Anti-MUC1 antibody-drug conjugate

Abstract

The present invention provides a pharmaceutical composition containing an antibody-drug conjugate useful for the treatment of cancer. [Solution] This disclosure relates to an antibody-drug conjugate for the cancer antigen MUC1. In particular, an antibody with improved antigen binding was obtained by deleting the glycosylation site in the CDR-H2 of a known anti-MUC1 antibody. The conjugate consists of an exatechcan derivative coupled to the anti-MUC1 antibody.

Description

[Technical Field]

[0001] This invention relates to the field of antibody-drug conjugates (ADCs). The ADCs of this invention comprise an anti-MUC1 antibody or a mutant anti-MUC1 antibody. An ADC having a mutant anti-MUC1 antibody with increased antigen-binding affinity is provided. In particular, in the mutant form of the humanized antibody PankoMab, asparagine 57 in the heavy chain variable region is substituted with another amino acid. This results in the deletion of the glycosylation site in the CDR2 region, increasing the antigen-binding affinity. The ADC showed significant antitumor efficacy. In specific embodiments, this invention relates to the therapeutic and diagnostic uses of this antibody-drug conjugate, as well as methods for producing such antibody-drug conjugates. [Background technology]

[0002] Antibodies against tumor-associated antigens are widely used anticancer therapies. Many anticancer antibodies are approved for human therapy today. Some of these antibodies act by blocking specific signaling pathways crucial for the survival or proliferation of certain cancer cells. Other anticancer antibodies activate a patient's immune response against target cancer cells, for example, by inducing antibody-dependent cell-mediated cytotoxicity (ADCC) via natural killer cells. This mechanism is induced by the binding of the antibody's Fc portion to Fc receptors on immune cells.

[0003] An interesting and important group of antibodies are those against mucin proteins. Mucins are a family of high molecular weight, highly glycosylated proteins produced by many epithelial tissues of vertebrates. Mucins can be subdivided into membrane-bound mucin proteins, which have hydrophobic transmembrane domains favorable for retention in the plasma membrane, and mucins that are secreted onto the mucosal surface or become components of saliva. The human mucin protein family consists of many family members, including membrane-bound MUC1.

[0004] Increased mucin production occurs in many adenocarcinomas, including cancers of the pancreas, lung, chest, ovaries, and colon. Mucins are also overexpressed in lung diseases such as asthma, bronchitis, chronic obstructive pulmonary disease, or cystic fibrosis. Two membrane mucins, MUC1 and MUC4, have been extensively studied for their pathological relevance in disease processes. Furthermore, mucins are being investigated for their potential as diagnostic markers. Several antibodies against mucin proteins (Clin. Cancer Res., November 1, 2011; Vol. 17 (No. 21): pp. 6822-6830; PLoS One, January 14, 2011; Vol. 6 (No. 1): e15921), particularly MUC1, are known in the art. However, there may still be room for improvement in their therapeutic efficacy.

[0005] From the above perspective, there is a need in this field to provide therapeutic anti-MUC1 antibodies with improved properties. [Overview of the project]

[0006] ADCs consist of three distinct components (antibody, linker, and drug / payload) responsible for the specific delivery of the payload to target cells. To date, four ADCs (gemtuzumab ozogamicin (Mylotarg®), inotuzumab ozogamicin (Besponsa®), brentuximab vedotin (Adcetris®), and trastuzumab emtansine (T-DM1; Kadcyla®)) have entered the market. In addition, more than 60 ADCs targeting a wide range of hematological and solid tumors are under development. ADCs have created a new paradigm in novel cancer chemotherapy, combining the specificity of monoclonal antibodies with the cytotoxic capabilities of small molecule drugs. Therefore, ADCs are expected to account for a large portion of future precision medicine and combination therapies. Consequently, the need for further ADCs, as well as means, methods, and uses related to the treatment and / or diagnosis of diseases, continues.

[0007] As for ADCs, those in which exatechcan is conjugated to an antibody (e.g., an anti-HER2 antibody) via a linker are known (WO2014 / 057687, WO2015 / 115091). However, ADCs in which exatechcan is conjugated to an anti-MUC1 antibody are not known.

[0008] The inventors have discovered that deletion of the glycosylation site in the heavy chain variable region of the anti-MUC1 antibody PankoMab did not eliminate antigen binding, but rather surprisingly increased the antibody's antigen affinity. This was particularly surprising because the glycosylation site is located in the second complementarity-determining region (CDR-H2) of the heavy chain variable region. The CDR is a region of the antibody that is directly involved in antigen binding and provides contact with the epitope. Therefore, it is generally expected that amino acid modifications of the CDR are detrimental to antigen binding affinity. In addition, the humanized PankoMab antibody contains a glycosylation site in CDR-H2 that maintains a large carbohydrate structure. This carbohydrate structure is directly present at the binding interface to the antigen and was therefore considered to be involved in antigen binding. However, as demonstrated in the examples, a PankoMab mutant (PM-N54Q) in which the glycosylation site is deleted by substituting the amino acid that maintains the carbohydrate structure shows increased antigen binding affinity. In addition, the inventors found that conjugates or antibody-drug conjugates (ADCs) containing PankoMab or a PankoMab variant (PM-N54Q) showed significant antitumor efficacy against MUC1-positive tumors, and that PM-N54Q-ADC showed significantly greater antitumor efficacy compared to PankoMab-ADC.

[0009] Therefore, in the first aspect, the present invention is a conjugate comprising an antibody conjugated to a cytotoxic agent, wherein the antibody is capable of binding to MUC1. (i) A heavy chain variable region including a complementarity-determining region (CDR) CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 2, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and (ii) Light chain variable region including complementarity-determining region (CDR) CDR-L1 having the amino acid sequence of SEQ ID NO: 4, CDR-L2 having the amino acid sequence of SEQ ID NO: 5, and CDR-L3 having the amino acid sequence of SEQ ID NO: 6 This includes information about conjugates.

[0010] In a second aspect, the present invention relates to a composition comprising a conjugate according to the present invention.

[0011] According to a third aspect, the present invention provides compositions or conjugates for use in pharmaceuticals, particularly in the treatment, prevention, or diagnosis of cancer.

[0012] In a fourth aspect, the present invention provides a method for treating cancer in a subject requiring such treatment, comprising the step of administering a therapeutically effective dose of the conjugate according to the present invention to a subject having cancer.

[0013] In a fifth aspect, the present invention provides a kit or device comprising a conjugate according to the present invention, as well as related methods useful for the diagnosis, detection, or monitoring of MUC1-related disorders such as cancer.

[0014] Other objects, features, advantages, and aspects of the present invention will be apparent to those skilled in the art from the following description and the appended claims. The scope and specific examples provided illustrate preferred embodiments of this application, but should be understood to be for illustrative purposes only. Various changes and modifications within the spirit and scope of the invention of this disclosure will be immediately apparent to those skilled in the art upon reading the following.

[0015] definition When used herein, the following expressions are generally intended to have the meanings set forth below, unless the context in which they are used indicates otherwise.

[0016] As used herein, the term "comprise" includes, in addition to its literal meaning, "consist essentially of" and "consist of", and specifically refers to them. Thus, the expression "comprise" refers to embodiments where the subject "comprises" the specifically listed elements and does not include further elements, as well as embodiments where the subject "comprises" the specifically listed elements and may or actually includes further elements. Similarly, the expression "have" should be understood as "comprise", and also includes the expressions "consist essentially of" and "consist of", and specifically refers to them. The term "consist essentially of" refers, where possible, in particular to embodiments where, in addition to the specifically listed elements of which the subject consists essentially, the subject includes further elements that are 20% or less, particularly 15% or less, 10% or less, or particularly 5% or less.

[0017] The term "antibody" refers particularly to a protein comprising at least two heavy chains and two light chains connected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (V H ) and a heavy chain constant region (C H ). Each light chain consists of a light chain variable region (V L ) and a light chain constant region (C L ). The heavy chain constant region includes three, or in the case of IgM or IgE antibodies, four heavy chain constant domains (C H1 , C H2 , C H3 , and C H4 ), the first constant domain C H1 being adjacent to the variable region and the second constant domain C H2 being separated therefrom by a hinge region. H ) and a heavy chain constant region (C H ). Each light chain consists of a light chain variable region (V L ) and a light chain constant region (C L ). The heavy chain constant region includes three, or in the case of IgM or IgE antibodies, four heavy chain constant domains (C H1 , C H2 , C H3 , and C H4 ), the first constant domain C H1 being adjacent to the variable region and the second constant domain C H2 being separated therefrom by a hinge region. H )で構成される。各軽鎖は、軽鎖可変領域(V L )および軽鎖定常領域(C L )で構成される。重鎖定常領域は、3つの、またはIgM型もしくはIgE型の抗体の場合は、4つの重鎖定常ドメイン(C H1 、C H2 、C H3 、およびC H4 )を含み、第1の定常ドメインC H1 は可変領域に隣接しており、ヒンジ領域により第2の定常ドメインC H2It may be connected to the light chain constant region. The light chain constant region consists of only one constant domain. The variable region can be further subdivided into a hypervariable region called the complementarity-determining region (CDR), which is dispersed into a more conserved region called the framework region (FR), with each variable region containing three CDRs and four FRs. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The heavy chain constant region may be of any type, such as a γ, δ, α, μ, or ε heavy chain. Preferably, the antibody heavy chain is a γ chain. Furthermore, the light chain constant region may be of any type, such as a κ or λ light chain. Preferably, the antibody light chain is a κ chain. The terms "γ(δ, α, μ, or ε) heavy chain" and "κ(λ) light chain" refer to antibody heavy chains or antibody light chains having a constant region amino acid sequence derived from a naturally occurring heavy chain or light chain constant region amino acid sequence, particularly a human heavy chain or light chain constant region amino acid sequence, respectively. In particular, the amino acid sequence of the constant domain of the γ-type (especially γ1-type) heavy chain is at least 95%, and especially at least 98%, identical to the amino acid sequence of the constant domain of the human γ (especially human γ1) antibody heavy chain. Furthermore, the amino acid sequence of the constant domain of the κ-type light chain is at least 95%, and especially at least 98%, identical to the amino acid sequence of the constant domain of the human κ antibody light chain. The constant region of the antibody can mediate the binding of immunoglobulins to various cells of the immune system (e.g., effector cells) and to host tissues or factors including the first component (C1q) of the classical complement system. The antibody may be, for example, a humanized antibody, a human antibody, or a chimeric antibody.

[0018] The antigen-binding portion of an antibody typically refers to the entire length of the antibody or one or more fragments that maintain its ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of the full-length antibody. Examples of antibody-binding fragments include: V L , V H , C L , and C H1Fab fragments are monovalent fragments consisting of domains; F(ab)2 fragments are bivalent fragments containing two Fab fragments, each binding to the same antigen, linked by disulfide crosslinks in the hinge region; V H and C H1 Fd fragment consisting of domains; V of a single arm of the antibody L and V H Fv fragments consisting of domains; and V H A dAb fragment consisting of domains.

[0019] The "Fab portion" of an antibody is, in particular, the heavy chain and light chain variable region (V H and V L ) and the first domain of the heavy chain and light chain constant region (C H1 and C L This refers to the portion of the antibody that contains region V. If the antibody does not contain all of these regions, the term "Fab portion" refers to the region V present in the antibody. H , V L , C H1 , and C L It merely refers to the part of the antibody that contains the antigen-binding activity of the antibody, obtained by digesting a natural antibody with papain. In particular, the Fab portion of the antibody includes its antigen-binding site or antigen-binding capacity. Preferably, the Fab portion includes at least the V of the antibody. H Includes the region.

[0020] The "Fc portion" of the antibody is, in particular, the heavy chain constant region 2, 3, and 4 (C) where applicable. H2 , C H3 , and C H4 This refers to the portion of the antibody that contains region C. In particular, the Fc portion contains two of each of these regions. If the antibody does not contain all of these regions, the term "Fab portion" refers to the region C present in the antibody. H2 , C H3 , and C H4 It merely refers to the one described above. Preferably, the Fc portion is at least the C of the antibody. H2The region includes. Preferably, the “Fc portion” refers to the portion of the antibody corresponding to a fragment of the antibody that does not contain antigen-binding activity, obtained by digesting a native antibody with papain. In particular, the Fc portion of the antibody is capable of binding to an Fc receptor and therefore includes, for example, an Fc receptor binding site or Fc receptor binding capacity.

[0021] When used herein, the terms “antibody” and “antibody construct” refer, in certain embodiments, to a group of antibodies or antibody constructs of the same type, respectively. In particular, all antibodies or antibody constructs in a group exhibit the features used to define the antibody or antibody construct. In certain embodiments, all antibodies or antibody constructs in a group have the same amino acid sequence. References to a particular type of antibody, such as an antibody capable of specifically binding to MUC1, refer, in particular, to a group of antibodies of that type.

[0022] The term “antibody,” as used herein, also includes fragments and derivatives of such antibodies. In particular, an “antibody fragment or derivative” is a protein or glycoprotein derived from such antibody that is capable of binding as an antibody to the same antigen, particularly to the same epitope. Thus, in this specification, an antibody fragment or derivative generally refers to a functional fragment or derivative. In particularly preferred embodiments, the antibody fragment or derivative includes a heavy chain variable region. It has been shown that the antigen-binding function of an antibody can be carried out by a fragment of the full-length antibody or a derivative thereof. Examples of antibody fragments include: (i) Fab fragments, which are monovalent fragments consisting of a variable region and the first constant domains of each heavy and light chain; (ii) F(ab)2 fragments, which are bivalent fragments containing two Fab fragments linked by disulfide crosslinks at the hinge region; (iii) Fd fragments, which consist of a variable region and the first constant domain CH1 of the heavy chain; (iv) Fv fragments, which consist of the heavy and light chain variable regions of a single arm of the antibody; (v) scFv fragments, which are Fv fragments consisting of a single polypeptide chain; (vi) (Fv)2 fragments, which consist of two Fv fragments linked together by covalent bonds; (vii) heavy chain variable domains; and (viii) attachments of heavy and light chain variable regions can occur only between molecules but not within molecules. A multibody consisting of a heavy chain variable region and a light chain variable region that are covalently linked together. Antibody derivatives include antibodies that bind to or compete with the same antigen as the parent antibody, but have a different amino acid sequence from the parent antibody from which they are derived. Such antibody fragments and antibody derivatives can be obtained using conventional techniques known to those skilled in the art.

[0023] A target amino acid sequence is "derived from" or "corresponding to" a reference amino acid sequence if the target amino acid sequence shares at least 75%, more preferably at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98%, or at least 99% homology or identity with the corresponding portion of the reference amino acid sequence over its entire length. "Corresponding portion" means, for example, that framework region 1 of the heavy chain variable region (FRH1) of the target antibody corresponds to framework region 1 of the heavy chain variable region of the reference antibody. In certain embodiments, a target amino acid sequence "derived from" or "corresponding to" a reference amino acid sequence is 100% homologous, or in particular 100% identical, to the corresponding portion of the reference amino acid sequence over its entire length. The "homology" or "identity" of an amino acid sequence or nucleotide sequence is preferably determined by the present invention over the entire length of the reference sequence, or over the entire length of the corresponding portion of the reference sequence corresponding to the sequence in which the homology or identity is defined. An antibody derived from a parent antibody, defined by one or more amino acid sequences such as a specific CDR sequence or a specific variable region sequence, is an antibody having an amino acid sequence such as a CDR sequence or a variable region sequence that is at least 75%, preferably at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98%, or at least 99% homologous or identical to the corresponding amino acid sequence of the parent antibody, and is particularly identical. In certain embodiments, an antibody derived from a parent antibody (i.e., its derivative) contains the same CDR sequence as the parent antibody, but the rest of the variable region sequence is different.

[0024] Furthermore, as used herein, the term “antibody” refers to a polyvalent and polyspecific antibody, that is, an antibody construct having more than two binding sites, each binding to the same epitope, as well as an antibody construct having one or more binding sites that bind to a first epitope, one or more binding sites that bind to a second epitope, and even further binding sites that optionally bind to further epitopes.

[0025] "Specific binding" preferably means that an agonist, such as an antibody, binds more strongly to a target, such as an epitope, that is specific to that target compared to binding to other targets. An example of a criterion for determining whether binding is specific is the dissociation constant (referred to herein as "K"). D One example is the agonist, which has a dissociation constant (K) lower than that of the second target. d When binding to the first target, the agonist binds more strongly to the first target compared to the second target. Preferably, the dissociation constant of the target to which the agonist specifically binds is 100, 200, 500, or 1000 times lower than the dissociation constant of the target to which the agonist does not specifically bind. Furthermore, the term "specific binding" specifically indicates the binding affinity between binding partners, and the affinity constant K a at least 10 6 M -1 Preferably at least 10 7 M -1 , more preferably at least 10 8 M -1 Therefore, antibodies specific to a particular antigen are, in particular, at least 10 6 M -1 Preferably at least 10 7 M -1 , more preferably at least 10 8 M -1 K a This refers to an antibody capable of binding to the antigen with an affinity having a certain affinity. For example, the term "anti-MUC1 antibody" specifically refers to an antibody that binds to MUC1 specifically, preferably at least 10 6 M -1 Preferably at least 107 M -1 , more preferably at least 10 8 M -1 K a This refers to an antibody capable of binding to MUC1 with a certain affinity.

[0026] The term "MUC1" refers to mucin-1, pleomorphic epithelial mucin (PEM), or anti-cancer agent. This term refers to the protein MUC1, also known as Proto-15-3, and more specifically, human MUC1 (accession number P15941). MUC1 is a member of the mucin family and encodes a membrane-bound glycosylated phosphoprotein. MUC1 has a core protein mass of 120–225 kDa, which increases to 250–500 kDa upon glycosylation. MUC1 extends 200–500 nm beyond the cell surface. This protein is anchored to the apical surface of many epithelial cells by a transmembrane domain. The extracellular domain contains a 20-amino acid variable number tandem repeat (VNTR) domain, the number of repeats varying from 20 to 120 depending on the individual. These repeats are rich in serine, threonine, and proline residues, enabling high levels of O-glycosylation. In certain embodiments, the term "MUC1" refers to tumor-associated MUC1 ("TA-MUC1"). TA-MUC1 is a variant of MUC1 found on cancer cells. This MUC1 differs from MUC1 found on non-cancer cells in that it is expressed at much higher levels, is more localized, and is glycosylated. In particular, TA-MUC1 is nonpolarly distributed across the entire cell surface of cancer cells, whereas in non-cancer cells, MUC1 exhibits strictly apical expression and is therefore inaccessible to systemically administered antibodies. Furthermore, TA-MUC1 possesses novel peptide epitopes in its MUC1 protein backbone and abnormal O-glycosylation that exposes novel carbohydrate tumor antigens such as Thomsen-Friedenreich antigen alpha (TFα).

[0027] "TFα," also known as Thomsen-Friedenreich antigen alpha or core-1, refers to the disaccharide Gal-β1,3-GalNAc, which is O-glycosidically linked in an alpha-anomeric configuration to the hydroxyamino acid serine or threonine of a protein in cancer cells.

[0028] The term "sialic acid" refers in particular to any N- or O-substituted derivative of neuraminic acid. This term can refer to both 5-N-acetylneuraminic acid and 5-N-glycolylneuraminic acid, but preferably only 5-N-acetylneuraminic acid. Sialic acid, in particular 5-N-acetylneuraminic acid, is preferably attached to the carbohydrate chain by 2,3- or 2,6-coupling. Preferably, the antibodies described herein contain both 2,3- and 2,6-coupled sialic acids.

[0029] In the present invention, "relative amount of glycan" refers to a specific percentage or percentage range of glycan attached to the antibody in an antibody preparation or to the antibody in an antibody-containing composition. In particular, the relative amount of glycan refers to a specific percentage or percentage range of all glycan contained in the antibody, and therefore attached to the peptide chain of the antibody in the antibody preparation or in an antibody-containing composition. 100% of glycan refers to all glycan attached to the antibody in an antibody preparation or to the antibody in an antibody-containing composition. For example, a relative amount of glycan holding bipartite GlcNAc of 10% refers to an antibody-containing composition in which 10% of all glycan contained in the antibody, and therefore attached to the antibody polypeptide chain in the composition, contains bipartite GlcNAc residues, but 90% of all glycan contained in the antibody, and therefore attached to the antibody polypeptide chain in the composition, does not contain bipartite GlcNAc residues. The corresponding reference amount of glycan equivalent to 100% may be any of the glycan structures attached to the antibody in the composition, or all N-glycans, i.e., all glycan structures attached to the asparagine residues of the antibody in the composition, or all complex glycans. The reference set of glycan structures is generally explicitly indicated or can be directly inferred from the context by those skilled in the art.

[0030] The term "N-glycosylation" refers to the asparagine residue of a protein polypeptide chain. This refers to all glycans attached to asparagine residues. These asparagine residues are generally part of an N-glycosylation site having the amino acid sequence Asn-Xaa-Ser / Thr (wherein Xaa may be any amino acid other than proline). Similarly, “N-glycan” is a glycan attached to an asparagine residue of a polypeptide chain. The terms “glycan,” “glycan structure,” “carbohydrate,” “carbohydrate chain,” and “carbohydrate structure” are generally used synonymously herein. N-glycans generally have a common core structure consisting of two N-acetylglucosamine (GlcNAc) residues and three mannose residues, with the structure Manα1,6-(Manα1,3-)Manβ1,4-GlcNAcβ1,4-GlcNAcβ1-Asn, where Asn is the asparagine residue of the polypeptide chain. N-glycans are subdivided into three distinct types: complex glycans, hybrid glycans, and high-mannose glycans.

[0031] The numerical values ​​shown herein, in particular the relative amounts of specific glycosylation properties, should preferably be understood as approximate. In particular, the values ​​are preferably higher and / or lower than 10% at most, and may be particularly higher and / or lower than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% at most.

[0032] The terms “antibody-drug conjugate” (ADC) or “conjugate,” as used herein, generally refer to an antibody or its antigen-binding fragment being linked to another agonist, such as a chemotherapeutic agent, toxin, immunotherapy agent, or imaging probe. The linkage may be covalent or non-covalent, such as through electrostatic interaction. Various linkers known in the art and described herein can be used to form antibody-drug conjugates. In addition, antibody-drug conjugates can be provided in the form of fusion proteins that can be expressed from polynucleotides encoding an immunoconjugate. As used herein, “fusion protein” refers to a protein created by joining two or more genes or gene fragments that originally encoded separate proteins (including peptides and polypeptides). Translation of the fusion gene results in a single protein with functional properties derived from each of the original proteins.

[0033] In a "conjugate," two or more compounds are linked together. In certain embodiments, at least some of the properties of each compound are maintained in the conjugate. Linking can be achieved by covalent or non-covalent bonds. Preferably, the compounds in the conjugate are linked by covalent bonds. The different compounds in the conjugate may be directly bonded to each other via one or more covalent bonds between the atoms of the compounds. Alternatively, the compounds may be bonded to each other via a chemical moiety such as a linker molecule, where the linker is covalently attached to the atoms of the compounds. If the conjugate consists of more than two compounds, such compounds may be linked in a chain conformation, for example, one attached to the next compound, or several compounds each attached to one central compound.

[0034] The term "nucleic acid" includes single-stranded and double-stranded nucleic acids, as well as ribonucleic acid and deoxyribonucleic acid. Nucleic acids may include naturally occurring and synthetic nucleotides, which may be naturally or synthetically modified, for example, by methylation, 5'-capping, and / or 3'-capping.

[0035] The term "expression cassette" specifically refers to a nucleic acid construct that enables and regulates the expression of a coding nucleic acid sequence introduced therein. An expression cassette may include a promoter, a ribosome binding site, an enhancer, and other regulatory elements that regulate gene transcription or mRNA translation. The exact structure of an expression cassette varies depending on the species or cell type. The expression cassette may vary as a function of the expression, but generally includes 5'-untranscribed sequences and 5'- and 3'-untranslated sequences involved in the initiation of transcription and translation, respectively, such as TATA boxes, capping sequences, and CAAT sequences. More specifically, the 5'-untranscribed expression regulatory sequence includes a promoter region containing a promoter sequence for the transcriptional regulation of a operably connected nucleic acid. The expression cassette may also include enhancer sequences or upstream activator sequences.

[0036] According to the present invention, the term “promoter” refers to a nucleic acid sequence located upstream (5') of the nucleic acid sequence to be expressed, which controls the expression of the sequence by providing a recognition and binding site for RNA polymerase. The “promoter” may also include further recognition and binding sites for further factors involved in the transcriptional regulation of the gene. Promoters can control the transcription of prokaryotic or eukaryotic genes. Furthermore, promoters may be “inducible,” meaning they may initiate transcription in response to an inducer, or they may be “constitutive,” meaning transcription is not regulated by an inducer. In the absence of an inducer, a gene under the control of an inducible promoter is not expressed or is expressed to a low degree. In the presence of an inducer, the gene is switched on or its transcription level increases. This is generally mediated by the binding of a specific transcription factor.

[0037] The term “vector” is used herein in its most common sense and includes, for example, any mediating vehicle for nucleic acids that enables the introduction of nucleic acids into prokaryotic and / or eukaryotic cells and, where appropriate, their integration into a genome. This type of vector is preferably replicated and / or expressed in cells. Vectors include plasmids, phagemids, bacteriophages, or viral genomes. The term “plasmid,” as used herein, generally refers to a construct of extrachromosomal genetic material, usually a circular double-stranded DNA, that can replicate independently of chromosomal DNA.

[0038] According to the present invention, the term “host cell” refers to any cell that can be transformed or transfected with an exogenous nucleic acid. According to the present invention, the term “host cell” includes prokaryotic cells (e.g., Escherichia coli) or eukaryotic cells (e.g., mammalian cells, particularly human cells, yeast cells, and insect cells). Mammalian cells, such as cells derived from humans, mice, hamsters, pigs, goats, or primates, are particularly preferred. The cells may be derived from a number of tissue types and include primary cells and cell lines. The nucleic acid may be present in the host cell in the form of a single copy or two or more copies, and in one embodiment, it is expressed in the host cell.

[0039] The term "patient" according to the present invention means a human, a non-human primate, or another animal, particularly mammals such as cattle, horses, pigs, sheep, goats, dogs, cats, or rodents such as mice and rats. In a particularly preferred embodiment, the patient is human.

[0040] The term "cancer" according to the present invention includes, in particular, leukemia, hemihydrocarcinoma, melanoma, carcinoma, teratoma, lymphoma, sarcoma, mesothelioma, neuroblastoma, glioma, rectal cancer, endometrial cancer, kidney cancer, adrenal cancer, thyroid cancer, hematological cancer, skin cancer, brain cancer, cervical cancer, intestinal cancer, liver cancer, colon cancer, stomach cancer, intestine cancer, head and neck cancer, gastrointestinal cancer, lymph node cancer, esophageal cancer, colorectal cancer, pancreatic cancer, ENT cancer, breast cancer, prostate cancer, bladder cancer, uterine cancer, ovarian cancer, and lung cancer, as well as their metastases. The term "cancer" according to the present invention also includes cancer metastases.

[0041] The term "tumor" refers to a group of cells or tissue formed by malregulated cell proliferation. Tumors may exhibit a partial or complete lack of structural organization and functional coordination with normal tissue, and typically form separate tissue masses, and may be either benign or malignant.

[0042] The terms "tumor" and "cancer" are used synonymously.

[0043] The term "metastasis" refers to the spread of cancer cells from their original site to another part of the body. The formation of metastasis is a very complex process that typically involves cancer cells detaching from the primary tumor, entering the body's circulation, and establishing and growing within normal tissue elsewhere in the body. When tumor cells metastasize, the new tumor is called a secondary tumor or metastatic tumor, and its cells are usually similar to those in the original tumor. This means, for example, that if breast cancer metastasizes to the lungs, the secondary tumor will consist of abnormal breast cells rather than abnormal lung cells. In that case, the tumor in the lungs is called metastatic breast cancer, not lung cancer.

[0044] The term “pharmaceutical composition” refers, in particular, to a composition suitable for administration to humans or animals, i.e., a composition containing pharmaceutically acceptable components. Preferably, a pharmaceutical composition contains an active compound or a salt or prodrug thereof, together with pharmaceutical excipients such as carriers, diluents, or buffers, preservatives, and tonicity modifiers. Numerical ranges described herein include numerical values ​​that define the range. The headings provided herein are not intended to limit the various aspects or embodiments of the invention that can be understood by referring to this specification as a whole. Subject matter described herein, in the case of a method, as comprising a particular step, or in the case of a composition, as comprising a particular component, refers to subject matter consisting of the respective step or component. It is preferable to select and combine preferred aspects and embodiments described herein, and specific subjects derived from each combination of preferred embodiments also belong to this disclosure. [Modes for carrying out the invention]

[0045] This invention is based on the development of a mutant (PM-N54Q) of the humanized anti-MUC1 antibody PankoMab in which the glycosylation site of CDR-H2 is deleted. The deletion of the glycosylation site was achieved by substituting amino acid Asn (asparagine) 57 (i.e., amino acid number 57 in SEQ ID NO: 11) in the heavy chain variable region with another amino acid, specifically Gln (glutamine). Asn 57 is the acceptor amino acid residue of the glycosylation site to which the carbohydrate structure is attached. Since the carbohydrate structure can only be translocated to the asparagine residue by host cell enzymes, substituting this asparagine residue with another residue eliminates the glycosylation. Surprisingly, the deletion of the glycosylation site of CDR-H2 in PankoMab was found to increase the antigen-binding affinity of the antibody.

[0046] In light of these findings, the present invention relates to a conjugate comprising an antibody conjugated to a cytotoxic agent, wherein the antibody is capable of binding to MUC1. (i) A heavy chain variable region including a complementarity-determining region (CDR) CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 2, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and (ii) Light chain variable region including complementarity-determining region (CDR) CDR-L1 having the amino acid sequence of SEQ ID NO: 4, CDR-L2 having the amino acid sequence of SEQ ID NO: 5, and CDR-L3 having the amino acid sequence of SEQ ID NO: 6 It provides a conjugate that includes [this].

[0047] Binding to MUC1 The antibody specifically binds to an epitope of MUC1. The epitope is located in the extracellular tandem repeat of MUC1. In certain embodiments, the antibody binds to MUC1 in a glycosylation-dependent manner. In particular, the antibody binds to an N-A threonine residue in the tandem repeat. Cetylgalactosamine (Tn), sialyl α2-6N-acetylgalactosamine (sTn), galactose β1-3N-acetylgalactosamine (TF), or galactose β1-3(sialyl α2-6)N-acetylgalactosamine (sTF), preferably glycosylated with Tn or TF, bind more strongly. Preferably, the carbohydrate moiety is bound to the threonine residue by an α-O-glycosidic bond. Epitopes in the tandem repeat domain of MUC1 include, in particular, the amino acid sequences PDTR (SEQ ID NO: 13) or PESR (SEQ ID NO: 14). Binding to this epitope is preferably glycosylation-dependent as described above, and binding is increased in particular when the carbohydrate moiety described above is attached to the threonine residue of sequences PDTR or PESR (SEQ ID NOs: 13 and 14), respectively.

[0048] The epitope is a tumor-associated MUC1 epitope (TA-MUC1). The TA-MUC1 epitope specifically refers to an MUC1 epitope that is present on tumor cells but not on normal cells, and / or, if present on tumor cells, is only accessible by antibodies in host circulation, and not accessible if present on normal cells. In certain embodiments, the binding of an antibody to cells expressing a TA-MUC1 epitope is stronger than the binding to cells expressing normal, non-tumor MUC1. Preferably, the binding is at least 1.5 times stronger, preferably at least 2 times stronger, at least 5 times stronger, at least 10 times stronger, or at least 100 times stronger. In the case of TA-MUC1 binding, the antibody preferably specifically binds to the glycosylated MUC1 tumor epitope, and the binding strength is at least 2 times, preferably 4 times, or 10 times, most preferably 20 times stronger, compared to binding to a non-glycosylated peptide of the same length and identical peptide sequence. The aforementioned binding can be assayed or determined by ELISA, RIA, or surface plasmon resonance (hereinafter referred to as "SPR") analysis. Examples of instruments used for SPR analysis include: BlAcore® (manufactured by GE Healthcare Bio-Sciences Crop.), ProteOn® (manufactured by Bio-Rad Laboratories, Inc.), DRX2 biosensor (manufactured by Dynamic Biosensors GmbH), SPR-Navi® (manufactured by BioNavis Oy Ltd.), Spreeta® (manufactured by Texas Instruments Inc.), SPRi-PlexII® (manufactured by Horiba, Ltd.), and Autolab SPR® (manufactured by Metrohm). The binding of antibodies to antigens expressed on the cell surface can be assayed by flow cytometry or other methods.

[0049] Furthermore, the antibody can exhibit antigen-binding properties similar to those of a reference antibody, which includes a heavy chain variable region having the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 10 and a light chain variable region having the amino acid sequence of SEQ ID NO: 12. Preferably, the reference antibody is a humanized antibody PankoMab. In particular, the antibody specifically binds to the same antigen as the reference antibody, preferably with a higher affinity. That is, the antibody preferably binds to the antigen with an affinity having a dissociation constant lower than, more preferably 10% lower, at least 20% lower, at least 30% lower, or at least 50% lower than, the dissociation constant of the reference antibody. Furthermore, the antibody preferably exhibits cross-specificity with a reference antibody, which includes a heavy chain variable region having the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 10 and a light chain variable region having the amino acid sequence of SEQ ID NO: 12. In particular, when a humanized antibody is present at a sufficiently high concentration, it can prevent the binding of the reference antibody to MUC1. This is possible if the binding of the reference antibody to MUC1 is inhibited when the antibody is already bound to the antigen MUC1.

[0050] Anti-MUC1 antibody Antibodies capable of binding to MUC1 include the complementarity-determining region (CDR) CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 2, and It includes a heavy chain variable region containing CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and a light chain variable region containing complementarity-determining region (CDR) CDR-L1 having the amino acid sequence of SEQ ID NO: 4, CDR-L2 having amino acid sequence 5, and CDR-L3 having the amino acid sequence of SEQ ID NO: 6.

[0051] In certain embodiments, the heavy chain variable region includes an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 9. In particular, the heavy chain variable region includes an amino acid sequence that is at least 95%, and especially at least 98%, identical to the amino acid sequence of SEQ ID NO: 9. In such embodiments, the heavy chain variable region still includes a CDR having the amino acid sequences of SEQ ID NOs: 1, 2, and 3. Therefore, any sequence deviation from SEQ ID NO: 9 is located in the framework region but not in the CDR. In particular, the heavy chain variable region includes the amino acid sequence of SEQ ID NO: 9.

[0052] In certain embodiments, CDR-H2 has the amino acid sequence of SEQ ID NO: 2, where the amino acid at position 8 of SEQ ID NO: 2 is selected from the group consisting of glutamine, alanine, valine, histidine, tryptophan, tyrosine, lysine, and arginine, particularly glutamine, histidine, tryptophan, tyrosine, lysine, and arginine. Preferably, the amino acid at position 8 of SEQ ID NO: 2 is glutamine, histidine, tryptophan, lysine, or arginine, particularly glutamine. In particular, CDR-H2 has the amino acid sequence of SEQ ID NO: 7.

[0053] In a particular embodiment, CDR-H2 has the amino acid sequence of SEQ ID NO: 8.

[0054] In specific embodiments, the heavy chain variable region includes an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 10. In particular, the heavy chain variable region includes an amino acid sequence that is at least 95%, and especially at least 98%, identical to the amino acid sequence of SEQ ID NO: 10. In such embodiments, the heavy chain variable region includes CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 7, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3. Therefore, any sequence deviation from SEQ ID NO: 10 is located in the framework region but not in the CDR. In particular, the heavy chain variable region includes the amino acid sequence of SEQ ID NO: 10.

[0055] In specific embodiments, the heavy chain variable region includes an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 11. In particular, the heavy chain variable region includes an amino acid sequence that is at least 95%, and especially at least 98%, identical to the amino acid sequence of SEQ ID NO: 11. In such embodiments, the heavy chain variable region includes CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 8, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3. Therefore, any sequence deviation from SEQ ID NO: 11 is located in the framework region but not in the CDR. In particular, the heavy chain variable region includes the amino acid sequence of SEQ ID NO: 11.

[0056] In certain embodiments, the light chain variable region includes an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 12. In particular, the light chain variable region includes an amino acid sequence that is at least 95%, and especially at least 98%, identical to the amino acid sequence of SEQ ID NO: 12. In such embodiments, the light chain variable region still includes a CDR having the amino acid sequences of SEQ ID NOs: 4, 5, and 6. Therefore, any sequence deviation from SEQ ID NO: 12 is located in the framework region but not in the CDR. In particular, the light chain variable region includes the amino acid sequence of SEQ ID NO: 12.

[0057] In a specific embodiment, the heavy chain variable region is at least 90 amino acids from the amino acid sequence of SEQ ID NO: 9. The CDR has an amino acid sequence that is % identical, and still has the amino acid sequences of SEQ ID NOs: 1, 2, and 3. The light chain variable region has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NOs: 12, and still has the amino acid sequences of SEQ ID NOs: 4, 5, and 6. In particular, the heavy chain variable region has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NOs: 9, and still has the amino acid sequences of SEQ ID NOs: 1, 2, and 3. The light chain variable region has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NOs: 12, and still has the amino acid sequences of SEQ ID NOs: 4, 5, and 6.

[0058] In a specific embodiment, the heavy chain variable region has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 10, and the CDR still has the amino acid sequences of SEQ ID NOs: 1, 7, and 3; the light chain variable region has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 12, and the CDR still has the amino acid sequences of SEQ ID NOs: 4, 5, and 6. In particular, the heavy chain variable region has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 10, and the CDR still has the amino acid sequences of SEQ ID NOs: 1, 7, and 3; the light chain variable region has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 12, and the CDR still has the amino acid sequences of SEQ ID NOs: 4, 5, and 6.

[0059] In a specific embodiment, the heavy chain variable region has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 11, and the CDR still has the amino acid sequences of SEQ ID NOs: 1, 8, and 3; the light chain variable region has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 12, and the CDR still has the amino acid sequences of SEQ ID NOs: 4, 5, and 6. In particular, the heavy chain variable region has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 11, and the CDR still has the amino acid sequences of SEQ ID NOs: 1, 8, and 3; the light chain variable region has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 12, and the CDR still has the amino acid sequences of SEQ ID NOs: 4, 5, and 6.

[0060] In specific embodiments, the heavy chain variable region includes an amino acid sequence that is at least 90% identical to the amino acid sequence represented by amino acid numbers 20-136 of SEQ ID NO: 20. In particular, the heavy chain variable region includes an amino acid sequence that is at least 95%, and especially at least 98%, identical to the amino acid sequence represented by amino acid numbers 20-136 of SEQ ID NO: 20. In such embodiments, the heavy chain variable region includes CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 2, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3. Therefore, any sequence deviation from the amino acid sequence represented by amino acid numbers 20-136 of SEQ ID NO: 20 is located in the framework region but not in the CDR. In particular, the heavy chain variable region includes the amino acid sequence represented by amino acid numbers 20-136 of SEQ ID NO: 20. In certain embodiments, the amino acid at position 76 of SEQ ID NO: 20 is selected from the group consisting of glutamine, alanine, valine, histidine, tryptophan, tyrosine, lysine, and arginine, particularly glutamine, histidine, tryptophan, tyrosine, lysine, and arginine. Preferably, the amino acid at position 76 of SEQ ID NO: 20 is glutamine, histidine, tryptophan, lysine, or arginine, particularly glutamine. In particular, CDR-H2 includes the amino acid sequence of SEQ ID NO: 7, and / or the heavy chain variable region includes the amino acid sequence represented by amino acid numbers 20-136 of SEQ ID NO: 23.

[0061] In a specific embodiment, the light chain variable region includes an amino acid sequence that is at least 90% identical to the amino acid sequence represented by amino acid numbers 21-133 of SEQ ID NO: 21. In particular, the light chain variable region includes an amino acid sequence that is at least 90% identical to the amino acid sequence represented by amino acid numbers 21-133 of SEQ ID NO: 21 It contains an amino acid sequence that is at least 95%, and especially at least 98%, identical. In such embodiments, the light chain variable region still contains a CDR having the amino acid sequences of SEQ ID NOs: 4, 5, and 6. Therefore, any sequence deviation from the amino acid sequence represented by amino acid numbers 21-133 of SEQ ID NO: 21 is located in the framework region but not in the CDR. In particular, the light chain variable region contains the amino acid sequence represented by amino acid numbers 21-133 of SEQ ID NO: 21.

[0062] In a specific embodiment, the heavy chain variable region has an amino acid sequence that is at least 90% identical to the amino acid sequence represented by amino acid numbers 20-136 of SEQ ID NO: 20, and the CDR still has the amino acid sequences of SEQ ID NOs: 1, 7, and 3; the light chain variable region has an amino acid sequence that is at least 90% identical to the amino acid sequence represented by amino acid numbers 21-133 of SEQ ID NO: 21, and the CDR still has the amino acid sequences of SEQ ID NOs: 4, 5, and 6. In particular, the heavy chain variable region has an amino acid sequence that is at least 95% identical to the amino acid sequence represented by amino acid numbers 20-136 of SEQ ID NO: 20, and the CDR still has the amino acid sequences of SEQ ID NOs: 1, 7, and 3; the light chain variable region has an amino acid sequence that is at least 95% identical to the amino acid sequence represented by amino acid numbers 21-133 of SEQ ID NO: 21, and the CDR still has the amino acid sequences of SEQ ID NOs: 4, 5, and 6.

[0063] In specific embodiments, the heavy chain includes an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 15. In particular, the heavy chain includes an amino acid sequence that is at least 95%, and especially at least 98%, identical to the amino acid sequence of SEQ ID NO: 15. In such embodiments, the heavy chain includes CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 2, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3. Therefore, any sequence deviation from SEQ ID NO: 15 is located in the framework region but not in the CDR. In particular, the heavy chain includes the amino acid sequence of SEQ ID NO: 15. In certain embodiments, the amino acid at position 57 of SEQ ID NO: 15 is selected from the group consisting of glutamine, alanine, valine, histidine, tryptophan, tyrosine, lysine, and arginine, and especially glutamine, histidine, tryptophan, tyrosine, lysine, and arginine. Preferably, the amino acid at position 57 of SEQ ID NO: 15 is glutamine, histidine, tryptophan, lysine, or arginine, particularly glutamine. In particular, CDR-H2 contains the amino acid sequence of SEQ ID NO: 7, and / or the heavy chain variable region contains the amino acid sequence represented by amino acid numbers 20-136 of SEQ ID NO: 22.

[0064] In specific embodiments, the heavy chain includes an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 19. In particular, the heavy chain includes an amino acid sequence that is at least 95%, and especially at least 98%, identical to the amino acid sequence of SEQ ID NO: 19. In such embodiments, the heavy chain includes CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 8, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3. Therefore, any sequence deviation from SEQ ID NO: 19 is located in the framework region but not in the CDR. In particular, the heavy chain includes the amino acid sequence of SEQ ID NO: 19.

[0065] In specific embodiments, the light chain includes an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 16. In particular, the light chain includes an amino acid sequence that is at least 95%, and especially at least 98%, identical to the amino acid sequence of SEQ ID NO: 16. In such embodiments, the light chain still includes a CDR having the amino acid sequences of SEQ ID NOs: 4, 5, and 6. Therefore, any sequence deviation from SEQ ID NO: 16 is located in the framework region but not in the CDR. In particular, the light chain includes the amino acid sequence of SEQ ID NO: 16.

[0066] In a specific embodiment, the heavy chain is at least 90% identical to the amino acid sequence of SEQ ID NO: 15. The CDR has the amino acid sequence of sequence numbers 1, 7, and 3, the light chain variable region has an amino acid sequence that is at least 90% identical to the amino acid sequence of sequence number 16, and the CDR still has the amino acid sequences of sequence numbers 4, 5, and 6. In particular, the heavy chain has an amino acid sequence that is at least 95% identical to the amino acid sequence of sequence number 15, the CDR still has the amino acid sequences of sequence numbers 1, 7, and 3, the light chain has an amino acid sequence that is at least 95% identical to the amino acid sequence of sequence number 16, and the CDR still has the amino acid sequences of sequence numbers 4, 5, and 6.

[0067] In a specific embodiment, the heavy chain has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 19, the CDR still has the amino acid sequences of SEQ ID NOs: 1, 8, and 3, the light chain has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 16, and the CDR still has the amino acid sequences of SEQ ID NOs: 4, 5, and 6. In particular, the heavy chain has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 19, the CDR still has the amino acid sequences of SEQ ID NOs: 1, 8, and 3, the light chain has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 16, and the CDR still has the amino acid sequences of SEQ ID NOs: 4, 5, and 6.

[0068] Antibodies include and encompass their modified forms. Modified forms of antibodies mean antibodies that have undergone chemical or biological modifications. Chemically modified forms include forms in which the amino acid backbone is conjugated with a chemical moiety, and forms having chemically modified N-linked or O-linked carbohydrate chains. The chemical moiety or form may be toxic or cytotoxic. Biologically modified forms include forms that have undergone post-translational modifications (e.g., N-linked or O-linked glycosylation, N-terminal or C-terminal processing, deamidation, aspartic acid isomerization, or methionine oxidation), and forms containing a methionine residue added to the N-terminus by expression using prokaryotic host cells. Such modified forms are also intended to include forms labeled to enable detection or isolation of the antibody or antigen, e.g., enzyme-labeled forms, fluorescently labeled forms, or affinity-labeled forms. Such modified forms of antibodies according to the present invention are useful for improving the stability or blood retention of the original antibody, reducing antigenicity, detecting or isolating antibodies or antigens, and so on.

[0069] In particular, the antibody may include one or more modifications selected from the group consisting of defucosylation, fucose reduction, N-linked glycosylation, O-linked glycosylation, N-terminal processing, C-terminal processing, deamidation, aspartic acid isomerization, methionine oxidation, substitution of two leucine (L) residues at positions 234 and 235 (according to the EU index) of the heavy chain with alanine (A) (LALA), amidation of a proline residue, and deletion or absence of one, two, or three amino acids at the carboxyl terminus. In specific embodiments, the antibody lacks one, two, or three carboxyl-terminal amino acids on one or both heavy chains, or lacks two carboxyl-terminal amino acids, and the carboxyl-terminal proline residue on one or both heavy chains is amidated.

[0070] Such modifications can be fabricated at any or desired location on the antibody. Alternatively, the same, two, or more different modifications can be fabricated at one, two, or more locations on the antibody.

[0071] For example, antibodies produced by culturing mammalian cells are known to lack the carboxyl-terminal lysine residue in their heavy chain (Journal of Chromatography A, Vol. 705: pp. 129-134 (1995)). Also, the two carboxyl terminals of the heavy chain The ruboxyl terminal amino acid residues (i.e., glycine and lysine) may be absent, and it is known that a newly located carboxyl-terminal proline residue may be amidated (Analytical Biochemistry, Vol. 360: pp. 75-83 (2007)). However, such absences or modifications of the heavy chain sequence do not affect the antibody's ability to bind to its antigen or its effector function (complement activation, antibody-dependent cytotoxicity, etc.).

[0072] In certain embodiments, the antibody comprises a deletion or absence of one or two amino acids at the carboxyl terminus of the heavy chain and has an amidated residue (e.g., an amidated proline residue at the carboxyl terminus of the heavy chain). However, the antibody is not limited to the types described above, as long as the deletion mutant maintains its ability to bind to the antigen.

[0073] In certain embodiments, the two heavy chains of the antibody may consist of any one type of heavy chain selected from the group consisting of full-length heavy chains and deletion mutant heavy chains, or any combination of any two types selected from them. The quantitative ratio of deletion mutant heavy chains depends on the type of cultured mammalian cells producing the antibody and the cell culture conditions.

[0074] In specific embodiments, the antibody may contain two heavy chains, both lacking one carboxyl-terminal amino acid residue.

[0075] In specific embodiments, the antibody comprises a heavy chain having an amino acid sequence represented by amino acid numbers 1 to 446 of SEQ ID NO: 15 or 22, and a light chain having an amino acid sequence represented by amino acid numbers 1 to 219 of SEQ ID NO: 16. In certain embodiments, the amino acid at position 57 of SEQ ID NO: 15 is selected from the group consisting of glutamine, alanine, valine, histidine, tryptophan, tyrosine, lysine, and arginine, particularly glutamine, histidine, tryptophan, tyrosine, lysine, and arginine. Preferably, the amino acid at position 57 of SEQ ID NO: 15 is glutamine, histidine, tryptophan, lysine, or arginine, particularly glutamine.

[0076] In a specific embodiment, the antibody comprises a heavy chain having an amino acid sequence represented by amino acid numbers 1 to 446 of SEQ ID NO: 19, and a light chain having an amino acid sequence represented by amino acid numbers 1 to 219 of SEQ ID NO: 16.

[0077] In a particular embodiment, the antibody competes for binding to TA-MUC1 with an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 10 and a light chain variable region having the amino acid sequence of SEQ ID NO: 12, or an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 11 and a light chain variable region having the amino acid sequence of SEQ ID NO: 12.

[0078] In certain embodiments, the antibody has the property of (a) specifically binding to MUC1, and / or (b) having the activity to be internally translocated into MUC1-expressing cells by binding to MUC1. In certain embodiments, the antibody comprises at least one antibody heavy chain. In particular, the antibody comprises two antibody heavy chains. The antibody heavy chain comprises, in particular, a VH domain, a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain. In certain other embodiments, the antibody heavy chain comprises the CH2 domain and the CH3 domain, but does not contain the CH1 domain. In further embodiments, one or more constant domains of the heavy chain may be substituted with other domains, in particular analogous domains such as albumin. The antibody heavy chain may be of any type comprising a γ chain, an α chain, an ε chain, a δ chain, and a μ chain, preferably a γ chain comprising a γ1 chain, a γ2 chain, a γ3 chain, and a γ4 chain, and particularly a γ1 chain. Thus, the antibody is preferably an IgG1 type antibody, an IgG3 type antibody, or These are IgG type antibodies, such as IgG4 type antibodies, and especially IgG1 type antibodies.

[0079] In particular, the antibody further comprises at least one antibody light chain, especially two antibody light chains. The antibody light chain particularly comprises a VL domain and a CL domain. The antibody light chain may be a κ chain or a λ chain, especially a κ chain.

[0080] In certain embodiments, the antibody comprises two antibody heavy chains and two antibody light chains. Specifically, the antibody comprises two γ1-type antibody heavy chains, each containing a VH domain, a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain, and two κ-type antibody light chains, each containing a VL domain and a CL domain.

[0081] In alternative embodiments, the antibody does not include an antibody light chain. In such embodiments, the light chain variable region may be fused to the N-terminus of the heavy chain variable region, or inserted into the C-terminus of the heavy chain variable region. A peptide linker may be present to connect the light chain variable region to the rest of the heavy chain.

[0082] In preferred embodiments, the antibody includes an Fc region. The antibody may also be an entire antibody comprising two heavy chains, each containing domains VH, CH1, hinge region, CH2, and CH3, and two light chains, each containing domains VL and CL. The antibody is capable of binding to one or more human Fcγ receptors, particularly human Fcγ receptor IIIA. In alternative embodiments, the antibody does not bind to or binds very little to human Fcγ receptor IIIA, and in particular, does not bind to or binds very little to any human Fcγ receptor. In such embodiments, the antibody does not include a glycosylation site in the CH2 domain.

[0083] In alternative embodiments, the antibody does not contain an Fc region. In such embodiments, the antibody is, in particular, a single-chain variable region fragment (scFv) or another antibody fragment that does not contain an Fc region.

[0084] Glycosylation of anti-MUC1 antibodies An anti-MUC1 antibody may contain a CH2 domain in one or more antibody heavy chains. Naturally occurring IgG-type human antibodies contain an N-glycosylation site in the CH2 domain. The CH2 domain present in the antibody may or may not contain an N-glycosylation site. In certain embodiments, the antibody does not contain a glycosylation site in the CH2 domain. In particular, the antibody does not contain an asparagine residue at the heavy chain position corresponding to position 297 in the IMGT / Eu numbering scheme. For example, the antibody may contain an Ala297 mutation in the heavy chain. In such embodiments, the antibody is preferably strongly reduced or completely lacking in the ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cell-mediated phagocytosis (ADCP) and / or complement-dependent cell-mediated cytotoxicity (CDC) by binding to the Fcγ receptor. In this regard, a strong reduction in capability refers to a reduction to 10% or less, particularly 3% or less, 1% or less, or 0.1% or less of activity compared to the same antibody having a common mammalian glycosylation pattern, such as the glycosylation pattern described herein, which can be obtained by producing it in a human cell line or CHO cell line and which contains an N-glycosylation site in its CH2 domain. In such embodiments, the antibody is particularly an IgG1 type antibody.

[0085] In alternative embodiments, the CH2 domain present in the antibody includes an N-glycosylation site. This glycosylation site is, in particular, the amino acid position corresponding to amino acid position 297 of the heavy chain according to the IMGT / Eu numbering system, and is an amino acid sequence motif (motive) Asn Xaa Ser / Thr (wherein Xaa may be any amino acid other than proline). ) has. The N-linked glycosylation of Asn297 is conserved in the homology region of mammalian IgG and other antibody isotypes. Since optional additional amino acids may be present in the variable region or other sequence modifications, the actual position of this conserved glycosylation site in the amino acid sequence of the antibody can vary. Preferably, the glycan attached to the antibody preferably has at least the following structure: Asn-GlcNAc-GlcNAc-Man-(Man-GlcNAc)2 The formula comprises a branched complex N-linked carbohydrate structure in which Asn is an asparagine residue of the polypeptide portion of the antibody, GlcNAc is N-acetylglucosamine, and Man is mannose. The terminal GlcNAc residue may further hold a galactose residue, and the galactose residue may optionally hold a sialic acid residue. Further GlcNAc residues (referred to as bifid GlcNAc) may be attached to the Man closest to the polypeptide. Fucose may be bound to the GlcNAc attached to Asn. In these embodiments, the antibody is particularly an IgG1 antibody.

[0086] In preferred embodiments, the antibody is free of N-glucoylneuraminic acid (NeuGc) or free of detectable amounts of NeuGc. Furthermore, the antibody is also preferably free of Galili epitopes (Galα1,3-Gal structures) or free of detectable amounts of Galili epitopes. In particular, the relative amount of glycans holding NeuGc and / or Galα1,3-Gal structures is less than 0.1% or even less than 0.02% of the total amount of glycans attached to the CH2 domain of the antibody in the antibody population.

[0087] In particular, the antibodies possess a human glycosylation pattern. Due to these glycosylation characteristics, there are no exogenous immunogenic non-human structures that induce side effects meaning unwanted side effects or disadvantages known to be caused by certain exogenous sugar structures, such as immunogenic non-human sialic acid (NeuGc) or Galili epitopes (Gal-Gal structures) known in rodent-producing systems, or other structures such as immunogenic high-mannose structures known to originate from yeast systems are avoided.

[0088] In specific embodiments, the antibody includes a glycosylation pattern having a detectable amount of glycan holding a bipartite GlcNAc residue. In particular, the relative amount of glycan holding a bipartite GlcNAc residue is at least 0.5%, particularly at least 1%, of the total amount of glycan attached to the glycosylation site of the antibody in the composition. Furthermore, in certain embodiments, the glycosylation pattern includes glycan holding at least one galactose residue in an amount relative to at least 25% of the total amount of glycan attached to the antibody in the composition. In particular, the relative amount of glycan holding at least one galactose residue is at least 30%, particularly at least 35%, or at least 40% of the total amount of glycan attached to the antibody in the composition. In specific embodiments, the glycosylation pattern includes glycan holding at least one sialic acid residue in an amount relative to at least 1% of the total amount of glycan attached to the antibody in the composition. In particular, the relative amount of glycan holding at least one sialic acid residue is at least 1.5%, particularly at least 2%, of the total amount of glycan attached to the antibody in the composition.

[0089] The antibody may have a glycosylation pattern with a large or small amount of core fucose. Reducing the amount of fucosylation increases the antibody's ability to induce ADCC. In certain embodiments, the relative amount of glycan holding core fucose residues is 40% or less, particularly 30% or less, or 20% or less, of the total amount of glycan attached to the antibody in the composition. In alternative embodiments, the relative amount of glycan holding core fucose residues is at least 60%, particularly at least 65%, or at least 70% of the total amount of glycan attached to the antibody in the composition.

[0090] The ability of an antibody to induce ADCC and the intensity of ADCC induction can be controlled by the presence or absence of a glycosylation site in the CH2 domain of an anti-MUC1 antibody and the presence or absence of fucose in the glycan structure of the glycosylation site. ADCC activity is increased by glycosylation of the Fc portion of the antibody and further increased by reducing the amount of fucosylation. In certain applications, fine-tuning of ADCC activity is important. Therefore, in certain situations, an antibody without a glycosylation site in the CH2 domain, an antibody with a glycosylation site in the CH2 domain and a large amount of fucosylation, or an antibody with a glycosylation site in the CH2 domain and a small amount of fucosylation may be most advantageous.

[0091] Production of anti-MUC1 antibodies Antibodies are preferably produced recombinantly in host cells. The host cells used for antibody production may be any host cells that can be used for antibody production. Preferred host cells are, in particular, eukaryotic host cells, especially mammalian host cells. Exemplary host cells include yeast cells such as the Pichia pastoris cell line, insect cells such as the SF9 and SF21 cell lines, plant cells, bird cells such as the EB66 duck cell line, rodent cells such as the CHO, NS0, SP2 / 0, and YB2 / 0 cell lines, and human cells such as the HEK293, PER.C6, CAP, CAP-T, AGE1.HN, Mutz-3, and KG1 cell lines.

[0092] In certain embodiments, the antibody is recombinantly produced in human blood cell lines, particularly in human myeloid leukemia cell lines. Preferred human cell lines and preferred production procedures that can be used for antibody production are described in WO2008 / 028686A2. In specific embodiments, the antibody is obtained by expressing it in human myeloid leukemia cell lines selected from the group consisting of NM-H9D8, NM-H9D8-E6, and NM-H9D8-E6Q12, and cell lines derived therefrom. These cell lines, in accordance with the requirements of the Budapest Convention, were deposited as accession numbers DSM ACC2806 (NM-H9D8; deposited on September 15, 2006), DSM ACC2807 (NM-H9D8-E6; deposited on October 5, 2006), and DSM ACC2856 (NM-H9D8-E6Q12; deposited on August 8, 2007) by Glycotope GmbH, Robert-Rossle-Str.10, 13125 Berlin (DE) under Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Inhoffenstraße 7B, 38124. Deposited at Braunschweig (DE), NM-H9D8 cells provide a glycosylation pattern with high sialylation, high bifidative GlycNAc, high galactosylation, and high fucosylation. NM-H9D8-E6 and NM-H9D8-E6Q12 cells provide a glycosylation pattern similar to that of NM-H9D8 cells, except for a very low degree of fucosylation. Other suitable cell lines include K562 (ATCC), a human myeloid leukemia cell line available at the American Cell Culture Lineage Preservation Center. Examples include CCL-243) and cell lines derived from the above-mentioned cells.

[0093] In further embodiments, the antibody is recombinantly produced in CHO cells. In particular, the antibody can be recombinantly produced in CHO dhfr- cell lines, such as the cell line with ATCC number CRL-9096.

[0094] Conjugate of anti-MUC1 antibody According to the present invention, the antibody is conjugated with one or more cytotoxic agents. The cytotoxic agents may be any cytotoxic agents suitable for conjugation with the antibody. If more than one cytotoxic agent is present in the antibody, these cytotoxic agents are identical. They may be different, or they may all be identical. The conjugation of the cytotoxic agent and the antibody can be achieved using any method known in the art. The cytotoxic agent may be attached to the antibody covalently or noncovalently, particularly by fusion or chemical coupling. In certain embodiments, the cytotoxic agent is attached to the antibody covalently, particularly via a linker moiety. The linker moiety may be any chemical entity suitable for attaching the cytotoxic agent to the antibody.

[0095] In addition to cytotoxic agents, the conjugate according to the present invention may also include further activators conjugated thereto. These further activators are preferably useful for the treatment, diagnosis, prognosis, and / or monitoring of diseases, particularly cancer. For example, the further activators can be selected from the group consisting of radionuclides, chemotherapeutic agents, antibodies or antibody fragments, particularly those with specificity different from anti-MUC1 antibodies, such as checkpoint antibodies that block or activate immunomodulatory targets, enzymes, interaction domains, detectable labels, toxins, cytolytic components, immunomodulators, immune effectors, MHC class I or class II antigens, and liposomes.

[0096] A specific preferred cytotoxic agent is a cytotoxic agent capable of killing cancer cells, such as a radionuclide or a chemotherapeutic agent. In a particular preferred embodiment, the chemotherapeutic agent is attached to an anti-MUC1 antibody that forms a conjugate. The chemotherapeutic agent is not particularly limited, as long as the compound has substituents or substructures that have an antitumor effect and can be attached to a linker structure. When part or all of the linker is cleaved in tumor cells, the chemotherapeutic agent or antitumor compound portion is released, and the chemotherapeutic agent exhibits an antitumor effect. When the linker is cleaved at the site of connection to the agonist, the chemotherapeutic agent is released in its original structure and exerts its original antitumor effect.

[0097] Specific examples of chemotherapeutic agents that can be conjugated as cytotoxic agents include alkylating agents such as cisplatin, antimetabolites, plant alkaloids and terpenoids, vinca alkaloids, podophyllotoxin, taxanes such as taxol, topoisomerase inhibitors such as irinotecan and topotecan, anti-cancer agents such as doxorubicin, or microtubule inhibitors such as mytansin / mytansinoids.

[0098] Chemotherapy agents can be selected from the following group in particular: V-ATPase inhibitors, apoptosis promoters, Bcl2 inhibitors, MCL1 inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule stabilizers, microtubule destabilizers, drastatin, mytansin, mytansinoids, amatoxins, methionine aminopeptidase, protein CRM1 nuclear export inhibitors, DPPIV inhibitors, proteasome inhibitors, mitochondrial phosphate transfer inhibitors, protein synthesis inhibitors, kinase inhibitors, CDK2 inhibitors, CDK9 inhibitors, kinesin inhibitors, HDAC inhibitors, topoisomerase I inhibitors, DNA damaging agents, DNA alkylating agents, DNA intercalators, DNA sulcus binding agents, DHFR inhibitors, microtubule formation inhibitors, microtubule stabilizers, actin stabilizers, topoisomerase II inhibitors, platinum compounds, ribosome inhibitors, RNA polymerase II inhibitors, and bacterial toxins. In specific embodiments, the chemotherapeutic agent attached to the anti-MUC1 antibody is selected from the group consisting of microtubule inhibitors such as mytansinoids, topoisomerase I inhibitors, DNA damaging agents, DNA alkylating agents, and DNA minor groove binding agents.

[0099] In some embodiments, the chemotherapeutic agent is a mytansin or mytansinoid. Specific examples of mytansinoids useful for conjugation include mytansinol, N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-mytansin (DM1), N2'-deacetyl-N2'-(4-mercapto-1-oxopentyl)-mytansin (DM3), and N2'-deacetyl-N2'-(4-methyl-4-methyl Capto-1-oxopentyl)-mytansin (DM4) is one example. In particular, DM1 or DM4 is attached to the anti-MUC1 antibody. In some embodiments, the chemotherapeutic agent attached to the anti-MUC1 antibody is a DNA subgroove binding agent, particularly pyrrolobenzodiazepine (PBD), pyrrolobenzodiazepine dimer (PBD dimer), duocalmycin, duocalmycin-hydroxybenzamide-azaindole (DUBA), seco-duocalmycin-hydroxybenzamide-azaindole (seco-DUBA), or doxorubicin. In some embodiments, the chemotherapeutic agent attached to the anti-MUC1 antibody is a DNA alkylating agent, particularly indolinobenzodiazepine or oxazolidinobenzodiazepine. In some embodiments, the chemotherapeutic agent attached to the anti-MUC1 antibody is a DNA damaging agent, particularly calitiamycin. In some embodiments, the chemotherapeutic agent attached to the anti-MUC1 antibody is a topoisomerase I inhibitor, particularly camptothecin, and 7-ethyl-10-hydroxy-camptothecin (SN-38), (S)-9-dimethylaminomethyl-10-hydroxycamptothecin (topotecan), (1S,9S)-1-amino-9-ethyl-5-fluoro-1,2,3,9,12,15-hexahydro-9-hydroxy-4-methyl-10H,13H-benzo[de]pyrano[3' These include derivatives such as [4':6,7]indolidino[1,2-b]quinoline-10,13-dione (exatequican (DX-8951)) and N-[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-1-yl]-2-hydroxyacetamide (DXd). In some embodiments, the chemotherapeutic agent attached to the anti-MUC1 antibody is a microtubule formation inhibitor, particularly tubulosin, anthamitocin, podophyllotoxin, or vinblastine. In some embodiments, the chemotherapeutic agent attached to the anti-MUC1 antibody is a microtubule stabilizer, particularly paclitaxel or epotilon.In some embodiments, the chemotherapeutic agent attached to the anti-MUC1 antibody is an actin stabilizer, particularly phalotoxin. In some embodiments, the chemotherapeutic agent attached to the anti-MUC1 antibody is a topoisomerase II inhibitor, particularly teniposide, XK469, razoxane, amsacrin, idarubicin, or mevalon. In some embodiments, the chemotherapeutic agent attached to the anti-MUC1 antibody is a platinum compound, particularly cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenantriplatin, picoplatin, or satraplatin. In some embodiments, the chemotherapeutic agent attached to the anti-MUC1 antibody is a ribosome inhibitor, particularly lysine, saporin, abrin, diphtheria toxin, or extracellular toxin A. In some embodiments, the chemotherapeutic agent attached to the anti-MUC1 antibody is an RNA polymerase II inhibitor, particularly amatoxin such as amanitin. In some embodiments, the chemotherapeutic agent attached to the anti-MUC1 antibody is a bacterial toxin, particularly anthrax toxin. Suitable antibody-drug conjugates are also described in the EP16151774.3 specification and the LU92659 specification, which is explicitly referenced therein.

[0100] In a preferred embodiment, the chemotherapeutic agent is (1S,9S)-1-amino-9-ethyl-5-fluoro-1,2,3,9,12,15-hexahydro-9-hydroxy-4-methyl-10H,13H-benzo[de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-10,13-dione (exatechcan (DX-8951)) or DXd.

[0101] Exatecine (DX-8951) is given by the following formula:

[0102] [ka] It is an antitumor compound represented by [the formula shown].

[0103] The compound can be readily obtained, for example, by the method described in U.S. Patent Application Publication No. 2016 / 0297890 or by other known methods, preferably with the amino group at position 1 used as the linking position to the linker structure. Furthermore, exateccan can be released in tumor cells, although a portion of the linker remains attached to it. However, the compound exhibits excellent antitumor effects even in such a state.

[0104] DXd is given by the following formula:

[0105] [ka] It is a compound represented by [this formula].

[0106] Because exatechcan or DXd has a camptothecin structure, it is known that in acidic aqueous media (e.g., pH 3), the equilibrium shifts to a ring-closed structure with a lactone ring formed, while in basic aqueous media (e.g., pH 10), the equilibrium shifts to an open-ring structure with an open lactone ring. Drug conjugates incorporating exatechcan residues corresponding to such ring-closed and open structures are expected to exhibit equivalent antitumor effects, and it goes without saying that any such drug conjugates fall within the scope of the present invention. In certain embodiments, the further activator is a polypeptide or protein. This polypeptide or protein may, in particular, be fused to the polypeptide chain of the antibody. In certain embodiments, the further activator, which is a polypeptide or protein, is fused to the C-terminus of the antibody light chain of the antibody. In embodiments in which the antibody comprises two antibody light chains, the further activator, which is a polypeptide or protein, may be fused to the C-terminus of each of the two antibody light chains. In further embodiments, the polypeptide or A further agonist, which is a protein, is fused to the C-terminus of the antibody's heavy chain. In embodiments in which the antibody comprises two antibody heavy chains, a further agonist, which is a polypeptide or protein, may be fused to the C-terminus of each of the two antibody heavy chains. The further agonists may be identical or different, and in particular, may have the same amino acid sequence. Preferred examples of such further agonists, which are polypeptides or proteins, may be selected from the group consisting of cytokines, chemokines, antibodies, antigen-binding fragments, enzymes, and interaction domains.

[0107] In certain embodiments, a further agonist, which is a polypeptide or protein, is a checkpoint antibody that blocks and / or induces an activation signal. Examples of targets include CD40, CD3, CD137 (4-1BB), OX40, GITR, CD27, CD278 (ICOS), CD154 (CD40 ligand), CD270 (HVEM), and CD258 (LIGHT) as activation targets, and CTLA4, PD1, CD80, CD244, A2AR, B7-H3 (CD276), B7-H4 (VTCN1), BTLA, IDO, KIR, LAG3, TIM-3, VISTA, and phosphatidylserine, as well as their corresponding ligands such as PDL1, as inhibitory targets. In a specific example, an anti-MUC1 antibody comprises two heavy chains and two light chains as described herein, with either an scFv fragment that specifically binds to CD3 fused to the C-terminus of each heavy chain, or an scFv fragment that specifically binds to PDL1 fused to the C-terminus of each light chain.

[0108] In further embodiments, the additional activator, which is a polypeptide or protein, is an immunomodulatory compound such as a chemokine, cytokine, or growth factor. Preferred cytokines in this regard include interferons such as interferon-α, interferon-β, and interferon-γ, as well as interleukins. Preferred growth factors include G-CSF and GM-CSF.

[0109] A concrete example of a linker is the following equations (a) to (f): (a)-(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, (b)-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, (c)-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-, (d)-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-, (e)-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, and (f)-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O- CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)- The structure can be represented by any of the following, where -(Succinimid-3-yl-N)- is: formula:

[0110] [ka] It has a structure represented by [this].

[0111] In a specific embodiment, the linker is given by the following equations (a) to (c): (a)-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-, (b)-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-, and (c)-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)- Includes a structure represented by any of the following.

[0112] In a preferred embodiment, the linker is given by the following formula (a): (a)-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)- Includes a structure represented by any of the following.

[0113] In an alternative embodiment, the conjugate has a drug-linker structure represented by the following formula, and the antibody is conjugated to the drug-linker structure represented by the following formula via a thioether bond, marked with an asterisk. * This represents the connection point with the antibody.

[0114] [ka]

[0115] In a preferred embodiment, the conjugate is given by the following formula:

[0116] [ka] It has a drug-linker structure represented by, In the formula, AB represents an antibody, y represents the average number of units of the drug-linker structure conjugated to the antibody per antibody, the antibody is conjugated to the drug-linker structure represented by the above formula by a thioether bond, the antibody represents the anti-MUC1 antibody described above, preferably the antibody is a combination of the heavy chain variable region and the light chain variable region or the heavy chain and light chain a)~d): (a) The heavy chain variable region has the amino acid sequence of SEQ ID NO: 10, and the light chain variable region has the amino acid sequence of SEQ ID NO: 12. (b) The heavy chain variable region has the amino acid sequence of SEQ ID NO: 11, and the light chain variable region has the amino acid sequence of SEQ ID NO: 12. (c) The heavy chain has the amino acid sequence of SEQ ID NO: 15, and the light chain has the amino acid sequence of SEQ ID NO: 16, and (d) The heavy chain has the amino acid sequence of SEQ ID NO: 19, and the light chain has the amino acid sequence of SEQ ID NO: 16. It is one of the following.

[0117] In the conjugates described above, the number of conjugated drug molecules (or cytotoxic agents) per antibody molecule is a crucial factor affecting their efficacy and safety. The production of antibody-drug conjugates (or conjugates) is carried out by specifying reaction conditions, such as the amounts of starting materials and reagents used in the reaction, so that a certain number of conjugated drug molecules are reached. Unlike the chemical reactions of low molecular weight compounds, a mixture containing varying numbers of conjugated drug molecules is usually obtained. The number of conjugated drug molecules per antibody molecule is defined and expressed as an average value, i.e., the average number of conjugated drug molecules. Furthermore, unless otherwise indicated, i.e., when referring to an antibody-drug conjugate containing a specific number of conjugated drug molecules within an antibody-drug conjugate mixture containing varying numbers of conjugated drug molecules, the number of conjugated drug molecules according to the present invention typically means an average value. The number of exatechcan molecules or DXd conjugated to the antibody molecule is controllable, and approximately 1 to 10 exatechcan molecules or 1 to 10 DXd can be conjugated as an average number of conjugated drug molecules per antibody. The number of exatechcan molecules or DXd is preferably 2 to 8, more preferably 4 to 8, and further Preferably 7 to 8, and more preferably 8. It should be noted that those skilled in the art can design a reaction to conjugate a required number of drug molecules to an antibody molecule based on the examples described in this application, thereby obtaining an antibody-drug conjugate having a controlled number of conjugated exatechcan molecules.

[0118] In the preferred embodiment described above, after the conjugate is migrated into the tumor cell, the linker portion is cleaved, and then DXd is released, exerting an antitumor effect. (Clinical Cancer Research, October 15, 2016; Vol. 22 (No. 20): pp. 5097-5108, Epub March 29, 2016).

[0119] Conjugates labeled with various radioactive or non-radioactive isotopes are also included in the present invention. One or more atoms constituting the conjugate of the present invention may contain atomic isotopes in ratios not found in nature. Examples of atomic isotopes include deuterium ( 2 H), tritium ( 3 H), Iodine-125 ( 125 I) and carbon-14 ( 14 C) is one example. Furthermore, conjugates contain tritium ( 3 H), Iodine-125 ( 125 I), carbon-14 ( 14 C), copper 64( 64 Cu), Zirconium-89 ( 89 Zr), Iodine-124 ( 124 I), Fluorine 18( 18 F), Indium-111 ( 111 In), carbon-11 ( 11 C), and iodine-131 ( 131 The conjugate may be radiolabeled with radioactive isotopes such as I). Radioisotope-labeled conjugates are useful as therapeutic or prophylactic agents, research reagents such as assay reagents, and diagnostic agents such as in vivo contrast agents. Regardless of radioactivity, any isotopic variant conjugate is within the scope of the present invention.

[0120] Nucleic acids, expression cassettes, vectors, cell lines, and compositions The antibody portion of the conjugate according to the present invention may be encoded by a nucleic acid. The nucleic acid sequence of the nucleic acid may have any nucleotide sequence suitable for encoding an antibody. However, preferably, the nucleic acid sequence is at least partially adapted to the specific codon usage frequency of the host cell or organism that is to express the nucleic acid, particularly the human codon usage frequency. The nucleic acid may be double-stranded or single-stranded DNA or RNA, preferably double-stranded DNA such as cDNA, or single-stranded RNA such as mRNA. The nucleic acid may be a single continuous nucleic acid molecule, or it may consist of several nucleic acid molecules, each encoding a different portion of the antibody. The nucleotide sequence of the heavy chain of the PankoMab variant (PM-N54Q) may be represented by SEQ ID NO: 17, and the nucleotide sequence of the light chain of the PankoMab variant (PM-N54Q) may be represented by SEQ ID NO: 18.

[0121] If the antibody is composed of more than one different amino acid chain, such as the light and heavy chains of the antibody, the nucleic acid may be a single nucleic acid molecule containing several coding regions, each encoding one of the amino acid chains of the antibody and preferably separated by regulatory elements such as IRES elements to produce separate amino acid chains, or the nucleic acid may be composed of several nucleic acid molecules, each containing one or more coding regions, each encoding one of the amino acid chains of the antibody. In addition to the coding region encoding the antibody, the nucleic acid may also contain further nucleic acid sequences or other modifications, which may, for example, encode other proteins, affect the transcription and / or translation of the coding region, affect the stability or other physical or chemical properties of the nucleic acid, or have no function at all.

[0122] The expression cassette or vector may include the nucleic acid and a promoter operably connected to the nucleic acid. In addition, the expression cassette or vector may affect further elements, particularly the transcription and / or translation of the nucleic acid, the amplification and / or reproduction of the expression cassette or vector, the integration of the expression cassette or vector into the host cell genome, and / or the copy number of the expression cassette or vector in the host cell. The expression cassettes may include elements that can be modified or adjusted. Suitable expression cassettes and vectors, each containing an expression cassette for expressing an antibody, are well known in the prior art and therefore do not require further explanation herein.

[0123] The host cell may contain nucleic acids, or expression cassettes or vectors. The host cell may be any host cell. The host cell may be an isolated cell or a cell contained in a tissue. Preferably, the host cell is a cultured cell, particularly a primary cell, a cell of an established cell line, preferably a tumor-derived cell. Preferably, the host cell is a bacterial cell such as Escherichia coli (E. coli), a yeast cell such as Saccharomyces cells, particularly S. cerevisiae, an insect cell such as Sf9 cells, or a mammalian cell, particularly a human cell such as tumor-derived human cells, a hamster cell such as CHO, or a primate cell. In a preferred embodiment, the host cell is derived from human myeloid leukemia cells. Preferably, the host cell is selected from the following cells or cell lines: K562, KG1, MUTZ-3, or cells or cell lines derived therefrom, or a mixture of cells or cell lines containing at least one of these aforementioned cells. The host cells are preferably selected from the group consisting of NM-H9D8, NM-H9D8-E6, NM H9D8-E6Q12, and cells or cell lines derived from any one of the host cells. These cell lines and their properties are described in detail in WO2008 / 028686A2. In further embodiments, the host cells are CHO dhfr-cell lines, such as the cell line of ATCC number CRL-9096. In preferred embodiments, the host cells are optimized to express glycoproteins having a specific glycosylation pattern, particularly antibodies. Preferably, the codon usage frequency of the coding regions of nucleic acids and / or promoters and further elements in the expression cassette or vector is compatible with the host cell type and, more preferably, optimized for the host cell type used. Preferably, the antibodies are produced by the host cells or cell lines as described above.

[0124] Methods for producing antibodies utilize host cells as described herein. These methods particularly include the steps of preparing a host containing nucleic acids encoding antibodies, culturing the host cells under conditions suitable for antibody expression, and obtaining antibodies expressed by the host cells. The antibodies described herein may or may not be obtained by these methods.

[0125] In another embodiment, the present invention provides a composition comprising a conjugate according to the present invention. Furthermore, the composition may further comprise one or more additional components selected from the group consisting of solvents, diluents, and excipients. Preferably, the composition is a pharmaceutical composition. In this embodiment, the components of the composition are preferably all pharmaceutically acceptable. The composition may be a solid composition or a fluid composition, in particular preferably an aqueous solution, emulsion, or suspension, or a lyophilized powder.

[0126] Use in pharmaceuticals Conjugates are particularly useful in pharmaceuticals, especially in the treatment, diagnosis, prognosis, detection, and / or monitoring of diseases, particularly those described herein, preferably cancer, infectious diseases, inflammatory diseases, graft-versus-host diseases, and immunodeficiency.

[0127] Accordingly, in further embodiments, the present invention provides conjugates or compositions for use in pharmaceuticals. Preferably, pharmaceutical use is in the treatment, prognosis, diagnosis, detection, and / or monitoring of diseases related to abnormal cell proliferation such as cancer, infectious diseases such as bacterial, viral, fungal, or parasitic infections, inflammatory diseases such as autoimmune diseases and inflammatory bowel disease, and diseases related to reduced immune activity such as immunodeficiency. In preferred embodiments, the disease is cancer.

[0128] Preferably, the cancer exhibits detectable expression of MUC1(TA-MUC1), which is detectable by immunohistochemistry, ELISA, RIA, enzyme immunospot (ELISPOT) assay, dot blotting, Octellony test, counter-immunoelectrophoresis (CIE), or in situ hybridization. The cancer particularly includes cells exhibiting MUC1(TA-MUC1) expression detectable by immunohistochemistry or in situ hybridization. The cancer may have been tested for MUC1(TA-MUC1) levels prior to administration of the anti-MUC1 antibody.

[0129] The present invention further provides kits and devices comprising the conjugate according to the present invention, as well as related methods useful for the diagnosis, detection, or monitoring of MUC1-related disorders such as cancer. In some embodiments, a sandwich ELISA kit for testing or diagnosis comprising the conjugate of the present invention is provided. This kit may further comprise one or more of the following: a solution of MUC1 (TA-MUC1) protein standard, a coloring reagent, a buffer solution for dilution, an antibody for the solid phase, an antibody for detection, and a washing solution. Preferably, the amount of conjugate bound to the antigen can be measured by applying methods such as absorbance, fluorescence, luminescence, or radioisotope (RI) methods. Preferably, an absorbance plate reader, fluorescence plate reader, luminescence plate reader, or RI liquid scintillation counter is used for measurement.

[0130] Antibodies can be used in immunohistochemistry (IHC) analysis.

[0131] Immunohistochemistry is not particularly limited in that it involves reacting tissue sections with antigen-binding antibodies (primary antibodies) and detecting the primary antibodies bound to the antigen.

[0132] Various forms of cancer, including metastases, can be treated with the conjugate according to the present invention. The cancer can be selected from the group consisting particularly of colon cancer, lung cancer, ovarian cancer, breast cancer (including tertiary pulmonary cancer), pancreatic cancer, cervical cancer, endometrial cancer, gastrointestinal cancer, kidney cancer, head and neck cancer, thyroid cancer, and urothelial cancer. Furthermore, the cancer can be selected from the group consisting particularly of gastric cancer, liver cancer, bladder cancer, skin cancer, prostate cancer, and hematological cancer. In certain embodiments, the cancer is metastatic cancer. The cancer may include any type of metastasis, such as skin metastases, lymph node metastases, lung metastases, liver metastases, peritoneal metastases, pleural metastases, and / or brain metastases. In certain embodiments, the cancer has an inflammatory phenotype. In such embodiments, any of the cancer types described above may be inflammatory cancers.

[0133] In certain embodiments, viral infections are caused by human immunodeficiency virus, herpes simplex virus, Epstein-Barr virus, influenza virus, lymphocytochoroidal meningitis virus, hepatitis B virus, or hepatitis C virus. Inflammatory diseases can be selected from inflammatory bowel disease, pelvic inflammatory disease, ischemic stroke, Alzheimer's disease, asthma, pemphigus vulgaris, and dermatitis / eczema. Autoimmune diseases can be selected from the group consisting of celiac disease, type 1 diabetes, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, vitiligo, psoriatic arthritis, atopic dermatitis, scleroderma, sarcoidosis, primary biliary cirrhosis, Guillain-Barré syndrome, autoimmune hepatitis, and ankylosing spondylitis. In certain embodiments, the disease involves or is associated with cells expressing MUC1, particularly TA-MUC1. For example, the cancer being treated includes MUC1-positive, particularly TA-MUC1-positive, meaning cancer cells that express MUC1, especially TA-MUC1.

[0134] In specific embodiments, the conjugate is used to treat cancer in combination with another therapeutic agent, particularly in combination with another anticancer agent. The therapeutic agent may be any known anticancer agent. Suitable anticancer therapeutic agents that can be combined with the conjugate according to the present invention may be chemotherapeutic agents, other antibodies, immunostimulants, cytokines, chemokines, and vaccines. Furthermore, conjugate therapy can be combined with radiotherapy, surgery, and / or traditional Chinese medicine.

[0135] The anticancer agents that can be used in combination with the conjugate can be selected from any chemotherapeutic agent, in particular, those known to be effective in treating MUC1-positive cancers. The type of chemotherapeutic agent also depends on the type of cancer being treated.The combination partner can be selected from the following group: taxanes such as paclitaxel (Taxol), docetaxel (Taxotere), and SBT-1214; cyclophosphamide; imatinib; pazopanib; capecitabine; cytarabine; vinorelbine; gemcitabine; anthracyclines such as daunorubicin, doxorubicin, epirubicin, idarubicin, barurubicin, and mitoxantrone; aminoglutethimide, testolactone (Teslac), and anastrozole. Aromatase inhibitors such as (Arimidex), letrozole (Femara), exemestane (Aromasin), borozole (Rivizor), formestan (Lentaron), fadrozole (Afema), 4-hydroxyandrostenedione, 1,4,6-androstatriene-3,17-dione (ATD), and 4-androsten-3,6,17-trione (6-oxo); irinotecan, topotecan, camptothecin, lamellarin D, etoposide (VP-16), teniposide Topoisomerase inhibitors such as doxorubicin, daunorubicin, mitoxantrone, amsacrin, ellipticin, oulintricarboxylic acid, and HU-331; platinum-based chemotherapeutic agents such as cis-diamminedichloroplatin(II) (cisplatin), cis-diammine(1,1-cyclobutanedicarboxylate)platin(II) (carboplatin), and [(1R,2R)-cyclohexane-1,2-diamine](ethandeoato-O,O')platin(II) (oxaliplatin); olaparib PARP inhibitors such as lucaparib and niraparib; TLR agonists such as imiquimod and reciquimod; as well as antimetabolites, particularly antifolic acid agents such as methotrexate, pemetrexed, larcitrexed, and pralatrexate; pyrimidine analogs such as fluorouracil, gemcitabine, phloxuridine, 5-fluorouracil, and tegafur-uracil; and purine analogs; selective estrogen receptor modifiers; and estrogen receptor downregulators.

[0136] Furthermore, therapeutic antibodies can also be used as additional combination partners. These therapeutic antibodies may be any antibodies useful for cancer therapy other than anti-MUC1 antibodies. In particular, additional antibodies have been approved for cancer treatment by authorities such as the U.S. Food and Drug Administration (FDA), the European Medicines Agency (EMA, formerly EMEA), and the Federal Institute for Pharmaceuticals and Medical Devices (BfArM). Further examples of antibodies that can be used in combination therapy include anti-EGFR antibodies such as cetuximab, tomuzotuximab, panitumumab, zaltumumab, nimotuzumab, matsuzumab, and nesitumumab; anti-HER2 antibodies such as trastuzumab, timigutuzumab, and pertuzumab; anti-VEGF antibodies such as bevacizumab (Avastin); anti-CD52 antibodies such as alemtuzumab (Campath); anti-CD30 antibodies such as brentuximab (Adcetris); anti-CD33 antibodies such as gemtuzumab (Mylotarg); and anti-CD20 antibodies such as rituximab (Rituxan, Mabthera), tositumomab (Bexxar), and ibritumomab (Zevalin). Further exemplary antibodies suitable for combination with the cancer therapies described herein include antibodies against antigens selected from the following group: Thomsen-Friedenreich antigens (TFα, TFβ), Tn, Lewis Y, CD44, folate receptor α, NeuGc-GM3 ganglioside, DLL-3, RANKL, PTK7, Notch-3, and F. Phosphorus A4, insulin-like growth factor receptor 1, activin receptor-like kinase-1, claudin-6, disiaroganglioside GD2, endoglin, transmembrane glycoprotein NMB, CD56, tumor-associated calcium signaling transducer 2, tissue factor, ectonucleodopyrophosphatase / phosphodiesterase 3, CD70, P-cadherin, mesothelin, 6-transmembrane prostatic epithelial antigen 1 (STEAP1), carcinoembryonic antigen-associated cell adhesion molecule 5 (CEACAM5), nectin 4, guanylyl cyclase C, solute transporter family 44 member 4 (SLC44A4), prostate-specific membrane antigen (PSMA), zinc transporter ZIP6 (LIV1(ZIP6)), SLIT and NTRK-like protein 6 (SLITRK6), trophoblast glycoprotein (TPBG; 5T4), Fyn3, carbonic anhydrase 9, NaPi2b, fibronectin extra-domain B B) Endothelin receptor ETB, VEGFR2 (CD309), tenascin c, collagen IV, and periostin.

[0137] Conjugates can be further combined with checkpoint antibodies, i.e., antibodies that block or activate immunomodulatory targets. This allows for the blocking of inhibitory signals in the immune response and / or the induction of activating signals. Examples of targets include CD40, CD3, CD137 (4-1BB), OX40, GITR, CD27, CD278 (ICOS), CD154 (CD40 ligand), CD270 (HVEM), and CD258 (LIGHT) as activating targets, and CTLA4, PD1, CD80, CD244, A2AR, B7-H3 (CD276), B7-H4 (VTCN1), BTLA, IDO, KIR, LAG3, TIM-3, VISTA, and phosphatidylserine, as well as their corresponding ligands such as PDL1, as inhibitory targets.

[0138] In further embodiments, the conjugate can be combined with treatment using immunomodulatory compounds such as chemokines, cytokines, growth factors, and vaccines. Preferred cytokines in this regard include interferons such as interferon-α, interferon-β, and interferon-γ, as well as interleukins. Preferred growth factors include G-CSF and GM-CSF.

[0139] Conjugates are preferably used to treat primary tumors, recurrent tumors and / or metastases of such tumors, particularly for pre-surgical, intra-surgical, or post-surgical treatment, and for the prevention or treatment of metastases. Conjugates are particularly intended to treat patients as adjuvant therapy. In certain embodiments, conjugates are intended to treat patients as neoadjuvant therapy or as neoadjuvant-adjuvant combination therapy. Furthermore, conjugates are intended to treat patients as symptomatic treatment.

[0140] Cancer therapy with conjugates preferably results in inhibition of tumor growth and, in particular, reduction of tumor size. Furthermore, the treatment prevents the development of further metastases and / or reduces the number of metastases. The treatment preferably results in increased progression-free survival and / or increased life expectancy and therefore increased overall survival.

[0141] The present invention further provides methods for treating, diagnosing, prognosing, detecting, and / or monitoring diseases using the conjugate according to the present invention. Embodiments and examples of the use of the conjugate in pharmaceuticals are also applicable to medical methods. In particular, a method is provided for treating a target disease in which such a disease is needed, comprising administering a therapeutically effective amount of the conjugate according to the present invention to the target.

[0142] For example, the present invention is a method for treating cancer of a target that requires it, and The present invention provides a method comprising administering a therapeutically effective dose of a conjugate by Akira to a subject having cancer. In a specific embodiment, the cancer is characterized by the expression of TA-MUC1. The cancer can be selected from the group consisting of ovarian cancer, breast cancer, pancreatic cancer, lung cancer, colon cancer, gastric cancer, liver cancer, kidney cancer, hematological cancer, endometrial cancer, thyroid cancer, leukemia, hemihydrocarcinoma, melanoma, carcinoma, teratoma, lymphoma, sarcoma, mesothelioma, neuroblastoma, glioma, rectal cancer, adrenal cancer, skin cancer, brain cancer, cervical cancer, intestinal cancer, intestinal cancer, head and neck cancer, gastrointestinal cancer, lymph node cancer, esophageal cancer, colorectal cancer, otorhinolaryngeal (ENT) cancer, prostate cancer, bladder cancer, uterine cancer, and metastases thereof.

[0143] Furthermore, the present invention provides a method for diagnosing, detecting, or monitoring cancer, comprising the step of bringing a test sample into contact with a conjugate according to the present invention.

[0144] Method for increasing MUC1 binding affinity Methods to increase the MUCI binding affinity of antibodies are: (i) A heavy chain variable region including a complementarity-determining region (CDR) CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 8, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and (ii) Light chain variable region including complementarity-determining region (CDR) CDR-L1 having the amino acid sequence of SEQ ID NO: 4, CDR-L2 having the amino acid sequence of SEQ ID NO: 5, and CDR-L3 having the amino acid sequence of SEQ ID NO: 6 It may include, The method includes the step of substituting the amino acid residue at position 8 of CDR-H2 with any amino acid residue other than asparagine to obtain CDR-H2 having the amino acid sequence of SEQ ID NO: 2.

[0145] Antibodies intended to increase MUC1 binding affinity are, in particular, antibodies capable of binding to MUC1, as described herein, except that they contain asparagine at position 8 of the CDR-H2 sequence.

[0146] In certain embodiments, the heavy chain variable region of an antibody intended to increase MUC1 binding affinity includes an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 11. In particular, the heavy chain variable region includes an amino acid sequence that is at least 95%, and especially at least 98%, identical to the amino acid sequence of SEQ ID NO: 11. In such embodiments, the heavy chain variable region still includes a CDR having the amino acid sequences of SEQ ID NOs: 1, 8, and 3. Therefore, any sequence deviation from SEQ ID NO: 11 is located in the framework region but not in the CDR. In particular, the heavy chain variable region includes the amino acid sequence of SEQ ID NO: 11.

[0147] In certain embodiments, the light chain variable region of an antibody intended to increase MUC1 binding affinity includes an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 12. In particular, the light chain variable region includes an amino acid sequence that is at least 95%, and especially at least 98%, identical to the amino acid sequence of SEQ ID NO: 12. In such embodiments, the light chain variable region still includes a CDR having the amino acid sequences of SEQ ID NOs: 4, 5, and 6. Therefore, any sequence deviation from SEQ ID NO: 12 is located in the framework region but not in the CDR. In particular, the light chain variable region includes the amino acid sequence of SEQ ID NO: 12.

[0148] In a specific embodiment, the heavy chain variable region of an antibody intended to increase MUC1 binding affinity has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 11, and the CDR still has the amino acid sequences of SEQ ID NOs: 1, 8, and 3; the light chain variable region has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 12, and the CDR still has the amino acid sequences of SEQ ID NOs: 4, 5, and 6. In particular, the heavy chain variable region The CDR has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 11, and the CDR still has the amino acid sequences of SEQ ID NOs: 1, 8, and 3. The light chain variable region has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 12, and the CDR still has the amino acid sequences of SEQ ID NOs: 4, 5, and 6.

[0149] For example, antibodies that aim to increase MUC1 binding affinity are anti-MUC1 antibodies, such as those described in WO2004 / 065423A2 or WO2011 / 012309A1. In particular, antibodies that aim to increase MUC1 binding affinity are gatipotuzumab or PankoMab.

[0150] Antibodies that increase MUC1 binding affinity are, in particular, antibodies capable of binding to MUC1, such as those described herein.

[0151] In certain embodiments, MUC1 binding is as described herein. An increase in TA-MUC1 binding affinity refers, in particular, to an increase of at least 10%, at least 20%, at least 33%, or at least 50%. In preferred embodiments, the MUC1 binding affinity is increased by at least 50%. The MUC1 binding affinity can be determined using surface plasmon resonance analysis or switchSENSE® technology (DRX2 biosensor, Dynamic Biosensors GmbH), as described in the examples, in particular, as described in Examples 4a and 4b, for example.

[0152] In certain embodiments, the step of substituting the amino acid residue at position 8 of CDR-H2 is achieved by introducing a mutation into the nucleic acid encoding the antibody, the mutation being introduced into the codon encoding the amino acid residue. The introduction of the mutation can be carried out by any method. Several preferred methods are known in the art, and those skilled in the art can carry out the work necessary to introduce the mutation. Subsequently, an antibody with increased MUC1 binding affinity can be obtained by expressing the mutant nucleic acid, for example, in a host cell. Nucleic acids, host cells, and methods for producing antibodies are described herein and can be used for methods to increase MUC1 binding affinity.

[0153] In a specific embodiment, a method for increasing the MUC1 binding affinity of an antibody is: (a) A step of preparing a nucleic acid encoding an antibody intended to increase MUC1 binding affinity, (b) A step of introducing a mutation into the nucleic acid to produce a mutant nucleic acid, wherein the mutation is introduced into the codon encoding the amino acid residue at position 8 of CDR-H2 such that the codon encodes any amino acid residue other than asparagine, and (c) The process includes the step of expressing mutant nucleic acids to produce antibodies with increased MUC1 binding affinity.

[0154] A method for producing antibodies with increased MUC1 binding affinity is: (a) (i) A heavy chain variable region including a complementarity-determining region (CDR) CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 8, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and (ii) Light chain variable region including complementarity-determining region (CDR) CDR-L1 having the amino acid sequence of SEQ ID NO: 4, CDR-L2 having the amino acid sequence of SEQ ID NO: 5, and CDR-L3 having the amino acid sequence of SEQ ID NO: 6 A step of preparing nucleic acids that encode antibodies containing, (b) Introducing a mutation into the nucleic acid to produce a mutant nucleic acid, wherein the mutation is an arbitrary mutation other than asparagine in the codon encoding the amino acid residue at position 8 of CDR-H2. Steps to be introduced to encode amino acid residues, and (c) A step of producing an antibody with increased MUC1 binding affinity by expressing mutant nucleic acid in host cells. It may include.

[0155] The embodiments, features, and examples described herein for other embodiments, in particular for methods of increasing the MUC1 binding affinity of an antibody, are also applicable to methods of producing an antibody with increased MUC1 binding affinity.

[0156] In a particular embodiment, a method for producing an antibody with increased MUC1 binding affinity further comprises the step (d) of processing the antibody with increased MUC1 binding affinity.

[0157] For example, the treatment of an antibody with increased MUC1 binding affinity may include isolating the antibody from a cell culture. Antibody isolation specifically refers to separating the antibody from the remaining components of the cell culture. Antibody isolation from the cell culture medium can be carried out, for example, by chromatography. Preferred methods and means for isolating antibodies are known in the art and can be readily applied by those skilled in the art.

[0158] Optionally, the obtained antibody may be subjected to further processing steps, such as modification steps, including chemical or enzymatic coupling of an additional agonist to the antibody, and / or formulation steps, to produce an antibody of a desired quality and composition. Such further processing steps and methods are generally known in the art.

[0159] In further embodiments, step (d) further includes providing a pharmaceutical formulation containing an antibody. Providing a pharmaceutical formulation containing an antibody, or formulating an antibody as a pharmaceutical composition, particularly involves exchanging the buffer solution or buffer solution components of the antibody-containing composition. Furthermore, this step may include lyophilizing the antibody. In particular, the antibody is transferred to a composition containing only pharmaceutically acceptable components.

[0160] Production method 1 An antibody-drug conjugate represented by formula (1) shown below, wherein the antibody is connected to the linker structure via a thioether, can be produced by reacting an antibody having a sulfhydryl group converted from a disulfide bond by reduction with compound (2), which can be obtained by known methods (e.g., by the method described in the published patent document, U.S. Patent Application No. 2016 / 297890 (e.g., the method described in paragraphs

[0336] to

[0374] ). This antibody-drug conjugate can be produced, for example, by the following method.

[0161] [ka] In the formula, AB represents an antibody (3a) having a sulfhydryl group. L1 has a structure represented by -(Succinimid-3-yl-N)-, L1' is given by the following formula:

[0162] [ka] This represents the malemidyl group represented by [this symbol].

[0163] -L1-LX has a structure represented by one of the following formulas. -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, and -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-

[0164] Among them, the most preferable are the following: -(Succinimid-3-yl-N)- CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-, and -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-

[0165] Even more preferable are the following: -(Succinimid-3-yl-N)- CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-, and -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH--CH2CH2CH2-C(=O)-

[0166] (NH-DX) is given by the following formula:

[0167] [ka] It has a structure represented by, This represents the group derived by removing one hydrogen atom from the amino group at position 1 of exatechican. In the reaction scheme described above (Equation 8), the compound of Equation (1) can be interpreted as having a structure in which one structural part from the drug to the linker end is connected to one antibody. However, this explanation is for convenience only, and in reality, there are many cases in which multiple of the aforementioned structural parts are connected to a single antibody molecule. The same applies to the description of the production method described below.

[0168] Specifically, the antibody-drug conjugate (1) can be produced by reacting a compound (2), which can be obtained by known methods (e.g., the method described in the patent publication, U.S. Patent Application No. 2016 / 297890 (e.g., the method described in paragraphs

[0336] to

[0374] ), with an antibody (3a) having a sulfhydryl group.

[0169] The provision of sulfhydryl groups to antibody (3a) can be achieved by methods well known to those skilled in the art (Hermanson, GT, Bioconjugate Techniques, pp. 56-136, 456-493, Academic Press (1996)). Examples of methods, but not limited to these, include: reacting the amino group of the antibody with Traut reagent; reacting the amino group of the antibody with N-succinimidyl S-acetylthioalkanoate, followed by hydroxylamine; reacting the antibody with N-succinimidyl 3-(pyridyldithio)propionate, followed by a reducing agent; and reducing the interchain disulfide bonds of the antibody to form sulfhydryl groups by reacting the antibody with a reducing agent such as dithiothreitol, 2-mercaptoethanol, or Tris(2-carboxyethyl)phosphine hydrochloride (TCEP).

[0170] Specifically, antibodies in which interchain disulfide bonds are partially or completely reduced can be obtained by reacting the antibody with TCEP in a buffer containing a chelating agent, using 0.3 to 3 molar equivalents of TCEP per interchain disulfide bond of the antibody as the reducing agent. Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA). The chelating agent can be used at concentrations of 1 mM to 20 mM. Solutions of sodium phosphate, sodium borate, or sodium acetate can be used as buffer solutions. As a specific example, antibodies (3a) having partially or completely reduced sulfhydryl groups can be obtained by reacting the antibody with TCEP at 4°C to 37°C for 1 to 4 hours.

[0171] It should be noted that the drug-linker moiety can be conjugated via a thioether bond by carrying out an addition reaction of a sulfhydryl group to the drug-linker moiety.

[0172] Next, using 2 to 20 molar equivalents of compound (2) per antibody (3a) having a sulfhydryl group, an antibody-drug conjugate (1) can be produced in which 2 to 8 drug molecules are conjugated per antibody. Specifically, a solution containing compound (2) dissolved therein may be added to a buffer containing the antibody (3a) having a sulfhydryl group and reacted. In this situation, sodium acetate solution, sodium phosphate, or sodium borate can be used as the buffer solution. The pH for the reaction is 5 to 9, and more preferably the reaction may be carried out at around pH 7. Organic solvents such as dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMA), or N-methyl-2-pyrrolidone (NMP) can be used as the solvent for dissolving compound (2). The reaction is carried out by adding a solution containing compound (2) dissolved in an organic solvent at a concentration of 1 to 20 vol / vol%, to an antibody (3a) having a sulfhydryl group. The reaction may also be carried out by adding the compound to a buffer solution. The reaction temperature is 0 to 37°C, more preferably 10 to 25°C, and the reaction time is 0.5 to 2 hours. The reaction can be terminated by inactivating the reactivity of the unreacted compound (2) with the thiol-containing reagent. The thiol-containing reagent is, for example, cysteine ​​or N-acetyl-L-cysteine ​​(NAC). More specifically, the reaction can be terminated by adding 1 to 2 molar equivalents of NAC to the compound (2) being used and incubating the resulting mixture at room temperature for 10 to 30 minutes.

[0173] Identification of antibody-drug conjugates The produced antibody-drug conjugate (e.g., antibody-drug conjugate (1)) can be identified by concentrating, exchanging buffers, purifying, and measuring the antibody concentration and the average number of conjugated drug molecules per antibody molecule, according to the common procedure described below.

[0174] 1. Common Procedure A: Concentration of an aqueous solution of antibody or antibody-drug conjugate A solution of antibody or antibody-drug conjugate was added to an Amicon Ultra (50,000 MWCO, Millipore Corporation) container, and the antibody or antibody-drug conjugate solution was concentrated by centrifugation using a centrifuge (Allegra X-15R, Beckman Coulter, Inc.) at 2000G to 4000G for 5 to 30 minutes.

[0175] 2. Common Procedure B: Measurement of Antibody Concentration Antibody concentrations were measured using a UV detector (Nanodrop 1000, Thermo Fisher Scientific Inc.) according to the manufacturer's specified method. In this regard, different 280 nm extinction coefficients were used for the antibodies (1.3 mL mg). -1 cm -1 ~1.8 mL mg -1 cm -1 ).

[0176] 3. Common Procedure C: Antibody Buffer Exchange NAP-25 columns using Sephadex G-25 support (catalog number 17-0852-02, GE Healthcare Japan Corporation) were equilibrated with phosphate buffer (50 mM, pH 6.0) containing sodium chloride (50 mM) and EDTA (2 mM) (referred to herein as PBS6.0 / EDTA) according to the method specified by the manufacturer. An aqueous solution of antibody was applied to each NAP-25 column in a volume of 2.5 mL, and the fraction (3.5 mL) obtained by eluting with 3.5 mL of PBS6.0 / EDTA was collected. This fraction was concentrated according to common procedure A. After measuring the antibody concentration using common procedure B, the antibody concentration was adjusted to 20 mg / mL using PBS6.0 / EDTA.

[0177] 4. Common Procedure D: Purification of Antibody-Drug Conjugates The NAP-25 column was equilibrated with any commercially available buffer such as acetate buffer (10 mM, pH 5.5; referred to herein as ABS) containing sorbitol (5%). An aqueous reaction solution (approximately 2.5 mL) of the antibody-drug conjugate was applied to the NAP-25 column, and then elution was performed with a specified amount of buffer by the manufacturer to collect the antibody fraction. The collected fraction was reapplied to the NAP-25 column, and the gel filtration purification step of eluting with the buffer solution was repeated a total of two or three times to obtain an antibody-drug conjugate excluding unconjugated drug linker and low molecular weight compounds (tris(2-carboxyethyl)phosphine hydrochloride (TCEP), N-acetyl-L-cysteine (NAC), and dimethyl sulfoxide).

[0178] 5. Common Procedure E: Measurement of Antibody Concentration of Antibody-Drug Conjugate and Average Number of Conjugated Drug Molecules per Antibody Molecule The conjugated drug concentration of the antibody-drug conjugate can be calculated by measuring the UV absorbance of an aqueous solution of the antibody-drug conjugate at two wavelengths of 280 nm and 370 nm and then performing the calculations shown below. The total absorbance at any given wavelength is equal to the sum of the absorbances of all light-absorbing chemical species present in the system [additivity of absorbance]. Therefore, based on the hypothesis that the molar absorption coefficients of the antibody and the drug do not change before and after conjugation of the antibody and the drug, the antibody concentration and the drug concentration of the antibody-drug conjugate are represented by the following equations.

[0179] Equation (1) A 280 =A D,280 +A A,280 =ε D,280 C D +ε A,280 C A Equation (1) A 370 =A D,370 +A A,370 =ε D,370 C D +ε A,370 C A Equation (2)

[0180] In this situation, A 280 represents the absorbance at 280 nm of an aqueous solution of the antibody-drug conjugate, and A 370 represents the absorbance at 370 nm of an aqueous solution of the antibody-drug conjugate, and A A,280 represents the absorbance of the antibody at 280 nm, and A A,370 represents the absorbance of the antibody at 370 nm, and A D,280 represents the absorbance of the conjugate precursor at 280 nm, and A D,370 represents the absorbance of the conjugate precursor at 370 nm, and ε A,280 represents the molar absorption coefficient of the antibody at 280 nm, and ε A,370 represents the molar absorption coefficient of the antibody at 370 nm, and ε D,280 represents the molar absorption coefficient of the conjugate precursor at 280 nm, and ε D,370 represents the molar absorption coefficient of the conjugate precursor at 370 nm, and C A represents the antibody concentration of the antibody-drug conjugate, and C D represents the drug concentration of the antibody-drug conjugate.

[0181] In this situation, for ε A,280 , ε A,370 , ε D,280 , and ε D,370 , values prepared preliminarily (estimated values based on calculations or measured values obtained by UV measurement of the compound) are used. For example, ε A,280 can be estimated from the amino acid sequence of the antibody by known calculation methods (Protein Science, 1995, Vol. 4, pp. 2411-2423). ε A,370 is generally zero. ε D,280 , and ε D,370 can be obtained according to the Lambert-Beer law (absorbance = molar concentration × molar absorption coefficient × cell optical path length) by measuring the absorbance of a solution in which the conjugate precursor used is dissolved at a certain molar concentration. C A and C D are the A 280 and A of an aqueous solution of the antibody-drug conjugate370 This can be determined by measuring and then solving the simultaneous equations (1) and (2) by substituting these values. Furthermore, C D to C A By dividing by this number, the average number of conjugated drug molecules per antibody can be determined.

[0182] 6. Common Procedure F: Measurement of the average number of conjugated drug molecules per antibody molecule in antibody-drug conjugates - (2) Furthermore, the average number of conjugated drug molecules per antibody molecule in an antibody-drug conjugate can be determined by high-performance liquid chromatography (HPLC) analysis using the following method, in addition to the "5. Common Procedure E" described above. Hereafter, a method for measuring the average number of conjugated drug molecules by HPLC when an antibody is conjugated to a drug linker via a disulfide bond will be described. Referring to this method, those skilled in the art can appropriately measure the average number of conjugated drug molecules by HPLC depending on the connection pattern between the antibody and the drug linker.

[0183] F-1. Preparation of samples for HPLC analysis (reduction of antibody-drug conjugates) Antibody-drug conjugate solution (approximately 1 mg / mL, 60 μL) is mixed with dithiolate. Mix with an aqueous solution of 100 mM (DTT). Cleave the mixture at 37°C for 30 minutes to cleave the disulfide bond between the light and heavy chains of the antibody-drug conjugate. Use the resulting sample for HPLC analysis.

[0184] F-2. HPLC analysis HPLC analysis will be performed under the following measurement conditions. HPLC device: Agilent1290 HPLC device (Agilent Technologies, Inc.) Detector: Ultraviolet absorption spectrometer (measurement wavelength: 280 nm) Column: ACQUITY UPLC BEH Phenyl (2.1 × 50 mm, 1.7 μm, 130 angstroms; Waters Corp., P / N 186002884) Column temperature: 80℃ Mobile phase A: Aqueous solution containing 0.10% trifluoroacetic acid (TFA) and 15% 2-propanol. Mobile phase B: Acetonitrile solution containing 0.075% TFA and 15% 2-propanol. Gradient program: 14% → 36% (0 min to 15 min), 36% → 80% (15 min to 17 min), 80% → 14% (17 min to 17.01 min), and 14% (17.01 min to 25 min) Sample injection: 10 μL or HPLC device: Agilent1290 HPLC device (Agilent Technologies, Inc.) Detector: Ultraviolet absorption spectrometer (measurement wavelength: 280 nm) Column: PLRP-S (2.1 × 50 mm, 8 μm, 1000 angstroms; Agilent Technologies, Inc., P / N PL1912-1802) Column temperature: 80℃ Mobile phase A: 0.04% aqueous TFA solution Mobile phase B: Acetonitrile solution containing 0.04% TFA Gradient program: 29% → 36% (0 min to 12.5 min), 36% → 42% (12.5 min to 15 min), 42% → 29% (15 min to 15.1 min), and 29% → 29% (15.1 min to 25 min) Sample injection: 15 μL

[0185] F-3. Data Analysis F-3-1. The light and heavy chains of the antibody are represented by Li and Hi, respectively, according to the number of conjugated drug molecules (i represents the number of conjugated drug molecules; that is, the number of conjugated drug molecules according to the present invention is represented by L0, L1, H0, H1, H2, H3, etc.).

[0186] Compared with the unconjugated antibody light chain (L0) and heavy chain (H0), the light chain (L1) conjugated to one drug molecule, the heavy chain (H1) conjugated to one drug molecule, the heavy chain (H2) conjugated to two drug molecules, and the heavy chain (H3) conjugated to three drug molecules show higher hydrophobicity in proportion to the number of conjugated drug molecules and thus have longer residence times. Therefore, such chains elute in the order of L0 and L1 or H0, H1, H2, and H3. The detection peaks can be assigned to any of L0, L1, H0, H1, H2, and H3 by comparing the residence times with L0 and H0.

[0187] F-3-2. Since the drug linker absorbs UV, the peak area value is corrected according to the following formula using the molar extinction coefficients of the light chain or heavy chain and the drug linker, depending on the number of conjugated drug linker molecules.

[0188] [Equation 1] Corrected value of the peak area of the light chain conjugated to i drug molecules (A Li )

[0189] [Number] ε L,280 : Molar extinction coefficient of the light chain at 280 nm ε D,280 : Molar extinction coefficient of the drug linker at 280 nm i: Number of conjugated drug molecules

[0190] [Equation 2] Corrected value of the peak area of the light chain conjugated to i drug molecules (A Hi )

[0191] [Number] ε H,280 : Molar extinction coefficient of the heavy chain at 280 nm ε D,280Molar extinction coefficient of drug linker at 280 nm i: Number of conjugated drug molecules

[0192] In this situation, the value estimated from the amino acid sequence of the light or heavy chain of each antibody using a known calculation method (Protein Science, 1995, Vol. 4, pp. 2411-2423) can be used as the molar extinction coefficient (280 nm) of the antibody's light or heavy chain. The actually measured molar extinction coefficient (280 nm) of the compound obtained by converting the maleimide group to succinimidothioether by reacting each drug linker with mercaptoethanol or N-acetylcysteine ​​was used as the molar extinction coefficient (280 nm) of the drug linker. The wavelength for absorbance measurement can be appropriately set by those skilled in the art, but preferably it is a wavelength that can measure the antibody peak, and more preferably it is 280 nm.

[0193] F-3-3. To sum the corrected peak area values, calculate the peak area ratio (%) of each chain according to the following formula.

[0194] [Formula 3]

[0195]

number

[0196] F-3-4. The average number of conjugated drug molecules per antibody molecule in an antibody-drug conjugate is calculated according to the following formula. Average number of conjugated drug molecules = (L0 peak area ratio × 0 + L0 peak area ratio × 1 + H0 peak area ratio × 0 + H1 peak area ratio × 1 + H2 peak area ratio × 2 + H3 peak area ratio × 3) / 100 × 2

[0197] It should be noted that in order to ensure the amount of the conjugate, multiple conjugates having conjugated drug molecules with almost the same (e.g., about ±1) average number, produced under the same conditions, can be mixed to prepare a new lot. In this case, the average number of drug molecules falls between the average numbers of drug molecules before mixing.

[0198] Specific embodiments The following describes specific embodiments of the antibody portion of the conjugate according to the present invention.

[0199] Embodiment 1. An antibody capable of binding to MUC1, (i) a heavy chain variable region comprising a complementarity determining region (CDR) CDR-H1 having the amino acid sequence of SEQ ID NO: 1, a CDR-H2 having the amino acid sequence of SEQ ID NO: 2, and a CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and (ii) a light chain variable region comprising a complementarity determining region (CDR) CDR-L1 having the amino acid sequence of SEQ ID NO: 4, a CDR-L2 having the amino acid sequence of SEQ ID NO: 5, and a CDR-L3 having the amino acid sequence of SEQ ID NO: 6 An antibody comprising.

[0200] Embodiment 2. The antibody according to Embodiment 1, wherein the amino acid at position 8 of CDR-H2 is selected from the group consisting of glutamine, alanine, valine, histidine, tryptophan, tyrosine, lysine, and arginine, particularly glutamine, histidine, tryptophan, tyrosine, lysine, and arginine, and particularly glutamine.

[0201] Embodiment 3. The antibody according to Embodiment 1, wherein the amino acid at position 8 of CDR-H2 is glutamine, histidine, arginine, tryptophan, or lysine.

[0202] Embodiment 4. The antibody according to any one of Embodiments 1 to 3, wherein CDR-H2 has the amino acid sequence of SEQ ID NO: 7.

[0203] Embodiment 5. The antibody according to Embodiment 1, wherein CDR-H2 has the amino acid sequence of SEQ ID NO: 8.

[0204] Embodiment 6. An antibody capable of binding to MUC1, (i) (a) Having an amino acid sequence that is at least 90% identical to the amino acid sequence of Sequence ID No. 9, (b) A heavy chain variable region comprising a complementarity-determining region (CDR) CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 2, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and (ii) (a) Having an amino acid sequence that is at least 90% identical to the amino acid sequence of Sequence ID No. 12, (b) Complementary determination region (CDR) CDR-L1 having the amino acid sequence of SEQ ID NO: 4, Light chain variable region including CDR-L2 having the amino acid sequence of SEQ ID NO: 5 and CDR-L3 having the amino acid sequence of SEQ ID NO: 6 Antibodies containing this substance.

[0205] Embodiment 7. An antibody capable of binding to MUC1, (i) (a) Having an amino acid sequence that is at least 95% identical to the amino acid sequence of Sequence ID No. 9, (b) A heavy chain variable region comprising a complementarity-determining region (CDR) CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 2, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and (ii) (a) Having an amino acid sequence that is at least 95% identical to the amino acid sequence of Sequence ID No. 12, (b) Light chain variable region including complementarity-determining region (CDR) CDR-L1 having the amino acid sequence of SEQ ID NO: 4, CDR-L2 having the amino acid sequence of SEQ ID NO: 5, and CDR-L3 having the amino acid sequence of SEQ ID NO: 6 Antibodies containing this substance.

[0206] Embodiment 8. The antibody according to Embodiment 6 or 7, wherein the amino acid at position 8 of CDR-H2 is selected from the group consisting of glutamine, alanine, valine, histidine, tryptophan, tyrosine, lysine, and arginine, and is particularly glutamine.

[0207] Embodiment 9. The antibody according to Embodiment 7 or 8, wherein the amino acid at position 8 of CDR-H2 is glutamine, histidine, arginine, tryptophan, or lysine.

[0208] Embodiment 10. An antibody capable of binding to MUC1, (i) (a) Having an amino acid sequence that is at least 90% identical to the amino acid sequence of Sequence ID No. 10, (b) A heavy chain variable region including a complementarity-determining region (CDR) CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 7, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and (ii) (a) Having an amino acid sequence that is at least 90% identical to the amino acid sequence of Sequence ID No. 12, (b) Light chain variable region including complementarity-determining region (CDR) CDR-L1 having the amino acid sequence of SEQ ID NO: 4, CDR-L2 having the amino acid sequence of SEQ ID NO: 5, and CDR-L3 having the amino acid sequence of SEQ ID NO: 6 Antibodies containing this substance.

[0209] Embodiment 11. An antibody capable of binding to MUC1, (i) (a) Having an amino acid sequence that is at least 95% identical to the amino acid sequence of Sequence ID No. 10, (b) A heavy chain variable region including a complementarity-determining region (CDR) CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 7, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and (ii) (a) Having an amino acid sequence that is at least 95% identical to the amino acid sequence of Sequence ID No. 12, (b) Light chain variable region including complementarity-determining region (CDR) CDR-L1 having the amino acid sequence of SEQ ID NO: 4, CDR-L2 having the amino acid sequence of SEQ ID NO: 5, and CDR-L3 having the amino acid sequence of SEQ ID NO: 6 Antibodies containing this substance.

[0210] Embodiment 12. An antibody capable of binding to MUC1, (i) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 9, and (ii) Light chain variable region having the amino acid sequence of Sequence ID No. 12 Antibodies containing this substance.

[0211] Embodiment 13. The antibody according to Embodiment 12, wherein the amino acid at position 57 of SEQ ID NO: 9 is selected from the group consisting of glutamine, alanine, valine, histidine, tryptophan, tyrosine, lysine, and arginine, and is particularly glutamine.

[0212] Embodiment 14. The antibody according to Embodiment 12, wherein the amino acid at position 57 of SEQ ID NO: 9 is glutamine, histidine, arginine, tryptophan, or lysine.

[0213] Embodiment 15. An antibody capable of binding to MUC1, (i) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 10, and (ii) Light chain variable region having the amino acid sequence of Sequence ID No. 12 Antibodies containing this substance.

[0214] Embodiment 16. An antibody capable of binding to MUC1, (i) A heavy chain variable region having an amino acid sequence represented by amino acid numbers 20-136 of SEQ ID NO: 20 or 23, and (ii) Light chain variable region having an amino acid sequence represented by amino acid numbers 21-133 of SEQ ID NO: 21 Antibodies containing this substance.

[0215] Embodiment 17. An antibody capable of binding to MUC1, (i) (a) Having an amino acid sequence that is at least 90% or at least 95% identical to the amino acid sequence of Sequence ID No. 15, (b) A heavy chain comprising a complementarity-determining region (CDR) CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 2 or 7, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and (ii) (a) Having an amino acid sequence that is at least 90% or at least 95% identical to the amino acid sequence of Sequence ID No. 16, (b) Light chain variable region including complementarity-determining region (CDR) CDR-L1 having the amino acid sequence of SEQ ID NO: 4, CDR-L2 having the amino acid sequence of SEQ ID NO: 5, and CDR-L3 having the amino acid sequence of SEQ ID NO: 6 Antibodies containing this substance.

[0216] Embodiment 18. An antibody capable of binding to MUC1, (i) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 22, and (ii) Light chain variable region having the amino acid sequence of SEQ ID NO: 16 Antibodies containing this substance.

[0217] Embodiment 19. An antibody capable of binding to MUC1, (i) (a) Having an amino acid sequence that is at least 90% or at least 95% identical to the amino acid sequence of SEQ ID NO: 19, (b) A heavy chain variable region comprising a complementarity-determining region (CDR) CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 8, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and (ii) (a) Having an amino acid sequence that is at least 90% or at least 95% identical to the amino acid sequence of Sequence ID No. 16, (b) Light chain variable region including complementarity-determining region (CDR) CDR-L1 having the amino acid sequence of SEQ ID NO: 4, CDR-L2 having the amino acid sequence of SEQ ID NO: 5, and CDR-L3 having the amino acid sequence of SEQ ID NO: 6 Antibodies containing this substance.

[0218] Embodiment 20. An antibody capable of binding to MUC1, (i) A heavy chain variable region having an amino acid sequence represented by amino acid numbers 20-460 of SEQ ID NO: 19, and (ii) Light chain variable region having an amino acid sequence represented by amino acid numbers 21-239 of SEQ ID NO: 16 Antibodies containing this substance.

[0219] Embodiment 21. An antibody according to any one of Embodiments 1 to 20, comprising at least one heavy chain including a heavy chain variable region, a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain.

[0220] Embodiment 22. An antibody according to any one of Embodiments 1 to 20, comprising two heavy chains, each containing a heavy chain variable region, a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain.

[0221] Embodiment 23. The antibody according to Embodiment 21 or 22, which is an IgG antibody, particularly an IgG1 antibody, an IgG2 antibody, or an IgG4 antibody.

[0222] Embodiment 24. An antibody according to any one of Embodiments 1 to 23, comprising at least one light chain including a light chain variable region and a CL domain.

[0223] Embodiment 25. An antibody according to any one of Embodiments 1 to 23, comprising two light chains, each containing a light chain variable region and a CL domain.

[0224] Embodiment 26. The antibody according to Embodiment 24 or 25, wherein the light chain is a κ-type light chain.

[0225] Embodiment 27. The antibody according to any one of Embodiments 1 to 26, wherein the CH2 domain does not contain an N-glycosylation site.

[0226] Embodiment 28. The antibody according to any one of Embodiments 1 to 26, wherein the CH2 domain of the antibody heavy chain contains an N-glycosylation site.

[0227] Embodiment 29. Features: (i) The relative amount of glycan holding the bifid GlcNAc residue is at least 0.5% of the total amount of glycan attached to the glycosylation site of the antibody in the composition. (ii) The relative amount of glycans holding at least one galactose residue is at least 30% of the total amount of glycans attached to the glycosylation sites of the antibody in the composition. (iii) The relative amount of glycans that hold core fucose residues is at least 60% of the total amount of glycans attached to the glycosylation sites of the antibody in the composition. The antibody according to Embodiment 28, having a glycosylation pattern comprising one or more of the above.

[0228] Embodiment 30. Features: (i) The relative amount of glycan holding the bifid GlcNAc residue is at least 0.5% of the total amount of glycan attached to the glycosylation site of the antibody in the composition. (ii) The relative amount of glycans holding at least one galactose residue is at least 30% of the total amount of glycans attached to the glycosylation sites of the antibody in the composition. (iii) The relative amount of glycans that hold core fucose residues is 40% or less of the total amount of glycans attached to the glycosylation sites of the antibody in the composition. The antibody according to Embodiment 28, having a glycosylation pattern comprising one or more of the above.

[0229] The following describes specific embodiments of the conjugate according to the present invention.

[0230] Embodiment 31. The antibody according to any one of Embodiments 1 to 30, comprising a further activating agent, preferably a cytotoxic agent, conjugated thereto.

[0231] Embodiment 32. The antibody according to Embodiment 31, wherein the cytotoxic agent is a chemotherapeutic agent coupled to the antibody.

[0232] Embodiment 33. The antibody according to Embodiment 32, wherein the chemotherapeutic agent is selected from the group consisting of microtubule inhibitors such as mytansinoids, topoisomerase I inhibitors, DNA damaging agents, DNA alkylating agents, and DNA sub-groove binding agents.

[0233] Embodiment 34. The antibody according to Embodiment 32, wherein the chemotherapeutic agent is selected from the group consisting of mytansinol, N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-mytansin (DM1), N2'-deacetyl-N2'-(4-mercapto-1-oxopentyl)-mytansin (DM3), and N2'-deacetyl-N2'-(4-methyl-4-mercapto-1-oxopentyl)-mytansin (DM4).

[0234] Embodiment 35. The antibody according to Embodiment 32, wherein the chemotherapeutic agent is selected from the group consisting of pyrrolobenzodiazepine (PBD), pyrrolobenzodiazepine dimer (PBD dimer), duocalmycin, duocalmycin-hydroxybenzamide-azaindole (DUBA), seco-duocalmycin-hydroxybenzamide-azaindole (seco-DUBA), and doxorubicin.

[0235] Embodiment 36. The antibody according to Embodiment 32, wherein the chemotherapeutic agent is selected from the group consisting of indolinobenzodiazepines and oxazolidinobenzodiazepines.

[0236] Embodiment 37. The antibody according to Embodiment 32, wherein the chemotherapeutic agent is calitiamycin.

[0237] Embodiment 38. The antibody according to Embodiment 32, wherein the chemotherapeutic agent is selected from the group consisting of camptothecin, 7-ethyl-10-hydroxy-camptothecin (SN-38), (S)-9-dimethylaminomethyl-10-hydroxycamptothecin (topotecan), (1S,9S)-1-amino-9-ethyl-5-fluoro-1,2,3,9,12,15-hexahydro-9-hydroxy-4-methyl-10H,13H-benzo[de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-10,13-dione (exatechcan (DX-8951)), and DXd.

[0238] Embodiment 39. The chemotherapeutic agent is of the following formula:

[0239] [ka] The antibody according to Embodiment 32, which is an antitumor compound represented by [the specified formula].

[0240] Embodiment 40. The chemotherapeutic agent is of the following formula:

[0241] [ka] The antibody according to Embodiment 32, which is an antitumor compound represented by [the specified formula].

[0242] Embodiment 41. The antibody according to Embodiment 31, wherein an additional activator, which is a polypeptide or protein, is fused to the polypeptide chain of the antibody.

[0243] Embodiment 42. The antibody according to Embodiment 41, comprising two antibody heavy chains and two antibody light chains, wherein a further activator, which is a polypeptide or protein, is fused to each of the C-terminuses of the antibody heavy chains or to each of the C-terminuses of the antibody light chains.

[0244] Embodiment 43. Further activators include cytokines, chemokines, other antibodies, and antigen-binding agents. An antibody according to embodiment 41 or 42, selected from the group consisting of a sex fragment, an enzyme, and a binding domain.

[0245] Embodiment 44. The antibody according to Embodiment 42, wherein a further activator is an scFv fragment that specifically binds to CD3, and one of the further activators is fused to the C-terminus of each antibody heavy chain.

[0246] Embodiment 45. The antibody according to Embodiment 42, wherein a further activator is an scFv fragment that specifically binds to PDL1, and one of the further activators is fused to the C-terminus of each antibody light chain.

[0247] Embodiment 46. A cytotoxic agent, preferably a topoisomerase I inhibitor such as DX-8951 or DXd, is provided by the following formulas (a) to (f): (a)-(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, (b)-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, (c)-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-, (d)-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-, (e)-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, and (f)-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)- It is conjugated to a linker having any structure selected from the group consisting of, The antibody is attached to the -(Succinimid-3-yl-N) terminus, while the antitumor compound uses the nitrogen atom of the amino group at position 1 as the attachment point, with the rightmost -(CH2)n in formulas (a) to (f). 2 -C(=O)-part(n 2 GGFG represents an amino acid sequence consisting of glycine-glycine-phenylalanine-glycine linked via peptide bonds, where GGFG is connected to the carbonyl group (which represents an integer of 1 or 3). -(Succinimid-3-yl-N)- is represented by the following formula:

[0248] [ka] It has a structure represented by, which is connected to the antibody at position 3, and is connected to the methylene group of the linker structure containing this structure at the nitrogen atom at position 1. The antibody according to any one of Embodiments 31 to 45.

[0249] Embodiment 47. A conjugate comprising an antibody according to any one of Embodiments 1 to 30, conjugated to a cytotoxic agent.

[0250] Embodiment 48. The conjugate according to Embodiment 47, wherein the cytotoxic agent is a chemotherapeutic agent.

[0251] Embodiment 49. The chemotherapeutic agent is a microtubule inhibitor, a topoisomerase I inhibitor, or a DNA damage inhibitor. A conjugate according to Embodiment 48, selected from the group consisting of a wounding agent, a DNA alkylating agent, and a DNA sub-groove binder.

[0252] Embodiment 50. The conjugate according to Embodiment 48, wherein the chemotherapeutic agent is selected from the group consisting of mytansinol, N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-mytansin (DM1), N2'-deacetyl-N2'-(4-mercapto-1-oxopentyl)-mytansin (DM3), and N2'-deacetyl-N2'-(4-methyl-4-mercapto-1-oxopentyl)-mytansin (DM4).

[0253] Embodiment 51. The conjugate according to Embodiment 48, wherein the chemotherapeutic agent is selected from the group consisting of pyrrolobenzodiazepine (PBD), pyrrolobenzodiazepine dimer (PBD dimer), duocalmycin, duocalmycin-hydroxybenzamide-azaindole (DUBA), seco-duocalmycin-hydroxybenzamide-azaindole (seco-DUBA), and doxorubicin.

[0254] Embodiment 52. The conjugate according to Embodiment 48, wherein the chemotherapeutic agent is selected from the group consisting of indolinobenzodiazepines and oxazolidinobenzodiazepines.

[0255] Embodiment 53. The conjugate according to Embodiment 48, wherein the chemotherapeutic agent is calitiamycin.

[0256] Embodiment 54. The conjugate according to Embodiment 48, wherein the chemotherapeutic agent is selected from the group consisting of camptothecin, 7-ethyl-10-hydroxy-camptothecin (SN-38), (S)-9-dimethylaminomethyl-10-hydroxycamptothecin (topotecan), (1S,9S)-1-amino-9-ethyl-5-fluoro-1,2,3,9,12,15-hexahydro-9-hydroxy-4-methyl-10H,13H-benzo[de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-10,13-dione (exatechcan (DX-8951)), and DXd.

[0257] Embodiment 55. The chemotherapeutic agent is of the following formula:

[0258] [ka] The conjugate according to Embodiment 48, which is an antitumor compound represented by [the specified formula].

[0259] Embodiment 56. The chemotherapeutic agent is of the following formula:

[0260] [ka] The conjugate according to Embodiment 48, which is an antitumor compound represented by [the specified formula].

[0261] Embodiment 57. The conjugate according to Embodiment 47, wherein a further activator, which is a polypeptide or protein, is fused to the polypeptide chain of the antibody.

[0262] Embodiment 58. The conjugate according to Embodiment 57, wherein the antibody comprises two antibody heavy chains and two antibody light chains, and a further activator, which is a polypeptide or protein, is fused to each of the C-terminuses of the antibody heavy chains or to each of the C-terminuses of the antibody light chains.

[0263] Embodiment 59. The conjugate according to Embodiment 57 or 58, wherein a further activator is selected from the group consisting of cytokines, chemokines, other antibodies, antigen-binding fragments, enzymes, and binding domains.

[0264] Embodiment 60. The conjugate according to Embodiment 58, wherein a further activator is an scFv fragment that specifically binds to CD3, and one of the further activators is fused to the C-terminus of each antibody heavy chain.

[0265] Embodiment 61. The conjugate according to Embodiment 58, wherein a further activator is an scFv fragment that specifically binds to PDL1, and one of the further activators is fused to the C-terminus of each antibody light chain.

[0266] Embodiment 62. The antibody is a compound of the following formulas (a) to (f): (a)-(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, (b)-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, (c)-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-, (d)-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-, (e)-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, and (f)-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)- It is conjugated to a further agonist or chemotherapeutic agent, preferably a topoisomerase I inhibitor such as DX-8951 or DXd, via a linker having any structure selected from the group consisting of the following: The antibody is attached to the -(Succinimid-3-yl-N) terminus, while the antitumor compound uses the nitrogen atom of the amino group at position 1 as the attachment point, with the rightmost -(CH2)n in formulas (a) to (f). 2 -C(=O)-part(n 2GGFG is linked via a peptide bond to the carbonyl group (where GGFG represents an integer of 1 or 3), and glycine-glycine-phenylalanine is linked via a peptide bond. This represents an amino acid sequence consisting of glycine, -(Succinimid-3-yl-N)- is represented by the following formula:

[0267] [ka] It has a structure represented by, which is connected to the antibody at position 3, and is connected to the methylene group of the linker structure containing this structure at the nitrogen atom at position 1. A conjugate according to any one of embodiments 47 to 61.

[0268] Embodiment 63. The antibody is a combination of the following heavy chain variable region and light chain variable region or heavy chain and light chain a) to f): (a) The heavy chain variable region has the amino acid sequence of SEQ ID NO: 10, and the light chain variable region has the amino acid sequence of SEQ ID NO: 12. (b) The heavy chain variable region has the amino acid sequence of SEQ ID NO: 11, and the light chain variable region has the amino acid sequence of SEQ ID NO: 12. (c) A heavy chain containing the amino acid sequence of SEQ ID NO: 15 or 22, and a light chain containing the amino acid sequence of SEQ ID NO: 16, (d) A heavy chain containing the amino acid sequence of SEQ ID NO: 19, and a light chain containing the amino acid sequence of SEQ ID NO: 16, (e) A heavy chain having an amino acid sequence represented by amino acid numbers 1 to 446 of SEQ ID NO: 15 or 22, and a light chain having an amino acid sequence represented by amino acid numbers 1 to 219 of SEQ ID NO: 16, (f) A heavy chain having the amino acid sequence represented by amino acid numbers 1-446 of SEQ ID NO: 19, and a light chain having the amino acid sequence represented by amino acid numbers 1-219 of SEQ ID NO: 16. It contains one of the following, and the antibody or its antigen-binding fragment is of the following formula (wherein the formula, asterisks * (This represents the connection point with the antibody.)

[0269] [ka] A conjugate according to any one of embodiments 47 to 61, which is conjugated by a thioether linkage to a drug linker represented by [the specified method].

[0270] Embodiment 64. The following formula:

[0271] [ka] It is represented by, In the formula, AB represents an antibody, which comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 10 and a light chain variable region having the amino acid sequence of SEQ ID NO: 12, y represents the average number of units of the drug-linker structure conjugated to the antibody per antibody, y being in the range of 1 to 10, 2 to 8, 3 to 8, 7 to 8, or 7.5 to 8, and the antibody is a conjugate or antibody conjugated by a thioether linkage to the drug-linker structure represented by the above formula.

[0272] Embodiment 65. The following formula:

[0273] [ka] It is represented by, In the formula, AB represents an antibody, which comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 11 and a light chain variable region having the amino acid sequence of SEQ ID NO: 12, y represents the average number of units of the drug-linker structure conjugated to the antibody per antibody, y being in the range of 1 to 10, 2 to 8, 3 to 8, 7 to 8, or 7.5 to 8, and the antibody is a conjugate or antibody conjugated by a thioether linkage to the drug-linker structure represented by the above formula.

[0274] Embodiment 66. The following formula:

[0275] [ka] It is represented by, In the formula, AB represents an antibody, and the antibody has the amino acid sequence of SEQ ID NO: 15 or 22. A conjugate or antibody comprising a chain and a light chain having the amino acid sequence of SEQ ID NO: 16, where y represents the average number of units of the drug-linker structure conjugated to the antibody per antibody, and y is in the range of 1 to 10, 2 to 8, 3 to 8, 7 to 8, or 7.5 to 8, wherein the antibody is conjugated to the drug-linker structure represented by the above formula via a thioether bond.

[0276] Embodiment 67. The following formula:

[0277] [ka] It is represented by, In the formula, AB represents an antibody, which comprises a heavy chain having the amino acid sequence of SEQ ID NO: 19 and a light chain having the amino acid sequence of SEQ ID NO: 16, y represents the average number of units of the drug-linker structure conjugated to the antibody per antibody, y being in the range of 1 to 10, 2 to 8, 3 to 8, 7 to 8, or 7.5 to 8, and the antibody is a conjugate or antibody conjugated by a thioether linkage to the drug-linker structure represented by the above formula.

[0278] Embodiment 68. A conjugate or antibody according to any one of Embodiments 31 to 67, wherein the antibody comprises one or more modifications selected from the group consisting of defucosylation, fucose reduction, N-linked glycosylation, O-linked glycosylation, N-terminal processing, C-terminal processing, deamidation, aspartic acid isomerization, methionine oxidation, substitution of two leucine (L) residues at positions 234 and 235 of the heavy chain with alanine (A) (LALA), amidation of a proline residue, and deletion or absence of one or two amino acids at the carboxyl terminus.

[0279] Embodiment 69. The conjugate or antibody according to Embodiment 68, wherein the antibody comprises a deletion or absence of one or two amino acids at the carboxyl terminus of the heavy chain.

[0280] Embodiment 70. The conjugate or antibody according to Embodiment 69, wherein the antibody comprises two heavy chains, both lacking one carboxyl-terminal amino acid residue.

[0281] Embodiment 71. The following formula:

[0282] [ka] It is represented by, In the formula, AB represents an antibody, which comprises a heavy chain having an amino acid sequence represented by amino acid numbers 1-446 of SEQ ID NO: 15 or 22, and a light chain having an amino acid sequence represented by amino acid numbers 1-219 of SEQ ID NO: 16, where y represents the average number of units of the drug-linker structure conjugated to the antibody per antibody, where y is in the range of 1 to 10, 2 to 8, 3 to 8, 7 to 8, or 7.5 to 8, and the antibody is a conjugate or antibody conjugated by a thioether linkage to the drug-linker structure represented by the above formula.

[0283] Embodiment 72. The following formula:

[0284] [ka] It is represented by, AB represents an antibody, which comprises a heavy chain having an amino acid sequence represented by amino acid numbers 1-446 of SEQ ID NO: 19 and a light chain having an amino acid sequence represented by amino acid numbers 1-219 of SEQ ID NO: 16, where y represents the average number of units of the drug-linker structure conjugated to the antibody per antibody, where y is in the range of 1 to 10, 2 to 8, 3 to 8, 7 to 8, or 7.5 to 8, and the antibody is a conjugate or antibody conjugated by a thioether bond to the drug-linker structure represented by the above formula.

[0285] Embodiment 73. A conjugate or antibody according to any one of Embodiments 31 to 72, wherein the number of conjugated drug molecules per antibody molecule is 8.

[0286] Embodiment 74. A pharmaceutical composition comprising an antibody or conjugate as described in any one of Embodiments 31 to 73, and one or more further components selected from the group consisting of solvents, diluents, and excipients.

[0287] Embodiment 75. An antibody or conjugate according to any one of Embodiments 31 to 73 or a pharmaceutical composition according to Embodiment 74, for use in pharmaceuticals.

[0288] Embodiment 76. An antibody or conjugate according to any one of Embodiments 31 to 73 or a pharmaceutical composition according to Embodiment 74 for use in the treatment, prognosis, diagnosis, detection, and / or monitoring of diseases associated with abnormal cell growth such as cancer; infections such as bacterial, viral, fungal, or parasitic infections; inflammatory diseases such as autoimmune diseases and inflammatory bowel disease; and diseases associated with reduced immune activity such as immunodeficiency.

[0289] Embodiment 77. An antibody, conjugate, or pharmaceutical composition according to Embodiment 76, for use in the treatment of cancer, particularly cancer expressing TA-MUC1, wherein the cancer is selected from the group consisting of ovarian cancer, breast cancer, pancreatic cancer, lung cancer, colon cancer, gastric cancer, liver cancer, kidney cancer, hematological cancer, endometrial cancer, thyroid cancer, leukemia, hemihydrocystic carcinoma, melanoma, carcinoma, teratoma, lymphoma, sarcoma, mesothelioma, neuroblastoma, glioma, rectal cancer, adrenal cancer, skin cancer, brain cancer, cervical cancer, intestinal cancer, intestinal cancer, head and neck cancer, gastrointestinal cancer, lymph node cancer, esophageal cancer, colorectal cancer, otorhinolaryngeal (ENT) cancer, prostate cancer, bladder cancer, uterine cancer, and metastases thereof.

[0290] Embodiment 78. An antibody, conjugate, or pharmaceutical composition according to Embodiment 76, for use in the treatment of an infectious disease, wherein the infectious disease is selected from the group consisting of bacterial infections, viral infections, fungal infections, and parasitic infections.

[0291] Embodiment 79. An antibody, conjugate, or pharmaceutical composition according to Embodiment 76, for use in the treatment of an autoimmune disease, wherein the autoimmune disease is selected from the group consisting of celiac disease, type 1 diabetes, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus.

[0292] Embodiment 80. A method for treating a target cancer that requires the same, comprising the step of administering to a target having cancer, in particular cancer expressing TA-MUC1, a therapeutically effective amount of the conjugate or antibody described in any one of Embodiments 31 to 73 or the composition described in Embodiment 74.

[0293] Embodiment 81. Cancers include ovarian cancer, breast cancer, pancreatic cancer, lung cancer, colon cancer, stomach cancer, liver cancer, kidney cancer, hematological cancer, endometrial cancer, thyroid cancer, leukemia, hemihydrocystic carcinoma, melanoma, A method for treating cancer according to Embodiment 80, selected from the group consisting of carcinoma, teratoma, lymphoma, sarcoma, mesothelioma, neuroblastoma, glioma, rectal cancer, adrenal cancer, skin cancer, brain cancer, cervical cancer, intestinal cancer, colon cancer, head and neck cancer, gastrointestinal cancer, lymph node cancer, esophageal cancer, colorectal cancer, otolaryngeal (ENT) cancer, prostate cancer, bladder cancer, uterine cancer, and metastases thereof. [Brief explanation of the drawing]

[0294] [Figure 1]This figure shows ELISA binding curves of anti-MUC1 antibodies against different MUC1 peptides. (A) shows antigen binding of PankoMab N54Q (PM-N54Q) lacking Fab glycosylation and PankoMab (PM) containing Fab glycosylation to the MUC1 peptide containing the epitope sequence PDTR. The threonine of the MUC1 peptide is glycosylated with Tn, sTn, TF, or sTF. (B) shows the binding of PankoMab and PM-N54Q to the MUC1 peptide containing the epitope sequence variant PESR. The serine of the MUC1 peptide is glycosylated with Tn. (C) shows the binding of PM-N54Q to the MUC1 peptide containing the epitope sequence PDTR. The threonine of the MUC1 peptide is glycosylated with Tn or not glycosylated. (D) shows the binding of several N54X variants to the Tn-PDTR MUC1 peptide compared to PankoMab containing Fab glycosylation, diluted from the cell culture supernatant of transiently transfected cells. (E) shows the binding curves of three purified N54X variants without Fab glycosylation to the Tn-PDTR, TF-PDTR, and non-glycosylated PDTR MUC1 peptides compared to PankoMab with Fab glycosylation. (F) shows the binding of two framework variants of PM-N54Q to the Tn-PDTR MUC1 peptide compared to PankoMab with Fab glycosylation. In the case of framework variant mf-a, nine amino acids are mutated in the VH framework and three amino acids in the VL framework, and in the case of mf-b, nine amino acids are mutated in the VH framework and four amino acids in the VL framework. [Figure 2] This figure shows the surface plasmon resonance (Biacore) binding of the anti-MUC1 antibody PM and PM-N54Q to the glycosylated PDTR-MUC1 peptide. The maximum binding signals of PM-N54Q and PankoMab at various concentrations are plotted against antibody concentration. [Figure 3]This figure shows the results of fluorescence proximity sensing using a DRX instrument. Binding and dissociation curves are shown. (A) PM with Fab glycosylation is compared to (B) PM-N54Q without Fab glycosylation. [Figure 4] This figure shows SDS acrylamide gels after electrophoretic separation of PM-N54Q and PankoMab under non-reducing conditions (left) and reducing conditions (right). Lane 1: PM-N54Q after the capture step; Lane 2: PM-N54Q after the finishing step; Lane 3: PankoMab after the capture step; Lane 4: PankoMab after the finishing step; Lane 5: Molecular weight marker. [Figure 5] This figure shows Coomassie blue stained gels from isoelectric focusing electrophoresis assays using PM-N54Q lacking Fab glycosylation and PankoMab with Fab glycosylation. Lane 1: PankoMab with Fab glycosylation; Lane 2: PM-N54Q lacking Fab glycosylation. [Figure 6] This figure shows the binding of an anti-MUC1 antibody to Fcγ receptor IIIa. By increasing the concentration of the antibody PM-N54Q or PankoMab, the rabbit anti-mouse coupling acceptor beads are extruded from the FcγRIIIa-loaded donor beads, thereby reducing the detected chemiluminescence. In Figure 6A, a low-fucosylated antibody was applied to the assay, and in Figure 6B, a high-fucosylated antibody was applied. [Figure 7] This figure shows the binding of anti-MUC1 antibodies PM-N54Q, PM-N54D, and PM with Fab glycosylation to tumor cell lines (A) CaOV-3 and (B) HSC-4, as analyzed by flow cytometry. [Figure 8]This figure shows the cytotoxic activity of control hIgG-ADC, naked PankoMab, and PankoMab-ADC against the cancer cell line MDA-MB-468, which expresses the TA-MUC1 protein; B) control hIgG-ADC, naked PankoMab, and PankoMab-ADC against the cancer cell line HCT-15, which does not express the TA-MUC1 protein; C) control hIgG-ADC, naked PankoMab, PankoMab-ADC, naked PM-N54Q, and PM-N54Q-ADC against the cancer cell line NCI-H441, which expresses the TA-MUC1 protein; and D) control hIgG-ADC, naked PankoMab, PankoMab-ADC, naked PM-N54Q, and PM-N54Q-ADC against the cancer cell line HPAC, which expresses the TA-MUC1 protein. Cells were treated with each compound over a 6-day period, and cell viability (%) was calculated using an ATP assay. Data represent mean ± standard deviation (N=3). [Figure 9] This figure shows the antitumor efficacy of control hIgG-ADC, naked PankoMab, and PankoMab-ADC against MDA-MB-468-conserving nude mice. A single dose of 3 mg / kg of control hIgG-ADC, naked PankoMab, or PankoMab-ADC, or a vehicle (acetic acid buffer solution), was administered intravenously to MDA-MB-468-conserving nude mice (N=6 / group). Estimated tumor volume data represent mean ± SEM. Arrows indicate the timing of administration. Estimated tumor volume 21 days after PankoMab-ADC administration was compared to the estimated tumor volume of the control hIgG-ADC group or the estimated tumor volume of the naked PankoMab-treated group by Student's t-test. ***P<0.001. [Figure 10]This figure shows the antitumor efficacy of control hIgG-ADC, naked PankoMab, and PankoMab-ADC against HCC70-conserving nude mice. A single dose of 10 mg / kg of control hIgG-ADC, naked PankoMab, or PankoMab-ADC, or a vehicle (acetic acid buffer solution), was administered intravenously to HCC70-conserving nude mice (N=6 / group). Estimated tumor volume data represent mean ± SEM. Arrows indicate the timing of administration. Estimated tumor volume 21 days after PankoMab-ADC administration was compared to the estimated tumor volume of the control hIgG-ADC-treated group or the naked PankoMab-treated group by Student's t-test. ***P<0.001. [Figure 11] This figure shows the antitumor efficacy of control hIgG-ADC, naked PM-N54Q, PankoMab-ADC, and PM-N54Q-ADC against HPAC-carrying nude mice. A single dose of 10 mg / kg of control hIgG-ADC, naked PM-N54Q, PankoMab-ADC, or PM-N54Q-ADC, or a vehicle (acetic acid buffer solution), was administered intravenously to HPAC-carrying nude mice (N=6 / group). Estimated tumor volume data represent mean ± SEM. Arrows indicate the timing of administration. Estimated tumor volumes 21 days after administration of PankoMab-ADC and PM-N54Q-ADC were compared to the estimated tumor volumes of the control hIgG-ADC-treated group using Dunnett's test. ***P<0.001. [Figure 12]This figure shows the antitumor efficacy of control hIgG-ADC, naked PankoMab, naked PM-N54Q, PankoMab-ADC, and PM-N54Q-ADC against NCI-H441-conserving nude mice. Naked PankoMab, naked PM-N54Q, control hIgG-ADC, PankoMab-ADC, and PM-N54Q-ADC at 10 mg / kg, or a vehicle (acetic acid buffer solution) were administered intravenously as a single dose to NCI-H441-conserving nude mice (N=6 / group). Estimated tumor volume data represent mean ± SEM. Arrows indicate the timing of administration. Estimated tumor volume 31 days after administration of PankoMab-ADC and PM-N54Q-ADC was compared to the estimated tumor volume of the control hIgG-ADC-treated group by Dunnett's test. ***P<0.001. [Figure 13] This figure shows the antitumor efficacy of control hIgG-ADC, naked PankoMab, naked PM-N54Q, PankoMab-ADC, and PM-N54Q-ADC against OVCAR-5-conserving nude mice. Control hIgG-ADC, naked PankoMab, naked PM-N54Q, PankoMab-ADC, and PM-N54Q-ADC, or a vehicle (acetic acid buffer solution), were administered intravenously as a single dose of 10 mg / kg to OVCAR-5-conserving nude mice (N=6 / group). Estimated tumor volume data represent mean ± SEM. Arrows indicate the timing of administration. Estimated tumor volumes 14 days after administration of PankoMab-ADC and PM-N54Q-ADC were compared to the estimated tumor volumes of the control hIgG-ADC-treated group using Dunnett's test. The estimated tumor volume 14 days after PM-N54Q-ADC administration was compared to the estimated tumor volume of the PankoMab-ADC treatment group using a Student's t-test. ***P<0.001. [Figure 14]This figure shows the antitumor efficacy of control hIgG-ADC, PankoMab-ADC, and PM-N54Q-ADC against HCT-15-conserving nude mice. A single dose of 10 mg / kg of control hIgG-ADC, PankoMab-ADC, PM-N54Q-ADC, or vehicle (acetic acid buffer solution) was administered intravenously to HCT-15-conserving nude mice (N=6 / group). Estimated tumor volume data represent mean ± SEM. Arrows indicate the timing of administration. Estimated tumor volume 21 days after administration of PankoMab-ADC and PM-N54Q-ADC was compared to the estimated tumor volume of the control hIgG-ADC-treated group using Dunnett's test. ***P<0.001. [Figure 15] This figure shows the amino acid sequence of the heavy chain of the humanized antibody PM N54Q (sequence number 15, but the amino acid at position 57 is Gln, i.e., sequence number 22). [Figure 16] This figure shows the amino acid sequences of the light chains of the humanized antibody PM N54Q and PankoMab (SEQ ID NO: 16). [Figure 17] This figure shows the amino acid sequence of the heavy chain of the humanized antibody PankoMab (SEQ ID NO: 19). [Figure 18] This figure shows the amino acid sequence of the heavy chain of the chimeric antibody PM N54Q (sequence number 20, but the amino acid at position 76 is Gln, i.e., sequence number 23). [Figure 19] This figure shows the amino acid sequence of the light chain of the chimeric antibody PM N54Q (SEQ ID NO: 21). [Examples]

[0295] Example 1: 1. Production of anti-MUC1 antibodies The nucleic acid sequence of the heavy chain of the humanized PankoMab antibody (see, for example, WO2011 / 012309) was modified by mutating the Asn54 codon (amino acid position 57 in SEQ ID NO: 11) using the Kabat / Eu numbering scheme to any amino acid other than Asn, particularly the Gln codon.

[0296] 1) Production of anti-MUC1 antibodies in human myeloid leukemia-derived cell lines A vector containing the coding sequences for the γ1 heavy chain and κ light chain of a mutant antibody was transfected into the human myeloid leukemia cell line NM-H9D8 (DSM ACC2806). Different αMUC1 antibodies containing N54X mutations (PankoMab N54X / PM-N54X, where X is any amino acid other than N / Asn) or amino acid mutations in the VH and VL framework sequences were expressed in the resulting clones to produce constructs with a human glycosylation pattern. The concentration of αMUC1 antibody in the supernatant was determined by Octet measurement using a protein A coated pin, or quantified by UV280 absorbance after purification by protein A chromatography. The binding characteristics of the different αMUC1 antibodies were determined by antigen-ELISA (see Example 2), and the selected purified antibodies were analyzed by scatchard analysis (see Example 3), Biacore (see Example 4a), and DRX. 2 Using switchSENSE (registered trademark) technology (Example 4b) (See reference) or analyzed by flow cytometry (Example 7).

[0297] In addition, PM-N54Q and non-mutant PankoMab with Fab glycosylation were also expressed in the human myeloid leukemia-derived cell line NM-H9D8-E6Q12 (DSM ACC2856), which expresses antibodies with reduced fucose content. These antibodies, along with the same antibodies expressed in NM-H9D8, were purified, and their binding behavior to Fc gamma receptor IIIA was analyzed in Example 6.

[0298] 2) Production of anti-MUC1 antibodies in CHO cell lines The PM-N54Q coding sequence (the heavy chain nucleotide sequence of PM-N54Q represented by SEQ ID NO: 17 and the light chain nucleotide sequence of PM-N54Q represented by SEQ ID NO: 18), synthesized by ThermoFisher Scientific's GeneArt™, was cloned into an expression vector, and the resulting plasmid was electro-transfected into CHO cells. PM-N54Q mutant antibodies were produced using a basic procedure, applying the principle of growing pooled cells under selective pressure. Anti-MUC1 antibodies (PM-N54Q) produced in the CHO cell line were used in Examples 8 and 9.

[0299] 2. PankoMab-ADC, N54Q-ADC, and DXd PankoMab-GEX, referring to a humanized anti-TA-MUC1 monoclonal antibody containing a glycosylation site in CDR2-H2 (Fab glycosylation), and PM-N54Q (Example 1-1), PankoMab-ADC, and PM-N54Q-ADC, referring to a humanized anti-TA-MUC1 monoclonal lacking Fab glycosylation, were produced by known methods such as WO2014 / 057687 and WO2015 / 115091. The PankoMab-GEX antibody contains a heavy chain containing SEQ ID NO: 19 and a light chain containing SEQ ID NO: 16, and therefore the PankoMab-GEX antibody is linked to the drug-linker of Formula 2. The PM-N54Q antibody mentioned above contains a heavy chain containing SEQ ID NO: 15 and a light chain containing SEQ ID NO: 16, and therefore the PM-N54Q antibody is linked to the drug-linker of Formula 2.

[0300] [ka]

[0301] Such PankoMab-ADC and PM-N54Q-ADC structures are represented by the following equation 5 (where y: the number of conjugated drug molecules per antibody molecule is 4 to 8, i.e., the average number of conjugated drug molecules per antibody (y): approximately 8), and AB represents PankoMab or PM-N54Q.

[0302] [ka]

[0303] The control hIgG-ADC consisted of a humanized IgG1 isotype control monoclonal antibody that does not bind to mammalian cells, as well as the same drug-linker as PnakoMab-ADC and PM-N54Q-ADC. The ADC payload (DXd) was produced by known methods such as WO2014 / 057687 and WO2015 / 115091.

[0304] Example 2: Antigen ELISA The antigen-binding properties of PankoMab N54X, in which the N-glycosylation site in the Fab region is knocked out, were compared with those of PankoMab, which has the N-glycosylation site in its Fab region.

[0305] The binding characteristics of the Fab deglycosylated form (PM-N54Q) of the MUC1-specific antibody PankoMab were analyzed using ELISA studies with various glycosylated and unglycosylated MUC1-derived tandem repeat peptides, compared with (glycosylated) PankoMab-GEX(registered trademark). In principle, both antibodies exhibit the same gradation when binding to glycosylated PDTR peptides (APPAHGVTSAPDT(X)-RPAPGSTAPPAHGVTSA) that have different glycosylations at T. That is, Galβ1-3GalNAc alpha The strongest binding was observed to the PDTR peptide holding (TF), followed by sialylated TF and GalNAc alpha (Tn)O-glycosylation. Siallylated GalNAc alpha Binding to (sTn)O-glycosylation was significantly lower. As PankoMab-GEX®, PM-N54Q showed only slight binding affinity to the non-glycosylated MUC1 PDTR peptide, demonstrating appropriate tumor specificity (Figure 1C).

[0306] However, GalNAc alpha In a TA-MUC1 antigen ELISA using a biotinylated glycopeptide that holds (Tn)O-glycan, PM-N54Q showed four times higher binding compared to PankoMab-GEX®. PM-N54Q is a sialylated GalNAc alpha When glycosylated with (sTn), it binds to the same MUC1 peptide approximately 7 times better. Galβ1-3GalNAc in threonine of the PDTR sequence alpha Binding to (TF) and sialylated TF (sTF) (Figure 1A) was twice as good with PM-N54Q.

[0307] Both antibodies show strongly reduced binding to the MUC1 peptide variant APPAHGVTSAPE-S(Tn)-RPAPGSTAPPAHGVTSA, which has Tn glycosylation at the serine, compared to binding to the PDT(Tn)R peptide. However, even in this case, Fab-deglycosylated PM-N54Q binds significantly more strongly than PankoMab-GEX® (Figure 1B).

[0308] Different Fab-deglycosylated PM-N54X mutants were compared to PankoMab, which has N-glycosylation in its Fab moiety. Initially, all mutants were compared directly from the supernatant without purification. Concentrations were determined by Octet. All PM-N54X mutants bound better than Fab-glycosylated PM. In addition, a clear trend dependent on the chemical properties of the amino acid side chain was observed. Carboxylic acid groups in the side chain showed the lowest binding enhancement. The best binding was observed with amino acids having one or two nitrogen atoms (as primary or secondary amines) (Figure 1D).

[0309] In addition, selected Fab deglycosylation mutants (PM-N54H, -W, and -Q) were purified by protein A chromatography and analyzed by ELISA (Figure 1E). Compared to PankoMab with Fab glycosylation, each mutant showed approximately 5-8 times improved binding to the TF-MUC1 peptide and approximately 2-3 times improved binding to the Tn-MUC1 peptide.

[0310] Furthermore, two different framework variants of PM-N54Q were analyzed by ELISA for binding to the Tn glycosylated PDTR-MUC1 peptide (see Figure 1F). Framework variant mf-a has nine amino acid mutations in the VH framework and three amino acid mutations in the VL framework, while variant mf-b has nine amino acid mutations in the VH framework and four amino acid mutations in the VL framework. Both mutant variants showed similar binding to the PM-N54Q antibody.

[0311] Example 3: Saturation binding analysis of anti-MUC1 antibodies against MCF-7 cells and ZR-75-1 cells Two factors are particularly important for the therapeutic suitability of an antibody: its affinity for tumor cells and the number of binding sites.

[0312] The binding of the MUC1-specific antibody PankoMab (PM-N54Q) to TA-MUC-1-positive human tumor cell lines was evaluated using radioisotope-labeled antibodies by saturated binding analysis against human breast cancer cell lines ZR-75-1 and MCF-7, compared to Fab-glycosylated PankoMab-GEX®. The antibody was chelated with 12-fold molar excess p-SCN-benzyl-DTPA in 50 mM sodium carbonate, 150 mM NaCl, pH 8.7 for 2 hours at 37°C, followed by overnight incubation at 2–8°C. Free chelating agents were removed by desalting column and total filtration (50 kDa cutoff, 6× buffer exchange to PBS). Chelated antibodies were incubated in 6 mM phosphate, 1.6 mM KCl, 80 mM NaCl, 0.2 M Na acetate, and 0.1 M HCl for 1 hour at 37°C without a carrier.111 The antibody was radiolabeled with In (2 μCi / μg antibody). The preparation was neutralized by adding 8-9 times the volume of 10× concentrated PBS. Approximately 1 / 50 volume of fetal bovine serum was added to the neutralized labeled antibody preparation. 1 × 10 for each cell binding method. 6 Cells were used. Several concentrations of labeled antibody were added to pelletized cells (30-1000 ng / 200 μL in 1% BSA / PBS). The resuspended cell-antibody mixture was measured with a gamma counter and incubated at 4°C for 1 hour. Antibody-bound cells were separated by centrifugation and washed with 1% BSA / PBS at 4°C for another 1 hour. The cell pellet was then subjected to further washing. 111 The In-labeled antibody was measured using a gamma counter. The evaluation was performed using Gra The analysis was performed using phPad Prism's "single-site specific ka" protocol. The obtained data are summarized in Table 1. The data indicate that PM-N54Q has high affinity for these tumor cells and a very large number of binding sites. Binding was more than 2.5 times higher than with PankoMab-GEX®, and the number of binding sites also increased slightly.

[0313] [Table 1]

[0314] Example 4a: Surface plasmon resonance (BiaCore) analysis The binding of the Fab-deglycosylated form (PM-N54Q) of the MUC1-specific antibody PankoMab to a glycosylated peptide derived from TA-MUC-1 was evaluated by surface plasmon resonance spectroscopy (Biacore). Streptavidin sensor chips were coated with biotinylated TA-MUC1 peptide (Tn glycosylated or unglycosylated). PankoMab and PM-N54Q were sequentially diluted 1:3 with HPS-EP from 3,600 to 4.9 nM. The dilutions were injected at 50 μL / min. The maximum binding at each concentration was determined as the response unit (RU) and evaluated using GraphPad Prism with "single-site specific binding". Figure 2 shows the binding curves with PM-N54Q compared to PankoMab-GEX®. Affinity (K) of PM-N54Q and PankoMab-GEX® are shown. D The values ​​were calculated to be 388 nM and 652 nM, respectively. Therefore, under this experimental setup, an almost twofold increase in affinity was detectable.

[0315] Example 4b: Fluorescence proximity detection method (DRX) 2 (Dynamic Biosensors) Fluorescence proximity sensing, using single-stranded DNA (96-mer) spotted on a chip and complementary DNA coupled to a ligand, is a novel method for determining binding constants and affinity. In this study, streptavidin was used as the ligand to capture the biotinylated TA-MUC1 peptide. Binding of PankoMab to the peptide resulted in a change in fluorescence. On-rate and off-rate can be calculated during binding and dissociation. Due to its higher sensitivity, it allows for monitoring of faster interactions compared to surface plasmon resonance. This yields binding dynamics that are different from SPR but more comparable to KinExA, an "optimal standard" method measured in liquid systems.

[0316] PankoMab and PM-N54Q were diluted in PE140 buffer from 300 nM to 3.67 nM in a 1:9 step ratio and applied to the peptide conjugated to the chip. Binding curves were evaluated by single exponential global fitting (using the instrument's software). Binding curves for PM and PM-N54Q are shown exemplarily in Figures 3A and 3B. Calculated affinities for PankoMab variants are shown in Table 2.

[0317] [Table 2]

[0318] Example 5: Biochemical Characterization Antibody purity and identity were analyzed using non-reducing and reduced SDS-PAGE. The band pattern of the non-reducing gel showed a major band at approximately 160 kDa, with theoretical artifacts of heavy and light chains and their combinations (approximately 25, 50-55, 75, 110, 135 kDa). The reduced gel showed distinct light and heavy chain bands at 25 and 50-55 kDa. Due to the lack of Fab glycosylation, PM-N54Q has a smaller heavy chain, as expected (see right side of Figure 4).

[0319] The charge profiles are clearly different, as shown by isoelectric focusing (IEF; see Figure 5). The Fab glycosylation is considerably sialylated, while the Fc glycosylation is minimally sialylated. Therefore, PankoMab-GEX® has more charged isoforms than PM-N54Q, which reflects a higher level of charged sialic acid in the Fab moiety.

[0320] Example 6: Fcγ receptor binding The FcγR binding assay for FcγRIIIa (CD16a) is based on PerkinElmer's AlphaScreen® technology. The AlphaScreen® platform relies on PerkinElmer's simple bead-based technology and does not require a washing step, making it a more efficient alternative to traditional ELISA.

[0321] In the receptor binding assay, His-tagged FcγRIIIa (Glycotope GmbH) is captured by Ni chelate donor beads. Anti-MUC1 antibody and rabbit anti-mouse coupling acceptor beads compete for binding to FcγR. In the case of interaction between FcγR and rabbit anti-mouse coupling acceptor beads, luminescence is produced when the donor and acceptor beads are in close proximity and laser excitation is performed at 680 nm. Maximum signal is achieved in the absence of competing substances. max ). If there is competition, the test antibody will bind to FcγR and signal max It is reduced in a concentration-dependent manner. Chemoluminescence was quantified by measuring from 520 to 620 nm using an EnSpire2300 multi-label reader (PerkinElmer) (AlphaScreen® method). All results are expressed as mean ± standard deviation of replicate samples. Data were evaluated and calculated using nonlinear curve fitting (sigmoidal dose-response variable slope) with GraphPad Prism 5 software. As a result, a concentration-dependent sigmoid curve was obtained. This curve has an upper plateau, a lower plateau, a slope, and EC. 50 It is determined by [the relevant law].

[0322] As shown in Figures 6A and 6B, the FcγRIIIa binding affinity to PankoMab N54Q and PankoMab was equivalent. In Figure A, a low-fucosylated antibody was applied to the assay, and in Figure B, a high-fucosylated antibody was applied. Therefore, Fab glycosylation Removal of the chromogenic compound did not affect the antibody receptor interaction.

[0323] Example 7: Binding to cellular TA-MUC1 N54Q and N54D were transiently expressed and purified by protein A chromatography. The binding of the two mutants to cell surface TA-MUC1 was compared with PM having Fab glycosylation using two different cancer cell lines. The tongue squamous cell carcinoma line HSC-4 moderately expresses TA-MUC1, while the ovarian cancer cell line CaOV-3 highly expresses it. Tumor cells were incubated with serially diluted antibodies, and the bound antibodies were detected using phycoerythrin conjugate goat anti-human IgG (heavy and light chain) antibodies. A human IgG control was included for background staining control. Binding was analyzed by flow cytometry.

[0324] The analyzed constructs PM, PM-N54Q, and PM-N54D showed potent and specific binding to TA-MUC1-expressing HSC-4 and CaOV-3 cells compared to human IgG1 control (Figure 7). TA-MUC1 high The binding of PM-N54D to CaOV-3 cells was comparable to that of PM with Fab glycosylation, but PM-N54Q showed slightly better binding (Figure 7A). When using HSC-4 cancer cells expressing TA-MUC1 at moderate levels, the mutant PM-N54Q showed significantly better binding to cellular TA-MUC1 compared to PM, while PM-N54D showed inferior binding compared to PM with Fab glycosylation (Figure 7B).

[0325] Example 8: Evaluation of the in vitro efficacy of PankoMab-ADC and PM-N54Q-ADC 8.1 Cell lines Human breast cancer cell line MDA-MB-468, human pancreatic cancer cell line HPAC, and human lung cancer cell line NCI-H441 were used as cells expressing TA-MUC1 at moderate to high levels. Human colorectal cancer cell line HCT-15 was used as a TA-MUC1-negative cell line. These cell lines were purchased from ATCC. Each cell line was cultured according to the instructions for use. The expression level of TA-MUC1 on each cancer cell line was confirmed by flow cytometry.

[0326] 8.2 Evaluation of the in vitro efficacy of PankoMab-ADC By using culture medium, 1.25 × 10 4 MDA-MB-468 suspensions were prepared to a concentration of cells / mL and added to each well of a 96-well plate with a black clear bottom at a concentration of 80 μL / well (1000 cells / well). In the case of blank wells, only culture medium was added to the well at a concentration of 80 μL / well (N=3). All cells were incubated overnight in MDA-MB-468 under appropriate conditions.

[0327] By using culture medium, 3.1 × 10 3 HCT-15 suspensions were prepared to a concentration of cells / mL and added to each well of a 96-well plate with a black clear bottom at a concentration of 80 μL / well (250 cells / well). In the case of blank wells, only culture medium was added to the well at a concentration of 80 μL / well (N=3). All cells were incubated overnight in HCT-15 under appropriate conditions.

[0328] The following day, each naked PankoMab, control hIgG-ADC, and PankoMab-ADC were triple-diluted in their respective culture media from 500 nM to 0.2 nM. 20 microliters of these dilutions were added to the appropriate wells (final concentration: 100 nM to 0.04 nM). For blank and untreated wells, only 20 μL of each culture medium was added to the well. All plates were incubated for 6 days under conditions appropriate for each cell line.

[0329] After incubation, the amount of ATP in each well was measured using the CellTiter-Glo luminescence cell survival assay (Promega). Luminescence was measured using a multi-label counter (ARVO X3, PerkinElmer Japan Co., Ltd.). This assay was performed in triple replicates.

[0330] The cell viability of each sample was calculated using the following equation. Cell viability (%)=100×(TB) / (CB) T: Luminescence intensity of the test well C: Average luminescence intensity of untreated wells B: Blankwell's average luminescence intensity

[0331] 8.3 Comparison of in vitro efficacy between PankoMab-ADC and PM-N54Q-ADC By using culture medium, 1.25 × 10 4 HPAC suspensions were prepared to a concentration of cells / mL and added to each well of a 96-well plate with a black clear bottom at a concentration of 80 μL / well (1000 cells / well). In the case of blank wells, only culture medium was added to the well at a concentration of 80 μL / well (N=3). All cells were incubated overnight in the HPAC under appropriate conditions.

[0332] By using culture medium, 1.25 × 10 4 NCI-H441 suspensions were prepared to a concentration of cells / mL and added to each well of a 96-well plate with a black clear bottom at 80 μL / well (1000 cells / well). In the case of blank wells, only culture medium was added to the well at 80 μL / well (N=3). All cells were incubated overnight in NCI-H441 under appropriate conditions.

[0333] The following day, each of the naked PankoMab, naked PM-N54Q, hIgG-ADC, PankoMab-ADC, and PM-N54Q-ADC was triple-diluted in their respective culture media from 500 nM to 0.2 nM. 20 microliters of these dilutions were added to the appropriate wells (final concentration: 100 nM to 0.04 nM). For blank and untreated wells, only 20 μL of each culture medium was added to the well. All plates were incubated for 6 days under conditions appropriate for each cell line.

[0334] After incubation, the amount of ATP in each well was measured using the CellTiter-Glo luminescence cell survival assay (Promega). Luminescence was measured using a multi-label counter (ARVO X3, PerkinElmer Japan Co., Ltd.). This assay was performed in triple replicates.

[0335] The cell viability of each sample was calculated using the following equation. Cell viability (%)=100×(TB) / (CB) T: Luminescence intensity of the test well C: Average luminescence intensity of untreated wells B: Blankwell's average luminescence intensity

[0336] The potency ratios of PankoMab-ADC versus PM-N54Q-ADC for cytotoxic activity against HPAC and NCI-H441, and their 95% confidence intervals (CIs), were calculated as a post-hoc analysis using 3-parameter logistic parallel-line analysis (common slope) with EXSUS ver. 8.1 (CAC Croit, Tokyo, Japan) based on SAS Release 9.4 (SAS Institute Japan, Tokyo, Japan). (Emax: 100, Emin: estimated value). Differences in cytotoxic activity were considered significant if the 95% confidence interval of the potency ratio did not include 1.

[0337] Example 9: Evaluation of the in vivo efficacy of PankoMab-ADC and PM-N54Q-ADC 9.1 Cell lines Human breast cancer cell lines MDA-MB-468 and HCC70, human pancreatic cancer cell line HPAC, and human lung cancer cell line NCI-H441 were used as tumor cells expressing TA-MUC1 at moderate to high levels. Human colorectal cancer cell line HCT-15 was used as TA-MUC1-negative tumor cells. These cell lines were purchased from ATCC. Human ovarian cancer cell line OVCAR-5 was purchased from the National Cancer Institute and used as TA-MUC1-low expressing tumor cells. Each cell line was cultured according to the instructions for use. The expression level of TA-MUC1 on each cancer cell line was confirmed by flow cytometry and IHC staining.

[0338] 9.2 Evaluation of the in vivo efficacy of PankoMab-ADC MDA-MB-468 cells were suspended in Matrigel (BD) and 1 × 10⁶ cells were prepared. 7 Cells were subcutaneously transplanted into the right side of each female nude mouse (day 0), and the mice were randomly grouped on day 20 (N=6). After grouping, each naked PankoMab solution, control hIgG-ADC solution, or PankoMab-ADC solution was administered intravenously as a single dose at a dose of 3 mg / kg. The vehicle (acetic acid buffer solution) group was established as the control group. After administration, the length and width of the tumors in each mouse were measured twice a week using a digital caliper over a period of 21 days.

[0339] HCC70 cells were suspended in physiological saline (Otsuka Pharmaceutical Co., Ltd.) and 1 × 10⁶ cells were prepared. 7 Cells were subcutaneously transplanted into the right side of each female nude mouse (day 0), and the mice were randomly grouped on day 19 (N=6). After grouping, each naked PankoMab solution, control hIgG-ADC solution, or PankoMab-ADC solution was administered intravenously as a single dose at a dose of 10 mg / kg. The vehicle (acetic acid buffer solution) group was established as the control group. After administration, the length and width of the tumors in each mouse were measured twice a week using a digital caliper for 21 days.

[0340] The estimated tumor volume for each mouse was calculated using the following equation. Estimated tumor volume (mm 3 ) = 1 / 2 × length (mm) × width (mm) 2

[0341] Furthermore, the tumor growth inhibition (TGI, %) for each group on the last measurement day of the vehicle treatment group was calculated using the following formula and rounded to an integer. TGI (%) = (1 - T / C) × 100 T: Mean estimated tumor volume (mm³) of naked PankoMab, control hIgG-ADC, or PankoMab-ADC 3 ) C: Mean estimated tumor volume (mm) of the vehicle treatment group 3 )

[0342] To evaluate the antitumor efficacy of PankoMab-ADC, the tumor volume of each mouse in the PankoMab-ADC-treated group at the last measurement day (MDA-MB-468: day 41, HCC70: day 40) was compared to the tumor volume of the control hIgG-ADC-treated group or the tumor volume of the naked PankoMab-treated group by Student's t-test. All statistical analyses were performed as post-hoc analyses using SAS System Release 9.2 (SAS Institute Inc.). A P-value of less than 0.05 was considered statistically significant.

[0343] 9.3 In vivo availability of PankoMab-ADC and PM-N54Q-ADC Comparison of effectiveness HPAC cells were suspended in physiological saline (Otsuka Pharmaceutical Co., Ltd.) and 3 × 10⁻⁶ cells were prepared. 6Cells were subcutaneously transplanted into the right side of each female nude mouse (day 0), and the mice were randomly grouped on day 11 (N=6). After grouping, each naked PM-N54Q solution, control hIgG-ADC solution, PankoMab-ADC solution, or PM-N54Q-ADC solution was administered intravenously as a single dose at a dose of 10 mg / kg. The vehicle (acetic acid buffer solution) group was established as the control group. After administration, the length and width of the tumors in each mouse were measured twice a week using a digital caliper over a period of 21 days.

[0344] NCI-H441 cells were suspended in Matrigel (BD) and 5 × 10⁻⁶ cells were prepared. 6 Cells were subcutaneously transplanted into the right side of each female nude mouse (day 0), and the mice were randomly grouped on day 7 (N=6). After grouping, each naked PankoMab solution or naked PM-N54Q solution was administered intravenously as a single dose at a dose of 10 mg / kg, and a control hIgG-ADC solution, PankoMab-ADC solution, or PM-N54Q-ADC solution was administered intravenously as a single dose at a dose of 3 mg / kg. A vehicle (acetic acid buffer solution) administration group was established as the control group. After administration, the length and width of the tumors in each mouse were measured twice a week using a digital caliper for 31 days.

[0345] OVCAR-5 cells were suspended in physiological saline (Otsuka Pharmaceutical Co., Ltd.) and 5 × 10⁻⁶ cells were prepared. 6 Cells were subcutaneously transplanted into the right side of each female nude mouse (day 0), and the mice were randomly grouped on day 12 (N=6). After grouping, a single intravenous dose of either naked PankoMab solution, naked PM-N54Q solution, control hIgG-ADC solution, PankoMab-ADC solution, or PM-N54Q-ADC solution was administered at a dose of 10 mg / kg. A vehicle (acetic acid buffer solution) group was established as the control group. After administration, the length and width of the tumors in each mouse were measured twice a week using digital calipers for 21 days.

[0346] HCT-15 cells were suspended in physiological saline (Otsuka Pharmaceutical Co., Ltd.) and 5 × 10 6Cells were subcutaneously transplanted into the right side of each female nude mouse (day 0), and the mice were randomly grouped on day 10 (N=6). After grouping, a single intravenous dose of either a control hIgG-ADC solution, PankoMab-ADC solution, or PM-N54Q-ADC solution was administered at a dose of 10 mg / kg. The vehicle (acetic acid buffer solution) group was established as the control group. After administration, the length and width of the tumors in each mouse were measured twice a week using a digital caliper for 21 days.

[0347] The tumor volume of each mouse was calculated using the following equation. Estimated tumor volume (mm 3 ) = 1 / 2 × length (mm) × width (mm) 2

[0348] Furthermore, the tumor growth inhibition (TGI, %) of each mouse on the last measurement day of the vehicle-treated group or the last day when all groups were still alive was calculated according to the following formula and rounded to an integer. TGI (%) = (1 - T / C) × 100 T: Mean estimated tumor volume (mm³) of naked PankoMab, naked PM-N54Q, control hIgG-ADC, PankoMab-ADC, or PM-N54Q-ADC. 3 ) C: Mean estimated tumor volume (mm) of the vehicle treatment group 3 )

[0349] To evaluate the antitumor efficacy of each compound against HPAC-retaining mice, NCI-H441-retaining mice, OVCAR-5-retaining mice, and HCT-15-retaining mice, the last measurement day of the control hIgG-ADC-treated group (HPAC: day 32, NCI-H441: day 38, HCT-15: day 32) or the last day of survival for all groups (OVCAR-5) was used. The tumor volume of each mouse at day 26 was compared to that of the control hIgG-ADC-treated group using Dunnett's test. In addition, the tumor volume of OVCAR-5-retaining nude mice at day 33 was compared between the PankoMab-ADC-treated group and the PM-N54Q-ADC-treated group using Student's t-test. All statistical analyses were performed as post-hoc analyses using SAS System Release 9.2 (SAS Institute Inc.). A p-value of less than 0.05 was considered statistically significant.

[0350] Example 10: Results 10.1 Cytotoxic activity of PankoMab-ADC against TA-MUC1-positive and TA-MUC1-negative cancer cell lines in vitro To investigate whether PankoMab-ADC exhibits target-dependent and drug-dependent cytotoxic activity against human cancer cell lines, the in vitro efficacy of naked PankoMab, control hIgG-ADC, and PankoMab-ADC against human breast cancer cells MDA-MB-468 (TA-MUC1 positive) and human colorectal cancer cells HCT-15 (TA-MUC1 negative) was evaluated. As shown in Figure 8, naked PankoMab and hIgG-ADC showed little activity against each cell line (IC). 50 Under these conditions, PankoMab-ADC showed dose-dependent cytotoxic activity against TA-MUC1-positive cells MDA-MB-468 (Figure 8A, IC). 50 (<10 nM). However, PankoMab-ADC did not show activity against TA-MUC1-negative HCT-15 cells (Figure 8B, IC). 50 (>100 nM). Based on these results, we concluded that PankoMab-ADC exhibits target-dependent and drug-dependent cytotoxic activity against TA-MUC1-positive cancer cell lines in vitro.

[0351] 10.2 Comparison of in vitro cytotoxic activity between PankoMab-ADC and PM-N54Q-ADC against TA-MUC1-positive cells To investigate whether improved antigen-binding affinity can contribute to enhanced cytotoxic activity, the in vitro efficacy of PankoMab-ADC and PM-N54Q-ADC against human pancreatic cancer cell line HPAC and human lung cancer cell line NCI-H441 was evaluated. The cytotoxic activity of PM-N54Q-ADC against these cells was 1.5 times more potent than that of PankoMab-ADC (Figures 8C and 8D). The potency ratio of PM-N54Q-ADC to PankoMab-ADC against HPAC was 1.917 (1.611~2.280, 95% CI), and the potency ratio against NCI-H441 was 1.663 (1.495~1.849, 95% CI of EC50). These data demonstrate that the cytotoxic activity of PM-N54Q-ADC is significantly more potent than that of PankoMab-ADC. These results suggest that improved antigen-binding affinity of PankoMab-ADC may contribute to a significant enhancement of its cell-killing activity.

[0352] 10.3 Antitumor efficacy of PankoMab-ADC against TA-MUC1-positive tumors To investigate whether PankoMab-ADC is effective not only in vitro but also in vivo, the antitumor efficacy of naked PankoMab, control hIgG-ADC, and PankoMab-ADC in MDA-MB-468-retaining mice was evaluated. As shown in Figure 9, naked PankoMab and control hIgG-ADC (3 mg / kg, single dose) did not show antitumor efficacy (TGI was -18% for both at day 41). In contrast, PankoMab-ADC (3 mg / kg, single dose) significantly inhibited tumor growth (TGI was 97% at day 41). Furthermore, PankoMab-ADC showed significant antitumor efficacy compared to control hIgG-ADC and naked PankoMab (P<0.001 for both at day 41). In terms of body weight change, no weight loss caused by drug treatment was observed in any of the drug treatment groups.

[0353] Furthermore, the antitumor efficacy of naked PankoMab, control hIgG-ADC, and PankoMab-ADC in HCC70-retaining mice was evaluated. As shown in Figure 10, naked PankoMab and control hIgG-ADC (10 mg / kg, single dose) showed weak antitumor efficacy in these xenograft models (TGI was 10% and 29%, respectively, at day 40). In contrast, PankoMab-ADC (10 mg / kg, single dose) significantly inhibited tumor growth (TGI was 95% at day 40). Moreover, PankoMab-ADC showed statistically significant antitumor efficacy compared to control hIgG-ADC (P<0.001 for both at day 40). In terms of body weight change, no weight loss induced by drug treatment was observed in any of the drug treatment groups. These results suggest that PankoMab-ADC possesses potent antitumor efficacy and demonstrated target-dependent and drug-dependent antitumor efficacy in various TA-MUC1-positive xenograft models.

[0354] 10.4 Comparison of antitumor efficacy of PankoMab-ADC and PM-N54Q-ADC against TA-MUC1-positive tumors in vivo To investigate whether PM-N54Q-ADC has equal or greater antitumor efficacy than PankoMab-ADC against TA-MUC1-positive tumor cells, the antitumor efficacy of PankoMab-ADC and PM-N54Q-ADC against various types of TA-MUC1-positive tumor cells was compared.

[0355] First, the inventors evaluated the in vivo efficacy against HPAC tumor cells and NCI-H441 tumor cells exhibiting moderate to high TA-MUC1 expression. As shown in Figure 11, naked PM-N54Q and control hIgG-ADC (10 mg / kg, single dose) showed weak antitumor efficacy against HPAC-retaining mice (TGI was 27% and 18%, respectively, at day 32). In contrast, PankoMab-ADC and PM-N54Q-ADC (10 mg / kg, single dose) significantly inhibited tumor growth (TGI was 93% for both at day 32). Furthermore, PankoMab-ADC and PM-N54Q-ADC (10 mg / kg, single dose) showed statistically significant antitumor efficacy compared to control hIgG-ADC (P<0.001 for both at day 32). Regarding weight changes, no weight loss induced by drug treatment was observed in any of the drug treatment groups.

[0356] As shown in Figure 12, naked PankoMab and naked PM-N54Q (10 mg / kg, single dose) showed weak antitumor efficacy against NCI-H441-retaining mice (TGI was 8% and 12%, respectively, at day 38). The control hIgG-ADC treatment group (3 mg / kg, single dose) showed antitumor efficacy over two weeks after administration, but tumor regrowth was observed at day 21 (TGI was 71% at day 38). In contrast, PankoMab-ADC and PM-N54Q-ADC (3 mg / kg, single dose) significantly inhibited tumor growth (TGI was 99% for both at day 38). Furthermore, PankoMab-ADC and PM-N54Q-ADC showed statistically significant antitumor efficacy compared to control hIgG-ADC (P<0.001, respectively, at day 38). Regarding weight changes, no weight loss induced by drug treatment was observed in any of the drug treatment groups.

[0357] Next, the inventors evaluated the in vivo efficacy of TA-MUC1 against OVCAR-5 tumor cells with low expression. As shown in Figure 13, naked PankoMab, PM-N54Q, and control hIgG-ADC (10 mg / kg, single dose) showed little antitumor efficacy against OVCAR5-retaining mice (TGI was 1%, 11%, and 3%, respectively, at day 26). In this model, PankoMab The antitumor efficacy of b-ADC (10 mg / kg, single dose) was limited (TGI was 37% at day 26), while PM-N54Q-ADC (10 mg / kg, single dose) showed potent antitumor efficacy (TGI was 73% at day 26). Furthermore, PankoMab-ADC and PM-N54Q-ADC showed statistically significant antitumor efficacy compared to the control hIgG-ADC (P=0.01 and P<0.001, respectively, at day 26). In addition, PM-N54Q-ADC showed statistically significant antitumor efficacy compared to PankoMab-ADC (P<0.001 at day 26). Regarding weight changes, no weight loss caused by drug treatment was observed in any of the drug treatment groups.

[0358] Finally, the inventors evaluated the in vivo efficacy against TA-MUC1-negative HCT-15 tumor cells.

[0359] As shown in Figure 14, naked PankoMab and PM-N54Q (10 mg / kg, single dose) showed little antitumor efficacy in this model (TGI was 7% and 4%, respectively, at day 32). Furthermore, PankoMab-ADC, PM-N54Q-ADC, and control hIgG-ADC also showed little antitumor efficacy in this model (TGI was 15%, 22%, and 26%, respectively, at day 32).

[0360] Based on these results, we concluded that the antitumor efficacy of PankoMab-ADC and PM-N54Q-ADC is target-dependent and drug-dependent. Furthermore, improved antigen-binding affinity can contribute to enhanced antitumor efficacy against TA-MUC1-positive tumor cells.

[0361] Identification of deposited biological materials The cell lines DSM ACC2806, DSM ACC2807, and DSM ACC2856 were deposited by Glycotope GmbH, Robert-Rossle-Str.10, 13125 Berlin (DE) to Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Inhoffenstraße 7B, 38124 Braunschweig (DE) on the dates shown in the table below.

[0362] [Table 3] SEQUENCE LISTING <110> Daiichi Sankyo Company, Limited <120> Anti-MUC1 antibody-drug conjugate <130> PD20A-0066D <150> EP18173253.8 <151> 2018-05-18 <160> twenty three <170> PatentIn version 3.5 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR-H1 <400> 1 Asn Tyr Trp Met Asn 1 5 <210> 2 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> CDR-H2 <220> <221> VARIANT <222> (8)..(8) <223> Xaa is any amino acid except Asn <400> 2 Glu Ile Arg Leu Lys Ser Asn Xaa Tyr Thr Thr His Tyr Ala Glu Ser 1 5 10 15 Val Lys Gly <210> 3 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> CDR-H3 <400> 3 His Tyr Tyr Phe Asp Tyr 1 5 <210> 4 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> CDR-L1 <400> 4 Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Ile Thr Tyr Phe Phe 1 5 10 15 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR-L2 <400> 5 Gln Met Ser Asn Leu Ala Ser 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR-L3 <400> 6 Ala Gln Asn Leu Glu Leu Pro Pro Thr 1 5 <210> 7 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> CDR-H2 <400> 7 Glu Ile Arg Leu Lys Ser Asn Gln Tyr Thr Thr His Tyr Ala Glu Ser 1 5 10 15 Val Lys Gly <210> 8 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> CDR-H2 <400> 8 Glu Ile Arg Leu Lys Ser Asn Asn Tyr Thr Thr His Tyr Ala Glu Ser 1 5 10 15 Val Lys Gly <210> 9 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> heavy chain variable region <220> <221> VARIANT <222> (57)..(57) <223> Xaa is any amino acid except Asn <400> 9 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Met Arg Leu Ser Cys Val Ala Ser Gly Phe Pro Phe Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Glu Ile Arg Leu Lys Ser Asn Xaa Tyr Thr Thr His Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr Arg His Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 10 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> heavy chain variable region <400> 10 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Met Arg Leu Ser Cys Val Ala Ser Gly Phe Pro Phe Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Glu Ile Arg Leu Lys Ser Asn Gln Tyr Thr Thr His Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr Arg His Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 11 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> heavy chain variable region <400> 11 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Met Arg Leu Ser Cys Val Ala Ser Gly Phe Pro Phe Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Glu Ile Arg Leu Lys Ser Asn Asn Tyr Thr Thr His Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr Arg His Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 12 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> light chain variable region <400> 12 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Asn Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30 Asn Gly Ile Thr Tyr Phe Phe Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn 85 90 95 Leu Glu Leu Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg <210> 13 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> epitope <400> 13 Pro Asp Thr Arg 1 <210> 14 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> epitope <400> 14 Pro Glu Ser Arg 1 <210> 15 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> heavy chain <220> <221> VARIANT <222> (57)..(57) <223> Xaa is any amino acid except Asn <400> 15 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Met Arg Leu Ser Cys Val Ala Ser Gly Phe Pro Phe Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Glu Ile Arg Leu Lys Ser Asn Xaa Tyr Thr Thr His Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr Arg His Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Gly Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 16 <211> 219 <212> PRT <213> Artificial Sequence <220> <223> light chain <400> 16 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Asn Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30 Asn Gly Ile Thr Tyr Phe Phe Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn 85 90 95 Leu Glu Leu Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 17 <211> 1401 <212> DNA <213> Artificial Sequence <220> <223> heavy chain <400> 17 atgaagcacc tgtggttctt tctgctgctg gtggccgctc ctagatgggt gctgtctgaa gtgcagctgg tggaatctgg cggaggattg gttcagcctg gcggctccat gagactgtct tgtgtggcct ctggcttccc cttctccaac tactggatga actgggtccg acaggcccct 180 ggcaaaggac tggaatgggt cggagagatc cggctgaagt ccaaccagta caccacacac 300. tacgccgagt ccgtgaaggg cagattcacc atctctcggg acgactccaa gaactccctg 360. tacctgcaga tgaacagcct gaaaaccgag gacaccgccg tgtactactg cacccggcac tactacttcg actactgggg ccagggcacc ctggtcacag tttcttccgc ttccaccag 420 ggacccagcg tgttccctct ggctccttcc cctctggcgg aacagctgct 480 ctgggctgcc tggtcaagga ctactttcct gagcctgtga ccgtgtcctg gaactctggc 540 gctctgacat ctggcgtgca cacctttcca gctgtgctgc agtcctccgg cctgtactct 600 ctgtcctctg tcgtgaccgt gccttccagc tctctgggaa cccagaccta catctgcaat 660 gtgaaccaca agccttccaa caccaaggtg gacaagaagg tggaacccaa gtcctgcgac 720 aagacccaca cctgtcctcc atgtcctgct ccagaactgc tcggcggacc ttccgtgttc 780 ctgtttcctc caaagcctaa ggacaccctg atgatcagca gaacccctga agtgacctgc 840 gtggtggtgg atgtgtctca cgaggacccc gaagtgaagt tcaattggta cgtggacggc 900 gtggaagtgc acaacgccaa gaccaagcct agagaggaac agtacaactc cacctacaga 960 gtggtgtccg tgctgaccgt gctgcaccag gattggctga acggcaaaga gtacaagtgc 1020 aaggtgtcca acaaggccct gcctgctcct atcgaaaaga ccatctccaa ggccaagggc 1080 cagcctaggg aaccccaggt ttacaccttg cctccaagca gggacgagct gaccaagaac 1140 caggtgtccc tgacctgcct cgtgaaggga ttctacccct ccgatatcgc cgtggaatgg 1200 gagtctaatg gccagcctga gaacaactac aagacaaccc ctcctgtgct ggactccgac 1260 ggctcattct tcctgtactc caagctgaca gtggacaagt ccagatggca gcagggcaac 1320 gtgttctcct gctccgtgat gcatgagggc ctgcacaacc actacaccca gaagtccctg 1380 tctctgagcc ccggcaaatg a 1401 <210> 18 <211> 720 <212> DNA <213> Artificial Sequence <220> <223> light chain <400> 18 atggttctgc agacacaggt gttcatctcc ctgctgctgt ggatctctgg cgcctacggc 60 gacatcgtga tgacccagtc tccactgagc aaccccgtga cacctggcga gcctgcctcc 120 atctcttgcc ggtcctctaa gtctctgctg cactccaacg gcatcaccta ctttttctgg tatctgcaga agcccggcca gtctcctcag ctgctgatct accagatgtc caacctggcc tctggcgtgc ccgatagtt ttccggctct ggctctggca ccgacttcac ccgagaatc tccagagtgg aagccgagga cgtggggcgtg tactactgtg cccagaacct ggaactgcct 360 cctacctttg gccagggcac caaggtgga atcaagcgga cagtggccgc tccttccgtg 420 tttatcttcc caccttccga cgagcagctg aagtccggca cagcttctgt cgtgtgcctg 480 540. ctgaacaact tctaccctcg ggagccag gtgcagtgga aggtggacaa tgccctgcag tccggcaact cccaagagtc tgtgaccgag caggactcca aggacagcac ctacagcctg tcctccacac tgaccctgtc caaggccgac tacgagaagc acaaggtgta cgcctgcgaa 660 gtgacccatc agggcctgtc tagccctgtg accaagtctt tcaaccgggg cgagtgctga 720 <210> 19 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> heavy chain <400> 19 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Met Arg Leu Ser Cys Val Ala Ser Gly Phe Pro Phe Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Glu Ile Arg Leu Lys Ser Asn Asn Tyr Thr Thr His Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr Arg His Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Gly Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 20 <211> 460 <212> PRT <213> Artificial Sequence <220> <223> heavy chain <220> <221> VARIANT <222> (76)..(76) <223> Xaa is any amino acid except Asn <400> 20 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Gly Gly Ser Met Arg Leu Ser Cys Val Ala Ser Gly Phe Pro Phe 35 40 45 Ser Asn Tyr Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Gly Glu Ile Arg Leu Lys Ser Asn Xaa Tyr Thr Thr His 65 70 75 80 Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser 85 90 95 Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr 100 105 110 Ala Val Tyr Tyr Cys Thr Arg His Tyr Tyr Phe Asp Tyr Trp Gly Gln 115 120 125 Gly Thr Leu Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val 130 135 140 Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr 145 150 155 160 Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr 165 170 175 Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val 180 185 190 Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser 195 200 205 Ser Thr Trp Pro Ser Gln Thr Val Thr Cys Asn Val Ala His Pro Ala 210 215 220 Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys 225 230 235 240 Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe 245 250 255 Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val 260 265 270 Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe 275 280 285 Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Lys Pro 290 295 300 Arg Glu Glu Gln Ile Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro 305 310 315 320 Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val 325 330 335 Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr 340 345 350 Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys 355 360 365 Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asn 370 375 380 Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro 385 390 395 400 Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser 405 410 415 Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala 420 425 430 Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His 435 440 445 His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 450 455 460 <210> 21 <211> 239 <212> PRT <213> Artificial Sequence <220> <223> light chain <400> 21 Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala Tyr Gly Asp Ile Val Met Thr Gln Ser Pro Leu Ser Asn Pro 20 25 30 Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser 35 40 45 Leu Leu His Ser Asn Gly Ile Thr Tyr Phe Phe Trp Tyr Leu Gln Lys 50 55 60 Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala 65 70 75 80 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 Thr Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr 100 105 110 Cys Ala Gln Asn Leu Glu Leu Pro Pro Thr Phe Gly Gln Gly Thr Lys 115 120 125 Val Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro 130 135 140 Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe 145 150 155 160 Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp 165 170 175 Gly Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp 180 185 190 Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys 195 200 205 Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys 210 215 220 Thr Ser Thr Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 225 230 235 <210> 22 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> heavy chain <400> 22 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Met Arg Leu Ser Cys Val Ala Ser Gly Phe Pro Phe Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Glu Ile Arg Leu Lys Ser Asn Gln Tyr Thr Thr His Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr Arg His Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Gly Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 23 <211> 460 <212> PRT <213> Artificial Sequence <220> <223> heavy chain <400> 23 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Gly Gly Ser Met Arg Leu Ser Cys Val Ala Ser Gly Phe Pro Phe 35 40 45 Ser Asn Tyr Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Gly Glu Ile Arg Leu Lys Ser Asn Gln Tyr Thr Thr His 65 70 75 80 Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser 85 90 95 Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr 100 105 110 Ala Val Tyr Tyr Cys Thr Arg His Tyr Tyr Phe Asp Tyr Trp Gly Gln 115 120 125 Gly Thr Leu Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val 130 135 140 Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr 145 150 155 160 Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr 165 170 175 Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val 180 185 190 Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser 195 200 205 Ser Thr Trp Pro Ser Gln Thr Val Thr Cys Asn Val Ala His Pro Ala 210 215 220 Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys 225 230 235 240 Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe 245 250 255 Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val 260 265 270 Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe 275 280 285 Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Lys Pro 290 295 300 Arg Glu Glu Gln Ile Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro 305 310 315 320 Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val 325 330 335 Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr 340 345 350 Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys 355 360 365 Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asn 370 375 380 Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro 385 390 395 400 Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser 405 410 415 Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala 420 425 430 Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His 435 440 445 His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 450 455 460

Claims

1. A pharmaceutical composition for use in combination with an anticancer agent, comprising an antibody-drug conjugate, Antibody-drug conjugates contain antibodies conjugated with cytotoxic agents. The antibody is capable of binding to MUC1 or TA-MUC1, and The antibody is, (i) A heavy chain variable region including a complementarity-determining region (CDR) CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 8, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and (ii) Light chain variable region including complementarity-determining region (CDR-L1) having the amino acid sequence of SEQ ID NO: 4, CDR-L2 having the amino acid sequence of SEQ ID NO: 5, and CDR-L3 having the amino acid sequence of SEQ ID NO: 6 Includes, The amino acid at position 8 of SEQ ID NO: 2 is selected from the group consisting of glutamine, histidine, tryptophan, tyrosine, lysine, arginine, aspartic acid, and glutamic acid, or CDR-H2 has the amino acid sequence of SEQ ID NO:

7. Pharmaceutical composition.

2. A pharmaceutical composition comprising an antibody-drug conjugate and an anticancer agent, Antibody-drug conjugates contain antibodies conjugated with cytotoxic agents. The antibody is capable of binding to MUC1 or TA-MUC1, and The antibody is, (i) A heavy chain variable region including a complementarity-determining region (CDR) CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 8, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and (ii) Light chain variable region including complementarity-determining region (CDR-L1) having the amino acid sequence of SEQ ID NO: 4, CDR-L2 having the amino acid sequence of SEQ ID NO: 5, and CDR-L3 having the amino acid sequence of SEQ ID NO: 6 Includes, The amino acid at position 8 of SEQ ID NO: 2 is selected from the group consisting of glutamine, histidine, tryptophan, tyrosine, lysine, arginine, aspartic acid, and glutamic acid, or CDR-H2 has the amino acid sequence of SEQ ID NO:

7. Pharmaceutical composition.

3. The pharmaceutical composition according to claim 1 or 2, wherein the antibody competes for binding to TA-MUC1 with an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 10 and a light chain variable region having the amino acid sequence of SEQ ID NO: 12, or an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 11 and a light chain variable region having the amino acid sequence of SEQ ID NO:

12.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the heavy chain variable region of the antibody has the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

9.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the heavy chain variable region of the antibody has the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

10.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the variable region of the antibody light chain has the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

12.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the heavy chain variable region of the antibody has the amino acid sequence of SEQ ID NO: 10, and the light chain variable region of the antibody has the amino acid sequence of SEQ ID NO:

12.

8. A pharmaceutical composition according to any one of claims 1 to 7, wherein the antibody comprises an Fc region.

9. The pharmaceutical composition according to claim 8, wherein the antibody is an IgG1 antibody, an IgG2 antibody, or an IgG4 antibody.

10. (a) The heavy chain of the antibody has the amino acid sequence of SEQ ID NO: 15, and the light chain of the antibody has the amino acid sequence of SEQ ID NO: 16, or (b) The heavy chain of the antibody has the amino acid sequence of amino acids 1-446 of SEQ ID NO: 15, and the light chain of the antibody has the amino acid sequence of SEQ ID NO:

16. A pharmaceutical composition according to any one of claims 1 to 9.

11. The pharmaceutical composition according to any one of claims 1 to 9, wherein the heavy chain of the antibody has the amino acid sequence of SEQ ID NO: 22, and the light chain of the antibody has the amino acid sequence of SEQ ID NO:

16.

12. The pharmaceutical composition according to any one of claims 1 to 10, wherein the heavy chain of the antibody has the amino acid sequence of amino acid numbers 1-446 of SEQ ID NO: 22, and the light chain of the antibody has the amino acid sequence of SEQ ID NO:

16.

13. The pharmaceutical composition according to any one of claims 1 to 3, wherein the heavy chain variable region of the antibody has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

11.

14. The pharmaceutical composition according to any one of claims 1-3, 13, wherein the variable region of the antibody light chain has the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

12.

15. A pharmaceutical composition according to any one of claims 1-3, 13, or 14, wherein the antibody comprises an Fc region.

16. The pharmaceutical composition according to claim 15, wherein the antibody is an IgG1 antibody, an IgG2 antibody, or an IgG4 antibody.

17. (a) The heavy chain of the antibody has the amino acid sequence of SEQ ID NO: 19, and the light chain of the antibody has the amino acid sequence of SEQ ID NO: 16, or (b) The heavy chain of the antibody has the amino acid sequence of amino acids 1-446 of SEQ ID NO: 19, and the light chain of the antibody has the amino acid sequence of SEQ ID NO:

16. A pharmaceutical composition according to any one of claims 1-3, 13-16.

18. The pharmaceutical composition according to any one of claims 1 to 17, wherein the antibody has the activity of being internally transferred to MUC1 or TA-MUC1 expressing cells by binding to MUC-1 or TA-MUC1.

19. The pharmaceutical composition according to any one of claims 1 to 18, wherein the cytotoxic agent is an antitumor agent.

20. The pharmaceutical composition according to any one of claims 1 to 19, wherein the cytotoxic agent is a chemotherapeutic agent.

21. The pharmaceutical composition according to claim 20, wherein the chemotherapeutic agent is selected from the group consisting of microtubule inhibitors, topoisomerase I inhibitors, DNA damaging agents, DNA alkylating agents, and DNA sub-groove binding agents.

22. The pharmaceutical composition according to claim 20 or 21, wherein the chemotherapeutic agent is a topoisomerase I inhibitor.

23. Topoisomerase I inhibitors are expressed by the following formula: 【Chemistry 1】 The pharmaceutical composition according to claim 21 or 22, which is an antitumor compound represented by [the specified formula].

24. The antibodies are expressed by the following formulas (a) to (f): (a)-(Succinimid-3-yl-N)-CH 2 CH 2 -C(=O)-GGFG-NH-CH 2 CH 2 CH 2 -C(=O)-, (b)-(Succinimid-3-yl-N)-CH 2 CH 2 CH 2 CH 2 CH 2 -C(=O)-GGFG-NH-CH 2 CH 2 CH 2 -C(=O)-, (c)-(Succinimid-3-yl-N)-CH 2 CH 2 CH 2 CH 2 CH 2 -C(=O)-GGFG-NH-CH 2 -O-CH 2 -C(=O)-, (d)-(Succinimid-3-yl-N)-CH 2 CH 2 CH 2 CH 2 CH 2 -C(=O)-GGFG-NH-CH 2 CH 2 -O-CH 2 -C(=O)-, (e)-(Succinimid-3-yl-N)-CH 2 CH 2 -C(=O)-NH-CH 2 CH 2 O-CH 2 CH 2 O-CH 2 CH 2 -C(=O)-GGFG-NH-CH 2 CH 2 CH 2 -C(=O)-,および (f)-(Succinimid-3-yl-N)-CH 2 CH 2 -C(=O)-NH-CH 2 CH 2 O-CH 2 CH 2 O-CH 2 CH 2 O-CH 2 CH 2 O-CH 2 CH 2 -C(=O)-GGFG-NH-CH 2 CH 2 CH 2 -C(=O)-, The cytotoxic agent is conjugated via a linker having one of the structures selected from the group consisting of the following: The antibody is attached to the -(Succinimid-3-yl-N) terminal, The cytotoxic agent is attached to the rightmost carbonyl group in formulas (a) to (f), GGFG represents an amino acid sequence consisting of glycine-glycine-phenylalanine-glycine linked via peptide bonds. -(Succinimid-3-yl-N) is represented by the following formula: 【Chemistry 2】 It has a structure represented by [this], which is connected to the antibody at position 3, and is connected to the methylene group of the linker structure containing this structure at the nitrogen atom at position 1. A pharmaceutical composition according to any one of claims 1 to 23.

25. The linker is defined by the following equations (a) to (c): (a)-(Succinimid-3-yl-N)-CH 2 CH 2 CH 2 CH 2 CH 2 -C(=O)-GGFG-NH-CH 2 -O-CH 2 -C(=O)-, (b)-(Succinimid-3-yl-N)-CH 2 CH 2 CH 2 CH 2 CH 2 -C(=O)-GGFG-NH-CH 2 CH 2 -O-CH 2 -C(=O)-, and (c)-(Succinimid-3-yl-N)-CH 2 CH 2 -C(=O)-NH-CH 2 CH 2 O-CH 2 CH 2 O-CH 2 CH 2 -C(=O)-GGFG-NH-CH 2 CH 2 CH 2 -C(=O)- The pharmaceutical composition according to claim 24, which is represented by any formula selected from the group consisting of the following.

26. The linker is given by the following equation (a): (a)-(Succinimid-3-yl-N)-CH 2 CH 2 CH 2 CH 2 CH 2 -C(=O)-GGFG-NH-CH 2 -O-CH 2 -C(=O)- The pharmaceutical composition according to claim 24 or 25, as represented by...

27. The antibody is expressed by the following formula (in the formula, asterisks indicate ＊ (This indicates the connection point with the antibody.) 【Transformation 3】 The pharmaceutical composition according to any one of claims 23-26, wherein a drug linker represented by is conjugated by a thioether bond.

28. The antibody-drug conjugate is expressed by the following formula: 【Chemistry 4】 It is represented by, In the formula, AB represents the antibody, y represents the average number of drug-linker units conjugated to the antibody per antibody. The pharmaceutical composition according to any one of claims 23 to 26, wherein the antibody is conjugated to a drug linker represented by the above formula via a thioether bond.

29. The pharmaceutical composition according to claim 27 or 28, wherein the antibody comprises a heavy chain variable region and a light chain variable region, or any one of the following combinations of a) to c) of a heavy chain and a light chain. (a) The heavy chain variable region has the amino acid sequence of SEQ ID NO: 10, and the light chain variable region has the amino acid sequence of SEQ ID NO:

12. (b) The heavy chain has the amino acid sequence of SEQ ID NO: 22, and the light chain has the amino acid sequence of SEQ ID NO:

16. (c) The heavy chain of the antibody has the amino acid sequence of amino acids 1-446 of SEQ ID NO: 22, and the light chain of the antibody has the amino acid sequence of SEQ ID NO:

16.

30. The pharmaceutical composition according to claim 27 or 28, wherein the antibody comprises a heavy chain variable region and a light chain variable region, or any one of the following combinations of a) to c) of a heavy chain and a light chain. (a) The heavy chain variable region has the amino acid sequence of SEQ ID NO: 11, and the light chain variable region has the amino acid sequence of SEQ ID NO:

12. (b) The heavy chain has the amino acid sequence of SEQ ID NO: 19, and the light chain has the amino acid sequence of SEQ ID NO:

16. (c) The heavy chain of the antibody has the amino acid sequence of amino acids 1-446 of SEQ ID NO: 19, and the light chain of the antibody has the amino acid sequence of SEQ ID NO:

16.

31. The pharmaceutical composition according to any one of claims 1 to 30, wherein the anticancer agent is a chemotherapy agent or a therapeutic antibody.

32. The pharmaceutical composition according to claim 31, wherein the chemotherapeutic agent is selected from taxanes; cyclophosphamide; imatinib; pazopanib; capecitabine; cytarabine; vinorelbine; gemcitabine; anthracyclines; aromatase inhibitors; topoisomerase inhibitors; platinum-based chemotherapeutic agents; PARP inhibitors; TLR agonists; and antimetabolites.

33. The taxane is paclitaxel (Taxol), docetaxel (Taxotere), or SBT-1214. Anthracyclines include daunorubicin, doxorubicin, epirubicin, idarubicin, barurubicin, or mitoxantrone. Aromatase inhibitors include aminoglutethimide, testactone (Teslac), anastrozole (Arimidex), letrozole (Femara), exemestane (Aromasin), borozole (Rivizor), formestan (Lentaron), phadrozole (Afema), 4-hydroxyandrostenedione, 1,4,6-androstatriene-3,17-dione (ATD), or 4-androsten-3,6,17-trione (6-oxo). The topoisomerase inhibitors are irinotecan, topotecan, camptothecin, lamelalin D, etoposide (VP-16), teniposide, doxorubicin, daunorubicin, mitoxantrone, amsacrin, ellipticin, oulintricarboxylic acid, or HU-331. Platinum-based chemotherapeutic agents include cis-diamminedichloroplatin(II) (cisplatin), cis-diammine(1,1-cyclobutanedicarboxylate)platin(II) (carboplatin), or [(1R,2R)-cyclohexane-1,2-diamine](ethandeoato-O,O')platin(II) (oxaliplatin). PARP inhibitors include olaparib, lucaparib, or niraparib. The TLR agonist is imiquimod or resikimod. An antimetabolite is an antifolate agent, pyrimidine analog, purine analog, selective estrogen receptor modifier, or estrogen receptor downregulator. The pharmaceutical composition according to claim 32.

34. The antifolic acid drugs are methotrexate, pemetrexed, larcitrexed, or pralatrexate. The pyrimidine analogs are fluorouracil, gemcitabine, phloxuridine, 5-fluorouracil, or tegafur-uracil. The pharmaceutical composition according to claim 33.

35. The pharmaceutical composition according to claim 31, wherein the therapeutic antibody is selected from anti-EGFR antibody; anti-HER2 antibody; anti-VEGF antibody; anti-CD52 antibody; anti-CD30 antibody; anti-CD33 antibody; and anti-CD20 antibody.

36. The anti-EGFR antibody is cetuximab, tomzotuximab, panitumumab, zaltumumab, nimotuzumab, matsuzumab, or necitumumab. The anti-HER2 antibody is trastuzumab, timigutuzumab, or pertuzumab. The anti-VEGF antibody is bevacizumab (Avastin). The anti-CD52 antibody is alemtuzumab (Campath). The anti-CD30 antibody is brentuximab (Adcetris). The anti-CD33 antibody is gemtuzumab (Mylotarg), The anti-CD20 antibody is rituximab (Rituxan, Mabthera), tositumomab (Bexxar), or ibritumomab (Zevalin). The pharmaceutical composition according to claim 35.

37. A pharmaceutical composition according to any one of claims 1 to 36, for the treatment of cancer.

38. The pharmaceutical composition according to claim 37, wherein the cancer is selected from the group consisting of ovarian cancer, breast cancer, pancreatic cancer, lung cancer, colon cancer, stomach cancer, liver cancer, kidney cancer, hematological cancer, endometrial cancer, thyroid cancer, leukemia, hemihydrocystic carcinoma, melanoma, carcinoma, teratoma, lymphoma, sarcoma, mesothelioma, neuroblastoma, glioma, rectal cancer, adrenal cancer, skin cancer, brain cancer, cervical cancer, intestinal cancer, intestinal cancer, head and neck cancer, gastrointestinal cancer, lymph node cancer, esophageal cancer, colorectal cancer, otolaryngeal (ENT) cancer, prostate cancer, bladder cancer, uterine cancer, and metastases thereof.

39. The pharmaceutical composition according to claim 38, wherein the cancer is selected from the group consisting of ovarian cancer, breast cancer, lung cancer, and bladder cancer.

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