WO2006119706A1 - Procede auxiliaire de diagnostic et traitement du cancer a la nucleoline - Google Patents

Procede auxiliaire de diagnostic et traitement du cancer a la nucleoline Download PDF

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WO2006119706A1
WO2006119706A1 PCT/CN2006/000942 CN2006000942W WO2006119706A1 WO 2006119706 A1 WO2006119706 A1 WO 2006119706A1 CN 2006000942 W CN2006000942 W CN 2006000942W WO 2006119706 A1 WO2006119706 A1 WO 2006119706A1
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nucleolin
antibody
cell
cells
angiogenesis inhibitor
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PCT/CN2006/000942
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English (en)
French (fr)
Inventor
Yongzhang Luo
Hubing Shi
Zhuobing Zhang
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Tsinghua University
Protgen Ltd.
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Priority to AU2006246197A priority Critical patent/AU2006246197B2/en
Priority to AT06722440T priority patent/ATE516499T1/de
Priority to CA002607832A priority patent/CA2607832A1/en
Priority to DK06722440.2T priority patent/DK1912066T3/da
Priority to JP2008510385A priority patent/JP4742142B2/ja
Priority to EP06722440A priority patent/EP1912066B1/en
Publication of WO2006119706A1 publication Critical patent/WO2006119706A1/zh
Priority to HK08110478.2A priority patent/HK1114903A1/xx
Priority to AU2010241351A priority patent/AU2010241351B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell

Definitions

  • the present invention relates to a novel method for identifying cancer subjects, particularly human patients, suitable for receiving anti-angiogenic cancer therapies.
  • the invention also relates to a method of searching for and screening for angiogenesis inhibitors.
  • the angiogenesis inhibitor is believed to be effective in inhibiting malignant growth of cells, and is particularly effective in angiogenesis-dependent cancers.
  • the present invention discloses a method for screening an angiogenesis inhibitor using nucleolin (hereinafter referred to as "NL").
  • NL nucleolin
  • the methods of the invention involve screening for an angiogenesis inhibitor that is similar in mechanism to endostatin (hereinafter referred to as "ES").
  • ES endostatin
  • the present invention is based on the following findings: NL is a specific receptor for ES and is involved in ES signaling pathways involved in the inhibition of angiogenic activity.
  • ES is the C-terminal globular domain of collagen XVIII with a molecular weight of 20 kDa. Initially it was isolated from the supernatant of murine endothelial cell culture medium because it inhibits the proliferation of capillary endothelial cells. In animal experiments, repeated use of ES can cause tumors to die out and not develop resistance. In addition, ES toxicity was shown to be low in both animal experiments and clinical settings. ES has a significant ability to inhibit endothelial cell proliferation, migration, adhesion, survival and induce apoptosis.
  • ES is considered to be an effective cancer treatment because ES kills tumors by inhibiting angiogenesis. Tumor cells need to spread through new blood vessels. Each increase in the tumor cell population must occur after the onset of new capillaries at the tumor. This phenomenon is almost universal: most human solid tumors or blood cancers are angiogenesis-dependent. Anti-angiogenic therapies have other advantages, including low toxicity, minimal drug resistance, and a longer period of tumor dormancy after repeated use of the therapy, during which no further treatment is required, see Boehm et al Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance. Nature (1997) 390: 404 ⁇ 07. However, the mechanism of action of ES is still unclear.
  • ES treatment is equally used in every cancer patient without considering the individual's sensitivity to the treatment.
  • the study found that in order for ES to effectively exert its function as an angiogenesis inhibitor to achieve the desired anticancer effect, a large amount of ES must be prepared and injected into a mammal or human suffering from cancer. Because of the high dose of ES required in this process, the price can be prohibitively expensive for the patient to afford. Therefore, there is an urgent need to find other methods to discover new angiogenesis inhibitors, which can be used economically and effectively for treating tumors. A number of clinical trials have been conducted in the hope of finding effective anti-angiogenic drugs. On the other hand, it would be a significant advancement to be able to select patients who are eligible for ES treatment with objective criteria.
  • cancer therapies vary widely for different patients, depending on a range of intrinsic or extrinsic factors. External factors include the stage of development of the cancer at the time of treatment (the sooner the finding is found to be beneficial for treatment and recovery) and the relative intensity of treatment, such as surgery, chemotherapy or radiation therapy. Intrinsic factors include the health of the patient's immune system, and a robust immune system can support long-term and high-intensity treatments to help patients recover quickly.
  • individualized medication Different individuals may have different tolerances and sensitivities to the same cancer drugs, prompting efforts to find ways to improve the effectiveness of cancer treatment. Therefore, due to individual differences, drugs that are effective for one patient are not necessarily effective for another patient.
  • Nucleoside (rmd e0 li n , NL) is a widely occurring non-histone, originally isolated from the nucleolus.
  • the amount of NL is regulated by Granzyme A and self-cleaving ability, and interestingly, it is also related to cell proliferation.
  • NL is also self-cleavable, its self-cleavage decreases as the rate of cell proliferation increases, and at the same time it is cleaved by an esterase Granzyme A released by cytotoxic lymphocytes (Chen et al., J. Biol. Chem., 1991, 266, 7754-7758; Fang and Yeh, Exp. Cell. Res" 1993, 208, 48-53; Pasternack et al., J. Biol. Chem., 1991, 266, 14703-14708). These cleavage and concomitant degradation constitute post-translational regulation of NL.
  • NL plays a key and fundamental role in cell proliferation, including: nucleolar chromatin alignment, pre-RNA packaging, rDNA transcription, and ribosome assembly. These activities of L are regulated by certain protein kinases such as CK2 and cdc2, which in turn are tightly regulated by other cyclins.
  • NL can also shuttle through cell surfaces, cytoplasm, and nucleus, and has the function of cell surface receptors. As a receptor for many viruses and cytokines, NL can trigger endocytosis of ligands when ligands bind to them.
  • L molecular weight is about 100-110 kDa, mainly in the nucleus of proliferating cells, L can self-degrade, and exhibits two bands of 70 and 50 kDa in immunoblotting.
  • NL is highly phosphoric acid And methylation, and can be ribosylated by adenosine diphosphate. Since the synthesis of NL is positively correlated with the increase in the rate of cell division, tumor cells and vigorously dividing cells contain a greater amount of NL. The sequence of NL was previously reported by Srivasta Va , et al. in Cloning and sequencing of the human nucleolin cDNA. FEBS Lett. 250 (1), 99-105 (1989).
  • NL also known as P92 or C23
  • P92 or C23 is the most abundant phosphorylated protein in actively growing nucleoli (Srivastava et al" FEBS Lett., 1989, 250, 99-105; Srivastava et al, J. Biol. Chem., 1990, 265, 14922-14931. It is known that NL is mainly involved in the biosynthesis of ribosomes (Ghisolfi et al., Mol. Biol. Rep., 1990, 14, 113-114; Sipos and Olson, Biochem. Biophys. Res.
  • NL is involved in the synthesis of ribosomes by transient binding of its own ribonucleic acid consensus sequence to ribosomal precursors (Bugler et al., J. Biol. Chem., 1987). G262, 10, 922, pp. NL can account for 5% of total nucleolar protein (Lapeyre et al, Proc. Natl. Acad. Sci. USA, 1987, 84, 1472-1476; Sapp et al., Eur. J.
  • the versatile nature of the above NL is derived from several structurally and functionally independent domains (Creancier et al., Mol. Biol. Cell., 1993, 4, 1239-1250; Sapp et al., Eur. J. Biochem ., 1989, 179, 541-548).
  • Three domains of NL are known: an N-terminal domain, an intermediate domain, and a C-terminal domain.
  • the N-terminal domain contains a sequence homologous to the HMG protein and can interact with chromatin (Erard et al., Eur. J. Biochem., 1988, 175, 525-530); the intermediate domain contains four RNA recognitions.
  • the motif can specifically bind to the short stem loop structure of 18S and 28S ribosomal RNA (Bugler et al., J. Biol. Chem., 1987, 262, 10922-10925); the C-terminal domain contains a base that can make RNA The region where the base is deposited (Ghisolfi et al., Mol. Biol. Rep., 1990, 14, 113-114; Ghisolfi-Nieto et al., J. Mol. Biol, 1996, 260, 34-53).
  • NL contains a binary nuclear localization signal that spans the N-terminus of the protein as well as the central region. This sequence helps the NL locate within the core. NL accumulates in the nucleus by interacting with other proteins (Schmidt-Zachmann and Nigg, J. Cell Sci., 1993, 105, 799-806).
  • NL According to the domain composition of NL, it is classified as an Ag-NOR protein (ie, an active ribosome gene localized in the nucleolar tissue region), also known as an active ribosome gene marker (Roussel et al, Exp. Cell. Res). ., 1992, 203, 259-269). There is evidence that the transcription of the ribosomal gene requires the Ag-NOR protein, and the expression of the Ag-NOR protein is also used to predict the rate of tumor growth.
  • NL has been purified from human erythroleukemia cells as a matrix adhesion domain (MAR) binding protein. Studies have found that L is involved in the process of anchoring chromatin loops to the nuclear matrix (Dickinson and Kohwi-Shigematsu, Mol. Cell. Biol., 1995, 15, 456-465).
  • MAR matrix adhesion domain
  • CK2 casein kinase II
  • L can be cleaved or cleaved by the esterase Granzyme A secreted by cytotoxic lymphocytes, and has a reduced self-cutting activity when the cells enter a vigorous proliferative phase (Chen et al, J. Biol. Chem., 1991, 266, 7754-7758; Fang and Yeh, Exp. Cell. Res., 1993, 208, 48-53; Pasternack et al., J. Biol. Chem., 1991, 266, 14703-14708).
  • the above cutting process and the consequent degradation constitute post-translational regulation of NL.
  • Anti-NL antibodies can be found in systemic connective tissue diseases including lupus erythematosus (SLE) (Minota et al., J. Immunol., 1990, 144, 1263-1269; Minota et al., J. Immunol., 1991, 146, 2249-2252) and patients with scleroderma-like chronic graft vs. host disease (Bell et al., Br. J. Dermatol., 1996, 134, 848-854) Found in serum. Therefore, pharmacological regulation of NL expression may be a suitable treatment for pathological conditions. Summary of invention
  • the present invention provides a kit for determining the sensitivity of a cancer subject to ES cancer therapy, including markers labeled NL and instructions for use.
  • the kit detects the amount of NL in a sample by marking the NL in the sample collected from the subject.
  • the subject is preferably a mammal, more preferably a human.
  • the labeled NL is the cell surface NL.
  • the marker comprises an antibody, preferably a polyclonal antibody, more preferably a monoclonal antibody.
  • the label comprises a nucleic acid molecule or probe, preferably a DNA probe, more preferably an RNA probe.
  • the invention further provides a method for determining the likelihood of successful administration of an ES cancer therapy to a subject, comprising detecting the level of expression of L in the sample collected from the subject. Determining whether the subject is sensitive to ES cancer therapy is primarily based on the level of expression of NL detected above.
  • the present invention provides methods for screening for angiogenesis inhibitors, particularly those molecules that have similar mechanisms of action to ES.
  • L is used as a target molecule, and a traditional methodological method is used to identify a component which can specifically bind to NL.
  • the molecule also has anti-angiogenic activity. Based on the fact that L is the receptor for ES on the cell surface, the molecules found by the above methods should have a similar mechanism of action as ES.
  • the invention provides a method of obtaining an effective angiogenesis inhibitor specific for L, comprising the steps of: screening a candidate molecule library for a series of specificities that are specific to NL using a suitable binding assay Sexually bound molecules; anti-angiogenic assays are used to verify the effectiveness of these molecules in inhibiting angiogenesis; finally, molecules that specifically bind to NL are screened for molecules that are effective in inhibiting angiogenesis based on the results of the activity assay.
  • the invention provides a method of screening for an angiogenesis inhibitor that inhibits endothelial cell proliferation and/or migration in vitro, comprising the steps of: using a pharmaceutically acceptable method to find specificity as NL Molecules that interact with NL for the target; verify the effectiveness of the molecules obtained in the previous step in inhibiting proliferation or migration of endothelial cells; harvest the molecules obtained by the above steps that inhibit endothelial cell proliferation or migration, and compare them with ES to inhibit blood vessels The effect that occurs.
  • the invention also provides methods of increasing the sensitivity of endothelial cells to ES.
  • the method introduces exogenous NL into the target endothelium, such that NL is overexpressed in the endothelial cells relative to the wild type, and preferred target cells should have no high levels of endogenous NL expression under normal conditions.
  • the method further proposes that the introduction of NL into the target cells can make these cells more sensitive to the anti-angiogenic activity of ES and effectively killed by ES.
  • the present invention also proposes that a target cell (or endothelial cell) expressing a large amount of L on the cell surface can be detected by an antibody against L, which is suitable for treatment with ES.
  • the present invention provides a method of enhancing sensitivity of a target cell to an angiogenesis inhibitor, comprising the steps of: introducing a foreign NL into a target cell to obtain a modified target cell capable of expressing exogenous NL, And the lethality of ES on these cells was examined.
  • the present invention provides a method of enhancing an anti-angiogenic effect of an angiogenesis inhibitor against a target endothelial cell, comprising the steps of: introducing a pharmaceutically effective amount of an exogenous NL gene into the target cell described above, NL is expressed in the cells while the above angiogenesis inhibitor is incubated with the transformed target cells to inhibit growth of the target cells.
  • the present invention provides a method for improving the effect of an angiogenesis inhibitor on controlling tumor growth in a patient, comprising the steps of: detecting the expression level of endogenous NL in a cancer patient by a feasible means; according to the patient's NL
  • the level of expression is used to evaluate the effectiveness of angiogenesis inhibitors. The higher the level of NL expression, the more likely it is that angiogenesis inhibitor therapy will achieve a successful therapeutic effect.
  • the angiogenesis inhibitor is ES.
  • the invention provides a diagnostic kit for determining individual sensitivity of a target cancer cell sensitive to angiogenesis inhibitor therapy, the kit comprising a molecule that specifically binds NL and a pharmaceutically acceptable carrier .
  • the molecule is a polyclonal antibody. More preferably, the molecule is a monoclonal antibody.
  • the invention provides a method of identifying a target tumor that is susceptible to treatment against an angiogenesis inhibitor, the method comprising producing an antibody against NL, screening the target tumor with the anti-NL antibody Samples, as well as tips obtained by specific interactions between the target tumor and the anti-NL antibody, identify target tumors that are sensitive to anti-angiogenesis inhibitor therapy.
  • the antibody is a polyclonal antibody. More preferably, the antibody is a monoclonal antibody.
  • the invention provides a diagnostic kit for determining a target tumor that is sensitive to anti-angiogenesis inhibitor therapy, comprising an antibody against L and a pharmaceutically acceptable carrier.
  • the invention provides a diagnostic kit for determining and selecting a cancer subject that is susceptible to treatment with an angiogenesis inhibitor, the kit comprising a label that binds to NL and prompts for its presence, and instructions for use, It can be used to test the level of L in a sample collected from a cancer subject.
  • the invention provides a method of inhibiting proliferation and/or migration of some endothelial cells in a cell sample, comprising ligating an anti-nucleolin antibody to a cytotoxic agent to form an anti-nucleoenzyme toxic antibody, and The anti-nucleolin toxic antibody is administered to the cell sample to inhibit proliferation and/or migration of the plurality of endothelial cells.
  • the cytotoxic agent is a cytokine. More preferably, the cytotoxic agent is a tumor necrosis factor.
  • the cell sample is obtained from a cancer patient.
  • FIG. 1 shows Human Microvascular Endothelial Cell (hereinafter referred to as Human Microvascular Endothelial Cell)
  • HMEC is a cell line that is sensitive to ES in terms of migration and proliferation.
  • a Cell migration assay was performed with HMEC under the treatment of ES at the indicated concentration, and PBS was used as a control.
  • Figure 2 shows L of ES binding to the cell surface. a, the ES binding protein isolated from the surface of HMEC was identified as N and its fragment.
  • ES binding proteins were isolated from HMEC cell membranes using a Ni-NTA affinity column preloaded with ES. The fraction eluted with PBS buffer containing 500 mM sodium chloride was subjected to SDS-PAGE analysis (left) and immunoblotted with NL monoclonal antibody (right).
  • b specifically binding NL to ES in vitro.
  • Co-immunoprecipitation was performed using recombinant NL and ES.
  • heparin interferes with the formation of complexes of ES-NL.
  • Co-immunoprecipitation was performed with recombinant NL and ES with or without heparin (200 nM) in vitro.
  • HMEC and ES specifically binds to HMEC via the cell surface L.
  • HMEC and ES as well as different concentrations of L antibody, were incubated for 30 minutes at room temperature and then washed three times with PBS buffer. The cells were subjected to SDS-PAGE analysis and immunoblotted with antibodies of ES. -- Actin was used as a control.
  • HEMC and ES 60 g/ml were incubated at 37 ° C and 5% C0 2 for different times. After washing with fresh medium, antibodies against NL and ES were used for co-immunoprecipitation and immunoblotting. --actin as a control. Colocalization of NL and ES on the surface of HMEC cells.
  • FIG. 1 shows that L is the receptor for ES.
  • a In the migration test of Human Umbilical Vascular Endothelial Cell (HUVEC), the inhibitory activity of ES was removed by the labeled concentration of recombinant NL, and PBS was used as a control.
  • b recombinant L itself has no effect on cell migration. A concentration of ES, NL, and a mixture of the two were added to the HUVEC migration assay. PBS was used as a control.
  • c In the migration test of Human Umbilical Vascular Endothelial Cell (HUVEC), the inhibitory activity of ES was removed by the labeled concentration of recombinant NL, and PBS was used as a control.
  • b recombinant L itself has no effect on cell migration. A concentration of ES, NL, and a mixture of the two were added to the HUVEC migration assay. PBS was used as a control.
  • RNA interference plasmids BS/ U6/1356 inhibited the expression of L, while BS U6/263 did not. Immunoblotting of myosin was used as a loading control.
  • Cell proliferation assay was performed with NL-deficient and control HMEC.
  • pBS/U6/1356 The plasmid-mediated method of RA interference inhibited the expression of NL to obtain L-deficient cells.
  • the blank pBSAJ6 plasmid-transfected cells served as controls.
  • the concentration of ES added was shown in the figure.
  • the number of cells was analyzed by MTT assay. 4 shows the signal network of NL-mediated ES.
  • ES is endocytosed by HMEC.
  • HMEC with or without (a) 10 g/ml biotin-labeled ES was incubated for 0.5 hour (b), 1 hour (c), 2 hours (d), 3 hours (e), and 7 hours (f).
  • Incubation of cells with NL antibodies blocked ES
  • Immunoblotting of NL was used as a loading control.
  • ik, NL and integrin ⁇ (mtegrin pl) on the surface of HMEC cells Co-localization.
  • Complete HEMC was stained with mouse anti-L, rabbit anti-integrin ⁇ , and indirect immunofluorescence was detected by laser scanning confocal microscopy. Scale, 10 ⁇ .
  • Figure 5 shows that the L distribution on the cell surface depends on the growth state of the cells. Ah, distribution of NL on the surface of HEMC cells. L on the surface of proliferating cells and quiescent cells was detected by indirect immunofluorescence using rabbit anti-NL.
  • DAPI indicates the position of nuclei in the visual field. Scale, 20 ⁇ .
  • a large complex comprising cell surface NL, integrins (such as integrins ( ⁇ 5 ⁇ 1) and other proteins) is involved in the signaling network of ES, which binds to myosin and is linked to actin fibers through it. Similarly, integrins are through some intracellular anchoring proteins talin, ⁇ -actinin, filagin, and vinculin and actin fiber bundles. There is no direct interaction between NL and integrins.
  • ES can compete with the extracellular matrix for binding to this complex, thereby inhibiting cell adhesion and migration. This binding can also trigger integrin-mediated Signal transduction. On the other hand, this complex mediates the endocytosis of ES, in which the myosin acts as a transporter.
  • ES may be released in the cytosol and inhibit L to make Bd-2.
  • the remaining ES is transported into the nucleus where it inhibits CK2-mediated NL phosphorylation and some downstream events.
  • Figure 7 shows colocalization of ES and NL in vivo.
  • ES and anti-NL antibodies were injected intravenously into mice bearing B16 F10 tumors. Control group was injected with biotinylated ES and purified rabbit IgG.
  • Figure 9 shows that the greater the amount of NL on the target endothelial cells, the stronger the anti-angiogenic activity of ES.
  • a Western blotting with L antibody to verify the inhibition of NL expression by RNA interference plasmids.
  • BS U6/1356 inhibited the expression of NL, while BS U6/263 and BS U6/1356C had no effect, and immunoblotting of actin was used as a loading control.
  • b Western blotting with L antibody to verify the inhibition of NL expression by RNA interference plasmids.
  • BS U6/1356 inhibited the expression of NL, while BS U6/263 and BS U6/1356C had no effect, and immunoblotting of actin was used as
  • L-deficient cells were obtained by inhibiting the expression of NL by the method of RNA interference mediated by BS/U6/1356 plasmid.
  • BS/U6/1356C plasmid transfected cells served as controls.
  • the addition of ES to the concentration of 10 g/tnL cells was analyzed by the MTT method.
  • the present invention is based on a surprising finding that L can act as a receptor for ES to mediate the biological activity of the latter, and that the amount of NL expression at the cytological level can predict the extent of ES potency.
  • NL is one of the most interesting of these proteins and has since proven to be a key member of the ES signaling network. NL was found to be a receptor for ES and mediates its activity in inhibiting angiogenesis.
  • the present invention provides a kit for determining the sensitivity of a cancer subject to ES cancer therapy, Method for achieving a successful likelihood of ES cancer therapy, a method for obtaining an effective angiogenesis inhibitor specific for NL, and a method for screening an angiogenesis inhibitor capable of inhibiting endothelial cell proliferation and/or migration in vitro a method for enhancing sensitivity of a target cell to an angiogenesis inhibitor, a method for enhancing an antiangiogenic effect of an angiogenesis inhibitor against a target endothelial cell, and a target cancer for determining sensitivity against an angiogenesis inhibitor treatment
  • a diagnostic kit for cells, a diagnostic kit for determining and selecting a cancer subject sensitive to angiogenesis inhibitor therapy, a method for improving the effect of an angiogenesis inhibitor on controlling tumor growth in a patient, and an identification for anti-angiogenesis A method of treating an inhibitor of a target cancer cell.
  • endostatin refers to a protein having a molecular weight of 18 to 20 kDa in non-reduction or reduction electrophoresis, respectively; "endostatin” also includes a precursor form of this 18 to 20 kDa protein.
  • conservative mutations in amino acid sequences frequently reported in the literature replace the original amino acid with a structurally, chemically similar amino acid, but this substitution has no significant change in the structure, conformation and activity of the original protein. Such conservative mutations are also within the scope of the appended claims.
  • endostatin also includes a protein or polypeptide which is shortened by the deletion of one or more amino acids at both ends or inside the original protein, but the protein or polypeptide still retains the activity of the original protein to inhibit endothelial cell proliferation.
  • the meaning of "endostatin” also includes an increased protein or polypeptide obtained by adding one or more amino acids to both ends or inside the original protein, but the protein or polypeptide still retains the original protein to inhibit endothelial cell proliferation. active.
  • These molecules can be used for labeling experiments, such as adding a cheese in the first place. The residue was subjected to 125 1 labeling. Labeling with other radioisotopes can also provide a molecular tool to kill target cells with ES receptors.
  • nucleolin refers to a protein whose size is preferably 100 kDa (with a precise molecular weight of 80 kDa without post-modification) by reductive gel electrophoresis.
  • nucleolin also includes a precursor form, a fragment form, and a modified protein or polypeptide form of the lOOkDa protein, as long as it is a protein or polypeptide having a sufficiently similar amino acid sequence and having a function of inhibiting endothelial cell proliferation.
  • nucleolipin Within the meaning of "nucleolipin”.
  • conservative mutations in amino acid sequences frequently reported in the literature replace the original amino acid with a structurally, chemically similar amino acid, but this substitution has no significant change in the structure, conformation and activity of the original protein.
  • These molecules can be used for labeling experiments, such as adding a tyrosine residue in the first position and labeling 125 1 .
  • Such conservative mutations are also within the scope of the appended claims.
  • NL-specific refers to the ability of NL to bind to an angiogenesis inhibitor and mediate the inhibitory activity of the inhibitor.
  • angiogenesis-dependent refers to tumors that require angiogenesis for growth or migration, including those with an increase in the number and density of blood vessels that require an increase in volume or mass (or both).
  • the term "subject” refers to any animal, such as a mammal, including but not limited to humans, non-human primates, rodents, pigs, rabbits and the like.
  • the subject receives a particular treatment, or undergoes a particular treatment, such as detecting the level of presence of a molecule.
  • sample is one of the broadest concepts including, but not limited to, biological and environmental samples. In one case, it refers to a specimen or culture obtained from a biological or environmental source. Biological samples can be obtained from animals, including humans, including liquids, solids, tissues and gases. Biological samples include blood products such as plasma, serum and the like. Environmental samples include environmental substances such as surface materials, soil, water, minerals, crystals or industrial samples. The above listed samples are not meant to limit the range of sample categories encompassed by the present invention.
  • the term "marker” includes chemical or biological molecules that exhibit the presence and amount of a target molecule by interacting with a target molecule.
  • labeling molecules include, but are not limited to, nucleic acid probes such as DNA probes or RNA probes, antibodies, radioisotopes, fluorescent dyes and the like.
  • instructions for use includes instructions for the use of the kit to direct the detection of target molecules, such as NL, in a sample.
  • suitable binding assays for screening specific L-related angiogenesis inhibitors include high pressure liquid chromatography, immunoprecipitation, fluorescence binding detection, capillary electrophoresis, and the like.
  • anti-angiogenic test refers to the effective degree of inhibition of angiogenesis by screening a series of candidate molecules.
  • different sources include natural or artificial (manual or automatic) synthetic proteins or more
  • the peptide can be easily and rapidly tested for its activity of inhibiting endothelial cell proliferation by a biological activity assay such as a bovine capillary endothelial cell proliferation test.
  • Other bioassays include chick embryo chorioallantoic membrane testing, mouse corneal neovascular growth inhibition testing, and detection of inhibitory effects of isolated or synthetic proteins on transplanted tumors.
  • the term “and/or” as used in the phrase “proliferation and/or migration” refers to two situations: 1) both proliferation and migration of endothelial cells are regulated; 2) endothelial cell proliferation or migration One is adjusted.
  • ligating refers to the attachment of an antibody to a cytotoxic agent, such as a cytokine molecule, using conventional, well-known biological or chemical techniques such as cross-linking or the like.
  • NL molecules can be used to prepare polyclonal and monoclonal antibodies that can be used to qualitatively and even quantitatively detect L in a particular target cell.
  • Appropriate labeling such as radioisotopes or fluorescently labeled NL can be used to detect ES in body fluids and tissues.
  • the method can be applied to the diagnosis and prognosis of diseases related to angiogenesis such as cancer.
  • the invention also encompasses methods for treating or preventing angiogenesis-related diseases or processes by enhancing the effects of ES on angiogenesis-dependent tumors, including but not limited to arthritis, tumors and the like.
  • methods such as flow cytometry and ELISA are used to quantify NL peptides.
  • Detection of NL-related nucleic acid molecules can utilize standard molecular biology techniques such as DNA probe hybridization, PCR, and the like. The various PCR and cloning steps described here can be found in the Guide to Molecular Cloning.
  • Northern blots can be used for the detection of NL RNA.
  • Northern blots include the isolation of RNA and the hybridization of complementary probes.
  • the RNA (or corresponding cDNA) is detected by hybridization of an oligonucleotide probe.
  • a series of tests using different hybridization and detection methods are feasible. For example, the TaqMan test (PE Biosystems, Foster City, Calif.; See eg, US Pat. Nos. 5, 962, 233, and 5, 538, 848, incorporated herein by reference). This test was performed during the PCR process.
  • the TaqMan assay utilized the 5'-3' exonuclease activity of AMPLITAQ GOLD DNA polymerase.
  • An oligonucleotide probe with a 5' reporter dye (such as a fluorescent dye) and a 3' quencher dye was introduced into the PCR system.
  • the 5'-3' of AMPLITAQ GOLD DNA polymerase exerts its 5'-3' exonuclease activity to degrade the sequence between the reporter dye and the quencher dye.
  • the separation of the reporter dye from the quencher dye enhances the fluorescent signal.
  • the fluorescent signal gradually accumulates and can be monitored by a fluorometer.
  • RT-PCR reverse transcription PCR
  • RNA is converted to complementary DNA, or "cDNA" by reverse transcriptase.
  • This cDNA serves as a template for PCR.
  • the PCR product can be detected by any suitable method including, but not limited to, gel electrophoresis as well as DNA-specific staining or labeled probe hybridization.
  • Quantitative RT-PCR is also used in some embodiments, which introduces a standardized mixture of competitive templates and is described in US Pat. Nos. 5,639,606, 5,643,765, and 5,876,978.
  • Detection of NL protein molecules can be performed by techniques known in the art, such as radioimmunoassay, ELISA, sandwich immunoassay, immunoradiometric assay, gel diffusion precipitation assay, immunodiffusion assay, in situ immunoassay
  • binding of an antibody is carried out by detecting a label on the primary antibody.
  • the primary antibody is detected indirectly by detecting a secondary antibody or other agent that binds to the primary antibody.
  • the secondary antibody is itself labeled. Many methods for detecting binding in immunoassays are known in the art and are included within the scope of the invention.
  • automated detection methods can be utilized. These automated methods are described in U.S. Pat. Nos. 5,885, 530, 4, 981, 785, 6, 159, 750, and 5, 358, 691, each incorporated herein by reference.
  • the analysis and presentation of the results is also automated. For example, some embodiments use software based on the presence or absence of certain cancer marker corresponding proteins to predict the likely consequences of the disease.
  • NL antibodies can also be used to screen cells rich in endogenous NL molecules to make them ideal targets for ES therapy.
  • NL or NL analogs are prepared using techniques known in the art, either polyclonal or monoclonal.
  • the antibody can be used in some well-known immunoassay formats, such as competitive, non-competitive immunoassays including ELISA, sandwich immunoassay, and radioimmunoassay to determine whether or not the endothelial cell proliferation inhibitor of the present invention is present in the body fluid.
  • Body fluid samples include, but are not limited to, blood, serum, ascites, pleural fluid, cerebrospinal fluid, urine, saliva, and other tissue mucus.
  • the present invention provides an isolated antibody that can be used in a diagnostic kit for detecting NL.
  • the invention provides a monoclonal antibody capable of specifically binding to NL.
  • the anti-NL antibody of the present invention may be any monoclonal or polyclonal antibody as long as it recognizes NL.
  • Antibodies can be prepared by conventional means using NL or an analog thereof as an antigen.
  • the invention encompasses the simultaneous use of monoclonal and polyclonal antibodies. Any suitable method can be used to prepare the antibodies used in the present invention, including but not limited to the methods described below.
  • the target protein, or together with a suitable carrier or diluent is injected into an animal (e.g., a mammal) under conditions such that the antibody is produced.
  • complete or incomplete Freund's adjuvant can be used.
  • the target protein is injected every two to six weeks for a total of two to ten injections.
  • Animals suitable for the method include, but are not limited to, primates, rabbits, dogs, pigs, mice, rats, sheep, goats, and the like.
  • an animal e.g., a mouse
  • the antibody titer has been determined
  • two or five days after the last immunization, the spleen or lymph node is taken, and the antibody-producing cells and myeloma are produced.
  • the cells are fused to obtain hybridoma cells capable of producing antibodies.
  • the titer of the antibody in the antiserum can be measured by reacting the antiserum with the labeled target protein and measuring the activity of the labeling reagent bound to the antibody.
  • Cell fusion can be carried out according to known methods, for example, the method described by Koehler and Milstein (Nature 256:495 [1975]).
  • Sendai virus (HVJ) or more preferably polyethylene glycol (PEG)
  • HVJ Sendai virus
  • PEG polyethylene glycol
  • Polyclonal antibodies can be prepared by any known method or modified method, including from a patient. For example, a complex of a carrier protein and an immunogen is prepared and used to immunize an animal in the same manner as in the preparation of the monoclonal antibody: described above. The antibody-containing fraction is obtained from the immunized animal, and the antibody is isolated and purified therefrom.
  • the present invention provides a method of inhibiting proliferation and/or migration of endothelial cells by producing a combined antibody comprising an anti-NL antibody linked to a cytotoxic agent such as a chemokine such as tumor necrosis factor alpha or the like.
  • a cytotoxic agent such as a chemokine such as tumor necrosis factor alpha or the like.
  • the anti-L antibody directs the cytotoxic agent to the endothelial cells, thereby allowing the cytotoxic agent such as tumor necrosis factor alpha to act on the Endothelial cells and destroy cell growth.
  • an antibody to another substance, such as a cytotoxic agent, to form a combined antibody (also known as an immunotoxic agent) are well known in the art.
  • a cytotoxic agent also known as an immunotoxic agent
  • Two major advances in the field of immunotoxicants are the use of recombinant DNA technology to produce recombinant toxic agents with better clinical properties and the production of single chains by fusing DNA elements encoding a combination of antibodies, growth factors or cytokines with a toxic agent gene.
  • Immunotoxic agent Two major advances in the field of immunotoxicants are the use of recombinant DNA technology to produce recombinant toxic agents with better clinical properties and the production of single chains by fusing DNA elements encoding a combination of antibodies, growth factors or cytokines with a toxic agent gene.
  • First generation immunotoxic agents were constructed by coupling a poison to a MAb or antibody fragment using a heterobifunctional cross-linker. It has also been discovered that genetic engineering can be used to replace the cell binding domain of a bacterial agent with the Fv portion or growth factor of the antibody.
  • cytokines are core small molecules that communicate between cells of the immune system, even between these cells and cells belonging to other tissue types. They are actively secreted by immune cells as well as other cell types.
  • the cytokines produced by immune cells form a subclass called lymphokines, which are usually localized, but sometimes have a systemic effect.
  • cytokines which have both stimulatory and inhibitory effects on lymphocytes and immune responses. Some of the more well-understood cytokines include: histamine, prostaglandin, TOF- ⁇ , a 1 and 1! ⁇ -6. There are three types of cytokines.
  • Another aspect of the invention is the use of antibodies to NL to screen for tumor cells or endothelial cells having a large amount of surface L expression. Finding such a group of patients is beneficial for the effective treatment of angiogenesis-related cancers, as patients with high levels of expression of cell surface NL are ideal candidates for ES tumor inhibition therapy.
  • the present invention proposes a method of introducing a foreign NL gene into a target cell so that it can express a cell surface L exceeding a normal level. These modified target cells become more sensitive to ES attacks due to elevated levels of NL.
  • the invention also provides an L detection and identification kit for cancer diagnosis.
  • the kit includes a specific antibody to NL in addition to the reagents and buffers for detection.
  • the kit includes an agent (e.g., an oligonucleotide probe or primer) that specifically detects NL mRNA or cDNA.
  • the kit includes all components for performing the assay, including controls, instructions for performing the assay, and software necessary to analyze and display the results.
  • the invention also includes the following kits:
  • the kit contains a marker that can measure NL, such as an antibody to L.
  • the antibody solution is prepared to detect L and its peptides from plasma, urine, tissue fluid, and cell culture media, and is further useful for rapidly and reliably, sensitively, and specifically determining and localizing ES.
  • Techniques used in these kits include, but are not limited to, competitive, non-competitive assays, radioimmunoassays, biological and chemiluminescence assays, fluorescence assays, dot blots, enzyme-linked immunosorbent assays such as ELISA, microtiterometry, rapid detection of urine samples. And blood-coated antibody-coated test strips, immunocytochemical techniques, etc.
  • the detection range, sensitivity, accuracy, reliability, specificity, and reproducibility of each kit were determined according to industry requirements familiar to and followed by those skilled in the art.
  • the diagnostic kit of the invention can also be used to localize ES in tissues and cells.
  • the NL immunohistochemistry kit includes instructions for use and NL molecules, which are preferably labeled or ligated with fluorescent molecules such as fluorescein or other reagents to develop primary antisera. Immunohistochemistry is a technique well known to those skilled in the art.
  • the kit can utilize optical and electron microscopy to localize ES in tissue or cells. It can be used for both scientific research and clinical applications. For example, the site of ES production is determined by detecting a tumor slice of a living tissue. This information is of great significance for the clinical diagnosis and treatment of tumors.
  • the invention is further illustrated by the following examples. The application of the present invention is not limited to these embodiments.
  • NL is an ES binding protein
  • HMEC human microvascular endothelial cells
  • Endothelial cells (HMEC or HUVEC, 2 ⁇ 10 4 cells per well) were inoculated into DMEM containing 0.5% fetal bovine serum (Hyclone) and 10 ng/ml VEGF (PeproTech EC) in the upper layer of TranswellTM plate (8 ⁇ m pore size, Costar). (Hyclone) in the medium.
  • a certain concentration of ES (provided by Protgen) and other reagents (NL and L antibodies) were added to the upper and lower layers of the plate.
  • the cells were allowed to migrate by continuing to culture for 6 hours at 37 ° C and 5% CO 2 .
  • Endothelial cells such as HMEC or HUVEC (IX 10 3 cells per well) were seeded into 96-well plates containing DMEM medium containing 0.5% fetal bovine serum and 10 ng/ml of bFGF (PeproTechEC). At the beginning of the experiment Different concentrations of ES and other reagents were added to each well to a final volume of 200 microliters. Endothelial cells were propagated for 48 hours at 37 ° C and 5% CO 2 . The original medium was then replaced with 100 microliters of DMEM medium containing no phenol red and 0.5 mg/ml MTT (Sigma).
  • the fractions eluted from the ES-conjugated Ni-NTA affinity column were collected and analyzed by 12% SDS-PAGE.
  • the main band was digested with sequencing-grade modified porcine trypsin (Promega).
  • the peptides were analyzed using MALTI-TOF using a Bruker Biflex linear time-of-flight mass spectrometer (Bmker Franzen) with a multi-probe SCOUT source, a super-nitrogen laser (337 nm) and a dual microchannel disc detector. Mass spectral data were aligned in the Swiss-Prot protein database to identify proteins. Indirect immunofluorescence
  • HMEC was incubated with ES (20 g/ml) for 1 hour at 37 °C and 5% C0 2 .
  • Cells were stained with antibodies without permeabilization and then treated with FITC-labeled goat anti-mouse secondary antibody (Santa Cruz) and TRITC-labeled goat anti-rabbit secondary antibody (Santa Cruz).
  • Observations and photographs were performed on an Olympus Fluoview laser scanning confocal imaging system (Olympus Inc.). Images were captured using multiple photomultiplier tubes conditioned by Fluoview 2.0 software (Olympus). Preparation of recombinant NL
  • the cDNA of NL was obtained from HMEC by RNA isolation and reverse transcription system (Promega). The sequence of NL was fused to polyhistidine (His) 6 and cloned into the methanol yeast expression vector pPIC9K (Invitrogen). The recombinant plasmid was linearized by restriction endonuclease Sail (Promega) and electrotransformed into methanol yeast strain GS115.
  • New Zealand white rabbits (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were immunized with 50 ⁇ g of L prepared by the above methanol yeast expression system.
  • the first immunization was mixed with Freund's complete adjuvant (Sangon) subcutaneously.
  • Freund's incomplete adjuvant (Sangon) containing 50 ⁇ g of NL was intramuscularly injected as a booster needle. Thereafter, respectively 4, 10, 22 weeks subcutaneous injection of the booster needle.
  • serum was taken and the antibody was purified using a Protein A column (Amersham Biosciences).
  • HMEC was used, and this cell line was in conformity as a good anti-angiogenic test model.
  • ES dose-dependently inhibited VEGF-induced HMEC migration with a half-inhibitory concentration (IC 50 ) of 4 g/ml (Fig. la), even ES with concentrations as low as (4 ng/ml).
  • HMEC has a 15% inhibition rate.
  • Similar results were obtained in the HMEC proliferation assay stimulated with bFGF (Fig. lb).
  • NL is a new receptor for ES. If NL is a receptor for ES, it should be able to mediate the activity of ES in anti-angiogenesis, such as inhibition of migration, proliferation and adhesion of endothelium. To identify the role of NL in mediating ES activity, competitive cell migration and proliferation assays were performed using ES, recombinant NL, and NL polyclonal antibodies, respectively. Since NL is an ES receptor isolated from HMEC, whether it has a similar effect on other widely accepted endothelial cells needs to be demonstrated. Therefore, human HUVECs isolated directly from the umbilical cord vein were subjected to competitive cell migration and proliferation assays.
  • This cell can be passed through a membrane with a pore size of 8 ⁇ under the stimulation of VEGF, and ES inhibits this migration.
  • Recombinant NL abolished the inhibition of ES in a dose-dependent manner (Fig. 3a), showing that recombinant NL is involved in the antiangiogenic activity of ES.
  • recombinant L itself has no effect on cell migration, excluding NL itself, which may stimulate the migration of endothelial cells.
  • Similar results were obtained for the HUVEC proliferation assay (Fig. 3c).
  • NL polyclonal antibodies antagonized the inhibition of ES on cell proliferation (Fig. 3c).
  • RNA expression (RNA,) (Sui, G. et al. Proc. Natl. Acad. Sci. USA 99, 2002, p5515-5520) was used to inhibit NL expression, followed by The effect of this change in expression on the important activity of ES in anti-angiogenesis, cell adhesion, was examined. The results indicate that HMEC is immobilized on ES and polylysine when NL expression is inhibited by RNA interference based on DNA vectors.
  • NL is a new receptor for ES and plays an important role in the signaling pathway of ES.
  • ES NL-mediated signaling pathways in ES
  • downstream events have also been investigated. Note that when ES is incubated with HMEC, the amount of ES-NL complex changes with time and reaches a maximum at around 2 hours (Fig. 2e), ES may be endocytosed by cell surface NL, such as HMEC, followed by some endocytosis ES is degraded by cells. This suggests a balance between ES endocytosis and degradation (Fig. 2e).
  • ES was labeled with biotin (Pierce) and immunofluorescence localization was performed. After incubation of HMEC with biotinylated ES for different times, biotinylated ES was stained with TRITC-labeled avidin (Pierce), and the endocytic process of ES was observed with a fluorescence microscope (Olympus).
  • FIG. 4a-f At 30 minutes of incubation, most of the endocytic ES was distributed in the cytoplasm and was less abundant (Fig. 4b). After 1 hour of incubation, the amount of endocytic ES increased and began to accumulate in the nucleus (Fig. 4c). The amount of endocytic ES reached a maximum at about 2 hours of incubation (Fig. 4d). At 3 hours, the ES accumulated in the nucleus began to disappear (Fig. 4e). At 7 hours, the ES was barely visible in the core (Fig. 4f).
  • a protein with a molecular weight of about 200 kDa was found and identified as non-muscle myosin by MALDI-TOF.
  • Myosin constitutes a large superfamily that participates in membrane dynamics and intramuscular muscles The organization of actin, which affects cell migration, adsorption, and endocytosis.
  • a fifth class of myosin with two "head" domains can transport vesicles, organelles and mRNA granules along actin fibers.
  • Cell surface NL was also found to bind ES and transport ES to the nucleus (Fig. 4a-f). Since myosin is an intracellular protein, it must bind to the intracellular domain of the cell surface L.
  • bFGF but not VEGF can stimulate phosphorylation of NL (Fig. 4h), which explains that bFGF can stimulate endothelial cell proliferation in the previous report and VEGF can stimulate endothelial cell migration.
  • ES can inhibit bFGF-stimulated CK2-mediated NL phosphorylation, and as a result, inhibits cell survival and proliferation.
  • ES affects cell migration and adhesion through NL-myosin complexes.
  • NL has been shown to be a novel receptor for ES and mediates ES inhibition of cell migration, proliferation and adhesion. Since integrins are also reported to be receptors for ES, it is interesting to see if there is an interaction between L and integrins. In the integrin family, Rehn and Sudhakar et al. reported that integrin ⁇ 5 ⁇ 1 is a receptor for ES. Therefore, indirect immunofluorescence assays were performed using the anti-inducible ⁇ ( ⁇ Santa Cruz) and NL rabbit anti-inflammation assays. Whether the NL of the cell surface and the integrin ⁇ 5 ⁇ 1 are localized together.
  • NL and non-muscle myosin are known to form a complex in the cell cortex, and this complex plays a key role in the motility and adhesion of endothelial cells. Therefore, it is speculated that ES may interfere with cell motility and adhesion through the NL-myosin complex. It is possible that L on the cell surface and integrin ⁇ 5 ⁇ 1 together with other proteins such as myosin form a large complex as a receptor for ES (see Figure 6).
  • ES specifically inhibits angiogenesis and tumor growth, and it was observed that ES was not toxic in animal experiments and showed only low toxicity in clinical trials. But the specific molecular mechanism behind this phenomenon is still unclear. It is speculated that NL mediates the specific activity of ES in anti-angiogenesis, which seems to contradict the previously reported NL is a ubiquitous protein in cells. To explain this contradiction, the abundance of NL on the surface of HMEC under different growth conditions was examined. The results showed that the abundance of NL on the surface of vigorously proliferating cells was much higher than that on the relatively stationary cell surface (Fig. 5 a-f).
  • Relatively stationary cells were obtained by serum starvation for 24 hours and their cell cycle was detected by flow cytometry (Becton Dickinson, Worldwide Inc., San Jose, CA) (Fig. 5 g-h).
  • the results of flow cytometry showed that the proportion of cells in the G1 phase increased by 24% after serum starvation, and the S phase decreased by 30% (Fig. 5 g-h).
  • the cells were not completely in the G1 phase, the amount of L on the cell surface was significantly reduced after 24 hours of serum starvation. It is speculated that the difference in NL abundance on the surface of this cell membrane leads to differences in the sensitivity of endothelial cells to ES.
  • ES specifically binds NL to selectively exert its anti-angiogenic activity on tumor tissues; on the other hand, ES is rare. Binding to normal organs, because normal organ cell surface NL is less, which in turn makes it no toxic when treating tumors with ES.
  • NL regulates the function of ES in cell migration assays
  • the cDNA of L was obtained from HMEC using the SV total RNA isolation system and the reverse transcription system (Promega) according to the manufacturer's recommended protocol.
  • the fusion sequence of NL and polyhistidine (ffis) 6 was obtained by PCR, and then subcloned into the expression vector pPIC9K (Invitrogen).
  • the vector was linearized by restriction endonuclease Sai l (Promega) and electroporated into methanol yeast strain GS115 according to the manufacturer's manual.
  • BMMY medium (10 g L yeast powder; 20 g/L peptone; 100 mmol/L potassium phosphate, pH 6.0; 13.4 g/L yeast nitrogen base; 40 mg/L biotin and daily supplementation to a final concentration of 0.5% methanol) were shaken for 3 days in shake flasks.
  • the medium supernatant was purified by a nickel ion affinity column (Qkgen) to obtain L.
  • the pH of the 1 L medium supernatant was adjusted to 8.0 and the upper 6 ml Ni-NTA column was washed and eluted according to the manufacturer's recommendations.
  • about 3 mg NL can be obtained from each liter of medium.
  • a polyclonal antibody to NL was prepared using NL obtained by this method.
  • HUVEC (2x10 4 per well) was inoculated into the upper chamber of Transwell filter (8 ⁇ well, Costar) containing DMEM medium, 0.5% fetal bovine serum, and 10 ng/ml bFGF (PeproTech EC).
  • ES (5 g/ml, Protgen) and recombinant NL (20 g/ml), or NL antibody (20 g/ml) were added at the beginning of the migration experiment.
  • PBS was also added to this migration assay system as a control.
  • the same DMEM medium and other reagents were also added to the lower chamber. Endothelial cells were migrated for 6 h at 37 ° C, 5% C0 2 .
  • Tumor tissue vascular surface NL is blocked by its antibodies and accelerates tumor growth
  • the cDNA of NL was obtained from HMEC using the SV total RNA isolation system and the reverse transcription system (Promega) according to the manufacturer's recommended protocol. PCR to obtain the fusion sequence of NL and polyhistidine (His) 6 , followed by sub Cloning into the expression vector pPIC9K (Invitrogen). The vector was linearized with restriction endonuclease Sal I (Promega) and electroporated into methanol yeast strain GS115 according to the manufacturer's recommendations.
  • BMMY medium (10 g / L yeast powder; 20 g / L peptone; 100 mmol / L potassium phosphate, pH 6.0; 13.4 g / L yeast nitrogen Base; 40 mg/L biotin and daily supplementation to a final concentration of 0.5% methanol) for 3 days in shake flasks.
  • the supernatant of the medium was purified by a nickel ion affinity column (Qiagen) to obtain NL.
  • the pH of the 1 L medium supernatant was adjusted to 8.0 and the upper 6 ml Ni-NTA column was washed and eluted according to the manufacturer's recommendations.
  • about 3 mg of NL can be obtained from each liter of medium.
  • a polyclonal antibody to NL was prepared using NL obtained by this method.
  • NL-specific angiogenesis inhibitors Screening of NL-specific angiogenesis inhibitors using an ES-Ni-NTA affinity chromatography column
  • the NL cDNA was obtained from HMEC using the SV total RNA isolation system and the reverse transcription system (Promega) according to the manufacturer's recommended protocol.
  • the fusion sequence of NL with polyhistidine (His) 6 was obtained by PCR, and then subcloned into the expression vector pPIC9K (Invitrogen).
  • the vector was linearized by restriction endonuclease Sal I (Promega) according to the manufacturer's recommendations and electrotransformed into methanol yeast strain GS115.
  • BMMY medium (10 g / L yeast powder; -20 g / L peptone; 100 mmol / L potassium phosphate, pH 6.0 ; 13.4 g / L yeast containing Nitrogen base; 40 mg/L biotin and daily supplementation to a final concentration of 0.5% methanol) were shaken for 3 days in shake flasks.
  • the medium supernatant was purified by a nickel ion affinity column (Qiagen) to obtain L.
  • the pH of the 1 L medium supernatant was adjusted to 8.0 and the upper 6 ml Ni-NTA column was washed and eluted according to the manufacturer's recommendations.
  • L was immobilized on a Ni-NTA nickel ion affinity column via a histidine tag fused at its N-terminus. This column can be used to high-throughput screening of proteins that bind to L. The resulting L-binding protein can be identified using MALDI-TOF binding peptide fingerprinting. Their biological activity during anti-angiogenesis can be detected by cell experiments, such as the cell migration and proliferation assays described above.
  • Example 9
  • the binding kinetics of ES and L were determined by the SPR method (method reference BIAcore 2000TM Biosensing System Manual), and the instrument was the BIAcore 2000TM biosensing system from Amersham Biotech.
  • the purified ES was diluted to 100 g/ml with 20 mM sodium acetate buffer (pH 6.5) and tested by using the oxy coupling method (l-ethyl-3-(dimethylaminopropyl) according to the method of the manual.
  • Carbodiimide, (-hydroxysuccinimide) was covalently immobilized on the CM5 sensor chip for research (Amersham Biotech).
  • ES 100 g/ml ES (20 mM sodium acetate buffer, pH 6.5) was injected into the activation.
  • the study was performed on a CM5 sensor chip until the response value reached 9000 units on the SPR instrument.
  • the surface of the unreacted chip was blocked with ethanolamine at pH 8.5 (Amersham Biotech).
  • the binding kinetics of ES to L SPR was 25 At °C, L dissolved in mobile phase HBS (10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, and 0.005% surfactant P20, pH 7.4) was injected into SPR 20 ⁇ l at a flow rate of 10 ⁇ /min.
  • the combined kinetic curve is the value of the concentration of L in the mobile phase as a function of time.
  • the raw data was analyzed by Amersham Biotech's analysis software BIAevaluation 3.1.
  • the binding rate constant k a of ES and NL, the dissociation rate constant k b and solution Balanced K D calculated by Langmu binding model (stoichiometry 1: 1).
  • mice Inoculation of mouse melanoma cells B16/F10 (ATCC) (2X 10 6 cells in 200 ⁇ of PBS) in 2 months old Balb/c mice (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) ) under the skin. Eight days later, co-localization experiments of ES and NL in mice were performed. The rabbit polyclonal antibodies labeled with biotin ES and NL were intravenously injected into tumor-bearing mice, respectively. The control group was injected with biotin-labeled ES and purified rabbit polyclonal antibody. One hour later, the mice were anesthetized, perfused from the heart with 20 ml of PBS, and then sacrificed.
  • mice Some normal tissues and tumors of the mice were removed, fixed, and sectioned. Sections were simultaneously detected with TRITC-labeled avidin (Pierce) and FITC-labeled secondary antibody (Santa Cruz) and observed under an Olympus Fluoview laser scanning confocal imaging system (Olympus Inc.). Colocalization of ES and NL was also observed in vivo.
  • the rabbit polyclonal antibodies labeled with biotin ES and L were sequentially injected intravenously into tumor-bearing mice. Control group was injected with biotin-labeled ES and purified rabbit polyclonal antibody Body. One hour later, some normal tissues and tumors of the mice were used for immunofluorescence detection.
  • RNA interference RNA interference
  • DNA vector-based RNA interference plasmid BS/U6 is constructed as in Dr. Shi and his colleagues ("A DNA vector-based RNAi technology to suppress gene expression in mammalian cells.” Sui, G. et al. Proc. Natl Acad. Sci. USA 99, 2002, p5515-5520):
  • the RNA interference sequence specific for L-specific BS U61356 is from 1356 to 1377 in the NL sequence, likewise, the L-specific RNA interference sequence BS/U6/ 263 is from 263 to 283 in the NL sequence (this interference sequence is inactive).
  • a random sequence BS/U61356C having the same base composition as the specific sequence was used in parallel as a control.

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Description

核仁素辅助的癌症诊断与治疗方法
相关申请
本申请要求享有 2005年 5月 12日提交的中国专利申请 200510011707.3的优先权。 技术领域
本发明涉及一种用于鉴别适于接受抗血管发生癌症疗法的癌症对象， 特别是人类患 者的新方法。 本发明还涉及一种能够搜索并筛选血管发生抑制剂的方法。 所述血管发生 抑制剂相信可以有效抑制细胞恶性生长， 尤其对血管发生依赖型的癌症效果更为显著。 本发明公开了用核仁素（nucleolin, 以下说简称 "NL" ) 筛选血管发生抑制剂的方法。 特 别地， 本发明的方法涉及筛选与血管内皮抑制素 （endostatin, 以下简称 "ES" ) 作用机 理相似的血管发生抑制剂。 本发明基于下列发现： NL是 ES的特异性受体， 并参与 ES抑 制血管发生活性相关的信号转导途径。
书
背景技术
一种新近发现的癌症疗法通过 ES来抑制肿瘤血管发生， 阻断肿瘤供血从而达到抑制 肿瘤生长的目的。 ES是胶原蛋白 XVIII的 C端球状结构域， 分子量 20 kDa。 最初它是从 鼠内皮瘤细胞培养基上清中分离出来的， 因为它可以抑制毛细血管内皮细胞的增殖。 在 动物实验中， 反复使用 ES可以促使肿瘤消亡并且不产生抗药性。 此外， 在动物实验和临 床中都显示 ES毒性很低。 ES有显著的抑制内皮细胞增殖、 迁移、 黏附、 生存的能力并 且可以诱导细胞凋亡。 虽然整连蛋白、 原肌球蛋白、 磷脂酰肌醇蛋白聚糖 (gtypican)和 E- 选择蛋白（E-selectin)被猜测作为 ES的受体调控细胞的迁移， β-catenin和 Shb则与 ES诱 导的内皮细胞 G1期停抑和凋亡相关， 但是对于 ES的确切分子扒理仍然存在很大的争 论， ES在动物实验和临床治疗中表现出来低毒型的具体原因还不清楚。 另一方面， 在动 物实验及临床中必须使用高浓度的 ES才能有抑瘤效果， 对这一事实现在还缺少充分的解 释。
ES被认为是一种有效的癌症治疗药物， 因为 ES通过抑制血管发生来杀死肿瘤。 肿 瘤细胞需要通过新生血管进行扩散。 肿瘤细胞群体的每一次增长都必须是汇集在肿瘤处 的新毛细血管发生之后方可发生。 这一现象几乎是普遍的： 多数人类的固体肿瘤或者血 癌都是血管发生依赖型的。 抗血管发生疗法还有其他的优势， 包括低毒性， 极小的耐药 性， 以及反复使用该疗法后可能会有较长时期的肿瘤休眠， 在此期间便不需要继续治 疗， 参考 Boehm et al.， Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance. Nature (1997) 390: 404~ 07。 然而， ES的作用机理至今仍不明确。 因此， ES治疗被同样地用在每一个癌症患者身上， 而并未考虑到个体对该治疗的敏感性。 同 时， 研究发现要让 ES有效地发挥其血管发生抑制剂的功能而达到预期的抗癌效果， 必须 制备大量的 ES并注射给患癌症的哺乳动物或人类。 由于该过程中需要高剂量的 ES， 其 价格可能会昂贵得让患者承受不起。 因而， 迫切需要找出其它方法以发现新的血管发生 抑制剂， 可以经济、 有效地用于治疗肿瘤。 已经进行了很多临床实验以期望找到有效的 抗血管发生药物。 另一方面， 如果能够以客观的标准选择出适合接受 ES治疗的患者， 那 将是一个显著的进步。
对于不同的患者来说， 癌症疗法的效果差异很大， 这取决于一系列内在或外在因 素。 外在因素包括实施治疗时癌症的发展阶段（越早发现越有利于治疗和恢复） 以及治 疗相对强度， 例如手术、 化疗或放疗。 内在因素包括患者免疫系统的健康程度， 强健的 免疫系统可以支撑时间长和强度大的治疗， 从而帮助患者快速康复。 在癌症治疗中， 甚 至整个医学界中， 正在被探讨的一个关键问题是个体化用药。 不同个体可能对同样的癌 症药物有不同的忍耐度和敏感度， 这促使人们去努力寻找提高 定癌症治疗效果的方 法。 因此， 由于个体差异， 对某一个患者有效的药物未必对另外一个患者也有效。 在癌 症治疗领域中， 人们试图通过患者的基因型来了解某一特定的药物是否对具有特定遗传 特征的患者有效。 同样， 为了实现 ES治疗的个体化应用， 最近有人试图通过基因芯片技 术获得基因表达谱来研究 ES为什么能够抑制内皮细胞血管发生， 参考 M. Mazzanti, et al, Genome Research, 14:1585-1593 (2004)。
核仁素 (rmde0lin，NL)是一个广泛存在的非组蛋白， 最初是从核仁中分离出来的。 NL 的量受 Granzyme A和自我切割能力的调控， 有趣的是， 它还和细胞的增殖有关系。 NL 也可以自切， 它的自切割随细胞进入增殖速度的加快而减少， 与此同时， 它还被细胞毒 淋巴细胞释放的一种酯酶 Granzyme A切割（Chen et al., J. Biol. Chem., 1991, 266, 7754- 7758; Fang and Yeh, Exp. Cell. Res" 1993, 208， 48-53; Pasternack et al., J. Biol. Chem., 1991, 266, 14703-14708)。 这些切割以及相伴的降解构成了 NL的翻译后调控。
作为一个多功能蛋白， NL在细胞增殖中起关键且基础的作用， 包括： 核仁染色质的 排列， pre-RNA的包装， rDNA的转录以及核糖体的组装。 L的这些活性被某些蛋白激 酶如 CK2和 cdc2所调控， 而后者又受到其它细胞周期蛋白的严格调控。 此外， NL还可 以在细胞表面、 细胞浆、 细胞核中穿梭， 并且具有细胞表面受体的功能。 作为很多病毒 和细胞因子的受体， 当配体与其结合时， NL可以触发配体的内吞。
Orrick et al (1973)曾经报道 L分子量大约 100-110 kDa, 主要存在于增殖细胞的细 胞核中， L可以自降解， 并在免疫印迹中呈现 70和 50 kDa的两条带。 NL被高度磷酸 化及甲基化， 并且可以被二磷酸腺苷核糖基化。 由于 NL的合成与细胞分裂速度的增加正 相关， 肿瘤细胞以及旺盛分裂的细胞中含有更大量的 NL。 NL的序列早先由 SrivastaVa, et al.在 Cloning and sequencing of the human nucleolin cDNA. FEBS Lett. 250 (1), 99-105 (1989) 中报道。
NL (亦称作 P92或 C23)是活跃生长的细胞核仁内最丰富的磷酸化蛋白 （Srivastava et al" FEBS Lett., 1989, 250, 99-105; Srivastava et al, J. Biol. Chem., 1990, 265, 14922- 14931 ) 。 已经知道 NL主要参与核糖体的生物合成（Ghisolfi et al., Mol. Biol. Rep., 1990, 14, 113-114; Sipos and Olson, Biochem. Biophys. Res. Commun., 1991, 177, 673-678) 。 NL 通过自身的核糖核酸蛋白共有序列与核糖体前体短暂结合， 从而参与了核糖体的合成 (Bugler et al., J. Biol. Chem., 1987, 262, 10922-10925; Ghisolfi-Nieto et al., J. Mol. Biol., 1996, 260, 34-53; Sapp et al., Eur. J. Biochem., 1989， 179, 541-548) 。 NL可以占总核仁蛋白 的 5% (Lapeyre et al, Proc. Natl. Acad. Sci. U.S.A., 1987, 84， 1472-1476; Sapp et al., Eur. J. Biochem., 1989, 179, 541-548) 。 有证据显示 L还参与了细胞动力学、 核的形成、 细胞 增殖和生长、 转录抑制、 DNA复制、 信号传导和染色质去压缩化， 参考综述（Tuteja and Tuteja, Crit. Rev. Biochem. Mol. Biol., 1998, 33, 407-436) 。
上述 NL的多功能的性质来源于它若干结构和功能上独立的结构域（Creancier et al., Mol. Biol. Cell., 1993, 4, 1239-1250; Sapp et al., Eur. J. Biochem., 1989, 179， 541-548) 。 已知 NL的三个结构域： N端结构域、 中间结构域以及 C端结构域。 N端结构域中包含与 HMG蛋白同源的序列， 可以与染色质相互作用 （Erard et al., Eur. J. Biochem., 1988, 175, 525-530) ； 中间结构域含有四个 RNA识别基序， 可以特异性结合 18S和 28S核糖体 RNA的短茎环结构 （Bugler et al., J. Biol. Chem., 1987, 262, 10922-10925 ) ； C端结构域含 有可以使 RNA内碱基去堆积的区域（Ghisolfi et al., Mol. Biol. Rep., 1990, 14， 113-114; Ghisolfi-Nieto et al., J. Mol. Biol, 1996, 260, 34-53) 。 NL含有二元核定位信号， 跨越蛋白 N端以及中部区域。 该序列帮助 NL定位到核内。 NL通过和其他蛋白相互作用在核内积 累 （Schmidt-Zachmann and Nigg, J. Cell Sci., 1993, 105, 799-806) 。
根据 NL的结构域组成特点将其归类为 Ag-NOR蛋白 （即定位于核仁组织区域的活 跃核糖体基因） ， 也称为活跃核糖体基因的标记 （Roussel et al, Exp. Cell. Res., 1992, 203, 259-269) 。 有证据表明核糖体基因的转录需要 Ag-NOR蛋白， Ag-NOR蛋白的表达也被 用来预测肿瘤生长的速率。 NL曾经作为基质粘附区域（MAR) 结合蛋白从人红白血病细胞中被纯化出来。 研究 发现， L参与了将染色质环状结构锚定到核基质的过程 (Dickinson and Kohwi- Shigematsu, Mol. Cell. Biol., 1995, 15, 456-465 )。
NL可以被高度磷酸化， 研究表明它是酪蛋白激酶 II (以下简称 CK2) (Csermely et al., J. Biol. Chem., 1993, 268, 9747-9752; Schneider and Issinger, Biochem. Biophys. Res.
Commun., 1988, 156, 1390-1397) 、 蛋白激酶 C-.xi. (Zhou et al., J. Biol. Chem., 1997, 272, 31130-31137) 以及 cdc2 (Belenguer et al" Mol. Cell. Biol., 1990, 10， 3607-3618 ) 的底物。 此外， 研究表明 NL的磷酸化状态调控 NL的亚细胞定位。
L可以自切割或者被细胞毒淋巴细胞分泌的酯酶 Granzyme A切割， 并且当细胞进 入旺盛增殖阶段时自切活性降低 （Chen et al, J. Biol. Chem., 1991， 266, 7754-7758; Fang and Yeh, Exp. Cell. Res., 1993, 208, 48-53; Pasternack et al., J. Biol. Chem., 1991, 266, 14703- 14708 ) 。 上述切割过程以及随之而来的降解构成了 NL的翻译后调控。
抗 NL的抗体可以在患有系统性结缔组织疾病包括红斑狼疮 （ SLE) (Minota et al., J. Immunol., 1990, 144, 1263-1269; Minota et al., J. Immunol., 1991， 146, 2249-2252) 和硬皮病 状慢性移植体对抗宿主疾病 （ scleroderma-like chronic graft vs. host disease ) ( Bell et al., Br. J. Dermatol., 1996, 134, 848-854) 的患者血清中发现。 因此， 对 NL的表达进行药理学调 控可能是病理状态下合适的治疗手段。 发明概述
本发明提供了一种用于确定癌症对象对 ES癌症疗法敏感度的试剂盒， 包括标记 NL 的标记物以及使用说明。 该试剂盒可以通过对从对象身上采集的样品中的 NL进行标记来 检测样品中 NL的含量。 所述对象优选是哺乳动物， 更优选是人类。 在本发明的优选实 施方案中， 被标记的 NL是细胞表面 NL。 在本发明的其他实施方案中， 所述标记物包括 抗体， 优选多克隆抗体， 更优选单克隆抗体。 在本发明的另一个实施方案中， 所述标记 物包括核酸分子或探针， 优选 DNA探针， 更优选 RNA探针。
本发明进一步提供了一种用于确定对对象实行 ES癌症疗法成功的可能性的方法， 包 括检测从所述对象采集的样品中 L的表达水平。 确定该对象是否对 ES癌症疗法敏感主 要依据上述检测到的 NL的表达水平。
本发明提供了筛选血管发生抑制剂， 尤其是筛选那些与 ES作用机理相似的分子的方 法。 本发明以 L作为靶分子， 运用传统的方法学， 鉴定出可以和 NL特异性结合的分 子， 同时该分子有抗血管发生的活性。 基于 L是 ES在细胞表面的受体这一事实， 用上 述方法发现的分子应该具有与 ES相似的作用机理。
在一个实施方案中， 本发明提供了一种获得有效的特异性针对 L的血管发生抑制剂 的方法， 包括如下步骤： 运用合适的结合试验在候选的分子库中筛选出一系列能与 NL特 异性结合的分子； 用抗血管发生分析去验证这些分子对于抑制血管发生的有效性； 最 后， 根据活性试验的结果在这些能与 NL特异性结合的分子中筛选出能有效抑制血管发生 的分子。
在另一个实施方案中， 本发明提供了一种筛选可以在体外抑制内皮细胞增殖和 /或迁 移的血管发生抑制剂的方法， 包括如下步骤： 运用药物学可接受的方法发现特异性以 NL 作为靶而与 NL相互作用的分子； 验证上一步获得的分子在抑制内皮细胞增殖或迁移中的 有效性； 收获上述步骤获得的能够抑制内皮细胞增殖或迁移的分子， 并与 ES比较它们在 抑制血管发生中的效果。
本发明还提供了增加内皮细胞对 ES的敏感度的方法。 所述方法将外源 NL引入靶内 皮细胞， 使得 NL在所述内皮细胞中相对于野生型过量表达， 优选的靶细胞应当在正常情 况下没有高水平的内源 NL表达。 所述方法进一步提出， 向靶细胞中引入 NL， 可以使这 些细胞对 ES的抗血管发生活性更敏感而被 ES有效杀伤。 本发明还提出可以用 L的抗 体检测细胞表面表达大量 L的靶癌细胞 （或内皮细胞） ， 这种癌症适宜用 ES治疗。
在进一步的实施方案中， 本发明提供一种增强靶细胞对血管发生抑制剂敏感度的方 法， 包括如下步骤： 将外源 NL导入靶细胞， 从而获得能够表达外源 NL的修饰的靶细 胞， 并且检测 ES对这些细胞的致死率。
在另一个实施方案中， 本发明提供一种增强血管发生抑制剂针对靶内皮细胞的抗血 管发生效果的方法， 包括如下步骤： 将药物学有效量的外源 NL基因导入上述的靶细胞， 使 NL在所述细胞中表达， 同时将上述血管发生抑制剂与经转化的靶细胞一起温育， 从而 抑制所述靶细胞的生长。
在一个实施方案中， 本发明提供一种提髙血管发生抑制剂控制患者肿瘤生长的效果 的方法， 包括如下步骤： 用可行的手段， 检测癌症患者内源 NL的表达水平； 根据该患者 的 NL表达水平来评价血管发生抑制剂对其有效程度， NL表达水平越高表明血管发生抑 制剂治疗越有可能取得成功的治疗效果。 优选地， 所述血管发生抑制剂是 ES。
在另一个实施方案中， 本发明提供一种确定对血管发生抑制剂治疗敏感的靶癌细胞 的个体敏感性的诊断试剂盒， 所述试剂盒包括特异结合 NL的分子和药物学可接受的载 体。 优选地， 所述分子是多克隆抗体。 更优选地， 所述分子是单克隆抗体。 在进一步的实施方案中， 本发明提供了一种鉴别对于抗血管发生抑制剂治疗敏感的 靶肿瘤的方法， 所述方法包括产生抗 NL的抗体、 使用所述抗 NL抗体筛选所述靶肿瘤的 样品、 以及通过所述靶肿瘤和所述抗 NL抗体之间的特异性相互作用所得到的提示来鉴别 对于抗血管发生抑制剂治疗敏感的靶肿瘤。 优选地， 所述抗体是多克隆抗体。 更优选 地， 所述抗体是单克隆抗体。
在另一个实施方案中， 本发明提供了一种用于确定对抗血管发生抑制剂治疗敏感的 靶肿瘤的诊断试剂盒， 包括针对 L的抗体和药物学可接受的载体。
在进一步的实施方案中， 本发明提供了一种确定并选择对抗血管发生抑制剂治疗敏 感的癌症对象的诊断试剂盒， 所述试剂盒包括可以结合 NL并提示其存在的标记物以及使 用说明书， 可以用来测试从癌症对象收集的样品中 L的水平。
在进一步的实施方案中， 本发明提供了一种抑制细胞样品中一些内皮细胞增殖和 /或 迁移的方法， 包括将抗核仁素抗体与胞毒剂连接， 从而形成抗核仁素毒性抗体， 以及将 所述抗核仁素毒性抗体施用于所述细胞样品， 从而抑制所述一些内皮细胞的增殖和 /或迁 移。 优选地， 所述胞毒剂是细胞因子。 更优选地， 所述胞毒剂是肿瘤坏死因子。 在一个 优选的实施方案中， 所述细胞样品得自癌症患者。 附图说明
图 1显示人微血管内皮细胞（Human Microvascular Endothelial Cell, 以下简称
"HMEC" ) 是在迁移和增殖方面对 ES敏感的细胞系。 a，在标示浓度的 ES的处理下， 用 HMEC进行细胞迁移试验， PBS作为对照。 b，在标示浓度的 ES的处理下， 进行 HMEC细胞增殖试验， PBS作为对照。 细胞的数目用 MTT法进行分析。 结果表示为平均 值 ± s.e.m.， n = 3 (a),以及 n = 5 (b)。 图 2显示 ES结合细胞表面的 L。 a，从 HMEC表面分离出来的 ES结合蛋白经鉴定为 N 及其片段。 如方法学部分描述的那样， ES结合蛋白用预载有 ES的 Ni-NTA亲和柱从 HMEC细胞膜中分离出来。 用含 500 mM氯化钠的 PBS缓冲液洗脱下来的组分进行 SDS- PAGE分析（左） 并用 NL的单克隆抗体做免疫印迹（右） 。 b，在体外 ES特异性地结合 NL。 用重组 NL和 ES做免疫共沉淀。 c，肝素干扰 ES-NL的复合物形成。 在体外加入或 不加肝素 （200 nM) 的情况下， 用重组 NL和 ES进行免疫共沉淀。 d，ES通过细胞表面 L特异性结合 HMEC。 HMEC与 ES, 以及不同浓度的 L抗体在室温下温育 30分钟， 然后用 PBS缓冲液洗三遍。 将细胞进行 SDS-PAGE分析并用 ES的抗体做免疫印迹。 β- 肌动蛋白作为对照。 e，将 HEMC与 ES (60 g/ml)在 37 °C和 5% C02下温育不同时 间。 用新鲜培养基冲洗后， 分别用 NL和 ES的抗体做免疫共沉淀和免疫印迹。 β-肌动蛋 白作为对照. 在 HMEC细胞表面 NL和 ES共定位。 完整的 HEMC用鼠抗 L、 兔抗 ES染色， 并用激光扫描共聚焦显微镜检测间接免疫荧光。 标尺， 20 μπι。 图 3显示 L是 ES的受体。 a，在人脐带血管内皮细胞（Human Umbilical Vascular Endothelial Cell, 以下简称 " HUVEC" )迁移试验中， 用标示浓度的重组 NL解除 ES的 抑制活性， PBS作为对照。 b，重组 L本身对细胞的迁移没有作用。 在 HUVEC的迁移试 验中分别加入标示浓度的 ES、 NL和两者的混合物。 PBS作为对照。 c，在 HUVEC的增殖 试验中分别加入标示浓度的 ES、 NL和 NL的抗体， PBS作为对照。 用 MTT法分析细胞 数目。 d，用 NL缺陷型和对照组 HMEC做细胞粘附试验。 用 pBS/U6/1356质粒介导的 RNA干扰的方法抑制 NL的表达来获得 NL缺陷型细胞。 空白的 PBS/U6质粒转染的细胞 作为对照。 结果为平均值± 5 .1^, 11 = 4 (3, (1) - 5( ，(;)。 e，用 NL的抗体进行免疫印迹以 验证 RNA干扰质粒对 NL表达的抑制作用。 BS/U6/1356可以抑制 L的表达， 而 BS U6/263却没有作用， 肌球蛋白的免疫印迹作为上样对照。 f，用 NL缺陷型和对照组 HMEC做细胞增殖试验。 用 pBS/U6/1356质粒介导的 R A干扰的方法抑制 NL的表达来 获得 L缺陷型细胞。 空白的 pBSAJ6质粒转染的细胞作为对照。 ES的加入浓度如图中所 示。 细胞的数目用 MTT法进行分析。 图 4显示 NL介导 ES的信号网络。 a-f，ES被 HMEC内吞。 HMEC加入或不加入 (a) 10 g/ml生物素标记的 ES分别温育 0.5小时 (b), 1小时（c), 2小时 (d), 3小时 (e),和 7小时 (f)。 内吞的 ES用 TRITC标记的抗生物素蛋白染色。 g，把细胞和 NL的抗体一起温育可以 阻断 ES的内吞。 标尺， 25 μηι.， h，在核中， ES可以抑制酪蛋白激酶 -2 (casein kinase-2, 以下简称 "CK2" ) 介导的 NL磷酸化。 磷酸化分析按照方法学部分所描述的进行。 磷酸 化的 NL用 SDS-PAGE和放射性自显影检测。 NL的免疫印迹作为上样对照。 i-k，在 HMEC细胞表面 NL和整联蛋白 βΐ (mtegrin pl ) 共定位。 完整的 HEMC用鼠抗 L, 兔 抗整联蛋白 βΐ染色， 并用激光扫描共聚焦显微镜检测间接免疫荧光。 标尺， 10 μιη。 图 5显示细胞表面的 L分布依赖于细胞的生长状态。 a-h，HEMC细胞表面 NL的分布。 增殖细胞和静止细胞表面的 L用兔抗 NL进行间接免疫荧光检测。 DAPI标明视野中细 胞核的位置。 标尺， 20 μπι。 增殖细胞所处的细胞周期 (g)和静止细胞 (h)的分别用流式细胞 仪检测， 相对静止期的细胞通过血清饥饿 24小时获得。 i-1,在荷瘤裸鼠中细胞表面 NL的 分布。 按方法学部分所描述的进行免疫组化染色。 心脏 (i)、 肾脏 (j)、 肺 (k)、 和肿瘤 (1)的 血管用箭头指示。 表达细胞表面 NL的内皮细胞被染成棕色。 标尺， 50 μπι。 图 6显示由 NL介导的 ES信号网络的模式图。 一个包含细胞表面 NL、 整联蛋白 （例如 整联蛋白 (Χ5β1 ) 和其它蛋白的大复合物参与到 ES的信号网络中， NL结合肌球蛋白， 并 通过它与肌动蛋白纤维相连。 与之相似， · 整联蛋白就是通过一些胞内锚定蛋白踝蛋白 (talin) , α-辅肌动蛋白 （actinin) ，细丝蛋白 （filamin) ，和钮带蛋白 （vinculin) 与肌动蛋 白纤维束相连。 没有发现 NL和整联蛋白有直接的相互作用。 ES可以与细胞外基质竞争 结合这个复合物， 以此来抑制细胞的粘附和迁移。 这种结合还可以触发整联蛋白介导的 信号传导。 另一方面， 这个复合物可以介导 ES的内吞， 在这个过程中肌球蛋白起到转运 蛋白的作用。 然后， ES可能在胞浆中释放出来， 并抑制 L使 Bd-2的 mRNA稳定的作 用。 剩下的 ES被转运到核内， 并在其中抑制 CK2介导的 NL磷酸化以及一些下游事件。 图 7显示体内 ES和 NL的共定位。 标记有生物素的 ES和抗 NL抗体被静脉注射到荷有 B16 F10肿瘤的小鼠体内。 对照组注射标记有生物素的 ES和纯化的兔 IgG。 在心脏 （a- c) 、 肝脏 (d-f) 、 肾脏 (g-i) 、 肿瘤 (j-l)及其对照组肿瘤 Cm-o) 中， 标记有生物素 的 ES和抗 NL抗体通过 TRITC缀合的抗生物素蛋白和 FITC缀合的二抗检测。 标尺 50
图 8显示通过 SPR (Surface plasmon resonance)方法测定的 ES和 NL的动力学结合曲线， 结 果显示： ES与 NL的亲和常数 KD = 2.32xlO'8 M。 图 9显示靶内皮细胞上 NL的量越多， ES的抗新生血管活性越强。 a，用 L的抗体进行 免疫印迹以验证 RNA干扰质粒对 NL表达的抑制作用。 BS U6/1356可以抑制 NL的表 达， 而 BS U6/263和 BS U6/1356C却没有作用， actin的免疫印迹作为上样对照。 b，用 NL 缺陷型和对照组 HMEC做细胞增殖试验。 用 BS/U6/1356质粒介导的 RNA干扰的方法抑 制 NL的表达来获得 L缺陷型细胞。 BS/U6/1356C质粒转染的细胞作为对照。 ES的加 入浓度为 lO g/tnL 细胞的数目用 MTT法进行分析。 发明详述
本发明基于一个令人'惊讶的发现： L可以作为 ES的受体介导后者的生物学活性的 发挥， 同时， NL表达量的多少在细胞学水平上可以预示 ES有效的程度。
目前在使用 ES进行癌症治疗的领域， 如果能够以客观的标准选择出适合接受 ES治 疗的患者， 那将是一个显著的进步。 本发明提出的一些方法和诊断试剂盒实现了这一目 标。
为了研究 ES的作用机理， 本发明人用固定化的 ES从 HMEC膜中分离能够结合 ES 的蛋白。 NL就是这些蛋白中很有趣的一个， 随后即被证明是 ES信号网络中的一个关键 的成员。 发现 NL作为 ES的受体并介导其在抑制血管发生方面的活性。
部分基于上述新发现， 即 NL是 ES的特异性受体并辅助 ES的抑制血管发生功能， 本发明提供一种用于确定癌症对象对 ES癌症疗法敏感度的试剂盒、 一种用于确定对对象 实行 ES癌症疗法成功的可能性的方法、 一种获得有效的特异性针对 NL的血管发生抑制 剂的方法、 一种筛选可以在体外抑制内皮细胞增殖和 /或迁移的血管发生抑制剂的方法、 一种增强靶细胞对血管发生抑制剂敏感度的方法、 一种增强血管发生抑制剂针对靶内皮 细胞的抗血管发生效果的方法、 一种用于确定对抗血管发生抑制剂治疗敏感的靶癌细胞 的诊断试剂盒、 一种确定并选择对抗血管发生抑制剂治疗敏感的癌症对象的诊断试剂 盒、 一种提高血管发生抑制剂控制患者肿瘤生长的效果的方法、 以及一种鉴别对于抗血 管发生抑制剂治疗敏感的靶癌细胞的方法。
如本文所用， 术语 "血管内皮抑制素"指一个在非还原或还原电泳中分子量分别表 现为 18到 20 kDa的蛋白； "血管内皮抑制素"还包括这个 18到 20· kDa蛋白的前体形 式， 片断形式， 以及修饰后的蛋白或多肽形式， 只要是具有充分类似的氨基酸序列， 并 具有抑制内皮细胞生长的功能均包括在 "血管内皮抑制素"的含义范围之内。 例如， 文 献中经常报道的氨基酸序列的保守突变， 即把原来的氨基酸用一个结构、 化学性质相似 的氨基酸替换， 但是这种替换对原来蛋白的结构， 构象和活性没有明显的改变。 此类保 守突变也落入附加的权利要求的保护范围之内。
术语 "血管内皮抑制素"还包括在原来蛋白的两端或者内部缺失一个或多个氨基酸 而得到缩短的蛋白或者多肽， 但是所述蛋白或者多肽仍然保留了原来蛋白抑制内皮细胞 增殖的活性。 "血管内皮抑制素" 的含义还包括在原来蛋白的两端或者内部增加了一个 或多个氨基酸而得到的增长的蛋白或多肽， 但是所述蛋白或者多肽仍然保留了原来蛋白 抑制内皮细胞增殖的活性。 这些分子可以用来进行标记实验， 例如在第一位增加一个酪 氨酸残基并进行 1251标记。 用其它的放射性同位素标记还可以提供一个分子工具来杀死具 有 ES受体的靶细胞。
类似的， 如本文所用， 术语 "核仁素 "是指一种通过还原凝胶电泳确定其大小优选 为 lOOkDa (不含后修饰的精确分子量为 80kDa)的蛋白质。 术语核仁素还包括所述 lOOkDa 蛋白质的前体形式， 片断形式， 以及修饰后的蛋白或多肽形式， 只要是具有充分类似的 氨基酸序列， 并具有抑制内皮细胞增殖的功能的蛋白或多肽均包括在 "核仁素"的含义 范围之内。 例如， 文献中经常报道的氨基酸序列的保守突变， 即把原来的氨基酸用一个 结构、 化学性质相似的氨基酸替换， 但是这种替换对原来蛋白的结构， 构象和活性没有 明显的改变。 这些分子可以用来进行标记实验， 例如在第一位增加一个酪氨酸残基并进 行 1251标记。 此类保守突变也落入附加的权利要求的保护范围之内。
术语 "NL特异性"指 NL结合血管发生抑制剂并介导该抑制剂抑制活性的能力。 术语 "血管发生依赖型"指那些生长或迁移需要血管发生的肿瘤， 包括那些体积或 质量 （或二者兼有） 的增加需要供血的血管的数量和密度的增加的肿瘤。
如本文所用， 术语 "对象"指任何动物， 例如哺乳动物， 包括但是不限于人， 非人 灵长类， 啮齿动物， 猪， 兔之类。 所述对象接受特定的治疗， 或者接受某种特定的处 理， 例如检测某种分子的存在水平。
如本文所用， 术语 "样品"是一个最广泛的概念， 包括但是不限于生物样品和环境 样品。 一种情形下， 它指由生物或环境来源获得的标本或者培养物。 生物样品可从动物 (包括人）处获得， 包括液体， 固体， 组织和气体。 生物样品包括血产品， 例如血浆， 血清之类。 环境样品包括环境物质， 例如表面物质， 土壤， 水， 矿物质， 晶体或者工业 样品。 上述所列样品并不意味着限定本发明所包括的样品类别范围。
如本文所用， 术语 "标记物"包括了化学的或者生物分子， 它们可以通过与靶分子 相互作用而显示靶分子的存在以及数量。 此类标记分子包括但是不限于， 核酸探针例如 DNA探针或 RNA探针、 抗体、 放射性同位素、 荧光染料之类。
如本文所用， 术语 "使用说明"包括了试剂盒的使用说明， 用来指导对样品中靶分 子， 例如 NL的检测。
如本发明中所用， 合适的筛选特异性 L相关血管发生抑制剂的结合实验方法包括高 压液相色谱， 免疫沉淀， 荧光结合检测， 毛细管电泳等等。
如本发明中所用， "抗血管发生试验"是指通过筛选一系列候选分子以发现它们抑 制血管发生的有效程度。 为了验证某个分子是否具有抑制新生血管生长的性质， 有多种 方法可供使用。 例如， 不同来源包括天然的或者人工 （手动或自动）合成的蛋白或者多 肽， 可以通过牛毛细血管内皮细胞增殖试验等生物活性测定方法来方便、 快捷地检验其 抑制内皮细胞增殖的活性。 其他的生物检测包括鸡胚尿囊膜测试， 小鼠角膜新生血管生 长抑制测试， 以及检测分离的或者合成的蛋白对移植肿瘤的抑制作用。 鸡胚尿囊膜测试 最早由 O'Reilly等在 "Angiogenic Regulation of Metastatic Growth" Cell, vol. 79 (2), Oct. 21, 1994， pp. 315-328中报道， 本发明中引用此实验方法。 更多的用来筛选血管发生抑制剂的 抗血管发生试验参见 Yu， et al., PNAS, Vol. 101, No. 21, pp 8005-8010 (2004), 该文献全文 并入本文作为参考。
如本文所用， 在短语 "增殖和 /或迁移"中所用术语 "和 /或"是指两种情况： 1 ) 内 皮细胞的增殖和迁移二者都被调节； 2) 内皮细胞增殖或迁移二者之一被调节。
如本文所用， 术语 "连接"是指用常规的、 众所周知的生物学或化学技术如交联等 等将抗体连接至胞毒剂如细胞因子分子上。
NL分子可以用来制备多克隆和单克隆抗体， 所述抗体可以用来定性甚至定量检测特 定靶细胞中的 L。 适当标记例如放射性同位素或者荧光标记的 NL可以用来检测体液和 组织中的 ES。 该方法可以应用于以血管发生相关疾病如癌症的诊断和预后。 本发明还包 括一些方法， 可以通过提高 ES对血管发生依赖型肿瘤的效果来治疗或者预防血管发生相 关的疾病或者过程， 包括但是不限于关节炎、 肿瘤等。
在本发明的一些实施方案中， 诸如流式细胞术和 ELISA之类的方法被用来定量 NL 肽。
对 NL相关的核酸分子的检测可以利用标准的分子生物学手段， 例如 DNA探针杂 交， PCR等。 此处描述的各类 PCR和克隆步骤可以参考《分子克隆实验指南》
( Sambrook et al, eds. Cold Spring Harbor Lab P bl. 1989, latest edition) 。 对 NL RNA的检 测可以利用 Northern印迹。 Northern印迹包括 RNA的分离以及互补探针的杂交。 在一些 具体实施方案中， RNA (或相应的 cDNA) 由寡聚核苷酸探针杂交检测。 一系列运用不同 杂交和检测手段的试验都是可行的。 例如， 某些实施方案中使用了 TaqMan试验（PE Biosystems, Foster City, Calif.; See e.g., U.S. Pat. Nos. 5,962,233以及 5,538,848，均并入本文 做为参考） 。 此试验是在 PCR过程中进行的。 TaqMan试验利用了 AMPLITAQ GOLD DNA聚合酶的 5'-3'外切酶活性。 一个带有 5'报告染料 （例如一种荧光染料） 以及一个 3'猝灭染料的寡聚核苷酸探针被引入 PCR体系。 在 PCR过程中， 如果探针与靶序列结 合， AMPLITAQ GOLD DNA聚合酶的 5'-3'便发挥其 5'-3'外切酶活性将报告染料与猝灭 染料之间的序列降解。 报告染料与猝灭染料的分离使得荧光信号增强。 随着每轮 PCR的 进行， 荧光信号逐渐累积并可以通过荧光计进行监测。 在某些其他的实施方案中， 反转录 PCR (RT-PCR)也被用来检测 RNA的表达。 在 RT-PCR中， RNA由反转录酶转化为互补的 DNA, 或 " cDNA" 。 该 cDNA即作为 PCR 的模板。 PCR产物可以用任何合适的方法检测， 包括但不限于凝胶电泳以及 DNA特异性 染色或者标记探针杂交。 在一些实施方案中也使用了定量 RT-PCR, 此方法引入了竞争性 模板的标准化混合物， 并在 U.S. Pat. Nos. 5,639,606, 5,643,765,以及 5,876,978中有描述
(均并入本文作为参考） 。
NL蛋白分子的检测可通过本领域已知的技术进行， 例如放射性免疫测定、 ELISA、 夹心法免疫测定、 免疫辐射度测定、 凝胶扩散沉淀反应、 免疫扩散测定、 原位免疫测定
(例如利用胶体金、 酶或放射性同位素标记） 、 Western印迹、 沉淀反应、 凝集测定 （例 如凝胶凝集测定、 血凝素凝集测定等等） 、 补体固定测定、 免疫荧光测定、 蛋白 A测定 以及免疫电泳测定等。
例如， 抗体的结合通过检测一抗上的标记进行。 在另外一个实施方案中， 通过检测 可以结合一抗的二抗或其他试剂来间接检测一抗。 更进一步的实施方案中， 二抗本身是 标记的。 本领域已知很多检测免疫测定中的结合的方法， 并且这些方法包括在本发明的 范围之内。
某些情形下， 可以利用自动化的检测手段。 这些自动化的方法在 U.S. Pat. Nos. 5,885,530， 4,981,785, 6,159,750,以及 5,358,691 (均被并入本文做参考） 中有描述。 在一些 实施方案中， 结果的分析及表述也是自动化的。 例如， 一些实施方案中使用了基于某些 癌症标记对应蛋白存在与否来预测疾病可能的后果的软件。
被动的抗体治疗通过 L特异性抗体来调节内皮细胞依赖性的过程如繁殖、 发育、 愈 伤和组织修复等。 另外， NL的抗体还可以用来筛选含有丰富内源性 NL分子的细胞使其 成为 ES治疗中的理想靶标。
NL或 NL类似物的特异性抗体采用本领域已知的技术方案制备， 可以是多克隆也可 以是单克隆抗体。 所述抗体可以用在众所周知的一些免疫测定形式中， 例如竞争性、 非 竞争性的免疫测定包括 ELISA、 夹心免疫测定以及放射性免疫测定等来判定体液中是否 '有本发明的内皮细胞增殖抑制因子。 体液样品包括但是不限于血液、 血清、 腹水、 胸腔 液、 脑脊液、 尿液、 唾液和其他组织粘液。
本发明提供了可以用于检测 NL的诊断试剂盒中的分离的抗体。 在优选的实施方案 中， 本发明提供能够特异性结合 NL的单克隆抗体。
本发明中抗 NL的抗体可以是任何单克隆或者多克隆抗体， 只要它能识别 NL。 抗体 可由 NL或其类似物作为抗原经常规手段制备。 本发明涵盖了同时使用单克隆和多克隆抗体。 任何合适的方法都可以用来制备本发 明使用的抗体， 包括但不限于下面所描述的方法。 例如， 为制备单克隆抗体， 将靶蛋 白， 或连同适当的载体或稀释剂， 在使得产生抗体的条件下注射进动物 （例如哺乳动 物） 体内。 为 7提高动物产生抗体的能力， 可以使用完全或不完全弗氏佐剂。 通常， 靶 蛋白每二到六周注射一次， 共注射二到十次。 适于本方法的动物包括但不限于灵长类， 兔子， 狗， 猪， 小鼠， 大鼠， 绵羊， 山羊等。
为了制备产生单克隆抗体的细胞， 选定已经确定了所产生抗体滴度的动物 （例如小 鼠） ， 最后一次免疫后二至五天， 取其脾脏或淋巴结， 其中产生抗体的细胞与骨髓瘤细 胞融合从而得到能够产生抗体的杂交瘤细胞。 测量抗血清中抗体的滴度可以采用如下方 法： 将抗血清与标记的靶蛋白反应， 进而测量与抗体结合的标记试剂的活性。 细胞融合 可以根据已知的方法进行， 例如， 由 Koehler和 Milstein所描述的方法 （Nature 256:495 [1975] ) 。 例如， 仙台病毒 （HVJ) ， 或更优选的聚乙二醇（PEG) 可以作为融合的促进 剂。
多克隆抗体可由任何已知的方法或经修饰的方法， 包括从患者身上获得， 来制备。 例如， 制备载体蛋白与免疫原的复合物并用来免疫动物， 方法与上面描述的单克隆抗体 ： 的制备过程中的相关部分相同。 从被免疫动物体内获得含有抗体的部分， 并将抗体从中 分离纯化出来。
本发明提供了通过产生含有与胞毒剂如趋化因子例如肿瘤坏死因子 α等等连接的抗 NL抗体的组合抗体来抑制内皮细胞的增殖和 /或迁移的方法。 当这种组合抗体被施用于包 括内皮细胞在内的一种细胞样品时， 所述抗 L抗体指导所述胞毒剂至所述内皮细胞， 从 而使所述胞毒剂如肿瘤坏死因子 α作用于所述内皮细胞并破坏细胞生长。
将抗体连接至另一种物质如胞毒剂以形成组合抗体 （也称为免疫毒剂） 的方法是本 领域所熟知的。 所述免疫毒剂领域的两项主要进展是使用重组 DNA技术产生具有更好的 临床性质的重组毒剂和通过将编码抗体、 生长因子或细胞因子的组合区域的 DNA元件与 毒剂基因融合而产生单链免疫毒剂。
第一代免疫毒剂通过使用异源双功能交联剂将毒剂偶联至 MAb或抗体片段而构建。 也发现了可以使用遗传工程将细菌毒剂的细胞结合结构域用抗体的 Fv部分或生长因子取 代。
如本领域所熟知的， 细胞因子是免疫系统细胞之间， 甚至这些细胞和属于其他组织 类型的细胞之间通讯的核心小分子。 它们由免疫细胞以及其他细胞类型活跃地分泌。 由 免疫细胞产生的细胞因子组成了称为淋巴因子的一个亚类， 其作用通常是局部的， 但是 有时候也可以具有全身性的作用。
已知有很多对淋巴细胞和免疫应答既具有刺激作用又具有抑制作用的细胞因子。一 些比较清楚地了解的细胞因子包括： 组胺、 前列腺素、 TOF— α、 一1和1!<—6。 存在 3 类细胞因子。
本发明的另一方面是， 用 NL的抗体去筛选具有大量表面 L表达的肿瘤细胞或内皮 细胞。 找到这样一组患者对于有效施行血管发生相关的癌症治疗是有益的， 因为具有高 水平表达细胞表面 NL的患者是接受 ES抑瘤治疗的理想对象。
为了使肿瘤细胞对 ES更加敏感， 本发明提出了一种方法： 将外源 NL基因导入靶细 胞， 从而使其能够表达超过正常水平的细胞表面 L。 这些修饰的靶细胞由于 NL水平的 升高， 会对 ES的攻击变得更敏感。
本发明还提供了用于癌症诊断的 L检测与鉴定试剂盒。 某些实施方案中， 所述试剂 盒除了用于检测的试剂和缓冲液之外， 还包括 NL的特异性抗体。 其他实施方案中， 所述 试剂盒包括特异性捡测 NL mRNA或者 cDNA的试剂 （例如寡聚核苷酸探针或引物） 。 优选的实施方案中， 所述试剂盒包括进行检测测定的所有成分， 包括对照， 进行测定的 指导， 以及必要的分析及展示结果的软件。
本发明还包括了如下试剂盒： 该试剂盒包含了可以测定 NL的标记， 例如 L的抗 体。 制备所述抗体溶液使其可以从血浆、 尿液、 组织液以及细胞培养基里检测 L及其肽 段， 并且进一步用于迅速、 可靠、 敏感、 及特异性地测定并定位 ES。 这些试剂盒使用的 技术包括但是不限于竞争性、 非竞争性测定， 放射性免疫测定， 生物及化学发光测定， 荧光测定， 点杂交， 酶联免疫吸附测定例如 ELISA, 微量滴定技术， 快速检测尿样和血 样的抗体包被的试纸条， 免疫细胞化学技术等。 每种试剂盒的检测范围， 灵敏性， 准确 度， 可靠性， 特异性和重现性均按照为本领域技术人员所熟悉及遵照的行业要求被确定 下来。
类似的， 本发明的诊断试剂盒也可以被用来定位组织和细胞中的 ES。 该 NL免疫组 化试剂盒包括使用说明及 NL分子， 所述 NL分子优选由荧光分子如荧光素或其他试剂标 记或连接以使初级抗血清显色。 免疫组织化学是本领域技术人员所熟知的技术。 所述试 剂盒可以利用光学和电子显微镜来对组织或细胞中的 ES进行定位。 它既可以用于科研也 可以应用于临床。 例如， 通过检测活体组织肿瘤切片来确定 ES生成的位点。 这一信息对 于肿瘤临床诊断和治疗有很重要的意义。 本发明通过以下的实施例进一步进行阐述。 本发明的应用并不周限于这些实施例。 相反， 必须清楚地说明本发明的实现手段可以有不同的实施方案、 修饰形式以及多种等 价形式。 通过阅读以下的描述， 本领域技术人员可以理解更多的实施手段而不背离本发 明的主题以及 /或者超出所附权利要求书的内容。 实施例
实施例 1
NL是 ES结合蛋白
为了研究 ES的作用机制， 固定化的 ES被用于直接从人微血管内皮细胞（HMEC) 分离 ES结合蛋白。 NL被鉴别为在 ES信号传导网络中的关键成员， 并且是该网络中最令 人感兴趣的成员。 本实施例发现 L是 ES的一个新的受体， 并且调节 ES在抗血管发生 中的活性。 方法学：
研究 NL和 ES相互作用的方法均为本领域巳知的成熟方法。 这些方法的详细描述如 -下， 其中除非另外说明， 内皮细胞（HMEC或者 HUVEC) (ATCC保藏号分别为 CRL 10636和 CRL-1730) ， ES购自 Protgen公司， NL使用毕赤酵母 (Invitrogen)表达， NL单 克隆抗体购自 Santa Cruz: 细胞迁移测定
内皮细胞（HMEC或者 HUVEC, 每孔 2X 104个细胞）接种到 Transwell™板（8 μιη孔径， Costar) 的上层含 0.5%胎牛血清（Hyclone) 以及 10 ng/ml VEGF (PeproTech EC) 的 DMEM (Hyclone)培养基中。 同时在板的上层和下层加入一定浓度的 ES (Protgen公司提供） 和其它试剂 （NL和 L抗体） 。 在 37 °C和 5% C02中继续培养 6 小时使细胞进行迁移。 用乙醇固定和曙红染色后， 每个孔随机选取 5个高倍放大 （400 倍）视野， 计算其中完全穿过膜迁移到板下层的细胞数并取平均值， 每一组重复三个 孔。 细胞增殖试验
接种内皮细胞， 如 HMEC或者 HUVEC (每孔 I X 103个细胞） 到 96孔板， 其中有 含有 0.5%胎牛血清和 10 ng/ml的 bFGF (PeproTechEC) 的 DMEM培养基。 在试验之初 向每个孔中添加不同浓度的 ES和其他试剂至终体积 200微升。 内皮细胞在 37 °C和 5% C02中增殖 48小时。 然后用 100微升不含酚红和含有 0.5 mg/ml MTT (Sigma) 的 DMEM培养基替换原有培养基。 继续在 37 °C和 5% C02中培养 4小时后， 用含有 0.05 M盐酸的异丙醇裂解细胞并检测在 570 nm下的光吸收。 利用 MALDI-TOF质谱鉴定所分离的蛋白
收集从结合有 ES的 Ni-NTA亲和柱 （Qiagen)洗脱下的组分并用 12% SDS-PAGE电 泳分析， 主带用测序级的经过修饰的猪胰蛋白酶（Promega) 酶切后， 得到的肽段用 MALTI-TOF进行分析， 所用仪器为 Bruker Biflex线性飞行时间质谱 （Bmker Franzen) ， 装有多探针 SCOUT源， 超氮激光 （337 nm) 以及双微通道盘检测器。 质谱数据在 Swiss- Prot蛋白数据库中进行比对分析以鉴定蛋白。 间接免疫荧光
HMEC与 ES (20 g/ml) —起于 37 °C和 5% C02温育 1个小时。 细胞不经过通透 化即用抗体染色， 然后分别加入 FITC标记的羊抗小鼠二抗 （Santa Cruz) 和 TRITC标记 的羊抗兔的二抗 （Santa Cruz) 处理。 在 Olympus Fluoview激光扫描共聚焦成像系统 (Olympus Inc.)上观察并照相。 图像用由 Fluoview 2.0软件 （Olympus)调节的多重光电 倍增管捕获。 重组 NL的制备
通过 RNA分离以及反转录系统 （Promega)从 HMEC中得到 NL的 cDNA。 NL的 序列与多聚组氨酸 (His)6融合后， 克隆到甲醇酵母表达载体 pPIC9K中 （Invitrogen) 。 该 重组质粒经限制性内切酶 Sai l (Promega) 线性化后电转化至甲醇酵母菌株 GS115
(Invitrogen) 。 经 G418 (Invitrogen)筛选得到的稳定转化子并按 Invitrogen公司的手册 中描述的方法于 30°C BMMY培养基中摇瓶培养 3天。 培养基上清经 Ni-NTA亲和柱
(Qiagen) 纯化得到 NL。
NL多克隆抗体的制备
用通过上述甲醇酵母表达系统制备的 50微克 L免疫新西兰大白兔（北京维通利华 实验动物技术有限公司） 。 初次免疫混合弗氏完全佐剂 （Sangon) 皮下注射， 14天后， 肌肉注射含有 50微克 NL的弗氏不完全佐剂 （Sangon)作为加强针。 此后， 分别在第 4, 10， 22周皮下注射加强针。 最后一次免疫 1周后， 取血清， 用蛋白 A柱（Amersham Biosciences)纯化抗体。 用甘氨酸一盐酸缓冲液（0.15M, pH 2.5)洗脱， 立即用 0.15M Tris调 pH到 6.8-7.2。 合并的组分用 0.2微米孔径的滤膜（Millipore)过滤除菌， 分装储 存在 -80°C。 细胞粘附试验
待检测的细胞血清饥饿 30分钟后， 接种到用 ES (20 μ§/ιη1) 和多聚赖氨酸
(Sigma) (50 g/ml) 包被的 96孔板上。 在 37。C和 5% C02中培养 1个小时后， 用新 鲜培养基小心洗掉没有粘附的细胞。 剩下的细胞用结晶紫（Amresco) (0.1 %溶于双蒸 水） 室温染色 25分钟。 用干净的水洗， 剩下的结晶紫用 0.5%的 Triton X-100 (Sangon) 溶解， 检测 570 nm的光吸收。 作为多种血管发生抑制因子的靶标， 内皮细胞被用作体外研究血管发生抑制作用的 模型。 然而结果往往变化很大且重复性较差， 原因可能是检测方法不完善或者细胞株不 稳定。 因此， 本实施例中使用 HMEC, 此细胞株符合作为一个良好的抗血管发生试验模 型。 细胞迁移试验中， ES剂量依赖型地抑制 VEGF诱导的 HMEC迁移， 其半数抑制浓度 (IC50) 为 4 g/ml (图 la) , 甚至浓度低至 （4 ng/ml) 的 ES都可以对 HMEC有 15%的 抑制率。 在用 bFGF刺激的 HMEC增殖试验中也可以得到类似的结果 （图 lb) 。 细胞增 殖和迁移实验的结果表明 HMEC是一株对 ES敏感的细胞株， 利用它进行的实验结果可 靠且重复性好。
HMEC对 ES的敏感性提示在这株细胞上可能存在 ES潜在的受体。 进一步的实验表 明， ES可以在生理条件下结合 HMEC (见图 2 d) 。 因此选择从 HMEC上分离 ES受 体。 重组 ES用其自带的组氨酸标签预先结合在镍柱上， 制备成 ES亲和柱。 粗提的 HMEC细胞质膜组分按照 Marshak等人的报道用 1% Triton X-100处理以释放膜蛋白。 处 理后的膜蛋白样品通过制务好的 ES亲和柱， 不结合 ES的蛋白用 PBS缓冲液从柱上洗 下， 结合 ES的蛋白挂在 ES亲和柱上。 收集每一组分并进行还原 SDS-PAGE分析。 对照 组的实验平行进行， 唯一的区别是使用未载有 ES的镍柱。 表观分子量为 110 kDa和 80 kDa的两个蛋白特异性地结合 ES亲和柱 （图 2a) ， 并随后用 MALDI-TOF结合肽段指紋 图谱鉴定为 NL (110 kDa) 和它降解的片段 （80 kDa) ， 此结果又通过 NL的单克隆抗 体 （Santa Cruz) 的免疫印迹得到进一步确证。 为了验证 ES和 NL的相互作用， 进行如下 研究： 体外免疫沉淀结果显示 ES和重组 L的相互作用具有特异性 （图 2b) ， 并且二者 形成一个复合物， 该过程可以被 200 mM肝素所破坏 （图 2c) 。 ES还可以通过 NL结合 HMEC, 因为 L的兔多克隆抗体 （根据 Ausubel et al, 1995制备）可以阻断此结合（图 2d) 。 用经 ES预温育的 HMEC进行的免疫沉淀的结果进一步证实了上述结论 （图 2e) 。 结果显示 ES与总内源性 NL (细胞表面， 细胞质和细胞核）在活细胞内形成复合 物。 此外， 激光扫描共聚焦荧光显微镜观察到了 ES和 NL在 HMEC表面的共定位 （图 2f-I) 。 总而言之， ES在体外或体内均特异性地结合 L， 说明 L是 ES的一个潜在受 体。 实施例 2
NL是 ES的一个新受体 如果 NL是 ES的受体， 它应该能够介导 ES在抗血管发生方面的活性， 例如抑制内 皮细胞的迁移、 增殖和粘附。 为了鉴定 NL在介导 ES发挥活性过程中的作用， 分别使用 ES、 重组 NL、 以及 NL的多克隆抗体进行了竞争性细胞迁移和增殖试验。 由于 NL是从 HMEC中分离出来的 ES受体， 它是否在其他被广泛接受的内皮细胞上具有类似的作用还 需要证明。 因此， 在从脐带静脉中直接分离的人 HUVEC进行竞争性细胞迁移和增殖试 验。 这种细胞可以在 VEGF的刺激下穿过一种孔径 8 μηι的膜， 而 ES抑制这种迁移。 重 组 NL可以以剂量依赖性的方式消除 ES的抑制作用 （图 3a) ， 显示了重组 NL与 ES的 抗新生血管活性有关。 如图 3b所示重组 L本身对细胞迁移没有作用， 排除 NL本身可 以剌激内皮细胞的迁移的可能。 HUVEC增殖试验也得到了类似的结果 （图 3c) 。 正如所 预期的， NL的多克隆抗体可以拮抗 ES对细胞增殖的抑制作用 （图 3c) 。 这些研究结果 强有力的证明了 NL是 ES抗血管发生过程中的受体。
为了进一步确定 NL是 ES的受体， 使用 RNA千扰（RNAi) ( Sui, G. et al. Proc. Natl. Acad. Sci. USA 99, 2002, p5515-5520) 对 NL的表达进行抑制， 随后检测了这种表达 变化对 ES在抗血管发生方面的一个重要活性——细胞粘附的影响。 结果表明， 当 NL的 表达被以基于 DNA载体的 RNA干扰抑制时， HMEC在固定化 ES和多聚赖氨酸
(Sigma) (一种人工合成的细胞外基质） 上的粘附显著降低 （图 3d) ， 尽管这种抑制是 不完全的 （图 3e) 。 Rehn等人报道， 内皮细胞在固定化 ES上的粘附对 ES的活性例如粘 着斑的形成和 FAK的磷酸化是关键的， 而这种粘附的丧失意味着 ES在内皮细胞上的功 能的丧失。 这一系列的研究证明了 NL是 ES发挥活性和内皮细胞对细胞外基质（ECM) 粘附所必需的。 此外还检测了当 NL的表达被以基于 DNA载体的 R A干扰抑制后， HMEC对 ES在抗血管发生方面的另一重要活性——细胞增殖的影响。 当 HMEC细胞膜 中的 NL的表达被抑制后， HMEC细胞的增殖不能被 ES抑制， 而 L的表达没有被抑制 的 HMEC细胞的增殖可以被 ES抑制 （图 3f) 。 综上所述， NL是 ES的一个新受体， 并 在 ES的信号传导通路中发挥了重要的作用。 实施例 3
NL介导 ES的信号传导通路 为了揭示 NL在 ES的信号传导通路中的确切作用， 其下游事件也被研究。 注意到当 ES与 HMEC温育时， ES-NL复合物的量随时间变化， 且在 2小时左右达到最大值（图 2e) ， ES可能通过细胞表面 NL被内吞， 如 HMEC， 随后一些内吞的 ES被细胞降解。 提示存在一个 ES内吞与降解之间的平衡 （图 2e) 。
为证实 ES的内吞是通过细胞表面 NL进行的， 用生物素 （Pierce)标记 ES并进行免 疫荧光定位。 HMEC与生物素标记的 ES温育不同时间后， 生物素标记的 ES被 TRITC标 记的抗生物素蛋白 （Pierce) 染色， 进而用荧光显微镜（Olympus)观察 ES的内吞过程
(图 4a-f) 。 温育 30分钟时， 多数内吞的 ES分布在胞质内且量较少 （图 4b) 。 温育 1 小时， 内吞的 ES量增加并且开始在核内积累 （图 4c) 。 内吞的 ES的量在温育 2小时左 右达到最大值 （图 4d) 。 3小时的时候， 积累在核内的 ES开始消失（图 4e) 。 7小时的 时候， 核内几乎看不到 ES (图 4f) 。 相比于免疫共沉淀的结果， 荧光实验中， 内吞的 ES 的量到达最大值的时间有一个延迟， 可能是因为生物素标记的 ES降解速率较慢。 重要的 是， 当 HMEC预先与 L的多克隆抗体温育后， 内吞的生物素标记 ES的量显著减少， 说 明 ES是通过细胞表面 NL被内吞的 （图 4d, g) 。 此外， 荧光显微镜下还可观察到细胞内 些小的亮点， 可能意味着 ES的内吞是通过膜泡进行的。 这些现象与 Wickstrom和 Christian等人先前的报道是一致的。
为解释 L如何介导 ES的内吞， 分别进行了交联和免疫共沉淀实验。 首先， 通过交 联试剂从活的 HMEC中分离到 ES, NL, 以及其它蛋白质的复合物是所希望的。 利用 BS3 (PIERCE公司的一种交联试剂） 得到了分子量远远大于 300 kDa的巨大复合物（数 据未显示） 。 尽管该复合物可以被 ES或者 L的抗体染色， 但是其组成部分却无法鉴 定。 另外一种方法是用 NL的多克隆抗体在 HMEC裂解液中进行免疫共沉淀以分离与 NL 相互作用的蛋白。 发现了一个分子量 200 kDa左右的蛋白， 并经 MALDI-TOF鉴定为非肌 肉肌球蛋白。 肌球蛋白组成一个很大的超家族， 参与到膜动力学过程以及膜下区域内肌 动蛋白的组织， 由此影响细胞迁移， 吸附以及内吞。 在这个超家族中， 拥有两个 "头" 结构域的第五类肌球蛋白可以沿肌动蛋白纤维运送膜泡， 细胞器和 mRNA颗粒。 还发现 细胞表面 NL可以结合 ES并将 ES运输到细胞核 （图 4a-f) 。 因为肌球蛋白是细胞内蛋 白， 它必然要与细胞表面 L的细胞内结构域结合。 推测这个 L-肌球蛋白复合物在 ES 的内吞过程中起到了转运载体的作用。 Shibata等人报道过相似的过程： 一种神经细胞的 生长因子， midkine, 也可以经 NL被内吞并定位于细胞核。 有趣的是， NL的 N端抗体可 以经细胞表面 NL被内吞到 Hep-2细胞内。 因此， 内吞似乎是一种普遍现象而且当配体结 合细胞表面 NL后变得不可避免。 区别在于 ES可以抑制细胞增殖而 L的抗体则不能 (图 3c)。
上述现象说明了尽管很多配体可以特异性结合到细胞表面 NL并且引发它们的内 吞， 它们后续的命运却是不同。 随后考察 ES被运送到细胞核后的功能。 Bouche等人报道 了 L可以促进一些对于细胞生存和增殖很重要的事件， 例如 DNA的转录以及核内核糖 体的生成， 只要其 N端的丝氨酸残基在 bFGF剌激下被某些激酶， 例如 CK2所磷酸化。 Folkman和他的同事们也报道了 bFGF所刺激的内皮细胞的迁移可以被 ES所抑制。 根据 Bouche等人的研究， bFGF剌激的 NL的磯酸化是由 CK2介导而非其它激酶， 例如封闭 的核内的 cdc2。 因此， 推测 ES通过抑制 bFGF刺激的 C 2介导的 NL磷酸化来抑制细胞 增殖。 为了证实此猜想， 分离出处于静止状态的 HMEC的核并在 ES存在或不存在的条 件下进行了磷酸化实验。 结果显示当 ES与 HMEC核预先温育后， NL的磷酸化被抑制 (图 4h) 。 此外， bFGF而非 VEGF可以剌激 NL的磷酸化（图 4h) ， 这解释了以前的 报道中 bFGF可以剌激内皮细胞的增殖而 VEGF可以剌激内皮细胞的迁移。 本实施例中所 观察到的现象表明 ES可以抑制 bFGF刺激的 CK2所介导的 NL磷酸化， 结果抑制了细胞 的生存和增殖。 实施例 4
ES通过 NL-肌球蛋白复合物影响细胞迁移和粘附 已经证明了 NL是 ES的一个新受体， 并介导 ES对细胞迁移、 增殖和粘附的抑制作 用。 由于整联蛋白也被报道是 ES的受体， 因此 L和整联蛋白之间是否存在相互作用是 令人感兴趣的。 在整联蛋白家族中， Rehn和 Sudhakar等人报道整联蛋白 α5β1是 ES的 受体， 因此， 使用整联蛋白 βΐ的鼠抗（Santa Cruz)和 NL的兔抗来做间接免疫荧光试 验以研究细胞表面的 NL和整联蛋白 οι5β1是否定位在一起。 通过激光扫描共聚焦显微 镜， 观察到细胞表面 NL和整联蛋白 βΐ在特定部位的共定位 （图 4 i-k) , 暗示 L和整 联蛋白 βΐ在细胞表面有某种相互作用。 由此， 尝试通过免疫共沉淀的方法， 使用 的 多克隆抗体来捕捉 NL和整联蛋白 βΐ的复合体。 但不幸的是在沉淀中没有检测到整联蛋 白 β1， 这暗示 NL和整联蛋白 βΐ之间的相互作用是间接的。 已知肌球蛋白参与膜的运动 以及肌动蛋白在细胞皮层的组织装配， 进而影响到细胞迁移、 粘附和内吞。 同时， 已知 NL和非肌肉肌球蛋白在细胞皮层形成一个复合物， 并且这种复合物对于内皮细胞的运 动性和粘附起着关键的作用。 因此， 推测 ES可能通过 NL-肌球蛋白复合物干扰细胞的运 动性和粘附。 有可能的是， 细胞表面的 L和整联蛋白 α5β1连同其他蛋白如肌球蛋白 一起形成一个大的复合物来作为 ES的受体（见图 6) 。 实施例 5
NL的分布为 ES的低毒性提供了基础
ES特异性地抑制血管发生和肿瘤生长， 并且观察到 ES在动物实验中没有毒性， 在 临床试验中仅显示低毒性。 但这种现象背后的具体分子机理却还不清楚。 推测 NL介导 ES在抗血管发生中的特异性的活性， 这似乎与过去所报道的 NL是细胞中的一种普遍存 在的蛋白相矛盾。 为了解释这一矛盾， 考察不同生长状态下 HMEC表面 NL的丰度， 结 果显示在旺盛增殖的细胞表面 NL的丰度要远远高于在相对静止的细胞表面（图 5 a-f) 。 相对静止的细胞是通过血清饥饿 24小时得到的， 它们所处的细胞周期通过流式细胞仪 (Becton Dickinson, Worldwide Inc., San Jose, CA)进行了检测 （图 5 g-h) 。 流式细胞仪 的检测结果显示血清饥饿后处于 G1期的细胞比例增加了 24 %， S期的减少了 30 % (图 5 g-h) 。 尽管细胞没有完全处于 G1期， 细胞表面的 L的量在血清饥饿 24小时后显著 减少。 推测这种细胞膜表面 NL丰度的不同导致了内皮细胞对 ES敏感度的不同。
为了研究是否类似的细胞学现象也会在动物体内的研究中发现， 对肿瘤组织和正常 器官细胞表面 NL的丰度进行了检测。 首先， 在接种了 Hela肿瘤的裸鼠 （北京维通利华 实验动物技术有限公司） 的背部皮下注射 NL的多克隆抗体， 注射部位远离接种的肿瘤， 然后使用免疫组织化学的手段观察这些抗体的分布 （图 5 i-l) 。 结果显示 NL的抗体只在 肿瘤诱生的内皮细胞上积累， 而在正常组织例如心脏、 肾和肺中没有发现 （图 5 i-D 。 这 些发现与 Folkman的抗血管发生理论非常的一致： 在成年人中， 除非血管内皮细胞被一 些内源性的血管发生剌激因子所上调， 否则它们会处于一种静止的状态而几乎不进行增 殖； 而正在增殖中的内皮细胞出现在某些生理或病理的血管发生过程中如肿瘤生长和转 移。 总之， NL， 也就是 ES的受体在内皮细胞表面的不同丰度为 ES在动物和临床试验中 的低毒性提供了一种解释。 因为在肿瘤诱生的血管内皮细胞膜表面的 L比正常组织的丰 富的多， 因此 ES特异性的结合 NL从而选择性的在肿瘤组织上发挥其抗血管发生的活 性； 另一方面， ES很少与正常器官结合， 因为正常器官细胞表面 NL较少， 这反过来也 使得在用 ES治疗肿瘤时不会带来毒性。 实施例 6
重组 NL在细胞迁移实验中调节 ES的功能 使用 SV总 RNA分离系统以及反转录系统 (Promega) 按照制造商建议的操作规程 从 HMEC中得到 L的 cDNA。 PCR得到 NL与多聚组氨酸（ffis)6的融合序列后， 继而亚 克隆到表达载体 pPIC9K (Invitrogen) 。 按照制造商手册上的建议， 该载体经限制性内切 酶 Sai l (Promega) 线性化后电转化甲醇酵母菌株 GS115。 经 G418 (Invitrogen)筛选 得到的稳定转化子于 30Ό， BMMY培养基 （10 g L酵母粉； 20 g/L蛋白胨； lOO mmol/L 磷酸钾， pH 6.0; 13.4 g/L酵母含氮碱基； 40 mg/L生物素以及每日补加至终浓度 0.5%的 甲醇） 中摇瓶培养 3天。 培养基上清经镍离子亲和柱 （Qkgen) 纯化得到 L。 将 1 L培 养基上清 pH调到 8.0后上 6 ml Ni-NTA柱并按照制造商的建议进行洗涤和洗脱。 最终可 从每升培养基获得约 3mg NL。 利用该方法得到的 NL制备了 NL的多克隆抗体。
HUVEC (2xl04每孔)） 接种到含有 DMEM培养基， 0.5%胎牛血清， 以及 10 ng/ml bFGF (PeproTech EC) 的 Transwell滤膜 （8 μιη孔， Costar) 的上室中。 ES (5 g/ml, Protgen)以及重组 NL (20 g/ml)， 或 NL的抗体（20 g/ml)于迁移实验开始时添加。 PBS 亦作为对照加入此迁移实验系统。 同样的 DMEM培养基与其它试剂也加在下室中。 内皮 细胞在 37°C， 5% C02的条件下迁移 6 h。 经乙醇和曙红固定染色后， 在光学显微镜下选 择五个不同的视野对完全迁移过滤膜的细胞进行计数并取其平均值。 结果显示重组人 NL 以剂量依赖的方式削弱 ES对细胞迁移的抑制作用， 说明重组 NL与 ES的抗血管发生活 性枏关。 类似地， NL的多克隆抗体阻断了 ES对细胞增殖的抑制作用。 实施例 7
肿瘤组织中血管表面 NL被其抗体封闭后肿瘤生长加速
使用 SV总 RNA分离系统以及反转录系统 （Promega) 按照制造商建议的操作规程 从 HMEC中得到 NL的 cDNA。 PCR得到 NL与多聚组氨酸（His)6的融合序列后， 继而亚 克隆到表达载体 pPIC9K (Invitrogen) 。 按照制造商的建议， 该载体经限制性内切酶 Sal I (Promega) 线性化后电转化甲醇酵母菌株 GS115。 经 G418 (Invitrogen)筛选得到的 稳定转化子于 30°C， BMMY培养基（10 g/L酵母粉； 20 g/L蛋白胨； 100 mmol/L磷酸 钾， pH 6.0; 13.4 g/L酵母含氮碱基； 40 mg/L生物素以及每日补加至终浓度 0.5%的甲 醇） 中摇瓶培养 3天。 培养基上清经镍离子亲和柱 （Qiagen) 纯化得到 NL。 将 1 L培养 基上清 pH调到 8.0后上 6 ml Ni-NTA柱并按照制造商的建议进行洗涤和洗脱。 最终可从 每升培养基获得约 3mgNL。 利用该方法得到的 NL制备了 NL的多克隆抗体。
Hela细胞接种至裸鼠 （北京维通利华实验动物技术有限公司）皮下。 从次日起， L 的抗体每三天一次缓慢注射到远离肿瘤接种部位的鼠背部皮下。 第七次注射后， 裸鼠被 处死， 称重肿瘤并测量三次其大小。 这些动物实验的结果显示当肿瘤组织中血管表面 NL 被抗体封闭后肿瘤生长显著加快， 说明 NL在调节肿瘤生长和血管发生中扮演重要角色。 实施例 8
利用 ES-Ni-NTA亲和层析柱筛选 NL-特异性血管发生抑制剂 使用 SV总 RNA分离系统以及反转录系统 （Promega) 按照制造商建议的操作规程 从 HMEC中得到 NL的 cDNA。 PCR得到 NL与多聚组氨酸（His)6的融合序列后， 继而亚 克隆到表达载体 pPIC9K (Invitrogen) 。 按照制造商的建议， 该载体经限制性内切酶 Sal I (Promega)) 线性化后电转化甲醇酵母菌株 GS115。 经 G418 (Invitrogen)筛选得到的 稳定转化子于 30°C， BMMY培养基（ 10 g/L酵母粉； -20 g/L蛋白胨； 100 mmol/L磷酸 钾， pH 6.0; 13.4 g/L酵母含氮碱基； 40 mg/L生物素以及每日补加至终浓度 0.5%的甲 醇） 中摇瓶培养 3天。 培养基上清经镍离子亲和柱 （Qiagen) 纯化得到 L。 将 1 L培养 基上清 pH调到 8.0后上 6 ml Ni-NTA柱并按照制造商的建议进行洗漆和洗脱。 最终可从 每升培养基获得约 3 mg NL。 L经其 N端融合的组氨酸标签固定在 Ni-NTA镍离子亲和 柱上。 此柱料可以用来高通量筛选与 L结合的蛋白。 所得到的 L结合蛋白可以利用 MALDI-TOF结合肽段指紋图谱鉴定。 它们在抗血管发生过程中的生物活性可由细胞实 验， 例如上面描述的细胞迁移与增殖实验， 来检测。 实施例 9
ES与 NL的体内共定位 SPR (Surface plasmon resonance)
ES与 L的结合动力学通过 SPR方法测定 （方法参考 BIAcore 2000™生物传感系 统手册） ， 仪器是 Amersham生物技术公司的 BIAcore 2000™生物传感系统。 纯化后的 ES用 20 mM醋酸钠缓冲液（pH 6.5)稀释成 100 g/ml, 并按照使用手册上的方法， 通过 使用氧基偶联试齐 U盒 (l-ethyl-3-(dimethylaminopropyl)carbodiimide， ( -hydroxysuccinimide) (Amersham生物技术公司）共价固定在研究用 CM5传感芯片上 （Amersham生物技术公 司） 。 100 g/ml的 ES (20 mM醋酸钠缓冲液， pH 6.5)注射到活化的研究用 CM5传感 芯片上， 直到在 SPR仪器上响应值达到 9000个单位。 没有反应的芯片表面用 pH 8.5的 乙醇氨封闭 （Amersham生物技术公司） 。 ES与 L的结合动力学 SPR在 25 °C进行， 溶解在流动相 HBS ( 10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, and 0.005% surfactant P20, pH 7.4) 中的 L以 10 μΐ/min的流速注射进 SPR 20 μ1。 ES与 NL的结合动力学曲线 就是在流动相中 L的浓度随时间变化的值。 原始数据通过 Amersham生物技术公司的分 析软件 BIAevaluation 3.1分析。 ES与 NL的结合率常数 ka,解离率常数 kb和解离平衡常 数 KD通过 Langmu 结合模型计算（化学计量比 1： 1 ) 。
- 实时 SPR是一种快速灵敏的测定两个分子间相互作用的实验方法。 用它来测定 ES 与 NL之间的亲和力。 通过对这些 ES与 NL的结合动力学曲线的分析， 计算出 ES与 NL 的解离常数 KD = 2.32x10— 8 M。
ES与 NL的体内共定位
将旺盛增殖期的小鼠黑色素瘤细胞 B16/F10 (ATCC) (2X 106 cells in 200 μΐ of PBS)接种在 2个月大的 Balb/c小鼠 （北京维通利华实验动物技术有限公司） 的皮下。 八 天后， 进行 ES与 NL在小鼠体内的共定位实验。 标记有生物素的 ES和 NL的兔多克隆 抗体分别先后被静脉注射到荷瘤小鼠体内。 对照组注射标记有生物素的 ES和纯化的兔多 克隆抗体。 一小时后， 小鼠被麻醉， 用 20 ml PBS从心脏灌流， 然后处死。 小鼠的一些正 常组织和肿瘤被取出、 固定、 切片。 切片同时用 TRITC标记的抗生物素蛋白 （Pierce)和 FITC标记的二抗 （Santa Cruz)检测， 在 Olympus Fluoview激光扫描共聚焦成像系统下 进行观察 （Olympus Inc.) 。 在体内也观测到了 ES和 NL的共定位。 标记有生物素的 ES和 L的兔多克隆抗体分 别先后被静脉注射到荷瘤小鼠体内。 对照组注射标记有生物素的 ES和纯化的兔多克隆抗 体。 一小时后， 小鼠的一些正常组织和肿瘤用于免疫荧光检测。 标记有生物素的 ES和 NL的兔多克隆抗体选择性地在肿瘤地血管表面聚集 （图 7j-l) 。 在心脏 （图 7a-c) 、 肝 脏（图 7d-f) 、 肾脏 （图 7g-i) 的血管壁上并没有发现标记有生物素的 ES和 NL的兔多 克隆抗体。 在小鼠肿瘤组织中， 标记有生物素的 ES和 NL的兔多克隆抗体很好地重叠在 了一起。 在对照肿瘤组织中， 并没有发现对照的 NL的兔多克隆抗体 （图 7m-o) 。 这些 结果表明， ES和 NL只在肿瘤血管表面共定位， 在正常组织内没有共定位。 实施例 10
靶内皮细胞上 NL的量与 ES的抗新生血管活性的关系 使用如实¾例 1中的体外内皮细胞增殖模型研究 ES的抗新生血管活性。 通过这个模 型研究 ES对 NL缺陷型内皮细胞增殖的影响， 从而确定内皮细胞上 NL的量与 ES的抗新 生血管活性的关系。
使用 RNA干扰 （RNAi)对 NL的表达进行抑制， 随后检测了这种表达变化对 ES在 抗新生血管发生方面的一个重要活性——细胞增殖的影响。 当 HMEC细胞转染了基于 DNA载体的 R A干扰质粒 BS/U6/1356后， HMEC细胞中的 NL的表达被抑制了 （图 9a) 。 在转染了对照质粒 BS/U6, BS/U6/263和 BS/U6/1356C的 HMEC细胞中的 NL的表 达没有被抑制 （图 9a) 。 还进一步检测了当 NL的表达被以基于 DNA载体的 RNA干扰 抑制后， HMEC对 ES在抗新生血管发生方面的另一重要活性——细胞增殖的影响。 结果 显示： 当 HMEC细胞中的 NL的表达被抑制后， HMEC细胞的增殖不能被 ES抑制， 而 NL的表达没有被抑制的 HMEC细胞的增殖可以被 ES抑制 （图 9b) 。 从这个结果可以看 出， 内皮细胞中 L的量与 ES的抑制新生血管发生的活性直接相关。 一方面， 可以通过 增加内皮细胞上 L的量来增加 ES的抑制新生血管发生的活性； 另一方面， 可以检测肿 瘤新生血管中 NL的量来预测 ES对这种肿瘤， 或者这个肿瘤患者的疗效。
基于 DNA载体的 RNA干扰质粒 BS/U6的构建如 Shi博士和他的同事的文章中 ( " A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. " Sui, G. et al. Proc. Natl. Acad. Sci. USA 99, 2002, p5515-5520)所述： 针对 L特异性的 RNA干扰序列 BS U61356是 NL序列中从 1356到 1377， 同样， 针对 L特异性的 RNA 干扰序列 BS/U6/263是 NL序列中从 263到 283 (这个干扰序列无活性） 。 同时一个与这 段特异性序列碱基组成相同的随机序列 BS/U61356C平行用来作为对照。 经 NCBI中查 找， 随机序列与任何已知序列无明显同源性。 质粒用 lipofectin™ (Invitrogen)转染细 胞。 所有上述提到的论文， 著作， 专利， 专利申请， 网站以及其它印刷的文档， 都被包括 在但不仅限于以下的参考文献列表中。
参考文献
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34. Bonnet, H. et al. Fibroblast Growth Factor-2 Binds to the Regulatory beta Subunit of CK2 and Directly Stimulates CK2 Activity toward NL. J. Biol. Chem. 271, 24781-24787 (1996).
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Claims

权 利 要 求
1. 一种评估是否对一个对象使用血管内皮抑制素进行癌症治疗的诊断试剂盒； 包括： a. 一种对核仁素进行标记的标记物； 以及
b. —种通过对得自所述对象的样品进行上述标记后， 测定该样品中核仁素含量的 使用指导。
2. 权利要求 1的试剂盒， 其中所述对象是哺乳动物。
3. 权利要求 1的试剂盒， 其中所述对象是人类。
4. 权利要求 1的试剂盒， 其中所述被标记的核仁素是细胞膜表面的核仁素。
5. 权利要求 1的试剂盒， 其中所述被标记的核仁素是全细胞中的核仁素。
6. 权利要求 1的试剂盒， 其中所述标记物包括抗体。
7. 权利要求 6的试剂盒， 其中所述抗体是单克隆抗体。
8. 权利要求 6的试剂盒， 其中所述抗体是多克隆抗体。
9. 权利要求 1的试剂盒， 其中所述标记物包括核酸分子。
10.权利要求 9的试剂盒， 其中所述核酸分子是 DNA分子。
11.权利要求 9的试剂盒， 其中所述核酸分子是 RNA分子。
12.—种评估使用血管内皮抑制素对对象进行癌症治疗的成功的可能性的方法； 包括- a. 对从所述对象中得到的样品进行关于核仁素表达的水平的筛选； 以及 b. 基于核仁素表达量， 决定是否对所述对象使用血管内皮抑制素进行癌症治 疗。
13.一种获得核仁素特异性的有效的血管发生抑制剂的方法； 包括：
a. 对待筛选的分子进行合适的结合实验， 从而得到一些核仁素特异性分子； b. 利用抗血管发生试验检验上步骤得到的各种分子抑制血管发生的效果； 以及 c 选择出能在抗血管发生试验中有效抑制血管发生的核仁素特异性分子。
14.权利要求 13的方法， 其中所述核仁素特异性血管发生抑制剂是蛋白质或肽。
15.权利要求 13的方法， 其中所述核仁素特异性血管发生抑制剂是小分子。
16.权利要求 13的方法， 其中所述核仁素特异性血管发生抑制剂用来治疗血管发生依赖 性疾病。
17.权利要求 16的方法， 其中所述血管发生依赖性疾病是癌症。
18.权利要求 16的方法， 其中所述血管发生依赖性疾病是内皮细胞疾病。
19.—种选择当在体外加入到增殖中的内皮细胞中时具有抑制内皮细胞增殖和 /或迁移的 血管发生抑制剂的方法， 包括如下步骤：
a. 使用药物学可接受的方法去发现与核仁素有特异性相互作用的分子； b. 检测 a步骤中得到的分子抑制内皮细胞增殖和 /或迁移的效果； 并且
c 确定所得到的能有效抑制内皮细胞增殖和 /或迁移的分子， 同时对比该分子的 抗血管发生的功能与血管内皮抑制素的抗血管发生的功能的效果。
20.权利要求 19的方法， 其中所述核仁素特异性分子是蛋白质或肽。
21.权利要求 19的方法， 其中所述核仁素特异性分子是小分子。
22.一种提高靶细胞对血管发生抑制剂的响应能力的方法， 包括- a. 导入外源性核仁素至靶细胞， 从而获得修饰后的表达更多核仁素的靶细胞； 以 及
b. 测定血管内皮抑制素对这些修饰过的靶细胞的杀伤率。
23.权利要求 22的方法， 其中所述靶细胞是癌细胞。
24.权利要求 22的方法， 其中所述靶细胞是内皮细胞。
25.权利要求 22的方法， 其中所述血管发生抑制剂是血管内皮抑制素。
26.权利要求 22的方法， 其中所述外源核仁素通过病毒载体导入靶细胞。
27.一种增强血管发生抑制剂对靶内皮细胞的抗血管发生效果的方法， 包括：
a. 将药物学有效量的外源核仁素分子导入所述靶细胞， 所述核仁素分子能够在 所述靶细胞中表达； 以及
b. 将所述靶细胞与所述血管发生抑制剂共温育， 从而引起对所述靶细胞生长的抑 制。
28.权利要求 27的方法， 其中所述血管发生抑制剂是血管内皮抑制素。
29.权利要求 27的方法， 其中所述靶内皮细胞是癌细胞。
30.一种检验靶细胞对于血管发生抑制剂的个体敏感度的诊断试剂盒， 包括- a. 特异性结合核仁素分子的分子； 以及
b. 一种药物学可接受的载体。
31.权利要求 30的诊断试剂盒， 其中所述血管发生抑制剂是血管内皮抑制素。
32.权利要求 30的诊断试剂盒， 其中所述靶细胞是癌细胞。
33.一种确定对抗血管发生抑制剂治疗敏感的靶肿瘤细胞的诊断试剂盒， 包括：
a. 一种针对核仁素的抗体； 以及
b. 一种药物学可接受的载体。
34.权利要求 33的诊断试剂盒， 其中所述抗体是多克隆抗体。
35.权利要求 33的诊断试剂盒， 其中所述抗体是单克隆抗体。
36.—种确定患者对血管内皮抑制素治疗敏感程度的方法； 包括： 取患者的一个样品， 与 核仁素抗体接触， 以及检测样品中核仁素与其抗体形成的复合物， 复合物的髙水平 存在提示血管内皮抑制素治疗成功的高可能性。
37.一种提高血管发生抑制剂控制患者携带的肿瘤生长的效果的方法， 包括：
a. 确定所述患者肿瘤样品中存在的内源性核仁素水平； 并且
b. 利用所述患者核仁素表达水平确定血管发生抑制剂对所述患者有抑瘤效果的 可能性， 较高水平的核仁素提示使用血管发生抑制剂治疗更容易成功。
38.权利要求 37的方法， 其中所述血管发生抑制剂是血管内皮抑制素。
39.权利要求 37的方法， 其中所述确定患者肿瘤样品中核仁素表达水平通过抗核仁素的 抗体与核仁素的免疫沉淀进行。
40.一种确定对抗血管发生抑制剂治疗敏感的靶癌细胞的方法， 包括- a. 制备一种抗核仁素的抗体；
b. 使所述抗核仁素抗体与从得自一个对象的样品接触； 以及
c. 根据在所述样品中存在的核仁素水平鉴别对血管发生抑制剂治疗敏感的靶癌细 胞， 高水平提示高敏感性。
41.权利要求 40的方法， 其中所述抗核仁素的抗体是多克隆抗体。
42.权利要求 40的方法， 其中所述抗核仁素的抗体是单克隆抗体。
43.权利要求 40的方法， 其中所述血管发生抑制剂是血管内皮抑制素。
44.一种抑制细胞样品中一些内皮细胞增殖和 /或迁移的方法， 包括：
a. 将抗核仁素抗体与胞毒剂连接， 从而形成抗核仁素毒性抗体； 以及
b. 将所述抗核仁素毒性抗体施用于所述细胞样品， 从而抑制所述一些内皮细胞的 增殖和 /或迁移。
45.权利要求 44的方法， 其中所述胞毒剂是细胞因子。
46.权利要求 44的方法， 其中所述胞毒剂是肿瘤坏死因子。
47.权利要求 44的方法， 其中所述细胞样品得自癌症患者。
PCT/CN2006/000942 2005-05-12 2006-05-11 Procede auxiliaire de diagnostic et traitement du cancer a la nucleoline WO2006119706A1 (fr)

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