CN105085632B

CN105085632B - A kind of polypeptide of special target EGFR albumen and its application

Info

Publication number: CN105085632B
Application number: CN201510583737.5A
Authority: CN
Inventors: 耿令令; 方巧君
Original assignee: National Center for Nanosccience and Technology China
Current assignee: National Center for Nanosccience and Technology China
Priority date: 2015-09-14
Filing date: 2015-09-14
Publication date: 2019-01-25
Anticipated expiration: 2035-09-14
Also published as: CN105085632A

Abstract

The present invention relates to a kind of polypeptide of special target EGFR (human epidermal growth factor acceptor 1) 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 EGFR 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

A kind of polypeptide of special target EGFR albumen and its application

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 EGFR 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.

EGF-R ELISA EGFR (Epidermal Growth Factor Receptor) is epidermal growth factor (EGF) receptor of cell Proliferation and signal transduction.EGFR belongs to one kind of ErbB receptor family, which includes EGFR (ErbB- 1), HER2/c-neu (ErbB-2), HER3 (ErbB-3) and HER4 (ErbB-4).EGFR is also referred to as HER1, ErbB1, mutation Or overexpression can generally cause tumour.EGFR belongs to tyrosine kinase receptor, molecular weight 170KDa.EGFR is located at cell membrane table Face, by being activated with ligand binding.Research shows that high expression or unconventionality expression in many entity tumors there are EGFR.EGFR It is related with the inhibition of the proliferation of tumour cell, angiogenesis, tumor invasion, transfer and Apoptosis.The overexpression of EGFR is being disliked Property tumour evolution in play an important role, spongiocyte, kidney, lung cancer, prostate cancer, cancer of pancreas, breast cancer etc. tissue in all There is the overexpression of EGFR.

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.

CN 102993272A discloses polypeptide and its application of a kind of targeting EGFR receptor, and the polypeptide is from EGFR master Want the small peptide EY of autophosphorylation action site¹⁰⁶⁸INQ (SEQ ID NO:1), DY¹¹⁴⁸QQD (SEQ ID NO:2), AEY¹¹⁷³LR (SEQ ID NO:3), the polypeptide is for modifying nano-lipid carrier.However it is high not enough with the affinity of EGFR, effect It is weaker.

Summary of the invention

The purpose of the present invention is to provide a kind of polypeptide, a kind of polypeptide of special target EGFR albumen and its application, especially It is that as derived from the peptide and targeting EGFR can prepared with the protein bound product of EGFR 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 of targeting EGFR 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, X_1-13For 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 or glutamine.

In the present invention, the polypeptide source be the polypeptide WP1 (its sequence are as follows: DTCPPLMLYNPTTYQM) based on EGFR and The storage capacity of foundation is 2 × 10⁵Polypeptide libraries:

Polypeptide WP1 (sequence: DTCPPLMLYNPTTYQM) is located at the extracellular region Section II structural domain of EGFR, is to form EGFR- The critical sites of EGFR dimer and EGFR-HER2 heterodimer.The positive cream of polypeptide energy specific bond Human epidermal growth factor receptor height expression Adenocarcinoma cell, Percentage bound are obviously improved.

As optimal technical scheme, the amino acid sequence of peptide of the present invention is selected from SEQ ID NO.1-SEQ ID NO.14 One of shown in amino acid sequence.

Amino acid sequence:

SEQ ID NO.1	KYFPPLALYNPTEYFY
		SEQ ID NO.2	DYFYPLAYYNPTTWQY
SEQ ID NO.3	KTIPYLGLYNPNTWRY
		SEQ ID NO.4	KKFYPLGWWNPNTWRY
SEQ ID NO.5	DTFYYLAYWNPNTWRY
		SEQ ID NO.6	DYIPYLALWNPNTWFY
SEQ ID NO.7	DKIPYLAWWNPNTWQQ
		SEQ ID NO.8	DYIPYLGYYNPTTWQY
SEQ ID NO.9	KTFPPLAYYNPTTWRY
		SEQ ID NO.10	DTFPYLGYWNPNTYRY
SEQ ID NO.11	KYIPPLALWNPTEWRQ
		SEQ ID NO.12	KKIYPLGYYNPNTYQQ
SEQ ID NO.13	DTIPYLAYWNPNTWRY
		SEQ ID NO.14	DYFPPLAWWNPNEYFQ

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.14.

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.14.

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.14 assembles.

Bivalent or multivalent as described above have the characteristic of the tumour cell of the targeting EGFR positive and to tumour cells With 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 connection molecule and/or polymer are polyethylene glycol (PEG), polyvinyl alcohol (PVA), cyclodextrin, gather In amide-amine type dendrimer (PAMAM), polylactic acid (PLA) or polylactic acid-ethanol amine (PLGA) any one or extremely Few two kinds of combination.

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.14 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.14 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.14 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.14 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 EGFR is overexpressed.

Preferably, the cancer is lung cancer, breast 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 EGFR albumen, can be used as target head and increases drug or be loaded with drug The carrier such as content of nano material, liposome in EGFR 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 EGFR 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 Human epidermal growth factor receptor positive breast cancer cells, Percentage bound polypeptide all more in the prior art It is obviously improved, it is little with the combination of EGFR low expression human embryo kidney (HEK) normal cell 293A cell, show the present invention as special The polypeptide of different targeting EGFR, to the selective combination of EGFR；

(3) polypeptide of the invention has the characteristic of targeting EGFR 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 Human epidermal growth factor receptor positive breast cancer cells (468 Cell line) actuating signal；Fig. 1 (B) is the SEQ ID that flow cyctometry method detects fluorescein isothiocynate (FITC) label Combination of the NO.1 to Human epidermal growth factor receptor positive breast cancer cells (468 cell line)；Fig. 1 (C) is that the detection of flow cyctometry method is different The SEQ ID NO.2 of thiocyanic acid fluorescein (FITC) label makees the combination of Human epidermal growth factor receptor positive breast cancer cells (468 cell line) With；Fig. 1 (D) is that flow cyctometry method detects the SEQ ID NO.3 of fluorescein isothiocynate (FITC) label to Human epidermal growth factor receptor sun The combination of property breast cancer cell (468 cell line)；Fig. 1 (E) is that flow cyctometry method detects fluorescein isothiocynate (FITC) combination of the SEQ ID NO.4 marked to Human epidermal growth factor receptor positive breast cancer cells (468 cell line)；Fig. 1 (F) is knot Conjunction rate histogram.

Fig. 2 (A) is that flow cyctometry method detects control group 0.01mM PBS to Human epidermal growth factor receptor feminine gender embryonic kidney normal cell (293A cell line) actuating signal；Fig. 2 (B) is that flow cyctometry method detects the SEQ ID NO.1 of FITC label to Human epidermal growth factor receptor 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.2 to Human epidermal growth factor receptor feminine gender embryonic kidney normal cell (293A cell line)；The flow cyctometry side Fig. 2 (D) It is combination of the SEQ ID NO.3 to Human epidermal growth factor receptor feminine gender embryonic kidney normal cell (293A cell line) that method, which detects FITC label,； Fig. 2 (E) is that flow cyctometry method detects the SEQ ID NO.4 of FITC label to Human epidermal growth factor receptor feminine gender embryonic kidney normal cell (293A Cell line) combination；Fig. 2 (F) 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 Human epidermal growth factor receptor positive breast cancer cells (468 cell line)；

Wherein, nucleus 33342 reagent dyeing of hoechst；A-C is SEQ ID NO.1 polypeptide and hoechst 33342 processed cells, wherein A is the binding site of SEQ ID NO.1 polypeptide, is primarily present in cell membrane, B is cell Core, the overlapping figure of C A, B；D-F is SEQ ID NO.2 polypeptide and the processed cell of hoechst 33342, and wherein D is SEQ The binding site of ID NO.2 polypeptide, is primarily present in cell membrane, and E is nucleus, the overlapping figure of F D, E；G-I is SEQ ID NO.3 polypeptide and the processed cell of hoechst 33342, wherein G is the binding site of SEQ ID NO.3 polypeptide, is primarily present In cell membrane, H is nucleus, the overlapping figure of I G, H；J-L is that SEQ ID NO.4 polypeptide and hoechst 33342 are processed Cell, wherein J is the binding site of SEQ ID NO.4 polypeptide, is primarily present in cell membrane, K is nucleus, the overlapping of L J, K Figure.

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 Human epidermal growth factor receptor feminine gender embryonic kidney normal cell (293A cell line)；

Wherein, nucleus 33342 reagent dyeing of hoechst；A-C is SEQ ID NO.1 polypeptide and hoechst 33342 processed cells, wherein A is the binding site of SEQ ID NO.1 polypeptide, and no binding signal, B is nucleus, C A, The overlapping figure of B；D-F is SEQ ID NO.2 polypeptide and the processed cell of hoechst 33342, and wherein D is SEQ ID NO.2 The binding site of polypeptide, no binding signal, E are nucleus, the overlapping figure of F D, E；G-I be SEQ ID NO.3 polypeptide and The processed cell of hoechst 33342, wherein G is the binding site of SEQ ID NO.3 polypeptide, and no binding signal, H is cell Core, the overlapping figure of I G, H；J-L is SEQ ID NO.4 polypeptide and the processed cell of hoechst 33342, and wherein J is SEQ The binding site of ID NO.4 polypeptide, no binding signal, K are nucleus, the overlapping figure of L J, K.

Fig. 5 (A) is that surface plasma resonance (SPR) method detects SEQ ID NO.1 to the Human epidermal growth factor receptor albumen of various concentration Combination；Fig. 5 (B) is that surface plasma resonance (SPR) method detects SEQ ID NO.2 to the Human epidermal growth factor receptor egg of various concentration White combination；Fig. 5 (C) is that surface plasma resonance (SPR) method detects SEQ ID NO.3 to the Human epidermal growth factor receptor 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 EGFR albumen.

Fig. 6 (A) is that surface plasma resonance (SPR) method detects SEQ ID NO.1-SEQ ID NO.4 and WP1 peptide to same The combination of the Human epidermal growth factor receptor albumen of one concentration (10 μ g/mL)；Fig. 6 (B) is that surface plasma resonance (SPR) method detects SEQ Human epidermal growth factor receptor 2 (HER2) albumen of ID NO.1-SEQ ID NO.4 and WP1 peptide to same concentration (10 μ g/mL) Combination；Fig. 6 (C) is that surface plasma resonance (SPR) method detects SEQ ID NO.1-SEQ ID NO.4 and WP1 peptide To the combination of the human serum albumins (HSA) of same concentration (10 μ g/mL).

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 is put into Solid-phase synthesis peptides pipe (hereinafter referred to as reactor), it is small that suitable DMF swelling half is added When more than.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, is used DMF, DCM are washed 3 times, from a small amount of resin (about 5~10mg) is taken in reactor in test tube, with ethanol washing 2 times, and ninhydrin method Color is detected and recorded, prepares to feed intake, is reacted 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 Human epidermal growth factor receptor positive breast cancer cells

One, experimental method

468 cell of human breast carcinoma EGFR overexpression cell line 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 of fluorescein isothiocynate (FITC) label is added NO.4, ultimate density are 50 μM/L, and control group is replaced more with the 0.01mM PBS (phosphate buffer pH7.4) with polypeptide equivalent Peptide.After being protected from light ice bath incubation 30 minutes, 1000g is centrifuged 4 minutes collection cells, and 1mL PBS is added and cleans, after repeated washing 3 times, 500 μ L PBS are added, mixes, uses flow cyctometry method fluorescence intensity and combining 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 Human epidermal growth factor receptor height expresses breast cancer cell, and Percentage bound is 97.3%, 96.1%, 93.2%, 81.9% respectively, and affinity interaction is significant, Illustrate that peptide exclusive use of the invention has affinity interaction to EGFR positive tumor cell, the polypeptide that can be used as targeting EGFR makes With.

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 Human epidermal growth factor receptor feminine gender embryonic kidney normal cell

One, experimental method

Human embryo kidney (HEK) EGFR down-regulated express cell strain 293A cell is collected, the H-DMEM containing 10% heat-inactivated fetal bovine serum is suspended in In culture solution, 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 different sulphur SEQ ID NO.1, the SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 of cyanic acid fluorescein (FITC) label are final dense Degree is 50 μM of ol/L, and control group replaces polypeptide with the 0.01mM PBS (phosphate buffer pH7.4) with polypeptide equivalent.It is protected from light ice bath After being incubated for 30 minutes, 1000g is centrifuged 4 minutes collection cells, and 1mL PBS is added and cleans, and after repeated washing 3 times, 500 μ L are added PBS 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 EGFR The combination of low expression human embryo kidney (HEK) normal cell 293A cell is little, and Percentage bound is all 3% hereinafter, illustrate peptide pair of the invention The selective combination of HER2, the polypeptide that can be used as special target EGFR use.

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 Human epidermal growth factor receptor positive breast cancer cells

One, experimental method

Human breast carcinoma EGFR overexpression cell line 468 is suspended in the culture of the RPMI1640 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 12 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 33342 reagent dyeing of hoechst dilutes using according to 1:500.It is protected from light ice bath It is incubated for 30 minutes.After repetition washes 3 times, 200 μ L PBS are added in PBS cleaning, are believed using laser confocal microscope observation fluorescence Number.

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 Human epidermal growth factor receptor positive breast cancer cells, illustrates that peptide of the invention is used alone to have to combine to EGFR positive tumor cell and make With the polypeptide that can be used as targeting EGFR 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 12 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 33342 reagent dyeing of hoechst dilutes using according to 1:500.It is protected from light ice bath It is incubated for 30 minutes.After repetition washes 3 times, 200 μ L PBS are added in PBS cleaning, are believed using laser confocal microscope observation fluorescence Number.

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 EGFR feminine gender embryonic kidney normal cell is not bound with, and illustrates that peptide of the invention is selectivity specificity knot to EGFR positive tumor cell It closes, the polypeptide that can be used as special target EGFR 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 Human epidermal growth factor receptor 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 0.625 μ g/mL, 1.25 μ g/mL, 2.5 μ The Human epidermal growth factor receptor albumen of g/mL, 5 μ g/mL and 10 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 Human epidermal growth factor receptor 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 EGFR albumen_DValue is respectively as follows: 9.41 × 10^-9M/L、 2.31×10^-8M/L、2.57×10^-8M/L、3.18×10^-8M/L illustrates that polypeptide of the invention has strong combination to EGFR, can It is used using the polypeptide as targeting EGFR.

Experimental example 6 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 same concentration Human epidermal growth factor receptor, HER2, HSA 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 Human epidermal growth factor receptor, HER2, HSA of 10 μ g/mL Three kinds of albumen record and analyze spr signal.

Two, experimental result

As seen from Figure 6, SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 peptide are to identical Tri- kinds of albumen of Human epidermal growth factor receptor, HER2, HSA of concentration have different spr signals, find out SEQ ID NO.1, SEQ according to SPR result ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 polypeptide have significant binding specificity to EGFR, and SEQ ID NO.1 is tied It is most strong to close signal, SEQ ID NO.2-SEQ ID NO.4 binding signal is close, as a result consistent with Fig. 5 result.SEQ ID NO.1- SEQ ID NO.4 is faint to HER2 and HSA protein binding signal, illustrates that peptide of the invention has strong specific binding to make EGFR With the polypeptide that can be used as targeting EGFR uses.

Experimental example 1-6'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 EGFR Breast cancer cell combines, and is incorporated into the cell membrane surface of EGFR protein expression, and the embryonic kidney normal cell with EGFR 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 EGFR albumen, and with EGFR albumen The raising of concentration, binding signal are stronger.In addition SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 are more Peptide has different spr signals from tri- kinds of Human epidermal growth factor receptor of same concentrations, HER2, HSA albumen, shows SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 polypeptide have significant binding specificity to EGFR.

From experimental example 1-6, it can be concluded that, peptide of the invention has the characteristic of targeting EGFR positive tumor cell, thus in reality In the application of border, it can be mutually conjugated or mix with the preparation that can kill cancer cell, for swelling using peptide of the invention as target polypeptide The targeted therapy of tumor, 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

1. a kind of peptide of special target EGFR 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 Human epidermal growth factor receptor height expresses positive breast cancer cells, and Percentage bound is 81.9% 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 EGFR 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 combinations.

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 11, 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 the drug in described pharmaceutical composition For nano material, any one in 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 5 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 EGFR is overexpressed.

21. purposes according to claim 20, which is characterized in that the cancer is lung cancer, breast cancer, gastric cancer, liver cancer, knot Any one in intestinal cancer, the carcinoma of the rectum, the cancer of the esophagus, leukaemia, bladder cancer or cervix cancer or nasopharyngeal carcinoma.