WO2012160990A1

WO2012160990A1 - Igf-1r-binding peptide

Info

Publication number: WO2012160990A1
Application number: PCT/JP2012/062092
Authority: WO
Inventors: 祐介飯森; 瑞恵山形; 智隆塩野; 芝　清隆
Original assignee: Hoya株式会社; 公益財団法人がん研究会
Priority date: 2011-05-25
Filing date: 2012-05-11
Publication date: 2012-11-29
Also published as: JP2014156401A

Abstract

Provided are: a substance capable of binding to IGF-1R in a specific manner and with high affinity; and a medicinal agent comprising the substance. A peptide which comprises at least the amino acid sequence represented by formula (1) and has 8-50 amino acid residues. Phe-X¹-X²-X³-X⁴-X⁵-X⁶-X⁷ (1) (wherein X¹ represents Tyr, Trp, Phe or His; X² represents Ser, Ala, Thr, Gly, Asn, Asp, Glu, Arg or Lys; X³ represents Met, Leu, Ile, Val, Ala, Phe, Tyr, Trp or Cys; X⁴ represents Leu, Ile, Val or Met; X⁵ represents Gln, Asn, Asp, Glu, Ala, Ser, Thr, Leu, Met, Lys or Arg; X⁶ represents Arg, Lys, His, Asn, Gln, Ser, Thr, Asp, Glu or Ala; and X⁷ represents Leu, Ile, Val or Met).

Description

IGF-1R binding peptide

The present invention relates to a peptide that specifically binds to insulin-like growth factor 1 receptor (IGF-1R) and a medicament containing the peptide.

Human insulin-like growth factor 1 receptor (IGF-1R) is a receptor belonging to the family of transmembrane protein tyrosine kinases, has high affinity for IGF-1, and binds weakly to IGF-2.
IGF-1R is a transmembrane heterotetramer protein and has a configuration in which two α chains outside the cell and two β chains straddling the membrane are disulfide bonded. When IGF-1 and IGF-2 bind to the extracellular domain of IGF-1R, the intracellular tyrosine kinase domain is activated, causing receptor autophosphorylation and substrate phosphorylation.

IGF-1R is known to promote tumor cell growth, transformation and survival. Known tumor cells include lung cancer, breast cancer, colon cancer, ovarian cancer, synovial sarcoma, pancreatic cancer, prostate cancer and the like. It is also known that the blood concentration and tissue concentration of IGF-1R correlate with an increased risk of many cancers.

Thus, IGF-1R is deeply involved in the development and development of various cancers, and anti-IGF-1R antibodies and IGF-1R antagonists have been reported as tools for the diagnosis and treatment of these cancers ( Patent Documents 1 to 3).

Japanese Patent No. 4473257 JP 2005-533493 A Special table 2009-542719

However, anticancer agents using anti-IGF-1R antibodies have not yet been approved as pharmaceuticals. In addition, regarding the IGF-1R antagonist of Patent Document 3, no specific substance is disclosed, and the invention has not been completed.

Therefore, an object of the present invention is to provide a substance that binds specifically and with high affinity to IGF-1R and a medicament containing the same.

Accordingly, the present inventors searched for phages that display peptides that specifically bind to IGF-1R using the phage display method. As a result, peptides having specific amino acid sequences specifically bound to recombinant human IGF-1R protein. And it was found that cells or tissues expressing IGF-1R can be specifically detected using this peptide or a labeled form thereof. Further, since the peptide or a labeled product thereof competitively inhibits the binding between IGF-1R and IGF-1 and suppresses the proliferation of cancer cells by IGF-1, the peptide or the labeled product is a cancer therapeutic agent. As a result, the present invention was completed.

That is, the present invention provides a peptide having 8 to 50 amino acids having at least an amino acid sequence represented by the following formula (1).

(Wherein X ¹ represents Tyr, Trp, Phe or His,
X ² represents Ser, Ala, Thr, Gly, Asn, Asp, Glu, Arg or Lys;
X ³ represents Met, Leu, Ile, Val, Ala, Phe, Tyr, Trp or Cys;
X ⁴ represents Leu, Ile, Val or Met;
X ⁵ represents Gln, Asn, Asp, Glu, Ala, Ser, Thr, Leu, Met, Lys or Arg;
X ⁶ represents Arg, Lys, His, Asn, Gln, Ser, Thr, Asp, Glu or Ala;
X ⁷ represents Leu, Ile, Val or Met)

The present invention also provides a medicine containing the above peptide or a label thereof.

Moreover, this invention provides the said peptide or its label for cancer diagnosis or cancer treatment.
Moreover, this invention provides use of the said peptide or its labeled body for cancer diagnostic agent or cancer therapeutic agent manufacture.
The present invention also provides a method for diagnosing or treating cancer, comprising administering the peptide or a labeled form thereof.

Since the peptide of the present invention has a specific and strong binding property to IGF-1R, this peptide or its label is a detection agent for cells or tissues expressing IGF-1R, particularly various cancer cells or cancer tissues. That is, it is useful as a diagnostic agent for cancer. The peptide of the present invention competitively inhibits the binding between IGF-1R and IGF-1 and suppresses the growth of cancer cells by IGF-1, and thus is useful as a cancer therapeutic agent expressing IGF-1R. It is.

The output titer / input titer ratio of the panning experiment with D12 library is shown. The output titer / input titer ratio of the panning experiment with C7C library is shown. The peptide sequence displayed by the phage recovered in 3 rounds of IGF-1R target panning is shown. The binding test result for the immobilized protein of S-011 phage is shown. The binding test result with respect to the solid-phased protein of M13KE phage is shown. The binding test result of S-011 phage to the liquid phase protein is shown. The bindability test result with respect to the liquid phase protein of M13KE phage is shown. The display peptide sequence of an alanine substituted phage is shown. The binding test result with respect to the solidification protein of an alanine substitution phage is shown. The result of examining the expression level of IGF-1R (Western blot) is shown. The actin expression level examination result (Western blot) is shown. The result of an in vitro binding test using MCF7 cells is shown. The in vitro binding test results using A-172 cells are shown. The competition test result of S-011 phage and peptide Pep02 is shown. The competition test result of S-011 phage and peptide Pep17 is shown. The MCF7 stained image of peptide Pep02-F is shown. The MCF7 stained image of peptide Pep17-F is shown. The display peptide sequences of amino acid substitution phage and short peptide phage are shown. The bindability test result with respect to the solid-phased protein of a 1 amino acid substitution phage is shown. The bindability test result with respect to the solid-phased protein of a 2 amino acid substitution phage is shown. The binding test result to the immobilized protein of the short peptide phage is shown. The competition test result of S-011 phage and rhIGF-1 is shown. The competition test result of S-011 phage and anti-IGF-1R antibody is shown. The growth inhibitory effect of peptide Pep02 with respect to the proliferation enhancement of Hela S3 by rhIGF-1 addition is shown. The growth inhibitory effect of peptide Pep02 with respect to the proliferation enhancement of MCF7 by rhIGF-1 addition is shown. The growth inhibitory effect of peptide Pep02 with respect to the anchorage independent growth enhancement of MCF7 by rhIGF-1 addition is shown.

The peptide of the present invention is a peptide having 8 to 50 amino acids having at least the amino acid sequence represented by the following formula (1).

In the above formula (1), X ¹ represents Tyr, Trp, Phe or His, among which Tyr, Trp or Phe is preferable, and Tyr is particularly preferable.
X ² represents Ser, Ala, Thr, Gly, Asn, Asp, Glu, Arg or Lys. Among these, Ser, Ala, Thr and Gly are preferred, Ser and Ala are more preferred, and Ser is particularly preferred.
X ³ represents Met, Leu, Ile, Val, Ala, Phe, Tyr, Trp, or Cys. Among them, Met, Leu, Ile, Val, Ala are preferable, and Met, Leu, Ile, Ala are more preferable. Met is particularly preferable.

X ⁴ represents Leu, Ile, Val or Met, but Leu is more preferable.
X ⁵ represents Gln, Asn, Asp, Glu, Ala, Ser, Thr, Leu, Met, Lys or Arg, and among them, Gln, Asn and Ala are preferable, Gln and Ala are more preferable, and Gln is particularly preferable.
X ⁶ represents Arg, Lys, His, Asn, Gln, Ser, Thr, Asp, Glu or Ala, preferably Arg, Lys, His, and Ala, more preferably Arg, Lys, and Ala, and particularly preferably Arg.
X ⁷ represents Leu, Ile, Val or Met, but Leu is more preferable.

Of the peptides of the present invention, peptides having at least the amino acid sequence represented by the formula (2) and having 10 to 50 amino acids are more preferable.

(Wherein X ^a represents Asp or Glu,
^Xb represents Pro, Val, Ile, Leu, Ala, Met, Trp, Tyr, Ser, Thr, Cys or Phe,
X ¹ to X ⁷ are the same as above)

Wherein (2), ^{X a} is show Asp or Glu, Asp are preferred. ^Xb represents Pro, Val, Ile, Leu, Ala, Met, Trp, Tyr, Ser, Thr, Lys, or Phe, but Pro, Val, Ile, Leu, Ala, and Met are preferable, and Pro, Val, Ile Is more preferable, and Pro is particularly preferable.

Among the peptides of the present invention, a peptide having at least an amino acid sequence represented by the formula (3) and having 10 to 50 amino acids is more preferable.

(Wherein ^Xc represents Ala, Gly, Ser, Thr, Asn, Val or Cys;
^Xd represents His, Tyr, Phe, Lys, Arg, Leu, Met or Ala;
X ¹ to X ⁷ are the same as above)

In the formula (3), ^Xc represents Ala, Gly, Ser, Thr, Asn, Val, or Cys. Among them, Ala, Gly, Ser, and Thr are preferable, Ala and Gly are more preferable, and Ala is particularly preferable. X ^d represents His, Tyr, Phe, Lys, Arg, Leu, Met or Ala, and among these, His, Tyr, Phe, Lys, Arg, Ala are preferred, His, Tyr, Phe, Ala are more preferred, His Is particularly preferred.

Furthermore, among the peptides of the present invention, a peptide having at least an amino acid sequence represented by the following formula (4) and having 12 to 50 amino acids is more preferable.

(Wherein, X ^a to X ^d and X ¹ to X ⁷ are the same as above)

The number of amino acids of the peptide of the present invention is 8 to 50, preferably 10 to 50, and more preferably 12 to 50. The upper limit of the number of amino acids is preferably 40, more preferably 30, more preferably 20, and particularly preferably 18.

The substitution of X ¹ to X ⁷ and X ^a to X ^d of the peptide of the present invention is based on the binding test results described in the examples below and the similar activity if conservative substitutions are shown. Here, typical conservative substitutions are shown in Table 1.

The peptide of the present invention can be produced by a recombinant technique using DNA encoding the amino acid sequence, but can also be produced by an organic synthetic chemical peptide synthesis method. Organic synthetic chemical peptide synthesis methods are performed by means of general protection of functional groups, activation of carboxyl groups, formation of peptide bonds, and deprotection of protecting groups. These reactions are preferably carried out by a solid phase method.

Among the peptides of the present invention, the peptide represented by SEQ ID NO: 2 is selected by screening a peptide having a binding property to IGF-1R by a phage display method from a phage library displaying a random peptide sequence. Can do. M13 is preferable as the phage used in the phage display method. To select those that bind strongly to IGF-1R from the phage library, the phage population is incubated with IGF-1R, the phage that did not bind to IGF-1R are washed away, and the bound phage is recovered. To determine what sequence of peptides the recovered phage displays, the relevant part of the phage genome may be sequenced. When the interaction between IGF-1R and the peptide is not so strong, a phage having a weak binding force is attached as a background. Therefore, after a series of flows of binding → washing → recovery, E. coli is again infected, a secondary library is prepared, and a panning operation is repeated by repeating the series of operations using this library. In the library obtained by repeating panning, the number of phages having high binding ability to IGF-1R increases. In this way, a phage having a strong binding property to the target IGF-1R can be selected.

The peptide of the present invention specifically binds to IGF-1R. Accordingly, the peptide of the present invention or a labeled form thereof is useful as a reagent for detecting cells or tissues expressing IGF-1R or IGF-1R. Here, the label of the peptide of the present invention may be a label capable of detecting a peptide bound to IGF-1R, and is a radioisotope, an affinity label (for example, biotin, avidin, etc.), an enzyme label (for example, Horseradish peroxidase, alkaline phosphatase, etc.), fluorescent labels (eg, FITC, rhodamine, etc.), paramagnetic atoms and the like. Among these labels, a fluorescent label or a label with a positron nuclide is more preferable for detecting cancer cells or cancer tissues expressing IGF-1R such as colorectal cancer.

The fluorescently labeled peptide of the present invention is useful for diagnosis of cancer expressing IGF-1R, for example, early cancer diagnosis. For example, in the diagnosis for the purpose of confirming the presence or absence of cancer tissue, after contacting the fluorescently labeled peptide to the target site by means such as spraying or injection, after removing the excess fluorescent component by washing treatment, Excitation light can be irradiated to the corresponding part, and the presence or absence of the fluorescently stained tissue can be confirmed macroscopically or microscopically.
In a more preferred form, the fluorescently labeled peptide of the present invention is used as a fluorescence contrast agent for cancer diagnosis by an endoscope. For example, the fluorescently labeled peptide is contacted with tissue by means such as endoscopically spraying. After that, a cleaning process is performed, and an excitation light is irradiated onto the corresponding part with an endoscope light source, and the presence or absence of the fluorescently stained tissue may be confirmed endoscopically. This makes it possible not only to be used for early cancer detection and diagnosis, but also by staining the site suspected to be lesioned endoscopically and magnifying the fluorescence to observe the boundary between the lesioned and non-lesioned sites. It can also be used to determine The type of endoscope is not particularly limited, but a fluorescent endoscope capable of irradiating excitation light for fluorescein as an endoscope light source or a confocal endoscope having an enlargement capability is preferable.
The fluorescent dye to be modified is not limited to fluorescein, and fluorescent dyes having different excitation wavelengths such as cyanine compounds may be used. When a cyanine compound such as indocyanine green is used as the fluorescent agent, the excitation wavelength is further shifted to the longer wavelength side compared to fluorescein, which is effective in confirming deeper lesions. Further, when the positron nuclide is modified with the peptide of the present invention, it can be detected by PET, SPECT or the like. Further, when gadolinium is modified with the peptide of the present invention, it can be detected by MRI. As a result, the peptide of the present invention can be used not only for cancer at sites that can be reached endoscopically, such as digestive organs, but also for cancer lesions throughout the body.

In addition, since the peptide of the present invention or a labeled product thereof competitively inhibits the binding between IGF-1R and IGF-1, and suppresses cancer growth of cancer cells by IGF-1, detection of cancer cells as described above, It can be used not only for cancer diagnosis but also for cancer treatment.

As a cell or tissue capable of detecting, diagnosing or treating the peptide of the present invention or a labeled form thereof, any cell or tissue expressing IGF-1R may be used. For example, cancer cell or cancer tissue, specifically lung cancer Breast cancer, colon cancer, osteosarcoma, cervical cancer, ovarian cancer, synovial sarcoma, pancreatic cancer, prostate cancer and the like.

When the peptide of the present invention or a labeled form thereof is used as a cancer detection agent, a cancer diagnostic agent, or a cancer therapeutic agent, the peptide of the present invention or a labeled form thereof can be used as it is, but various administration forms together with a pharmaceutically acceptable carrier. It can be set as the composition suitable for. Examples of the composition include injectable agents, spray agents, oral administration agents, rectal agents and the like. Examples of the pharmaceutically acceptable carrier include water, physiological saline, various buffers, excipients, disintegrants, binders, lubricants, and the like.

The dose when the peptide of the present invention or a labeled product thereof is used as a cancer therapeutic agent varies depending on symptoms, body weight, etc., but is usually preferably 0.1 mg to 1000 mg per day for an adult.

Next, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.

Example 1 (phage display)
[procedure]
1. Panning experiment-round 1
200 μL of 5 μg / mL Recombinant human Insulin Like Growth Factor-1 Receptor (rhIGF-1R, R & D Systems) was added to a 96-well plate (1 μg / well) and incubated at 4 ° C. overnight (solid phase on the plate). ). The non-solid phase protein in the well was removed, Blocking buffer (5 mg / mL BSA (bovine serum albumin) / TBS (50 mM Tris-HCL / 150 mM NaCl)) was added at 300 μL / well, and allowed to stand at 37 ° C. for 1 hour. Incubation was followed by 3 washes with 200 μL / well of 0.1% TBST (0.1% Tween20 / TBS).
100 μL of 0.1% TBST was added, and 10 μL each of D12 phage library (1 × 10 ¹³ PFU / mL) and C7C phage library (2 × 10 ¹³ PFU / mL) were added.

The peptide-displaying phage library (NEW ENGLAND BioLabs Inc.) utilizes two types, and the D12 library displays 12-residue linear random peptides, and 2.7 × 10 ⁹ different peptide sequences. It has a phage library. The C7C library presents a 7-residue cyclic random peptide and is a phage library having 1.2 × 10 ⁹ different peptide sequences.
After shaking incubation with a seesaw at room temperature for 1 hour, peptide-displayed phage (hereinafter abbreviated as phage) was bound to the immobilized rhIGF-1R, the phage solution was removed, and 0.1% TBST 200 μL / Washed 10 times with well. Phage was eluted by adding 100 μL of 1 mg / mL BSA / 0.2M Glycine-HCl (pH 2.2) and shaking at room temperature for 10 minutes. The eluate was recovered and neutralized by adding 15 μL of 1M Tris-HCl (pH 9.1) to the recovered solution. A part of the collected eluate was used to measure the phage titer.

2. The phage eluate obtained by the operation of amplification 1 of the recovered phage was subjected to ER2738 logarithmically growing in 20 mL of LB [F'lacl ^q Δ (lacZ) M15proA ⁺ B ⁺ zzf :: Tn10 (TetR) fhuA2supEthiΔ (lac-proAB) Δ (hsdMS-mcrB) 5 (r _k -m _k -McrBC-)] was infected and incubated for 4 hours 30 minutes at 37 ° C. with vigorous stirring using a shaker. The phage-infected bacterial culture was transferred to a 50 mL centrifuge tube, and the sample was centrifuged at 8,900 × g at 4 ° C. for 10 minutes using a micro-cooled centrifuge. After centrifugation, the supernatant was collected in a new tube for the purpose of removing ER2738 bacteria. Add 3.6 mL (1/5 volume) of PEG / NaCl (20% Polyethylene glycol 6,000, 2.5 M NaCl) solution to the recovered phage solution, stir well with a mixer, and incubate at 4 ° C. for 16 hours. Then, phages were precipitated. In order to collect the precipitated phage, the supernatant was removed by centrifugation at 8,900 × g for 10 minutes at 4 ° C. in a micro-cooled centrifuge. In order to completely remove the supernatant, it was centrifuged once more and the supernatant was removed. 1 mL of ice-cold TBS was added to the precipitated phage, suspended, and transferred to a microtube. The phage suspension was centrifuged at 16,000 × g for 5 minutes at 4 ° C. using a compact high speed cooling centrifuge. The supernatant was collected in another tube, the residue that was not suspended was removed, 200 μL of PEG / NaCl was added to the collected solution, and the mixture was stirred with a mixer. The solution was incubated on ice for 1 hour to precipitate the phage. The solution was centrifuged at 16,000 × g for 10 minutes at 4 ° C. in a high-speed centrifuge to precipitate phages and remove the supernatant. This centrifugation step was performed again, and the supernatant was completely removed. To the obtained phage precipitate, 200 μL of 0.02% NaN ₃ / TBS is completely suspended, suspended in a compact refrigerated centrifuge at 4 ° C., 5 minutes, 16,000 × g, and the supernatant is removed. The residue that was not suspended was removed by recovery. The titer of the recovered phage concentrate was measured.

3. Panning experiment-

Round

2, 3
Second and third panning experiments were performed using the concentrated phage solution. The second panning experiment differs from the first operation in that the amount of added phage was 2 × 10 ¹¹ PFU / well and the washing solution was 0.3% TBST (0.3% Tween 20 / TBS). The third panning experiment differs from the first operation in that the amount of added phage was 2 × 10 ¹¹ PFU / well and the washing solution was 0.5% TBST (0.5% Tween 20 / TBS).

4). Titer measurement LB In 3 mL of LB, ER2738 bacteria were cultured until reaching the logarithmic growth phase (OD600; to 0.5), and 10 μL of phage solution diluted to the required concentration was added to 200 μL of ER2738 culture solution. The mixed solution was thoroughly stirred with a mixer, incubated at room temperature for 5 minutes, mixed with 4 mL of dissolved top agar solution, and seeded on an LB / IPTG / Xgal plate.

After incubating the phage-infected E. coli seed plate at 37 ° C. for 16 hours, the number of blue plaques obtained was counted. The phage number was calculated using the phage dilution factor.

[result]
Changes in the ratio of the input titer (phage force added to the target molecule) and the output titer (phage force eluted from the target molecule after washing) in the experiment using the phage library were measured using the D12 library. FIG. 1 shows the results and FIG. 2 shows the results of the C7C library.

As a result of IGF-1R target panning experiment using D12 library up to 3 rounds, when comparing the output titer / input titer ratio, an increase of about 87 times of 1 round was observed in 3 rounds. . Therefore, it was expected that phages showing IGF-1R specific binding were selected.
IGF-1R targeted panning experiments using the C7C library proceeded to 4 lands, but only an increase of about 2-fold was observed compared to 1 round.

Example 2 (sequence analysis)
[procedure]
Phage obtained from three rounds of panning experiments using the D12 library was cloned according to a conventional method (Page Display A Laboratory Manual, Cole Spring Harbor Laboratory Press, 2001). The base sequence of the part was determined. For the determination of the base sequence, a primer corresponding to the complementary strand of the base sequence located 96 residues downstream from the presented peptide region [-96 gIII sequencing primer (5′- ^HO CCCTCATTAGTAGCGTAACG-3 ′) (SEQ ID NO: 1), S1259A , NEB] by the dideoxyterminate method (CEQ DTCS Quick start kit, Beckmann). A capillary sequencer (CEQ2000, Beckman) was used for electrophoresis of reaction products and data analysis.

[result]
The presented peptide sequence predicted from the determined nucleotide sequence is shown in FIG.

Among them, the peptide sequence DPFYSMLQRLAH (SEQ ID NO: 2) displayed by the obtained S-011 phage was 9 clones having the same sequence among 12 clones examined in 3 rounds (75% ). Only one clone of each of S-012 phage (SEQ ID NO: 3), S-017 phage (SEQ ID NO: 4), and S-020 phage (SEQ ID NO: 5) was obtained.
Therefore, it was revealed that S-011 phage was selected as the number of pannings progressed. The reason why a specific phage clone is selected is that the phage clone shows a strong binding property to the target molecule.

Example 3 (Phage binding test (protein immobilization conditions))
[procedure]
Add target protein rhIGF-1R and IGF-1R structurally similar protein, Recombinant human Epidermal Factor Receptor (rhEGFR, R & D Systems), BSA as standard protein to 96 μm microplate at 1 μg / well. It was solidified by leaving it overnight. The protein solution was removed, 300 μl of Blocking buffer was added, and the wells were blocked by incubating at 37 ° C. for 1 hour. After removing the blocking buffer, the wells were washed three times with 200 μl of a washing solution 0.5% TBST, and finally 100 μl of 0.5% TBST was added. An amplification phage solution (S-011 or M13KE (peptide non-displaying phage)) was added to the wells so as to be 1 × 10 ¹⁰ PFU and mixed by pipetting. The reaction was gently shaken for 1 hour at room temperature. After removing the reaction solution and washing the well 10 times with 200 μl of 0.5% TBST, 100 μl of 0.2 M Glycine-HCl (pH 2.2) was added to the well, and the mixture was stirred by pipetting. Gently shake for minutes. The eluate was collected from the wells into a microtube and neutralized by adding 15 μl of 1M Tris-HCl (pH 9.1) to obtain a target-binding phage solution. The binding ability of the recovered phage was determined by titration.

[result]
FIG. 4 shows the change in the ratio between the input titer and the output titer in the binding test by the solid-phase method, and FIG. 5 shows the result of the M13KE phage.
It was revealed that the binding of S-011 to IGF-1R was 9963 times higher than that of EGFR, which is an IGF-1R structure-analogous protein, and 7130 times higher than that of BSA, which is a standard protein.
In M13KE phage, no difference in binding was observed between the immobilized proteins, and all of them were low values.

From the results shown in FIG. 4, it was revealed that S-011 phage specifically binds to IGF-1R under protein-immobilized conditions.
Further, comparing the binding properties of S-011 phage and M13KE phage to IGF-1R, it was revealed that S-011 was 2892 times higher. From these results, it was suggested that the peptide sequence displayed by the S-011 phage may be involved in the binding to IGF-1R.

Example 4 (Phage binding test (protein solution phase conditions))
[procedure]
Dispense 200 μl of 0.01% PBST (0.01% Tween20 / PBS) into a microtube, add 10 μl of magnetic beads coated with Protein A (Dynabeads Protein A, invitrogen), mix by pipetting, and mix the magnet with a micro tube. The supernatant was removed by pressing against the side of the tube. Thereafter, the magnet was separated from the microtube, 200 μl of 0.01% PBST was added and mixed by pipetting, the magnet was pressed against the microtube, and the supernatant was removed. The above process was repeated three times to wash the magnetic beads. 200 μl of 0.01% PBST was added, transferred to a new microtube, and the supernatant was removed using a magnet. Add 200 μl of Monoclonal Anti Human IGF-1R antibody (R & D Systems) adjusted to 25 μg / mL with 5 mg / mL BSA / 0.01% PBST, mix with Voltex, and then magnetically use a microtube stirrer at room temperature for 1 hour. Reacted with beads. After the reaction, the supernatant was removed while pressing the magnet against the microtube, then the magnet was separated from the microtube, 200 μl of 0.01% PBST was added and mixed by pipetting, and the magnet was pressed against the microtube. The supernatant was removed. The above process was repeated 3 times and washed. 200 μl of 0.01% PBST was added, transferred to a new microtube, and the supernatant was removed using a magnet. 200 μl of rhIGF-1R prepared to 5 μg / mL with 5 mg / mL BSA / 0.01% PBST was added, mixed with Vortex, and reacted with magnetic beads using a microtube stirrer for 1 hour at room temperature. After the reaction, the supernatant was removed while pressing the magnet against the microtube, then the magnet was separated from the microtube, 200 μl of 0.01% PBST was added and mixed by pipetting, and the magnet was pressed against the microtube. The supernatant was removed. The above process was repeated three times, and after washing, 200 μl of 0.01% PBST was added and transferred to a new microtube. Amplified phage solution (S-011 or M13KE) was added to the microtube so as to be 1 × 10 ¹⁰ PFU, and mixed by pipetting. The reaction was performed at room temperature for 1 hour using a microtube stirrer. The reaction solution was removed using a magnet and washed 5 times with 200 μl of 0.01% PBST. 200 μl of 0.01% PBST was added, transferred to a new microtube, and the supernatant was removed using a magnet. 100 μl of 0.2M Glycine-HCl (pH 2.2) was added to the microtube, stirred by pipetting, and then stirred at room temperature for 10 minutes using a microtube stirrer. Thereafter, pipetting was performed, and the supernatant was collected into a new microtube using a magnet and neutralized by adding 15 μl of 1 M Tris-HCl (pH 9.1) to obtain a target-binding phage solution. The binding ability of the recovered phage was determined by titration.

[result]
Changes in the ratio between the input titer and the output titer in the binding test by the liquid phase method are shown in FIG. 6 for the results of S-011 phage and in FIG. 7 for the results of M13KE phage.
The S-011 phage is strongly bound to the magnetic beads bound with IGF-1R and is 74 times higher than the magnetic beads bound with the IGF-1R antibody, compared to the other magnetic beads. 427 times higher binding was shown.
In M13KE phage, there was no big change in the binding property depending on the magnetic bead carrier, and all showed low values.

As shown in FIG. 6, it was revealed that the S-011 phage specifically binds to IGF-1R.
In addition, comparing the binding properties of S-011 phage and M13KE phage to IGF-1R, it became clear that S-011 was 402 times higher, and the peptide displayed by S-011 phage was able to bind to IGF-1R. It was suggested that it was greatly involved.
The above results are similar to those obtained under the solid-phase conditions in Example 3. The possibility that S-011 phage specifically recognizes IGF-1R regardless of the presence of IGF-1R. There is.

Example 5 (Alanine-substituted phage production)
[procedure]
1. Using a site-directed mutagenesis KOD-Plus-Mutageness kit (SMK-101, TOYOBO), a phage in which the amino acid in the peptide display portion of M13KE phage was substituted with alanine was prepared. An oligonucleotide primer containing a desired mutation in the nucleotide sequence of the displayed peptide sequence was commissioned and synthesized (Nippon Genetics Research Laboratories). The synthetic primer is a desalted oligonucleotide purified by HPLC. The primer synthesized this time was designed to have a length of 24-27 bp and a GC content of 50-60%. In the KOD-Plus-Mutageness Kit used this time, the phosphorylation of the PCR product can be carried out simultaneously with the self-ligation of the PCR product, so that the primer is not phosphorylated. Template plasmid DNA was purified from S-011 phage-infected ER2738 bacteria using QIAGEN Plasmid kit.

Using template plasmid DNA, primer, dNTPs, KOD-plus-, [94 ° C., 2 minutes] → {[98 ° C., 10 seconds] → [68 ° C., 7.5 minutes]} × 8 cycles → [4 ° C. , Hold] conditions. For the PCR reaction, a thermal cycler (PCR Thermal Cycler Dice, TAKARA) was used. PCR reaction conditions were set based on the amplification size.

The template plasmid was digested by DpnI treatment, and the PCR product was self-ligated by T4 Polynucleotide Kinase treatment.

2. Transformed XL-1 Blue competent cells were lysed on ice. 10 μL of the self-ligated solution was added to 60 μL of XL-1 Blue competent cells and mixed. After incubating on ice for 30 minutes, it was incubated on a heat block at 42 ° C. for 90 seconds and incubated on ice for 2 minutes. In a 15 mL conical tube, 1 μL of the XL-Blue bacterium was added to 100 μL of ER2738 O / N culture and mixed. The remaining XL-1 Blue bacteria were placed in a 15 mL conical tube. 4 mL of top agar was added to each of the above samples and mixed by vortexing.
The E. coli sample was seeded on an LB / IPTG / Xgal plate and incubated at 37 ° C. for 16 hours.

3. Sequence analysis of mutant The blue plaque on the obtained LB / IPTG / Xgal plate was cloned according to a conventional method (Page Display A Laboratory Manual, Cole Spring Harbor Laboratory Press, 2001), and the nucleotide sequence of the displayed peptide portion of the phage It was determined.

[result]
The alanine substituted phages of SEQ ID NOs: 6 to 16 shown in FIG. 8 were obtained.

Example 6 (Alanine-substituted phage binding test)
[procedure]
The target protein rhIGF-1R was added to a 96-well microplate at 1 μg / well and allowed to stand overnight at 4 ° C. for immobilization. The protein solution was removed, 300 μl of Blocking buffer was added, and the wells were blocked by incubating at 37 ° C. for 1 hour. After removing the blocking buffer, the wells were washed three times with 200 μl of a washing solution 0.5% TBST, and finally 100 μl of 0.5% TBST was added. Amplified phage solution (S-011 phage or alanine-substituted phage prepared in Example 5) was added to the well so as to be 1 × 10 ¹⁰ PFU and mixed by pipetting. The reaction was gently shaken for 1 hour at room temperature. After removing the reaction solution and washing the well 10 times with 200 μl of 0.5% TBST, 100 μl of 0.2M Glycine-HCl (pH 2.2) was added to the well and stirred by pipetting. Gently shake for 10 minutes. The eluate was collected from the wells into a microtube and neutralized by adding 15 μl of 1M Tris-HCl (pH 9.1) to obtain a target-binding phage solution. The binding ability of the recovered phage was determined by titration.

[result]
FIG. 9 shows the change in the ratio between the input titer and the output titer in the alanine-substituted phage binding test.
As can be seen from FIG. 9, there are five types of phages with decreased binding compared to S-011, the first D (aspartic acid), the third F (phenylalanine), the fourth Y (tyrosine), the seven A phage in which the 10th L (leucine) and the 10th L (leucine) are substituted with alanine.
In particular, it was revealed that the binding ability of the phage in which the seventh L (leucine) was substituted with alanine was most lowered.

There are five types of phages in which the amino acid of S-011 phage is substituted with alanine, and the binding to IGF-1R is lower than that of S-011 phage. The amino acid sequence of the peptide displayed by S-011 phage Among them, amino acids considered to be involved in binding to IGF-1R are the first D (aspartic acid), the third F (phenylalanine), the fourth Y (tyrosine), the seventh L ( Leucine) and tenth L (leucine).
Of the above amino acids, the amino acid most likely to be involved in binding to IGF-1R was the seventh L (leucine).

Example 7 (Western blot)
[procedure]
Cultured cells MCF7; human breast cancer cells (DS Pharmabiomedical), A-172 cells; human neuroblastoma cells (DS PharmaBiomedical), Hela S3: human cervical cancer cells (DS PharmaBiomedical)) A cell lysate was prepared according to the Laemmli method and subjected to SDS-PAGE. A-431 Whole Cell Lysate (Santa Cruz Biotechnology) was used as a control cell sample. Proteins were transferred from the polyacrylamide gel after electrophoresis to a PVDF membrane, and blocking was performed using 5% skim milk / TBST (0.05% Tween-20 / TBS). For IGF-1R detection, 1 μg / mL IGF-1Rα (N20) (Santa Cruz Biotechnology) was used as the primary antibody, and Goat anti-rabbit IgG-HRP (BioRad) was used as the secondary antibody. In β-actin detection, Monoclonal Anti-β-Actin, Clone AC-15 (SIGMA-ALDRICH) was used as the primary antibody, and Goat anti-mouse IgG-HRP (BioRad) was used as the secondary antibody. Amersham ^™ ECL Plus Western Blotting Detection System (GE Healthcare), which is a detection agent, was added to the PVDF membrane after the antibody reaction, and a band was detected using a chemiluminescence detection analyzer (LumiVison PRO, Taitec).

[result]
As a result of evaluating the expression level of IGF-1Rα in each cell, it was revealed that it was highly expressed in Hela S3 and MCF7 (FIG. 10). This time, the amount of β-actin in the cell samples used was as follows, and there was no significant difference in the amount of protein between samples (FIG. 11).

Example 8 (Bindability test (in vitro))
From Example 7, a phage peptide binding test was carried out targeting the MCF7 cell line in which high expression of IGF-1R was observed and the A-172 cell line in which expression was not observed.

[procedure]
1. Cells were seeded at 2 × 10 ⁶ cells / well in 6-well culture plates of MCF7 cells and cultured overnight at 37 ° C. and 5% CO ₂ conditions. After incubating the plate at 4 ° C. for 30 minutes, the medium in each well was removed and washed twice with 1 mL of 1% BSA / PBS. S-011 and S-011_L7A (SEQ ID NO: 12) phage and M13KE phage were diluted in 1 mL of 10% FBS medium (growth medium) to a titer of 1 × 10 ¹⁰ PFU, added to each well, and 4 ° C. And incubated for 60 minutes. After the reaction, the reaction solution was removed and washed 10 times with 1 mL of 1% BSA / PBS to remove unbound phage. 100 μl of 0.05% trypsin / 0.53 mM EDTA solution was added, incubated at 37 ° C. for 5 minutes, and 900 μl of growth medium was added to obtain a recovered phage solution. The titer of each recovered phage solution was measured.

[result]
FIG. 12 shows the ratio between the input titer and the output titer of each phage. The titer ratio of S-011 phage was 170 times higher than that of M13KE phage. In contrast, the titer ratio of S-011_L7A phage was 1.6 times that of M13KE phage.

[procedure]
2. Cells were seeded at 7 × 10 ⁵ cells / well in A-172 cell 6-well culture plates and cultured overnight at 37 ° C. and 5% CO ₂ . After incubating the plate at 4 ° C. for 30 minutes, the medium in each well was removed and washed twice with 1 mL of 1% BSA / PBS. S-011 and S-011_L7A (SEQ ID NO: 12) phage and M13KE phage were diluted in 1 mL of growth medium to a titer of 1 × 10 ¹⁰ PFU, added to each well, and incubated at 4 ° C. for 60 minutes. After the reaction, the reaction solution was removed and washed 10 times with 1 mL of 1% BSA / PBS to remove unbound phage. 100 μl of 0.05% trypsin / 0.53 mM EDTA solution was added, incubated at 37 ° C. for 5 minutes, and 900 μl of growth medium was added to obtain a recovered phage solution. The titer of each recovered phage solution was measured.

[result]
FIG. 13 shows the ratio between the input titer and the output titer of each phage. The titer ratio of S-011 phage peptide was 3.0 times that of M13KE phage. The titer ratio of S-011_L7A phage was 2.7 times that of M13KE phage.
From the results shown in FIG. 13, it can be seen that in A-172 cells not expressing IGF-1R, the phage peptide displaying the peptide showed about two times higher binding than the peptide non-displaying phage. However, specific binding due to the peptide sequence is not observed, and it is considered that cells bind nonspecifically.
On the other hand, from the results of FIG. 12, in MCF7 cells that strongly express IGF-1R, the phage peptide displaying the peptide of SEQ ID NO: 2 was compared with the phage peptide of SEQ ID NO: 12 or the peptide non-displaying phage. It was strongly tied to the superiority. This advantage is considered to be due to the binding of the peptide of SEQ ID NO: 2 to IGF-1R expressed in MCF7 cells.

Example 9 (competitive test)
[procedure]
The target protein rhIGF-1R was added to a 96-well microplate at 1 μg / well and allowed to stand overnight at 4 ° C. for immobilization. The protein solution was removed, 300 μl of Blocking buffer was added, and the wells were blocked by incubating at 37 ° C. for 1 hour. After removing the blocking buffer, the wells were washed three times with 200 μl of a washing solution 0.5% TBST, and finally 100 μl of 0.5% TBST was added. The S-011 amplified phage solution was added to the wells to 1 × 10 ¹⁰ PFU, and then the synthetic peptide Pep02 (purity 90% < , HPLC grade, AnyGen, Korea) and synthetic peptide Pep17 (GAASTRYLHELI: SEQ ID NO: 17) unrelated to the binding of IGF-1R with a purity of 90% <, HPLC grade, AnyGen, Korea, 0, 1 nM, 100 nM, respectively. It added so that it might become a density | concentration of 1 micromol and 10 micromol, and mixed by pipetting. The reaction was gently shaken for 1 hour at room temperature. The reaction solution was removed, and the wells were washed 10 times with 200 μl of 0.5% TBST. Thereafter, 100 μl of 0.2M Glycine-HCl (pH 2.2) was added to the well, and the mixture was stirred by pipetting, and then gently shaken at room temperature for 10 minutes. The eluate was collected from the wells into a microtube and neutralized by adding 15 μl of 1M Tris-HCl (pH 9.1) to obtain a target-binding phage solution. The binding ability of the recovered phage was determined by titration.

[result]
The change in the ratio of the input titer and the output titer in the competition test is shown in FIG. 14 for the results of S-011 phage and Pep02, and in FIG. 15 for the results of S-011 phage and Pep17.
As can be seen from FIG. 14, the recovery rate of the S-011 phage has dropped to the right when 0.1 μM of Pep02 is added, and Pep02 has a concentration of S-011 phage binding to IGF-1R. It became clear that it inhibited in a dependent manner.
Referring to FIG. 15, the recovery rate of S-011 phage remains high with no change even when the concentration of Pep17 added increases.

Pep17 was not involved in the binding of S-011 phage to IGF-1R at all, so there was no difference in the recovered amount of S-011 phage even at high concentrations, but a synthetic peptide having a peptide sequence displayed by S-011 phage Pep02 was found to inhibit the binding between S-011 phage and IGF-1R in a concentration-dependent manner. From this result, it is considered that there is a high possibility that the peptide displayed by the S-011 phage binds to the same site of the S-011 phage and IGF-1R even with the peptide alone.

Example 10 (In vitro imaging)
[procedure]
Human breast adenocarcinoma cells MCF7 were seeded in a glass bottom dish (φ35 mm, Mat Tek Corp.) so as to be 400 × 10 ⁴ Cells / Dish, and cultured at 37 ° C. under CO ₂ conditions for 48 hours. The cultured dish was incubated at 4 ° C. for 30 minutes, and after removing the culture solution, it was washed 3 times with 1 mL of 4 ° C. growth medium. Peptides (Pep02-F, Pep18-F (LASFMFLDQPYR: SEQ ID NO: 18)) in which an aminocaproic acid linker is linked to the N-terminal of the peptide and FITC is labeled on the opposite side of the linked linker (purity 90% <, HPLC grade AnyGen, Korea) was prepared to 100 μM in growth medium, 1 mL was added and incubated at 4 ° C. for 1 hour. The peptide solution was removed, washed 10 times with 1 mL of growth medium at 4 ° C., and finally 1 mL of medium was added. Observation was carried out using a confocal microscope (Leica SP2), and the excitation light was irradiated with a laser wavelength of 488 nm, and fluorescence was observed.

[result]
FIG. 16 shows the staining property of MCF7 of Pep02-F observed with a confocal microscope.
The imaging conditions were an image taken with a gain value of 700 Volt using excitation light of 488 nm, fluorescence receiving side set to 515 to 600 nm, objective lens using a 63 × oil lens (HCX PL APO CS 63x OIL, Leica). .
Although the cell gap of MCF7 is strongly stained, it can be observed that the cytoplasm, nucleus, and other organelles are not stained.
FIG. 17 shows the staining of Pep18-F MCF7 observed with a confocal microscope.
The shooting conditions are the same as for Pep02-F.
MCF7 is not stained at all. Even when the Gain value was maximized, the staining of the cells could not be observed.

When the staining of Pep02-F and Pep18-F was compared, the staining ability of MCF7 could be confirmed only with Pep02-F. That is, Pep02-F may specifically stain the cell gap of MCF7.
In addition, from the results of WB, MCF7 is highly likely to express IGF-1R, and Pep02-F staining is also in the intercellular space. Therefore, Pep02-F binds to IGF-1R expressed by MCF7. It is thought that there is.

Example 11 (Production of amino acid-substituted phage)
[procedure]
1. Using site-directed mutagenesis KOD-Plus-Mutageness kit (SMK-101, TOYOBO), the amino acid in the peptide display part of M13KE phage was replaced with another amino acid, and several amino acids of the display peptide were removed. The phage (short peptide-displaying phage) was prepared. An oligonucleotide primer containing a desired mutation in the nucleotide sequence of the displayed peptide sequence was commissioned and synthesized (Nippon Genetics Research Laboratories). The synthetic primer is a desalting oligo purified by HPLC. The primer synthesized this time was designed to have a length of 20 to 27 bp and a GC content of about 50 to 60%. In the KOD-Plus-Mutageness Kit used this time, the phosphorylation of the PCR product can be carried out simultaneously with the self-ligation of the PCR product, so that the primer is not phosphorylated. Hereinafter, reference will be made to the procedure for preparing alanine-substituted phage (Example 5).

2. Reference is made to the procedure for producing transformed alanine-substituted phage (Example 5).

3. Sequence analysis of mutants The blue plaques on the obtained LB / IPTG / Xgal plate were cloned according to a conventional method (Page Display A Laboratory Multiple, Cole Spring Harbor Laboratory Press, 2001), and the nucleotide sequence of the displayed peptide part of the phage It was determined.

[result]
Amino acid-substituted phages of SEQ ID NOs: 19 to 34 shown in FIG. 18 were obtained.

Example 12 (Binding test of amino acid-substituted phage)
[procedure]
The target protein rhIGF-1R was added to a 96-well microplate at 1 μg / well and allowed to stand overnight at 4 ° C. for immobilization. The protein solution was removed, 300 μl of Blocking buffer was added, and the wells were blocked by incubating at 37 ° C. for 1 hour. After removing the blocking buffer, the wells were washed three times with 200 μl of a washing solution 0.5% TBST, and finally 100 μl of 0.5% TBST was added. Amplified phage solution (S-011 phage or amino acid-substituted phage prepared in Example 11) was added to the well so as to be 1 × 10 ¹⁰ PFU, and mixed by pipetting. The reaction was gently shaken for 1 hour at room temperature. After removing the reaction solution and washing the well 10 times with 200 μl of 0.5% TBST, 100 μl of 0.2M Glycine-HCl (pH 2.2) was added to the well and stirred by pipetting. Gently shake for 10 minutes. The eluate was collected from the wells into a microtube and neutralized by adding 15 μl of 1M Tris-HCl (pH 9.1) to obtain a target-binding phage solution. The binding ability of the recovered phage was determined by titration.

[result]
1. The change in the ratio of the values of the input titers and output titer binding test of one amino acid substitution phage single amino acid substitutions phage 19.
Among the one amino acid substituted phages, there were 10 phages that showed less change compared to the binding properties of S-011 phage. Among them, the phage showing the binding of 1 to 1/10 times that of S-011 has the seventh L (leucine) substituted with I (isoleucine), V (valine), and M (methionine). Case (SEQ ID NO: 19 to 21). Subsequently, in the case of a phage exhibiting a binding property of 1/10 to 1/100 times, the fourth Y (tylosin) is substituted with W (tryptophan), F (phenylalanine), and H (histidine) (SEQ ID NO: 22 to 24) and the first D (aspartic acid) is replaced with E (glutamic acid) (SEQ ID NO: 25) and the tenth L (leucine) is replaced with I (isoleucine), V (valine), M (methionine) (SEQ ID NO: 26 to 28).

From the above results, it was found that the binding property to IGF-1R changes depending on the amino acid to be substituted. Comparing the amino acids with changes, the amino acids with little change in binding by substituting the fourth Y are W, F and H, and W and F have an aromatic benzene ring like Y Things became clear. H does not have an aromatic group but has a five-membered ring, so that structural similarity with Y, W, and F is observed. Similarly, when the seventh and tenth Ls having an alkyl side chain were substituted, it was also revealed that they had similar structures such as I, V, and M. However, from the results of the binding test of the alanine-substituted phage of Example 6, it is considered that at least V or higher alkyl side chains are necessary since the binding is reduced when A is substituted.

2. The change in the input titer and output titer value of a ratio of binding test amino acid two substitutions phages acid two substitutions phages is shown in FIG. 20.
A two-amino acid-substituted phage showing 1/10 to 1 / 100-fold binding compared to the binding of S-011 is a phage in which the first D is replaced with E and the seventh L is replaced with I (D1E / L10I, SEQ ID NO: 29), phage with 4th Y replaced with W and 7th L replaced with I (Y4W / L7I, SEQ ID NO: 30), 7th L replaced with I and 10th L replaced with V Phage (L7I / L10V, SEQ ID NO: 31).

The binding of phages with two amino acid substitutions combined with a pattern with little change in binding by substitution of one amino acid is consistent with the binding result of single substitution and retains binding to IGF-1R. It became clear that.

3. FIG. 21 shows the change in the ratio between the input titer and the output titer in the binding test of the S-011 short peptide phage S-011 short peptide phage.
The S-011 short peptide phage showing 1/10 to 1 / 100-fold binding compared to the binding of S-011 is the first to the 10th L from D (D1-L10, SEQ ID NO: 32). And a phage displaying the third F to the 12th H (F3-H12, SEQ ID NO: 33). Subsequently, the phage with a small change in binding was a phage displaying the third F to the tenth L (F3-L10, SEQ ID NO: 34).

From the above results, it was found that the minimum necessary site for binding to IGF-1R in the S-011 sequence is from the third F to the tenth L (F3-L10). Although not shown, it is also clear from the fact that phages covered with a part of the F3-L10 sequence (Examples D1-L7, L7-H12, etc.) have lost significant binding. .

Example 13 (competition test between rhIGF-1 and S-011 phage)
[procedure]
The target protein rhIGF-1R was added to a 96-well microplate at 1 μg / well and allowed to stand overnight at 4 ° C. for immobilization. The protein solution was removed, 300 μl of Blocking buffer was added, and the wells were blocked by incubating at 37 ° C. for 1 hour. After removing the blocking buffer, the wells were washed three times with 200 μl of a washing solution 0.5% TBST. Add 100 μl of rhIGF-1 (R & D Systems) diluted to each concentration (0, 1, 10 μM) with 0.5% TBST or Monoclonal Anti-human IGF-1R Antibodies (R & D systems) to 20 μg / well. And gently shaken at room temperature for 1 hour. The S-011 phage amplification solution was added to the well so as to be 1 × 10 ¹⁰ PFU and mixed by pipetting. The reaction was gently shaken for 15 minutes at room temperature. After removing the reaction solution and washing the well 10 times with 200 μl of 0.5% TBST, 100 μl of 0.2M Glycine-HCl (pH 2.2) was added to the well and stirred by pipetting. Gently shake for 10 minutes. The eluate was collected from the wells into a microtube and neutralized by adding 15 μl of 1M Tris-HCl (pH 9.1) to obtain a target-binding phage solution. The binding ability of the recovered phage was determined by titration.

[result]
The change in the ratio between the input titer and the output titer in the competitive test is shown in FIG. 22 for the rhIGF-1 and S-011 phage results, and in FIG. 23 for the Anti-hIGF-1R mAb and the S-011 phage results. .
It can be seen that the binding of S-011 phage to IGF-1R decreases as the concentration of IGF-1 added increases.
Addition of Anti-hIGF-1R mAb did not affect the binding between S-011 phage and IGF-1R.

From the above results, the binding of S-011 phage decreased depending on the concentration of IGF-1 added, indicating that IGF-1 inhibited the binding of S-011 phage to IGF-1R. It became clear. That is, since it is considered that S-011 phage can inhibit the binding of IGF-1 to IGF-1R, the peptide presented by S-011 phage may be an inhibitor of IGF-1.

Example 14 (cancer cell growth inhibition test 1)

[procedure]
Each cell is suspended in a culture medium without addition of serum, and 900 μL each in a 12-well plate so as to have a predetermined number of cells (Hela S3: 5 × 10 ⁴ cells / well, MCF7: 10 × 10 ⁴ cells / well). Sowing. Pre-culture was performed in a CO ₂ incubator (37 ° C.). Next, 50 μL of a peptide (Pep02, Pep12) diluted with a serum-free culture solution to a predetermined concentration was added to each well and incubated at 4 ° C. for 30 minutes. Add 50 μL of rhIGF-1 solution diluted with serum-free medium to a final concentration of 10 ng / mL for Hela S3 and 100 ng / mL for MCF7, and incubate for 2 days in a CO ₂ incubator (37 ° C.). went. The growth rate after culture was evaluated by the MTT test. The MTT test was performed using CellTiter 96 (Promega). 45 μL of MTT solution (dye solution) was added to each well, mixed, and reacted in a CO ₂ incubator for 4 hours. After the reaction, 300 μL of a solubilization solution (stop solution) was added to each well and mixed. After sealing, the mixture was allowed to stand overnight at room temperature, and then the absorbance value at 570 nm was measured using a microplate reader.

[result]
It was confirmed that cell growth was enhanced for both Hela S3 and MCF7 when rhIGF-1 was added and cultured compared to the case where the peptide and rhIGF-1 were not added.
24 and 25 show the results of co-culturing with rhIGF-1 and peptide added, and graphing the absorbance when no peptide was added as 100% growth rate. 24 and 25, the growth rate was suppressed in a concentration-dependent manner when Pep02 was added, and was suppressed to about 80 to 90% when 100 μM was added. On the other hand, when Pep12 was added, it remained flat regardless of the addition concentration. From this result, it was found that Pep02 suppresses the growth promoting effect of rhIGF-1 on cancer cells.

Example 15 (cancer cell growth inhibition test 2)
The effect of Pep02 on anchorage independent growth was tested.

[procedure]
Each cell is suspended in a serum-free culture medium, and 90 μL each in an ultra-low adhesion surface culture plate (Ultra Low Attachment Microplate, CORNING) so as to have a predetermined cell number (MCF: 5 × 10 ⁴ cells / well). Sowing. Pre-culture was performed in a CO ₂ incubator (37 ° C.). 5 μL of peptides (Pep02, Pep12) diluted with a serum-free culture solution at a predetermined concentration were added to each well, and incubated at 4 ° C. for 30 minutes. 5 μL of rhIGF-1 solution diluted with a serum-free culture solution was added to a final concentration of 100 ng / mL, and the cells were cultured in a CO ₂ incubator (37 ° C.) for 2 days. The growth rate after culture was evaluated by the MTT test. The MTT test was performed using CellTiter 96 (Promega). 15 μL of MTT solution (dye solution) was added to each well, mixed, and reacted in a CO ₂ incubator for 4 hours. After the reaction, 100 μL of a solubilization solution (stop solution) was added to each well and mixed. After sealing, the mixture was allowed to stand overnight at room temperature, and then the absorbance value at 570 nm was measured using a microplate reader.

[result]
It was confirmed that the cell growth of MCF7 was enhanced when rhIGF-1 was added and cultured compared to the case where the peptide and rhIGF-1 were not added.
rhIGF-1 and peptide were added and co-cultured, and the absorbance when no peptide was added was plotted as a 100% growth rate. The results are shown in FIG. From FIG. 26, the growth rate was suppressed in a concentration-dependent manner when Pep02 was added, and was suppressed to about 80% when 100 μM was added. On the other hand, Pep12 was level regardless of the addition concentration.
It was confirmed that the Pep02 peptide suppresses the cancer cell proliferation promoting effect by rhIGF-1 in both anchorage-dependent and anchorage-independent growth.

Claims

A peptide having 8 to 50 amino acids having at least an amino acid sequence represented by the following formula (1).

(Wherein X 1 represents Tyr, Trp, Phe or His,
X 2 represents Ser, Ala, Thr, Gly, Asn, Asp, Glu, Arg or Lys;
X 3 represents Met, Leu, Ile, Val, Ala, Phe, Tyr, Trp or Cys;
X 4 represents Leu, Ile, Val or Met;
X 5 represents Gln, Asn, Asp, Glu, Ala, Ser, Thr, Leu, Met, Lys or Arg;
X 6 represents Arg, Lys, His, Asn, Gln, Ser, Thr, Asp, Glu or Ala;
X 7 represents Leu, Ile, Val or Met)
The peptide according to claim 1, which has at least an amino acid sequence represented by the following formula (2) and has 10 to 50 amino acids.

(Wherein X a represents Asp or Glu,
Xb represents Pro, Val, Ile, Leu, Ala, Met, Trp, Tyr, Ser, Thr, Cys or Phe,
X 1 to X 7 are the same as above)
The peptide according to claim 1 or 2, which has at least an amino acid sequence represented by the following formula (3) and has 10 to 50 amino acids.

(Wherein Xc represents Ala, Gly, Ser, Thr, Asn, Val or Cys;
Xd represents His, Tyr, Phe, Lys, Arg, Leu, Met or Ala;
X 1 to X 7 are the same as above)
The peptide according to any one of claims 1 to 3, which has at least an amino acid sequence represented by the following formula (4) and has 12 to 50 amino acids.

(Wherein, X a to X d and X 1 to X 7 are the same as above)
The peptide according to any one of claims 1 to 4, wherein X 2 is Ser, X 3 is Met, X 5 is Gln, and X 6 is Arg.
X a is Asp, X b is claim 2, 4 or 5, wherein the peptide is Pro.
6. The peptide according to claim 3, 4 or 5, wherein Xc is Ala and Xd is His.
The peptide according to any one of claims 1 to 7, wherein the upper limit of the number of amino acids is 30.
The peptide according to any one of claims 1 to 7, which has IGF-1R binding property.
A medicament comprising the peptide according to any one of claims 1 to 9 or a labeled form thereof.
A cancer cell or cancer tissue detection agent comprising the peptide according to any one of claims 1 to 9 or a label thereof.
A cancer diagnostic agent comprising the peptide according to any one of claims 1 to 9 or a labeled form thereof.
A cancer therapeutic agent comprising the peptide according to any one of claims 1 to 9 or a labeled form thereof.
The peptide according to any one of claims 1 to 9 or a labeled body thereof for cancer diagnosis or cancer treatment.
Use of the peptide according to any one of claims 1 to 9 or a labeled product thereof for the manufacture of a cancer diagnostic agent or a cancer therapeutic agent.
A method for diagnosing or treating cancer, comprising administering the peptide according to any one of claims 1 to 9 or a labeled form thereof.