KR20220032276A - A complex comprising an antibody to Digoxigenin and use thereof - Google Patents

Abstract

The present disclosure relates to a complex in which an anti-digoxigenin antibody is coupled with a conjugate of digoxigenin and a binder, a method for preparing the corresponding complex, and a use of the complex. According to an aspect of the present disclosure, a complex (drug-conjugated Oligobody: DOligobody) including: an antibody specifically recognizing digoxigenin or an antigen-binding fragment; and a conjugate (drug-conjugated oligomer: DOligomer) including digoxigenin and a physiologically active substance or a detection label is expected to control a concentration of the physiologically active substance or the detection label in the human body or to increase the in vivo half-life of the drug, as needed.

Description

A complex comprising an antibody to digoxigenin, and their use {A complex comprising an antibody to Digoxigenin and use thereof}

The present disclosure relates to a complex in which an anti-digoxigenin antibody is bound to a conjugate of digoxigenin and a conjugated substance, a method for preparing the complex, and use of the complex.

Various physiologically active ingredients such as compounds, ligands, and cytotoxic substances or detection markers lose their efficacy through various mechanisms, such as being degraded or converted into other components in the body. In particular, since most substances are decomposed and excreted through the kidneys, thereby losing their efficacy, research to increase the in vivo half-life to enhance the bioavailability of physiologically active ingredients or detection markers is in progress.

In this regard, there have been attempts to develop an antibody-drug conjugate (antibody-drug conjugate). For example, a drug to be delivered to a target tissue, etc. is covalently linked to an antibody that binds to a target tissue, cell, protein, etc. (Antibody-drug conjugates—an emerging class of cancer treatment. Nikolaos Diamantis, Udai Banerji. Br J Cancer. 2016 Feb 16; 114(4): 362-367.). In the meantime, attempts have been made to conjugate polyethylene glycol (PEG) to increase the half-life of the drug as well (PEGylation, successful approach to drug delivery. Francesco M. Veronese, Gianfranco Pasut. Drug Discovery Today. 2005 Nov 1; 10(21): 1451-1458.).

However, it is very difficult to deliver the desired drug to the inside of the desired tissue by penetrating the capillaries because the drug that is directly connected to the antibody or polyethylene glycol and reaches the target tissue or cell becomes a very large molecule in the form connected to these. Accordingly, there is a need to develop a new type of drug delivery system that can increase the residence time of the drug in the human body and at the same time deliver the drug well to a target tissue or cell.

On the other hand, digoxigenin (Digoxigenin) is a kind of steroid found extracted from a plant called digitalis (Digitalis purpurea), it corresponds to aglycone, a non-carbohydrate part of the glycoside of digoxin (Digoxin). Digoxigenin is generally known as a hapten, a substance that does not have immunogenicity, and is frequently used in the field of molecular biology using this property.

Digoxigenin is used for similar purposes to other widely used haptens, such as 2,4-dinitrophenol, biotin, and fluorescein. In order to measure biomolecules such as nucleic acids, they are chemically bound to the biomolecules. Among them, biotin, fluorescein and digoxigenin are known as the most commonly used haptens.

Biotin has the advantage of having the strongest binding force among them by binding to avidin, but there is a disadvantage that non-specific binding to avidin may occur because some tissues such as the liver may contain endogenous biotin. .

In addition, the fluorescein hapten has the advantage of directly checking the bound biomolecules with a fluorescence microscope or indirectly using an anti-fluorescein antibody, but background problems may arise when viewing data using a microscope. And the signal doesn't last long.

On the other hand, since digoxigenin is widely used in the field of molecular biology, various antibodies targeting it have already been commercialized. Anti-digoxigenin antibodies having high avidity and specific binding affinity are used in various biological immunoassays (eg, ELISA). Antibodies are directly or indirectly labeled with dyes, enzymes or fluorescent substances for the purpose of visualization or measurement of substances.

In this respect, digoxigenin is used as various types of immuno-labelling, especially as a standard immunohistochemical label for in situ hybridization experiments. In this experiment, digoxigenin is incorporated into RNA by conjugation to a specific species of RNA nucleoside triphosphate (usually uridine).

On the other hand, with respect to the antibody against digoxigenin, which is used only for the purpose of hapten in the field of molecular biology, modification on the premise of use in the human body has not been made.

Republic of Korea Patent No. 10-1648960 Republic of Korea Patent No. 10-1827962

The inventors of the present disclosure paid attention to digoxigenin, which was conventionally used as a biochemical detection means, while examining a system capable of increasing the residence time in a drug delivery system, and digoxigenin and a humanized antibody system therefor were administered to the human body. It has been found that it can be used for drug delivery systems that increase the half-life of drugs. To this end, the conventional anti-digoxigenin mouse antibody was developed as a humanized antibody and as a result of earnest efforts to construct a drug delivery system using the same, the invention of the present disclosure was developed, and the effect of increasing the human half-life was confirmed.

In order to solve the above problems, one aspect of the present disclosure is an anti-digoxigenin antibody, specifically an antibody or antigen-binding fragment thereof that specifically recognizes digoxigenin; And it provides a complex comprising a conjugate comprising digoxigenin and a drug.

An antibody or antigen-binding fragment thereof that specifically recognizes digoxigenin according to an aspect of the present disclosure; And digoxigenin and a physiologically active substance or a conjugate containing a detection label (Drug-conjugated Oligomer; DOligomer) through the complex (Drug-conjugated Oligobody; DOligobody), if necessary, the concentration of the physiologically active substance or detection label in the human body It is expected to be able to control the drug or increase the in vivo half-life of the drug.

1 ( FIGS. 1A to 1C ) shows a mouse antibody (mAb[21H8]), a chimeric antibody (chimeric[21H8]), a humanized antibody (humab[21H8]-v1, humab[21H8]-v2, humab[21H8]-v3, humab[21H8]-v4, humab[21H8]-v5, humab[21H8]-v6, humab[21H8]-v7, humab[21H8]-v8, humab[21H8]-v9, humab Dig of [21H8]-v10, humab[21H8]-v11, humab[21H8]-v12, humab[21H8]-v13, humab[21H8]-v14, humab[21H8]-v15, humab[21H8]-v16) It is a graph of the result of confirming the binding force with oxygenin (DIG) by ELISA experiment.
2 is a diagram illustrating the structure of a complex (Drug-conjugated Oligobody; DOligobody) by binding the antibody prepared in the present disclosure and the DIG-aptamer-MMAE conjugate.
3A is a diagram of a compound structure (DIG-aptamer) in which digoxigenin and an aptamer are connected through a linker, and B is a diagram of a DIG-aptamer-MMAE conjugate structure in which MMAE is bonded to the corresponding DIG-aptamer.
FIG. 4 is a diagram analyzing the purity of the hapten-aptamer compound and the synthesized hapten-aptamer-drug conjugate (DIG-aptamer-MMAE) by detecting and analyzing the purity using reversed-phase analytical HPLC. Specifically, FIG. 4A is the HPLC analysis result of the hapten-aptamer compound (DIG-SQ7-1(8)-SH), and FIG. 4B is the hapten-aptamer-drug conjugate (DIG-SQ7-1(8). )-MMAE) is the result of HPLC analysis.
Figure 5 confirms the pancreatic cancer cell killing efficacy of the hapten-aptamer-drug conjugate (DIG-aptamer-MMAE), Figure 5a is confirmed through optical microscope imaging, Figure 5b is the result of confirming the cell death rate through ATP assay It is a graph.
6 ( FIGS. 6A to 6C ) is a graph of the result of confirming the binding force of the complex (DOligobody) to CFPAC-1, a human pancreatic cancer cell line, by binding the antibody prepared in the present disclosure with the DIG-aptamer-MMAE conjugate by FACS experiment.
7 is a graph comparing the binding rate of DOligobody, which is a complex of the present disclosure, based on FACS results.
8 is a schematic diagram illustrating the outline of a pharmacokinetic experiment of a complex DOligobody comprising an anti-digoxigenin antibody.
9 is a conjugate containing digoxigenin and an aptamer (DOligomer) and an anti-digogenin antibody of the humab[21H8]-v5 antibody, the humab[21H8]-v6 antibody, and the humab[21H8]-v8 antibody It is a graph disclosing the pharmacokinetic results for the complex DOligobody.
10 is a conjugate comprising digoxigenin and an aptamer (DOligomer) and an anti-digoxigenin antibody of the humab[21H8]-v5 antibody, the humab[21H8]-v6 antibody, and the humab[21H8]-v8 antibody This is a table summarizing the half-life measured through the pharmacokinetic results of the complex DOligobody.
11 is a graph showing the pharmacokinetic results for a complex DOligobody comprising an anti-digogenin antibody of humab[21H8]-v5 antibody, humab[21H8]-v6 antibody, and humab[21H8]-v8 antibody.
12 is a humab[21H8]-v5 antibody, a humab[21H8]-v6 antibody, and an anti-digoxigenin antibody of the humab[21H8]-v8 antibody. The half-life measured through the pharmacokinetic results for the complex DOligobody. This is an organized table.
Figure 13a is a negative control group administered with a carrier (Vehicle) to the pancreatic cancer orthotopic animal model; The experimental group administered with the complex DOligobody (DIG-SQ7-1(8)-MMAE + humab[21H8]-v8; and the conventional anticancer drug Gemcitabine) containing a complex DOligobody containing an anti-digogenin antibody It is a view explaining a method for measuring the volume of a pancreatic tumor Fig. 13b is a time frame showing cells and the timing of each treatment in the experiment of the orthotopic animal model.
14 is a negative control group administered with a carrier (Vehicle) in an orthotopic animal model of pancreatic cancer; an experimental group administered with a complex DOligobody (DIG-SQ7-1(8)-MMAE + humab[21H8]-v8) containing an anti-digoxigenin antibody; And it is a diagram measuring the size (Luminesence intensity) of the pancreatic tumor of the positive control group administered with the existing anticancer drug Gemcitabine through IVIS fluorescence imaging equipment for 5 weeks. 14A is a graph showing the size of a pancreatic tumor measured using IVIS fluorescence imaging equipment, and FIG. 14B is an imaging photograph measured by the IVIS fluorescence imaging equipment in the negative control group, the experimental group, and the positive control group on the 30th day.
15a is a negative control group administered with a carrier (Vehicle) in an orthotopic animal model of pancreatic cancer; an experimental group administered with a complex DOligobody (DIG-SQ7-1(8)-MMAE + humab[21H8]-v8) containing an anti-digoxigenin antibody; And it is an abdominal MRI measurement image for confirming the pancreatic tumor of the positive control group administered with the existing anticancer drug Gemcitabine, Figure 15b is a graph showing the average pancreatic tumor volume confirmed above.
16 is a negative control group administered with a carrier (Vehicle) in an orthotopic animal model of pancreatic cancer; an experimental group administered with a complex DOligobody (DIG-SQ7-1(8)-MMAE + humab[21H8]-v8) containing an anti-digoxigenin antibody; and a diagram showing the results of performing TUNEL assay by extracting pancreatic tissue after completion of the animal experiment of the positive control group administered with the existing anticancer drug Gemcitabine.
17 is a negative control group administered with a carrier (Vehicle) in an orthotopic animal model of pancreatic cancer; an experimental group administered with a complex DOligobody (DIG-SQ7-1(8)-MMAE + humab[21H8]-v8) containing an anti-digoxigenin antibody; And it is a graph measuring the body weight change during the animal experiment period of the positive control group administered with the existing anticancer drug Gemcitabine.
18 is a negative control group administered with a carrier (Vehicle) in an orthotopic animal model of pancreatic cancer; an experimental group administered with a complex DOligobody (DIG-SQ7-1(8)-MMAE + humab[21H8]-v8) containing an anti-digoxigenin antibody; And it is a graph showing the results of measuring the hepatotoxicity index (ALT, AST, TBIL) and renal toxicity index (BUN, CRE) with the serum of the positive control group administered with the existing anticancer drug Gemcitabine. Specifically, FIG. 18A is a result of analyzing blood collected 3 days after administration of each administration group once, and FIG. 18B is a result of analyzing blood collected after multiple administration (five times) of each administration group.

Embodiments or aspects of the present disclosure are exemplified for the purpose of explaining the technical spirit of the present disclosure. The scope of the rights according to the present disclosure is not limited to the embodiments or aspects presented below or specific descriptions thereof.

In addition, those skilled in the art to which this disclosure pertains will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific aspects of the disclosure described herein. Such equivalents are intended to be encompassed by this disclosure.

All technical and scientific terms used in this disclosure, unless otherwise defined, have the meanings commonly understood by one of ordinary skill in the art to which this disclosure belongs. All terms used in the present disclosure are selected for the purpose of more clearly describing the present disclosure and not to limit the scope of the present disclosure.

As used in this disclosure, expressions such as "comprising", "including", "having", etc. are open-ended terms connoting the possibility of including other embodiments, unless otherwise stated in the phrase or sentence in which the expression is included. (open-ended terms).

Expressions such as “consisting only of” a corresponding component used in the present disclosure should be understood as closed-ended terms excluding the possibility of including other components in addition to the corresponding component.

Expressions in the singular in this disclosure may include the meaning of the plural unless otherwise stated, and the same applies to expressions in the singular in the claims.

In the present disclosure, "antibody" refers to a substance produced by stimulation of an antigen in the immune system, and the type thereof is not particularly limited. Antibodies herein include animal antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies. Also, as used herein, the term "antibody" also includes an antigen-binding fragment of an antibody having antigen-binding ability, for example, Fab.

In the present disclosure, "chimeric antibody" refers to an antibody derived from an animal in which an antibody variable region or a complementarity determining region (CDR) thereof is different from the rest of the antibody. Such an antibody may be, for example, an antibody in which the antibody variable region is derived from a non-human animal (eg, mouse, rabbit, poultry, etc.) and the antibody constant region is a human-derived antibody. Such chimeric antibodies can be prepared by methods such as genetic recombination known in the art.

The antibody may be a polyclonal antibody or a monoclonal antibody. The antibody may be selected from all subtypes of immunoglobulins (eg, IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM, etc.). The antibody in the form of IgG may be in the form of an IgG1, IgG2, IgG3, or IgG4 subtype, such as an IgG1 or IgG2 subtype.

As used herein, an "antigen-binding fragment" of a chain (heavy or light chain) of an antibody or immunoglobulin comprises a portion of an antibody that lacks some amino acids compared to the full-length chain, but is capable of specifically binding to an antigen. will be. Such antigen-binding fragments can be said to be biologically active in the sense that they can specifically bind to a target antigen, or compete with other antibodies or antigen-binding fragments for binding to a specific epitope. Specifically, the antigen-binding fragment is an antibody fragment comprising one or more complementarity determining regions, such as scFv, (scFv) ₂ , scFv-Fc, Fab, Fab' and F(ab') ₂ Selected from the group consisting of may be, but is not limited thereto. Such biologically active fragments may be produced by recombinant DNA techniques, or may be produced, for example, by enzymatic or chemical cleavage of intact antibodies.

In the present disclosure, "heavy chain" refers to a full-length heavy chain comprising a variable region domain VH comprising an amino acid sequence of a sufficient variable region to confer specificity for an antigen, and three constant domain domains CH1, CH2 and CH3, and fragments thereof refers to all

In the present disclosure, "light chain" refers to both a full-length light chain including a variable region domain VL and a constant region domain CL comprising an amino acid sequence of a variable region sufficient to confer specificity to an antigen, and a fragment thereof.

In the present disclosure, the term "complementarity determining region" refers to a site that confers antigen-binding specificity among the variable regions of an antibody.

In the present disclosure, "anti-digoxigenin humanized antibody or antigen-binding fragment thereof" refers to a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 5; and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 10. For example, an anti-digoxigenin humanized antibody or antigen-binding fragment thereof of the present disclosure can be

a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2; and an anti-digoxigenin humanized antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7;

b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3; and an anti-digoxigenin humanized antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7;

c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4; and an anti-digoxigenin humanized antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7;

d) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5; and an anti-digoxigenin humanized antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7;

e) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2; and an anti-digoxigenin humanized antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8;

f) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3; and an anti-digoxigenin humanized antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8;

g) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4; and an anti-digoxigenin humanized antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8;

h) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5; and an anti-digoxigenin humanized antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8;

i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2; and an anti-digoxigenin humanized antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 9;

j) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3; and an anti-digoxigenin humanized antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 9;

k) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4; and an anti-digoxigenin humanized antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 9;

l) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5; and an anti-digoxigenin humanized antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 9;

m) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2; and an anti-digoxigenin humanized antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10;

n) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3; and an anti-digoxigenin humanized antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10;

o) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4; and an anti-digoxigenin humanized antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10, or

p) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5; And anti-digoxigenin humanized antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10

can be

In the present disclosure, a "humanized antibody" is an antibody containing a sequence derived from a non-human immunoglobulin that specifically binds to a specific antigen, and a complementarity determining region (CDR) that determines the binding affinity to an antigen in the non-human immunoglobulin. It is a human immunoglobulin replaced with the sequence of On the other hand, in the humanized antibody, human immunoglobulin Fv framework region (FR) residues may be replaced with corresponding non-human FR residues, or which are not found in non-human immunoglobulin or CDR or FR sequences of human immunoglobulin. residues may be included. These modifications may be made to further enhance and optimize antibody performance. In general, a humanized antibody is one in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin, and all or substantially all of the FR residues have at least one, typically two variable, residues of a human immunoglobulin consensus sequence. It may include substantially all of the domains.

In the present disclosure, the anti-digoxigenin humanized antibody or antigen-binding fragment thereof may include the amino acid sequence of any one of SEQ ID NOs: 2 to 5, or the amino acid sequence of any one of SEQ ID NOs: 7 to 10. The present disclosure provides in one aspect about 80% or more, specifically about 90% or more, more specifically about 95% or more, even more specifically about 97, with any one of SEQ ID NOs: 2-5, or SEQ ID NOs: 7-10. It provides an antibody comprising an amino acid sequence having at least % homology, most specifically at least about 99% homology, and substantially any one of the amino acid sequence of SEQ ID NOs: 2 to 5, or SEQ ID NO: Any amino acid sequence constituting the antibody having the same or corresponding binding activity to the antibody comprising the amino acid sequence of any one of 7 to 10 is included without limitation. In addition, if it is an amino acid sequence having such homology, it is apparent that amino acid sequences in which some sequences are deleted, modified, substituted or added are also included in the scope of the present disclosure.

In the present disclosure, "conjugate" refers to a molecule different from digoxigenin, in particular, a chimeric molecule of a bioactive substance or detection label described below. In the present disclosure, a conjugate means that digoxigenin and another molecule are covalently conjugated, and the physiologically active substance is, for example, a chemotherapeutic drug, an enzyme, a peptide, a cytotoxin, or an affinity ligand. can Specifically, in the conjugate of the present disclosure, digoxigenin and a bioactive substance or a detection label may be covalently conjugated through an aptamer. Covalently conjugated means that molecules are directly covalently bonded to each other or are indirectly covalently linked to each other through a linker, a space, a connecting moiety, or the like. The conjugate to which digoxigenin and a physiologically active substance or detection label of the present disclosure are bound may also be referred to as a drug-conjugated oligomer (DOligomer), meaning a drug-conjugated oligomer.

In the present disclosure, an aptamer is a short single-stranded oligonucleotide having a property of binding to a target target with high affinity and specificity, and may mean having a unique three-dimensional structure. The aptamer specifically binds to a target tissue, target cell, target carbohydrate, target lipid, or target protein of interest, and the target tissue, target cell, target carbohydrate, target lipid, or target protein is specific for a specific condition. It can be a tissue, cell, carbohydrate, lipid, or protein, eg, an infected tissue, an infected cell, an infection-specific carbohydrate, an infection-specific lipid, an infection-specific protein; inflamed tissue, inflammatory cells, inflammation-specific carbohydrates, inflammation-specific lipids, inflammation-specific proteins; tumor tissue, tumor cells, tumor-specific carbohydrates, tumor-specific lipids, or tumor proteins; and the like, but is not limited thereto. In the present disclosure, the aptamer may include, for example, a nucleotide sequence of any one of SEQ ID NOs: 11 to 23 that binds to cancer cells, particularly pancreatic cancer tissue or pancreatic cancer cells.

On the other hand, a technology for combining an aptamer and a drug has been variously developed in the technical field to which the present disclosure pertains. For example, in Gray, et al. (PNAS; 2018. 5. 1.; 115 (18) pp4761-4766), an aptamer (E3; RNA) and MMAE/MMAF are combined through a linker to treat prostate cancer. The construction is disclosed in Kratschmer, et al. In (Mol Ther Nucleic Acids; 2018. 3. 2.; 10: pp227-236), an aptamer (E07 or Waz; DNA) that targets EGFR or TfR and MMAE/MMAF are combined through a linker to treat pancreatic cancer. The construction is disclosed, Yoon, et al. In (Mol Ther Nucleic Acids; 2017. 3. 17.; 6: pp80-88), -H2AX-binding aptamer (P19; RNA) and MMAE/DM1 are combined through sticky sequence (SE) to treat pancreatic cancer A configuration is disclosed. See also Liu, et al. (J Transl Med; 2012. 7. 20.; 10: p148) discloses a configuration used for treating breast cancer by binding DM1 with an aptamer (HB5; DNA) that targets HER2, Dou, et al. (Int J Nanomedicine; 2018. 2. 5.; 13: pp763-776) discloses a composition used for breast cancer treatment by linking a HER3 targeting DNA aptamer and doxorubicin with DSPE-PEG or liposomes, Huang, etc. In (Chembiochem; 2009. 3. 23.; 10(5): pp862-868), an aptamer (sgc8; DNA) targeting PTK7 (protein tyrosine kinase 7) and doxorubicin were combined through a hydrazone linker to prevent leukemia. A configuration using it is disclosed in Liu, et al. (Cell Prolif; 2019. 1.; 52(1): e12511) discloses a configuration in which PTK7-targeting aptamers (sgc8-TDNs) and doxorubicin form a complex for use in leukemia. This is summarized in Table 1 below.

literature Aptamer target linker drug target disease Gray, et al. E3 (RNA) MC-VC-PAB MMAE, MMAF prostate cancer Kratschmer, et al. E07,Waz (DNA) EGFR, TfR MC-VC-PAB MMAE, MMAF pancreatic cancer Yoon, et al. P19 (RNA) -H2AX sticky sequence (SE) MMAE, DM1 pancreatic cancer Liu, et al. HB5 (DNA) HER2 DM1 breast cancer Dou, et al. (DNA) HER3 DSPE-PEG, liposomes doxorubicin breast cancer Huang, et al. sgc8 (DNA) PTK7 hydrazone linker doxorubicin leukemia Liu, et al. sgc8c-TDNs PTK7 Nano doxorubicin leukemia

According to the knowledge of the relevant technical field as described above, various aptamers can be cooked and selected according to the purpose, and a drug suitable for the purpose can be selected and bound to the aptamer.

As a result of the inventors of the present disclosure trying to develop an aptamer that binds to cancer cells, it was confirmed that the aptamers of SEQ ID NOs: 11 to 40 bind to cancer cells (refer to Korean Patent Application Laid-Open No. 10-2020-0078303). In one aspect, the present disclosure provides about 80% or more, specifically about 90% or more, more specifically about 95% or more, even more specifically about 97% or more, most of the nucleotide sequence of any one of SEQ ID NOs: 11 to 40. Specifically, it provides a DNA aptamer comprising a nucleotide sequence having a sequence homology of about 99% or more, and a DNA aptamer substantially comprising any one nucleotide sequence of SEQ ID NOs: 11 to 40 as a sequence having such homology. The nucleotide sequence constituting the aptamer having the same or corresponding binding activity to the tamer is included without limitation. In addition, if it is a nucleotide sequence having such homology, it is obvious that a nucleotide sequence in which some sequences are deleted, modified, substituted or added is also included in the scope of the present disclosure. In one aspect, the present disclosure may be an aptamer consisting of the nucleotide sequence of any one of SEQ ID NOs: 11 to 40.

On the other hand, the aptamer of the present disclosure may include a linker if necessary, and the linker is, for example, a C6 linker, MC-VC-PAB, sticky sequence (SE), DSPE-PEG, liposome, hydrazone linker, Oligo dT, Oligo dA, DNA hairpin, Poly A linker containing 3GC repeat, pyrimidine sequence (4nt), 5S rRNA 3-way binding, RCA, SH-mediated, CopA-CopT binding, Au®MgO nanoflowers, globular AuNPs , AuNP, AgNP, DNA scaffold, biotin-sterptavidin binding, polyU linker, and the like, but is not limited thereto. The linker may be conjugated to the aptamer by a technique widely used in the art (Vorobyeva M, et al., Molecules. 2016.).

In one aspect of the present disclosure, the chemotherapeutic drug or cytotoxic agent is a DNA production inhibitor (eg, calicheamicin, pyrrolobenzodiazepine (PBD), duocarmycin, anthracycline/Nemorubicin), doxorubicin , irinotecan, amatoxin), microtubule inhibitors (auristatin, maytansine, tubulin), inhibitors of topoisomerase I (SN-38), inhibitors of topoisomerase II, platinum coordination compounds, alkylating agents, Photosensitizers (e.g. 5-Aminolaevulinic acid (ALA), Benzoporphyrin derivative monoacid ring A (BPD-MA), Chlorine, Tetra (m-hydroxyphenyl) chlorin (mTHPC), Lutetium texaphyrin, MMAE (monomethyl auristatin E), MMAF) (monomethyl auristatin F), may be one selected from the group consisting of DM4 and DM1, but is not limited thereto.

ller A, 등, Adv Drug Deliv Rev Actions., 2018. 9.;134:94-106.).In one aspect of the present disclosure, the detection label is a fluorescent substance, for example, a fluorescent dye, a fluorescent protein including a tetracysteine fluorescent motif, Cy3, Cy5, Cy5-FITC, GFP, YFP, CFP, and RFP, or a fluorescent protein that emits fluorescence. Nanoparticles, ¹⁸ F, ⁶⁴ Cu, ^99m Tc, ¹¹¹ In ¹²⁴ I, ¹²⁵ I, ¹³¹ I, ¹⁸⁸ Re and a radiolabeled material containing a radioactive isotope selected from the group consisting of ²¹¹ At, for example ¹⁸ F- It may be selected from the group consisting of FDG (2-deoxy-2-[ ¹⁸ F]fluro-D-glucose), Magnetic NPs, Quantum Dots, Microbubbles, and glass dosimeter, but is limited thereto doesn't happen The detection label directly or indirectly bound to the aptamer may be conjugated to the aptamer by a technique widely used in the art (Bouvier-M).

ller A, et al., Adv Drug Deliv Rev Actions., 2018. 9.; 134:94-106.).

In the present disclosure, “complex” refers to a complex in which an anti-digoxigenin humanized antibody or antigen-binding fragment thereof of the present disclosure is bound to a conjugate comprising digoxigenin of the present disclosure. The complex may be such that the antibody or antigen-binding fragment thereof and the conjugate have non-peptidic covalent bonds (eg, disulfide bonds) and/or non-covalent interactions (eg, hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions, etc.). The complex in which the conjugate of the present disclosure and the anti-digoxigenin antibody are bound may also be referred to as a drug-conjugated oligobody (DOligobody), meaning a drug-binding oligobody. Specifically, in the present disclosure, the complex comprises (a) an anti-digoxigenin humanized antibody or antigen-binding fragment thereof, (b) digoxigenin, (c) a cytotoxin or detection label, and (d) a linker. As a complex including an aptamer that may include, the (b) digoxigenin and (c) a cytotoxin or detection label are shared through an aptamer that may include (d) a linker It may be a complex in the form of a conjugate that has been physically joined.

In the present disclosure, "homology" refers to the degree of the same degree of nucleotides or amino acid residues between sequences after aligning both sequences to the maximum extent in a specific comparison region in the amino acid sequence or nucleotide sequence of a gene encoding a protein. . When the homology is sufficiently high, the expression product of the gene may have the same or similar activity. The percentage (%) of the sequence identity can be determined using a known sequence comparison program (eg, Blast (NCBI), etc.).

In one aspect of the present disclosure, the term "about" is used to include errors in manufacturing processes included in specific values or slight numerical adjustments included in the scope of the technical spirit of the present disclosure. For example, the term “about” means a range of ±10%, ±5% in one aspect, and ±2% in another aspect of the value to which it refers. In the field of this disclosure, this level of approximation is appropriate unless a value is specifically stated to require a narrower range.

Proteins or antibodies in the present disclosure may be expressed and produced through methods known in the art to which the present disclosure pertains. Suitable host cells for expressing and producing a protein or antibody in the present disclosure include higher eukaryotic cells described herein, including vertebrate host cells. Propagation of vertebrate cells in culture has become a common technique. Examples of useful mammalian host cell lines include monkey kidney cells (CV1), monkey kidney CV1 line transformed by SV40 (COS-7), human embryonic kidney line (293 cells), baby hamster kidney cells (BHK), Chinese Hamster Ovary Cells (CHO), Mouse Sertoli Cells (TM4), African Green Monkey Kidney Cells (VERO-76), Human Cervical Carcinoma Cells (HELA), Dog Kidney Cells (MDCK), Buffalo Rat Hepatocytes (BRL 3A), human lung cells (W138), human hepatocytes (Hep G2), mouse mammary tumors (MMT 060562), TRI cells, MRC 5 cells, and FS4 cells. The host cells used for this purpose may be cultured in various media. Commercially available media, such as Ham's F10 medium, minimal essential medium, RPMI-1640, Dulbecco's Modified Eagle Medium (DMEM), and any other medium used in the art to which this disclosure pertains, can be used for culturing host cells. can be used for Any of these media may contain, if desired, hormones and/or growth factors (eg insulin, transferrin or epidermal growth factor), salts (eg sodium chloride, calcium, magnesium and phosphate), buffers (eg HEPES), Nucleotides (eg, adenosine and thymidine), antibiotics (eg, GENTAMYCIN ^™ drugs), trace elements, and glucose or equivalent energy sources and the like may be supplemented. Any other essential/auxiliary supplements may also be included at appropriate concentrations known to those of ordinary skill in the art. Other culture conditions such as temperature, pH, CO ₂ concentration, etc. may be set to appropriate conditions known in relation to the host cell selected for protein expression, which will be apparent to those skilled in the art.

The protein or antibody in the present disclosure may be purified by any protein or antibody purification method known in the art. For example, hydrophobic interaction, immune affinity or fractionation on an ion-exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica, or chromatography on anion or cation-exchange resin, chromatofocusing, SDS-PAGE, ammonium sulfate A variety of known purification methods including precipitation, gel filtration, or genetic engineering methods (tag, etc.) may be used, and a plurality of purification methods may be arbitrarily combined to increase protein purity.

In the present disclosure, the protein or antibody purified by the above method may have a high purity level that can be administered to animals, particularly humans, for experimental, clinical, and therapeutic purposes.

When the complex comprising the anti-digoxigenin humanized antibody or antigen-binding fragment thereof of the present disclosure and the conjugate comprising digoxigenin of the present disclosure is prepared, the antigen to which the humanized anti-digoxigenin antibody and digoxigenin are bound The mole ratio is 1:2, but is not limited thereto.

A method for preparing a complex of the present disclosure may include, for example, i) (b) digoxigenin and (c) cytotoxin or a detection label (d) a linker and covalently via an aptamer bonding to prepare a conjugate; and ii) (a) binding an anti-digoxigenin humanized antibody or antigen-binding fragment thereof to a conjugate in which digoxigenin and a cytotoxin or detection label are covalently conjugated via an aptamer It may include the step of

In the present disclosure, the pharmaceutical/detection composition including the complex may be formulated and administered in a manner consistent with medical practice. Factors contemplated in this regard include the disease being treated or detected, the particular animal being treated or detected, the clinical condition of the individual subject, the cause of the disease, the site of delivery of the substance, the method of administration, the regimen of administration, and other factors known to the clinician. do. In the present disclosure, the formulation may include a liquid formulation, a freeze-dried powder formulation, and the like. The pharmaceutical/detection composition of the present disclosure may be prepared in the form of an ampoule, a vial, a bottle, a cartridge, a reservoir, a lyoject, or a pre-filled syringe, and may be prepared in a single dosage form or a multiple dosage form. .

The "administration dose", "pharmaceutically effective amount", "therapeutically effective amount" or "effective dosage" of the complex being administered will be determined by the above considerations and is the minimum amount necessary for the prevention, amelioration, detection or treatment of a particular disease. In the present disclosure, the "administration dose", "pharmaceutically effective amount", "therapeutically effective amount" or "effective dose" of the complex is, for example, from about 0.001 mg/kg to about 500 mg/kg. may be, but is not limited thereto. The pharmaceutical/detection composition of the present disclosure is periodically administered once a day, three times a week, twice a week, once a week, three times a month, twice a month, or once a month, etc. by the judgment of an experienced clinician. may be administered, and may be administered aperiodically in situations such as acute progression of disease, urgency of detection, etc., but is not limited thereto.

In the present disclosure, the pharmaceutical/detection composition may be prepared by mixing the complex of the present disclosure with a physiologically acceptable carrier, excipient or stabilizer using standard methods known in the art. Acceptable carriers include saline or buffers such as phosphate, citrate and other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides (comprising less than about 10 amino acid residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides and other carbohydrates such as glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt forming counterions such as sodium; and/or nonionic surfactants such as TWEEN ^™ , PLURONICS ^™ , or PEG.

The pharmaceutical/detection composition of the present disclosure may contain a pharmaceutically acceptable salt in an approximately physiological concentration. Optionally, the formulations of the present disclosure may contain a pharmaceutically acceptable preservative. Specifically, the preservative concentration may be between 0.1 and 2.0% (typically v/v). In the present disclosure, the preservative may be one known in the pharmaceutical industry, and specifically, benzyl alcohol, phenol, m-cresol, methylparaben, propylparaben, or a combination thereof. The pharmaceutical/detection composition of the present disclosure may include a pharmaceutically acceptable surfactant at a concentration of 0.005 to 0.02%.

In the present disclosure, the pharmaceutical/detection composition may further contain one or more active compounds necessary for treating or detecting a target disease, specifically, an active compound having complementary activities that do not adversely affect each other with the complex of the present disclosure. The compound may be contained in the composition in an amount effective to achieve the intended purpose.

In one aspect of the present disclosure, an anti-digoxigenin humanized antibody or antigen-binding fragment thereof of the present disclosure; and a conjugate in which digoxigenin and a chemotherapeutic drug or cytotoxin are covalently conjugated through an aptamer that binds to cancer cells; provides a pharmaceutical composition for treating cancer comprising a conjugated complex do. Specifically, the pharmaceutical composition for treating cancer of the present disclosure comprises (a) an anti-digoxigenin humanized antibody or antigen-binding fragment thereof, (b) digoxigenin, (c) cytotoxin, and (d) A complex including an aptamer that may include a linker, wherein (b) digoxigenin and (c) cytotoxin are covalently covalent through an aptamer that may include (d) a linker It may include a complex in the form of a conjugate to which it is conjugated. Meanwhile, the aptamer may include any one of the nucleotide sequences of SEQ ID NOs: 11 to 40, but is not limited thereto.

In one aspect of the present disclosure, an anti-digoxigenin humanized antibody or antigen-binding fragment thereof of the present disclosure; and a conjugate in which digoxigenin and a detection label are covalently conjugated through an aptamer that binds to cancer cells. Specifically, the composition for detecting cancer cells of the present disclosure may include (a) an anti-digoxigenin humanized antibody or antigen-binding fragment thereof, (b) digoxigenin, (c) a detection label, and (d) a linker. A complex comprising an aptamer, wherein (b) digoxigenin and (c) detection label (d) a complex in the form of a conjugate covalently conjugated via an aptamer that may include a linker may include. Meanwhile, the aptamer may include any one of the nucleotide sequences of SEQ ID NOs: 11 to 40, but is not limited thereto.

In one aspect of the present disclosure, there is provided a cancer treatment method or cancer cell detection method comprising administering a pharmaceutical/detection composition comprising the complex of the present disclosure.

According to a known method, the pharmaceutical/detection composition comprising the complex in the present disclosure can be administered intravenously, intramuscularly, intraperitoneally, intracerebrospinal fluid, subcutaneously, intraarticularly, intrasynovially, intrathecally, oral, topical, subcutaneous, or by an inhalation route or the like to a human or animal subject.

Hereinafter, the configuration and effect of the present disclosure will be described in more detail with reference to Examples and Test Examples. However, these Examples and Test Examples are provided only for the purpose of illustration to help the understanding of the present disclosure, and the scope and scope of the present disclosure are not limited by the following examples.

In addition, embodiments of the present disclosure will be described with reference to the accompanying drawings. In the accompanying drawings, the same or corresponding components may be assigned the same reference numerals. In addition, in the description of the embodiments below, overlapping description of the same or corresponding components may be omitted. However, even if descriptions regarding components are omitted, it is not intended that such components are not included in any embodiment.

[Example]

Example 1. Preparation of humanized antibody of anti-digoxigenin antibody

The inventors of the present disclosure sought to prepare humanized antibodies based on mouse antibodies. Specifically, an anti-digoxigenin mouse antibody having a heavy chain variable region comprising SEQ ID NO: 1 and a light chain variable region comprising SEQ ID NO: 6 was obtained.

By analyzing the mouse antibody sequence, a specific sequence was humanized and a specific sequence was maintained, thereby reducing the possibility of immunogenicity and performing a humanization process to maintain binding ability as much as possible.

※ Mouse holding antibody optimization method

The mouse monoclonal antibody used as a holding antibody in the present disclosure is very usefully used as a diagnostic reagent or basic research because it can be prepared by setting various target antigens and can be mass-produced, but it is used in the human body for the purpose of treating diseases. Repeated administration induces an immune response (HAMA, human anti-mouse antibody response) in the body, resulting in side effects and reduced effectiveness. Therefore, it cannot be used as a therapeutic agent. In order to solve this problem, there is a need for a humanized antibody manufacturing technology that makes a mouse monoclonal antibody similar to a human antibody so that there is no problem even when administered to the human body.

An antibody consists of a variable region and a constant region, which in turn are complementarity determining regions (CDRs) that directly bind antigen and framework regions (FRs) supporting the CDR loops. framework regions). Humanized antibodies are primarily prepared by a CDR grafting method in which the CDR loops of a mouse antibody are grafted into a human antibody (chimerization). However, when only CDR grafting is performed, the affinity of the humanized antibody is lowered, so by substituting several key framework region amino acid residues that are thought to affect the structure of the CDRs with those of the mouse antibody, the affinity of the original mouse antibody is similar to that of the original mouse antibody. The process of increasing affinity to the level is performed (humanization).

Specific implementation was requested by Absolute Antibody's Prometheus ^TM service and humanized, and a total of 16 types of humanized antibody candidates were produced. The sequences of the heavy chain variable region and the light chain variable region of the thus-prepared antibody are summarized in Table 2 below.

ID Format Human
Germline order with human variable region sequences
% sequence identity VH-m (SEQ ID NO: 1) mouse - QVTLKESGPGILQPSQTLSLTCSLSGFSLTTSGMGVGWIRQSSGKGLEWLANIWWYDTKYYNAALKSRLTISKDTSKNQVFLKIVSVDTADTATYYCGRIHYNGSRFGDYWGQGTTLTVSS 69.1 VH-h1 (SEQ ID NO: 2) humanization IGHV2-5*05 QVTLKESGPTLVKPTQTLTLTCTLSGFSLTTSGMGVGWIRQPPGKALEWVLANIWWYDTKYYNASLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCGRIHYNGSRFGDYWGQGTLVTVSS 89.7 VH-h2 (SEQ ID NO: 3) humanization IGHV2-5*05 QITLKESGPTLVKPTQTLTLTCTLSGFSLTTSGMGVGWIRQPPGKALEWLANlWWYDTKYYNASLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCGRlHYNGSRFGDYWGQGTLVTVSS 89.7 VH-h3 (SEQ ID NO: 4) humanization IGHV4-38-2*02 QVTLQESGPGLVKPSETLSLTCTLSGFSLTTSGMGVGWIRQPPGKGLEWLANIWWYDTKYYNASLKSRVTISKDTSKNQVSLKLSSVTAADTAVYYCGRIHYNGSRFGDYWGQGTLVTVSS 77.8 VH-h4 (SEQ ID NO: 5) humanization IGHV4-38-2*02 QVQLQESGPGLVKPSETLSLTCTLSGFSLTTSGMGVGWIRQPPGKGLEWIANIWWYDTKYYNASLKSRVTISKDTSKNQVSLKLSSVTAADTAVYYCGRIHYNGSRFGDYWGQGTLVTVSS 79.8 VL-m (SEQ ID NO: 6) mouse - DVVMTQTPLTLSVTFGQPASISCKSSQSLLYTNGKTYLMWLLQRPGQSPKRLIYLVSTLDSGVPGRFSGSGSGTDFTLKISRVEAEDLGVYYCLOTIHFPYSFGGGTKLEIK 79.0 VL-h1 (SEQ ID NO: 7) humanization IGKV2D-30*01 DVVMTQSPLSLPVTLGQPASISCRSSQSLLYTNGKTYLMWLQQRPGQSPRRLIYLVSTLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQTTHFPYSFGQGTKLEIK 88.0 VL-h2 (SEQ ID NO: 8) humanization IGKV2D-30*01 DVVMTQSPLSLPVTLGQPASISCRSSQSLLYTNGKTYLMWLQQRPGQSPRRLIYLVSTWDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQTTHFPYSFGQGTKLEIK 89.0 VL-h3 (SEQ ID NO: 9) humanization IGKV4-1*01 DVVMTQSPDSLAVSLGERATINCKSSQSLLYTNGKTYLMWLQQKPGQPPKRLIYLVSTLDSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCLQTTHFPYSFGQGTKLEIK 82.2 VL-h4 (SEQ ID NO: 10) humanization IGKV4-1*01 DVVMTQSPDSLAVSLGERATINCKSSQSLLYTNGKTYLMWLQQKPGQPPKRLIYLVSTRDSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCLQTTHFPYSFGQGTKLEIK 82.2

Accordingly, one chimeric antibody and 16 humanized antibodies were prepared by combining the heavy chain variable region and the light chain variable region of Table 2 above. The variable region combinations of the prepared humanized antibodies are summarized in Table 3 below.

clone name Format heavy chain variable region light chain variable region mAb[21H8] mouse IgG1 VH-m VL-m chimeric[21H8] Chimeric IgG1 VH-m VL-m humab[21H8]-v1 Humanized human IgG1 VH-h1 VL-h1 humab[21H8]-v2 Humanized human IgG1 VH-h1 VL-h2 humab[21H8]-v3 Humanized human IgG1 VH-h1 VL-h3 humab[21H8]-v4 Humanized human IgG1 VH-h1 VL-h4 humab[21H8]-v5 Humanized Human IgG1 VH-h2 VL-h1 humab[21H8]-v6 Humanized human IgG1 VH-h2 VL-h2 humab[21H8]-v7 Humanized human IgG1 VH-h2 VL-h3 humab[21H8]-v8 Humanized human IgG1 VH-h2 VL-h4 humab[21H8]-v9 Humanized human IgG1 VH-h3 VL-h1 humab[21H8]-v10 Humanized Human IgG1 VH-h3 VL-h2 humab[21H8]-v11 Humanized human IgG1 VH-h3 VL-h3 humab[21H8]-v12 Humanized human IgG1 VH-h3 VL-h4 humab[21H8]-v13 Humanized human IgG1 VH-h4 VL-h1 humab[21H8]-v14 Humanized human IgG1 VH-h4 VL-h2 humab[21H8]-v15 Humanized Human IgG1 VH-h4 VL-h3 humab[21H8]-v16 Humanized Human IgG1 VH-h4 VL-h4

A gene having a nucleotide sequence encoding a combination of the heavy chain variable region and the light chain variable region was cloned into an antibody expression plasmid (Absolute Antibody cloning and expression vector), and codon optimization was carried out to facilitate expression in human cells. The correct sequence was confirmed by Sanger sequencing using DNASTAR Lasergene software, and the size was confirmed once more through plasmid DNA prep, and a sufficient amount of high-purity plasmid DNA for transfection was obtained.

The prepared high-purity plasmid DNA was transiently transfected into a HEK 293 (human fetal kidney cell 293) cell line. Cells were transiently transfected with vectors expressing heavy and light chains and cultured for 6 days. The cultures were centrifuged at 4000 rpm and filtered with a 0.22 μM filter. Antibodies were eluted once via Protein A affinity chromatography with pH 3.0 citrate buffer followed by pH 9.0 0.5M Tris neutralization buffer. The eluted protein was buffer exchanged with PBS using a desalting column. The obtained antibody concentration was confirmed by UV spectroscopy, and the antibody was concentrated if necessary.

The prepared mouse antibody (mAb[21H8]), chimeric antibody (chimeric[21H8]), humanized antibody (humab[21H8]-v1, humab[21H8]-v2, humab[21H8]-v3, humab[21H8]- v4, humab[21H8]-v5, humab[21H8]-v6, humab[21H8]-v7, humab[21H8]-v8, humab[21H8]-v9, humab[21H8]-v10, humab[21H8]-v11 , humab[21H8]-v12, humab[21H8]-v13, humab[21H8]-v14, humab[21H8]-v15, and humab[21H8]-v16) were evaluated for binding to digoxigenin.

Example 2. Anti-Digoxigenin Humanized Antibody Binding Affinity Confirmation

The prepared mouse antibody (mAb[21H8]), chimeric antibody (chimeric[21H8]), humanized antibody (humab[21H8]-v1, humab[21H8]-v2, humab[21H8]-v3, humab[21H8]- v4, humab[21H8]-v5, humab[21H8]-v6, humab[21H8]-v7, humab[21H8]-v8, humab[21H8]-v9, humab[21H8]-v10, humab[21H8]-v11 , humab[21H8]-v12, humab[21H8]-v13, humab[21H8]-v14, humab[21H8]-v15, humab[21H8]-v16) were tested for binding to digoxigenin through ELISA experiments. did The ELISA experiment was performed under the following conditions.

※ ELISA test method

1) 100 μL of digoxigenin-BSA (CellMosaic ^TM , Cat.No. CM52107) at a concentration of 4 μg/mL is plated on an Immuno Clear plate (Thermo Scientific ^TM , Cat No. 468667) and left at 4° C. overnight. .

2) Wash the plate 4 times with 150 μL of PBS + 0.05% Tween-20 (PBS-T).

3) Treat the plate with 100 μL of Blocking solution (PBS + 3% BSA (Calbiochem ^® , Cat.No. 126609)) and allow to stand at room temperature for 2 hours.

4) After removing the blocking solution from the plate, each antibody (mAb[21H8], chimeric[21H8], humab[21H8]-v1-v16) was added at various concentrations (from 53.33 nM to 1/3 serial dilution, that is, 17.7 nM, 5.92 nM, 1.97 nM, 0.66 nM, 0.22 nM, 0.07 nM, 0.024 nM) in 50 μL and allow to stand at room temperature for 1 hour.

5) Wash the plate 4 times with 150 μL of PBS-T.

6) Treat with 50 μL of secondary antibody (HRP-conjugated IgG) and allow to stand at room temperature for 1 hour. Specifically, sheep anti-mouse IgG (whole molecule), HRP (Sigma, Cat. No. A6782) and rabbit anti-human IgG Fc secondary antibody, HRP (invitrogen, Cat. No. 31423) were each 1/5000 Use diluted.

7) Wash the plate 4 times with 150 μL of PBS-T.

8) Treat the plate with 100 μL of 1-Step™ Ultra TMB-ELISA Substrate (ThermoFisher Scientific, Cat. No. 34029) and incubate for 10 minutes.

9) Stop the reaction using 40 μL of 2.5 NH ₂ SO ₄ .

10) Measure OD value at absorbance at 450 nm using Synergy ^TM H1 Hybrid Multi-Mode Reader (BioTek).

The results are summarized in FIG. 1 ( FIGS. 1A to 1C ) and Table 4 below.

mAb[21H8] chimeric[21H8] humab[21H8]-v1 humab[21H8]-v2 humab[21H8]-v3 humab[21H8]-v4 humab[21H8]-v5 humab[21H8]-v6 humab[21H8]-v7 K _D value (nM) 28.42 11.21 5.75 8.99 6.77 7.21 7.04 9.03 13.24 humab[21H8]-v8 humab[21H8]-v9 humab[21H8]-v10 humab[21H8]-v11 humab[21H8]-v12 humab[21H8]-v13 humab[21H8]-v14 humab[21H8]-v15 humab[21H8]-v16 K _D value (nM) 4.84 14.48 9.55 10.88 17.89 14.31 17.05 9.09 12.14

*K _D : Equilibrium dissociation constant (the smaller the value, the greater the binding affinity of the ligand to the target)

As can be seen from the above experiment, it can be seen that the binding force of the humanized antibody is significantly improved. For example, it can be seen that humab[21H8]-v1 has about 5 times stronger binding than mAb[21H8], and humab[21H8]-v3 is about 3 times stronger than that of mAb[21H8]. In particular, it was confirmed that humab[21H8]-v8 had about 6 times stronger binding than mAb[21H8].

The purpose of humanizing antibodies from other animals is to reduce the immunogenicity in the human body, and the binding force is lowered in the general process. The humanized antibody of the present disclosure showed exceptional properties having superior binding ability than the mouse antibody. This is because the mouse antibody used as the holding antibody in the present disclosure did not undergo the binding affinity optimization process, so it seems that the binding power was increased during the humanization and optimization process. Accordingly, in the present disclosure, an antibody that is unexpectedly humanized and has excellent antigen-binding ability was obtained.

Example 3. Digoxigenin-aptamer-MMAE Conjugate (DOligomer) Preparation and Identification

Example 3-1. Preparation of digoxigenin-aptamer-MMAE conjugate (DOligomer)

As the aptamer, the aptamer SQ7-1(8) (SEQ ID NO: 11), which binds to pancreatic cancer cells, and SQ7-1(8)-Rev (SEQ ID NO: 41), which are in the opposite direction, were used as the control aptamer. A conjugate was prepared in which digoxigenin was bound to the 5' end of the aptamer and monomethyl auristatin E (MMAE) was bound to the 3' end. A specific manufacturing method is as follows.

It was provided by synthesizing an aptamer modified with digoxigenin at the 5' end and S-S at the 3' end by Integrated DNA Technology (IDT; USA) (FIG. 3A).

To break the 3' thiol C3 SS linker of the aptamer, 0.1 M TEAA (Triethylammonium acetate) and 1 mM TCEP (Tris-(2-carboxylethyl) phosphine hydrochloride) were added and reacted at 70° C. for 5 minutes, followed by reaction at room temperature. The reaction was carried out for 2 hours. The remaining TCEP was removed with PBS and 2 mM EDTA (Ethylenediaminetetraacetic acid) using Amicon Ultra 3K spin columns (Milipore, Billerica, MA).

The DIG-aptamer and MC-Val-Cit-PAB-MMAE (Levena Biopharma, San Diego, CA) of 5 times the aptamer concentration (molar) were reacted at room temperature for 16 hours. Thereafter, residual MMAE was removed with PBS using Amicon Ultra 3K spin columns (Milipore, Billerica, MA).

Example 3-2. Synthesis confirmation of digoxigenin-aptamer-MMAE conjugate (DOligomer)

The synthesized digoxigenin-aptamer-MMAE (FIG. 3B) was e2695 (Waters, USA) high-performance using reversed-phase analytical HPLC on a 4.6 x 50 mm Xbridge C18 column (Waters, Milford, MA) Liquid chromatography (HPLC) was used to detect and analyze at 260 nm with a UV detector (Waters, USA).

During analysis, the column temperature was maintained at 65 °C, and 0.1 M TEAA at pH 7.0 (eluent A) and Acetonitrile (eluent B) were used as mobile phases. Analysis conditions were 90% A and 10% B flow from the initial stage to 5 minutes, and then 1 mL/minute of eluent B was flowed with a linear gradient from 20 to 70% for 5-15 minutes.

30 μM of DIG-SQ7-1(8)-SH aptamer was injected in a volume of 10 μL, and as a result of the analysis, a peak was confirmed at a retention time of 6.21 minutes (FIG. 4 A), and 10 μL of the 30 μM DOligomer prepared above As a result of analysis by injection by volume, a peak was confirmed at a retention time of 10.02 minutes (FIG. 4B). This means that the synthesis between MC-Val-Cit-PAB-MMAE (Levena Biopharma, San Diego, CA) and DIG-SQ7-1(8)-SH was well done, and digoxigenin-aptamer-MMAE ( It was confirmed that the DIG-SQ7-1(8)-MMAE) conjugate was synthesized with a purity of 98%.

On the other hand, a technology for combining an aptamer and a drug has been variously developed in the technical field to which the present disclosure pertains. Those of ordinary skill in the art, through the specific examples and conjugation methods disclosed in the present disclosure (Table 1), and the knowledge known in the art, according to the purpose, various aptamers and substances (bioactive substances or detection labels) etc.) can be selected and connected.

Example 3-3. Confirmation of binding force and apoptosis effect of digoxigenin-aptamer-MMAE conjugate (DOligomer)

The ability of the synthesized digoxigenin-aptamer-MMAE conjugate (DOligomer) to inhibit pancreatic cancer cell proliferation or apoptosis was confirmed by the following method.

※Hapten-aptamer-drug conjugate (DIG-aptamer-MMAE) test method for inhibition of pancreatic cancer cell proliferation

1) After seeding the pancreatic cancer cell line (CFPAC-1) at 3,000 cells/well in a 96-well-plate, incubate for 24 hours.

2) After washing with serum free media, block with 1 mg/ml salmon sperm ssDNA at 37°C for 1 hour.

3) After treatment with the hapten-aptamer-drug conjugate (DIG-aptamer-MMAE) by concentration (0.5, 1, 2 μM), incubated for 48 hours in a 37°C CO ₂ incubator, re-treated at the same concentration, 48 time to incubate.

4) Observe cell shape through an optical microscope and analyze by ATP assay (Cell Titer Glo, Promega).

As a result, as can be seen in FIGS. 5a and 5b, when DIG-SQ7-1(8)-MMAE containing SQ7-1(8) aptamer targeting pancreatic cancer was treated at a concentration of 1 μM or more, pancreatic cancer cell proliferation In the case of DIG-SQ7-1(8)-Rev-MMAE, which contains SQ7-1(8)-Rev, a reverse sequence aptamer that does not target pancreatic cancer, while causing inhibition and death, even at high concentrations (2 μM), pancreatic cancer It did not show significant cell proliferation inhibitory and apoptotic effects. VC-MMAE must be cut by cathepsin B, a protease that is known to be highly expressed in cancer cells, in which the VC portion can inhibit the proliferation of cancer cells by free MMAE action, and DIG-SQ7-1(8)-MMAE After binding to the pancreatic cancer cell membrane, it is internalized into the cell, and the VC portion is cut by the cathepsin B enzyme in the lysosome, indicating that free MMAE exerts a cell proliferation inhibitory effect. Considering that 1 μM of free MMAE in FIG. 5a exhibits pancreatic cancer cell proliferation inhibition and apoptosis effects, it can be seen that only drugs delivered to a specific target by an aptamer have specific efficacy. That is, it was confirmed that the effect of the drug can be sufficiently delivered through the DIG-aptamer-drug conjugate of the present disclosure, and in particular, the drug can be delivered specifically to a specific cell or tissue targeted by the aptamer (target-specific drug delivery). ability).

Example 4. Preparation of a complex DOligobody comprising an anti-digoxigenin antibody and confirmation of binding force

Example 4-1. Preparation of complex DOligobody comprising anti-digoxigenin antibody

The mouse antibody (mAb[21H8]), chimeric antibody (chimeric[21H8]), humanized antibody (humab[21H8]-v1, humab[21H8]-v2, humab[21H8]-v3, humab prepared in Example 1 above [21H8]-v4, humab[21H8]-v5, humab[21H8]-v6, humab[21H8]-v7, humab[21H8]-v8, humab[21H8]-v9, humab[21H8]-v10, humab[ 21H8]-v11, humab[21H8]-v12, humab[21H8]-v13, humab[21H8]-v14, humab[21H8]-v15, humab[21H8]-v16) and the DIG prepared in Example 3-1 -aptamer-MMAE conjugate (DOligomer) was combined to prepare a complex (DOligobody).

Specifically, 250 nM of the DIG-aptamer-MMAE conjugate and 125 nM of the antibody prepared in Example 1 were mixed in a binding buffer (Binding buffer: DPBS (Hyclone #SH30028.02) with 0.1% BSA (Merk #126593), 0.1 Dissolve in mg/mL yeast tRNA (Sigma #R9001), and 5 mM MgCl ₂ (Biosesang #M3101) to a total volume of 100 μL. The mixture was incubated on a rotator at room temperature for 30 minutes to bind the antibody and the DIG-aptamer-MMAE conjugate to prepare a complex (DOligobody). The structure of the thus prepared DOligobody is shown in FIG. 2 .

Example 4-2. Confirmation of binding affinity of the complex (DOligobody) comprising the anti-digoxigenin humanized antibody through FACS experiment

Aptamer SQ7-1(8) contained therein for the complex DOligobody prepared in Example 4-1 The binding force was confirmed using CFPAC-1, a target human pancreatic cancer cell line. This was measured using a flow cytometer (FACS, Fluorescence-activated cell sorting). It is the aptamer in the conjugate that has binding to pancreatic cancer cells, and FACS was analyzed using a secondary antibody to anti-digoxigenin antibody (mouse or human). It can be confirmed that the complex (DOligobody) of the present disclosure to which the anti-digoxigenin antibody is bound was normally prepared.

Specifically, CFPAC-1 cells were obtained by trypsinization, and prepared in a round bottom tube at a concentration of 3 x 10 ⁵ cells. 100 μL of the complex DOligobody mixture prepared in Example 3-2 was added to each tube, and then incubated at 4° C. for 30 minutes.

Then, the cells in the tube were washed three times with 500 μL of a washing buffer (Washing buffer: Binding buffer of Example 3-2 + 0.1% NaN ₃ included).

A secondary antibody was attached to the washed cells. Specifically, Alexa Fluor ^TM 488 anti-mouse IgG (H+L) (Invitrogen #A21202) prepared from a donkey was diluted 1/500, and Anti-Human IgG (Fc specific)-FITC antibody prepared from a goat (Sigma #F9512) was diluted to 1/500 and incubated at 4°C for 15 minutes.

Then, the cells in the tube were washed three times with 500 μL of a washing buffer (Washing buffer: Binding buffer of Example 3-2 + 0.1% NaN ₃ included).

Samples for FACS were prepared by treating the washed cells with a solution of 7-AAD (BD Pharmingen ^TM #559925: 1/100 dilution) added to 300 μL of binding buffer.

The prepared samples were analyzed using a FACSVerse ^™ (BD Biosciences, San Jose, USA) instrument. The analysis result is shown in FIG. 6 ( FIGS. 6A to 6C ).

The results of comparing the binding rate of the DOligobody complex of the present disclosure based on the FACS results are shown in FIG. 7 . According to the results, it can be confirmed that the binding rate of the complex to the human pancreatic cancer cell line, CFPAC-1, is superior when the humanized antibody is bound to the DIG-aptamer-MMAE conjugate than when the original mouse antibody is bound to the DIG-aptamer-MMAE conjugate. In particular, in the case of humanized antibodies such as humab[21H8]-v6, it was confirmed that the binding rate of the complex to cancer cells was superior to that of the chimeric antibody. This shows that the difference in the binding force between the digoxigenin-aptamer-MMAE conjugate and the anti-digoxigenin antibody also affects the binding force of the entire complex to pancreatic cancer cells. That is, since mouse and chimeric antibodies have weaker binding force to the conjugate than the humanized antibody, the binding force to the pancreatic cancer cells targeted by the aptamer in the conjugate is also weaker.

Example 5. Pharmacokinetic study of complex DOligobody containing anti-digoxigenin antibody

anti-digoxigenin For the complex DOligobody containing the antibody, pharmacokinetic analysis was performed through animal experiments. In Balb/c mice, ① DIG-SQ7-1(8)-MMAE conjugate DOligomer (control), ② DOligobody containing humab[21H8]-v5 antibody, ③ DOligobody containing humab[21H8]-v6 antibody, or ④ DOligobody containing humab[21H8]-v8 antibody was administered at 8 mg/kg. At the time of administration, at 0.5 hours, 1 hour, 4 hours, 8 hours, 12 hours, 24 hours, 48 hours, and 72 hours after administration, about 50 to 100 μL of blood is drawn using a heparin-coated capillary tube. Orbital vein blood was drawn (FIG. 8).

After standing the collected blood on ice for 30 minutes, the supernatant (plasma) was separated by centrifugation, and the concentration of aptamer and humanized antibody in the blood was analyzed using digoxigenin ELISA and human IgG ELISA kit. .

※ Digoxigenin ELISA method

1) Anti-digoxigenin (Abcam, cat. Ab420) at a concentration of 2 μg/mL was plated on an Immuno Clear plate (Thermo Scientific ^TM , Cat. No. 468667) (100 μL each), 4°C, overnight.

2) After washing 3 times with PBS+0.05% Tween-20 (PBS-T) (150 μL), blocking with 100 μL 3% BSA (Calbiochem®, Cat.No. 126609) at 37°C for 1 hour ).

3) After removing the blocking solution, 50 μL of each 1/10 diluted blood sample was incubated at 37°C for 1 hour.

4) After washing 3 times with PBS-T (150 μL), incubate 50 μL HRP-conjugated digoxigenin (1/4000 dilution) at 37°C for 1 hour.

5) After washing 3 times with PBS-T (150 μL), add 100 μL 1-Step ^TM Ultra TMB-ELISA Substrate (Thermo Fisher Scientific, Cat.No. 34029), and incubate for 15 minutes.

6) Add 2.5 NH ₂ SO ₄ (40 μL).

7) OD value measurement at 450 nm absorbance using Synergy ^TM H1 Hybrid Multi-Mode Reader (BioTek).

※ Human IgG ELISA method

-The experiment was performed according to the manual of the Human IgG total ELISA Kit (Invitrogen, Cat. BMS2091). Specifically, it is as follows.

1) Dilute the blood sample 1/10,000 with assay dilution buffer.

2) Standard is prepared at 0.2, 0.1, 0.05, 0.025, 0.013, and 0.003 μg/mL by serial dilution with assay dilution buffer.

3) HRP-conjugate is diluted 1/100 with assay dilution buffer.

4) Wash the plate once with wash buffer.

5) Add 100 μL of blood sample dilution or standard and 100 μL of HRP-conjugate to each well, and react at room temperature for 1 hour and 30 minutes.

6) Wash 4 times with wash buffer

7) Add 100 μL of TMB substrate and react at room temperature for 30 minutes.

8) After adding 100 μL of stop solution, measure OD value at absorbance at 450 nm using Synergy ^TM H1 Hybrid Multi-Mode Reader (BioTek).

As a result of the analysis through the above experiment, the PK graph for DOligomer including digoxigenin was confirmed as shown in FIG. 9 . On the other hand, the plasma concentration of the antibody after intravenous administration is divided into a distribution phase and an elimination phase, and the result of calculating the half-life (t1/2) of DOligomer ① DIG-SQ7-1(8 )-MMAE conjugated DOligomer (control) was injected for only 0.02 hours, while ② DOligomer in DOligobody containing humab[21H8]-v5 antibody was 9.2 hours, ③ in DOligobody containing humab[21H8]-v6 antibody. The DOligomer of 4 was confirmed to be 16.8 hours, and the DOligomer in the DOligobody containing the humab[21H8]-v8 antibody was 38.5 hours (FIG. 10).

The PK graph for human IgG is as shown in FIG. 11, and when DOligobody comprising the humab[21H8]-v5 antibody, the humab[21H8]-v6 antibody, and the humab[21H8]-v8 antibody was injected, anti-digoxigenin As a result of calculating the half-life (t1/2) of the antibody, ② anti-digoxigenin antibody in DOligobody containing humab[21H8]-v5 antibody was 35 hours, ③ in DOligobody containing humab[21H8]-v6 antibody. Anti-digoxigenin antibody was measured at 32 hours, ④ anti-digoxigenin antibody in DOligobody containing humab[21H8]-v8 antibody was measured at 143 hours (FIG. 12).

As can be seen from the above data, it was confirmed that the half-life of DOligomer in the body was significantly increased by the DOligobody fusion of the present disclosure. In addition, it was confirmed that the half-life of the humab[21H8]-v8 antibody was the highest among the anti-digoxigenin antibodies.

Example 6. Efficacy evaluation and safety evaluation of a complex DOligobody comprising an anti-digoxigenin antibody

Example 6-1. Efficacy evaluation and safety evaluation of complex DOligobody containing anti-digoxigenin antibody Animal experiments

anti-digoxigenin Efficacy and safety were evaluated through animal experiments on the complex DOligobody containing the antibody. The specific experimental method is as follows.

※ Experiment to confirm the effect of DOligobody using a mouse model of pancreatic cancer orthotopic metastasis

1) Prepare 6-week-old female Athymic nude mice.

2) After anesthetizing the mouse by inhalation using Isofluoran, make a very small incision in the skin where the left spleen is visible. When the spleen is exposed, fix the spleen with a cotton swab and take out the attached pancreas with tweezers Inject pancreatic cancer cells (CFLAC-1/Luci, 1 x 10 ⁶ cell/ 30 μL) into the tail of After injection, after hemostasis with a cotton swab for a certain period of time, the organ is returned to its original position and the surgical site is finished with a clip and disinfected.

3) Measurement of tumor growth through IVIS imaging and MRI.

4) After 2 weeks, using IVIS fluorescence imaging equipment, each group was divided according to luminescence.

5) DIG-aptamer-drug/anti-digoxigenin antibody complex (DOligobody [DIG-SQ7-1(8)-MMAE + humab[21H8]-v8]) at a concentration of 20 mg/kb once a week; Administered intraperitoneally for 5 weeks. Gemcitabine, a standard treatment for pancreatic cancer patients, was administered twice a week at 52 μM, the same number of moles as DOligobody (FIG. 13b).

6) The tumor suppression effect was observed through IVIS imaging and MRI for 5 weeks.

7) After the animal experiment is over, euthanize, collect blood, remove tumors and organs, and fix them.

※ TUNEL assay

1) After the animal test is over, the tumor is removed.

2) Prepare frozen cells (Frozen block) from the excised tumor tissue.

3) After fixing the frozen section with 4% formaldehyde at room temperature for 10 minutes, the slides were dried and washed three times with PBS for 5 minutes each.

4) After reacting in the permeabilization solution (0.1% Triton X-100 in PBS, 0.1% sodium citrate) at room temperature for 3 minutes, wash 3 times with PBS for 5 minutes each.

5) Drop 50 μL of the TUNEL reaction mixture (TUNEL reaction mixture; Enzyme Solution: Label Solution = 1:9) onto the fixed tissue, block light in an incubator at 37 ° C, and react for 1 hour.

6) After the reaction, the slides were washed twice with PBS for 5 minutes each. For nuclear staining, 50 μL of Hoest 33342 (Beyotime, C1022, China) diluted 1:500 in PBS was dropped onto the slide, and then reacted at room temperature for 5 minutes.

7) After the reaction was completed, the sample was washed with PBS, mounted, and checked with a fluorescence microscope (ZEISS, Germany).

※ Assessment of liver and kidney toxicity of DOligobody

After the animal experiment was completed, hepatotoxicity indicators (ALT, AST, TBIL) and nephrotoxicity indicators (BUN, CRE) were analyzed using Fuji Dri-chem 3500i (Fujifilm, Kanagawa, Japan) with the serum obtained from the blood from the inferior vena cava. did

Example 6-2. Efficacy evaluation and safety evaluation of complex DOligobody containing anti-digoxigenin antibody Animal test results

The evaluation results of the DOligobody effect/toxicity of the present disclosure using the pancreatic cancer orthotopic mouse model performed in the Example 6-1 method are as follows.

※ Evaluation of tumor suppression efficacy of complex DOligobody containing anti-digoxigenin antibody

In this embodiment, the drug bound to the complex containing the anti-digogenin antibody is MMAE, a cytotoxic substance, and the bound aptamer is SQ7-1(8), which is an aptamer targeting pancreatic cancer cells. Therefore, in order to confirm that the present complex properly targets aptamer-mediated tissue and drug delivery occurs properly, whether the volume of orthotopic transplanted tumor tissue is suppressed in the pancreatic cancer orthotopic mouse model, or apoptosis in the tumor tissue You can check what is happening.

First, in the pancreatic cancer orthotopic mouse model, the tumor size was observed weekly through the IVIS fluorescence imaging equipment (transplanted pancreatic cancer cells are CFPAC-1/Luci-expressing luciferase cells), and after 30 days, the tumor was measured using MRI equipment. was measured.

In the case of the experimental group administered with the DOligobody of the present disclosure during the measurement period of 5 weeks, the tumor size (Luminesence intensity) decreased compared to the negative control group (Vehicle; carrier group) or the Gemcitabine administration group (administered at the same molar concentration as DOligobody). was seen ( FIGS. 14A and 14B ).

As a result of measurement using MRI on the 30th day after the last administration, it was confirmed that the tumor volume was reduced by 50% in the DOligobody-treated group compared to the negative control group ( FIGS. 15a and 15b ).

That is, it was possible to confirm the excellent tumor suppression efficacy of DOligobody through the pancreatic cancer orthotopic mouse model.

※ Confirmation of mechanism of action of anticancer effect of complex DOligobody containing anti-digoxigenin antibody

In the pancreatic cancer orthotopic mouse model, it was confirmed that the DOligobody of the present disclosure had excellent tumor suppression efficacy. Accordingly, the inventors of the present disclosure performed a TUNEL assay to confirm that the tumor suppressive efficacy of the DOligobody is due to apoptosis caused by the delivered MMAE. TUNEL assay is an experimental method that uses an enzyme called TdT (Terminal deoxynucleotidyl transferase) to attach UTP (uridine triphosphate) to the ends of DNA fragments to confirm whether DNA fragmentation, a phenomenon that occurs during cell death, has occurred. When DNA fragmentation occurs, cleavage occurs in the double helix structure, the N-terminus is exposed, and TUNEL is bound.

As can be seen from the results of FIG. 16 , it can be seen that a lot of cell death occurred in the tumors of the experimental group to which the DOligobody of the present disclosure was administered. Therefore, it was confirmed that the DOligobody of the present disclosure normally performs a drug delivery function and exhibits excellent efficacy as a drug delivery system.

※ Evaluation of liver and kidney toxicity of complex DOligobody containing anti-digoxigenin antibody

Whether the DOligobody administered in the pancreatic cancer orthotopic mouse model showed no liver or kidney toxicity was confirmed through weight change and indicators. Body weight was measured twice a week during the administration period (FIG. 17), and the corresponding index was measured at the time point 3 days after single administration (FIG. 18A) and at the end of the experiment (FIG. 18B) after multiple administration (5 times).

As can be seen in FIG. 17 , body weight during the administration period did not show a significant difference or change in all of the negative control group, the experimental group, and the positive control group.

As a result of analysis of GPT/ALT and GOT/ALT, which are indicators of liver toxicity, all of them were measured below the reference values (250 U/L and 300 U/L, respectively), and TBIL levels were also measured below the reference values (1.2 mg/dL), so there was no liver toxicity. was confirmed. In addition, it was confirmed that both the BUN level and CRE level, which are indicators of renal toxicity, were measured within the normal range below the standard values (40 mg/dL and 1.0 mg/dL, respectively).

Through the above experiment, the anti-digoxigenin antibody of the present disclosure and digoxigenin-aptamer-targeting the drug to the target cell by using the complex DOligobody comprising the conjugate (DOligomer) as a carrier, And it was confirmed that it can increase the half-life of the drug in the body. A person skilled in the art uses the complex DOligobody in which the anti-digoxigenin antibody of the present disclosure and the digoxigenin-aptamer-drug conjugate are combined as a drug delivery system to appropriately select an aptamer that binds to a target cell or substance and the drug to be delivered can be selected to effectively deliver the drug into the body.

From the above description, those skilled in the art to which the present disclosure pertains will be able to understand that the present disclosure may be implemented in other specific forms without changing the technical spirit or essential features of the present disclosure. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present disclosure should be construed as including all changes or modifications derived from the meaning and scope of the claims to be described later rather than the above detailed description, and equivalent concepts thereof, to be included in the scope of the present disclosure.

<110> National Cancer Center JP BIO A Inc. Kookmin University Industry Academy Cooperation Foundation <120> A complex comprising an antibody to Digoxigenin and use thereof <130> KC201047 <160> 41 <170> KoPatentIn 3.0 <210> 1 <211> 121 <212> PRT <213> Artificial Sequence < 220> <223> Mus musculus VH-m <400> 1 Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Leu Ser Gly Phe Ser Leu Thr Thr Ser 20 25 30 Gly Met Gly Val Gly Trp Ile Arg Gln Ser Ser Gly Lys Gly Leu Glu 35 40 45 Trp Leu Ala Asn Ile Trp Trp Tyr Asp Thr Lys Tyr Tyr Asn Ala Ala 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Phe Leu Lys Ile Val Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Gly Arg Ile His Tyr Asn Gly Ser Arg Phe Gly Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Thr Leu Thr Val Ser Ser 115 120 <210> 2 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Humanized VH-h1 <400> 2 Gln Val Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Leu Ser Gly Phe Ser Leu Thr Thr Ser 20 25 30 Gly Met Gly Val Gly Trp Ile Arg Gln Pro Gly Lys Ala Leu Glu 35 40 45 Trp Val Leu Ala Asn Ile Trp Trp Tyr Asp Thr Lys Tyr Tyr Asn Ala 50 55 60 Ser Leu Lys Ser Arg Leu Thr Ile Thr Lys Asp Thr Ser Lys Asn Gln 65 70 75 80 Val Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr 85 90 95 Tyr Cys Gly Arg Ile His Tyr Asn Gly Ser Arg Phe Gly Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 3 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Humanized VH-h2 <400> 3 Gln Ile Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Leu Ser Gly Phe Ser Leu Thr Thr Ser 20 25 30 Gly Met Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45 Trp Leu Ala Asn Leu Trp Trp Tyr Asp Thr Lys Tyr Tyr Asn Ala Ser 50 5 5 60 Leu Lys Ser Arg Leu Thr Ile Thr Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Gly Arg Leu His Tyr Asn Gly Ser Arg Phe Gly Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 4 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Humanized VH-h3 <400 > 4 Gln Val Thr Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Leu Ser Gly Phe Ser Leu Thr Thr Ser 20 25 30 Gly Met Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45 Trp Leu Ala Asn Ile Trp Trp Tyr Asp Thr Lys Tyr Tyr Asn Ala Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Gly Arg Ile His Tyr Asn Gly Ser Arg Phe Gly Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 5 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Humanized VH-h4 <400> 5 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Leu Ser Gly Phe Ser Leu Thr Thr Ser 20 25 30 Gly Met Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Ala Asn Ile Trp Trp Tyr Asp Thr Lys Tyr Tyr Asn Ala Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Gly Arg Ile His Tyr Asn Gly Ser Arg Phe Gly Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 6 <211> 111 <212> PRT <213> Artificial Sequence < 220> <223> Mus musculus VL-m <400> 6 Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Phe Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Thr 20 25 30 Asn Gly Lys Thr Tyr Leu Met Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Thr Leu Asp Ser Gly Val Pro 50 55 60 Gly Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Leu Thr Ile 85 90 95 His Phe Pro Tyr Ser Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 7 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Humanized VL-h1 <400> 7 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Tyr Thr 20 25 30 Asn Gly Lys Thr Tyr Leu Met Trp Leu Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Leu Val Ser Thr Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Leu Gln Thr 85 90 95 Thr His Phe Pro Tyr Ser Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 8 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Humanized VL-h2 <400> 8 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Tyr Thr 20 25 30 Asn Gly Lys Thr Tyr Leu Met Trp Leu Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Leu Val Ser Thr Trp Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Leu Gln Thr 85 90 95 Thr His Phe Pro Tyr Ser Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 9 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Humanized VL-h3 < 400> 9 Asp Val Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Tyr Thr 20 25 30 Asn Gly Lys Thr Tyr Leu Met Trp Leu Gln Gln Lys Pro Gly Gln Pro 35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Thr Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75 80 Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Leu Gln Thr 85 90 95 Thr His Phe Pro Tyr Ser Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 10 <211> 112 <212> PRT <213 > Artificial Sequence <220> <223> Humanized VL-h4 <400> 10 Asp Val Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Tyr Thr 20 25 30 Asn Gly Lys Thr Tyr Leu Met Trp Leu Gln Gln Lys Pro Gly Gln Pro 35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Thr Arg Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75 80 Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Leu Gln Thr 85 90 95 Thr His Phe Pro Tyr Ser Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 11 <211> 32 <212> DNA_RNA <213> Artificial Sequence <220> <223> internal 2'-O-methyl-modified aptamer SQ7-1(8) <220> <223> internal 2'- O-methyl-modified aptamers (from 1to 4 and 29 to 32) <220> <221> modified_base <222> (1)..(4) <223> 2'-O-methyl-modified bases from 1 to 4 < 220> <221> modified_base <222> (29)..(32) <223> 2'-O-methyl-modified bases from 29 to 32 <400> 11 guuggtatat acttctttag cttggaacca ac 32 <210> 12 <211> 80 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer SQ7 <400> 12 agcagcacag aggtcagatg atgttggtat atacttcttt agcttggaac caactcttgc 60 cctatgcgtg ctaccgtgaa 80 <210> 13 <211> 32 <212> DNA <213> Artificial Sequence <212> 220> <223> DNA aptamer SQ7-1 <400> 13 gttggtatat acttctttag cttggaacca ac 32 <210> 14 <211> 32 <212> DNA_RNA <213> Artificial Sequence <220> <223> internal 2'-O-methyl- modified aptamer SQ7-1(1) <220> <223> internal 2'-O-methyl-modified aptamers (from 1 to 6) <400> 14 guugguatat acttctttag cttggaacca ac 32 <210> 15 <211> 32 <212> DNA_RNA <213> Artificial Sequence <220> <223> internal 2'-O-methyl-modified aptamer SQ7-1(2) <220> <223> internal 2'-O-methyl-modified aptamers (from 7 to 11) <220> <221> modified_base <222> (7)..(11) <223> 2'-O-methyl-modified bases from 7 to 11 <400> 15 gttggtauau acttctttag cttggaacca ac 32 <210> 16 <211> 32 <212> DNA_RNA <213> Artificial Sequence <220> <223> internal 2'-O-methyl-modified aptamer SQ7-1(3) <220> <223> internal 2'-O-methyl- modified aptamers (from 14 to 18) <220> <221> modified_base <222> (14)..(18) <223> 2'-O-methyl-modified bases from 14 to 18 <400> 16 gttggtatat actucuuuag cttggaacca ac 32 <210> 17 <211> 32 <212> DNA_RNA <213> Artificial Sequence <220> <223> internal 2'-O-methyl-modified aptamer SQ7-1(4) <220> <223> internal 2'- O-methyl-modified aptamer (from 21 to 26) <220> <221> modified_base <222> (21)..(26) <223> 2'-O-methyl-modified bases from 21 to 26 <400> 17 gttggtatat acttctttag cuuggaacca ac 32 <210> 18 <211> 32 <212> DNA_RNA <213> Artificial Sequ ence <220> <223> internal 2'-O-methyl-modified aptamer SQ7-1(5) <220> <223> internal 2'-O-methyl-modified aptamers (from 27 to 32) <220> <221 > modified_base <222> (27)..(32) <223> 2'-O-methyl-modified bases from 27 to 32 <400> 18 gttggtatat acttctttag cttggaacca ac 32 <210> 19 <211> 32 <212> DNA_RNA <213> Artificial Sequence <220> <223> internal 2'-O-methyl-modified aptamers SQ7-1(6) <220> <223> internal 2'-O-methyl-modified aptamers (from 7 to 26) < 220> <221> modified_base <222> (7)..(26) <223> 2'-O-methyl-modified bases from 7 to 26 <400> 19 gttggtauau acuucuuuag cuuggaacca ac 32 <210> 20 <211> 32 <212> DNA_RNA <213> Artificial Sequence <220> <223> internal 2'-O-methyl-modified aptamers SQ7-1(1,5) <220> <223> internal 2'-O-methyl-modified aptamers ( from 1 to 6 and 27 to 32) <220> <221> modified_base <222> (1)..(6) <223> 2'-O-methyl-modified bases from 1 to 6 <220> <221> modified_base <222> (27)..(32) <223> 2'-O-methyl-modified bases from 27 to 32 <400> 20 guugguatat acttctttag cttggaac ca ac 32 <210> 21 <211> 32 <212> DNA_RNA <213> Artificial Sequence <220> <223> internal 2'-O-methyl-modified aptamer SQ7-1(7) <220> <223> Artificial Sequence <220> <221> modified_base <222> (1)..(5) <223> 2'-O-methyl-modified bases from 1 to 5 <220> <221> modified_base <222> (27)..( 32) <223> 2'-O-methyl-modified bases from 27 to 32 <400> 21 guuggtatat acttctttag cttggaacca ac 32 <210> 22 <211> 32 <212> DNA_RNA <213> Artificial Sequence <220> <223> internal 2'-O-methyl-modified aptamer SQ7-1(9) <220> <223> Artificial Sequence <220> <221> modified_base <222> (1)..(3) <223> 2'-O- methyl-modified bases from 1 to 3 <220> <221> modified_base <222> (30)..(32) <223> 2'-O-methyl-modified bases from 30 to 32 <400> 22 guuggtatat acttctttag cttggaacca ac 32 <210> 23 <211> 79 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer SQ7a <400> 23 agcagcacag aggtcagatg atgttggtat atacttcttt agcttggaac caactcttct 60 cctatgcgtg ctaccgtga <211> 80 <210> 24 212> DNA <213> Artificial Sequence <220> <223> DNA aptamer SQ7b <400> 24 agcaacacag aggtcagatg atgttggtat atacttcttt agcttggaac ccactcttgt 60 cctatgcgtg ctaccgtgaa 80 <210> 25 <211> 80 <212> DNA <213> Artificial Sequence <220> <223> DNA aptam> agcagcacag aggtcagatg cttgggctat ctcttattca tgctgttcca ccgctctcgg 60 cctatgcgtg ctaccgtgaa 80 <210> 26 <211> 79 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer SQ2 <400> 26 agcagcacag aggtcagatg aaccgcgatc tcatctgtac gctcacccgg tcgaagggac 60 ctatgcgtgc taccgtgaa 79 <210> 27 <211> 80 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer SQ3 <400> 27 agcagcacag aggtcagatg tgggggcgat tacgacccgg cgaaattaat agatctgccg 60 cctatgcgtg ctaccgtgaa 80 <210> > DNA <213> Artificial Sequence <220> <223> DNA aptamer SQ4 <400> 28 agcagcacag aggtcagatg agtgtccaac gtcggcgaaa ttaataggtt ggaacgaacg 60 cctatgcgtg ctaccgtgaa 80 <210> 29 <211> 80 <212> DNA <213> Artificial Sequence <223> DNA aptamer SQ5 <400> 29 agcagcacag aggtcagatg tcatggcaaa acgtccctac gctacgatta gttaggtcga 60 cctatgcgtg ctaccgtgaa 80 <210> 30 <211> 80 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer SQ6 <400> 30 agcagcacag aggtcagatg gcttgccaca acgtgcgact 210> 30 <211> 80 <212> DNA <213> Artificial Sequence <220> <223> <211> 80 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer SQ8 <400> 31 agcagcacag aggtcagatg cttgggctat ttcttattca tgctgttcca ccgctctcgg 60 cctatgcgtg 213 ctaccgtgaa 80 <210> 32 <211> > Artificial Sequence <220> <223> DNA aptamer SQ9 <400> 32 agcagcacag aggtcagatg attcaacttt gaataagtcc agtgacttct aacatagtgg 60 cctatgcgtg ctaccgtgaa 80 <210> 33 <211> 80 <212> DNA <213> Artificial Sequence <220> <223> aptamer SQ10 <400> 33 agcagcacag aggtcagatg ctcttgaagg gatacattgc ccttcgatgc ttgtctgatc 60 cctatgcgtg ctaccgtgaa 80 <210> 34 <211> 80 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence aptcagcaagq11 aggact aptamer SQ10 <213> Artificial Sequence <220> <223> aggact agatatcaac cggtaggcct 60 cctatgcgtg ctaccgtgaa 80 <210> 35 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer SQ8-1 <400> 35 cagcacagag gtcagatgct tgggctattt cttattcatg ctg 43 <210> 36 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer SQ8-2 <400> 36 cagcacagag gtcagatgct tgggct 26 <210> 37 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer SQ8-3 <400> 37 catgctgttc caccgctctc ggcctatgcg tg 32 <210> 38 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer SQ8-4 <400> 38 agcagcacag aggtcagatg cttgggct 28 <210> 39 <211> 23 <212> DNA <213 > Artificial Sequence <220> <223> DNA aptamer SQ8-5 <400> 39 ctcggcctat gcgtgctacc gtg 23 <210> 40 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer SQ8-6 <400> 40 caccgctctc ggcctatgcg tgctaccgtg 30 <210> 41 <211> 32 <212> DNA_RNA <213> Artificial Sequence <220> <223> internal 2'-O-methyl-modified aptamer SQ7-1(8)-Rev < 220> <223> internal 2'-O-methyl-modified aptamer (from 1 to 4 and 29 to 32) <220> <221> modified_base <222> (1)..(4) <223> 2'-O-methyl-modified bases from 1 to 4 <220> <221> modified_base <222> (29)..(32) <223> 2'-O-methyl-modified bases from 29 to 32<400> 41 caaccatata tgaagaaatc gaaccttggu ug 32

Claims (13)

(a) an anti-digoxigenin humanized antibody or antigen-binding fragment thereof;
(b) digoxigenin;
(c) a cytotoxin or detection label, and
(d) a complex comprising an aptamer which may comprise a linker,
The complex in the form of a conjugate in which (b) digoxigenin and (c) a cytotoxin or detection label are covalently conjugated via an aptamer that may include (d) a linker. The method according to claim 1, wherein the anti-digoxigenin humanized antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 5; And SEQ ID NO: 7 to comprising a light chain variable region comprising an amino acid sequence selected from the group consisting of 10, the complex. The complex of claim 1, wherein the anti-digoxigenin humanized antibody or antigen-binding fragment thereof is one of:
a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2; and an anti-digoxigenin humanized antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7;
b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3; and an anti-digoxigenin humanized antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7;
c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4; and an anti-digoxigenin humanized antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7;
d) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5; and an anti-digoxigenin humanized antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7;
e) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2; and an anti-digoxigenin humanized antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8;
f) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3; and an anti-digoxigenin humanized antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8;
g) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4; and an anti-digoxigenin humanized antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8;
h) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5; and an anti-digoxigenin humanized antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8;
i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2; and an anti-digoxigenin humanized antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 9;
j) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3; and an anti-digoxigenin humanized antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 9;
k) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4; and an anti-digoxigenin humanized antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 9;
l) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5; and an anti-digoxigenin humanized antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 9;
m) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2; and an anti-digoxigenin humanized antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10;
n) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3; and an anti-digoxigenin humanized antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10;
o) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4; And an anti-digoxigenin humanized antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10, and
p) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5; and an anti-digoxigenin humanized antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10. The complex according to claim 1, wherein the anti-digoxigenin humanized antibody is in the form of IgG1, IgG2, IgG3 or IgG4. The complex according to claim 1, wherein the antigen-binding fragment of the antibody is selected from the group consisting of scFv, (scFv) ₂ , scFv-Fc, Fab, Fab' and F(ab') ₂ . The complex of claim 1, wherein the aptamer specifically binds to a target tissue, target cell, target carbohydrate, target lipid, or target protein. 7. The method of claim 6, wherein the target tissue, target cell, target carbohydrate, target lipid, or target protein is selected from the group consisting of an infected tissue, an infected cell, an infection-specific carbohydrate, an infection-specific lipid, an infection-specific protein; inflamed tissue, inflammatory cells, inflammation-specific carbohydrates, inflammation-specific lipids, inflammation-specific proteins; tumor tissue, tumor cells, tumor-specific carbohydrates, tumor-specific lipids, or tumor proteins. The complex according to claim 1, wherein the aptamer comprises any one of the nucleotide sequences of SEQ ID NOs: 11 to 40. According to claim 1, wherein the cytotoxic substance is a DNA production inhibitor, calicheamicin, pyrrolobenzodiazepine (PBD), duocarmycin, anthracycline / nimorubicin (Anthracycline / Nemorubicin), doxorubicin, irinotecan, amatoxin, micro tubul inhibitor, auristatin, maytansine, tubulin, inhibitor of topoisomerase I (SN-38), inhibitor of topoisomerase II, platinum coordination compound, alkylating agent, photosensitizer, 5-Aminolaevulinic acid (ALA) ), Benzoporphyrin derivative monoacid ring A (BPD-MA), chlorine, Tetra (m-hydroxyphenyl) chlorin (mTHPC), Lutetium texaphyrin, MMAE (monomethyl auristatin E), MMAF (monomethyl auristatin F), from the group consisting of DM4 and DM1 One of the selected ones, the complex. The fluorescent protein, fluorescent nanoparticle, ¹⁸ F, ¹²⁴ I, ¹²⁵ I, ¹³¹ I according to claim 1, wherein the detection label is a fluorescent substance, a fluorescent dye, a tetracysteine fluorescent motif, GFP, YFP, CFP, or RFP. , and a radiolabel containing a radioisotope selected from the group consisting of ²¹¹ At, ¹⁸ F-FDG (2-deoxy-2-[ ¹⁸ F]fluro-D-glucose), and selected from the group consisting of a free dosimeter to become a complex. A method for preparing the composite according to claim 1, comprising:
i) preparing a conjugate by covalently conjugating (b) digoxigenin and (c) a cytotoxin through (d) an aptamer that may include a linker; and
ii) (a) binding an anti-digoxigenin humanized antibody or antigen-binding fragment thereof to the conjugate. (a) an anti-digoxigenin humanized antibody or antigen-binding fragment thereof;
(b) digoxigenin;
(c) a cytotoxin, and
(d) a complex comprising an aptamer which may comprise a linker,
The composition for cancer treatment comprising a complex in which (b) digoxigenin and (c) cytotoxin are covalently conjugated via an aptamer that may include (d) a linker. (a) an anti-digoxigenin humanized antibody or antigen-binding fragment thereof;
(b) digoxigenin;
(c) a cytotoxin, and
(d) a complex comprising an aptamer which may comprise a linker,
The composition for detecting cancer cells comprising a complex in which (b) digoxigenin and (c) cytotoxin are covalently conjugated via an aptamer that may include (d) a linker.

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