Use of alpha-enolase antagonists for the treatment of angiogenesis-related diseases - Patents.com

Abstract

A method for treating angiogenesis-associated diseases is provided, the method comprising administering to a subject in need thereof an effective amount of an alpha-enolase (enolase-1, ENO-1) antagonist.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 303,421, filed January 26, 2022, which is incorporated by reference herein for all purposes.

[0002] 2. Background of the Invention FIELD OF THEINVENTION The present disclosure relates to the medical field of angiogenesis. More specifically, the present disclosure relates to the use of alpha-enolase (enolase-1, ENO-1) antagonists for inhibiting angiogenesis and treating and / or preventing angiogenesis-related diseases, particularly age-related macular degeneration and diabetic retinopathy. [Background technology]

[0003] Angiogenesis is the formation of new capillaries from pre-existing blood vessels. When dysregulated, neovascularization contributes to many malignant, ischemic, inflammatory, infectious, and immune diseases. These angiogenesis-related diseases include, but are not limited to, cancer, vascular neoplasms and malformations (angiofibromas, arteriovenous malformations, hemangiomatosis), cardiovascular diseases, pulmonary vascular diseases and central nervous system diseases (atherosclerosis, vascular adhesions, vascular dementia, restenosis / reperfusion injury, pulmonary fibrosis, Alzheimer's disease), ophthalmic diseases (corneal neovascular disease, retinal neovascular disease, retinal angiomatous proliferation, polypoidal choroidal vasculopathy, neovascular age-related macular degeneration, diabetic retinopathy, diabetic macular edema, neovascular glaucoma, ischemic retinopathy, retrolental fibroplasia, or retinopathy of prematurity), or chronic inflammatory diseases (Crohn's disease, diabetes, psoriasis, pyogenic granuloma, periodontitis, rheumatoid arthritis, systemic sclerosis), etc.

[0004] Angiogenesis is mediated by a complex multistep process consisting of a series of cellular events that lead to the formation of new blood vessels. Angiogenic factors such as vascular endothelial growth factors (VEGF) induce increased proliferation and migration of vascular endothelial cells. In order for endothelial cells to invade the interstitium of adjacent tissues, proteolysis of the basement membrane is required, which requires the cooperative activity of the plasminogen activator system and matrix metalloproteinases. Therefore, VEGF inhibitors can be highly effective in treating diseases caused by angiogenesis. For example, anti-VEGF therapy can induce tumor regression and prevent blindness associated with neovascular age-related macular degeneration and proliferative diabetic retinopathy. However, primary and secondary non-responsiveness occurs when using anti-VEGF therapy. The development of new methods to inhibit angiogenesis is still desired.

[0005] Tissue hypoxia is sensed by hypoxia-inducible factor-1α (HIF-1α) and influences angiogenesis in multiple ways. HIF-1α functions as a master transcription factor that activates the transcription of genes encoding glycolytic enzymes, VEGF, and other proteins important for maintaining oxygen homeostasis. Glycolysis, via HIF-1α, controls the proliferation and migration of vascular endothelial cells.

[0006] ENO-1 is a multifunctional protein that was initially discovered as a cytoplasmic enzyme in the glycolytic pathway. Adenosine triphosphate (ATP) is involved in the growth and proliferation of vascular endothelial cells. Under hypoxic conditions, glycolysis is a compensatory adaptive process of energy metabolism to obtain the energy required for vital activities. Under hypoxic conditions, ENO-1 expression has been shown to increase under the control of HIF-1α. Activation of the glycolytic pathway via ENO-1 improves the energy imbalance in hypoxic cells, promotes transcription and proliferation, inhibits cell apoptosis, and promotes angiogenesis.

[0007] ENO-1 has also been found to be expressed on the cell surface as a plasminogen receptor. Increased expression of the plasminogen receptor protein is known to induce the cascade reaction of the urokinase plasminogen activation system, resulting in the degradation of the extracellular matrix, which is necessary for angiogenesis. Summary of the Invention

[0008] The present invention relates to an alpha-enolase (enolase-1, ENO-1) antagonist targeting ENO-1 and its use, the ENO-1 antagonist has the binding ability to ENO-1, for example human ENO-1 antibody, as an antigen-binding structural domain, and neutralizes the biological effect of ENO-1. The ENO-1 antagonist can bind to free ENO-1 protein and ENO-1 protein on the cell surface, and has an important application prospect in the treatment of angiogenesis-related diseases.

[0009] In some embodiments of the present disclosure, the angiogenesis-related disease or disorder may be any condition that results from the aberrant activation or expression of ENO-1 protein.

[0010] In some embodiments of the present disclosure, the angiogenesis-related disease or disorder may include the following (1) to (5): (1) ocular neovascular diseases (such as retinal neovascular diseases, neovascular age-related macular degeneration, diabetic retinopathy, and retinopathy of prematurity), which are diseases characterized by the invasion of new blood vessels into the retina or cornea, resulting in irreversible visual imperfections; (2) rheumatoid arthritis and osteoarthritis; cells in the subchondral space and vascular channels, chondrocytes, secrete various growth factors that induce angiogenesis, which may result in the pathogenesis of synovitis and subsequent destruction of articular cartilage; (3) inflammation and inflammatory diseases; during inflammation, inflammatory cytokines (such as cyclooxygenase 2, prostaglandin E2, thromboxane A2), inflammatory cells (such as macrophages, T cells, mast cells), and hypoxia-inducible factor (HIF) induce the expression of pro-angiogenic factors that cause angiogenesis. The newly formed vasculature in turn promotes the infiltration of inflammatory cells, further exacerbating inflammation. (4) Hereditary diseases such as Osler-Weber-Rendu disease and hereditary hemorrhagic telangiectasia, in which multisystem vascular dysplasia is associated with impaired angiogenesis leading to severe bleeding. (5) Tumor formation and metastasis; tumor cells secrete various pro-angiogenic factors that promote the formation of new blood vessels through the angiogenic process. Neovascularization is important in tumor growth by providing adequate nutrients and oxygen to tumor cells, and is compensated for with tumor metastasis by providing a route for tumor migration and invasion.

[0011] In some embodiments of the disclosure, administration is by oral, parenteral, buccal, vaginal, rectal, inhalation, insufflation, sublingual, intramuscular, subcutaneous, topical, intranasal, intraperitoneal, intrathoracic, intravenous, epidural, intrathecal, intraventricular, intraocular, or intravitreal route.

[0012] In some embodiments of the present disclosure, the subject is a mammal. In preferred embodiments, the subject is a human.

[0013] In some embodiments of the present disclosure, the ENO-1 antagonist can be a nucleic acid, such as DNA or RNA, that is delivered to a cell and expressed as a peptide intracellular protein. For example, the ENO-1 antagonist can be a nucleic acid that can be transcribed and translated, for example, as an anti-ENO-1 antibody or a binding fragment thereof. Furthermore, the nucleic acid can have or not have a secretory signal peptide, and thus the protein or peptide transcribed and translated from the nucleic acid can be secreted, non-secreted, or a combination thereof. In some embodiments of the present disclosure, the nucleic acid can be delivered to a subject via any known method or vehicle, such as a viral vector, a polymer, or a liposome. In some embodiments of the present disclosure, the nucleic acid can 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.

[0014] Compared with the prior art, the invention disclosed herein has the following beneficial effects: According to the present disclosure, the angiogenesis inhibitory effect of ENO-1 antagonist, such as ENO-1 monoclonal antibody, is demonstrated by in vitro experiments, that is, ENO-1 antagonist is a potential treatment for angiogenesis-related diseases, such as neovascular age-related macular degeneration, through a novel mechanism of inhibition of plasmin activation and glycolysis reprogramming.

[0015] Those skilled in the art will understand that the pharma- ceutically effective amount depends on many factors, such as the condition of the patient, age, disease state, route of administration, etc., and that such an effective amount may be determined based on these factors in routine practice without undue experimentation.

[0016] Other aspects of the invention disclosed herein will become apparent from the following description. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 is a set of photographs showing increased surface ENO-1 expression in VEGF-A stimulated HUVEC.

[0018] [Diagram 2] FIG. 1 shows the reduction in plasmin activity and cell migration of VEGF-A stimulated HUVEC treated with ENO-1 antibody.

[0019] [Diagram 3] FIG. 1 shows the reduction of tube formation in VEGF-A stimulated HUVEC treated with ENO-1 antibody.

[0020] [Figure 4] FIG. 1 shows that ENO-1 antibody reduced vascular sprouting in a rat aortic ring model of angiogenesis.

[0021] [Diagram 5] FIG. 1 shows that ENO-1 antibody inhibited angiogenesis in vivo in a Matrigel plug assay.

[0022] [Figure 6] FIG. 1 shows that ENO-1 antibody inhibited in vivo angiogenesis in a subcutaneous xenograft model of prostate cancer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] general definition

[0024] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA and immunology which are within the skill of one of ordinary skill in the art and are fully explained 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)".

[0025] The term "ENO-1 antagonist" refers to a molecule that has binding capacity to ENO-1 as an antigen-binding structural domain and neutralizes the biological action of ENO-1.

[0026] The terms "antibody" and "immunoglobulin" are used interchangeably in the broadest sense and include monoclonal antibodies (e.g., full-length or intact monoclonal antibodies), polyclonal antibodies, monovalent antibodies, polyvalent antibodies, multispecific antibodies (e.g., bispecific antibodies, so long 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.

[0027] The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for a particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments, called complementarity-determining regions (CDRs) or hypervariable regions, both in the light and heavy chain variable domains. The more highly conserved parts of the variable domains are called the framework (FR). Native heavy and light chain variable domains each contain four FR regions, mostly in a beta-sheet structure, connected by three CDRs, which form loops that connect the beta-sheet structure and, in some cases, form part of the beta-sheet structure. The CDRs of each chain are held in close proximity by the FR regions and contribute to the formation of the antigen-binding site of antibodies with the CDRs of the other chain (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in binding between the antibody and the antigen, but exhibit various effector functions, such as the participation of the antibody in antibody-dependent cellular cytotoxicity.

[0028] The antibody can be full length or can include a fragment (or fragments) of an antibody having an antigen-binding portion, 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 (see, e.g., Ward et al, Nature, 341:544-546 (1989)), CDRs, diabodies, triabodies, tetrabodies, linear antibodies, single chain antibody molecules, and multispecific antibodies formed from antibody fragments. Also encompassed by the invention are single chain antibodies produced by joining antibody fragments using recombinant methods, or synthetic linkers. This point is described in Bird et al. Science, 1988, 242:423-426; Huston et al., Proc. Natl. Acad. Sci. USA, 1988, 85:5879-5883.

[0029] As used herein, "treatment" or "treating" refers to a clinical intervention that seeks to alter the natural course of the individual or cell being treated, and treatment can be performed for prophylaxis or during the course of clinical pathological treatment. Desirable effects of treatment include prevention of disease onset or recurrence, alleviation of symptoms, reduction of direct or indirect pathological consequences of the disease, prevention or reduction of inflammation and / or tissue / organ damage, reduction in the rate of disease progression, improvement or amelioration of the disease state, and remission or improvement of prognosis. In some embodiments, the antibodies of the present disclosure are used to delay the onset of a disease or disorder.

[0030] An "individual" or "subject" is a vertebrate. In some embodiments, the vertebrate is a mammal. Mammals include, but are not limited to, farm animals (such as cows), sport animals, pets (such as cats, dogs, horses), primates, mice, rats, etc. In some embodiments, the vertebrate is a human.

[0031] An "effective amount" refers to an amount effective at a dosage and for a period of time necessary to achieve a desired therapeutic or prophylactic result. The "effective amount" of a substance / molecule of the present disclosure may vary depending on factors such as an individual's disease state, age, sex, and weight, as well as the ability of the substance / molecule to elicit a desired response in an individual. An effective amount is an amount in which the therapeutically beneficial effects outweigh the toxic or adverse 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.

[0032] The term "angiogenesis" as used herein refers to the biological process of new blood vessels sprouting from pre-existing vessels and plays an important role in disease development and progression (see Folkman J. N Engl J Med 1995, 333:1757-1763). Angiogenesis is a complex process in which endothelial cells function as building blocks for blood vessel expansion. Angiogenesis is controlled by a delicate balance of pro-angiogenic and anti-angiogenic molecules.

[0033] In certain diseases, the control of angiogenesis is altered. Many of these diseases involve pathological angiogenesis (i.e., inappropriate, excessive, or unwanted blood vessel formation), which supports the pathology and often contributes to the cell and tissue damage associated with such diseases. Angiogenesis-related diseases (i.e., diseases involving pathological angiogenesis) are numerous and diverse. These include, but are not limited to, various forms of tumors, chronic inflammatory diseases, and neovascularization diseases. In one embodiment, angiogenesis-related diseases can include retinal neovascular diseases, neovascular age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity, or cancer.

[0034] General reviews of the role of angiogenesis in angiogenesis-related diseases can be found in Moses et al., 1991, BioTechol. 9:630-633; Leek et al., 1994, J. Leuko. Biol. 56:423-435; and Beck et al., 1997, FASEB J. 11:365-373.

[0035] In one embodiment, the ENO-1 antagonist may be administered in conjunction with an anti-angiogenic agent for the treatment of angiogenesis-related diseases. Many anti-angiogenic agents have been isolated or developed. These include cartilage-derived factor (Moses et al., 1990, 248:1408-1410; Oikawa et al., 1990, Cancer Lett. 51:181-186), antiangiogenic steroids (Folkman et al., 1983, Science 221:719-725; Crum et al., 1985, Science 230:1375-1378; Oikawa et al., 1988, Cancer Lett. 43:85-92), and antiangiogenic vitamin D analogs (Oikawa et al., 1989, Cancer Lett. 48:157-162; Oikawa et al., 1990, Eur. J. Pharmacol. 178:247-50), angiostatin (O'Reilly et al., These include endostatin (O'Reilly et al., 1997, Cell 88:277-285), and verostatin (Oikawa et al., 1989, Cancer Lett. 48:157-162; Oikawa et al., 1990, Eur. J. Pharmacol. 178:247-50). Consistent with the view that pathological angiogenesis is the etiology of angiogenesis-related diseases, a number of antiangiogenic agents have been demonstrated to have beneficial therapeutic activity against these diseases.

[0036] The medicament of the present disclosure may be applied locally or systemically.The medicament of the present disclosure may also be administered in combination or with cofactors.If the medicament of the present disclosure is a substance that is normally present at the target site, the medicament of the present disclosure may be administered in an amount sufficient to restore normal levels, or in an amount that increases levels above normal levels at the target site.

[0037] Medicaments of the present disclosure may be supplied to the target site from an exogenous source or may be produced in vivo by cells at the target site or by cells of the same organism as the target site.

[0038] Medicaments of the present disclosure may be in any physiologically suitable formulation. They may be administered to an organism by injection, topically, by inhalation, orally, or by any other effective means.

[0039] The above-described medicaments and methods may also be used to suppress or inhibit pathological angiogenesis, for example 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, urogenital tract, tumors, or wounds.

[0040] Methods for treating angiogenesis-related diseases

[0041] The present disclosure provides a method for treating an angiogenesis-related disease, such as retinal neovascular disease, neovascular age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity, or cancer. The method generally comprises administering to a subject in need thereof an effective amount of an alpha-enolase (enolase-1, ENO-1) antagonist. In another aspect, the present disclosure further provides the use of an ENO-1 antagonist in the manufacture of a medicament for treating an angiogenesis-related disease.

[0042] In some embodiments, the ENO-1 antagonist used in the methods may include, but is not limited to, any of the following: (1) An anti-ENO-1 antibody or a binding fragment thereof. (2) A nucleic acid of an anti-ENO-1 antibody or a binding fragment thereof that is delivered into a cell and expressed as an anti-ENO-1 antibody or a binding fragment thereof within the cell.

[0043] The following specific examples are used to illustrate embodiments of the present disclosure, but those skilled in the art will recognize that these examples are for illustrative purposes and that other modifications and variations are possible without departing from the scope of the present disclosure.

[0044] Examples Example 1. Preparation of anti-ENO-1 antibody

[0045] In the embodiment of the present disclosure, the common method for producing anti-ENO-1 antibodies includes obtaining hybridomas that produce monoclonal antibodies against ENO-1. Methods for producing monoclonal antibodies are known in the art and will not be detailed here. In brief, mice are challenged with an antigen (ENO-1) together with a suitable adjuvant. The spleen cells of the immunized mice are then harvested and fused with hybridomas. Positive clones may be identified for their ability to bind to ENO-1 antigen using any known method, such as ELISA.

[0046] In one embodiment, the anti-ENO-1 antibody of the present disclosure may be a murine or humanized anti-ENO-1 antibody, or an scFv or Fab fragment thereof. An exemplary anti-ENO-1 antibody comprises 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).

[0047] In an embodiment of the disclosure, the antibody may be a murine antibody. Alternatively, the antibody may be a chimeric antibody (e.g., a human constant region combined with a murine variable region) or a humanized antibody (e.g., a murine CDR grafted into a human immunoglobulin framework region) or a fully human antibody.

[0048] Monoclonal antibodies may be humanized by obtaining CDR sequences from hybridomas and cloning the CDR sequences into human framework sequences to produce humanized antibodies. Common methods known in the art may be used to identify CDR sequences. The CDR regions in the present invention are identified by the Kabat numbering scheme. First, anti-ENO-1 hybridomas (e.g., mouse hybridomas) are generated. Such hybridomas may be generated by standard protocols for generating monoclonal antibodies. The total RNA of the hybridomas is then subjected to PCR using, for example, TRIzol (R) The DNA was isolated using a first strand cDNA synthesis kit (Superscript III) and oligo (dT 20 ) primer or Ig-3′ constant region primer, cDNA was synthesized from total RNA.

[0049] Next, the heavy and light chain variable regions of the immunoglobulin genes were cloned from the cDNA. For example, the VH and VL variable regions of the anti-ENO-1 mAb were amplified from mouse hybridoma cDNA by PCR using the mouse Ig-5' primer set (Novagen). The PCR products were amplified using the CloneJet TMThey may be directly cloned into a suitable vector (e.g., pJET1.2 vector) using a PCR cloning kit (Ferments). The pJET1.2 vector contains lethal insertions, and the target gene will only withstand the selection conditions if it is cloned into this lethal region. This facilitates the selection of recombinant colonies. Finally, the target clones were selected from the recombinant colonies, and the DNA of these clones was isolated and sequenced. Immunoglobulin (IG) sequences can be analyzed on the website of the international immunogenetics (ImMunoGeneTics: IMGT) information system.

[0050] Antibody expression and purification

[0051] For antibody production, the isolated clones may be expressed in any suitable cells. As an example, F293 cells (Life technologies) were transfected with anti-ENO-1 mAb expression plasmid and cultured for 7 days. Anti-ENO-1 antibodies were purified from the culture medium using a Protein A affinity column (GE). Protein concentration may be measured using a Bio-Rad protein assay kit and analyzed by 12% SDS-PAGE according to procedures known in the art or according to the manufacturer's instructions.

[0052] Example 2. In vitro effects of ENO-1 antagonists on VEGF-A stimulated endothelial cells

[0053] Primary human umbilical vein endothelial cells (HUVECs) grown on 4-well slides were treated with vascular endothelial growth factor A (VEGF-A) for 4 h, fixed in fixation buffer (BioLegend) for 15 min at 4 °C, blocked with 0.1% bovine serum albumin for 1 h, and incubated with anti-ENO1 primary antibody (Abnova, catalog number: H00002023-M01) for 1 h. After a brief wash in phosphate-buffered saline (PBS), the slides were incubated with Alexa Fluor488-conjugated goat anti-mouse IgG secondary antibody (Invitrogen) for 1 h. After a brief wash in PBS, cell nuclei were stained with DAPI (Sigma) for 5–10 min and mounted with Fluoromount-G mounting medium (Southern Biotech). Images were acquired using a Nikon microscope. FIG. 1 shows that VEGF-A treatment increased the surface expression of ENO-1 on HUVECs as examined by immunofluorescence staining.

[0054] HUVEC cells stimulated with VEGF-A (PeproTech) for 4 hours were washed twice with PBS and then cultured at 10 6Cells were resuspended in PBS at 100 cells / ml and preincubated for 1 h at 37°C with 30 μM human Glu-plasminogen (Sigma) in the absence or presence of various concentrations of ENO-1 antibody or control antibody human IgG1. After incubation, cells were washed three times with PBS, resuspended in PBS, and then incubated with 1.5 nM tissue plasminogen activator (Sigma) and 0.1 mM plasmin substrate Chromogenix S-2251 (Diapharma) for 2.5 h at 37°C. Plasmin activity was determined by measuring absorbance at 405 nm. Figure 2A shows that ENO-1 antibody reduced VEGF-A-induced plasmin activation in a dose-dependent manner. TXA (tranexamic acid), a lysine analogue developed to saturate plasminogen binding sites and prevent the formation of plasmin, was used as a positive control. For migration assays, 900 μl of medium containing 10% fetal bovine serum (FBS) was added to the bottom well, and then a coating-free insert (Corning) was placed in the bottom well. HUVEC cells treated with VEGF-A for 4 h were resuspended in medium containing 2% FBS and seeded into the transwell in the absence or presence of various concentrations of ENO-1 antibody, control antibody human IgG1, or TXA. Cells were allowed to migrate for 18 h at 37°C. Cells remaining on the top of the insert were gently wiped off with a cotton swab. Cells on the bottom side of the insert were fixed with methanol for 10 min and then stained with 1% crystal violet for an additional 2 h. The inserts were gently washed with PBS and dried. Bound crystal violet was eluted with 33% acetic acid, and the absorbance at 590 nm was measured. Since plasmin is known to regulate cell migration, Figure 2B shows that ENO-1 antibody dose-dependently reduced VEGF-A-induced migration of HUVEC cells, which was consistent with the results in Figure 2A.

[0055] HUVEC cells grown on Matrigel-coated 96-well plates were stimulated with VEGF-A for 24 h in the absence or presence of various concentrations of ENO-1 antibody, control human IgG1, or TXA. The formation of capillary-like tubes was observed and quantified by manual node counting. Figure 3 shows that ENO-1 antibody treatment dose-dependently reduces tube formation in VEGF-A-stimulated HUVEC. These results suggest an in vitro antiangiogenic effect of ENO-1 antagonist antibody on activated endothelial cells.

[0056] Example 3. Ex vivo effect of ENO-1 antagonistic antibodies on VEGF-A stimulated vascular sprouting

[0057] To examine the antiangiogenic effect of ENO-1 antagonists, an ex vivo rat aortic ring sprouting assay was used. Isolated thoracic aortic ring segments (1 mm) immersed in Matrigel were treated with VEGF-A or VEGF-A plus various doses of ENO-1 antibody for 4 days. Human IgG1 (hIgG1) and a VEGF monoclonal antibody (bevacizumab) were used as negative and positive controls, respectively. As shown in Figure 4, VEGF-A treatment significantly increased the number of sprouted microvessels compared to the control group, and a complex network was formed especially inside the ring after VEGF-A treatment. The growth of new microvessels was significantly inhibited in both the ENO-1 antibody and bevacizumab treatment groups.

[0058] Example 4. In vivo angiogenesis inhibitory effect of ENO-1 antagonist

[0059] The antiangiogenic effect of ENO-1 antagonists in vivo was accessed using a tumor cell-induced angiogenesis Matrigel plug assay. Matrigel mixed with RPMI8226 (BCRC no. 60384) human myeloma cells (1:1) with or without ENO-1 antibody was implanted subcutaneously into the right flank of BALB / c nude mice (Lasco). Seven days after treatment, the plugs were removed and the angiogenic response was quantified by measuring hemoglobin levels in the plugs using an enzyme-linked immunosorbent assay (ELISA). The results in Figure 5 show that ENO-1 antagonists significantly reduced the hemoglobin content of the plugs compared to the control group.

[0060] A subcutaneous xenograft model was established in male BALB / c nude mice (Lasco) using the androgen-independent human prostate cancer cell line PC-3 (ATCC No. CRL-1435). Before inoculation, PC-3 cells were washed with phosphate-buffered saline (PBS) and resuspended in PBS and Matrigel at a 1:1 ratio to a final concentration of 10 7 Cells (10 6 PC-3 cells (100 / μl) were implanted subcutaneously into the right flank of mice. Three days after PC-3 cell implantation, mice were randomized and injected intraperitoneally with ENO-1 antibody (30 mg / kg) twice weekly. Tumors were harvested on day 23 and intratumoral angiogenesis was measured by immunofluorescence staining for CD31 expression. Figure 6 shows a trend toward decreased CD31 expression, indicating the in vivo antiangiogenic activity of ENO-1 antagonists.

[0061] In summary, ENO-1 antagonists significantly attenuated angiogenesis by decreasing plasmin activity, cell migration, tube formation, and angiogenesis, either in vitro or in vivo.

[0062] Unless otherwise defined, all technical and scientific terms and abbreviations used herein have the same meaning as commonly understood by one of ordinary skill in the art of the present invention. Any compositions, methods, kits, and means for conveying information similar or equivalent to those described herein can be used to practice the present invention, and preferred compositions, methods, kits, and means for conveying information are disclosed herein.

[0063] All references cited herein are incorporated by reference to the fullest extent permitted by law. The discussion of these references is intended merely to summarize the assertions made by their authors. The citation of any document (or portion of a document) is not an admission that it is relevant prior art. Applicants reserve the right to challenge the accuracy and pertinence of the cited documents.

Claims

1. 1. A method for treating an angiogenesis-associated disease, comprising: The method comprises administering to a subject in need thereof an effective amount of an alpha-enolase (enolase-1, ENO-1) antagonist.

2. The method of claim 1, wherein the ENO-1 antagonist is an anti-ENO-1 antibody or a binding fragment thereof.

3. The method of claim 2 , wherein the antibody is a monoclonal antibody.

4. The antibody or binding fragment thereof HCDR1 (GYTFTSCVMN: SEQ ID NO: 1), HCDR2 (YINPYNDGTKYNEKFKG: SEQ ID NO: 2), and a heavy chain variable domain comprising the three complementary regions of HCDR3 (EGFYYGNFDN: SEQ ID NO: 3); LCDR1 (RASENIYSYLT: SEQ ID NO: 4), LCDR2 (NAKTLPE: SEQ ID NO:5), and and a light chain variable domain comprising the three complementary regions of LCDR3 (QHHYGTPYT: SEQ ID NO: 6).

5. The method of claim 2 , wherein the antibody or binding fragment thereof is a murine antibody, a human antibody, a chimeric antibody, a humanized antibody, or an antibody fragment thereof.

6. The method of claim 1, wherein the ENO-1 antagonist is a nucleic acid that is delivered intracellularly and expressed as an intracellular protein or peptide.

7. The method of claim 6, wherein the ENO-1 antagonist is secreted, non-secreted, or a combination thereof.

8. The method of claim 6, wherein the ENO-1 antagonist is delivered via a viral vector, a polymer, and / or a liposome.

9. The method, wherein the angiogenesis-related disease comprises retinal neovascular disease, neovascular age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity, or cancer.

10. Use of an ENO-1 antagonist according to any one of claims 1 to 8 in the manufacture of a medicament for treating an angiogenesis-related disease.

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