WO2021025352A1 - Virus anticancéreux exprimant le peptide dérivé du fgf2 ou de l'api5, et son utilisation - Google Patents

Virus anticancéreux exprimant le peptide dérivé du fgf2 ou de l'api5, et son utilisation Download PDF

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Publication number
WO2021025352A1
WO2021025352A1 PCT/KR2020/009856 KR2020009856W WO2021025352A1 WO 2021025352 A1 WO2021025352 A1 WO 2021025352A1 KR 2020009856 W KR2020009856 W KR 2020009856W WO 2021025352 A1 WO2021025352 A1 WO 2021025352A1
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Prior art keywords
cancer
virus
api5
anticancer
fgf2
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PCT/KR2020/009856
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English (en)
Korean (ko)
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이병일
김건영
배승현
봉승민
송보민
장현철
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국립암센터
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Publication of WO2021025352A1 publication Critical patent/WO2021025352A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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

Definitions

  • the peptide may inhibit the binding of API5 (apoptosis inhibitor 5) and FGF2 (fibroblast growth factor-2).
  • the cancer showing the anticancer effect of the anticancer virus of the present invention is any one selected from the group consisting of gastric cancer, lung cancer, cervical cancer, colon cancer, breast cancer, prostate cancer, melanoma, liver cancer, pancreatic cancer, ovarian cancer, thyroid cancer, and bladder cancer. It can exhibit anticancer effects on the above cancers.
  • the cancer is more preferably any one or more selected from the group consisting of cervical cancer, liver cancer, kidney cancer, and pancreatic cancer.
  • composition of the present invention may be used alone or in combination with surgery, radiation therapy, hormone therapy, chemotherapy, and methods using biological response modifiers.
  • the anticancer adjuvant may additionally include a Bromodomain and Extra-Terminal motif (BET) inhibitor, and the BET inhibitor is the same as described in the pharmaceutical composition, so the description is replaced with the above description.
  • BET Bromodomain and Extra-Terminal motif
  • the virus expressing the FGF2 or API5-derived peptide of the present invention is involved in mRNA transport, thereby inhibiting the resistance of anticancer drugs, and consequently, can suppress the survival of cancer cells.
  • the BRD4 inhibitor is used in combination, synergy is generated and more effectively It can inhibit survival. Therefore, the virus expressing the FGF2 or API5-derived peptide of the present invention can be effectively used as an anticancer virus or a composition for inhibiting anticancer drug resistance including the anticancer virus, a pharmaceutical composition for use in preventing or treating cancer, or an anticancer adjuvant,
  • a method for preventing or treating cancer including administering a composition containing the anticancer virus to a cancer patient can also be effectively used.
  • API5-segment 2 shows the results of confirming the binding strength of the API5 derived peptide (API5-segment 2) to FGF2 and the binding force of the FGF2-derived peptide (FGF2-segment 1) to API5 expressed in the anticancer virus. It was confirmed that the API5-derived peptide directly binds to FGF2, and the FGF2-derived peptide directly binds to API5.
  • 25 shows an experimental method for confirming the anticancer effect in cancer cells by preparing a lentivirus expressing the peptide derived from API5 or FGF2 in the present invention.
  • API5 full length (1-504) and low molecular weight (LMW) FGF2 were each cloned into pET 28b vector and expressed in E. coli.
  • LMW low molecular weight
  • FGF2 a codon-optimized synthetic gene was used for E. coli, and cysteine, which forms a disulfide bond, was replaced with serine to facilitate E. coli expression (C211S/C229S, Uniport P09038). Based on -4, the amino acid substitution did not affect the function and structure of FGF2, so it was named FGF2 in the present invention).
  • GST-API5 wild type (GST-API5 WT) and mutant (GST-API5 3Mut (E184A/D185A/E190A)) were used for pulldown assay, and GST (Glutathione-S-Transferase) was used as a control.
  • GST Glutathione-S-Transferase
  • Protein was prepared. Each of the three proteins, 2 each, 10 ⁇ g each into a tube containing 50 ⁇ l of a total of 6 glutathione-agarose resins, was incubated at 4° C. for 2 hours, and then PBS (phosphatebuffered saline) buffer was added.
  • FGF2 WT C211S/C229S
  • FGF2 mutations FGF2 3Mut, C211S/C229S/R262A/T263A/K271A
  • sample buffer 250 mM Tris-HCl pH 6.8, 10% SDS, 0.05% bromophenol blue). blue
  • a vector capable of inhibiting the expression of API5 inherent in the CRISPR/Cas9 system was guided to the CRISPR V2 vector (Plasmid #52961, Addgene), and guided RNA (5′-CACCGTGTTTTGCAGGGACTTTAGG-3′, SEQ ID NO: 5). It was produced by introducing. Then, mutant 3Mut (E184A/D185A/E190A) and 4Mut (D145A/E184A/D185A/E190A) unable to bind API5 WT and FGF2 were added to the pCAG-Flag-IRES-Blasticidin vector to express wild-type and mutant API5. Each was inserted.
  • a lentivirus containing API5 guided RNA, Cas9, and puromycin resistance genes at the same time was prepared, transfected into HeLa cells, and treated with puromycin to allow only the transfected cells to survive.
  • a recombinant vector capable of simultaneously expressing Flag-API5 normal and mutant and blasticidine resistance genes was inserted using Lipofectamine 3000 (Thermefisher), and treated with blasticidine. Primary transformed cells were selected.
  • Samples prepared in advance in 6 tubes were added respectively, stored at 4°C for 2 hours, centrifuged at 10,000 rpm for 1 minute, discarded the supernatant, and added 500 ⁇ l of the remaining resin in PBS buffer into 6 tubes, mixed, and then at 10,000 rpm. After centrifugation for 1 minute, the supernatant was discarded, and the washing method was repeated twice.
  • FISH was performed using Cy3-labeled Oligo(dT)50 (FISH probe, Genelink, USA), and experiments were conducted using Stellaris RNA-FISH kit (LGC Bioresearch Technologies), and cells (shAPI5, shAPI5/API5 WT, shAPI5/API5) were used.
  • 3Mut was cultured by adding 10% fetal bovine serum and 1% penicillin streptomycin (Gibco, USA) to MEM/EBSS (Hyclone, USA) medium. Treated and untreated were cultured, respectively, and after 3 days, 10 ⁇ M Actinomycin D was treated for 2 hours. Mock was transformed with a Tet-pLKO-puro empty vector expressing shRNA and was used as a control.
  • DAPI (4',6-diamidino-2-phenylindole) was dissolved at 5 ng/ml in 1X Wash Buffer A containing 10% formamide, and 1 ml of the DAPI solution was added to each well at 37°C. Stored for 30 minutes. After 30 minutes, the DAPI solution was discarded, and 1 mL of 1X Wash Buffer B in the RNA-FISH kit was added to each well and treated at room temperature for 5 minutes.
  • the two cell lines were treated with doxycycline for at least 4 days and separated into single cells by trypsin treatment, and then 2 ⁇ 10 3 cells per well were dispensed into a 96-well plate. The next day, after treatment with 10 ⁇ M cisplatin, cell viability was confirmed through MTT analysis 48-72 hours later.
  • Example ⁇ 1-2> the binding force between the API5 or FGF2 derived peptide and FGF2 or API5 was measured using the same surface plasmon resonance (SPR) spectrophotometry as in Example ⁇ 1-2>.
  • SPR surface plasmon resonance
  • the API5-derived peptide affects the expression of MYC or CCND1 in the same manner as in 3 of Example ⁇ 2-3>.
  • the sequence used as a control is SEQ ID NO: 8 (DRQLQLSTLQRML), and the polynucleotide sequence encoding it is SEQ ID NO: 9.
  • SEQ ID NO: 1 is a sequence derived from API5 that is directly related to the binding of API5 and FGF2, and is a sequence of 9 amino acids.
  • SEQ ID NO: 8 is a control sequence, a 13 amino acid sequence.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Veterinary Medicine (AREA)
  • Biochemistry (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Virology (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Epidemiology (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biotechnology (AREA)
  • Immunology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biomedical Technology (AREA)
  • General Chemical & Material Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Mycology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne un virus anticancéreux exprimant un peptide dérivé du FGF2 ou de l'API5, et son utilisation. Le virus exprimant un peptide dérivé du FGF2 ou de l'API5 selon la présente invention peut participer au transport d'ARNm pour inhiber la résistance à des médicaments anticancéreux et par conséquent inhiber la viabilité de cellules cancéreuses, et peut générer une synergie lorsqu'Il est utilisé en combinaison avec des inhibiteurs de BRD4 pour inhiber plus efficacement la viabilité de cellules cancéreuses. Ainsi, le virus exprimant un peptide dérivé du FGF2 ou de l'API5 selon la présente invention peut être utilisé efficacement en tant que virus anticancéreux ou dans une composition contenant le virus anticancéreux pour inhiber la résistance aux médicaments anticancéreux, et dans une composition pharmaceutique ou un adjuvant anticancéreux destiné à être utilisé dans la prévention ou le traitement du cancer. De plus, une méthode de prévention ou de traitement du cancer comprenant l'administration d'une composition contenant le virus anticancéreux à un patient cancéreux peut également être efficacement utilisée.
PCT/KR2020/009856 2019-08-08 2020-07-27 Virus anticancéreux exprimant le peptide dérivé du fgf2 ou de l'api5, et son utilisation WO2021025352A1 (fr)

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KR1020190097019A KR102241134B1 (ko) 2019-08-08 2019-08-08 Fgf2 또는 api5 유래 펩타이드를 발현하는 항암 바이러스 및 이의 용도
KR10-2019-0097019 2019-08-08

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20130102990A (ko) * 2012-03-09 2013-09-23 고려대학교 산학협력단 항암면역우회암의 표시인자 및 무력화 표적으로서의 ａｐｉ５의 용도
WO2017216620A1 (fr) * 2016-06-13 2017-12-21 Uniwersytet Wrocławski Conjugués de facteur de croissance des fibroblastes 2 humain (fgf2) et de médicament cytotoxique pour le traitement ciblé de cancers liés au fgfr
KR101845739B1 (ko) * 2014-10-08 2018-04-05 부산대학교 산학협력단 신규한 암용해 바이러스, 이의 제작 방법 및 이의 용도

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20130102990A (ko) * 2012-03-09 2013-09-23 고려대학교 산학협력단 항암면역우회암의 표시인자 및 무력화 표적으로서의 ａｐｉ５의 용도
KR101845739B1 (ko) * 2014-10-08 2018-04-05 부산대학교 산학협력단 신규한 암용해 바이러스, 이의 제작 방법 및 이의 용도
WO2017216620A1 (fr) * 2016-06-13 2017-12-21 Uniwersytet Wrocławski Conjugués de facteur de croissance des fibroblastes 2 humain (fgf2) et de médicament cytotoxique pour le traitement ciblé de cancers liés au fgfr

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BONG SEOUNG MIN, BAE SEUNG-HYUN, SONG BOMIN, GWAK HYERAN, YANG SEUNG-WON, KIM SUNSHIN, NAM SEUNGYOON, RAJALINGAM KRISHNARAJ, OH SE: "Regulation of mRNA export through API5 and nuclear FGF2 interaction", NUCLEIC ACIDS RESEARCH, INFORMATION RETRIEVAL LTD., GB, vol. 48, no. 11, 19 June 2020 (2020-06-19), GB, pages 6340 - 6352, XP055777478, ISSN: 0305-1048, DOI: 10.1093/nar/gkaa335 *
BONG, SEOUNG MIN ET AL.: "Crystallization and preliminary X-ray analysis of API5-FGF2 complex", BIO DESIGN, vol. 6, no. 4, 2018, pages 92 - 95, XP009525786, ISSN: 2288-7105 *
HAN SOL JANG, ET AL.: "API5 induces cisplatin resistance through FGFR signaling in human cancer cells", EXPERIMENTAL AND MOLECULAR MEDICINE, SEOUL, KR, vol. 49, no. 9, 1 September 2017 (2017-09-01), KR, pages 1 - 10, XP055703427, ISSN: 1226-3613, DOI: 10.1038/emm.2017.130 *

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