KR102503143B1 - Antibody-drug conjugate comprising antibody against human DLK1 and its use - Google Patents

Abstract

The present invention provides novel antibody-drug conjugates (ADCs) targeting DLK1, active metabolites of these ADCs, methods for preparing these ADCs, uses of these ADCs for the treatment and/or prevention of diseases, and It also relates to the use of these ADCs for producing a medicament for the treatment and/or prevention of diseases, more particularly hyperproliferative and/or angiogenic diseases such as cancer diseases, and more particularly, antibodies that bind to DLK1 or their It relates to an antibody-drug conjugate comprising an antigen-binding fragment and a pharmaceutical composition comprising the same.

Description

Antibody-drug conjugate comprising antibody against human DLK1 and its use {Antibody-drug conjugate comprising antibody against human DLK1 and its use}

The present invention provides novel antibody-drug conjugates (ADCs) targeting DLK1, active metabolites of these ADCs, methods for preparing these ADCs, uses of these ADCs for the treatment and/or prevention of diseases, and It also relates to the use of these ADCs for producing a medicament for the treatment and/or prevention of diseases, more particularly hyperproliferative and/or angiogenic diseases such as cancer diseases, and more particularly, antibodies that bind to DLK1 or their It relates to an antibody-drug conjugate comprising an antigen-binding fragment and a pharmaceutical composition comprising the same.

Cancer refers to a disease caused by an abnormally grown mass due to the autonomous excessive growth of body tissues, and is a result of uncontrolled cell growth in various tissues. Early-stage tumors can be removed by surgical and radiotherapy measures, and metastasized tumors are usually palliatively treated with chemotherapeutic agents.

Most of the chemotherapeutic agents administered parenterally can induce serious effects of unwanted side effects and even toxicity as a result of systemic administration, and thus improved and selective application of these chemotherapeutic agents in tumor cells or immediate adjacent tissues is expected. The focus has been on the development of novel chemotherapeutic agents to simultaneously increase efficacy and minimize toxicity/side effects.

Antibody-drug conjugate (ADC) is a target-oriented new technology that binds toxins or drugs to antibodies that bind to antigens and then releases toxic substances from the inside of the cells, leading to the death of cancer cells. Because it accurately delivers drugs to target cancer cells and releases them only under specific conditions with minimal effect on healthy cells, it is a technology that is more effective than antibody therapeutics themselves and can greatly reduce the risk of side effects compared to existing anticancer drugs.

The basic structure of these antibody-drug conjugates consists of "antibody-linker-small molecule drug (toxin)". Here, the linker not only plays a functional role in linking the antibody and the drug, but also stably reaches the target cell during circulation in the body, and then the antibody-drug conjugate enters the cell to cause antibody-drug dissociation (e.g., hydrolysis by enzymes). result), the drug should be well released and show the drug effect on the target cancer cells. That is, the linker plays a very important role in terms of safety such as efficacy and systemic toxicity of the antibody-drug conjugate depending on the stability of the linker (Discovery Medicine 2010, 10(53): 329-39).

The inventors of the present invention developed a linker containing an effective self-immolative group that is more stable in plasma, stable during body circulation, and can easily release a drug from cancer cells to exhibit a drug effect. Patents have been secured (Korean Registered Patent No. 1,628,872, etc.). On the other hand, the use of monoclonal antibodies for cancer treatment has been met with considerable success. Monoclonal antibodies are suitable for target-directed addressing of tumor tissues and tumor cells. Antibody drug conjugates have become a powerful new therapeutic option for the treatment of lymphoma and solid cancers, and immunomodulatory antibodies have also recently achieved significant clinical success. The development of therapeutic antibodies is based on a deep understanding of cancer serology, protein engineering techniques and their actions, mechanisms of resistance, and interactions between the immune system and cancer cells.

The definition of antigens expressed on the cell surface of human cancer refers to a wide range of targets that are overexpressed or mutated and selectively expressed compared to normal tissues. A key problem is the identification of suitable antigens for antibody-based therapies. These therapeutics mediate changes in antigen or receptor function (i.e. function as stimulators or antagonists), modulate the immune system through Fc and T cell activation, and target specific drugs through the delivery of antibodies that target specific antigens. exert an effect Molecular technologies capable of altering antibody pharmacokinetics, function, size and immunostimulatory properties are emerging as key elements in the development of novel antibody-based therapies. Evidence from clinical trials of therapeutic antibodies in cancer patients indicates the development of optimized antibodies, including affinity and binding of the antibodies to the target antigen, choice of antibody structure, and therapeutic approach (signal transduction blockade or immune action function). It emphasizes the importance of approaches for selection.

In this regard, studies on antibodies using DLK1 as an antigen are being conducted. Human-derived DLK1 (delta-like 1 homolog (Drosophila)) is a one-time transmembrane membrane protein consisting of 383 amino acids in length, and the protein has 6 EGF-like repeats (epidermal growth factor-like repeat) in the extracellular region. ) domain.

DLK1 is commonly referred to as a gene called DLK1 due to the homology of amino acid sequence with Delta, a ligand of the Notch receptor, which is a cell differentiation control factor. is also called DLK1 is also a membrane protein, but is well known as a protein that has a separate function by shedding the extracellular domain from the cell membrane by tumor necrosis factor alpha convertingenzyme (TACE).

DLK1 is highly expressed in undifferentiated fetal cells with high proliferative potential. In particular, it shows high expression in fetal liver, kidney, skeletal muscle, brain, etc., but expression is not confirmed in most tissues after birth, preadipocyte, pancreatic islet cell, thymic somatic cell ( It is expressed only in specific cells such as thymic stromal cells and adrenal gland cells.

The function of DLK1 is known as Pref-1 (preadipocyte factor-1), which inhibits adipocyte differentiation, and has been studied the most. Apart from the ability to inhibit adipocyte differentiation, DLK1 inhibits the differentiation of hematopoietic stem cells, controls the differentiation of lymphoid progenitor cells, and is related to wound healing. is also known

In addition, it has been reported that DLK1 is expressed at a high frequency also in various cancers and tumors. So far, as types of cancers whose expression has been confirmed, among solid cancers, neuroendocrine tumors, neuroblastomas, gliomas, type 1 neurofibromatosis, small cell lung cancer, liver cancer, kidney cancer, ovarian cancer, colorectal cancer, breast cancer and pancreatic cancer are known. In hematologic malignancies, myelodysplastic syndrome and acute myelogenous leukemia are known. As a study on the relationship between DLK1 and cancer, the expression of DLK1 is overexpressed in brain cancer cells (glioma), and when the cDNA of DLK1 is overexpressed in brain cancer cells, the proliferation of brain cancer cells increases, leading to an increase in migration. It has been reported that the expression of DLK1 in liver cancer is higher than that of normal hepatocytes, and that the size of tumors decreases when the expression of DLK1 is reduced through siRNA experiments.

Under this technical background, the inventors of the present invention have developed an anti-DLK1 antibody that exhibits excellent binding ability to DLK1 as a result of efforts to develop an antibody that specifically binds to DLK1, and to further enhance the effect of the antibody in plasma. A linker containing an effective self-immolative group that is stable during body circulation and is easily released from cancer cells to exhibit drug efficacy is applied to anti-cancer therapeutic antibodies that specifically bind to DLK1. Thus, it was confirmed that a novel antibody-drug conjugate (ADC) targeting DLK1 effective for the treatment and/or prevention of hyperproliferative and/or metastatic cancer diseases could be provided, and the present invention was completed.

The present invention is intended to provide a novel antibody-linker drug conjugate targeting DLK1 or a salt or solvate thereof.

The present invention is to provide an antibody-drug conjugate comprising an antibody that specifically binds to DLK1 and a drug that binds thereto, and a pharmaceutical composition comprising the same.

In the present invention, a linker technology including a self-killing group that is more stable in plasma and circulated in the body and can be easily released from cancer cells to maximize drug efficacy is incorporated, so that the drug and/or toxin can be targeted. It is intended to provide an antibody-linker-drug (toxin) system that can stably reach cells and effectively exert drug effects while greatly reducing toxicity.

In one aspect of the present invention, there is provided a conjugate having the structure of Formula I, or a pharmaceutically acceptable salt or solvate thereof:

[Formula I] Ab - (A) _y

From here,

Ab is an anti-DLK1 antibody or antigen-binding fragment thereof;

A is independently a chemical moiety comprising one or more active agents and a linker, and

The linker connects the antibody and the active agent,

Said y is an integer from 1 to 20.

The anti-DLK1 antibody or antigen-binding fragment thereof, that is, the antibody or antigen-binding fragment thereof that binds to DLK1 is at least one heavy chain CDR1 selected from the group consisting of SEQ ID NOs: 2, 16, 30, 44, 58, 72 and 86, SEQ ID NO: At least one heavy chain CDR2 selected from the group consisting of 4, 18, 32, 46, 60, 74 and 88, and at least one heavy chain CDR3 selected from the group consisting of SEQ ID NOs: 6, 20, 34, 48, 62, 76 and 90 heavy chain variable region; and

At least one light chain CDR1 selected from the group consisting of SEQ ID NOs: 9, 23, 37, 51, 65, 79, 93, 115 and 121, one selected from the group consisting of SEQ ID NOs: 11, 25, 39, 53, 67, 81 and 95 and a light chain variable region including at least one light chain CDR2 selected from the group consisting of SEQ ID NOs: 13, 27, 41, 55, 69, 83, 97, 116 and 125.

Antibodies according to the present invention can specifically bind, for example, to the extracellular domain of human DLK1.

As used herein, the term "antibody" refers to an anti-DLK1 antibody that specifically binds to DLK1, particularly the extracellular domain of human DLK1 protein. The scope of the present invention includes not only complete antibody forms specifically binding to DLK1, but also antigen-binding fragments of the antibody molecules.

A complete antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is linked to the heavy chain by disulfide bonds. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and subclasses include gamma 1 (γ1), gamma 2 (γ2), and gamma 3 (γ3). ), gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2). The constant region of the light chain has kappa (κ) and lambda (λ) types.

An antigen-binding fragment of an antibody or antibody fragment refers to a fragment having an antigen-binding function, and includes Fab, F(ab'), F(ab')2, and Fv. Among antibody fragments, Fab has a structure having variable regions of light and heavy chains, constant regions of light chains, and a first constant region (CH1) of heavy chains, and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain CH1 domain. An F(ab')2 antibody is produced by forming a disulfide bond between cysteine residues in the hinge region of Fab'. Fv is a minimal antibody fragment having only the heavy chain variable region and the light chain variable region. Recombinant technology for generating Fv fragments is disclosed in PCT International Publication Patent Applications WO88/10649, WO88/106630, WO88/07085, WO88/07086 and WO88/09344. has been initiated Double-chain Fv (two-chain Fv) has a heavy chain variable region and light chain variable region connected by a non-covalent bond, and single-chain Fv (scFv) is generally a heavy chain variable region and a light chain variable region through a peptide linker. They are covalently linked or directly linked at the C-terminus, so that they can form a dimer-like structure like double-chain Fv. Such antibody fragments can be obtained using proteolytic enzymes (for example, Fab can be obtained by restriction digestion of whole antibodies with papain, and F(ab')2 fragments can be obtained by digestion with pepsin), and gene It can also be produced through recombinant technology.

In one embodiment, an antibody according to the present invention is in the form of an Fv (eg scFv) or a complete antibody. In addition, the heavy chain constant region may be selected from any one isotype of gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε). For example, the constant region is gamma 1 (IgG1), gamma 3 (IgG3) or gamma 4 (IgG4). Light chain constant regions can be of the kappa or lambda type.

As used herein, the term "heavy chain" refers to a full-length heavy chain comprising a variable region domain VH comprising an amino acid sequence having sufficient variable region sequence for imparting specificity to an antigen and three constant region domains CH1, CH2 and CH3. and fragments thereof. In addition, as used herein, the term "light chain" refers to a full-length light chain and fragments thereof comprising a variable region domain VL and a constant region domain CL comprising an amino acid sequence having a sufficient variable region sequence to impart specificity to an antigen. I mean everything.

Antibodies of the invention include monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain Fvs (scFV), single chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFV) and Anti-idiotypic (anti-Id) antibodies, or epitope-binding fragments of the antibodies, and the like, but are not limited thereto.

The monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the same thing except for possible naturally occurring mutations that may be present in minor amounts in the individual antibodies comprising the population. Monoclonal antibodies are highly specific, directed against a single antigenic site. In contrast to conventional (polyclonal) antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. For example, monoclonal antibodies useful in the present invention may be made by hybridoma methods, or may be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see US Pat. No. 4,816,567). In addition, monoclonal antibodies can be isolated from phage antibody libraries.

In one embodiment of the present invention, 7 types of monoclonal human antibodies specifically binding to DLK1 were prepared by panning a native human single chain Fv library using a phage display method.

“Phage display” is a technique in which a variant polypeptide is displayed as a fusion protein with at least a portion of an envelope protein on the surface of a phage, eg, a filamentous phage particle. The usefulness of phage display lies in the fact that large libraries of randomized protein variants can be targeted and rapidly and efficiently sorted for sequences that bind with high affinity to the target antigen. Displaying peptide and protein libraries on phage has been used to screen millions of polypeptides for those with specific binding properties.

Phage display technology has provided a powerful tool for generating and selecting novel proteins that bind specific ligands (eg antigens). Using phage display technology, large libraries of protein variants can be generated and sequences that bind with high affinity to the target antigen can be rapidly sorted. A nucleic acid encoding a variant polypeptide is fused with a nucleic acid sequence encoding a viral envelope protein, eg, a gene III protein or a gene VIII protein. Monovalent phage display systems have been developed in which a nucleic acid sequence encoding a protein or polypeptide is fused with a nucleic acid sequence encoding a portion of a gene III protein. In monovalent phage display systems, gene fusions are expressed at low levels and the wild-type gene III protein is also expressed to maintain particle infectivity.

Demonstrating expression of peptides on the filamentous phage surface and expression of functional antibody fragments in the periplasm of E. coli is important for developing antibody phage display libraries. Libraries of antibodies or antigen-binding polypeptides have been prepared in a number of ways, for example by altering a single gene by inserting a random DNA sequence or by cloning a series of related genes. The library can be screened for expression of antibodies or antigen-binding proteins with desired characteristics.

Phage display technology has several advantages over conventional hybridoma and recombinant methods for producing antibodies with desired characteristics. This technology enables the generation of large antibody libraries with diverse sequences in a short time without using animals. Production of hybridomas or production of humanized antibodies may require a production period of several months. In addition, since no immunization is required, the phage antibody library can generate antibodies against toxic or low antigenic antigens. Phage antibody libraries can also be used to generate and identify novel therapeutic antibodies.

Techniques for generating human antibodies from immunized, non-immunized human, germline sequences, or naïve B cell Ig repertoires can be used using phage display libraries. A variety of lymphoid tissues can be used to prepare naïve or non-immune antigen-binding libraries.

A technique capable of identifying and isolating high-affinity antibodies from phage display libraries is important for isolating novel antibodies for therapeutic use. Isolation of a high affinity antibody from a library may depend on the size of the library, the efficiency of production in bacterial cells and the diversity of the library. The size of the library is reduced by inefficient production due to improper folding of the antibody or antigen-binding protein and the presence of stop codons. Expression in bacterial cells may be inhibited if the antibody or antigen-binding domain does not fold properly. Expression can be improved by alternating mutations of residues on the surface of the variable/constant interface or selected CDR residues. The sequence of the framework region is one element for providing proper folding when generating an antibody phage library in bacterial cells.

In high-affinity antibody isolation, it is important to generate a diverse library of antibodies or antigen-binding proteins. CDR3 regions have been found to be those that often participate in antigen binding. Since the CDR3 region on the heavy chain varies considerably in size, sequence, and structural conformation, various libraries can be prepared using it.

Diversity can also be generated by randomizing the CDR regions of the variable heavy and light chains using all 20 amino acids at each position. The use of all 20 amino acids may result in a high diversity of variant antibody sequences and increase the chance of identifying novel antibodies.

"Epitope" refers to a protein determinant to which an antibody can specifically bind. An epitope usually consists of a group of chemically active surface molecules, such as amino acids or sugar side chains, and usually has specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and non-stereotypic epitopes are distinguished by loss of binding to the former but not to the latter in the presence of a denaturing solvent.

Such “humanized” forms of non-human (eg, murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are produced in a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, which retains the desired specificity, affinity, and capacity for residues from a hypervariable region of the recipient. It is a human immunoglobulin (recipient antibody) in which residues from the hypervariable region of

The "human antibody" is a molecule derived from human immunoglobulin, and means that all amino acid sequences constituting the antibody, including complementarity determining regions and structural regions, are composed of human immunoglobulin.

Some of the heavy and/or light chains are identical to or homologous to corresponding sequences in antibodies that are derived from a particular species or belong to a particular antibody class or subclass, while the remaining chain(s) are from another species or belong to another antibody class or subclass. Included are “chimeric” antibodies (immunoglobulins) that are identical to or homologous to the corresponding sequence in an antibody belonging to a subclass, as well as fragments of such antibodies that exhibit the desired biological activity.

“Antibody variable domain” as used herein refers to the light and heavy chain portions of an antibody molecule that include the amino acid sequences of the complementarity determining regions (CDRs; i.e., CDR1, CDR2, and CDR3), and the framework regions (FR). . VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain.

"Complementarity determining regions" (CDRs; ie, CDR1, CDR2, and CDR3) refer to the amino acid residues of antibody variable domains that are required for antigen binding. Each variable domain typically has three CDR regions identified as CDR1, CDR2 and CDR3.

In the present invention, the antibody or antigen-binding fragment thereof that binds to DLK1 may specifically include the CDR sequences shown in Table 1 below. Among them, anti-DLK1 antibodies of two types (18A5, 27F7) and another two types of 18A5 mutant antibodies (18A5_LS_1A10, 18A5_AM_1A12) can bind to cells in which DLK1 is overexpressed and target DLK1 expressed on the surface of cancer cells. It was confirmed in Korean Application No. 10-2018-0107639 that it can be developed as an anti-DLK1 antibody-drug conjugate capable of killing cancer cells.

[Table 1]

A "framework region" (FR) is a variable domain residue other than a CDR residue. Each variable domain typically has four FRs identified as FR1, FR2, FR3 and FR4.

According to one embodiment of the present invention, at least one heavy chain FR1 selected from the group consisting of SEQ ID NOs: 1, 15, 29, 43, 57, 71, 85 and 119, SEQ ID NOs: 3, 17, 31, 45, 59, 73 and at least one heavy chain FR2 selected from the group consisting of 87, at least one heavy chain FR3 selected from the group consisting of SEQ ID NOs: 5, 19, 33, 47, 61, 75 and 89; at least one heavy chain FR4 selected from the group consisting of 91, at least one light chain FR1 selected from the group consisting of SEQ ID NOs: 8, 22, 36, 50, 64, 78, 92, 117 and 120, SEQ ID NOs: 10, 24, 38, 52; At least one light chain FR2 selected from the group consisting of 66, 80, 94 and 122, at least one light chain FR3 selected from the group consisting of SEQ ID NO: 12, 26, 40, 54, 68, 82, 96, 118 and 123, or SEQ ID NO: 14 , 28, 42, 56, 70, 84, 98 and may include one or more light chain FR4 selected from the group consisting of 125.

An “Fv” fragment is an antibody fragment that contains the complete antibody recognition and binding site. This region consists of a dimer of one heavy-chain variable domain and one light-chain variable domain in tight, virtually covalent association, for example a scFv.

The “Fab” fragment contains the variable and constant domains of the light chain and the variable and first constant domains (CH1) of the heavy chain. F(ab')2 antibody fragments generally comprise a pair of Fab fragments covalently linked near their carboxy termini by hinge cysteines between them.

A “single-chain Fv” or “scFv” antibody fragment comprises the VH and VL domains of an antibody, which domains are present in a single polypeptide chain. The Fv polypeptide may further include a polypeptide linker between the VH and VL domains that allows the scFv to form a desired structure for antigen binding.

Antibodies according to the present invention are monovalent or divalent and contain single or double chains.

Functionally, the binding affinity of the antibody to the extracellular domain of DLK1 is in the range of 10 ⁻⁵ M to 10 ⁻¹² M. For example, the binding affinity is 10 ^-6 M to 10 ^-12 M, 10 ^-7 M to 10 ^-12 M, 10 ^-8 M to 10 ^-12 M, 10 ^-9 M to 10 ^-12 M, 10 ^-5 M to 10 ^-11 M, 10 ^-6 M to 10 ^-11 M, 10 ^-7 M to 10 ^-11 M, 10 ^-8 M to 10 ^-11 M, 10 ^-9 M to 10 ^-11 M, 10 ^-10 M to 10 ^-11 M, 10 ^-5 M to 10 ^-10 M, 10 ^-6 M to 10 ^-10 M, 10 ^-7 M to 10 ^-10 M, 10 ^-8 M to 10 ^-10 M, 10 ^-9 M to 10 ^-10 M, 10 ^-5 M to 10 ^-9 M, 10 ^-6 M to 10 ^-9 M, 10 ^-7 M to 10 ^-9 M, 10 ^-8 M to 10 ^-9 M, 10 ^-5 M to 10 ^-8 M, 10 ^-6 M to 10 ^-8 M, 10 ^-7 M to 10 ^-8 M, 10 ^-5 M to 10 ^-7 M, 10 ^-6 M to 10 ^-7 M or 10 ^-5 M to 10 ^-6 M.

In addition, the antibody according to the present invention is an antibody having increased affinity for an antigen. The term "affinity" refers to the ability of an antibody to specifically recognize and bind to a specific site of an antigen, and a high degree of affinity together with specificity of an antibody to an antigen is an important factor in an immune response. Affinity can be determined using any of a variety of assays known in the art, such as radioimmunoassay (RIA) and ELISA, and can be expressed in a variety of quantitative values. The affinity of an antibody for an antigen can generally be expressed by a dissociation constant (Kd) of a specific antibody-antigen interaction. The lower the Kd value, the higher the affinity of the antibody for the antigen. For example, since the Kd value of the antibody of the present invention is 0.52 for the 18A5 antibody and 0.22 for the 27F7 antibody, it can be seen that the antibody is a high-affinity antibody that specifically binds to human DLK1.

The antibody or antigen-binding fragment thereof that binds to the extracellular domain of DLK1 is a sequence having 90% or more sequence homology with a sequence selected from the group consisting of SEQ ID NOs: 99, 101, 103, 105, 107, 109, 111 and 127 It may include a heavy chain variable region comprising a. The antibody or antigen-binding fragment thereof that binds to the extracellular domain of DLK1 may include a heavy chain variable region selected from the group consisting of SEQ ID NOs: 99, 101, 103, 105, 107, 109, 111 and 127.

In addition, the antibody or antigen-binding fragment thereof that binds to the extracellular domain of DLK1 is at least 90% in sequence with a sequence selected from the group consisting of SEQ ID NOs: 100, 102, 104, 106, 108, 110, 112, 126 and 128. It may include a light chain variable region including sequences having homology. The antibody or antigen-binding fragment thereof that binds to the extracellular domain of DLK1 may include a light chain variable region selected from the group consisting of SEQ ID NOs: 100, 102, 104, 106, 108, 110, 112, 126, and 128.

The antibody or antibody fragment of the present invention may include not only the sequence of the anti-DLK1 antibody of the present invention described herein, but also a biological equivalent thereof to the extent that it can specifically recognize the extracellular domain of DLK1. For example, additional changes may be made to the amino acid sequence of the antibody to further improve its binding affinity and/or other biological properties. Such modifications include, for example, deletions, insertions and/or substitutions of residues in the amino acid sequence of the antibody. Such amino acid variations are made based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape and type of amino acid side chain substituents revealed that arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have similar shapes. Accordingly, based on these considerations, arginine, lysine and histidine; alanine, glycine and serine; And phenylalanine, tryptophan and tyrosine are biologically functional equivalents.

Considering the mutations having the above-described biologically equivalent activity, the antibodies of the present invention or the nucleic acid molecules encoding them are construed to include sequences showing substantial identity with the sequences described in SEQ ID NOs. The above substantial identity is at least 90% when the sequence of the present invention and any other sequence described above are aligned so as to correspond as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art. It refers to sequences exhibiting homology, most preferably at least 95% homology, 96% or more, 97% or more, 98% or more, 99% or more homology.

Alignment methods for sequence comparison are known in the art. The NCBI Basic Local Alignment Search Tool (BLAST) is accessible from NBCI, etc., and can be used in conjunction with sequence analysis programs such as blastp, blasm, blastx, tblastn, and tblastx on the Internet. The BLAST is accessible at www.ncbi.nlm.nih.gov/BLAST/. A sequence homology comparison method using this program can be found at www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

Based on this, the antibody or antigen-binding fragment thereof of the present invention is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% compared to the specified sequence described in the specification or the whole. , 99%, or greater homology. Such homology can be determined by sequence comparison and/or alignment by methods known in the art. For example, a sequence comparison algorithm (eg, BLAST or BLAST 2.0), manual alignment, or visual inspection can be used to determine the percent sequence homology of a nucleic acid or protein of the invention.

In another aspect, the present invention relates to a nucleic acid encoding the antibody or antigen-binding fragment thereof.

The antibody or antigen-binding fragment thereof can be recombinantly produced by isolating a nucleic acid encoding the antibody or antigen-binding fragment thereof of the present invention. The nucleic acid is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or further expression. Based on this, the present invention relates to a vector comprising the nucleic acid in another aspect.

"Nucleic acid" has the meaning of comprehensively including DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are basic structural units in nucleic acids, include not only natural nucleotides, but also analogs in which sugar or base sites are modified. . The sequences of nucleic acids encoding the heavy and light chain variable regions of the present invention may be modified. Such modifications include additions, deletions, or non-conservative or conservative substitutions of nucleotides.

DNA encoding the antibody is readily isolated or synthesized using conventional procedures (eg, by using oligonucleotide probes capable of specifically binding to DNA encoding the heavy and light chains of the antibody). Many vectors are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

As used herein, the term "vector" refers to a plasmid vector as a means for expressing a gene of interest in a host cell; cosmid vector; and viral vectors such as bacteriophage vectors, adenoviral vectors, retroviral vectors, and adeno-associated viral vectors. In the vector, the nucleic acid encoding the antibody is operably linked to a promoter.

"Operably linked" means a functional linkage between a nucleic acid expression control sequence (eg, a promoter, signal sequence, or array of transcriptional regulator binding sites) and another nucleic acid sequence, whereby the control sequence is linked to the other nucleic acid. It will regulate the transcription and/or translation of the sequence.

When prokaryotic cells are used as hosts, a strong promoter capable of promoting transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.), a ribosome binding site for initiation of translation, and a transcription/translation termination sequence. Further, for example, when a eukaryotic cell is used as a host, a promoter derived from the genome of a mammalian cell (eg, metallotionine promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or mammalian Promoters derived from animal viruses, such as adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, tk promoter from HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter from HIV , the promoter of Moloney virus, the promoter of Epstein Barr virus (EBV) and the promoter of Rouss sarcoma virus (RSV) can be used, and usually has a polyadenylation sequence as a transcription termination sequence.

In some cases, vectors may be fused with other sequences to facilitate purification of antibodies expressed therefrom. Sequences to be fused include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6x His (hexahistidine; Quiagen, USA).

The vector contains an antibiotic resistance gene commonly used in the art as a selectable marker, for example, for ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline. There is a resistance gene.

In another aspect, the present invention relates to a cell transformed with the above-mentioned vector. Cells used to generate the antibodies of the present invention may be, but are not limited to, prokaryotic, yeast or higher eukaryotic cells.

Escherichia coli, strains of the genus Bacillus such as Bacillus subtilis and Bacillus thuringiensis, Streptomyces , Pseudomonas (e.g. Pseudomonas putida ), Proteus Prokaryotic host cells such as Proteus mirabilis and Staphylococcus (eg, Staphylocus carnosus ) can be used.

However, animal cells are of greatest interest, and examples of useful host cell lines are COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO/-DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2/0, NS-0 , U20S, or HT1080, but is not limited thereto.

In another aspect of the present invention, (a) culturing the cells; and (b) recovering the antibody or antigen-binding fragment thereof from the cultured cells.

The cells can be cultured in various media. Among commercially available media, it can be used as a culture medium without limitation. All other necessary supplements known to those skilled in the art may also be included in suitable concentrations. Culture conditions such as temperature, pH, etc. are already in use with host cells selected for expression and will be apparent to those skilled in the art.

The antibody or antigen-binding fragment thereof may be recovered by, for example, centrifugation or ultrafiltration to remove impurities, and the resultant product may be purified using, for example, affinity chromatography. Additional other purification techniques may be used, such as anion or cation exchange chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, and the like.

In one aspect of the invention, the linker between the antibody and the active agent may be cleavable.
In one aspect of the present invention, the linker includes a self-immolative group or a cleavable group having the structure of Formula II:

[Formula II]

delete

In the above formula,

G is sugar, sugar acid, or sugar derivatives;

W is -C(O)-, -C(O)NR'-, -C(O)O-, -S(O) ₂ NR'-, -P(O)R''NR'-, -S (O) NR'-, or -PO ₂ NR'-;
Here, C(O), S, or P is directly connected to the phenyl ring, and R' and R'' are each independently hydrogen, (C ₁ -C ₈ )alkyl, (C ₃ -C ₈ )cycloalkyl, (C ₁ -C ₈ )alkoxy, (C ₁ -C ₈ )alkylthio, (C ₁ -C ₈ )alkylamino, (C ₃ -C ₂₀ )heteroaryl, or (C ₆ -C ₂₀ ) aryl,

each Z is independently (C ₁ -C ₈ )alkyl, halogen, cyano or nitro;

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n is an integer from 0 to 3;

m is 0 or 1;

R ₁ and R ₂ are each independently hydrogen, (C ₁ -C ₈ )alkyl or (C ₃ -C ₈ )cycloalkyl, or

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R ₁ and R ₂ together with the carbon atoms to which they are attached form a (C ₃ -C ₈ )cycloalkyl ring;
In the above formula, the tilde is a site connected to the rest of the linker,
* indicates a site connected to an active agent (drug or toxin).
In one aspect of the invention, the sugar or sugar acid is a monosaccharide.

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In one aspect of the present invention, G is a glucuronic acid moiety or a compound having the structure of formula (IIIa):

[Formula (IIIa)]

From here,

R ₃ is hydrogen or a carboxyl protecting group;

Each R ₄ is independently hydrogen or a hydroxyl protecting group.

In one aspect of the present invention, said R ₃ is hydrogen and each R ₄ is hydrogen.

In addition, each of R ₁ and R ₂ is hydrogen.

In addition, the W is -C (O) -, -C (O) NR '- or -C (O) O-, more specifically, the W is -C (O) NR'-, where C ( O) is linked to the phenyl ring, and NR' is attached to the rest of the linker.

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In addition, the n is 0, 1, 2 or 3, more specifically 0, 1 or 2, more specifically 0.
In one aspect of the invention, the linker contains at least one branching unit (BR) and at least one connection unit.
In one aspect of the invention, at least one branching unit is an alkylene having from 1 to 100 carbon atoms, wherein the carbon atoms of the alkylene are one or more hetero groups selected from the group consisting of N, O and S atoms, and alkylenes may be further substituted with one or more alkyls having from 1 to 20 carbon atoms.

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Specifically, at least one branching unit is a C ₁ -C ₅₀ alkylene or 1-50 membered heteroalkylene, and may satisfy one or more of the following:

(i) contains one or more unsaturated bonds;

(ii) two atoms in the Branching Unit are substituted with a divalent substituent such as a substituent; This completes heteroarylene.

(iii) the alkylene _{is substituted with one or more C 1-20 alkyl} .

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In addition, the at least one branching unit is a nitrogen-containing 1-50 membered heteroalkylene, the linker includes two or more atoms of a hydrophilic amino acid, and the nitrogen may form a peptide bond with a carbonyl of the hydrophilic amino acid.

In one aspect of the invention, at least one branching unit is a hydrophilic amino acid.

In one aspect of the invention, the hydrophilic amino acid can be arginine, aspartate, asparagine, glutamate, glutamine, histidine, lysine, ornithine, proline, serine, or threonine.

In addition, the hydrophilic amino acid may covalently bind the oxime of the linker to the polyethylene glycol unit of the linker.

In one aspect of the invention, a hydrophilic amino acid may be an amino acid comprising a side chain having residues that carry a charge at neutral pH in aqueous solution.

In one aspect of the invention, the hydrophilic amino acid is aspartate or glutamate.

In one aspect of the invention, the hydrophilic amino acid is ornithine or lysine.

In one aspect of the invention, the hydrophilic amino acid is arginine.

In one aspect of the invention, at least one Linking Unit is represented by Formula VIII or Formula IX:

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[Formula VIII]

-(CH ₂ ) _r (V(CH ₂ ) _p ) _q -

[Formula IX]

-(CH ₂ CH ₂ X) _w -

V is a single bond, -O-, -S-, -NR ²¹ -, -C(O)NR ²² -, -NR ²³ C(O)-, -NR ²⁴ SO ₂ - or -SO ₂ NR ²⁵ - ego;

X is -O-, C ₁ -C ₈ alkylene or -NR ²¹ -;

R ²¹ to R ²⁵ are each independently hydrogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkyl C ₆ -C ₂₀ aryl, or C ₁ -C ₆ alkylC ₃ -C ₂₀ heteroaryl;

r is an integer from 0 to 10;

p is an integer from 0 to 10;

q is an integer from 1 to 20; and

w is an integer from 1 to 20;

In one aspect of the present invention, q may be 4 to 20, more specifically 6 to 20. In addition, q may be 2 to 12, more specifically 2, 5, or 11. Also, r may be 2. Also, p may be 2. Also, V may be -O-.

More specifically, r is 2, p is 2, q is 2, 5 or 11, and V may be -O-.

Also, in one aspect of the present invention, X may be -O-. Also, w may be an integer of 6 to 20.

More specifically, X is -O-, and w may be 6 to 20.

또는

의 구조를 갖으며, 여기서 n은 1 내지 12이다.In one aspect of the invention, the at least one linking unit is at least one polyethylene glycol unit,

or

Has a structure, where n is 1 to 12.

In one aspect of the invention, at least one linking unit is 1 to 12 -OCH ₂ CH ₂ -units, or 3 to 12 -OCH ₂ CH ₂ -units, or 5 to 12 -OCH ₂ CH ₂ -units, or 6 to 12 -OCH ₂ CH ₂ -units, or 3 -OCH ₂ CH ₂ -units.

In one aspect of the invention, at least one linking unit is -(CH ₂ CH ₂ X)w-;

wherein X is a single bond, -O-, (C ₁ -C ₈ )alkylene, or -NR ₂₁ -;

R ₂₁ is hydrogen, (C ₁ -C ₆ )alkyl, (C ₁ -C ₆ )alkyl(C ₆ -C ₂₀ )aryl, or (C ₁ -C ₆ )alkyl(C ₃ -C ₂₀ )heteroaryl; ,

w is an integer from 1 to 20, specifically 1, 3, 6 or 12;

In one aspect of the present invention, X is -O-, and w is an integer from 6 to 20.

In one aspect of the present invention, the linker is 1,3-dipolar cycloaddition reactions, hetero-Diels-Alder reactions, nucleophilic substitution reactions, Bonding units formed by non-aldol type carbonyl reactions, addition to carbon-carbon multiple bond, oxidation reactions or click reactions additionally includes

In one aspect of the invention, the linking unit is formed by a reaction between acetylene and an azide, or between an aldehyde or ketone group and a hydrazine or alkoxyamine.

In one aspect of the invention, the binding unit,

,

,

또는

이고,

,

,

or

ego,

From here,

L ₁ is a single bond or an alkylene having 1 to 30 carbon atoms;

R ₁₁ is hydrogen or alkyl having 1 to 10 carbon atoms, specifically methyl.

In one aspect of the invention, L ₁ is a single bond, or an alkylene having 11 carbon atoms, or an alkylene having 12 carbon atoms.

In addition, in one aspect of the present invention, the binding unit

또는

or

을 포함하고,

including,

V is a single bond, -O-, -S-, -NR ²¹ -, -C(O)NR ²² -, -NR ²³ C(O)-, -NR ²⁴ SO ₂ -, or -SO ₂ NR ²⁵ -;

R ²¹ to R ²⁵ are independently hydrogen, C _1-6 alkyl, C _1-6 alkyl C _6-20 aryl, or C _1-6 alkyl C _3-20 heteroaryl;

r is an integer from 1 to 10;

p is an integer from 0 to 10;

q is an integer from 1 to 20; and

L ¹ is a single bond.

In one aspect of the present invention, r may be 2 or 3. Also, p may be 1 or 2. Also, q may be 1 to 6.

More specifically, in the binding unit, r is 2 or 3; p is 1 or 2; q may be 1 to 6.
In addition, in one aspect of the present invention, the antibody conjugate comprising the binding unit

,

,

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,

,

또는

or

일 수 있고, 여기서 Ab는 DLK1에 특이적으로 결합하는 항체 또는 이의 항원 결합단편이고, B는 활성제이며, n은 0 내지 20의 정수이다.

, wherein Ab is an antibody or antigen-binding fragment thereof that specifically binds to DLK1, B is an active agent, and n is an integer from 0 to 20.

또는

이다.In one aspect of the invention, the branching unit is

or

am.

In one aspect of the invention, the linker may include 3 to 50 heteroalkylenes including oximes,

The oxygen atom of the oxime is on the side of the linker linked to W, and the carbon atom of the oxime is on the side of the linker linked to Ab; or

The carbon atom of the oxime may be on the side of the linker connected to W, and the oxygen atom of the oxime may be on the side of the linker connected to Ab.

의 구조를 갖는 최소 하나 이상의 이소프레닐 유도체 유닛을 추가로 포함할 수 있으며, 여기에서 n은 최소 2 이상이다.In one aspect of the invention, the linker is

It may further include at least one isoprenyl derivative unit having a structure of, where n is at least 2 or more.

In one aspect of the invention, at least one isoprenyl derivative unit is a substrate of an isoprenoid transferase or a product of an isoprenoid transferase.

In one aspect of the present invention, the isoprenyl derivative unit of the linker is covalently bonded to the antibody via a thioether bond, and the thioether bond includes a sulfur atom of a cysteine of the antibody.

In addition, the isoprenyl derivative unit may covalently bond the oxime included in the linker to the antibody.

In one aspect of the invention, the antibody comprises an amino acid motif recognized by an isoprenoid transferase and the thioether bond comprises a sulfur atom of a cysteine of the amino acid motif.

In one aspect of the present invention, the antibody or antigen-binding fragment thereof that binds to DLK1 includes an amino acid motif recognized by isoprenoid transferase, and the thioether bond includes a sulfur atom of a cysteine of the amino acid motif.

In one aspect of the invention, the amino acid motif is a sequence selected from the group consisting of CXX, CXC, XCXC, XXCC, and CYYX, wherein C represents cysteine; Y independently represents an aliphatic amino acid; X independently at each occurrence represents glutamine, glutamate, serine, cysteine, methionine, alanine, or leucine; The thioether linkage includes the sulfur atom of the cysteine of the amino acid motif.

In one aspect of the invention, the amino acid motif is a CYYX sequence, and Y is independently in each case alanine, isoleucine, leucine, methionine, or valine.

In one aspect of the invention, the amino acid motif is a CVIM (cysteine-valine-isoleucine-methionine) or CVLL (cysteine-valine-leucine-leucine) sequence.

In one aspect of the invention, one or more of the 1 to 10 amino acids preceding the amino acid motif may each independently be selected from glycine, arginine, aspartic acid and serine. For example, in one aspect of the invention, at least one of the seven amino acids preceding the amino acid motif is glycine. Alternatively, 3 or more of the 7 amino acids preceding the amino acid motif are each independently selected from glycine, arginine, aspartic acid and serine. Alternatively, 1 to 10 amino acids preceding the amino acid motif are glycine, specifically at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids preceding the amino acid motif are glycine (G) am.

In one aspect of the invention, the antibody may comprise the amino acid sequence of GGGGGGGCVIM.

In one aspect of the invention, the linker comprises one or more branched linkers covalently linked to the Ab,

i) each branched linker comprises a branching unit (BR) covalently linked to the Ab by a main linker (PL);

ii) each branched linker binds the first active agent to the branch unit and includes a second linker (SL) and a first branch (B1) comprising a cleavable group; and

iii) each branched linker comprises a) a second branch (B2) where the second active agent is covalently linked to the branch unit by a second linker (SL) and a cleavage group (CG); or b) a second branch wherein the polyethylene glycol unit is covalently linked to the branch unit;

Each of the above cleavage groups may be hydrolyzed to release the active agent from the antibody conjugate.

,

, 또는

으로부터 선택되는 분지형 연결기를 포함하고,In one aspect of the invention, the branched linker is

,

, or

Including a branched linking group selected from,

Wherein L ² , L ³ , L ⁴ are each independently a direct bond or -(CH ₂ ) _n -, where n is an integer from 1 to 30;

,

,

또는

이며,G ¹ , G ² , G ³ are independently directly bonded;

,

,

or

is,

Wherein R ³⁰ is hydrogen or C _1-30 alkyl, R ⁴⁰ is hydrogen or L ₅ -COOR ₆ , where L ₅ is a direct bond or -(CH ₂ ) _n -, where n is 1 to 10 is an integer of, and R ₆ is hydrogen or C _1-30 alkyl.

More specifically, an antibody conjugate comprising a branched linker has the following structure.

wherein B and B' represent active agents which may be the same or different;

n each independently represents an integer from 0 to 30;

f each independently represents an integer from 0 to 30;

Combined with Ab on the side of the wavy line.

In one aspect of the present invention, n is an integer from 1 to 20, more specifically an integer from 1 to 10, or an integer from 4 to 20.

In one aspect of the invention, the linker comprises an oxime and at least one polyethylene glycol unit covalently bonds the oxime to the active agent.

In one aspect of the invention, the cleavage group can be cleaved within a target cell and can release one or more active agents.

In one aspect of the invention, it comprises at least one branched linker covalently linked to the Ab; and two or more active agents covalently linked to a branched linker.

Specifically, one branched linker may bind to Ab.

In addition, two or more branched linkers may bind to Ab, and each branched linker may bind to two or more active agents. More specifically, a three-branched linker may be combined with Ab. Alternatively, a four-branched linker may be combined with Ab.

In one aspect of the invention, each branched linker is associated with two or more identical or different active agents. In one aspect of the invention, each active agent is linked to the branched linker by a cleavable bond.

In one aspect of the invention, each branched linker comprises a branched linker, each active agent is coupled to the branched linker via a secondary linker, and the branched linker is coupled to the antibody via a primary linker. .

In one aspect of the present invention, the branched linking group may be a nitrogen atom. Alternatively, the branched linking group may be an amide, and the primary linker or secondary linker may include a carbonyl of the amide. Alternatively, the branched linking group may be a lysine unit.

Also, in one aspect of the invention, the active agent may be a chemotherapeutic agent or a toxin.

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In addition, the active agent may be an immune modulatory compound, an anticancer agent, an antiviral agent, an antibacterial agent, an antifungal agent, an antiparasitic agent, or a combination thereof, and may be selectively used among the active agents listed below:

(a) erlotinib, bortezomib, fulvestrant, sutent, letrozole, imatinib mesylate, PTK787/ZK 222584, oxaliplatin ( oxaliplatin), 5-fluorouracil, leucovorin, rapamycin, lapatinib, lonafarnib, sorafenib, gefitinib , AG1478, AG1571, thiotepa, cyclophosphamide, busulfan, improsulfan, piposulfan, benzodopa, carboquone, Meturedopa, uredopa, ethylenimine, altretamine, triethylenemelamine, triethylenephosphoramide, triethiylenethiophosphoramide , trimethylolomelamine, bullatacin, bullatacinone, camptothecin, topotecan, bryostatin, callystatin, CC-1065, ADO adozelesin, carzelesin, bizelesin, cryptophycin 1, cryptophycin 8, dolastatin, duocarmycin, KW- 2189, CB1-TM1, eleutherobin, pancratistatin , sarcodictyin, spongestatin, chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, carmu Carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimnustine, calicheamicin, calicheamicin gamma 1 (calicheamicin gamma 1), calicheamicin omega 1, dynemicin, dynemicin A, clodronate, esperamicin, neocarzinostatin chromophore, alacinomysins, actinomycin, antrmycin, azaserine, bleomycins, cactinomycin, kara carabicin, carninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubucin, 6-dia Crude-5-oxo-L-norleucine (6-diazo-5-oxo-L-norleucine), doxorubicin (doxorubicin), mo morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, liposomal doxorubicin, deoxydoxorubicin (deoxydoxorubicin), epirubicin, esorubicin, marcellomycin, mitomycin C, mycophenolic acid, nogalamycin, olibomycin ( olivomycins), peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, 5-fluorouracil, denopterin, methotrexate, Pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thiguanine, ancitabine ), azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, eno enocitabine, floxuridine, calusterone, dromostanolone, propionate ), epitiostanol, mepitiostane, testolactone, aminoglutethimide, mitotane, trilostane, folinic acid, aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene ), edatraxate, defofamine, demecolcine, diaziquone, elfornithine, elliptinium acetate, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansine, ansamitocins, mitoguazone, mito mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, 2-ethylhydrazide (2-ethylhydrazide), procarbazine, polysaccharide-k, razoxane, rhizoxin, sizofiran, spirogermanium, tenua Tenuazonic acid, triaziquone, 2,2',2''-trichlorotriethylamine (2,2',2''-trichlorotriethylam ine), T-2 toxin, verracurin A, roridin A, anguidine, urethane, vindesine, dacarbazine, mannomustine (mannomustine), mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside, cyclophosphamide, thiotepa , paclitaxel, paclitaxel, albumin-engineered nanoparticle formulation of paclitaxel, docetaxel, chlorambucil, gemcitabine, 6-thioguanine, mercaptopurine, cisplatin, carboplatin ), vinblastine, platinum, etoposide, ifosfamide, mitoxantrone, vincristine, vinorelbine, novantrone, tenyne teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronate, CPT-11, topoisomerase inhibitor RFS 2000 , difluoromethylornithine, retinoic acid, capecitabine, or a pharmaceutically acceptable salt, solvate or acid thereof;

(b) monokines, lymphokines, traditional polypeptide hormones, parathyroid hormones, thyroxine, relaxin, prorelaxin; glycoprotein hormone, follicle stimulating hormone, thyroid stimulating hormone, luteinizing hormone, hepatic growth factor fibroblast growth factor, prolactin (prolactin), placental lactogen, tumor necrosis factor-α, tumor necrosis factor-β, mullerian-inhibiting substance, mouse gonadotropin-associated Peptide (mouse gonadotropin-associated peptide), inhibin, activin, vascular endothelial growth factor, thrombopoietin, erythropoietin, osteoinductive factor factor), interferon, interferon-α, interferon-β, interferon-γ, colony stimulating factor (CSF), macrophage-CSF, granulocyte-macrophage-CSF, granulocyte-CSF, interleukin (IL), IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL- 11, IL-12, tumor necrosis factor, TNF-α, TNF-β, polypeptide factor, LIF , a kit ligand, or a combination thereof;

(c) diphtheria toxin, botulinum toxin, tetanus toxin, decentretoxin, cholera toxin, amanitin, α-amanitin, pyrrolobenzodiazepine, pyrrolobenzodiazepine derivatives, tetrodotoxin, brevetoxin, siguatoxin ciguatoxin, ricin, AM toxin, auristatin, tubulysin, geldanamycin, maytansinoid, calicheamycin, daunomycin (daunomycin), doxorubicin, methotrexate, vindesine, SG2285, dolastatin, dolastatin analog, auristatin, cryptophycin, camptothecin (camptothecin), rhizoxin, rhizoxin derivatives, CC-1065, CC-1065, analogs or derivatives, duocarmycin, enediyne antibiotic, esperamicin ), epothilone, toxoid, or combinations thereof;

(d) an affinity ligand, wherein the affinity ligand is a substrate, inhibitor, activator, neurotransmitter, radioisotope, or combination thereof;

(e) radioactive label, 32P, 35S, fluorescent dye, electron dense reagent, enzyme, biotin, streptavidin, dioxigenin, hapten, an immunogenic protein, a nucleic acid molecule with a sequence complementary to a target, or a combination thereof;

(f) immunomodulatory compound, anti-cancer agent, anti-viral agent, anti-bacterial agent, anti-fungal agent agent), and an anti-parasitic agent, or a combination thereof;

(g) tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone ( onapristone or toremifene;

(h) 4(5)-imidazole, aminoglutethimide, megestrol acetate, exemestane, letrozole Ehsms anastrozole

(i) flutamide, nilutamide, bicalutamide, leuprolide, goserelin, or troxacitabine;

(j) aromatase inhibitors;

(k) protein kinase inhibitors;

(l) lipid kinase inhibitors;

(m) antisense oligonucleotides;

(n) ribozymes;

(o) vaccines; and

(p) anti-angiogenic agents

In another aspect of the present invention, the use of an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof in the preparation of a drug for the prevention or treatment of hyperproliferation, cancer or angiogenic disease is provided.

In another aspect of the present invention, a pharmaceutical composition containing an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof as an active ingredient for preventing or treating hyperproliferation, cancer or angiogenic disease is administered to a subject Thus, a method for preventing or treating hyperproliferative, cancer, or angiogenic disease is provided, and a method for administering a combination of one or more anti-hyperproliferative, cytostatic, or cytotoxic substances is also provided.

In another aspect of the invention, the active agent is a pyrrolobenzodiazepine (PBD) dimer,

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The linker and the antibody are linked through the N10 or N10' position of the pyrrolobenzodiazepine dimer.

-C(O)O*, -S(O)O*, -C(O)*, -C(O)NR*, -S( O) any one selected from the group consisting of ₂ NR*, -P(O)R'NR*, -S(O)NR*, and -PO ₂ NR* groups is attached;

Where * is the part to which the linker is attached,

wherein R, and R' are each independently H, OH, N ₃ , CN, NO ₂ , SH, NH ₂ , ONH ₂ , NHNH ₂ , halo, substituted or unsubstituted C _1-8 alkyl, substituted or Unsubstituted C _3-8 cycloalkyl, substituted or unsubstituted C _1-8 alkoxy, substituted or unsubstituted C _1-8 alkylthio, substituted or unsubstituted C _3-20 heteroaryl, substituted or unsubstituted C _5-20 aryl or C _1-8 alkylamino;

wherein, when C _1-8 alkyl, C _3-8 cycloalkyl, C _1-8 alkoxy, C _1-8 alkylthio, C _3-20 heteroaryl, or C _5-20 aryl is substituted, H, OH, N ₃ , CN, NO ₂ , SH, NH ₂ , ONH ₂ , NNH ₂ , halo, C _1-6 alkyl, C _1-6 alkoxy and C _6-12 aryl;

A pyrrolobenzodiazepine dimer prodrug, a pharmaceutically acceptable salt or solvate thereof, can be used as an active agent.

In one aspect of the invention, pyrrolobenzodiazepine dimer precursors are provided. When administered in the form of a precursor according to the present invention, it is necessary to convert into an effective drug by an additional reaction when exposed to blood, so the possibility of side effects that may occur when the linker is unexpectedly decomposed can be prevented in advance, It has advantages over existing PBD drugs in that toxicity to normal cells is reduced and the drug is more stable.

In addition, when preparing antibody-drug conjugates, in the case of antibody-drug conjugates prepared by conventional methods, the content of impurities is high and the exposed imine group is attacked by nucleophiles, resulting in the production of drugs with unwanted structures. On the other hand, the antibody-drug conjugate prepared by the method according to the present invention has the advantage of easy separation due to its high purity, and has been shown to have improved physical properties compared to existing PBDs or PBD dimers.

In one aspect of the present invention, the pyrrolobenzodiazepine dimer precursor is characterized in that it has the structure of Formula X or Formula XI:

A pyrrolobenzodiazepine dimer precursor, a pharmaceutically acceptable salt or solvate thereof:

[Formula X]

,

,

[Formula XI]

,

,

In the above formula,

the dotted line indicates the optional presence of a double bond between C1 and C2, between C2 and C3, between C'1 and C'2, or between C'2 and C'3;

R ^X1 and R ^X1' are independently H, OH, =O, =CH ₂ , CN, R ^m , OR ^m , =CH-R ^m' =C(R ^m' ) ₂ , O-SO ₂ -R ^m , CO ₂ R ^m , COR ^m , halo and dihalo;

R ^m' is selected from R ^m , CO ₂ R ^m , COR ^m , CHO, CO ₂ H, and halo;

Each R ^m is independently C _1-12 alkyl, C _2-12 alkenyl, C _2-12 alkynyl, C _5-20 aryl, C _5-20 heteroaryl, C _3-6 cycloalkyl, 3 to 7 -is selected from the group consisting of -membered heterocyclyl, 3 to 7-membered heterocycloalkyl, and 5 to 7-membered heteroaryl;

R ^X2 _, R ^X2' R ^X3 , R ^X3' , R ^X5 , and R ^X5' are independently H, R ^m , OH, OR ^m , SH, SR ^m , NH ₂ , NHR ^m , NR ^m ₂ , NO ₂ , Me ₃ Sn and halo;

R ^X4 and R ^X4' are independently H, R ^m , OH, OR ^m , SH, SR ^m , NH ₂ , NHR ^m , NR ^m ₂ , NO ₂ , Me ₃ Sn, halo, C _1-6 alkyl, C _{1- 6} alkoxy, C _2-6 alkenyl, C _2-6 alkynyl, C _3-6 cycloalkyl, 3-7 membered heterocycloalkyl, C _5-12 aryl, 5-7 membered heteroaryl, -CN, -NCO, -OR ⁿ , -OC(O)R ⁿ , -OC(O)NR ⁿ R ^n' , -OS(O)R ⁿ , -OS(O) ₂ R ⁿ , -SR ⁿ , -S( O)R ⁿ , -S(O) ₂ R ⁿ , -S(O)NR ⁿ R ^n' , -S(O) ₂ NR ⁿ R ^n' , -OS(O)NR ⁿ R ^n' , -OS (O) ₂ NR ⁿ R ^n' , -NR ⁿ R ^n' , -NR ⁿ C(O)R ^o , -NR ⁿ C(O)OR ^o , -NR ⁿ C(O)NR ^o R ^o' , -NR ⁿ S(O)R ^o , -NR ⁿ S(O) ₂ R ^o , -NR ⁿ S(O)NR ^o R ^o' , -NR ⁿ S(O) ₂ NR ^o R ^o' , -C (O)R ⁿ , -C(O)OR ⁿ and -C(O)NR ⁿ R ^n' ;

Y and Y' are independently selected from O, S, and N(H);

delete

R ^X6 is independently selected from C _3-12 alkylene, C _3-12 alkenylene, or C _3-12 heteroalkylene;

R ^X7 and R ^X7' are independently H, C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _3-6 cycloalkyl, 3 to 7-membered heterocycloalkyl, C ₆ - ₁₀ aryl, 5 to 7-membered heteroaryl, -OR ^r , -OC(O)R ^r , -OC(O)NR ^r R ^r' , -OS(O)R ^r , -OS(O) ₂ R ^r , -SR ^r , -S(O)R ^r , -S(O) ₂ R ^r , -S(O)NR ^r R ^r' , -S(O) ₂ NR ^r R ^r' , -OS(O) NR ^r R ^r' , -OS(O) ₂ NR ^r R ^r' , -NR ^r R ^r' , -NR ^r C(O)R ^s , -NR ^r C(O)OR ^s , -NR ^r C( O)NR ^s R ^s' , -NR ^r S(O)R ^s , -NR ^r S(O) ₂ R ^s , -NR ^r S(O)NR ^s R ^s' , -NR ^r S(O) ₂ is selected from NR ^s R ^s , -C(0)R ^r , -C(0)OR ^s or -C(0)NR ^r R ^r' ;

R ^X8 each and R ^X8' is independently H, C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _3-6 heteroalkyl, 3 to 7-membered heterocycloalkyl, C _5-10 aryl , 5 to 7-membered heteroaryl, -S(O)R ^m , -S(O) ₂ R ^m , -S(O)NR ^m R ^m' , -S(O) ₂ NR ^m R ^m' , - NR ^m R ^m' , -NR ^m C(O)R ^m , -NR ^m C(O)OR ⁿ , -NR ^m C(O)NR ⁿ R ^n' , -NR ^m S(O)R ⁿ , - NR ^m S(O) ₂ R ⁿ , -NR ^m S(O)NR ⁿ R ^n' , -NR ^m S(O) ₂ NR ⁿ R ^n' , -C(O)R ^m , -C(O) OR ^m and -C(O)NR ^m R ^m' ;

delete

Z ^a is selected from OR ^X12a , NR ^X12a R ^X12a , or SR ^X12a ;

Z ^b is selected from OR ^X13a , NR ^X13a R ^X13a , or SR ^X13a ;

Z ^a' is selected from OR ^X12a' , NR ^X12a' R ^X12a' , or SR ^X12a' ;

Z ^b' is OR ^X13a' , NR ^X13' R ^X13a' , or SR ^X13a' ;

Each R ^X12a , R ^X12a' , R ^X13a and R ^X13a' is independently H, C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _3-6 cycloalkyl, 3 to 7- is selected from membered heterocycloalkyl, C _5-10 aryl, 5 to 7-membered hetearyl, -C(O)R ^X15a , -C(O)OR ^X15a and -C(O)NR ^X15a R ^X15a ';

Each of R ^X15a and R ^X15a 'is independently C _1-12 alkyl, C _2-12 alkenyl, C _2-12 alkynyl, C _5-20 aryl, C _5-20 heteroaryl, C _3-6 cycloalkyl , 3 to 7-membered heterocyclyl, 3 to 7-membered heterocycloalkyl, and 5 to 7-membered heteroaryl;

or wherein R ^X12a and R ^X13a are optionally taken together with the atoms to which they are attached to form a 3 to 7-membered heterocyclyl, a 3 to 7-membered heterocycloalkyl, or a 3 to 7-membered heteroaryl, and R ^X12a' and R ^X13a' are optionally taken together with the atoms to which they are attached to form a 3 to 7 membered heterocyclyl, a 3 to 7 membered heterocycloalkyl, or a 3 to 7 membered heteroaryl.

delete

In addition, the R ^m is additionally C _1-12 alkyl, C _2-12 alkenyl, C _2-12 alkynyl, C _5-20 aryl, C _5-20 heteroaryl, C _3-6 cycloalkyl, 3 to 7 -membered heterocyclyl, 3 to 7-membered heterocycloalkyl, or 5 to 7-membered heteroaryl.

delete

In addition, the R ^X4 or R ^X4' is additionally C _1-6 alkyl, C _1-6 alkoxy, C _2-6 alkenyl, C _2-6 alkynyl, C _3-6 cycloalkyl, 3 to 7-membered heterocycloalkyl, C _{5 -12} aryl or 5 _to 7-membered heteroaryl, additionally one or more C _1-6 alkyl, _{C 1-6 alkoxy, C 2-6 alkenyl, C 2-6 alkynyl , C 3-} C ₆ cycloalkyl, 3 to 7-membered heterocycloalkyl, C _{5 -10} aryl, 5 to 7-membered heteroaryl, -OR ^p , -OC(O)R ^p , -OC(O)NR ^p R ^p' , -OS(O )R ^p , -OS(O) ₂ R ^p , -SR ^p , -S(O)R ^p , -S(O) ₂ R ^p , -S(O)NR ^p R ^p' , -S(O) ₂ NR ^p R ^p' , -OS(O)NR ^p R ^p' , -OS(O) ₂ NR ^p R ^p' , -NR ^p R ^p' , -NR ^p C(O)R ^q , -NR ^p C(O)OR ^q , -NR ^p C(O)NR ^q R ^q' , -NR ^p S(O)R ^q , -NR ^p S(O) ₂ R ^q , -NR ^p S(O)NR ^q R ^q' , -NR ^p S(O) ₂ NR ^q R ^q' , -C(O)R ^p , -C(O)OR ^p or -C(O)NR ^p R ^p ;

delete

R ^X7 or R ^X7' is additionally C _1-6 alkyl, C _2-6 alkenyl _, C _2-6 alkynyl, C _3-6 cycloalkyl, 3 to 7-membered heterocycloalkyl, C _6-10 aryl , 5 to 7-membered heteroaryl, additionally C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _3-6 cycloalkyl, 3 to 7-membered heterocycloalkyl, C ₆ - ₁₀ aryl, 5 to 7-membered heteroaryl, -OR ^t , -OC(O)R ^t , -OC(O)NR ^t R ^t' , -OS(O)R ^t , -OS(O) ₂ R ^t , -SR ^t , -S(O)R ^t , -S(O) ₂ R ^t , -S(O)NR ^t R ^t' , -S(O) ₂ NR ^t R ^t' , -OS(O) NR ^t R ^t' , -OS(O) ₂ NR ^t R ^t' , -NR ^t R ^t' , -NR ^t C(O)R ^u , -NR ^t C(O)OR ^u , -NR ^t C( O)NR ^u R ^u' , -NR ^t S(O)R ^u , -NR ^t S(O) ₂ R ^u , -NR ^t S(O)NR ^u R ^u' , -NR ^t S(O) ₂ NR ^u R ^u' , -C(O)R ^t , -C(O)OR ^t or -C(O)NR ^t R ^t' ,

delete

R ⁿ , R ⁿ ', R ^o , R ^o ', R ^p , R ^p ', R ^r , R ^r' , R ^s , R ^s ', R ^t , R ^t ', R ^u and ^Ru ' independently H, C _1-7 alkyl, C _2-7 alkenyl, C _2-7 alkynyl, C _3-13 cycloalkyl, 3-7 membered heterocycloalkyl, C _5-10 aryl, and 5-7 -membered heteroaryls.

Also, more specifically, R ^X1 and R ^X1' are independently selected from R ^m ;

R ^m is selected from C _1-6 alkyl, C _2-6 alkenyl, C _5-7 aryl and C _3-6 heteroaryl.

In addition, R ^X2 _, R ^X2' _, R ^X3 , R ^X3' , R ^X5 , and R ^X5' are independently selected from H or OH.

In addition, the R ^X4 and R ^X4' are independently selected from R ^m ;

R ^m is C _1-6 alkoxy.

In addition, the R ^X4 and R ^X4 is independently any one selected from the group consisting of methoxy, ethoxy and butoxy.

In addition, Y and Y' are O.

In addition, R ^X6 is C _3-12 alkylene, C _3-12 alkenylene or C _3-12 heteroalkylene, and R ^X6 is -NH ₂ , -NHR ^m , -NHC(O)R ^m , -NHC(O)CH ₂ -[OCH ₂ CH ₂ ] _r -R ^XX , or -[CH ₂ CH ₂ O] _n -R ^XX is substituted; , where n is an integer from 1 to 6;

Wherein R ^XX is H, OH, N ₃ , CN, NO ₂ , SH, NH ₂ , ONH ₂ , NHNH ₂ , halo, C _1-8 alkyl, C _3-8 cycloalkyl, C _1-8 alkoxy, C _{1 -8} alkylthio, C _3-20 heteroaryl, C _5-20 aryl or C _1-8 alkylamino.

Further, in one aspect of the present invention, the antibody conjugate is a compound of formula XII or formula XIII comprising a pyrrolobenzodiazepine dimer as an active agent:

delete

[Formula XII]

,

,

[Formula XIII]

,

,

wherein X ^a and X ^a' are independently selected from a direct bond or C _1-6 alkylene;

, 또는

로부터 선택되며;Z ^X' and Z ^X are independently C _1-8 alkyl, halogen, cyano, nitro,

, or

is selected from;

wherein each of R ⁸⁰ , R ⁹⁰ and R ¹⁰⁰ is selected from hydrogen, C _1-8 alkyl, C _2-6 alkenyl, and C _1-6 alkoxy, or from -(CH ₂ ) _m -OCH ₃ , wherein

, 및

로 이루어진 군으로부터 선택되는 어느 하나이고, m is an integer from 1 to 12;
n is an integer from 0 to 3;
The remaining substituents and other symbols are the same as those defined for general formulas I, X, and XI and general formula II above.
More specifically, the Z ^{X '} and Z ^X are independently

, and

Any one selected from the group consisting of

delete

Each of R ⁸⁰ , R ⁹⁰ and R ¹⁰⁰ is any one selected from the group consisting of hydrogen, C _1-3 alkyl, C _1-3 alkoxy, -(CH ₂ )m-OCH ₃ ,

m is an integer from 1 to 3;

The antibody conjugate according to the present invention can be prepared from the following compounds:

,

,

,

,

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and

The present invention also provides a pharmaceutical composition for preventing or treating hyperproliferative disease or angiogenic disease, comprising an antibody conjugate to which the above-described pyrrolobenzodiazepine dimer is bound, or a pharmaceutically acceptable salt or solvate thereof.

The present invention also provides a use of the above-described pyrrolobenzodiazepine dimer-linked antibody conjugate, a pharmaceutically acceptable salt or solvate thereof, for use as a pharmaceutical composition for preventing or treating hyperproliferative or angiogenic diseases.

Also, one or more therapeutic co-agents; And a pharmaceutically acceptable excipient may be additionally included.

In one aspect of the present invention, the therapeutic co-agent is an agent that exhibits a preventive, ameliorative, or therapeutic effect on hyperproliferative disease, or an agent that can reduce the expression of side effects that appear when administering a proliferative disease treatment agent, or enhances immunity It may be an agent that exhibits an effect, but is not limited thereto, and when applied in the form of a compounding agent together with pyrrolobenzodiazepine, exhibits a therapeutically useful effect, further improves the stability of pyrrolobenzodiazepine, and/or This means that any agent that can reduce side effects that may appear during administration of benzodiazepines and/or maximize the therapeutic effect through the enhancement of immunity can be applied in combination.

In one aspect of the present invention, the hyperproliferative disease refers to a disease associated with cell proliferation in which undesirably excessive or abnormal cells, such as neoplasia or hyperplastic growth, are undesirably uncontrolled, whether in vitro or in vivo. The hyperproliferative disease may be selected from the group consisting of neoplasia, tumor, cancer, leukemia, psoriasis, bone disease, fibroproliferative disorder, and atherosclerosis. Examples of neoplasms and tumors include histiocytoma, glioma, astrocytoma, osteoma, and the like.

In one aspect of the invention, the cancer is lung cancer, small cell lung cancer, gastrointestinal cancer, colorectal cancer, bowel cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma And it may be selected from the group consisting of melanoma, but it can be applied to any type of carcinoma for which pyrrolobenzodiazepine can exhibit a therapeutic effect.

The present invention also relates to the treatment of hyperproliferative disease in a subject having a hyperproliferative disease comprising administering to the subject an effective amount of an antibody conjugate to which a pyrrolobenzodiazepine dimer is linked, or a pharmaceutically acceptable salt or solvate thereof for treating the hyperproliferative disease. Provided is a method for preventing or treating hyperproliferative diseases of

In one aspect of the present invention, there is provided a method for preventing or treating cancer, comprising administering the pharmaceutical composition to a patient.

In one aspect of the present invention, there is provided a method for preventing or treating angiogenic disease, comprising administering the pharmaceutical composition to a patient.

The present invention is suitable for use in providing a PBD compound to a target location in a subject. Conjugates according to the present invention release an active PBD compound that does not contain any linker moiety, and nothing that could affect the reactivity of the PBD compound is present.

[Justice]

The following definitions apply herein:

As used herein, “conjugates” refer to cell binding agents that are covalently bound to one or more molecules of a cytotoxic compound. Here, "cell binding agent" is a molecule having affinity for a biological target, and may be, for example, a ligand, a protein, an antibody, specifically a monoclonal antibody, a protein or antibody fragment, a peptide, an oligonucleotide, an oligosaccharide, In other words, binding agents function to direct biologically active compounds to biological targets. In one aspect of the invention, conjugates can be designed to target tumor cells via cell surface antigens. The antigen may be a cell surface antigen that is overexpressed or expressed in an abnormal cell type. Specifically, the target antigen may be expressed only on proliferating cells (eg, tumor cells). Target antigens can usually be selected based on differential expression between proliferative and normal tissues. In the present invention, the antibody is linked to a linker.

As used herein, an "antibody" is an immunoglobulin molecule capable of specifically binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" refers to an intact polyclonal or monoclonal antibody, as well as any antigen-binding portion of an intact antibody that retains the ability to specifically bind to a given antigen (e.g., "antigen-binding fragments") or single chains thereof, fusion proteins including antibodies, and any other modified arrangement of immunoglobulin molecules comprising antigen recognition sites, including but not limited to, Fab; Fab';F(ab') ₂ Fd fragment; Fv fragment; single domain antibody (dAb) fragments; an isolated complementarity determining region (CDR); single chain (scFv) and single domain antibodies (e.g., shark and camelid antibodies), maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs and bis-scFvs ( See, eg, Hollinger and Hudson, 2005, Nature Biotechnology 23(9): 1126-1136).

Antibodies include any class of antibody, such as IgG, IgA or IgM (or subclasses thereof), and the antibody need not be of any particular class. Depending on the amino acid sequence of the constant region of the heavy chain of the antibody, immunoglobulins can be assigned to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, several of which can be further divided into subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. can be classified. The heavy chain (HC) constant domains corresponding to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma and mu, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known. Antibodies of the present invention can be prepared using techniques well known in the art, such as recombinant techniques, phage display techniques, synthetic techniques, or combinations of the foregoing techniques or other techniques readily known in the art.

An “isolated antibody” as used herein refers to an antibody that is substantially free of other antibodies having a different antigenic specificity, and may be substantially free of other cellular material and/or chemicals.

As used herein, "biological target" refers to an antigen located on the surface of a tumor, cancer cell, or extracellular matrix.

As used herein, "linker" refers to a compound that covalently binds a cytotoxic compound to an antibody. In one aspect of the present invention, linkers disclosed in PCT/US2016/063564 and PCT/US2016/063595 may be used as the linker.

As used herein, a “therapeutic agent” is an agent that exerts cytotoxic, cytostatic and/or immunomodulatory effects on proliferative diseases, eg, cancer cells or activated immune cells. Examples of therapeutic agents include cytotoxic agents, chemotherapeutic agents, cytostatic agents, and immunomodulatory agents.

A “chemotherapeutic agent” herein is a chemical compound useful in the treatment of cancer.

As used herein, “subject” is intended to include humans and non-human animals, particularly mammals. An example of a subject includes a human subject, such as a concept that includes a normal subject or a human patient having a disorder described herein, more specifically cancer. "Non-human animal" refers to all vertebrates, eg, non-mammals (eg, chickens, amphibians, reptiles) and mammals, eg, non-human primates, livestock and/or useful for agriculture. animals (eg sheep, dogs, cats, cows, pigs, etc.) and rodents (eg mice, rats, hamsters, guinea pigs, etc.). In certain embodiments, the subject is a human patient.

“Treatment” or “treat” as used herein refers to both therapeutic treatment and prophylactic or prophylactic measures. Those in need of treatment include those already with the disease, and those prone to have the disease or those in which the disease is to be prevented. Thus, when used in reference to a disease or subject in need of treatment, the term includes arresting or slowing the progression of a disease, preventing symptoms, reducing the severity of a disease and/or symptoms, or reducing the duration of a disease, compared to an untreated subject. However, it is not limited to this.

As used herein, “administration” or “administering” refers to providing, contacting, and/or delivering a compound or compounds by any suitable route to achieve a desired effect. Administration can be oral, sublingual, parenteral (eg intravenous, subcutaneous, intradermal, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional or intracranial injection), transdermal, topical, administration via buccal, rectal, vaginal, intranasal, ophthalmic, inhalation and implantation.

In the present specification, "unsubstituted or substituted" refers to a parent group that may be unsubstituted or may be substituted, "substituted" refers to a parent group having one or more substituents, and the substituent refers to a parent group. group) or a chemical moiety fused to a parent group.

As used herein, "halo" refers to fluorine, chlorine, bromine, iodine, and the like.

As used herein, "alkyl" is a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of an aliphatic or alicyclic, saturated or unsaturated (unsaturated, completely unsaturated) hydrocarbon compound, examples of saturated alkyl include methyl, ethyl, propyl, butyl , pentyl, hexyl, heptyl, etc. Examples of saturated straight-chain alkyl include methyl, ethyl, n-propyl, n-butyl, n-pentyl (amyl), n-hexyl, n-heptyl, etc. Examples of saturated branched-chain alkyl may include isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, neopentyl and the like.

As used herein, "alkoxy" means -OR [wherein R is an alkyl group], and examples thereof include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, isobutoxy, tert-butoxy; and the like.

In this specification, "aryl" means a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound having a ring atom.

As used herein, "alkenyl" is an alkyl having one or more carbon-carbon double bonds, and examples of unsaturated alkenyl groups include ethenyl (vinyl, -CH=CH ₂ ), 1-propenyl (-CH=CH-CH ₃ ), 2-propenyl, isopropenyl, butenyl, pentenyl, hexenyl and the like.

In this specification, "alkynyl" is an alkyl group having at least one carbon-carbon triple bond, and examples of unsaturated alkynyl groups include ethynyl and 2-propynyl.

As used herein, "carboxy" refers to -C(=0)OH.

As used herein, "formyl" refers to -C(=0)H.

As used herein, “aryl” refers to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound. For example, "C _5-7 aryl" means a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, wherein the moiety has 5 to 7 ring atoms, and "C _5-7 aryl"_"10aryl" means a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, wherein the moiety has 5 to 10 ring atoms. Here, the prefix (C _5-7 , C _5-10, etc.) refers to a number of ring atoms or a range of ring atoms, whether carbon atoms or heteroatoms. For example, "C _5-6 aryl" relates to an aryl group having 5 or 6 ring atoms. Here, the ring atoms may all be carbon atoms as in the "carboaryl group". Examples of carboaryl groups include, but are not limited to, those derived from benzene, naphthalene, azulene, anthracene, phenanthrene, naphthacene and pyrene. Examples of aryl groups comprising fused rings in which at least one is an aromatic ring include groups derived from indane, indene, isoindene, tetralin, acenaphthene, fluorene, phenalene, acephenanthrene and aceantrene, but Not limited. Alternatively, the ring atoms may include one or more heteroatoms as in "heteroaryl group".

As used herein, "heteroaryl" is an aryl containing one or more heteroatoms, examples of which include pyridine, pyrimidine, benzothiophene, furyl, dioxalanyl, pyrrolyl, oxazolyl, pyridyl, pyridazinyl, and pyrimidyl. Nil et al., more specifically benzofuran, isobenzofuran, indole, isoindole, indolizine, indoline, isoindoline, purine (adenine or guanine), benzimidazole, indazole, benzoxazole, benzisoxazole, Benzodioxole, benzofuran, benzotriazole, benzothiofuran, benzothiazole, C ₉ having two fused rings derived from benzothiadiazole, chromene, isochromene, chromane, isochromane, benzo Two fused rings derived from dioxane, quinoline, isoquinoline, quinolizine, benzoxazine, benzodiazine, pyridopyridine, quinoxaline, quinazoline, cinnoline, phthalazine, naphthyridine, pteridine C ₁₀ , C ₁₁ having two fused rings derived from benzodiazepine, C ₁₃ having 3 fused rings derived from carbazole, dibenzofuran, dibenzothiophene, carboline, perimidine, pyridoindole, Three fused rings derived from acridine, xanthene, thioxanthene, oxantrene, phenoxathiine, phenazine, phenoxazine, phenothiazine, thianthrene, phenanthridine, phenanthroline, and phenazine and C ₁₄ having.

In this specification, "cycloalkyl" is an alkyl group which is a cyclyl group, and relates to a monovalent moiety obtained by removing a hydrogen atom from an alicyclic ring atom of a cyclic hydrocarbon compound. Examples of cycloalkyl groups include, but are not limited to, those derived from:

saturated monocyclic hydrocarbon compounds: cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, methylcyclopropane, dimethylcyclopropane, methylcyclobutane, dimethylcyclobutane, methylcyclopentane, dimethylcyclopentane and methylcyclohexane;

unsaturated monocyclic hydrocarbon compounds: cyclopropene, cyclobutene, cyclopentene, cyclohexene, methylcyclopropene, dimethylcyclopropene, methylcyclobutene, dimethylcyclobutene, methylcyclopentene, dimethylcyclopentene and methylcyclohexene; and

Saturated heterocyclic hydrocarbon compounds: norcarane, norfinan, norbornane.

In this specification, "heterocyclyl" relates to a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound.

A prefix (eg, C _1-12 , C _3-8 , etc.) herein refers to a number of ring atoms or a range of ring atoms, whether carbon atoms or heteroatoms. For example, the term "C _3-6 heterocyclyl" as used herein relates to a heterocyclyl group having 3 to 6 ring atoms.

Examples of monocyclic heterocyclyl groups include, but are not limited to, those derived from:

N ₁ : aziridine, azetidine, pyrrolidine, pyrroline, 2H- or 3H-pyrrole, piperidine, dihydropyridine, tetrahydropyridine, azepine;

N ₂ : imidazolidine, pyrazolidine, imidazoline, pyrazoline, piperazine;

O ₁ : oxirane, oxetane, oxolane, oxol, oxane, dihydropyran, pyran, oxepin;

O ₂ : dioxolane, dioxane and dioxepane;

O ₃ : trioxane;

N ₁ O ₁ : tetrahydrooxazole, dihydrooxazole, tetrahydroisoxazole, dihydroisoxazole, morpholine, tetrahydrooxazine, dihydrooxazine, oxazine

S ₁ : thilan, thietane, thiolane, thian, thiepan;

N ₁ S ₁ : thiazoline, thiazolidine, thiomorpholine;

N ₂ O ₁ : oxadiazine;

O ₁ S ₁ : oxathiol, oxathiane; and

N ₁ O ₁ S ₁ : oxathiazine.

As used herein, "prodrug" refers to pyrrolobenzodiazepine by the action of an enzyme or gastric acid under physiological conditions in vivo (eg, enzymatic oxidation, reduction, and/or hydrolysis, etc.). A compound that can be directly or indirectly converted into a zepine drug.

In the present specification, as the "pharmaceutically acceptable salt", an acid addition salt formed by a pharmaceutically acceptable free acid may be used, and an organic acid or an inorganic acid may be used as the free acid.

The organic acid is not limited thereto, but citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, metasulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, Includes glutamic acid and aspartic acid. In addition, the inorganic acid includes, but is not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid.

For example, when a compound has a functional group that is or can be an anion (e.g. -COOH can be -COO-), a salt can be formed with an appropriate cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Na ⁺ and K ⁺ , alkaline earth metal cations such as Ca ²⁺ and Mg ²⁺ and other cations such as Al ³⁺ . Examples of suitable organic cations include, but are not limited to, ammonium ions (ie, NH ₄ ⁺ ) and substituted ammonium ions (eg, NH ₃ R ⁺ , NH ₂ R ₂ ⁺ , NHR ₃ ⁺ , NR ₄ ⁺ ).

Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine , phenylbenzylamine, choline, meglumine and tromethamine, as well as amino acids such as lysine and arginine. An example of a typical quaternary ammonium ion is N(CH ₃ ) ₄ ⁺ .

When a compound is cationic or has a functional group that can be cationic (eg -NH ₂ can be -NH ₃ ⁺ ), it can form a salt with an appropriate anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfurous acid, nitric acid, nitrous acid, phosphoric acid and phosphorous acid, and the like.

Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetioxybenzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphorsulfonic acid, cinnamic acid, citric acid, edetic acid, ethane Disulfonic acid, ethanesulfonic acid, fumaric acid, glutethonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxymaleic acid, hydroxynaphthalenecarboxylic acid, isethionic acid, lactic acid, lactobionic acid, lauric acid, maleic acid, Malic acid, methanesulfonic acid, mucoic acid, oleic acid, oxalic acid, palmitic acid, palmic acid, pantothenic acid, phenylacetic acid, phenylsulfonic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, toluenesulfonic acid and valeric acid etc. can be cited as an example. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose, and the like.

In this specification, "solvate" refers to a molecular complex between the compound according to the present invention and solvent molecules, and examples of solvates include water, isopropanol, ethanol, methanol, dimethyl sulfoxide (dimethylsulfoxide), ethyl acetate, acetic acid, ethanolamine, or a mixed solvent thereof, and the compound according to the present invention combined, but is not limited thereto.

It may be convenient or desirable to prepare, purify and/or handle equivalent solvates of the active compounds. The term “solvate” is used herein in its conventional sense to refer to a complex of a solute (eg, an active compound, a salt of an active compound) and a solvent. When the solvent is water, the solvate may conveniently be referred to as a hydrate, such as a monohydrate, dihydrate, trihydrate, and the like.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may include macromolecules that are usually slowly metabolized, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, lipid aggregates, and the like. Acceptable carriers can be appropriately selected and used by those skilled in the art.

The composition containing a pharmaceutically acceptable carrier may be in various oral or parenteral formulations. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations contain at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. in one or more compounds. mixed and prepared In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

Liquid preparations for oral administration include suspensions, solutions for oral administration, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. there is.

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

The pharmaceutical composition is selected from the group consisting of injections, tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. It can have any one formulation that is.

For intravenous, dermal or subcutaneous injection, etc., the active ingredient may be in the form of an aqueous solution that is pyrogen-free and has an acceptable pH, isotonicity and stability for parenteral administration. One skilled in the art will be able to prepare suitable solutions using isotonic vehicles such as, for example, aqueous sodium chloride solution, Ringer's solution, lactated Ringer's solution, and the like, and preservatives, stabilizers, buffers, antioxidants, or other additives may be included as desired. Solid forms suitable for injection can also be prepared as emulsions or in the form of the polypeptide encapsulated in liposomes.

As used herein, the phrase "effective amount" or "therapeutically effective amount" refers to the amount necessary (relative to dosage and duration and means of administration) to achieve the desired therapeutic result. An effective amount is at least the smallest amount of active agent necessary to confer a therapeutic benefit to a subject and is less than a toxic amount. For example, dosages may range from about 100 ng to about 100 mg/kg per patient, more typically from about 1 μg/kg to about 10 mg/kg. When the active compound is a salt, ester, amide, prodrug or the like, the dosage is calculated based on the parent compound, so the actual weight used is proportionally increased. The pyrrolobenzodiazepine compound according to the present invention may be formulated to contain 0.1 mg to 3000 mg, 1 mg to 2000 mg, or 10 mg to 1000 mg of the active ingredient per dosage form, but is not limited thereto. The active ingredient can be administered to obtain a peak plasma concentration of active compound of about 0.05 μM to 100 μM, 1 μM to 50 μM, or 5 μM to 30 μM. eg by intravenous injection of a 0.1 w/v% to 5 w/v% solution of the active ingredient, optionally in saline.

The concentration of active compound in a pharmaceutical composition can be determined by absorption, inactivation and excretion rates of the drug and other factors known to those skilled in the art. The dosage may vary depending on the severity of the symptom/disease. In addition, the dosage and administration regimen for a specific patient can be adjusted according to the professional judgment of the administration supervisor in comprehensive consideration of the patient's severity of symptoms/disease, necessity, age, reactivity to drugs, etc., and the concentrations suggested in the present invention The ranges are exemplary only and are not intended to limit the embodiments of the claimed compositions thereto. Also, the active ingredient may be administered once, or smaller doses may be administered in several divided doses.

The precursor compounds, or precursor-linker compounds, precursor-linker-antibody conjugate compounds according to the present invention can be used to treat proliferative diseases, especially cancer diseases. The term "proliferative disorder" refers to unwanted or uncontrolled cell proliferation of undesirable, excessive or abnormal cells, such as neoplastic or hyperplastic growth, either in vitro or in vivo. Proliferative diseases include, for example, neoplasms, tumors, cancers, leukemias, psoriasis, bone diseases, fibroproliferative disorders, atherosclerosis, and the like, and may include, but are not limited to, benign, premalignant or malignant cell proliferation. . The cancer may be lung cancer, small cell lung cancer, gastrointestinal cancer, colorectal cancer, bowel cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, or melanoma. It is not limited.

The antibody-drug conjugate according to the present invention includes an anti-DLK1 antibody that specifically binds to DLK1-expressing cells and is internalized into cells, thereby enabling effective, specific and selective delivery of a drug, and stably binding the drug to the antibody Including a self-killing device that is more stable in plasma and circulated in the body, and can easily release the drug from cancer cells to maximize drug efficacy. By incorporating a linker technology to provide a drug-linker-antibody system in which drugs and/or toxins can stably reach target cells to effectively exert drug effects while greatly reducing toxicity.

1 is a diagram confirming the efficacy of the ADC according to the present invention in a patient-derived tumor animal model.

Hereinafter, the present invention will be described in more detail through Examples and Experimental Examples.

The following Examples and Experimental Examples are intended to aid understanding of the present invention, and are not intended to limit the scope of the present invention thereto.

<Example 1> Preparation of compounds 1, 2, 3 and 4

,

,

,

,

,

,

.

.

The compounds 1, 2, 3 and 4 were prepared by the method described in International Patent Publication WO2017-089895.

The structures of MMAE in the compounds 1, 2, 3 and 4 are as follows.

<Example 2> Preparation of compounds 5, 6, 7 and 8

,

,

,

,

.

.

The compounds 5, 6 and 7 were prepared by the method described in Korean Patent Publication No. 10-2018-0110645.

The compound 8 was prepared by the method described in Korean Application No. 10-2019-0000514.

<Example 3> Preparation of ADC

The ADC was prepared through the following two steps, and the commonly used LCB14-0511 and LCB14-0606 were prepared by the method described in Korean Patent Publication No. 10-2014-0035393. The structural formulas of LCB14-0511 and LCB14-0606 are as follows:

Step 1: Preparation of prenylated antibody

A prenylation reaction mixture of the antibody (18A5) prepared by the method described in Korean Application No. 10-2018-0107639 was prepared and reacted at 30 ° C. for 16 hours. The reaction mixture was a buffer solution (50 mM Tris-HCl (pH 7.4), 5 mM MgCl ₂ , 10 μM ZnCl ₂ , 0.144 mM DTT). After completion of the reaction, the prenylated antibody was desalted with a G25 Sepharose column (AKTA purifier, GE healthcare) equilibrated with PBS buffer.

Step 2: drug-conjugation method

<Conjugation by oxime bond formation>

The oxime bond formation reaction mixture between the prenylated antibody and the linker-drug was 100 mM Na-acetate buffer pH 5.2, 10% DMSO, 20 μM antibody and 200 μM linker-drug (in house, the compound of Examples 1 and 2). 1, 2, 3, 4, 5, 6 and 8) were prepared by mixing and gently stirred at 30 ° C. After 6 hours or 24 hours of reaction, excess low-molecular-weight compounds were removed through FPLC (AKTA purifier, GE healthcare), and protein fractions were collected and concentrated.

<Conjugation by click reaction>

The reaction mixture for generating a click bond between the prenylated antibody and linker-drug is 10% DMSO, 20 μM antibody and 200 μM linker-drug (in house, Compound No. 7 of Example 2), 1 mM copper (II) sulfate pentachloride It was prepared by mixing hydrate, 2 mM (BimC ₄ A) ₃ (Sigma-Aldrich 696854), 10 mM sodium ascorbate and 10 mM aminoguanidine hydrochloride, and treated with 2.0 mM EDTA after reaction at 25 ° C for 3 hours. Reacted for 30 minutes. After completion of the reaction, excess low-molecular-weight compounds were removed through FPLC (AKTA purifier, GE healthcare), and protein fractions were collected and concentrated.

[Table 2] List of ADC manufactures

<Experimental Example 1> In vitro Cytotoxicity assessment

The cytostatic activity of the drugs listed in Table 3 below and the ADC prepared in <Example 3> against cancer cell lines was measured. As cancer cell lines, commercially available human pancreatic cancer cell lines MIA-PaCa2 (DLK1 negative to normal) and MIA-PaCa2-DLK1 overexpressing cell lines were used. As the drug, MMAF-OMe was used, and as the ADC, the ADCs in Table 2 were used. Each cancer cell line was seeded in a 96-well plate at 4,000-5,000 per well for the 72-hour treatment group, 2,000-3,000 per well for the 96-hour treatment group, and 800-1,000 per well for the 168-hour treatment group. After culturing for 24 hours, the drug and ADC were treated at concentrations of 0.0015-10.0 nM (three-fold serial dilution). After 72/96/168 hours, the number of viable cells was quantified using SRB (Sulforhodamine B) dye.

[Table 3]

Comparison of cytotoxicity of ADC samples

*NT: not tested.

It was confirmed that most of the antibody-drug complexes in the pancreatic cancer cell line in which DLK1 was overexpressed exhibited significantly higher cytotoxicity than in the pancreatic cancer cell line in which DLK1 was not expressed. In a cytotoxicity test that reacted at the same time in pancreatic cancer cell lines in which DLK1 was overexpressed, pyrrolobenzodiazepine antibody-drug complexes (ADC5, 7, 8) were more effective than auristatin-based antibody-drug complexes (ADC1, 2, 3, 4). It was confirmed that showed stronger cytotoxicity. In addition, it was confirmed that the cytotoxicity of the pyrrolobenzodiazepine-based antibody-drug conjugates increased as the reaction time increased, compared to the auristatin-based antibody-drug conjugates.

<Experimental Example 2> In vivo Efficacy evaluation

The efficacy of the ADC prepared in <Example 3> was evaluated in a patient-derived tumor animal model. Specifically, a patient-derived small cell lung cancer mouse model (PDX model) was provided by Champions Oncology. PBS as a control group and ADC4 as an experimental group were intravenously administered to the tail of the PDX model under three conditions of 6 mpk/Q4D*4, 6 mpk/QW*4, and 9 mpk/QW*4, respectively, and then the tumor size was measured.

As a result, as shown in FIG. 1, in the 6 mpk/Q4D*4 group and the 9 mpk/QW*4 group, all 8 experimental animals had tumors disappeared (CR, complete remission), and in the 6 mpk/QW*4 group, the experiment It was confirmed that tumors disappeared in 6 out of 8 animals.

<110> legochembioscience ybiologics <120> Antibody-drug conjugate comprising antibody binding to antibody against human DLK1 and its use <130> pb2020-032 <150> KR 10-2019-0025987 <151> 2019-03-06 <160> 128 <170> KoPatentIn 3.0 <210> 1 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 1 Gln Val Gln Leu Val Glu Ser Gly Gly Thr Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 2 Gly Phe Thr Phe Ser Ser His Ala 1 5 <210> 3 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 3 Met Ser Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Val Ser 1 5 10 15 Ser <210> 4 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 4 Ile Thr Lys Ser Gly Ser Gly Thr 1 5 <210> 5 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence nce <400> 5 Tyr Ser Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 1 5 10 15 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 6 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 6 Thr Arg Glu Gly Leu Gly Tyr Tyr Tyr Gly Met Asp Val 1 5 10 <210> 7 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 7 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 1 5 10 <210> 8 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 8 Gln Leu Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Ile Ile Ser Cys Thr Gly Ser 20 25 < 210> 9 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 9 Ser Ser Asn Ile Gly Ala Gly Tyr Asp 1 5 <210> 10 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 10 Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Arg Leu Leu Ile 1 5 10 15 Tyr <210> 11 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 11 Gly Ser Thr 1 <210> 12 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 12 Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly 1 5 10 15 Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln Ala Glu Asp Glu Ala 20 25 30 Asp Tyr Tyr Cys 35 <210> 13 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence < 400> 13 Gln Ser Tyr Asp Asn Ser Leu Ser Ala His Tyr Val 1 5 10 <210> 14 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 14 Phe Gly Thr Gly Thr Lys Val Thr Val Leu 1 5 10 <210> 15 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 15 Gln Val Gln Leu Val Gln Ser Gly Gly Ala Val Val Gln Pro Gly His 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ala Se r 20 25 <210> 16 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 16 Gly Phe Lys Phe Lys Asp Tyr Gly 1 5 <210> 17 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 17 Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu Ala 1 5 10 15 Val <210> 18 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 18 Ile Ser His Asp Gly Arg Asn Lys 1 5 <210> 19 <211> 38 <212> PRT <213> Artificial Sequence <220 > <223> Artificial Sequence <400> 19 Asn Tyr Ala Asp Ser Val Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn 1 5 10 15 Ser Lys Asn Thr Leu Ser Phe Gln Met Asn Ser Leu Arg Ala Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 20 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 20 Val Arg Asp Trp Ser Tyr Ala Phe Asp Ile 1 5 10 <210 > 21 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial S sequence <400> 21 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 22 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 22 Asp Ile Gln Met Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Asn Ile Thr Cys Arg Ala Ser 20 25 <210> 23 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 23 Gln Asp Ile Ser Arg Arg 1 5 <210> 24 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 24 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 1 5 10 15 Tyr <210> 25 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 25 Gly Ala Ala 1 <210> 26 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 26 Ser Leu Gln Ser Ala Val Ala Ser Arg Phe Ser Gly Ser Gly Ser Gly 1 5 10 15 Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala 20 25 30 As n Tyr Tyr Cys 35 <210> 27 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 27 Gln Gln Ile Tyr Thr Thr Pro Leu Thr 1 5 <210> 28 < 211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 28 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 1 5 10 <210> 29 <211> 25 <212> PRT < 213> Artificial Sequence <220> <223> Artificial Sequence <400> 29 Gln Met Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 30 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 30 Gly Phe Thr Phe Asp Asp Tyr Ala 1 5 <210> 31 <211> 17 <212> PRT <213 > Artificial Sequence <220> <223> Artificial Sequence <400> 31 Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 1 5 10 15 Gly <210> 32 <211> 8 <212> PRT <213 > Artificial Sequence <220> <223> Artificial Sequence <400> 32 Ile Ser Trp Asn Ser Gly Ser I le 1 5 <210> 33 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 33 Gly Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 1 5 10 15 Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 34 <211> 15 <212> PRT <213> Artificial Sequence <220> <223 > Artificial Sequence <400> 34 Thr Lys Gly Pro Gly Leu Ala Thr Gly Lys Val Tyr Phe Asn Ser 1 5 10 15 <210> 35 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 35 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 36 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 36 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 20 25 <210> 37 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 37 Gln Arg Ile Ser Ser Trp 1 5 <210> 38 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 38 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Arg Ala Pro Lys Leu Leu Ile 1 5 10 15 His <210> 39 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 39 Ser Ala Ser 1 <210> 40 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 40 Thr Leu His Asn Gly Val Pro Ser Arg Phe Ser Gly Ser Ala Ser Gly 1 5 10 15 Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala 20 25 30 Ile Tyr Tyr Cys 35 <210> 41 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 41 Gln Gln Gly His Ser Phe Pro Tyr Thr 1 5 <210> 42 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 42 Phe Gly Gln Gly Thr Lys Leu Asp Ile Lys 1 5 10 <210> 43 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 43 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 44 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 44 Gly Phe Thr Phe Ser Ser Tyr Trp 1 5 <210> 45 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 45 Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Val Trp Val Ser 1 5 10 15 Arg <210> 46 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 46 Ile Ser Pro Asp Gly Ser Ser Thr 1 5 <210> 47 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 47 Thr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 1 5 10 15 Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 48 <211> 15 <212> PRT <213> Artificial Sequence <220> <223 > Artificial Sequence <400> 48 Ala Arg Gly Tyr Ser Pro Lys Tyr Pro Thr Val Gly Leu Asp Val 1 5 10 15 <210> 49 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 49 Trp Gly Gln Gly Thr Thr Ile Thr Val Ser Ser 1 5 10 <210> 50 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 50 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Ser Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser 20 25 <210> 51 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 51 Glu Ser Leu Leu His Ser Asn Gly Asn Thr Tyr 1 5 10 <210> 52 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 52 Leu Thr Trp Leu Gln Gln Arg Pro Gly Gln Pro Pro Arg Leu Leu Ile 1 5 10 15 His <210> 53 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 53 Lys Ile Ser 1 <210> 54 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 54 Asn Arg Phe Ser G ly Val Pro Asp Arg Phe Ser Gly Ser Gly Ala Gly 1 5 10 15 Thr Asp Phe Thr Leu Gln Ile Thr Arg Val Glu Thr Glu Asp Val Gly 20 25 30 Val Tyr Tyr Cys 35 <210> 55 <211> 9 <212 > PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 55 Val Gln Thr Thr Gln Trp Pro Trp Thr 1 5 <210> 56 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 56 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 1 5 10 <210> 57 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 57 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Arg Val Ser Cys Lys Val Ser 20 25 <210> 58 <211> 8 <212> PRT <213> Artificial Sequence < 220> <223> Artificial Sequence <400> 58 Gly Tyr Ser Leu Ser Glu Phe Pro 1 5 <210> 59 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 59 Ile His Trp Val Arg Gln Ala Pro Arg Met Gly Leu Glu Trp Met Gly 1 5 10 15 Gly <210> 60 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 60 Ser Tyr Pro Glu Asp Gly Glu Thr 1 5 <210> 61 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 61 Leu Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Glu Asp Thr 1 5 10 15 Ser Thr Asp Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 62 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 62 Ala Arg Leu Asn Tyr Phe Glu Ser Thr Asp Tyr Trp Val Asp Ala Phe 1 5 10 15 Asp Ile <210> 63 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 63 Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 1 5 10 <210> 64 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 64 Gln Leu Val Leu Thr Gln Pro Tyr Ser Val Ser Glu Ser Pro Gly Lys 1 5 10 15 Thr Ile Thr Ile Ser Cys Thr Arg Ser 20 25 <210> 65 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 65 Ser Gly Ser Ile Ala Ser Asn Phe 1 5 <210> 66 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 66 Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Pro Val Ile 1 5 10 15 Tyr <210> 67 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 67 Glu Asp Asn 1 <210> 68 <211> 38 <212> PRT <213> Artificial Sequence <220> < 223> Artificial Sequence <400> 68 Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Ile Asp Ser 1 5 10 15 Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly Val Met Thr Glu Asp 20 25 30 Glu Ala Asp Tyr Tyr Cys 35 <210> 69 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 69 Gln Ser Tyr Asp Ser Gly Ser Ser Trp Val 1 5 10 <210> 70 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 70 P he Gly Gly Gly Thr Lys Leu Thr Val Leu 1 5 10 <210> 71 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 71 Gln Met Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Thr Leu Ser Cys Asp Ala Thr 20 25 <210> 72 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400 > 72 Gly Phe Asn Phe Gly Ser Tyr Tyr 1 5 <210> 73 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 73 Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu Ala 1 5 10 15 His <210> 74 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 74 Ile Ser Ser Thr Gly Arg Thr Ile 1 5 <210> 75 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 75 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 1 5 10 15 Ala Lys Ser Ser Leu Asp Leu Gln Met Asn Ser Leu Arg Ala G lu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 76 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 76 Ala Arg Asp Gln Gly Tyr Pro Phe Gly Met Asp Val 1 5 10 <210> 77 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 77 Trp Gly His Gly Thr Thr Thr Val Thr Val Ser Ser 1 5 10 < 210> 78 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 78 Gln Leu Val Leu Thr Gln Pro Ser Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser 20 25 <210> 79 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 79 Ser Ser Asn Ile Gly Ala Gly Tyr Asp 1 5 < 210> 80 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 80 Val Asp Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile 1 5 10 15 Tyr < 210> 81 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Artifici al Sequence <400> 81 Gly Asn Thr 1 <210> 82 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 82 Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly 1 5 10 15 Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln Ala Glu Asp Asp Ser 20 25 30 Asp Tyr Tyr Cys 35 <210> 83 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 83 Gln Ser Tyr Asp Ser Ser Leu Ser Ala Trp Val 1 5 10 <210> 84 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 84 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 1 5 10 <210> 85 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 85 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 86 <211> 8 <212> PRT <213> Artificial Sequence <220> <223 > Artificial Sequence <400> 86 Gly Phe Thr Phe Ser Ser Tyr Ala 1 5 <210> 87 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 87 Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala 1 5 10 15 Val <210> 88 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 88 Ile Tyr Ser Gly Gly Ser Thr 1 5 <210> 89 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 89 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 1 5 10 15 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 90 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 90 Ala Arg Glu Gly Ser Tyr Asp Val Met Thr Tyr Thr Arg Ile Gly Gly 1 5 10 15 Tyr Phe Asp Tyr 20 <210> 91 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 91 Trp Gly Gln Gly Ala Leu Val Thr Val Ser Ser 1 5 10 <210> 92 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 92 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 20 25 <210> 93 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Artifi cial Sequence <400> 93 Gln Gly Ile Ser Asp Trp 1 5 <210> 94 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 94 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 1 5 10 15 Tyr <210> 95 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 95 Ala Ala Ser 1 <210> 96 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 96 Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 1 5 10 15 Thr Glu Phe Ser Leu Thr Ile Ser Asn Leu Gln Pro Glu Asp Phe Ala 20 25 30 Thr Tyr Tyr Cys 35 <210> 97 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 97 Gln Gln Ala Asn Ser Phe Pro Leu Thr 1 5 <210> 98 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 98 Phe Gly Pro Gly Thr Lys Val Glu Ile Lys 1 5 10 <210> 99 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 99 Gln Val Gln Leu Val Glu Ser Gly Gly Thr Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser His 20 25 30 Ala Met Ser Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Thr Lys Ser Gly Ser Gly Thr Tyr Ser Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Glu Gly Leu Gly Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln 100 105 110 Gly Thr Thr Thr Val Thr Val Ser Ser 115 120 <210> 100 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 100 Gln Leu Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Ile Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Arg Leu 35 40 45 Leu Ile Tyr G ly Ser Thr Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Asn Ser 85 90 95 Leu Ser Ala His Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu 100 105 110 <210> 101 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence < 400 > 101 Gln Val Gln Leu Val Gln Ser Gly Gly Ala Val Val Gln Pro Gly His 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Lys Phe Lys Asp Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Ala Val Ile Ser His Asp Gly Arg Asn Lys Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Ser 65 70 75 80 Phe Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Asp Trp Ser Tyr Ala Phe Asp Ile Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 1 15 <210> 102 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 102 Asp Ile Gln Met Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Asn Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Arg Arg 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ala Ser Leu Gln Ser Ala Val Ala Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Asn Tyr Tyr Cys Gln Gln Ile Tyr Thr Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 103 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 103 Gln Met Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Lys Gly Pro Gly Leu Ala Thr Gly Lys Val Tyr Phe Asn Ser Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 104 <211> 107 <212> PRT <213> Artificial Sequence <220> <223 > Artificial Sequence <400> 104 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Arg Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Arg Ala Pro Lys Leu Leu Ile 35 40 45 His Ser Ala Ser Thr Leu His Asn Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Ala Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Ile Tyr Tyr Cys Gln Gln Gly His Ser Phe Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Asp Ile Lys 100 105 <210> 105 <211> 122 <212> P RT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 105 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Val Trp Val 35 40 45 Ser Arg Ile Ser Pro Asp Gly Ser Ser Thr Thr Thr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Ser Pro Lys Tyr Pro Thr Val Gly Leu Asp Val Trp 100 105 110 Gly Thr Thr Ile Thr Val Ser Ser 115 120 <210> 106 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 106 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Ser Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Glu Ser Leu Leu His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Thr Trp Leu Gln Gl n Arg Pro Gly Gln Pro 35 40 45 Pro Arg Leu Leu Ile His Lys Ile Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Gln Ile 65 70 75 80 Thr Arg Val Glu Thr Glu Asp Val Gly Val Tyr Tyr Cys Val Gln Thr 85 90 95 Thr Gln Trp Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 107 <211> 125 <212> PRT <213 > Artificial Sequence <220> <223> Artificial Sequence <400> 107 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Arg Val Ser Cys Lys Val Ser Gly Tyr Ser Leu Ser Glu Phe 20 25 30 Pro Ile His Trp Val Arg Gln Ala Pro Arg Met Gly Leu Glu Trp Met 35 40 45 Gly Gly Ser Tyr Pro Glu Asp Gly Glu Thr Leu Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Glu Asp Thr Ser Thr Asp Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Asn Tyr Phe Glu Ser Thr Asp Tyr Trp Val Asp Ala Phe 100 1 05 110 Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 125 <210> 108 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 108 Gln Leu Val Leu Thr Gln Pro Tyr Ser Val Ser Glu Ser Pro Gly Lys 1 5 10 15 Thr Ile Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Ile Ala Ser Asn 20 25 30 Phe Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Pro Val 35 40 45 Ile Tyr Glu Asp Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly 65 70 75 80 Val Met Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser 85 90 95 Ser Ser Ser Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 109 <211> 119 <212> PRT <213> Artificial Sequence <220 > <223> Artificial Sequence <400> 109 Gln Met Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Thr Leu Ser Cys Asp Ala Thr G ly Phe Asn Phe Gly Ser Tyr 20 25 30 Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Ala His Ile Ser Ser Thr Gly Arg Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Ser Leu Asp 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gln Gly Tyr Pro Phe Gly Met Asp Val Trp Gly His Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 110 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 110 Gln Leu Val Leu Thr Gln Pro Ser Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Asp Val Asp Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Gly Asn Thr Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu 65 70 75 80 Gln Ala Glu Asp Asp Ser Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser 85 90 95 Leu Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 111 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 111 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Glu Gly Ser Tyr Asp Val Met Thr Tyr Thr Arg Ile Gly Gly Tyr 100 105 110 Phe Asp Tyr Trp Gly Gln Gly Ala Leu Val Thr Val Ser Ser 115 120 125 <210> 112 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 112 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asp Trp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Ser Leu Thr Ile Ser Asn Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Leu 85 90 95 Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys 100 105 <210> 113 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 113 agggggccgt gggggccgct gaatgcttcc cggcctgc 38 <210> 114 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 114 tagcggccga cgcggccaac tggccctcgg tgaggagagg <1 1 11502> > 6 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 115 Gln Gly Ile Ser Ser Ala 1 5 <210> 116 <211> 9 <212> PRT <213> Artificial Sequence <220 ><22 3> Artificial Sequence <400> 116 Gln Gln Ser Tyr Thr Thr Thr Pro Leu Thr 1 5 <210> 117 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 117 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 20 25 <210> 118 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 118 Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 1 5 10 15 Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Gln Pro Glu Asp Phe Ala 20 25 30 Thr Tyr Tyr Cys 35 <210> 119 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 119 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 120 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 120 Asp Ile Gln Met Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 20 25 <210> 121 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 121 His Asp Ile Ser Ser Ser Ser 1 5 <210> 122 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 122 Leu Ala Trp Tyr Gln Gln Lys Ser Gly Lys Ala Pro Lys Leu Leu Ile 1 5 10 15 Tyr <210> 123 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 123 Asn Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 1 5 10 15 Thr Asp Phe Ser Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala 20 25 30 Thr Tyr Tyr Cys 35 <210> 124 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence < 400> 124 Gln Gln Ser Thr Thr Thr Val Leu Thr 1 5 <210> 125 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 125 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 1 5 10 <210> 126 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 126 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 127 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 127 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Lys Phe Lys Asp Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Ala Val Ile Ser His Asp Gly Arg Asn Lys Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Ser 65 70 75 80 Phe Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Asp Trp Ser Tyr Ala Phe Asp Ile Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 128 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 128 Asp Ile Gln Met Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser His Asp Ile Ser Ser Ser Ser 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Ser Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Asn Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Thr Val Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105

Claims (59)

An antibody conjugate of general formula I or a pharmaceutically acceptable salt thereof:
[Formula I]
Ab - (A) _y
In the above formula,
Ab is an antibody or antigen-binding fragment thereof that specifically binds to DLK1 (delta-like 1 homolog (Drosophila)) containing a heavy chain variable region and a light chain variable region;
The antibody or antigen-binding fragment thereof that specifically binds to DLK1 includes a heavy chain variable region and a light chain variable region of any one of the following (a) to (i);
(a) a heavy chain CDR1 of SEQ ID NO: 2, a heavy chain CDR2 of SEQ ID NO: 4, a heavy chain variable region comprising heavy chain CDR3 of SEQ ID NO: 6, a light chain CDR1 of SEQ ID NO: 9, a light chain CDR2 of SEQ ID NO: 11, and a light chain CDR3 of SEQ ID NO: 13 A light chain variable region comprising;
(b) heavy chain CDR1 of SEQ ID NO: 16, heavy chain CDR2 of SEQ ID NO: 18, heavy chain variable region comprising heavy chain CDR3 of SEQ ID NO: 20, light chain CDR1 of SEQ ID NO: 23, light chain CDR2 of SEQ ID NO: 25, and light chain CDR3 of SEQ ID NO: 27 A light chain variable region comprising;
(c) a heavy chain variable region comprising the heavy chain CDR1 of SEQ ID NO: 30, the heavy chain CDR2 of SEQ ID NO: 32, and the heavy chain CDR3 of SEQ ID NO: 34, the light chain CDR1 of SEQ ID NO: 37, the light chain CDR2 of SEQ ID NO: 39, and the light chain CDR3 of SEQ ID NO: 41 A light chain variable region comprising;
(d) heavy chain CDR1 of SEQ ID NO: 44, heavy chain CDR2 of SEQ ID NO: 46, heavy chain variable region comprising heavy chain CDR3 of SEQ ID NO: 48, light chain CDR1 of SEQ ID NO: 51, light chain CDR2 of SEQ ID NO: 53, and light chain CDR3 of SEQ ID NO: 55 A light chain variable region comprising;
(e) a heavy chain CDR1 of SEQ ID NO: 58, a heavy chain CDR2 of SEQ ID NO: 60, a heavy chain variable region comprising heavy chain CDR3 of SEQ ID NO: 62, a light chain CDR1 of SEQ ID NO: 65, a light chain CDR2 of SEQ ID NO: 67, and a light chain CDR3 of SEQ ID NO: 69 A light chain variable region comprising;
(f) a heavy chain CDR1 of SEQ ID NO: 72, a heavy chain CDR2 of SEQ ID NO: 74, a heavy chain variable region comprising heavy chain CDR3 of SEQ ID NO: 76, a light chain CDR1 of SEQ ID NO: 79, a light chain CDR2 of SEQ ID NO: 81, and a light chain CDR3 of SEQ ID NO: 83 A light chain variable region comprising;
(g) heavy chain CDR1 of SEQ ID NO: 86, heavy chain CDR2 of SEQ ID NO: 88, heavy chain variable region comprising heavy chain CDR3 of SEQ ID NO: 90, light chain CDR1 of SEQ ID NO: 93, light chain CDR2 of SEQ ID NO: 95, and light chain CDR3 of SEQ ID NO: 97 A light chain variable region comprising;
(h) heavy chain CDR1 of SEQ ID NO: 16, heavy chain CDR2 of SEQ ID NO: 18, heavy chain variable region comprising heavy chain CDR3 of SEQ ID NO: 20, light chain CDR1 of SEQ ID NO: 115, light chain CDR2 of SEQ ID NO: 95, and light chain CDR3 of SEQ ID NO: 116 A light chain variable region comprising; or
(i) heavy chain CDR1 of SEQ ID NO: 16, heavy chain CDR2 of SEQ ID NO: 18, heavy chain variable region comprising heavy chain CDR3 of SEQ ID NO: 20, light chain CDR1 of SEQ ID NO: 121, light chain CDR2 of SEQ ID NO: 39, and light chain CDR3 of SEQ ID NO: 124 A light chain variable region comprising;
A is a chemical residue each independently including at least one active agent and a linker, wherein the linker has a self-immolative group of Formula II below at a linking site with each active agent,
[Formula II]

in the above formula
G is sugar, sugar acid, or sugar derivatives, and W is -C(O)-, -C(O)NR'-, -C(O)O-, -S (O)2NR'-, -P(O)R''NR'-, -S(O)NR'-, or -PO2NR'-, wherein C(O), S, or P is a phenyl ring ( phenyl ring), and R' and R'' are each independently hydrogen, C _1-8 alkyl, C _3-8 cycloalkyl, C _1-8 alkoxy, C _1-8 alkylthio, C _1-8 alkylamino, C _3-20 heteroaryl, or C _6-20 aryl;
each Z is independently C _1-8 alkyl, halogen, cyano or nitro;
n is an integer from 0 to 3;
m is 0 or 1;
R1 and R2 are each independently hydrogen, C _1-8 alkyl or C _3-8 cycloalkyl, or R1 and R2 together with the carbon atoms to which they are attached form a C _3-8 cycloalkyl ring;
In the above formula, the wave sign indicates a site connected to the rest of the linker, and the * sign indicates a site linked to the active agent;
Said y is an integer from 1 to 20.
The method of claim 1, wherein the Ab is
at least one heavy chain FR1 selected from the group consisting of SEQ ID NOs: 1, 15, 29, 43, 57, 71, 85 and 119;
at least one heavy chain FR2 selected from the group consisting of SEQ ID NOs: 3, 17, 31, 45, 59, 73 and 87;
at least one heavy chain FR3 selected from the group consisting of SEQ ID NOs: 5, 19, 33, 47, 61, 75 and 89;
at least one heavy chain FR4 selected from the group consisting of SEQ ID NOs: 7, 21, 35, 49, 63, 77 and 91;
at least one light chain FR1 selected from the group consisting of SEQ ID NOs: 8, 22, 36, 50, 64, 78, 92, 117 and 120;
at least one light chain FR2 selected from the group consisting of SEQ ID NOs: 10, 24, 38, 52, 66, 80, 94 and 122;
at least one light chain FR3 selected from the group consisting of SEQ ID NOs: 12, 26, 40, 54, 68, 82, 96, 118 and 123; or
An antibody conjugate comprising at least one light chain FR4 selected from the group consisting of SEQ ID NOs: 14, 28, 42, 56, 70, 84, 98 and 125, or a pharmaceutically acceptable salt thereof.
The antibody of claim 1, wherein the Ab comprises a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NOs: 101, 109, and 127 or a sequence having 90% or more sequence homology thereto. A conjugate or a pharmaceutically acceptable salt thereof.
The method of claim 1, wherein the Ab comprises a light chain variable region comprising a sequence selected from the group consisting of SEQ ID NOs: 102, 110, 126 and 128 or a sequence having 90% or more sequence homology thereto. An antibody conjugate or a pharmaceutically acceptable salt thereof.
According to claim 1, wherein A is an antibody conjugate having the structure of the following general formula II or a pharmaceutically acceptable salt thereof;
[Formula Ⅱ]

In the above formula,
G is a glucuronic acid moiety,

)ego,
wherein R ³ is hydrogen or a carboxyl protecting group, R ⁴ are each independently hydrogen or a hydroxyl protecting group;
Ab binds to the tilde;
B is an active agent;
R ¹ and R ² are each independently hydrogen, C ₁ -C ₈ alkyl or C ₃ -C ₈ cycloalkyl;
W is -C(O)-, -C(O)NR'-, -C(O)O-, -SO ₂ NR'-, -P(O)R''NR'-, -SONR'- or -PO ₂ NR'-, wherein C, S or P is directly bonded to a phenyl ring, N is bonded to L, and R' and R'' are each independently hydrogen; C ₁ -C ₈ alkyl, C ₃ -C ₈ cycloalkyl, C ₁ -C ₈ alkoxy, C ₁ -C ₈ alkylthio, C ₁ -C ₈ alkylamino, C ₃ -C ₂₀ heteroaryl or C ₆ -C ₂₀ aryl;
each Z is independently C ₁ -C ₈ alkyl, halogen, cyano or nitro;
n is an integer from 0 to 3;
L comprises at least one of the following A) and B):
A) C ₁ -C ₅₀ alkylene or 1-50 membered heteroalkylene, and satisfies one or more of the following conditions:
(i) L contains one or more unsaturated bonds;
(ii) two atoms in L are substituted with a divalent substituent, which completes the heteroarylene;
(iii) the alkylene _is substituted with one or more C _1-20 alkyl;
B) An isoprenyl derivative unit of the general formula III which can be recognized by an isoprenoid transferase.
[Formula Ⅲ]

.
delete delete delete delete delete delete delete The antibody conjugate according to claim 1 or 5, wherein the linker in A or L is covalently bonded to Ab via a thioether bond, and the thioether bond includes a sulfur atom of a cysteine of Ab. A pharmaceutically acceptable salt.
14. The method of claim 13, wherein the Ab contains an amino acid motif recognized by isoprenoid transferase at the C-terminus of the Ab, and the thioether bond includes a sulfur atom of a cysteine of the amino acid motif. An antibody conjugate to or a pharmaceutically acceptable salt thereof.
15. The method of claim 14, wherein the amino acid motif is a CYYX sequence,
where C is cysteine,
Y is an aliphatic amino acid selected from the group consisting of alanine, isoleucine, leucine, methionine and valine;
An antibody conjugate or a pharmaceutically acceptable salt thereof, characterized in that X is an amino acid selected from the group consisting of glutamine, glutamate, serine, cysteine, methionine, alanine and leucine.
The antibody conjugate according to claim 15, wherein the amino acid motif is a CVIM (cysteine-valine-isoleucine-methionine) or CVLL (cysteine-valine-leucine-leucine) sequence, or a pharmaceutically acceptable salt thereof.
The antibody conjugate according to claim 14, wherein at least one of 1 to 10 amino acids preceding the amino acid motif is selected from the group consisting of glycine, proline, aspartic acid, arginine and serine, or a pharmaceutically acceptable antibody thereof. salt.
The antibody conjugate according to claim 17, wherein each of 1 to 10 amino acids preceding the amino acid motif is glycine (G), or a pharmaceutically acceptable salt thereof.
The antibody conjugate according to claim 18, wherein the Ab comprises the amino acid sequence GGGGGGGCVIM at the C-terminus, or a pharmaceutically acceptable salt thereof.
6. The antibody conjugate according to claim 1 or 5, wherein the linker in A or L comprises at least one isoprenyl derivative unit of the following general formula III that can be recognized by isoprenoid transferase, or Pharmaceutically acceptable salts thereof:
[Formula Ⅲ]

.
The method according to claim 1 or 5, wherein the linker in A or L is a 3 to 50 membered heteroalkylene containing oxime,
The oxygen atom of the oxime is on the side of the linker connected to W or L, and the carbon atom of the oxime is on the side of the linker connected to Ab or L; or
The carbon atom of the oxime is on the side of the linker linked to W or L, and the oxygen atom of the oxime is on the side of the linker or L linked to Ab. An antibody conjugate or a pharmaceutically acceptable salt thereof.
22. The antibody conjugate or pharmaceutically acceptable salt thereof according to claim 21, characterized in that at least one isoprenyl unit covalently binds the oxime to Ab.
According to claim 1 or 5, wherein the linker of A or L is

or

An antibody conjugate or a pharmaceutically acceptable salt thereof comprising a.
delete According to claim 1 or 5, wherein the linker of A or L is

or

An antibody conjugate comprising 1 to 12 polyethylene glycol units represented by or a pharmaceutically acceptable salt thereof.
delete 26. The antibody conjugate according to claim 25, wherein the linker in A or L comprises an oxime, and 1 to 20 polyethylene glycol units covalently bond the oxime to the active agent, or a pharmaceutically acceptable salt thereof.
delete delete The antibody conjugate according to claim 1 or 5, wherein the linker or L in A further comprises a binding unit represented by the following general formula IV, V, VI or VII, or a pharmaceutically acceptable salt thereof. :
[Formula IV]

,
[Formula V]

,
[Formula VI]

, and
[Formula VII]

In the above formula,
L ¹ is a single bond or C ₁ -C ₃₀ alkylene;
R ¹¹ is hydrogen or C ₁ -C ₁₀ alkyl.
31. The antibody conjugate according to claim 30, wherein L ¹ is a single bond or C ₁₁ or C ₁₂ alkylene, or a pharmaceutically acceptable salt thereof.
31. The antibody conjugate or pharmaceutically acceptable salt thereof according to claim 30, wherein 1 to 20 polyethylene glycol units covalently link L ¹ of the binding unit to the active agent, and L ¹ is a direct bond.
According to claim 5,

Any one selected from the group consisting of
An antibody conjugate or a pharmaceutically acceptable salt thereof, characterized in that n is an integer from 0 to 20.
The antibody conjugate according to claim 20, wherein the isoprenoid transferase is farnesyl protein transferase (FTase) or geranylgeranyl transferase (GGTase), or a pharmaceutically acceptable salt thereof.
The method of claim 1 or 5, wherein the linker in A or L comprises one or more branched linking groups,
The branched connector is

,

, or

ego,
Wherein L ² , L ³ , L ⁴ are each independently a direct bond or -(CH ₂ ) _n -, where n is an integer from 1 to 30;
G ¹ , G ² , G ³ are each independently directly bonded;

,

,

or

is,
In the above formula, R ³⁰ is hydrogen or C _1-30 alkyl;
R ⁴⁰ is hydrogen or L ₅ -COOR ₆ ;
wherein L ₅ is a direct bond or -(CH ₂ ) _n -;
Here, n is an integer from 1 to 10, R ₆ is hydrogen or C _1-30 alkyl,
An antibody conjugate or a pharmaceutically acceptable salt thereof, characterized in that 1 to 6 identical or different active agents are covalently linked to one or more branches of the branched linking group.
delete 36. The antibody conjugate according to claim 35 comprising the following structural formula or a pharmaceutically acceptable salt thereof:

wherein B and B' represent active agents which may be the same or different;
two n and f each independently represent an integer from 0 to 30;
Combined with Ab on the side of the wavy line.
delete delete delete delete delete delete delete The antibody conjugate according to claim 1 or 5, wherein the active agent is a chemotherapeutic agent or a toxin, or a pharmaceutically acceptable salt thereof.
The antibody conjugate or pharmaceutically acceptable salt thereof according to claim 1 or 5, wherein the active agent is an immunomodulatory compound, an anticancer agent, an antiviral agent, an antibacterial agent, an antifungal agent, an antiparasitic agent, or a combination thereof.
delete According to claim 1 or 5,
The active agent is a pyrrolobenzodiazepine dimer;
The antibody conjugate or a pharmaceutically acceptable salt thereof, characterized in that the linker connects Ab to the N10 position of the pyrrolobenzodiazepine dimer.
delete delete delete delete delete delete 49. The antibody conjugate according to claim 48 having the structure of Formula XII or Formula XIII, or a pharmaceutically acceptable salt thereof:
[Formula XII]

,
[Formula XIII]

,
In the above formula,
The dotted line indicates that a double bond may exist between C1 and C2, between C2 and C3, between C1' and C2', or between C2' and C3';
R ^X1 and R ^X1' are each independently H, OH, =O, =CH ₂ , CN, R ^m , OR ^m , =CH-R ^m' =C(R ^m' ) ₂ , O-SO ₂ -R ^m , CO ₂ R ^m , COR ^m , halo and dihalo;
R ^X2 _, R ^X2' R ^X3 , and R ^X3' are each independently selected from H, R ^m , OH, OR ^m , NR ^m ₂ , NO ₂ , and halo;
R ^X4 and R ^X4' are each independently selected from H, R ^m , OH, OR ^m , SH, SR ^m , NH ₂ , NHR ^m , NR ^m ₂ , halo, and C _1-6 alkyl;
R ^X5 , and R ^X5' are each independently selected from H, R ^m , OH, OR ^m , SH, SR ^m , NH ₂ , NHR ^m , NR ^m ₂ , NO ₂ , and halo;
Y and Y' are each independently selected from O, S, and N(H);
R ^X6 is independently selected from C _3-12 alkylene, C _3-12 alkenylene, or C _3-12 heteroalkylene, and R ^X6 is -NH ₂ , -NHR ^m , -NHC(O)R ^m , -NHC(O)CH ₂ -[OCH ₂ CH ₂ ] _n -R ^XX , or -[CH ₂ CH ₂ O] _n -R ^XX can be;
Wherein R ^XX is H, OH, N ₃ , CN, NO ₂ , SH, NH ₂ , ONH ₂ , NHNH ₂ , halo, C _1-8 alkyl, C _{3-8 cycloalkyl, C 1-8 alkoxy} , C _{1 -8} alkylthio, C _3-20 heteroaryl, C _5-20 aryl or C _1-8 alkylamino, where n is an integer from 1 to 6;
R ^X7 and R ^X7' are each independently H, C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, -C(O)R ^r , -C(O)OR ^s or -C (O)NR ^r R ^r' ;
R ^r , R ^r' , and R ^s are each independently H, C _1-7 alkyl, C _2-7 alkenyl, C _2-7 alkynyl, C _3-13 cycloalkyl, 3 to 7-membered heterocyclo selected from alkyl, C _5-10 aryl, and 5 to 7-membered heteroaryl;
R ^X8 And R ^X8' are each independently H, C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, -C(O)R ^m , -C(O)OR ^m and -C(O ) NR ^m R ^{m '} ,
R ^m' is selected from R ^m , CO ₂ R ^m , COR ^m , CHO, CO ₂ H, and halo;
R ^m are each independently H, C _1-12 alkyl, C _2-12 alkenyl, C _2-12 alkynyl, C _5-20 aryl, C _5-20 heteroaryl, C _3-6 cycloalkyl, 3 to 6 7-membered heterocyclyl, 3 to 7-membered heterocycloalkyl, and 5 to 7-membered heteroaryl;
Z ^a is selected from OR ^X12a , NR ^X12a R ^X12a , or SR ^X12a ;
Z ^b is selected from OR ^X13a , NR ^X13a R ^X13a , or SR ^X13a ;
Z ^a' is selected from OR ^X12a' , NR ^X12a' R ^X12a' , or SR ^X12a' ;
Z ^b' is OR ^X13a' , NR ^X13a' R ^X13a' , or SR ^X13a' ;
R ^X12a , R ^X12a', R ^X13a and R ^X13a' are each independently H, C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _3-6 cycloalkyl, 3 to 7- is selected from membered heterocycloalkyl, C _5-10 aryl, 5 to 7-membered heteroaryl, -C(O)R ^X15a , -C(O)OR ^X15a and -C(O)NR ^X15a R ^X15a' ;
Here, R ^X15a and R ^X15a' are each independently C _1-12 alkyl, C _2-12 alkenyl, C _2-12 alkynyl, C _5-20 aryl, C _5-20 heteroaryl, C _3-6 cyclo selected from alkyl, 3 to 7 membered heterocyclyl, 3 to 7 membered heterocycloalkyl, and 5 to 7 membered heteroaryl;
or R ^X12a and R ^X13a are taken together with the atoms to which they are attached to form a 3 to 7-membered heterocyclyl, a 3 to 7-membered heterocycloalkyl, or a 3 to 7-membered heteroaryl, and R ^X12a' and R ^X13a' are optionally taken together with the atoms to which they are attached to form a 3 to 7-membered heterocyclyl, a 3 to 7-membered heterocycloalkyl, or a 3 to 7-membered heteroaryl;
X and X' are -C(O)O-*, where * indicates the point of attachment to the linker side;
X ^a and X ^a' are each methylene;
Z ^X' and Z ^X are each independently C _1-8 alkyl, halogen, cyano, nitro,

, or

is selected from;
Each of R ⁸⁰ , R ⁹⁰ and R ¹⁰⁰ is selected from hydrogen, C _1-8 alkyl, C _2-6 alkenyl, C _1-6 alkoxy, or -(CH ₂ ) _m -OCH ₃ , where m is An integer from 1 to 12,
The G and G' are each independently a glucuronic acid moiety,

), wherein R ³ is hydrogen or a carboxyl protecting group, R ⁴ are each independently hydrogen or a hydroxyl protecting group;
W is -C(O)-, -C(O)NR'-, -C(O)O-, -SO ₂ NR'-, -P(O)R''NR'-, -SONR'- or -PO ₂ NR'-, wherein C, S or P is directly bonded to a phenyl ring, N is bonded to L, and R' and R'' are each independently hydrogen, C _{1 -8} alkyl, C _3-8 cycloalkyl, C _1-8 alkoxy, C _1-8 alkylthio, C _1-8 alkylamino, C _3-20 heteroaryl or C _6-20 aryl;
L is as defined in claim 5;
n is an integer from 0 to 3;
Ab is the same as the definition of claim 1.
The antibody conjugate according to claim 1 or 5, characterized in that it is prepared from any one of the following compounds, or a pharmaceutically acceptable salt thereof:

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

and

.
A pharmaceutical composition for preventing or treating hyperproliferation, cancer or angiogenic disease comprising the antibody conjugate of claim 1.
58. The pharmaceutical composition of claim 57, further comprising a pharmaceutically effective amount of a chemotherapeutic agent.
58. The method of claim 57, wherein the cancer is lung cancer, small cell lung cancer, gastrointestinal cancer, colorectal cancer, bowel cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma And a pharmaceutical composition, characterized in that any one selected from the group consisting of melanoma.

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