JP6789573B2 - Peptides with glioma-specific accumulation and their use - Google Patents

Description

The present invention relates to peptides having glioma-specific accumulation and their use.
The present application claims priority based on Japanese Patent Application No. 2015-21959 filed in Japan on October 28, 2015, the contents of which are incorporated herein by reference.

The incidence of primary brain tumors is said to be 10 to 15 per 100,000 population per year, of which 1/4 is a group of tumors called gliomas. The 5-year survival rate of anaplastic astrocytoma (grade 3), which is classified as a high-grade group among gliomas, is about 23%, and that of glioblastoma (glioblastoma) (grade 4) is about 10%. And remarkably low. Since glioma infiltrates existing brain tissue as a characteristic of tumor lesions, the lesion area including the tumor boundary as precise as possible in confirmation of diagnosis by biopsy, craniotomy surgery, and radiation therapy Diagnostic information is needed. Examples of examinations or diagnostic methods that can provide the highest accuracy as image information for current central nervous system diseases (intracerebral lesions) include computed tomography (CT) and magnetic resonance imaging in combination with a contrast medium. Examples thereof include inspection methods such as a resonance imaging (MRI) and a positron emission tomography (PET) method. Examples of a method for obtaining more precise diagnostic imaging information include a fluorodeoxyglucose (FDG) -PET method and the like.

By the way, in the trend in the medical field using peptides as biomaterials, cell membrane penetrating (cell-absorbing) peptides such as Tat, penetratin, and polyargine have been attracting attention.
However, since these peptides are widely and non-selectively absorbed without distinction between normal cells or normal tissues and tumor cells or tumor tissues, the treatment of malignant tumors requiring targeted drug transport DDS (Drag Delivery). It is difficult to apply it to the System) tool because it causes serious side effects. In particular, cell membrane penetrating (cell-absorbing) peptides such as Tat, which are widely used in experimental systems worldwide, are known to have the property of causing accumulation in the liver (see, for example, Non-Patent Document 1).
In contrast, cyclic RGD is the only peptide that has been medicinalized. Cyclic RGD is the alpha _v beta ₃ integrin has been reported to be highly expressed in endothelial cells of the neovasculature or in existing vessels (and some tumor cells) has been targeted, the point of action on the vascular hyperpermeability Since it has, it is applied as an imaging agent or a DDS agent not only alone but also in combination with other pharmaceuticals (see, for example, Patent Document 1).

Japanese Patent No. 5721140

Vives E., et al., A Truncated HIV-1 Tat Protein Basic Domain Rapidly Translocates through the Plasma Membrane and Accumulates in the Cell Nucleus, J. Biol. Chem., 272, 16010-16017, 1997.

Glioma is still very well recognized as one of the malignant tumors that is difficult to treat and has a low survival rate, and an effective treatment method is required.
In addition, in the above-mentioned examination or diagnosis method for central nervous system diseases (lesions in the brain), there is a risk of physical burden and complications of administration of a contrast medium to a patient. Furthermore, the FDG-PET examination is a method for detecting the inside of cells or tissues into which FDG has been taken up and reflects glucose metabolism activity, and therefore, particularly in the brain in which an increase in glucose metabolism activity is constantly observed. , Depiction of intracerebral lesions is difficult.
Further, since the cyclolic RGD described in Patent Document 1 is not a peptide that targets tumor cells and tumor tissues themselves, there is still room for improvement in terms of medical technology.

The present invention has been made in view of the above circumstances, and provides a novel peptide that acts directly on a glioma and has specific accumulation.

That is, the present invention includes the following aspects.
[1] The following peptide (a) or (c).
(A) A peptide consisting of an amino acid sequence containing the sequence represented by any of SEQ ID NOs: 1, 2 and 3.
( C) A peptide consisting of an amino acid sequence containing a sequence having 90 % or more identity with the sequence represented by any of SEQ ID NOs: 1, 2 and 3, and having glioma-specific accumulation.
[2] The peptide according to [1], which is a peptide composed of L-amino acids.
[3] A nucleic acid comprising the peptide according to [1] or [2].
[4] A vector containing the nucleic acid according to [3].
[5] A carrier comprising the peptide according to [1] or [2].
[6] The carrier according to [5], further comprising a labeling substance or a modifying substance.
[7] The carrier according to [6], wherein the labeling substance is a stable isotope, a radioisotope or a fluorescent substance.
[8] The carrier according to [6] or [7], wherein the modifying substance is a sugar chain or polyethylene glycol.
[9] A pharmaceutical composition comprising the carrier according to any one of [5] to [8] and a physiologically active substance.
[10] The pharmaceutical composition according to [9], which is used for treating or diagnosing a cerebrospinal tumor caused by glioma.

According to the present invention, it is possible to provide a novel peptide having glioma-specific accumulation. In addition, glioma can be detected easily, with high sensitivity and selectively.

It is a fluorescence micrograph of various glioma cells to which various peptides were added in Test Example 1. It is a fluorescence micrograph of the primary astrocyte cells and the primary glioblastoma cell to which various peptides were added in Test Example 2. 3 is a fluorescence micrograph of primary neurons and primary glioblastoma cells supplemented with various peptides in Test Example 3. 3 is a graph showing the fluorescence intensity in various tissues of mice in which human glioma cells in Test Example 4 were subcutaneously transplanted and intravenously injected with FITC-labeled PEP1ΔNS peptide, Peptide1 or Peptide2. In addition, it is a fluorescence micrograph of bright field and dark field of various tissues of a mouse in which human glioma cells in Test Example 4 were subcutaneously transplanted and intravenously injected with FITC-labeled PEP1ΔNS peptide, Peptide1 or Peptide2. It is a fluorescence micrograph of a light field and a dark field of the right cerebral hemisphere of a mouse in which human glioma cells in Test Example 5 were transplanted into the right cerebral hemisphere and Peptide2 was injected from the tail vein. It is a fluorescence micrograph showing the result of HE-staining a slice of the right cerebral hemisphere of a mouse in which human glioma cells in Test Example 5 were transplanted into the right cerebral hemisphere and Peptide2 was injected from the tail vein.

[Peptide with glioma-specific accumulation]
In one embodiment, the present invention provides any of the following peptides (a) to (c).
(A) A peptide consisting of an amino acid sequence containing the sequence represented by any of SEQ ID NOs: 1, 2 and 3.
(B) In the amino acid sequence represented by any of SEQ ID NOs: 1, 2 and 3, the amino acid sequence comprises a sequence in which one or several amino acids are deleted, substituted or added, and is specific to a glioma. Peptides with a high degree of accumulation,
(C) A peptide consisting of an amino acid sequence containing a sequence having 60% or more identity with the sequence represented by any of SEQ ID NOs: 1, 2 and 3, and having glioma-specific accumulation.

The peptide of this embodiment is a novel peptide having glioma-specific accumulation.

The present inventors have found a novel peptide having glioma-specific accumulation by the in-betro-virus (IVV) method, and have completed the present invention.
In the IVV method, puromycin, which is a kind of antibiotic, is bound to the 3'end of mRNA via a PEG (polyethylene glycol) spacer, and a cell-free translation reaction is performed using it as a template, so that the protein and mRNA become puromycin. A simple mRNA-protein linking molecule, IVV, covalently linked via is constructed. The present inventors constructed an IVV library by independently producing IVV. From this constructed IVV library, IVV containing a protein that binds to bait is caught in vitro, and then the mRNA linked to the IVV is reverse-transcribed, amplified by PCR, and the base sequence is obtained. By decoding, the interacting proteins can be identified in very small amounts (more than 1000 times more sensitive than mass spectrometry).

The peptide of the present embodiment includes the peptide of (a) below.
(A) A peptide consisting of an amino acid sequence containing the sequence represented by any one of SEQ ID NOs: 1, 2 and 3.

The amino acid sequence represented by SEQ ID NO: 1, 2 or 3 in (a) above is the sequence represented by the following amino acid sequence.
RQAXXLTV (SEQ ID NO: 1)
RCWYAVLYP (SEQ ID NO: 2)
RRIHXFPLH (SEQ ID NO: 3)
[In the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3 above, X represents an aspartic acid residue (D) or a glutamic acid residue (E). ]

The peptide (a) above has glioma-specific accumulation. Further, the peptide of the present embodiment has glioma-specific accumulation even if it is a peptide consisting only of the amino acid sequence represented by SEQ ID NO: 1, 2 or 3.

As used herein, the term "glioma" is also referred to as a glioma and means a tumor that develops from a glial cell. In addition, "glial cells" is a general term for cells that are not nerve cells that make up the nervous system (brain, spinal cord, etc.), and plays a role of nourishing nerves and supporting nerve activity.
There are various types and grades of glioma, which can be classified as shown in Table 1, for example.

In Table 1, "malignancy", also referred to as grade, is evaluated based on abnormal microscopic findings of cells or prediction of proliferation and metastasis rate. The malignancy can be classified as shown in Table 2 (Reference: Yoichi Nakazato, New WHO Classification of Brain Tumors, Neurosurgery 36: 473-491,2008).

In Table 2, MIB-1 is one of the clones of the anti-Ki-67 antibody and is a cell proliferation marker indicating the degree of proliferation. In addition, Ki-67 is known to correlate well with the malignancy and prognosis of tumors such as degree of differentiation, vascular invasion and lymph node metastasis in many tumors such as breast cancer, gastric cancer, colon cancer and uterine cancer, and cell proliferation. Very useful as a marker.

The above-mentioned gliomas are often a mixture of various types or grades of gliomas, and even if they have the same disease name, the effects of anticancer agents, the effects of radiotherapy, the prognosis of life, etc. are different. In addition, a mixture of various types or grades of glioma can make diagnosis difficult by biopsy of only a portion of the tumor.
The peptide (a) has specific accumulation in grade 3 and 4 glioblastomas (gliomas), which are particularly high-grade gliomas, among all the above-mentioned types or grades of glioma. Therefore, by using the above-mentioned peptide (a) as a carrier as described later, high-grade glioblastoma (gliomablastoma) can be detected with high sensitivity and selectively. In addition, high-grade glioblastoma (glioblastoma) can be treated.

As used herein, "glioma-specific agglomeration" means the property of being highly absorbed and agglomerated by gliomas as compared to normal tissues in vivo and tumor cells of other strains.

In the amino acid sequence represented by any of SEQ ID NOs: 1, 2 and 3, X is preferably an amino acid residue having basicity, and is an aspartic acid residue (D) or a glutamic acid residue (E). It is more preferable, and it is further preferable that it is an aspartic acid residue (D).

Among the amino acid sequences represented by SEQ ID NO: 1 or SEQ ID NO: 3 in (a) above, more specifically, the amino acid sequence represented by SEQ ID NO: 4 or 5 below can be mentioned.
RQADDRLTV (SEQ ID NO: 4)
RRIHDFPLH (SEQ ID NO: 5)

The peptide of the present embodiment includes the peptide of the following (b) as a peptide functionally equivalent to the peptide of the above (a).
(B) In the amino acid sequence represented by any of SEQ ID NOs: 1, 2 and 3, the amino acid sequence comprises a sequence in which one or several amino acids are deleted, substituted or added, and is specific to a glioma. Peptide with a high degree of accumulation.

Here, the number of amino acids that may be deleted, substituted, or added is preferably 1 or more and 15 or less, more preferably 1 or more and 10 or less, and particularly preferably 1 or more and 5 or less.
Furthermore, the peptide (b) has glioma-specific accumulation.

The peptide of the present embodiment contains the peptide of the following (c) as a peptide functionally equivalent to the peptide of the above (a).
(C) A peptide consisting of an amino acid sequence having 60% or more identity with the amino acid sequence represented by any one of SEQ ID NOs: 1, 2 and 3, and having glioma-specific accumulation.

In order to be functionally equivalent to the peptide of (a) above, it has 60% or more identity. As such identity, 70% or more is preferable, 80% or more is more preferable, 85% or more is further preferable, 90% or more is particularly preferable, and 95% or more is most preferable.
Furthermore, the peptide (c) has a glioma-specific accumulation.

Here, the sequence identity of the target amino acid sequence with respect to the reference amino acid sequence can be obtained, for example, as follows. First, the reference amino acid sequence and the target amino acid sequence are aligned. Here, each amino acid sequence may include a gap so as to maximize the sequence identity. Subsequently, the number of matching amino acids in the reference amino acid sequence and the target amino acid sequence can be calculated, and the sequence identity can be determined according to the following formula (1).
"Sequence identity (%)" = [Number of matched amino acids] / [Total number of amino acids in the target amino acid sequence] x 100 (1)

The peptides (a) to (c) may have a cyclic structure. Due to the cyclic structure, it is easily absorbed only by the glioma. Further, the peptides (a) to (c) may be composed of L-amino acid, D-amino acid, or a combination thereof, and are preferably peptides composed of L-amino acid.
The L-amino acid is a naturally occurring amino acid, and the D-amino acid is the one in which the chirality of the L-amino acid residue is inverted. It may also be chemically modified to increase glioma-specific integration or to optimize other physical properties.

The peptides (a) to (c) may further have cysteine residues at the N-terminal and C-terminal. Specifically, the amino acid sequence represented by the following SEQ ID NO: 6, 7 or 8 can be mentioned.
CRQADDRLTVC (SEQ ID NO: 6)
CRCWYAVLYPC (SEQ ID NO: 7)
CRRIHDFPLHC (SEQ ID NO: 8)
By providing cysteine residues at the N-terminal and C-terminal, the peptide of the present embodiment can take a cyclic form by utilizing the disulfide bond between thiol groups contained in the cysteine residue.

[Nucleic acid encoding peptide]
In one embodiment, the invention provides nucleic acids encoding the peptides described above.

According to the nucleic acid of the present embodiment, a peptide having glioma-specific accumulation can be obtained.

Examples of the nucleic acid encoding the above peptide include a nucleic acid consisting of the base sequence represented by any of SEQ ID NOs: 9, 10 and 11, or a base sequence represented by any of SEQ ID NOs: 9, 10 and 11. Nucleotide sequence of a combination encoding each amino acid that is a constituent of a peptide having 80% or more, for example 85% or more, for example 90% or more, for example 95% or more identity, and having a glioma-specific accumulation. Nucleic acids and the like including any of the above can be mentioned. The base sequence represented by SEQ ID NO: 9 is the base sequence of the nucleic acid encoding the peptide consisting of the amino acid sequence represented by the above SEQ ID NO: 4, and the base sequence represented by SEQ ID NO: 10 is the above. It is a base sequence of a nucleic acid encoding a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2, and the base sequence represented by SEQ ID NO: 11 encodes a peptide consisting of the amino acid sequence represented by SEQ ID NO: 5 above. It is a base sequence of a nucleic acid.

Here, the sequence identity of the control base sequence with respect to the reference base sequence can be determined, for example, as follows. First, the reference base sequence and the target base sequence are aligned. Here, each base sequence may include a gap so as to maximize the sequence identity. Subsequently, the number of matching bases in the reference base sequence and the target base sequence can be calculated, and the sequence identity can be determined according to the following formula (2).
"Sequence identity (%)" = [number of matched bases] / [total number of bases in the target base sequence] x 100 (2)

[Vector containing nucleic acid encoding peptide]
In one embodiment, the invention provides a vector containing the nucleic acids described above.

According to the vector of this embodiment, a peptide having glioma-specific accumulation can be obtained.

The vector of this embodiment is preferably an expression vector. The expression vector is not particularly limited, and for example, a plasmid derived from Escherichia coli such as pBR322, pBR325, pUC12 and pUC13; a plasmid derived from bacillus such as pUB110, pTP5 and pC194; a plasmid derived from yeast such as pSH19 and pSH15; Bacteriophages; viruses such as adenovirus, adeno-associated virus, lentivirus, vaccinia virus, baculovirus, retrovirus, hepatitis virus; and vectors modified thereto can be used.

In the above-mentioned expression vector, the promoter for expressing the above-mentioned peptide is not particularly limited, and for example, animal cells such as EF1α promoter, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, HSV-tk promoter and the like are used. A promoter for expression as a host, a promoter for expression using a plant cell as a host such as a 35S promoter of Califlower Mosaic Virus (CaMV), a REF (rubber election promoter) promoter, a polyhedrin promoter, an insect cell such as a p10 promoter, etc. A promoter for expression as a host can be used. These promoters can be appropriately selected depending on the host expressing the above-mentioned peptides.

The expression vector described above may further have a multicloning site, enhancer, splicing signal, polyA addition signal, selectable marker, origin of replication and the like.

[Career]
In one embodiment, the invention provides a carrier comprising the peptides described above.

According to the carrier of the present embodiment, the target substance can be easily and efficiently transported to the glioma.

The brain is protected against toxic substances by the presence of two barrier systems, the Blood-Cerebrospinal Fluid Barrier (BBB) and the Blood-Cerebrospinal Fluid Barrier (BCSFB). The surface area of BBB is about 5000 times larger than the surface area of BCSFB and is considered to be the main pathway for serum ligand uptake. In addition, the brain endothelium that constitutes the BBB poses a major obstacle to the use of drugs that are considered to be effective in many diseases of the central nervous system. Also, in principle, it is believed that only lipophilic molecules smaller than about 500 daltons can pass through the BBB, i.e. from blood to the brain. However, the carrier of the present embodiment can pass through the BBB when administered intravenously to a test animal (various mammals including humans or non-human animals, preferably humans), resulting in glioma in the brain. Reach and selectively absorbed.

The carrier of the present embodiment preferably further comprises a labeling substance or a modifying substance. In addition, the carrier of the present embodiment may include both a labeling substance and a modifying substance. The labeling substance or modifying substance may be physically or chemically bound to the above-mentioned peptide, either directly or via a linker. Specifically, it may be a coordination bond, a covalent bond, a hydrogen bond, a hydrophobic interaction, or a physical adsorption, and any known bond, linker, and bond method can be adopted. Further, the binding position may be either the N-terminal or the C-terminal of the above-mentioned peptide.

Examples of the labeling substance include stable isotopes, radioactive isotopes, fluorescent substances, positron emission tomography (PET) nuclei, single photon emission computed tomography (SPECT) nuclei, and SPECT nuclei. Examples thereof include magnetic resonance imaging (MRI) contrasting agents, computed tomography (CT) contrasting agents, and magnetic materials. Of these, stable isotopes, radioactive isotopes or fluorescent substances are preferable. By providing the above-mentioned labeling substance, it is possible to easily and highly sensitively confirm whether or not the target substance has been transported to the glioma.

Examples of stable isotopes include ¹³ C, ¹⁵ N, ² H, ¹⁷ O, and ¹⁸ O. Examples of the radioactive isotope include ³ H, ¹⁴ C, ¹³ N, ³² P, ³³ P, ³⁵ S, ¹²⁵ I, ¹¹¹ In, ⁶⁴ Cu, and ¹⁸ F. When the labeling substance is a stable isotope or a radioisotope, the above-mentioned peptide may be prepared by using a stable isotope-labeled amino acid or a radioisotope-labeled amino acid. Amino acids labeled with stable isotopes or radioactive isotopes include 20 types of amino acids (alanine, arginine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine). , Valine, tryptophan, cysteine, asparagine, glutamic acid), and is not particularly limited as long as it is an amino acid contained in the above-mentioned peptide. Further, the amino acid may be L-form or D-form, and can be appropriately selected as needed.

The stable isotope-labeled or radioisotope-labeled above-mentioned peptide is prepared by expressing the above-mentioned vector containing a nucleic acid encoding the above-mentioned peptide in a system in which a stable isotope-labeled amino acid or a radioisotope-labeled amino acid is present. can do. Examples of the system in which the stable isotope-labeled amino acid or the radioactive isotope-labeled amino acid is present include a cell-free peptide synthesis system and a live cell peptide synthesis system in which the stable isotope-labeled amino acid or the radioactive isotope-labeled amino acid is present. .. That is, in a cell-free peptide synthesis system, a peptide may be synthesized using a stable isotope-unlabeled amino acid or a radioisotope-unlabeled amino acid in addition to a stable isotope-labeled amino acid or a radioactive isotope-labeled amino acid, or a live cell peptide synthesis system. In, by culturing cells transformed with the above vector containing the above-mentioned peptide-encoding nucleic acid in the presence of a stable isotope-labeled amino acid or a radioisotope-labeled amino acid, the above-mentioned above-mentioned peptide-encoding nucleic acid is contained. The above-mentioned peptides labeled with stable isotopes or radioactive isotopes can be prepared from the vector.

Expression of the above stable isotope-labeled or radioisotope-labeled peptide using a cell-free peptide synthesis system is the above-mentioned vector containing a nucleic acid encoding the above-mentioned peptide or the above-mentioned stable isotope-labeled amino acid or radioisotope. In addition to labeled amino acids, stable isotope-unlabeled amino acids or radioactive isotope-unlabeled amino acids required for the synthesis of the above-mentioned peptides labeled with stable isotopes or radioactive isotopes, cell extracts for cell-free peptide synthesis, It can be carried out using an energy source (a high-energy phosphate bond-containing substance such as ATP, GTP, creatine phosphate, etc.) or the like. The reaction conditions such as temperature and time can be appropriately selected and carried out. For example, the temperature is 20 to 40 ° C., preferably 23 to 37 ° C., and the reaction time is 1 to 24 hours, preferably 1 to 24 hours. 10 to 20 hours.

As used herein, the term "cell extract for cell-free peptide synthesis" refers to a translation system involved in the synthesis of proteins such as ribosomes and tRNA, or plant cells and animal cells containing components necessary for the transcription system and translation system. It means fungal cells and extracts from bacterial cells. Specific examples thereof include cell extracts of Escherichia coli, wheat germ, rabbit reticulocyte, mouse L-cell, Ehrlich ascites cancer cell, HeLa cell, CHO cell, budding yeast and the like. The preparation of such cell extracts can be described, for example, in Pratt, J. et al. M. Et al., Translation and translation-a practical approach (1984), pp. According to the method described in 179-209, the above cells are disrupted using a French press, glass beads, an ultrasonic disruptor or the like, and a buffer solution containing several kinds of salts for solubilizing protein components and ribosomes is prepared. In addition, it can be homogenized and centrifuged to precipitate insoluble components.

In addition, the expression of the above-mentioned peptides labeled with stable isotopes or radioisotopes using a cell-free peptide synthesis system is described, for example, in Premium Expression Kit (manufactured by Cellfree Science) equipped with a wheat germ extract, Escherichia coli extraction. RTS 100 with liquid, E.I. Commercially available kits such as Escherichia coli HY Kit (manufactured by Roche Applied Science) and Cell-free-kun Quick (manufactured by Taiyo Nippon Sanso) may be used as appropriate. When the expressed stable isotope-labeled or radioisotope-labeled above-mentioned peptide is insoluble, it may be appropriately solubilized with a protein denaturing agent such as guanidine hydrochloride or urea. The above-mentioned peptides labeled with stable isotopes or radioisotopes are further prepared by fractionation treatment by fractionation centrifugation, sucrose density gradient centrifugation, etc., or purification treatment using affinity column, ion exchange chromatography, etc. You can also do it.

Expression of the above-mentioned stable isotope-labeled or radioisotope-labeled peptide using a live cell peptide synthesis system introduces the above-mentioned vector containing a nucleic acid encoding the above-mentioned peptide into a living cell and nourishes the living cell. And antibiotics, as well as stable isotope-labeled amino acids or radioactive isotope-labeled amino acids, stable isotope-labeled peptides or radioactive isotope-labeled peptides necessary for the synthesis of the above stable isotope-labeled amino acids or radioactive isotope-labeled peptides. This can be done by culturing in a culture solution containing a labeled amino acid or the like. Here, the living cell is not particularly limited as long as it is a living cell capable of expressing the above-mentioned vector containing the nucleic acid encoding the above-mentioned peptide, for example, a mammalian cell line such as a Chinese hamster ovary (CHO) cell, or a mammalian cell line. Living cells such as Escherichia coli, yeast cells, insect cells, and plant cells can be mentioned, and Escherichia coli is preferable from the viewpoint of convenience and cost effectiveness. The expression of the above-mentioned vector containing the nucleic acid encoding the above-mentioned peptide is incorporated into an expression vector designed to be expressed in each living cell by gene recombination technology, and the expression vector is introduced into the living cell. It can be carried out. In addition, the above-mentioned vector containing the nucleic acid encoding the above-mentioned peptide can be introduced into living cells by a method suitable for the living cells to be used, for example, electroporation method, heat shock method, calcium phosphate method, lipofection. Method, DEAE dextran method, microinjection method, particle gun method, method using virus, FuGENE (registered trademark) 6 Transfection Reagent (manufactured by Roche), Lipofectamine 2000 Reagent (manufactured by Invitrogen), Lipofectamine LTX Reagent A method using a commercially available transfection reagent such as Lipofectionamine 3000 Reagent (manufactured by Invitrogen) can be mentioned.

The above-mentioned stable isotope-labeled or radioisotope-labeled peptide expressed by the live cell peptide synthesis system disrupts or extracts live cells containing the above-mentioned stable isotope-labeled or radioisotope-labeled peptide. Can be prepared by. Examples of the crushing treatment include a freeze-thaw method, a French press, glass beads, a homogenizer, a physical crushing treatment using an ultrasonic crushing device, and the like. Further, as the extraction treatment, for example, an extraction treatment using a protein denaturing agent such as guanidine hydrochloride or urea can be mentioned. The above-mentioned peptides labeled with stable isotopes or radioisotopes are further subjected to fractionation treatment by fractionation centrifugation, sucrose density gradient centrifugation, etc., purification treatment using affinity column, ion exchange chromatography, etc. It can also be prepared.

Examples of the fluorescent substance include known quantum dots, indocyanine green, 5-aminolevulinic acid (5-ALA; metabolite protoporphyrin IX (PP IX)), near-infrared fluorescent dye (for example, Cy5.5, Cy7, AlexaFluoro, etc.). ), Other known fluorescent dyes (for example, GFP, FITC (Fluorescein), TAMRA, etc.). The above-mentioned fluorescent substance-labeled peptide contains the above-mentioned vector containing a fluorescent substance and a nucleic acid encoding the above-mentioned peptide. May be prepared by the above-mentioned cell-free peptide synthesis system or live cell peptide synthesis system without using stable isotope-labeled amino acids or radioisotope-labeled amino acids.

The nuclides for PET and SPECT are preferably, for example, ¹¹ C, ¹³ N, ¹⁵ O, ¹⁸ F, ⁶⁶ Ga, ⁶⁷ Ga, ⁶⁸ Ga, ⁶⁰ Cu, ⁶¹ Cu, ⁶² Cu, ⁶⁷ Cu, ⁶⁴ Cu, ⁴⁸ V. , Tc-99m, ²⁴¹ Am, ⁵⁵ Co, ⁵⁷ Co, ¹⁵³ Gd, ¹¹¹ In, ¹³³ Ba, ⁸² Rb, ¹³⁹ Ce, Te-123m, ¹³⁷ Cs, ⁸⁶ Y, ⁹⁰ Y, ^185/187 Re, ^186/188 Examples thereof include Re, ¹²⁵ I, or a complex thereof, or a combination thereof. The above-mentioned peptide labeled with a PET nuclide or a SPECT nuclide may be prepared by preparing the above-mentioned vector containing the nucleic acid encoding the above-mentioned peptide by the above-mentioned cell-free peptide synthesis system or live cell peptide synthesis system.
Examples of the MRI contrast agent, CT contrast agent and magnetic material include gadolinium, Gd-DTPA, Gd-DTPA-BMA, Gd-HP-DO3A, iodine, iron, iron oxide, chromium, manganese, or a complex thereof, or a chelate thereof. Examples include a complex. The above-mentioned peptide labeled with an MRI contrast agent, CT contrast agent or magnetic material can be physically or chemically obtained by directly or via a linker between the MRI contrast agent, CT contrast agent or magnetic material and the above-mentioned peptide. It may be prepared by combining them. Specifically, it may be a coordination bond, a covalent bond, a hydrogen bond, a hydrophobic interaction, or a physical adsorption, and any known bond, linker, and bond method can be adopted.

Examples of the modifying substance include sugar chains and polyethylene glycol (PEG). By providing the above-mentioned modifying substance, the target substance can be easily and efficiently absorbed into glioma cells. The above-mentioned peptide modified with the modifying substance may be prepared by physically or chemically binding the modifying substance and the above-mentioned peptide directly or via a linker. Specifically, it may be a coordination bond, a covalent bond, a hydrogen bond, a hydrophobic interaction, or a physical adsorption, and any known bond, linker, and bond method can be adopted.

In the carrier of the present embodiment, the target substance can be appropriately selected depending on the intended use. For example, when used for imaging of glioma, the above-mentioned labeling substance is provided as the target substance as described later. In addition, when used for the treatment or diagnostic use of glioma, a physiologically active substance can be provided as a target substance as described later. The target substance may be physically or chemically bound to the above-mentioned peptide, either directly or via a linker. Specifically, it may be a coordination bond, a covalent bond, a hydrogen bond, a hydrophobic interaction, or a physical adsorption, and any known bond, linker, and bond method can be adopted. Further, the binding position may be either the N-terminal or the C-terminal of the above-mentioned peptide.

Further, in the carrier of the present embodiment, when the target substance is a protein, a fusion protein containing the target substance and the above-mentioned peptide can be produced, for example, by the following method. First, a host is transformed with an expression vector containing a nucleic acid encoding a fusion protein. Subsequently, the host is cultured to express the fusion protein. Conditions such as the composition of the medium, the temperature and time of culturing, and the addition of the inducer can be determined by those skilled in the art according to known methods so that the transformant grows and the fusion protein is efficiently produced. Further, for example, when an antibiotic resistance gene is incorporated into an expression vector as a selection marker, a transformant can be selected by adding an antibiotic to the medium. Subsequently, the fusion protein expressed by the host is purified by an appropriate method to obtain the fusion protein.

The host is not particularly limited as long as it is a living cell capable of expressing an expression vector containing a nucleic acid encoding a fusion protein, and a mammalian cell line such as a Chinese hamster ovary (CHO) cell or a virus (for example, adenovirus) is used. Viral cells such as viruses, adenovirus, lentivirus, vaccinia virus, baculovirus, retrovirus, hepatitis virus and other viruses), bacteria (for example, Escherichia coli) and other microorganisms, yeast cells, insect cells and plant cells Can be mentioned.
Furthermore, in the carrier of the present embodiment, an expression vector containing the nucleic acid encoding the above-mentioned fusion protein may be directly introduced into the glioma and expressed.

[Pharmaceutical composition]
In one embodiment, the present invention provides a pharmaceutical composition comprising the carriers described above and a bioactive substance.

According to the pharmaceutical composition of the present embodiment, glioma-induced cerebrospinal tumors can be selectively treated.

In the present specification, the "physiologically active substance" is not particularly limited as long as it is effective for the treatment of cerebral spinal cord tumors caused by glioma, and specifically binds to, for example, a drug such as an anticancer agent, nucleic acid, or glioma. Examples thereof include antibodies, antibody fragments, aptamers and the like.
As the "physiologically active substance", a molecular-targeted drug having glioma-selective cytotoxic activity is preferable, but since glioma-selective accumulation is performed by the above-mentioned carrier, even cytotoxic drugs used as conventional anticancer agents can be used. Good.
In addition, the physiologically active substance may be physically or chemically bonded to the above-mentioned carriers directly or via a linker. Specifically, it may be a coordination bond, a covalent bond, a hydrogen bond, a hydrophobic interaction, or a physical adsorption, and any known bond, linker, and bond method can be adopted. In addition, the binding position between the physiologically active substance and the above-mentioned carrier can be appropriately selected as needed. Further, in the pharmaceutical composition of the present embodiment, the above-mentioned carrier may contain the above-mentioned labeling substance or modifying substance.

Examples of the nucleic acid include siRNA, miRNA, antisense, and artificial nucleic acids that compensate for their functions (for example, BNA (Bridged Nucleic Acid) and the like).

The antibody can be produced, for example, by immunizing a rodent animal such as a mouse with a peptide derived from glioma as an antigen. It can also be prepared, for example, by screening a phage library. Examples of the antibody fragment include Fv, Fab, scFv and the like.

Aptamers are substances that have a specific binding ability to glioma. Examples of the aptamer include nucleic acid aptamers and peptide aptamers. Nucleic acid aptamers having a specific binding ability to glioma can be selected by, for example, the systematic evolution of ligand by exponential evolution (SELEX) method or the like. In addition, peptide aptamers having a specific binding ability to glioma can be selected by, for example, the Two-hybrid method using yeast.

In the present specification, "cerebral spinal cord tumor caused by glioma" means a brain tumor and spinal cord tumor caused by glioma, and the type and malignancy thereof are as described above.

The pharmaceutical composition of the present embodiment comprises glioma-induced cerebrospinal tumor diagnosis, glioma-induced cerebrospinal tumor therapeutic effect diagnosis, pathological analysis, glioma-induced cerebrospinal tumor treatment, or glioma-induced cerebrospinal tumor. It can be used for diagnosis of associated diseases, pathological analysis, treatment, and diagnosis of therapeutic effect. Examples of the diagnostic method using the pharmaceutical composition of the present embodiment include PET, SPECT, CT, MRI, endoscopic diagnosis, fluorescence detector diagnosis, and the like.

<Dose>
The pharmaceutical composition of the present embodiment takes into consideration the age, sex, body weight, symptoms, treatment method, administration method, treatment time, etc. of the test animal (various mammals including human or non-human animals, preferably human). Adjusted as appropriate.
For example, when the pharmaceutical composition of the present embodiment is intravenously injected (Intravenous: iv) by an injection, 5 mg or more per 1 kg of body weight per 1 kg of a test animal (preferably human) is administered. It is preferable to administer the amount of peptide, more preferably the amount of peptide of 5 mg or more and 20 mg or less, and particularly preferably the amount of peptide of 5 mg or more and 15 mg or less.

As for the number of administrations, it is preferable to administer once to several times per week on average.
Examples of the administration form include intra-arterial injection, intravenous injection, subcutaneous injection, intranasal injection, transbronchial, intramuscular, percutaneous, or oral methods known to those skilled in the art, and intravenously. Injection is preferred. As described above, the pharmaceutical composition of the present embodiment can pass through the BBB and can pass through the brain when administered to a test animal (various mammals including humans or non-human animals, preferably humans) by intravenous injection. It can reach the glioma within and selectively treat glioma-induced cerebrospinal tumors.

<Composition component>
The pharmaceutical composition of this embodiment comprises a therapeutically effective amount of the carrier and bioactive agent described above, as well as a pharmaceutically acceptable carrier or diluent. Pharmaceutically acceptable carriers or diluents are excipients, diluters, bulking agents, disintegrants, stabilizers, preservatives, buffers, emulsifiers, fragrances, colorants, sweeteners, thickeners, flavors. Examples include agents, solubilizers, additives and the like. By using one or more of these carriers, pharmaceutical compositions in the form of injections, liquids, capsules, suspensions, emulsions, syrups and the like can be prepared.
A colloidal dispersion system can also be used as the carrier. The colloidal dispersion system is expected to have an effect of enhancing the in vivo stability of the peptide and an effect of enhancing the transferability of the peptide to a specific organ, tissue or cell. Examples of the colloidal dispersion system include polyethylene glycol, polymer composites, polymer aggregates, nanocapsules, microspheres, beads, oil-in-water emulsifiers, micelles, mixed micelles, and lipids including liposomes. Liposomal or artificial membrane vesicles, which have the effect of efficiently transporting peptides to the organs, tissues, or cells of the body, are preferred.

Examples of formulation in the pharmaceutical composition of the present embodiment include those used orally as sugar-coated tablets, capsules, elixirs, and microcapsules as needed.
Alternatively, aseptic solutions with water or other pharmaceutically acceptable liquids, or those used parenterally in the form of injections of suspensions can be mentioned. Furthermore, pharmacologically acceptable carriers or diluents, specifically sterile water or saline, vegetable oils, emulsifiers, suspensions, surfactants, stabilizers, flavoring agents, excipients, vehicles, etc. Examples thereof include those formulated by appropriately combining with an antiseptic, a binder and the like and mixing in a unit dose form required for generally accepted pharmaceutical practice.

Additives that can be mixed with tablets and capsules include, for example, binders such as gelatin, corn starch, traganth gum, gum arabic, excipients such as crystalline cellulose, swelling such as corn starch, gelatin and alginic acid. Agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavors such as peppermint, reddish oil or cherry are used. When the dispensing unit form is a capsule, the above-mentioned material can further contain a liquid carrier such as fat or oil.

Aseptic compositions for injection can be formulated according to routine formulation practices using vehicles such as distilled water for injection.
Aqueous solutions for injection include, for example, saline, isotonic solutions containing glucose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, and suitable solubilizers such as. Alcohols, specifically ethanol, polyalcohols such as propylene glycol, polyethylene glycol and nonionic surfactants such as Polysorbate 80 (TM), HCO-50 may be used in combination.

Examples of the oily liquid include sesame oil and soybean oil, and benzyl benzoate and benzyl alcohol may be used in combination as solubilizing agents. In addition, a buffer (for example, phosphate buffer, sodium acetate buffer, etc.), a soothing agent (for example, procaine hydrochloride, etc.), a stabilizer (for example, benzyl alcohol, phenol, etc.), an antioxidant, etc. are blended. You may. The prepared injection solution is usually filled in a suitable ampoule.

When it is an injection, it can also be prepared as an aqueous or non-aqueous diluent, suspension, or emulsion as described above. Such sterilization of the injection can be performed by filtration sterilization with a filter, blending of a bactericidal agent or the like. Injectables can be produced in the form of errand preparation. That is, a sterile solid composition can be prepared by a freeze-drying method or the like, and the composition can be dissolved in distilled water for injection or another solvent before use.

<Treatment method>
One aspect of the present invention provides a pharmaceutical composition comprising the above-mentioned carriers and bioactive substances for the treatment of cerebrospinal tumors caused by glioma.
Also, one aspect of the invention provides a pharmaceutical composition comprising a therapeutically effective amount of the aforementioned carrier and bioactive agent, as well as a pharmaceutically acceptable carrier or diluent.
In addition, one aspect of the present invention provides a therapeutic agent for glioma-induced cerebrospinal tumors, which comprises the pharmaceutical composition.
Also, one aspect of the invention provides the use of the carriers and bioactive substances described above for the manufacture of therapeutic agents for glioma-induced cerebrospinal tumors.
In addition, one aspect of the present invention provides a method for treating a glioma-induced cerebrospinal tumor, which comprises administering an effective amount of the above-mentioned carrier and bioactive substance to a patient in need of treatment.

[Methods for imaging glioma]
In one embodiment, the present invention provides a method for imaging a glioma, using the carriers described above.

According to the method of the present embodiment, glioma can be detected easily, with high sensitivity and selectively.

In the method of this embodiment, the carrier described above preferably comprises a labeling substance. Further, it may be provided with a modifying substance. Examples of the labeling substance and the modifying substance include the same substances as those described above.

For example, when the above-mentioned carrier containing the labeling substance is added to the glioma, the amount of the above-mentioned carrier containing the labeling substance added is preferably 1 μM or more and 4 μM or less in the culture solution. In addition, it is possible to evaluate whether or not it is accumulated in the glioma 30 minutes or more and 3 hours or less after the addition.
Further, for example, when the above-mentioned carrier having a fluorescent substance as a labeling substance is injected intravenously (Intravenous: iv) by an injection, the weight of 1 kg per administration is given to the test animal (preferably human). It is preferable to administer an amount of 5 mg or more of the peptide, more preferably an amount of 5 mg or more and 20 mg or less of the peptide, and particularly preferably an amount of 5 mg or more and 15 mg or less of the peptide.
Further, for example, when the above-mentioned carrier having a stable isotope, a nuclide for PET or a nuclide for SPECT as a labeling substance is injected intravenously (Intravenous: iv) by an injection, the stable isotope to be used and the nuclide for PET are used. Alternatively, the dose may be determined from the radiation dose according to the type of SPECT nuclide.

In the method of the present embodiment, examples of the above-mentioned carrier detection method including a labeling substance include PET, SPECT, CT, MRI, detection by an endoscope, detection by a fluorescence detector, and the like.

Hereinafter, the present invention will be described with reference to Examples, but the present invention is not limited to the following Examples.

[Example 1] Peptide synthesis A protein-RNA chimeric random type having a uniquely prepared 9-amino acid residue peptide as a phenotype and an mRNA coding sequence as a corresponding genotype via puromycin. Using a peptide library (in-peptide library; IVVL), each peptide (Peptide 1-3) shown in Table 3 below was separated and identified according to a known IVV (mRNA) method. In addition, each of the identified peptides derived from IVVL was synthesized under the FITC (Fluorescein isothiocyanate) label and subjected to hydrochloride treatment. In addition, r9 (9-residue continuous D-arginine) is a non-selective membrane-permeable peptide currently widely used.
All of these were obtained through consignment synthesis to Sigma-Aldrich Japan (Genosis Division).

The cell lines and origins of the various cells used in Test Examples 1 to 5 below are as shown in Table 4 below. These are those that the inventor has subcultured and maintained in the laboratory.

The various cells shown in Table 4 above were cultured in RPMI 1640 medium containing 10% FBS (RPMI 1640 medium).

[Test Example 1] Confirmation test of accumulation of peptide in glioma cells In primary glioblastoma cells, U87MG cells, Gli36 cells, SF767 cells, T98 cells, SK-AO2 cells, SK-MG-1 cells, U251 cells and A172 cells. , Peptide 1, 2 and 3 prepared in Example 1 were added to the medium so as to be 4 μM, respectively. The cells were cultured at 37 ° C. for 60 minutes. Subsequently, the uptake of each peptide in living cells was visually evaluated with an inverted fluorescence microscope. Before microscopic examination, the culture supernatant to which the peptide was added is removed, washed 3 times with 1 × PBS (-), then trypsin-treated to remove adherent cells, and immediately transferred to a new 96-well plate and reconstituted in a new culture medium. After suspension, microscopic examination was performed. The results are shown in FIG.

From FIG. 1, fluorescence was detected in all cells of Peptide 1, 2 and 3. In particular, fluorescence was strongly detected in cells to which Peptide2 was added. From the above, it was clarified that Peptide2 has high permeability into glioma cells.

[Test Example 2] Confirmation test of peptide accumulation in normal astrocyte cells Peptides 1, 2, 3, and r9 (4 μM) prepared in Example 1 were added to primary astrocyte cells and glioblastoma cells at 37 ° C. The cells were cultured for 120 minutes. Subsequently, the uptake of each peptide in living cells was visually evaluated with an inverted fluorescence microscope by the same method as in Test Example 1. The results are shown in FIG. In FIG. 2, "Bright Field" is an image taken in a bright field, and "FITC" is an image taken in a dark field under 488 nm wavelength green fluorescence excitation conditions.

From FIG. 2, fluorescence of r9 was detected in both the primary astrocytes cells and the primary glioblastoma cells. On the other hand, in the primary 1, 2 and 3, fluorescence was detected only in the primary glioblastoma cells.

[Test Example 3] Confirmation test of accumulation of peptide in normal nerve cells Peptide 1, 2, 3, and r9 (4 μM) prepared in Example 1 were added to primary glioblastoma cells and primary glioblastoma cells, and 120 at 37 ° C. Incubated for minutes. Subsequently, the uptake of each peptide in living cells was visually evaluated with an inverted fluorescence microscope by the same method as in Test Example 1. The results are shown in FIG. In FIG. 3, "Bright Field" is an image taken in a bright field, and "FITC" is an image taken in a dark field under 488 nm wavelength green fluorescence excitation conditions.

From FIG. 3, fluorescence of r9 was detected in both the primary neurons and the primary glioblastoma cells. On the other hand, in the primary 1, 2 and 3, fluorescence was detected only in the primary glioblastoma cells.
From Test Examples 1 to 3, it was clarified that Peptides 1, 2 and 3 have glioma cell-specific accumulation.

[Test Example 4] Evaluation test of peptide accumulation in various tissues in human glioma cell transplanted mice NOD-SCID mice in which 1 × 10 ⁶ human glioma cells (primary glioma cells, derived from white women) were subcutaneously transplanted (Japan) A 6-week-old female mouse purchased from Charles River Co., Ltd.) was prepared as a human glioblastoma cell transplantation model. Thirty days after transplantation of human glioma cells, FITC-labeled PEP1ΔNS peptide was intravenously (iv) injected as a Peptide1, Peptide2 or control prepared in Example 1 at 300 μg per 20 g of mouse body weight. The PEP1 peptide is a peptide having accumulation in human glioblastoma cells discovered by the present inventors and Mr. Higa and Mr. Matsushita of Ryukyu University, and the PEP1ΔNS peptide is an N-terminal aspartic acid and C from this PEP1 peptide. It is a peptide consisting of 8 amino acid residues with the terminal serine removed (Reference: Moritoshi H., et al., Identification of a novel cell-penetrating peptide targeting human glioblastoma cell lines as a cancer-homing transporter, Biochem. Biophys . Res. Commun., 457, 206-212, 2015.). The amino acid sequence of the PEP1ΔNS peptide is shown in SEQ ID NO: 13. 30 minutes after the administration, the subcutaneous tumor was resected and the abdomen was opened, and the distribution and fluorescence intensity of the peptide in the tumor lesion and the normal organ group were observed under a fluorescent stereomicroscope in a freshly resected state. The results are shown in FIG. In FIG. 4, brain is the brain, heart is the heart, kidney is the kidney, liver is the liver, lung is the lung, and s. muscle means skeletal muscle, splen means spleen, and tumor means malignant tumor. Further, in FIG. 4, "Bright Field" is an image taken in a bright field, and "FITC" is an image taken in a dark field under 488 nm wavelength green fluorescence excitation conditions.

The graph of FIG. 4 is a graph showing the ratio of the fluorescence intensity detected in various tissues when the fluorescence detected in each tissue is quantified in Test Example 4 and the fluorescence detected in the normal brain is 1.0. Is.
From FIG. 4, the absorption ratio of the PEP1ΔNS peptide to the target glioblastoma was about 18 times the S / N ratio (Signal / noiseratio) when the absorption into the normal brain was 1.0. On the other hand, constant intensity fluorescence was detected in normal liver, kidney, and skeletal muscle tissue. On the other hand, Peptide1 had an S / N ratio of 8 times, but its absorption into normal organ systems such as kidney and skeletal muscle tissue was suppressed to 1/2 or less as compared with PEP1ΔNS. .. Furthermore, in Peptide2, a strong fluorescent signal with an S / N ratio of 70 times or more was detected in the target malignant tumor (glioblastoma), confirming excellent target tumor accumulation.

From the above, it was confirmed that Peptide2 has well-suppressed absorption into normal tissue systems and higher accumulation in glioma cells.

[Test Example 5] Evaluation test of peptide accumulation in the brain in human glioma cell transplanted mice NOD-SCID mice in which 5 × 10 ⁵ human glioma cells (primary glioma cells, derived from white women) were transplanted into the right cerebral hemisphere ( A 6-week-old female mouse purchased from Charles River Japan Co., Ltd.) was prepared as a human glioblastoma cell transplantation model. Three weeks after transplantation of human glioma cells, 200 μg of Peptide 2 prepared in Example 1 was administered from the tail vein to 20 g of mouse body weight. Immediately 30 minutes after administration, the mouse brain was surgically opened and a mouse brain was taken out, and a tissue section containing an infiltrated part of glioma cells in the brain was prepared and observed under a fluorescent stereomicroscope. After fluorescence observation, a histopathological specimen of a tissue section of the brain was prepared and stained with hematoxylin-Eosin (HE). By confirming the tissue section of the brain after HE staining under an optical microscope, it was verified that the portion emitting the green fluorescent signal in the freshly excised brain corresponds to the glioblastoma cell infiltrated portion. The results are shown in FIGS. 5A and 5B. In FIG. 5A, lt. The lobe is the left cerebral hemisphere, rt. robe means the right cerebral hemisphere. Further, in FIG. 5A, "Bright Field" is an image taken in a bright field, and "FITC" is an image taken in a dark field under 488 nm wavelength green fluorescence excitation conditions.

From FIG. 5B, it was confirmed that glioma cells infiltrated into the right cerebral hemisphere.
Further, from FIG. 5A, it was confirmed that the fluorescence signal was well detected in the infiltrated part, and that the glioblastoma cell infiltrated part and the fluorescence detection part were in agreement.
From this, it was clarified that Peptide2 administered from the tail vein can cross the blood-brain barrier (BBB) and accumulate in glioma cells in the right cerebral hemisphere.

According to the present invention, it is possible to provide a novel peptide having glioma-specific accumulation. In addition, glioma can be detected easily, with high sensitivity and selectively.

Claims (10)

The peptide of (a) or (c) below.
(A) A peptide consisting of an amino acid sequence containing the sequence represented by any of SEQ ID NOs: 1, 2 and 3.
( C) A peptide consisting of an amino acid sequence containing a sequence having 90 % or more identity with the sequence represented by any of SEQ ID NOs: 1, 2 and 3 and having glioma-specific accumulation. The peptide according to claim 1, which is a peptide composed of L-amino acids. A nucleic acid comprising encoding the peptide according to claim 1 or 2. A vector comprising the nucleic acid according to claim 3. A carrier comprising the peptide according to claim 1 or 2. The carrier according to claim 5, further comprising a labeling substance or a modifying substance. The carrier according to claim 6, wherein the labeling substance is a stable isotope, a radioactive isotope, or a fluorescent substance. The carrier according to claim 6 or 7, wherein the modifying substance is a sugar chain or polyethylene glycol. A pharmaceutical composition comprising the carrier according to any one of claims 5 to 8 and a physiologically active substance. The pharmaceutical composition according to claim 9, which is used for treating or diagnosing a cerebrospinal tumor caused by glioma.