WO2021225423A1 - 신규 핵산 리간드 및 그의 식별방법 - Google Patents
신규 핵산 리간드 및 그의 식별방법 Download PDFInfo
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Definitions
- the present disclosure relates to novel nucleic acid ligands and methods for their identification.
- An aptamer is a single-stranded nucleic acid (DNA or RNA) fragment having a property of binding with high affinity and specificity to various types of targets, from small molecule compounds to proteins.
- the aptamer can specifically bind only to a target molecule, and its binding force is very excellent at a level of several uM to several nM.
- the aptamer specifically recognizes and binds to a target, similar to a monoclonal antibody. Therefore, it can be used as a replacement for monoclonal antibodies, such as diagnostic reagents, analytical reagents, and harmful substances detection, and can be applied to biosensors, bioimaging, drug delivery, and therapeutics.
- SELEX systematic evolution of ligands by exponential enrichment
- SELEX is a technology first developed in 1990. It is a process of repeatedly screening oligonucleotides binding to a desired target from a nucleic acid library having a random sequence to obtain a nucleic acid aptamer having high binding ability to “one specific target” have. That is, in the SELEX method, oligonucleotides bound to the target are separated from those not bound to the target from a pool of SELEX nucleic acid libraries containing 10 14 to 10 16 single-stranded random oligonucleotides usually composed of 40 to 100 nucleotides.
- oligonucleotides bound to the target are amplified by PCR, and then the amplified oligonucleotides are used again as a library pool. By repeating the above selection process several times, oligonucleotides that do not bind to a target are excluded, so that an aptamer that specifically binds to "only one target" with high affinity is obtained.
- Non-Patent Document 1 Non-Patent Document 1
- Non-Patent Document 1 After the first classic SELEX, Negative SELEX and Counter SELEX were developed, and after that, IP-SELEX, Capture-SELEX, Cell-SELEX, CE-SELEX, M-SELEX, AFM-SELEX, AEGIS-SELEX, Animal-SELEX The same technology was additionally developed (Non-Patent Document 1).
- the SELEX method and the aptamer research direction that have been developed so far is a precondition that does not change that the oligonucleotide binds to only one target. This is to find an oligonucleotide having a specific binding affinity for only one target, that is, an aptamer.
- Various additional studies have been made to use the aptamer developed and studied by the existing SELEX method for various purposes, but the following problems have been raised in using the aptamer as a drug.
- Non-Patent Document 2 The aptamer obtained by the conventional SELEX technology has a fatal disadvantage to be used as a drug in an actual living body due to its physicochemical properties. That is, instability due to nuclease degradation and rapid renal clearance have been a major obstacle to clinical application of aptamers (Non-Patent Document 2). In order to solve these problems, various studies have been conducted.
- Aptamers have a serum half-life of about 5 minutes and an intracellular half-life of about 1 hour due to degradation by nucleases commonly present in living bodies (Non-Patent Document 3).
- the aptamer can be chemically modified, for example, by modifying both ends of an oligonucleotide, the sugar structure of a nucleic acid, or a phosphodiester backbone. The method is known.
- the aptamer has an average diameter of less than 5 nm and a molecular weight of about 6-30 kDa.
- Non-Patent Document 2 the aptamer is formulated in a form such as polyethylene glycol (PEG), protein, cholesterol, fatty acid, or conjugated with macromolecules such as liposomes, so that it is not easily filtered by the kidneys, thereby extending the residence time in the body technique is known.
- PEG polyethylene glycol
- aptamers which are single-stranded oligonucleotides, naturally form various three-dimensional shapes such as helices and loops, and such three-dimensional three-dimensional structures are involved in target recognition and binding.
- the affinity and specificity of the aptamer for the target is sensitively affected by its structure. Therefore, it can be sufficiently expected that the modification of the aptamer using a macromolecule in the post-SELEX process will have a negative effect on the properties and folding structure of the aptamer.
- the aptamer when the aptamer is modified or used together with other substances, there may be a problem in that the quality or performance of the aptamer varies for each batch that manufactures the aptamer, and as a result, it is difficult to control the quality of the aptamer, so it is difficult to manufacture an actual drug. However, the consistency of the effect cannot be guaranteed. If high cost is involved in quality control, the advantage of aptamer that production cost is low is halved.
- aptamers studied so far could not penetrate the cell membrane by themselves and be actively delivered into the cytoplasm. Accordingly, most aptamers have been developed to target only membrane proteins or secreted proteins, and the selection of aptamers targeting proteins present in membranes of cytoplasm or intracellular organelles was mostly made on purified proteins. In order to improve the cell permeability of the aptamer, transferrin protein, cholesterol, and transporters such as liposomes were used. As above, in order to utilize aptamer as a drug that is actually applied to clinical practice, there are various problems that need to be overcome, and various attempts have been made to overcome these problems, but cases of failure in clinical practice are continuing. So far, only one approved aptamer drug is Macuzen. Therefore, there is a continuing need for new substances that can be used and realized clinically as actual drugs by solving the problems of aptamers.
- the aptamer has the property of binding only to one target.
- two or more aptamers having specific binding affinity to different targets are combined with each other. Research is continuously being conducted.
- the dual specific aptamer consists of two distinct aptamers, and due to these properties, for example, it can promote or regulate an immune response between a functional molecule and a target cell, so continuous research has been mainly conducted in the field of immuno-cancer drugs (non-patented).
- Literature 5 The dual specific aptamer consists of two distinct aptamers, and due to these properties, for example, it can promote or regulate an immune response between a functional molecule and a target cell, so continuous research has been mainly conducted in the field of immuno-cancer drugs (non-patented). Literature 5).
- each single-specific aptamer constituting the dual-specific aptamer since each single-specific aptamer constituting the dual-specific aptamer has a unique structure that specifically binds to each target, they may interfere with each other when linked with or without a linker. It can affect the specific binding affinity of the tamer. Therefore, simply binding two aptamers specific to different targets does not maintain binding affinity for both targets, nor can it be guaranteed. Accordingly, in order to obtain a properly functioning dual-specific aptamer, there is a difficulty in finding an aptamer that does not interfere with each other's structure without affecting each other's structure through numerous trials and errors.
- the dual-specific aptamer has a problem in that it is difficult to mass-produce, and the ease of manufacture, which is one of the advantages of the aptamer, may be lost.
- the length of the aptamer increases, it is difficult to isolate a purely purified aptamer having a desired sequence during manufacture or synthesis, and accordingly, an increase in impurities occurs, making quality control difficult. Therefore, in order to satisfy the large-scale production process in which the purity of the final product and the synthesis yield must be considered, an aptamer of as short a length as possible is desirable.
- the dual-specific aptamer is two aptamers linked by a linker, which is a practical limitation in commercialization of therapeutic agents when considering mass production.
- the dual specific aptamer since the dual specific aptamer has the disadvantages (in vivo instability, rapid renal clearance, inability to deliver into cells, etc.) of the above-mentioned aptamer as a drug, it is more difficult to be utilized as a drug.
- Patent Document 1 Korean Patent Publication No. 2019-0126356
- Patent Document 2 US Patent No. 8,969,318
- Non-Patent Document 1 Zhang et al., Recent Advances in Aptamer Discovery and Applications, Molecules 2019, 24, 941
- Non-Patent Document 2 Zhou, J.; Rossi, J. Aptamers as Targeted Therapeutics: Current Potential and Challenges. Nat. Rev. Drug Discovery 2017, 16, 181-202.
- Non-Patent Document 3 Dougan, H.; Lyster, D. M.; Vo, C. V.; Stafford, A.; Weitz, J. I.; Hobbs, J. B. Extending the Lifetime of Anticoagulant Oligodeoxynucleotide Aptamers in Blood. Nucl. Med. Biol. 2000, 27, 289-297.
- Non-Patent Document 4 Povsic, T. J., et al. J ALLERGY CLIN IMMUNOL, 2016, 138(6), 1712
- Non-Patent Document 5 M. M. Soldevilla, H. Villanueva, F. Pastor, “Aptamers: A Feasible Technology in Cancer Immunotherapy”, Journal of Immunology Research, vol. 2016, Article ID 1083738, 12 pages, 2016. https://doi.org/0.1155/2016/1083738
- the present inventors have completed the present disclosure by discovering a new nucleic acid ligand that goes beyond the conventional aptamer concept.
- an aptamer is defined as a nucleic acid molecule that binds to a single target, such as a monoclonal antibody.
- the inventors of the present disclosure have solved the problems of conventional aptamers by discovering novel nucleic acid ligands capable of having specific binding affinity to two or more different targets.
- a nucleic acid ligand according to an embodiment of the present disclosure can be obtained by using a conventional SELEX method, but it is not known in the art that a nucleic acid ligand capable of binding to two or more different targets can be searched for. .
- researchers or scientists engaged in the technical field of the present disclosure thought that it was impossible to find a nucleic acid ligand capable of binding to two or more different targets using the existing SELEX technology. This is because, in the conventional SELEX technology, it is common knowledge to remove or exclude oligonucleotides that do not bind to a desired target or to remove or exclude oligonucleotides that bind to other targets.
- the nucleic acid ligand of the present disclosure does not conform to the conventional definition of an aptamer, and is a breakthrough that does not follow the development flow or common knowledge of the aptamer technology.
- An object of the present disclosure is to provide a novel nucleic acid ligand that temporarily solves the limitations of the above existing aptamers in clinical application.
- An object of the present disclosure is to provide a novel method for preparing a novel nucleic acid ligand or a novel screening method.
- the present disclosure provides a nucleic acid ligand that does not exist as a novel nucleic acid ligand capable of having specific binding affinity to two or more different targets, wherein the binding sites for two or more targets among the nucleic acid ligands are separated from each other.
- the nucleic acid ligand according to an embodiment of the present disclosure is not a dual specific aptamer provided in a form in which two or more aptamers are linked to each other.
- a single nucleic acid ligand that binds to a plasma protein (eg, albumin) as a first target and to a cancer cell specific molecule as a second target uses the first target, i.e., when administered to the blood. , it binds to the albumin protein in plasma, and thus can reach cancer tissues by avoiding degradation or renal excretion by nucleases in the body. Then, after reaching the cancer tissue, it enters the cell by a pathway such as endocytosis and binds to cancer cell-specific molecules in the cell to exert the intended therapeutic effect.
- the nucleic acid ligand according to an embodiment of the present disclosure solves problems such as in vivo instability, rapid renal clearance, and inability to deliver into cells of the conventional aptamer.
- An embodiment of the present disclosure is a nucleic acid ligand having specific binding affinity for two or more different targets, wherein each target has a three-dimensional structure, and is not a linkage of a plurality of aptamers (non -coupling), and may relate to a non-natural nucleic acid ligand.
- An embodiment of the present disclosure is a nucleic acid ligand having specific binding affinity for two or more different targets, wherein each target has a three-dimensional structure, and the nucleic acid ligand binds to one target all or part of the nucleic acid sequence forming the site forms all or part of the nucleic acid sequence forming the binding site for another target.
- One embodiment of the present disclosure is a nucleic acid ligand having specific binding affinity for two or more different targets, wherein each target has a three-dimensional structure, It may relate to a non-naturally occurring nucleic acid ligand, wherein the binding sites do not exist separately from each other.
- An embodiment of the present disclosure is a nucleic acid ligand having specific binding affinity for two or more different targets, wherein each target has a three-dimensional structure, and binding sites for two or more targets are one It may relate to a non-natural nucleic acid ligand, which is formed from a single nucleic acid ligand of
- the portion of the nucleic acid sequence forming the binding site for the target is from about 1% to about 99%, from about 5% to about 95%, from about 10% to about 90% of the total sequence of the non-natural nucleic acid ligand. %, from about 20% to about 80%, from about 30% to about 70%, or from about 40% to about 60%.
- the non-natural nucleic acid ligand may have specific binding affinity for two different targets.
- the target may be selected from the group consisting of cells, viruses and proteins.
- one of the targets may be a plasma protein and the other may be a therapeutic target.
- the plasma protein is selected from the group consisting of albumin, alpha 1 globulin, alpha 2 globulin, beta globulin, gamma globulin, immunoglobulin A, immunoglobulin G, lipoprotein, fibrinogen, transferrin and transdiretin. it may be
- the therapeutic target may be a protein present in a cell or a protein present in a cell membrane.
- the therapeutic target is a membrane protein, a transmembrane protein, a glycoprotein, an immune antibody, a virus, a viral envelope glycoprotein, a viral enzyme, a secreted protein, a serine proteolytic enzyme, a peptide hormone, a neurotransmitter , hormone, dehydrogenase, cytokine, E3 ubiquitin ligase, neuropeptide, hydrolase, serine protease inhibitor, human hydrolase, chemokine protein, methyl converting enzyme, oxidase, growth factor, bacteria, bacterial protein , may be selected from the group consisting of intracellular proteins, extracellular matrix, receptors, transcription factors, and tumor proteins.
- the therapeutic target is 4-1BB, acetylcholine receptor, alpha thrombin, amylin, angiopoietin 1, angiopoietin 2, AXL, BCL-2, BMPR-1R, BRD1, BRD2, BRD3, BRD4, BRDT, BTLA, calcitonin gene-related peptide, CBP (CREB-binding protein), CCK4/PTK7, CD16a, CD16b, CD19, CD20, CD200, CD200R, CD27, CD28, CD3, CD30, CD32A, CD32B, CD33, CD4, CD40L, CD52, CD80, CD94, CSF1R, CTLA-4, DDR1, DDR2, E2F1, EGFR, EPH, ERBB2, FGF, FGFR, ghrelen, GITR, glypican3, gonadotropin-releasing hormone 1, HIV gp120, HIV-1 integrase, HIV-1 reverse transcripta
- CBP
- the therapeutic target may be selected from the group consisting of KRAS G12D, KRAS G12V, KRAS G12C, KRAS G12A, KRAS G12S, KRAS G12R, KRAS G13D, KRAS Q61H, and combinations thereof.
- the non-natural nucleic acid ligand may consist of about 100 nucleotides or less.
- the nucleotides may be selected from the group consisting of DNA nucleotides, RNA nucleotides, modified nucleotides thereof, and combinations thereof.
- the non-natural nucleic acid ligand may consist of one nucleic acid sequence selected from the group consisting of SEQ ID NO: 6 to SEQ ID NO: 183.
- the nucleic acid may be selected from the group consisting of single-stranded RNA, double-stranded RNA, single-stranded DNA and double-stranded DNA.
- An embodiment of the present disclosure may relate to a pharmaceutical composition for preventing or treating cancer comprising the non-natural nucleic acid ligand according to the embodiment.
- the non-natural nucleic acid ligand may have a specific binding affinity to a plasma protein as a first target and a therapeutic target protein as a second target.
- the pharmaceutical composition may be administered to a patient intravenously.
- the cancer may be a solid cancer.
- the cancer may be one that expresses the PD-L1 protein on the surface of cancer cells.
- the cancer expressing PD-L1 protein on the surface of the cancer cells is brain tumor, lung cancer, colon cancer, small intestine cancer, stomach cancer, testicular cancer, thyroid cancer, cervical cancer, skin cancer, bladder cancer, ovarian cancer, kidney cancer, liver cancer , at least one selected from the group consisting of pancreatic cancer, urothelial cell carcinoma, and breast cancer,
- the cancer may be a cancer cell having a KRAS mutant protein.
- the cancer in which the cancer cell has a KRAS mutant protein is lung cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, gallbladder cancer, biliary tract cancer, skin cancer, stomach cancer, brain tumor, kidney cancer And it may be one or more selected from the group consisting of acute myeloid leukemia.
- the non-natural nucleic acid ligand may be a nucleic acid ligand consisting of one nucleic acid sequence selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 84, and SEQ ID NO: 100.
- An embodiment of the present disclosure may relate to a diagnostic composition comprising the non-natural nucleic acid ligand according to the embodiment.
- An embodiment of the present disclosure may relate to a contrast agent comprising the non-natural nucleic acid ligand according to the embodiment.
- An embodiment of the present disclosure may relate to a radiopharmaceutical comprising the non-natural nucleic acid ligand according to the embodiment.
- An embodiment of the present disclosure may relate to a composition for diagnosing cancer comprising the non-natural nucleic acid ligand according to the embodiment.
- An embodiment of the present disclosure comprises the step of sequentially contacting one or more nucleic acid ligand candidate sequences with two or more different targets, wherein each step comprises a nucleic acid ligand candidate sequence having specific binding affinity with the target. It may relate to a method for identifying a non-natural nucleic acid ligand having specific binding affinity for two or more different targets, comprising identifying, wherein each target has a three-dimensional structure.
- the method comprises the steps of (a) contacting at least one first nucleic acid ligand candidate sequence with a target and identifying at least one second nucleic acid ligand candidate sequence that specifically binds the target; and (b) (i) at least one second nucleic acid ligand candidate sequence obtained from step (a) or (ii) at least one second nucleic acid ligand candidate sequence obtained from step (a). contacting the candidate sequence with a target different from that of step (a), and identifying one or more fourth nucleic acid ligand candidate sequences that specifically bind to the different target.
- step (b) comprises binding a random sequence consisting of a plurality of nucleotides to at least one of both ends of the at least one second nucleic acid ligand candidate sequence obtained from step (a) to bind the at least one third nucleic acid ligand It may further include the step of preparing a candidate sequence.
- the random sequence is bound to either end of the second nucleic acid ligand candidate sequence, and the second nucleic acid ligand candidate sequence or a portion thereof in the third nucleic acid ligand candidate sequence is amplified of a nucleic acid It may be used as a primer sequence for
- the random sequence may consist of about 20 to about 40 consecutive nucleotides, or the third nucleic acid ligand candidate sequence may consist of about 40 to about 100 consecutive nucleotides.
- the method may be performed by systematic evolution of ligands by exponential enrichment (SELEX).
- An embodiment of the present disclosure provides a method for identifying nucleic acid ligands by systematic evolution of ligands by exponential enrichment (SELEX) starting from one nucleic acid library, wherein the method includes each other having a three-dimensional structure in each SELEX round.
- SELEX systematic evolution of ligands by exponential enrichment
- the method includes each other having a three-dimensional structure in each SELEX round.
- For two or more different targets comprising selecting one target from the group consisting of two or more different targets and selecting a nucleic acid ligand having a specific binding affinity for the selected target in each SELEX round It may relate to a method of identifying a nucleic acid ligand having a specific binding affinity.
- the method may be to perform SELEX performed in one or more rounds on one target and then perform SELEX performed in one or more rounds on another target.
- the method comprises the steps of (i) performing one or more SELEX rounds on a target; (ii) performing one or more SELEX rounds on a target different from the target of step (i) using the nucleic acid library obtained from step (i); (iii) performing step (i) again using the nucleic acid library obtained from step (ii); and (iv) repeating steps (i) to (iii).
- the method may further comprise performing negative SELEX or count SELEX on other traits of the target.
- said SELEX consists of classical SELEX, Advanced SELEX, IP-SELEX, Capture-SELEX, Cell-SELEX, CE-SELEX, M-SELEX, AFM-SELEX, AEGIS-SELEX, and Animal-SELEX. It may be one or more selected from the group.
- the method may further comprise assessing the specific binding affinity of the identified nucleic acid ligand candidate sequence to the target.
- the target may be selected from the group consisting of cells, viruses and proteins.
- one of the targets may be a plasma protein and the other may be a therapeutic target protein.
- the non-natural nucleic acid ligand may consist of about 40 to about 100 nucleotides.
- the method may comprise removing, from the identified nucleic acid ligand candidate sequence, nucleotides not involved in specific binding affinity for the target.
- An embodiment of the present disclosure may relate to a method of detecting a target in a sample, comprising contacting the target from the sample with a nucleic acid ligand according to an embodiment.
- An embodiment of the present disclosure may relate to a non-natural nucleic acid ligand prepared by a method comprising the method according to the embodiment.
- novel nucleic acid ligand according to an embodiment of the present disclosure may exhibit excellent specific binding affinity for two or more different targets as a single nucleic acid ligand.
- the novel nucleic acid ligand according to an embodiment When the novel nucleic acid ligand according to an embodiment is administered to the body as a drug, it specifically binds to plasma proteins and can exhibit the effect that it can be delivered into cells through routes such as excellent in vivo stability, slow renal clearance, and endocytosis. have. Due to these characteristics, the novel nucleic acid ligand can exhibit excellent therapeutic effects for diseases dependent on therapeutic targets, and can be utilized as drugs applied to actual clinical practice.
- the novel nucleic acid ligand according to an embodiment may be used for diagnosis, bio-imaging, drug-discovery tool, assay reagent, biosensor, food inspection, or target (eg, : Protein) It can be used in the purification field.
- the novel nucleic acid ligand does not require use with other macromolecules (eg, PEG) after preparation to achieve in vivo stability or slow renal clearance, so that such molecules may affect the structure of the nucleic acid ligand or The problem of reducing affinity or specificity for the target can be eliminated. Accordingly, the nucleic acid ligand can maintain the excellent therapeutic efficacy confirmed in the experiment as it is as a drug applied clinically.
- macromolecules eg, PEG
- the novel nucleic acid ligand according to one embodiment has a relatively short sequence length (eg, about 20 to about 100, preferably about 25 to about 65 oligonucleotide sequences) and does not need to be used in combination with other substances. . Accordingly, there is no problem in that the quality or performance of the nucleic acid ligand varies from batch to batch in the mass production process, quality control is easy, and it has the effect of satisfying the purity and synthesis yield for use as a drug after mass production. As a result, the novel nucleic acid ligand according to an embodiment can be efficiently mass-produced using only an automatic synthesizer.
- a relatively short sequence length eg, about 20 to about 100, preferably about 25 to about 65 oligonucleotide sequences
- the novel nucleic acid ligand according to an embodiment may bind to a three-dimensional structure of a cell, virus, or protein, and may exhibit an excellent therapeutic effect to be achieved by such binding.
- FIG. 1 shows a schematic flowchart for obtaining a single nucleic acid ligand having a first target protein as a plasma protein and a second target protein as a cancer cell specific protein according to an embodiment.
- FIG. 2 shows an example of a schematic schematic diagram of a first selection method, which is one of methods for preparing a nucleic acid ligand according to an embodiment.
- FIG. 3 shows an example of a schematic schematic diagram of a second selection method, which is one of methods for preparing a nucleic acid ligand according to an embodiment.
- FIG. 4 shows another example of a schematic schematic diagram of a second selection method, which is one of methods for preparing a nucleic acid ligand according to an embodiment.
- FIG. 5 shows another example of a schematic schematic diagram of a second selection method, which is one of methods for preparing a nucleic acid ligand according to an embodiment.
- Figure 6a shows the results of a bead-based ELISA binding assay analyzing the binding force to plasma proteins (human serum albumin) of a nucleic acid library pool obtained through primary SELEX.
- 6B shows the results of a bead-based ELISA binding assay in which the binding ability of a nucleic acid library pool obtained through primary SELEX to plasma protein (immunoglobulin G) was analyzed.
- Figure 6c shows the results of the bead-based ELISA binding assay (ELISA binding assay) analysis of the binding force to the plasma protein (transferrin) of the nucleic acid library pool obtained through the primary SELEX.
- FIG. 7 shows the results of a filter binding assay analyzing the binding ability of a nucleic acid library pool obtained through primary SELEX to plasma protein (albumin).
- Figure 8a shows the results of analyzing the binding force of the nucleic acid library pool obtained through the secondary SELEX to the KRAS G12D mutant protein.
- Figure 8b shows the results of analyzing the binding force of the nucleic acid library pool obtained through the secondary SELEX to the KRAS G12V mutant protein.
- Figure 8c shows the results of analyzing the binding affinity to the KRAS G12C mutant protein of the nucleic acid library pool obtained through the secondary SELEX.
- Figure 8d shows the results of analyzing the binding affinity to BCL2 of the nucleic acid library pool (pool) obtained through the secondary SELEX.
- Figure 9a shows the results of analyzing the binding affinity to human serum albumin (HSA) of the clonal nucleic acid ligand selected from the secondary SELEX nucleic acid library pool (pool).
- HSA human serum albumin
- 9B shows the results of analyzing the binding affinity of the clonal nucleic acid ligand selected from the secondary SELEX nucleic acid library pool to the PD-L1 protein.
- Figure 10a shows the results of analyzing the binding affinity to human serum albumin (HSA) of the clonal nucleic acid ligand selected from the secondary SELEX nucleic acid library pool (pool).
- HSA human serum albumin
- 10B shows the results of analyzing the binding affinity of the clonal nucleic acid ligand selected from the secondary SELEX nucleic acid library pool to the KRAS G12D protein.
- 11A to 11D show Dnase stability results for human serum albumin (HSA) and KRAS G12D target nucleic acid ligands for human serum albumin (HSA) and KRAS G12D protein and 2D predicted structures of each individual nucleic acid ligand, according to an embodiment.
- (-) is a negative control without adding the target protein and nuclease
- (+) is a positive control containing only nuclease
- 1 is the experimental group with human serum albumin and nuclease.
- 2 shows the results of the experimental group in which KRAS G12D protein and nuclease were added.
- FIGS 12A to 12C show Dnase stability results for human serum albumin (HSA) and KRAS G12D target nucleic acid ligands for human serum albumin (HSA) and KRAS G12D protein and 2D predicted structures of each individual nucleic acid ligand, according to an embodiment.
- (-) is a negative control that does not add the target protein and nuclease
- (+) is a positive control that contains only nuclease
- 1 is the experimental group in which human serum albumin and nuclease are added.
- 2 shows the results of the experimental group in which KRAS G12D protein and nuclease were added.
- FIG. 13 shows the results of UPLC analysis before and after purification of the nucleic acid ligand AD005 according to one embodiment.
- 15A and 15B show the results of stability in serum in various species of human serum albumin (HSA) and KRAS G12D target nucleic acid ligands according to an embodiment.
- HSA human serum albumin
- KRAS G12D target nucleic acid ligands according to an embodiment.
- FIG. 16 shows the results of confirming stability after reacting human serum albumin (HSA) and a KRAS G12D target nucleic acid ligand with a KRAS mutant or wild-type protein and DNase treatment according to an embodiment.
- HSA human serum albumin
- FIG. 17 shows the results of a serum half-life test of human serum albumin (HSA) and a KRAS G12D target nucleic acid ligand according to an embodiment.
- HSA human serum albumin
- FIG. 18 shows the results of analysis of binding affinity for KRAS G12D of a nucleic acid ligand capable of specifically binding to human serum albumin (HSA) and a KRAS G12D mutant protein according to an embodiment.
- HSA human serum albumin
- 19A to 19D show the results of microscopic confirmation of the intracellular delivery effect of human serum albumin (HSA) and KRAS G12D target nucleic acid ligand according to an embodiment.
- HSA human serum albumin
- KRAS G12D target nucleic acid ligand colocalize with 70KDa dextran-TMR, a macropinocytosis marker, through stereoscopic analysis.
- FIG. 21 shows cancer cell survival rates according to treatment with human serum albumin (HSA) and a KRAS G12D target nucleic acid ligand according to an embodiment.
- HSA human serum albumin
- HSA human serum albumin
- KRAS G12D target nucleic acid ligand show cancer cell survival rates according to treatment with human serum albumin (HSA) and a KRAS G12D target nucleic acid ligand according to an embodiment.
- HSA human serum albumin
- KRAS G12D target nucleic acid ligand prepared according to an embodiment after treatment with an endocytosis inhibitor.
- 24A and 24B show the inhibitory effect of human serum albumin (HSA) and KRAS G12D target nucleic acid ligand on ERK phosphorylation, which is a downstream protein of the KRAS signal transduction pathway, according to an embodiment.
- HSA human serum albumin
- KRAS G12D target nucleic acid ligand on ERK phosphorylation, which is a downstream protein of the KRAS signal transduction pathway, according to an embodiment.
- HSA human serum albumin
- KRAS G12D target nucleic acid ligands according to an embodiment.
- 26A to 26C show the tumor suppression effect of human serum albumin (HSA) and KRAS G12D target nucleic acid ligand in xenograft mice injected with MIA PaCa-2 pancreatic cancer cell line according to an embodiment.
- HSA human serum albumin
- KRAS G12D target nucleic acid ligand in xenograft mice injected with MIA PaCa-2 pancreatic cancer cell line according to an embodiment.
- 27A and 27C show in vivo safety results according to changes in mouse organ weight when human serum albumin (HSA) and KRAS G12D target nucleic acid ligand are administered according to an embodiment.
- HSA human serum albumin
- KRAS G12D target nucleic acid ligand are administered according to an embodiment.
- HSA human serum albumin
- KRAS G12D target nucleic acid ligand show the tumor suppression effect according to the administered dose of human serum albumin (HSA) and KRAS G12D target nucleic acid ligand according to an embodiment.
- FIG. 29 shows the results of mouse organ weight change according to the administration dose of human serum albumin (HSA) and the KRAS G12D target nucleic acid ligand, and in vivo safety results according to this, according to an embodiment.
- HSA human serum albumin
- liver damage index according to the administered dose of human serum albumin (HSA) and KRAS G12D target nucleic acid ligand according to an embodiment.
- FIG. 31 shows the measurement results of leukocytes, red blood cells and platelets of human serum albumin (HSA) and KRAS G12D target nucleic acid ligands according to an embodiment.
- HSA human serum albumin
- FIG. 32 shows the PD-1/PD-L1 binding inhibitory effect at the protein level of human serum albumin (HSA) and a PD-L1 target nucleic acid ligand according to an embodiment.
- HSA human serum albumin
- FIG 33 shows the effect of inhibiting PD-1/PD-L1 binding at the protein level of human serum albumin (HSA) and a PD-L1 target nucleic acid ligand according to an embodiment.
- HSA human serum albumin
- HSA human serum albumin
- A, B, and C As used herein, “A, B, and C,” “A, B, or C,” “A, B, and/or C,” or “at least one of A, B, and C,” “A, B , or at least one of C,” “at least one of A, B, and/or C,” “at least one selected from A, B, and C,” “at least one selected from A, B, or C,” “A The expression “at least one selected from , B, and/or C” may mean each listed item or all possible combinations of the listed items.
- At least one selected from A and B means (1) at least one of A, (2) at least one of A, (3) at least one of B, (4) B, (5) at least one of A and B (6) at least one of A and B, (7) at least one of B and A, and (8) both A and B.
- the term “about” or “approximately” may refer to the usual error range for the respective value, as is well known to those skilled in the art. In the context of a numerical value or range described herein, it is ⁇ 20%, ⁇ 15%, ⁇ 10%, ⁇ 9%, ⁇ 8%, ⁇ 7% of a numerical value or range recited or claimed in one embodiment. , ⁇ 6%, ⁇ 5%, ⁇ 4%, ⁇ 3%, ⁇ 2%, or ⁇ 1%.
- aptamer refers to an oligonucleotide having a specific binding affinity for only one target.
- SELEX SELEX technology
- SELEX method SELEX process
- a nucleic acid that interacts with a target in a preferred manner eg, by binding to a protein
- It may refer to a method in which the process of selecting and amplifying the selected nucleic acid are combined.
- SELEX method is an in vitro method for screening nucleic acid molecules capable of binding to a target molecule with high specificity, and includes U.S. Patent No. 5,475,096 (“Nucleic Acid Ligands”) and U.S. Patent No. 5,270,163 (WO 91). /19813, "Nucleic Acid Ligands").
- the present disclosure relates, in one embodiment, to novel non-naturally occurring nucleic acid ligands, which are a new class of nucleic acid compounds previously thought impossible to exist.
- new nucleic acid ligand As used herein, the terms “new nucleic acid ligand”, “nucleic acid ligand”, “non-natural nucleic acid ligand”, and “new non-natural nucleic acid ligand” may be used interchangeably with each other. This may refer to a nucleic acid molecule having specific binding affinity for two or more different targets.
- non-naturally occurring means that it does not occur in nature, and in this respect, the novel nucleic acid ligand according to an embodiment of the present disclosure may not be a nucleic acid having a known physiological function that binds to a target. have.
- the novel nucleic acid ligand may have specific binding affinities for two or more different targets.
- a novel nucleic acid ligand may have specific binding affinities for two, three, four, five, six, or more different targets.
- the novel nucleic acid ligand may have specific binding affinities for two different targets.
- the term "specific binding affinity" means that a nucleic acid ligand binds its target with a generally much higher affinity than binding to other non-target components or components present in a mixture or sample. can do.
- the plurality of nucleic acid ligand candidate sequences may be selected from a nucleic acid library or nucleic acid mixture having specific binding affinity to two or more different targets (see FIGS. 8A to 8D ), and in one embodiment The novel nucleic acid ligand according to the method may be selected by being confirmed to have specific binding affinity to two or more different targets from among the plurality of nucleic acid ligand candidate sequences selected in this way ( FIGS. 9A, 9B, 10A, and FIG. see 10b).
- the target may have a three-dimensional structure. In one embodiment, the target does not include a polynucleotide that binds to a nucleic acid via a mechanism that primarily relies on Watson/Crick base pairing or triple helix binding.
- the novel nucleic acid ligand can interact with the target by three-dimensionally binding to the target, recognizing the three-dimensional structure of the target, or forming a three-dimensional structure by itself.
- the novel nucleic acid ligand can bind to a target, recognize the three-dimensional structure of the target, or interact with the target to modulate the properties of each target.
- the novel nucleic acid ligand according to an embodiment may bind to a target protein and inhibit the activity of the protein, block the signal transduction pathway, or inhibit the activity of proteins downstream of the signaling pathway.
- bonding refers to, for example, pi-stacking, electrostatic interactions, hydrophobic interactions, ionic interactions and/or hydrogen bonding interactions under physiological conditions. It may refer to an association between two molecules due to an action or the like (eg, a stable association between a nucleic acid ligand and a target).
- the term “modulation” may refer to changing a functional property, biological activity, and/or biological mechanism (or pathway) of a target. For example, it may be to up-regulate (eg, when activating or stimulating), down-regulating (eg, when inhibiting or inhibiting), or otherwise changing a property, activity, and/or mechanism of a target.
- the target is a cell
- the cell's property to be regulated is cell viability, cell proliferation, gene expression, cell morphology, cell activation, phosphorylation, calcium mobilization, degranulation, cell migration, and/or cell It may be fragmentation.
- the novel nucleic acid ligand is not an existing aptamer that binds only to one specific target, but is also a bispecific aptamer in which a plurality of different aptamers are linked or bound to each other. aptamer) is not.
- the novel nucleic acid ligand is non-coupling of a plurality of aptamers.
- the term “connector” may refer to linking, fusion, bonding, or conjugation of two or more separate aptamers to each other.
- the linker may mean a dimer, trimer, tetramer, or more multimer of any monomer aptamer, and a dual specific aptamer in which two or more different aptamers are linked or bound.
- Linkers may or may not include a linker.
- a novel nucleic acid ligand may be one in which all or a portion of a nucleic acid sequence forming a binding site for one target forms all or a portion of a nucleic acid sequence forming a binding site for another target.
- the nucleic acid sequence forming the binding site for the target may include a sequence of a 5' or 3' primer for amplification of a nucleic acid ligand or a portion thereof.
- the portion of the nucleic acid sequence forming the binding site for the target is about 1% to about 99%, about 5% to about 95%, about 10% to about 90%, about 20% of the total sequence of the nucleic acid ligand. % to about 80%, from about 30% to about 70%, or from about 40% to about 60%, but is not limited thereto.
- a portion of the nucleic acid sequence forming the binding site for the target may include one or more nucleotides constituting a nucleic acid ligand, but is not limited thereto.
- the novel nucleic acid ligand may be one in which binding sites for two or more targets among the nucleic acid ligands are not separately separated from each other.
- the presence of a target binding site separated from each other may mean an aptamer multimer, a dual specific aptamer, or other complex/conjugate/conjugate composed of a plurality of aptamers.
- the novel nucleic acid ligand may be one in which binding sites for two or more targets are formed from one single nucleic acid ligand or from one single nucleic acid ligand.
- the tertiary structure of the nucleic acid ligand formed for binding to the target is formed from a sequence of one nucleic acid ligand or a single nucleic acid ligand, rather than formed by combining those formed from separate sequences or from separate sequences. may mean that Accordingly, the novel nucleic acid ligand corresponds to a substance that is different and distinct from the dual specific aptamer.
- the novel nucleic acid ligand may consist of no more than about 100 nucleotides. Specifically, the novel nucleic acid ligand may consist of 100 or less, 90 or less, 80 or less, 70 or less, 60 or less, 50 or less, or 40 or less nucleotides. In one embodiment, the novel nucleic acid ligands are 10 to 105, 15 to 100, 20 to 95, 25 to 90, 30 to 85, 35 to 80, 40 to 75 , 45 to 70, 50 to 65, or 55 to 60 nucleotides.
- the nucleotides may be selected from the group consisting of DNA nucleotides, RNA nucleotides, modified nucleotides thereof, and combinations thereof.
- Modified nucleotide as used in the present disclosure can mean analogs, substituents, or esters of naturally occurring nucleotides (A, G, T/U, and C) and encompasses modified nucleotides readily available to those of ordinary skill in the art. It can mean a wide range of concepts. For example, a modified nucleotide has a substitution at the C-5 position (e.g., a C-5 modified pyrimidine) or the saccharides (ribose or deoxyribose) that make up the nucleotide are modified, or both of these modifications. Branches may mean nucleotides.
- a C-5 modified pyrimidine may refer to a C-5 modified amino carbonyl pyrimidine, e.g., 5-(N-benzylcarboxyamide)-2'-deoxyuridine (Bz-dU), 5-(N-isobutylcarboxyamide)-2'-deoxyuridine (iBu-dU), 5-(N-phenethylcarboxyamide)-2'-deoxyuridine (Pe) -dU), 5-(N-thiophenylmethylcarboxyamide)-2'-deoxyuridine (Th-dU), 5-(N-[2-(1H-indol-3yl)ethyl]carboxyamide) -2'-deoxyuridine (Trp-dU), 5-(N-[1-(3-trimethylammonium)propyl]carboxyamide)-2'-deoxyuridine chloride, 5-(N-naphthyl) methylcarboxyamide)-2'-deoxyuridine (N
- the modified nucleotide with the 2' position modification of the ribose sugar is 2'-deoxy-2'-fluorouridine (U f ), 2'-deoxy-2'-fluoro Rocytidine (C f ), 2'-hydroxyadenosine (A r ), 2'-methoxyadenosine (A m ), 2'-methoxyguanosine (G m ) does not
- a modified nucleotide having both a C-5 modification and a 2' position modification of a ribose sugar is 5-(N-benzylcarboxyamide)-2'-O-methyluridine, 5-( N-benzylcarboxyamide)-2'-fluorouridine, 5-(N-isobutylcarboxyamide)-2'-O-methyluridine, 5-(N-isobutylcarboxyamide)-2'-fluoro Louridine, 5-(N-[2-(1H-indol-3yl)ethyl]carboxyamide)-2'-O-methyluridine, 5-(N-[2-(1H-indole-3yl) )ethyl]carboxyamide)-2'-O-fluorouridine, 5-(N-naphthylmethylcarboxyamide)-2'-O-methyluridine, 5-(N-naphthylmethylcarboxyamide)- 2'-fluorouridine, 5-(N-n
- a modified nucleotide having both a C-5 modification and a 2' position modification of a ribose sugar is 5-(N-benzylcarboxyamide)-2'-O-methylcytidine, 5-( N-benzylcarboxyamide)-2'-fluorocytidine, 5-(N-isobutylcarboxyamide)-2'-O-methylcytidine, 5-(N-isobutylcarboxyamide)-2'-fluoro Rocytidine, 5-(N-[2-(1H-indol-3yl)ethyl]carboxyamide)-2'-O-methylcytidine, 5-(N-[2-(1H-indole-3yl) )ethyl]carboxyamide)-2'-O-fluorocytidine, 5-(N-naphthylmethylcarboxyamide)-2'-O-methylcytidine, 5-(N-naphthylmethylcarboxyamide)
- the modified nucleotide is 5-(N-benzylcarboxyamide)-2'-deoxyuridine (Bz-dU) or 5-(N-naphthylmethylcarboxyamide)-2'-deoxyuridine It may be din (Nap-dU), and accordingly, at least one of the nucleotides constituting the novel nucleic acid ligand may be such a modified nucleotide.
- the novel nucleic acid ligand may consist of one nucleic acid sequence selected from the group consisting of SEQ ID NO: 6 to SEQ ID NO: 183.
- the novel nucleic acid ligand may be selected from the group consisting of single-stranded RNA, double-stranded RNA, single-stranded DNA and double-stranded DNA.
- the novel nucleic acid ligand may be used by binding biotin to the front of the 5' end or the back of the 3' end, if necessary.
- the novel nucleic acid ligand is a fluorescent material (eg, FAM, VIC, HEX, BHQ-1, Cy5, Cy3, Rodamine, or Texas Red, etc.) in front of the 5' end or behind the 3' end, if necessary. can be used in combination.
- a fluorescent material eg, FAM, VIC, HEX, BHQ-1, Cy5, Cy3, Rodamine, or Texas Red, etc.
- the present disclosure may relate to a non-natural nucleic acid ligand prepared by a method comprising the method according to an embodiment.
- the present disclosure may relate to a pharmaceutical composition for preventing or treating cancer, including the novel nucleic acid ligand according to an embodiment.
- the novel nucleic acid ligand may be delivered into cancer cells in which a therapeutic target exists by a pathway such as endocytosis.
- the novel nucleic acid ligand may bind to a plasma protein present in the body of a subject to which the pharmaceutical composition is administered.
- the pharmaceutical composition containing the novel nucleic acid ligand can exhibit excellent in vivo stability and slow renal clearance (excellent residence time in the body), and the novel nucleic acid ligand reaches cancer tissue and then through the pathway such as endocytosis. It can enter cancer cells.
- subject used in the present disclosure refers to mammals such as horses, sheep, pigs, goats, dogs, etc. meaning, but preferably a human.
- endocytosis refers to a cellular action in which a cell transports a substance outside the cell membrane into the cell membrane. Endocytosis encloses foreign substances with a cell membrane and enters the cells, then forms vesicles within the cells to decompose the inhaled substances, and then recycles them or discharges them to the outside. Endocytosis can be divided into macropinocytosis, receptor-mediated endocytosis involving clathrin protein, caveolae endocytosis, and the like depending on the activation method or the type of protein involved.
- the pharmaceutical composition may further comprise a plasma protein.
- the pharmaceutical composition may be a form in which a plasma protein and a novel nucleic acid ligand are combined or a form of a novel nucleic acid ligand alone, and the plasma protein may be a protein existing outside the body of a subject to be treated.
- the novel nucleic acid ligand may be delivered into cancer cells in which a therapeutic target exists by the macropinocytosis pathway.
- the term "macropinocytosis” is one type of endocytosis, and the cell membrane protrudes out of the cell by actin, a cytoskeletal protein, and a pocket can be formed during the process of contracting again. , the formed sac can be separated as it enters the cell to form vesicles (macropinosomes) with a diameter of about 0.2 ⁇ m to 5 ⁇ m.
- Macropinocytosis plays an important role in tumor metabolism, which has recently been attracting attention. Through macropinocytosis, nutrients necessary for rapid growth of tumor cells (eg, glutamine, albumin, etc.) can be supplied to the inside of cancer cells and used for metabolism. That is, the inside of the vesicle formed by macropinocytosis may contain extracellular fluid, extracellular molecules, and the like. Macropinocytosis may be activated by mutation of an oncogene or protein of cancer cells.
- the oncogene may be an SRC gene or a KRAS gene.
- macropinocytosis may be activated by mutation of a KRAS gene or protein in cancer cells.
- the mutation of the KRAS protein may be a mutation in the 12th, 13th, and 61st amino acids of the wild-type KRAS protein, for example, KRAS G12D, KRAS G12V, KRAS G12C, KRAS G12A, KRAS G12S, KRAS G12R, KRAS G13D, or KRAS Q61H.
- the nucleic acid ligand may increase the efficiency of its delivery into the cell by macropinocytosis by binding to the plasma protein. That is, the nucleic acid ligand bound to the plasma protein may have a superior effect of being delivered into the cell compared to the nucleic acid ligand not bound to the plasma protein (see FIG. 25 ).
- the nucleic acid ligand according to an embodiment of the present disclosure may be delivered into the cytoplasm through various mechanisms of endocytosis. Macropinocytosis, which is attracting attention as a major pathway for supplying essential nutrients necessary for rapid proliferation of cancer cells, as well as other endocytosis pathways may be involved in the delivery of nucleic acid ligands.
- the nucleic acid ligand delivered into the cytoplasm binds to a therapeutic target protein and disrupts the signal transduction pathway, thereby inhibiting the proliferation of cancer cells.
- the nucleic acid ligand delivered into the cell binds to a KRAS mutant protein (eg, KRAS G12D) inside the cancer cell and inhibits phosphorylation of proteins downstream of the KRAS signaling pathway (ERK, etc.), thereby causing excessive cell growth , proliferation, and division.
- a KRAS mutant protein eg, KRAS G12D
- ERK phosphorylation of proteins downstream of the KRAS signaling pathway
- the nucleic acid ligand according to an embodiment of the present disclosure may be used for cancer expressing a KRAS mutant protein (eg, lung cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, gallbladder cancer, biliary tract cancer, skin cancer, stomach cancer) , brain tumor, kidney cancer, or acute myeloid leukemia) can be prevented or treated.
- a KRAS mutant protein eg, lung cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, gallbladder cancer, biliary tract cancer, skin cancer, stomach cancer
- a KRAS mutant protein eg, lung cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, gallbladder cancer, biliary tract cancer, skin cancer, stomach cancer
- treatment may refer to any action in which the symptoms of a subject having a disease are improved or are beneficially changed.
- prevention may refer to any action that suppresses or delays a disease.
- the novel nucleic acid ligand may be administered in a pharmaceutically effective amount.
- the novel nucleic acid ligand is from about 1 ug to about 100 ug, from about 5 ug to about 80 ug, from about 10 ug to about 70 ug, from about 15 ug to about 60 ug, from about 20 ug to about 50 ug, or It may be administered in a dose of about 30 ug to about 40 ug, and the administration may be administered once or dividedly several times a day.
- the term “administration” refers to introducing the pharmaceutical composition according to an embodiment to a subject by any suitable method.
- the administration route may be oral or parenteral administration through any general route as long as it can reach the target tissue.
- the term "pharmaceutically effective amount” means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the weight, sex, age, and health status of a patient. , severity, indication, drug activity, sensitivity to drug, administration time, administration route and excretion rate, duration of treatment, factors including concomitant drugs, and other factors well known in the medical field.
- the cancer may be a solid cancer.
- the cancer may be one that expresses the PD-L1 protein on the surface of cancer cells.
- the cancer is selected from the group consisting of brain tumor, lung cancer, colon cancer, small intestine cancer, stomach cancer, testicular cancer, thyroid cancer, cervical cancer, skin cancer, bladder cancer, ovarian cancer, kidney cancer, liver cancer, pancreatic cancer, urothelial cell cancer, and breast cancer There may be more than one.
- the pharmaceutical composition may be an immune anticancer agent.
- the novel nucleic acid ligand included in the immune anticancer agent binds to CD3d/e or 4-1BB on the surface of cytotoxic T cells to activate cytotoxic T cells, thereby exhibiting an immune anticancer effect.
- the cancer may be expressed by the BCL-2 protein.
- the cancer may be expressed by a KRAS mutant protein.
- a KRAS mutant protein is at least one selected from the group consisting of lung cancer, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, gallbladder cancer, biliary tract cancer, skin cancer, stomach cancer, brain tumor, kidney cancer and acute myeloid leukemia.
- the pharmaceutical composition for preventing or treating cancer may inhibit the division or proliferation of the aforementioned cancer cells, or induce apoptosis of cancer cells.
- the pharmaceutical composition for preventing or treating cancer comprises a novel nucleic acid ligand consisting of one nucleic acid sequence selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 84, and SEQ ID NO: 100 It may be included as an active ingredient.
- the pharmaceutical composition may be administered orally or administered parenterally according to a desired method, and examples of parenteral administration routes include intravenous, intradermal, intrathoracic, intramuscular, subcutaneous, topical, transdermal, transdermal. mucosal, intraperitoneal, and rectal administration.
- the pharmaceutical composition may be administered by inhalation.
- Inhalation administration may be administration using a pharmaceutical preparation that can be inhaled through the respiratory tract, nasal passages, etc., including respirable particles or droplets containing a nucleic acid ligand, but is not limited thereto.
- a dry powder inhaler device DPI
- pMDI pressurized metered dose inhaler
- the pharmaceutical composition may be formulated for oral or parenteral use.
- the pharmaceutical composition may be a solid preparation for oral administration (eg, tablets, pills, powder, granules, and capsules), and a liquid preparation for oral administration (eg, suspension, internal solution, emulsion, liposome formulation, etc.) , and syrup), or parenteral preparations (eg, injections, non-aqueous solutions, suspensions, emulsions, nasal sprays, transdermal absorption formulations, freeze-dried formulations, suppositories, etc.).
- parenteral preparations eg, injections, non-aqueous solutions, suspensions, emulsions, nasal sprays, transdermal absorption formulations, freeze-dried formulations, suppositories, etc.
- the pharmaceutical composition is a pharmaceutically acceptable salt, diluent, excipient, carrier, buffer, adjuvant or conventionally used in the pharmaceutical composition at a level that does not affect the action of the novel nucleic acid ligand. It may further include a surfactant, but is not limited thereto. Such ingredients may aid in the formulation of pharmaceutical compositions.
- the term “pharmaceutically acceptable” may mean that a substance is non-toxic and does not interact with the action of an active ingredient of a pharmaceutical composition, for example, approved by the regulatory agency of each country or each It may mean listed in the national pharmacopeia.
- pharmaceutically acceptable salt may refer to a product that contains an ionic bond and is conventionally produced by reacting a compound with an acid or base suitable for administration to a subject.
- the adjuvant is, for example, a cationic lipid such as citric acid, hydrochloric acid, tartaric acid, stearic acid, polyethylene glycol, polypropylene glycol, ethanol, sodium bicarbonate, distilled water, hyaluronic acid, lipofectamine, starch, gelatin, talc, ascorbic acid, olive oil, palm oil, methylcellulose, titanium oxide, sodium-carboxymethyl cellulose, sweetener, preservative, etc., but are limited thereto no.
- a cationic lipid such as citric acid, hydrochloric acid, tartaric acid, stearic acid, polyethylene glycol, polypropylene glycol, ethanol, sodium bicarbonate, distilled water, hyaluronic acid, lipofectamine, starch, gelatin, talc, ascorbic acid, olive oil, palm oil, methylcellulose, titanium oxide, sodium-carboxymethyl cellulose, sweetener, preservative, etc., but are limited thereto no
- the present disclosure may relate to a method for preventing or treating cancer, comprising administering a novel nucleic acid ligand according to an embodiment to a subject in need thereof in a pharmaceutically effective amount.
- the present disclosure may relate to the use of the novel nucleic acid ligand according to an embodiment for the manufacture of a medicament for preventing or treating cancer.
- the present disclosure may relate to a novel nucleic acid ligand according to an embodiment for use in preventing or treating cancer.
- the present disclosure may relate to a composition for diagnosing cancer including the novel nucleic acid ligand according to the embodiment.
- the present disclosure may relate to a kit for diagnosing cancer including the novel nucleic acid ligand according to the embodiment.
- the present disclosure may relate to a method for diagnosing cancer, comprising administering the novel nucleic acid ligand according to the embodiment to a subject in need thereof.
- the present disclosure may relate to the use of the novel nucleic acid ligand according to an embodiment for the manufacture of a medicament for diagnosing cancer.
- the present disclosure may, in one embodiment, relate to a novel nucleic acid ligand according to an embodiment for use in diagnosing cancer.
- the present disclosure may relate to a diagnostic composition comprising a novel nucleic acid ligand according to an embodiment.
- the novel nucleic acid ligand according to an embodiment may be used as a diagnostic reagent, an analytical reagent, and a hazardous substance detection.
- a method of detecting a target in a sample using a novel nucleic acid ligand according to an embodiment which may relate to a method comprising the step of (a) contacting the target from the sample with the novel nucleic acid ligand according to an embodiment .
- the detection method may further include (b) determining whether a binding between the target and the nucleic acid ligand is formed after step (a) to detect the presence of the target in the sample.
- the present disclosure may relate to a contrast agent or a diagnostic radiopharmaceutical comprising the novel nucleic acid ligand according to an embodiment.
- the novel nucleic acid ligand may be bound to a material commonly used for acting as a contrast agent or diagnostic radiopharmaceutical, for example, may be bound to a radioactive isotope.
- the present disclosure may relate to a method for identifying a novel non-natural nucleic acid ligand having specific binding affinity for two or more different targets.
- the method may comprise sequentially contacting one or more nucleic acid ligand candidate sequences with two or more different targets.
- each of the steps may include identifying a nucleic acid ligand candidate sequence having specific binding affinity to a target.
- the method comprises (a) contacting one or more first nucleic acid ligand candidate sequences with a target and identifying one or more second nucleic acid ligand candidate sequences that specifically bind to the target; and (b) (i) at least one second nucleic acid ligand candidate sequence obtained from step (a) or (ii) at least one second nucleic acid ligand candidate sequence obtained from step (a). contacting the candidate sequence with a target different from step (a), and identifying at least one fourth nucleic acid ligand candidate sequence that specifically binds the different target.
- the method may further include isolating a sequence specifically bound to the target after the step of contacting the target.
- the isolated sequence may be identified as a sequence that specifically binds to the target through a step of evaluating specific binding affinity for the target.
- the nucleic acid ligand candidate sequence may refer to a nucleic acid molecule included in a nucleic acid library or a mixture of nucleic acid molecules.
- a nucleic acid ligand candidate sequence may refer to a contiguous nucleotide molecule consisting of DNA nucleotides, RNA nucleotides, modified nucleotides thereof, and combinations thereof.
- the first nucleic acid ligand candidate sequence may consist of a 5'-primer binding sequence, a random sequence, and a 3'-primer binding sequence.
- the 5'-primer binding sequence or the 3'-primer binding sequence may be appropriately selected according to the type of nucleic acid ligand or target to be selected, and is not limited to the primer binding sequence or primer sequence described in the present disclosure.
- the first nucleic acid ligand candidate sequence may consist of the nucleic acid sequence of SEQ ID NO: 1 (GCTGGTGGTGTGGCTG-N(40)-CAGGCAGACGGTCACTCA).
- the first nucleic acid ligand candidate sequence may be 10 14 to 10 30 , 10 16 to 10 28 , 10 20 to 10 26 , or 10 22 to 10 25 .
- the first nucleic acid ligand candidate sequence may be included in the SELEX nucleic acid library pool.
- the nucleic acid ligand candidate sequence obtained in step (a) may be used as it is.
- the third nucleic acid ligand candidate sequence may be a random sequence consisting of a plurality of nucleotides bound to one or more of both ends of the second nucleic acid ligand candidate sequence. wherein the random sequence bound to the second nucleic acid ligand candidate sequence consists of about 10 to about 50, about 15 to about 45, about 20 to about 40, or about 25 to about 35 contiguous nucleotides.
- step (b) comprises binding a random sequence consisting of a plurality of nucleotides to at least one of both ends of the at least one second nucleic acid ligand candidate sequence obtained in step (a) to thereby bind one or more third nucleic acid ligand candidate sequences. It may further include the step of manufacturing.
- the random sequence that binds to the second nucleic acid ligand candidate sequence is about 10 to about 50, about 15 to about 45, about 20 to about 40, or about 25 to about 35 contiguous sequences. may consist of nucleotides.
- the random sequence is bound to either end of the second nucleic acid ligand candidate sequence, and the second nucleic acid ligand candidate sequence or a portion thereof in the third nucleic acid ligand candidate sequence is used for amplification of the nucleic acid. It can be used as a primer sequence.
- the length of the nucleic acid ligand candidate sequence can be prevented from becoming excessively long, and a novel nucleic acid ligand capable of achieving the objects and effects of the present disclosure can be obtained.
- random sequence and “random sequence” can be used interchangeably and may refer to a sequence in which a nucleic acid sequence having a desired length is randomly constructed.
- the third nucleic acid ligand candidate sequence is about 40 to about 100, about 50 to about 90, about 55 to about 85, about 60 to about 80, about 65 to about 75 It may consist of consecutive nucleotides.
- the method according to an embodiment may be performed by systematic evolution of ligands by exponential enrichment (SELEX).
- the present disclosure provides a method for identifying nucleic acid ligands by SELEX (Systematic evolution of ligands by exponential enrichment) starting from one nucleic acid library, wherein the method includes different methods having a three-dimensional structure in each SELEX round. specific for two or more different targets, comprising selecting one target from the group consisting of two or more targets and selecting a nucleic acid ligand having a specific binding affinity for the selected target in each SELEX round It may relate to a method for identifying a non-naturally occurring nucleic acid ligand having a specific binding affinity.
- SELEX Systematic evolution of ligands by exponential enrichment
- the method according to an embodiment may be to perform SELEX performed in one or more rounds for another target after completing SELEX performed in one or more rounds for one target.
- the SELEX for one target may be performed in about 4 rounds to about 10 rounds, or about 5 rounds to about 8 rounds, but is not limited thereto.
- a method comprises the steps of (i) performing one or more SELEX rounds on one target; (ii) performing one or more SELEX rounds on a target different from the target of step (i) using the nucleic acid library obtained from step (i); (iii) performing step (i) again using the nucleic acid library obtained from step (ii); and (iv) repeating steps (i) to (iii).
- the SELEX for one target may be performed 1 round, 2 rounds, 3 rounds, 4 rounds, 5 rounds, 6 rounds, 7 rounds, 8 rounds, 9 rounds, 10 rounds, or more.
- the method according to an embodiment may further include a negative selection step (eg, negative SELEX) or a count selection step (eg, count SELEX) in order to select a sequence having excellent binding ability to a trait of the target.
- a negative selection step eg, negative SELEX
- a count selection step eg, count SELEX
- the negative selection step is a method of removing non-specific binding to a target
- the count selection step is a method of increasing specificity for a target through binding to another trait (eg, a mutant) of a target other than the target.
- the count selection step may be selecting a sequence that specifically binds to one trait of the target by separating it from the other trait of the target.
- the sequence having affinity for the wild-type KRAS protein is first separated from the library sequence and removed, and the remaining library sequence is used to target the target. It may be to obtain a sequence that specifically binds only to a mutation of the phosphorus KRAS protein (eg, KRAS G12D).
- the count selection step may be to separate and select a sequence that specifically binds to one protein of the therapeutic target from a similar family protein including the therapeutic target.
- the count selection step in order to isolate a sequence that specifically binds to the BCL2 protein, first, a sequence having affinity for BCL2L1, a family protein having a similar structure, is separated from the library sequence and removed, and the remaining library sequence may be used to obtain a sequence that specifically binds only to the target BCL2 protein.
- the method according to an embodiment may further include performing negative SELEX or count SELEX for other traits of the target.
- the SELEX is classical SELEX (classic SELEX), Advanced SELEX, IP-SELEX, Capture-SELEX, Cell-SELEX, CE-SELEX, M-SELEX, AFM-SELEX, AEGIS-SELEX, and Animal-SELEX It may be one or more selected from the group consisting of, but is not limited thereto.
- the method according to an embodiment may further comprise evaluating the specific binding affinity of the identified nucleic acid ligand candidate sequence to the target.
- the step of assessing specific binding affinity may include one or more of a bead-based ELISA binding assay, a filter binding assay, an assessment of Dnase stability to a target, an ELISA-based determination of avidity (K d ), and an assessment of stability or half-life in serum. It may be performed by, but is not limited thereto.
- the target may be two different targets.
- the method according to an embodiment may include removing nucleotides not involved in specific binding affinity for a target from the identified nucleic acid ligand candidate sequence.
- the present disclosure includes sequentially performing SELEX (Systematic Evolution of Ligands by Exponential Enrichment) on two or more different targets, wherein each SELEX has a specific binding affinity for each target target. It may relate to a method of identifying a non-natural nucleic acid ligand having specific binding affinity for two or more different targets, which is to select a nucleic acid ligand having a
- the method comprises the steps of (a) performing SELEX on one target; (b) performing SELEX on a target different from the target of step (a) using the nucleic acid library obtained from step (a), wherein the target is two or more different targets having a three-dimensional structure
- each SELEX may be a method of identifying a non-natural nucleic acid ligand that selects a nucleic acid ligand having a specific binding affinity to a target.
- the method comprises the steps of: (a) contacting a first nucleic acid library with one target to first select a plurality of nucleic acid ligand candidate sequences that bind to the target; (b) contacting the plurality of firstly selected nucleic acid ligand candidate sequences with other targets to secondarily select a plurality of nucleic acid ligand candidate sequences that bind to different targets, wherein the targets are two or more different targets It may be a method for identifying phosphorus, non-natural nucleic acid ligands.
- the present disclosure may, in one embodiment, relate to a method of detecting a target in a sample, the method comprising contacting the target from the sample with a nucleic acid ligand according to an embodiment.
- a target may refer to any compound to which a nucleic acid can act in a desired manner.
- a target may be a protein, peptide, nucleic acid, carbohydrate, lipid, polysaccharide, glycoprotein, hormone, receptor, antigen, antibody, virus, pathogen, toxic agent, substrate, metabolite, transition state analog, cofactors, inhibitors, drugs, dyes, nutrients, growth factors, cells, tissues and/or any fragment, component or portion thereof.
- the target may be altered within a range that does not substantially change its identity.
- the target is a protein
- it may be a minor modification or alternation in the amino acid sequence, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation ) or any other manipulation or alteration (eg, conjugation with a display component that does not materially change the nature of the target).
- the target may have a three-dimensional structure.
- the three-dimensional structure may mean a biological three-dimensional structure.
- the target may be selected from the group consisting of cells, viruses and proteins.
- the cell is a prokaryotic cell (eg, a bacterium), a eukaryotic cell (eg, a mammalian cell), a parasitic cell (eg, a malaria cell, a leishmania cell, a Cryptosporidium cell, or an amoeba cell), or a fungal cell.
- a prokaryotic cell eg, a bacterium
- a eukaryotic cell eg, a mammalian cell
- a parasitic cell eg, a malaria cell, a leishmania cell, a Cryptosporidium cell, or an amoeba cell
- a fungal cell e.g, a fungal cell.
- a prokaryotic cell eg, a bacterium
- a eukaryotic cell eg, a mammalian cell
- a parasitic cell eg, a malaria cell, a leishmania cell, a Cryptosporidium cell, or an am
- the cell may be a cancer cell.
- Such cells may be derived from any cancerous or precancerous tumor.
- one of the targets may be a plasma protein and the other of the targets may be a therapeutic target.
- the therapeutic target may be a therapeutic target protein.
- the plasma protein is not limited as long as it is a protein present in the plasma of an organism, for example, albumin, alpha 1 globulin, alpha 2 globulin, beta globulin, gamma globulin, immunoglobulin A, immunoglobulin G, lipoprotein , fibrinogen, transferrin, and may be selected from the group consisting of transdiletin, but is not limited thereto.
- the novel nucleic acid ligand according to an embodiment of the present disclosure can bind to plasma proteins and exhibit excellent in vivo stability and slow renal clearance (ie, increase in residence time in the body), and further enter cells by pathways such as endocytosis. can be transmitted.
- the novel nucleic acid ligand exhibits resistance to nucleotide degrading enzymes such as Dnase in serum conditions by specifically binding to plasma proteins, thereby exhibiting excellent effects in in vivo stability and half-life.
- the novel nucleic acid ligand specifically binds to plasma proteins, thereby delaying renal filtration, thereby reducing the clearance rate and increasing the residence time of the nucleic acid ligand in the body.
- the novel nucleic acid ligand can exhibit excellent therapeutic effect by specifically binding to plasma proteins and flowing into cells, for example, into cancer cells.
- therapeutic target is a target for treating a disease or disease, and if it is a target that can exhibit a therapeutic effect by interacting with a novel nucleic acid ligand according to an embodiment of the present disclosure, the type is not limited .
- the therapeutic target may be a protein involved in intracellular signal transduction.
- the therapeutic target may be a protein present in a cell, a protein present in a cell membrane, or a mutant thereof.
- the novel nucleic acid ligand specifically binds to a plasma protein and then can reach a target tissue by avoiding degradation or renal excretion by nucleases in the body, and then target by a pathway such as endocytosis. It can enter the cell and bind to a therapeutic target (eg, intracellular protein) present in the cell to exert the intended therapeutic effect.
- a therapeutic target eg, intracellular protein
- the novel nucleic acid ligand that enters the cell may be separated from the plasma protein by an intracellular digestion process, and then bind to a therapeutic target to exhibit an intended therapeutic effect.
- the novel nucleic acid ligand can specifically bind to a plasma protein and then reach the target tissue by avoiding degradation or renal excretion by nucleases in the body, and thereafter, the therapeutic agent present in the cell membrane of the target tissue It may bind or interact with a target (eg, a cell membrane protein), or bind or interact with a therapeutic target present in the cell membrane after dissociation from the plasma protein.
- a target eg, a cell membrane protein
- the therapeutic target is a membrane protein, a transmembrane protein, a glycoprotein, an immune antibody, a virus, a viral envelope glycoprotein, a viral enzyme, a secreted protein, a serine protease, a peptide hormone, a neurotransmitter, a hormone, Dehydrogenase, cytokine, E3 ubiquitin ligase, neuropeptide, hydrolase, serine protease inhibitor, human hydrolase, chemokine protein, methyl converting enzyme, oxidase, growth factor, bacteria, bacterial protein, intracellular It may be selected from the group consisting of proteins, extracellular matrix, receptors, transcription factors, and tumor proteins, but is not limited thereto.
- the therapeutic target is 4-1BB, acetylcholine receptor, alpha thrombin, amylin, angiopoietin 1, angiopoietin 2, AXL, BCL-2, BMPR-1R, BRD1, BRD2, BRD3 , BRD4, BRDT, BTLA, calcitonin gene-related peptide, CBP (CREB-binding protein), CCK4/PTK7, CD16a, CD16b, CD19, CD20, CD200, CD200R, CD27, CD28, CD3, CD30, CD32A, CD32B, CD33 , CD4, CD40L, CD52, CD80, CD94, CSF1R, CTLA-4, DDR1, DDR2, E2F1, EGFR, EPH, ERBB2, FGF, FGFR, ghrelen, GITR, glypican3, gonadotropin-releasing hormone 1, HIV gp120, HIV-1 integrase, HIV-1 reverse
- the therapeutic target may be at least one of a protein present in a cell, or a mutation thereof.
- the therapeutic target present in the cell is selected from the group consisting of KRAS, BCL2, MYC, PP2A, BRD1, BRD2, BRD3, BRD4, BRDT, CBP, E2F1, MDM2, MDMX, PPP2CA, PPM1D, STAT3 and IDH1. may be at least one.
- the therapeutic target may be a KRAS mutant protein.
- the KRAS mutation may be a mutation in amino acids 12, 13, and 61 of the KRAS wild-type protein.
- the KRAS mutation may be KRAS G12D, KRAS G12V, KRAS G12C, KRAS G12A, KRAS G12S, KRAS G12R, KRAS G13D, KRAS Q61H, or a combination thereof, preferably KRAS G12D KRAS G12C, KRAS G12V, or a combination thereof.
- the signaling pathway is continuously stimulated because the KRAS protein activated by growth factors is not inactivated but always retains GTP in an activated state. Due to this, cell division protein, cell growth protein, cyclin or CDK protein is constantly produced, so that cell growth, proliferation, division, etc. continuously occur, and as a result, a tumor may be formed.
- the novel nucleic acid ligand specifically or selectively binds to a KRAS mutant protein (eg, KRAS G12D) inside a cancer cell and inhibits phosphorylation of proteins downstream of the KRAS signaling pathway (eg, ERK), thereby causing excess cells It can inhibit growth, proliferation and division. Therefore, the novel nucleic acid ligand is effective in preventing or treating cancers expressing the KRAS mutant protein (eg, lung cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, gallbladder biliary tract cancer, acute myeloid leukemia, etc.) can represent cancers expressing the KRAS mutant protein (eg, lung cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, gallbladder biliary tract cancer, acute myeloid leukemia, etc.) can represent cancers expressing the KRAS mutant protein (
- the therapeutic target may be a protein present in a cell membrane or a mutant thereof.
- therapeutic targets present in cell membranes are PD-L1, PD-L2, CD40L, LAG3, TIM3, TIGIT, BTLA, CD52, SLAMF7, 4-1BB, OX-40, ICOS, GITR, CD27, CD28 , CD16, CD3, CD20, EGFR family, AXL, CSF1R, DDR1, DDR2, EPH receptor family, FGFR family, VEGFR family, IGF1R, LTK, PDGFR family, RET, KIT, at least selected from the group consisting of NTRK1, and NTRK2 can be one
- the novel nucleic acid ligand binds to PD-L1 on the surface of cancer cells and inhibits the binding of T-cell surface proteins, PD-1 and PD-L1, thereby blocking the inhibition of T cells so that T cells attack cancer cells.
- the novel nucleic acid ligand may exhibit a preventive or therapeutic effect on cancers expressing PD-L1 on the surface of cancer cells (eg, bladder cancer, lung cancer, breast cancer, ovarian cancer, urothelial cell cancer, skin cancer, etc.).
- the novel nucleic acid ligand binds to CD3d/e or 4-1BB on the surface of cytotoxic T cells and activates cytotoxic T cells, thereby exhibiting an immune anticancer effect. Therefore, the novel nucleic acid ligand activates cytotoxic T cells and thus can be applied to immuno-cancer therapy (eg, multiple myeloma, solid cancer, metastatic non-small cell cancer, kidney cancer, lung cancer, head and neck cancer, liver cancer, breast cancer, stomach cancer, esophageal cancer, ovarian cancer, treatment of Hodgkin's lymphoma, bladder cancer, or melanoma).
- immuno-cancer therapy eg, multiple myeloma, solid cancer, metastatic non-small cell cancer, kidney cancer, lung cancer, head and neck cancer, liver cancer, breast cancer, stomach cancer, esophageal cancer, ovarian cancer, treatment of Hodgkin's lymphoma, bladder cancer, or melanoma.
- the following example was performed to obtain a nucleic acid ligand according to an embodiment of the present disclosure.
- nucleic acid library pool having binding affinity to the first target from the nucleic acid library pool using the SELEX technology (primary SELEX)
- secondary SELEX was performed for the nucleic acid ligands capable of binding to a plurality of targets were finally obtained (see FIG. 2 ).
- a specific method for selecting a novel nucleic acid ligand is described below.
- Single-stranded nucleic acid library template [ Biotin-GCTGGTGGTGTGGCTG-N(40)-CAGGCAGACGGTCACTCA (SEQ ID NO: 1)] was prepared.
- the prepared nucleic acid library template is theoretically a mixture of DNA having 1 x 10 24 different nucleotide sequences.
- the template of this single-stranded nucleic acid library was synthesized using an automatic DNA synthesizer, and was used for the experiment after PAGE purification (Trilink, USA).
- the library template and 500 ⁇ L resin were mixed in 5 M NaCl solution and reacted for 1 hour at room temperature to immobilize.
- 20 ⁇ M 5' primer (GAGTGACCGTCTGCCTG (SEQ ID NO: 2)
- 0.5 mM dNTP dATP, dGTP, dCTP, Nap-dUTP or Bz-dUTP
- 0.25 U/ ⁇ L KOD XL DNA polymerase (Merck, USA)
- 120 mM Tris-HCl pH 7.8, 1X KOD buffer was added and the enzymatic reaction was performed at 70° C. for 2 hours to prepare double-stranded DNA containing modified nucleic acids.
- the double-stranded DNA was denatured using 20 mM NaOH to separate the resin and single-stranded DNA, and the separated supernatant was used as a Neutralization buffer (700 mM HCl, 180 mM HEPES, 0.45% (v/v) Tween 20) solution. neutralized.
- a Neutralization buffer 700 mM HCl, 180 mM HEPES, 0.45% (v/v) Tween 20
- a protein containing a histidine tag (6xHis-tag) among recombinant proteins was used as the target protein for SELEX.
- the target protein was immobilized on the beads using Dynabead (Thermo Scientific, USA), which is a magnetic bead that is surface-modified with cobalt and binds to the His-tag of the protein.
- target protein and 50 ⁇ l of his-tag magnetic beads dissolved in buffer SB18 (40 mM HEPES, 102 mM NaCl, 5 mM KCl, 5 mM MgCl 2 , and 0.05% (v/v) Tween 20; hereinafter the same) was reacted in a thermomixer (Thermomixer; Enpendorf, Germany) at 25° C., 1200 rpm, for 30 minutes, and then washed three times with buffer SB18 using a magnet to prepare the first target protein immobilized on the beads.
- buffer SB18 40 mM HEPES, 102 mM NaCl, 5 mM KCl, 5 mM MgCl 2 , and 0.05% (v/v) Tween 20; hereinafter the same
- a thermomixer Thermomixer; Enpendorf, Germany
- the first target protein is human serum albumin (HSA; Abcam, UK, product name: Recombinant human serum albumin Protein (His tag), catalog number: ab217817) corresponding to plasma protein, immunoglobulin G (IgG; Abcam, UK, product name). : Recombinant Human IgG1 protein (His tag), catalog number: ab219660), or transferrin (TF; Abcam, UK, product name: Recombinant human Transferrin protein (Active), catalog number: ab155698).
- HSA human serum albumin
- IgG Abcam, UK, product name: Recombinant human serum albumin Protein (His tag), catalog number: ab217817) corresponding to plasma protein, immunoglobulin G (IgG; Abcam, UK, product name).
- IgG immunoglobulin G
- TF transferrin
- Abcam Abcam, UK, product name: Recombinant human Transferrin protein (Active), catalog number: ab155698).
- the synthesized nucleic acid library pool was placed in buffer SB18, and the temperature was gradually lowered while reacting from 95°C to 37°C for 10 seconds every 1°C to induce the formation of a tertiary structure of the nucleic acid library.
- protein competition buffer (1 ⁇ M prothrombin, 1 ⁇ M casein) was mixed with the above reaction solution, added to 10 ⁇ g of magnetic beads, and reacted at 37° C., 1200 rpm, for 10 minutes using a thermomixer. Then, the supernatant was transferred to the Dynabead on which the plasma protein was immobilized, and a nucleic acid library pool that binds to the plasma protein was selected.
- the modified nucleic acid library which had been subjected to non-specific negative selection, was reacted with magnetic beads immobilized with plasma proteins and a thermomixer at 37° C., 1200 rpm, for 1 hour. After washing the nucleic acid library and the target protein-magnetic bead complex 5 times with buffer SB18, 2 mM NaOH solution was added to recover the nucleic acid library pool bound to the target protein, and then neutralized with 8 mM HCl solution.
- the library bound to plasma proteins was amplified using QPCR (quantitative PCR, CFX real time PCR detection system; BioRad, USA) for use in the next round of SELEX.
- QPCR buffer [5' primer (GAGTGACCGTCTGCCTG (SEQ ID NO: 2)), 3' primer (Biotin-GCTGGTGGTGTGGCTG (SEQ ID NO: 3)), 1X KOD buffer, KOD XL DNA polymerase, 1 mM dNTP] in the nucleic acid library recovered in the above SELEX process
- one cycle under the conditions of 96 ° C. 15 sec, 55 ° C. 10 sec, and 68 ° C. 30 min, and 30 cycles at 96 ° C. 15 sec, and 72 ° C. 1 min condition were repeated to bind to plasma proteins.
- Double-stranded oligo DNA was prepared by amplifying the nucleic acid library.
- the double-stranded oligo DNA prepared through QPCR was mixed with 25 ⁇ l Myone TM streptavidin magnetic beads (Thermo Scientific, USA; hereinafter the same) at room temperature for 10 minutes and fixed, and 20 mM NaOH solution was added to remove the anti-sense strand.
- a nucleic acid library pool containing the modified nucleic acid was synthesized using an enzyme in the same manner as in the preparation of the library containing the modified nucleic acid, and used in the next round. The SELEX round was repeated a total of 8 times.
- a kinetic challenge was performed in which a nucleic acid library pool and a target protein were bound, and then reacted with 10 mM dextran sulfate. It is the same as the round of qPCR amplification and anti-sense recovery.
- a bead-based ELISA binding assay was performed to confirm the binding affinity to plasma proteins of the nucleic acid library pool in which the SELEX round was performed.
- buffer [(5' primer (GAGTGACCGTCTGCCTG (SEQ ID NO: 2)), 3' primer (Biotin-GCTGGTGGTGTGGCTG (SEQ ID NO: 3)), 1X KOD buffer, KOD XL DNA polymerase, 0.2 mM dNTP] to prepare a mixture, and repeating 20 cycles with 96 °C 15 seconds, and 72 °C 1 bun conditions to amplify the nucleic acid library pool (pool) and oligo double-stranded DNA.
- oligonucleotide duplexes of DNA 25 ⁇ l Myone TM streptavidin After mixing with Dean magnetic beads for 10 minutes at room temperature and fixing, 20 mM NaOH solution was added to remove the sense strand from the solution. MgCl 2 , 1 mM EDTA, and 0.05% (v/v) Tween 20; After washing twice using the same hereinafter), additional washing was performed using 16 mM NaCl.
- Washed magnetic beads-anti sense conjugate with 2.5 ⁇ M 5' primer Biotin-GAGTGACCGTCTGCCTG (SEQ ID NO: 4)
- 0.5 mM dNTP dATP, dGTP, dCTP, Nap-dUTP or Bz-dUTP
- 0.25 U/ ⁇ L KOD XL DNA polymerase 120mM Tris-HCl pH7.8, and 1X KOD buffer
- enzymatic reaction was performed at 68°C for 1 hour to prepare a nucleic acid library pool containing modified nucleic acid and biotin.
- the supernatant was removed using a magnet and washed 3 times with buffer SB17.
- 20 mM NaOH was added, followed by neutralization with 80 mM HCl.
- the prepared 1 pmol of biotin-binding nucleic acid library pool was put into buffer SB18, and the temperature was gradually lowered while reacting from 95°C to 37°C for 10 seconds every 1°C to induce the formation of a tertiary structure of DNA.
- Plasma proteins were prepared by diluting four-fold from 200 nM, and after binding to a structure-stabilized nucleic acid library pool at 37° C. for 30 minutes, 10 ⁇ l histidine pull-down beads (Thermo Scientific, USA) were added and combined at 25° C. for 30 minutes.
- Figure 6a is a target binding force result obtained from a nucleic acid library pool including Bz-dU (top of the figure) and Nap-dU (bottom of the figure) for albumin, a first target protein, respectively
- Figure 6b is for immunoglobulin G.
- Target binding results obtained from a nucleic acid library pool containing Bz-dU and Nap-dU for transferrin, respectively
- FIG. 6C is a nucleic acid library pool containing Bz-dU and Nap-dU for transferrin, respectively. It is the result.
- FIGS. 6A to 6C regardless of the type of modified nucleic acid or target protein used for library construction, the existence of a nucleic acid library pool showing binding affinity to the target protein in all the first SELEX steps was confirmed.
- a filter binding assay was performed as another method of measuring the binding force of the target protein and the nucleic acid library pool undergoing the SELEX round.
- the ends of the first round 6 and 8 nucleic acid library pools were labeled with ⁇ -32 P ATP (Perkin Almer, USA) and TdT (Terminal deoxynucleotidyl transferase; New England Biolabs, USA).
- a sense single-stranded nucleic acid library pool was prepared after amplifying the nucleic acid library pool obtained through the SELEX process in the same manner as in 2-2-1 above. Then, 1 pmole of the nucleic acid library pool, 0.25 ⁇ L ⁇ - 32 P ATP, 0.25 ⁇ L TdT, and NEBuffer TM 4 (New England Biolabs, USA) were mixed and reacted at 37° C. for 30 minutes, and at 70° C. for 10 minutes. treatment to inactivate the TdT enzyme. The labeled nucleic acid library pool was purified using a Micro spin G50 column (GE Healthcare, USA) and then quantified using a beta counter device.
- 20,000 cpm of the nucleic acid library pool was put into 100 ⁇ L buffer SB18, and the temperature was gradually lowered while reacting from 95° C. to 37° C. for 10 seconds every 1° C. to induce the formation of a tertiary structure of the nucleic acid library pool.
- 30 ⁇ L of a structure-stabilized labeled nucleic acid library pool was added and reacted at 37° C. for 30 minutes.
- 5.5 ⁇ L of zorbax resin (Agilent, USA) was added to the mixture of the nucleic acid library pool and the target protein, followed by further reaction for 5 minutes.
- the sample was transferred to a MultiScreen-HV Filter Plate (Merck Millipore, USA) moistened with 50 ⁇ L of buffer SB18 in advance, the solution was removed by vacuum, and the membrane filter was washed with 100 ⁇ L of buffer SB18. After exposing the filter plate to an image plate for 16 hours, images were quantified with FLA-5100 (Fujifilm, Japan).
- the binding affinity was obtained using the value obtained through the filter binding assay using Sigmaplot 11 analysis software (Systat Soft, USA), Bmax represents the ratio of the bound oligo DNA to the input, and Kd (dissociation constant) is It shows affinity, and Nap lib refers to a nucleic acid library pool that has not been subjected to SELEX and was used as a negative control (see FIG. 7 ).
- the target binding affinity of the nucleic acid library pools of the 6th and 8th rounds was evaluated using the above filter binding assay. method and is shown in FIG. 7 .
- FIG. 7 the existence of a nucleic acid library pool exhibiting binding ability to albumin protein was confirmed in both rounds.
- binding force to the albumin protein did not appear.
- Secondary SELEX was performed on the therapeutic target protein using the round nucleic acid library pool in which binding to plasma proteins was confirmed through the above-described primary SELEX and binding assay.
- the second SELEX method is the same as the first SELEX method, but a count selection process was added if necessary to increase the binding specificity of the therapeutic target protein.
- the therapeutic target proteins used in the Examples below are as follows.
- KRAS wild-type protein Abcam, product name: Recombinant Human KRAS protein (His tag), catalog number: ab96817
- KRAS G12D protein Signal chem, product name: KRAS (G12D) Protein (R06-32BH), catalog number: R06-32BH
- KRAS G12C protein Signal chem, product name: KRAS (G12C) Protein (R06-32DH), catalog number: R06-32DH
- KRASG12V protein Signal chem, product name: KRAS (G12V) Protein (R06-32CH), catalog number: R06-32CH
- PD-L1 protein R&D SYSTEMS, product name: Recombinant Human PD-L1/B7-H1 His-tag Protein, catalog number: 9049-B7-100
- BCL-2 protein R&D SYSTEMS, product name: Recombinant Human Bcl-2 (minus C-Terminus) Protein, CF, catalog number: 827-BC-050
- 4-1BB protein R&D SYSTEMS, product name: Recombinant Human 4-1BB/TNFRSF9 Fc Chimera Protein, CF , catalog number: 838-4B-100
- CD3d/e protein Prosci, product name: CD3 delta/epsilon Heterodimer Recombinanat Protein (Fc Chimera), catalog number: 96-164
- a mixture of the prepared nucleic acid library pool and protein competition buffer was added to the thus-prepared complex and reacted at 37° C., 1200 rpm for 10 minutes. Then, the nucleic acid library pool of the supernatant not bound to the wild-type protein. was transferred to beads on which the mutant target protein was immobilized, and a nucleic acid library pool that binds to the therapeutic target protein was selected. Secondary SELEX for the therapeutic target protein was also performed 5 to 8 rounds in the same manner, and dynamic induction using dextran sulfate was not performed.
- PD-L1, KRAS G12D, KRAS G12V, KRAS G12C, BCL-2, 4-1BB, and CD3d/e were used as the second target protein, respectively.
- the results for PD-L1, KRAS G12D, KRAS G12V, KRAS G12C, and BCL-2 are shown in FIGS. 8A to 8D.
- FIGS. 8A to 8D the existence of a nucleic acid library pool exhibiting binding affinity to the second target protein was confirmed regardless of the type of the first target protein and the type of the second target protein.
- the TOPO TA Cloning kit (Thermo Scientific, USA) was used to clone and transform E. coli TOP10 cells.
- the plasmid nucleic acid of a single colony grown on an agar medium is amplified using an RCA (rolling circle amplification) kit (Engenomics, Korea), and after confirming whether the cloned PCR product is inserted, use the RCA sample of the clone into which the single sequence is inserted.
- nucleotide sequencing Solgent, Korea was performed with the BigDye terminator cycle sequencing kit (Thermo Scientific, USA) and capillary electrophoresis. For example, at least about 50 clones were sequenced for each sample, and each nucleic acid sequence present in the nucleic acid library pool selected through SELEX was finally confirmed.
- Nucleic acid sequences that have been analyzed were analyzed using the Multalin website or our software, and sequences with the highest expression frequency (eg, about 5 to about 10) were selected.
- a 5' primer (GAGTGACCGTCTGCCTG (SEQ ID NO: 2)
- a biotin-binding 3' primer Biotin-GCTGGTGGTGTGGCTG (SEQ ID NO: 3)
- DNA polymerization It was synthesized using an enzyme and corresponds to a clonal nucleic acid ligand. 2-2-1 of Example 1 described above. The binding ability of the clonal nucleic acid ligand to the target protein was measured in the same manner as in the bead-based ELISA binding assay of the item (eg, see FIGS. 9A, 9B, 10A, and 10B).
- the binding force to each target protein was measured using a clonal nucleic acid ligand that specifically binds to HSA and PD-L1 after sequencing has been completed (see FIGS. 9A and 9B ).
- S019-A1 #2 clones and S019-A1 #3 clones with confirmed binding affinity were selected.
- Nucleic acid ligands of SEQ ID NO: 99 and SEQ ID NO: 100 were synthesized using these clones, and were used in the following examples (see FIGS. 9A and 9B ).
- the sequences for which the binding affinity for the first target protein and the second target protein were confirmed were selected, and the thus-selected full-length nucleic acid ligand was synthesized in a form including the modified nucleic acid using an automatic DNA synthesizer, and the following operation was carried out. In the example, it was used as a full-length nucleic acid ligand.
- the full-length nucleic acid ligand selected by the method of Example 1 is the nucleic acid ligands AP013, AP027, AP014, AA001, AB001, AD001, AD002, AD003, AD009, AV001, AV006, AX001, ID001, IC001, IC007 listed in Table 1. , IV001, TD001, TD005, TD009, and TC001.
- a sequence having binding affinity to the first target was selected from a first nucleic acid library pool using SELEX technology (primary SELEX), and a second nucleic acid library pool was prepared by binding a random sequence to the selected sequence. . Then, using a second nucleic acid library pool (pool), a sequence having binding ability to the second target was selected by performing secondary SELEX, and finally a nucleic acid ligand capable of binding to a plurality of targets was prepared (Figs. 3 to 3). see Fig. 5).
- a specific method for preparing a nucleic acid ligand is the same as that of Example 1, but the process of selecting a nucleic acid library pool specific for the second target protein is different as follows.
- a sequence confirmed for binding to plasma protein from items 1. and 2. of Example 1 is selected.
- a total of 64 to 74 consecutive nucleic acid sequences are prepared by linking 25 to 35 consecutive random sequences to the selected nucleic acid sequence, a primer binding site for nucleic acid amplification at the 3' end, and biotin in front of the 5' end
- This conjugated single-stranded nucleic acid library template [Biotin-selective sequence-N (25-35 pieces)-CAGGCAGACGGTCACTCA] was prepared.
- the prepared nucleic acid library template is theoretically a mixture of nucleic acids having 1 x 10 24 different nucleotide sequences.
- the primer binding site at the 5' end was omitted, and the 5' primer was replaced with a selection sequence.
- the template of this single-stranded nucleic acid library was synthesized using an automatic DNA synthesizer, and was used for the experiment after PAGE purification (Trilink, USA).
- nucleic acid ligands AD032, AD034, AP030, and AP031 listed in Table 1.
- nucleic acid ligands selected by the methods of Examples 1 and 2 are shown in Table 1 below.
- the nucleic acid ligands thus prepared were analyzed and purified by reverse-phase C18 column on Waters Prep150 (Waters, USA) and Waters ACQUITY UPLC H-Class Bio PLUS System (Waters, USA).
- mass spectrometry of purified nucleic acid ligands was performed with a Waters Xevo G2-XS Q-TOF System (Waters, USA). The mass spectrometry results are shown in Table 2.
- the portion indicated by 6 means 5-[N-(1-naphthylmethyl)carboxamide]-2'-deoxyuridine (Nap-dU).
- the portion indicated by n in SEQ ID NOs: 6 to 183 of the attached sequence listing means 5-[N-(1-naphthylmethyl)carboxamide]-2'-deoxyuridine (Nap-dU) do.
- a truncation process for optimization was performed using the full-length nucleic acid ligand selected by the method of Examples 1 and 2.
- the 2D structure of the full-length nucleic acid ligand was predicted using the Mfold web server (http://www.unafold.org/mfold/applications/dna-folding-form.php), and the full-length nucleic acid ligand was selected based on this prediction.
- Optimized nucleic acid ligand candidates similar to the clone sequence (the full-length nucleic acid ligand serving as the parent) were synthesized at the minimum length by reducing the length from both ends.
- the 2D prediction structures of the nucleic acid ligands AD001 to AD015 are shown in FIGS. 11A to 11D and 12A to 12C.
- Nucleic acid ligands were synthesized using a general automatic DNA synthesis method using Mermade 12 or Mermade 48 (Bio Automation, USA). The four-step process of [Deblocking -> Coupling -> Capping -> Oxidation] was sequentially synthesized from the 3' end to the 5' end by 1 mer (1 nucleotide each) in one cycle. If necessary, biotin or a fluorescent substance (FAM or Cy5) was bound to the front of the 5' end of the nucleic acid ligand. Biotin and FAM were synthesized in an automatic synthesizer, and Cy5 was synthesized by amine coupling after synthesizing a 5'-amine nucleic acid ligand. The synthesized nucleic acid ligand or nucleic acid ligand to which biotin or fluorescent material (FAM or Cy5) is bound was purified by HPLC and used in the following examples.
- Nucleic acid ligands prepared according to Example 3 together with full-length nucleic acid ligands are shown in Table 2 below. Optimized nucleic acid ligands until the next full-length nucleic acid ligand all use the same full-length nucleic acid ligand (eg, AP001-AP006 and AP021-AP026 nucleic acid ligands use AP013 full-length nucleic acid ligand).
- nucleic acid ligands thus prepared were analyzed and purified by reverse-phase C18 column on Waters Prep150 (Waters, USA) and Waters ACQUITY UPLC H-Class Bio PLUS System (Waters, USA).
- mass spectrometry of purified nucleic acid ligands was performed with a Waters Xevo G2-XS Q-TOF System (Waters, USA). The mass spectrometry results are shown in Table 2.
- UPLC analysis results before and after purification of the nucleic acid ligand AD005 according to an embodiment of the present disclosure are shown in FIG. 13
- mass spectrometry results are shown in FIG. 14 .
- nucleic acid ligands having biotin bound to the 5' end were used except for the nucleic acid ligands AP030 and AP031.
- nucleic acid degrading enzymes exist in serum, it is well known that oligonucleic acid molecules are degraded within a few hours when an oligonucleic acid material is reacted with serum.
- a nucleic acid ligand bound to a plasma protein such as albumin is not degraded because it is resistant to the degradation action of a nuclease.
- the nucleic acid ligand binds to a target protein other than a plasma protein, it may exhibit resistance to the degradation action of a nucleic acid degrading enzyme.
- the selected full-length nucleic acid ligand is reacted under conditions of exposure to a nucleic acid degrading enzyme, the degree of degradation of the nucleic acid ligand is determined to evaluate the presence or absence of binding to the first and second target proteins, and furthermore, the stability of the nucleic acid ligand in serum was evaluated.
- the structure of the nucleic acid ligand that binds to the target and exhibits the desired effect by binding to the target protein in the blood or the binding site of the nucleic acid ligand to the target is maintained to determine whether it is a nucleic acid ligand capable of exhibiting effective efficacy can be checked
- nucleic acid ligand dissolved in buffer SB18 (selected according to the method of Examples 1 and 2), DMEM medium, nuclease reaction buffer (DNaseI RNase-free, Thermoscientific, USA) and sterile DW Mix.
- the nucleic acid ligand mixture thus prepared was reacted for 10 seconds at every 1°C from 95°C to 37°C, and the temperature was gradually lowered to induce the formation of a tertiary structure of the nucleic acid ligand.
- the nucleic acid ligand and the target protein were bound by reaction at 4° C. for 30 minutes. At this time, the target protein was not added to the positive control group and the negative control group.
- 1 unit of a nuclease DNaseI, Thermo Scientific, USA was added to activate the action of the nuclease at 37°C for 16 hours. At this time, no nucleic acid degrading enzyme was added to the negative control group.
- nuclease was inactivated by heating at 85°C, and electrophoresis was performed on a 10% urea-polyacrylamide gel (Urea-Polyacrylamide gel).
- the stability of the nucleic acid ligand to the nucleic acid degrading enzyme was confirmed by comparing the band intensity of each control group and each experimental group. From these results, it can be confirmed whether the nucleic acid ligand excellently binds to the target protein, and nucleic acid ligand candidates exhibiting excellent stability to nucleases can be selected.
- FIGS. 11A-11D and 12A-12C Dnase stability electrophoresis results for AD001, AD002, AD003, and AD009 nucleic acid ligands are shown in FIGS. 11A-11D and 12A-12C.
- Binding force (K d ) was measured to further confirm the binding affinity to the target of the nucleic acid ligand showing the result of binding to each target protein.
- the binding ability of the nucleic acid ligand was measured using an ELISA (Enzyme Linked Immunosorbent assay) method.
- ELISA Enzyme Linked Immunosorbent assay
- the experimental method proceeds in the following steps.
- Blocking Buffer PierceTM Protein-Free (PBS) Blocking Buffer, Thermo Scientific, USA.
- HRP Streptavidin-Horseradish Peroxidase (HRP) Conjugate, Thermo Scientific, USA
- HRP Streptavidin-Horseradish Peroxidase (HRP) Conjugate, Thermo Scientific, USA
- TMB TMB Substrate Solution, Thermo Scientific, USA
- the target protein was diluted 4 times from 500 nM and the nucleic acid ligand was used from 500 nM.
- Table 3 shows the binding affinity of the measured nucleic acid ligands to each target protein. It can be confirmed that the nucleic acid ligand showing an electrophoresis result that binds to all target proteins exhibits binding affinity for each target protein.
- 2 pmole nucleic acid ligand dissolved in buffer SB18 was reacted from 95°C to 37°C for 10 seconds at every 1°C, and the temperature was gradually lowered to induce the formation of a tertiary structure of the nucleic acid ligand.
- the stabilized nucleic acid ligand was added to 96% human serum, it was reacted at 37° C. for 0, 3, 6, 9, 12, 15, and 18 hours to induce degradation of the nucleic acid ligand. After each reaction time, heating at 85° C. to inactivate the nuclease and electrophoresis on a 10% urea-polyacrylamide gel.
- a control group to which human serum was not added was included as a loading control to measure the band intensity of the control group and the experimental group. Using the measured values, the intensity trend line was confirmed by time, and the half-life was confirmed by substituting the intensity value into the trend formula. If it deviates from the trend line, additional experiments were performed under conditions of 0, 24, 48, 72, and 96 hours.
- the electrophoresis results over time for the AD001 to AD015 nucleic acid ligands are shown in FIGS. 11A to 11D and 12A to 12C.
- the serum half-life (hr) for each target protein measured in this way is shown in Table 3. According to Table 3, it can be seen that the nucleic acid ligand according to an embodiment of the present disclosure has a high half-life in serum, which means that the nucleic acid ligand according to an embodiment of the present disclosure has excellent binding ability to plasma proteins, and thus excellent in vivo This means that it can show stability.
- Example 3 For some of the optimized nucleic acid ligands prepared in Example 3, a Dnase stability test for the target protein was performed in the same manner as in Example 4. Table 3 shows the results of representative optimized nucleic acid ligands.
- the binding affinity (K d ) to the target protein was measured in the same manner as in Example 5 for the optimized nucleic acid ligand whose binding to the target protein was confirmed according to the DNA stability experiment, and the half-life in plasma was measured in the same manner as in Example 6. Confirmed. The results obtained in this way are shown in Table 3.
- nucleic acid ligands indicated in bold and underlined in Table 3 are the original full-length nucleic acid ligands based on the optimization, and the nucleic acid ligands below them correspond to the optimized truncated nucleic acid ligands.
- candidate nucleic acid ligands for in vitro and in vivo experiments on a desired therapeutic target can be selected.
- Example 6 It was carried out in the same manner as in Example 6, but after reacting with serum for 24 hours, electrophoresis was performed. Stability experiments of nucleic acid ligands for sera from various animals were performed. This is because it is necessary to select a nucleic acid ligand that shows stability in both animal and human serum in order to expect the effect in animal experiments to be shown in humans. Stability experiments were performed on HSA/KRAS target nucleic acid ligands AD001 to AD015, and the electrophoresis results are shown in FIGS. 15A and 15B. Additionally, AP013, an HSA/PD-L1 target nucleic acid ligand, was also tested.
- nucleic acid ligand according to an embodiment of the present disclosure is not degraded in human serum and is stably maintained.
- nucleic acid ligands such as AD005, AD008 and AD011 are human serum, bovine serum, and excellent stability in various serum conditions of rat serum. From the above results, it could be confirmed that the nucleic acid ligand according to an embodiment of the present disclosure binds to plasma proteins and is stably present in serum.
- the nucleic acid ligand AD005 according to an embodiment of the present disclosure which specifically binds to HSA/KRAS G12D, exhibits resistance to nucleases even when treated with DNase when reacted with KRAS G12D or KRAS G12C ( 16).
- the reason why it also binds to KRAS G12C is that G12D and G12C are mutated at one point, so their structures are very similar.
- AD005 showed excellent resistance to nucleases even when reacted with human serum albumin, one of the targets.
- AD005 had increased resistance to nuclease as the molar ratio of the target protein increased, which means that as the amount of the target protein increases, the nucleic acid ligand binds more to the target protein, preventing degradation by the nuclease. Because it can be avoided.
- nucleic acid ligand according to an embodiment of the present disclosure shows strong binding to only some KRAS mutations (G12D and G12C), and as a result, stability to nucleases is increased. Moreover, it was confirmed that the nucleic acid ligand according to an embodiment of the present disclosure selectively exhibits binding depending on the presence or absence of mutation in the KRAS protein.
- the nucleic acid sequence of aptamer KD003 is ACC5G55GCAGCACC5A55CA5ACGGACGC5C5C5 (SEQ ID NO: 184).
- the part denoted by 5 means 5-(N-benzylcarboxamide)-2'-deoxyuridine (Bz-dU).
- the UPLC instrument was a Waters ACQUITY UPLC H-Class Bio System (Water Corporation, USA), and the analytical column was ZORBAX RR Eclipse Plus C18, 95 ⁇ , 3.5 ⁇ m, 4.6 ⁇ 100 mm (Agilent Technologies, USA). installed. At this time, the flow rate was 1.7 mL/min, and as the mobile phase, A: 15 mM TEA, 38 mM HFIP in Water, B: Acetonitrile was used for analysis by gradient method. The detection of nucleic acid ligands or aptamers was performed using a PDA (UV 260 nm) detector.
- PDA UV 260 nm
- the half-life of the nucleic acid ligand according to an embodiment of the present disclosure is about 5 times longer than that of the control nucleic acid aptamer (KD003) that does not bind to plasma protein (see FIG. 17 ).
- the nucleic acid ligand according to an embodiment of the present disclosure exhibits excellent binding ability to plasma proteins, and thus has excellent stability in actual plasma.
- the binding force of the nucleic acid ligand was measured using the bio-layer interferometry (BLI) method.
- BLI is an experimental method to monitor the binding action between molecules by measuring the interference of light caused by the change in the thickness of the protein layer adsorbed to the end of the biosensor.
- the experiment was performed using Octet Red 96e (ForteBio, USA) equipment. did.
- the nucleic acid ligand having biotin bound to the 5' end was reacted in buffer SB18 from 95°C to 37°C for 10 seconds at every 1°C while gradually lowering the temperature to induce the formation of a tertiary structure of the nucleic acid ligand.
- the binding force measurement was carried out in the following five steps: (1) measuring the baseline in buffer SB18 using a streptavidin-coated biosensor (60 seconds), (2) strepping the nucleic acid ligand prepared by the above method Binding to the tavidin biosensor (180 sec), (3) measuring the second baseline in buffer SB18 (120 sec), (4) preparing a target protein in buffer SB18 and nucleic acid bound to the biosensor interacting with the ligand (300 s), (5) and finally inducing dissociation of the target protein in buffer SB18 (600 s). All binding force measurement steps were performed at 37 ° C., and the nucleic acid ligand was diluted twice from 500 nM and the target protein was used from 500 nM.
- nucleic acid ligand AD005 SEQ ID NO: 24
- the dissociation constant value of the nucleic acid ligand AD005 for KRAS G12D was measured to be 35.5 nM, but for wild-type KRAS (KRAS WT) and other types of mutant protein (KRAS G12V) within the tested concentration range, no significant results were obtained.
- the binding affinity is expected to be low (see Figure 18).
- the nucleic acid ligand AD005 according to an embodiment of the present disclosure exhibits a relatively strong binding affinity to KRAS G12D and KRAS G12C, whereas wild-type KRAS (KRAS WT) and a different type of mutant protein (KRAS) G12V), it was confirmed that the binding force did not appear or was very weak. These results are consistent with the results of the Dnase stability test in item 1.
- AD005 and AD011 were finally selected as target nucleic acid ligands for KRAS G12D protein present in cancer cells, and the following in vitro efficacy experiment was performed.
- the AsPC-1 pancreatic cancer cell line was purchased from ATCC (USA) and used in the experiment, and was cultured using a medium containing 10% FBS and 1% antibiotic in ATCC modified RPMI-1640 (Thermo Scientific, USA). The medium was changed once every 2-3 days, and the subculture was performed after washing the cells with PBS, adding 0.05% trypsin-EDTA, incubating for 1 minute at 37° C. It was cultured after passage at a ratio of 1:5.
- MIA PaCa-2 pancreatic cancer cell line was also purchased from ATCC and used in the experiment, and cultured using a medium containing 10% FBS and 1% antibiotic in DMEM. Medium replacement and subculture were performed in the same manner as AsPC-1. All cells were periodically checked for mycoplasma contamination by PCR using a kit (Biomax, Korea), and in vitro experiments were performed only with cells in which no contamination was detected.
- TMR Tetramethylrhodamine
- FAM carboxyfluorescein
- FITC fluorescein isothiocyanate
- a round 10mm cover glass was placed in each well of a 24-well plate, and sterilized under UV for 30 minutes after sterilization using 500ul of 70% ethanol. After the sterilized cover slip was washed with PBS, MIA PaCa-2 cells were added at 4 to 10 x 10 4 cells/well and cultured.
- Cells were cultured in a cell incubator for 48 hours, adhered well to a cover glass, and cultured for 16 hours after changing to a medium free of FBS and glutamine. Thereafter, 1 mg/ml of 70 KDa dextran-TMR, 2 nmole of BSA-FITC and BSA albumin (Sigma-Aldrich, USA, product number: A8806) were treated with FAM-binding nucleic acid ligand and incubated for 1 hour. After the reaction, the cells were washed 5 times with cold PBS on ice and treated with 4% paraformaldehyde for 30 minutes to fix the cells.
- DAPI 4,',6-diamino-2-phenylindole
- a sample was prepared by dropping a drop of mount media on the slide glass, removing PBS from the cover glass in which the cells were grown, and standing on the mount medium of the slide glass.
- the finished sample was observed in a confocal microscope (FV3000RS Olympus, Japan) at a magnification of 100X using a filter of each fluorescent material, and the results are shown in FIGS. 19A to 19D and 20A and 20B.
- albumin introduced into the cytoplasm was observed as a cluster with a diameter of about 1 ⁇ m, and it was confirmed that it colocalized with 70KDa dextran-TMR, a macropinocytosis marker (FIG. 19(b)). This means that most albumin is introduced into cells by macropinocytosis under the experimental conditions of nutrient deficiency.
- nucleic acid ligand according to an embodiment of the present disclosure can be delivered into cells by macropinocytosis by binding to albumin.
- the cell growth inhibitory effect of nucleic acid ligand treatment was confirmed using the WST assay.
- This method is a method using the property that WST treated in cells is changed to orange formazan by dehydrogenase present in the mitochondrial electron transport system of the cell.
- the cancer cells expressing the KRAS mutant protein (AsPC-1 cells 2 x 10 4 cells/well, MIA PaCa-2 cells 8 x 10 3 cells/well) After culturing for 24 hours to attach cells, the medium was replaced with a medium not containing FBS or glutamine, and macropinocytosis was activated by culturing for 24 hours.
- the nucleic acid sequence of aptamer KD006 is G5G5CACC5G55GCAGCACC5A55CA5ACGGACGC5C5C5 (SEQ ID NO: 185).
- the part denoted by 5 means 5-(N-benzylcarboxamide)-2'-deoxyuridine (Bz-dU).
- AD005 the cell growth inhibitory effect of AsPc-1, a KRASG12D mutant cell line, and MiaPaCa-2, a KRASG12C mutant cell line, was significantly reduced in a concentration-dependent manner. When treated, it was confirmed that the inhibitory effect was increased.
- AD005 a nucleic acid ligand according to an embodiment of the present disclosure, excellently inhibits cell growth by binding to a plasma protein and a KRAS mutant protein.
- nucleic acid ligand according to an embodiment of the present disclosure is introduced into the cell not only by macropinocytosis but also by various endocytosis pathways (see FIG. 23 ).
- the KRAS mutant protein When the KRAS mutant protein is activated by the stimulation of a growth factor such as epidermal growth factor (EGF), it continuously maintains the activated state and induces phosphorylation of the ERK protein downstream of the signaling pathway. However, if the nucleic acid ligand delivered into the cell binds to the KRAS mutant protein and inhibits the signal transduction process, the phosphorylation level of the downstream ERK protein is estimated to decrease, and this was verified by the Western blot method.
- a growth factor such as epidermal growth factor (EGF)
- cells were added to a 35mm dish (3 x 10 5 AsPC-1 cells were added), cultured and adhered, and then replaced with a medium free of FBS and glutamine to activate macropinocytosis while culturing for 16 hours.
- Cells were treated with the nucleic acid ligand bound to the plasma protein prepared by the above method, and then cultured for 3 hours, and 200 ng/ml human EGF protein (Merck, Germany) was treated 15 minutes before the end of the culture to activate the KRAS signaling pathway. did.
- the negative control group was not treated with anything, and the positive control group was treated with only EGF.
- Proteins extracted and quantified from cells were mixed with SDS loading dye and reacted at 85° C. for 10 minutes to prepare a sample for analysis. After putting on a 10% SDS-PAGE gel prepared with 15 ⁇ g of protein for each sample, electrophoresis was performed at 4° C. for 50 V, 15 minutes, 150 V, and 1 hour. The electrophoresis-completed gel protein was transferred to a polyvinylidene fluoride (PVDF) membrane (Bio Red, USA) activated with methanol, and was carried out at 4° C. at 250 mA for 2 hours.
- PVDF polyvinylidene fluoride
- the membrane was washed 3 times with distilled water and reacted in TBS-T buffer containing 5% skim milk for 1 hour to block non-specific proteins.
- Primary antibodies ERK, p-ERK1/2 and GAPDH (Cell Signaling Technologies, USA) were diluted 1/1000 and reacted at 4°C for 16 hours, washed 3 times with TBS-T, and rabbit IgG-HRP antibody ( Santa Cruz Biotechnology, USA) diluted with 1/2000 and reacted at room temperature for 1 hour. After washing 3 times with TBS-T, chemiluminescence solution was treated, and expression of each protein was imaged using Fusion Solo (Bilver, France) imaging equipment.
- the phosphorylated protein (p-ERK) was detected in each experiment, the antibody on the membrane was removed using a stripping buffer. Then, after washing 3 times with TBS-T, the internal standardized protein was detected using a chemiluminescent solution after skim-milk blocking, GAPDH primary antibody reaction, and HRP secondary antibody reaction as above. The band intensity of the phosphorylated protein (p-ERK) was normalized to each GAPDH.
- nucleic acid ligand that binds albumin and KRAS mutant protein.
- nucleic acid ligands AD005 and AD011 continuously exhibited excellent phosphorylation inhibitory effects from 1 hour to 24 hours after treatment (see FIGS. 24A and 24B ).
- the nucleic acid ligand according to an embodiment of the present disclosure binds to KRAS, an intracellular therapeutic target, and inhibits phosphorylation of downstream proteins, thereby preventing or treating diseases that may be caused by the signal transduction pathway of KRAS. It can be predicted that
- nucleic acid ligand according to an embodiment of the present disclosure can exhibit a therapeutic effect through the route shown in FIG. 25 .
- the experiment below was performed to confirm whether these effects were also observed in vivo.
- a mouse pancreatic cancer tumor model (Balb/c-nu) was prepared.
- SC Subcutaneous Injection
- PBS a nucleic acid ligand according to an embodiment of the present disclosure
- a positive control drug (Gemcitabine) were each injected intravenously (Intravenous Injection, IV) to compare tumor suppression efficacy.
- the number of experimental animals in each experimental group was 4 animals.
- the AD005 nucleic acid ligand of the present disclosure was administered at a much lower concentration (1/100 times) than that of the positive control drug (Gemcitabine), it exhibited a significantly superior tumor suppression effect compared to the positive control drug.
- the positive control drug (Gemcitabine) showed a decrease in body weight from 8 days after drug administration, but the nucleic acid ligand according to an embodiment of the present disclosure showed that the body weight of the mouse was normally increased (see FIGS. 26A to 26C ).
- mice showing abnormal dynamics were sacrificed and organs were weighed to confirm acute toxicity.
- chronic toxicity was confirmed by measuring the organ weight of the sacrificed mice after the last injection of the drug.
- the positive control drug (Gemcitabine) showed a serious side effect of spleen enlargement, but the AD005 nucleic acid ligand of the present disclosure did not change the size of the spleen as in mice not injected with the drug. In addition, it was confirmed that there were no major side effects in the heart, liver, lung, kidney, etc. This shows that the nucleic acid ligand according to an embodiment of the present disclosure exhibits superior tumor suppression efficacy than the positive control drug and has almost no toxicity or side effects (see FIGS. 27A and 27C ).
- a mouse pancreatic cancer tumor model (Balb/c-nu) was first prepared.
- PBS a nucleic acid ligand
- AD005 nucleic acid ligand
- Gemcitabine a positive control drug
- Nucleic acid ligands of different doses (5, 10, 20, 50 ug) were administered once every 2 days 7 times (Day 0, 2, 4, 6, 8, 12), and positive control drug (1 mg) was administered once every 3 days 5 (Day 0, 3, 6, 9, 12) was injected. Tumor size was measured every 2 days using the equation [1/2 x longest diameters x shortest diameter].
- the nucleic acid ligand according to an embodiment of the present disclosure exhibited a significantly superior tumor suppression effect as the dose increased.
- the difference in the effect was not large, and it was confirmed that the tumor suppression efficacy did not increase when the dose exceeded a certain amount (see FIGS. 28a and 28b ).
- the positive control drug (Gemcitabine) showed serious side effects such as enlarged liver and enlarged spleen.
- the size of the spleen and liver of the nucleic acid ligand according to an embodiment of the present disclosure did not change as in mice not injected with the drug even when the dose was increased.
- liver damage indicators As a result of measuring the liver damage indicators (AST and ALT) of the positive control drug, liver damage occurred severely, but the nucleic acid ligand according to an embodiment of the present disclosure is a liver damage indicator like a mouse that did not inject the drug even though the dose was increased. The results did not change (see Figure 30).
- the positive control drug showed a side effect of decreasing white blood cell (WBC), red blood cell (RED) and platelet (Platelet) levels, but the nucleic acid ligand according to an embodiment of the present disclosure did not show abnormal levels with increasing dose (FIG. 31) Reference).
- the PD-1/PD-L1 binding inhibitory effect of the nucleic acid ligand binding to the prepared albumin and PD-L1 protein was confirmed.
- Binding inhibition at the protein level was performed by ELISA using a purified protein-based PD-1/PD-L1 inhibitor screening assay kit (BP, USA).
- the product includes immunobuffer 1, PD-1 protein, PD-L1 protein, blocking buffer, and chemiluminescent solution for use in experiments.
- a commercially available PD-L1 neutralizing antibody (BPS, USA) was used as a positive control as an inhibitory activity measurement method designed to decrease the fluorescence signal as the inhibitory activity increased.
- purified PD-L1 protein according to the product manual was fixed on a 96-well plate, and then each well was washed with Immuno Buffer 1 a total of 3 times to remove unbound PD-L1 protein.
- the nucleic acid ligand according to an embodiment of the present disclosure is structurally stabilized in buffer SB18 at each experimental concentration, 5 ⁇ L of the nucleic acid ligand is added to the well to which the PD-L1 protein is fixed, and 20 ⁇ L of biotin-binding PD- prepared according to the instructions 1 protein was added to each well.
- Inhibition of PD-1/PD-L1 binding at the cellular level was performed using a cell-based PD-1/PD-L1 inhibition bioassay (Promega, USA). This is an assay method based on a luciferase reporter. Jurkat effector cells expressing PD-1 and luciferase genes, TCR activator and CHO-K1 expressing PD-L1 After mixing the cells, the inhibitor (nucleic acid ligand or neutralizing antibody) was treated and cultured to test the PD-1/PD-L1 binding inhibitory activity at the cell level.
- This system is a cell-based inhibitory activity measurement method designed to increase the fluorescence signal as the inhibitory activity increases, and the above-mentioned PD-L1 neutralizing antibody was used as a positive control.
- nucleic acid ligands AP013 and AP031 bind to PD-L1 and inhibit the binding to PD-1, thereby neutralizing the immune cell attack evasion pathway of cancer cells.
- nucleic acid ligand according to an embodiment of the present disclosure can be utilized for the treatment of PD-L1 overexpressing cancer (see FIG. 34).
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Abstract
Description
핵산 리간드 코드 | 제1 타겟 | 제2 타겟 | 서열번호 | 핵산 염기 서열 |
AP013 | HSA | PD-L1 | 서열번호 84 | GAGTGACCGTCTGCCTGACAACAACGAG6GCGAAC6G6AC6AC66GA6AGAGACCC6CAGCCACACCACCAGCC |
AP027 | HSA | PD-L1 | 서열번호 98 | GAGTGACCGTCTGCCTG6CAA6CAC6GACGAAA6G6AC6AC66GA6AGAGC6AACC6CAGCCACACCACCAGCC |
AP014 | HSA | PD-L1 | 서열번호 85 | GAGTGACCGTCTGCCTGGAGCA6GG6CACA6G6AC6AC66GA6AGGAGCCCG6GCC6CAGCCACACCACCAGCC |
AA001 | HSA | CD3d/e | 서열번호 6 | GAGTGACCGTCTGCC6GGA6CC6GCCC6666AA6CCGAC666AA6GGCCACGC6CACCAGCCACACCACCAGCC |
AB001 | HSA | 4-1BB | 서열번호 13 | GAGTGACCGTCTGCC6G6C6CCAACC6CGCC666ACAAC6A6A6C6GACGAAC6AACACAGCCACACCACCAGCC |
AD001 | HSA | KRAS G12D | 서열번호 20 | GAGTGACCGTCTGCCTG6G6GAGAA66A6G6ACC66AACGA6CA6CCGAAGG6666GACCACAGCCACACCACCAGCC |
AD002 | HSA | KRAS G12D | 서열번호 21 | GAGTGACCGTCTGCCTG6G6C6GAGGGACCC66666AAGGGCA66A6CAGCA6GCC6CACACAGCCACACCACCAGCC |
AD003 | HSA | KRAS G12D | 서열번호 22 | GAGTGACCGTC6GCCTGGAC6AGCA6AAG6CCGGA6GGCAGC666A6GCCGACCA6CAGCCACACCACCAGCC |
AD009 | HSA | KRAS G12D | 서열번호 28 | GAGTGACCGTCTGCC6GCCCCGCC6C66C6GAAG66AACA6G6GC6CCGAGA6GG6ACAGCCACACCACCAGCC |
AD032 | HSA | KRAS G12D | 서열번호 42 | CAGG6CGAGC6G6ACGCG6G6G6GACC6GAAGACG6A6AAC6666ACAGAA6C6ACA |
AD034 | HSA | KRAS G12D | 서열번호 43 | CAGG6CGAGC6G6ACGCG6G6G6GACC6GCCCCAC66C6GACA66AGAC6G6G6AGA |
AV001 | HSA | KRAS G12V | 서열번호 119 | GAGTGACCGTCTGCCTGAGCAGGAGGA6G6GACACAAC666GCA666CAAGCC6GA6CAGCCACACCACCAGCC |
AV006 | HSA | KRAS G12V | 서열번호 124 | GAGTGACCGTCTGCCTG6GAGAA66A6G6ACC66AACGA6CA6CCGAAGG6666GACCAGCCACACCACCAGCC |
AX001 | HSA | BCL-2 | 서열번호 173 | GAGTGACCGTCTGCCTG6ACGA6AAC6GAG6G6A6GCA66AGACC6666G6GACCG6CAGCCACACCACCAGCC |
ID001 | IgG | KRAS G12D | 서열번호 139 | GAGTGACCGTCTGCCTGGAC6AGCA6AAG6CCGGA6GGCAGC666A6GCCGACCA6CAGCCACACCACCAGCC |
IC001 | IgG | KRAS G12C | 서열번호 128 | GAGTGACCGTCTGCCTGAAAG6CCGGA6GGCAGC666ACA6GCAGAAC6AACG6ACACAGCCACACCACCAGCC |
IC007 | IgG | KRAS G12C | 서열번호 134 | GAGTGACCGTCTGCCTGGACCAGCA6AAG6CCGGA6GGCAGC666A6GCCGACCA6CAGCCACACCACCAGCC |
IV001 | IgG | KRAS G12V | 서열번호 178 | GAGTGACCGTCTGCCTG6G6GAGAA66A6G6ACCC66AACGA6CA6CCGAAGG6666GACCACAGCCACACCACCAGCC |
TD001 | TF | KRAS G12D | 서열번호 149 | GAGTGACCGTCTGCCTGGGAC6AGCA6AAG6CCGGA6GGCAGC666A6GCCGACCA6CAGCCACACCACCAGCC |
TD005 | TF | KRAS G12D | 서열번호 153 | GAGTGACCGTCTGCCTGAAAG6CCGGA6GGCAGC666ACAGGCAGAAC6AACG6ACACAGCCACACCACCAGCC |
TD009 | TF | KRAS G12D | 서열번호 157 | GAGTGACCGTCTGCCTGGA6A6GCC6C66C6GCAG66ACGG6G6GA6CGCCCAA6AGCAGCCACACCACCAGCC |
TC001 | TF | KRAS G12C | 서열번호 144 | GAGTGACCGTCTGCCTGGAC6AGCATAAG6CCGGA6GGCAGC666A6GCCGACCA6CAGCCACACCACCAGCC |
AP030 | HSA | PD-L1 | 서열번호 99 | 6CCCAGG6CGAGC6G6ACGCG6G6G6GACCAC6G6AC6AC66GA6AGAGGCAC6GCC6 |
AP031 | HSA | PD-L1 | 서열번호 100 | 6CCCAGG6CGAGC6G6ACGCG6G6G6GACC66AC6GA6AA6CACC66GAA6AG6G6AC |
핵산 리간드 코드 | 제1 타겟 | 제2 타겟 | 서열번호 | 핵산 염기 서열 | 분자량 계산값
(Da) |
분자량 측정값
(Da) |
AP013 | HSA | PD-L1 | 서열번호 84 | GAGTGACCGTCTGCCTGACAACAACGAG6GCGAAC6G6AC6AC66GA6AGAGACCC6CAGCCACACCACCAGCC | 24409.37708 | 24408.9492 |
AP001 | HSA | PD-L1 | 서열번호 72 | GCC6GACAACAACGAG6GCGAAC6G6AC6AC66GA6AGAGACCC6CAGCC | 17252.21971 | 17252.0977 |
AP002 | HSA | PD-L1 | 서열번호 73 | ACAACAACGAG6GCGAAC6G6AC6AC66GA6AGAGACCC6 | 14033.67385 | 14033.1553 |
AP003 | HSA | PD-L1 | 서열번호 74 | CAACGAG6GCGAAC6G6AC6AC66GA6AGAGACCC6 | 12805.45465 | 12804.9521 |
AP004 | HSA | PD-L1 | 서열번호 75 | CAACGAG6GCGAAC6G6AC6AC66GA6AGAGA | 11465.21672 | 11464.7471 |
AP005 | HSA | PD-L1 | 서열번호 76 | CGAG6GCGAAC6G6AC6AC66GA6AGAGA | 10550.05513 | 10549.5020 |
AP006 | HSA | PD-L1 | 서열번호 77 | CGAG6GCGAAC6G6AC6AC66GA6AG | 9594.887391 | 9593.9219 |
AP021 | HSA | PD-L1 | 서열번호 92 | ACCGTCTGCCTGACAACAACGAG6GCGAAC6G6AC6AC66GA6AGAGACCC6CAGCCACACCACCAGCC | 22805.11586 | 22805.3711 |
AP022 | HSA | PD-L1 | 서열번호 93 | GTCTGCCTGACAACAACGAG6GCGAAC6G6AC6AC66GA6AGAGACCC6CAGCCACACCACCAGCC | 21913.9655 | 21913.3750 |
AP023 | HSA | PD-L1 | 서열번호 94 | GAGTGACCGTCTGCCTGACAACAACGAG6GCGAAC6G6AC6AC66GA6AGAGACCC6C | 19584.56662 | 19584.5176 |
AP024 | HSA | PD-L1 | 서열번호 95 | GTCTGCCTGACAACAACGAG6GCGAAC6G6AC6AC66GA6AGAGACCC6C | 17089.15504 | 17089.0684 |
AP025 | HSA | PD-L1 | 서열번호 96 | GTCTGCCTGACAACAACGAG6GCGAAC6G6AC6AC66GA6AGAGACCC6 | 16800.10867 | 16800.0156 |
AP026 | HSA | PD-L1 | 서열번호 97 | GTCTGCCTGACAACAACGAG6GCGAAC6G6AC6AC66GA6AGAG | 15146.81313 | 15146.5127 |
AP027 | HSA | PD-L1 | 서열번호 98 | GAGTGACCGTCTGCCTG6CAA6CAC6GACGAAA6G6AC6AC66GA6AGAGC6AACC6CAGCCACACCACCAGCC | 24881.52207 | 24881.1738 |
AP015 | HSA | PD-L1 | 서열번호 86 | GCCTG6CAA6CAC6GACGAAA6G6AC6AC66GA6AGAGC6AACC6CAGCC | 17555.31194 | 17555.2031 |
AP016 | HSA | PD-L1 | 서열번호 87 | 6CAA6CAC6GACGAAA6G6AC6AC66GA6AGAGC6AACC6 | 14505.81884 | 14505.3877 |
AP017 | HSA | PD-L1 | 서열번호 88 | CAC6GACGAAA6G6AC6AC66GA6AGAGC6AACC6 | 12644.45964 | 12644.9463 |
AP018 | HSA | PD-L1 | 서열번호 89 | CAC6GACGAAA6G6AC6AC66GA6AGAGC6 | 10967.15287 | 10966.6768 |
AP019 | HSA | PD-L1 | 서열번호 90 | 6GACGAAA6G6AC6AC66GA6AGAGC6 | 10076.00251 | 10075.2451 |
AP020 | HSA | PD-L1 | 서열번호 91 | ACGAAA6G6AC6AC66GA6AGAG | 8511.706006 | 8511.6914 |
AP014 | HSA | PD-L1 | 서열번호 85 | GAGTGACCGTCTGCCTGGAGCA6GG6CACA6G6AC6AC66GA6AGGAGCCCG6GCC6CAGCCACACCACCAGCC | 24777.44422 | 24776.7734 |
AP007 | HSA | PD-L1 | 서열번호 78 | GCC6GGAGCA6GG6CACA6G6AC6AC66GA6AGGAGCCCG6GCC6CAGCC | 17620.28685 | 17620.1641 |
AP008 | HSA | PD-L1 | 서열번호 79 | GAGCA6GG6CACA6G6AC6AC66GA6AGGAGCCCG6GCC6 | 14401.74099 | 14401.2188 |
AP009 | HSA | PD-L1 | 서열번호 80 | CA6GG6CACA6G6AC6AC66GA6AGGAGCCCG6GCC6 | 13430.57834 | 13430.0586 |
AP010 | HSA | PD-L1 | 서열번호 81 | CA6GG6CACA6G6AC6AC66GA6AGGAGCCCG | 11577.23545 | 11577.7646 |
AP011 | HSA | PD-L1 | 서열번호 82 | CA6GG6CACA6G6AC6AC66GA6AGGAGC | 10670.09018 | 10669.5986 |
AP012 | HSA | PD-L1 | 서열번호 83 | CA6GG6CACA6G6AC6AC66GA6AG | 9409.88115 | 9409.8633 |
AP045 | HSA | PD-L1 | 서열번호 109 | CA6GG6CACA6G6AC6AC66GA6AGGAGCCCG6GCC6CAGCC | 14939.82759 | 14939.0322 |
AP046 | HSA | PD-L1 | 서열번호 110 | GG6CACA6G6AC6AC66GA6AGGAGCCCG6GCC6CAGCC | 13864.6248 | 13863.8174 |
AP047 | HSA | PD-L1 | 서열번호 111 | G6CACA6G6AC6AC66GA6AGGAGCCCG6GCC6CAGCC | 13535.57228 | 13535.0977 |
AP048 | HSA | PD-L1 | 서열번호 112 | 6CACA6G6AC6AC66GA6AGGAGCCCG6GCC6CAGCC | 13206.51976 | 13205.7412 |
AP050 | HSA | PD-L1 | 서열번호 113 | AC6AC66GA6AGGAGCCCG6GCC6CAGCC | 10253.96285 | 10253.0781 |
AP051 | HSA | PD-L1 | 서열번호 114 | C6AC66GA6AGGAGCCCG6GCC6CAGCC | 9940.905245 | 9940.8955 |
AP052 | HSA | PD-L1 | 서열번호 115 | GAGCCCG6GCC6CAGCC | 5873.139405 | 5872.1543 |
AP053 | HSA | PD-L1 | 서열번호 116 | AGCCCG6GCC6CAGCC | 5544.086882 | 5543.0952 |
AP054 | HSA | PD-L1 | 서열번호 117 | GCC6GGAGCA6GG6CACA6G6AC6AC66GA6AGGAGCCCG6GCC6CA | 16713.14157 | 16713.1035 |
AP055 | HSA | PD-L1 | 서열번호 118 | GCC6GGAGCA6GG6CACA6G6AC6AC66GA6AGGAGCCCG6GCC6C | 16400.08397 | 16400.043 |
AA001 | HSA | CD3d/e | 서열번호 6 | GAGTGACCGTCTGCC6GGA6CC6GCCC6666AA6CCGAC666AA6GGCCACGC6CACCAGCCACACCACCAGCC | 24777.44422 | 24777.6914 |
AA002 | HSA | CD3d/e | 서열번호 7 | GCCTGGA6CC6GCCC6666AA6CCGAC666AA6GGCCACGC6CACCAGCC | 17595.29697 | 17595.1602 |
AA003 | HSA | CD3d/e | 서열번호 8 | GA6CC6GCCC6666AA6CCGAC666AA6GGCCACGC6CAC | 14545.80388 | 14545.2637 |
AA004 | HSA | CD3d/e | 서열번호 9 | 6GCCC6666AA6CCGAC666AA6GGCCACGC6CAC | 12852.50219 | 12851.9795 |
AA005 | HSA | CD3d/e | 서열번호 10 | 6GCCC6666AA6CCGAC666AA6GGCCACG | 11199.20665 | 11198.7344 |
AA006 | HSA | CD3d/e | 서열번호 11 | 6GCCC6666AA6CCGAC666AA6GGC | 9979.003771 | 9979.1611 |
AA007 | HSA | CD3d/e | 서열번호 12 | GCCC6666AA6CCGAC666AA6 | 8558.753545 | 8558.7832 |
AB001 | HSA | 4-1BB | 서열번호 13 | GAGTGACCGTCTGCC6G6C6CCAACC6CGCC666ACAAC6A6A6C6GACGAAC6AACACAGCCACACCACCAGCC | 25195.59154 | 25195.1484 |
AB002 | HSA | 4-1BB | 서열번호 14 | GCCTG6C6CCAACC6CGCC666ACAAC6A6A6C6GACGAAC6AACACAGCC | 17700.32864 | 17700.1992 |
AB003 | HSA | 4-1BB | 서열번호 15 | 6C6CCAACC6CGCC666ACAAC6A6A6C6GACGAAC6AACA | 14650.83554 | 14650.4082 |
AB004 | HSA | 4-1BB | 서열번호 16 | AACC6CGCC666ACAAC6A6A6C6GACGAAC6AACA | 12837.49881 | 12836.9912 |
AB005 | HSA | 4-1BB | 서열번호 17 | AACC6CGCC666ACAAC6A6A6C6GACGAAC | 11136.1808 | 11135.7344 |
AB006 | HSA | 4-1BB | 서열번호 18 | 6CGCC666ACAAC6A6A6C6GACGAAC | 9931.972837 | 9931.1016 |
AB007 | HSA | 4-1BB | 서열번호 19 | 6CGCC666ACAAC6A6A6C6GACGA | 9329.868854 | 9329.8477 |
AD001 | HSA | KRAS G12D | 서열번호 20 | GAGTGACCGTCTGCCTG6G6GAGAA66A6G6ACC66AACGA6CA6CCGAAGG6666GACCACAGCCACACCACCAGCC | 26685.87207 | 26685.7910 |
AD002 | HSA | KRAS G12D | 서열번호 21 | GAGTGACCGTCTGCCTG6G6C6GAGGGACCC66666AAGGGCA66A6CAGCA6GCC6CACACAGCCACACCACCAGCC | 25867.68811 | 25867.6348 |
AD003 | HSA | KRAS G12D | 서열번호 22 | GAGTGACCGTC6GCCTGGAC6AGCA6AAG6CCGGA6GGCAGC666A6GCCGACCA6CAGCCACACCACCAGCC | 24457.40326 | 24457.0605 |
AD004 | HSA | KRAS G12D | 서열번호 23 | GCCTGGAC6AGCA6AAG6CCGGA6GGCAGC666A6GCCGACCA6CAGCC | 16962.14036 | 16962.0449 |
AD005 | HSA | KRAS G12D | 서열번호 24 | GAC6AGCA6AAG6CCGGA6GGCAGC666A6GCCGACCA6 | 13912.64727 | 13912.1592 |
AD006 | HSA | KRAS G12D | 서열번호 25 | GAC6AGCA6AAG6CCGGA6GGCAGC666A6GCCG | 12235.3405 | 12234.8760 |
AD007 | HSA | KRAS G12D | 서열번호 26 | GCA6AAG6CCGGA6GGCAGC666A6GCCGACC | 11409.17794 | 11408.7393 |
AD008 | HSA | KRAS G12D | 서열번호 27 | GCA6AAG6CCGGA6GGCAGC666A6GC | 9899.928681 | 9899.9736 |
AD016 | HSA | KRAS G12D | 서열번호 35 | GAC6AGCA6AAG6CCGGA6GGCAGC666A | 10558.03373 | 10557.3926 |
AD017 | HSA | KRAS G12D | 서열번호 36 | GAC6AGCA6AAG6CCGGA6GGCAG | 8536.633329 | 8535.7422 |
AD018 | HSA | KRAS G12D | 서열번호 37 | GAC6AGCA6AAG6CCGGA6 | 6947.371777 | 6946.603 |
AD020 | HSA | KRAS G12D | 서열번호 38 | AG6CCGGA6GGCAGC666A6GCCGACCA6 | 10510.01126 | 10509.3818 |
AD024 | HSA | KRAS G12D | 서열번호 39 | GCA6AAG6CCGGA6GGCAGC666A6G | 9610.882306 | 9609.9199 |
AD025 | HSA | KRAS G12D | 서열번호 40 | AG6CCGGA6GGCAGC666A6GC | 8182.615762 | 8181.6958 |
AD026 | HSA | KRAS G12D | 서열번호 41 | 6AGCA6AAG6CCGGA6GGCAGC666A6GC | 10686.08509 | 10685.0693 |
AD059 | HSA | KRAS G12D | 서열번호 50 | GAC6AGCA6AAG6CCGGA6GGCAGC666A6GCCGACCA | 13439.54846 | 13438.7393 |
AD060 | HSA | KRAS G12D | 서열번호 51 | GAC6AGCA6AAG6CCGGA6GGCAGC666A6GCCGACC | 13126.49086 | 13125.6611 |
AD061 | HSA | KRAS G12D | 서열번호 52 | GAC6AGCA6AAG6CCGGA6GGCAGC666A6GCC | 11906.28798 | 11905.8672 |
AD063 | HSA | KRAS G12D | 서열번호 53 | GAC6AGCA6AAG6CCGGA6GGCAGC666A6G | 11328.19523 | 11327.3516 |
AD064 | HSA | KRAS G12D | 서열번호 54 | GAC6AGCA6AAG6CCGGA6GGCAGC666A6 | 10999.1427 | 10998.335 |
AD065 | HSA | KRAS G12D | 서열번호 55 | GAC6AGCA6AAG6CCGGA6GGCAGC | 8793.689874 | 8792.627 |
AD066 | HSA | KRAS G12D | 서열번호 56 | CA6AAG6CCGGA6GGCAGC666A6G | 9281.829783 | 9281.8711 |
AD067 | HSA | KRAS G12D | 서열번호 57 | A6AAG6CCGGA6GGCAGC666A6 | 8663.730885 | 8662.6592 |
AD068 | HSA | KRAS G12D | 서열번호 58 | AAG6CCGGA6GGCAGC666 | 7091.418059 | 7090.4351 |
AD069 | HSA | KRAS G12D | 서열번호 59 | AG6CCGGA6GGCAGC666 | 6778.360451 | 6777.4009 |
AD070 | HSA | KRAS G12D | 서열번호 60 | AAG6CCGGA6GGCAGC66 | 6618.319254 | 6617.3169 |
AD071 | HSA | KRAS G12D | 서열번호 61 | GAC6AGCA6AAG6CC | 5471.120488 | 5470.1323 |
AD072 | HSA | KRAS G12D | 서열번호 62 | GAC6AGCA6AAG6C | 5182.074113 | 5181.0767 |
AD073 | HSA | KRAS G12D | 서열번호 63 | GAC6AGCA6AAG6 | 4893.027738 | 4892.04 |
AD078 | HSA | KRAS G12D | 서열번호 64 | AGC666A6GCCGACCA6 | 6393.306865 | 6393.3403 |
AD079 | HSA | KRAS G12D | 서열번호 65 | GC666A6GCCGACCA6 | 6080.249257 | 6079.2754 |
AD009 | HSA | KRAS G12D | 서열번호 28 | GAGTGACCGTCTGCC6GCCCCGCC6C66C6GAAG66AACA6G6GC6CCGAGA6GG6ACAGCCACACCACCAGCC | 25026.52588 | 25025.8418 |
AD010 | HSA | KRAS G12D | 서열번호 29 | GCCTGCCCCGCC6C66C6GAAG66AACA6G6GC6CCGAGA6GG6ACAGCC | 17531.26298 | 17531.1484 |
AD011 | HSA | KRAS G12D | 서열번호 30 | CCCCGCC6C66C6GAAG66AACA6G6GC6CCGAGA6GG6A | 14481.76989 | 14481.2471 |
AD012 | HSA | KRAS G12D | 서열번호 31 | CCCCGCC6C66C6GAAG66AACA6G6GC6CCGAGA | 12564.40963 | 12563.9199 |
AD013 | HSA | KRAS G12D | 서열번호 32 | CC6C66C6GAAG66AACA6G6GC6CCGAGA | 11079.1716 | 11078.6982 |
AD014 | HSA | KRAS G12D | 서열번호 33 | CCCCGCC6C66C6GAAG66AACA6G6GC6CC | 11280.18936 | 11279.7344 |
AD015 | HSA | KRAS G12D | 서열번호 34 | CC6C66C6GAAG66AACA6G6GC6CC | 9794.951341 | 9794.9541 |
AD080 | HSA | KRAS G12D | 서열번호 66 | 6C66C6GAAG66AACA6G6GC6CCGAGA6GG6A | 12418.43912 | 12417.5723 |
AD081 | HSA | KRAS G12D | 서열번호 67 | 6C66C6GAAG66AACA6G6GC6CCGAGA6GG6 | 12105.38151 | 12104.5459 |
AD083 | HSA | KRAS G12D | 서열번호 68 | C66C6GAAG66AACA6G6GC6CCGAGA6GG6A | 11945.34031 | 11945.0078 |
AD084 | HSA | KRAS G12D | 서열번호 69 | 6CCGAGA6GG6A | 4595.965045 | 4594.9746 |
AD085 | HSA | KRAS G12D | 서열번호 70 | CCCCGCC6C66C6GAAG66AACA6G6GC6CCGAGA6GG6 | 14168.71228 | 14167.8818 |
AD086 | HSA | KRAS G12D | 서열번호 71 | CCCCGCC6C66C6GAAG66AACA6G6GC6CCGAGA6GG | 13695.61348 | 13694.8086 |
AD032 | HSA | KRAS G12D | 서열번호 42 | CAGG6CGAGC6G6ACGCG6G6G6GACC6GAAGACG6A6AAC6666ACAGAA6C6ACA | 20523.88859 | 20523.3672 |
AD043 | HSA | KRAS G12D | 서열번호 162 | CAGG6CGAGC6G6ACGCG6G6G6GACC6GAAGAC | 12107.28913 | 12106.8447 |
AD044 | HSA | KRAS G12D | 서열번호 163 | CAGG6CGAGC6G6ACGCG6G6G6GACC6GAAGACG6A6A | 14008.65448 | 14008.1396 |
AD045 | HSA | KRAS G12D | 서열번호 164 | CAGG6CGAGC6G6ACGCG6G6G6GACC6GAAGACG6A6AAC666 | 16030.05488 | 16029.8789 |
AD046 | HSA | KRAS G12D | 서열번호 165 | CAGG6CGAGC6G6ACGCG6G6G6GACC6GAAGACG6A6AAC6666ACAG | 17747.3678 | 17747.3984 |
AD047 | HSA | KRAS G12D | 서열번호 166 | CAGG6CGAGC6G6ACGCG6G6G6GACC6GAAGACG6A6AAC6666ACAGAA6C6 | 19608.727 | 19608.5332 |
AD048 | HSA | KRAS G12D | 서열번호 167 | AAGACG6A6AAC6666ACAGAA6C6ACA | 10317.06414 | 10316.5586 |
AD049 | HSA | KRAS G12D | 서열번호 168 | ACC6GAAGACG6A6AAC6666ACAGAA6C6ACA | 12010.36583 | 12009.8408 |
AD050 | HSA | KRAS G12D | 서열번호 169 | G6G6GACC6GAAGACG6A6AAC6666ACAGAA6C6ACA | 13943.72101 | 13943.1904 |
AD051 | HSA | KRAS G12D | 서열번호 170 | CGCG6G6G6GACC6GAAGACG6A6AAC6666ACAGAA6C6ACA | 15653.01761 | 15652.9365 |
AD052 | HSA | KRAS G12D | 서열번호 171 | C6G6ACGCG6G6G6GACC6GAAGACG6A6AAC6666ACAGAA6C6ACA | 17530.37173 | 17530.3730 |
AD053 | HSA | KRAS G12D | 서열번호 172 | 6CGAGC6G6ACGCG6G6G6GACC6GAAGACG6A6AAC6666ACAGAA6C6ACA | 19263.67956 | 19263.7363 |
AD034 | HSA | KRAS G12D | 서열번호 43 | CAGG6CGAGC6G6ACGCG6G6G6GACC6GCCCCAC66C6GACA66AGAC6G6G6AGA | 20483.84472 | 20483.1777 |
AD036 | HSA | KRAS G12D | 서열번호 44 | CAGG6CGAGC6G6ACGCG6G6G6GACC6GCCCCAC66C6GACA66AGAC6G6G | 19055.57818 | 19054.5293 |
AD037 | HSA | KRAS G12D | 서열번호 45 | CAGG6CGAGC6G6ACGCG6G6G6GACC6GCCCCAC66C6GACA66AGAC6G6 | 18726.52565 | 18725.4512 |
AD038 | HSA | KRAS G12D | 서열번호 46 | CAGG6CGAGC6G6ACGCG6G6G6GACC6GCCCCAC66C6GACA66AGAC6G | 18253.42685 | 18253.7031 |
AD039 | HSA | KRAS G12D | 서열번호 47 | CAGG6CGAGC6G6ACGCG6G6G6GACC6GCCCCAC66C6GACA66AGA | 17162.22915 | 17161.3594 |
AD040 | HSA | KRAS G12D | 서열번호 48 | CAGG6CGAGC6G6ACGCG6G6G6GACC6GCCCCAC66C6GACA66AG | 16849.17154 | 16848.0254 |
AD041 | HSA | KRAS G12D | 서열번호 49 | 6ACGCG6G6G6GACC6GCCCCAC66C6GACA66AG | 12764.45697 | 12763.2266 |
AV001 | HSA | KRAS G12V | 서열번호 119 | GAGTGACCGTCTGCCTGAGCAGGAGGA6G6GACACAAC666GCA666CAAGCC6GA6CAGCCACACCACCAGCC | 24809.47177 | 24809.8281 |
AV002 | HSA | KRAS G12V | 서열번호 120 | CTGAGCAGGAGGA6G6GACACAAC666GCA666CAAGCC6GA6CAGCCACACCACCAGCC | 20469.77032 | 20469.3262 |
AV003 | HSA | KRAS G12V | 서열번호 121 | CTGAGCAGGAGGA6G6GACACAAC666GCA666CAAGCC6GA6CAG | 16287.06999 | 16287.0889 |
AV004 | HSA | KRAS G12V | 서열번호 122 | AGCAGGAGGA6G6GACACAAC666GCA666CAAGCC6GA6 | 14433.76854 | 14433.3174 |
AV005 | HSA | KRAS G12V | 서열번호 123 | CCGTCTGCCTGAGCAGGAGGA6G6GACACAAC6 | 11071.03097 | 11070.5186 |
AV006 | HSA | KRAS G12V | 서열번호 124 | GAGTGACCGTCTGCCTG6GAGAA66A6G6ACC66AACGA6CA6CCGAAGG6666GACCAGCCACACCACCAGCC | 25281.61676 | 25281.3516 |
AV007 | HSA | KRAS G12V | 서열번호 125 | GTCTGCCTG6GAGAA66A6G6ACC66AACGA6CA6CCGAAGG6666GACC | 17961.39473 | 17961.2266 |
AV008 | HSA | KRAS G12V | 서열번호 126 | 6GAGAA66A6G6ACC66AACGA6CA6CCGAAGG6666GACCA | 15508.01752 | 15507.9736 |
AV009 | HSA | KRAS G12V | 서열번호 127 | A66A6G6ACC66AACGA6CA6CCGAAGG6666GACC | 13437.64084 | 13437.1006 |
AX001 | HSA | BCL-2 | 서열번호 173 | GAGTGACCGTCTGCCTG6ACGA6AAC6GAG6G6A6GCA66AGACC6666G6GACCG6CAGCCACACCACCAGCC | 25457.65288 | 25457.5684 |
AX002 | HSA | BCL-2 | 서열번호 174 | CGTCTGCCTG6ACGA6AAC6GAG6G6AT6GCA66AGACC6666G6GACCG6C | 18730.52325 | 18730.3633 |
AX003 | HSA | BCL-2 | 서열번호 175 | GAG6G6A6GCA66AGACC6666G6GACCG6CAGCCACACCAC | 15107.88511 | 15107.7080 |
AX004 | HSA | BCL-2 | 서열번호 176 | A66AGACC6666G6GACCG6CAGCCACACCACCAG | 12388.37351 | 12388.8965 |
AX005 | HSA | BCL-2 | 서열번호 177 | A6AAC6GAG6G6A6GCA66AGACC6666G6 | 11655.34013 | 11654.8096 |
ID001 | IgG | KRAS G12D | 서열번호 139 | GAGTGACCGTCTGCCTGGAC6AGCA6AAG6CCGGA6GGCAGC666A6GCCGACCA6CAGCCACACCACCAGCC | 24288.3505 | 24287.6738 |
ID002 | IgG | KRAS G12D | 서열번호 140 | CTGCCTGGAC6AGCA6AAG6CCGGA6GGCAGC666A6GCCGACCA6CAGCCAC | 18157.33676 | 18157.2598 |
ID003 | IgG | KRAS G12D | 서열번호 141 | CTGCCTGGAC6AGCA6AAG6CCGGA6GGCAGC666A6GCC | 14039.62423 | 14039.1328 |
ID004 | IgG | KRAS G12D | 서열번호 142 | CTGGAC6AGCA6AAG6CCGGA6GGCAGC666A6GCC | 12828.43291 | 12827.9570 |
ID005 | IgG | KRAS G12D | 서열번호 143 | CTGGAC6AGCA6AAG6CCGGA6GGCAGC666A6GCCGACCA6CAGCCACACCACCAGCC | 19948.64904 | 19948.5723 |
IC001 | IgG | KRAS G12C | 서열번호 128 | GAGTGACCGTCTGCCTGAAAG6CCGGA6GGCAGC666ACA6GCAGAAC6AACG6ACACAGCCACACCACCAGCC | 24449.38323 | 24448.9531 |
IC002 | IgG | KRAS G12C | 서열번호 129 | GAGTGACCGTCTGCCTGAAAG6CCGGA6GGCAGC666ACA6GCAGAAC6AACG6ACA | 19335.52639 | 19335.3242 |
IC003 | IgG | KRAS G12C | 서열번호 130 | GAGTGACCGTCTGCCTGAAAG6CCGGA6GGCAGC666ACA6GCA | 14985.73896 | 14985.2871 |
IC004 | IgG | KRAS G12C | 서열번호 131 | AAAG6CCGGA6GGCAGC666ACA6GCAGAAC6AACG6ACA | 14073.68 | 14073.1436 |
IC005 | IgG | KRAS G12C | 서열번호 132 | ACCGTCTGCCTGAAAG6CCGGA6GGCAGC666ACA6GCA | 13381.47774 | 13381.0195 |
IC006 | IgG | KRAS G12C | 서열번호 133 | CTGCCTGAAAG6CCGGA6GGCAGC666ACA6GCA | 11857.22882 | 11856.7051 |
IC007 | IgG | KRAS G12C | 서열번호 134 | GAGTGACCGTCTGCCTGGACCAGCA6AAG6CCGGA6GGCAGC666A6GCCGACCA6CAGCCACACCACCAGCC | 24104.29807 | 24103.6348 |
IC008 | IgG | KRAS G12C | 서열번호 135 | CTGCCTGGACCAGCA6AAG6CCGGA6GGCAGC666A6GCCGACCA6CAG | 16793.0876 | 16793.0137 |
IC009 | IgG | KRAS G12C | 서열번호 136 | CTGCCTGGACCAGCA6AAG6CCGGA6GGCAGC666A6GCCGACCA | 15388.83229 | 15388.7598 |
IC010 | IgG | KRAS G12C | 서열번호 137 | CTGCCTGGACCAGCA6AAG6CCGGA6GGCAGC666A6GCC | 13855.5718 | 13855.0996 |
IC011 | IgG | KRAS G12C | 서열번호 138 | CTGGACCAGCA6AAG6CCGGA6GGCAGC666A6GCC | 12644.38048 | 12643.9121 |
IV001 | IgG | KRAS G12V | 서열번호 178 | GAGTGACCGTCTGCCTG6G6GAGAA66A6G6ACCC66AACGA6CA6CCGAAGG6666GACCACAGCCACACCACCAGCC | 26974.91845 | 26974.8516 |
IV002 | IgG | KRAS G12V | 서열번호 179 | CTG6G6GAGAA66A6G6ACCC66AACGA6CA6CCGAAGG6666GACCACAGCCACACCACCAGCC | 22635.217 | 22634.6035 |
IV003 | IgG | KRAS G12V | 서열번호 180 | G6ACCC66AACGA6CA6CCGAAGG6666GACCACAGCCACACCACCAGCC | 17108.19003 | 17108.0723 |
IV004 | IgG | KRAS G12V | 서열번호 181 | 66AACGA6CA6CCGAAGG6666GACCACAGCCACACCACCAGCC | 15125.84197 | 15125.4365 |
IV005 | IgG | KRAS G12V | 서열번호 182 | 66AACGA6CA6CCGAAGG6666GACCACAGCCACACCAC | 13616.59271 | 13616.0742 |
IV006 | IgG | KRAS G12V | 서열번호 183 | 6G6GAGAA66A6G6ACCC66AACGA6CA6CCGAAGG6666GACCA | 16599.21522 | 16599.1641 |
TD001 | TF | KRAS G12D | 서열번호 149 | GAGTGACCGTCTGCCTGGGAC6AGCA6AAG6CCGGA6GGCAGC666A6GCCGACCA6CAGCCACACCACCAGCC | 24617.40302 | 24616.8574 |
TD002 | TF | KRAS G12D | 서열번호 150 | CTGCCTGGGAC6AGCA6AAG6CCGGA6GGCAGC666A6GCCGACCA6 | 16375.03605 | 16374.9404 |
TD003 | TF | KRAS G12D | 서열번호 151 | GGAC6AGCA6AAG6CCGGA6GGCAGC666A6GCCGACCA6 | 14241.69979 | 14241.1777 |
TD004 | TF | KRAS G12D | 서열번호 152 | CTGCCTGGGAC6AGCA6AAG6CCGGA6GGCAG | 10967.03228 | 10966.5381 |
TD005 | TF | KRAS G12D | 서열번호 153 | GAGTGACCGTCTGCCTGAAAG6CCGGA6GGCAGC666ACAGGCAGAAC6AACG6ACACAGCCACACCACCAGCC | 24305.33695 | 24304.8672 |
TD006 | TF | KRAS G12D | 서열번호 154 | GAGTGACCGTCTGCCTGAAAG6CCGGA6GGCAGC666ACAGGCAGAAC6AACG | 17803.21971 | 17803.1973 |
TD007 | TF | KRAS G12D | 서열번호 155 | ACCGTCTGCCTGAAAG6CCGGA6GGCAGC666ACAGGCAGAAC6AACG | 16198.9585 | 16198.8730 |
TD008 | TF | KRAS G12D | 서열번호 156 | CTGCCTGAAAG6CCGGA6GGCAGC666ACAGGCAG | 12042.23506 | 12041.7080 |
TD009 | TF | KRAS G12D | 서열번호 157 | GAGTGACCGTCTGCCTGGA6A6GCC6C66C6GCAG66ACGG6G6GA6CGCCCAA6AGCAGCCACACCACCAGCC | 25081.5317 | 25080.6152 |
TD010 | TF | KRAS G12D | 서열번호 158 | CTGCCTGGA6A6GCC6C66C6GCAG66ACGG6G6GA6CGCCCAA6AGCAGCCACACC | 19841.66832 | 19841.5605 |
TD011 | TF | KRAS G12D | 서열번호 159 | A6GCC6C66C6GCAG66ACGG6G6GA6CGCCCAA6AGCAGCCACACC | 16593.12313 | 16593.0039 |
TD012 | TF | KRAS G12D | 서열번호 160 | A6GCC6C66C6GCAG66ACGG6G6GA6CGCCCAA6AGCAGCCAC | 15701.97277 | 15701.9824 |
TD013 | TF | KRAS G12D | 서열번호 161 | GGAC6AGCA6AAG6CCGGA6GGCAGC666A6GCCGACCA6CAGCCACACCACCAGCC | 19355.55663 | 19355.4766 |
TC001 | TF | KRAS G12C | 서열번호 144 | GAGTGACCGTCTGCCTGGAC6AGCATAAG6CCGGA6GGCAGC666A6GCCGACCA6CAGCCACACCACCAGCC | 24119.29773 | 24118.6152 |
TC002 | TF | KRAS G12C | 서열번호 145 | CTGCCTGGAC6AGCATAAG6CCGGA6GGCAGC666A6GCCGACCA6CAGCCAC | 17988.284 | 17988.2266 |
TC003 | TF | KRAS G12C | 서열번호 146 | CTGGAC6AGCATAAG6CCGGA6GGCAGC666A6GCCGACCA6 | 14665.73944 | 14665.2207 |
TC004 | TF | KRAS G12C | 서열번호 147 | CTGCCTGGAC6AGCATAAG6CCGGA6GGCAGC666A6GCC | 13870.57146 | 13869.9961 |
TC005 | TF | KRAS G12C | 서열번호 148 | CTGGAC6AGCATAAG6CCGGA6GGCAGC666A6GCC | 12659.38015 | 12658.9131 |
AP030 | HSA | PD-L1 | 서열번호 99 | 6CCCAGG6CGAGC6G6ACGCG6G6G6GACCAC6G6AC6AC66GA6AGAGGCAC6GCC6 | 20367.74236 | 20366.1543 |
AP031 | HSA | PD-L1 | 서열번호 100 | 6CCCAGG6CGAGC6G6ACGCG6G6G6GACC66AC6GA6AA6CACC66GAA6AG6G6AC | 20703.85739 | 20702.7285 |
AP033 | HSA | PD-L1 | 서열번호 101 | CAGG6CGAGC6G6ACGCG6G6G6GACC66AC6GA6AA6CACC66GAA6AG6G6AC | 20089.8044 | 20088.1133 |
AP034 | HSA | PD-L1 | 서열번호 102 | AGC6G6ACGCG6G6G6GACC66AC6GA6AA6CACC66GAA6AG6G6AC | 17738.39767 | 17737.1855 |
AP035 | HSA | PD-L1 | 서열번호 103 | 6ACGCG6G6G6GACC66AC6GA6AA6CACC66GAA6AG6G6AC | 16005.08983 | 16005.373 |
AP036 | HSA | PD-L1 | 서열번호 104 | G6G6G6GACC66AC6GA6AA6CACC66GAA6AG6G6AC | 14311.78815 | 14311.8096 |
AP037 | HSA | PD-L1 | 서열번호 105 | ACC66AC6GA6AA6CACC66GAA6AG6G6AC | 11576.28164 | 11576.7568 |
AP038 | HSA | PD-L1 | 서열번호 106 | 6CCCAGG6CGAGC6G6ACGCG6G6G6GACC66AC6GA6 | 13967.6568 | 13967.8672 |
AP039 | HSA | PD-L1 | 서열번호 107 | 6CCCAGG6CGAGC6G6ACGCG6G6G6GACC66AC6GA6AA6CA | 15668.9748 | 15668.9502 |
AP040 | HSA | PD-L1 | 서열번호 108 | 6CCCAGG6CGAGC6G6ACGCG6G6G6GACC66AC6GA6AA6CACC66GAA6A | 18934.58932 | 18934.2793 |
핵산 리간드 코드 | 제1 타겟 | 제2 타겟 | 서열번호 | 혈청 반감기
t 1/2 (hr) |
제1 타겟 결합 친화도 | 제2 타겟 결합 친화도 | ||
K d(nM) | R 2 | K d(nM) | R 2 | |||||
AP013 | HSA | PD-L1 | 서열번호 84 | 72이상 | 19.05 | 92 | 73.97 | 99.6 |
AP001 | HSA | PD-L1 | 서열번호 72 | 72이상 | 20.88 | 95.3 | 42.74 | 98.1 |
AP021 | HSA | PD-L1 | 서열번호 92 | 72이상 | 15.57 | 91.7 | 129.53 | 98.9 |
AP022 | HSA | PD-L1 | 서열번호 93 | 72이상 | 16.61 | 90.0 | 66.56 | 99.3 |
AP023 | HSA | PD-L1 | 서열번호 94 | 72이상 | 51.72 | 95.2 | 93.21 | 95.7 |
AP024 | HSA | PD-L1 | 서열번호 95 | 72이상 | 22.29 | 95.0 | 33.26 | 99.8 |
AP027 | HSA | PD-L1 | 서열번호 98 | 72이상 | 2.17 | 83.9 | 353.64 | 94.70 |
AA001 | HSA | CD3d/e | 서열번호 6 | 6.42 | 9.62 | 89.6 | 3.17 | 74.5 |
AA003 | HSA | CD3d/e | 서열번호 8 | 72이상 | 4.32 | 97.5 | 7.63 | 65 |
AA004 | HSA | CD3d/e | 서열번호 9 | 11.7 | 9.54 | 98.1 | 10.21 | 96.4 |
AA005 | HSA | CD3d/e | 서열번호 10 | 15.6 | 8.27 | 97.0 | 39.42 | 93.0 |
AA006 | HSA | CD3d/e | 서열번호 11 | 4.1 | 15.54 | 93 | 14.28 | 97.0 |
AA007 | HSA | CD3d/e | 서열번호 12 | 13.5 | 2.03 | 96 | 15.05 | 96.0 |
AB001 | HSA | 4-1BB | 서열번호 13 | 72이상 | 8.39 | 97.7 | 0.04 | 96.4 |
AB002 | HSA | 4-1BB | 서열번호 14 | 47.01 | 2.92 | 98.4 | 0.48 | 99.1 |
AB003 | HSA | 4-1BB | 서열번호 15 | 8.3 | 19.77 | 87.3 | 0.87 | 99.4 |
AB004 | HSA | 4-1BB | 서열번호 16 | 6.4 | 101.51 | 87.3 | 0.05 | 98.9 |
AB005 | HSA | 4-1BB | 서열번호 17 | 5.3 | 53.52 | 84.1 | 0.12 | 99.0 |
AB006 | HSA | 4-1BB | 서열번호 18 | 6.7 | 277.62 | 98.8 | 0.44 | 94.6 |
AB007 | HSA | 4-1BB | 서열번호 19 | 2.5 | 185.52 | 80.7 | 0.49 | 93.0 |
AD001 | HSA | KRAS G12D | 서열번호 20 | 22.0 | 8.40 | 94.1 | 8.12 | 97.3 |
AD002 | HSA | KRAS G12D | 서열번호 21 | 58.62 | 1.30 | 96.7 | 0.55 | 93.6 |
AD003 | HSA | KRAS G12D | 서열번호 22 | 49.81 | 13.01 | 91.2 | 13.79 | 91.3 |
AD004 | HSA | KRAS G12D | 서열번호 23 | 72이상 | 190.44 | 89.7 | 16.59 | 97.4 |
AD005 | HSA | KRAS G12D | 서열번호 24 | 30.1 | 121.90 | 94.9 | 22.92 | 92.8 |
AD006 | HSA | KRAS G12D | 서열번호 25 | 17.4 | 938.84 | 75.9 | 77.56 | 93.3 |
AD007 | HSA | KRAS G12D | 서열번호 26 | 12.1 | 535.83 | 97.0 | 151.68 | 98.4 |
AD008 | HSA | KRAS G12D | 서열번호 27 | 17.2 | 66.86 | 96.6 | 286.65 | 97.1 |
AD009 | HSA | KRAS G12D | 서열번호 28 | 72이상 | 0.65 | 82.6 | 0.51 | 82.7 |
AD010 | HSA | KRAS G12D | 서열번호 29 | 53.73 | 4.49 | 99.0 | 8.18 | 97.7 |
AD011 | HSA | KRAS G12D | 서열번호 30 | 72이상 | 5.46 | 99.0 | 9.46 | 97.6 |
AD012 | HSA | KRAS G12D | 서열번호 31 | 10.8 | 28.74 | 98.9 | 23.09 | 99.2 |
AD013 | HSA | KRAS G12D | 서열번호 32 | 11.12 | 47.02 | 99.7 | 3.99 | 98 |
AD014 | HSA | KRAS G12D | 서열번호 33 | 10.89 | 28.78 | 98.6 | 1.95 | 95 |
AD015 | HSA | KRAS G12D | 서열번호 34 | 10.65 | 36.84 | 98.6 | N/A | N/A |
AD032 | HSA | KRAS G12D | 서열번호 42 | 72이상 | 0.68 | 96.3 | 15.51 | 95.0 |
AD043 | HSA | KRAS G12D | 서열번호 162 | 72이상 | 4.18 | 98.3 | 263.23 | 79.0 |
AD044 | HSA | KRAS G12D | 서열번호 163 | 48.7 | 2.93 | 98.8 | 1577.76 | 80.5 |
AD045 | HSA | KRAS G12D | 서열번호 164 | 45.53 | 0.73 | 99.6 | 20.03 | 97.0 |
AD046 | HSA | KRAS G12D | 서열번호 165 | 18.26 | 0.65 | 94.9 | 5.38 | 88.0 |
AD050 | HSA | KRAS G12D | 서열번호 169 | 72이상 | 5.55 | 92.1 | 454.57 | 95.0 |
AD051 | HSA | KRAS G12D | 서열번호 170 | 34.0 | 5.95 | 97.4 | 514.12 | 94.5 |
AD052 | HSA | KRAS G12D | 서열번호 171 | 14.29 | 3.06 | 97.7 | 4.93 | 95.0 |
AD053 | HSA | KRAS G12D | 서열번호 172 | 72.00 | 1.37 | 99.4 | 1.03 | 93.7 |
AD034 | HSA | KRAS G12D | 서열번호 43 | 9.3 | 0.59 | 96.9 | 472.54 | 91.1 |
AV001 | HSA | KRAS G12V | 서열번호 119 | 72이상 | 11.21 | 80.8 | 58.01 | 93.3 |
TD001 | TF | KRAS G12D | 서열번호 149 | 72이상 | No binding | No binding | N/A | N/A |
TD003 | TF | KRAS G12D | 서열번호 151 | 13.30 | 141.29 | 78.5 | 2720.49 | 94.2 |
TD005 | TF | KRAS G12D | 서열번호 153 | 72.00 | No binding | No binding | No binding | No binding |
TD006 | TF | KRAS G12D | 서열번호 154 | 72.00 | 967.50 | 82.3 | 628.34 | 91.2 |
TD007 | TF | KRAS G12D | 서열번호 155 | 7.5 | 431.36 | 84.0 | 2118.95 | 84.3 |
TD009 | TF | KRAS G12D | 서열번호 157 | 11.745 | No binding | No binding | 2481.58 | 92.6 |
TD012 | TF | KRAS G12D | 서열번호 160 | 18.10 | 14.92 | 84.0 | 1311.19 | 96.1 |
TD013 | TF | KRAS G12D | 서열번호 161 | 14.0 | 645.60 | 92 | 1545.48 | 94.0 |
KRAS WT | KRAS G12D | KRAS G12C | KRAS G12V | |
실험 1 | No Binding | 35.50 nM | - | No Binding |
실험 2 | No Binding | 22.92 nM | 2.50 nM | No Binding |
Claims (49)
- 서로 다른 2개 이상의 타겟에 대한 특이적인 결합 친화성을 가지는 핵산 리간드로서,상기 각각의 타겟은 3차원 구조를 가지는 것이고,복수의 압타머의 연결체가 아닌(non-coupling),비천연 핵산 리간드.
- 서로 다른 2개 이상의 타겟에 대한 특이적인 결합 친화성을 가지는 핵산 리간드로서,상기 각각의 타겟은 3차원 구조를 가지는 것이고,상기 핵산 리간드에서 하나의 타겟에 대한 결합 부위를 형성하는 핵산 서열의 전부 또는 일부가 다른 타겟에 대한 결합 부위를 형성하는 핵산 서열의 전부 또는 일부를 형성하는 것인,비천연 핵산 리간드.
- 서로 다른 2개 이상의 타겟에 대한 특이적인 결합 친화성을 가지는 핵산 리간드로서,상기 각각의 타겟은 3차원 구조를 가지는 것이고,상기 핵산 리간드 중 2개 이상의 타겟에 대한 결합 부위가 서로 별개로 분리되어 존재하지 않는,비천연 핵산 리간드.
- 서로 다른 2개 이상의 타겟에 대한 특이적인 결합 친화성을 가지는 핵산 리간드로서,상기 각각의 타겟은 3차원 구조를 가지는 것이고,2개 이상의 타겟에 대한 결합부위가 하나의 단일 핵산 리간드에서 형성되는 것인,비천연 핵산 리간드.
- 제2항에 있어서,상기 타겟에 대한 결합 부위를 형성하는 핵산 서열의 일부는 비천연 핵산 리간드 전체 서열의 약 1% 내지 약 99%, 약 5% 내지 약 95%, 약 10% 내지 약 90%, 약 20% 내지 약 80%, 약 30% 내지 약 70%, 또는 약 40% 내지 약 60%인,비천연 핵산 리간드.
- 제1항 내지 제4항 중 어느 한 항에 있어서,서로 다른 2개의 타겟에 대해 특이적인 결합 친화성을 가지는,비천연 핵산 리간드.
- 제1항 내지 제4항 중 어느 한 항에 있어서,상기 타겟은 세포, 바이러스 및 단백질로 이루어진 군으로부터 선택된 것인,비천연 핵산 리간드.
- 제6항에 있어서,상기 타겟 중 어느 하나는 혈장 단백질이고, 다른 하나는 치료적 타겟인,비천연 핵산 리간드.
- 제8항에 있어서,상기 혈장 단백질은 알부민, 알파1 글로불린, 알파2 글로불린, 베타글로불린, 감마글로불린, 면역글로불린 A, 면역글로불린 G, 지질단백질, 피브리노겐, 트랜스페린 및 트렌스디레틴으로 이루어진 군에서 선택되는 것인,비천연 핵산 리간드.
- 제8항에 있어서,상기 치료적 타겟은 세포내에 존재하는 단백질 또는 세포막에 존재하는 단백질인,비천연 핵산 리간드.
- 제8항에 있어서,상기 치료적 타겟은 막 단백질, 막관통 단백질, 당 단백질, 면역 항체, 바이러스, 바이러스 외피 당 단백질, 바이러스 효소, 분비 단백질, 세린 단백질 가수분해 효소, 펩타이드 호르몬, 신경 전달 물질, 호르몬, 탈수소효소, 사이토카인, E3 유비퀴틴 리가아제, 신경 펩타이드, 가수분해효소, 세린단백질가수분해효소 억제제, 인간 가수분해 효소, 케모카인 단백질, 메틸 전환 효소, 산화효소, 성장 인자, 세균, 세균성 단백질, 세포내 단백질, 세포외 기질, 수용체, 전사인자, 및 종양 단백질로 이루어진 군에서 선택되는 것인,비천연 핵산 리간드.
- 제8항에 있어서,상기 치료적 타겟은 4-1BB, 아세틸콜린 수용체, 알파 트롬빈, 아밀린, 안지오포이에틴 1, 안지오포이에틴 2, AXL, BCL-2, BMPR-1R, BRD1, BRD2, BRD3, BRD4, BRDT, BTLA, 칼시토닌 유전자-관련 펩티드, CBP(CREB-결합 단백질), CCK4/PTK7, CD16a, CD16b, CD19, CD20, CD200, CD200R, CD27, CD28, CD3, CD30, CD32A, CD32B, CD33, CD4, CD40L, CD52, CD80, CD94, CSF1R, CTLA-4, DDR1, DDR2, E2F1, EGFR, EPH, ERBB2, FGF, FGFR, 그렐렌, GITR, 글리피칸3, 생식샘자극호르몬 분비호르몬 1, HIV gp120, HIV-1 인테그레이즈, HIV-1 역전사 효소, HRAS, HVEM, ICOS, IDH1, IGF1R, 면역글로불린 E, 인터페론-감마, KIT, KRAS, LAG3, LFA, L-셀렉틴, LTK, MDM2, MDMX, 뮤신 1, MYC, 뉴로텐신 1, 뉴트로필 엘라스테이즈, NF-Kb, NKG2D, 노시셉틴, NTRK1, NTRK2, OX-40, PD-1, PDGF, PDGFR 패밀리, PD-L1, PD-L2, 포스포리파아제 A2, 플라스미노겐 활성화 억제제 1, PP2A, PPM1D, PPP2CA, 단백질 티로신 인산가수분해효소, P-셀렉틴, PSMA, 호흡기 세포융합 바이러스, RET, SDF1b, SetDB1, SLAMF7, 스타필로코커스 엔테로톡신 B, STAT3, 테나신, TGFBR, TIGIT, TIM3, TNFSF7, 티로시나아제, VCAM-1, VEGF, VEGFR 패밀리, α vβ 3 인테그린, 및 이들의 돌연변이형으로 이루어진 군에서 선택되는 것인,비천연 핵산 리간드.
- 제8항에 있어서,상기 치료적 타겟은 KRAS G12D, KRAS G12V, KRAS G12C, KRAS G12A, KRAS G12S, KRAS G12R, KRAS G13D, KRAS Q61H 및 이들의 조합으로 이루어진 군으로부터 선택된 것인,비천연 핵산 리간드.
- 제1항 내지 제4항 중 어느 한 항에 있어서,약 100개 이하의 뉴클레오티드로 이루어지는,비천연 핵산 리간드.
- 제14항에 있어서,상기 뉴클레오티드는 DNA 뉴클레오티드, RNA 뉴클레오티드, 이들의 변형된 뉴클레오티드, 및 이들의 조합으로 이루어진 군으로부터 선택되는 것인,비천연 핵산 리간드.
- 제1항 내지 제4항 중 어느 한 항에 있어서,서열번호 6 내지 서열번호 183으로 이루어진 군으로부터 선택된 하나의 핵산 서열로 이루어진,비천연 핵산 리간드.
- 제1항 내지 제4항 중 어느 한 항에 있어서,상기 핵산은 단일 가닥 RNA, 이중 가닥 RNA, 단일 가닥 DNA 및 이중 가닥 DNA로 이루어진 군으로부터 선택된,비천연 핵산 리간드.
- 제1항 내지 제4항 중 어느 한 항에 따른 비천연 핵산 리간드를 포함하는,암 예방 또는 치료를 위한 제약 조성물.
- 제18항에 있어서,상기 비천연 핵산 리간드가 제1 타겟인 혈장 단백질 및 제2 타겟인 치료적 타겟 단백질에 특이적인 결합 친화성을 가지는 것인,암 예방 또는 치료를 위한 제약 조성물.
- 제18항에 있어서,환자에게 정맥 투여 되는 것인, 암 예방 또는 치료를 위한 제약 조성물.
- 제18항에 있어서,상기 암은 고형암인,암 예방 또는 치료를 위한 제약 조성물.
- 제18항에 있어서,상기 암은 암세포 표면에 PD-L1 단백질을 발현하는 것인,암 예방 또는 치료를 위한 제약 조성물.
- 제22항에 있어서,상기 암은 뇌종양, 폐암, 대장암, 소장암, 위암, 고환암, 갑상선암, 자궁경부암, 피부암, 방광암, 난소암, 신장암, 간암, 췌장암, 요로상피세포암, 및 유방암으로 이루어진 군으로부터 선택되는 하나 이상인,암 예방 또는 치료를 위한 제약 조성물.
- 제18항에 있어서,상기 암은 암세포가 KRAS 돌연변이 단백질을 가지는 것인,암 예방 또는 치료를 위한 제약 조성물.
- 제24항에 있어서,상기 암은 폐암, 대장암, 췌장암, 유방암, 난소암, 자궁내막암, 자궁경부암, 방광암, 담낭 담도암, 피부암, 위암, 뇌종양, 신장암 및 급성골수성백혈병으로 이루어진 군으로부터 선택되는 하나 이상인,암 예방 또는 치료를 위한 제약 조성물.
- 제18항에 있어서,상기 비천연 핵산 리간드는 서열번호 24, 서열번호 27, 서열번호 30, 서열번호 84, 및 서열번호 100로 이루어진 군으로부터 선택된 하나의 핵산 서열로 이루어진 핵산 리간드인,암 예방 또는 치료를 위한 제약 조성물.
- 제1항 내지 제4항 중 어느 한 항에 따른 비천연 핵산 리간드를 포함하는 진단용 조성물.
- 제1항 내지 4항 중 어느 한 항에 따른 비천연 핵산 리간드를 포함하는 조영제.
- 제1항 내지 제4항 중 어느 한 항에 따른 비천연 핵산 리간드를 포함하는 방사성 의약품.
- 제1항 내지 제4항 중 어느 한 항에 따른 비천연 핵산 리간드를 포함하는,암 진단을 위한 조성물.
- 하나 이상의 핵산 리간드 후보 서열을 서로 다른 2개 이상의 타겟과 순차적으로 접촉시키는 단계를 포함하고,상기 각 단계는 타겟과 특이적인 결합 친화성을 가지는 핵산 리간드 후보 서열을 식별하는 것을 포함하고,상기 각 타겟은 3차원 구조를 가지는 것인,서로 다른 2개 이상의 타겟에 대해 특이적인 결합 친화성을 가지는 비천연 핵산 리간드를 식별하는 방법.
- 제31항에 있어서,상기 방법은(a) 하나 이상의 제1 핵산 리간드 후보 서열을 타겟과 접촉시키고, 상기 타겟과 특이적으로 결합하는 하나 이상의 제2 핵산 리간드 후보 서열을 식별하는 단계; 및(b) (i) 단계 (a)로부터 얻어진 하나 이상의 제2 핵산 리간드 후보 서열 또는 (ii) 단계 (a)로부터 얻어진 하나 이상의 제2 핵산 리간드 후보 서열을 이용하여 제조된 하나 이상의 제3 핵산 리간드 후보 서열을 단계 (a)와 상이한 타겟과 접촉시키고, 상기 상이한 타겟과 특이적으로 결합하는 하나 이상의 제4 핵산 리간드 후보 서열을 식별하는 단계를 포함하는 것인,비천연 핵산 리간드를 식별하는 방법.
- 제32항에 있어서,상기 단계 (b)는단계 (a)로부터 얻어진 하나 이상의 제2 핵산 리간드 후보 서열의 양 말단 중 하나 이상에 복수개의 뉴클레오티드로 이루어진 랜덤 서열을 결합시켜 하나 이상의 제3 핵산 리간드 후보 서열을 제조하는 단계를 더 포함하는 것인,비천연 핵산 리간드를 식별하는 방법.
- 제33항에 있어서,상기 랜덤 서열이 상기 제2 핵산 리간드 후보 서열의 양 말단 중 어느 하나에 결합되고,상기 제3 핵산 리간드 후보 서열에서 상기 제2 핵산 리간드 후보 서열 또는 그 일부가 핵산의 증폭을 위한 프라이머 서열로 사용되는 것인,비천연 핵산 리간드를 식별하는 방법.
- 제33항에 있어서,상기 랜덤 서열은 약 20개 내지 약 40개의 연속적인 뉴클레오티드로 이루어지거나, 또는상기 제3 핵산 리간드 후보 서열은 약 40 내지 약 100개의 연속적인 뉴클레오티드로 이루어진,비천연 핵산 리간드를 식별하는 방법.
- 제31항에 있어서,상기 방법은 SELEX(Systematic evolution of ligands by exponential enrichment)로 수행되는 것인,비천연 핵산 리간드를 식별하는 방법.
- 하나의 핵산 라이브러리로부터 출발하여 SELEX(Systematic evolution of ligands by exponential enrichment)에 의해 핵산 리간드를 식별하는 방법으로서,상기 방법은 각 SELEX 라운드에서 3차원 구조를 가지는 서로 다른 2개 이상의 타겟으로 이루어진 군으로부터 하나의 타겟을 선택하는 단계 및각 SELEX 라운드에서 상기 선택된 타겟에 특이적인 결합 친화성을 가지는 핵산 리간드를 선별하는 단계를 포함하는,서로 다른 2개 이상의 타겟에 대해 특이적인 결합 친화성을 가지는 비천연 핵산 리간드를 식별하는 방법.
- 제36항 또는 제37항에 있어서,하나의 타겟에 대해 1회 이상의 라운드로 수행되는 SELEX를 완료한 뒤 다른 타겟에 대한 1회 이상의 라운드로 수행되는 SELEX를 수행하는 것인,비천연 핵산 리간드를 식별하는 방법.
- 제36항 또는 제37항에 있어서,(i) 하나의 타겟에 대해 1회 이상의 SELEX 라운드를 수행하는 단계;(ii) 단계 (i)로부터 수득된 핵산 라이브러리를 이용하여 단계 (i)의 타겟과 다른 타겟에 대해 1회 이상의 SELEX 라운드를 수행하는 단계;(iii) 단계 (ii)로부터 수득된 핵산 라이브러리를 이용하여 다시 단계 (i)을 수행하는 단계; 및(iv) 단계 (i) 내지 (iii)을 반복하는 단계를 포함하는 것인,비천연 핵산 리간드를 식별하는 방법.
- 제36항 또는 제37항에 있어서,타겟의 다른 형질에 대한 네거티브 SELEX 또는 카운트 SELEX를 수행하는 단계를 더 포함하는,비천연 핵산 리간드를 식별하는 방법.
- 제3436 또는 제37항에 있어서,상기 SELEX는 고전적 SELEX, Advanced SELEX, IP-SELEX, Capture-SELEX, Cell-SELEX, CE-SELEX, M-SELEX, AFM-SELEX, AEGIS-SELEX, 및 Animal-SELEX로 이루어진 군으로부터 선택된 하나 이상인,비천연 핵산 리간드를 식별하는 방법.
- 제31항 내지 제37항 중 어느 한 항에 있어서,식별된 핵산 리간드 후보 서열의 타겟에 대한 특이적 결합 친화성을 평가하는 단계를 더 포함하는,비천연 핵산 리간드를 식별하는 방법.
- 제31항 내지 제37항 중 어느 한 항에 있어서,상기 타겟은 세포, 바이러스 및 단백질로 이루어진 군으로부터 선택된 것인,비천연 핵산 리간드를 식별하는 방법.
- 제31항 내지 제37항 중 어느 한 항에 있어서,상기 타겟이 서로 다른 2개의 타겟인비천연 핵산 리간드를 식별하는 방법.
- 제44항에 있어서,상기 타겟 중 어느 하나는 혈장 단백질이고, 다른 하나는 치료적 타겟인,비천연 핵산 리간드를 식별하는 방법.
- 제31항 내지 제37항 중 어느 한 항에 있어서,상기 비천연 핵산 리간드는 약 40개 내지 약 100개의 뉴클레오티드로 이루어지는 것인,비천연 핵산 리간드를 식별하는 방법.
- 제31항 내지 제37항 중 어느 한 항에 있어서,상기 식별된 핵산 리간드 후보 서열에서, 타겟에 대한 특이적인 결합 친화성에 관여하지 않은 뉴클레오티드를 제거하는 단계를 포함하는,비천연 핵산 리간드를 식별하는 방법.
- 샘플에서 타겟을 검출하는 방법으로서,상기 샘플로부터의 타겟을 제1항 내지 제4항 중 어느 한 항의 핵산 리간드와 접촉시키는 것을 포함하는 방법.
- 제31항 내지 제37항 중 어느 한 항의 방법을 포함하는 방법으로 제조된 비천연 핵산 리간드.
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