reduce, delay, or eliminate the need for dialysis in the subject. In one embodiment, the subject has a kidney disease or a disease selected from the group consisting of chronic kidney disease, chronic renal failure, glomerular disease (eg, focal segmental glomerulosclerosis), lupus nephritis, nephrotic syndrome, diabetic nephropathy, tubulointerstitial disease and glomerulonephritis (for example, SZ-glomerulopathy). In one embodiment, the subject has proteinuria, and the MA5P-2 inhibitory agent is administered in an amount effective to reduce the severity of proteinuria in the subject. In one embodiment, the MABR-2 inhibitory agent is administered in an amount and for a period of time effective to achieve at least a 20 95 reduction (eg, at least a 30 95 reduction, or at least a 40 95 reduction, or at least a 50 95 reduction) in 24-hour excretion of urinary protein compared to the subject's baseline 24-hour urinary protein excretion before treatment. In one embodiment, the subject suffers from a kidney disease or disorder associated with proteinuria selected from the group consisting of nephrotic syndrome, preeclampsia, eclampsia, toxic kidney injury, amyloidosis, angiocollagenous diseases (eg, systemic lupus erythematosus), dehydration, glomerular diseases (for example, membranous glomerulonephritis, focal segmental glomerulonephritis, CZ3-glomerulopathy, minimal change disease, lipoid nephrosis), intense physical activity, stress, benign orthostatic (postural) proteinuria, focal segmental glomerulosclerosis, IdA-nephropathy (for example, diseases Berger's), ICHM nephropathy, membranoproliferative glomerulonephritis, membranous nephropathy, minimal change disease, sarcoidosis, Alport syndrome, diabetes mellitus (diabetic nephropathy), drug-induced toxicity (eg, induced by NSAIDs, nicotine, penicillamine, lithium carbonate, gold, and other heavy metals , inhibitors

ACE inhibitors, antibiotics (eg Adriamycin) or opiates (eg heroin) or other nephrotoxins); Fabry disease, infections (for example, HIV, syphilis, hepatitis A, B or C, post-streptococcal infection, urinary schistosomiasis); aminoaciduria, Fanconi syndrome, hypertensive nephrosclerosis, interstitial nephritis, sickle cell disease, hemoglobinuria, multiple myeloma, myoglobinuria, organ rejection (eg, kidney transplant rejection), Ebola hemorrhagic fever, patellar nail syndrome, familial fever, NEGIR syndrome, systemic lupus erythematosus, granulomatosis Wegener's, rheumatoid arthritis, glycogen storage disease type 1 (Ghirke's disease), syndrome

Goodpasture, Schönlein-Henoch purpura, urinary tract infection that has spread to the kidneys, Sjogren's syndrome, and postinfectious glomerulonephritis. In one embodiment, the subject suffers from IdA nephropathy. In one embodiment, the subject suffers from membranous nephropathy.

In another aspect of the present invention, there is provided a method of preventing or reducing kidney damage in a subject suffering from a disease or condition associated with proteinuria, which comprises administering an amount of an MABR-2 inhibitory agent effective to reduce or prevent proteinuria in the subject. In one of the variants, the MAZR-2 inhibitory agent is a MA5R-2 antibody or its fragment. In one embodiment, the inhibitory

MA5R-2 agent is a monoclonal MAB5bR-2 antibody or its fragment, which specifically binds to the 5EO part of IJU MO:6. In one embodiment, the MA5P-2 inhibitory agent selectively inhibits activation of the complement lectin pathway without substantially inhibiting activation

Std-dependent complement. In one embodiment, the disease or condition associated with proteinuria is selected from the group consisting of nephrotic syndrome, preeclampsia, eclampsia, toxic kidney injury, amyloidosis, vascular collagen diseases (eg, systemic lupus erythematosus), dehydration, glomerular diseases ( for example, membranous glomerulonephritis, focal segmental glomerulonephritis, SZ-glomerulopathy, minimal change disease, lipoid nephrosis), intense physical activity, stress, benign orthostatic (postural) proteinuria, focal segmental glomerulosclerosis, IdA-nephropathy (for example, diseases

Berger), IDM nephropathy, membranoproliferative glomerulonephritis, membranous nephropathy, minimal change disease, sarcoidosis, Alport syndrome, diabetes

(diabetic nephropathy), drug-induced toxicity (eg, induced by NSAIDs, nicotine, penicillamine, lithium carbonate, gold and other heavy metals, inhibitors

ACE inhibitors, antibiotics (eg Adriamycin) or opiates (eg heroin)); diseases

Fabry, infections (for example, HIV, syphilis, hepatitis A, B or C, post-streptococcal infection, urinary schistosomiasis); aminoaciduria, Fanconi syndrome, hypertensive nephrosclerosis, interstitial nephritis, sickle cell disease, hemoglobinuria, multiple myeloma, myoglobinuria, organ rejection (for example, kidney transplant rejection), Ebola hemorrhagic fever, nail patella syndrome, familial fever, HER IR syndrome, systemic lupus erythematosus, Wegener's granulomatosis, rheumatoid arthritis, glycogen storage disease type 1, Goodpasture syndrome, Schönlein-Henoch purpura, urinary tract infection that has spread to the kidneys, Sjogren's syndrome, and postinfectious glomerulonephritis. In one embodiment, the MABR-2 inhibitory agent is administered in an amount and for a period of time effective to achieve at least a 20 95 reduction (eg, at least a 30 95 reduction, or at least a 40 95 reduction, or at least a 50 95 reduction) in 24-hour excretion urinary protein compared to the subject's baseline 24-hour urinary protein excretion before treatment.

In another aspect of the present invention, there is provided a method of inhibiting the progression of chronic kidney disease, which comprises administering to a subject in need thereof an amount of an inhibitory

MABR-2 is an agent effective in reducing or preventing renal fibrosis, such as tubulointerstitial fibrosis. In one embodiment, the MA5P-2 inhibitory agent is a MA5P-2 antibody or its fragment. In one embodiment, the inhibitory

The MA5P-2 agent is a monoclonal MA5P-2 antibody or its fragment that specifically binds to the 5EO part of the IUO MO:6. In one embodiment, the MAZR-2 inhibitory agent selectively inhibits activation of the complement lectin pathway without substantially inhibiting activation

Std-dependent complement. In one embodiment, the subject in need had proteinuria prior to administration of the MA5R-2 inhibitory agent, wherein administration of the inhibitory agent

MA5BR-2 agent reduces proteinuria in the subject. In one embodiment, the inhibitory

The MA5R-2 agent is administered in an amount and for a period of time that is effective to achieve at least a 20 95 reduction (eg, at least a 30 95 reduction or at least

Of a 40 95 decrease, or at least a 50 95 decrease) in 24-hour urinary protein excretion compared to the subject's baseline 24-hour urinary protein excretion before treatment. In one embodiment, the MA5P-2 inhibitory agent is administered in an amount effective to reduce, delay, or eliminate the need for dialysis in the subject.

In another aspect of the present invention, there is provided a method of protecting the kidneys from injury in a subject who has received, is receiving, or will receive treatment with one or more nephrotoxic agents, which comprises administering an amount of an MABR-2 inhibitory agent effective to prevent or alleviate drug-induced nephropathy. In one embodiment, the MABR-2 inhibitory agent is a MABR-2 antibody or its fragment. In one of the variants, the inhibitory MA5P-2 agent is a monoclonal MA5P-2 antibody or its fragment, which specifically binds to a part of ZEO IU MO:6. In one embodiment, the MABR-2 inhibitory agent selectively inhibits activation of the lectin pathway of complement without substantially inhibiting C1d-dependent complement activation.

In one of the aspects of the invention, there is provided a method of treating a person suffering from immunoglobulin A nephropathy (IAM), which includes administering to the subject a composition containing an amount of an MA5R-2 inhibitory antibody or an antigen-binding fragment thereof effective to inhibit MABR-2- dependent complement activation. In one embodiment, the subject suffers from steroid-dependent UTI. In one embodiment, the MA5BR-2 inhibitory antibody is a monoclonal antibody or its fragment that specifically binds to human MA5P-2. In one embodiment, the antibody or its fragment is selected from the group consisting of a recombinant antibody, an antibody with reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody. In one embodiment, the MA5BR-2 inhibitory antibody essentially does not inhibit the activation of the classical pathway. In one embodiment, the inhibitory MA5R-2 antibody suppresses the deposition of SZB in 90 95 human serum with an IC value of 30 nM or less. In one embodiment, the method additionally includes identifying a subject who has steroid-dependent (DAM) prior to the step of administering to the subject a composition containing an amount of MABR-2 inhibitory antibody or its antigen-binding fragment effective to improve kidney function. In one embodiment, the MABR-2 inhibitory antibody or its antigen-binding fragment is administered in an amount effective to improve kidney function. are effective to achieve at least a 20 95 reduction in 24-hour urinary protein excretion compared to the subject's baseline 24-hour urinary protein excretion prior to treatment.In one embodiment, the composition is administered in an amount sufficient to improve renal function and reduce dose of corticosteroids in the specified subject. In one embodiment, the MABZR-2 inhibitory antibody or antigen-binding fragment thereof contains a heavy chain variable region containing SOC-NI, SOB-H2, and SOC-NH of the amino acid sequence listed in ZEO IU MO:67, the variable region of the light chain containing SOB-I 1, SOB-12 and SOC-YZ of the amino acid sequence given in ZEO IU MO:70.

In another aspect of the invention, a method of treating a person suffering from membranous nephropathy (MN) is provided, which comprises administering to the subject a composition containing an amount of an inhibitory

MA5P-2 antibody or its antigen-binding fragment, effective for suppressing MAP-2-dependent complement activation. In one embodiment, the subject suffers from steroid-dependent MN. In one of the variants, the inhibitory MA5R-2 antibody is a monoclonal antibody or its fragment that specifically binds to human MA5BR-2. In one of the variants, the MABZR-2 inhibitory antibody or its antigen-binding fragment is administered in an amount effective to improve kidney function. In one embodiment, the MA5BR-2 inhibitory antibody or antigen-binding fragment thereof is administered in an amount and for a period of time effective to achieve at least a 20 95 reduction in 24-hour urinary protein excretion compared to baseline 24-hour protein excretion urine in the subject before treatment. In one embodiment, the composition is administered in an amount sufficient to improve kidney function and reduce the dose of corticosteroids in the subject. In one embodiment, the MA5BR-2 inhibitory antibody or its antigen-binding fragment contains a heavy chain variable region containing SOVB-

NO, SOB-H2 and SOVB-NZ of the amino acid sequence given in ZEO IU MO:67, the variable region of the light chain containing SOK-I1, SOB-12 and SOK-IZ of the amino acid sequence given in 5ZEO IU MO:70.

In another aspect of the invention, a method of treating a person suffering from lupus nephritis (LU) is provided, which comprises administering to the subject a composition containing an amount of an inhibitory

MA5P-2 antibody or its antigen-binding fragment, effective for suppressing MAP -

From 2-dependent complement activation. In one embodiment, the subject suffers from steroid-dependent HF. In one embodiment, the MA5BR-2 inhibitory antibody is a monoclonal antibody or its fragment that specifically binds to human MA5BR-2. In one embodiment, the MABR-2 inhibitory antibody or its antigen-binding fragment is administered in an amount effective to improve kidney function. In one embodiment, the MABR-2 inhibitory antibody or antigen-binding fragment thereof is administered in an amount and for a period of time that is effective to achieve at least a 20 95 reduction in 24-hour urinary protein excretion compared to baseline 24-hour urinary protein excretion. urine in the subject before treatment. In one embodiment, the composition is administered in an amount sufficient to improve kidney function and reduce the dose of corticosteroids in the subject. In one of the variants, the MAZR-2 inhibitory antibody or its antigen-binding fragment contains a variable region of the heavy chain containing SOB-

NO, SOB-H2 and SOK-NZ of the amino acid sequence given in ZEO IU MO:67, and the variable region of the light chain containing SOK-I1, SOV-12 and SOK-IZ of the amino acid sequence given in 5ZEO IU MO:70.

In another aspect, the invention provides a method of reducing proteinuria in a human sufferer

ICHAM, which includes administering to a subject a composition that contains an amount of an MA5ZR-2 inhibitory antibody or an antigen-binding fragment thereof containing a heavy chain variable region that contains SOB-NI, SOB-H2, and SOB-NH of the amino acid sequence listed in HEO

IU MO:67, and the variable region of the light chain, which contains SOK-I1, SOV-1I2 and SOK-I Z

BO of the amino acid sequence given in 5EO IU MO:70, according to the following dosage schemes: a) administration of approximately 4 mg/kg (ie, from 3.6 to 4.4 mg/kg) of the specified antibody to a subject suffering from IDA, intravenously once a week for a treatment period of at least 12 weeks; p) administering from about 180 mg to about 725 mg (i.e., 162 to 797 mg) of said antibody to a subject suffering from UTI intravenously once a week for a treatment period of at least 12 weeks, wherein the method reduces proteinuria in said subject subject

DESCRIPTION OF THE DRAWINGS The above aspects and many of the accompanying advantages of the present invention will become more clear with reference to the following detailed description with reference to the attached drawings, in which: fig. 1 is a diagram illustrating the genomic structure of human MAB5R-2; fig. 2A is a schematic diagram illustrating the structure of human protein domains

MA5R-2; fig. 28 is a schematic diagram illustrating the structure of human protein domains

MAr19; fig. C is a diagram illustrating the mouse MA5R-2 knockout strategy; fig. 4 is a diagram illustrating the construction of the MA5R-2 mini-gene; in fig. BA presents results demonstrating that MABR-2 deficiency results in loss of C4-mediated activation of the lectin pathway as determined by the absence of deposits

C46p on mannan, as described in example 2; in fig. 58 presented results demonstrating that MABR-2 deficiency leads to loss of C4-mediated activation of the lectin pathway as determined by the absence of deposits

C4p on zymosan, as described in example 2; in fig. 5C shows the results demonstrating the relative levels of C4 activation in serum samples obtained from MABR-2-/-, MA5R-2-/- and wild-type lines, determined by deposition

C46b on mannan and on zymosan, as described in example 2; in fig. b presents results demonstrating that addition of mouse recombinant MABR-2 to MABR-2-/- serum samples restores C4-mediated activation of the lectin pathway, which is dependent on protein concentration as determined by deposition

C46p on mannan, as described in example 2; in fig. 7 presents the results demonstrating that the classical pathway is functional in the MA5P-2-/- line as described in example 8; in fig. BA presents the results that demonstrate that the anti-MA5BR-2 antibody Bar2 Mo 11 inhibits the formation of C3-convertase, as described in example 10; in fig. 88 presents the results that demonstrate that the anti-MA5P-2 antibody Rab2 Mo 11 binds to native rat MA5P-2, as described in example 10; in fig. 8C presents the results demonstrating that the anti-MA5R-2 antibody Par2 Mo 41

Zo inhibits cleavage of C4, as described in example 10; in fig. 9 presents results demonstrating that all tested anti-MABR-2 Rabr2 antibodies, which inhibited the formation of C3-convertase, also inhibited the cleavage of Ca4, as described in example 10; fig. 10 is a diagram illustrating recombinant polypeptides derived from rat

MAB5P-2, which were used to map the epitopes of blocking MABR-2 antibodies Par2, as described in example 11; in fig. 11 presents the results demonstrating the binding of anti-MABR-2 Bab2 MoMo and 60 to rat MA5R-2 polypeptides, as described in example 11; in fig. 12A graphically shows the level of MAC deposition in the presence or absence of 40 human monoclonal anti-MABR-2 antibody (OM5646) under lectin pathway-specific assay conditions, demonstrating that OM5646 inhibits lectin-mediated MAC deposition with an IC value of about 1 nM, as described in example 12; in fig. 128 graphically shows the level of MAC deposition in the presence or absence of a human monoclonal anti-MABR-2 antibody (OM5646) under classical pathway-specific test conditions, indicating that OM5646 does not inhibit classical pathway-mediated MAC deposition as described in Example 12; in fig. 12C graphically shows the level of MAC deposition in the presence or absence of a human monoclonal anti-MABR-2 antibody (OM5646) under alternative pathway-specific test conditions, indicating that OM5646 does not inhibit alternative pathway-mediated MAC deposition as described in Example 12; in fig. 13 graphically shows the pharmacokinetic (PC) profile of a human monoclonal anti-MAB5R-2 antibody (OM5646) in mice showing the concentration of OM5646 (mean value for n-Z animals/group) as a function of time after administration of the indicated dose as described in Example 12 ; in fig. 14A graphically shows the pharmacodynamic (PO) response of monoclonal anti-

MABR-2 human antibody (0M5646), determined by the decrease in activity of the lectin pathway of the complement system in mice after intravenous administration, as described in example 12; in fig. 148 graphically shows the pharmacodynamic (PO) response of the human monoclonal MABR-2 bo antibody (0M5646), determined by reducing the activity of the lectin pathway of the complement system in mice after subcutaneous administration, as described in example 12; in fig. 15 graphically shows the results of computer analysis of images of kidney tissue sections stained with Sirius red; tissue sections were obtained from wild-type mice and MABR-2-/- mice 7 days after unilateral ureteral obstruction (USO) and pseudo-operated wild-type mice and MA5R-2-/- mice, as described in example 14; in fig. 16 graphically shows the results of computer analysis of images of kidney tissue sections stained with antibodies to the E4/80 macrophage marker; tissue sections were obtained from wild-type mice and MA5BR-2-/- mice 7 days after unilateral ureteral obstruction (USO) and pseudo-operated wild-type mice and MA5P-2-/- mice, as described in example 14; in fig. 17 graphically shows the relative expression levels of collagen-4 mRNA, measured quantitatively

PCR (cPCR) in kidney tissue sections obtained from wild-type Her MABR-2-/- mice 7 days after unilateral ureteral obstruction (USO) and sham-operated wild-type mice and

MAB5BR-2-/- mice, as described in example 14; in fig. 18 graphically shows the relative mRNA expression levels of transforming growth factor beta-1 (TaEr-1), measured by PCR in sections of kidney tissues obtained from wild-type mice and MA5P-2-/- mice 7 days after unilateral ureteral obstruction (USO) and pseudo-operated wild-type mice and MA5P-2-/- mice, as described in example 14; in fig. 19 graphically shows the relative expression levels of interleukin-b mRNA (1I/-6), measured

PCR in kidney tissue sections obtained from wild-type mice and MABR-2-/- mice 7 days after unilateral ureteral obstruction (USO) and sham-operated wild-type mice and

MAB5BR-2-/- mice, as described in example 14; in fig. 20 graphically shows the relative expression levels of interferon-γ mRNA measured by qPCR in kidney tissue sections obtained from wild-type and MA5R-2-/- mice 7 days after unilateral ureteral obstruction (USO) and sham-operated wild-type and MA5R- mice 2-/- mice as described in example 14; in fig. 21 graphically shows the results of computer analysis of images of sections of kidney tissues stained with Sirius red; tissue sections were obtained 7 days after unilateral ureteral obstruction (USO) in wild-type mice receiving inhibitory

MAB5R-2 antibody and isotype control antibody, as described in example 15;

From in fig. 22 graphically shows the content of hydroxyproline in kidneys obtained 7 days after unilateral ureteral obstruction (USO) in wild-type mice that received inhibitory

MABR-2 antibody, in comparison with the level of hydroxyproline in the tissue of the occluded kidneys obtained from wild-type mice that received the isotype control, Ids4, as described in example 15; in fig. 23 graphically shows the total amount of serum proteins (mg/ml), measured on the 15th day of studying the effect of protein overdose in control wild-type mice (n-2) that received only saline, wild-type mice that received VZA (n- b), and MA5P-2-/- mice receiving B5A (n:-6b), as described in example 16; in fig. 24 graphically shows the total amount of protein (mg) excreted in urine collected during 24 hours, on the 15th day of studying the effects of protein overdose in control wild-type mice (p-2), receiving only saline, wild-type mice , which received VZA (p:-b), and MA5R-2-/- mice, which received VZA (p-b), as described in example 16; in fig. 25 shows representative sections of kidney tissues, stained with hematoxylin and eosin (HE), on the 15th day of the study of the level of protein overdose in the following groups of mice: (panel A) control wild-type mice; (panel B) MA5R-2-/- control mice, (panel C) wild-type mice treated with VZA; and (panel Y) MA5P-2-/- mice receiving bovine serum albumin (BSA) as described in example 16; in fig. 26 graphically shows the results of computer analysis of images of kidney tissue sections stained with an antibody to the macrophage marker E4/80, showing the average stained area of macrophages (90), where the tissue sections were obtained on day 15 of the study of the effects of protein overdose in wild-type control mice (n-2), wild-type mice receiving VZA (n-b), and MA5R-2-/- mice receiving VZA (n-5), as described in example 16; in fig. 27A graphically shows the analysis of the correlation between the level of macrophages and proteinuria in each wild-type mouse (n-b) that received VZA, in the form of a graph of the dependence of the total amount of secreted protein, measured in a 24-hour urine sample, on the infiltration of macrophages (average colored area , 95), as described in example 16; in fig. 27B graphically shows the analysis of the correlation between the level of macrophages and proteinuria in each MA5R-2-/- mouse (n-5) receiving B5A, in the form of a graph of the dependence of the total amount of secreted protein, measured in a 24-hour urine sample, on the infiltration of macrophages (average stained area, 9b5), as described in example 16;

in fig. 28 graphically shows the results of computer analysis of images of tissue sections stained with anti-TaeEr antibody (measured as the area of stained anti-TOEr antibodies, expressed as a percentage), in wild-type mice that received B5A (p-4) and MABR- 2-/- mice receiving VZA (n:-5) as described in example 16; in fig. 29 graphically shows the results of computer analysis of images of tissue sections stained with anti-TMEo; antibody (measured as the area of stained anti-TMEc; antibodies) in wild-type mice treated with B5A (n--4) and MA5R-2-/- mice treated with BZA (n:-5) as described in example 16; in fig. 30 graphically shows the results of computer analysis of images of tissue sections stained with anti-1II-6 antibody (measured as the area of stained anti-1II-6 antibodies) in control wild-type mice, control MABR-2-/- mice, wild-type mice type receiving VZA (n-7) and MABR-2-/- mice receiving VZA (n-7), as described in example 16; in fig. 31 graphically shows the frequency of TOMEI -positive apoptotic cells, counted in 20 consecutively selected fields of view under high magnification (HPE) in tissue sections of the renal cortical substance obtained from control wild-type mice (n-1), control MAP -2-/- mice (p-1), wild-type mice receiving B5A (p-b), and MAZR-2-/- mice receiving

VZA (n-7), as described in example 16; in fig. 32 shows representative HE-stained tissue sections obtained on day 15 after B5A treatment of the following groups of mice: (Panel A) wild-type control mice, saline-treated mice; (panel B) mice receiving an isotype control antibody and (panel C) wild-type mice receiving an inhibitory MA5P-2 antibody as described in example 17; in fig. 33 graphically shows the frequency of TOME! -positive apoptotic cells, counted in consecutively selected 20 fields of view under high magnification (HPE) in tissue sections of the renal cortical substance, obtained from control wild-type mice that received control physiological solution and VBA (n-8), wild-type mice that received an isotype control antibody and BBA (n-8), and wild-type mice that received an inhibitory MA5BR-2 antibody and BBA (n-7), as described in example 17; in fig. 34 graphically shows the results of computer analysis of images of tissue sections,

Of those stained with anti-TYEr antibody (measured as the area of stained anti-TyeR antibodies, 90), in wild-type mice that received B5A and saline (n-8), wild-type mice that received VZA and an isotype control antibody (n -7), and wild-type mice that received VZA and inhibitory MA5bR-2 antibody (n-8), as described in example 17; in fig. 35 graphically shows the results of computer analysis of images of tissue sections stained with anti-TMEo antibody (measured as the area of stained anti-TMEo antibodies, 90), in wild-type mice that received B5A and saline (n-8), wild-type mice type that received VZA and isotype control antibody (n-7), and wild-type mice that received VZA and inhibitory MA5bR-2 antibody (n-8), as described in example 17; in fig. 36 graphically shows the results of computer analysis of images of tissue sections stained with anti-1Й -6 antibody (measured in the form of the area of stained anti-1Й -6 antibodies, Uro) in wild-type mice that received B5A and physiological solution (p-8 ), mice receiving

B5A and an isotype control antibody (p-7), and wild-type mice that received B5A and an inhibitory

MA5BR-2 antibody (n-8), as described in example 17; in fig. 37 shows representative HE-stained tissue sections obtained on day 14 after treatment with Adriamycin alone or saline (control) for the following groups of mice: (Panels A-1, A-2, A-3) wild-type control mice, that received only physiological solution; (panels B-1, B-2, B-3) wild-type mice treated with Adriamycin; and (panels C-1, b-2, C-3) MA5P-2-/- mice treated with Adriamycin as described in Example 18; in fig. 38 graphically shows the results of computer analysis of images of kidney tissue sections stained with an antibody to the macrophage marker E4/80, showing the average stained area of macrophages (95) on day 14 after treatment with Adriamycin alone or saline (wild-type control mice), for the following groups of mice: wild-type control mice that received only physiological solution; wild-type mice treated with adriamycin;

MA5P-2-/- mice receiving only saline and MAZR-2-/- mice receiving Adriamycin, where ""p-0.007, as described in example 18; in fig. 39 graphically shows the results of computer analysis of images of Sirius red-stained kidney tissue sections showing stained areas of collagen deposition (96) on day 14 after treatment with adriamycin or saline alone (wild-type control mice) for the following groups of mice: wild-type control mice that received only physiological solution; wild-type mice treated with adriamycin; MA5P -2-/- mice receiving only saline and MABR-2-/- mice receiving Adriamycin, where "xr-0.005, as described in example 18; in fig. 40 graphically shows the urinary albumin/creatinine ratio (URC) in two IdA patients during a twelve-week study with weekly treatment with an inhibitory MA5R-2 antibody (OM5646) as described in example 19; in fig. 41 shows a graph of the dependence of the value of ЧАСскК (mg/g) against time for four IDAM patients who received OM5646 from the initial moment to day 120, as described in example 21; in fig. 42 graphically shows the change in protein levels in the 24-hour urine collection after treatment relative to baseline on day 1 before treatment for four patients with ISAM receiving

OM5646, as described in example 21; and in fig. 43 graphically shows the mean change in protein levels in the 24-hour post-treatment urine collection relative to the pre-treatment baseline for four patients with ISAM receiving

OM5646, as described in example 21.

DESCRIPTION OF THE LIST OF SEQUENCES

ZEO IO MO:1 - cDNA of human MAr19;

ZEO IO MO:C2 - human MAr119 protein (with leader sequence);

ZEO IO MO:Z - human protein MAr!19 (mature);

ZEO IU MO:4 - cDNA of human MA5R-2;

ZEO IO MO:5 - human MA5BR-2 protein (with leader sequence);

ZEO I MO:6 - human protein MA5R-2 (mature);

ZEO IO MO:7 - hDNA of human MA5R-2 (exons 1-6).

ANTIGENS (RELATED TO THE MATURE MA5BR-2 PROTEIN)

ZEO I MO:8 - sequence of SIOVI (aa 1-121);

ZEO I MO:9 - sequence of SOVESE (aa 1-166);

ZEO IU MO:10 - COSESSESIVIES (aa 1-293);

ZEO IO MO:11 - ESE-plot (aa 122-166);

ZEO IO MO:12 - serine protease domain (aa 429-671);

ZEO IO MO:13 - inactive domain of serine protease (aa 610-625 with replacement of Zegb18 with Aa);

ZEO IU MO:14 - THREE ORKMURERMUESKI (SIOVI peptide);

ZEO IU MO:15 - TARROURKI KI METNEOGG EI ZNI SEMOERMKI Z5OAKMI ATI CO (SIOVI peptide);

ZEO IU MO:16 - TEKZOUZM (the central part of the connecting section of the MVI);

ZEO IU MO:17 - EM5I a55I OITEAZOUZMEKRETOE (binding site of MVI);

ZEO IU MO:18 - IPOESOMARO (ESRBE peptide);

ZEO Ir MO: 19 - AMMI SAS EZOSKOSSKORZOSAY M (the central part that binds serine protease).

DETAILED DESCRIPTION

PEPTIDE INHIBITORS

ZEO IU MO:20 - full-size kKDNK MVI;

ZEO IU MO:21 - full-length MVI protein;

ZEO IU MO:22 - OBK-X-OR (binding consensus);

ZEO Ir MO:23 - OSKI S;

ZEO Ir MO:24 - SI V SO SRO SOKI RO S;

ZEO Ir MO:25 - aRO SRO SGA SO SRO SKI SRO SRO SRO;

ZEO IR MO26 -- akranratkaEXSERIOOSI ON OSROSK AROSE;

ZEO I MO:27 - vANLOV5BOSEXAOBROSROSROSKMORKOEOSBO (human N-ficolin);

ZEO Ir MO28 - asosviGgosdovrkaeEdatchakvaEVNaROSROSKASROS RMaAOsSEO (human ficolin p35);

ZEO IU MO:29 - GOKAI EP/'RMEMTIKAMEREIIMRY (C4 cleavage site).

INHIBITORS OF EXPRESSION

ZEO IU MO:Z30 - cDNA of SOVI-ESEE domain (nucleotides 22-680 in ZEO IU MO 4);

ZEO IU MO:31 - U-СсССсайСАССАТОАДСЦСТОСТОСТТОоStTasyaaas-z, 12-45 nucleotides in

ZEO IU MO:4, which includes the site of initiation of MA5R-2 translation (semantic);

ZEO IR MO:32 - У-(44АСАТАССТТ ССИаСТОСОаASTОСААСОПСАСАДСИ-3", 361-396 nucleotides in

ZEO IU MO:4, coding region containing MVI-binding site MA5R-2 (sense);

ZEO Ir MO:33 - U-AVSAdaSSSTaAATASSSASOvyssostatossada-Z3", 610-642 nucleotides in

ZEO IU MO:4, the coding region containing the SOVII domain.

CODING PRIMERS

ZEO Ir MO:34 - bsasasАToSATaAdaasStTastaАСsstTo (5-PCR for SIV); 60 ZEO IU MO:35 - aiAATTOSTASaIstaSATA (3-PCR for SIV);

ZEO IU MO:36 - ayAATTSOSTASASIaassasst (3-PCR for SOVIESBE);

ZEO and MO:37 - aiAATTOSTAaTASstTasAT (3-PCR for SOVIETSESOVIA);

ZEO IU MO:38-47 - cloning primers for humanized antibody;

ZEO IU MO:48 - a peptide bond of 9 amino acids.

EXPRESSION VECTOR

ZEO IU MO:49 is an insertion of the MA5R-2 mini-gene; 5EO IU MO:50 is cDNA of mouse MA5R-2;

ZEO IU MO:51 is a murine MA5R-2 protein (with a leader sequence);

ZEO IU MO:52 is a mature mouse MA5R-2 protein;

ZEO IU MO:53 is the cDNA of rat MA5R-2;

ZEO IU MO:54 is a rat MA5R-2 protein (with a leader sequence);

ZEO IU MO:55 is a mature rat MABR-2 protein;

ZEO 10 MO:56-59 are oligonucleotides for site-directed mutagenesis of human MA5R-2, which are used to obtain human MABR-2A;

ZEO 10 MO:60-63 are oligonucleotides for site-directed mutagenesis of mouse MA5R-2, which are used to obtain mouse MAZR-2A;

ZEO 10 MO:64-65 are oligonucleotides for site-directed mutagenesis of rat MA5R-2, which are used to obtain rat MABR-2A;

ZEBEO IU MO:6b is a DNA encoding the variable region of the heavy chain (MH) 17020 asz5МНн2г1М11М. (OM5646) (without signal peptide);

ZEBEO IU MO:67 is a polypeptide 17020 asz5MnH2g1M11MI (0M5646) of the variable section of the heavy chain (MH);

ZEO IU MO:68 is a polypeptide of 17M1bts of the variable region of the heavy chain (MH);

ZEO IU MO:69 is DNA encoding the variable region of the light chain (MI) 17020 aszBungtmM11MI (OM5646);

ZEBEO IU MO:70 is a polypeptide 17020 asz5MnH2g1M11MI (0M5646) of the variable part of the light chain (Mi);

ZEO IO MO:71 is a polypeptide 17M16 as17M9 of the variable region of the light chain (MU);

ZEO IU MO:72 is ZOMI-2I. (full size);

ZEO IU MO:73 is ZOMI-2M (shortened version of medium length);

ZEO IU MO:74 is ZOMI-25 (short version, short);

ZEO IU MO:75 is a mature polypeptide containing MH-M2abb6-5OaMI-2-M and the constant region of human IDS4 with a mutation in the hinge region;

ZEO IU MO:76 is a mature polypeptide containing MH-M2abb6-5OaMI-2-C and the constant region of human Ids4 with a mutation in the hinge region;

ZEO IYUO MO:77 is a mature polypeptide containing MI -M2abBb-5СО2MI-2-M and the constant region of the lambda chain of human Id;

ZEO IYUO MO:78 is a mature polypeptide containing MI-M2abBb6-5C2MI-2-C and the constant region of the lambda chain of human Id;

ZEO IU MO:79 is a peptide linker (10 aa);

ZEO IU MO:80 is a peptide linker (6 aa);

ZEO IU MO:81 is a peptide linker (4 aa);

ZEO I MO:82 is a polynucleotide encoding a polypeptide containing MH-Ma2arb-

ZOMI-2-M and the constant region of human Ids4 with a mutation in the hinge region;

ZEO IU MO:83 is a polynucleotide encoding a polypeptide containing MH-Ma2arb-

ZOAMI-2-C and the constant region of human Ids4 with a mutation in the hinge region;

ZEBEO IU MO:84 represents a polynucleotide encoding a polypeptide containing MI -Magarb-

BO ZOMI-2-M and the constant region of the human Id lambda chain;

ZEBEO IU MO:85 represents a polynucleotide encoding a polypeptide containing MI -Magabrb-

ZOAMI-2-C and the constant region of the lambda chain of human Id.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the unexpected discovery of the authors of the present invention that inhibition of mannan-binding lectin-associated serine protease-2 (MABR-2), a key regulator of the lectin pathway of the complement system, significantly reduces inflammation and fibrosis in various animal models of fibrotic disease, including a unilateral obstruction model ureter (SHO), protein overdose model, and renal fibrosis model in adriamycin-induced nephropathy. Thus, the inventors have demonstrated that inhibition of MA5R-2-mediated activation of the lectin pathway provides an effective therapeutic approach to alleviate, treat, or prevent renal fibrosis, such as tubulointerstitial inflammation and fibrosis, regardless of the underlying cause. This description also shows that the use of an inhibitory MAB5R-2 antibody (OM5646) effectively improves kidney function and reduces the need for corticosteroids in people suffering from immunoglobulin

A-nephropathy (IdAM) and membranous nephropathy (MN).

I. DEFINITION

Unless otherwise specified, all terms used in this specification have the same meaning as would be understood by a person skilled in the art to which the present invention belongs. The following definitions are intended to clarify the terms used in the description and formula of this invention.

As used herein, the term "MABR-2-dependent complement activation" refers to MA5BR-2-dependent activation of the lectin pathway, which occurs under physiological conditions (ie, in the presence of Sam) and which leads to the formation of C3-convertase C4p2a of the lectin pathway and the accumulation of the cleavage product C3, such as SZBr, and then formation

C5-convertase C4B2a(C3p)n, which was determined to be the main cause of opsonization.

As used herein, the term "alternative pathway" refers to complement activation, which is initiated by, for example, zymosan from the cell walls of fungi and yeast, lipopolysaccharide (LPS) of the outer membrane of gram-negative bacteria and rabbit erythrocytes, as well as a variety of pure polysaccharides, rabbit erythrocytes, viruses, bacteria , tumor cells of animals, parasites and damaged cells, and which is believed to arise as a result of spontaneous proteolytic cleavage of complement factor S3 with the formation of

SZZ

As used herein, the term "lectin pathway" refers to complement activation that occurs through the specific binding of serum and non-serum carbohydrate-binding proteins, including mannan-binding lectin (MB),

SI -11th ficolin (H-ficolin, M-ficolin or I-ficolin).

As used herein, the term "classical pathway" refers to complement activation, which is triggered by an antibody bound to a foreign particle, and which requires binding to the C14 recognition molecule.

As used herein, the term "MAZR-2 inhibitory agent" means any agent that binds to or directly interacts with MA5BR-2 and effectively inhibits MABR-2-dependent complement activation, including anti-MA5BR-2 antibodies and their MAB5BR-2-binding fragments, natural and synthetic peptides, small molecules, soluble MABR-2 receptors, expression inhibitors and selected natural inhibitors; as well as peptides that, upon activation of the lectin pathway, compete with MABR-2 for binding to other recognition molecules (for example, MVGI, H-ficolin, M-ficolin, or I-ficolin), while this term does not include antibodies that bind bind to other recognition molecules. The MA5BR-2 inhibitory agents used in the present invention can reduce the level of MA5BR-2-mediated complement activation by more than 20 95, such as more than 50 95, such as more than 9095. In one embodiment, the MA5BR-2 inhibitory agent reduces the level of MA5BR-2-mediated complement activation is greater than 9095 (ie, the level of MABR-2-mediated complement activation will be only 10 95 or less as a result).

As used herein, the term "fibrosis" refers to the formation or presence of excess connective tissue in an organ or tissue. Fibrosis can occur as a repair or replacement response to a stimulus such as tissue injury or inflammation. A distinctive feature of fibrosis is the production of excess extracellular matrix.

The normal physiological response to injury leads to the formation of connective tissue as a component of the healing process, but this formation of connective tissue can be prolonged and become pathological, changing the architecture and function of the tissue. At the cellular level, epithelial cells and fibroblasts proliferate and differentiate into myofibroblasts, leading to matrix contraction, increased stiffness, microvascular compression, and hypoxia.

As used herein, the term "treating fibrosis in a mammal suffering from or at risk of developing a disease or disorder caused or complicated by fibrosis and/or inflammation" means stopping, attenuating, alleviating or suppressing fibrosis in said mammal.

As used herein, the term "proteinuria" means the presence of protein in human urine in an abnormal amount, for example, in an amount greater than 0.3 g of protein in human urine collected in 24 hours, or in a concentration greater than 1 g/L. In some embodiments, the proteinuric subject is a subject, such as a subject with 60 immunoglobulin A (IdA) nephropathy, who has protein in the urine greater than 1.0 g of protein in a 24-

hourly urine collection.

As used herein, the term "improvement in proteinuria" or "reduction in proteinuria" refers to a reduction in a proteinuric subject's 24-hour urinary protein excretion of at least 20 95 , such as at least 30 95 , such as at least 40 95 , for example by at least 50 95 or more compared to the subject's baseline 24-hour urinary protein excretion before treatment with the MA5R-2 inhibitory agent. In one embodiment, treatment with a MAB5bR-2 inhibitory agent according to a method of the invention is effective in reducing proteinuria in a human such that there is a greater than 2095 reduction in 24-hour urinary protein excretion or, for example, a greater than 3095 reduction in 24-hour urinary protein excretion. of protein with urine, or, for example, more than 40 95 reduction of 24-hour protein excretion with urine, or, for example, more than 50 95 reduction of 24-hour protein excretion with urine.

As used herein, the term "antibody" includes antibodies and fragments thereof obtained from any antibody-producing mammal (eg, mouse, rat, rabbit, and primate, including human) or obtained by hybridoma, phage selection, recombinant expression, or transgenic animals (or by other methods of obtaining antibodies or fragments of antibodies), where these antibodies or their fragments specifically bind to the target polypeptide, such as, for example, MA5P-2, its polypeptides or sections. This does not mean that the term "antibody" is limited to the sources of origin of antibodies specified in this description or the methods of their production (for example, by means of hybridoma, phage selection, recombinant expression, transgenic animals, peptide synthesis, etc.). Exemplary antibodies include polyclonal, monoclonal and recombinant antibodies; multivalent, multispecific antibodies (for example, bispecific antibodies, trispecific antibodies); humanized antibodies; mouse antibodies; chimeric antibodies; monoclonal antibodies "mouse-human", "mouse-primate", "primate-human"; and anti-idiotypic antibodies, which may be intact antibodies or fragments thereof. As used in this description, the term "antibody" covers not only intact polyclonal or monoclonal antibodies, but also their fragments (such as AB, Rab, Rar', K(ab)»2, Em), single-chain fragments (5Cem), their synthetic variants , natural variants, hybrid proteins that include part of the antibody together with the antigen-binding one

With a fragment with the desired specificity, humanized antibodies, chimeric antibodies and any modified configuration of immunoglobulin molecules containing an antigen-binding site or a fragment (epitope-recognizing region) with the desired specificity. "Monoclonal antibody" means a homogeneous population of antibodies in which the monoclonal antibody consists of amino acids (naturally occurring and non-naturally occurring) that participate in the selective binding of an epitope. Monoclonal antibodies are highly specific for the target antigen. The term "monoclonal antibody" covers not only intact monoclonal antibodies and full-length monoclonal antibodies, but also their fragments (such as Rab, Rab E(ar)g, Em), single-chain fragments (5cEm), their variants, recombinant proteins containing antigens binding part, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any modified configuration of an immunoglobulin molecule containing an antigen-binding fragment (an epitope-recognizing region) with the desired specificity and ability to bind to an epitope. This does not mean that this term is limited to the sources of origin of antibodies specified in this description or the methods of their production (for example, by means of hybridoma, phage selection, recombinant expression, transgenic animals, etc.). This term includes whole immunoglobulins as well as fragments, etc., described above under the definition of "antibody".

As used herein, the term "antibody fragment" refers to a portion derived from or belonging to a full-length antibody, such as, for example, an anti-MA5R-2 antibody, typically including the antigen-binding or variable region of that antibody.

Representative examples of antibody fragments include Rab, Rab, E(ab), Kia) and Emx fragments, 5cEm fragments, diatyl, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

The "single-chain Em" or "5cEm" antibody fragment used in this description contains the MH and Mi domains of the antibody, and these domains are present in one polypeptide chain. As a rule, the Em polypeptide additionally contains a polypeptide linker between the MH and MI domains, which allows 5cEm to form the required structure for antigen binding.

As used herein, the term "chimeric antibody" is a recombinant protein that contains variable domains and complementarity-determining regions derived from a non-human (eg, rodent) antibody, while the rest of the antibody molecule is derived from a human antibody.

As used herein, the term "humanized antibody" is a chimeric antibody containing a minimal sequence that corresponds to specific complementarity-determining regions derived from a non-human immunoglobulin transplanted into a human antibody framework. Humanized antibodies, as a rule, are recombinant proteins, in which only the complementarity-determining regions of the antibodies are of non-human origin.

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

As used herein, the term "membrane attack complex" (MAC) refers to a complex of five terminal components of complement (C5p, combined with Cb, C7, C8, and C9) that embeds itself in and destroys membranes (also called C56B-9 ).

As used herein, the term "subject" includes all mammals, including, without limitation, humans, non-human primates, dogs, cats, horses, sheep, goats, cows, rabbits, pigs and rodents.

Amino acid residues used in this description have the following abbreviations: alanine (Aia; A), asparagine (Azp; M), aspartic acid (Azr; 0), arginine (Ago; K), cysteine (Suv;

C), glutamic acid (Sim; E), glutamine (Sp; C), glycine (Su; c), histidine (Niv; H), isoleucine (Pe; I), leucine (Genes; I), lysine (Huz; K), methionine (Me; M), phenylalanine (Re; E), proline (Pho; P), serine (5eg; 5), threonine (TAg; T), tryptophan (Tgr; MU), tyrosine (TuUg; U ) and valine (may; M).

In a broader sense, natural amino acids can be divided into groups based on the chemical properties of the side chains of the corresponding amino acids. "Hydrophobic" amino acids are Pe, Hei, Mei, Re, Tgr, Tug, Mai, Aa, Sub5 or Rgo. "Hydrophilic" amino acids are Su, Avp, Sp, Zeg, TPg, Ar, Sim, Guz, Agud or Ni. This group of amino acids can also be divided into subclasses. "Uncharged hydrophilic" amino acids are zZeg, TNg,

Ap or Sip. "Acid" amino acids are Sim or Ar. The "basic" amino acids are Huz,

Agud or Niv.

As used herein, the term "conservative amino acid substitution" is represented by amino acid substitutions within one of the following groups: (1) glycine, alanine, valine, leucine, and isoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine, (4) ) aspartate

Zo and glutamate, (5) glutamine and asparagine, and (6) lysine, arginine, and histidine.

As used herein, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term also covers such oligonucleotides that consist of naturally occurring nucleotides, sugars, and covalent internucleoside (backbone) bonds, as well as oligonucleotides that have modifications that do not occur in nature.

As used herein, the term "epitope" refers to a region on a protein (e.g., a protein

MA5P-2), to which the antibody binds. "Overlapping epitopes" include at least one (eg, two, three, four, five or six) common amino acid residue(s), including linear and non-linear epitopes.

As used herein, the terms "polypeptide", "peptide" and "protein" are interchangeable and mean any chain of amino acids linked to a peptide, regardless of length or post-translational modification. The MA5BR-2 protein disclosed herein may contain or be a wild-type protein or may be a variant having no more than 50 (e.g., no more than 1, 2, 3,4, 5,6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40 or 50) conservative amino acid substitutions. Conservative substitutions usually include substitutions within one of the following groups: glycine and alanine; valine, isoleucine and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine and threonine; lysine, histidine and arginine; and phenylalanine and tyrosine.

In some embodiments, the human MABR-2 protein can have an amino acid sequence that is identical by 70 to 95 or more (eg, 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 95) human MABR-2 protein having the amino acid sequence presented in 5EO IU MO:5.

In some embodiments, peptide fragments can be at least 6 (eg, 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, Ab, 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, 400, 450, 500 or 600 or more) amino acid residues (for example, at least 6 contiguous amino acid residues from ZEO IU MO:5). In some embodiments, the antigenic peptide fragment is a protein

Human MAB5R-2 is less than 500 (eg, 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, 60 48, 47, Ab, 45, 44, 43, 42, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21,

20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7 or 6) amino acid residues (for example, less than 500 contiguous amino acid residues anywhere in the ZEO IU MO sequence: 5).

The percentage (95) of amino acid sequence identity is defined as the percentage of amino acids in the candidate sequence that are identical to amino acids in the control sequence after aligning the sequences and introducing gaps (gaps), if necessary, to achieve the maximum percentage of sequence identity. Alignment to determine percent sequence identity can be accomplished in a variety of ways within the skill of the art, such as using publicly available software such as VIABT, VIGABT-2, ACOM, AGISM-2, or Medaiidn (OMAZTAK). Appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the compared sequences, can be determined by known methods.

As used in this application, the term "approximately" before a numerical value indicates that value plus or minus 10 95 of the stated value.

I. BRIEF DESCRIPTION OF THE INVENTION

As disclosed in this description, the authors of the invention determined the central role of the lectin pathway in the initiation and progression of the disease of renal tubular pathology, which implies the key role of the activation of the lectin pathway in the pathophysiology of a wide range of kidney diseases, including IDA-nephropathy, SZ-glomerulopathy and other glomerulonephritis. This specification also states that the authors of this invention have discovered that inhibition of mannan-binding lectin-associated serine protease-2 (MA5P-2), a key regulator of the lectin pathway of the complement system, significantly reduces inflammation and fibrosis in various animal models of fibrotic disease, including a model of unilateral ureteral obstruction (OO), protein overdose model, and renal fibrosis model in adriamycin-induced nephropathy. Thus, the inventors have shown that inhibition of MA5R-2-mediated activation of the lectin pathway provides an effective therapeutic approach for alleviating, treating, or preventing renal fibrosis, such as tubulointerstitial fibrosis, regardless of the underlying cause.

Lectins (MV, M-ficolin, H-ficolin, I-ficolin and SI-11) are specific molecules

From recognition, which trigger the complement system according to the mechanism of implementation of innate immunity, and such a system includes the lectin pathway of initiation and the loop of amplification of the terminal pathway associated with it, which enhances lectin-initiated activation of the terminal effector molecules of complement. C1d is a specific recognition molecule that triggers the complement system by the mechanism of the implementation of acquired immunity, and such a system includes the classical initiation pathway and the associated terminal pathway amplification loop, which enhances C1d-initiated activation of terminal complement effector molecules. The authors refer to these two main complement activation systems as the lectin-dependent complement system and the C1d-dependent complement system, respectively.

In addition to its important role in immune defense, the complement system contributes to tissue damage in many clinical conditions. Thus, there is a very urgent need to develop therapeutically effective complement inhibitors to prevent the development of these side effects. Establishing the fact of the possibility of inhibition of the MAZR-2-mediated lectin pathway, while not affecting the classical pathway, provides an opportunity for the highly desired specific inhibition of the activation of only the complement system, which causes a specific pathology, without completely inhibiting the complement's ability to protect the immune system. So, for example, in pathological conditions in which complement activation is mediated mainly by the lectin-dependent complement system, the specific suppression of only this system is preferable. To regulate the processing of the immune complex and to ensure the protection of the host from infection, the system of C1id-dependent complement activation must remain unchanged.

A preferred bleaching component to target in the development of therapeutic agents for specific inhibition of the lectin-dependent complement system is MABR-2. Of all the known protein components of the lectin-dependent complement system (MVI, H-ficolin, M-ficolin, I-ficolin, MAZR-2, Сб2-С9, factor B, factor O, and properdin), only MA5R-2 is unique and necessary for its functioning of this lectin-dependent complement system. Lectins (MV, H-ficolin, M-ficolin, I-ficolin and SI-11) are also unique components of the lectin-dependent complement system. However, the loss of any one of these lectin components will not necessarily suppress the activation of this system, which is due to an excess amount of lectin. All five lectins must be inhibited to guarantee inhibition of activation of the lectin-dependent complement system. Furthermore, since MV and ficolins are also known to have complement-independent opsonin activity, inhibition of lectin function would result in the loss of this valuable host defense mechanism against infection. In contrast, such complement-independent opsonin activity of the lectin would remain unaffected if MABR-2 is the target for inhibition. Another advantage of MA5BR-2 as a therapeutic target for inhibiting the activation of the lectin-dependent complement system is that the concentration of MABR-2 in plasma, in comparison with any other complement proteins, is the lowest (500 ng/ml), and therefore for complete suppression will be sufficient low concentrations of high-affinity MABR-2 inhibitors (MoiIeg-Kiiviepzep M. ei ai.,..

And so on. Meiypoav, 282:159-167, 2003).

As disclosed in example 14 of this description, in an animal model of fibrotic kidney disease (unilateral ureteral obstruction of the SHO), it was determined that kidney disease in mice lacking the MABR-2 gene (MABR-2-/-) was significantly less severe compared to with wild-type control animals, as evidenced by inflammatory cell infiltrates (75-95 reduction) and histological markers of fibrosis such as collagen deposition (one-third reduction). Example 15 further shows that wild-type mice systemically injected with a monoclonal anti-MAB-2 antibody that selectively blocks the lectin pathway while leaving the classical pathway unaffected were protected from renal fibrosis compared to wild-type mice injected with an isotype control antibody . These results suggest that the lectin pathway is a key factor in causing kidney disease and once again demonstrate that a lectin pathway-blocking MA5P-2 inhibitor, such as an anti-MA5P-2 antibody, is effective as an antifibrotic agent. In Example 16, the protein overdose model further shows that wild-type mice receiving bovine serum albumin (B5A) developed proteinuric nephropathy, while MABR-2-/- mice receiving the same dose of VZA developed kidney damage to a lesser extent. Example 17 shows that wild-type mice systemically injected with a monoclonal anti-MA5R-2 antibody that selectively blocks the lectin pathway while leaving the classical pathway unaffected were protected from kidney damage in a protein overdose model. Example 18 shows that MAZR-2-/- mice exhibited renal inflammation and tubulointerstitial injury in an Adriamycin-induced nephrological model of renal fibrosis to a lesser extent compared to wild-type mice. Example 19 shows

Therefore, in the phase 2 open clinical study of kidney diseases, which is currently being conducted, patients with IDA nephropathy who were administered anti-MA5R-2 antibody had a clinically significant and statistically significant decrease in the ratio of albumin/creatinine in urine (SHASK) by the end of treatment ) throughout the study, as well as a decrease in the level of protein in the urine collected during the 24-hour period, compared to the initial level. Example 19 also shows that in the same phase 2 study in patients with membranous nephropathy who were administered anti-

MAB5BR-2 antibody, a decrease in TIME during treatment was also observed. Example 20 shows that in an ongoing phase 2 open-label renal study, at the end of treatment, 4 of 5 patients with lupus nephritis (LU) treated with anti-MA5R-2 antibody had a clinically significant decrease in protein levels at 24- hourly urine collection relative to the initial level.

According to the above, the present invention relates to the use of MA5BR-2 inhibitory agents, such as MA5BR-2 inhibitory antibodies, as antifibrotic agents, the use of inhibitory

MA5P-2 agents for the manufacture of a medicament for the treatment of a fibrotic condition and methods of preventing, treating, ameliorating, or ameliorating a fibrotic condition in a human in need thereof, wherein said method comprises administering to said patient an effective amount of an MA5P-2 inhibitory agent (e.g., an anti-MA5P -2 antibodies).

The methods of the invention may be used to prevent, treat, alleviate, or reverse a fibrotic condition in a human suffering from any disease or disorder caused or complicated by fibrosis and/or inflammation, including kidney disease (e.g., chronic kidney disease, IDA nephropathy , SZ-glomerulopathy and other glomerulonephritis), lungs (for example, idiopathic pulmonary fibrosis, cystic fibrosis, bronchiectasis), liver (for example, cirrhosis, non-alcoholic fatty liver disease), heart (for example, myocardial infarction, atrial fibrosis, valvular fibrosis, endomyocardial fibrosis ), brain (eg, stroke), skin (eg, excessive wound healing, scleroderma, systemic sclerosis, keloids), vascular system (eg, atherosclerotic vascular disease), bowel (eg, Crohn's disease), eyes (eg, anterior subcapsular cataract , opacification of the posterior capsule), soft tissue structures of the musculoskeletal system (for example, cellular capsulitis, Dupuytren's contracture, myelofibrosis), reproductive organs (for example, endometriosis, Peyronie's disease) and some infectious diseases (for example, alpha virus, because hepatitis C and hepatitis B).

I. THE ROLE OF MAB5BR-2 IN DISEASES AND CONDITIONS CAUSED OR COMPLICATED

FIBROSIS

Fibrosis is the formation or presence of an excess amount of connective tissue in an organ or tissue, usually formed in response to injury or damage.

A hallmark of fibrosis is the production of excess extracellular matrix after injury. In the kidneys, fibrosis is characterized as a progressive deleterious deposition of connective tissue on the kidney parenchyma, which inevitably leads to a decline in kidney function regardless of the primary kidney disease that causes their initial damage. The so-called epithelial-mesenchymal transition (EMT), a change in cellular characteristics in which tubular epithelial cells transform into mesenchymal fibroblasts, is the main mechanism of renal fibrosis. Fibrosis affects almost all tissues and organ systems and can occur as a repair or replacement response to a stimulus such as tissue injury or inflammation. The normal physiological response to injury leads to the accumulation of connective tissue, but if this process becomes pathological, the replacement of highly differentiated cells by scarring of the connective tissue alters the structure and function of the tissue. At the cellular level, epithelial cells and fibroblasts proliferate and differentiate into myofibroblasts, leading to matrix contraction, increased stiffness, microvascular compression, and hypoxia. There is currently no effective treatment or therapy for fibrosis, but both animal studies and anecdotal reports in humans suggest that fibrotic tissue lesions can be reversed (Tatre apa 7eisregud, Mai. Kemu. Merpgoi., Moi. 10: 226-237, 2014).

Many diseases are the result of fibrosis that causes the progression of organ failure, including diseases of the kidneys (eg, chronic kidney disease, IdA nephropathy, SZ glomerulopathy and other glomerulonephritis), lungs (eg, idiopathic pulmonary fibrosis, cystic fibrosis, bronchiectasis), liver (eg, cirrhosis, nonalcoholic fatty liver disease), heart (eg, myocardial infarction, atrial fibrosis, valvular fibrosis, endomyocardial fibrosis), brain (eg, stroke), skin (eg, excessive wound healing, scleroderma, systemic sclerosis, keloids) , vascular system (for example, atherosclerotic vascular disease), intestines (for example, Crohn's disease), eyes (for example, anterior

(e.g. subcapsular cataract, opacification of the posterior capsule), soft tissue structures of the musculoskeletal system (eg, cellular capsulitis, Dupuytren's contracture, myelofibrosis), reproductive organs (eg, endometriosis, Peyronie's disease) and some infectious diseases (eg, alpha -virus, hepatitis C and hepatitis B, etc.).

Although fibrosis occurs in many tissues and in many diseases, there are common molecular and cellular mechanisms in its pathology. Deposition of the extracellular matrix by fibroblasts is accompanied by infiltrates of immune cells, mainly mononuclear cells (see MUupp T., Mai. Keum. Ittipoi. 4(8):583-594, 2004, incorporated into this description by reference). An active inflammatory response leads to the expression of growth factors (TOEDV, MESBE, hepatocyte growth factor, connective tissue growth factor), cytokines and hormones (endothelin,

I. -4, 1-6, 1-13, chemokines), enzymes of destruction (elastase, matrix metalloproteinases, cathepsins) and proteins of the extracellular matrix (collagen, fibronectin, integrins).

In addition, the complement system becomes active in numerous fibrotic diseases.

Complement components, including the membrane attack complex, have been identified in numerous fibrotic tissue samples. For example, the components of the lectin pathway were detected in fibrous lesions of the kidneys (Zaotiga eyi a!., Mergpop., 92(3):702-4 (2002), Zayu evi a!., I iryv, 29(13):1378-86 (2011), i.e., Sipy. Ehr. Ittipoi., 174(1):152-60 (2013)); liver diseases (Kepzep ei ai., Nerayuiodu, 50(6):1809-17 (2009)); and lung disease (OiIezep ei ai., Siip.

And so on. 121(3):324-31 (2006)).

It was established that excessive activation of the complement system is one of the key factors in the development of immune complex-mediated, as well as antibody-independent, glomerulonephritis. However, there is convincing evidence that the uncontrolled activation of complement /l7 5yi is associated with the pathophysiological progression of TI fibrosis in non-glomerular disease (Skidd K.9., u. Ittipoi. 171:3319-3324, 2003, MaikK A. ei ai.,

Zetip Merpgoi. 33:575-585, 2013, Maiyegtp O.N. ei ai., Siip. U. At. wasps Merygoi 10:P1636-1650, 2015). Strong pro-inflammatory signals caused by local activation of complement can be initiated by complement components filtering into the proximal tubule and further into the interstitial space, or by abnormal synthesis of complement components by tubular or other resident and infiltrating cells, or by altered expression of complement regulatory proteins in kidney cells , or the absence or loss or increase of functional mutations in the regulatory components of complement

(Mainet O.V. ei ai., Sip. U. At. os. Merngoi. 10:P1636-1650, 2015, Zpeegip M.5. ei a!., EABEV .. 22:1065-1072, 2008). For example, in mice, a deficiency of the regulatory protein/gene (Sitgu) associated with the complement regulatory protein SC! leads to complement activation in the form of tubulointerstitial (TI) damage, followed by inflammation and fibrosis, characteristic of the damage observed in human TI diseases ( Maik A. ei ai., Zetip Mernpgoi. 33:575-585, 2013, Vao I. ei ai., 9). At. boss. Merpegoi 18:811-822, 2007). The effect of tubular epithelial cells on anaphylatoxin SZa leads to an epithelial-mesenchymal transition (Tvapa 7. eyi a. U. At. bos. MerpgoiYi. 20:593-603, 2009). Recently, it was shown that blocking CZa signaling only through the CZa receptor reduces TI fibrosis of the kidneys in animals with proteinuric and non-proteinuric diseases (Tzapa 2. ei ai., 9. At. os. Mernpgoi. 20:593-603, 2009, Vao I. ei ai., Kidpeu Ipi. 80:524-534, 2011).

As disclosed in this description, the authors of the invention have determined the central role of the lectin pathway in the initiation and progression of renal tubular pathology, which implies the key role of lectin pathway activation in the pathophysiology of a wide range of kidney diseases, including

IDA-nephropathy, SZ-glomerulopathy and other glomerulonephritis (Epao M. ei ai!., Merpgoi. Oiaiuviv

Tgapzriapi, 13:1984-1990, 1998; Nizapo 5. ei a!., At. U. Kidpeu Riv. 45:295-302, 2005; Booz A. eiyi aI., y. At. boss. Merngo!. 17:1724-1734, 2006; Pi | .Yi... ei ai., SiIip. Ex. And so on. 174:152-160, 2013;

Mrs. K. ei ai., Tkapzriapi Merngoi. Oivuviv, 14:881-886, 1999; RisKetgipod ei ai., Kidpeu Ipiegpaiopai, 84:1079-1089, 2013), diabetic nephropathy (Nomipa R. ei ai., Oiabese5, 54:1523-1527, 2005), ischemic reperfusion injury (Azdagi E. ei aI., REABEV u. 28:3996-4003, 2014) and transplant rejection (Vegdeg 5. R. ei ai., At. 9. Tgapzriapi. 5:1361-1366, 2005).

As further shown in this specification, the inventors have demonstrated that inhibition

MABR-2 reduces inflammation and fibrosis in mouse models of tubulointerstitial disease. Therefore, MA5R-2 inhibitory agents are expected to be useful in the treatment of renal fibrosis, including tubulointerstitial inflammation and fibrosis, proteinuria, IdA nephropathy, SZ glomerulopathy and other glomerulonephritis and ischemic reperfusion injury of the kidney.

Kidney diseases and disorders

According to the National Kidney Foundation, 26 million American adults are affected

With chronic kidney disease (CKD). Most patients have progressive disease leading to renal failure that requires treatment with erythropoiesis-stimulating drugs, dialysis, or kidney transplantation for survival. There are several drugs that can treat the main symptom of CKD, hypertension, but there are currently no drugs that address its root cause.

Studies have shown that progressive kidney damage is caused by capillary hypertension in the substructures of the kidney known as nephrons (MUpimogiy U.A., Appai!5. Asad. oi Mea., moyo. 34(1):2005). As the nephrons (filtering units of the kidney) are damaged or destroyed in this process, inflammation occurs and tissue scarring occurs, resulting in the nephrons being replaced by non-functional scar tissue. As a result, the kidney's ability to filter blood decreases over time. This is called renal fibrosis, which is a common process of progressive kidney disease. Regardless of the nature of the initial lesion, renal fibrosis is considered a common final process by which kidney disease progresses to end-stage renal disease. Attenuation of renal fibrosis can be determined by one or more of the following parameters: assessment of interstitial volume, IM collagen deposition, and/or levels of mRNA containing genes that control connective tissue growth. The compounds and methods described in this application are useful in the treatment of renal fibrosis.

Fibrosis and inflammation of the kidneys are characteristic signs of the late stage of kidney disease of almost any etiology (see Voog ei ai., Voog R. ei ai., U. oi At. bos. Merpgoiodu, 18:1508-1515, 2007; and Spemaiieg ei ai., Kidpeu Ipegaiiopai, 75:1145-1152, 2009). Renal failure can be caused by a heterogeneous group of disorders. Progressive kidney dysfunction leads to proteinuria and kidney failure. As the patient's health deteriorates, dialysis may be required simply to prevent kidney damage and multisystem failure. Over time, kidney damage and kidney failure can progress to end-stage renal disease (ESRD), which is complete or near-complete, irreversible loss of kidney function. Depending on the form of kidney disease, kidney function may be lost within days or weeks, or it may slowly and gradually deteriorate over decades. After the patient's disease progressed to

TSNN, dialysis (hemodialysis or peritoneal dialysis) is required to prevent death of the patient.

Patients must be maintained on some form of dialysis or given a kidney transplant.

Components of the lectin pathway were detected in fibrous lesions in kidney diseases (Zaiotiga ei ai., Mergpop., 92(3):702-4 (2002), Zayu ei a!., Hyryv, 20(13):1378-86 (2011) ), Qi ei ai., Sip. Ehr. Ittipoi., 174(1):152-60 (2013)). In IA nephropathy, patients with glomerular deposition of MVI had more pronounced proteinuria, reduced renal function, lower levels of serum albumin, more severe histology, and increased blood pressure compared to patients without MVI deposition. (I name ei ai., Siip Ehr Ittipoi. (1):1152-60). In patients with lupus nephritis (Za ei aI., Gyry5, 20(13):1378-86, 2011) and chronic renal failure (Zayotiga ei aiYi., Mergpop 92(3):702-4, 2002) elevated levels were also observed MVI and lectin pathway activity.

It was also demonstrated that C5 deficiency leads to a significant attenuation of the main components of renal fibrosis in a non-proteinuric model of primary tubulointerstitial lesion, namely unilateral ureteral obstruction (USO) (Voog R. ei ai., 9. oi At. Bob. oi

Merpgoiodu, 18:1508-1515, 2007). There is also a report of increased expression of the C3 gene in wild-type mice after SHO and a significant decrease in collagen deposits in C3-/- mice after SHO compared to wild-type mice, which indicates the role of complement activation in renal fibrosis (Geat et al., Moi . Ittipoi. 48:1666-1733, 2011: Arzigas). However, prior to the discovery by the authors of this invention described in this application, the complement components involved in renal fibrosis were not clearly defined. As described in Examples 14-17 of this application, the authors of this invention unexpectedly determined that MABR-2 deficiency or blockade with an MABR-2 inhibitory antibody that selectively blocks the lectin pathway without affecting the classical pathway clearly protects mice from renal fibrosis in various animal models. models of kidney disease.

Accordingly, in some embodiments, there is provided a method of inhibiting renal fibrosis in a subject suffering from a renal disease or disorder caused or complicated by fibrosis and/or inflammation, which comprises administering an MA5R-2 inhibitory agent, such as an anti-

MA5BR-2 antibody to a subject in need thereof. The method includes administering a composition that contains an amount of a MABR-2 inhibitor effective to inhibit renal fibrosis in a subject suffering from a kidney disease or disorder caused or complicated by fibrosis and/or inflammation.

The MABR-2 inhibiting composition can be administered locally in the area of fibrosis, for example

By local application of the composition during surgery or by local injection either directly into the area of fibrosis, or remotely, for example using a catheter.

Alternatively, the MABR-2 inhibitory agent can be administered to the subject systemically, such as intra-arterially, intravenously, intramuscularly, by inhalation, nasally, subcutaneously or by other means of parenteral administration, or, in the case of non-peptidergic agents, oral administration is possible. The introduction can be repeated, as determined by the doctor, until the condition is alleviated or until control over this condition is achieved.

In some embodiments, MAZR-2 inhibitory agents (eg, anti-MA5R-2 antibodies) are administered in combination with one or more agents or treatments appropriate for the underlying kidney disease or condition. In some embodiments, the implementation of inhibitory

MA5P-2 agents (for example, anti-MA5P-2 antibodies) are administered in combination with dialysis or plasmapheresis. In some embodiments, MAZR-2 inhibitory agents (eg, anti-MA5R-2 antibodies) are used to reduce the frequency of required dialysis or plasmapheresis. In some other embodiments, MAZR-2 inhibitory agents (eg, anti-MA5R-2 antibodies) are used in combination with kidney transplantation. In some other embodiments, MAZR-2 inhibitory agents (eg, anti-MA5R-2 antibodies) are used to control renal failure and prevent further deterioration of kidney function in patients awaiting kidney transplantation.

By way of example, in some embodiments, anti-MA5bR-2 antibodies are used to inhibit renal fibrosis and thereby treat or alleviate (including treating or alleviating disease symptoms) glomerular diseases such as focal segmental glomerulosclerosis and nephrotic syndrome. Examples of treatable symptoms include, but are not limited to, hypertension, proteinuria, hyperlipidemia, hematuria, and hypercholesterolemia.

In some embodiments, the MABR-2 inhibitory agent suppresses tubulointerstitial fibrosis. In some embodiments, the treatment includes improving kidney function, reducing proteinuria, improving hypertension, and/or reducing renal fibrosis. In some embodiments, the implementation of the treatment includes (i) delaying or preventing the progression of renal failure, fading of renal function, or the development of TSNN; (ii) withholding, reducing the frequency or eliminating the need for dialysis; or (ii) delaying or eliminating the need for a kidney transplant. for Some specific kidney diseases and disorders caused or complicated by fibrosis and/or inflammation are described below.

In some embodiments, the kidney disease caused or complicated by fibrosis and/or inflammation is a glomerular disease, such as focal segmental glomerulosclerosis (E5055). Glomerular diseases affect the glomeruli, allowing proteins and sometimes red blood cells to enter the urine. Sometimes glomerular disease also prevents the kidneys from cleaning waste products, so they begin to accumulate in the blood. Symptoms of glomerular disease include proteinuria, hematuria, decreased glomerular filtration rate, hypoproteinemia, and edema. Several different diseases can lead to the development of glomerular disease. This can be the direct result of an infection or drug that is toxic to the kidneys, or the result of a disease that affects the whole body, such as hypertension, diabetes, or lupus. TON is a glomerular disease, but even this particular condition characterized by scarring of the kidney can have multiple causes. In patients with TON, as a rule, end-stage renal failure is reached within 5-20 years, although patients with aggressive forms of the disease reach TON after 2-3 years.

In some embodiments, the kidney disease caused or complicated by fibrosis and/or inflammation is diabetic nephropathy (DN), which belongs to the field of medicine that requires immediate solutions. Diabetic nephropathy is kidney disease or kidney dysfunction that is complicated by diabetes. The condition is worsened by high blood pressure, high blood sugar, and high cholesterol and lipid levels. The exact cause of diabetic nephropathy is unknown. However, without being bound by a particular theory, it is believed that uncontrolled high blood sugar results in kidney damage such as fibrosis and tissue scarring. In humans, DN manifests as a clinical syndrome consisting of albuminuria, a gradually decreasing glomerular filtration rate (GFR) and an increased risk of cardiovascular disease. Diabetic albuminuria is associated with the development of characteristic histopathological features, including thickening of the glomerular basement membrane (GBM) and expansion of the mesangial matrix. As albuminuria and renal failure develop, glomerulosclerosis, arteriolar hyalinosis, and tubulointerstitial fibrosis develop.

Accordingly, in one embodiment, the present disclosure provides methods of treating diabetic nephropathy that include administering an effective amount of an inhibitory

MA5P-2 agent (eg, MAZP-2 inhibitory antibody) to a subject in need thereof. In some embodiments, the treatment includes reducing one or more symptoms of diabetic nephropathy. In some embodiments, the treatment includes reducing, slowing, or eliminating the need for dialysis. In some embodiments, the treatment includes reducing, delaying, or eliminating the need for a kidney transplant. In some embodiments, treatment includes delaying, preventing, or halting the progression of diabetic nephropathy to renal failure or end-stage renal disease.

In some embodiments, the kidney disease caused or complicated by fibrosis or inflammation is lupus nephritis. As detailed below, lupus nephritis, which is a serious complication of systemic lupus erythematosus (SLE), is another example of renal fibrosis that can be treated with MA5BR-2 inhibitory agents (eg, anti-MA5R-2 antibodies).

Accordingly, in one embodiment, the present disclosure includes methods of inhibiting renal fibrosis in a subject suffering from a renal disease or disorder caused or complicated by fibrosis and/or inflammation, which comprise administering an effective amount of an MABB-2 inhibitory agent (e.g., an MABB inhibitor -2 antibodies). In some embodiments, the renal disease or disorder complicated by fibrosis and/or inflammation is selected from the group consisting of chronic kidney disease, chronic renal failure, glomerular disease (e.g., focal segmental glomerulosclerosis), immune complex disorder (e.g., IDA nephropathy , membranous nephropathy), lupus nephritis, nephrotic syndrome, diabetic nephropathy, tubulointerstitial lesions and SZ-glomerulopathy or other types of glomerulonephritis.

Ways to prevent or treat kidney damage caused by drug-induced toxicity

Another cause of kidney damage includes drug-induced toxicity. For example, nephrotoxins can have a direct toxic effect on tubular epithelial cells.

As disclosed herein, the inventors have demonstrated that MA5R-2 deficient mice are protected from Adriamycin-induced nephropathy. because Nephrotoxins include, without limitation, therapeutic medicinal substances (eg,

cisplatin, gentamicin, cephaloridine, cyclosporine, amphotericin, adriamycin), radiocontrast dye, pesticides (eg, paraquat), and environmental pollutants (eg, trichlorethylene and dichloroacetylene). Other examples include puromycin aminonucleoside (RAM); aminoglycosides such as gentamicin; cephalosporins such as cephaloridine; calcineurin inhibitors such as tacrolimus or sirolimus. Drug-induced nephrotoxicity can also be caused by nonsteroidal anti-inflammatory drugs, antiretroviral drugs, anticytokines, immunosuppressants, oncological drugs, or ACE inhibitors. In addition, drug-induced nephrotoxicity can be caused by abuse of nalgesine, ciprofloxacin, clopidogrel, cocaine, soh-2 inhibitors, diuretics, foskamet, gold, ifosfamide, immunoglobulin, Chinese herbs, interferon, lithium, mannitol, mesalamine, mitomycin, nitrosourea, penicillamine , penicillin, pentamidine, quinine, rifampin, streptozocin, sulfonamides, ticlopidine, triamterene, valproic acid, doxorubicin, glycerin, cidofovir, tobramycin, neomycin sulfate, colistimethate, vancomycin, amikacin, cefotaxime, cisplatin, acyclovir, lithium, interleukin nom-2, cyclosporine or indinavir .

Accordingly, in one embodiment, a subject at risk of developing or suffering from kidney damage may receive one or more therapeutic drugs that exhibit nephrotoxicity. These subjects can be administered MA5P-2 inhibitors of the invention before or simultaneously with such therapeutic agents. Similarly, MABR-2 inhibitors can be administered after administration of a therapeutic agent to treat or reduce the likelihood of developing nephrotoxicity.

Diseases and conditions associated with proteinuria

It has been established that a violation of glomerular filtration of protein leads to proteinuria and accelerates the progressive loss of nephrons, which occurs in all chronic kidney diseases (Ketil7li apa Vegpapi, Mem Epd. U. Mei., moi. 339(20):11448-1456, 1998).

For example, in the study described by Eadu ei ai., At. IN). Heaven 135:719-33, 1989, glomerular filtration of albumin was invariably accompanied by the development of interstitial lesions and scarring. Edau et al., 1989, also noted that the deposition of complement CZ3 on

From the luminal surface of the proximal tubule was observed in rats with nephropathy caused by an overdose of protein, indicating that components of the complement system filtered in the glomeruli can cause interstitial damage. It was demonstrated that depletion of complement or lack of Sb alleviated tubulointerstitial damage in proteinuric animal models, such as mesangioproliferative glomerulonephritis, adriamycin nephropathy, 5/6 nephrectomy, and puromycin-aminonucleoside nephrosis (Voog et al., Ei et al., U. oi At. of Merpgoiodu: U9AZM, 18:11508-1515, 2007). Human studies have shown that proteinuria is an independent prognostic factor for the progression of chronic kidney disease, and that the reduction of proteinuria is renal protective (Kiddepepii R. yei a!., U. At. os. Merngoi. 23:1917-1928, 2012).

Accordingly, in one embodiment, the present invention relates to methods of preventing or reducing proteinuria and/or preventing or reducing kidney damage in a subject suffering from a disease or condition associated with proteinuria, which comprise administering an amount of an MA5P-2 inhibitory agent (e.g., MA5R-2 inhibitory antibody) effective for reducing or preventing proteinuria in a subject In one embodiment, the disease or condition associated with proteinuria is selected from the group consisting of nephrotic syndrome, preeclampsia, eclampsia, toxic kidney injury, amyloidosis, vascular-collagenous diseases (for example, systemic lupus erythematosus), dehydration, glomerular diseases (for example, membranous glomerulonephritis, focal segmental glomerulonephritis, C3-glomerulopathy, minimal change disease, lipoid nephrosis), intense physical activity, stress, benign orthostatic (postural) proteinuria , focal segmental glomerulosclerosis, IDA nephropathy (eg, Berger's disease), ICH nephropathy, membranoproliferative glomerulonephritis, membranous nephropathy, minimal change disease, sarcoidosis, Alport syndrome, diabetes mellitus (diabetic nephropathy), drug-induced toxicity (eg, NSAID-induced, nicotine, penicillamine, lithium carbonate, gold and other heavy metals, inhibitors

ACE inhibitors, antibiotics (eg Adriamycin) or opiates (eg heroin)); diseases

Fabry, infections (for example, HIV, syphilis, hepatitis A, B or C, post-streptococcal infection, urinary schistosomiasis); aminoaciduria, Fanconi syndrome, hypertensive nephrosclerosis, interstitial nephritis, sickle cell disease, hemoglobinuria, multiple myeloma, myoglobinuria, organ rejection (for example, kidney transplant rejection), Ebola hemorrhagic fever, nail patella syndrome, familial fever, HER R syndrome, systemic lupus erythematosus , Wegener's granulomatosis, rheumatoid arthritis, glycogen storage disease type 1, Goodpasture syndrome, Schönlein-Henoch purpura, urinary tract infection that has spread to the kidneys, Sjogren's syndrome, and postinfectious glomerulonephritis.

Liver disease

Fibrosis of the liver, also called hepatic fibrosis, is caused by the accumulation of scar tissue in the liver and is characteristic of most types of liver disease. The replacement of healthy liver tissue with scar tissue reduces the ability of the liver to function properly.

If the scarring condition is not treated, liver fibrosis can progress to cirrhosis and fulminant liver failure, a life-threatening condition. The main causes of liver fibrosis are alcohol abuse, chronic hepatitis C virus infection, nonalcoholic steatohepatitis, and hepatotoxicity (eg, drug-induced liver injury caused by acetaminophen or another drug).

Components of the lectin pathway were found in fibrotic lesions of the liver (Kepzep ei a!., Neraiuiodu, 50(6):1809-17 (2009)). For example, in nonalcoholic steatohepatitis (also known as fatty liver disease), the activation of proteins of the complement system is widespread, and their expression is associated with the severity of the disease (Kepzep ei ai., Neraf!odu, 50(6):1809-17 (2009) , and, in addition to deposits of SZ and SU, the accumulation of MVI was detected, which confirms the activation of the lectin pathway.

Accordingly, in some embodiments, there is provided a method of inhibiting hepatic fibrosis in a subject suffering from a liver disease or disorder caused or complicated by fibrosis and/or inflammation, which comprises administering to the subject in need thereof an inhibitory

MA5BR-2 agent, such as a MA5R-2 inhibitory antibody. The method includes administering a composition that contains an amount of a MA5BR-2 inhibitor effective to inhibit hepatic fibrosis in a subject suffering from a liver disease or disorder caused or complicated by fibrosis and/or inflammation.

The MABR-2 inhibitory composition can be administered locally to the area of fibrosis, for example by local application of the composition during surgery or by local injection or

From directly to the area of fibrosis, or remotely, for example, with the help of a catheter.

Alternatively, the MABR-2 inhibitory agent can be administered to the subject systemically, such as intra-arterially, intravenously, intramuscularly, by inhalation, nasally, subcutaneously or by other means of parenteral administration, or, in the case of non-peptidergic agents, oral administration is possible. Administration can be repeated, as determined by the doctor, until the condition is alleviated or until control is achieved.

In some embodiments, MABR-2 inhibitory agents (eg, MABR-2 inhibitory antibodies) are administered in combination with one or more agents or treatments that are appropriate for the underlying liver disease or condition.

In some embodiments, the liver disease or disorder caused or complicated by fibrosis and/or inflammation is selected from the group consisting of: cirrhosis, non-alcoholic fatty liver disease (steatohepatitis), liver fibrosis secondary to alcohol abuse, liver fibrosis secondary to acute or chronic hepatitis, biliary disease, and toxic liver injury (eg, hepatotoxicity from drug-induced liver injury caused by acetaminophen or another drug).

Lung disease

Pulmonary fibrosis is the formation or development of excess fibrous connective tissue in the lungs, where normal lung tissue is replaced by fibrous tissue. This scarring leads to lung stiffness and impaired lung structure and function.

In humans, pulmonary fibrosis is thought to result from repeated damage to the tissue inside and between the tiny air sacs (alveoli) in the lungs. In experimental studies, aspects of the human disease were reproduced in various animal models.

For example, a foreign agent such as bleomycin, fluorescein isothiocyanate, silicon oxide, or asbestos can be introduced into the animal's trachea (Spagaye-Keptapi et al., Apita! Moadei5 oi

Ryitopagu Ribgozi5. Meipodz Moi. Mea., 2005, 117:251-259).

Accordingly, in some embodiments, there is provided a method of inhibiting pulmonary fibrosis in a subject suffering from a pulmonary disease or disorder caused or complicated by fibrosis and/or inflammation, which comprises administering to the subject in need thereof an inhibitory

MA5BR-2 agent, such as a MA5BR-2 inhibitory antibody. This method includes the administration of a composition containing an amount of MABR-2 inhibitor effective for inhibiting pulmonary fibrosis,

reduction of fibrosis in the lungs and/or improvement of lung function. Improvement in lung function symptoms includes improvement in lung function and/or capacity, reduction in fatigue, and improvement in oxygen saturation.

In some embodiments, the present invention relates to a method of treating, inhibiting, preventing or ameliorating pulmonary fibrosis in a subject suffering from cystic fibrosis, which comprises administering to the subject in need thereof a MABR-2 inhibitory agent, such as a MABR5BR-2 inhibitor antibody.

The MABR-2 inhibitory composition can be administered locally to the area of fibrosis, for example by local application of the composition during surgery or by local injection either directly into the area of fibrosis, or remotely, for example by means of a catheter.

Alternatively, the MABR-2 inhibitory agent can be administered to the subject systemically, for example intra-arterially, intravenously, intramuscularly, by inhalation, nasally, subcutaneously or by other means of parenteral administration or, in the case of non-peptidergic agents, oral administration is possible. Administration can be repeated, as determined by the doctor, until the condition is alleviated or until control is achieved.

In some embodiments, MABR-2 inhibitory agents (eg, MABR-2 inhibitory antibodies) are administered in combination with one or more agents or treatments appropriate for the underlying lung disease or condition.

Some specific lung diseases and disorders caused or complicated by fibrosis and/or inflammation are described below.

In some embodiments, the lung disease caused or complicated by fibrosis or inflammation is chronic obstructive pulmonary disease (COPD). COPD is a disease in which the walls of the airways are fibrosed with an accumulation of myofibroblasts and collagen, and it is the leading cause of disability and the fourth leading cause of death in the United States. With COPD, the airflow is blocked and the patient's breathing becomes difficult. COPD is caused by damage to the respiratory tract, which ultimately prevents the exchange of oxygen and carbon dioxide in the lungs. COPD includes chronic obstructive bronchitis and emphysema, and often both. Patients with COPD, whose lungs are already damaged and lung function is already impaired, are at increased risk of complications related to

With bacterial and viral infections.

Accordingly, in one embodiment, the present invention relates to methods of treating chronic obstructive pulmonary disease (COPD) comprising administering to a subject in need thereof an amount of a MAZR-2 inhibitory agent (eg, an anti-MA5R-2 antibody) effective to suppression and/or reduction of lung fibrosis. In some embodiments, the treatment includes reducing one or more symptoms of COPD. Symptoms of COPD and/or pulmonary fibrosis include, but are not limited to, coughing up mucus, shortness of breath (dyspnea) that may worsen with moderate activity, fatigue, frequent respiratory infections, wheezing, chest tightness, irregular heartbeat (arrhythmia), need for ventilator apparatus and oxygen therapy, right-sided heart failure or pneumonia (swelling of the heart and heart failure due to chronic lung disease), pneumonia, pneumothorax, severe weight loss, and inadequate nutrition. Symptoms also include decreased lung function as assessed by testing using one or more standard lung function tests.

In some embodiments, the lung disease caused or complicated by fibrosis and/or inflammation is scleroderma-associated pulmonary fibrosis. As described in more detail below, pulmonary fibrosis associated with scleroderma is another example of pulmonary fibrosis that can be treated with MA5BR-2 inhibitors (eg, MABR-2 inhibitory antibodies).

In some embodiments, the lung disease or lung dysfunction caused or complicated by fibrosis and/or inflammation is selected from the group consisting of chronic obstructive pulmonary disease, cystic fibrosis, scleroderma-associated pulmonary fibrosis, bronchiectasis, and pulmonary hypertension.

Cardiovascular diseases

The cause of a number of different cardiovascular pathologies is a common fibrotic process. Excessive deposition of fibrous tissue in the heart leads to cardiac pathology, in which the production of extracellular matrix proteins changes the structure, architecture, and shape of the heart and affects its contractile function (Kpap apa zperraga, Ittipoiiodu, 118:10-24, 2006).

Research suggests that fibrosis may contribute significantly to cardiac dysfunction in ischemic, dilated, and hypertrophic cardiomyopathy. For example, it was shown that patients with chronic atrial fibrillation had higher levels of interstitial myocardial fibrosis compared to controls (Kpap apa Zperraga, Ittipoiiodu, 118:10-24, 2006).

As another example, it was established that in the majority of cases of arrhythmogenic right ventricular cardiomyopathy (RVCD) in the United States, fat infiltration and scarring (fibrofatty

AKPSH) (VygKe ei ai., Sigsshiatsop, 97:1571-1580, 1998). In a study that examined the histopathological characteristics of the ventricular myocardium in people with ACPH, it was found that extensive fibrosis was present in biopsy specimens from pediatric patients with ACPH (Mihpikama T. ei a!I., Sagdiomazsshag Raipoiodu, my. 8(4 ):185-189, 1999).

Accordingly, in some embodiments, there is provided a method of preventing, treating, stopping, inhibiting and/or reducing fibrosis and/or inflammation in a subject suffering from a cardiac or vascular disease or disorder caused or complicated by fibrosis or inflammation, which comprises administering a sub to a subject in need thereof, an MA5BR-2 inhibitory agent, such as an MA5BR-2 inhibitory antibody. This method includes administration of a composition that contains an amount of MABR-2 inhibitor effective to inhibit cardiac and/or vascular fibrosis and/or improve cardiac and/or vascular function.

In some embodiments, the present invention relates to a method of treating, inhibiting, preventing, or attenuating fibrosis in a subject suffering from valvular fibrosis, which comprises administering to the subject in need thereof an MA5R-2 inhibitory agent, such as an MABR-2 inhibitory antibody .

The MABR-2 inhibitory composition can be administered locally to the area of fibrosis, for example by local application of the composition during surgery or by local injection either directly into the area of fibrosis, or remotely, for example by means of a catheter.

Alternatively, the MABR-2 inhibitory agent can be administered to the subject systemically, such as intra-arterially, intravenously, intramuscularly, by inhalation, nasally, subcutaneously or by other means of parenteral administration, or, in the case of non-peptidergic agents, oral administration is possible. Administration can be repeated, as determined by the doctor, until the condition is alleviated or until control is achieved.

In some embodiments, MA5BR-2 inhibitory agents (eg, MABR-2 inhibitory antibodies) are administered in combination with one or more agents or treatments appropriate for the underlying cardiac or vascular disease or condition.

In some embodiments, the cardiac or vascular disease or disorder caused or complicated by fibrosis and/or inflammation is selected from the group consisting of: cardiac fibrosis, myocardial infarction, atrial fibrosis, endomyocardial fibrosis, arrhythmogenic right ventricular cardiomyopathy (RVCD), vascular disease, atherosclerotic vascular disease, vascular stenosis, restenosis, vasculitis, phlebitis, deep vein thrombosis and abdominal aortic aneurysm.

Chronic infectious diseases

Chronic infectious diseases such as hepatitis C and hepatitis B cause tissue inflammation and fibrosis, and high activity of the lectin pathway can be harmful. MA5BR-2 inhibitors can be useful in such diseases. For example, it was found that the levels

MVI and MA5R-1 are important prognostic parameters of the severity of liver fibrosis in hepatitis C virus infection (Vhom/p ei ai., Siip. Ehr. Ittipoi., 147(1):90-8, 2007, Zaadapau ei ai., Agar. 3 .

Savzygoepiegoi. 12(2):68-73, 2011; Zaeeai ei ai., Sip. Ex. And so on. 174(2):265-73, 2013). It was previously shown that MA5BR-1 is a powerful activator of MA5R-2 and the lectin pathway (Meduyegi et al.,

U. Vioi. Spet., 29:288(13):8922-34, 2013). Alphaviruses such as chikungunya virus and Ross virus

River, cause a strong inflammatory response in the host, leading to arthritis and myositis, and this pathology is mediated by MVI. and activation of the lectin pathway (Sypp ei ai., Rio5. Raipod. 8(3):e1002586, 2012).

Accordingly, in some embodiments, the disclosure provides a method of preventing, treating, stopping, inhibiting, and/or reducing fibrosis and/or inflammation in a subject suffering from or having previously suffered from a chronic infectious disease that causes inflammation and/or fibrosis, which comprises administering to a subject in need thereof, an MA5R-2 inhibiting agent, such as

MA5P-2 antibody.

The MABR-2 inhibitory composition can be administered locally to the area of fibrosis, for example by local application of the composition during surgery or by local injection either directly into the area of fibrosis, or remotely, for example by means of a catheter.

Alternatively, the MABR-2 inhibitory agent can be administered to the subject systemically, such as intra-arterially, intravenously, intramuscularly, by inhalation, nasally, subcutaneously or by other means of parenteral administration, or, in the case of non-peptidergic agents, oral administration is possible. The introduction can be repeated, as determined by the doctor, 60 times until the condition is alleviated or until control is achieved.

In some embodiments, MABR-2 inhibitory agents (eg, MABR-2 inhibitory antibodies) are administered in combination with one or more agents or treatments that are appropriate for the underlying chronic infectious disease.

In some embodiments, the chronic infectious disease that causes inflammation of mAbo fibrosis is selected from the group consisting of: alpha virus, hepatitis A, hepatitis B, hepatitis C, tuberculosis, HIV, and influenza.

Autoimmune diseases

Scleroderma is a chronic autoimmune disease characterized by fibrosis, vascular changes and autoantibodies. There are two main forms: limited systemic scleroderma and diffuse systemic scleroderma. Skin symptoms of limited systemic scleroderma affect the hands, shoulders and face. Patients with this form of scleroderma often have one or more of the following complications: calcinosis, Raynaud's phenomenon, esophageal dysfunction, sclerodactyly, and telangiectasia. Diffuse systemic scleroderma progresses rapidly and affects a large area of the skin and one or more internal organs, often the kidneys, esophagus, heart, and/or lungs.

Scleroderma affects small blood vessels, known as arterioles, in all organs.

Initially, the endothelial cells of the arterioles die as a result of apoptosis along with the smooth muscle cells. These cells are replaced by collagen and other fibrous material. Inflammatory cells, in particular СОр4и-- helper T-cells, penetrate into the arteriole and cause further damage.

Skin manifestations of scleroderma can be painful, lead to avoidance of use of the affected area (eg hands, fingers, toes, feet, etc.-) and can lead to disfigurement of such areas. Ulcers may appear on the skin, and such ulcers may be prone to infection or even gangrene. Ulcerated skin can be difficult to heal or slow to heal. Difficulty in healing skin ulcers can be especially difficult in patients with impaired blood circulation, for example with the phenomenon

Reynaud. In some embodiments, the methods of the present invention are used to treat scleroderma, such as skin symptoms of scleroderma. In some embodiments, the treatment of scleroderma includes the treatment of skin ulcers, such as digital ulcers. Administration of an MABR-2 inhibitory agent, such as an anti-MA5BR-2 antibody, is possible

To be used to reduce fibrotic and/or inflammatory symptoms of scleroderma in affected tissues and/or organs.

In addition to skin symptoms/manifestations, scleroderma can also affect the heart, kidneys, lungs, joints, and digestive tract. In some embodiments, treatment of scleroderma includes treatment of disease symptoms in any one or more of these tissues, such as by reducing fibrotic and/or inflammatory symptoms. Lung problems are among the most serious complications of scleroderma and are responsible for most of the deaths associated with the disease. The two predominant lung conditions associated with scleroderma are pulmonary fibrosis and pulmonary hypertension. A patient with lung involvement may have either or both of these conditions. Pulmonary fibrosis associated with scleroderma is one example of pulmonary fibrosis that can be treated with inhibitors

MA5BR-2. Scleroderma that affects the lungs causes scarring (pulmonary fibrosis). This type of pulmonary fibrosis occurs in approximately 70-95% of patients with scleroderma, although its progression is usually slow and symptoms vary widely among patients in terms of severity. In patients who have symptoms associated with pulmonary fibrosis, these symptoms include a dry cough, shortness of breath, and decreased exercise capacity.

Approximately 16 to 95 patients with some level of pulmonary fibrosis develop severe pulmonary fibrosis. In patients with severe pulmonary fibrosis, there is a significant decrease in lung function and alveolitis.

In some embodiments, the methods of the present invention are used to treat scleroderma, such as scleroderma-associated pulmonary fibrosis. Administration of MA5P-2 inhibitory agents, such as MA5P-2 inhibitory antibodies, can be used to reduce the fibrotic symptoms of scleroderma in the lungs. For example, these methods can be used to improve lung function and/or reduce the risk of death from scleroderma.

Patients with scleroderma can also often have kidney disease. Renal fibrosis associated with scleroderma is an example of renal fibrosis that can be treated by administration of MABR-2 inhibitory agents such as anti-MA5BR-2 antibodies. In some embodiments, the methods of the present invention are used to treat scleroderma, such as scleroderma-associated renal fibrosis. In one embodiment, administration of MABZR-2 inhibitory antibodies can be used to reduce the fibrous symptoms of scleroderma in the kidneys. For example, these methods can be used to improve kidney function, reduce protein in the urine, reduce the severity of arterial hypertension, or reduce the risk of kidney crisis, which can lead to fatal kidney failure.

Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disorder characterized by spontaneous B- and T-cell autoreactivity and multiorgan immune damage and can affect the skin, joints, kidneys, and other organs. Almost all people with SLE have joint pain and most develop arthritis. The joints of the fingers, hands, wrists and knees are often affected. Common symptoms of SLE include: arthritis; fatigue; general discomfort, anxiety or feeling unwell (malaise); joint pain and swelling; muscle pain; nausea and vomiting; and skin rash. In addition, symptoms may also include: abdominal pain; blood in the urine; discoloration of the fingers when squeezed or in the cold; numbness and tingling; and red spots on the skin. Some patients with SLE have lung or kidney involvement.

Without being limited to theory, inflammation and/or fibrosis in the lungs and kidneys leads to damage to these organs and the development of symptoms associated with lung and/or kidney damage. In some cases, patients with SLE develop a certain kidney condition called lupus nephritis.

In some embodiments, the present invention relates to methods of treating SLE that include the administration of an effective amount of an MABZR-2 inhibitory agent, such as an anti-MA5R-2 antibody.

Administration of MA5P-2 inhibitory antibodies can be used to reduce one or more symptoms of SLE. In some embodiments, administration of anti-MA5bR-2 antibodies is used to treat SLE in a patient with lupus nephritis. In such cases, treatment of SLE includes treatment of lupus nephritis, for example by reducing the symptoms of lupus nephritis. In some embodiments, the treatment comprises treating the skin symptoms of SLE. In some embodiments, the treatment includes reducing one or more symptoms of lupus nephritis. In some embodiments, the treatment includes reducing, slowing, or eliminating the need for dialysis. In some embodiments, the treatment includes reducing, delaying, or eliminating the need for a kidney transplant. In some embodiments, the treatment includes delaying or preventing the progression of lupus nephritis to renal failure or end-stage renal disease.

Lupus nephritis is an inflammation of the kidneys and a serious complication of systemic lupus erythematosus (SLE). As for the kidneys, lupus nephritis can lead to a disabling loss of their function. Patients with lupus nephritis may eventually develop kidney failure and may require dialysis or a kidney transplant. Related complications that can also be treated by the methods of the invention include interstitial nephritis and nephrotic syndrome. Symptoms of lupus nephritis include: blood in the urine, foamy urine, high blood pressure, protein in the urine, fluid retention, and swelling. Other symptoms include signs and symptoms of kidney fibrosis and/or kidney failure. If the disease is not treated, lupus nephritis can lead to kidney failure and even end-stage renal failure.

Accordingly, in some embodiments, there is provided a method of preventing, treating, stopping, inhibiting, and/or reducing fibrosis and/or inflammation in a subject suffering from an autoimmune disease that is caused or complicated by fibrosis and/or inflammation, which comprises administering to the subject , which requires a MABZR-2 inhibitory agent, such as a MABZR-2 inhibitory antibody. This method includes administering a composition that contains an amount of MA5P-2 inhibitor effective to inhibit fibrosis.

The MABR-2 inhibitory composition can be administered locally to the area of fibrosis, for example by local application of the composition during surgery or by local injection either directly into the area of fibrosis, or remotely, for example by means of a catheter.

Alternatively, the MABR-2 inhibitory agent can be administered to the subject systemically, such as intra-arterially, intravenously, intramuscularly, by inhalation, nasally, subcutaneously or by other means of parenteral administration, or, in the case of non-peptidergic agents, oral administration is possible. Administration can be repeated, as determined by the doctor, until the condition is alleviated or until control is achieved.

In some embodiments, MABR-2 inhibitory agents (eg, MA5BR-2 inhibitory antibodies) are administered in combination with one or more agents or treatments that are appropriate for the underlying autoimmune disease.

In some embodiments, the autoimmune disease caused or complicated by fibrosis and/or inflammation is selected from the group consisting of scleroderma and systemic lupus erythematosus (SLE). for Diseases and conditions of the central nervous system

In some embodiments, there is provided a method of preventing, treating, stopping, inhibiting and/or reducing fibrosis and/or inflammation in a subject suffering from a disease or disorder of the central nervous system caused or complicated by fibrosis and/or inflammation, which comprises administering to the subject a subject in need thereof, an MA5P-2 inhibitory agent, such as an anti-MA5P-2 antibody. This method includes administering a composition that contains an amount of MABR-2 inhibitor effective to inhibit fibrosis and/or inflammation.

The MABR-2 inhibitory composition can be administered locally to the area of fibrosis, for example by local application of the composition during surgery or by local injection either directly into the area of fibrosis, or remotely, for example by means of a catheter.

Alternatively, the MABR-2 inhibitory agent can be administered to the subject systemically, such as intra-arterially, intravenously, intramuscularly, by inhalation, nasally, subcutaneously or by other means of parenteral administration, or, in the case of non-peptidergic agents, oral administration is possible. Administration can be repeated, as determined by the doctor, until the condition is alleviated or until control is achieved.

In some embodiments, MAZP-2 inhibitory agents (eg, anti-MA5P-2 antibodies) are administered in combination with one or more agents or treatments that are appropriate for the underlying disease or disorder of the central nervous system.

In some embodiments, the disease or disorder of the central nervous system caused or complicated by fibrosis and/or inflammation is selected from the group consisting of: stroke, traumatic brain injury, and spinal cord injury.

Skin diseases or conditions

In some embodiments, there is provided a method of preventing, treating, stopping, inhibiting and/or reducing fibrosis and/or inflammation in a subject suffering from a skin disease or disorder caused or complicated by fibrosis and/or inflammation, which comprises administering to the subject, that needs it, an MAZR-2 inhibitory agent, such as an MAZR-2 inhibitory antibody. This method includes administering a composition that contains an amount of MA5bR-2 inhibitor effective to inhibit fibrosis and/or inflammation.

The MABR-2 inhibitory composition can be administered locally to the area of fibrosis, for example by topical application of the composition to the skin or topical application during surgery

By either local injection or directly, or remotely, for example by means of a catheter.

Alternatively, the MABR-2 inhibitory agent can be administered to the subject systemically, such as intra-arterially, intravenously, intramuscularly, by inhalation, nasally, subcutaneously or by other parenteral route of administration, by topical application, or, in the case of non-peptidergic agents, by oral administration. Administration can be repeated, as determined by the doctor, until the condition is alleviated or until control is achieved.

In some embodiments, MABR-2 inhibitory agents (eg, MABR-2 inhibitory antibodies) are administered in combination with one or more agents or treatments that are appropriate for the underlying skin disease or disorder.

In some embodiments, the skin disease or disorder caused or complicated by fibrosis and/or inflammation is selected from the group consisting of skin fibrosis, wound healing, scleroderma, systemic sclerosis, keloids, connective tissue diseases, scarring, and hypertrophic scars.

Diseases and conditions of bones and soft tissues of the musculoskeletal system

In some embodiments, there is provided a method of preventing, treating, stopping, inhibiting and/or reducing fibrosis and/or inflammation in a subject suffering from a bone or soft tissue disease or disorder caused or complicated by fibrosis and/or inflammation, which includes administering to a subject in need thereof an MA5R-2 inhibitory agent, such as an MA5BR-2 inhibitory antibody. This method includes administering a composition that contains an amount of MA5P-2 inhibitor effective to inhibit fibrosis and/or inflammation.

The MABR-2 inhibitory composition can be administered locally to the area of fibrosis, for example by topical application of the composition to the bone or soft tissue structure or topical application during surgery or local injection or directly, or remotely, for example via a catheter. Alternatively, the MABR-2 inhibitory agent can be administered to the subject systemically, such as intra-arterially, intravenously, intramuscularly, by inhalation, nasally, subcutaneously or by other parenteral route of administration, by topical application, or, in the case of non-peptidergic agents, by oral administration. Administration can be repeated, as determined by the doctor, until the condition is alleviated or until control is achieved.

In some embodiments, MABR-2 inhibitory agents (eg, MABR-2 inhibitory antibodies) are administered in combination with one or more agents or treatments that are appropriate for the underlying bone or soft tissue disease or disorder.

In some embodiments, the bone or soft tissue disease or disorder caused or complicated by fibrosis and/or inflammation is selected from the group consisting of osteoporosis and/or osteopenia associated with, for example, cystic fibrosis, myelodysplastic conditions with increased bone fibrosis, adhesive capsulitis, Dupuytren's contracture and myelofibrosis.

Joint diseases or conditions

In some embodiments, there is provided a method of preventing, treating, stopping, inhibiting and/or reducing fibrosis and/or inflammation in a subject suffering from a joint disease or disorder caused or complicated by fibrosis and/or inflammation, which comprises administering to the subject, in need thereof, an MA5R-2 inhibiting agent, such as an inhibitor

MABR-2 antibody. This method includes administering a composition that contains an amount of an inhibitor

MA5P-2, which is effective in suppressing fibrosis and/or inflammation.

The MABR-2 inhibitory composition can be administered locally to the area of fibrosis, for example by local application of the composition to the joint, or local application during surgery or local injection or directly, or remotely, for example by means of a catheter.

Alternatively, the MABR-2 inhibitory agent can be administered to the subject systemically, such as intra-arterially, intravenously, intramuscularly, by inhalation, nasally, subcutaneously or by other parenteral route of administration, by topical application, or, in the case of non-peptidergic agents, by oral administration. Administration can be repeated, as determined by the doctor, until the condition is alleviated or until control is achieved.

In some embodiments, MAZP-2 inhibitory agents (eg, anti-MA5P-2 antibodies) are administered in combination with one or more agents or treatments that are appropriate for the underlying joint disease or disorder.

In some embodiments, the joint disease or disorder caused or complicated by fibrosis and/or inflammation is arthrofibrosis.

Diseases or conditions of the digestive system

In some embodiments, there is provided a method of preventing, treating, stopping, inhibiting and/or reducing fibrosis and/or inflammation in a subject suffering from a disease or disorder of the digestive system caused or complicated by fibrosis and/or inflammation that

20 includes administering to a subject in need thereof an MA5R-2 inhibitory agent, such as

MABR-2 antibody. This method includes administering a composition that contains an amount of an inhibitor

MA5P-2, which is effective in suppressing fibrosis and/or inflammation.

The MABR-2 inhibitory composition can be administered locally to the area of fibrosis, for example by local application of the composition during surgery or by local injection or directly, or remotely, for example by means of a catheter. Alternatively, the MABR-2 inhibitory agent can be administered to the subject systemically, such as intra-arterially, intravenously, intramuscularly, by inhalation, nasally, subcutaneously or by other parenteral route of administration, by topical application, or, in the case of non-peptidergic agents, by oral administration. Administration can be repeated, as determined by the doctor, until the condition is alleviated or until control is achieved.

In some embodiments, MAZP-2 inhibitory agents (eg, anti-MA5P-2 antibodies) are administered in combination with one or more agents or treatments that are appropriate for the underlying disease or disorder of the digestive system.

In some embodiments, the digestive disease or disorder caused or complicated by fibrosis and/or inflammation is selected from the group consisting of Crohn's disease, ulcerative colitis, and pancreatic fibrosis.

Eye diseases or conditions

In some embodiments, there is provided a method of preventing, treating, stopping, inhibiting and/or reducing fibrosis and/or inflammation in a subject suffering from an ocular disease or disorder caused or complicated by fibrosis and/or inflammation, which comprises administering to the subject , which requires a MA5R-2 inhibitory agent, such as an inhibitor

MABR-2 antibody. This method includes administering a composition that contains an amount of an inhibitor

MA5P-2, which is effective in suppressing fibrosis and/or inflammation.

The MABR-2 inhibitory composition can be administered locally to the area of fibrosis, for example 55 by local application of the composition during surgery or by local injection or directly, or remotely, for example by means of a catheter. Alternatively, the MABR-2 inhibitory agent can be administered to a subject systemically, such as intra-arterially, intravenously, intramuscularly, by inhalation, nasally, subcutaneously or by other parenteral route of administration, by topical application to the eye (e.g., eye drops), or, in in the case of 60 non-peptidergic agents, oral administration is possible. Administration can be repeated, as determined by the doctor, until the condition is alleviated or until control is achieved.

In some embodiments, MABR-2 inhibitory agents (eg, MABR-2 inhibitory antibodies) are administered in combination with one or more agents or treatments that are appropriate for the underlying ocular disease or disorder.

In some embodiments, the ocular disease or disorder caused or complicated by fibrosis and/or inflammation is selected from the group consisting of anterior subcapsular cataract, posterior capsule opacification, macular degeneration, and retinal and vitreous retinopathy.

Diseases or conditions of the reproductive organs

In some embodiments, there is provided a method of preventing, treating, stopping, inhibiting and/or reducing fibrosis and/or inflammation in a subject suffering from a reproductive organ disease or disorder caused or complicated by fibrosis and/or inflammation, which comprises administering to the subject , which requires an MA5R-2 inhibitory agent, such as an MA5BR-2 inhibitory antibody. This method includes administering a composition that contains an amount of MA5P-2 inhibitor effective to inhibit fibrosis and/or inflammation.

The MABR-2 inhibitory composition can be administered locally to the area of fibrosis, for example by local application of the composition during surgery or by local injection or directly, or remotely, for example by means of a catheter. Alternatively, the MABR-2 inhibitory agent can be administered to the subject systemically, such as intra-arterially, intravenously, intramuscularly, by inhalation, nasally, subcutaneously or by other parenteral route of administration, by topical application, or, in the case of non-peptidergic agents, by oral administration. Administration can be repeated, as determined by the doctor, until the condition is alleviated or until control is achieved.

In some embodiments, MABR-2 inhibitory agents (eg, MABR-2 inhibitory antibodies) are administered in combination with one or more agents or treatments that are appropriate for the underlying reproductive disease or disorder.

In some embodiments, the reproductive disease or disorder caused or complicated by fibrosis and/or inflammation is selected from the group consisting of: endometriosis and Peyronie's disease.

From Trauma-related scarring

In some embodiments, there is provided a method of preventing, treating, stopping, inhibiting and/or reducing fibrosis and/or inflammation in a subject suffering from a disease or condition resulting from trauma-related scarring, which comprises administering to the subject , which requires a MA5BR-2 inhibitory agent, such as a MAB5BR-2 inhibitory antibody.

This method includes administering a composition that contains an amount of MABR-2 inhibitor effective to inhibit fibrosis and/or inflammation.

The MABR-2 inhibitory composition can be administered locally to the area of fibrosis, for example by local application of the composition during surgery or by local injection or directly, or remotely, for example by means of a catheter. Alternatively, the MABR-2 inhibitory agent can be administered to the subject systemically, such as intra-arterially, intravenously, intramuscularly, by inhalation, nasally, subcutaneously or by other parenteral route of administration, by topical application, or, in the case of non-peptidergic agents, by oral administration. Administration can be repeated, as determined by the doctor, until the condition is alleviated or until control is achieved.

In some embodiments, MABR-2 inhibitory agents (eg, MABR-2 inhibitory antibodies) are administered in combination with one or more agents or treatments that are appropriate for the underlying disease or disorder.

In some embodiments, the trauma-related scarring is selected from the group consisting of: surgical complications (eg, surgical adhesions, in which scar tissue can form between internal organs, causing contracture, pain, and can lead to infertility); chemotherapy drug-induced fibrosis, radiation-induced fibrosis and scarring associated with burns.

Additional diseases and disorders caused or complicated by fibrosis and/or inflammation

In some embodiments, there is provided a method of preventing, treating, stopping, inhibiting and/or reducing fibrosis and/or inflammation in a subject suffering from a disease or disorder caused and/or complicated by fibrosis and/or inflammation selected from the group consisting of of organ transplantation, breast fibrosis, muscle fibrosis, retroperitoneal fibrosis, thyroid fibrosis, lymph node fibrosis, bladder fibrosis and pleural fibrosis, which comprises administering to a subject in need thereof an MA5R-2 inhibiting agent such as as an inhibitory MA5P-2 antibody. This method includes administering a composition that contains an amount of MABR-2 inhibitor effective to inhibit fibrosis and/or inflammation.

The MABR-2 inhibitory composition can be administered locally to the area of fibrosis, for example by local application of the composition during surgery or by local injection or directly, or remotely, for example by means of a catheter. Alternatively, the MABR-2 inhibitory agent can be administered to a subject systemically, such as intra-arterially, intravenously, intramuscularly, by inhalation, nasally, subcutaneously or by other parenteral route of administration, by topical application to the eye (e.g., eye drops), or, in in the case of non-peptidergic agents, oral administration is possible. Administration can be repeated, as determined by the doctor, until the condition is alleviated or until control is achieved.

In some embodiments, MA5BR-2 inhibitory agents (eg, MABR-2 inhibitory antibodies) are administered in combination with one or more agents or treatments that are appropriate for the underlying disease or disorder.

In some embodiments of any of the various methods and pharmaceutical compositions described herein, the MA5R-2 inhibitory antibody selectively inhibits the lectin pathway without affecting the classical pathway.

In various aspects, the present invention relates to methods of inhibiting the adverse effects of fibrosis and/or inflammation, which include administering an MA5R-2 inhibitory agent to a subject in need thereof. MA5P-2 inhibitory agents are administered in an amount effective to inhibit MA5P-2-dependent complement activation in a living subject. In practicing this aspect of the invention, typical MABR-2 inhibitory agents include: molecules that inhibit the biological activity of MABR-2 (such as small molecule inhibitors, anti-MA5BR-2 antibodies (eg, inhibitory MA5R-2 antibodies) or blocking peptides that interact with MAB5R-2 or disrupt the protein-biliuin interaction) and molecules that reduce the expression of MA5BR-2 (such as MABR-2 antisense nucleic acid molecules, MABR-2-specific RNAi molecules and MA5BR-2 ribozymes), thereby preventing MA5BR -2-activation of the complement lectin pathway.

MAZR-2 inhibitory agents can be used alone as primary therapy or in combination with other therapeutic agents as adjuvant therapy to enhance the therapeutic effect of other medicinal products.

ZO Suppression of MABR-2-dependent complement activation is characterized by at least one of the following changes in the component of the complement system, which occur as a result of the introduction of an MA5βR-2 inhibitory agent according to the methods of the invention: suppression of the generation or production of products of MA5Р-2-dependent activation of complement C4B, СЗа, Сба and/or С5Б5-9 (МАС) (measured, for example, as described in example 2), a decrease in the level of cleavage of Са and deposition of С4р (measured, for example, as described in example 2) or a decrease in the level of cleavage of СЗЗ and deposition of СЗБ (measured, for example, as described in example 2).

In accordance with this invention, MA5P-2 inhibitory agents are used that are effective in suppressing fibrosis and/or inflammation and exhibit detectable antifibrotic activity and/or induce a reduction in fibrosis. In the context of the invention, antifibrotic activity may include at least one or more of the following: (1) reducing inflammation, for example, as assessed by the activation and recruitment of macrophages and endothelial cells; recruitment and activation of lymphocytes and/or eosinophils by secretion of a number of cytokines/chemokines; release of cytotoxic mediators and fibrogenic cytokines; (2) decrease in cell proliferation, ECM synthesis (ECM) or angiogenesis; and/or (3) a reduction in the level of collagen deposition compared to fibrotic activity in the absence of an MAZP-2 inhibitory agent.

Evaluation of an antifibrotic agent, such as an MA5bR-2 inhibitory agent, can be performed by detection by any method known to one of ordinary skill in the art. For example, the evaluation of an antifibrotic agent can be carried out using the SHO model (as described in this application in examples 12 and 14). If the estimated anti-fibrotic activity and/or the reduction or reduction of fibrosis is evaluated with the MAZR-2 inhibitory agent, the MA5BR-2 inhibitory agent is considered to be used as a drug to prevent, treat, stop and/or inhibit fibrosis.

Assessment of fibrosis can be done periodically, for example weekly or monthly. Thus, the increase/decrease of fibrosis and/or the presence of antifibrotic activity can be assessed periodically, for example weekly or monthly. This assessment is preferably performed at multiple time points for a given subject or at one or more time points for a given subject and a healthy control. Evaluation can be done at regular intervals, such as weekly or monthly. Therefore, the assessment can be carried out regularly, for example weekly or monthly. If the evaluation reveals a reduction in fibrosis or the presence of antifibrotic activity, the MA5R-2 inhibitory agent, such as an MAZR-2 inhibitory antibody, is considered to exhibit detectable antifibrotic activity and/or induce a reduction or decrease in fibrosis.

MABR-2 inhibitory agents, in the case of practical application of this aspect of the invention, include, for example, MA5BR-2 antibodies and fragments thereof, MABR-2 inhibitory peptides, small molecules, soluble MAZR-2 receptors and expression inhibitors. MABZR-2 inhibitory agents can suppress the system of MA5bR-2-dependent complement activation, blocking the biological function

MA5R-2. For example, an inhibitory agent can effectively block MA5P-2 protein-protein interaction, prevent MA5PR-2 dimerization or assembly, block Ca binding, interfere with the active site of the MA5P-2 serine protease, or reduce MA5P-2 protein expression.

In some embodiments, MABR-2 inhibitory agents selectively inhibit MA5R-2-dependent complement activation without affecting the functional activity of the C1d-dependent complement activation system.

In one embodiment, the MA5P-2 inhibitory agent used in the methods of the invention is a specific MA5P-2 inhibitory agent that specifically binds to a ZE IU MO:6-containing polypeptide with a binding affinity of at least 10 times higher than the affinity of binding to other antigens in the complement system. In another embodiment, the inhibitory MA5P-2 agent specifically binds to the polypeptide containing ZE IU MO:6 with a binding affinity that is at least 100 times greater than the binding affinity to other antigens in the complement system. In one embodiment, the MA5BR-2 inhibitory agent specifically binds to at least one of the domains

SSRI-SSR2 (aa 300-431 in ZEO IU MO:6) or the MA5R-2 serine protease domain (aa 445-682 in

ZBO IU MO:6b) and inhibits MA5R-2-dependent complement activation. In one of the variants, the MA5ZR-2 inhibitory agent is a MA5BR-2 monoclonal antibody or its fragment that specifically binds to MA5R-2. The binding affinity of an MAZP-2 inhibitory agent can be determined using a suitable binding assay.

Polypeptide MA5R-2 has a molecular structure similar to the structure of MABR-1, MAB5R-3 and C1g and C15, proteases of the C1 complement system. The cDNA molecule presented in 5EO IU MO:4 encodes a typical example of MA5P-2 (consisting of the amino acid sequence presented in zHEO

IO MO:5) and provides a human MA5R-2 polypeptide with a leader sequence (aa 1-15) that is cleaved after secretion to form the mature form of human MABR-2 (5EO IU MO:6). As shown in fig. 2, the human MA5P-2 gene contains twelve exons. The cDNA of human MA5P-2 is encoded by exons B, C, O, EE, 0, H, I, y, K and M. As a result of alternative splicing, a protein of 20 kDa is formed, called MVI-associated protein 19 (MAp19, also called 5MAP) (ZEO IU MO:2), coded (5EO IU MO), obtained from exons B, C, O and E, as shown in fig. 2. The cDNA molecule presented in 5EO IU MO:50 encodes mouse MA5P-2 (consisting of the amino acid sequence presented in 5EO IUO MO:51) and provides a mouse MA5P-2 polypeptide with a leader sequence, as a result of which cleavage after secretion the mature form of murine MABR-2 (5EBEO IU MO:52) is formed. The cDNA molecule presented in 5EBEO I MO:53 encodes rat MABR-2 (consisting of the amino acid sequence presented in 5EO IU MO:54) and provides a rat MAZR-2 polypeptide with a leader sequence, the cleavage of which after secretion produces a mature form of rat MAZR-2 (ZEO I MO:55).

Those skilled in the art will understand that the sequences disclosed in ZEO IU MO:4, 5ZEO IU

MO:50 and BEO IU MO:53 are single alleles of human, mouse and rat MA5R-2, respectively, and that allelic variants and alternative splicing are possible. Allelic variants of the nucleotide sequences given in ZEO IU MO:4, 5EBEO IU MO:50 and 5EBO IO MO:53, including those containing silencing mutations and those in which mutations lead to changes in the amino acid sequence, are within the limits of this . Allelic variants of the MABR-2 sequence can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures.

Domains of the human MABR-2 protein (ZEO IU MO:6) are shown in fig. 1 and 2A and include domains

M-terminal C1g/C15//SE of sea urchin/bone morphogenetic protein (BMP) domain (aa 1-121 in ZEO IO MO:6), epidermal growth factor-like domain (aa 122-166), second domain of BMP (aa 167-293), as well as tandem domains of complement regulatory proteins.

Alternative splicing of the MA5BR-2 gene leads to the formation of MAp19, shown in fig. 1.

MAp19 is a non-enzymatic protein containing the M-terminal SOBI-ESE region of MABR-2 with four additional residues (EOBI) derived from exon E shown in fig. 1.

It was shown that some proteins are able to bind or interact with MA5BR-2 through 60 protein-protein interactions. For example, it is known that MA5BR-2 is able to bind and form

Sag-dependent complexes with MV lectin proteins, H-ficolin and I-ficolin. It was shown that each MAZR-2/lectin protein complex is capable of activating complement by

MA5BR-2-dependent cleavage of C4 and C2 proteins (IKeda K. ei ai., 9. Vioi. Speth. 262:7451-7454, 1987; Maizivnya M. ei a!., U. Ehr. Med. 176:1497- 2284, 2000; Maizivpya M. ei ai., 9. Ittipoi. 168:3502-3506, 2002). Studies have shown that the SOVI-ESE domains of MABR-2 are important for the binding of MABR-2 to MVI (Tpiviyep5 MM ei ai., U. Ittipoi. 166:5068, 2001). It was also shown that the SOVIESESIVI domains mediate the dimerization of MABR-2, which is necessary for the formation of an active MVI complex (UMaiiv5 V. ei ai., Vioi. Spet. 275:30962-30969, 2000).

It follows that MABR-2 inhibitory agents are characterized by their ability to bind or interact with MA5R-2 target sites that play an important role in the process of MABR-2-dependent complement activation.

ANTI-MAB5BR-2 ANTIBODIES

In some embodiments of this aspect of the present invention, the MA5R-2 inhibitory agent includes an anti-MA5bR-2 antibody that inhibits the MAZR-2-dependent complement activation system.

Anti-MA5BR-2 antibodies suitable for carrying out this aspect of the present invention include polyclonal, monoclonal or recombinant antibodies derived from any antibody-producing mammal, and these antibodies can be multispecific, chimeric, humanized, anti-idiotypic antibodies and antibody fragments. Antibody fragments include Rab, Rar', E(abr)g, K(ar)", HEm-fragments, csEm-fragments and single-chain antibodies, described below.

Anti-MABR-2 antibodies can be screened for the ability to suppress the system

MABR-2-dependent complement activation and antifibrotic activity and/or inhibition of renal damage associated with proteinuria or Adriamycin-induced nephropathy, using the assay described in this application. A number of anti-MA5R-2 antibodies have been described in the literature, several of which are listed in Table 1. For example, as shown in the present application in Examples 10 and 11, anti-MA5R-2 antibodies Gar2 have been found to block MA5ZR-2- dependent complement activation. As shown in example 12 and described in M/O 2012/151481, incorporated herein by reference, fully human anti-MABR-2 antibodies 5cEm (eg, OM5646) were determined to block MA5ZR-2-dependent complement activation. As described in example 13, as well as in M/O 024141442, which is incorporated herein by reference, anti-MABR-2 antibodies carrying the 5OMI-2 peptide and fragments thereof exhibiting inhibitory

MAB5BR-2 activity, were obtained by fusion with the amino acid sequence of the peptide

ZOMI-2 (ZEO IO MO:72, 73 or 74) at the amino or carboxyl end of the heavy and/or light chain of a human anti-MA5R-2 antibody (eg, OM5646-512:3MI-2).

Accordingly, in one embodiment, the MA5BR-2 inhibitory agent for use in the methods of the invention comprises a human antibody, such as, for example, OM5646. Accordingly, in one of the variants, the MABR-2 inhibitory agent for use in the compositions and methods according to the claimed invention contains a human antibody that binds a polypeptide consisting of human MAZR-2 (ZEO IU MO:6), and the antibody contains: (I) (a) variable section of the heavy chain, containing: i) SOK-NI of the heavy chain, containing the amino acid sequence from aa 31-35 in 5EBO IU MO:67; and ii) SOC-H2 heavy chain containing the amino acid sequence from aa 50-65 in 5EO IU MO:67; and ii) SOC-NZ of a heavy chain containing the amino acid sequence from aa 95-107 in ZEO IU MO:67; and (5) a variable region of the light chain containing: Her) SOK-Y1 light chain containing the amino acid sequence from aa 24-34 in 5EO IO MO:70; and her) SOK-1 2 of the light chain, containing the amino acid sequence from aa 50-56 in 5BO IU MO:70; and her) SOMK-IZ of the light chain, containing the amino acid sequence from aa 89-97 in ZEO IU MO:70; or (II) a variant thereof comprising a heavy chain variable region with at least 9095 identity (eg, with at least 91 95, at least 92 95, at least 93 95, at least 94 9565, at least 9595, at least 9695, at least 9795, at least 9895, at least 99 95 identity) with 5EO IU MO:67; and a light chain variable region with at least 90 95 identity (eg, with at least 9195, at least 9295, at least 93 965, at least 94 95, at least 95 95, at least 96 95, at least 97 965, at least 98 96, at least 99 95 identity) with BZEO IU MO:70.

In some embodiments, the method includes administering to the subject a composition that contains an amount of an MABR-2 inhibitory antibody or an antigen-binding fragment thereof that contains a heavy chain variable region that contains the amino acid sequence represented in 5EO IU MO: b7, and a variable the region of the light chain, which contains the amino acid sequence presented in ZEO IU MO:70. 60 In some embodiments, the method includes administering to the subject a composition that contains a MA5R-2 inhibitory antibody or an antigen-binding fragment thereof that specifically recognizes at least a portion of an epitope on human MA5R-2 recognized by a reference antibody

OM5646, containing the variable region of the heavy chain as specified in ZEO IU MO:67 and the variable region of the light chain as specified in 5EO IU MO:70. In one embodiment, the MABR-2 inhibitory agent used in the methods of the invention contains the human antibody OM5646.

TABLE 1

MA5ZR-2-SPECIFIC ANTIBODIES ARE GIVEN AS AN EXAMPLE

And so on! 37:803-811, 2000

Fragment of recombinant Moi Ieg-Kgibiyepzep M.., ei ai., 9. oi human SSR1/2-5R Rat MoAB (subclass IdS1) Ittipoi. Meiyoav, 282:159-167,

Mod 885 2003

MAM | Moieg-Ktisiepzep M., ei ai., u. oi (cross-reaction with Rat MoAB (subclass IdS1) will go Meipod5, 282:159-167,

MABR-2

Mouse MoAB (M-term.) Ittipoi!. 35:409, Arius! 1998

Rat MoAbB: Mitoar101, cell line 03050904 (ESASS)

Mouse MoAbz:

MitoAB104 produced by a hybridoma cell line

MOo545UM035 (054М2)

MitoABIt08, produced by . - by a hybridoma cell line (full-size, | //545УМО29 (О5М7) UO 2004/106384 iv-labeled).

MitoOoAb109 is produced by a hybridoma cell line

МмОо545УМ046 (05472)

MitoAbI110 produced by the hybridoma cell line mmO545u5M048 (05M2 (full-length) ERarg

ANTI-MABR-2 ANTIBODIES WITH REDUCED EFFECTOR FUNCTION

In some embodiments of this aspect of the present invention, anti-MA5P-2 antibodies have reduced effector function to reduce inflammation that may occur upon activation of the classical complement pathway. It was found that the ability of molecules to trigger the classical pathway of complement activation is related to the Es region of the molecule (YuOipsap A.K. ei ai., Maishge, 332:738-740 1988). Ids molecules, in which the E-region was removed by enzymatic cleavage, are deprived of this effector function (see Nagpom/, Apiirodiev: A Iarogayugu

Mapiai!, Soii Zrgipd Nagrog IGaroghaiogu, Mem; ZhMogK, 1988). According to this, antibodies with reduced effector function can be obtained as a result of removing the Es region of a molecule containing a genetically engineered Es sequence that has minimal effector function, this molecule can belong to both the human Ids2 isotype and the Id isotype .

Antibodies with reduced effector function can be produced by standard molecular biological manipulations of the E region of Ids heavy chains, as described in this application and shown in UoiIiTe eii ai., ps! Chem. And so on. 10:241-250, 11993, and Koadgidtse5 ei ai., U. Ittipoi. 151:6954-6961, 1998. Antibodies with reduced effector function also include human isotypes Ids2 and Id4, which have a reduced ability to activate complement or interact with E-receptors (Kameishi UM. ei ai., Appi. Chem. Ettipoi. 9 :457-492, 1991; Izsaasz U.O. ei ai., U. Ittipoi. 148:3062-3071, 1992; map de M/ipKkei U.sh. ei ai., Ittipoi. Todau, 14:215-221 , 1993). Humanized or fully human antibodies specific for human

MABR-2, including isotypes IdD52 or Id054, can be obtained by one of several methods known to a specialist in this technical field, as described in Matsdpap T.9. ей ай., Маййге

Journal of Physiology, 16:535-539, 1998.

OBTAINING ANTI-MA5BR-2 ANTIBODIES

Anti-MABR-2 antibodies can be obtained using MABR-2 polypeptides (for example, full-length MABR-2) or antigenic peptides carrying the MA5ZR-2 epitope (for example, a part of the MAZR-2 polypeptide). Immunogenic peptides can consist of only five amino acid residues. For example, the MA5P-2 polypeptide containing the complete amino acid sequence 5EO IU MO:b can be used to induce anti-MA5P-2 antibodies that can be used in the method of the present invention. It is known that specific domains of MAB5bR-2 involved in protein-protein interactions, such as the SIOVI and SOVIESE domains, as well as the region containing the active site of the serine protease, can be expressed as recombinant polypeptides as described in Example 3 and used as antigens In addition, peptides that contain a region consisting of at least 6 amino acids of the MABR-2 polypeptide (5EO I

MO:6), can also be used to induce MA5BR-2 antibodies. Additional examples of produced MA5P-2 antigens capable of inducing MA5P-2 antibodies are listed below in Table 2. MA5P-2 peptides and polypeptides used to generate antibodies can be isolated as natural polypeptides or recombinant or synthetic peptides and catalytically inactive. recombinant polypeptides, such as MA5P-2A, as described further in this application. In some embodiments of this aspect of the present invention, anti-MA5P-2 antibodies are produced using a transgenic line of mice as described below in this application.

Antigens capable of producing anti-MA5R-2 antibodies also include fusion polypeptides, such as polypeptides obtained by fusing MA5R-2 or its part with an immunoglobulin polypeptide or a maltose-binding protein. Polypeptide immunogen can be represented by a full-sized molecule or its part. If a portion of the polypeptide is represented by a hapten, such portion may preferably be attached or linked for immunization to a macromolecular carrier (such as fissurella hemocyanin (CIN), bovine serum albumin (B5A) or tetanus toxoid).

TABLE 2

MA5BR-2 5EO AND MO ANTIGENS:

ZEO IU MO:b6 Human MA5R-2 protein

ZEO IU MO:51 Mouse MA5R-2 protein, SIiIVI domain of human MABR-2

FAILURE OF MOV aa 1-121 in BEO IYU MO:6), Domains of the SOVIET human MA5R-2

FAILURE of the Ministry of Health aa 1-166 in the ZEO IU of the Ministry of Health:6), Domains of the SOVIET SESSION of the human MABR-2

ZO Yur MOLO aa 1-293 in BEO IYU MO:6), ESE domain of human MABR-2

FAILURE MO aa 122-166 in 5EO IU MO:6), Serine protease domain of human MA5R-2

ZEO about Mol aa 429-671 in 5BEO IYU MO:6

EO IU MOH protease form with an inactivated serine oblique domain of AUNI. aa 610-625 in 5EO IO MO:6 with mutant Zeg 618)

ZEO IU MO: 14 p.m

THREE SRKM/RERMESVI. Human SOVI peptide

ZEO IU MO:15:

TARROMNI AND METNEOI EI. Human SOVI peptide

ZNI SEMOBGMHKI 55Y(; 1AKMI ATI SSO

ZEO IO MO:16: ; . .

TEVEPUEM MVI is a binding site in the human SOVI domain

TABLE 2

MA5BR-2 ANTIGENS

ZEO and MO:18

AMMI SAaGESSSKOZSnOrzasatsiAi mm

POLYCLONAL ANTIBODIES

Polyclonal anti-MABR-2 antibodies can be obtained by immunizing animals with the MA5BR-2 polypeptide or its immunogenic region by methods well known to a person skilled in the art. See, for example, Sgeep ei ai., Rgodisiop oi Roiiusiopa! Apiizega, v

Ititipospetisa! RgoiosoYi5 (Mapzop, ea.), p. 105. The immunogenicity of the MABR-2 polypeptide can be increased with the help of an adjuvant, including mineral gels such as aluminum hydroxide or Freund's adjuvant (complete or incomplete), surfactants such as lysolecithin, high molecular weight alcohols - pluronics, polyanions, oil emulsions, fissurela hemocyanin and dinitrophenol. Animals such as horses, cows, dogs, chickens, rats, mice, rabbits, guinea pigs, goats or sheep are usually used to produce polyclonal antibodies. Alternatively, the anti-MA5R-2 antibody used in the present invention may also be derived from a great ape. The general method of obtaining diagnostically and therapeutically useful antibodies in baboons can be found, for example, in SoiYidepregud ei ai., publication of the international patent application UMO 91/11465, and in Go5tap Mei ai., Ipi. U. Sapseg, 46:310, 1990.

Then, using standard well-known procedures, serum containing immunologically active antibodies is obtained from the blood of such immunized animals.

MONOCLONAL ANTIBODIES

In some embodiments, the MA5BR-2 inhibitory agent is a monoclonal anti-

MABR-2 antibody. Monoclonal anti-MA5BR-2 antibodies are highly specific epitope directed against a single epitope of MA5R-2. As used herein, the modifier "monoclonal" indicates the nature of the antibody obtained from an essentially homogeneous population of antibodies, and does not imply the production of antibodies by any particular method. Monoclonal antibodies can be obtained by any method that provides for the production of antibody molecules in the culture of a stable cell line, such as, for example, the hybridoma method described in Kopieg O. ei aiYi,, Maishige, 256:495, 1975, or they can be obtained by recombinant DNA methods (see, for example, US patent No. 4,816,567, Sariju). Monoclonal antibodies can also be isolated from a phage library of antibodies by the methods described in Siaskzop T. ei a!., Makighe, 352:624-628, 1991, and Magkz 9.0. ei ai., U. Moi. Vioi 222:581-597, 1991. Similar antibodies may belong to

From any class of immunoglobulins, including Ids, IM, IDE, IdA, to, and any of their subclasses.

For example, monoclonal antibodies can be obtained by injecting a suitable mammal (for example, a mouse of the VAI.V/c line) a composition that contains the MABZR-2 polypeptide or its part. After a predetermined period of time, splenocytes are isolated from the mouse and resuspended in a culture medium. Then fusion of splenocytes with an immortalized cell line is performed to form a hybridoma. The resulting hybridomas are grown in cell culture and screened for the ability to produce monoclonal antibodies to

MABR-2. Further in this description, examples of obtaining monoclonal anti-MA5R-2 antibodies are given (see also Sigtepi Rgoiosoi5 ip Ittipoioudu, Moi. 1, dhopp Umieu 5 5op5, radez 2.5.1-2.6.7, 1991).

Human monoclonal antibodies can be obtained from transgenic mice genetically engineered to produce human antibodies in response to an antigenic stimulus. In this method, elements of the human immunoglobulin heavy and light chain loci are introduced into mouse lines derived from embryonic stem cell lines that contain targeted disruptions of the endogenous immunoglobulin heavy and light chain loci. Transgenic mice can synthesize human antibodies specific for human antigens such as antigens

MABR-2 described in the present invention and these mice can be used to generate hybridomas secreting antibodies to human MA5R-2 by fusing B cells obtained from such animals with suitable myeloma cell lines by the conventional method of Koehler-

Milstein, as described below in this application. Transgenic mice with the human immunoglobulin genome are commercially available (eg, from Abdepih, Ips., Egetopi, SA, and Medageh,

Ips., Appapaaiye, M.)U.). Methods of obtaining human antibodies from a transgenic mouse are described in the scientific literature, for example, Step 1.Y. Yei aYi., Mayte Sepei, 7:13, 1994; And Opregu M. ei ai.,

Maishge, 368:856, 1994; and Tauiog I.O. ей ай., Ипи. Etc. 6:579, 1994.

Monoclonal antibodies can be isolated and purified from the hybridoma culture by various well-known methods. Such separation methods include affinity chromatography on sepharose with protein A, size exclusion chromatography, and ion exchange chromatography (see, for example, Soyidap pp. 2.7.1-2.7.12 and pp. 2.9.1-2.9.3; Vaipev5 ei ai., " Riniiisaiop oi Ittipodiobiip S (du, v

Meiposd3z ip MoiIesshciag Vioiodu, Te Nytapa Rgez5, Ips., Moi. 10, p. 79-104, 1992).

After receiving polyclonal, monoclonal antibodies or antibodies produced by phages, they are first tested for specific binding to MABR-2. A number of different methods known to a person skilled in the art can be used to determine antibodies that specifically bind to MABR-2. Illustrative examples of assays performed by standard methods include Western blot or immunoprecipitation assays (e.g., as described in

Ah! Yezire! ei ai), immunoelectrophoresis, enzyme-linked immunosorbent assay (ELISA), dot blotting, inhibition assay, or competition assay (as described in Nagyom/ and I apa, Apiiibodieye: A I abrogafgu

Mapia!i, Soii Zrgipd Nagrog Garogaiogu Rgez55, 1988). After identifying antibodies capable of specifically binding to MABR-2, the anti-MABR-2 antibodies are tested for the ability to function as a MABR-2 inhibitory agent by one of several assay methods, such as, for example, a C4 lectin-specific cleavage assay (described in example 2), SZB deposition analysis (described in example 2) or C46B deposition analysis (described in example 2).

The binding affinity of anti-MA5R-2 monoclonal antibodies can be easily determined by a person skilled in the art (see, for example, 5saispaga A., MU Asai. z5si. 51:660-672, 1949).

In one embodiment, the monoclonal anti-MA5BR-2 antibodies used in the methods of the present invention bind to MA5R-2 with a binding affinity of 100 nmol, preferably 10 nmol and more preferably 2 nmol.

CHIMERIC/HUMANIZED ANTIBODIES

Monoclonal antibodies used in the method of the present invention include chimeric antibodies in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies that are derived from a particular species or belong to a particular class or subclass of antibodies, while the other part of the chain (chains) is identical or homologous to the corresponding sequences of antibodies that are isolated from another species or belong to another class or subclass of antibodies, as well as fragments of such antibodies (patent

US Mo 4,816,567, Saryu; and Moitizop 5.I... ei ai., Rhos. Magician! Asad 5 OBA, 81:6851-6855, 1984).

One of the forms of the chimeric antibody used in the present invention is represented by a humanized monoclonal anti-MABR-2 antibody. Humanized forms of non-human (eg, murine) antibodies are represented by chimeric antibodies that contain a minimal sequence isolated from a non-human immunoglobulin. Humanized monoclonal antibodies are made by transferring non-human (eg, murine) complementarity-determining regions (CDOs) from the variable regions of the heavy and light chains of the mouse immunoglobulin to the human variable domain. Typically, remnants of human antibodies are then substituted into the framework regions of the non-human prototypes. Moreover, humanized antibodies may contain residues that are not detected in the recipient's antibodies or in the donor's antibodies. These modifications are carried out in order to further improve the effectiveness of antibodies. In general, a humanized antibody may contain substantially all of at least one and typically two variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the E-framework regions are regions of the sequence human immunoglobulin. A humanized antibody may also contain at least part of the constant region of an immunoglobulin (EC), usually a human immunoglobulin. A detailed description can be found in ChShkopez ei ai., Mayige, 321:522-525, 1986; Neisptapp ei a!., Maishige, 332:323-329, 1988; Rgezia, St. Or. Bigisi Bio!., 2:593-596, 1992.

Humanized antibodies used in the present invention include human monoclonal antibodies that include at least the MA5BR-2-binding site of the SOCNZ. In addition, Pc sites can be replaced in such a way that IdA or IdM as well as human db antibodies are produced. Thus, humanized antibodies will find, in particular, clinical application, since they will specifically recognize human MA5P-2, without causing an immune response in a person against the antibody itself. As a result, they are better suited for intravenous administration to humans, especially when repeated or prolonged administration is required.

An example of obtaining a humanized anti-MA5R-2 antibody from a mouse monoclonal 60 anti-MABR-2 antibody is given in example 6. Methods of obtaining humanized monoclonal antibodies are also described, for example, in Shdope5 R.T. ei ai., Mayihe, 321:522, 1986;

Sapeg R. ei ai!., Rgos. Magician. Asad si. BOTH, 89:4285, 1992; Zapapy ..5., Stii. Wow Vioyesn. 12:437, 1992; Bipdeg I.I. ei ai., U. Ittip. 150:2844, 1993; Bytsanig (unit), Apiibodu Epdipeegipud Rgoioso!v,

Nytapa Rgezz, Ips., 1995; Keiieu, "Epidipeegipd Tnegareshis Apiirodiev", ip Rgoieip Epdipeegipd:

Rgipsirieh apa Rgasiise, Siveiapa ei ai. (widow), UMUPeu doip in 5Bopv, Ips., radez5 399-434, 1996; and in U.S. Patent No. 5,693,762, Occup, 1997. In addition, there are commercial organizations that synthesize humanized antibodies from specific regions of the antibody, such as Antibodies

Miem, SA).

RECOMBINANT ANTIBODIES

Anti-MAB5BR-2 antibodies can also be obtained using recombinant methods.

For example, human antibodies can be produced using a human immunoglobulin expression library (available, for example, in Bigayadepe, Sogr., I a doPa, CA) to produce human antibody fragments (MN, Mi, Em, Ba, Rab or P(ab) 2). These fragments are then used to construct fully human antibodies by methods similar to those used to produce chimeric antibodies.

ANTIIDIOTYPIC ANTIBODIES

After identifying anti-MA5P-2 antibodies with the desired inhibitory activity, these antibodies can be used to generate anti-idiotypic antibodies that are similar to a portion of MA5P-2 by methods well known in the art. See, for example, Sgeepzrap M.5. Yei AI., EABEV 9. 17:437, 1993. For example, antibodies that bind to MA5R-2 and competitively inhibit MA5R-2-protein interactions necessary for complement activation can be used to produce anti-idiotypic antibodies that have similarity with the MVI-binding site of the MA5R-2 protein and, therefore, bind and neutralize the binding of MA5R-2 ligands, such as, for example, MVI.

IMMUNOGLOBULIN FRAGMENTS

MABR-2 inhibitory agents used in the method of the invention include not only intact immunoglobulin molecules, but also well-known fragments, including Rab, Rab", E(ab)",

Kab)" and Em-fragments, csEm-fragments, dimers, linear antibodies, single-chain antibody molecules and multispecific antibodies formed from antibody fragments.

It is well known that only a small part of the antibody molecule, the paratope, is involved in the binding of the antibody to its epitope (see, for example, Siagk M/.K., Te Ehregitepai! Roipaaiop5 oi Modet Ittipoidou, UMieu 5 Bop5, Ips., Moscow State University, 1986). Sections of pEs and Es antibodies are effectors of the classical pathway of complement activation, but do not participate in antigen binding.

An antibody from which the pEs region has been enzymatically cleaved or that was produced without the pEs region is labeled with an E(ab)g fragment and contains both antigen-binding regions of an intact antibody. The isolated fragment E(ab)" belongs to the bivalent monoclonal fragment due to the presence of two antigen-binding sites. Similarly, an antibody from which the Es region has been enzymatically cleaved or that has been produced without the Es region is labeled

Eab fragment and contains one of the antigen-binding regions of the intact antibody molecule.

Fragments of antibodies can be obtained by proteolytic hydrolysis, such as pepsin or papain hydrolysis of whole antibodies, by standard methods. For example, antibody fragments can be obtained by enzymatic digestion of antibodies with pepsin to obtain fragment 55, designated K(ab)". This fragment can be further cleaved with a thiol-reducing agent to yield 3.55 Pa' of monovalent fragments.

Optionally, the cleavage reaction can be carried out using a group blocking sulfhydryl groups obtained as a result of cleavage of disulfide bonds. Alternatively, enzymatic digestion with pepsin yields two monovalent Rab fragments and an Es fragment. These methods are described, for example, in US Pat. No. 4,331,647,

Soideprego; Mibzopoiiyi A. eyi ai., Agsp. Viospet. Viorpuz. 89230, 1960; VV Ropeg, Viospet. .. 73:119, 1959; Edeitap ei ai!., in Meypod5 ip Eplutoiodo 1:422, Asadetis Rgez5, 1967; and in Soiidap, p. 2.8.1-2.8.10 and 2.10.-2.10.4.

In some embodiments, the use of antibody fragments with a missing Es region is preferable because it avoids the activation of the classical complement pathway, which is initiated by Es binding to the Es receptor. There are several ways of production

MoAr, which does not affect the interaction of the Esu receptor. For example, the E region of a monoclonal antibody can be removed chemically by partial cleavage with the participation of proteolytic enzymes (for example, ficin cleavage), with the production, for example, of antigen-binding fragments of antibodies, such as fragments of Rab or K(ab)" (Maiapi M .

MY. And so on. 28:69-71, 1991). Alternatively, the y4 isotype of human IDS, which does not bind to Esu receptors, can be used to create a humanized antibody, as described in this application. Antibodies, single-chain antibodies and antigen-binding domains that lack

E-domain can also be created by the recombinant methods described in this application.

FRAGMENTS OF SINGLE-CHAIN ANTIBODIES

Alternatively, it is possible to create MABR-2-specific molecules that bind one polypeptide chain, in which the Em regions of the heavy and light chains are linked together. Em-fragments can be connected with the help of a peptide linker to form a single-chain antigen-binding protein (5SEm). Such single-chain antigen-binding proteins are obtained by creating a structural gene containing DNA sequences encoding MN and Mi domains bound to an oligonucleotide. The structural gene is inserted into an expression vector, which is then introduced into a host cell, such as a BE. soybeans

Recombinant host cells synthesize a single polypeptide chain with a peptide linker connecting two M-domains. Methods of obtaining 5sem are described, for example, in MUpyShomu ei a!., "Meshod5: A Sotrapiop io Meipodvz ip Epgutoiodu", 2:97, 1991; Viga ei a!., 5siepse, 242:423, 1988; US patent Mo 4,946,7 78, I adpeg; RaskK R. ei a!i., Vio/TesppoiYodu, 11:1271, 1993.

As an illustrative example, MABZR-2-specific 5cEm can be obtained by exposing lymphocytes to MA5R-2 polypeptide and selecting antibody libraries by the phage display method in phage or similar vectors (for example, by using immobilized or tagged proteins or peptides of MABZ-2) . Genes encoding polypeptides containing potential domains that bind MA5P-2 polypeptides can be obtained by screening any peptide libraries displayed on phage or bacteria such as E. soya. These random peptide phage libraries can be used to screen for peptides that interact with MAZP-2.

Methods for generating and screening such random peptide display libraries are well known (see US Pat. No. 5,223,409, Agapeg I; US Pat. Mo. 4,946,778, Agapeg I; US Pat. Moe 5,403,484, Agapeg I; US Pat. Moe 5,571,698, Agapeg I; and Kau et al. .,, Rpade bizriau oireriide5 apa

Rgoivip5 Asadetis Rgev5, Ips., 1996), and random peptide display libraries and sets for screening such libraries are commercially available, for example, from SI OMTESN I abogayutgiev, Ips. (Rayo Ayu, Saiy.), Ipiygodep Ips. (Zap Oiedo, Saiyi.), Mem/ Epdiapa Vioiarv, Ips. (Irem/isp, Ma55.) and

Rpaptasia I KV Viotesppoiodu Ips. (Rizsaiamau, M.U.).

Another form of anti-MABR-2 antibody fragment used in this aspect of the present invention is a peptide encoding a single complementarity determining region (CDR) that

Zo binds to the epitope on the MA5P-2 antigen and inhibits MABR-2-dependent complement activation.

SOC peptides ("minimum recognition units") can be obtained by engineering genes encoding the SOC antibody of interest. Such genes are obtained, for example, by the method of polymerase chain reaction for the synthesis of a variable region from the RNA of an antibody-producing cell (see, for example, I aggisk ei aiI., Meipodv5: A Sotrapiop oyu Meinosdz ip Epgutoiodu 2:106, 1991; Soshpepau-I isK , "Sepeiis Mapirshiaiop oi Moposiopa! Apiiroadiev", v

Moposiopai! Apiirodiev: Rhodisip, Epaipeehipd apa Siipisa! Ariisatiop, Vice-President of the Republic. (Edv.), council 166,

Satrygidde Opimegeyu Rgevzv5, 1995; and UMaga ei ai., "Sepeiis Mapirshianiop apa Ehrgevviop oi

Apiirodiev", in Moposiopai! Apiirodiev: Rgipsirie5 apa Arriisaiop5, Viksy eyi a!. (ed5.), rade 137, UMiIeu-

I iv5, Ips., 1995).

The anti-MA5BR-2 antibodies described in this application are administered to a subject in need to inhibit MA5R-2-dependent complement activation. In some embodiments, the inhibitory implementation

MA5BR-2 agent is a human or humanized monoclonal anti-MAB5R-2 antibody with reduced effector function.

PEPTIDE INHIBITORS

In some embodiments of this aspect of the present invention, the MAZP-2 inhibitory agent contains isolated peptide inhibitors of MA5P-2, including isolated natural peptide inhibitors and synthetic peptide inhibitors that inhibit the MAZP-2-dependent complement activation system.

As used herein, the term "isolated MA5R-2 peptide inhibitors" refers to peptides that inhibit MA5R-2-dependent complement activation by binding to MA5PR-2, competing with MA5R-2 for binding to another recognition molecule ( for example, MVIG., H-ficolin, M-ficolin or I-ficolin) upon activation of the lectin pathway and/or by direct interaction with MA5BR-2, thus inhibiting MA5P-2-dependent complement activation, while these inhibitors are essentially clean and practically do not contain other substances with which they can be in natural conditions, so much so that they are suitable for use for their intended purpose.

Peptide inhibitors are successfully used intravenously to inhibit protein-protein interactions and the catalytic site. For example, peptide inhibitors of adhesion molecules, structurally related to GEA-1, have recently been approved for clinical use in coagulopathy (Optap E.M. Yei aI., Yeigorvap Neap -. 16:50-55, 1995). Short linear bo peptides (30 amino acids) have been shown to have the ability to prevent or inhibit integrin-dependent adhesion

(Migawata 0. ei ai.,, 9. Viospet. 120:445-51, 1996). Longer peptides, from 25 to 200 amino acid residues, have also been successfully used to block integrin-dependent adhesion (Pope G. ei ai., 9. Vioi. Spet. 271(47):29953-57, 1996). Generally, longer peptide inhibitors have higher affinity and/or lower dissociation rates than shorter peptides and, therefore, may be more effective inhibitors. It was also shown that cyclic peptide inhibitors are effective integrin inhibitors in the treatment of human inflammatory diseases (Daskhop O.U. ei ai., U. Med. Spet. 40:3359-68, 1997). One method of obtaining cyclic peptides involves the synthesis of peptides in which the terminal amino acids of the peptide are cysteines, which allows the peptide to exist in a cyclic form due to the formation of disulfide bonds between the terminal amino acids, which has been shown to improve binding affinity and half-life /l u//o in the treatment of hematopoietic neoplasias (for example, US patent Mo 6,649,592, I agvop).

SYNTHETIC PEPTIDE INHIBITORS MA5R-2

MABR-2 inhibitory peptides used in the methods of this aspect of the present invention are amino acid sequences that mimic target regions important for function

MA5BR-2. The sizes of the inhibitory peptides used in practice in the methods of the present invention range from about 5 amino acids to about 300 amino acids. Table C lists exemplary inhibitory peptides that may be used in the practice of this aspect of the present invention. A candidate MA5R-2 inhibitory peptide can be tested for its ability to function as a MA5R-2 inhibitory agent in one or more assays, including, for example, a C4 lectin-specific cleavage assay (described in Example 2) and a SZB deposition assay (described in Example 2 ).

In some embodiments, the MABR-2 inhibitory peptides are derived from MAZR-2 polypeptides and are selected from the full-length mature MABZR-2 protein (ZEO IU MO:6) or from a specific domain of the MA5R-2 protein, such as, for example, the SIVI domain (ZEO IU MO:8), SOVIECE domain (ZEO IU MO:9), ESE domain (5EO IO MO:11) and serine protease domain (5EO IO MO:12). As described earlier, it has been shown that regions of COSESSIVIA are required for dimerization and binding to MVI. (Tpivyep5 ei ai., zirga). In particular, in a study on the identification of a person carrying a homozygous substitution of Azr105 for СІu105, which leads to the loss of МА5Р-2 from the MVI complex, it was shown that

The peptide sequence TRKZOMM (5EO I MO:16) in the SOVI domain of MAZR-2 is involved in binding to MVI. (Ziepdaaga-Redegzep K. eyi ai., Mem Epdiapa U). Mea. 349:554-560, 2003).

In some embodiments, peptides inhibiting MABR-2 were obtained from lectin peptides that bind to MA5R-2 and participate in the activation of the lectin pathway of complement.

Several different lectins have been identified that participate in this pathway, including mannan-binding lectin (MVI), I-ficolin, M-ficolin, and H-ficolin (Ikeda K. ei ai., "VioiI. Spec. 262: 7451-7454, 1987; Maiziznia M. ei a!., U. Ehr. Med. 176:1497-2284, 2000; Maiziznia M. eiai.,".

And so on. 168:3502-3506, 2002). These lectins are present in serum as oligomers or homotrimeric subunits, each of which has M-terminal collagen-like fibers with hydrocarbon recognition domains. These different lectins have been shown to bind to MA5P-2, and the lectin/MmMA5P-2 complex activates complement through cleavage of C4 and C2 proteins. H-ficolin has an amino acid terminal region of 24 amino acids, a collagen-like domain with 11 repeats

Spiu-Haa-Uaa, neck domain of 12 amino acids and fibrinogen-like domain of 207 amino acids (Maizibpya M. ei ai., U. Ittipoi. 168:3502-3506, 2002). N-ficolin binds to SISMAs and agglutinates human erythrocytes coated with LPS obtained from 5. urpitigpit, 5. tippezoia and E. soy.

It was shown that H-ficolin binds to MABR-2 and MAb 19 and activates the lectin pathway of complement. /and. I -ficolin/P35 also binds to SISMAc and has been shown to form bonds with

MA5R-2 and MAr19 in human serum, and this complex activates the lectin pathway of complement (Maizikhpya M. ei ai., 9U. Ittipoi. 164:2281, 2000). Accordingly, the MABR-2 inhibitory peptides used in the present invention may contain a region consisting of at least 5 amino acids selected from the MVI protein (5EO IYUO MO:21), the H-ficolin protein (accession number in Seprapk

MM 173452), protein M-ficolin (accession number in Seprapk 000602) and protein I-ficolin (accession number Seprapk MM 015838).

More specifically, the scientists identified the MA5R-2 binding site in MVI, consisting of 12 triplets Siu-x-U "YAKO according to TC SEC ER OSY NI GO RUSSIA KIS ROS MOSS RBa 5О0СА

RKa oka roa ke" (ZEO IU MO:26), which are located between the hinge and neck areas in the C-terminal part of the collagen-like domain of the MVR (Umayiyev MK. ei ai., U. VioYi. Spet. 279:14065, 2004). This the MA5P-2 binding site is also highly conserved in human H-ficolin and human I-ficolin. A consensus binding site has been described that is present in all three lectin proteins, containing the amino acid sequence "OSK-X-SR" (ZEO I MO:22), where the letter "O" means hydroxyproline, and the letter "X" is a hydrophobic residue (Umayii5 ei ai.,

2004, v5irga). Accordingly, in some embodiments, the MABR-2 inhibitory peptides used in this aspect of the present invention are at least 6 amino acids in length and contain ZEO IU MO:22. It was shown that peptides obtained from MVI, which include the amino acid sequence "BI V OO SRO SKI SRO a" (5EO IO MO:24), bind to MAZR-2 ip myo (UMaiyy5, ei ai,, 2004, 5irga ). To enhance binding to MABR-2, peptides flanked by two triplets of SPO at each end can be synthesized ("YARO SPO SI B

SO aRO KI RO SOR OSR 0", 5EO IYUO MO:25) to increase the probability of the formation of triple helices detected in the native MVI protein. (as described in Umayyy5 M. ei ai., U. Vioi. Spet. 279:14065, 2004 ).

MA5R-2 inhibitory peptides can also be derived from human H-ficolin, which includes the sequence "SCHAO 250 SEC BAO SRO SRO SRO YAKM RK SBO 200" (ZEO IU MO:27), from the consensus MABR-2-binding site in N-ficolini. Also included are peptides derived from human I-ficolin, which includes the sequence "Я4СО СИ1О pAO OK SEA stM OKA SEN

SRO SRO KA PRO RM PSAO SES" (ZEO IU MO:28), from the consensus MAZR-2-binding site in I-ficolin.

MABR-2 inhibitory peptides can also be derived from the C4 cleavage site, such as "OVAGEIP RMAMTICAMAREI ME!" (ZEO IU MO:29), which is a C4 cleavage site associated with

C-terminal part of antithrombin PI (Siomeg 6.1. Yei ai., Moi. Ittipoi. 251261 (1988)).

TABLE C

EXAMPLES OF MAZR-2 INHIBITING PEPTIDES ARE GIVEN

ZEO Ir MO:22 rak-kh-aR, . . ,; mon pct Synthetic peptide, consensus binding site with a hydrophobic aminoxylot residue of aiv-sa o-aPO-SKI-aPO-a MABR-2

SROSRO formation of triple helices

ZEO Ir MO:26 ekravratkaEkKaEROOSI YOU. .

OSROSKISROSMOSRUZSSZOSRK | Lodsk MVR triplets 1-17 soka, aroako5

Human N-ficolin (Naiaka) kMmaRKkaEoaro

ZEO Ir MO:28

EVNaROSROSKASROSRMYAdOSsSEO

TABLE C

EXAMPLES OF MAZR-2 INHIBITING PEPTIDES ARE GIVEN

Bosch 00

KZ5AUMSTKI MSMO

ZEO IU MO:73

TSEROTTEKOKSMTonOSOaBOOKZA ZOMI-2M (short version, medium length)

USsTKkImUSsMO

Peptides derived from the C4 cleavage site, as well as other peptides inhibiting the MA5R-2 serine protease site, can be chemically modified so that they become irreversible protease inhibitors. For example, suitable modifications may include, without limitation, halomethyl ketones (Bg, CI, I, E) at the C-terminal end, A5p or Sim, or may be attached to functional side chains; haloacetyl (or other α-haloacetyl) groups on amino groups or other functional side chains; epoxide- or imine-containing groups on amino- or carboxy-terminal ends or functional side chains; or esters, imidates at amino- or carboxy-terminal ends or functional side chains. Such modifications can provide an advantage in the irreversible inhibition of the enzyme due to the covalent attachment of the peptide. This can lead to a decrease in the effective dose and/or a decrease in the frequency of administration of the peptide inhibitor.

In addition to the above-described inhibitory peptides, MABR-2 inhibitory peptides used in the method according to the present invention include peptides containing the MA5P-2-binding site of the SOCZ in the anti-MA5P-2 MoAB obtained by the method described in this application.

The sequence of regions of SOC for use in peptide synthesis can be determined by methods known in the field. The variable region of the heavy chain is a peptide, the length of which is usually between 100 and 150 amino acids. The variable region of the light chain is a peptide, the length of which is usually between 80 and 130 amino acids. SOC sequences within the variable regions of the heavy and light chains include sequences consisting of only about 3-25 amino acids that can be easily sequenced by one skilled in the art.

Those skilled in the art know that essentially homologous variants of the MA5P-2 inhibitory peptides described above will also exhibit MA5P-2 inhibitory activity. Illustrative options include, without limitation, peptides containing insertions, deletions, substitutions and/or additional amino acids at the carboxy-terminal or amino-terminal regions of the claimed peptides, and mixtures thereof. Accordingly, such homologous peptides having MAB5BR-2 inhibitory activity are also contemplated for use in the methods of the present invention. Described

Zo peptides may also include repetitive motifs and other modifications with conservative substitutions. Conservative variants are described in this application and include the replacement of one amino acid with another amino acid of similar charge, similar size, similar hydrophobicity, etc.

MABR-2 inhibitory peptides can be modified to increase solubility and/or obtain a maximum positive or negative charge to increase similarity with a segment in an intact protein. The derivative may or may not have the exact primary amino acid structure of the peptide disclosed herein, provided that the derivative remains functionally active in inhibiting MABR-2. Modifications may include replacement of an amino acid with one of the well-known twenty amino acids or any other amino acid, derivatized or substituted amino acid with additional desired characteristics such as resistance to enzymatic cleavage, or an O-amino acid, or replacement with another molecule or compound such as a carbohydrate, that mimics the natural conformation and function of an amino acid, amino acids or peptide; amino acid deletion; insertion of one of the twenty well-known amino acids or any other amino acid, derivatized or substituted amino acid with additional desired characteristics, such as resistance to enzymatic cleavage, or a U-amino acid, or replacement by another molecule or compound, such as a carbohydrate that mimics the natural conformation its function of amino acid, amino acids or peptide; or replacement with another molecule or compound, such as a carbohydrate or nucleic acid monomer, that mimics the native conformation, charge distribution, and function of the parent peptide. Peptides can also be modified by acetylation or amidation.

The synthesis of derivative inhibitory peptides can be based on known methods of peptide biosynthesis, carbohydrate biosynthesis, etc. As a starting point, the skilled person can use a suitable computer program to determine the conformation of the peptide of interest.

After determining the conformation of the peptide disclosed in this description, the expert, using modeling methods, can determine the type of substitutions that are necessary at one or another site to obtain a derivative that retains the basic conformation and charge distribution characteristic of the parent peptide, but which may have the characteristics , absent from the parent peptide or improved in comparison with the characteristics of the parent peptide. After identification of candidate molecule derivatives, these derivatives can be tested using the assays described herein to determine their ability to function as MA5P-2 inhibitory agents.

SCREENING OF MA5BR-2 INHIBITING PEPTIDES

Molecular modeling and rational molecular design methods can be used to obtain and screen peptides that mimic the molecular structure of the key MABR-2 binding sites and inhibit MABR-2-dependent complement activation. The molecular structures used for modeling include sites

SOC of anti-MA5R-2 monoclonal antibodies, as well as target regions known to be important for MA5R-2 function, including the region required for dimerization, the region involved in MV binding, and the serine protease active site as described above. Methods for identifying peptides that bind to a specific target are well known in the art.

For example, molecular imprinting can be used to help design macromolecular structures, such as peptides that bind to a specific molecule. See, for example, Zpea K.)., "Moiesshiag Itriipiipud oi Zupipeiis Meimogk Roiutetv: Te Oe Momo zupipeviv oi Masgotoiiiesciag Vipaipu apa Saia|uiis 5pev", TAIR, 2:(5), 1994.

As an illustration, below is an example of one of the methods of obtaining mimetics of MA5P-2-binding peptides. Functional monomers of the known MABR-2-binding peptide or the binding site of anti-MA5R-2 antibodies, which inhibit MAZR-2 activity (matrix)

Zo is subjected to polymerization. The matrix is then removed, followed by the polymerization of a second class of monomers at the point where the matrix was removed to create a new molecule exhibiting one or more of the required properties analogous to those of the matrix. In addition to obtaining peptides, other MABR-2-binding molecules that are MABR-2 inhibiting agents can be obtained in this way, such as polysaccharides, nucleosides, medicinal substances, nucleoproteins, lipoproteins, carbohydrates, glycoproteins, steroids, lipids and other biological active substances. This method can be used to create a wide variety of biological mimetics, which are more stable than their natural prototypes, because they are obtained by the method of free radical polymerization of functional monomers, resulting in a compound with a framework that is not prone to biochemical decomposition.

SYNTHESIS OF PEPTIDES

MA5BR-2 inhibitory peptides can be prepared by methods well known in the art, such as the solid-phase synthesis method first described by McGill, in U. Ateg. Spent Goiter. 85:2149-2154, 1963. Automated synthesis can be performed according to the manufacturer's instructions using the 431A Reriyide Zupipezigeg apparatus by Arriey Viozuet5 (Bobieg Sipu, Saiy). Other methods are described, for example, in Vodap57Ku M. ei ai., Reriide Zupipevzib5, zesopa eaiiop, dop UMieu 5 Bop5, 1976, as well as in other works known to specialists in this field of technology.

Peptides can also be produced by standard genetic engineering techniques known to those skilled in the art. For example, a peptide can be obtained as a result of enzymatic synthesis by introducing a nucleic acid encoding a peptide into an expression vector that expresses DNA and translates this DNA into a peptide in the presence of the necessary amino acids. The peptide is then purified using chromatography or electrophoresis, or a carrier protein that can be fused and further cleaved from the peptide by inserting into the expression vector the nucleic acid sequence encoding the carrier protein together with the peptide coding sequence. The fusion protein-peptide can be isolated using chromatography, electrophoresis, or immunological methods (for example, binding the carrier protein to resin with an antibody). The peptide can be cleaved chemically or enzymatically, for example with hydrolase.

MA5BR-2 inhibitory peptides used in the method according to the invention can also be produced in recombinant host cells using conventional methods. For the expression of the sequence encoding MABR-2 inhibitory peptides, the nucleic acid molecule encoding the peptide must be functionally linked to the regulatory sequences controlling transcriptional expression in the expression vector, and then introduced into the host cell. In addition to transcriptional regulatory sequences, such as promoters and enhancers, expression vectors may include translational regulatory sequences and a marker gene that are suitable for selection of cells carrying the expression vector.

Nucleic acid molecules encoding the MA5BR-2 inhibitory peptide can be synthesized using "genetic machines" using protocols such as the phosphoramide method. If the chemical synthesis of double-stranded DNA is required for an application such as gene synthesis or gene fragment synthesis, then each complementary strand is synthesized separately. Obtaining short genes (from 60 to 80 base pairs) will not cause complications and can be performed by synthesizing complementary strands followed by annealing. To obtain longer genes, synthetic genes (double-stranded) are assembled into a modular form from single-stranded fragments with a length of 20 to 100 nucleotides. An overview of the synthesis of polynucleotides can be found, for example, in sSiisK apa Razhiegpak,

Moyesshiag VioyesNpoiodu, Rhipsiriez apa Arriisaiopv oi Vesotbripapi OMA, AZM Rgezz, 1994; PaKiga

K. ei aI., Appy. Chem. Viospet. 53:323, 1984; and Siytiye 5. eyi aiYi., Rgos. Magician! Asai. si. OBA, 87:633, 1990.

LOW MOLECULAR INHIBITORS

In some embodiments, MA5P-2 inhibitory agents are low molecular weight inhibitors, including natural and synthetic low molecular weight substances such as peptides, peptidomimetics, and non-peptide inhibitors (including oligonucleotides and organic substances). Low molecular weight MABR-2 inhibitors can be obtained on the basis of the molecular structure of the variable regions of anti-MA5R-2 antibodies.

Low molecular weight inhibitors can also be designed and created on the basis of the MABR-2 crystal structure using programs for computer modeling of medicinal substances (Kipiya 1.0. ei ai., Zsiepse, 257:1078, 1992). The crystal structure of rat MA5R-2 is described (Reiipregu N. ei aI., EMVO 4. 22:2348-2359, 2003). Using the method described by Kipi7 ei aY., the coordinates of the MABR-2 crystal structure are entered into a computer program, such as SOSK, which outputs a list of low-molecular-weight structures that, as

It is assumed that they connect with MAZR-2. The use of such computer programs is well known to a person skilled in the art. For example, the crystal structure of a protease inhibitor

NIM-1 was used to identify unique non-peptide ligands that are inhibitors of the NIM-1 protease by evaluating the compatibility of substances from the Satrbgidde StguvziaPodgarpis database with the binding site of the enzyme using the SOSK program (Kipig 1.0. ei ai., y. Moi. Vioi

Z5 0 M/:269-288, 1982; Oeziapai" V.G. her and I., RMAB, 87:6644-6648, 1990).

A list of small molecular structures identified by computer programs as potential MA5P-2 inhibitors are screened in a MA5P-2 binding assay such as that described in Example 10. Those small molecules that are identified to bind with MA5P-2, then evaluated in a functional assay such as described in Example 2 to determine their ability to inhibit MA5P-2-dependent complement activation.

SOLUBLE RECEPTORS MAZBR-2

Other suitable MABR-2 inhibitory agents are believed to include soluble receptors

MAB5R-2, which can be obtained by methods well known to a person skilled in the art.

MA5R-2 EXPRESSION INHIBITORS

In another embodiment of this aspect of the invention, the MA5P-2 inhibitory agent is an inhibitor of MA5P-2 expression capable of inhibiting MA5BR-2-dependent complement activation. In the practical application of this aspect of the implementation of this aspect of the invention, typical inhibitors of MA5BR-2 expression include MA5R-2 antisense nucleic acid molecules (such as antisense mRNA, antisense DNA, or antisense oligonucleotides), ribozymes

MA5P-2 and MA5P-2 RNAi molecules.

Antisense RNA and DNA molecules directly block MA5BR-2 mRNA translation by hybridizing with MAB5R-2 mRNA, preventing the translation of MABR-2 protein. The antisense nucleic acid molecule can be designed in various ways and it is able to interfere with the expression of MABR-2. For example, an antisense nucleic acid molecule can be constructed by inverting the coding region (or its part) of the MA5BR-2 cDNA (ZEO IU

MO:4) relative to its normal orientation during transcription, which ensures the transcription of its complement.

Typically, the antisense nucleic acid molecule is identical to at least part of the target gene or genes. However, to suppress the expression of the target gene, the nucleic acid does not necessarily have to be absolutely identical to its nucleotide sequence. Usually,

the shorter the antisense nucleic acid sequence, the higher the homology it should have. The minimum percentage of identity is usually not less than about 65 95, but a higher percentage allows to achieve more effective inhibition of the expression of the endogenous sequence. A percent identity substantially greater than about 80 95 is usually preferred, although values in the range of about 95 95 to complete identity are most preferred.

The antisense nucleic acid molecule does not necessarily have to have an intron or exon of the same structure as that of the target gene; non-coding segments of a target gene can be as effective in achieving antisense inhibition of target gene expression as coding segments. A DNA sequence of 8 or more nucleotides can be used as an antisense nucleic acid molecule, although a longer sequence is preferred. In this invention, a typical example of an inhibitory MABR-2 agent is a molecule of antisense nucleic acid MA5R-2, which is at least 90 95 identical to the complementary cDNA of MA5R-2, consisting of the nucleic acid sequence presented in 5EBEO IJOO MoO:4.

The nucleic acid sequence presented in 5EBO IU MO:4 encodes the MABR-2 protein, which consists of the amino acid sequence presented in zEO IU MOB.

Targeting antisense oligonucleotides to bind to MA5BR-2 mRNA is another mechanism that can be used to reduce MA5R-2 protein synthesis.

For example, the synthesis of polygalacturonase and acetylcholine muscarinic receptor type 2 is inhibited by antisense oligonucleotides targeting the corresponding sequences

mRNA (US Pat. No. 5,739,119, Spepo, and US Pat. No. 5,759,829, ZpemltakKeg). In addition, an example of antisense repression was demonstrated for nuclear protein cyclin, multidrug resistance gene (MOC1), ISAM-1, E-selectin, Z5TK-1, striatal receptor

GABA" and human ESE (see, e.g., US Pat. Mo. 5,801,154, Wagaspipi; US Pat. Mo. 5,789,573, WaKeg; US Pat. Mo. 5,718,709, Sopvidipe; and US Pat. Mo. 5,610,288, Keibepviewip).

A system has been described that allows one skilled in the art to determine which oligonucleotides can be used for the purposes of this invention, which includes probing suitable sites in a target mRNA by RNase H digestion as an indicator of the availability of sequences in transcripts. Professor M. ei a!., Mysieis Asiaz Kez. 26:5079-5085, 1998; Pisua her

Zo a!., Mysieis Asidb5 Nev5. 29:3665-3673, 2001. A mixture of antisense oligonucleotides complementary to specific regions of the MA5R-2 transcript is added to cell extracts expressing MAB5R-2, such as hepatocytes, and hybridized to create RNase H-sensitive sites. This method can be combined with sequence selection using a computer program that can predict the selection of optimal sequences for antisense compositions based on their relative ability to form dimers, hairpins, or other secondary structures that could reduce or prevent specific binding with the target

mRNA in the host cell. The analysis of these secondary structures and the selection of target sites can be performed with the help of the computer program FO ISO for the analysis of primers (KUsPIiyK I., 1997) and the computer program VIGABTM 2.0.5 (Akb5spytsi 5.E. ei a!., Mysi . Acid5 Kev5. 25:3389-3402, 1997).

Antisense compounds directed to the target sequence preferably contain from about 8 to about 50 nucleotides. Antisense oligonucleotides containing from about 9 to about 35 nucleotides are particularly preferred. The authors of the invention consider all oligonucleotide compositions in the range from 9 to 35 nucleotides (that is, having a length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides) as the most preferred for the methods of the invention based on the use of antisense oligonucleotides. The most preferred target regions of MA5R-2 mRNA are those located in the immediate vicinity of the translation initiation codon A!Ys, and sequences that are essentially complementary to the 5'-regions of the mRNA, for example between -10 and 410 regions of the nucleotide sequence of the MAZR-2 gene (ZEO IU MO:4). Examples of MABR-2 expression inhibitors are presented in TABLE 4.

TABLE 4

EXAMPLES OF MABR-2 EXPRESSION INHIBITORS

MO:4 (ZEO IU MO:4), the governing body of the Soviet Union

ZEO IU MO:Z1 Nucleotides 12-45 of the sequence ZEO IU MO:4, 5-SsapsaSASASSATOAsaSsTastad including the translation initiation site MA5R-2 sostoStasas-3' sense

ZEO IU MO:32 Nucleotides 361-396 of the sequence BEO IU MO:4, 5-VASATTASSTTOSOSTOSOSASTO of the coding region containing the MVI-binding site

SAAdSOaAsSAds-3! MA5R-2 (meaningful)

ZEO IU MO:ZZ3. . And in-ASSASSSSSTOAATSSASSOSSS Pucleotides of the sequence of BEOTSU MOM,

STATSSSAAAA-Z du area containing the domain

As indicated above, the term "oligonucleotide" used herein means an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term also includes such oligonucleotides that consist of natural nucleotides, sugars and covalent internucleoside (backbone) bonds, as well as oligonucleotides that have modifications that do not occur in nature. Such modifications make it possible to introduce certain desired properties that natural oligonucleotides do not have, such as reducing toxic properties, improving resistance to nuclease cleavage, and increasing cellular uptake. In illustrative embodiments, the antisense compounds according to the invention differ from native DNA by modifications of the phosphodiester backbone designed to extend the life cycle of the antisense oligonucleotide, in which the phosphate substituents are replaced by phosphorothioates. Similarly, one or both ends of the oligonucleotide can be substituted with one or more acridine derivatives that intercalate between adjacent pairs of nucleotides in the nucleic acid chain.

Another alternative to the use of antisense elements is the use of "RNA interference" (RNAi). Double-stranded RNA (dlRNA) can provoke gene silencing in mammals. The natural function of RNAi and cosuppression is apparently the protection of the genome from the invasion of mobile genetic elements, such as retrotransposons and viruses, producing aberrant RNAs or dlRNAs in the host cell upon their activation (see, for example, Uepzep .. ei ai., Mai. Sepei. 21:209-12, 1999). A double-stranded RNA molecule can be obtained in the process of synthesis of two chains

RNAs capable of forming double-stranded RNA molecules, each of which is approximately 19 to 25 (eg, 19-23) nucleotides in length. For example, a dlRNA molecule used in the methods of the invention may contain RNA corresponding to the sequence and its complement shown in Table 4. Preferably, at least one RNA chain has a 3 "sticky" end of 1-5 nucleotides. Synthesized RNA chains are combined under conditions that form a double-stranded molecule. The RNA sequence may contain at least parts of 8 nucleotides of the sequence zEO IU MO:4 with a total length of 25 nucleotides or less.

Designing miRNA sequences for a given target is well known to a person skilled in the art

From technology. There are commercial organizations engaged in the construction of miRNA sequences that guarantee at least 70 95 knockdown of expression (Oiadep, MyIepsia, Saiyu).

DlRNA can be introduced in the form of a pharmaceutical composition by known methods, by means of which the nucleic acid is introduced into the required target cell. Well-known methods of gene transfer include the phosphate-calcium method, the OEAE-dextran method, electroporation, microinjection, and methods using viruses. Such methods are described in A!yzirei ei ai., Sttepi Rgoiyusoi5 ip Moiiesshiag Vioidodu, dopp UMieu b Bopv, Ips., 1993.

Ribozymes, such as ribozymes targeting MA5BR-2 mRNA, can also be used to reduce the amount and/or biological activity of MA5R-2. Ribozymes are catalytic RNA molecules capable of cleaving nucleic acid molecules with a sequence completely or partially homologous to the ribozyme sequence. It is possible to construct ribozyme transgenes encoding RNA ribozymes that form specific pairs with the target RNA and cleave the phosphodiester backbone in a specific region, resulting in functional inactivation of the target RNA. When carrying out such splitting, the ribozyme itself does not change and, thus, is capable of recycling and splitting other molecules. The inclusion of ribozyme sequences in antisense RNA determines the activity of RNA cleavage,

thus increasing the activity of antisense constructions.

The ribozymes used in the invention usually contain a hybridizing region of at least nine nucleotides, which is complementary to the nucleotide sequence of at least part of the MA5R-2 target mRNA, and a catalytic region that is capable of cleaving the target mRNA (see EPA Mo 0 321 201; UMO 88/04300; Nazeioni.). ei aI., Maishge, 334:585-591, 1988;

Eeedog M.U. hey ay!., Rgos. May). Asad axis OBA, 87:1668-1672, 1990; Sesi T.V. eyi ai., App. Wow

Viospet. 55:599-629, 1986).

Ribozymes can either be targeted directly to cells in the form of oligonucleotides

RNAs that include ribozyme sequences, or be introduced into the cell as an expression vector encoding the required ribozyme RNA. Ribozymes can be used and applied in much the same way as described for antisense polynucleotides.

Antisense RNA and DNA, ribozymes and RNAi molecules used in the methods according to the invention can be obtained by any method known in the field for the synthesis of DNA and RNA molecules. These include methods of chemical synthesis of oligodeoxyribonucleotides and oligoribonucleotides, widely known in the art, such as solid-phase phosphoramide chemical synthesis. Alternatively, RNA molecules can be obtained using /l Uygo and /n by transcription of DNA sequences encoding antisense RNA molecules. Such DNA sequences can be introduced into a wide range of vectors that include suitable RNA polymerase promoters, such as T7 or 5Rb polymerase promoters. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be stably introduced into cell lines.

Various well-known modifications of DNA molecules can be introduced to increase stability and half-life. Suitable modifications include, without limitation, the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5'-mMabo C3-termini of the molecule or the use of phosphorothioate or 2-O-methyl instead of phosphodiester linkages within the oligodeoxyribonucleotide framework.

M. PHARMACEUTICAL COMPOSITIONS and METHODS OF DELIVERY

DOSAGE

In another aspect of the present invention, compositions for suppressing side effects are provided

MA5R-2-dependent complement activation in a subject suffering from a disease or condition as disclosed herein, which contain a therapeutically effective amount of an MAZR-2 inhibitory agent and a pharmaceutically acceptable carrier. MABR-2 inhibitory agents can be administered to a subject in need thereof in therapeutically effective doses to treat or ameliorate conditions associated with MA5BR-2-dependent complement activation. By a therapeutically effective dose is meant an amount of MABR-2 inhibitory agent sufficient to alleviate the symptoms associated with the disease or condition.

The toxicity and therapeutic efficacy of MABR-2 inhibitory agents can be determined by standard pharmaceutical procedures using experimental animal models, such as the MABR-2-/- mouse model expressing the human MAZBR-2 transgene described in Example 1. Using such animal models, the value MOABEI (no observed adverse effect level) and MEO (minimum effective dose) can be determined by standard methods. The dose ratio between the effects of MOAEB and MEO is the therapeutic ratio, which is expressed as the ratio of MOAEB/MEO. MABR-2 inhibitory agents exhibiting high therapeutic ratios or rates are most preferred. Data obtained from cell culture and animal studies can be used to determine the range of doses for humans. Doses of the MA5BR-2 inhibitory agent are preferably in the range of concentrations observed in the bloodstream that include

MEO with minimal or no toxicity. The dose may vary within this range depending on the dosage form or route of administration used.

In some embodiments, the therapeutic efficacy of MA5R-2 inhibitory agents for treating, inhibiting, ameliorating, or preventing fibrosis in a mammal suffering from or at risk of developing a disease or disorder caused or complicated by fibrosis and/or inflammation is determined by one or more of the following: a decrease in the renal tissue of one or more markers of inflammation or scarring (for example, TOEDr-1, STER, 1-6, apoptosis, fibronectin, laminin, collagens, EMT, infiltrating macrophages); reducing the level of release in urine or plasma of soluble markers of inflammation and fibrotic kidney disease (for example, by measuring renal excretory function).

For the composition of any compound, a therapeutically effective dose can be estimated using 60 animal models. For example, the dose can be adjusted in an animal model in such a way that

to reach the range of concentrations circulating in the plasma that includes MEO. Quantitative levels of the MA5BR-2 inhibitory agent in plasma can also be measured, for example, using high performance liquid chromatography.

In addition to studying toxicity, the effective dose can also be estimated by the amount of MA5R-2 protein in a living subject and the binding affinity of the MAZR-2 inhibitory agent. It was shown that Eq

Serum MA5R-2 in healthy individuals is low, in the range of 500 ng/ml, and MAZR-2 levels in a particular subject can be determined by quantitative MABR-2 assays as described in

My Ieg-Kgibiepzep M. ei ai., U. Ittipoi. Meijosv5, 282:159-167, 2003.

Of course, the dose of administered compositions containing MAZR-2 inhibitory agents varies depending on factors such as the subject's age, body weight, height, gender, general health, and medical history. By way of illustration, MA5R-2 inhibitory agents, such as anti-MA5R-2 antibodies, can be administered in a dose range of from about 0.010 to 10.0 mg/kg, preferably from 0.010 to 1.0 mg/kg, more preferably from 0.010 to 0.1 mg/kg body weight of the subject. In some embodiments, the composition contains a combination of anti-MA5R-2 antibodies and MAZR-2 inhibitory peptides.

The therapeutic efficacy of the MAZR-2 inhibiting compositions and methods of the invention for a given subject, as well as suitable doses, can be determined by analyzing complement reactions, well known to a person skilled in the art. Complement generates numerous specific products. Over the past ten years, accurate and specific assays have been developed and are commercially available for most of these activation products, including the small activation fragments CZa, Cda, and C5a and the large activation fragments iC3Pr, C4a, Bb, and 5C56-9. Most of these tests use monoclonal antibodies that react to new antigens (neoantigens) located on these fragments, but not to the native proteins from which they were formed, which makes these tests quite accessible and specific. In most cases, the ELISA method is used, although radioimmunoassay is still sometimes used for SZa and Sba. The latter analysis is used to measure the level of both unprocessed fragments and their "ezAgo" fragments, which are the main forms found in the bloodstream. Unprocessed fragments and Sbadezlyu are quickly removed through binding to cell surface receptors and are therefore present in very low concentrations, while SZadezlyu does not bind to cells and accumulates in the plasma. Measurement of SZa is a sensitive, independent indicator of activation

From complement. An alternative way of complement activation can be determined by measuring the fragment of Vr. Detection of soluble products of activation of the membrane attack pathway, 5C5B-9, is evidence that complement is fully activated. Since both lectin and classical activation pathways generate the same activation products, C4a and C44, measurement of these two fragments provides no information as to which of the two complement activation pathways generated the activation products.

Inhibition of MABR-2-dependent complement activation is characterized by at least one of the following changes in the component of the complement system, which occur as a result of the introduction of an MA5bR-2 inhibitory agent according to the methods of the invention: inhibition of the generation or production of products of the MA5R-2-dependent complement activation system: C4B . , as described in example 10).

ADDITIONAL AGENTS

In some embodiments, methods of preventing, treating, stopping, and/or suppressing fibrosis and/or inflammation include administration of an MA5BR-2 inhibitory agent (eg, an MA5R-2 inhibitory antibody) as part of a therapeutic regimen with one or more other drugs, biologics, or therapeutic agents. treatment methods suitable for suppressing fibrosis and/or inflammation. In some embodiments, an additional drug, biological substance, or therapeutic method of treatment is suitable for specific symptoms associated with a disease or disorder caused or complicated by fibrosis and/or inflammation. As an example, MA5P-2 inhibitory antibodies can be administered as part of a therapeutic regimen together with one or more immunosuppressants such as methotrexate, cyclophosphamide, azathioprine, and mycophenolate mofetil. As another example, inhibitors

MA5P-2 antibodies can be administered as part of a therapeutic regimen together with one or more agents designed to increase blood flow (eg, nifedipine, amlodipine, diltiazem, felodipine, or nicardipine). As another example, MA5BR-2 inhibitory antibodies can be administered as part of a therapeutic regimen together with one or more agents designed to reduce fibrosis, such as α-penicillamine, colchicine, ROMA, relaxin, cyclosporine, TOEr blockers, and/or p38 MARK- blockers As an additional example, MA5R-2 inhibitory antibodies can be administered as part of a therapeutic regimen along with steroids or bronchodilators.

Compositions and methods using inhibitory MA5P-2 agents (for example, inhibitors

MA5BR-2 antibodies), may optionally include one or more additional therapeutic agents that are able to enhance the activity of the MABR-2 inhibitory agent or that provide related therapeutic functions, providing an additional or synergistic effect.

For example, in the context of treating a subject suffering from a disease or disorder caused or complicated by fibrosis and/or inflammation, one or more inhibitory agents

MABR-2 can be administered in combination (including co-administration) with one or more additional antifibrotic agents and/or one or more anti-inflammatory agents and/or immunosuppressants.

MA5P-2 inhibitory agents (eg, MAZP-2 inhibitory antibodies) can be used in combination with other therapeutic agents, such as general immunosuppressants such as corticosteroids, immunosuppressants, or cytotoxic agents and/or antifibrotic agents.

PHARMACEUTICAL CARRIERS and MEANS OF DELIVERY

Typically, the MA5BR-2 inhibitory agent compositions of the present invention, combined with any other selected therapeutic agent, are contained in a suitable pharmaceutically acceptable carrier. The carrier is non-toxic, biocompatible, and chosen to avoid adverse effects of the biological activity of the MA5BR-2 inhibitory agent (and any other therapeutic agent included in the combination). Typical pharmaceutically acceptable carriers for peptides are described in US Pat. No. 5,211,657, Matada. Anti-MAB5R-2 antibodies and inhibitory peptides used in the present invention can be formulated into solid, semi-solid, gel-like, liquid and gaseous medicinal forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, inhalations, etc. injections for oral, parenteral or surgical administration. The present invention also provides for local administration of compositions by coating medical instruments, etc.

Suitable vehicles for parenteral administration by injection, infusion or irrigation and local delivery are distilled water, phosphate-buffered saline,

Ringer's, normal or lactated, 10-glucose solution, Hank's solution or propanediol. In addition, sterile non-volatile oils can be used as a solvent or suspending medium.

Any biocompatible oil can be used for this purpose, including synthetic mono-

Zo or diglycerides. In addition, fatty acids such as oleic acid can be used to prepare injectable preparations. The carrier and the agent can be mixed in the form of liquid solutions, suspensions, gels, pastes or balms capable or not capable of polymerization.

Carriers may also contain a delivery agent to slow (ie, prolong, delay, or control) the delivery of the agent(s) or to enhance the delivery, absorption, stabilization, or pharmacokinetic parameters of the therapeutic agent(s). Such a delivery vehicle may include, without limitation, microparticles, microspheres, nanospheres, or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organics, inorganic compounds, polymeric or copolymeric hydrogels, and polymeric micelles. Suitable delivery systems based on hydrogels and micelles include REO:RNB'REO copolymers and copolymer/cyclodextrin complexes described in UUO 2004/009664 A2, and REO and

REO/cyclodextrin complexes described in US Patent Application Publication No. 2002/0019369

A1. Such hydrogels can be administered by local injection into the area of intended exposure either subcutaneously or intramuscularly to provide a sustained release of the agent.

In the case of intra-articular delivery, the MAZR-2 inhibitory agent can be delivered in the above-described injectable liquid or gel carriers, the above-described injectable sustained-release delivery vehicles, or hyaluronic acid or derivative-based carriers.

In the case of oral administration of non-peptidergic agents, the MA5P-2 inhibitory agent may be delivered in inert excipients or solvents such as sucrose, corn starch, or cellulose.

For local administration, the MA5R-2 inhibitory agent may be delivered as an ointment, lotion, cream, gel, drops, suppository, spray, liquid, or powder, or in gel or microcapsule delivery systems via a transdermal patch.

Various nasal and pulmonary delivery systems, including aerosols, metered-dose inhalers, dry powder inhalers, and nebulizers, are under development and may be suitably adapted to deliver the drug of the present invention by aerosol, inhaler, or nebulizer delivery, respectively.

In the case of intrathecal (IT) or intracerebroventricular (ICM) delivery, appropriate sterile delivery systems (eg, liquids, gels, suspensions, etc.) may be used.

Compositions of the present invention may also include biocompatible excipients such as dispersing or wetting agents, suspending agents, solvents, buffers, penetration enhancing agents, emulsifiers, binders, thickeners, flavoring agents (for oral administration).

PHARMACEUTICAL CARRIERS FOR ANTIBODIES AND PEPTIDES

More specifically, with respect to anti-MA5bR-2 antibodies and inhibitory peptides, the exemplary compositions may be administered parenterally as metered injections of a solution or suspension of the compound in a physiologically acceptable solvent with a pharmaceutical carrier, which may be a sterile liquid such as water, oil, saline, glycerin or ethanol. Compositions containing anti-MA5BR-2 antibodies and inhibitory peptides may additionally contain auxiliary substances, such as moisturizing or emulsifying agents, surface-active substances, pH-adjusting substances, etc. Additional components of pharmaceutical compositions include a hydrocarbon mixture ( such as animal, vegetable or synthetic origin), such as soybean oil and mineral oil. For injectable solutions, the preferred liquid carriers are generally glycols such as propylene glycol and polyethylene glycol.

Anti-MABR-2 antibodies and inhibitory peptides can also be administered in the form of slow-absorbing injectables or implanted preparations that can be formulated with the possibility of slow or pulsatile release of active agents.

PHARMACEUTICALLY ACCEPTABLE CARRIERS FOR EXPRESSION INHIBITORS

With respect to the expression inhibitors used in the methods of the invention, compositions are provided that contain an expression inhibitor as described above and a pharmaceutically acceptable carrier or solvent. The compositions may additionally contain a colloidal dispersion system.

Pharmaceutical compositions containing expression inhibitors may include, without exception, compositions containing solutions, emulsions and liposomes. Such compositions can be obtained from various components, including, without limitation, pre-prepared liquids, self-emulsified solids and self-emulsified semi-solids. Preparation of such compositions typically involves combining the expression inhibitor with one or more of the following: buffers, antioxidants, low molecular weight polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose or dextrin, chelating agents such as EOTA, glutathione and other stabilizers and

From fillers. Neutral buffered saline or saline mixed with non-specific serum albumin are examples of suitable solvents.

In some embodiments, the compositions can be prepared and formulated in the form of emulsions, which are usually heterogeneous systems of one liquid substance dispersed in another in the form of microdroplets (see .), Magsek UOekKeg, Ips., Moscow State University, 1988). Examples of natural emulsifying agents used in emulsion formulations are gum arabic, beeswax, lanolin, lecithin, and phosphatides.

In one of the variants, the compositions containing nucleic acids can be formulated in the form of microemulsions. Microemulsion, as specified in this description, is a system of water, oil and amphiphilic substance, which is a single optically homogeneous and thermodynamically stable liquid composition (see Mo5oi, in Rpappaseshisa! bozade ERogtv, MoI1.1).

The method according to the invention can also include the use of liposomes for transfer and delivery of antisense oligonucleotides to the required area.

Pharmaceutical compositions and formulations of expression inhibitors for topical application may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Traditional pharmaceutical carriers can also be used, for example based on water, powders or oils, thickeners, etc.

METHODS OF ADMINISTRATION

Pharmaceutical compositions containing MA5R-2 inhibitory agents can be administered in different ways

BO methods, depending on which of the methods of administration, local or systemic, is more suitable for the disease to be treated. Further, the compositions of the present invention can be delivered by coating or incorporating the compositions into the surface of an implantable medical device or into the device itself.

SYSTEM DELIVERY

As used herein, the term "systemic delivery" or "systemic administration" includes, without limitation, oral and parenteral routes of administration, including intramuscular (IM), subcutaneous, intravenous (IM), intraarterial, inhalation, sublingual, buccal , topical application, transdermal, nasal, rectal, vaginal and other methods of administration that provide effective dispersion of the delivered agent 60 in one or multiple areas of intended therapeutic effect.

Preferred methods of systemic delivery of these compositions include intravenous, intramuscular, subcutaneous and inhalation. It will be understood that the precise method of systemic administration of selected agents used in the particular compositions of the present invention may be determined by taking into account the propensity of that agent to undergo metabolic transformation in a manner associated with a given route of administration. For example, the most suitable route of administration for peptidergic agents is any route other than oral.

Antibodies and polypeptides inhibiting MABR-2 can be delivered to a subject in need by any suitable method. Methods of delivery of MABR-2 antibodies and polypeptides include oral, pulmonary, parenteral (eg, intramuscular, intraperitoneal, intravenous (IM) or subcutaneous injection), inhalation (eg, finely divided powder), transdermal, nasal, vaginal, rectal or subcutaneous administration, and can be prepared in dosage forms suitable for each of the methods of administration.

As an illustrative example, antibodies and peptides inhibiting MABR-2 can be introduced into a living organism by application to a mucous membrane that has the ability to absorb polypeptides, such as the mucosa of the nose, gastrointestinal tract and rectum. Polypeptides are usually applied to an absorbent mucosa in combination with a permeability-enhancing agent (see, for example, I ee M.N.H.., Stii. Chem. Teg. Ogyd Saitieg Zub. 5:69, 1988; I ee M .N.G..,

U. SopigoPei Veivaze, 13:213, 1990; ee M.N.G., Ed., Reriiide apa Rgoieip Ogyd OeiYimegu, Magsei

OekKeg, Mem mok (1991); Oeroeg A.O. ей яй., U. SopigoPei Keyease, 13:241, 1990). Example,

ZTONE is a synthetic derivative of fusidic acid, a steroidal surface-active substance, which in its structure is similar to salts of bile acids and which is used as an agent that enhances permeability during nasal delivery (I ee M/.A., Viorpagt. 22, Mom./ Oees. 1990).

Antibodies and polypeptides inhibiting MABR-2 can be administered in combination with another molecule, such as a lipid, to protect the polypeptides from enzymatic degradation. For example, the covalent attachment of polymers, in particular polyethylene glycol (PES), was used to protect some proteins from enzymatic hydrolysis in the body and, thus, extend their half-life (Epegide5 R. Yei ai., U. SopigoPei Veilease, 11:139, 1990) . Many polymeric systems for the delivery of proteins have been described (Vaye U.N. ei ai., U9. Sopigoii KeiiIease, 9:271, 1989; Nogi E.

From her ai., Rnapt. Not known 6:813, 1989; MatakKaucha I. eyi ai., U. Rnapt. si. 79:505, 1990; Moznpinigo I. ei ai., u.

Sopigoiyed Veyease, 10:195, 1989; Azapo M. evy ai.,. Sopigoii Veiliase, 9:111, 1989; Kozepbiaii 9. ei ai., u. Sopigoiyed Veyease, 9:195, 1989; Makipo K., U. Sopigoei Nei'ease 12:235, 1990; TaKaKita

U. ei a!., U. Rpagt. 5 78:117, 1989; Takakiga U. eyi ai., 9. Rnatt. 5 78:219, 1989).

Liposomes with increased serum stability and increased half-life have recently been developed (see, for example, US Pat. No. 5,741,516, UMerb). In addition, various methods of producing liposomes and liposome-like preparations as potential drug carriers have been considered (see, for example, US Patent Mo 5,567,434, 520Ka; US Patent Mo 5,552,157, Madi; US Patent Mo 5,565,213, Makatogi; US Patent Mo 5,738,868, ZPPipKagepko ; and US patent Mo 5,795,587, sao).

For transdermal application, antibodies and polypeptides inhibiting MA5ZR-2 can be combined with other suitable ingredients, such as carriers and/or adjuvants. Such ingredients are used regardless of their nature, except that they should be pharmaceutically acceptable for the intended administration and should not reduce the effect of the active ingredients of the composition. Examples of suitable bases include creams, ointments, gels or suspensions containing or not containing purified collagen. Antibodies and polypeptides inhibiting MA5P-2 can also be impregnated into transdermal patches, bandages and bandages, preferably in liquid or semi-liquid form.

The compositions according to the present invention can be administered systemically at specified time intervals necessary to maintain the required level of therapeutic effect. For example, the compositions can be administered by subcutaneous injection every two to four weeks or at less frequent intervals. The dosage scheme is determined by the doctor taking into account various factors that can affect the effect of the combination of agents. These factors include the degree of progression of the disease to be treated, the patient's age, gender and body weight, and other clinical factors.

The dose of each individual agent will vary depending on the MA5R-2 inhibitory function of the agent included in the formulation, as well as the presence and nature of any drug delivery vehicle (eg, sustained release delivery vehicle). In addition, the dosage can be adjusted taking into account the frequency of administration and the pharmacokinetic behavior of the agent(s) being delivered.

LOCAL DELIVERY As used herein, the term "topical" encompasses the application of a drug to or around the area of intended local effect and may include, for example, topical delivery to the skin or other affected tissue, ophthalmic delivery, intrathecal (IT), intracerebroventricular (IM), intra-articular , intracavitary, intracranial, or intravesical administration, placement, or irrigation. Local administration may be more advantageous in terms of administration of lower doses, prevention of systemic side effects, and for more precise control of delivery time and concentration of active agents at the site of local delivery. Local administration involves known concentrations in the target area, regardless of individual differences between patients in terms of metabolism, circulation, etc. Improved dosage control is also provided by direct delivery.

Local delivery of an MA5R-2 inhibitory agent can be achieved in the context of surgical treatments for diseases or disorders caused or complicated by fibrosis and/or inflammation, for example during procedures such as surgery.

TREATMENT SCHEMES

For prophylactic purposes, pharmaceutical compositions containing an MA5BR-2 inhibitory agent (eg, an MABR-2 inhibitory antibody) are administered to a subject predisposed to or at risk of developing a disease or disorder caused or complicated by fibrosis and/or inflammation in an amount , sufficient to inhibit fibrosis and/or inflammation, thereby eliminating or reducing the risk of developing symptoms of this condition. In some embodiments, the pharmaceutical compositions are administered to a subject suspected of having a disease or disorder caused or complicated by fibrosis and/or inflammation, or already suffering from such a disease or disorder, in a therapeutically effective amount sufficient to alleviate or at least partially reduce symptoms of this state. Both prophylactically and therapeutically, compositions containing an MABR-2 inhibitory agent can be administered in multiple doses until a sufficient therapeutic effect is achieved. The use of MA5P-2 inhibitory compositions according to the present invention can be carried out by a single administration of the composition or a limited series of administrations for the treatment of an acute condition associated with fibrosis and/or inflammation. Alternatively, the composition may be administered at defined intervals over an extended period of time to treat a chronic condition associated with

With fibrosis and/or inflammation.

For both prophylactic and therapeutic purposes, compositions containing a MAZR-2 inhibitory agent can be administered in multiple doses until a sufficient therapeutic effect is achieved. In one embodiment, the MA5R-2 inhibitory agent comprises an MABR-2 inhibitory antibody, which preferably can be administered to an adult patient (eg, an adult patient with an average body weight of 70 kg) at a dose ranging from 0.1 to 10,000 mg, more preferably from 1.0 to 5000 mg, more preferably from 10.0 to 2000 mg, more preferably from 10.0 to 1000 mg and even more preferably from 50.0 to 500 mg. For pediatric patients, the dose can be adjusted in proportion to the patient's body weight. The use of MABR-2 inhibitory compositions according to the present invention can be carried out by a single administration of the composition or a limited series of administrations for the treatment of a subject suffering from or at risk of developing a disease or disorder caused or complicated by fibrosis and/or inflammation. Alternatively, the composition may be administered at specified intervals, such as daily, twice weekly, once weekly, every other week, once monthly, twice monthly, over an extended period of time to treat a subject suffering from or at risk of developing a disease or a disorder caused or complicated by fibrosis and/or inflammation.

Both prophylactically and therapeutically, compositions containing a MA5P-2 inhibitory agent can be administered in multiple doses until a sufficient therapeutic effect is achieved.

In some embodiments, a subject is identified as being at risk of developing a disease or disorder caused or complicated by fibrosis or inflammation by determining that the subject has one or more symptoms of renal dysfunction, as assessed, for example, by measuring creatinine levels in serum creatinine, serum creatinine clearance, urinary nitrogen, urinary protein, and/or by measuring one or more biomarkers associated with kidney disease or injury.

Methods for assessing renal function are well known in the art and include, without limitation, measurement of systemic blood pressure and glomerular capillary pressure, proteinuria (eg, albuminuria), microscopic and macroscopic hematuria, serum creatinine (eg, according to one of the estimation formulas renal function in humans, a creatinine level equal to 2.0 mg/dL corresponds to 50 96 normal kidney function, and 4.0 mg/dL to 25 95), because a decrease in glomerular filtration rate (for example, creatinine clearance rate) and the degree tubular damage. For example, assessment of renal function may include assessment of at least one renal function using biological and/or physiological parameters such as serum creatinine level, creatinine clearance rate, 24-hour protein secretion, glomerular filtration rate, urinary creatinine to albumin ratio, albumin excretion rate (for example, determining the degree of kidney fibrosis by measuring the deposition of collagen and/or fibronectin).

E. EXAMPLES

The following examples are only illustrative of the best mode of practical implementation of the present invention and should not be construed as limiting the present invention. All literature sources mentioned in this application are incorporated into this description by reference.

EXAMPLE 1

This example describes the creation of a mouse line deficient in MABR-2 (МА5Р-2-/-), but complete in MAр19 (МАр19-/).

Materials and methods

The targeting vector pKOo-MTKkMU-1901 was created to destroy three exons encoding the C-terminal end of mouse MABR-2, including the exon encoding the serine protease domain, as shown in Fig. 3. RKO-MTKMU-1901 was used for transfection of mouse E5 cell line E14.1a (5M129 Oia;)". Clones resistant to neomycin and sensitive to thymidine kinase were selected. 600 E5 clones were screened, and of these, four different clones containing the necessary selective targeting and recombination events were identified and verified by Southern blot analysis, as shown in Fig. 3. Chimeras were obtained from these four positive clones by embryo transfer. Then the chimeras were backcrossed with the C57/VIb6 background phenotype to obtain male transgenic individuals. Then transgenic males were crossed with females to obtain HE1 from 50 95 progeny showing heterozygosity for the disrupted MA5BR-2 gene. Heterozygous mice were crossed with each other to obtain homozygous offspring, deficient in MA5BR-2, with the resulting ratio between heterozygous mice and wild-type mice of 1:2:1, respectively.

Results and phenotype

The resulting homozygous MABR-2-/- deficient mice, which appeared

Viable and fertile were tested for MABR-2 deficiency using Southern blot analysis to confirm the correct target event, Northern blot analysis to confirm the absence of MABR-2 mRNA, and Western blot analysis to confirm the absence MA5P-2 protein (data not shown). Presence and absence of Mart9 mRNA

MA5BR-2 mRNA was further confirmed by time-resolved PCR-RF method on the device

PopSusieg. As expected, MAZR-2-/- mice continued to express Mar19, MA5R-1, and MAB5BR-3 mRNA and proteins (data not shown). MABR-2-/- mice were analyzed for presence and excess

Properdin, factor B, factor 0, C4, C2, and C3 mRNAs were detected by HydpiSusieg analysis and were found to be identical to the levels found in wild-type control littermates (data not shown). Plasma from homozygous MA5ZR-2-/- mice was completely deficient in lectin-mediated complement activation as described in Example 2 below.

Generation of MA5R-2-/- line on pure background genotype C57VI 6

МА5Р-2-/- mice were subjected to backcrossing with the pure C57ВИ 6 line for nine generations before using the МА5Р-2-/- line as an experimental animal model.

A transgenic mouse line, which is a mouse MAZR-2-/-, MAr19-/- line and which expresses a human MABR-2 transgene (mouse MA5BR-2 knockout and human MA5R-2 knockin), was obtained as follows.

Materials and methods

A mini-gene encoding human MA5R-2, named "mini-pMABR-2" (5EO IO MO:49), was created, as shown in fig. 4, which includes the promoter region of the human MAB5R-2 gene, including the first three exons (exon 1 - exon 3), followed by a cDNA sequence that is a coding sequence of 8 consecutive exons, providing the coding for the full-length MA5R-2 protein that is triggered endogenous promoter. The mini-LMA5R-2 construct was injected into fertilized MABR-2-/- eggs. to replace the deficient murine MABR-2 gene with transgenically expressed human MA5R-2.

EXAMPLE 2

This example shows that MABR-2 is required for complement activation through the lectin pathway.

Materials and methods

Assay of cleavage of C4, specific for the lectin pathway, because Assay of cleavage of C4 is described in Regieegzep ei ai.,.. Ipttipoi. Meiypoaz, 257:107 (2001), in which the activation of the lectin pathway initiated by lipoteichoic acid (I TA) from o. asheiv, which binds to I-ficolin. The analysis described by Reyegzep ei ai. (2001) adapted to measure activation of the lectin pathway by MVI by plating

LPS and mannan or zymosan before the addition of serum obtained from MA5R-2-/- mice as described below. The assay was also modified to exclude the possibility of cleavage of C4 by the classical pathway. This was achieved by using a sample dilution buffer containing TM Masi, which provided high affinity binding of the lectin pathway recognition components to their ligands, but prevented the activation of endogenous C4, thus excluding the involvement of the classical pathway as a result of the dissociation of the C1 complex. Briefly, in a modified assay, serum samples (diluted with high-salt buffer (1M wt) were added to ligand-coated plates, followed by the addition of a constant amount of purified CA in physiological salt buffer. Bound recognition complexes containing MABR-2 , split C4, leading to deposition of C4p.

Methods of analysis 1) Mips Makhizogo titration microplates (MahibZogoF, Mips, cat. Mo 442404, Rizpeg

Zsiepiis) were coated with 1 µg/ml mannan (M7504 5bidta) or any other ligand (as listed below) diluted in coating buffer (15 mM MaCO3, 35 mM

Mmanso", pH 9.6).

The following reagents were used in the analysis: a) mannan (1 μg per well of mannan (M7504 Zidt) in 100 μl of coating buffer); p) zymosan (1 μg per well of zymosan (bidta) in 100 μl of coating buffer); c) G TA (1 μg per well in 100 μl of coating buffer or 2 μg per well in 20 μl of methanol); a) 1 μg of H-ficolin-specific Mab 4H5 in a coating buffer; f) RZA from Aegosossis migidapz (2 μg per well in 100 μl of coating buffer);

I) 100 μl per well of 5. asheikh О5ЗМ20233, fixed in formalin (ОЮ55о-0.5), in buffer for coating. 2) The tablet was incubated overnight at 4 °C.

C) After overnight incubation, the remaining protein binding sites were saturated by

From incubation of plates with 0.1 95 NBA-TV5 blocking buffer (0.1 95 (v/o) NZA in 10 mM Ttiv-SI, 140 mM Mass, 1.5 mM Mam», pH 7.4) for 1- 3 hours, and then the tablets were washed three times with a TV5/Tween/Sag mixture: (TV5 with 0.05 95 Tween 20 and 5 mM Sasi», 1 mM Mas», pH 7.4). 4) Serum samples to be tested were diluted in MVI-binding buffer (1M mass), and diluted samples were added to plates and incubated overnight at 4 °C. Wells to which only buffer was added were used as negative controls. 5) After overnight incubation at 4 "C, the tablets were washed three times with a mixture of TV5/Ivin/Sag:. Then, human C4 (1 μg/ml diluted in BB5 (4 mM barbital, 145 mM Masi, 2

MM Sasi", 1 mM Masi", pH 7.4) 100 μl per well) and incubated for 90 minutes at 37 "C.

Then the tablets were washed again three times with a TV5/Tween/Sag mixture. 6) C4p deposition was determined using alkaline phosphatase-conjugated chicken anti-human C4c (diluted in a ratio of 1:1000 in TV5/Ivin/Ca?), which was added to tablets and incubated for 90 min. at room temperature. Then the tablets were washed again three times with a TV5/Tween/Sag mixture. 7) Alkaline phosphatase was detected by adding 100 μl of p-nitrophenyl phosphate substrate solution, incubating at room temperature for 20 minutes and reading the optical density of OO on a titration microplate reader.

The results

In fig. 5BA-B shows the deposition of C46 on mannan (Fig. 5A) and zymosan (Fig. 58) in diluted sera obtained from MABR-2-/- (crosses), MABR-2-/- (closed circles) and MABR-2 -/- (closed triangles) mice. In fig. 5C shows the relative activity of C4-convertase on plates coated with zymosan (white columns) or mannan (dark columns) for MABR-2-/- mice (n-5) and MABR-2-/- mice (n-4) relative of wild-type mice (n-5), measured by the deposition of C4Bb, normalized relative to the serum of wild-type mice. Error bars represent standard deviation. As shown in fig. 5A-C, in mannan- and zymosan-coated tablets, plasma obtained from MABR-2-/- mice is completely deficient in lectin-mediated complement activation. These results clearly demonstrate that MA5BR-2, and not MABR-1 or MABBR-3, is the effector component of the lectin pathway.

Recombinant MABR-2 restores lectin-mediated C4 activation in 60 serum from MA5R-2-/- mice

To establish the fact that the absence of MA5BR-2 in MABR-2-/- mice is the direct cause of the loss of C4 activation dependent on the lectin pathway, the effect of adding recombinant MABR-2 protein to serum samples was evaluated using the C4 cleavage assay described above. Recombinant proteins, functionally active mouse MABR-2 and catalytically inactive mouse MA5P-2A (in which the serine residue in the active site of the serine protease domain is replaced by an alanine residue), were obtained and purified as described below in example 3.

Pooled serum obtained from four MABR-2-/- mice was pre-incubated with increasing concentrations of recombinant mouse MA5P-2 or inactive recombinant mouse MA5P-2A, and C4-convertase activity was assessed as described above.

The results

As shown in fig. 6, addition of functionally active mouse recombinant protein

MABR-2 (shown as white triangles) in serum from MABR-2-/- mice restored lectin pathway-dependent C4 activation in a protein concentration-dependent manner, whereas the catalytically inactive murine MABR-2A protein (shown as asterisks) did not C4 activation. The results shown in Fig. b, normalized relative to C4 activation observed in the pooled serum of wild-type mice (indicated by a dashed line).

EXAMPLE Z

This example shows the recombinant expression and production of recombinant full-length human, rat, and mouse MA5P-2 proteins, polypeptides derived from MA5P-2, and catalytically inactive mutant forms of MA5P-2.

Expression of full-length human, rat, and mouse MABR-2

The full-length cDNA sequence of human MABR-2 (5EO IU MO:4) was also subcloned into the mammalian pCI-Meo expression vector (Rgoteda), which drives eukaryotic expression under control

SMM of the enhancer/promoter region (described in Kashiytap K..). ei ai., Mysivs Acid5 Kezeagsi, 19:4485-90, 1991; Kashitap, Meinoad5 ip Epgutoiodu, 185:531-66 (1991)). Full-sized mouse

cDNA (ZEO IU MO:50) and rat MABR-2 cDNA (5EO IU MO:53) were subcloned into the pEO expression vector. MA5BR-2 expression vectors were then transfected into adherent Chinese hamster ovary cell lines Okhvi1 according to the standard transfection procedure using calcium phosphate described in Mapii5ei a!., 1989. Cells transfected with such constructs developed very

It is slow, which can be explained by the cytotoxicity of the encoded protease.

In another approach, a mini-gene construct (ZEO IU MO:49) containing human MA5bR-2 cDNA driven by an endogenous promoter was transiently transfected into Chinese hamster ovary (CHO) cells. Human MABR-2 protein secreted into the culture medium was isolated according to the scheme described below.

Expression of full-length catalytically inactive MABR-2

Justification

MABR-2 was activated by autocatalytic cleavage after binding of MVI recognition subcomponents. or ficolins (or I-ficolin, H-ficolin, or M-ficolin) with their corresponding carbohydrate molecules. Autocatalytic cleavage leading to the activation of MA5P-2 often occurs during the MA5P-2 isolation procedure from serum or during purification after recombinant expression. In order to obtain a more stable protein preparation for use as an antigen, a catalytically inactive form of MA5R-2, designated as MA5R-2A, was created by replacing the serine residue present in the catalytic triad of the protease domain with an alanine residue in rats (5EO IU MO:55 5egb617 on AiIab17 ); in mice (ZEO IU MO:52 zZegb617 on AIab17) or in humans (ZEO IU MO:6 Zegb18 on AiIab18).

To obtain catalytically inactive human and mouse MA5R-2A proteins, site-directed mutagenesis was carried out using oligonucleotides presented in Table 5.

Oligonucleotides shown in Table 5 were created by annealing with a region of human and mouse cDNA encoding enzymatically active serine, and the oligonucleotide contained a mismatch to replace the serine codon with the alanine codon. For example, PCR oligonucleotides

ZEO IU MO5:56-59 was used in combination with lod cDNA MABR-2 (5EO IUO MO:4) to amplify the region from the start codon to the enzymatically active serine and from serine to the stop codon to obtain a complete open read from the mutated MABR- 2A containing the Zegb18/AIab18 mutation. PCR products were purified after agarose gel electrophoresis and banding, and single adenosine overlaps were obtained using a standard amplification procedure. Then MABR-2A with added adenosine was cloned into the vector ROEM-T

Eaz and transformed into E. so).

Catalytically inactive rat protein MAZR-2A was obtained by subjecting 5EO IU MO:64 and ZEO

IO MO:65 kinase action and annealing by combining these two oligonucleotides in equal molar volumes, heating at a temperature of 100 "C for 2 minutes and slowly cooling at room temperature. The fragment obtained as a result of annealing contained P5I and Xbvai1 compatible ends and was inserted instead of the ReI-Khra!1! cDNA fragment of wild-type rat MA5BR-2 (ZEO IU

MO:53) with the preparation of rat MA5R-2A. 5v-aldsstadSsa,asAaa,asvsAa,aasaSATTASTaTttt-3 (5EO I MO:64);

B-STABAAAASASTAATOSSSSSToSstTasSsaTtSASSSTOSTasA-Z (5EO IYU MO:65).

Then, each of the human, mouse and rat MA5P-2A was subcloned into mammalian expression vectors REO or psiI-Meo and transfected into the Chinese hamster ovary OHVI1 cell line as described below.

In another approach, the catalytically inactive form of MA5BR-2 was obtained by the method described in

Spep ei ai., U. Vioi. Spet., 276(28); 25894-25902, 2001. Briefly, the plasmid containing the full-length cDNA of human MA5R-2 (described in TPiei ei a. Maishge, 386:506, 1997) was cut with the restriction enzymes Khpoi and EsoK1, and the cDNA of MABR-2 (given in this description in 5EO IU MO:4) were cloned at the appropriate restriction sites into the baculovirus transfer vector rEabziVavje (I iite

Tesppoodie5, Moscow State University). Then, Zegb618 in the active site of MA5R-2 serine protease was replaced by AIab18 by substituting double-stranded oligonucleotides encoding the peptide region from 610 to 625 amino acids (ZEO IYUO MO:13) with the natural region from 610 to 625 amino acids to obtain the full-length MA5R-2 polypeptide with an inactive protease domain

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

To ensure the secretion of different domains of MAZR-2, the following designs were obtained using the MABR-2 signal peptide (residues 1-15 in 5EBO IU MO:5). The construct expressing the SOVI domain of human MABR-2 (Z5EBEO IU MO:8) was obtained by PCR amplification of the region encoding residues 1-121 of MABR-2 (ZEO IU MO:6) (corresponding to the M-terminal domain

SYVI) The construct expressing the SIVIEESE domain of human MABR-2 (5EBEO IU MO:9) was obtained by PCR amplification of the region encoding residues 1-166 of MABR-2 (5EO IU MO:6) (corresponding to the M-terminal domain of SIVIEE). A structure expressing a domain

Human MABR-2 (5EBEO IU MO:10) was obtained by PCR amplification of the region encoding residues 1-293 of MABR-2 (5EBEO IU MO:6) (corresponds to the M-terminal domain

SOVIESESESIVII) O. The above domains were amplified by PCR using Mepiv

From polymerase and pB5-MABR-2 as a matrix, according to established PCR methods. In the sequence of the 5'-primer of the sense primer (5-СсССсСасSATОСАТОАаСсОаСсТастаАСссСто-3'

ZBO IU MO:34) introduced a Watney restriction site (underlined) at the 5'-end of the PCR product.

The antisense primers for each MA5P-2 domain, shown below in Table 5, were prepared by introducing a stop codon (bold), followed by the addition of an Esoki site (underlined) at the end of each PCR product. After amplification, the DNA fragments were treated with Watni and EsokiI restriction enzymes and cloned into the corresponding regions of the rEaziVas1 vector. The resulting constructs were characterized by restriction mapping and confirmed by dlDNA sequencing.

TABLE 5

PCR PRIMERS FOR MAB5BR-2 5EO IO MO:8 STE ASOSTO IN (voi | 5-SOAATTOSOSTOSOSA

GRAY (aa 1-121 in ZEO IU MO:b) MO:z4 AND (5EO and MO:З35)

ZEO and MO:9 5-bss, pAToSATada '

MO:6 MO:34 I

ZEO I MO0 5-bss, pAToSATada '

IO MO:6b MO:34 I

EO IU MOA 5-ATYAVOASOYAOSTO, STaA B-TTAAAAATSASTAATTAT

Human MAYER-? sostostasasstto-5' (5EO | aTtotToSaldto-z' (5EO I

IO MO:56) LIMABR-2 direct MO:59) LIMABR-2 reverse

EO IU MOA 5-bdadaatadSOasSA B-atassostostasatsA "DNA of human MABR-? sado, aasysSAS-Z3 (5EO IU SsSteSta-3 (5EO IO MO:57)

MO:58) IMABR-2 Aa direct |PMAB5R-2 Aa reverse

TABLE 5

PCR PRIMERS FOR MAB5BR-2 tptMAB-2 forward MO:63) tpMAB5bR-2 reverse

EO IU MO5O0 5B-ssssosssta7asiato 5-staSsAaadsatadSasAa

cDNA of mouse MA5R-2 Aestotasda-35' (5EO I ssascyza-3' (5EO IU MO:61)

MO:62) tMA5bR-2 Aa direct |tMABR-2 Aa reverse

Eukaryotic expression of recombinant MABR-2 and production of enzymatically inactive mouse, rat and human MAZR-2A proteins

The above-described MABR-2 and MABR-2A expression constructs were transfected into OKHV1 cells using a standard calcium-phosphate transfection procedure (Mapiai5 ei a!., 1989). MA5P-2A was prepared in a serum-free nutrient medium to exclude contamination of preparations by other serum proteins. After a day, confluent cells were collected from nutrient media (in total, the process was carried out four times). The average level of recombinant MABZR-2A for each of the three species was approximately 1.5 mg/liter of culture medium.

Purification of MA5R-2A protein

MA5P-2A (a mutant form of Zeg-AIa, described above) was purified by affinity chromatography on MVR-A-agarose columns. This strategy allows for rapid cleanup without the use of external markers. MABR-2A (100-200 ml of nutrient medium, diluted with an equal volume of loading buffer (50 mM Ttgi5-CI, pH 7.5, containing 150 mM Maci and 25

MM Sasig) was loaded into a column with MVR-agarose (4 ml) for affinity chromatography, pre-equilibrated with 10 ml of loading buffer. After washing with an additional 10 ml of loading buffer, the protein was eluted in 1 ml fractions with 50 mm Tti5-CI, pH 7.5, containing 1.25 M Masi and 10 mm EOTA. Fractions containing МА5Р-2А were identified using О5-electrophoresis in a polyacrylamide gel. If necessary, MAZR-2A was additionally purified using ion exchange chromatography on a MopoO column (ME. 5/5). The protein was dialyzed with 50 mM

Tiov-CI, pH 7.5, containing 50 mM Mass, and loaded into a column equilibrated with the same buffer. After washing, the bound MABR-2A was eluted with 10 ml of 0.05-1M mass gradient solution.

The results

The MABR-2A protein yield was 0.25-0.5 mg per 200 ml of medium. The molecular mass determined by the MAGOI-M5 method was 77.5 kDa, which exceeded the calculated value for the unmodified polypeptide (73.5 kDa) due to glycosylation. The attachment of glycans to each of the M-glycosylation sites explains the observed mass. MABR-2A migrated in the form of

From one band in 5ZO5-polyacrylamide gels, indicating that it was not subjected to proteolytic degradation in the process of biosynthesis. The weighted average molecular weight, determined by equilibrium ultracentrifugation, is consistent with the calculated value for glycosylated polypeptide homodimers.

RECEIVING RECOMBINANT HUMAN MAZBR-2 POLYPEPTIDES

Another method of obtaining recombinant derivatives of MA5P-2 and MABR-2A polypeptides is described in TViepe M.M. ей ай., у. And so on. 166:5068-5077, 2001. Briefly, cells of the insect Broaoriega tidiregda (Keadu-Riadne 519 cells, obtained from Momadep, Madizop, UM) are grown and maintained in serum-free nutrient medium 519001 (Iite Tespoiodie5) containing 50 Mo/ml penicillin and 50 mg/ml of streptomycin (Se Tesppoiodiee5). Cells of the insect G/sporivia pi (Nidp Rime) (obtained from Daim/ida Spgoroshek, IpziSshshii de Vioiodie BigisiigaIe, Sogeporie, Egapse) are maintained in TS100 medium (Iie Thesppoiodiev5) containing 10,925 EC5 (Botipidie JuOshvseneg,

Vgytaiy, Egapse), supplemented with 50 IU/ml of penicillin and 50 mg/ml of streptomycin. Recombinant baculoviruses are obtained using the Vas-O-Vas system (I ee Thesppoiodie5). Bacmid DNA is purified using the Siadep tidirger purification system (Oiadep) and used to transfect insect 519 cells using Selfectin in 51900 I EM medium (ie

Thesppoodie5), according to the manufacturer's protocol. Recombinant viral particles are collected after 4 days, titrated by the virus plaque method, and amplified as described in Kipd apa Rob5zee, Te

Vasshomigiv Ehrghezviop Zuvziet: And I arogayugu Sshcide, Spartap apa Nai! Che., I opaop, pp. 111-114, 1992.

Nidp ime cells (1.75x107 cells/175-cm" of tissue culture in a flask) were infected with recombinant viruses containing MA5R-2 polypeptides by multiple infection, 2 times, in 51900 I 5EM medium at 28 C for 96 hours. Supernatants were collected by centrifugation and added disopropyl phosphorofluoridate to obtain a final concentration of 1 mM.

MABR-2 polypeptides were secreted in cell culture into the nutrient medium.

Culture supernatants were dialyzed against 50 mM Masi, 1 mM Sasi, 50 mM triethanolamine hydrochloride, pH 8.1, and loaded at 1.5 ml/min. into an O-zZerpagose Ravi Riom/ (Ateg5Nat RNaptasia Vioyesi) column (2.8x12 cm) equilibrated with the same buffer.

Elution was performed using 1.2 liters of a linear gradient in 350 mM Mass in the same buffer. Fractions containing recombinant MA5P-2 polypeptides were identified by Western blot analysis, precipitated by adding (МН4)25О54 to 60 95 (by mass) and left overnight at 4 7b. The sediments were resuspended in 145 mM Mass, 1 mM Sas, 50 mM triethanolamine hydrochloride, pH 7.4, and applied to a T5K 53000 BUMO column (7.5x600 mm) (Tozopaa5, MopidotegumiPe, RA), equilibrated with the same buffer. Then the purified polypeptides were concentrated to 0.3 mg/ml by the method of ultrafiltration in Misgoser microconcentrators (threshold value MUU-10000) (Riygop,

Kapvivip, Septapu).

EXAMPLE 4

This example describes the preparation of polyclonal antibodies to MA5P-2 polypeptides.

Materials and methods

MA5R-2 antigens

Polyclonal antiserum against human MABR-2 was obtained by immunization of rabbits with the following isolated MA5BR-2 polypeptides: human MABR-2 (ZEO IO MO:6), isolated from serum; recombinant human MA5P-2 (5EO IUO MO:b6), MA5P-2A containing an inactive protease domain (ZEO IU MO:13), described in example 3; and recombinant SOVI (ZEO IU MO:8),

SOVESRI (ZEO IO MO:9) and SOVESESOVI! (ZEO and MO:10), expressed as described in example 3.

Polyclonal antibodies

Six-week-old rabbits primed with BSO (vaccine against bacillus Calmette-Guérin) were immunized by injecting 100 μg of MA5R-2 polypeptide at a concentration of 100 μg/ml in physiological solution. Injections were carried out every 4 weeks with a titer of antibodies which

Zo was monitored by ELISA as described in Example 5. Culture supernatants were collected for antibody purification using protein A affinity chromatography.

EXAMPLE 5

This example describes a method of obtaining murine monoclonal antibodies against rat or human MABR-2 polypeptides.

Materials and methods

Male A/U mice (Nagiap, Noisiop, Tex.) aged 8-12 weeks were injected subcutaneously with 100 μg of human or rat GMA5R-2 or gMA5R-2A polypeptides (prepared as described in Example 3) in complete Freund's adjuvant (Oiiso I arogaiogie5, Yueigoi, Misp.) in 200 μl of phosphate-buffered physiological solution (РВ5), pH 7.4. Mice received two subcutaneous injections of 50 μg of human or rat GMA5R-2 or gMAZR-2A polypeptide in incomplete Freund's adjuvant at two-week intervals. In the fourth week, the mice received an injection of 50 μg of human or rat polypeptide ГМА5Р-2 or гМА5Р-2A in the PVE, and after 4 days the mice were used for fusion.

For each fusion, single-cell suspensions were prepared from the spleen of an immunized mouse and used for fusion with 5pg/0 myeloma cells. The fusion of 5x108 5p2/0 cells and 5x108 spleen cells was carried out in a medium containing 50 96 polyethylene glycol (M.M/. 1450) (Kodak,

Vospezieg, M.M.) and 5595 dimethyl sulfoxide (bidta SPetis! So., 5. Gotsi5, Mo.). Then the concentration of cells was brought up to 1.5x10? of spleen cells in 200 μl of suspension in the medium

Ibsome ((sirso, Sgapa Ivziapa, M.M.), supplemented with 1095 fetal serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.1 mM hypoxanthine, 0.4 μM aminopterin, and 16 μm thymidine. Two hundred microliters of cellular suspensions were added to each well of approximately twenty 96-well microplates Ten days later, culture supernatants were screened by ELISA for their ability to react with purified MA5R-2 factor.

ELISA analysis

Wells of IptypoiFf 2 microplates (Oupayesi I arogaigie5, SpapiShu, Ma.) were coated by adding 50 μl of purified PMAZR-2 or rat gHMABR-2 (or gMABR-2A) at a concentration of 50 ng/ml overnight at room temperature. The low concentration of MA5BR-2 for coating provides an opportunity for the selection of high-affinity antibodies. After removing the coating solution by shaking the plate, 200 μl of BG OTTO (non-fat dry milk) in RV5 was added to each well for one hour to block non-specific sites. An hour later, the wells were washed with РВ5BT buffer (РВЗ with a content of 0.0595 Tween 20). Fifty microliters of culture supernatants from each confluence were collected and mixed with 50 μl VI OTTO and then added to separate wells of microplates. After an hour of incubation, the wells were washed with RV5T. Bound mouse antibodies were then detected by reaction with goat anti-mouse Ide (Es-specific) conjugated with horseradish peroxidase (HPR) (daskbzop IttypoKezeagsp

And arogagogieh, MuUeb5i Sgome, Ra.), and diluted in a ratio of 1:2000 in VIOTTO. A peroxidase substrate solution containing 0.1 95 3,3,5,5-tetramethylbenzidine (Zidta, I otsi5, Mo.) and 0.0003 95 hydrogen peroxide (Zidta) was added to the wells and left for 30 minutes to develop the color . The reaction was completed by adding 50 μl of 2M He25O»x to each well. The optical density of the reaction mixture at 450 nm was determined using the VioTek EGISA Keadeg device (VioTekf Ipvigitepiv, UMipoo5ki, MI.).

MA5P-2 binding analysis

Culture supernatants that gave a positive result in the analysis of MA5R-2 by the ELISA method, as described above, can be tested in a binding assay to determine the binding affinity of inhibitory MA5ZR-2 agents to MAZR-2. A similar analysis can be used to determine the ability of an inhibitory agent to bind to other antigens in the complement system.

Wells of a polystyrene microplate (96-well plates for binding media,

Sogpipd Soviag, Satgidde, MA) were coated with MAB5R-2 (20 ng/100 μl per well, Admapsei Kezeagsp

Tesppoiodu, Zap Oiedo, CA) in phosphate-buffered saline (PB5), pH 7.4, overnight at 4 °C. After removal of MA5R-2 solution, the wells were blocked with PB5 containing 1 95 bovine serum albumin (BBA; bidta SPpetisa !), for 2 hours at room temperature. Wells without MABR-2 coating were used as controls. Aliquots of hybridoma supernatants or purified anti-MA5BR-2 MoAB with varying concentrations in blocking solution were added to the wells. After two hours of incubation at room temperature, the wells were washed abundantly PB5. MABR-2-bound anti-MA5BR-2 MoABr was detected by adding peroxidase-conjugated goat anti-mouse Ids (Zidta SPpetisai) to the blocking solution, which was incubated for 1 hour at room temperature. The plate was rinsed again with PB5, after which it was added 100 μl of 3,3,5,5'-tetramethylbenzidine (TMB) substrate (Kigkedaaga apa

From Regtu I arogayogie5, sSaipegzrygu, MO). The TMB reaction was stopped by adding 100 μl of 1M phosphoric acid, and the plate was read at 450 nm using a microplate reader (ZRESTKA MAH 250, Moiseshtiag Oemise5, Ippumaye, CA).

The culture supernatants from the positive wells were then tested for the ability to inhibit complement activation in a functional assay such as the C4 cleavage assay as described in Example 2. Thereafter, the cells from the positive wells were cloned by serial dilutions. MOaABs were again tested by ELISA for their ability to react with AMA5P-2 as described above. Selected hybridomas were grown in spinner flasks, and spent culture supernatant was collected for antibody purification by protein A affinity chromatography.

EXAMPLE 6

This example describes the preparation and production of humanized murine anti-MA5R-2 antibodies and antibody fragments.

A murine anti-MABR-2 monoclonal antibody was obtained from male mice of the A/) line as described in Example 5. The murine antibody was then subjected to a humanization process as described below to reduce its immunogenicity by replacing the murine constant regions with human counterparts to produce chimeric Ids and Ear fragment of an antibody that can be used to inhibit the side effects of MA5P-2-dependent complement activation in humans, according to the present invention. 1. Cloning of anti-MA5R-2 variable region genes from mouse hybridoma cells

Total RNA was isolated from hybridoma cells secreting anti-MA5BR-2 MoAB (obtained as described in Example 7) using EMA?2oi according to the manufacturer's protocol (Vioiesi,

Noisiop, Tech). The first strand of cDNA was synthesized on the basis of total RNA, using oligo-8T as a primer. PCR was performed using 3'-primers obtained from the constant C region of immunoglobulin and sets of degenerate primers obtained from the leader peptide or the first framework region of mouse MUn or Mk genes as 5'-primers. Anchored PCR was performed as described in Spep and Riaizisaz5 (Spep RE, zsapa. u. Ittipoi. 35:539-549, 1992). To clone the Mk gene, a double-stranded cDNA was obtained using the Moi-MAKI primer (5-tassasSsastatAdaatastatsttt-3 5EO IO MO:38). The annealed adapters AO1 (5-vaAAATTSASTSYaTTATTSTOSSOA-3 ZEO IU MO:39) and AO2 (5-TSSOSASAATAASO,adiAta-3 ZEO Ibo MO:40) were ligated to both 5'- and 3i"ends of double-stranded cDNA. 3' adapters -ends were removed by cleavage of Moi. The cleavage product was then used as a template in PCR with oligonucleotide AYU as the 5'-primer and MAK2 (5-SATTFAAAaST t TassaatAaAAdaTtTa,atto-3

ZBO IYUO MO:41) as a 3-primer. DNA fragments with a length of approximately 500 base pairs were cloned into the ryC19 vector. Several clones were selected for sequence analysis to confirm that the cloned sequence covers the required murine immunoglobulin constant region. Oligonucleotides MoOi-MAKI and MAK? was obtained from the Mk region and was located at sites 182 and 84 bp, respectively, below the first base pair of the S-Carr gene.

Clones containing full-length Uk and leader peptide were selected.

To clone the Mn gene, double-stranded cDNA was obtained using the primer Moi1-mdata p-ssSssa,asSs,asbAastasTSsApAatatASCA-V ZEO IYUO MO:42). Annealed adapters

ABI and AOB2 were ligated to both 5- and 3'-ends of double-stranded cDNA. Adapters at the 3' ends were removed by Moi cleavage. Then the cleavage product was used as a matrix in PCR with the oligonucleotide AOI and MAC2 (5-ССССИСТАаStТтСАСТас!sstoАсаасААААТА-Z' 5EO Ir

MO:43) as primers. DNA fragments with a size of 500 to 600 p.o. cloned in figC19.

Oligonucleotides Мой1-МАС1 and МАС2 were obtained from the mouse Su.7.1 region and were located in the regions 180 and 93 bp, respectively, below the first pair of bases in the mouse Su.7.1 gene.

Clones containing full-length Mn and leader peptide were selected. 2. Construction of expression vectors for chimeric MA5P-2 Id and Bar

The cloned Mn and Mk genes described above were used as a template in the PCR reaction to add the Kozak consensus sequence to the 5'-end and the splicing donor to the 3'-end of the nucleotide sequence. After analyzing the sequences for the absence of PCR errors, the genes Mn and

Mk was incorporated into cassettes of expression vectors containing human S.u! and S. Karra, respectively, with the receipt of romM2peouN-pySu! and reM2peom-piSu. Plasmid DNA vectors of heavy and light chains purified in a C5CI gradient were used for transfection of CO5 cells by the electroporation method. After 48 hours of incubation, the culture supernatant was assayed by ELISA to confirm the presence of approximately 200 ng/ml chimeric to. Total RNA was isolated from the collected cells. The first strand of cDNA was synthesized on the basis of total RNA, using oligo-QDT as a primer. This cDNA was used as a template in PCR to obtain DNA fragments for Ba and Carr. For the Ba gene, PCR was performed using 5'-

From AApchAAHasttasSasSASSATOaaATTaaStTatasAAST-3 (ZEO IU MO:44) as a 5-primer and a 3-primer isolated from SNI (5-sSsatspATSSTSAAAASTTTSOTTatoSASSTTOI-3 5EO IU MO:45).

It was confirmed that the DNA sequence contains a complete Mn and the SNI domain of human IDS1.

After cleavage with the appropriate enzymes, the DNA fragments for Ba were inserted into the Nip restriction sites! and Vatni in the cassette of the expression vector remg2antpt-tO5 to obtain rouUganptva. Plasmid remM2 is commercially available and consists of DNA segments obtained from different sources: rVvkzZ22 DNA (thin line) contains the rvK322 DNA replication start site (rVE ogy) and the gene for lactamase resistance to ampicillin (Atr); 5M40 DNA, shown in broad strokes and labeled, contains the 5M40 DNA replication initiation site (5440 og), an early promoter (5' to the apii and peo genes) and a polyadenylation signal (3 to the apii and peo genes). The polyadenylation signal obtained from 5M40 (pA) was also placed at the 3'-end of the Ha gene.

For the Carr gene, PCR was performed using 5-

AAASAAADIaSsTTasSsasSASSATATtTSsTSASTASTSTOST-3 (5ZBO IU MO:46) as a 5-primer and a 3-primer obtained from Sk (5-SOSbSsSSATOSTSTOSTSTSTAASASTST-3 BEO 10 MO:47).

It was confirmed that the DNA sequence contains regions of full-length Mk and human Sk.

After cleavage by appropriate DNA restriction enzymes, fragments for Carr were inserted into Nipapy and Watni restriction sites in the remM2peo-TO5 expression vector cassette to obtain roM2peokK. Expression of both Ga and Carr genes was initiated using an enhancer and a promoter obtained from NSMU. Since the ha gene does not include the cysteine amino acid residue involved in the formation of a disulfide bond between the chains, this recombinant chimeric Rab contains non-covalently linked heavy and light chains. This whimsical Bar is designated as cEab.

To obtain a recombinant Bar with a disulfide bond between the heavy and light chains, the above BJ gene can be extended by including the sequence encoding nine additional amino acids (ERK5SOCTN ZEO IU MO:48) from the hinge region of human IDS1. The Be(EI-Watni) DNA segment encoding 30 amino acids at the 3'-end of the Ba gene can be replaced by DNA segments encoding extended Ba, resulting in rUganptga/Zdaa. 3. Expression and purification of chimeric anti-MA5R-2 Id

To obtain cell lines secreting chimeric anti-MABR-2 IDS, MBO cells were transfected with purified DNA plasmids reMm2peoMN-pyS 1 and rem2peom-pyS-Karra by electroporation. Transfected cells were selected in the presence of 0.7 mg/ml (3418. Cells were grown in a 250 ml spinner flask with medium containing serum.

The culture supernatant obtained after centrifugation of 100 ml of culture was loaded into a 10 ml RKOZBER-A column (Viorgosezzipo, Ips., Rgipseiop, M.9U.). The column was washed with 10 volumes of the RV5 layer. The bound antibody was eluted with 50 mM citrate buffer, pH 3.0. An equal volume of 1M Nerevz, pH 8.0, was added to the fraction containing the purified antibody to bring the pH to 7.0. Residual salts were removed by replacing the buffer with PB5 by ultrafiltration through a Miyiroge membrane (threshold MMU: 3000). The protein concentration of the purified antibody was determined by the BSA method (Riegse). 4. Expression and purification of chimeric anti-MA5R-2 Rab

To obtain cell lines secreting chimeric anti-MA5BR-2 Rab, CHO cells were transfected with purified DNA plasmids romganptva (or ramg2haptva/aa) and rem2peoKcarra by electroporation. Transfected cells were selected in the presence of 5418 and methotrexate.

The selected cell lines were propagated at elevated concentrations of methotrexate. Cells were represented by single cells subcloned by the method of serial dilutions. Then, high-performance cell lines obtained from subcloned single cells were grown in 100 ml of culture in serum-free medium with constant stirring.

Chimeric anti-MA5BR-2 Rab was purified by affinity chromatography using mouse anti-idiotypic MoAB to MABR-2 MoAB. An anti-idiotypic MABR-2 MoAB can be produced by immunizing mice with murine anti-MABR-2 MoAB conjugated to fissurella hemocyanin (CIN) and then screened for specific binding to the MoAb that can compete with human MA5BR-2. For purification, 100 ml of culture supernatant of CHO cells producing cEab or cEar/9aa was loaded onto an affinity column with bound antiidiotypic

MABR-2 MoAr. Then the column was thoroughly washed with PB5 to elute the bound Bar with 50 mM diethylamine, pH 11.5. Residual salts were removed by buffer exchange as described above.

The concentration of purified Rab protein was determined by the BSA method (Riegse).

The ability of chimeric MAZR-2 IdS, cRab and sbar/daa to inhibit the MABR-2-dependent pathway of complement activation can be determined by the inhibition assay described in example 2.

EXAMPLE 7

This example describes a C4/1 Uygo cleavage assay used as a functional screen to identify MA5βR-2 inhibitory agents capable of inhibiting

MA5P-2-dependent activation, with the help of I-ficolin/P35, H-ficolin, M-ficolin or mannan.

C4 cleavage analysis

The analysis of cleavage of C4 is described in Reiegzep, ei ai., 9. Ittipoi. Meiosis5, 257:107 (2001), in which activation of the lectin pathway initiated by lipoteichoic acid (I TA) from 5. ashmgeiiv, which binds to I-ficolin, was measured.

Reagents

The formalin-fixed preparation of 5. asheoiv (05М20233) was prepared as follows: bacteria were grown overnight at 37 "С in a medium based on tryptone-soy agar with the addition of blood, washed three times with РВ5, then fixed for 1 hour at room temperature in РВ5Б/О ,5 96 formalin and washed PB5 three times before resuspension in buffer for immobilization (15 mM MagSO3, 35 mM Manso», pH 9.6).

Analysis

The wells of the Myps Makhizogr microplate (Mayidepe Myps Ipiegpaiopai, Kospevzieeg, MU) were covered with 100 μl of the preparation fixed in formalin 5. asheikh Yu5M20233 (00550-0.5) in a buffer for immobilization with 1 μg of I-ficolin. After overnight incubation, the wells were blocked with a 0.1 96 solution of human serum albumin (HA) in TV5 (10 mM Tgtgi5-HCI, 140 mM wt, pH 7.4), then washed with TV5 containing 0.05 95 Tween 20 and 5 mM Sasi" (washing buffer). Human serum samples were dissolved in 20 mM Ttg5-HCI, 1 M Mass, 10 mm Sas, 0.05 95 Tiop X-100, 0.1 95 NZA, pH 7.4, which prevented the activation of endogenous C4 and led to the dissociation of the C1 complex (consisting of C1d, Stig and C15). MABR-2 inhibitory agents, including anti-MA5bR-2 MoAB and inhibitory peptides, were added to serum samples at various concentrations. Diluted samples were applied to a plate and incubated overnight at 4 °C. After 24 hours, the plates were thoroughly washed with washing buffer, then 0.1 μg of purified human

C4 (obtained as described in Bodaz AMU., MeShshoa5 Eplutoi!. 223:46, 1993) in 100 μl of 4 mM barbital, 145 mM Mass, 2 mM Sasi", 1 mM Mosi", pH 7.4. After 1.5 hours of incubation at 37 "C, the plates were washed again, and the deposition of C4p was determined using alkaline phosphatase-conjugated chicken anti-human C4c (Ittipzuziet, Orrzaia, Zuuedep), and was measured by a colorimetric method using the p-nitrophenyl phosphate substrate.

Analysis of C4 on mannan

The above assay was adapted to measure activation of the lectin pathway by MVI by coating the plate with LPS and mannan before adding serum mixed with various MA5bR-2 inhibitory agents.

Analysis of C4 by H-ficolin (NakKaia Ad)

The above assay was adapted to measure activation of the lectin pathway by H-ficolin by coating the plate with LPS and H-ficolin before adding serum mixed with various MA5P-2 inhibitory agents.

EXAMPLE 8

The following analysis demonstrates the presence of activation of the classical pathway in wild-type mice and

MA5R-2-/- mice.

Methods

Immune complexes were obtained by coating microplates (Makhizogo, Mips, cat. Mo 442404, Riepeg Zsieepiyis) with a 0.1 95 solution of human serum albumin in 10 mM

Tgi5, 140 mM Masi, pH 7.4, for 1 hour at room temperature, followed by overnight incubation at 4 °C with sheep anti-target antiserum (5soycyzp Apibodu Rhodisiop Opii,

Sapike, ZsoMPapa), diluted in a ratio of 1:1000 with a TV5/Twin/Sa?: mixture. Serum samples were obtained from wild-type mice and MA5R-2-/- mice and added to coated tablets.

Control samples were prepared in which C14 was extracted from serum samples of wild-type mice and

MA5R-2-/- mice. C14-depleted mouse serum was prepared using protein A-bound YuupareadbzF microspheres (Oupa! Vioyesp, O5Io, Mogmau) coated with rabbit antihuman IdsS Steed (Oako, Siozigir, YuOeptagk) according to the manufacturer's instructions.

Tablets were incubated for 90 minutes at 37 °C. Bound SZB was detected using polyclonal anti-human SZs antibodies (Shako A 062) diluted in TV5/Ivin/Ca-e at a ratio of 1:1000. The secondary antibody was goat anti-rabbit Ido.

The results

In fig. 9 shows the relative levels of SZB deposition on coated tablets in wild-type serum, MABR-2-/- serum, C1d-depleted wild-type serum, and Sta-depleted MA5BR-2-/- serum. These results indicate that in mice line MABR-2-/- classical pathway

Zo remains unaffected.

EXAMPLE 9

The following analysis was used to test the ability of the MA5BR-2 inhibitory agent to block the classical pathway of activation by studying the action of the MA5R-2 inhibitory agent under conditions of initiation of the classical pathway by immune complexes.

Methods

To evaluate the effect of the MA5R-2 inhibitory agent under the conditions of complement activation, in which the initiation of the classical pathway is carried out by immune complexes, three serum samples, 50 μl each, containing 90 95 МН5, were incubated at 37 "С in the presence of 10 μg/ml of immune complex (IS) or

PB5, and three more samples (-/-IS) containing 200 nM anti-properdin monoclonal antibodies were incubated in parallel at 37 "C. After two hours of incubation at 37 "C, 13 mM EDTO was added to all samples to stop further complement activation, after which all samples were immediately cooled to 5 "С. Then the samples were stored at -70 "С until the moment of their use in the analysis of complement activation products (СЗа and 5С25р0-9) using an ELISA kit (Opsidei, cat. MoMo

АО15 and АО09), according to the manufacturer's instructions.

EXAMPLE 10

This example describes the identification of high-affinity Bar2 fragments of anti-MA5R-2 antibodies that block the activity of MA5R-2.

Prerequisites and rationale

MABR-2 is a protein complex with a number of separate functional domains, including: a site (sites) of binding for MVI. and ficolins, the catalytic site of serine protease, the binding site for the C2 proteolytic substrate, the binding site for the C4 proteolytic substrate, the MA5P-2 cleavage site for zymogen self-activation

MABR-2 and two Ca-binding sites. Identified Bar2 antibody fragments that have high affinity for MAB5R-2, and these identified Bar2 fragments were tested in a functional assay to determine their ability to block functional activity

MA5R-2.

To block the functional activity of MABR-2, the antibody or the Par2-fragment of the antibody must bind or interfere with the structural epitope on MA5R-2, which is necessary for the functional activity of MAZR-2. Therefore, many or all of the high-affinity binding anti-MA5R-bo 2 Bar2 have no ability to inhibit the functional activity of MABR-2 unless they bind to structural epitopes on MABR-2 that are directly involved in the functional activity of MA5R- 2.

A functional assay measuring inhibition of Lectin pathway S3-convertase formation was used to assess the "blocking activity" of anti-MA5BR-2 PAP2. It is known that the primary physiological role of МА5Р-2 in the activation of complement through the lectin pathway consists in the formation of the next functional component of lectin-dependent complement activation, known as S3-convertase of the lectin pathway. C3-convertase of the lectin pathway is a critical enzyme complex (C4rsSga) that carries out proteolytic cleavage of C3 into

SZa and SZ3B. MA5BR-2 is not a structural component of the NW-convertase of the lectin pathway (C4rsC2a); however, the functional activity of MAZR-2 is necessary for the formation of two protein components (C4p, C2a), which contain the C3-convertase of the lectin pathway. In addition, all individual types of functional activity of MA5R-2, listed above, are necessary for the formation with the participation of

MA5BR-2 NW-convertase of the lectin pathway. For this reason, the preferred method of analysis for evaluating the "blocking activity" of anti-MA5R-2 Rabr2 is considered to be a functional assay that allows measurement of inhibition of Lectin pathway C3-convertase formation.

Formation of high-affinity Bar2

To create high-affinity Bar2 to the rat protein MABZR-2 (5EBEO IO MO:55), a phage display library of human antibody light and heavy chain sequences and an automated antibody selection technology were used to identify Rabr2g that react with selected ligands of interest. The amount of rat protein is known

MABR-2 (-1 mg, »85 95 purity) was used for antibody screening. Three cycles of amplification were used to select antibodies with the best affinity. Approximately 250 different variants expressing antibody fragments were selected for screening by ELISA. High-affinity variants were further sequenced to determine the uniqueness of the different antibodies.

Fifty unique anti-MA5R-2 antibodies were purified, and 250 μg of each purified antibody

Ear2 was used to characterize MA5P-2 binding affinity and functional testing of the complement pathway, as described in more detail below.

Assays used to assess inhibitory (blocking) activity of anti-MABR-2

Earg

Zo 1. Analysis to measure the inhibition of formation of SZ-convertase of the lectin pathway

Prerequisites

C3-convertase of the lectin pathway is an enzymatic complex (C4rsC2a) that carries out proteolytic cleavage of CVD into two potential pro-inflammatory fragments, anaphylatoxin CZa and opsonin CZ3B. The formation of SZ-convertase is a key step in the lectin 35 pathway in terms of mediating inflammation. MABR-2 is not a structural component of C3-convertase (C4B0BS2a) of the lectin pathway, so anti-MABR-2 antibodies (or Bar2) will not directly inhibit the activity of pre-existing C3-convertase. However, to obtain two protein components (C4B, C2a), which contain C3-convertase of the lectin pathway, the activity of serine protease MABR-2 is required. Therefore, anti-MABR-2 Papp2, which inhibits the functional activity of MA5P-2 (ie, blocks anti-MA5P-2 Papp2), will inhibit the formation of the C3-convertase of the lectin pathway. SZ contains an unusual and highly reactive thioester group in its structure. During the cleavage of SZB by SZ-convertase in this assay, the thioester group on SZ3p can form a covalent bond with hydroxyl groups or amino groups on macromolecules immobilized at the bottom of the plastic wells with the help of 45 ester or amide bonds, which facilitates the detection of SZB by the ELISA method.

Yeast mannan is known as an activator of the lectin pathway. In the next method of measuring the formation of C3-convertase, plastic wells coated with mannan were incubated for min. at 37 "C with diluted rat serum to activate the lectin pathway. The wells were then washed and analyzed for the presence of SZB immobilized in the wells by standard ELISA methods. The amount of SZB formed in this assay is a direct reflection of the amount of SZ-convertase formation of the lectin pathway. Anti-MA5R-2 Papp2 at selected concentrations were tested in this assay for their ability to inhibit the formation of SZ-convertase and the subsequent formation of SZB.

Methods 9b-well plates of Sovziag Meadisht Vipaipud were incubated overnight at 5 "C with mannan diluted in 50 mM carbonate buffer (pH 9.5) in the amount of 1.0 μg/50 μl/well. After overnight incubation, each well was washed three times with 200 μl PB5. The wells were then blocked with 100 μl/well of 1 95-n bovine serum albumin in PB5 and incubated for one hour at room temperature with gentle agitation. Each well was washed three times with 200 μl PB5. The anti-60 MA5R-2 Par2 samples were diluted to the selected concentrations with a buffer of SMUV containing Ca and Md"- (4.0

MM of barbital, 141 mM Masi, 1.0 mM Masi, 2.0 mM Sasi, 0.1 95 gelatin, pH 7.4), at 5 "С. 0.5 95 rat serum was added to the above samples at 5 "C, and 100 μl was introduced into each well. The tablets were covered with a lid and incubated for 30 minutes in a water bath at 37 "C for complement activation. The reaction was stopped by transferring the tablets from a water bath with a temperature of 37 "C to a container containing a mixture of ice and water. Each well was washed five times with 200 μl of PB5-Tween 20 (0.05 95 Tween 20 in PB5), then washed twice with 200 μl of PB5. The primary antibody (rabbit anti-human SZs, VAKO A0yOb2) with a dilution of 1:10,000 in the amount of 100 μl/well was added to РВ5 containing 2.0 mg/ml bovine serum albumin and incubated for 1 hour at room temperature with gentle agitation. Each well was washed five times with 200 μl of PB5. The secondary antibody (peroxidase-conjugated goat anti-rabbit dO, Ategisap Osaieh AT02RI) with a dilution of 1:10,000 in the amount of 100 μl/well was added to RV5 containing 2.0 mg/ml bovine serum albumin and incubated for 1 hour at room temperature. temperature on a shaker with gentle stirring. Each well was washed five times with 200 μl of PB5. Peroxidase substrate TMB (KigkKedaaga 45 Reggtu

And arogafogie5) was added in the amount of 100 μl/well and incubated at room temperature for 10 minutes. The peroxidase reaction was stopped by adding 1.0 M HzPOx in the amount of 100 μl/well and measuring ОХг50. 2. Analysis to measure inhibition of MAP-2-dependent cleavage of C4

Prerequisites

The serine protease activity of MABR-2 is highly specific, and only two protein substrates have been identified for MABR-2, C2 and C4. When C4 is split, C4a and C4p are formed.

Anti-MABR-2 Babr2 can bind to structural epitopes on MABR-2 that are directly involved in cleavage of C4 (for example, MA5P-2-binding site for C4; MA5P-2-catalytic site of serine protease) and thus thus suppressing functional activity

MA5R-2, which leads to the cleavage of C4.

Yeast mannan is known as an activator of the lectin pathway. In the method described below for measuring MABR-2 C4-cleaving activity, mannan-coated plastic wells were incubated for 30 minutes at 37°C with diluted rat serum to activate the lectin pathway. Since the primary antibodies used in this ELISA method recognize

From C4 alone, diluted rat serum was also supplemented with human C4 (1.0 μg/ml). The wells were then washed and analyzed for the presence of human C4B immobilized on the wells by standard ELISA methods. The amount of C4b5 formed in this analysis is a measure of MA5BR-2-dependent activity of C4 cleavage. Anti-MA5P-2 Rabr2 at selected concentrations were tested in this assay for their ability to inhibit Ca cleavage.

Methods 9b-well plates of Sovziag Meadisht Vipaipud were incubated overnight at 5 "С with mannan diluted in 50 mM carbonate buffer (pH 9.5) in the amount of 1.0 μg/50 μl/well. Each well was washed three times with 200 μl РВ5 . Wells were then blocked with 100 μl/well of 1 95-n bovine serum albumin in PB5 and incubated for one hour at room temperature with gentle agitation. Each well was washed three times with 200 μl of PB5. Anti-MA5R-2 samples

Ear2 was diluted to the selected concentrations with CMV buffer containing Ca and Mod (4.0 mM barbital, 141 mM Mass, 1.0 mM Maosi, 2.0 mM Saci, 0.1 95 gelatin, pH 7.4), at 5 "C. 1.0 μg/ml human C4 (OcideI!)M was also injected into these samples. 0.5 96 rat serum was added to the above samples at 5 "C, and 100 μl was introduced into each well. The tablets were covered with a lid and incubated for 30 minutes in a water bath at 37 "C for complement activation.

The reaction was stopped by transferring the tablets from a 37 °C water bath to a container containing a mixture of ice and water. Each well was washed five times with 200 μl of РВ5-Tween 20 (0.05 95 Tween 20 in РВ5), then each well washed twice with 200 μl of PB5 Biotin-conjugated chicken antihuman C4 (Ittipzuviet AV, Orrzaia, Zmedep) diluted 1:700 in the amount of 100 μl/well was added to PB5 containing 2.0 mg/ml bovine serum albumin (B5A ), and incubated for 1 hour at room temperature with gentle agitation.

Each well was washed five times with 200 μl of PB5. 0.1 μg/ml peroxidase-conjugated streptavidin (Riegse Spetisai! 221126) in an amount of 100 μl/well was added to PB5 containing 2.0 mg/ml B5A and incubated for 1 hour at room temperature on a shaker at low mixing Each well was washed five times with 200 μl of PB5. Peroxidase substrate TMV (Kigkedaaga 8. Regtu I arogaiyugie5) was added in the amount of 100 μl/well and incubated at room temperature for 16 minutes. The peroxidase reaction was stopped by adding 1.0M

NzRoOx in the amount of 100 μl/well and measured ОЯзво. 3. Analysis of binding of anti-rat MA5P-2 Rabr2 to native rat MA5P-2 60

Prerequisites

MABR-2 is normally present in plasma in the form of dimeric complexes of MABR-2, which also include specific lectin molecules (mannose-binding protein (MVI) and ficolins). As a consequence, if there is an interest in studying the binding of anti-MA5R-2 Rab2 to the physiologically relevant form of MABR-2, it is important to develop a binding assay that utilizes the interaction of Rab2 not with purified recombinant MA5R-2, but with native MA5R- 2 in plasma. In this binding assay, the native MABR-2-MVIG complex from 10 9u5 rat serum is first immobilized in mannan-coated wells. Then the binding affinity of different anti-MABR-2 Rab2 relative to the immobilized native MABR-2 is studied by the standard ELISA method.

Methods 9b-well plates of Soviag Nidp Vipaiipud were incubated overnight at 5 "С with mannan diluted in 50 mM carbonate buffer (pH 9.5) in the amount of 1 μg/50 μl/well. Each well was washed three times with 200 μl of РВ5. Wells blocked with 0.5 965 skimmed milk powder in RVZT (RVZ with 0.05 95 Tween 20) in an amount of 100 μl/well and incubated for one hour at room temperature with gentle agitation.Each well was washed three times with 200 μl of wash buffer TV5/Ivin /Ca"" (saline solution, buffered with Tris, 0.05 95 Tween 20 containing 5.0

MM Sasi", pH 7.4). 10 95 rat serum was prepared on ice in immobilization buffer with a high salt content (20 mM Tti5, 1.0 M Masi, 10 mM Sasig, 0.05 965 Tiyop X100, 0.1 95 (v/o) bovine serum albumin, pH 7 ,4). 100 μl/well was added and incubated overnight at 5 °C. The wells were washed three times with 200 μl of washing buffer TV5/TIvin/Ca". Then the wells were washed twice with 200 μl of PB5. 100 μl/well of the selected concentration of anti-MABR-2 Rarg, diluted in the buffer of SUV containing Ca»: and Md"" (4.0 mM barbital, 141 mM Masi, 1.0 mM Mosi», 2.0 mM Sasi», 0.195 gelatin, pH 7.4), was added and incubated for one hour at room temperature with gentle agitation.Each well was washed five times with 200 μl

RV5. HER-conjugated goat anti-Par2 (Viodepesiz cat. Mo 0500-0099), diluted 1:5000 in 2.0 mg/ml bovine serum albumin in PB5, was added in the amount of 100 μg/well and incubated for one hour at room temperature with gentle stirring. Each well was washed five times with 200 μl of PB5. Peroxidase

The TMB substrate (Kigkedaaga 8. Reitu I arogayogie5) was added in the amount of 100 μl/well and incubated at room temperature for 70 minutes. The peroxidase reaction was stopped by adding 1.0 M NzRoOx in the amount of 100 μl/well and measured ОХг50.

The results

Approximately 250 different Rabrgs that reacted with high affinity to rat MA5BR-2 bileu were selected for screening by ELISA. These high-affinity Rar2s were sequenced to determine the uniqueness of the various antibodies, and 50 unique anti-MA5BR-2 antibodies were purified for further analysis. 200 μg of each purified Bar2 antibody was used to characterize MABR-2 binding affinity and functional testing of the complement pathway.

The results of this analysis are presented below in Table 6.

TABLE 6

ANTI-MABR-2 EAV2, BLOCKING THE ACTIVATION OF COMPLEMENT THROUGH THE LECTIN PATHWAY. CZ3-convertase Cleavage of C4 11112786. 055 | yuyu l147 11111 77111766 | .ЮюЮюрю092 |... 45 юЮущЩщ | m 22601716 11111181 1111111 mosSsS 11268 Y11111117120 11117166 1111101 8811171 YH11111118011171 11111125 11111111

TABLE 6

ANTI-MABR-2 EAV2, BLOCKING THE ACTIVATION OF COMPLEMENT THROUGH THE LECTIN PATHWAY. CZ3-convertase Cleavage of C4 22871160 25 |... m s

As shown in Table 6 above, of the 50 anti-MA5R-2 Rabr2s tested, seventeen of the Rar2s were identified as MA5R-2 blocking RAr2s that could potentially inhibit C3-convertase formation with ICs equal to or less than 10 nmol Rar2 (34 95- positive success rate). Eight of the seventeen identified Bar2s have IC30 values in the subnanomolar range. In addition, all seventeen MA5bR-2 blocking Papp2s shown in Table 6 show essentially complete inhibition of C3-convertase formation in the C3-convertase lectin pathway assay. In fig. BA graphically shows the results of the analysis of the formation of SZ-convertase for the antibody

Bar2 Mo 11, which is a typical representative of the Papp2 antibodies tested, the results of which are shown in Table 6. This fact is important because it suggests the theoretical possibility that "blocking" Papp2 may only partially inhibit the function of MA5P-2, even when each molecule of MA5P -2 binds to Bar2g.

Although mannan is a known activator of the lectin pathway, it is theoretically possible that the presence of anti-mannan antibodies in rat serum may also activate the classical pathway and generate S3 via the S3 convertase of the classical pathway. However, each of the seventeen blocking anti-MA5R-2 Papp2 listed in this example can potentially inhibit the formation of SZB (295 95), thus demonstrating the specificity of this assay for the SZ convertase of the lectin pathway.

Binding assays were also performed for all seventeen Bar2 blockers in order to calculate the apparent Ka for each of them. Results of the binding analysis of anti-rat MABR-2

Ear2 with native rat MA5P-2 for six blocking Rabr2 are also presented in Table 6. In Fig. 88 graphically shows the results of the binding analysis of the Bar2 Mo 11 antibody. A similar binding analysis was also performed for other ERab2g, the results of which are presented in Table 6.

Basically, the apparent Kαs obtained for the binding of each of the six Bar2s to native MA5R-2 completely correspond to the ICs for Par2 in the functional analysis of the S3-convertase. There is evidence that MABR-2 undergoes conformational changes from an inactive to an active form upon activation of its protease activity (Reipregod ei aI., EMVO . 22:2348-59 (2003); Sa! ei ai., 9. Vioi.

Spent 280:33435-44 (2005)). In normal rat plasma used in the formation assay

SZ-convertase, MABR-2 is present mainly in the "inactive" conformation of the zymogen. On

In contrast, in the binding analysis MA5BR-2 is present as part of the complex with MVI bound to immobilized mannan; therefore, MABR-2 is rather represented by an "active" conformation (Regiegzep ei ai.,». Ittipoi. Meiosis, 257:107-16, 2001). Therefore, one should not expect a clear relationship between ISvo and Ka for each of the seventeen Rabb2 blockers tested in these two functional assays, as Bar2 will bind to different conformational forms of MABR-2 in each assay. However, with the exception of Bar2 Mo 88, there appears to be a fairly close correspondence between ICzo and apparent Ka for each of the other sixteen Rabr2s tested in the two tests (see Table 6).

Some Par2 blockers were evaluated for their ability to inhibit MAZR-2-dependent C4 cleavage.

In fig. 8C graphically shows the results of the analysis of C4 cleavage, demonstrating inhibition in the case of Bar2 Mo 41, with ICvo-0.81 nM (see Table 6). As shown in fig. 9, all of the Papp2s tested inhibited C4 cleavage with ICvho values similar to those obtained in the S3-convertase assay (see Table 6).

Although mannan is a known activator of the lectin pathway, it is theoretically possible that the presence of anti-mannan antibodies in rat serum may also activate the classical pathway and thus generate C46 via C15-mediated cleavage of C4.

However, several anti-MA5R-2 Rab2gs were identified that effectively inhibited formation

C4p (29595), thereby demonstrating the specificity of this assay for MABR-2-mediated cleavage of C4. C4, like SSZ, contains an unusual and highly reactive thioester group in its structure. In this analysis, during cleavage of C4 with the participation of MABR-2, the thioester group on C45 can form a covalent bond with hydroxyl groups or amino groups on macromolecules immobilized at the bottom of plastic wells with the help of ester or amide bonds, which facilitates the detection of C4B by ELISA .

These studies clearly demonstrate the creation of high-affinity Bar2 for the rat protein

MABR-2, which functionally block the activity of both C4- and C3-convertase, thereby preventing the activation of the lectin pathway.

EXAMPLE 11

This example describes the mapping of epitopes of several blocking anti-rat MA5P-2 antibodies, PaP2, obtained by the method described in example 10.

Methods

As shown in fig. 10, the following proteins, all with M-terminal bhNiv-tags, were expressed in CHO cells using the pEO4 vector: rat MABR-2A, full-length MABR-2 protein inactivated by replacing serine with alanine in the active site (5613A); rat MABR-2K, full-length MA5R-2 protein, modified to reduce autoactivation (8424K);

SOVI-P, the M-terminal fragment of rat MABR-2, which contains only SYVI domains,

ESEE-like and SOVII; and

SOVI/ESEE-like, M-terminal fragments of rat MAZ5R-2 that contain only domains

SIiVI and ESE-like.

These proteins were purified from culture supernatants by nickel-affinity chromatography as previously described (Spep et al., 9. Viol. Spet. 276:25894-02 (2001)).

C-terminal polypeptide (SSRII-5P), containing SSRII and the serine protease domain of rat MA5R-2, was expressed in BE. soy/ in the form of protein fused with thioredoxin with the help of rTthgiz (Ipmigodep). Protein was purified from cell lysates using Tpioprop affinity resin.

The protein fused to thioredoxin was expressed in empty rtgihRiz5 as a negative control.

All recombinant proteins were dialyzed in TV buffer, and their concentrations were determined by measuring OO at 280 nm.

Dot blot analysis

Serial dilution of the five recombinant MA5P-2 polypeptides described above and shown in fig. 10 (and the thioredoxin polypeptide as a negative control for the SSRII-serine polypeptide

From protease), applied to a nitrocellulose membrane. The amount of protein applied was in the range from 100 ng to 6.4 pg, in fivefold dilutions. In subsequent experiments, the amount of protein applied was in the range from 50 ng to 16 pg, also in fivefold dilutions. Membranes were blocked with 595 nonfat dry milk in TV5 (blocking buffer), then incubated with 1.0 μg/ml anti-MA5BR-2 Papp2 in blocking buffer (containing 5.0 mM Ca). Bound Ghar2 was determined using HER-conjugated anti-human EPar (ArO/Zegoyes; diluted 1/10000) and an ESI detection kit. (Ateg5pat). One membrane was incubated with polyclonal rabbit anti-human MABR-2 AB (described in 5iomega ei ai., U. Ittipoi. 163:6848-59 (1999)) as a positive control. In this case, bound AB was detected using NCR-conjugated goat anti-rabbit Ide (Bako; diluted 1/2000).

MA5P-2 binding analysis

ELISA plates were coated with recombinant MABR-2A or SOVI-Y polypeptide in a carbonate buffer (pH 9.0) in the amount of 1.0 μg per well overnight at 4 °C. The wells were blocked with 1 95 B5A in TV5, then serial dilutions of anti- MA5bR-2 Rabr2 in TV5 containing 5.0 mM

Sa. The tablets were incubated for one hour at room temperature. After triple washing with TV5/Ivin/Ca, NCR-conjugated anti-human Bar (ABrOr/Zegoyes) diluted 1/10000 in TV5/Sag: was added, and the plates were again incubated for one hour at room temperature. The bound antibody was detected using the TMB kit, peroxidase substrate (Viogai).

The results

The results of a dot blot analysis demonstrating the reactivity of BGar2 with various MA5R-2 polypeptides are presented below in Table 7. The numerical values given in Table 7 indicate the amount of protein applied required to obtain a signal of approximately 1/2 intensity from the maximum value. As can be seen, the positive control AB (serum polyclonal anti-human MABR-2, obtained from rabbits) recognized all polypeptides (the only exception is the chimeric thioredoxin protein).

TABLE 7

REACTIVITY OF DIFFERENT RECOMBINANT POLYPEPTIDES

RAT MA5BR-2 IN DOT BLOT ANALYSIS is similar 60 | o4n | ObO4ni | mA | ma | mV YAYAOK 7.66 | o4n | mA | mA | 2nd bn | mV YAZH 7.86 | ob4n | mA | mA | He | mA I'm the control

MA-no reaction. The positive control antibody is a polyclonal anti-human MA5R-2 serum obtained from rabbits.

All Rar2 reacted with MA5BR-2A as well as MA5BR-2K (data not shown). Majority

Bar2 recognized the SSRII-5R polypeptide, but not the M-terminal fragments. The exceptions are Rabr2 Mo 60 and Bar2 Mo 57. Bar2 Mo 60 recognizes MA5BR-2A and the SOVI-HER fragment, but does not recognize the SOVI/LESE-like polypeptide or the SSRII-5R polypeptide, which suggests its binding to an epitope on SOVI, or overlapping GRAY! of the ESE-like domain. Bar2 Mo 57 recognizes MA5R-2A, but does not recognize any of the MA5R-2 fragments tested, indicating that this BGar2 antibody recognizes an epitope on SSRI. Bar2 Mo 40 and Mo 49 bind only to complete MA5R-2A. In the ELISA binding assay shown in FIG. 11, Bar2 Mo 60 also bound to the SIVI-II polypeptide, although with a slightly lower apparent affinity.

These results demonstrate the unique ability of Bar2 to block multiple regions of the protein

MA5R-2.

EXAMPLE 12

This example describes the identification by phage display of fully human 5cEm antibodies that bind to MA5R-2 and inhibit lectin-mediated complement activation, while not affecting the classical (C1d-dependent) pathway component of the immune system.

A brief overview

Fully human, high-affinity MABR-2 antibodies were identified by screening a phage display library. Fragments of the variable regions of the light and heavy chains of antibodies were isolated both in the 5cm format and in the full-length Id format. Human antibodies to

MABR-2 are useful in inhibiting cellular damage associated with lectin-mediated activation of the complement pathway, while not affecting the classical (Std-dependent) pathway of the component of the immune system. In some embodiments, the provided MABR-2 inhibitory antibodies have the following characteristics: (a) high affinity for human MABR-2 (eg, Co-10 nM or less) and (B) inhibit MAZR-2-dependent

ZO complement activity in 90 95 human serum with ISvo equal to 30 nM or less.

Methods

Expression of full-length catalytically inactive MA5R-2

The full-length cDNA sequence of human MABR-2 (ZEO IU MO:4), encoding the human MAZR-2 polypeptide with a leader sequence (ZEO IU MO:5), was subcloned into the expression vector pCI-Meo of mammals (Rgoteda), which triggers expression in eukaryotes under management of the SMMU of the enhancer/promoter region (described in Kayytap K.). ей айЙ., Mysieis Asiae Kezeagsy,

19:4485-90, 1991; Kashtap, Meipodz ip Epgutoiodu, 185:531-66 (1991)).

To obtain the catalytically inactive human protein MA5BR-2A, site-directed mutagenesis was carried out as described in 5 2007/0172483, which is incorporated herein by reference. PCR products were purified after agarose gel electrophoresis and banding, and single adenosine overlaps were obtained using a standard amplification procedure. Then MABR-2A with added adenosine was cloned into the vector pOEM-T Eava and transformed into BE. soybeans Human MAZR-2A was additionally subcloned into the mammalian expression vector REO or rsCI-Meo.

The above-described MABR-2 and MABR-2A expression constructs were transfected into OKH1 cells using a standard calcium-phosphate transfection procedure (Mapii5 et al., 1989).

MABR-2A was obtained in a serum-free nutrient medium to exclude contamination of preparations by other serum proteins. After a day, confluent cells were collected from nutrient media (in total, the process was carried out four times). The average level of recombinant MABR-2A was approximately 1.5 mg/liter of culture medium. MABR-2A (the mutant form of 5eg-AIIa, described above) was purified by affinity chromatography on columns with MVR-

A-agarose.

ELISA method for MA5BR-2A on 5cSem candidate clones identified by penning/5cSem transformation and screening using a filter

The phage display library of the sequences of the variable regions of the light and heavy chains of human immunoglobulin was subjected to antigen penning, followed by automated screening and selection for the detection of high-affinity 5cEm antibodies to the human MA5BR-2 protein. Three cycles of panning of the 5sEM phage library relative to NI5-label or biotin-labeled MA5R-2A were performed. In the third cycle of penning, elution was carried out first with

MVI, and then with TEA (alkaline). To track the specific enrichment of phages displaying 5cEm fragments relative to the target MABR-2A, ELISA was performed for polyclonal phages against immobilized MA5R-2A. The 5cEm genes from the Z penning cycle were cloned into the RNase expression vector and screened through a fine filter to search for specific clones targeting MABR-2A.

Bacterial colonies containing plasmids encoding 5cM-fragments from the Z penning cycle,

Zo was collected, placed on nitrocellulose membranes and grown overnight in a non-inducing medium to obtain master plates. A total of 18,000 colonies from cycle Z penning, half from competitive elution and half from subsequent TEA elution, were selected and analyzed. Screening of the phagemid 5sEm library against MABZR-2A followed by 5sEm transformation and screening using a filter yielded 137 positive clones. 108/137 clones were positive in the ELISA analysis for MA5R-2 binding (data not shown), of which 45 clones were further analyzed for the ability to block the activity of MABR-2 in normal human serum.

Assay to measure inhibition of lectin pathway SZ-convertase formation

A functional analysis measuring the inhibition of the formation of S3-convertase of the lectin pathway was used to evaluate the "blocking activity" of candidate CEM clones MA5R-2.

The activity of the MAZR-2 serine protease is required for the production of two protein components (C4B, Cga), which contain the S3-convertase of the lectin pathway. Therefore, MABR-2 sems that inhibit the functional activity of MA5R-2 (ie, block MAZR-2 sems) will inhibit the formation of S3-convertase of the lectin pathway. SZ contains an unusual and highly reactive thioester group in its structure. When SZ3 is cleaved by SZ-convertase in this assay, the thioester group on SZ3p can form a covalent bond with hydroxyl groups or amino groups on macromolecules immobilized at the bottom of plastic wells with the help of ester or amide bonds, which facilitates the detection of SZB by ELISA.

Yeast mannan is known as an activator of the lectin pathway. In the next method to measure N-convertase formation, mannan-coated plastic wells were incubated with diluted human serum to activate the lectin pathway. The wells were then washed and analyzed for the presence of SZB immobilized in the wells by standard ELISA methods. Number

The SZp formed in this assay is a direct reflection of the failure of the Lectin pathway SZ-convertase formation. 5sEM-clones MA5P-2 in selected concentrations were tested in this analysis for the ability to suppress the formation of SZ-convertase and the subsequent formation of SZB.

Methods 45 candidate clones identified as described above were expressed, purified, and diluted to the same stock concentration, which was diluted again with Ca and Md-containing CMV buffer (4.0 mM barbital, 141 mM Maci, 1 .0 mM Mosi", 2.0 mM Sasi", 0.1 95 gelatin, pH 7.4), to ensure that all clones had the same amount of buffer. 5cM clones were tested in triplicate at a concentration of 2 μg/ ml. The positive control was OM5100 Par2, which was tested at a concentration of 0.4 μg/ml. The formation of SZs was monitored in the presence and absence of 5cEm/Ida clones.

Mannan was diluted to a concentration of 20 μg/ml (1 μg/well) in 50 mM carbonate buffer (15

MM MagbOz-35 mM MansSozaya 1.5 mM Mam»), pH 9.5, and coated the ELISA tablets with it, leaving it overnight at 4 °C. The next day, the mannan-coated tablets were washed three times with 200 μl of РВ5.

Then 100 μl of 1 95 blocking H5A solution was added to the wells and incubated for 1 hour at room temperature. Plates were washed three times with 200 μl PB5Z and stored on ice with 200 μl PB5Z until samples were added.

Normal human serum was diluted to a concentration of 0.5 95 in SamosmvB buffer, and 5cEm clones or the positive control OM5100 Papp2 were added to this buffer in triplicate at concentrations of 0.01 μg/ml, 1 μg/ml (OM5100 control only), and 10 μg /ml and pre-incubated for 45 minutes on ice before adding to the blocked ELISA plate.

The reaction was initiated by incubation for one hour at 37 C, which was stopped by transferring the tablets to an ice bath. Deposits of SZr were detected using a rabbit antibody to o-mouse SZs, after which NKR-conjugated goat anti-o-rabbit antibody was used. A buffer without antibodies served as a negative control (no antibodies - the maximum level of SZB deposits), and a buffer with EDTO (no SZ3B deposition) served as a positive control. Background was determined using the same assay except that the wells did not contain mannan. The background signal relative to mannan-free plates was subtracted from the signals in mannan-containing wells. The threshold value criterion was set equal to half the activity of the irrelevant 5cEm clone (MAM) and the pure buffer.

The results

Based on the criteria of the threshold value, a total of 13 clones blocking the activity of MAZR-2 were identified. All 13 clones providing »50 95 inhibition of the pathway were selected and sequenced, yielding 10 unique clones. All ten clones were found to have a light chain of the same subclass, AZ, but a heavy chain of three different subclasses: МН2, UNZ, and

MNVb In the functional analysis, five out of ten candidate 5cEu clones had ICzo values in NM less than the target criterion, 25 nM, using 0.5 95 human serum.

To identify antibodies with improved efficiency, three maternal 5cM-clones, defined as described above, were subjected to the light chain shuffling method. This process involved the creation of a combinatorial library consisting of Mn from each maternal clone mated to a library of native human light lambda chains (Mi) obtained from six healthy donors. This library was then screened to identify 5cM clones with improved binding affinity and/or functionality.

TABLE 8

Comparison of the functional efficiency according to ISvo (NM) of the main daughter clones and their corresponding maternal clones (all in 5cEm format) 1 95 human serum, 90 95 human 90 95 human 5cSEU-clone analysis SZ serum, serum, (ISvo NM) analysis SZ analysis C4

ISvo NM) ISvo NM)

LYAMmIbts7777777717171711111111176811111171 11111111 ma | msSshS

Below are the sequences of the heavy chain variable region (HCH) for the parent clones and daughter clones shown in Table 8 above.

СО (31-35 (NI), 50-65 (Н2) and 95-107 (НЗ)) according to Kabvai are highlighted in bold; and SOC (26-32 (NO), 52-56 (H2) and 95-101 (NZ)) according to Spoiilia are underlined.

Variable section of heavy chain (MH) 17020 35МН-21М11М (5EO ИО МО:67, coded

ZEO and MO:66)

OMTIKEBOARMI MKRTETI TI TSTUBAV5I ZNAKMOaU5UMvORROKAI EM .ANIEZ5ZOEKVUVT5I.

KOAIGTIZKOTZKMOMMI TMTMMORUUUOTATUUSAVIVVOSIIUUMUSOSTI UTUB5, variable section of heavy chain (MN) 417M9 (ZEO IV MO:68)

OMOG OO5aryy MKRBOTI 5I TSAIZIO5UZZTZAAMMMIMOVOZROVYa EM OaVTUUVZKUMUMOU

AM5UKZAITIMROTZKMOGBI OY M5MTREOTAMUUSANORESUREOM/SOSTMUTUBZ5.

Presented below are the sequences of the variable region of the light chain (MI) for the parent clones and daughter clones shown in Table 8 above.

SO (24-34 (I 1); 50-56 (I 2) and 89-97 (I 3)) according to Kabvai are highlighted in bold; and SOC (24-34 (11); 50-56 (12) and 89-97 (13)) are underlined. These sections are the same, regardless of the numbering system, according to Kabaya or Spoipi.

Variable section of light chain (MI) 17020t a3521M11 M5EO IYUO MO:70, coded

ZEO IU MO:69)

ORMGTORRBOY BMBROOTABITOZOEKI JUICE AUMUOOOKROOBZRMI UMMOVKOVRZaYaRENE5SO

ZMZamtTATI TIza TGOAMOEAVUMSOAMOZZTAMESSSITKI TMI, variable section of the light chain (MI) 17M1bt a17M9 (ZEO IV MO:71)

ZUEEPORROUZMARSOTATITSAaOM SKKAUNMUOOARIOOARMI MIMOOZOVRza! ROVEZABM

ZAMTATI TITASEAVREAOUMSMOIATONUURGHIASITKI TM AADOISEOKI IZE.

Anti-MABR-2 antibodies OM5100 and Modr a3521M11M| (containing the heavy chain variable region represented in ZEO IU MO:67 and the light chain variable region represented in 5EO IUO MO:70, also called "OM5646" and "tABb"), both of which demonstrated binding of human MABR- 2 with high affinity and have the ability to block the functional activity of complement, were analyzed for epitope binding using dot-blot analysis. The results show that antibodies OM5646 and OM5100 are highly specific for MA5P-2 and do not bind to MA5P-1/3. No antibody associated with

MAb19 or MA5BR-2 fragments did not contain the SSRI1I domain of MABR-2, which allowed us to conclude that the binding sites include SSRI1.

It was determined that the anti-MA5P-2 antibody OM5646 actively binds to the recombinant

MA5P-2 (Ka from 60 to 250 μM) with a selectivity that exceeds 5000 times that for binding

C15, C1g or MA5R-1 (see table 9 below).

TABLE 9

Binding affinity and specificity of the interaction of anti-MABR-2 antibody OM5646 with MA5R-2, according to the results of solid-phase ELISA "average value 50; n-12.

OM5646 specifically blocks components of lectin-dependent activation

Methods

The effect of OM5646 on membrane attack complex (MAC) deposition was analyzed using specific conditions for the lectin pathway, the classical pathway, and the alternative pathway. For this purpose, a kit was used for complement screening UMeziab Sotr300 (Umeziab, Ipa, zugedep), according to the manufacturer's instructions.

The results

In fig. 12A graphically shows the level of MAC deposition in the presence or absence of anti-MA5P-2 antibody (OM5646) under conditions specific to the lectin pathway. In fig. 128 graphically shows the level of MAC deposition in the presence or absence of anti-MA5R-2 antibody (OM5646) under conditions specific to the classical pathway. In fig. 12C graphically shows the level of MAC deposition in the presence or absence of anti-MA5R-2 antibody (OM5646) under alternative pathway-specific conditions.

As shown in fig. 12A, OM5646 blocks lectin-mediated deposition activation

MAC with an IC value of approximately 1 nM. However, OM5646 had no effect on deposition

MAC, which occurs as a result of activation mediated by the classical pathway (Fig. 128) or alternative pathway (Fig. 1253).

Pharmacokinetics and pharmacodynamics of OM5646 after intravenous (IM) or subcutaneous (52) administration to mice

The pharmacokinetics (PK) and pharmacodynamics (PDO) of OM5646 were evaluated in a 28-day study

RK/PO in mice with a single dose. The study tested dose levels of 5 mg/kg and 15 mg/kg OM5646 administered subcutaneously (5C), as well as a dose level of 5 mg/kg OM5646 administered intravenously (IM).

As for the OM5646 LCD profile, in fig. 13 graphically shows the concentration of OM5646 (average number of p-Z animals per group) as a function of time after administration of OM5646 in the indicated dose. As shown in fig. 13, at 5 mg/kg of 5C, the maximum concentration of OM5646 in plasma, equal to 5-6 μg/ml, was reached approximately 1-2 days after dosing. Bioavailability

OM5646 at 5 mg/kg 5C was approximately 60 95. In fig. 13 also shows that at 15 mg/kg 5C, the maximum plasma concentration of OM5646, equal to 10-12 μg/ml, was reached approximately 1-2 days after dosing. In all groups, OM5646 was slowly eliminated from the systemic circulation with a terminal half-life of approximately 8-10 days. The profile of OM5646 appeared to be typical of human antibodies in mice.

RO activity of OM5646 is graphically shown in fig. 14A and 14B. In fig. 14A and 14B show the PO response (decrease in systemic activity of the lectin pathway) for each mouse in groups receiving 5 mg/kg IM (Fig. 14A) and 5 mg/kg 5C (Fig. 148). The dashed line indicates baseline in the assay (maximal inhibition, intact mouse serum injected /l mto with excess

OM5646 before analysis). As shown in fig. 14A, after IM administration of 5 mg/kg OM5646, lectin pathway activity of the complement system immediately decreased to almost undetectable levels, and recovery of lectin pathway activity over the 28-day observation period was moderate. As shown in fig. 1488, in mice that received a dose of 5 mg/kg 0M5646 5C, a time-dependent inhibition of the activity of the lectin pathway was observed. Activity of the lectin pathway decreased to almost undetectable levels within 24 hours of drug administration and remained at a low level for at least 7 days.

Lectin pathway activity gradually increased over time but did not return to predose levels during the 28-day observation period.

The time-dependent profile of lectin pathway activity observed after administration of 15 mg/kg 5C was similar to that for the 5 mg/kg 5C dose (data not shown), indicating saturation of the PO endpoint. The data also indicate that doses of 5 mg/kg OM5646 administered once weekly either IM or 5C are sufficient to achieve sustained inhibition

On the activity of the lectin pathway of the complement system in mice.

EXAMPLE 13

This example describes the production of recombinant antibodies that inhibit MA5P-2, containing a variable region of the heavy chain and/or light chain, which contains one or more SOCs that specifically bind to MABR-2, and at least one nuclear peptide sequence ZOMI (also called anti-MA5bR-2 antibody carrying the ZOMI peptide or its antigen-binding fragment).

Prerequisites/reasoning

The creation of specific inhibitors of MA5P-2, called 5OMI-2, is described in Neda ei ai., 9. Vioi!.

Spent 287:20290 (2012); and Neda Yeyi AI., РМА5, 109:10498 (2012), which are included in this description as a reference. 5OMI-2 is a 36 amino acid peptide that was selected from a phage library of bsRiviosegs dgedap protease inhibitor 2 variants, in which six of the eight positions of the protease-binding loop were completely randomized. The subsequent evolution of Ugoda resulted in monospecific inhibitors with single-digit Ki values in the nM range (Nea eyi ai., 9. VioiI., Spet., 287:20290, 2012). Structural studies have shown that the optimized protease-binding loop forms the primary binding site that determines the specificity of the two inhibitors. The amino acid sequences of the extended secondary and internal binding regions are common to both inhibitors and contribute to the contact region (He)a ei ai., 2012.

U. Visi. Spet., 287:20290). Mechanically, ZOMI-2 blocks the activation of the lectin pathway of complement, without affecting the classical pathway (Neda Yei Ai., 2012. Rhos. May. Asaid. 5si. 109:10498).

Amino acid sequences of zOMI-2 inhibitors are given below:

ZOMI-2I,, full-size:

HEMTSEROTTEKOKSMTSsASABOOaKZAMSTKI MUSMO (5EO IYU MO:72),

ZOMI-2GI, medium length:

TSEROTTEKOKSMTsASABORAKZAMSTKI MSMO (ZEO I MO:73),

ZOMI-2I,, short: toneSavzrakvzAMSTKIMSMO (5EO IO MO:74).

As described in this example, as well as in UMO 2014/144542, anti-MAB5R-2 antibodies carrying the ZOMI-2 peptide and their fragments were obtained by fusion with the amino acid sequence of the ZOMI-2 peptide (ZEO IO MO:72, 73 or 74) at the amino or carboxyl end of the heavy and/or 60 light chain of the human anti-MA5R-2 antibody. Anti-MA5BR-2 peptide-carrying antibodies

ZOMI-2 and their fragments have increased inhibitory activity compared to the anti-MABR-2 antibody with a bare backbone, which does not contain the 5OMI-2 peptide sequence, according to the results of the C3B or C4Bb deposition assay using human serum, as described in UMO 2014/144542, and also have increased inhibitory activity compared to anti-

MA5BR-2 antibody with a bare skeleton, measured in a mouse model /l m/imo. Methods of obtaining anti-MA5BR-2 antibody carrying the 5OMI-2 peptide are described below.

Methods

Expression constructs were obtained to encode four illustrative anti-MA5R-2 antibodies carrying the 5ZOMI-2 peptide, where the ZOMI-2 peptide was fused to either the M- or C-terminus of the heavy or light chain of a representative MABR-2 inhibitory antibody OM5646 ( as described in example 12).

TABLE 10

FUSION MA5BR-2 ANTIBODY/ZOMI-2 nIi-m2 bolemderoomeme | 70017101 n-ema-5emMmi2-m 0 | vami2 | - | - | - 1 75570 n-ma-5ami2s | 7 - | 5aMmi2 | /- | - | 76570 - ma-zamiaM HER 7777-1717 - | in5Bami2 | - | 67-77 p-ma-zemiaS HER 7777-7177 - 171 - | Bema | 67-78

Designation in table 10: "H-M": the amino end of the heavy chain; "H-C"-carboxyl end of the heavy chain; "| -M" is the amino end of a light chain; "| -бС"" the carboxyl end of the light chain; "M2g" hosts MABR-2 AR (0M5646).

For the M-terminal fusions shown in Table 10, a peptide linker (oTobooobOo55 5EO IU MO:79) was added between the 5OMI-2 peptide and the variable region.

For the C-terminal fusions shown in Table 10, a peptide linker (HAАСО5О' ZEO

IO MO:80) between the constant region and the 5SOEMI-2 peptide, and the second peptide "Z5OA" (ZEO IU MO:81) was added at the C-end of the fused polypeptide to protect the C-terminal peptides of 5OMI-2 from degradation.

The amino acid sequences are shown below for the following representative MA5R - 2 antibody/FZOMI-2 fusions:

H-M2arb-5OMI-2-M (ZEO IO MO:75, coded 5EO IO MO:82):

HEMTSERSOTTEKOXMTSASOaZOaKKKBZAUSTK USMOSTISOsSoSOvOOMTI KEZORMI MKRTET

EtstotUzaevy znakmauvzUvoRROKAI EM ANIEEZ5ZOEKOZMUVT5I KOZA TİZKOTZKMOMMI TM

MMORMOTATUUSAVIVNNSSISVUMUSOSTI MTGU55AZTKaROSZMERI ARSZAZTZEZTAAG SS MKOMER

ERMTUBM/MZSIAI TZaMNTERAMI O55O11 U5I 55 URZBII TKU SMMONKRUOMTKUMOKAMEZK

УЯАРСОРРОРАРЕРИ САРБЗМУЕГРРКРКОТИ МИЗВТРЕУТСУУМОУЗОБЕОРЕМОЕМИУ MMrОaМЕМНМА

KTKRAEEEOEMZTUVUMUMI TM NOVU ME KEUKSKUSMKai RZBIEKTIZKAKYOORAVEROMMUTI RRB

OBEMTKMOM5I TS UKEEMRZOIAMEM EZMOOREMMUKTTRRMI YUOBORAZERI BY TMOKZAMOvS

MUMEZSZUMNEAIII NMNUTOKOI 51 5I SC 491 aa protein, aa 1-36-5025MI-2 (underlined), aa 37-46-linker (highlighted in italics); aa 47- 164-variable section of the MABR-2 heavy chain ab Mo 6 (underlined); aa 165-491-Ids4 constant region with a mutation in the hinge region.

H-M2arb-5OMI-2-S (ZEO IO MO:76, coded 5EO IO MO:83):

OMTKEBZOARMI MKRTET TTSTUzaIaE5I BDaEKMOamu5UVORROKASEUM ANIEZZOEKOZUVTOI.

KZASTIZKOT5KMOMMI TMTMMORUYUVTATUUSAVIV NOSI SOSTI UTU55AZTKOaROZMUERI ARS

ZAZTZEZTAAI JSC UKOMERERUTUBUUMZSIAI TZAUNTERAMI O55O0I 51 55 GURZBUITKTMT

SMUONKRUHMTKUOKAMEZKUSRRORRARARERI ORBZMUBI ERRKRKOTI MIZVAVTREMTSUUMOMBOE

OREMOEEMUMUramMEMMNMAKTKRAEEOEMZTUVUMMI TM NOBUULI MSKEUKOSKUZMKYai RZOEKTI

ZKAKYORVEROMUTI RRZOEEEMTKMOMUBI TS UKIEMRZOIAMEZHM EZMSOOREMMUKTTRRMI O502

ZE MZA TMOKZAUMOB,MMEZSZUMNEAIII NMNUTOKOBI 5I BI AKAASSOSI EMTSERSTTEKOX mmmtovsa,avzrakvAUStTkIUSMOssA 491 aa protein, aa 1-118-variable section of heavy chain MA5BR-2 ab Mo 6 (underlined);

aa 119-445-|3654 constant region with a mutation in the hinge region; aa 446-451-first linker (in italics); aa 452-487-5(02MI-2; aa 488-491-second linker (highlighted in italics).

Г-М2арбв-5СаМИ-2-М (5EO IO MO:77, coded ZEO IU MO:84):

HEMISERSOTTRKOKSOMTs!SsaOSKBZAMSTKI MSMOSTEISOSOOOBORMI TORRBI 5M5ROYUT

ABITOVIEK arKUAUMMOOKREOBRMI UMUOSKOVRZSIRENEZOZMZSIMTATI TIBSTOAMOEA

VUMSOAMroZGTAMgasaTtk TM aORKAARZMTI ERRZEBEI OAMKATI MSG IBOEMROAMTMAMKA

P5ZRUKASMETTTTROKOZMMKMUAAABZUZMI ZI TREOMUKZNAZUZSOMTNEVZTUEKTUMARTESZ (258 aa protein, aa 1-36-5025MI-2 (underlined); aa 37-46-linker (highlighted in italics); aa 47-152-variable section of the MABR-2 light chain ar Mo 6 (underlined); aa 153-258-constant section of human Id lambda).

H-M2arb-5IaMI-2-S (5EO IO MO:78, coded ZEO IU MO:85):

ORMITORRBOY BM5ROOTABITOZZSEKG SOKMAMUUOOOKRIOBRMI UMUOVSKOVARZIaIREVEZSO

ZMZamMTATI TIZS TOAMOEAVUMSOAM O551TAMgSaIITtkeTM SORKAARBZMTI ERRZBZEBY OAMKA

TMSPIZOEUREIAMTMAMKAOZZRUKASMETTTRUKOZMMKUAABZOZMUI ZI TREOSMUKZNAZUZSOMTN

EVZTUEKTMARTESZAASSSI EMTISERSOTTEKOXMT SnNSSOSZVIakKZAUSTK USMOSESA (258 aa protein, aa 1-106-variable region of the light chain MABR-2 ar Mo 6 (underlined); aa 107-212-constant region of human Id lambda; aa 213-21v8-first linker; aa 219- 254-5(2MI-2; aa 255-258-the second linker.

Functional analysis

Four anti-MA5R-2-502MI-2 fusion antibody constructs were transiently expressed in Ehri293E cells (Ipmigodep), purified by protein A affinity chromatography, and tested in 10 95 normal human serum for inhibition of SZB deposition in a mannan-coated microsphere assay. as described below.

Testing of MAZR-2-5ОМИ-2 fusions in the analysis of SZBB deposition using mannan-coated microspheres

MA5BR-2-5OEMI-2 fusion antibodies were evaluated for their ability to inhibit the lectin pathway in the SZBbB deposition assay on mannan-coated microspheres. This assay, which allows determination of the degree of activity by flow cytometry, has a higher resolution than the UmeziarfF assay. Analysis of the lectin pathway using microspheres was performed as follows

In this way: mannan was adsorbed on polystyrene microspheres with a diameter of 7 µM (Vapo5

And arogafogye5, Rizpegv5, IM, OA) overnight at 4 "С in a carbonate-bicarbonate buffer (pH 9.6). Microspheres were washed with РВ5 and exposed to 1095 human serum or 10 95 serum pre-incubated with antibodies or inhibitors. A mixture of serum- the microspheres were incubated at room temperature for one hour with agitation. After incubation in serum, the microspheres were washed, and the deposition of C3Ob on the microspheres was measured by detection with an anti-C3c rabbit polyclonal antibody (Yuko Mogpipe Ategisa,

Sagrippiegia, SA, YUBA) and RE-Sub-conjugated goat anti-rabbit secondary antibody (Zoshegp Vioiesi, Vippipdpat, AG, O5A). After the staining procedure, the microspheres were analyzed using a flow cytometer. The microspheres were analyzed as a homogenous population using forward and side scatter, and NWB deposition appeared as

EI of C-positive particles (PI-3 or "PI-3-channel" indicates the third or red channel on the cytometer). Population geometric mean fluorescence intensity (MEI) for each experimental condition was plotted against antibody/inhibitor concentration to assess inhibition of the lectin pathway.

ISvo values were calculated using the SgarpRai RKIZM software.

In particular, the value of ISvo was obtained by applying a non-linear (four-parameter), with variable angle of inclination fitting curves of the dependence of Id (antibody) on the average intensity of fluorescence, obtained as a result of cytometric analysis.

The results are shown in Table 11.

TABLE 11

Deposition of SZB (assay using mannan-coated microspheres) in 10 95 human serum

The results

A control anti-MABR-2 antibody with a "bare" core that does not contain ZOMI (tAr Mo 6) was inhibitory in this assay with an IC50 value of 3.63 nm, which is consistent with the inhibition results observed in Example 12. It is noteworthy that , that, as shown in Table 11, all 5OMI-2-MA5BR-2 antibody fusions that were tested improved the efficacy of the MABR-2-backbone antibody in this assay, indicating that increased valence may also be beneficial in suppressing deposition of C3r.

Testing MABR-2-5024MI-2 fusions in a mannan-coated microsphere assay to study C46 deposition with 10 95 human serum

C4b deposition analysis was performed using 10 95 human serum, under the same conditions of analysis as described above for SZB0 deposition analysis, with the following modifications.

Analysis for the detection of CA and flow cytometry analysis was performed by staining the deposition reaction with an anti-C45b mouse monoclonal antibody (1:500, OpcideI) and by staining with a secondary goat anti-mouse E(ar')g2 conjugated with PE-Su5 ( 1200, boshpet

Vioyesi)), before flow cytometry analysis.

The results

Both fusions 50OM5-2-carrying anti-MABR-2-M-terminal antibody (Н-М2-5СаМИ-2-М:

ISvo-0.34 NM, I-M2-5OMI-2-M: ISvo-0.41 nM) had increased efficiency in comparison with the anti-MA5R-2-ostov antibody (NI-M2: ISvo-0.78 NM) .

Similarly, the fusion of a single 5OM-2-bearing C-terminal anti-MM5-2 antibody (H-

M2-5OMI-2-S: ISvo-0.45 nM and G-M2-52MI-2O: ISvo-0.47 NM) had increased activity compared to the anti-MA5R-2-ostov antibody (NI -M2: ISvo -1.2 nM).

Testing MA5BR-2-52MI-2 fusions in the SZB deposition assay with mannan-coated microspheres with 10,956 mouse serum

The deposition assay of SZB with mannan-coated microspheres was performed as described above with 1095 mouse serum. Similar to the results observed in the case of using human serum, it was established that in mouse serum the fusion of ZOMI-2-

Anti-MABR-2 carriers have increased efficiency compared to the anti-MABR-2 antibody.

A brief overview of the results. The results in this example demonstrate that all mergers

ZOAMI-2-MABR-2 antibody, which were tested, improved the efficiency of the anti-

MA5R-2-rest.

EXAMPLE 14

This example presents results obtained using a unilateral ureteral obstruction (USO) renal fibrosis model in MAZR-2-/- deficient and MAZR-2-/-- sufficient mice to assess the role of the lectin pathway in renal fibrosis.

Prerequisites/reasoning

Kidney fibrosis and inflammation are characteristic features of late-stage kidney disease.

Renal tubulointerstitial fibrosis is a progressive process involving persistent cell injury, aberrant healing, activation of resident and infiltrating renal cells, cytokine release, inflammation, and phenotypic activation of renal cells to produce extracellular matrix. Renal tubulointerstitial (TI) fibrosis is a common endpoint of multiple renal pathologies and represents a key target for potential therapies aimed at preventing progressive renal functional impairment in chronic kidney disease (CKD). Renal TI insufficiency is closely related to a decrease in kidney function in glomerular diseases (Kizaop K.A. ei ai., I apsei. 1:363-366, 1968;

ZesNaipysk I.I. ei ai., Nit. Raiipoi 1:631-640, 1970; Maiy K.A., At. U. Kia. Oiv. 20:1-17, 1992) and is characteristic of CKD, in which there is an accumulation of myofibroblasts, and the potential space between tubules and peritubular capillaries is blocked by a matrix consisting of collagens and other proteoglycans. The question of the origin of TI myofibroblasts remains quite controversial, but fibrosis is usually preceded by inflammation, which is first characterized by the accumulation of T lymphocytes and then macrophages (Gi U. ei aI., Mai. Chem. Merpgoi. 7:684-696, 2011;

BinyivYia 9U.5., u. Siip. Ipmevi 124:2299-2306, 2014).

The OO rodent model generates progressive renal fibrosis, a hallmark of progressive kidney disease of almost any etiology (Spemaiieg ei ai., Kidpeu Ipiegpaiopai, 75:1145-1152, 2009). There are reports of increased expression of the SZ gene in wild-type mice after OO and a significant decrease in collagen deposits in C3-/- knockout mice after OO compared to wild-type mice, which indicates the role of complement activation in renal fibrosis (Reat et al.,

MY. And so on. 48:1666-1733, 2011). There are also reports that C5 deficiency led to a significant weakening of the main components of kidney fibrosis in a model of tubulointerstitial injury (Voog R. ei ai., U. oi At. bos. oi Merpgoiodu, 18:1508-1515, 2007). However, prior to the research described in this application and performed by the inventors, the specific complement components involved in renal fibrosis were not clearly defined. Thus, the following study was conducted to evaluate MA5R-2 (-/-) and MABR-2 (1/k) mice in a unilateral ureteral obstruction (USO) model.

Methods

The MA5R-2-/- mouse was obtained as described in example 1, and subjected to backcrossing for 10 generations with C57VI/yu. Wild-type (M/T) C57VI/6 mice and homozygous MA5R-2-deficient (MABR-2-/-) mice on the C57VI/6 background were kept in standardized conditions with a 12/12 day/night cycle, while the mice were fed standard food pellets, water and food were freely available. Ten-week-old mice, b per group, were anesthetized with 2.595 isoflurane at 1.5 l/min. oxygen Right ureters in two groups of ten-week-old male mice

C56/VI 6, wild type and MA5R-2-/- were surgically ligated. The right kidney was exposed through a lateral incision 1 cm long. The right ureter was completely blocked at two points using polyglactin thread 6/0). Anesthesia was performed intraoperatively with buprenorphine every 12 hours up to 5 doses depending on the degree of pain. Local anesthesia

Bupivacaine was administered once during surgery.

Mice were killed 7 days after surgery, and kidney tissues were collected, fixed, and embedded in paraffin blocks. Blood was collected from mice by cardiac puncture under anesthesia, and after nephrectomy, the mice were exsanguinated. Blood was left on ice to coagulate for 2 hours, and serum was separated by centrifugation and stored frozen in 35 aliquots at -80 20.

Immunohistochemistry of kidney tissue

To measure the degree of renal fibrosis by collagen deposition, 5-micron paraffin sections of kidneys were stained with picrosirius red, a collagen-specific stain, as described in

Mupinakeg R. ei a!., Vazis Vez. Sagaiyo!. 89:397-410, 1994. Briefly, kidney sections were deparaffinized, 40 rehydrated, and collagen stained for 1 hour with an aqueous solution of picrosirium red (0.5 g of Sirius Red, Zidt, Juogzei OK) in 500 mL of a saturated aqueous picric acid solution. The slides were washed twice with acidified water (0.5 95 glacial acetic acid in distilled water) for 5 minutes each time, then dehydrated and prepared for examination. 45 To measure the degree of inflammation, as evidenced by macrophage infiltration, kidney sections were stained with the macrophage-specific antibody E4/80 as follows. Formalin-fixed, paraffin-embedded 5-micron kidney sections were deparaffinized and rehydrated.

Antigen unmasking was carried out in citrate buffer at 95 °C for 20 minutes, followed by blocking of endogenous peroxidase activity by incubation in C 95 NgO» 50 for 10 minutes. Tissue sections were incubated in blocking buffer (10 95 heat-inactivated normal goat serum with 1 95 bovine serum albumin in phosphate-buffered saline (PB5)) for 1 hour at room temperature, followed by blocking with avidin/biotin. Tissue sections were washed with PB5 after each stage three times for 5 minutes. Primary antibody to macrophage E4/80 (Zapia Stgy7, Raiiav, TX, 55 OBA), diluted 1:100 in blocking buffer, was applied for 1 hour. Biotinylated goat anti-rat secondary antibody, diluted 1:200, was then applied for 30 minutes, followed by application of horseradish peroxidase (HPR)-conjugated enzyme for minutes Color staining was developed under the influence of diaminobenzidine (AB) substrate (uUesiog I arch, Regegrogotsdp SHK) for 10 minutes, and the slides were washed in water, dehydrated and 60 prepared for examination without counterstaining to facilitate computer analysis.

Image analysis

The percentage of staining of the cortical layer of the kidneys was determined as described in Rygpe55 R.M. hey ay.,

U. Siip. Rainpoi 50:118-122, 1997. Briefly, 24-bit color images were taken from consecutive non-overlapping fields of the renal cortical substance immediately below the renal capsule around the entire periphery of the kidney slice. After each capture, the image was used

MIN image to extract the red channel from it as an 8-bit monochrome image. The non-uniformity of the background illumination was subtracted using a pre-recorded image of the illuminated field of the microscope without a slice placed on it. A fixed threshold was set for the image to determine the area of the image corresponding to positive staining. The percentage of black pixels was then calculated, and after all images around the kidney were measured in this way, the average percentage was recorded, providing a value corresponding to the percentage of stained area in the kidney section.

Analysis of gene expression

The expression of several genes related to renal inflammation and fibrosis in mouse kidneys was measured by quantitative PCR (qPCR) as follows. Total RNA was isolated from the renal cortical substance using Ttgi2o!iFf (TeptoRib5peg Zsiepiis, RaibIeu, SHOK), according to the manufacturer's instructions. Extracted RNA was treated with a Tigro DNA-free kit (Tpeptogizop 5ssepiiis) to eliminate DNA contamination, and then first-strand cDNA was synthesized using the AMM reverse transcription system (Rgoteda, Maadizop, UM, O5A). Integrity

cDNA was confirmed by a single qPCR reaction using the TadMap SARON Avzzau kit (Arriei Viosuziet5, Raizieu, OK) followed by a qPCR reaction using Sybiot Tadmap Agtau 96-well plates (Ie Thesppoiodie5, Raizieu, OK).

Twelve genes were studied in this analysis:

Collagen type IM alpha-1 (soi4o--1; analysis ID: MipO1210125 t/);

Transforming growth factor beta-1 (TaErd-1; analysis ID: MtO1178820 t/!);

Cadherin-1 (San-1; analysis ID: Mt0O1247357 t/!);

Fibronectin 1 (Ep1; analysis ID: MtO1256744 ti);

Interleukin-6 (II -6; analysis ID Mt00446191 t/);

From Interleukin-10 (1-10; analysis ID MtO0439614 t/);

Interleukin-12a (II -12a; ID analysis MtoO0434165 pt);

Vimentin (Mitt; analysis ID MtO1333430 t);

Actinin alpha-1 (Assip-1; analysis ID MtOo1304398 1);

Wasp tumor necrosis factor; (TMEs, analysis ID Mt0O0443260 91);

Complement component C (C3; analysis ID MtO0437838 t/!);

Interferon-gamma (Np-y; analysis ID MtO1168134).

The following constitutive control genes were used:

Glyceraldehyde-3-phosphate dehydrogenase (SARON, analysis ID Mitp99999915 91);

Glucuronidase beta (Sy5r; analysis ID MitO0446953 tt!);

Eukaryotic 185 rRNA (185; analysis ID H»599999901 51);

Hypoxanthineguanine phosphoribosyltransferase (HRET; analysis ID MipO0446968 1). 20 μl of the reaction agents were amplified using Ravi Opimegzai's TadMap mix

Mazhieg Mih (Arriiei Viozuvzietv5), for 40 cycles. Real-time PCR amplification data were analyzed using Ariyed Viozuviet5 7000 505 m1.4 software.

The results

After unilateral ureteral obstruction (USO), an influx of inflammatory cells, in particular macrophages, is observed in the occluded kidneys, followed by the rapid development of fibrosis, which is evidenced by the accumulation of collagen along with the dilatation of the tubules and the thinning of the proximal tubular epithelium (see Spemaiyeg E.I... ei ai ., Kiapeu Ipi. 75:1145-1152, 2009).

In fig. 15 graphically shows the results of computer analysis of images of kidney tissue sections stained with Sirius red, where tissue sections were obtained from wild-type mice and

MABR-2-/- mice 7 days after ureteral obstruction (UOO) and from sham-operated control mice. As shown in fig. 15, kidney sections of wild-type mice 7 days after ureteral obstruction showed a significantly greater amount of collagen deposits compared to MA5 P-2-/- mice (p-value 0.0096). The mean values and the standard error of the mean for SHO-operated mice in the wild-type and MA5R-2 -/- groups were 24.79-1.908 (n-b) and 16.58-41.3 (n-b), respectively. As further shown in FIG. 15, tissue sections from sham-operated control wild-type mice and sham-operated control MA5R-2 -/- mice showed very low levels of collagen staining, as expected. because in fig. 16 graphically shows the results of computer analysis of images of kidney tissue sections stained with antibodies specific for the macrophage marker E4/80, where tissue sections were obtained from wild-type mice and MAZR-2-/- mice 7 days after ureteral obstruction or from sham-operated controls mice As shown in fig. 16, compared with wild-type mice, tissues obtained from SHO-kidneys of MABR-2-/- mice showed significantly less macrophage infiltration 7 days after ureteral obstruction (95 area of macrophages stained in M/T: 2.23- -0.4, against MABR-2-/-: 0.53--0.06, p-0.0035). As further shown in FIG. 16, tissue sections from pseudooperated wild-type mice and pseudooperated MA5R-2-/- mice did not show detectable staining of macrophages.

Analysis of the expression of a number of different genes associated with renal inflammation and fibrosis was performed on kidney tissue sections obtained from wild-type mice and MABR-2-/- mice 7 days after ureteral obstruction and from sham-operated wild-type and MAZR-2 mice -/- mice.

The data shown in Fig. 17-20, represent the Godot relative abundance in a sample of pseudooperated wild-type mice, and the bars represent the standard error of the mean. Regarding the results of the expression analysis of the genes associated with fibrosis in fig. 17 graphically shows the relative mRNA expression levels of IM collagen type alpha-1 (collagen-4), measured by qPCR in sections of kidney tissues obtained from wild-type mice and MABR-2-/- mice 7 days after ureteral obstruction and from sham-operated controls mice In fig. 18 graphically shows the relative mRNA expression levels of transforming growth factor beta-1 (TOaEr-1), measured by PCR, in sections of kidney tissues obtained from wild-type mice and MABR-2-/- mice 7 days after ureteral obstruction and from sham-operated controls mice As shown in fig. 17 and 18, occluded kidneys of wild-type mice showed a significantly increased expression of fibrosis-related genes collagen type IM (Fig. 17) and TOEr-1 (Fig. 18) in comparison with pseudo-operated kidneys of wild-type mice, which indicates that , that these fibrosis-related genes were induced after SHO injury in wild-type mice, as expected. In contrast, as further shown in FIG. 17 and 18, the occluded kidneys of MABR-2-/- mice subjected to SHO-injury showed a significant decrease in the expression of type IM collagen (Fig. 17, p-0.0388) and a significant decrease in the expression of taggr-1 (Fig. 18, p -0.0174) compared to wild-type mice subjected to SHO injury.

As for the results of the analysis of the expression of genes related to inflammation, in fig. 19 graphically shows the relative expression levels of interleukin-b mRNA (1-6), measured by PCR in sections

From kidney tissues obtained from wild-type mice and MABR-2-/- mice 7 days after ureteral obstruction and from sham-operated control mice. In fig. 20 graphically shows the relative expression levels of interferon-γ mRNA, measured by PCR, in kidney tissue sections obtained from wild-type mice and MAZR-2-/- mice 7 days after ureteral obstruction and from sham-operated control mice. As shown in fig. 19 and 20, occluded kidneys in wild-type mice showed significantly increased expression of the inflammation-related genes interleukin-b (Fig. 19) and interferon-y (Fig. 20) in comparison with sham-operated kidneys of wild-type mice, indicating that , that these inflammation-related genes are induced after SHO injury in wild-type mice. In contrast, as further shown in FIG. 19 and 20, the occluded kidneys of MA5P -2-/- mice subjected to SHO lesions showed a significant decrease in the expression of interleukin-b (Fig. 19, p-0.0109) and interferon-y (Fig. 20, p-0, 0182) in comparison with wild-type mice exposed to SHO-injury.

It should be noted that the expression of Mit, Asip-1, TMEso, SZ, and 11-10 genes is significantly enhanced in SHO-kidneys obtained from both wild-type mice and MA5R-2-/- mice, without a significant difference in the expression levels of these of specific genes between wild-type mice and MABR-2-/- mice (data not shown). Expression levels of Sap-1 and 1/-12a genes did not change in the occluded kidneys of animals in any of the groups (data not shown).

Discussion

The OSHO rodent model is recognized to induce early, active, and profound injury in the occluded kidney with reduced renal blood flow, interstitial inflammation, and rapid fibrosis within one to two weeks after obstruction and has been widely used to understand the general mechanisms and mediators of inflammation and fibrosis in the kidney ( see for example

Spemayeg VG, Kidpeu Ipi. 75:1145-1152, 2009; Map of N. ei ai., Ogyd Oibvsom. Todau iv. Modeiv, 7:13-19, 2010).

The results described in this example demonstrate a significant reduction in collagen deposition and the level of macrophage infiltration in SHOSO-operated kidneys in MABR-2 (-/-) mice compared to control (1/5) wild-type mice. Unexpected results indicating a significant reduction of kidney damage both at the histological level and at the level of gene expression in

MA5P-2-/- animals, show that the lectin pathway of complement activation affects the development of inflammation and fibrosis in the occluded kidney. Without being bound by a particular theory, it is believed that the lectin pathway influences the pathophysiology of fibrotic disease by initiating and maintaining pro-inflammatory stimuli that perpetuate a vicious cycle in which cell damage causes inflammation, which in turn causes further cell damage, scarring, and tissue loss. . Based on these results, inhibition or blockade of MABR-2 by an inhibitor is expected to have a prophylactic and/or therapeutic effect, manifested in inhibition or prevention of renal fibrosis, as well as inhibition or prevention of fibrosis in general (ie, independent of tissue or organ).

EXAMPLE 15

This example describes the analysis of the effectiveness of a monoclonal inhibitory antibody

MA5R-2 in the model of unilateral ureteral obstruction (USO), a mouse model of renal fibrosis.

Prerequisites/reasoning

Amelioration of tubulointerstitial fibrosis, a common endpoint of many renal pathologies, represents an important target for therapies aimed at preventing the progression of renal disease. Given the paucity of new and existing therapies targeting inflammatory profibrotic pathways in kidney disease, there is an urgent need to develop new therapies. Many patients with proteinuric kidney disease have tubulointerstitial inflammation and progressive fibrosis, which are closely related to the decline in kidney function. Proteinuria itself causes tubulointerstitial inflammation and leads to the development of proteinuric nephropathy (Vhyp5Kii! M.u. ei aiI., U. At. os. MerpgoiY. 15:504-505, 2004). Regardless of the primary kidney disease, tubulointerstitial inflammation and fibrosis are invariably observed in patients with progressive renal failure and are closely correlated with a decrease in excretory function (Kizaop E.A. ei ai., I apsei. 1:363-366, 1968; ZsNnaipyskK I.I. Yei aI., Nyt. Raiyoi. 1:631-640, 1970). Therapies capable of interrupting common key cellular pathways leading to fibrosis will find widespread use in kidney disorders.

As described in example 14, in the SHSO-model of non-proteinuric renal fibrosis, it was established that MAZR-2-/- mice have a significantly lower degree of renal fibrosis and inflammation compared to wild-type control animals, as evidenced by inflammatory cell infiltrates (75 9b) and histological markers of fibrosis, such as collagen (reduced by one third), thereby proving the key role of the lectin pathway in renal fibrosis.

As described in example 13, it was also shown that the synthesized monoclonal anti-MABRI-

ZO 2 antibody (0OM5646-502MI-2 fusion protein containing the ZOMI-2 peptide fused to the C-terminus of the OM5646 heavy chain), which specifically blocks the function of the lectin pathway in humans, blocks the lectin pathway in mice. In this example, OM5646-524MI-2 was analyzed in the murine SHSO model of renal fibrosis in wild-type mice to determine the ability of a specific inhibitor

MA5P-2 suppresses kidney fibrosis.

Methods

This study evaluated the effects of an MA5R-2 inhibitory antibody (10 mg/kg OM5646-502MI-2) compared with a human Id(4) isotype control antibody (10 mg/kg ET904) and a delivery medium control in male M/Y C57VI /6 mice. Antibodies (10 mg/kg) were administered to groups of 9 mice by intraperitoneal (i.r.) injection on day 7, day 4, and day 1 before OO surgery and then on 2 Day after surgery Blood samples were taken before the administration of antibodies, and the functional activity of the lectin pathway was assessed at the end of the experiment.

The OO operation, tissue collection and staining with gray mustache red and macrophage-specific antibody E4/80 were performed by the methods described in example 14.

The content of hydroxyproline in the kidneys of mice was measured using a specific test kit for colorimetric analysis (Zidta), according to the manufacturer's instructions.

To assess the pharmacodynamic effect of the inhibitory tAr-MABR-2 in mice, the activity of the lectin pathway of the complement system was assessed by quantifying the lectin-induced activation of SZ in minimally diluted serum samples collected at the specified time after i.r. administration of MABR-2 or control tAr mice. Briefly, polystyrene microspheres with a diameter of 7 μM (Vapoz Garogayogie5, Rizpeg IM, OBA) were coated with mannan by overnight incubation with 30 μg/ml mannan (Zidta) in sodium bicarbonate buffer (pH 9.6), then washed, blocked with 1 95 fetal bovine serum in RV5Z and resuspended in RV5 to a final concentration of 1 x 108 myrospheres/ml. Complement deposition reactions were initiated by adding 2.5 μl of mannan-coated microspheres (7,250,000 microspheres) to 50 μl of minimally diluted mouse serum samples (final serum concentration 90–95), followed by incubation for 40 minutes at 4 C. After termination of the deposition reaction by adding 250 μl of ice-cold buffer for flow cytometry (EB:PB5 containing 0.1 95 fetal bovine serum), the microspheres were collected by centrifugation and washed two more times with 300 μl of ice-cold EB. bo For quantitative assessment of lectin-induced activation of SZ microspheres were incubated for 1 hour at 4 2С with 50 μl of rabbit anti-human SZs antibody (OSAko, Sagrepiegia, SA, OBA) diluted in EV. After two washings with EB to remove unbound material, the microspheres were incubated for 30 minutes at 4 C with 50 μl of goat anti-rabbit antibody conjugated with RE-Su5 (ZoshPpegp Violiesi, Vigptipopat, AG, OA), diluted in EB. After two EV washes to remove unbound material, the microspheres were resuspended in EV and analyzed using a BAS Sairig cytometer.

The results

Assessment of collagen deposition

In fig. 21 graphically shows the results of computer analysis of images of kidney tissue sections stained with Sirius red, where the tissue sections were obtained 7 days after ureteral obstruction in wild-type mice that received either an inhibitory MA5R-2 antibody or an isotype control antibody. As shown in fig. 21, tissue sections from kidneys harvested 7 days post-obstruction (SHOC) obtained from wild-type mice receiving MAZR-2 inhibitory antibody showed a significant decrease (p-0.0477) in collagen deposition compared to the amount of collagen deposition in tissue sections from occluded kidneys obtained from wild-type mice receiving Idsya4 isotype control.

Estimation of hydroxyproline content

Hydroxyproline was measured in kidney tissues as an indicator of collagen content. Hydroxyproline is a parameter that is a very telling indicator of the pathophysiological progression of the disease induced in this model.

In fig. 22 graphically shows the content of hydroxyproline in kidneys collected 7 days after obstruction (SOO), obtained from wild-type mice that received either an inhibitory MA5BR-2 antibody or an isotype control antibody. As shown in fig. 22, tissues of the occluded kidneys of mice receiving MABR-2 inhibitory antibody showed a significant decrease in hydroxyproline, an indicator of collagen content, in comparison with the kidneys of mice receiving tAr isotype control, Ids4 (p-0.0439).

Assessment of inflammation

In occluded kidneys from wild-type animals that received an isotype control antibody and wild-type animals that received an inhibitory MA5R-2 antibody, an abundant infiltrate of macrophages was observed. Careful quantification revealed no significant difference in the percentage of stained macrophages between these two groups (data not shown). However, despite equivalent numbers of infiltrating macrophages, occluded kidneys from animals injected with MABR-2 inhibitory antibodies developed significantly less fibrosis as judged by Sirius red staining compared with occluded kidneys from isotype control animals. this result is consistent with the findings that occluded kidney tissues from mice receiving the MAbR-2 inhibitory antibody had significantly less hydroxyproline than the kidneys of mice receiving the isotype control tAb, Idso4.

Discussion

The results described in this example showed that the use of an inhibitory MA5BR-2 antibody provides protection against renal fibrosis in the SHO model, which is consistent with the results described in example 14, which demonstrate that renal fibrosis and inflammation in MAZR-2-/ - mice in

OSHOO-models turned out to be of a significantly lower degree compared to wild-type mice.

The results in this example demonstrate reduced fibrosis in mice that received an inhibitory MA5R-2 antibody. The finding that fibrosis in SHO kidneys is reduced in animals with reduction or blockade of the MA5R-2-dependent activity of the lectin pathway is a very significant new finding. Taken together, the results presented in example 14 and this example demonstrate a beneficial effect of MA5ZR-2 inhibition on renal tubulointerstitial inflammation, tubular cell injury, profibrotic cytokine release, and scarring.

Reducing renal fibrosis remains a key goal of renal treatment. The SHO model is a model of a severe condition with accelerated renal fibrosis, and an intervention that reduces fibrosis in this model, such as the use of MAB5bR-2 inhibitory antibodies, is likely to be used to inhibit or prevent renal fibrosis. The results of the SHO model can probably also be attributed to kidney diseases characterized by glomerular and/or proteinuric tubule damage.

EXAMPLE 16

This example presents the results obtained using a model of renal fibrosis, inflammation, and tubulointerstitial injury against the background of protein overdose-induced proteinuria in MA5ZR-2-/- mice and wild-type mice to assess the role of the lectin pathway in proteinuric nephropathy.

Prerequisites/reasoning

Proteinuria is a risk factor for the development of kidney fibrosis and loss of excretory function regardless of the primary kidney disease (Tguddmazop K. ei ai.,.). IPIEGP May 254:216-224, 2003,

Matv M., At. IN). Merngoi 25:77-94, 2005). The concept of proteinuric nephropathy describes the toxic effects of excess protein entering the proximal tubules as a result of impaired glomerular selective selectivity (Vhyp5KiiY M.9., u. At. bos. Merpgoi. 15:504-505, 2004,

Vaiipez VA.9U., Maishte Vehu. MerNgoiY 7:177-180, 2011). This phenomenon, common to many glomerular diseases, results in a proinflammatory scarring environment in the kidney and is characterized by changes in proximal tubular cell growth, apoptosis, gene transcription, and inflammatory cytokine production as a consequence of dysregulated signaling pathways stimulated by proteinuric tubular fluid. Proteinuric nephropathy is generally recognized as a key contributing factor to the progressive renal damage characteristic of various primary renal pathologies.

Chronic kidney disease affects more than 15 95 adults in the United States and is the cause of approximately 750,000 deaths worldwide annually (I o7apo K. ei ai., I apsei. moi. 380,

I65itse 9859:2095-2128, 2012). Proteinuria is an indicator of chronic kidney disease, as well as a factor contributing to the progression of the disease. Many patients with proteinuric kidney disease have tubulointerstitial inflammation and progressive fibrosis, which are closely related to the decline in kidney function. Proteinuria by itself causes tubulointerstitial inflammation and leads to the development of proteinuric nephropathy (Vhyp5kKiiY Me...ei ai., U. At. Bob.

Merpgoi 15:504-505, 2004). In proteinuric diseases of the kidneys, an excessive amount of albumin and other macromolecules is filtered through the glomeruli and reabsorbed by the proximal tubular epithelial cells. This triggers a vicious inflammatory cycle mediated by complement activation, leading to cytokine and leukocyte infiltrates that cause interstitial tubular injury and fibrosis, thereby increasing proteinuria and leading to loss of renal function and ultimately progression to end-stage renal disease. (see, for example, Siagk eyi aiYi., Sapadiap Meais!

From Avzosiaiop doshsgtpai, 178:173-175, 2008). Treatments that modulate this vicious cycle of inflammation and proteinuria are expected to improve outcome in chronic kidney disease.

Given the positive effect of MABR-2 inhibition in the SHO model of tubulointerstitial injury, the following experiment was performed to determine whether inhibition could

MABR-2 reduces renal injury in a protein overdose model. In this study, protein overdose was used to induce proteinuric kidney disease as described in Izpoia et al., Eygoreap Kepai Avzosiyop, 21:591-597, 2006.

Methods

A MA5P-2-/- mouse was obtained as described in example 1 and backcrossed for 10 generations with VAI V/s. In this study, we compared the results obtained for wild-type mice and MABR-2-/- WAIV/c mice in a model of proteinuria induced by an overdose of protein.

A week before the start of the experiment, one kidney was removed from the mice before the protein overdose to obtain an optimal response. The proteinuria-inducing agent was bovine serum albumin with a low level of endotoxin (VZA, Zidta), which was administered in normal saline to UMT (n-:7) and MABR-2-/- mice (n--7) in the following doses: one a dose of 2 mg BBA/g, 4 mg B5A/g, 6 mg B5A/g, 8 mg VZA/g, 10 mg B5A/g and 12 mg B5A/g body weight and 9 doses of 15 mg B5A/g body weight , a total of 15 days was administered i.r. 15 doses Control mice

MMT (n-:4) and MABR-2-/- (n-4) mice received a saline solution administered only intraperitoneally. After the administration of the last dose, the animals were placed one at a time in metabolic cages for 24 hours to collect urine. Blood was collected by cardiac puncture under anesthesia, the blood was left to coagulate on ice for 2 hours, and the serum was separated by centrifugation. Serum and urine samples were collected at the end of the experiment on the 15th day, stored and frozen for analysis.

Mice were sacrificed 24 hours after the last VZA administration on day 15, and various tissues were collected for analysis. Kidneys were harvested and processed for HE staining and immunostaining. Immunohistochemical staining was performed as follows. Formalin-fixed, paraffin-embedded 5-micron sections of kidney tissue from each mouse were deparaffinized and rehydrated. Antigen unmasking was carried out in citrate buffer at 95 °C for 20 minutes followed by tissue incubation in C 95 H2Og for 10 minutes. Then the tissues were incubated in blocking buffer (10 95 serum from the species in which the secondary antibody was raised and 195 B5A in PB5) with 10 95 avidin solution for 1 hour at room temperature. Tissue sections were washed with PB5 after each stage three times for 5 minutes. Then the primary antibody was used in blocking buffer with 10 95 biotin solution for 1 hour at a concentration of 1:100 for antibodies E4/80 (Zapia Sgy7, cat. Mo 56-25830), TaEr (Zapia

Sgy, cat. Mo 5s-7892), 1-6 (Zapia Sgy7, cat. Mo 50-1265) and concentrations of 1:50 for TMEo antibody; (Zapia Stgy7, cat. Mo 50s-1348). Then a biotinylated secondary antibody was applied for 30 minutes at a concentration of 1:200 for sections with E4/80, TOEr and 11-6 and 1:100 for sections with TMEo, followed by the application of the HER enzyme conjugate for another 30 minutes. Staining was performed using a kit with a diaminobenzidine (SAB) substrate (Mesiog I para) for 10 minutes, and the slides were washed in water, dehydrated, and prepared for examination without contrast staining to facilitate computer analysis. Stained renal cortical tissue sections were analyzed by digital image capture followed by quantification using automated image analysis software.

Apoptosis was assessed in tissue sections by staining with the end label NAOTR. using terminal deoxynucleotidyl transferase (TOMEI) as follows.

Apoptotic cells in kidney sections were stained using a peroxidase kit

ArortTadF Regohidase (MiProge) as follows. Paraffin-embedded, formalin-fixed kidney sections from each mouse were deparaffinized, rehydrated, and then protein permeabilized with proteinase K (20 μg/ml) applied to each sample for 15 min at room temperature. Between stages, the samples were washed with RV5. Endogenous peroxidase activity was extinguished by incubating the tissues in C 95 H2gO" for 10 minutes. The tissues were then incubated in equilibration buffer followed by incubation with Tat enzyme for 1 hour at 37 ab. After washing in stop/wash buffer for 10 minutes, the anti-digoxigenin conjugate was applied for 30 minutes at room temperature, followed by washing.

Staining was carried out with the help of a set with ЯАВ-substrate for 4 minutes, followed by washing in water. Tissues were counterstained in hematoxylin and fixed in OVC. The frequency of TOMEI-stained (brown) apoptotic cells

Zo was counted manually in 20 consecutively selected fields of view of the cortical substance under high magnification with the help of a light microscope.

The results

Assessment of proteinuria

To confirm the presence of proteinuria in mice, total serum protein was analyzed on day 15, and total secreted protein was measured in urine samples collected within 24 hours on day 15 of the study.

In fig. 23 graphically shows the total amount of serum proteins (mg/ml) measured on day 15 in control wild-type mice (p-2) that received only saline, wild-type mice that received B5A (p-b), and MABR-2-/- mice treated with VZA (p-b).

As shown in fig. 23, administration of VZA increased total serum protein levels in both wild-type and MABR-2-/- groups by more than twofold compared to the concentration in the saline-only control group, while between groups, that received treatment, no significant difference was observed.

In fig. 24 graphically shows the total amount of protein (mg) excreted in urine collected during 24 hours on the 15th day of the study in control wild-type mice (n-2) that received only saline, wild-type mice that received VZA (n -b), and MA5P-2-/- mice receiving B5A (p-6). As shown in fig. 24, on day 15 of this study, an approximately sixfold increase in total urinary protein was observed in the VZA-treated groups compared to a sham-treated control group that received only saline. The results shown in fig. 23 and 24, suggest that the proteinuria model works as expected.

Assessment of histological changes in the kidneys

In fig. 25 shows representative NUE-stained sections of kidney tissues collected on day 15 of the protein overdose study from mice of the following groups: (panel A) wild-type control mice; (panel B) MABR-2-/- control mice; (panel C) wild-type mice receiving B5A; and (panel Y) MABR-2-/- mice treated with VZA. As shown in fig. 25, a significantly higher degree of tissue preservation was observed in the MABR-2-/- overdose group (panel 0) compared to the wild-type overdose group (panel C) at the same level of protein overdose provocation. For example, Bowman's capsules in wild-type mice treated with VZA (overdose) (panel C) were significantly enlarged compared to Bowman's capsules in wild-type controls (panel A). On the contrary, in capsules

Bowman's cells of MA5R-2-/- mice (overdose) receiving the same amount of B5A (panel 0) maintained a morphology similar to that observed in control MAZR-2-/- mice (panel B) and wild-type control mice ( panel A). As further shown in FIG. 25, large cast protein structures accumulate in the proximal and distal tubules of renal regions in wild-type (panel C), which are larger and more numerous compared to those in MA5R-2-/- mice (panel 0).

It should also be noted that analysis of kidney sections in this study using a transmission electron microscope showed that mice treated with VZA had a generalized lesion at the ciliary margins in the distal and proximal cells of the renal tubule with cell contents and nuclei embedded in the lumen of the tubule. On the contrary, the fabric of

MA5R-2-/- mice receiving VZA was preserved.

Assessment of macrophage infiltration in the kidneys

In order to measure the degree of inflammation, which is evidenced by the infiltration of macrophages, tissue sections of the collected kidneys were also stained with an antibody to the macrophage marker E4/80 by the methods described in Voog ei ai., U. oi At. wasps MerpgoYodu, 18:1508-1515, 2007.

In fig. 26 graphically shows the results of computer analysis of images of kidney tissue sections stained with an antibody to the macrophage marker E4/80, showing the average stained area of macrophages (95), where tissue sections were obtained on day 15 of the protein overdose study in wild-type control mice ( n-:-2), wild-type mice receiving B5A (n-b), and MA5P-2-/- mice receiving VZA (n-5). As shown in fig. 26, sections of kidney tissues stained with an antibody to macrophage markers P4/80 indicate that, although both groups receiving

VZA, showed a significant increase in macrophage infiltration in the kidney (measured as 95 area of E4/80 antibody staining) compared to sham-treated wild-type control mice, a significant decrease in macrophage infiltration was observed in tissue sections from MABR-2-/- mice, which received B5A, compared with macrophage infiltration in tissue sections from wild-type mice receiving BZA (p-value 0.0345).

In fig. 27A graphically shows the analysis of the correlation between the level of macrophages and proteinuria in each wild-type mouse (p-6) that received VZA, in the form of a graph of the dependence of the total amount of secreted protein, measured in urine from a 24-hour sample, on the degree of macrophage infiltration (95 average colored area). As shown in fig. 27A, most samples from wild-type kidneys showed a positive correlation between the level of proteinuria and the degree of macrophage infiltration.

In fig. 27B graphically shows the analysis of the correlation between the level of macrophages and proteinuria in each MA5R-2-/- mouse (n-5) that received B5A, in the form of a graph of the dependence of the total amount of secreted protein, measured in the urine from a 24-hour sample, on the degree infiltration of macrophages (95 average stained area). As shown in fig. 27B, the positive correlation observed in wild-type mice between the level of proteinuria and the degree of macrophage infiltration (shown in Fig. 27A) was not observed in MA5ZR-2-/- mice. Without being bound by a particular theory, these results may indicate a clearance mechanism for inflammation at high levels of proteinuria in MABR-2-/- mice.

Assessment of cytokine infiltration

Interleukin-b (1-6), transforming growth factor beta (TGF) and tumor necrosis factor alpha (TMEo) are pro-inflammatory cytokines that are known to be activated in the proximal tubules of wild-type mice in the proteinuria model (Arraye M. ei ai. .

Tgaperiapiayop, ONisia! Rybiisayop oi She Eigoreap Oiaiiuzizapa Tgapzriapi Avzzosiaiop - Eigoreap

Wow! Azzosiaiop, 12:51-56, 1997). Kidney tissue sections were stained with cytokine-specific antibodies as described above.

In fig. 28 graphically shows the results of computer analysis of images of tissue sections stained with anti-TIEB antibody (measured as 95 area of stained anti-TaeEr antibodies) in wild-type mice that received B5A (p-4) and MA5R-2-/ - receiving mice

VZA (p-5). As shown in fig. 28, a significant increase in TOERD staining was observed in the group of wild-type mice receiving VZA (overdose) compared to the group of MABR-2-/- mice receiving B5A (overdose) (p-:0.026).

In fig. 29 graphically shows the results of computer analysis of images of tissue sections stained with anti-TMEo antibody (measured as 95 area of stained anti-TMEo antibodies) in wild-type mice receiving B5A and MABR-2-/- mice receiving VBA (p-5). As shown in fig. 29, a significant increase in TMEo staining; was observed in the bo wild-type group receiving B5A (overdose) compared to the MA5BR-2-/- group receiving VZA (overdose) (p-0.0303).

In fig. 30 graphically shows the results of computer analysis of images of tissue sections stained with anti-1І-6 antibody (measured as 95 area of stained anti-1ІІ-6 antibodies) in control wild-type mice, control MABR-2-/- mice, mice wild-type mice receiving B5A (n-7) and MA5BR-2-/- mice receiving B5A (n-:7). As shown in fig. 30, in the wild-type group receiving B5A, a very significant increase in HER-6 staining was observed compared to the MA5R-2-/- group receiving VZA (p-0.0016).

Assessment of apoptosis

Apoptosis was assessed in tissue sections by staining with the terminal label NAOTR. with the use of terminal deoxynucleotidyl transferase (TOMEI), and the frequency of stained

TOMEI - apoptotic cells were counted in 20 consecutively selected fields of view of the cortical substance under high magnification (HPE).

In fig. 31 graphically shows the frequency of TOMEI-positive apoptotic cells, counted in 20 consecutively selected fields of view under high magnification (HPRE), in tissue sections of the renal cortical substance in control wild-type mice (n-1), control MA5R-2-/- mice ( p-1), wild-type mice receiving B5A (p-b), and MABR-2-/- mice receiving VZA (p-7).

As shown in fig. 31, a significantly higher level of cortical apoptosis was observed in kidneys obtained from wild-type mice receiving VZA compared to kidneys obtained from MA5R-2-/- mice receiving VZA (p-0.0001).

General overview of results and conclusions

The results of this example indicate that the MABR-2-/- mouse has reduced kidney damage in the protein overdose model. Therefore, MA5R-2 inhibitory agents, such as MABR-2 inhibitory antibodies, would be expected to suppress or prevent the vicious cycle of inflammation and proteinuria and improve outcomes in chronic kidney disease.

EXAMPLE 17

This example describes the analysis of a monoclonal MABR-2 inhibitory antibody for efficacy in reducing and/or preventing renal inflammation and tubulointerstitial injury in a model of protein overdose-induced proteinuria in wild-type mice.

Prerequisites/reasoning

As described in Example 16, in a model of protein overdose-induced proteinuria, MA5P-2-/- mice were found to exhibit significantly better outcomes (eg, less tubulointerstitial injury and less renal inflammation) than wild-type mice, which has refers to the pathogenic role of the lectin pathway in proteinuric kidney disease.

As described in Example 13, a monoclonal MAZBR-2 inhibitory antibody (O0M5646-5(22MI-2)) was created, which specifically blocks the function of the lectin pathway in humans, and was also shown to block the lectin pathway in mice. In this example, it was evaluated the effectiveness of MABR-2 inhibitory antibody OM5646-5(22MI-2 in a mouse model of proteinuria caused by protein overdose regarding the reduction and/or prevention of renal inflammation and tubulointerstitial damage in wild-type mice.

Methods

This study evaluated the effect of an inhibitory MA5R-2 antibody (10 mg/kg OM5646-502MI-2) compared with an anti-human Ids4 isotype control antibody (10 mg/kg ET904) and a saline control.

Similar to the study described in Example 16, this study used protein overdose to induce proteinuric kidney disease (Iz5poia ei ai.,

Eshgoreap Vepai Azzosiaiyop, 21:591-597, 2006). Proteinuria was induced in Vair/s mice with one kidney removed by daily i.p. injection of escalating doses (from 2 to 15 g/kg) of bovine serum albumin with a low endotoxin content (B5A) for 15 days, as described in example 16.

Treatment with antibodies was carried out by i.r. injections twice a week starting 7 days before the induction of proteinuria throughout the study. This dosing schedule was chosen based on previous RK/PO studies and pharmacological studies demonstrating sustained inhibition of the lectin pathway (data not shown). Mice were sacrificed on day 15, and kidneys were collected and processed for NUE staining and immunostaining. Stained renal cortical tissue sections were analyzed by digital image capture followed by quantification using automated image analysis software.

Evaluation of immunohistochemical staining and apoptosis was performed as described in example 16. bo Results

Assessment of proteinuria

To confirm the presence of proteinuria in mice, the total amount of protein excreted in the urine was analyzed in urine samples collected within 24 hours, on the 15th day (the end of the experiment). Total protein levels in urine samples were found to be, on average, increased almost sixfold in the B5A-treated groups compared to the non-B5A-treated controls (data not shown), confirming the presence of proteinuria in the B5A-treated mice. VZA No significant differences were observed in protein levels between groups receiving B5A.

Assessment of histological changes

In fig. 32 shows typical sections of NUE-stained tissues for the following groups of mice on day 15 after VZA treatment: (Panel A) saline-treated wild-type control mice; (panel B) control mice receiving isotype antibody; and (panel C) wild-type mice receiving an inhibitory MA5R-2 antibody.

As shown in fig. 32, the group receiving MA5bR-2 inhibitory antibody (panel C) showed a significantly higher degree of tissue preservation compared to the wild-type group receiving saline (panel A) or isotype control (panel B), while levels of protein overdose provocation.

Assessment of apoptosis

Apoptosis was assessed in tissue sections by staining the terminal label of NAOTR using terminal deoxynucleotidyl transferase (TDMA), and the frequency of stained

TOMEI - apoptotic cells were counted in 20 consecutively selected fields of view of the cortical substance under high magnification (HPE). In fig. 33 graphically shows the frequency of TOMEI - positive apoptotic cells, counted in 20 consecutively selected fields of view under high magnification (HPE) in tissue sections of the renal cortical substance in wild-type control mice that received saline and VZA (n-8), wild-type mice , which received an isotype control antibody and B5A (n-8), and wild-type mice that received an inhibitory MA5R-2 antibody and VB5A (n-7). As shown in fig. 33, a very significant reduction in the level of apoptosis in the cortical substance was observed in kidneys obtained from the MA5BR-2 inhibitory antibody group compared with the saline and isotype control group

Zo (p-0.0002 for saline control against MA5P-2 inhibitory antibody; p-0.0052 for isotype control against MA5P-2 inhibitory antibody).

Assessment of cytokine infiltration

Interleukin-b (II-6), transforming growth factor-beta (TGF), and tumor necrosis factor-alpha (TMEo), which are proinflammatory cytokines known to be activated in the proximal tubule in wild-type mice in a model of proteinuria.

In fig. 34 graphically shows the results of computer analysis of images of tissue sections stained with anti-TIEV antibody (measured as 95 area of stained anti-TaeEr antibodies) in wild-type mice that received VZA and saline (p-8), wild-type mice , which received B5ZA and an isotype control antibody (n-:7), and wild-type mice that received BZA and an inhibitory MA5R-2 antibody (n--8). As shown in fig. 34, quantitative determination of TOEr stained areas showed a significant decrease in Tr levels in mice receiving MA5BR-2 inhibitory antibody compared to groups receiving saline and isotype control antibodies (p-values 0.0324 and 0.0349, respectively) .

In fig. 35 graphically shows the results of computer analysis of images of tissue sections stained with anti-TMEo antibody (measured as 95 area of stained anti-TMEo antibodies) in wild-type mice that received VZA and saline (p-8), wild-type mice , which received VZA and an isotype control antibody (n-7), and wild-type mice that received B5A and an inhibitory MA5R-2 antibody (n-8). As shown in fig. 35, the analysis of stained sections showed a significant decrease in the level of TMEs; in the group receiving the MAZR-2 inhibitory antibody compared to the control group receiving saline (p-0.011) and the isotype control group (p-:0.0285).

In fig. 36 graphically shows the results of computer analysis of images of tissue sections stained with anti-1I-6 antibody (measured in the form of 95 area of stained anti-1II-6 antibodies) in wild-type mice that received B5A and physiological solution (p-8) , mice receiving

B5A and isotype control antibody (p-7), and wild-type mice that received VZA and an inhibitory

MA5P-2 antibody (p-8). As shown in fig. 36, the analysis of the stained areas showed a significant decrease in the level of II/-b in the group receiving MABR-2 inhibitory antibody, in comparison with the control group receiving physiological solution (p-0.0269), as well as the group receiving isotypic control (p-:0.0455).

General overview of results and conclusions

The results in this example demonstrate that the use of an MA5R-2 inhibitory antibody provides protection against renal injury in a protein overdose model, which is consistent with the results described in example 16, which demonstrates that MABR-2-/- mice have reduced renal injury in the model proteinuria.

EXAMPLE 18

This example presents results obtained using a model of renal fibrosis, inflammation, and tubulointerstitial injury in adriamycin-induced nephrology in MA5R-2-/- and wild-type mice to assess the role of the lectin pathway in proteinuric nephropathy.

Prerequisites/reasoning

Adriamycin is an antacycline anticancer antibiotic used to treat a wide range of cancers, including hematologic malignancies, soft tissue sarcomas, and many types of carcinoma. Adriamycin-induced nephropathy is a well-known model of chronic kidney disease in rodents, which allowed a better understanding of the progression of chronic proteinuria (I ee apa Naitikh5, Merpgoiodu, 16:30-38, 2011). The type of structural and functional damage in adriamycin-induced nephropathy is very similar to the type of chronic proteinuric kidney disease in humans (Rirrip et al., Ategisap

Wow! oi Repai Rpuzioiodu, 296:6213-29, 2009).

Adriamycin-induced nephropathy is characterized by damage to podocytes followed by glomerulosclerosis, tubulointerstitial inflammation, and fibrosis. Many studies have shown that Adriamycin-induced nephropathy is modulated by both immune and non-immune mechanisms (I ee apa Nagtiz, Merpgoiodu, 16:30-38, 2011). Adriamycin-induced nephropathy has several advantages as a model of kidney disease. First, it is a highly reproducible and predictable model of kidney injury, as it is characterized by the induction of kidney injury within days of drug administration, which facilitates experimental design as the injury develops consistently over time. It is also a model in which the extent of tissue damage is severe despite acceptable mortality (5,905) and morbidity (loss of body weight). Therefore, due to the severity and

Because of the time-course development of kidney damage in Adriamycin-induced nephropathy, this model is suitable for testing therapeutic methods that protect against kidney damage.

As described in Examples 16 and 17, in a protein overdose-induced model of proteinuria, it was found that MA5R-2-/- mice and mice receiving an inhibitory MAZR-2 antibody showed significantly improved outcomes (eg, less degree of tubulointerstitial injury and less degree kidney inflammation) compared to wild-type mice, suggesting a pathogenic role of the lectin pathway in proteinuric kidney disease.

In this example, MA5BR-2-/- mice were analyzed compared to wild-type mice in an adriamycin (AM)-induced nephrology model to determine whether the deficiency

MABR-2 to reduce and/or prevent the development of renal inflammation and tubulointerstitial injury caused by Adriamycin.

Methods 1. Optimization of dose and time point

First, an experiment was conducted to determine the dose of Adriamycin and the time point at which the YAB/s mice develop a level of renal inflammation suitable for testing a therapeutic intervention.

One dose of adriamycin (10.5 mg/kg) was administered IM to three groups of wild-type VAH V/s mice (n-8).

Mice were sacrificed at three time points: one week, two weeks, and four weeks after administration of Adriamycin. Only physiological solution was administered to control mice.

The results

All mice in the three groups showed signs of glomerulosclerosis and proteinuria, determined by NAE staining, with a gradual increase in the degree of tissue inflammation, as measured by the degree of macrophage infiltration in the kidney (data not shown). The degree of tissue damage was moderate in the one-week group, moderate in the two-week group, and severe in the four-week group (data not shown).The two-week time point was chosen for the remainder of the study. 2. Analysis of Adriamycin-induced nephropathy in wild-type mice and MAZR-2-/- mice

To clarify the role of the complement lectin pathway in adriamycin-induced nephropathy, a group of MA5P-2-/- (BAI V/s) mice was compared with wild-type mice (BAI B/s) at the same dose of adriamycin. MA5R-2-/- mice were crossed with VAG V/s mice for 10 generations.

Wild-type mice (n-8) and MABR-2-/- mice (n-8) were injected IM with adriamycin (10.5 mg/kg) and three mice of each line were injected with saline alone as a control. All mice were sacrificed two weeks after treatment and tissues were collected. The degree of histopathological damage was assessed by NaE staining.

The results

In fig. 37 shows typical sections of NUE-stained tissues for the following groups of mice on the 14th day after treatment with Adriamycin alone or saline (control): (panels A-1, A-2, A-3) wild-type control mice receiving only saline solution; (panels B-1, B-2, B-3) wild-type mice receiving Adriamycin; and (panels C-1, 0-2, C-3) MABR-2-/- mice treated with Adriamycin. Each photograph (eg, panel A-1, A-2, A-3) represents an individual mouse.

As shown in fig. 37, in the MABR-2-/- group, which received adriamycin, a much higher degree of tissue preservation is observed compared to the wild-type group, which received the same dose of adriamycin.

In fig. 38 graphically shows the results of computer analysis of images of kidney tissue sections stained with an antibody to the macrophage marker E4/80, showing the average stained area of macrophages (95) for the following groups of mice on day 14 after treatment with Adriamycin alone or saline (wild-type control). : wild-type control mice that received only saline; wild-type mice treated with adriamycin;

MA5P-2-/- mice receiving only saline and MA5P-2-/- mice receiving adriamycin. As shown in fig. 38, in MABR-2-/- mice treated with Adriamycin, the degree of macrophage infiltration is reduced ("p-0.007") compared to wild-type mice treated with Adriamycin.

In fig. 39 graphically shows the results of computer analysis of images of Sirius red-stained kidney tissue sections showing the area of stained collagen deposition (95) for the following groups of mice at day 14 after treatment with adriamycin or saline alone (wild-type control): wild-type control mice , who received only physiological solution; wild-type mice treated with adriamycin; MA5P -2-/- mice that received only saline and MABR-2-/- mice that received adriamycin. As

Zo is shown in fig. 39, in MABR-2-/- mice treated with Adriamycin, the degree of collagen deposition is reduced (""p-0.005) compared to wild-type mice treated with Adriamycin.

Results and conclusions

Alleviation of renal tubulointerstitial inflammation is a key goal in the treatment of kidney diseases. The results presented in this description show that the lectin pathway of complement activation makes a significant contribution to the development of renal tubulointerstitial inflammation. As further described herein, an MA5R-2 inhibitory agent, such as an

MABR-2 antibody can be used as a new therapeutic approach to treat proteinuric nephropathy, Adriamycin-induced nephropathy and to reduce renal tubulointerstitial inflammation.

EXAMPLE 19

This example describes the initial results of an ongoing phase 2 clinical trial to evaluate the safety and clinical efficacy of a fully human monoclonal inhibitory

MABR-2 antibodies in adults with steroid-dependent immunoglobulin-A nephropathy (SID) Its in adults with steroid-dependent membranous nephropathy (SMD).

Prerequisites

Chronic kidney disease affects more than 20 million people in the United States (Ogamu? R. yi ai., App. Ipiet. Mea. 162(11), ITS1-16, 2015). Glomerulonephritis (SM), including

ISAM and MN are kidney diseases in which the glomeruli are damaged and which often lead to end-stage renal failure and dialysis. There are several types of primary OM, the most common of which is ADAM. Many of these patients have persistent renal inflammation and progressive deterioration. Often these patients are treated with corticosteroids or immunosuppressants, which are characterized by numerous serious long-term adverse effects. Many patients continue to deteriorate even with these treatments. There is no treatment method for IDAM or MN.

IDA-nephropathy

Immunoglobulin A nephropathy (IAM) is an autoimmune kidney disease that causes internal inflammation of the kidneys and their damage. IDAM is the most common primary glomerulonephritis worldwide (Madivigopi et al., Kidpeu Ipi. 88(5):974-89, 2015). With an annual incidence rate of approximately 2.5 per 100,000 people, it is estimated that 1 in 1,400 people in the United States will develop UTI. In 40 95 patients with IDAM, the disease will progress to end-stage renal disease (ESRD) within 20 years after diagnosis (Sorro K., r'Atiso a., U. Merptgoi. 18(5):503-12, 2005; Khie eyi a!., Rio5 Ope, 7(6):e38904 (2012)). As a rule, patients have microscopic hematuria with mild and moderate proteinuria and various levels of renal failure (Muuai kK.)). ei a!., M. Epoai. U. Mea. 368(25):2402-14, 2013). Clinical markers such as impaired renal function, long-term hypertension and severe proteinuria (more than 1 g per day) are associated with a poor prognosis (Soyo M. ei ai., Tgapzriapi. Merpgoi. Oiai. 24(10):3068-74 , 2009; Vepoikh BE. vii a!., U. At. bos. Merngo!. 22(4):752-61, 2011). Proteinuria is the strongest prognostic factor that does not depend on other risk factors according to the results of several large non-experimental and prospective studies (Sorro K. eyi a1., U. Merpgoi. 18(5):503-12, 2005; Vavisn N.M. her ai., 9). At. boss. Mernpgoi. 18(12):3177-83, 2007). According to estimates, 15-20 95 patients reach TONN within 10 years after the onset of the disease, if they do not receive treatment (S'Atiso 0., At. y. Kidpeu biv. 36(2):227-37, 2000).

A diagnostic feature of IDAM is the predominance of deposition of either only IdA or in combination with

IDS, MI, or both in the glomerular mesangium. In IDA, a kidney biopsy reveals glomerular deposition of mannan-binding lectin (MVI), a key recognition molecule for the activation of MABR-2, an effector enzyme of the lectin pathway of the complement system.

Glomerular deposits of MVI, which are usually localized together with IdA and indicate complement activation, and a high level of MVI. in the urine are associated with an unfavorable prognosis in AMI, while these patients show more serious histological changes and mesangial proliferation than in patients without MVI deposition. or with high levels of MVI. in the urine (Maizida

M. eyi ai., Mergpop, 80(4):408-13, 1998; (ish I. eyi ai., SiIip. Ehr. Ittipoi. 169(2):148-155, 2012;

Voo5 A. ei ai., u). At. wasps Merpgoi 17(6):1724-34, 2006; Pi G.S. ei ai., Siip. Ex. And so on. 174(1):152-60, 2013). The frequency of remission is also significantly lower in patients with delayed MVI. (Hi

AHEM. eia)., Siip. Ex. And so on. 174(1):152-60, 2013).

Current approaches to the treatment of UTI are based on attempts to slow, stop, or delay the deterioration of kidney function. In Clinical Practice Guide for Glomerulonephritis "Kidney Disease: Improving Overall Outcomes" (Te Kidpeu Oiveaze: Itrgomipud SpPobai

Ochisotesis (COISO)) is a recommended scheme for the treatment of MIDA, according to which primary attention should be paid to blood pressure control by blockade of the renin-angiotensin system

Zo (KAB) YKOISO Umogk Sgoyr, 2012). For patients with persistent daily proteinuria of 1 g or more despite maximally tolerated antihypertensive doses and well-controlled blood pressure, recommended treatment includes corticosteroids and/or other immunosuppressants such as cyclophosphamide, azathioprine, or mycophenolate mofetil. According to the guideline for clinical practice in glomerulonephritis "Kidney Disease: Improving Overall Outcomes" (COISO) (Ipi Bos. oi Merpgoi. 2(2):1139-274, 2012) it is recommended to administer corticosteroids to patients with proteinuria at a dose that is greater than or equal to 1 g/day, with a usual duration of treatment of 6 months. For patients with sickle-cell AMI (defined as the presence of sickle cells in 25,095 glomeruli) and rapidly deteriorating renal clearance, another immunosuppressant (eg, cyclophosphamide) may be added to corticosteroids. However, even with aggressive immunosuppressive treatment, which is associated with serious long-term consequences, progressive deterioration of kidney function is observed in some patients. To date, there is no EOA-approved treatment for AMI, and even with the use of angiotensin-converting enzyme (ACE) inhibitors or angiotensin receptor blockers (ARBs) to control blood pressure, some patients continue to have elevated proteinuria. None of these methods have been shown to stop or even slow the progression of MI in patients at risk of rapid disease progression. Alternative therapies that could reduce or eliminate the need for long-term corticosteroid and/or immunosuppressive therapy would clearly address unmet medical needs.

Membranous nephropathy

The annual incidence rate of membranous nephropathy (MN) is approximately 10-12 per 1,000,000 people. Patients with MN can have a variable disease course, but approximately 25 to 95 will develop end-stage renal disease.

Membranous nephropathy is an immunomodulated glomerular disease and one of the most common causes of nephrotic syndrome in adults. The disease is characterized by the formation of immune deposits, primarily Ids54, on the outer layer of the glomerular basement membrane, which contain podocyte antigens and antibodies specific to these antigens, which leads to complement activation. Initial manifestations of MEN are associated with nephrotic syndrome: proteinuria, hypoalbuminemia, hyperlipidemia, and edema. Although MN may resolve spontaneously without treatment, one-third of patients experience progressive loss of kidney function and disease progression to TON after an average of 5 years after diagnosis. Corticosteroids are often used to treat MN, and there is a need to develop alternative treatment methods. In addition, patients considered to be at moderate risk of progression, depending on the severity of proteinuria, receive prednisone in combination with cyclophosphamide or a calcineurin inhibitor, and these two treatments used together are often associated with serious systemic side effects.

Methods

Two phase 1 clinical trials in healthy volunteers demonstrated that both intravenous and subcutaneous administration of the MABR-2 inhibitory antibody, OM5646, resulted in sustained inhibition of the lectin pathway.

This case study describes the interim results of a currently ongoing phase 2, uncontrolled, multicenter study of the MABR-2 inhibitory antibody, OM5646, in subjects with IMAD and MN. Inclusion criteria required that all patients in this study, regardless of subtype of kidney disease, be maintained on a stable dose of corticosteroids for at least 12 weeks prior to enrollment (ie, patients were steroid dependent). The study is a non-comparative pilot study with a 12-week treatment and a b-week follow-up period.

According to the study plan, it is necessary to recruit approximately four patients per disease. The study is designed to evaluate the ability of OM5646 to improve renal function (eg, reduce the severity of proteinuria) and reduce corticosteroid requirements in patients with (DAM and MN. To date, 2 patients with IDA nephropathy and 2 patients with membranous nephropathy have completed treatment in this study.

To enter the study, each subject had to have a high level of protein in the urine, despite constant treatment with a stable dose of corticosteroids. These criteria are selected for patients who are unlikely to improve spontaneously during the study.

The age of subjects at the time of selection was 218, and subjects were included in the study only if they were diagnosed with one of the following: UTI on renal biopsy or primary MN on renal biopsy. Enrolled patients also had to meet all of the following

The inclusion criteria were: (1) mean urinary albumin/creatinine ratio of »0.6 in three samples collected consecutively and daily before each of the two visits during the selection period; (2) taking prednisone at a dose of 210 mg or an equivalent dose for at least 12 weeks before visit 1 in the selection period; (3) with background immunosuppressive treatment (eg, cyclophosphamide, mycophenolate mofetil) taking a stable dose for at least 2 months before visit 1 in the selection period with no expected dose change during the study; (4) estimated glomerular filtration rate (GFR) of 230 mL/min/1.73 me, calculated using the MOCO equation"; (5) receiving stable, optimized therapy with angiotensin-converting enzyme inhibitors (ACEIs) and/or angiotensin receptor blockers ( ACS), systolic blood pressure "150 mm Hg and diastolic blood pressure "90 mm Hg in a relaxed state; (b) no use of belimumab, etsulizumab, or rituximab in the b months prior to visit 1 in the selection period; and ( 7) lack of kidney transplantation. "Equation of MOCO: es (ml/min./1.73 m2e)-175 x (5Obg)1y154 x (Age) 203 x (0.742 for women) x (1.122 for African Americans). Note: 5Cg-creatinine level in blood serum should be expressed in mg/dL.

The monoclonal antibody used in this study, OM5646, is a fully human IdD54 monoclonal antibody that binds and inhibits human MABR-2. MABR-2 is an effector enzyme of the lectin pathway. As shown in example 12, OM5646 efficiently binds to recombinant MA5P-2 (apparent equilibrium dissociation constant in the range of 100

PM) and exhibits more than 5000-fold selectivity to homologous proteins C15, C1ig and MABR-1. In functional assays, OM5646 inhibits the activity of the human lectin pathway with nanomolar efficacy (50-95 inhibitory concentration (IC) is approximately 3 nM), but has no significant effect on the classical pathway. OM5646 administered by either intravenous (IM) or subcutaneous (5C) injection to mice, nonhuman primates, and humans resulted in high plasma concentrations that were associated with inhibition of lectin pathway activation in analysis of ex m/o.

In this study, the drug substance OM5646 was given at a concentration of 100 mg/ml, which was further diluted for IM administration. The corresponding calculated volume is 100 mg/ml of solution

OM5646 for injection was withdrawn from the vial using a syringe to prepare the dose. The infusion bag was inserted within four hours after preparation.

The study included screening (28 days), treatment (12 weeks) and follow-up (6 weeks) periods as shown in the study design below.

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At screening before the first dose of OM5646, screened patients provided three urine samples (collected once daily) in each of two three-day periods to establish baseline urinary albumin:creatinine ratios. After the end of the selection period, selected subjects were administered IM OM5646 at 4 mg/kg once a week for 12 weeks (treatment period). After the last dose of OM5646, there was a b-week observation period.

During the first 4 weeks of treatment with OM5646, subjects continued to take a stable dose of corticosteroids, which was taken before the start of treatment. At the end of the first 4 weeks of the 12-week period, subjects were tapered (ie, the corticosteroid dose was reduced) over 4 weeks, if possible, followed by 4 weeks in which the resulting reduced corticosteroid dose was continued. The goal was to reduce the dose of prednisone to 2 mg (or equivalent) daily. During this period, dose tapering was stopped in patients who experienced deterioration of renal function, as assessed by the investigator. Subjects were treated with OM5646 on the background of a gradual reduction in the corticosteroid dose and for a full 12 weeks. Patients were then followed for additional weeks after the last dose of OM5646. A gradual reduction in the dose of corticosteroids and treatment with OM5646 made it possible to assess whether the reception allows

OM5646 to reduce the doses of corticosteroids necessary to maintain stable kidney function.

The key measures of effectiveness in this study are the change in urinary albumin/creatinine ratio (UAC) and 24-hour protein level from baseline before the start of 12 weeks. Measurement of protein or albumin in the urine is commonly used to assess kidney damage, and persistently high levels of protein in the urine correlate with progression of kidney disease. sASC is used in clinical trials to assess proteinuria.

Performance evaluation

The analyzed cCASC value is defined as the average of all values obtained at some point in time. The planned number of CHASK is three for each planned time point. The initial value of pASK was determined as the average value of the analyzed values at two visits.

The results

In fig. 40 graphically shows the sASCs for two patients with (DAM) during a 12-week study with weekly treatment with the MABR-2 inhibitory antibody (OM5646) at 4 mg/kg. As shown in Fig. 40, the change from baseline is statistically significant at the time point " a" (p-0.003); time point "p" (p-0.007) and time point "c" (p-0.033), according to the untransformed analysis. Table 12 presents data for protein in the 24-hour urine collection of patients with IDAM, receiving OM5646.

TABLE 12

Protein in 24-hour urine collection (mg/day) in IDAM patients receiving OM5646 (mg/24 hours) (mg/24 hours)

As shown in fig. 40 and in table 12, in the course of the study, clinically and statistically significant improvement in kidney function was observed in patients with (DAM). Table 12). As shown by the data of sASC in Fig. 40, the mean initial level of sASC was 1264 mg/g and at the end of treatment reached 525 mg/g (p-0.011), decreasing to 128 mg/g at the end of the observation period. In Fig. 40 further showed that the treatment effect was maintained throughout the observation period.Measurement of 24-hour urinary protein excretion was monitored by the AUC, with a mean decrease from 3156 mg/24 hours to 1119 mg/24 hours (p-0.017).Treatment effects in two patients were highly concordant.Both patients had a reduction of approximately 2000 mg/day, and both patients achieved partial remission (defined as greater than 50 95 reduction in protein excretion in the 24-hour urine collection and/or terminal protein excretion of less than 1000 mg/day ; complete remission was defined as protein excretion less than 300 mg/day). The magnitude of the 24-hour decrease in proteinuria in both patients with IdA nephropathy was associated with a significant improvement in kidney survival. Both patients with IdA-nephropathy were also tolerant to a significant reduction in the doses of steroids they took, with a decrease in the daily dose to "5 mg (from 60 to 0 mg, from ZO to 5 mg).

In two patients with MN, a decrease in CKAS was also observed during treatment with OM5646. In one patient with MN, the level of cACE decreased from 1003 to 69 mg/g, and this low level was maintained during the observation period. In another patient with MN, there was a decrease in HRaSq from 1323 to 673 mg/g with a variable course after treatment. The first patient with MN showed a marked decrease in 24-hour urine protein (10,771 mg/24 hours at the start of treatment to 325 mg/24 hours on day 85), resulting in partial and almost complete remission, while the other remained practically unchanged (4272 mg/24 hours at the beginning of treatment to 4502 mg/24 on day 85). Steroid levels in two patients with MN were gradually reduced from 30 to 15 mg and from 10 to 5 mg.

Thus, consistent improvements in renal function were observed in patients with AMI and MN treated with the MA5BR-2 inhibitory antibody, OM5646. The treatment effects of OM5646 in patients with AMI are robust and consistent, suggesting a strong signal of efficacy. These

The effects were confirmed by the results in patients with MN. The temporal profile and magnitude of changes in PAsSqC during treatment were consistent in all four patients with MI and MN. No significant safety issues were observed. The patients in this study were a difficult-to-treat group, and the therapeutic effect in these patients suggests the efficacy of an MA5P-2 inhibitory antibody such as OM5646 in patients with AMI and MN, such as patients with steroid-dependent AMI and MN (ie patients receiving treatment with a stable dose of corticosteroids before treatment with an inhibitory MA5R-2 antibody), including patients with an increased risk of rapid progression to end-stage renal disease.

According to the above, in one of the variants of implementation of the invention, a method of treating a person suffering from DAM or MN is provided, which includes administering to the subject a composition that contains an amount of MABR-2 inhibitory antibody effective for inhibiting MAZR-2-dependent complement activation. In one embodiment, the method includes administering to a person suffering from ICH or MN an amount of MABR-2 inhibitory antibody sufficient to improve kidney function (eg, reduce proteinuria). In one embodiment, the subject suffers from steroid-dependent DM. In one embodiment, the subject suffers from steroid-dependent MN. In one embodiment, an MA5BR-2 inhibitory antibody is administered to a subject suffering from steroid-dependent AMI or steroid-dependent MN in an amount sufficient to improve renal function and/or reduce the dose of corticosteroids in said subject.

In one embodiment, the method additionally includes the identification of a subject suffering from steroid-dependent UTI before the step of administering to the subject a composition containing an amount of MABR-2 inhibitory antibody effective for inhibiting MABR-2-dependent complement activation.

In one embodiment, the method additionally includes identifying a subject having steroid-dependent MH before the step of administering to the subject a composition that contains an amount of inhibitory

MA5P-2 antibody, effective in inhibiting MA5P-2-dependent complement activation.

According to any of the embodiments disclosed herein, an MAZR-2 inhibitory antibody exhibits at least one or more of the following characteristics: said antibody binds human MAbR-2 with a Ko of 10 nM or less, said antibody binds an epitope in the SSRI1 domain

MAB5BR-2, the specified antibody inhibits the deposition of SZB in the analysis /l m/o in 1 95 human serum with an IC5o of 10 nM or less, the specified antibody inhibits the deposition of SZB in 90 95 human serum with an IC5o of 30 nM or less, where the antibody is a fragment an antibody selected from the group consisting of Em, Bar, Rab", E(ar)" and E(ar)g2, where the antibody is a single-chain molecule, where the specified antibody is an Ids2 molecule, where the specified antibody is an IdDO1 molecule , where the specified antibody is an Ids4i molecule, where the Ids4 molecule contains the mutation 5228P. In one of the variants of implementation, the antibody binds to

MA5P-2 and selectively inhibits the lectin pathway without intrinsically inhibiting the classical pathway (ie, inhibits the lectin pathway while leaving the classical complement pathway intact).

In one embodiment, the MA5R-2 inhibitory antibody is administered in an amount effective to improve at least one or more clinical parameters related to kidney function, such as reduction of proteinuria (eg, reduction of shACE and/or reduction of protein concentration in 24-hour urine collection, such as a greater than 20 95 decrease in protein excretion in a 24-hour urine collection, or such as a greater than 30 95 decrease in protein excretion in a 24-hour urine collection, or such as a greater than 40 95 decrease in protein excretion in a 24-hour collection urine, or such as a greater than 50 95 decrease in protein excretion in a 24-hour urine collection).

In some embodiments, the method comprises administering a MA5R-2 inhibitory antibody to a subject suffering from IDA (such as steroid-dependent IDA) via a catheter (e.g., intravenously) for a first period of time (e.g., at least one day to a week or two weeks, or three weeks, or four weeks or more) followed by administration of an MABR-2 inhibitory antibody to the subject subcutaneously for a second period of time (eg, a chronic phase for at least two weeks or more).

In some embodiments, the method comprises administering an MA5R-2 inhibitory agent to a subject suffering from MN (such as steroid-dependent MN) via a catheter (eg, intravenously) for a first period of time (eg, at least one day to a week or two weeks , or three weeks, or four weeks, or more) followed by the introduction of an inhibitor

MA5P-2 antibodies to the subject subcutaneously during a second period of time (eg, chronic phase for at least two weeks or more).

In some embodiments, the method comprises administering an MA5P-2 inhibitory antibody to a subject suffering from AMI (such as steroid-dependent AMI) or MH (such as steroid-dependent

MN) intravenously, intramuscularly or subcutaneously. Treatment can be chronic and administered daily monthly, but preferably at least every two weeks or at least once a week, such as twice a week or three times a week.

In one embodiment, the method comprises treating a subject suffering from UTI (such as steroid-dependent UTI) or MN (such as steroid-dependent MN), which comprises administering to the subject a composition comprising an amount of an MABR-2 inhibitory antibody or antigen-binding fragment containing the variable region of the heavy chain, which contains SOB-NI, SOB-H2 and

SOB-NZ of the amino acid sequence presented in ZEO IU MO:67, and the variable region of the light chain, which contains SOK-I1, SOV-12 and SOK-I3 of the amino acid sequence presented in 5EO IU MO:70. In some embodiments, the composition comprises an inhibitor

MA5P-2 antibody containing (I) (a) variable region of the heavy chain, containing: i) SOK-NI1 of the heavy chain, containing the amino acid sequence from aa 31-35 in 5EO IU MO:67; and iii) SOC-

H2 of the heavy chain containing the amino acid sequence from aa 50-65 in 5EO IYUO MO:67; and hers)

SOV-NZ of a heavy chain containing the amino acid sequence from aa 95-107 in 5EO IU MO:67; and (B) a variable region of the light chain, containing: i) SOK-Y1 of the light chain, containing the amino acid sequence from aa 24-34 in 5EO IYU MO:70; and i) SOK-12 of the light chain, containing the amino acid sequence from aa 50-56 in 5EBO IYUO MO:70; and ii) SOK-I From the light chain containing the amino acid sequence from aa 89-97 in 5EBEO IU MO:70; or (II) a variant thereof comprising a heavy chain variable region with at least 90 95 identity (eg, at least 91 95, at least 92 95, at least 93 95, at least 94 9565, at least 9595, at least 9695, at least 9795, at least 9895, at least 99 95 identity) with ZEO IU MO:67, and a variable region of the light chain with an identity of at least 9095 (for example, at least 91 956, at least 92 956, at least 93 95,

at least 94 95, at least 95 95, at least 96 95, at least 97 965, at least 98 96, at least 99 95 identity) with BZEO IU MO:70.

In some embodiments, the method includes administering to the subject a composition that contains an amount of an MA5BR-2 inhibitory antibody or an antigen-binding fragment thereof that contains a heavy chain variable region that contains an amino acid sequence represented in

ZEO IU MO:67, and a variable region of the light chain that contains the amino acid sequence presented in ZEO IU MO:70.

In some embodiments, the method includes administering to the subject a composition that contains a MA5P-2 inhibitory antibody or an antigen-binding fragment thereof that specifically recognizes at least a portion of an epitope on human MA5P-2 recognized by a reference antibody

OM5646 containing the heavy chain variable region as specified in ZEO IU MO:67 and the light chain variable region as specified in ZEO IU MO:70.

In some embodiments, the method comprises administering to a subject suffering from or at risk of developing AMI (such as steroid-dependent AMI) or MN (such as steroid-dependent MN) a composition that comprises a MAZR-2 inhibitory antibody or an antigen-binding fragment comprising a heavy chain variable region that contains the amino acid sequence represented by 5EO IU MO:b7 and a light chain variable region that contains the amino acid sequence represented by 5EO IUO MO:70 at a dose of 1 to 10 mg/kg (i.e., 1, 2, 3,4, 5,6, 7, 8, 9, or 10 mg/kg) at least once a week (eg, at least twice a week or at least three times a week) for at least 3 weeks or for at least 4 weeks, or for at least 5 weeks, or for at least b weeks, or for at least 7 weeks, or for at least 8 weeks, or for at least 9 weeks, or for at least 10 weeks, or for at least 11 weeks, or for at least 12 weeks.

EXAMPLE 20

This example describes the preliminary results of an ongoing phase 2 clinical trial evaluating the safety and clinical efficacy of a fully human monoclonal mAb-2 inhibitory antibody in adults with steroid-dependent lupus nephritis (SLU).

From the background

Chronic kidney disease affects more than 20 million people in the United States

States (Ogam/? R. ei ai., App. Ipiegp. Mei. 162(11); ITS1-16, 2015). Glomerulonephritis (M), including IdAM, MN and VN, are kidney diseases in which the glomeruli are damaged, often leading to end-stage renal failure and dialysis. Many of these patients have persistent renal inflammation and progressive deterioration. Often, such patients are treated with corticosteroids or immunosuppressants, which are characterized by numerous long-term serious adverse effects. Even with these treatments, many patients continue to deteriorate.

Lupus nephritis

The main complication of systemic lupus erythematosus (SLE) is nephritis, also known as lupus nephritis, which is classified as a secondary form of glomerulonephritis. Later in the course of the disease, almost 60 95 adults with SLE develop one or another form of kidney damage (Koda-Kitrbie ei a!., Koda-Kitrie apa Moipd'5 Arriiei Tnegareshisv: Te siipisa! ive oi agdv, 10 Ei, Pirripsoi UMiate 8 UMiKipe: Radev 792-9, 2012) with a frequency of 20-70 per 100,000 people in

USA. Lupus nephritis is often accompanied by other active symptoms in patients

SLE, including fatigue, dyspnea, rash, arthritis, serositis, or central nervous system disease (Rizei5Ku 0.5. ei aiYi.,, Mei. Siip. Mohi. At. 81(1):113-28, 1997). Some patients have asymptomatic lupus nephritis; however, laboratory abnormalities such as elevated serum creatinine, low albumin, or protein or sediment in the urine are suggestive of active lupus nephritis on regular monitoring. Autoimmunity plays an important role in the pathogenesis of lupus nephritis. These autoantibodies form intravascular pathogenic immune complexes that are deposited in the glomeruli. Autoantibodies can also bind to antigens already located in the basal membrane of the glomeruli, forming immune complexes / l 5. Immune complexes contribute to the inflammatory response, activating complement and attracting inflammatory cells (O'Adayi M.O. ei ai.,

Giriv perngivv: raiypoiodu apa raipodepeviv: UMaiyase 0.3. Nanpp, Yuybroiv' I priv Eguipetaio5iv, 77

EYA RPiiadeIrpa: Iirripsoi MUyShiatve and UMiKIipe: pp. 1094-111, 2007). Thus, complement activation mediated by the immune complex plays a key role in the pathogenesis of lupus nephritis. C44 deposits are present in renal tissue and are usually associated with 60 immune complex deposits, C1d and SZ, initiating the classical pathway. In some cases, C4a deposits are present without C1d, which indicates the possible involvement of the lectin pathway (Cat

M.K. ei ai., Ipi. U. Siip. Ex. Raina 6(10):2157-67, 2013).

Additional confirmation of the important role of the lectin pathway is the fact that deposition

MVI occur in lesions of the skin of patients with SLE (Umaiyit G.M. ei aI., Nyt. Ittipoi. 75(7):629-32, 2014). In addition, persistent deposition of MVI and ficolin was observed in most kidney biopsies in patients with lupus nephritis (Mizipaga K.M. ei ai., Nyt. Ittipoi. 74(8):907-10, 2013). Postponement of MVI. in the kidneys, it was most evident in patients with high proteinuria.

In addition, MVI levels. in plasma were significantly higher in patients with SLE than in healthy controls, and MVI levels. correlated with disease activity, suggesting that MVI levels. may be a biomarker of SLE disease activity (Rapaa A.K. ei ai., Agiipgyi5 Kezv.

Tag. 14(5):K218, 2012). Corticosteroids are the main traditional treatment option for patients with mild lupus nephritis. In more severe cases, high doses of prednisone, methylprednisolone, mycophenolate mofetil, cyclophosphamide, azathioprine, and cyclosporine were used in clinical practice. Treatment options for SLE and lupus nephritis are associated with a high rate of complications and mortality. Side effects, especially with long-term use of corticosteroids, limit the patient's compliance with the treatment regimen, with subsequent effects on its effectiveness. There is a need to develop treatment regimens that are more easily tolerated by patients.

Methods

As described above in Example 19, two phase 1 clinical studies in healthy volunteers demonstrated that both intravenous and subcutaneous administration of an inhibitor

The MA5BR-2 antibody, OM5646, resulted in persistent suppression of the lectin pathway.

This example describes interim results from an ongoing phase 2, uncontrolled, multicenter study of the MA5BR-2 inhibitory antibody, OM5646, in patients with lupus nephritis (LU). According to the study inclusion criteria, all patients in this study, regardless of subtype of kidney disease, must have received a stable dose of corticosteroids for at least 12 weeks prior to study enrollment (ie, patients are steroid dependent). The study is a simple (non-comparative) pilot study with a 12-week treatment course and a b-week period

From observation.

The study is designed to evaluate the ability of OM5646 to improve renal function (eg, reduce proteinuria) and reduce the need for corticosteroids in patients with HF. To date, 5 patients with lupus nephritis (LU) have completed treatment in the study.

At the beginning of the study, each subject should have a high level of protein in the urine, despite continued treatment with a stable dose of corticosteroids. These criteria make it possible to select patients who are unlikely to show spontaneous improvement during the study period.

At the time of screening, the age of the patients was 218 years, and only those patients who were diagnosed with lupus nephritis on renal biopsy were included in the study. Patients included in the study also had to meet all of the following inclusion criteria: (1) a mean urinary albumin/creatinine ratio of 20.6 in three samples collected consecutively and daily before each of the 2 visits during the screening period; (2) taking 210 mg of prednisone or an equivalent dose for at least 12 weeks before Screening 1; (3) in the case of immunosuppressive therapy (eg, cyclophosphamide, mycophenolate mofetil), taking a stable dose for at least 2 months before screening visit 1 with no expected dose change during the study; (4) estimated glomerular filtration rate (ESR) of 30 ml/min/1.73 me, calculated by the MOCO equation"; (5) receipt of stable, optimized treatment with angiotensin-converting enzyme inhibitors (ACEIs) and/or receptor blockers under the guidance of a physician angiotensin-converting enzyme (ACE), and a systolic blood pressure of "150 mmHg and a diastolic blood pressure of "90 mmHg at rest; (6) no use of belimumab, eculizumab, or rituximab within 6 months of screening visit 1; and (7) no history of kidney transplantation." MOCO equation: es (ml/min/1.73m2)-175 x (5017157 x (Age) 203 x (0.742 (for women)) x (1.212 (for African Americans)) . Note: The serum creatinine level should be expressed as mg/dL. 60 The monoclonal antibody used in this study, OM5646, is a fully human Idea monoclonal antibody that binds to and inhibits human MABR-2. MA5R-2 is an effector enzyme of the lectin pathway.As shown in example 12, OM5646 actively binds to recombinant MAB5bR-2 (the apparent equilibrium dissociation constant is 100 pM) and exhibits more than 5000-fold selectivity over homologous proteins C15, C1g, and MA5R-1.

In functional assays, OM5646 inhibits the human lectin pathway with nanomolar activity (concentration leading to 50–95 inhibition (IC) is approximately 3 nM), but does not show a significant effect on the classical pathway. In ex u/mo assay, high concentrations

OM5646 in plasma administered by intravenous (IM) or subcutaneous (5C) injection to mice, nonhuman primates, and humans resulted in inhibition of lectin pathway activation.

In this study, the drug substance OM5646 was given at a concentration of 100 mg/ml, which was then diluted for intravenous administration. The corresponding calculated volume of OM5646 100 mg/ml injection solution was withdrawn from the vial with a syringe for preparing the dose.

The infusion bag was administered within four hours after preparation.

The study consists of screening (28 days), treatment (12 weeks) and follow-up (6 weeks) periods as shown in the study design below. : хайфораны" ! : kah. Pizhuevvynkh Oposterezhemykh game "era Doguvknia, | the works of EE Emma

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Uyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuyuuuuu

During the screening period and before administration of the first dose of OM5646, subjects who met the study inclusion criteria provided three urine samples (collected once daily) on three consecutive days in each of two urine collection periods for three consecutive days to determine baseline protein levels in 24-hour urine collection and albumin-creatinine ratio in urine. After the screening period, subjects meeting the inclusion criteria received

OM5646 at a dose of 4 mg/kg intravenously once a week for 12 weeks (treatment period). After the last dose of OM5646, there was a b-week observation period.

During the first 4 weeks of treatment with OM5646, subjects received a stable dose of corticosteroids received before the study. At the end of the first 4 weeks of the 12-week treatment period, subjects received a tapering dose of corticosteroids (ie, the dose of corticosteroids was tapered), if possible, for 4 weeks, followed by 4 weeks during which subjects continued to receive the final dose of corticosteroids. The goal was to reduce the daily dose of prednisone to 6 mg (or an equivalent dose). During this period, the dose reduction was stopped for those subjects in whom, according to the investigator, deterioration of renal function was observed. Subjects received OM5646 treatment during corticosteroid tapering and throughout the 12-week treatment period. Patients were then followed for an additional 6 weeks after the last dose. Corticosteroid dose reduction and OM5646 treatment assessed the ability of OM5646 to reduce the corticosteroid dose required to maintain stable renal function.

Performance analysis

The key measure of effectiveness in this study is the change in 24-hour protein levels from baseline over 12 weeks. Measurement of protein or albumin in the urine is commonly used to assess kidney damage, and persistently high levels of protein in the urine correlate with progression of kidney disease. Partial remission is defined as a greater than 50 95 reduction in 24-hour urinary protein excretion.

The results

Table 13 presents the level of protein in the 24-hour urine collection (mg/day) for five patients with VN receiving OM5646.

TABLE 13

Protein level in 24-hour urine collection (mg/day) in patients with HF treated with OM5646

Average

Time taken | Patient Mo 1 |Patient Mo 2| Patient Mo Z | Patient Mo 4 | Patient Mo 5 sample value (mg/24 h) | (mg/24 h.) | (mg/24 h.) | (mg/24 h.) | (mg/24 h.) | (patients Mo 2-

Note: patient Mo 1 had an unexpected exacerbation of systemic disease during treatment. 5 As shown in Table 13, patients with HF had a clinically and statistically significant improvement in kidney function throughout the study. As shown in Table 13, four out of five patients with 3 BH experienced a significant (average 69 percent) reduction in 24-hour urinary protein excretion during treatment. The fifth patient (patient Mo 1) had a sudden exacerbation of the systemic disease with a marked increase. For most of the respondents with lupus, their steroid doses were reduced.

Thus, a significant improvement in kidney function was observed in four out of five patients with

HNs that received the inhibitory MA5BR-2 antibody, OM5646. The results of treatment with OM5646 in patients with VN were stable and reliable, indicating a strong signal of efficacy.

No significant safety issues were observed. In this study, patients represent a difficult-to-treat group, and it is assumed that the therapeutic effect observed in these patients is a predictor of the effectiveness of the inhibitory

MAB5BR-2 antibodies, such as OM5646, in patients with VN, such as patients with steroid-dependent VN (ie, patients receiving a stable dose of corticosteroids prior to initiation of treatment with an inhibitory MA5R-2 antibody), including patients at risk of rapid disease progression kidneys to the terminal stage.

In accordance with the foregoing, in one embodiment of the invention, a method of treating a human subject suffering from HF is provided, which comprises administering to the subject a composition that contains an amount of an MA5R-2 inhibitory antibody effective to inhibit MAZR-2-dependent complement activation . In one embodiment, the method includes administering to a human subject suffering from HF an amount of MA5R-2 inhibitory antibody sufficient to improve kidney function (for example, to reduce proteinuria). In one embodiment, the subject suffers from steroid-dependent HF. In one embodiment, an MA5R-2 inhibitory antibody is administered to a subject suffering from steroid-dependent BH in an amount sufficient to improve kidney function in said subject and/or reduce the dose of corticosteroids.

In one embodiment, the method additionally includes the identification of a human subject suffering from steroid-dependent BH before the step of administering to the subject a composition containing an amount of MABR-2 inhibitory antibody effective for inhibiting MABR-2-dependent complement activation.

According to any of the embodiments disclosed in this application, the MABR-2 inhibitory antibody exhibits at least one or more of the following characteristics: said antibody binds human MABR-2 with a C of 10 nM or less, said antibody binds an epitope in domain SSR1 of MAB5R-2, the specified antibody inhibits the deposition of SZB in /l mitgo analysis of 1 95 human serum at an IC of 10 nM or less, the specified antibody inhibits the deposition of SZB in 90 of 95 human serum at an IC of 30 nM or less, and the antibody is an antibody fragment , selected from the group consisting of Em, Bar, Rab", E(ab)" and E(ab)", and the antibody is a single-chain molecule, and the specified antibody is a molecule

Id9052, and the specified antibody is an IDS1 molecule, and the specified antibody is an Ids4 molecule, where the Ids4 molecule contains the 5228P mutation. In one embodiment, the antibody binds to MA5P-2 and selectively inhibits the lectin pathway and essentially does not inhibit the classical pathway (ie, inhibits the lectin pathway while leaving the classical complement pathway unchanged).

In one embodiment, the MA5BR-2 inhibitory antibody is administered to a subject suffering from

BH, in an amount effective to improve at least one or more clinical parameters related to kidney function, such as attenuating proteinuria (eg,

decrease in 24-hour urine protein concentration and/or decrease in 24-hour urinary protein excretion, for example, decrease in 24-hour urinary protein excretion by more than 20 95 or decrease in 24-hour urinary protein excretion by more than 30 95, or decrease in 24-hour urinary protein excretion urinary protein by more than 40 95, or, for example, a decrease in 24-hour urinary protein excretion by more than 5095). In some embodiments, an MABR-2 inhibitory antibody is administered to a subject suffering from BH in an amount effective to produce at least partial remission of proteinuria (ie, a greater than 50 95 reduction in 24-hour urinary protein excretion compared to baseline).

In some embodiments, the method comprises administering an MAZP-2 inhibitory antibody to a subject suffering from HF (such as steroid-dependent HF) via a catheter (e.g., intravenously) for a first period of time (e.g., at least one day per week, two weeks, three weeks or four weeks or more) followed by administration to the subject of an inhibitory MA5BR-2 antibody subcutaneously for a second period of time (eg, a chronic phase lasting at least two weeks or more).

In some embodiments, the method includes administering an inhibitory MAbβR-2 antibody to a subject suffering from HF (such as steroid-dependent HF), intravenously, intramuscularly, or subcutaneously. The treatment may be prolonged and carried out daily or monthly, but preferably at least every two weeks or at least once a week, for example twice a week or three times a week.

In one embodiment, the method comprises treating a subject suffering from VN (such as steroid-dependent VN), which comprises administering to the subject a composition comprising an amount of an MABR-2 inhibitory antibody or antigen-binding fragment thereof comprising a variable region of severe chain, which contains SOK-NI, SOB-H2 and SOB-NZ of the amino acid sequence given in ZEO IU MO:67, and a variable section of the light chain, which contains

SOVv-11, SOV-I2 and SOV-IIZ of the amino acid sequence given in ZEO IU MO:70. In some embodiments, the composition contains an inhibitory MABR-2 antibody containing (I) (a) a variable region of the heavy chain, which contains: ) SOK-NI of the heavy chain, containing the amino acid sequence from 31-35 of the sequence zBEO IU MO:67; and i) SOC-H2 heavy chain containing the amino acid sequence from 50-65 of the ZEO IU MO:67 sequence; and iii) SOK-NZ

From the heavy chain, containing the amino acid sequence of 95-107 5EO IU MO:67, and B) the variable section of the light chain, which contains: Ї) SOC-Y1 of the light chain, containing the amino acid sequence of 24-34 5EO IU MO:70 ; and ii) SOK-12 of the light chain, containing the amino acid sequence of 50-56 5EO IYUO MO:70; and its) SOK-I From a light chain containing the amino acid sequence from 89-97 ZEO IO MO:70, or (I) a variant thereof comprising a heavy chain variable region with at least 9095 identity (eg, with at least 91 95, at least 92 95, at least 93 95, at least 94 9565, at least 9595, at least 9695, at least 9795, at least 9895, at least 99 95 identity) with 5EO IU MO:67; and a light chain variable region with at least 90 95 identity (eg, with at least 9195, at least 9295, at least 93 965, at least 94 96, at least 95 956, at least 96 956, at least 97 906, at least 98 956, at least 99 95 identity) with BZEO IU MO:70.

In some embodiments, the method comprises administering to a subject suffering from BH (such as steroid-dependent BH) a composition comprising an amount of an MABR-2 inhibitory antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising an amino acid sequence, given in ZEO IU MO:67, and a variable region of the light chain that contains the amino acid sequence given in ZEO IU MO:70.

In some embodiments, the method comprises administering to a subject suffering from HF (such as steroid-dependent HF) a composition comprising an MABR-2 inhibitory antibody or antigen-binding fragment thereof that specifically recognizes at least a portion of an epitope on human MAZ5R-2 recognized reference antibody OM5646, containing the variable region of the heavy chain, as specified in ZEO IYUO MO:67, and the variable region of the light chain, as specified in 5EO IYU MO:70.

In some embodiments, the method comprises administering to a subject suffering from or at risk of developing VN (such as steroid-dependent VN) a composition that comprises an MABR-2 inhibitory antibody or an antigen-binding fragment thereof comprising a heavy chain variable region that comprises the amino acid sequence given in 5EO IU MO:67 and a variable region of the light chain that contains the amino acid sequence given in 5EO IU MO:70 at a dose of 1 to 10 mg/kg (ie 1, 2, 3, 4, 5 , 6, 7, 8, 9, or 10 mg/kg) at least once per week (eg, at least twice per week or at least three times per week) for a period of 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.

EXAMPLE 21

This example describes additional results from an ongoing Phase 2 clinical trial evaluating the safety and clinical efficacy of a fully human monoclonal MAbR-2 inhibitory antibody, OM5646, in reducing proteinuria in adult patients with glomerulopathies, including IdAM. , as described in example 19.

Methods

As described in Example 19, Phase 2 involved patients with DAM receiving corticosteroids at study enrollment; all patients received OM5646 in an open-label fashion, and two patients with ICH had positive results. To date, two more patients with IMAD have completed the dosing phase using the methods described in Example 19, for a total of four patients with IMAD have completed the study.

To be included in this study, patients with AMI had to have (1) biopsy-diagnosed AMI, (2) hACE > 0.6 g/h, (3) eSR 530 ml/min/1.73 m, (4) controlled blood pressure during treatment with a stable dose of ACE/AKA, and (5) taking a stable dose of the steroid prednisone 210 mg for at least 12 weeks.

All four adult patients with AMI who were treated with OM5646 in this study had previously diagnosed renal insufficiency with an estimated glomerular filtration rate (eGFR) of 30 to 46 mL/mg/1.73 m: and a 24-hour protein level of 2.44 up to 4.87 g/24 hours. Before the start of the study, all patients received stable blockade of the renin-angiotensin system (RAS) and at least 3 months of corticosteroid treatment.

All patients received OM5646 IM once weekly for 12 weeks. Patients underwent a four-week run-in period, followed by OM5646 treatment, which consisted of four weeks of stable steroid dose, four weeks of taper, if possible, and four weeks of tapered steroid.

After treatment with OM5646, patients were observed for a further two weeks included in the study. After the study was completed, the researcher continued to observe the patients.

To evaluate the effectiveness, the following parameters were measured in the study: (1) ratio

Urine albumin/creatinine (URC), measured 6 times before treatment (baseline) and 3 times at each performance assessment during treatment and follow-up; and (2) 24-hour urine protein levels measured once before OM5646 treatment and once 2-4 weeks after completion of OM5646 treatment. According to the protocol, the corticosteroid dose was tapered between weeks 4 and 8 if clinically appropriate.

The results

Four patients with DAM underwent a two-week follow-up period after treatment. Table 14 presents the demographic and baseline characteristics of these patients.

TABLE 14

Demographics and initial characteristics 11111111 | Patient! | Patient2 | Patient 3 | Patient 4 posi zone | more | above | I'm going 8 years, 5 months, 5 months, 2 years of diagnosis of ESEV mm Hg. Art. ESEV - estimated glomerular filtration rate; Z5A - standard surface area (1.73 m); SHASV - albumin/creatinine ratio in urine.

All patients had a marked reduction in proteinuria during treatment with OM5646.

Statistically and clinically significant improvements were manifested both in the value of HRSC and in 24-hour protein levels, as shown in Fig. 41 and 42.

In fig. 41 shows a graph of the dependence of the value of ХАСскК (mg/g) on time for four IDAM patients who received OM5646, from the initial moment to day 120. As shown in fig. 41, from the initial moment to the end of the study, the average value of cASA decreased by 1.13 g/hl-0.27 (decrease by 77 95, p-0.026). As further shown in FIG. 41, at the time of the last visit in the observation period after treatment with OM5646, the value of PASK in each patient decreased by 94, 86, 47 and 89 95 (patients 1-4, respectively) relative to the initial level.

In fig. 42 graphically shows the change in protein levels in the 24-hour urine collection after treatment relative to baseline on day 1 before treatment in four patients with UTI receiving

OM5646. As shown in fig. 42, the protein level in the 24-hour urine collection decreased by 54, 81, 63, and 95 95 (patients 1-4, respectively) relative to baseline.

In fig. 43 graphically shows the mean change in protein levels in the 24-hour post-treatment urine collection relative to the pre-treatment baseline for four patients with ISAM receiving

OM5646. As shown in fig. 43, the average level of protein in the 24-hour urine collection decreased by 2.87-1.08 g/day (decrease by 73 95; p-0.013).

All patients were able to discontinue corticosteroids during or soon after the study, indicating that the effect of OM5646 on proteinuria is unlikely to be related to corticosteroids. Estimated glomerular filtration rates (es) (calculated by the formula for modifying the diet in kidney disease) were stable throughout the treatment period and the follow-up period.

OM5646 was well tolerated by all patients.

Thus, in phase 2 of this open-label clinical trial, there was a significant and sustained reduction

ASA was observed in all patients with AMI who received OM5646 for 12 weeks. 24-hour proteinuria was significantly reduced in all patients. The observed degree of proteinuria reduction was associated with significant improvements in kidney disease prognosis and clinical outcomes (IpKeg G.A. ei ai., At. 9. Kidpeu Oiv. 68(3):392-401 (2016)). The results demonstrating a strong reduction in proteinuria allowing steroid withdrawal after treatment with OM5646, an anti-MA5R-2 monoclonal antibody that inhibits the lectin pathway of complement, support the potential of OM5646 as a therapeutic

From a means to improve the results of treatment of IdA-glomerulopathy. Effects of OM5646 in patients with

The ISAM is stable and reliable, indicating its effectiveness in this population.

EXAMPLE 22

Preservation of remission after completion of OM5646 treatment in patients with IDA nephropathy (CHAM)

Prerequisites

According to Examples 19 and 21, in the phase 2 studies in patients with AMI, four patients with AMI were treated with OM5646, a fully human monoclonal antibody that inhibits MABR-2 activity. As described in Examples 19 and 21, all patients received OM5646 IM once weekly within 12 weeks. According to Example 21, after treatment with OM5646, the study patients were followed up for an additional 6 weeks, and all patients with IAH treated with OM5646 showed a partial remission (defined as a decrease of more than 50 95 in 24-hour urinary protein excretion and/ or final protein excretion less than 1000 mg/day). As described in this example, after completion of the study, these four patients were followed up to assess the duration of remission after treatment with OM5646.

Methods

Following the completion of the Phase 2 clinical trials described in Example 21, the investigator observed the condition of 4 patients with IDA who received OM5646. Endpoints in the study were hACE and 24-hour proteinuria. As described in Example 21, at the end of the study, all four patients with DAM were in partial remission. At the next observation, the ratio of protein to creatinine in urine (urinary creatinine) was measured. Each value of iRSE was converted to AUC (ratio of albumin/creatinine in urine) by multiplying by 0.64 (see pao eyi ai., Siip. )U. At. wasps Merpgoi 11:947-55, 2016).

The results

All patients experienced partial remission after treatment with OM5646. The average age of three women and one man was 42 years; three of them were European and one Asian.

The mean eSR was 41 mL/min/1.73 m7, and the mean steroid dose administered was 55 mg. The follow-up period after the last dose of OM5646 ranged from 2 to 10 months. As described in example 21, during the study the average value of hChSSK decreased by 77 95 (p-0.026). During follow-up, partial 60 remission was maintained in three patients (decrease in SHR by 54, 93, and 78,95 after 12, 12, and 5 months, respectively). In one patient, the value of SCA was 88 95 from the initial level after 7 months. During follow-up, three patients also had an improvement in esEC by 7, 13, and 7 ml/min./1.73 me. eSMC in the fourth patient was stable. All patients stopped taking steroids. OM5646 was well tolerated.

Thus, as described in Example 21, proteinuria was significantly reduced in ISAM patients during the 12-week OM5646 treatment and b-week post-treatment follow-up period included in the study. This reduction in proteinuria persisted up to 10 months after the end of treatment. These data support the use of OM5646 as a therapeutic agent to improve outcomes in IchA glomerulopathy.

In an update from the investigator on the status of the four patients described in this example, after approximately one year of follow-up after a single 12-week course of OM5646 treatment, it was reported that three of the four patients maintained a reduction in proteinuria. In these three patients, CSFs remained reduced by 14, 23, and 24 95 relative to the baseline values of the patients before OM5646 treatment. In addition, improvements in estimated glomerular filtration rate (ESR), a measure of kidney function, were observed in 3 of 4 patients after the study. In the patient with the most pronounced decrease in kidney function, an improvement in ES was observed from 30 ml/min.11.73 m? to 47 ml/min./1.73 m", i.e. the improvement was 57 95.

Thus, a sustained reduction in proteinuria after completion of one course of treatment

OM5646 remained impressive during one year of follow-up. The improvement observed in esEC was unexpected, especially after one year of follow-up, as it was predicted that a longer period of time would be required. As described above, two of the four patients had a slight increase in eSEC, and one patient had a dramatic response of 5095. The improvements observed in eSEC indicate that the additional beneficial effect of OM5646 in patients is a potential eliminating or significantly delaying the need for dialysis, as well as reducing the risk of complications associated with the progression of chronic kidney disease.

In accordance with the foregoing, in one embodiment, the invention relates to a method of reducing proteinuria in a human subject suffering from AMI, which comprises administering to the subject

From the MABR-2 inhibitory antibody or its antigen-binding fragment containing the variable region of the heavy chain, which contains SOK-NI, SOV-H2 and SOB-NZ of the amino acid sequence given in ZEO IU MO:67, and the variable region of the light chain, which contains

SOVv-11, SOV-I2 and SOV-I From the amino acid sequence given in ZEO IU MO:70, according to the following dosage schemes: c) administration of approximately 4 mg/kg (ie from 3.6 to 4.4 mg/kg ) of the specified antibody to a subject suffering from IDA, intravenously once a week during a treatment period of at least 12 weeks; or a) administering from about 180 mg to about 725 mg (i.e., 162 to 797 mg) of said antibody to a subject suffering from UTI intravenously once weekly for a treatment period of at least 12 weeks, wherein the method reduces proteinuria in the specified human subject.

In one embodiment, the dose of MABR-2 inhibitory antibody is about 4 mg/kg (ie, 3.6 to 4.4 mg/kg), such as about 3.6 mg/kg, about 3.7 mg/kg, about 3.8 mg/kg, about 3.9 mg/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 dose of the MA5R-2 inhibitory antibody is a fixed dose of from about 180 mg to about 725 mg (ie, from 160 to 800 mg or from about 300 to 500 mg, such as from about 300 mg to about 400 mg), e.g. about 160 mg, about 165 mg, about 170 mg, about 175 mg, about 180 mg, about 185 mg,

BO 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, approximately 385 mg, approximately 390 mg, approximately 395 mg, approximately 400 mg, approximately 405 mg, approximately 410 mg, approximately 415 mg, approximately 420 mg, approximately 425 mg, approximately 430 mg, approximately 435 mg, approximately 440 mg , 60 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, the treatment period is 12 weeks.

In one embodiment, the treatment period is followed by a break (i.e., a period without administration of a MA5P-2 inhibitor) of at least 2 months, or a break of at least 3 months, or a break of at least 4 months, or a break of at least 5 months, or a break of at least b months or longer, such as a break of at least 7 months, or a break of at least 8 months, or a break of at least 9 months, or a break of at least 10 months, or a break of at least 11 months, or a break of at least 12 months or longer.

In some embodiments, the method additionally includes periodic monitoring of urinary protein levels in the subject during a treatment and/or break period and, optionally, resuming treatment with an inhibitory MA5bR-2 antibody upon detection of a relapse of proteinuria.

In some embodiments, the method is effective to reduce the subject's proteinuria (DAM) by at least 30 95, such as at least 40 956 or at least 5095 or more than 5095, from baseline (before treatment) according to the results

From the end of the treatment period and/or at the end of the break.

In some embodiments, the method is effective for increasing the estimated glomerular filtration rate (EFR) in a subject suffering from MI.

In some embodiments, the subject suffering from UTI has proteinuria greater than 1 g protein/24-hour urinary protein excretion prior to treatment, and the method is effective to reduce proteinuria in the subject by at least 30 95 , such as at least 40 95 or at least 50 95, or more than 50 95, from baseline (before treatment), according to results at the end of the treatment period and/or at the end of the break, and/or to reduce proteinuria to less than 1 g protein/24-hour excretion protein in the urine, according to the results at the end of the treatment period and/or at the end of the break.

In some embodiments, the subject suffering from UTI has proteinuria greater than 1 g protein/24-hour urinary protein excretion despite maximum tolerated doses of antihypertensive agents and well-controlled blood pressure prior to treatment, and the method is effective for reducing the proteinuria in the subject by at least 3095, such as at least 40 95 or at least 50 95, or more than 50 95, from the baseline (pre-treatment) level determined at the end of the treatment period and/or at the end of the break, and/or to reduce proteinuria to less than 1 g of protein/24-hour urinary protein excretion determined at the end of the treatment period and/or at the end of the break.

In some embodiments, the subject suffering from IDdAM has not been treated with steroids for at least one year. In some embodiments, the subject suffering from AMI is treated with steroids during at least part of the 12-week period of treatment with OM5646.

In some embodiments, the subject suffering from UTI receives steroid treatment for at least part of the 12-week treatment period with OM5646, and the method is effective to reduce proteinuria and reduce or eliminate the need for steroid treatment until the end of the treatment period and/or until the end of the break.

Other implementation options

All publications, patent applications and patents mentioned in this specification are incorporated herein by reference.

Various modifications and variants of the described methods and compositions according to the invention will be obvious to specialists in this field of technology without deviating from the scope and essence of the invention. Although the invention is described 60 with respect to specific preferred embodiments, it is to be understood that the claimed invention is not to be limited in any way to such specific embodiments.

In accordance with the above, the invention offers the following implementation options. 1A. A method of treating, inhibiting, alleviating or preventing fibrosis in a mammal suffering from or at risk of developing a disease or disorder caused or complicated by fibrosis and/or inflammation, which comprises administering to the subject an amount of a MAZR-2 inhibitory agent effective to inhibit fibrosis. 2A. The method according to paragraph TA, where the MABR-2 inhibitory agent is a MA5BR-2 antibody or its fragment.

BY. The method according to paragraph 2A, where the inhibitory MA5P-2 agent is a monoclonal

MA5BR-2 antibody or its fragment, which specifically binds to part of ZEO IU MO:6. 4A. The method according to paragraph 2A, where the MABR-2 antibody or its fragment specifically binds to a polypeptide containing 5EO IU MO:6 with an affinity that is 10 times greater than the affinity of binding to another antigen in the complement system.

BA The method according to paragraph 2A, wherein the antibody or fragment thereof is selected from the group consisting of a recombinant antibody, an antibody with reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody. bA. The method of paragraph TA, wherein the MA5BR-2 inhibitory agent selectively inhibits activation of the lectin pathway of complement without significantly inhibiting C1d-dependent activation of complement.

AND. The method according to paragraph TA, wherein the MABR-2 inhibitory agent is administered subcutaneously, intraperitoneally, intramuscularly, intraarterially, intravenously, or by inhalation. 8A. The method according to any of paragraphs 1A-7A, wherein the disease or disorder caused or complicated by fibrosis and/or inflammation is associated with ischemia reperfusion injury. 9A. The method according to any of paragraphs 1A-7A, wherein the disease or disorder caused or complicated by fibrosis and/or inflammation is not associated with ischemia reperfusion injury. 10A. The method according to any of paragraphs 1A-7A, where before the introduction of the inhibitory MA5R-2

The subject had proteinuria from the agent, and administration of the MABR-2 inhibitory agent reduced the subject's proteinuria. 11A. The method according to any one of paragraphs TA-7A, wherein the subject suffers from a disease or disorder caused or complicated by renal fibrosis and/or inflammation. 12A. The method of paragraph 11A, wherein the MA5BR-2 inhibitory agent is administered in an amount effective to inhibit tubulointerstitial fibrosis. 13A. The method of paragraph 11A, wherein the MA5R-2 inhibitory agent is administered in an amount effective to reduce, delay, or eliminate the need for dialysis in the subject. 14A. The method of paragraph 114A, wherein the disease or disorder is selected from the group consisting of chronic kidney disease, chronic renal failure, glomerular disease (eg, focal segmental glomerulosclerosis), immune complex disorder (eg, IDA nephropathy, membranous nephropathy), lupus nephritis, nephrotic syndrome, diabetic nephropathy, tubulointerstitial lesions and glomerulonephritis (for example, SZ-glomerulopathy). 15A. The method according to any one of paragraphs TA-7A, wherein the subject suffers from a disease or disorder caused or complicated by fibrosis and/or inflammation of the lungs. 16A. The method of paragraph 15A, wherein the disease or disorder is selected from the group consisting of chronic obstructive pulmonary disease, cystic fibrosis, scleroderma-associated pulmonary fibrosis, bronchiectasis, and pulmonary hypertension. 17A. The method according to any one of paragraphs TA-7A, wherein the subject suffers from a disease or disorder caused or complicated by fibrosis and/or inflammation of the liver. 18A. The method of paragraph 17A, wherein the disease or disorder is selected from the group consisting of: cirrhosis, non-alcoholic fatty liver disease (steatohepatitis), liver fibrosis secondary to alcohol abuse, liver fibrosis secondary to acute or chronic hepatitis, biliary disease, and toxic liver damage (eg, drug-induced hepatotoxicity caused by acetaminophen or another drug such as a nephrotoxin). 19A. The method according to any one of paragraphs TA-7A, wherein the subject suffers from a disease or disorder caused or complicated by fibrosis and/or inflammation of the heart. 20A. The method of paragraph 19A, wherein the disease or disorder is selected from the group consisting of cardiac fibrosis, myocardial infarction, valvular fibrosis, atrial fibrosis,

endomyocardial fibrosis, arrhythmic right ventricular cardiomyopathy (AKIPS). 21A. The method according to any one of paragraphs TA-7A, wherein the subject suffers from a disease or disorder caused or complicated by vascular fibrosis. 22A. The method of paragraph 21A, wherein the disease or disorder is selected from the group consisting of vascular disease, atherosclerotic vascular disease, vascular stenosis, restenosis, vasculitis, phlebitis, deep vein thrombosis, and abdominal aortic aneurysm. 23A. The method according to any one of paragraphs TA-7A, wherein the subject suffers from a disease or disorder caused or complicated by skin fibrosis. 24A. The method of paragraph 23A, wherein the disease or disorder is selected from the group consisting of excessive wound healing, scleroderma, systemic sclerosis, keloids, connective tissue diseases, scarring, and hypertrophic scars. 25A. The method according to any one of paragraphs TA-7A, wherein the subject suffers from a disease or disorder caused or complicated by joint fibrosis. 26A. The method of paragraph 25A, wherein the disease or disorder is arthrofibrosis. 27A. The method according to any one of paragraphs TA-7A, wherein the subject suffers from a disease or disorder caused or complicated by fibrosis of the central nervous system. 28. The method of paragraph 27A, wherein the disease or disorder is selected from the group consisting of stroke, traumatic brain injury, and spinal cord injury. 29A. The method according to any one of paragraphs TA-7A, wherein the subject suffers from a disease or disorder caused or complicated by fibrosis of the digestive system.

ZOA. The method of paragraph 29A, wherein the disease or disorder is selected from the group consisting of Crohn's disease, pancreatic fibrosis, and ulcerative colitis.

Z1A. The method according to any one of paragraphs 1A-7A, wherein the subject suffers from a disease or disorder caused or complicated by ocular fibrosis.

Z32A. The method of paragraph C1A, wherein the disease or disorder is selected from the group consisting of anterior subcapsular cataract, posterior capsule opacification, macular degeneration, and retinal and vitreous retinopathy.

From ZZA. The method according to any of paragraphs 1A-7A, wherein the subject suffers from a disease or disorder caused or complicated by fibrosis of a bone or soft tissue structure of the musculoskeletal system. 34A. The method according to paragraph ZZA, wherein the disease or disorder is selected from the group consisting of osteoporosis and/or osteopenia associated with cystic fibrosis, myelodysplastic conditions with increased bone fibrosis, cellular capsulitis, contracture

Dupuytren's disease and myelofibrosis.

Z5A. The method according to any one of paragraphs 1A-7A, wherein the subject suffers from a disease or disorder caused by or complicated by fibrosis of the reproductive organs.

ZbA The method of paragraph C35A, wherein the disease or disorder is selected from the group consisting of endometriosis and Peyronie's disease.

Z7A. The method according to any one of paragraphs TA-7A, wherein the subject suffers from a chronic infectious disease caused or complicated by fibrosis and/or inflammation.

Z8A. The method according to paragraph 37A, wherein the infectious disease is selected from the group consisting of alpha virus, hepatitis A, hepatitis B, hepatitis C, tuberculosis, HIV and influenza.

Z9A. The method according to any one of paragraphs TA-7A, wherein the subject suffers from an autoimmune disease caused or complicated by fibrosis and/or inflammation. 40A. The method of paragraph C9A, wherein the autoimmune disease is selected from the group consisting of scleroderma and systemic lupus erythematosus (SLE).

A1A. The method according to any one of paragraphs 1A-7A, wherein the subject suffers from trauma-related scarring. 42A. The method of paragraph 41A, wherein the trauma-related scarring is selected from the group consisting of: surgical complications (eg, surgical adhesions in which scar tissue can form between internal organs, causing contracture, pain, and can lead to infertility), chemotherapy-induced fibrosis, radiation-induced fibrosis and burn-related scarring. 4 FOR. The method according to any of paragraphs 1A-7A, wherein the disease or disorder caused or complicated by fibrosis and/or inflammation is selected from the group consisting of organ transplantation, breast fibrosis, muscle fibrosis, retroperitoneal fibrosis, fibrosis of the thyroid gland, fibrosis of the lymph nodes, fibrosis of the bladder and 60 pleural fibrosis.

18. A method of preventing or reducing kidney damage in a subject suffering from a disease or condition associated with proteinuria, which comprises administering an amount of an MA5R-2 inhibitory agent effective to reduce or prevent proteinuria in the subject. 28. The method according to paragraph 1B, where the MABZR-2 inhibiting agent is an inhibitor

MA5P-2 antibody or its fragment.

ZV. The method of paragraph 18 or 28, wherein the MA5R-2 inhibitory agent is administered in an amount and for a period of time effective to achieve at least a 20 95 reduction in 24-hour urinary protein excretion compared to baseline 24-hour urinary protein excretion in subject before treatment. 4B. The method according to any of paragraphs 18-3B, wherein the disease or condition associated with proteinuria is selected from the group consisting of nephrotic syndrome, preeclampsia, eclampsia, toxic renal injury, amyloidosis, vascular collagen diseases (eg, systemic red lupus), lupus nephritis, dehydration, glomerular diseases (for example, membranous glomerulonephritis, focal segmental glomerulonephritis, SZ-glomerulopathy, minimal change disease, lipoid nephrosis), intense physical activity, stress, benign orthostatic (postural) proteinuria, focal segmental glomerulosclerosis, IdA -nephropathy (eg, Berger's disease), ICHM nephropathy, membranoproliferative glomerulonephritis, membranous nephropathy, minimal change disease, sarcoidosis, Alport syndrome, diabetes mellitus (diabetic nephropathy), drug-induced toxicity (eg, induced by NSAIDs, nicotine, penicillamine, lithium carbonate , gold and other heavy metals, inhibitors

ACE inhibitors, antibiotics (eg Adriamycin) or opiates (eg heroin) or other nephrotoxins); Fabry's disease, infections (for example, HIV, syphilis, hepatitis A, B or C, post-streptococcal infection, urinary schistosomiasis); aminoaciduria, Fanconi syndrome, hypertensive nephrosclerosis, interstitial nephritis, sickle cell disease, hemoglobinuria, multiple myeloma, myoglobinuria, organ rejection (eg, kidney transplant rejection), Ebola hemorrhagic fever, patellar nail syndrome, familial fever, NEGIGR syndrome, systemic lupus erythematosus, granulomatosis Wegener's, rheumatoid arthritis, glycogen storage disease type 1, Goodpasture's syndrome, purpura

From Schönlein-Henoch, urinary tract infection that has spread to the kidneys, Sjogren's syndrome and post-infectious glomerulonephritis. 5B. The method according to any of paragraphs 18-3B, wherein the disease or condition associated with proteinuria is IdA nephropathy (ie, Berger's disease). 68. The method of any of paragraphs 18-3B, wherein the disease or condition associated with proteinuria is membranous nephropathy. 7B. The method according to any of paragraphs 18-3B, wherein the disease or condition associated with proteinuria is lupus nephritis. 10. A method of inhibiting the progression of chronic kidney disease, which includes administering to a subject in need thereof an amount of an MABR-2 inhibitory agent effective to reduce or prevent tubulointerstitial fibrosis of the kidneys. 26. The method according to paragraph 1C, where the MA5BR-2 inhibiting agent is an inhibitor

MA5P-2 antibody or its fragment.

ZS. The method of paragraph 1C, wherein the subject in need thereof has proteinuria prior to the administration of the MABR-2 inhibitory agent, and the administration of the MABR-2 inhibitory agent reduces proteinuria in the subject such that the subject has at least a 20 95 reduction 24-hour urinary protein excretion in comparison with the initial 24-hour urinary protein excretion in the specified subject before the start of treatment. 4C. The method of paragraph 1C, wherein the MABR-2 inhibiting agent is administered in an amount effective to reduce, delay, or eliminate the need for dialysis in the subject. 10. A method of protecting the kidneys from kidney damage in a subject who has received, is receiving, or will receive treatment with one or more nephrotoxic agents, which comprises administering an amount of an MA5R-2 inhibitory agent effective to prevent or alleviate drug-induced nephropathy. 20. The method according to paragraph 10, where the MA5BR-2 inhibiting agent is an inhibitor

MA5P-2 antibody or its fragment. 30. The method according to paragraph 1cC, where the MA5R-2 inhibitory agent is administered before the administration of the specified nephrotoxic agent. 40. The method according to paragraph 1C, in which the MA5R-2 inhibitory agent is administered together with the specified nephrotoxic agent. 60 50. The method according to paragraph 1cC, where the MAB5bR-2 inhibiting agent is administered after the specified nephrotoxic agent for the treatment of nephrotoxicity. 1E. A method of treating a person suffering from immunoglobin A nephropathy (IADM), which includes administering to the subject a composition containing an amount of an MABR-2 inhibitory antibody or an antigen-binding fragment thereof effective to inhibit MABR-2-dependent complement activation. 2EB. The method according to paragraph 1E, wherein the subject suffers from steroid-dependent IDM.

ZE. The method according to paragraph 1E or 2E, where the inhibitory MA5bR-2 antibody is a monoclonal antibody or its fragment that specifically binds to human MA5R-2. 4E. The method according to any of paragraphs 1E-ZE, where the antibody or its fragment is selected from the group consisting of a recombinant antibody, an antibody with reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody.

BE. The method according to any one of paragraphs 1E-4E, wherein the MA5R-2 inhibitory antibody does not substantially inhibit the classical pathway. 6E. The method according to any of paragraphs 1E-5E, wherein the inhibitory MAB5R-2 antibody inhibits the deposition of SZB in 90 95 human serum with an IC of 30 nM or less.

THAT. The method according to paragraph 2E, which further comprises identifying a person having a steroid-dependent (DAM) prior to the step of administering to the subject a composition containing an amount of inhibitory

MABR-2 antibody or its antigen-binding fragment, effective for improving kidney function. 8E. The method according to any of paragraphs 1E-7E, wherein the MA5R-2 inhibitory antibody or antigen-binding fragment thereof is administered in an amount effective to improve kidney function.

OE. The method of paragraph 8E, wherein the MABR-2 inhibitory antibody or antigen-binding fragment thereof is administered in an amount and for a period of time effective to achieve at least a 20 95 reduction in 24-hour urinary protein excretion compared to baseline 24-hour excretion protein in the subject's urine before treatment. 10. The method according to paragraph TE, wherein the composition is administered in an amount sufficient to improve kidney function and reduce the dose of corticosteroids in said subject. 11E. The method according to any of paragraphs 1E-10E, wherein the MA5BR-2 inhibitory antibody or antigen-binding fragment thereof contains a heavy chain variable region that contains SOVB-

From NI, SOB-H2 and SOK-NZ of the amino acid sequence given in ZEO IU MO:67, and the variable region of the light chain, which contains SOK-I1, SOB-I2 and SOB-II From the amino acid sequence given in ZEO IU MO: 70. 1R. A method of treating a person suffering from membranous nephropathy (MN), which includes administering to the subject a composition containing an amount of MABR-2 inhibitory antibody or antigen-binding fragment thereof effective to inhibit MABR-2-dependent complement activation. 2. The method according to paragraph 1, wherein the subject suffers from steroid-dependent MN.

ZE. The method according to paragraph 1E or 2PE, where the inhibitory MA5BR-2 antibody is a monoclonal antibody or its fragment that specifically binds to human MABR-2. 4R. The method according to any of paragraphs 1E-ZE, where the antibody or its fragment is selected from the group consisting of a recombinant antibody, an antibody with reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody.

BE. The method according to any of paragraphs 1R-4E, wherein the MA5R-2 inhibitory antibody does not substantially inhibit the classical pathway. 6R. The method according to any of paragraphs 1BE-5E, wherein the inhibitory MA5R-2 antibody inhibits the deposition of SZB in 90 95 human serum with an IC of 30 nM or less.

THAT. The method according to paragraph MR, wherein the method further comprises identifying a human subject having steroid-dependent MN before the step of administering to the subject a composition that contains an amount of an MABR-2 inhibitory antibody or antigen-binding fragment thereof effective to improve kidney function . 8. The method according to any of paragraphs 1E-7E, wherein the MA5R-2 inhibitory antibody or its antigen-binding fragment is administered in an amount effective to improve kidney function.

OP. The method of paragraph 8E, wherein the MABR-2 inhibitory antibody or antigen-binding fragment thereof is administered in an amount and for a period of time effective to achieve at least a 20 95 reduction in 24-hour urinary protein excretion compared to baseline 24-hour excretion protein in the subject's urine before treatment.

TORUS. The method according to paragraph 1E or 2E, wherein the composition is administered in an amount sufficient to improve renal function and reduce the dose of corticosteroids in said subject. 117. The method according to any of paragraphs 1E-10E, where the MA5R-2 inhibitory antibody or antigen-binding fragment thereof contains a heavy chain variable region that contains SOC-

NO, SOB-H2 and SOC-NZ of the amino acid sequence given in ZEO IU MO:67, and the variable region of the light chain, which contains SOC-I1, SOB-I2 and SOB-II From the amino acid sequence given in ZEO IU MO:70 . 16. A method of treating a human subject suffering from lupus nephritis (LU), which includes administering to the subject a composition containing an amount of an MA5R-2 inhibitory antibody or an antigen-binding fragment thereof effective to inhibit MA5R-2-dependent activation complement 26. The method according to paragraph 1c, where the subject suffers from steroid-dependent VL.

Coll. The method according to paragraph 10 or 20, where the inhibitory MA5R-2 antibody is a monoclonal antibody or its fragment that specifically binds to human MABR-2. 406. The method according to any of paragraphs 105-305, wherein the antibody or fragment thereof is selected from the group consisting of a recombinant antibody, an antibody with reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody. 50. The method of any one of paragraphs 105-405, wherein the MA5R-2 inhibitory antibody does not substantially inhibit the classical pathway. 66. The method according to any one of paragraphs 10-56, wherein the MAB5bR-2 inhibitory antibody inhibits the deposition of SZB in 90 95 human serum with an IC of 30 nM or less. 76. The method according to paragraph 10, wherein the method additionally includes identifying a human subject having steroid-dependent HF before the step of administering to the subject a composition that contains an amount of an MABR-2 inhibitory antibody or an antigen-binding fragment thereof effective to improve kidney function. 80. The method according to any of paragraphs 10-70, wherein the MA5R-2 inhibitory antibody or antigen-binding fragment thereof is administered in an amount effective to improve kidney function. 90. The method according to paragraph 8Cb, where the MABR-2 inhibitory antibody or its antigen-binding fragment is administered in an amount and for a period of time that is effective to achieve at least a 20 95 reduction in 24-hour urinary protein excretion compared to the initial 24- hourly urinary protein excretion in the subject before treatment. 1020. The method according to paragraph 10 or 20, wherein the composition is administered in an amount sufficient to improve renal function and reduce the dose of corticosteroids in said subject.

Zo 116. The method according to any of paragraphs 105-100, wherein the MA5R-2 inhibitory antibody or antigen-binding fragment thereof comprises a heavy chain variable region that contains SOC-

NO, SOB-H2 and SOB-NZ of the amino acid sequence given in ZEO IU MO:67, and the variable region of the light chain, which contains SOK-I1, SOB-I2 and SOB-II From the amino acid sequence given in ZEO IU MO:70 .

Although illustrative embodiments have been shown and described, it will be understood that various changes may be made without departing from the spirit and scope of the invention.

LIST OF SEQUENCES

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Te bIy TigoGuz 5eg Azp Tig Ma! Tys IIe Tig Rpe Mai Tig Ar s1y 279 275 280 isa bra vas sas asa ves TyivbR aavr as sas tas asv avs asa all sav 915 b5eg b1u Azr Nih Tig s1u Ter Guz I1e Ni5 Tug Tig 5eg Tig A1a sp 285 298 295

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З5 BEsvsssass tvEsvassass astvsssasaa ssasstvves vvEESstTasti vstsstvsse 1920 svsavvstas visstvsass vtaasavsv sasstvstsa vRivavvvav vysvstvev 1980 ssssaasvsa ssstEstsstv vratasssvv vvstsstsav vessayesevst vEStTvsssa 29040 7 bEBRTvsSvva vevssivReS stIRvasastv vvyvstsstTa vevssstv sstssavsts 2186 sssttstsav ssstvstTss sststsavsa vsavvsisa isavtvvssas sstvssstav 2160 sastvavasi aaststaasa yesssastvev tTasstvvies sasstrevsy strReEvaasss 2220 sIsayesevtavs sasevravav SsevRev tayes tassstsviE sstServastv veESSsSTvIT 2280 ssstvsSastiv hevevrRaseves savrtvststv veveSseiRevS avsssssss TvIResvssiv 2340 7 assstvstss sssvastsvv EEESSTESSTSE seVRETSTSE ssESvsSsSTsST stvayeststs 24080 tssssavavsa vavsststav sstsssstv avstssvvsi vsssavsave isa vraavssa 2460

Bavssavesii vstvevSsstSsa vEsSsSerVII vveESSsStTravai vsyvTvsss aastsssaett 2520 sasssassaye vevassaasta abaaasstvv sssssssa sstvstvssv srivIsIstv 2580

BEBIvBRAvv VISERVares eVIRERESsVS vSESSSTST vstsssts sesasawsst 2640 saevaaassss avvEstvtvR vavsstssts sasevvessa sasevissti vvssisasss 2790 sSEVESstivRa avaevveevsa stEvarvssvv avargevvtaa vvsstsvsIs vavtssavv 2760 ? sssssavavrvs ivavsssava vtaaesteva assasssssa sEsareviEsii vvstvvavv 2820 avsstIvasss asavravrvava sassstivvva vgatstayassasi varvvvtaae sterisssss 2880 vsaaaessav vevvIvaesss sastvsssa tavrvsasaws saseivvaav aaresavesa 2940 asvEsvvevs tsst Ssastes stavavevssi sasaastesaa aevsssssa svsavstvv z60o

BEBIVBRvv vIvvTasvvv asevvrassa avsst(issti raarvasavra vsssavsssa ze6o 7 asassssvss ssvEvvsavs avsayesasvi viyissavrsva vraarvavav sassavasts 3120 aveEsaevayes astevesvssi SrRaaseissa avaasavsss savvesaave vBisaaaasav 3180

BeEvRaaavvvv vivaivavav ayesstEs sevaievEess tssavvaass areevvsiwe 3240

STIVVESTTRv STReviIisSveR vBtTavrvavass sasvaesvaae aaastiseveva aisastevvev 3300

Test taav ввгаессавв вевавстіссва ареввсссті аввсісваве аваевстсст 3360 7 сс ссс вааетсссав врасссавва вавтівтсссті ЕЕСССТСЕЕ ССТРевТВИСС 3420 саессассс свсссссвсС sstvesSavav stvbRIvRaas savtvstst avsssstas 3480

Zo STVEEVESesS vaststvessi savrvrasassa ssasesissss TevReRIvIv aryvavvvss 3540

TvEvsvstss aeysssvavtv svssTvyiy savstaaavs sIsaaavrsaa vagaaassss 3600 sIsSeEsStaavs vvsssstsav ssassevv vvysvSSsve TESsStvReRTa vesstsarev 3660 7 vBstvvssass tvsavrvvsss avssaasss arevaevsav aeyvisssavs sasaiessstv 37 20 tssssssss sEvstssssa avesayessas sststvETR vIvsvavevs tvRagsavravvv 3780 sasvssaavii sastsssstv ssstYessssts stgssavsss YavTvsStsssvv ssavvists 3840 asssavavrvi stveReEvavsii vaastasss vvssvtaiss saaaststss 3900 avEsvsasti 3960 7.

Tssvsavsa Tvvsssssas sasvieYesssv isasssstsvev Tvassssass Esttsarve 4140 sIsStaevvav vysaarvvvvsESsvRav tasaarivyv vbrRavresavav SRReEevavevv 4200 sassssaayes sasvevsstvv vESevssis TeestIviss stEvaaaasesi barbarians 4260 7 vEsbast tsss tsssssssav ssavasssav vsawsvsts sssavstets ayevavstest 4320 gEstsavayeyi saaasavrasa vagaavaasa Yevsssayess ver vaava sasevssssa 4380 savvaesvaa asaaaaaavsa asasevivas sassassei vEsasavaye aayesavrvava dadda ssasasavrvs Tvvaavrayess sasastavav sasavsvavs aavsevv sts avrayessttvv 4580

Tevaavsevsa vavsvssts YesstRvavii vsaavvvavst vbavavtvta vevststs 4560 vBevSsavvasi avrvaavvvas assawvteta vieviestiva veivIstvRaves avsavstest 4620 aagvvvaavs asssvivsss tsstIsavsav sasssavsaye stEsassasti saesstssaa 4680 ssasssa есс ассаесасті саестсеттас ссасссасс есвссастс аесстеств 4740 сассаесст саесааей саесааесас саассааей саесаааесас саассааес ааессааесс асасааасат 4800 саассааесс тестасстас сааессастаес саеассаєся естаясавса Аесстстасс 4860 ассааессте свіесввіса tssa yessayes yessayssats 4980 "21905 8 "2115 136 "212" Protein "2135 Noto zariep5 "4005 8

Met Age Gay Ge TPg Gay Ge s1u Ge Gay Suh s1u beg Mai Aia Tig 1 5 189 15

Zo

Rgo Gets o1u Rgo Gu5 Ter Rgo fight Rgo Ma! Ripe s1u Age Gay Aa 5eg 20 25 3a

Rgo b1u Rpe Rgo o1u bij Tug ATa Ap Azr bip b1y Age Age Ter Ts 35 40 45

Ge Tyg ATa Rgo Rgo b1u Tug Ag Gay Ag Gay Tug Ripe Tig Ni Re 50 55 va

Azr Gay 01 Getz beg No Gay Suz bI1y Tug Azr Ripe Ma! Guz Gei beg 65 78 75 80 b5eg b1u ATa uh Mai Ge Aia Tig Ge Suzh s1u bI1p o01y beg Tig Ar 85 90 95

Te bij As ATa Rgo b1u Gu5 Azr TPg Rpe Tug beg Ge o1u b5eg 5eg 189 195 119

Hey Azr IIe Tig Re Age beg Azr Tug 5eg Azp bi Gu5 Rgo Rpe Tpg 115 128 125 o1u Re bIy ATa Rie Tug A1a Aa

1308 135 "2105 9 "2115 181 "212" Protein "2135 Noto zariep5 "4005 9

Met Age Gay Ge TPg Gay Ge s01u Ge Gay Suh o01u beg Mai A1a Tig 1 5 16 15

Rgo Gets o1u Rgo Gu5 Ter Rgo fight Rgo Ma! Ripe s1u Age Gei Aa 5eg 29 25 36

Rgo b1u Rpe Rgo o1u bIy Tug Aia Azp Azr b1p b1y Ag» Age Ter Tvg 35 40 45

Ge Tyg ATa Rgo Rgo b1u Tug Ag Gay Ag Gay Tug Re Tig No Re 50 55 60

Azr Gay 01 Ge 5eg Ni5 Ge Suh bT1y Tug Azr Rpe Mai Guz Getsy 5beg 65 70 75 80

Zo b5eg b1u ATia Guz Mai Ge A1ia Tipg Ge Suz b1u b1p 01 beg Tpg Az5r 85 90 95

Te bij As ATa Rgo sb1u Guz Azr Tig Re Tug 5eg Ge o1u 5eg 5eg 166 185 116

Hey Azr IIe Tig Re Age beg Azr Tug beg Azp bi Gu5 Rgo Re Tpg 115 129 125 so1u Re 01 ATa Rpe Tug Aia Aia b1y Azr I1e Azr 01y Su b1p Mai1 1308 135 140

A1a Rgo s1u bI1y ATA Rgo Tiig Su Ar No No Su5 No Autop No Ge 145 158 155 160 s1u o01u Rpe Tug Su beg Sub Age Aia sb1u Tug Mai Gets No Age Azp 165 176 175

Guz Age Tig Su 5eg 188 "2105 16

"2115 293 "212" Protein "2135 Noto zariep5 "4005 186

Met Age Gay Ge TPg Gay Ge s1u Ge Gay Suh s1u beg Mai Aia Tig 1 5 16 15

Rgo Gets o1u Rgo Gu5 Ter Rgo fight Rgo Ma! Ripe s1u Age Gei Aa 5eg 29 25 36

Rgo b1u Rpe Rgo o1u bij Tug ATa Ap Azr bip b1y Age Age Ter Ts 35 40 45

Ge Tyg ATa Rgo Rgo b1u Tug Ag Gay Ag Gay Tug Re Tig Ni Re 58 55 60

Azr Gay 01 Getz beg No Gay Suz bI1y Tug Azr Ripe Ma! Guz Gei beg 65 70 75 80 b5eg b1u ATia Guz Mai Ge A1ia Tipg Ge Suz b1u b1p 01 beg Tpg Az5r 85 90 95

Zo

Te bij As ATa Rgo b1u Gu5 Azr TPg Rpe Tug beg Ge o1u b5eg 5eg 166 185 116

Hey Azr IIe Tig Re Age beg Azr Tug beg Azp bi Gu5 Rgo Re Tpg 115 1208 125 so1u Re 01 ATa Rpe Tug Aia Aia b1y Azr I1e Azr 01y Su b1p Mai1 138 135 140

A1a Rgo s1u bI1y ATA Rgo Tiig Su Ar No No Su5 No Autop No Ge 145 158 155 160 s1u o01u Rpe Tug Su beg Sub Age Aia sb1u Tug Mai Gets No Age Azp 165 176 175

Guz Age Ts o Su5 beg ATa Ge Su5x 5eg b1u beep Ma! Rpe Tig bIp Age 188 185 199 b5eg b1u bij Hey b5eg beg Rgo biy Tug Rgo Age Rgo Tug Rgo Guz Ge 195 290 295 b5eg 5eg Su5 Tig Tug 5eg I1e beg Ge 01 bI1y s1u Rpe beg Ma! Pe

2168 215 229

IGets Azr Rpe Mai bij b5eg Rpe Azr Ma! sb1y Tpg No Rgo would Tpg Ge 225 230 235 240

Sub Rgo Tug Azr Rpe Ge Guz I1e b1p Tig Ar Age b1y 01y Nih s1u 245 258 255

Rgo Rpe Suh 01u Gu5 Tyg Gay Rgo Nib5 Age I1e b1y TPg Gu b5eg Avp 260 265 279

Tig o Mai TiG I1e Tis Rpe Mai Tig Azr oTi 5eg su Azr Nih5 Tig o1u 275 2808 285

Ter Guz Pe Ni Tug 2908 "2105 11 "2115 41 "212" Protein "2135 Noto zariep5 "4005 11

Zo bI1y Ar IT1e Azr sb1y Suh5 sb01p Mai ATia Rgo s1u ob01y ATa Rgo Tig Su 1 5 16 15

Ar No No Sub No Azp Nih Gets b1u b01u Re Tug Su5 5eg Su5 Ag 29 25 36

A1i1a o1u Tug Mai Gets Ni Age Ap Gu 35 40 "2105 12 "2115 242 "2125 Protein "2135 Noto zariep5 "4005 12

I1e Tug s1u s1u b1p Gu5 AITA Gu5 Rgo sb1u Azr Rpe Rgo Ter bip Mai 1 5 16 15

Yiets I1e Ge s1u o1u Tpg Tpg A1a AIia s1u AIa Hey Getsy Tug Author Azp 20 25 36

Ter Mai Gei Tig Ai1a A1a Ni5 Aia Ma! Tug 01 b1p Guz No Ar AIa 35 40 45 b5eg A1i1a Gay Azr I1e Age Mei b1u TPg Gay Gu5 Age Gets b5eg Rgo No 50 55 60

Tug Tig bIp Aia Ter 5eg 010 Aia Ma! Rpe I1e Ni5 bIy o1u Tug Tig 65 70 75 80

No Azr ATia s1u Rpe Azr Ap Azr I1Ie Aia Ge Pe iu5 Getsy Avzp Avp 85 90 95

Guz Ma! Ma! ITIe Abp beg Ap I1e Tyg Rgo IIe Su Gets Rgo Age Guz 166 185 116 bi ATa bij beg Rpe Mei Age Tyg Ar Azr Ie o1u Tyg ATa 5eg s1u 115 129 125

Ter b1u Ge Ts bip As b1u Re Gay Aia Ag» Ap Ge Mei Tug Mai 1308 135 140

Azr IT1e Rgo I1e Mai A5r Ni5 bIp Huz Suh5 Tig Aia ATa Tug osi Guz 145 158 155 160

Zo

Rgo Rgo Tug Rgo Age b1u 5eg Ma! Tpyg ATa Ap Mee Hey Su5h Aia Oo1Uu 165 176 175 ey 61 beg s1u o1u Guz Azr beg Sub Agev b1u Azr 5eg o1u o1u A1Ta 188 185 199 ei Ma! Rpe Gay Ar beg bi Tyg bIy Ag» Ter Rpe Mai o1u o1u I1e 195 290 295

Mmai beg Ter s1u beg Mee Azp Suz b1u b01y Aia s1u bIp Tug s1u Mai 2168 215 229

Tug Tig Guz Ma! II1e Azp Tug I1e Rgo Tgr I1e bi Azp I1e I1e 5eg 225 230 235 240

Azr Rpe "2105 13 "2115 16 "212" Protein "2135 Artificial sequence

"223" Synthetic "4005 13 o1u Guz Azr 5eg Su Ag" b1u Azr ATia s1u s1u AIa Hec Mai Re Ge 1 5 18 15 "2105 14 "2115 15 "212" Protein "2135 Artificial sequence "220" "223" Synthetic " 4005 14

Te Rgo Gets s1u Rgo Gu Ter Rgo bI1y Rgo Ma! Rie o1u Age Gei 1 5 18 15 "2105 15 "2115" 43 "212" Protein "2135 Artificial sequence "220"

From "223" Synthetic "4005 15

Te ATa Rgo Rgo bI1u Tug Agv Gay Age Gay Tug Re Tig Ni Re Ar 1 5 18 15

Yech bIy Ge 5eg Ni Ge Suz b1y Tug Azr Rpe Mai Guz Gay b5eg beg 20 25 30 o1u ATa Gu Mai Ge ATa Tig Gay Su o1u b1p 35 4 "2105 16 "2115 8 "212" Protein "2135 Artificial sequence "220" " 223" Synthetic "4005 16

Te Rpe Ag» b5eg Ar Tug beg Azp 1 5

"2105 17 "2115 25 "212" Protein "2135 Artificial sequence "220" "223" Synthetic "4005 17

Rpe Tug beg Ge 01u 5eg 5eg Gets Azr I1e Tig Rpe Age 5eg Ar Tug 1 5 18 15 b5eg Azp bij Gu5 Rgo Rpe Tyg o1u РPE "2105 18 20 "2115 9 "212" Protein "2135 Artificial sequence "220" 25 "223 » Synthetic «4005 18

Ie Azr bI1y Su5 sI1p Mai AIa Rgo s1u

From 1 5 "2105 19 "2115 25 "2125 Protein "2135 Artificial sequence "220" "223" Synthetic "4005 19

A1a Ahp Mei Hey Su5 Ai1a o1u Ge b0I1y beg oIu s1u Guz Ar 5eg Su 1 5 18 15

Age b1u Azr beg oO1u s1u A1a Getsy Mai 20 25 "2105 29 "2115" 9606 "2125 DNA "2135 Noto zariep5 "220" "221" Coding sequence "222" (51)..(797)

"4005 29

Met 5eg 1 stv ES ssa sa sts sst sts stK ss stv avt as vyivb vsa vse s 194

Ge Rpe Rgo 5eg Gets Rgo Hey Hey Ge Ge 5eg Mei Ma! Aia Aa 5eg 5 189 15

Zhtas sa vaa asti ate ass ate

Tug 5eg bIy Tig Mai Tig Su5 obT1y Azr ATa bip Guz Tig Su Rgo A1a 20 25 3a

Iv asst wss tvI avs istI ssa vvs as aas wes Ts ssa wes aaa vaye 290

Mmai IIe A1a Su5x beg beg Rgo b1u I1e Azp s01u Rpe Rgo Ou Guz Az5r 35 40 45 58

B SBI wai ves ass aar bra vaa aar vve vaa ssa vs saa vv sts 248 so1u Age Azr sb1u Tpg Gu5 o1u b1y Gu5 s1u b1y Rgo o1u bip o1u Ge 55 va 65 ava ves iYita sav wes sss ssT vva aav TS vve ssT ssa vva aae ssa 296

Age b1u Ge beep o01u Rgo Rgo sI1u Guh5 Ge Oo1u Rgo Rgo s1u Ap Rgo 76 75 80

BVB SST ST vve isa ssa bra ssa aar vBsS saa aaa vva vas ss vva 344 o1u Rgo beg o1u 5eg Rgo s1u Rgo Guz o1u bI1p Guz oTu Ar Rgo o01u 85 90 95

Zo aaa aut sse wai vvi wat aut avs stv vsI vss isa raa ava aaa vs 392

Huz 5eg Rgo Azr bI1u Ar beg b5eg Hey AIia AI1a 5eg 01y Age Huz A1Ta 196 195 119

З5 stv saa asa raa ase vsa svBI ass aaa aav svv ss ass EU Ts stv to

Hey b1p Tys biy Mee ATa Age I1e Guz Gu5 Ter Ge Tig Re 5eg Gey 115 1209 125 130 ves aaa saa HER BR aas aav SS s stv ass aas 55Bi raa asa aib 488 s1u Gu5x b1p Ma! s1u Azp uh Re Rpe Gay TPg Azp b1u bIii Ie Met 135 140 145 ass t waa aaa yiv aav vss Tv vysS aav SS sav vss ts viv 536

Tig Rpe oBITy Guz Mai Guz ATia Ge Suzh Ma! Guz Rie b1p AIia b5eg Mai 1509 155 169 vss ass sss avv aai vsiY Bvbsa vav aai vva vss as sav aae ss ats 584

A1a Tys Rgo Age Ahp A1i1a A1i1a bij Ahp b01u Aia I1e b1p Ahp Ge Pe 165 178 175 aav vav vaa vss TES stv vBS ass asti rai var aav asa vaa vve sav 632

Guz bIy b1y A1ia Rpe Gey sb1u I1e Tpg Azr by uh Tpg oii o1u s1p 186 185 199

TE BIB vaye stv asa vva aai ara stv ass tas asa aas ev aas vav 680

Rpe Mai Ar Ge Tig b1u Ap Ag» Gay Tig Tug Ts Azp Ter Up bi 195 299 295 219

BVi vaa sss aas aati vsti vv SI vae vaa vae HER mouth 6 hundred siv 728 o1u obTy Rgo Azp Azp ATia s1u beg Azr bTy Ar Su Mai Gey Getsi Ge 215 220 225 aaa aae ves sav web aai vas vs sss o tvsS ss ass tss SAYE STV VSS 776

Guz Azp b1u bip Ter Azp Ar Mai Rgo Sub5 5eg Tig beg Ni Getsy A1Ta 230 235 240 vBES Tv wav Es sst ass va avvysataye sastsavvss stСSETvEs 827

Mmayi Suz s1y Rpe Rgo I1e 245 -essstastvsa asssasawvs ssasawtaev stEsSvaaaava bEaaastayeae saayetsstssts 887 asayessavta Sv EssstY TetvReeSsaayei sastaaaaaay vayesastaas avrsassaasa 947 aavsaayeaae avt 960 "2105 21 "2115 24 8 "212" Protein "2135 Noto zariep5 "4005 21

Mat beg Gay Rpe Rgo beg Gay Rgo Ge Gay Gay Gay beg Me Mai A1Ta 1 5 18 15

Zo A1a b5eg Tug beg bi Tig Mai Tis Su5 bI Ar ATi1a b1p Gu Tis Su 20 25 30

Rgo Aia Ma! Iie Aia Su5 beg beg Rgo b1u IIe Azp o01u Rie Rgo o01u 35 40 45

Guz Azr b1u Ag» Azr o1u Tig Guz b1u bIy Gu5x o1u 01 Rgo o1u sp 58 55 va so1u Ge Age s1u Ge bip s1u Rgo Rgo s1u Guz Gets o1u Rgo Rgo o1u 65 78 75 va

Azp Rgo s1u Rgo beg b1u beg Rgo o1u Rgo Huz s1u bIp Huz o1u A5r 85 90 95

Rgo b1u Gu beg Rgo Azr b1u Azr 5eg beg Geu A1a A1a 5eg siIy Age5 188 185 118

Guz ATia Ge bip Tig bi Met A1i1a Age ITe Gu5 Gu5 Ter Ge Tig Re 115 129 125 b5eg Ge oI1u Guz bI1p Ma! s01u Azp Guz Re Rpe Ge Tpg Azp o1u axles 138 135 140

Ie Mee Tig Re si Guz Mai Guz ATa Hey Suh Mai Guz Rpe b1p A1a 145 1509 155 160 b5eg Ma! Aia Tpyg Rgo Ag" Ahp Ai1a A1i1a biy Ahp b01u Aia Ie b1p Azp 165 178 175

Yeets I1e Guz 01 01 A1ia Rpe Gey b1u I1e Tpg Azr bIy Guz Tyg sy 1809 185 199 s1u b1ip Rpe Mai Azr Gay Tpg o1u Ap Ag Gay Tyg o Tus Ts Azp Ter 195 290 295

Ap bii s01u bT1y Rgo Ap Azp Aia b1u 5eg Ar s01y Azr Su Mai Gei 216 215 229

Ge Ge Guz Azp b1u b01p Ter Azp Ar Mai Rgo Su5 5eg Tig 5eg Niv 225 230 235 240

Ge Ai1a Mai Su bI1y Rpe Rgo I1e 245

From "2105 22 "2115 6 "212" Protein "2135 Artificial sequence "220" "223" Synthetic "220" "221" MI5S EEATIVE "222" (1)..(1) "223" in which X is in position 1 is hydroxyproline "220" "221" MI5S EEATIVE "222" (4)..(4) "223" in which X in position 4 is a hydrophobic residue "4005 22

Haa o1u Guz Haa s1u Rgo 1 5 "2105 23 "2115 5 "212" Protein "2135 Artificial sequence

"223" Synthetic "220" "221" MI5S EEATIVE "222" (1)..(1) "223" in which X is hydroxyproline "4005 23

Haa o1u Guz Getsy a1u 1 5 "2105 24 "2115 16 "212" Protein "2135 Artificial sequence "220" "223" Synthetic "220" "221" MI5S EEATIVE "222" (9)..(15) "223" in which X in position 9 is hydroxyproline

Zo "4005 24 so1u Ge Age s1u Ge bip s01u Rgo Haa s1u Guh5 Gec o1u Rgo Haa o1u 1 5 18 15 "2105 Y -725 "2115 27 "212" Protein "2135 Artificial sequence "220" "223" Synthetic "220" "221" MI5S EEATIVE "2225 (3)..(27) "223" with X in positions 3, 6, 15, 21, 24, 27 is hydroxyproline "4005 KZh225

O1u Rgo Haa s1u Rgo Haa s1u Ge Age s1u Gay beep o1u Rgo Haa o1u 1 5 18 15

Guz Ge o1u Rgo Haa s1u Rgo Haa s1u Rgo Haa 20 25

"2105 26 "2115 53 "2125 Protein "2135 Artificial sequence "220" "223" Synthetic "220" "221" ti5s Beaige "222" (26)..(26) "223" Haa can be any amino acid, occurring in nature "220" "221" ti5s Beaeige "2225" (32)..(32) "223" Haa can be any amino acid occurring in nature "220" "221" ti5s Beaeige "222" ( (35).

From "223" Haa can be any naturally occurring amino acid "220" "221" ti5s Beayige "222" (50)..(58) "223" Haa can be any naturally occurring amino acid "4005 726 s1u Guz Azr s1u Age Azr b1u Tig Gu sb1u bI1y Guh o1u sy Rgo o1u 1 5 18 15 beep o01u Hey Age s1u Hey beep s01u Rgo Haa s1u Gu5 Gets o1u Rgo Haa 20 25 30 o1u Azp Haa o1u Rgo beg s1u 5 eg Haa o1u Rgo Guz Ou bI1p Guz o1u 35 4 45

Azr Haa s1u Gu 5eg 58 "2105 27 "2115 33 "212" Protein "213" Artificial sequence

"223" Synthetic "220" "221" MI5S EEATIVE "2225" (3)..(33) "223" with X in positions 3, 6, 12, 18, 21, 30, 33 is hydroxyproline "4005 27 so1u ATia Haa o1u 5eg Haa s1u s1y Guz o1u ATia Haa o1u Rgo sbIp o1u 1 5 18 15

Rgo Haa s1u Rgo Haa s1u Guh Mee o1u Rgo uh s01u o1y Haa s1u Ar 20

Haa 25 "2105 28 "2115" 45 "212" Protein "2135 Artificial sequence

From "220" "223" Synthetic "220" "221" MI5S EEATIVE "2225 (3)..(45) "223" with X in positions 3, 6, 9, 27, 30, 36, 42, 45 is hydroxyproline "4005 28 o1u Su5z Haa o1u Ge Haa s1u AIia Haa o1u Azr Guz o1u osi Aia o1Uu 1 5 18 15

Te Azp b1u Guz Ag" b1u bi Age b1u Rgo Haa o1u Rgo Haa Oo1u Guz 20 25 30

A1ia s1u Rgo Haa o1u Rgo Azhp s1u Aia Haa o1u s1y Haa 35 4 45 "2105 29 "2115 24 "212" Protein "2135 Artificial sequence "220"

"223" Synthetic "4005 29

Ge bip Age ATa Ge bi I1e Hey Rgo Azp Ag» Ma! Tpg Pe Guz ATa 1 5 189 15

Azp Ag Rgo Rpe Ge Ma! Rpe Pe 20 "2195 36 "2115 559 "2125 DNA "2135 Noto zariep5 "4005" 36 asvavvsts Evassstsssii vevsstTsTv TRTRreSTSsvRe TRESSasss SESeReSSSV 6o aavevvevsstv aasstvsvssi seRvSsvsstIv vsaiiissssv vsttYissave in гавтатвсс 120 ааевассавве авсевсвств вассстваси всасссссв встассвсст всвсстстас 188 тТЕсасссаст сваствва встстсссас сИстесвави асвасттсві саавствавс 2408

TseveBevSssa avvsivstivvs sasestvyvs RevSsavrava vsasavasas vvgavsevvrss 300 sstvvsaavrve asasesesta stsvstvvvs tssavstve asayetasstye ssvstssvas 360

Zo tastssaasv avaavssvii saseeeviiis vavvsstisi asesavssva vrgasatsvas 420 vavivssavv ieevssssReReE avavvssss asstvsvass assastvssa saassasstv 480

З5 BESEVETTST astvstsstv ssesvsavvs tasvisstvs assetaasa vsvsasstvs 540

Esavssstvi vstsssevss 559 "2195 31 "211" 34 "2125 DNA "2135 Artificial sequence "220" "223" Synthetic "4005 31 svevSasass ayevarvstvs tvassstsststi ves 34 "2195 32 "2115" 33 "2125 DNA "2135 Artificial sequence "220" " 223" Synthetic

"4005 32 vasayesasst essvsEssva stssaasvav aav 33 "2195 33 "2115" 33 "2125 DNA "2135 Artificial sequence "220" "223" Synthetic "4005 33 avsavssstv aastasssasv vssvtatsss aaa 33 "210" 34 "2115 26 "2125 DNA "2135 Artificial sequence "220" "223" Synthetic "400" 34 svevaessaye vavestvstv asstis 26 "21905 35

From "2115 19 "2125 DNA "2135 Artificial sequence "220"

C5 "223" Synthetic "4005 35 vvaayesssta vvvsata 19 "2195 36 "2115 19 "2125 DNA "2135 Artificial sequence "220" "223" Synthetic "4005 36 vvaayesssta savevsvs 19 "2195 37 "2115 19 "2125 DNA "2 135 Artificial sequence "220" "223" Synthetic

"4005" 37 vvaayesssta vtatvevai 19 "2105" 38 "2115" 25 "2125" DNA "2135 Artificial sequence "220" "223" Synthetic "4005" 38

TesSEVSSESE vIarvivstv ST 25 "2105 39 "2115" 23 "212" DNA "2135 Artificial sequence "220" "223" Synthetic "400" 39 vvaayessast sveTAyessts vva 23 "210" 40 "2115 17 "2125" DNA "2135 Artificial sequence " 220" "223" Synthetic "400" 40 tssvaavaata asvavtv 17 "210" 41 "2115" 29 "2125" DNA "2135 Artificial sequence "220" "223" Synthetic "400" 41 sayesvaaavs Ese eRetav aavttvs 29 "2105" 42 " 2115" 27 "2125" DNA "2135 Artificial sequence "220"

"223" Synthetic "4005 42 svsvvssvsa vsIvsisava vEvtava 27 "219" 43 "2115 28 "2125 DNA "2135 Artificial sequence "220" "223" Synthetic "400" 43 svvtaavstje sastvvsisa vevaaata 28 "2105 44 "2115 37 " 2125 DNA « 2135 Artificial sequence "220" "223" Synthetic "400" 44 aavgaavstev ssvssassaei vrgastivevstv Yevvaast 37

From "21905 45 "2115 31 "2125 DNA "2135 Artificial sequence "220" "223" Synthetic "4005 45 svevassssts aaastetstje vIssassttv v 31 "21905 46 "2115" 36 "2125 DNA "2135 Artificial sequence "220" "223" Son thematic "4005 46 aavgaaavstyeE vssvssassa ЕвЕСstsase avstst 36 "21905 47 "2115 26 "2125 DNA "2135 Artificial sequence

"223" Synthetic "4005 47 svevayesstye sEssstIstaa sastse 26 "2195 48 "2115 9 "2125 Protein "2135 Artificial sequence "220" "223" Synthetic "400" 48 b1y Rgo Guz beg Su Ar Guz Tig Ni 1 5 "2195 49 " 211" 4966 "2125 DNA "2135 Noto 5ariepo "4005 49

From vBsSISvaasss sevavresava vvieEiieRIveE sIsasasstv Taayessssavs astteesvav 120 vBstvavesav vIvsayesvsi EsEveesisave aviysaaras savsstvvvs aasasareva 188 vassssayes ystasaaaaa asaaaaaasa abasaaareve rgasaaaaaaa aaaaaaaavas 2408 aavgasayeraa yssaevav va savavseiiev aaravraavs avsavsstsa aavtsstvva 300 avstvvaara asavasaaas avrvesvaaa baasevsstv vaaavsaasti SES 360

ЕС ТТ varte avistsasts TRISsvIssavr vstvrRavtvs avseivsva 420 tstIsvvatsa sEvsaassyas svsstsssav vysisaavrsaa ЕССЕsSsStvss tsavsstsss 480 vavtavvv vaesaesaavi eesvsestvss asasstvae vasessvta ES tavtav 540 avaevevaeeee ysassaevsi vvi savvstv vistsaassi sssaasstsv Evgayessasss 600 asEsvvsstY ssaaavtvs yeveRvaysasa veestasaavrss assvavsssa vsaaaaaaas 66a astEsstaavs steveEsaareva asservaasi vyv EsSassa vvisstESstTv asavvvieta 720 avaaestavs savsstvave stvesasvv ivvBsS Isasas stEvsaayessss 780 becomes vassivsavsa stEvsastvaa vssevvResaa savraasava tssaaaaaaa 1920 avevavgvvvi vravrevvsava vsavvasee vEssavvs evEsvETtass tavvissvas 1986

Тсстввстсс savavsavsso ЕВТСссстевСсссты всствваасии стравсаввс ЕеРрРависате 11406 vavisvayesis ssavaassss avavisavvv arrestivvrev savrevvsavrv Yisastvrasa 120806 aasavaesaa avvyavass avvtavvvs ivsavassav vssavrvssav stvevrasevvs 1260 "7 asassayev vtavrvivvvs vsssasawss tssstvsarv BIVIReReIR vRavsasawvv 1320 sSTvvEvsssT sssvsssi vssstvsssa vssstvssiv asstiststve vstTstrRIv 1380

TVEESTSeEVEv vssasssst EevvSsSsssvaa BIVvSssStvaa SSTVTVETSV ReESESSTVVS 1440 assssssvvs TEtssavrvvv avtaevssaa Yevassarvav sevesvstvva ssstvastvs 1586 assssssvvs tTassvsstvs vststasti sasssasees vasstvvavs tstEsssasst 1560 7 stIvsvavtas vastEesvIsa avvsessvis avvaservav vvstyaevvvET iSstSsarevis 1620

BevvevevIsss saarvavtav ssaveviisa vvvasasstv vravsavevev ssavvstivv 1680 ssavvavvva vgayesavvssi vevvistsvss tEsastsssti vEvasasstv assssasaws 17406

Tvavsisvve vessaavrvsv sIvevSssasvs YivyveseResSa vravavsasa vasasevavs 1880

BevsSsssTvB saavvasase sstastsvs TevvSstssav sstevasaeii asststssvst 1860 7 ssvastastis saasvaav ssvIisasev vevyissvavvs stESstaysvsa vssvareviv 1920 avssaavare vevIisstIvsaa sayestsavts Evsvsavstvstvivveev taasstvis 1980

З5 Tsgavvessave savssstvss tEtsavsess ssassttsss savrvvsarvv vgavavvssis 29040

Tevsstvasa isasssasaa Yevsaaavrass aaaasawssva Yevasstssat sasatsevvs 2186

Tivavsvssaa sEstvavssa vevaisstvav vasavsaiesv sstsaaviva svsavevast 2160 7 BVSSsvBeSsvS avsavstsas vsstvtaasi ssavsastei vReevavvssva veveststva 2220 tEsaestvave Isavrvavtss aarevssavss arevvsaasas vevsvaaasts taestssast 2280 aaaas tasaa aaayesavsiv vevseievivv TeseEsasstv vgaayesssavs tastavvvav 2340 vBstvavesav vavaayesesti Evaasstvsv arvyvvavvs ivsavsvaas avavayetesa 24080 ssastasassi ssavsstvve srasavavsi avastssvIs Isaaaaaasa aaaaaaaaa 2460 7 asvasvsavrv vevssvavvvs ssaestasa v strRasaaav Seevvssstv ssavsevvav 2580

SSSSSSSST veEstvasii vastssssts asstavstve SrRasstsvve ssevasasiv 2790 aaasstsssas TvevBEssTaasa vavvEvayesvh Esvsasseee stsssavsvs EvstvRgavs 2760 tsSvsavsvas sstavvsstv taavvivati vevsssvvsas savtisssvsa ssstavrasav 2820 vasvavvsst і ssEstvavvi ssastistvav visaesvevaes ssstvRevavv ausssavvsi 2880 ? вваессссвссо ЖСЕСТССССТС ствасевссии всстввссстИ вссЕССстсссс savasayetsva 2940 svavtivssav vyvevssssvR vavavvssss sasstvsvas sassasstvss asaaassst z60o

BBVSEVTEsS tastvstssti vssevsvsave stasvisstv sassvtiaasa avsesassiv ze6o stIsavssstv EvsEsSsvvSS avvisttisas ssavravvssi veeeRevavsisa vsawsstva 3120 asasssasvv ssvtassssa aasstssav svsastsas avsayesavss TRrRavrvavvv 3180 7 vEssavtvis as ествваси Есе ЕвРавис стЕСsвасвсв ravasasass seraaassst 3240 вЕВЕссстас васттсстса аваессааас аврасавараа rgaасаеввсс саесстввв 3300

Baavasayes ssssasarvra Tiraaaasaa aarsaaasasv vIvgassayesa ssettvEsas 3360 avayevaayesa revavassasa savvstvrraa vasssastas asvavsasaw sesasestiv 3420 sssttayessv ayevvsvssas staayevvssa svt Esassi vsesaavssa aasasatsstya 3480 7 Baaavasavs sstssaesya ssesva vas SeRestayavav sEsstvsaav visasttvss 3540 ssEvaaayss EEtastvsav sv Esavaa avaevevayesi EvRevassevvs saasevsssvs 3600

From BIvsavsasti vEsvastiv vssstststva yvayestass avivvssvav TevRavtasat 3660 sasavvyssi vvavsvassa sstasaaavs vsvRaesssav tasavstvsv aaravgasstt 3720 stasasaayeve aaavsvaayee ayevvsaasa esseiv yvav vstvaevvai str vvasvav 3780 7 sIssaaaarra raaaayesass sssavsstv Yevavsstvsi EvREvrRastaye savsssvsas 3840 aasavrvarareve svtasasasasee vgarevsaaa veesaaaassti vevsvaets stsvvsaavt 3900 sstIvasaesa vevRievaassa savsavsave Tvesastttisa tasvasaasti vevevisstaas 3960 avsevstsaye vssvistasv avrsaaavasa Yevaev Sayess вссстврРаса тЕсваасввв 4020 сассстівааа авастаяса stEсаесасас асаавсствв стваавств ЕСе satasa 4de8a 7 tvaavvetaye astsaevaev sIevstssvrRa saasvasata vsastvayesa aastvaaasa 4140 saaavtisevta ayesaasavsa asassasvss tastsevistv ssaaraaaav aavstvaats 4200 sE-taevavv asavaevasa Yeseraastvs asstevaitee vvastaasss aaagvvv 4260 stv sStava aaestaaevs asessvasae assvassevsi vassaesaaa aasetsvs 4320

Tvsasaevaa aavssasssti assaaaveee aavsesaasti vstaasaevs STRESS ve 4380 7 stIgavaaavri Reve vSsaave asavstsav avvivasavs vravevvsas TevyvESTs dadda avayesavsvaa asavravravvi veBESSeiveve avvaatavte EsstvRevIi ssasvaayetv 4580

TeReBaavsa vvyasavtaev vavistasas aaaavstayei aastasaees sstvrvaesva 4560

Bvaasayeaayes aveyvassye aaseseyesvsv sstEvsavisa avvayesstts asesaraa 4620 ayevsstvsva avasstievs avsvasvivv sisravaavs astsayesayessi asevtvrvasa 4680

Tevsavtsvi Testsssss aaaavavaasav assessavvsv avvstvi saestestssa 4740 stEvssavtje taayessssavs sEsasssaessi vastisaavree vasaebaaass asvravgavitva 4800 savisaeste Evsssasssa vEvtaaevss astestsaaa tasaesttsa stassesaaa 4860 aavssavtst seESEssatas tevstvItvv sasteeststvta aastesstvE ssaevstst 4920

Tv Taaa sttvEisITta ysraaaaaaaaaaaaaaaaaaa 4960 "2195 56 "2115" 2898 "2125 DNA "213" mouse "220" "221" Coding sequence "222" (33)..(2099) "4005 56

53

Zo Mee Ag Gei Ge I1e Rpe Gei 1 5

VE stv ve aut Her BIR vBss asa st stv vv isa aav vv ss 1801

Oo1u Gay Ge Ter 5eg iey Mai Aia Tig Gay Gay o1u beg Guz Ter Rgo 10 15 20

Bvaa sst vTa s vve SBS STV vv SS SST ves yisS ssa vav aav tat 149 bi Rgo Ma! Rpe s1u Ag» Hey Mai beg Rgo b1u Re Rgo would Gu Tug 25 3a 35

БСИ vas sai saa vai sva ss treb asa stv asi vsa sss o sst ves tas 197

A1a Azr Ni5h bip Ar Age 5eg Ter Ts o Ge Tyg ATa Rgo Rgo su Tug 40 45 58 55 svs sv svsS ss Tas Es ass sas TES ras sv vaa ss TsT Tas svs 245

Age Gay Agv Ge Tug Re Tpg Nib5 Rpe Ar Ge bI1y Gay b5eg Tug Age va 65 78

Ts vav tae vas HER BIS aav se avs isa vve ass aav vv stv vss 293

Suz bIy Tug Azr Rpe Mai Gu Gay 5eg beg b1u Tig Guz Mai Gay A1Ta 75 80 85 asa sb HER Be sav vav avi asa vas asi vav sav vsa ss ves aaye 341

Tig Ge Suz s1u bIip b01y beg Tig Azr Tig bIy b1ip Aia Rgo o1u Ap 96 95 189 vas ass zhis tas sa stv vBvi sss avs sta aav vys ass ts sas yss 389

Azr Tyg Rpe Tug beg Ge b1u Rgo beg Ge Guz Ma! Tig Rpe No 5eg 195 119 115 vas tas ss aaye wav aav ssv SS asa vve TS wav vss ts tae vsa 437

Azr Tug b5eg Azp biIy Guz Rgo Rpe Tpg s1u Rpe bi AI1a Rpe Tug Aa 1209 125 130 135 all vav vai vav vai vai vva vsa ava vse ST stv vva vva sa vyas ss 485

A1і1a fight Ar Mai Ar b1y Sub Ag» Ma! beg Gets b1u Azr 5eg Mai Rgo 1406 145 1509

TV vas sae tae vs sas aas as Te ves ves Tas tat yivs Tss vs 533

Suz Azr No Tug Suz Niz Azp Tug Ge s1u s1u Tug Tug Su 5eg Suzv 155 169 165 ava vs vs tas as ss sas sav aas aav sas asv yivs sa vss st 581

Age A1i1a s01u Tug I1e Ge Ni5 b1ip Azp Gu5 Ni Tg Su 5eg Aa Gets 179 175 1809

TVI isa ves sav vv hersS asa vva ava ST vve tae ss avt avs ss 629

Suz 5eg b1u beep Ma! Ripe Tpg s1u Ag" beg O01u Tug Gay 5eg 5eg Rgo 185 199 195 wav tas ssv sav ssa as sss o aav ss ss avs vs ass tas avs ats 677 bI1y Tug Rgo bIp Rgo Tug Rgo Guz Ge beg beg Suz Tig Tug beg Pe 299 295,219,215

Age Hey bi Azr b1u Rpe b5eg Ma! I1e Ge Ar Rpe Ma! b1y 5eg Rpe 229 225 230

Zo vaye vii wav asv sas sstI raa vss o sav vs sss tai vas ss ss aav 773

Azr Mai si Tig Ni Rgo oboT Aia bIp Su Rgo Tug Azr 5eg Getsy Gu 235 240 245 as saa asa vas aav web waa sas vs ssa TT 5 rReBbB aav asev siv 821

I1e b1p Tag Ar Guz o01u bij Nib5 01u Rgo Rpe Sub5 o1u Gu Tig Ge 2509 255 260 ssSTI sss abwv asi waa asi vas avs sas aav vv ass ass ass 1 vss 869

Rgo Rgo Age I1e bi Tyg Ar beg Nibh Guz Ma! Tyg I1e Tpg Rpe ATa 265 279 275 asty vas vav yse BR aas sas asa ves yiv aar asa sas tas asa avs 917

Tig Azr bIy 5eg b1u Ap No Tpg b1u Ter Guz Ie No Tug Tig 5eg 280 285 298 295 asa all seB sss TvsS ssT vaye ssa asb all ssa ss aaiyei vvs at 965

Te AtTa Ag» Rgo Sub Rgo Ar Rgo Tpg ATa Rgo Rgo Azhp o01u beg I1e 399 395 319 isa sst vyiv saa vss ase tai vis stv aav vas avv EE ST vis S 1813 b5eg Rgo Ma! beep Aia Tig Tug Mai Gay Guz A5zr Age Re beg Ma1 РПе 315 320 325

Ts aav asa ves Ts wav st st sav saa vv ST v sss st aaa Isa 1861

Suz Guz Tpg b1u Rpe bI1y Ge Hey b1p s1u 5eg Mai Rgo Getsy Gu 5eg 330 335 340 ts oast SE vys yviY sav aaa vat vva ST yivbrR vas svv ssv atv ssa 1189

Rpe Tpg ATa Mai Sub5x b1p Guz Azr o1u beg Tgr Azr Age Rgo Mee Rgo

345 359 355 wav yvs avs a act vate sv ves sstT sss vaye vas sta sss aaye vs 1157 bi Suzh beg ITIe IIe Ar Su s1u Rgo Rgo Azr A5r Geiy Rgo Azp o01u 360 365 379 375 saye vv vas tae as asa vBS ssT saa vib asti ass tas aaa vs viv 1285

No Mai Azr Tug IIie Tpg s1u Rgo b1p Ma! Tag Tig Tug Guz A1a Mai 389 385 399 as sav tas avs TieiY vraa wav asti (s tTas asa ase avs avs aae vv

Te bI1p Tug beg Suz bI1y 01 Tig Rpe Tug Tig Mei 5eg 5eg Azp s1u 395 489 4985 aaa taye vv EV vav all vvae vva TS yveB ase avs yss aaa vra vaa 1301

Guz Tug Mai Suz bI1y A1ia Ar s1u Rpe Tgr TPg 5eg 5eg Guz o1u sy 416 415 4209 aaa sts sss o ssv VES ІІ vav ssti VE І8БІ vRB stv yss asa sas ass 1349

Guz Gay Rgo Rgo Mai Su5h bi Rgo Ma! Suh o1u Ge beg Tig No TAg 425 430 435 asa vva vva svs asa her vva vve sav sst vsa aav ss BE vas 1397

I1e o01u s1u Age5 I1e Ma! s1u o1u b1p Rgo A1ia Gu5 Rgo o01u Azr Rie 440 445 456 455

SSSE TVB saa BIS TTB Sat STR vv saa asti asa bsa bsa vsa vv vsa 1445

Rgo Ter bIp Mai Ge Gay Ge sb1u b1p Tig Tig Aia AT1a Aia o1u AIa 460 465 479

Zo

STE asa sayes vas aae se vBIs sta asa vss vsE sae vs vta tae wav 1493

Yech IIe Ni5 Ar Ap Tgr MaiYi Ge Tpg Aia Aia Nih5 A1a Ma! Tug si 475 486 485

Z5 aaa ava aev all all ss Tss stv aas as sva aev ves ats ss aaa 1541

Guz Age Mei Aia A1a beg beg Hey Azp I1e Ag" Mei o1u Pe Gey Guz 498 495 589 avv ses ysa sstE sae tas asti saa vss yiveB sss wav raa ass her ata 1589

Age Gay beg Rgo Nih Tug Tpg b1ip Aia Ter Rgo bi b1y I1e Rpe Pe 585 519 515 saye waa ves tas ast sas BE ST vvYI YEI vas aae vas ata all her6 1637

Ni5 bIy o1u Tug Tyg Nih b1u Aia s1u Ripe Ar Azp Azr IIe A1a Ge 528 525 539 535 asseaaa ss aavr aas aaa vs asa ass aas vva avs ayes asv sst vi 1685

Te Guz Gay Guz Azp Guz Ma! Tig I1e Azp s1u beg I1e Mei Rgo Mai1 540 545 558

Tvbs sta ss sva aaa waa vs vsa yss ytTa ayeb ava asa vas s ast 1733

Sub Ge Rgo Ag» Gu5 bi Aia A1i1a 5eg Ge Me Age Tyg Ar Re Tpg 555 569 565

Ba ast BIV VSI ES TB REB yTa ass sav aav vve SET ST vs ava 1781 s1u Tig Mai A1a si1u Ter o1u Ge Tig b1p Gu5h o1u Hey Hey Aa Age 578 575 589 aas sta aev HER web ras ata ssa as vs vas sas saa aaa T8th ass 1829

A5zp o Gets Met Rpe Mai Azr IIe Rgo I1e A1a A5r Ni5 bIp Guz Suz Tig 585 599 595 ass vyv Tai waa aav ss tat ssa vva vta ava vsta avs all aas at 1877

Tys Mai Tug bIy Gu5 Ge Tug Rgo sb1u Ma! Age May beg A1a Azp May wav 605 619 615

STS TT vST vBS HerTa vav asi v5Bi ves aav vas avs ivsS ava vv vas 1925

Hey Suz Aia s01u Ge b1y Tig b1u s1u Gu5 Ar beg Sub Age O1u Azr 620 625 630 avi vg85 ReB vSsa and vv ІІ sta vas aai vav asa sav sva Tvv her 1973 b5eg b1u b1u Aia Hey Mai Re Gay Ar Azp biy Tig bip Age Ter RPe 635 640 645

BE vBa vva asa ET ss TR vvI yss as aase s vve all vsa vs 29021 mai s1u s1u I1e Mai b5eg Ter os1u beg I1e Azp Sukh s01u Aia Ai1a o1u 6509 655 6v6O tae vv vys sav Tas asa aaa vys as aas tae as sss tv aae wav 2069 b1ip Tug s01u Mai Tug Tig Guz Ma! Ie Azp Tug IIe Rgo Ter Azp s01y 665 679 675 aas aba aba avi aaye s taa 2899

Azp I1e I1e beg Azp RPe 680 685 "2105 51

From "2115" 685 "212" Protein "2135 mouse "4005 51

Met Ag" Gay Ge I1e Rpe Gay s1u Ge Gay Ter beg Gets Mai Aia Tig 1 5 18 15

Hey Hey o01u beg Gu5 Ter Rgo fight Rgo Ma! Rpe o1u Age Ge Mai 5eg 20 25 30

Rgo b1u Re Rgo bi Gu Tug ATa Ar Ni5 bip Azr Age 5eg Ter Tig 35 4 45

Ge Tyg ATa Rgo Rgo b1u Tug Ag Gay Ag Gay Tug Re Tig Ni Re 58 55 va

Azr Hey bi Ge beg Tug Age Su5 biy Tug Azr Rpe Mai Gu Getsy 5eg 65 78 75 va b5eg b01u TyIg Guz Mai Ge A1ia Tig Ge Suz b1u b1p 01 beg Tpg A5r 85 90 95

Tig bI bI1p ATa Rgo s1u Ap Azr Ts Re Tug 5eg Ge o1u Rgo 5beg 166 185 116

Hey Goose Ma! Tys Rpe Ni beg Azr Tug beg Azp bi Gu5 Rgo Re TEig 115 1208 125 s1u Rpe bi A1i1a Rpe Tug Ai1a Aia bIy Azr Ma! Azr bIy Su5 Age Mai 138 135 140 b5eg Ge oI1u Azr beg Ma! Rgo Su Ar No Tug Su5 No Azp Tug Ge 145 158 155 160 s1u o1u Tug Tug Suz b5eg Sub Age Aia s1u Tug I1e Gets No bIp Up 165 176 175

Guz Ni Tis Sub beg ATa Ge Su5x 5eg b1u beep Ma! Rpe Tyg o1u Age 188 185 199 b5eg b1u Tug Gay 5eg beg Rgo biy Tug Rgo bip Rgo Tug Rgo Guz Ge 195 290 295 b5eg b5eg Su5 TYg o Tug beg I1e Age Gay bij Azr b1u Re 5eg Ma! I1e

From 216,215,229

IGets Azr Rpe Mai bij b5eg Rpe Azr Mai sb1y Tpg No Rgo would A1a sp 225 230 235 240

Suz Rgo Tug Azr beg Ge Guz Ie bIp Tig Azr Guz s1u oYi No o1u 245 258 255

Rgo Rpe Suh 01u Gu Tyg Gay Rgo Rgo Age I1e b1y TPg Ar 5eg No 260 265 279

Guz Ma! Tys IIe Tys Rpe A1ia TPg Ar biy b5eg 01u Azp Nih Tyg o1u 275 2808 285

Ter Guz I1e Ni5 Tug Tiig beg Tg ATa Ag» Rgo Su Rgo Azr Rgo TEpg 298 295 309

A1a Rgo Rgo Azp s1u beg I1e beg Rgo Ma! b1ip Aia Tig Tug Mai Ge 395 316 315 329

Guz Azr Age Re beg Mai Re Su5 Guh Tpg o1u Re o1y Gets Gay sp 325 339 335 o1u beg Mai Rgo Gay Guz beg Rpe Tpg ATa Mai Su5 bIp Guz Ar o01u 340 345 359 b5eg Ter Ar Aga Rgo Mei Rgo bij Su5x beg I1e I1e Ar Su5 Oo1u Rgo 355 zva 365

Rgo Azr Ar Gets Rgo Ap b1u Ni5 Ma! Azr Tug I1e Tpg s1u Rgo b1p 3708 375 389

Mmai Tys Tys Tug Guz Aia Mai IIe bip Tug beg Su5x 01 bI1y Tpg Rpe 385 390 395 4ev

Tug Ts Me 5eg beg Azp b1u Guz Tug Ma! Suz b1y A1a Azr s1u Rpe 405 416 415

Ter Ts beg o b5eg Guz s1u bTy Guz Ge Rgo Rgo Mai Suz si Rgo Mai 420 425 430

Suz s1u Gets beg Tpg Nih Tig I1e s1u s1u Ag» I1e Mai o1u o1u sIp 435 44o 445

Zo Rgo Aia Gu Rgo b1u Azr Rpe Rgo Ter bip Mai Gay Gay Gay s1u b1p 4506 455 460

Tig Ts ATa A1a DIia s1u DIa Ge I1e Ni5 A5r Azhp Tgr Mai Gey Tig 465 470 475 480

A1a A1i1a Nib5 Aia Mai Tug bij Gub5 Ag" Mei Aia A1a 5eg 5eg Getsy Avp 485 490 495

I1e Ag Mee s1u I1e Gay Gu5 Agv Gets 5eg Rgo Ni5 Tug Tig b1ip ATa 580 505 518

Ter Rgo bTiy 010 I1e Rpe I1e Ni5 bi s01u Tug Tig Ni5 o1u A1a o1u 515 529 525

Rpe Azr Ahp Ar I1e Aia Hey Ie Guh5 Ge Gu5 Azp Guz Ma! Tys Pe 539 535 540

Azp b1u b5eg IT1e Mei Rgo Ma! Su5 Gets Rgo Age Huz b1y A1a Aa 5eg 545 559 555 560

Ge Mee Ag» Tyg Azr Rpe Tyg b1u Tpyg Mai Aia o1u Ter o01u Getsy Tpg 565 5708 575 beep Gu5x s1u Ge Ge A1ia Ag» Azp Ge Mee Rpe Ma! Azr I1e Rgo Pe 589 585 599

A1a Azr Nih b1p Guz Suz Tig Tygs Mai Tug oTy Guz Ge Tug Rgo a1u 595 6o0 605

Mma1 Ag» Mai beg ATa Azp Mee Gay Su5x Aia o01u Gay b1y Tpg s1u s1u 618 615 6206

Guz Azr beg Sub Ag» b1u Ar beg b1u s01u Aia Ge Mai Re Getsy Ar 625 630 635 640

Azp bij Ts bip Ase Ter Rpe Ma! s1u o01u I1e Ma! 5eg Ter s1u 5eg 645 6509 655

Ie Azp Su5x s1u A1a ATia s1u b1p Tug o01u Mai Tug Tig Guz Mai I1e 6v6O 665 679

A5zp Tug I1e Rgo Ter Azp bI1y Azp IIe I1e beg Ap RPe 675 680 685

From "2105 52 "2115 676 "212" Protein "213" mouse "4005 52

Te Ge Gets s01u beg Gu5 Ter Rgo bI1y Rgo Ma! Rpe o1u Age Hey Mai! 1 5 18 15 b5eg Rgo b1u Rpe Rgo bi Gu Tug ATa Ar Ni bip Ar Age 5eg Tgr 20 25 30

Te Se Te ATa Rgo Rgo b1u Tug Ag Gay Ag Ge Tug Re Tig No 35 4 45

Rpe Azr Ge bi Ge beg Tug Ag» Su5 bi Tug Azr RPpe Mai Gu Gets 58 55 va b5eg 5eg b1u Tyg Guz Ma! Ge Aia TPg Ge Suz s1u bIp b01y beg Tig 65 78 75 80

Azr Tyg b1y b1p ATa Rgo b1u Azp Ar Tyg Rpe Tug beg Hey so1u Rgo 85 90 95 b5eg Ge Guz Ma! Tig Rpe Ni beg Ar Tug beg Azp bIy Guz Rgo Re 166 185 116

Te b1u Rpe bij Aia Rpe Tug Ai1a A1a b1ii Ar Mai Aztr o1Yi Su Age 115 1208 125

Mmai beg Ge b1u Azr beg Mai Rgo Su Azr Ni Tug Su Ni Azp Tug 1308 135 140

Genes s1u s1u Tug Tug Su5 beg Su5 Age AIia s1u Tug Te Gets No sIp 145 158 155 160

A5zp Guz Ni Tpg Sub beg Aia Ge Su5z 5eg b1u b1p Ma! Re Tig o1u 165 178 175

Age beg 01u Tug Ge 5eg 5eg Rgo bIy Tug Rgo bIp Rgo Tug Rgo Gu 188 185 199

Hey b5eg beg Su5 Tig o Tug beg I1e Age Hey bij Azr o1u Rie 5eg Mai! 195,290,295

Zo

IIe Ge Azr Rpe Mai obTy 5eg Ripe Azr Ma! sb1y Tig Ni Rgo sy A1a 2168 215 229 bIip Su5 Rgo Tug Azr beg Hey uh Ie b1p Tpg Azr Guh5 o1u sy Ni 225 230 235 240 s1u Rgo Rpe Suzh sb1u Gu5 Tpg Gets Rgo Rgo Age I1e biy Tig Ar 5eg 245 2509 255

Nib5 Guz Ma! Tyg ITe Tpg Rpe Aia Tpg Azr bIy beg b1u Ap Ni Tig 260 265 279 s1u Ter Guz I1e Ni Tug Tyg b5eg Tyg A1Ta Age Rgo Sub Rgo Ar Rgo 275 2808 285

Tig ATa Rgo Rgo Azp s1u beg I1e beg Rgo Ma! b1ip A1ia Tig Tug Mai1 2908 295 309

Hey Guz Azr Ag» Rpe beg Mai Re Su Guh Tyg o1u Re o1y Gay Gays 395 316 315 329 b1ip o1u beg Mai Rgo Hey Guz beg Ripe Tpg ATa Mai Su5 bIp Guz Ar

325 339 335 s1u beg Ter Azr Age Rgo Mei Rgo bi Suh 5eg I1e I1e A5zr Suh Oo1u 340 345 359

Rgo Rgo Azr Ar Ge Rgo Azp s1u Nih5 Ma! Azr Tug I1e Tig s1u Rgo 355 zva 365 b1ip Mai Tig Tis o Tug Guz ATia Ma! Te b1ip Tug beg Su5 o01y si Tig 3708 375 389

Rpe Tug Tyg Mezh beg beg Azp s1u Guh Tug Mai Suh o1y Ai1a Azr o01u 385 399 395 4ev

Rpe Ter Tig beg beg Guz b1u bIy Gu Ge Rgo Rgo Ma! Su Siy Rgo 405 416 415

Mmmy! Suh o1u Ge beg Tpg No Tpg I1e o01u o1u Age I1e Ma! s1u o1u 420 425 430 b1ip Rgo Aiia Guh5 Rgo o0T1u Azr Re Rgo Tgr bIp Ma! Gay Gays Gay o1u 435 44o 445

Zo b1ip Tys Tpg ATa Aia ATia s1u ATa Ge IIe Ni Azr Azp Ter Mai Ge 4506 455 460

Tipe AtTa Ai1a Ni5 Aia Mai Tug bii Guz Ag" Mei ATa A1a 5eg 5eg Gey 465 470 475 480

Azp I1e Ag» Mei sb1u I1e Gay Gu5 Agv Gay 5eg Rgo No Tug Tig bIp 485 490 495

A1a Ter Rgo 010 b01y I1e Rpe I1e Ni5 01 s1u Tug Tig Ni o1u Aa 589 595 518

Oo1u Re Ar Azp Ar I1e DIa Ge ITIe Guz Gay Guz Azp Guz Ma! Tig 515 529 525

I1e Ahp b1u 5eg I1e Me Rgo Mai Su5 Hey Rgo Ag» Gu5 b1y AIa A1Ta 539 535 540 b5eg Ge Mee Ag» Tpg Ar Re Tpg s1u Tig Mai Aia o1u Ter o1u Ge 545 550 555 560

Te b1ip uh o1u Ge Ge ATa Age Ap Ge Me Re Mai Ar I1e Rgo 565 579 575

I1e ATa Ar Ni5 b1p Gu5 Sub Tig Tig Mai Tug bi Gu Gets Tug Rgo 589 585 599 s1u Mai Ag Ma! 5eg A1a Ahp Mei Ge Su5x A1i1a o1u Ge bIy Tig o1u 595 6o0 605 o1u Guz Azr 5eg Su Ag» b1u Ar beg b1u sb1u AIa Gets Mai Re Ge 618 615 6206

Azr Azp bi Ts bip Ave Ter Rpe Ma! s1u o1u I1e Ma! 5eg Ter 01u 625 630 635 640 b5eg I1e Azp Suh b1u A1i1a DAia s1u b1p Tug s1u Mai Tug Tig Guz Mai 645 6509 655

I1e Ahp Tug I1e Rgo Ter Azp bi Azhp I1e I1e 5eg A5zp Рппе 6в6О 665 679 «2105 53

From "2115 2891 "2125 DNA "2135 rat "220" "221" Coding sequence "222" (18)..(2067) "4005 53

TvEsSasasa asv avv sta stv ass all sev BI stv SII yeveB avt in Riv 51

Met Ag Gay Gay I1e Mai Ge s1u Gay Ge Ter 5eg Getsy Mai! 1 5 18 vBss asa st TTB vBS ss aav yiv SST wav sst va s ReB SsvS siv 99

A1a Tpg Ge Ge o1u 5eg Guz Ter Rgo bi Rgo Ma! Rpe s1u Ag» Gets 15 20 25 30

WEB исСсС ств вБсСс Тs ssa vav aav tai veBvBs aas sasi sav vaye sva іss 147

Ma! 5eg Ge ATa Rpe Rgo bij Guh Tug 01u Ap Ni5 b1p Ar Age 5eg 35 4 45

TV asv st asti all sss o sstT ves TS svs sv svS ss Tas i-s ass 195

Ter Ts Ge Te ATa Rgo Rgo b1u Rpe Ag» Hey Age Ge Tug Re TAg 58 55 va sas ts aas stv vaa ss ST Tas ses yivsS wav tae vas Ye vs aav 243

No Rpe Ahp Hey biy Ge beg Tug Age Su5 biy Tug Ar Rie Ma! Guz 65 78 75

ТІiv5 ass sa vevB ass aav vv sta vss asv stv HER vve sav vav avt 291

Yeets Tig beg b1u Tig Guz Mai Ge A1a Tig Ge Su5 s01u bip o1iiy 5beg 80 85 90 asa vaye asti wav svv ssa sstYi ves aae vas ass ts tas isa stv vvi 339

Te Azr Ts bii As ATa Rgo b1u Azp Azr Tpg Rpe Tug 5eg Getsy s1u 95 189 195 119 sss avs sta aav vis ass ts sas ss vas as ss aai wav aav ssa 387

Rgo beg Ge Guz Ma! Thoukeys no beaver azr tight bug bard 01I Gu RGO 115 1209 125 eating OAS VVA Was Vsd TSA Tai BCA All of you all

Rpe Tig b1u Rpe oboI1y Aia Rpe Tug Aia Ai1a sy Azr Mai Ar sy Su 130 135 140 ava asa yss stv vva vas sa vs ss BI vas saye taye vs sas aas 483

Age Ts obeg Ge b1u Azr 5eg Ma! Rgo Sub Ar Ni Tug Su Ni Ap 145 158 155

Tas sv ves ves tas tas ves ss yivsS sva yiv ves tas ak sv sas 531

Tug Heya s1u s1u Tug Tug Su5 beg Suz Ag» Ma! o1u Tug Pe Getsy No 166 165 179 sav aas aav saye ass vs sa vss SES TviY isa ves sav viv -4is as 579 b1p Azp Guz Ni Tig Sub b5eg Aia Gei Suz beg b1u bIp Mai Re Tig 175 1809 185 199

Be avv sii ves TT sts aut avs ssiY vav tTas ssa sav ssa tas sss 627

Zo O1u Age beg b1u Rpe Hey beg b5eg Rgo bij Tug Rgo beep Rgo Tug Rgo 195 299 295 aaa ss tss avs vs vBss Tas aas as svs stv wav waa wes ts aut 675

Guz Hey beg beg Su5x ATa Tug Azp I1e Ag» Ge bi bi o1u Re 5eg

З5 216 215 229 assoass stv vas

Te Tig Hey Azr Rpe Mai b1y beg Rpe Azr Mai oTy Mei Ni Rgo s1y 225 238 235 vss sav vs sss tas vas ss ss aav assi saa asa vas aav ave vaa 771

A1a b1ip Sub Rgo Tug Azr 5eg Gay Gu5 Ie bip TPyg Ar Guz Age bIy 248 245 2509

Tas vs sse TE BI vrRrrR aav as stv sss sss arv as! waa aas you 819

Tug b1u Rgo Rpe Su5 o1u Gu5 Tg Gets Rgo Rgo Age I1e bi Tig A5r 255 260 265 279 avs aas aav vv ass ayess ass her ass ass vas vav isa vve aas sas 867 b5eg Azp Gu5 Ma! Tpg I1e Tig Rpe Tig Tpg Ar bij b5eg o01u Ap Ni 275 280 285 asa ves iІeB5 aav asa sas tas asa avs asa vsa sav sss vs ssT vat 915

Tig o1u Ter Guz IT1e Ni5h Tug Tipg beg Tig ATa biIip Rgo Su Rgo Az5r 298 295 389 ssa asb all ssa sst aai vBE sas as sa sstY yiv saa vss asv tah 963

Rgo Tyg ATia Rgo Rgo Ap b01u Ni5 I1e beg Rgo Ma! beep Aia Tig Tug 395 316 315

BEsS st aavr vas avs ESE ST VES Ts v aav asti wes Ts wav st 1811

Mmai Hey Guz Azr beg Rpe beg Ma! Rpe Su Guz Tig s01u Re o Ge 329 325 330 sv saa tu STE v sss sv aav ysa es asti ST vys tvBY sav aaa 19859

Yiets bIp s1u 5eg Mai Rgo Gets Guz beg Rpe Tpg ATa Mai Su bIp Guz 335 340 345 359 vaye vva ST ve vas ssv ssv ata ssa vav yivs avs ak ak vas vi 1187

Azr b1u beg Tgr Ar Age Rgo I1e Rgo bi Suh5 5eg I1e I1e A5r Su 355 360 365 vs o sSST sss ovaye vas sta sss aae ves sas vBivB vas tae as asa vs 1155

O1u Rgo Rgo Azr Azr Hey Rgo Azp b1u Ni5 Ma! Azr Tug I1e Tig o1u 379 375 zva

SsSTY waa vib ass ass tas aaa vBSsTY vv as sav as avs Her waa wav 1283

Rgo bI1y Mai Tiig Tig o Tug Guz ATia Mai I1e bIp Tug beg Su5 sy o1y 385 399 395 asieE ss tas asa ayesbv avs avs aae vevi aaa tae all her wav vst vat 1251

Tys Re Tug Tig Mezh 5eg beg Azp b1u Guz Tug Mai Su5z o1y A1Ia Ar 460 4985 416 vva iys ter asbv avs yss aaa vva vaa aaa yss ss ssv v vs aav 1299

Oo1u Re Ter TyYg 5eg beg Guz oO1u bT1y Guz 5eg Hey Rgo Mai Su Guz 415 4209 425 430

From SST VES TBI vva stv ysSs asa sas asti isa vra vs sei ata as vva 1347

Rho Ma! Subh o1u Ge 5eg TPg Ni5 Tyg o beg o01u b1u Ag5 I1e Pe O1Uu 435 440 445 vva sav ssti all aavr ssti vvi vas SE sst yivB saa BIS Tv yya siv 1395 s1u b1p Rgo A1i1a Guh5 Rgo b1u Ar Ripe Rgo Tgr bip Mai Getsy Getsy Ge 456 455 4606

BVY waa asi asa vsa vsa vv vs siiY ata saye vas vas te vis sta 1443 o1u oTy Tyg Tys ATa ATa s1u AIa Ge IIe Ni Ar Azr Tgr Mai Gei 465 470 475 asa all vst saie all wa tai ver aaa asa wav all asv ss Iss stv 1491

Tig AT1a ATia Ni5 Dia Ma! Tug o1u Guz Tig bI Aia Mei beg 5eg Ge 480 485 498 you ass svs aev wes ass sts aaa ave ss Tss sts as as as ast saa 1539

Azr I1e Age Mei o1u I1e Hey Gu5 Ag» Ge beg Hey I1e Tug Tig bIp 495 589 585 518

BSS TvB ssa vav vv vvs HER ais saiye waa vev tas ast sas vva vv vv 1587

A1a Ter Rgo 610 Aia Ma! Rpe I1e Ni5 01 s1u Tug Tig No o1u Aa 515 528 525

BV ESY you aaye you asta vsa stv asi aaa ss aabv aas aaa vs asa 1635 s1u Rpe Azr Azp Azr I1e Aia Hey I1e Guz Hey Gu5 Azp Guz Ma! Tyg 539 535 548 ass aas ava aas as asv ssv ase sv sta ssa avra aaa vaa vBsT vsa 1683

I1e Azp Age Azp I1e Me Rgo I1e Su Gay Rgo Age Gu b1y Aa A1Ta

545 550 555 ss her and ayebv aaa asa you ss vBIiY vra asi Her ST tes yBR vvv herTa 1731 b5eg Ge Mee Guz Tpg Azr Rpe Ma! s1u Tig Mai Aia su Ter o1u Ge 560 565 578 ass sav aav vve TET ST vBSTI ava aas sta asv EI vy6 vas ata ssa 1779

Te b1ip uh b1u Rpe Ge ATa Age Azp Ge Me Re Mai Ar I1e Rgo 575 589 585 598 as in you sas saa aaa BE BvBst asti all tae asa aav sav sss tas 1827

Ie Mai Azr Nih sb1p Guz Suzh AIia Tig ATa Tug Tig Guz bIp Rgo Tug 595 6aa 605 ssa vva vsa aaa viv asi vBii aas aev sts TR VSI ves sta vas svs 1875

Rgo b1u ATia Guh Mai! Tyg Mai Ahp Mei Ge Sub A1a s1u Gay A5r Age 619 615 6209

BVY ves aar vas avs yiv6s ava vei vas avs vra vve vsa a yiv TS 1923 s1u o01u Guz Ar beg Sub Ag" b1u Ar 5eg b1u o01u AIia Gets Mai Rpe 625 639 635 sta vas aai vaa asa ava ievB sav YII ve vra vva aia 1971

Hey Azr Ap biy Tig o bip Ase Ter Rpe Ma! s1u o01u I1e Mai 5eg Ter 640 645 659

BE st as aas BI ve BRE isSa waa sav tae web vs tas ase aaa 2019 o1u beg I1e Azp Suh s01u s1u beg 01 bIip Tug o1u Mai Tug Tig Guz 655 66a 665 679

Zo

BEsS asv aas tae as sss tvBe as wav aas aba aba aai aae s taa 2067

Mmai Tpg Azp Tug IShe Rgo Ter I1e bTy Azp IIe I1e A5p Azp Rie 675 680 685

Z5 tTEtvsaaaaa aaaaaaaaaaaaaaa 2091 "2105 54 "211" 685 40 "2125 Protein "213" rat "4005 54 45 Met Ag" Hey Ge I1e Ma! Ge o1u Ge Ge Ge Ter beg Gets Mai Aia Tig 1 5 189 15

Hey Hey o01u beg Gu5 Ter Rgo fight Rgo Ma! Rpe o1u Age Ge Mai 5eg 50 20 25 3a

Hey ATia Rpe Rgo biy Gu Tug b1u Ap Nib5 bip Azr Age beg Tgr TApg

Ge Tig ATa Rgo Rgo b1u Re Ag Gay Ag Gay Tug Re Tig Ni Re 58 55 va

Abp Ge bi Ge beg Tug Age Su5 bii Tug Azr Rpe Mai Guz Gets Tig 65 70 75 80 b5eg b01u TyYg Guz Mai Ge A1ia Tig Ge Suz b1u b1p 01 beg Tpg A5r 85 90 95

Te bij As ATa Rgo b1u Ap Azr Tyg Rpe Tug beg Gets O1u Rgo 5eg 166 185 116

Hey Guz Ma! Tig Re Ni beg Azr Tug 5eg Ap bi Guz Rgo Ripe Tig 115 129 125 s1u Rpe 610 A1i1a Rpe Tug Ai1a Aia b1y Azr Mai Azr bIy Su5 Age Tig 1308 135 140 b5eg Ge oI1u Azr beg Ma! Rgo Su Ar Ni Tug Su5 Ni Azp Tug Ge 145 158 155 160 s1u o1u Tug Tug Suz beg Sub Age Ma! s1u Tug I1e Gets Ni bIp Up 165 176 175

Zo Guz Ni Tis Sub beg ATa Ge Su5x 5eg b1u beep Ma! Rpe Tig o1u Age 188 185 199 b5eg b1u Re Ge beg 5eg Rgo bTy Tug Rgo bIp Rgo Tug Rgo Guz Ge 195 290 295 b5eg b5eg Su5x AIia Tug Ahp I1e Age Hey bii bij b01u Re beg I1e Tpg 216 215 229

IGets Azr Rpe Mai bij b5eg Rpe Azr Ma! s1y Mee No5 Rgo would A1a sp 225 230 235 240

Suz Rgo Tug Azr beg Ge Guz I1e b1p Tpg Ar Guz Age b1y Tug sb1u 245 258 255

Rgo Rpe Suh 01u Gu5 Tyg Gay Rgo Rgo Age I1e bij TPg Ar 5eg Avp 260 265 279

Guz Ma! Tig I1e Tig Rpe Tig Tig Azr 01 beg s01u Azp Nih Tig s1u 275 2808 285

Ter Guz She Ni Tug Tig beg Tig ATia b1p Rgo Su Rgo Ar Rgo Tpg 2908 295 309

A1a Rgo Rgo Azp b01u Ni5 IIe beg Rgo Ma! b1p Aia Tig Tug Mai Ge 395 316 315 329

Guz Azr beg Rpe beg Ma! Rpe Su Guz Tyg s01u Re sy Gay Gey bIp 325 339 335 o1u beg Mai Rgo Ge Guz b5eg Rpe Tyg Aia Mai Su5 bIp Guz Azr o01u 340 345 359 b5eg Ter Azr Ag Rgo I1e Rgo bij Su5x beg I1e I1e Ar Su5 Oo1u Rgo 355 zva 365

Rgo Azr Azr Gay Rgo Azp b1u Ni5 Ma! Azr Tug I1e Tig s1u Rgo s1y 379 375 389

Mmai Tys Tys Tug Guz Aia Mai IIe bip Tug beg Su5x 01 bI1y Tpg Rpe 385 399 395 4ev

Tug Ts Me 5eg beg Azp b1u Guz Tug Ma! Su5 bI1y A1a Azr s1u Re 405 416 415

Zo

Ter Ts beg b5eg Guz s1u bi Guz beg Gets Rgo Mai Suz Guz Rgo Mai 420 425 430

Suz s1u Gets b5eg Typg No Tig b5eg b1u o1u Ag I1e I1e s1u o1u bIp 435 44o 445

Rgo Aia Guz Rgo o1u Azr Ripe Rgo Tgr bIp MaiYi Gey Ge s1u o1y 450 455 460

Tig Ts ATa Aia o1u A1ia Gay I1e Ni Ar Azr Tgr MaiYi Gay Tig A1a 465 470 475 480

A1a Ni5 A1a Mai Tug s1u Guz Tig fight A1ia Mee 5eg beg Gay Azr Pe 485 490 495

Age Mee o1u IIe Gets Guz Ag Gay beg Ge I1e Tug Tg sp Aa Ter 580 505 518

Fight Aia Ma! Rpe I1e Ni5x siy s1u Tug Tig Ni5 sb1u A1a s1u Rpe 515 529 525

Azr Azp Azr I1e ATia iey IIe Guz Hey Guz Azp Guz Ma! Tig I1e Ap

539 535 540

Age Azp IT1e Mei Rgo I1e Suh5 Hey Rgo Age Gu5 b1y A1a A1a 5eg Gets 545 559 555 560

Mezhe Guz Tpyg Azr Rpe Mai s1u Tig Mai Aia s1u Ter o1u Gei Tig s1Ip 565 5708 575

Guz b1u Re Gets ATia Age Ap Gay Mee Rpe Mai Azr I1e Rgo I1e Mai! 5808 585 599

Azr Ni5 b1p Guz Sub AI1a Tig Aia Tug Tys Guz b1p Rgo Tug Rgo o1u 595 ve 605

A1ia Guz Mai Tig Mai Azp Mee Gay Su5 Aia o1u Gay Azr Age s1u O1Uu 618 615 620

Guz Azr beg Sub Ag» b1u Ar beg b1u s01u Aia Ge Mai Re Getsy Ar 625 630 635 640

Azp bij Ts bip Ase Ter Rpe Ma! s1u s01u I1e Ma! 5eg Ter o1u 5eg 645 659 655

Zo

Ie Azp Su5x os1u s1u beg 01 b1p Tug o01u Mai Tug Tig Guz Mai TApg 66O 665 670

A5zp Tug I1e Rho Ter I1e bT1y Azp I1e I1e A5p Ap Rpe 675 680 685 "2105 555 "2115 670 "212" Protein "2135 rat "4005 -h55

Te Ge Gets s01u beg Gu5 Ter Rgo bI1y Rgo Ma! Rpe o1u Age Hey Mai! 1 5 16 15 b5eg Ge Aia Rpe Rgo bi Gu Tug b1u Azp Nib5 bip Ar Age 5eg Tgr 29 25 36

Tys Sei Tapg ATa Rgo Rgo biu Rpe Age Ge Ag» Ge Tug Rpe Tig No 35 40 45

Rpe Azp Ge bi Ge beg Tug Ag» Sub bi Tug Azr RPpe Mai Gu Ge

50 55 60

Tig o5eg o1u Tig Guz Mai Ge Aia Tpg Ge Suz s1u bIp b01y beg Tig 65 78 75 80

Azr Ts bi Asv ATa Rgo b1u Azp Ar Tiig Re Tug 5eg Ge Oo1u Rgo 85 90 95 b5eg Ge Guz Ma! Tig Rpe Ni beg Ar Tug beg Azp bIy Guz Rgo Re 166 185 116

Te b1u Rpe bij Aia Ripe Tug A1і1a A1іa b1ii Ar Mai! Arsi Su Age 115 1208 125

Te 5eg Ge b1u Ar beg Mai Rgo Su5 Ar Ni Tug Sub Ni Azp Tug 1308 135 140 ei sb1u s1u Tug Tug Su5 5eg Sub Ag» Mai o1u Tug I1e Getsy Ni b1Ip 145 1509 155 160

A5zp Guz Ni Tpg Sub beg Aia Ge Su5z 5eg b1u b1p Ma! Re Tig o1u 165 176 175

Zo

Age beg b1u Re Gets 5eg 5eg Rgo biy Tug Rgo bIp Rgo Tug Rgo Gu 188 185 199

Hey 5eg beg Sub Aia Tug Azhp I1e Age Hey biy bi o1u Ripe b5eg I1e 195 290 295

Te Ge Azr Rpe Mai bi beg Rpe Azr Ma! bii Mei Ni5 Rgo b1y A1Ta 2168 215 229 bIip Su5 Rgo Tug Azr beg Ge Guh Ie b1p Tig Ar Guz Age o1y Tug 225 230 235 240 s1u Rgo Rpe Suzh sb1u Gu5 Tpg Gets Rgo Rgo Age I1e bii Tig Ar 5eg 245 258 255

A5zp Guz Ma! Tpg Ie Teg Re Tpg Tiig Azr 01 b5eg b1u Azp Ni Tpg 260 265 279 o1u Ter Guz She Ni Tug Tig b5eg Tig Aia bIp Rgo Sub Rgo Ar Rgo 275 2808 285

Tig ATa Rgo Rgo Azp s1u Ni5 I1e beg Rgo Ma! b1p A1a Tig Tug Mai 2908 295 309

Gay Guz Azr beg Rpe beg Mai Re Su5 Guh Tig o1u Re o1y Gay Gays 395 316 315 329 b1ip o1u beg Mai Rgo Gay Guz beg Ripe Tpg ATa Mai Suh5 bI1p Guz Ar 325 330 335 s1u beg Ter Azr Age Rgo I1e Rgo bi Suh5x 5eg I1e I1e A5zr Sukh Oo1u 340 345 359

Rgo Rgo Azr Ar Ge Rgo Azp s1u Nih5 Ma! Azr Tug IIe Tig o1u Rgo 355 zva 365 bIii Mai Tig Tys o Tug Guz ATia Ma! Te b1ip Tug beg Su5 o01y si Tig 3708 375 389

Rpe Tug Tig Met beg b5eg Azp sI1u Gu5 Tug Mai Su5 biIy A1a Azr o1u 385 399 395 4ev

Rpe Ter Tyg beg beg Guz o1u si Gub5 beg Hey Rgo Mai Su Gu Rgo

From 485 416 415

Mmmy! Suh o1u Ge beg Tpg No Tpg beg b1u o1u Age I1e I1e s1u O1Uu 420 425 430 b1ip Rgo Aiia Guh5 Rgo o0T1u Azr Re Rgo Tgr bIp Ma! Gay Gays Gay o1u 435 44o 445 bI1y Tys Ts ATa ATa o1u Aia Gay I1e No Ar Ar Ter Mai Gay Tig 4506 455 460

A1i1a Ai1a Nib5 Aia Mai Tug s1u Gu5 Tpyg bi Aia Mee 5eg 5eg Gets A5r 465 470 475 480

I1e Ag Mee s1u I1e Gay Gu5 Age Ge b5eg Gay I1e Tug Tig b1ip A1Ta 485 499 495

Ter Rgo bT1y A1ia Mai Rpe I1e Ni5 bi s01u Tug Tig Ni5 o1u A1a o1u 580 505 518

Rpe Azr Azp Azr I1e Aia Hey I1e Gu5 ep Guz Azp Guz Ma! Tyg Pe 515 529 525

Abzp Age Abp IT1e Mei Rgo I1e Su5 Gets Rgo Age Guz b1y A1a Aa 5eg 5308 535 5409

Ge Mee Guz Tiig Azr Rpe Mai s1u Tig Mai Aia s1u Ter (1u Gei Tig 545 559 555 569 bip Gu5 b1u Rpe Ge A1ia Ag» Azp Ge Mee Rpe Ma! Azr I1e Rgo Pe 565 579 575

Mmai Azr Nih b1p Guz Suz Aia Tig Aia Tug Tpg Guz bIp Rgo Tug Rgo 589 585 599 s1u ATa Guz Ma! Tpg Mai Ahp Mei Ge Su A1a o1u Hey Azr Age O1u 595 6o0 605 o1u Guz Azr 5eg Su Ag» b1u Ar beg b1u s1u Aia Ge Mai Rpe Ge 618 615 6206

Azr Azp bi Ts bip Ave Ter Rpe Ma! s1u o1u I1e Ma! 5eg Ter 01u 625 630 635 640

Zo b5eg I1e Ahzp Su5 bI1u b01u 5eg bi beep Tug b1u Ma! Tug Tug Gu Mai 645 6509 655

Tys o Azp Tug I1e Rgo Ter I1e 010 A5p I1e IT1e Ap Ap Rpe 6v6O 665 670 "2105 56 "2115 28 "2125 DNA "2135 Artificial sequence "220" "223" Noto b5ariep5 "4005 56 asvavvsts Evassstssti vvevssTT s 28 "2105 57 " 2115 23 "2125 DNA "2135 Artificial sequence "220" "223" Noto b5ariep5 "4005 57 вЕВСсСссСТсСс ТвСсвЕсСасст siv 23

"2105 58 "2115 23 "2125 DNA "2135 Artificial sequence "220" "223" Noto b5ariep5 "4005 58 savavrvivas vsavrvavvvve sas 23 "2105 59 "2115 27 "2125 DNA "2135 Artificial sequence "220" "223" Noto b5 Ariep5 « 4005 59

EsSaaaayesas taayesaevs stsvats 27 "2105 60 "2115 22 "2125 DNA "2135 Artificial sequence "220" "223" mouse "4005" 60 asvavvstas Esassesssya vo 22 "2105 61 "2115 23 "2125 DNA "2135 Artificial sequence "220" "223 » mouse «4005 61 svssavvvi vasvsavvv v88 23 «2105 62 «2115» 23 «2125 DNA «2135 Artificial sequence «220» «223» mouse «4005 62 ssssssstvsS savststtv 23 «2195 63 «2115 29 «2125 DNA « 2135 Artificial sequence "220" "223" mouse "4005" 63

EsSavgaaaesa seEsayesaevs estsaayess 29 "210" 64 "2115 29 "2125 DNA "2135 Artificial sequence "220" "223" rat "400" 64

Bvagvivasvs avvagvvvsa ESavtv 29 "2195 65 "2115 37

From "2125 DNA "2135 Artificial sequence "220" "223" rat "4005 65 stavaaasas taaevsssst sstvsvIsas ststvsa 37 "2195 66 "2115" 354 "2125 DNA "2135 Artificial sequence "220" "223" Synthetic "4005" 66 savvIsassi Wow ivvysstvIv stvrRIvaaas ssasavavas sstsasvsiv 6o asstvsassv ESTISTREVBII stEsastsavs avreevtaaa ee ееviIvav stevatsssv 120 savsssssav vevaavvsssi vravtivvsti vsasasayessi sssraviva sraaaatss 188

Tasavvasae sestraavrav savvstsass ayestssaavre asasstssaa aaassarete 2408 vEssttasaa Yevassaasae gevassstviv vasasavssa svtaesastv Evsasserata 300 svasvyivrav vgaaeesvassta stvevevssav vvaassstvv isasstvssts stsa 354

"2105 67 "2115 118 "2125 Protein "2135 Artificial sequence "220" "223" Synthetic "4005 67 b1ip Mai Tyg Gay Guz b1y b5eg 01u Rgo Mai Ge Mai Gu5 Rgo Tyg sy 1 5 18 15

Te Se Ts Ge Te Subh Ts o Mai 5eg b1u Rpe b5eg Gets 5eg Age b1u 20

Guz Mee s1u Ma! 5eg Ter Te Age sp Rgo Rgo o1u Guz ATa Hey sy 4 45 25 Ter Ge Aia Ni5 I1e Rpe beg beg Azr biy Gu5 beg Tug Age Tg 5eg 58 55 va

Hey Guz beg Ag Hey Tig I1e beg Guz Azr Tig beg Gu5 Azp b1ip Mai

From 65 78 75 80

Mmai Ge Typg Mee TyYg o Azp Mei Azr Rgo Mai Azr Tig A1a Tig Tug Tug 85 90 95 35

Suz A1a Ag» I1e Ag» Age b1u b1u I1e Azr Tug Ter o1u bIp o1u Tig 188 185 118 ей Ma! Tig Mai 5eg 5eg 115 "2105 68 "2115 121 "212" Protein "2135 Artificial sequence "220" "223" Synthetic "4005 68 bip Mai b1p Ge b1p b1p beg s01u Rgo s1u Gay Mai Gu5 Rgo 5eg bip 1 5 18 15

Tig Ge beg Ge Tig Suz A1ia I1e beg s1u Azr beg Ma! run 5eg Tig

20 25 30 b5eg A1i1a Aia Ter Azp Ter I1e Age b1p 5eg Rgo beg Age O1u Hey would 35 40 45

Ter Ge s01u Ag Tiig o Tugs Tug Agsv b5eg Guz Ter Tug Azp Azr Tug Aa 58 55 va

Whoa! 5eg Mai Gu5 beg Age I1e Tpyg ITe Ap Rgo Azr TPg 5eg Gu Avp 65 78 75 va b1ip Re beg Hey b1p Hey Ap beg Ma! Tig Rgo bIy Azr Tig A1a Mai 85 90 95

Tug Tug Sub Aia Ag» Ar Rgo Rpe b1u Ma! Rgo Rie Azr Te Ter s1u 188 185 118 b1ip o1u Tpyg Me Mai TyIg Mai beg 5eg 115 1208 «2105 69 «2115 186

Zo "2125 Protein "2135 Artificial sequence "220" "223" Synthetic "4005 69 b1ip Rgo Mai Gay Tpg bI1p Rgo Rgo b5eg ey b5eg Ma! beg Rgo (in SP 1 5 18 15

Tig AT1a beg I1e Tpg Suzh beg o1u by Guz Ge o1u Azr Guz Tug AIa 20 25 30

Tug Ter Tug b1p bIip Guz Rgo s1u b1p 5eg Rgo Ma! Ge Mai Mee Tug 35 4 45 bIip Ar Guz b1p Age Rgo beg b1u I1e Rgo bij Ag» Rpe 5eg Oo1u 5eg 58 55 va

A5p beg s01u A5zp Tig ATia Tig o Ge Tyg I1e beg s01u Tpg bip A1a Mee 65 78 75 80

Azr bij ATa Ar Tugs Tugs Sub b1p Aia Ter Azr 5eg b5eg Tig A1Ia Mai 85 90 95

Rpe s01u s1u o1u Tig Guz Gay Tpg Mai Ge 189 195 "2195 76 "2115 324 "2125 DNA "2135 Artificial sequence "220" "223" Synthetic "4005 70 tsstavevavs Svassta assstsvvyv yesavivvsss sarvasavas vvsssssate 6o asstvyvs vRe ravasaassi SRevBaaraaa svsvyvsasi vertassavsa vavrvssavvs 1206 savvssstv YivYITvvIsae stairaevrai avsvassvvs sstIsarevai ssstrassva 188

ЕСЕ sStvsSsST ssaasstvv vaasasvvss assstvassa esasastavevv svaavsseve 2408 vaevavvssv astayesaesye esavrvyivsve vasayesevsta stvaesaevye vievistSsevs 300 vBvavevassa avstsassvi ssta 324 "21905 71

From "2115 126 "212" Protein "2135 Artificial sequence "220"

З5 "223" Synthetic "4005 71 b5eg Tug bIy Ge IIe b1p Rgo Rgo beg Mai 5eg Mai A1a Rgo o1u bIp 40 1 5 16 15

Te ATa Ts I1e Tig Suh ATa 01u Ar Ahp Gay o1u Guz Guz Age Mai 20 25 3a 45

No Ter Tug beep b1ip Ag» Rgo b1u b1p Ai1a Rgo Mai Ge Mai I1e Tug 4a 45

Ar Azr beg Ar Age Rgo 5eg s1u I1e Rgo Ar Age Rpe 5eg ATa 5eg 58 55 va

Abzp beg b1u Ahp Tig ATa Tig Gay Ts I1e Tig Age s1u sb1y Aia Oo1Uu 65 78 75 80

Azr bij ATa Ar Tusb Tugs Suz b1p Mai Ter Azr I1e AIa Tig Aor Ni

85 90 95

Mmai Ma! Rpe s1u oI1u s1u Tig Guz Gay Tig Mai Ge A1a Aia Ai1a o01u 188 185 116 b5eg 01 bI1p uh Ge Ie 5eg Oo1y 115 129 "2105 72 "2115 36 "212" Protein "2135 Artificial sequence "220" "223" Synthetic "4005 72

Yech bIy Mai Tiig Suz 01 Rgo s1u Tiig Tys Rpe Guz Ar Guz Su Avp 1 5 18 15

Te Suz Aga Sub b1u beg Azr b1u Guz beg Aia Mai Sub Tig Guz Ge 20 25 30

Zo Ter Su Ap b1p "2105 73 35 "2115 33 "212" Protein "2135 Artificial sequence "220" "223" Synthetic "4005 73

Te Suz bii Rgo b1u Tig Tys Re Guz Azr Guz Sub Azp Tig Su Age 1 5 18 15

Suz s1u beg Azr o1u Guz beg ATa Mai Su5 Tig Guz Gay Ter Su Azp 20 25 30 b1p "2105 74 "2115" 29 "212" Protein "2135 Artificial sequence

"223" Synthetic "4005 74

Te Suz Aga Sub b1u beg Azr b1u Guz beg Aia Mai Su Tpg Gu Gets 1 5 18 15

Ter Suz Azp b1Ip 20 "2105 75 "2115" 491 "212" Protein "2135 Artificial sequence "220" "223" Synthetic "4005 75

Hey bIy Ma! Te Sukhz b1y Rgo sb1u Tyg Tys Re Guz Ar Guz Su Azp 1 5 18 15

Te Suz Aga Sub b1u beg Azr b1u Guz beg Aia Mai Sub Tig Guz Ge

From 20 25 36

Ter Suz Azp b1p s1u Tpg s1u s1u o1u beg o1u 5eg beg 5eg b1p Mai1 4 45 35

Tig Ge Guz 01 beg s1u Rgo Ma! Ge Mai Guz Rgo Tyg si Tpg Ge 58 55 va

Te Se Te Suh Ts Mai beg b1u Rpe 5eg Ge beg Age Oo1u Guz Mee 65 78 75 va s1u Mai 5eg Ter I1e Ag» bip Rgo Rgo s1u Gu Aa Hey si Ter Ge 85 90 95

A1a Ni5 I1e Rpe beg 5eg Azr bij Guz beg Tug Age TPg 5eg Getsy Gu 188 185 116 b5eg Agv Gay Tpg o I1e beg Gu5 Azr Tg o beg Guz Azp bip Mai Mai Ge 115 129 125

Tig Met TiYg o Azp Mezh Azr Rgo Ma! Azr Tig Ai1a Tig Tug Tug Suz Aa 138 135 140

Age I1e Ag Ag b1u b1u I1e Azr Tug Ter o01u b1p s1u Tig Getsy Mai 145 158 155 160

Tyg Mai beg 5eg AIa 5eg Tpg Guz b1u Rgo beg Mai Rpe Rgo Gey Aa 165 176 175

Rgo Sub beg Age beg Tg 5eg bij beg Tig ATa A1a Hey s1u Su Gets 188 185 199

Mmai Guz Azr Tug Re Rgo fight Rgo Mai Tiig Mai 5eg Ter Azp 5eg O1u 195 290 295

A1a Hey Tpg beg o0I1u Ma! Ni5 Tipg Rpe Rgo DAIia Mai Ge beep 5eg 5eg 2168 215 229

O1u Gei Tug beg Ge beg 5eg Ma! Mai Tig Mai Rgo 5eg 5eg 5eg Ge 225 230 235 240 o1u Tig Guz Tys o Tug Tys Su Azp Mai Azr Ni Guz Rgo 5eg Azp Tig 245 258 255

Zo Guz Mai Azr Guz Ag» Mai 01 beg Guz Tug O1u Rgo Rgo Su Rgo Rgo 260 265 279

Suz Rgo Aia Rgo bij Rpe Ge s01u b1u Rgo beg Mai Rpe Gay Rpe Rgo 275 2808 285

Rgo Guz Rgo Guz Azr Tig Gay Mee IT1e beg Age Tig Rgo bIy Mai Tig 2908 295 309

Suz Mai Mai Mai Azr Mai beg bI1p 01 Azr Rgo bi Mai b1ip Re Ap 395 316 315 329

Ter Tug Mai Azr s1u Mai b1y Mai Ni5 Ahp Aia Gu5 Tig Gu Rgo Age 325 339 335 bij 010 b1p Rpe Ap 5eg Tipg Tug Ag» Ma! Mai 5eg Mai Gets Tig Mai 340 345 359

Ge Ni bip Azr Ter Ge Azp o1u Guz 01 Tug Guz Suz Guz Mai beg 355 zvo 365

A5zp Guz s1u Hey Rgo beg beg I1e oTy Guz Tig I1e beg Guz Aa Gu 3708 375 389 s1u b1p Rgo Age fight Rgo beep Ma! Tug Tig Hey Rgo Rgo 5eg beep b1y 385 390 395 4ev bIy Mee Tig o Guz Azp beep Mai beg Ge Tyg Suh Ge Mai Guz s1u Re 485 416 415

Tug Rgo beg Azr IIe A1i1a Mai o0ii Ter sb1y beg Azp b1u bip Rgo sy 420 425 430

Abzp Azp o Tug Guz Ts Te Rgo Rgo Mai Ge Ar 5eg Azr o1u 5eg Rie 435 dda 445

Rpe Gay Tug beg Age Gay TPg Mai Azr Guz 5eg Age Tgr b1p siy o1u 450 455 460

A5p Ma! Rpe beg Sub beg Ma! Met Ni5 01 DAIa Ge Ni Ap Ni Tug 465 470 475 4808

Tig bI1p Guz 5eg Ge 5eg Ge beg Ge o1u Guz 485 499

Zo "2105 76 "2115" 491 "212" Protein "2135 Artificial sequence "220" "223" Synthetic "4005 76 b1ip Mai Tyg Gay Guz b1y b5eg 01u Rgo Mai Ge Mai Gu5 Rgo Tyg sy 1 5 18 15

Te Se Ts Ge Te Subh Ts o Mai 5eg b1u Rpe b5eg Gets 5eg Age b1u 20 25 30

Guz Mee s1u Ma! 5eg Ter Te Age sp Rgo Rgo o1u Guz ATa Gay si 35 40 45

Ter Ge Aia Ni5 I1e Rpe beg beg Azr biy Gu5 beg Tug Age Tg 5eg 58 55 va

Hey Guz beg Ag Hey Tig I1e beg Guz Azr Tig beg Gu5 Azp b1ip Mai 65 78 75 va

Mmai Ge Typg Mee TyYg o Azp Mei Azr Rgo Mai Azr Tig A1a Tig Tug Tug 85 90 95

Suz A1a Ag» I1e Ag» Age b1u b1u I1e Azr Tug Ter o1u bIp o1u Tig 166 185 116 ей Ma! Tis Mai beg beg ATa beg Tig Guz o1u Rgo beg Ma! Rpe Rgo 115 1208 125

Gay A1Ta Rgo Sub beg Age beg Tg 5eg bij 5eg Tig ATa ATa Getsy s1u 138 135 140

Suz Ge Mai Guz Azr Tug Rpe Rgo 01 Rgo Ma! Tig Mai beg Ter Azp 145 158 155 160 b5eg b01u ATa Ge Tig b5eg b1u Ma! Ni5 Tig Rpe Rgo A1Ia Mai Gey sp 165 176 175 b5eg 5eg o01u Ge Tug 5eg ep beg beg Ma! Ma! Tig Mai Rgo 5eg 5eg 188 185 199

Zo b5eg Ge b1u Ts o Gu5 Tiig o Tug Ts Su5 Azp Mai Ar Ni Gu Rgo 5eg 195 290 295

Azp Te Guz Mai Ar Guz Age Ma! bIy b5eg Gu5 Tug s1u Rgo Rgo Su 216 215 229

Rgo Rgo Suz Rgo Aia Rgo b1y Rpe Hey b1u o1u Rgo beg Ma! Rpe Gaia 225 230 235 240

Rpe Rgo Rgo Gu Rgo Guz Azr TYg Ge Mei I1e 5eg Age Tig Rgo b1y 245 258 255

Mmai Tig Suz Mai Mai Mai Azr Mai beg b1p by Azr Rgo by Mai sp 260 265 279

Rpe Azp Ter Tug Mai Azr sI1u Mai b1y Mai Ni5 Ahzp AIa Gu5 Tig Gu 275 2808 285

Rgo Ag» bi bi bip Rpe Azp beg Tyg o Tug Age Mai Ma! 5eg Mai Gets 298 295 309

Tig Mai Getsy Ni bIp Azr Ter Ge Azp s1u Guz si Tug Guz Su Tuz 395 316 315 329

Mai beg Azp Guz o1u Ge Rgo beg beg I1e bI1y iu5 Tig Pe 5eg Gu 325 330 335

A1i1a Guz o1u beep Rgo Age bi Rgo beep Ma! Tug Tig Gay Rgo Rgo 5eg 340 345 3509 b1ip bTiy b1y Me Tipg Guz Azp bip Mai beg Gey Tig Su Gey Mai Guz 355 zvo 365 s1u Rpe Tug Rgo 5eg Azr I1e Aia Mai sb1y Ter biy 5eg Azp o1u bIp 379 375 38 0

Rgo beats Azp Azp Tubsb Guz Ts Te Rgso Rgo Mai Ge Azr 5eg A5r o1u 385 390 395 4ev b5eg Rpe Rpe Gay Tug beg Age Gay Tg Mai Ar Guz 5eg Age Ter bIp 485 416 415 bI1y o1u Ap Ma! Rpe beg Suh beg Mai Mei Ni5 o1y Aa Gey No Up 420 425 430

Zo

No5 Tug Tig b1I1p Guz 5eg Ge beg Ge 5eg Ge o1u Guz Aia A1a o1u 435 dda 445 s1u beg b1u Ge bi Ma! Te Suh bI1y Rgo b1u Tys Tys Rpe Gu Azr 450 455 460

Guz Suz Azp Tyg o Sub5 Age Sub5 b1u beg Azr b1u Gu5 beg ATa Mai Su 465 470 475 480

Tig Guz Ge Ter Suz Azp bIp o1u 5eg o1u A1a 485 490 "2105 77 "2115 258 "212" Protein "2135 Artificial sequence "220" "223" Synthetic "4005 77

Yech bIy Mai Tig Suz 01 Rgo s01u Tig Tys Rpe Guz Azr Guz Su Azp 1 5 18 15

Te Suz Aga Sub b1u beg Azr b1u Guz beg Aia Mai Su Tpg Gu Gets 29 25 36

Ter Suz Azp b01p sb1u Tpyg sb1u s1u s1u 5eg b1u 5eg 5eg beg bIp Rgo 35 40 45

Uai Gay Tig beep Rgo Rgo beg Ge beg Mai beg Rgo b1u beep Tig A1Ta 50 55 60 b5eg I1e Tig Suzh beg s1u bi Guz Ge s1u Ar Guz Tug AIa Tug Ter 65 78 75 80

Tug bI1p bI1p Guz Rgo s1u b1p b5eg Rgo Ma! Hey Mai Mee Tug b1p Ar 85 90 95

Guz b1p Ag Rgo beg b01u I1e Rgo bij Ag» Rpe beg o1u beg Ap 5eg 166 185 116 o1u Azp Tyg ATa Tyg Gay Tyg Ie b5eg s01u Tyg bIp Aia Met A5r s1y 115 1208 125

Zo A1Ta Azr Tug Tug Su5 b1p A1i1a Ter Azr beg beg Tg A1a Mai! Re o1u 1308 135 140 so1u o1u Tig Guz Ge Tig Mai Gay o1u b1p Rgo Guz ATa AIa Rgo 5eg 145 1509 155 160

Mai Tig Ge Rpe Rgo Rgo beg beg b1y 01 Ge b1p AIa Ap Guz AIa 165 176 175

Tig Ge Mai Suz Ge I1e beg Azr Rpe Tug Rgo s1u A1ia Ma! Tig Mai 188 185 199

A1a Ter Guz Aia Azr beg beg Rgo Mai Guz Aia s1u Ma! o01y Tag Tig 195 290 295

Te Rgo beg Guz bip beg Abp Azp Guz Tug Ai1a Aia beg beg Tug Ge 2168 215 229 b5eg Ge Tyg Rgo bii bip Ter Gu5 5eg Nib5 Ag» beg Tug beg Su bIp 225 230 235 240

Mmai Tig Nih 01 o1u beg Tig Mai bi Guz Tig Mai A1a Rgo Tig si 245 258 255

Sub 5eg "2105 78 "2115 258 "212" Protein "2135 Artificial sequence "220" "223" Synthetic "4005 78 b1ip Rgo Mai Gay Tpg bI1p Rgo Rgo b5eg ey b5eg Ma! beg Rgo (in SP 1 5 18 15

Tig AT1a beg I1e Tpg Suzh beg o1u by Guz Ge o1u Azr Guz Tug AIa 20 25 30

Tug Ter Tug beep b1ip Gu Rgo b1u b1p beg Rgo Ma! Ge Mai Mee Tug 4 45 bIip Ar Guz b1p Age Rgo beg b1u I1e Rgo bij Ag» Rpe 5eg Oo1u 5eg

From 58 55 60

A5p beg s01u A5zp Tig ATia Tig o Ge Tyg I1e beg s01u Tpg bip A1a Mee 65 78 75 va 35

Azr bij ATa Ar Tugs Tugs Sub b1p Aia Ter Azr 5eg b5eg Tig A1Ia Mai 85 90 95

Rpe s01u sb1u o1u Tig Guz Ge Tig Mai Ge o1u bIp Rgo Guz Aa A1Ta 188 185 118

Rgo beg Mai Tig Gets Rpe Rgo Rgo b5eg beg bi b1y Gay b1p Aa Ap 115 129 125

Guz Aia TyYg Ge Mai Suzh Ge I1e beg Azr Rpe Tug Rgo o1u A1a Mai 138 135 140

Tig Mai ATia Ter Guz A1i1a Ar beg beg Rgo Ma! Guz Aia o1u Mai sy 145 1509 155 160

Tis Te Ts Rgo beg Guz b1p beg Up Azp Guz Tug AIi1a AIa 5eg 5eg 165 178 175

Tug Ge 5eg Ge Tpg Rgo bi b1ip Ter Gu5 beg Nib5 Age beg Tug 5eg 1809 185 199

Suz b1p Ma! Tpg Ni5 01 s01u beg Tpg Mai bIy uh Tyg Mai A1a Rgo 195 290 295

Te bi Suh 5eg Aia Aia s1u b1u 5eg s01u Hey b1y Ma! Tig Su5 sy 216 215 220

Rgo b1u Tig Tig Re Guz Azr Guz Sub Azp Tig Su Age Su o1u 5eg 225 230 235 240

Azr s01u Guz beg ATa Mai Suh Tpg Guz Getsy Ter Suz Azp bip s1u beg 245 2509 255

Oo1u A1Ta "2105 79 "2115 16 "212" Protein "2135 Artificial sequence

Zo "220" "223" Synthetic "4005 79 so1u Tig o1u o1u s1u beg o1u 5eg 5eg beg 1 5 18 "2105 88 "2115 6 "212" Protein "2135 Artificial sequence "220" "223" Synthetic "4005 88

A1a Aia o1u o1u beg s1u 1 5 "2105 81 "2115 4 "212" Protein "2135 Artificial sequence "220"

"223" Synthetic "400" 81 o1u beg s1u A1a 1 "2105 82 "211" 1533 "2125 DNA "2135 Artificial sequence "220" "223" Synthetic "4005 82 asvayevesst ESVESTESTSE vSESSTvVII vvsaeysstaye sssaevssas ssavvsstyev 6o

Baavivasvi vyvavsssev aasvasayeses aaarasaavi vsaatastee sei esev 120 tsavaevevva aassevsevI svsasaaaav ststevBIvta assavrevsas sevBEvravve 188

TsveBvayessa vstsasavvi sassteYevaav vavsstvvis stRTvsTvRviI vaaasssasa 2408 7 vavassstsa svstvasstv sassvistsi vevEsstsas ysavsavvee taaaasevv 300 veEvavsivva yssvIsavss sssavevaav vssstvvBavi vyvsEsvsasa sayessteets 360

Zo avivasvaaa aassstasavye vasayesvstv aaravsaves ysassaests saarvasass 420 tssaaaaass avvsevissI Tasaayevass aasaevvass stvivRvasas avssasvtae 480 tastvyvsas vvastasvasv vRvavrvaayei vastastvve vssavrevaas sstevEsasst 540 7 vESTTsSstsav sstssassa а веввсссаесс весстТсссссс Tввсвссств stssavravs 600 asstssvavra vsaavssvs sstevvstIvsS sIvrRIsaarve astasessss sraasseviv 66a asevivisSvi vraastsavrv svssstrass avrsevsvivs asassttsss vvstvIssta 720 savtissstsav vasestasts sstsavsavs vyvvRi vassvya vssstssav savsttevvs 780 asvaavassi asasstvsaa setavaisas aavsssavsa asassaavvi vrasaavava 840 7 vEsvavissa aasaevviss ssaevsssa ssaevsssav sasstvavte ssterereva 980 ssayesavtist tssEvESsss sssaaaasss aavrvasasts tsayesvaests ssevasssst 960

Bavvisasvi esvIRRivvi vRrRasvivravs savvaavrass ssvavvissa vissaastvv 1920 tasvsvvasv vsvIvvBavvi vsasaasevss aavrasaaavs sesevRvarva vsavsssaas 1986 avsasvtass vyvivvvyasav svisstsass vIisstvsass avrvastvvsii vaaseesaav 11406 7 vavtasaavi vsaavvtstis saasaaavvs stsssvisst ssayesvaaa aassayestss 120806 aaavssaaaav vevsavssssv avavssasaw vIvtasasss vssssayes ssawvavvav 1260 asvassaavra assawvyasav sstvasstvs stvrIisaaav vEssstasss savsvasats 1320 vBssvTvRavi verRavravsaa Ter esawss in varaaasaasi asaavassas vsstsssviv 1380 stvvastssv asevstsstE stsstsStas avsavvstaa ssvIRrasaa havsavviev 1440 savvavvvrva aevEstests aevstsssvtv asvsayesvavv sEstTesasaa ssstasasa 1586 savaavavss EstssstvssS tstseRERaaa va 1533 «2105 83 «211» 153 3 «2125 DNA «2135 Artificial sequence «220» «223» Synthetic «4005 83 6o vEsasstEeEeva avvavsstvv isstvIivsTvR virRaaasssa savavasssti sasvstvass 120

TvsassvEsti stIREVTYSISS astsavsavrv verRiaaaastev vyvyvavstv vayessvisav 188 sssssavrrva avvsstvva vivevBSEivsa sasayestes-i svavivasva aaaayesstas 2408 avvasayesvs Yivaavravsawye vstsassayes tsaavrvasa sstIssaaaaa ssarvtvvis 30 07

З5 aavvvsssaye ssvisttsss sstIRESsvsss Tvstssavrva vsasstssva rgavsasawss 480

USSR вси всстввіса вгвастастсс ссваассвв evasevivis vrivrraastsa 540 bvsvsstva ssavsevsvi vsasasstts ssevstviss tasavsissts avvasestas 600 7 tssstsavsa vsvyevevEvas svEvssstIss avsavstievvR vsasvaaras stasasstvs 66a aasvtavaes asaavssssav saas assaav vIevRasaavra vavsevavis saaatayeev 720 sssssaevss sassaevsss avsasstvav ESSSTveReE vgassaesavti seyesstvess 780 sssssaaaas ssaarvasas tstsaevayes tsssvvasss stvavvisas viesvivRiv 840 vBEvvRasvyva vsavrvaavra ssssvavvisste savesaasti vvtTase svvra TevsvIvvav 960

Tssasaaaav vsstsssvis stssaisvrav aaaaassaesti ssaaaarssaa arevssss 1986 svavavssas avvyvstasas sstvssssa ysssavrave avrayesrassaa vaassavvis 11406 avsstvassy vsstvvysaa avvstEestas sssavsvasa ysssvirRva vIRevavavs 120806 aaevveves avs sevavaasaa stasaavass asvsstsssv YevstRvasts svasevstss 1260

HESTESSsSTsST asavsavevsi aasseivvras aaravsavrvi vesavvavrv vaaevists 1320 tEsaevstssv Tvaesvsayeva vvststvsas aassastasa sasavaavrav sststsssiv 1380

TsSIsStsvvva aavssevstvv vReTavtvvi iYisivrRaavryva svyvSvavss seraasvasa 1440

TESaaavrasa avivsaastas sei seeTvesS vRIisavaye vraaassevs vviIstesasa 1586 aavststvvi vtaassavee tavtevivsyi tva 1533 "210" 84 "211" 834 "2125 DNA "2135 Artificial sequence "220" "223" Synthetic "400" 84 asvaevest ЕСВЕСТЕСТСЕ вСЕСССТРВИЙ ввсаессстае tssaevssas ssavvssiiv 6o vaavivasvi vIvgavsssvv aasvasayess aaavasaavi всаастастте всев series 120 tsavaevevva aassevsevI svsasaaaav ststevBIvta assavrevsas sevBEvravve 188

From TsEvvaessa vstisasavss avvstvasi savssssssti sastvissvI vissssavrva 2408 savasavssa vsayesasstv sEstevavrav aaayessevve abaaasaevs ttastevtat 300 savsavaavs savvssavts ssstIviviYiiv vIsaevtaes aavraebaasa vsevssstsa 360 vevaessstv avsvaessts tvvst ssaas sver vRaasa savssastsi vrassayesavs 420 vBevassssavv stasevvaeva vestvastyaei tastvysarv seiveevasav savsastvse 480 vIiaksssevse vgavevassaa vSEvassvis stavessavs staarvsevs vsssisevis 540 assstvEEess svssstsstsS travrvavstye saavssaasa avrvssasasti essays sts 600 asaaavtivasi Estasssvvr avssvivasa vievssivva avrvsavastav savsssvis 66a aagvsevrav yivevassas sasssstss aaaasaavrsa asasaavrta sesevssavs 720 avstaestva vssEvasvss rRavsavrstev aavisssasa vaavstasav svssavvis 780 asvsaevaav gravsassvi vevaraav race vievssssta savayevets atav 834 "2195 85 "211" 834 "2125" DNA "2135 Artificial sequence "220" "223 » Synthetic

4005 85

188

TssssTVIve TevIsaevsta isaarasaaa savsevsssi savrvvaesss EvRavsvatts 2408

TstvvsStssa asistvrvaa sasavssasi stvassayesa vsevrvasssa vstatevat 300 vavvstvasti astastvisa gevseiveeRvas avsavsastv sevtTastisvv seravvvass 360 aavstirassv isstavvssa vsstaarese vsessstssvE isassstvii sssvssstss 420

TstIvavrvavs tEsaavssaa saavvessasa stvevEviivIs ysasaavtva ststasssv 480 vvavssviiva savssssiv raavrvsvai avsssssv Esaavresvvre avtievavass 540 assasasssi ssaaasaare saasaasaav Tasesvvssa vsawstaesii vavsstvasv 600 ssEvavsavi vevaavssssa aavstas avstvssave isasvsaiieiva arregavsass 66a

BIgvaavaava savtivevsss tTasavaaevyi isavssestv vivRvTavtvev TEYseRRaaviv 720 asvyvivivavs ssvvaasvas astsaaavas aavtivsaasta seseiyesevIv sevIESavat 780 vBevaaaysev sevistvsas aaavststve TRiaassave vtavvivivs eatav 834

Zo

Claims (7)

FORMULA OF THE INVENTION 1. A method of treating a human subject suffering from steroid-dependent lupus nephritis (SLE), which includes administering to the subject a composition containing a MABR-2 inhibitory monoclonal antibody or an antigen-binding fragment thereof that specifically binds to MAZR- 2 in humans and inhibits MA5BR-2-dependent complement activation, in an amount effective to improve kidney function, while the MA5BR-2 inhibitory antibody or its anti-hesogen binding fragment contains a variable region of the heavy chain containing SOBK-NI, SOB-H2, etc. SOB-NZ of the amino acid sequence given in the form of ХЭО ИЮО MO: 67, and the variable region of the light chain containing SOC-I1, СОВ-12 and СОК-ИЗ3 of the amino acid sequence given in the form of 5EO IЮ MO: 69. 2. The method according to claim 1, in which the antibody or its fragment is selected from the group consisting of a recombinant antibody, an antibody with reduced effector function, a chimeric antibody, a humanized antibody, and a human antibody. 3. The method according to claim 1, in which the MA5R-2 inhibitory antibody essentially does not inhibit the classical pathway. 4. The method according to claim 1, in which the inhibitory MAB5bR-2 antibody suppresses the deposition of SZB in 90 95 human serum with an IC of 30 NM or less. 5. The method according to claim 1, which additionally includes the identification of a human subject having steroid-dependent BH before the stage of receiving by the subject a medicinal product containing an amount of an MABR-2 inhibitory antibody or an antigen-binding fragment thereof effective to improve function kidney 6. The method according to claim 1, in which the medicinal product containing the MA5R-2 inhibitory antibody or antigen-binding fragment thereof is administered in an amount and for a period of time effective to achieve at least a 20 95 reduction in 24-hour protein excretion from urine compared to the subject's baseline 24-hour urinary protein excretion before treatment. 7. The method according to claim 1, in which the drug should be administered in an amount sufficient to improve kidney function and reduce the dose of corticosteroids in said subject. kah Ya sus, hiii SV er -podyuny t their i BEJ usya VIV vi ; Z sh- SKK nn an Mk SHE o i I VER i ME noz I Mart UT AYAdOomO I zhi KK i her. uh gi TU E YAN de Kaya i RIO IL MAV sh y kl i pe o i pet hu sya v se dona VI BZH B s OVK KO Z ny ko MU o" VMMYA 55 BULL MY sk PI a 1 M zhe o Ah i " Ky i KK B in - OK mo fe EESHINNYE sd 1 shu VOHSHIYA MM BSHSHYA OS fi BO VOSHEiiya MKK nn Shan y V len ne shi 00 Mk OBKOMU AK, ch i. х Э У е ran rodit pt dyk et ох з Х х 2 ТА Е 7 zhit en kt che ОХ х и Ж й и и ей в t ОХ те х х и КИ Я A y sy t kv y She EM et eV i I U Kei i i dk sht MU HE VE V puh e v Pe PO PEKI zaz p she VKA V ro OMVO OK KV V V V duzhnuyuyutnchyuyukn OB pon ee v i i i : ? su kan i i ee 3 i Ebe-obibnyi i EB obezmishova pen - miv seevat kine »erinova pritevza SEC Pe shi i . Fig. 1 TS MAKAR. UMH. 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M2/- Fig as Y Her : y x : E y : x. sh How I STILL but and cha. i kih y h ee Be h i i kh ze z YAKE dk v a F: pev zhuk ah KS Z a - ge meh y Esh: I E -e ho ikh koh KZ v roja: ko sho nya KK. PI en , . | ; oh x b he V frooiyuktsro ooo r o yur ii or kuro yusuyuyu koo ; ! i and Ba Vi and the points are not working Fig. 6 in on a a a Z b and B ! "o v y V 8 pna a popeeoo fe ЖК eta ТК КАК КК енны nt chn і zh rt I contron E і ш і І І фоні Vern nem VE KY control donya schy ; - ; i x ZD Ki te - i Zh x inve M abbneni po ST i mu: i piuokti i st j ve fony dnyni Kn pip i- «fen OO PIDNOS osenii SA | m xx MAKUHA NAVAANA CANADA ANAAATA KAH KZ : sword y y y y y SHE h s Кк х Х Fo sia zh х Shrot kn; o I MK zhh sch ch sleep that KUM UT Ko pud one nyutdyalchyayunkttt nn ME B Her same Z I Z I Z I 3. yud dilutions of serum Fig. 7 what i t. EOM same I in Not or them aa Met, Suppression of formation J. I 5 juicers F! B Ж Z che ikh i h zv o grad in 4 k EN g ! Z Complete oppression of us yuVYS POYITIRSHYANYA SHOV MU VO x pa i Zhe 5 7 i Zh y" : B Dt rt ru rt rt rok r r u rn kur ke freon unrderde tr rr" kare KI NM Fig. va, Binding of Bara Me to native MAZA-2 o and ssh 4 d 0.30 and K 4 ozh 4 iv s Ve as VOM Ko eOVENM y / He is . 60024 6 8 2 34 8 8 o harem Yam Fig. 8c iv E io a i ; vla" aa Moi, Suppression of posture 41, Suppression of post-planing SYA t h y shk I y y regarding: DON y 4 schen, y y kashenya same ""ai as Z 7 ED : - - Х I pak KO In them ; dati that VL h o i h u uUh echo -« at. Is5O OV nm y: fa sho her PD t Te M o i o shuy 7 ; m TK TD "in EE ttodktete ek kre ik: pekuche rr rkfkkevsseichki svoe rekiresvfnu scha ve ! titi yur 1 ZO - ry V EB EB 4 Ed Me NM - : PIENE dos R ; i.e. Suppression of post-natal SA: PE 3 and Kukya. I Z VIKO tk y ; i i i Z stgNAi сх сх ! and the use is M gavo "a i Z - : "a 1 i -- su Y GK i Z i z 4 i ki OO v. 4 th day od rota - i EZei N: i - i ro! - : more X g pieces and ! i : : rain r. NE t i Uh i zu. 1 kii i Sh»ka i i Hri i skzhddnknu TYA Z OBO Z 4 ron pn V, Ri i i OBOI i ni och, z vy i ti KE: ; of NC. ; OH NO! LLC BOB. BO shi shi she Ge! BO EN OO dec I and her 33rd Her those : ! IN NE : heh and OB OO: BO. that рН ши ши ш g E BOB. BO i she X 1 i hi 1 i KI i N : x i re i i i 1: 1 i i ti : i : I ; IS . shi shi shshn sh Fo m nen shi BUT azh ON i dao r: AND FIGHTS - Z Z Ri 1 BOR Ki ! i EE i i I Z N Ri 3: ri YAN I i IZ i i KU i i her 1 PN: KI : i 1 i: i 5-5 i I: i: nya i i i i i i NE ; this is BO NT NO and Я BO BNO NO 4 Z ZI 3: Ri NOT her! GO Z Ii! her i i I 13 KI: Ii i those N NE: in the present I shi she Domenne un rntrin un BNO SHE - i atria snrchy in unissSH Ii EN Ii IN; x 7: 7 fot tyuttu shik zn gegukeive x Her a 472 "5 85 that Torba errvnereytn x x gu ; And with; 5 2 her that ka da tsu p ZE OBO W x . and MAMO Co. ANTAMABRYA EVNO Fig. 8 go Ebb-event gray honeycomb OWL Berinova pritease mininn-- | | and i nka MNNNNNN! ; E and sh o Mashche. a Monte eeucht en uyuyu Ah eh nzhyuutchnch nki kny eteteieye tn kichiyuye de knchteyeyayayainnny 00 Danya yu Z ZNA ash ponos o pesosechno chno pre lokonntosts, submitted uchadnnknnnachnnnn by Her and her 3rd SHE W kih AACAAA ALANA mA Maky AKULA ASU M CHAYNA AKA yu plchhahat And AHAMAAAAAAA Ahh same channel sus YANNNNN! - OWL. В Z dyllchchyucha tea with fury NnNNNN- and Ann SIVUESE- like SHI 7 SV Ve Fig. 10 in ek eku geneni g KI, th KK check Za -th. slu For tying OR AB MO and Mobo with popshentids shuryacheago MAVRO x DU u U UK U UK KU KK UK U U KT U U UK U UT Ku uk chuk uk : і 1 : to ; 1 чик Я и ж : che pa - she U Loudnynny shKh- ET m t -- ! zhen KEDY Genome - SHCHOMASHVOA chu 5 - dr Ye pe det i! Opa I dyan cutting. VO MAVROA i oyu 5 vl se ; taken yana ХО SuVI Sa i хх и я "its po z fak YAVI SuVIYA Smoked tt рн Kt p t More p i i nn a ny p i nn nn i i i p m a a V r a ! i i V zd ttto ko sttechn o sos endo stvosskh tusrnstknttst eto ns rotettsetevstsnkchnnnnu from 20 40 vo vo zdo TAshchi Ibn Fig. 11 Lectin pathway ZE. че - у б х ! them! Sh- "k "12 "I "V V -y "T from 646 pAV, M! Fig. 12A Kyvsychny way o, : xx OO. i 8-2 i o "ii "A i "V "V "V -i io BYab pa, M! Fig. 128 Alternative way that x ha - Z 3 o. 9 nk: same Z Z Z: wow "TE " "o V "V -Y iosz 6546 tA, MI Fig. 12C 100000 cosvyosp: en 5 MIZH: -ae 5 MKU , i 15 MIZH 5O t 100003 Lyu de za nn Penya - 0005 no b 200 405 BOYU oo time Fig.13 in mkZ X - / coffee VII A a. 8 "o 5 MKG M M OBIVI E : an Intact E and "in" intact 4 (OM5846) F in 10 is o i - shk. in w I p. 1 more shin I V as sko - SCHE - ey still something same V De zheozhk zhk om lk as ЖК du zvez zhh A A HO HM KKOJKOKYU lk lk r o vane nn 2 5 pa 00 what what 0 time Fig/lL4d I. I am I ! K le HPK ACTS 5 LIMITS OF M VOLUMES) about ; tav |ntactni sh I she Ziztezitni i ASV d -V- intact « (0M5846) 2 1069 in i She. ru and ah - ; where E and "o ho zhk he v. legal notice x 7 from Z and B | sh sk and shcha 0.04 feeeeeetenyueteeesoeonh sosen sosoesesosososefose sego sego sesesefres vot s so segoososssenyu ih "83 BO shyu chas ii Fig148v sho sk "SO operated x 403 as "Poevdooperovani sa ! run nn flow kekehuh kto, x Z Fe - s Eh What s u sec xx Z kV khzhunnuyu vo. . with 05 "g? х No» o Z sere shcha i shcha. scha u ze EAZ; ho t | -Ш- zvo, ЗВ with | There is also a wild type of MAO Fig. 15. in Pseudo-operated Vo i - same mya u 5" g it Fo ee g Wild type MASCHEEE i" i same 035 Fig 16 Collagen M/ type in ! front e MT, poevdoopertsvani ha . everything Ov 5 I MABRAYA, pseudosperous -E | koe t a NOYU Fo 15 so also MAO KSOMU about | zh vo zon EE x U zh en I seeenfestnya sx s Y "I that oo : KU sce Ka Ko Gaya her s s 58 U Kozy B F ia Zhe IC 5 "U yah SCHE Group of mice works Fig. 17 TOB det! From the day of 5.5. frannklkky kakinkiny for - x5 ? th MT, povadobperovani -- "Coll. t; uz :i vv zae sh MAVR-?, pseudo-slepovani o -VE- y a VTSHYU Х х " mmavvo ko sho s ev s Ya a u "a Z a "FW nnntruenyuittnnnrtktsensyunortsttotoopeyunoyernpovnsonnkh . In ЗХ sa I I I s «BU 3 ir what KU I in I «E F -? I s, y Ko i y Group of mice tre svia Fig. 18 Interlacen o sikuk Kk Ne Z with fuknncnzhnkzhklannuknky tki 2 kN hg e M. pseudo-suppressed Bo also with the same MAVR-V, poeudosperated Zh. t MAE KOTSIU v. НВ и ; Fo - "Z Doctrine nkne ryn minrok sneyany I g ih Sai do E z "o K Bu I zh "o Z Kz "V SYA S a shchei that so I I -- - dA ke " i u still ; . and in the Group of Muscles zR«ODi0V Fig. 19 interferon gamma - interferon gamma fe shcha rkchknnnnonya "M, povadoovanovan: Z zv for z MABR-E, pseudo-operated ke ot in V ISHO shock che - " CHE ЖЖ in How: "v vnut innnnnsnnryunnnnnnornnnnh . 2 cha Z oo I a s a zay t is ' «i i a I a I as tih ; from them sx and vupa mice AH g, ze 0185 Fig. 20 Obstruction of sleep with red nips, snarky after 7 divuls MY p x "Ж ву funk rental Э X 7 x ; BE. I ve SHE and be CEH and AKA koh za eh llanyi KO slko In m or "I have I y - a In zaepoyetu Fig. 21 The pdrocnproline content in the kidneys, collected 7 days after the nurse's CO. t 135 in what and B in with more. - And puree in the hook 3 ooo them. e Same spria - and "vv z shko b she ZE zhu hi 5 ha is I ssh with "E K- de sey M KU zone eo3 o Fig. 22 The total amount of proteinase in the serum of F in EE in Zh and MT control 0. - sho VO - wo AT KU stu me MEDIC v zh ve ee E sh v zh «v. s-, Mt with the same Ba! In p. оеососососо осо рого кекс сокососок ех осо роюсосососососное КМ ех » ah how a m Groups Fig.23 The total number of beeps excreted in the urine 125 hours! same 5 5 t VO : : y y from 2004 » i M 5 Sho from MAVR i. 0 Za Zhe I va x 7 5 100 shi Z i ! y TM «in Zh z to sk k still z pe « - in U z Gozhpya Fig. 24 Dikmy tive VADA ia KO OH vno dk KK - . sk d OO still MO UN in MOV KK KO OS A M KI HM UKH U M still a OO MK o Okh s u . SS Fr. OO V OO i MOSCOW 0 heh x A OO M KB Z Z HU KO OO V OK he - SUKHO OO xx OO OO VE OK Oh OM KO p V V VO g what fa OH o year ZOZ OO EK 5 V V BO a, OO 3 WHAT ZO EV Z ss g UC po OO OH COKO PO ih AK KK Z zh MK HMK MK MOH OO OH KK Ч ОВ ZA KK щ VO OO KO o o nut VO o M V OO VC v o - s s KV ee OKO VC OK OK o OKO OK FORGING OK KK В ВХ Z s s zhishen ne 0 B . i TOM ZHK ZO OVK OKKO OH MMK odv tiimn KO o pen M VO oo koka s B BV s. in V OH AK in OBU ZKU OO OV OKH OM m ЗХ xx OK M ZO i OK OK HM OK V I do OO M OO a V V BU o UK UA OO OK g OKO On -X Z OO o OO U KE ON M SO OH Oh y i M me OO p. M VOK OO i u what KO V OK o s MO a KB ee MO V OKKO M KK SS. He Z BO Ee OK OK PAM OO VV Z HOM OO. OK KK Kh Fig. 25 inekltvanii man weights, B copper Number of macrophages, RUVO antibodies, shk MI rherchu Fu I Fk UT against MAVR-DU" at. this V r « o ; Ях Ж p: ladies 4 no fueevu I ve o zh WE KNYSHL zi hny Uch wo higher M a MARIA she seekyefmtntnya i e I p Ж t du same a v-o m p . SHO pa and her o U and I and BeYa e zu k o; and I of Group Z Fig. 26 infiltration of macrophages, the correlation is with proteinuria (wild type) r Frya e ' dok I 5 Shk that g OT Хух хх de a KU сх В infiltration of macrophages IMean 25 stained area) Fig. 27A p macrophage infiltration, correlation with proteinuria (MABR-Y 5 o. I OB | same BOB in what i g shova za e a 2 a ke Z 5 oo M sla infiltration of macrophages (Average 5 stained coat) Fig. 27 B Transforming growth factor de Za 7X zhk oon i - MABR M . 7 ch hovyn Z Z V: ooo shk and z 5 kohli o 154 tr r 3 shcha; Vo. ke J that "in "g Groups of Christians. 026 Fig. 28 Tumor necrosis factor al --- i i - fononttottyuuttyuotyunetesnuyuetnny a A Ne « v o MABR o i. is --- x E a B KU zenenokne : che x K u ka zhre0. 0303 Fig. 29 interleukin 6 and fen .e MI control of VoYao and MABR-OU Control is « che ik zh t MAV. and "ka is x s ko Z zo " cho NK) BOOOVA onyuntuyutttnnya tributary of Chi Ko a V KU seg Kk "8 same -h i schyh Mar I Group KN Fig. 30 ApoptoZ Z - Z VO kah still early a. M control k kv I MAR control GU I. still Ye Zh and Mp Zh! zhk to - -- t MAR 2. i: dk zani 5 men BoOVOK- t "DE x x m zo them where" their 2 "I zh che shck h v da Groups kzhvzhnyi ОИ Fig. 31 Patopopenic changes, coloring NeE A V S s o s s g PEK o. o /i s SO s V UK V ZH M PMK OPP i KU SHYH xx what XX s MKK o s s KO V OM M V KK M V Z ВХ V pi i EE o o . Oh s s s oh oh oh OO s s what s o o s 0. 0. s o s pon Control group that Group that atomized Group that received received physiological solution and isotype control MAZR-E TA fig.32 that Apoptase re B dk not NO . 5 Fig. 33 LLC SE she V phonetic e Control, physiologic solution with growth, also iZotylny control Z Z 7 SUK zy fo a z « a MABR-I MA V | in k And we Z I i ! in what ho k i KZ s zho" her their s o oyu - she 7 8 Z Tu sh o z -kh KO she o OK "In my " " her Z - ke cho - Groups tr: 0.5324 preva Fig. 34 TKA we ik o TMA yalsa Sholshch, x that I as a pediment - / Kantolna, fPhysiopogic solution Zh E-: nnnnnnnnnn In nnnnnnnn sho iotiynYah komtyl 5 ah ; NN B vu a « MAER-o TA Kesh 3 - осн - s Ж r z Ж y "de ЖК and в ж : vo ! kah B I still aiv and. : : shi Z shh M what s shcha WHAT t a ; e y Ash U bu i U Ya k still her d zi M I a KU A Eh che soodya Groups of mice zhu TI hz OIV Fig. 35 xi interleukin b zhd i ne aka etc. ki still Vk z -j a 0 voantrolne, diaaleapchniM solution tp u nn i sk Ye I I, h p nIi k x . what is the motive control of them | ZAM di g her and a MA sha mo ozh ; ф 5 Ям эх в SOKU з я я Эх ; Z ИЗ ж и S е и Fo Ж V dunnyastnttetyustskevotertyutttsyuvsststetevsttozoienttssya -Eh ky Z ak I "o s gure MY "Eh schu ЖЖ "U se skh Tsya ke still MU schu xx still cho sa 5 chu Z eh sho Ne cho No o UK z Z ME M Groups of mice zho gu rebdeeo «er«edlMAV Fig. 36 plan of pits and sections of KOVI and Advriamycin-induced nephropathy (inhibition of NAE) of OVT juvenile ptUva, which is attached to the caddy and behind the chest. that is easy to calculate BO MAU - VOMS KO VV with 5 KOVO VC oka -l VEK forging sh 0000000 sh 00000 shcha OO OM OM B oh o VO in OKH o XX p. OO U o. KKU OO MO ME s shsh-- 0 s I M MEM U 0. Z OO OKO OO KK h V a PO oo OO ВХ as OKKO ВХ OKOM ZOM z Х КК MM ОХ КК о claim 5, POV V o not i p i OKKO KK XX Khx U MK MKM dz KO ХК В Za sov TO zh xx oh vlUv x sa oso PK - tyu DOBKOOH : ХК я - s s s MO V o E KO o o V V E KN OA o V OO zh OH OK V o OO V KKZ o p Okh o OVO OH o i OO V s s s OKO o OV Me A OK ZHK MM M KMU KK СХ pe o KV OK KK Ko KAC KK OV s OO KO po He EK p.: OV KK : A ZO OV OEOE OO ! . s s s Z XX ZO OM OM sh-z VOVK VO Z ZHK 5.5 VOK KK o; OK Ve Az sg y sg y og KK MOH KK OK s x OO 5 M OKH HU xx KOVO OKH U vah OKH MO MOB Х ОО M КК ОО ОО ОО ОО MOM OM OO ОК ОО их хх ОО ХК ООН их КМ ОО M Я ОО OA U OK ZU also ZO SO MOH Z KOH M SK OO MOM OMVO ; OH U OKKO ZO sh oo OK still OO M MO 5 KO NK OO i p B 000 shsh,. 0000 Fig. 37 macrofan infiltration Ж ж ок х й ! frekolkyuk uk yuyu mts y sh v o . ЧО з шщ З о / и ШХИ s v ih i i 8. хх ж m sekkyuKu de и шх шх р se и «ke S Ж dao o В их чех чех че че Fig. 38 c Deposition of collagen still in U -k vu. : i Donu emu ma muku kk That's it. - - th 5 C zhk i vo - F : z. eat and ochno Z «y - 1 ee men X Zv sovsvovve, : shchi i «y. еВ х х и х З в в. and o - z cha g dayu che che che oo a JHE U che s : schu cho v Fig. 39 Albumincreatin ratio (ASC in DAM of patients who received (OMOBYAEI g voo dl st ta-weekly picudane e5vo in ; ; Men, roko Patient same eye Patient? i AH F : 7 M x : ee U-n-n "V : Masses , : еВ а за и че за NO Change from the original level Sub-transformed: а: web: реВ ОВ; s rev Change from the original level: я: ре од в: вео ов сечо Fig. 40 has a regu she penenevrnia ban 00 OoBymyMmymMiyM,. : U U x. : ho U h ee GatsENKT KE | Ше ше У х Ше пам 2 щ I Duh ммчечкктт я B шх же -- щ y : х M х K- za» DPasient Z Ж Koko і pi . A. pen pn pi o IN pinni PUT Givtsivnt 4 ' h ich U Z pen nn kdd Z pn finan : Te Pd - Sha : p i oevudttn, : Hzhnu vas s au koni : Ah Uchyuml i teh Uha Positive oment Denyoi 00 Deney? 00 Denyb 00 Deny and 00 Deny chu Fig. 41