CN105085631B - A kind of polypeptide of special target HER2 albumen and its application - Google Patents
A kind of polypeptide of special target HER2 albumen and its application Download PDFInfo
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Abstract
The present invention relates to a kind of polypeptide of special target HER2 (human epidermal growth factor receptor 2) albumen and its applications, and the polypeptide is as shown in following general formula: X1X2X3X4X5LX6X7X8NPX9X10X11X12X13；The invention further relates to a kind of nucleotide sequences for encoding polypeptide, and express the expression vector and host cell of the polypeptide；The invention further relates to the polypeptide, its bivalent formed or multivalent and its medical composition and its uses formed as target polypeptide with the preparation and Imaging agent that can kill cancer cell, polypeptide of the present invention can play targeting to HER2 positive tumor cell, selectivity is strong, and peptide of the present invention can be prepared using chemically synthesized method, purity is high, molecular weight be small, high specificity, non-immunogenicity and safe and reliable.
Description
Technical field
The present invention relates to field of medicinal chemistry, and in particular to a kind of polypeptide more particularly to a kind of special target HER2 albumen
Polypeptide and its application.
Background technique
Cancer is a global major causes of death, and data are shown: the whole world increases about 14,100,000 cases of cancer newly within 2012,
Number of cancer deaths is up to 8,200,000, in contrast, data in 2008 are respectively 12,700,000 and 7,600,000.It is diagnosed in world wide
Most common cancer is followed successively by lung cancer (1,800,000,13%), breast cancer (1,700,000,11.9%) and colorectal cancer (1,400,000,9.7%),
Main lethal cancer is lung cancer (1,600,000,19.4%), liver cancer (800,000,9.1%) and gastric cancer (700,000,8.8%).International cancer
Disease research institution is according to available data, it is expected that due to population in the world growth and aging, and to before 2025, the whole world increases cancer newly every year
Disease case load will be up to 19,300,000.2012, more than half cancer new cases and number of cancer deaths's hair of whole world sum
It is raw in low developed area, respectively 56.8% and 64.9%.
The treatment method of malignant tumour mainly has operative treatment, chemotherapy, three kinds of radiotherapy at present, and wherein chemotherapy is to exist in recent years
Method with fastest developing speed in oncotherapy.But it is thin also to have killed human normal while killing tumor cell for chemotherapeutics
Born of the same parents, toxic side effect are big.Therefore, anti-tumor drugs targeting and molecular probe research are imperative.
Targeting cancer therapy is successfully on condition that find the target spot for the treatment of accurately.HER2 (human epidermal growth factor receptor 2, also known as
ErbB2) albumen is the transmembrane glycoprotein receptor tyrosine kinase of a 185kDa, is positioned at cell membrane, has extracellular ligand knot
Close area, single membrane span area and tyrosine kinase activity area intracellular.HER2 participates in signal transduction pathway, causes phosphorylating kinase after activation
Cascade reaction causes cell to grow and break up.There is the mistake of HER2 in the kinds of tumors such as breast cancer, oophoroma, gastric cancer and uterus
Degree expression.HER2 has become the important target for the treatment of cancer.
Polypeptide is easy to largely synthesize, and molecular weight is small, and tissue permeability is strong, while can prevent the non-specific of reticuloendothelial system
Property intake.In addition, polypeptide can be chemically modified to change its affinity, charge, hydrophobicity, stability and dissolubility, can pass through
Optimization modification repeatedly uses in vivo.In cancer target therapy, polypeptide drug and diagnostic probe show very strong excellent
More property is a kind of attractive substitute of antibody.In application aspect, the cooperation of polypeptide and radionuclide can be used as spy
Needle is applied to molecular imaging；Some naturally occurring polypeptides have been used as agent delivery；The derivative of the peptide of reproductive hormone and they
It can be used for neoplasm targeted therapy.Therefore high efficiency finds the weight that valuable active peptides have become cancer diagnosis and treatment faces
Want task.
104130315 A of CN discloses a kind of polypeptide of special target HER2 (human epidermal growth factor receptor 2) albumen,
The polypeptide is as shown in following general formula: X1X2X3X4X5X6X7RX8YWX9X10X11X12X13X14X15X16X17RX18X19X20X21YX22, energy
Targeting is risen to HER2 positive cell.Although it discloses the polypeptide for treating cancer have targeting, however, its with
The interaction that HER2 is combined is not high enough, and targeting is also relatively poor.
Summary of the invention
The purpose of the present invention is to provide a kind of polypeptide, a kind of polypeptide of special target HER2 albumen and its application, especially
It is that as derived from the peptide and HER2 can be targeted in preparation with the protein bound product of HER2 and aforementioned polypeptides or its derived product
Purposes in anti-tumor drug or Imaging agent.
In order to achieve that object of the invention, the invention adopts the following technical scheme:
In a first aspect, the present invention provides a kind of peptide for targeting HER2 albumen, amino acid sequence formula are as follows: X1X2X3X4X5LX6X7X8NPX9X10X11X12X13
The amino acid residue that one-letter symbol represents in above-mentioned peptide chain is defined as follows: L is leucine, and N is asparagine, P
For proline, X1-13For variable amino acid residue.
As optimal technical scheme, X1It is charge residue residue, preferably lysine or aspartic acid；X2It is polarity
Amino acid residue, preferably lysine, threonine or tyrosine；X3It is aromatic series or aliphatic amino acid residue, preferably benzene
Alanine or isoleucine；X4It is armaticity or heterocyclic amino acid residue, preferably tyrosine or proline；X5Be armaticity or
Heterocyclic amino acid residue, preferably tyrosine or proline；X6It is aliphatic amino acid residue, preferably glycine or the third ammonia
Acid；X7It is aliphatic or aromatic amino acid residue, preferably leucine, tyrosine or tryptophan；X8It is that aromatic amino acid is residual
Base, preferably tyrosine or tryptophan；X9It is polar amino acid residues, preferably threonine or asparagine；X10It is polarity ammonia
Base acid residue, preferably threonine or glutamic acid；X11It is aromatic amino acid residue, preferably tyrosine or tryptophan；X12It is
Armaticity or polar amino acid residues, preferably phenylalanine, arginine or glutamine；X13It is armaticity or polar amino acid
Residue, preferably tyrosine, lysine or glutamine.
In the present invention, the polypeptide source is the polypeptide sequence DTCPPLMLYNPTTYQM based on HER2 homologous protein EGFR
And the storage capacity established is 2 × 105Polypeptide libraries:
The polypeptide sequence DTCPPLMLYNPTTYQM of EGFR is to form EGFR-EGFR dimer and the different dimerization of EGFR-HER2
The critical sites of body, with the protein extracellular HER2, Section II structural domain is combined.Polypeptide of the present invention and HER2 extracellular region Section II
Structural domain combines, and the polypeptide energy specific bond people HER2 high expresses positive breast cancer cells, and Percentage bound is 99% or more.
As optimal technical scheme, the amino acid sequence of peptide of the present invention is selected from SEQ ID NO.1-SEQ ID NO.59
One of shown in amino acid sequence.
Amino acid sequence:
| SEQ ID NO.1 | DTFPYLGWWNPNEYRY |
| SEQ ID NO.2 | KTIYYLGYYNPNEYRY |
| SEQ ID NO.3 | DYIPYLAYYNPNTYFQ |
| SEQ ID NO.4 | KKIPYLGWWNPTTWQQ |
| SEQ ID NO.5 | DKFPYLALYNPTTWRQ |
| SEQ ID NO.6 | DKFPYLALYNPTTYFY |
| SEQ ID NO.7 | DKFYPLGYYNPTEWFQ |
| SEQ ID NO.8 | DKFYYLAYWNPNEYRQ |
| SEQ ID NO.9 | DTFPPLGWWNPTTWFY |
| SEQ ID NO.10 | DKFPPLGLYNPNEYFY |
| SEQ ID NO.11 | DTFYYLAWWNPNEWRQ |
| SEQ ID NO.12 | DTFYYLGYWNPTTYRQ |
| SEQ ID NO.13 | DTIYYLAWWNPTTWFY |
| SEQ ID NO.14 | DYFPPLGWYNPTTWFY |
| SEQ ID NO.15 | DYFPPLGYYNPNEWQY |
| SEQ ID NO.16 | DYFPYLAWWNPNEWQQ |
| SEQ ID NO.17 | DYFPYLAYWNPNEWQY |
| SEQ ID NO.18 | DYFPYLAYWNPNTWRY |
| SEQ ID NO.19 | DYFPYLGLYNPNEWFY |
| SEQ ID NO.20 | DYFYYLAWWNPNEYFQ |
| SEQ ID NO.21 | DYFYYLAWWNPTTWFQ |
| SEQ ID NO.22 | DYFYYLGYWNPNEYFY |
| SEQ ID NO.23 | DYIPPLALWNPNEYFY |
| SEQ ID NO.24 | DYIPYLALWNPTTYFQ |
| SEQ ID NO.25 | DKFPYLALWNPTTYFQ |
| SEQ ID NO.26 | DYIPYLAYYNPTEYFQ |
| SEQ ID NO.27 | DYIYYLGLWNPTEYQQ |
| SEQ ID NO.28 | DYIYYLGLWNPTTWFY |
| SEQ ID NO.29 | DYIYYLGYWNPTEWRK |
| SEQ ID NO.30 | DYIYYLGYYNPTTYQQ |
| SEQ ID NO.31 | KKFPPLALYNPNTWFQ |
| SEQ ID NO.32 | KKFPPLGYYNPTEYRY |
| SEQ ID NO.33 | KKFPYLAYWNPTTYRY |
| SEQ ID NO.34 | KKFPYLGYYNPTEYRQ |
| SEQ ID NO.35 | KKFYPLGWWNPNEWRY |
| SEQ ID NO.36 | KKFYYLGYWNPNEWQY |
| SEQ ID NO.37 | KKFYYLGYWNPNEWRQ |
| SEQ ID NO.38 | KKFYYLGYWNPNTWFQ |
| SEQ ID NO.39 | KKIPPLAYYNPNTWFY |
| SEQ ID NO.40 | KKIPPLGWWNPNTWRY |
| SEQ ID NO.41 | KKIPYLAWWNPTTYRQ |
| SEQ ID NO.42 | KKIPYLAYYNPNTWQQ |
| SEQ ID NO.43 | DKFPYLALYNPTEWQQ |
| SEQ ID NO.44 | KKIYYLALWNPTEYRY |
| SEQ ID NO.45 | KKIYYLAWWNPTEYFQ |
| SEQ ID NO.46 | KKIYYLGYYNPNEWRQ |
| SEQ ID NO.47 | KKIYYLGYYNPTEWRQ |
| SEQ ID NO.48 | KTFYYLGWWNPNEWRQ |
| SEQ ID NO.49 | KTFYYLGYWNPNEWQY |
| SEQ ID NO.50 | KTIPYLAWWNPNTWFQ |
| SEQ ID NO.51 | DKFPPLGYWNPTTWQQ |
| SEQ ID NO.52 | KYFPPLGWWNPNTYQY |
| SEQ ID NO.53 | KYFPPLGYWNPNTWQY |
| SEQ ID NO.54 | KYFPYLGWWNPTTWQY |
| SEQ ID NO.55 | KYFYYLAYWNPTTYRQ |
| SEQ ID NO.56 | KYFYYLGLWNPNEWFQ |
| SEQ ID NO.57 | KYIPPLAWWNPTEYQY |
| SEQ ID NO.58 | KYIPPLGWWNPTEYRY |
| SEQ ID NO.59 | KYIYYLALYNPNTWRY |
Second aspect, the present invention also provides a kind of DNA fragmentations, and it includes the amino of the peptide of coding aforementioned present invention general formula
Acid sequence.
As optimal technical scheme, the DNA fragmentation includes to encode the present invention such as SEQ ID NO.1-SEQ ID
The amino acid sequence of peptide shown in one of NO.59.
The third aspect, the present invention also provides a kind of expression vector, the encoding amino acid sequence including at least one copy
DNA fragmentation described in second aspect of the present invention for peptide shown in general formula.
As optimal technical scheme, expression vector of the invention is including at least one encoding amino acid sequence copied
DNA fragmentation described in the second aspect of the present invention of peptide shown in SEQ ID NO.1-SEQ ID NO.59.
Fourth aspect, the present invention also provides a kind of protokaryon or eukaryotic host cell, which contains such as the present invention
Expression vector described in the third aspect.
5th aspect, the present invention also provides a kind of bivalent or multivalents, as described in the general formula 1 of first aspect present invention
Peptide described in peptide and SEQ ID NO.1-SEQ ID NO.59 assembles.
As described above bivalent or multivalent are the characteristics of the tumour cell with the targeting HER2 positive and thin to tumour
Born of the same parents have lethality.
As optimal technical scheme, bivalent of the invention or multivalent are covalent by connection molecule and/or polymer
Connection formed or by with connection molecule and/or mixed with polymers, be not covalently linked formation.
Preferably, the polymer is polyethylene glycol (PEG), polyvinyl alcohol (PVA), cyclodextrin, polyamidoamine branch
In shape macromolecule (PAMAM), polylactic acid (PLA) or polylactic acid-ethanol amine (PLGA) any one or at least two mixing.
6th aspect, the present invention still further provide a kind of pharmaceutical composition, the amino including first aspect present invention
Acid sequence is the bivalent or more of peptide described in general formula, the peptide as described in one of SEQ ID NO.1-SEQ ID NO.59 or the peptide
Valence body is as target polypeptide, and can kill the preparation of cancer cell.
As optimal technical scheme, peptide, bivalent or multivalent of the present invention are thin with that can kill cancer as target polypeptide
The preparation of born of the same parents is mutually conjugated or mixes.
Preferably, the preparation is chemicals, bio-pharmaceutical, Nano medication, the radioactivity medicine that can kill cancer cell
Object, photo-thermal therapy or optical dynamic therapy medicine wrap up any one in the carriers of these drugs；
It is further preferred that the preparation is alkylating agent, antimetabolite, antitumor natural drug, antitumor antibiosis
Element, hormone and metal complex or tumour radiotherapy target any one in marker.
It is further preferred that the carrier is nano material, and any one in liposome or oiliness compound, Huo Zheyou
Mixture composed by a variety of oiliness compounds.
The present invention use by general formula as described in one of SEQ ID NO.1-SEQ ID NO.59 the bivalent of peptide and the peptide
Or the high molecular materials such as multivalent and nano material, liposome conjugation, peptide, bivalent or multivalent of the present invention can make
The compound generated after conjugation is more stably transported to target cell in body.Peptide, bivalent or multivalence of the present invention
Body can also be mixed with the mixture of oiliness compound or a variety of oiliness compounds, and peptide of the present invention can also make gained
To mixture target cell is more stably transported in body.
7th aspect, the present invention still further provide another pharmaceutical composition, and described pharmaceutical composition includes this
The amino acid sequence of invention first aspect is peptide described in general formula, peptide or the peptide described in SEQ ID NO.1-SEQ ID NO.59
Bivalent or multivalent；And Imaging agent.
Preferably, the peptide, bivalent or multivalent are mutually conjugated or mix with Imaging agent.
Preferably, the Imaging agent is in radionuclide, radioisotope labeling thing or molecular image preparation
Any one.
Eighth aspect is peptide, SEQ ID described in general formula the present invention also provides the amino acid sequence of first aspect present invention
The bivalent or multivalent of peptide or the peptide described in NO.1-SEQ ID NO.59 are in preparation for treating, preventing or diagnosing cancer
The drug of disease or the purposes in Imaging agent.
As optimal technical scheme, cancer of the present invention is the cancer that HER2 is overexpressed.
Preferably, the cancer is breast cancer, lung cancer, gastric cancer, liver cancer, colon and rectum carcinoma, the cancer of the esophagus, leukaemia, wing
Guang cancer or cervix cancer.
Peptide of the present invention has the function of targeting HER2 albumen, can be used as target head and increase drug or be loaded with drug
The carrier such as content of nano material, liposome in HER2 positive cell, then add pharmaceutically acceptable auxiliary material or adjuvant
Novel more effective targeted anticancer medicine is made.
Compared with prior art, the invention has the following advantages:
(1) polypeptide of the invention can play targeting to HER2 positive cell, and selectivity is strong, and polypeptide of the invention can be with
It is prepared using chemically synthesized method, purity is high, molecular weight is small, high specificity, non-immunogenicity, securely and reliably；
(2) polypeptide of the invention can be in conjunction with people HER2 positive breast cancer cells, and Percentage bound is low with HER2 all 99% or more
The combination for expressing human embryo kidney (HEK) normal cell 293A cell is little, shows polypeptide of the peptide of the present invention as special target HER2,
To the selective combination of HER2；
(3) polypeptide of the invention has the characteristic of targeting HER2 positive tumor cell, also has to tumour cell with killing
The characteristic of overstrain can be used for the targeted therapy of tumour or the molecular imaging of target tumor and diagnosis as target polypeptide.
Detailed description of the invention
Fig. 1 (A) is that flow cyctometry method detects control group 0.01mM PBS to people's HER2 positive breast cancer cells
The actuating signal of (SKBR3 cell line)；Fig. 1 (B) is that flow cyctometry method detects fluorescein isothiocynate (FITC) label
Combination of the Anti-HER 2 to people HER2 positive breast cancer cells (SKBR3 cell line)；Fig. 1 (C) is flow cyctometry side
Method detects the SEQ ID NO.1 of fluorescein isothiocynate (FITC) label to people HER2 positive breast cancer cells (SKBR3 cell
System) combination；Fig. 1 (D) is the SEQ ID NO.2 that flow cyctometry method detects fluorescein isothiocynate (FITC) label
To the combination of people HER2 positive breast cancer cells (SKBR3 cell line)；Fig. 1 (E) is that flow cyctometry method detects different sulphur
The SEQ ID NO.3 of cyanic acid fluorescein (FITC) label makees the combination of people HER2 positive breast cancer cells (SKBR3 cell line)
With；Fig. 1 (F) is that flow cyctometry method detects the SEQ ID NO.4 of fluorescein isothiocynate (FITC) label to people's HER2 sun
The combination of property breast cancer cell (SKBR3 cell line)；Fig. 1 (G) is Percentage bound histogram.
Fig. 2 (A) is that flow cyctometry method detects control group 0.01mM PBS to people's HER2 feminine gender embryonic kidney normal cell
The actuating signal of (293A cell line)；Fig. 2 (B) is that flow cyctometry method detects the Anti-HER 2 of FITC label to people HER2
The combination of negative embryonic kidney normal cell (293A cell line)；Fig. 2 (C) is that flow cyctometry method detects FITC label
Combination of the SEQ ID NO.1 to people's HER2 feminine gender embryonic kidney normal cell (293A cell line)；Fig. 2 (D) is flow cyctometry
Method detects combination of the SEQ ID NO.2 to people's HER2 feminine gender embryonic kidney normal cell (293A cell line) of FITC label；
It is SEQ ID NO.3 to people's HER2 feminine gender embryonic kidney normal cell (293A that Fig. 2 (E) flow cyctometry method, which detects FITC label,
Cell line) combination；Fig. 2 (F) is that flow cyctometry method detects the SEQ ID NO.4 of FITC label to people's HER2 feminine gender
The combination of embryonic kidney normal cell (293A cell line)；Fig. 2 (G) is Percentage bound histogram.
Fig. 3 be immunofluorescence method detect FITC label SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3,
SEQ ID NO.4 is respectively to the combination of people HER2 positive breast cancer cells (SKBR3 cell line)；
Wherein, nucleus hoechst reagent dyeing；A-D is that SEQ ID NO.1 polypeptide and hoechst are processed thin
Born of the same parents, wherein A is the binding site of SEQ ID NO.1 polypeptide, is primarily present in cell membrane, and B is nucleus, and C is that bright-field is entire
Cell, D are the overlapping figure of A-C；E-H is SEQ ID NO.2 polypeptide and the processed cell of hoechst, and wherein E is SEQ ID
The binding site of NO.2 polypeptide is primarily present in cell membrane, and F is nucleus, and G is the entire cell of bright-field, and H is the overlapping of E-G
Figure；I-L is SEQ ID NO.3 polypeptide and the processed cell of hoechst, and wherein I is the bound site of SEQ ID NO.3 polypeptide
Point is primarily present in cell membrane, and J is nucleus, and K is the entire cell of bright-field, and L is the overlapping figure of I-K；M-P is SEQ ID
NO.4 polypeptide and the processed cell of hoechst, wherein M is the binding site of SEQ ID NO.4 polypeptide, is primarily present in cell
Film, N are nucleus, and O is the entire cell of bright-field, and P is the overlapping figure of M-O.
Fig. 4 be immunofluorescence method detect FITC label SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3,
SEQ ID NO.4 is respectively to the combination of people's HER2 feminine gender embryonic kidney normal cell (293A cell line)；
Wherein, nucleus hoechst reagent dyeing；A-D is that SEQ ID NO.1 polypeptide and hoechst are processed thin
Born of the same parents, wherein A is the binding site of SEQ ID NO.1 polypeptide, and B is nucleus, and C is the entire cell of bright-field, and D is the overlapping of A-C
Figure；E-H is SEQ ID NO.2 polypeptide and the processed cell of hoechst, and wherein E is the bound site of SEQ ID NO.2 polypeptide
Point, F are nucleus, and G is the entire cell of bright-field, and H is the overlapping figure of E-G；I-L is at SEQ ID NO.3 polypeptide and hoechst
The cell managed, wherein I is the binding site of SEQ ID NO.3 polypeptide, and J is nucleus, and K is the entire cell of bright-field, L I-
The overlapping figure of K；M-P is SEQ ID NO.4 polypeptide and the processed cell of hoechst, and wherein M is SEQ ID NO.4 polypeptide
Binding site, N are nucleus, and O is the entire cell of bright-field, and P is the overlapping figure of M-O.
Fig. 5 (A) is that surface plasma resonance (SPR) method detects SEQ ID NO.1 to people's HER2 albumen of various concentration
Combination；Fig. 5 (B) is that surface plasma resonance (SPR) method detects SEQ ID NO.2 to people's HER2 egg of various concentration
White combination；Fig. 5 (C) is that surface plasma resonance (SPR) method detects SEQ ID NO.3 to the people HER2 of various concentration
The combination of albumen；Fig. 5 (D) is that surface plasma resonance (SPR) method detects SEQ ID NO.4 to the people of various concentration
The combination of HER2 albumen.
Specific embodiment
The invention will now be further described with reference to specific embodiments, it will be understood by those skilled in the art that described
Embodiment is only intended to illustrate the present invention, and limits the scope of the invention without being intended to.The scope of the present invention is by appended
Claim specifically limits.
The synthesis of the polypeptide of the present invention of embodiment 1
1) laboratory apparatus and material
Dimethylformamide (DMF), piperidines, resin, methylene chloride (DCM), ninhydrin reaction reagent (ninhydrin, Victoria C,
Phenol), tetramethylurea hexafluorophosphate (HBTU), hexahydropyridine (piperidines), tri isopropyl silane (TIS), dithioglycol
(EDT), anhydrous ether, trifluoroacetic acid (TFA), N-methylmorpholine (NMM), methanol, various amino acid, Solid-phase synthesis peptides pipe.
2) solution is prepared
It is deprotected liquid --- hexahydropyridine: DMF=1:4
Reaction solution --- NMM:DMF=1:24
Lysate --- TFA (92.5%), TIS (2.5%), EDT (2.5%), H2O (2.5%)
Ninhydrin test fluid --- ninhydrin: Victoria C: phenol=1:1:1
3) experimental procedure
It weighs resin and puts into Solid-phase synthesis peptides pipe (hereinafter referred to as reactor), suitable DMF swelling half is added
Hour or more.DMF is taken out, Fmoc deprotection reaction is carried out with deprotection liquid, is placed on shaking table 10 minutes.Deprotection liquid is taken out,
It is washed 3 times with DMF, DCM, from a small amount of resin (about 5~10mg) is taken in reactor in test tube, with ethanol washing 2 times, ninhydrin
Method detects and records color, prepares to feed intake, and reacts into amino acid condensation.Respectively according to SEQ ID NO.1, SEQ ID NO.2,
SEQ ID NO.3, SEQ ID NO.4 peptide amino acid sequence sequence take corresponding amino acid, HBTU (amino acid: HBTU=1:1),
It is dissolved, is put into reactor with reaction solution, is stirred to react.After 1-2 hours, from taken in reactor a small amount of resin in test tube,
With ethanol washing 2 times, ninhydrin method is detected.The liquid in reactor is taken out, is respectively washed with DMF, DCM 2 times, first ammonia is obtained
Peptide resin after the condensation of base acid.Above " Fmoc deprotection --- amino acid condensation " the reaction step is repeated to gained peptide resin
Suddenly, until the last one amino acid end of reaction, obtain Serial No. SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3,
The peptide of SEQ ID NO.4.After completion of the reaction, DMF, DCM are respectively washed resin 2-3 times, and methanol is washed twice, continue to drain 15-20 points
Clock.The part peptide resin synthesized is taken out in reactor, the cracking in lysate (lysate elder generation ice bath 20 minutes) at room temperature
Two hours.After resin is filtered, it is evaporated in revolving instrument, is washed 3 times with anhydrous ether (ice bath).Thick peptide uses preparative reversed-phase HPLC
Purifying detects purity > 90% using HPLC.Obtained pure peptide is identified using mass spectrum (MS, electrospray).
To the last one peptide synthesis, takes out part peptide resin and add fluorescein isothiocynate (FITC) fluorescent marker.First will
Fmoc-e-Acp-OH is linked on polypeptide by amino acid couplings method, then appropriate HBTU and FITC is taken to be dissolved in fluorescence coupling solvent
In, after 3-5 hours, ninhydrin surveys solution testing.It cracked, purified and is reflected using above-mentioned same method after marking successfully
It is fixed.
SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 peptide related physicochemical character such as table 1
With shown in table 2.
Table 1
Table 2
1 flow cyctometry method of experimental example detects SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID
NO.4 is respectively to the combination of people's HER2 positive breast cancer cells
One, experimental method
Human breast carcinoma HER2 overexpression cell line SKBR3 is collected, is suspended in containing 10% heat-inactivated fetal bovine serum
In RPMI1640 culture solution, cell density is in 1x106/ mL or so is sub-packed in the EP pipe of four 1.5mL, 200 μ L/ pipe.Respectively
SEQ ID NO.1, the SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 of fluorescein isothiocynate (FITC) label is added
With Anti-HER 2 (ebioscience), ultimate density is 50 μM/L, 0.01mM PBS (phosphorus of the control group with polypeptide equivalent
Acid buffer pH7.4) replace polypeptide.After being protected from light ice bath incubation 30 minutes, 1000g is centrifuged 4 minutes collection cells, and 1mL is added
PBS is cleaned, and after repeated washing 3 times, 500 μ L PBS are added, mixes, uses flow cyctometry method fluorescence intensity and combination
Ratio.
Two, experimental result
As seen from Figure 1, SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 peptide are combinable
People HER2 high expresses breast cancer cell, and Percentage bound illustrates that peptide of the invention is used alone to the HER2 positive all 99% or more
Tumour cell has affinity interaction, and the polypeptide that can be used as targeting HER2 uses.
2 flow cyctometry method of experimental example detects SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID
NO.4 is respectively to the combination of people's HER2 feminine gender embryonic kidney normal cell
One, experimental method
Human embryo kidney (HEK) HER2 down-regulated express cell strain 293A is collected, the H-DMEM culture containing 10% heat-inactivated fetal bovine serum is suspended in
In liquid, cell density is in 1x106/ mL or so is sub-packed in four 1.5mL EP pipes.200 μ L/ pipe.It is separately added into isothiocyanic acid
SEQ ID NO.1, the SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, Anti-HER 2 of fluorescein (FITC) label
(ebioscience), ultimate density is 50 μM of ol/L, 0.01mM PBS (phosphate buffer of the control group with polypeptide equivalent
PH7.4 polypeptide) is replaced.After being protected from light ice bath incubation 30 minutes, 1000g is centrifuged 4 minutes collection cells, and 1mL PBS cleaning, weight is added
After cleaning 3 times again, 500 μ L PBS are added, mixes, uses flow cyctometry method fluorescence intensity and combining ratio.
Two, experimental result
As seen from Figure 2, SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 peptide and HER2
The combination of low expression human embryo kidney (HEK) normal cell 293A cell is little, illustrates that peptide of the invention makees the selective combination of HER2
With the polypeptide that can be used as special target HER2 uses.
3 immunofluorescence method of experimental example detects SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID
NO.4 is respectively to the combination of people's HER2 positive breast cancer cells
One, experimental method
Human breast carcinoma HER2 overexpression cell line SKBR3 is suspended in the training of the RPMI1640 containing 10% heat-inactivated fetal bovine serum
In nutrient solution, it is inoculated in three confocol capsules with the density of 3000-5000/ware.After culture 24 hours, in the capsule that exhausts
Culture medium, be then respectively adding containing fluorescein isothiocynate (FITC) label 50 μM of ol/L SEQ ID NO.1, SEQ
ID NO.2, SEQ ID NO.3, SEQ ID NO.4 polypeptide 200 μ L of culture medium, control group uses containing PBS (phosphorus with polypeptide equivalent
Acid buffer pH7.4) culture medium, nucleus hoechst reagent dyeing dilutes using according to 1:200.It is protected from light ice bath incubation
30 minutes.PBS cleaning, after repetition washes 3 times, is added 200 μ L PBS, observes fluorescence signal using laser confocal microscope.
Two, experimental result
As seen from Figure 3, SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 peptide can act on
In the surface of people's HER2 positive breast cancer cells, illustrates that peptide of the invention is used alone to have to combine to HER2 positive tumor cell and make
With the polypeptide that can be used as targeting HER2 uses.
4 immunofluorescence method of experimental example detects SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID
NO.4 is respectively to the combination of people's HER2 feminine gender embryonic kidney normal cell
One, experimental method
HER2 feminine gender human embryo kidney (HEK) normal cell strain 293A is suspended in the culture of the H-DMEM containing 10% heat-inactivated fetal bovine serum
In liquid, it is inoculated in three confocol capsules with the density of 3000-5000/ware.After culture 24 hours, in the capsule that exhausts
Culture medium is then respectively adding SEQ ID NO.1, the SEQ ID of 50 μM of ol/L containing fluorescein isothiocynate (FITC) label
NO.2, SEQ ID NO.3, SEQ ID NO.4 polypeptide 200 μ L of culture medium, control group uses containing PBS (phosphoric acid with polypeptide equivalent
PH of buffer 7.4) culture medium, nucleus hoechst reagent dyeing dilutes using according to 1:200.It is protected from light ice bath and is incubated for 30
Minute.PBS cleaning, after repetition washes 3 times, is added 200 μ L PBS, observes fluorescence signal using laser confocal microscope.
Two, experimental result
As seen from Figure 4, SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 peptide and people
HER2 feminine gender embryonic kidney normal cell is not bound with, and illustrates that peptide of the invention is selectivity specificity knot to HER2 positive tumor cell
It closes, the polypeptide that can be used as special target HER2 uses.
Experimental example 5 surface plasma resonance (SPR) method detects SEQ ID NO.1, SEQ ID NO.2, SEQ ID
NO.3, SEQ ID NO.4 are respectively to the affinity of people's HER2 albumen
One, experimental method
By the SEQ ID NO.1 of 1mg/mL, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 peptide and 0.01mM
It on PBS point to chip, is incubated overnight, is then cleaned ten minutes with 0.1mM PBS, 0.01mM PBS is clear under 4 DEG C of wet conditions
It washes ten minutes, is finally cleaned ten minutes with deionized water, and be repeated once, immersed in the 0.01mM PBS containing 5% milk, 4 DEG C
Under the conditions of be incubated overnight, then cleaned ten minutes with 0.1mM PBS, 0.01mM PBS clean ten minutes, finally use deionized water
Cleaning ten minutes, and is repeated once, with being dried with nitrogen, upper machine.Mobile phase passes sequentially through 1.25 μ g/mL, 2.5 μ g/mL, 5 μ g/
People's HER2 albumen of mL, 10 μ g/mL and 20 μ g/mL record and analyze spr signal.
Two, experimental result
As seen from Figure 5, the SPR of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 peptide
Signal is gradually increased with the increase of people's HER2 protein concentration, and SEQ ID NO.1, SEQ ID is calculated according to SPR result
NO.2, SEQ ID NO.3 and SEQ ID NO.4 polypeptide are to the dissociation constant K of HER2 albumenDValue is respectively as follows: 1.86 × 10-8M/L、
8.12×10-8M/L、9.06×10-8M/L、2.67×10-7M/L illustrates that polypeptide of the invention has strong combination to HER2, can
It is used using the polypeptide as targeting HER2.
Experimental example 1-5's the result shows that, on a cellular level, the two kinds of experiments test of flow cyctometry and immunofluorescence technique
Show that SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 polypeptide are specifically positive with HER2
Breast cancer cell combines, and is incorporated into the cell membrane surface of HER2 protein expression, and the embryonic kidney normal cell with HER2 feminine gender
Obviously do not combine；On a molecular scale, surface plasma resonance (SPR) experiment similarly confirms SEQ ID NO.1, SEQ
ID NO.2, SEQ ID NO.3, SEQ ID NO.4 polypeptide have strong combination with HER2 albumen, and with HER2 albumen
The raising of concentration, binding signal are stronger.
From experimental example 1-5 it can be concluded that, polypeptide of the invention have targeting HER2 positive tumor cell characteristic, thus
In practical application, it can be mutually conjugated or mix with the preparation that can kill cancer cell, be used for using peptide of the invention as target polypeptide
The targeted therapy of tumour, or can be mutually conjugated or mix with developer, molecular imaging and diagnosis for target tumor.
The Applicant declares that the present invention illustrates the process method of the present invention through the above embodiments, but the present invention not office
It is limited to above-mentioned processing step, that is, does not mean that the present invention must rely on the above process steps to be carried out.Technical field
Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to raw material selected by the present invention
Addition, selection of concrete mode etc., all of which fall within the scope of protection and disclosure of the present invention.
Claims (21)
1. a kind of peptide of special target HER2 albumen, which is characterized in that the amino acid sequence of the peptide is selected from SEQ ID NO.1-
Amino acid sequence shown in one of SEQ ID NO.4；Wherein, the peptide energy specific bond people HER2 high expression positive breast cancer is thin
Born of the same parents, and Percentage bound is 99% or more.
2. a kind of DNA fragmentation, which is characterized in that it includes the nucleotide sequences of peptide described in coding claim 1.
3. a kind of expression vector, which is characterized in that the expression vector contains the as claimed in claim 2 of at least one copy
DNA fragmentation.
4. a kind of host cell, which is characterized in that the host cell contains expression vector as claimed in claim 3.
5. bivalent or multivalent that peptide according to claim 1 is formed, which is characterized in that the bivalent or multivalence
Body has the characteristic of targeting HER2 positive tumor cell.
6. bivalent according to claim 5 or multivalent, which is characterized in that the bivalent or multivalent is to pass through
Connection molecule and/or polymer, which are covalently attached, to be formed.
7. bivalent according to claim 5 or multivalent, which is characterized in that the bivalent or multivalent is to pass through
With connection molecule and/or mixed with polymers, it is not covalently linked formation.
8. bivalent according to claim 6 or 7 or multivalent, which is characterized in that the connection molecule or polymer are
Appointing in polyethylene glycol, polyvinyl alcohol, cyclodextrin, polyamidoamine dendrimer, polylactic acid or polylactic acid-ethanol amine
It anticipates a kind of or at least two mixing.
9. a kind of pharmaceutical composition, which is characterized in that described pharmaceutical composition includes peptide described in claim 1 and can kill
The preparation of cancer cell.
10. pharmaceutical composition according to claim 9, which is characterized in that described pharmaceutical composition includes claim 5 institute
The bivalent or multivalent stated and the preparation that cancer cell can be killed.
11. pharmaceutical composition according to claim 9 or 10, which is characterized in that the peptide, bivalent or multivalent conduct
Target polypeptide is mutually conjugated or mixes with the preparation that can kill cancer cell.
12. pharmaceutical composition according to claim 9, which is characterized in that the preparation is the change that can kill cancer cell
Learn any one in drug, bio-pharmaceutical, Nano medication, radiopharmaceutical, photo-thermal therapy or optical dynamic therapy medicine.
13. pharmaceutical composition according to claim 12, which is characterized in that the preparation is alkylating agent, antimetabolite
It is any one in object, antitumor natural drug, antitumor antibiotics, hormone, metal complex or tumour radiotherapy targeting marker
Kind.
14. pharmaceutical composition according to claim 12, which is characterized in that the carrier of drug is in described pharmaceutical composition
Any one in nano material, liposome or oiliness compound, or the mixture as composed by a variety of oiliness compounds.
15. a kind of pharmaceutical composition, which is characterized in that described pharmaceutical composition includes peptide described in claim 1 and imaging system
Agent.
16. pharmaceutical composition according to claim 15, which is characterized in that described pharmaceutical composition includes claim 6
The bivalent or multivalent and Imaging agent.
17. pharmaceutical composition according to claim 15 or 16, which is characterized in that the peptide, bivalent or multivalent with
Imaging agent is mutually conjugated or mixes.
18. pharmaceutical composition according to claim 17, which is characterized in that the Imaging agent be radionuclide,
Any one in radioisotope labeling thing or molecular image preparation.
19. peptide, bivalent or multivalent described in any one of claim 1 or claim 5-8 are being prepared for treating, in advance
Anti- or diagnosis cancer drug or the purposes in Imaging agent.
20. purposes according to claim 19, which is characterized in that the cancer is the cancer that HER2 is overexpressed.
21. purposes described in 9 or 20 according to claim 1, which is characterized in that the cancer is breast cancer, lung cancer, gastric cancer, liver
Any one in cancer, colon and rectum carcinoma, the cancer of the esophagus, leukaemia, bladder cancer or cervix cancer or nasopharyngeal carcinoma.
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| CN105693860B (en) * | 2016-03-03 | 2019-08-27 | 国家纳米科学中心 | The polypeptide of selectively targeted HER2 albumen and its application |
| CN110981936B (en) * | 2018-09-28 | 2021-10-12 | 北京京东方技术开发有限公司 | Peptoid compound, preparation method thereof, oligomer, pharmaceutical composition and kit |
| CN111393509B (en) * | 2020-03-30 | 2022-03-29 | 国家纳米科学中心 | Target specific polypeptide and application thereof |
| CN111848746A (en) * | 2020-08-08 | 2020-10-30 | 四川大学华西医院 | Binding protein for targeted binding to HER2, and preparation method and application thereof |
| CN112358531B (en) * | 2020-11-09 | 2022-05-27 | 国家纳米科学中心 | Polypeptide targeting HER2 protein and application thereof |
| CN113307849A (en) * | 2020-12-15 | 2021-08-27 | 北京理工大学 | Stapler peptide of targeting tumor stem cell marker CD133 and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005003156A1 (en) * | 2003-07-04 | 2005-01-13 | Affibody Ab | Polypeptides having binding affinity for her2 |
| CN104130315A (en) * | 2014-07-25 | 2014-11-05 | 国家纳米科学中心 | Polypeptide for specifically targeting human epidermal growth factor receptor 2 (HER2) protein |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005003156A1 (en) * | 2003-07-04 | 2005-01-13 | Affibody Ab | Polypeptides having binding affinity for her2 |
| CN104130315A (en) * | 2014-07-25 | 2014-11-05 | 国家纳米科学中心 | Polypeptide for specifically targeting human epidermal growth factor receptor 2 (HER2) protein |
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