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WO2019117691A1 - Peptides se liant à cd44v6 et utilisation correspondante - Google Patents

Peptides se liant à cd44v6 et utilisation correspondante Download PDF

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Publication number
WO2019117691A1
WO2019117691A1 PCT/KR2018/016022 KR2018016022W WO2019117691A1 WO 2019117691 A1 WO2019117691 A1 WO 2019117691A1 KR 2018016022 W KR2018016022 W KR 2018016022W WO 2019117691 A1 WO2019117691 A1 WO 2019117691A1
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Prior art keywords
cancer
cd44v6
peptide
v6pep
cells
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PCT/KR2018/016022
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English (en)
Korean (ko)
Inventor
이병헌
칸파티마
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Industry Academic Cooperation Foundation of KNU
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Industry Academic Cooperation Foundation of KNU
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Priority claimed from KR1020180161981A external-priority patent/KR102194025B1/ko
Application filed by Industry Academic Cooperation Foundation of KNU filed Critical Industry Academic Cooperation Foundation of KNU
Priority to US16/772,810 priority Critical patent/US11192920B2/en
Priority to EP18889460.4A priority patent/EP3733685A4/fr
Publication of WO2019117691A1 publication Critical patent/WO2019117691A1/fr
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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 present invention relates to peptides that bind to CD44v6 and uses thereof for inhibiting cancer metastasis.
  • CD44 generally binds to the primary ligand hyaluronic acid (HA) and has a molecular weight in the range of 80 to 200 kDa.
  • HA primary ligand hyaluronic acid
  • the heterogeneity of the CD44 protein is due to selective splicing of the 10 variable exons and additional post-translational modifications.
  • the human CD44 gene is located on the short arm of chromosome 11p13, and all CD44 proteins are encoded on chromosome 2 in mice.
  • the CD44 chromosome consists of 20 exons, 10 of which are called CD44 variants and play a role in splicing, and "v” is abbreviated as variant and is named CD44v (1v-10v).
  • the isomer controls and participates in cell differentiation, cell migration and cellular behavior by mediating contact between the cell and the extracellular matrix, which is essential for maintaining tissue integrity. Because of these important functions, they tend to be related to pathological conditions, including tumor progression and metastasis.
  • Specific CD44 variants have been shown to be highly expressed in certain cancer metastatic stem cells.
  • CD44v6 is known to be involved in many cancers, and CD44v6-v10, which has a high frequency of mutation, has been reported to be involved in metastatic proliferation, for example, exon v6 promotes cancer cell metastasis.
  • the role of CD44v6 peptide in metastatic cancer is shown in Fig. According to previous reports, in the activation of c-Met of epithelial cells by binding of hepatocyte growth factor (HGF), HGF was found to contain mutant exon v6 in that it was dependent on CD44 exon v6 containing isomers The CD44 isomer plays a role in the transcriptional determinant.
  • HGF hepatocyte growth factor
  • CD44v6 in metastasis is known to mediate the action of HGF by interacting with c-Met receptor tyrosine kinase (RTK).
  • RTK c-Met receptor tyrosine kinase
  • the inhibitory action of the v6 peptide inhibits the cooperative receptor action between CD44v6 and c-Met to inhibit tumor cell invasion and metastasis, suggesting the possibility of developing a more stable and systematically usable compound.
  • CD44v6-specific antibodies CD44v6 siRNAs
  • CD44 variant v6-specific peptides CD44v6 isomers play a dual role in c-Met dependent signaling. According to several reports, an increase in CD44 isoforms in several human tumors is associated with a poor prognosis. Expression of CD44v6 isoform was confirmed in advanced non-Hodgkin's lymphoma and was associated with a poor prognosis. Expression of CD44v6 and CD44v8-v10 in colorectal cancer and ovarian cancer was also associated with poor prognosis, which may be considered as a precise diagnostic factor.
  • CD44v6 and CD44v7-v8 were associated with poor prognosis, and expression of CD44v6 and CD44v5 was also upregulated in gastric cancer.
  • humanized monoclonal anti-blocking antibodies have also been developed and applied to clinical trials, but the development of peptides capable of binding to and blocking CD44v6 is highly required.
  • the present invention relates to a peptide which binds to CD44v6 and its use, which comprises a peptide specifically binding to CD44v6 consisting of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, a cancer diagnostic composition comprising the peptide as an active ingredient, A pharmaceutical composition for preventing or treating cancer, a health functional food composition for cancer prevention or improvement, and a composition for drug delivery.
  • the present invention provides a peptide specifically binding to CD44v6 consisting of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.
  • the present invention also provides a polynucleotide encoding the peptide, a recombinant vector comprising the polynucleotide, and a transformant transformed with the recombinant vector.
  • the present invention also provides a composition for cancer diagnosis comprising the peptide as an active ingredient.
  • the present invention also provides a pharmaceutical composition for preventing or treating cancer comprising the peptide as an active ingredient.
  • the present invention also provides a health functional food composition for preventing or ameliorating cancer comprising the peptide as an active ingredient.
  • the present invention also provides a drug delivery composition comprising the peptide as an active ingredient.
  • the present invention relates to a peptide binding to CD44v6 and its use for inhibiting cancer metastasis using the peptide.
  • the peptide of the present invention specifically binds to CD44v6 and inhibits it, thereby inhibiting the movement and metastasis of cancer cells.
  • the peptide of the present invention is relatively stable in the serum and shows a high possibility as an anticancer therapeutic agent for inhibiting metastasis in accordance with progression and migration of cancer in the future.
  • Figure 1 shows the results of screening of CD44v6 binding peptides using phage display.
  • Figure 2 shows the CD44v6 specific cell binding results of v6Pep-1 and v6Pep-2.
  • Figure 3 shows HGF-induced phosphorylation inhibition results of c-Met and Erk by v6Pep-1 and v6Pep-2.
  • Figure 4 shows the inhibition of the internalization of HGF-induced c-Met by v6Pep-1 and v6Pep-2 and the pull-down of CD44v6.
  • Figure 5 shows the results for CD44v6 peptide targeting CD44v6-expressing human breast tumor cells in vivo.
  • e The result of analysis of the tumor tissue section by confocal microscope.
  • Figure 6 shows the results for inhibition of metastasis of the human breast cancer model by CD44v6 peptide.
  • (a) Represents a medication schedule.
  • Figure 7 shows the results for inhibition of metastasis of the human breast cancer model by CD44v6 peptide.
  • (a) and (b) are photographed whole body X-ray and bioluminescence imaging results.
  • (c) represents the lung weight on the 22nd day.
  • Figure 8 shows the results for CD44v6 peptide selectively targeting CD44v6-expressing mouse breast tumor cells in vivo.
  • Figure 9 shows the results for a CD44v6 peptide that blocks the metastasis of mouse tumor cells in vivo.
  • (c) and (d) are quantification results of whole proton fluxes in whole body and lung regions.
  • Figure 10 shows the results for a CD44v6 peptide that blocks the metastasis of mouse tumor cells in vivo.
  • (a) shows the result of tumor volume change after tumor inoculation.
  • Figure 11 shows the results for a CD44v6 peptide that blocks metastasis of mouse tumor cells in vivo.
  • Figure 12 shows the results for a CD44v6 peptide that blocks the metastasis of mouse tumor cells in vivo.
  • the primary tumor tissues were analyzed by immunofluorescence staining with phosphorylated c-Met (P-met) and c-Met antibodies.
  • P-met phosphorylated c-Met
  • c-Met antibodies phosphorylated c-Met antibodies
  • FIG. 13 is a schematic diagram showing the role of CD44v6 peptide in metastatic cancer.
  • the present inventors screened phage display peptide libraries with cells transiently overexpressing CD44v6. After multiple screenings, the selected phage clones were sequenced. Of these, two clones were found to bind most selectively to CD44v6 expressing cells compared to control clones. The peptides blocked RTK activation and inhibited tumor growth and metastatic spread in MDA-MB-231 and 4T1 breast cancer mice and also demonstrated significant effects in combination with crizotinib. As a result, the present inventors have successfully screened peptides that can specifically bind to CD44v6 and effectively inhibit them, and confirmed the possibility of the peptide as an anti-cancer therapeutic agent, thus completing the present invention.
  • the present invention provides a peptide specifically binding to CD44v6 consisting of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. Specifically, the peptides can inhibit phosphorylation of c-Met and block signal transduction between CD44v6 and c-Met.
  • the peptides of the present invention can be readily prepared by chemical synthesis known in the art (Creighton, Proteins, Structures and Molecular Principles, W. H. Freeman and Co., NY, 1983). Typical methods include liquid or solid phase synthesis, fractional condensation, F-MOC or T-BOC chemistry (see, for example, Chemical Approaches to the Synthesis of Peptides and Proteins, Williams et al., Eds., CRC Press, Boca Raton Florida A Practical Approach, Atherton & Sheppard, Eds., IRL Press, Oxford, England, 1989).
  • the peptides of the present invention can also be produced by genetic engineering methods.
  • a DNA sequence encoding the peptide is synthesized according to a conventional method.
  • DNA sequences can be synthesized by PCR amplification using appropriate primers.
  • the DNA sequence may be synthesized by standard methods known in the art, for example, using an automated DNA synthesizer (e.g., marketed by Biosearch or Applied Biosystems).
  • the constructed DNA sequence is operatively linked to the DNA sequence and contains one or more expression control sequences (e.g., promoters, enhancers, etc.) that regulate the expression of the DNA sequence , And the host cells are transformed with the recombinant expression vector formed therefrom.
  • expression control sequences e.g., promoters, enhancers, etc.
  • the resulting transformant is cultured under appropriate medium and conditions so that the DNA sequence is expressed, and the substantially pure peptide encoded by the DNA sequence is recovered from the culture.
  • the recovery can be performed using methods known in the art (e.g., chromatography).
  • substantially pure peptide herein is meant that the peptide according to the invention is substantially free of any other proteins derived from the host.
  • the peptide represented by the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 includes a functional variant thereof.
  • &Quot; Functional variant " means any similar sequence in which substitution of some amino acids occurs at amino acid positions that do not affect the properties of the peptides of the invention that specifically bind to CD44v6.
  • the present invention also provides a polynucleotide encoding said peptide.
  • the " polynucleotide " is a polymer of deoxyribonucleotides or ribonucleotides present in single-stranded or double-stranded form.
  • the polynucleotide includes a nucleotide sequence encoding the peptide as well as a sequence complementary to the nucleotide sequence.
  • the complementary sequence includes not only perfectly complementary sequences but also substantially complementary sequences.
  • polynucleotide may be modified. Such modifications include addition, deletion or non-conservative substitution or conservative substitution of nucleotides.
  • the polynucleotide encoding the amino acid sequence is also interpreted to include a nucleotide sequence that exhibits substantial identity to the nucleotide sequence. The above substantial identity is determined by aligning the nucleotide sequence with any other sequence as much as possible and analyzing the aligned sequence using algorithms commonly used in the art to obtain a sequence having at least 80% homology, At least 90% homology or at least 95% homology.
  • the present invention also provides a recombinant vector comprising the polynucleotide.
  • the present invention provides a transformant transformed with said recombinant vector.
  • &quot means a DNA molecule that is replicated by itself, which is used to carry the clone gene (or another fragment of the clone DNA).
  • " recombinant vector " means a plasmid, viral vector or other medium known in the art capable of expressing an inserted nucleic acid in a host cell.
  • the polynucleotide encoding the peptide of the invention may be operably linked.
  • the recombinant vector generally comprises a replication origin that is capable of propagating in a host cell, at least one expression control sequence (e.g., a promoter, enhancer, etc.) that regulates expression, a selectable marker, and a sequence operably linked to an expression control sequence And a polynucleotide encoding the peptide of the invention.
  • the transformant may be one which has been transformed by the recombinant vector.
  • the transformants are produced by recombinant vectors containing polynucleotides encoding the peptides of the invention by methods known in the art such as, but not limited to, transient transfection, microinjection, Transfection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection, (Wu et al., J. Org. Immunol. Immunol. Immunol. Immunol. Immun. J. Immunol. Immun. Bio. Chem., 267: 963-967, 1992; Wu and Wu, J. Bio. Chem., 263: 14621-14624, 1988).
  • the present invention also provides a composition for cancer diagnosis comprising the peptide as an active ingredient.
  • the cancer may be a cancer overexpressing CD44v6, more preferably the cancer overexpressing CD44v6 is lung cancer, brain tumor, breast cancer, liver cancer, skin cancer, esophageal cancer, testicular cancer, kidney cancer, colon cancer, rectal cancer, stomach cancer, But are not limited to, renal cancer, bladder cancer, ovarian cancer, cholangiocarcinoma, gallbladder cancer, uterine cancer, cervical cancer, prostate cancer, head and neck cancer, pancreatic cancer or squamous cell carcinoma.
  • diagnosis means identifying the presence or characteristic of a pathological condition.
  • diagnosis is to identify the presence or characteristics of the cancer.
  • the diagnosis of cancer using the peptide of the present invention can be made by detecting the binding of the peptide of the present invention to the tissue or cells directly obtained by blood, urine or biopsy.
  • the peptide of the present invention can be provided in a labeled state. That is, they may be provided by linking (e.g., covalently binding or bridging) to a detectable label.
  • the detectable label is a chromogenic enzyme (e.g., peroxidase (peroxidase), alkaline phosphatase (alkaline phosphatase)), radioactive isotopes (for example: 124 I, 125 I, 111 In, 99 mTc, 32 P, 35 S), A chromophore, a luminescent material or a fluorescent material such as FITC, RITC, rhodamine, cyanine, Texas Red, fluorescein, phycoerythrin, Quantum dots), and the like.
  • a chromogenic enzyme e.g., peroxidase (peroxidase), alkaline phosphatase (alkaline phosphatase)
  • radioactive isotopes for example: 124 I, 125 I, 111 In, 99 mTc, 32 P, 35 S
  • a chromophore e.g., a luminescent material or a fluorescent material
  • the detectable label may be an antibody epitope, a substrate, a cofactor, an inhibitor or an affinity ligand. Such labeling may be performed during the synthesis of the peptide of the present invention, or may be performed in addition to the peptide already synthesized.
  • a fluorescent substance is used as a detectable label
  • the cancer can be diagnosed by fluorescence-based tomography (FMT).
  • FMT fluorescence-based tomography
  • the peptide of the present invention labeled with a fluorescent substance can be circulated into the blood and the fluorescence by the peptide can be observed by fluorescence tomography. If fluorescence is observed, it is diagnosed as cancer.
  • the present invention also provides a pharmaceutical composition for preventing or treating cancer comprising the peptide as an active ingredient.
  • the present invention also provides a pharmaceutical composition for preventing or treating cancer, comprising a peptide and an anticancer agent as an active ingredient.
  • the anticancer agent is selected from the group consisting of crizotinib, doxorubicin, paclitaxel, vincristine, daunorubicin, vinblastine, actinomycin-D, docetaxel, etoposide, teniposide, bisantrene, homoharringtonine, Gleevec (STI-571), cisplatin, 5-fluorouracil, adriamycin, methotrexate, busulfan, But are not limited to, chlorambucil, cyclophosphamide, melphalan, nitrogen mustard, or nitrosoourea.
  • the cancer may be a cancer overexpressing CD44v6, more preferably the cancer overexpressing CD44v6 is lung cancer, brain tumor, breast cancer, liver cancer, skin cancer, esophageal cancer, testicular cancer, kidney cancer, colon cancer, rectal cancer, stomach cancer, But are not limited to, renal cancer, bladder cancer, ovarian cancer, cholangiocarcinoma, gallbladder cancer, uterine cancer, cervical cancer, prostate cancer, head and neck cancer, pancreatic cancer or squamous cell carcinoma.
  • the pharmaceutical composition can inhibit cancer metastasis.
  • the pharmaceutical composition of the present invention may be prepared by using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the active ingredients.
  • the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, Or a solubilizing agent such as a flavoring agent can be used.
  • the pharmaceutical composition of the present invention may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the active ingredient for administration.
  • Acceptable pharmaceutical carriers for compositions that are formulated into a liquid solution include sterile water and sterile water suitable for the living body such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
  • compositions of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, suspending agents or injectable solutions or suspensions .
  • the pharmaceutical compositions of the present invention may be formulated and administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, percutaneous, intranasal, inhalation, topical, rectal, ≪ / RTI >
  • the effective amount of the active ingredient of the pharmaceutical composition of the present invention means the amount required for prevention or treatment of the disease.
  • the present invention is not limited to the particular type of the disease, the severity of the disease, the kind and amount of the active ingredient and other ingredients contained in the composition, the type of formulation and the patient's age, body weight, general health status, sex and diet, Rate of administration, duration of treatment, concurrent medication, and the like.
  • the composition of the present invention can be administered in an amount of 0.1 ng / kg to 10 g / kg once a day to several times a day in the case of an adult.
  • the present invention also provides a health functional food composition for preventing or ameliorating cancer comprising the peptide as an active ingredient.
  • the health functional food composition of the present invention may be provided in the form of powder, granules, tablets, capsules, syrups or beverages.
  • the health functional food composition may be used in combination with other food or food additives in addition to the active ingredient, Can be suitably used.
  • the amount of the active ingredient to be mixed can be suitably determined according to its use purpose, for example, prevention, health or therapeutic treatment.
  • the effective dose of the active ingredient contained in the health functional food composition may be used in accordance with the effective dose of the pharmaceutical composition.
  • the active ingredient can be used in an amount exceeding the above range since there is no problem in terms of safety.
  • the type of the health food includes meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, Drinks, alcoholic beverages and vitamin complexes.
  • the present invention also provides a drug delivery composition comprising the peptide as an active ingredient.
  • the peptide according to the present invention can be used as an intelligent drug delivery vehicle for selectively delivering a drug to cancer tissues.
  • the peptide of the present invention When used in combination with a conventionally known drug to treat cancer, the peptide of the present invention selectively transmits the drug only to cancer tissues and cancer cells, thereby increasing the efficacy of the drug, Can significantly reduce the side effects.
  • the above drug may be used as an anticancer agent, and any anticancer agent that can be linked to the peptide of the present invention is not limited as long as it is conventionally used for the treatment of cancer.
  • any anticancer agent that can be linked to the peptide of the present invention is not limited as long as it is conventionally used for the treatment of cancer.
  • the linkage between the anticancer agent and the peptide of the present invention can be carried out by a method known in the art, for example, through covalent bonding, crosslinking and the like.
  • the peptide of the present invention can be chemically modified to the extent that its activity is not lost if necessary.
  • Human embryonic kidney cells HEK293 (ATCC), human breast cancer cells MDA-MB231 (ATCC), human prostate cancer cell PANC-1 (KCLB) and human cervical cancer cell HELA (ATCC) were cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) .
  • DMEM Dulbecco's modified Eagle's medium
  • MDA-MB-231-Red-Fluc-GFP (Bioware) and 4T1-Red-Fluc-GFP (Bioware) were cultured in DMEM supplemented with 10% fetal calf serum (FCS, PAA Laboratories).
  • MCF-7 was cultured in RMPI (Invitrogen) supplemented with 10% FCS.
  • Cells were cultured in a humidified incubator (85%) at 5% CO 2 , 37 ° C, under sterile conditions. All experiments were performed on a sterile clean bench. At 80% confluency, the adherent cells were passaged.
  • VFF-18 Mouse monoclonal CD44v6 (VFF-18) was purchased from Abcam, rabbit monoclonal antibody (AB51037) was purchased from Abcam, and the human monoclonal antibody to CD44 (VFF-7) was purchased from Santa Cruz Human monoclonal antibody (sc-7297) was purchased from Santa Cruz. Antibodies against Erk 1 (K-23) were purchased from Santa Cruz, and phospho-Erk phospho-p44 / 42 and phospho-c-Met (D26) ). Recombinant human HGF (R & D Systems, Wiesbaden, Germany) was activated overnight with 5% FCS.
  • a sequence encoding human CD44V6 was introduced using the PCR subcloning method.
  • the forward primer was GGACTTTCCAAAATGTCG and the reverse primer was ATTAGGACAAGGCTGGTGGG.
  • the expression vector was transformed with E. coli DH5 ⁇ and the DNA was isolated using a Mini-Prep Kit (Dokdo-Preparation) to obtain a high concentration of DNA for transient transfection.
  • HEK293 cells were transiently transfected in a 6-well plate with Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Transformants were inoculated 24 hours prior to infection, 2 ⁇ 10 5 cell density of cells in 6-well plates. To transfect a well, 10 ⁇ l Lipofectamine 2000 reagent was diluted in 250 ⁇ l DMEM and reacted at room temperature for 5 minutes. Without serum, 4 ⁇ g of vector DNA was filled with 250 ⁇ l DMEM and mixed with Lipofectamine 2000 solution. The mixture was allowed to react at room temperature for 20 minutes (500 ⁇ l total per well). The old media of cells was immediately removed and replaced with fresh serum-free 1.5 ml DMEM medium.
  • Lipofectamine 2000 Invitrogen
  • DNA-transfection reagent-mixture was added to the cells. After 6 hours of incubation, the medium was replaced with a pre-warmed growth medium containing serum. Before starting the experiment, the newly inserted protein was expressed in the cells.
  • CD44v6 Peptides The bio-activity of the T7 hydrophobic library for screening Panning (Bio-panning)
  • Phage peptide libraries based on the T7 415-1b phage vector designated CX7C were designed according to the manufacturer's instructions (Novagen, Madison, WI).
  • the phage library has approximately 1 x 10 9 plaque-forming units (pfu). That is, transfected HEK-293 cells (GFP) were dispensed into 35 mm dishes and cultured confluently at 60-70%. For transient transfection of HEK-293 cells, cells were treated with Lipofectamine 2000 (Invitrogen). Prior to the start of the experiment, the cells were allowed to express a new insertion protein (GFP) expressing CD44v6 for 24-48 hours.
  • GFP transfected HEK-293 cells
  • the phage library of 1 x 10 9 plaque-forming units (pfu) was cultured in transfected HEK 293 cells for 1 hour at 4 ° C and the phages bound to the cells were incubated with 500 ⁇ L of BL21 bacteria (OD: 1 ) Culture medium. The eluate was used for titration and the remaining eluted phage were cultured in non-transfected cells for 30 min at 4 < 0 > C.
  • the unbound phages were then washed with DMEM containing 10 mg / ml bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • the phages bound to the cells were eluted with 500 ⁇ L of BL21 bacterial (OD: 1) culture medium at room temperature for 10 minutes.
  • the eluate was used for titration and the remaining eluted phage clone was dissolved in 10 ml of LB medium for the next cycle of amplification and the procedure was repeated 5 times.
  • the stepwise dilutions of the eluates were inoculated with E. coli cultured in LB medium for 2 hours at 37 ° C, and the number of colonies was counted by inverting the phage.
  • phages were first cultured and transfected into transfected cells.
  • a direct screening method was used to use all the phage clones in the next round as an amplification and phage enrichment method.
  • the drainage reduction of phage clones decreased by 1.9 ⁇ 10 1 fold in each of the 5 rounds.
  • DNA and amino acid sequence analysis of the DNA inserts of each of the 70 phage clones collected in the above was carried out by an automated DNA sequencer (Genotech Inc., Daegeon, Korea) using each primer (Macrogen). Amino acid sequences deduced from nucleotide sequences were aligned using the Clustal W program to find amino acid motifs shared between consensus sequences or peptides. Some of these peptides were randomly selected and BLAST searches were performed on the NCBI protein database to investigate proteins with high homology to each peptide sequence.
  • Serum-deficient (24 h) cells were incubated with growth factor HGF (25-50 ng / mL) for 10 min at 37 ° C. If indicated, cells were treated with CD44v6 specific peptide or control peptide (50 ⁇ g / ml) for 10 min at 37 ° C before induction, and the cells were washed with ice-cold PBS.
  • biotinylated CD44v6 peptide was incubated with monomeric avidin magnetic beads (Bioclone Inc.) for 30-60 minutes at room temperature with gentle rotation for peptide pull down prior to incubation with the cell lysate.
  • Cells were lysed using a cell lysis reagent (Thermo scientific) containing protease inhibitor. After the initial incubation of the biotinylated peptide + avidin beads (complex 1), the cells were washed with PBS and the complexes were incubated with the cell lysate for 30-60 minutes with gentle rotation at room temperature.
  • biotinylated peptide + avidin bead + cell lysate (complex 2) was eluted with 1X blocking buffer / elution buffer for 5 to 10 minutes after elution and eluted with CD44v6 antibody (Millipore) for pull- Western blot analysis was performed.
  • the MDA-MB-231 at a concentration of 2.5 ⁇ 10 5 cells per well was dispensed in a 12-well plate. After 24 hours, a scratch was made on the confluent cell layer using a sterile pipette tip. The medium was replaced with fresh medium containing HGF and HGF at 25 ng / ml was treated at 37 ° C for 10 minutes to induce growth factor. After the induction of HGF, 5 ⁇ g / ml CD44v6 peptide (each) and a combination of two peptides (v6Pep-1 + v6Pep-2) or 5 ⁇ g / ml of control peptide were treated at 37 ° C. for 10 minutes, , 48 hours and 60 hours, respectively (original magnification, ⁇ 100). The computer program ImageJ was used for quantitative evaluation and quantified the area covered by the cells in the scratch.
  • v6-1 5'-AGU AGU ACA ACG GAA ATT-39; v62: 59-GGA UAU CGC CAAACA CCC ATT-3 'or pool of non-specific control siRNA.
  • the control siRNA 50-CUACGCCAAUUUCGU (dTdT) 30) and glyceraldehyde-3 phosphate dehydrogenase (GAPDH) siRNA (50-UGUGAACCAUGAGAAGUA (dTdT) -30) were purchased from Bioneer. Transfection was performed using Lipofectamine 2000 (Invitrogen, Düsseldorf, Germany). Two transfections were performed at 24 hour intervals. Cells were cultured for 24 hours in a serum-deficient state for 48 hours after the first transfection and then further treated.
  • mice 6- to 8-week-old Balb / c female mice were purchased from Orient Bio. Under the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Kyungpook National University, mice were kept and maintained.
  • IACUC Institutional Animal Care and Use Committee
  • the MDA-MB-231 cells (1 ⁇ 10 5 cells) was dispensed on the slide 4-well chamber. After the complete adhesion, the cell culture medium was changed to a fresh medium containing FITC-labeled peptide (10 ⁇ M) and incubated at 37 ° C. for 1 hour. The cells were washed with PBS for 3 to 5 minutes three times and then washed with CD44v6 antibody cruz), followed by Alexa 594-labeled goat anti-mouse IgG secondary antibody.
  • the nuclei were stained with DAPI and mounted on glass slides with a fading reagent, followed by observation of v6Pep-1 and v6Pep-2 accumulation labeled with FITC under confocal microscopy. Localization of FITC-labeled peptides in cells was observed under a confocal microscope (Zeiss, Jena, Germany).
  • In vivo NIRF imaging was performed using an IVIS Lumina III Imaging System (Perkin Elmer, Waltham, MA) under inhalation anesthesia.
  • IVIS Lumina III Imaging System Perkin Elmer, Waltham, MA
  • in vivo fluorescence images were taken at various time points (1, 2, 4 and 4 respectively) before and after injection. Mice were sacrificed after in vivo imaging and tumors and control organs were separated for further imaging using the IVIS Lumina III imaging system.
  • mice were euthanized 6 hours after CO 2 infusion. All major organs (liver, kidney, spleen, heart and lungs) were isolated with tumor tissue, washed with PBS and ex vivo fluorescence images were taken using IVIS Lumina III imaging system. The fluorescence intensities in the ROI of each organ were analyzed. Tumor tissues were fixed overnight with 4% paraformaldehyde and rapidly frozen. Tissue sections (8 ⁇ m thick) were prepared with cryo-microtome, stained with CD44v6 antibody (Santa Cruz) and stained with goat anti-mouse IgG secondary antibody labeled with Alexa 594. Control peptides and v6Pep-1 and v6Pep-2 tumor accumulation were observed by confocal microscopy after the nuclei were stained with DAPI.
  • MDA-MB-231 cells (5x10 3 cells / well in 96-well plate) were incubated with v6Pep-1, v6Pep-2 and v6Pep- Gt; 37 C < / RTI > for 4 hours. After replacing the serum-free medium with the culture medium containing 10% FBS, the cells were cultured for 24 hours and 48 hours and evaluated for cytotoxicity using CCK-8 assay (Dojindo, Kumamoto, Japan).
  • the cultured samples were diluted 100-fold and subjected to C18 reverse phase FPLC with a linear gradient of acetonitrile (Vydac protein and peptide C18, 0.1% trifluoroacetate in water for equilibrium and 0.1% trifluoroacetate in acetonitrile for equilibration) ≪ / RTI > To confirm the identity of the peaks from the profile of the C18 reversed phase FPLC, each peak was collected, vacuum dried, and analyzed by mass spectrometry (MS) using a MALDI-TOF mass spectrometer.
  • MS mass spectrometry
  • mice blood was collected and hematological factors were analyzed in DGMIF (Daegu-Gyeongbuk Medical Innovation Foundation) (Daegu, Korea).
  • DGMIF Daegu-Gyeongbuk Medical Innovation Foundation
  • HEK-293 cells transiently transfected with the CD44v6-GFP plasmid.
  • HEK-293 transfected cells were stained with CD44 variant 6 antibody (Fig. 1a). After confirmation by immunofluorescence analysis, transient transfection effects were also confirmed by western blotting at different time intervals of 24 h and 48 h, confirming transfection at each time point as compared to non-transfected HEK-293 cells .
  • the inventors In order to screen the CD44v6 specific peptides by direct screening methods, the inventors have confirmed the aggregate titer of all the phage clones in each round, which is significantly increased compared to the untransfected titres, 3.11 x 10 & lt ; 1 > times (Fig. 1B). In the 3rd, 4th and 5th rounds, 20 phage clones were randomly selected and the peptide-coding DNA inserts of phage clones were sequenced. Phage clones binding to CD44v6 expressing cells with high specificity were selected by phage binding ELISA, and several clones were found to bind in several expression and non-expression cell lines.
  • phage clones Compared with other phage clones, high levels of binding of clone-1 and clone-2 to CD44v6 were confirmed by phage ELISA and phage immunofluorescence staining, confirming the binding specificity and effect of phage clones.
  • Two phage clones showed high binding specificity in CD44v6 expressing cells.
  • the peptide sequence of the two clones is as follows. CNLNTIDTC (v6Pep-1, SEQ ID NO: 1), CNEWQLKSC (v6Pep-2, SEQ ID NO: 2).
  • Example 2 > CD44v6-specific cell binding of v6Pep-1 and v6Pep-2
  • MDA-MB-231 cells were cultured for 24 hours under the condition of serum deficiency and then applied to the experiment.
  • siRNAs v6-1 and v6-2 were transfected and maintained at different time intervals.
  • whole cell extracts were fractionated by SDS-PAGE, transferred to membranes, treated with 1 ⁇ g / ml of CD44v6 and CD44 antibody, and reacted overnight at 4 ° C.
  • Western blot analysis to confirm siRNA inhibition showed that CD44v6 specific siRNA inhibition was evident at 24 h, 48 h and 72 h, while wild-type CD44 was unchanged (Fig. 2a).
  • the CD44v6 isoform acts as a co-receptor for c-Met.
  • c-Met activation and signal transduction can be blocked by CD44v6 antibodies and peptides.
  • CD44v6 peptides v6Pep-1 and v6Pep-2
  • v6Pep1 + 2 the combination of two peptides
  • MDA-MB-231 and 4T1 cells were cultured under serum deprivation conditions (24 hours) and induced with growth factor HGF (25-50 ng / mL) for 10 minutes at 37 ° C. Prior to induction, cells were treated with CD44v6 specific peptide or control peptide (100 ng / ml) for 10 min at 37 < 0 > C. To detect activated Erk and activated c-Met, cells were lysed and applied to Western blot analysis using antibodies against phosphorylated Erk and phosphorylated c-Met.
  • CD44v6 and c-Met in order to determine the co-receptor function of CD44v6 and c-Met, it was dispensed on a 12-well plate for MDA-MB-231 at a concentration of 2.5 ⁇ 10 5 cells per well. After 24 hours, a scratch was made on the confluent cell layer using a sterile pipette tip. The medium was replaced with fresh medium containing HGF and HGF at 25 ng / ml was treated at 37 ° C for 10 minutes to induce growth factor.
  • the inventors have verified the internalization of the FITC-labeled peptide.
  • the internalization of the peptides was confirmed by confocal microscopy after reacting two peptides in MDA-MB-231 cells at 37 DEG C for 1 hour.
  • FITC-v6Pep-1 and FITC-v6Pep-2 were internalized into the cells in the range of 1-10 mu m and both were located in the cytoplasm.
  • CD44v6 peptide has high specificity with CD44v6 and its co-receptor c-MET.
  • MDA-MB-231 and 4T1 cells were incubated with different concentrations of v6Pep-1, v6Pep-2 and a combination of the two peptides (v6Pep-1 + v6Pep-2) for 24 hours to confirm the cytotoxicity of the CD44v6 peptide .
  • the combination of v6Pep-1, v6Ppep-2 and the two peptides did not affect cell viability.
  • MDA-MB-231 cells were xenografted with immunodeficient female nude mice to produce animal models.
  • MDA-MB231 cell suspension (5 x 106 cells) was subcutaneously injected with PBS into the right side of 5-week-old BALB / c female mice to prepare a tumor xenograft mouse.
  • In vivo fluorescence images were taken before or after injection at multiple time points (1, 2, 4, and 6 h, respectively) and flamma 675 was injected at the same concentration and method.
  • NIRF image signals were scanned and acquired using an IVIS imaging system (Caliper Life Sciences, Massachusetts).
  • IVIS imaging system Caliper Life Sciences, Massachusetts.
  • In vivo image analysis showed that v6Pep-1 and v6Pep-2 in MDA-MB-231 xenografted mice were located in tumor tissues at different time intervals and lasted for more than 6 h (FIGS. 5A and 5C).
  • the control peptides showed high non-specific tissue localization, and no accumulation could be confirmed at the tumor location.
  • v6Pep-1 and v6Pep-2 showed better tumor accumulation than the control peptides.
  • the fluorescence intensity of the target tumors showed a high accumulation in the v6Pep-1 and v6Pep-2 injected mice compared to the control peptides (Fig. 5b and Fig. 5d).
  • Control peptide-injected mice showed high accumulation in the liver and kidney compared to v6Pep-1 and v6Pep-2, and negligible levels in the lungs and spleen.
  • immunohistochemical analysis of the tissues revealed that v6Pep-1 and v6Pep-2 were co-located with CD44v6 in the tumor tissue, which was detected by staining with anti-CD44v6 antibody.
  • v6Pep-1 and v6Ppe-2 confirmed the antitumor effect of v6Pep-1 and v6Ppe-2 in combination with the c-Met inhibitor compound crizotinib in the MDA-MB-231 tumor model.
  • CD44v6 peptides are stable in the serum without degradation for more than 24 hours.
  • Systemic administration of v6Pep-1 + crizotinib and v6Pep-2 + crizotinib significantly inhibited metastasis compared to saline and control peptide treated groups, while v6Pep-1, v6Pep-2 and Cre- Each single administration of crizotinib also inhibited metastatic growth (Figure 6b).
  • tumor cells were xenografted with immunodeficient female nude mice to produce animal models.
  • 4T1 cell suspension (5 x 106 cells) was transplanted subcutaneously with PBS into breast fat pads of 5-week old BALB / c wild-type female mice to prepare tumor xenograft mice.
  • In vivo fluorescence images were taken before or after injection at multiple time points (1, 2, 4, and 6 h, respectively) and flamma 675 was injected at the same concentration and method.
  • NIRF image signals were scanned and acquired using an IVIS imaging system (Caliper Life Sciences, Massachusetts).
  • IVIS imaging system Caliper Life Sciences, Massachusetts.
  • v6Pep-1 and v6Pep-2 in 4T1 orthotopic mice were located in tumor tissues at different time intervals and lasted for more than 6h (Figs. 8A and 8C).
  • the control peptides showed high non-specific tissue localization, and no accumulation could be confirmed at the tumor location.
  • v6Pep-1 and v6Pep-2 showed better tumor accumulation than the control peptides. This clearly demonstrates that improved pharmacokinetic properties may not be an important factor in determining the tumor targeting activity of the peptides.
  • Control peptide-injected mice showed high accumulation in the liver and kidney compared to v6Pep-1 and v6Pep-2, and negligible levels in the lungs and spleen.
  • immunohistochemical analysis of the tissues revealed that v6Pep-1 and v6Pep-2 were co-located with CD44v6 in the tumor tissue, which was detected by staining with anti-CD44v6 antibody. Consistent with in vivo and ex vivo results, immunohistochemical analysis of tumor tissues indicated that v6Pep-1 and v6Pep-2 versus control peptides were associated with CD44v6 overexpressing tumor tissue (Fig. 8e).
  • the present inventors have confirmed the antitumor effect of v6Pep-1 and v6Ppe-2 in combination with crizotinib in the 4T1 tumor model.
  • Systemic administration of v6Pep-1 + crizotinib and v6Pep-2 + crizotinib significantly inhibited metastasis compared to the saline-treated group, and v6Pep-1, v6Pep-2 and crizotinib crizotinib alone inhibited tumor and metastatic growth slightly (Figure 9b).
  • Figure 9b As a result of quantification of total proton flux (proton number / second, p / s) in whole body and lung regions, flux was significantly reduced in the treated group (FIG.
  • In vivo antitumor activity was also measured in the 4T1 transgenic mouse model.
  • In vivo treatment similar to biochemical distribution and pharmacokinetic results, was performed by intravenously injecting 14.2 mg / kg of CD44v6 peptide three times a week and oral administration of crizotinib 25 mg / kg three times per week .
  • Intravenous administration of v6Pep-1 + crizotinib and v6Pep-2 + crizotinib in 4T1 mice significantly inhibited tumor growth, but a slight decrease in tumor growth was detected in the single treatment group (Fig. 10A).
  • the tumors grew more aggressively in the treated group with PBS and control peptide, and reached a size of 1,409 mm 3 after 28 days.
  • tumor volume has decreased slightly in CD44v6 peptide or crizotinib alone treated mice.
  • the weight of the resected tumor at the end of treatment was significantly reduced compared to the control (Fig. 10d).
  • inhibited metastatic growth in the PBS (saline) or single treatment group versus treated group showed negligible metastatic mass or micro mass (FIG. 10c).
  • C-Met activation in tumors using phosphorylated-c-Met staining was shown to be completely inhibited by the combination of CD44v6 peptide and crizotinib, and was partially inhibited in the single treatment group (Fig. 12A). Furthermore, caspase 3 detected more apoptotic cells in the tumor tissues of v6Pep-1 + crizotinib and v6Pep-2 + crizotinib treated group, but not the control group (Fig. 12b). These results suggest that v6Pep-1 + crizotinib and v6Pep-2 + crizotinib, which are specific for tumor location, penetrate various biological barriers and promote intracellular entry, followed by induction of large cell death .

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Abstract

La présente invention concerne des peptides se liant à CD44V6 et une utilisation pour inhiber la formation de métastases cancéreuses l'utilisant. Les peptides de la présente invention se lient spécifiquement à CD44v6 et l'inhibent et présentent ainsi l'effet d'inhibition de la migration et de la formation de métastases de cellules cancéreuses. Les peptides de la présente invention ont été obtenus par sélection de deux types de peptides (v6Pep-1 et v6Pep-2) qui se lient bien à des cellules dans lesquelles des protéines CD44v6 humaines sont hautement exprimées à l'aide de la technologie d'affichage de peptide phage et il a été découvert que les peptides interfèrent avec la liaison entre c-Met et CD44v6 et inhibent la migration de cellules cancéreuses. Les peptides de la présente invention sont relativement stables dans un sérum et présentent une possibilité élevée en tant qu'agent thérapeutique anticancéreux pour inhiber la formation de métastases provoquée par l'évolution et la migration d'un cancer dans le futur.
PCT/KR2018/016022 2017-12-15 2018-12-17 Peptides se liant à cd44v6 et utilisation correspondante Ceased WO2019117691A1 (fr)

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CN112266409B (zh) * 2020-10-28 2022-05-13 南开大学 依托泊苷自组装纳米纤维多肽、制备方法及应用

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