WO2019134526A1 - 膜型金属蛋白酶抑制蛋白和包含其的药物和药物组合物及各自的用途 - Google Patents

膜型金属蛋白酶抑制蛋白和包含其的药物和药物组合物及各自的用途 Download PDF

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WO2019134526A1
WO2019134526A1 PCT/CN2018/122305 CN2018122305W WO2019134526A1 WO 2019134526 A1 WO2019134526 A1 WO 2019134526A1 CN 2018122305 W CN2018122305 W CN 2018122305W WO 2019134526 A1 WO2019134526 A1 WO 2019134526A1
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prαtace
cells
bhk
pharmaceutical composition
cancer
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PCT/CN2018/122305
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English (en)
French (fr)
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李明辉
姜冰洁
刘坚
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西交利物浦大学
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Priority claimed from CN201810004200.2A external-priority patent/CN108314727B/zh
Priority claimed from CN201811257903.2A external-priority patent/CN109394786A/zh
Application filed by 西交利物浦大学 filed Critical 西交利物浦大学
Priority to US16/960,010 priority Critical patent/US11851475B2/en
Publication of WO2019134526A1 publication Critical patent/WO2019134526A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/8146—Metalloprotease (E.C. 3.4.24) inhibitors, e.g. tissue inhibitor of metallo proteinase, TIMP
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/33—Fibroblasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A61K38/57—Protease inhibitors from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors

Definitions

  • the present application relates to the field of biomedical technology, and in particular to a membrane type metalloproteinase inhibitory protein having inhibitory effects on proliferation of human renal cancer cells, and pharmaceutical and pharmaceutical compositions comprising the same, and respective uses thereof.
  • the current technologies for treating cancer can be roughly divided into three categories:
  • Chemotherapy The most common side effects of chemotherapy include nausea and vomiting, hair loss, immunosuppression, and loss of appetite.
  • Radiotherapy The side effects of radiotherapy are usually slightly less than those of chemotherapy. Skin reactions such as peeling, erythema and fibrosis are side effects associated with radiation therapy. In addition, this treatment requires specialized treatment equipment and hospital environments.
  • Immunotherapy Immune-related symptoms such as fatigue, fever, weakness, nausea, body aches and high blood pressure are common side effects of immunotherapy.
  • Membrane Type 1-Matrix Metalloproteinase (MT1-MMP) and TNF- ⁇ Converting Enzyme (TACE) are zinc-dependent transmembrane peptide hydrolase enzymes, both of which are involved.
  • the decomposition and remodeling of the extracellular matrix (ECM) plays an important role in the evolution of tumor invasion and metastasis by releasing various active factors required to induce cell growth.
  • MT1-MMP can degrade a variety of extracellular matrices and basement membrane macromolecules (such as collagens I, II and III, laminin, fibronectin, fibrin and some important adhesion molecules)
  • the signaling molecules required to activate cancer cell invasion and spread (such as multi-ligand glycans, CD44, RANKL, MUC1, etc.) play a key role in promoting tumor invasion and metastasis.
  • TACE is a member of the A Disintegrin and Metalloproteinase family, also known as ADAM-17.
  • the activity of TACE is mainly manifested in the following aspects: by hydrolyzing extracellular extracellular membrane precursors, TACE can release many ligands and growth factors (such as TNF- ⁇ , TGF- ⁇ , HG-EGF), receptors (TNFR II). , EGFR, IL-6R, Notch-1), adhesion molecules (ICAM-1, V-CAM-1, L-selectin) and prions associated with neurodegenerative diseases and infectious spongiform encephalopathy. Through these molecules for signal transduction, TACE can affect the occurrence, progression and metastasis of tumors.
  • TACE can affect the occurrence, progression and metastasis of tumors.
  • TIMPs endogenous metalloproteinase tissue inhibitors
  • TIMPs Through its wedge-shaped N-terminal domain, TIMPs can form a tight 1:1 conjugate with the zinc ion binding site in the catalytically active region of the target protease (PDB crystal case #4ILW, #3V96, #3CKI, etc.), thereby exerting MMP Inhibition.
  • PDB crystal case #4ILW, #3V96, #3CKI, etc. the target protease
  • MMP Inhibition MT1-MMP is sensitive to the inhibitory activity of TIMP-2, -3 and -4, but to TIMP-1.
  • TIMP-3 is the only TIMP member that inhibits TACE activity.
  • other TIMPs are highly soluble secreted proteins.
  • TIMP-1 is the first member of the TIMP family isolated from human lymphocytes in 1980. It has a molecular weight of approximately 25 kDa and is a secreted glycoprotein with no significant inhibitory activity on MT1-MMP and TACE. . At the same time, studies have shown that TIMP-1, as a cell growth factor, can enhance the activity of cancer cells.
  • TIMP-1 Disadvantages of existing proteins (ie, original TIMP-1): (1) The original TIMP-1 is a soluble secreted protein. Whether intracellular or extracellular, there is no evidence that TIMP-1 interacts with MT1-MMP and TACE. (2) TIMP-1 has no inhibitory effect on MT1-MMP and TACE. Correlation experiments determined that TIMP-1 has a K i app for MT1-MMP or TACE greater than 150 nM. (3) TIMP-1 can enhance cell activity and greatly limit its value in cancer research.
  • the problem to be solved by the present application is to provide a modified and improved membrane type metalloproteinase inhibitor protein T1 Pr ⁇ TACE according to the prior art metalloproteinase inhibitor protein TIMP.
  • the first aspect of the present application provides an edit-modified membrane type metalloproteinase inhibitor protein T1 Pr ⁇ TACE having an amino acid sequence as shown in Seq ID No. 1.
  • the second aspect of the present application provides the use of the above membrane type metalloproteinase inhibitor protein T1 Pr ⁇ TACE for the preparation of a tumor drug having a MT1-MMP or TACE endonuclease as a potential target.
  • a third aspect of the present application provides a medicament comprising a membrane type metalloproteinase inhibitor protein T1 Pr ⁇ TACE and a pharmaceutically acceptable excipient.
  • the excipient is a carrier, a solvent, an emulsifier, a dispersing agent, a wetting agent, a binder, a stabilizer, a coloring agent, and a perfume.
  • the drug is an injection, a tablet, a capsule, a granule, a drop, a granule or an ointment.
  • the preparation of the pharmaceutical composition targeting MT1-MMP or TACE endonuclease can be prepared by adding a universal medicinal auxiliary component to a protein T1 Pr ⁇ TACE having a purity of 98% or more.
  • the T1 Pr ⁇ TACE protein can be formulated as a solution, emulsion, suspension, dispersion, or in a suitable pharmaceutical solvent or carrier, such as a cyclodextrin, according to conventional methods known in the art for preparing various dosage forms.
  • a suitable pharmaceutical solvent or carrier such as a cyclodextrin
  • the pharmaceutical compositions of the embodiments can be administered by a suitable route of delivery, such as oral, parenteral, rectal, nasal, topical, or ocular routes, or by inhalation.
  • the composition is formulated for intravenous or oral administration.
  • the drug may be provided in the form of a solid, such as a tablet or capsule, or as a solution, emulsion or suspension.
  • Oral tablets may include active ingredients in admixture with pharmaceutically acceptable compatible excipients such as diluents, disintegrating agents, binding agents, lubricants, sweetening agents, flavoring agents, coloring agents And preservatives.
  • suitable inert fillers include sodium carbonate and calcium carbonate, sodium and calcium phosphate, lactose, starch, sugar, glucose, methylcellulose, magnesium stearate, mannitol, sorbitol, and the like.
  • Exemplary liquid oral vehicles include ethanol, glycerin, water, and the like.
  • Exemplary disintegrants include starch, polyvinylpyrrolidone (PVP), sodium starch glycolate, microcrystalline cellulose, and alginic acid.
  • the binding agent can include starch and gelatin.
  • the lubricant if present, may be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated by materials such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract or may be coated by enteric coating.
  • Oral formulations may be presented in the form of discrete units, such as capsules, cachets or tablets, each containing a predetermined amount of active ingredient; in the form of powders or granules; in solution or in aqueous liquid or nonaqueous In the form of a suspension in a liquid; or in the form of an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
  • the active ingredient can also be formulated as a bolus, electuary or paste.
  • Oral capsules include hard and soft gelatin capsules.
  • the active component can be combined with a solid, semi-solid or liquid diluent.
  • the preparation of soft gelatin capsules can be achieved by mixing the active ingredient with water, oils such as peanut oil or olive oil, liquid paraffin, mixtures of mono- and diglycerides of short-chain fatty acids, polyethylene Alcohol 400, or propylene glycol.
  • Tablets may be made by compression or molding, and optionally one or more accessory ingredients may be added to the tablets.
  • Tablets can be made by compressing the active ingredient in a free-flowing form such as a powder or granules in a suitable machine, and optionally incorporating a binder (eg, povidone, gelatin, Hydroxypropyl methylcellulose), lubricants, inert diluents, preservatives, decomposers (eg, sodium starch glycolate, crospovidone, croscarmellose sodium), surfactants or dispersion Agent.
  • Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and may be formulated such that the active ingredient therein is released slowly or controlled, for example by using hydroxypropyl methylcellulose in varying proportions, Obtain the desired release profile.
  • the oral solution can be in the form of a suspension, solution, emulsion, or syrup, or can be lyophilized or presented as a dry product which can be reconstituted with water or other suitable vehicle before being administered.
  • Such liquid compositions may optionally comprise the following materials: pharmaceutically acceptable excipients such as suspending agents (for example, sorbitol, methylcellulose, sodium alginate, gelatin, hydroxyethylcellulose, carboxylate) Methylcellulose, aluminum stearate gel, etc.; non-aqueous carrier such as oil (for example, almond oil or fractionated coconut oil), propylene glycol, ethanol or water; preservative (for example, methyl or propyl pair) Hydroxybenzoic acid or sorbic acid; a wetting agent such as lecithin; and a flavoring or coloring agent if needed.
  • pharmaceutically acceptable excipients such as suspending agents (for example, sorbitol, methylcellulose, sodium alginate, gelatin, hydroxyethyl
  • compositions can be formulated as follows: buffered to a suitable pH and isotonic sterile aqueous solution or suspension, or A parenterally acceptable oil.
  • suitable aqueous carriers include Ringer's solution and isotonic sodium chloride.
  • Such forms can be presented as unit dosage forms, such as ampoules or disposable injection devices; multiple dosage forms, for example, a suitable dose of vial from which it can be taken; or a solid form or pre-concentrate that can be used in the preparation of injectable formulations.
  • Formulations suitable for parenteral administration include the following forms: aqueous and non-aqueous sterile injectable solutions, including buffers, bacteriostatic agents, and blood to the intended recipient Isotonic solutes; and aqueous and non-aqueous sterile suspensions which may contain suspending and thickening agents.
  • the formulations may be stored in unit or multi-dose containers, such as sealed ampoules and vials, and may be stored in a lyophilized (lyophilized) state, just prior to use, by the addition of a sterile liquid carrier, such as water for injection.
  • the temporary injections and suspensions can be prepared from the sterile powders, granules and tablets described above.
  • Preferred unit dosage formulations are those which comprise a daily dose or unit, daily sub-dose or an appropriate amount of the active ingredient.
  • the T1 Pr[alpha] TACE doses administered to an individual (e.g., a human) described herein can vary depending on the particular composition, method of administration, and the particular stage of the condition. The amount should be sufficient to produce the desired reaction. In some embodiments, the amount of the composition is a therapeutically effective amount. In some embodiments, the amount of the composition is a prophylactically effective amount. In some embodiments, the total amount of T1 Pr ⁇ TACE in the composition is lower than the level that induces a toxicological effect (ie, the level is above a clinically acceptable level of toxicity), or is potential when the composition is administered to an individual. The level of side effects can be controlled or tolerated.
  • the amount of protein T1 Pr ⁇ TACE in the drug is in any of the following ranges: from about 0.5 mg to about 5 mg, from about 5 mg to about 10 mg, from about 10 mg to about 15 mg, from about 15 mg to about 20 mg, about 20 mg. To about 25 mg, from about 20 mg to about 50 mg, from about 25 mg to about 50 mg, from about 50 mg to about 75 mg, from about 50 mg to about 100 mg, from about 75 mg to about 100 mg, from about 100 mg to about 125 mg.
  • the T1 Pr ⁇ TACE protein is administered in the medicament in an amount of at least about 0.1 mg/kg, 0.5 mg/kg, 1 mg/kg, 2.5 mg/kg, 5 mg/kg, 7.5 mg/kg, 10 mg/day. Kg, 15 mg/kg or 20 mg/kg.
  • the effective dose of T1 Pr ⁇ TACE protein may vary depending on the mode of administration, the age and weight of the patient, the severity of the disease, and other related factors.
  • the recommended dose for oral administration is 10-2000 mg/time, 1-2 times a day; parenteral
  • the recommended dose for administration is 5-500 mg/time, once a day.
  • T1 Pr ⁇ TACE retains the original basic protein structure of TIMP-1, a large number of editing improvements have been made to its "MMP-binding ridge" (the actual binding site with the target MMP) and the structure at the carboxy terminus.
  • MMP-binding ridge the actual binding site with the target MMP
  • the modified T1 Pr ⁇ TACE has both inhibitory effects on MT1-MMP and TACE.
  • the experimental data show that T1 Pr ⁇ TACE can effectively inhibit the tumorigenic effect of human kidney cancer cells (Caki-1) in mice.
  • T1 Pr ⁇ TACE has a very efficient inhibitory effect.
  • the K i app was experimentally determined to be as low as 7.70 nM (inhibiting MT1-MMP) and 0.14 nM (inhibiting TACE).
  • the newly developed T1 Pr ⁇ TACE was successfully expressed in cells and embedded in the cell membrane surface. Subsequent immunofluorescence experiments further indicated that T1 Pr ⁇ TACE located on the membrane surface was more easily bound to MT1-MMP or TACE.
  • a fourth aspect of the present application provides an antitumor pharmaceutical composition
  • the final concentration of BHK-21 cells (named BHK+T1 Pr ⁇ TACE cells) expressing the T1 Pr ⁇ TACE protein is 300,000 to 625,000/mL.
  • the mass percentage of the artificial base film is 30% by weight to 60% by weight.
  • the antitumor pharmaceutical composition further comprises 2 to 10% of fetal bovine serum in Dulbecco's medium (DMEM).
  • DMEM Dulbecco's medium
  • the anti-tumor composition comprises: BHK-21 cells expressing T1 Pr ⁇ TACE protein at a final concentration of 300,000-625,000 /mL; 30 wt%-60% artificial basement membrane; and 5% fetal Dulbecco's medium (DMEM) of bovine serum.
  • DMEM fetal Dulbecco's medium
  • the final concentration of BHK-21 cells expressing the T1 Pr ⁇ TACE protein is 500,000 to 625,000/mL.
  • the artificial base film has a mass percentage of 50% by weight to 60% by weight, and more preferably, the artificial base film is Matrigel.
  • a fifth aspect of the present invention provides the use of the above pharmaceutical composition for the preparation of a medicament for treating or preventing a tumor selected from the group consisting of kidney cancer, cervical cancer, breast cancer, lung cancer, colon cancer, ovarian cancer, liver cancer, gastric cancer and leukemia.
  • the kidney cancer comprises renal cell carcinoma.
  • the pharmaceutical composition functions at a temperature of 24-37 °C.
  • a method for preparing or using the pharmaceutical composition of the present application mixing a certain amount of BHK+T1 Pr ⁇ TACE cells with a volume of 30-60% ice artificial basement membrane to prepare a mixture, and then adding a certain amount of cancer cells, The concentration of both cells was between 300,000 and 625,000/mL, and inoculated into the culture plate until the artificial basement membrane was coagulated at a temperature of 24-37 ° C. Finally, Dulbecco's Modified with 2 to 10% fetal bovine serum was added. Eagle Medium (DMEM) culture medium; the above cancer cells are preferably labeled with Red Fluorescence Protein (RFP).
  • DMEM Eagle Medium
  • RFP Red Fluorescence Protein
  • the artificial basement membrane is a reconstituted basement membrane extract that is typically used primarily as a simulated in vivo microenvironment for 3D culture of in vitro cells.
  • a soluble 3D medium with abundant colloidal network structure extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma rich in extracellular matrix proteins.
  • Matrigel contains a large number of macromolecular proteins in the extracellular matrix, such as laminin (60%), collagen (30%), endactin (8%) and heparan sulfate glycoprotein, and similar FGF. , EGF, TGF- ⁇ and other large growth factors that stimulate cells and signaling.
  • Matrigel can mimic the structure, composition, physical properties and functions of the cell basement membrane to a large extent, thus providing favorable conditions for in vitro culture of cells. Growth conditions (Hughes, Postovit et al. 2010, Benton, Kleinman et al. 2011, Benton, Arnaoutova et al. 2014).
  • BHK-21 is a cell line derived from the fibroblasts of the newborn hamster kidney, first established by Stoker and Macpherson in 1961 (Stoker and Macpherson, 1961). Since BHK-21 is very sensitive to viral infections, this cell has been widely used for many years in the breeding and biopharmaceutical production of hosts.
  • BHK-21 Baby hamster kidney
  • the final concentration is 30-60%, and the concentration of BHK-21 is between 300,000 and 625,000/mL). Studies have shown that within this specific concentration range
  • the BHK-21 composition is suitable for capturing, encapsulating and inhibiting the spread and growth of cancer cells in vitro, ultimately leading to their death.
  • Figure 1 is a schematic representation of the amino acid sequence of T1 Pr ⁇ TACE and the original TIMP-1.
  • FIG. 2 shows a graphical representation of the inhibitory mechanism of T1 Pr ⁇ TACE on MT1-MMP and TACE, demonstrating the mechanism of action of T1 Pr ⁇ TACE .
  • the T1 Pr ⁇ TACE protein can be broadly divided into two relatively independent and functionally distinct domains:
  • the first is the inhibitory domain, which can be tightly bound to the enzyme active sites of MT1-MMP (K i app 7.70nM, Table 1) and TACE (K i app 0.14nM, Table 1) by blocking the substrate. It is digested to efficiently inhibit the activity of metalloproteinases.
  • the second is a transmembrane domain containing a GPI anchor chain that allows free TIMP to be embedded on the cell membrane surface, closer to MT1-MMP and TACE (also located on the membrane surface).
  • Fig. 3 is a non-penetrating immunofluorescence experiment diagram of (cell membrane), wherein Fig. 3A shows a non-penetrating immunofluorescence map of T1 Pr ⁇ TACE and MT1-MMP on the surface of the cell membrane; Fig. 3B shows that T1 Pr ⁇ TACE and TACE are on the surface of the cell membrane. Combine non-penetrating immunofluorescence maps.
  • Figure 4 is a graph showing the inhibitory effect of T1 Pr ⁇ TACE and TIMP-1 on MT1-MMP enzymatic gelatin.
  • Figures 5A and 5B show changes in the concentration of extracellular active HB-EGF and TNF-[alpha].
  • Figure 6 shows the effect of expression of TIMP-1 and T1 Pr ⁇ TACE on the tumor-forming volume of cancer cells in NOD/SCID mice ( heterocellular tumor formation model).
  • FIG. 7 is a schematic diagram showing the amino acid sequence of T1 Pr ⁇ TACE of the present application.
  • Figure 8 is an immunostaining map showing the T1 Pr ⁇ TACE recombinant protein located on the surface of BHK+T1 Pr ⁇ TACE cells.
  • Figure 9 shows the BHK+T1 Pr ⁇ TACE of Example 7: The composition encapsulates and inhibits CaKi-1 cells.
  • Figure 10 shows the effect of BHK+T1 Pr ⁇ TACE group, BHK-21 group and control group on Caki-1 cells after 37 days of culture under inverted fluorescence microscope. It was shown that BHK+T1 Pr ⁇ TACE cells were more effective in wrapping and limiting the growth of CaKi-1 cells than unmodified BHK-21 cells.
  • Figure 11 shows the survival counts of CHK-1 cells after B78+T1 Pr ⁇ TACE group, BHK-21 group and control group after 37 days of culture.
  • FIG 12 shows that the fluorescence intensity of cancer cells in the BHK+T1 Pr ⁇ TACE group is much lower than that in the BHK-21 group under an inverted fluorescence microscope. It is shown that the BHK+T1 Pr ⁇ TACE composition is more effective in inhibiting the proliferation and spread of HeLa cells than the unmodified BHK-21 cell composition.
  • Figure 13 is a graph showing the survival counts of HeLa cells after B37 +T1 Pr ⁇ TACE group, BHK-21 group and control group after 37 days of culture.
  • NOD-SCID mouse (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.)
  • 2.1 drugs puromycin; basement membrane From BD Bioscience; BHK-21 cell line (Shanghai cell bank GNHa10), CaKi-1 cell (Shanghai cell bank TCHu135), HeLa cell (Shanghai cell bank TCHu187); the culture medium is 5% fetal bovine serum, penicillin-streptococcus Solution (1X) and DMEM medium.
  • TIMP-1 antibody (Abeam), MT1-MMP antibody (Abeam), TACE antibody (Acam), Alexa Fluor 488 anti-mouse secondary antibody (Invitrogen), Alexa Fluor555 anti-rabbit secondary antibody (Invitrogen), DAPI sealant ( Invitrogen), 38% paraformaldehyde (Sigma), green fluorescent gelatin (ThermoScientific: Oregon green 488), lipofectamine transfection kit (Invitrogen), propylene glycol methyl ether acetate (PMA), phosphate substrate (Sigma), TNF - ⁇ Elisa kit (Sino Bio), EcoR I endonuclease (ThermoScientific), Xho I endonuclease (ThermoScientific), Apa I endonuclease (ThermoScientific), Nde I endonuclease (ThermoScientific), related gene sequences and primers
  • the obtained two PCR products were digested with Nde I and Xho I, and inserted into the same pRSET-c expression vector (Invitrogen) to obtain a recombinant plasmid carrying the gene of interest.
  • the recombinant plasmid was sequenced (Shanghai Shenggong Technology Co., Ltd.), and the inserted target gene showed no mutation.
  • the inhibitory domains of the recombinant plasmids TIMP-1 and T1 Pr ⁇ TACE were transformed into BL21(DE3)pLys competent cells, respectively, and the two proteins of interest were obtained using the E. coli protein expression system.
  • the kinetic analysis of the two purified proteins was performed by LS-55 fluorescence spectrometer with MT1-MMP and TACE as the targeting proteins, respectively.
  • T1 Pr ⁇ TACE The inhibitory effect of T1 Pr ⁇ TACE on MT1-MMP and TACE is much stronger than that of original TIMP-1.
  • Table 1 shows the inhibitory effects of the T1 Pr ⁇ TACE inhibitory domain and the original TIMP-1 on the metalloproteinases MT1-MMP and TACE, respectively.
  • the same test method was used for all experiments, that is, the change of the fluorescent polypeptide substrate (Mca-KPLGL-Dpa-AR-NH2, R&D Systems) was measured using an LS-55 fluorescence spectrometer to calculate the corresponding inhibitory effect.
  • the T1 Pr ⁇ TACE inhibition domain K i app 7.70 nM and 0.14 nM, respectively
  • K i app >150 nM was much more potent than the original TIMP-1
  • T1 Pr ⁇ TACE and MT1-MMP (or TACE) bind to each other on the cell membrane surface:
  • TIMP-1 gene sequence (GenBank: S68252.1) for transfection of cells, PCR amplification using the following primers, forward primer: 5'-GCAGCA GAATTC ACCATGGCCCCCTTTGAGCCCCTGGCT-3' (EcoR I digested) The site, as indicated by Seq ID No. 6, and the reverse primer: 5'-TCAACTG GGGCCC TTAGGCTATCTGGGACCGCAGGGACTG-3' (labeled Apa I restriction site, as indicated by Seq ID No. 7), gave a PCR product.
  • the forward primer is the same as TIMP-1
  • reverse primer 15'-GGCCTGAGATTCCCTCTCGTACTGGGCTATCTGGGACCGCAGGGACTG-3', as shown by Seq ID No. 8; 25'-CACAGGTGGGGAGGAGAAGAGGACCATGCTCGATCCTCTCTGGTAATAGGCCTGAGATTCCCTCTCGTACTG-3'; Seq ID No. 9; 35'-TAAAC GGGCCC TCATCCCACTATTAGGAAGATGAGGAAAGAGATCAGGAGGATCACAGGTGGGGAGGAGAAG AGGAC-3', as shown by Seq ID No. 10; PCR products were obtained by sequential action of one forward primer and three reverse primers.
  • the obtained two PCR products were digested with EcoR I and Apa I, respectively, and ligated with the pLVX-Puro (Takara) expression vector which was digested with the same enzyme to obtain a recombinant plasmid carrying the gene of interest.
  • the recombinant plasmid was sequenced and the inserted gene of interest was not mutated.
  • the above mixed solution was separately added to Lenti-X 293T cells seeded in a 10 cm cell culture dish, and cultured at 37 ° C, 5% carbon dioxide for 3 days, and then the supernatant culture liquid was collected.
  • CaKi-1 cells were infected with a lentivirus containing the gene of interest, and then the cells were screened with puromycin antibiotic to obtain CaKi-1 cells stably expressing TIMP-1 and T1 Pr ⁇ TACE .
  • T1 Pr ⁇ TACE and MT1-MMP (or TACE) bind to each other on the surface of cell membrane
  • T1 Pr ⁇ TACE exists only on the surface of the cell membrane, and binds to and aggregates with MT1-MMP or TACE on the surface of the membrane.
  • T1 Pr ⁇ TACE inhibits MT1-MMP decomposition of animal gelatin matrix:
  • HT1080 cells were infected with the lentivirus successfully packaged in the above Example 2.1.
  • the HT1080 cells stably expressing TIMP-1 and T1 Pr ⁇ TACE were obtained in the same manner.
  • T1 Pr ⁇ TACE can inhibit TACE more efficiently, thereby effectively blocking the activation and release of TNF- ⁇ and HB-EGF by (TACE):
  • HT1080 cells Three groups of HT1080 cells were inoculated into 24-well plates, which were control group, TIMP-1 (stable expression) group and T1 Pr ⁇ TACE (stable expression) group. Each group of cells was inoculated with 3 wells, and the number of cells in each well was 1 x 10 5 , culture overnight.
  • the culture medium containing HB-EGF (-PMA/+PMA 200 ng/ml) was added to a diethanolamine buffer with an alkaline phosphate substrate, and then transferred to a 96-well plate, and placed at 37 ° C until the color change.
  • the microplate reader reads the absorbance of HB-EGF.
  • TNF- ⁇ (-PMA/+PMA 200 ng/ml) was measured by TNF- ⁇ ELISA kit (Sino Bio).
  • HT1080 cells stably expressing T1 Pr ⁇ TACE and TIMP-1 were used as samples to detect changes in the concentrations of extracellular TNF- ⁇ and HB-EGF, thereby reflecting the strength of TACE enzyme activity in each cell line.
  • the results of the two groups showed that the inhibitory effect of T1 Pr ⁇ TACE on TACE was stronger than that of the original TIMP-1.
  • DMEM resuspend the cells so that the concentration of each group of cells reached 2x10 6 /ml.
  • mice were randomly divided into 3 groups: control group, TIMP-1 group and T1 Pr ⁇ TACE group, with 8 mice in each group.
  • mice were continuously cultured for 35 days, and the size of the tumors was measured twice a week (the vernier caliper).
  • mice were sacrificed after 35 days and the tumors were weighed.
  • TIMP-1 8 mice cells there are formed six different volumes (volume between 20mm 3 to 248mm 3) tumors. However, no cell formation was observed in the control group and the mice implanted with T1 Pr ⁇ TACE cells (*p ⁇ 0.05). T1 Pr ⁇ TACE does not have the effect of promoting cell growth, unlike the original TIMP-1.
  • T1 Pr ⁇ TACE sequence for transfection of cells, PCR amplification using the following primers, forward primer: 5'-GCAGCAGAATTCACCATGGCCCCCTTTGAGCCCCTGGCT-3', as shown in Seq ID No. 6, reverse primer in the order of 15'-GGCCTGAGATTCCCTCTCGTACTGGGCTATCTGGGACCGCAGGGACTG -3, as shown by Seq ID No. 8, 25'-CACAGGTGGGGAGGAGAAGAGGACCATGCTCGATCCTCTCTGGTAATAGGCCTGAGATTCCCTCTCGTACTG-3', as shown by Seq ID No. 9, 35'-TAAACGGGCCCTCATCCCACTATTAGGAAGATGAGGAAAGAGATCAGGAGGATCACAGGTGGGGAGGAGAAG AGGAC-3', as shown by Seq ID No. 10, to obtain a PCR product ;
  • the PCR product and the pLVX-Puro (Takara) expression vector were subjected to restriction enzyme digestion with EcoR I and Apa I, and then the desired fragment was inserted into a vector to obtain a recombinant plasmid carrying the gene of interest. After sequencing, the recombinant plasmid with the target gene not mutated is taken up;
  • Lenti-X virus packaging reagent (Takara 631276) and 10 ⁇ g T1 Pr ⁇ TACE DNA were mixed in proportion, and then the mixture was infected with Lenti-X 293T cells to obtain a lentivirus containing T1 Pr ⁇ TACE ;
  • BHK-21 cells were infected with the obtained T1 Pr ⁇ TACE lentivirus, and then the cells were screened with puromycin to obtain BHK-21 cells stably expressing T1 Pr ⁇ TACE , and named as BHK+T1 Pr ⁇ TACE cells;
  • BHK-21 cells BHK+T1 Pr ⁇ TACE stably expressing T1 Pr ⁇ TACE were subjected to TIMP-1 immunostaining (antibody Abcam ab1827) under non-penetrating conditions.
  • TIMP-1 immunostaining antibody Abcam ab1827
  • the upper row BHK-21 cell nuclei were stained with 4',6-diamine-2-phenylindole (DAPI).
  • Lower panel The left panel shows that no TIMP-1 expression was detected on the cell surface of the control group; right panel: contrary to the control group, a large amount of TIMP recombinant protein was detected at the cell membrane edge of BHK+T1 Pr ⁇ TACE (arrow display ).
  • BHK+T1 Pr ⁇ TACE Method for encapsulation and inhibition of renal cell carcinoma (CaKi-1) by composition :
  • BHK+T1 Pr ⁇ TACE cells were cultured in complete medium containing 10 ug/ml Puromycin, and Caki-1 cells were cultured in complete medium containing 5 ug/ml Blasticidin to obtain a sufficient number of cells for use;
  • the concentration of cells and matrigel can be adjusted within the range of protection.
  • BHK+T1 Pr ⁇ TACE group A sufficient number of BHK+T1 Pr ⁇ TACE cells and RFP-labeled Caki-1 cells were cultured. 50,000 BHK+T1 Pr ⁇ TACE cells were resuspended in 40 ul of cold Matrigel, then mixed with the same volume of culture medium containing 50,000 Caki-1 cells, and after mixing, 8 ul of the cells were dropped and placed at 37 ° C until solidification. Finally, Dulbecco's medium supplemented with 5% fetal calf serum was incubated for 37 days.
  • BHK-21 group 50,000 unmodified BHK-21 cells were mixed with Matrigel's composition and the same amount of RFP-labeled Caki-1 cells, and then subjected to the same experimental procedure as the BHK+T1 Pr ⁇ TACE group.
  • Control group 50,000 RFP-labeled Caki-1 cells were individually resuspended in 40 ul of culture medium, then mixed with the same volume of ice Matrigel mixture, and 8 ul was taken out and inoculated onto the culture plate. After solidification, complete culture solution was added. Continuous culture for 37 days.
  • the BHK-21 spheroid expressing T1 Pr ⁇ TACE inhibited CaKi-1 cells significantly better than unmodified BHK-21 cells, and BHK-21 cells expressing T1 Pr ⁇ TACE inhibited CaKi-1 proliferation.
  • the aspect has a more significant effect than the unmodified BHK-21 cells.
  • Control group 50,000 HeLa cells resuspended in 40 ul of culture medium were mixed with the same volume of liquid Matrigel, and then 8 ul was inoculated into the culture plate for culture. After 28 days of culture, photographs and statistics were performed.
  • BHK+T1 Pr ⁇ TACE Under inverted fluorescence microscopy, as shown in Figure 12, HeLa cells were used as target cancer cells.
  • the fluorescence brightness of BHK+T1 Pr ⁇ TACE group was much lower than that of BHK-21 group.
  • BHK+T1 Pr ⁇ TACE cells were more than unmodified BHK- 21 cells can more effectively encapsulate and limit the growth of HeLa cells, so the BHK+T1 Pr ⁇ TACE composition can inhibit the proliferation and spread of HeLa cells more effectively than the unmodified BHK-21 cell composition.

Abstract

本申请公开一种膜型金属蛋白酶抑制蛋白T1Pr αTACE以及用途，其可以用于制备以MT1-MMP或TACE蛋白内切酶为靶点的药物。本申请公开一种抗肿瘤的药物组合物，其包括表达T1Pr αTACE蛋白的BHK-21细胞和人工基底膜。

Description

膜型金属蛋白酶抑制蛋白和包含其的药物和药物组合物及各自的用途 技术领域
本申请涉及生物医药技术领域，具体涉及具有抑制人肾癌细胞增殖作用的膜型金属蛋白酶抑制蛋白和包含其的药物和药物组合物及各自的用途。
背景技术
目前治疗癌症的技术大致可以分为三类：
1.化学疗法：化疗最常见的副作用包括恶心呕吐、脱发、免疫抑制以及食欲不振等。
2.放射疗法：放疗的副作用通常比化疗的要轻微一些。脱皮、红斑和纤维化等皮肤反应均是与放疗相关的副作用。此外，这种治疗方法还需要专门的治疗设备和医院等环境。
3.免疫疗法：疲劳、发热、虚弱、恶心、身体疼痛和高血压等与免疫相关的症状都是免疫疗法的常见副作用。
因此，现有技术治疗癌症的方法存在很多缺陷和弊端。
膜型基质金属蛋白酶-1(Membrane Type 1-Matrix Metalloproteinase，MT1-MMP)和TNF-α转化酶(TNF-αConverting Enzyme，TACE)属锌离子依赖性跨膜內肽水解酶类，二者皆参与细胞外基质(Extracellular Matrix，ECM)的分解和重塑，通过释放诱导细胞生长所需的各种活性因子，在肿瘤侵袭和转移的演进过程中扮演着极重要角色。在催化作用方面，MT1-MMP可降解多种细胞外基质和基底膜大分子(如胶原蛋白I、II和III型，层粘连蛋白，纤连蛋白，纤维蛋白以及一些重要的黏附分子)并同时激活癌细胞侵袭和扩散所需的信号传导分子(如多配体聚糖、CD44、RANKL、MUC1等)，在促进肿瘤侵袭转移中起着关键性作用。
TACE原属解聚素金属蛋白酶ADAM(A Disintegrin and Metalloproteinase) 家族的一员，又名ADAM-17。TACE的活性主要表现在以下方面：通过水解细胞外各种跨膜前体，TACE能释放出许多配体和生长因子(如TNF-α、TGF-α、HG-EGF)，受体(TNFR II、EGFR、IL-6R、Notch-1)，粘附分子(ICAM-1、V-CAM-1、L-selectin)以及与神经退行性疾病和传染性海绵状脑病相关的朊病毒。通过这些分子进行信号传导，TACE可以影响肿瘤的发生、进程及转移过程。
在正常的生理条件下，MMP和ADAM蛋白酶的活性是由内源性的金属蛋白酶组织抑制因子(TIMPs)来进行调节的。目前已知的人类TIMPs有四种，即TIMP-1、2、3和4。TIMPs均是小分子蛋白，分子量介于21-25kDa之间。在结构上，TIMPs是由2个蛋白结构域组成，即：由5个反向平行β-链组成的N端结构域(分子量约15kDa)以及分子量约为8kDa，无固定结构的C端结构区域(Protein Data Bank结晶例#1BR9，#1UEA)。通过其楔形的N端结构域，TIMPs能与靶蛋白酶催化活性区内的锌离子结合位点形成紧密的1∶1结合物(PDB结晶例#4ILW，#3V96，#3CKI等)，从而发挥MMP抑制作用。尽管所有的TIMPs都有相似的三级结构，但每个TIMP却有其独特的MMP抑制特性。例如MT1-MMP对TIMP-2、-3和-4的抑制活性很敏感，而对TIMP-1则完全相反。有趣的是，TIMP-3是唯一能抑制TACE活性的TIMP成员。除了TIMP-3，其他TIMPs均为高度可溶性分泌型蛋白。
TIMP-1是在1980年从人类淋巴细胞中分离出来的TIMP家族中的第一位成员，分子量约为25kDa，属分泌糖蛋白的一种，且对MT1-MMP和TACE都没有明显的抑制活性。同时，研究表明TIMP-1作为一种细胞生长因子，可以增强癌细胞的活性。
现有蛋白(即原始TIMP-1)的缺点：(1)原始TIMP-1是一种可溶性分泌蛋白。无论是在胞内还是胞外，尚未有任何证据表明TIMP-1可以和MT1-MMP及TACE相互作用。(2)TIMP-1对MT1-MMP和TACE没有抑制效果。相关实验测定，TIMP-1针对MT1-MMP或TACE的K i app均大于150nM。(3)TIMP-1可以增强细胞活性，极大地限制了其在癌症研究中的价值。
发明内容
以下是对本文详细描述的主题的概述。本概述并非是为了限制权利要求的保护范围。
本申请所要解决问题是根据现有技术金属蛋白酶抑制蛋白TIMP，提供一种编辑改良后的膜型金属蛋白酶抑制蛋白T1 PrαTACE。
本申请第一方面提供一种编辑改良后的膜型金属蛋白酶抑制蛋白T1 PrαTACE，其氨基酸序列如Seq ID No.1所示。
本申请第二方面提供上述膜型金属蛋白酶抑制蛋白T1 PrαTACE用于制备以MT1-MMP或TACE蛋白内切酶为潜在靶点的肿瘤药物的用途。
本申请第三方面提供一种药物，其包含膜型金属蛋白酶抑制蛋白T1 PrαTACE以及药学上可接受的赋形剂。
本申请优选的技术方案中，所述赋形剂为载体、溶剂、乳化剂、分散剂、湿润剂、粘合剂、稳定剂、着色剂、香料。
本申请优选的技术方案中，所述药物为针剂、片剂、胶囊剂、冲剂、滴剂、颗粒剂或软膏剂。
上述技术方案中，所述制备以MT1-MMP或TACE蛋白内切酶为靶点的药物组合物可通过纯度98％(质量百分数)以上的蛋白T1 PrαTACE添加通用的药用辅助成分，制成片剂、胶囊剂、冲剂、滴剂、冻干物、颗粒剂、软膏剂或针剂。
药学制剂和给药
根据在本领域中已知的用于制备各种剂型的常规方法，T1 PrαTACE蛋白可被配制成溶液、乳液、悬浮液、分散液，或诸如环糊精之类的在合适药物溶剂或载体中的包合配合物，或被配制成丸剂、片剂、锭剂、栓剂、小药囊、糖衣丸、颗粒剂、粉剂、重组粉剂、或连同固体载体的胶囊。实施方式中的药物组合物可通过适当的递送路径被施用，例如口服、肠胃外、直肠、鼻部、局部、或眼部途径，或通过吸入。优选地，组合物被配制成用于静脉内或口服给药。
对于口服给药，药物可以以固体的形式来提供，例如片剂或胶囊，或被配 制成溶液、乳液或悬浮液。口服片剂可包括与药学上可接受的相容赋形剂混合的活性组分，所述赋形剂例如稀释剂、崩解剂、结合剂、润滑剂、甜味剂、调味剂、着色剂和防腐剂。适当的惰性填充剂包括碳酸钠和碳酸钙、磷酸钠和磷酸钙、乳糖、淀粉、糖、葡萄糖、甲基纤维素、硬脂酸镁、甘露醇、山梨醇等。示例性的液体口服赋形剂包括乙醇、甘油、水等。示例性崩解剂包括淀粉、聚乙烯基吡咯烷酮(PVP)、淀粉羟乙酸钠、微晶纤维素和藻酸。结合剂可包括淀粉和明胶。润滑剂一如果存在的话一可以是硬脂酸镁、硬脂酸或滑石。如果需要，片剂可通过诸如单硬脂酸甘油酯或二硬脂酸甘油酯之类的材料而被包衣，以延缓在胃肠道中的吸收，或可通过肠溶衣而被包衣。口服制剂可以以下列形式存在：以离散单位的形式，例如胶囊、扁囊剂或片剂，各包含预定量的活性组分；以粉剂或颗粒剂的形式；以溶液或在水性液体或非水性液体中的悬浮液形式；或以水包油液体乳液或油包水液体乳液的形式。活性组分也可被制成大丸剂、药糖剂或糊剂。
口服胶囊包括硬的和软的明胶胶囊。为制备硬明胶胶囊，活性组分可与固体、半固体或液体稀释剂混合。软明胶胶囊的制备可通过将活性组分与以下物质混合而实现：水，诸如花生油或橄榄油之类的油，液体石蜡，短链脂肪酸的甘油单酯与甘油二酯的混合物，聚乙二醇400，或丙二醇。
可通过压制或模制而制成片剂，且可任选一种或多种辅助组分加入到片剂中。可通过在适当的机器中压制以诸如粉末或颗粒之类的自由流动形式存在的活性组分而制得制片剂，且可向其中任选混入粘合剂(例如，聚维酮、明胶、羟丙基甲基纤维素)、润滑剂、惰性稀释剂、防腐剂、分解剂(例如，淀粉羟乙酸钠、交联聚维酮、交联羧甲基纤维素钠)、表面活性剂或分散剂。可通过在适当的机器中对被惰性液体稀释剂润湿的粉状化合物的混合物进行模制而制得模制片剂。片剂可任选被包衣或刻痕，且可被配制成使其中的活性组分缓慢或受控制地被释放，这例如可通过按不同比例使用羟丙基甲基纤维素而实现，以获得所需的释放曲线。
口服液可具有以下形式：悬浮液、溶液、乳液、或糖浆，或可被冻干或呈现为干燥产品，其可在被服用之前使用水或其他适当的载体重新配制。这类液体组合物可任选包含下列物质：药学上可接受的诸如悬浮剂之类的赋形剂(例如，山梨醇、甲基纤维素、藻酸钠、明胶、羟乙基纤维素、羧甲基纤维素、硬脂酸铝凝胶等)；诸如油之类的非水性载体(例如，杏仁油或分馏椰子油)，丙二醇，乙醇或水；防腐剂(例如，甲基或丙基对羟基苯甲酸或山梨酸)；诸如卵磷脂之类的润湿剂；以及一如果需要一调味剂或着色剂。
对于包括静脉内、肌肉内、腹膜内、鼻内或皮下途径在内的肠胃外施用，可将组合物制成如下形式：缓冲至适当pH值和等渗性的无菌水溶液或悬浮液，或肠胃外可接受的油。适当的水性载体包括林格氏溶液和等渗氯化钠。这类形式可被呈现如下：单位剂量形式，例如安瓿或一次性注射装置；多剂量形式，例如从中可抽取适当剂量的小瓶；或可用于制备可注射制剂的固体形式或预浓缩物。适用于包括静脉内给药在内的肠胃外给药的制剂包括以下形式：可包含抗氧化剂的水性和非水性无菌注射液，缓冲液，抑菌剂，以及使制剂与预期接受者的血液等渗的溶质；以及可包含悬浮剂和增稠剂的水性和非水性无菌悬浮液。制剂可被存放在单位剂量或多剂量容器中，例如密封安瓿和小瓶，且可被存储在冷冻干燥(冻干)状态下，仅需在使用之前即刻加入无菌液态载体，例如注射用水。临时注射液和悬浮液可由先前所述的无菌粉剂、颗粒剂和片剂类型制得。
优选的单位剂量制剂是指那些包含一活性组分的每日剂量或单位、每天亚剂量或适当分量的制剂。
剂量和给药途径
本文描述的施用给个体(例如，人类)的T1 PrαTACE剂量可因特定组合物、施用方法、以及病情的特定阶段而变化。其量应足以产生期望的反应。在一些实施方式中，组合物的量是治疗有效量。在一些实施方式中，组合物的量是预防有效量。在一些实施方式中，T1 PrαTACE在组合物中的总量低于诱导毒理学效 应的水平(即该水平高于在临床上可接受的毒性水平)，或处于当组合物被施用给个体时潜在副作用可被控制或容忍的水平。
在一些实施方式中，蛋白T1 PrαTACE在所述药物中的量在以下任一范围内：约0.5mg至约5mg，约5mg至约10mg，约10mg至约15mg，约15mg至约20mg，约20mg至约25mg，约20mg至约50mg，约25mg至约50mg，约50mg至约75mg，约50mg至约100mg，约75mg至约100mg，约100mg至约125mg。
在一些实施方式中，T1 PrαTACE蛋白在药物中的给药量至少约为一天0.1mg/kg、0.5mg/kg、1mg/kg、2.5mg/kg、5mg/kg、7.5mg/kg、10mg/kg、15mg/kg或20mg/kg。
T1 PrαTACE蛋白有效的剂量可以根据给药方式、病人的年龄体重、病情严重程度以及其它相关的因素而改变，口服给药时推荐剂量为10-2000mg/次，每日1-2次；肠胃外给药推荐剂量为5-500mg/次，每日1次。
虽然蛋白T1 PrαTACE保留了TIMP-1原有的基本蛋白结构，但是对其“MMP-结合脊”(与目标MMP的实际结合部位)以及羧基端的结构进行了大量的编辑改良。从而使改良后的T1 PrαTACE同时具有对MT1-MMP和TACE的抑制作用。除此之外，本实验数据表明，T1 PrαTACE可以有效抑制人肾癌细胞(Caki-1)在小鼠体内的成瘤效果。
本申请的蛋白T1 PrαTACE的优点在于：
(1)原始TIMP-1对MT1-MMP和TACE并没有明显的抑制效果。而T1 PrαTACE则具有十分高效的抑制作用。经实验测定，K i app可低至7.70nM(抑制MT1-MMP)和0.14nM(抑制TACE)。和水溶性的原始TIMP-1不同，新开发的T1 PrαTACE成功在细胞中表达，并嵌入了细胞膜表面。后续的免疫荧光实验进一步表明位于膜表面的T1 PrαTACE更容易和MT1-MMP或TACE相结 合。
(2)小鼠异种细胞成瘤模型实验表明，不同于原始TIMP-1，T1 PrαTACE不会促进细胞生长(即失去了增强细胞活性的功能)。
(3)小鼠移植人肾癌细胞(Caki-1)的模型试验进一步表明，T1 PrαTACE具有有效抑制人肾癌细胞在小鼠体内生长、增殖并向远端转移的作用。
本申请第四方面提供一种抗肿瘤的药物组合物，其包括表达T1 PrαTACE蛋白的BHK-21细胞和人工基底膜。
本申请优选的技术方案中，表达T1 PrαTACE蛋白的BHK-21细胞(名为BHK+T1 PrαTACE细胞)的最终浓度为300,000～625,000/mL。
本申请优选的技术方案中，人工基底膜的质量百分比为30wt％-60wt％。
本申请优选的技术方案中，所述抗肿瘤的药物组合物还包括2～10％的胎牛血清的Dulbecco′s培养液(DMEM)。
本申请优选的技术方案中，所述抗肿瘤的组合物包括：最终浓度为300,000～625,000/mL的表达T1 PrαTACE蛋白的BHK-21细胞；30wt％-60％的人工基底膜；和5％胎牛血清的Dulbecco′s培养液(DMEM)。
本申请优选的技术方案中，表达T1 PrαTACE蛋白的BHK-21细胞的最终浓度为500,000～625,000/mL。
本申请优选的技术方案中，人工基底膜质量百分比为50wt％-60wt％，更优选地，所述人工基底膜为
Figure PCTCN2018122305-appb-000001
基质胶。
本申请第五方面提供上述药物组合物在制备治疗或预防肿瘤的药物中的用途，所述肿瘤选自肾癌、宫颈癌、乳腺癌、肺癌、结肠癌、卵巢癌、肝癌、胃癌和白血病。
优选地，所述肾癌包括肾细胞癌。
本申请优选的技术方案中，所述的药物组合物在24-37℃温度下发挥作用。
本申请的药物组合物的制备方法或使用方法：将一定数量的BHK+T1 PrαTACE 细胞与一定体积的30-60％冰的人工基底膜混合，制备成混合物，然后再加入一定数量的癌细胞，使这两种细胞的浓度均处于300,000～625,000/mL之间，并接种到培养板中直至人工基底膜在24-37℃温度内凝固，最后加入2～10％胎牛血清的Dulbecco′s Modified Eagle Medium(DMEM)培养液培养；上述的癌细胞优选用红色荧光蛋白(Red Fluorescence Protein；RFP)标记过。
人工基底膜是一种再造的基底膜提取物，通常主要作为模拟体内微环境用于体外细胞的3D培养。
Figure PCTCN2018122305-appb-000002
最初是从富含胞外基质蛋白的Engelbreth-Holm-Swarm(EHS)小鼠肉瘤中提取出来的具有丰富的胶质网状结构的可溶性的3D培养基。由于基质胶中含有大量的细胞外基质中的大分子蛋白，如层粘连蛋白(60％)、胶原蛋白(30％)、内肌动蛋白(8％)和硫酸类肝素糖蛋白，以及类似FGF，EGF，TGF-β等大量的刺激细胞和信号传导必需的生长因子，基质胶能够在很大程度上模拟体内细胞基底膜的结构、组成、物理特性和功能，从而对细胞的体外培养提供有利的生长条件(Hughes，Postovit et al.2010，Benton，Kleinman et al.2011，Benton，Arnaoutova et al.2014)。
BHK-21是一种源于新生仓鼠肾脏的成纤维细胞的细胞株，由Stoker和Macpherson在1961年首先建株(Stoker and Macpherson，1961)。由于BHK-21对病毒感染非常敏感，此细胞多年来被广泛用于病毒繁殖和生物制药生产的宿主。
在本专利申请中，公开了一种能用于捕捉并抑制癌细胞在体外生长的组合物，命名为“
Figure PCTCN2018122305-appb-000003
baby hamster kidney(BHK)-21”。如其名所示，其由基底膜提取物
Figure PCTCN2018122305-appb-000004
和BHK-21细胞所组成(
Figure PCTCN2018122305-appb-000005
的终浓度为30-60％，BHK-21的浓度则介于300,000至625,000/mL之间)。经研究证明，这个特定浓度比例范围内的
Figure PCTCN2018122305-appb-000006
BHK-21组合物适合用于捕获、包裹并抑制癌细胞在体外的扩散与生长，最终导致它们的死亡。
在本专利申请中，描述了如何通过引入T1 PrαTACE从而进一步增强
Figure PCTCN2018122305-appb-000007
BHK-21组合物的功效。为了达到此目的，用经过基因修饰而能稳定表达T1 PrαTACE蛋白的BHK-21细胞突变体(该突变体命名为“BHK+T1 PrαTACE”) 来代替正常的BHK-21细胞，以提升
Figure PCTCN2018122305-appb-000008
BHK-21组合物对癌细胞的抑制能力。证据显示，经改良后的
Figure PCTCN2018122305-appb-000009
BHK+T1 PrαTACE组合物在包裹和杀死癌细胞方面比以前所建立的模型更加有效。
附图说明
图1为T1 PrαTACE和原始TIMP-1的氨基酸序列示意图。
图2显示了T1 PrαTACE对MT1-MMP和TACE的抑制机理图解，展示了T1 PrαTACE的作用机制。T1 PrαTACE蛋白可大致分为两个相对独立且功能不同的结构域：
(1)第一个为抑制结构域，可以和MT1-MMP(K i app 7.70nM，表1)及TACE(K i app 0.14nM，表1)的酶活性位点紧密结合，通过阻止底物被酶解，从而高效抑制金属蛋白酶的活性。
(2)第二个为跨膜结构域，包含一个GPI锚链，使游离的TIMP嵌入到细胞膜表面，更接近(同样位于膜表面的)MT1-MMP和TACE。
图3为(细胞膜)非穿透性免疫荧光实验图，其中，图3A为T1 PrαTACE和MT1-MMP在细胞膜表面相互结合非穿透性免疫荧光图；图3B为T1 PrαTACE和TACE在细胞膜表面相互结合非穿透性免疫荧光图。
图4为T1 PrαTACE和TIMP-1对MT1-MMP酶解明胶的抑制效果对比图。
图5A和5B为胞外活性HB-EGF和TNF-α浓度的变化。
图6显示了TIMP-1和T1 PrαTACE的表达对癌细胞在NOD/SCID小鼠(异种细胞成瘤模型)中成瘤体积的影响。
图7为本申请的T1 PrαTACE氨基酸序列示意图。
图8为免疫染色图显示位于BHK+T1 PrαTACE细胞表面的T1 PrαTACE重组蛋白。
图9显示实施例7的BHK+T1 PrαTACE：
Figure PCTCN2018122305-appb-000010
组合物对CaKi-1细胞的包裹和抑制作用。
图10为倒置荧光显微镜下，BHK+T1 PrαTACE组、BHK-21组和对照组在37 天培养后对Caki-1细胞的作用。显示BHK+T1 PrαTACE细胞比未修饰的BHK-21细胞能更有效的包裹和限制CaKi-1细胞的生长。
图11为BHK+T1 PrαTACE组、BHK-21组和对照组在37天培养后对Caki-1细胞作用后，存活计数。
图12为倒置荧光显微镜下，显示BHK+T1 PrαTACE组癌细胞的荧光亮度远远地低于BHK-21组。显示BHK+T1 PrαTACE组合物比未修饰的BHK-21细胞组合物能更有效的抑制HeLa细胞的增殖和扩散。
图13为BHK+T1 PrαTACE组、BHK-21组和对照组在37天培养后对HeLa细胞作用后，存活计数图。
具体实施方式
以下结合附图描述本申请具体实施方式。
一、材料
1.实验动物
NOD-SCID小鼠(北京维通利华实验动物技术有限公司)
2.药品和试剂：
2.1药品：puromycin；基底膜
Figure PCTCN2018122305-appb-000011
来自BD Bioscience；BHK-21细胞株(上海细胞库GNHa10)、CaKi-1细胞(上海细胞库TCHu135)、HeLa细胞(上海细胞库TCHu187)；其培养液为5％胎牛血清、青霉素-链霉素溶液(1X)和DMEM培养液。
2.2试剂：TIMP-1抗体(Abcam)、MT1-MMP抗体(Abcam)、TACE抗体(Abcam)、Alexa Fluor488抗鼠二抗(Invitrogen)、Alexa Fluor555抗兔二抗(Invitrogen)、DAPI封片剂(Invitrogen)、38％多聚甲醛(Sigma)、绿色荧光明胶(ThermoScientific：Oregon green 488)、lipofectamine转染试剂盒(Invitrogen)、丙二醇甲醚醋酸酯(PMA)、磷酸盐底物(Sigma)、TNF-αElisa试剂盒(Sino Bio)、EcoR I内切酶(ThermoScientific)、Xho I内切酶(ThermoScientific)、Apa I内 切酶(ThermoScientific)、Nde I内切酶(ThermoScientific)，相关基因序列及引物由生工合成。
2.3仪器：LS-55荧光光谱仪(PerkinElmer Life Sciences)、C1-Si激光共聚焦显微镜(Nikon)、正置荧光显微镜(Nikon)、倒置荧光显微镜(Nikon)、多功能酶标仪(ThermoScientific)；FACSAria II流式细胞分选仪(BD Bioscience)、自动细胞计数器(Invitrogen)。
二、实施例
实施例1T1 PrαTACE对MT1-MMP和TACE的抑制效果：
根据文献的方法：
1.Lee MH，Rapti M，Knauper V，Murphy G.Threonine 98，the pivotal residue of tissue inhibitor of metalloproteinases(TIMP)-1 in metalloproteinase recognition.J.Biol.Chem.2004；279(17)：17562-69.
2.Lee MH，Rapti M，Murphy G.Unveiling the surface epitopes that render tissue inhibitor of metalloproteinase-1 inactive against membrane type 1-matrix metalloproteinase.J.Biol.Chem.2003；278(41)：40224-30.具体操作如下：
1.从R&D公司购买MT1-MMP(R&D，918-MP-010)和TACE(R&D，930-ADB-010)蛋白。
2.制备合成TIMP-1基因序列(GenBank：S68252.1)，用以下引物(生工合成)进行PCR扩增，正向引物：5′-GAAC CATATGTGCACCTGTGTACCACCCCACCCA-3′(已标出Nde I酶切位点，如Seq ID No.3所示)，反向引物：5′-TCAACTG CTCGAGTTAATGATGATGATGATGATGATGATGGGCTATCTGGGACCGCAGGGACTG-3′(标出Xho I酶切位点，如Seq ID No.4所示)，得到PCR产物。
3.制备合成同样目的的含抑制区域的胞外部分的T1 PrαTACE序列，正向引 物：5′-GAAC  CATATGTGCACCTGTTCCCCACCCCACCCA-3′，如Seq ID No.5所示，反向引物与TIMP-1序列相同，得到PCR产物。
4.将得到的两种PCR产物进行Nde I和Xho I酶切，分别插入经过同样酶切的pRSET-c表达载体(Invitrogen)，得到携带目的基因的重组质粒。对重组的质粒进行测序(上海生工技术有限公司)，插入的目的基因没有出现突变。
5.测序后的重组质粒TIMP-1和T1 PrαTACE的抑制结构域分别转化到BL21(DE3)pLys感受态细胞内，利用E.coli蛋白表达系统来得到这两种目的蛋白。
6.分别以MT1-MMP和TACE为靶向蛋白，用LS-55荧光光谱仪对这两种纯化后的蛋白进行动力学分析。
结果：如下表1所示：
表1
新型T1 PrαTACE对MT1-MMP及TACE的抑制效果
(K i app，单位：nM)
Figure PCTCN2018122305-appb-000012
T1 PrαTACE对MT1-MMP和TACE的抑制效果远强于原始TIMP-1
表1显示了T1 PrαTACE抑制结构域和原始TIMP-1分别对金属蛋白酶MT1-MMP以及TACE的抑制效果。所有实验均使用了同样的检测方法，即使用LS-55荧光光谱仪测量荧光多肽底物(Mca-K-P-L-G-L-Dpa-A-R-NH2，R&D Systems)的变化，从而计算相应的抑制效果。不论是针对MT1-MMP还是TACE，T1 PrαTACE抑制结构域(K i app分别为7.70nM和0.14nM)的抑制效果都要远强于原始TIMP-1(K i app均＞150nM)。
实施例2T1 PrαTACE与MT1-MMP(或TACE)在细胞膜表面相互结合聚集作用：
1.制备包装携带目的蛋白编码基因的慢病毒：
a.制备TIMP-1基因序列(GenBank：S68252.1)用于转染细胞，用以下引物进行PCR扩增，正向引物：5′-GCAGCA GAATTCACCATGGCCCCCTTTGAGCCCCTGGCT-3′(已标出EcoR I酶切位点，如Seq ID No.6所示)，反向引物：5′-TCAACTG GGGCCCTTAGGCTATCTGGGACCGCAGGGACTG-3′(标出Apa I酶切位点，如Seq ID No.7所示)，得到PCR产物。
b.制备同样用途的T1 PrαTACE序列，正向引物与TIMP-1相同，反向引物：依次①5′-GGCCTGAGATTCCCTCTCGTACTGGGCTATCTGGGACCGCAGGGACTG-3′，如Seq ID No.8所示；②5′-CACAGGTGGGGAGGAGAAGAGGACCATGCTCGATCCTCTCTGGTAATAGGCCTGAGATTCCCTCTCGTACTG-3′；如Seq ID No.9所示；③5′-TAAAC GGGCCCTCATCCCACTATTAGGAAGATGAGGAAAGAGATCAGGAGGATCACAGGTGGGGAGGAGAAG AGGAC-3′，如Seq ID No.10所示；由1个正向引物和3个反向引物依次作用得到PCR产物。
c.将得到的两种PCR产物进行EcoR I和Apa I酶切，分别与经过同样酶切的pLVX-Puro(Takara)表达载体连接，得到携带目的基因的重组质粒。对重组的质粒进行测序，插入的目的基因没有出现突变。
d.测序之后，用Maxiprep质粒抽提试剂盒(AXYGEN)提取大量的目的DNA，纯化备用。
e.0.5ml无菌超纯水溶解Lenti-X病毒包装试剂(Takara 631276)，然后取10μg TIMP-1和T1 PrαTACE DNA分别与Lenti-X病毒包装试剂混合。
f.将上述混合溶液分别加入到接种在10cm细胞培养皿的Lenti-X 293T细胞 中，37℃，5％二氧化碳条件下培养3天，然后收集上清培养液。
g.用含有目的基因的慢病毒的培养液感染CaKi-1细胞，然后用puromycin抗生素对细胞进行筛选，得到稳定表达TIMP-1和T1 PrαTACE的CaKi-1细胞。
2.T1 PrαTACE与MT1-MMP(或TACE)在细胞膜表面相互结合聚集作用
根据文献的方法：
Jiang B，Zhang Y，Liu J，et al.Ensnaring membrane type 1-matrix metalloproteinase(MT1-MMP)with tissue inhibitor of metalloproteinase(TIMP)-2 using the haemopexin domain of the protease as a carrier：a targeted approach in cancer inhibition.Oncotarget，2017，8(14)：22685。具体操作如下：
(1)接种稳定表达TIMP-1和T1 PrαTACE的CaKi-1细胞到腔式载玻片上，37℃，5％二氧化碳条件下培养2天，
(2)4％多聚甲醛固定细胞10分钟，然后用PBS冲洗3遍，
(3)用含有5％BSA的PBS封闭载玻片，放置室温2个小时，
(4)在载玻片上加入两种不同来源的抗体TIMP-1+MT1-MMP/TIMP-1+TACE，4℃孵育过夜，
(5)PBS浸洗载玻片3次，每次5分钟，
(6)同时加入两种不同荧光的二抗，室温孵育2个小时，
(7)PBS浸洗3次之后，封片备用，
(8)用激光共聚焦显微镜观察准备好的载玻片。
结果：(细胞膜)非穿透性免疫荧光实验应用了稳定表达TIMP-1和T1 PrαTACE的CaKi-1细胞系。原始TIMP-1可溶解于水，因此不存在于细胞膜表面。如图3A、3B所示，T1 PrαTACE则只存在于细胞膜表面，并且和膜表面的MT1-MMP或者TACE相互结合、聚集。
实施例3T1 PrαTACE抑制MT1-MMP分解动物明胶基质：
根据文献的方法：
Jiang B，Zhang Y，Liu J，et al.Ensnaring membrane type 1-matrix  metalloproteinase(MT1-MMP)with tissue inhibitor of metalloproteinase(TIMP)-2 using the haemopexin domain of the protease as a carrier：a targeted approach in cancer inhibition.Oncotarget，2017，8(14)：22685。具体操作如下：
1.用上述实施例2.1.包装成功的慢病毒感染HT1080细胞，以同样的方法得到稳定表达TIMP-1和T1 PrαTACE的HT1080细胞，
2.用0.5mg/ml荧光明胶包被腔式载玻片，然后室温放置2个小时，
3.PBS轻揉地冲洗载玻片2次，然后用4％多聚甲醛固定明胶10分钟，
4.重复冲洗步骤5次，以确保充分地去除多聚甲醛，
5.接种分别稳定表达TIMP-1和T1 PrαTACE的HT1080细胞，培养1天，
6.同样方法固定细胞，然后PBS冲洗3次，
7.冷的甲醇穿透细胞膜20分钟(低温条件下)，PBS冲洗3遍，
8.含5％BSA+0.3％Triton X-100的封闭液封闭载玻片之后，孵育MT1-MMP抗体，4℃过夜，
9.孵育荧光二抗，室温2小时，封片备用，
10.用正置荧光显微镜观察和拍照。
结果：该实验以能够稳定表达TIMP-1和T1 PrαTACE的人纤维肉瘤HT1080细胞为样本。将其接种在以荧光明胶为基底的载玻片上，并且过夜培养。实验结果表明，如图4所示，稳定表达T1 PrαTACE的细胞对明胶的酶解能力受到明显抑制，远低于表达TIMP-1的细胞。
实施例4和原始TIMP-1相比较，T1 PrαTACE可以更高效地抑制TACE，从而有效阻碍TNF-α和HB-EGF被(TACE)激活和释放：
根据文献的方法：
Duan J X，Rapti M，Tsigkou A，Lee MH.Expanding the activity of tissue inhibitors of metalloproteinase(TIMP)-1 against surface-anchored metalloproteinases by the replacement of its C-terminal domain：Implications for anti-cancer effects[J].PloS one，2015，10(8)：e0136384。具体操作如下：
1.接种三组HT1080细胞到24孔板中，分别为对照组、TIMP-1(稳定表达)组和T1 PrαTACE(稳定表达)组，每组细胞接种3个孔，每个孔的细胞数为1x 10 5，培养过夜。
2.转染0.1μg HB-EGF/TNF-α质粒，培养2天。
3.吸掉旧的培养液，然后每组细胞一个孔加新鲜培养液，另一个孔加含有PMA(200ng/ml)的培养液，培养3小时，三组细胞做同样的处理。
4.收集每个孔的培养液(含有HB-EGF/TNF-α)，贮存备用。
5.把含有HB-EGF的培养液(-PMA/+PMA 200ng/ml)加入到有碱性磷酸盐底物的二乙醇胺缓冲液中，然后转移到96孔板，放置37℃直到颜色变化。
6.酶标仪读取HB-EGF的吸光度。
7.TNF-α(-PMA/+PMA 200ng/ml)的浓度由TNF-α ELISA试剂盒(Sino Bio)检测。
结果：胞外活性TNF-α和HB-EGF浓度的变化表明，T1 PrαTACE对TACE的抑制效果明显优于原始TIMP-1。
该离体实验使用能够稳定表达T1 PrαTACE和TIMP-1的HT1080细胞为样本，检测胞外TNF-α和HB-EGF的浓度变化，从而反映各细胞系中TACE酶活性的强弱。两组实验结果综合表明，T1 PrαTACE对TACE的抑制效果比原始TIMP-1更强。
实施例5 T1 PrαTACE(在载体实验中)不具有促进细胞生长的作用：
材料：T175培养瓶(Coming)、细胞计数薄片(Invitrogen)、1ml注射器
试剂：0.4％台盼蓝溶液(Sigma)、DMEM
仪器：自动细胞计数器(Invitrogen)、游标卡尺、天平
方法：
1.培养空白CaKi-1细胞、稳定表达原始TIMP-1和T1 PrαTACE的CaKi-1细胞，直到达到实验所需要的细胞数。
2.分别收集细胞，然后用0.4％台盼蓝溶液染色计数。
3.DMEM重悬细胞，使每组细胞的浓度均达到2x10 6/ml。
4.NOD/SCID小鼠随机分成3组，对照组、TIMP-1组、T1 PrαTACE组，每组8只小鼠。
5. 0.1ml/处注射到小鼠腹部皮下，左右腹各一处。
6.持续培养小鼠35天，一周内测量两次瘤体的大小(游标卡尺测)。
7. 35天之后处死小鼠，取瘤称重。
结果：如图6所示，被植入TIMP-1细胞的8只小鼠中，有6只都形成了不同体积(体积在20mm 3到248mm 3之间)的肿瘤。但是在对照组和被植入了T1 PrαTACE细胞的小鼠体内均未发现有细胞成瘤(*p＜0.05)。T1 PrαTACE不具有促进细胞生长的作用，不同于原始的TIMP-1。
实施例6制备表达T1 PrαTACE蛋白的BHK-21细胞(名为BHK+T1 PrαTACE细胞)及检测
1.制备T1 PrαTACE序列用于转染细胞，用以下引物进行PCR扩增，正向引物：5′-GCAGCAGAATTCACCATGGCCCCCTTTGAGCCCCTGGCT-3′，如Seq ID No.6所示，反向引物依次为①5′-GGCCTGAGATTCCCTCTCGTACTGGGCTATCTGGGACCGCAGGGACTG-3，如Seq ID No.8所示，②5′-CACAGGTGGGGAGGAGAAGAGGACCATGCTCGATCCTCTCTGGTAATAGGCCTGAGATTCCCTCTCGTACTG-3′，如Seq ID No.9所示，③5′-TAAACGGGCCCTCATCCCACTATTAGGAAGATGAGGAAAGAGATCAGGAGGATCACAGGTGGGGAGGAGAAG AGGAC-3′，如Seq ID No.10所示，得到PCR产物；
2.将PCR产物和pLVX-Puro(Takara)表达载体进行EcoR I和Apa I酶切处理，然后把目的片段插入载体中，得到携带目的基因的重组质粒。测序之后，取目的基因未突变的重组质粒备用；
3.把Lenti-X病毒包装试剂(Takara 631276)和10μg T1 PrαTACE DNA按比例混合，然后将其混合液感染Lenti-X 293T细胞，得到含有T1 PrαTACE的慢病毒；
4.用得到的T1 PrαTACE慢病毒感染BHK-21细胞，然后用puromycin对细胞进行筛选，得到稳定表达T1 PrαTACE的BHK-21细胞，命名为BHK+T1 PrαTACE细胞；
5.将筛选过的BHK+T1 PrαTACE细胞接种到腔式载玻片上，然后进过固定、封闭和冲洗一系列处理，最后用TIMP-1抗体(Abcam，ab1827)孵育过夜；
6.PBS浸洗3次，孵育荧光二抗，室温2小时；
7.洗去多余的二抗，封片，然后用正置荧光显微镜观察。
结果：在非穿透性条件下，对稳定表达T1 PrαTACE的BHK-21细胞(BHK+T1 PrαTACE)进行TIMP-1免疫染色(抗体为Abcam ab1827)。如图8所示，上排图：用4′，6-二胺-2-苯基吲哚(DAPI)对BHK-21细胞核染色。下排图：左图显示，在对照组的细胞表面未检测到有TIMP-1表达；右图：与对照组相反，BHK+T1 PrαTACE的细胞膜边缘能被测出大量的TIMP重组蛋白(箭头显示)。
实施例7
BHK+T1 PrαTACE：
Figure PCTCN2018122305-appb-000013
组合物对肾细胞癌(CaKi-1)的包裹与抑制作用方法：
1.用购买的RFP慢病毒(吉满生物科技有限公司)感染Caki-1细胞，然后经过blasticidin和流式细胞分选仪双重筛选，得到高表达RFP的Caki-1细胞；
2.用含10ug/ml Puromycin的完全培养液培养BHK+T1 PrαTACE细胞，含5ug/ml Blasticidin的完全培养液培养Caki-1细胞，得到足够数量的细胞备用；
3.用自动细胞计数器分别对两种细胞进行计数；
4.以40ul液体状的Matrigel重悬50,000个BHK+T1 PrαTACE细胞，然后再加入同体积的含有50,000个Caki-1细胞的培养液，充分混合，最后取8ul该混合液滴入24孔培养板中；
5.放置到37℃直至Matrigel凝固，然后加入完全培养液，连续培养37天；
6.用倒置荧光显微镜观察并拍照记录。
结果：如图9所示，经过37天，BHK+T1 PrαTACE：
Figure PCTCN2018122305-appb-000014
组合物和CaKi-1细胞形成了茧状球形体。
另外，对于细胞和基质胶的浓度，可以在保护范围内变动调整。
对照例1
未经修饰的BHK-21和BHK+T1 PrαTACE球状体分别对CaKi-1细胞的抑制作 用
方法：
BHK+T1 PrαTACE组：培养足够数量的BHK+T1 PrαTACE细胞和RFP标记的Caki-1细胞。将50,000个BHK+T1 PrαTACE细胞重悬在40ul冷的Matrigel中，然后与同体积的含有50,000个Caki-1细胞的培养液混合，混匀之后取出8ul滴入培养板，放置37℃直至凝固，最后加入5％胎牛血清的Dulbecco′s培养液联系培养37天。
BHK-21组：将50,000个未修饰的BHK-21细胞与Matrigel的组合物和相同数量的RFP标记的Caki-1细胞混合，然后进行与BHK+T1 PrαTACE组相同的实验操作。
对照组：将50,000个RFP标记的Caki-1细胞独自重悬在40ul培养液中，然后与同体积的冰的Matrigel的混合液混合，再取出8ul接种到培养板上，凝固后加入完全培养液连续培养37天。
在培养结束后，对球状体内荧光细胞进行计数，分析归纳取其平均值。
结果一：
如图10所示，表达T1 PrαTACE的BHK-21球状体对CaKi-1细胞的抑制作用明显优于未修饰过的BHK-21细胞，表达T1 PrαTACE的BHK-21细胞在抑制CaKi-1增殖生长方面比未修饰的BHK-21细胞有着更显著的效果。
结果二：
在培养结束后，对球状体内荧光标记癌细胞进行计数，分析归纳取其平均值。
细胞计数结果如表2显示，BHK+T1 PrαTACE细胞组最后存活的CaKi-1细胞是未修饰的BHK-21细胞组的十分之一(p＝0.00132)。所以更进一步的证明了BHK+T1 PrαTACE细胞能更有效的抑制CaKi-1细胞的增殖。
表2
Figure PCTCN2018122305-appb-000015
综合荧光显微镜图片所示的结果和实验结束时仍然存活的CaKi-1细胞计数结果，数据表示，BHK+T1 PrαTACE在抑制CaKi-1增殖方面比未修饰的BHK-21细胞更加有效。
对照例2
方法：BHK+T1 PrαTACE组和BHK-21组与对照例1相同；
对照组：将重悬在40ul培养液中的5万个HeLa细胞与同体积的液体状的 Matrigel混合，然后取8ul接种到培养板上进行培养，培养28天后，进行拍照及统计。
结果一：
倒置荧光显微镜下，如图12所示，以HeLa细胞为靶癌细胞，BHK+T1 PrαTACE组癌细胞的荧光亮度远远地低于BHK-21组，BHK+T1 PrαTACE细胞比未修饰的BHK-21细胞能更有效的包裹和限制HeLa细胞的生长，所以BHK+T1 PrαTACE组合物比未修饰的BHK-21细胞组合物能更有效的抑制HeLa细胞的增殖和扩散。
结果二：
如图13所示，培养28天后，对球状体内的存活的HeLa细胞进行计数，分析归纳，取其平均值。
如表3细胞计数，结果显示，BHK+T1 PrαTACE细胞组存活的HeLa细胞数量极低于BHK-21组(P＜0.0001)。
表3
Figure PCTCN2018122305-appb-000016
该对照例的两组结果共同的证明了，BHK+T1 PrαTACE组合物在抑制HeLa增殖方面比未修饰的BHK-21细胞组合物有更加明显的效用。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解，本发明不受上述实例的限制，上述实例和说明书中描述的只是说明本发明的原理，在不脱离本发明精神和范围的前提下本发明还会有各种变化和改进，这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等同物界定。

Claims (15)

  1. 一种膜型金属蛋白酶抑制蛋白T1 PrαTACE，其氨基酸序列如Seq ID No.1所示。
  2. 权利要求1所述的膜型金属蛋白酶抑制蛋白T1 PrαTACE用于制备以MT1-MMP或TACE蛋白内切酶为潜在靶点的肿瘤药物的用途。
  3. 一种药物，其包含权利要求1所述的膜型金属蛋白酶抑制蛋白T1 PrαTACE以及药学上可接受的赋形剂。
  4. 根据权利要求3所述的药物，其中，所述赋形剂为载体、溶剂、乳化剂、分散剂、湿润剂、粘合剂、稳定剂、着色剂、香料。
  5. 根据权利要求3所述的药物，其中，所述药物为针剂、片剂、胶囊剂、冲剂、滴剂、颗粒剂或软膏剂。
  6. 一种抗肿瘤的药物组合物，其包括表达权利要求1所述的T1 PrαTACE蛋白的BHK-21细胞和人工基底膜。
  7. 根据权利要求6所述的药物组合物，其中，所述表达T1 PrαTACE蛋白的BHK-21细胞的最终浓度为300,000～625,000/mL。
  8. 根据权利要求6所述的药物组合物，其中，所述人工基底膜的质量百分比为30wt％-60wt％。
  9. 根据权利要求6所述的药物组合物，其中，所述抗肿瘤的药物组合物还包括2～10％的胎牛血清的Dulbecco′s培养液(DMEM)。
  10. 根据权利要求6所述的药物组合物，其中，所述药物组合物包括：最终浓度为300,000～625,000/mL的表达T1 PrαTACE蛋白的BHK-21成纤维细胞；30wt％-60％的人工基底膜；和5％胎牛血清的Dulbecco′s培养液。
  11. 根据权利要求6所述的药物组合物，其中，所述人工基底膜为
    Figure PCTCN2018122305-appb-100001
    基质胶。
  12. 根据权利要求6所述的药物组合物，其中，将培养在含胎牛血清Dulbecco′s培养液的BHK+T1 PrαTACE细胞和基质胶混合后使用。
  13. 如权利要求6-12中任一项所述的药物组合物在制备治疗或预防肿瘤的 药物中的用途。
  14. 根据权利要求13所述的用途，其中，所述肿瘤选自肾癌、宫颈癌、乳腺癌、肺癌、直肠癌、卵巢癌、肝癌、胃癌和白血病。
  15. 根据权利要求14所述的用途，其中，所述肾癌包括肾细胞癌。
PCT/CN2018/122305 2018-01-03 2018-12-20 膜型金属蛋白酶抑制蛋白和包含其的药物和药物组合物及各自的用途 WO2019134526A1 (zh)

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