CN108912212A - A kind of polypeptide and its application with CD105 specific binding - Google Patents
A kind of polypeptide and its application with CD105 specific binding Download PDFInfo
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- 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
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Abstract
The present invention provides polypeptide and its application that a kind of pair of involucrin (Endoglin/CD105) has specific binding, peptide (nABP296) can specifically bind recombined human CD105 albumen, the peptide molecular weight is small, with good biocompatibility, it is safe and non-toxic, deficiency present in CD105 antibody can be made up.The alternative CD105 antibody of polypeptide provided by the invention is applied, such as it can be used for preparing the probe that CD105 positive cell is separated from tissue, such as osteosarcoma cell, vascular endothelial cell or mescenchymal stem cell etc., it can be also used for preparation target tumor imaging or visual preparation in vivo or in vitro, can be labeled and be applied to detection tumour cell.Polypeptide nABP296 provided by the invention is a kind of non-antibody binding proteins, which can be used as the delivery vehicle of antineoplaston drug.The polypeptide and anti-tumor drug or other reagents are formed into conjugate, CD105 albumen can be targeted, anti-tumor drug or other reagents orientation are transferred to CD105 positive tumor cell.
Description
Technical field
The present invention relates to a kind of non-antibody binding peptides, and in particular to a kind of special with involucrin (Endoglin/CD105)
Property combine polypeptide and its application.
Background technique
CD105 is also referred to as Endoglin, is the I type membrane glycoprotein that TGF-β receptor complex is formed on cell membrane.
The 180kDa homodimer transmembrane protein that CD105 is made of 633 amino acid.CD105 is hydrophobic by big extracellular domain
Property transmembrane domain and short intracellular domain composition.CD105 outer domains are with high-affinity combination TGF-β -1 and TGF-β -
3, form TGF-β receptor complex.It is adjusting cell Proliferation, breaks up, migration, plays an important role in adherency and angiogenesis,
This survives and shifts most important for tumour growth.CD105 is mainly in mescenchymal stem cell (MSC), tumor neovasculature
Endothelial cell, the tumor cells expressions such as osteosarcoma, angiosarcoma and leukaemia.CD105 antibody is used for detecting tumor marked micro- blood
Pipe density separates MSC and the conjugated drug for targeted therapy.Osteosarcoma is one of most common primary malignant tumor, main needle
To elongated cylindrical bone.It is reported that about 5% osteosarcoma occurs in Maxillary region.It occurs in teenager and children, annual to send out
Patient's number is about ten thousand people of 3-450, and can actively be transferred to lung in early stage.In a research, it was reported that some
Osteosarcoma cell expresses mescenchymal stem cell marker (CD105), more more aggressive than other osteosarcoma cell lines.Therefore,
CD105 antibody may be the marker for identifying osteosarcoma grade malignancy.
But the complexity of antibody producing, thermal instability and it is poor to the infiltration of cancer microenvironment the defects of limit
The application of antibody in vivo.Non-antibody binding proteins (nABP) are artificial synthesized thermostabilization peptides.The peptide is to tumor microenvironment
Osmosis is effective, because its very little and having positive charge.The advantages of due to this peptide, it has caused huge pass
Note, and have been used in various antitumor researchs and neoplasm targeted therapy.
Summary of the invention
It is provided the purpose of the present invention is to provide polypeptide and its application that a kind of couple of CD105 has specific binding
Polypeptide is a kind of non-antibody binding proteins.
The present invention is to reach its purpose, and the technical solutions adopted are as follows：
First aspect present invention provides a kind of polypeptide with involucrin (Endoglin/CD105) specific binding, described
The amino acid sequence of polypeptide is as shown in sequence 1.
The present invention also provides a kind of polynucleotides, and it includes the nucleotide sequences of the coding such as polypeptide of sequence 1 above.
The present invention also provides a kind of carriers, and it includes polynucleotides described above.
The present invention also provides a kind of host cells comprising carrier described above or the polynucleotides.
The present invention also provides a kind of pharmaceutical preparation, the pharmaceutical preparation includes following component：Polypeptide described above with control
The compound that treatment drug molecule and/or the label that can be detected are formed by connecting；The polypeptide can be with therapeutic agent molecule and/or label
Form conjugate.The connection of the two is also possible to covalent bond or non-covalent bond mode connection.
Preferably, the label being detected includes one of fluorescent reagent, nucleic or radiation reagent or a variety of；This
A little labels can specifically use respective markers object known in the art, such as fluorescent reagent rhodamine B, FITC (the different sulphur of 5-
Cyanic acid fluorescein) etc..
The pharmaceutical preparation, wherein the anticancer agent is to have anticancer to the tumour of CD105 protein positive expression feature
Activity, the anti-tumor neovascularization medicine are the drug inhibited to neonate tumour blood vessel；The specific such as adriamycin of drug,
Taxol, cis-platinum and cyclophosphamide etc..
Preferably, the tumour both can be swollen for osteosarcoma, angiosarcoma and leukaemia of CD105 protein positive expression etc.
Oncocyte, or CD105 protein negative expresses tumour, but its tumor neogenetic blood vessels expresses CD105 albumen.
The pharmaceutical preparation can may include the carrier pharmaceutically allowed or auxiliary as needed in practical applications
Material, those skilled in the art can be specifically chosen required carrier or auxiliary material as needed, such as can be normal for pharmaceutical field
Diluent, excipient, adhesive, wetting agent, disintegrating agent, sorbefacient, surfactant, absorption carrier and lubricant
Etc., this is not repeated.The dosage form of pharmaceutical preparation is not particularly limited, according to specific needs the dosage form needed for selecting,
Such as, but not limited to capsule, soft capsule, tablet, oral solution, dispersible tablet, pulvis, injection or dripping pill etc..
The present invention also provides a kind of application, the polypeptide or the pharmaceutical preparation for tumor imaging or swell in preparation
Application in the reagent of tumor new vessels imaging, the tumour and tumor neogenetic blood vessels are CD105 positive expression.Tumor imaging
Or tumor neogenetic blood vessels imaging includes but is not limited to positron emission tomography art (PET), single photon emission computerized tomgraphy art
(SPECT), near-infrared (NIR) optical imagery or magnetic resonance imaging (MRI).The tumour is such as, but not limited to osteosarcoma, blood vessel
Sarcoma or leukaemia etc..
The present invention also provides another kinds to apply, and the polypeptide or the pharmaceutical preparation are being prepared for inhibiting or preventing
Purposes in the drug of growth of tumour cell, the tumour cell are the cancer cell of CD105 positive expression, such as, but not limited to bone
Sarcoma, angiosarcoma or leukaemia etc..
The present invention also provides another kinds to apply, and the polypeptide or the pharmaceutical preparation are being prepared for inhibiting or preventing
Purposes in the drug of neonate tumour blood vessel, it is thin which is formed by tumor neovasculature tumor vascular endothelium
Born of the same parents have CD105 positive expression feature.
The present invention also provides another kinds to apply, and the polypeptide or the pharmaceutical preparation are being prepared for diagnosing, preventing
Or applied in treatment tumour or tumor neovasculature reagent, the tumour or tumor neogenetic blood vessels are CD105 positive expression.Institute
It states tumour and is such as, but not limited to osteosarcoma, angiosarcoma or leukaemia etc..
The present invention also provides another kinds to apply, and the polypeptide is in preparation for marking, identifying, be enriched with, sort or purify
It is applied in the preparation of CD105 positive cell；Or, the polypeptide is in the label of CD105 positive cell, identification, enrichment, sorting or pure
It is applied in change method.CD105 positive cell is such as, but not limited to osteosarcoma cell, vascular endothelial cell or mescenchymal stem cell
Deng.
The present invention also provides another kinds to apply, and the polypeptide or the pharmaceutical preparation are being prepared for targeting CD105
It is applied in the reagent of positive cell.
Present inventor identifies 13 kinds in conjunction with CD105 in the course of the research, using M13 phage display library
New peptide.In 13 kinds of peptides, nABPK296 peptide (sequence is as shown in the sequence 1 of sequence table) is to CD105 positive osteosarcoma cell line
MNNG has affinity more higher than other peptides, and has lower affinity to CD105 negative cells system Cal27,
NABPK296 peptide can specificity combination CD105 positive cell.It is swollen that nABPK296 can show the MNNG in MNNG tumor-bearing mice
Tumor.In addition, inventor has found that nABP296 can be marked from animal MNNG Xenograft Tumor Models and osteosarcoma under study for action
The osteosarcoma tissue of patient is sliced.
Technical solution provided by the invention has the advantages that：
Polypeptide (nABP296) provided by the invention can specifically bind recombined human CD105 albumen, which has molecular weight
It is small, the characteristics of CD105 positive cell can be specifically bound, while also there is good biocompatibility, and it is safe and non-toxic, it can be more
Mend deficiency present in CD105 antibody.The alternative CD105 antibody of polypeptide provided by the invention is applied, such as can be used for
The probe for separating CD105 positive cell from tissue, such as osteosarcoma cell or MSC etc. are prepared, can be also used for preparation in body
Interior or targeting tumor imaging or visual preparation can be labeled and be applied to detection tumour cell.
Polypeptide nABP296 provided by the invention is a kind of non-antibody binding proteins, is only made of 12 amino acid, to tumour
The penetration capacity of microenvironment is better than antibody, and due to effective penetration capacity and targeting, which can be used as antineoplaston
The delivery vehicle of drug.The polypeptide and anti-tumor drug or other reagents are formed into conjugate, CD105 albumen can be targeted, will be resisted
Tumour medicine or other reagents orientation are transferred to CD105 positive tumor cell.
Detailed description of the invention
In Fig. 1:A figure is the semi-quantitative RT-PCR analysis of CD105 level in MNNG and Cal27 cell line.B figure be MNNG and
The immunofluorescence analysis that CD105 is expressed in Cal27 cell line.C figure is in flow cytometry MNNG and Cal27 cell line
CD105 expression.
In Fig. 2：A figure is flow cytometry of 13 kinds of peptides to the binding affinity of MNNG cell line.B figure is in figure A
The average fluorescent strength (MFI) of flow cytometry is analyzed.C figure is nABP296 and nABP297 is thin to the streaming of MNNG and Cal27
The analysis of born of the same parents' art.D figure be MNNG and Cal27 in nABP296 immunofluorescence analysis.E figure be nABP296 to CD105 albumen and
The ELISA of the binding affinity of CD106 albumen is measured, and wherein CD106 albumen is used as compareing.F figure is small 24 hours and 48
When interior nABP296 to the cytotoxicity assay of MNNG.
Fig. 3 is the chemical structure of nABP296.
Fig. 4 is external visualization measurement.Wherein：A figure is nABP296 and is originated from the tumor biopsy of MNNG tumor-bearing mice
CD105 antibody common location.B figure is the tumor tissue section from Patients with Osteosarcoma.C figure is the tumor tissues from Tongue Cancer Patients
Slice.
Fig. 5 is the internal and in vitro imaging of MNNG tumor-bearing mice.Wherein, A figure is small in intravenously application nABP296 peptide 1
When after MNNG tumor-bearing mice in-vivo imaging.B figure is the body of the tumour cut off at 1.5 hours from the mouse for carrying MNNG tumour
Outer imaging.C figure is the frozen section of the organ cut off from MNNG tumor-bearing mice.
Specific embodiment
For a better understanding of the technical solution of the present invention, below with reference to the embodiment content that the present invention is further explained,
But the contents of the present invention are not limited only to following embodiment.
Used main approaches include phage display library biopanning, ELISA measurement, polypeptide in embodiment
Synthesis, RT-PCR, flow cytometry, immunofluorescence analysis, cytotoxicity experiment, cell proliferation experiment, mouse living imaging etc.,
Experimental implementation is that those skilled in the art's routine experiment operation grasped or the experiment carried out according to product description are grasped
Make, does not repeat one by one.It is the conventional reagent that commercial channel purchase obtains if agents useful for same is not specified.It is related
It is mass percent if the percentage of solution is not specified.
Statistical analysis is carried out using 5 software of GraphPad Prism, P value<0.05 is considered significant.
The screening and synthesis of embodiment 1, CD105 non-antibody binding proteins
It mainly include library screening, ELISA experiment and sequencing analysis.
1, phage display peptide library biopanning：
Phage display peptide library biopanning is carried out according to standardization program.
Using business building phage display peptide library (Ph.D.-12 phage display library, NEB, Beverly, MA,
USA) carry out following experiment.
By be dissolved in 100 μ g/ml of the concentration in sterile PBS recombined human CD105 albumen (R&DSystems, Minnesota,
USA it) is added to the MaxiSorp plate (Thermo Fisher Scientific, MA, USA) of sterilizing, is incubated overnight at 4 DEG C.
It is closed 1 hour with TBST (0.1% tween) washing flat board 6 times, and using the PBS containing 1%BSA at 4 DEG C.By M13 phage display technology
Show that peptide library combines 1 hour at room temperature, and unbonded bacteriophage is washed 10 times with TBST (0.1% tween).In conjunction with phagocytosis
Body is eluted with elution buffer, and the bacteriophage of elution is amplified for next round biopanning.So washed in a pan altogether by three-wheel biology
After choosing, target M13 bacteriophage is enriched with.Table 1 list three-wheel biopanning as a result, after three-wheel biopanning, counterweight
There is group people CD105 albumen the M13 bacteriophage of high-affinity to be enriched with.
The rate of recovery of the every wheel biopanning of table 1
Seen from table 1, after three-wheel biopanning, the rate of recovery of M13 bacteriophage is from 4.9 × 10-6Rise to 4.5 × 10-5.It has been enriched in conjunction with the M13 bacteriophage of recombined human CD105 albumen, has been used for subsequent experimental.
2, Phage-ELISA binding analysis and sequencing
In order to identify the monoclonal in combination with CD105, ELISA measurement has been carried out to M13 bacteriophage monoclonal.Random selection
16 monoclonal phages obtained after elutriation are used for ELISA binding analysis.By the nothing of the recombined human CD105 albumen of 100 μ g/ml
Bacterium PBS liquid is added in the MaxiSorp plate of 16 independent sterilizings, and is incubated overnight at 4 DEG C.It is washed with 0.1%TBST flat
Plate 6 times, and closed 1 hour using the PBS (i.e. Block buffer) containing 1%BSA at 4 DEG C.16 monoclonal phages are carried out
It expands and is added in plate, be incubated at room temperature 1 hour.After 0.1%TBST repeatedly washing flat board, by with rabbit-anti M13
The goat anti-rabbit igg (1 of phage antibody and HRP conjugation:20；Abcam, Cambridge, UK) it is incubated with to detect combination
Bacteriophage.Use ABTS/H2O2Substrate measures the amount of the HRP of combination, monitors absorbance at 405nm.Use primer 5'-
CCCTCATAGTTAGCGTAACG-3'(NEB, Beverly, MA, USA) measure institute in 13 positive compatibility monoclonal phages
The DNA sequence dna of insertion.
Combination activity of 16 kinds of monoclonal M13 bacteriophages to people's recombinant C D105 albumen and NC (Block buffer, control group)
ELISA measurement result be shown in Table 2, A-P successively represents the correspondence experimental result of 16 kinds of monoclonal M13 bacteriophages in table.
The associativity (light absorption value) of 2 M13 bacteriophage of table and CD105 albumen
As can be found from Table 2, there is compatibility of 13 kinds of monoclonal M13 bacteriophages in experimental group with CD105 albumen, compare
For the Block buffer of control group, there is higher binding affinity, as shown in table 2, it was demonstrated that 13 monoclonal phages
There is high-affinity to CD105.
13 monoclonal phages that positive findings are presented in ELISA measurement are thus obtained after screening, table 3 is listed
The correspondence polypeptide sequence and property of positive findings, it is nABP295 to nABP307 that these polypeptides are numbered respectively.
The property of 3 polypeptide of table
2 Peptide systhesis of embodiment
13 polypeptides obtained by commercial company (Shanghai Qiangyao Biotechnology Co., Ltd.) synthetic example 1, more
N-terminal connection FITC (5-isothiocyanate fluorescein) of peptide is for marking.By the standby peptide of mass spectrum (MS) system of identification, and pass through
High performance liquid chromatography (HPLC) measurement purity (>95%).The chemical structure of nABP296 is shown in Fig. 3.
The peptide of FITC label is stored in -20 DEG C.When in use, peptide is dissolved in sterile water with 1mg/kg and is wanted according to experiment
It asks and is diluted to required concentration.
Embodiment 3 is used to combine the determination of the cell line of identification
MNNG and Cal27 cell line is obtained from Chinese Shanghai Chinese Academy of Sciences Type Tissue Collection.With DF (Sigma,
USA)+5% fetal calf serum (FBS) cultivates MNNG cell, cultivates Cal27 cell with DMEN (Sigma, USA)+10%FBS.It will be thin
Born of the same parents are inoculated in 10cm2Flask, at 37 DEG C, 5%CO2It is cultivated in 90% relative humidity conditions.
By RT-PCR, the CD105 in immunofluorescence and Flow cytometry MNNG and Cal27 is expressed, wherein using
CD105 primer be (F：CACCACAGCGGAAAAAGGTG；R：GCCGGTTTTGGGTATGGGTA)(Synbio
Technologies, Suzhou, China)；CD105 antibody used in immunofluorescence experiment is (1:200；Invitrogen,
Waltham, MA, USA)；CD105 antibody used in flow cytometry tests is (MACS, Bergisch Gladbach, moral
State).
In RT-PCR experiment, 300ng total serum IgE is extracted respectively from tumour cell MNNG and Cal27, and few using TaKaRa
Poly- (dT) primer (Takara Bio, Japan) and reverse transcriptase (Toyobo Life Science, Japan) are translated into mutually
It mends DNA and is used for PCR amplification.CD105 primer used in PCR amplification is (F：CACCACAGCGGAAAAAGGTG；R：
GCCGGTTTTGGGTATGGGTA) (Suzhou Hong Xun Biotechnology Co., Ltd)；PCR reaction condition is：95 DEG C 5 minutes, 95 DEG C
30 seconds, 30 circulation, 60 DEG C 30 seconds, 72 DEG C 1 minute, 72 DEG C extend 10 minutes, be finally maintained at 16 DEG C.It is solidifying in 1% agarose
Electrophoresis detection PCR product on glue, under uv illumination ethidium bromide (EB) dyeing show.
In immunofluorescence experiment, MNNG and Cal27 is seeded in 1 × 10^5 cell densities with glass slide respectively
6 hole culture bottles in, and at 37 DEG C, 5%CO2With overnight incubation under 90% relative humidity.Second day, by cell in 4%PFA
Fix 15 minutes.It is closed 1 hour at room temperature with 5% bovine serum albumin(BSA) (BSA, Guangzhou Xiang Bo Bioisystech Co., Ltd).
By cell and CD105 antibody (1:200；Invitrogen, Waltham, MA, USA) it is incubated overnight together at 4 DEG C.Unbonded
Antibody elutes 3 removals by PBS.Fluor568 containing Alexa (1 is used at room temperature：1000；Invitrogen, Waltham,
MA, USA) Block buffer of donkey anti-mouse IgG (H+L) secondary antibody of label is further processed cell 1 hour.It is washed later with PBS
It washs, is redyed nucleus using DAPI, observed under laser confocal scanning microscope (CLSM) (Leica, Germany).
In flow cytometry tests, by MNNG and Cal27 cell respectively in CD105 antibody (1:10, MACS, Germany) in
4 DEG C are incubated for 10 minutes.Cell precipitation is washed 3 times with PBS, cell precipitation is suspended in 500 μ l PBS and is used
FACSCalibur (BD, USA) analysis.
In Fig. 1, A figure is the semi-quantitative RT-PCR analysis of CD105 level in MNNG and Cal27 cell line as a result, in MNNG
In have CD105 cDNA express, do not have in Cal27 CD105 cDNA express.B figure is in MNNG and Cal27 cell line
The immunofluorescence analysis result of CD105 expression；As shown in red fluorescence, CD105 is located at the surface of MNNG cell line.In Cal27
There is no CD105 fluorescence in cell line.C figure is the CD105 expression in flow cytometry MNNG and Cal27 cell line.
According to RT-PCR, the result (Fig. 1) of immunofluorescence and flow cytometry, it is seen that MNNG height expresses CD105 albumen,
And Cal27 is not then, thus MNNG cell line is the CD105 positive, Cal27 cell line is CD105 feminine gender.It will be used below
The two cell lines are tested to identify the peptide for having high binding affinity to the CD105 of endoglin expression.
4 flow cytometry of embodiment identifies the associativity of two kinds of peptides
By the experimental result of embodiment 3, the external combination that polypeptide is analyzed using CD105 positive cell line MNNG is determined
Property.
Resulting 13 kinds of peptides are screened before use, and the detection of peptide associativity is carried out by flow cytometry.By MNNG cell with 5
×105A cell density is incubated for 30 minutes in the peptide of 100 μ l concentration, 100 μ g/ml in 4 DEG C.Cell precipitation is washed with PBS
3 times.Cell precipitation is suspended in 500 μ l PBS and is tested and analyzed using FACSCalibur (BD, USA).
In Fig. 2, A figure is flow cytometry of 13 kinds of peptides to the binding affinity of MNNG cell line, should the result shows that
NABP296 and nABP297 peptide has higher binding affinity to MNNG cell line；B figure is to scheme flow cytometry in A to be averaged
Fluorescence intensity (MFI) analysis, the results showed that nABP296 and nABP297 MFI with higher.By the above flow cytometry results
As it can be seen that two kinds of peptide nABP296 and nABP297 to CD105 positive cell line MNNG have high-affinity, affinity be higher than other 11
Kind peptide (result is shown in Fig. 2A, B).
In order to determine whether both peptides nABP296 and nABP297 have specific affinity to CD105, inventor makes
The specific binding of nABP296 and nABP297 peptide Yu CD105 albumen is analyzed by flow cytometry with MNNG and Cal27.It is real
It is as follows to test process：By MNNG and Cal27 cell with 1 × 10 in nABP296 the and nABP297 peptide that concentration is respectively 100 μ g/ml
The cell density of ^6 is incubated for 30 minutes in 4 DEG C；Cell precipitation is washed 3 times with PBS；Cell precipitation is suspended in 500 μ l PBS
In and using FACSCalibur (BD, USA) analyze.
In Fig. 2, C figure is nABP296 and nABP297 is to the flow cytometry of MNNG and Cal27, in this result
In, nABP297 has higher combination affine MNNG to affinity difference, nABP296 is not bound between MNNG and Cal27
Power is then lower to the affinity of Cal27.According to Flow Cytometry Assay result as it can be seen that between nABP296 and MNNG have than
The higher joint efficiency of Cal27, and the joint efficiency of nABP297 and MNNG or Cal27 does not have difference.
The binding specificity of 5 Immunofluorescence test nABP296 peptide of embodiment
MNNG and Cal27 cell is seeded in the 6 hole flasks with glass slide respectively with 1 × 10^5 cell densities
In, at 37 DEG C, 5%CO2With overnight incubation under 90% relative humidity.Second day, flask is washed twice in PBS, Zhi Hou
It is incubated for 30 minutes in the nABP296 peptide of 100 μ g/ml concentration in 4 DEG C.Later, the unbonded peptide of removal is washed with PBS, and 4%
15 minutes are fixed in PFA.1 is closed at room temperature with 5% bovine serum albumin(BSA) (BSA, Guangzhou Xiang Bo Bioisystech Co., Ltd)
Hour.By cell and CD105 antibody (1:200；Invitrogen, Waltham, MA, USA) it is incubated overnight together at 4 DEG C.It does not tie
The peptide and antibody of conjunction are eluted 3 times by PBS.Fluor containing Alexa 568 (1 is used at room temperature：1000；Invitrogen,
Waltham, MA, USA) Block buffer of donkey anti-mouse IgG (H+L) secondary antibody of label is further processed cell 1 hour.Later
It is washed with PBS, observes knot with DAPI staining cell core, and under laser confocal scanning microscope (CLSM) (Leica, Germany)
Fruit.
In Fig. 2, D figure be MNNG and Cal27 in nABP296 immunofluorescence analysis.NABP296 can be with as the result is shown
CD105 antibody common location in MNNG, CD105 and nABP296 cannot be in conjunction with Cal27.From immunofluorescence assay result
As it can be seen that the CD105 antibody common location in nABP296 and MNNG cell line, and to being not bound with property of Cal27 (Fig. 2 D).
The specific binding of embodiment 6ELISA measurement nABP296 peptide
NABP296 and CD105 albumen are subjected to ELISA binding assay, and use CD106 albumen as control.It will be dissolved in
Concentration in sterile PBS is respectively the recombined human CD105 albumen of 100 μ g/ml and CD106 albumen be added separately to 6 it is independent
In the MaxiSorp plate of sterilizing, and it is incubated overnight at 4 DEG C.With 0.1%TBST washing flat board 6 times, and using containing 1%BSA's
PBS is closed 1 hour at 4 DEG C.It is incubated at room temperature 1 hour with the nABP296 peptide of 100 μ g/ml of concentration later.Unbonded peptide is logical
It crosses PBS and washs 10 removals.Absorbance is measured at 490nm using Victor X5 (PerkinElmer, Singapore)
(OD)。
E figure is ELISA measurement of the nABP296 to the binding affinity of CD105 albumen and CD106 albumen in Fig. 2, wherein
CD106 albumen is used as compareing, and visible CD105 protein groups OD value is higher than CD106 protein groups in figure.From experimental result as it can be seen that
NABP296 is higher than reference protein (Fig. 2 E) to the affinity of CD105 albumen.
7 cytotoxicity experiment of embodiment
MNNG cell is seeded in 100 μ l complete mediums of 96 orifice plates with 1 × 10^5 cell densities.At 37 DEG C,
5%CO2Under atmosphere after overnight incubation, cell is incubated in the various concentration nABP296 solution of 0.5 μM of ol to 5 μM of ol.24
Hour and 48 hours, -8 (CCK-8, Dojindo, Japan) solution of 10 μ l Cell counting Kit is added into cell and trains again
It supports 1 hour.Using VICTOR TM X5Multilabel Plate Reader (PerkinElmer, Singapore) in 450nm
Absorbance is measured, survival rate is calculated.
It is cytotoxicity assay of the nABP296 to MNNG in 24 hours and 48 hours that F is schemed in Fig. 2.As the result is shown
NABP296 does not have cytotoxicity (p to MNNG cell line>0.05).
By the result of above embodiments as it can be seen that nABP296 alternative combination CD105 positive cell line MNNG, and with
MNNG cell line bio-compatible can speculate that nABP296 has the potentiality for targeting human osteosarcoma in vivo.
8 vitro binding assay of embodiment
Osteosarcoma and tongue cancer derive from Zhongshan University's brilliance School of Stomatology postoperative patients.It is swollen using heterograft
The tumor biopsy of tumor model, Patients with Osteosarcoma and Tongue Cancer Patients carries out vitro binding assay.Use cryostat slicer
(HM560, German MICROM) cuts tumour.All patients both provide informed consent form before tissue collecting, and all patients are
With the sample being intended in scientific research using them.This research is ratified through School of Stomatology Ethics Committee of Zhongshan University, approval
Code is ERC-2017-11.
Histotomy is closed non-at room temperature with 5% bovine serum albumin(BSA) (BSA, Guangzhou Xiang Bo Bioisystech Co., Ltd)
Specific binding site 1 hour, then using the nABP296 peptide and CD105 antibody (mouse monoclonal for containing 50 μM respectively；1：
200；Abcam, Cambridge, UK) Block buffer in 4 DEG C be incubated overnight.Unbonded peptide and antibody passes through PBS elution 3
It is secondary.With Alexa Fluor 568 (1：1000；Invitrogen, Waltham, MA, USA) label donkey anti-mouse IgG (H+L)
Secondary antibody is further processed cell 1 hour at room temperature.After being washed again with PBS, with DAPI staining cell core, and in CLSM
Result is analyzed under (Leica, Germany).
In Fig. 4, A figure is nABP296 and the CD105 antibody common location in the tumor biopsy from MNNG tumor-bearing mice；B figure
For the tumor tissue section from Patients with Osteosarcoma, it is seen that osteosarcoma slice can be marked with nABP296 peptide and CD105 antibody.C
Figure is the tumor tissue section from Tongue Cancer Patients, it is seen that does not have CD105 antibody fluorescence in the slice but has faint nABP296
Fluorescence.
The above result shows that nABP296 can combine the osteosarcoma external from Xenograft Tumor Models and patient.Cause
And the alternative CD105 of nABP296 is for diagnosing and marking osteosarcoma.
The experiment of 9 Binding in vivo of embodiment
The animal operational version of this experiment is examined by Zhongshan University's animal welfare and the welfare committee and approval.It will be suspended in
MNNG cell in DF is with 1 × 10^7/ml of cell density subcutaneous transplantation into 5 week old BALB/c NOD mouse.Work as heterograft
The volume of tumour reaches about 200mm3When, 0.1 μM of nABP296 intravenous administration in the sterile Milli-Q water of 100 μ l will be dissolved in
To mouse.Using observing mouse in IVIS spectrometer in vivo imaging system.In 1 hour shoot photo (PerkinELmer,
Akron, Ohio, USA).Mouse is euthanized in 1.5 hours, cuts tumour from the mouse for carrying MNNG tumour, and use
IVIS spectrometer is imaged in imaging system in vivo.Organ is sliced, and nucleus is dyed by DAPI.As a result
By CLSM (Leica, Germany) analysis.
A figure is the in-vivo imaging of the MNNG tumor-bearing mice after intravenous application nABP296 peptide 1 hour in Fig. 5, it is seen that in liver
Dirty, the tumor region of kidney and phlebography can detecte fluorescence.The projecting tissue of the fluorescence detected in tumour.NC
Fluorescence is not detected in group (NC group is to inject the control group of 100 μ l sterile salines).B figure is at 1.5 hours from carrying
The external imaging of the tumour of the mouse excision of MNNG tumour, it is seen that the nABP296 of FITC label accumulates in xenograft tumours
(Fig. 5 B).C figure is the frozen section of the organ cut off from MNNG tumor-bearing mice, and fluorescence is detected in tumor tissue section, and
And there is no FITC fluorescence (Fig. 5 C) in heart sections.It is swollen in MNNG heterograft that these results further demonstrate nABP296 peptide
The intracorporal high-affinity of tumor and specificity.
By testing above as it can be seen that in the present invention, inventor has found that (sequence is shown in by the peptide nABP296 of 12 amino acid a kind of
The sequence 1 of sequence table), recombined human CD105 albumen can be specifically bound.CD105 be for MSC (mescenchymal stem cell) separation and
The biomarker of enrichment.CD105+ phenotype cells, such as CD105+ table are separated using the alternative CD105 antibody of peptide nABP296
Type MSC (mescenchymal stem cell).NABP296 peptide provided by the present invention is relatively short, and molecular weight is small, and can specifically bind
CD105 positive cell line MNNG.NABP296 has special sexual compatibility and good biocompatibility, can solve internal use
Problem present in CD105 antibody.NABP296 is alternatively arranged as separating the novel spy of CD105 positive cell such as MSC from tissue
Needle.
Angiogenesis is the process of neovascularization in tissue, is required for growth and metastasis of tumours.CD105 is in blood
Pipe plays a crucial role in generating.Therefore, CD105 is overexpressed in the tumour of active proliferation.The internal targeting of CD105 can be used for swelling
Tumor PET imaging and visualization.And nABP296 peptide of the invention can position osteosarcoma (Fig. 5) in MNNG tumor-bearing mice.
NABP296 peptide is a kind of external artificial peptides of relatively short synthesis, it does not have animal heterogeneous format or immune response.
NABP296 can be safely used for human body to cytotoxic.NABP296 can be by fluorescence-activation, therefore can easily apply
In detection CD105 positive tumor cell and tumor neogenetic blood vessels with CD105 positive expression.Due to its advantage, nABP296 peptide
Have in terms of the metastases early diagnosis and tracking that have CD105 positive expression feature as patient's osteosarcoma etc. using latent
Power.
NABP296 peptide of the invention is a kind of non-antibody binding peptide, is only made of 12 amino acid.To tumor microenvironment
Penetration capacity is better than antibody.Due to effective penetration capacity and targeting, the polypeptide be used as antineoplaston drug or
The delivery vehicle of anti-tumor neovascularization medicine.In immunofluorescence experiment, we have found nABP296 in cytoplasm.Therefore,
Deducibility nABP296 can be with penetrating cell film.Therefore, nABP296 can be formed with anti-tumor drug or other therapeutic agents and is conjugated
Anti-tumor drug or therapeutic agent are transferred to the targeting tool of CD105 positive tumor cell as targeting CD105 protein by object.
To sum up, new type of peptides nABP296 provided by the present invention can specifically bind recombined human CD105 albumen, not only can be
Osteosarcoma slice and MNNG Xenograft Tumor Models of the labeled in vitro from patient can also be moved in label MNNG xenogenesis in vivo
Plant tumor model.Thus, which can be used for in-vivo diagnostic and osteosarcoma imaging；NABP296 apply also for MSC separation and
CD105 targeted therapy.
It will be understood by those skilled in the art that under the introduction of this specification, the present invention can be made some modifications or
Adjustment.These modifications or adjustment should also be as within the scope of the claims in the present invention.
Sequence table
<110>The Stomatologial Hospital of Zhongshan University
Guangzhou generation Pharmaceutical Technology Co., Ltd
<120>A kind of polypeptide and its application with CD105 specific binding
<130> DP1F180953GZ
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 12
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 1
Trp Ile Tyr Asp Thr Thr Arg Val Ile Val Pro Gly
1 5 10
Claims (12)
1. a kind of polypeptide with CD105 specific binding, which is characterized in that the amino acid sequence of the polypeptide is as shown in sequence 1.
2. a kind of polynucleotides, which is characterized in that it includes the nucleotide sequences for encoding polypeptide as described in claim 1.
3. a kind of carrier, which is characterized in that it includes polynucleotides as claimed in claim 2.
4. a kind of host cell, which is characterized in that it includes carrier as claimed in claim 3 or multicore as claimed in claim 2
Thuja acid.
5. a kind of pharmaceutical preparation, which is characterized in that the pharmaceutical preparation includes following component：Polypeptide described in claim 1 with
The compound that therapeutic agent molecule and/or the label that can be detected are formed by connecting；
Preferably, the label being detected includes one of fluorescent reagent, nucleic or radiation reagent or a variety of；
Preferably, the therapeutic agent molecule is anticancer agent or anti-tumor neovascularization medicine.
6. pharmaceutical preparation according to claim 5, which is characterized in that the anticancer agent is to CD105 protein positive expression
The tumour of feature has the drug of anticancer activity, and the anti-tumor neovascularization medicine is inhibited to neonate tumour blood vessel
Drug, the new vessels of tumour involved in the neonate tumour blood vessel have CD105 protein positive expression feature；
Preferably, the tumour of the CD105 protein positive expression feature is osteosarcoma, angiosarcoma or leukaemia.
7. pharmaceutical preparation according to claim 5 or 6, which is characterized in that further include the carrier or auxiliary material pharmaceutically allowed.
8. polypeptide described in claim 1 or the described in any item pharmaceutical preparations of claim 5-7 are used for tumor imaging in preparation
Or the application in the reagent of tumor neogenetic blood vessels imaging, the tumour or tumor neogenetic blood vessels have CD105 positive expression feature.
9. polypeptide described in claim 1 or the described in any item pharmaceutical preparations of claim 5-7 are in preparation for inhibiting or hindering
The only purposes in the drug of neonate tumour blood vessel or growth of tumour cell, the tumour or tumor neogenetic blood vessels have CD105 sun
Property expression characteristic.
10. the application of polypeptide described in claim 1 or the described in any item pharmaceutical preparations of claim 5-7, which is characterized in that
It is applied in preparation for diagnosing, preventing or treat in tumour or tumor neovasculature reagent, the new life of the tumour or tumour
Blood vessel has CD105 positive expression feature.
11. the application of polypeptide described in claim 1, which is characterized in that in preparation for marking, identifying, be enriched with, sort or pure
Change and is applied in the preparation of CD105 positive cell；
Or, the polypeptide is applied in the label of CD105 positive cell, identification, enrichment, sorting or purification process.
12. polypeptide described in claim 1 or the described in any item pharmaceutical preparations of claim 5-7 are in preparation for targeting
It is applied in the reagent of CD105 positive cell.
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| PCT/CN2018/103544 WO2020000634A1 (en) | 2018-06-28 | 2018-08-31 | Polypeptide specifically binding to cd105 and use thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111358960A (en) * | 2019-12-02 | 2020-07-03 | 哈尔滨医科大学 | Double-targeting polypeptide and application thereof in resisting tumors and inhibiting tumor angiogenesis |
| CN112876538A (en) * | 2021-02-04 | 2021-06-01 | 福建医科大学 | Polypeptide targeting neovascular marker CD105 and application thereof |
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| CN102388067A (en) * | 2008-09-19 | 2012-03-21 | 米迪缪尼有限公司 | Targeted binding agents directed to cd105 and uses thereof |
| CN106928355A (en) * | 2015-12-30 | 2017-07-07 | 广西医科大学 | A kind of CD105 nano antibodies Nb184 |
| CN107236025A (en) * | 2017-03-30 | 2017-10-10 | 中山大学 | The polypeptide combined with CD56 molecular specificities and its application |
| CN107353326A (en) * | 2017-05-09 | 2017-11-17 | 中山大学附属口腔医院 | Non-antibody binding proteins and its application with reference to the acceptors of PD 1 |
| CN107921144A (en) * | 2015-06-20 | 2018-04-17 | 杭州多禧生物科技有限公司 | The auspicious statin analog of Australia and its conjugation conjugate with cell-binding molecules |
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2018
- 2018-06-28 CN CN201810687443.0A patent/CN108912212B/en active Active
- 2018-08-31 WO PCT/CN2018/103544 patent/WO2020000634A1/en active Application Filing
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102388067A (en) * | 2008-09-19 | 2012-03-21 | 米迪缪尼有限公司 | Targeted binding agents directed to cd105 and uses thereof |
| CN107921144A (en) * | 2015-06-20 | 2018-04-17 | 杭州多禧生物科技有限公司 | The auspicious statin analog of Australia and its conjugation conjugate with cell-binding molecules |
| CN106928355A (en) * | 2015-12-30 | 2017-07-07 | 广西医科大学 | A kind of CD105 nano antibodies Nb184 |
| CN107236025A (en) * | 2017-03-30 | 2017-10-10 | 中山大学 | The polypeptide combined with CD56 molecular specificities and its application |
| CN107353326A (en) * | 2017-05-09 | 2017-11-17 | 中山大学附属口腔医院 | Non-antibody binding proteins and its application with reference to the acceptors of PD 1 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111358960A (en) * | 2019-12-02 | 2020-07-03 | 哈尔滨医科大学 | Double-targeting polypeptide and application thereof in resisting tumors and inhibiting tumor angiogenesis |
| CN111358960B (en) * | 2019-12-02 | 2021-04-06 | 哈尔滨医科大学 | Double-targeting polypeptide and application thereof in resisting tumors and inhibiting tumor angiogenesis |
| CN112876538A (en) * | 2021-02-04 | 2021-06-01 | 福建医科大学 | Polypeptide targeting neovascular marker CD105 and application thereof |
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| WO2020000634A1 (en) | 2020-01-02 |
| CN108912212B (en) | 2019-08-30 |
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