WO2024038918A1 - Aptamère de liaison à un anticorps simple brin - Google Patents

Aptamère de liaison à un anticorps simple brin Download PDF

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WO2024038918A1
WO2024038918A1 PCT/JP2023/030053 JP2023030053W WO2024038918A1 WO 2024038918 A1 WO2024038918 A1 WO 2024038918A1 JP 2023030053 W JP2023030053 W JP 2023030053W WO 2024038918 A1 WO2024038918 A1 WO 2024038918A1
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scfv
aptamer
seq
ggggs
peptide linker
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PCT/JP2023/030053
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Japanese (ja)
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竜太郎 浅野
一典 池袋
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国立大学法人東京農工大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • the present invention relates to an aptamer that specifically binds to a single chain antibody.
  • scFv also called single-chain Fv or single-chain antibody
  • scFvs are stabilized with peptide linkers, can be prepared using microorganisms, and are advantageously used as pharmaceuticals and sensing elements.
  • blinatumomab a bispecific antibody whose constituent unit is scFv, has already been approved in Japan.
  • scFv is a small molecule, it has a short half-life in the body, and pharmacokinetic analysis is difficult because there is no method to specifically detect the administered scFv. Additionally, there are concerns about antigenicity with the histidine tags often used for purification in scFvs. Therefore, only a few pharmaceutical products using scFv have been approved in Japan.
  • scFv can be used as a molecular target drug by being modified with a medical drug such as an anticancer drug, or as a detection element by being modified (labeled) with a labeling substance such as a fluorescent substance.
  • Modification of proteins such as scFv usually requires the use of amino coupling via lysine residues or thioether bonding via cysteine residues. In the former case, many lysine residues are commonly present on the protein surface, so there is concern that protein function may be reduced due to modification, and the amino group at the N-terminus is also modified, resulting in Application to scFvs with an antigen-binding site is less suitable.
  • Non-patent Document 1 discloses an aptamer that binds to the Fc region of an IgG antibody, such an aptamer cannot be used to detect scFv that does not have an Fc region.
  • An object of the present invention is to provide an aptamer that specifically binds to a single chain antibody scFv.
  • scFv single-chain Fv
  • GGGGS peptide linker
  • the present invention includes the following.
  • scFv single chain Fv
  • GGGGS peptide linker
  • the oligonucleotide is at least selected from the group consisting of biotin, a thiol group, polyethylene glycol, a fluorescent substance, a redox probe, a radioisotope, an enzyme, a carrier, an electron carrier, a cytotoxic substance, and a medical drug.
  • [6] The aptamer according to any one of [1] to [5] above, wherein the oligonucleotide is DNA or RNA.
  • GGGGS peptide linker
  • [10] Contacting the aptamer according to any one of [1] to [6] above with a sample containing scFv to generate an scFv-aptamer complex, and separating the scFv-aptamer complex from the sample.
  • a method for purifying scFv having a peptide linker (GGGGS) 3 comprising: [11] A peptide linker ( A method for detecting scFv having GGGGS) 3 (SEQ ID NO: 3). [12] Introducing nucleotide mutations into the aptamer described in [2] above, measuring the binding ability of the resulting mutant to scFv having a peptide linker (GGGGS) 3 (SEQ ID NO: 3), and 1.
  • a method for screening an aptamer comprising an oligonucleotide that specifically binds to an scFv having a peptide linker (GGGGS) 3 (SEQ ID NO: 3), the method comprising selecting as an aptamer a mutant that specifically binds to the scFv. [13] Contacting the aptamer according to any one of [1] to [6] above with an scFv having a peptide linker (GGGGS) 3 (SEQ ID NO: 3) to generate an scFv-aptamer complex, How to qualify scFv.
  • scFv-aptamer complex comprising an scFv having a peptide linker (GGGGS) 3 (SEQ ID NO: 3) bound to the aptamer according to any one of [1] to [6] above.
  • scFv single chain Fv having peptide linker
  • FIG. 1 schematically shows vector maps of pRA-528 scFv (A) and pRA-528 Fv (B).
  • Figure 2 shows the protein coding sequence in pRA-528 scFv (SEQ ID NO: 1) and the amino acid sequence encoded thereby (SEQ ID NO: 2). Each region from pelB to the His tag shown in the vector map of FIG. 1A is shown relative to the sequence in FIG. 2.
  • Figure 3 is a photograph showing the results of Ni-NTA (nickel-nitrilotriacetic acid) affinity chromatography (column chromatography) of a 528 scFv sample at different stages of purification.
  • Ni-NTA nickel-nitrilotriacetic acid
  • the samples applied to each lane are ordered from left to right: molecular weight marker, 1: before filtration, 2: before column chromatography, 3: column chromatography flow-through solution, 4: washing solution, 5: 50 mM imidazole. /PBS, 6: 150mM imidazole/PBS, 7: 300mM imidazole/PBS-1, 8: 300mM imidazole/PBS-2, and 9: 500mM imidazole/PBS.
  • the samples applied to each lane were, in order from left to right, molecular weight marker, 1: before filtration, 2: before column chromatography, 3: column chromatography flow-through solution, 4: washing solution, 5: 50 mM imidazole. /PBS, 6: 150mM imidazole/PBS, 7: 300mM imidazole/PBS-1, 8: 300mM imidazole/PBS-2, and 9: 500mM imidazole/PBS.
  • Figure 6 shows a chromatogram obtained by gel filtration chromatography of purified 528 Fv.
  • FIG. 7 is a graph showing the results of an evaluation test of the binding ability of aptamer candidate sequences to scFv by ELONA.
  • FIG. 8 shows the results of an evaluation test of the binding ability of aptamer candidate sequences P2-63 and P1-12 to scFv by dot blot analysis.
  • A shows binding to 20 pmol of antibody protein scFv, Fv, and bevacizumab;
  • B shows binding to 20 pmol, 10 pmol, and 5 pmol of antibody protein scFv and Fv.
  • FIG. 9 is a sensorgram showing the results of an evaluation test of the binding ability of aptamer candidate sequences P2-63 and P1-12 to scFv by BLI.
  • A shows changes in the binding and dissociation states of P2-63 and P1-12 to scFv and Fv.
  • Figure 10 shows the results of evaluating the binding ability of aptamers to 528 scFv, 528 Fv, and anti-Hb scFv-SpyTag. From left to right, 528 scFv, anti-Hb scFv-SpyTag, 528 Fv, and frozen-thawed 528 Fv spots are shown.
  • FIG. 11 shows the results of measuring the antigen-binding activity of scFv bound to an aptamer by ELISA using an HRP-modified anti-c-Myc antibody.
  • Figure 12 shows concentration-dependent binding of aptamer (P1-12) to 528 scFv bound to sEGFR on the plate.
  • FIG. 13 shows the results of CD spectrum measurement of the aptamer.
  • A shows the results of P1-12
  • B shows the results of P2-63.
  • Figure 14 schematically shows vector maps of scFv expression vectors pRA-anti-Hb scFv-SpyTag (A), pRA-anti-Hb scFv-SnT (B), and pRA-HB125 scFv (C).
  • Figure 15 shows the protein coding sequence (SEQ ID NO: 29) and the amino acid sequence encoded thereby (SEQ ID NO: 30) in pRA-anti-Hb scFv-SpyTag. Each region from pelB to the His tag shown in the vector map of FIG. 14A is shown relative to the sequence in FIG. 15.
  • Figure 16 shows the protein coding sequence (SEQ ID NO: 31) and the amino acid sequence encoded thereby (SEQ ID NO: 32) in pRA-anti-Hb scFv-SnT. Each region from pelB to the His tag shown in the vector map of FIG. 14B is shown relative to the sequence in FIG. 16.
  • Figure 17 shows the protein coding sequence (SEQ ID NO: 33) and the amino acid sequence encoded thereby (SEQ ID NO: 34) in pRA-HB125 scFv. Each region from pelB to the His tag shown in the vector map of FIG. 14C is shown relative to the sequence in FIG. 17.
  • FIG. 18 shows the results of measuring the antigen binding activity of anti-Hb scFv-SnT antibody in the aptamer-anti-Hb scFv-SnT antibody conjugate by ELISA.
  • the vertical axis shows chemiluminescence intensity, and the horizontal axis shows aptamer concentration.
  • Figure 19 shows the results of SWV measurements on the binding of different concentrations of svFv to aptamers on electrodes with immobilized aptamers P1-12 or P2-63.
  • FIG. 20 shows changes in peak current values measured by SWV with respect to scFv concentration.
  • FIG. 21 shows the results of SWV measurements using 10-fold dilutions for binding of different concentrations of svFv to aptamers on electrodes with immobilized aptamers P1-12 or P2-63.
  • FIG. 22 shows changes in peak current values measured by SWV using a 10-fold diluted solution with respect to scFv concentration.
  • the present invention relates to aptamers and their uses for single chain Fv (scFv), in particular for scFv with a peptide linker (GGGGS) 3 (SEQ ID NO: 3).
  • an "aptamer” refers to a single-stranded nucleic acid that specifically binds to a target substance, or a ligand containing the single-stranded nucleic acid.
  • the nucleic acids that make up aptamers can be produced relatively inexpensively through chemical synthesis, can be stored and transported at room temperature in a dry state, and can be easily chemically modified, which are advantages that antibodies, which are proteins, do not have. .
  • the invention relates to an aptamer comprising an oligonucleotide that specifically binds to a single chain Fv (scFv) with a peptide linker (GGGGS) 3 (SEQ ID NO: 3).
  • Natural antibodies usually have a Y-shaped structure in which two heavy chains and two light chains are linked by disulfide bonds as a basic unit, and 1 to 5 of these structures are assembled to form a single molecule (immunoglobulin molecule). ) and is characterized by having a unique antigen-binding activity.
  • the heavy chain (H chain) and light chain (L chain) of antibodies each have a variable region (heavy chain variable domain, light chain variable domain) at their N-terminus, and the heavy chain variable domain and light chain variable domain Together they form the antigen binding site.
  • Each heavy and light chain variable domain contains three complementarity determining regions (CDRs) (CDR1-CDR3) and four framework regions (FR1-FR4), with six sets of CDRs each It determines the specificity of an antibody to its antigen.
  • CDRs are also called hypervariable regions and have large sequence diversity, but framework region sequences are highly conserved.
  • Framework regions FR1 to FR4 are located alternately with CDR1 to CDR3 in order from the N-terminal side of each heavy chain and light chain variable domain, and FR4 is present at the C-terminus of each.
  • a constant region (CH1 to CH3, CL) with high sequence conservation exists adjacent to the C-terminus of the heavy chain and light chain variable domains.
  • a single chain Fv is a single chain polypeptide made by linking the heavy chain variable domain and light chain variable domain with a peptide linker, and is also called a single chain antibody (single chain antibody fragment). ing.
  • Peptide linker (GGGGS) 3 (SEQ ID NO: 3), also called GS linker, is widely used as a peptide linker for scFv.
  • An scFv with (GGGGS) 3 as a peptide linker typically has a heavy chain variable domain and a light chain variable domain with a peptide linker at their termini (usually the C-terminus of the heavy chain variable domain and the N-terminus of the light chain variable domain).
  • (GGGGS) 3 Peptide linker (GGGGS) 3 is a peptide consisting of the amino acid sequence shown by SEQ ID NO: 3.
  • a polypeptide sequence in which a heavy chain variable domain and a light chain variable domain are linked via a peptide linker (GGGGS) 3 at their ends means that the heavy chain variable domain and the light chain variable domain are linked at their ends. Not only are the polypeptide sequences linked directly to the amino acid sequence (GGGGS) 3 at their termini, but also the heavy and light chain variable domains are linked at their termini via the amino acid sequence (GGGGS) 3 .
  • scFv having a peptide linker (GGGGS) 3 refers to an scFv having a peptide linker consisting of the amino acid sequence (GGGGS) 3 , as well as a peptide linker having an additional amino acid sequence in addition to the amino acid sequence (GGGGS) 3 .
  • scFvs with any peptide linker comprising the amino acid sequence (GGGGS) 3 , such as scFvs.
  • peptide linkers include, but are not limited to, at least 15 amino acids, preferably 15-50 amino acids, such as 15-40 amino acids, 15-35 amino acids, 15-30 amino acids, 15-20 amino acids, or It may consist of 15 amino acids.
  • the present invention relates to an aptamer using an oligonucleotide capable of specifically recognizing and binding to an scFv having (GGGGS) 3 as a peptide linker.
  • the scFv to which the aptamer of the present invention binds may be derived from a mammalian antibody or a recombinant antibody having a framework region thereof.
  • the scFv to which the aptamer of the present invention binds is preferably, but not limited to, one derived from a human antibody or a humanized antibody.
  • the scFv to which the aptamer of the invention binds can be derived from IgG, IgA, IgE, IgD, or IgM, but is typically derived from IgG.
  • the scFv to which the aptamer of the present invention binds is an scFv having a peptide linker (GGGGS) 3. Specifically, the heavy chain variable domain and light chain variable domain of an antibody are linked via a peptide linker (GGGGS) 3 .
  • the scFv to which the aptamer of the present invention binds is a mammalian antibody (e.g., a primate antibody such as a human antibody, a rodent antibody such as a mouse antibody) or a recombinant antibody having a framework region thereof (e.g., a humanized antibody).
  • a mammalian antibody e.g., a primate antibody such as a human antibody, a rodent antibody such as a mouse antibody
  • a recombinant antibody having a framework region thereof e.g., a humanized antibody
  • the derived heavy chain variable domain and light chain variable domain may comprise polypeptide sequences linked via a peptide linker (GGGGS) 3 .
  • the scFv to which the aptamer of the invention binds contains or is conjugated to other polypeptides such as tag sequences (including, but not limited to, Spy tag, C-myc tag, His tag, and/or Snoop tag). It's okay.
  • tag sequences including, but not limited to, Spy tag, C-myc tag, His tag, and/or Snoop tag. It's okay.
  • Examples of scFv having a peptide linker (GGGGS) 3 to which the aptamer of the present invention binds include polypeptides (scFv) comprising the amino acid sequence shown in SEQ ID NO: 2, 30, 32, or 34, and SEQ ID NO: 2, 30.
  • scFv amino acid sequence at positions 1 to 22
  • tag sequences Spy tag, C-myc Examples include, but are not limited to, polypeptides (scFv) that include an amino acid sequence excluding the amino acid sequence (scFv), His tag, and/or Snoop tag.
  • aptamer of the present invention “specifically binds" to a target (an scFv having a peptide linker (GGGGS) 3 , particularly a peptide linker (GGGGS) 3 region in an scFv having a peptide linker (GGGGS) 3 ). It means having the ability to bind selectively and with sufficiently high affinity to maintain stable binding.
  • the aptamers of the invention are able to bind selectively (with higher affinity) to scFv with a peptide linker (GGGGS) 3 compared to Fv.
  • the aptamer of the present invention comprises an oligonucleotide that specifically binds to a single-stranded Fv (scFv) having a peptide linker (GGGGS) 3 (SEQ ID NO: 3), preferably as a single-stranded nucleic acid component (typically the only one). (as a nucleic acid component).
  • oligonucleotide refers to a nucleotide polymer (polynucleotide) of 80 bases or less in length.
  • the oligonucleotide constituting the aptamer of the present invention has a length of 80 bases or less, preferably 50 bases or less, preferably 20 bases or more, more preferably 30 to 40 bases, still more preferably 30 to 37 bases, e.g.
  • the oligonucleotide can be 30-35 bases long, 30-33 bases long, or 33-35 bases long.
  • the oligonucleotide that specifically binds to the above-mentioned single-stranded Fv (scFv) constituting the aptamer of the present invention has a base sequence represented by SEQ ID NO: 14 or 24 (in particular, the entire base sequence). It may contain a base sequence having a sequence identity of 60% or more, preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, for example 95% or more.
  • the oligonucleotide constituting the aptamer of the present invention includes or consists of the base sequence shown in SEQ ID NO: 14 or 24.
  • oligonucleotides may be included at the 5' end (half of the 5' end) and/or at the 3' end (half of the 3' end), for example, at or near the 5' end (1 or 2 of the 5' end). may be included in Such oligonucleotides further preferably contain 60% or more, preferably 70% or more, more preferably 80% or more, even more preferably It may contain a base sequence having sequence identity of 90% or more, for example 95% or more.
  • the above oligonucleotide constituting the aptamer of the present invention may be DNA, RNA, or a hybrid thereof.
  • sequence of SEQ ID NO: 14 or 24 is described as a DNA sequence, but if the oligonucleotide constituting the aptamer of the present invention is RNA or a hybrid of DNA and RNA, the RNA or RNA Regarding the part, "t" (thymine) in the base sequence shown by SEQ ID NO: 14 or 24 in the sequence listing shall be read as "u" (uracil).
  • the oligonucleotide constituting the aptamer of the present invention contains a base sequence in which "t" (thymine) in the base sequence shown by SEQ ID NO: 14 or 24 in the sequence listing is replaced with "u” (uracil).
  • t thymine
  • u uracil
  • the oligonucleotide may be chemically modified. However, the chemical modification does not eliminate the ability of the oligonucleotide to specifically bind to the scFv with the peptide linker (GGGGS) 3 .
  • the oligonucleotide may be labeled, typically with a labeling substance that allows detection, and such a label is also included in chemical modification.
  • the oligonucleotide may be bound to other substances (eg, non-nucleic acid substances), and such cases are also included in chemical modification.
  • the oligonucleotides constituting the aptamer of the present invention include, for example, low-molecular label molecules such as biotin, desthiobiotin, avidin, streptavidin, and neutravidin, phosphate groups, phosphorothioate groups, thiol groups, amino groups, aminoallyl groups, etc.
  • biocompatible polymers such as polyethylene glycol (PEG) and its derivatives, fluorescein (FAM) and its derivatives, various Alexa Fluor (R) dyes, rhodamine derivatives such as carboxytetramethylrhodamine (TAMRA), Cy3 and Cy5 fluorescent substances including fluorescent dyes such as cyanine dyes, quenchers used in combination with fluorescent substances, redox probes such as methylene blue and chlorogenic acid, radioactive isotopes such as yttrium -90 , indium -111 , and iodine -131 , and hoses.
  • PEG polyethylene glycol
  • FAM fluorescein
  • R Alexa Fluor
  • Cy3 and Cy5 fluorescent substances including fluorescent dyes such as cyanine dyes, quenchers used in combination with fluorescent substances, redox probes such as methylene blue and chlorogenic
  • Enzymes including labeled enzymes such as radish peroxidase (HRP), carriers such as magnetic beads, gold particles, latex particles, nanofibers, electron carriers such as phenazine methosulfate (PMS), or drugs (e.g. anticancer with one or more arbitrary substances or chemical structural moieties (atoms or atomic groups such as functional groups, etc.) It may be chemically modified by being bonded or introduced.
  • Substances that enable detection such as the above-mentioned low-molecular labeling molecules, fluorescent substances, quenchers, labeled enzymes, and radioactive isotopes, can be used as labeling substances to label oligonucleotides.
  • the oligonucleotide constituting the aptamer of the present invention may contain at least one of a modified nucleotide and an artificial base, and in that case, it is chemically modified by containing it.
  • a "modified nucleotide” is a nucleotide in which the sugar moiety, phosphate moiety, and/or base moiety of a natural nucleotide is chemically modified.
  • a modified nucleotide may be a nucleotide containing a modified nucleoside.
  • modified nucleotides include halogenated bases such as 2'-fluorinated pyrimidine, methylated bases such as 2'-O-methylated bases, modified nucleosides such as deoxyuridine, inosine, and 2-amino-deoxyadenosine. These include, but are not limited to, nucleotides containing nucleotides. When the oligonucleotide constituting the aptamer of the present invention contains a modified nucleotide, the modified nucleotide may be present in place of a corresponding natural (ie, unmodified) nucleotide.
  • the oligonucleotide in the aptamer of the present invention includes the base sequence shown by SEQ ID NO: 14 or 24 and also includes a modified nucleotide
  • the modified nucleotide corresponds to the base sequence shown by SEQ ID NO: 14 or 24.
  • the oligonucleotide comprising the base sequence shown by SEQ ID NO: 14 or 24.
  • the oligonucleotide comprising the base sequence shown by SEQ ID NO: 14 or 24.
  • the term "artificial base” refers to a nucleobase that is neither a natural base nor a chemically modified natural base, such as 7-(2-thienyl), which can be used in combination as an artificial base pair.
  • the oligonucleotide constituting the aptamer of the present invention contains an artificial base
  • the artificial base may be present at any position in the oligonucleotide.
  • the oligonucleotide constituting the aptamer of the present invention contains the base sequence shown by SEQ ID NO: 14 or 24 and also contains an artificial base
  • the artificial base may be any one of the base sequences shown by SEQ ID NO: 14 or 24.
  • the oligonucleotide may be inserted at the position shown in SEQ ID NO: 14 or 24.
  • biomolecules such as "chemically modified” or “modified” oligonucleotides, nucleotides, nucleosides, etc. are not limited to those obtained by modifying biomolecules after production, but are in a modified state. It also includes biomolecules produced in
  • the oligonucleotide constituting the aptamer of the present invention is preferably guanine-rich and capable of forming a guanine quadruplex (G4) structure, and is capable of forming a parallel guanine quadruplex (G4) structure. is even more preferable.
  • oligonucleotides that have formed a parallel G4 structure generally exhibit a positive peak near 260 to 270 nm (e.g., 265 nm), which is characteristic of a parallel G4 structure, and a positive peak at 235 to 245 nm (e.g., 240 nm). A negative peak is shown nearby.
  • the aptamer of the invention is capable of binding to an scFv with a peptide linker (GGGGS) 3 while forming a guanine quadruplex (G4) structure, in particular a parallel guanine quadruplex (G4) structure. .
  • oligonucleotides constituting the aptamer of the present invention can be synthesized by those skilled in the art using conventional methods, for example, using an automatic nucleic acid synthesizer.
  • the aptamer of the present invention can specifically bind to an scFv having a peptide linker (GGGGS) 3 through the oligonucleotide constituting it, and preferably specifically binds to a structure containing a peptide linker (GGGGS) 3 of an scFv. Can be combined.
  • the aptamer of the present invention can also bind to scFvs having different antigen binding properties, and preferably has a more general binding ability to scFvs.
  • the aptamer of the present invention can specifically bind to various scFvs having a peptide linker (GGGGS) 3 without inhibiting their antigen binding activity.
  • the aptamer of the present invention can modify an scFv having a peptide linker (GGGGS) by specifically binding to the scFv.
  • the aptamer of the present invention can bind to an scFv having a peptide linker (GGGGS) 3 to generate (form) an scFv-aptamer complex (scFv-aptamer conjugate).
  • the present invention also provides a kit for the detection, modification or purification of scFv having a peptide linker (GGGGS) 3 (SEQ ID NO: 3), which includes the aptamer of the present invention.
  • the scFv detection, modification, or purification kit of the present invention may contain, in addition to the aptamer of the present invention, instructions for use (for scFv detection, modification, or purification) indicating the scFv detection, modification, or purification method.
  • the kit for scFv detection, modification or purification of the present invention may also contain other reagents such as buffers for use during scFv detection, modification or purification.
  • the present invention also provides a device for detection or purification of scFv having a peptide linker (GGGGS) 3 (SEQ ID NO: 3), which comprises the aptamer of the present invention and a support to which the aptamer is bound (e.g., immobilized).
  • the support may be, but is not limited to, a substrate, an electrode, a column carrier, etc. used as a carrier for a nucleic acid array such as a microarray.
  • the scFv detection or purification device of the present invention can be, for example, a nucleic acid array, an electrochemical sensor, a chromatography column, etc.
  • the present invention also provides a method for detecting or purifying scFv having a peptide linker (GGGGS) 3 (SEQ ID NO: 3) using the aptamer of the present invention.
  • the invention also provides the use of the aptamer of the invention in the detection or purification of scFv with a peptide linker (GGGGS) 3 (SEQ ID NO: 3).
  • the scFv detection method of the present invention can be carried out using, for example, the scFv detection kit or device of the present invention.
  • the scFv purification method of the present invention can be carried out using, for example, the scFv purification kit or device of the present invention.
  • the scFv detection method of the present invention can also be used for pharmacokinetic analysis of scFv having a peptide linker (GGGGS) 3 (SEQ ID NO: 3) as an antibody drug.
  • the aptamer of the present invention can also be used for pharmacokinetic analysis of scFv having a peptide linker (GGGGS) 3 (SEQ ID NO: 3).
  • the scFv detection method of the present invention comprises contacting (e.g., mixing) an aptamer of the present invention containing a labeled oligonucleotide with a sample (e.g., sample solution) containing the scFv-
  • the method includes generating an aptamer complex and detecting a label signal derived from the scFv-aptamer complex.
  • an aptamer of the invention comprising a labeled oligonucleotide is incubated in solution with a sample comprising an scFv, thereby incubating the aptamer with a peptide linker (GGGGS) 3 (sequence number 3) to generate an scFv-aptamer complex, and a label signal derived from the scFv-aptamer complex, more specifically, a label derived from the labeling of the aptamer in the scFv-aptamer complex.
  • GGGGS peptide linker
  • the label signal to be detected can be easily recognized by those skilled in the art based on the label of the aptamer used and the measurement system.
  • the labeling signal when using an aptamer labeled with a fluorescent substance, the labeling signal is typically the fluorescence from the fluorescent substance, and when using an aptamer labeled with a radioisotope, the labeling signal is typically the fluorescence from the fluorescent substance. is the radioactivity from its radioactive isotope.
  • the labeling signal when using a biotin-labeled aptamer, is typically chemiluminescence in a chemiluminescent measurement system using HRP-labeled avidin or an avidin derivative (streptavidin, etc.) and a chemiluminescent substrate.
  • the labeling signal when using a biotin-labeled aptamer is obtained by combining HRP-labeled avidin or an avidin derivative (streptavidin, etc.) with a chromogenic substrate such as 3,3',5,5'-tetramethylbenzidine (TMB).
  • TMB 3,3',5,5'-tetramethylbenzidine
  • the scFv detection method of the present invention comprises, for example, a nucleic acid array or a chromatography column comprising a support to which an aptamer of the present invention containing a labeled oligonucleotide is bound (e.g., immobilized). It can be carried out using In one embodiment, the scFv detection method of the invention uses an aptamer of the invention that is bound (e.g., immobilized) to a carrier, such as a magnetic bead, and comprises a labeled oligonucleotide; The method may include separating the generated scFv-aptamer complex from free components in solution using the method.
  • a carrier such as a magnetic bead
  • the scFv detection method of the present invention may also include a further step of detecting the scFv-aptamer complex or scFv.
  • the scFv detection method of the present invention can also be used for the quantification of scFv having a peptide linker (GGGGS) 3 (SEQ ID NO: 3).
  • the scFv detection method of the present invention includes an electrode immobilized with an aptamer of the present invention containing an oligonucleotide modified by binding to an electron mediator, and a sample containing the scFv (for example, a sample solution). contacting (e.g., mixing) to form an scFv-aptamer complex on the electrode and detecting a change in current, etc. at the electrode due to the formation of the scFv-aptamer complex; Changes in current, etc. at the electrode indicate the presence of the scFv with the peptide linker (GGGGS) 3 (SEQ ID NO: 3).
  • GGGGS peptide linker
  • the scFv detection method of the present invention can also be used for the quantification of scFv having a peptide linker (GGGGS) 3 (SEQ ID NO: 3).
  • the scFv detection method of the invention can be carried out using a device such as an electrochemical sensor comprising an electrode to which an aptamer of the invention is bound (e.g., immobilized). .
  • a method of purifying an scFv of the invention comprises contacting (e.g., mixing) an aptamer of the invention with a sample (e.g., a sample solution) containing an scFv to produce an scFv-aptamer complex; The method comprises isolating the scFv-aptamer complex from the sample.
  • an aptamer of the invention is coupled to an scFv with a peptide linker (GGGGS) 3 (SEQ ID NO: 3) by incubating the aptamer of the invention in solution with a sample containing the scFv.
  • the scFv purification method of the present invention can be carried out using, for example, a nucleic acid array, a chromatography column, etc. provided with a support to which the aptamer of the present invention is bound (e.g., immobilized). can.
  • the scFv purification method of the present invention uses an aptamer of the present invention bound (e.g., immobilized) to a carrier such as, for example, magnetic beads, and utilizes the carrier to produce scFv.
  • the method may involve separating the aptamer complex from free components in solution.
  • the separated scFv-aptamer complex may be further subjected to a purification step.
  • the scFv may be separated from the scFv-aptamer complex by cleaving or decomposing the aptamer, and the scFv may be further extracted and purified.
  • the scFv purification method of the present invention may further include a step of detecting the scFv-aptamer complex or scFv.
  • enzyme-linked oligonucleotide assay ELONA
  • enzyme-linked immunosorbent assay Enzyme-linked immunosorbent assay; ELISA
  • biolayer interferometry BBI
  • immunoblot immunoprecipitation
  • immunocapture flow cytometry
  • protein array technology spectroscopy
  • mass spectrometry mass spectrometry
  • chromatography surface plasmon resonance (SPR)
  • fluorescence Any nucleic acid or protein detection technique can be used, such as fluorescence extinction and fluorescence energy transfer (FRET).
  • the present invention also provides a method for screening an aptamer comprising an oligonucleotide that specifically binds to an scFv having a peptide linker (GGGGS) 3 (SEQ ID NO: 3) using the aptamer of the present invention. More specifically, the screening method of the present invention involves introducing a nucleotide mutation into the aptamer of the present invention (more specifically, the above-mentioned oligonucleotide constituting the aptamer of the present invention), and introducing a peptide linker of the resulting mutant.
  • GGGGS peptide linker
  • GGGGS 3 (SEQ ID NO: 3) is measured, and a variant that specifically binds to that scFv is specifically bound to the scFv that has a peptide linker (GGGGS) 3 (SEQ ID NO: 3).
  • the aptamer of the present invention for example, 60% or more of the base sequence (especially the entire base sequence) shown in SEQ ID NO: 14 or 24 of the aptamer of the present invention, preferably contains a nucleotide sequence having a sequence identity of 70% or more, more preferably 80% or more, still more preferably 90% or more, for example 95% or more, for example, the nucleotide sequence shown in SEQ ID NO: 14 or 24, or the nucleotide sequence thereof It is preferable to introduce nucleotide mutations into an oligonucleotide consisting of
  • nucleotide mutations into the aptamer of the present invention can be carried out by synthesizing nucleic acids using an automatic nucleic acid synthesizer based on the base sequence into which mutations have been introduced, or by mutagenesis techniques using nucleic acid amplification techniques such as PCR. You can also do that.
  • nucleotide mutations may be introduced into the aptamer of the present invention using in silico maturation (Savory et al., Biosens. Bioelectron., 26, (2010) 1386-1391).
  • Determination of the binding ability of aptamer variants to scFv may be performed, for example, by ELONA, dot blot analysis, or biolayer interferometry (BLI), as described in the Examples below, or by other methods. It may be performed by a biomolecular interaction measurement assay. Depending on the measurement method used, it is also preferable, for example, to label the oligonucleotide of the aptamer or to bind it to a purification carrier.
  • the screening method of the present invention efficiently selects and obtains aptamers with superior binding properties to scFv having a peptide linker (GGGGS) 3 (SEQ ID NO: 3) by performing sequence evolution based on the aptamer of the present invention. We can provide a method for doing so.
  • the aptamer obtained using the screening method of the invention can be used as a further aptamer of the invention.
  • the reaction of the aptamer of the present invention with the scFv in the present invention is performed under any potassium concentration.
  • it may be carried out at a potassium concentration of 0 to 100 mM or 10 to 100 mM.
  • scFv can also be modified using the aptamer of the present invention.
  • the invention comprises contacting (e.g., mixing) an aptamer of the invention with an scFv having a peptide linker (GGGGS) 3 (SEQ ID NO: 3) to generate an scFv-aptamer complex. It also provides a method for modifying. According to this method, scFv can be modified by adding an aptamer.
  • aptamers of the invention comprising oligonucleotides that are not chemically modified may be used to modify scFvs.
  • an aptamer of the invention comprising a chemically modified oligonucleotide (chemically modified aptamer of the invention) is contacted with an scFv having a peptide linker (GGGGS) 3 (SEQ ID NO: 3).
  • An scFv with that chemical modification can be made by (eg, mixing) to generate an scFv-aptamer complex.
  • the present invention involves contacting ( e.g. , mixed Also provided are methods of chemically modifying scFvs or producing scFvs having chemical modifications, including generating scFv-aptamer conjugates. Chemical modification is as described above.
  • the aptamer of the invention comprising a chemically modified oligonucleotide
  • the aptamer of the invention comprising a labeled oligonucleotide (labeled aptamer of the invention) is linked to a peptide linker (GGGGS). ) 3 (SEQ ID NO: 3) to generate an scFv-aptamer complex.
  • an aptamer of the invention comprising an oligonucleotide conjugated to a drug, such as a cytotoxic substance or a medical drug (an aptamer of the invention conjugated to a drug), is linked to a peptide linker (GGGGS) 3 (SEQ ID NO: A drug can be bound to an scFv by contacting it with an scFv having 3) to generate an scFv-aptamer complex.
  • a drug such as a cytotoxic substance or a medical drug
  • an aptamer of the invention comprising an oligonucleotide conjugated to a carrier or a biocompatible polymer or the like (an aptamer of the invention conjugated to a carrier or a biocompatible polymer or the like) is linked to a peptide linker (GGGGS) 3 ( By contacting the scFv having SEQ ID NO: 3) to generate an scFv-aptamer complex, a carrier, a biocompatible polymer, or the like can be bound to the scFv.
  • the invention also provides the use of an aptamer of the invention for modifying, eg chemically modifying, an scFv with a peptide linker (GGGGS) 3 (SEQ ID NO: 3).
  • any composition containing the aptamer of the present invention can be used.
  • the present invention also provides compositions for the detection, modification or purification of scFv with a peptide linker (GGGGS) 3 (SEQ ID NO: 3), comprising the aptamer of the invention.
  • a composition comprising an aptamer of the invention may be a reagent composition.
  • Compositions containing aptamers of the invention may further contain additives such as carriers, excipients, or mediums such as buffers, and other additives such as pH adjusters, stabilizers, etc. It may further contain additives.
  • the present invention also provides scFv-aptamer complexes (scFv-aptamer conjugates) comprising an scFv with a peptide linker (GGGGS) 3 (SEQ ID NO: 3) linked (i.e., conjugated) with an aptamer of the invention. do.
  • the invention comprises an scFv with a peptide linker (GGGGS) 3 (SEQ ID NO: 3) conjugated to an aptamer of the invention comprising a chemically modified oligonucleotide (e.g. a labeled oligonucleotide).
  • scFv-aptamer conjugates having chemical modifications (eg, labeled).
  • the invention provides a peptide linker (GGGGS) conjugated to an aptamer of the invention comprising an oligonucleotide conjugated to a drug, such as a cytotoxic substance or a medical drug (aptamer of the invention conjugated to a drug).
  • a drug such as a cytotoxic substance or a medical drug
  • an scFv-aptamer conjugate with a drug comprising an scFv having the following: 3 SEQ ID NO: 3
  • the drug's efficacy can be imparted to scFv having a peptide linker (GGGGS) 3 (SEQ ID NO: 3).
  • the scFv-aptamer complex of the present invention having a drug bound to the aptamer of the present invention can be used as an active ingredient of a medicament (eg, a pharmaceutical composition) based on the drug's efficacy.
  • a medicament eg, a pharmaceutical composition
  • Such a medicament may contain, in addition to the scFv-aptamer conjugate of the invention, pharmaceutically acceptable additives.
  • pharmaceutically acceptable additives include pharmaceutical carriers, excipients, surfactants, binders, disintegrants, lubricants, solubilizing agents, suspending agents, coating agents, coloring agents, and flavoring agents. Examples include, but are not limited to, flavoring agents, preservatives, buffers, pH adjusters, diluents, stabilizers, propellants, antioxidants, thickeners, and the like.
  • Example 1 Preparation of single chain antibody 528 scFv
  • the anti-human epidermal growth factor receptor (EGFR) was derived from the humanized monoclonal antibody 528, which has a proven track record of use as a pharmaceutical and sensing element.
  • Single chain antibody 528 scFv was produced by recombinant methods and purified by Ni-NTA affinity chromatography.
  • the 528 scFv expression vector, pRA-528 scFv was provided by Mr. Hayato Kimura (Tokyo University of Agriculture and Technology, Japan).
  • the vector map of pRA-528 scFv is shown in FIG. 1A, and the protein coding sequence (SEQ ID NO: 1) in pRA-528 scFv and the amino acid sequence encoded thereby (SEQ ID NO: 2) are shown in FIG.
  • SEQ ID NO: 1A and 2 528 scFv includes a polypeptide sequence in which a heavy chain variable region (VH) and a light chain variable region (VL) are linked via a peptide linker (GGGGS) 3 .
  • Escherichia coli BL21 (DE3) strain was transformed with pRA-528 scFv and cultured on LB agar medium (Merck) at 37°C for 18 hours.
  • the obtained colonies were inoculated into an LB liquid medium containing ampicillin at a final concentration of 100 ⁇ g/mL, and precultured at 28° C. and 170 rpm for 18 hours.
  • the resulting culture solution was centrifuged at 4500 xg and 4°C for 15 minutes to remove bacterial cells.
  • the obtained culture supernatant was filtered through a nitrocellulose membrane.
  • the obtained filtrate was added to a Ni-Sepharose column (CV: 1 mL), and Ni-NTA affinity chromatography was performed using elution buffers of various concentrations.
  • the purity of the flow-through liquid and each elution fraction obtained by Ni-NTA affinity chromatography was confirmed by SDS-PAGE analysis.
  • Figure 3 shows the results of SDS-PAGE analysis after Ni-NTA affinity chromatography. In the 300 mM imidazole/PBS (pH 7.4) fraction, a dark band was observed near the theoretical molecular weight (30 ⁇ 10 3 ) of 528 scFv.
  • Fv fragment antibody 528 Fv corresponding to 528 scFv was recombinantly produced using a 528 Fv expression vector in a manner similar to that described above for 528 scFv, followed by Ni-NTA affinity chromatography followed by gel filtration chromatography.
  • the vector map of pRA-528 Fv, which is a 528 Fv expression vector, is shown in Figure 1B.
  • Figure 5 shows the SDS-PAGE analysis results of 528 Fv after Ni-NTA affinity chromatography.
  • the results of gel filtration chromatography of 528 Fv are shown in Figure 6.
  • 528 Fv has the same heavy and light chain variable domains as scFv, but unlike scFv, it does not have a peptide linker (GGGGS) 3 (SEQ ID NO: 3) connecting the variable domains. In the Examples described below, 528 Fv was used for competitive selection and comparison with 528 scFv.
  • aptamer candidate sequences that recognize 528 scFv were screened by the SELEX method from a random DNA library containing a 30 base long random region.
  • the SELEX method selects sequences that bind to a ligand from a nucleic acid library with random sequences, and then exponentially amplifies them using PCR to obtain a next-generation nucleic acid library that collects only sequences with high binding affinity to the ligand. This is a technique in which this operation is repeated (multiple rounds) to identify a nucleic acid sequence that specifically binds to a ligand.
  • each DNA library, as well as 5' block DNA and Rv primer having sequences complementary to the priming sequence were added with 100 mM NaCl. It was added to 100 mM potassium phosphate buffer (hereinafter referred to as PPB(Na+); sometimes referred to as SPPB) and mixed. The resulting mixture was heated at 95°C for 10 minutes on a heat block and then slowly cooled to 25°C over 30 minutes to fold the nucleic acid and generate folded DNA.
  • PPB(Na+) 100 mM potassium phosphate buffer
  • 528 scFv prepared in Example 1 1 ⁇ L of 528 scFv prepared in Example 1 was dropped onto a nitrocellulose membrane (Amersham TM Protran (R) Premium 0.45 NC Nitrocellulose Western Blotting Membrane, GE Healthcare) and fixed by adsorption. Furthermore, 528 Fv prepared in Example 1 was dropped as a competitive protein for competitive selection next to the position where 528 scFv was dropped on the nitrocellulose membrane.
  • the nitrocellulose membrane was coated with PPB(Na+)-T (PPB(Na+) containing 2% (w/v) bovine serum albumin (BSA) and 0.05% (v/v) Tween. 20; sometimes referred to as SPPB-T) for 1 hour, and blocking was performed (in addition, from the third round onwards, blocking was performed using human serum instead of BSA).
  • PPB(Na+)-T PPB(Na+)-T
  • the nitrocellulose membrane was shaken for 1 hour in a solution in which the folded DNA generated as described above was diluted with PPB(Na+) to 1 mL.
  • the scFv adsorption region of the nitrocellulose membrane was cut out, followed by phenol-chloroform extraction, followed by ethanol precipitation to recover single-stranded DNA.
  • the recovered single-stranded DNA was dissolved in buffer TE (Tris-EDTA) to prepare a recovered DNA library.
  • the amount of recovered DNA was calculated by performing quantitative PCR (real-time PCR) on the recovered DNA library.
  • Table 2 shows the composition of the PCR reaction solution used.
  • a reaction solution (Table 2) was prepared using serially diluted DNA (standard material) of known concentration as template DNA, and incubated at 98°C for 1 minute, then at 98°C for 10 seconds, at 50°C for 30 seconds, and at 72°C. Perform 40 cycles of 20 seconds at 4°C, and then perform nucleic acid amplification by PCR under the PCR reaction conditions of holding at 4°C. From the Cq (quantification cycle) value (sometimes called the Ct (threshold cycle) value), a standard curve is generated. It was created. Furthermore, prepare a reaction solution (Table 2) using the recovered DNA library of unknown concentration as template DNA, perform nucleic acid amplification by PCR under the same reaction conditions as above, and apply the obtained Cq value to the prepared standard curve. The amount of recovered DNA in the recovered DNA library was calculated.
  • the single-stranded DNA constituting the recovered DNA library was amplified by PCR.
  • Table 3 shows the composition of the PCR reaction solution used.
  • Primers Fw primer and Bio-Rv primer
  • the 3' primer complementary to the 3' priming sequence is biotinylated.
  • the biotinylated DNA library obtained by nucleic acid amplification was incubated with avidin beads, and the DNA (single strand) bound to the collected beads was eluted with 0.15M sodium hydroxide (NaOH). After making the solution neutral using 2M hydrogen chloride (HCl), DNA was precipitated by adding 100% isopropanol, an aqueous sodium acetate solution, and Perin Mate (Nippon Gene), a coprecipitant for alcohol precipitation, and centrifuging it. After washing with % ethanol and centrifugation, the supernatant was discarded and air-dried. A DNA library to be used in the next round (second round) was prepared by dissolving the obtained DNA in TE.
  • HCl 2M hydrogen chloride
  • PCR amplification was set to 25 cycles, and in the SELEX method using the library having library sequence 2, PCR amplification was set to 28 cycles, and the same SELEX method as above was performed. was carried out until the fourth round.
  • the amount of recovered DNA was measured at the end of each round, and the recovery rate was calculated as the ratio (%) of the amount of recovered DNA (number of moles) to the number of moles of the DNA library used.
  • the amount of reagents used in the first to fourth rounds, the amount of recovered DNA, and the recovery rate are summarized in Tables 4 and 5. The success of the SELEX method was demonstrated by the increase in recovery rate as the number of rounds increased.
  • single-stranded DNA in the DNA library was PCR amplified using the Fw primer and Rv primer shown in Table 1 to obtain double-stranded DNA.
  • the purified double-stranded DNA and vector were mixed at a molar ratio of 1:1 using pGEM (R) -T Easy Vector Systems (Promega), and the two were ligated by reacting overnight at 16°C. .
  • Add the obtained ligation product (derived from library sequence 1: 2.5 ⁇ L, derived from library sequence 2: 7.5 ⁇ L) to 50 ⁇ L of E. coli Jet Competent Cell (DH5 ⁇ ) (ByoDynamics Laboratory Inc.) and leave it on ice for 5 minutes. Transformation was performed by.
  • reaction buffer and enzyme mix of the kit were added and the mixture was reacted at 30°C for 12 hours, followed by rolling cycle amplification by reacting at 65°C for 10 minutes to prepare template DNA for sequencing.
  • sequencing was performed using this template DNA, it was possible to obtain 16 single-stranded DNA sequences from the library having library sequence 1 and 79 from the library having library sequence 2, for a total of 95 single-stranded DNA sequences. Sequence analysis was performed by aligning the random sequence portions sandwiched between the priming sequences of these single-stranded DNA sequences.
  • Example 3 Evaluation of binding ability of aptamer candidate sequence to scFv and Fv
  • the binding ability of the aptamer candidate sequence obtained in Example 2 to scFv and Fv was evaluated.
  • 528 scFv and 528 Fv were added to the plate by adding 100 ⁇ L of 100 nM 528 scFv and 100 nM 528 Fv (prepared in Example 1) to separate wells of a 96-well plate and incubating for 90 minutes at 37°C. Fixed by adsorption. After washing with PPB(Na+)-T, 2% (w/v) skim milk dissolved in PPB(Na+)-T was added to the plate and incubated at room temperature for 1 hour with shaking at 800 rpm. was blocked.
  • Example 2 20 pmol each of 528 scFv and 528 Fv prepared in Example 1 were placed on a nitrocellulose membrane (Amersham TM Protran (R) Premium 0.45 NC Nitrocellulose Western Blotting Membrane, GE Healthcare), and as a negative control, bevacizumab (anti-VEGF human 1 pmol of monoclonal antibody) was added dropwise and fixed by adsorption. The nitrocellulose membrane was then shaken for 1 hour in 2% (w/v) skim milk dissolved in PPB(Na+)-T for blocking.
  • a nitrocellulose membrane Amersham TM Protran (R) Premium 0.45 NC Nitrocellulose Western Blotting Membrane, GE Healthcare
  • bevacizumab anti-VEGF human 1 pmol of monoclonal antibody
  • the nitrocellulose membrane was shaken for 1 hour in an aptamer solution (final concentration 2 ⁇ M) diluted with PPB(Na+) to 1 mL. After washing with PPB(Na+)-T, the nitrocellulose membrane was shaken for 1 hour in neutravidin-HRP conjugate (Thermo Fisher Scientific) diluted to 0.2 ng/ ⁇ L. After washing with PPB(Na+)-T in the same manner as above, Immobilon Western chemiluminescent HRP substrate (Millipore) was added dropwise to the above adsorption/immobilization site and incubated for 5 minutes at room temperature and shielded from light. Chemiluminescence was measured with an image analyzer ImageQuant TM LAS 4000 mini (GE Healthcare).
  • streptavidin sensor chip A biosensor chip having streptavidin as a surface modification molecule (streptavidin sensor chip) was immersed in PPB(Na+)-T for 10 minutes. After making it hydrophilic, biotinylated aptamer candidate sequences (biotinylated P1-12 and biotinylated P2-63) were immobilized on a streptavidin sensor chip via biotin-avidin interaction.
  • biotinylated aptamer candidate sequences immobilized on a streptavidin sensor chip and biotinylated aptamer candidate sequences in solution were The binding and dissociation signals between 528 scFv and 528 Fv (prepared in Example 1) were measured. The results showed that for both P2-63 and P1-12, the level of binding to scFv was much higher compared to the level of binding to Fv ( Figure 9).
  • P2-63 and P1-12 are aptamers selected from different DNA libraries, but both are G-rich sequences consisting of consecutive G sequences, and under physiological conditions, guanine quadruplex (G-quadruplex; ) structure, which is thought to form a stable higher-order structure.
  • G-quadruplex guanine quadruplex
  • Example 4 Evaluation of the binding ability of aptamer to other scFv
  • anti-Hb scFv-SpyTag was further used as an scFv with binding ability to a different antigen to create aptamer P2.
  • the binding ability of -63 and P1-12 to scFv was evaluated.
  • the anti-Hb scFv-SpyTag used was an scFv derived from an anti-hemoglobin (Hb) mouse monoclonal antibody, and was produced as follows.
  • Escherichia coli BL21 (DE3) was transformed with the Spy tag (or SpyTag)-added anti-Hb scFv expression vector pRA-anti Hb scFv-ST (hereinafter also referred to as "pRA-anti-Hb scFv-SpyTag”), and ampicillin (final concentration 100 ⁇ g/mL) on an LB agar medium plate and incubated overnight at 28°C.
  • the obtained colonies were normalized and precultured in 3 mL of LB medium (ampicillin was added at a final concentration of 100 ⁇ g/mL).
  • the culture solution after preculture was inoculated into a total of 600 mL of LB medium, and cultured for 40 hours.
  • the culture supernatant was subjected to ammonium sulfate precipitation using 60% (w/v) ammonium sulfate.
  • the precipitate obtained by centrifugation was condensed, dialyzed, and purified using a histidine (His) tag by Ni-NTA affinity chromatography.
  • the eluted fraction in which a band was observed near the theoretical molecular weight on SDS-PAGE was concentrated and subjected to gel filtration chromatography.
  • the anti-Hb scFv-SpyTag thus obtained was used below.
  • the vector map of pRA-anti-Hb scFv-SpyTag is shown in Figure 14A, and the protein coding sequence (SEQ ID NO: 29) and the amino acid sequence encoded thereby (SEQ ID NO: 30) in pRA-anti-Hb scFv-SpyTag are shown in Figure 15. .
  • the anti-Hb scFv-SpyTag contains a polypeptide sequence in which a heavy chain variable region (VH) and a light chain variable region (VL) are linked via a peptide linker (GGGGS) 3 . .
  • 528 scFv and 528 Fv prepared in Example 1, frozen and thawed 528 Fv, and anti-Hb scFv-SpyTag were placed on a nitrocellulose membrane (Amersham TM Protran (R) Premium 0.45 NC Nitrocellulose Western Blotting Membrane, GE Healthcare). was added dropwise in 10 pmol portions and fixed by adsorption. The nitrocellulose membrane was then blocked in 2% (w/v) skim milk dissolved in PPB(Na+)-T by shaking for 1 hour. After washing with PPB(Na+)-T, it was shaken for 1 hour in an aptamer solution (P2-63 or P1-12) diluted with PPB(Na+) to a final concentration of 1 ⁇ M.
  • P2-63 or P1-12 aptamer solution
  • Aptamers P2-63 and P1-12 were able to bind to multiple types of scFv with different complementarity-determining regions (CDRs). It was thought to specifically recognize the conserved structure of the linker (GGGGS) 3 region.
  • Example 5 Evaluation of the influence of aptamer binding on 528 scFv activity Using soluble EGFR (sEGFR), it was determined whether the binding of aptamers P2-63 and P1-12 affects the binding activity of 528 scFv to sEGFR. This was investigated using enzyme-linked immunosorbent assay (ELISA).
  • sEGFR soluble EGFR
  • CHO cells with high sEGFR-His expression carrying a His-tagged sEGFR expression vector were incubated in a CO 2 incubator in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin (PC) + streptomycin (SM). (Serum medium) Using 15 mL, the cells were grown in a 75 cm 2 culture flask until reaching confluence. Proliferating cells (stable producing strain) were suspended in 10 mL of the above serum medium, and then added to a roller bottle containing 90 mL of the above serum medium. Culture was carried out at 37°C and 5% CO2 while rotating the roller bottle.
  • FBS fetal bovine serum
  • PC penicillin + streptomycin
  • the supernatant was removed by suction.
  • the cells were washed with 50 mL of phosphate buffer (PBS), and 250 mL of CHO cell serum-free medium CHO-S-SFM/PC/SM was added. After culturing for 4 days, the culture solution was collected and centrifuged at 300 ⁇ g for 5 minutes to obtain a culture supernatant. The culture supernatant was centrifuged at 10,000 ⁇ g for 20 minutes to remove floating cells. The supernatant after centrifugation was collected, and 1M Tris-HCl (pH 8.0) was added to the final concentration of 50mM.
  • PBS phosphate buffer
  • CHO-S-SFM/PC/SM CHO cell serum-free medium
  • the 3' end of the aptamer was modified with biotin via a complementary strand and diluted to different concentrations with PPB(Na+). Equal volumes of the 200 nM 528 scFv solution were mixed at 1.5 Aptamer-scFv antibody conjugates were prepared by mixing in a mL tube and reacting at room temperature for 1 hour with shaking at 600 rpm.
  • BM chemiluminescent ELISA substrate 100 ⁇ L was added to the plate, incubated for 10 minutes at room temperature and protected from light, and chemiluminescence was measured using a plate reader Varioskan (R) Flash (Thermo Fisher Scientific).
  • Figure 11 shows the measurement results based on 528 scFv detection via the c-myc tag using the HRP-modified anti-c-Myc antibody.
  • aptamer concentrations 0. nM, 1 nM, 10 nM, 100 nM, and 1000 nM
  • little change was observed in chemiluminescence levels, which indicate the amount of 528 scFv bound to sEGFR on the plate.
  • the chemiluminescence level was higher and significantly different compared to when sEGFR was not immobilized on the plate (negative control; aptamer concentration 100 nM).
  • 528 scFv was able to bind to sEGFR regardless of aptamer concentration. That is, the aptamer was able to bind to 528 scFv without inhibiting the binding of 528 scFv to sEGFR.
  • aptamers P2-63 and P1-12 can bind to scFv without inhibiting the antigen-binding activity of scFv. It was also shown that scFv bound to an antigen (here, sEGFR) can be detected based on the binding (complexation) of aptamers P2-63 and P1-12 to scFv.
  • Aptamers P1-12 and P2-63 were diluted to 2 ⁇ M with buffer, heated to 95°C for 10 minutes, and then gradually cooled to 25°C over 30 minutes to fold the nucleic acids.
  • CD spectra were measured at wavelengths of 220 nm to 300 nm using a circular dichroism spectrophotometer. The results are shown in FIG.
  • Example 7 Evaluation of the influence of aptamer binding to other scFvs on scFv antigen binding activity Using anti-Hb scFv-SnT as an additional scFv, the binding ability of aptamers P2-63 and P1-12 to scFv was evaluated. .
  • the anti-Hb scFv-SnT used was an scFv derived from the same anti-hemoglobin (Hb) mouse monoclonal antibody clone used in Example 4 to which a tag such as a Snoop tag (SnoopTag or SnT) was added.
  • Anti-Hb scFv-SnT was prepared by recombinant production using E.
  • anti-Hb scFv-SnT contains a polypeptide sequence in which a heavy chain variable region (VH) and a light chain variable region (VL) are linked via a peptide linker (GGGGS) 3 . .
  • Hb hemoglobin
  • Thermo Fisher Scientific 100 ⁇ L of 100 nM hemoglobin (Hb) (Thermo Fisher Scientific) was added to a 96-well plate and incubated overnight at 4°C to adsorb and immobilize Hb onto the plate. After washing with PPB(Na+)-T, 2% (w/v) skim milk dissolved in PPB(Na+)-T was added to the plate and incubated at room temperature for 1 hour with shaking at 600 rpm. , the plate was blocked.
  • Hb hemoglobin
  • An aptamer-anti-Hb scFv-SnT antibody conjugate was prepared by mixing equal volumes of anti-Hb scFv-SnT solutions in a 1.5 mL tube and reacting at room temperature for 1 hour while shaking at 600 rpm.
  • BM chemiluminescent ELISA substrate 100 ⁇ L was added to the plate, incubated for 10 minutes at room temperature and protected from light, and chemiluminescence was measured using a plate reader Varioskan (R) Flash (Thermo Fisher Scientific).
  • Figure 18 shows the measurement results based on anti-Hb scFv-SnT detection via the c-myc tag using the HRP-modified anti-c-Myc antibody. Regardless of the final aptamer concentrations used (0 nM, 1 nM, 10 nM, 100 nM, and 500 nM), little change was observed in chemiluminescence levels, which indicate the amount of BH5 scFv-SnT bound to Hb on the plate. Ta. The chemiluminescence level was significantly higher than when no Hb was immobilized on the plate ("No Hb" in Figure 18; negative control; aptamer final concentration 100 nM), and a significant difference was observed.
  • anti-Hb scFv-SnT was able to bind to Hb regardless of the aptamer concentration. That is, the aptamer was able to bind to anti-Hb scFv-SnT without inhibiting the binding of anti-Hb scFv-SnT to antigen Hb.
  • Example 8 Electrochemical detection of scFv using an aptamer-immobilized electrode by square wave voltammetry Using HB125 scFv as an additional scFv, electrochemical detection of scFv using an aptamer-immobilized electrode was performed.
  • the HB125 scFv used was an anti-insulin antibody-derived scFv with a C-myc tag and a His tag added.
  • HB125 scFv was prepared by recombinant production using E. coli transformed with the HB125 scFv expression vector pRA-HB125 scFv.
  • the vector map of pRA-HB125 scFv is shown in FIG.
  • HB125 scFv includes a polypeptide sequence in which a heavy chain variable region (VH) and a light chain variable region (VL) are linked via a peptide linker (GGGGS) 3 .
  • the gold disk electrode was polished 50 times on each side in a figure-eight pattern using an alumina polishing pad onto which 0.05 ⁇ m polishing alumina was dropped. After polishing, it was washed with ultrapure water (Milli-Q (R) water), and then the gold disk electrode was immersed in ultrapure water and ultrasonically cleaned for 10 minutes. After ultrasonic cleaning, a mixed solution of potassium hydroxide (KOH) with a final concentration of 50 mM and hydrogen peroxide (H 2 O 2 ) with a final concentration of 25% was dropped onto the surface of the gold disk electrode. After standing for 10 minutes, the gold disk electrode surface was further cleaned by sweeping the potential in the range of -0.35 V to -1.35 V in 0.5 M NaOH.
  • KOH potassium hydroxide
  • H 2 O 2 hydrogen peroxide
  • Aptamers P1-12 and P2-63 were each heated at 95°C for 10 minutes, and then slowly cooled to 25°C over 30 minutes to cause folding, thereby preparing a folded aptamer solution.
  • Gold rinsed with ultrapure water was placed in a 1 ⁇ M fold aptamer solution containing an equimolar amount of tris(2-carboxyethyl)phosphine (TCEP; Fujifilm Wako Pure Chemical Industries, Ltd.) dissolved in Tris-HCl.
  • TCEP tris(2-carboxyethyl)phosphine
  • the aptamer-immobilized electrode was blocked by immersing it in a 1 mM 6-mercaptohexanol (6-MCH)/20 mM Tris-HCl solution for 2 hours.
  • the blocked gold disk electrodes were rinsed with ultrapure water and then stored in PPB(Na+) at 4°C until use.
  • HB125 scFv (anti-insulin) was added to the reaction solution (PPB(Na+) containing 10 mM potassium ferricyanide) to a final concentration of 10, 50, 100, 500, or 1000 nM, and after incubation for 30 minutes with stirring.
  • Square wave voltammetry (SWV) measurements were performed.
  • SWV measurement 2 mL of PPB(Na+)/10mM potassium ferricyanide solution was used as the reaction solution, and the aptamer-immobilized electrode prepared above, platinum wire, and Ag/AgCl reference electrode were used as the working electrode, counter electrode, and reference electrode, respectively.
  • a three-electrode method was used.
  • the potentiostat was VSP-150 (Bio-Logic Science Instruments), and SWV measurements were made in the range 0 to 0.4 V (vs. Ag/AgCl) with an amplitude of 150 mV, steps of 10 mV, and measurements at 20 Hz. carried out.
  • SWV measurement was performed in the same manner as above using a 10-fold dilution of LB medium with PPB(Na) as a reaction solution instead.
  • the composition of the LB medium used was as follows: 10 g of soy peptone (Gibco Phytone TM pepton), 5 g of yeast extract (Gibco Bacto TM Yeast Extract), 5.844 g of NaCl (1 L of ultrapure water). During).
  • the present invention can be used to specifically detect scFv. Since natural scFv does not exist in vivo, a versatile aptamer that specifically binds to scFv with a peptide linker (GGGGS) 3 (SEQ ID NO: 3) is useful for general detection of scFv antibody drugs (e.g., pharmacokinetics). It is also considered useful for analysis). Furthermore, the aptamer of the present invention can be used as a detection element for scFv or as a capture means for scFv purification by subjecting the oligonucleotide to various chemical modifications.
  • GGGGS peptide linker
  • the aptamer of the present invention can be used as a general-purpose tool for performing various chemical modifications on scFv.
  • the aptamer of the present invention can also be used in a drug delivery system by binding an oligonucleotide to a medical drug or the like and using it in combination with an scFv that binds to a target site.

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Abstract

La présente invention concerne un aptamère contenant un oligonucléotide (par exemple, un oligonucléotide contenant une séquence de bases qui présente au moins 90 % d'identité de séquence avec la séquence de bases représentée par SEQ ID NO : 14 ou 24) qui se lie particulièrement à un Fv à chaîne unique (scFv) qui présente un lieur peptidique (GGGGS)3 (SEQ ID NO : 3) ; une composition le contenant ; un kit et un dispositif ; un complexe scFv-aptamère qui contient le scFv lié à l'aptamère ; et un procédé de détection ou d'affinement du scFv ou un procédé de modification du scFv qui utilise ledit aptamère.
PCT/JP2023/030053 2022-08-19 2023-08-21 Aptamère de liaison à un anticorps simple brin WO2024038918A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2014509849A (ja) * 2011-03-07 2014-04-24 シャリテ−ウニヴェルジテーツメディツィン・ベルリン 自己免疫疾患の治療及び／又は診断におけるアプタマーの使用
JP2017529872A (ja) * 2014-08-04 2017-10-12 ベルリン キュアーズ ホールディング アクチェンゲゼルシャフト 自己抗体関連疾患に対する使用のためのアプタマー
JP2018027024A (ja) * 2016-08-15 2018-02-22 国立大学法人東京農工大学 アプタマー及び抗体検出方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2014509849A (ja) * 2011-03-07 2014-04-24 シャリテ−ウニヴェルジテーツメディツィン・ベルリン 自己免疫疾患の治療及び／又は診断におけるアプタマーの使用
JP2017529872A (ja) * 2014-08-04 2017-10-12 ベルリン キュアーズ ホールディング アクチェンゲゼルシャフト 自己抗体関連疾患に対する使用のためのアプタマー
JP2018027024A (ja) * 2016-08-15 2018-02-22 国立大学法人東京農工大学 アプタマー及び抗体検出方法

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Title
IKEBUKURO KAZUNORI, RYUTARO ASANO, DAIMEI MIURA, WAKANA HAYASHI : "Development of the technology to visualize viruses by luminescence using antibody and aptamer", PROCEEDINGS OF THE SPRING ANNUAL MEETING OF THE CHEMICAL SOCIETY OF JAPAN, vol. 103, 22 March 2023 (2023-03-22), pages K402 - 3am, XP093140232 *

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