Anti-ephrin-b2 blocking antibodies for treatment of fibrotic diseases

Abstract

To provide a novel treatment method for treating organ fibrosis in a subject.SOLUTION: A method for treating organ fibrosis in a subject comprises administering to a subject a therapeutically effective amount of one or more antibodies or antigen binding fragments thereof that bind to and block soluble ephrin-B2 ectodomain.SELECTED DRAWING: None

Description

[Technical field]

[0001] Priority claim This application is a continuation of U.S. Provisional Patent Application No. 62 / 785,8 filed on December 28, 2018. No. 73, the entire contents of which are incorporated herein by reference. Be absorbed.

[0002] Technical Field Described herein is a soluble ephrin-B2 (sEphrin-B2) ectodomain. Organ fibrosis can be prevented using antibodies or antigen-binding fragments that bind to and block Methods and compositions for treating [Background technology]

[0003] The ability of organs to regenerate after injury declines with age. In the elderly, chronic tissue damage is An abnormal wound healing response characterized by the development of scar tissue or fibrosis and subsequent organ failure results. Summary of the Invention [Problem to be solved by the invention]

[0004] A maladaptive wound healing response to chronic tissue injury leads to organ fibrosis. Fibrosis is Excessive extracellular matrix (ECM) deposition and tissue remodeling by activated myofibroblasts This is accompanied by loss of proper tissue structure and organ function. The furin-B2 pathway is a major driver of myofibroblast activation 16 As shown herein In addition to being associated with the development of fibrosis, this pathway specifically mediates the expression of neutralizing antibodies. A strategy to directly block ephrin-B2 using Thus, anti-ephrin-B2 antibodies can be specifically targeted for therapeutic intervention. The body is intended to treat fibrosis, such as pulmonary fibrosis in patients with idiopathic pulmonary fibrosis (IPF). At the time of diagnosis, pulmonary fibrosis is by definition established. Anti-ephrin-B2 antibodies have been shown to be effective in treating early and late stage disease. Progressive pulmonary fibrosis, including pulmonary fibrosis, as well as fibrosis in other organs (e.g., systemic fibrosis / It can be used to treat conditions such as scleroderma, or liver fibrosis or cirrhosis. [Means for solving the problem]

[0005] Thus, provided herein is a method for treating organ fibrosis in a subject, comprising administering to the subject a The method comprises identifying a subject having organ fibrosis and detecting soluble ephrin-B2 and / or soluble ephrin-B3 in a subject having organ fibrosis. One or more antibodies or their antigen-binding flags that bind to and block the target domain. The method includes administering a therapeutically effective amount of a ment.

[0006] In some embodiments, the organ fibrosis is pulmonary (e.g., idiopathic pulmonary fibrosis), cutaneous, renal, fibrosis, liver fibrosis or cirrhosis, systemic sclerosis or desmoplastic tumors.

[0007] In some embodiments, treatment involves reducing fibrosis and / or restoring normal function of an organ. brings about a recovery or approach of

[0008] In some embodiments, the subject has pulmonary fibrosis, and the therapeutically effective amount reduces pulmonary fibrosis and and improved lung function, e.g. improved oxygenation and / or normalization of forced vital capacity (FVC) results.

[0009] In some embodiments, the subject has pulmonary fibrosis, e.g., as determined by chest x-ray or chest coagulation. Fibrosis on computed tomography (CT) or high-resolution CT (HRCT) scans The patient has a paroxysmal pattern of bronchial and bibasilar expiratory crackles.

[0010] In some embodiments, the subject has systemic sclerosis (SSc), e.g., the skin of the fingers. Thickening, fingertip lesions, telangiectasias, abnormal nailfold capillaries, interstitial lung disease or pulmonary arterial disease He has hypertension, Raynaud's phenomenon and SSc-associated autoantibodies.

[0011] In some embodiments, the subject has liver fibrosis or cirrhosis, e.g., by biopsy or Imaging, e.g., ultrasound (US), computed tomography (CT), fibros Fibrosis has been detected in can or magnetic resonance imaging (MRI).

[0012] In some embodiments, the antibody is clone B11 or 2B1 antibody.

[0013] In some embodiments, the antibody is a monoclonal chimeric, deimmunized or humanized antibody. It is a body.

[0014] Unless otherwise specified, all technical and scientific terms used herein refer to the The methods and materials have the same meaning as commonly understood by one of ordinary skill in the art to which they pertain. Other suitable methods and techniques described herein and known in the art for use in the present invention may also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, Tori and other references mentioned herein are incorporated by reference in their entireties. In case of conflict, the present specification, including definitions, will control.

[0015] Other features and advantages of the invention are set forth in the following detailed description, drawings, and claims. This will be clear from the surrounding area. [Brief description of the drawings]

[0016] [Figure 1A-B] Ephrin-B2 is elevated in IPF fibroblasts. (A, B) Ephrin-B2 mRNA and protein expression levels in human lung fibroblasts from healthy control donors (n=3) and individuals with IPF (n=3). [Figure 2A-C] Fibroblast-specific ephrin-B2 KO mice are protected from bleomycin-induced pulmonary fibrosis. (A) Masson's trichrome staining of lung sections from control wild-type (WT) and fibroblast-specific ephrin-B2 KO mice 14 days after PBS or bleomycin (BLM) challenge. (B) Hydroxyproline content in WT (left lane) and KO (right lane). (C) α-SMA and collagen type I protein expression. n=6 mice for all groups. [Figure 3A-C] Ephrin-B2 ectodomain is shed into the alveolar space upon lung injury. (A) Western blot showing ephrin-B2 expression levels in whole lung homogenates from WT mice harvested 14 days after PBS or bleomycin challenge. Arrows indicate the appearance of a low molecular weight band (approximately 50 kDa). (B) Western blot showing cleaved sEphrin-B2 levels in bronchoalveolar lavage fluid from PBS (left bar) and bleomycin (right bar) challenged mice after 14 days of treatment. (C) Concentrations of sEphrin-B2 as determined by ephrin-B2 ELISA in bronchoalveolar lavage fluid from WT mice at different time points in the model. [Figure 4A-F]Soluble ephrin-B2 ectodomain is sufficient to cause myofibroblast formation and tissue fibrosis. (A) Domain structure of full-length ephrin-B2 protein and recombinant soluble ephrin-B2 ectodomain fused to Fc. (B) Effect of ephrin-B2-Fc or IgG-Fc control on α-SMA and type I collagen protein expression in mouse lung fibroblasts. (C) WT mice were treated for 14 days by daily subcutaneous injection of either pre-clustered ephrin-B2-Fc (n=6) or IgG-Fc as control (n=6). H&E and Masson's trichrome staining are shown. (D-F) Quantification of skin thickness (D) and hydroxyproline (E), dermal fibrosis markers (left bar: IgG-Fc, right bar: ephrin-B2-Fc), as well as western blot showing α-SMA and type I collagen levels in skin (F). [Diagram 5] Effect of control (left bar) or anti-ephrin-B2 neutralizing antibody (B11, right bar) on TGF-β-induced α-SMA expression in pulmonary fibrosis. [Figure 6] Treatment strategy with anti-ephrin-B2 blocking antibodies. WT mice are treated with bleomycin or saline by intratracheal instillation as well as anti-ephrin-B2 blocking antibodies or control antibodies as indicated. [Figure 7A-C] Anti-ephrin-B2 antibody prevents myofibroblast activation and bleomycin-induced pulmonary fibrosis in mice. (A) Representative images of Masson's trichrome staining (type I collagen) of lung sections from mice treated with anti-ephrin-B2 antibody (B11 clone) or control IgG2a antibody 21 days after PBS or bleomycin challenge (from n=6 mice per group). (B) Hydroxyproline content (collagen levels) measured in mouse lungs (n=6 for all groups). (C) α-SMA mRNA expression assessed by real-time PCR in whole lung homogenates (n=6 for all groups). [Figure 8A-C]Anti-ephrin-B2 antibody prevents TGF-β-induced myofibroblast activation and fibrotic phenotype of lung fibroblasts from patients with IPF. (A) Concentration of sEphrin-B2 determined by ELISA in conditioned medium of primary lung fibroblasts from control (healthy) donors (n=5) and individuals with IPF (n=5). (B) Effect of anti-ephrin-B2 antibody (B11 clone) on TGF-β-induced α-SMA protein expression in primary lung fibroblasts from control donors (n=3) using GAPDH as a loading control. n=3 for all groups. Anti-ephrin-B2 antibody prevents TGF-β-induced α-SMA protein expression (P<0.05). (C) Effect of anti-ephrin-B2 antibodies (B11 and 2B1 clones) on α-SMA protein expression and phosphorylated SMAD3 in primary lung fibroblasts from control donors (n=3) and individuals with IPF (n=3), using GAPDH as a loading control. n=3 for all groups. [Figure 9A-B] sEphrin-B2 levels are elevated in BAL and plasma from IPF patients. (A) Concentrations of sEphrin-B2 in bronchoalveolar lavage fluid (A) and plasma (B) from control donors (n=30) and IPF patients (n=30) assessed by ELISA. **P<0.01 [Figure 10] sEphrin-B2 levels correlate with clinical prognosis in patients with IPF. Elevated plasma sEphrin-B2 is associated with increased mortality in patients with IPF (n=30, *P<0.03). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Identification of novel therapeutic strategies aimed at reducing tissue fibrosis and promoting regeneration of damaged tissue. The determination of fibrosis is a major unmet clinical need in regenerative medicine. Revealing a novel molecular mechanism and investigating the role of ADAM10-soluble ephrin in scar-forming myofibroblasts Targeting the IL-B2 pathway reverses established pulmonary fibrosis and restores organ function This discovery has implications for various human fibrotic diseases, such as idiopathic pulmonary fibrosis (IDF). New drugs for the treatment of systemic sclerosis (scleroderma), liver cirrhosis, renal fibrosis and desmoplastic tumors Potential therapeutic targets have been identified.

[0018] Targeting the ADAM10-sEphrin-B2 pathway in pulmonary fibrosis Chronic lung disease is one of the leading causes of death in the United States. Idiopathic pulmonary fibrosis (IPF) is a A common condition that consistently results in progressive decline in lung function and causes significant morbidity and mortality. It is a lung disease. 1~3 Patients with IPF suffer from a terminal disease characterized by progressive lung scarring (fibrosis). suffer from an irreversible and ultimately fatal interstitial lung disease that permanently impairs the ability to breathe 4、 5 Recent epidemiological studies have demonstrated that IPF affects many more people than previously recognized. It has been shown to have 6~8 The prevalence of IPF in the United States has risen to 100,000 in recent years. Estimates range from 10-60 cases per 000 people. 6 This is in the United States Approximately 130,000 people are diagnosed with IPF in the United States, and 34,000 people die each year. There is a possibility that some people may develop 9 This indicates that the prognosis for IPF is poor. Median time from diagnosis is 2 to 5 years 10 Current treatments are primarily based on two recent developments: Injected antifibrotic drugs (pirfenidone and nintedanib) or some IPF Moderately slows the decline in lung function in patients 11、12 but halts disease progression or relies on symptomatic treatment that cannot be reversed.

[0019] IPF is associated with unacceptably high morbidity and mortality. Developments include improved understanding of the biological processes involved in the pathogenesis of pulmonary fibrosis and the application of these A more complete identification of the molecular mediators that control the process will be required. Fibroblast activation plays a key role in the progressive scarring that underlies the development and progression of pulmonary fibrosis Step 3、13 Myofibroblasts promote collagen synthesis and α-SMA (α-sm The expression of oth muscle actin was increased, which remodeled the ECM. Confers a ringing hypercontractile phenotype 14 As a result, myofibroblast activity Targeting the molecular pathways that cause the progression of IPF may be a promising therapeutic strategy. Have 3、13~15 .

[0020] The ADAM10-sEphrin-B2 pathway has recently been implicated in patients with IPF and in mouse models of pulmonary fibrosis. Identified as a key driver of myofibroblast activation in DELTA 16 Efri -B2 is a transmembrane ligand highly expressed in quiescent lung fibroblasts 16、17 However, its profibrotic effect is regulated by an activation step that occurs upon lung injury. The study showed that after lung injury, the external dosing of full-length ephrin-B2 in quiescent lung fibroblasts was increased The main one is the disintegrin and metalloproteinase ADAM10. It is proteolytically cleaved to produce the biologically active molecule, soluble ephrin-B2 (sEphrin- Once released, ephrin-B2 is released into the autocrine / Activating EphB4 receptor signaling in a paracrine manner to inhibit dormant This study demonstrated that s-ephrin produces profibrotic signaling in fibroblasts with EphB2 / EphB4 receptor signaling transforms quiescent fibroblasts into activated myofibroblasts and that this is sufficient to cause tissue fibrosis in mice. Furthermore, genetic deletion of ephrin-B2 specifically in lung fibroblasts has been shown to suppress Mice receiving the antibody showed significant protection from bleomycin-induced pulmonary fibrosis. Administration of sEphrin-B2 antibodies reverses established fibrosis. The strategy of blocking the production of ephrin-B2 by directly blocking ephrin-B2 may be a promising strategy to prevent fibrosis. It serves as a novel therapeutic strategy.

[0021] Treatment method As shown herein, soluble ephrin-B2 inhibits scar-forming muscle in the lung and skin. is sufficient to cause fibroblast activation. Pathologies associated with myofibroblast activation The mechanisms underlying this are thought to be conserved between organs (e.g., Rockey et al., N Engl J Med. 2015 Mar 19;372(12):1138-49). Thus, myofibroblast fibril Targeting pathways involved in maintaining the fibrotic state may provide a global anti-fibrotic strategy. Represents fibrotic targets. Zeisberg and Kalluri, Am J Physiol Cell Physiol. 2013 Feb 1;30 See also 4(3):C216-25.

[0022] The methods described herein are directed to treating organ fibrosis, such as pulmonary (e.g., idiopathic pulmonary fibrosis), skin for the treatment of renal fibrosis, liver fibrosis or cirrhosis, systemic sclerosis and desmoplastic tumors. Generally, the method includes a method for producing a soluble ephrin-B2 as described herein. A therapeutically effective amount of an antibody that binds to and blocks the ectodomain is administered to a subject in need of such treatment. The antibody can be administered to a subject determined to have or be in need of the antibody. Sterically hinder the binding of antibodies (e.g., clone B11) and / or soluble ephrinB2 The clone may be harmful (eg, clone 2B1).

[0023] As used in this context, "treat" means to ameliorate at least one symptom of organ fibrosis. Organ fibrosis often results in scarring and thickening of tissue and loss of function. Treatment may result in a reduction in fibrosis and restoration or activation of normal organ function. For example, to treat lung-related conditions, Administering a therapeutically effective amount of the compounds described herein reduces pulmonary fibrosis and improves pulmonary function. Improved lung function, e.g. improved oxygenation and / or normalization of forced vital capacity (FVC) It is likely.

[0024] The method may be used in any subject with organ fibrosis. Methods for identifying or diagnosing IPF are known in the art, e.g., , Raghu et al., Am J Respir Crit Care Med. 2018 Sep 1;198(5):e44-e68 and Martin ez et al., Lancet Respir Med. 2017 Jan;5(1):61-71, van den Hooge for scleroderma n et al., Arthritis Rheum. 2013 Nov;65(11):2737-47 and liver fibrosis and cirrhosis For details, see Lurie et al., World J Gastroenterol. 2015 Nov 7;21(41):11567-83 and L See i et al., Cancer Biol Med. 2018 May; 15(2): 124-136.

[0025] An "effective amount" is an amount sufficient to effect beneficial or desired results. A therapeutic amount is an amount that achieves the desired therapeutic effect. This amount is the same as a prophylactically effective amount. A prophylactically effective amount may be any amount that is sufficient to prevent the onset of a disease or disease symptoms. The amount of the compound that is needed to treat a disease or condition when administered according to a given treatment protocol. Prevent, delay or reduce the risk of developing the disease or symptoms associated with the disease or condition or otherwise sufficient to result in amelioration of an approved characteristic of a disease or condition. It may also refer to the amount of Ephrin-B2 antibody. An effective amount refers to one or more administrations, applications or A therapeutically effective amount (i.e., an effective dose) of a therapeutic compound may be administered in any one or more doses. Depending on the therapeutic compound, the compositions may be administered from one or more times per day to once or twice a week. may be administered once up to multiple times, e.g., once every other day. Those skilled in the art will appreciate that certain factors, e.g. Including but not limited to, the severity of the disease or disorder, previous treatments, overall health and and / or age and the presence of other diseases may affect the dosage and It will be appreciated that the timing of administration may be influenced. Treatment of a subject with the therapeutic compounds described herein can include a single treatment or a series of treatments. good.

[0026] Dosage, toxicity, and therapeutic efficacy of therapeutic compounds can be determined in cell cultures or experimental animals, e.g. For example, LD50 (the dose lethal to 50% of the population) and ED50 (the dose lethal to 50% of the population) The amount of the compound that is effective for the treatment of a patient can be determined by standard pharmaceutical procedures to determine a therapeutically effective dose. The dose ratio between the therapeutic and non-therapeutic effects is the therapeutic index and can be expressed as the ratio LD50 / ED50. Compounds that exhibit high therapeutic indices are preferred. Compounds that exhibit toxic side effects are However, it is possible to use other drugs that may be useful in reducing side effects by minimizing potential damage to uninfected cells. Attention has been given to the design of delivery systems that target such compounds to the affected tissue site to reduce We should pay attention to this.

[0027] Data obtained from cell culture assays and animal studies are not supported for use in humans. A wide range of doses can be formulated. The dose of such compounds can be adjusted to minimize toxicity. Dosages preferably lie within a range of circulating concentrations that include little or no ED50. The amount of the active ingredient may vary within this range depending on the dosage form and the route of administration used. For any compound used in the method, the therapeutically effective dose is initially estimated from cell culture assays. Doses can be determined in animal models based on IC50s (i.e., Achieve a circulating plasma concentration range that includes the concentration of the test compound that achieves half-maximal inhibition of symptoms. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography. It can be measured by

[0028] sEphrinB2 (sEphrinB2) ANTIBODIES - PHARMACEUTICAL COMPOSITIONS AND METHODS OF ADMINISTRATION Binding of ephrin-B2 to EphB receptors is mediated by highly conserved portions of the ephrin-B2 ectodomain. The crystal structure has recently been solved (Toth et al., Dev C ell. 2001 Jul;1(1):83-92; Qin et al., J Biol Chem. 2010 Jan 1;285(1):644-54; Him anen et al., Nature. 2001 Dec 20-27;414(6866):933-8). We have The ephrin-B2 ectodomain, which is necessary for activation, is shed upon lung injury. and the soluble ectodomain are biologically active in lung fibroblasts and bind to EphB receptors. We found that ephrin-B can bind to and activate signal transduction. 2 Ectodomain-mediated EphB receptor activation induces myofibroblast activation, Disrupting protein-protein interactions may provide antifibrotic properties for the treatment of organ fibrosis. There is a possibility that it may have a medical application as a therapeutic. One approach to this is to use blocking antibodies against the ephrin-B2 ectodomain, It neutralizes the binding and activation of EphB receptors (i.e., EphB3 and EphB4). Highly specific ephrin-binding domains that inhibit receptor signaling B2 blocking antibodies are being developed 26 Thus, the method includes the step of: and inhibiting receptor signaling by administering a therapeutically effective amount of an antibody or antigen-binding portion thereof to The method may include administering a composition comprising the

[0029] The methods described herein include administering to the patient a pharmaceutical composition comprising an sEphrin-B2 antibody as an active ingredient. As used herein, the term "antibody" refers to an immunoglobulin molecule or an antibody thereof. Examples of antigen-binding portions of immunoglobulin molecules include F(ab) and F(ab')2 fragments, which retain antigen-binding ability. Polyclonal, monoclonal, recombinant, chimeric, deimmunized or humanized, fully human, It may be a non-human (e.g., murine) or single chain antibody. , antibodies have effector functions and can fix complement. Antibodies or antibody flags The modifications include any post-translational modifications known in the art and commonly applied to antibodies. provided that the modified antibody or fragment is human or It maintains the binding specificity to mouse ephrin B2. Modifications include PEGylation and phosphorylation. , methylation, acetylation, ubiquitination, nitrosylation, glycosylation, ADP-ribosylation Alternatively, or in addition, the antibody or fragment may be Further, a detectable label that can be used to detect binding in an immunoassay is provided. Labels that can be used include radioactive labels, fluorophores, chemiluminescent labels, and the like. Labels, enzyme labels (e.g., alkaline phosphatase or horseradish peroxidase) In some embodiments, the antibody may be a nucleotide, a nucleotide sequence ... The antibody has reduced or no ability to bind to Fc receptors. For example, the antibody It may be an isotype or subtype, fragment or other variant. This does not support binding to Fc receptors, e.g., mutated or deleted Fc receptors. In addition to the ephrinB2 antibodies described above, other antibodies may be generated. Methods for producing ribozymes and fragments thereof are known in the art, see, for example, Harlow et. al., editors, Antibodies: A Laboratory Manual (1988); Goding, Monoclonal Antibodies dies: Principles and Practice, (NY Academic Press 1983); Howard and Kaser, Mak ing and Using Antibodies: A Practical Handbook (CRC Press; 1st edition, Dec 13, 2006); Kontermann and Dubel, Antibody Engineering Volume 1 (Springer Protocols) (Springer; 2nd ed., May 21, 2010); Lo, Antibody Engineering: Methods and Protoco ls (Methods in Molecular Biology) (Humana Press; Nov 10, 2010); and Dubel, Handb ook of Therapeutic Antibodies: Technologies, Emerging Developments and Approved Therapeutics, (Wiley-VCH; 1 edition September 7, 2010). The sequence of genotype B2 is available from GenBank accession numbers NM_004093.3 (nucleic acid) and N An exemplary sequence of the full-length human ephrin B2 precursor is shown in Table 1. The columns are as follows:

[0030] [ka]

[0031] In some embodiments, the EphrinB2 ectodomain comprises amino acid 2 of SEQ ID NO:1. Contains or consists of 9 to 165 (bold above).

[0032] A pharmaceutical composition typically includes a pharma- ceutically acceptable carrier. A "pharmaceutical acceptable carrier" is a diluent, saline, solvent, or dispersion medium that is compatible with pharmaceutical administration. Supplementary activity includes, but is not limited to, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. A photoactive compound may be incorporated into the composition.

[0033] A pharmaceutical composition is typically formulated to be compatible with its intended route of administration. Examples of routes include parenteral, e.g., intravenous, intradermal, subcutaneous, intraperitoneal, oral (e.g., inhalation, nasal, These include intracavitary, transdermal (topical), transmucosal and rectal administration.

[0034] Methods for formulating suitable pharmaceutical compositions are known in the art and are described, for example, in Remington: Science and Practice of Pharmacy, 21st ed., 2005 and the series Drugs and the Pharmacology See the book "The Mechanical Sciences: a Series of Textbooks and Monographs" (Dekker, NY). For example, solutions or suspensions used for parenteral, intradermal or subcutaneous application may be components, sterile diluents, such as water for injection, saline solution, fixed oils, polyethylene glycols, Glycerin, propylene glycol or other synthetic solvents, antibacterial agents such as benzyl alcohol or methylparaben, antioxidants such as ascorbic acid or sodium bisulfite chelating agents, e.g., ethylenediaminetetraacetic acid; buffers, e.g., acetates, citrates or or phosphate and an osmolality adjuster, such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral preparations are packaged in ampoules, disposable syringes or vials made of glass or plastic. It may be packaged in multi-dose vials.

[0035] Pharmaceutical compositions suitable for injectable use can be prepared by the extemporaneous preparation of sterile injectable solutions or dispersions. The composition may comprise sterile aqueous solutions (if water soluble) or dispersions and sterile powders for the preparation of the pharmaceutical composition. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS) In all cases, the composition must be sterile and syringable. It should be fluid enough to be easily absorbed. It should be stable under the conditions of manufacture and storage. and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier may be, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, etc.), Solvents, including but not limited to liquid polyethylene glycols and liquid polyethylene glycols, and suitable mixtures thereof. Alternatively, it may be a dispersion medium. Suitable fluidity may be achieved, for example, by coating, e.g. lecithin. by using dispersants, maintaining the required particle size, and by using surfactants. Prevention of the action of microorganisms can be achieved by the use of various antibacterial and antifungal agents, e.g., paraben. This is achieved by chlorobutanol, phenol, ascorbic acid and thimerosal. In many cases, isotonic agents, such as sugars, polyalcohols, for example mannitol, sorbitol, It is preferable that the composition contains teflon, sodium chloride, etc. The absorption of the injectable composition can be prolonged by The composition may include agents that delay absorption, such as aluminum monostearate and gelatin. This can be achieved by including

[0036] Sterile injectable solutions may contain the active compound in combination with one or a combination of ingredients as enumerated above. The required amount can be mixed with a suitable solvent and then sterilized by filtration, if necessary. Dispersions may be prepared in a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. The sterile powder for the preparation of a sterile injectable solution is prepared by incorporating the active compound into the powder. In the case of the powder, the preferred methods of preparation are vacuum drying and freeze drying, which results in an activated A powder of the mixture and any additional desired ingredients from a previously sterile filtered solution thereof is obtained. can be.

[0037] Oral compositions generally include an inert diluent or an edible carrier. The active compound may be incorporated with excipients and formulated into tablets, troches, or capsules, e.g. The oral composition may also be used as a mouthwash. The composition may be formulated with a fluid carrier for the administration of pharma- ceutical compatible binding agents and / or azido groups. The pharmaceutical composition may be included as part of the composition. The lozenges and the like may contain the following ingredients: binders, such as microcrystalline cellulose, gum tragacanth or or gelatin, excipients such as starch or lactose, disintegrants such as alginic acid , Primogel or corn starch, lubricants such as magnesium stearate Sium or Sterotes; lubricants, e.g. colloidal silicon dioxide; sweeteners, e.g. For example, sucrose or saccharin or flavoring agents such as peppermint, methyl salicylate may contain any of the following compounds: .

[0038] For administration by inhalation, the compounds are administered in a suitable propellant, e.g. a gas, e.g. carbon dioxide. in the form of an aerosol spray from a pressurized container or dispenser or nebulizer containing Such a method may be described in U.S. Pat. No. 6,468,798. This includes those that:

[0039] Systemic administration of the therapeutic compounds described herein can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, appropriate permeation media should be used to penetrate the barrier to be permeated. A penetrant is used in the formulation. Such penetrants are generally known in the art, and include, for example, For transmucosal administration, surfactants, bile salts and fusidic acid derivatives are included. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. The active compound may be formulated into an ointment, salve, gel or other formulation, as is generally known in the art. is formulated into a cream.

[0040] The pharmaceutical compositions may also be formulated as suppositories (e.g., in a conventional suppository base, such as cocoa) for rectal delivery. They may also be prepared in the form of retention enemas (using butter and other glycerides).

[0041] In one embodiment, the therapeutic compound is a compound that protects the therapeutic compound from rapid elimination from the body. With a carrier, the composition may be administered in a controlled release formulation, including, for example, implants and microencapsulated delivery systems. The composition is formulated with biodegradable biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, etc. Polyesters, polyglycolic acid, collagen, polyorthoesters and polylactic acid may also be used. Such formulations may be prepared using standard techniques or may be prepared, for example, by Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (with monoclonal antibodies against cell antigens) are also available commercially. (including liposomes targeted to selected cells) may also be used as pharma- ceutical acceptable carriers. These may be used in a manner known to those skilled in the art, for example, in U.S. Pat. They can be prepared as described in US Pat. No. 4,811.

[0042] The pharmaceutical compositions may be included in a container, pack, or dispenser together with instructions for administration. Thus, for example, the methods described herein may include Also included are devices, such as inhalers, that contain the sEphrinB2 antibodies used in the methods. EXAMPLES

[0043] The present invention is further described in the following examples, which are incorporated herein by reference in their entirety. It is not intended to limit the scope.

[0044] Ephrin-B2 is elevated in IPF fibroblasts. We previously reported elevated expression of genes related to fibroblast migration and activation in the lungs of patients with is shown in 18 Putative genes regulating activation and migration in IPF fibroblasts To identify, we analyzed gene expression in pulmonary fibrosis isolated from individuals with IPF versus controls. Publicly available microarrays were used to compare the results with those of healthy lung fibroblasts, which were used as controls. Analyzing the data set 19、20 , a gene that encodes the transmembrane protein ephrin-B2 We found that its offspring, EFNB2, was significantly increased in IPF lung fibroblasts. EFNB2 (Gene Expression Omnibus accession number GSE17 twenty four) 19 encodes the transmembrane protein ephrin-B2, which binds to the surface of adjacent cells It belongs to a family of ephrin ligands that bind to Eph receptors in 21 Ligand The ephrin family is structurally composed of phosphatidylinositol-linked ephrin-A ligands. (ephrin-A1-6) and transmembrane ephrin-B ligands (ephrin-B1-3) Classified as 17、21 Both ephrin-A and -B ligands bind to the surface of neighboring cells. Binds to Eph receptors and initiates biochemical signaling 21 All ephrin-A and Among the ephrin-B ligands, ephrin-B2 is the most highly expressed ephrin in lung fibroblasts. Ligand and expression is upregulated in lung fibroblasts from patients with IPF 19 . I We also found that expression of ephrin-B2, but not other members of the ephrin family of ligands, Expression of IL-1 in pulmonary IPF fibroblasts as demonstrated by mRNA and protein analysis The results confirmed that the levels of pulmonary fibrosis in isolated pulmonary fibrosis patients were significantly higher than those in control subjects (Figure 1A, B).

[0045] Fibroblast-specific ephrin-B2-deficient mice are protected from bleomycin-induced pulmonary fibrosis We next investigated whether fibroblast ephrin-B2 is required for the development of fibrosis in vivo. Overall, mice lacking ephrin-B2 exhibited cardiovascular events during mid-gestation. We investigated the condition in collagen-expressing cells, e.g., fibroblasts, because they die when their body is deficient in collagen. We generated mice that were selectively deficient in Efnb2. Mice carrying Efnb2 (Efnb2 loxP / loxP mice) were cultured in Col1 a2 (collagen, type I, alpha 2) mouse promoter Mice expressing tamoxifen-inducible Cre recombinase driven by Col1a2 -CreERT mice). homozygous for the b2 allele and hemizygous for the Col1a2-Cre transgene The offspring were syngeneic (Efnb2loxP / loxP;Col1a2-CreERT mice). Tamoxifen treatment of fibroblasts caused the deletion of Efnb2 and E The creation of fnb2 conditional knockout mice was induced. Littermates treated with ephrin-B2 alone were used as controls. Stan blots showed that lung fibroblasts from ephrin-B2 KO mice were significantly higher than those from control mice. Our study demonstrated that expression of β-lactamase was significantly lower in extracts derived from the lineage. Fibroblast-specific ephrin-B2 knockout mice showed histological (mass) changes in collagen accumulation. Intrichrome staining), biochemical (hydroxyproline level for collagen content) ) and molecular (type I collagen and α-SMA, markers of myofibroblast differentiation) evaluation. The titer of 100 mg / kg / day reduced the onset of bleomycin-induced pulmonary fibrosis compared to wild-type (WT) mice. It was shown to exhibit significant protection (Figures 2A-C).

[0046] Ephrin-B2 ectodomain is shed during lung injury. Although α-di did not increase the expression of full-length transmembrane ephrin-B2 (approximately 60 kDa) in control lungs, We found that this resulted in the production of a low molecular weight band (approximately 50 kDa) that was not seen in the previous study ( (Figure 3A, arrow indicates 50 kDa band). Ephrin-B ligands shed their ectodomains. To release active proteins via 22-25 We report that bleomycin injury is associated with Proteolytic cleavage of ephrin-B2 results in ephrin-B2, referred to herein as sEphrin-B2. This leads to the generation of a soluble 50 kDa form of B2, which may contribute to the pathogenesis of pulmonary fibrosis. Our results suggest that ectodomain-specific anti-ephrin-B2 monoclonal antibodies Western blot and enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies and acellular leukocytes from bronchoalveolar lavage (BAL) fluid of WT mice after bleomycin challenge. The results showed that sEphrin-B2 expression in the cell supernatant was significantly increased (Fig. 3B, C). These results indicate that the ephrin-B2 ectodomain is shed following lung injury.

[0047] Soluble ephrin-B2 ectodomain induces myofibroblast activation and tissue fibrosis ephrin-B2 directly functions as a profibrotic mediator To test whether this is the case, we transfected fibroblasts with recombinant ephrin-B2 ectodomain-F c, which replaces the transmembrane and C-terminal domains of the full-length ephrin-B2 protein. The ectodomain of ephrin-B2 is fused to the Fc domain of the pre-clamp complex (Figure 4A). Treatment of primary mouse lung fibroblasts with activated ephrin-B2-Fc resulted in a 100% increase in the fibroblast population compared with control I. Significantly increased α-SMA and type I collagen protein expression compared to glucagon-Fc treatment. This supports a direct profibrotic effect (Fig. 4B). Preclustered ephrin-B2-Fc (100 μg / kg) or control IgG-Fc was administered subcutaneously daily for two weeks, and the skin thickness, hydroxyproline content and α - Significant increase in dermal fibrosis as assessed by increased expression of SMA and type I collagen These data suggest that ephrin-B2 ectodomain is involved in the regulation of the ephrin-B2 signaling pathway. is sufficient to cause myofibroblast activation and tissue fibrosis in vivo It has been shown that:

[0048] Therapeutic antibodies against ephrin-B2 for the treatment of pulmonary fibrosis in IPF. Our study demonstrated that subcutaneous injection of ephrin-B2 ectodomain induces myofibroblast activation. This demonstrated that the expression of α-aminobutyric acid in mice was sufficient to induce tissue fibrosis in vivo. Taken together, our results suggest that therapeutic inhibition of sEphrin-B2 signaling may be an effective treatment for represents a novel strategy to reduce pulmonary fibrosis by preventing myofibroblast activation. It has been shown that blocking ephrin-B2 signaling may be effective in treating Therapeutic strategies have previously been developed for cancer treatment. 26、27 However, its anti-fibrotic effect is had not been investigated until now.

[0049] Activation of EphB4 receptor by sEphrin-B2 ectodomain induces myofibroblast activation Therefore, disrupting this protein-protein interaction could be a promising treatment for IPF. This may have potential medical applications as an anti-fibrotic treatment for the treatment of epidermal growth factor receptor β (ESR)-induced fibrosis. One approach to inhibit delivery would be to develop a blocking antibody against the ephrin-B2 ectodomain. This neutralizes EphB4 receptor binding and activation. Neutralizing ephrin-B2 binding to the EphB4 receptor using a display library We identified a potent anti-ephrin-B2 antibody (clone B11) that 26 Recent research has melanoma and breast cancer 26、27 and xenograft models 26 In preclinical models B11 has been identified as a powerful research tool in

[0050] Blockade of sEphrin-B2 by B11 neutralizing antibody prevents TGF-β-induced myofibroblast formation To investigate the therapeutic efficacy of ephrin-B2 blocking antibodies in vitro, we B11 In the presence or absence of anti-ephrin-B2 blocking antibody (100 μg / mL = 3 μM) α-SM in primary cultured human lung fibroblasts treated with TGF-β for 48 hours in As shown in Figure 5, B11 pretreatment significantly increased myofibroblast activation in the lungs. Significantly reduces TGF-β-induced α-SMA expression in fibroblasts.

[0051] Blockade of sEphrin-B2 with B11 neutralizing antibody reverses pulmonary fibrosis in a mouse model Based on the above findings, we conclude that therapeutic inhibition of sEphrin-B2 by neutralizing antibodies is We hypothesized that inhibiting myofibroblast activation in vivo may be able to treat pulmonary fibrosis. To investigate the therapeutic efficacy of sEphrin-B2 blocking antibodies in vivo, we Ephrin-B2 blocking antibody (clone B11) inhibits pulmonary fibrosis in an isin-induced pulmonary fibrosis model In this mouse model of pulmonary fibrosis, we investigated the ability of C57Bl / 6 mice to reverse the disease. The rats developed robust pulmonary fibrosis 21 days after bleomycin challenge. Male and female mice were used to address sex-based differences. Sicor Pharmaceuticals) by standard methods in our laboratory. Mice were administered intratracheally (it). A sublethal dose of 1.2 units / kg was used. This is sufficient to induce pulmonary fibrosis without causing mortality.

[0052] In this “therapeutic strategy,” ephrin-B2 blocking antibodies were administered to bleomycin challenge Dosing will begin on day 14 and the duration of the study will continue through day 21. For this study, ephrin- B2 blocking antibodies were administered twice weekly in 0.2 ml of PBS until a total dose of 20 mg / kg was reached. Control C57Bl mice were injected intravenously with a control IgG2a antibody (Clone). 10 mice per group. The timing of bleomycin and ephrin-B2 blocking antibody administration is shown in Figure 6. vinegar.

[0053] Blinded histological analysis demonstrated no bleomycin in mice receiving a control IgG2a antibody. Pulmonary parenchymal fibrosis developed 21 days after isin challenge was induced by ephrin-B2 blocking antibody (CL1). The results showed that the inflammatory response was alleviated in mice treated with chlorpromazine (Fig. 7A). The pulmonary fibrosis model was associated with a significant decrease in lung hydroxyproline levels (Fig. 7B). To gain insight into the mechanism of action of ephrin-B2 blocking antibodies in delta-D, we used qPCR Therefore, we evaluated myofibroblast formation in vivo. In vitro studies have shown that it inhibits TGF-β-induced α-SMA expression in blastoma cells. Therapeutic treatment of mice with ephrin-B2 blocking antibodies reduced the expression of α-SMA. It has been shown to reverse established pulmonary fibrosis by administering We show that the activated cell state of fibroblasts is regulated by ephrin-B2 signaling. is doing.

[0054] Statistical Analysis. Differences in all other outcomes were analyzed by randomized block ANOVA as described above. Results were tested for statistical significance using the mean ± SEM. P<0.05 was considered significant for all comparisons.

[0055] Blockade of sEphrin-B2 with a neutralizing antibody inhibits the activity of human lung fibroblasts from patients with IPF To determine the relevance of our study to human disease, we ADAM10-s expression in fibroblasts isolated from the lungs of individuals with and healthy controls We investigated the role of phospho-B2 signaling. IPF lung fibroblasts were compared with normal in vitro had substantially higher concentrations of sEphrin-B2 in the culture medium compared to lung fibroblasts. (Figure 8A), suggesting that this pathway may be activated in human disease and may be useful for therapeutic intervention. The authors demonstrate that ephrin-B2 blocking antibodies can be targeted to the tumor. To characterize this, we first investigated the TGF-β-induced inflammatory responses in primary cultured human lung fibroblasts. We investigated the effect of the B11 ephrin-B2 antibody on myofibroblast activation induced by IFN-γ. As shown in 8B, anti-ephrin-B2 antibody suppressed TGF-α expression in primary cultured healthy lung fibroblasts. We then demonstrated that anti-ephrin-B2 antibodies prevent IP α-SMA protein expression. To investigate whether the activated phenotype of fibrotic lung fibroblasts isolated from patients with F. As shown in Figure 8C, α-SMA significantly increased the expression of α-SMA in IPF fibroblasts compared to control healthy fibroblasts. Ephrin-B2 was elevated in IPF fibroblasts by anti-ephrin-B2 antibody (clone B11) at 48 Treatment with time significantly reduced α-SMA levels (Figure 8C), which was consistent with the anti-ephrin-B2 treatment. Treatment directly down-regulates profibrogenic mechanisms in fibroblasts from patients with IPF Furthermore, IPF fibroblasts were treated with a second anti-ephrin-B2 antibody (clone 2B 1), which binds to the ephrin-B2 ectodomain as previously shown, but not to the Ep As shown in Figure 8C, the anti-ephrin-B2 antibody ( Treatment of fibrotic IPF fibroblasts with clone 2B1) was comparable to that of clone B11. Notably, anti-ephrin-B2 treatment reduced α-SMA levels in While examining the molecular pathways involved, we found that the B11 ephrin-B2 antibody inhibited the proliferation and proliferation of healthy fibroblasts. Compared with IPF fibroblasts, expression of ribonuclease, a surrogate marker of the canonical TGF-β pathway, was These results suggest that B 11The antifibrotic effect of ephrin-B2 antibodies is not mediated by regulation of the canonical TGF-β pathway. In contrast, the 2B1 ephrinB2 antibody did not inhibit IPF fibroblasts. down-regulated phosphorylated SMAD3 levels, indicating that the anti-fibrotic effect of this antibody is related to classical TG. This indicates that the direct regulation of the F-β pathway is involved. Both 2B1 ephrin-B2 antibodies, despite their likely different mechanisms of action, It has antifibrotic activity in human IPF fibroblasts.

[0056] sEphrin-B2 levels in plasma and bronchoalveolar lavage fluid in patients with IPF The natural history of IPF is highly variable and disease progression varies among individual patients. Speed ​​is difficult to predict 28 Clinical, histopathological and radiological analyses were consistent with IPF. can predict mortality in patients with 10 , can predict disease progression There are no clinically applicable biomarkers. Blood Biomarkers in IPF are being investigated in the hope of improving prediction of disease progression. 29~32 . epithelium Biomarkers of injury, e.g. KL-6 33、34 and biomarkers of endothelial activation, For example, VEGF 35 or VCAM-1 36、37 predicts poor survival in IPF Although it has been found that myofibroblast activation in IPF is predictive, no biomarkers have been reported. Our results suggest that sEphrin-B2 levels are a predictor of IPF. Bronchoalveolar lavage fluid from 16 individuals was compared with samples from 8 healthy volunteers. We then showed that the concentration of IL-1 in patients with IPF was significantly increased compared to those with IPF (Figure 9A). Plasma ephrin-B2 concentrations were compared in 30 individuals and 30 gender-matched nonsmoking controls. In comparison, ephrin-B was significantly higher in individuals with IPF compared to samples from controls. We observed significantly higher plasma concentrations of 2 (Figure 9B). Ephrin-B2 may serve as a novel myofibroblast prognostic biomarker in IPF It has been shown that

[0057] Elevated plasma s-ephrin-B2 levels are associated with increased mortality in patients with IPF. To determine the association of our biomarkers with the progression of IPF, we Whether plasma s-ephrin-B2 levels correlate with disease severity and prognosis in IPF As shown in Figure 11, the plasma levels in patients with IPF were Our data suggest that higher plasma s-ephrin-B2 levels at diagnosis correlate with clinical prognosis. Patients with a baseline of 100 mg / kg / day will experience a rapid decline in lung function, which may lead to patient death. It has been shown that

[0058] References

[0059] [Table 1-1]

[0060] [Table 1-2]

[0061] [Table 1-3]

[0062] Other embodiments The present invention has been described in conjunction with its detailed description, and the foregoing description is for illustrative purposes only. It is not intended to limit the scope of the invention, the scope of which is set forth in the appended claims. It should be understood that other aspects, advantages, and modifications are set forth in the following claims. It is within the range.

Claims

1. 1. A method of treating organ fibrosis in a subject, comprising: Identifying a subject with organ fibrosis; and One or more antibodies that bind to and block the soluble ephrin-B2 ectodomain. or an antigen-binding fragment thereof. The method comprising:

2. The organ fibrosis is selected from the group consisting of lung (e.g., idiopathic pulmonary fibrosis), skin, kidney, liver, or 2. The method of claim 1, wherein the disease is cirrhosis, systemic sclerosis, or desmoplastic tumor.

3. The treatment reduces fibrosis and / or restores or approaches normal function of the organ. The method of claim 1 ,

4. The subject has pulmonary fibrosis, and the therapeutically effective amount reduces pulmonary fibrosis and improves lung function, e.g.

2. The method of claim 1, which results in improved oxygenation, for example.

5. The method of claim 1 , wherein the subject has pulmonary fibrosis.

6. The subject is examined by chest x-ray or chest computed tomography (CT) or high resolution C The patient had a pattern of fibrosis on T (HRCT) scan and bibasilar expiratory crackles. The method according to claim 5 .

7. 10. The method of claim 1, wherein the subject has systemic sclerosis.

8. The subject has symptoms such as finger skin thickening, fingertip lesions, telangiectasia, abnormal nailfold capillaries, interstitial lung Claims: Having pulmonary arterial hypertension, Raynaud's phenomenon and SSc-associated autoantibodies Item 7. The method according to item 7.

9. 10. The method of claim 1, wherein the subject has liver fibrosis or cirrhosis.

10. The subject may be examined by biopsy or imaging, e.g. ultrasound (US), computed tomography, (CT), FibroScanning or Magnetic Resonance Imaging (MRI) The method of claim 9, wherein a disease is detected.

11. The method according to any one of claims 1 to 10, wherein the antibody is clone B11 antibody.

12. The method according to any one of claims 1 to 11, wherein the antibody is clone 2B1 antibody.

13. The antibody according to claim 1, wherein the antibody is a monoclonal, chimeric, deimmunized or humanized antibody.

13. The method according to any one of claims 12 to 13.

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