WO2012160990A1 - Peptide de liaison à igf-1r - Google Patents

Peptide de liaison à igf-1r Download PDF

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Publication number
WO2012160990A1
WO2012160990A1 PCT/JP2012/062092 JP2012062092W WO2012160990A1 WO 2012160990 A1 WO2012160990 A1 WO 2012160990A1 JP 2012062092 W JP2012062092 W JP 2012062092W WO 2012160990 A1 WO2012160990 A1 WO 2012160990A1
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phage
peptide
igf
ala
cancer
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PCT/JP2012/062092
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English (en)
Japanese (ja)
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祐介 飯森
瑞恵 山形
智隆 塩野
芝　清隆
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Hoya株式会社
公益財団法人がん研究会
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Publication of WO2012160990A1 publication Critical patent/WO2012160990A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57423—Specifically defined cancers of lung
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57434—Specifically defined cancers of prostate
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • 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.
  • IGF-1R insulin-like growth factor 1 receptor
  • IGF-1R Human insulin-like growth factor 1 receptor
  • IGF-1R Human insulin-like growth factor 1 receptor
  • IGF-1R Human insulin-like growth factor 1 receptor
  • IGF-2 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 ⁇ chains outside the cell and two ⁇ chains straddling the membrane are disulfide bonded.
  • 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.
  • 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).
  • anticancer agents using anti-IGF-1R antibodies have not yet been approved as pharmaceuticals.
  • IGF-1R antagonist of Patent Document 3 no specific substance is disclosed, and the invention has not been completed.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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).
  • 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
  • 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.
  • 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.
  • 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
  • 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.
  • a peptide having at least an amino acid sequence represented by the formula (3) and having 10 to 50 amino acids is more preferable.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the fluorescently labeled peptide of the present invention is used as a fluorescence contrast agent for cancer diagnosis by an endoscope.
  • 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.
  • 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.
  • 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.
  • a cyanine compound such as indocyanine green
  • the excitation wavelength is further shifted to the longer wavelength side compared to fluorescein, which is effective in confirming deeper lesions.
  • the positron nuclide is modified with the peptide of the present invention, it can be detected by PET, SPECT or the like.
  • 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.
  • 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.
  • any cell or tissue expressing IGF-1R may be used.
  • 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.
  • 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.
  • Example 1 (phage display) [procedure] 1. Panning experiment-round 1 200 ⁇ L of 5 ⁇ g / mL Recombinant human Insulin Like Growth Factor-1 Receptor (rhIGF-1R, R & D Systems) was added to a 96-well plate (1 ⁇ g / 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 ⁇ L / well, and allowed to stand at 37 ° C. for 1 hour.
  • BSA bovine serum albumin
  • TBS 50 mM Tris-HCL / 150 mM NaCl
  • 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.
  • peptide-displayed phage (hereinafter abbreviated as phage) was bound to the immobilized rhIGF-1R, the phage solution was removed, and 0.1% TBST 200 ⁇ L / Washed 10 times with well. Phage was eluted by adding 100 ⁇ L 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 ⁇ L of 1M Tris-HCl (pH 9.1) to the recovered solution. A part of the collected eluate was used to measure the phage titer.
  • phage peptide-displayed phage
  • 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 ⁇ (lacZ) M15proA + B + zzf :: Tn10 (TetR) fhuA2supEthi ⁇ (lac-proAB) ⁇ (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.
  • 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 ⁇ L 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.
  • phage precipitate 200 ⁇ L 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.
  • 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).
  • 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.
  • 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 ⁇ m microplate at 1 ⁇ g / well. It was solidified by leaving it overnight. The protein solution was removed, 300 ⁇ l 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 ⁇ l of a washing solution 0.5% TBST, and finally 100 ⁇ l of 0.5% TBST was added.
  • rhIGF-1R Recombinant human Epidermal Factor Receptor
  • 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 ⁇ l of 0.5% TBST, 100 ⁇ l 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 ⁇ l 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.
  • 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
  • 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.
  • 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 ⁇ l of 0.01% PBST (0.01% Tween20 / PBS) into a microtube, add 10 ⁇ l 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 ⁇ l 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.
  • PBST 0.01% Tween20 / PBS
  • 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%.
  • 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.
  • PCR reaction 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.
  • a thermal cycler PCR Thermal Cycler Dice, TAKARA
  • 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.
  • Transformed XL-1 Blue competent cells were lysed on ice. 10 ⁇ L of the self-ligated solution was added to 60 ⁇ L 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 ⁇ L of the XL-Blue bacterium was added to 100 ⁇ L 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.
  • Example 6 (Alanine-substituted phage binding test) [procedure]
  • the target protein rhIGF-1R was added to a 96-well microplate at 1 ⁇ g / well and allowed to stand overnight at 4 ° C. for immobilization.
  • the protein solution was removed, 300 ⁇ l 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 ⁇ l of a washing solution 0.5% TBST, and finally 100 ⁇ l 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 ⁇ l of 0.5% TBST, 100 ⁇ l 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 ⁇ l 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.
  • FIG. 9 shows the change in the ratio between the input titer and the output titer in the alanine-substituted phage binding test.
  • FIG. 9 shows the change in the ratio between the input titer and the output titer in the alanine-substituted phage binding test.
  • 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.
  • phages 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 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).
  • IGF-1R detection 1 ⁇ g / mL IGF-1R ⁇ (N20) (Santa Cruz Biotechnology) was used as the primary antibody, and Goat anti-rabbit IgG-HRP (BioRad) was used as the secondary antibody.
  • ⁇ -actin detection Monoclonal Anti- ⁇ -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 TM 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).
  • 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.
  • reaction solution was removed and washed 10 times with 1 mL of 1% BSA / PBS to remove unbound phage. 100 ⁇ l of 0.05% trypsin / 0.53 mM EDTA solution was added, incubated at 37 ° C. for 5 minutes, and 900 ⁇ l of growth medium was added to obtain a recovered phage solution. The titer of each recovered phage solution was measured.
  • 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.
  • the titer ratio of S-011_L7A phage was 1.6 times that of M13KE phage.
  • reaction solution was removed and washed 10 times with 1 mL of 1% BSA / PBS to remove unbound phage. 100 ⁇ l of 0.05% trypsin / 0.53 mM EDTA solution was added, incubated at 37 ° C. for 5 minutes, and 900 ⁇ l of growth medium was added to obtain a recovered phage solution. The titer of each recovered phage solution was measured.
  • 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.
  • 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 ⁇ g / well and allowed to stand overnight at 4 ° C. for immobilization.
  • the protein solution was removed, 300 ⁇ l 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 ⁇ l of a washing solution 0.5% TBST, and finally 100 ⁇ l 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
  • 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 Human breast adenocarcinoma cells MCF7 were seeded in a glass bottom dish ( ⁇ 35 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 ⁇ M 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.
  • 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). .
  • HCX PL APO CS 63x OIL, Leica 63 ⁇ oil lens
  • 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.
  • Pep02-F 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%.
  • 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.
  • Example 12 (Binding test of amino acid-substituted phage) [procedure]
  • the target protein rhIGF-1R was added to a 96-well microplate at 1 ⁇ g / well and allowed to stand overnight at 4 ° C. for immobilization.
  • the protein solution was removed, 300 ⁇ l 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 ⁇ l of a washing solution 0.5% TBST, and finally 100 ⁇ l 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 ⁇ l of 0.5% TBST, 100 ⁇ l 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 ⁇ l 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.
  • the one amino acid substituted phages there were 10 phages that showed less change compared to the binding properties of S-011 phage.
  • 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).
  • 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).
  • 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).
  • 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).
  • 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 ⁇ g / well and allowed to stand overnight at 4 ° C. for immobilization.
  • the protein solution was removed, 300 ⁇ l 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 ⁇ l of a washing solution 0.5% TBST.
  • the eluate was collected from the wells into a microtube and neutralized by adding 15 ⁇ l 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.
  • Each cell is suspended in a culture medium without addition of serum, and 900 ⁇ L 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 ⁇ L 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.
  • a peptide Pep02, Pep12
  • Example 15 (cancer cell growth inhibition test 2) The effect of Pep02 on anchorage independent growth was tested.
  • Each cell is suspended in a serum-free culture medium, and 90 ⁇ L 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 ⁇ L 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.
  • rhIGF-1 solution 5 ⁇ L 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 ⁇ L 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 ⁇ L 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.

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Abstract

L'invention concerne : une substance apte à se lier à IGF-1R d'une manière spécifique et avec une haute affinité ; et un agent médicinal comprenant la substance. L'invention concerne un peptide qui comprend au moins la séquence d'acides aminés représentée par la formule (1) et qui comporte 8-50 résidus d'acides aminés. Phe-X1-X2-X3-X4-X5-X6-X7 (1) (où X1 représente Tyr, Trp, Phe ou His ; X2 représente Ser, Ala, Thr, Gly, Asn, Asp, Gly, Arg ou Lys ; X3 représente Met, Leu, Ile, Val, Ala, Phe, Tyr, Trp ou Cys; X4 représente Leu, Ile, Val ou Met; X5 représente Gln, Asn, Asp, Glu, Ala, Ser, Thr, Leu, Met, Lys ou Arg; X6 représente Arg, Lys, His, Asn, Gln, Ser, Thr, Asp, Glu ou Ala; et X7 représente Leu, Ile, Val ou Met).
PCT/JP2012/062092 2011-05-25 2012-05-11 Peptide de liaison à igf-1r WO2012160990A1 (fr)

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CN103113457A (zh) * 2013-02-26 2013-05-22 张惠中 拮抗肽sa-12及其在治疗乳腺癌药物中的应用

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* Cited by examiner, † Cited by third party
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JP2004512010A (ja) * 2000-03-29 2004-04-22 ディージーアイ・バイオテクノロジーズ・エル・エル・シー インスリン及びｉｇｆ−１受容体のアゴニスト及びアンタゴニスト
KR20100035240A (ko) * 2008-09-26 2010-04-05 (주)케어젠 성장인자―관련 펩타이드 및 그의 용도

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004512010A (ja) * 2000-03-29 2004-04-22 ディージーアイ・バイオテクノロジーズ・エル・エル・シー インスリン及びｉｇｆ−１受容体のアゴニスト及びアンタゴニスト
KR20100035240A (ko) * 2008-09-26 2010-04-05 (주)케어젠 성장인자―관련 펩타이드 및 그의 용도

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103113457A (zh) * 2013-02-26 2013-05-22 张惠中 拮抗肽sa-12及其在治疗乳腺癌药物中的应用
CN103113457B (zh) * 2013-02-26 2014-06-18 中国人民解放军第四军医大学 拮抗肽sa-12及其在制备用于治疗乳腺癌的药物中的应用

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