WO2020147508A1 - Polypeptide pour l'inhibition de métastases tumorales et de tumeurs osseuses, et son utilisation - Google Patents

Polypeptide pour l'inhibition de métastases tumorales et de tumeurs osseuses, et son utilisation Download PDF

Info

Publication number
WO2020147508A1
WO2020147508A1 PCT/CN2019/126726 CN2019126726W WO2020147508A1 WO 2020147508 A1 WO2020147508 A1 WO 2020147508A1 CN 2019126726 W CN2019126726 W CN 2019126726W WO 2020147508 A1 WO2020147508 A1 WO 2020147508A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
pharmaceutically acceptable
none
acceptable salt
bone
Prior art date
Application number
PCT/CN2019/126726
Other languages
English (en)
Chinese (zh)
Inventor
胡国宏
连丞
彭方理
李晓逊
Original Assignee
中国科学院上海营养与健康研究所
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国科学院上海营养与健康研究所 filed Critical 中国科学院上海营养与健康研究所
Publication of WO2020147508A1 publication Critical patent/WO2020147508A1/fr

Links

Images

Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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
    • C07K2319/00—Fusion polypeptide
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/20—Screening for compounds of potential therapeutic value cell-free systems

Definitions

  • the invention relates to the field of biomedicine, in particular to a polypeptide for inhibiting tumor metastasis and bone tumors and its application.
  • the main clinical drugs used to treat bone metastasis are bisphosphonates, neutralizing antibodies and small molecule inhibitors, all of which act to achieve therapeutic effects by inhibiting the differentiation and maturation of osteoclasts.
  • bisphosphonates can reduce bone pain, the duration of treatment is not clear and there is no evidence to prove that it is helpful to extend the life of patients.
  • Neutralizing antibody drugs include Denosumab, which inhibits RANKL.
  • Neutralizing antibodies and small molecule inhibitors are difficult to popularize because of their high prices. Therefore, the development of low-cost, safe, and high-efficiency drugs is of great significance.
  • Giant cell tumors of bone account for about 6% of all primary bone tumors. The age of onset is 20-40 years old. The incidence of women is higher than that of men. At the same time, the incidence of Asian countries is higher than that of European and American countries.
  • Giant cell tumor of bone originates from the mesenchymal tissue in the bone marrow and is mainly composed of three cell types: spindle-shaped stromal cells, monocytes and multinucleated giant cells. Multinucleated giant cells have many properties similar to osteoclasts and are considered to be the main effector cells responsible for osteolysis. Therefore, giant cell tumor of bone is a kind of osteolytic tumor, which has similar osteolytic phenomena and symptoms caused by osteoclasts to the osteolytic metastases of breast cancer and other tumors. Similar to bone metastases, the current treatments for giant cell tumor of bone are mainly bisphosphonates and denosumab.
  • the purpose of the present invention is to provide a safe and effective polypeptide medicine.
  • an isolated polypeptide or a pharmaceutically acceptable salt thereof characterized in that the polypeptide or a pharmaceutically acceptable salt thereof has the structure shown in Formula I:
  • X1 is none or any peptide
  • X2 is none or any peptide
  • the length of the polypeptide or a pharmaceutically acceptable salt thereof is ⁇ 100aa, preferably ⁇ 70aa, more preferably ⁇ 50aa, more preferably, ⁇ 40aa, more preferably, ⁇ 30aa, more preferably, ⁇ 20aa ; More preferably, it is 5aa, 6aa, 7aa, 8aa, 9aa, 10aa, 11aa, 12aa, 13aa, 14aa, 15aa, 16aa, 17aa, 18aa, 19aa, or 20aa.
  • polypeptide or a pharmaceutically acceptable salt thereof has (a) inhibiting tumor metastasis; and/or (b) inhibiting bone tumor; and/or (c) inhibiting osteoclast differentiation and maturation activity.
  • the tumor is selected from the group consisting of breast cancer, lung cancer, stomach cancer, liver cancer, colon cancer, multiple myeloma, kidney cancer, pancreatic cancer, melanoma, lymphoma, thyroid cancer, or a combination thereof .
  • the tumor metastasis is selected from the group consisting of breast cancer bone metastasis, lung cancer bone metastasis, gastric cancer bone metastasis, liver cancer bone metastasis, colon cancer bone metastasis, multiple myeloma bone metastasis, kidney cancer bone metastasis, Pancreatic cancer bone metastasis, melanoma bone metastasis, lymphoma bone metastasis, thyroid cancer bone metastasis, or a combination thereof.
  • the bone tumor is selected from the following group: osteoclast bone tumor, giant cell tumor of bone, aneurysmal bone cyst, bone fibrous dysplasia, or a combination thereof.
  • the peptide fragment includes a tag protein.
  • the length of X1 is 1-80aa, preferably, 1-30aa, more preferably, 1-20aa, more preferably, 1-10aa.
  • the length of X2 is 1-65aa, preferably, 1-30aa, more preferably, 1-20aa, and more preferably, 1-10aa.
  • the X1 or X2 includes natural or unnatural amino acids.
  • the polypeptide has a cell penetrating element.
  • the length of the cell penetrating element is 4-20 amino acids, preferably 5-15 amino acids.
  • a cyclic peptide is formed between X1 and X2.
  • At least one pair of disulfide bonds are optionally formed between X1 and X2.
  • polypeptide is an N-mer.
  • N-mer has the following structure of formula II:
  • X1 and X2 are defined as described above; L1 is no or connecting peptide; n is 1-10, preferably, 1-7, more preferably, 1-5; each "-" is independently a connecting peptide or Peptide bond.
  • the length of L1 is 1-30aa, preferably, 1-20aa, and more preferably, 1-10aa.
  • polypeptide or a pharmaceutically acceptable salt thereof has the structure of Formula III:
  • X1a is none or D
  • X2a is none or T or I
  • X3a is none or H or K or T;
  • X4a is none or I or V
  • X5a is none or I or L
  • X6a is none or K or D or R;
  • X7a is none or A
  • X8a is none or Q or K or H
  • X9a is none or S or Y or C
  • X1b is none or I
  • X2b is none or K
  • X3b is none or Y
  • X4b is none or F or Y;
  • X5b is none or L or M
  • X6b is none or T
  • the length of the polypeptide or a pharmaceutically acceptable salt thereof is ⁇ 100aa, preferably ⁇ 70aa, more preferably ⁇ 50aa, more preferably, ⁇ 40aa, more preferably, ⁇ 30aa, more preferably, ⁇ 20aa ; More preferably, it is 5aa, 6aa, 7aa, 8aa, 9aa, 10aa, 11aa, 12aa, 13aa, 14aa, 15aa, 16aa, 17aa, 18aa, 19aa, or 20aa.
  • polypeptide or a pharmaceutically acceptable salt thereof has (a) inhibiting tumor metastasis; and/or (b) inhibiting bone tumor; and/or (c) inhibiting osteoclast differentiation and maturation activity.
  • sequence of the polypeptide is shown in SEQ ID NO.: 1-8.
  • polypeptide of Formula I or Formula III has ⁇ 50%, ⁇ 60%, ⁇ 70%, ⁇ 80% compared with the polypeptide of SEQ ID NO.: 1-8, ⁇ 90% identity (or homology).
  • the polypeptide represented by formula I or formula III retains at least ⁇ 50%, ⁇ 60%, ⁇ 70%, ⁇ 80 of the biological activity of the polypeptide shown in SEQ ID No.: 1-8 %, ⁇ 90%, ⁇ 100%, such as 80-500%, preferably 100-400%.
  • the biological activity refers to (a) inhibiting tumor metastasis; and/or (b) inhibiting bone tumors; and/or (c) inhibiting osteoclast differentiation and maturation activity.
  • polypeptide is artificially synthesized.
  • polypeptide is not the polypeptide shown in SEQ ID NO.: 1-8.
  • polypeptide is selected from the following group:
  • amino acid sequence shown in SEQ ID NO:1-8 is formed by the substitution, deletion or addition of 1-5 (preferably 1-3, more preferably 1-2) amino acid residues, and A polypeptide derived from (a) having the activity of (a) inhibiting tumor metastasis; and/or (b) inhibiting bone tumors; and/or (c) inhibiting osteoclast differentiation and maturation.
  • polypeptide is a polypeptide shown in SEQ ID NO.: 1-8 through 1-3, preferably 1-2, more preferably 1 amino acid substitution or deletion; and/ or
  • It is formed by adding 1-5, preferably 1-4, more preferably 1-3, and most preferably 1-2 amino acids.
  • the length of the polypeptide is 5-150 amino acids, preferably, 5-100 aa, more preferably, 5-90, more preferably, 5-40, more preferably, 5-25, more preferably, 5-20, more preferably, 5-15, more preferably, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.
  • the derivative polypeptide retains ⁇ 50%, ⁇ 60%, ⁇ 70%, ⁇ 80%, ⁇ 90%, ⁇ 100%, such as 80-500%, preferably 100-400 % SEQ ID NO: 1-8 (a) inhibit tumor metastasis; and/or (b) inhibit bone tumor; and/or (c) inhibit osteoclast differentiation and maturation activity.
  • the identity of the derivative polypeptide with SEQ ID NO: 1-8 is ⁇ 50%, preferably, ⁇ 60%, more preferably, ⁇ 70%, more preferably, ⁇ 80% , More preferably, ⁇ 90%.
  • the present invention also provides (a) inhibition of tumor metastasis; and/or (b) inhibition of bone tumors; and/or (c) inhibition of osteoclast differentiation and maturation activity, dimers and multimers of polypeptides represented by formula I or formula III
  • the dimer and multimeric forms have (a) inhibiting tumor metastasis; and/or (b) inhibiting bone tumors; and/or (c) inhibiting osteoclast differentiation and maturation activity.
  • a fusion protein including:
  • the peptide segment includes a carrier protein.
  • the carrier protein is selected from the following group: Fc fragment, human serum albumin (HSA), CTP, transferrin, or a combination thereof.
  • the peptide segment is modified.
  • the modification includes polyethylene glycol (PEG) modification.
  • PEG polyethylene glycol
  • an isolated nucleic acid which encodes the polypeptide of the first aspect of the present invention or a pharmaceutically acceptable salt thereof.
  • a pharmaceutical composition including:
  • the polypeptide retains ⁇ 70%, 75%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, or 200 % SEQ ID NO: 1-8 (a) inhibit tumor metastasis; and/or (b) inhibit bone tumor; and/or (c) inhibit osteoclast differentiation and maturation activity.
  • the drug is administered by a mode selected from the group consisting of intravenous, intratumor, intracavity, subcutaneous or hepatic artery administration (such as injection, drip, etc.).
  • the pharmaceutical preparation is selected from the group consisting of tablets, capsules, injections, granules, sprays, and freeze-dried agents.
  • the pharmaceutical preparation is an injection.
  • the polypeptide is administered to the mammal at a dose of 0.01-100 mg/kg body weight (each time or daily).
  • the polypeptide according to the first aspect of the present invention or a pharmaceutically acceptable salt thereof for preparing a composition or preparation is used in (a) Inhibit tumor metastasis; and/or (b) inhibit bone tumor; and/or (c) inhibit osteoclast differentiation and maturation.
  • the composition includes a pharmaceutical composition.
  • the tumor is selected from the group consisting of breast cancer, lung cancer, stomach cancer, liver cancer, colon cancer, multiple myeloma, kidney cancer, pancreatic cancer, melanoma, lymphoma, thyroid cancer, or a combination thereof .
  • the tumor metastasis is selected from the group consisting of breast cancer bone metastasis, lung cancer bone metastasis, gastric cancer bone metastasis, liver cancer bone metastasis, colon cancer bone metastasis, multiple myeloma bone metastasis, kidney cancer bone metastasis, Pancreatic cancer bone metastasis, melanoma bone metastasis, lymphoma bone metastasis, thyroid cancer bone metastasis, or a combination thereof.
  • the bone tumor is selected from the following group: osteoclast bone tumor, giant cell tumor of bone, aneurysmal bone cyst, bone fibrous dysplasia, or a combination thereof.
  • a method for screening (a) inhibiting tumor metastasis; and/or (b) inhibiting bone tumors; and/or (c) inhibiting the differentiation and maturation activity of osteoclasts, comprising the steps :
  • test substance if the test substance binds to the CTSB protein, it indicates that the test substance that binds to the CTSB protein is a candidate substance.
  • the method further includes step (b): administering the candidate substance determined in step (a) to a non-human mammal, and determining its (a) tumor metastasis to the non-human mammal; and /Or (b) bone tumor; and/or (c) inhibition or treatment of osteoclast differentiation and maturation.
  • the tumor metastasis is selected from the group consisting of breast cancer bone metastasis, lung cancer bone metastasis, gastric cancer bone metastasis, liver cancer bone metastasis, colon cancer bone metastasis, multiple myeloma bone metastasis, kidney cancer bone metastasis, Pancreatic cancer bone metastasis, melanoma bone metastasis, lymphoma bone metastasis, thyroid cancer bone metastasis, or a combination thereof.
  • the bone tumor is selected from the following group: osteoclast bone tumor, giant cell tumor of bone, aneurysmal bone cyst, bone fibrous dysplasia, or a combination thereof.
  • the method is non-diagnostic and non-therapeutic.
  • test substance is selected from the group consisting of polypeptides, compounds, or combinations thereof.
  • a method of (a) inhibiting tumor metastasis; and/or (b) inhibiting bone tumors; and/or (c) inhibiting the differentiation and maturation of osteoclasts which includes the steps of: Administration of a therapeutically effective amount of the polypeptide according to the first aspect of the present invention or a pharmaceutically acceptable salt thereof, and/or the fusion protein according to the second aspect of the present invention, and/or the pharmaceutical combination according to the fourth aspect of the present invention Things.
  • the subject is a human or non-human mammal.
  • the non-human mammal includes rodents (such as mice, rats, rabbits) and primates (such as monkeys).
  • the method is non-diagnostic and non-therapeutic.
  • a method for treating bone tumors which includes the steps of: administering to a subject in need a therapeutically effective amount of the polypeptide according to the first aspect of the present invention or a pharmaceutically acceptable salt thereof, and/or The fusion protein according to the second aspect of the present invention, and/or the pharmaceutical composition according to the fourth aspect of the present invention.
  • the subject is a human or non-human mammal.
  • the non-human mammal includes rodents (such as mice, rats, rabbits) and primates (such as monkeys).
  • FIG. 1 A schematic diagram showing the structure of CST6 (also known as Cystatin E/M) and related peptides, and the binding of CST6 to its downstream target protease CTSB.
  • FIG. 1A Schematic diagram of the structure of the cysteine protease inhibitor Cystatin E/M (CST6) and its effective truncated GQ86, DQ51, GM-30, AY-11 and truncated negative control DR-9, where CST6, GQ86 and DQ51 are obtained by prokaryotic expression and purification; DR-9, GM-30 and AY-11 are obtained by artificial synthesis (Gill Biochemical Company).
  • CST6 cysteine protease inhibitor Cystatin E/M
  • FIG. 1B Alignment of partial sequences of CST6 in mammals. The yellow part is the conserved sequence, and the red box is the key QLVAG site for functioning. The serial number of human CST6 protein in Genbank is AAH31334.1, and other serial numbers have been indicated.
  • FIG. 1C CST6 protein structure diagram.
  • the key site QLVAG that binds to the CST6 target protein CTSB is shown in red.
  • W135 locus is shown in pink.
  • FIG. 1D The structure diagram of the binding of CTSB and CST6 family protein CSTA.
  • the active cleft site of the CTSB protein (top left) is shown in purple.
  • the QLVAG homology region of CSTA (bottom right) and CST6 is shown in red; the homology site of W135 of CST6 is shown in pink.
  • FIG. 1E Binding structure diagram of CTSB and its inhibitor CA-074. CA-974 is displayed in red.
  • FIG. 1 Shows that CST6 functions by inhibiting downstream CTSB.
  • CTSL Cathepsin L
  • FY Cathepsin L
  • CTSL enzyme activity was detected by BioVision's kit (Catalog#K142-100).
  • CTSL inhibitor Z-FY(t-Bu)-DMK (abbreviated as FY) cannot inhibit the differentiation of Raw264.7 into mature osteoclasts.
  • the multinucleated giant TRAP+ positive cells are mature osteoclasts.
  • FIG. 2C CTSB inhibitor CA-074 Me inhibits the enzymatic activity of CTSB in osteoclast precursor cells.
  • CTSB enzyme activity was detected by BioVision's kit (Catalog#K140-100).
  • FIG. 2D CA-074 Me, an inhibitor of Cathepsin B, can inhibit osteoclast differentiation.
  • CA-074 Me or its solvent control DMSO was added to Raw264.7 cell culture medium, and then osteoclast differentiation was observed by TRAP staining.
  • the multinucleated giant TRAP+ positive cells are mature osteoclasts.
  • Figure 3 Shows that CST6 recombinant protein and GQ86 polypeptide have the function of inhibiting CTSB enzyme activity.
  • FIG. 3A Western blotting experiments to identify recombinantly expressed CST6 wild-type and mutant (mutated with CTSB binding key sites) protein and GQ86 and DQ51 polypeptides. Both protein and peptide carry 6 tandem His tags. His antibody was used for western blotting.
  • FIG. 3B Coomassie brilliant blue staining experiment to identify recombinantly expressed CST6 wild-type and mutant (mutant that binds to the key site of CTSB) proteins and GQ86 and DQ51 polypeptides.
  • FIG. 3C The recombinantly expressed CST6 protein and polypeptide GQ86 can inhibit the enzymatic activity of CTSB.
  • Figure 4 Shows that the CST6 recombinant protein and GQ86 and DQ51 polypeptides have the function of inhibiting the differentiation and maturation of osteoclasts, but the CST6 mutant protein cannot inhibit the differentiation of osteoclasts.
  • FIG. 4A In vitro induction of osteoclasts from primary mouse bone marrow cells.
  • CST6 mutant protein (CST6-mutant), CST6 recombinant protein, GQ86, and DQ51 were added to primary mouse bone marrow cells at concentrations of 8nM, 16nM, and 32nM, respectively, and TRAP staining was performed 7 days after the induction of differentiation.
  • the black arrow points to a wine-red multinucleated cell with more than three nuclei as a mature osteoclast. Scale bar, 100 ⁇ m.
  • FIG. 4B Corresponding mature osteoclast count.
  • Figure 5 Shows that synthetic short peptides GM-30 and AY-11 containing active sites can inhibit osteoclast differentiation in vitro, but short peptides DR-9 that do not contain active sites cannot inhibit osteoclast differentiation.
  • FIG. 5A In vitro induction of osteoclasts from primary mouse bone marrow cells. GQ86, DR-9, GM-30 and AY-11 were added to the primary mouse bone marrow cells at a concentration of 32 nM. After 7 days of differentiation, the TRAP staining results showed that the black arrow points to wine-red multinucleated cells with more than three nuclei. Each cell is a mature osteoclast. Scale bar, 100 ⁇ m.
  • FIG. 5B Corresponding mature osteoclast count. *, p ⁇ 0.05.
  • FIG. 6 Shows that CST6 recombinant protein and GQ86 and DQ51 polypeptides inhibit the bone metastasis of breast cancer tumors in mice, but the CST6 mutant protein cannot inhibit bone metastasis.
  • a mouse model of bone metastasis was constructed by injecting breast cancer cell line SCP2 into the left ventricle of the mouse. SCP2 cells are labeled with F-Luciferase, which can quantify bone metastasis through bioluminescence in vivo imaging.
  • the protein and peptide administration concentration is 1 mg/kg/day, and the administration method is tail vein injection.
  • FIG. 6A Bioluminescence in vivo imaging (top) and X-ray (bottom) analysis of control and bone metastasis after CST6 or GQ86 administration.
  • the white arrow points to the site of bone loss.
  • Figure 6B Quantification of bone metastasis signals in control and CST6 and GQ86 administration mice within four weeks. *, p ⁇ 0.05.
  • FIG. 6C Body weight changes of control and CST6 and GQ86 administration mice in the first and fourth weeks. ns, the statistical difference is not significant; *, p ⁇ 0.05.
  • Figure 6D Survival curve of control and CST6 and GQ86 mice within 38 days. *, p ⁇ 0.05 compared with control.
  • FIG. 6E Bioluminescence in vivo imaging (top) and X-ray and micro-CT (bottom) analysis of control and CST6 mutant, GQ86 and DQ51 administration of bone metastases in mice at the fifth week.
  • the white arrow points to the site of bone loss.
  • Figure 6F Quantification of bone metastasis signals in mice administered with CST6-Mutant, GQ86 and DQ51 within four weeks. ns, the statistical difference is not significant; **, p ⁇ 0.01.
  • FIG. 6G Body weight changes of mice administered with CST6-Mutant, GQ86 and DQ51 in the first and fourth weeks. ns, the statistical difference is not significant; *, p ⁇ 0.05.
  • FIG. 6H Survival curves of mice administered with CST6-Mutant, GQ86 and DQ51 within 38 days. *, p ⁇ 0.05 compared with control.
  • Figure 8 Shows the therapeutic effects of CST6, GQ86, DQ51 and GM30 on giant cell tumor of bone.
  • the conditioned medium and corresponding protein or peptide drugs from the primary cell samples of patients with giant cell tumor of bone were added to the primary mouse bone marrow for in vitro culture, and the ability of the giant cell tumor of bone samples to induce osteoclasts was analyzed.
  • FIG 8A The experiment of the cells from the patient sample of giant cell tumor of bone No. 2 inducing osteoclasts from primary mouse bone marrow. While adding the conditioned medium of giant cell tumor cells of bone, 32nM CST6, GQ86 and GM30 were added respectively. After 7 days of induction of differentiation, the TRAP staining results showed that the wine-red multinucleated cells indicated by the black arrows are mature osteoclasts. CST6, GQ86 and GM30 all inhibit the osteoclast differentiation induced by giant cell tumor of bone. Scale bar, 100 ⁇ m.
  • Figure 8B Mature osteoclast count corresponding to Figure 8A.
  • FIG 8C The experiment of inducing osteoclasts from primary mouse bone marrow by cells from patient sample of giant cell tumor of bone No. 4.
  • the experimental method is the same as Figure 8A.
  • Figure 8D Mature osteoclast count corresponding to Figure 8C.
  • the inventors prepared for the first time a class of CST6 protein-derived products that have (a) inhibit tumor metastasis (such as breast cancer bone metastasis); and/or (b) inhibit bone tumors (such as bone giant cells). Tumor); and/or (c) a small molecule polypeptide (such as peptide DQ51, GM30, etc.) with a molecular weight of less than 16kD (such as 6kD or 3kD) that inhibits the differentiation and maturation of osteoclasts.
  • tumor metastasis such as breast cancer bone metastasis
  • bone tumors such as bone giant cells
  • Tumor such as a small molecule polypeptide
  • a small molecule polypeptide such as peptide DQ51, GM30, etc.
  • 16kD such as 6kD or 3kD
  • the present invention integrates a variety of different technologies such as protein polypeptide production technology, and successfully developed an effective (a) inhibition of tumor metastasis (such as breast cancer bone metastasis); and/or (b) inhibition of bone tumors (such as bone giant cells) Tumor); and/or (c) a polypeptide that inhibits osteoclast differentiation and maturation, and the polypeptide of the present invention is safe and has little toxic and side effects on biological tissues.
  • tumor metastasis such as breast cancer bone metastasis
  • bone tumors such as bone giant cells
  • Tumor such as bone giant cells Tumor
  • CST6 also known as Cystatin E/M, is a member of the cysteine protease inhibitor superfamily. In terms of structure and function, it has greater similarity with Cystatin type II proteins, and is also a tightly bound semi- As a cystine protease inhibitor, human CST6 acts as a protease inhibitor, exists in various human body fluids and exosomes, and is encoded and expressed by the CST6 gene. Like most cystatin genes, the three exons of the human CST6 gene are separated by two introns. Exon 1 is 294 bp long and contains the 5'-untranslated region (5'-UTR) of the coding sequence and the initiation ATG codon. Exon 2 is 126bp long.
  • Exon 3 is 188bp long, contains a TGA stop codon, 3'-UTR and a typical aataaa polyadenylation signal, followed by 20bp.
  • the lengths of intron 1 and intron 2 are 541 and 365 bp, respectively.
  • the human CST6 gene is transcribed into a messenger RNA (mRNA) containing 607 nucleotides (nt), with no other transcription products.
  • the transcript consists of 53 nt 5'-UTR, 447 nt coding sequence and 107 nt 3'-UTR.
  • accession number of the gene sequence of wild-type human CST6 protein is NM_001323, and the accession number of its protein sequence is NP_001314.
  • accession number of the gene sequence of the wild-type mouse CST6 protein is NM_028623, which has 85% homology with the gene sequence of the wild-type human CST6 protein, and the accession number of the protein sequence is NP_082899, which is similar to wild-type human CST6.
  • the homology of the protein sequence of the protein is 70%.
  • polypeptide of the present invention refers to having (a) inhibiting tumor metastasis; and/or (b) Inhibiting bone tumors; and/or (c) A protein or polypeptide of an amino acid sequence (formula I, formula III) that inhibits osteoclast differentiation and maturation activity.
  • the term also includes variants conforming to Formula I and Formula III with CTSB inhibitory activity. These variants include (but are not limited to) the addition of one or several (usually within 5, preferably within 3, and more preferably within 2) amino acids at the N-terminal.
  • the substitution of amino acids with similar or similar properties usually does not change the function of the protein. Adding one or several amino acids to the N-terminus usually does not change the structure and function of the protein.
  • the term also includes the polypeptides of the present invention or pharmaceutically acceptable salts thereof in monomer and multimeric forms.
  • the present invention also includes active fragments, derivatives and analogs of the polypeptides of the present invention.
  • fragment refers to a polypeptide that substantially retains the activity of inhibiting CTSB protein.
  • polypeptide fragments, derivatives or analogs of the present invention can be (i) a polypeptide with one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) in one or more A polypeptide with substitution groups in three amino acid residues, or (iii) a polypeptide formed by fusing the polypeptide of the present invention with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) an additional amino acid
  • the sequence is fused to the polypeptide sequence to form a polypeptide (the protein fused to the leader sequence, secretory sequence, or 6His tag sequence). According to the teachings herein, these fragments, derivatives and analogs are within the scope of those skilled in the art.
  • a preferred type of active derivative means that compared with the amino acid sequence of formula I and III, there are at most 5, preferably at most 3, more preferably at most 2, and most preferably 1 amino acid is composed of similar or similar properties. Amino acids are replaced to form polypeptides. These conservative variant polypeptides are best produced by amino acid substitutions according to Table 1a.
  • the invention also provides analogs of the polypeptides of the invention.
  • the difference between these analogs and the natural polypeptide of the present invention may be the difference in the amino acid sequence, the difference in the modified form that does not affect the sequence, or both.
  • Analogs also include analogs with residues different from natural L-amino acids (such as D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (such as ⁇ , ⁇ -amino acids).
  • Cys can form disulfide bonds with unnatural Hcy. It should be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above.
  • Modified (usually without changing the primary structure) forms include: chemically derived forms of polypeptides in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those produced by glycosylation modifications during the synthesis and processing of the polypeptide or in further processing steps. This modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation (such as a mammalian glycosylase or deglycosylase). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). It also includes polypeptides that have been modified to improve their resistance to proteolysis or optimize their solubility.
  • the polypeptide of the present invention has at least one internal disulfide bond (introduced intrachain disulfide bond).
  • the existence of the internal disulfide bond not only does not affect its inhibitory activity, but also helps to extend the half-life and increase the inhibitory activity.
  • it can be formed by conventional methods in the art, such as combining cysteine or homocysteine sulfhydryl groups under oxidizing conditions to form disulfide bonds.
  • a preferred polypeptide of the present invention includes SEQ ID NO.: 1-8.
  • polypeptides of the present invention also include polypeptides modified from the polypeptides shown in SEQ ID NO.: 1-8.
  • the polypeptide of the present invention can also be used in the form of a salt derived from a pharmaceutically or physiologically acceptable acid or base.
  • These salts include (but are not limited to) salts formed with the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, citric acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, succinic acid, oxalic acid, fumaric acid, maleic acid Acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, or isethionic acid.
  • Other salts include: salts with alkali metals or alkaline earth metals (such as sodium, potassium, calcium, or magnesium), and in the form of esters, carbamates, or other conventional "prodrugs".
  • the invention also relates to polynucleotides encoding the polypeptides of the invention.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA can be a coding strand or a non-coding strand.
  • the sequence of the coding region encoding the mature polypeptide may be the same as the sequence of the coding region or a degenerate variant.
  • the full-length nucleotide sequence of the polypeptide of the present invention or its fragments can usually be obtained by PCR amplification, recombination or artificial synthesis.
  • the DNA sequence encoding the polypeptide (or fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.
  • the present invention also relates to a vector containing the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or the polypeptide coding sequence of the present invention.
  • the present invention also includes polyclonal antibodies and monoclonal antibodies or antibody fragments specific to the polypeptides of the present invention, especially monoclonal antibodies.
  • the term "substantially identical” refers to two or more sequences or subsequences that have at least about 80%, such as at least about 85 %, about 90%, about 95%, about 98%, or about 99% of the nucleotide or amino acid residues are identical to a specific reference sequence, as determined by using the following sequence comparison method and/or by visual inspection.
  • the polypeptide of the present invention can be a recombinant polypeptide or a synthetic polypeptide.
  • the polypeptide of the present invention can be chemically synthesized or recombinant.
  • the polypeptide of the present invention can be artificially synthesized by conventional methods, or can be produced by recombinant methods.
  • a preferred method is to use liquid phase synthesis technology or solid phase synthesis technology, such as Boc solid phase method, Fmoc solid phase method or a combination of the two methods.
  • Solid-phase synthesis can quickly obtain samples, and an appropriate resin carrier and synthesis system can be selected according to the sequence characteristics of the target peptide.
  • the preferred solid phase carrier in the Fmoc system is the Wang resin connected with the C-terminal amino acid in the peptide.
  • the Wang resin structure is polystyrene, and the arm between the amino acid is 4-alkoxybenzyl alcohol; 25% hexahydropyridine is used /Dimethylformamide is treated at room temperature for 20 minutes to remove the Fmoc protecting group and extend from the C-terminus to the N-terminus one by one according to the given amino acid sequence.
  • the synthesized proinsulin-related peptide is cleaved from the resin with trifluoroacetic acid containing 4% p-methylphenol and the protective group is removed.
  • the crude peptide can be separated by ether precipitation after the resin is filtered off.
  • the desired peptide is purified by gel filtration and reverse phase high pressure liquid chromatography.
  • the preferred resin is PAM resin connected with the C-terminal amino acid in the peptide.
  • the PAM resin structure is polystyrene, and the arm between the amino acid is 4-hydroxymethyl phenylacetamide; synthesized in Boc
  • the protective group Boc is removed with TFA/dichloromethane (DCM) and neutralized with diisopropylethylamine (DIEA/dichloromethane.
  • the peptide chain condensation is completed Afterwards, treated with hydrogen fluoride (HF) containing p-cresol (5-10%) at 0°C for 1 hour to cut the peptide chain from the resin while removing the protective group.
  • HF hydrogen fluoride
  • acetic acid containing A small amount of mercaptoethanol
  • the solution is lyophilized and further separated and purified with molecular sieve Sephadex G10 or Tsk-40f, and then purified by high pressure liquid phase to obtain the desired peptide.
  • Various couplings known in the field of peptide chemistry can be used Reagents and coupling methods to couple each amino acid residue, for example, dicyclohexylcarbodiimide (DCC), hydroxybenzotriazole (HOBt) or 1,1,3,3-tetraurea hexafluorophosphate can be used (HBTU) for direct coupling.
  • DCC dicyclohexylcarbodiimide
  • HOBt hydroxybenzotriazole
  • HBTU 1,1,3,3-tetraurea hexafluorophosphate
  • the polypeptide of the present invention is prepared by solid-phase synthesis according to its sequence, purified by high performance liquid chromatography to obtain high-purity target peptide freeze-dried powder, and stored at -20°C.
  • Another method is to use recombinant technology to produce the polypeptide of the present invention.
  • the polynucleotide of the present invention can be used to express or produce a recombinant polypeptide of the present invention.
  • polynucleotide (or variant) of the present invention encoding the polypeptide of the present invention, or use a recombinant expression vector containing the polynucleotide to transform or transduce a suitable host cell;
  • the recombinant polypeptide can be expressed in the cell or on the cell membrane or secreted out of the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other characteristics. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitation agent (salting out method), centrifugation, osmotic disruption, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment treatment with protein precipitation agent (salting out method)
  • centrifugation osmotic disruption
  • ultra-treatment ultra-centrifugation
  • molecular sieve chromatography gel filtration
  • adsorption layer Analysis ion exchange chromatography
  • polypeptide of the present invention is relatively short, it is possible to consider connecting multiple polypeptides in series to obtain a multimeric expression product after recombinant expression, and then forming the required small peptides by methods such as restriction enzyme digestion.
  • cell penetrating element and “cell penetrating peptide” are used interchangeably and both refer to the ability to effectively penetrate the inhibitory polypeptide into the cell without any damage to the cell without affecting the inhibitory polypeptide. Active small peptides.
  • the present invention also provides a pharmaceutical composition, which contains (a) a safe and effective amount of the polypeptide of the present invention or a pharmaceutically acceptable salt thereof; and (b) a pharmaceutically acceptable carrier or excipient .
  • the amount of the polypeptide of the present invention or its pharmaceutically acceptable salt is usually 10 micrograms to 100 mg/dose, preferably 100 to 1000 micrograms/dose.
  • an effective dose is about 0.01 mg/kg to 50 mg/kg, preferably 0.05 mg/kg to 10 mg/kg body weight of the polypeptide of the present invention or a pharmaceutically acceptable salt thereof.
  • the polypeptide of the present invention or a pharmaceutically acceptable salt thereof can be used singly or together with other therapeutic agents (for example, formulated in the same pharmaceutical composition).
  • the pharmaceutical composition may also contain a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to a carrier used for the administration of a therapeutic agent.
  • pharmaceutical carriers that do not themselves induce the production of antibodies that are harmful to the individual receiving the composition, and do not have excessive toxicity after administration. These vectors are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable excipients can be found in Remington’s Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).
  • Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, adjuvants and combinations thereof.
  • the pharmaceutically acceptable carrier in the therapeutic composition may contain liquids such as water, saline, glycerol and ethanol.
  • these carriers may also contain auxiliary substances, such as wetting or emulsifying agents, and pH buffering substances.
  • the therapeutic composition can be made into an injectable, such as a liquid solution or suspension; it can also be made into a solid form suitable for being formulated into a solution or suspension in a liquid carrier before injection.
  • an injectable such as a liquid solution or suspension
  • it can also be made into a solid form suitable for being formulated into a solution or suspension in a liquid carrier before injection.
  • composition of the invention can be administered by conventional routes, including (but not limited to): intratumoral, intramuscular, intravenous, subcutaneous, intradermal, or topical administration.
  • routes including (but not limited to): intratumoral, intramuscular, intravenous, subcutaneous, intradermal, or topical administration.
  • the objects to be prevented or treated can be animals; especially humans.
  • various dosage forms of the pharmaceutical composition can be used according to the use situation. It is preferably an intravenous preparation or an intratumor injection.
  • compositions can be formulated by mixing, diluting or dissolving according to conventional methods, and occasionally adding suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic Isotonicities, preservatives, wetting agents, emulsifiers, dispersants, stabilizers and co-solvents, and the formulation process can be carried out in a usual manner according to the dosage form.
  • suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic Isotonicities, preservatives, wetting agents, emulsifiers, dispersants, stabilizers and co-solvents, and the formulation process can be carried out in a usual manner according to the dosage form.
  • the preparation of eye drops can be carried out by dissolving the polypeptide of the present invention or a pharmaceutically acceptable salt thereof together with the basic substance in sterile water (a surfactant is dissolved in sterile water) to adjust the osmotic pressure And the pH to the physiological state, and appropriate pharmaceutical additives such as preservatives, stabilizers, buffers, isotonic agents, antioxidants, and thickeners can be added optionally, and then completely dissolved.
  • sterile water a surfactant is dissolved in sterile water
  • appropriate pharmaceutical additives such as preservatives, stabilizers, buffers, isotonic agents, antioxidants, and thickeners can be added optionally, and then completely dissolved.
  • the pharmaceutical composition of the present invention can also be administered in the form of a sustained-release formulation.
  • the polypeptide of the present invention or a pharmaceutically acceptable salt thereof can be incorporated into a pill or microcapsule with a sustained-release polymer as a carrier, and then the pill or microcapsule is surgically implanted into the tissue to be treated.
  • sustained-release polymers ethylene-vinyl acetate copolymers, polyhydrometaacrylate, polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymers, Lactic acid-glycolic acid copolymers and the like are preferably exemplified by biodegradable polymers such as lactic acid polymers and lactic acid-glycolic acid copolymers.
  • the dosage of the polypeptide of the present invention or its pharmaceutically acceptable salt as an active ingredient can be adjusted according to the weight, age, sex, and degree of symptoms of each patient to be treated. Reasonably determine.
  • polypeptide of the present invention and its derivative polypeptides have small molecular weight, small toxic and side effects on biological tissues, and high safety.
  • the polypeptide of the present invention can effectively inhibit tumor metastasis; and/or bone tumor; and/or osteoclast differentiation and maturation.
  • polypeptide of the present invention has good stability.
  • polypeptide of the present invention has high specificity.
  • the reagents or materials used in the embodiments of the present invention are all commercially available products.
  • polypeptide sequences obtained by screening are shown in the following table:
  • the 5'-end primer (CST6-F) of the human CST6 gene cDNA sequence (used to clone the human CST6 gene mature peptide coding sequence):
  • 5'-CATGCCATGGCGCGTTCGAACCTCC-3' (SEQ ID NO.: 9); wherein the 5'-end has an Ncol restriction site;
  • the 3'-end primer (CST6-R) of the cDNA sequence of the human CST6 gene (used to clone the mature peptide coding sequence of the human CST6 gene):
  • the primer contains a restriction site Xhol and a stop codon.
  • RNA Extraction of total RNA: Digest the human breast cancer cell line MDA-MB-231 from the culture dish with trypsin, collect the cells in a 1.5ml EP tube, and then lyse with 1ml Trizol. Vibrate for 30s. Add 0.2mL chloroform, shake vigorously for 30s, and room temperature for 2min. Centrifuge at 13000-13300rpm at 4°C for 15min. Suck the upper colorless water phase and transfer it to another EP tube. Add an equal volume of isopropanol and place it at -20°C for 30 minutes. Centrifuge at 13000-13300 rpm for 10 min at 4°C. Discard the supernatant, add 1 mL of 75% ethanol, and shake.
  • the reverse transcription PCR program is as follows:
  • the Q5 enzyme amplifies the target gene fragment.
  • the amplification system and procedures are as follows:
  • the digestion system is as follows:
  • T4 ligase (NEB) to ligate the purified vector DNA and insert DNA according to the ratio of the number of molecules of 1:10 at 16°C overnight.
  • connection system is as follows
  • Bacteria detection uses the laboratory's self-made pTaq enzyme, the PCR program is the same as that of gene fragment amplification, and the PCR system is as follows:
  • GQ86 and DQ51 use the same method as CST6 to construct GQ86/pET28a(+) and DQ51/pET28a(+) plasmids ( Figure 1A). The only difference is the use of different primers, where the primer GQ86-F:
  • 5'-CATGCCATGGGAGAACTCCGGGACCTGTCG-3' (SEQ ID NO.: 11); wherein the 5'-end has an Ncol restriction site;
  • the primer GQ86-R The primer GQ86-R:
  • the primer contains a restriction site Xhol and a stop codon.
  • the primer DQ51-F The primer DQ51-F:
  • the primer DQ51-R The primer DQ51-R:
  • the primer contains a restriction site Xhol and a stop codon.
  • the supernatant was sucked off, leaving 50-100ul of the bacterial solution, which was blown evenly, and then spread evenly on the Kanar-resistant LB solid plate with a coating rod.
  • the colonies on the plate grow to a size of about 0.5 mm in diameter visible to the naked eye, pick up the monoclonal colonies with a small pipette tip, and pipette into the EP tube pre-added with 400 ⁇ l of kana-resistant LB liquid medium. Shake the bacteria in a constant temperature shaker at 37°C and 250 rpm for 2 hours.
  • the protein was detected by Western blotting with His antibody to detect whether it contained the target protein (Figure 3A), the whole protein was detected by Coomassie brilliant blue staining ( Figure 3B), and the concentration of purified protein was detected by BCA. Finally, use endotoxin high-efficiency removal and purification resin (Yusheng Biotechnology, 20518ES10) to remove endotoxin, and store at -80°C for later use.
  • the MEGA software was used to align the sequence of CST6 in some mammals.
  • the key functional site of CST6, QLVAG, is very conserved in mammals ( Figure 1B).
  • CTSB enzyme activity was detected by BioVision's kit (Catalog#K140-100).
  • CTSL enzyme activity was detected by BioVision's kit (Catalog#K142-100) ( Figure 2A, 2C). Shows that inhibition of CTSB enzyme activity, but not CTSL enzyme activity, can inhibit osteoclast differentiation and maturation ( Figure 2B, 2D). Combined with the experiment in Figure 3C, it is confirmed that CTS6 inhibits the function of downstream CTSB through its key active site.
  • ⁇ -MEM culture medium containing 20% FBS, RANKL 50-100ng/ml, M-CSF 25ng/ml
  • conditioned medium of primary cultured cells of patients with giant cell tumor of bone final concentration is 10-20%) .
  • the cells are changed once every three days, and the experiment ends on the sixth day.
  • the medium used is the same as before.
  • Anesthesia 1% sodium pentobarbital is injected intraperitoneally for anesthesia, and the required anesthetic dose is calculated at the amount of 30-40 mg/kg for each nude mouse. Sterilize the front chest wall with 75% alcohol. Touch the most obvious part of the apex of the heart, about 3mm to the left of the sternum and the second intercostal space, aim at the center and enter the needle at 45 degrees to the body. If you can see bright red blood spurting out, it means The needle has entered the left ventricle. After slowly pushing the cell suspension in, the needle is quickly withdrawn.
  • the amount of injected cells is: inject at a concentration of 5.0 ⁇ 10 5 cells/ml and at a volume of 0.1 ml/head.
  • D-Luciferin was injected at the bottom of 0.1ml/mouse at a dose of 5mg/ml (nude mice intravenous injection is the same), and Berthold Imaging System imaging was performed weekly ( Figure 6A, Figure 6E) and photographed. After the injection, continue to raise, eat freely, observe the life state and general conditions of the nude mice; closely observe the vital signs and state of the nude mice within 24 hours after the injection.
  • CST6, GQ86 and DQ51 were administered by tail vein injection at a concentration of 1 mg/kg, and the administration cycle was once a day.
  • the results of the experiment were presented in two times. The first time was administered with TGE buffer-Control, CST6 and GQ86 (Figure 6A-6D); the second time was administered with TGE buffer-Control, CST6-Mutant, GQ86 and DQ51 ( Figure 6E- 6H).
  • Berthold Imaging System imaging every week ( Figure 6A, Figure 6E), take pictures, use the built-in software Indigo2.0 to quantify the fluorescence signal (Figure 6B, Figure 6F), weigh its weight (Figure 6C, Figure 6G), and finally count 40 Survival curve within days (Figure 6D, Figure 6H).
  • CST6 and GQ86 were administered via tail vein injection at concentrations of 25mg/kg, 50mg/kg, 90mg/kg, 120mg/kg and 200mg/kg. Observe the symptoms of experimental animals within 24 hours after one administration and record the number of deaths. Finally, using the modified Kou’s method, the median lethal dose (LD50) of CST6 was 126.61 mg/kg, and the median lethal dose (LD50) of GQ86 was 142.23 mg/kg (Figure 7).

Landscapes

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

Abstract

La présente invention concerne un polypeptide pour l'inhibition de métastases tumorales et de tumeurs osseuses, et son utilisation. Spécifiquement, le polypeptide de la présente invention ou un sel pharmaceutiquement acceptable de celui-ci présente la structure de formule I ou de formule III. Le polypeptide de la présente invention ou un sel pharmaceutiquement acceptable de celui-ci peut inhiber significativement (a) les métastases tumorales; et/ou (b) les tumeurs osseuses; et/ou (c) la différenciation et la maturation des ostéoclastes.
PCT/CN2019/126726 2019-01-17 2019-12-19 Polypeptide pour l'inhibition de métastases tumorales et de tumeurs osseuses, et son utilisation WO2020147508A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910044856.1A CN111440227B (zh) 2019-01-17 2019-01-17 一种抑制肿瘤转移及骨肿瘤的多肽及其应用
CN201910044856.1 2019-01-17

Publications (1)

Publication Number Publication Date
WO2020147508A1 true WO2020147508A1 (fr) 2020-07-23

Family

ID=71614161

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/126726 WO2020147508A1 (fr) 2019-01-17 2019-12-19 Polypeptide pour l'inhibition de métastases tumorales et de tumeurs osseuses, et son utilisation

Country Status (2)

Country Link
CN (1) CN111440227B (fr)
WO (1) WO2020147508A1 (fr)

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BERQUIN, I.M. ET AL.: "Cysteine Proteases and Tumor Progression", PERSPECTIVES IN DRUG DISCOVERY AND DESIGN, vol. 2, no. 3, 31 July 1995 (1995-07-31) *
BROEMME, D. ET AL.: "Tight-Binding Inhibition of Cathepsin S by Cystatins", BIOMEDICA BIOCHIMICA ACTA, vol. 50, no. 4-6, 1 January 1991 (1991-01-01) *
JIA, HONG ET AL.: "Characterization of a Cysteine Proteinase Inhibitor Induced during Neuronal Cell Differentiation", JOURNAL OF NEUROCHEMISTRY, vol. 81, 31 December 2012 (2012-12-31), XP055725892 *
PREMACHANDRA, H.K. ET AL.: "Expression Profile of Cystatin B Ortholog from Manila Clam (Ruditapes Philippin arum) in Host Pathology with respect to Its Structural and Functional Properties", FISH SHELLFISH IMMUNOL, vol. 34, no. 6, 30 June 2013 (2013-06-30), XP055725896 *
WANG, BING ET AL.: "Cathepsin S Controls Angiogenesis and Tumor Growth via Matrix- derived Angiogenic Factors", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 281, 2 March 2006 (2006-03-02), XP055419964, DOI: 10.1074/jbc.M509134200 *

Also Published As

Publication number Publication date
CN111440227B (zh) 2023-02-17
CN111440227A (zh) 2020-07-24

Similar Documents

Publication Publication Date Title
JP4006021B2 (ja) ケモカインの生物学的活性の強化法
US8603969B2 (en) Pancreatic polypeptide family motifs and polypeptides comprising the same
JP6267190B2 (ja) 悪液質の予防用または治療用組成物
ES2277676T3 (es) Antagonistas de los peptidos glp-2 intestinotroficos.
JP2003511071A (ja) Ｊｎｋシグナル導入経路の細胞透過性ペプチドインヒビター
BRPI0718566A2 (pt) Peptídeo, análogo do mesmo, composição farmacêutica, usos de um análogo de peptídeo, e de uma molécula de ácido nucléico, de um vetor de expressão, ou de uma célula hospedeira, molécula de ácido nucléico, vetor de expressão, célula hospedeira, métodos para produzir o análogo de peptídeo-2 tipo-glucagon (glp-2), e, kit terapêutico.
EP2094288A2 (fr) Mutants résistants aux protéases du facteur dérivé des cellules stromales de type 1 utilisés dans la réparation d'une lésion tissulaire
JP2010526527A (ja) 抗腫瘍活性を有する新規なポリペプチド
JP2002526073A (ja) 新規のヒト生長分化因子のコード配列、そのｄｎａ配列によりコードされるポリペプチド、およびこれらの製造方法。
US20200062811A1 (en) Yap protein inhibiting polypeptide and application thereof
EP2870174A1 (fr) Peptides pénétrant dans une cellule pour cibler eif4e
WO2006041205A1 (fr) Promoteur d'angiogenese
CN105524139B (zh) 高活性的肿瘤抑制剂及其制法和应用
CN102482323B (zh) 新型肽及其应用
SK287523B6 (sk) Použitie CC chemokínového mutanta, farmaceutický prostriedok s obsahom chemokínového mutanta, skrátený a mutovaný humánny RANTES a spôsob jeho výroby
WO2020147508A1 (fr) Polypeptide pour l'inhibition de métastases tumorales et de tumeurs osseuses, et son utilisation
WO2014022271A1 (fr) Procédé de traitement de cancer métastatique
JPS6371195A (ja) 新規ペプチド
US20130237476A1 (en) Adipose tissue targeted peptides
KR101323669B1 (ko) 암세포 특이적 세포괴사 유도 및 암 소멸 효과를 나타내는 세포사 유도 융합 펩타이드
CN117756909A (zh) 改进的抗衰老化合物及其在癌症治疗中的用途
CN113018418B (zh) 微小RNA31前体编码多肽miPEP31在制备高血压药物中的应用
CN116785402B (zh) 预防和/或治疗乳腺癌的多肽及其应用
WO2023035817A1 (fr) Protéine mutante fgf21 et son utilisation
WO2023061487A1 (fr) Polypeptide pour inhiber trpm8 et son utilisation

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19909821

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19909821

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 17/11/2021)

122 Ep: pct application non-entry in european phase

Ref document number: 19909821

Country of ref document: EP

Kind code of ref document: A1