US20170322213A1 - Peptides for targeting gastric cancer, and medical use tehreof - Google Patents

Peptides for targeting gastric cancer, and medical use tehreof Download PDF

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US20170322213A1
US20170322213A1 US15/527,717 US201515527717A US2017322213A1 US 20170322213 A1 US20170322213 A1 US 20170322213A1 US 201515527717 A US201515527717 A US 201515527717A US 2017322213 A1 US2017322213 A1 US 2017322213A1
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peptide
gastric cancer
cancer tissue
targeting
cancer
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Eun kyung Choi
Seong-Yun Jeong
Si Yeol SONG
Kyoung Jin LEE
Seol Hwa SHIN
Eun Jin JU
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University of Ulsan Foundation for Industry Cooperation
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University of Ulsan Foundation for Industry Cooperation
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Priority claimed from PCT/KR2015/007943 external-priority patent/WO2016080633A1/ko
Assigned to UNIVERSITY OF ULSAN FOUNDATION FOR INDUSTRY COOPERATION reassignment UNIVERSITY OF ULSAN FOUNDATION FOR INDUSTRY COOPERATION ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CHOI, EUN KYUNG, JEONG, SEONG-YUN, JU, EUN JIN, LEE, KYOUNG JIN, SHIN, Seol Hwa, SONG, SI YEOL
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
    • 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
    • G01N33/57446—Specifically defined cancers of stomach or intestine
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • 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

Definitions

  • the present invention relates to a peptide for targeting gastric cancer, a composition for diagnosing gastric cancer based on performance of irradiation using the peptide, and drug delivery use of the peptide.
  • cancer refers to cell proliferation that is not inhibited, and cancer destroys the structure and function of normal cells and organs. In this regard, it is significantly important to diagnose and treat cancer.
  • drug delivery systems or targeted therapies that selectively deliver drugs to cancer cells and cancer tissues are technologies that have received much attention, because even if the same amount of an anticancer agent is used, drug efficacy may be increased while side effects of drugs on normal tissues may be significantly reduced at the same time.
  • selective delivery of virus to cancer cells can increase treatment efficacy and reduce severe side effects.
  • antigens that are mainly specific to tumor cells and antibodies that target such antigens have been developed up to date. However, in the case of antibodies, there are problems including concerns of immune response and low efficiency of penetration into tissues.
  • peptides In the case of peptides, a molecular weight thereof is so small that there is less concern of an immune responses and the penetration of peptides into tissues is easy. Therefore, if cancer-targeting peptides are coupled with existing anticancer drugs, such resulting products can be utilized as intelligent drug vehicles that selectively deliver drugs to tumors.
  • the present invention unlike screening methods that have been studied at the existing cell culture levels, establishes mouse models transplanted with a cancer tissue of an actual human, to thereby divide them into an irradiated population and a non-irradiated population as a control group.
  • a method of screening a peptide that specifically binds to each population above is disclosed to provide a novel peptide for targeting gastric cancer and a medical use of such a novel peptide.
  • the present invention provides a peptide for targeting gastric cancer and a polynucleotide encoding the peptide, the peptide including an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6.
  • the present invention provides a peptide for targeting gastric cancer, the peptide including an amino acid selected from the group consisting of SEQ ID NOs: 1 to 3.
  • the present invention provides a composition including the peptide for diagnosing gastric cancer and a composition including the peptide for diagnosing radio-reactive gastric cancer.
  • the present invention provides a composition including the peptide for delivering a drug.
  • present invention relates to a peptide for targeting gastric, a composition for diagnosing radioresponsiveness-dependent gastric cancer using the peptide, and a drug delivery use of the peptide.
  • a functional peptide capable of targeting cancer has been discovered so as to establish personalized diagnosis and treatment for individual patients.
  • Animal models similar to cancer microenvironments of actual patients having cancer are prepared and divided into irradiated populations and non-irradiated populations as a control group, to thereby test target efficiency for respective peptides that are selected by screening peptides specifically binding to the respective populations.
  • the present invention can be finally utilized in the technical development of image diagnosis for predicting responsiveness to radiotherapy, and accordingly, the development of customized targeted therapeutic agents.
  • FIG. 1 is an image showing a method of establishing an irradiated animal model after transplanting an actual patient's gastric cancer tissue into a mouse according to Example 1 of the present invention, and also showing confirmation results of the established animal model.
  • FIG. 1A shows a patient's gastric cancer tissue distributed from the Bio Research Center (BRC, Korea)
  • FIG. 1B shows a NOD/SCID mouse that undergoes heterotrophic transplantation into the flanks of the mouse
  • FIG. 1C shows a cancer tissue cut into pieces each having a size of 3 ⁇ 3 mm to be used for subculturing, when the size of the cancer tissue of FIG. 1B is increased up to 500 mm 3 ,
  • FIG. 1A shows a patient's gastric cancer tissue distributed from the Bio Research Center (BRC, Korea)
  • FIG. 1B shows a NOD/SCID mouse that undergoes heterotrophic transplantation into the flanks of the mouse
  • FIG. 1C shows a cancer tissue cut into pieces each having a size of 3 ⁇ 3
  • FIG. 1D shows a mouse model prepared in a way that a nude mouse is anesthetized via intraperitoneal injection and undergoes heterotrophic transplantation of one piece of the cut tissues subcutaneously on the both thighs
  • FIG. 1E shows irradiation of 10 grays (Gy) of radiation over the thigh portions where the cancer cell is formed, when the size of the cancer tissue is increased up to 150-200 mm 3 .
  • a control group is not subjected to irradiation.
  • FIG. 2 shows a biopanning scheme for identifying a sequence of a peptide, which targets a gastric cancer tissue of an irradiated in vivo patient, by using an M13 phage display method according to embodiments of the present invention.
  • FIG. 3 shows results of comparing phage concentrations obtained by eluting phages after extracting heart, lung, liver, spleen, kidney, and tumor during biopanning process performed five times.
  • FIG. 4 shows results of a linking proportion between a Cy5.5 fluorescent probe and a phage by calculating phages of the same concentration after a discovered peptide-expressing phage of the present invention is amplified in terms of linking with the Cy5.5 fluorescent probe, and by calculating region of interest (ROI) values in connection with linking between the Cy5.5 fluorescent probe and the phage.
  • ROI region of interest
  • FIG. 5 shows results confirming specific binding to in vivo gastric cancer tissue based on images on the 2 nd day after injecting a peptide phage labeled with a Cy5.5 fluorescent probe into each mouse mode.
  • FIG. 6 shows results confirming fluorescence intensity of cancer tissue after only cancer tissue is extracted and also confirming places where fluorescence is located on cancer tissue that is equally divided, as in vivo image confirmation is completed on the 2 nd day.
  • FIG. 7 shows a schematic diagram for observing changes in targeting ability of a peptide phage of the present invention as being selected depending on irradiation.
  • mouse models in which the size of the cancer tissue is increased up to 150-200 mm 3 are divided into 1) irradiated mouse models and 2) irradiated mouse models with 2 grays (Gy) of radiation.
  • Gy grays
  • FIG. 8 shows results verifying specific binding ability of a peptide sequence discovered in each population through immunohistochemistry.
  • FIG. 9 shows preparation of a liposome, a drug encapsulation process and optimization thereof.
  • FIG. 10 shows results of linking a peptide to a liposome including to a liposome including a drug encapsulated therein.
  • FIG. 10A is a schematic diagram showing linking of a peptide to a liposome including a drug encapsulated therein and also shows a chemical constitutional formula representing actually linked residues
  • FIG. 10B shows results of a reduction test to calculate the number of —SH residues in a liposome before being linked to a peptide and also shows results confirming stability through verification of the size distribution after being linked to a peptide.
  • FIG. 11 shows in vivo imaging results for verifying targeting ability of a material in which a peptide is linked to a liposome including a drug encapsulated therein. It is confirmed that only a liposome linked to a peptide is targeted in an irradiated mouse.
  • FIG. 12 shows in vivo tumor growth delay results for verifying possibility of a material in which a peptide is linked to a liposome including a drug encapsulated therein to be used as an anticancer drug.
  • a liposome linked to a peptide and including a drug encapsulated therein is proved to be effective in treating tumors in an irradiated group.
  • FIG. 13 shows results confirming targeting ability of a selected peptide upon irradiation to mice transplanted with other patient's gastric cancer tissue according to Example 8 of the present invention.
  • the present invention provides a peptide for targeting gastric cancer, the peptide including an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 TO 6.
  • the above-mentioned amino acid sequences are shown in Table 1.
  • the peptide of the present invention is a low-molecular weight peptide consisting of 7 amino acids. Such a low-molecular weight peptide is small in size so that it can be stabilized three-dimensionally.
  • a low-molecular weight peptide has the advantage of being able to easily pass through a membrane and to recognize a target molecule deep in tissues. Since the stability of the low-molecular weight peptide of the present invention is secured through local injection and the immunoreactivity can be minimized, there is an advantage that cancer can be diagnosed early.
  • the mass production of the low-molecular weight peptide of the present invention is relatively easy compared that of an antibody, and the toxicity of the low-molecular weight peptide of the present invention is weak.
  • the low-molecular weight peptide of the present invention is has an advantage of a strong binding force to a target material compared to an antibody, and do not undergo denaturation during thermal/chemical treatment.
  • the low-molecular weight peptide can be used as a fused protein as being attached to other proteins.
  • the low-molecular weight peptide can be also used as being attached to a high-molecular weight protein chain, and accordingly, can be used as a diagnosis kit and a drug delivery carrier.
  • the low-molecular weight peptide of the present invention can be easily prepared according to the chemical process known in the art (Creighton, Proteins; Structures and Molecular Principles, W. H. Freeman and Co., NY, 1983).
  • chemical process known in the art
  • liquid or solid phase synthesis, fractional condensation, F-MOC or T-BOC chemical method, or the like may be used (Chemical Approaches to the Synthesis of Peptides and Proteins, Williams et al., Eds., CRC Press, Boca Raton Fla., 1997; A Practical Approach, Athert on & Sheppard, Eds., IRL Press, Oxford, England, 1989), but the method is not limited thereto.
  • the low-molecular weight peptide of the present invention can be prepared according to a genetic engineering method.
  • a DNA sequence encoding the sequence low-molecular weight peptide is prepared.
  • a DNA sequence can be prepared by PCR amplification using an appropriate primer.
  • a DNA sequence can be synthesized using, for example, an automatic DNA synthesizer (manufactured by Biosearch or AppliedBiosystems).
  • Such a synthesized DNA sequence is inserted to a vector including one or more expression control sequences (for example: a promoter, an enhancer, or the like) that are operatively linked with the DNA sequence to control expression of the DNA sequence, and then, a host cell is transformed with a recombinant expression vector prepared therefrom.
  • a resulting transformant is cultured in an appropriate medium under suitable conditions to allow the expression of the DNA sequence, so that substantially pure peptides that are encoded by the DAN sequence are recovered from the culture. Such recovery may be performed according to a method known in the art (for example, chromatography).
  • substantially pure peptides used herein refers to peptides that do not substantially include any protein derived from the host.
  • the present invention provides a peptide for targeting gastric cancer, the peptide being specific to an irradiated gastric cancer tissue and including an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3.
  • a peptide including an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3 specifically binds to a gastric cancer tissue, in particular, an irradiated gastric cancer tissue.
  • target refers to ability to specifically bind only to a gastric cancer tissue, especially an irradiated gastric cancer tissue, without binding to other normal tissues.
  • a gastric cancer-specific peptide can specifically bind to the inside or outside of a gastric cancer tissue.
  • the present invention provides a polynucleotide encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3.
  • polynucleotide refers to a single-stranded or double-stranded polymer of deoxyribonucleotides or ribonucleotides. Such a polynucleotide includes a RNA genome sequence, a DNA sequence (for example, gDNA and cDNA), and a RNA sequence transcribed from the DNA sequence. Unless otherwise mentioned, a polynucleotide includes an analog of a natural polynucleotide.
  • the polynucleotide includes not only a nucleotide sequence that encodes the peptide for targeting gastric cancer, but also a sequence complementary to the nucleotide sequence, wherein such a complementary sequence includes not only a perfectly complementary sequence, but also a substantially complementary sequence.
  • polynucleotide may be subjected to modifications. Such modifications include addition, deletion, non-conservative substitution, or conservative substitution of a nucleotide.
  • the polynucleotide encoding the amino acid sequence is also interpreted to include a nucleotide sequence that exhibits substantial identity to the nucleotide sequence.
  • the substantial identity is obtained by aligning the nucleotide sequence with any other sequences to the greatest extent and by analyzing the aligned sequence using algorithms commonly used in the art, and in this regard, the substantial identity may indicate a sequence having at least 80% homology, at least 90% homology, or at least 95% homology with the aligned sequence.
  • the present invention provides a composition for diagnosing gastric cancer, the composition including a peptide including an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6.
  • the present invention provides a composition for radio-sensitive diagnosing gastric cancer, the composition including a peptide being specific to an irradiated gastric cancer tissue and including an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3.
  • diagnosis refers to identification of the presence or characteristic of a pathological condition.
  • diagnosis is to identify the presence or characteristic of gastric cancer.
  • the diagnosis of gastric cancer using the peptide of the present invention may be performed by detecting binding of the peptide of the present invention to a corresponding tissue or cell directly obtained from blood, urine, or biopsy.
  • the peptide of the present invention can be provided in a labeled state. That is, the peptide provided herein may be linked to a detectable label (for example, via covalent binding or cross-linking).
  • the detectable label may be a chromogenic enzyme (for example, peroxidase and alkaline phosphatase), a radioactive isotope (for example, 124 I, 125 I, 111 In, 99 mTc, 32 P, and 35 S), a chromophore, or a luminescent material or a fluorescent material (for example, FITC, RITC, rhodamine, cyanine, Texas Red, fluorescein, phycoerythrin, or quantum dots).
  • a chromogenic enzyme for example, peroxidase and alkaline phosphatase
  • a radioactive isotope for example, 124 I, 125 I, 111 In, 99 mTc, 32 P, and 35 S
  • a chromophore or a luminescent material or a fluorescent material (for example, FITC, RITC, rhodamine, cyanine, Texas Red, fluorescein, phycoeryth
  • the detectable label may be an antibody-epitope, a substrate, a cofactor, an inhibitor, or a affinity ligand. Such labeling may be performed during the synthesis of the peptide of the present invention, or may be additionally performed on a peptide that is already synthesized.
  • a fluorescent material is used as a detectable label
  • cancer may be diagnosed according to fluorescence mediated tomography (FMT).
  • FMT fluorescence mediated tomography
  • the peptide of the present invention labeled with a fluorescent material may be circulated into the blood, and the fluorescence by the peptide may be observed by FMT. If fluorescent is observed, it is diagnosed as cancer.
  • the present invention provides a composition for delivering a drug, the composition including the peptide for targeting gastric cancer.
  • the peptide of the present invention may be used as an intelligent drug delivery vehicle that selectively delivers a drug to a cancer tissue. If the peptide of the present invention is used in combination with drugs of the related art in terms of treatment of cancer, the peptide of the present invention selectively delivers a drug only to a cancer tissue and a cancer cell, so that drug efficacy may be increased while drug side effects on a normal tissue may be significantly reduced at the same time.
  • any anticancer drug that is conventionally used in the treatment of cancer can be used so long as the anticancer drug is able to be linked to the peptide of the present invention.
  • the drug include cisplatin, 5-fluorouracil, adriamycin, methotrexate, vinblastine, busulfan, chlorambucil, cyclophosphamide, melphalan, nitrogen mustard, nitreosourea, taxol, paclitaxel, docetaxel, 6-mercapropurine, 6-thioguanine, bleomycin, daunorubicin, doxorubicin, epirubicin, idarubicin, mitomycin-C, and hydroxyurea.
  • linking of the anticancer drug to the peptide of the present invention may be performed by a method known in the art, for example, covalent bonding, cross linking, or the like.
  • the peptide of the present invention may be, if necessary, subjected to chemical modifications to the extent that the activity thereof is not lost.
  • an ideal animal model of cancer similar to actual patient's cancer microenvironments is prepared. Then, to establish an animal model that can confirm influence of an irradiation-dependent cancer tissue, first, a mouse model transplanted with a cancer tissue that was extracted from an actual patient having gastric cancer was established. Regarding the establishment of such an animal model, the cancer tissue extracted from a patient was cultured in an NOD/SCID mouse. Once the cancer tissue was found in the NOD/SCID mouse, subculturing was carried out by using a Balb/c nude mouse, beginning from the next subculturing.
  • the cancer tissue was cut into pieces each having a size of 3 ⁇ 3 mm.
  • each of both flanks of the NOD/SCID mouse was transplanted with a piece of the cut cancer tissues, treated with a mixed solution of 100 ⁇ l of 100 IU/ml penicillin, 100 ⁇ g/ml streptomycin, 100 ⁇ g/ml gentamicin, and 2.5 ⁇ g/ml amphotericin B antibiotics, and then, sutured.
  • the NOD/SCID mouse was recovered on a heating pad (for about 2 hours). Afterwards, the formation and growth of tumors were observed every week.
  • the cancer tissues When the cancer tissues grew to a size of 400-500 mm 3 , the cancer tissues were separated and cut into pieces each having a size of 3 ⁇ 3 mm for subculturing.
  • a nude mouse was anesthetized via intraperitoneal injection of anesthetics, and a piece of the cut cancer tissues was transplanted subcutaneously on the right thigh of the nude mouse.
  • a transplantation site was changed from the flank to the both thighs so that irradiation can be locally done without affecting other organs during irradiation. That is, a mouse model in which a cancer tissue that underwent subculturing and was re-transplanted up to four times was formed was established.
  • the growth of the cancer tissue was observed for about a month (4 weeks), and when the size of the cancer tissue was increased to about 200 mm 3 , only the cancer tissue was locally irradiated with 10 grays (Gy) of radiation. After having recovery time for no longer than 24 hours, in vivo peptide screening was performed.
  • a mouse model that was not irradiated among the same mouse models was used to screen a peptide. The results of the establishment of such a mouse model are shown in FIG. 1 .
  • a method for identifying a peptide having high specificity during in vivo peptide screening using a random loop peptide library was designed.
  • a loop peptide library manufactured to have about 2.7 billion different amino acids sequences via random array of 7 amino acids [(i.e., a library fused with an M13 phage gp3 minor coat protein)-Ph.DTM phage display peptide library kit, New England Biolabs (NEB)] was purchased.
  • a M13 phage peptide library i.e., a library fused with an M13 phage gp3 minor coat protein and consisting of 7 amino acids having about 2.7 billion different amino acids sequences
  • e M13 phage peptide library was circulated in vivo for 15 minutes (also known as a method of binding an in vivo cancer tissue with an M13 phage peptide library).
  • a peptide-expressing phage specifically binding to the cancer tissue was selected with different washing conditions.
  • FIG. 2 Such a screening scheme is shown in FIG. 2 .
  • FIG. 2 In detail, FIG.
  • an M13 phage screening scheme for screening a cancer tissue-targeting peptide wherein (1) a mouse model in which a cancer tissue was formed on the right hind leg was established, (2) a phage library in which a loop peptide library consisting of 7 amino acids was expressed on a surface of a M13 phage was injected into the tail vein of the mouse to allow circulation of the phage library, (3) phages were washed under a variety of washing conditions, and (4) phages were obtained by eluting finally targeted phages. The eluted phages infected Escherichia coli , and were injected again into the tail vein of the mouse to allow circulation of the phages.
  • a process of screening phages having high specificity and a strong binding strength was repeatedly performed (also known as biopanning). Biopanning was performed five times per cycle so that a phage expressing a sequence of a peptide specifically binding to the patient's in vivo gastric cancer tissue was obtained. To confirm whether the peptide-expressing phage actually targeted the cancer tissue only, other in vivo organs were also subjected to comparison. That is, for every biopanning, phages were eluted from each of extracted heart, lung, liver, spleen, kidney, and cancer tissue, and the phage concentration was measured for comparison. The results of the comparison are shown in FIG. 3 .
  • phage plaques were selected randomly from each of an irradiated population (experimental group) and a non-irradiated population (control group), and the M13 phage genomic DNA (single-stranded circular DNA) was separated and purified to identify a gene sequence, thereby identifying an amino acid sequence of a peptide expressed in a phage-surface protein (e.g., a gp3 minor coat protein) and targeting the cancer tissue.
  • Table 1 shows a summary of sequences discovered from each of the irradiated (experimental group) and the non-irradiated population (control group) by using the Clustal X program for sequence analysis.
  • Peptide sequence Irradiated P1 TVRTSAD (SEQ ID NO: 1) population (10 P2 RYVGTLF (SEQ ID NO: 2) Gy) P3 NRGDRIL (SEQ ID NO: 3) Non-irradiated W1 NWGDRIL (SEQ ID NO: 4) population as W2 QRSLPSL (SEQ ID NO: 5) control W3 DVWHSAY (SEQ ID NO: 6)
  • a process of linking a fluorescent probe was performed first.
  • 1 ⁇ g/ ⁇ l of N-hydroxysuccinimide esters of Cy5.5 (Amersham) was added to 1 mL of bicarbonate buffer (pH 8.3) having the phage concentration of 10 11 plaque forming units (pfu), and then, in a condition where a dark environment was maintained, 3 a phage-surface protein was linked to the Cy5.5 fluorescent probe at room temperature for 3 hours.
  • loop peptide-expressing phages to which the Cy5.5 fluorescent probe was linked were each obtained by precipitation with 170 ⁇ l of 20% (w/v) PEG 8000/2.5 M NaCl solution and purification.
  • an IVIS spectrum imaging system Xenogen
  • ROI region of interest
  • the pieces of the extracted cancer tissue were collected independently, and phages bound to the cancer tissues were eluted.
  • the concentration of each of the eluted phages was measured according to titering, and due to different size and weight of the extracted cancer tissue, the weight of each cancer tissue was also measured in terms of establishing numerical standardization. In this regard, the amount of the identified phages was calculated relative to the weight of the cancer tissue.
  • the in vivo imaging and the ex vivo imaging were confirmed by measuring ROI values that were determined using the IVIS spectrum (Xenogen) program, and the results thereof are shown in Table 2.
  • Example 3 To confirm whether 3 peptide sequences identified in Example 3 were responsive to cancer microenvironments during irradiation, the targeting efficiency of these irradiation-dependent peptide sequences was confirmed.
  • targeting of the peptide which was dependent upon cancer microenvironments was subjected to verification. Accordingly, as in the mouse model established in Example 1, mice in which tumor was formed by transplantation of a patient's gastric cancer tissue were selected. Among the selected mice, only some of them were irradiated to thereby establish a control group and an experimental group.
  • Example 7 In the same manner as in Example 3, the selected phages expressing the peptide were amplified and fluorescent labeling was also performed thereon, The same sample was injected into an irradiated mouse group and a control group thereof, thereby obtaining images for the last two days. Consequently, when comparing targeting in the irradiated mouse group with that in the control group, the two groups showed differences in the targeting efficiency.
  • the imaging measurement results of the present embodiment are shown in FIG. 7 . As shown in FIG. 7 , the control group including the mouse model transplanted with the patient's gastric cancer tissue showed low targeting efficiency, whereas the irradiated mouse model including the same mouse model transplanted with the patient's gastric cancer tissue showed specific binding ability through images.
  • each population was injected via the tail vein.
  • cancer tissues of each population were extracted to prepare paraffin blocks and slices.
  • the extracted cancer tissues were immersed in a formaldehyde solution at room temperature for 24 hours in terms of for immobilization.
  • paraffin was added to the solution through penetration to form paraffin blocks.
  • microtome was used to manufacture slices having a thickness of 3 ⁇ m.
  • an unmasking process was performed so that structures of various proteins immobilized to the tissue slices were recovered to restore sites where antibodies normally bind.
  • lipids constituting a liposome such as dipalmitoylphosphatidylcholine (DPPC, concentration of 50 mM), dipalmitoylphosphatidylglycerol (DPPG-Na, concentration of 50 mM), N-[3-(2pyridinyldithio)-1-oxopropyl]-L- ⁇ -dipalmitoylphosphatidylcholine (DPPE-PDP, concentration of 50 mM), cholesterol (concentration of 200 mM), and cholesterol—PEG (concentration of 200 mM), were each dissolved in an organic solvent containing methanol and chloroform (prepared at a ratio of 1:1).
  • DPPC dipalmitoylphosphatidylcholine
  • DPPG-Na dipalmitoylphosphatidylglycerol
  • DPPE-PDP concentration of 50 mM
  • cholesterol concentration of 200 mM
  • cholesterol—PEG concentration of 200 mM
  • DPPG-Na which does not melt at room temperature was completely dissolved at 55° C. for more than 30 minutes.
  • Each of the five dissolved lipids was added to a round-bottom flask so as to prepare a mixed solution containing DPPC:DPPE-PDP:DPPG-Na:cholesterol-PEG:cholesterol at a ratio of 15:15:30:4:36.
  • the round-bottom flask was rotated at 55° C., and was pressurized for about 2 to 3 hours to volatilize the organic solvent therefrom. Meanwhile, a thin lipid film was formed within the round-bottom flask.
  • a nitrogen gas extruder and a poly-carbonate filter were used to filter the liposome through a filter with fine holes, wherein the fine holes used herein had a diameter of 800 nm, 400 nm, 200 nm, and 100 nm in the stated order.
  • a filter having a diameter of 200 nm and a filter having a diameter of 100 nm were used twice or several times for extrusion.
  • PBS pH 7.0
  • the resulting liposome was dissolved in the buffer such that the inside of the liposome had pH 4.0 and outside thereof had pH 7.0.
  • the liposome resulting from the completion of buffer replacement and doxorubicin dissolved in buffer having pH 7.0 were mixed in a round-bottom flask. Then, to encapsulate the drug dissolved in the buffer having pH 7.0 within the liposome having pH 4.0 by a concentration gradient, the round-bottom flask was rotated in a bath at 60° C. for 20 minutes. Then, to isolate the remaining non-encapsulated drug, a pure liposome including the drug encapsulated therein was purified using a Sephadex column. According to the Dinamic light scattering; DLS method, the drug delivery carrier was optimized by measuring the size and stability of the finally prepared drug-encapsulated liposome. The results of the drug delivery process and optimization thereof described above are shown in FIG. 9 .
  • the peptide having ‘TVRTSAD’ sequence was synthesized by a request, and ligated with the drug-encapsulated liposome of Example 6. Accordingly, a peptide in which the C-terminal of the ‘TVRTSAD’ was linked with a Cy7 fluorescent probe and the N-terminal of the ‘TVRTSAD’ was free from any process to be linked with a liposome was prepared by a request from AnyGen Inc. (South Korea). The residue of the N-terminal of the prepared peptide was processed to be linked with a thiol group (—SH) of a liposome via a disulfide bond.
  • —SH thiol group
  • the number of the thiol group of the liposome was counted, and a liposomal reduction test was conducted so as to link the liposome to the peptide depending on the ratio of the thiol group.
  • DTT 1 mM was added and pyridine 2-thione was measured at OD 343 nm, to count the number of the thiol group of the liposome.
  • the liposome and the peptide were mixed at a molar ratio of 1:1.5 to allow a reaction for 2 hours at room temperature.
  • the liposome linked with a pure peptide was purified using a Sephadex column. Afterwards, to verify that there is no change in the size and stability of the liposome before and after being linked with the peptide, the verification was demonstrated according to the Dinamic light scattering; DLS method, and the results are shown in FIG. 10 .
  • Example 6 To verify whether the drug carrier of Example 6 in which the ‘TVRTSAD’ sequence was linked to the drug-encapsulated liposome actually played a function in the living body, in vivo imaging was performed.
  • the radio-sensitive xenograft mouse model of Example 1 and the control group were each injected through the vein tail of the mouse, and images were measured for 2 days immediately after the injection, thereby confirming that images showing in vivo circulation of the peptide and the targeting of the peptide only in the cancer tissue while the targeting to other organs and tissues gradually disappeared.
  • the peptide was proved to completely target the in vivo gastric cancer tissue.
  • the results of the in vivo imaging are shown in FIG. 11 .
  • in vivo tumor growth delay was also performed to validate the peptide as a target drug delivery carrier.
  • the radio-sensitive xenograft mouse model of Example 1 and the control group were established, and once the tumor size was increased to about 100 mm 3 , grouping was performed thereon.
  • a total of 5 groups i.e., 1; PBS, 2; irradiation (2 Gy), 3; DOX(2 mg/kg)+irradiation (2 Gy), 4; LP-DOX (2 mg/kg)+irradiation (2 Gy), 5; P1(peptide)-LP-DOX (2 mg/kg)+irradiation (2 Gy), were prepared.
  • test group was designated as Group 5 while the control groups were designated as Groups 1 to 4 for observation.
  • the test group i.e., Group 5
  • the results of validation of a new concept anticancer drug are shown in FIG. 12 .
  • the material linked with the corresponding peptide and the drug-encapsulated liposome were verified to be utilized in the in vivo imaging and the in vivo tumor growth delay, and accordingly, the possibility of the material as a new concept anticancer drug that can simultaneously diagnose and treat cancer was proved.
  • gastric cancer tissues each extracted from different patients were prepared to establish a mouse model.
  • gastric cancer tissues of other patients were prepared, wherein all the gastric cancer tissues used herein were characterized as adenocarcinoma.
  • mice models including each of the corresponding gastric cancer tissues was established, and some of them were irradiated to thereby establish a control group and an experimental group.
  • the in vivo imaging which is the same method as the one used for confirming targeting efficiency in Example 4, the irradiation-dependent targeting efficiency of the peptide was verified.
  • the amplification of phages expressing the selected peptide and the fluorescent labeling were performed in the same manner as in Example 3. Then, the completed sample was injected into each of the irradiated test mouse group and the control group, and images thereof were confirmed on the 2 nd day of the observation.
  • the two gastric cancer tissues of other patients also showed selective accumulation of peptide-phage only in the tumors of the irradiated test mouse group, in the same manner as in the existing gastric cancer tissue of the patient.
  • the results of the imaging measurement of the corresponding embodiments are shown in FIG. 13 .
  • the peptide-expressing phage did not target the cancer tissue when there is no irradiation applied to the cancer tissue, whereas the peptide-expressing phage targeted the cancer tissue when irradiation is applied to the cancer tissue.
  • the peptide sequence selected in the corresponding technology was clearly verified as the peptide sequence selectively targeting the gastric cancer upon irradiation, and that the targeting ability of the corresponding peptide is not limited to the patient's gastric cancer tissue only. That is, as the peptide sequence exhibiting selective targeting ability only in the case where the gastric cancer tissue of other patients are irradiated, it was verified that the scope of application of the peptide of the present invention is not limited in clinical applications.

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US10925980B2 (en) 2014-08-04 2021-02-23 Case Western Reserve University Molecular probes and methods of use

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US10925980B2 (en) 2014-08-04 2021-02-23 Case Western Reserve University Molecular probes and methods of use

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US10124073B2 (en) * 2014-08-04 2018-11-13 Case Western Reserve University Targeting peptides and methods of use
US10653801B2 (en) 2014-08-04 2020-05-19 Case Western Reserve University Targeting peptides and methods of use
US10925980B2 (en) 2014-08-04 2021-02-23 Case Western Reserve University Molecular probes and methods of use
US11738099B2 (en) 2014-08-04 2023-08-29 Case Western Reserve University Molecular probes and methods of use

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