WO2020000634A1 - 一种与cd105特异性结合的多肽及其应用 - Google Patents

一种与cd105特异性结合的多肽及其应用 Download PDF

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WO2020000634A1
WO2020000634A1 PCT/CN2018/103544 CN2018103544W WO2020000634A1 WO 2020000634 A1 WO2020000634 A1 WO 2020000634A1 CN 2018103544 W CN2018103544 W CN 2018103544W WO 2020000634 A1 WO2020000634 A1 WO 2020000634A1
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tumor
polypeptide
cells
neovascularization
protein
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PCT/CN2018/103544
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French (fr)
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汪华
李小龙
张雁
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中山大学附属口腔医院
广州一代医药科技有限公司
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Publication of WO2020000634A1 publication Critical patent/WO2020000634A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0056—Peptides, proteins, polyamino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • 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
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia

Definitions

  • the invention relates to a non-antibody binding peptide, in particular to a polypeptide specifically binding to endothelin (Endoglin / CD105) and its application.
  • CD105 also known as endoglin, is a type I membrane glycoprotein that forms a TGF- ⁇ receptor complex on the cell membrane.
  • CD105 is a 180 kDa homodimer transmembrane protein composed of 633 amino acids.
  • CD105 consists of a large extracellular domain, a hydrophobic transmembrane domain, and a short intracellular domain.
  • the CD105 external domain binds TGF- ⁇ -1 and TGF- ⁇ -3 with high affinity to form a TGF- ⁇ receptor complex. It plays an important role in regulating cell proliferation, differentiation, migration, adhesion and angiogenesis, which is essential for tumor growth, survival and metastasis.
  • CD105 is mainly expressed in mesenchymal stem cells (MSC), endothelial cells of tumor neovascularization, tumor cells such as osteosarcoma, angiosarcoma and leukemia.
  • CD105 antibody is used to label tumor microvessel density, isolate MSCs and conjugate drugs for targeted therapy.
  • Osteosarcoma is one of the most common primary malignancies, mainly targeting long cylindrical bones. About 5% of osteosarcomas have been reported in the maxillofacial region. It occurs in adolescents and children, and it affects about 34.5 million people each year, and can be actively transferred to the lungs at an early stage.
  • CD105 antibody may be a marker to identify the malignancy of osteosarcoma.
  • Non-antibody binding protein is a synthetic thermostable peptide. This peptide is effective in penetrating the tumor microenvironment because it is small and has a positive charge. Due to the advantages of this peptide, it has attracted great attention and has been used in various antitumor studies and tumor-targeted therapies.
  • the object of the present invention is to provide a polypeptide having specific binding to CD105 and its application.
  • the provided polypeptide is a non-antibody binding protein.
  • a polypeptide that specifically binds to endothelin (Endoglin / CD105) is provided.
  • the amino acid sequence of the polypeptide is shown as sequence 1 in the sequence listing.
  • the invention also provides a polynucleotide comprising a nucleotide sequence encoding a polypeptide as described in Sequence 1 above.
  • the present invention also provides a vector comprising the polynucleotide described above.
  • the present invention also provides a host cell comprising the vector or the polynucleotide described above.
  • the invention also provides a pharmaceutical preparation, which comprises the following components: a compound in which the polypeptide described above is linked to a therapeutic drug molecule and / or a detectable label; the polypeptide can be linked to a therapeutic drug molecule and And / or labeling to form a conjugate.
  • the connection between the two can also be covalent or non-covalent bonding.
  • the detectable labels include one or more of a fluorescent reagent, a nuclide, or a radioactive reagent; these labels may each specifically adopt a corresponding label well known in the art, such as the fluorescent reagent rhodamine B, FITC (5-fluorescein isothiocyanate) and so on.
  • the therapeutic drug molecule is an anticancer agent or an antitumor neovascular agent
  • the anticancer agent is an agent having anticancer activity against a tumor with a positive expression characteristic of CD105 protein
  • the antitumor neovascular agent is an anticancer agent.
  • Drugs that inhibit tumor angiogenesis drugs such as adriamycin, paclitaxel, cisplatin, cyclophosphamide, and the like.
  • the tumor may be tumor cells such as osteosarcoma, angiosarcoma, and leukemia with positive expression of CD105 protein, or CD105 protein-negative expression tumor, but the tumor neovascularization expresses CD105 protein.
  • tumor cells such as osteosarcoma, angiosarcoma, and leukemia with positive expression of CD105 protein, or CD105 protein-negative expression tumor, but the tumor neovascularization expresses CD105 protein.
  • the pharmaceutical preparation may include a pharmaceutically acceptable carrier or auxiliary material as required, and those skilled in the art may specifically select the required carrier or auxiliary material according to need, for example, it may be a dilution commonly used in the pharmaceutical field.
  • Agents, excipients, adhesives, wetting agents, disintegrating agents, absorption enhancers, surfactants, adsorption carriers, lubricants, etc. will not be described in detail here.
  • the dosage form of the pharmaceutical preparation is not particularly limited, and a desired dosage form may be selected according to specific needs, such as, but not limited to, capsules, soft capsules, tablets, oral liquids, dispersible tablets, powders, injections, dripping pills, and the like.
  • the present invention also provides an application in which the polypeptide or the pharmaceutical preparation is used in preparing a reagent for tumor imaging or tumor neovascularization, and both the tumor and the tumor neovascularization are positive for CD105.
  • Tumor imaging or tumor neovascularization includes, but is not limited to, positron emission tomography (PET), single photon emission tomography (SPECT), near infrared (NIR) optical imaging, or magnetic resonance imaging (MRI).
  • PET positron emission tomography
  • SPECT single photon emission tomography
  • NIR near infrared
  • MRI magnetic resonance imaging
  • the tumor is, for example, but not limited to, osteosarcoma, angiosarcoma, or leukemia, and the like.
  • the present invention also provides another application, the use of the polypeptide or the pharmaceutical preparation in the preparation of a medicament for inhibiting or preventing the growth of tumor cells, which are cancer cells with positive expression of CD105, such as but not Limited to osteosarcoma, angiosarcoma or leukemia, etc.
  • the invention also provides another application, the use of the polypeptide or the pharmaceutical preparation in the preparation of a medicament for inhibiting or preventing tumor angiogenesis, tumor vascular endothelial cells of tumor angiogenesis formed by tumor angiogenesis With CD105 positive expression characteristics.
  • the present invention also provides another application.
  • the polypeptide or the pharmaceutical preparation is used in preparing a reagent for diagnosing, preventing or treating a tumor or tumor neovascularization, and the tumor or tumor neovascularization is positive for CD105.
  • the tumor is, for example, but not limited to, osteosarcoma, angiosarcoma, or leukemia, and the like.
  • the present invention also provides another application, wherein the polypeptide is used in preparing a preparation for labeling, identifying, enriching, sorting, or purifying CD105-positive cells; or, the polypeptide is used for labeling, identifying, Application in enrichment, sorting or purification methods.
  • CD105-positive cells are, for example, but not limited to, osteosarcoma cells, vascular endothelial cells, or mesenchymal stem cells.
  • the present invention also provides another application.
  • the polypeptide or the pharmaceutical preparation is used in preparing a reagent for targeting CD105 positive cells.
  • the inventors of the present application identified 13 new peptides that bind to CD105 using the M13 phage display library.
  • the nABPK296 peptide (the sequence is shown in Sequence 1 of the Sequence Listing) has a higher affinity for the CD105-positive osteosarcoma cell line MNNG than other peptides, and a lower affinity for the CD105-negative cell line Cal27, and The peptide specifically binds CD105-positive cells.
  • nABPK296 can visualize MNNG tumors in MNNG tumor-bearing mice.
  • the inventors found in the study that nABP296 can mark osteosarcoma tissue sections derived from animal MNNG xenograft tumor models and osteosarcoma patients.
  • the polypeptide (nABP296) provided by the present invention can specifically bind to recombinant human CD105 protein.
  • the peptide has the characteristics of small molecular weight and can specifically bind to CD105-positive cells; meanwhile, it also has good biocompatibility, safety and non-toxicity, and can make up for The deficiency of CD105 antibody.
  • the polypeptide provided by the present invention can be used instead of the CD105 antibody, for example, it can be used to prepare probes for separating CD105 positive cells from tissues, such as osteosarcoma cells or MSCs (mesenchymal stem cells), etc., and can also be used to prepare in vivo or In vitro targeted tumor imaging or visualization preparations that can be labeled and used to detect tumor cells.
  • the polypeptide nABP296 provided by the present invention is a non-antibody binding protein, consisting of only 12 amino acids, and has better penetrating ability to the tumor microenvironment than antibodies. Due to its effective penetrating ability and targeting effect, the polypeptide can be used as an antibody Delivery vehicle for tumor treatment drugs.
  • the polypeptide is conjugated with an anti-tumor drug or other agent to target the CD105 protein, and the anti-tumor drug or other agent is directed to CD105-positive tumor cells.
  • Panel A is a semi-quantitative RT-PCR analysis of CD105 levels in MNNG and Cal27 cell lines.
  • Panel B is an immunofluorescence analysis of CD105 expression in MNNG and Cal27 cell lines.
  • Panel C shows CD105 expression in MNNG and Cal27 cell lines by flow cytometry.
  • Panel A is a flow cytometric analysis of the binding affinities of 13 peptides to the MNNG cell line.
  • Panel B is the mean fluorescence intensity (MFI) analysis of flow cytometry in Panel A.
  • Panel C is a flow cytometric analysis of nABP296 and nABP297 on MNNG and Cal27.
  • Panel D is the immunofluorescence analysis of nABP296 in MNNG and Cal27.
  • Panel E is an ELISA measurement of the binding affinity of nABP296 to CD105 protein and CD106 protein, where CD106 protein was used as a control.
  • Panel F shows the cytotoxicity of nABP296 to MNNG at 24 and 48 hours.
  • Figure 3 shows the chemical structure of nABP296.
  • Figure 4 shows the results of in vitro visualization experiments. Among them: Panel A shows the colocalization of nABP296 with CD105 antibody in tumor sections derived from MNNG tumor-bearing mice. Panel B is a tumor tissue section from an osteosarcoma patient. Panel C is a tumor tissue section from a tongue cancer patient.
  • Figure 5 shows in vivo and ex vivo imaging of MNNG tumor-bearing mice.
  • Panel A is an in vivo imaging of MNNG tumor-bearing mice 1 hour after intravenous administration of nABP296 peptide.
  • Panel B is an in vitro imaging of tumors excised from mice bearing MNNG tumors at 1.5 hours.
  • Panel C shows frozen sections of organs excised from MNNG tumor-bearing mice.
  • the main research methods used in the examples include phage display library biological panning, ELISA measurement, peptide synthesis, RT-PCR, flow cytometry, immunofluorescence analysis, cytotoxicity experiments, cell proliferation experiments, mouse in vivo imaging, etc.
  • the experimental operations are all conventional experimental operations mastered by those skilled in the art or experimental operations carried out according to product specifications, and will not be repeated one by one.
  • the reagents used are conventional reagents that can be purchased from commercial sources.
  • the percentages of the solutions involved are mass percentages unless otherwise specified.
  • Phage display peptide library biological panning
  • Phage display peptide library biopanning was performed according to standard procedures. Experiments were performed using a commercially constructed phage display peptide library (Ph.D.-12 phage display library, NEB, Beverly, MA, USA).
  • Recombinant human CD105 protein (R & D Systems, Minnesota, USA) at a concentration of 100 ⁇ g / ml in sterile PBS was added to a sterilized MaxiSorp plate (Thermo Fisher Scientific, MA, USA) and incubated overnight at 4 ° C. The plate was washed 6 times with TBST (0.1% Tween) and blocked with 1% BSA in PBS for 1 hour at 4 ° C. The M13 phage display peptide library was bound for 1 hour at room temperature, and the unbound phage was washed 10 times with TBST (0.1% Tween).
  • the bound phage was eluted with an elution buffer, and the eluted phage was amplified for the next round of biopanning. After three rounds of biological panning in this way, the target M13 phage was enriched. Table 1 lists the results of three rounds of biopanning. After three rounds of biopanning, M13 phages with high affinity for recombinant human CD105 protein were enriched.
  • M13 phage monoclonals were subjected to an ELISA assay.
  • the 16 monoclonal phages obtained after panning were randomly selected for ELISA binding analysis.
  • 100 ⁇ g / ml of recombinant human CD105 protein in sterile PBS was added to 16 separate sterilized MaxiSorp plates and incubated overnight at 4 ° C.
  • the plate was washed 6 times with 0.1% TBST and blocked with 1% BSA in PBS (ie, blocking buffer) at 4 ° C for 1 hour.
  • Sixteen monoclonal phages were amplified and added to the plate, and incubated at room temperature for 1 hour.
  • bound phages were detected by incubation with rabbit anti-M13 phage antibodies and HRP-conjugated goat anti-rabbit IgG (1:20; Abcam, Cambridge, UK). The amount of bound HRP was measured using an ABTS / H 2 O 2 substrate, and the absorbance was detected at 405 nm.
  • the primers 5'-CCCTCATAGTTAGCGTAACG-3 '(NEB, Beverly, MA, USA) were used to determine the DNA sequences inserted into the 13 positive affinity monoclonal phages.
  • Example 2 The 13 peptides obtained in Example 1 were synthesized by a commercial company (Shanghai Qiangyao Biotechnology Co., Ltd.), and FITC (fluorescein isothiocyanate) was connected to the N-terminus of the peptide for labeling.
  • the prepared peptides were identified by mass spectrometry (MS), and the purity (> 95%) was determined by high performance liquid chromatography (HPLC).
  • MS mass spectrometry
  • HPLC high performance liquid chromatography
  • FITC-labeled peptides were stored at -20 ° C for subsequent experiments. In use, the peptide is dissolved in sterile water at 1 mg / kg and diluted to the required concentration according to experimental requirements.
  • MNNG and Cal27 cell lines were obtained from the Type Culture Collection of the Chinese Academy of Sciences in Shanghai, China.
  • MNNG cells were cultured with DF (Sigma, USA) + 5% fetal bovine serum (FBS), and Cal27 cells were cultured with DMEN (Sigma, USA) + 10% FBS.
  • the cells were seeded in a 10 cm 2 flask and cultured in an environment of 37 ° C., 5% CO 2 and 90% relative humidity.
  • CD105 in MNNG and Cal27 was detected by RT-PCR, immunofluorescence and flow cytometry.
  • the CD105 primers used were (F: CACCACAGCGGAAAAAGGTG; R: GCCGGTTTTGGGTATGGGTA) (Synbio, Technologies, Suzhou, China); in immunofluorescence experiments
  • the CD105 antibody used was (1: 200; Invitrogen, Waltham, MA, USA); the CD105 antibody used in flow cytometry experiments was (MACS, Bergisch Gladbach, Germany).
  • RNA was extracted from tumor cells MNNG and Cal27, respectively, and transformed into complementary using TaKaRa oligo (dT) primers (Takara Bio, Japan) and reverse transcriptase (Toyobo Life Science, Japan). DNA is used for PCR amplification.
  • TaKaRa oligo (dT) primers Takara Bio, Japan
  • reverse transcriptase Toyobo Life Science, Japan
  • the CD105 primers used for PCR amplification are (F: CACCACAGCGGAAAAAGGTG; R: GCCGGTTTTGGGTATGGGTA) (Suzhou Hongxun Biotechnology Co., Ltd.); PCR reaction conditions are: 95 ° C for 5 minutes, 95 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 1 Minutes, 30 cycles, extended at 72 ° C for 10 minutes, and finally maintained at 16 ° C.
  • PCR products were detected by electrophoresis on a 1% agarose gel, and ethidium bromide (EB) staining appeared under UV irradiation.
  • EB ethidium bromide
  • MNNG and Cal27 were seeded at a cell density of 1 ⁇ 10 ⁇ 5 in a 6-well culture flask with a glass slide, and cultured at 37 ° C, 5% CO 2 and 90% relative humidity overnight. . The next day, the cells were fixed in 4% PFA for 15 minutes. Block with 5% bovine serum albumin (BSA, Guangzhou Xiangbo Biotechnology Co., Ltd.) for 1 hour at room temperature. Cells were incubated with CD105 antibody (1: 200; Invitrogen, Waltham, MA, USA) at 4 ° C overnight. Unbound antibody was removed by elution 3 times in PBS.
  • BSA bovine serum albumin
  • Cells were further treated with blocking buffer containing Alexa Fluor 568 (1: 1000; Invitrogen, Waltham, MA, USA) labeled donkey anti-mouse IgG (H + L) secondary antibody for 1 hour at room temperature. After washing with PBS, the nuclei were counterstained with DAPI and observed under a laser confocal scanning microscope (CLSM) (Leica, Germany).
  • CLSM laser confocal scanning microscope
  • MNNG and Cal27 cells were incubated in CD105 antibody (1:10, MACS, Germany) at 4 ° C for 10 minutes. The cell pellet was washed three times with PBS, and the cell pellet was suspended in 500 ⁇ l of PBS and analyzed using FACSCalibur (BD, USA).
  • panel A shows the results of semi-quantitative RT-PCR analysis of CD105 levels in MNNG and Cal27 cell lines. There was cDNA expression of CD105 in MNNG and no cDNA expression of CD105 in Cal27.
  • Panel B shows the results of immunofluorescence analysis of CD105 expression in MNNG and Cal27 cell lines; as shown by red fluorescence, CD105 is located on the surface of the MNNG cell line. There is no CD105 fluorescence in the Cal27 cell line.
  • Panel C shows CD105 expression in MNNG and Cal27 cell lines by flow cytometry.
  • the 13 peptides obtained in the previous screening were used for peptide binding detection by flow cytometry.
  • MNNG cells were incubated at a cell density of 5 ⁇ 10 5 cells in 100 ⁇ l of a peptide having a concentration of 100 ⁇ g / ml at 4 ° C. for 30 minutes.
  • the cell pellet was washed 3 times with PBS.
  • Cell pellets were suspended in 500 ⁇ l of PBS and assayed using FACSCalibur (BD, USA).
  • Panel A is a flow cytometry analysis of the binding affinities of 13 peptides to the MNNG cell line. The results show that the nABP296 and nABP297 peptides have higher binding affinity to the MNNG cell line.
  • Panel B is the flow cytometer in Panel A. The average fluorescence intensity (MFI) analysis showed that nABP296 and nABP297 had higher MFI. From the results of the above flow cytometry, it can be seen that the two peptides nABP296 and nABP297 have high affinity for the CD105-positive cell line MNNG, and the affinity is higher than the other 11 peptides (the results are shown in Figures A and B in Figure 2).
  • MNNG and Cal27 were incubated in nABP296 and nABP297 peptides at a concentration of 100 ⁇ g / ml, respectively, at a cell density of 1 ⁇ 10 ⁇ 6 for 30 minutes at 4 ° C; the cell pellet was washed 3 times with PBS; and the cell pellet Resuspend in 500 ⁇ l PBS and analyze using FACSCalibur (BD, USA).
  • C is a flow cytometry analysis of nABP296 and nABP297 on MNNG and Cal27.
  • NABP296 has a higher binding affinity on MNNG and Cal27.
  • Has a lower affinity According to the results of flow cytometry measurement, it can be seen that nABP296 and MNNG have higher binding efficiency than Cal27, and there is no difference in the binding efficiency between nABP297 and MNNG or Cal27.
  • MNNG and Cal27 cells were seeded at a cell density of 1 ⁇ 10 ⁇ 5 cells in 6-well flasks with glass slides, and cultured overnight at 37 ° C, 5% CO 2 and 90% relative humidity. The next day, the flask was washed twice in PBS, and then incubated in nABP296 peptide at a concentration of 100 ⁇ g / ml for 30 minutes at 4 ° C. After that, unbound peptides were removed by washing with PBS and fixed in 4% PFA for 15 minutes. Block with 5% bovine serum albumin (BSA, Guangzhou Xiangbo Biotechnology Co., Ltd.) for 1 hour at room temperature.
  • BSA bovine serum albumin
  • D is the immunofluorescence analysis of nABP296 in MNNG and Cal27.
  • the results showed that nABP296 could co-localize with CD105 antibody in MNNG, and neither CD105 nor nABP296 could bind Cal27. It can be seen from the results of the immunofluorescence assay that nABP296 is co-localized with the CD105 antibody in the MNNG cell line and has no binding to Cal27 (Fig. 2D).
  • ELISA binding assay was performed for nABP296 and CD105 protein, and CD106 protein was used as a control.
  • Recombinant human CD105 protein and CD106 protein at a concentration of 100 ⁇ g / ml in sterile PBS were added to six independent sterilized MaxiSorp plates and incubated at 4 ° C. overnight. Plates were washed 6 times with 0.1% TBST and blocked with 1% BSA in PBS for 1 hour at 4 ° C.
  • the nABP296 peptide was then incubated at a concentration of 100 ⁇ g / ml for 1 hour at room temperature. Unbound peptides were removed by washing 10 times in PBS.
  • the absorbance (OD) was measured at 490 nm using Victor X5 (PerkinElmer, Singapore).
  • Figure E in Figure 2 is an ELISA measurement of the binding affinity of nABP296 to CD105 protein and CD106 protein, in which CD106 protein is used as a control. It can be seen that the OD value of the CD105 protein group is higher than that of the CD106 protein group. It can be seen from the experimental results that nABP296 has a higher affinity for the CD105 protein than the control protein ( Figure 2E).
  • MNNG cells were seeded at a cell density of 1 ⁇ 10 ⁇ 5 in 100 ⁇ l of complete medium in a 96-well plate. After culturing overnight at 37 ° C. in a 5% CO 2 atmosphere, the cells were incubated in nABP296 solutions of different concentrations from 0.5 ⁇ Mol to 5 ⁇ Mol. At 24 hours and 48 hours, 10 ⁇ l of Cell Counting Kit-8 (CCK-8, Dojindo, Japan) solution was added to the cells and cultured for an additional hour. The absorbance was measured at 450 nm using a VICTOR TM X5 Multilabel Plate Reader (PerkinElmer, Singapore) to calculate the survival rate.
  • VICTOR TM X5 Multilabel Plate Reader PerkinElmer, Singapore
  • Panel F in Figure 2 shows the cytotoxicity of nABP296 to MNNG within 24 hours and 48 hours. The results showed that nABP296 was not cytotoxic to MNNG cell lines (p> 0.05).
  • nABP296 can selectively bind to the CD105 positive cell line MNNG and is biocompatible with the MNNG cell line. It can be speculated that nABP296 has the potential to target human osteosarcoma in vivo.
  • Osteosarcoma and tongue cancer tissues were derived from patients after surgery at Guanghua School of Stomatology, Sun Yat-sen University. Xenograft tumor models, tumor sections from osteosarcoma patients and tongue cancer patients were used for in vitro binding experiments. The tumor was cut using a cryostat microtome (HM560, MICROM, Germany). All patients provided informed consent prior to tissue collection, and all patients agreed to use their samples in scientific research. This study was approved by the Ethics Committee of the School of Stomatology, Sun Yat-sen University, and the approval number is ERC-2017-11.
  • Tissue sections were blocked with 5% bovine serum albumin (BSA, Guangzhou Xiangbo Biotechnology Co., Ltd.) for 1 hour at room temperature, and then nABP296 peptide and CD105 antibody (mouse monoclonal; 1 containing 50 ⁇ M) were used. : 200; Abcam, Cambridge, UK) blocking buffer was incubated at 4 ° C overnight. Unbound peptides and antibodies were eluted three times with PBS. Donkey anti-mouse IgG (H + L) secondary antibody labeled with Alexa Fluor 568 (1: 1000; Invitrogen, Waltham, MA, USA) was further treated for 1 hour at room temperature. After washing with PBS, the nuclei were stained with DAPI and the results were analyzed under CLSM (Leica, Germany).
  • BSA bovine serum albumin
  • nABP296 peptide and CD105 antibody mouse monoclonal; 1 containing 50 ⁇ M
  • panel A shows the colocalization of nABP296 with CD105 antibodies in tumor sections derived from MNNG tumor-bearing mice;
  • panel B shows tumor tissue sections from osteosarcoma patients. It can be seen that osteosarcoma sections can be labeled with nABP296 peptide and CD105 antibody .
  • Panel C is a tumor tissue section from a tongue cancer patient. It can be seen that there is no CD105 antibody fluorescence but weak nABP296 fluorescence in this section.
  • nABP296 can bind osteosarcoma from xenograft tumor models and patients in vitro. Therefore, nABP296 can replace CD105 for the diagnosis and labeling of osteosarcoma.
  • MNNG cells suspended in DF were transplanted subcutaneously at a cell density of 1 ⁇ 10 ⁇ 7 / ml into 5-week-old BALB / c NOD mice.
  • 0.1 ⁇ M nABP296 dissolved in 100 ⁇ l of sterile Milli-Q water was administered intravenously to mice. Mice were observed in an in vivo imaging system using an IVIS spectrometer. Photos were taken within 1 hour (PerkinELmer, Akron, Ohio, USA).
  • mice were euthanized within 1.5 hours, tumors were excised from MNNG tumor-bearing mice, and imaged in an in vivo imaging system using an IVIS spectrometer. Organs were frozen and sectioned, and nuclei were stained by DAPI. Results were analyzed by CLSM (Leica, Germany).
  • Figure A in Figure 5 is an in vivo imaging of MNNG tumor-bearing mice 1 hour after intravenous administration of nABP296 peptide. It can be seen that fluorescence can be detected in liver, kidney and venography tumor areas. The fluorescence detected in tumors is higher than in surrounding tissues. No fluorescence was detected in the NC group (the NC group was a control group injected with 100 ⁇ l of sterile normal saline).
  • Panel B is an in vitro imaging of tumors excised from mice bearing MNNG tumors at 1.5 hours. It can be seen that FITC-labeled nABP296 accumulated in xenograft tumors ( Figure 5B).
  • Panel C is a frozen section of an organ excised from a MNNG tumor-bearing mouse, fluorescence was detected in the tumor tissue section, and no FITC fluorescence was detected in the heart section (Figure 5C).
  • nABP296 12 amino acid peptide nABP296 (for the sequence, see Sequence 1 in the Sequence Listing), which can specifically bind to the recombinant human CD105 protein.
  • CD105 is a biomarker for isolation and enrichment of MSC (mesenchymal stem cells).
  • the use of peptide nABP296 can replace CD105 antibodies to isolate CD105 + phenotype cells, such as CD105 + phenotype MSCs (mesenchymal stem cells).
  • the nABP296 peptide provided by the present invention is relatively short, has a small molecular weight, and can specifically bind to the CD105-positive cell line MNNG.
  • nABP296 has specific affinity and good biocompatibility, which can solve the problems of using CD105 antibody in vivo.
  • nABP296 can also be used as a new probe to isolate CD105-positive cells such as MSC from tissues.
  • Angiogenesis is the process of new blood vessel formation in tissues and is necessary for tumor growth and metastasis.
  • CD105 plays a key role in angiogenesis. Therefore, CD105 is overexpressed in actively proliferating tumors. CD105 in vivo targeting can be used for tumor PET imaging and visualization.
  • the nABP296 peptide of the present invention can locate osteosarcoma in MNNG tumor-bearing mice ( Figure 5).
  • the nABP296 peptide is a relatively short synthetic in vitro artificial peptide, which has no animal heterologous form or immune response. nABP296 is non-toxic to cells, so it can be safely used in humans.
  • nABP296 can be activated by fluorescence, so it can be conveniently used to detect CD105 positive tumor cells and tumor neovascularization with CD105 positive expression. Due to its advantages, the nABP296 peptide has application potential in early diagnosis and tracking of tumor metastases with CD105 positive expression characteristics, such as osteosarcoma.
  • nABP296 peptide of the present invention is a non-antibody binding peptide and consists of only 12 amino acids. It has better penetrating ability to tumor microenvironment than antibody. Due to its effective penetrating ability and targeting effect, the polypeptide can be used as a vehicle for antitumor therapy drugs or antitumor neovascular drugs. In immunofluorescence experiments, we found nABP296 in the cytoplasm. Therefore, it can be concluded that nABP296 can penetrate cell membranes. Therefore, nABP296 can form conjugates with antitumor drugs or other therapeutic agents, and can be used as a targeting tool for targeting CD105 proteins to transfer antitumor drugs or therapeutic agents to CD105 positive tumor cells.
  • the novel peptide nABP296 provided by the present invention can specifically bind to recombinant human CD105 protein, and can not only label osteosarcoma slices and MNNG xenograft tumor models from patients in vitro, but also label MNNG xenograft tumor models in vivo. Therefore, the peptide can be used for in vivo diagnosis and imaging of tumors (such as osteosarcoma with positive expression of CD105, etc.); nABP296 can also be used for MSC isolation and CD105 targeted therapy.

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Abstract

提供一种对内皮蛋白(Endoglin/CD105)具有特异性结合的多肽，用于制备从组织中分离CD105阳性细胞的探针，例如骨肉瘤细胞、血管内皮细胞或间充质干细胞等，还可制备在体内或体外靶向肿瘤成像或可视化的制剂，可被标记并应用于检测肿瘤细胞。所述多肽为一种非抗体结合蛋白，该多肽可用作抗肿瘤治疗药物的运载工具，可与抗肿瘤药物或其他试剂形成缀合物，可靶向CD105蛋白，将抗肿瘤药物或其他试剂定向转移至CD105阳性肿瘤细胞。

Description

一种与CD105特异性结合的多肽及其应用 技术领域
本发明涉及一种非抗体结合肽，具体涉及一种与内皮蛋白(Endoglin/CD105)特异性结合的多肽及其应用。
背景技术
CD105也称为内皮糖蛋白，是在细胞膜上形成TGF-β受体复合物的I型膜糖蛋白。CD105是由633个氨基酸组成的180kDa同型二聚体跨膜蛋白。CD105由大的胞外结构域，疏水性跨膜结构域和短的胞内结构域组成。CD105外部结构域以高亲和力结合TGF-β-1和TGF-β-3，形成TGF-β受体复合物。它在调节细胞增殖，分化，迁移，粘附和血管生成中起重要作用，这对于肿瘤生长，存活和转移至关重要。CD105主要在间充质干细胞(MSC)、肿瘤新生血管的内皮细胞，骨肉瘤、血管肉瘤和白血病等肿瘤细胞表达。CD105抗体用于标记检测肿瘤微血管密度，分离MSC和用于靶向治疗的缀合药物。骨肉瘤是最常见的原发恶性瘤之一，主要针对长圆柱形骨。据报道大约5％的骨肉瘤发生在颌面部。它发生在青少年和儿童中，每年发病人数约为3-450万人，并可在早期阶段积极转移至肺部。在一项研究中，据报道有一部分骨肉瘤细胞表达间充质干细胞标志物(CD105)，其比其他骨肉瘤细胞系更具侵袭性。因此，CD105抗体可能是鉴定骨肉瘤恶性程度的标志物。
但是，抗体生产的复杂性，热不稳定性以及对癌症微环境的渗透差等缺陷限制了抗体在体内的应用。非抗体结合蛋白(nABP)是人工合成的热稳定肽。该肽对肿瘤微环境的渗透作用是有效的，因为它很小且具有正电荷。由于这种肽的优点，它已经引起了巨大的关注，并已被用于各种抗肿瘤研究和肿瘤靶向治疗中。
发明内容
本发明的目的在于提供一种对CD105具有特异性结合的多肽及其 应用，所提供的多肽为一种非抗体结合蛋白。
本发明为达到其目的，采用的技术方案如下：
本发明第一方面提供一种与内皮蛋白(Endoglin/CD105)特异性结合的多肽，所述多肽的氨基酸序列如序列表中序列1所示。
本发明还提供一种多核苷酸，其包含编码如上文序列1的多肽的核苷酸序列。
本发明还提供一种载体，其包含上文所述的多核苷酸。
本发明还提供一种宿主细胞，其包括上文所述的载体或所述的多核苷酸。
本发明还提供一种药物制剂，所述药物制剂包括如下组分：上文所述的多肽与治疗药物分子和/或可被检测的标记连接而成的化合物；该多肽可与治疗药物分子和/或标记形成缀合物。二者的连接也可以是共价键或非共价键方式连接。
优选的，所述可被检测的标记包括荧光试剂、核素或放射试剂中的一种或多种；这些标记均可以具体采用本领域所熟知的相应标记物，例如荧光试剂罗丹明B、FITC(5-异硫氰酸荧光素)等等。
在一些实施方案中，所述治疗药物分子为抗癌剂或抗肿瘤新生血管药，抗癌剂为对CD105蛋白阳性表达特征的肿瘤具有抗癌活性的药物，所述抗肿瘤新生血管药为对肿瘤血管新生具有抑制作用的药物；药物具体如阿霉素、紫杉醇、顺铂和环磷酰胺等等。
优选的，所述肿瘤既可以为CD105蛋白阳性表达的骨肉瘤、血管肉瘤和白血病等肿瘤细胞，也可以为CD105蛋白阴性表达肿瘤，但是其肿瘤新生血管表达CD105蛋白。
所述的药物制剂，在实际应用中，可根据需要而可以包括药学上允许的载体或辅料，本领域技术人员可根据需要而具体选择所需要的载体或辅料，例如可以为药学领域常用的稀释剂、赋形剂、粘合剂、润湿剂、崩解剂、吸收促进剂、表面活性剂、吸附载体和润滑剂等等，对此不作赘述。药物制剂的剂型不作特别限定，可根据具体需要而选择所需的剂型，例如但不限于胶囊、软胶囊、片剂、口服液、分散片、粉剂、注射液或滴丸等等。
本发明还提供一种应用，所述的多肽或所述的药物制剂在制备用于肿瘤成像或肿瘤新生血管成像的试剂中的应用，所述肿瘤和肿瘤新生血管均为CD105阳性表达。肿瘤成像或肿瘤新生血管成像包括但不限于正电子发射断层显像术(PET)、单光子发射断层显像术(SPECT)、近红外(NIR)光学成像或磁共振成像(MRI)。所述肿瘤例如但不限于骨肉瘤、血管肉瘤或白血病等等。
本发明还提供另一种应用，所述的多肽或所述的药物制剂在制备用于抑制或阻止肿瘤细胞生长的药物中的用途，所述肿瘤细胞为CD105阳性表达的癌细胞，例如但不限于骨肉瘤、血管肉瘤或白血病等等。
本发明还提供另一种应用，所述的多肽或所述的药物制剂在制备用于抑制或阻止肿瘤血管新生的药物中的用途，该肿瘤血管新生所形成的肿瘤新生血管的肿瘤血管内皮细胞具有CD105阳性表达特征。
本发明还提供另一种应用，所述的多肽或所述的药物制剂在制备用于诊断、预防或治疗肿瘤或肿瘤新生血管的试剂中应用，所述肿瘤或肿瘤新生血管为CD105阳性表达。所述肿瘤例如但不限于骨肉瘤、血管肉瘤或白血病等等。
本发明还提供另一种应用，所述的多肽在制备用于标记、鉴定、富集、分选或纯化CD105阳性细胞的制剂中应用；或，所述多肽在CD105阳性细胞的标记、鉴定、富集、分选或纯化方法中应用。CD105阳性细胞例如但不限于骨肉瘤细胞、血管内皮细胞或间充质干细胞等。
本发明还提供另一种应用，所述的多肽或所述的药物制剂在制备用于靶向CD105阳性细胞的试剂中应用。
本申请发明人在研究过程中，使用M13噬菌体展示文库鉴定了13种与CD105结合的新肽。在13种肽中，nABPK296肽(序列见序列表的序列1所示)对CD105阳性骨肉瘤细胞系MNNG具有比其他肽更高的亲和力，而对CD105阴性细胞系Cal27具有更低的亲和力，nABPK296肽可特异性的结合CD105阳性细胞。nABPK296可以显现MNNG荷瘤小鼠中的MNNG肿瘤。此外，发明人在研究中发现nABP296可以标记源自动物MNNG异种移植肿瘤模型和骨肉瘤患者的骨肉瘤组织切片。
本发明提供的技术方案具有如下有益效果：
本发明提供的多肽(nABP296)可特异性结合重组人CD105蛋白，该肽具有分子量小，可特异性结合CD105阳性细胞的特点；同时还具有很好的生物相容性，安全无毒，可以弥补CD105抗体所存在的不足。本发明提供的多肽可替代CD105抗体进行应用，例如可以用于制备从组织中分离CD105阳性细胞的探针，例如骨肉瘤细胞或MSC(间充质干细胞)等，还可以用于制备在体内或体外靶向肿瘤成像或可视化的制剂，可被标记并应用于检测肿瘤细胞。
本发明提供的多肽nABP296为一种非抗体结合蛋白，仅由12个氨基酸组成，对肿瘤微环境的穿透能力优于抗体，由于有效的穿透能力和靶向作用，该多肽可用作抗肿瘤治疗药物的运载工具。将该多肽与抗肿瘤药物或其他试剂形成缀合物，可靶向CD105蛋白，将抗肿瘤药物或其他试剂定向转移至CD105阳性肿瘤细胞。
附图说明
图1中:A图为MNNG和Cal27细胞系中CD105水平的半定量RT-PCR分析。B图为MNNG和Cal27细胞系中CD105表达的免疫荧光分析。C图为流式细胞术分析MNNG和Cal27细胞系中的CD105表达。
图2中：A图为13种肽对MNNG细胞系的结合亲和力的流式细胞术分析。B图为图A中流式细胞术的平均荧光强度(MFI)分析。C图为nABP296和nABP297对MNNG和Cal27的流式细胞术分析。D图为MNNG和Cal27中nABP296的免疫荧光分析。E图为nABP296对CD105蛋白和CD106蛋白的结合亲和力的ELISA测定，其中CD106蛋白被用作对照。F图为在24小时和48小时内nABP296对MNNG的细胞毒性测定。
图3为nABP296的化学结构。
图4为体外可视化实验结果。其中：A图为nABP296与源自MNNG荷瘤小鼠的肿瘤切片中的CD105抗体共定位。B图为来自骨肉瘤患者的肿瘤组织切片。C图为来自舌癌患者的肿瘤组织切片。
图5为MNNG荷瘤小鼠的体内和离体成像。其中，A图为在静脉内施用nABP296肽1小时后MNNG荷瘤小鼠的体内成像。B图为在1.5小时时从携带MNNG肿瘤的小鼠切除的肿瘤的体外成像。C图为从MNNG荷 瘤小鼠切除的器官的冷冻切片。
具体实施方式
为了更好的理解本发明的技术方案，下面结合实施例进一步阐述本发明的内容，但本发明的内容并不仅仅局限于以下实施例。
实施例中所用到的主要研究方法包括噬菌体展示文库生物淘选、ELISA测定，多肽合成，RT-PCR，流式细胞术，免疫荧光分析，细胞毒性实验、细胞增殖实验、小鼠活体成像等，实验操作均为本领域技术人员所掌握的常规实验操作或根据产品说明书而开展的实验操作，不作一一赘述。所用试剂若未特别说明，均为能从商业渠道购买获得的常规试剂。所涉及的溶液的百分比若未特别说明，均为质量百分比。
统计分析采用GraphPad Prism 5软件进行，P值<0.05被认为是显著的。
实施例1、CD105非抗体结合蛋白的筛选与合成
主要包括文库筛选、ELISA实验和测序分析。
1、噬菌体展示肽库生物淘选：
噬菌体展示肽库生物淘选按照标准程序进行。使用商业构建的噬菌体展示肽库(Ph.D.-12噬菌体展示文库，NEB，Beverly，MA，USA)开展实验。
将溶于无菌PBS中的浓度100μg/ml的重组人CD105蛋白(R&D Systems，Minnesota，USA)加入到灭菌的MaxiSorp平板(Thermo Fisher Scientific，MA，USA)，在4℃孵育过夜。用TBST(0.1％吐温)洗涤平板6次，并使用含1％BSA的PBS在4℃封闭1小时。将M13噬菌体展示肽库在室温下结合1小时，并将未结合的噬菌体用TBST(0.1％吐温)洗涤10次。结合噬菌体用洗脱缓冲液洗脱，洗脱的噬菌体被扩增用于下一轮生物淘选。如此共经过三轮生物淘选后，目标M13噬菌体被富集。表1列出了三轮生物淘选的结果，经过三轮生物淘选后，对重组人CD105蛋白具有高亲和力的M13噬菌体被富集。
表1 每轮生物淘选的回收率
Figure PCTCN2018103544-appb-000001
由表1可见，经过三轮生物淘选后，M13噬菌体的回收率从4.9×10 -6升至4.5×10 -5。结合重组人CD105蛋白的M13噬菌体已被富集，用于后续实验。
2、噬菌体ELISA结合分析及测序
为了鉴定可结合CD105的单克隆，对M13噬菌体单克隆进行了ELISA测定。随机选择淘选后得到的16个单克隆噬菌体用于ELISA结合分析。将100μg/ml的重组人CD105蛋白的无菌PBS液加入到16个独立的灭菌的MaxiSorp平板中，并在4℃孵育过夜。用0.1％TBST洗涤平板6次，并使用含1％BSA的PBS(即封闭缓冲液)在4℃封闭1小时。将16个单克隆噬菌体进行扩增并加入到平板中，在室温下孵育1小时。用0.1％TBST反复洗涤平板后，通过与兔抗M13噬菌体抗体和HRP缀合的山羊抗兔IgG(1:20；Abcam，Cambridge，UK)一起孵育来检测结合的噬菌体。使用ABTS/H 2O 2底物来测量结合的HRP的量，在405nm处检测吸光度。使用引物5'-CCCTCATAGTTAGCGTAACG-3'(NEB，Beverly，MA，USA)测定13个阳性亲和性单克隆噬菌体中所插入的DNA序列。
16种单克隆M13噬菌体对人重组CD105蛋白和NC(封闭缓冲液，对照组)的结合活性的ELISA测定结果见表2所示，表格中A-P依次代表16种单克隆M13噬菌体的对应实验结果。
表2 M13噬菌体与CD105蛋白的结合性(吸光值)
Figure PCTCN2018103544-appb-000002
从表2可以发现，有13种单克隆M13噬菌体在实验组中与CD105蛋白的亲和性，相比与对照组的封闭缓冲液而言，具有更高的结合亲和力，如表2所示，证实13个单克隆噬菌体对CD105具有高亲和力。
因而经筛选后获得了13个在ELISA测定中呈现阳性结果的单克隆噬菌体，表3列出了阳性结果的对应多肽序列和性质，这些多肽依次分别编号为nABP295到nABP307。
表3 多肽的性质
Figure PCTCN2018103544-appb-000003
Figure PCTCN2018103544-appb-000004
实施例2多肽合成
通过商业公司(上海强耀生物科技有限公司)合成实施例1得到的13个多肽，在多肽的N末端连接FITC(5-异硫氰酸荧光素)用于标记。通过质谱(MS)鉴定制备的肽，并且通过高效液相色谱法(HPLC)测定纯度(>95％)。在图3示出了nABP296的化学结构。
FITC标记的肽储存在-20℃，用于后续实验。在使用时，将肽以1mg/kg溶于无菌水中并根据实验要求稀释至所需浓度。
实施例3用于结合鉴定的细胞系的确定
MNNG和Cal27细胞系获自中国上海中国科学院典型培养物保藏中心。用DF(Sigma，USA)+5％胎牛血清(FBS)培养MNNG细胞，用DMEN(Sigma，USA)+10％FBS培养Cal27细胞。将细胞接种于10cm 2烧瓶，在37℃，5％CO 2和90％相对湿度环境中培养。
通过RT-PCR，免疫荧光和流式细胞术检测MNNG和Cal27中的CD105表达，其中用到的CD105引物为(F：CACCACAGCGGAAAAAGGTG；R：GCCGGTTTTGGGTATGGGTA)(Synbio Technologies，Suzhou，China)；免疫荧光实验中使用的CD105抗体为(1:200；Invitrogen，Waltham，MA，USA)；流式细胞术实验中使用的CD105抗体为(MACS，Bergisch  Gladbach，德国)。
RT-PCR实验中，从肿瘤细胞MNNG和Cal27中分别提取300ng总RNA，并使用TaKaRa寡聚(dT)引物(Takara Bio，日本)和逆转录酶(Toyobo Life Science，Japan)将其转化为互补DNA用于PCR扩增。PCR扩增所用的CD105引物为(F：CACCACAGCGGAAAAAGGTG；R：GCCGGTTTTGGGTATGGGTA)(苏州泓迅生物科技有限公司)；PCR反应条件为：95℃5分钟，95℃30秒、60℃30秒、72℃1分钟、30个循环，72℃延伸10分钟，最后保持在16℃。在1％琼脂糖凝胶上电泳检测PCR产物，在UV照射下溴化乙锭(EB)染色显现。
免疫荧光实验中，将MNNG和Cal27分别以1×10^5个的细胞密度接种在具有载玻片的6孔培养瓶中，并在37℃，5％CO 2和90％相对湿度下培养过夜。第二天，将细胞在4％PFA中固定15分钟。用5％牛血清白蛋白(BSA，广州祥博生物技术有限公司)在室温下封闭1小时。将细胞与CD105抗体(1:200；Invitrogen，Waltham，MA，USA)一起在4℃孵育过夜。未结合的抗体通过PBS洗脱3次去除。在室温下用含Alexa Fluor 568(1：1000；Invitrogen，Waltham，MA，USA)标记的驴抗小鼠IgG(H+L)二抗的封闭缓冲液进一步处理细胞1小时。之后用PBS洗涤，使用DAPI将细胞核复染，在激光共聚焦扫描显微镜(CLSM)(Leica，德国)下观察。
流式细胞术实验中，将MNNG和Cal27细胞分别在CD105抗体(1:10，MACS，德国)中于4℃孵育10分钟。将细胞沉淀用PBS洗涤3次，将细胞沉淀悬浮于500μl PBS中并使用FACSCalibur(BD，USA)分析。
图1中，A图为MNNG和Cal27细胞系中CD105水平的半定量RT-PCR分析结果，在MNNG中有CD105的cDNA表达，在Cal27中没有CD105的cDNA表达。B图为MNNG和Cal27细胞系中CD105表达的免疫荧光分析结果；如红色荧光所示，CD105位于MNNG细胞系的表面。在Cal27细胞系中没有CD105荧光。C图为流式细胞术分析MNNG和Cal27细胞系中的CD105表达。
根据RT-PCR，免疫荧光和流式细胞术的结果(图1)，可见MNNG高度表达CD105蛋白，而Cal27则不是，因而MNNG细胞系是CD105阳性的，Cal27细胞系是CD105阴性的。以下将使用这两个细胞系进行实验来鉴定对细胞膜表达的CD105具有高结合亲和力的肽。
实施例4流式细胞术鉴定两种肽的结合性
由实施例3的实验结果，确定采用CD105阳性细胞系MNNG来分析多肽的体外结合性。
使用前面筛选所得的13种肽通过流式细胞术进行肽结合性检测。将MNNG细胞以5×10 5个的细胞密度在100μl浓度100μg/ml的肽中于4℃孵育30分钟。将细胞沉淀用PBS洗涤3次。将细胞沉淀悬浮于500μl PBS中并使用FACSCalibur(BD，USA)进行检测分析。
图2中，A图为13种肽对MNNG细胞系的结合亲和力的流式细胞术分析，该结果表明nABP296和nABP297肽对MNNG细胞系具有更高的结合亲和力；B图为图A中流式细胞术的平均荧光强度(MFI)分析，结果表明nABP296和nABP297具有较高的MFI。由以上流式细胞术结果可见，两种肽nABP296和nABP297对CD105阳性细胞系MNNG具有高亲和力，亲和力高于其他11种肽(结果见图2中的图A、B)。
为了确定这两种肽nABP296和nABP297对CD105是否具有特异性亲和力，发明人使用MNNG和Cal27通过流式细胞术来分析nABP296和nABP297肽与CD105蛋白的特异性结合。实验过程如下：将MNNG和Cal27细胞在浓度分别为100μg/ml的nABP296和nABP297肽中以1×10^6的细胞密度于4℃孵育30分钟；将细胞沉淀用PBS洗涤3次；将细胞沉淀悬浮于500μl PBS中并使用FACSCalibur(BD，USA)分析。
图2中，C图为nABP296和nABP297对MNNG和Cal27的流式细胞术分析，在这个结果中，nABP297对MNNG和Cal27之间没有结合亲和力差异，nABP296对MNNG具有更高的结合亲和力，对Cal27的亲和力则更低。根据流式细胞术测定结果可见，nABP296与MNNG之间具有 比Cal27更高的结合效率，而nABP297与MNNG或Cal27的结合效率没有差异。
实施例5免疫荧光检测nABP296肽的结合特异性
将MNNG和Cal27细胞分别以1×10^5个的细胞密度接种在具有载玻片的6孔烧瓶中，在37℃，5％CO 2和90％相对湿度下培养过夜。第二天，将烧瓶在PBS中洗涤两次，之后在100μg/ml浓度的nABP296肽中于4℃孵育30分钟。之后，用PBS洗涤去除未结合的肽，并在4％PFA中固定15分钟。用5％牛血清白蛋白(BSA，广州祥博生物技术有限公司)在室温下封闭1小时。将细胞与CD105抗体(1:200；Invitrogen，Waltham，MA，USA)一起在4℃孵育过夜。未结合的肽和抗体通过PBS洗脱3次。在室温下用含Alexa Fluor 568(1：1000；Invitrogen，Waltham，MA，USA)标记的驴抗小鼠IgG(H+L)二抗的封闭缓冲液进一步处理细胞1小时。之后用PBS洗涤，用DAPI染色细胞核，并在激光共聚焦扫描显微镜(CLSM)(Leica，德国)下观察结果。
图2中，D图为MNNG和Cal27中nABP296的免疫荧光分析。结果显示nABP296可以与MNNG中的CD105抗体共定位，CD105和nABP296都不能与Cal27结合。从免疫荧光测定结果中可见，nABP296与MNNG细胞系中的CD105抗体共定位，并且对Cal27没有结合性(图2D)。
实施例6 ELISA测定nABP296肽的特异性结合
将nABP296与CD105蛋白进行ELISA结合测定，并使用CD106蛋白作为对照。将溶于无菌PBS中的浓度分别为100μg/ml的重组人CD105蛋白和CD106蛋白分别加入到6个独立的灭菌的MaxiSorp平板中，并在4℃孵育过夜。用0.1％TBST洗涤平板6次，并使用含1％BSA的PBS在4℃封闭1小时。之后用浓度100μg/ml的nABP296肽在室温下孵育1小时。未结合的肽通过PBS洗涤10次去除。使用Victor X5(PerkinElmer，Singapore)在490nm处测量吸光度(OD)。
图2中E图为nABP296对CD105蛋白和CD106蛋白的结合亲和力 的ELISA测定，其中CD106蛋白被用作对照，图中可见CD105蛋白组OD值高于CD106蛋白组。从实验结果可见，nABP296对CD105蛋白的亲和力高于对照蛋白(图2E)。
实施例7细胞毒性实验
将MNNG细胞以1×10^5个的细胞密度接种在96孔板的100μl完全培养基。在37℃，5％CO 2气氛下培养过夜后，将细胞在0.5μMol至5μMol的不同浓度nABP296溶液中孵育。在24小时和48小时，向细胞中加入10μl细胞计数试剂盒-8(CCK-8，Dojindo，Japan)溶液并再培养1小时。使用VICTOR TM X5Multilabel Plate Reader(PerkinElmer，Singapore)在450nm测定吸光度，计算存活率。
图2中图F为在24小时和48小时内nABP296对MNNG的细胞毒性测定。结果显示nABP296对MNNG细胞系没有细胞毒性(p>0.05)。
由以上实施例的结果可见，nABP296可选择性结合CD105阳性细胞系MNNG，并与MNNG细胞系生物相容，可推测nABP296具备在体内靶向人骨肉瘤的潜力。
实施例8体外结合实验
骨肉瘤和舌癌组织来源于中山大学光华口腔医学院手术后患者。使用异种移植肿瘤模型、骨肉瘤患者和舌癌患者的肿瘤切片进行体外结合实验。使用低温恒温切片机(HM560，德国MICROM)切割肿瘤。所有患者在组织收集前都提供了知情同意书，所有患者都同意在科学研究中使用他们的样本。本研究经中山大学口腔医学院伦理委员会批准，批准文号是ERC-2017-11。
组织切片用5％牛血清白蛋白(BSA，广州祥博生物技术有限公司)在室温下封闭非特异性结合位点1小时，然后使用分别含50μM的nABP296肽和CD105抗体(小鼠单克隆；1：200；Abcam，Cambridge，UK)的封闭缓冲液于4℃孵育过夜。未结合的肽和抗体通过PBS洗脱3次。用Alexa Fluor 568(1：1000；Invitrogen，Waltham，MA， USA)标记的驴抗小鼠IgG(H+L)二抗在室温下进一步处理细胞1小时。再用PBS洗涤后，用DAPI染色细胞核，并在CLSM(Leica，德国)下分析结果。
图4中，A图为nABP296与源自MNNG荷瘤小鼠的肿瘤切片中的CD105抗体共定位；B图为来自骨肉瘤患者的肿瘤组织切片，可见骨肉瘤切片可以用nABP296肽和CD105抗体标记。C图为来自舌癌患者的肿瘤组织切片，可见该切片中没有CD105抗体荧光但是有微弱nABP296荧光。
以上结果表明，nABP296可以结合来自异种移植肿瘤模型和患者体外的骨肉瘤。因而，nABP296可替代CD105用于诊断和标记骨肉瘤。
实施例9体内结合实验
本实验的动物使用方案由中山大学动物伦理和福利委员会审查和批准。将悬浮于DF中的MNNG细胞以细胞密度1×10^7/ml皮下移植到5周龄BALB/c NOD小鼠中。当异种移植肿瘤的体积达到大约200mm 3时，将溶于100μl无菌Milli-Q水中的0.1μM nABP296静脉施用给小鼠。使用IVIS光谱仪在体内成像系统中观察小鼠。在1小时内拍摄照片(PerkinELmer，Akron，Ohio，USA)。小鼠在1.5小时内被安乐死，从携带MNNG肿瘤的小鼠中切下肿瘤，并使用IVIS光谱仪在体内成像系统中成像。将器官冷冻切片，并通过DAPI对细胞核进行染色。结果由CLSM(Leica，德国)分析。
图5中A图为在静脉内施用nABP296肽1小时后MNNG荷瘤小鼠的体内成像，可见在肝脏，肾脏和静脉造影的肿瘤区域可以检测到荧光。在肿瘤中检测到的荧光高于周围组织。NC组(NC组是注射100μl无菌生理盐水的对照组)未检测到荧光。B图为在1.5小时时从携带MNNG肿瘤的小鼠切除的肿瘤的体外成像，可见FITC标记的nABP296在异种移植肿瘤中累积(图5B)。C图为从MNNG荷瘤小鼠切除的器官的冷冻切片，在肿瘤组织切片中检测到荧光，并且心脏切片 中没有FITC荧光(图5C)。这些结果进一步证明了nABP296肽在MNNG异种移植肿瘤体内的高亲和力和特异性。
由以上实验可见，本发明中，发明人发现了一种12个氨基酸的肽nABP296(序列见序列表的序列1)，可特异性结合重组人CD105蛋白。CD105是用于MSC(间充质干细胞)分离和富集的生物标志物。使用肽nABP296可替代CD105抗体分离CD105+表型细胞，例如CD105+表型MSC(间充质干细胞)。本发明所提供的nABP296肽相对较短，分子量小，且可特异性结合CD105阳性细胞系MNNG。nABP296具有特异性亲和性和良好生物相容性，可以解决体内使用CD105抗体所存在问题。nABP296还可作为从组织中分离CD105阳性细胞例如MSC的新型探针。
血管生成是组织中新血管形成的过程，对于肿瘤生长和转移是必需的。CD105在血管生成中起关键作用。因此，CD105在活跃增殖的肿瘤中过表达。CD105的体内靶向可用于肿瘤PET成像和可视化。而本发明的nABP296肽可以在MNNG荷瘤小鼠中定位骨肉瘤(图5)。nABP296肽是一种相对较短的合成的体外人造肽，它没有动物异源形式或免疫反应。nABP296对细胞无毒，因此可以安全地用于人体。nABP296可被荧光激活，因此可方便的应用于检测CD105阳性肿瘤细胞及具有CD105阳性表达的肿瘤新生血管。由于其优点，nABP296肽在成为患者骨肉瘤等具备CD105阳性表达特征的肿瘤转移早期诊断和追踪方面具备应用潜力。
本发明的nABP296肽为一种非抗体结合肽，仅由12个氨基酸组成。对肿瘤微环境的穿透能力优于抗体。由于有效的穿透能力和靶向作用，该多肽可被用作抗肿瘤治疗药物或抗肿瘤新生血管药的运载工具。在免疫荧光实验中，我们在细胞质中发现了nABP296。因此，可推断nABP296可以穿透细胞膜。因此，nABP296可以与抗肿瘤药物或其他治疗剂形成缀合物，用作靶向CD105蛋白质将抗肿瘤药物或治疗剂转移至CD105阳性肿瘤细胞的靶向工具。
综上，本发明所提供的新型肽nABP296能特异性结合重组人CD105 蛋白，不仅可在体外标记来自患者的骨肉瘤切片和MNNG异种移植肿瘤模型，亦可在在体内标记MNNG异种移植肿瘤模型。因而，该肽可用于体内诊断和肿瘤(例如CD105阳性表达的骨肉瘤等)成像；nABP296还可应用于MSC分离和CD105靶向治疗。
本领域技术人员可以理解，在本说明书的教导之下，可对本发明做出一些修改或调整。这些修改或调整也应当在本发明权利要求所限定的范围之内。
Figure PCTCN2018103544-appb-000005

Claims (12)

  1. 一种与CD105特异性结合的多肽，其特征在于，所述多肽的氨基酸序列如序列1所示。
  2. 一种多核苷酸，其特征在于，其包含编码如权利要求1所述的多肽的核苷酸序列。
  3. 一种载体，其特征在于，其包含权利要求2所述的多核苷酸。
  4. 一种宿主细胞，其特征在于，其包括权利要求3所述的载体或权利要求2所述的多核苷酸。
  5. 一种药物制剂，其特征在于，所述药物制剂包括如下组分：权利要求1所述的多肽与治疗药物分子和/或可被检测的标记连接而成的化合物；
    优选的，所述可被检测的标记包括荧光试剂、核素或放射试剂中的一种或多种；
    优选的，所述治疗药物分子为抗癌剂或抗肿瘤新生血管药。
  6. 根据权利要求5所述的药物制剂，其特征在于，所述抗癌剂为对CD105蛋白阳性表达特征的肿瘤具有抗癌活性的药物，所述抗肿瘤新生血管药为对肿瘤血管新生具有抑制作用的药物，所述肿瘤血管新生所涉及的肿瘤的新生血管具有CD105蛋白阳性表达特征；
    优选的，所述CD105蛋白阳性表达特征的肿瘤为骨肉瘤、血管肉瘤或白血病。
  7. 根据权利要求5或6所述的药物制剂，其特征在于，还包括药学上允许的载体或辅料。
  8. 权利要求1所述的多肽或权利要求5-7任一项所述的药物制剂在制备用于肿瘤成像或肿瘤新生血管成像的试剂中的应用，所述肿瘤或肿瘤新生血管具有CD105阳性表达特征。
  9. 权利要求1所述的多肽或权利要求5-7任一项所述的药物制剂在制备用于抑制或阻止肿瘤血管新生或肿瘤细胞生长的药物 中的用途，所述肿瘤或肿瘤的新生血管具有CD105阳性表达特征。
  10. 权利要求1所述的多肽或权利要求5-7任一项所述的药物制剂的应用，其特征在于，在制备用于诊断、预防或治疗肿瘤或肿瘤新生血管的试剂中应用，所述肿瘤或肿瘤的新生血管具有CD105阳性表达特征。
  11. 权利要求1所述的多肽的应用，其特征在于，在制备用于标记、鉴定、富集、分选或纯化CD105阳性细胞的制剂中应用；
    或，所述多肽在CD105阳性细胞的标记、鉴定、富集、分选或纯化方法中应用。
  12. 权利要求1所述的多肽或权利要求5-7任一项所述的药物制剂在制备用于靶向CD105阳性细胞的试剂中应用。
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