CN105330725B - A kind of pH response and polypeptide and its application for targeting human tumour vascular markers VEGFR2 - Google Patents

Abstract

The present invention provides a kind of pH response and special target human tumour vascular markers VEGFR2 polypeptide and the polypeptide derived from and the application in anti-tumor drug or Imaging agent can prepared with the protein bound product of VEGFR2 and aforementioned polypeptides or product derived from it.The amino acid sequence formula of the polypeptide are as follows: CX₁X₂DX₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁LSX₁₂GM.The polypeptide can play targeting to VEGFR2 positive cell, it is responded with pH, selectivity is strong, it can be used as target head to increase drug or be loaded with the carrier such as content of nano material, liposome in VEGFR2 positive cell of drug, then add pharmaceutically acceptable auxiliary material or novel more effective targeted anticancer medicine is made in adjuvant.And polypeptide provided by the invention is easily chemically modified and adjusts its affinity, charge, hydrophilic and hydrophobic, stability and dissolubility, can also be prepared using chemically synthesized method, purity is high, molecular weight is small, high specificity, non-immunogenicity, securely and reliably.

Description

A kind of pH response and polypeptide and its application for targeting human tumour vascular markers VEGFR2

Technical field

The present invention relates to selectively targeted polypeptides, specifically, being related to a kind of pH response and special target human tumour blood vessel The polypeptide of marker VEGFR2 and its application.

Background technique

Tumor vascular formation is the process that blood circulation is established in the microvessel growth and tumour of tumor cell induction. When gross tumor volume reaches 1mm³When, tumour shows angiogenesis phenotype and supplements blood vessel from surrounding substrate.The generation of solid tumor, Growth and infiltration are responsive to new vessels in tumour with transfer and are formed, and provide oxygen and nutriment for it, have met metabolism life It is long to need.The microenvironment of tumour makes pH gradually become 5.5~7.4 by 7.4.

As that studies elaboration of tumour mechanism deepens continuously, critical role of the Tumor angiogenesis in tumor development and The effect of antiangiogenesis therapy tumour becomes a completely new field of oncotherapy.Therefore currently, it is raw for new vessels At neoplasm targeted therapy become current malignant solid tumor research hot spot.

VEGF is the vascular endothelial cell mitogen of high special, is the currently known strongest angiogenic growth of effect The factor and increase vasopermeability the factor, mainly with vascular endothelial cell receptor ining conjunction with promotion endothelial cell division growth, The effect of migration and reconstructing blood vessel.Therefore, VEGF R2 (VEFGR2) is as one of its receptor, as new The target spot for the oncotherapy that angiogenic generates.VEGFR2 is made of 1357 amino acid residues, the transmembrane glycoprotein of 150kDa Receptor tyrosine kinase is positioned at cell membrane, has extracellular ligand binding domain, single membrane span area and tyrosine kinase activity intracellular Area.VEFGR2 high is expressed on the interior cutaneous vessel abundant of solid tumor surface, for the invasion of tumour, transfer and tumor neogenetic blood The formation of pipe plays an important role, and directly results in and takes part in tumor cell invasion and penetrate the process that basilar memebrane shifts.

Currently, the targeting diagnosis and therapeutic agent for VEFGR2 are mainly antibody drug.Although it is with special target The disadvantages of property, but cumbersome there is also preparing, stability is poor, somewhat expensive, and penetration power is weak.Therefore, tumour is turned in order to improve The specificity and accuracy for moving diagnosing and treating, make up the defect of antibody, and there is an urgent need to seek to design small molecule for VEGFR2 Probe, using the effective ways as detection and treating cancer.

Compared to antibody, polypeptide is easy to largely synthesize, and molecular weight is small, at low cost, good biocompatibility, in addition, polypeptide by Amino acid determines polypeptide thousand poor ten thousand in sequence, conformation, function as basic unit, the diversity of amino acid range combination Not.Moreover, polypeptide, which is easily chemically modified, adjusts its affinity, charge, hydrophilic and hydrophobic, stability and dissolubility.These features make Polypeptide drug and diagnostic probe be administered in cancer target, in terms of show very strong superiority, or even it is aobvious The trend of substitution antibody class diagnosis and treatment reagent is shown.Therefore, using these characteristics, high-throughput filter out in polypeptide libraries has PH response and the polypeptide with specific target tropism, then develop into cancer diagnosis, neoplasm targeted therapy drug becomes present face The vital task faced.

Summary of the invention

In order to solve the problems in the existing technology, the object of the present invention is to provide pH response and special target human tumours And it can be with the protein bound product of VEGFR2 and aforementioned polypeptides derived from the polypeptide of vascular markers VEGFR2 and the polypeptide Or its derivative product is preparing the application in anti-tumor drug or Imaging agent.

In order to achieve the object of the present invention, technical scheme is as follows:

It is described more present invention firstly provides a kind of pH response and the polypeptide of special target human tumour vascular markers VEGFR2 The amino acid sequence formula of peptide are as follows:

CX₁X₂DX₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁LSX₁₂GM

Wherein, C is cysteine；X₁It is hydrophilic amino acid, preferably serine or glutamic acid；X₂It is hydrophilic amino Acid, preferably lysine or aspartic acid；D is aspartic acid；X₃It is neutral or hydrophilic amino acid, preferably leucine or paddy Propylhomoserin；X₄It is acidic amino acid or nonpolar amino acid, preferably glutamic acid or proline；X₅It is aromatic amino acid, preferably For tryptophan；X₆It is basic amino acid, preferably histidine；X₇It is alkalinity perhaps acidic amino acid preferably lysine or paddy Propylhomoserin；X₈It is neutral amino acid, preferably asparagine or threonine；X₉It is neutral or aromatic amino acid, preferably asparagus fern acyl Amine or tyrosine；X₁₀It is aromatic series or neutral amino acid, preferably phenylalanine or serine；X₁₁For nonpolar amino acid, Preferably proline；L is leucine；S is serine；X₁₂For nonpolar amino acid, preferably proline and phenylalanine；G is Glycine；M is methionine.

Polypeptide of the present invention and tumor cell surface vascular markers VEGFR2 binding force are strong, and have good spy It is anisotropic.

Preferably, working as X₁When for serine, X₂For lysine or aspartic acid, X₃For glutamic acid, X₄For paddy ammonia Acid, X₅For tryptophan, X₆For histidine, X₇For lysine or glutamic acid, X₈For asparagine or threonine, X₉Asparagine or junket Propylhomoserin, X₁₀For phenylalanine or serine, X₁₁For proline, X₁₂For proline or phenylalanine；

Work as X₂When for lysine, X₁For glutamic acid or serine, X₃For glutamic acid, X₄For glutamic acid, X₅For tryptophan, X₆For histidine, X₇For lysine or glutamic acid, X₈For threonine, X₉Asparagine, X₁₀For serine or phenylalanine, X₁₁For Proline, X₁₂For phenylalanine or proline.

The advantage designed in this way is so that X₃, X₄Fixed aspartic acid forms α in acidic environment in energy binding sequence Helical structure so that polypeptide has the function of Premeabilisation of cells.

The present invention provides a preferred polypeptide, amino acid sequence CSKDEEWHKNNFPLSPGM on the basis of the above (SEQ ID No.1).It not only has the function of selectively targeted VEGFR2 albumen, but also has optimal pH response function, Conducive to peptide molecule under the slightly sour environment of tumour specific recognition tumor vessel marker, to identify tumor sites.

Polypeptide of the present invention also contains substituent group, and the substituent group is alkoxy, alkanoyl or amide groups.

Further, the amino acid residue for forming the polypeptide is L-type and/or D type.Preferably, the amino acid is residual Base is L-type.

Invention further provides the bivalent/multivalents formed by foregoing polypeptides.

Bivalent/the multivalent has the characteristic and targets neoplastic cells table of targeting VEGFR2 interior cutaneous vessel cell The characteristic of face vascular markers VEGFR2 albumen.

Optionally, the bivalent/multivalent is to be covalently attached to be formed by connection molecule.

Optionally, the bivalent/multivalent is to be not covalently linked formation by mixing with polymer.

The polymer can be selected according to specific purposes, such as can be polyethylene glycol (PEG) or cyclodextrin etc..

The present invention also provides a kind of pharmaceutical composition, it includes the preparation that can kill cancer cell and foregoing polypeptides and/or Aforementioned bivalent/multivalent.

Further, the polypeptide, bivalent/multivalent are as target polypeptide, with the preparation phase that can kill cancer cell Conjugation or mixing.

For example, the preparation be the chemicals that can kill cancer cell, bio-pharmaceutical, Nano medication, radiopharmaceutical, Photo-thermal therapy or optical dynamic therapy medicine wrap up any one in the carriers of these drugs.

Further, the preparation is alkylating agent, antimetabolite, antitumor natural drug, antitumor antibiotics, swashs Element, metal complex or tumour radiotherapy target any one in marker.

Polypeptide of the present invention, bivalent/multivalent can make the pharmaceutical composition generated after conjugation in body more Steadily it is transported to target cell.

Polypeptide of the present invention, bivalent/multivalent can also be mixed with oiliness compound or a variety of oiliness compounds It closes object to mix, polypeptide of the present invention, bivalent/multivalent can also make obtained mixture more stable in body Ground is transported to target cell.

Preferably, the carrier is nano material, any one in liposome or oiliness compound, or by a variety of oil Mixture composed by property compound.

Polypeptide of the present invention, bivalent/multivalent have the function of targeting VEFGR2 albumen, can be used as target head increasing Dosing object or the carrier such as content of nano material, liposome in VEGFR2 positive cell for being loaded with drug, then add pharmacy Novel more effective targeted anticancer medicine is made in upper acceptable auxiliary material or adjuvant.

The present invention also provides a kind of Imaging agents, and it includes imaging agent and foregoing polypeptides and/or aforementioned bivalent/multivalence Body.

The polypeptide, bivalent/multivalent are mutually conjugated or mix with Imaging agent.

Preferably, the imaging agent is appointing in radionuclide, radioisotope labeling thing or molecular image preparation It anticipates one kind.

The present invention also provides a kind of polypeptide coupling, the polypeptide coupling is obtained by foregoing polypeptides and carrier conjugation； The carrier is drug, toxin, cell factor, radioactive element, carrier protein, antibody, enzyme, agglutinin, fluorophor, quantum Point or high absorptivity chromophore.

The carrier can be selected according to specific purpose, for example, carrier can be for drug or cell factor etc., it can By drug or cell factor target cancer cell, it is also possible to radioactive element, carrier protein, antibody, enzyme, agglutinin, fluorophor, Quantum dot high absorptivity chromophore living is as carrier.

Preferably, the carrier is antibody or fluorophor；More preferably, the antibody is the anti-of phycoerythrin label Source of people VEGFR2 antibody；The fluorophor is fluorescein isothiocynate.

The present invention still further provides a kind of for detecting people's solid tumor vascular surface cell or blood vessel endothelium is thin The kit of intracellular growth factor acceptor 2 albumen, it includes foregoing polypeptides or foregoing polypeptides conjugates.

The present invention also provides foregoing polypeptides or bivalent/multivalent in preparation for treating, preventing or diagnosing cancer Application in drug or Imaging agent.

Further, the cancer is the cancer of tumor surface blood vessel V EFGR2 protein overexpression.For example, the cancer is Any one in breast cancer, lung cancer, gastric cancer, liver cancer, colon and rectum carcinoma, cancer of pancreas, oophoroma or kidney.

In addition, the present invention has pH responsiveness by the experimental verification polypeptide, it can be specific in acid condition Identify VEGFR2 albumen.

The beneficial effects of the present invention are:

The present invention provides a kind of pH response and the polypeptides and the polypeptide of special target human tumour vascular markers VEGFR2 It is derivative and anti-tumor drug can prepared with the protein bound product of VEGFR2 and aforementioned polypeptides or product derived from it Or the application in Imaging agent.

Polypeptide of the present invention can play targeting to VEGFR2 positive cell, have pH response, selectivity is strong, Ke Yizuo Increase drug for target head or is loaded with the carrier such as content of nano material, liposome in VEGFR2 positive cell of drug, then It adds pharmaceutically acceptable auxiliary material or novel more effective targeted anticancer medicine is made in adjuvant.

And polypeptide provided by the invention is easily chemically modified and adjusts its affinity, charge, hydrophilic and hydrophobic, stability and dissolution Property, it can also be prepared using chemically synthesized method, purity is high, molecular weight is small, high specificity, non-immunogenicity, safety Reliably.It compensates for antibody drug in the prior art and prepares cumbersome, the disadvantages of stability is poor, somewhat expensive, penetration power is weak.

Detailed description of the invention

Fig. 1 is the peptide library building chemical formula schematic diagram for screening VEGFR2 target polypeptide.

Fig. 2 is screening VEGFR2 target polypeptide schematic illustration.

Fig. 3 is that surface plasma resonance imaging (SPRi) method detection STP is right respectively at pH 7.4 and pH5.8 environment The combination of VEGFR2 albumen.

Fig. 4 is the VEGFR2 albumen on the surface specific recognition people interior cutaneous vessel cell line HUVEC under pH5.8 environment STP Site.

Wherein:

Fig. 4-(a)~(d) is STP special with the VEGFR2 albumen on the surface VEGFR2 positive cell HUVEC under pH5.8 environment The opposite sex combines, Fig. 4-(e)~(h) be STP under pH5.8 environment STP and VEGFR2 negative cells 293T without combination.

Fig. 4-(i)~(l) is VEGFR2 of the control peptide TP under pH7.4 environment with the surface VEGFR2 positive cell HUVEC Protein-specific combine, Fig. 4-(m)~(p) be control peptide TP under 7.4 environment of pH with VEGFR2 negative cells 293T without knot It closes.

Fig. 4-(a), (e), (i), (m) be the polypeptide with FITC and cell combination fluorogram.Fig. 4-(b), (f), (j), (n) be antibody and cell combination fluorogram.Fig. 4-(c), (g), (k), (o) be the superposition of preceding fluorescence twice figure.Fig. 4-(d), It (h), (l), is (p) light field figure.

Fig. 5 is that specificity of the STP respectively under pH7.4 and pH5.8 environment respectively with VEGFR2 positive cell HUVEC is tied It closes.

Wherein, Fig. 5-(a)~(d) be STP under pH7.4 environment with HUVEC without combination, Fig. 5-(e)~(h) is that STP exists It is specifically bound under 5.8 environment of pH with HUVEC.

Fig. 5-(i)~(l) and Fig. 5-(m)~(p) be control peptide TP under pH7.4 and pH5.8 environment respectively with VEGFR2 The VEGFR2 protein-specific on the surface positive cell HUVEC combines.

Fig. 5-(a) is (e) (m) the polypeptide fluorogram with FITC (i).Fig. 5-(b) is (f) (n) band FITC (j) The fluorogram of polypeptide and cell combination.Scheming-5 (c) is (g) (o) Fig. 5-(b) (k), (f), (j), (n) is superimposed again with light field Figure.Fig. 5-(d) is (p) (h) that fluorescence intensity is quantitatively schemed (l).

Fig. 6 is the Cell permeable that dynamic cellular verifies the STP under pH5.8 environment.

Specific embodiment

The preferred embodiment of the present invention is described in detail below in conjunction with embodiment.It will be appreciated that following real Providing merely to play the purpose of explanation for example is applied, is not used to limit the scope of the present invention.The skill of this field Art personnel without departing from the spirit and purpose of the present invention, can carry out various modifications and replace to the present invention.

Experimental method used in following embodiments is conventional method unless otherwise specified.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

The building and the screening of APN target polypeptide of 1 polypeptide libraries of embodiment

1. laboratory apparatus and material

N,N dimethylformamide (DMF), methylene chloride (DCM), N-methylmorpholine (NMM), ninhydrin, vitamin C, benzene Phenol, tetramethylurea hexafluorophosphate (HBTU), hexahydropyridine, piperidines, trifluoroacetic acid (TFA), tri isopropyl silane (TIS), second Two mercaptan (EDT), anhydrous ether, resin, methanol, various Fmoc protected amino acids, Peptide systhesis pipe, shaking table, vacuum pump, rotation Turn evaporimeter, mentioned reagent and material obtain from commercial channels.

2. solvent is prepared

Deprotection solvent --- hexahydropyridine: n,N dimethylformamide=1:4

Reaction solution --- N-methylmorpholine: n,N dimethylformamide=1:24

Lysate --- trifluoroacetic acid (92.5%), tri isopropyl silane (2.5%), dithioglycol (2.5%), ultrapure water (2.5%).

Ninhydrin test fluid --- ninhydrin: vitamin C: phenol=1:1:1

The synthesis of " 3. one object of a pearl " polypeptide libraries

Using Fmoc Solid-phase peptide synthesis synthesis polypeptide library, change constructed by the peptide library of VEGFR2 target polypeptide is screened Formula is as shown in Figure 1, principle is as shown in Figure 2.Specific method be in such a way that mixing is split point by amino acid randomly idol one by one It is linked on solid-phase resin, then removes side chain protecting group under strong acid, and then screened.

Weigh the Tentagel-NH of 900mg₂Resin is recycled according to above-mentioned solid phase Peptide systhesis program, is sequentially added The HBTU of 650mg Met and equivalent, 520mg Gly and the HTBU of equivalent are successively reacted.It is to after the reaction was completed, resin is equal It is divided into 2 parts, the HTBU of 295mg Pro and equivalent is added in a wherein pipe, 340mg Phe and equivalent is added in another pipe HTBU be coupled respectively, after being coupled, 2 pipe resins mix, be deprotected, cleaning.1g Ser and equivalent is added HBTU, 620mg Leu and the HBTU of equivalent successively carry out two circulations of reaction.To after the reaction was completed, resin is divided into 2 parts, The HTBU of 455mg Pro and equivalent is added in a wherein pipe, HTBU points of 620mg Tyr and equivalent are added in another pipe It is not coupled, after being coupled, 2 pipe resins is mixed, are deprotected, cleaning.Resin is divided into 3 parts again, to every pipe point Not Jia Ru the HTBU of 356mg Thr, 347mg Phe, 510mg Ser and equivalent be coupled, after being coupled, 3 pipe trees Rouge mixing, is deprotected, cleaning.Resin is divided into 3 parts again, is separately added into 537mg Asn, 413mg Tyr, 510mg to every pipe Ser and the HBTU of equivalent are coupled, and after being coupled, 3 pipe resins are mixed, are deprotected, cleaning.Resin is divided into again 2 parts, 535mg Thr is separately added into every pipe, and the HBTU of 805mg Asn and equivalent is coupled, after being coupled, 2 pipes Resin mixing, is deprotected, cleaning.Resin is divided into 5 parts again, is separately added into 254mg Lys, 214mg Arg to every pipe, 230mgGlu, 190mg Leu, 306mg Ser and the HBTU of equivalent are coupled, and after being coupled, 5 pipe resins are mixed, Deprotection, cleaning.Resin is divided into 3 parts again, is separately added into 356mg Arg to every pipe, 423mg Lys, 560mg His with etc. The HBTU of amount is coupled, and after being coupled, 3 pipe resins are mixed, are deprotected, cleaning.Resin is divided into 2 parts again, to Every pipe is separately added into 710mg Trp, and the HBTU of 475mg Leu and equivalent is coupled, and after being coupled, 2 pipe resins are mixed It closes, is deprotected, cleaning.Resin is divided into 5 parts again, is separately added into 230mg Glu, 214mg Arg, 222mg Asp to every pipe, 182mg Pro, 214mg Thr and the HBTU of equivalent are coupled, and after being coupled, 5 pipe resins are mixed, deprotection, clearly It washes.Resin is divided into 5 parts again, is separately added into 322mg Asn, 330mg His, 334mg Glu, 190mg Leu to every pipe, 248mg Tyr and the HBTU of equivalent are coupled, and after being coupled, 5 pipe resins are mixed, are deprotected, cleaning.It adds 1.11g Asp and the HBTU of equivalent are coupled, and after being coupled, are deprotected, cleaning.Resin is divided into 5 parts again, to every Pipe is separately added into 190mg Ile, and the HBTU progress of 254mg Lys, 220mg Asp, 182mg Pro, 306mg Ser and equivalent is even Connection mixes 5 pipe resins after being coupled, and is deprotected, cleaning.Resin is divided into 4 parts again, is separately added into every pipe 382mg Ser, 287mg Glu, 237mg Leu, 267mg Thr and the HBTU of equivalent are coupled, after being coupled, 4 Pipe resin mixing, is deprotected, cleaning.It is finally coupled to the secondary HBTU that 1.12g Cys and equivalent is added of pipe, is finished wait be coupled Afterwards, it is deprotected, cleaning.

The lysate into above-mentioned resin deviates from side chain protecting group, and vacuum is drained, and obtains the dry resin for being loaded with peptide library It is spare.

4. the screening with the polypeptide of VEGFR2 specific binding

The peptide pearl in polypeptide libraries is cleaned 3 times with 1 × PBS；Peptide pearl is mixed in DL instrument with 5% skim milk It closes (37 DEG C, 2h)；It is cleaned 3 times with 1 × PBS again, then with the VEGFR2 albumen of biotin labeling and peptide storehouse pearl in DL instrument Upper 37 DEG C of mixing are incubated for 2h, are cleaned 3 times with PBS, the peptide library after incubation is set in 1.5mL EP pipe, EP pipe is placed in magnetic frame On.Positive polypeptides, which are affected by magnetic forces, is adsorbed in EP pipe side wall, and negative polypeptide is since gravitational settling is in EP tube bottom.Positive microballon with After the receptor protein of biotin labeling is incubated for, ositive peptide pearl specific recognition albumen, the magnetic bead of marked by streptavidin passes through knowledge Other biotin and identify ositive peptide pearl.It is magnetic to be captured by magnetic field to have that ositive peptide pearl surface will coat one layer of magnetic bead, Interaction is as shown in Figure 2.

The peptide pearl of EP bottom of the tube is transferred in another EP pipe with liquid-transfering gun.The peptide pearl sub-elected through magnetic field is chosen one by one Out, on the micro-array chip for dialling in integral chip, using hydrogen bromide lysisin situ, through MALDI-TOF-MS (ground substance assistant laser Desorption ionization flight time mass spectrum) identification acquisition corresponding sequence information, part flag F ITC, MALDI- are recombined by sequence TOF identification and HPLC purifying are used for follow-up test.

Polypeptide sequence of the invention is made through chemical synthesis are as follows: STP:CSKDEEWHKNNFPLSPGM.

Experimental example 1 detects the affinity interaction of polypeptide and VEGFR2 albumen by surface plasma resonance imaging (SPRi) method

While chip lysisin situ, SPR chip is being placed above, polypeptide is being printed on chip, in 4 DEG C of wet items It is incubated overnight under part, then cleans 10min with 10 × PBS, then clean 10min with 1 × PBS, finally clean 2 with deionized water Secondary, each 10min immerses in 1 × PBS containing 5% milk, is incubated overnight under the conditions of 4 DEG C, is then cleaned with 10 × PBS 10min, 1 × PBS clean 10min, are finally cleaned 2 times, each 10min with deionized water, with being dried with nitrogen, machine on cartridge chip (Plexera HT surface plasma resonance imaging system).

Mobile phase passes sequentially through 1 × PBS, 2 × PBS, 1.25 μ g/mL, 2.5 μ g/mL, 5 μ g/mL, 10 μ g/mL and 20 μ g/ The VEGFR2 purifying protein of mL records and analyzes SPRi signal.

The pH of PBS is tuned into 5.8 with hydrochloric acid, is repeated the above steps.

As seen from Figure 3, the SPRi signal of STP is gradually increased with the increase of VEGFR2 protein concentration, illustrates this The polypeptide of invention has strong combination to VEGFR2 in pH5.8, and the polypeptide that can be used as targeting VEGFR2 is ground for relevant Study carefully application.

2 STP of experimental example interacts under pH5.8 environment with the specificity of VEGFR2

Fluorescein isothiocynate (FITC) conjugate of STP is obtained using solid phase synthesis process, more in the STP recombined Continue to be coupled ε-aminocaproic acid on blaa polypeptide.In the solution that pyridine/n,N dimethylformamide/methylene chloride ratio is 1:5:7 In, FITC and peptide pearl hybrid reaction are stayed overnight.

STP-FITC conjugate is obtained after lysate cracks, control peptide TP is coupled with FITC according to the method described above, is obtained TP-FITC conjugate is as control.Subsequent experimental is used for using MALDI-TOF identification and HPLC purifying.

Human vascular endothelial system HUVEC and human renal epithelial cell line 293T are incubated at respectively containing 10% fetal calf serum In H-DMEM culture solution, with 1 × 10⁵The round Glass bottom culture dish (Φ=35mm) of cell concentration implantation of/mL, 37 DEG C, 5%CO₂Carefully After cultivating for 24 hours in born of the same parents' incubator, culture solution is discarded, the 200 μ L H-DMEM training containing 5 μ L antibody is separately added into two kinds of cells It supports in base, after being protected from light 4 DEG C of incubation 2h, discards antibody-solutions, the H-DMEM culture containing 1 μm of ol/L Hoechst 33342 is added Base after being protected from light 37 DEG C of incubation 15min, discards solution, the 200 μ L of polypeptide solution of 1mg/mL pH5.8 is added, is protected from light 4 DEG C of incubations After 10min, polypeptide solution is discarded, and washed 3 times with the H-DMEM culture medium of the pH5.8 of pre-cooling.Control peptide takes same method, It is operated under pH7.4 environment.

With the fluorescence distribution in laser confocal microscope (ZEISS LSM 710, Germany) detection cell.As a result such as Fig. 4 Shown, the HUVEC cell that STP is added under pH5.8 environment can observe that apparent fluorescence, antibody are also specifically bound HUVEC cell surface, and 293T cell surface does not have fluorescence.Under pH7.4 environment, the HUVEC cell that control peptide TP is added can To observe apparent fluorescence, antibody is also specifically bound in HUVEC cell surface, and 293T cell surface does not have fluorescence.Together Shi Liyong Hoechst 33342 (a kind of blue fluorescent dyes for showing nucleus) carries out nuclear location, the results showed that in 10min When, STP and TP polypeptide is incorporated on the cell membrane of HUVEC cell, this is identical as VEGFR2 expressive site.

The above results explanation, the identification of STP-FITC and HUVEC are sequence specificity, STP-FITC and high VEGF expression R2 HUVEC cell be specific binding, and the 293T cell of VEGFR2 feminine gender is not specifically bound then.

The pH of experimental example 3 STP and VEGFR2 responds interaction

Human vascular endothelial system HUVEC is incubated in the H-DMEM culture solution containing 10% fetal calf serum, with 1 × 10⁵/mL Cell concentration be implanted into round Glass bottom culture dish (Φ=35mm), 37 DEG C, 5%CO₂After being cultivated for 24 hours in cell incubator, discard Culture solution.The H-DMEM culture medium containing 1 μm of ol/L Hoechst 33342 is added to discard molten after being protected from light 37 DEG C of incubation 15min The 200 μ L of polypeptide solution of 1mg/mL pH7.4 is added in liquid, after being protected from light 4 DEG C of incubations 10min, discard polypeptide solution, and with being pre-chilled The H-DMEM culture medium of pH7.4 is washed 3 times.Repetitive operation is under pH5.8 environment.Control peptide takes same method, exists respectively It is operated under pH7.4 and pH5.8 environment.

With the fluorescence distribution in laser confocal microscope (ZEISS LSM 710, Germany) detection cell.As a result such as Fig. 5 Shown, the HUVEC cell that STP is added under pH5.8 environment can observe apparent fluorescence, and be added under pH7.4 environment The HUVEC cell of STP does not have fluorescence.Control peptide TP has all shown fluorescence in both environments, but glimmering under pH7.4 environment Luminous intensity is stronger.

The above results explanation, STP-FITC have pH responsiveness, can specific recognition VEGFR2 egg in acid condition It is white.

The Cell permeable of 4 dynamic cellular of experimental example verifying STP under pH5.8 environment

Human vascular endothelial system HUVEC is incubated in the H-DMEM culture solution containing 10% fetal calf serum, with 1 × 10⁵/mL Cell concentration be implanted into round Glass bottom culture dish (Φ=35mm), 37 DEG C, 5%CO₂After being cultivated for 24 hours in cell incubator, discard Culture solution.The H-DMEM culture medium containing 1 μm of ol/L Hoechst 33342 is added to discard molten after being protected from light 37 DEG C of incubation 15min Liquid is added the 200 μ L of polypeptide solution of 1mg/mL pH 5.8, is protected from light 4 DEG C and is incubated for 0min, 5min, 10min, 15min respectively, After 20min, polypeptide solution is discarded, and washed 3 times with the H-DMEM culture medium of the pH5.8 of pre-cooling.Control peptide takes same method, It is operated under pH5.8 environment.

With the fluorescence distribution in laser confocal microscope (ZEISS LSM 710, Germany) detection cell.As a result such as Fig. 6 Shown, under pH5.8 environment, as time increases, polypeptide starts to be incorporated in cell membrane surface, after 15min, STP-FITC It progresses into cytoplasm, it is more obvious that born of the same parents' situation is entered in 20min.There is no displays to enter and leave under identical condition for control peptide The case where born of the same parents.

The above results explanation, STP-FITC not only has VEGFR2 targeting, but also has pH responsiveness, can be in acidity Under the conditions of slowly enter cell interior.

From experimental example 1-4, it can be concluded that, polypeptide of the invention not only has targeting VEGFR2 protein positive tumour cell table The characteristic of face blood vessel, and there is pH responsiveness, cell interior can be slowly entered in acid condition.Thus in practical application In, it can be mutually conjugated or mix with the preparation that can kill cancer cell, for tumour using polypeptide of the invention as target polypeptide Targeted therapy.

2 bivalents of embodiment/multivalent preparation and its functional verification

Quadrivalent is prepared using Fmoc-Lys (Fmoc)-OH as starting point, lysine is subjected to deprotection 10min, vacuum first It filters, uses n,N dimethylformamide respectively, methylene chloride cleans three times, and Fmoc-Lys (Fmoc)-OH is added and is coupled, even It after connection, is deprotected, cleaning.A certain number of Pro, Ser, Leu, Pro, Phe, Asn, Asn, Lys, His are added again, Trp, Glu, Glu, Asp, Lys, Ser, Cys and the HTBU of equivalent are successively reacted.After coupling reaction, remove-insurance is carried out Shield, cleaning.It is protected from light coupling FITC fluorescent dye again, is deprotected, cleaning.The finally lysate into above-mentioned resin, abjection side chain are protected Group is protected, vacuum is drained, and quadrivalent polypeptide is obtained.

It carries out cell experiment and verifies its function, by human vascular endothelial system HUVEC and human renal epithelial cell line 293T points It is not incubated in the H-DMEM culture solution containing 10% fetal calf serum, with 1 × 10⁵The cell concentration of/mL is implanted into round glass bottom culture Ware (Φ=35mm), 37 DEG C, 5%CO₂After being cultivated for 24 hours in cell incubator, culture solution is discarded, is separately added into and contains in two kinds of cells There is the H-DMEM culture medium of 1 μm of ol/L Hoechst 33342, after being protected from light 37 DEG C of incubation 15min, discards solution, 1mg/mL is added 200 μ L of polypeptide solution, after being protected from light 4 DEG C of incubation 10min, discard polypeptide solution, and washed 3 times with the H-DMEM culture medium of pre-cooling.

The preparation of 3 Imaging agent of embodiment

The wang-Gly resin for weighing 300mg recycles according to Solid phase peptide synthssis program, sequentially adds 300mg Pro, 510mg Ser, 320mg Leu, 300mg Pro, 350mg Phe, 540mg Asn, 540mg Asn, 420mg Lys, 560mg His, 470mg Trp, 380mg Glu, 380mg Glu, 370mg Asp, 420mg Lys, 510mg Ser and the HTBU of equivalent according to It is secondary to be reacted.It after coupling reaction, is deprotected, cleaning.Acp is added to be reacted, after coupling, is deprotected, cleaning. FITC is added to carry out being protected from light coupling reaction, after reaction, is deprotected, cleaning.The finally lysate into above-mentioned resin deviates from side Chain blocking group, vacuum are drained, and obtain crude product polypeptide fluorescence conjugate, then carry out HPLC purifying, obtain purity up to 95% it is more Peptide Imaging agent, can be used for cell and animal imaging.

The preparation of 4 polypeptide coupling of embodiment

By taking the preparation of polypeptide fluorescence conjugate as an example, the wang-Gly resin of 300mg is weighed, according to Solid phase peptide synthssis program Circulation, sequentially adds 300mg Pro, 510mg Ser, 320mg Leu, 300mg Pro, 350mg Phe, 540mg Asn, 540mg Asn, 420mg Lys, 560mg His, 470mg Trp, 380mg Glu, 380mg Glu, 370mg Asp, 420mg Lys, 510mg Ser and the HTBU of equivalent are successively reacted.It after coupling reaction, is deprotected, cleaning.Continuously add ε-ammonia Base caproic acid is reacted, in the solution that pyridine/n,N dimethylformamide/methylene chloride ratio is 1:5:7, by FITC and peptide Pearl hybrid reaction is stayed overnight.After reaction, it is deprotected, cleaning.The finally lysate into above-mentioned resin deviates from Side chain protective group Group, vacuum are drained, and crude product polypeptide fluorescence conjugate is obtained.

The preparation of 5 kit of embodiment

Polypeptide fluorescein isothiocynate (FITC) conjugate is obtained using solid phase synthesis process, micro- in the polypeptide recombined Continue to be coupled ε-aminocaproic acid on pearl.It, will in the solution that pyridine/n,N dimethylformamide/methylene chloride ratio is 1:5:7 FITC and peptide pearl hybrid reaction are stayed overnight.Polypeptide FITC conjugate is obtained after lysate cracks, using MALDI-TOF identification and HPLC purifying is used for subsequent experimental.

Human vascular endothelial system HUVEC and human renal epithelial cell line 293T are incubated at respectively containing 10% fetal calf serum In H-DMEM culture solution, with 1 × 10⁵The round Glass bottom culture dish (Φ=35mm) of cell concentration implantation of/mL, 37 DEG C, 5%CO₂Carefully After cultivating for 24 hours in born of the same parents' incubator, culture solution is discarded, the 200 μ L H-DMEM training containing 5 μ L antibody is separately added into two kinds of cells It supports in base, after being protected from light 4 DEG C of incubation 2h, discards antibody-solutions, the H-DMEM culture containing 1 μm of ol/L Hoechst 33342 is added Base discards solution after being protected from light 37 DEG C of incubation 15min, and the 200 μ L of polypeptide solution of 1mg/mL is added, after being protected from light 4 DEG C of incubation 10min, Polypeptide solution is discarded, and is washed 3 times with the H-DMEM culture medium of pre-cooling.It can carry out cell imaging experiment.

Although above the present invention is described in detail with a general description of the specific embodiments, On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.

Claims (10)

1. the polypeptide of a kind of pH response and special target human tumour vascular markers VEGFR2, which is characterized in that the polypeptide Amino acid sequence is CSKDEEWHKNNFPLSPGM.

2. bivalent/the multivalent formed by polypeptide described in claim 1, which is characterized in that the bivalent/multivalent is The polypeptide is covalently attached by connection molecule or by mixing with polymer, and formation is not covalently linked.

3. a kind of pharmaceutical composition, which is characterized in that preparation and polypeptide described in claim 1 comprising cancer cell can be killed And/or bivalent/multivalent as claimed in claim 2.

4. a kind of Imaging agent, which is characterized in that include imaging agent and polypeptide described in claim 1 and/or claim 2 institute Bivalent/the multivalent stated.

5. a kind of polypeptide coupling, which is characterized in that the polypeptide coupling is by polypeptide described in claim 1 and carrier conjugation It obtains；The carrier is toxin, cell factor, radioactive element, carrier protein, antibody, enzyme, agglutinin, fluorophor, quantum Point or high absorptivity chromophore.

6. polypeptide coupling according to claim 5, which is characterized in that the carrier is antibody or fluorophor.

7. polypeptide coupling according to claim 6, which is characterized in that the antibody is the anti-human source of phycoerythrin label VEGFR2 antibody；The fluorophor is fluorescein isothiocynate.

8. a kind of for detecting the examination of people's solid tumor vascular surface cell or vascular endothelial cell growth factor R-2 albumen Agent box, which is characterized in that include polypeptide described in claim 1 or the described in any item polypeptide couplings of claim 5-7.

9. polypeptide described in claim 1 or bivalent/multivalent as claimed in claim 2 preparation for treating, prevent or The drug for diagnosing cancer or the application in Imaging agent.

10. application according to claim 9, which is characterized in that the cancer is tumor surface blood vessel V EFGR2 albumen mistake The cancer of expression.