WO2024038918A1

WO2024038918A1 - Single-stranded antibody-binding aptamer

Info

Publication number: WO2024038918A1
Application number: PCT/JP2023/030053
Authority: WO
Inventors: 竜太郎浅野; 一典池袋
Original assignee: 国立大学法人東京農工大学
Priority date: 2022-08-19
Filing date: 2023-08-21
Publication date: 2024-02-22

Abstract

The present invention pertains to: an aptamer which contains an oligonucleotide (for example, an oligonucleotide containing a base sequence which has at least 90% of the sequence identity of the base sequence represented by SEQ ID NO 14 or 24) which specifically binds to a single-chain Fv (scFv) which has a peptide linker (GGGGS)3 (SEQ ID NO 3); a composition containing the same; a kit and device; a scFv-aptamer complex which contains scFv bonded to the aptamer; and an scFv detection or refinement method or a method for modifying the scFv which uses said aptamer.

Description

Single chain antibody binding aptamer

The present invention relates to an aptamer that specifically binds to a single chain antibody.

Although conventional antibody drugs have been shown to have certain therapeutic effects, they do not easily lead to a complete cure, especially in cancer treatment, and their therapeutic effects are limited. For this reason, the development of completely non-natural artificial antibodies with higher functionality has become a global trend.

scFv (also called single-chain Fv or single-chain antibody), which is the antigen-binding domain of an antibody, the heavy chain variable domain and light chain variable domain of Fv, are linked by a peptide linker, and is the most commonly used artificial antibody. There is one. scFvs are stabilized with peptide linkers, can be prepared using microorganisms, and are advantageously used as pharmaceuticals and sensing elements. As a pharmaceutical, blinatumomab, a bispecific antibody whose constituent unit is scFv, has already been approved in Japan. On the other hand, since 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.

For example, 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. In the latter case, since a pair of disulfide bonds usually exists in the heavy chain variable domain and light chain variable domain that constitute the scFv, modifications that involve the introduction of new cysteine residues may affect disulfide bond formation. , mismultiplication, etc. may significantly reduce the yield and function of the desired scFv. Therefore, conventional protein modification techniques are not suitable for scFv modification.

Furthermore, no general-purpose technology for specifically detecting scFv has been reported. In order to detect a specific antibody drug separately from antibodies endogenous to a living body, for example, anti-idiotype antibodies that recognize the antigen-binding site of antibodies (Non-patent Document 1) and anti-idiotype nucleic acid aptamers (Non-Patent Document 2) are used. However, anti-idiotype antibodies and anti-idiotype nucleic acid aptamers are not easy to obtain, and they are not versatile because they can only detect specific antibodies. Although 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.

Therefore, it is desired to develop tools that can specifically modify or detect scFv.

International publication WO2020/081510

An object of the present invention is to provide an aptamer that specifically binds to a single chain antibody scFv.

As a result of intensive studies to solve the above problems, the present inventors discovered an oligonucleotide that can specifically bind to a single-chain Fv (scFv) having a peptide linker (GGGGS) ₃ (SEQ ID NO: 3). The present invention has now been completed.

That is, the present invention includes the following.
[1] An aptamer containing an oligonucleotide that specifically binds to a single chain Fv (scFv) having a peptide linker (GGGGS) ₃ (SEQ ID NO: 3).
[2] The aptamer according to [1] above, wherein the oligonucleotide includes a base sequence having 90% or more sequence identity to the base sequence shown by SEQ ID NO: 14 or 24.
[3] The aptamer according to [1] or [2] above, wherein the oligonucleotide is chemically modified.
[4] The aptamer according to any one of [1] to [3] above, wherein the oligonucleotide is labeled.
[5] 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. The aptamer according to [3] above, which is chemically modified by one bond or introduction, and/or by containing at least one of a modified nucleotide and an artificial base.
[6] The aptamer according to any one of [1] to [5] above, wherein the oligonucleotide is DNA or RNA.
[7] A composition for detecting, modifying, or purifying scFv having a peptide linker (GGGGS) ₃ (SEQ ID NO: 3), which includes the aptamer according to any one of [1] to [6] above.
[8] A kit for detecting, modifying or purifying scFv having a peptide linker (GGGGS) ₃ (SEQ ID NO: 3), which includes the aptamer according to any one of [1] to [6] above.
[9] A device for detecting or purifying scFv having a peptide linker (GGGGS) ₃ (SEQ ID NO: 3), comprising the aptamer according to any one of [1] to [6] above and a support to which it is bound.
[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 (SEQ ID NO: 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.
[14] The method according to [13] above, wherein the aptamer includes the oligonucleotide that has been chemically modified, and the method is a method of chemically modifying scFv.
[15] An 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.

This specification includes the disclosure content of Japanese Patent Application No. 2022-131326, which is the basis of the priority of this application.

According to the present invention, it is possible to specifically bind to the peptide linker (GGGGS) ₃ region in a single chain Fv (scFv) having peptide linker (GGGGS) ₃ , and thereby it can be used for a wide range of scFv modification, detection, etc. aptamer.

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. 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. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a sample is applied to a polyacrylamide gel (SuperSep ^TM Ace, 10-20% 17 wells), electrophoresis is performed, and the gel after electrophoresis is transferred to CBB ( Coomassie brilliant blue). The arrow and square frame indicate the band of 528 scFv. Figure 4 shows a chromatogram obtained by gel filtration chromatography of purified 528 scFv. Figure 5 is a photograph showing the results of Ni-NTA affinity chromatography (column chromatography) of a 528 Fv sample at different purification stages. 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. The vertical axis shows the chemiluminescence level, and the horizontal axis shows the aptamer candidate sequence. Filled bar: scFv, shaded bar: Fv. Protein (antibody): 100 nM, aptamer candidate sequence: 100 nM. 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. A: P2-63, B: P1-12. 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. A: P2-63, B: P1-12. 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. A: P2-63, B: P1-12. FIG. 20 shows changes in peak current values measured by SWV with respect to scFv concentration. A: P2-63, B: P1-12. 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. A: P2-63, B: P1-12. FIG. 22 shows changes in peak current values measured by SWV using a 10-fold diluted solution with respect to scFv concentration. A: P2-63, B: P1-12.

The present invention will be explained in detail below.
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).

In the present invention, an "aptamer" (sometimes referred to as a nucleic acid 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. .

More specifically, 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.

When an antibody is digested with the proteolytic enzyme papain, the disulfide bond (hinge site) that connects the H chains is cleaved, and the Y-shaped structure of the antibody is divided into three fragments. The two antibody fragments at the N-terminus are called Fab, and the one antibody fragment at the C-terminus is called Fc. On the other hand, the set of one heavy chain variable domain and one light chain variable domain that constitutes the antigen-binding site is called Fv. A single chain Fv (scFv) 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. In addition, in the present invention, "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 . Additional amino acid sequences (for example, but not limited to, 1-20, 1-15, or 1-20) between the heavy chain variable domain terminus and/or the light chain variable domain terminus and the amino acid sequence (GGGGS) ₃ Also included are polypeptide sequences in which a sequence of 5 amino acids) is present. Such further amino acid sequences may form part of the peptide linker together with the amino acid sequence (GGGGS) ₃ . In the present invention, "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 . Also included are scFvs with any peptide linker comprising the amino acid sequence (GGGGS) ₃ , such as scFvs. Such 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 . a polypeptide sequence (typically comprising a heavy chain variable domain and a light chain variable domain, and the C-terminus of the heavy chain variable domain and the N-terminus of the light chain variable domain are linked via a peptide linker (GGGGS) ₃₎ . polypeptide sequence). 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). 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. 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. , 32, or 34, excluding the amino acid sequence at positions 1 to 22 (pelB signal sequence) (scFv), as well as 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.

With respect to the 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). Regarding the aptamer of the present invention, "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.

In one embodiment, 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. In a preferred embodiment, the oligonucleotide constituting the aptamer of the present invention includes or consists of the base sequence shown in SEQ ID NO: 14 or 24.

In one embodiment, the oligonucleotide constituting the aptamer of the present invention has a base sequence CG _n (n=5 to 7), specifically at least one base sequence CGGGGGGG, CGGGGGG, and CGGGGG, preferably a base sequence It may include at least one of the sequences CGGGGGGG and CGGGGG. The oligonucleotide constituting the aptamer of the present invention has a base sequence CG _n (n=5 to 7), specifically at least one of the base sequences CGGGGGGG, CGGGGGG, and CGGGGG, preferably at least one of CGGGGGGG and CGGGGG. 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. In the sequence listing, the 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). That is, 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). In this case, the term "oligonucleotide containing the base sequence shown by SEQ ID NO: 14 or 24" is also included. "Oligonucleotide consisting of the base sequence shown by SEQ ID NO: 14 or 24" and "60% or more, preferably 70% or more, more preferably 80% or more, even more preferably, the base sequence shown by SEQ ID NO: 14 or 24 is similarly interpreted as "an oligonucleotide containing a base sequence having a sequence identity of 90% or more, for example 95% or more."

In the aptamer of the present invention, 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 . In one embodiment, the oligonucleotide may be labeled, typically with a labeling substance that allows detection, and such a label is also included in chemical modification. In one embodiment, 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. functional groups, 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. 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.

Further, 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. In the present invention, 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. Examples of 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. That is, for example, when 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. It may be present as a substitute for at least one of the natural nucleotides, and in that case, it is also included in the scope of "the oligonucleotide comprising the base sequence shown by SEQ ID NO: 14 or 24.""Oligonucleotide consisting of the base sequence shown by SEQ ID NO: 14 or 24" and "60% or more, preferably 70% or more, more preferably 80% or more, even more preferably, the base sequence shown by SEQ ID NO: 14 or 24 is similarly interpreted as "an oligonucleotide containing a base sequence having a sequence identity of 90% or more, for example 95% or more." In the present invention, 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. Examples include, but are not limited to, imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px), 5SICS and NaM, and the like. When 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. For example, when 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. In one embodiment, the oligonucleotide constituting the aptamer of the present invention, which includes the base sequence CG _n (n=5 to 7), specifically, at least one of the base sequences CGGGGGGG, CGGGGGG, and CGGGGG, comprises an artificial base. If so, the artificial base can be inserted at a position that does not interrupt the respective base sequence CG _n . Regarding the present invention, 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. In CD spectrum analysis, 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. In a preferred embodiment, 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. .

The 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. In a preferred embodiment, 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). provide. 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. In the present invention, 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).

In one embodiment, 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. In a typical embodiment, more specifically, 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. By detecting the signal, the scFv with the peptide linker (GGGGS) ₃ (SEQ ID NO: 3) can be detected. It is preferable to detect the label signal after the generated scFv-aptamer complex is separated from free components in the solution by washing or the like. 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. For example, 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. For example, when using a biotin-labeled aptamer, the labeling signal is typically chemiluminescence in a chemiluminescent measurement system using HRP-labeled avidin or an avidin derivative (streptavidin, etc.) and a chemiluminescent substrate. . For example, 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). In the color reaction measurement system used, the color development intensity is typically measured. In one embodiment, 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. 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).

In one embodiment, 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). 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). In one embodiment, 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). .

In one embodiment, 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. In an exemplary embodiment, more specifically, 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. to generate an scFv-aptamer complex, and the scFv-aptamer complex is separated from free components in the solution by washing etc., thereby purifying the scFv having the peptide linker (GGGGS) ₃ (SEQ ID NO: 3). be able to. In one embodiment, 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. In one embodiment, 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.

In the scFv detection or purification method of the present invention, enzyme-linked oligonucleotide assay (ELONA), enzyme-linked immunosorbent assay (Enzyme-linked immunosorbent assay; ELISA), biolayer interferometry (BLI), immunoblot, immunoprecipitation, immunocapture, flow cytometry, protein array technology, spectroscopy, 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).

Furthermore, 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) 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). This includes selecting an aptamer (or aptamer candidate) containing an oligonucleotide that In one embodiment, in the screening method of the present invention, 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

Introducing 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. Alternatively, 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. In a preferred embodiment, the aptamer obtained using the screening method of the invention can be used as a further aptamer of the invention.

In one embodiment, the reaction of the aptamer of the present invention with the scFv in the present invention (e.g., the step of contacting the aptamer of the present invention with the scFv to generate an scFv-aptamer complex) is performed under any potassium concentration. For example, it may be carried out at a potassium concentration of 0 to 100 mM or 10 to 100 mM.

In the present invention, 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. In one embodiment, aptamers of the invention comprising oligonucleotides that are not chemically modified may be used to modify scFvs. In another embodiment, for example, 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. In one embodiment of the method using the aptamer of the invention comprising a chemically modified oligonucleotide, for example, 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. In one embodiment, 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. In one embodiment, for example, 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).

In the above method of the present invention, 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. In one embodiment, 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). Also provided are scFv-aptamer conjugates having chemical modifications (eg, labeled). Chemical modification is as described above. In one embodiment, 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). Also provided is an scFv-aptamer conjugate with a drug comprising an scFv having the following: ₃ (SEQ ID NO: 3). By using the aptamer of the present invention bound to a drug, 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. Such a medicament, eg, a pharmaceutical composition, may contain, in addition to the scFv-aptamer conjugate of the invention, pharmaceutically acceptable additives. Examples of 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.

Hereinafter, the present invention will be explained in more detail using Examples. However, the technical scope of the present invention is not limited to these Examples.

[Example 1] Preparation of single chain antibody 528 scFv In this example, 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. As shown in FIGS. 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 μg/mL, and precultured at 28° C. and 170 rpm for 18 hours. Add 1 mL of the preculture solution to Auto induction medium (ampicillin 100 μg/mL, 0.5% glycerol, 0.05% glucose, 0.2% β-lactose, 50 mM KH ₂ PO ₄ , 25 mM (NH ₄ ) ₂ SO ₄ , 50 mM Na ₂ HPO ₄ , 1 mM MgSO ₄ ) and cultured with shaking at 20° C. and 170 rpm for 40 hours in a baffled flask.

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.

A fraction of 300 mM imidazole/PBS (pH 7.4) was collected and subjected to gel filtration chromatography to collect only the monomer. The elution buffer used was 20 mM potassium phosphate buffer (PPB) + 200 mM sodium chloride (NaCl). The results of gel filtration chromatography are shown in FIG. In the obtained chromatogram, a single peak was observed near the theoretical molecular weight. Furthermore, the fractions that showed peaks in the chromatogram were concentrated and subjected to SDS-PAGE analysis, and a single band was confirmed near the theoretical molecular weight of 528 scFv. These results indicated that 528 scFv was successfully prepared uniformly. The single chain antibody 528 scFv thus prepared was used in the following examples.

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. We succeeded in homogeneous preparation of 528 Fv by purification by graphography. 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.

[Example 2] Aptamer Screening In this example, 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.

Two types of DNA libraries (library 1 with library sequence 1 and library sequence 2 with library sequence 2) have priming sequences (primer binding sequences for nucleic acid amplification) at both ends and single-stranded DNA with 30 mer random sequences between them. Library 2) was used for screening. Table 1 shows the single-stranded DNA sequences constituting each of the two types of DNA libraries and the oligo DNA used in the SELEX method in combination with each DNA library as described below.

In the first round of the SELEX method, in order to select DNA that binds to 528 scFv, 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.

1 μL 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.

Next, 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). After washing with 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. After further washing with PPB(Na+)-T, 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.

Next, 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) having sequences complementary to the respective priming sequences on the 5' and 3' sides of the single-stranded DNA in the recovered DNA library were used for PCR. The 3' primer (Bio-Rv primer) complementary to the 3' priming sequence is biotinylated.

Prepare the reaction solution (Table 3) and perform the PCR reaction at 98°C for 1 minute, then at 98°C for 15 seconds, at 50°C for 30 seconds, at 72°C for 20 seconds for 25 cycles, and then hold at 4°C. Nucleic acid amplification was performed by PCR.

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 Etatin 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.

Using the obtained DNA library, in the same way as the first round, selection of 528 scFv-binding DNA was performed, followed by extraction of recovered DNA, amplification by PCR, and recovery and purification using avidin beads as a second round. Furthermore, the SELEX method was repeated in the same manner until the fourth round.

DNA samples collected after 28 cycles of PCR amplification in each round of the SELEX method were electrophoresed. As a result, in SELEX using the library with library sequence 1, a smeared band was observed that was considered to be nonspecific amplification due to PCR, and it was observed that nonspecific amplification increased as the number of rounds increased. . On the other hand, in SELEX using the library having library sequence 2, a single band was obtained near the target base length in all rounds, confirming that non-specific amplification was low. Furthermore, in the SELEX method using the library having library sequence 2, the amplification efficiency by PCR using the same number of cycles was increased compared to the SELEX method using the library having library sequence 1.

Based on these results, in the SELEX method using the library having library sequence 1, 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.

Next, 94 single-stranded DNAs were sequenced for each of the DNA libraries obtained in the fourth round of the SELEX method using the library having library sequence 1 and the library having library sequence 2.

Specifically, 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. After purification, 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 μL, derived from library sequence 2: 7.5 μL) to 50 μL of E. coli Jet Competent Cell (DH5α) (ByoDynamics Laboratory Inc.) and leave it on ice for 5 minutes. Transformation was performed by. 900 μL of SOC medium was added to E. coli and cultured for 30 minutes. 100 μL of X-gal diluted 4 times with SOC medium (final concentration 5 ng/μL) was applied onto an ampicillin-containing agar plate medium, and the mixture was allowed to stand at room temperature for 30 minutes. Thereafter, the transformed E. coli competent cells were spread on the plate and cultured at 37°C for 16 hours. A total of 94 white colonies were selected and aliquoted into a 96-well plate, sample buffer from illustra TempliPhi DNA Amplification Kit (Cytiva) was added, and heat treated at 95°C for 3 minutes. Further, the 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. When 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.

As a result, 13 sequences with overlapping or common motifs were found (Table 6). These sequences were designated as aptamer candidate sequences.

[Example 3] Evaluation of binding ability of aptamer candidate sequence to scFv and Fv In this example, the binding ability of the aptamer candidate sequence obtained in Example 2 to scFv and Fv was evaluated.

i) Evaluation of the binding ability of aptamer candidate sequences to scFv by enzyme-linked oligonucleotide assay (ELONA) Regarding the 13 aptamer candidate sequences obtained in Example 2, library sequence 2 (SEQ ID NO: 9) in Table 1 was used. Oligonucleotides with priming sequences shown at both ends (CTATCTATGGTGAGTCCT [SEQ ID NO: 27] and CTAAGTACACACGCATCA [SEQ ID NO: 28]) added to the 5' and 3' ends, respectively, 5' block DNA-2 (SEQ ID NO: 10), and Bio Biotin modification was performed by mixing -Rv primer 2 (SEQ ID NO: 13).

528 scFv and 528 Fv were added to the plate by adding 100 μL 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. After washing with PPB(Na+)-T, 100 μL of a biotin-modified aptamer candidate sequence diluted to 100 nM with PPB(Na+) was added to each well and incubated at room temperature for 1 hour while shaking at 800 rpm. After washing with PPB(Na+)-T, 100 μL of streptavidin-alkaline phosphatase (Promega) diluted 1:1000 was added, incubated at room temperature for 1 hour while shaking at 800 rpm, and treated with PPB(Na+) in the same manner as above. - Washed with T. Add 100 μL of alkaline phosphatase substrate CDP-Star ^TM Substrate (0.25 mM Ready-To-Use) with Nitro-Block-I ^TM Enhancer (Thermo Fisher Scientific) to each well and incubate for 10 minutes at room temperature and protected from light. did. Subsequent chemiluminescence was measured with a plate reader Varioskan ^(R) Flash (Thermo Fisher Scientific).

The results are shown in Figure 7. In the two aptamer candidate sequences P2-63 and P1-12, the chemical bond strength to 528 scFv was significantly higher than that to 528 Fv. P2-63 and P1-12 were observed to exhibit scFv selective binding.

ii) Evaluation of scFv-binding ability of aptamer candidate sequences by dot blot analysis Aptamer candidate sequences P2-63 and P1-12, which showed scFv-selective binding, were further evaluated for their scFv-binding ability by dot blot analysis.

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. After washing with PPB(Na+)-T, the nitrocellulose membrane was shaken for 1 hour in an aptamer solution (final concentration 2 μM) 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/μL. 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 ^™ LAS 4000 mini (GE Healthcare).

The results are shown in Figure 8A. Comparing the chemiluminescence intensities on the nitrocellulose membrane, strong chemiluminescence was observed only in the scFv-immobilized spots for both P2-63 and P1-12, indicating scFv-selective binding (Figure 8A ). No chemiluminescence was observed in the immobilized bevacizumab spots as a negative control (FIG. 8A).

Furthermore, 20 pmol, 10 pmol, and 5 pmol of each of 528 scFv and 528 Fv were adsorbed and immobilized on the nitrocellulose membrane, and dot blot analysis was performed in the same manner as above. As a result, the chemiluminescence intensity at the spot increased in a scFv concentration-dependent manner for both P2-63 and P1-12 (FIG. 8B). P2-63 and P1-12 were shown to specifically bind to scFv in a scFv concentration-dependent manner.

iii) Evaluation of the binding ability of aptamer candidate sequences to scFv by biolayer interferometry (BLI) 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. Using the biomolecular interaction analysis system Personal Assay Octet ^(R) N1 System (SARTORIUS), 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).

In the above evaluation tests i) and ii), binding ability was evaluated by binding an aptamer candidate sequence in solution to scFv or Fv immobilized on a plate or nitrocellulose membrane. On the other hand, in the evaluation test iii), the binding ability of the immobilized aptamer candidate sequence with scFv or Fv in solution was evaluated. As mentioned above, selective binding of P2-63 and P1-12 to scFv was confirmed in all evaluation tests i) to iii), so P2-63 and P1-12 were It was shown that it can specifically bind not only to scFv immobilized on a support but also to free (soluble) scFv in solution. This result indicates that P2-63 and P1-12 do not recognize conformational changes in scFv caused by immobilization, and therefore can be widely used as aptamers for scFv with peptide linker (GGGGS) ₃ . shows.

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.

[Example 4] Evaluation of the binding ability of aptamer to other scFv In addition to the 528 scFv prepared in Example 1, 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. First, 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 μg/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 μg/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. Washing and elution buffers were 20 mM potassium phosphate buffer (PPB) + 0.5 M NaCl (pH 7.4) containing imidazole at concentrations of 0, 50, 150, 200, 300, or 500 mM, respectively. 5, 5, and 5 CV (1 CV=1 mL) were used. 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. . As shown in Figures 14A and 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 μM. After washing with PPB(Na+)-T, it was shaken for 1 hour in neutravidin-HRP conjugate (Thermo Fisher Scientific) diluted to 0.2 ng/μL. 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 ^™ LAS 4000 mini (GE Healthcare).

The results are shown in Figure 10. For both P2-63 and P1-12, chemiluminescence was observed in the spots where 528 scFv and anti-Hb scFv-SpyTag were immobilized, respectively. No chemiluminescence was observed in spots fixed with 528 Fv or frozen and thawed 528 Fv. This result showed that aptamers P2-63 and P1-12 can bind not only to 528 scFv but also to another scFv.

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).

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. After confirming that the sides of the roller bottle were confluent, 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. After purification using a His tag by Ni-NTA affinity chromatography, the eluted fraction in which a band was observed near the theoretical molecular weight in SDS-PAGE was concentrated and subjected to gel filtration chromatography. The sEGFR thus obtained was used below.

100 μL of 100 nM sEGFR was added to a 96-well plate and fixed by adsorption onto the plate by incubating at 37°C for 90 minutes. 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.

By the method described in Example 3, 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.

After washing the plate with PPB(Na+)-T, 100 μL each of the aptamer-scFv antibody conjugate was added and allowed to react at room temperature for 1 hour while shaking at 600 rpm. Add 100 μL of HRP-modified anti-c-Myc antibody (Cosmo Bio Inc.) diluted 1000 times or 100 μL of neutravidin-HRP conjugate (Thermo Fisher Scientific) diluted to 0.2 ng/μL, and incubate at 600 rpm. Plates were incubated for 1 hour at room temperature with shaking and then washed with PPB(Na+)-T as above. 100 μL of BM chemiluminescent ELISA substrate (Thermo Fisher Scientific) 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. Regardless of the aptamer concentrations used (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). Thus, 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.

Measurements based on detection of biotin-modified aptamers via avidin-biotin interaction using neutravidin-HRP conjugates showed that chemiluminescence levels increased in an aptamer concentration-dependent manner (Figure 12). This confirmed that the aptamer bound to 528 scFv bound to sEGFR on the plate to form a complex (conjugate).

The above results showed that 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.

[Example 6] Analysis of secondary structure of aptamer by CD spectrum measurement CD spectrum measurement was performed to investigate the secondary structure of aptamers P1-12 and P2-63 under various potassium concentration conditions.

A 100 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 7.4) containing 0 mM, 10 mM, or 100 mM potassium was prepared. Aptamers P1-12 and P2-63 were diluted to 2 μM 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.

In the CD spectrum of P1-12, at potassium concentrations of 10 mM and 100 mM, a positive peak around 265 nm and a negative peak around 240 nm, which are characteristics of a parallel G4 structure, are seen, indicating the formation of a parallel G4 structure. (Fig. 13A). On the other hand, no obvious peak was observed at a potassium concentration of 0 mM, and no secondary structure was formed (FIG. 13A). This result showed that P1-12 forms a parallel G4 structure in the presence of potassium ions.

In the CD spectrum of P2-63, a positive peak was observed near 265 nm and a negative peak was observed near 240 nm at any potassium concentration, indicating the formation of a parallel G4 structure (Figure 13B). Furthermore, the molar ellipticity increased in a potassium concentration-dependent manner (FIG. 13B). This indicates that while P2-63 forms a parallel G4 structure regardless of the presence or absence of potassium, it becomes easier to form a parallel G4 structure depending on the potassium concentration.

[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. coli transformed with the expression vector pRA-anti-Hb scFv-SnT. The vector map of pRA-anti-Hb scFv-SnT is shown in FIG. 14B, and the protein coding sequence (SEQ ID NO: 31) and the amino acid sequence encoded by it (SEQ ID NO: 32) in pRA-anti-Hb scFv-SnT are shown in FIG. 16. . As shown in Figure 14B and Figure 16, 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 . .

100 μL 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.

Aptamer dilution solutions in which the 3' ends of aptamers (P1-12, P2-63) were modified with biotin via complementary strands by the method described in Example 3 and diluted to different concentrations with PPB (Na+), and 200 nM 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.

After washing the plate with PPB(Na+)-T, 100 μL each of the aptamer-anti-Hb scFv-SnT antibody conjugate was added and allowed to react at room temperature for 1 hour while shaking at 600 rpm. Add 100 μL of HRP-modified anti-c-Myc antibody (Cosmo Bio Inc.) diluted 1000 times or 100 μL of neutravidin-HRP conjugate (Thermo Fisher Scientific) diluted to 0.2 ng/μL, and incubate at 600 rpm. Plates were incubated for 1 hour at room temperature with shaking and then washed with PPB(Na+)-T as above. 100 μL of BM chemiluminescent ELISA substrate (Thermo Fisher Scientific) 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. Thus, 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.

The above results further supported that aptamers P2-63 and P1-12 can bind to scFv without inhibiting the antigen-binding activity of scFv.

[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. 14C, and the protein coding sequence (SEQ ID NO: 33) and the amino acid sequence encoded thereby (SEQ ID NO: 34) in pRA-HB125 scFv are shown in FIG. 17. As shown in FIG. 10C and FIG. 17, 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 μm 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. 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 μM fold aptamer solution containing an equimolar amount of tris(2-carboxyethyl)phosphine (TCEP; Fujifilm Wako Pure Chemical Industries, Ltd.) dissolved in Tris-HCl. The aptamer was immobilized on the gold disk electrode by soaking the disk electrode overnight at 25°C. 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. For 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.

As a result of SWV measurement, in both electrodes immobilized with aptamer P1-12 or P2-63, the peak current value derived from potassium ferricyanide, which was observed around 0.2 V, decreased as the HB125 scFv concentration increased (Figure 19). The results showed that the flux of free ferricyanine changed with scFv binding to the aptamer. Furthermore, high linearity in peak current value changes was obtained in the range of 10 nM to 1 μM (Figure 20). 19 and 20 show that scFv can be electrochemically detected using the aptamer of the present invention.

Furthermore, 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).

As a result, in both electrodes immobilized with aptamer P1-12 or P2-63, the peak current value derived from potassium ferricyanide, which was observed around 0.2 V, decreased as the HB125 scFv concentration increased (Figure 21). Furthermore, high linearity in peak current value changes was obtained in the range of 10 nM to 1 μM (Figure 22). 21 and 22 show that scFv can be electrochemically detected using the aptamer of the present invention even in a cell culture medium. This indicates that the aptamer of the present invention is also useful for monitoring the production process in the scFv production process by culturing scFv-producing cells.

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. Alternatively, 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.

All publications, patents, and patent applications cited herein are incorporated by reference in their entirety.

Claims

An aptamer comprising an oligonucleotide that specifically binds to a single chain Fv (scFv) having a peptide linker (GGGGS) 3 (SEQ ID NO: 3).
The aptamer according to claim 1, wherein the oligonucleotide includes a base sequence having 90% or more sequence identity to the base sequence shown in SEQ ID NO: 14 or 24.
The aptamer according to claim 2, wherein the oligonucleotide is chemically modified.
The aptamer according to claim 3, wherein the oligonucleotide is labeled.
The oligonucleotide has at least one bond 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. The aptamer according to claim 3, wherein the aptamer has been chemically modified by or by introduction, and/or by containing at least one of a modified nucleotide and an artificial base.
The aptamer according to claim 1 or 2, wherein the oligonucleotide is DNA or RNA.
A composition for detecting, modifying or purifying scFv having a peptide linker (GGGGS) 3 (SEQ ID NO: 3), comprising the aptamer according to any one of claims 1 to 5.
A kit for detecting, modifying or purifying scFv having a peptide linker (GGGGS) 3 (SEQ ID NO: 3), comprising the aptamer according to any one of claims 1 to 5.
A device for detecting or purifying scFv having a peptide linker (GGGGS) 3 (SEQ ID NO: 3), comprising the aptamer according to any one of claims 1 to 5 and a support to which it is bound.
A peptide linker comprising contacting an aptamer according to any one of claims 1 to 5 with a sample containing an scFv to produce an scFv-aptamer complex, and separating the scFv-aptamer complex from the sample. A method for purifying scFv having (GGGGS) 3 (SEQ ID NO: 3).
A peptide linker (GGGGS) 3 (GGGGS) comprising contacting the aptamer according to claim 4 with a sample containing an scFv to produce an scFv-aptamer complex, and detecting a labeling signal derived from the scFv-aptamer complex. Method for detecting scFv having SEQ ID NO: 3).
A nucleotide mutation is introduced into the aptamer according to claim 2, and the binding ability of the resulting mutant to an scFv having a peptide linker (GGGGS) 3 (SEQ ID NO: 3) is measured, and the aptamer specifically binds to the scFv. 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 a variant as an aptamer.
Modifying an scFv comprising contacting an aptamer according to any one of claims 1 to 5 with an scFv having a peptide linker (GGGGS) 3 (SEQ ID NO: 3) to generate an scFv-aptamer complex. Method.
14. The method according to claim 13, wherein the aptamer comprises the oligonucleotide that has been chemically modified, and the method is a method of chemically modifying scFv.
An scFv-aptamer complex comprising an scFv with a peptide linker (GGGGS) 3 (SEQ ID NO: 3) coupled to an aptamer according to any one of claims 1 to 5.