WO2014019520A1 - 一种肿瘤血管特异性结合多肽及其应用 - Google Patents

一种肿瘤血管特异性结合多肽及其应用 Download PDF

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WO2014019520A1
WO2014019520A1 PCT/CN2013/080568 CN2013080568W WO2014019520A1 WO 2014019520 A1 WO2014019520 A1 WO 2014019520A1 CN 2013080568 W CN2013080568 W CN 2013080568W WO 2014019520 A1 WO2014019520 A1 WO 2014019520A1
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polypeptide
seq
tumor
vegfr
amino acid
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PCT/CN2013/080568
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English (en)
French (fr)
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朱毅敏
原丽华
王安欣
濮科锋
陈丽莎
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中国科学院苏州纳米技术与纳米仿生研究所
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Priority claimed from CN201210270640.5A external-priority patent/CN102775474B/zh
Priority claimed from CN 201210268468 external-priority patent/CN102757482B/zh
Application filed by 中国科学院苏州纳米技术与纳米仿生研究所 filed Critical 中国科学院苏州纳米技术与纳米仿生研究所
Publication of WO2014019520A1 publication Critical patent/WO2014019520A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids

Definitions

  • Tumor vascular specific binding polypeptide and application thereof
  • the present invention relates to the field of biomedicine, and in particular to a tumor blood vessel VEGFR-2 factor specific binding polypeptide and use thereof. Background technique
  • Tumor-targeted therapy technology has the characteristics of strong specificity, significant effect, and basically does not damage normal tissues.
  • Monoclonal antibodies are currently known as tumor targeting molecules for tumor targeted therapy.
  • 8 antibodies have been approved by FM for clinical treatment of tumors.
  • the antibody still has the following problems in the treatment of solid tumors:
  • the cells of the solid tumor are encapsulated by a dense matrix, which makes it difficult for the antibody to penetrate the cells of the solid tumor; the internal pressure of the solid tumor is high, which hinders the entry of the antibody into the tumor, resulting in antibody treatment.
  • the efficacy of large-volume solid tumors is not ideal; because of the large amount of antibodies required to treat tumors, high purity of the product is required, so the production cost of antibodies is high and expensive.
  • using bevacizumab for 10 months will cost about 4 $40,000.
  • antibody tumor therapy has achieved some success, scientists are still trying to find other biomolecules for targeted therapy of tumors.
  • the polypeptide is considered to have at least the same effect as the targeted therapeutic treatment of the antibody, and the polypeptide has the advantages of small molecular weight, low antigenicity, rapid action, easy in vitro and biosynthesis, and can have the function of ordinary proteins, so the polypeptide can be directly It is used as an anti-tumor drug and can also be used as a targeting molecule in combination with other methods.
  • the tumor vascular endothelial growth factor (VEGF) receptor (VEGFR) is unique to tumor sites and can be an ideal target for vascular targeted therapy.
  • VEGF tumor vascular endothelial growth factor
  • VEGFR-2 tumor vascular endothelial growth factor-2
  • FLK-1 or KDR tumor vascular endothelial growth factor-2
  • Vascular endothelial growth factor receptor-2 its unique s ignal ing and spec i fi c 1 igand, VEGF-E. Cancer Sc i 2003 ; 94 (9) : 751- 756, and Prade CR, Suni la ES, Kuttan G.
  • VEGF Vascular Endothelial Growth Factor
  • VEGF Receptors in Tumor Angiogenes are and Mal ignanc i es. Integrat ive Cancer Therapi es 2005; 4 (4): 315.
  • VEGFR-2 is mainly expressed in tumor vascular endothelial cells, but in tumor epithelial cells. No expression or low expression. This suggests that VEGFR-2 plays a very important role in promoting angiogenesis and regeneration, so the development of inhibitors against VEGFR-2 will effectively inhibit tumor growth.
  • monoclonal antibodies against VEGFR-2 have been developed, see Prewett M, Huber J, Li Y, Sant iago A, 0' Connor W, King K, Overholser J, Hooper A, Pytowski B, Witte L.
  • Ant Ivascular Endothelial Growth Factor Receptor (Fetal Liver Kinase 1) Monoclonal Ant ibody Inhibits Tumor Angiogenesi s and Growth of Several Mouse and Human Tumors. Cancer Research, Volume 59: AACR; 1999. p 5209-5218. However, the toxic side effects and long-term effects of the monoclonal antibody still need to be further identified.
  • the invention provides a tumor vascular-specific binding polypeptide that targets VEGFR-2.
  • an amino acid sequence which is FF/YEXWGVK In a first aspect of the invention, there is provided an amino acid sequence which is FF/YEXWGVK. In a second aspect of the invention, there is provided a VEGFR-2 binding active polypeptide, said polypeptide having the following amino acid core sequence: FF/YEXWGVK.
  • the polypeptide has a length of 25 a, preferably 20 a.
  • the structural formula of the polypeptide is as follows:
  • Z1 is a peptide having no or 1-7 amino acids
  • XI is F or Y
  • X2 is any amino acid, (preferably any of the 20 natural amino acids);
  • Z2 is a peptide having no or 1-5 amino acids.
  • the Z1 or Z2 is a hydrophilic amino acid.
  • Z1 is a peptide of 1-5 amino acids, preferably 1-3, more preferably 1-2;
  • X2 is selected from the group consisting of A, M, I, S, E, L, T, R;
  • Z2 is a peptide of 1-4 amino acids, preferably 1-3, more preferably 1-2.
  • the invention provides a polypeptide, the polypeptide having a sequence selected from the group consisting of:
  • the derivative polypeptide is any one of SEQ ID ⁇ 0.: 1-16, after 1-3, preferably 1-2, more preferably 1 amino acid substitution, deletion and / or a polypeptide formed after addition and having VEGFR-2 binding activity.
  • sequence of the derivative polypeptide is SRLVGDFFGTWGVKY (SEQ ID NO.: 17)
  • polypeptide is selected from the group consisting of:
  • SEQ ID NO.: 2 a polypeptide formed by 1-3, preferably 1-2, more preferably 1-3 amino acid substitutions, deletions and/or additions and having VEGFR-2 binding activity . ;
  • said having VEGFR-2 binding activity means in an assay for detecting the proportion of bacteria having a red fluorescent label by flow cytometry
  • Pb Pl/P0, where P0 is the binding fluorescence of the test peptide before elution to VEGFR-2, and P1 is the binding fluorescence of the test peptide after elution to VEGFR-2.
  • the derivative polypeptide retains 70% of the VEGFR-2 binding activity of the polypeptide of SEQ ID NO: 1.
  • the derivative polypeptide retains 70% of the VEGFR-2 binding activity of the polypeptide of SEQ ID NO: 2.
  • the derivative polypeptide is 80% identical to SEQ ID NO: 1-16, preferably 90%; more preferably 95%.
  • an isolated polynucleotide encoding the amino acid sequence of the first aspect of the invention, the polypeptide of the second or third aspect of the invention.
  • a pharmaceutical composition comprising the pharmaceutical composition
  • the pharmaceutical composition is in the form of an injection, a solution, a powder, a spray or the like.
  • an active ingredient selected from the group consisting of:
  • the active ingredient is used to prepare a medicament for treating a tumor or binding to VEGFR-2.
  • the tumor blood vessel expresses VEGFR_2.
  • the tumor is a malignant tumor.
  • the tumor comprises melanoma, non-small cell lung cancer, glioma, breast cancer or other tumors highly expressing VEGFR2.
  • the use of the amino acid sequence according to the first aspect of the invention, or the polypeptide of the second or third aspect of the invention, for the preparation of a tumor localization diagnostic reagent or kit is provided.
  • the reagent or kit further comprises magnetic particles capable of binding to the polypeptide, Light particles and other multimodal nanoparticles.
  • the magnetic particles comprise magnetic nanoparticles or superparamagnetic nanoparticles.
  • the reagent is a contrast agent.
  • the tumor blood vessel expresses VEGFR_2.
  • the tumor comprises melanoma, non-small cell lung cancer, glioma, breast cancer or other tumors that highly express VEGFR2.
  • a method of treating a tumor comprises administering a pharmaceutical composition according to the fifth aspect of the invention to a subject in need thereof.
  • a method for tumor localization diagnosis comprising the steps of:
  • the contrast agent is a superparamagnetic nanoparticle, that is, Fe 2 O 3 having a hydration radius of 15 nm.
  • the administration includes intravenous injection, oral administration, local injection, intraluminal injection, and enema.
  • Figure 1 shows the use of bacterial surface display technology to screen for a polypeptide sequence that specifically binds to VEGFR-2, 96% of which have a conserved sequence of FF/YEXWGVK.
  • Figure 2 shows the construction of a library of predisposed bacterial peptides around conserved sequences, and a model was constructed:
  • Z1 and Z2 are peptides of five hydrophilic amino acids), and the polypeptide library which binds to VEGFR-2 with higher affinity and specificity is screened by using the library of the preferred polypeptide.
  • Figure 3 shows a fluorescence micrograph of the core polypeptide and the control polypeptide. It can be seen from the figure that the chemically synthesized rhodamine-conjugated core sequence FF/YEXWGVK can target HUVEC cells with high expression of VEGFR-2 receptor and the rhodamine-conjugated control polypeptide does not bind to HUVEC cells.
  • Figure 4 shows the affinity of the SEQ ID No. 2 polypeptide and its competitive binding relationship to VEGF using the SPR method.
  • Figure 4A shows the response of the chip covering VEGFR-2;
  • Figure 4B shows the response of different concentrations of the SEQ ID No. 2 polypeptide through the immobilized VEGFR2 protein chip;
  • Figure 4C shows that the SEQ ID No. 2 polypeptide competes with VEGF sexual binding to VEGFR-2;
  • Figure 4D shows that VEGF can compete with VEGFR-2 for competitive binding to the SEQ ID No. 2 polypeptide.
  • Figure 5 shows that the SEQ ID No. 2 polypeptide can inhibit the proliferation of HUVEC cells induced by VEGF in a concentration-dependent manner.
  • Figure 6 shows the specificity and guiding effect of the SEQ ID No. 2 polypeptide.
  • Figure 6A shows SEQ ID No. 2
  • the polypeptide (K5) can specifically target cells that highly express VEGFR-2;
  • Figure 6B shows that the SEQ ID No. 2 polypeptide (K5) can be used to isolate VEGFR-2 expressing cells after coupling with magnetic beads;
  • Figure 6C shows It was confirmed by MRI imaging that the superparamagnetic nanoparticles could be specifically bound to HUVEC cells, and this binding was concentration-dependent.
  • Figure 7 shows the specificity and guiding effect of the polypeptide of SEQ ID No. 14.
  • Figure 7A shows that SEQ ID No. 14 polypeptide (VI) can specifically target melanoma cell B16;
  • Figure 7B SEQ ID No. 14 polypeptide (VI) can be used to isolate melanoma cell B16 after coupling with magnetic beads;
  • 7C shows nuclear magnetic imaging images of different concentrations of superparamagnetic nanoparticles without coupling/coupling the polypeptide of SEQ ID No. 14 (VI). It was confirmed from the MRI image of Fig. 7C that the polypeptide of SEQ ID No. 14 (VI) can direct superparamagnetic nanoparticles to specifically bind to HUVEC cells, and this binding is concentration-dependent.
  • Figure 8 shows that the polypeptide of SEQ ID No. 2 can inhibit the growth of tumor tissues in mice, and it was confirmed by CD31 immunohistochemistry that inhibition of tumor growth was caused by inhibition of tumor blood vessels. detailed description
  • the polypeptide of FF/YEXWGVK (SEQ ID NO.: 1) can specifically bind to VEGFR-2 specific to tumor blood vessels, and it was confirmed that a polypeptide derived from a certain range according to the core polypeptide also has binding activity.
  • the polypeptide and the polypeptide derived therefrom can be used for tumor localization and development, and the polypeptide has a remarkable effect of inhibiting tumor angiogenesis, thereby achieving the purpose of treating tumor.
  • the polypeptide of the invention has a small molecular weight and can penetrate a variety of natural barriers of the human body to achieve the target tumor location. On the basis of this, the present invention has been completed. Sequence diagram of the present invention
  • NVSNVSNVSNVSTTY (TYY/TAY) G table C or G, S for C or G, Y for T or ARNNSTGAGGNGTNAARNVSNVSNV C, R for A or G
  • core sequence refers to an amino acid sequence having VEGFR-2 binding activity (FF/YEXWGVK, such as SEQ ID A protein or polypeptide of NO: 1).
  • polypeptide having a core sequence refers to a protein or polypeptide having SEQ ID NO.: 1 as a core sequence and having VEGFR-2 binding activity.
  • a preferred polypeptide having a core sequence has the following structural formula:
  • Z1 is a peptide having no or 1-7 amino acids, preferably Z1 is a peptide of 1-5 amino acids, more preferably 1-3, most preferably 1-2;
  • XI is F or Y
  • X2 is any amino acid, preferably any of the 20 natural amino acids; preferably, X2 is selected from the group consisting of A, M, I, S, E, L, T, R;
  • Z2 is a peptide having no or 1-5 amino acids, preferably a peptide of 1-4 amino acids, more preferably 1-3, most preferably 1-2.
  • Z1 or Z2 is a hydrophilic amino acid.
  • a preferred polypeptide having a core sequence is selected as the polypeptides set forth in SEQ ID NO.: 1-16.
  • derived polypeptide refers to a variant having the VEGFR-2 binding activity, having the sequence of any of SEQ ID NOS: 1-6 as the original sequence.
  • variants include, but are not limited to, 1-3 (usually 1-2, more preferably 1) amino acid deletions, insertions and/or substitutions, as well as at the C-terminus and compared to the original sequence. / or N-terminal addition or deletion of one or several (usually 3 or less, preferably 2 or less, more preferably 1 or less) amino acids.
  • the function of the protein is generally not altered.
  • the addition or deletion of one or more amino acids at the C-terminus and/or N-terminus will generally not alter the structure and function of the protein.
  • the surgery Also included are the polypeptides of the invention in monomeric and multimeric forms.
  • the term also includes both linear as well as non-linear polypeptides (such as cyclic peptides).
  • polypeptide of the present invention refers to a generic term for "core sequence”, “polypeptide having a core sequence”, and "derived polypeptide”.
  • the invention also includes active fragments, derivatives and analogs of the polypeptides of the invention shown.
  • fragment refers to a polypeptide that substantially retains VEGFR-2 binding activity.
  • a polypeptide fragment, derivative or analog of the invention may be (i) a polypeptide having one or several conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) at one or more a polypeptide having a substituent group in one amino acid residue, or (iii) a polypeptide formed by fusing a core sequence with another compound (such as a compound that extends half-life of the polypeptide, such as polyethylene glycol), or (iv) an additional amino acid sequence A polypeptide formed by fusion of the polypeptide sequence (subsequent protein formed by fusion with a leader sequence, a secretory sequence or a tag sequence such as 6His).
  • a preferred class of active derivatives means that up to 3, preferably up to 2, more preferably up to 1 amino acid are replaced by amino acids of similar or similar nature to the amino acid sequence of Formula I or the corresponding original sequence. And forming a polypeptide.
  • These conservative variant polypeptides are preferably produced by amino acid substitution according to Table 1.
  • the invention also provides analogs of the polypeptides of the invention.
  • the difference between these analogs and the natural polypeptide may be a difference in amino acid sequence, a difference in the modification form which does not affect the sequence, or a combination thereof.
  • Analogs also include analogs having residues other than the native L-amino acid (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, ⁇ -amino acids). It is to be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above.
  • Modifications include: chemically derivatized forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those produced by glycosylation modifications in the synthesis and processing of the polypeptide or in further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylation enzyme or a deglycosylation enzyme. Modified forms also include sequences having phosphorylated amino acid residues such as phosphotyrosine, phosphoserine, phosphothreonine. Also included are polypeptides modified to increase their resistance to proteolytic properties or to optimize solubility properties.
  • the polypeptides of the invention may also be used in the form of a salt derived from a pharmaceutically or physiologically acceptable acid or base.
  • These salts include, but are not limited to, salts formed with: hydrochloric acid, hydrobromic acid, sulfuric acid, citric acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, succinic acid, oxalic acid, fumaric acid, malay Acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, or isethionic acid.
  • Other salts include: salts with alkali or alkaline earth metals such as sodium, potassium, calcium or magnesium, as well as esters, carbamates or other conventional "prodrugs". Coding sequence
  • the invention also relates to polynucleotides encoding the polypeptides of the invention, respectively.
  • a preferred nucleotide sequence for the core sequence of the coding sequence is:
  • SEQ ID NO: 23 TTCTACGAANNSTGGGGAGTTAAA. It encodes the core fragment shown by SEQ ID NO: 1.
  • SEQ ID NOS. 2-16 and other derived polypeptides the coding sequences can be readily determined by those skilled in the art based on codon correspondence.
  • the polynucleotide of the present invention may be in the form of sputum or RNA.
  • the DNA can be a coding strand or a non-coding strand.
  • the coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 22, SEQ ID NO: 23 or a degenerate variant.
  • a "degenerate variant" in the present invention refers to a polypeptide encoding a sequence having the sequence of SEQ ID NO: 1, but corresponding to the coding in SEQ ID NO: 22 and SEQ ID NO: 23.
  • the full length sequence encoding a nucleotide of the present invention or a fragment thereof can be usually obtained by a PCR amplification method, a recombinant method or a synthetic method.
  • DNA sequences encoding the polypeptides of the present invention (or fragments thereof, or derivatives thereof) have been obtained by chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (e.g., vectors) and cells known in the art.
  • the invention also relates to vectors comprising the polynucleotides of the invention, and to host cells genetically engineered using the vectors of the invention or the polypeptide coding sequences of the invention.
  • the invention also encompasses monoclonal and polyclonal antibodies, particularly monoclonal antibodies, of the polypeptides of the invention.
  • the bacterial surface display random peptide library used in the present invention is a Patrick s. Daugherty experiment from the Department of Chemical Engineering, University of California, Santa Barbara. Room purchased. Inoculate at least 10 times the library capacity of the frozen peptide library from the surface of the frozen bacteria to 100 mL of SOC medium containing 34 ⁇ g/mL chloramphenicol and 2 mg/mL glucose at 37, 200 rpm. Incubate overnight under conditions.
  • a certain amount of bacteria was taken from the overnight cultured bacteria at a ratio of 1:50, and added to 5 mL of chloramphenicol LB medium containing 34 ⁇ g/mL, and cultured at 37 ° C, 200 rpm for 2 hours. 0D 6 . . The value is between 0. 5-0. 6.
  • the arabinose was added in an amount of 200 ⁇ g/mL, and cultured at room temperature (about 25 ° C) and 200 rpm for about 1 h, which was 0 D 6 . . The value reached 0. 8-1. 0, and the total number of bacteria was calculated according to a 0D equal to IX 10 9 cells.
  • the bacterium containing an amount of about 1 X 1 CT bacteria was taken out from the above-mentioned induced bacterial solution, and after centrifugation, it was resuspended in PBS buffer having a value of 7.4.
  • magnetic beads equal to a ratio of about 100:1 (number ratio)
  • streptavidin-coated magnetic microbeads were added for negative sorting of magnetic beads.
  • streptavidin-coated magnetic beads and magnetic frames were purchased from Invitrogen, and the streptavidin-coated magnetic beads were sold under the trade name Dynabeads® My0neTM, trade number 656-01.
  • Dynabeads® My0neTM trade number 656-01
  • VEGFR-2 protein is biotinylated.
  • the VEGFR-2 protein (65bp-2493bp encoding nucleotide) is commercially available from PR0SPEC. PKA-242.
  • the biotinylated VEGFR-2 protein was mixed with the negatively selected bacteria in a ratio of protein: bacteria equal to about 500:1 (number ratio), so that the concentration of VEGFR-2 protein in the mixture was 10 nM, in a silent rotation Incubate for 45 minutes at 4 ° C on the mixer. After the end of the incubation, the cells were centrifuged at 3,800 rpm and 4 ° C, the supernatant was removed, and the pellet was resuspended in 1 mL of PBS buffer solution.
  • the magnetic beads bacteria in a ratio of about 1: 100 (number ratio), the magnetic beads were mixed with the bacteria resuspended in PBS buffer, washed with PBS buffer for 3 times, resuspended in 1 mL LB, and taken out 10 ⁇ L is diluted 100 times and its 0D 6 is measured. . After the value, the solution was coated on a solid LB plate and cultured overnight at 37 ° C. On the next day, the bacterial library capacity after magnetic bead sorting was calculated based on the number of bacterial clones grown. The remaining bacteria were resuspended in 5 mL of SOC medium and cultured overnight at 37 and 200 rpm for flow sorting.
  • the pellet was resuspended in 100 L of SAPE stain for 45 minutes. After the incubation, centrifugation was carried out for 5 minutes at 4, 3000 rpm, and the supernatant was discarded. The pellet was resuspended in 600 ⁇ L of PBS buffer at pH 7.4 so that the concentration of bacteria in PBS buffer was approximately 5 X 106 to 1 X 10 7 per ml. The bacteria resuspended in PBS buffer were sorted by flow cytometry.
  • the surface of the bacteria combined with biotinylated VEGFR-2, gp, surface display can be specific Bacteria that bind to a polypeptide of the VEGFR-2 protein emit a higher fluorescence intensity upon excitation by a 488 nm source than bacteria that do not bind to the VEGFR-2 protein.
  • the bacteria of high fluorescence value are sorted by the sorting function of the flow cytometer, that is, the bacteria having the surface bound to the biotinylated VEGFR-2 protein are sorted.
  • the sorted bacterial-protein complex was inoculated into 5 mL of S0C medium for overnight culture. During the culture, the protein was degraded, the bacteria were enriched, and the enriched bacteria were again brought into the flow sorting process.
  • a polypeptide having entangled with any five hydrophilic amino acids on both sides of the amino acid sequence of SEQ ID NO.: 1 was ligated to the N-terminus of CPX.
  • the sequence encoding the above polypeptide was amplified by PCR.
  • the template for the PCR reaction is plasmid PB33CPX-SAP.
  • N represents any nucleotide of A, T, C, G, V represents A, C or G, S represents C or G, and Y represents or (, R represents or 6.
  • the PCR amplification product was digested and ligated into alajGFP1 downstream of the same pB33CPX vector (Bessette, PH et al. Rapid isolat of high-affinity protein binding pept ides using bacterial di splay. (2004) Protein Eng. Des. Sel. 17, 731-739) on the multiple cloning site.
  • the transformed pB33CPX plasmid was transformed into E.
  • the bacterial peptide library comprises from about 2 X 10 8 polypeptides.
  • the successful construction of a biased bacterial display library was performed in the same manner as the random display library for the screening of VEGFR-2 binding polypeptides.
  • the peptide was analyzed by a surface plasmon resonance instrument Biacore 3000 for the affinity between the polypeptide and VEGFR-2.
  • the specific process is as follows: First, inject 50 ⁇ L of NHS/EDC mixture (0.05 mol/L NHS and 0.2 mol/L EDC, mixed by volume ratio of 1:1 before use) at a flow rate of 10 ⁇ L/min for activation of the chip.
  • the present invention also innovatively uses the SPR method for competitive binding assays of polypeptides and VEGF to VEGFR-2. 5. Method for detecting the effect of cell proliferation of the polypeptide of the present invention
  • MTT methyl thiazolyl tetrazolimu assay was used to detect the inhibition of cell proliferation by peptides.
  • Primary HUVEC cells or B16 melanoma cells were seeded in 96-well plates at 5000 cells per well. Before starting the experiment, the cells were first adhered for 12 h, then replaced with serum-free culture and cultured for another 24 h. Some wells were supplemented with 30 ng/mL recombinant human VEGF165, and others were spiked with 2 ⁇ g/mL-100 ⁇ g/mL in addition to the equivalent amount of VEGF165.
  • the polypeptide was coupled to a magnetic polystyrene pellet (magnetic beads) using Invitrogen's DYNAL kit, product number 650.11.
  • Invitrogen's DYNAL kit product number 650.11.
  • DYNAL kit product number 650.11
  • HUVEC, B16 and HLF were digested and resuspended in 1 mL of PBS buffer to prepare a suspension containing approximately 2 ⁇ 10 6 cells, and the magnetic beads-conjugated polypeptide was added to the cell suspension at 4°. C was incubated on a silent mixer for 10 minutes, separated by a magnetic stand purchased from Invitrogen, and the amount of cells in the separated precipitate and supernatant was measured. According to the results of the above two experiments, it was confirmed that the polypeptide was expressed in vitro. Targeting of cells of the VEGFR-2 protein. The polypeptide superparamagnetic nanoparticles (iron core diameter 6.
  • HUVEC, B16 and HLF cells were planted in 6-well plates, and about 5 ⁇ 10 5 cells were seeded per well.
  • the medium was DMEM supplemented with 10% fetal bovine serum.
  • Fe particles were added to different pores at a concentration of 128 ⁇ g. /mL of the above superparamagnetic nanoparticles linked to different polypeptides. After incubation with the cells for 2 h at 37 ° C, the PBS buffer was rinsed 4 times to remove those superparamagnetic nanoparticles that were not bound to the cells.
  • the cells were collected by centrifugation at 800 rpm for 3 minutes, resuspended in 0.6 mL of PBS buffer and 0.6 mL of 2% paraformaldehyde fixative, placed at 4 ° C for 1 h, washed 3 times with PBS buffer, and centrifuged at 800 rpm for 3 minutes. Add 300 ⁇ L of PBS buffer and 200 ⁇ L of 2% low melting point agarose to the pellet, mix and add to the nucleus tube. The above-mentioned incubated cells were examined by a nuclear magnetic imager to further verify the targeting of the polypeptide.
  • mice In 3-4 weeks old nude mice, lxlO 6 U87MG suspension cells were injected under the armpits. After 14 days, the mice were sacrificed to the appropriate size and the mice were sacrificed. The tumor tissue pieces were removed and placed in a Petri dish with 13 ⁇ 4 7.4 PBS. After rinsing three times, the tumor tissue was cut into a size of 1 mm with a surgical scissors, and the tumor tissue cut into small pieces was pushed into the hind leg of the mouse by the inoculation needle. In order to prevent the tissue pieces from falling out, the inoculation needle was at least entered into the mouse. Subcutaneous lcm far.
  • mice were randomized on the third day after inoculation of tissue blocks.
  • One group was used to inject the experimental group of the polypeptide K5 (SEQ ID NO.: 2) or K11 (SEQ ID NO.: 14), and the other control group was injected with the same volume of physiological saline, which was injected every other day. Tumor volume was recorded every two days during the injection, and the mice were sacrificed and photographed after 21 days of continuous injection. Tumor tissues from the experimental group and the control group were subcutaneously placed in 4% paraformaldehyde for subsequent paraffin section and immunohistochemistry.
  • the paraformaldehyde-fixed tumor tissue was embedded in paraffin and cut into a wax band of 5 ⁇ m thickness. Paraffin sections were dewaxed by xylene and dehydrated into gradient alcohol. After the end of dehydration, the endogenous peroxidase activity was blocked with 3% hydrogen peroxide at 37 ° C for 10 minutes, and then subjected to antigen retrieval by boiling at pH 95 ° C for 20 minutes at 95 ° C; then 5% BSA was added dropwise. The unreacted site was blocked by reaction at 37 ° C for 30 min.
  • rabbit anti-mouse monoclonal antibody CD31 (bs- 0195R, Beij ing biosynthesi s biotechnology., LTD) was added dropwise at 4 ° C overnight, the concentration of primary antibody was 1 ⁇ g / mL; I3 ⁇ 47. 4 PBS buffer rinse After the end, HRP-labeled goat anti-rabbit IgG (HA1001, Huaan Bio) was added dropwise, and reacted at 37 ° C for 30 minutes, rinsed in PBS buffer (13 ⁇ 47.
  • MB (AR1022, Boster Bio) was developed at room temperature for 20 min, washed with distilled water; Hematoxylin (AR0005, Bude Bio) was lightly counterstained for 60 seconds, washed with distilled water and immersed in hydrochloric acid alcohol for differentiation. After being taken out, rinsed under tap water for 20 min; again dehydrated with gradient alcohol. The xylene was twice transparent and then added in the middle of the material. A drop of 90% glycerol and 10% PBS was used to seal the plate with nail polish, photographed under a microscope and observed to be stained yellow-brown areas, which are tumor vascular sites. 8.
  • polypeptide of the present invention can prepare using conventional methods, such as biosynthesis, total chemical synthesis, and the like.
  • a preferred method is the use of liquid phase synthesis techniques or solid phase synthesis techniques such as Boc solid phase method, Fmoc solid phase method or a combination of both methods.
  • Solid phase synthesis allows rapid sample acquisition, depending on the sequence of the peptide of interest The appropriate resin carrier and synthesis system are selected.
  • a preferred solid phase support in the Fmoc system is a Wang resin linked to a C-terminal amino acid in the peptide, a Wang resin structure is polystyrene, and an arm between the amino acids is 4-decyloxybenzyl alcohol; using 25% hexahydropyridine /Dimethylformamide was treated at room temperature for 20 minutes to remove the Fmoc protecting group and extended from the C-terminus to the N-terminus according to the given amino acid sequence. After the completion of the synthesis, the synthesized proinsulin-related peptide was cleaved from the resin with trifluoroacetic acid containing 4% p-methylphenol, and the protective group was removed.
  • the resin was removed by filtration and the diethyl ether was precipitated to obtain a crude peptide. After the solution of the obtained product was lyophilized, the desired peptide was purified by gel filtration and reverse phase high pressure liquid chromatography.
  • the resin is a PAM resin to which a C-terminal amino acid in the peptide is attached, the PAM resin structure is polystyrene, and the arm between the amino acid is 4-hydroxymethyl phenylacetamide;
  • the protecting group Boc was removed with TFA/dichloromethane (DCM) and neutralized with diisopropylethylamine (DIEA/dichloromethane.
  • the p-cresol (5-10%) containing hydrogen fluoride (HF) is treated at 0 ° C for 1 hour, and the peptide chain is cleaved from the resin while removing the protecting group. 50-80% The peptide is extracted with acetic acid (containing a small amount of mercaptoethanol), and the solution is further lyophilized and further purified by molecular sieve Sprint haddex G10 or Tsk-40f, and then purified by high pressure liquid phase to obtain the desired peptide. It can be known in the field of peptide chemistry.
  • Various coupling agents and coupling methods are coupled to each amino acid residue, and for example, dicyclohexylcarbodiimide (DCC), hydroxybenzotriazole (HOBt) or 1, 1, 3, 3-tetra can be used. Urea hexafluorophosphate 03 ⁇ 411;) for direct coupling.
  • DCC dicyclohexylcarbodiimide
  • HOBt hydroxybenzotriazole
  • 1, 1, 3, 3-tetra 1, 1, 3, 3-tetra
  • Urea hexafluorophosphate 03 ⁇ 411; for direct coupling.
  • the synthesized short peptide the purity and structure can be confirmed by reversed phase high performance liquid chromatography and mass spectrometry.
  • the biosynthetic method has the following steps:
  • the polynucleotide sequence can be inserted into a recombinant expression vector.
  • Methods well known to those skilled in the art can be used to construct expression vectors encoding the polypeptide encoding a purine sequence and a suitable transcriptional/translational control signal.
  • Vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences, can be used to transform appropriate host cells to enable expression of the active polypeptides of the invention.
  • the host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell.
  • Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl 2 method, and the procedures used are well known in the art.
  • Another method is to use MgCl 2 .
  • Conversion can also be carried out by electroporation if desired.
  • the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like.
  • the obtained transformant can be cultured by a conventional method to express the active polypeptide of the present invention.
  • the medium used in the culture may be selected from various conventional media depending on the host cell used.
  • the cultivation is carried out under conditions suitable for the growth of the host cells.
  • the selected promoter is induced by a suitable method (e.g., temperature conversion or chemical induction) and the cells are cultured for a further period of time.
  • the polypeptide of the invention can be expressed intracellularly, or on the cell membrane, or secreted extracellularly. If necessary, the physical, chemical and other properties can be utilized to separate and purify the activity by various separation methods. Peptide. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the peptides of the polypeptide K5, RSRNAFFELWGVKRETPG (SEQ ID NO.: 14) and the polypeptide of the GGSSSGSSGGGSSGS (SEQ ID NO: . 21) of the polypeptide of the YDGNSFYEMWGVKPASES (SEQ ID NO.: 2) were respectively synthesized by Shanghai Shenggong Biotechnology Co., Ltd. In the test described below.
  • Pharmaceutical composition and method of administration were respectively synthesized by Shanghai Shenggong Biotechnology Co., Ltd. In the test described below.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising (a) a safe and effective amount of a polypeptide of the present invention or a pharmaceutically acceptable salt thereof; and (b) a pharmaceutically acceptable carrier or excipient .
  • the amount of the polypeptide of the present invention is usually from 10 ⁇ g to 100 mg / dose, preferably from 100 to 1000 ⁇ g / dose.
  • an effective dose is from about 0.01 mg/kg to 50 mg/kg, preferably from 0.05 mg/kg to 10 mg/kg body weight of the polypeptide of the invention.
  • the polypeptides of the invention may be used alone or in combination with other therapeutic agents (e.g., formulated in the same pharmaceutical composition).
  • the pharmaceutical composition may also contain a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to a carrier for the administration of a therapeutic agent.
  • pharmaceutical carriers which do not themselves induce the production of antibodies harmful to the individual receiving the composition and which are not excessively toxic after administration. These vectors are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N. J. 1991).
  • Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, adjuvants, and combinations thereof.
  • the pharmaceutically acceptable carrier in the therapeutic composition may contain a liquid such as water, saline, glycerol and ethanol.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers.
  • the therapeutic composition can be formulated as an injectable preparation, such as a liquid solution or suspension; it can also be prepared as a solid form of a liquid carrier suitable for formulation into a solution or suspension prior to injection.
  • compositions of the invention may be administered by conventional routes including, but not limited to, intratumoral, intraluminal, intramuscular, intravenous, subcutaneous, intradermal, enema or topical administration.
  • the subject to be prevented or treated may be an animal; especially a human.
  • compositions can be formulated by mixing, diluting or dissolving according to conventional methods, and occasionally adding suitable pharmaceutical additives such as excipients, disintegrating agents, binders, lubricants, diluents, buffers, isotonicity Isotonic ities preservatives, wetting agents, emulsifiers, dispersing agents, stabilizers and co-solvents, and the formulation process can be carried out in a customary manner depending on the dosage form.
  • suitable pharmaceutical additives such as excipients, disintegrating agents, binders, lubricants, diluents, buffers, isotonicity Isotonic ities preservatives, wetting agents, emulsifiers, dispersing agents, stabilizers and co-solvents, and the formulation process can be carried out in a customary manner depending on the dosage form.
  • the preparation of the intravenous solution can be carried out by dissolving the polypeptide of the present invention or a pharmaceutically acceptable salt thereof together with the essential substance in sterile water (a surfactant is dissolved in a sterile water) to adjust the osmotic pressure and pH.
  • a suitable pharmaceutical additive such as a preservative, a stabilizer, a buffer, an isotonicity agent, an antioxidant, and a tackifier may be optionally added, and then completely dissolved.
  • compositions of the invention may also be administered in the form of sustained release agents.
  • the core sequence or its salt can be uncomfortable
  • the pellet or microcapsule is supported by a sustained release polymer, and then the pellet or microcapsule is surgically implanted into the tissue to be treated.
  • the sustained-release polymer include ethylene-vinyl acetate copolymer, polyhydroxymethacrylate (polyhydrometaacrylate polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymer, A lactic acid-glycolic acid copolymer or the like is preferably exemplified by a biodegradable polymer such as a lactic acid polymer and a lactic acid-glycolic acid copolymer.
  • the dose of the polypeptide of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient may be based on the weight, age, sex, and degree of symptoms of each patient to be treated. Determine it reasonably.
  • the total dose applied per day in most cases is from 1 mg to 200 g per person, preferably from 10 mg to 100 g per person.
  • the type of tumor that can be detected and treated with the polypeptide of the present invention is not particularly remarkable, and representative examples include, but are not limited to, non-small cell lung cancer and glioma.
  • Solid tumors are preferred, especially tumor types that overexpress VEGFR-2.
  • VEGFR-2 is highly expressed in tumor blood vessels, and VEGFR-2 is also overexpressed in tissues at wound healing, and is not expressed or expressed in other normal tissues. During solid tumor formation, overexpression of VEGFR-2 ensures tumor angiogenesis and provides an energy supply for rapid tumor growth.
  • the polypeptide of the present invention specifically binds to VEGFR-2 which is specifically expressed by tumor blood vessels, whereby when the polypeptide of the present invention is coupled to a contrast agent, it exhibits a phenomenon of concentration in nuclear magnetic resonance or other imaging examination. According to this, the tumor site or tumor metastasis site can be judged.
  • a preferred contrast agent is a superparamagnetic nanoparticle, i.e., Fe 2 O 3 having a hydration radius of 15 nm. It will be appreciated that there are other contrast agents that can be conjugated to the polypeptides of the invention, which can be obtained experimentally by those skilled in the art in light of the teachings of the present invention.
  • the polypeptide of the present invention is coupled to the contrast agent and can reach the location of the tumor by intravenous injection, oral administration, local injection, intraluminal injection, enema, and the like.
  • the main advantages of the invention include -
  • polypeptide of the present invention specifically binds to tumor blood vessel VEGFR-2, and effectively inhibits tumor growth;
  • the polypeptide of the present invention can be prepared as a contrast agent, specifically identifying a tumor growth site, thereby performing tumor localization diagnosis;
  • polypeptide of the present invention has a small molecular weight and is easily permeable to various physiological barriers;
  • (d) can be prepared by solid phase synthesis, with high purity, high yield and low cost;
  • the polypeptide of the present invention is expected to be developed into a medicament for the diagnosis or treatment of tumors (especially solid tumor types expressing VEGFR-2).
  • the invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention.
  • the experimental methods in the following examples which do not specify the specific conditions are usually produced according to the conditions described in the conventional conditions, for example, Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturing conditions. Manufacturer's recommendation conditions of. Unless otherwise stated, percentages and parts are by weight and parts by weight.
  • Example 1 Preliminary screening of polypeptides that bind well to VEGFR-2 and obtain their core sequences
  • the bacterial liquid is collected and the collected bacterial liquid is inoculated into 5 mL LB medium and cultured overnight. From the overnight cultured bacteria, 10 w L was coated on LB solid medium and cultured overnight at 37 °C. 50 monoclonal bacteria were randomly selected from the plate, numbered clonel-clone50, inoculated into 5 mL LB liquid medium, cultured overnight at 37 and 200 rpm, and peptides were cultured separately for the overnight cultured bacteria. Sequencing, here, sequencing of the peptides at Shanghai Shenggong Bioengineering Co., Ltd.
  • a biased peptide library for flow screening was constructed according to the method described in General Method 2, and a successful biased bacterial display library was constructed for the screening of VEGFR-2 binding polypeptides in the same manner as the random display library.
  • the peptide affinity test was carried out in accordance with General Method 4, and the fluorescence binding data of Fig. 2 was obtained.
  • the core peptide FF/YEXWGVK and the control polypeptide C1 (SEQ ID NO.: 21), which were coupled with rhodamine, were synthesized in Shanghai Botai Biotechnology Co., Ltd., and 100 ⁇ M fluorescent peptide and 4% paraformaldehyde cultured overnight were used.
  • the fixed HUVEC cells were incubated for 1 hour at 37 ° C and observed and photographed by fluorescence microscopy (Nikon Ti, Japan ). It can be seen from Fig. 3 that the core polypeptide FF/YEXWGVK binds to HUVEC cells with high expression of VEGFR2.
  • the control polypeptide was completely unbound.
  • Example 5 Chemical synthesis of SEQ ID No.
  • RESULTS The injection of 50 VEGF protein at a concentration of 25 nM and entering the PBS washing procedure showed that the RU value was 77. 9; when the VEGF protein was injected, it did not enter the PBS washing process but directly injected the peptide K5 (SEQ ID NO. 2: The RU value read by the instrument is 46.1.
  • the VEGFR-2 conjugated chip was injected with 3.57 uM of peptide K5 (SEQ ID NO.: 2) and injected with VEGF protein.
  • the RU value decreased from 200. 8 to 163.64.
  • a combination of two reductions in RU values indicates that the binding site of K5 (SEQ ID NO.: 2) to VEGFR2 is consistent with the binding site of VEGF to VEGFR-2.
  • Figure 6B shows the amount of cells in the pellet and supernatant separated by a magnetic stand, the ordinate indicates the percentage of cells in the pellet and supernatant and the total number of cells, and the HLF C1 in the abscissa indicates that the beads are coupled.
  • polypeptide obtained by co-incubation of polypeptide C1 (SEQ ID NO.: 21) with human lung fibroblasts, HLF K5 (representing that peptide K5 (SEQ ID NO.: 2) coupled with magnetic beads is associated with human lung fibroblasts
  • HUVEC K5 indicates a cell in which magnetic beads-conjugated polypeptide K5 (SEQ ID NO.: 2) was incubated with HUVEC
  • HUVEC C1 indicates a polypeptide-coupled polypeptide CI (SEQ ID NO.: 21).
  • the cells obtained by co-incubation with HUVEC, HLF and HUVEC represent cells that were not co-incubated with the polypeptide, respectively.
  • FIG. 6C The results of nuclear magnetic imaging after coupling of the K5 (SEQ ID NO.: 2) polypeptide with the superparamagnetic nanoparticles are shown in Fig. 6C.
  • the three T2-weighted images in the figure were imaged from HUVEC cells incubated with polypeptide K5 (SEQ ID NO.: 2) in order from top to bottom, and incubated with peptide K5 (SEQ ID NO.: 2).
  • polypeptide K5 (SEQ ID NO.: 2) according to the present invention can be used as a contrast agent in combination with magnetic particles for the localization and development of tumors.
  • Example 10 Coupling of VI polypeptide-carboxy magnetic beads and in vitro functional identification
  • FIG. 7A shows that magnetic beads are distributed in a photograph labeled only with B16 VI, indicating that the magnetic peptide-conjugated polypeptide VI (SEQ ID NO.: 14) binds to melanoma cells, polypeptide V1 (SEQ ID NO.: 14) Has a good role in targeting VEGFR2.
  • Figure 7B shows that almost all of the B16 cells incubated with the magnetic beads-conjugated polypeptide V1 (SEQ ID NO.: 14) were present in the pellet.
  • Figure 7C shows the results of T2-weighted imaging of different concentrations of superparamagnetic nanoparticles and the results of nuclear magnetic imaging after co-incubation with B16 cells after coupling with V1 (SEQ ID NO.: 14) polypeptide.
  • the results of these nuclear magnetic imaging indicate that the peptide V1 (SEQ ID NO.: 14) can increase the absorption of contrast agent by B16 cells when coupled with superparamagnetic nanoparticles, and the polypeptide V1 (SEQ ID NO.: 14) can be used as a tumor.
  • An effective guiding molecule for angiography is an effective guiding molecule for angiography.
  • the polypeptide V1 (SEQ ID NO.: 14) according to the present invention can be used as a contrast agent in combination with magnetic particles for the localization and development of tumors.
  • Example 11 Inhibition of Tumor Volume and Vascular Proliferation of Tumor-bearing Mice by Peptide K5 (SEQ ID NO.: 2) The inhibition of tumor volume in tumor-bearing mice by the polypeptide as shown in Figure 8 The tumor volume of mice was significantly larger than that of the experimental group injected with peptide K5 (SEQ ID NO.: 2), tumor volume fraction. In addition, 58 3 and 17. 06 mm 3 CD31 immunohistochemistry results showed that the vascular density of the experimental group was significantly lower than that of the control group. All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the the the the the the the In addition, it is to be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

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Abstract

本发明提供了序列为FF/YEXWGVK的多肽及其衍生物。该多肽及其衍生物能够特异性结合VEGFR-2，可作为造影剂特异性标记肿瘤发生部位，并显著抑制肿瘤血管的新生，发挥抑制或治疗肿瘤的作用。

Description

一种肿瘤血管特异性结合多肽及其应用
技术领域
本发明涉及生物医药领域， 具体地， 涉及一种肿瘤血管 VEGFR-2因子特异性结 合多肽及其应用。 背景技术
据世界卫生组织统计， 每年有大约 500 万人死于癌症。 癌症已成为摧毁人类 健康、 剥夺人类生命的最危险的疾病之一， 因此癌症的预防、 诊断及治疗任务十 分艰巨。
传统的肿瘤治疗方法存在各种弊端， 例如， 手术治疗只适用于体积较大、 影像 技术可检测的良性实体瘤， 对于体积较小的肿瘤或者转移性肿瘤无能为力； 放射 疗法对局部组织的损伤较大； 化学疗法需要全身给药， 针对性差， 给患者造成巨 大的毒副作用。
鉴于以上肿瘤治疗方法中的缺陷，研究人员一直努力开发可用于肿瘤早期定位 诊断及有效治疗的药物或办法。肿瘤靶向分子的出现为解决这一难题提供了思路， 肿瘤靶向治疗技术具有特异性强、 效果显著、 基本不损伤正常组织的特点。
单克隆抗体是目前已知的、 最早应用于肿瘤靶向治疗的肿瘤靶向分子， 目前已 有 8种抗体被 FM批准应用于肿瘤的临床治疗。 但是， 目前抗体治疗实体瘤仍存 在以下问题： 因实体肿瘤的细胞被致密的基质包裹， 导致抗体难以穿透实体肿瘤 的细胞； 实体肿瘤内压力较高， 阻碍了抗体进入肿瘤内部， 致使抗体治疗大体积 实体肿瘤的疗效不理想； 由于治疗肿瘤的抗体需要量大， 要求产品纯度高， 所以 抗体的生产成本高、 价格昂贵， 据 Genentech报告， 使用贝伐单抗治疗 10个月将 花费大约 4. 4万美元。 虽然目前抗体肿瘤治疗取得一定成效， 但科学家们仍然在 努力寻找其他的生物分子对肿瘤进行靶向治疗。
多肽被认为具有至少相同于抗体的靶向引导治疗的作用，而且多肽具有分子量 小、 抗原性低、 作用迅速、 较易体外及生物合成等优点， 并且可以具有普通蛋白 质的功能， 因此多肽可以直接用作抗肿瘤药物， 也可以与其他方法合用而作为靶 向引导分子使用。
肿瘤血管内皮生长因子 （VEGF ) 的受体 （VEGFR ) 是肿瘤部位所特有的， 可作 为血管靶向治疗的理想靶点。 其中， 在所有的 VEGFR中， 仅肿瘤血管内皮生长因 子 -2 ( VEGFR-2 , 又称 FLK-1或 KDR) 既在细胞的生长和分化中起重要作用又与内 皮细胞的增殖和血管的生成有关， 请参见 Shibuya M. Vascular endothel ial growth factor receptor- 2 : its unique s ignal ing and spec i fi c 1 igand, VEGF- E. Cancer Sc i 2003 ; 94 (9) : 751- 756， 以及 Prade印 CR， Suni la ES， Kuttan G. Express ion of Vascular Endothel ial Growth Factor (VEGF) and VEGF Receptors in Tumor Angiogenes i s and Mal ignanc i es. Integrat ive Cancer Therapi es 2005； 4 (4) : 315。
在肿瘤组织中， VEGFR-2主要表达于肿瘤血管内皮细胞， 而在肿瘤上皮细胞中 不表达或低表达。这说明 VEGFR-2在促进血管发生及再生中起着非常重要的作用， 所以开发针对 VEGFR-2的抑制剂将有效抑制肿瘤的生长。 目前， 已经开发出针对 VEGFR- 2的单克隆抗体， 请参见 Prewett M, Huber J, Li Y, Sant iago A, 0' Connor W, King K, Overholser J, Hooper A, Pytowski B， Witte L. Ant ivascular Endothel ial Growth Factor Receptor (Fetal Liver Kinase 1) Monoclonal Ant ibody Inhibits Tumor Angiogenesi s and Growth of Several Mouse and Human Tumors. Cancer Research, Volume 59 : AACR ; 1999. p 5209-5218。 但该单克隆 抗体的毒副作用及长期效应仍需进一步鉴定。
因此， 本领域迫切需要开发靶向 VEGFR-2的多肽。 发明内容
本发明提供了一种靶向于 VEGFR-2的肿瘤血管特异性结合多肽。
本发明第一方面， 提供了一种氨基酸序列， 所述氨基酸序列为 FF/YEXWGVK。 本发明第二方面， 提供了一种具有 VEGFR-2结合活性多肽， 所述的多肽具有以下 氨基酸核心序列： FF/YEXWGVK。
在另一优选例中， 所述多肽的长度 25aa， 较佳地 20aa。
在另一优选例中， 所述多肽的结构式如下：
Z1-FX1EX2WGVK-Z2
式中，
Z1为无或 1-7个氨基酸的肽段；
XI为 F或 Y;
X2为任一氨基酸， （较佳地为 20种天然氨基酸中任一种）； 和
Z2为无或 1-5个氨基酸的肽段。
在另一优选例中， 所述 Z1或 Z2为亲水性氨基酸。
在另一优选例中， Z1为 1-5个氨基酸的肽段， 较佳地 1-3个， 更佳地 1-2个； 和
X2选自 A、 M、 I、 S、 E、 L、 T、 R; 禾口 /或
Z2为 1-4个氨基酸的肽段， 较佳地为 1-3个， 更佳地为 1-2个的肽段。
本发明第三方面， 提供了一种多肽， 所述多肽的序列选自下组：
(a) SEQ ID NO. 1所示的序列 (FF/YEXWGVK)
(b) SEQ ID NO. 2所示的序列 (YDGNSFYEMWGVKPASES)
(c) SEQ ID NO. 3所示的序列 (MAKGDFFERWGVKEM)
(d) SEQ ID NO. 4所示的序列 (ENVVVNTFYEMWGVK)
(e) SEQ ID NO. 5-16所示的序列; (对应于 K1- K4， K6-K13)
(f) SEQ ID NO. 1-16的衍生多肽。
在另一优选例中， 所述的衍生多肽为 SEQ ID Ν0.： 1-16中任一序列， 经过 1-3个， 较佳地 1-2个， 更佳地 1个氨基酸取代、 缺失和 /或添加后形成的且具有 VEGFR-2结 合活性的多肽。
在另一优选例中， 所述衍生多肽的序列为 SRLVGDFFGTWGVKY (SEQ ID NO.： 17)
GNFGQNFNERWGVIAD (SEQ ID NO.： 18)
在另一优选例中， 所述的多肽选自下组：
(i) 氨基酸序列如 SEQ ID NO.： 2所示的多肽；
(i i) SEQ ID NO.： 2经过 1-3个， 较佳地 1-2个， 更佳地 1-3个氨基酸取代、 缺 失和 /或添加后形成的且具有 VEGFR-2结合活性的多肽。 ；
在另一优选例中， 所述的具有 VEGFR-2结合活性指在运用流式细胞仪检测带有红 色荧光标记的细菌的比例的试验中，
(a) 结合荧光数值 1000， 较佳地 1200， 更佳地 1700， 最佳地 2000; 和 / 或
(b) 结合洗脱前后的荧光比值 Pb 0. 3， 较佳地， 0. 5， 更佳地 0. 6， 最佳地 ^0. 7，
其中 Pb = Pl/P0， 式中 P0为洗脱前的测试多肽与 VEGFR— 2的结合荧光值， P1为 洗脱后测试多肽与 VEGFR— 2的的结合荧光值。
在另一优选例中，所述的衍生多肽保留了 70% SEQ ID NO: 1的所示多肽的 VEGFR-2 结合活性。
在另一优选例中，所述的衍生多肽保留了 70% SEQ ID N0: 2的所示多肽的 VEGFR-2 结合活性。
在另一优选例中， 所述的衍生多肽与 SEQ ID N0: 1-16的相同性 80%， 较佳地 90%； 更佳地 95%。
本发明第四方面， 提供了一种分离的多核苷酸， 所述的多核苷酸编码如本发明第 一方面所述的氨基酸序列、 本发明第二、 或第三方面所述的多肽。
本发明第五方面， 一种药物组合物， 所述的药物组合物含有
(a) 活性成分， 该活性成分选自：
(al)本发明第一方面所述的氨基酸序列或其药学上可接受的盐； 和 /或
(a2)本发明第二或第三方面所述的多肽或其药学上可接受的盐； 以及
(b) 药学上可接受的载体。
在另一优选例中， 所述的药物组合物的剂型为针剂、 溶液剂、 粉剂、 喷雾剂等。 本发明第六方面， 提供了一种活性成分的用途， 该活性成分选自：
(al)本发明第一方面所述的氨基酸序列或其药学上可接受的盐； 和 /或
(a2)本发明第二或第三方面所述的多肽或其药学上可接受的盐；
所述的活性成分用于制备治疗肿瘤或与 VEGFR-2结合的药物。
在另一优选例中， 所述的肿瘤血管表达 VEGFR_2。
在另一优选例中， 所述的肿瘤为恶性肿瘤。
在另一优选例中， 所述的肿瘤包括黑色素细胞瘤、 非小细胞肺癌、 神经胶质瘤、 乳腺癌或者其它高表达 VEGFR2的肿瘤
本发明第七方面， 提供了如本发明第一方面所述氨基酸序列、 或本发明第二或第 三方面所述多肽的用途， 用于制备肿瘤定位诊断的试剂或试剂盒。
在另一优选例中， 所述的试剂或试剂盒还包括能与所述多肽结合的磁性粒子， 荧 光粒子及其他多模态纳米粒子等。
在另一优选例中， 所述的磁性粒子包括磁性纳米粒子或者超顺磁纳米粒子 在另一优选例中， 所述的试剂为造影剂。
在另一优选例中， 所述的肿瘤血管表达 VEGFR_2。
在另一优选例中， 所述的肿瘤包括黑色素细胞瘤、 非小细胞肺癌、 脑胶质瘤、 乳 腺癌或者其它高表达 VEGFR2的肿瘤。
本发明第八方面， 提供了一种治疗肿瘤的方法， 给需要的对象施用本发明第五方 面所述的药物组合物。
本发明第九方面， 提供了一种肿瘤定位诊断的方法， 包括步骤：
(i)向需要的对象施用造影剂，所述造影剂偶联有本发明第一方面所述的氨基酸序 列、 和 /或本发明第二或第三方面所述多肽；
(i i)对所述的造影剂进行显影；
(i i i)基于显影结果对肿瘤进行定位， 其中， 如果造影剂发生浓聚， 则所述的浓聚 部位就是肿瘤的位置。
在另一优选例中， 所述的造影剂是超顺磁铁纳米颗粒即水合半径 15nm的 Fe203。 在另一优选例中， 所述的施用包括静脉注射、 口服、 局部注射、 腔内注射、 灌肠。 应理解， 在本发明范围内中， 本发明的上述各技术特征和在下文（如实施例） 中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。 限于篇幅， 在此不再一一累述。 附图说明
图 1显示了运用细菌表面展示技术筛选得到可与 VEGFR-2特异结合的多肽序 列， 其中 96%的多肽序列拥有保守序列 FF/YEXWGVK。
图 2显示了围绕保守序列构建倾向性细菌多肽库， 构建了模型：
Z1-FF/YEXWGVK-Z2
其中 Z1和 Z2是 5个亲水性氨基酸的肽段）， 利用该倾向性多肽库筛选得到亲 和性和特异性更高的可与 VEGFR-2结合的多肽序列。
图 3 显示核心多肽与对照多肽的荧光显微镜图片。 从图中可以看出化学合成 的与罗丹明偶联的核心序列 FF/YEXWGVK可以靶向高表达 VEGFR-2受体的 HUVEC 细胞而与罗丹明偶联的对照多肽则与 HUVEC细胞没有结合。
图 4显示了运用 SPR方法测定 SEQ ID No. 2多肽的亲和性及其与 VEGF的竞争 性结合关系。 图 4A显示了芯片覆盖 VEGFR-2的响应情况； 图 4B显示不同浓度的 SEQ ID No. 2多肽流经固定了 VEGFR2蛋白芯片的响应情况； 图 4C显示了 SEQ ID No. 2多肽可与 VEGF竞争性结合 VEGFR-2 ; 图 4D显示了 VEGF可与 SEQ ID No. 2 多肽竞争性结合 VEGFR_2。
图 5显示了 SEQ ID No. 2多肽可以浓度依赖地抑制 VEGF引起的 HUVEC细胞的 增殖作用。
图 6显示了 SEQ ID No. 2多肽的特异性及引导作用。 图 6A显示 SEQ ID No. 2 多肽 （K5 ) 可以特异性的靶向高表达 VEGFR-2的细胞； 图 6B显示 SEQ ID No. 2 多肽 （K5 ) 与磁珠偶联后可以用于分离表达 VEGFR-2的细胞； 图 6C显示经 MRI 影像证实可以引导超顺磁纳米粒特异地结合于 HUVEC细胞， 且这种结合具有浓度 依赖性。
图 7显示了 SEQ ID No. 14多肽的特异性及引导作用。 图 7A显示 SEQ ID No. 14 多肽 （VI ) 可以特异性的靶向黑色素瘤细胞 B16 ; 图 7B SEQ ID No. 14多肽 （VI ) 与磁珠偶联后可以用于分离黑色素瘤细胞 B16 ; 图 7C示出不同浓度的超顺磁纳米 粒子在不偶联 /偶联 SEQ ID No. 14多肽 （VI ) 的核磁成像图片。 由图 7C的 MRI 影像证实 SEQ ID No. 14多肽 （VI ) 可以引导超顺磁纳米粒特异地结合于 HUVEC 细胞， 且这种结合具有浓度依赖性。
图 8显示了 SEQ ID No. 2多肽可以抑制肿瘤组织在小鼠体内的生长， 并且经过 CD31免疫组化实验证实对肿瘤生长的抑制是通过对肿瘤血管的抑制导致的。 具体实施方式
本发明人经过广泛而深入地研究， 首次意外地发现， 具有核心序列为
FF/YEXWGVK (SEQ ID NO.： 1)的多肽， 可以特异性地与肿瘤血管中特有的 VEGFR-2结 合， 并且证实了， 根据该核心多肽进行一定范围内衍生的多肽， 同样具有结合活性。 实验证明， 该多肽及其衍生多肽可用于肿瘤定位显影， 且该多肽具有显著的抑制肿瘤 血管新生作用， 从而达到治疗肿瘤的目的。 本发明多肽分子量小， 可穿透人体多种自 然屏障， 从而达到目标肿瘤位置。 在此基础上， 完成了本发明。 本发明序列示意表
SEQ ID 序列 备注
NO.：
1 FF/YEXWGVK 核心序列
2 YDGNSFYEMWGVKPASES K5
3 MAKGDFFERWGVKEM 优先权 2 (201210268468. X)中的序列 1
4 ENVVVNTFYEMWGVK 优先权 2 (201210268468. X)中的序列 2
5 DRGNAFFEAWGVKSPSSS K1
6 QTRNAFFEAWGVKSPSGA K2
7 PQGNSFFEAWGVKAPSNS K3
8 AGYASFFEMWGVKTPSES K4
9 YQSSRFFEIWGVKSPSPS K6
10 QAGSSFFEIWGVKSPESS K7
11 NSSNSFFESWGVKASTQQ K8
12 NYPNNFFESWGVKPSGSW K9
13 NPGSSFFEEWGVKERTSP K10 14 RSRNAFFELWGVKRETPG Kl l优先权文件中的 VI
15 NSGNTFFETWGVKGYTPG K12
16 DAGYNFFEAWGVKPTTTG K13
17 SRLVGDFFGTWGVKY
18 GNFGQNFNERWGVIAD
19 CGTAGCTGGCCAGTCTGGCCAGNVS N代表 A、 T、 C, G中的任意核苷酸， V代
NVSNVSNVSNVSTTY (TYY/TAY) G表 、 C或 G， S代表 C或 G， Y代表 T或 ARNNSTGAGGNGTNAARNVSNVSNV C, R代表 A或 G
SNVSNVSGGAGGGCAGTCTGGGCAG TC
20 GGCTGAAAATCTTCTCTC
21 GGSSSGSSGGGSSGS C1
22 TTCTTCGAA顺 STGGGGCGTTAAA 对应的氨基酸序列 FFEXWGVK
23 TTCTACGAA顺 STGGGGAGTTAAA 对应的氨基酸序列 FYEXWGVK 活性多肽
在本发明中， 术语 "核心序列"、 "核心肽段"、 "核心多肽"、 "核心片段" 可互换使用，都指具有 VEGFR-2结合活性的氨基酸序列（FF/YEXWGVK,如 SEQ ID NO : 1 所示）的蛋白或多肽。
术语 "具有核心序列的多肽" 指具有 SEQ ID NO.： 1作为核心序列的且具有 VEGFR-2结合活性的蛋白或多肽。
一种优选的具有核心序列的多肽具有如下结构式：
Z1-FX1EX2WGVK-Z2
式中， Z1为无或 1-7个氨基酸的肽段， 较佳地 Z1为 1-5个氨基酸的肽段， 更佳 地 1-3个， 最佳地 1-2个；
XI为 F或 Y;
X2为任意氨基酸， 较佳地为 20种天然氨基酸中任一种； 较佳地， X2选自 A、 M、 I、 S、 E、 L、 T、 R;
Z2为无或 1-5个氨基酸的肽段， 较佳地， 为 1-4个氨基酸的肽段， 更佳地为 1-3 个， 最佳地为 1-2个。
在另一优选例中， Z1或 Z2为亲水性氨基酸。
一种优选的具有核心序列的多肽选择如 SEQ ID NO.： 1- 16所示的多肽。
如本文所用， 术语 "衍生多肽" 、 指具有 VEGFR-2结合活性的、 以 SEQ ID NO: 1- 16中任一序列作为原序列的变异形式。 这些变异形式包括（但并不限于）： 与原 序列相比， 1-3个（通常为 1-2个， 更佳地 1个）氨基酸的缺失、 插入和 /或取代， 以及在 C末端和 /或 N末端添加或缺失一个或数个（通常为 3个以内， 较佳地为 2 个以内， 更佳地为 1个以内）氨基酸。 例如， 在本领域中， 用性能相近或相似的氨 基酸进行取代时， 通常不会改变蛋白质的功能。 又比如， 在 C末端和 /或 N末端添 加或缺失一个或数个氨基酸通常也不会改变蛋白质的结构和功能。 此外， 所述术 语还包括单体和多聚体形式本发明多肽。该术语还包括线性以及非线性的多肽（如 环肽）。
如本文所用， 术语 "本发明多肽"指的是 "核心序列" 、 "具有核心序列的多 肽" 以及 "衍生多肽" 的总称。
本发明还包括所示本发明多肽的活性片段、 衍生物和类似物。 如本文所用， 术 语 "片段" 、 "衍生物" 和 "类似物" 是指基本上保持 VEGFR-2结合活性的多肽。 本发明的多肽片段、 衍生物或类似物可以是（i)有一个或几个保守或非保守性氨基 酸残基 (优选保守性氨基酸残基)被取代的多肽， 或（ii)在一个或多个氨基酸残基 中具有取代基团的多肽， 或（iii)核心序列与另一个化合物（比如延长多肽半衰期 的化合物， 例如聚乙二醇）融合所形成的多肽， 或（iv)附加的氨基酸序列融合于此 多肽序列而形成的多肽（与前导序列、 分泌序列或 6His等标签序列融合而形成的 然后蛋白）。 根据本文的教导， 这些片段、 衍生物和类似物属于本领域熟练技术人 员公知的范围。
一类优选的活性衍生物指与式 I的氨基酸序列或相应的原序列相比， 有至多 3 个， 较佳地至多 2个， 更佳地至多 1个氨基酸被性质相似或相近的氨基酸所替换 而形成多肽。 这些保守性变异多肽最好根据表 1进行氨基酸替换而产生。
表 1
最初的残基 代表性的取代 优选的取代
Ala (A) Val； Leu； lie Val
Arg (R) Lys； Gin； Asn Lys
Asn (N) Gin； His； Lys； Arg Gin
Asp (D) Glu Glu
Cys (C) Ser Ser
Gin (Q) Asn Asn
Glu (E) Asp Asp
Gly (G) Pro; Ala Ala
His (H) Asn； Gin； Lys； Arg Arg lie (I) Leu； Val Met Ala; Phe Leu
Leu (L) lie; Val Met Ala; Phe lie
Lys (K) Arg Gin Asn Arg
Met (M) Leu Phe lie Leu
Phe (F) Leu； Val lie Ala ; Tyr Leu
Pro (P) Ala Ala
Ser (S) Thr Thr
Thr (T) Ser Ser
Trp (W) Tyr； Phe Tyr
Tyr (Y) Trp； Phe； Thr； Ser Phe
Val (V) He; Leu; Met; Phe; Ala Leu 发明还提供本发明多肽的类似物。 这些类似物与天然多肽的差别可以是氨基酸 序列上的差异， 也可以是不影响序列的修饰形式上的差异， 或者兼而有之。 类似 物还包括具有不同于天然 L-氨基酸的残基（如 D-氨基酸）的类似物， 以及具有非天 然存在的或合成的氨基酸（如 β、 Υ -氨基酸）的类似物。 应理解， 本发明的多肽并 不限于上述例举的代表性的多肽。
修饰（通常不改变一级结构）形式包括： 体内或体外的多肽的化学衍生形式如乙 酰化或羧基化。 修饰还包括糖基化， 如那些在多肽的合成和加工中或进一步加工 步骤中进行糖基化修饰而产生的多肽。 这种修饰可以通过将多肽暴露于进行糖基 化的酶（如哺乳动物的糖基化酶或去糖基化酶）而完成。 修饰形式还包括具有磷酸 化氨基酸残基（如磷酸酪氨酸， 磷酸丝氨酸， 磷酸苏氨酸）的序列。 还包括被修饰 从而提高了其抗蛋白水解性能或优化了溶解性能的多肽。
本发明多肽还可以以由药学上或生理学可接受的酸或碱衍生的盐形式使用。这 些盐包括（但不限于）与如下酸形成的盐： 氢氯酸、 氢溴酸、 硫酸、 柠檬酸、 酒石 酸、 磷酸、 乳酸、 丙酮酸、 乙酸、 琥珀酸、 草酸、 富马酸、 马来酸、 草酰乙酸、 甲磺酸、 乙磺酸、苯磺酸、 或羟乙磺酸。其他盐包括： 与碱金属或碱土金属（如钠、 钾、钙或镁）形成的盐， 以及以酯、氨基甲酸酯或其他常规的"前体药物"的形式。 编码序列
本发明还涉及分别编码本发明多肽的多核苷酸。
一种优选的编码序列核心序列的核苷酸序列是：
(SEQ ID NO : 22) TTCTTCGAANNSTGGGGCGTTAAA; 或
(SEQ ID NO : 23) TTCTACGAANNSTGGGGAGTTAAA。 其编码了 SEQ ID NO : 1所示的 核心片段。 对于 SEQ ID NO.： 2-16及其他衍生多肽， 其编码序列可基于密码子对 应关系， 由本领域技术人员轻易地确定。
本发明的多核苷酸可以是匪形式或 RNA形式。 DNA可以是编码链或非编码链。 编码成熟多肽的编码区序列可以与 SEQ ID N0 : 22、 SEQ ID N0 : 23所示的编码区序 列相同或者是简并的变异体。 如本文所用， 以核心序列为例， "简并的变异体" 在本发明中是指编码具有 SEQ ID N0 : 1序列的多肽， 但与 SEQ ID NO : 22及 SEQ ID NO : 23中相应编码区序列有差别的核酸序列。
本发明的编码核苷酸全长序列或其片段通常可以用 PCR扩增法、 重组法或人工 合成的方法获得。 目前， 已经可以完全通过化学合成来得到编码本发明多肽（或其 片段， 或其衍生物）的 DNA序列。 然后可将该 DNA序列引入本领域中已知的各种现 有的 DNA分子（或如载体）和细胞中。
本发明也涉及包含本发明的多核苷酸的载体， 以及用本发明的载体或本发明多 肽编码序列经基因工程产生的宿主细胞。
另一方面， 本发明还包括本发明多肽的单克隆抗体和多克隆抗体， 尤其是单克 隆抗体。 通用方法
1.从随机的细菌多肽库筛选可与 VEGFR-2特异结合的多肽序列的方法 本发明所使用的细菌表面展示随机肽库是从美国加州大学圣巴巴拉分校化学 工程系的 Patrick s. Daugherty实验室购得。 从冻存的细菌表面展示随机肽库中 取至少 10倍库容量的菌接种到 lOOmL含 34 μ g/mL氯霉素和 2mg/mL葡萄糖的 S0C 培养基中， 在 37 、 200转 /分的条件下过夜培养。从过夜培养的细菌中按照 1 : 50 的比例取出一定量的细菌加入 5mL含 34 μ g/mL的氯霉素 LB培养基中， 在 37°C、 200转 /分的条件下培养 2小时至 0D6。。值在 0. 5-0. 6之间。以 200 μ g/mL的量加入 阿拉伯糖， 在室温 （约 25°C ) 、 200转 /分的条件下诱导培养约 lh， 其 0D6。。值达 到 0. 8-1. 0， 按照一个 0D等于 I X 109个细胞计算细菌总数。 从上述经诱导的菌液 中取出包含大约 1 X 1CT个细菌的量的菌液， 离心后用 1¾值为 7. 4的 PBS缓冲液 重悬。 按照细菌：磁珠等于大约 100 : 1 (个数比） 的比例， 加入链霉亲和素包被的 磁珠 ( Streptavidin-coated magnetic microbeads )进行磁珠的阴性分选。 其中， 链霉亲和素包被的磁珠和磁架均从 Invitrogen公司购得，链霉亲和素包被的磁珠 的商品名为 Dynabeads® My0ne™ ， 商品号为 656-01。 经磁珠分选， 那些不与链霉 亲和素 （Streptavidin) 结合的细菌吸附进入下一轮的磁珠阳性分选。 使用从 Invitrogen 公司购得的商品号为 F6347的蛋白荧光生物素标记试剂盒
( Fluoreporter mini- biotin- xx protein label ing Kit ) 寸 VEGFR- 2蛋白进行 生物素化 ( biotinylated) ， 其中， VEGFR- 2蛋白 (编码核苷酸 265bp-2493bp ) 是从 PR0SPEC公司购得， 商品号为 PKA-242。
按照蛋白：细菌等于大约 500 : 1 (个数比） 的比例将生物素化的 VEGFR-2蛋白 与经阴性选择的细菌混合， 使混合液中 VEGFR-2蛋白的浓度为 10 nM， 在静音旋 转混合器上 4°C共同孵育 45分钟。 孵育结束后在 3800转 /分、 4°C的条件下离心， 移除上清液， 并将沉淀重新悬浮在 lmL PBS缓冲溶液中。 按照磁珠：细菌为大约 1 : 100 (个数比） 的比例将磁珠与重新悬浮在 PBS缓冲液中的细菌混合， 用 PBS 缓冲液清洗沉淀 3次后用 1 mL LB重悬， 取出 10 μ L稀释 100倍， 在测量其 0D6。。 值之后， 涂布在固体 LB平板上， 37°C过夜培养， 第二天根据长成的细菌克隆数量 计算磁珠分选后的细菌库容量。 剩下的细菌重新悬浮在 5 mL的 S0C培养基中， 在 37 、 200转 /分的条件下过夜培养， 以用于流式分选。
取 100 过夜培养的细菌接种到 5 mL的 LB培养基中， 在 37°C、 200转 / 分的条件下培养约 2h至 0D6。。值在 0. 4-0. 6之间， 以 200 μ g/mL的量加入阿拉伯 糖， 室温下诱导细菌表达。 按照蛋白： 细菌等于大约 10 : 1 (个数比） 的比例取相 应的生物素化的 VEGFR-2蛋白与离心后的细菌混合， 在 4°C、 温和旋转 （例如， 20转 /分） 的条件下共同孵育 45分钟。 然后， 在 4 、 3000转 /分的条件下离心 5 分钟， 弃除上清。 将沉淀重新悬浮在 100 L SAPE染液中共孵育 45分钟。 孵育 结束后， 在 4 、 3000转 /分的条件下离心 5分钟， 弃除上清。 将沉淀重新悬浮在 PH值为 7. 4的 600 μ L的 PBS缓冲液中， 使细菌在 PBS缓冲液中的浓度为大约每 毫升 5 X 106至 1 X 107个。利用流式细胞仪对重新悬浮在 PBS缓冲液中的细菌进行 分选。 其中， 表面结合有生物素化的 VEGFR-2的细菌， gp， 表面展示有可以特异 性结合 VEGFR-2蛋白的多肽的细菌， 在 488nm光源激发下发出的荧光强度比没有 结合 VEGFR-2蛋白的细菌的荧光强度高。 通过调节流式细胞仪的电压， 利用流式 细胞仪的分选功能将高荧光值的细菌分选出来， 即， 将表面结合有生物素化的 VEGFR-2蛋白的细菌分选出来。 将分选出来的细菌 -蛋白复合体接种到 5mL S0C培 养基中过夜培养， 培养期间蛋白降解， 细菌得以富集， 将富集的细菌再次进入流 式分选流程。
2.构建偏向性的细菌表面展示随机肽库进行 VEGFR-2结合多肽的进一步筛 选方法
将 SEQ ID NO.： 1的氨基酸序列两侧分别连接有任意 5个亲水氨基酸的多肽连 接到 CPX的 N端。 编码上述多肽的序列用 PCR方法扩增得到。 PCR反应的模板是 质粒 PB33CPX-SAP印，该质粒的详细信息请见 Rice J， J. and Daugherty, PS. (2008) Directed Evolution of a Biterminal Bacterial Display Scaffold Improves the Display of Diverse Peptides, Protein Eng. Des Sel. 2008, 21 (7)， 435-42。
正向引物（SEQ ID NO.： 19)
SNVSNVSNVSNVSGGAGGGCAGTCTGGGCAGTC;
其中， N代表 A、 T、 C、 G中的任意核苷酸， V代表 A、 C或 G， S代表 C或 G， Y代表了或（， R代表 或6。
反向引物（SEQ ID NO.： 20)
GGCTGAAAATCTTCTCTCo
PCR扩增产物用 酶切后连接到经相同酶切的 pB33CPX载体中下游的 alajGFPl (Bessette, P. H. et al. Rapid isolat ion of high-affinity protein binding pept ides using bacterial di splay. (2004) Protein Eng. Des. Sel. 17, 731-739 ) 多克隆位点上。 改造后的 pB33CPX质粒转化入大肠杆菌 MC1061菌 株 [F- araD139 △ (ara- leu) 7696 galE15 galK16A (lac) X74 rpsL (StrR) hsdR2 (rK-mKt) mcrA mcrBl] (Casadaban and Cohen, 1980)， 从而完成细菌表面展示 随机肽库的构建。 该细菌展示肽库包含大约 2 X 108种多肽。 构建成功的偏向型的 细菌展示库采用与随机展示库相同的方法进行 VEGFR-2结合多肽的筛选工作。
3.本发明多肽理化性质的表征方法
细菌表面展示多肽特异性及亲和力的初步测试
确定了多肽序列后， 还需要了解多肽与靶分子间的亲和力情况， 因此发展了 多肽细菌与靶分子间亲和力的测定方法。 将生物素化的 VEGFR-2与经阿拉伯糖诱 导的细菌及 SAPE共同孵育结束后， 延长 PBS清洗时间至 15分钟。 清洗结束后用 流式细胞仪分别检测每个细菌与 VEGFR-2沉淀的荧光数值，作为多肽 -细菌亲和力 测定的初步结果。 有了亲和力的初步数据之后， 接着在 VEGFR-2与细菌及 SAPE 孵育过程中加入 20%的 IgG或者血清来检测多肽与 VEGFR2的特异性。 孵育结束后 利用流式细胞仪检测每个细菌克隆的荧光数值。 4.游离多肽的亲和力测定方法
多肽采用表面等离子体共振仪 Biacore 3000来分析多肽和 VEGFR-2间的亲和 力。 具体过程如下： 首先以 10μ L/min的流速进样 50 μ L NHS/EDC混合液 （0.05 mol/L NHS 和 0.2 mol/L EDC，使用前按体积比 1: 1 混合） ， 用于活化芯片表面 葡萄糖的羧基； 活化结束后注射醋酸钠稀释的 VEGFR-2蛋白， 按照 10μ ΐ7πήη的 流速进样 5 分钟， 待固定信号达到 2000 RU以上后用 20 的 1M 盐酸乙醇胺 封闭 7分钟， ， 本实施例中达到了 3465.2 RU。 之后将待检测的靶向多肽溶解在 PBS 缓冲液 （ΡΗ=7· 4) 中， 并配成 40μ Μ， 20 μ Μ， 10 μ Μ， 5 μ Μ 和 2.5 μ Μ 的 浓度梯度， 以流速 10μ L/min 进样。 每个循环包括 1 min 的休息时间以检测基 线稳定， 2 min 注射样品， 1 min 解离期。 多肽注射结束后用 100 mM, PH 2.0 的 Glycine再生， 注射体积是 210 μ L, 速度是 30 μ L/min。 本发明还创新性的 应用 SPR的方法进行多肽和 VEGF与 VEGFR-2的竞争性结合试验。 5.本发明多肽细胞增殖的影响的检测方法
用 MTT(methyl thiazolyl tetrazolimu)实验来检测多肽对细胞增殖的抑制 作用。 按照每孔 5000个细胞在 96孔板中种 HUVEC原代细胞或者 B16黑色素瘤细 胞。 开始实验前， 细胞先贴壁 12h， 然后换成无血清培养继续培养另外 24h。 一些 孔加入 30 ng/mL的重组人 VEGF165, 另外一些孔除了加入等量的 VEGF165外， 还 会加入浓度在 2 μ g/mL-100 μ g/mL的多肽。 共同孵育 48h后， 每孔加入 20 μ L 浓度为 5 mg/ml的 ΜΤΤ， 和细胞在 37°C共同孵育 4h。 之后， 移去上清加入 150 L 的 DMS0溶解之前形成的甲瓒。 VICTOR X4酶标仪 （PerkinElmer Life and Analytical Sciences, Boston, MA, USA) 上记录 490 nm处的光吸收值。 实验结果 以成活率来表示。
6.本发明多肽的肿瘤靶向作用测定方法
使用 Invitrogen公司的商品号为 650. 11的 DYNAL试剂盒将多肽与磁性聚苯乙 烯小球偶联 （磁珠） 。 具体偶联过程参见试剂盒的使用说明书。
分别取 10 偶联有磁珠的多肽悬浮在 200 w L PBS缓冲液中， 加入种植在 12孔板中的高表达 VEGFR-2的人脐静脉内皮细胞 （HUVEC) ， B16细胞和人肺成 纤维细胞 HLF中， 其中， 细胞种植密度为每孔 1万个， 培养基为含 10%胎牛血清 的 DMEM。 然后， 在 4°C孵育 7-10分钟， PBS冲洗 4次， 每次 5分钟， 在显微镜下 观察连接有磁珠的多肽与细胞的结合情况； 与磁珠偶联后的多肽在与细胞表面表 达的 VEGFR-2结合后， 在磁力作用下就可以将这部分细胞捕捉下来。根据此原理， 将 HUVEC, B16和 HLF消化后用 1 mL PBS缓冲液重悬， 制成包含大约 2xl06个细 胞的悬液， 将偶联有磁珠的多肽加入此细胞悬液中， 4°C在静音混合器上孵育 10 分钟， 用从 Invitrogen公司购买的磁力架进行分离， 测量分离得到的沉淀和上清 液中的细胞量， 根据上述两个实验的结果， 确定多肽在体外对表达有 VEGFR-2蛋 白的细胞的靶向作用。 将多肽超顺磁纳米粒子 （铁芯直径 6. 7 ± 0. 3讓， 水合半径为 15 nm的 Fe203 颗粒）进行偶联。 在 6孔板中种植 HUVEC, B16和 HLF细胞， 每孔种植大约 5xl05 个细胞， 培养基为加入 10%胎牛血清的 DMEM, 第二天在不同孔中分别加入 Fe粒子 浓度为 128 μ g/mL的上述连接有不同多肽的超顺磁纳米粒子。 在 37°C下与细胞共 同孵育 2h后， PBS缓冲液冲洗 4遍以去除那些未结合到细胞的超顺磁纳米粒子。 800rpm离心 3分钟收集细胞， 重悬在 0. 6 mL PBS缓冲液和 0. 6 mL 2%多聚甲醛固 定液中， 4°C放置 lh， PBS缓冲液冲洗 3遍， 800 rpm离心 3分钟。 向沉淀中加入 300 μ L PBS缓冲液和 200 μ L融化的浓度为 2%的低熔点琼脂糖， 混匀后加入核磁 管中。 通过核磁成像仪对上述经孵育的细胞进行检测， 来进一步验证多肽的靶向 作用。
7.本发明多肽的肿瘤血管抑制作用检测方法
在 3-4周龄裸鼠的腋下注射 lxlO6个 U87MG悬浮细胞， 14天后待肿瘤体积长 到合适大小后处死小鼠， 将肿瘤组织块取出放在培养皿中用 1¾ 7. 4的 PBS冲洗三 次后用手术剪将肿瘤组织剪成 1个毫米大小， 用接种针将切成小块的肿瘤组织推 入小鼠的后腿皮下， 为了防止组织块掉出， 接种针至少要进入小鼠皮下 lcm远。
组织块接种后的第三天对小鼠进行随机分组。一组是用来注射多肽 K5 (SEQ ID NO. : 2)或 K11 (SEQ ID NO.： 14)的实验组， 另外的对照组则注射同样体积的生理 盐水， 隔天注射。 注射过程中每两天记录一次肿瘤体积， 连续注射 21天后处死小 鼠并拍照。 并从实验组和对照组小鼠皮下拨出肿瘤组织放进 4%多聚甲醛固定， 用 于后面的石蜡切片及免疫组化实验。
将经多聚甲醛固定的肿瘤组织包埋进石蜡中， 切成 5 μ πι厚度的蜡带。 石蜡 切片经二甲苯脱蜡后进入梯度酒精中脱水。 脱水结束后用 3%双氧水在 37°C 10分 钟封闭内源过氧化物酶活性， 之后用 PH6. 0的枸橼酸在 95°C煮沸 20分钟进行抗 原修复； 接下来滴加 5% BSA， 37°C反应 30min封闭未反应的位点。 之后滴加兔抗 鼠的单克隆抗体 CD31 ( bs- 0195R, Beij ing biosynthesi s biotechnology. , LTD) 4°C反应过夜， 一抗的浓度为 1 μ g/mL; I¾7. 4的 PBS缓冲液冲洗结束后滴加 HRP 标记羊抗兔 IgG ( HA1001 , 华安生物） ， 37°C反应 30分钟， PBS缓冲液 （1¾7. 4) 冲洗、 MB (AR1022 , 博士德生物）室温显色 20min， 蒸馏水洗涤； 苏木素（AR0005, 博士德生物）轻度复染 60秒， 蒸馏水洗涤后浸入盐酸酒精中进行分化， 取出后在 自来水下冲洗 20min; 再次进入梯度酒精脱水， 二甲苯两次透明后在材料中间滴 加一滴 90%甘油和 10% PBS的保存液后用指甲油封片， 显微镜下拍照并观察被染 成黄褐色的区域， 这些区域为肿瘤血管部位。 8.本发明多肽的制备方法
本领域的普通技术人员可以使用常规方法制备本发明多肽， 如生物合成， 全化 学合成等方法。
一种优选的方法是使用液相合成技术或固相合成技术， 如 Boc固相法、 Fmoc固 相法或是两种方法联合使用。 固相合成可快速获得样品， 可根据目的肽的序列特 征选用适当的树脂载体及合成系统。 例如， Fmoc系统中优选的固相载体如连接有 肽中 C端氨基酸的 Wang树脂， Wang树脂结构为聚苯乙烯， 与氨基酸间的手臂是 4-垸氧基苄醇； 用 25%六氢吡啶 /二甲基甲酰胺室温处理 20分钟， 以除去 Fmoc保 护基团，并按照给定的氨基酸序列由 C端逐个向 N端延伸。合成完成后，用含 4%对 甲基苯酚的三氟乙酸将合成的胰岛素原相关肽从树脂上切割下来并除去保护基， 可过滤除树脂后乙醚沉淀分离得到粗肽。 将所得产物的溶液冻干后， 用凝胶过滤 和反相高压液相层析法纯化所需的肽。 当使用 Boc系统进行固相合成时， 优选树 脂为连接有肽中 C端氨基酸的 PAM树脂， PAM树脂结构为聚苯乙烯， 与氨基酸间的 手臂是 4-羟甲基苯乙酰胺； 在 Boc合成系统中， 在去保护、 中和、 偶联的循环中， 用 TFA/二氯甲垸（DCM)除去保护基团 Boc并用二异丙基乙胺（DIEA/二氯甲垸中和。 肽链缩合完成后， 用含对甲苯酚（5-10%)的氟化氢（HF)，在 0°C下处理 1小时， 将肽 链从树脂上切下， 同时除去保护基团。 以 50-80%乙酸 (含少量巯基乙醇)抽提肽， 溶液冻干后进一步用分子筛 S印 hadex G10或 Tsk-40f 分离纯化， 然后再经高压液 相纯化得到所需的肽。 可以使用肽化学领域内已知的各种偶联剂和偶联方法偶联 各氨基酸残基， 例如可使用二环己基碳二亚胺（DCC)， 羟基苯骈三氮唑（HOBt)或 1， 1，3，3-四脲六氟磷酸酯0¾11；)进行直接偶联。 对于合成得到的短肽， 其纯度与 结构可用反相高效液相和质谱分析进行确证。
在一个优选的实施例中， 生物合成法有以下步骤：
(1) .用编码本发明活性多肽或其衍生物的多核苷酸 (或其变异体），或用含有该 多核苷酸的重组表达载体转化或转导合适的宿主细胞；
(2) .在合适的培养基中培养的宿主细胞；
(3) .从培养基或细胞中分离、 纯化本发明的多肽。
本发明中， 多核苷酸序列可插入到重组表达载体中。本领域的技术人员熟知的 方法能用于构建含所述多肽的编码匪序列和合适的转录 /翻译控制信号的表达 载体。 包含上述适当 DNA序列以及适当启动子或者控制序列的载体， 可以用于转 化适当的宿主细胞， 以使其能够表达本发明的活性多肽。 宿主细胞可以是原核细 胞， 如细菌细胞； 或是低等真核细胞， 如酵母细胞； 或是高等真核细胞， 如植物 细胞。
用重组 DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为 原核生物如大肠杆菌时， 能吸收 DNA的感受态细胞可在指数生长期后收获， 用 CaCl2法处理， 所用的步骤在本领域众所周知。 另一种方法是使用 MgCl2。 如果需 要， 转化也可用电穿孔的方法进行。 当宿主是真核生物， 可选用如下的 DNA转染 方法： 磷酸钙共沉淀法， 常规机械方法如显微注射、 电穿孔、 脂质体包装等。
获得的转化子可以用常规方法培养， 表达本发明的活性多肽。 根据所用的宿主 细胞， 培养中所用的培养基可选自各种常规培养基。 在适于宿主细胞生长的条件 下进行培养。 当宿主细胞生长到适当的细胞密度后， 用合适的方法（如温度转换或 化学诱导）诱导选择的启动子， 将细胞再培养一段时间。
本发明多肽可在细胞内、 或在细胞膜上表达、 或分泌到细胞外。 如果需要， 可利用其物理的、 化学的和其它特性通过各种分离方法分离和纯化所述的活性多 肽。 这些方法是本领域技术人员所熟知的。 这些方法的例子包括但并不限于： 常 规的复性处理、 用蛋白沉淀剂处理（盐析方法）、 离心、 渗透破菌、 超处理、 超离 心、 分子筛层析（凝胶过滤）、 吸附层析、 离子交换层析、 高效液相层析 (HPLC)和 其它各种液相层析技术及这些方法的结合。 在上海生工生物有限公司分别合成序 列为 YDGNSFYEMWGVKPASES (SEQ ID NO.： 2)的多肽 K5， RSRNAFFELWGVKRETPG (SEQ ID NO.： 14)的多肽 VI和 GGSSSGSSGGGSSGS (SEQ ID NO : . 21)的多肽 CI用于下述的试 验。 药物组合物和施用方法
另一方面， 本发明还提供了一种药物组合物， 它含有（a)安全有效量的本发明 多肽或其药学上可接受的盐； 以及 (b)药学上可接受的载体或赋形剂。本发明多肽 的数量通常为 10微克 -100毫克 /剂， 较佳地为 100-1000微克 /剂。
为了本发明的目的，有效的剂量为给予个体约 0. 01毫克 /千克至 50毫克 /千克， 较佳地 0. 05毫克 /千克至 10毫克 /千克体重的本发明多肽。 此外， 本发明的多肽 可以单用， 也可与其他治疗剂一起使用（如配制在同一药物组合物中）。
药物组合物还可含有药学上可接受的载体。 术语 "药学上可接受的载体"指用 于治疗剂给药的载体。 该术语指这样一些药剂载体： 它们本身不诱导产生对接受 该组合物的个体有害的抗体， 且给药后没有过分的毒性。 这些载体是本领域普通 技术人员所熟知的。 在 Remington' s Pharmaceutical Sci ences (Mack Pub. Co.， N. J. 1991)中可找到关于药学上可接受的赋形剂的充分讨论。 这类载体包括（但并 不限于）： 盐水、 缓冲液、 葡萄糖、 水、 甘油、 乙醇、 佐剂及其组合。
治疗性组合物中药学上可接受的载体可含有液体， 如水、 盐水、 甘油和乙醇。 另外， 这些载体中还可能存在辅助性的物质， 如润湿剂或乳化剂、 pH缓冲物质等。
通常， 可将治疗性组合物制成可注射剂， 例如液体溶液或悬液； 还可制成在注 射前适合配入溶液或悬液中、 液体载体的固体形式。
一旦配成本发明的组合物， 可将其通过常规途径进行给药， 其中包括（但并不 限于）： 瘤内、 腔内、 肌内、 静脉内、 皮下、 皮内、 灌肠或局部给药。 待预防或治 疗的对象可以是动物； 尤其是人。
当本发明的药物组合物被用于实际治疗时，可根据使用情况而采用各种不同剂 型的药物组合物。
这些药物组合物可根据常规方法通过混合、稀释或溶解而进行配制， 并且偶尔 添加合适的药物添加剂， 如赋形剂、 崩解剂、 粘合剂、 润滑剂、 稀释剂、 缓冲剂、 等渗剂（isotonic ities 防腐剂、 润湿剂、 乳化剂、 分散剂、 稳定剂和助溶剂， 而且该配制过程可根据剂型用惯常方式进行。
例如， 静脉注射液的配制可这样进行： 将本发明多肽或其药学上可接受的盐与 基本物质一起溶解于无菌水（在无菌水中溶解有表面活性剂）中， 调节渗透压和酸 碱度至生理状态， 并可任意地加入合适的药物添加剂如防腐剂、 稳定剂、 缓冲剂、 等渗剂、 抗氧化剂和增粘剂， 然后使其完全溶解。
本发明的药物组合物还可以缓释剂形式给药。例如， 核心序列或其盐可被惨入 以缓释聚合物为载体的药丸或微囊中， 然后将该药丸或微囊通过手术植入待治疗 的组织。 作为缓释聚合物的例子， 可例举的有乙烯-乙烯基乙酸酯共聚物、 聚羟基 甲基丙烯酸酯（polyhydrometaacrylate 聚丙烯酰胺、 聚乙烯吡咯垸酮、 甲基纤 维素、 乳酸聚合物、 乳酸 -乙醇酸共聚物等， 较佳地可例举的是可生物降解的聚合 物如乳酸聚合物和乳酸-乙醇酸共聚物。
当本发明的药物组合物被用于实际治疗时，作为活性成分的本发明多肽或其药 学上可接受的盐的剂量， 可根据待治疗的每个病人的体重、 年龄、 性别、 症状程 度而合理地加以确定。施用时，极大多数病例一般每天应用的总剂量为每人 lmg〜 200g， 较佳地为 10mg〜100g。
可用本发明多肽进行检测和治疗的肿瘤类型没有特别显著， 代表性例子包括 (但并不限于）： 非小细胞肺癌及脑胶质瘤。 优选实体瘤， 尤其是过表达 VEGFR-2 的肿瘤类型。 VEGFR-2在肿瘤血管中高表达， 另外在伤口愈合处的组织也可见 VEGFR-2的过度表达， 在其它正常组织中不表达或者表达量极低。 在实体肿瘤形 成过程， VEGFR-2的过度表达保证了肿瘤新生血管的生成， 从而为肿瘤的快速生 长提供能量供应。 肿瘤定位诊断方法
本发明多肽可特异性结合肿瘤血管特异性表达的 VEGFR-2 , 由此， 当本发明多 肽偶联于造影剂后， 可在核磁共振或其他影像学检查中显现出浓聚现象。 据此可 以判断肿瘤发生部位或肿瘤转移部位。
在本发明中， 一种优选的造影剂是超顺磁铁纳米颗粒， 即水合半径 15nm的 Fe203。 应理解， 还有其他可用的、 可偶联于本发明多肽的造影剂， 本领域技术人 员可根据本发明的教导进行实验获得。
优选地， 本发明多肽与造影剂偶联后可通过静脉注射、 口服、 局部注射、 腔内 注射、 灌肠等部位到达肿瘤所在位置。 本发明的主要优点包括-
(a)本发明多肽可特异性结合肿瘤血管 VEGFR-2 , 有效抑制肿瘤生长；
(b)本发明多肽可制备成造影剂， 特异性识别肿瘤生长部位， 从而进行肿瘤定 位诊断；
(c)本发明多肽分子量小， 易于透过各种生理屏障；
(d)可通过固相合成的方法制备， 纯度高， 产量大， 成本低；
因此本发明多肽有望开发成药物， 用于诊断或治疗肿瘤（尤其是表达 VEGFR-2 的实体肿瘤类型）。 下面结合具体实施例， 进一步阐述本发明。 应理解， 这些实施例仅用于说明本 发明而不用于限制本发明的范围。 下列实施例中未注明具体条件的实验方法， 通 常按照常规条件， 例如 Sambrook等人， 分子克隆： 实验室手册（New York： Cold Spring Harbor Laboratory Press, 1989)中所述的条件， 或按照制造厂商所建议 的条件。 除非另外说明， 否则百分比和份数是重量百分比和重量份数。 实施例 1 初步筛选与 VEGFR-2结合活性良好的多肽并获得其核心序列
采用通用方法 1 , 经过 6轮流式分选的筛选， 细菌与多肽的结合效率达到 60% 时， 收集菌液并将收集的菌液接种到 5 mL LB培养基中， 过夜培养。 从过夜培养 的细菌中取出 10 w L涂布在 LB固体培养基上， 在 37°C过夜培养。 从平板上随机 挑选 50个单克隆细菌， 编号为 clonel-clone50， 分别接种到 5 mL LB液体培养 基中， 在 37 、 200转 /分的条件下过夜培养， 并对过夜培养的细菌分别进行多肽 的测序， 这里， 在上海生工生物工程有限公司对多肽进行测序。
结果： 如图 1所示， 其中 SEQ ID NO.： 3的序列和 SEQ ID NO.： 4的序列出现 的频率最高（分别为 80 %以及 10 % )， 其次为 SEQ ID NO.： 17和 SEQ ID NO.： 18 所示的序列（均为 3 % )。
结论： 由图 1可见， SEQ ID NOs.： 3-4、 SEQ ID NOs.： 17-18所示的多肽含有 相同的核心序列即 FF/YEXWGVK。 实施例 2 按实施例 1中所获得的核心序列进行偏向性展示肽库构建
按照通用方法 2中所述实施方法构建用于流式筛选的偏向性肽库， 构建成功 的偏向型的细菌展示库采用与随机展示库相同的方法进行 VEGFR-2结合多肽的筛 选工作。
结果： 经过 4轮筛选之后， 一共得到 40个单克隆细菌， 按照实施例 1中的方 法对细菌表面展示多肽进行测序。 实施例 3 多肽-细菌与 VEGFR2亲和力的初步测试
按照通用方法 4进行多肽亲和力测试， 结果得到图 2的荧光结合数据。
结果： 图 2可见， 筛选所得的多肽的荧光结合数值均大于 1000， 部分甚至大 于 2000， 而洗脱前 /洗脱后的荧光比值均在 0. 3以上， 部分甚至达到 0. 5-0. 7。
结论： 由此可见， 含有本发明核心序列的肽段均具备 VEGFR-2结合活性， 因 此可以推断， 这些可以结合 VEGFR-2的肽段具有相同或相似的抑制表达 VEGF-2 的肿瘤的活性。 实施例 4 核心序列的亲和力测试
分别在上海波泰生物有限公司合成与罗丹明偶联的核心多肽 FF/YEXWGVK和 对照多肽 C1 (SEQ ID NO.： 21)， 取 100 u M的荧光多肽与过夜培养的经 4%多聚甲 醛固定的 HUVEC细胞在 37°C共孵育 1小时候后通过荧光显微镜（Nikon Ti， Japan ) 观察并拍照， 由图 3可以看出核心多肽 FF/YEXWGVK与高表达 VEGFR2的 HUVEC细 胞有一定的结合， 而对照多肽则完全没有结合。 实施例 5 化学合成 SEQ ID No. 2多肽与 VEGFR-2亲和力 d) 的测定 按照通用方法 4经 Bi acore 3000测试， 结果如图 4中的 A和 B， 经过计算 ( Karlsson, Robert, et al. , " Experimental Design for Kinet ic Analysis of Protein― Protein Interactions with surface plasmon resonance biosensors " Journal of Immunological Methods, 200, (1997)， 121-133· ) 多肽 K5 (SEQ ID NO.： 2)的 Λί值是 228 ηΜ ο 实施例 6 竞争结合试验
通过 SPR技术（通用方法 4)来测定是否可以竞争性的结合 VEGF与 VEGFR-2的 结合位点，我们采取的方式是在已经偶联了 VEGFR-2的芯片上首先按照常规方法 完成多肽 K5 (SEQ ID NO.： 2)和 VEGF与 VEGFR-2的结合实验。
结果： 注射 50 浓度为 25 nM的 VEGF蛋白并进入 PBS清洗程序后仪器读 出的 RU值是 77. 9； 当 VEGF蛋白注射结束后不进入 PBS清洗过程而是直接注射多 肽 K5 (SEQ ID NO.： 2)， 此时仪器读出的 RU值为 46. 1。 反过来， 在 VEGFR-2偶联 芯片上先注射 3. 57 uM的多肽 K5 (SEQ ID N0.： 2)再注射 VEGF蛋白， RU值从 200. 8 降至 163. 64。 综合两次 RU值的减少情况说明 K5 (SEQ ID NO.： 2)与 VEGFR2的结 合位点与 VEGF与 VEGFR-2的结合位点一致。
结论： 如图 4的 C和 D所示多肽 K5 (SEQ ID NO.： 2)在一定程度上干扰了 VEGF 与 VEGFR-2的结合。 实施例 7 多肽对细胞增殖的影响
MTT (methyl thiazolyl tetrazol imu)实验结果如图 5所示， 随着多肽 K5 (SEQ
ID NO.： 2)浓度的增高细胞的增殖得到了明显抑制。 实施例 8 K5 (SEQ ID NO.： 2)多肽 -羧基磁珠的偶联及体外功能鉴定
K5 (SEQ ID NO.： 2)多肽的体外功能的验证结果如图 6所示（通用方法 5)。 其 中， 图 6A中标注有 HUVEC C1的照片示出了偶联有磁珠的多肽 C1与人脐静脉内皮 细胞 （HUVEC) 的结合情况， 标注有 HUVEC K5的照片示出了偶联有磁珠的多肽 K5 与人脐静脉内皮细胞 （HUVEC) 的结合情况， 标注有 HLF C1的照片示出了偶联有 磁珠的多肽 C1与人肺成纤维细胞的结合情况， 标注有 HLF K5的照片示出了偶联 有磁珠的多肽 K5 (SEQ ID NO.： 2)与人肺成纤维细胞的结合情况。 从图 6A中可以 看出， 仅标注有 HUVEC K5的照片中分布有磁珠， 这说明偶联有磁珠的多肽 K5 (SEQ ID NO.： 2)与人脐静脉内皮细胞 （HUVEC) 结合， 多肽 K5 (SEQ ID NO.： 2)具有很 好地靶向于 VEGFR2的作用。
图 6B示出了经磁力架分离得到的沉淀和上清液中的细胞量， 纵坐标表示沉淀 和上清中细胞数量与总细胞数的百分比， 横坐标中的 HLF C1表示偶联有磁珠的多 肽 C1 (SEQ ID NO.： 21)与人肺成纤维细胞共同孵育得到的细胞， HLF K5 (表示偶 联有磁珠的多肽 K5 (SEQ ID NO.： 2)与人肺成纤维细胞共同孵育得到的细胞， HUVEC K5表示偶联有磁珠的多肽 K5 (SEQ ID NO.： 2)与 HUVEC共同孵育得到的细胞， HUVEC C1表示偶联有磁珠的多肽 CI (SEQ ID NO.： 21)与 HUVEC共同孵育得到的细胞， HLF 和 HUVEC分别表示未与多肽进行共同孵育的细胞。 如图 6B中所示， 与偶联有磁珠的多肽 K5 (SEQ ID NO.： 2)共同孵育的 HUVEC 细胞几乎全部存在于沉淀中， 这是由于偶联有磁珠的多肽 K5 (SEQ ID NO.： 2)与 HUVEC细胞特异性地结合， 通过磁力架吸附磁珠将 HUVEC细胞带入沉淀中。 这进 一步地说明了多肽 K5 (SEQ ID NO.： 2)具有很好地靶向性， 另外， 也进一步验证 了根据本发明的多肽 K5 (SEQ ID NO.： 2)靶向于 VEGFR-2蛋白。 实施例 9 K5 (SEQ ID NO.： 2)多肽用于肿瘤的定位显影
K5 (SEQ ID NO.： 2)多肽与超顺磁纳米粒子偶联后的核磁成像结果如图 6C所 示。 该图中的三个 T2加权图像从上至下依次分别为与多肽 K5 (SEQ ID NO.： 2)共 同孵育后的 HUVEC细胞的成像图片、 与多肽 K5 (SEQ ID NO.： 2)共同孵育后的 HLF 细胞的成像图片以及与多肽 CI (SEQ ID NO.： 21)共同孵育后的 HUVEC细胞成像图 片。
结论： T2信号强度随着铁离子浓度的降低而降低。 HUVEC细胞与偶联了多肽 K5 (SEQ ID NO.： 2)的超顺磁纳米粒子孵育后的 T2加权图片相较于偶联了 CI (SEQ ID NO.： 21)多肽的颜色更加暗。 另外无论是和 C1 (SEQ ID NO.： 21)多肽还是多 肽 K5 (SEQ ID NO.： 2)偶联的超顺磁纳米粒子在和 HLF细胞孵育后都没有信号的 差异。 这些核磁成像的结果说明用多肽 K5 (SEQ ID NO.： 2)与超顺磁纳米粒子偶 联后可以提高 HUVEC细胞对造影剂的吸收， 并且多肽 K5 (SEQ ID NO.： 2)可以作 为肿瘤血管成像一个有效的引导分子。
因此， 根据本发明的多肽 K5 (SEQ ID NO.： 2)可以与磁性粒子结合作为造影剂 而用于肿瘤的定位显影。 实施例 10 VI多肽 -羧基磁珠的偶联及体外功能鉴定
VI (SEQ ID NO.： 14)多肽的体外功能测试结果如图 7所示。 图 7A显示仅标注 有 B16 VI的照片中分布有磁珠， 这说明偶联有磁珠的多肽 VI (SEQ ID NO.： 14) 与黑色素瘤细胞结合， 多肽 V1 (SEQ ID NO.： 14)具有很好地靶向于 VEGFR2的作 用。 图 7B显示与偶联磁珠的多肽 V1 (SEQ ID NO.： 14)共同孵育的 B16细胞几乎 全部存在于沉淀中。 图 7C显示不同浓度的超顺磁纳米粒子的 T2加权成像结果及 与 V1 (SEQ ID NO.： 14)多肽偶联后与 B16细胞共孵育后的核磁成像结果。
结论： T2信号强度随着铁离子浓度的降低而降低。 这些核磁成像的结果说明 用多肽 V1 (SEQ ID NO.： 14)与超顺磁纳米粒子偶联后可以提高 B16细胞对造影剂 的吸收， 并且多肽 V1 (SEQ ID NO.： 14)可以作为肿瘤血管成像一个有效的引导分 子。
因此， 根据本发明的多肽 V1 (SEQ ID NO.： 14)可以与磁性粒子结合作为造影 剂而用于肿瘤的定位显影。 实施例 11 多肽 K5 (SEQ ID NO.： 2)对荷瘤小鼠肿瘤体积及血管增殖的抑制 多肽对荷瘤小鼠肿瘤体积的抑制结果如图 8小鼠肿瘤消减部分所示多肽对照 组的小鼠肿瘤体积明显大于注射多肽 K5 (SEQ ID NO.： 2)的实验组， 肿瘤体积分 别是 58 3和 17. 06 mm3 CD31 免疫组化结果所示多肽实验组的血管密度明显低 于对照组的血管密度。 在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单 独引用作为参考那样。 此外应理解， 在阅读了本发明的上述讲授内容之后， 本领 域技术人员可以对本发明作各种改动或修改， 这些等价形式同样落于本申请所附 权利要求书所限定的范围。

Claims

权 利 要 求
1. 一种氨基酸序列， 其特征在于， 所述氨基酸序列为 FF/YEXWGVK。
2.一种具有 VEGFR-2结合活性多肽， 其特征在于， 所述的多肽具有以下氨基酸核 心序列： FF/YEXWGVK。
3. 如权利要求 2所述的多肽， 其特征在于， 所述多肽的结构式如下：
Z1-FX1EX2WGVK-Z2
式中，
Z1为无或 1-7个氨基酸的肽段；
XI为 F或 Y;
X2为任一氨基酸， （较佳地为 20种天然氨基酸中任一种）； 和
Z2为无或 1-5个氨基酸的肽段。
4. 如权利要求 2所述的多肽， 其特征在于， Z1为 1-5个氨基酸的肽段， 较佳地 1-3个， 更佳地 1-2个； 和 /或
X2选自 A、 M、 I、 S、 E、 L、 T、 R; 禾口 /或
Z2为 1-4个氨基酸的肽段， 较佳地为 1-3个， 更佳地为 1-2个的肽段。
5. 一种多肽， 其特征在于， 所述多肽的序列选自下组：
(a) SEQ ID NO.： 1所示的序列；
(b) SEQ ID NO.： 2所示的序列;
(c) SEQ ID NO.： 3所示的序列；
(d) SEQ ID NO.： 4所示的序列；
(e) SEQ ID NO.： 5- 16所示的序列； 。
(f) SEQ ID NO.： 1-16的衍生多肽。
6. 如权利要求 5所述的多肽， 其特征在于， 所述的衍生多肽为 SEQ ID NO.： 1-16 中任一序列， 经过 1-3个， 较佳地 1-2个， 更佳地 1个氨基酸取代、 缺失和 /或添加 后形成的且具有 VEGFR-2结合活性的多肽。
7.一种分离的多核苷酸， 其特征在于， 所述的多核苷酸编码如权利要求 1所述的 氨基酸序列、 权利要求 2或 5所述的多肽。
8. 一种药物组合物， 其特征在于， 所述的药物组合物含有
(a) 活性成分， 该活性成分选自：
(al)权利要求 1所述的氨基酸序列或其药学上可接受的盐； 和 /或
(a2)权利要求 2或 5所述的多肽或其药学上可接受的盐； 以及 (b) 药学上可接受的载体。
9. 一种活性成分的用途， 该活性成分选自：
(al)权利要求 1所述的氨基酸序列或其药学上可接受的盐； 和 /或
(a2)权利要求 2或 5所述的多肽或其药学上可接受的盐；
其特征在于， 所述的活性成分用于制备治疗肿瘤或与 VEGFR-2结合的药物。
10. 如权利要求 1所述氨基酸序列、 或如权利要求 2或 5所述多肽的用途， 其特 征在于， 用于制备肿瘤定位诊断的试剂或试剂盒。
11. 一种治疗肿瘤的方法， 其特征在于， 给需要的对象施用权利要求 8所述的药 物组合物。
12. 一种肿瘤定位诊断的方法， 其特征在于， 包括步骤：
(i)向需要的对象施用造影剂， 所述造影剂偶联有权利要求 1所述的氨基酸序列、 和 /或权利要求 2或 5所述多肽；
(i i)对所述的造影剂进行显影；
(i i i)基于显影结果对肿瘤进行定位， 其中， 如果造影剂发生浓聚， 则所述的浓聚 部位就是肿瘤的位置。
PCT/CN2013/080568 2012-07-31 2013-07-31 一种肿瘤血管特异性结合多肽及其应用 WO2014019520A1 (zh)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101838311A (zh) * 2010-01-20 2010-09-22 暨南大学 内皮细胞生长因子vegf抗原表位的模拟短肽7b及其应用
CN101838313A (zh) * 2010-01-20 2010-09-22 暨南大学 内皮细胞生长因子vegf抗原表位的模拟短肽9b及其应用
CN102757482A (zh) * 2012-07-31 2012-10-31 中国科学院苏州纳米技术与纳米仿生研究所 一种多肽及其应用
CN102775474A (zh) * 2012-07-31 2012-11-14 中国科学院苏州纳米技术与纳米仿生研究所 一种多肽及其应用

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CN101838311A (zh) * 2010-01-20 2010-09-22 暨南大学 内皮细胞生长因子vegf抗原表位的模拟短肽7b及其应用
CN101838313A (zh) * 2010-01-20 2010-09-22 暨南大学 内皮细胞生长因子vegf抗原表位的模拟短肽9b及其应用
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