Use of alpha-enolase antagonists in the treatment of fibrous diseases

Abstract

The present disclosure relates to the use of an effective amount of an alpha-enolase (enolase-1, ENO-1) antagonist in the manufacture of a medicament for treating a fibrotic disease. The ENO-1 antagonist significantly inhibited body weight loss and lung weight gain, as well as fibrotic lesions and collagen deposition in the lung. The ENO-1 antagonist also significantly reduced cell migration, collagen, and TGF-β secretion in mouse primary pulmonary myofibroblasts.

Description

[Background technology]

[0001] Cross-reference with related applications This application claims priority to U.S. Provisional Application No. 63 / 235,486, filed on 20 August 2021, which is incorporated herein by reference for any purpose.

[0002] Background of the Invention Field of Invention This disclosure relates to the field of fibrous diseases and connective tissue disorders. More specifically, this disclosure relates to the use of alpha-enolase (enolase-1, ENO-1) antagonists for the treatment and / or prevention of fibrous diseases, particularly idiopathic pulmonary fibrosis.

[0003] Background technology Fibrous diseases include the formation of excessive fibrous connective tissue in organs or tissues during repair or reaction processes. These fibrotic diseases include idiopathic pulmonary fibrosis (IPF), pulmonary hypertension, pulmonary fibrosis, emphysema, non-alcoholic steatohepatitis, pancreatic fibrosis, intestinal fibrosis, cardiac fibrosis, myelofibrosis, arthralgia fibrosis, interstitial lung disease, non-specific interstitial pneumonia (NSIP), usual interstitial pneumonia (UIP), endocardial fibrosis, mediastinal fibrosis, retroperitoneal fibrosis, progressive giant fibrosis (complication of coal worker's pneumoconiosis), nephrogenic systemic fibrosis, Crohn's disease, old myocardial infarction, scleroderma / systemic sclerosis, neurofibromatosis, Hermansky-Pudlak syndrome, diabetic nephropathy, renal fibrosis, hypertrophic cardiomyopathy (HCM), hypertension-associated nephropathy, and focal segmental glomerulosclerosis. This includes glomerulosclerosis (FSGS), radiation-induced fibroids, uterine leiomyomas (uterine fibroids), alcoholic liver disease, hepatic steatosis, hepatic fibrosis, cirrhosis, hepatitis C virus (HCV) infection, chronic organ transplant rejection, fibrotic skin conditions, keloid scars, Dupuytren's contracture, Ehlers-Danlos syndrome, bullous epidermolysis dystrophy, oral submucosal fibrosis, and fibroproliferative disorders.

[0004] ENO-1 is a multifunctional protein initially discovered as an important enzyme in the glycolysis pathway. Under normal conditions, ENO-1 is expressed in the cytoplasm. However, it has also been found to be expressed as a plasminogen receptor on the cell surface of many cancer cells, as well as on activated hematopoietic cells such as neutrophils, lymphocytes, and monocytes. Upregulation of the plasminogen receptor protein is known to induce a cascade reaction of the urokinase plasminogen activation system, leading to the degradation of the extracellular matrix. [Overview of the Initiative]

[0005] This disclosure relates to an alpha-enolase (enolase-1, ENO-1) antagonist that targets ENO-1, and its use. The ENO-1 antagonist has an antigen-binding domain that can bind to ENO-1, such as a human ENO-1 antibody, and neutralizes the biological effects of ENO-1. The ENO-1 antagonist can bind to free ENO-1 protein and ENO-1 protein on the cell surface, and has important application prospects in the treatment of fibrous diseases.

[0006] In certain embodiments of this disclosure, the fibrotic disease or disorder may be any condition resulting from abnormal activation or expression of the ENO-1 protein. Examples of such diseases include fibrosis of the liver, intestines, kidneys, skin, epidermis, endothelium, muscles, tendons, cartilage, heart, pancreas, lungs, uterus, nervous system, testes, penis, ovaries, adrenal glands, arteries, veins, colon, intestines (e.g., small intestine), biliary tract, soft tissues (e.g., mediastinum or retroperitoneum), bone marrow, joints, eyes, stomach, or any combination thereof.

[0007] In certain embodiments of this disclosure, fibrotic diseases or disorders include idiopathic pulmonary fibrosis (IPF), pulmonary hypertension, pulmonary fibrosis, emphysema, non-alcoholic steatohepatitis, pancreatic fibrosis, intestinal fibrosis, cardiac fibrosis, myelofibrosis, arthralgia, interstitial lung disease, nonspecific interstitial pneumonia (NSIP), usual interstitial pneumonia (UIP), endocardial fibrosis, mediastinal fibrosis, retroperitoneal fibrosis, progressive giant fibrosis (complication of coal worker's pneumoconiosis), nephrogenic systemic fibrosis, Crohn's disease, old myocardial infarction, scleroderma / systemic sclerosis, neurofibromatosis, Her Mansky-Pudlak syndrome, diabetic nephropathy, renal fibrosis, hypertrophic cardiomyopathy (HCM), hypertension-associated nephropathy, focal segmental glomerulosclerosis (FSGS), radiation-induced fibrosis, uterine leiomyoma (uterine fibroid), alcoholic liver disease, hepatic steatosis, hepatic fibrosis, cirrhosis, hepatitis C virus (HCV) infection, chronic organ transplant rejection, fibrotic skin conditions, keloid scars, Dupuytren's contracture, Ehlers-Danlos syndrome, epidermolysis bullous dystrophy, oral submucosal fibrosis, and fibroproliferative disorders may be mentioned. In preferred embodiments, fibrotic diseases or disorders may include idiopathic pulmonary fibrosis, pulmonary hypertension, emphysema, non-alcoholic steatohepatitis, pancreatic fibrosis, renal fibrosis, intestinal fibrosis, cardiac fibrosis, myelofibrosis, articular fibrosis, or systemic sclerosis.

[0008] In some embodiments of the present disclosure, the ENO-1 antagonist may be an anti-ENO-1 antibody or a binding fragment thereof. In some embodiments of the present disclosure, the ENO-1 antagonist may also be an anti-ENO-1 chimeric antigen receptor (CAR) comprising an antigen-binding domain, a hinge region, a transmembrane domain, and a signaling domain, wherein the antigen-binding domain is at least a portion of an anti-ENO-1 antibody.

[0009] In some embodiments of this disclosure, the antigen-binding domain may include the amino acid sequences shown in SEQ ID NO: 1 (GYTFTSCVMN), SEQ ID NO: 2 (YINPYNDGTKYNEKFKG), SEQ ID NO: 3 (EGFYYGNFDN), SEQ ID NO: 4 (RASENIYSYLT), SEQ ID NO: 5 (NAKTLPE), and SEQ ID NO: 6 (QHHYGTPYT). Preferably, the antibody may include a heavy chain variable domain having three complementary regions: HCDR1 (GYTFTSCVMN: SEQ ID NO: 1), HCDR2 (YINPYNDGTKYNEKFKG: SEQ ID NO: 2), and HCDR3 (EGFYYGNFDN: SEQ ID NO: 3), and a light chain variable domain having three complementary regions: LCDR1 (RASENIYSYLT: SEQ ID NO: 4), LCDR2 (NAKTLPE: SEQ ID NO: 5), and LCDR3 (QHHYGTPYT: SEQ ID NO: 6).

[0010] In other embodiments of the present disclosure, the antigen-binding domain may include the amino acid sequences of SEQ ID NO: 7 (GYTFTSXVMN, where X is any amino acid other than cysteine), SEQ ID NO: 2 (YINPYNDGTKYNEKFKG), SEQ ID NO: 3 (EGFYYGNFDN), SEQ ID NO: 4 (RASENIYSYLT), SEQ ID NO: 5 (NAKTLPE), and SEQ ID NO: 6 (QHHYGTPYT). Preferably, the antibody may include a heavy chain variable domain having three complementary regions: HCDR1(GYTFTSXVMN, where X is any amino acid other than cysteine: SEQ ID NO: 7), HCDR2(YINPYNDGTKYNEKFKG: SEQ ID NO: 2), and HCDR3(EGFYYGNFDN: SEQ ID NO: 3), and a light chain variable domain having three complementary regions: LCDR1(RASENIYSYLT: SEQ ID NO: 4), LCDR2(NAKTLPE: SEQ ID NO: 5), and LCDR3(QHHYGTPYT: SEQ ID NO: 6).

[0011] In some embodiments of the present disclosure, the hinge region may include a CD8 alpha hinge region (TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD: SEQ ID NO: 8), and optionally the transmembrane domain may include a CD8 alpha transmembrane domain (IYIWAPLAGTCGVLLLSLVIT: SEQ ID NO: 9) and / or a CD28 transmembrane domain (FWVLVVVGGVLACYSLLVTVAFIIFWV: SEQ ID NO: 10), and optionally the signal The signaling domain may include CD3 zeta (RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR: SEQ ID NO: 11), and optionally the signaling domain may further include 4-1BB, the intracellular domain of CD28, DAP10, OX40, or a combination thereof, and optionally the anti-ENO-1 CAR may further include a signal peptide, and optionally the signal peptide may include IgG kappa light chain signal peptide, CD8 alpha signal peptide, GM-CSF signal peptide, HSA signal peptide, IgG heavy chain signal peptide, IgG light chain signal peptide, CD33 signal peptide, IL-2 signal peptide, or insulin signal peptide.

[0012] In some embodiments of this disclosure, the anti-ENO-1 CAR may comprise a signal peptide, an anti-ENO-1 antibody, a CD8 alpha hinge region, a CD8 alpha transmembrane region, 4-1BB, and a CD3 zeta.

[0013] In some embodiments of this disclosure, the ENO-1 antagonist may be an immunocomplex that specifically binds to ENO-1 and has the general formula Ab-(LD)m(I), where Ab is an anti-ENO-1 antibody or its binding fragment, L is a linker or direct binding, D is a therapeutic agent or label, and m is an integer from 1 to 12. The antibody may preferably be a monoclonal antibody. The antibody may preferably be a mouse antibody, a human antibody, a chimeric antibody, a humanized antibody, or a fragment thereof.

[0014] In certain embodiments of this disclosure, the therapeutic agent may include antifibrotic agents, immunomodulators, radioisotopes, and toxins. Preferably, the ENO-1 antagonist can be used in combination with at least one therapeutically active agent having known antifibrotic activity, the agent being pirfenidone or a receptor tyrosine kinase inhibitor (RTKI), e.g., nintedanib, sorafenib, and other RTKIs, or an angiotensin II (AT1) receptor blocker, or a CTGF inhibitor, or a TGF-β and BMP activation pathway including activators of the latent TGF-β complex, e.g., MMP2, MMP9, THBS1, or cell surface integrins, TGF3 receptor type I (TGFBRI) or type II (TGFBRII) and their ligands, e.g., TGF3, activin, inhibin, Nodal, anti-mydylerian hormone, GDF, or BMP, or co-coreceptor (I The selection is made from components of SMAD-dependent canonical pathways, including type II receptors, regulatory or inhibitory SMAD proteins, or members of SMAD-independent or non-covalent pathways, including various branches of MAPK signaling, TAK1, Rho-like GTPase signaling pathways, phosphatidylinositol-3 kinase / AKT pathways, and TGF-β-induced EMT processes, or any member of canonical and non-canonical Hedgehog signaling pathways, or WNT pathways, or anti-fibrotic compounds that readily inhibit the Notch pathway, which is prone to affecting TGF-β signaling.

[0015] In some embodiments of the present disclosure, the ENO-1 antagonist may be administered orally, parenterally, buccally, vaginally, rectally, by inhalation, pneumoperitoneum, sublingually, intramuscularly, subcutaneously, topically, intranasally, intraperitoneally, intrathoracically, intravenously, epidurally, intrathecally, or intraventricularly, or by injection into the joint.

[0016] In some embodiments of this disclosure, the subject is a mammal. In one preferred embodiment, the subject is a human.

[0017] In some embodiments of this disclosure, the ENO-1 antagonist may be a nucleic acid, such as DNA or RNA, that is delivered to cells and expressed as an intracellular protein of a peptide. For example, the ENO-1 antagonist may be a nucleic acid that can be transcribed and translated as, for example, an anti-ENO-1 antibody or its binding fragment. Furthermore, the nucleic acid may or may not have a secretory signaling peptide, and therefore the protein or peptide transcribed and translated from the nucleic acid may be secretory, non-secretory, or a combination thereof. In some embodiments of this disclosure, the nucleic acid can be delivered to a target via any known method or medium, such as a viral vector, polymer, or liposome. In some embodiments of this disclosure, the nucleic acid may be substituted with known modified nucleotides, such as pseudo-UTP, 1-Me pseudo-UTP, 5-methoxy-UTP, N1-ethyl pseudo-UTP, 5-methyl-CTP, or N4-acetyl-CTP, to enhance expression efficiency.

[0018] Compared to the prior art, the invention disclosed herein has the following beneficial effects. According to this disclosure, in vivo and in vitro experiments demonstrate the anti-fibrotic and anti-inflammatory effects of ENO-1 antagonists, such as ENO-1 mAb HuL217. That is, ENO-1 antagonists are a potential treatment for fibrotic diseases, such as IPF.

[0019] A pharmaceutically effective amount depends on many factors such as the patient's condition, age, disease state, route of administration, etc., and it will be understood by those skilled in the art that such an effective amount can be determined based on these factors in routine medical practice without performing excessive experiments.

[0020] Other aspects of the invention disclosed herein will become apparent from the following description.

Brief Description of the Drawings

[0021] [Figure 1] It is a diagram showing overexpression of ENO-1 in the lungs of human fibrotic lungs and bleomycin-induced pulmonary fibrosis mouse models. Details are described in Example 2.

[0022] [Figure 2] It is a diagram showing the in vivo antifibrotic effect of HuL217, which suppresses weight loss and lung weight increase in bleomycin-induced pulmonary fibrosis model mice. Details are described in Example 3.

[0023] [Figure 3] It is a diagram showing the in vivo antifibrotic effect of ENO-1 mAb HuL217, which reduces the Ashcroft score and inflammatory score of lung sections in bleomycin-induced pulmonary fibrosis model mice. Details are described in Example 3.

[0024] [Figure 4] It is a diagram showing the in vivo antifibrotic effect of ENO-1 mAb HuL217, which reduces collagen in the lungs and TGF-β in bronchoalveolar lavage fluid from bleomycin-induced pulmonary fibrosis model mice. Details are described in Example 3.

[0025] [Figure 5] It is a diagram showing the in vivo antifibrotic effect of ENO-1 mAb HuL217, which reduces myofibroblasts (alpha-SMA positive) in the lungs from bleomycin-induced pulmonary fibrosis model mice. Details are described in Example 3.

[0026] [Figure 6] This figure shows the in vivo anti-inflammatory effect of ENO-1 mAb HuL217, which reduces monocyte and neutrophil recruitment in the lungs of a bleomycin-induced pulmonary fibrosis model mouse. Further details are described in Example 3.

[0027] [Figure 7] This figure shows the in vitro anti-fibrotic effect of ENO-1 mAb HuL217, which reduces the migration of TGF-β stimulated primary mouse and human lung fibroblasts. Further details are described in Example 4.

[0028] [Figure 8] This figure shows the in vitro anti-fibrotic effect of ENO-1 mAb HuL217, which reduces collagen, TGF-β1, and VEGF secretion in TGF-β1-stimulated primary human normal fibroblasts. Further details are described in Example 4.

[0029] [Figure 9] This figure shows the in vitro anti-fibrotic effect of ENO-1 mAb HuL217, which reduces collagen, TGF-β1, and VEGF secretion in primary human IPF fibroblasts. Further details are described in Example 4. [Modes for carrying out the invention]

[0030] General definition

[0031] Unless otherwise specified, the implementation of this invention utilizes prior art in molecular biology, microbiology, recombinant DNA, and immunology, which are within the scope of the art of the art. Such art is well described in the literature.For example, there are documents such as "Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 1989)", "DNA Cloning, Volumes I and II (D. N. Glover ed., 1985)", "Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987)", "Immobilized Cells And Enzymes (IRL Press, 1986)", "B. Perbal, A Practical Guide To Molecular Cloning (1984)", "the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.)", "Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory)", "Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987)", "Antibodies: A Laboratory Manual, by Harlow and Lane s (Cold Spring Harbor Laboratory Press, 1988)", and "Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986)".

[0032] The terms “antibody” and “immunoglobulin” are used interchangeably in their broadest sense and include monoclonal antibodies (e.g., full-length or intact monoclonal antibodies), polyclonal antibodies, monovalent antibodies, multivalent antibodies, and multispecific antibodies (e.g., bispecific antibodies insofar as they exhibit the desired biological activity), and may also include certain antibody fragments (described in more detail herein). Antibodies may be chimeric, human, humanized, and / or affinity-matured.

[0033] The term "variable" refers to the fact that the sequences of specific portions of the variable domain vary widely among antibodies, and that these portions are used for the binding and specificity of each particular antibody to a particular antigen. However, variability is not evenly distributed throughout the variable domain of an antibody. It is concentrated in three segments, all called complementarity-determining regions (CDRs) or hypervariable regions within the light chain and heavy chain variable domains. The more highly conserved portions of the variable domain are called frameworks (FRs). The native heavy chain and light chain variable domains each consist of four FR regions, most of which take the form of a beta-sheet structure, linked by three CDRs, forming loops that link the beta-sheet structures, and in some cases forming parts of the beta-sheet structure. The CDRs of each chain are held in close proximity by the FR region and, together with the CDR of the other chain, contribute to the formation of the antibody's antigen-binding site (see "Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)"). The constant domain does not directly participate in antibody-antigen binding, but exhibits various effector functions, such as the antibody's involvement in antibody-dependent cytotoxicity.

[0034] Antibodies may be full-length or may contain antibody fragments (or multiple fragments) having antigen-binding moieties, including, but not limited to, Fab, F(ab')2, Fab', F(ab)', Fv, single-chain Fv (scFv), bivalent scFv (bi-scFv), trivalent scFv (tri-scFv), Fd, dAb fragments (e.g., "Ward et al, Nature, 341:544-546 (1989)"), CDR, diabody, triabody, tetrabody, linear antibodies, single-chain antibody molecules, and polyspecific antibodies formed from antibody fragments. Single-chain antibodies or synthetic linkers produced by conjugating antibody fragments using recombinant methods are also included in this invention. (See "Bird et al. Science, 1988, 242:423-426. Huston et al, Proc. Natl. Acad. Sci. USA, 1988.") It is listed as "85:5879-5883".

[0035] Here, “treatment” means a clinical intervention that seeks to alter the natural course of the individual or cell being treated, and can be performed for preventive purposes or during the course of clinicopathological treatment. Desired effects of treatment include prevention of disease onset or recurrence, relief of symptoms, reduction of direct or indirect pathological consequences of the disease, prevention or reduction of inflammation and / or tissue / organ damage, reduction of the rate of disease progression, improvement or mitigation of the disease state, and remission or improved prognosis. In some embodiments, the antibodies of this disclosure are used to delay the onset of a disease or disorder.

[0036] The "individual" or "subject" is a vertebrate. In some embodiments, the vertebrate is a mammal. Examples of mammals include, but are not limited to, agricultural animals (cattle, etc.), sports animals, pets (cats, dogs, horses, etc.), primates, mice, rats, etc. In certain embodiments, the vertebrate is a human.

[0037] "Effective amount" refers to the amount effective in the dosage and duration necessary to achieve the desired therapeutic or prophylactic result. Note that the "effective amount" of the substance / molecule of the present disclosure may vary depending on factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the substance / molecule to elicit the desired response in the individual. Also, the effective amount is the amount at which the therapeutically beneficial effect outweighs the toxic or harmful effects of the substance / molecule. In one embodiment, the effective amount of the anti-ENO-1 antibody is 1 to 1000 mg / kg, preferably 5 to 100 mg / kg, more preferably 10 to 50 mg / kg, for example, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg / kg.

[0038] As used herein, the term "therapeutic agent" refers to a substance that inhibits or blocks the function of cells and / or causes cell destruction. This term includes anti-fibrotic agents, radioisotopes (e.g., 211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 60 C, and radioisotopes of lutetium-177, strontium-89, and samarium ( 153 Sm), immunomodulatory agents, and toxins, and examples of toxins include toxins such as low molecular weight toxins or enzyme-active toxins derived from bacteria, fungi, plants, or animals, as well as synthetic analogs and derivatives thereof.

[0039] The terms "fibrotic state," "fibroproliferative state," "fibrotic disease," "fibroproliferative disorder," "fibrotic disorder," and "fibroproliferative disorder" are interchangeable and refer to conditions, diseases, or disorders characterized by dysregulated proliferation or activity of fibroblasts and / or pathological or excessive accumulation of collagenous tissue. Typically, any such disease, disorder, or condition can be treated with the administration of compounds having antifibrotic effects. Fibrotic diseases include, but are not limited to, idiopathic pulmonary fibrosis (IPF), pulmonary hypertension, pulmonary fibrosis, emphysema, non-alcoholic steatohepatitis, pancreatic fibrosis, intestinal fibrosis, cardiac fibrosis, myelofibrosis, arthralgia, interstitial lung disease, nonspecific interstitial pneumonia (NSIP), usual interstitial pneumonia (UIP), endocardial fibrosis, mediastinal fibrosis, retroperitoneal fibrosis, progressive giant fibrosis (complication of coal worker's pneumoconiosis), nephrogenic systemic fibrosis, Crohn's disease, old myocardial infarction, scleroderma / systemic sclerosis, neurofibromatosis, and Herm Examples include Ansky-Pudlak syndrome, diabetic nephropathy, renal fibrosis, hypertrophic cardiomyopathy (HCM), hypertension-related nephropathy, focal segmental glomerulosclerosis (FSGS), radiation-induced fibrosis, uterine leiomyoma (uterine fibroid), alcoholic liver disease, hepatic steatosis, hepatic fibrosis, cirrhosis, hepatitis C virus (HCV) infection, chronic organ transplant rejection, fibrotic skin conditions, keloid scarring, Dupuytren's contracture, Ehlers-Danlos syndrome, bullous epidermolysis dystrophy, oral submucosal fibrosis, or fibroproliferative disorders.

[0040] The term "idiopathic pulmonary fibrosis" as used herein refers to a chronic, progressive, and usually fatal lung disease that is thought to be the result of a chronic inflammatory process.

[0041] The term "antifibrotic agent" as used herein refers to a substance known to have antifibrotic activity. This term includes pirfenidone or receptor tyrosine kinase inhibitors (RTKIs), such as nintedanib, sorafenib, and other RTKIs, or angiotensin II (AT1) receptor blockers, or CTGF inhibitors, or TGF-β and BMP activation pathways including activators of the latent TGF-β complex, such as MMP2, MMP9, THBS1, or cell surface integrins, TGF3 receptor type I (TGFBRI) or type II (TGFBRII) and their ligands, such as TGF3, activin, inhibin, nodal, anti-mydylerian hormone, GDF, or BMP, or co-receptors (type III receptor). The selection is made from components of SMAD-dependent canonical pathways, including regulatory or inhibitory SMAD proteins (also known as SMAD receptors), or members of SMAD-independent or non-covalent pathways, including various branches of MAPK signaling, TAK1, Rho-like GTPase signaling pathways, phosphatidylinositol-3 kinase / AKT pathways, and TGF-β-induced EMT processes, or any member of canonical and non-canonical Hedgehog signaling pathways, or WNT pathways, or anti-fibrotic compounds that readily inhibit the Notch pathway, which is prone to affecting TGF-β signaling.

[0042] The pharmaceuticals disclosed herein may be applied topically or systemically. Furthermore, the pharmaceuticals disclosed herein may be administered in combination or with cofactors. If the pharmaceuticals disclosed herein are substances normally present at the target site, they may be administered in an amount sufficient to restore normal levels, or in an amount that raises levels above normal levels at the target site.

[0043] The pharmaceutical product of this disclosure may be supplied to a target site from an exogenous source, or it may be produced in vivo by cells of the target site or by cells of the same organism as the target site.

[0044] The pharmaceuticals disclosed herein may be any physiologically appropriate formulations. These may be administered to a living organism by injection, topically, by inhalation, orally, or by any other effective means.

[0045] Furthermore, the pharmaceuticals and methods described above for suppressing or inhibiting the formation and maintenance of excessive fibrosis may also be used to suppress or inhibit the formation of inappropriate fibrosis. For example, these may be used to treat or prevent conditions occurring in the liver, kidneys, lungs, heart and pericardium, eyes, skin, mouth, pancreas, gastrointestinal tract, brain, breast, bone marrow, bone, genitourinary tract, tumors, or wounds.

[0046] In general, these may be used to treat or prevent fibrosis resulting from conditions including, but are not limited to, rheumatoid arthritis, lupus, pathogenic fibrosis, fibrotic diseases, fibrotic lesions such as those formed after Schistosoma japonicum infection, radiation injury, autoimmune diseases, Lyme disease, chemotherapy-induced fibrosis, HIV or infection-induced focal segmental sclerosis, spinal surgery scarring resulting in incomplete back syndrome, post-abdominal adhesion scarring, and fibrocystic formation.

[0047] Embodiments of this disclosure relate to antibody-drug conjugates containing ENO-1 antibodies and their use in the treatment of fibrotic diseases. ENO-1 is a multifunctional protein that has been found to be expressed on the cell surface of many cancer cells as a plasminogen receptor and on activated hematopoietic cells such as neutrophils, lymphocytes, and monocytes. Therefore, ADCs based on antibodies against ENO-1 may be useful as diagnostic and / or therapeutic agents.

[0048] However, rapid internalization of therapeutic antibodies and a lack of ADCC activity can lead to antibody ineffectiveness or resistance. Therefore, it is necessary to enhance the therapeutic effect of anti-ENO-1 therapeutic agents. One approach is to conjugate a payload with the anti-ENO-1 antibody (i.e., an antibody-drug conjugate).

[0049] In embodiments of this disclosure, the anti-ENO-1 antibody or its conjugated fragment may be conjugated to a drug, diagnostic agent, or therapeutic agent. Accordingly, the term “antibody-drug conjugate (ADC)” as used herein may mean the antibody portion (which may be the whole antibody or its conjugated fragment) conjugated to a payload (which may be a drug, diagnostic agent, or therapeutic agent). An exemplary ADC is described in WO2021 / 228044A1, which is incorporated herein by reference in its entirety.

[0050] Methods for treating fibrous diseases

[0051] This disclosure provides a method for treating fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF). This method generally involves administering an effective amount of an alpha-enolase (enolase-1, ENO-1) antagonist to a subject in need.

[0052] In some embodiments, the ENO-1 antagonist used in the method may include, but is not limited to, any of the following: (1) Anti-ENO-1 antibody or its conjugated fragment. (2) An anti-ENO-1 chimeric antigen receptor (CAR) comprising an antigen-binding domain, a hinge region, a transmembrane domain, and a signaling domain, wherein the antigen-binding domain is at least a portion of an anti-ENO-1 antibody. (3) An immune complex having the general formula Ab-(LD)m (I), where Ab is an anti-ENO-1 antibody or its binding fragment, L is a linker or direct binding agent, D is a therapeutic agent or label, and m is an integer from 1 to 12, which specifically binds to ENO-1. (4) The nucleic acid of an anti-ENO-1 antibody, its binding fragment, or anti-ENO-1 CAR, which is delivered into a cell and expressed as an anti-ENO-1 antibody or its binding fragment or as an anti-ENO-1 CAR within the cell.

[0053] The embodiments of this disclosure will be illustrated using the following specific examples. Those skilled in the art will understand that these examples are for illustrative purposes only and can be otherwise modified and adapted without departing from the scope of this disclosure. [Examples]

[0054] Example 1. Preparation of anti-ENO-1 antibody

[0055] In embodiments of this disclosure, a general method for producing an anti-ENO-1 antibody involves obtaining a hybridoma that produces a monoclonal antibody against ENO-1. Methods for producing monoclonal antibodies are known in the art and will not be described in detail here. Briefly, mice are subjected to antigen (ENO-1) challenge with a suitable adjuvant. Subsequently, spleen cells are collected from the immunized mice and fused with the hybridoma. The ability of positive clones to bind to the ENO-1 antigen may be identified using any known method, such as ELISA. In one embodiment, the anti-ENO-1 antibody is HuL217.

[0056] Antibody-drug conjugates can specifically target ENO-1. These ADCs can use any antibody that specifically binds to ENO-1. For example, the ADC of the claimed invention may use a mouse or humanized anti-ENO-1 antibody, or its scFv or Fab fragment. An exemplary anti-ENO-1 antibody, such as HuL217, may comprise a heavy chain variable domain having three complementary regions: HCDR1(GYTFTSCVMN: SEQ ID NO: 1), HCDR2(YINPYNDGTKYNEKFKG: SEQ ID NO: 2), and HCDR3(EGFYYGNFDN: SEQ ID NO: 3), and a light chain variable domain having three complementary regions: LCDR1(RASENIYSYLT: SEQ ID NO: 4), LCDR2(NAKTLPE: SEQ ID NO: 5), and LCDR3(QHHYGTPYT: SEQ ID NO: 6). Another exemplary anti-ENO-1 antibody may comprise a heavy chain variable domain having three complementary regions: HCDR1(GYTFTSXVMN, where X is any amino acid other than cysteine: SEQ ID NO: 7), HCDR2(YINPYNDGTKYNEKFKG: SEQ ID NO: 2), and HCDR3(EGFYYGNFDN: SEQ ID NO: 3), and a light chain variable domain having three complementary regions: LCDR1(RASENIYSYLT: SEQ ID NO: 4), LCDR2(NAKTLPE: SEQ ID NO: 5), and LCDR3(QHHYGTPYT: SEQ ID NO: 6).

[0057] In embodiments of this disclosure, the antibody may be a mouse antibody. Alternatively, the antibody may be a chimeric antibody (e.g., a human constant region conjugated to a mouse variable region), a humanized antibody (e.g., a mouse CDR grafted onto a framework region of human immunoglobulin), or a complete human antibody.

[0058] Monoclonal antibodies may be humanized by obtaining a CDR sequence from a hybridoma, cloning the CDR sequence to a human framework sequence, and producing a humanized antibody. Common methods known in the art can be used to identify the CDR sequence. The CDR region in this invention is identified by the Kabat numbering scheme. First, anti-ENO-1 hybridomas (e.g., mouse hybridomas) were prepared. Such hybridomas may be prepared using standard protocols for producing monoclonal antibodies. Next, the total RNA of the hybridoma was isolated using, for example, TRIzol® reagent. Then, for example, a first-strand cDNA synthesis kit (Superscript III) and oligonucleotides (dT) were used. 20 cDNA was synthesized from total RNA using primers or Ig-3' constant region primers.

[0059] Next, the heavy and light chain variable regions of immunoglobulin genes were cloned from cDNA. For example, the VH and VL variable regions of anti-ENO-1mAb were amplified by PCR using a mouse Ig-5' primer set (Novagen) from mouse hybridoma cDNA. The PCR product may be directly cloned into a suitable vector (e.g., pJET1.2 vector) using the CloneJet® PCR cloning kit (Ferments). The pJET1.2 vector contains lethal insertions, and only if the target gene is cloned into this lethal region will it withstand the selection criteria. This facilitates the selection of recombinant colonies. Finally, the target clones were selected from the recombinant colonies, the DNA of these clones was isolated, and their nucleotide sequences were analyzed. The nucleotide sequences of immunoglobulins (IG) can be analyzed on the international ImMunoGeneTics information system (IGMT) website.

[0060] Antibody expression and purification

[0061] For antibody production, isolated clones may be expressed in any suitable cells. As an example, F293 cells (Life Technologies) were transfected with an anti-ENO-1 mAb expression plasmid and cultured for 7 days. The anti-ENO-1 antibody was purified from the culture medium using a protein A affinity column (GE). Protein concentrations may be measured using a Bio-Rad protein assay kit and analyzed by 12% SDS-PAGE according to known procedures in the art or manufacturer's instructions.

[0062] Example 2. Overexpression of ENO-1 in human and mouse fibrotic lung

[0063] In this example, ENO-1 immunohistochemistry (IHC) staining was used to determine whether ENO-1 was abnormally expressed in fibrotic lung. Formalin-fixed, paraffin-embedded (FFPE) lung tissue samples, shown in Figure 1A, were purchased from commercial suppliers. One normal human lung FFPE tissue section was obtained from BioChain Institute (Newark, California, USA) and US Biomax (Darwood, Maryland, USA). Three human fibrotic lung FFPE slides were obtained from OriGene Technologies (Rockville, Maryland, USA). ENO-1 expression was elevated in human fibrotic lung but not in normal lung. Quantitative results are shown in Figure 1B.

[0064] Bleomycin-induced pulmonary fibrosis in C57BL / 6 mice is the most common experimental model of idiopathic pulmonary fibrosis, a fibrotic disease in humans. Male C57BL / 6 mice aged 7-9 weeks were administered a single intratracheal dose of bleomycin (3 mg / kg). After 21 days, lungs were collected for ENO-1 IHC staining. Figure 1C shows that ENO-1 was overexpressed in the bleomycin group (BLM) compared to the placebo group. Quantitative results are shown in Figure 1D.

[0065] Example 3. IPF disease model using ENO-1 antibody

[0066] HuL217 was evaluated in C57BL / 6 mice with bleomycin-induced pulmonary fibrosis. Male C57BL / 6 mice aged 7-9 weeks received a single intratracheal dose of bleomycin (3 mg / kg). The mice were randomly divided into three groups: 4 mice in the placebo group and 7 mice in either the bleomycin + vehicle group or the bleomycin + HuL217 group. Day 0 was designated as the bleomycin challenge day. Mice in the HuL217 group received intravenous injections on days 1, 7, 13, and 19. The results showed that HuL217 administration attenuated weight loss and lung weight increase (Figure 2), Ashcroft and inflammation scores (Figure 3), and lung collagen content and TGF-β levels in bronchial alveolar lavage fluid (BALF) (Figure 4) compared to the bleomycin group. Furthermore, HuL217 treatment reduced the accumulation of myofibroblasts in the lungs of bleomycin-inducible mice (Figure 5). To investigate the anti-inflammatory effects of HuL217, flow cytometry was used to analyze the recruitment of monocytes and neutrophils to the lungs in BALF. Figure 6 shows that HuL217 (intravenously administered 2 hours before bleomycin injection) reduced the recruitment of monocytes and neutrophils to the lungs of bleomycin-inducible mice on day 4.

[0067] Example 4. In vitro effect of ENO-1 antibody against pulmonary fibroblasts

[0068] Primary mouse lung fibroblasts were isolated from male C57BL / 6 mice, and primary human lung fibroblasts were purchased from Lonza, Inc. (London, UK). Both cell lines were treated with TGF-β to induce migration. Figure 7 shows that HuL217 dose-dependently reduced the migratory ability of primary lung fibroblasts treated with TGF-β. The in vitro antifibrotic effect of HuL217 was further investigated using primary human lung fibroblasts isolated from normal (NHLF) or IPF patients (DHLF-IPF). HuL217 was able to dose-dependently reduce the secretion of collagen, TGF-β1, and VEGF in TGF-β stimulated NHLF (Figure 8) or DHLF-IPF (Figure 9).

[0069] In summary, in bleomycin-treated mice, HuL217 significantly attenuated weight loss and lung weight gain, as well as pulmonary fibrotic lesions and collagen deposition. Elevations of TGF-β and monocytes were also reduced in BALF. HuL217 was able to significantly reduce cell migration, collagen, and TGF-β secretion in primary mouse lung myofibroblasts.

[0070] Unless otherwise defined, all technical and scientific terms and abbreviations used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. Any composition, method, kit, and means of conveying information that are similar or equivalent to those described herein may be used to carry out the present invention, and preferred compositions, methods, kits, and means of conveying information are disclosed herein.

[0071] All references cited herein are incorporated herein by reference to the extent permitted by law. The consideration of these references is intended merely to summarize the claims made by their authors. No reference (or any part of a reference) is recognized as prior art. The applicant reserves the right to challenge the accuracy and appropriateness of the cited documents.

Claims

1. The use of an effective amount of anti-alpha-enolase (enolase-1, ENO-1) antibody or its conjugated fragment for the manufacture of a pharmaceutical product for the treatment of pulmonary fibrosis disease, The anti-ENO-1 antibody or its conjugated fragment is HCDR1 (GYTFTSCVMN: Sequence ID 1), HCDR2 (YINPYNDGTKYNEKFKG: Sequence ID No. 2), and A heavy chain variable domain having three complementary regions including HCDR3 (EGFYYGNFDN: SEQ ID NO: 3), LCDR1 (RASENIYSYLT: Sequence ID 4), LCDR2 (NAKTLPE: Sequence ID No. 5), and Including a light chain variable domain having three complementary regions including LCDR3 (QHHYGTPYT: SEQ ID NO: 6), The aforementioned use.

2. The anti-ENO-1 antibody or its conjugated fragment is It forms an immune complex that specifically binds to ENO-1, represented by the general formula Ab-(L-D)m(I). The use according to claim 1, wherein Ab is an anti-ENO-1 antibody or a conjugated fragment thereof, L is a linker or direct conjugate, D is a therapeutic agent or label, and m is an integer from 1 to 12.

3. The use according to claim 2, wherein the antibody is a mouse antibody, a human antibody, a chimeric antibody, a humanized antibody, or an antibody fragment thereof.

4. The use according to claim 2, wherein the therapeutic agent comprises an antifibrotic agent, an immunomodulator, a radioisotope, and a toxin.

5. The use according to claim 4, wherein the antifibrotic agent comprises pirfenidone or a receptor tyrosine kinase inhibitor (RTKI), or an angiotensin II (AT1) receptor blocker, or a CTGF inhibitor, or an antifibrotic compound that inhibits the TGF-β and BMP activation pathway, or a component of the SMAD-dependent canonical pathway, or a member of the SMAD-independent or non-covalent pathway.

6. The use according to claim 2, wherein the label includes a diagnostic reagent or an imaging reagent.

7. The use according to claim 1, wherein the anti-ENO-1 antibody or its conjugated fragment is delivered via a viral vector, polymer, and / or liposome.

8. The use according to claim 1, wherein the pulmonary fibrosis disease is selected from the group consisting of idiopathic pulmonary fibrosis (IPF), pulmonary hypertension, pulmonary fibrosis, interstitial lung disease, nonspecific interstitial pneumonia (NSIP), and usual interstitial pneumonia (UIP).

9. A composition for treating pulmonary fibrosis, The composition comprises an anti-ENO-1 antibody or a conjugated fragment thereof. The anti-ENO-1 antibody or its conjugated fragment is HCDR1 (GYTFTSCVMN: Sequence ID 1), HCDR2 (YINPYNDGTKYNEKFKG: Sequence ID No. 2), and A heavy chain variable domain having three complementary regions including HCDR3 (EGFYYGNFDN: SEQ ID NO: 3), LCDR1 (RASENIYSYLT: Sequence ID 4), LCDR2 (NAKTLPE: Sequence ID No. 5), and Including a light chain variable domain having three complementary regions including LCDR3 (QHHYGTPYT: SEQ ID NO: 6), The aforementioned composition.

10. The composition according to claim 9, wherein the pulmonary fibrosis disease is selected from the group consisting of idiopathic pulmonary fibrosis (IPF), pulmonary hypertension, pulmonary fibrosis, interstitial lung disease, nonspecific interstitial pneumonia (NSIP), and conventional interstitial pneumonia (UIP).

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