WO2020103961A2 - 一种脑部肿瘤靶向肽及其应用 - Google Patents

一种脑部肿瘤靶向肽及其应用

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WO2020103961A2
WO2020103961A2 PCT/CN2020/072652 CN2020072652W WO2020103961A2 WO 2020103961 A2 WO2020103961 A2 WO 2020103961A2 CN 2020072652 W CN2020072652 W CN 2020072652W WO 2020103961 A2 WO2020103961 A2 WO 2020103961A2
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tumor targeting
brain tumor
brain
peptide
blood
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PCT/CN2020/072652
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French (fr)
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WO2020103961A3 (zh
WO2020103961A9 (zh
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林坚
陈龙
李成鹏
李朝刚
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江苏集萃分子工程研究院有限公司
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Priority to US17/295,566 priority Critical patent/US20220331440A1/en
Publication of WO2020103961A2 publication Critical patent/WO2020103961A2/zh
Publication of WO2020103961A3 publication Critical patent/WO2020103961A3/zh
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1058—Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/40—Vectors comprising a peptide as targeting moiety, e.g. a synthetic peptide, from undefined source
    • C12N2810/405—Vectors comprising RGD peptide

Definitions

  • the invention relates to the field of medicine and bioengineering, in particular to a protein detection or therapeutic drug, in particular to a peptide capable of efficiently crossing the blood-brain barrier and targeting brain tumors, as well as its in vivo imaging detection and target in brain tumors Use in therapy.
  • the brain is a high-level nerve center of human beings, and has a unique protective mechanism, the blood-brain barrier, which limits the entry of various substances into the brain.
  • the brain and the outside world have strict blocking mechanisms of the blood-brain barrier, which will make it difficult for most currently effective drugs to reach the target, and it is difficult to effectively treat brain diseases.
  • RGD is a short peptide sequence containing arginine-glycine-aspartic acid (Arg-Gly-Asp), which serves as a recognition site for the interaction between integrin and its ligand, mediating between cells and extracellular matrix and cells Interaction.
  • Tumor cells or neovascularization can specifically express certain integrins such as ⁇ v ⁇ 3, which can bind RGD peptide with a certain affinity and become a new target for tumor therapy. Therefore, the application of RGD peptides in tumor therapy has become a research hotspot.
  • This short peptide not only has the ability to cross the blood-brain barrier, but also has a specific role in targeting brain tumors. At the same time, other short peptides targeting highly expressed antigens on the tumor surface may also have similar functions.
  • Pb001 short peptide can be used as a short peptide carrier that efficiently crosses the blood-brain barrier, and can transport other drugs through the blood-brain barrier, which will greatly improve the scope and efficacy of brain drugs.
  • the receptor pathway mediated by the receptor LRP1 can be selected, and the skeleton can be selected by referring to the Kunitz region of the conserved active center of the blood-brain barrier protein of the bovine islet inhibitor and ⁇ -amyloid peptide.
  • phage display technology artificially synthesize a high-throughput phage display library in vitro, obtain new independent intellectual property rights efficient blood-brain barrier short peptides from the foundation, use mouse in vivo screening technology, optimize the screening process, and obtain high efficiency through three rounds of screening Short peptides that cross the blood-brain barrier.
  • a targeting molecule that efficiently crosses the blood-brain barrier, and its C-terminus contains the tumor targeting region sequence.
  • the present invention provides a brain tumor targeting molecule, which is characterized by comprising a blood-penetrating brain barrier region and a tumor targeting region, and the blood-penetrating brain barrier region includes SEQ ID NO: 1.
  • the tumor targeting area may have different molecular sizes.
  • a basic principle is that the higher the hydrophilicity, the smaller the molecular size suitable for the brain tumor targeting area; the hydrophilic The lower the sex, the larger the molecular size suitable for the brain tumor targeting area.
  • the tumor targeting region as a brain tumor targeting peptide may include no more than 100, 50, 40, 30, 20 , 15, or 10 amino acids.
  • the brain tumor targeting molecule can be obtained by connecting the blood-penetrating brain barrier region with any polypeptide having tumor targeting function.
  • the tumor targeting region includes any one or a combination of CGKRK (SEQ ID NO: 3) and ATWLLPPR (SEQ ID NO: 4).
  • amino acid sequence of the brain tumor targeting molecule is TFYGGRPKRNNFLRGIRCGKRK (SEQ ID NO: 5) or TFYGGRPKRNNFLRGIRATWLLPPR (SEQ ID NO: 6)
  • the brain tumor targeting molecule of the present invention is characterized in that the tumor targeting area includes RGD sequence.
  • the RGD sequence is a linear RGD polypeptide, a cyclic GRD polypeptide or a peptidomimetic compound with an arginine-glycine-aspartate tripeptide sequence as the active center.
  • the sequence of the cyclic RGD polypeptide is CRGDKGPDC (SEQ ID NO: 7), wherein two cysteine forms a disulfide bond.
  • the brain tumor targeting molecule of the present invention is characterized in that the amino acid sequence of the brain tumor targeting peptide is shown in SEQ ID NO: 2.
  • amino acid sequence of the brain tumor targeting peptide is TFYGGRPKRNNFLRGIRCRGDKGPDC (SEQ ID NO: 8)
  • the present invention also provides a nucleic acid encoding the brain tumor targeting molecule.
  • the present invention provides a construct comprising the encoding nucleic acid, the construct comprising a nucleic acid expression cassette and a vector.
  • the vectors of the present invention include prokaryotic expression vectors and eukaryotic expression vectors.
  • the present invention also provides a host cell containing the encoding nucleic acid and nucleic acid construct.
  • the host cells of the present invention include prokaryotic host cells and eukaryotic host cells; the prokaryotic host cells include E. coli, and the eukaryotic host cells include Pichia pastoris, Saccharomyces cerevisiae, insect cells and the like.
  • the present invention also provides the use of the brain tumor targeting molecule in the preparation of a brain tumor diagnostic reagent.
  • the brain tumor targeting molecule is also connected with an active substance, and the active substance is a diagnostic marker.
  • the diagnostic markers include fluorescence, isotopes, radioactive substances, and the like.
  • the brain tumor diagnostic reagent is used for brain tumor imaging or brain tumor positioning.
  • the present invention also provides the use of the brain tumor targeting molecule in the preparation of brain tumor therapeutic drugs.
  • the brain tumor targeting molecule is also connected with an active substance, and the active substance is a therapeutic drug.
  • the therapeutic drugs include chemotherapy drugs, radioactive substances and the like.
  • the brain tumor targeting molecule is used for brain tumor targeted therapy.
  • the active substances connected to brain tumor targeting molecules can have different molecular sizes.
  • a basic principle is that the higher the hydrophilicity, the more suitable for the activity of connecting brain tumor targeting molecules The smaller the molecular size of the substance; the lower the hydrophilicity, the larger the molecular size of the active substance suitable for attachment to brain tumor targeting molecules.
  • the brain tumor targeting molecule connected with active substances such as diagnostic or therapeutic drugs of the present invention at least partially retains the ability to penetrate the blood-brain barrier of the forebrain tumor targeting molecule linked to the active substance.
  • the part of the blood-brain barrier included in the Pb001 peptide provided by the present invention is obtained by in vivo screening of animal models. Compared with in vitro screening methods, in vivo screening of animal models is closer to the actual process of crossing the blood-brain barrier in humans, so It is ensured that the Pb001 peptide of the present invention has an efficient blood-brain barrier function in vivo.
  • the present invention combines the high-efficiency blood-brain-brain barrier peptide obtained by screening with RGD to achieve the functions of blood-brain-brain barrier and tumor targeting, and provides a new carrier and platform for the diagnosis and treatment of brain tumors.
  • the Pb001 peptide of the present invention is composed of only 21 amino acids.
  • the molecule is relatively small and has great prospects for transformation. Therefore, it can carry various diagnostic imaging agents, radiotherapy agents, and chemotherapeutic agents to target brain tumors.
  • Figure 1 Optimal enrichment time after phage display peptide administration in mouse brain
  • FIG. 3 Enrichment diagram of fluorescently labeled Pb001 peptide brain
  • Figure 7 Localization and enrichment of fluorescently labeled Pb001 peptide in brain tumors
  • Figure 8 Enrichment multiples of fluorescently labeled brain tumor targeting peptides at brain tumor sites
  • FIG. 11 PBL001 inhibits U87MG glioma cell proliferation at different concentrations in vitro
  • Figure 12 PBL001 inhibits tumor proliferation in U87MG glioma mouse model in vivo
  • Example 1 Screening peptides that efficiently cross the blood-brain barrier by using animal in vivo screening methods
  • the peptide library kun-M was designed and constructed. Use this library for high-throughput screening.
  • the screening methods are as follows:
  • mice were anesthetized with 5% chloral hydrate, the surface of the mice was wiped with alcohol, disinfected, and 100ml of saline was flowed through the heart.
  • the brain was carefully dissected and the brain mesh homogenate was broken up by ultrasound, centrifuged, 0.45 ⁇ m filter membrane, take the supernatant solution and mix with the ER2738 bacterial liquid cultured to the logarithmic phase, mix and incubate at 37 °C for 4h.
  • the phage-infected clone can be taken and sent for sequencing analysis to show the sequence of the polypeptide Nanobody.
  • the highest frequency sequence to be screened out TFYGGRPKRNNFLRGIR.
  • the tumor targeting region sequence is connected to the C-terminus of TFYGGRPKRNNFLRGIR to obtain a brain tumor targeting peptide that penetrates the blood-brain barrier.
  • the brain tumor targeting peptide Pb001 that penetrates the blood-brain barrier is designed and synthesized:
  • TFYGGRPKRNNFLRGIRCRGDKGPDC two cysteine to form a disulfide bond
  • TFYGGRPKRNNFLRGIRCGKRK TFYGGRPKRNNFLRGIRATWLLPPR.
  • Label Pb001 with CY5.5 fluorescence use Lumiprobe brand Cyanine5.5 NHS ester labeling reagent to fluorescently label short peptides, one short peptide is labeled with one fluorescent molecule.
  • Pb001-CY5.5 can be clearly enriched in the brain 4h after the tail vein injection.
  • the heart flows through 100ml of normal saline at a rate of 5ml / min, wash off the fluorescence interference in the blood, take the mouse brain, and other organs to observe, it can be seen that the brain has obvious fluorescence, which is higher than the muscle heart.
  • the fluorescence of the mouse brain can still be clearly seen, and the fluorescence value is only reduced by 40% (Figure 4). It shows that short peptides can cross the blood-brain barrier and have the ability to efficiently cross the blood-brain barrier, and the short peptide fluorescence has a strong residence time in the brain and a good half-life.
  • the U87 tumorigenic cell line is a cell line transformed with the luciferase gene, and the tumor imaging effect can be observed in real time using a mouse imaging device.
  • a short peptide solution labeled with fluorescent molecules was injected into the tail vein.
  • Angiochem a short peptide for efficient blood-brain barrier barrier angiopep2 reported in an article, was selected as a reference control. It is currently reported that the short peptide has excellent application in blood-brain barrier and brain tumor targeting.
  • fluorescent cy5.5 molecules of equal fluorescence equivalent were used.
  • the IVIS small animal live imaging system checked the brain fluorescence. 100ul of 5mg / ml luciferase reaction substrate was injected intraperitoneally. After 5 minutes, the small animal imaging system checked the brain tumor.
  • the pb001 short peptide can be enriched in the brain of glioma model mice after being connected with fluorescence.
  • the heart can flow through 100ml of normal saline at a rate of 5ml / min to wash off the fluorescence interference in the blood.
  • Figure 5 shows that the amount of brain enrichment of the fluorescent-labeled Pb001 peptide is superior to the fluorescent-labeled reference peptide Angiopep2.
  • Figure 6 shows that the fluorescently labeled Pb001 peptide is 1.2 times the fluorescent intensity of the fluorescently labeled reference peptide Angiopep2.
  • Figure 7 shows that the fluorescently labeled Pb001 peptide has obvious fluorescence accumulation at the brain tumor site, that is, Pb001 not only can cross the blood-brain barrier, but also has a better enrichment for brain tumors.
  • Example 3 Enrichment multiple of brain tumor targeting peptide in the brain
  • TFYGGRPKRNNFLRGIR TFYGGRPKRNNFLRGIRCRGDKGPDC (two cysteines form a disulfide bond)
  • TFYGGRPKRNNFLRGIRCGKRK TFYGGRPKRNNFLRGIRATWLLPPR
  • Lumiprobe brand Cyanine5.5 NHS ester labeling reagent to fluorescently label each short peptide
  • each short peptide is labeled with a fluorescent molecule.
  • Pb001 labeled with CY5.5 as in Example 2 is injected into the tail vein respectively Into different U87 brain tumor-bearing mice, 2 hours later, the mice were dissected and brains were taken. Calculate the fluorescence intensity value of the brain tumor location and the unit area of other locations in the brain, and the division of the two is the enrichment factor of the corresponding polypeptide at the tumor location.
  • TFYGGRPKRNNFLRGIRCGKRK and TFYGGRPKRNNFLRGIRATWLLPPR peptides contain different tumor targeting sequences (CGKRK, ATWLLPPR) and RGD sequence binding targets, so it can be seen that tumor targeting peptides for different receptors on the surface of brain tumors can be connected to the blood-brain barrier
  • the C-terminal of the peptide TFYGGRPKRNNFLRGIR makes it have both the ability to penetrate the blood-brain barrier and the ability to target tumors.
  • TFYGGRPKRNNFLRGIRCRGDKGPDC does not increase brain tumor enrichment as significantly as the other three synthetic peptides.
  • the possible reason is that the cyclic peptide structure of the tumor targeting sequence CRGDKGPDC has a certain effect on the structure of the TFYGGRPKRNNFLRGIR peptide. The effect can be eliminated by adjusting the connecting sequence between the two peptide sequences.
  • TFYGGRPKRNNFLRGIRCRGDKGPDC in Figure 8 also show that the blood-brain barrier peptide TFYGGRPKRNNFLRGIR can not only carry shorter peptides (such as short peptides of 5 amino acids or less), but also suitable for carrying larger molecules and longer ones. Peptide segments, cyclic peptide structures, etc. cross the blood-brain barrier, even if they are connected to a cyclic peptide consisting of 10 amino acids, they do not significantly affect the efficiency of crossing the blood-brain barrier.
  • Example 4 Synthesis and characterization of peptide paclitaxel coupled drug PBL001 in vitro and in vivo
  • the synthesized peptide Pb001 was dissolved in DMF (N, N-dimethylformamide), and the paclitaxel molecule was synthesized according to the literature (British Journal of Pharmacology, 2008, 155 (2): 185-197.) To synthesize 2'-NHS-Paclitaxel. Pb001, 2'-NHS-Paclitaxel, triethylamine (or N, N-diisopropylethylamine) are mixed in a ratio of 1: 5: 5, react at 37 degrees for 3 hours, after purification by HPLC, PBL001, mass spectrometry The result is shown in Fig. 9, and the schematic structural diagram is shown in Fig. 10. Each PBL001 molecule contains 2 paclitaxel molecules, with a theoretical molecular weight of 4339.75.

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Abstract

本发明涉及一种脑部肿瘤的靶向肽，其编码核酸，以及包含所述编码核酸的载体、宿主细胞。所述靶向肽连接诊断标记可用于脑部肿瘤诊断，特别是脑部肿瘤成像；所述靶向肽连接治疗性药物可携带药物直达脑部肿瘤病灶实现脑部肿瘤的治疗。

Description

一种脑部肿瘤靶向肽及其应用 技术领域
本发明涉及医药及生物工程领域，特别涉及一种蛋白质检测或治疗药物，具体涉及一种能够高效穿越血脑屏障、靶向脑部肿瘤的肽，以及其在脑部肿瘤的活体成像检测、靶向治疗中的用途。
背景技术
大脑是人的高级神经中枢，具有独特的保护机制血脑屏障，该屏障限制了多种物质进入到脑部。脑部与外界有严格的物质限制进入的血脑屏障的阻挡机制，会导致大部分目前有效药物难以到达作用靶点，而难以对脑部疾病进行有效治疗。
RGD为含有精氨酸-甘氨酸-天冬氨酸(Arg―Gly―Asp)的短肽序列，作为整合素和其配体相互作用的识别位点，介导细胞与细胞外基质及细胞之间的相互作用。肿瘤细胞或者新生血管可以特异表达某些整合素如αvβ3，能以一定的亲和力结合RGD肽，成为肿瘤治疗的新靶点。因此，RGD肽在肿瘤治疗中的应用已成为研究热点，此短肽不仅具有穿越血脑屏障的能力，还具有一定的特异靶向脑部肿瘤的作用。同时，其他一些靶向肿瘤表面高表达抗原的短肽也可能具有类似的功能。
目前脑部肿瘤治疗药物受限于血脑屏障的作用，能较好穿越血脑屏障的药物只有替莫唑胺，但其作用有效性有限。Pb001短肽可作为一个高效穿越血脑屏障短肽载体，能将其它药物载运通过血脑屏障，这将大大提高脑部药物的使用范围与疗效。
发明内容
为解决上述问题，基于受体介导途径思路，能通过受体LRP1介导的受体途径，参考牛胰岛抑制剂以及β-淀粉样肽跨血脑屏障蛋白保守的活性中心kunitz区域，选择骨架，利用噬菌体展示技术，体外人工合成构建高通量噬菌体展示文库，从基础获取新型自主知识产权高效穿血脑屏障短肽，利用小鼠体内筛选技术，优化筛选流程，通过三轮筛选，得到高效穿过血脑屏障短肽。在此基础上，进一步设计获得高效穿过血脑屏障的靶向分子，其C端含有肿瘤靶向区序列。
一方面，本发明提供一种脑部肿瘤靶向分子，其特征在于：包括穿血脑屏障 区和肿瘤靶向区，所述穿血脑屏障区包括SEQ ID NO:1。
根据亲水性等因素的不同，所述肿瘤靶向区可以具有不同的分子大小，一个基本的原则是，亲水性越高、适合作为脑部肿瘤靶向区的分子大小越小；亲水性越低、适合作为脑部肿瘤靶向区的分子大小越大。作为举例，对于亲水性分子(如线性肽、环状肽等肽类衍生物)而言，作为脑部肿瘤靶向肽的肿瘤靶向区可包括不超过100、50、40、30、20、15、或10个氨基酸。
本领域技术人员可以通过常规实验检测和验证肿瘤靶向区与SEQ ID NO:1所述穿血脑屏障肽经肽键连接后穿过血脑屏障的能力和效率。本发明SEQ ID NO:1所述穿血脑屏障区与肿瘤靶向区连接后，至少部分地保留了SEQ ID NO:1的穿血脑屏障能力。
所述至少部分地保留SEQ ID NO:1的穿血脑屏障能力，例如保留SEQ ID NO:1的至少10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、或98％的穿血脑屏障能力。
所述脑部肿瘤靶向分子可通过以下方式得到，将所述穿血脑屏障区与任意具有肿瘤靶向功能的多肽相连接。
在具体的实施方式中，所述肿瘤靶向区包括：CGKRK(SEQ ID NO：3)、ATWLLPPR(SEQ ID NO：4)中的任意一种或两种的组合。
进一步，所述脑部肿瘤靶向分子的氨基酸序列为TFYGGRPKRNNFLRGIRCGKRK(SEQ ID NO：5)或TFYGGRPKRNNFLRGIRATWLLPPR(SEQ ID NO：6)
本发明所述脑部肿瘤靶向分子，其特征在于：所述肿瘤靶向区包括RGD序列。
进一步，所述RGD序列为以精氨酸-甘氨酸-天冬氨酸三肽序列为活性中心的线性RGD多肽、环状GRD多肽或拟肽化合物。在具体实施方式中，所述环状RGD多肽的序列为CRGDKGPDC(SEQ ID NO：7)，其中，两个半胱氨酸形成二硫键。
本发明所述脑部肿瘤靶向分子，其特征在于，所述脑部肿瘤靶向肽的氨基酸序列如SEQ ID NO:2所示。
在另一实施方式中，所述脑部肿瘤靶向肽的氨基酸序列为TFYGGRPKRNNFLRGIRCRGDKGPDC(SEQ ID NO：8)
第二方面，本发明还提供编码所述脑部肿瘤靶向分子的核酸。
第三方面，本发明提供包含所述编码核酸的构建体，所述构建体包括核酸表 达盒、载体。
本发明所述载体包括原核表达载体和真核表达载体。
第四方面，本发明还提供一种含有所述编码核酸、核酸构建体的宿主细胞。
本发明所述宿主细胞包括原核宿主细胞和真核宿主细胞；所述原核宿主细胞包括大肠杆菌，所述真核宿主细胞包括毕赤酵母、酿酒酵母、昆虫细胞等。
第五方面，本发明还提供所述脑部肿瘤靶向分子在制备脑部肿瘤诊断试剂中的用途。
其中，所述脑部肿瘤靶向分子还连接有活性物质，所述活性物质为诊断标记。
所述诊断标记包括荧光、同位素、放射物质等。
其中，所述脑部肿瘤诊断试剂用于脑部肿瘤成像或脑部肿瘤定位。
第六方面，本发明还提供所述脑部肿瘤靶向分子在制备脑部肿瘤治疗药物中的用途。
其中，所述脑部肿瘤靶向分子还连接有活性物质，所述活性物质为治疗药物。
所述治疗药物包括化疗药、放射物质等。
其中，所述脑部肿瘤靶向分子用于脑部肿瘤靶向治疗。
根据亲水性等因素的不同，连接到脑部肿瘤靶向分子的活性物质可以具有不同的分子大小，一个基本的原则是，亲水性越高、适合连接到脑部肿瘤靶向分子的活性物质的分子大小越小；亲水性越低、适合连接到脑部肿瘤靶向分子的活性物质的分子大小越大。
本领域技术人员可以通过常规实验检测和验证活性物质(如肿瘤诊断或治疗性药物等)与脑部肿瘤靶向分子连接后的穿过血脑屏障的能力和效率。优选地，本发明与诊断或治疗药物等活性物质连接后的脑部肿瘤靶向分子，至少部分地保留了连接活性物质前脑部肿瘤靶向分子的穿血脑屏障能力。
所述至少部分地保留连接活性物质前脑部肿瘤靶向肽的穿血脑屏障能力，例如保留本发明所述连接活性物质前脑部肿瘤靶向分子的至少10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、或98％的穿血脑屏障能力。
与现有技术相比，本发明的技术方案具有以下优点：
第一、本发明提供的Pb001肽包含的穿血脑屏障部分是通过动物模型活体筛选获得的，与体外筛选方式相比，动物模型活体筛选更接近人体内穿过血脑屏障 的真实过程，因此确保了本发明Pb001肽具有高效的体内穿血脑屏障功能。
第二、本发明将筛选获得的高效穿血脑屏障肽与RGD联合同时实现了穿血脑屏障和靶向肿瘤的作用，为脑部肿瘤的诊断和治疗提供了新的载体和平台。
第三、本发明所述Pb001肽仅由21个氨基酸组成，分子较小、改造的前景大，因而可以携带各种诊断成像剂、放疗剂、化疗剂靶向至脑部肿瘤处。
附图说明
通过阅读下文优选实施方式的详细描述，各种其他的优点和益处对于本领域普通技术人员将变得清楚明了。附图仅用于示出优选实施方式的目的，而并不认为是对本发明的限制。在附图中：
图1：小鼠脑部噬菌体展示肽给药后最佳富集时间
图2：合成Pb001肽的质谱检测图
图3：荧光标记的Pb001肽脑部富集图
图4：荧光标记的Pb001肽脑内存留荧光强度
图5：荧光标记的Pb001肽穿血脑屏障与肿瘤定位
图6：荧光标记的Pb001肽在脑部的荧光数值强度
图7：荧光标记的Pb001肽在脑部肿瘤定位富集
图8：荧光标记的各脑肿瘤靶向肽在脑部肿瘤部位的富集倍数
图9：Pb001偶联化药紫杉醇的多肽紫杉醇偶联物PBL001质谱结果
图10：PBL001结构示意图
图11：PBL001体外不同浓度下抑制U87MG脑胶质瘤细胞增殖
图12：PBL001体内抑制U87MG脑胶质瘤小鼠模型中肿瘤增殖
图13：PBL001延长脑胶质瘤模型小鼠生存期
具体实施方式
下面将参照附图更详细地描述本公开的示例性实施方式。虽然附图中显示了本公开的示例性实施方式，然而应当理解，可以以各种形式实现本公开而不应被这里阐述的实施方式所限制。相反，提供这些实施方式是为了能够更透彻地理解本公开，并且能够将本公开的范围完整的传达给本领域的技术人员。
根据本发明的实施方式，提出以下实施例
实施例一、利用动物体内筛选法，筛选高效率穿越血脑屏障肽
基于活性中心kunitz区域的序列分析，设计构建了噬菌体展示的多肽库的多肽库kun-M。利用该库进行高通量筛选。
筛选方法如下：
1成年balb/c小鼠(18-22g)，取噬菌体库TBS稀释到100ul/1011PFU，尾静脉注射，根据前期预实验确定接种后24h噬菌体在脑的富集脑血比最高(图1)。
2在注射后24h，5％的水合氯醛麻醉小鼠，酒精擦拭小鼠表面，消毒，心脏贯流100ml生理盐水，小心解剖取脑，将脑网状体匀浆超声打碎，离心，过0.45μm滤膜，取上清溶液与培养到对数期的ER2738菌液混匀，37℃感染培养4h。
3 12000rpm离心20min，取菌液上清，用PEG/NaCl的方法富集噬菌体，将该富集的库用于下一轮的筛选。
4如上重复至少三轮。在看到输出噬菌体的滴度出现明显富集，可取噬菌体感染克隆，送测序分析展示多肽纳米抗体的序列。
得筛选出的最高频序列：TFYGGRPKRNNFLRGIR。
在TFYGGRPKRNNFLRGIR的C端连接肿瘤靶向区序列，以得到穿血脑屏障的脑肿瘤靶向肽。
在此基础上设计合成穿血脑屏障的脑肿瘤靶向肽Pb001：
TFYGGRPKRNNFLRGIRSRGD。
另设计合成多肽TFYGGRPKRNNFLRGIRCRGDKGPDC(两个半胱氨酸形成二硫键)，TFYGGRPKRNNFLRGIRCGKRK，TFYGGRPKRNNFLRGIRATWLLPPR。
实施例二、脑肿瘤靶向肽的合成及功能验证
1化学合成多肽及荧光标记
合成多肽TFYGGRPKRNNFLRGIRSRGD(MW:2467.78PI:11.83)，无标记多肽质谱检测结果如图2所示，根据图2计算分子量为823.71*3-3＝2468,与多肽Pb001的分子量符合。
将Pb001标记CY5.5荧光，采用Lumiprobe品牌的Cyanine5.5 NHS ester标记试剂将短肽进行荧光标记，一个短肽标记上一个荧光分子。
2荧光标记Pb001在正常小鼠脑部的富集
准确稀释调节标记短肽的浓度，用生理盐水将其溶解，调整稀释连接荧光短 肽到相同额荧光当量，尾静脉注射裸鼠100μl，在不同时间点，利用IVIS小动物活体成像系统分不同时间点时间点观测成像。
如图3所示，在尾静脉注射后4h可以明显观测到Pb001-CY5.5在脑部富集。取小鼠，5ml/min的速度心脏贯流100ml生理盐水，洗掉血液中的荧光干扰，取小鼠脑，以及其他器官观察，可以看出脑部有明显荧光，且高于肌肉心脏等。在过夜16h后，依然能清晰的看到小鼠脑部荧光存在，荧光值仅降低了40％(图4)。说明短肽能穿过血脑屏障的阻拦，具有高效的穿血脑屏障的能力，并且短肽荧光在脑部有较强的存留时间，有较好的半衰期。
3建立肿瘤模型，查看荧光短肽在肿瘤的富集。
培养u87脑胶质瘤细胞，待细胞长到足够量，取细胞，选取8周的裸鼠，脑部定位，定位注射5×10 5个细胞，进行脑部成瘤。U87成瘤细胞株为转入了荧光素酶基因的细胞株，可用小鼠成像仪进行实时肿瘤成瘤效果观测。
在肿瘤模型鼠成瘤稳定后，尾静脉注射标有荧光分子的短肽溶液100μl。选取一个文章报道的angiochem公司高效穿血脑屏障短肽Angiopep 2做为一个参考对照，目前报道该短肽具有优秀的在穿血脑屏障以及脑部肿瘤靶向应用。阴性对照采用等荧光当量的荧光cy5.5分子。尾静脉注射后，IVIS小动物活体成像系统查看脑部荧光。腹腔注射100ul 5mg/ml的荧光素酶反应底物，5min后小动物成像系统查看脑部肿瘤情况。
pb001短肽连上荧光后，能在脑胶质瘤模型小鼠脑部富集，5ml/min的速度心脏贯流100ml生理盐水，洗掉血液中的荧光干扰，取小鼠脑，比较小鼠脑部荧光。图5表明荧光标记的Pb001肽脑部富集量优于荧光标记的参考肽Angiopep2。图6表明荧光标记的Pb001肽为荧光标记的参考肽Angiopep2荧光强度的1.2倍。
图7表明荧光标记Pb001肽在脑部肿瘤部位有明显的荧光聚集，即Pb001不仅能穿越血脑屏障，且对脑部肿瘤有较好的富集。
实施例三、脑肿瘤靶向肽在脑部的富集倍数
利用多肽固相合成分别合成多肽TFYGGRPKRNNFLRGIR，TFYGGRPKRNNFLRGIRCRGDKGPDC(两个半胱氨酸形成二硫键)，TFYGGRPKRNNFLRGIRCGKRK，TFYGGRPKRNNFLRGIRATWLLPPR。采用Lumiprobe品牌 的Cyanine5.5 NHS ester标记试剂将各短肽分别进行荧光标记，每个短肽上标记一个荧光分子，标记完成后与实施例二中同样标记了CY5.5的Pb001分别尾静脉注射入不同的荷U87脑瘤的小鼠中，2小时后，解剖小鼠，并取脑。分别计算脑瘤位置和脑部其他位置单位面积的荧光强度值，二者相除即为相应多肽在肿瘤位置的富集倍数。
如图8所示，相对于没有连接肿瘤靶向肽的TFYGGRPKRNNFLRGIR肽，其他肽都展示出了更好的肿瘤富集。其中，TFYGGRPKRNNFLRGIRCGKRK与TFYGGRPKRNNFLRGIRATWLLPPR肽中所含有的肿瘤靶向序列(CGKRK，ATWLLPPR)与RGD序列结合的靶标不同，因此可知，针对脑瘤表面不同受体的肿瘤靶向肽都可以连接至穿血脑屏障肽TFYGGRPKRNNFLRGIR的C端，使其同时具备穿血脑屏障能力和肿瘤靶向能力。
与TFYGGRPKRNNFLRGIR肽相比，TFYGGRPKRNNFLRGIRCRGDKGPDC的脑部肿瘤富集提高幅度不如另外三种合成多肽显著，可能的原因是所述肿瘤靶向序列CRGDKGPDC的环肽结构对TFYGGRPKRNNFLRGIR肽的结构产生了一定的影响，应当可通过调整两段肽序列间的连接序列消除影响。另外，图8中关于TFYGGRPKRNNFLRGIRCRGDKGPDC的实验结果还表明穿血脑屏障肽TFYGGRPKRNNFLRGIR不仅可携带较短的肽(如长度小于等于5个氨基酸的短肽)，同样也适合携带更大的分子、更长的肽段、环肽结构等穿过血脑屏障，即使与包括10个氨基酸的环肽连接也并未显著影响其穿血脑屏障的效率。
实施例四、多肽紫杉醇偶联药物PBL001的合成及体外、体内表征
1 PBL001的合成
合成好的多肽Pb001溶解于DMF(N，N-二甲基甲酰胺),紫杉醇分子依据文献(British Journal of Pharmacology,2008,155(2):185-197.)合成2’-NHS-Paclitaxel。Pb001，2’-NHS-Paclitaxel，三乙胺(或N,N-二异丙基乙胺)三者按1:5:5比例混合，37度反应3小时，HPLC纯化后即得PBL001，质谱结果如图9所示，结构示意图如图10所示。每个PBL001分子包含2个紫杉醇分子，理论分子量为4339.75。
2 PBL001体外抑制U87MG脑胶质瘤的增殖
第一天在96孔板内接种2000个U87MG细胞，第二天以不同浓度的紫杉醇 (PTX)或PBL001处理细胞，两天后测定细胞活性，得到如图11所示肿瘤增殖抑制曲线。图11的结果表明将pb001短肽与紫杉醇偶联获得的PBL001，并没有降低紫杉醇的抗脑胶质瘤活性，甚至还略有增强，推测可能的原因是修饰后的PBL001更易穿透细胞膜。
3 PBL001体内抑制U87MG脑胶质瘤小鼠模型中肿瘤增殖
第一天在6-8周裸鼠右侧尾状核部位接种10 6个U87MG细胞，三天后开始进行PBL001尾静脉给药，U87MG细胞稳转了荧光素酶的基因，通过荧光素酶的化学发光测定脑瘤的增殖，如图12所示。图12的结果表明PBL001在小鼠模型中对脑胶质瘤增殖有明显抑制作用。
4 PBL001延长脑胶质瘤模型小鼠生存期
第一天在6-8周裸鼠右侧尾状核部位接种10 6个U87MG细胞，三天后开始进行溶剂或等剂量紫杉醇或PBL001尾静脉给药，记录各组小鼠的生存期，如图13所示。图13的结果表明与溶剂组或紫杉醇组相比，PBL001在小鼠模型中能显著延长脑胶质瘤小鼠的生存期，与紫杉醇相比PBL001的毒副作用更低。
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到的变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以所述权利要求的保护范围为准。

Claims (14)

  1. 一种脑部肿瘤靶向分子，其特征在于：包括穿血脑屏障区和肿瘤靶向区，所述穿血脑屏障区包括SEQ ID NO:1。
  2. 如权利要求1所述脑部肿瘤靶向分子，其特征在于，所述肿瘤靶向区包括：CGKRK、ATWLLPPR、CRGDKGPDC中的任意组合。
  3. 如权利要求1所述脑部肿瘤靶向分子，其特征在于，所述肿瘤靶向区包括RGD序列。
  4. 如权利要求3所述脑部肿瘤靶向分子，其特征在于，所述RGD序列为以精氨酸-甘氨酸-天冬氨酸三肽序列为活性中心的线性RGD多肽、环状RGD多肽或拟肽化合物。
  5. 如权利要求4所述脑部肿瘤靶向分子，其特征在于，所述肿瘤靶向区的氨基酸序列为CRGDKGPDC，其中两个半胱氨酸形成二硫键。
  6. 如权利要求1所述的脑部肿瘤靶向分子，其特征在于，所述脑部肿瘤靶向肽的氨基酸序列如SEQ ID NO：5、SEQ ID NO：6或SEQ ID NO：8所示。
  7. 如权利要求1所述脑部肿瘤靶向分子，其特征在于，所述脑部肿瘤靶向肽的氨基酸序列如SEQ ID NO:2所示。
  8. 编码权利要求1-7所述脑部肿瘤靶向分子的核酸。
  9. 包含权利要求8所述核酸的核酸构建体，所述构建体包括表达盒、载体。
  10. 含有权利要求8所述核酸或权利要求9所述核酸构建体的宿主细胞。
  11. 权利要求1-7所述脑部肿瘤靶向分子在制备脑部肿瘤诊断试剂中的用途。
  12. 权利要求1-7所述脑部肿瘤靶向分子在制备脑部肿瘤治疗药物中的用途。
  13. 如权利要求11或12所述的用途，其特征在于所述脑部肿瘤靶向分子还连接有活性物质，所述活性物质为诊断标记或肿瘤治疗药物。
  14. 如权利要求13所述用途，其特征在于所述脑部肿瘤靶向分子连接的活性物质包括荧光、同位素、放射物质、化疗药等。
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