WO2023046147A1 - 针对dkk1的适体及其用途 - Google Patents

针对dkk1的适体及其用途 Download PDF

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WO2023046147A1
WO2023046147A1 PCT/CN2022/121254 CN2022121254W WO2023046147A1 WO 2023046147 A1 WO2023046147 A1 WO 2023046147A1 CN 2022121254 W CN2022121254 W CN 2022121254W WO 2023046147 A1 WO2023046147 A1 WO 2023046147A1
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aptamer
dkk1
identity
cancer
preferably less
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PCT/CN2022/121254
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English (en)
French (fr)
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张戈
于媛媛
何伊欣
朱倖莹
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安沛治疗有限公司
张戈
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Priority to CN202280043227.7A priority Critical patent/CN117916377A/zh
Publication of WO2023046147A1 publication Critical patent/WO2023046147A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7115—Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers

Definitions

  • the invention relates to the field of biomedicine. Specifically, the present invention relates to an aptamer against DKK1 and its use, especially its use in treating DKK1-related diseases such as DKK1-related cancers.
  • Dickkopf-1 (DKK1), a member of the dickkopf protein family, has been shown to be a negative regulator of Wnt signaling, which has a central role in bone development and formation.
  • DKK1 By binding LRP5 (LRP6) and kremen proteins, DKK1 prevents LRP5 or LRP6 from binding to members of the Wnt pathway, thereby preventing Wnt-mediated signal transduction, thereby inhibiting bone formation.
  • DKK1 Wnt antagonist DKK1
  • DKN-01 A humanized monoclonal therapeutic antibody against DKK1, called DKN-01, has been used in phase II trials to treat esophagogastric and other types of cancer in combination with chemotherapy. Therefore, DKK1 may be a promising target for tumor immunotherapy.
  • therapeutic antibodies have several major issues, including high immunogenicity (Padhi, Jang et al., 2011; Padhi, Allison et al., 2014), high production costs (Baker, 2015; Bradbury and Pluckthun 2015; Groff , Brown et al., 2015), unstable and requires continuous cold chain transport and storage (Jayasena, 1999). Therefore, non-immunogenic, easy-to-manufacture, low-cost and high-stability alternative anti-DKK1 agents are desired for anti-tumor therapy.
  • Aptamers have attracted extensive attention due to their high binding affinity and specificity and low immunogenicity. Aptamers are short single-stranded oligonucleotides that bind to their targets through conformational complementarity (Ellington and Szostak, 1990; Tuerk and Gold, 1990). Compared with therapeutic antibodies, aptamers have similar affinity and specificity, but have some important advantages. For immunogenicity, aptamers are not recognized as foreign by the immune system and do not stimulate negative immune responses because of their low molecular weight (Keefe, Pai et al., 2010).
  • aptamers In terms of production and cost, aptamers can be identified in vitro under various selection conditions and can be easily synthesized by chemical methods, so the production cost is lower and the risk is lower (Banerjee, 2010). In terms of stability, aptamers have an unlimited shelf life since they are temperature insensitive and do not require any special cooling during transport, thus not requiring a continuous cold chain (Jayasena, 1999).
  • VEGF vascular endothelial growth factor
  • the present invention provides an aptamer against DKK1, the aptamer
  • ii) comprise at least 30, at least 35, at least 40, at least 45, at least 50 or more contiguous nucleotides of any of SEQ ID NO: 1-20; or
  • iii) comprising at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity to any of SEQ ID NO:45-64 , a nucleotide sequence that is at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical; or
  • iv) comprising at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity to any of SEQ ID NO:87-105 , a nucleotide sequence that is at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical,
  • the aptamer specifically binds to DKK1.
  • the aptamer comprises the sequence of any one of SEQ ID NOs: 1-64 and 87-105.
  • the aptamer comprises the nucleotide sequence shown in SEQ ID NO:5, 20, 35-38, 49, or 64, preferably the nucleotide sequence shown in SEQ ID NO:37.
  • the aptamer comprises one or more modifications that confer increased nuclease resistance to the aptamer and/or enhance the in vivo half-life of the aptamer.
  • the aptamer comprises a 2'-methoxy (2'-OMe) modification and a 3' inverted deoxythymidine (3'idT) modification.
  • the aptamer has an effect on DKK1 of less than 350 nM, preferably less than 150 nM, preferably less than 100 nM, preferably less than 50 nM, preferably less than 30 nM, preferably less than 20 nM, preferably less than 10 nM, preferably less than 5 nM, preferably less than 1 nM or less
  • the K d preferably less than 350 nM, preferably less than 150 nM, preferably less than 100 nM, preferably less than 50 nM, preferably less than 30 nM, preferably less than 20 nM, preferably less than 10 nM, preferably less than 5 nM, preferably less than 1 nM or less The K d .
  • the aptamer is capable of inhibiting the biological activity of DKK1. In some embodiments, the aptamer is capable of blocking DKK1 antagonism in a cell-based Wnt signaling assay. In some embodiments, the aptamer inhibits the biological activity of DKK1 with an IC50 value of less than 2000 nM, preferably less than 1500 nM, preferably less than 1000 nM, preferably less than 800 nM or less, such as inhibiting the antagonism of DKK1 on the Wnt signaling pathway.
  • the present invention provides a method for treating DKK1-related diseases, the method comprising administering a therapeutically effective amount of the aptamer against DKK1 of the present invention to a subject in need, such as a human.
  • the DKK1-associated disease is a DKK1-associated cancer, such as myeloma (e.g., multiple myeloma with osteolytic lesions, hilar cholangiocarcinoma multiple myeloma, etc.), breast cancer, colon cancer , melanoma, hepatocellular carcinoma, epithelial cancer, esophageal cancer, gastric cancer, gastroesophageal cancer, hilar cholangiocarcinoma, brain cancer, lung cancer, prostate cancer or pancreatic cancer, and any metastases thereof.
  • myeloma e.g., multiple myeloma with osteolytic lesions, hilar cholangiocarcinoma multiple myeloma, etc.
  • breast cancer colon cancer
  • melanoma hepatocellular carcinoma
  • epithelial cancer esophageal cancer
  • gastric cancer gastric cancer
  • gastroesophageal cancer hilar cholangio
  • the DKK1-associated disease is selected from the group consisting of osteoporosis, osteopenia, osteomalacia, osteogenesis imperfecta (OI), avascular osteonecrosis, rheumatoid arthritis, bone fractures, osteoarthritis, and bone marrow tumor.
  • the present invention provides a pharmaceutical composition comprising at least one aptamer against DKK1 of the present invention, and a pharmaceutically acceptable carrier or excipient.
  • the present invention provides the use of the aptamer against DKK1 of the present invention or the pharmaceutical composition of the present invention in the preparation of medicines, wherein the medicines are used to treat DKK1-related diseases.
  • the DKK1-associated disease is a DKK1-associated cancer, such as myeloma (e.g., multiple myeloma with osteolytic lesions, hilar cholangiocarcinoma multiple myeloma, etc.), breast cancer, colon cancer , melanoma, hepatocellular carcinoma, epithelial cancer, esophageal cancer, gastric cancer, gastroesophageal cancer, hilar cholangiocarcinoma, brain cancer, lung cancer, prostate cancer or pancreatic cancer, and any metastases thereof.
  • myeloma e.g., multiple myeloma with osteolytic lesions, hilar cholangiocarcinoma multiple myeloma, etc.
  • breast cancer colon cancer
  • melanoma hepatocellular carcinoma
  • epithelial cancer esophageal cancer
  • gastric cancer gastric cancer
  • gastroesophageal cancer hilar cholangio
  • the DKK1-associated disease is selected from the group consisting of osteoporosis, osteopenia, osteomalacia, osteogenesis imperfecta (OI), avascular osteonecrosis, rheumatoid arthritis, bone fractures, osteoarthritis, and bone marrow tumor.
  • Figure 1 shows the determination of the binding ability of Ni-NTA magnetic agarose beads to DKK1 protein.
  • Two experiments were performed to determine the binding ability of Ni-NTA magnetic agarose beads to DKK1 protein.
  • the apparent molecular weight of DKK1 is about 40KDa. This condition (0.6 nmol DKK1 + 6 ⁇ l 5% beads) showed the highest binding capacity.
  • Figure 2 shows the agarose gel (3%) electrophoresis analysis of the polymerase chain reaction (PCR) products of the aptamer pools screened in rounds 1 to 20.
  • the first DNA ladder.
  • the DNA band exists at about 80bp.
  • Figure 3 shows the phylogenetic tree and alignment of aptamer candidates.
  • the sequences of the twenty aptamers were clustered into two groups.
  • Group 1 contains eight guanine-rich sequences, called the G-rich group;
  • group 2 includes twelve sequences belonging to other nucleotides, called the non-G-rich group.
  • Figure 4 shows the specificity of representative aptamer candidates. 1 [mu]M of each aptamer/RS was used to determine specificity for DKK1 using an enzyme-linked oligonucleotide assay (ELONA) and the absorbance at 450 nm was detected. ****P ⁇ 0.0001; **P ⁇ 0.005; *P ⁇ 0.05.
  • Figure 5 shows the affinities of representative aptamer candidates. Affinity to DKK-1 was determined using specific concentrations of each aptamer and absorbance at 450 nm was measured.
  • Figure 6 shows the inhibitory effect of representative aptamer candidates. Aptdkk-5, 19, and 20 showed higher ratios compared with controls, suggesting their potential to undo the repression of Wnt signaling by DKK1.
  • Figure 7 shows the inhibitory potency of representative aptamer candidates.
  • the data for aptdkk-19 do not fit the fitted curve.
  • Figure 8 shows the specificity of truncated aptdkk5. a) 100 nM and b) 1 nM of each truncated aptdkk5 were used to determine specificity for DKK-1 by comparing their absorbance at 450 nm with that of native aptdkk5. *P ⁇ 0.05; NS, no significant difference.
  • Figure 9 shows the affinity of truncated aptdkk5. Gradient concentrations of each aptamer were used to determine the binding affinity for DKK1 and the absorbance at 450 nm was determined.
  • FIG. 11 Expression levels of DKK1 and defective DKK1 in HEK293T cells measured by SDS-PAGE gel electrophoresis. S4, pCS2-DKK1-His-flag; S3, pCS2-DKK1- ⁇ C-His-flag; S2, pCS2-DKK1- ⁇ L2&C-His-flag.
  • FIG. 12 Binding capacity of full-length and truncated DKK1. Fluorescent images of aptDKK5s binding to DKK1 mutants. S4, pCS2-DKK1-His-flag; S3, pCS2-DKK1- ⁇ C-His-flag; S2, pCS2-DKK1- ⁇ L2&C-His-flag; positive Ctrl, pure protein DKK1His6; negative Ctrl, beads only.
  • Figure 14 Affinity of modified aptddk5s.
  • the affinity to DKK-1 was determined by ELONA using a certain concentration of each aptamer, and the absorbance at 450 nm was detected.
  • Figure 15 Serum stability assessment of modified aptdkk5s compared to unmodified aptdkk5s. All aptamers were treated with 10% and 100% fetal bovine serum from 0 to 72 hours, respectively.
  • FIG. 18 Agarose gel (3%) electrophoretic analysis of polymerase chain reaction (PCR) products of aptamer pools from rounds 21 to 30 of selection. Front lane, DNA ladder. The DNA band is about 80bp.
  • PCR polymerase chain reaction
  • FIG. 20 Binding curves of representative aptamer candidates. Affinity to DKK-1 was determined by BLI using concentrations of each aptamer.
  • nucleotide refers to ribonucleotides or deoxyribonucleotides, or modified forms and analogs thereof. Nucleotides include species that include purines (e.g., adenine, hypoxanthine, guanine, and their derivatives and analogs) and pyrimidines (e.g., cytosine, uracil, thymine, and their derivatives and the like) things).
  • purines e.g., adenine, hypoxanthine, guanine, and their derivatives and analogs
  • pyrimidines e.g., cytosine, uracil, thymine, and their derivatives and the like
  • nucleic acid As used herein, “nucleic acid”, “oligonucleotide” and “polynucleotide” are used interchangeably to refer to polymers of nucleotides and include DNA, RNA, DNA/RNA hybrids and nucleic acids of these species, Modification of oligonucleotides and polynucleotides, including the addition of various entities or moieties at any position of the nucleotide unit.
  • polynucleotide “oligonucleotide” and “nucleic acid” include double or single stranded molecules.
  • nucleic acid, oligonucleotide and polynucleotide are broader terms than the term aptamer, thus the terms nucleic acid, oligonucleotide and polynucleotide include but are not limited to aptamers.
  • aptamer refers to a non-naturally occurring nucleic acid that has a desired effect on a target molecule. Desired effects include, but are not limited to, binding to the target, catalytically altering the target, reacting with the target in a manner that modifies or alters the target or the functional activity of the target, covalently linking the target, and promoting the target. The reaction between the target and other molecules.
  • the effect is specific binding affinity for a target molecule (e.g., DKK1), which is a three-dimensional chemical structure rather than a polynucleotide, through independent Watson/Crick base pairing or triplex binding.
  • the mechanism of helix formation is used to bind the aptamer, wherein the aptamer is not a nucleic acid with a known physiological function to be bound by the target molecule.
  • "specific binding" of an aptamer to its target means that said aptamer binds to the target, usually with much higher affinity than it binds to other non-target components in the mixture or sample. its target.
  • Sequence "identity” has an art-recognized meaning, and the percent sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the entire length of a polynucleotide or polypeptide, or along a region of the molecule.
  • identity is well known to those of skill (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988) ).
  • a number of algorithms are available to determine percent sequence identity.
  • An example of an algorithm suitable for determining percent sequence identity is the algorithm used in the Basic Local Alignment Search Tool (hereinafter "BLAST"), see e.g. Altschul et al., J. Mol. Biol. 215:403-410, 1990 and Altschul et al., Nucleic Acids Res., 15:3389-3402, 1997.
  • BLAST Basic Local Alignment Search Tool
  • NCBI National Center for Biotechnology Information
  • the DKK1 described herein is preferably human DKK1, for example, the DKK1 whose amino acid sequence is shown in SEQ ID NO:73.
  • the invention provides aptamers directed against DKK1.
  • the aptamer specifically binds DKK1.
  • the aptamer comprises at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity to any of SEQ ID NOs: 1-20 nucleotide sequences that are identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical.
  • the aptamer is at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical to any of SEQ ID NOs: 1-20 identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or at least about 99% identity.
  • the aptamer comprises at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40 of any of SEQ ID NO: 1-20 , at least 45, at least 50, at least 55, at least 60, at least 65 or more contiguous nucleotides.
  • the aptamer is composed of at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50 or more contiguous nucleotides.
  • the aptamer comprises at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity to SEQ ID NO:5 Nucleotide sequences that are identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical.
  • the aptamer is at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical to SEQ ID NO: 5 Nucleotide sequence compositions that are identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical.
  • the aptamer comprises at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, At least 50, at least 55, at least 60, at least 65 or more contiguous nucleotides.
  • the aptamer is composed of at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45 of SEQ ID NO:5 , consisting of at least 50 or more consecutive nucleotides.
  • the aptamer comprises at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity to SEQ ID NO: 19 Nucleotide sequences that are identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical.
  • the aptamer is at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical to SEQ ID NO: 19 Nucleotide sequence compositions that are identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical.
  • the aptamer comprises at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, At least 50, at least 55, at least 60, at least 65 or more contiguous nucleotides.
  • the aptamer is composed of at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45 of SEQ ID NO: 19 , consisting of at least 50 or more consecutive nucleotides.
  • the aptamer comprises at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity to SEQ ID NO: 20 Nucleotide sequences that are identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical.
  • the aptamer is at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical to SEQ ID NO: 20 Nucleotide sequence compositions that are identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical.
  • the aptamer comprises at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, At least 50, at least 55, at least 60, at least 65 or more contiguous nucleotides.
  • the aptamer is composed of at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45 of SEQ ID NO: 20 , consisting of at least 50 or more consecutive nucleotides.
  • the aptamer comprises at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity to any of SEQ ID NOs: 45-64 nucleotide sequences that are identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical.
  • the aptamer comprises or consists of any nucleotide sequence in SEQ ID NO: 45-64.
  • the aptamer comprises or consists of the nucleotide sequence shown in SEQ ID NO: 49, 63 or 64.
  • the aptamer comprises at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity to any of SEQ ID NOs: 87-105 nucleotide sequences that are identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical.
  • the aptamer comprises or consists of any nucleotide sequence of SEQ ID NO: 87-105.
  • the aptamer comprises any nucleotide sequence in SEQ ID NO:87-90, 93-96 and 98-105 or consists of SEQ ID NO:87-90, 93-96 and 98- Any nucleotide sequence in 105.
  • the aptamer comprises any nucleotide sequence in SEQ ID NO: 1-20. In some embodiments, the aptamer consists of any nucleotide sequence in SEQ ID NO: 1-20.
  • the aptamer comprises any nucleotide sequence in SEQ ID NO: 21-44. In some embodiments, the aptamer consists of any nucleotide sequence in SEQ ID NO:21-44.
  • the aptamer comprises any nucleotide sequence of SEQ ID NO:74-86. In some embodiments, the aptamer consists of any nucleotide sequence in SEQ ID NO:74-86.
  • the aptamer comprises the nucleotide sequence of any one of SEQ ID NO:1-44, more preferably, the aptamer comprises any of SEQ ID NO:5, 19 or 20-44 A nucleotide sequence, more preferably, the aptamer comprises a nucleotide sequence of one of SEQ ID NO:5, 20 or 35-38.
  • the aptamer consists of any nucleotide sequence of SEQ ID NO: 1-44, more preferably, the aptamer consists of SEQ ID NO: 5, 19 or 20-44 One of the nucleotide sequences, more preferably, the aptamer consists of one of the nucleotide sequences of SEQ ID NO:5, 20 or 35-38. In some preferred embodiments, the aptamer comprises the nucleotide sequence described in SEQ ID NO: 37 or consists of the nucleotide sequence described in SEQ ID NO: 37.
  • the aptamer of the present invention has an effect on DKK1 of less than about 350 nM, preferably less than about 150 nM, preferably less than about 100 nM, preferably less than about 50 nM, preferably less than about 30 nM, preferably less than about 20 nM, preferably less than about 10 nM,
  • a Kd (dissociation constant) of less than about 5 nM, preferably less than about 1 nM or less is preferred.
  • the aptamer of the present invention has about 0.01 nM to about 350 nM, preferably about 0.01 nM to about 150 nM, preferably about 0.01 nM to about 100 nM, preferably about 0.01 nM to about 50 nM, preferably about 0.01 nM to DKK1.
  • Kd dissociation constant of nM to about 30 nM, preferably about 0.01 nM to about 20 nM, preferably about 0.01 nM to about 10 nM, preferably about 0.01 nM to about 5 nM, preferably about 0.01 nM to about 1 nM.
  • the K d is determined, for example, by enzyme-linked oligonucleotide assay (ELONA) or biofilm interferometry (BLI).
  • the aptamers of the invention inhibit the biological activity of DKK1.
  • “Inhibition” means that the biological activity of DKK1 is reduced in the presence of the aptamer, for example by at least about 10%, at least about 20%, at least about 30%, at least about 40%, compared to the absence of the aptamer. %, at least about 50%, at least about 60%, at least about 70%, at least about 80%, even at least about 90%.
  • biological activity means an effect on one or more cellular or extracellular processes, which may affect physiological or pathophysiological processes.
  • Biological activities of DKK1 include, but are not limited to, antagonizing the Wnt signaling pathway.
  • the aptamer of the present invention can inhibit the antagonism of DKK1 on the Wnt signaling pathway.
  • the aptamers of the invention can block DKK1 antagonism in cell-based Wnt signaling assays.
  • the aptamers of the present invention inhibit DKK1 with an IC50 value of less than about 2000 nM, preferably less than about 1500 nM, preferably less than about 1000 nM, preferably less than about 800 nM, preferably less than about 400 nM, preferably less than about 200 nM or less biological activities, such as inhibiting the antagonism of DKK1 on the Wnt signaling pathway.
  • the aptamer of the present invention has an IC value of about 200 nM to about 2000 nM, preferably about 200 nM to about 1500 nM, preferably about 200 nM to about 1000 nM, preferably about 200 nM to about 800 nM or preferably about 200 nM to about 400 nM Inhibiting the biological activity of DKK1, such as inhibiting the antagonism of DKK1 on the Wnt signaling pathway.
  • the IC50 value is determined in vitro by a TCF-Wnt induced luciferase activity assay in HEK293T cells.
  • the aptamers of the present invention may further comprise one or more modifications.
  • the modification is one that confers increased nuclease resistance on the aptamer and/or increases the in vivo half-life of the aptamer.
  • the aptamer is capped at the 3' end with inverted deoxythymidine, ie, 3' inverted deoxythymidine (3'idT) modification.
  • the modification may also include substituting a modified nucleotide for one or more naturally occurring nucleotides.
  • the modified nucleotides include, but are not limited to, 2'-fluoro, 2'-methoxyethyl, 2'-methoxy and/or 2'-acryloxy modified nucleotides (i.e. ribose The hydroxyl group at the 2' position is substituted by fluorine, methoxyethyl, methoxy or propyleneoxy, etc.).
  • the modified nucleotides may also include a C-5 modified pyrimidine.
  • C-5 modified pyrimidine refers to a pyrimidine modified at the C-5 position.
  • C-5 modified pyrimidines can enhance the nuclease resistance of oligonucleotides and are known in the art, for example, refer to International Patent Application WO2011/130195 and references thereto.
  • the modification is a 2'-methoxy (2'-OMe) modification.
  • the modification such as 2'-methoxyl (2'-OMe) modification, is carried out at one or more, such as 4 nucleotides, of the 5' and/or 3' end of the aptamer.
  • the modifications also include internucleotide modifications, such as those with uncharged linkages (e.g., methylphosphonate, phosphotriester, phosphoamidate, carbamate, etc.) and those with charged linkages.
  • Internucleotide modification of charged bonds e.g., phosphorothioate, phosphorodithioate, etc.
  • internucleotide modification with intercalators e.g., acridine, psoralen, etc.
  • chelating agents e.g., metals, radioactive metals, boron, oxidizing metals, etc.
  • internucleotide modifications containing alkylating agents e.g., boron, oxidizing metals, etc.
  • the modification may also include pegylation modification (PEG modification).
  • PEG modification pegylation modification
  • the half-life of the aptamer, eg in vivo, can be extended by conjugation with PEG.
  • the PEG has a molecular weight of about 1 kDa to about 100 kDa, such as about 10 kDa to about 80 kDa, about 20 kDa to about 60 kDa, about 30 kDa to about 50 kDa, about 40 kDa.
  • the PEG can be conjugated to the 5' end of the aptamer.
  • the PEG can be conjugated to the 3' end of the aptamer.
  • the modification may also include fatty acid modification or/or coumarin modification.
  • the aptamers shown can be conjugated to fatty acids and/or coumarin derivatives (e.g., to the 5' end of the aptamer molecule).
  • Suitable fatty acids include, but are not limited to, dodecanedioic acid, palmitic acid (PA), tetradecanedioic acid, hexadecandioic acid, stearic acid (SA), octadecanedioic acid, lauric acid, eicosanedioic acid, Pentaenoic acid (EPA), docosahexaenoic acid (DHA), arachidonic acid (ARA).
  • PA palmitic acid
  • SA stearic acid
  • SA octadecanedioic acid
  • lauric acid eicosanedioic acid
  • Pentaenoic acid EPA
  • DHA
  • Suitable coumarin derivatives include, but are not limited to, 4-hydroxycoumarin, 3-acetyl-6-carboxycoumarin, warfarin, (2-oxo-2H-chromen-3-yl)acetic acid, [(8-Acetyl-4-methyl-2-oxo-2H-chromen-7-yl)oxy]acetic acid, coumarin-3-carboxylic acid, N-(4-methyl-7-coumarin element) oxalic acid amide, 7-(carboxymethyl)-4-methylcoumarin, 7-methoxycoumarin-3-carboxylic acid, 6-methoxy-2-oxo-2H-chromene- 3-Carboxylic acid.
  • the aptamer may comprise a combination of the modifications described above.
  • the aptamer may comprise a 2'-methoxy (2'-OMe) modification and a 3' inverted deoxythymidine (3'idT) modification.
  • the in vivo half-life of the modified aptamers of the invention is at least about 2 times, at least about 5 times, at least about 10 times, at least about 25 times, at least about 50 times, at least about 100 times, at least about 200 times or more.
  • the present invention provides a method for treating a disease with the aptamer against DKK1 of the present invention, the method comprising administering a therapeutically effective amount of the aptamer against DKK1 of the present invention to a subject in need.
  • the diseases treated by the aptamer against DKK1 of the present invention are, for example, DKK1-related diseases, such as DKK1-mediated diseases.
  • the DKK1-associated disease is characterized by an aberrant expression, eg overexpression, of DKK1.
  • DKK1-associated disease includes DKK1-associated cancers.
  • a “DKK1-associated cancer” is a cancer characterized by aberrant expression, eg, overexpression, of DKK1.
  • the DKK1-related cancer is, for example, selected from myeloma (for example, multiple myeloma with osteolytic lesions, hilar cholangiocarcinoma multiple myeloma, etc.), breast cancer, colon cancer, melanoma , hepatocellular, epithelial, esophageal, gastric, gastroesophageal, hilar cholangiocarcinoma, brain, lung, prostate, or pancreatic cancer, and any metastases thereof.
  • myeloma for example, multiple myeloma with osteolytic lesions, hilar cholangiocarcinoma multiple myeloma, etc.
  • breast cancer colon cancer
  • melanoma hepatocellular, epithelial,
  • DKK1-associated disease includes bone-related diseases, eg, bone-related diseases associated with abnormal osteoblast or osteoclast activity.
  • Diseases characterized by abnormal osteoblast or osteoclast activity include, but are not limited to: primary and secondary osteoporosis, osteopenia, osteomalacia, osteogenesis imperfecta (OI), avascular necrosis ( osteonecrosis), bone fractures and implant healing (dental implants and hip implants), bone loss due to other conditions (for example, associated with HIV infection, cancer and arthritis).
  • Other “DKK1-associated diseases” include, but are not limited to: rheumatoid arthritis, osteoarthritis, arthritis, and osteolytic lesions.
  • the subject can be any animal (domesticated, domestic or wild), including but not limited to cats, dogs, horses, pigs and cows, and is preferably a human subject.
  • the terms patient, individual and subject are used interchangeably.
  • the subject can be male or female.
  • treating means that the subject's symptoms are partially or completely relieved, or remain unchanged after treatment.
  • treatment includes prophylaxis, treatment and/or cure.
  • Prevention refers to preventing an underlying disease and/or preventing worsening of symptoms or development of a disease.
  • therapeutically effective amount or “therapeutically effective dose” refers to an amount of a substance, compound, material, or composition comprising a compound that is at least sufficient to produce a therapeutic effect when administered to a subject. Thus, it is the amount necessary to prevent, cure, ameliorate, arrest or partially arrest the symptoms of a disease or disorder.
  • therapeutic effect means an effect resulting from treatment of a subject that alters, usually ameliorates or improves the symptoms of, or cures a disease or condition.
  • Dosage regimens utilizing the aptamer against DKK1 are selected according to a variety of factors including, for example, the type, category, age, weight, sex, and medical condition of the patient; the severity of the condition to be treated; administering pathway; the function of the patient's kidney and liver; and the specific aptamer or salt thereof against DKK1 used.
  • a physician of ordinary skill can readily determine and prescribe the effective amount of the composition required to prevent, combat or inhibit the progression of the condition.
  • the dosage regimen of the aptamer against DKK1 is about 1 ⁇ g/kg body weight to about 100 mg/kg body weight per day.
  • Exemplary treatment regimens entail dosing once daily, once every two days, once a week, twice a week, once every two weeks, once every three weeks, once every four weeks, once a month, every Once every 3 months, once every 3-6 months, or the initial dosing interval is slightly shorter (such as once a week to once every three weeks), and the later dosing interval is increased (such as once a month to once every 3-6 months ).
  • Dosing frequency and interval can be determined by those skilled in the art according to the pharmacokinetic parameters of the aptamer.
  • the present invention also provides a pharmaceutical composition, which includes at least one aptamer against DKK1 of the present invention, and a pharmaceutically acceptable carrier or excipient.
  • the pharmaceutical composition is used, for example, to treat DKK1-related diseases.
  • aptamers described herein can be used in any pharmaceutically acceptable dosage form, including but not limited to injectable dosage forms, liquid dispersions, gels, sprays, ointments, creams, lyophilized formulations, dry powders, tablets, Capsules, controlled release formulations, fast melt formulations, delayed release formulations, extended release formulations, pulsatile release formulations, mixed immediate release and controlled release formulations, and the like.
  • the aptamers described herein can be formulated: (a) by means of a drug selected from the group consisting of oral, pulmonary, intravenous, intraarterial, intrathecal, intraarticular, rectal, ophthalmic, colonic, parenteral, intracisternal, intravaginal (b) any one selected from liquid dispersion, gel, spray, ointment, cream, tablet, sachet and capsule (c) a dosage form selected from any one of lyophilized formulations, dry powders, fast-melt formulations, controlled release formulations, delayed release formulations, extended release formulations, pulsatile release formulations and mixed immediate release and controlled release formulations ; or (d) any combination thereof.
  • a drug selected from the group consisting of oral, pulmonary, intravenous, intraarterial, intrathecal, intraarticular, rectal, ophthalmic, colonic, parenteral, intracisternal, intravaginal
  • a dosage form selected from any one of lyophilized formulation
  • Solutions or suspensions for parenteral, intradermal or subcutaneous administration may contain one or more of the following components: (1) sterile diluents such as water for injection, saline solutions, fixed oils, polyethylene glycol Glycols, glycerin, propylene glycol, or other synthetic solvents; (2) antimicrobials, such as benzyl alcohol or methylparaben; (3) antioxidants, such as ascorbic acid or sodium sulfite; (4) chelating agents, such as ethylenediaminetetra Acetic acid; (5) buffers such as acetate, citrate or phosphate; and (5) substances for tonicity adjustment such as sodium chloride or glucose. pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use may include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the composition should be sterile and should be fluid to the extent that easy syringability exists.
  • the pharmaceutical compositions should be stable under the conditions of manufacture and storage and should be protected against the contaminating action of microorganisms such as bacteria and fungi.
  • stable as used herein means maintained in a state or condition suitable for administration to a patient.
  • the carrier can be a solvent or dispersion medium including, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof.
  • Proper fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents such as sugars, polyalcohols (eg, mannitol or sorbitol), and inorganic salts (eg, sodium chloride) in the compositions.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent delaying absorption, such as aluminum monostearate and gelatin.
  • Sterile disposables can be prepared by incorporating an active agent (eg, an aptamer against DKK1) in the required amount with one or a combination of ingredients enumerated above in an appropriate solvent, as required, followed by filtered sterilization. Injection solution.
  • an active agent eg, an aptamer against DKK1
  • dispersions are prepared by incorporating at least one aptamer directed against DKK1 into a sterile vehicle that contains a basic dispersion medium and any other desired ingredients.
  • exemplary methods of preparation include vacuum drying and freeze-drying, both of which will yield a powder of the aptamer against DKK1 as well as from its previous sterile filtration. Any additional desired components of the solution.
  • Oral compositions generally include an inert diluent or an edible carrier. For example, they may be enclosed in gelatin capsules or compressed into tablets.
  • the aptamer against DKK1 can be incorporated into excipients and used in the form of tablets, lozenges or capsules.
  • Pharmaceutically compatible binding agents and/or adjuvant materials may be included as part of the composition.
  • the drug is delivered as an aerosol spray from a pressurized container or dispenser containing a suitable propellant, such as a gas (such as carbon dioxide), an aerosolized liquid, or dry powder from a suitable device. said compound.
  • a suitable propellant such as a gas (such as carbon dioxide)
  • an aerosolized liquid or dry powder from a suitable device. said compound.
  • penetrants are used in the formulation that are appropriate to the barrier to be permeated. Such penetrants are generally known in the art and include, for example, for transmucosal administration, detergents, bile salts and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active agents are formulated into ointments, salves, gels or creams as is well known in the art.
  • the agents may also be prepared in the form of suppositories (eg, using conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • an aptamer directed against DKK1 is formulated for topical administration.
  • topical administration refers to the delivery of an aptamer against DKK1 to the aptamer against DKK1 by contacting (directly or otherwise) a formulation comprising the aptamer against DKK1 with all or part of the skin (epidermis) of an animal. animal.
  • the term encompasses several routes of administration including, but not limited to, topical and transdermal administration. A general requirement for these modes of administration is efficient delivery to the target tissue or layer.
  • topical application is used as a means of penetrating the epidermis and dermis and ultimately achieving systemic delivery of an aptamer directed against DKK1.
  • topical administration is used as a means of selectively delivering aptamers against DKK1 to the epidermis or dermis of animals or specific layers thereof.
  • the aptamer against DKK1 can be formulated into pharmaceutically acceptable ointments, creams, lotions, ophthalmic ointments, eye drops, ear drops, impregnated dressings, and aerosols, medicated Powders, medicated binders, foams, and may contain suitable conventional additives or excipients including, for example, preservatives or solvents to aid penetration of the drug and emollients in ointments, gels and creams.
  • Such topical formulations may also contain compatible conventional carriers, such as ethanol or oleyl alcohol for emulsions. Such carriers may constitute from about 1% to about 98% by weight of the formulation, more typically such carriers will constitute up to about 80% by weight of the formulation. Specific formulations for local delivery of aptamers have been described in the prior art.
  • aptamers to DKK1 are prepared with a carrier that will prevent rapid removal from the body.
  • controlled release formulations including implants and microencapsulated delivery systems, may be used.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.
  • suspensions of said aptamers directed against DKK1 may be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil or synthetic fatty acid esters such as ethyl oleate, triglycerides, or liposomes.
  • Non-lipid polycationic amino acid polymers can also be used for delivery.
  • the suspension may also contain suitable stabilizers or agents to increase the solubility of the compounds and to allow for the preparation of highly concentrated solutions.
  • Dosage unit form as used herein refers to physically discrete units suitable as unitary dosages for a subject to be treated; each unit contains a predetermined amount of an aptamer against DKK1 calculated to produce the desired therapeutic effect together with the required drug carrier.
  • the specification of the dosage unit forms of the aptamers directed against DKK1 described herein is determined by and directly dependent on the unique characteristics of the specific aptamers directed against DKK1 and the particular therapeutic effect to be achieved and the formulation of such active agents for the treatment of an individual inherent limitations of the technology.
  • a pharmaceutical composition comprising at least one aptamer directed against DKK1 may comprise one or more pharmaceutical excipients.
  • excipients include, but are not limited to, binders, fillers, lubricants, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants, effervescent agents and other excipients. Such excipients are known in the art.
  • Exemplary excipients include: (1) Binders, including various celluloses and cross-linked polyvinylpyrrolidone, microcrystalline cellulose (such as Avicel PH101 and Avicel PH102), silicified microcrystalline cellulose (ProSolv SMCC TM ), yellow Achillea gum and gelatin; (2) fillers such as various starches, lactose, lactose monohydrate, anhydrous lactose; (3) disintegrants such as alginic acid, Primogel, corn starch, lightly cross-linked polyvinyl Pyrrolidine, potato starch, corn starch and modified starches, croscarmellose sodium, crospovidone, sodium starch glycolate and mixtures thereof; (4) Lubricants, including those acting on the powder to be compressed Fluidity agents, including magnesium stearate, colloidal silicon dioxide (such as Aerosil200, talc), stearic acid, calcium stearate and silica gel; (5) glidants, such as colloidal silicon dioxide; (6)
  • Protein-SELEX A Ni-NTA bead-based SELEX method was used to screen for aptamers of DKK1. Briefly, His6-tagged DKK-1 was first incubated with Ni-NTA beads to form protein-bound Ni-NTA magnetic beads. This complex was then incubated with a ssDNA random library at room temperature to form ssDNA-protein-bound Ni-NTA magnetic bead complexes. After washing, the ssDNA-protein-bound Ni-NTA magnetic bead complex was used as a template for PCR with forward primer and biotinylated reverse primer. Regenerate ssDNA using streptavidin-coated magnetic beads according to the manufacturer's protocol. The ssDNA pool was used for the next round of SELEX.
  • the evolved ssDNA pool was incubated with Ni-NTA beads or non-target proteins for reverse selection. Unbound ssDNA from the reverse selection negative selection was then collected and applied to DKK1-bound Ni-NTA magnetic beads. ssDNA products from rounds 10, 16, 18 and 20 were used to determine the degree of enrichment of the selection process compared to the initial random library. Cloning and sequencing of the ssDNA library with the highest binding affinity.
  • Enzyme-Linked Oligonucleotide Assay 160 ng per well of DKK1 and negative targets were coated by hydrophobic interaction into 96-well microtiter plates overnight at 4°C. Non-binding sites in the wells were blocked with BSA for 1 hr at room temperature, and then washed 5 min x 4 times with DNA binding buffer. Add the appropriate concentration of biotinylated aptamer candidate/RS to each well and incubate for 45 min at room temperature with constant gentle shaking. After binding, the plate was washed 5 min x 4 times with wash buffer to remove non-specific and weakly bound sequences.
  • HRP streptavidin-horseradish peroxidase
  • TCF-wnt-induced luciferase activity assay HEK293T cells were co-transfected with corresponding plasmids including TOPFlash, FOPFlash, Wnt3a and DKK1 constructs. An equal amount of vector was used for each transfection. 24 hours after transfection, the medium was changed. Cells were treated with conditioned medium containing 1 ⁇ M of the corresponding aptamer candidate. To determine inhibitory potency, cells were treated with gradient concentrations of aptamer candidates. Twenty-four hours after treatment, cells were lysed with 100 ⁇ l of lysis buffer and 20 ⁇ l was used for analysis. Calculations were performed according to the protocol (Dual Luciferase Reporter Assay, Promega).
  • DNA detection The band density of all aptamer samples was determined by a molecular imager (Bio-Rad) (Klussmann, Nolte et al. 1996, Siller-Matula, Merhi et al. 2012).
  • Ni-NTA Magnetic Sepharose Beads are supplied as a 5% (v/v) suspension with a binding capacity of 300 ⁇ g protein ( ⁇ 24 kDa) per mL of suspension for 6x His-tagged proteins.
  • the amount and ratio of recombinant His6-DKK1 and NTA magnetic beads were optimized for immobilization of 0.2 nmol DKK1 on magnetic beads in the subsequent first round of SELEX. 0.8nmol protein + 10 ⁇ l magnetic beads, 0.6nmol protein + 10 ⁇ l magnetic beads, 0.4nmol protein + 10 ⁇ l magnetic beads, 0.8nmol protein + 6 ⁇ l magnetic beads, 0.6nmol protein + 6 ⁇ l magnetic beads and 0.4nmol protein + 6 ⁇ l magnetic beads at 4°C Incubate for 60 minutes.
  • Recombinant human DKK-1 with His6 tag will be immobilized on Ni-NTA magnetic beads and used as forward screening target for forward screening. Blank Ni-NTA beads and other His6-tagged proteins will be used as negative selection targets for negative selection.
  • the pool of ssDNA collected after each round of forward selection was amplified by PCR and checked by agarose gel electrophoresis before ssDNA regeneration ( Figure 2). Monitor PCR cycles to avoid non-specific by-products in PCR. 20 rounds of SELEX were performed.
  • the ssDNA pool from the 10th and final round was sequenced by Next Generation Sequencing. There were no enriched sequences in round 10, while enriched sequences appeared in the last round (Table 1). Therefore, the 20 most frequently occurring potential sequences were identified in the final enriched ssDNA pool and named Aptdkk-1-20 (Table 2). According to the results of multiple sequence alignment using Cluster Omega, these sequences can be divided into two groups (Fig. 3). Group 1 is clearly guanine-rich (shown in red box), while group 2 is non-G-rich sequences (shown in blue box).
  • G-rich sequences often form specialized G-quadruplex structures that have a stable helical shape and contain guanine tetrads that can be formed by one, two, or four strands.
  • G-rich sequences were then analyzed by QGRS for potential G groups and G-scores for their tendency to form unimolecular quadruplexes. Typically, the size and distribution of gaps in the predicted QGRS are considered when calculating the G-score. Sequences with higher scores will be better candidates for G-quadruplexes. According to the analysis results, all these sequences have two G tetrads, which can form two G chains (Table 3). G scores are around 19-20. Furthermore, the distribution of intervals is shown in a similar pattern. This suggests that their ability to form stable G-quadruplexes is very similar.
  • Aptamer candidates with significant specificity and candidate 3 as a negative control were used to determine the binding affinity to DKK1, except for candidates 1, 2, 4 and 14 at concentrations of (0, 62.5 nM, 125 nM, 250 nM, 500nM, 1000nM, 2000nM, 4000nM), the concentrations of candidates 7, 10, 15 and 16 were (0, 23.44nM, 46.86nM, 93.75nM, 187.5nM, 375nM, 750nM, 1500nM), the other candidates were at (0 , 0.18nM, 0.74nM, 2.96nM, 11.84nM, 47.35nM, 189.39nM, 757.58nM).
  • the binding curves for each candidate are shown in Figure 5 and Table 5. Eleven aptamer candidates showed high affinity (low Kd values) for DKK1. Six candidates (aptdkk-5, 13, 20, 19, 6 and 1) were G-enriched. The other five sequences (aptdkk-9, 18, 12, 17 and 8) were not G-rich.
  • the data for candidate 19 did not fit the fitted curve for the tested concentrations, indicating a higher IC50 ( Figure 7). This result indicated that both aptdkk-5 and aptdkk-20 exhibited an inhibitory effect on DKK1 inhibition of Wnt signaling, whereas aptdkk-19 had a relatively weaker effect.
  • apt5-1-50, apt5-1-47, apt5-1-44, apt5-1-41, apt5-1-38, apt5-1-35, apt5-1-65, apt5- 1-62, apt5-1-59 and apt5-1-53 showed no significant/slight difference in absorbance compared to native aptdkk5 (Fig. 8b).
  • apt5-1-44, apt5-1-41, apt5-1-38 and apt5-1-35 were selected to determine the binding affinity to DKK1.
  • Aptamer candidates with significant specificities (apt5-1-44, apt5-1-41, apt5-1-38, and apt5-1-35) and the original aptdkk5 as a positive control were used to determine the binding affinity to DKK1, where the concentrations of original aptdkk5 and apt5-1-44 are (0.15625nM, 0.625nM, 1.25nM, 2.5nM, 5nM, 10nM, 50nM, 100nM), apt5-1-41, apt5-1-38 and apt5-1- The concentrations of 35 were (0, 1.5625 nM, 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM). The binding curves for each candidate are shown in Figure 9. Eleven aptamer candidates showed high affinity (low Kd values) for DKK1. Among the four candidates, apt5-1-38 showed the highest affinity to DKK1, so it was selected as the best aptamer for further study.
  • aptdkk5s significantly binds to full-length DKK1, but hardly binds to other truncated DKK1s, indicating that the C domain (178-266aa) of DKK1 may be the binding domain of DKK1 and aptdkk5s ( Figure 12 ).
  • IDT modifications of aptdkk5s were used for the following characterizations, including dkk5s-2G-indT and dkk5s-OM-indT. Both modified and original aptdkk5s showed remarkably high absorbance (>2), whereas all modified aptdkk5s had slightly lower absorbance than native aptdkk5s ( Figure 13), suggesting a rather high specificity of aptdkk5s modification.
  • the modified aptamer and the original aptdkk5s as a positive control were used to determine the binding affinity to DKK1 at different concentrations (0, 15.625 nM, 31.25 nM, 62.5 nM, 125 nM, 250 nM, 500 nM, 1000 nM) by ELONA.
  • the modified aptdkk5s (dkk5s-2G-indT and dkk5s-OM-indT) both showed high affinity for DKK1, but slightly lower (higher Kd values) compared to the original aptdkk5s (Fig. 14).
  • Serum metabolic stability of modified and unmodified aptamers was assessed in fetal bovine serum. Aptamers were treated with 10% and 100% fetal bovine serum from 0 to 72 hours (Fig. 15).
  • Raw aptdkk5s start to degrade after 16 hours of incubation in 10% FBS and from 2 hours in 100% FBS.
  • Dkk5s-2G-indT began to degrade after 72 hours of incubation in 10% FBS and from 8 hours in 100% FBS.
  • Dkk5s-OM-indT began to degrade after 48 hours of incubation in 10% FBS and from 8 hours in 100% FBS.
  • the serum stability of the modified aptdkk5s was significantly improved compared with the unmodified one, in other words, both the 3′-end inverted dT and O-methyl modification of IDT enhanced the serum metabolic stability.
  • Example 14 Inhibition potency of chemically modified aptdkk5s
  • DKK-1 was determined by ELONA using 1 ⁇ M of each aptamer/RS. The results showed that most of the aptamers showed significantly higher absorbance compared to the vector, while candidates 3, 7, 8 and 12 had higher absorbance compared to the previously identified aptddk5s (Fig. 19).
  • the binding affinity to DKK1 was determined by BLI using the candidate aptamers. DKK1 protein was applied at gradient concentrations of 0 nM, 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM, and 200 nM. In addition to candidates 5, 6 and 11 (Table 9, Figure 20), 16 candidates were detected with high affinity, with Kd values ranging from 1.67 nM to 23 nM.

Abstract

属于生物医药领域。具体而言，提供针对DKK1的适体及其用途，特别是其用于治疗DKK1相关疾病例如DKK1相关癌症中的用途。

Description

针对DKK1的适体及其用途 技术领域
本发明涉及生物医药领域。具体而言，本发明涉及针对DKK1的适体及其用途，特别是其用于治疗DKK1相关疾病例如DKK1相关癌症中的用途。
发明背景
Dickkopf-1(DKK1)是dickkopf蛋白家族的成员，已被证明是Wnt信号传导的负调节因子，其在骨发育和形成中具有核心作用。通过结合LRP5(LRP6)和kremen蛋白，DKK1阻止LRP5或LRP6与Wnt通路的成员结合，从而阻止Wnt介导的信号转导，从而抑制骨形成。
在临床研究中，已在患者血清或肿瘤中的一系列癌症中检测到Wnt拮抗剂DKK1水平升高，并且这通常与不良预后相关。此外，DKK1促进免疫抑制性肿瘤微环境并通过抑制T细胞的方式诱导肿瘤逃避免疫监视。名为DKN-01的针对DKK1的人源化单克隆治疗性抗体与化学疗法相结合，已在II期试验中用于治疗食管胃癌和其他类型的癌症。因此，DKK1可能是肿瘤免疫治疗的一个有希望的靶点。然而，治疗性抗体有几个主要问题，包括高免疫原性(Padhi,Jang et al.,2011；Padhi,Allison et al.,2014)，生产成本高(Baker,2015；Bradbury and Pluckthun 2015；Groff,Brown et al.,2015)，不稳定，需要连续冷链运输和储存(Jayasena,1999)。因此，对于抗肿瘤疗法而言，期望无免疫原性、容易生产、低成本和高稳定性的替代性抗DKK1剂。
适体(aptamer)因其高结合亲和力和特异性以及低免疫原性而受到广泛关注。适体是通过构象互补而与其靶标结合的短单链寡核苷酸(Ellington and Szostak,1990；Tuerk and Gold,1990)。与治疗性抗体相比，适体具有相似的亲和力和特异性，但是具有一些重要的优点。对于免疫原性，适体不会被免疫系统识别为外来的，并且因为低分子量而不刺激负面的免疫应答(Keefe,Pai et al.,2010)。就生产和成本而言，适体可以在各种选择条件下在体外鉴定，并且可以通过化学方法容易地合成，因此生产成本更低，风险更低(Banerjee,2010)。就稳定性而言，适体具有无限的保存期限，因为它们温度不敏感，并且在运输过程中没有任何冷却的特殊要求，因此不需要连续的冷链(Jayasena,1999)。Pegaptanib，一种针对血管内皮生长因子(VEGF)的适体，已成功用于临床治疗年龄相关性黄斑变性(Jellinek,Green et al.,1994；Ruckman,Green et al.,1998；Ng and Adamis 2006；Que-Gewirth and Sullenger 2007)。
因此，期望开发针对DKK1的适体来代替单克隆抗体用于治疗DKK1相关癌症。
发明简述
在一方面，本发明提供一种针对DKK1的适体，所述适体
i)包含与SEQ ID NO:1-20中的任一具有至少大约90％相同性、至少大约91％相同性、至少大约92％相同性、至少大约93％相同性、至少大约94％相同性、至少大约95％相同性、至少大约96％相同性、至少大约97％相同性、至少大约98％相同性或至少大约99％相同性的核苷酸序列；或者，
ii)包含SEQ ID NO:1-20中的任一中的至少30个、至少35个、至少40个、至少45个、至少50个或更多个连续的核苷酸；或者
iii)包含与SEQ ID NO:45-64中的任一具有至少大约90％相同性、至少大约91％相同性、至少大约92％相同性、至少大约93％相同性、至少大约94％相同性、至少大约95％相同性、至少大约96％相同性、至少大约97％相同性、至少大约98％相同性或至少大约99％相同性的核苷酸序列；或者
iv)包含与SEQ ID NO:87-105中的任一具有至少大约90％相同性、至少大约91％相同性、至少大约92％相同性、至少大约93％相同性、至少大约94％相同性、至少大约95％相同性、至少大约96％相同性、至少大约97％相同性、至少大约98％相同性或至少大约99％相同性的核苷酸序列，
其中所述适体特异性结合DKK1。
在一些实施方案中，所述适体包含SEQ ID NO:1-64和87-105中的任一的序列。
在一些实施方案中，所述适体包含SEQ ID NO:5、20、35-38、49、或64所示的核苷酸序列，优选包含SEQ ID NO:37所示核苷酸序列。
在一些实施方案中，所述适体包含一种或多种昂赋予所述适体增强的核酸酶抗性和/或增强所述适体的体内半衰期的修饰。
在一些实施方案中，所述适体包含2’-甲氧基(2’-OMe)修饰和3’反向脱氧胸苷(3’idT)修饰。
在一些实施方案中，所述适体对DKK1具有小于350nM，优选小于150nM，优选小于100nM，优选小于50nM，优选小于30nM，优选小于20nM，优选小于10nM，优选小于5nM，优选小于1nM或更小的K d。
在一些实施方案中，所述适体能够抑制DKK1的生物学活性。在一些实施方案中，所述适体能在基于细胞的Wnt信号测试法中阻断DKK1的拮抗作用。在一些实施方案中，所述适体以小于2000nM，优选小于1500nM，优选小于1000nM，优选小于800nM或更小的IC50值抑制DKK1的生物学活性，例如抑制DKK1对Wnt信号通路的拮抗作用。
在另一方面，本发明提供一种治疗DKK1相关疾病的方法，该方法包括给有需要的对象施用治疗有效量的本发明的针对DKK1的适体，所述对象例如是人。
在一些实施方案中，所述DKK1相关疾病为DKK1相关癌症，例如骨髓瘤(例如，伴随溶骨性病变的多发性骨髓瘤、肝门部胆管癌多发性骨髓瘤等)、乳腺癌、结肠癌、黑色素瘤、肝细胞癌、上皮癌、食道癌、胃癌、胃食管癌、肝门部胆管癌、脑癌、肺癌、前列腺癌或胰癌，及其任何转移瘤。
在一些实施方案中，所述DKK1相关疾病选自骨质疏松症、骨质减少、骨软化、成骨不全(OI)、缺血性骨坏死、类风湿关节炎、骨折、骨关节炎和骨髓瘤。
在另一方面，本发明提供一种药物组合物，其包含至少一种本发明的针对DKK1的适体，和药学上可接受的载体或赋形剂。
在另一方面，本发明提供本发明所述的针对DKK1的适体或本发明所述的药物组合物在制备药物中的用途，其中所述药物用于治疗DKK1相关疾病。
在一些实施方案中，所述DKK1相关疾病为DKK1相关癌症，例如骨髓瘤(例如，伴随溶骨性病变的多发性骨髓瘤、肝门部胆管癌多发性骨髓瘤等)、乳腺癌、结肠癌、黑色素瘤、肝细胞癌、上皮癌、食道癌、胃癌、胃食管癌、肝门部胆管癌、脑癌、肺癌、前列腺癌或胰癌，及其任何转移瘤。
在一些实施方案中，所述DKK1相关疾病选自骨质疏松症、骨质减少、骨软化、成骨不全(OI)、缺血性骨坏死、类风湿关节炎、骨折、骨关节炎和骨髓瘤。
附图简述
图1示出测定Ni-NTA磁性琼脂糖珠对DKK1蛋白的结合能力。进行了两个实验来确定Ni-NTA磁性琼脂糖珠对DKK1蛋白的结合能力。在还原条件下的SDS-PAGE中，DKK1的表观分子量约为40KDa。该条件(0.6nmol DKK1+6μl 5％珠子)显示出最高的结合能力。
图2示出第1轮至第20轮筛选的适体池的聚合酶链反应(PCR)产物的琼脂糖凝胶(3％)电泳分析。第一道，DNA梯带。DNA条带大约存在于80bp。
图3示出适体候选物的进化树和比对。二十个适体的序列聚集成两组。第1组包含8个富含鸟嘌呤的序列，称为富含G的组；第2组包括十二个属于其他核苷酸的序列，称为非富含G的组。
图4示出代表性适体候选物的特异性。使用酶联寡核苷酸测定(ELONA)将1μM的每个适体/RS用于确定对DKK1的特异性，并检测450nm处的吸光度。****P<0.0001；**P<0.005；*P<0.05。
图5示出代表性适体候选物的亲和力。使用特定浓度的每种适体来测定对DKK-1的亲和力，并测定450nm处的吸光度。
图6示出代表性适体候选物的抑制作用。与对照组相比，aptdkk-5、19和20显示出更高的比率，这表明它们有可能解除DKK1对Wnt信号的抑制。
图7示出代表性适体候选物的抑制效力。aptdkk-5发挥较高的抑制效力，IC 50＝754±29nM，而aptdkk-20的抑制效力较低，IC 50＝1504±39nM。aptdkk-19的数据不符合拟 合曲线。
图8示出截短的aptdkk5的特异性。a)100nM和b)1nM的每个截短的aptdkk5用于确定对DKK-1的特异性，将其在450nm处的吸光度与原始aptdkk5的吸光度进行比较。*P<0.05；NS，无显著差异。
图9示出截短的aptdkk5的亲和力。梯度浓度的每个适体用于确定对DKK1的结合亲和力，并确定450nm处的吸光度。
图10.Aptdkk5s突变体的特异性。使用1μM每种适体通过ELONA测定对DKK-1的特异性，检测450nm处的吸光度。****P<0.0001；**P<0.005；*P<0.05。
图11.通过SDS-PAGE凝胶电泳测量的HEK293T细胞中DKK1、缺陷型DKK1的表达水平。S4，pCS2-DKK1-His-flag；S3，pCS2-DKK1-ΔC-His-flag；S2，pCS2-DKK1-ΔL2&C-His-flag。
图12.全长和截短的DKK1的结合能力。aptdkk5s与DKK1突变体结合的荧光图像。S4，pCS2-DKK1-His-flag；S3，pCS2-DKK1-ΔC-His-flag；S2，pCS2-DKK1-ΔL2&C-His-flag；阳性Ctrl，纯蛋白DKK1His6；阴性Ctrl，只有珠。
图13.修饰的aptdkk5s的特异性。使用1μM每种适体通过ELONA测定对DKK-1的特异性，检测450nm处的吸光度。
图14.修饰的aptddk5s的亲和力。使用一定浓度的每种适体通过ELONA来测定对DKK-1的亲和力，检测450nm处的吸光度。
图15.修饰的aptdkk5s与未修饰的aptdkk5s相比的血清稳定性评估。分别用10％和100％胎牛血清从0到72小时处理所有适体。
图16.aptdkk5-OM-indT的抑制效力。每组分析一式三份。
图17.序列富集趋势曲线。每轮中DNA池的富集度是通过每轮中适体的类型除以测序读段的数量来计算的。饱和富集被认为发生在第24轮。
图18.第21至第30轮选择的适体池的聚合酶链反应(PCR)产物的琼脂糖凝胶(3％)电泳分析。前泳道，DNA阶梯。DNA条带大约在80bp。
图19.第30轮代表性适体候选物的特异性。使用1μM每中适体/RS通过酶联寡核苷酸测定法(ELONA)测定对DKK-1的特异性，检测450nm处的吸光度。
图20.代表性适体候选物的结合曲线。使用一定浓度的每种适体通过BLI来确定对DKK-1的亲和力。
发明详述
除非另有指示或定义，否则所有所用术语均具有本领域中的通常含义，该含义将为本领域技术人员所了解。参考例如标准手册，如Sambrook et al.,“Molecular Cloning:A Laboratory Manual”；Lewin,“Genes VIII”；及Roitt et al.,“Immunology”(第8版)，以及本文中引用的一般现有技术；此外，除非另有说明，否则未具体详述的所有方法、步骤、技术及操作均可以且已经以本身已知的方式进行，该方式将为本领域技术人员所了 解。亦参考例如标准手册、上述一般现有技术及其中引用的其他参考文献。
定义
如本文所用，术语“核苷酸”是指核糖核苷酸或脱氧核糖核苷酸，或其修饰的形式及其类似物。核苷酸包括种类，其包括嘌呤(例如，腺嘌呤、次黄嘌呤、鸟嘌呤和它们的衍生物和类似物)以及嘧啶(例如，胞嘧啶、尿嘧啶、胸腺嘧啶和它们的衍生物和类似物)。
在本文中，互换地使用“核酸”、“寡核苷酸”和“多核苷酸”来指核苷酸的聚合物，并包括DNA、RNA、DNA/RNA杂交体和这些种类的核酸、寡核苷酸和多核苷酸的修饰，其中包括在所述核苷酸单位的任何位置上附加各种实体或部分。术语“多核苷酸”、“寡核苷酸”和“核酸”包括双或单链分子。核酸、寡核苷酸和多核苷酸是比术语适体更宽的术语，因此术语核酸、寡核苷酸和多核苷酸包括适体但不限于适体。
如本文所用，“适体”指具有期望的对靶分子的作用的非天然存在的核酸。期望的作用包括但不限于与所述靶结合、催化改变所述靶、以修饰或改变所述靶或所述靶的功能活性的方式与靶反应、共价地连接所述靶和促进所述靶与其他分子间的反应。在一些实施方案中，所述作用是针对靶分子(例如DKK1)的特异性结合亲和力，这样的靶分子是三维化学结构而不是多核苷酸，其通过不依赖于Watson/Crick碱基配对或三重螺旋形成的机制来结合所述适体，其中所述适体不是具有被所述靶分子结合的已知生理功能的核酸。在本上下文中，适体针对其靶(例如DKK1)的“特异性结合”是指所述适体通常以比其结合至混合物或样品中其他的非靶组分高得多的亲和力来结合至其靶。
序列“相同性”具有本领域公认的含义，并且可以利用公开的技术计算两个核酸或多肽分子或区域之间序列相同性的百分比。可以沿着多核苷酸或多肽的全长或者沿着该分子的区域测量序列相同性。(参见，例如：Computational Molecular Biology,Lesk,A.M.,ed.,Oxford University Press,New York,1988；Biocomputing:Informatics and Genome Projects,Smith,D.W.,ed.,Academic Press,New York,1993；Computer Analysis of Sequence Data,Part I,Griffin,A.M.,and Griffin,H.G.,eds.,Humana Press,New Jersey,1994；Sequence Analysis in Molecular Biology,von Heinje,G.,Academic Press,1987；and Sequence Analysis Primer,Gribskov,M.and Devereux,J.,eds.,M Stockton Press,New York,1991)。虽然存在许多测量两个多核苷酸或多肽之间的相同性的方法，但是术语“相同性”是技术人员公知的(Carrillo,H.&Lipman,D.,SIAM J Applied Math 48:1073(1988))。许多算法可用于确定序列相同性百分比。适合于确定序列相同性百分比的算法的一个实例是在基本局部比对搜索工具(以下为“BLAST”)中使用的算法，见例如Altschul等人，J.Mol.Biol.215:403-410,1990和Altschul等人，Nucleic Acids Res.,15:3389-3402,1997。进行BLAST分析的软件是通过国家生物技术信息中心(以下为“NCBI”)公开地可获得的。在使用从NCBI可获得的软件(如针对核酸序列的BLASTN)来确定序列相同性所使用的默认参数在McGinnis等人Nucleic Acids Res.,32:W20-W25,2004中有所描述。
针对DKK1的适体
本发明人基于蛋白质-SELEX技术，采用DKK1作为靶蛋白正向筛选，并采用不相关蛋白进行负筛选，最终选择出以高亲和力特异性地结合DKK1的适体。本文所述DKK1优选是人DKK1，例如，氨基酸序列示于SEQ ID NO:73的DKK1。
示例性人DKK1氨基酸序列：
Figure PCTCN2022121254-appb-000001
因此，在一方面，本发明提供针对DKK1的适体。在一些实施方案中，所述适体特异性结合DKK1。
在一些实施方案中，所述适体包含与SEQ ID NO:1-20中的任一具有至少大约90％相同性、至少大约91％相同性、至少大约92％相同性、至少大约93％相同性、至少大约94％相同性、至少大约95％相同性、至少大约96％相同性、至少大约97％相同性、至少大约98％相同性或至少大约99％相同性的核苷酸序列。在一些实施方案中，所述适体由与SEQ ID NO:1-20中的任一具有至少大约90％相同性、至少大约91％相同性、至少大约92％相同性、至少大约93％相同性、至少大约94％相同性、至少大约95％相同性、至少大约96％相同性、至少大约97％相同性、至少大约98％相同性或至少大约99％相同性的核苷酸序列组成。在一些实施方案中，所述适体包含SEQ ID NO:1-20中的任一中的至少10个、至少15个、至少20个、至少25个、至少30个、至少35个、至少40个、至少45个、至少50个、至少55个、至少60个、至少65个或更多个连续的核苷酸。在一些实施方案中，所述适体由与SEQ ID NO:1-20中的任一中的至少10个、至少15个、至少20个、至少25个、至少30个、至少35个、至少40个、至少45个、至少50个或更多个连续的核苷酸组成。
在一些实施方案中，所述适体包含与SEQ ID NO:5具有至少大约90％相同性、至少大约91％相同性、至少大约92％相同性、至少大约93％相同性、至少大约94％相同性、至少大约95％相同性、至少大约96％相同性、至少大约97％相同性、至少大约98％相同性或至少大约99％相同性的核苷酸序列。在一些实施方案中，所述适体由与SEQ ID NO:5具有至少大约90％相同性、至少大约91％相同性、至少大约92％相同性、至少大约93％相同性、至少大约94％相同性、至少大约95％相同性、至少大约96％相同性、至少大约97％相同性、至少大约98％相同性或至少大约99％相同性的核苷酸序列组成。在一些实施方案中，所述适体包含SEQ ID NO:5中的至少10个、至少15个、至少20个、至少25个、至少30个、至少35个、至少40个、至少45个、至少50个、至少55个、至少60个、至少65个或更多个连续的核苷酸。在一些实施方案中，所述适体由与SEQ ID NO:5中的至少10个、至少15个、至少20个、至少25个、至少30个、至少 35个、至少40个、至少45个、至少50个或更多个连续的核苷酸组成。
在一些实施方案中，所述适体包含与SEQ ID NO:19具有至少大约90％相同性、至少大约91％相同性、至少大约92％相同性、至少大约93％相同性、至少大约94％相同性、至少大约95％相同性、至少大约96％相同性、至少大约97％相同性、至少大约98％相同性或至少大约99％相同性的核苷酸序列。在一些实施方案中，所述适体由与SEQ ID NO:19具有至少大约90％相同性、至少大约91％相同性、至少大约92％相同性、至少大约93％相同性、至少大约94％相同性、至少大约95％相同性、至少大约96％相同性、至少大约97％相同性、至少大约98％相同性或至少大约99％相同性的核苷酸序列组成。在一些实施方案中，所述适体包含SEQ ID NO:19中的至少10个、至少15个、至少20个、至少25个、至少30个、至少35个、至少40个、至少45个、至少50个、至少55个、至少60个、至少65个或更多个连续的核苷酸。在一些实施方案中，所述适体由与SEQ ID NO:19中的至少10个、至少15个、至少20个、至少25个、至少30个、至少35个、至少40个、至少45个、至少50个或更多个连续的核苷酸组成。
在一些实施方案中，所述适体包含与SEQ ID NO:20具有至少大约90％相同性、至少大约91％相同性、至少大约92％相同性、至少大约93％相同性、至少大约94％相同性、至少大约95％相同性、至少大约96％相同性、至少大约97％相同性、至少大约98％相同性或至少大约99％相同性的核苷酸序列。在一些实施方案中，所述适体由与SEQ ID NO:20具有至少大约90％相同性、至少大约91％相同性、至少大约92％相同性、至少大约93％相同性、至少大约94％相同性、至少大约95％相同性、至少大约96％相同性、至少大约97％相同性、至少大约98％相同性或至少大约99％相同性的核苷酸序列组成。在一些实施方案中，所述适体包含SEQ ID NO:20中的至少10个、至少15个、至少20个、至少25个、至少30个、至少35个、至少40个、至少45个、至少50个、至少55个、至少60个、至少65个或更多个连续的核苷酸。在一些实施方案中，所述适体由与SEQ ID NO:20中的至少10个、至少15个、至少20个、至少25个、至少30个、至少35个、至少40个、至少45个、至少50个或更多个连续的核苷酸组成。
在一些实施方案中，所述适体包含与SEQ ID NO:45-64中的任一具有至少大约90％相同性、至少大约91％相同性、至少大约92％相同性、至少大约93％相同性、至少大约94％相同性、至少大约95％相同性、至少大约96％相同性、至少大约97％相同性、至少大约98％相同性或至少大约99％相同性的核苷酸序列。在一些实施方案中，所述适体包含SEQ ID NO:45-64中的任一核苷酸序列或由SEQ ID NO:45-64中的任一核苷酸序列组成。在一些优选实施方案中，所述适体包含SEQ ID NO:49、63或64所示核苷酸序列或由SEQ ID NO:49、63或64所示核苷酸序列组成。
在一些实施方案中，所述适体包含与SEQ ID NO:87-105中的任一具有至少大约90％相同性、至少大约91％相同性、至少大约92％相同性、至少大约93％相同性、至少大约94％相同性、至少大约95％相同性、至少大约96％相同性、至少大约97％相同性、至少大约98％相同性或至少大约99％相同性的核苷酸序列。在一些实施方案中，所述适体包 含SEQ ID NO:87-105中的任一核苷酸序列或由SEQ ID NO:87-105中的任一核苷酸序列组成。在一些实施方案中，所述适体包含SEQ ID NO:87-90、93-96和98-105中的任一核苷酸序列或由SEQ ID NO:87-90、93-96和98-105中的任一核苷酸序列组成。
在一些实施方案中，所述适体包含SEQ ID NO:1-20中的任一核苷酸序列。在一些实施方案中，所述适体由SEQ ID NO:1-20中的任一核苷酸序列组成。
在一些实施方案中，所述适体包含SEQ ID NO:21-44中的任一核苷酸序列。在一些实施方案中，所述适体由SEQ ID NO:21-44中的任一核苷酸序列组成。
在一些实施方案中，所述适体包含SEQ ID NO:74-86中的任一核苷酸序列。在一些实施方案中，所述适体由SEQ ID NO:74-86中的任一核苷酸序列组成。
在一些优选实施方案中，所述适体包含SEQ ID NO:1-44中的任一的核苷酸序列，更优选地，所述适体包含SEQ ID NO:5、19或20-44之一的核苷酸序列，更优选地，所述适体包含SEQ ID NO:5、20或35-38之一的核苷酸序列。在一些优选实施方案中，所述适体由SEQ ID NO:1-44中的任一的核苷酸序列组成，更优选地，所述适体由SEQ ID NO:5、19或20-44之一的核苷酸序列组成，更优选地，所述适体由SEQ ID NO:5、20或35-38之一的核苷酸序列组成。在一些优选实施方案中，所述适体包含SEQ ID NO:37所述核苷酸序列或由SEQ ID NO:37所述核苷酸序列组成。
在一些实施方案中，本发明的所述适体对DKK1具有小于约350nM，优选小于约150nM，优选小于约100nM，优选小于约50nM，优选小于约30nM，优选小于约20nM，优选小于约10nM，优选小于约5nM，优选小于约1nM或更小的K d(解离常数)。在一些实施方案中，本发明的所述适体对DKK1具有约0.01nM-约350nM，优选约0.01nM-约150nM，优选约0.01nM-约100nM，优选约0.01nM-约50nM，优选约0.01nM-约30nM，优选约0.01nM-约20nM，优选约0.01nM-约10nM，优选约0.01nM-约5nM，优选约0.01nM-约1nM的K d(解离常数)。所述K d例如通过酶联寡核苷酸测定法(ELONA)测定或生物膜干涉技术(BLI)测定。
在一些实施方案中，本发明的所述适体抑制DKK1的生物学活性。“抑制”是指与不存在所述适体相比，DKK1的生物学活性在所述适体存在下被降低，例如降低至少约10％，至少约20％，至少约30％，至少约40％，至少约50％，至少约60％，至少约70％，至少约80％，甚至至少约90％。
如本文所用，术语“生物学活性”表示对一或多种细胞或细胞外的过程的作用，其可以影响生理或病生理过程。DKK1的生物学活性包括但不限于拮抗Wnt信号通路。
在一些实施方案中，本发明的所述适体能够抑制DKK1对Wnt信号通路的拮抗作用。例如，本发明的所述适体能在基于细胞的Wnt信号测试法中阻断DKK1的拮抗作用。
在一些实施方案中，本发明的所述适体以小于约2000nM，优选小于约1500nM，优选小于约1000nM，优选小于约800nM，优选小于约400nM，优选小于约200nM或更小的IC50值抑制DKK1的生物学活性，例如抑制DKK1对Wnt信号通路的拮抗作用。 在一些实施方案中，本发明的所述适体以约200nM-约2000nM，优选约200nM-约1500nM，优选约200nM-约1000nM，优选约200nM-约800nM或优选约200nM-约400nM的IC50值抑制DKK1的生物学活性，例如抑制DKK1对Wnt信号通路的拮抗作用。在一些实施方案中，所述IC50值在体外通过HEK293T细胞中的TCF-Wnt诱导萤光素酶活性测定法测定。
在一些实施方案中，本发明的所述适体还可以包含一或多种修饰。例如，所述修饰是赋予所述适体增强的核酸酶抗性和/或增强所述适体的体内半衰期的修饰。
所述修饰包括例如3’和5’的修饰，如3’和5’加帽。在一些实施方案中，所述适体在3’末端用反向脱氧胸苷加帽，即3’反向脱氧胸苷(3’idT)修饰。
所述修饰还可以包括用经修饰的核苷酸取代一或多个天然存在的核苷酸。例如，所述经修饰的核苷酸包括但不限于2’-氟、2’-甲氧乙基、2’-甲氧基和/或2’丙烯氧基修饰的核苷酸(即核糖的2’位置羟基被氟、甲氧乙基、甲氧基或丙烯氧基等取代)。所述经修饰的核苷酸还可以包括C-5修饰的嘧啶。术语“C-5修饰的嘧啶”是指C-5位上有修饰的嘧啶。C-5修饰的嘧啶能够增强寡核苷酸的核酸酶抗性，且是本领域已知的，例如可以参见国际专利申请WO2011/130195及其引用的文献。在一些优选实施方式中，所述修饰是2’-甲氧基(2’-OMe)修饰。在一些实施方式中，在适体的5’和/或3’端的一或多个，例如4个核苷酸进行所述修饰，例如2’-甲氧基(2’-OMe)修饰。
所述修饰还包括核苷酸间的修饰，例如具有不带电荷的键(例如甲基膦酸酯、磷酸三酯、磷酸胺酯、氨基甲酸酯等)的核苷酸间修饰和具有带电荷的键(例如硫代磷酸酯、二硫代磷酸酯等)的核苷酸间修饰，有嵌入剂(例如吖啶、补骨脂素等)的核苷酸间修饰，含有螯合剂(例如金属、放射性金属、硼、氧化性金属等)的核苷酸间修饰，含有烷化剂的核苷酸间修饰和有修饰的键(例如阿尔法异头核酸等)的核苷酸间修饰。
所述修饰还可以包括聚乙二醇化修饰(PEG修饰)。通过与PEG缀合可以延长适体的半衰期例如体内半衰期。在一些实施方式中，所述PEG的分子量为大约1kDa至大约100kDa，例如大约10kDa至大约80kDa，大约20kDa至大约60kDa，大约30kDa至大约50kDa，大约40kDa。在一些实施方案中，所述PEG可以缀合至适体的5’端。在一些实施方案中，所述PEG可以缀合至适体的3’端。
所述修饰还可以包括脂肪酸修饰或/或香豆素修饰。例如，所示适体可以与脂肪酸和/或香豆素衍生物缀合(例如缀合至所述适体分子的5’端)。合适的脂肪酸包括但不限于十二烷二酸、棕榈酸(PA)、十四烷二酸、十六烷二酸、硬脂酸(SA)、十八烷二酸、月桂酸、二十碳五烯酸(EPA)、二十二碳六烯酸(DHA)、花生四烯酸(ARA)。合适的香豆素衍生物包括但不限于4-羟基香豆素、3-乙酰-6-羧基香豆素、华法林、(2-氧代-2H-色烯-3-基)乙酸、[(8-乙酰-4-甲基-2-氧代-2H-色烯-7-基)氧]乙酸、香豆素-3-羧酸、N-(4-甲基-7-香豆素)草酸酰胺、7-(羧甲基)-4-甲基香豆素、7-甲氧香豆素-3-羧酸、6-甲氧基-2-氧代-2H-色烯-3-羧酸。
在一些实施方案中，所述适体可以包含上述多种修饰的组合。例如，所述适体可以 包含2’-甲氧基(2’-OMe)修饰和3’反向脱氧胸苷(3’idT)修饰。
在一些实施方案中，本发明的修饰的适体的体内半衰期是相应的未修饰的适体的体内半衰期的至少约2倍、至少约5倍、至少约10倍、至少约25倍、至少约50倍、至少约100倍、至少约200倍或更多倍。
疾病治疗
在另一方面，本发明提供通过本发明的针对DKK1的适体治疗疾病的方法，该方法包括给有需要的对象施用治疗有效量的本发明的针对DKK1的适体。
通过本发明的针对DKK1的适体治疗的疾病例如是DKK1相关疾病，例如DKK1介导的疾病。在一个实施方案中，所述DKK1相关疾病特征在于DKK1异常表达例如过表达的疾病。
如本文所使用，“DKK1相关疾病”包括DKK1相关癌症。在一个实施方案中，“DKK1相关癌症”为特征在于DKK1异常表达例如过表达的癌症。在一个实施方案中，所述DKK1相关癌症例如选自骨髓瘤(例如，伴随溶骨性病变的多发性骨髓瘤、肝门部胆管癌多发性骨髓瘤等)、乳腺癌、结肠癌、黑色素瘤、肝细胞癌、上皮癌、食道癌、胃癌、胃食管癌、肝门部胆管癌、脑癌、肺癌、前列腺癌或胰癌，及其任何转移瘤。
如本文所使用，“DKK1相关疾病”包括骨相关疾病，例如与异常的成骨细胞或破骨细胞活性相关的骨相关疾病。由异常的成骨细胞或破骨细胞活性表征的疾病包括但不限于：原发性和继发性骨质疏松症、骨质减少、骨软化、成骨不全(OI)、缺血性坏死(骨坏死)、骨折和植入物愈合(牙种植体和髋植入物)、由于其它病症的骨丧失(例如，与HIV感染、癌症和关节炎相关)。其它“DKK1相关疾病”包括但不限于：类风湿性关节炎、骨关节炎、关节炎，以及溶骨性病变。
所述对象可以是任何动物(驯养的、家畜或野生的)，其包括但不限于猫、狗、马、猪和牛，并优选人类对象。如本文中所用，术语患者、个体和对象可以互换地使用。在一些实施方案中，所述对象可以是男性或女性。
如本文所用，“治疗”患有疾病的对象表示所述对象的症状部分或全部缓解，或者在治疗后保持不变。因此，治疗包括预防、治疗和/或治愈。预防指防止潜在疾病和/或防止症状恶化或疾病发展。
如本文所用，“治疗有效量”或“治疗有效剂量”指施用于对象之后至少足以产生疗效的物质、化合物、材料或包含化合物的组合物的量。因此，其为防止、治愈、改善、阻滞或部分阻滞疾病或病症的症状所必需的量。如本文所用，“疗效”表示由对象的治疗所导致的效果，其改变、通常改良或改善疾病或疾病状况的症状，或者治愈疾病或疾病状况。
按照多种因素选择利用所述针对DKK1的适体的剂量方案，所述因素包括例如，所述患者的类型、种类、年龄、体重、性别和医疗病症，；所要治疗的病症的严重性；施用途径；所述患者的肾与肝的功能；和所使用的特定的针对DKK1的适体或其盐。普通 熟练的医生可以容易地确定并指定预防、对抗或抑制所述病症进展所需要的组合物的有效量。
通常，所述针对DKK1的适体的剂量方案为每天大约1μg/kg体重至约100mg/kg体重。
示例性的治疗方案需要每天给药一次、每两天给药一次、每周给药一次、每周给药两次、每两周一次、每三周一次、每四周一次、每月一次、每3个月一次、每3-6个月一次、或起始给药间隔略短(如每周一次至每三周一次)后期给药间隔加长(如每月一次至每3-6个月一次)。给药频率和间隔可以由本领域技术人员根据适体的药代动力学参数确定。
药物组合物
在另一方面，本发明还提供药物组合物，其包括至少一种本发明的针对DKK1的适体，和药学上可接受的载体或赋形剂。所述药物组合物例如用于治疗DKK1相关的疾病。
本文所描述的适体可以任何药学上可接受的剂量形式使用，包括但不限于以可注射剂量形式、液体分散体、凝胶、喷雾、软膏、霜剂、冻干制剂、干粉、片剂、胶囊、控制释放制剂、速熔制剂、延迟释放制剂、延长释放制剂、脉动释放制剂、混合的立即释放和控制释放制剂等。具体地，本文所描述的适体可以配制为：(a)通过选自口腔、肺、静脉内、动脉内、鞘内、内关节、直肠、眼、结肠、胃肠外、脑池内、阴道内、腹腔内、局部、颊、鼻和局部施用中的任一项来施用；(b)选自液体分散体、凝胶、喷雾、软膏、霜剂、片剂、香囊和胶囊中任一项的剂量形式；(c)选自冻干制剂、干粉、速熔制剂、控制释放制剂、延迟释放制剂、延长释放制剂、脉动释放制剂和混合的立即释放和控制释放制剂中任一项的剂量形式；或(d)它们的任意组合。
用于肠胃外、皮内或皮下的施用的溶液或悬浮液可以包含一或多种的以下组分：(1)无菌稀释剂，例如用于注射的水、盐溶液、固定油、聚乙二醇、甘油、丙二醇或其他合成溶剂；(2)抗菌剂，例如苯甲醇或对羟苯甲酯；(3)抗氧化剂，例如抗坏血酸或亚硫酸钠；(4)螯合试剂，例如乙二胺四乙酸；(5)缓冲液，例如乙酸盐、柠檬酸盐或磷酸盐；和(5)用于调节张力的物质，例如氯化钠或葡萄糖。用酸或碱(如盐酸或氢氧化钠)可以调整pH。肠胃外的制剂可以封装在玻璃或塑料制成的安瓿瓶、一次性注射器或多剂量小瓶中。
适于注射使用的药物组合物可以包括无菌的水性溶液(其中是水溶的)或分散体和用于临时制备无菌注射溶液或分散体的无菌粉末。对于静脉内施用，适合的载体包括生理盐水、抑菌水、或磷酸盐缓冲盐水(PBS)。在所有情况下，所述组合物应该是无菌的并且其流动程度应该为易于注射。在制造和储存的条件下所述药物组合物应该是稳定的并应当被保护以防微生物如细菌和真菌的污染作用。如本文中所用的术语“稳定的”意为保持在适合于给患者施用的状态或条件。
所述载体可以是溶剂或分散介质，包括例如水、乙醇、多元醇(例如，甘油、丙二 醇、液体聚乙二醇等)及其适合的混合物。例如通过使用如卵磷脂的包衣，通过在分散体的情况下维持所需的颗粒大小和通过使用表面活性剂可以保持适当的流动性。通过各种抗菌和抗真菌的试剂，例如，对羟基苯甲酯、氯丁醇、苯酚、抗坏血酸，硫柳汞等可以实现防止微生物的作用。在许多情况下，优选在所述组合物中包括等渗试剂，例如糖、多元醇(如甘露醇或山梨醇)和无机盐(如氯化钠)。通过在所述组合物中包括延迟吸收的物质如单硬脂酸铝和明胶可以带来可注射组合物的延长的吸收。
可以通过在适当的溶剂中用以上所列举的成分的一种或组合(如需要的)以需要的量并入活性剂(例如，针对DKK1的适体)接着过滤灭菌来制备无菌的可注射溶液。通常，通过向含有基本分散介质和任何其他所需成分的无菌载体并入至少一种针对DKK1的适体来制备分散体。在用于制备无菌可注射溶液的无菌粉末的情况下，制备的示例性方法包括真空干燥和冷冻干燥，这两者会获得所述针对DKK1的适体的粉末以及来自之前其无菌过滤溶液的任何额外的所需的成分。
口服组合物通常包括惰性的稀释剂或可食用的载体。例如，可以将它们装入明胶胶囊或压成片剂。针对口服治疗施用的目的，所述针对DKK1的适体可以并入赋形剂并以片剂、锭剂或胶囊的形式使用。可以包括药物相容的结合剂和/或佐剂材料作为所述组合物的一部分。
对于通过吸入的施用，以来自含有适合的推进物(例如气体(如二氧化碳)、雾化的液体或来自适合的装置的干粉末)的加压容器或分配器的气溶胶喷雾的形式来递送所述化合物。对于经粘膜或经皮的施用，在制剂中使用对要渗透的屏障是适当的渗透剂。这样的渗透剂通常是本领域已知的，并包括例如，用于经粘膜施用的去污剂、胆汁盐和夫西地酸衍生物。通过使用鼻腔喷雾剂或栓剂可以实现经粘膜的施用。对于经皮的施用，所述活性剂配制成如本领域所熟知的软膏、油膏、凝胶或霜剂。也可以栓剂(例如用常规的栓剂基质，如可可脂和其它甘油酯)或用于直肠递送的保留灌肠剂的形式来制备所述试剂。
在一个实施方案中，针对DKK1的适体配制为用于局部施用。如本文所用，“局部施用”是指通过将(直接地或其他方式)包含所述针对DKK1的适体的制剂与动物的全部或部分皮肤(表皮)接触来递送针对DKK1的适体至所述动物。该术语包括了几种施用途径，包括但不限于局部施用和经皮施用。针对这些施用模式的普遍需求是有效递送至靶组织或层。一方面，使用局部施用作为渗透所述表皮和真皮的方式并最终实现针对DKK1的适体的全身性递送。另一方面，使用局部施用作为选择性地递送针对DKK1的适体至动物的表皮或真皮或其特定的层的方式。
对于局部施用，所述针对DKK1的适体可以配制成药学上可接受的软膏剂、霜剂、洗剂、眼用软膏剂、滴眼剂、滴耳剂、浸渍敷料、和气雾剂、含药粉末、含药粘合剂、泡沫，并且可以含有适当的常规添加剂或赋形剂，包括，例如防腐剂或帮助药物渗透的溶剂和软膏剂、凝胶和霜剂中的软化剂。这种局部制剂也可以含有兼容的常规载体，例如用于乳液的乙醇或油醇。这样的载体可能构成所述制剂的大约1％至大约98％的重量， 更通常地，这样的载体会构成所述制剂的高达大约80％的重量。针对适体的局部递送的具体制剂在现有技术中已有描述。
在一个实施方案中，用会防止从身体迅速除去的载体制备针对DKK1的适体。例如，可以使用控制释放制剂，包括植入和微囊化的递送系统。可以使用可生物降解的、生物兼容的聚合物，例如乙烯乙酸乙烯酯、聚酐、聚乙醇酸、胶原、聚原酸酯和聚乳酸。这种制剂的制备方法对本领域技术人员会是显而易见的。
脂质体悬浮液(包括以被感染的细胞为靶的具有针对病毒抗原的单克隆抗体的脂质体)也可以用作药学上可接受的载体。这些可以根据本领域技术人员已知的方法制备。
此外，可以制备所述针对DKK1的适体的悬浮液作为适当的油性注射悬浮液。适合的亲脂性溶剂或载体包括脂肪油(如芝麻油)或合成脂肪酸酯(如油酸乙酯、甘油三酯)或脂质体。非脂的聚阳离子氨基酸聚合物也可以用于递送。任选地，所述悬浮液还可以包括适合的稳定剂或试剂以增加所述化合物的溶解度并允许用于制备高浓缩的溶液。
在某些情况下，以剂量单位来配制口服或胃肠外的组合物对便于施用和剂量均一可能是特别有利的。如本文中所用的剂量单位形式是指物理上离散的单位，适合作为用于要治疗的对象的单一剂量；每单位含有经计算能产生期望的治疗效果的预定量的针对DKK1的适体以及所需的药物载体。本文所描述的针对DKK1的适体的剂量单位形式的说明决定于并直接依赖于特定针对DKK1的适体的唯一的特性和所要实现的特定的治疗效果和配制此类用于治疗个体的活性剂的技术的固有限制。
包含至少一种针对DKK1的适体的药物组合物可以包括一或多种药物赋形剂。这样的赋形剂的实施例包括但不限于，结合剂、填充剂、润滑剂、悬浮剂、甜味剂、调味剂、防腐剂、缓冲剂、润湿剂、崩解剂、泡腾剂和其它赋形剂。这样的赋形剂在本领域中是已知的。示例性赋形剂包括：(1)结合剂，包括各种纤维素和交联聚乙烯吡咯烷酮、微晶纤维素(如Avicel PH101和Avicel PH102)、硅化微晶纤维素(ProSolv SMCC TM)、黄蓍胶和明胶；(2)填充剂，例如各种淀粉、乳糖，乳糖单水合物、无水乳糖；(3)崩解剂如藻酸、Primogel、玉米淀粉、轻度交联的聚乙烯基吡咯烷、马铃薯淀粉、玉米淀粉和修饰的淀粉、交联羧甲基纤维素钠、交聚维酮、乙醇酸淀粉钠及它们的混合物；(4)润滑剂，包括作用于所要压缩的粉末的流动性的试剂，包括硬脂酸镁、胶状二氧化硅(如Aerosil200、滑石)、硬脂酸、硬脂酸钙和硅胶；(5)助流剂，如胶状二氧化硅；(6)防腐剂，如山梨酸钾、对羟基苯甲酸甲酯、对羟基苯甲酸丙酯、苯甲酸及其盐、其他对羟基苯甲酸的酯(如对羟基苯甲酸丁酯)、醇(如乙醇或苯甲醇)、酚类化合物(如苯酚)或季铵化合物(如氯化苯甲烃胺)；(7)稀释剂，如药学上可接受的惰性填料，如微晶纤维素、乳糖、磷酸氢钙、糖类和/或任何以上的混合物；稀释剂的实施例包括微晶纤维素，如Avicel PH101和Avicel PH102；乳糖如乳糖单水合物、无水乳糖和Pharmatose DCL21；磷酸氢钙如Emcompress甘露醇、淀粉、山梨醇、蔗糖和葡萄糖；(8)甜味剂，包括任何天然或人造的甜味剂，如蔗糖、糖精蔗糖、木糖醇、糖精钠、甜蜜素、阿斯巴甜和安赛蜜；(9)调味剂，如薄荷、水杨酸甲酯、橙调味剂、Magnasweet(商标MAFCO)、泡泡糖香料、水果 香料等；和(10)泡腾剂，包括泡腾剂对，如有机酸和碳酸盐或重碳酸盐。
实施例
下面将通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所描述的实施例范围中。
材料与方法
蛋白质-SELEX：基于Ni-NTA珠的SELEX方法用于筛选DKK1的适体。简而言之，His6标记的DKK-1首先与Ni-NTA珠一起孵育，形成蛋白质结合的Ni-NTA磁珠。然后将此复合物与ssDNA随机文库在室温下孵育以形成ssDNA-蛋白质结合的Ni-NTA磁珠复合物。洗涤后，ssDNA-蛋白质结合的Ni-NTA磁珠复合物用作模板，和正向引物和生物素化的反向引物一起进行PCR。根据制造商的方案，使用链霉亲和素包被的磁珠再生ssDNA。ssDNA池用于下一轮SELEX。从第四轮开始，进化的ssDNA池与Ni-NTA珠或非靶蛋白一起孵育以进行反向筛选。然后收集来自反向筛选负向筛选的未结合ssDNA并应用于结合DKK1的Ni-NTA磁珠。来自第10、16、18和20轮的ssDNA产品用于确定与初始随机文库相比选择过程的富集程度。克隆和测序具有最高结合亲和力的ssDNA库。
酶联寡核苷酸测定法(ELONA)：通过疏水相互作用将每孔160ng DKK1和阴性靶标包被到96孔微量滴定板中4℃过夜。孔中的非结合位点在室温下用BSA封闭1小时，然后用DNA结合缓冲液洗涤5分钟x4次。将适当浓度的生物素化的适体候选物/RS加入每个孔中，并在室温下持续轻柔摇动孵育45分钟。结合后，将板用洗涤缓冲液洗涤5分钟x4次以去除非特异性的和弱结合的序列。将100μl链霉亲和素-辣根过氧化物酶(HRP)(1:10000稀释到PBST+0.1％BSA中)加入每个孔中，孵育30分钟，并用结合缓冲液洗涤4次。将作为HRP底物的50μl 3,3',5,5'-四甲基联苯胺(TMB)添加到每个孔中并孵育20分钟。加入50μl 2M H 2SO 4终止反应。用酶标仪(Molecular Device i3x)测量450nm处的吸光度。用Origin软件分析数据。非线性曲线拟合模型logistic用于绘制结合曲线并计算K D。
TCF-wnt诱导的荧光素酶活性测定：HEK293T细胞与相应的质粒共转染，相应的质粒包括TOPFlash、FOPFlash、Wnt3a和DKK1构建体。每次转染使用等量的载体。转染后24小时，更换培养基。用含有1μM相应适体候选物的条件培养基处理细胞。为了测定抑制效力，用具有梯度浓度的适体候选物处理细胞。处理后24小时，细胞用100μl裂解缓冲液裂解，取20μl用于分析。根据方案(双荧光素酶报告基因测定，Promega)进行计算。
结合能力测定：转染48h后，将培养基收集在15ml离心管中，3000rpm离心10min。将2ml上清液与30μl NTA磁珠在室温下孵育1小时(Saiyed，Telang等人，2003)。未结合的蛋白质将通过用PBS洗涤珠两次来去除。之后，珠-DKK1复合物与250nM FAM标记的aptdkk5s一起在20μl的总体积中在室温下孵育45分钟。孵育后，珠复合物用PBS洗涤两次以去除未结合的DNA。然后将在倒置荧光显微镜下检查荧光信号。
DNA检测：通过分子成像仪(Bio-Rad)测定所有适体样品的条带密度(Klussmann，Nolte等人1996，Siller-Matula，Merhi等人2012)。
实施例1.重组His6-DKK1固定于NTA磁珠
Ni-NTA磁性琼脂糖珠以5％(v/v)悬浮液提供，其对6x His标记的蛋白质的结合能力为每毫升悬浮液300μg蛋白质(～24kDa)。为了针对在随后的第一轮SELEX在磁珠上固定0.2nmol DKK1，优化了重组His6-DKK1和NTA磁珠的量和比例。0.8nmol蛋白质+10μl磁珠、0.6nmol蛋白质+10μl磁珠、0.4nmol蛋白质+10μl磁珠、0.8nmol蛋白质+6μl磁珠、0.6nmol蛋白质+6μl磁珠和0.4nmol蛋白质+6μl磁珠在4℃下孵育60分钟。孵育后，通过SDS-PAGE凝胶分析蛋白质珠复合物。根据结果，最优化的条件是0.6nmol His6-DKK1和6μL NTA琼脂糖磁珠(图1)。因此，该条件用于以下的研究。
实施例2.靶向全长DKK1的适体的SELEX
带有His6标签的重组人DKK-1将固定在Ni-NTA磁珠上并用作正向筛选正向筛选靶标。空白Ni-NTA珠子和其他带有His6标签的蛋白质将用作负向筛选负向筛选靶标。每轮正向筛选后收集的ssDNA池通过PCR扩增，并在ssDNA再生前通过琼脂糖凝胶电泳检查(图2)。监测PCR循环以避免PCR中的非特异性副产物。进行了20轮SELEX。
实施例3.测序数据分析
第10轮和最后一轮的ssDNA池通过Next Generation Sequencing进行测序。第10轮没有富集序列，而最后一轮出现富集序列(表1)。因此，在最终富集的ssDNA池中识别出出现率最高的20个潜在序列并将其命名为Aptdkk-1-20(表2)。根据使用Cluster Omega的多序列比对结果，这些序列可以分为两组(图3)。第1组显然富含鸟嘌呤(以红框显示)，而第2组是非富含G的序列(以蓝框显示)。
表1.来自最终轮的富集结果
Figure PCTCN2022121254-appb-000002
表2.来自最终轮的具有最高富集的序列(未包含保守引物区)
Figure PCTCN2022121254-appb-000003
实施例4.G-组模式的预测
富含G的序列通常形成特殊的G四链体结构，该结构具有稳定的螺旋形状，并包含可以由一条、两条或四条链形成的鸟嘌呤四分体。然后通过对于潜在G组的QGRS和它们形成单分子四链体的趋势的G分数来分析富含G的序列。通常，在计算G分数时考虑了预测的QGRS中间隔(gap)的大小和分布。得分越高的序列将成为G-四链体的更好候选者。根据分析结果，所有这些序列都有两个G四分体，可以形成两条G链(表3)。G分数都在19-20左右。此外，间隔的分布以类似的模式显示。这表明它们形成稳定的G-四链体的能力非常相似。
表3.富含G的组的QGRS分析。
该组的序列显示出长度和QGRS的G分数的相似性。
Figure PCTCN2022121254-appb-000004
实施例5.代表性适体候选物的特异性
从出现率最高的20个潜在序列(表4)中，使用酶联寡核苷酸测定(ELONA)使用1μM的每个适体/RS来确定对DKK-1的特异性，并检测450nm处的吸光度。与空白对照相比，除候选物3外的大多数适体候选物显示出明显更高的吸光度(图4)。
表4. 20个潜在候选物(包含保守引物区)
Figure PCTCN2022121254-appb-000005
实施例6.代表性适体候选物的亲和力
具有显著特异性的适体候选物和作为阴性对照的候选物3用于确定对DKK1的结合亲和力，其中除了候选物1、2、4和14的浓度为(0、62.5nM、125nM、250nM、500nM、1000nM、2000nM、4000nM)，候选物7、10、15和16的浓度为(0、23.44nM、46.86nM、 93.75nM、187.5nM、375nM、750nM、1500nM)，其他候选物浓度为(0、0.18nM、0.74nM、2.96nM、11.84nM、47.35nM、189.39nM、757.58nM)。每个候选物的结合曲线如图5和表5所示。11个适体候选物显示出对DKK1的高亲和力(低K d值)。六个候选物(aptdkk-5、13、20、19、6和1)富含G。其他五个序列(aptdkk-9、18、12、17和8)不富含G。
表5.代表性适体候选物的特异性和亲和性的总结
Figure PCTCN2022121254-appb-000006
实施例7.代表性适体候选物的抑制作用
与未处理组相比，用aptdkk-5、19和20处理的细胞显示出更高的荧光素酶信号，表明它们有可能释放DKK1对Wnt信号传导的抑制(图6)。然而，用其他适体候选物处理的细胞显示出与未处理组相似的荧光素酶信号。因此，aptdkk-5、19和20可以抑制DKK1对Wnt信号的拮抗作用。
实施例8.代表性适体候选物的抑制效力
根据计算，aptdkk-5的抑制效力为IC 50＝754±29nM，aptdkk-20的抑制效力为IC 50＝1504±39nM。候选物19的数据不符合测试浓度的拟合曲线，表明IC 50更高(图7)。 该结果表明aptdkk-5和aptdkk-20均表现出对DKK1抑制Wnt信号传导的抑制作用，而aptdkk-19的作用相对较弱。
实施例9.截短的aptdkk-5的特异性
从aptdkk5的24个截短序列和原始aptdkk5(表6)中，使用酶联寡核苷酸测定(ELONA)用100nM/1nM每个适体/RS确定对DKK-1的特异性，并检测在450nm的吸光度。当浓度为100nM时，apt5-1-38、apt5-1-35、apt5-1-62和apt5-1-59与原始aptdkk5相比，吸光度没有显著差异/略有差异(图8a)。当浓度为1nM时，apt5-1-50、apt5-1-47、apt5-1-44、apt5-1-41、apt5-1-38、apt5-1-35、apt5-1-65、apt5-1-62、apt5-1-59和apt5-1-53与原始aptdkk5相比，吸光度没有显著差异/略有差异(图8b)。在考虑这些适体的特异性和长度后，选择apt5-1-44、apt5-1-41、apt5-1-38和apt5-1-35来确定与DKK1的结合亲和力。
表6.截短的Aptdkk-5的序列
Figure PCTCN2022121254-appb-000007
Figure PCTCN2022121254-appb-000008
实施例10.截短后的aptdkk-5的亲和力
具有显著特异性的适体候选物(apt5-1-44、apt5-1-41、apt5-1-38和apt5-1-35)和作为阳性对照的原始aptdkk5用于确定与DKK1的结合亲和力，其中原始aptdkk5和apt5-1-44的浓度为(0.15625nM、0.625nM、1.25nM、2.5nM、5nM、10nM、50nM、100nM)，apt5-1-41、apt5-1-38和apt5-1-35的浓度为(0、1.5625nM、3.125nM、6.25nM、12.5nM、25nM、50nM)。每个候选物的结合曲线如图9所示。11个适体候选物对DKK1显示出高亲和力(低K d值)。在这四个候选物中，apt5-1-38对DKK1显示出最高的亲和力，因此它被选为用于进一步研究的最佳适体。
实施例11.截短的Aptdkk5(Aptdkk5s)与DKK1结合位点的研究
从aptdkk5s的13个突变序列和原始aptdkk5s(实施例10的apt5-1-38)(表7)中，每种适体/RS的1μM用于通过ELONA确定对DKK-1的特异性，检测450nm处的吸光度。与空白对照相比，大多数候选适体显示出显著较低的吸光度，尤其是aptdkk5s-3、aptdkk5s-9和aptdkk5s-11(图10)，表明这些可能是aptdkk5s与DKK1的结合位点。为了鉴定DKK1结合至aptdkk5s的结构域，在带有His标签的载体pCS2中成功构建了用于HEK293T细胞转染的野生型DKK1质粒或3个截短的DKK1(SEQ ID NO:73的残基1-91、1-142和1-178)质粒。蛋白-珠复合物用于SDS-PAGE凝胶电泳和pCS2-DKK1-His-flag(S4)、pCS2-DKK1-ΔC-His-flag(S3)和pCS2-DKK1-ΔC&L2-His-flag(S2)的表达得到验证(图11)。从荧光图像的结果来看，aptdkk5s与全长DKK1显著结合，而与其他截短的DKK1几乎不结合，表明DKK1的C结构域(178-266aa)可能是DKK1与aptdkk5s的结合结构域(图12)。
表7.aptdkk5s突变体的序列
Figure PCTCN2022121254-appb-000009
实施例12.化学修饰aptdkk5s的特异性和亲和力
aptdkk5s的IDT修饰应用于以下表征，包括dkk5s-2G-indT和dkk5s-OM-indT。修饰的和原始的aptdkk5s均显示出显著的高吸光度(>2)，而所有修饰的aptdkk5s的吸光度均略低于原始aptdkk5s的吸光度(图13)，这表明aptdkk5s修饰的特异性相当高。随后，修饰的适体和作为阳性对照的原始aptdkk5s用于通过ELONA确定与不同浓度(0、15.625nM、31.25nM、62.5nM、125nM、250nM、500nM、1000nM)的DKK1的结合亲和力。修饰的aptdkk5s(dkk5s-2G-indT和dkk5s-OM-indT)都显示出对DKK1的高亲和力，但与原始aptdkk5s相比略低(更高的Kd值)(图14)。
实施例13.化学修饰aptdkk5s的血清稳定性
在胎牛血清中评估修饰和未修饰适体的血清代谢稳定性。从0到72小时用10％和100％胎牛血清处理适体(图15)。原始的aptdkk5s在10％FBS中孵育16小时后开始降解，在100％FBS中从2小时开始降解。Dkk5s-2G-indT在10％FBS中孵育72小时后开始降解，在100％FBS中从8小时开始降解。Dkk5s-OM-indT在10％FBS中孵育48小时后开始降解，在100％FBS中从8小时开始降解。简而言之，与未修饰的相比，修饰的aptdkk5s的血清稳定性显著提高，换句话说，IDT的3'-末端倒置dT和O-甲基修饰均增强了血清代谢稳定性。
实施例14.化学修饰aptdkk5s的抑制效力
从细胞测定结果来看，修饰的aptdkk-5(aptdkk5-OM-indT)的抑制效力为IC50＝383±64nM，而原始aptdkk-5的抑制效力为IC50＝754±29nM，如前所述(图16)。结果表明修饰的aptdkk-5对DKK1对Wnt信号的抑制具有更高的抑制作用。
实施例15.针对全长DKK1的适配体的进一步SELEX
根据最近二十轮SELEX对DKK1的数据分析结果，二十轮SELEX序列的富集不 够。因此，在更苛刻的条件下进行针对DKK1的额外10轮SELEX，以获得具有更好富集的ssDNA池(图17、图18)。随后，在第30轮的富集ssDNA库中鉴定出19个潜在序列，并命名为D30-1至D30-20(表8)。
表8.第30轮最高富集的序列
SEQ ID NO: 名称 序列
87 D30-1 ATGTCCATTTGCGAGTAGGTGTTTGAGTGAGGTGA
88 D30-2 CATTGTCTCAGCGCATGTGTTCAGTTCTTGTGTGT
89 D30-3 CTTCTTGTCCCTCGATGTGTTCAGTGTTTGTGTGT
90 D30-4 TGCTGAACTTTACGTGATTCTAGTCGCTGACCGCG
91 D30-5 ATAGGTTGGGTAGGTTGGTTGCTTTTTTTGTCGCG
92 D30-6 TCAGTGTCGTAAAGTGTGTCCCTTTCTTTACGCGC
93 D30-7 TGTAGATAGAGGGGTTGGTGTGGTTGGCGCTATCC
94 D30-8 TGCATCTTCCTTCTTCGATTCTATGCTCGGCCGCG
95 D30-9 CTGTTCTAACGTGGTTGACCCCCCTTTGCTTTGAC
96 D30-10 TCCTCTTCCTTTGGCTAGGTTGGTTAGGTTGGTGC
97 D30-11 TGGTGTGGGGGTGGGTGGTGGGTTGTATCGTTTGA
98 D30-12 TCGTGCTTGATTCCATGCTTGGCCGCCTTCTCGTG
99 D30-13 TATGGTTTCCCTACATGTGTTCAGTGTTTGTGTGT
100 D30-14 ATGCCCCTCCTCTGTTGAACTGTTTACCCCTTTGA
101 D30-15 AATTTCGATAGTCCATGTGTTCAGTGCCTGTGTGT
102 D30-16 ATGTAAATCGATTCCTCTGGCCGCATCGCGCTTTA
103 D30-17 CAGTCTTAGTGCTCATGTGTTCAGTGTTTGTGTGT
104 D30-18 TCTGCTTTCACGTACTAGGTTGGTTAGGTTGGTGT
105 D30-19 TACCTTCTGCTCGATGTGTTCAGTGTGTTGTGTGT
实施例15.第30轮代表性适配体候选物的特异性和亲和力
从第30轮中出现率最高的19个潜在序列中，使用1μM的每种适体/RS通过ELONA来确定对DKK-1的特异性。结果表明，与载体相比，大多数适体显示出显著更高的吸光度，而候选3、7、8和12与先前鉴定的适体aptddk5s相比具有更高的吸光度(图19)。接下来，使用候选适体通过BLI确定与DKK1的结合亲和力。DKK1蛋白以0nM、3.125nM、6.25nM、12.5nM、25nM、50nM、100nM、200nM的梯度浓度应用。除了候选5、6和11(表9，图20)外，检测到16个候选具有高亲和力，Kd值范围为1.67nM至23nM。
表9.代表性适体候选物的特异性和亲和力总结
Figure PCTCN2022121254-appb-000010
Figure PCTCN2022121254-appb-000011
参考文献：
1.H Kagey M,Xi H.Rationale for targeting the Wnt signalling modulator Dickkopf-1 for oncology.Br J Pharmacol(2017)174:4637–4650.doi:10.1111/bph.13894
2.D’Amico L,Mahajan S,Capietto AH,Yang Z,Zamani A,Ricci B,Bumpass DB,Meyer M,Su X,Wang-Gillam A,et al.Dickkopf-related protein 1(Dkk1)regulates the accumulation and function of myeloid derived suppressor cells in cancer.J Exp Med(2016)213:827–840.doi:10.1084/jem.20150950
3.Malladi S,MacAlinao DG,Jin X,He L,Basnet H,Zou Y,De Stanchina E,Massagué J.Metastatic Latency and Immune Evasion through Autocrine Inhibition of WNT.Cell(2016)165:45–60.doi:10.1016/j.cell.2016.02.025
4.Arend R,Castro C,Matulonis U,Hamilton E,Gunderson C,LyBarger K,Goodman H,Duska L,Mahdi H,ElNaggar A,et al.76 Patients(PTS)with recurrent gynecologic cancer whose tumors have activating wnt pathway mutations respond better to DKN-01,a DICKKOPF-1(DKK1)inhibitor.Int J Gynecol Cancer(2019)29:A40.doi:10.1136/ijgc-2019-igcs.76
5.Arend RC,Castro CM,Matulonis UA,Hamilton E,Gunderson CC,Lybarger KSS,Kagey M,Sirard C,Birrer MJ.Safety and efficacy of a DKK1 inhibitor(DKN-01)as monotherapy or in combination with paclitaxel in patients with Wnt activated recurrent gynecologic malignancies.Gynecol Oncol(2019)154:34.doi:10.1016/j.ygyno.2019.04.080
6.Jayasena SD.Aptamers:An emerging class of molecules that rival antibodies in diagnostics.Clin Chem(1999)45:1628–1650.doi:10.1093/clinchem/45.9.1628
7.Boylan MO,Athanassiou M,Houle B,Wang Y,Zarbl H.Activation of tumor suppressor genes in nontumorigenic revertants of the HeLa cervical carcinoma cell line.Cell Growth Differ(1996)
8.Largy E,Mergny J-L,Gabelica V.“Role of Alkali Metal Ions in G-Quadruplex Nucleic Acid Structure and Stability,”in doi:10.1007/978-3-319-21756-7_7
9.Routh ED,Creacy SD,Beerbower PE,Akman SA,Vaughn JP,Smaldino PJ.A G-quadruplex DNA-affinity approach for purification of enzymatically active G4 resolvase1.J Vis Exp(2017)doi:10.3791/55496
10.Sundquist WI,Klug A.Telomeric DNA dimerizes by formation of guanine tetrads between hairpin loops.Nature(1989)doi:10.1038/342825a0
Figure PCTCN2022121254-appb-000012
Figure PCTCN2022121254-appb-000013
Figure PCTCN2022121254-appb-000014
Figure PCTCN2022121254-appb-000015
Figure PCTCN2022121254-appb-000016
Figure PCTCN2022121254-appb-000017
Figure PCTCN2022121254-appb-000018

Claims (16)

  1. 一种针对DKK1的适体，所述适体
    i)包含与SEQ ID NO:1-20中的任一具有至少大约90％相同性、至少大约91％相同性、至少大约92％相同性、至少大约93％相同性、至少大约94％相同性、至少大约95％相同性、至少大约96％相同性、至少大约97％相同性、至少大约98％相同性或至少大约99％相同性的核苷酸序列；或者，
    ii)包含SEQ ID NO:1-20中的任一中的至少10个、至少15个、至少20个、至少25个、至少30个、至少35个、至少40个、至少45个、至少50个或更多个连续的核苷酸；或者
    iii)包含与SEQ ID NO:45-64中的任一具有至少大约90％相同性、至少大约91％相同性、至少大约92％相同性、至少大约93％相同性、至少大约94％相同性、至少大约95％相同性、至少大约96％相同性、至少大约97％相同性、至少大约98％相同性或至少大约99％相同性的核苷酸序列；或者
    iv)包含与SEQ ID NO:87-105中的任一具有至少大约90％相同性、至少大约91％相同性、至少大约92％相同性、至少大约93％相同性、至少大约94％相同性、至少大约95％相同性、至少大约96％相同性、至少大约97％相同性、至少大约98％相同性或至少大约99％相同性的核苷酸序列，
    其中所述适体特异性结合DKK1。
  2. 权利要求1的适体，其中所述适体包含SEQ ID NO:1-64和87-105中的任一的序列。
  3. 权利要求1的适体，其中所述适体包含SEQ ID NO:5、20、35-38、49、或64所示的核苷酸序列，优选包含SEQ ID NO:37所示核苷酸序列。
  4. 权利要求1-3中任一项的适体，其中所述适体对DKK1具有小于350nM，优选小于150nM，优选小于100nM，优选小于50nM，优选小于30nM，优选小于20nM，优选小于10nM，优选小于5nM，优选小于1nM更小的K d。
  5. 权利要求1-4中任一项的适体，其中所述适体能够抑制DKK1的生物学活性。
  6. 权利要求1-5中任一项的适体，其中所述适体能在基于细胞的Wnt信号测试法中阻断DKK1的拮抗作用。
  7. 权利要求1-6中任一项的适体，其中所述适体以小于2000nM，优选小于1500nM，优选小于1000nM，优选小于800nM或更小的IC50值抑制DKK1的生物学活性，例如 抑制DKK1对Wnt信号通路的拮抗作用。
  8. 权利要求1-7中任一项的适体，其中所述适体包含一种或多种昂赋予所述适体增强的核酸酶抗性和/或增强所述适体的体内半衰期的修饰。
  9. 权利要求1-8中任一项的适体，其中所述适体包含2’-甲氧基(2’-OMe)修饰和3’反向脱氧胸苷(3’idT)修饰。
  10. 一种治疗DKK1相关疾病的方法，该方法包括给有需要的对象施用治疗有效量的权利要求1-9中任一项的针对DKK1的适体，所述对象例如是人。
  11. 权利要求10的方法，其中所述DKK1相关疾病为DKK1相关癌症，例如骨髓瘤(例如，伴随溶骨性病变的多发性骨髓瘤、肝门部胆管癌多发性骨髓瘤等)、乳腺癌、结肠癌、黑色素瘤、肝细胞癌、上皮癌、食道癌、胃癌、胃食管癌、肝门部胆管癌、脑癌、肺癌、前列腺癌或胰癌，及其任何转移瘤。
  12. 权利要求10的方法，其中所述DKK1相关疾病选自骨质疏松症、骨质减少、骨软化、成骨不全(OI)、缺血性骨坏死、类风湿关节炎、骨折、骨关节炎和骨髓瘤。
  13. 一种药物组合物，其包含至少一种权利要求1-9中任一项的针对DKK1的适体，和药学上可接受的载体或赋形剂。
  14. 权利要求1-9中任一项的针对DKK1的适体或权利要求13的药物组合物在制备药物中的用途，其中所述药物用于治疗DKK1相关疾病。
  15. 权利要求14的用途，其中DKK1相关疾病为DKK1相关癌症，例如骨髓瘤(例如，伴随溶骨性病变的多发性骨髓瘤、肝门部胆管癌多发性骨髓瘤等)、乳腺癌、结肠癌、黑色素瘤、肝细胞癌、上皮癌、食道癌、胃癌、胃食管癌、肝门部胆管癌、脑癌、肺癌、前列腺癌或胰癌，及其任何转移瘤。
  16. 权利要求14的用途，其中所述DKK1相关疾病选自骨质疏松症、骨质减少、骨软化、成骨不全(OI)、缺血性骨坏死、类风湿关节炎、骨折、骨关节炎和骨髓瘤。
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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101400406A (zh) * 2006-01-13 2009-04-01 诺瓦提斯公司 使用Dickkopf-1和/或-4抗体的组合物和方法
US20160154011A1 (en) * 2013-07-15 2016-06-02 King's College London Dickkopf (DKK) Proteins as Biomarkers for Cognitive Decline Associated with Alzheimer's Disease
CN107760686A (zh) * 2016-08-23 2018-03-06 上海伯豪医学检验所有限公司 Dkk‑1蛋白的核酸适配体及其应用
US20190022094A1 (en) * 2016-01-22 2019-01-24 Yale University Compositions and methods for inhibiting dkk-1
CN109576272A (zh) * 2018-07-02 2019-04-05 广西医科大学 一种dkk-1核酸适配体及其应用
WO2020149644A1 (ko) * 2019-01-15 2020-07-23 (주)바이오니아 Dkk1 유전자를 표적으로 하는 이중나선 올리고뉴클레오티드, 이를 포함하는 구조체 및 이를 포함하는 탈모 예방 또는 발모용 조성물
CN111712573A (zh) * 2018-02-12 2020-09-25 安沛治疗有限公司 针对骨硬化蛋白的适体及其用途

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101400406A (zh) * 2006-01-13 2009-04-01 诺瓦提斯公司 使用Dickkopf-1和/或-4抗体的组合物和方法
US20160154011A1 (en) * 2013-07-15 2016-06-02 King's College London Dickkopf (DKK) Proteins as Biomarkers for Cognitive Decline Associated with Alzheimer's Disease
US20190022094A1 (en) * 2016-01-22 2019-01-24 Yale University Compositions and methods for inhibiting dkk-1
CN107760686A (zh) * 2016-08-23 2018-03-06 上海伯豪医学检验所有限公司 Dkk‑1蛋白的核酸适配体及其应用
CN111712573A (zh) * 2018-02-12 2020-09-25 安沛治疗有限公司 针对骨硬化蛋白的适体及其用途
CN109576272A (zh) * 2018-07-02 2019-04-05 广西医科大学 一种dkk-1核酸适配体及其应用
WO2020149644A1 (ko) * 2019-01-15 2020-07-23 (주)바이오니아 Dkk1 유전자를 표적으로 하는 이중나선 올리고뉴클레오티드, 이를 포함하는 구조체 및 이를 포함하는 탈모 예방 또는 발모용 조성물

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HE LEI, ZHAO YUNWANG; LI BAOLIN; AN XIAOGANG: "Screening and Identification of Aptamers of Liver Cancer Serum Specific Marker Dickkopf-1", JOURNAL OF MOLECULAR IMAGING, vol. 42, no. 4, 31 December 2019 (2019-12-31), pages 528 - 533, XP093055126, ISSN: 1647-4500, DOI: 10.12122/j.issn.1674-4500.2019.04.25 *
ZHOU YIN, LI WENSHUAI, TSENG YUJEN, ZHANG JUN, LIU JIE: "Developing slow-off dickkopf-1 aptamers for early-diagnosis of hepatocellular carcinoma", TALANTA, ELSEVIER, AMSTERDAM, NL, vol. 194, 1 March 2019 (2019-03-01), NL , pages 422 - 429, XP093055127, ISSN: 0039-9140, DOI: 10.1016/j.talanta.2018.10.014 *

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