JP2003018994A - Peptide-based compound binding to receptor-type tyrosine kinase ret - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a peptide-based compound capable of selectively transforming or targeting a receptor-type tyrosine kinase RET expression cell. SOLUTION: This peptide-based compound has an amino acid sequence: Gly Xaa Gly Xbb (Gly is glycine; and Xaa and Xbb are each a positively charged amino acid), or contains the amino acid sequence, and binds to the receptor-type tyrosine kinase RET.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a peptide-type compound that binds to a receptor tyrosine kinase; RET (Oncogene 5, 97-102, 1990), and particularly to the extracellular domain of the RET gene product showing a specific expression pattern. Peptide compounds that bind to. Further, the present invention relates to the peptide compound, a method for selectively transforming RET-expressing cells and a method for identifying, collecting and preparing the RET-expressing cells, and a carrier for a diagnostic agent or a therapeutic agent based on these methods. Is.

[0002]

RET is a receptor tyrosine kinase and is known to function as a co-receptor for glial cell-derived neurotrophic factor (GDNF) (Cell
85, 1113-1124, 1996; Nature 382, 80-83, 1996).
As an extracellular domain of RET, a protein of about 100 kDa is expressed in a form in which an arm is extended outside the expressing cell. R
The expression of ET is characteristic, and in the nervous system tissues, it is found in Schwann cells, neuroblastoma cells, and neurons constituting the substantia nigra tissue responsible for Parkinson's disease (Nature 3
81, 785-789, 1996; Nature 387, 717-721, 1997; Natu
re 387, 721-724, 1997). In addition, RET is expressed in approximately 20 to 30% of CD34-positive cells that are hematopoietic stem cells (Blood 8
9, 2925-2937, 1997). Further, conventionally, an antibody has been used for the identification / preparation of RET-expressing cells, and as a target for RET cells, for example, a virus is mainly used for gene transfer to Parkinson's disease or gene transfer to hematopoietic stem cells. Vector systems have been used.

[0003]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a peptide that specifically binds with a RET molecule as a target protein, as a new cell targeting method.
Another object of the present invention is to selectively transform the peptide and RET-expressing cells with the peptide and R.
It is intended to identify, collect and prepare ET-expressing cells, and to provide a diagnostic or therapeutic carrier based on these.

[0004]

Means for Solving the Problems As a result of intensive investigations, the present inventors have found peptides, which are the above-mentioned problems, that specifically bind to the RET molecule as a target protein. It has become possible to selectively transform RET-expressing cells, identify, collect and prepare RET-expressing cells, and provide a carrier for a diagnostic agent or a therapeutic agent based on these. That is, the present invention relates to the following (1) to (7). (1) Formula (1)

[0005] Gly Xaa Gly Xbb (1)

(Wherein Gly represents glycine, Xaa and
Xbb may be the same or different and each represents an amino acid capable of being positively charged) or a peptide compound which contains this amino acid sequence and which binds to the receptor tyrosine kinase RET.

(2) The peptide compound according to (1), wherein the positively charged amino acids Xaa and Xbb are amino acids selected from arginine, lysine and histidine, respectively. (3) The peptide compound according to (1), wherein the amino acid sequence is represented by any of the amino acid sequences of SEQ ID NOs: 1 to 3 and 5 to 13. (4) By treating a cell with the peptide compound according to any one of (1) to (3) above, the binding or uptake of the peptide compound into the cell is observed, and if desired, A method for identifying, collecting or preparing a RET-expressing cell, which comprises collecting the incorporated cells. (5) A method for introducing a foreign substance into a cell, which comprises contacting the cell with the peptide compound according to any one of (1) to (3) and the foreign substance. (6) A carrier comprising the peptide compound according to any one of (1) to (3) above. (7) The carrier according to (6), which is for diagnosis or treatment.

According to the present invention, cancer can be diagnosed and treated by using the peptide compound of the present invention for cancer cells which highly express the RET gene. Also,
By using this peptide compound, RET-expressing cells can be identified and collected. Furthermore, the property of the peptide compound of the present invention that it is taken up into cells after binding to RET can be used to induce transformation of RET-expressing cells.

With the features of the present invention as described above, it becomes possible to introduce a gene into the substantia nigra tissue cells forming a responsible lesion in Parkinson's disease, and to develop a therapeutic method. In addition, gene transfer to hematopoietic stem cells enables radical gene therapy for diseases such as metabolic diseases.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION Next, embodiments of the present invention will be specifically described. The peptide-based compound of the present invention has an amino acid sequence represented by the above formula (1) or contains this amino acid sequence, and the receptor tyrosine kinase RE
It is a peptide compound having the property of binding to T. That is, the peptide-based compound of the present invention is a peptide in which a plurality of amino acids are further added to the above-mentioned amino acid sequence, or a linker such as biotin at the N-terminus and C-terminus, as long as it has a property of binding to the receptor tyrosine kinase RET. It also includes a combination of. The number of amino acids to be added may be plural as long as it binds to RET of the peptide, but in the present invention, it is 100 or less, preferably 30 or less, particularly preferably 10 or less.
In the above amino acid sequence, Xaa and Xbb may be the same or different and each may be a positively charged amino acid, and specifically, basic amino acids such as arginine, lysine and histidine are preferable. Examples of the peptide compound in the present invention include peptides having the amino acid sequences represented by SEQ ID NOs: 1 to 3 and 5 to 13 and biotinylated products thereof.

This peptide compound can be obtained by synthesizing by a known peptide synthesis method or the like. The peptide synthesis method may be, for example, either a solid phase synthesis method or a liquid phase synthesis method, and after the reaction, the peptide of the present invention can be produced by combining ordinary separation / purification methods. In addition, modification of a specific amino acid in a peptide can be performed by, for example, Nucleic Acid Res.
487-6500, 1982., Methods in Enzymol., 217, 218-22
7, 1993., 217, 270-278, 1993, etc., and site-specific mutagenesis techniques can be used. In addition, biotinylation is performed by, for example, a method of binding to the peptide using biotin-N-hydroxysuccinimide ester.

In order to achieve the above-mentioned object of the present invention, the present inventors prepared a RET recombinant protein, and then identified a peptide that binds to the RET recombinant protein from a random peptide library, which will be described below. In addition, the following amino acid notation is used in the meaning usually used. That is, Gly is glycine, Ser is serine, Lys is lysine, Ala is alanine, Arg is arginine, A
sp means aspartic acid and His means histidine.

First, RET recombinant protein was prepared using a protein expression system using yeast and purified by anion column chromatography (Exp. Hemat
ol. 13, 591-596, 1985). Next, the peptide that binds to this recombinant protein was purified from a random peptide library manufactured by Invitrogen. It consists of a pool of E. coli designed to express one of 12 randomly aligned amino acids outside of one E. coli cell. Furthermore, E. coli that binds to the RET recombinant protein was purified, and the amino acid sequence of the peptide expressed by the E. coli was identified. The identified amino acid sequence is "Gl
y Ser Lys Ala Gly Arg Gly Arg Asp Arg Arg Arg ''
(SEQ ID NO: 1).

Next, this amino acid sequence was examined, and within this sequence, "Lys Ala Gly Arg Gly Arg Asp Arg" (SEQ ID NO: 2) was named RBP-1 (RET binding peptide-1) and further examined. . A peptide with a biotinylated modification at the N-terminus of RBP-1 was synthesized and analyzed for its ability to bind to RET-expressing cells. As a method, cells were fixed with methanol, biotinylated RBP-1 was allowed to bind to the cells, and the presence or absence of binding was observed using a FITC-conjugated avidin under a fluorescence microscope. As a result, it was revealed that it binds to NB-39-nu cells, which is one of the neuroblastoma cell lines. On the other hand, binding was not observed in RET-negative cells such as HeLa cells and HT-1080 cells.

In order to clarify whether RBP-1 is taken up by the endoplasmic reticulum after binding to RET, RBP-1 was added to cultured cells.
A biotinylated peptide was added to the N-terminal of -1. After culturing for 24 hours, RBP-incorporated into cells
Detection of 1 was performed using FITC-conjugated avidin. As a result, uptake was observed in NB-39-nu cells.

Further, the possibility of introducing a foreign gene by RBP-1 was clarified. A plasmid DNA expressing a luciferase gene as a reporter plasmid DNA
Was used. By allowing a complex consisting of streptavidin, plasmid DNA and biotinylated RBP-1 to act on NB-39-nu cells, the expression of the luciferase gene was induced about 200 times that of the control.

"Gly Arg Gly Arg Asp Arg" was used to identify smaller peptides that retain the action of RBP-1.
A peptide consisting of 6 amino acids of (SEQ ID NO: 3) (RBP
-2) was synthesized and biotinylated at the N-terminus RBP-2
Was added to the culture medium. As a result, NB- as well as RBP-1
It was selectively taken up by 29-nu cells.

Further, changes in action by substituting the second and fourth basic amino acids from the N-terminus with neutral amino acids were analyzed. That is, "Gly Ala Gly Ala Asp Arg" (SEQ ID NO: 4) (RBP-3) was synthesized, and the presence or absence of binding to NB-39-nu cells was analyzed. As a result, RBP-3 is NB-39-n
It was revealed that it was not taken up by u cells.

Next, in order to analyze the binding ability with less amino acids, "Gly Arg Gly Arg" (SEQ ID NO:
5) The peptide of (RBP-4) was synthesized, and the binding ability to NB-39-nu cells was analyzed. As a result, RBP-4 bound to NB-39-nu cells, and when added to the culture medium, RBP-4
It was revealed to be taken up by ET-expressing cells.

From the above results, the binding ability to RET is determined by 4 amino acids of "Gly Arg Gly Arg", and the binding ability is lost by substituting a neutral amino acid at the position indicated by Arg. From Gly Xaa Gly Xbb
The four-amino acid peptide Xaa and Xbb each consisting of a positively charged amino acid are the basic peptides provided by the present invention.

The above-mentioned positively charged amino acid is any one of Arg, Lys and His, or a combination of the two. That is, the peptide of the present invention also corresponds to a peptide consisting of the following four amino acids. Gly Arg Gly Arg (SEQ ID NO: 5) Gly Arg Gly Lys (SEQ ID NO: 6) Gly Arg Gly His (SEQ ID NO: 7) Gly Lys Gly Arg (SEQ ID NO: 8) Gly Lys Gly Lys (SEQ ID NO: 9) Gly Lys Gly His (SEQ ID NO: 10) Gly His Gly Arg (SEQ ID NO: 11) Gly His Gly His (SEQ ID NO: 12) Gly His Gly Lys (SEQ ID NO: 13)

In the present invention, the use of a peptide that binds to RET enables identification, collection and preparation of RET-expressing cells. That is, a biological sample predicted to express RET is taken out by surgery or biopsy, and the peptide of the present invention is brought into contact with this to obtain R.
It enables identification and collection of ET-expressing cells. and again,
CD expressing RET using peptide for RET
It is also possible to prepare 34-positive cells from normal bone marrow cells.

According to the present invention, cancer can be diagnosed and treated by using the peptide of the present invention for cancer cells that express the RET gene at a high rate. For example, for malignant tumors such as neuroblastoma, the peptide can be applied to diagnostic imaging by binding a radioisotope or a magnetic substance. Further, by binding a cytotoxic compound to a peptide, it is possible to treat RET-expressing cancer cells with a selective disorder. Furthermore,
By combining target gene therapy for these RET-expressing malignant tumor cells and a peptide that induces cell death, it becomes possible to develop a highly advanced cancer therapeutic method.

The property of the peptide of the present invention to be taken up into cells after binding to RET makes it possible to induce transformation of RET-expressing cells. That is, when a peptide that binds to RET, a complex of a peptide and an exogenous gene, or a conjugate of a peptide and an exogenous protein is used, selective transformation of RET-expressing cells becomes possible, resulting in neurological disorders and metabolic disorders. It is useful for the treatment of For example, gene transfer into substantia nigra tissue cells forming responsible lesions in Parkinson's disease becomes possible, and development of therapeutic methods becomes possible. Parkinson's disease is a neurological disease caused by deficient production of catecholamines in the brain, and it is considered that replacement therapy by introducing and expressing a gene that induces catecholamine production may be a radical therapy for improving symptoms. A therapeutic effect is expected by administration of a complex of the peptide of the present invention and a gene that induces the production of catecholamines.

Further, by introducing the gene into hematopoietic stem cells,
Radical gene therapy for diseases such as metabolic diseases becomes possible. Since it was revealed that RET is expressed in about 20 to 30% of CD34-positive cells that are hematopoietic stem cells, it becomes possible to prepare hematopoietic stem cells and to introduce genes into these cells, which results in metabolic diseases. It is thought that gene therapy for will be possible.

The method for identifying, collecting or preparing RET-expressing cells of the present invention is to treat the cells with the above-mentioned peptide compounds to observe the uptake of the peptide compounds into the cells, and bind or incorporate them as desired. RET-expressing cells are identified and collected. Conditions for treating the cells include, for example, 37 ° C and 5% CO ₂ for 10 minutes to 24 hours.
Incubate for hours. The amount of the above peptide compound added to the medium is, for example, a concentration of 1 to 30 μg / mL. Examples of RET expressing cells include neuroblastoma NB-38-nu cells. For example, binding and uptake are observed by a fluorescence microscope using a fluorescent dye such as FITC for RBP-1 peptide.

The introduction of the foreign substance into the cell of the present invention is carried out by causing the above-mentioned peptide compound and the foreign substance to act on the cell. Examples of foreign substances include genes, cytotoxic compounds, various proteins, and the like. The method for introducing the foreign substance into the cell is carried out, for example, by collecting bone marrow cells or nerve cells and allowing the peptide-gene complex to act ex vivo.

The carrier of the present invention comprises the above peptide compound. This carrier plays a role as a carrier of an exogenous substance to the target site, and is used as a complex or a conjugate with the exogenous substance, and usually for diagnosis or treatment.
Examples of foreign substances include radioisotopes, magnetic substances and fluorescent substances for diagnostic purposes, and genes, cytotoxic compounds and various proteins for therapeutic purposes. For example, for diagnostic use, it can be applied to diagnostic imaging by binding a radioisotope or a magnetic substance to a peptide. Also, for therapeutic use, RET by administration of, for example, a complex or conjugate of the peptide and the cytotoxic compound.
It is possible to treat malignant tumors that express the above, and to treat Parkinson's disease by administering a complex of the peptide and a gene that induces the production of catecholamine.

[0029]

EXAMPLES Examples of the present invention will be shown below, but the present invention is not limited thereto. Example 1: Expression of RET recombinant protein Part of the region encoding the extracellular domain of RET cDNA (part from amino acids 331 to 584: Human Gene Therapy 11, 995)
-1004, 2000, Tumor Research 24, 1-13, 1989, Oncoge
ne 5, 97-102, 1990) was amplified by PCR and the expression plasmid containing zeocin resistance gene (pICZ ?: Invitrogen)
Built in. SMD1168, which is a yeast cell, was introduced by electroporation to obtain zeocin (100 μg / ml) resistant colonies. Cells were expanded from each colony and protein expression was induced with methanol. And the molecular weight is about
A 45 kDa RET protein was obtained. This protein was purified by anion exchange column chromatography to give 5 μg / ml.
Protein was coated onto a 6 cm plate. With this,
RET from Invitrogen random peptide library
Bound E. coli was selected and purified. A plasmid was prepared from each E. coli and, after determining the nucleotide sequence, the encoded amino acid was deduced and identified (SEQ ID NO: 1).

Example 2: RET binding peptide RBP-
Synthesis of 1 RBP-1 was synthesized by a solid phase method using a peptide sequencer, and purified and confirmed using high performance liquid chromatography. Example 3: Uptake into RET-expressing cells by addition of RBP-1 Biotinylated RBP-1 (SEQ ID NO: 2) was added at a concentration of 1 μg / ml to NB-39-nu cells (RET expressing cells) and HT1080 cells (RET.
Non-expressing cells) and cultured overnight, then avidinated FIT
The uptake of the peptide was observed at C. As a result, clear peptide uptake was observed in NB-39-nu cells. On the other hand, peptide uptake was not observed in HT1080 cells, which are RET non-expressing cells, even at the same concentration (1 μg / ml). Further, in RBP-2 (SEQ ID NO: 3), uptake by RET-expressing cells was observed, whereas in RBP-3 (SEQ ID NO: 3),
No uptake was observed in (SEQ ID NO: 4).

Example 4: Expression of a foreign gene introduced into a neuroblastoma cell using RBP-1 A foreign gene was expressed using RBP-1. Biotinylated plasmid DNA (pSV40 / lucif) having biotinylated RBP-1, streptavidin, and luciferase gene.
NB-39-nu cells (RET expressing cells)
And HT1080 cells (cells not expressing RET) were added.
After 60 hours, luciferase activity in each cell was measured. RBP-1 was used in an amount of 100 ng. As a control, streptavidin / plasmid DN
A was used. As shown in FIG. 1, neuroblastoma cell NB-3
In 9-nu, luciferase activity was observed due to a complex of RBP-1, streptavidin, and biotinylated plasmid DNA. The addition of RBP-1 increased the activity of luciferase by about 200 times. On the other hand, almost no increase in luciferase activity was observed in HT1080 cells.

[0032]

INDUSTRIAL APPLICABILITY As described above, by using the peptide compound of the present invention, it becomes possible to target and selectively introduce RET-expressing cells. That is, the peptide of the present invention enables diagnosis and treatment of cancer cells that highly express the RET gene. Moreover, by using the peptide of the present invention, it becomes possible to identify and collect cells expressing RET. Furthermore, after binding to the peptide RET,
The property of being taken up intracellularly can be used to induce transformation on RET expressing cells. Due to such features of the present invention, it becomes possible to introduce a gene into the substantia nigra tissue cells forming a responsible lesion in Parkinson's disease and to develop a therapeutic method. Further, by introducing a gene into hematopoietic stem cells, radical gene therapy for diseases such as metabolic diseases can be performed.

[0033]

[Sequence list] SEQUENCE LISTING <110> NIPPON KAYAKU KABUSHIKI KAISHA <120> Peptide compounds binding to a receptor tyrosine kinase RET <130> <140> <141> <150> <151> <160> 13 <170> <210> 1 <211> 12 <212> PRT <213> Artificial Sequence <223> twelve peptides binding selectively to cells expressing RET, a rece ptor tyrosine kinase <400> 1 Gly Ser Lys Ala Gly Arg Gly Arg Asp Arg Arg Arg  1 5 10 <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <223> Octa peptides binding selectively to cells expressing RET, a recept or tyrosine kinase <400> 2 Lys Ala Gly Arg Gly Arg Asp Arg  1 5 <210> 3 <211> 6 <212> PRT <213> Artificial Sequence <223> Hexa eptides binding selectively to cells expressing RET, a recept or tyrosine kinase <400> 3 Gly Arg Gly Arg Asp Arg  1 5 <210> 4 <211> 6 <212> PRT <213> Artificial Sequence <223> Hexa peptides with mutated amino acids without activity of binding  to cells expressing RET, a receptor tyrosine kinase <400> 4 Gly Ala Gly Ala Asp Arg  1 5 <210> 5 <211> 4 <212> PRT <213> Artificial Sequence <223> Tetra peptides binding selectively to cells expressing RET, a recep tor tyrosine kinase <400> 5 Gly Arg Gly Arg <210> 6 <211> 4 <212> PRT <213> Artificial Sequence <223> Tetra peptides binding selectively to cells expressing RET, a recep tor tyrosine kinase <400> 6 Gly Arg Gly Lys <210> 7 <211> 4 <212> PRT <213> Artificial Sequence <223> Tetra peptides binding selectively to cells expressing RET, a recep tor tyrosine kinase <400> 7 Gly Arg Gly His <210> 8 <211> 4 <212> PRT <213> Artificial Sequence <223> Tetra peptides binding selectively to cells expressing RET, a recep tor tyrosine kinase <400> 8 Gly Lys Gly Arg <210> 9 <211> 4 <212> PRT <213> Artificial Sequence <223> Tetra peptides binding selectively to cells expressing RET, a recep tor tyrosine kinase <400> 9 Gly Lys Gly Lys <210> 10 <211> 4 <212> PRT <213> Artificial Sequence <223> Tetra peptides binding selectively to cells expressing RET, a recep tor tyrosine kinase <400> 10 Gly Lys Gly His <210> 11 <211> 4 <212> PRT <213> Artificial Sequence <223> Tetra peptides binding selectively to cells expressing RET, a recep tor tyrosine kinase <400> 11 Gly His Gly Arg <210> 12 <211> 4 <212> PRT <213> Artificial Sequence <223> Tetra peptides binding selectively to cells expressing RET, a recep tor tyrosine kinase <400> 12 Gly His Gly His <210> 13 <211> 4 <212> PRT <213> Artificial Sequence <223> Tetra peptides binding selectively to cells expressing RET, a recep tor tyrosine kinase <400> 13 Gly His Gly Lys

[Brief description of drawings]

FIG. 1 is a neuroblastoma cell (NB-39-using a complex consisting of RBP-1, streptavidin, and biotinylated plasmid DNA.
(nu cells) and HT1080 cells are shown in FIG.

[Explanation of symbols] indicates HT1080 cells, and indicates NB-39-nu cells. SA /
DNA is a control, streptavidin /
Shows plasmid DNA.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 3/00 A61P 25/16 4H045 25/16 35/00 35/00 C07K 5/103 C07K 5/103 7 / 06 7/06 7/08 7/08 C12Q 1/02 C12N 5/10 C12N 15/00 ZNAA C12Q 1/02 5/00 BF term (reference) 4B024 AA01 AA11 CA04 DA02 EA04 GA11 HA01 HA11 HA17 4B063 QA18 QA19 QQ08 QQ43 QQ79 QR33 QR48 QR59 QR77 QS05 QS36 QX02 4B065 AA90X AA90Y AB01 BA02 CA24 CA44 CA46 4C076 AA95 CC01 CC21 CC27 EE41 FF02 4C084 AA13 AA17 A20 AA27 A20 AA27 A20 AA27 AA27 A20 AA27 AB27 A20 AA27 A20 AA27 A20 AA27 A20 AA17 A20 A4

Claims (7)

[Claims] 1. A formula (1) Gly Xaa Gly Xbb (1) (in the formula, Gly represents glycine, and Xaa and Xbb may be the same or different and each represents a positively charged amino acid). A peptide-based compound having or including an amino acid sequence, which binds to the receptor tyrosine kinase RET. 2. The positively charged amino acids Xaa and Xbb
Is an amino acid selected from arginine, lysine and histidine, respectively. 3. Amino acid sequences of SEQ ID NOs: 1-3, 5-13
The peptide compound according to claim 1, which is represented by any one of the amino acid sequences of. 4. Treating a cell with the peptide compound according to any one of claims 1 to 3, observing binding or uptake of the peptide compound into the cell, and binding or uptake as desired. R which is characterized by collecting the dead cells
Methods for identifying, harvesting or preparing ET expressing cells. 5. A method for introducing a foreign substance into a cell, which comprises contacting the peptide-based compound according to any one of claims 1 to 3 with a foreign substance with a cell. 6. A carrier comprising the peptide compound according to any one of claims 1 to 3. 7. The carrier according to claim 6, which is used for diagnosis or treatment.