Antibody-drug conjugates including antibody against human DLK1, and use thereof

Abstract

To provide an antibody-drug conjugate comprising an antibody binding to DLK1 or an antigen-binding fragment thereof, and a pharmaceutical composition comprising the same.SOLUTION: Provided is an antibody-drug conjugate, or a pharmaceutically acceptable salt or solvate thereof, comprising an antibody or an antigen-binding fragment thereof, specifically binding to DLK1 (Delta-like 1 homolog (Drosophila)), and comprising a heavy chain variable region and a light chain variable region having CDR1, CDR2, and CDR3 consisting of specific amino acid sequences.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to novel antibody drug conjugates (ADCs) targeting DLK1, active metabolites of such ADCs, methods for preparing such ADCs, the use of such ADCs in the treatment and / or prevention of diseases, and the use of such ADCs in the manufacture of a medicament for the treatment and / or prevention of diseases, more particularly the use of such ADCs in the manufacture of a medicament for the treatment and / or prevention of proliferative and / or angiogenic diseases, such as cancer. More particularly, the present invention relates to antibody drug conjugates comprising an antibody or antigen-binding fragment thereof that binds to DLK1, and pharmaceutical compositions comprising same. [Background technology]

[0002] Cancer is a disease caused by uncontrolled cell proliferation in various tissues, resulting in an abnormal mass of uncontrolled cell growth in the tissues of the body. Early stage cancer tumors can be removed by surgery or radiation therapy, while metastatic tumors are generally treated palliatively using chemotherapy.

[0003] Many chemotherapy agents administered parenterally may induce undesirable side effects and severe toxicity as a result of systemic administration. Therefore, there is a growing interest in the development of novel chemotherapy agents that can improve efficacy and minimize toxicity and side effects by selectively acting on tumor cells and nearby tissues.

[0004] Antibody-drug conjugates (ADCs) are a new targeting technology in which toxins or drugs are attached to antibodies, which then bind to antigens and release the toxins or drugs into tumor cells, causing cell death. This technology specifically delivers drugs to targeted cancer cells while minimizing the effects on healthy cells, and releases the drugs only under specific conditions, making it more effective than antibody drugs and significantly reducing the risk of side effects compared to conventional anticancer drugs.

[0005] The basic structure of an antibody-drug conjugate is "antibody-linker-small molecule drug (toxin)". Here, the linker not only plays a functional role of linking the antibody and the drug, but also needs to ensure that the drug is properly released by dissociation of the antibody-drug (e.g., as a result of enzymatic hydrolysis) after circulating in the body and reaching the target cells, and exerts its effectiveness against the target cancer cells. In other words, the stability of the linker plays a very important role in the efficacy and systemic toxicity of the antibody-drug conjugate (Discovery Medicine 2010, 10(53): 329-39).

[0006] The present inventors have developed and patented a linker containing an effective self-immolative group. This linker is more stable in plasma and circulation, and can easily release drugs and exert its effectiveness in cancer cells (e.g., Korean Patent No. 1,628,872). Meanwhile, cancer treatment using monoclonal antibodies has also achieved great success. Monoclonal antibodies are suitable for targeting tumor tissues and tumor cells. Antibody-drug conjugates have become a novel and powerful option in the treatment of lymphomas and solid tumors, and recently, immune-regulating antibodies have achieved great success in clinical trials. The development of therapeutic antibodies is based on a deep understanding of cancer serology, protein engineering technology, mechanisms of action and resistance, and the interactions between the immune system and cancer cells.

[0007] Antigens expressed on the surface of human cancer cells have been defined as a wide range of targets, including those that are overexpressed, mutated, or selectively expressed compared to normal tissues. A key issue is identifying antigens suitable for antibody-based therapy. These therapeutics mediate changes in antigen or receptor function (i.e., as stimulators or antagonists), modulate the immune system through Fc and T cell activation, and exert their efficacy by delivery of specific drugs that bind to antibodies targeting specific antigens. Molecular techniques that can alter the pharmacokinetics, function, size, and immune stimulation of antibodies have emerged as key elements in developing new antibody-based therapies. Evidence from clinical trials of therapeutic antibodies in cancer patients highlights the importance of approaches to select optimal antibodies, including the affinity and binding of the antibody to the target antigen, the choice of antibody structure, and the therapeutic approach (blocking signaling or immune function).

[0008] Related research is underway on antibodies whose antigen is DLK1. Human DLK1 (Delta-like 1 homolog (Drosophila)) is a single-pass transmembrane protein containing a total length of 383 amino acids. This protein has six EGF (epidermal growth factor-like repeat) domains in the extracellular domain.

[0009] DLK1 is commonly referred to as the gene DLK1 due to its amino acid sequence homology with Delta, a ligand of the Notch receptor, a cell differentiation regulator, and is also known as Pref-1, pG2, SCP-1, and ZOG. DLK1 is a transmembrane protein, but it has been well reported that the extracellular domain is shed from the cell membrane by TACE (tumor necrosis factor alpha converting enzyme) and is a protein with separate functions.

[0010] DLK1 is highly expressed in undifferentiated fetal cells with high proliferation potential, particularly in the fetal liver, kidney, skeletal muscle, and brain. However, after birth, its expression is not confirmed in most tissues, and it is expressed only in specific cells such as preadipocytes, pancreatic islet cells, thymic stromal cells, and adrenal cells.

[0011] DLK1 has been most widely studied as a factor that inhibits adipocyte differentiation, Pref-1 (Preadipocyte factor-1). In addition to its ability to inhibit adipocyte differentiation, DLK1 has been reported to inhibit hematopoietic stem cell differentiation, regulate lymphatic progenitor cell differentiation, and be involved in wound healing.

[0012] DLK1 has also been reported to be frequently expressed in various cancers and tumors. To date, DLK1 expression has been confirmed in solid cancers such as neuroendocrine tumors, neuroblastomas, gliomas, type 1 neurofibromatosis, small cell lung cancer, liver cancer, kidney cancer, ovarian cancer, colorectal cancer, breast cancer, and pancreatic cancer, and in blood cancers such as myelodysplastic syndrome and acute myeloid leukemia. nIn studies on the relationship between DLK1 and cancer, it has been reported that DLK1 is overexpressed in brain cancer cells (gliomas), and that overexpressing DLK1 cDNA in brain cancer cells increases the proliferation and migration of brain cancer cells, and that the expression of DLK1 in liver cancer is elevated compared to normal liver cells, and that tumor size decreases when the expression of DLK1 is reduced by siRNA experiments.

[0013] Under such technical background, the inventors of the present invention have made efforts to develop an antibody that specifically binds to DLK1, and as a result, have developed an anti-DLK1 antibody that exhibits excellent binding ability to DLK1. In order to further enhance the effect of the antibody, the inventors have confirmed that it is possible to provide a novel antibody-drug conjugate (ADC) that targets DLK1 and is effective in treating and / or preventing cancer diseases by applying a linker containing an effective self-immolative group to the anti-DLK1 antibody, which is more stable in plasma and circulation and enables the drug to be easily released and effective in cancer cells, and have completed the present invention. Summary of the Invention [Problem to be solved by the invention]

[0014] An object of the present invention is to provide a novel antibody-drug conjugate, or a salt or solvent thereof, that targets DLK1.

[0015] Another object of the present invention is to provide a drug-antibody conjugate comprising an antibody that specifically binds to DLK1 and a drug that binds to the antibody, and a pharmaceutical composition comprising the same.

[0016] Another object of the present invention is to provide a method for preventing or treating a proliferative disease, cancer or angiogenic disease, comprising the step of administering to an individual a pharmacologic effective amount of an antibody that specifically binds to DLK1 and a drug that binds thereto.

[0017] Another object of the present invention is to provide the use of an antibody-drug conjugate comprising a pharma- ceutical effective amount of an antibody that specifically binds to DLK1 and a drug that binds thereto, as a pharmaceutical composition for preventing or treating a proliferative disease, cancer or angiogenic disease.

[0018] Yet another object of the present invention is to provide an antibody-linker-drug (toxin) system that allows drugs and / or toxins to safely reach target cells and effectively exert their efficacy while substantially reducing toxicity by a grafting technique of a linker containing a self-immolative group that is highly stable in plasma and circulation, and that easily releases the drug in cancer cells to maximize its efficacy. [Means for solving the problem]

[0019] One aspect of the invention relates to an antibody-conjugate of general formula I, or a pharma- ceutically acceptable salt or solvate thereof: [General formula I] Ab-(X)y [In formula: Ab is an anti-DLK1 antibody or an antigen-binding fragment thereof; X is independently a chemical moiety comprising at least one active agent and a linker; The linker connects the antibody and the active agent, y is an integer between 1 and 20. to provide.

[0020] The anti-DLK1 antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof that binds to DLK1, and comprises a heavy chain variable region comprising at least one heavy chain CDR1 selected from the group including SEQ ID NOs: 2, 16, 30, 44, 58, 72, and 86, at least one heavy chain CDR2 selected from the group including SEQ ID NOs: 4, 18, 32, 46, 60, 74, and 88, and at least one heavy chain CDR3 selected from the group including SEQ ID NOs: 6, 20, 34, 48, 62, 76, and 90; and a light chain variable region comprising at least one light chain CDR1 selected from the group comprising SEQ ID NOs: 9, 23, 37, 51, 65, 79, 93, 115, and 121, at least one light chain CDR2 selected from the group comprising SEQ ID NOs: 11, 25, 39, 53, 67, 81, and 95, and at least one light chain CDR3 selected from the group comprising SEQ ID NOs: 13, 27, 41, 55, 69, 83, 97, 116, and 125.

[0021] The antibody according to the present invention can specifically bind to, for example, the extracellular domain of human DLK1.

[0022] The term "antibody" as used herein refers to 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 the complete antibody form that specifically binds to DLK1, but also the antigen-binding fragment of the antibody molecule.

[0023] Intact antibodies have a structure with two full-length heavy chains and two full-length light chains, with each light chain linked to a heavy chain by a disulfide bond. Heavy chain constant regions include gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types, with gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2) subclasses. Light chain constant regions include kappa (κ) and lambda (λ) types.

[0024] Antigen-binding fragment of an antibody or antibody fragment means 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 light and heavy chain variable regions, a light chain constant region, and the first constant region (CH1) of the heavy chain, and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing at least one cysteine ​​residue at the C-terminus of the heavy chain CH1 domain. F(ab')2 antibodies are generated when the cysteine ​​residues in the hinge region of Fab' form a disulfide bond. Fv is the smallest antibody fragment having only a heavy chain variable region and a light chain variable region, and recombinant techniques for preparing Fv fragments are disclosed in PCT International Publication Nos. WO88 / 10649, WO88 / 106630, WO88 / 07085, WO88 / 07086, and WO88 / 09344. In a two-chain Fv, the heavy chain variable region and the light chain variable region are linked by a non-covalent bond, and in a single-chain Fv (scFv), the heavy chain variable region and the light chain variable region are generally linked by a covalent bond via a peptide linker or directly at the C-terminus, so that they can form a dimer-like structure like a two-chain Fv. Such antibody fragments can be obtained using proteolytic enzymes (for example, Fab can be obtained by restrictive cleavage of a whole antibody with papain, and F(ab')2 fragments can be obtained by cleavage with pepsin), or can be produced using genetic recombination techniques.

[0025] In an embodiment, the antibody according to the invention is in Fv format (e.g., scFv) or in full antibody format. Furthermore, the heavy chain constant region can be of any one of the isotypes selected from gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε). For example, the constant region is gamma 1 (IgG1), gamma 3 (IgG3) or gamma 4 (IgG4). The light chain constant region can be of kappa or lambda type.

[0026] The term "heavy chain" as used herein refers to a full-length heavy chain comprising a variable region domain VH and three constant region domains CH1, CH2, and CH3, each of which contains an amino acid sequence having sufficient variable region sequence to confer antigen specificity, and any of its fragments. The term "light chain" as used herein refers to a full-length light chain comprising a variable region domain VL and a constant region domain CL, each of which contains an amino acid sequence having sufficient variable region sequence to confer antigen specificity, and any of its fragments.

[0027] Antibodies of the present invention include, but are not limited to, 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 these antibodies.

[0028] A monoclonal antibody refers to an antibody obtained from a substantially homogeneous antibody population, i.e., an antibody obtained from a substantially homogeneous antibody population, except possibly for naturally occurring mutations that may be present in minor amounts among the individual antibodies populating the population. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Unlike conventional (polyclonal) antibody preparations that contain different antibodies against different determinants, each monoclonal antibody is directed against a single determinant on the antigen. For example, monoclonal antibodies useful in the present invention can be produced by the hybridoma method or can be produced using recombinant DNA methods in bacteria, eukaryotic animal cells or plant cells (see U.S. Pat. No. 4,816,567). Additionally, monoclonal antibodies can be isolated from phage antibody libraries.

[0029] In one embodiment of the present invention, a phage display method was used to pan a naive human single-chain Fv library (naive human scFv library) to prepare seven monoclonal human antibodies that specifically bind to DLK1.

[0030] "Phage display" is a technique in which mutant polypeptides are displayed on the surface of phages, e.g., phage particles, as fusion proteins with at least a portion of a coat protein. The utility of phage display is that it allows rapid and efficient sorting of large libraries of randomized protein mutants for sequences that bind with high affinity to a target antigen. Displaying peptide and protein libraries on phage has been used to screen millions of polypeptides to identify those with specific binding properties.

[0031] Phage display technology has provided a powerful tool for generating and screening novel proteins that bind to a specific ligand (e.g., antigen). 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. Nucleic acids encoding mutant polypeptides are fused to nucleic acid sequences encoding viral coat proteins, such as gene III protein or gene VIII protein. Monovalent phage display systems have been developed in which a nucleic acid sequence encoding a protein or polypeptide is fused to a nucleic acid sequence encoding a portion of the gene III protein. In monovalent phage display systems, the gene fusion is expressed at low levels and the wild-type gene III protein is also expressed to maintain particle infectivity.

[0032] Demonstrating expression of peptides on the surface of filamentous phages and expression of functional antibody fragments in the peripheral cytoplasm of E. coli is key in developing an antibody phage display library. Libraries of antibodies or antigen-binding polypeptides have been produced in a number of ways, for example, by altering a single gene by inserting random DNA sequences or by cloning a series of related genes. Libraries can be screened for expression of antibodies or antigen-binding proteins with the desired characteristics.

[0033] Phage display technology has several advantages over conventional hybridoma and recombinant methods for producing antibodies with desired characteristics. Such technology allows the generation of large antibody libraries with diverse sequences in a short time without the use of animals. The production of hybridomas and humanized antibodies can take several months of production time. In addition, since no immunization is required, phage antibody libraries can generate antibodies against antigens that are toxic or have low antigenicity. Furthermore, phage antibody libraries can be used to generate and identify novel therapeutic antibodies.

[0034] Phage display libraries can be used to generate human antibodies from immunized, non-immunized humans, germline sequence, or naive B cell Ig repertoires. Various lymphoid tissues can be used to generate undetected or non-immunogenic antigen-binding libraries.

[0035] The technique of identifying and isolating high affinity antibodies from phage display libraries is important for isolating novel antibodies for therapeutic use. Isolating high affinity antibodies from libraries can 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 improper folding of the antibody or antigen binding protein and inefficient production due to the presence of stop codons. Expression in bacterial cells can be suppressed if the antibody or antigen binding domain is not folded properly. Expression can be improved by mutating alternating residues at the variable / constant interface surface and selected CDR residues. The sequence of the framework region is one element to provide proper folding when generating antibody phage libraries in bacterial cells.

[0036] In high affinity antibody isolation, it is important to generate a diverse library of antibodies or antigen binding proteins. It has been found that CDR3 regions often participate in antigen binding. The CDR3 regions on the heavy chain are highly diverse in size, sequence and structural conformation, so that diverse libraries can be produced using the CDR3 regions.

[0037] 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, which may generate a greater variety of variant antibody sequences and increase the chances of identifying novel antibodies.

[0038] "Epitope" refers to a protein determinant to which an antibody can specifically bind. Epitopes are usually composed of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and generally have specific three-dimensional structural features as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.

[0039] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (acceptor antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate that retains the desired specificity, affinity, and capacity.

[0040] The above-mentioned "human antibody" refers to a molecule derived from human immunoglobulin, and all of the amino acid sequences constituting the antibody, including the complementarity determining regions and structural regions, are composed of human immunoglobulin.

[0041] The other chains, e.g., portions of the heavy and / or light chains, are identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remaining chains are identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, including "chimeric" antibodies (immunoglobulins), as well as fragments of such antibodies that exhibit the desired biological activity.

[0042] As used herein, "antibody variable domain" refers to the light and heavy chain portions of an antibody molecule that contain the amino acid sequences of the complementarity determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) and framework regions (FRs). VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain.

[0043] "Complementarity determining region" (CDR; i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of an antibody variable domain which are necessary for antigen binding. Each variable domain typically has three CDR regions identified as CDR1, CDR2, and CDR3.

[0044] 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, two types of anti-DLK1 antibodies (18A5, 27F7) and two other types of 18A5 mutant antibodies (18A5_LS_1A10 and 18A5_AM_1A12) can bind to cells in which DLK1 is overexpressed, and it has been confirmed in Korean Patent Application No. 10-2018-0107639 that they can be developed as anti-DLK1 antibody-drug conjugates that can target DLK1 expressed on the surface of cancer cells to cause apoptosis of cancer cells.

[0045] [Table 1] TIFF2025076435000003.tif213154TIFF2025076435000004.tif209154TIFF2025076435000005.tif125155

[0046] "Framework regions" (FR) are those variable domain residues other than the CDR residues. Each variable domain typically has four FRs identified as FR1, FR2, FR3 and FR4.

[0047] In an embodiment of the invention, at least one heavy chain FR1 selected from the group comprising SEQ ID NOs: 1, 15, 29, 43, 57, 71, 85 and 119, at least one heavy chain FR2 selected from the group comprising SEQ ID NOs: 3, 17, 31, 45, 59, 73 and 87, at least one heavy chain FR3 selected from the group comprising SEQ ID NOs: 5, 19, 33, 47, 61, 75 and 89, at least one heavy chain FR4 selected from the group comprising SEQ ID NOs: 7, 21, 35, 49, 63, 77 and 91, at least one heavy chain FR5 selected from the group comprising SEQ ID NOs: 8, 22, 35, 49, 63, 77 and 91, at least one heavy chain FR6 selected from the group comprising SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 75 and 89, at least one heavy chain FR7 selected from the group comprising SEQ ID NOs: 10, 11, 12, at least one light chain FR1 selected from the group including SEQ ID NOs: 6, 50, 64, 78, 92, 117, and 120; at least one light chain FR2 selected from the group including SEQ ID NOs: 10, 24, 38, 52, 66, 80, 94, and 122; at least one light chain FR3 selected from the group including SEQ ID NOs: 12, 26, 40, 54, 68, 82, 96, 118, and 123; and at least one light chain FR4 selected from the group including SEQ ID NOs: 14, 28, 42, 56, 70, 84, 98, and 125.

[0048] An "Fv" fragment is an antibody fragment that contains a complete antigen recognition and binding site. Such a region consists of a dimer of one heavy- and one light-chain variable domain in tight, substantially covalent association, e.g., as an scFv.

[0049] The "Fab" fragment contains the variable and constant domains of the light chain and the variable and first constant domain (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.

[0050] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of antibody, but these domains are present in a single polypeptide chain. The Fv polypeptide can further comprise a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.

[0051] The antibodies of the present invention may be monovalent or divalent and include single chain or double chain antibodies.

[0052] Functionally, the binding affinity of the antibody to the extracellular domain of DLK1 is 10 -5 M~10 -12 For example, the binding affinity is in the range of 10 -6 M~10 -12 M, 10 -7 M~10 -12 M, 10 -8 M~10 -12 M, 10 -9 M~10 -12 M, 10 -5 M~10 -11 M, 10 -6 M~10 -11 M, 10 -7 M~10 -11 M, 10 -8 M~10 -11 M, 10 -9 M~10 -11 M, 10 -10 M~10 -11 M, 10 -5 M~10 -10 M, 10 -6 M~10 -10 M, 10 -7 M~10 -10 M, 10 -8 M~10 -10 M, 10 -9 M~10 -10 M, 10 -5 M~10 -9 M, 10 -6 M~10 -9 M, 10 -7 M~10 -9 M, 10 -8 M~10 -9 M, 10 -5 M~10-8 M, 10 -6 M~10 -8 M, 10 -7 M~10 -8 M, 10 -5 M~10 -7 M, 10 -6 M~10 -7 M or 10 -5 M~10 -6 It's M.

[0053] The antibody according to the present invention is also an antibody with increased affinity for the antigen. The term "affinity" refers to the ability to specifically recognize and bind to a specific site of an antigen. The high affinity as well as specificity of these antibodies are important factors in immune reactions. 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 indicated by the dissociation constant (Kd) of a particular antibody-antigen interaction. A lower Kd value means a higher affinity of the antibody for the antigen. For example, the Kd value of the antibody according to the present invention is 0.52 for the 18A5 antibody and 0.22 for the 27F7 antibody. This indicates that these are high affinity antibodies that specifically bind to human DLK1.

[0054] An antibody or antigen-binding fragment thereof that binds to the extracellular domain of DLK1 can comprise a heavy chain variable region comprising a sequence having at least 90% sequence identity to a sequence selected from the group including SEQ ID NOs: 99, 101, 103, 105, 107, 109, 111, and 127. An antibody or antigen-binding fragment thereof that binds to the extracellular domain of DLK1 can comprise a heavy chain variable region selected from the group including SEQ ID NOs: 99, 101, 103, 105, 107, 109, 111, and 127.

[0055] In addition, an antibody or antigen-binding fragment thereof that binds to the extracellular domain of DLK1 can comprise a light chain variable region comprising a sequence having at least 90% sequence identity with a sequence selected from the group including SEQ ID NOs: 100, 102, 104, 106, 108, 110, 112, 126, and 128. An antibody or antigen-binding fragment thereof that binds to the extracellular domain of DLK1 can comprise a light chain variable region selected from the group including SEQ ID NOs: 100, 102, 104, 106, 108, 110, 112, 126, and 128.

[0056] 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 its biological equivalents, so long as it specifically recognizes DLK1. For example, the amino acid sequence of the antibody may be further altered to further improve the binding affinity and / or other biological properties of the antibody. Such alterations include, for example, deletion, insertion and / or substitution of residues in the amino acid sequence of the antibody. Such amino acid mutations are made based on the relative similarity of the amino acid side chain substitutes, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape and type of amino acid side chain substitutes reveals that arginine, lysine and histidine are all positively charged residues; alanine, glycine and serine have similar sizes; and phenylalanine, tryptophan and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine are biologically functional equivalents.

[0057] Considering the above-mentioned biologically equivalent mutations, the antibody of the present invention or the nucleic acid molecule encoding the same is also understood to include sequences that show substantial identity to the sequences described in SEQ ID NO. The above-mentioned substantial identity means a sequence that shows at least 90% homology, most preferably at least 95% homology, at least 96%, at least 97%, at least 98%, or at least 99% homology when the above-mentioned sequence of the present invention is aligned with any other sequence so as to maximize its correspondence and the aligned sequences are analyzed using an algorithm commonly used in the art. Alignment methods for sequence comparison are well known in the art. The NCBI Basic Local Alignment Search Tool (BLAST) is accessible at NCBI and can be used in conjunction with sequence analysis programs such as blastp, blasm, blastx, tblastn, and tblastx on the Internet. BLAST can be accessed at www.ncbi.nlm.nih.gov / BLAST / . A sequence homology comparison method using this program is as follows: www.ncbi.nlm.nih.gov / BLAST / blast_help.html It can be confirmed from.

[0058] On this basis, the antibodies or antigen-binding fragments thereof of the present invention can have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the sequences explicitly set forth herein or to the entirety thereof. Such homology can be determined by sequence comparison and / or alignment by methods known in the art. For example, percent sequence homology of the nucleic acids or proteins of the present invention can be determined using sequence comparison algorithms (i.e., BLAST or BLAST 2.0), manual alignment, or visual inspection.

[0059] In another aspect, the invention relates to a nucleic acid encoding the antibody or antigen-binding fragment thereof.

[0060] Nucleic acids encoding the antibodies or antigen-binding fragments thereof of the invention can be isolated to recombinantly produce the antibodies or antigen-binding fragments thereof. The nucleic acids are isolated and inserted into replicable vectors for further cloning (amplification of the DNA) or for further expression. Based on this, the present invention in yet another aspect relates to vectors comprising the nucleic acids.

[0061] The term "nucleic acid" encompasses DNA (gDNA and cDNA) and RNA molecules, and the basic building block of a nucleic acid includes not only natural nucleotides but also analogs in which the sugar or base moiety is modified. The sequences of the nucleic acids encoding the heavy and light chain variable regions of the present invention can be modified. Such modifications include addition, deletion, or non-conservative or conservative substitution of nucleotides.

[0062] DNA encoding the antibody is readily isolated or synthesized using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to DNA encoding the heavy and light chains of the antibody). Many vectors are available. The vector components generally include, but are not limited to, any 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.

[0063] The term "vector" as used herein refers to a means for expressing a gene of interest in a host cell, and includes plasmid vectors, cosmid vectors, bacteriophage vectors, adenovirus vectors, retrovirus vectors, adeno-associated virus vectors, and other viral vectors. In the vector, the nucleic acid encoding the antibody is operably linked to a promoter.

[0064] "Operably linked" refers to the functional association of a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or an array of transcriptional regulator binding sites) with another nucleic acid sequence such that the control sequence controls the transcription and / or translation of the other nucleic acid sequence.

[0065] When a prokaryotic cell is used as the host, it generally contains a strong promoter capable of driving 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, T7 promoter, etc.), a ribosome binding site for initiating translation, and a transcription / translation termination sequence. Furthermore, for example, when a eukaryotic cell is used as the host, a promoter derived from the genome of a mammalian cell (e.g., metallothionine promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter), or a mammalian promoter derived from a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Rous sarcoma virus (RSV) promoter) can be used, which generally has a polyadenylation sequence as a transcription termination sequence.

[0066] In some cases, the vector may be fused to other sequences to facilitate purification of the antibodies expressed therefrom, such as glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6xHis (hexahistidine; Qiagen, USA).

[0067] Such vectors contain antibiotic resistance genes commonly used in the art as selectable markers, such as genes for resistance to ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline.

[0068] In another aspect, the present invention relates to a cell transformed with the above mentioned vector. The cells used to produce the antibodies of the present invention can be, but are not limited to, prokaryotic, yeast or higher eukaryotic cells.

[0069] Prokaryotic host cells such as strains of Bacillus, such as Escherichia coli, Bacillus subtilis, and Bacillus thuringiensis, Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis, and Staphylococcus (e.g., Staphylococcus carnosus) can be used.

[0070] However, of greatest interest are animal cells, and examples of useful host cell lines can be, but are not limited to, 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, MRC5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S, or HT1080.

[0071] In yet another aspect, the present invention relates to a method for producing an antibody or antigen-binding fragment thereof, comprising the steps of: (a) culturing cells; and (b) recovering the antibody or antigen-binding fragment thereof from the cultured cells.

[0072] The cells can be cultured in various media. Commercially available media can be used as the culture medium without limitation. Any other necessary supplements known to those skilled in the art may be included at appropriate concentrations. The culture conditions, such as temperature, pH, etc., are already used with the host cell selected for expression and will be apparent to those skilled in the art.

[0073] In recovering the antibody or antigen-binding fragment thereof, impurities can be removed, for example, by centrifugation or ultrafiltration, and the resultant product can be purified, for example, by affinity chromatography. Additional purification techniques, such as anion or cation exchange chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, etc., can also be used.

[0074] In one aspect of the invention, the linker between the antibody and the active agent may be cleavable.

[0075] In one embodiment of the invention, the linker has the following formula IIa:

[0076] [ka] [In the formula, G is a sugar, sugar acid or sugar derivative; W is -C(O)-, -C(O)NR'-, -C(O)O-, -S(O)NR'-, -P(O)R''NR'-, -S(O)NR'- or -PONR'-, and when C(O), S or P is directly connected to a phenyl ring, R' and R'' are each independently hydrogen, (C1-C8) alkyl, (C3-C8) cycloalkyl, (C1-C8) alkoxy, (C1-C8) alkylthio, mono- or di-(C1-C8) alkylamino, (C3-C 20 ) Heteroaryl or (C6-C 20 ) aryl, each Z is independently (C1-C8) alkyl, halogen, cyano, or nitro; n is an integer from 0 to 3, m is 0 or 1; L is not present, or Contains at least one branching unit (BR) and at least one connector unit, R1 and R2 are each independently hydrogen, (C1-C8) alkyl, or (C3-C8) cycloalkyl, or R1 and R2 together with the carbon atom to which they are attached form a (C3-C8) cycloalkyl ring; and In the above formula, "~" represents the region that binds to the antibody, and * represents the region that binds to the drug or toxin. It has the structure shown below.

[0077] In one aspect of the invention, the sugar or sugar acid is a monosaccharide.

[0078] In one aspect of the invention, G is a glucuronic acid moiety or a group represented by the following formula (IIIa):

[0079] [ka] [In the formula, R3 is hydrogen or a carboxyl protecting group; each R4 is independently hydrogen or a hydroxyl protecting group. It is a compound having the structure shown below.

[0080] In one embodiment of the invention, R3 is hydrogen and each R4 is hydrogen.

[0081] Further R1 and R2 are each hydrogen.

[0082] Further, each Z is independently (C1-C8) alkyl, halogen, cyano, or nitro.

[0083] Further, W is -C(O)-, -C(O)NR'-, or -C(O)O-. More particularly, W is -C(O)NR'-, where C(O) is linked to the phenyl ring and NR' is linked to L.

[0084] Further, n is 0, 1, 2 or 3, specifically 0, 1 or 2, and more specifically 0.

[0085] More specifically, G is represented by the following formula (IIIa):

[0086] [ka] [In the formula, R3 is hydrogen or a carboxyl protecting group; each R4 is independently hydrogen or a hydroxyl protecting group; W is -C(O)NR'-, where C(O) is linked to a phenyl ring and NR' is linked to L, each Z is (C1-C8) alkyl, halogen, cyano or nitro, n is 0, m is 1, and R1 and R2 are each hydrogen. It is a compound having the structure shown below.

[0087] In one aspect of the invention, at least one branching unit is an alkylene having 1 to 100 carbon atoms, the carbon atoms of which are optionally substituted with one or more heteroatoms selected from the group consisting of N, O, and S, and the alkylene is optionally further substituted with one or more alkyls having 1 to 20 carbon atoms.

[0088] Specifically, at least one branching unit is C1-C 50 alkylene or heteroalkylene of 1 to 50 atoms, (i) the branching units contain at least one unsaturated bond; (ii) two atoms in the branching unit are identical to those in the substituents, thereby completing a heteroarylene; (iii) the branching unit is a heteroalkylene of 1 to 50 atoms; (iv) Alkylene has at least one C1- 20 Being substituted by alkyl At least one of the following may be satisfied.

[0089] Additionally, at least one branching unit is a heteroalkylene of 1 to 50 atoms containing nitrogen, and the linker contains at least two atoms of a hydrophilic amino acid, and the nitrogen can form a peptide bond with a carbonyl of the hydrophilic amino acid.

[0090] In one aspect of the invention, at least one branching unit is a hydrophilic amino acid.

[0091] In one aspect of the invention, the hydrophilic amino acid can be arginine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, lysine, ornithine, proline, serine, or threonine.

[0092] Additionally, a hydrophilic amino acid may covalently attach the oxime of the linker to the polyethylene glycol unit of the linker.

[0093] In one aspect of the invention, a hydrophilic amino acid can be an amino acid that contains a side chain that has a moiety that carries a charge in aqueous solution at neutral pH.

[0094] In one aspect of the invention, the hydrophilic amino acid is aspartic acid or glutamic acid.

[0095] In one aspect of the invention, the hydrophilic amino acid is ornithine or lysine.

[0096] In one aspect of the invention, the hydrophilic amino acid is arginine.

[0097] In one embodiment of the invention, at least one branch unit is -C(O)-, -C(O)NR'-, -C(O)O-, -S(O)NR'-, -P(O)R''NR'-, -S(O)NR'- or -PONR'-, where R' and R'' are each independently hydrogen, (C1-C8) alkyl, (C3-C8) cycloalkyl, (C1-C8) alkoxy, (C1-C8) alkylthio, mono- or di-(C1-C8) alkylamino, (C3-C8) alkoxy, (C1-C8) alkylthio, mono- or di-(C1-C8) alkylamino, (C3-C8) alkoxy, (C3-C8) alkylthio, mono- or di-(C1-C8) alkylamino, (C3-C8) alkoxy, (C3-C8) alkylthio, mono- or di-(C3 ... 20 ) Heteroaryl or (C6-C 20 ) aryl.

[0098] In one embodiment of the invention, at least one branching unit is -C(O)NR'-, where R' is hydrogen.

[0099] In one embodiment of the invention, at least one connecting unit has general formula VIII or general formula IX: [General formula VIII] -(CH2) r (V(CH2) p ) q - [General formula IX] -(CH2CH2X) w - [In the formula, V is a single bond, -O-, -S-, -NR 21 -, -C(O)NR 22 -, NR 23 C(O)-, NR 24 SO2- or -SO2NR 25 - and X is -O-, C1-C8 alkylene or -NR 21 - and R 21 ~R 25 are each independently hydrogen, (C1-C6) alkyl, (C1-C6) alkyl(C6-C 20 )aryl or (C1-C6)alkyl(C3-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; w is an integer from 1 to 20. It is represented by:

[0100] In one embodiment of the present invention, q can be 4 to 20, more specifically, 6 to 20. Furthermore, q can be 2 to 12, more specifically, 2, 5 or 11. Furthermore, it can be 2. Furthermore, p can be 2. Furthermore, V can be -O-.

[0101] More specifically, r can be 2, p can be 2, q can be 2, 5 or 11, and V can be --O--.

[0102] In a further embodiment of the present invention, X may be -O-. Furthermore, w may be an integer of 6 to 20.

[0103] More specifically, X can be -O-, and w can be 6-20.

[0104] In one embodiment of the present invention, at least one connection unit comprises at least one

[0105] [ka] [In the formula, n is 1 to 12] The polyethylene glycol unit is represented by:

[0106] In one embodiment of the present invention, at least one connecting unit can be 1 to 12 -OCH2CH2- units, 3 to 12 -OCH2CH2- units, 5 to 12 -OCH2CH2- units, 6 to 12 -OCH2CH2- units or 3 -OCH2CH2- units.

[0107] In one embodiment of the invention, at least one connecting unit is -(CH2CH2X) w - and X is a single bond, -O-, (C1-C8) alkylene or -NR 21 - and R 21 is hydrogen, (C1-C6) alkyl, (C1-C6) alkyl(C6-C 20 )aryl or (C1-C6)alkyl(C3-C 20 ) heteroaryl; and w is an integer of 0 to 20, specifically, 1, 3, 6, or 12.

[0108] In one embodiment of the present invention, X is —O— and w is an integer of 6-20.

[0109] In one aspect of the invention, the linker further comprises a linking unit formed by a 1,3-dipolar cycloaddition reaction, a heteroDiels reaction, a nucleophilic substitution reaction, a non-aldol type carbonyl reaction, an addition to a carbon-carbon multiple bond, an oxidation reaction, or a Click reaction.

[0110] In one aspect of the invention, the linking units are formed by reaction between an acetylene and an azide or between an aldehyde or ketone group and a hydrazine or an alkoxyamine.

[0111] In one aspect of the invention, the linking unit is

[0112] [ka] [In the formula, L1 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. It is.

[0113] In one embodiment of the invention, L1 is a single bond, an alkylene having 11 carbon atoms, or an alkylene having 12 carbon atoms.

[0114] Further, in one aspect of the invention, the linking unit is

[0115] [ka] [V is a single bond, -O-, -S-, -NR 21 -, -C(O)NR 22 -, NR 23 C(O)-, NR 24 SO2- or -SO2NR 25 - and R 21 ~R 25 are each independently hydrogen, C1-C6 alkyl, C1-C6 alkylC6-C 20 Aryl or C1-C6 alkyl C3-C 20is 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; L 1 is a single bond] Includes.

[0116] In one embodiment of the invention, r can be 2 or 3. Additionally, p can be 1 or 2. Additionally, q can be 1 to 6.

[0117] More specifically, in the linking unit, r can be 2 or 3, p can be 1 or 2, and q can be 1 to 6.

[0118] In a further aspect of the invention, the linking unit is

[0119] [ka] [wherein Ab is an anti-DLK1 antibody, B is an active agent, and n is an integer from 0 to 20] It is.

[0120] In one aspect of the invention, the branching unit is

[0121] [ka] It is.

[0122] In one embodiment of the invention, the linker may include 3-50 heteroalkylenes, including oximes, the oxygen atom of the oxime is on the side of L that is linked to W and the carbon atom of the oxime is on the side of L that is linked to Ab, or The carbon atom of the oxime is on the side of L that is linked to W, and the oxygen atom of the oxime is on the side of L that is linked to Ab.

[0123] In one aspect of the invention, the linker has the structure

[0124] [ka] wherein n is at least 2. The amino acid sequence may further include at least one isoprenyl unit having the formula:

[0125] In one aspect of the invention, at least one isoprenyl unit is an isoprenoid transferase substrate or a product of an isoprenoid transferase.

[0126] In one aspect of the invention, the isoprenyl unit of the linker is covalently attached to the antibody by a thioether bond, the thioether bond involving the sulfur atom of a cysteine.

[0127] Additionally, the isoprenyl unit may covalently attach an oxime contained in the linker to the antibody.

[0128] In one embodiment of the invention, the antibody comprises an amino acid motif recognized by an isoprenoid transferase and the thioether bond involves the sulfur atom of a cysteine ​​of the amino acid motif.

[0129] In one embodiment of the invention, the antibody binding to DLK1 or an antigen-binding fragment thereof comprises an amino acid motif recognized by an isoprenoid transferase and the thioether bond involves the sulfur atom of a cysteine ​​of the amino acid motif.

[0130] 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, where C represents cysteine, Y at each occurrence independently represents an aliphatic amino acid, and X at each occurrence independently represents glutamine, glutamic acid, serine, cysteine, methionine, alanine or leucine, and the thioether bond involves the sulfur atom of the cysteine ​​of the amino acid motif.

[0131] In one aspect of the invention, the amino acid motif is the sequence CYYX, where Y in each occurrence is independently alanine, isoleucine, leucine, methionine, or valine.

[0132] In one embodiment of the invention, the amino acid motif is a CVIM or CVLL sequence.

[0133] In one aspect of the invention, at least one of the 1 to 10 amino acids preceding the amino acid motif may be independently selected from glycine, arginine, aspartic acid, and serine. For example, in one aspect of the invention, at least one of the 7 amino acids preceding the amino acid motif is glycine. Alternatively, at least three of the 7 amino acids preceding the amino acid motif are independently selected from glycine, arginine, aspartic acid, and serine. Alternatively, the 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.

[0134] In one aspect of the invention, the antibody may comprise the amino acid sequence GGGGGGCVIM.

[0135] In one aspect of the invention, L comprises at least one branched linker covalently attached to Ab, i) each branched linker comprises a branching unit covalently linked to an Ab by a primary linker (PL); ii) each branched linker comprises a first branch (B1) that connects a first active agent to a branching unit and comprises a second linker (SL) and a cleavage group (CG); iii) each branched linker comprises a) a second branch (B2) in which a second active agent is covalently attached to the branching unit by a second linker (SL) and a cleavage group (CG) or b) a second branch in which a polyethylene glycol moiety is covalently attached to the branching unit; Each cleavable group is hydrolyzed to release the active agent from the antibody conjugate.

[0136] In one aspect of the invention, the branched linker is

[0137] [ka]

[0023] wherein L2, L3 and L4 each independently represent a direct bond or -C n H 2n n is an integer from 1 to 30; G 1 , G 2 and G 3 each independently represents a direct bond,

[0138] [ka] and R 30 is hydrogen or C 1~30 is alkyl] It is.

[0139] More particularly, the branched linker is

[0140] [ka]

[0033] where B and B' are active agents, which may be different or the same; Each n independently represents an integer of 0 to 30, Each f independently represents an integer of 0 to 30; L indicates binding to Ab] Includes.

[0141] In one embodiment of the present invention, n is an integer of 0-20, more specifically, an integer of 1-10 or 4-20.

[0142] In one embodiment of the invention, L comprises an oxime and at least one polyethylene glycol unit covalently attaches the oxime to the active agent.

[0143] In one aspect of the invention, the cleavable group can be cleaved within a target cell, where the cleavable group can release one or more active agents.

[0144] In one embodiment of the invention, the linker comprises at least one branched linker covalently bonded to the Ab and at least two active agents covalently linked to the branched linker.

[0145] Specifically, one branched linker may be attached to the Ab.

[0146] In addition, two or more branched linkers may be attached to the Ab, and each branched linker may be attached to at least two active agents. More specifically, three branched linkers may be attached to the Ab. Alternatively, four branched linkers may be attached to the Ab.

[0147] In one aspect of the invention, each branched linker is linked to at least two of the same or different active agents. In one aspect of the invention, each active agent is linked to the branched linker by a cleavable bond.

[0148] In one embodiment of the invention, each branched linker comprises a branching unit, each active agent is attached to the branching unit by a second linker, and the branching unit is attached to the antibody by a first linker.

[0149] In one embodiment of the present invention, the branching unit can be a nitrogen atom. Additionally, the branching unit can be an amide, and the first linker or the second linker can include a carbonyl of the amide. Alternatively, the branching unit can be a lysine unit.

[0150] In yet another aspect of the invention, the linker is It may comprise (a) at least one branching unit, (b) at least one connecting unit, (c) at least one binding unit (BU) and at least one trigger unit (TU).

[0151] Here, the connection unit connects a trigger unit and a bond unit, a trigger unit and a branch unit, or a branch unit and a bond unit, At least one trigger unit is capable of releasing at least one drug or toxin, and a branching unit links a connecting unit to a trigger unit or a connecting unit to another connecting unit.

[0152] In one embodiment of the invention, the trigger unit has the following formula (IIb):

[0153] [ka] [In the formula, G is a sugar, sugar acid or sugar derivative; W is -C(O)-, -C(O)NR'-, -C(O)O-, -S(O)NR'-, -P(O)R''NR'-, -S(O)NR'- or -PONR'-, in the case of C(O), S or P is directly linked to the phenyl ring, and R' and R'' are each independently hydrogen, (C1-C8) alkyl, (C3-C8) cycloalkyl, (C1-C8) alkoxy, (C1-C8) alkylthio, mono- or di-(C1-C8) alkylamino, (C3-C 20 ) Heteroaryl or (C6-C 20 ) aryl, W is linked to a connecting or branching unit; each Z is independently (C1-C8) alkyl, halogen, cyano, or nitro; n is an integer from 1 to 3; m is 0 or 1; R1 and R2 are each independently hydrogen, (C1-C8) alkyl, or (C3-C8) cycloalkyl, or R1 and R2 together with the carbon atom to which they are attached form a (C3-C8) cycloalkyl ring. It has the structure shown below.

[0154] In one aspect of the invention, the sugar or sugar acid is a monosaccharide.

[0155] In one embodiment of the present invention, G has the following formula (IIIa):

[0156] [ka] [In the formula, R3 is hydrogen or a carboxyl protecting group; each R4 is independently hydrogen or a hydroxyl protecting group. It is a compound having the structure shown below.

[0157] In one embodiment of the invention, R3 is hydrogen and each R4 is hydrogen.

[0158] In one embodiment of the invention, W is -C(O)NR'-, where C(O) is linked to a phenyl ring and NR' is linked to L.

[0159] In one aspect of the invention, Z is hydrogen.

[0160] In one embodiment of the invention, R1 and R2 are each hydrogen.

[0161] In one aspect of the invention, the connecting unit is -(CH2) r (V(CH2) p ) q -, -((CH2) p V) q -, -(CH2) r (V(CH2) p ) q Y-, -((CH2) p V) q (CH2) r -, -Y((CH2) p V) q -or-(CH2) r (V(CH2) p ) q YCH2- [In the formula, r is an integer from 0 to 10; p is an integer from 1 to 10; q is an integer from 1 to 20; V and Y are independently a single bond, -O-, -S-, or -NR 21 -, -C(O)NR 22-, NR 23 C(O)-, NR 24 SO2- or -SO2NR 25 - and R 21 ~R 25 are each independently hydrogen, (C1-C6) alkyl, (C1-C6) alkyl(C6-C 20 )aryl or (C1-C6)alkyl(C3-C 20 ) heteroaryl] It is expressed as:

[0162] In one embodiment of the invention, r is 2.

[0163] In one embodiment of the invention, p is 2.

[0164] In one embodiment of the present invention, q is an integer of 6-20.

[0165] In one aspect of the invention, q is 2, 5 or 11.

[0166] In one aspect of the present invention, V and Y are each independently -O-,

[0167] [ka] [In the formula, L1, L2 and L3 each independently represent a direct bond or -C n H 2n - and n is an integer from 1 to 30; G1, G2 and G3 each independently represent a direct bond;

[0168] [ka] and R3 is hydrogen or C1-C 30 is alkyl, R4 is hydrogen or L4-COOR5, and L4 is a direct bond or -C n H 2n-, n is an integer from 1 to 10, and R5 is hydrogen or C1 to C 30 is alkyl] It is.

[0169] In one aspect of the invention, the branching unit is

[0170] [ka] [In the formula, L1 is a direct bond or an alkylene having 1 to 30 carbon atoms, R 11 is hydrogen or alkyl having 1 to 10 carbon atoms, specifically methyl; L2 is an alkylene having 1 to 30 carbon atoms; The branching unit links the connecting unit and the antibody. It is.

[0171] In one embodiment of the invention, L1 is an alkylene having 12 carbon atoms.

[0172] In one aspect of the invention, R 11 is methyl.

[0173] In one embodiment of the invention, L2 is an alkylene having 11 carbon atoms.

[0174] In one aspect of the invention, the branching unit is

[0175] [ka] It is.

[0176] In one aspect of the invention, the isoprenyl unit of the linker is covalently attached to the antibody by a thioether bond, the thioether bond involving the sulfur atom of a cysteine.

[0177] In one aspect of the invention, the antibody comprises an amino acid motif recognized by an isoprenoid transferase and the thioether bond involves the sulfur atom of a cysteine ​​of the amino acid motif.

[0178] 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, where C represents cysteine, Y at each occurrence independently represents an aliphatic amino acid, and X at each occurrence independently represents glutamine, glutamic acid, serine, cysteine, methionine, alanine or leucine, and the thioether bond involves the sulfur atom of the cysteine ​​of the amino acid motif.

[0179] In one aspect of the invention, the amino acid motif is the sequence CYYX, where Y in each occurrence is independently alanine, isoleucine, leucine, methionine, or valine.

[0180] In one embodiment of the invention, the amino acid motif is a CVIM or CVLL sequence.

[0181] In one aspect of the invention, at least one of the seven amino acids preceding the amino acid motif may each independently be selected from glycine, arginine, aspartic acid, and serine.

[0182] In one aspect of the invention, at least three of the seven amino acids preceding the amino acid motif are independently selected from glycine, arginine, aspartic acid, and serine.

[0183] Alternatively, between 1 and 10 amino acids preceding the amino acid motif are glycines. Specifically, at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids preceding the amino acid motif are glycines.

[0184] In one aspect of the invention, the antibody may comprise the amino acid sequence GGGGGGCVIM.

[0185] Additionally, in one aspect of the invention, the active agent may be a chemotherapeutic agent or a toxin.

[0186] Additionally, the active agent may be an immunomodulatory compound, an anti-cancer agent, an antiviral agent, an antibacterial agent, an antifungal agent, an antiparasitic agent, or a combination thereof, and the active agents listed below may alternatively be used: (a) erlotinib, bortezomib, fulvestrant, sutent, letrozole, imatinib mesylate, PTK787 / ZK 222584, oxaliplatin, 5-fluorouracil, leucovorin, rapamycin, lapatinib, lonafarnib, sorafenib, gefitinib, AG1478, AG1571, thiotepa, cyclophosphamide, busulfan, improsulfan, piposulfan, benzodopa, carboquone, meturedopa, uredopa, ethyleneimine, altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramine mido, teimethyloromelanin, bullatacin, bullataciionone, camptothecin, topotecan, bryostatin, kallistatin, CC-1065, adozelesin, carzelesin, bizelesin, cryptophycin 1, cryptophycin 8, dolastatin, duocarmycin, KW-2189, CB1-TM1, eleutherobin, pancratistatin, sarcodictyin, spongistatin, chlorambucil, chlornaphag , chlorophosphamide, estramustine, ifosfamide, mechlorethamine, melphalan, novembichine, phenesterine, prednimustine, trofosfamide, uracil mustard, carmustine, chlorozotoxin, fotemustine, lomustine, nimustine, ranimustine, calicheamicin, calicheamicin gamma 1, calicheamicin omega 1, dynemicin, dynemicin A, clodronate, esperamicin, neocarzinostatin chromophores, aclacinomycin, actinomycin, anthrymycin, azaserine, bleomycin, catcinomycin, carabicin, carninomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubucin, 6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin,Cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, liposomal doxorubicin, deoxydoxorubicin, epirubicin, esorubicin, marcelomycin, mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomigrin, streptozocin, tudercidin, ubenimex, zinostatin, Zorubicin, 5-fluoDLK acyl, denopterin, methotrexate, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thiguianine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, calsterone, dromostanolone, propionate, epithiostanol, mepitiostane, testolactone, aminoglutethimide, mitotane , trilostane, folinic acid, aceglatone, aldophosphamide glycosides, aminolevulinic acid, eniluracil, amsacrine, bestravcil, bisantrene, edatrexate, defofamine, democolcine, diaziquone, elfornithine, elliptinium acetate, etoglucide, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansine, ansamitocins, mitoguazone, mitoxantrone, mopi mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, rosoxantrone, 2-ethylhydrazide, procarbazine, polysaccharide-k, razoxane, rhizoxin, schizophyllan, spirogermanium, tenuazonic acid, triaqizuone, 2,2',2''-trichlorotriethylamine, T-2 toxin, verracurin A, roridin A, anguidine, urethane, vindesine, dacarbazine, mannomustine,mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside, cyclophosphamide, thiotepa, paclitaxel, albumin engineered nanoparticle formulations of paclitaxel, docetaxel, chlorambucil, gemcitabine, 6-thioguanine, mercaptopurine, cisplatin, carboplatin, vinblastine, platinum, etoposide, ifosfamide, mitoxantrone, vincristine, vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronate, CPT-11, topoisomerase inhibitor RFS 2000, difluoromethylornithine, retinoic acid, capecitabine or a pharma- ceutically acceptable salt, solvate or acid thereof, (b) monokines, lymphokines, traditional polypeptide hormones, parathyroid hormone, thyroxine, relaxin, pDLKelaxin, glycoprotein hormones, follicle-stimulating hormone, thyroid-stimulating hormone, luteinizing hormone, hepatic growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor, tumor necrosis factor-α, tumor necrosis factor-β, Müllerian inhibitory factor, mouse gonadotropin-related peptide, inhibin, activin, vascular endothelial growth factor, thrombopoietin, erythropoietin, bone morphogenetic 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, kit ligand or mixtures thereof; (c) diphtheria toxin, botulinum toxin, tetanus toxin, dysentery toxin, cholera toxin, amanitin, α-amanitin, pyrrolobenzodiazepines, pyrrolobenzodiazepine derivatives, indolinobenzodiazepines, pyridobenzodiazepines, tetrodotoxin, brevetoxin, ciguatoxin, ricin, AM toxin, auristatin, tubulysin, geldanamycin, maytansinoids, calicheamicin, daunomycin, doxorubicin, methotrexate, vindesine, SG2285, dolastatin, dolastatin analogs, auristatin, cryptophycin, camptothecin, rhizoxin, rhizoxin derivatives, CC-1065, CC-1065 analogs or derivatives, duocarmycins, enediyne antibiotics, esperamicin, epothilones, toxoids or mixtures thereof. (d) an affinity ligand which is a substrate, an inhibitor, an active agent, a neurotransmitter, a radioisotope, or a mixture thereof; (e) a radioactive label, 32P, 35S, a fluorescent dye, an electron-dense reagent, an enzyme, biotin, streptavidin, dioxygenin, a hapten, an immunogenic protein, a nucleic acid molecule having a sequence complementary to a target, or a mixture thereof; (f) an immunomodulatory compound, an anticancer agent, an antiviral agent, an antibacterial agent, an antifungal agent, an antiparasitic agent, or mixtures thereof; (g) tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxyfene, ketoxifene, LY117018, onapristone or toremifene, (h) 4(5)-imidazole, aminoglutethimide, megestrol acetate, exemestane, letrozole or anastrozole, (i) flutamide, nilutamide, bicalutamide, leuprolide, goserelin or troxacitabine, (j) aromatase inhibitors, (k) a protein kinase inhibitor, (l) lipid kinase inhibitors, (m) antisense oligonucleotides, (n) ribozyme, (o) Vaccines; and (p) Antiangiogenic agents.

[0187] Yet another aspect of the invention provides the use of an antibody-drug conjugate, or a pharma- ceutically acceptable salt or solvate thereof, in connection with the preparation of a medicament for the prevention or treatment of a proliferative, cancer or angiogenic disease.

[0188] Yet another aspect of the present invention provides a method for a drug for the prevention or treatment of a proliferative, cancer or angiogenic disease by administering to an individual with a pharmaceutical composition for the prevention or treatment of a proliferative, cancer or angiogenic disease, the pharmaceutical composition comprising an antibody-drug conjugate or a pharma- ceutically acceptable salt or solvate thereof as an active ingredient, the pharmaceutical composition being mixed with and administered at least one antiproliferative, cytostatic or cytotoxic agent.

[0189] Yet another aspect of the present invention relates to compounds of the general formula Ia: [General formula Ia] Ab-(Linker-D) n [In the formula, Ab is an anti-DLK1 antibody, Linker is a linker, D is a pyrrolobenzodiazepine dimer as the active agent, The linker and antibody are linked via the N10 or N10' position of the pyrrolobenzodiazepine dimer. The present invention provides an antibody-drug conjugate represented by the formula:

[0190] wherein the pyrrolobenzodiazepine dimer prodrug, or a pharma- ceutically acceptable salt or solvate thereof is bonded independently at the N10 and N10' positions of the pyrrolobenzodiazepine dimer to any one selected from the group consisting of -C(O)O*, -S(O)O*, -C(O)*, -C(O)NR*, -S(O)NR*, -(P(O)R')NR*, -S(O)NR* and -PONR* groups; * is the region where the linker is attached, R and R' are each independently H, OH, N3, CN, NO2, SH, NH2, ONH2, NHNH2, 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 mono- or di-C 1~8 is alkylamino, C 1~8 Alkyl, C 3~8 Cycloalkyl, C 1~8 Alkoxy, C 1~8 Alkylthio, C 3~20 Heteroaryl or C 5~20 When aryl is substituted, the substitutions are H, OH, N3, CN, NO2, SH, NH2, ONH2, NNH2, halo, C 1~6 Alkyl, C 1~6 Alkoxy and C 6~12 With substituents selected from the group including aryl.

[0191] A pyrrolobenzodiazepine dimer prodrug, a pharma- ceutically acceptable salt or solvate thereof may be used as the active pharmaceutical agent.

[0192] In one aspect of the present invention, pyrrolobenzodiazepine dimer precursors are provided, which, when administered in the form of the precursors according to the present invention, require additional reactions for conversion to the active drug upon exposure to blood, and have the advantages over conventional PBD drugs in that they avoid possible adverse reactions that may occur if the linker is unintentionally dissolved, have reduced toxicity to normal cells, and are more stable drugs.

[0193] Furthermore, when preparing antibody-drug conjugates, the antibody-drug conjugates prepared using conventional methods have high impurity content, and the exposed imine groups are susceptible to nucleophilic attack, which poses the risk of producing drugs with undesired structures. On the other hand, the antibody-drug conjugates prepared using the methods of the present invention have the advantage of being easy to isolate due to high purity, and further have improved physical properties over conventional PBDs or PBD dimers.

[0194] In one aspect of the invention, the pyrrolobenzodiazepine dimer precursor has the following general formula X or general formula XI:

[0195] [ka] or a pharma- ceutically acceptable salt or solvate thereof.

[0196] In the above formula, The dotted lines represent the possible 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, =CH2CN, and R m OR m , =CH-R m’ , =C(R m ')2, O-SO2-R m , CO2R m , C.O.R. m , halo, and dihalo; R m’ is R m , CO2R m , C.O.R. m , CHO, COH 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, C3~6 selected from the group consisting of cycloalkyl, 3- to 7-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 H and R independently m , O.H., O.R. m , S.H., S.R. m , NH2, NHR m , N.R. m 2, NO2, Me3SN and halo; R X4 and R X4’ are H and R independently m , O.H., O.R. m , S.H., S.R. m , NH2, NHR m , N.R. m 2, NO2, Me3SN, 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)2R n , -SR n , -S(O)R n , -S(O)2R n , -S(O)NR n R n ', -S(O)2NR n R n ',-OS(O)NR n R n ',-OS(O)2NR n R n ', -NR n R n ', -NR n C(O)Ro , -NR n C(O)OR o , -NR n C(O)NR o R o ', -NR n S(O)R o , -NR n S(O)2R o , -NR n S(O)NR o R o ', -NR n S(O)2NR o R o ', -C(O)R n , -C(O)OR n and -C(O)NR n R n ' is selected from R X and R X ' is independently H, OH, N3, CN, NO2, SH, NH2, ONH2, NHNH2, 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 mono- or di-C 1~8 alkylamino; Y and Y' are independently selected from O, S and N(H); R x6 is independently C 3~12 Alkylene, C 3~12 Alkenylene or C 3~12 heteroalkylene; R X7 and R X7’ are H, C independently 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 3~6 Cycloalkyl, 3-7 membered heterocycloalkyl, C 6~10 Aryl, 5-7 membered heteroaryl, -OR r , -OC(O)R r , -OC(O)NR r R r',-OS(O)R r , -OS(O)2R r , -SR r , -S(O)R r , -S(O)2R r , -S(O)NR r R r ', -S(O) 2 NR r R r ',-OS(O)NR r R r ',-OS(O)2NR 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)2R s , -NR r S(O)NR s R s ', -NR r S(O)2NR s R s , -C(O)R r , -C(O)OR s or -C(O)NR r R r ' is selected from Each R r , R r’ , R s and R s’ are H, C independently 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- to 7-membered heteroaryl; Each R X8 and R X8’ are H, C independently 1~6 Alkyl, C 2~6 Alkenyl, C 2~6Alkynyl, C 3~6 Heteroalkyl, 3-7 membered heterocycloalkyl, C 5~10 Aryl, 5-7 membered heteroaryl, -S(O)R m , -S(O)2R m , -S(O)NR m R m ', -S(O)2NR 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)2R n , -NR m S(O)NR n R n ', -NR m S(O)2NR n R n ', -C(O)R m , -C(O)OR m and -C(O)NR m R m '; is selected from Z a OR X12a , N.R. X12a R X12a or SR X12a is selected from among Z b OR X13a , N.R. X13a R X13a or SR X13a is selected from among Z a’ OR X12a , N.R. X12a R X12a or SR X12a is selected from among Z b’ OR X13a’ , N.R. X13a’ R X13a’ or SR X13a’ is selected from among Each R X12a , R X12a’ , R X13a’ and R x13a’ are H, C independently 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 3~6 Cycloalkyl, 3-7 membered heterocycloalkyl, C 5~10 Aryl, 5-7 membered heteroaryl, -C(O)R X15a , -C(O)OR X15a and -C(O)NR X15a R X15a’ is selected from Each 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 selected from cycloalkyl, 3- to 7-membered heterocyclyl, 3- to 7-membered heterocycloalkyl, and 5- to 7-membered heteroaryl; R X13a and R X14a optionally together with the atom to which they are attached form a 3- to 7-membered heterocyclyl, a 3- to 7-membered heterocycloalkyl, or a 3- to 7-membered heteroaryl; R X13a’ and R X14a’ optionally together with the atom to which they are attached form a 3- to 7-membered heterocyclyl, a 3- to 7-membered heterocycloalkyl, or a 3- to 7-membered heteroaryl; R n , R n’ , R o , R o’ , R p and R p’ Each of the groups is 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 It is selected from among aryl and 5- to 7-membered heteroaryl.

[0197] Further 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 selected from the group consisting of cycloalkyl, 3- to 7-membered heterocyclyl, 3- to 7-membered heterocycloalkyl, and 5- to 7-membered heteroaryl; R m is C 1~12 Alkyl, C 2~12 Alkenyl, C 2~12 Alkynyl, C 5~20 Aryl, C 5~20 Heteroaryl, C 3~6 Further substituted cycloalkyl, 3- to 7-membered heterocyclyl, 3- to 7-membered heterocycloalkyl or 5- to 7-membered heteroaryl.

[0198] Further R x4 and R x4’ are H and R independently m , O.H., O.R. m , S.H., S.R. m , NH2, NHR m , N.R. m R m’ , NO2, Me3Sn, 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)2R n , -SR n , -S(O)R n , -S(O)2R n , -S(O)NR n R n ', -S(O)2NR nR n ',-OS(O)NR n R n ',-OS(O)2NR 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)2R o , -NR n S(O)NR o R o ',-NR n S(O)2NR o R o ', -C(O)R n , -C(O)OR n and -C(O)NR n R n ' is selected from R X4 or R X4’ is 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 or 5-7 membered heteroaryl, 1~6 Alkyl, C 1~6 Alkoxy, C 2~6 Alkenyl, C 2~6 Alkynyl, C 3~6 Cycloalkyl, 3-7 membered heterocycloalkyl, C 5~10 Aryl, 5-7 membered heteroaryl, -OR p , -OC(O)R p , -OC(O)NR p R p ',-OS(O)R p , -OS(O)2R p , -SR p , -S(O)Rp , -S(O)2R p , -S(O)NR p R p ', -S(O)2NR p R p ',-OS(O)NR p R p ',-OS(O)2NR 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)2R q , -NR p S(O)NR q R q ', -NR p S(O)2NR q R q ', -C(O)R p , -C(O)OR p or -C(O)NR p R p is further substituted with

[0199] Further R X7 and R X7’ are H, C independently 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 3~6 Cycloalkyl, 3-7 membered heterocycloalkyl, C 6~10 Aryl, 5-7 membered heteroaryl, -OR r , -OC(O)R r , -OC(O)NR r R r ',-OS(O)R r , -OS(O)2R r , -SR r , -S(O)R r , -S(O)2R r , -S(O)NR r Rr ', -S(O)2NR r R r ',-OS(O)NR r R r ',-OS(O)2NR 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)2R s , -NR r S(O)NR s R s ', -NR r S(O)2NR s R s , -C(O)R r , -C(O)OR s or -C(O)NR r R r ' is selected from R X7 and R X7’ is independently C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 3~6 Cycloalkyl, 3-7 membered heterocycloalkyl, C 6~10 aryl or 5-7 membered heteroaryl; C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 3~6 Cycloalkyl, 3-7 membered heterocycloalkyl, C 6~10 Aryl, 5-7 membered heteroaryl, -OR t , -OC(O)R t , -OC(O)NR t R t ',-OS(O)R t , -OS(O)2R t , -SR t , -S(O)Rt , -S(O)2R t , -S(O)NR t R t ', -S(O)2NR t R t ',-OS(O)NR t R t ',-OS(O)2NR 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)2R u , -NR t S(O)NR u R u ', -NR t S(O)2NR u R u ', -C(O)R t , -C(O)OR t or -C(O)NR t R t ', which is further replaced by R r , R r’ , R s , R s’ , R t , R t’ , R u and R u’ are H, C independently 1~7 Alkyl, C 2~7 Alkenyl, C 2~7 Alkynyl, C 3~13 Cycloalkyl, 3-7 membered heterocycloalkyl, C 5~10 It is selected from among aryl and 5- to 7-membered heteroaryl.

[0200] Further R X1 and R X1’ are independently R m is selected from R mis C 1~6 Alkyl, C 2~6 Alkenyl, C 5~7 Aryl and C 3~6 heteroaryl.

[0201] Further R X2 , R X2’ , R X3 , R X3’ , R X5 and R X5 ' are independently selected from H or OH.

[0202] Further R X4 and R X4’ are independently R m is selected from R m is C 1~6 It is an alkoxy.

[0203] Further R X4 and R X4’ is independently any one selected from the group consisting of methoxy, ethoxy and butoxy.

[0204] Further, Y and Y' are O.

[0205] Further R x6 is C 3~12 Alkylene, C 3~12 Alkenylene or C 3~12 Heteroalkylene, R x6 -NH2, -NHR m , -NHC(O)R m , -NHC(O)R m , -NHC(O)CH2-[OCH2CH2] n -R XX or -[CH2CH2O] n -R XX is replaced by R XX H, OH, N3, CN, NO2, SH, NH2, ONH2, NHNH2, Halo, C 1~8 Alkyl, C 3~8 Cycloalkyl, C 1~8 Alkoxy, C 1~8Alkylthio, C 3~20 Heteroaryl, C 5~20 Aryl or mono- or di-C 1~8 is alkylamino, n is an integer from 1 to 6.

[0206] In a further aspect of the invention, the active agent has the general formula XII or the general formula XIII:

[0207] [ka] It is a pyrrolobenzodiazepine dimer represented by the formula:

[0208] X a and X a’ are independently a bond or C 1~6 alkylene, Z X’ and Z X are independently hydrogen, C 1~8 Alkyl, halogen, cyano, nitro,

[0209] [ka] or -(CH2) m -OCH3, Each R 80 , R 90 and R 100 is hydrogen, C 1~8 Alkyl, C 2~6 Alkenyl and C 1~6 alkoxy; m is 0 ~ It is an integer number equal to 12.

[0210] Z X’ and Z X are independently hydrogen,

[0211] [ka] and -(CH2) m-OCH3, Each R 80 , R 90 and R 100 is hydrogen, C 1~3 Alkyl and C 1~3 alkoxy; m is an integer from 1 to 6; The active agent is

[0212] [ka] TIFF2025076435000029.tif112170TIFF2025076435000030.tif212160TIFF2025076435000031.tif217160TIFF2025076435000032.tif227162TIFF2025076435000033.tif238162TIFF2025076435000034.tif225162TIFF2025076435000035.tif46162.

[0213] As used herein, a "conjugate" refers to a cell-binding agent that is covalently linked to one or more molecules of a cytotoxic compound. A "cell-binding agent" is a molecule, such as a ligand, protein, or antibody, particularly a monoclonal antibody, protein, or antibody fragment, that has affinity for a biological target, and the binding agent serves to target a biologically active compound to the biological target. In one aspect of the present invention, the conjugate can be designed to target tumor cells via a cell surface antigen. The antigen can be a cell surface antigen that is overexpressed or expressed in abnormal cell types. In particular, the target antigen can be expressed only in proliferating cells (e.g., tumor cells). The target antigen can be selected based on its differential expression between proliferating and normal tissues in general. In the present invention, the ligand is attached to a linker.

[0214] As used herein, an "antibody" refers to an immunoglobulin molecule capable of specifically binding to a target, such as a carbohydrate, polynucleotide, lipid or polypeptide, via at least one antigen recognizing site located in the variable region of the immunoglobulin molecule. The term "antibody", as used herein, includes not only complete monoclonal or polyclonal antibodies, but also a given antigen-binding portion (e.g., an "antigen-binding fragment") of an complete antibody or single chain thereof that has the ability to specifically bind to a particular antigen, fused proteins that contain antibodies, or any other modified configuration of an immunoglobulin molecule that contains an antigen recognition site, including, but not limited to, Fab, Fab', F(ab')2, Fd fragments, Fv fragments, single domain antibody (dAb) fragments, isolated complementarity determining regions (CDRs), single chain (scFv) and single domain antibodies (e.g., shark and camel antibodies), maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR or bis-scFv (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology 23(9):1126-1136).

[0215] The antibody includes any category of antibody, e.g., IgG, IgA, or IgM (or subcategory thereof), and the antibody is not required to be of a particular type. Depending on the amino acid sequence of the constant domain of the antibody's heavy chain, immunoglobulins can be assigned to different categories. There are five major types of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further classified into subcategories (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain (HC) constant domains corresponding to the different categories are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the different categories of immunoglobulins are well known in the art. The antibodies of the present invention can be prepared using techniques well known in the relevant art, e.g., recombinant techniques, phage display techniques, synthetic techniques, combinations of the above techniques, or other techniques readily known in the relevant art.

[0216] As used herein, an "isolated antibody" refers to an antibody that is substantially free of different antibodies having specificity for other antigens, and may be substantially free of other cellular material and / or chemicals.

[0217] As used herein, a "biological target" refers to an antigen located on the surface of a tumor, cancer cell, or the extracellular matrix.

[0218] As used herein, "linker" refers to a compound that covalently binds a cytotoxic compound to a ligand. In one aspect of the present invention, the linkers disclosed in PCT / US2016 / 063564 and PCT / US2016 / 063595 can be used as linkers.

[0219] As used herein, a "therapeutic agent" refers to an agent that exerts a cytotoxic, cell growth inhibitory and / or immunomodulatory effect on cancer cells or activated immune cells. Examples of therapeutic agents include cytotoxic agents, chemotherapeutic agents, cell growth inhibitors and immunomodulators.

[0220] As used herein, a "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer.

[0221] As used herein, "subject" includes humans and non-human animals, particularly mammals. Examples of subjects include human subjects, such as human patients with disorders, such as those described herein, such as cancer, or normal subjects. "Non-human animals" refers to all vertebrate animals, such as non-mammals (e.g., birds, amphibians, reptiles) and mammals, such as non-human primates, domestic animals and / or animals (e.g., sheep, dogs, cats, pigs, etc.) and rodents (e.g., mice, rats, hamsters, guinea pigs, etc.). In certain embodiments, the subject is a human patient. In certain examples, the subject is a human patient.

[0222] As used herein, "treating" or "treating" refers to both therapeutic treatment and prophylactic or preventative measures. Subjects in need of treatment include those who already have the disorder, those who are susceptible to the disorder, or those in whom the disorder needs to be prevented. Thus, the term, when used in relation to a disease or a subject in need of treatment, includes, but is not limited to, inhibiting or slowing the progression of the disease, remission of the disease, prevention of symptoms, and reducing the severity of the disease and / or symptoms.

[0223] As used herein, "administer" or "administering" refers to providing and / or contacting and / or delivering a compound(s) by any suitable route to achieve a desired effect. Administration may be oral, sublingual, parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular, intraarticular, intraarterial, intrathecal, intrasternal, intraspinal, rectal, nasal, buccal, rectal, vaginal, nasal, ocular, inhalation and by implantation.

[0224] As used herein, "unsubstituted or substituted" refers to a parent group which may be unsubstituted or substituted, and "substituted" refers to a parent group which has at least one substituent, where a substituent refers to a chemical moiety that is covalently attached to or fused to the parent group.

[0225] As used herein, "halo" refers to fluorine, chlorine, bromine, or iodine, and the like.

[0226] As used herein, "alkyl" refers to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of an aliphatic or alicyclic, saturated or unsaturated (unsaturated, fully unsaturated) hydrocarbon compound. Examples of saturated alkyls include methyl, ethyl, butyl, n-pentyl (amyl), n-hexyl, n-heptyl, etc., and saturated cyclic alkyl groups such as methyl, ethyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, and neopentyl.

[0227] As used herein, "alkoxy" refers to --OR, where R is an alkyl group, examples of which include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, isobutoxy, and tert-butoxy.

[0228] As used herein, "aryl" refers to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound having ring atoms.

[0229] In the present invention, "alkenyl" is an alkyl having at least one carbon-carbon double bond. Examples of unsaturated alkenyl groups include ethenyl (vinyl, -CH=CH2), 1-propenyl (-CH=CH-CH3), 2-propenyl, isopropenyl, butenyl, pentenyl, and hexenyl.

[0230] In the present invention, "alkynyl" refers to an alkyl group having at least one carbon-carbon triple bond; examples of unsaturated alkynyl groups include ethenyl and 2-propynyl.

[0231] As used herein, "carboxy" refers to -C(=O)OH.

[0232] As used herein, "formyl" refers to -C(=O)H.

[0233] 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 moiety having 5 to 7 ring atoms, a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, and "C 5~10 "Aryl" means a moiety having 5 to 10 ring atoms, a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound. 5~7 , C 5~10 ) refers to the number of ring atoms or range of numbers of ring atoms, whether carbon atoms or heteroatoms. For example, "C 5~6 "Aryl" refers to an aryl group having 5 or 6 ring atoms, where the ring atoms can all be carbon atoms as in "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 containing fused rings, at least one of which is aromatic, include, but are not limited to, those derived from indane, indene, isoindene, tetralin, acenaphthene, fluorene, phenalene, acesphenanthrene, and aceantrene. Alternatively, the ring atoms can contain one or more heteroatoms as in "heteroaryl group".

[0234] In the present invention, "heteroaryl" refers to aryl containing one or more heteroatoms, such as pyridine, pyrimidine, benzothiophene, furyl, dioxolanyl, pyrrolyl, oxazolyl, pyridyl, pyridazinyl, and more specifically, benzofuran, isobenzofuran, indole, isoindole, indolizine, indoline, isoindoline, purine (adenine or guanine), benzimidazole, indazole, benzoxazole, benzisoxazole, benzodioxole, benzofuran, benzotriazole, benzothiofuran, benzothiazole, C9 having two condensed rings derived from benzothiazole, chromene, isochromene, chroman, isochroman (is-chroman), benzodioxane, quinoline, isoquinoline, quinolizine, benzoxazine, benzodiazine, pyridopyridine, quinoxaline, quinazoline, cinnoline, phthalazine, naphthyridine, C1 having two condensed rings derived from pteridine. 10 , C with two condensed rings derived from benzodiazepines 11 C, which has three fused rings derived from carbazole, dibenzofuran, dibenzothiophene, carboline, pyrimidine, and pyridoindole 13 , acridine, xanthene, thioxanthene, phenoxathiin, phenazine, phenoxazine, phenothiazine, thianthrene, phenanthridine, phenanthroline and C with three condensed rings derived from phenazine 14 Refers to...

[0235] As used herein, "cycloalkyl" refers to an alkyl group that is a cycloalkyl 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 hydrocarbons: 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 the saturated heterocyclic hydrocarbon compounds: norcarane, norferone and norbornene.

[0236] As used herein, "heterocyclyl" refers to a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound.

[0237] In the present invention, the prefixes (e.g., C 1~12 , C 3~8 etc.) refers to the number of ring atoms or range of numbers of ring atoms, whether carbon atoms or heteroatoms. For example, as used herein, "C 3~6 The term "heterocyclyl" refers to a heterocyclyl group having 3 to 6 ring atoms.

[0238] Examples of monocyclic heterocyclyl groups include, but are not limited to, those derived from: N1: aziridine, azetidine, pyrrolidine, pyrroline, 2H- or 3H-pyrrole, piperidine, dihydropyridine, tetrahydropyridine, azepine, N2: Imidazolidine, pyrazolidine, imidazoline, pyrazoline, piperazine, O1: oxirane, oxetane, oxolane, oxole, oxane, dihydropyran, pyran, oxepine; O2: dioxolane, dioxane and dioxepane, O3: Trioxane, N1O1: Tetrahydrooxazole, dihydrooxazole, tetrahydroisoxazole, dihydroisoxazole, morpholine, tetrahydrooxazine, dihydrooxazine, S1: Thiiran, thietane, thiolane, thiane, thiepane, N1S1: thiazoline, thiazolidine, thiomorpholine, N2O1: oxadiazine, O1S1: oxathiol, oxathiane, N1O1S1: Oxathiazine.

[0239] In the present invention, a "prodrug" refers to a compound that can be converted directly or indirectly into a pyrrolobenzodiazepine drug under in vivo physiological conditions (e.g., enzymatic oxidation, reduction and / or hydrolysis) by the action of enzymes or gastric acid.

[0240] In the present invention, a "pharmaceutically acceptable salt" can be an acid addition salt formed with a pharmaceutically acceptable free acid, the free acid being an organic acid or an inorganic acid.

[0241] Organic acids include, but are not limited to, citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, glutamic acid, and aspartic acid, and inorganic acids include, but are not limited to, hydrochloric acid, bromic acid, sulfuric acid, and phosphoric acid.

[0242] For example, if a compound has a functional group that is anionic or can be anionic (e.g., -COOH can be -COO-), then a suitable cation can be used to form a salt. Examples of suitable inorganic cations include, but are not limited to, Na + and K + Alkaline earth cations, e.g., Ca 2+ and Mg 2+ and other cations, e.g., Al 3+ Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH4 + ) and substituted ammonium ions (e.g., NHR + , NH2R2 + , NHR3 + , NR4 + ) are mentioned.

[0243] Some examples of suitable substituted ammonium ions are 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 common quaternary ammonium ion is N(CH3)4 + There is.

[0244] The compound may have a cation or a functional group that may be a cation (e.g., -NH2 may be -NH3 + In the case of phosphate groups, which may be phosphate groups, salts may be formed with suitable anions. 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, nitric acid, nitrous acid, phosphoric acid, and phosphorous acid.

[0245] Examples of suitable organic anions include, but are not limited to, those derived from organic acids such as 2-acetoxybenzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphorsulfonic acid, cinnamic acid, citric acid, including, but not limited to, disulfonic acid, ethanesulfonic acid, fumaric acid, glutaronic acid, gluconic acid, glutamic acid, glycolic acid, hydroxymaleic acid, hydroxynaphthalene carboxylic acid, isethionic acid, lactic acid, including, but not limited to, malic acid, methanesulfonic acid, mucilous acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, pantothenic acid, phenylacetic acid, phenylsulfonic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, and tartaric acid. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethylcellulose, and the like.

[0246] As used herein, the term "solvate" refers to a molecular complex between a compound of the present invention and a solvent molecule, examples of which include, but are not limited to, a compound of the present invention bound to water, isopropanol, ethanol, methanol, dimethylsulfoxide, ethyl acetate, acetic acid, ethanolamine, or a mixed solvent.

[0247] It may be convenient or desirable to prepare, purify and / or handle the solvate corresponding to the active compound.The term "solvate" is used herein in its conventional sense to refer to a complex of solute (e.g., active compound and salt of active compound) and solvent.When the solvent is water, the solvate may be conveniently referred to as hydrate, for example, monohydrate, dihydrate or trihydrate.

[0248] The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers include large macromolecules that are slowly metabolized, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates, etc. Such pharmaceutically acceptable carriers can be appropriately selected and used by those skilled in the art.

[0249] The composition containing a pharma- ceutically acceptable carrier may be of various oral or parenteral formulations, in which case diluents or excipients, such as bulking agents, bulking fillers, binders, wetting agents, disintegrating agents or surfactants, are commonly used.

[0250] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., which may contain at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin. In addition to simple excipients, lubricants, such as magnesium stearate, talc, etc., may also be used.

[0251] Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, flavoring agents, preservatives, etc. may be included.

[0252] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. Examples of non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil and injectable esters such as ethyl oleate. Examples of suppository bases include Witepsol (registered trademark), macrogol, Tween 61, cacao paper, laurin, glycerogelatin, etc.

[0253] The pharmaceutical compositions may be in the form of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, frozen solutions, and may have one formulation.

[0254] For intravenous, intradermal or subcutaneous injection, the active ingredient may be in the form of an acceptable aqueous solution for parenteral administration, which may be pyrogen-free and have suitable pH, isotonicity and stability.Those skilled in the art can prepare suitable solutions using isotonic vehicles such as sodium chloride solution, Ringer's solution or lactated Ringer's solution, and may also include preservatives, stabilizers, buffers, antioxidants and other additives as necessary.Solid forms suitable for injection may also be prepared as emulsions or in the form of polypeptides encapsulated in liposomes.

[0255] As used herein, the phrase "effective amount" or "therapeutically effective amount" refers to the amount (in terms of dose and duration and means of administration) required to achieve the desired therapeutic result. An effective amount is at least the minimum amount of active agent required to impart a therapeutic benefit to a subject, and is less than a toxic amount. For example, the amount administered may range from about 100 ng to about 100 mg / kg, more usually from about 1 μg / kg to about 10 mg / kg per patient. In cases where the active compound is a salt, ester, amide, or prodrug, the dosage is calculated based on the parent compound, and therefore the actual weight used will be increased proportionately. The pyrrolobenzodiazepine compounds of the present invention may be formulated to contain, but are not limited to, 0.1 mg to 3000 mg, 1 mg to 2000 mg, or 10 mg to 1000 mg of active ingredient per unit dosage form. The active ingredient may be administered to provide a peak plasma concentration of the active compound of about 0.05 μM to 100 μM, 1 μM to 50 μM, or 5 μM to 30 μM. For example, administration may optionally be by intravenous injection of a 0.1% to 5% w / v solution of the active ingredient in saline solution.

[0256] In the pharmaceutical composition, the concentration of the active compound is determined by the absorption, inactivation and excretion rate of the drug and other factors not known to those skilled in the art. The dose administered may vary depending on the severity of the symptoms or disease. Furthermore, the dose and administration regimen for a particular patient may be adjusted according to the professional judgment of the person administering, taking into account general considerations such as the patient's symptoms and the extent of the disease, the need for the drug, age and response to the drug. The concentration ranges described in the present invention are intended to be merely examples, and the method of implementing the claimed compositions is not limited thereto. Furthermore, the active ingredient may be administered once, or smaller doses may be administered over multiple doses.

[0257] Prodrug compounds or prodrug-linker compounds or prodrug-linker-ligand conjugate compounds can be used to treat proliferative diseases, particularly cancer. The term "proliferative disease" refers to excessive, abnormal, undesirable, or unregulated cell growth, such as hyperplasia or cytogenesis, either in vitro or in vivo. Non-limiting examples of proliferative diseases include neoplasms, tumors, cancer, leukemia, psoriasis, bone disease, fibrous dysplasia, and pharyngeal atherosclerosis. Non-limiting examples of cancer include lung cancer, small cell lung cancer, gastrointestinal cancer, colon cancer, intestinal 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 melanoma.

[0258] Advantages of the invention The antibody-drug conjugate of the present invention, which comprises an anti-DLK1 antibody that specifically binds to DLK1-expressing cells and is internalized into the cells, has the advantage that the drug can be efficiently, selectively and specifically delivered, and the drug can be stably bound to the antibody to exhibit intended cytotoxicity while maintaining in vivo stability. In particular, by the grafting technique of a linker containing a self-immolative group, the present invention provides a drug-linker-ligand system that allows the drug and / or toxin to stably reach the target cells and efficiently exhibit drug effects with substantially reduced toxicity. [Brief description of the drawings]

[0259] [Figure 1] FIG. 1 confirms the efficacy of the ADC of the invention in a patient-derived tumor animal model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0260] BEST MODE FOR CARRYING OUT THE PRESENTINVENTION The present invention will now be described in more detail with reference to embodiments and examples.

[0261] The following embodiments and examples are intended to aid in the understanding of the present invention, and are not intended to limit the scope of the present invention thereto.

[0262] [Example 1] Preparation of Compounds 1, 2, 3 and 4

[0263] [ka] TIFF2025076435000037.tif71157

[0264] Compounds 1, 2, 3 and 4 were prepared using the methods described in International Patent Publication WO2017-089895.

[0265] In compounds 1, 2, 3, and 4, the structure of MMAE is:

[0266] [ka]

[0267] [Example 2] Preparation of Compounds 5, 6, 7 and 8

[0268] [ka]

[0269] Compounds 5, 6 and 7 were prepared using the method described in Korean Patent Publication No. 10-2018-0110645.

[0270] [ka]

[0271] Compound 8 was prepared using the method described in Korean Patent Application No. 10-2019-0000514.

[0272] [Example 3] Preparation of ADCs The ADCs were prepared according to the following two steps, and the commonly used LCB14-0511 and LCB14-0606 were prepared using the method described in Korean Patent Publication No. 10-2014-0035393. The structural formulas of LCB14-0511 and LCB14-0606 are as follows:

[0273] [ka]

[0274] Step 1: Preparation of prenylated antibodies The prenylation reaction mixture of the antibody (18A5) was prepared using the method described in Korean Patent Application No. 10-2018-0107639 and reacted at 30°C for 16 hours. The reaction mixture consisted of 24 μM antibody (18A5), 200 nM FTase (Calbiochem No. 344145), and a buffer solution (50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 10 μM ZnCl2, 0.144 mM DTT) containing 0.144 mM LCB14-0511 or LCB14-0606. After the reaction was completed, the prenylated antibody was desalted using a G25 Sepharose column (AKTA purifier, GE healthcare) equilibrated with PBS buffer solution.

[0275] Step 2: Drug conjugation methods <Conjugation via oxime bond formation> The mixture for the oxime bond formation reaction between the prenylated antibody and the linker-drug was prepared by mixing 100 mM Na acetate buffer solution pH 5.2, 10% DMSO, 20 μM antibody and 200 μM linker-drug (in-house, compounds 1, 2, 3, 4, 5, 7 and 8 obtained from embodiments 1 and 2) and gently stirring at 30° C. After reacting for 6 or 24 hours, the FLPC (AKTA purifier, GE healthcare) process was carried out to remove the excess small compounds used. The protein fraction was collected and concentrated.

[0276] <Conjugation by Click Reaction> The mixture for the oxime bond formation reaction between the prenylated antibody and the linker-drug was 10% DMSO, 20 μM antibody and 200 μM linker-drug (in-house, compound 6 obtained from embodiment 2), 1 mM copper(II) sulfate pentahydrate, 2 mM (BimC4A)3 (Sigma-Aldrich 696854), 10 mM sodium ascorbate and 10 mM aminoguanidine hydrochloride, reacted at 25°C for 3 hours, then treated with 2.0 mM EDTA and reacted for 30 minutes. After the reaction, the FLPC (AKTA purifier, GE healthcare) process was carried out to remove the excess small compounds used. The protein fraction was collected and concentrated.

[0277] [Table 2]

[0278] Experimental Example 1: In vitro cytotoxicity evaluation The cancer cell line cell proliferation inhibitory activity of the drugs listed in Table 3 below and the ADC prepared in the above <Embodiment 3> was measured. For this purpose, commercially available human pancreatic cancer cell line MIA-PaCa2 (DLK1 negative or normal) and MIA-PaCa-2-DLK1 overexpressing cell line were used. As a drug, MMAF-OMe was used as ADC, and the ADCs in Table 2 were used. In a 96-well plate, 4,000-5,000 cells of each cancer cell line for the 72-hour treatment group, 2,000-3,000 cells for the 96-hour treatment group, and 800-1,000 cells for the 168-hour treatment group were seeded in each well. After 24 hours of culture, they were treated with drugs and ADCs at concentrations of 0.0015-10.0 nM (diluted three-fold serially). After 72, 96, and 168 hours, respectively, the number of viable cells was measured using SRB (sulforhodamine B) dye.

[0279] [Table 3]

[0280] Most of the antibody-drug conjugates were confirmed to have substantially improved cytotoxicity in pancreatic cancer cell lines with DLK1 overexpression than in pancreatic cancer cell lines without DLK1 expression. In pancreatic cancer cell lines overexpressing DLK1, pyrrolobenzodiazepine-based antibody-drug conjugates (ADC5, 7 and 8) were confirmed to show stronger cytotoxicity than auristatin-based antibody-drug conjugates (ADC1, 2, 3 and 4). Furthermore, pyrrolobenzodiazepine-based antibody-drug conjugates were confirmed to show stronger cytotoxicity compared to auristatin-based antibody-drug conjugates when using longer reaction times.

[0281] Experimental Example 2: In vivo efficacy evaluation In a patient-derived tumor animal model, the efficacy of the ADC prepared in <Embodiment 3> was evaluated. Specifically, a patient-derived small cell lung cancer mouse model (PDX model) was provided by Champions Oncology. The control group of the PDX model was PBS administered intravenously in the tail, and the experimental groups were administered ADC4 at 6mpk / Q4D*4, 6mpk / QW*4 and 9mpk / QW*4, and then the tumor size was measured.

[0282] As a result, as shown in Figure 1, tumors disappeared from all eight experimental animals in each of the 6mpk / Q4D*4 group and the 9mpk / QW*4 group (CR, complete remission), and tumors disappeared in six of the eight experimental animals in the 6mpk / QW*4 group. [Industrial Applicability]

[0283] Novel antibody-drug conjugates (ADCs) targeting DLK1 and pharmaceutical compositions comprising the same may be used in the manufacture of drugs for the treatment and / or prevention of proliferative and / or angiogenic diseases, such as cancer.

[0284] [Sequence table] SEQUENCE LISTING <110> LIGACHEM BIOSCIENCES, INC. Y-BIOLOGICS INC. <120> Antibody-drug conjugates including antibody against human DLK1, and use thereof <130> PA25-013 <150> KR 10-2019-0025987 <151> 2019-03-06 <150> KR 10-2020-0027373 <151> 2020-03-04 <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 <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 Ser 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 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 Asn 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 Ile 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 Gly 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 Phe 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 Glu 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 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> Artificial 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> Artificial 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 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 Gly 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 115 <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> PRT <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 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 Gln 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 Gln 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 100 105 110 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 Gly 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 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 40 <210> 115 <211> 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> <223> Artificial Sequence <400> 116 Gln Gln Ser Tyr 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 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 Tyr 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 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

1. An antibody conjugate of general formula I, or a pharma- ceutically acceptable salt or solvate thereof: [General formula I] Ab-(X)y [In formula: Ab is an antibody or antigen-binding fragment thereof that specifically binds to DLK1 (Delta-like 1 homolog (Drosophila)) comprising a heavy chain variable region and a light chain variable region, and the antibody or antigen-binding fragment thereof that binds to DLK1 is At least one heavy chain CDR1 selected from the group comprising SEQ ID NOs: 2, 16, 30, 44, 58, 72 and 86; At least one heavy chain CDR2 selected from the group comprising SEQ ID NOs: 4, 18, 32, 46, 60, 74 and 88; and a heavy chain variable region comprising at least one heavy chain CDR3 selected from the group comprising SEQ ID NOs: 6, 20, 34, 48, 62, 76 and 90; and At least one light chain CDR1 selected from the group comprising SEQ ID NOs: 9, 23, 37, 51, 65, 79, 93, 115 and 121; At least one light chain CDR2 selected from the group comprising SEQ ID NOs: 11, 25, 39, 53, 67, 81 and 95; and a light chain variable region comprising at least one light chain CDR3 selected from the group comprising SEQ ID NOs: 13, 27, 41, 55, 69, 83, 97, 116 and 125. Including, X is independently a chemical moiety comprising at least one active agent and a linker, the linker connecting the antibody and the active agent; and y is an integer from 1 to 20.

2. 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 comprising SEQ ID NOs: 7, 21, 35, 49, 63, 77 and 91; At least one light chain FR1 selected from the group comprising SEQ ID NOs: 8, 22, 36, 50, 64, 78, 92, 117 and 120; At least one light chain FR2 selected from the group comprising SEQ ID NOs: 10, 24, 38, 52, 66, 80, 94 and 122; At least one light chain FR3 selected from the group comprising SEQ ID NOs: 12, 26, 40, 54, 68, 82, 96, 118 and 123; At least one light chain FR4 selected from the group consisting of SEQ ID NOs: 14, 28, 42, 56, 70, 84, 98 and 125 2. An antibody or antigen-binding fragment thereof that specifically binds to DLK1 according to claim 1 .

3. An antibody or antigen-binding fragment thereof that specifically binds to DLK1 as described in claim 1, comprising a heavy chain variable region comprising an array having at least 90% sequence identity with an array selected from the group consisting of SEQ ID NOs: 99, 101, 103, 105, 107, 109, 111 and 127.

4. An antibody or antigen-binding fragment thereof that specifically binds to DLK1 as described in claim 1, comprising a light chain variable region comprising an array having at least 90% sequence identity to an array selected from the group consisting of SEQ ID NOs: 100, 102, 104, 106, 108, 110, 112, 126 and 128.

5. The linker has the structure of general formula II: 【Chemistry 1】 [In the formula, G is a glucuronic acid moiety or 【Chemistry 2】 and R 3 is hydrogen or a carboxyl protecting group, Each R 4 are independently hydrogen or a hydroxyl protecting group; Ab is an antibody or antigen-binding fragment thereof that specifically binds to DLK1; B is an active agent, R 1 and R 2 are each independently hydrogen, (C1-C8) alkyl, or (C3-C8) cycloalkyl; W is -C(O)-, -C(O)NR'-, -C(O)O-, -SONR'-, -P(O)R''NR'-, -SONR'- or -PO 2 NR'-, where C, S or P is directly bonded to the phenyl ring, NR' is bonded to L, and R' and R'' are each independently hydrogen, (C1-C8)alkyl, (C3-C8)cycloalkyl, (C1-C8)alkoxy, (C1-C8)alkylthio, mono- or di-(C1-C8)alkylamino, (C3-C20)heteroaryl or (C6-C20)aryl; Each Z is independently (C 1 ~C 8 ) alkyl, halogen, cyano or nitro; n is an integer from 1 to 3, L is either A) or B) of the following: A) C 1 ~C 50 alkylene or heteroalkylene of 1 to 50 atoms, (i) L contains at least one unsaturated bond; (ii) two atoms in L are replaced with the same divalent substituents as in Substituent, thereby completing a heteroarylene; (iii) L is a heteroalkylene of 1 to 50 atoms; (iv) The alkylene has at least one C 1～20 Being substituted by alkyl At least one of the following is satisfied: B) A compound of the general formula III, which can be recognized by isoprenoid transferase: 【Chemistry 3】 Contains at least one isoprenyl derivative unit of Either one of the following:

2. An antibody or antigen-binding fragment thereof that specifically binds to DLK1 according to claim 1, having the following structure:

6. L is a nitrogen-containing heteroalkylene of 1 to 50 atoms; 6. The antibody conjugate of claim 5, or a pharma- ceutically acceptable salt or solvate thereof, wherein the linker comprises at least two atoms of a hydrophilic amino acid, the nitrogen of which forms a peptide bond with the carbonyl of a hydrophilic amino acid selected from the group comprising arginine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, lysine, ornithine, proline, serine, or threonine.

7. 6. The antibody conjugate of claim 5, or a pharma- ceutically acceptable salt or solvate thereof, wherein W is -C(O)NR'- and the nitrogen of W is a nitrogen atom of a hydrophilic amino acid selected from the group consisting of arginine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, lysine, ornithine, proline, serine, or threonine.

8. 8. The antibody conjugate of claim 6 or 7, or a pharma- ceutically acceptable salt or solvate thereof, wherein the amino acid covalently bonds the oxime of the linker to the polyethylene glycol unit of the linker.

9. 8. The antibody conjugate or pharma- ceutically acceptable salt or solvate thereof of claim 6 or 7, wherein the hydrophilic amino acid is an amino acid selected from the group consisting of aspartic acid, glutamic acid, ornithine, lysine and arginine, the side chain comprising a moiety that exhibits a neutral pH charge in aqueous solution.

10. 6. The antibody conjugate of claim 5, or a pharma- ceutically acceptable salt or solvate thereof, wherein the linker comprises a peptide, the peptide comprising 2 to 20 amino acids and comprising at least one hydrophilic amino acid selected from the group comprising alanine, aspartic acid, asparagine, glutamic acid, glutamine, glycine, lysine, ornithine, proline, serine, and threonine, and comprising at least one hydrophilic amino acid selected from the group consisting of aspartic acid, glutamic acid, ornithine, lysine, and arginine that comprises a side chain having a moiety that exhibits a neutral pH charge in aqueous solution.

11. The antibody conjugate or a pharma- ceutically acceptable salt or solvate thereof of claim 10, wherein W is -C(O)NR'- and the nitrogen of W is the nitrogen atom of the N-terminal amino acid of the peptide.

12. 11. The antibody conjugate of claim 10, or a pharma- ceutically acceptable salt or solvate thereof, wherein the peptide covalently bonds the oxime of the linker to the polyethylene glycol unit of the linker.

13. 6. The antibody conjugate of claim 5, or a pharma- ceutically acceptable salt or solvate thereof, wherein L is covalently bound to the antibody by a thioether bond, the thioether bond comprising a sulfur atom of a cysteine ​​of the antibody.

14. 14. The antibody conjugate of claim 13, or a pharma- ceutically acceptable salt or solvate thereof, wherein the antibody comprises an amino acid at the C-terminus of the antibody that can be recognized by an isoprenoid transferase, and the thioether bond comprises a sulfur atom of a cysteine ​​of the antibody.

15. The amino acid motif is the CYYX sequence, C is cysteine, Y is an aliphatic amino acid selected from the group consisting of alanine, isoleucine, leucine, methionine and valine; X is any one selected from the group consisting of glutamine, glutamic acid, serine, cysteine, methionine, alanine, and leucine; 15. The antibody conjugate of claim 14, or a pharma- ceutically acceptable salt or solvate thereof, wherein the thioether bond comprises a sulfur atom of a cysteine ​​of the amino acid motif.

16. 16. The antibody conjugate or a pharma- ceutically acceptable salt or solvate thereof of claim 15, wherein the amino acid motif is a CVIM or CVLL sequence.

17. 17. The antibody conjugate of any one of claims 14 to 16, or a pharma- ceutically acceptable salt or solvate thereof, wherein at least one of the 1 to 10 amino acids preceding the amino acid motif is selected from among glycine, proline, aspartic acid, arginine and serine.

18. 18. The antibody conjugate of claim 17, or a pharma- ceutically acceptable salt or solvate thereof, wherein the 1 to 10 amino acids preceding the amino acid motif are each glycine.

19. 6. The antibody conjugate of claim 5, or a pharma- ceutically acceptable salt or solvate thereof, wherein L comprises the amino sequence GGGGGGCVIM at the C-terminus.

20. L is a group of the general formula III, which can be recognized by an isoprenoid transferase: 【Chemistry 4】 or a pharma- ceutically acceptable salt or solvate thereof, comprising at least one isoprenyl derivative unit of the formula:

21. 6. The antibody conjugate of claim 5, or a pharma- ceutically acceptable salt or solvate thereof, wherein L is 3-50 heteroalkylene containing an oxime, and the oxygen atom of the oxime is on the side of L linked to W and the carbon atom of the oxime is on the side of L linked to Ab, or the carbon atom of the oxime is on the side of L linked to W and the oxygen atom of the oxime is on the side of L linked to Ab.

22. 22. The antibody conjugate of claim 20 or 21, or a pharma- ceutically acceptable salt or solvate thereof, wherein L comprises an oxime and at least one isoprenyl unit covalently links the oxime to Ab.

23. L 【Chemistry 5】 or 【Chemistry 6】 6. The antibody conjugate of claim 5, or a pharma- ceutically acceptable salt or solvate thereof.

24. L is of general formula VIII or general formula IX: [General formula VIII] -(CH 2 ) r (V(CH 2 ) p ) q - [General formula IX] -(CH 2 CH 2 X) w - [In the formula, 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 - and X is -O-, C 1 ~C 8 Alkylene or -NR 21 - and R 21 ~R 25 are independently 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; 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. or a pharma- ceutically acceptable salt or solvate thereof.

25. L 【Chemistry 7】 or a pharma- ceutically acceptable salt or solvate thereof, comprising 1 to 20 polyethylene glycol units represented by

26. -OCH where L is 1 to 12 2 CH 2 26. The antibody conjugate of claim 25, or a pharma- ceutically acceptable salt or solvate thereof, comprising a - unit.

27. 26. The antibody conjugate of claim 25, or a pharma- ceutically acceptable salt or solvate thereof, wherein L comprises an oxime and 1 to 20 polyethylene glycol units covalently attach the oxime to the active agent.

28. 6. The antibody conjugate of claim 5, or a pharma- ceutically acceptable salt or solvate thereof, wherein L further comprises a linking unit formed by a 1,3-dipolar cycloaddition reaction, a hetero Diels reaction, a nucleophilic substitution reaction, a non-aldol type carbonyl reaction, an addition to a carbon-carbon multiple bond, an oxidation reaction, or a Click reaction.

29. 29. The antibody conjugate of claim 28, or a pharma- ceutically acceptable salt or solvate thereof, wherein the linking unit is formed by reaction between an acetylene and an azide, or between an aldehyde or ketone group and a hydrazine or an alkoxyamine.

30. L is of the following general formula IV, V, VI or VII: 【Chemistry 8】 [In the formula, L 1 is a single bond or C 1 ~C 30 is alkylene, R 11 is hydrogen or C 1 ~C 10 is alkyl] or a pharma- ceutically acceptable salt or solvate thereof, comprising a linking unit represented by:

31. L 1 is a single bond or C 11 Or C 12 31. The antibody conjugate of claim 30, or a pharma- ceutically acceptable salt or solvate thereof, which is alkylene.

32. L 【Chemistry 9】 [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 - and R 21 ~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 0 to 10; p is an integer from 0 to 10; q is an integer from 1 to 20; L 1 is a single bond] or a pharma- ceutically acceptable salt or solvate thereof.

33. L 【Chemistry 10】 [Ab is an antibody or an antigen-binding fragment thereof that specifically binds to DLK1, B is an active agent, n is an integer between 0 and 20. The antibody conjugate of claim 32, or a pharma- ceutically acceptable salt or solvate thereof, comprising any one selected from the group comprising:

34. The antibody conjugate or a pharma- ceutically acceptable salt or solvate thereof according to claim 5, wherein the isoprenoid transferase is farnesyl protein transferase (FTase) or geranylgeranyl transferase (GGTase).

35. L comprises at least one branched linker covalently attached to Ab; i) each branched linker comprises a branching unit (BR) covalently linked to the Ab by a principal linker (PL); ii) each branch linker comprises a first branch (B1) that connects a first active agent to a branch unit and comprises a second linker (SL) and a cleavage group (CG); iii) each branched linker comprises: a) a second branch (B2) in which a second active agent is covalently attached to the branching unit by a second linker (SL) and a cleavage group (CG); or b) a second branch in which a polyethylene glycol moiety is covalently attached to the branching unit; iv) The antibody conjugate of claim 5, or a pharma- ceutically acceptable salt or solvate thereof, wherein each cleavage group is hydrolyzed to release the active drug from the antibody conjugate.

36. Branched linker 【Chemistry 11】 [L 2 , L 3 and L 4 each independently represents a direct bond or -C n H 2n where n is an integer from 1 to 30; G 1 , G 2 and G 3 are independently a direct bond, 【Chemistry 12】 and R 30 is hydrogen or C 1～30 is alkyl] 36. The antibody conjugate of claim 35, which is:

37. 【Chemical 13】 [0033] where B and B' are active agents, which may be different or the same; Each n independently represents an integer from 0 to 30, Each f independently represents an integer of 0 to 30; L indicates binding to Ab] 37. The antibody conjugate of claim 36, comprising:

38. 38. The antibody conjugate of claim 37, or a pharma- ceutically acceptable salt or solvate thereof, wherein L comprises the oxime and 1 to 20 polyethylene glycol units which covalently link the oxime to the active agent.

39. 36. The antibody conjugate of claim 35, or a pharma- ceutically acceptable salt or solvate thereof, wherein the cleavable group can be cleaved within a target cell, the cleavable group being capable of releasing one or more active agents.

40. 6. The antibody conjugate of claim 5, or a pharma- ceutically acceptable salt or solvate thereof, wherein L comprises 1 to 6 branched linkers covalently bonded to Ab and 2 to 6 identical or different active agents covalently linked to the branched linkers.

41. 41. The antibody conjugate of claim 40, or a pharma- ceutically acceptable salt or solvate thereof, wherein each active agent is attached to the branched linker by a cleavable bond.

42. 41. The antibody conjugate of claim 40, or a pharma- ceutically acceptable salt or solvate thereof, wherein each branched linker comprises a branching unit, each active agent is attached to the branching unit by a second linker, and the branching unit is attached to the Ab by a first linker.

43. 43. The antibody conjugate or a pharma- ceutically acceptable salt or solvate thereof of claim 42, wherein the branching unit is a nitrogen unit or a lysine unit.

44. 43. The antibody conjugate of claim 42, or a pharma- ceutically acceptable salt or solvate thereof, wherein the branching unit is an amide and the first linker or the second linker comprises a carbonyl of the amide.

45. 6. The antibody conjugate or a pharma- ceutically acceptable salt or solvate thereof of claim 5, wherein the active agent is a chemotherapeutic agent or a toxin.

46. 6. The antibody conjugate of claim 5, or a pharma- ceutically acceptable salt or solvate thereof, 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.

47. The active agent is: (a) erlotinib, bortezomib, fulvestrant, sutent, letrozole, imatinib mesylate, PTK787 / ZK 222584, oxaliplatin, 5-fluorouracil, leucovorin, rapamycin, lapatinib, lonafarnib, sorafenib, gefitinib, AG1478, AG1571, thiotepa, cyclophosphamide, busulfan, improsulfan, piposulfan, benzodopa, carboquone, meturedopa, uredopa, ethyleneimine, altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramine mido, teimethyloromelanin, bullatacin, bullataciionone, camptothecin, topotecan, bryostatin, kallistatin, CC-1065, adozelesin, carzelesin, bizelesin, cryptophycin 1, cryptophycin 8, dolastatin, duocarmycin, KW-2189, CB1-TM1, eleutherobin, pancratistatin, sarcodictyin, spongistatin, chlorambucil, chlornaphag , chlorophosphamide, estramustine, ifosfamide, mechlorethamine, melphalan, novembichine, phenesterine, prednimustine, trofosfamide, uracil mustard, carmustine, chlorozotoxin, fotemustine, lomustine, nimustine, ranimustine, calicheamicin, calicheamicin gamma 1, calicheamicin omega 1, dynemicin, dynemicin A, clodronate, esperamicin, neocarzinostatin chromophores, aclacinomycin, actinomycin, anthrymycin, azaserine, bleomycin, catcinomycin, carabicin, carninomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubucin, 6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin,Cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, liposomal doxorubicin, deoxydoxorubicin, epirubicin, esorubicin, marcelomycin, mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomycin, streptozocin, tudercidin, ubenimex, zinostatin, zorubicin Syn, 5-fluoDLKacil, denopterin, methotrexate, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thiguianine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, calsterone, dromostanolone, propionate, epithiostanol, mepitiostane, testolactone, aminoglutethimide, mitotane, trilosta , folinic acid, aceglatone, aldophosphamide glycosides, aminolevulinic acid, eniluracil, amsacrine, bestravcil, bisantrene, edatrexate, defofamine, democolcine, diaziquone, elfornithine, elliptinium acetate, etoglucide, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansine, ansamitocins, mitoguazone, mitoxantrone, mopidanol idanmol), nitraerine, pentostatin, phenamet, pirarubicin, rosoxantrone, 2-ethylhydrazide, procarbazine, polysaccharide-k, razoxane, rhizoxin, sizofiran, spirogermanium, tenuazonic acid, triaqizuone, 2,2',2''-trichlorotriethylamine, T-2 toxin, verracurin A, DLKidin A, anguidine, urethane, vindesine, dacarbazine, mannomustine,mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside, cyclophosphamide, thiotepa, paclitaxel, albumin engineered nanoparticle formulations of paclitaxel, docetaxel, chlorambucil, gemcitabine, 6-thioguanine, mercaptopurine, cisplatin, carboplatin, vinblastine, platinum, etoposide, ifosfamide, mitoxantrone, vincristine, vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronate, CPT-11, topoisomerase inhibitor RFS 2000, difluoromethylornithine, retinoic acid, capecitabine or a pharma- ceutically acceptable salt, solvate or acid thereof, (b) monokines, lymphokines, traditional polypeptide hormones, parathyroid hormone, thyroxine, relaxin, pDLKelaxin, glycoprotein hormones, follicle-stimulating hormone, thyroid-stimulating hormone, luteinizing hormone, hepatic growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor, tumor necrosis factor-α, tumor necrosis factor-β, Müllerian inhibitory factor, mouse gonadotropin-related peptide, inhibin, activin, vascular endothelial growth factor, thrombopoietin, erythropoietin, bone morphogenetic 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, kit ligand or mixtures thereof; (c) diphtheria toxin, botulinum toxin, tetanus toxin, dysentery toxin, cholera toxin, amanitin, α-amanitin, pyrrolobenzodiazepines, pyrrolobenzodiazepine derivatives, indolinobenzodiazepines, pyridobenzodiazepines, tetrodotoxin, brevetoxin, ciguatoxin, ricin, AM toxin, auristatin, tubulysin, geldanamycin, maytansinoids, calicheamicin, daunomycin, doxorubicin, methotrexate, vindesine, SG2285, dolastatin, dolastatin analogs, auristatin, cryptophycin, camptothecin, rhizoxin, rhizoxin derivatives, CC-1065, CC-1065 analogs or derivatives, duocarmycins, enediyne antibiotics, esperamicin, epothilones, toxoids or mixtures thereof. (d) an affinity ligand which is a substrate, an inhibitor, an active agent, a neurotransmitter, a radioisotope, or a mixture thereof; (e) a radioactive label, 32P, 35S, a fluorescent dye, an electron-dense reagent, an enzyme, biotin, streptavidin, dioxygenin, a hapten, an immunogenic protein, a nucleic acid molecule having a sequence complementary to a target, or a mixture thereof; (f) an immunomodulatory compound, an anticancer agent, an antiviral agent, an antibacterial agent, an antifungal agent, an antiparasitic agent, or mixtures thereof; (g) tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxyfene, ketoxifene, LY117018, onapristone or toremifene, (h) 4(5)-imidazole, aminoglutethimide, megestrol acetate, exemestane, letrozole or anastrozole, (i) flutamide, nilutamide, bicalutamide, leuprolide, goserelin or troxacitabine, (j) aromatase inhibitors, (k) a protein kinase inhibitor, (l) lipid kinase inhibitors, (m) antisense oligonucleotides, (n) ribozyme, (o) Vaccines; and (p) Antiangiogenic agent 6. The antibody conjugate of claim 5, or a pharma- ceutically acceptable salt or solvate thereof, selected from:

48. 2. The antibody conjugate of claim 1, or a pharma- ceutically acceptable salt or solvate thereof, wherein the active agent is a pyrrolobenzodiazepine dimer and the linker links the Ab to the N10 or N10' position of the pyrrolobenzodiazepine dimer.

49. The activator is a pyrrolobenzodiazepine dimer, the pyrrolobenzodiazepine dimer is substituted at the N10 position by X or at the N'10 position by X', X or X' connecting the pyrrolobenzodiazepine dimer to a linker; X and X' are each independently -C(O)O*, -S(O)O-*, -C(O)-*, -C(O)NR X -*, -S(O) 2 NR X -*, -P(O)R'NR X -*, -S(O)NR X -* or -PO 2 NR X -*, R X C 1～8 Alkyl, C 3～8 Cycloalkyl, C 3～20 Heteroaryl or C 5～20 is aryl, R X '+ is OH, N 3 , C.N., S.H., 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 amino; 49. The antibody conjugate of claim 48, or a pharma- ceutically acceptable salt or solvate thereof, wherein * is the attachment site between the pyrrolobenzodiazepine dimer and the linker.

50. The pyrrolobenzodiazepine dimer has the following general formula X or general formula XI: 【Chemistry 14】 [In the formula, The dotted lines represent 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 C.N., R. m OR m , =CH-R m’ , =C(R m ') 2 , O-SO 2 -R m , CO 2 R m , C.O.R. m , halo, and dihalo; R m’ is R m , CO 2 R m , C.O.R. 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 selected from the group consisting of cycloalkyl, 3- to 7-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 H and R independently m , O.H., O.R. m , S.H., S.R. m , N.H. 2 , N.H.R. m , N.R. m 2 , NO 2 , Me 3 Selected from among SN and halo; R X4 and R X4’ are H and R independently m , O.H., O.R. m , S.H., S.R. m , N.H. 2 , N.H.R. m , N.R. m 2 , NO 2 , Me 3 SN, Haro, C 1～6 Alkyl, C 1～6 Alkenyl, C 2～6 Alkynyl, C 2～6 Aryl, C 3～6 Heteroaryl, C 3～6 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 5～12 Aryl, 5- to 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 ' is selected from R X and R X ' is independently H, OH, or N 3 , CN, NO 2 , S.H., N.H. 2 , O.N.H. 2 , N.H.N.H. 2 , Haro, 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 mono- or di-C 1～8 alkylamino; Y and Y' are independently selected from O, S and N(H); R x6 is independently C 3～12 Alkylene, C 3～12 Alkenylene or C 3～12 heteroalkylene; R X7 and R X7’ are H, C independently 1～6 Alkyl, C 2～6 Alkenyl, C 2～6 Alkynyl, C 3～6 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 6～10 Aryl, 5-7 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 ', -NRr S (O) 2 NR s R s , -C(O)R r , -C(O)OR s or -C(O)NR r R r ' is selected from Each R r , R r’ , R s and R s’ are H, C independently 1～7 Alkyl, C 2～7 Alkenyl, C 2～7 Alkynyl, C 3～13 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 5～10 aryl and 5- to 7-membered heteroaryl; Each R X8 and R X8’ are H, C independently 1～6 Alkyl, C 2～6 Alkenyl, C 2～6 Alkynyl, C 3～6 Heteroalkyl, 3- to 7-membered heterocycloalkyl, C 5～10 Aryl, 5-7 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 ' is selected from Z a OR X12a , N.R. X12a R X12a or SR X12a is selected from among Z b OR X13a , N.R. X13a R X13a or SR X13a is selected from among Z a’ OR X12a , N.R. X12a R X12a or SR X12a is selected from among Z b’ OR X13a’ , N.R. X13a’ R X13a’ or SR X13a’ is selected from among Each R X12a , R X12a’ , R X13a’ and R x13a’ are H, C independently 1～6 Alkyl, C 2～6 Alkenyl, C 2～6 Alkynyl, C 3～6 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 5～10 Aryl, 5-7 membered heteroaryl, -C(O)R X15a , -C(O)OR X15a and -C(O)NR X15a R X15a’ is selected from Each 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 selected from cycloalkyl, 3- to 7-membered heterocyclyl, 3- to 7-membered heterocycloalkyl, and 5- to 7-membered heteroaryl; R X13a and R X14a optionally together with the atom to which they are attached form a 3- to 7-membered heterocyclyl, a 3- to 7-membered heterocycloalkyl, or a 3- to 7-membered heteroaryl; R X13a’ and R X14a’ optionally together with the atom to which they are attached form a 3- to 7-membered heterocyclyl, a 3- to 7-membered heterocycloalkyl, or a 3- to 7-membered heteroaryl; R n , R n’ , R o , R o’ , R p and R p’ Each of the groups is independently H, C 1～7 Alkyl, C 2～7 Alkenyl, C 2～7 Alkynyl, C 3～13 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 5～10 aryl and 5- to 7-membered heteroaryl. or a pharma- ceutically acceptable salt or solvate thereof.

51. R m C independently 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; R m C 1～12 Alkyl, C 2～12 Alkenyl, C 2～12 Alkynyl, C 5～20 Aryl, C 5～20 Heteroaryl, C 3～6 51. The antibody conjugate of claim 50, or a pharma- ceutically acceptable salt or solvate thereof, which is further substituted with cycloalkyl, 3- to 7-membered heterocyclyl, 3- to 7-membered heterocycloalkyl, or 5- to 7-membered heteroaryl.

52. R x4 and R x4’ are H and R independently m , O.H., O.R. m , S.H., S.R. m , N.H. 2 , N.H.R. m , N.R. m R m’ , 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- to 7-membered heterocycloalkyl, C 5～12 Aryl, 5- to 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 ' is selected from R X4 or R X4’ 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, 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 5～10 Aryl, 5-7 membered heteroaryl, -OR p , -OC(O)R p , -C(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 51. The antibody conjugate of claim 50, or a pharma- ceutically acceptable salt or solvate thereof, further substituted by:

53. 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, 5-7 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 NR s R s , -C(O)R r , -C(O)OR s or -C(O)NR r R r ' is selected from 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, or 5- to 7-membered heteroaryl; 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, 5-7 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 ' and R r , R r’ , R s , R s’ , R t , R t’ , R u and R u’ are independently H, C 1～7 Alkyl, C 2～7 Alkenyl, C 2～7 Alkynyl, C 3～13 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C 5～10 51. The antibody conjugate of claim 50, or a pharma- ceutically acceptable salt or solvate thereof, wherein: R is selected from among aryl and 5- to 7-membered heteroaryl.

54. R x6 C 3～12 Alkylene, C 3～12 Alkenylene or C 3～12 Heteroalkylene, R x6 Ga-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 is replaced by R XX H, OH, N 3 , CN, NO 2 , S.H., N.H. 2 , O.N.H. 2 , N.H.N.H. 2 , Haro, 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 mono- or di-C 1～8 51. The antibody conjugate of claim 50, or a pharma- ceutically acceptable salt or solvate thereof, wherein n is alkylamino and n is an integer from 1 to 6.

55. The active agent is of general formula XII or general formula XIII: 【Chemistry 15】 [X a and X a’ are independently a bond or C 1～6 alkylene; Z X’ and Z X are independently hydrogen, C 1～8 Alkyl, halogen, cyano, nitro, 【Chemistry 16】 or -(CH 2 ) m -OCH 3 is selected from among Each R 80 , R 90 and R 100 is hydrogen, C 1～8 Alkyl, C 2～6 Alkenyl and C 1～6 alkoxy; m is an integer between 0 and 12. or a pharma- ceutically acceptable salt or solvate thereof.

56. The active agent 【Chemistry 17】 【change】 【change】 【change】 【change】 【change】 【change】 or a pharma- ceutically acceptable salt or solvate thereof, selected from the group comprising:

57. A pharmaceutical composition for the prevention or treatment of a proliferative, cancer or angiogenic disease comprising the antibody conjugate of claim 1.

58. 58. The pharmaceutical composition of claim 57, further comprising a pharma- ceutically effective amount of a chemotherapeutic agent.

59. 58. The pharmaceutical composition of claim 57, wherein the cancer is selected from the group comprising lung cancer, small cell lung cancer, gastrointestinal cancer, colon cancer, intestinal 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 melanoma.

60. 13. A method for preventing a proliferative, cancer or angiogenic disease, comprising administering to an individual a pharma- ceutically effective amount of the conjugate of claim 1.

61. 13. A method for treating a proliferative, cancer or angiogenic disease, comprising administering to an individual a pharma- ceutically effective amount of the conjugate of claim 1.

62. 13. Use of the conjugate according to claim 1 as a pharmaceutical composition for the prevention or treatment of a proliferative, cancer or angiogenic disease.

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