WO2010031361A1 - 抑制病理性血管发生作用的组合物 - Google Patents

抑制病理性血管发生作用的组合物 Download PDF

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WO2010031361A1
WO2010031361A1 PCT/CN2009/074086 CN2009074086W WO2010031361A1 WO 2010031361 A1 WO2010031361 A1 WO 2010031361A1 CN 2009074086 W CN2009074086 W CN 2009074086W WO 2010031361 A1 WO2010031361 A1 WO 2010031361A1
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lect2
cancer
protein
composition
inhibiting
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French (fr)
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郭明良
吴玉琳
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TTY Biopharm Co Ltd
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TTY Biopharm Co Ltd
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Priority to JP2011527194A priority Critical patent/JP2012502934A/ja
Priority to EP09814081A priority patent/EP2347761A4/en
Priority to US13/120,101 priority patent/US20120071405A1/en
Priority to CN2009801372193A priority patent/CN102159238A/zh
Publication of WO2010031361A1 publication Critical patent/WO2010031361A1/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1808—Epidermal growth factor [EGF] urogastrone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1833—Hepatocyte growth factor; Scatter factor; Tumor cytotoxic factor II
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1858—Platelet-derived growth factor [PDGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • composition for inhibiting pathological angiogenesis Composition for inhibiting pathological angiogenesis
  • the present invention relates to a method of inhibiting angiogenesis, and in particular to a disease which inhibits angiogenesis by LECT2 protein and/or is associated with angiogenesis. Background technique
  • LECT2 Leukocyte cell-derived chemotaxin 2
  • LECT2 is a novel neutrophil chemokine that has been induced by phytohemagglutinin in human T cell leukemia SWK3 cells. It was purified from the medium and was a 16-kDa protein (S OC/H Biophys Acta 1396: 105-13 (1998)). Furthermore, by isolating the cDNA encoding LECT2, it was found that its mRNA specifically expresses in the liver of human or fetal liver and human liver cancer cell line Biophys Res Commun 237 : 1 16-20 (1997)).
  • LECT2 is a chondromodulin-II cartilage regulator, a bovine protein that stimulates the proliferation of chondrocytes and osteoblasts (J Bio Chem 125:436-42 (1999)).
  • ConA-induced hepatitis is an animal model of human autoimmune hepatitis. It can be induced by cytokines and cytotoxic molecules associated with immune cells (such as CD4+ T cells and macrophages) (J Clin Invest 90: 196 (1992)).
  • cytokines and cytotoxic molecules associated with immune cells such as CD4+ T cells and macrophages
  • Angiogenesis refers to the creation of new blood vessels that occur when a healthy individual is injured, to repair the wound and restore the tissue to a blood supply.
  • the process of angiogenesis is strictly controlled by a variety of positive and negative regulatory factors. However, in many disease states, the patient's angiogenic effects are out of control.
  • Angiogenesis is an important process in the growth of newborns, and it is also important for repairing wounds, but it is also a pathological feature of many clinical diseases, including tissue inflammation, arthritis, tumor growth, and diabetes. Retinopathy, macular degeneration caused by retinal angiogenesis, and similar diseases. These clinical manifestations associated with angiogenesis are also known as angiogenic diseases.
  • angiogenic diseases usually include cancer (including solid tumors and blood tumors), cardiovascular diseases (eg, arteriosclerosis), chronic inflammation (eg, Rheumatoid arthritis or Crohn's disease), diabetes (eg, diabetic retinopathy), psoriasis, endometrial tissue ectopic, and obesity. Therefore, there is a need in the medical community for a method and composition for effectively inhibiting angiogenesis to treat or prevent such diseases. Summary of the invention
  • the present invention provides a method of inhibiting pathological angiogenesis comprising administering to a subject an effective amount of a LECT2 protein or analog thereof, and a pharmaceutically acceptable carrier.
  • the invention further provides a composition for inhibiting angiogenesis comprising an effective amount of LECT2 protein or an analog thereof, and a pharmaceutically acceptable carrier.
  • the invention further provides a method of treating and/or inhibiting a cell proliferative disorder comprising administering to the individual an effective amount of a LECT2 protein or analog thereof, and a pharmaceutically acceptable carrier.
  • the invention further provides a composition for treating and/or inhibiting a cell proliferative disorder comprising an effective amount of a LECT2 protein or analog thereof, and a pharmaceutically acceptable carrier.
  • the present invention further provides a method of inhibiting pathological angiogenesis comprising administering to a subject an effective amount of a LECT2 protein or analog thereof, and a pharmaceutically acceptable carrier.
  • Figure 1A shows immunohistochemical staining analysis of microvascular density (CD34 staining) of human liver cancer.
  • Figure 1B shows the relationship between LECT2 protein expression and HCC microvessel density.
  • Figure 2A shows similar growth curves for vehicle control cells (SK-Hepl/Neo), overexpressing LECT2 cells (SK-Hepl/LECT2), and wild-type cells (SK-Hepl/WT).
  • Figure 2B shows that the mean tumor volume of the SK-Hepl Neo group was significantly greater than that of the SK-Hepl/LECT2 group.
  • Figure 2C shows that the microvessel density (CD31 staining) of the SK-Hepl/LECT2 group was smaller than that of the vector control group (SK-Hepl/Neo).
  • Figure 3 A shows that the growth curve of vector control cells (BNL Neo), overexpressing LECT2 cells (BNL/LECT2) and wild type cells (BNL/WT) is similar.
  • Figure 3B shows that the mean tumor volume of the BNL/Neo group was significantly greater than that of the BNL/LECT2 group.
  • Figure 3C shows that LECT2 inhibits microvessel density (CD31 staining:) in BNL cells in situ tumors.
  • Figure 4A shows the BNL/Neo group, the BNL Neo+rLECT2 group, and the BNL/LECT2 group, respectively. Tumor volume.
  • Figures 4B-4C show that the mean tumor volume in the BNL Neo group was significantly greater than in the BNL/Neo+ rLECT2 group and the BNL/LECT2 group.
  • Figure 5 shows that conditioned medium overexpressing LECT2 cells disrupts the production of new blood vessels.
  • Figure 6A shows that LECT2 conditioned medium inhibits the formation of tubular structures.
  • FIGB shows the LECT2 expression levels of HCC transformants (PLC PRF/5 and Huh-7) in which endogenous LECT2 (shLECT2-l and shLECT2-2) were inhibited.
  • Figure 6C shows that conditioned medium of PLC/PRF/5 and Huh-7 cells inhibiting the production of endogenous LECT2 promotes the formation of vascular tubular structures in vascular endothelial cells compared to control cells.
  • Figure 7 shows that LECT2 conditioned medium significantly inhibits the movement of vascular endothelial cells.
  • Figure 8 shows that rLECT2 protein significantly inhibits VEGF-A-induced tubular structure formation compared to cells treated with VEGF-A (50 ng/mL).
  • Figures 9A-9B show that inhibition of LECT2 recombinant protein by CAM mode inhibits VEGF-A-induced angiogenesis.
  • Figure 10 shows that LECT2 recombinant protein reduces VEGF-A-induced vascular endothelial cell migration.
  • Figure 11 shows that LECT2 recombinant protein inhibits vascular endothelial cell-induced tubular structure formation in vascular endothelial cells.
  • Figure 12 shows that LECT2 recombinant protein inhibits the formation of vascular tubular structures induced by conditioned medium in vascular endothelial cells.
  • Figures 13A-13B show that the mean tumor volume of the B16F1/Neo group was significantly greater than that of the B16F1/LECT2 group.
  • Figure 13C shows that the microvessel density of the B16F1/LECT2 group is smaller than that of the B16F1/Neo group.
  • FIG 14 shows that LECT2 protein inhibits tumor growth of A549 cells in mice.
  • the present invention provides a composition for inhibiting pathological angiogenesis and/or cell proliferative diseases.
  • This composition comprises an effective amount of a LECT2 protein or analog thereof, and a pharmaceutically acceptable carrier.
  • LECT2 or “LECT2 protein” as used in the present invention refers to all forms of leukocyte-derived chemokine 2 protein, including active or inactive forms. Any analog/fragment of a biologically active LECT2 protein is within the scope of the invention. The analog/fragment can be conjugated to a carrier molecule, such as an immunoprotein, for a variety of applications, including increasing the valency of the protein binding site.
  • the LECT 2 protein analogs of the invention may also be substituted, deleted, added or inserted into one or more of the original proteins. Amino acids are obtained, and they still retain at least the active portion of the original protein.
  • the "LECT2 variant” or “variant protein” described in the present invention includes at least 60% of the same sequence as the LECT2 protein, and the sequence similarity is defined by the amino acid sequence of the protein to maximize the same portion and minimum. Sequence differences.
  • the LECT2 protein of the invention also includes a modified LECT2 protein, which is similar to natural
  • LECT2 protein Those skilled in the art can modify peptide or DNA sequences by conventional techniques. For example, modification of a protein sequence can include altering, replacing, substituting, inserting or deleting an amino acid in the coding sequence. The methods used to alter, replace, substitute, insert or delete sequences are generally known techniques. Preferably, the sequence retains the activity of the LECT2 protein after alteration, substitution, substitution, insertion or deletion.
  • recombinant protein means that the protein comprises one or more recombinant moieties.
  • Site-specific recombinases are enzymes present in various viruses and bacteria, which simultaneously have the characteristics of limiting endonuclease and ligase. This recombination enzyme recognizes specific DNA sequences and alters DNA fragments.
  • LECT2 protein LECT2 protein analog/fragment or LECT2 recombinant protein
  • Animal cells may include monkey COS cells, CHO cells, human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed primate cells, normal diploid cells, Native tissue-derived cell lines, native grafts, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells cultured in vitro.
  • Lower eukaryotes include yeast, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida or any expressible Yeast of heterologous protein.
  • Prokaryotes may include Escherichia coli, Bacillus subtilis, Salmonella typhimurmm, or any bacterium that expresses a heterologous protein. If the protein is expressed by yeast or bacteria, the expressed protein can be further modified, for example, by silicidation or glycosylation at an appropriate position to make the protein functional.
  • the composition is administered to a tissue having a pathological angiogenic effect, the composition comprising an effective amount of a LECT2 protein or a nucleotide vector expressing an active LECT2 protein.
  • This tissue can be any tissue or organ that has an angiogenic effect, including the brain, skin, muscles, stomach, connective tissue, joints, bones, and the like.
  • Pathological angiogenesis can be induced by growth factors, including vascular endothelial growth factor (VEGF), platelet growth factor (PDGF;), fibroblasts. Growth factors (FGF;), epidermal growth factor (EGF), and hepatocyte growth factor (HGF).
  • VEGF vascular endothelial growth factor
  • PDGF platelet growth factor
  • FGF growth factors
  • EGF epidermal growth factor
  • HGF hepatocyte growth factor
  • LECT2 protein, LECT2 protein analog/fragment, or LECT2 recombinant protein of the present invention can also inhibit the formation of neovascular tubular structures, endothelial cell migration and proliferation, tumor vascular invasion or other diseases associated with angiogenesis.
  • Diseases associated with angiogenesis include, but are not limited to, inflammatory diseases, chronic rheumatoid arthritis, and psoriasis, diseases associated with abnormal vascular invasion, such as diabetic retinopathy, neovascular glaucoma, vascular restenosis, and Cell proliferative diseases, such as diseases associated with tumors or cancer (for example, solid tumors, solid tumor metastasis, liver cancer, lung cancer, breast cancer, colon cancer, gastric cancer, and melanoma).
  • diseases associated with tumors or cancer for example, solid tumors, solid tumor metastasis, liver cancer, lung cancer, breast cancer, colon cancer, gastric cancer, and melanoma.
  • cell proliferative disease refers to multicellular tissue damage caused by abnormal proliferation of one or more cell populations in multicellular tissues.
  • Cell proliferative diseases can occur in different animals and humans.
  • Cell proliferative diseases include, but are not limited to, cancer, vascular proliferative diseases, and fibrotic diseases.
  • the LECT2 protein of the present invention and/or the pharmaceutical composition comprising the LECT2 protein can be administered orally, parenterally, by inhalation, rectally, vaginally, intradermally, transdermally or topically, and the pharmaceutical unit can comprise a conventional non-toxic pharmaceutically acceptable carrier. , adjuvants and vehicles, and can be given by a peristaltic tool.
  • the LECT2 protein of the present invention and/or the pharmaceutical composition comprising the LECT2 protein can be administered at one time, multiple times or continuously within 24 hours.
  • suitable known means may be employed, including, but not limited to, intravenous drip, intravenous pump, implantable syringe pump or topical administration.
  • the duration of treatment can be adjusted according to different conditions, for example, the course and severity of angiogenesis.
  • the LECT2 protein alone or in combination with other agents of the invention is until the end of pruritus, or continued treatment for life.
  • the invention provides a pharmaceutical composition that inhibits pathological angiogenesis and/or cell proliferative disorders.
  • This pharmaceutical composition comprises an effective amount of the LECT2 protein or an analog thereof, and a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers include solvents, dispersions, coatings, antibacterial/antifungal agents, isotonic agents, absorption delaying agents, and the like.
  • the invention further provides a method of inhibiting pathological angiogenesis comprising administering to a subject an effective amount of a LECT2 protein or analog thereof, and a pharmaceutically acceptable carrier.
  • liver cancer hepatocellular carcinoma
  • the cells were washed 3 times with PBST, and diluted with 1:500 of CD34 antibody at room temperature, and biotin-labeled anti-mouse IgG secondary antibody treatment (Vector Laboratories, Inc., Burlingame, CA)1 Hours, bound antibodies were detected by ABC kit (Vector Laboratories Inc.).
  • diaminobenzidine diammobenzidine staining, washing, and finally to German hematoxylin ⁇ : cyan (Delafield's hematoxylin) after anti-staining (counterstain) and counted analysis results.
  • microvessel density was measured by staining for endothelial cell antigen and the degree of angiogenesis was measured.
  • the microvessel density (MVD) was measured by counting endothelial cells stained with CD34 antibody. Three highly vascularized regions in each tumor were analyzed at high magnification, and the total number of microvessels in each region was counted, as well as the average number of microvessels per tumor.
  • Figure 1A shows the results of analysis of human HCC microvessel density, which can be stained with CD34 antibodies.
  • the endothelial cell CD34 marker was analyzed by immunohistochemical staining for liver tumors, confirming the presence of many endothelial cells around the tumor cells.
  • LECT2 protein is opposite to the density of microvessels.
  • the liver cancer cells of the patients were divided into two groups, including high LECT2 protein expression (LECT2/p-actin ratio > 0.45) and low LECT2 protein expression (LECT2/p-actin ratio ⁇ 0.45).
  • high LECT2 protein expression levels are highly correlated with low microvessel density. It can be seen that in 14 human liver cancer samples, the expression level of LECT2 protein and microvessel density showed an opposite trend.
  • a human liver cancer cell line that does not endogenously express the LECT2 protein is used: SK-Hepl.
  • the cDNA of human LECT2 was constructed into the pSecTag2A eukaryotic expression vector to obtain an expression vector overexpressing the LECT2 protein, and the hygromycin B gene was contained downstream of the same promoter.
  • This LECT2 protein expression vector was transfected into liver cancer cells (SK-Hepl) with Lipofectamine 2000 reagent (Invitrogen Life tech.).
  • HCC cells were modeled in vivo in xenograft tumor models.
  • 8 week old thymidine mothers purchased from the National Animal Center
  • the rats were randomly divided into two groups, including a cell line (SK-Hepl/LECT2) overexpressing LECT2 protein and a control cell line (SK-Hepl/Neo).
  • SK-Hepl/LECT2 overexpressing LECT2 protein
  • SK-Hepl/Neo a control cell line
  • the volume of the tumor is expressed by the width X length ⁇ ⁇ .5.
  • the size of the tumor was measured in a double angle clamp.
  • mice were able to detect significant tumors for the first time 10 days after injection of cells.
  • the average tumor volume of the mice injected with the SK-Hepl/Neo cell strain was significantly larger than that of the SK-Hepl/LECT2 cell line.
  • immunohistochemical staining was used to analyze the effect of LECT2 on microvessel density in paraffin sections of tumor tissue in situ by CD31 antibody.
  • the tumors produced by the stable and highly expressed LECT2 cells were 3 times smaller than the control group per mm 2 unit volume. This example shows that LECT2 protein can inhibit the growth of human hepatoma tumor SK-Hepl cells in mice.
  • mice tumor cells overexpressing LECT2 protein were injected into BALB/c mice and analyzed.
  • the analysis procedure of this example was the same as that of Example 2 except that SK-Hepl human hepatoma cells and mice were replaced with BNL mouse hepatoma cells and BALB/c mice, respectively.
  • Mice BALB/c mice, 8 weeks old were injected subcutaneously with BNL mouse hepatoma cells (BNL/LECT2) or control BNL cells (BNL/Neo) stably and highly expressing LECT2, and each mouse was injected 1 ⁇ 10 5 Cells.
  • BNL/LECT2 BNL mouse hepatoma cells
  • BNL/Neo control BNL cells
  • the growth curves of the wild-type cell line (BNL/WT), the control cell line (BNL/Neo), and the monolayer cultured cells overexpressing the LECT2 protein cell line (BNL/LECT2) were similar.
  • the tumor volume of mice injected with BNL/Neo cell line was significantly larger than that of BNL/LECT2 cell line, and the microvessel density in the orthotopic tumor tissue was analyzed by CD31 antibody, and the microvessel density was high by LECT. Protein expression was inhibited (Fig. 3C).
  • the results of this example show that LECT2 protein can inhibit the growth of mouse liver cancer tumor BNL cells in BALB/c mice.
  • This example analyzes whether LECT2 recombinant protein can also affect mice transplanted with HCC tumors, and evaluates the effect of LECT2 recombinant protein on BALB/c mice injected with BNL/Neo cells.
  • LECT2 recombinant protein (1.25 mg/kg body weight) or 1X PBS buffer (50 L) was injected into the tumor for 10 days.
  • the result is shown in Figure 4A. Shown.
  • the mean volume of tumors in the BNL/Neo group was 35 days after injection of cells.
  • FIG. 4C shows the tumor weight of each group of xenografted BALB/c mice. Similarly, the tumor weights of the BNL/LECT2+rLECT2 group and the BNL/LECT2 group were significantly smaller than those of the BNL/Neo group. It can be seen that the LECT2 recombinant protein can inhibit the growth of mouse liver cancer tumor BNL cells in BALB/c mice.
  • Example 5 Effect of conditioned medium of human HCC cells expressing LECT2 on angiogenesis
  • a chick chorioallantoic membrane assay (CAM assay) was used to evaluate the effect of conditioned medium of SK-Hepl cells overexpressing LECT2 on angiogenesis.
  • This culture was obtained from SK-Hepl cells transfected with LECT2 or vector.
  • the cancer cells were cultured in DMEM medium containing no fetal bovine serum for 48 hours.
  • the conditioned medium of SK-Hepl cells transfected with LECT2 and the vector was centrifuged at a low speed.
  • CAM analysis of chicken embryos can be used as a suitable model when studying vascular responses in a variety of regulatory factors during in vivo angiogenesis.
  • the fertilized eggs were cultured at 100. F, humidity controlled incubator and air circulation for 14 days.
  • a small hole was made above the egg, located above the main vascular area, separating the chicken embryo chorion from the eggshell membrane.
  • a conditioned culture with a total volume of 20 was dropped onto a small disc of 5 mm in diameter, and a small disc was placed over the chicken embryo chorion (3 chicken embryos per group).
  • the small hole above the egg is sealed with a breathable tape and placed back in the incubator. Eggs were carefully opened 3 days after treatment (Day 12) and 5 days after treatment (Day 14).
  • Angiogenesis in chicken embryonic chorion was assessed by measuring vessel length and density. The number of blood vessels in the control and experimental groups was compared to analyze the effect of LECT2 recombinant protein on angiogenesis, and the number of blood vessels was analyzed by Image-Pro Plus 4.5 software. Referring to Figure 5, the conditioned medium overexpressing the LECT2 protein inhibits the angiogenesis of the capillary bed in the chicken embryo chorion. From the CAM analysis, it was found that for the conditioned medium overexpressing the LECT2 protein, the addition of the LECT2 antibody prior to the binding of the LECT2 protein in the conditioned medium promoted angiogenesis compared to the control group to which the IgG neutralizing antibody was added. effect.
  • Matrigel analysis is an extracellular interstitial (ECM) assay that provides a physiological environment for analyzing endothelial cell morphology, biochemical function, and gene expression.
  • ECM extracellular interstitial
  • VECs vascular endothelial cells
  • vascular endothelial cells were plated in Matrigel-coated 24-well culture plates and treated with conditioned medium of SK-Hepl or HCC36 cells transfected with LECT2 or Neo for 6 hours.
  • the formation of the tubular structure was observed with an inverted phase contrast microscope. Under the observation of the microscope, the tubular networks under six randomly selected fields of view were compared.
  • the SK-Hepl/LECT2 conditioned medium or the HCC36/LECT2 conditioned medium can inhibit the Matrigel layer compared to the control medium (SK-Hepl/Neo conditioned medium or HCC36/Neo conditioned medium).
  • LECT2 recombinant protein is an important factor in the regulation of angiogenesis in the treatment of angiogenesis, it inhibits the expression of endogenous LECT2 (shLECT2-l and shLECT2-2) in PLC/PRF/5 and Huh-7 cells.
  • endogenous LECT2 shLECT2-l and shLECT2-2
  • transformants in which endogenous LECT2 was inhibited were analyzed by Western blotting.
  • conditioned medium of PLC/PRF/5 and Huh-7 cells inhibited by endogenous LECT2 promoted tubular structure formation in vascular endothelial cells. From this, it was revealed that the cell-conditioned medium in which LECT2 was inhibited promoted the formation of a tubular structure in vascular endothelial cells.
  • Vascular endothelial cells were cultured in (7 x 104 cells/well) M199 medium in a 24-well culture plate.
  • HCC conditioned medium (SK-Hepl / LECT2 CM) containing overexpressing LECT2 and vehicle control HCC conditioned medium (SK-Hepl Neo CM) or endogenous LECT2 inhibited HCC conditioned medium (Huh-7) /shLECT2-2 CM) and vehicle control medium HCC conditioned medium (Huh-7/shLuc CM).
  • the LECT2 conditioned medium inhibits the migration of vascular endothelial cells, and the Huh-7 conditioned medium (Huh-7/shLECT2-2 CM), which is inhibited by endogenous LECT2, promotes the repair of vascular endothelial cells. From this, it can be seen that the HCC conditioned medium overexpressing LECT2 can inhibit the mobilization ability of vascular endothelial cells.
  • Example 8 Effect of LECT2 Recombinant Protein on Vascular Endothelial Growth Factor-Induced Tubular Structure
  • vascular endothelial cells Place vascular endothelial cells on Matrigel in a 24-well plate with VEGF-A (50 ng/mL),
  • PDGF (50 ng/mL), bFGF (30 ng/mL), EGF (50 ng/mL), HGF (40 ng/mL) or recombinant protein containing LECT2 (200 ng/mL) for 6 hours, and analysis The formation of the tube structure. From the results, it was revealed that the LECT2 recombinant protein can effectively inhibit the angiogenesis of the vascular tubular structure induced by VEGF-A, and the inhibition rate was 52% as compared with the VEGF-A (50 ng/mL) group, as shown in Fig. 8.
  • LECT2 recombinant protein can inhibit the formation of endothelial cell tubular structure, especially VEGF-A, induced by vascular growth factors VEGF-A, PDGF, bFGF, EGF and HGF.
  • VEGF-A vascular growth factors
  • PDGF vascular growth factors
  • bFGF vascular growth factors
  • EGF EGF
  • HGF HGF
  • Chicken embryonic chorion of 9-day large chicken embryos was treated with VEGF-A, and different concentrations of LECT2 recombinant protein (0, 1.25, 2.5 and 5 nM) or Fc-Tag (control group) (3 chicken embryos per group) ), dripped in a small disc with a diameter of 5 mm, the total volume is 20 L, the small disc covers the chicken embryonic chorion, and the chicken embryo chorion is treated with VEGF-A and LECT2 recombinant protein for 3 days ( That is, the 12th day) or 5 days (ie, the 14th day).
  • VEGF-A (50 ng/mL) significantly promoted the microvascular bed region of chorioamnose compared to the control group.
  • Fig. 9B it was revealed from the results of CAM analysis that on day 12, 2.5 nM and 5 nM of LECT2 recombinant protein significantly inhibited angiogenesis induced by VEGF-A. On day 14, 5 nM of LECT2 recombinant protein inhibited the chorioamnose-induced microvascular bed induced by VEGF-A. Thus, the LECT2 recombinant protein inhibits VEGF-A-induced angiogenesis.
  • Example 10 Effect of LECT2 Recombinant Protein on VEGF-A Induced Angiogenesis in Vitro
  • This example analyzes the effect of LECT2 recombinant protein on vascular endothelial cell migration and angiogenesis.
  • the movement of vascular endothelial cells is one of the important factors of angiogenesis, and therefore the ability of rLECT2 protein to VEGF-A-induced vascular endothelial cell migration was analyzed.
  • Different concentrations of LECT2 recombinant protein (0 200 ng/mL) and LECT2 recombinant protein (200) were treated simultaneously with 2% FBS (control), VEGF-A (50 ng/mL), and VEGF-A (50 ng/mL).
  • VEGF-A (50 ng/mL) promotes the movement of vascular endothelial cells.
  • the LECT2 recombinant protein was dose-dependent in inhibiting VEGF-A-induced movement.
  • the LECT2 recombinant protein in particular at a dose of 200 ng/mL, significantly inhibited VEGF-A-induced vascular endothelial cell migration. Since the LECT2 recombinant protein inhibits VEGF-A-induced cell migration, the LECT2 recombinant protein is further analyzed for its response to angiogenesis during angiogenesis.
  • LECT2 recombinant protein 0-200 ng/mL
  • BSA control
  • VEGF-A 50 ng/mL
  • the LECT2 recombinant protein was dose-dependent with the disruption of the VEGF-A-induced microtubule network. It can be seen that the LECT2 recombinant protein can inhibit the vascular endothelial cell-like microtubule structure induced by VEGF-A in vitro.
  • Example 11 Effect of LECT2 recombinant protein on the formation of vascular tubular structures induced by different cancer cell conditioned medium
  • This example analyzes the effect of LECT2 recombinant protein on angiogenesis of different cancer cell lines.
  • the vascular endothelial cells were placed on Matrigel in a 24-well plate and treated with conditioned medium of various cancer cell lines for 6 hours.
  • the cancer cells included liver cancer cells (SK-Hepl and Huh-7) and lung cancer cells (A549 and H1299).
  • breast cancer cells (MB-MDA-231 and MCF7), gastric cancer cells (MKN and N87), rectal cancer cells (HCT116) and mouse melanoma cells (B16F1), or cancer cell strain conditioned medium combined with rLECT2 (5 nM) treatment 6 hours.
  • Figure 12 shows the results of treatment of vascular endothelial cells with cancer cell conditioned medium alone or with rLECT2 protein (5 nM) for 6 hours. It can be seen that LECT2 recombinant protein can inhibit the formation of vascular tubular structures in SK-Hepl, A549, MCF-7 and B16F1 cell conditioned medium.
  • Example 12 Effect of LECT2 protein on tumorigenesis and angiogenesis of melanoma and lung cells
  • a mouse melanoma cell line B16F1 overexpressing LECT2 was constructed. After screening with hygromycin B, the expression level of LECT2 was analyzed, and the effect of LECT2 protein on tumor growth and angiogenesis was evaluated by in vivo xenogenic melanoma transplantation model.
  • B16F1 cells (B16Fl/Neo and B16F1/LECT2) were injected subcutaneously into C57BL/6 female rats of sputum age, each Only 5 ⁇ 10 5 cells were injected into the mice. Referring to Figure 13A, on day 18 after injection of cells, the mean tumor volume of mice of B16F1/Neo cells was significantly greater than that of mice injected with B16F1/LECT2 cells.
  • Lung cancer A549 cells were subcutaneously injected into 7-week-old mother rats, and 5 to 10 6 cells were injected per mouse. Twenty days after the injection of ⁇ 549 cells, D-luciferin (20 mg/mL/mouse per kg body weight) was intraperitoneally injected, the mice were anesthetized, and photographed with a Berthold NightOWL imaging system for 5 minutes. Referring to Figure 14, even for a short exposure (30 seconds), the color image showed that all A549/Luc/Neo tumor mice had bright spots on the back of the skin, but this spot was small in A549/Luc/LECT2 tumors. In mice, it is significantly smaller, indicating that LECT2 can inhibit the growth of lung cancer cells in mouse tumors. It can be seen that LECT2 protein can effectively inhibit the tumor growth of A549 cells in rats.

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Description

抑制病理性血管发生作用的组合物 技术领域
本发明涉及一种抑制血管发生作用的方法， 且特别涉及以 LECT2蛋白 抑制血管发生作用和 /或与血管发生作用相关的疾病。 背景技术
白细胞衍生趋化素 2(Leukocyte cell-derived chemotaxin 2 ; 以下筒称 LECT2)为一种新颖的嗜中性白细胞趋化因子， 其已由经植物凝集素 (phytohemagglutinin)诱导的人类 T细胞白血病 SWK3细胞的培养基中纯化出 来， 为 16-kDa的蛋白质 (S OC/H Biophys Acta 1396 : 105-13 (1998))。 此外， 藉由分离编码 LECT2的 cDNA, 得知其 mRNA在成人或胎儿的肝脏及人类 肝癌细胞林中特异性地表达 Biophys Res Commun 237 : 1 16-20 (1997))。 LECT2为一种 chondromodulin-II软骨调节因子， 其为一种可刺激 软骨细胞 (chondrocytes)及成骨细胞 (osteroblasts)增生的牛蛋白（J Bio Chem 125 :436-42 (1999))。 目前， 已有文献指出老鼠 LECT2的表达在刀豆素 A(Con A)诱发的肝炎中会短暂地降低，此刀豆素 A(Con A)诱发的肝炎为一种人类自 体免疫肝炎的动物模式， 其可被免疫细胞 (如 CD4+ T细胞及巨噬细胞)相关 的细胞激素及细胞毒杀分子所诱发 (J Clin Invest 90: 196 (1992))。 然而， 目前 并没有任何文献揭示 LECT2蛋白可抑制血管发生作用。
血管发生作用是指生成新的血管， 其发生于当健康个体受到伤害时， 得 以修复创伤并使组织恢复血流的供应。血管发生作用的过程是受到各种正向 及负向的调控因子严格控制。 然而， 在许多疾病状态中， 患者的血管发生作 用却失去控制。
血管发生作用对于新生儿的生长中为一重要的过程， 其对于修复创伤亦 非常重要， 但同时也为许多临床上疾病的一种病理特征， 这些疾病包括组织 发炎、 关节炎、 肿瘤生长、 糖尿病视网膜病变、 视网膜血管发生所引起的黄 斑部退化， 以及类似的疾病。 这些与血管发生作用有关的临床表现又称为血 管发生疾病 (angiogenic diseases)„ 血管发生疾病通常包括癌症（包含实体瘤及 血液肿瘤）， 心血管疾病 (如， 动脉硬化）， 慢性发炎 (如， 类风湿性关节炎或 克隆氏症）， 糖尿病 (如， 糖尿病性视网膜病变）， 牛皮癣、 子宫内膜组织异位 及肥胖症。 因此，医学界亟需一种方法及组合物来有效抑制血管发生作用以治疗或 防止上述疾病。 发明内容
本发明提供一种抑制病理性血管发生作用的方法， 包括给予个体有效量 的 LECT2蛋白或其类似物， 以及药学上可接受的载体。
本发明另提供一种抑制血管发生作用的组合物， 包括有效量的 LECT2 蛋白或其类似物， 以及药学上可接受的载体。
本发明更提供一种治疗和 /或抑制细胞增生性疾病的方法，包括给予个体 有效量的 LECT2蛋白或其类似物， 以及药学上可接受的载体。
本发明更提供一种治疗和 /或抑制细胞增生性疾病的组合物，包括有效量 的 LECT2蛋白或其类似物， 以及药学上可接受的载体。
本发更提供一种抑制病理性血管发生的方法， 包括给予个体有效量的 LECT2蛋白或其类似物， 以及药学上可接受的载体。
为了让本发明的上述和其它目的、 特征、 和优点能更明显易懂， 下文特 举优选实施例， 并配合所附图示， 作详细说明如下： 附图说明
图 1A显示人类肝癌微血管密度 (CD34染色)的免疫组织化学染色分析。 图 1B显示 LECT2蛋白表达量与 HCC微血管密度之间的关系。
图 2A 显示载体对照组细胞 (SK-Hepl/Neo)、 过量表达 LECT2 细胞 (SK-Hepl/LECT2)及野生型细胞 (SK-Hepl/WT)的生长曲线类似。
图 2B显示 SK-Hepl Neo组的平均肿瘤体积明显大于 SK-Hepl/LECT2 组。
图 2C显示 SK-Hepl/LECT2组的微血管密度 (CD31染色)小于载体控制 组 (SK-Hepl/Neo)。
图 3 A 显示载体对照组细胞（BNL Neo)、 过量表达 LECT2 细胞 (BNL/LECT2)与野生型细胞 (BNL/WT)的生长曲线类似。
图 3B显示 BNL/Neo组的平均肿瘤体积明显大于 BNL/LECT2组。
图 3C显示 LECT2可抑制 BNL细胞原位性肿瘤中的微血管密度 (CD31 染色：)。
图 4A分别显示 BNL/Neo组、 BNL Neo+rLECT2组及 BNL/LECT2组的 肿瘤体积。
图 4B-4C显示 BNL Neo组的平均肿瘤体积明显大于 BNL/Neo+ rLECT2 组及 BNL/LECT2组。
图 5显示过量表达 LECT2细胞的条件培养液可破坏新血管的生成。 图 6A显示 LECT2条件培养液可抑制血管管状结构的形成。
图 6B显示内生性 LECT2(shLECT2-l及 shLECT2-2)被抑制的 HCC转化 株 (PLC PRF/5及 Huh-7)的 LECT2表达量。
图 6C显示相较于对照组细胞， 内生性 LECT2被抑制的 PLC/PRF/5与 Huh-7细胞的条件培养液可促进血管内皮细胞中的血管管状结构的生成。
图 7显示 LECT2条件培养液显著地抑制血管内皮细胞的移动。
图 8显示相较于经 VEGF-A (50 ng/mL)处理的细胞， rLECT2蛋白可明显 抑制 VEGF-A诱导的管状结构生成。
图 9A-9B显示以 CAM模式证实 LECT2重组蛋白可抑制 VEGF-A诱导 的血管发生作用。
图 10显示 LECT2重组蛋白可减少 VEGF-A诱导的血管内皮细胞移动。 图 11显示 LECT2重组蛋白可抑制血管内皮细胞内因 VEGF-A诱导的管 管状结构形成。
图 12显示 LECT2重组蛋白可抑制血管内皮细胞内条件培养液所诱导的 血管管状结构的形成。
图 13A-13B显示 B16Fl/Neo组的平均肿瘤体积明显大于 B16F1/LECT2 组。
图 13C显示 B16F1/LECT2组的微血管密度小于 B16Fl/Neo组。
图 14显示 LECT2蛋白可抑制 A549细胞在棵鼠内的肿瘤生长。 具体实施方式
本发明提供一种抑制病理性血管发生作用和 /或细胞增生疾病的组合物。 此组合物包括有效量的 LECT2蛋白或其类似物， 以及药学上可接受的载体。
本发明中所述的 "LECT2"或" LECT2蛋白"指白细胞衍生趋化素 2蛋白的 所有形式， 包括活性或失活的形式。 任何具有生物活性的 LECT2蛋白的类 似物 /片断皆在本发明的范畴中。 类似物 /片断可与载体分子， 如免疫蛋白接 合以进行各种的应用， 包括增加蛋白质接合位的价数。此外，本发明的 LECT 2蛋白类似物也可藉由于原始蛋白中置换、 缺失、 增加或插入一个或复数个 氨基酸来获得， 且其仍保有至少原始蛋白的活性部份。
本发明中所述的" LECT2 变体"或"变体蛋白"包括至少有 60%序列与 LECT2蛋白相同的蛋白质，序列的相似性以蛋白质的氨基酸序列来界定， 以 最大化相同部份及最小化序列差异。
本发明的 LECT2蛋白也包括经修饰的 LECT2蛋白， 其类似于天然的
LECT2蛋白。 熟悉此技术领域的人员可以一般传统的技术对肽或 DNA序列 进行修饰。 例如， 蛋白质序列的修饰可包括改变、 置换、 取代、 插入或缺失 编码序列中的一个氨基酸。 改变、 置换、 取代、 插入或缺失序列所使用的方 法为一般已知的技术。 序列在改变、 置换、 取代、 插入或缺失后， 优选仍保 有 LECT2蛋白的活性。
本发明中所述的"重组蛋白"是指蛋白质包括一个或复数个重组部份。 特 定位置重组酶 (Site-specific recombinases)为存在于各种病毒及细菌中的一种 酶， 其同时具有限制内切核酸酶 (endonuclease)及连接酶 (ligase)的特性。 此重 组酶可辨识特定 DNA序列并改变 DNA片断。
在本发明中 , LECT2蛋白、 LECT2蛋白类似物 /片断或 LECT2重组蛋白
(rLECT2)可在动物细胞、 低等真核生物或原核生物中表达。 动物细胞可包括 猴 COS细胞、 CHO细胞、 人类肾脏 293细胞、 人类表皮 A431细胞、 人类 Colo205细胞、 3T3细胞、 CV-1细胞、 其它经转化的灵长类细胞、 正常的二 倍体细胞、 由体外培养的原生组织衍生的细胞株、 原生移植体、 HeLa细胞、 老鼠 L细胞、 BHK、 HL-60、 U937、 HaK或 Jurkat细胞。 低等真核生物包括 酵母菌 , 口酉良酒 酵母 (Saccharomyces cerevisiae)、 粟酒 裂殖酵母 (Schizosaccharomyces pombe)、克鲁维酵母属菌株 (Kluyveromyces strains)、念 珠菌属 (Candida)或任何可表达异源性蛋白的酵母菌。原核生物可包括大肠杆 菌（Escherichia coli)、 枯草芽孢杆菌（Bacillus subtilis)、 鼠伤寒沙门氏菌 (Salmonella typhimurmm) . 或任何可表达异源性蛋白的细菌。 若以酵母菌或 细菌来表达蛋白质， 则可进一步修饰所表达的蛋白， 例如， 于适当的位置上 进行磚酸化或糖基化使蛋白质具有功能。
在本发明中，将组合物投予至组织中，此组织具有病理性血管发生作用， 此组合物包括有效量的 LECT2蛋白或表达活性 LECT2蛋白的核苷酸载体。 此组织可为任何发生血管发生作用的组织或器官， 包括脑、皮肤、 肌肉、 胃、 结締组织、 关节、 骨骼及类似组织。 病理性血管发生作用可受生长因子所诱 导， 包括血管内皮生长因子 (VEGF)、 血小板生长因子 (PDGF；)、 成纤维细胞 生长因子 (FGF；)、 表皮生长因子 (EGF)、 及肝细胞生长因子 (HGF)。
此外， 本发明的 LECT2蛋白、 LECT2蛋白类似物 /片断、 或 LECT2重 组蛋白也可抑制新生血管管状结构的生成、 内皮细胞的移动与增生、 肿瘤的 血管侵入或其它与血管发生作用有关的疾病。 血管发生作用有关的疾病包 括， 但不限于， 发炎性疾病、 慢性风湿性关节炎及牛皮癣、 与不正常血管侵 入有关的疾病， 例如， 糖尿病性视网膜病变、 新生血管型青光眼、 血管再狭 窄、 及细胞增生性疾病， 如与肿瘤或癌症有关的疾病 (；例如， 实体瘤、 实体 瘤转移、 肝癌、 肺癌、 乳癌、 大肠癌、 胃癌及黑色素瘤）。
本发明所述的 "细胞增生性疾病 "是指在多细胞组织中发生一个或复数 个细胞群不正常增生所导致的多细胞组织伤害。 细胞增生性疾病可发生于不 同的动物及人类。 细胞增生性疾病包括， 但不限于， 癌症、 血管增生性疾病 及纤维化疾病。
本发明的 LECT2蛋白和 /或包含 LECT2蛋白的药学组合物可以口服，非 肠胃、 吸入、 直肠、 阴道、 皮内、 经皮或局部给予， 药剂单位可包括传统无 毒的药学上可接受的载体、 佐剂及载具， 且可藉由蠕动性工具给予。
本发明的 LECT2蛋白和 /或包含 LECT2蛋白的药学组合物可一次给予， 24小时内多次给予或连续给予。 当注射的方式为连续给予时， 可选用适合的 已知方式， 包括， 但不限于， 静脉注射点滴、 静脉注射泵、 埋植式注射泵或 局部给予。 治疗的时间可依不同的情况做适当的调整， 例如， 血管发生作用 的病程及严重性。 在以 LECT2蛋白单独或合并本发明其它药剂直到搔痒症 结束， 或持续治疗终身。
在另一实施方案中，本发明提供一种抑制病理性血管发生作用和 /或细胞 增生性疾病的药学组合物。 此药学组合物包括有效量的 LECT2蛋白或其类 似物， 以及药学上可接受的载体。 药学上可接受的载体包括溶剂、 分散剂、 涂层、 抗菌剂 /抗真菌剂、 等渗剂、 吸收延迟剂及其类似物。
在另一实施方案中， 本发明另提供一种抑制病理性血管发生的方法， 包 括给予个体有效量的 LECT2蛋白或其类似物， 以及药学上可接受的载体。 实施例
实施例 1 LECT2蛋白对人类微血管密度的影响
首先， 以免疫组织化学染色法分析 14个人类肝癌 (HCC)组织的 LECT2 蛋白表达及内皮细胞的生物标志， 此人类肝癌组织由台湾大学医学院附设医 院提供。 简言之， 从肝癌患者取得原发性肝癌肿瘤组织， 以甲醛固定及石蜡 包埋后进行免疫染色。 脱蜡、 脱水处理后， 以 PBST清洗 3次， 于室温下， 以 1 : 500稀释的 CD34抗体， 再以生物素标定抗小鼠 IgG二抗处理 (Vector Laboratories, Inc., Burlingame, CA)1小时, 以 ABC试剂盒 (Vector Laboratories Inc.)侦测结合的抗体。 将此切片以二氨基联苯胺 (diammobenzidine)染色、 清 洗， 最后以德式苏木^ :青 (Delafield's hematoxylin)进行反染色 (counterstain)后分 析并计数结果。藉由对内皮细胞抗原染色来测定微血管密度并测量血管发生 的程度， 微血管密度 (MVD)的测定是通过计数被 CD34抗体染色的内皮细胞 来表示。 对每个肿瘤中三个高度血管化的区域以高放大倍率进行分析， 计数 每个区域的微血管总数， 以及每个肿瘤的微血管平均数目。 图 1A显示人类 HCC微血管密度的分析结果， 肿瘤的位置可被 CD34抗体染色。在所有的肿 瘤样本中， 以免疫组织化学染色法分析肝肿瘤的内皮细胞 CD34标志， 证实 在肿瘤细胞的周围存在许多的内皮细胞， 此结果与临床所发现的高度血管化 肿瘤相符。 参照图 IB, LECT2蛋白的表达量与微血管的密度呈相反的趋势。 将病患的肝癌细胞区分成 2组， 包括高 LECT2蛋白表达量 (LECT2/p-肌动蛋 白比〉 0.45)及低 LECT2 蛋白表达量 (LECT2/p-肌动蛋白比 < 0.45)。 在统计 上， 高 LECT2蛋白表达量与低微血管密度上具有高度的相关性。 由此可知， 在 14个人类肝癌样本中， LECT2蛋白表达量与微血管密度呈现相反的趋势。 实施例 2 LECT2蛋白对裸鼠肿瘤生长的影响
在本实施例中 , 使用没有内源性表达 LECT2蛋白的人类肝癌细胞株： SK-Hepl。 将人类 LECT2的 cDNA构建至 pSecTag2A真核表达载体中， 以 获得过量表达 LECT2蛋白的表达载体，且在相同启动子的下游含有潮霉素 B (hygromycin B)基因。以 Lipofectamine (脂质转染胺） 2000试剂（Invitrogen Life tech.)将此 LECT2蛋白的表达载体转染至肝癌细胞 (SK-Hepl)中。经潮霉素 B 筛选后， 可获得过量表达 LECT2蛋白的细胞株 (SK-Hepl/LECT2)， 过量表达 LECT2蛋白细胞株 (SK-Hepl /LECT2)的 LECT2蛋白表达量至少高于对照组 细胞株 (SK-Hepl/Neo)5倍以上。由图 2A可知，对照组细胞株 (SK-Hepl/Neo)、 野生型细胞株（SK-Hepl/WT)以及过量表达 LECT2 蛋白 细胞株 (SK-Hepl/LECT2)的单层培养细胞的生长曲线类似。 本实施例所有的实验皆 重复 3次。 误差长条图具 95%的信赖区间。 为了分析 LECT2蛋白功能对肿 瘤细胞及血管发生作用的影响， 以活体内异种移植肿瘤模式， 将 HCC细胞 以皮下注射方式至棵鼠背部。 简言之， 将 8 周龄的去胸腺母棵鼠 (购自国家 动物中心)养殖于无病原菌的环境中， 并持续哏食及提供饮水。 将棵鼠随机 分为 2组， 包括分别注射过量表达 LECT2蛋白的细胞株 (SK-Hepl/LECT2) 及对照组细胞株 (SK-Hepl/Neo)。 在注射后的第 10天， 当肿瘤变成明显可见 时， 每隔一天测量肿瘤的大小。 肿瘤的体积以宽度 X长度 χθ.5表示。 肿瘤的 大小以双角规形夹进行测量。 所有的小鼠均于注射细胞后 10天可首度测到 明显的肿瘤。 参照图 2Β, 在注射细胞 20天后， 注射 SK-Hepl/Neo细胞株的 棵鼠肿瘤平均体积明显大于注射 SK-Hepl/LECT2细胞株的肿瘤平均体积。 此外， 另利用免疫组织化学染色法， 以 CD31抗体分析原位肿瘤组织的石蜡 切片中， LECT2对微血管密度的影响。由图 2C可知，稳定且高度表达 LECT2 细胞所生成的肿瘤，每 mm2单位体积下微血管数小于对照组 3倍。 由此实施 例可知 LECT2蛋白可抑制棵鼠体内人类肝癌肿瘤 SK-Hepl细胞的生长。 实施例 3 LECT2蛋白对 BALB/c小鼠肿瘤生长的影响
此外， 将过量表达 LECT2蛋白的肿瘤细胞注射至 BALB/c小鼠后， 并 进行分析。 除了分别将 SK-Hepl人类肝癌细胞与棵鼠置换为 BNL老鼠肝癌 细胞及 BALB/c小鼠外，本实施例的分析过程与实施例 2相同。小鼠 (BALB/c 小鼠， 8 周龄）以皮下注射稳定且高表达 LECT2 的 BNL 老鼠肝癌细胞 (BNL/LECT2)或对照组 BNL细胞 (BNL/Neo)， 每只老鼠注射 1 χ 105个细胞。 本实施例的结果与实施例 2 的结果类似。 参照图 3Α， 野生型细胞株 (BNL/WT)、 对照组细胞株 (BNL/Neo)及过量表达 LECT2 蛋白细胞株 (BNL/LECT2)的单层培养细胞的生长曲线类似。 参照图 3B, 注射 BNL/Neo 细胞株的小鼠肿瘤体积明显大于注射 BNL/LECT2细胞林的肿瘤体积， 且在 以 CD31抗体分析原位肿瘤组织中的微血管密度后发现， 微血管密度会受到 高 LECT蛋白表达后而被抑制（图 3C)。 本实施例的结果显示 LECT2蛋白可 抑制 BALB/c小鼠体内老鼠肝癌肿瘤 BNL细胞的生长。 实施例 4 LECT2重组蛋白对 BALB/c小鼠肿瘤生长的影响
本实施例分析 LECT2重组蛋白是否同样可影响被移植 HCC肿瘤的小 鼠， 评估 LECT2重组蛋白对注射 BNL/Neo细胞的 BALB/c小鼠的影响。 当 BNL/Neo组的肿瘤生长至约 150 mm3时，分别将 LECT2重组蛋白（1.25 mg/kg 体重)或 1X PBS緩沖液 (50 L)注射至肿瘤内， 持续注射 10天， 结果如图 4A 所示。 参照图 4B-4C, 在注射细胞后 35天， BNL/Neo组的肿瘤平均体积明
BNL/LECT2+rLECT2组。 图 4C显示每组异种肿瘤移植 BALB/c小鼠的肿瘤 重量。 同样地， BNL/LECT2+rLECT2组与 BNL/LECT2组的肿瘤重量明显小 于 BNL/Neo组。 由此可知， LECT2重组蛋白可抑制 BALB/c小鼠体内小鼠 肝癌肿瘤 BNL细胞的生长。 实施例 5 表达 LECT2的人类 HCC细胞条件培养液对血管发生作用的 影响
在本实施例中， 利用鸡胚胎绒毛膜分析 (chick chorioallantoic membrane assay; CAM assay)来评估过量表达 LECT2的 SK-Hepl细胞的条件培养液对 血管发生作用的影响。此培养液来自于转染 LECT2或载体的 SK-Hepl细胞。 将癌细胞培养于不含胎牛血清的 DMEM培养液中 48小时。 将转染 LECT2 及载体的 SK-Hepl细胞的条件培养液以低转速离心过滤。当研究体内血管发 生作用过程中， 对于多种调控因子的血管反应时， 鸡胚胎的 CAM分析可作 为一种适合的模式。 将受精卵培养于 100。F, 湿度控制的培养箱且空气流通 的环境下 14天。 在第 9天时， 在蛋的上方凿出一个小洞， 位于主要血管区 域的上方， 使鸡胚胎绒毛膜与蛋壳膜分开。 以 SK-Hepl/Neo 条件培养液、 SK-Hepl /LECT2 条件培养液、 或预处理抗 LECT2 或抗 IgG 抗体的 SK-Hepl/LECT2条件培养液进行处理。将总体积为 20 的条件培养液滴至 直径 5mm的小圓片上， 其小圓片覆于鸡胚胎绒毛膜 (每组处理 3个鸡胚)。将 蛋上方的小洞以透气胶布封合， 并放回培养箱中。 于处理后 3 天 (第 12天) 及处理后 5天 (第 14天)， 小心地打开鸡蛋。 藉由测量血管长度与密度来评估 鸡胚胎绒毛膜中的血管生成。 比较对照组与实验组中的血管数量来分析 LECT2重组蛋白对血管发生作用的影响， 以 Image-Pro Plus 4.5软件分析血 管的数量。 参照第 5图， 过量表达 LECT2蛋白的条件培养液可抑制鸡胚胎 绒毛膜中毛细管床的血管发生作用。 由 CAM 分析中发现， 对于过量表达 LECT2蛋白的条件培养液而言， 与添加 IgG 中和抗体的对照组相比， 添加 LECT2抗体预先竟争结合条件培养液中 LECT2蛋白后， 则会促进血管发生 作用。 由此可知， 在 CAM的分析中， 过量表达 LECT2的 SK-Hepl细胞的 条件培养液会抑制血管发生作用。 实施例 6 过量表达 LECT2的 HCC细胞条件培养液对管状结构形成的 影响
在体外， 内皮细胞在 6小时内会自然地于 matrigel上对准且形成二维空 间微管状网络。 Matrigel 分析为一种细胞外间质 (ECM)分析， 其提供生理环 境用以分析内皮细胞的形态、 生化功能与基因表达。 将血管内皮细胞 (HUVECs)置于 Matrigel上以分化及形成类微管状结构。 在 Matrigel上， 微 管状的形成需要细胞 -间质的相互反应、 细胞间的沟通以及细胞的移动。 为 分析体外 LECT2重组蛋白质对血管发生作用的影响， 将血管内皮细胞置于 覆盖 Matrigel的 24孔培养板中， 并分别以转染 LECT2或 Neo的 SK-Hepl 或 HCC36细胞的条件培养液处理 6小时， 以倒置相差显微镜来观察管状结 构的形成。 在显微镜的观察下， 分别比较 6个随机选取视野下的管状网絡。 参照图 6A,相较于对照组的培养液 (SK-Hepl/Neo条件培养液或 HCC36/Neo 条件培养液)， SK-Hepl/LECT2条件培养液或 HCC36/LECT2条件培养液可 抑制 Matrigel层上管状结构的形成。 将血管内皮细胞培养于 Matrigel覆盖的 24孔板中， 并以 VEGF- A处理 6小时， 作为正对照组。 为证明 LECT2重组 蛋白为治疗血管发生作用中调控血管发生作用的重要因子， 进而抑制 PLC/PRF/5与 Huh-7 细胞中内源性的 LECT2(shLECT2-l 与 shLECT2-2)表 达。 参照图 6B， 由 Western印迹法分析内源性 LECT2被抑制的转化株。 参 照图 6C, 内源性 LECT2被抑制的 PLC/PRF/5与 Huh-7细胞的条件培养液可 促进血管内皮细胞中的管状结构生成能力。 由此可知， LECT2被抑制的细胞 条件培养液可促进血管内皮细胞中的管状结构生成。 实施例 Ί 过量表达 LECT2蛋白的 HCC细胞的条件培养液对细胞移动 的影响
将血管内皮细胞培养于 24孔培养板的 (7x104 cells/well)M199培养基中。
24小时后， 更换为含 5%胎牛血清的培养基至血管内皮细胞单层膜， 并以蓝 色微量吸管尖刮除一条直线以获得不含细胞的单层膜。 将含有过量表达 LECT2的 HCC条件培养液 (SK-Hepl /LECT2 CM)及载体对照组 HCC条件培 养液 (SK-Hepl Neo CM) , 或内源性 LECT2 被抑制的 HCC 条件培养液 (Huh-7/shLECT2-2 CM)及载体对照组 HCC条件培养液 (Huh-7/shLuc CM)。 于 37。C下培养 14小时后， 以倒置相差显微镜观察内皮细胞的移动能力， 并 分别测量 4个随机选取视野下， 细胞移动后的程度。 藉由 Image Pro-Plus 4.5 软件分析所获得影像。 每个孔板分别观察 4个视野， 并测量刮除细胞后的伤 口宽度。 参照图 7, 在培养于 50%的 LECT2转染或内源性 LECT2被抑制的 条件培养液后 14个小时， 血管内皮细胞会移动至伤口区域。 LECT2条件培 养液会抑制血管内皮细胞的移动， 且内源性 LECT2被抑制的 Huh-7条件培 养液 (Huh-7/shLECT2-2 CM)会促进血管内皮细胞的修复反应。 由此可知， 过 量表达 LECT2的 HCC条件培养液可抑制血管内皮细胞的移动能力。 实施例 8 LECT2重组蛋白对血管内皮生长因子诱导的管状结构生成的 影响
将血管内皮细胞置于 24孔板的 Matrigel上， 并以 VEGF-A(50 ng/mL)、
PDGF(50 ng/mL)、 bFGF(30 ng/mL)、 EGF(50 ng/mL)、 HGF(40 ng/mL)或含 LECT2重组蛋白（200 ng/mL)处理 6小时， 并分析类啟管结构的形成。 由结 果可知， LECT2重组蛋白可有效地抑制 VEGF-A所诱导的血管管状结构的 新生， 与 VEGF-A (50 ng/mL)组相比， 其抑制率为 52%， 如图 8所示。 由此 可知， LECT2 重组蛋白可不同程度抑制血管生长因子 VEGF-A、 PDGF、 bFGF、 EGF、 HGF诱发内皮细胞管状结构的生成， 特别是 VEGF-A的作用。 实施例 9 LECT2重组蛋白对 VEGF-A诱导的血管发生作用的影响 以 CAM分析 LECT2重組蛋白对 VEGF-A诱导的血管发生作用的影响。 将 9天大鸡胚胎的鸡胚胎绒毛膜以 VEGF-A, 以及不同浓度的 LECT2重组 蛋白（0、 1.25、 2.5与 5 nM)或 Fc-Tag (对照组)处理 (每组处理 3个鸡胚胎）， 滴 于直径为 5 mm的小圓片中， 其总体积为 20 L， 小圓片覆盖于鸡胚胎绒毛 膜上， 在以 VEGF-A及 LECT2重组蛋白处理鸡胚胎绒毛膜后 3天 (即第 12 天)或 5天 (即第 14天)。 参照图 9A， 相较于对照组， VEGF-A (50 ng/mL)明 显促进绒毛膜尿嚢的微血管床区域。 参照图 9B, 由 CAM分析结果可知， 在 第 12天， 2.5 nM与 5 nM的 LECT2重组蛋白可明显抑制由 VEGF-A诱导的 血管发生作用。 在第 14天， 5 nM的 LECT2重组蛋白可抑制由 VEGF-A诱 导的绒毛膜尿嚢微血管床。 由此可知， LECT2重组蛋白可抑制 VEGF-A诱 导的血管发生作用。 实施例 10 LECT2重组蛋白对体外 VEGF-A诱导的血管发生作用的影响 本实施例分析 LECT2重组蛋白对血管内皮细胞移动及血管生成的影响。 血管内皮细胞的移动为血管发生作用的重要因子之一， 因此分析 rLECT2蛋 白对于 VEGF-A诱导的血管内皮细胞移动的能力。分别以 2% FBS (对照组)、 VEGF-A (50 ng/mL)、 VEGF-A (50 ng/mL)同时处理不同浓度 LECT2重组蛋 白（0 200 ng/mL)、以及 LECT2重组蛋白（200 ng/mL)处理 14小时来进行内皮 细胞移动。 VEGF-A (50 ng/mL)可促进血管内皮细胞的移动。 由图 10可知， LECT2重组蛋白与抑制 VEGF-A诱导的移动具有剂量依赖性。 此外， 尤其 200 ng/mL剂量的 LECT2重组蛋白具明显抑制 VEGF-A诱导的血管内皮细 胞移动。 由于 LECT2重组蛋白可抑制 VEGF-A诱导的细胞移动， 因此进一 步分析 LECT2重组蛋白在血管生成时对抗血管发生作用的反应。 在此分析 中， 以 0.1% BSA (对照组)或 VEGF-A (50 ng/mL)合并不同浓度的 LECT2重 组蛋白 （0-200 ng/mL)来分析类微管结构的形成。 由图 11可知， LECT2重组 蛋白与瓦解 VEGF-A诱导的微管网络具有剂量依赖性。 由此可知， LECT2 重组蛋白可抑制体外 VEGF-A诱导的血管内皮细胞类微管结构。 实施例 11 LECT2重组蛋白对不同癌细胞条件培养液诱导的血管管状结 构形成的影响
本实施例分析 LECT2重组蛋白对不同癌细胞株的血管发生作用的影响。 将血管内皮细胞置于 24孔板的 Matrigel上， 并以各种癌细胞株的条件培养 液处理 6 小时， 癌细胞包括肝癌细胞 (SK-Hepl 及 Huh-7)、 肺癌细胞 (A549 及 H1299)、 乳癌细胞 (MB-MDA-231及 MCF7)、 胃癌细胞 (MKN及 N87)、 直肠癌细胞 (HCT116)及老鼠黑色素瘤细胞 (B16F1), 或癌细胞株条件培养液 合并 rLECT2(5 nM)处理 6小时。 图 12显示单独以癌细胞条件培养液， 或合 并 rLECT2蛋白（5 nM)处理血管内皮细胞 6小时的结果。 由此可知， LECT2 重组蛋白可抑制 SK-Hepl、 A549、 MCF-7与 B16F1细胞条件培养液诱导内 皮细胞的血管管状结构生成。 实施例 12 LECT2蛋白对黑色素瘤及肺细胞肿瘤发生及血管发生作用的 影响
首先，构建过量表达 LECT2的老鼠黑色素瘤细胞株 B16F1。以潮霉素 B (hygromycin B)筛选后， 分析 LECT2的表达量， 并利用活体内异种黑色素瘤 移植模式来评估 LECT2蛋白对肿瘤生长及血管发生作用的影响。 将 B16F1 细胞 (B16Fl/Neo及 B16F1/LECT2)皮下注射至 Ί周龄的 C57BL/6母鼠中，每 只小鼠注射 5χ 105细胞。 参照图 13A, 在注射细胞后第 18天， B16Fl/Neo 细 胞之小鼠的平均肿瘤体积明显大于注射 B16F1/LECT2细胞的小鼠肿瘤体积。 在第 24天后， 将小鼠牺牲， 取出皮下肿瘤并拍照， 如第 13B所示。 此外， 以免疫组织化学染色法分析过量表达 LECT2蛋白的肿瘤细胞对异种肿瘤移 植体内血管发生作用与血管数的影响。 参照图 13C, 以甲醛固定肿瘤标本， 以石蜡包埋并以特定血管染色法 (对内皮标志 CD31进行免疫组织化学染色) 将切片染色。 由统计结果可知， 载体对照组细胞的肿瘤， 每 mm2体积单位血 管数是转染 LECT2细胞组的 2倍。 由此可知， LECT2可抑制 C57BL/6J小 鼠体内肿瘤中 B16F1黑色素瘤细胞的生长及血管发生作用。 实施例 13 LECT2蛋白对棵鼠肿瘤生长的影响
将肺癌 A549细胞皮下注射至 7周龄的母棵鼠中， 每只小鼠注射 5χ 106 个细胞。 在注射 Α549细胞后 20天， 腹腔注射 D-luciferin (20 mg/mL/小鼠每 公斤体重）， 将小鼠麻醉， 并以 Berthold NightOWL影像系统摄影 5分钟。 参 照图 14 , 即使是短时间的曝光 (30秒）， 彩色影像亦显示所有 A549/Luc/Neo 肿瘤小鼠的背部皮下皆具有明亮的光点， 但此光点在 A549/Luc/LECT2肿瘤 小鼠中则明显较小， 表示 LECT2可抑制棵鼠肿瘤的肺癌细胞生长。 由此可 知， LECT2蛋白可有效地抑制 A549细胞在棵鼠的肿瘤生长。
虽然本发明已以优选实施例揭露如上， 然其并非用以限定本发明， 任何 本领域技术人员 ,在不脱离本发明的精神和范围内 ,当可作些许更动与润饰， 因此本发明的保护范围当视后附之申请专利范围所界定者为准。

Claims

权利要求书
I. 一种在个体中抑制病理性血管发生作用的组合物， 包括有效量的
LECT2蛋白或其类似物， 以及药学上可接受的载体。
2. 权利要求 1所述的抑制病理性血管发生作用的组合物，其中该 LECT2 蛋白类似物为 LECT2重组蛋白。
3. 权利要求 1所述的抑制病理性血管发生作用的组合物，其中该 LECT2 蛋白或其类似物为血管发生作用因子抑制剂。
4. 权利要求 3所述的抑制病理性血管发生作用的组合物，其中该血管发 生作用因子选自下组： 血管内皮生长因子 (VEGF)、 血小板生长因子 (PDGF)、 成纤维细胞生长因子 (FGF)、 表皮生长因子 (EGF)及肝细胞生长因子 (HGF)。
5. 权利要求 3所述的抑制病理性血管发生作用的组合物，其中该血管发 生作用因子为血管内皮生长因子抑制剂。
6. 权利要求 1所述的抑制病理性血管发生作用的组合物，其中该 LECT2 蛋白或其类似物为内皮细胞移行抑制剂。
7. 权利要求 1所述的抑制病理性血管发生作用的组合物，其中该 LECT2 蛋白或其类似物为肿瘤血管侵入抑制剂。
8. 权利要求 1所述的抑制病理性血管发生作用的组合物，其中该病理性 血管发生作用伴随细胞增生性疾病。
9. 权利要求 8所述的抑制病理性血管发生作用的组合物，其中该细胞增 生性疾病为癌症。
10. 权利要求 9所述的抑制病理性血管发生作用的组合物， 其中该癌症 选自下组： 肝癌、 肺癌、 乳癌、 大肠癌、 胃癌及黑色素瘤。
II. 权利要求 9所述的抑制病理性血管发生作用的组合物， 其中该癌症 为肺癌或肝癌。
12. 权利要求 1所述的抑制病理性血管发生作用的组合物， 其中该个体 为哺乳类动物。
13. 权利要求 1所述的抑制病理性血管发生作用的组合物， 其中该个体 为人类。
14. 一种在个体中治疗和 /或抑制细胞增生性疾病的组合物， 包括有效量 的 LECT2蛋白或其类似物， 以及药学上可接受的载体。
15. 权利要求 14所述的治疗和 /或抑制细胞增生性疾病的组合物， 其中 该 LECT2蛋白类似物为 LECT重组蛋白。
16. 权利要求 14所述的治疗和 /或抑制细胞增生性疾病的组合物， 其中 该细胞增生性疾病为癌症。
17. 权利要求 16所述的治疗和 /或抑制细胞增生性疾病的组合物， 其中 该癌症选自下组； 肝癌、 肺癌、 乳癌、 大肠癌、 胃癌及黑色素瘤。
18. 权利要求 16所述的治疗和 /或抑制细胞增生性疾病的组合物， 其中 该癌症为申癌或肝癌。
19. 权利要求 14所述的治疗和 /或抑制细胞增生性疾病的组合物， 其中 该 LECT2蛋白或其类似物抑制该个体的肿瘤生长。
20. 权利要求 14所述的治疗和 /或抑制细胞增生性疾病的组合物， 其中 该 LECT2蛋白或其类似物为肿瘤内皮细胞移行抑制剂。
21. 权利要求 14所述的治疗和 /或抑制细胞增生性疾病的组合物， 其中 该 LECT2蛋白或其类似物为肿瘤血管侵入抑制剂。
22. 权利要求 14所述的治疗和 /或抑制细胞增生性疾病的组合物， 其中 该个体为哺乳类动物。
23. 权利要求 14所述的治疗和 /或抑制细胞增生性疾病的组合物， 其中 该个体为人类。
24. 一种 LECT2 蛋白或其类似物用于制备抑制病理性血管发生作用的 医药组合物的用途。
25. 权利要求 24所述的 LECT2蛋白或其类似物的用途， 其中该 LECT2 蛋白类似物为 LECT2重组蛋白。
26. 一种 LECT2蛋白或其类似物用于制备治疗和 /或抑制细胞增生性疾 病的医药组合物的用途。
27. 权利要求 26所述的 LECT2蛋白或其类似物的用途， 其中该细胞增 生性疾病为癌症。
28. 权利要求 27所述的 LECT2蛋白或其类似物的用途， 其中该癌症选 自下组： 肝癌、 肺癌、 乳癌、 大肠癌、 胃癌及黑色素瘤。
29. 权利要求 27所述的 LECT2蛋白或其类似物的用途， 其中该癌症为 肺癌或肝癌。
30. 权利要求 26所述的 LECT2蛋白或其类似物的用途， 其中该 LECT2 蛋白类似物为 LECT2重组蛋白。
31. 一种抑制病理性血管发生的方法， 包括给予个体有效量的 LECT2 蛋白或其类似物， 以及药学上可接受的载体。
32. 权利要求 31所述的抑制病理性血管发生的方法， 其中该 LECT2蛋 白类似物为 LECT2重组蛋白。
33. 权利要求 31所述的抑制病理性血管发生的方法， 其中该 LECT2蛋 白或其类似物抑制血管发生因子的活性。
34. 权利要求 33所述的抑制病理性血管发生的方法，其中该血管发生因 子选自下组： 血管内皮生长因子 (VEGF)、 血小板生长因子 (PDGF)、 成纤维 细胞生长因子 (FGF)、 表皮生长因子 (EGF)及肝细胞生长因子 (HGF)。
35. 权利要求 33所述的抑制病理性血管发生的方法，其中该血管发生因 子为血管内皮生长因子。
36. 权利要求 31所述的抑制病理性血管发生的方法， 其中该 LECT2蛋 白或其类似物抑制内皮细胞的移行。
37. 权利要求 31所述的抑制病理性血管发生的方法， 其中该 LECT2蛋 白或其类似物抑制肿瘤的血管侵入。
38. 权利要求 31所述的抑制病理性血管发生的方法，其中该病理性血管 发生伴随细胞增生性疾病。
39. 权利要求 38所述的抑制病理性血管发生的方法，其中该细胞增生性 疾病为癌症。
40. 权利要求 39所述的抑制病理性血管发生的方法，其中该癌症选自下 组： 肝癌、 肺癌、 乳癌、 大肠癌、 胃癌及黑色素瘤。
41. 权利要求 39所述的抑制病理性血管发生的方法，其中该癌症为肺癌 或肝癌。
42. 权利要求 31所述的抑制病理性血管发生的方法，其中该个体为哺乳 类动物。
43. 权利要求 31 所述的抑制病理性血管发生的方法， 其中该个体为人 类。
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