Anti-digoxigenin humanized antibody and its uses

Abstract

The present invention relates to a humanized antibody having excellent binding affinity for digoxigenin and its uses. When using an antibody or an antigen-binding fragment thereof that specifically recognizes digoxigenin according to an embodiment of the present invention, it can be expected to regulate the concentration of a drug in vivo as needed or increase the half-life of the drug in a living body by means of a complex (DOligobody) containing the same.

Description

Technical Field

[0001] The present invention relates to a humanized antibody having excellent binding affinity for digoxigenin and uses thereof. Background Art

[0002] A variety of physiologically active components such as compounds, ligands, and cytotoxic substances lose their pharmacological effects in the body through various mechanisms such as decomposition or conversion into other components. In particular, most substances are excreted through the kidneys after decomposition and lose their effects. Therefore, research is continuously being conducted to increase the utilization rate of physiologically active components in the body by increasing their half-life in the living body.

[0003] Related thereto, attempts have been made to develop antibody-drug conjugates. For example, a drug that is desired to be delivered to a relevant tissue or the like is conjugated to an antibody that binds to a target tissue, cell, or 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 this process, attempts have also been made to link polyethylene glycol (PEG) in order to increase the half-life of the drug in the body (PEGylation, successful approach to drug delivery. Francesco M. Veronese, Gianfranco Pasut. Drug Discovery Today. 2005 Nov 1;10(21):1451-1458.).

[0004] However, the drug that directly reaches the target tissue or cell after being linked to an antibody or polyethylene glycol becomes a very large molecule due to its linked form, and thus it is difficult to transport the desired drug into the desired tissue through capillaries. Therefore, there is a need to develop a new form of drug transporter that can transport a drug to a target tissue or cell while increasing the residence time of the drug in the human body.

[0005] On the other hand, digoxigenin is a steroid discovered after being extracted from a plant called Digitalis purpurea and is the aglycone as the non-saccharide part in the glycoside of digoxin. Digoxigenin is generally considered a hapten as a substance that does not have immunogenicity and is often used in the field of molecular biology because of such properties.

[0006] Digoxigenin is used in a similar way to other widely used haptens such as 2,4-dinitrophenol, biotin, fluorescein, etc. Generally, in experiments, it is chemically bound to relevant biomolecules in order to assay biomolecules such as proteins and nucleic acids. Among them, biotin, fluorescein, and digoxigenin are the most commonly used haptens.

[0007] Biotin has the advantage of having the strongest binding force among them due to its binding to avidin, but since some tissues such as the liver may contain endogenous biotin, it has the disadvantage of possible non-specific binding to avidin.

[0008] Also, in the case of the fluorescein hapten, it has the advantage that the bound biomolecules can be directly confirmed by a fluorescence microscope or indirectly confirmed using anti-fluorescein antibodies, but background problems may occur when observing data using a microscope and the signal duration is not long.

[0009] On the other hand, digoxigenin has been widely used in the field of molecular biology, etc., and various antibodies based on it have been commercialized. Anti-digoxigenin antibodies with high binding force and specific binding properties are used in various biological immunoassays (such as enzyme-linked immunosorbent assay (ELISA)). Antibodies can be directly or indirectly labeled with dyes, enzymes, or fluorescent substances for the visualization or detection of substances.

[0010] Therefore, digoxigenin is used as a standard immunohistochemical marker for various immuno-labels, especially for in situ hybridization. In related experiments, digoxigenin is incorporated into ribonucleic acid by binding to a specific species (usually uridine) of ribonucleic acid triphosphate.

[0011] On the other hand, in the field of molecular biology, modification of antibodies to digoxigenin used only for the purpose of haptens on the premise of use in the human body has not been achieved.

[0012] Prior Art Documents

[0013] Patent Document 1: Korean Patent No. 10-1648960

[0014] Patent Document 2: Korean Patent No. 10-1827962 Summary of the Invention

[0015] Technical Problem

[0016] In the process of the present inventor's research on a system capable of increasing the residence time in a drug delivery system, attention was paid to digoxigenin, which has been used as a biochemical detection tool in the past, and it was found that the digoxigenin and anti-digoxigenin humanized antibody system can be used for the purpose of a drug delivery system that increases the drug half-life. For this reason, as a result of continuously working on developing the existing anti-digoxigenin mouse antibody into a humanized antibody, an antibody that can be used in humans and has a more excellent antigen-binding ability than the mouse antibody was developed, and its effect was confirmed.

[0017] Technical solution

[0018] In order to solve the above problems, an embodiment of the present invention provides an anti-digoxigenin antibody. Specifically, an antibody or an antigen-binding fragment thereof that specifically recognizes digoxigenin is provided.

[0019] Effect of the invention

[0020] It is expected that through a complex (Drug-conjugated Oligobody; DOligobody) containing an antibody or an antigen-binding fragment thereof that specifically recognizes digoxigenin according to an embodiment of the present invention, the concentration of a bioactive substance in the human body can be adjusted as needed or the half-life of a drug in the living body can be increased. Description of the drawings

[0021] Figure 1( Figures 1a to 1c ) is a graph showing the results of confirming the binding ability of the mouse antibody (mAb[21H8]), chimeric antibody (chimeric[21H8]), humanized antibodies (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) prepared in the present invention to digoxigenin (DIG) by an enzyme-linked immunosorbent assay experiment.

[0022] Figure 2 It is a diagram showing the structure of a complex in which the antibody prepared in the present invention binds to a digoxigenin-aptamer-monomethyl auristatin E (MMAE) conjugate.

[0023] Figure 3( Figures 3a to 3c) It is a graph of the results of a fluorescence-activated cell sorting (FACS) experiment to confirm the binding affinity of the complex (DOligobody) formed by the antibody in the present invention and the digoxigenin-aptamer-monomethyl auristatin E conjugate to CFPAC-1, a human pancreatic cancer cell line.

[0024] Figure 4 It is the result of comparing the binding rate of the complex DOligobody of the present invention based on the fluorescence-activated cell sorting results.

[0025] Figure 5 It is a schematic diagram for explaining the outline of a pharmacokinetic experiment involving the anti-digoxigenin antibody complex DOligobody.

[0026] Figure 6 It is a graph showing the pharmacokinetic results of the conjugate (DOligomer) containing digoxigenin and aptamer and the complex DOligobody containing anti-digoxigenin antibodies such as humab[21H8]-v5 antibody, humab[21H8]-v6 antibody, and humab[21H8]-v8 antibody.

[0027] Figure 7 It is a table organizing the half-lives determined from the pharmacokinetic results of the conjugate (DOligomer) containing digoxigenin and aptamer and the complex DOligobody containing anti-digoxigenin antibodies such as humab[21H8]-v5 antibody, humab[21H8]-v6 antibody, and humab[21H8]-v8 antibody.

[0028] Figure 8 It is a graph showing the pharmacokinetic results of the complex DOligobody containing anti-digoxigenin antibodies such as humab[21H8]-v5 antibody, humab[21H8]-v6 antibody, and humab[21H8]-v8 antibody.

[0029] Figure 9 It is a table organizing the half-lives determined from the pharmacokinetic results of the complex DOligobody containing anti-digoxigenin antibodies such as humab[21H8]-v5 antibody, humab[21H8]-v6 antibody, and humab[21H8]-v8 antibody. Detailed implementation mode

[0030] The examples or implementation modes of the present invention are illustrated for the purpose of explaining the idea of the present invention. The scope of the rights of the present invention is not limited to the examples or implementation modes disclosed below or the specific descriptions related thereto.

[0031] Moreover, those of ordinary skill in the art to which the present invention pertains can recognize or confirm, through ordinary experiments, most of the equivalents of the specific embodiments of the present invention described in the present invention. All such equivalents are included in the present invention.

[0032] Unless otherwise defined, all technical and scientific terms used in the present invention have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains. All terms used in the present invention are selected only to more clearly illustrate the present invention and are not intended to limit the present invention.

[0033] Expressions such as "comprising", "including", and "having" used in the present invention shall be understood as open-ended terms that may also include other embodiments if there is no other definition in the sentences or paragraphs in which they are expressed.

[0034] Expressions such as "consisting only of" used for the relevant structures in the present invention shall be understood as closed-ended terms that exclude the possibility of including other structures except the relevant structures.

[0035] Unless otherwise defined, singular expressions described in the present invention may include plural meanings, and this also applies to the singular expressions described in the claims of the invention.

[0036] The "antibody" in the present invention refers to a substance formed by the stimulation of an antigen within the immune system, and its type is not particularly limited. The antibodies in this specification include all animal antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies. Moreover, the antibodies in this specification also include antigen-binding fragments of antibodies that retain the ability to bind to antigens, such as Fab.

[0037] The "chimeric antibody" in the present invention refers to an antibody in which the variable region or its complementarity determining region (CDR) of the antibody is derived from an animal different from the rest of the antibody. For example, such an antibody may be an antibody in which the variable region of the antibody is derived from an animal other than humans (such as mice, rabbits, poultry, etc.), and the constant region of the antibody is derived from humans. Such chimeric antibodies can be prepared by methods such as genetic recombination well-known in the art to which the present invention pertains.

[0038] The above-mentioned antibody can be a polyclonal antibody or a monoclonal antibody. The above-mentioned antibody can be selected from immunoglobulins of all subtypes (such as IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM, etc.). The antibody in the form of IgG can be of the IgG1, IgG2, IgG3 or IgG4 subtype. For example, it can be in the form of the IgG1 or IgG2 subtype.

[0039] The "antigen-binding fragment" of the antibody or immunoglobulin chain (heavy chain or light chain) used in the present invention, compared with the full-length chain, lacks some amino acids but contains a part of the antibody capable of specifically binding to the antigen. Such an antigen-binding fragment can specifically bind to the target antigen, or it can be said to have biological activity in competing with other antibodies or antigen-binding fragments for binding to a specific epitope. Specifically, the above-mentioned antigen-binding fragment can be an antibody fragment containing one or more of the above-mentioned complementary determining regions. For example, it can be selected from the group consisting of scFv, (scFv)2, scFv-Fc, Fab, Fab' and F(ab')2, but is not limited thereto. Such a biologically active fragment can be produced by recombinant deoxyribonucleic acid (DNA) technology, or can be produced by, for example, enzymatically or chemically cleaving the intact antibody.

[0040] The "heavy chain" in the present invention refers to the full-length heavy chain including the variable domain VH containing an amino acid sequence sufficient to confer specificity for the antigen and three constant domain CH1, CH2 and CH3 and its fragments.

[0041] The "light chain" in the present invention refers to the full-length light chain including the variable domain VL containing an amino acid sequence sufficient to confer specificity for the antigen and the constant domain CL and its fragments.

[0042] The "complementary determining region" in the present invention refers to the site in the variable region of the antibody that confers binding specificity to the antigen.

[0043] The "digoxigenin - specific humanized antibody or its antigen - binding fragment" in the present invention may comprise: a heavy - chain variable region comprising an amino - acid sequence selected from the group consisting of Sequence 2 to Sequence 5, and a light - chain variable region comprising an amino - acid sequence selected from the group consisting of Sequence 7 to Sequence 10. For example, the digoxigenin - specific humanized antibody or its antigen - binding fragment of the present invention may be: a) a digoxigenin - specific humanized antibody or its antigen - binding fragment, comprising: a heavy - chain variable region comprising the amino - acid sequence of Sequence 2 and a light - chain variable region comprising the amino - acid sequence of Sequence 7; b) a digoxigenin - specific humanized antibody or its antigen - binding fragment, comprising: a heavy - chain variable region comprising the amino - acid sequence of Sequence 3 and a light - chain variable region comprising the amino - acid sequence of Sequence 7; c) a digoxigenin - specific humanized antibody or its antigen - binding fragment, comprising: a heavy - chain variable region comprising the amino - acid sequence of Sequence 4 and a light - chain variable region comprising the amino - acid sequence of Sequence 7; d) a digoxigenin - specific humanized antibody or its antigen - binding fragment, comprising: a heavy - chain variable region comprising the amino - acid sequence of Sequence 5 and a light - chain variable region comprising the amino - acid sequence of Sequence 7; e) a digoxigenin - specific humanized antibody or its antigen - binding fragment, comprising: a heavy - chain variable region comprising the amino - acid sequence of Sequence 2 and a light - chain variable region comprising the amino - acid sequence of Sequence 8; f) a digoxigenin - specific humanized antibody or its antigen - binding fragment, comprising: a heavy - chain variable region comprising the amino - acid sequence of Sequence 3 and a light - chain variable region comprising the amino - acid sequence of Sequence 8; g) a digoxigenin - specific humanized antibody or its antigen - binding fragment, comprising: a heavy - chain variable region comprising the amino - acid sequence of Sequence 4 and a light - chain variable region comprising the amino - acid sequence of Sequence 8; h) a digoxigenin - specific humanized antibody or its antigen - binding fragment, comprising: a heavy - chain variable region comprising the amino - acid sequence of Sequence 5 and a light - chain variable region comprising the amino - acid sequence of Sequence 8; i) a digoxigenin - specific humanized antibody or its antigen - binding fragment, comprising: a heavy - chain variable region comprising the amino - acid sequence of Sequence 2 and a light - chain variable region comprising the amino - acid sequence of Sequence 9; j) a digoxigenin - specific humanized antibody or its antigen - binding fragment, comprising: a heavy - chain variable region comprising the amino - acid sequence of Sequence 3 and a light - chain variable region comprising the amino - acid sequence of Sequence 9; k) a digoxigenin - specific humanized antibody or its antigen - binding fragment, comprising: a heavy - chain variable region comprising the amino - acid sequence of Sequence 4 and a light - chain variable region comprising the amino - acid sequence of Sequence 9; l) a digoxigenin - specific humanized antibody or its antigen - binding fragment, comprising: a heavy - chain variable region comprising the amino - acid sequence of Sequence 5 and a light - chain variable region comprising the amino - acid sequence of Sequence 9; m) a digoxigenin - specific humanized antibody or its antigen - binding fragment, comprising: a heavy - chain variable region comprising the amino - acid sequence of Sequence 2 and a light - chain variable region comprising the amino - acid sequence of Sequence 10; n) a digoxigenin - specific humanized antibody or its antigen - binding fragment, comprising: a heavy - chain variable region comprising the amino - acid sequence of Sequence 3 and a light - chain variable region comprising the amino - acid sequence of Sequence 10;o) A humanized antibody against digoxigenin or an antigen-binding fragment thereof, comprising: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10; and p) A humanized antibody against digoxigenin or an antigen-binding fragment thereof, comprising: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10.;

[0044] The "humanized antibody" in the present invention is an antibody comprising a sequence derived from a non-human immunoglobulin that specifically binds to a specific antigen, and is a human immunoglobulin in which the sequences of complementarity-determining regions (CDRs) that determine the binding affinity for the antigen in the non-human immunoglobulin are replaced. On the other hand, the residues of the human immunoglobulin Fv framework region (FR) of the above-mentioned humanized antibody may be replaced by corresponding non-human framework region residues, or may contain residues not found in either the complementarity-determining regions or the framework regions of the non-human immunoglobulin or the human immunoglobulin. The performance of the antibody can be optimized by making the above-mentioned modifications. Generally, all or substantially all of the complementarity-determining regions of the humanized antibody correspond to the regions of the non-human immunoglobulin, and all or substantially all of the framework regions may contain residues of at least one human immunoglobulin consensus sequence, typically, may contain substantially all of the two variable regions.

[0045] In the present invention, the humanized antibody against digoxigenin or an antigen-binding fragment thereof may comprise any one of the amino acid sequences of SEQ ID NOs: 2 to 5, or may comprise any one of the amino acid sequences of SEQ ID NOs: 7 to 10. One embodiment of the present invention provides an antibody comprising an amino acid having a homology of about 80% or more, specifically, an amino acid having a homology of about 90% or more, more specifically, an amino acid having a homology of about 95% or more, even more specifically, an amino acid having a homology of about 97% or more, and most specifically, an amino acid having a homology of about 99% or more, with any one of the amino acid sequences of SEQ ID NOs: 2 to 5 or SEQ ID NOs: 7 to 10. A sequence having such homology may unrestrictedly comprise an amino acid sequence constituting an antibody that is substantially the same as or has a corresponding binding activity to the antibody comprising any one of the amino acid sequences of SEQ ID NOs: 2 to 5 or SEQ ID NOs: 7 to 10. And, as long as it is an amino acid sequence having such homology, even an amino acid sequence with a partial sequence deletion, modification, substitution, or addition is included within the scope of the present invention.

[0046] The "conjugate" in the present invention refers to a chimeric molecule of digoxigenin and other molecules, particularly a chimeric molecule of digoxigenin and the bioactive substance described below. The conjugate in the present invention refers to a conjugate in which digoxigenin and other molecules are covalently bound. For example, the above bioactive substance may be a chemotherapeutic drug, an enzyme, a peptide, a cytotoxin, an affinity ligand, or a detection label. Specifically, the digoxigenin and the bioactive substance of the conjugate of the present invention can be covalently bound through an aptamer. The meaning of covalent binding means that the molecules are directly covalently bound to each other, or are indirectly connected in a covalent binding manner through a linker, a spacer, a connecting portion, etc. The conjugate in which the digoxigenin of the present invention is bound to the bioactive substance can also be referred to as a DOligomer (Drug-conjugated Oligomer) having the meaning of an oligomer to which a drug is bound.

[0047] The aptamer in the present invention is a short single-stranded oligonucleotide, which has the property of binding to the item list with high affinity and specificity, respectively representing the meaning of having a unique three-dimensional structure. The aptamer specifically binds to a target tissue, target cell, target carbohydrate, target lipid or target protein. The relevant target tissue, target cell, target carbohydrate, target lipid or target protein can be a specific tissue, cell, carbohydrate, lipid or protein in a specific disease. For example, it can be an infected tissue, infected cell, infected specific carbohydrate, infected specific lipid, infected specific protein; an inflamed tissue, inflamed cell, inflamed specific carbohydrate, inflamed specific lipid, inflamed specific protein; a tumor tissue, tumor cell, tumor specific carbohydrate, tumor specific lipid or tumor specific protein, etc., but not limited thereto. The aptamer in the present invention contains any one of the base sequences from sequence 11 to sequence 40 that binds to, for example, cancer cells, especially pancreatic cancer tissue or pancreatic cancer cells. As a result of the present inventors' efforts to develop aptamers that bind to cancer cells, it was confirmed that the aptamers of sequences 11 to 40 bind to cancer cells (refer to Korean Patent Publication No. 10-2020-0078303). One embodiment of the present invention provides a deoxyribonucleic acid aptamer containing a base sequence having a sequence homology of about 80% or more with any one of the base sequences from sequence 11 to sequence 40. Specifically, a deoxyribonucleic acid aptamer having a base sequence with a sequence homology of about 90% or more, more specifically, a deoxyribonucleic acid aptamer having a base sequence with a sequence homology of about 95% or more, even more specifically, a deoxyribonucleic acid aptamer having a base sequence with a sequence homology of about 97% or more, and most specifically, a deoxyribonucleic acid aptamer having a base sequence with a sequence homology of about 99% or more. A sequence having such homology can unrestrictedly contain the base sequence constituting an aptamer having the same or corresponding binding activity as the deoxyribonucleic acid aptamer containing any one of the base sequences from sequence 11 to sequence 40. And as long as it is a base sequence having such homology, even an amino acid sequence with partial sequence deletion, deformation, substitution or addition is included within the scope of the present invention. One embodiment of the present invention can be an aptamer formed by any one of the base sequences from sequence 11 to sequence 40.

[0048] The chemotherapeutic agent or cytotoxic substance according to an embodiment of the present invention may be selected from the group consisting of, but not limited to, deoxyribonucleic acid generation inhibitors (e.g., calicheamicin, pyrrolobenzodiazepine (PBD), duocarmycin, anthracycline / nemorubicin, doxorubicin, irinotecan, amanitin), tubulin inhibitors (auristatin, maytansine, tubulysin), 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), chlorin, tetra(m-hydroxyphenyl)chlorin (mTHPC), lutetium texaphyrin, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), DM4, and DM1).

[0049] The detection marker according to an embodiment of the present invention may be selected from the group consisting of: fluorescent substances such as fluorescent dyes, tetracysteine fluorescent motifs, fluorescent proteins including GFP, YFP, CFP, and RFP, or fluorescent nanoparticles, radioactive markers including radioactive isotopes in tissues selected from the group consisting of 18 F, 124 I, 125 I, 131 I, and 211 At, such as 2-deoxy-2- 18 F]fluoro-D-glucose ( 18 F-FDG, 2-deoxy-2- 18 F]fluro-D-glucose), and glass dosimeters. However, it is not limited thereto.

[0050] The "complex" in the present invention refers to a complex formed by the anti-digoxigenin humanized antibody of the present invention or its antigen-binding fragment binding to a conjugate containing the digoxigenin of the present invention. The complex can be formed by binding the above-mentioned antibody or its antigen-binding fragment to the conjugate through covalent binding of non-peptide bonds (such as disulfide bonds) and / or non-covalent interactions (such as hydrogen bonds, ionic bonds, van der Waals forces, and hydrogen interactions, etc.). The complex formed by the conjugate of the present invention binding to the anti-digoxigenin antibody can also be referred to as DOligobody (Drug-conjugated Oligobody) having the meaning of a complex bound with a drug.

[0051] The "homology" in the present invention refers to the degree of identity of bases or amino acid residues between two sequences after aligning the two sequences in the most consistent manner in a specific comparison region in the amino acid sequence or base sequence of the gene encoding the protein. When the homology is high enough, the expression products of related genes can have the same or similar activities. The percentage (%) of the above sequence homology can be determined using well-known sequence comparison programs (such as Blast (National Center for Biotechnology Information (NCBI)) in the United States, etc.).

[0052] In one embodiment of the present invention, the term "about" is used for the purpose of including errors in the preparation process included in the specific value and slight numerical adjustments included within the scope of the technical concept of the present invention. For example, the term "about" refers to a range of ±10% of the stated value, in one embodiment, it refers to a range of ±5%, and in another embodiment, it refers to a range of ±2%. In the field of the present disclosure, as long as a narrower range is not specifically mentioned for the value, an approximate value at this level is appropriate.

[0053] The proteins or antibodies of the present invention can be expressed or produced by methods well-known in the technical field to which the present invention pertains. Suitable host cells for expressing and producing the proteins or antibodies in the present invention include higher eukaryotic cells such as vertebrate host cells described in the present application. The proliferation of vertebrate cells in culture medium has become a common technique. Examples of useful mammalian host cells include monkey kidney cells (CV1), monkey kidney CV1 cell line transformed by SV40 (COS-7), human embryonic kidney cell 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 cancer cells (HELA), dog kidney cells (MDCK), BRL rat hepatocytes (BRL3A), human lung cells (W138), human hepatocytes (Hep G2), mouse mammary tumors (MMT 060562), TRI cells, MRC 5 cells, and FS4 cells, etc., but are not limited thereto.

[0054] The host cells used to produce the proteins or antibodies of the present invention can be cultured in a variety of media. Commercially available media such as, for example, Ham's F10 medium, Minimal Essential Medium, RPMI-1640, Dulbecco's Modified Eagle's Medium (DMEM), etc., and any other media used in the technical field to which the present invention pertains can be used to culture the host cells. Any of the above media can be supplemented, as needed, with hormones and / or growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium chloride, magnesium chloride and phosphates), buffers (such as N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES)), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN TM drug), trace elements and glucose or an equivalent energy source. It is also possible to include any other necessary / supplementary supplements at appropriate concentrations known to those of ordinary skill in the relevant technical field. In addition, culture conditions such as temperature, pH, CO2 concentration, etc. can also be set to appropriate conditions related to the host cells selected for protein expression, which will be obvious to those of ordinary skill in the relevant technical field.

[0055] The proteins or antibodies in the present invention can be purified by any protein or antibody purification method known in the technical field to which the present invention pertains. For example, a variety of known purification methods can be used, including hydrophobic interaction, immunoaffinity or fractionation on an ion exchange column, ethanol precipitation, reverse-phase high performance liquid chromatography (HPLC), chromatography on a silica column, chromatography on an anion or cation exchange resin, ion exchange focusing chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), ammonium sulfate precipitation, gel filtration or genetic engineering methods (such as tags, etc.). In order to improve the purity of the protein, any number of purification methods can be combined and used.

[0056] In the present invention, the proteins or antibodies purified by the above methods can have a purity high enough to be administered for experimental, clinical and therapeutic purposes in animals, especially in humans.

[0057] When preparing a complex in which the anti-digoxigenin humanized antibody or its antigen-binding fragment of the present invention binds to the conjugate containing digoxigenin of the present invention, the molar ratio of the antigen of the anti-digoxigenin humanized antibody to the conjugate containing digoxigenin is 1:2, but it is not limited thereto.

[0058] In the present invention, the pharmaceutical / detection composition containing the above complex can be formulated and administered in a manner consistent with medical practice. Factors to be considered in this regard include the disease to be treated or detected, the specific animal to be treated or detected, the clinicopathological status of the individual subject, the cause of the disease, the site of substance delivery, the method of administration, the dosing regimen, and other factors known in the art.

[0059] In the present invention, the dosage form may include a liquid dosage form or a freeze-dried powder dosage form, etc. The pharmaceutical / detection composition of the present invention can be prepared in the form of an ampoule, vial, bottle, box, storage box, lyogect, or prefilled syringe, etc., and can be prepared as a single-dose type or a multi-dose type.

[0060] The "dosage administered", "pharmaceutically effective amount", "therapeutically effective amount", or "effective administered amount" of the complex to be administered is determined by the above considerations and is the minimum amount required to prevent, ameliorate, detect, or treat a specific disease. For example, the "dosage administered", "pharmaceutically effective amount", "therapeutically effective amount", or "effective administered amount" of the above complex in the present invention can be about 0.001 mg / kg to about 500 mg / kg, but is not limited thereto. The pharmaceutical / detection composition of the present invention can be administered periodically 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., according to the judgment of clinical experience, and can be administered aperiodically according to the acute course of the disease, the urgency of detection, etc., but is not limited thereto.

[0061] In the present invention, the pharmaceutical / detection composition can be prepared by mixing the complex of the present invention with a physiologically acceptable carrier, excipient, or stabilizer and using standard methods known in the technical field to which the present invention pertains. Acceptable carriers include physiological saline or buffers, such as phosphates, citrate silicates, and other organic acids; antioxidants, such as ascorbic acid; low molecular weight polypeptides (including about less than 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 dextrin; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium ions; and / or nonionic surfactants, such as TWEEN TM , PLURONICS TM or polyethylene glycol (PEG).

[0062] The pharmaceutical / detection composition of the present invention can contain salts at approximately physiological concentrations. The dosage forms of the present invention can optionally contain any pharmaceutically acceptable preservatives. Specifically, the concentration of the preservative can be from 0.1% to 2.0% (typically, v / v). The preservatives in the present invention can be those well-known in the pharmaceutical industry. Specifically, they can be benzyl alcohol, phenol, m-cresol, methyl paraben, propyl paraben, or a combination thereof. The pharmaceutical / detection composition of the present invention can contain a pharmaceutically acceptable surfactant at a concentration of from 0.005% to 0.02%.

[0063] The pharmaceutical / detection composition of the present invention can also contain one or more active compounds required for treating or detecting the target disease. Specifically, it can also contain active substances with complementary activities that do not have an adverse effect on the complexes of the present invention. The composition can contain an effective amount of the above compounds to achieve the intended purpose.

[0064] In one embodiment of the present invention, there is provided a pharmaceutical composition for treating cancer, which contains a complex formed by the following parts: the anti-digoxigenin humanized antibody of the present invention or its antigen-binding fragment; and a conjugate formed by covalently linking a nucleic acid aptamer that binds digoxigenin and a chemotherapeutic drug or a cytotoxin to cancer cells in a shared manner. For example, the above nucleic acid aptamer can contain any one of the base sequences from Sequence 11 to Sequence 40, but is not limited thereto.

[0065] In one embodiment of the present invention, there is provided a composition for detecting cancer cells, which contains a complex formed by the following parts: the anti-digoxigenin humanized antibody of the present invention or its antigen-binding fragment; and a conjugate formed by covalently linking a nucleic acid aptamer that binds digoxigenin and a detection-labeled cancer cell in a shared manner. For example, the above nucleic acid aptamer can contain any one of the base sequences from Sequence 11 to Sequence 40, but is not limited thereto.

[0066] In one embodiment of the present invention, there is provided a method for treating cancer or detecting cancer cells, which includes the step of administering a pharmaceutical / detection composition containing the complex of the present invention.

[0067] In the present invention, the pharmaceutical / detection composition containing the above complex can be administered to human or animal subjects by intravenous, intramuscular, intraperitoneal, intrathecal, subcutaneous, intra-articular, intra-synovial, intradural, oral, topical, subcutaneous, or inhalation routes according to well-known methods.

[0068] Hereinafter, the structure and effects of the present invention will be described more specifically through examples and test examples. However, these examples and test examples are provided for illustrative purposes only to assist in understanding the present invention, and the scope and range of the present invention are not limited to the following examples.

[0069] Furthermore, embodiments of the present invention will be described with reference to the accompanying drawings. In the drawings, the same or corresponding structural elements may be given the same reference numerals. Also, in the following description of the embodiments, repeated descriptions of the same or corresponding structural elements may be omitted. However, omitting the relevant description of such a structural element does not mean that the structural element is not included in a certain embodiment.

[0070] Embodiment

[0071] Embodiment 1. Preparation of a humanized antibody against digoxigenin

[0072] The inventors of the present invention prepared a humanized antibody based on a mouse antibody. Specifically, a mouse antibody against digoxigenin having a heavy chain variable region containing Sequence 1 and a light chain variable region containing Sequence 6 was obtained.

[0073] The sequences of such mouse antibodies were analyzed, and a humanization process was carried out in which specific sequences were humanized while maintaining specific sequences to reduce the possibility of immunogenicity and maintaining the binding ability as much as possible.

[0074] ※ Method for optimizing mouse backbone antibodies

[0075] The mouse monoclonal antibodies used as backbone antibodies in the present invention can be prepared by variously setting the target antigens and can be mass-produced, so they are effectively used in diagnostic reagents or basic research. However, when repeatedly administered to the human body for the purpose of treating diseases, they induce an immune reaction (human anti-mouse antibody response (HAMA)) and cause side effects and reduce the effect. Therefore, they cannot be used as therapeutic agents. To solve the above problems, a preparation technique for preparing a humanized antibody similar to a human antibody from a mouse monoclonal antibody is required so that there are no problems when administered to humans.

[0076] An antibody is composed of a variable region and a constant region. The variable region is composed of complementarity determining regions (CDRs) that directly bind to an antigen and framework regions (FRs) that support the loops of the complementarity determining regions. A humanized antibody is prepared by a grafting method (chimerization) in which the loops of the complementarity determining regions of a mouse antibody are transplanted into a human antibody all at once. However, in the case of simply grafting the complementarity determining regions, the affinity of the humanized antibody decreases. Therefore, several major framework region amino acid residues that are considered to affect the structure of the complementarity determining regions are replaced with the amino acid residues of the mouse antibody, and a process of increasing the affinity to a level similar to that of the original mouse antibody (humanization) is carried out.

[0077] The specific implementation process relies on Prometheus of Absolute Antibody Company TM For humanization of the service source, a total of 16 humanized antibody candidate groups were prepared. The sequences of the heavy chain variable region and the light chain variable region of the prepared antibodies are shown in Table 1 below.

[0078] Table 1

[0079]

[0080]

[0081] Then, a chimeric antibody and 16 humanized antibodies were prepared by combining the heavy chain variable region and the light chain variable region in Table 1 above. The variable region combinations of the prepared humanized antibodies are shown in Table 2 below.

[0082] Table 2

[0083] 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

[0084] The gene with the base sequence encoding the combination of the above heavy chain variable region and light chain variable region was cloned into an antibody expression plasmid (Absolute Antibody cloning and expression vector), and the codons were optimized for good expression in human cells. The accurate sequence was confirmed by Sanger sequencing using DNASTAR Lasergene software, and the size was reconfirmed by plasmid DNA prep, so as to obtain a sufficient amount of high-purity plasmid deoxyribonucleic acid (DNA) for transfection. The prepared high-purity plasmid deoxyribonucleic acid was transiently transfected into the HEK 293 (human embryonic kidney cell 293) cell line. After the cells were transiently transfected with the vectors expressing the heavy chain and the light chain, they were cultured for 6 days. After centrifuging the culture at a speed of 4000 rpm, it was filtered using a 0.22 μM filter. First, the antibody was eluted using a citrate buffer at pH 3.0 by Protein A affinity spectrometry, and then the antibody was eluted using a 0.5 M Tris neutralization buffer at pH 9.0. The eluted protein was exchanged using a phosphate buffer solution (PBS) with a desalting column. The obtained antibody was confirmed using ultraviolet spectroscopy (UV spectroscopy), and the antibody was concentrated as needed.

[0085] The binding ability of the mouse antibody (mAb[21H8]), chimeric antibody (chimeric[21H8]), and 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) prepared above to digoxigenin was confirmed.

[0086] Example 2. Confirmation of the binding capacity of anti-digoxigenin humanized antibodies

[0087] The 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) prepared above were confirmed by enzyme-linked immunosorbent assay. The binding capacity of 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) to digoxigenin. The enzyme-linked immunosorbent assay was performed under the following conditions.

[0088] ※Enzyme-linked immunosorbent assay experimental method

[0089] 1) Immuno Clear plates (Thermo Scientific TM , Cat No.468667) were inoculated into each well with 100 μL of 4 μg / mL digoxigenin-bovine serum albumin (BSA) (CellMosaic TM , Cat.No.CM52107) and then allowed to stand at 4°C overnight.

[0090] 2) Wash the above culture plates 4 times with 150 μL of phosphate buffered solution + 0.05% Tween-20 (PBS-T).

[0091] 3) Treat the above culture plates with 100 μL of blocking solution (phosphate buffered solution + 3% bovine serum albumin Cat.No.126609)) and then let it stand at room temperature for 2 hours.

[0092] 4) After removing the blocking solution from the above culture plates, treat them with 50 μL of each antibody (mAb[21H8], chimeric[21H8], humab[21H8]-v1 to v16) at various concentrations (serially diluted 1 / 3 starting from 53.33 nM, i.e., 17.7 nM, 5.92 nM, 1.97 nM, 0.66 nM, 0.22 nM, 0.07 nM, 0.024 nM) and then let it stand at room temperature for 1 hour.

[0093] 5) Wash the above culture plates 4 times with 150 μL of phosphate buffered solution + 0.05% Tween-20.

[0094] 6) Treat with 50 μL of secondary antibody (immunoglobulin G (IgG) conjugated with horseradish peroxidase (HRP)) and then let it stand at room temperature for 1 hour. Specifically, dilute goat anti-mouse IgG (whole molecule), horseradish peroxidase (Sigma, Cat.No.A6782) and rabbit anti-human IgG Fc secondary antibody, horseradish peroxidase (Invitrogen, Cat.No.31423) to 1 / 5000 for use.

[0095] 7) Wash the above culture plates 4 times with 150 μL of phosphate buffered solution + 0.05% Tween-20.

[0096] 8) Treat the above culture plates with 100 μL of 1-Step TM Ultra TMB - Enzyme-Linked Immunosorbent Assay Substrate (ELISA Substrate) (ThermoFisher Scientific, Cat.No.34029) and incubate for 10 minutes.

[0097] 9) Stop the reaction with 40 μL of 2.5N H2SO4.

[0098] 10) Use Synergy TMThe absorbance of the H1 Hybrid Multi-Mode Reader (BioTek) was measured at 450 nM to determine the OD value.

[0099] The above results are organized as shown in Figure 1 ( Figures 1a to 1c ) and Table 3 below.

[0100] Table 3

[0101]

[0102] *K D : Equilibrium dissociation constant (the smaller the value, the greater the ligand binding affinity for the target). Through the above experiments, it can be confirmed that the binding force of the humanized antibody becomes significantly excellent. For example, it can be seen that the binding force of humab[21H8]-v1 is about 5 times stronger than that of mAb[21H8], and the binding force of humab[21H8]-v3 is about 3 times stronger. In particular, it can be confirmed that the binding force of humab[21H8]-v8 is about 6 times stronger than that of mAb[21H8].

[0103] The purpose of humanizing the antibodies of other animals is to reduce the immunogenicity in the human body, and usually the binding force is reduced during this process. However, the humanized antibodies of the present invention exhibit the exceptional property of having a better binding force than mouse antibodies. The mouse antibody used as the backbone antibody in the present invention does not undergo the process of optimizing the binding force. Therefore, this indicates that the binding force is improved during the humanization and optimization processes. Thus, unexpectedly, the humanized antibody obtained at any time in the present invention has an even better antigen-binding force.

[0104] Example 3. Preparation of the complex DOligobody containing an anti-digoxin ligand antibody

[0105] Example 3-1. Digoxin ligand-nucleic acid aptamer-monomethyl auristatin E conjugate (DOligomer)

[0106] The nucleic acid aptamer used was the nucleic acid aptamer SQ7-1(8) (Sequence 11) that binds to pancreatic cancer cells. A conjugate was prepared by binding digoxin ligand to the 5'-end of the above nucleic acid aptamer and monomethyl auristatin E to the 3'-end. The specific method is as follows.

[0107] A nucleic acid aptamer synthesized by Integrated DNA Technology (IDT; USA) with digoxin ligand bound to the 5'-end and the 3'-end modified to a disulfide bond (S-S) was obtained.

[0108] To cleave the 3'-thiol C3 S-S linker of the aptamer, 0.1 M triethylammonium acetate (TEAA) and 1 mM tris-(2-carboxylethyl)phosphine hydrochloride (TCEP) were added and reacted at 70 °C for 5 minutes, followed by reaction at room temperature for 2 hours. The remaining tris-(2-carboxylethyl)phosphine hydrochloride (TCEP) was removed using phosphate buffer solution and 2 mM ethylenediaminetetraacetic acid (EDTA) through an Amicon Ultra 3K spin column (Millipore Corporation, Billerica, MA).

[0109] The above DIG-aptamer was reacted with 5-fold molar concentration of MC-Val-Cit-PAB-MMAE (Levena Biopharma, San Diego, CA) at room temperature for 16 hours. Then, the remaining monomethyl auristatin E was removed using phosphate buffer solution through an Amicon Ultra 3K spin column (Millipore Corporation, Billerica, MA).

[0110] The synthesized digoxigenin-aptamer-monomethyl auristatin E was analyzed by reverse-phase high-performance liquid chromatography on an Xbridge C18 column (4.6 mm × 50 mm) using a Waters e2695 high-performance liquid chromatography (HPLC) instrument with a UV detector (Waters Corporation, USA) at a wavelength of 260 nM.

[0111] During the analysis, the column temperature was maintained at 65 °C, and 0.1 M triethylammonium acetate at pH 7.0 (eluent A) and acetonitrile (eluent B) were used as the mobile phase. The analysis conditions were as follows: from the start to 5 minutes, 90% of eluent A and 10% of eluent B were flowed, and then from 5 minutes to 15 minutes, eluent B was flowed at a rate of 1 mL / minute with a linear gradient from 20% to 70%.

[0112] The result of analysis by injecting 10 μL of 30 μM DIG-SQ7-1(8)-SH nucleic acid aptamer showed that a peak was confirmed at a retention time of 6.21 minutes. The result of analysis after injecting 10 μL of the prepared 30 μM DOligomer showed that a peak was confirmed at a retention time of 10.02 minutes. This indicates that good synthesis occurred between MC-Val-Cit-PAB-MMAE (Lianning Biotech Co., San Diego, California) and DIG-SQ7-1(8)-SH, and the purity of the synthesized digoxigenin-nucleic acid aptamer-monomethyl auristatin E (DIG-SQ7-1(8)-MMAE) conjugate was confirmed to be 98%.

[0113] Example 3-2. Preparation of a complex (DOligobody) of a digoxigenin-nucleic acid aptamer-monomethyl auristatin E conjugate and an anti-digoxigenin humanized antibody

[0114] The mouse antibody (mAb[21H8]), chimeric antibody (chimeric[21H8]), and humanized antibodies (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) prepared in Example 1 above were combined with the digoxigenin-nucleic acid aptamer-monomethyl auristatin E conjugate (DOligomer) prepared in Example 3-1 to prepare a complex (DOligobody).

[0115] Specifically, 250 nM of digoxigenin-aptamer-monomethyl auristatin E conjugate and 125 nM of the antibody prepared in Example 1 above were dissolved in binding buffer (Binding buffer: Dulbecco's phosphate buffered saline (DPBS) (Hyclone Laboratories, #SH30028.02) containing 0.1% bovine serum albumin (Merck, #126593), 0.1 mg / mL of yeast transfer ribonucleic acid (yeast tRNA) (Sigma, #R9001), and 5 mM of magnesium chloride (MgCl2) (Biosesang, #M3101)) to make a total volume of 100 μL. The above mixture was incubated on a rotator at room temperature for 30 minutes to allow the antibody to bind to the digoxigenin-aptamer-monomethyl auristatin E conjugate to prepare a complex (DOligobody). The structure of the DOligobody thus prepared is as Figure 2 shown.

[0116] Example 3-3. Confirming the binding affinity of the complex (DOligobody) containing the anti-digoxigenin humanized antibody by fluorescence-activated cell sorting (FACS) experiment

[0117] For the complex DOligobody prepared in Example 3-2 above, the binding affinity was confirmed using the human pancreatic cancer cell line CFPAC-1 targeted to the aptamer SQ7-1(8) contained therein. The measurement was performed using a flow cytometer (fluorescence-activated cell sorting (FACS)). The aptamer in the conjugate has binding affinity for pancreatic cancer cells, and FACS analyzes using a secondary antibody against the anti-digoxigenin antibody (mouse or human). Therefore, from this result, it can be confirmed whether the complex (DOligobody) of the present invention in which the DIG-aptamer-MMAE conjugate binds to the anti-digoxigenin antibody was properly prepared.

[0118] Specifically, after obtaining CFPAC-1 cells by trypsin treatment, a cell concentration of 3×10 5 was prepared in a round-bottom test tube. After adding 100 μL of the complex DOligobody mixture prepared in Example 3-2 to each test tube, the mixture was incubated at 4°C for 30 minutes.

[0119] Then, the cells in the above test tubes were washed 3 times with 500 μL of washing buffer (Washing buffer: containing the binding buffer of Example 3-2 + 0.1% sodium azide (NaN3)).

[0120] Add a secondary antibody to the cells washed above. Specifically, Alexa Fluor TM 488 anti-mouse immunoglobulin G (anti-mouse IgG) (H+L) (Invitrogen, #A21202) was diluted to 1 / 500, and anti-human immunoglobulin G (Anti-Human IgG) (Fc specific)-FITC antibody (antibody) (Sigma, #F9512) prepared in goats was diluted to 1 / 500. After treatment, the cells were cultured at 4 °C for 15 minutes.

[0121] Then, wash the cells in the above test tube 3 times with 500 μL of wash buffer (containing the binding buffer of Example 3-2 above + 0.1% sodium azide).

[0122] Use a solution prepared by adding 7-AAD (BD Pharmingen TM , #559925: diluted to 1 / 100) to 300 μL of binding buffer to treat the cells washed above to prepare a sample for fluorescence-activated cell sorting.

[0123] Use FACS Verse TM (BD Biosciences, San Jose, USA) to analyze the sample prepared above. The results of the above analysis are shown in Figure 3 ( Figures 3a to 3c ).

[0124] The results of comparing the binding rate of DOligobody as the complex of the present invention based on the relevant fluorescence-activated cell sorting results are as Figure 4 shown. According to the relevant results, it can be confirmed that compared with the case of binding the original mouse antibody to digoxigenin-aptamer-monomethyl auristatin E conjugate, in the case of binding the humanized antibody to digoxigenin-aptamer-monomethyl auristatin E conjugate, the binding rate of the complex to the human pancreatic cancer cell line CFPAC-1 is more excellent. Especially in the case of humanized antibodies such as humab[21H8]-v6, it is confirmed that the binding rate of the complex to cancer cells is more excellent than that of chimeric antibodies. This indicates that the difference in the binding force between the digoxigenin-aptamer-monomethyl auristatin E conjugate and the anti-digoxigenin antibody also affects the binding force of the entire complex to pancreatic cancer cells. That is, compared with humanized antibodies, the binding force of mouse and chimeric antibodies to the conjugate is weaker, indicating that the binding force of the aptamer in the conjugate to the targeted pancreatic cancer cells is also weak.

[0125] Example 4. Drug of Complex DOligobody Containing Anti-Digoxigenin Antibody

[0126] Kinetic experiment

[0127] Pharmacokinetic analysis of the complex DOligobody containing anti-digoxigenin antibody was performed through animal experiments. The following DOligobodies were administered to Balb / c mice at a dose of 8 mg / kg: ① DIG-SQ7-1(8)-MMAE conjugate DOligomer (control group); ② DOligobody containing humab[21H8]-v5 antibody; ③ DOligobody containing humab[21H8]-v6 antibody; or ④ DOligobody containing humab[21H8]-v8 antibody. Blood samples of 50 μL to 100 μL were collected from the eyes and veins using heparin-coated capillary tubes at the time of administration, 0.5 hour, 1 hour, 4 hours, 8 hours, 12 hours, 24 hours, 48 hours, and 72 hours after administration.

[0128] After the above-collected blood was allowed to stand on ice for 30 minutes, the supernatant (plasma) was separated by centrifugation, and the concentrations of the aptamer and the humanized antibody in the blood were analyzed using a digoxigenin enzyme-linked immunosorbent assay kit and a human immunoglobulin G enzyme-linked immunosorbent assay (IgG ELISA) kit.

[0129] ※ Digoxigenin enzyme-linked immunosorbent assay method

[0130] 1) Anti-digoxigenin (2 μg / mL concentration, Abcam, cat. Ab420) (100 μL each) was coated in an Immuno Clear plate (Thermo Scientific TM , Cat. No. 468667) and incubated overnight at 4°C.

[0131] 2) After washing 3 times with phosphate buffer solution + 0.05% Tween 20 (150 μL), 100 μL of 3% bovine serum albumin Cat. No. 126609) was used to block for 1 hour at 37°C.

[0132] 3) After removing the blocking solution, 50 μL of each blood sample diluted 1 / 10 was incubated at 37°C for 1 hour.

[0133] 4) After washing 3 times with phosphate buffer solution + 0.05% Tween 20 (150 μL), incubate for 1 hour at 37 °C in 50 μL of digoxigenin conjugated to horseradish peroxidase (HRP-conjugated digoxigenin) (diluted 1 / 4000).

[0134] 5) After washing 3 times with phosphate buffer solution + 0.05% Tween 20 (150 μL), add 100 μL of 1-Step TM Ultra TMB - ELISA Substrate (Thermo Fisher Scientific, Cat. No. 34029) and incubate for 15 minutes.

[0135] 6) Add 2.5 N sulfuric acid (H2SO4) (40 μL).

[0136] 7) Measure the OD value at an absorbance of 450 nM using a Synergy TM H1 Hybrid Multi-Mode Microplate Reader (BioTek).

[0137] ※ Human Immunoglobulin G Enzyme-Linked Immunosorbent Assay (IgG ELISA) Method

[0138] Perform the experiment according to the instructions of the Human IgG total ELISA Kit (Invitrogen, Cat. BMS2091). Specifically as follows.

[0139] 1) Dilute the blood sample 1 / 10000 with assay dilution buffer.

[0140] 2) Serially dilute the standard sample with assay dilution buffer to 0.2 μg / mL, 0.1 μg / mL, 0.05 μg / mL, 0.025 μg / mL, 0.013 μg / mL, and 0.003 μg / mL.

[0141] 3) Dilute the HRP-conjugate 1 / 100 with assay dilution buffer.

[0142] 4) Wash the microtiter plate once with wash buffer.

[0143] 5) Add 100 μL of the blood sample or standard sample and 100 μL of the HRP-conjugate to each well and react at room temperature for 1 hour 30 minutes.

[0144] 6) Wash 4 times with wash buffer.

[0145] 7) Add 100 μL of tetramethylbenzidine (TMB) substrate and react at room temperature for 30 minutes.

[0146] 8) After adding 100 μL of stop solution, measure the OD value at an absorbance of 450 nM using a Synergy TM H1 multimode microplate reader (Biotium).

[0147] The pharmacokinetic (PK) curve of digoxigenin confirmed by the results of the above experimental analysis is as Figure 6 shown. On the other hand, after intravenous administration, the plasma concentration of the antibody is divided into a distribution phase and an elimination phase, and the results of calculating the half-life (t1 / 2) are confirmed as follows: ① DIG-SQ7-1(8)-MMAE conjugate DOligomer (control group) is only 0.02 hours; ② DOligobody containing humab[21H8]-v5 antibody is 9.2 hours; ③ DOligobody containing humab[21H8]-v6 antibody is 16.8 hours; ④ DOligobody containing humab[21H8]-v8 antibody is 38.5 hours ( Figure 7 ).

[0148] The pharmacokinetic curve of human immunoglobulin G is as Figure 8 shown, and the results of calculating the half-life (t1 / 2) of DOligobody containing humab[21H8]-v5 antibody, humab[21H8]-v6 antibody and humab[21H8]-v8 antibody are as follows: ② DOligobody containing humab[21H8]-v5 antibody is 35 hours; ③ DOligobody containing humab[21H8]-v6 antibody is 32 hours; ④ DOligobody containing humab[21H8]-v8 antibody is 143 hours ( Figure 9 ).

[0149] It can be confirmed from the above data that the in vivo half-life of the DOligobody of the present invention is significantly increased.

[0150] Through the above experiments, it can be confirmed that a complex DOligobody containing a conjugate (DOligomer) formed by the anti-digoxin ligand antibody of the present invention and digoxin ligand-aptamer-drug can be used as a carrier to increase the targeting of the drug to target cells and the half-life of the drug in vivo. Those of ordinary skill in the art to which the present invention pertains can use the complex DOligobody of the present invention as a drug delivery vehicle to appropriately select an aptamer that binds to a target cell or substance and select a drug to be delivered into the body to effectively deliver the drug into the body.

[0151] Those skilled in the art to which the present invention pertains should understand that, through the above description, it can be implemented in other specific forms without changing the technical idea or essential features of the present invention. In this regard, it should be understood that the above-described embodiments are only for illustrative purposes in general and not for limiting purposes. The scope of the present invention is not limited to the above detailed description, and all changes or variations derived from the meaning, scope of the appended claims for invention, and their equivalent concepts should be construed as being included within the scope of the present invention. <110> National Cancer Center JP BIO A INC. Industrial-Academic Cooperation Foundation of Konkuk University <120> Anti-digoxin ligand humanized antibody and its uses <130> POPC232046PCT <150> KR10-2020-0109727 <151> 2020-08-28 <150> PCT / KR2020 / 013221 <151> 2020-09-28 <160> 40 <170> PatentIn version 3.2 <210> 1 <211> 121 <212> PRT <213> Artificial sequence <220> <223> Mouse muscle 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 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 55 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> Mouse Muscle 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 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 Gln 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 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 <213> Artificial Sequence <220> <223> 2'-O-methyl inserted modified nucleic acid aptamer SQ7-1(8) <220> <221> Modified Base <222> (1)..(4) <223> 2’-O-methyl modified bases 1 to 4 <220> <221> Modified Base <222> (29)..(32) <223> 2’-O-methyl modified bases 29 to 32 <400> 11 guuggtatat acttctttag cttggaacca ac 32 <210> 12 <211> 80 <212> DNA <213> Artificial sequence <220> <223> Deoxyribonucleic acid aptamer SQ7 <400> 12 agcagcacag aggtcagatg atgttggtat atacttcttt agcttggaac caactcttgc 60 cctatgcgtg ctaccgtgaa 80 <210> 13 <211> 32 <212> DNA <213> Artificial sequence <220> <223> Deoxyribonucleic acid aptamer <400> 13 gttggtatat acttctttag cttggaacca ac 32 <210> 14 <211> 32 <212> DNA <213> Artificial sequence <220> <223> 2'-O-methyl inserted modified nucleic acid aptamer SQ7-1(1) <220> <221> Modified base <222> (1)..(6) <223> 2’-O-methyl modified bases 1 to 6 <400> 14 guugguatat acttctttag cttggaacca ac 32 <210> 15 <211> 32 <212> DNA <213> Artificial sequence <220> <223> 2'-O-methyl inserted modified nucleic acid aptamer SQ7-1(2) <220> <221> Modified base <222> (7)..(11) <223> 2’-O-methyl modified bases 7 to 11 <400> 15 gttggtauau acttctttag cttggaacca ac 32 <210> 16 <211> 32 <212> DNA <213> Artificial sequence <220> <223> 2'-O-methyl inserted modified aptamer SQ7-1(3) <220> <221> Modified base <222> (14)..(18) <223> 2’-O-methyl modified bases 14 to 18 <400> 16 gttggtatat actucuuuag cttggaacca ac 32 <210> 17 <211> 32 <212> DNA <213> Artificial sequence <220> <223> 2'-O-methyl inserted modified aptamer SQ7-1(4) <220> <221> Modified base <222> (21)..(26) <223> 2’-O-methyl modified bases 21 to 26 <400> 17 gttggtatat acttctttag cuuggaacca ac 32 <210> 18 <211> 32 <212> DNA <213> Artificial sequence <220> <223> 2'-O-methyl inserted modified aptamer SQ7-1(5) <220> <221> Modified base <222> (27)..(32) <223> 2’-O-methyl modified bases 27 to 32 <400> 18 gttggtatat acttctttag cttggaacca ac 32 <210> 19 <211> 32 <212> DNA <213> Artificial sequence <220> <223> 2'-O-methyl inserted modified aptamer SQ7-1(6) <220> <221> Modified base <222> (7)..(26) <223> 2’-O-methyl modified bases 7 to 26 <400> 19 gttggtauau acuucuuuag cuuggaacca ac 32 <210> 20 <211> 32 <212> DNA <213> Artificial sequence <220> <223> 2'-O-methyl inserted modified aptamer SQ7-1(1,5) <220> <221> Modified base <222> (1)..(6) <223> 2’-O-methyl modified bases 1 to 6 <220> <221> Modified base <222> (27)..(32) <223> 2’-O-methyl modified bases 27 to 32 <400> 20 guugguatat acttctttag cttggaacca ac 32 <210> 21 <211> 32 <212> DNA <213> Artificial sequence <220> <223> 2'-O-methyl inserted modified aptamer SQ7-1(7) <220> <221> Modified base <222> (1)..(5) <223> 2’-O-methyl modified bases 1 to 5 <220> <221> Modified base <222> (27)..(32) <223> 2’-O-methyl modified bases 27 to 32 <400> 21 guuggtatat acttctttag cttggaacca ac 32 <210> 22 <211> 32 <212> DNA <213> Artificial sequence <220> <223> 2'-O-methyl inserted modified aptamer SQ7-1(9) <220> <221> Modified base <222> (1)..(3) <223> 2’-O-methyl modified bases 1 to 3 <220> <221> Modified base <222> (30)..(32) <223> 2’-O-methyl modified bases 30 to 32 <400> 22 guuggtatat acttctttag cttggaacca ac 32 <210> 23 <211> 79 <212> DNA <213> Artificial sequence <220> <223> Deoxyribonucleic acid aptamer SQ7a <400> 23 agcagcacag aggtcagatg atgttggtat atacttcttt agcttggaac caactcttct 60 cctatgcgtg ctaccgtga 79 <210> 24 <211> 80 <212> DNA <213> Artificial sequence <220> <223> Deoxyribonucleic acid 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> Deoxyribonucleic acid aptamer SQ1 <400> 25 agcagcacag aggtcagatg cttgggctat ctcttattca tgctgttcca ccgctctcgg 60 cctatgcgtg ctaccgtgaa 80 <210> 26 <211> 79 <212> DNA <213> Artificial sequence <220> <223> Deoxyribonucleic acid 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> Deoxyribonucleic acid aptamer SQ3 <400> 27 agcagcacag aggtcagatg tgggggcgat tacgacccgg cgaaattaat agatctgccg 60 cctatgcgtg ctaccgtgaa 80 <210> 28 <211> 80 <212> DNA <213> Artificial sequence <220> <223> Deoxyribonucleic acid aptamer SQ4 <400> 28 agcagcacag aggtcagatg agtgtccaac gtcggcgaaa ttaataggtt ggaacgaacg 60 cctatgcgtg ctaccgtgaa 80 <210> 29 <211> 80 <212> DNA <213> Artificial sequence <220> <223> Deoxyribonucleic acid 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> Deoxyribonucleic acid aptamer SQ6 <400> 30 agcagcacag aggtcagatg gcttgccaca acgtgcgacg ctatgactaa ttcggcgacc 60 cctatgcgtg ctaccgtgaa 80 <210> 31 <211> 80 <212> DNA <213> Artificial sequence <220> <223> Deoxyribonucleic acid aptamer SQ8 <400> 31 agcagcacag aggtcagatg cttgggctat ttcttattca tgctgttcca ccgctctcgg 60 cctatgcgtg ctaccgtgaa 80 <210> 32 <211> 80 <212> DNA <213> Artificial sequence <220> <223> Deoxyribonucleic acid 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> Deoxyribonucleic acid 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> Deoxyribonucleic acid aptamer SQ11 <400> 34 agcagcacag aggtcagatg gtgccaacct gaagtgactg agatatcaac cggtaggcct 60 cctatgcgtg ctaccgtgaa 80 <210> 35 <211> 43 <212> DNA <213> Artificial sequence <220> <223> Deoxyribonucleic acid aptamer SQ8-1 <400> 35 cagcacagag gtcagatgct tgggctattt cttattcatg ctg 43 <210> 36 <211> 26 <212> DNA <213> Artificial sequence <220> <223> Deoxyribonucleic acid aptamer SQ8-2 <400> 36 cagcacagag gtcagatgct tgggct 26 <210> 37 <211> 32 <212> DNA <213> Artificial sequence <220> <223> Deoxyribonucleic acid aptamer SQ8-3 <400> 37 catgctgttc caccgctctc ggcctatgcg tg 32 <210> 38 <211> 28 <212> DNA <213> Artificial sequence <220> <223> Deoxyribonucleic acid aptamer SQ8-4 <400> 38 agcagcacag aggtcagatg cttgggct 28 <210> 39 <211> 23 <212> DNA <213> Artificial sequence <220> <223> Deoxyribonucleic acid aptamer SQ8-5 <400> 39 ctcggcctat gcgtgctacc gtg 23 <210> 40 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Deoxyribonucleic acid aptamer SQ8-6 <400> 40 caccgctctc ggcctatgcg tgctaccgtg 30

Claims

1. A digoxigenin - specific humanized antibody or its antigen - binding fragment, characterized in that, Specifically binds to digoxigenin and comprises: A heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 5; and A light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 7 to SEQ ID NO:

10.

2. The digoxigenin - specific humanized antibody or its antigen - binding fragment according to claim 1, characterized in that, The above-mentioned digoxigenin-specific humanized antibody or its antigen-binding fragment is one of the following digoxigenin-specific humanized antibodies or its antigen-binding fragments: a) A digoxigenin-specific humanized antibody or its antigen-binding fragment, comprising: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7; b) A digoxigenin-specific humanized antibody or its antigen-binding fragment, comprising: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7; c) A digoxigenin-specific humanized antibody or its antigen-binding fragment, comprising: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7; d) A digoxigenin-specific humanized antibody or its antigen-binding fragment, comprising: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7; e) A digoxigenin-specific humanized antibody or its antigen-binding fragment, comprising: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8; f) A digoxigenin-specific humanized antibody or its antigen-binding fragment, comprising: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8; g) A digoxigenin-specific humanized antibody or its antigen-binding fragment, comprising: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8; h) A digoxigenin-specific humanized antibody or its antigen-binding fragment, comprising: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8; i) A digoxigenin-specific humanized antibody or its antigen-binding fragment, comprising: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 9; j) A digoxigenin-specific humanized antibody or its antigen-binding fragment, comprising: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 9; k) A digoxigenin-specific humanized antibody or its antigen-binding fragment, comprising: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 9; l) A digoxigenin-specific humanized antibody or its antigen-binding fragment, comprising: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 9; m) A digoxigenin-specific humanized antibody or its antigen-binding fragment, comprising: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10; n) A digoxigenin-specific humanized antibody or its antigen-binding fragment, comprising: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10; o) A digoxigenin - specific humanized antibody or an antigen - binding fragment thereof, comprising: a heavy - chain variable region comprising the amino - acid sequence of SEQ ID NO: 4 and a light - chain variable region comprising the amino - acid sequence of SEQ ID NO: 10; and p) A digoxigenin - specific humanized antibody or an antigen - binding fragment thereof, comprising: a heavy - chain variable region comprising the amino - acid sequence of SEQ ID NO: 5 and a light - chain variable region comprising the amino - acid sequence of SEQ ID NO:

10.

3. The digoxigenin - specific humanized antibody or its antigen - binding fragment according to claim 1, characterized in that, The above - mentioned digoxigenin - specific humanized antibody is in the form of immunoglobulin G1, immunoglobulin G2, immunoglobulin G3 or immunoglobulin G4.

4. The digoxigenin - specific humanized antibody or its antigen - binding fragment according to claim 1, characterized in that, The antigen - binding fragment of the above - mentioned antibody is selected from the group consisting of scFv, (scFv)2, scFv - Fc, Fab, Fab' and F(ab')2.

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