Anti-MUC1 antibody-drug conjugates

Abstract

To provide methods for producing antibody-drug conjugates against the cancer antigen MUC1.SOLUTION: This method includes the steps of: reacting an antibody capable of binding to MUC1 with a compound represented by (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX); and conjugating a drug-linker moiety to the antibody by the technique of forming a thioether bond with the disulfide bonds located in the hinge region of the antibody. The-(NH-DX) is a group represented by the formula shown in the image below, and -GGFG- denotes a tetrapeptide residue of -Gly-Gly-Phe-Gly-.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the field of antibody drug conjugates (ADCs). Mutant anti-MUC1 antibodies with increased antigen binding affinity. ADCs containing different anti-MUC1 antibodies are provided. In particular, the humanized antibody PankoMab In this mutant form, asparagine 57 in the heavy chain variable region is replaced by another amino acid. This results in the deletion of glycosylation sites in the CDR2 region, increasing antigen-binding affinity. The ADC demonstrated significant anti-tumor efficacy. In a specific embodiment, the present invention provides Therapeutic and diagnostic uses of drug conjugates and of such antibody drug conjugates The present invention relates to a method for producing a steroid. [Background technology]

[0002] Antibodies against tumor-associated antigens are widely used anti-cancer therapeutic agents. Anti-cancer antibodies have been approved for human therapy. Some of these antibodies inhibit the survival of certain cancer cells. They act by blocking certain signaling pathways that are important for growth or proliferation. Antibodies can be used to induce antibody-dependent cellular cytotoxicity (ADCC), for example, via natural killer cells. ), which activates the patient's immune response against the targeted cancer cells. is induced by the binding of the Fc portion of an antibody to an Fc receptor on an immune cell.

[0003] An interesting and important group of antibodies are those directed against mucin proteins. Mucins are the proteins that bind to vertebrate muscles. A high molecular weight, highly glycosylated protein produced by many epithelial tissues of the body. Mucins are a family of proteins that contain hydrophobic transmembrane domains that favor retention in the plasma membrane. Mucin proteins are membrane-bound due to the presence of phospholipids, while mucin proteins are secreted or lysed at the mucosal surface. Human mucin proteins can be subdivided into mucins, which are secreted and become components of saliva. The family consists of many family members, including membrane-bound MUC1.

[0004] Increased mucin production occurs in many adenocarcinomas, including those of the pancreas, lung, breast, ovary, and colon. Mucins are essential for the development of lung diseases such as asthma, bronchitis, chronic obstructive pulmonary disease, or cystic fibrosis. Two membrane mucins, MUC1 and MUC4, are also overexpressed. Mucins have also been widely studied for their potential as diagnostic markers. Several antibodies against mucin proteins (Clin. Cancer R es., 2011 Nov. 1; Vol. 17(Issue 21): pp. 6822-30, PLoS One, January 14, 2011; Vol. 6 (1): e15921), and MUC1 in particular is However, their therapeutic efficacy still leaves room for improvement. obtain.

[0005] In view of the above, the art provides therapeutic anti-MUC1 antibodies with improved properties. The need exists. Summary of the Invention

[0006] ADCs consist of three distinct components that are responsible for specifically delivering the payload to target cells: To date, four ADCs (Gemtu) have been developed. Inotuzumab ozogamicin (Mylotarg®), Inotuzumab ozogamicin ( Besponsa®), brentuximab vedotin (Adcetris®), Trademark), trastuzumab emtansine (T-DM1; Kadcyla®) In addition, it targets a wide range of hematological cancers and solid tumors. There are more than 60 ADCs in development that represent a new class of novel cancer chemotherapy drugs. The group created a platform that combines the specificity of monoclonal antibodies with the cytotoxicity of small molecule drugs. Therefore, ADCs are expected to be a major part of the future of precision medicine and combination therapies. Therefore, there are many possibilities for providing additional ADCs and for the treatment and / or management of diseases. There is a continuing need for tools, methods, and uses related to the detection and treatment of cancer.

[0007] As an ADC, exatexanthan is attached to an antibody (e.g., an anti-HER2 antibody) via a linker. Conjugated ADCs are known (WO2014 / 057687, WO20 However, exatexanthan is conjugated to anti-MUC1 antibodies. The ADCs being tested are not publicly known.

[0008] The present inventors have demonstrated that the glycosylation site of the heavy chain variable region of the anti-MUC1 antibody PankoMab is deleted. We found that the loss of α-glucan did not abolish antigen binding, but rather unexpectedly increased the antigen affinity of the antibody. This is because the glycosylation site is located in the second complementarity-determining region (CDR-H2) of the heavy chain variable region. This was particularly surprising because the CDRs are located in the epitopes directly involved in antigen binding. The CDRs are the regions of the antibody that provide contact with the target site. Therefore, modifications of amino acids in the CDRs are generally The addition of these modifications is expected to be detrimental to antigen binding affinity. Antibodies contain glycosylation sites in CDR-H2 that carry large carbohydrate structures. The compound structure is directly at the binding interface to the antigen, and therefore is involved in antigen binding. However, as demonstrated in the examples, PankoM in which glycosylation sites have been deleted by substituting retained amino acids The ab mutant (PM-N54Q) exhibits increased antigen binding affinity. , conjugates containing PankoMab or PankoMab mutant (PM-N54Q) Antibody-drug conjugates (ADCs) showed significant anti-cancer activity against MUC1-positive tumors. PM-N54Q-ADC demonstrated tumor efficacy compared to PankoMab-ADC. We found that the compound showed significant antitumor efficacy.

[0009] Thus, in a first aspect, the present invention provides a method for the treatment of cancer comprising administering to a patient an antibody conjugated to a cytotoxic agent. a conjugate comprising an antibody capable of binding to MUC1; (i) 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 a heavy chain variable region comprising a CDR-H3 comprising: (ii) a 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. Light chain variable region containing CDR-L3 The present invention relates to a conjugate comprising:

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

[0011] According to a third aspect, the present invention provides a method for the treatment, prevention or diagnosis of cancer in medicine, The present invention provides a composition or conjugate according to the invention for use in

[0012] In a fourth aspect, the present invention is a method for treating cancer in a subject in need thereof. administering to a subject having cancer a therapeutically effective amount of a conjugate according to the present invention. The present invention provides a method comprising:

[0013] In a fifth aspect, the present invention provides a kit or device comprising a conjugate according to the invention. and related compounds useful for diagnosing, detecting, or monitoring MUC1-associated disorders, such as cancer. A method is provided.

[0014] Other objects, features, advantages, and aspects of the present invention will become apparent to those skilled in the art from the following description and accompanying drawings. However, the following description and the accompanying claims will be more fully understood. The ranges and specific examples are intended to represent preferred embodiments of the present application, but are provided for illustrative purposes. It should be understood that the present disclosure is provided for illustrative purposes only. Various changes and modifications within the scope of the present invention will become readily apparent to those skilled in the art upon reading the following. Deaf.

[0015] definition As used herein, the following expressions generally apply regardless of the context in which they are used: Except where indicated, the terms "a," "b," "c," "d," "e," "f," "g," "h," "i," "j," "ma," "ma," "ma" and "ma" are preferably intended to have the meanings indicated below. are.

[0016] The term "comprises" as used herein means In addition to the meaning of "consist essentially of" "consist of" and "consist of" are also included and specifically refer to them. Thus, the expression "comprises" means that the subject matter is included in the list of items specifically recited. "Comprises" an element refers to an embodiment that does not include additional elements, and the subject matter "comprises" the elements specifically recited, Similarly, "having" refers to an embodiment that may and / or does include an element. The expression "have" should be understood as "comprise" "consist essentially of" and "or This also includes the expression "consist of" and specifically refers to them. The term "consist essentially of" is a possible In particular, the subject matter may contain elements in addition to those specifically listed from which the subject matter essentially consists. but 20% or less, especially 15% or less, even 10% or less, or in particular 5% or less of a further element Refers to an embodiment.

[0017] The term "antibody" particularly refers to at least two amino acids connected by disulfide bonds. Each heavy chain contains a heavy chain variable region (V H ) and and heavy chain constant region (C H Each light chain is composed of a light chain variable region (V L ) and light chain constant Area (C L The heavy chain constant region consists of three, or IgM or IgE type In the case of antibodies, there are four heavy chain constant domains (C H1 , C H2 , C H3 , and C H4 )of and a first constant domain C H1 The variable region is flanked by a hinge region, which defines the second variable region. Common domain C H2 The light chain constant region may consist of only one constant domain. The variable regions are composed of more conserved regions called framework regions (FR). The hypervariable regions are called complementarity-determining regions (CDRs), which are distributed over a wide range of regions. Each variable region can be further subdivided, and contains three CDRs and four FRs. The heavy and light chain variable regions contain a binding domain that interacts with an antigen. The heavy chain may be of any type, such as gamma, delta, alpha, mu, or epsilon heavy chain. The heavy chain of the antibody is a γ chain. Furthermore, the light chain constant region may be a κ or λ light chain. The light chain of the antibody may be of any type. Preferably, the light chain of the antibody is a κ chain. The terms "κ (or ε) heavy chain" and "κ (λ) light chain" refer to naturally occurring Heavy or light chain constant region amino acid sequences, particularly human heavy or light chain constant region amino acid sequences It refers to an antibody heavy chain or an antibody light chain having a constant region amino acid sequence derived from the antibody. The amino acid sequence of the constant domain of the human gamma (particularly human gamma 1) heavy chain is the constant domain of the human gamma (particularly human gamma 1) antibody heavy chain. It is at least 95%, particularly at least 98%, identical to the amino acid sequence of the common domain. In particular, the amino acid sequence of the constant domain of the κ-type light chain is the constant domain of the human κ antibody light chain. The amino acid sequence of the antibody is at least 95%, particularly at least 98% identical to the amino acid sequence of the antibody. , various cells of the immune system (e.g., effector cells) and the first component of the classical complement system ( can mediate the binding of immunoglobulins to host tissues or factors, including C1q 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 comprises a full-length or fragment thereof that retains the ability to specifically bind to an antigen. The antigen-binding function of an antibody is performed by a fragment of a full-length antibody. Examples of antibody binding fragments include the following: :V L , V H , C L , and C H1 Fab fragments are monovalent fragments consisting of domains; each Contains two Fab fragments that bind to the same antigen and are linked by a disulfide bridge in the hinge region F(ab)2 fragment, a bivalent fragment; V H and C H1 Fd fragment consisting of domains fragment; single arm V of antibody L and V H Fv fragments consisting of domains; and V H dAb fragments consisting of domains.

[0019] The "Fab portion" of an antibody is specifically composed of heavy and light chain variable regions (V H and V L ) and The first domain of the heavy and light chain constant regions (C H1 and C L ) refers to the portion of an antibody that contains If an antibody does not contain all of these regions, the term "Fab portion" may be used to refer to the antibody. Area V of the body H , V L , C H1 , and C L It simply refers to the The "Fab portion" is the antigen-binding portion of an antibody obtained by digesting a natural antibody with papain. In particular, the Fab portion of an antibody is the portion of the antibody that contains the antigen-binding activity. Preferably, the Fab portion comprises at least the V of the antibody. H Includes the area.

[0020] The "Fc portion" of an antibody comprises, in particular, heavy chain constant regions 2, 3, and, where applicable, 4 (C H2 , C H3 , and C H4 In particular, the Fc portion refers to the portion of an antibody that contains these regions. It contains two of each. If an antibody does not contain all of these regions, it is called the "Fab region." The term "moiety" refers to the region C present in an antibody. H2 , C H3 , and C H4 It refers to the Preferably, the Fc portion comprises at least the C portion of the antibody. H2 Preferably, " The "Fc portion" is a portion of the antibody that has antigen-binding activity and is obtained by digesting a natural antibody with papain. In particular, the Fc portion of an antibody refers to the portion of an antibody that binds to an Fc receptor. and thus, for example, an Fc receptor binding site or an Fc receptor binding site. Includes combined capabilities.

[0021] The terms "antibody" and "antibody construct" as used herein refer to certain In embodiments, each refers to a population of antibodies or antibody constructs of the same type. All antibodies or antibody constructs are subject to the same characteristics used to define the antibody or antibody construct. In certain embodiments, all of the antibodies or antibody constructs in the population have the same amino acid sequence. Certain types of antibodies, such as antibodies capable of specifically binding to MUC1, have a sequence similar to that of the nucleotide sequence ... Reference to specifically refers to a population of antibodies of that type.

[0022] The term "antibody" as used herein also includes fragments and derivatives of said antibodies. In particular, a "fragment or derivative" of an antibody is derived from said antibody and is directed against the same antigen, in particular the same enzyme. It is a protein or glycoprotein that can bind to a target antigen as an antibody. In general, a fragment or derivative of an antibody is referred to herein as a functional fragment or derivative. In a highly preferred embodiment, the antibody fragment or derivative comprises a heavy chain variable region. It has been shown that the binding function can be performed by fragments of a full-length antibody or derivatives thereof. Examples of antibody fragments include: (i) the variable region and each heavy chain and (ii) a Fab fragment, which is a monovalent fragment consisting of the first constant domain of the light chain and the second constant domain of the light chain; F(a), a bivalent fragment containing two Fab fragments linked by a disulfide bridge. b) 2 fragments; (iii) Fd fragment consisting of the variable region and the first constant domain CH1 of the heavy chain (iv) an Fv fragment consisting of the heavy and light chain variable regions of a single arm of an antibody; (v) a single (vi) scFv fragments, which are Fv fragments consisting of polypeptide chains covalently linked together; (vii) a heavy chain variable domain; and (viii) association of heavy and light chain variable regions can occur only intermolecularly, but not intramolecularly; A polymer consisting of a heavy chain variable region and a light chain variable region covalently linked together so that Multibody. An antibody derivative is a molecule that specifically binds to the same antigen as the parent antibody. an antibody that competes with or has an amino acid sequence that differs from the parent antibody from which it is derived Such antibody fragments and antibody derivatives can be prepared using conventional techniques known to those skilled in the art. This is obtained.

[0023] The target amino acid sequence is determined by determining whether the target amino acid sequence is identical to the reference amino acid sequence over its entire length. at least 75%, more preferably at least 80%, at least 8 5%, at least 90%, at least 93%, at least 95%, at least 97%, If the reference sequence shares at least 98% or at least 99% homology or identity with the reference sequence, A "corresponding portion" is "derived from" or "corresponding to" an amino acid sequence of a target antibody, for example. The framework region 1 of the heavy chain variable region (FRH1) of the reference antibody is In certain embodiments, the reference amino acid sequence A target amino acid sequence that is "derived from" or "corresponding to" a reference amino acid sequence is one that is "derived from" or "corresponding to" a reference amino acid sequence ... It is 100% homologous, or in particular 100% identical, to the corresponding portion of the amino acid sequence. "Homology" or "identity" of amino acid or nucleotide sequences is preferably determined by the over the entire length of the reference sequence or corresponding to the sequence to which homology or identity is defined The CD is determined by the present invention over the entire length of the corresponding portion of the reference sequence. R sequence or specific variable region sequences. Antibodies derived from a parent antibody have, in particular, at least 75% identical amino acid sequence to the corresponding amino acid sequence of the parent antibody. 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, especially identical, amino acids such as CDR sequences or variable region sequences In certain embodiments, an antibody derived from a parent antibody (i.e., A derivative of a variable region (a CDR sequence of a parent antibody) contains the same CDR sequences as the parent antibody but differs in the sequence of the remainder of the variable region.

[0024] The term "antibody" as used herein also includes multivalent and multispecific antibodies, That is, antibody constructs with more than two binding sites that each bind to the same epitope. and one or more binding sites that bind to the first epitope and one or more binding sites that bind to the second epitope. one or more binding sites that bind to the epitope, and optionally bind to a further epitope The term "antibody construct" refers to an antibody construct that has even further binding sites.

[0025] "Specific binding" preferably refers to the binding of an agent, such as an antibody, to a target that binds specifically to the target compared to its binding to another target. This means that the binding is more potent to a target, such as an epitope, that is specific to the target. Examples of criteria for determining whether a molecule is heterologous include the dissociation constant (referred to herein as "K D "It's called The agent may have a dissociation constant (K) lower than the dissociation constant of the second target. d ) binds to a first target more strongly than a second target. Preferably, the dissociation constant of the target to which the agent specifically binds is 100-fold, 200-fold, 500-fold, or 1000-fold lower than the dissociation constant of the target Furthermore, the term "specific binding" specifically refers to the binding affinity between binding partners, affinity constant K a is at least 10 6 M -1 , preferably at least 10 7 M -1 ,twist Preferably at least 10 8 M -1 An antibody specific to a particular antigen is to at least 106 M -1 , preferably at least 10 7 M -1 , and more preferably less At most 10 8 M -1 K a an antibody capable of binding to said antigen with an affinity having For example, the term "anti-MUC1 antibody" refers to an antibody that specifically binds to MUC1 and Preferably, at least 10 6 M -1 , preferably at least 10 7 M -1 , more preferred At least 10 8 M -1 K a Antibodies capable of binding to MUC1 with affinity Point to the body.

[0026] The term "MUC1" refers to mucin-1, polymorphic epithelial mucin (PEM), or cancer anti- The protein MUC1, also known as genotype 15-3, specifically human MUC1 (accession number 15-3) MUC1 is a member of the mucin family and is membrane-bound. MUC1 encodes a glycosylated phosphoprotein. It has a protein mass that increases to 250-500 kDa upon glycosylation. MUC1 extends 200-500 nm beyond the cell surface. This protein It is anchored to the apical surface of many epithelial cells by a transmembrane domain. 20 amino acid variable number tandem repeat (VNTR) Includes a tunable number tandem repeat) domain and the number of repeats The number of repeats varies from 20 to 120 in different individuals. It is rich in serine, threonine, and proline residues that allow glycosylation. In certain embodiments, the term "MUC1" refers to tumor-associated MUC1 ("TA-MUC1"). 1). TA-MUC1 is MUC1 present on cancer cells. This MUC1 is a non-GMO protein with significantly higher expression levels, localization, and glycosylation. It is different from MUC1 present on cancer cells. In particular, TA-MUC1 is expressed on the surface of cancer cells. In non-cancer cells, MUC1 is expressed in a strictly apical manner, whereas in non-cancer cells, MUC1 is expressed in a non-polarized manner across the entire cell surface. It is not accessible to systemically administered antibodies. identified a novel peptide epitope in the MUC1 protein scaffold and Thomsen-free Abnormal O2 exposes novel carbohydrate tumor antigens, such as Denreich antigen alpha (TFα) -Glycosylated.

[0027] "TFα" is also known as Thomsen-Friedenreich antigen alpha or core-1. alpha- to the hydroxyamino acids serine or threonine in proteins in cancer cells It refers to the O-glycosidically linked disaccharide Gal-β1,3-GalNAc in the anomeric configuration. vinegar.

[0028] The term "sialic acid" specifically refers to any N- or O-substituted derivative of neuraminic acid. The term refers to 5-N-acetylneuraminic acid and 5-N-glycolylneuraminic acid. Although it can refer to both 5-N-acetylneuraminic acid and 5-N-acetylneuraminic acid, it preferably refers only to 5-N-acetylneuraminic acid. Sialic acid, in particular 5-N-acetylneuraminic acid, is preferably 2,3- or 2,6- -linkage. Preferably, the antibodies described herein have a 2 Both 2,3- and 2,6-coupled sialic acids exist.

[0029] The "relative amount of glycans" according to the present invention refers to the amount of glycans present in or on the antibody of an antibody preparation, respectively. A specific percentage or percentage range of glycans attached to antibodies in a composition containing In particular, the relative amounts of glycans contained in the antibody and therefore in the antibody preparation. The specific patterns of all glycans attached to the peptide chains of antibodies in a composition containing antibodies are 100% of the glycans are present in the antibody Refers to all glycans attached to an antibody in a preparation or in a composition comprising the antibody. For example, a relative abundance of 10% of glycans bearing a bisecting GlcNAc indicates that the antibody and thus all of the antibodies attached to the polypeptide chains in the composition. 10% of the glycans in antibodies contain a bisecting GlcNAc residue, therefore 90% of all glycans attached to antibody polypeptide chains in the composition are This refers to a composition containing an antibody that does not contain any GlcNAc residues. is all glycan structures attached to antibodies in the composition, or or all N-glycans, i.e., those attached to asparagine residues of the antibodies in the composition. The glycan may be any glycan structure that is compatible with the glycan structure of the present invention, or any complex glycan. The reference group for the can structure is generally either explicitly stated or readily apparent to those skilled in the art from the context. It can be reasonably estimated.

[0030] The term "N-glycosylation" refers to the cleavage of an asparagine residue in the polypeptide chain of a protein. These asparagine residues are generally amino The amino acid sequence Asn-Xaa-Ser / Thr (where Xaa is any amino acid except proline) Similarly, "N-glycosylation site" is a site containing an N-glycosylation site having a hydroxyl group (which may be a hydroxyl group). A "glycan" is a glycan attached to an asparagine residue of a polypeptide chain. "glycan," "glycan structure," "carbohydrate," "carbohydrate chain," and "carbohydrate structure." The terms N-glycans and N-glycans are generally used interchangeably herein. Manα1,6-(Manα1,3-)Manβ1,4-GlcNAcβ1,4-Glc NAcβ1-Asn, where Asn is an asparagine residue in the polypeptide chain. A covalent bond consisting of an N-acetylglucosamine (GlcNAc) residue and three mannose residues N-glycans have a common core structure. N-glycans are of three different types: complex glycans, They are further subdivided into hybrid glycans and high-mannose glycans.

[0031] The values ​​given herein, particularly the relative amounts of specific glycosylation features, are preferably should be understood as an approximation. In particular, the figures are preferably at most 10% higher. and / or lower, especially up to 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% , or may be higher and / or lower than 1%.

[0032] The term "antibody drug conjugate" (ADC) or "conjugate" is used herein to refer to When used, it generally involves the combination of an antibody or antigen-binding fragment thereof with a chemotherapeutic agent, toxin, immunotherapeutic agent, or This refers to the linking of a fluorophore to a fluorophore, a fluorophore agent, and another agent, such as an imaging probe. The bond may be a covalent bond or a non-covalent interaction such as electrostatic forces. Various linkers known in the art and described herein can be used to link antibody drugs. In addition, they can be used to form antibody-drug conjugates. The immunoconjugate may be a fusion protein that can be expressed from a polynucleotide encoding the immunoconjugate. As used herein, a "fusion protein" can be provided in the form of a fusion protein. "Proteins" originally coded for separate proteins (including peptides and polypeptides). created by joining two or more genes or gene fragments that were previously The translation of the fusion gene results in the functional properties of each of the original proteins. This results in a single protein with the same properties.

[0033] In a "conjugate," two or more compounds are linked together In certain embodiments, at least some of the properties of each compound are imparted to the conjugate. The linkage can be achieved by covalent or non-covalent bonding. Preferably, the compounds of the conjugate are linked by a covalent bond. Different compounds are directly bonded to each other through one or more covalent bonds between atoms of the compounds. Alternatively, the compounds may be connected to each other via chemical moieties such as linker molecules. The linker may be covalently attached to an atom of the compound. If the duct consists of more than two compounds, such compounds may be, e.g. One is attached to the next compound, or several compounds each attached to one central compound. They may be attached or linked in a chain conformation.

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

[0035] The term "expression cassette" refers specifically to a nucleic acid sequence that allows expression of a coding nucleic acid sequence introduced therein. An expression cassette refers to a nucleic acid construct that can activate and regulate a gene. inflammasome binding sites, enhancers, and other molecules that regulate gene transcription or mRNA translation The exact structure of the expression cassette may vary depending on the species or cell type. Although this may vary as a function of the sequence, it generally contains a TATA box, capping sequence, and 5'-nontranscribed sequences, such as the CAAT sequence and the 5'-nontranscribed sequences, which are involved in the initiation of transcription and translation, respectively. More specifically, the 5'-non-transcribed expression control sequence comprises: a promoter region comprising a promoter sequence for transcriptional control of the operably linked nucleic acid; The expression cassette may also contain an enhancer sequence or an upstream activator sequence. Good too.

[0036] According to the present invention, the term "promoter" refers to a promoter located upstream of a nucleic acid sequence to be expressed. (5') of the sequence by providing a recognition and binding site for RNA polymerase. A "promoter" refers to a nucleic acid sequence that controls the expression of a gene. The promoter may contain additional recognition and binding sites for factors such as: The promoter can control the transcription of a gene in a mammalian or eukaryotic organism. may be "inducible," i.e., capable of initiating transcription in response to an inducing agent; or Transcription may be "constitutive" if it is not controlled by an inducing agent. If the promoter is not activated, the gene under its control will not be expressed or will be expressed poorly. In the presence of an inducer, the gene is either switched on or Increased transcription levels are generally mediated by the binding of specific transcription factors. can be.

[0037] The term "vector" is used herein in its most general sense, e.g., Introduction of nucleic acids into prokaryotic and / or eukaryotic cells and, where appropriate, integration into the genome This type of vector includes any intermediate vehicle for said nucleic acid that allows for the expression of said nucleic acid. Preferably, the vector is replicated and / or expressed in a cell. The term "plasmid" includes a plasmid, bacteriophage, or viral genome. As used herein, generally refers to a gene that can replicate independently of chromosomal DNA. Pertaining to the construction of extrachromosomal genetic material, usually a circular DNA duplex.

[0038] According to the present invention, the term "host cell" refers to a cell that has been transformed or transfected with exogenous nucleic acid. The term "host cell" refers to any cell into which a vector can be injected. and prokaryotic cells (e.g., E. coli) or eukaryotic cells (e.g., mammalian cells, particularly human cells). , yeast cells, and insect cells). Human, mouse, hamster, pig, goat, or Mammalian cells, such as cells derived from primates, are particularly preferred. Cells are available in many tissue types. The nucleic acid may be derived from a single copy or from two or more cells, including primary cells and cell lines. The gene may be present in the host cell in a higher copy form, and in one embodiment, It is expressed in the cells.

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

[0040] The term "cancer" according to the present invention includes, in particular, leukemia, hemi-malignant tumors, melanoma, carcinoma, teratoma, Lymphoma, sarcoma, mesothelioma, neuroblastoma, glioma, rectal cancer, endometrial cancer, kidney cancer , adrenal cancer, thyroid cancer, blood cancer, skin cancer, brain cancer, cervical cancer, intestinal cancer (int estinal cancer), liver cancer, colon cancer, stomach cancer, intestinal cancer ine cancer), head and neck cancer, gastrointestinal cancer, lymph node cancer, esophageal cancer, colorectal cancer Cancer, pancreatic cancer, ear, nose and throat (ENT) cancer, breast cancer, prostate cancer, bladder cancer, uterine cancer, egg Cancers according to the present invention include uterine cancer, pulmonary cancer, and metastases thereof. The term includes cancer metastasis.

[0041] The term "tumor" refers to a group of cells or tissues formed by misregulated cell growth. Tumors show a partial or complete lack of structural organization and functional coordination with normal tissue. They can be either benign or malignant, usually forming a discrete mass of tissue.

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

[0043] The term "metastasis" means the spread of cancer cells from their original site to another part of the body. The formation of metastases is a highly complex process, usually involving the detachment of cancer cells from the primary tumor. and to enter the systemic circulation and to colonize and multiply in normal tissues elsewhere in the body. When tumor cells metastasize, the new tumor is called a secondary or metastatic tumor. The cells are usually similar to those in the original tumor. This is the case, for example, when breast cancer metastasizes to the lungs. This means that the secondary tumor is made up of abnormal breast cells rather than abnormal lung cells. In this case, the lung tumor 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. It refers to a composition containing pharmaceutically acceptable ingredients. Preferably, the pharmaceutical composition does not contain a carrier, diluent, or or together with pharmaceutical excipients such as buffers, preservatives, and tonicity adjusting agents, the active compound or Numerical ranges set forth herein include the numbers defining the range. The headings provided herein are incorporated by reference in their entirety. It is not intended to be limiting of the various aspects or embodiments of the present invention that may be realized. According to the embodiment, a method may be characterized as including certain steps or a composition may be characterized as including certain steps. The subject matter described herein as including certain components is The present invention refers to a subject matter consisting of a group or component. It is preferred to combine the above embodiments, and specific examples derived from each combination of preferred embodiments are given below. The subject matter also belongs to this disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0045] The present invention provides a humanized anti-MUC1 antibody P1 in which the glycosylation site of CDR-H2 is deleted. Based on the development of a mutant of ankoMab (PM-N54Q), the deletion of the glycosylation site , amino acid Asn (asparagine) 57 of the heavy chain variable region (i.e., amino acid Asn of SEQ ID NO: 11) This is achieved by replacing the amino acid (amino acid number 57) with another amino acid, specifically Gln (glutamine). Asn57 is the acceptor of the glycosylation site where the carbohydrate structure is attached. The carbohydrate structure is transferred to the asparagine residue by a host cell enzyme. Therefore, by substituting this asparagine residue with another residue, The deletion of the glycosylation site in CDR-H2 of PankoMab abolishes glycosylation. Surprisingly, it was found to increase the antigen binding affinity of the antibody.

[0046] In light of these findings, the present invention provides a composition comprising an antibody conjugated to a cytotoxic agent. a conjugate, wherein the antibody is capable of binding to MUC1; (i) 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 a heavy chain variable region comprising a CDR-H3 comprising: (ii) a 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. Light chain variable region containing CDR-L3 The present invention provides a conjugate comprising:

[0047] Binding to MUC1 The antibody specifically binds to an epitope of MUC1. The epitope is located on the extracellular region of MUC1. In certain embodiments, the antibody is In particular, the antibody binds to MUC1. Cetylgalactosamine (Tn), sialyl α2-6N-acetylgalactosamine (sTn ), galactose β1-3N-acetylgalactosamine (TF), or galactose β 1-3(sialyl α2-6)N-acetylgalactosamine (sTF), preferably Tn or TF, binds more strongly if it is glycosylated. The moiety is attached to a threonine residue by an α-O-glycosidic bond. The epitope in the tandem repeat domain is particularly the amino acid sequence PDTR (SEQ ID NO:1). 3) or PESR (SEQ ID NO: 14). Binding to this epitope is preferably is glycosylation dependent as described above, in particular the carbohydrate moiety as described above , to the threonine residue of the sequence PDTR or PESR (SEQ ID NOs: 13 and 14), respectively If attached, binding increases.

[0048] The epitope is the tumor-associated MUC1 epitope (TA-MUC1). 1 epitope is specifically present on tumor cells but not on normal cells and / or It is only accessible to antibodies in the host circulation when present on tumor cells. refers to epitopes of MUC1 that are not accessible when present on normal cells. In certain embodiments, binding of the antibody to a cell expressing the TA-MUC1 epitope is determined by: The binding is stronger than that to cells expressing normal, non-tumor MUC1. The binding is at least 1.5 times stronger, preferably at least 2 times stronger, and at least at least 5 times more potent, at least 10 times more potent, or at least 100 times more potent For TA-MUC1 binding, the antibody preferably binds to a glycosylated MUC1 tumor epitope. The binding strength is higher than that of non-glycosylated peptides of the same length and sequence. at least 2-fold, preferably 4-fold, or 10-fold, greater binding compared to the silyl peptide 10-fold, most preferably 20-fold increase in binding. The binding is measured by ELISA, RIA, or surface plate assay. It can be assayed or determined by SPR analysis or the like. Examples of equipment used in SPR analysis include: BlAcore(TM)(GE Healthcare Bio-Sciences C rop.), ProteOn™ (Bio-Rad Laboratories, Inc.), DRX2 biosensor (Dynamic Biosensors Gm bH), SPR-Navi (trademark) (BioNavis Oy Ltd.), Spr eeta (trademark) (manufactured by Texas Instruments Inc.), SPRi-P lexII™ (Horiba, Ltd.), and Autolab SPR™ Binding of antibodies to antigens expressed on the cell surface is determined by flow cytometry. It can be assayed by cytometry or the like.

[0049] Additionally, the antibody may comprise a heavy chain variable region having the amino acid sequence of SEQ ID NO:11 or SEQ ID NO:10. and antigen binding of a reference antibody comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 12. Preferably, the reference antibody is a humanized antibody P In particular, the antibody specifically binds to the same antigen as the reference antibody, preferably The antibody preferably binds to said antigen with a higher affinity than the reference antibody. Lower than the dissociation constant, more preferably 10% lower, at least 20% lower, at least 30% lower % lower, or at least 50% lower, affinity to the antigen. Preferably, the antibody has a heavy chain containing the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 10. cross-reacting with a reference antibody comprising a light chain variable region and a light chain variable region having the amino acid sequence of SEQ ID NO: 12 In particular, the humanized antibody exhibits specificity against MUC1 when present at sufficiently high concentrations. The binding of the reference antibody can be blocked because the antibody is already bound to the antigen MUC1. This is possible if the binding of the reference antibody to MUC1 is disrupted.

[0050] Anti-MUC1 antibody The antibody capable of binding to MUC1 is a complementary molecule having the amino acid sequence of SEQ ID NO:1. constant region (CDR) CDR-H1, CDR-H2 having the amino acid sequence of SEQ ID NO: 2, and a heavy chain variable region comprising CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and a CDR-H4 having the amino acid sequence of SEQ ID NO: 4; a complementarity-determining region (CDR) CDR-L1 having the amino acid sequence of No. 4; a light chain variable region comprising CDR-L2 having the amino acid sequence of SEQ ID NO: 6, and CDR-L3 having the amino acid sequence of SEQ ID NO: 6; Includes the area.

[0051] In certain embodiments, the heavy chain variable region has at least 9 amino acid sequences identical to the amino acid sequence of SEQ ID NO: 9. In particular, the heavy chain variable region comprises an amino acid sequence having an amino acid sequence identical to that of SEQ ID NO: 9. These include amino acid sequences that are at least 95%, particularly at least 98%, identical to the In embodiments, the heavy chain variable region comprises CDRs having the amino acid sequences of SEQ ID NOs: 1, 2, and 3. Therefore, any sequence deviation from SEQ ID NO: 9 still includes the framework In particular, the heavy chain variable region is located in the amino acid sequence of SEQ ID NO: 9, but not in the CDRs. Contains the acid sequence.

[0052] In certain embodiments, CDR-H2 has the amino acid sequence of SEQ ID NO: 2, The amino acids at position 8 of No. 2 are glutamine, alanine, valine, histidine, and tryptophan. , tyrosine, lysine, and arginine, especially glutamine, histidine, and tryptophan , tyrosine, lysine, and arginine. The amino acid at position 8 of No. 2 is glutamine, histidine, tryptophan, lysine, or amino acid. In particular, CDR-H2 has the amino acid sequence of SEQ ID NO: 7. Has.

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

[0054] In a specific embodiment, the heavy chain variable region has at least 9 amino acid sequences identical to the amino acid sequence of SEQ ID NO: 10. In particular, the heavy chain variable region contains an amino acid sequence that is 0.000% identical to the amino acid sequence of SEQ ID NO: 10. Such sequences include amino acid sequences that are at least 95%, particularly at least 98%, identical to the sequence. In embodiments, the heavy chain variable region comprises CDR-H1 having the amino acid sequence of SEQ ID NO: 1, the sequence 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 CDR-H3. Therefore, any sequence deviations from SEQ ID NO: 10 are within the framework In particular, the heavy chain variable region is located in the base region but not in the CDRs. Contains the amino acid sequence.

[0055] In a specific embodiment, the heavy chain variable region has at least 9 amino acid sequences identical to the amino acid sequence of SEQ ID NO: 11. In particular, the heavy chain variable region contains an amino acid sequence that is 0.000% identical to the amino acid sequence of SEQ ID NO: 11. Such sequences include amino acid sequences that are at least 95%, particularly at least 98%, identical to the sequence. In embodiments, the heavy chain variable region comprises CDR-H1 having the amino acid sequence of SEQ ID NO: 1, the sequence 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 CDR-H3. Therefore, any sequence deviations from SEQ ID NO: 11 are within the framework In particular, the heavy chain variable region is located in the base region but not in the CDRs. Contains the amino acid sequence.

[0056] In certain embodiments, the light chain variable region has at least one amino acid sequence identical to that of SEQ ID NO: 12. 90% identical amino acid sequence. In particular, the light chain variable region contains the amino acid sequence of SEQ ID NO: 12. It includes an amino acid sequence that is at least 95%, particularly at least 98%, identical to the sequence. In certain embodiments, the light chain variable region comprises a C nucleotide sequence having the amino acid sequence of SEQ ID NOs: 4, 5, and 6. Therefore, any sequence deviations from SEQ ID NO: 12 are in-frame. The light chain variable region is located in the framework region but not in the CDRs. In particular, the light chain variable region is SEQ ID NO: 12 It contains the amino acid sequence of

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

[0058] In a specific embodiment, the heavy chain variable region has at least 9 amino acid sequences identical to the amino acid sequence of SEQ ID NO: 10. 1, 7, and 3, and the CDRs have amino acid sequences that are 0% identical to those of SEQ ID NOs: 1, 7, and 3. and the light chain variable region still has the amino acid sequence of SEQ ID NO: 12 at least 90% identical to that of SEQ ID NO: 13. The CDRs have the amino acid sequences of SEQ ID NOs: 4, 5, and 6. In particular, the heavy chain variable region has at least 9 amino acid sequences identical to those of SEQ ID NO: 10. 5% identical amino acid sequence, and the CDRs are identical to the amino acid sequences of SEQ ID NOs: 1, 7, and 3. and the light chain variable region still has the amino acid sequence of SEQ ID NO: 12 at least 95% identical to the amino acid sequence of SEQ ID NO: 13. The CDRs have the amino acid sequences of SEQ ID NOs: 4, 5, and 6. Still have.

[0059] In a specific embodiment, the heavy chain variable region has at least 9 amino acid sequences identical to the amino acid sequence of SEQ ID NO: 11. 1, 8, and 3, and the CDRs have amino acid sequences that are 0% identical to those of SEQ ID NOs: 1, 8, and 3. and the light chain variable region still has the amino acid sequence of SEQ ID NO: 12 at least 90% identical to that of SEQ ID NO: 13. The CDRs have the amino acid sequences of SEQ ID NOs: 4, 5, and 6. In particular, the heavy chain variable region has at least 9 amino acid sequences identical to those of SEQ ID NO: 11. 5% identical amino acid sequence, and the CDRs are identical to the amino acid sequences of SEQ ID NOs: 1, 8, and 3. and the light chain variable region still has the amino acid sequence of SEQ ID NO: 12 at least 95% identical to the amino acid sequence of SEQ ID NO: 13. The CDRs have the amino acid sequences of SEQ ID NOs: 4, 5, and 6. Still have.

[0060] In a specific embodiment, the heavy chain variable region comprises amino acids 20-136 of SEQ ID NO:20. In particular, the amino acid sequence of the present invention is at least 90% identical to the amino acid sequence represented by The chain variable region has the amino acid sequence represented by amino acid numbers 20 to 136 of SEQ ID NO: 20. These include amino acid sequences that are at least 95%, particularly at least 98%, identical. In one embodiment, the heavy chain variable region comprises a 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, a CDR-H3 having the amino acid sequence of SEQ ID NO: 3, a CDR-H4 having the amino acid sequence of SEQ ID NO: 4, a CDR-H5 having the amino acid sequence of SEQ ID NO: 5, a CDR-H6 having the amino acid sequence of SEQ ID NO: 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. R-H3. Therefore, the amino acid sequence represented by amino acid numbers 20 to 136 of SEQ ID NO: 20 is Any sequence deviations to the amino acid sequence are located in the framework regions but not in the CDRs. In particular, the heavy chain variable region is represented by amino acid numbers 20 to 136 of SEQ ID NO: 20. In certain embodiments, the amino acid sequence at position 76 of SEQ ID NO: 20 is are glutamine, alanine, valine, histidine, tryptophan, tyrosine, lysine, and arginine, especially glutamine, histidine, tryptophan, tyrosine, lysine, and arginine. Preferably, the amino acid at position 76 of SEQ ID NO: 20 is selected from the group consisting of: The amino acids are glutamine, histidine, tryptophan, lysine, or arginine, especially glutamine. In particular, CDR-H2 comprises the amino acid sequence of SEQ ID NO: 7 and / or or the heavy chain variable region is amino acid sequence 20 to 136 of SEQ ID NO: 23. Contains arrays.

[0061] In a specific embodiment, the light chain variable region comprises amino acids 21-133 of SEQ ID NO:21. In particular, the amino acid sequence of the present invention is at least 90% identical to the amino acid sequence represented by the following: The chain variable region has the amino acid sequence represented by amino acid numbers 21 to 133 of SEQ ID NO: 21. These include amino acid sequences that are at least 95%, particularly at least 98%, identical. In one embodiment, the light chain variable region comprises CDRs having the amino acid sequences of SEQ ID NOs: 4, 5, and 6. Therefore, the amino acid sequence represented by amino acid numbers 21 to 133 of SEQ ID NO: 21 is still included. Any sequence deviations to the amino acid sequence are located in the framework regions but not in the CDRs. In particular, the light chain variable region is represented by amino acid numbers 21 to 133 of SEQ ID NO: 21. The amino acid sequence includes the amino acid sequence

[0062] In a specific embodiment, the heavy chain variable region comprises amino acids 20-136 of SEQ ID NO:20. and the CDRs are , SEQ ID NOs: 1, 7, and 3, and the light chain variable region still has the amino acid sequences of SEQ ID NOs: At least 90% identical to the amino acid sequence represented by amino acid numbers 21 to 133 of No. 21 and the CDRs have the amino acid sequences of SEQ ID NOs: 4, 5, and 6. In particular, the heavy chain variable region has the amino acid sequence 20 to 136 of SEQ ID NO: 20. and the CDRs have an amino acid sequence that is at least 95% identical to the amino acid sequence shown in still have the amino acid sequences of SEQ ID NOs: 1, 7, and 3, and the light chain variable region is SEQ ID NO: 2 1. The amino acid sequence represented by amino acid numbers 21 to 133 of The CDRs have the amino acid sequences of SEQ ID NOs: 4, 5, and 6. It has.

[0063] In a specific embodiment, the heavy chain comprises a sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 15. In particular, the heavy chain comprises an amino acid sequence which is at least 9 amino acid sequences of SEQ ID NO: 15. In such an embodiment, the amino acid sequence is at least 5%, particularly at least 98% identical. The chain comprises 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 deviations relative to SEQ ID NO: 15 are located in the framework regions but not in the C The heavy chain is not located in the DR. In particular, the heavy chain comprises the amino acid sequence of SEQ ID NO: 15. In embodiments, the amino acid at position 57 of SEQ ID NO: 15 is selected from glutamine, alanine, valine, histidine, ribonucleotides, and the like. thymine, tryptophan, tyrosine, lysine, and arginine, especially glutamine, histidine, thritol, tryptophan, tyrosine, lysine, and arginine. Preferably, the amino acid at position 57 of SEQ ID NO: 15 is selected from glutamine, histidine, tripeptide, and ribonucleotides. CDR-H2 is preferably a nucleotide of the CDR-H1 amino acid sequence selected from the group consisting of ribonucleotides, lysine, arginine, and glutamine. and / or the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 22. It includes the amino acid sequence represented by the amino acid numbers 20 to 136.

[0064] In a specific embodiment, the heavy chain comprises a sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 19. In particular, the heavy chain comprises an amino acid sequence which is at least 9 amino acid sequences of SEQ ID NO: 19. In such an embodiment, the amino acid sequence is at least 5%, particularly at least 98% identical. The chains include 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 deviations relative to SEQ ID NO: 19 are located in the framework regions but not in the C It is not located in the DR. In particular, the heavy chain comprises the amino acid sequence of SEQ ID NO:19.

[0065] In a specific embodiment, the light chain has an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 16. In particular, the light chain comprises an amino acid sequence which is at least 9 amino acid sequences of SEQ ID NO: 16. In such an embodiment, the amino acid sequence is at least 5%, particularly at least 98% identical. The chains still contain CDRs having the amino acid sequences of SEQ ID NOs: 4, 5, and 6. Therefore, any sequence deviations relative to SEQ ID NO: 16 are located in the framework regions, but not in the C It is not located in the DR. In particular, the light chain comprises the amino acid sequence of SEQ ID NO:16.

[0066] In a specific embodiment, the heavy chain comprises a sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 15. and the CDRs have the amino acid sequences of SEQ ID NOs: 1, 7, and 3. and the light chain variable region is at least 90% identical to the amino acid sequence of SEQ ID NO: 16. The CDRs have 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: 15. The CDRs still have the amino acid sequences of SEQ ID NOs: 1, 7, and 3. and 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 CDRs still have the amino acid sequences of SEQ ID NOs: 4, 5, and 6.

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

[0068] The term "antibody" includes and encompasses modified forms thereof. Modified forms of antibodies may be modified chemically or biologically. Chemically modified forms refer to antibodies in which the amino acid backbone has been modified with chemical moieties. and chemically modified N-linked or O-linked The chemical moiety or form may be toxic or cytotoxic, and may include forms having carbohydrate chains attached thereto. Biologically modified forms may include post-translational modifications (e.g., N-linked or O-linked). glycosylation, N- or C-terminal processing, deamidation, aspartic acid aberration forms that have undergone cleavage (or methionine oxidation), and using prokaryotic host cells. Examples include a form containing a methionine residue added to the N-terminus upon expression. Such modified forms may also be labeled to allow for detection or isolation of the antibody or antigen. may include modified forms, such as enzyme-labeled, fluorescent-labeled, or affinity-labeled forms. It is contemplated that such modified forms may be used to allow for the detection or isolation of the antibody or its antigen. and other labelled forms, such as enzyme-labelled forms, fluorescent-labelled forms and affinity-labelled forms. Such modified forms of the antibodies of the invention are intended to include those forms which are stable to the original antibody. These are useful for improving the activity or blood retention, reducing antigenicity, detecting or isolating antibodies or antigens, etc. be.

[0069] In particular, the antibodies may be defucosylated, fucose-reduced, N-linked glycosylated, O-linked glycosylated, or Sylation, N-terminal processing, C-terminal processing, deamidation, aspartic acid isomerism oxidation of methionine, and cleavage of heavy chain positions 234 and 235 (according to EU index). The two leucine (L) residues were replaced by alanine (A) (LALA), and the proline residue was replaced by alanine (A). Midation and deletion or absence of one, two, or three amino acids at the carboxyl terminus and optionally, one or more modifications selected from the group consisting of: In this study, antibodies consist of one, two, or three carboxyl termini of one or both heavy chains. one heavy chain lacking the terminal amino acid or two carboxyl-terminal amino acids or the carboxyl-terminal proline residues of both heavy chains are amidated.

[0070] Such modifications can be made at any or desired position in the antibody. Instead, the same or two or more different modifications may be used, one or more of which may be It can be made in one or two or more positions.

[0071] For example, antibodies produced in mammalian cell culture may contain carboxyl-terminal fragments of their heavy chains. It is known to lack a methyl group residue (Journal of Chromatogr Aphy A, 705:129-134 (1995)). They may lack the carboxyl-terminal amino acid residues (i.e., glycine and lysine) and It is also known that the proline residue located at the carboxyl terminus is amidated in the presence of α-amylated proline (An Analytical Biochemistry, vol. 360, pp. 75-83 (2007 However, such deletions or modifications of the heavy chain sequence may prevent the antibody from binding to its antigen. The ability to bind to the antibody also influences its effector functions (complement activation, antibody-dependent cellular cytotoxicity, etc.). ) is not affected.

[0072] In certain embodiments, the antibody comprises one or two amino acids at the carboxyl terminus of the heavy chain. amidated residues (e.g., amides at the carboxyl-terminal position of the heavy chain) However, antibodies have shown that deletion mutants do not bind to antigens. As long as the ability is maintained, it is not limited to the types described above.

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

[0074] In a specific embodiment, the antibodies are both missing one carboxyl-terminal amino acid residue. The antibody may comprise two heavy chains.

[0075] In specific embodiments, the antibody comprises a sequence identical to amino acids 1-446 of SEQ ID NO: 15 or 22. and a heavy chain having an amino acid sequence represented by amino acid numbers 1 to 219 of SEQ ID NO: 16. In one particular embodiment, the light chain has an amino acid sequence represented by SEQ ID NO: 1 The amino acid at position 57 of 5 is glutamine, alanine, valine, histidine, or tryptophan. , tyrosine, lysine, and arginine, especially glutamine, histidine, and tryptophan , tyrosine, lysine, and arginine. The amino acid at position 57 of No. 15 is glutamine, histidine, tryptophan, lysine, or is arginine and especially glutamine.

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

[0077] In certain embodiments, the antibody 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, or an antibody having the amino acid sequence of SEQ ID NO: 1 a heavy chain variable region having the amino acid sequence of SEQ ID NO:1 and a light chain variable region having the amino acid sequence of SEQ ID NO:12 It competes with an antibody containing the variable region for binding to TA-MUC1.

[0078] In certain embodiments, the antibody has the following properties: (a) it specifically binds to MUC1; and and / or (b) are internalized into MUC1-expressing cells by binding to MUC1. In certain embodiments, the antibody has the property of having at least one 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 a CH2 domain and a CH3 domain, but In a further embodiment, one or more constant domains of the heavy chain do not include the H1 domain. may be replaced by other domains, especially similar domains such as albumin. Antibody heavy chains may be of any type, including gamma, alpha, epsilon, delta, and mu chains. Preferably, the γ chain includes γ1 chain, γ2 chain, γ3 chain, and γ4 chain, and particularly γ1 chain. Therefore, the antibody is preferably an IgG1 type antibody, an IgG3 type antibody, or IgG type antibodies such as IgG4 type antibodies, particularly IgG1 type antibodies.

[0079] In particular, the antibody further comprises at least one antibody light chain, in particular two antibody light chains. The chains contain, inter alia, a VL domain and a CL domain. Antibody light chains can be kappa or lambda chains. It may be a κ chain, in particular.

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

[0081] In alternative embodiments, the antibody does not comprise an antibody light chain. The variable region may be fused to the N-terminus of the heavy chain variable region or may be fused to the N-terminus of the heavy chain variable region. Inserted at the C-terminus: a peptide linker that connects the light chain variable region to the rest of the heavy chain may be present.

[0082] In a preferred embodiment, the antibody comprises an Fc region, in particular an Fc region, each of which comprises the domains VH, Two heavy chains, each containing a CH1, hinge region, CH2, and CH3 domain The antibody may be a whole antibody comprising two light chains comprising a VL and a CL. or multiple human Fcγ receptors, particularly human Fcγ receptor IIIA. In an alternative embodiment, the antibody does not bind or significantly binds to human Fcγ receptor IIIA. In particular, it does not bind or binds significantly to any human Fcγ receptor. In such embodiments, the antibody does not contain a glycosylation site, particularly in the CH2 domain. Not at all.

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

[0084] Glycosylation of anti-MUC1 antibodies The anti-MUC1 antibody may comprise a CH2 domain in one or more of the antibody heavy chains. Naturally occurring human antibodies of the IgG type contain N-glycosylation sites in the CH2 domain. The CH2 domain 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 has an address at the heavy chain position corresponding to position 297 according to the IMGT / Eu numbering system. For example, the antibody contains an Ala297 mutation in the heavy chain. In such embodiments, the antibody preferably binds to an Fcγ receptor. Antibody-dependent cellular cytotoxicity (ADCC) and / or antibody-dependent cellular phagocytosis potent ability to induce ADCP and / or complement-dependent cytotoxicity (CDC) In this respect, a strong reduction in capabilities is especially , which contains an N-glycosylation site in its CH2 domain and can be expressed in human or CHO cell lines. glycosylation patterns that can be obtained by producing, for example, The same antibody has a typical mammalian glycosylation pattern, such as a glycosylation pattern similar to that of Compared to the body, 10% or less, especially 3% or less, 1% or less, or a reduction to 0.1% or less activity. In such an embodiment, the antibody is in particular an IgG1 type antibody.

[0085] In an alternative embodiment, the CH2 domain present in the antibody contains an N-glycosylation site. This glycosylation site is located specifically at amino acid position 1 of the heavy chain according to the IMGT / Eu numbering system. The amino acid position corresponding to 297 has the amino acid sequence motif Asn Xaa Ser / Thr (wherein Xaa can be any amino acid except proline) ) N-linked glycosylation of Asn297 is found in mammalian IgG as well as other antibodies. Conserved in regions of isotype homology. Optional for variable regions or other sequence modifications. This conserved amino acid sequence of the antibody may have additional amino acids present. The actual location of the glycosylation site may vary. Preferably, the glycans attached to the antibody The compound preferably has at least the following structure: Asn-GlcNAc-GlcNAc-Man-(Man-GlcNAc)2 wherein Asn is an asparagine residue in the polypeptide portion of the antibody; and NAc is N-acetylglucosamine and Man is mannose. The terminal GlcNAc residue carries an additional galactose residue. The galactose residues may optionally carry sialic acid residues. The GlcNAc residue (called the bisecting GlcNAc) is closest to the Man Fucose may be attached to GlcNAc attached to Asn. In such an embodiment, the antibody is in particular an IgG1 type antibody.

[0086] In a preferred embodiment, the antibody does not contain N-glycol neuraminic acid (NeuGc). Furthermore, the antibody also preferably contains no or detectable amounts of NeuGc. Does not contain or has detectable amounts of the lili epitope (Galα1,3-Gal structure) It does not contain Galili epitopes, especially NeuGc and / or Galα1,3- The relative abundance of glycans bearing Gal structures attached to the CH2 domains of antibodies in a population of antibodies less than 0.1% or even less than 0.02% of the total amount of glycans present.

[0087] In particular, the antibody has a human glycosylation pattern. , both of which contain known immunogenic non-human sialic acid (NeuGc) in rodent production systems. is due to certain exogenous sugar structures such as the Galili epitope (Gal-Gal structure). Inducing unwanted or adverse side effects known to cause Foreign immunogenic non-human structures are not known to exist or are derived, for example, from yeast systems. Other structures such as the immunogenic high mannose structures that are known to be present in the ribozyme are avoided.

[0088] In a specific embodiment, the antibody detects a detectable amount of glycoprotein bearing a bisecting GlcNAc residue. In particular, glycosylation patterns bearing bisecting GlcNAc residues The relative amount of glycans refers to the total amount of glycans attached to the glycosylation sites of the antibodies in the composition. In addition, in certain embodiments, The glycosylation pattern is determined by measuring at least the total amount of glycans attached to the antibodies in the composition. The glycans contain at least 25% of the relative amount of glycans carrying at least one galactose residue. The relative amount of glycans bearing at least one galactose residue is determined by the relative amount of the antibody in the composition. at least 30%, in particular at least 35%, of the total amount of glycans attached to In a specific embodiment, the glycosylation pattern is at least 40%. at least one glycan in a relative amount of at least 1% of the total amount of glycans attached to the antibody It contains glycans bearing sialic acid residues. In particular, it contains glycans bearing at least one sialic acid residue. The relative amount of glycans attached to the antibodies in the composition is at least 1 / 2 of the total amount of glycans attached to the antibodies in the composition. 1.5%, especially at least 2%.

[0089] Antibodies can have glycosylation patterns with either high or low amounts of core fucose. Reducing the amount of fucosylation increases the ability of the antibody to induce ADCC. In certain embodiments, the relative amount of glycans bearing a core fucose residue in the composition 40% or less of the total amount of glycans attached to the antibody, especially 30% or less, or 20% or less. The relative amount of glycans bearing a core fucose residue is determined by the amount of glycans attached to the antibodies in the composition. At least 60%, in particular at least 65%, or at least 70% of the total volume of the can do.

[0090] The presence or absence of glycosylation sites in the CH2 domain of the anti-MUC1 antibody and the The presence or absence of fucose in the glycan structure at the glycosylation site determines whether the antibody is capable of ADCC. The ADCC activity can be controlled by controlling the ability of the antibody to induce ADCC and the strength of the ADCC induction. The glycosylation of the Fc portion of the antibody is increased, and the amount of fucosylation of said glycosylation is reduced. In certain applications, fine-tuning of ADCC activity is important. Therefore, in certain circumstances, it is necessary to have a glycosylation site in the CH2 domain. antibodies with no glycosylation site in the CH2 domain and with extensive fucosylation Antibodies with glycosylation sites in the IgG1 or CH2 domain and low levels of fucosylation , may be most advantageous.

[0091] Anti-MUC1 antibody production The antibodies are preferably produced recombinantly in host cells. The host cell may be any host cell that can be used for antibody production. The cell is particularly a eukaryotic host cell, especially a mammalian host cell. Exemplary host cells include yeast cells, such as Pichia pastoris cell lines, S Insect cells such as F9 and SF21 cell lines, plant cells, and chicken cells such as the EB66 duck cell line cells, rodent cells such as CHO, NS0, SP2 / 0, and YB2 / 0 cell lines, Ravi HEK293, PER.C6, CAP, CAP-T, AGE1.HN, Mutz- 3, and human cells such as the KG1 cell line.

[0092] In certain embodiments, the antibody is expressed in human blood cell lines, particularly human myeloid leukemia cell lines. A preferred human cell line that can be used for antibody production is Suitable production procedures are described in WO2008 / 028686A2. In embodiments, the antibodies are NM-H9D8, NM-H9D8-E6, and NM-H9D8- E6Q12, and human myeloid leukemia cell lines derived therefrom. These cell lines comply with the requirements of the Budapest Treaty. Therefore, accession number DSM ACC2806 (NM-H9D8; September 2006 Deposited on January 15th), DSM ACC2807 (NM-H9D8-E6; January 2006) Deposited on 05), and DSM ACC2856 (NM-H9D8-E6Q12 (Deposited August 8, 2007) and is registered with Glycotope GmbH, Robe rt-Rossle-Str.10, 13125 Berlin (DE) by Deut sche Sammlung von Mikroorganismen und Ze llkulturen(DSMZ),Inhoffenstraβe 7B,38124 The NM-H9D8 cells were deposited in Braunschweig (DE). lysylation, highly bisecting GlycNAc, highly galactosylated, and highly fucosylated. The glycosylation patterns of NM-H9D8-E6 cells and NM-H9D 8-E6Q12 cells are similar to NM-H9D8 cells except that they have a much lower degree of fucosylation. Other suitable cell lines include those that provide glycosylation patterns similar to those of the American A human myeloid leukemia cell line, K562 (ATCC 94214), is housed in the American Type Culture Collection. CCL-243) as well as cell lines derived from those mentioned above.

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

[0094] Anti-MUC1 antibody conjugates According to the present invention, the antibody is conjugated to one or more cytotoxic agents. The cytotoxic agent may be any cytotoxic agent suitable for conjugation to an antibody. When more than one cytotoxic agent is present on the antibody, those cytotoxic agents may be the same or different. The combination of the cytotoxic agent and the antibody may be the same or different, and in particular may all be the same. Conjugation can be achieved using any method known in the art. Cytotoxic agents may be covalently attached to the antigen, particularly by fusion or chemical coupling, or non-covalently attached to the antigen. In certain embodiments, the cytotoxic agent may be attached to the body, particularly to the linker moiety. The linker moiety attaches the cytotoxic agent to the antibody. The compound may be any chemical entity suitable for forming a complex.

[0095] In addition to the cytotoxic agent, the conjugate according to the invention may also contain a cytotoxic agent conjugated thereto. The additional agent may preferably be a therapeutic agent for treating a disease, particularly cancer. are useful in the treatment, diagnosis, prognosis, and / or monitoring of The agent may be a radionuclide, a chemotherapeutic agent, an antibody or antibody fragment, particularly an antibody with a different characteristic than an anti-MUC1 antibody. heterologous, e.g., checkpoint antibodies, enzymes that block or activate immunoregulatory targets , interaction domains, detectable labels, toxins, cytolytic components, immunomodulators, immune effectors an MHC class I or class II antigen, and a liposome. It is possible.

[0096] Certain preferred cytotoxic agents are those that kill cancer cells, such as radionuclides, or chemotherapeutic agents. In certain preferred embodiments, the chemotherapeutic agent is a cytotoxic agent capable of The chemotherapeutic agent is attached to an anti-MUC1 antibody forming a conjugate. It has an antitumor effect and has a substituent or moiety that can be connected to a linker structure. When a part or the whole of the linker is cleaved in tumor cells, The chemotherapeutic or anti-tumor compound moiety is released, and the chemotherapeutic agent exerts an anti-tumor effect. When the cleavage site is broken at the site of attachment to the agent, the chemotherapeutic agent is released in its original structure and It 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, irinotecan, and topoisomerase inhibitors such as topotecan, anti-cancer drugs such as doxorubicin, or microtubule inhibitors such as maytansine / maytansinoids.

[0098] The chemotherapeutic agent may in particular be selected from the group consisting of: V-ATPa se inhibitor, pro-apoptotic agent, Bcl2 inhibitor, MCL1 inhibitor, HSP90 inhibitor , IAP inhibitors, mTor inhibitors, microtubule stabilizers, microtubule destabilizers, dolastatins, Maytansine, maytansinoids, amatoxin, methionine aminopeptidase, tan Protein CRM1 nuclear export inhibitor, DPPIV inhibitor, proteasome inhibitor, mitochondrial Inhibitor of phosphoryl transfer reaction in doria, protein synthesis inhibitor, kinase inhibitor, CDK 2 inhibitors, CDK9 inhibitors, kinesin inhibitors, HDAC inhibitors, topoisomerase I inhibitors agents, DNA damaging agents, DNA alkylating agents, DNA intercalators, DNA minor groove binders , DHFR inhibitor, microtubule formation inhibitor, microtubule stabilizer, actin stabilizer, topoisomerase inhibitor polymerase II inhibitors, platinum compounds, ribosome inhibitors, RNA polymerase II inhibitors, and bacterial toxins. In a specific embodiment, the chemotherapeutic agent attached to the anti-MUC1 antibody is a Microtubule inhibitors such as itansinoids, topoisomerase I inhibitors, DNA damaging agents, DNA alkylating agents, and DNA minor groove binders.

[0099] In some embodiments, the chemotherapeutic agent is maytansine or a maytansinoid. Specific examples of maytansinoids useful for inductive administration include maytansinol, N2'-Deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine (DM1), N2'-deacetyl-N2'-(4-mercapto-1-oxopentyl)- Maytansine (DM3), and N2'-deacetyl-N2'-(4-methyl-4-methyl) In particular, DM1 or or DM4 is attached to the anti-MUC1 antibody. The chemotherapeutic agents that are attached are DNA minor groove binders, particularly pyrrolobenzodiazepines (PBDs), ... Parabenzodiazepine dimer (PBD dimer), duocarmycin, duocarmycin Benzyl-hydroxybenzamido-azaindole (DUBA), seco-duocarmyl syn-hydroxybenzamido-azaindole (seco-DUBA), or doxo In some embodiments, the chemotherapeutic agent attached to the anti-MUC1 antibody is DNase. A alkylating agents, especially indolinobenzodiazepines or oxazolidinobenzodiazepines In some embodiments, the compound is oxazolidinobenzodiazepine. The chemotherapeutic agent attached to the anti-MUC1 antibody is a DNA damaging agent, specifically calicheamicin. In some embodiments, the chemotherapeutic agent attached to the anti-MUC1 antibody is a topoisomer. Enzyme I inhibitors, especially camptothecin and 7-ethyl-10-hydroxy-camptothecin 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-benzyl Nzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10 ,13-dione (exatexicane (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]indolizino[1,2-b]quinolin-1-yl]-2-hydroxya and derivatives thereof, such as cetoamide (DXd). In some embodiments, the anti-MUC1 antibody The attached chemotherapeutic agent is an inhibitor of microtubule formation, particularly tubulysin, ansamitocin, , podophyllotoxin, or vinblastine. The chemotherapeutic agent attached to the antibody is a microtubule stabilizer, particularly paclitaxel or epothilone. In some embodiments, the chemotherapeutic agent attached to the anti-MUC1 antibody is an actin stabilizers, particularly phallotoxins. In some embodiments, the anti-MUC1 antibody attached The chemotherapeutic agents used are topoisomerase II inhibitors, particularly teniposide, XK469, and razoxane. In some embodiments, the anti-MU antibody is cycloheximide, amsacrine, idarubicin, or mevalon. The chemotherapeutic agents attached to the C1 antibody include platinum compounds, particularly cisplatin, carboplatin, Oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picop In some embodiments, the compound attached to the anti-MUC1 antibody is platin, or satraplatin. The chemotherapeutic agents used are ribosome inhibitors, especially ricin, saporin, abrin, and diphtheria toxin. or exotoxin A. In some embodiments, the chemotherapy agent attached to the anti-MUC1 antibody is The therapeutic agent is an RNA polymerase II inhibitor, in particular an amatoxin, such as amanitin. In some embodiments, the chemotherapeutic agent attached to the anti-MUC1 antibody is a bacterial toxin, particularly a Suitable antibody drug conjugates are described in EP16151774.3 and and the LU92659 specification expressly 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]indolizino[1,2 -b]quinoline-10,13-dione (exatexicane (DX-8951)) or DX d.

[0101] Exatexican (DX-8951) has the following formula:

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

[0103] The compound can be prepared, for example, by the method described in U.S. Patent Application Publication No. 2016 / 0297890. It can be easily obtained by the method or other known methods, and preferably, the amino group at the 1-position is It can be used as a linking point for a linker structure. It can be released in the tumor cell, but with a portion of the linker still attached to it. However, this compound exhibits excellent antitumor effects even under such conditions. do.

[0104] DXd is represented by the following formula:

[0105] [ka] It is a compound represented by the formula:

[0106] Exatexican or DXd has a camptothecin structure and is therefore suitable for use in acidic aqueous media (e.g. At pH levels of around 3, for example, the equilibrium shifts to the closed structure where the lactone ring is formed. In a basic aqueous medium (for example, at about pH 10), the lactone ring is opened (opened) It is known that the equilibrium shifts to the closed and open ring structures. Drug conjugates incorporating the exatexican moiety also exhibited equivalent antitumor activity. It is expected that such drug conjugates will be used in various ways, and all such drug conjugates are within the scope of the present invention. It goes without saying that in certain embodiments, the additional agent is a polypeptide or a tag. This polypeptide or protein is particularly suitable for use in the polypeptide chains of antibodies. In certain embodiments, the polypeptide or protein The additional agent is fused to the C-terminus of the antibody light chain of the antibody. In embodiments comprising the additional agent that is a polypeptide or protein, the additional agent is a polypeptide or protein. In a further embodiment, the polypeptide or The additional agent, which is a protein, is fused to the C-terminus of the antibody heavy chain of the antibody. In embodiments in which the antibody comprises two antibody heavy chains, a further construct that is a polypeptide or protein may be used. The therapeutic agent may be fused to the C-terminus of each of the two antibody heavy chains. They may be the same or different, and in particular may have the same amino acid sequence. Suitable examples of such additional agents that are polypeptides or proteins are cytokines. from the group consisting of kinases, chemokines, antibodies, antigen-binding fragments, enzymes, and interaction domains. You may choose from:

[0107] In certain embodiments, the additional agent that is a polypeptide or protein has an active These are checkpoint antibodies that block and / or induce activation signals. Examples of targets include CD40, CD3, CD137 (4-1BB), and O as activation targets. X40, GITR, CD27, CD278 (ICOS), CD154 (CD40 ligand) ), CD270 (HVEM), and CD258 (LIGHT), as inhibitory targets TLA4, PD1, CD80, CD244, A2AR, B7-H3(CD276), B7 -H4(VTCN1), BTLA, IDO, KIR, LAG3, TIM-3, VISTA These include phosphatidylserine and their corresponding ligands, such as PDL1. In a specific example, the anti-MUC1 antibody comprises two heavy chains and The scFv fragments that specifically bind to CD3 are fused to the C-terminus of each heavy chain. Alternatively, an scFv fragment that specifically binds to PDL1 is fused to the C-terminus of each light chain. are combined.

[0108] In a further embodiment, the additional agent that is a polypeptide or protein is a chemotherapeutic agent. Preferred serotonin receptors in this regard are immunomodulatory compounds such as steroids, cytokines, or growth factors. Interferon-α, interferon-β, and interferon-β are interferon-α and interferon-β. Suitable anti-inflammatory agents include interferons such as interferon-γ, and interleukins. Growth factors include G-CSF and GM-CSF.

[0109] Specific examples of linkers include those represented 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-N H-CH2CH2CH2-C(=O)-, and (f)-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O- CH2CH2O-CH 2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)- In the formula, -(Succinimid-3-yl-N)- is a structure represented by the following formula: formula:

[0110] [ka] It has a structure represented by:

[0111] In specific embodiments, the linker has the following formulas (a)-(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-N H-CH2CH2CH2-C(=O)- The structure includes any of the following:

[0112] In a preferred embodiment, the linker has the following formula (a): (a)-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)- The structure includes any of the following:

[0113] In an alternative embodiment, the conjugate comprises a drug-linker structure represented by the formula: The antibody is linked to a drug linker structure represented by the following formula via a thioether bond: It is gated and marked with an asterisk * represents the point of attachment to the antibody.

[0114] [ka]

[0115] In a preferred embodiment, the conjugate has the following formula:

[0116] [ka] and having a drug-linker structure represented by where AB represents the antibody, and y represents the unit of the drug-linker structure conjugated to the antibody. The antibody is a drug-linker structure represented by the formula above. conjugated via a thioether bond, the antibody representing the anti-MUC1 antibody described above; Preferably, the antibody has the following combinations of heavy and light chain variable regions or heavy and light chains: Combined 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: having the amino acid sequence of 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: having the amino acid sequence of 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 having the sequence (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 Having an array It is one of the following.

[0117] In the conjugates described above, the number of conjugated drug molecules (or The number of antibodies (cytotoxic agents) is an important factor affecting their efficacy and safety. -The production of drug conjugates (or conjugates) involves the production of conjugated drug molecules. Reaction conditions, such as the amounts of starting materials and reagents used in the reaction, to achieve a certain number of children Unlike chemical reactions of low molecular weight compounds, A mixture containing a suitable number of conjugated drug molecules per antibody molecule is obtained. The number of conjugated drug molecules is calculated as the mean value, i.e., the average number of conjugated drug molecules and unless otherwise indicated, i.e., various numbers of conglomerates. A specific number of conjugates in an antibody-drug conjugate mixture having conjugated drug molecules Except when referring to an antibody-drug conjugate having a conjugated drug molecule, The number of conjugated drug molecules according to the invention is usually meant as an average value. The number of exatexanthan molecules or DXd conjugated to one antibody can be controlled. The average number of conjugated drug molecules per exon is approximately 1–10. A DXd molecule or 1 to 10 DXds can be conjugated. The number of CAN molecules or DXd is preferably 2 to 8, more preferably 4 to 8, and even more preferably Preferably, it is 7 to 8, and even more preferably 8. Those skilled in the art will understand the examples of the present application. Based on this description, we designed a reaction to conjugate the required number of drug molecules to an antibody molecule. The antibody can be designed to have a controlled number of conjugated exatexin molecules. It should be noted that it is possible to obtain an organo-drug conjugate.

[0118] In the preferred embodiment, the conjugate is internalized in tumor cells and then phosphorylated. The car moiety is cleaved, and then DXd is released, exerting its antitumor effect. (Clinic al Cancer Research, 2016 Oct 15;Vol. 22(Issue 20): pp. 5097–5108, Epub 2016 March 29).

[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 have a ratio of atoms that is 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 also mentioned. The conjugate is a tritium ( 3 H), iodine-125( 125 I), carbon-14 ( 14 C) , copper 64( 64 Cu), Zirconium 89 ( 89 Zr), Iodine-124 ( 124 I), 18 18 F), Indium 111 ( 111 In), carbon-11 ( 11 C), and Yo Usine-131( 131 It may be radiolabeled with a radioisotope such as I. The labeled conjugates can be used as therapeutic or prophylactic agents, assay reagents, and other research reagents. and as diagnostic agents such as in vivo diagnostic imaging agents. Regardless, all isotopic variant conjugates are 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 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. Preferably, however, the nucleic acid sequence is expressed in a host cell or organism in which it is desired to express the nucleic acid. At least partially adapted to a particular codon usage of a substance, especially human codon usage The nucleic acid may be double-stranded or single-stranded DNA or RNA, preferably double-stranded DNA such as cDNA. A nucleic acid may be a single-stranded RNA, such as a nucleic acid molecule, a nucleic acid fragment, or a single-stranded RNA, such as mRNA. or may be composed of several nucleic acid molecules, each encoding a different portion of the antibody. The nucleotide sequence of the heavy chain of PankoMab variant (PM-N54Q) may be represented by SEQ ID NO: 17, and the PankoMab variant (PM-N54Q) The nucleotide sequence of the light chain may be represented by SEQ ID NO:18.

[0121] Antibodies are made up of more than one different amino acid chain, such as the antibody's light and heavy chains. In this case, the nucleic acids may, for example, each encode one of the amino acid chains of the antibody, preferably separate separated by regulatory elements such as IRES elements to produce a chain of amino acids The nucleic acid may be a single nucleic acid molecule containing several coding regions, or the nucleic acid may be a single nucleic acid molecule containing several coding regions, each of which is The fragment contains several coding regions each encoding one of the amino acid chains of the antibody. The nucleic acid molecule may also contain a nucleic acid sequence in addition to the coding region encoding the antibody. The sequences may, for example, encode other proteins, and transcription and / or transcription of the coding regions may be controlled. may affect translation, may affect the stability or other physical or chemical properties of the nucleic acid Additional nucleic acid sequences or other sequences that may have an effect or may have no function at all. It may also include modifications of:

[0122] The expression cassette or vector comprises the nucleic acid and a promoter operably linked to the nucleic acid. In addition, the expression cassette or vector may contain further elements. In particular, transcription and / or translation of nucleic acids, amplification and expression cassettes or vectors and / or reproduction, integration of the expression cassette or vector into the host cell genome, and and / or affect the copy number of the expression cassette or vector in the host cell, and The antibody may contain a gene encoding the antibody, and / or a gene encoding the antibody may contain a gene encoding the antibody. Suitable expression cassettes and vectors containing the respective expression cassettes are known in the art. It is well known and therefore does not require further explanation here.

[0123] The host cell may contain the nucleic acid, or the expression cassette or vector. The cell may be any host cell. The host cell may be an isolated cell or a recombinant cell. Preferably, the host cells are cultured cells, particularly primary cells. Preferably, the host cell is a colon cell, a cell of an established cell line, or preferably a tumor-derived cell. Bacterial cells such as E. coli and Saccharomyces Cells, especially yeast cells such as S. cerevisiae, Sf9 cells, etc. Any insect cells or mammalian cells, especially human cells such as tumor-derived human cells, CHO etc. In a preferred embodiment, the host cell is a human hamster cell, or a primate cell. Derived from myeloid leukemia cells. Preferably, the host cells are the following cells or cell lines: K5 62, KG1, MUTZ-3, or cells or cell lines derived therefrom, or The host cell is selected from a mixture of cells or cell lines comprising at least one of the above-mentioned cells. The main cells are preferably NM-H9D8, NM-H9D8-E6, NM-H9D8-E6 Q12, and a cell or cell line derived from any one of the above host cells. Such cell lines and their properties are described in WO2008 / 028686A2. In a further embodiment, the host cell is a strain of the genus Streptococcus aureus (Streptococcus mutans) having ATCC number CRL-90. In a preferred embodiment, the host cell is a CHO dhfr- cell line, such as the cell line 96. is optimal for expressing glycoproteins with specific glycosylation patterns, especially antibodies. Preferably, the nucleic acid and / or promoter in the expression cassette or vector is The codon usage of the coding region of the promoter and further elements depends on the type of host cell and its suitability. Preferably, the vector is suitable for the host cell type being used. Preferably, the antibody is produced by a host cell or cell line as described above.

[0124] The method for producing antibodies uses host cells as described herein. The method includes, inter alia, providing a host cell containing nucleic acid encoding the antibody, the host cell being suitable for expression of the antibody. culturing the host cells under conditions, and obtaining the antibody expressed by the host cells. The antibodies described herein may be obtained or obtainable by the methods. That's fine.

[0125] In another aspect, the present invention provides a composition comprising a conjugate according to the present invention. The composition may further comprise one or more selected from the group consisting of solvents, diluents, and excipients. It may contain further ingredients. Preferably, the composition is a pharmaceutical composition. In this embodiment, the components of the composition are preferably all pharmaceutically acceptable. liquid or fluid compositions, in particular preferably aqueous solutions, emulsions or suspensions, Alternatively, it may be a freeze-dried powder.

[0126] Use in medicine The conjugates are particularly useful in medicine, in particular for the treatment of diseases, in particular those diseases as described herein. Therapy, diagnosis and treatment of diseases, preferably cancer, infectious diseases, inflammatory diseases, graft-versus-host disease, and immunodeficiency The present invention is useful in the diagnosis, prognosis, detection, and / or monitoring of cancer.

[0127] Thus, in a further aspect, the present invention provides a conjugate for use in medicine. Preferably, the pharmaceutical use is for the treatment of disorders such as cancer. Cell proliferation-related diseases, bacterial, viral, fungal, or parasitic infections infectious diseases such as bronchitis, inflammatory diseases such as autoimmune diseases and inflammatory bowel disease, and immune deficiencies Treatment, prognosis, diagnosis, detection, and / or monitoring of diseases associated with reduced immune activity, such as In a preferred embodiment, the disease is cancer.

[0128] Preferably, the cancer is detected by immunohistochemistry, ELISA, RIA, enzyme-linked immunosorbent assay (ELISA), or the like. ELISPOT assay, dot blot, Ochterlony test, or Detected by immunochromatographic electrophoresis (CIE) or in situ hybridization. Cancers, in particular, show detectable expression of MUC1 (TA-MUC1). MUC1 detectable by histochemical or in situ hybridization The cancer contains cells that exhibit MUC1 (TA-MUC1) expression. 1 (TA-MUC1) levels may be tested.

[0129] The present invention relates to kits and devices comprising the conjugates according to the present invention, and to methods for treating cancer and other diseases. Further provided are related methods useful for diagnosing, detecting, or monitoring any MUC1-associated disorder. In some embodiments, a test or diagnostic sample comprising a conjugate of the invention is provided. A cross-sectional ELISA kit is provided. This kit detects MUC1 (TA-MUC1) Protein standard solutions, coloring reagents, buffer solutions for dilution, antibodies for solid phase, and antibodies for detection The solution may further comprise one or more of an antibody, a washing solution, etc. The amount of originally bound conjugate can be determined by absorbance, fluorescence, luminescence, or radioactivity. It can be measured by applying a method such as an isotope (RI) method. Absorbance plate readers, fluorescence plate readers, luminescence plate readers, and RI liquid scintillation counters and the like are used for measurement.

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

[0131] Immunohistochemistry is a technique that involves reacting tissue sections with antigen-binding antibodies (primary antibodies). There is no particular limitation as long as it includes the step of detecting the primary antibody bound to the antigen and the primary antibody bound to the antigen.

[0132] Various forms of cancer, including metastases, can be treated with the conjugates according to the invention. Cancers include, in particular, colon cancer, lung cancer, ovarian cancer, breast cancer including triple-negative breast cancer, and pancreatic cancer. , cervical cancer, endometrial cancer, gastrointestinal cancer, kidney cancer, head and neck cancer, thyroid cancer, and Furthermore, the cancer may be selected from the group consisting of gastric cancer, liver cancer, and urothelial cancer. Cancers can be selected from liver, bladder, skin, prostate, and blood cancers. In certain embodiments, the cancer is metastatic cancer. The cancer may include skin metastasis, lymph node metastasis, and the like. Any type of metastasis, including lung metastasis, liver metastasis, peritoneal metastasis, pleural metastasis, and / or brain metastasis In certain embodiments, the cancer has an inflammatory phenotype. In certain embodiments, any of the cancer types listed above may be an inflammatory cancer.

[0133] In certain embodiments, the viral infection is a virus such as human immunodeficiency virus, herpes simplex virus, or Epstein-Barr virus, influenza virus, lymphocytic choriomeningitis virus It is caused by the hepatitis B virus, hepatitis C virus, or hepatitis B virus. , inflammatory bowel disease, pelvic inflammatory disease, ischemic stroke, Alzheimer's disease, asthma, and psoriasis vulgaris The autoimmune disease may be selected from celiac disease, smallpox, and dermatitis / eczema. 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 can be selected from the group consisting of MUC1, For example, the tumor may contain or be associated with cells that express TA-MUC1. MUC1-positive, especially TA-MUC1-positive, i.e., MUC1-positive, especially TA-MUC1-positive, This includes cancer cells in which the gene is expressed.

[0134] In a specific embodiment, the conjugate is used for treatment in combination with another therapeutic agent. It is used to treat cancer, especially in combination with another anti-cancer drug. The therapeutic agent may be any known anti-cancer agent. Suitable anti-cancer therapeutic agents that can be administered include chemotherapeutic agents, other antibodies, immunostimulants, cytokines, and the like. The conjugates may be used in therapeutics such as steroids, chemokines, and vaccines. It may be combined with radiation therapy, surgery, and / or traditional Chinese medicine.

[0135] Anti-cancer drugs that can be used in combination with the conjugates include any chemotherapeutic agent, In particular, the agent is selected from chemotherapeutic agents known to be effective in treating MUC1-positive cancers. The type of chemotherapy agent used also depends on the cancer being treated. The agonist may be selected from the group consisting of: paclitaxel (Taxol); ), docetaxel (Taxotere), and taxanes such as SBT-1214; Rofosfamide; imatinib; pazopanib; capecitabine; cytarabine; vinorelbine; Gemcitabine; Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, Valproicin anthracyclines such as bicine and mitoxantrone; aminoglutethimide, tes Trakton (Teslac), anastrozole (Arimidex), letrozole ( Femara), exemestane (Aromasin), vorozole (Rivizor) , formestane (Lentaron), fadrozole (Afema), 4-hydroxybenzoates Androstenedione, 1,4,6-androstatriene-3,17-dione (ATD ), and aromatase such as 4-androstene-3,6,17-trione (6-oxo). enzyme inhibitors; irinotecan, topotecan, camptothecin, lamellarin D, etoposide ( VP-16), teniposide, doxorubicin, daunorubicin, mitoxantrone, am Topoisome inhibitors such as cyclin, ellipticine, aurintricarboxylic acid, and HU-331 Platinum methylase inhibitors: cis-diamminedichloroplatinum(II) (cisplatin), cis- Diammine(1,1-cyclobutanedicarboxylato)platinum(II) (carboplatin) , and [(1R,2R)-cyclohexane-1,2-diamine](ethanedioato-O ,O') Platinum-based chemotherapy agents such as platinum(II) (oxaliplatin); olaparib PARP inhibitors such as rucaparib, niraparib, and imiquimod; which TLR agonists; as well as antimetabolites, especially methotrexate, pemetrexed, Antifolates such as raltitrexed and pralatrexate, fluorouracil (fl fluorouracil), gemcitabine, floxuridine, 5-fluorouracil, and pyrimidine analogues, such as tegafur-uracil, and purine analogues, selective estrogens, Gen receptor modulators, and estrogen receptor down-regulators.

[0136] Additionally, therapeutic antibodies can also be used as further combination partners. The antibody of interest may be any antibody useful in cancer therapy other than an anti-MUC1 antibody. Further antibodies are approved by the U.S. Food and Drug Administration (FDA), the European Medicines Agency (EMA, formerly EME A), and the Federal Institute for Pharmaceuticals and Medical Devices (BfArM, Bundesinstitut Fur Arzneimittel und Medizinprodukte) etc. It has been approved by the authorities for the treatment of cancer. It can also be used in combination therapy. Examples of suitable antibodies include cetuximab, tomuzotuximab, and paclitaxel. Anti-E, such as nitumumab, zalutumumab, nimotuzumab, matuzumab, and necitumumab GFR antibodies; trastuzumab, timigutuzumab, and Anti-HER2 antibodies such as pertuzumab; anti-VEGF antibodies such as bevacizumab (Avastin) Antibodies; anti-CD52 antibodies such as alemtuzumab (Campath); brentuximab (A anti-CD30 antibodies such as gemtuzumab (Mylotarg); D33 antibody; and rituximab (Rituxan, Mabthera), anti-CD20 antibodies such as Bexxar and ibritumomab (Zevalin) Additional exemplary antibodies suitable for combination with the cancer therapies described herein include: Antibodies against antigens selected from the group consisting of: Thomsen-Friede Tn, Lewis Y, CD44, folate receptor α, NeuG c-GM3 ganglioside, DLL-3, RANKL, PTK7, Notch-3, F Phosphorin A4, insulin-like growth factor receptor 1, activin receptor-like kinase-1, claude tyrosine-6, disialoganglioside GD2, endoglin, transmembrane glycoprotein NMB, CD56, tumor-associated calcium signal transducer 2, tissue factor, ectonuclease Odopyrophosphatase / phosphodiesterase 3, CD70, P-cadherin, mesothelial cell membrane protein Lin, six-transmembrane prostate epithelial antigen 1 (STEAP1), carcinoembryonic antigen-related 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 (SLIT TRK6), trophoblast glycoprotein (TPBG; 5T4), Fyn3, carbonic anhydrase 9, NaPi2b, fibronectin extra domain B tra-domain B), endothelin receptor ETB, VEGFR2 (CD309) , tenascin c, collagen IV, and periostin.

[0137] The conjugates act as checkpoint antibodies, blocking or activating immunoregulatory targets. It can be further combined with an antibody that inhibits the inhibitory signals of the immune response. can inhibit and / or induce activation signals. Examples of targets include CD40, CD3, and CD137 (4-1BB) as activation targets; OX40, GITR, CD27, CD278 (ICOS), CD154 (CD40 Rigan CD270 (HVEM), and CD258 (LIGHT), as inhibitory targets CTLA4, PD1, CD80, CD244, A2AR, B7-H3(CD276), B 7-H4(VTCN1), BTLA, IDO, KIR, LAG3, TIM-3, VIST A, and phosphatidylserine, as well as their corresponding ligands such as PDL1 Examples include:

[0138] In further embodiments, the conjugate is a chemokine, cytokine, growth factor, and In this regard, the present invention may be combined with treatment with immunomodulatory compounds such as steroids and vaccines. Suitable cytokines include interferon-α, interferon-β, and interferon-β. Interferons such as interferon-γ, as well as interleukins. Suitable growth factors include G-CSF and GM-CSF.

[0139] The conjugate is preferably administered to a primary tumor, a recurrent tumor of such a tumor, and / or is used in the treatment of metastases, especially before, during, or after surgery. and for the prevention or treatment of metastasis. The conjugates are particularly useful in adjuvant therapy. In certain embodiments, the conjugate is for treating a patient as As neoadjuvant or combined neoadjuvant-adjuvant therapy Additionally, the conjugates are intended to treat patients as a palliative treatment. It is intended to be used for this purpose.

[0140] Cancer therapy with the conjugate preferably involves the inhibition of tumor growth, and in particular the reduction of tumor size. Furthermore, treatment may prevent the development of further metastases and / or The treatment preferably results in an increase in progression-free survival and / or lifespan. resulting in increased mortality and therefore increased overall survival.

[0141] The present invention relates to the use of conjugates according to the present invention to treat, diagnose, prognose, detect, or Further provided are methods for monitoring and / or determining the activity of conjugates in pharmaceuticals. The embodiments and examples of use also apply to medical methods, especially to patients in need thereof. A method for treating a disease in an animal, comprising administering a therapeutically effective amount of a conjugate according to the present invention to said animal. and administering to an elephant.

[0142] For example, the present invention provides a method for treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the compound of the present invention. and administering to a subject having cancer a therapeutically effective amount of a conjugate according to the present invention. In a specific embodiment, the cancer is characterized by expressing TA-MUC1. Cancers include ovarian cancer, breast cancer, pancreatic cancer, lung cancer, colon cancer, stomach cancer, and liver cancer. , kidney cancer, blood cancer, endometrial cancer, thyroid cancer, leukemia, hemiplegia, melanoma, carcinoma, and rare tumours, lymphomas, sarcomas, mesothelioma, neuroblastoma, glioma, rectal cancer, adrenal cancer, skin Brain cancer, cervical cancer, intestinal cancer, bowel cancer, head and neck cancer, digestive tract cancer, lymph node cancer, Esophageal cancer, colorectal cancer, ear, nose and throat (ENT) cancer, prostate cancer, bladder cancer, uterine cancer, and metastases thereof.

[0143] Additionally, the present invention provides a method for diagnosing, detecting, or monitoring cancer, comprising: A method is provided that includes contacting a test sample with a conjugate according to the present invention.

[0144] Methods for increasing MUC1 binding affinity A method for increasing the MUCI binding affinity of an antibody includes: (i) 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 a heavy chain variable region comprising a CDR-H3 comprising: (ii) a 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. Light chain variable region containing CDR-L3 and The method involves substituting any amino acid residue other than asparagine for the amino acid residue at position 8 of CDR-H2. to provide a CDR-H2 having the amino acid sequence of SEQ ID NO:2. nothing.

[0145] Antibodies that aim to increase MUC1 binding affinity should have a specific amino acid sequence at position 8 of the CDR-H2 sequence. An antibody capable of binding to MUC1, as described herein, but containing a sparagine. It is the body.

[0146] In certain embodiments, the heavy chain variable region of the antibody for which MUC1 binding affinity is to be increased is The region comprises 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 has at least 95%, particularly at least 95%, identical to the amino acid sequence of SEQ ID NO: 11. In such embodiments, the heavy chain variable region comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: still contain CDRs having amino acid sequences of Nos. 1, 8, and 3. Any sequence deviations relative to number 11 are located in the framework regions but not in the CDRs. In particular, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:11.

[0147] In certain embodiments, the light chain variable region of the antibody for which MUC1 binding affinity is to be increased is The region comprises 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 has at least 95%, particularly at least 95%, identical to the amino acid sequence of SEQ ID NO: 12. In such embodiments, the light chain variable region comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: still contain CDRs having amino acid sequences of Nos. 4, 5, and 6. Any sequence deviations relative to number 12 are located in the framework regions but not in the CDRs. In particular, the light chain variable region comprises the amino acid sequence of SEQ ID NO:12.

[0148] In a specific embodiment, the heavy chain variable region of the antibody for which MUC1 binding affinity is to be increased has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 11, and C The DR still has the amino acid sequence of SEQ ID NOs: 1, 8, and 3, and the light chain variable region is having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 12, still have 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 C The DR still has the amino acid sequence of SEQ ID NOs: 1, 8, and 3, and the light chain variable region is having an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 12, still have the amino acid sequences of SEQ ID NOs: 4, 5, and 6.

[0149] For example, antibodies intended to increase MUC1 binding affinity are described in WO2004 / 06542 3A2 or an anti-MUC1 antibody such as those described in WO2011 / 012309A1. Antibodies that attempt to increase MUC1 binding affinity include gatipotuzumab ( uzumab) or PankoMab.

[0150] Antibodies that increase MUC1 binding affinity include, in particular, antibodies that bind to MUC1, as described herein. It is an antibody capable of binding to

[0151] In certain embodiments, the MUC1 binding is as described herein. The increase in C1 binding affinity is particularly at least 10%, at least 20%, at least 33% %, or at least a 50% increase in MUC1 binding affinity. The MUC1 binding affinity is increased by at least 50%. , for example, by surface plasmon resonance analysis or swit as described in Examples 4a and 4b. chSENSE® technology (DRX2 biosensor, Dynamic Bios The determination can be performed using a ion exchange sensor (manufactured by sensors GmbH).

[0152] In certain embodiments, the step of substituting the amino acid residue at position 8 of CDR-H2 comprises: This is achieved by introducing mutations into the nucleic acid encoding the antibody, the mutations The mutation is introduced into the codon encoding the amino acid residue. Several suitable methods are known in the art and will be readily apparent to those skilled in the art. The necessary steps for introducing the mutation can then be carried out. , for example, by expression in a host cell to obtain an antibody with increased MUC1 binding affinity. Nucleic acids, host cells, and methods for producing antibodies are described herein. and can be used for methods of increasing MUC1 binding affinity.

[0153] In a specific embodiment, the method for increasing the MUC1 binding affinity of an antibody comprises: (a) A step of preparing nucleic acid encoding an antibody for which MUC1 binding affinity is to be increased. Top, (b) introducing a mutation into the nucleic acid to produce a mutant nucleic acid, the mutation being in the CDR-H 2, wherein the codon encoding the amino acid residue at position 8 of the introducing the amino acid residues encoding the amino acid sequence; (c) expressing the mutant nucleic acid to produce an antibody with increased MUC1 binding affinity. Includes step.

[0154] A method for producing an antibody with increased MUC1 binding affinity includes: (a) (i) 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. a heavy chain variable region comprising a CDR-H3 that (ii) a 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 light chain variable region containing CDR-L3 having providing a nucleic acid encoding an antibody comprising: (b) introducing a mutation into the nucleic acid to produce a mutant nucleic acid, the mutation being in the CDR-H 2, wherein the codon encoding the amino acid residue at position 8 of the introducing the amino acid residues encoding the amino acid sequence; (c) expressing the mutant nucleic acid in a host cell increases MUC1 binding affinity. Producing the isolated antibody may also include:

[0155] For other embodiments, particularly for methods of increasing the MUC1 binding affinity of an antibody, the present invention The embodiments, features, and examples described herein relate to antibodies with increased MUC1 binding affinity. The same applies to methods of producing bodies.

[0156] In certain embodiments, the method for producing an antibody with increased MUC1 binding affinity comprises: The method further comprises a step (d) of treating the antibody with increased UC1 binding affinity.

[0157] For example, treatment of an antibody with increased MUC1 binding affinity can be performed by isolating the antibody from a cell culture. Isolating the antibody may include, inter alia, separating the antibody from the remaining components of the cell culture. The separation of antibodies from the cell culture medium can be carried out, for example, by chromatographic methods. Suitable methods and means for isolating antibodies are known in the art. This is well known and can be easily applied by those skilled in the art.

[0158] Optionally, the resulting antibody can be further modified, for example by chemically or enzymatically injecting an additional agent into the antibody. and / or modification steps such as coupling to produce antibodies of desired quality and composition. The compound may be subjected to further processing steps, such as formulation steps for regeneration. The processing steps and methods are generally known in the art.

[0159] In a further embodiment, step (d) comprises providing a pharmaceutical formulation comprising the antibody. Further included are providing a pharmaceutical formulation comprising the antibody or formulating the antibody as a pharmaceutical composition. This may include, inter alia, exchanging the buffer or buffer components of the antibody-containing composition. Additionally, this step may include lyophilizing the antibody. is transferred into a composition containing only pharmaceutically acceptable ingredients.

[0160] Production method 1 An antibody-drug conjugate represented by formula (1) shown below, wherein the antibody The antibody-drug conjugate is connected to the linker structure via a thioether. Antibodies with sulfhydryl groups converted from disulfide bonds by reducing the antibody can be obtained by known methods (see, for example, patent publications, U.S. Patent Application No. 2016 The method described in the specification of / 297890 (for example, the method described in paragraphs

[0336] to

[0374] ) (2) (which can be obtained by the method (a) above) with This antibody-drug conjugate can be produced, for example, by the following method. Cut.

[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 of the following formula:

[0162] [ka] It represents a maleimidyl group represented by the following formula:

[0163] -L1-LX has a structure represented by any 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-CH2C H2CH2-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 following are more preferred: -(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-CH2C H2CH2-C(=O)-

[0165] More preferred 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--CH2 CH2CH2-C(=O)-

[0166] (NH-DX) has the following formula:

[0167] [ka] and having a structure represented by It is derived by removing one hydrogen atom from the amino group at position 1 of exatexicane. In the reaction scheme (formula 8) described above, the compound of formula (1) is and a structure in which one structural portion from the end of the linker to the end of the linker is connected to one antibody. However, this explanation is provided for convenience and may be used in multiple ways. In reality, there are many cases where the above structural parts are connected to one antibody molecule. The same applies to the description of the production method given below.

[0168] Specifically, the antibody-drug conjugate (1) can be prepared by known methods (e.g., see patent publications). Compound (2), which can be obtained by the method described in U.S. Patent Application No. 2016 / 297890 (for example, the method described in paragraphs

[0336] to

[0374] ), is treated with a sulfhydryl compound (2). The antibody (3a) can be produced by reacting the antibody (3a) with an aryl group.

[0169] Provision of sulfhydryl groups to antibody (3a) can be achieved by methods well known to those skilled in the art. (Hermanson, GT, Bioconjugate Technique es, pp. 56-136, pp. 456-493, Academic Press(1 996)). Examples of methods include, but are not limited to, the following: Reaction of Raut's reagent with the amino groups of antibodies; N-succinimidyl S-acetylthiol Reaction of alkanoates with the amino groups of antibodies, followed by reaction with hydroxylamine N-succinimidyl 3-(pyridyldithio)propionate was reacted with the antibody. followed by reaction with a reducing agent; or reduced compounds such as Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) The antibody is reacted with a denaturing agent to break down the interchain disulfide bonds of the antibody so that sulfhydryl groups are formed. To give back.

[0170] Specifically, antibodies in which the interchain disulfide bonds have been partially or completely reduced are 0.3 to 3 molar equivalents of TCEP per interchain disulfide bond was used as a reducing agent. It can be obtained by reacting a reducing agent with an antibody in a buffer containing a chelating agent. Examples of retarders include ethylenediaminetetraacetic acid (EDTA) and diethylenetriamine. Chelating agents include dibenzodibenzofuran (DTPA) and dibenzofuran (DTPA). Chelating agents are used at concentrations of 1 mM to 20 mM. Other suitable solutions include sodium phosphate, sodium borate, or sodium acetate. Any solution can be used as a buffer solution. Antibody (3a) with fully reduced sulfhydryl groups was incubated for 1-4 h at 4°C to 37°C. It can be obtained by reacting the antibody with TCEP for a period of time.

[0171] By carrying out the addition reaction of the sulfhydryl group to the drug-linker moiety, the drug -It is noted that the linker moiety can be conjugated with a thioether bond It should be.

[0172] Then, 2 to 20 molar equivalents of the compound per one sulfhydryl-containing antibody (3a) (2) to prepare antibodies conjugated with 2 to 8 drug molecules per antibody. The drug conjugate (1) can be produced. Specifically, A solution containing compound (2) is added to a buffer solution containing antibody (3a) having a sulfhydryl group. In this situation, sodium acetate solution, sodium phosphate, or Sodium borate can be used as a buffer solution. The pH for the reaction is The reaction may be carried out at a pH of 5 to 9, more preferably around 7. dimethylformamide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMA), Alternatively, an organic solvent such as N-methyl-2-pyrrolidone (NMP) may be used to dissolve compound (2). The reaction can be carried out in an organic solvent at a concentration of 1-20% by volume. A solution containing the dissolved compound (2) was added to a solution containing an antibody (3a) having a sulfhydryl group. The reaction may be carried out by adding the compound to a buffer solution containing the compound. The reaction temperature is 0 to 37°C, more preferably The reaction temperature is about 10 to 25°C, and the reaction time is 0.5 to 2 hours. The reactivity of 2) with thiol-containing reagents can be terminated by inactivating the thiol. The ol-containing reagent is, for example, cysteine ​​or N-acetyl-L-cysteine ​​(NAC). More specifically, the reaction involves adding 1 to 2 molar equivalents of NAC to the compound ( 2) and incubate the resulting mixture at room temperature for 10 to 30 minutes. It can be terminated by

[0173] Identification of antibody-drug conjugates The produced antibody-drug conjugate (e.g., antibody-drug conjugate (1)) Concentrate, buffer exchange, purify, and concentrate the antibody to 100% RI according to the common procedures described below. The drug-antibody conjugates were subjected to a measurement of the average number of drug molecules conjugated per antibody molecule. It is possible to identify the conjugate (1).

[0174] 1. Common Procedure A: Concentration of Aqueous Solutions of Antibody or Antibody-Drug Conjugate Amicon Ultra (50,000MWCO, Millipore Corp. Add a solution of antibody or antibody-drug conjugate to a ration container and The antibody-drug conjugate solution was centrifuged (Allegra X-15R, Be Centrifuge (2000G-4000G) using a centrifuge (Beckman Coulter, Inc.). The mixture was concentrated by centrifugation at 40°C for 5 to 30 minutes.

[0175] 2. Common Procedure B: Measurement of antibody concentration UV detector (Nanodrop 1000, Thermo Fisher Scientific) Antibody concentrations were measured using a kit (Protein Biosciences, Inc.) according to the method specified by the manufacturer. Measurements were carried out using different 280 nm extinction coefficients for the antibodies (1.3 mL). mg -1 cm -1 ~1.8mLmg -1 cm -1 ).

[0176] 3. Common Procedure C: Buffer Exchange of Antibodies NAP-25 columns (catalog no. 1) are made with Sephadex G-25 support. 17-0852-02, GE Healthcare Japan Corporati on) was dissolved in sodium chloride (50 mM) and Phosphate buffer (50 mM, pH 6.0) containing EDTA (2 mM) (referred to herein as PBS The antibody solution was then loaded onto one NAP-25 column. Apply 2.5 mL per column, then elute with 3.5 mL of PBS 6.0 / EDTA. The fraction (3.5 mL) was collected and concentrated according to the common procedure A. After measuring the antibody concentration using Procedure B, use PBS6.0 / EDTA to The concentration was adjusted to 20 mg / mL.

[0177] 4. Common Procedure D: Purification of Antibody-Drug Conjugates The NAP-25 column was loaded with acetate buffer (10 mM, pH 5.0) containing sorbitol (5%). The antibody-drug mixture was equilibrated with any commercially available buffer, such as PBS (5; herein referred to as ABS). The aqueous reaction solution of the product conjugate (approximately 2.5 mL) was applied to a NAP-25 column. Then, elution was performed with the amount of buffer specified by the manufacturer to collect the antibody fraction. The collected fractions were reapplied to the NAP-25 column and eluted with a buffer solution. The filter filtration purification step was repeated a total of two or three times to remove unconjugated drug linker and and low molecular weight compounds (tris(2-carboxyethyl)phosphine hydrochloride (TCEP) , N-acetyl-L-cysteine ​​(NAC), and dimethyl sulfoxide) -drug conjugates were obtained.

[0178] 5. Common Procedure E: Antibody Concentration and Conjugate Per Antibody Molecule of Antibody-Drug Conjugates Determination of the average number of conjugated drug molecules The conjugated drug concentration of the antibody-drug conjugate is determined by the The UV absorbance of the aqueous solution of acetone was measured at two wavelengths, 280 nm and 370 nm, and then This can be calculated by performing the calculations shown below.

[0179] The total absorbance at any given wavelength is the sum of the absorbances of all light-absorbing species present in the system. Therefore, the molar extinction coefficients of the antibody and drug are equal to the sum of the absorbances [additive absorbances]. Based on the hypothesis that the antibody-drug conjugates do not change before and after conjugation with the drug, The antibody and drug concentrations in the drugate are expressed by the following equations: A 280 =A D,280 +A A,280 =ε D,280 C D +ε A,280 C A etc. Formula (1) A 370 =A D,370 +A A,370 =ε D,370 C D +ε A,370 C A etc. Formula (2)

[0180] In this situation, A 280 is the absorbance at 280 nm of an aqueous solution of an antibody-drug conjugate represents luminous intensity, A 370 is the absorbance at 370 nm of an aqueous solution of the antibody-drug conjugate. Represents A A,280 represents the absorbance of the antibody at 280 nm, and A A,370 is 370n represents the absorbance of the antibody in m, A D,280 is the absorbance of the conjugate precursor at 280 nm represents luminous intensity, A D,370 represents the absorbance of the conjugate precursor at 370 nm, and ε A,280 represents the molar extinction coefficient of the antibody at 280 nm, and ε A,370 is 370nm represents the molar extinction coefficient of the antibody at 1000 kJ / s, and ε D,280 is the conjugate precursor at 280 nm represents the molar extinction coefficient, ε D,370 is the molar absorbance of the conjugate precursor at 370 nm represents the optical coefficient, C A represents the antibody concentration of the antibody-drug conjugate, and C D antibody-drug The drug concentration of the monoconjugate is shown.

[0181] In this situation, ε A,280 , ε A,370 , ε D,280 , and ε D,370 Regarding For example, preliminary values ​​(calculated estimates or UV measurements of the compound) For example, ε A,280 is calculated by a known method (Protein Science, 1995, Vol. 4, pp. 2411-2423) It can be deduced from the amino acid sequence. A,370 is generally zero. D,280 ,oh Yobi ε D,370 The conjugate precursor used is dissolved in a certain molar concentration. By measuring the absorbance of the solution containing the compound, we can obtain the Beer-Lambert law (absorbance = molar concentration). × molar extinction coefficient × cell path length) C A and C D antibody-drug Aqueous solution of the product conjugate 280 and A 370 and then assign these values can be determined by solving the simultaneous equations (1) and (2) by Furthermore, C D C A Conjugated drug per antibody by dividing by The average number of molecules can be determined.

[0182] 6. Common Procedure F: Conjugated Antibody-Drug Conjugates per Antibody Molecule Measurement of the average number of drug molecules (2) Also, the number of conjugated drug molecules per antibody molecule of the antibody-drug conjugate In addition to the above "5. Common Procedure E," the average number of This can be determined by high performance liquid chromatography (HPLC) analysis. When conjugated to a drug linker by a bond, the conjugated drug molecule The method for determining the average number by HPLC is described. If you are a pharmaceutical company, you can select the conjugated drug according to the connection pattern between the antibody and the drug linker. The average number of molecules can be suitably determined by HPLC.

[0183] F-1. Preparation of Samples for HPLC Analysis (Reduction of Antibody-Drug Conjugates) The antibody-drug conjugate solution (approximately 1 mg / mL, 60 μL) was diluted with dithiothreitol. The mixture was mixed with an aqueous solution of diethyl ether (DTT) (100 mM, 15 μL). The mixture was then incubated at 37°C for 30 minutes. By incubating for 1 hour, dissociation between the light and heavy chains of the antibody-drug conjugate was observed. The sulfide bond is cleaved, and the resulting sample is used for HPLC analysis.

[0184] F-2. HPLC analysis The HPLC analysis is carried out under the following measurement conditions: HPLC equipment: Agilent 1290 HPLC equipment (Agilent Techn ologies, Inc.) Detector: UV absorption spectrometer (measurement wavelength: 280 nm) Column: ACQUITY UPLC BEH Phenyl (2.1 x 50 mm, 1. 7 μm, 130 Å; Waters Corp., P / N 1860028 84) Column temperature: 80℃ Mobile phase A: Contains 0.10% trifluoroacetic acid (TFA) and 15% 2-propanol. aqueous solution Mobile phase B: Acetonitrile containing 0.075% TFA and 15% 2-propanol liquid Gradient program: 14% → 36% (0 min to 15 min), 36% → 80% (15 min to 1 7 min), 80% → 14% (17 min to 17.01 min), and 14% (17.01 min to 25 minutes) Sample injection: 10 μL or HPLC equipment: Agilent 1290 HPLC equipment (Agilent Techn ologies, Inc.) Detector: UV absorption spectrometer (measurement wavelength: 280 nm) Column: PLRP-S (2.1 × 50 mm, 8 μm, 1000 Å; Ag ilent 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) minutes to 15 minutes), 42% to 29% (15 minutes to 15.1 minutes), and 29% to 29% (1 5.1 to 25 minutes) Sample injection: 15 μL

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

[0186] Compared to unconjugated antibody light chain (L0) and heavy chain (H0), one drug molecule Light chain (L1) bound to one drug molecule, heavy chain (H1) bound to two drug molecules The heavy chain bound to the hydroxylase (H2), and the heavy chain bound to three drug molecules (H3) form the conjugate. The more drug molecules gated, the more hydrophobic they are and therefore the longer their residence time. Thus, these chains are L0 and L1 or H0, H1, H2 and The detected peaks are identified by comparing the retention times of L0 and H0. Therefore, it can be assigned to any of L0, L1, H0, H1, H2, and H3. .

[0187] F-3-2. Because the drug linker absorbs UV, the light or heavy chain and the drug linker The molar extinction coefficient of the conjugated drug-linker molecule is calculated according to the following formula: Correct the peak area value according to the number of

[0188] [Formula 1] The corrected peak area of ​​the light chain bound to i drug molecules (A Li )

[0189]

number

[0190] [Formula 2] The corrected peak area of ​​the light chain bound to i drug molecules (A Hi )

[0191]

number

[0192] In this situation, the known calculation method (Protein Science, 1995, Vol. 4) , pp. 2411-2423) from the amino acid sequence of the light or heavy chain of each antibody. The value obtained can be used as the molar extinction coefficient (280 nm) of the antibody light or heavy chain. Each drug linker can be reacted with mercaptoethanol or N-acetylcysteine. The maleimide group was converted to a succinimide thioether by the addition of methyl groups. The determined molar extinction coefficient (280 nm) was compared with the molar extinction coefficient (280 nm) of the drug linker. The wavelength for measuring absorbance can be appropriately determined by a person skilled in the art. Preferably, it is a wavelength at which the antibody peak can be measured, more preferably 280 nm. m.

[0193] F-3-3. To sum the peak area correction values, calculate the peak area ratio (%) of each chain as follows: It is calculated according to the formula:

[0194] [Formula 3]

[0195]

number

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

[0197] To ensure the amount of conjugate, almost identical samples were produced under similar conditions. Multiple conjugates having an average number of conjugated drug molecules (e.g., on the order of ±1) It should be noted that gates can be mixed to prepare new lots. In this case, the average number of drug molecules falls between the average numbers of drug molecules before mixing.

[0198] Specific Embodiments Below are described specific embodiments of the antibody portion of the conjugate according to the invention. do.

[0199] Embodiment 1. An antibody capable of binding to MUC1, comprising: (i) 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 a heavy chain variable region comprising a CDR-H3 comprising: (ii) a 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. Light chain variable region containing CDR-L3 An antibody comprising:

[0200] Embodiment 2. The amino acid at position 8 of CDR-H2 is selected from glutamine, alanine, valine, and hyaluronan. stidine, tryptophan, tyrosine, lysine, and arginine, especially glutamine, selected from the group consisting of stigmine, tryptophan, tyrosine, lysine, and arginine 2. The antibody of embodiment 1, wherein the amino acid sequence is glutamine.

[0201] Embodiment 3. The amino acid at position 8 of CDR-H2 is glutamine, histidine, arginine 2. The antibody of embodiment 1, wherein the amino acid sequence is phenylalanine, tryptophan, or lysine.

[0202] Embodiment 4. The antibody of embodiment 1, wherein CDR-H2 has the amino acid sequence of SEQ ID NO: 7. 3. An antibody described in any one of claims 2 to 3.

[0203] Embodiment 5. The method of embodiment 1, wherein CDR-H2 has the amino acid sequence of SEQ ID NO: 8. The antibodies listed.

[0204] Embodiment 6. An antibody capable of binding to MUC1, comprising: (i) (a) having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 9 , (b) 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. a heavy chain variable region comprising a CDR-H3 that (ii) (a) a nucleic acid sequence having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 12; death, (b) a 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. Light chain variable region containing CDR-L3 An antibody comprising:

[0205] Embodiment 7. An antibody capable of binding to MUC1, comprising: (i) (a) having an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 9 , (b) 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. a heavy chain variable region comprising a CDR-H3 that (ii) (a) a nucleic acid having an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 12; death, (b) a 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. Light chain variable region containing CDR-L3 An antibody comprising:

[0206] Embodiment 8. The amino acid at position 8 of CDR-H2 is selected from glutamine, alanine, valine, hyaluronan, and hydroxylase. stidine, tryptophan, tyrosine, lysine, and arginine, especially glutamine, selected from the group consisting of stigmine, tryptophan, tyrosine, lysine, and arginine 8. The antibody of embodiment 6 or 7, wherein the amino acid sequence is glutamine.

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

[0208] Embodiment 10. An antibody capable of binding to MUC1, comprising: (i) (a) a nucleic acid sequence having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 10; death, (b) 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. a heavy chain variable region comprising a CDR-H3 that (ii) (a) a nucleic acid sequence having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 12; death, (b) a 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. Light chain variable region containing CDR-L3 An antibody comprising:

[0209] Embodiment 11. An antibody capable of binding to MUC1, comprising: (i) (a) a nucleic acid sequence having an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 10; death, (b) 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. a heavy chain variable region comprising a CDR-H3 that (ii) (a) a nucleic acid having an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 12; death, (b) a 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. Light chain variable region containing CDR-L3 An antibody comprising:

[0210] Embodiment 12. An antibody capable of binding to MUC1, comprising: (i) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 9, and (ii) a light chain variable region having the amino acid sequence of SEQ ID NO: 12 An antibody comprising:

[0211] Embodiment 13. The amino acid at position 57 of SEQ ID NO: 9 is selected from glutamine, alanine, valine, histidine, tryptophan, tyrosine, lysine, and arginine, especially glutamine; Selected from the group consisting of histidine, tryptophan, tyrosine, lysine, and arginine 13. The antibody of embodiment 12, wherein the amino acid sequence is a nucleotide sequence selected from the group consisting of methyl, methyl, propyl, methyl ...

[0212] Embodiment 14. The amino acid at position 57 of SEQ ID NO: 9 is selected from glutamine, histidine, arginine, and arginine. 13. The antibody of embodiment 12, wherein the amino acid sequence is nin, tryptophan, or lysine.

[0213] Embodiment 15. An antibody capable of binding to MUC1, comprising: (i) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 10, and (ii) a light chain variable region having the amino acid sequence of SEQ ID NO: 12 An antibody comprising:

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

[0215] Embodiment 17. An antibody capable of binding to MUC1, comprising: (i) (a) at least 90% or at least 95% identical to the amino acid sequence of SEQ ID NO: 15 and having an amino acid sequence (b) 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 the amino acid sequence of SEQ ID NO: 3 a heavy chain comprising a CDR-H3 having the sequence: (ii) (a) at least 90% or at least 95% identical to the amino acid sequence of SEQ ID NO: 16 and having an amino acid sequence (b) a 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. Light chain variable region containing CDR-L3 An antibody comprising:

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

[0217] Embodiment 19. An antibody capable of binding to MUC1, comprising: (i) (a) at least 90% or at least 95% identical to the amino acid sequence of SEQ ID NO: 19 and having an amino acid sequence (b) 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. a heavy chain variable region comprising a CDR-H3 that (ii) (a) at least 90% or at least 95% identical to the amino acid sequence of SEQ ID NO: 16 and having an amino acid sequence (b) a 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. Light chain variable region containing CDR-L3 An antibody comprising:

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

[0219] Embodiment 21. A heavy chain variable region, a CH1 domain, a hinge region, a CH2 domain, and 21. Any one of embodiments 1 to 20, comprising at least one heavy chain comprising a CH3 domain and a CH4 domain. The antibody according to item 1.

[0220] Embodiment 22. Each of a heavy chain variable region, a CH1 domain, a hinge region, and a CH2 domain 21. The method of any one of embodiments 1 to 20, comprising two heavy chains comprising a CH3 domain, and a CH4 domain. The antibody described.

[0221] Embodiment 23. IgG type antibody, particularly IgG1 type antibody, IgG2 type antibody, or IgG 23. The antibody of embodiment 21 or 22, which is a type 4 antibody.

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

[0223] Embodiment 25. A human immunodeficiency virus (HIV) antibody comprising two light chains, each comprising a light chain variable region and a CL domain. 24. The antibody of any one of embodiments 1 to 23.

[0224] Embodiment 26 The antibody of embodiment 24 or 25, wherein the light chain is a kappa light chain.

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

[0226] Embodiment 28. An embodiment in which the CH2 domain of the antibody heavy chain comprises an N-glycosylation site 27. An antibody described in any one of 1 to 26.

[0227] Embodiment 29. The following features: (i) The relative amount of glycans bearing a bisecting GlcNAc residue determines the glycosylation of antibodies in the composition. at least 0.5% of the total amount of glycans attached to the sylation site; (ii) the relative amount of glycans carrying at least one galactose residue in the composition; at least 30% of the total amount of glycans attached to the glycosylation sites of the antibody thing, (iii) The relative amount of glycans bearing core fucose residues is determined by the glycosylation of the antibodies in the composition. At least 60% of the total amount of glycans attached to the sylation site 29. The antibody of embodiment 28, having a glycosylation pattern having one or more of: .

[0228] Embodiment 30. The following features: (i) The relative amount of glycans bearing a bisecting GlcNAc residue determines the glycosylation of antibodies in the composition. at least 0.5% of the total amount of glycans attached to the sylation site; (ii) the relative amount of glycans carrying at least one galactose residue in the composition; at least 30% of the total amount of glycans attached to the glycosylation sites of the antibody thing, (iii) The relative amount of glycans bearing core fucose residues is determined by the glycosylation of the antibodies in the composition. 40% or less of the total amount of glycans attached to sylation sites 29. The antibody of embodiment 28, having a glycosylation pattern having one or more of: .

[0229] Below are described specific embodiments of the conjugates according to the invention.

[0230] Embodiment 31. A further agent, preferably a cell injury agent, conjugated thereto. 31. The antibody of any one of embodiments 1 to 30, comprising an anti-inflammatory agent.

[0231] Embodiment 32. A method in which the cytotoxic agent is a chemotherapeutic agent coupled to an antibody. The antibody of embodiment 31.

[0232] Embodiment 33. The chemotherapeutic agent is a microtubule inhibitor, such as a maytansinoid, a topoisomerase inhibitor, or a chemotherapeutic agent. A group consisting of enzyme I inhibitors, DNA damaging agents, DNA alkylating agents, and DNA minor groove binders 33. The antibody of embodiment 32, selected from:

[0233] Embodiment 34. The chemotherapeutic agent is maytansinol, N2'-deacetyl-N2'-( 3-mercapto-1-oxopropyl)-maytansine (DM1), N2'-deacetyl -N2'-(4-mercapto-1-oxopentyl)-maytansine (DM3), and N2'-Deacetyl-N2'-(4-methyl-4-mercapto-1-oxopentyl)- 33. The antibody of embodiment 32, selected from the group consisting of maytansine (DM4).

[0234] Embodiment 35. The chemotherapeutic agent is a pyrrolobenzodiazepine (PBD), a pyrrolobenzodiaze ... Azepine dimer (PBD dimer), duocarmycin, duocarmycin-hydroxy Dibenzamide-azaindole (DUBA), seco-duocarmycin-hydro From hydroxybenzamide-azaindole (seco-DUBA) and doxorubicin 33. The antibody of embodiment 32, selected from the group consisting of:

[0235] Embodiment 36. The chemotherapeutic agent is an indolinobenzodiazepine or an oxazolidinone. 33. The antibody of embodiment 32, wherein the antibody is selected from the group consisting of benzodiazepines.

[0236] Embodiment 37. The antibody of embodiment 32, wherein the chemotherapeutic agent is calicheamicin. .

[0237] Embodiment 38. The chemotherapeutic agent is camptothecin, 7-ethyl-10-hydroxy- Amputatecin (SN-38), (S)-9-dimethylaminomethyl-10-hydroxyca Amphothecin (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]indolizino[1,2-b]chino Phosphorus-10,13-dione (exatexicane (DX-8951)), and DXd 33. The antibody of embodiment 32, wherein the antibody is selected from the group consisting of:

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

[0239] [ka] 33. The antibody of embodiment 32, wherein the antibody is an anti-tumor compound represented by

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

[0241] [ka] 33. The antibody of embodiment 32, wherein the antibody is an anti-tumor compound represented by

[0242] Embodiment 41. Additionally, the additional agent, which is a polypeptide or protein, is an anti- 32. The antibody of embodiment 31, wherein the antibody is fused to a polypeptide chain of the same kind.

[0243] Embodiment 42. A polypeptide or tag comprising two antibody heavy chains and two antibody light chains. The additional agent, which is a protein, is attached to the C-terminus of each of the antibody heavy chains or the antibody light chain. 42. The antibody of embodiment 41, wherein the antibody is fused to the C-terminus of each of:

[0244] Embodiment 43. The additional agent is a cytokine, a chemokine, another antibody, an antigen-binding 43. The method of claim 41, wherein the nucleotide sequence of the present invention is selected from the group consisting of a functional fragment, an enzyme, and a binding domain. The antibody described in

[0245] Embodiment 44. The additional agent is an scFv fragment that specifically binds to CD3, 43. The method of embodiment 42, wherein one of the additional agents is fused to the C-terminus of each antibody heavy chain. The antibodies listed.

[0246] Embodiment 45. The further agent is an scFv fragment that specifically binds to PDL1. 43. The method of claim 42, wherein one of the additional agents is fused to the C-terminus of each antibody light chain. The antibody described.

[0247] Embodiment 46. A cytotoxic agent, preferably a topoisomerase inhibitor such as DX-8951 or DXd. Somerset I inhibitors are represented by the following formulae (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-N H-CH2CH2CH2-C(=O)-, and (f)-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2 CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)- and conjugated thereto via a linker having any structure selected from the group consisting of It is being The antibody is attached to the -(Succinimid-3-yl-N) terminal, and the antitumor compound is attached to the 1-amino The nitrogen atom of the -(CH2)n group is the connecting point, and the rightmost -(CH2)n 2 -C (=O)-part(n 2 represents an integer of 1 or 3), GGF is a glycine-glycine-phenylalanine amino acid that is linked via a peptide bond. represents the amino acid sequence consisting of guanine-glycine, -(Succinimid-3-yl-N)- is a compound of the formula:

[0248] [ka] and is connected to the antibody at its 3-position and has a structure represented by the following formula: is connected to a methylene group of a linker structure comprising the structure 46. ​​The antibody of any one of embodiments 31 to 45.

[0249] Embodiment 47. Any of embodiments 1 to 30 conjugated to a cytotoxic agent. A conjugate comprising the antibody described in any one of claims 1 to 4.

[0250] Embodiment 48. The conjugate of embodiment 47, wherein the cytotoxic agent is a chemotherapeutic agent. Route.

[0251] Embodiment 49. The chemotherapeutic agent is a microtubule inhibitor, a topoisomerase I inhibitor, a DNA damage inhibitor. an embodiment selected from the group consisting of a DNA damaging agent, a DNA alkylating agent, and a DNA minor groove binding agent. 49. The conjugate according to embodiment 48.

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

[0253] Embodiment 51. The chemotherapeutic agent is a pyrrolobenzodiazepine (PBD), a pyrrolobenzodiaze ... Azepine dimer (PBD dimer), duocarmycin, duocarmycin-hydroxy Dibenzamide-azaindole (DUBA), seco-duocarmycin-hydro From hydroxybenzamide-azaindole (seco-DUBA) and doxorubicin 49. The conjugate of embodiment 48, selected from the group consisting of:

[0254] Embodiment 52. The chemotherapeutic agent is an indolinobenzodiazepine or an oxazolidinone. 49. The conjugate of embodiment 48, wherein the conjugate is selected from the group consisting of benzodiazepines.

[0255] Embodiment 53. The composition of embodiment 48, wherein the chemotherapeutic agent is calicheamicin. Jugate.

[0256] Embodiment 54. The chemotherapeutic agent is camptothecin, 7-ethyl-10-hydroxy- Amputatecin (SN-38), (S)-9-dimethylaminomethyl-10-hydroxyca Amphothecin (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]indolizino[1,2-b]chino Phosphorus-10,13-dione (exatexicane (DX-8951)), and DXd 49. The conjugate of embodiment 48, wherein the conjugate is selected from the group consisting of:

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

[0258] [ka] 49. The conjugate of embodiment 48, wherein the antitumor compound is represented by

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

[0260] [ka] 49. The conjugate of embodiment 48, wherein the antitumor compound is represented by

[0261] Embodiment 57. The additional agent that is a polypeptide or protein is a polypeptide of an antibody. 48. The conjugate of embodiment 47, which is fused to a peptide chain.

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

[0263] Embodiment 59. The additional agent is a cytokine, a chemokine, another antibody, an antigen-binding 59. The method of claim 57 or 58, wherein the nucleotide sequence is selected from the group consisting of a functional fragment, an enzyme, and a binding domain. The conjugate according to claim 1.

[0264] Embodiment 60. The additional agent is an scFv fragment that specifically binds to CD3, 59. The method of embodiment 58, wherein one of the additional agents is fused to the C-terminus of each antibody heavy chain. Conjugates listed above.

[0265] Embodiment 61. The further agent is an scFv fragment that specifically binds to PDL1. 59. The method of embodiment 58, wherein one of the additional agents is fused to the C-terminus of each antibody light chain. The conjugate described herein.

[0266] Embodiment 62. The antibody has 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-N H-CH2CH2CH2-C(=O)-, and (f)-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2 CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)- and a linker having any structure selected from the group consisting of: is a chemotherapeutic agent, preferably a topoisomerase I inhibitor such as DX-8951 or DXd It is conjugated to The antibody is attached to the -(Succinimid-3-yl-N) terminal, and the antitumor compound is attached to the 1-amino The nitrogen atom of the -(CH2)n group is the connecting point, and the rightmost -(CH2)n 2 -C (=O)-part(n 2 represents an integer of 1 or 3), GGF is a glycine-glycine-phenylalanine amino acid that is linked via a peptide bond. represents the amino acid sequence consisting of guanine-glycine, -(Succinimid-3-yl-N)- is a compound of the formula:

[0267] [ka] and is connected to the antibody at its 3-position and has a structure represented by the following formula: is connected to a methylene group of a linker structure comprising the structure 62. The conjugate according to any one of embodiments 47 to 61.

[0268] Embodiment 63. The antibody comprises a heavy chain variable region and a light chain variable region, or a heavy chain and a light chain variable region. Combinations a) to f) below: (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: having the amino acid sequence of 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: having the amino acid sequence of No. 12; (c) a heavy chain comprising the amino acid sequence of SEQ ID NO: 15 or 22, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 16 a light chain comprising the amino acid sequence (d) a heavy chain comprising the amino acid sequence of SEQ ID NO: 19, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 16 light chains, including (e) an amino acid sequence represented by amino acid numbers 1 to 446 of SEQ ID NO: 15 or 22 and a heavy chain having an amino acid sequence represented by amino acid numbers 1 to 219 of SEQ ID NO: 16. a light chain having a sequence, and (f) a heavy chain having an amino acid sequence represented by amino acid numbers 1 to 446 of SEQ ID NO: 19; and an amino acid sequence represented by amino acid numbers 1 to 219 of SEQ ID NO: 16. Light chain and wherein the antibody or antigen-binding fragment thereof has the following formula: * teeth , which represents the attachment point to the antibody)

[0269] [ka] and the drug linker is conjugated via a thioether bond to a drug linker represented by 62. The conjugate of any one of embodiments 47 to 61.

[0270] Embodiment 64. A compound of the formula:

[0271] [ka] is represented by wherein AB represents an antibody, the 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, wherein y is a conjugate of the antibody. represents the average number of condensed drug-linker units per antibody, and y is a number between 1 and range from 10 to 10, range from 2 to 8, range from 3 to 8, range from 7 to 8, or or 7.5 to 8, and the antibody is linked to a drug-linker structure represented by the above formula. A conjugate or antibody that is conjugated with a thioether bond.

[0272] Embodiment 65. A compound of the formula:

[0273] [ka] is represented by wherein AB represents an antibody, the 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, wherein y is a conjugate of the antibody. represents the average number of condensed drug-linker units per antibody, and y is a number between 1 and range from 10 to 10, range from 2 to 8, range from 3 to 8, range from 7 to 8, or or 7.5 to 8, and the antibody is linked to a drug-linker structure represented by the above formula. A conjugate or antibody that is conjugated with a thioether bond.

[0274] Embodiment 66. A compound of the formula:

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

[0276] Embodiment 67. A compound of the formula:

[0277] [ka] is represented by wherein AB represents an antibody, the antibody comprising 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, and y is a drug conjugated to the antibody. represents the average number of units of the mono-linker structure per antibody, and y ranges from 1 to 10. , 2 to 8 range, 3 to 8 range, 7 to 8 range, or 7.5 to 8 and the antibody is linked to the drug linker structure represented by the formula above by a thioether bond. A conjugate or antibody conjugated with

[0278] Embodiment 68. The antibody is defucosylated, fucose-reduced, N-linked glycosylated, O- Linkage glycosylation, N-terminal processing, C-terminal processing, deamidation, asparagi isomerization of phosphate, oxidation of methionine, and two leucines at positions 234 and 235 of the heavy chain. Substitution of (L) residues with alanine (A) (LALA), amidation of proline residues, and a deletion or absence of one or two amino acids at the carboxyl terminus 68. The conjugate of any one of embodiments 31 to 67, comprising one or more modifications. ate or antibody.

[0279] Embodiment 69. The antibody has a deletion of one or two amino acids at the carboxyl terminus of the heavy chain. or the absence thereof.

[0280] Embodiment 70. The antibody comprises two nucleotides, both of which lack one carboxyl-terminal amino acid residue. 70. The conjugate or antibody of embodiment 69, comprising a heavy chain of:

[0281] Embodiment 71. A compound of the formula:

[0282] [ka] is represented by In the formula, AB represents an antibody, and the antibody is an antibody having amino acid numbers 1 to 446 of SEQ ID NO: 15 or 22. and amino acid numbers 1 to 21 of SEQ ID NO: 16. 9, and y is a light chain conjugated to the antibody represents the average number of drug-linker structural units per antibody, and y ranges from 1 to 10. range, 2 to 8 range, 3 to 8 range, 7 to 8 range, or 7.5 to 8, and the antibody is linked to the drug linker structure represented by the formula above by a thioether bond. A conjugate or antibody conjugated with a nucleotide sequence.

[0283] Embodiment 72. A compound of the formula:

[0284] [ka] is represented by AB represents an antibody, which is represented by amino acid numbers 1 to 446 of SEQ ID NO: 19. a heavy chain having an amino acid sequence represented by amino acid numbers 1 to 219 of SEQ ID NO: 16; and y is a drug-linker conjugated to the antibody. represents the average number of structural units per antibody, y ranging from 1 to 10, 2 to 8, in the range of 3 to 8, in the range of 7 to 8, or in the range of 7.5 to 8 wherein the antibody is conjugated to the drug linker structure represented by the formula above via a thioether bond. Conjugates or antibodies that are

[0285] Embodiment 73. The number of conjugated drug molecules per antibody molecule is 8. 73. The conjugate or antibody of any one of embodiments 31 to 72.

[0286] Embodiment 74. The antibody or conjugate of any one of embodiments 31 to 73. and one or more selected from the group consisting of solvents, diluents, and excipients. A pharmaceutical composition comprising additional ingredients.

[0287] Embodiment 75. Any one of embodiments 31 to 73 for use in medicine. or the pharmaceutical composition of embodiment 74.

[0288] Embodiment 76. Diseases associated with abnormal cell growth, such as cancer; bacterial infections, viral infections infections, such as rheumatoid, fungal, or parasitic infections; autoimmune diseases and inflammatory bowel disease Treatment, prognosis, and diagnosis of inflammatory diseases and diseases associated with reduced immune activity, such as immunodeficiency 74. Any of embodiments 31 to 73 for use in detecting and / or monitoring. 75. The antibody or conjugate according to any one of embodiments 74 or the pharmaceutical composition according to embodiment 74.

[0289] Embodiment 77. For use in treating cancer, particularly cancers that express TA-MUC1. The cancers are ovarian cancer, breast cancer, pancreatic cancer, lung cancer, colon cancer, stomach cancer, liver cancer, kidney cancer, Liver cancer, blood cancer, endometrial cancer, thyroid cancer, leukemia, hemiplegoma, melanoma, carcinoma, teratoma , lymphoma, sarcoma, mesothelioma, neuroblastoma, glioma, rectal cancer, adrenal cancer, skin cancer, Brain cancer, cervical cancer, intestinal cancer, bowel cancer, head and neck cancer, digestive tract cancer, lymph node cancer, esophageal cancer Cancer, colorectal cancer, ear, nose and throat (ENT) cancer, prostate cancer, bladder cancer, uterine cancer, and 77. The antibody, conjugate of embodiment 76, selected from the group consisting of: , or a pharmaceutical composition.

[0290] Embodiment 78. For use in treating an infectious disease, the infectious disease being a bacterial infection, a viral infection, Embodiment 76. The method according to claim 76, wherein the infection is selected from the group consisting of a virulent infection, a fungal infection, and a parasitic infection. 2. The antibody, conjugate, or pharmaceutical composition according to claim 1.

[0291] Embodiment 79. For use in treating an autoimmune disease, wherein the autoimmune disease is a seborrheic dermatitis. Acute myelopathy, type 1 diabetes, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis 77. The anti-inflammatory drug of embodiment 76, selected from the group consisting of rheumatoid arthritis, ... and systemic lupus erythematosus. a compound, conjugate, or pharmaceutical composition.

[0292] Embodiment 80. A method for treating cancer in a subject in need thereof, comprising: In particular, a subject with a cancer that expresses TA-MUC1 may receive any one of the methods of any of embodiments 31 to 73. a therapeutically effective amount of the conjugate or antibody described in paragraph 74 or the composition described in embodiment 74. Administering

[0293] Embodiment 81. The cancer is ovarian cancer, breast cancer, pancreatic cancer, lung cancer, colon cancer, stomach cancer, Liver cancer, kidney cancer, blood cancer, endometrial cancer, thyroid cancer, leukemia, hemiplegia, melanoma, Carcinoma, teratoma, lymphoma, sarcoma, mesothelioma, neuroblastoma, glioma, rectal cancer, adrenal cancer , skin cancer, brain cancer, cervical cancer, intestinal cancer, bowel cancer, head and neck cancer, gastrointestinal cancer, lymphatic esophageal cancer, colorectal cancer, ear, nose and throat (ENT) cancer, prostate cancer, bladder cancer, 81. The method of claim 80, wherein the cancer is selected from the group consisting of uterine cancer, uterine cancer, and metastases thereof. A method for treating [Brief explanation of the drawings]

[0294] [Figure 1] Figure 1 shows ELISA binding curves of anti-MUC1 antibodies to different MUC1 peptides. (A) shows antigen binding of PankoMab N54Q (PM-N54Q) lacking Fab glycosylation and PankoMab (PM) containing Fab glycosylation to a MUC1 peptide containing the epitope sequence PDTR. The threonine of the MUC1 peptide is glycosylated with Tn, sTn, TF, or sTF. (B) shows binding of PankoMab and PM-N54Q to a MUC1 peptide containing the epitope sequence variant PESR. The serine of the MUC1 peptide is glycosylated with Tn. (C) shows binding of PM-N54Q to a MUC1 peptide containing the epitope sequence PDTR. The threonine of the MUC1 peptide is either glycosylated with Tn or not glycosylated. (D) Binding of several N54X mutants to the Tn-PDTR MUC1 peptide compared to PankoMab with Fab glycosylation diluted from cell culture supernatant of transiently transfected cells. (E) Binding curves of three purified N54X mutants without Fab glycosylation compared to PankoMab with Fab glycosylation to Tn-PDTR, TF-PDTR, and unglycosylated PDTR MUC1 peptide. (F) Binding of two framework mutants, PM-N54Q, to the Tn-PDTR MUC1 peptide compared to PankoMab with Fab glycosylation. Framework mutant mf-a contains nine mutated amino acids in the VH and three in the VL framework, while mf-b contains nine mutated amino acids in the VH and four in the VL framework. [Figure 2] 1 shows surface plasmon resonance (Biacore) binding of anti-MUC1 antibodies PM and PM-N54Q to glycosylated PDTR-MUC1 peptide. The maximum binding signal of various concentrations of PM-N54Q and PankoMab is plotted against antibody concentration. [Figure 3]Figure 1 shows the results of fluorescence proximity sensing on 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] SDS-acrylamide gel of electrophoretic separation of PM-N54Q and PankoMab under non-reducing (left) and reducing (right) conditions. Lane 1: PM-N54Q after the capture step; Lane 2: PM-N54Q after the polishing step; Lane 3: PankoMab after the capture step; Lane 4: PankoMab after the polishing step; Lane 5: molecular weight marker. [Figure 5] Coomassie blue stained gel of an isoelectric focusing assay with PM-N54Q lacking Fab glycosylation and PankoMab with Fab glycosylation: Lane 1: PankoMab with Fab glycosylation; Lane 2: PM-N54Q without Fab glycosylation. [Figure 6] Figure 6 shows anti-MUC1 antibody binding to Fcγ receptor IIIa. Increasing concentrations of antibody PM-N54Q or PankoMab push the rabbit anti-mouse coupling acceptor beads away from the FcγRIIIa-loaded donor beads, thereby reducing the detected chemiluminescence. In Figure 6A, a hypofucosylated antibody was applied to the assay, and in Figure 6B, a hyperfucosylated antibody was applied. [Figure 7] FIG. 1 shows 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 analyzed by flow cytometry. [Figure 8]FIG. 10 shows the cytotoxic activity of: A) control hIgG-ADC, naked PankoMab, and PankoMab-ADC against the cancer cell line MDA-MB-468, which expresses TA-MUC1 protein; B) control hIgG-ADC, naked PankoMab, and PankoMab-ADC against the cancer cell line HCT-15, which does not express 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 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 TA-MUC1 protein. Cells were treated with each compound for 6 days, and cell viability (%) was calculated by ATP assay. Data represent mean ± SD (N=3). [Figure 9] Figure 1 shows the antitumor efficacy of control hIgG-ADC, naked PankoMab, and PankoMab-ADC in MDA-MB-468-bearing nude mice. A single dose of 3 mg / kg of control hIgG-ADC, naked PankoMab, or PankoMab-ADC, or vehicle (acetate buffer solution), was administered intravenously to MDA-MB-468-bearing nude mice (N=6 / group). Estimated tumor volume data represent the mean ± SEM. Arrows indicate the timing of administration. Estimated tumor volumes 21 days after administration of PankoMab-ADC were compared with those of the control hIgG-ADC or naked PankoMab-treated groups by Student's t-test. ***P<0.001. [Figure 10]Figure 1 shows the antitumor efficacy of control hIgG-ADC, naked PankoMab, and PankoMab-ADC in HCC70-bearing nude mice. A single dose of 10 mg / kg of control hIgG-ADC, naked PankoMab, or PankoMab-ADC, or vehicle (acetate buffer solution), was administered intravenously to HCC70-bearing nude mice (N=6 / group). Estimated tumor volume data represent the mean ± SEM. Arrows indicate the timing of administration. Estimated tumor volume 21 days after administration of PankoMab-ADC was compared with that of the control hIgG-ADC-treated group or the naked PankoMab-treated group by Student's t-test. ***P<0.001. [Figure 11] Figure 1 shows the antitumor efficacy of control hIgG-ADC, naked PM-N54Q, PankoMab-ADC, and PM-N54Q-ADC in HPAC-bearing nude mice. A single dose of 10 mg / kg of control hIgG-ADC, naked PM-N54Q, PankoMab-ADC, and PM-N54Q-ADC, or vehicle (acetate buffer solution) was administered intravenously to HPAC-bearing nude mice (N=6 / group). Estimated tumor volume data represent the mean ± SEM. Arrows indicate the timing of administration. Estimated tumor volumes 21 days after administration of PankoMab-ADC and PM-N54Q-ADC were compared with those of the control hIgG-ADC-treated group by Dunnett's test. ** *P<0.001. [Figure 12]Figure 1 shows the antitumor efficacy of control hIgG-ADC, naked PankoMab, naked PM-N54Q, PankoMab-ADC, and PM-N54Q-ADC in NCI-H441-bearing nude mice. A single dose of 10 mg / kg of naked PankoMab, naked PM-N54Q, control hIgG-ADC, PankoMab-ADC, and PM-N54Q-ADC at a dose of 3 mg / kg, or vehicle (acetate buffer solution) was administered intravenously to NCI-H441-bearing nude mice (N=6 / group). Estimated tumor volume data represent the mean ± SEM. Arrows indicate the timing of administration. Estimated tumor volumes 31 days after administration of PankoMab-ADC and PM-N54Q-ADC were compared with those of the control hIgG-ADC-treated group by Dunnett's test. ***P<0.001. [Figure 13] Figure 1 shows the antitumor efficacy of control hIgG-ADC, naked PankoMab, naked PM-N54Q, PankoMab-ADC, and PM-N54Q-ADC in OVCAR-5-bearing nude mice. A single dose of 10 mg / kg of control hIgG-ADC, naked PankoMab, naked PM-N54Q, PankoMab-ADC, and PM-N54Q-ADC, or vehicle (acetate buffer solution) was administered intravenously to OVCAR-5-bearing nude mice (N=6 / group). Estimated tumor volume data represent the mean ± SEM. Arrows indicate the timing of administration. Estimated tumor volumes 14 days after administration of PankoMab-ADC and PM-N54Q-ADC were compared with those of the control hIgG-ADC-treated group by Dunnett's test. The estimated tumor volume 14 days after administration of PM-N54Q-ADC was compared with the estimated tumor volume in the PankoMab-ADC-treated group by Student's t-test. ***P<0.001. [Figure 14]Figure 1 shows the antitumor efficacy of control hIgG-ADC, PankoMab-ADC, and PM-N54Q-ADC in HCT-15-bearing nude mice. A single dose of 10 mg / kg of control hIgG-ADC, PankoMab-ADC, and PM-N54Q-ADC, or vehicle (acetate buffer solution), was administered intravenously to HCT-15-bearing nude mice (N=6 / group). Estimated tumor volume data represent the mean ± SEM. Arrows indicate the timing of administration. Estimated tumor volumes 21 days after administration of PankoMab-ADC and PM-N54Q-ADC were compared with those of the control hIgG-ADC-treated group by Dunnett's test. ***P<0.001. [Figure 15] FIG. 1 shows the amino acid sequence of the heavy chain of humanized antibody PM N54Q (SEQ ID NO: 15, but the amino acid at position 57 is Gln, ie, SEQ ID NO: 22). [Figure 16] FIG. 1 shows the amino acid sequences of the light chains of humanized antibodies PM N54Q and PankoMab (SEQ ID NO: 16). [Figure 17] FIG. 1 shows the amino acid sequence of the heavy chain of humanized antibody PankoMab (SEQ ID NO: 19). [Figure 18] FIG. 1 shows the amino acid sequence of the heavy chain of chimeric antibody PM N54Q (SEQ ID NO: 20, but the amino acid at position 76 is Gln, i.e., SEQ ID NO: 23). [Figure 19] FIG. 2 shows the amino acid sequence of the light chain of chimeric antibody PM N54Q (SEQ ID NO: 21). [Example]

[0295] Example 1: 1. Anti-MUC1 antibody production The code for Asn54 (amino acid position 57 of SEQ ID NO: 11) according to the Kabat / Eu numbering system By mutating the codon for any amino acid other than Asn, specifically Gln, The nucleic acid sequence of the heavy chain of the humanized PankoMab antibody (see, for example, WO2011 / 012309) (see reference) was modified.

[0296] 1) Production of anti-MUC1 antibodies in human myeloid leukemia-derived cell lines Vectors containing the coding sequences for the γ1 heavy chain and the κ light chain of the mutant antibody were transfected into human myeloid leukocytes. The leukemia-derived cell line NM-H9D8 (DSM ACC2806) was transfected. N54X mutation (PankoMab N54X / PM-N54X, where X is other than N / Asn) or amino acid changes in the framework sequences of VH and VL. Different αMUC1 antibodies containing the variants were expressed in the resulting clones and compared with the human glycosylated protein. The concentration of αMUC1 antibody in the supernatant was adjusted to 100% by Protein A concentration. The results were determined by Octet measurements using a dowel pin or by Protein A chromatographic analysis. After purification by fluoroscopy, the results were quantified by UV280 absorbance. The binding properties of the antibodies were determined by antigen-ELISA (see Example 2) and were also analyzed by the immunohistochemistry of selected antibodies. The antibodies were analyzed by Scatchard analysis (see Example 3) and by Biacore (see Example 4). 4a), DRX 2 switchSENSE® technology (Example 4b ), or by flow cytometry (Example 7).

[0297] In addition, the non-mutated PankoMa with PM-N54Q and Fab glycosylation b) Human myeloid leukemia-derived cell line NM-H9D8 expressing antibodies with reduced fucose. The same gene was expressed in NM-H9D8. These antibodies, along with the same antibody, were purified and their binding to Fc gamma receptor IIIA was assayed. The behavior was analyzed in Example 6.

[0298] 2) Production of anti-MUC1 antibodies in CHO cell lines Synthesized by GeneArt™ from ThermoFisher Scientific The PM-N54Q coding sequence (heavy chain of PM-N54Q represented by SEQ ID NO: 17) and the nucleotide sequence of the light chain of PM-N54Q represented by SEQ ID NO: 18. The resulting plasmid was then transferred to CHO cells. The pooled cells were then grown under selective pressure. The PM-N54Q mutant antibody was produced using the general procedure. The anti-MUC1 antibody (PM-N54Q) was used in Examples 8 and 9.

[0299] 2. PankoMab-ADC, N54Q-ADC, and DXd Humanized anti-TA-MU containing a glycosylation site in CDR2-H2 (Fab glycosylation) PankoMab-GEX refers to the C1 monoclonal antibody, and Fab glycosylation PM-N54Q (Example 1-1), which refers to a humanized anti-TA-MUC1 monoclonal lacking , PankoMab-ADC, and PM-N54Q-ADC, The recombinant proteins were produced by known methods such as those described in WO2015 / 115091 and WO2015 / 115091. the Mab-GEX antibody comprises a heavy chain comprising SEQ ID NO: 19 and a light chain comprising SEQ ID NO: 16; Thus, the PankoMab-GEX antibody is linked to a drug-linker of formula 2. The above-mentioned PM-N54Q antibody comprises a heavy chain comprising SEQ ID NO: 15 and a sequence comprising SEQ ID NO: 16. The PM-N54Q antibody comprises a light chain and is therefore linked to a drug-linker of Formula 2.

[0300] [ka]

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

[0302] [ka]

[0303] The control hIgG-ADC is a humanized IgG1 isotype control that does not bind to mammalian cells. Monoclonal antibodies, as well as PnakoMab-ADC and PM-N54Q-ADC The ADC payload (DXd) consisted of the same drug-linker as in WO2014 The recombinant human leukocytes were produced by known methods, such as those described in WO2015 / 115091 and WO2015 / 115091.

[0304] Example 2: Antigen ELISA PankoMab N5, in which the N-glycosylation site in the Fab portion has been knocked out The antigen-binding properties of 4X were compared with those of PankoMat, which has an N-glycosylation site in its Fab portion. Compared with b.

[0305] MUC1 specific compared to (glycosylated) PankoMab-GEX® The binding characteristics of the Fab deglycosylated form (PM-N54Q) of the antibody PankoMab were determined using EL In ISA studies, various glycosylated and non-glycosylated MUC1-derived In principle, both antibodies were analyzed using tandem repeat peptides. Glycosylated PDTR peptide (APPAHGVTSAPDT) with different glycosylation The same gradation is obtained by binding to (X)-RPAPGSTAPPAHGVTSA). In other words, Galβ1-3GalNAc alpha (TF)-containing PDTR peptide The strongest binding was observed for TF, followed by sialylated TF and GalNAc alpha (Tn) O-glycosylated. Sialylated GalNAc alpha (sTn)O-g Binding to glycosylated mAbs was significantly lower. ), PM-N54Q exhibits binding to the non-glycosylated MUC1 PDTR peptide. It showed only modest affinity and appropriate tumor specificity (Fig. 1C).

[0306] However, GalNAc alpha (Tn) Biotinylated sugar bearing O-glycan In the TA-MUC1 antigen ELISA using peptides, PankoMab-GEX (registered trademark) A four-fold higher binding was found for PM-N54Q compared to PM-N54Q (registered trademark). Sialylated GalNAc alpha When glycosylated with (sTn), it showed approximately 7-fold better affinity. It binds to the same MUC1 peptide. Galβ1-3Ga at the threonine in the PDTR sequence lNAc alpha Binding to TF and sialylated TF (sTF) (Fig. 1A) , the PM-N54Q was twice as good.

[0307] Both antibodies showed a significant increase in Tn glycosylation at serine compared to binding to the PDT(Tn)R peptide. MUC1 peptide mutant APPAHGVTSAPE-S(Tn)-RPAPGS It shows strongly reduced binding to TAPPAHGVTSA. However, in this case However, the Fab deglycosylated PM-N54Q was more effective than PankoMab-GEX®. The binding was significantly stronger than that of the IgG4-dependent agonist (Figure 1B).

[0308] A different Fab deglycosylated PM-N54X mutant was synthesized by adding N-glycosylation to the Fab portion. First, all mutants were compared with PankoMab with cosylation from the supernatant. The PM-N54X mutant was directly compared without purification. The concentrations were determined by Octet. In addition, the chemical characteristics of the amino acid side chains of the Fab-glycosylated PMs were significantly different from those of the Fab-glycosylated PMs. A clear gender-dependent trend was visible. Carboxylic acid groups in the side chain showed the lowest binding enhancement. The best bonds have one or two nitrogens (as primary or secondary amines). This was observed with amino acids (Fig. 1D).

[0309] In addition, selected Fab deglycosylation mutants (PM-N54H, -W, and -Q) ) was purified by protein A chromatography and analyzed by ELISA (Figure 1E). TF-MUC1 peptide compared to PankoMab with Fab glycosylation, respectively. The binding enhancement for the Tn-MUC1 peptide was approximately 5-8 fold, and for the Tn-MUC1 peptide was approximately 2 fold. ~3 times.

[0310] Furthermore, two different framework mutants of PM-N54Q were tested against Tn glycosylated P Binding to the DTR-MUC1 peptide was analyzed by ELISA (see Fig. 1F The framework variant mf-a has nine amino acid mutations in the VH and The mutant mf-b has three amino acid mutations in the VH. There are four amino acid mutations in the VL framework and four amino acid mutations in the VL framework. Both variants show similar binding compared to the PM-N54Q antibody.

[0311] Example 3: Saturation of Anti-MUC1 Antibody on MCF-7 and ZR-75-1 Cells combined analysis Two factors determine the therapeutic suitability of an antibody: its affinity for tumor cells and the number of binding sites. This is particularly important.

[0312] PankoMab (a MUC1-specific antibody) was developed against TA-MUC-1-positive human tumor cell lines. Binding of the deglycosylated form of Fab (PM-N54Q) was compared with the Fab glycosylated PankoM Compared to ab-GEX®, human breast cancer cell lines ZR-75-1 and MCF The antibody was evaluated using a radiolabeled antibody in a saturation binding assay against 5-7. 12-fold molar excess of pS in 0 mM sodium carbonate, 150 mM NaCl, pH 8.7 Chelation with CN-benzyl-DTPA for 2 hours at 37°C followed by incubation overnight at 2-8°C. The free chelator was separated by desalting column and dead-end filtration (50 kDa cap). The chelating antibody was removed by buffer exchange (6x buffer exchange to PBS, 6 mM phosphate, 1.6 mM KCl, 80 mM NaCl, 0.2 M Na acetate, 0.1 M HCl 1 for 1 hour at 37°C, carrier-free 111 Radiolabeled with In (2 μCi / μg antibody). The preparation was neutralized by adding 8-9 volumes of 10x concentrated PBS. A volume of fetal bovine serum was added to the neutralized labeled antibody preparation. 0 6 Several concentrations of labeled antibodies were added to the pelleted cells (1% (30-1000ng / 200µL in BSA / PBS). The antibody-bound cells were counted in a gamma counter and incubated for 1 hour at 4°C. The cells were separated by centrifugation and washed with 1% BSA / PBS at 4°C for another hour. The combined 111 The In-labeled antibody was measured using a gamma counter. The results were obtained using the "1 site specific ka" option in phPad Prism. The data are shown in Table 1. The data show that PM-N54Q has a high affinity for these tumor cells. This indicates a very large number of binding sites. The binding site number was 2.5-fold higher than that of the control, and the number of binding sites was also slightly increased.

[0313] [Table 1]

[0314] Example 4a: Surface Plasmon Resonance (BiaCore) Analysis Panko, a MUC1-specific antibody against TA-MUC-1-derived glycosylated peptides Binding of the deglycosylated form of the Fab (PM-N54Q) of the MAb was analyzed by surface plasmon resonance. The streptavidin sensor chip was attached to a biotinylated Coated with TA-MUC1 peptide (Tn glycosylated or non-glycosylated) PankoMab and PM-N54Q were treated with HPS-EP at concentrations ranging from 3,600 to 4.9 nM. The dilutions were injected at 50 μL / min. The maximum binding of each concentration was determined by Each was determined as a response unit (RU) and analyzed using GraphP Ad Prism. Figure 2 shows the results compared with PankoMab-GEX (registered trademark). The binding curves for PM-N54Q and PankoMa were obtained using the same method. b-GEX® affinity (K D ) were 388 nM and 652 nM, respectively. Therefore, an almost two-fold increase in affinity was detectable in this experimental setting. It was.

[0315] Example 4b: Fluorescence proximity sensing (DRX) 2 Dynamic Biosensor by s) Single-stranded DNA (96-mer) spotted on the chip and coupled to a ligand Fluorescence proximity sensing using tagged complementary DNA has been used to determine binding constants and affinities. In this study, streptavidin was used to bind biotinylated TA-MUC1. The peptide was used as a ligand for capturing. PankoMab against the peptide Binding of the antibody resulted in a change in fluorescence. The on-rate and off-rate were measured during binding and dissociation. It is more rapid compared to surface plasmon resonance due to its higher sensitivity. This is different from SPR, which is measured in a liquid system. This results in binding kinetics more comparable to KinExA, the "gold standard" method used.

[0316] PankoMab and PM-N54Q were diluted from 300 nM to 3.6 nM in PE140 buffer. The peptides were diluted in 1:9 steps to 7 nM and applied to the chip-bound peptides. Binding curves were plotted. , was evaluated (in the instrument software) by single-exponential global fitting. The binding curves of PM and PM-N54Q are exemplarily shown in Figures 3A and 3B. The calculated affinities of the ankoMab variants are shown in Table 2.

[0317] [Table 2]

[0318] Example 5: Biochemical characterization Non-reducing and reducing SDS-PAGE is used to analyze the purity and identity of the antibody. The band pattern of the non-reducing gel showed a major band at approximately 160 kDa, with heavy and light chains and their combination theoretical artifacts (approximately 25, 50-55, 75, 110, 13 The reducing gel shows distinct light chain bands at 25 and 50-55 kDa and The heavy chain band is shown. Due to the lack of Fab glycosylation, PM-N54Q, as expected, It has a smaller heavy chain (see right side of Figure 4).

[0319] The charge profiles are clearly different as shown by isoelectric focusing (IE F; see Figure 5). Fab glycosylation is heavily sialylated, whereas Fc glycosylation is The sylation is only minimally sialylated. GEX® has more charged isoforms than PM-N54Q, which , reflecting the higher levels of negatively charged sialic acid in the Fab portion.

[0320] Example 6: Fcγ receptor binding FcγR binding assay for FcγRIIIa (CD16a) was performed using PerkinElmer Based on AlphaScreen(R) technology. The target platform relies on PerkinElmer's simple bead-based technology. It is a more efficient alternative to traditional ELISA because it does not require a washing step.

[0321] In receptor binding assays, His-tagged FcγRIIIa (Glycotope Gm bH) is captured by Ni-chelate donor beads. The weakly coupled acceptor beads compete for binding to FcγR. For interaction with rabbit anti-mouse conjugated acceptor beads, donor and acceptor Close proximity of the beads results in emission of light upon laser excitation at 680 nm. When there is no max In case of competition, test The body binds to FcγR and transmits the signal max is reduced in a concentration-dependent manner. 520-620n using the ire2300 multi-label reader (PerkinElmer) By measuring m (AlphaScreen® method), chemiluminescence All results are expressed as the mean ± standard deviation of replicate samples. Data are presented in Gr Nonlinear curve fitting (sigmoido) with aphPad Prism 5 software sigmoidal dose-response variab The concentration-dependent signal was evaluated and calculated using the α-value slope. A morphologic curve was obtained. This curve has an upper plateau, a lower plateau, a slope, and an EC5 It is specified by 0.

[0322] As shown in Figures 6A and 6B, PankoMab N54Q and Pank The FcγRIIIa binding affinity for the oMab was comparable. In Figure B and Figure C, a hyperfucosylated antibody was applied in the assay. Removal of the carboxyl group did not affect 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 examined in two different cancer cells. The tongue squamous cell carcinoma line HSC- 4 moderately expresses TA-MUC1, and the ovarian cancer cell line CaOV-3 highly expresses it. Tumor cells were incubated with serially diluted antibodies, and bound antibodies were analyzed by phycoerythrocytometry. Detection was performed using a phosphoconjugated goat anti-human IgG (heavy and light chain) antibody. A human IgG control was included to control for background staining. Binding was measured using flow cytometry. The results were analyzed by chromatometry.

[0324] The analyzed constructs PM, PM-N54Q, and PM-N54D were compared with a human IgG1 control. In comparison, the strong effect on TA-MUC1-expressing HSC-4 cells and CaOV-3 cells was TA-MUC1 exhibits specific binding with strong binding (Figure 7). high P for CaOV-3 cells Binding of PM-N54D was comparable to that of PM with Fab glycosylation, but PM-N5 4Q showed slightly better binding (Figure 7A). Using HSC-4 cancer cells expressing PM-N54Q, the mutant PM-N54Q showed increased cell proliferation compared to PM-N54Q. Although PM-N54D clearly had better binding to cellular TA-MUC1, PM-N54D b showed inferior binding compared to PM with glycosylation (Fig. 7B).

[0325] Example 8: In vitro analysis of PankoMab-ADC and PM-N54Q-ADC oEvaluation of effectiveness 8.1 Cell lines The human breast cancer cell line MDA-MB expresses TA-MUC1 moderately to highly. -468, the human pancreatic cancer cell line HPAC, and the human lung cancer cell line NCI-H441 The 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 manufacturer's instructions. The expression level of TA-MUC1 on each cancer cell line was confirmed by flow cytometry. Ta.

[0326] 8.2 Evaluation of in vitro efficacy of PankoMab-ADC 1.25 x 10 cells by using culture medium 4 MD to have a concentration of cells / mL Prepare an A-MB-468 suspension and infuse it into a black smear at 80 μL / well (1,000 cells / well). The medium was added to each well of a 96-well plate with a clear bottom. 1 μL / well was added to the wells (N=3). All cells were MDA-MB-468. The mixture was incubated overnight under appropriate conditions.

[0327] 3.1 x 10 cells by using culture medium 3 HCT to have a concentration of cells / mL Prepare a 15% PBS suspension and inject it into a black, clear-bottom 96-well plate at 80 μL / well (250 cells / well). For blank wells, 80 μL of medium alone was added to each well of the plate. All cells were incubated overnight in the appropriate conditions for HCT-15. I was invited.

[0328] The next day, each naked PankoMab, control hIgG-ADC, and PankoM The ab-ADC was serially diluted 3-fold in each culture medium from 500 nM to 0.2 nM. Twenty microliters of the diluted solution was added to the appropriate wells (final concentration: 100 nM For blank and untreated wells, 20 μL of culture medium alone was added. All plates were incubated for 6 days under conditions appropriate for each cell line. did.

[0329] After incubation, the amount of ATP in each well was measured using CellTiter-Glo Luminometer. Luminescence was measured by using a luminescence cell viability assay (Promega). The application was carried out using a multi-label counter (ARVO X3, PerkinElmer Japan The assay was performed in triplicate.

[0330] The cell viability of each sample was calculated by the following equation: Cell viability (%)=100×(TB) / (CB) T: luminescence intensity of the test well C: Mean luminescence intensity of untreated wells B: Average luminescence intensity of blank wells

[0331] 8.3 In vitro activity of PankoMab-ADC and PM-N54Q-ADC Comparison of efficacy 1.25 x 10 cells by using culture medium 4 HP to have a concentration of cells / mL Prepare the AC suspension and infuse it into a black, clear-bottom 96-well plate at 80 μL / well (1,000 cells / well). For blank wells, 80 μL of medium alone was added to each well of the plate. All cells were incubated overnight under conditions appropriate for HPAC. I installed it.

[0332] 1.25 x 10 cells by using culture medium 4 NC to have a concentration of cells / mL Prepare I-H441 suspension, 80uL / well (1000 cells / well) in a black clear bottom The medium was added to each well of a 96-well plate. For blank wells, 80 μL of medium alone was added. All cells were added to wells at 1000 x g / well (N=3). The mixture was incubated overnight at RT.

[0333] The next day, each naked PankoMab, naked PM-N54Q, and hIgG-AD C, PankoMab-ADC, and PM-N54Q-ADC were treated with 500 nM to 0. The solution was serially diluted 3-fold in each culture medium to 2 nM. 20 microliters of each diluted solution was added. The blank wells and the 100 nM-0.04 nM-containing wells were added to the appropriate wells (final concentration: 100 nM-0.04 nM). For treated and untreated wells, only 20 μL of culture medium was added to each well. The cells 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 CellTiter-Glo Luminometer. Luminescence was measured by using a luminescence cell viability assay (Promega). The application is a multi-label counter (ARVO X3, PerkinElmer Japan The assay was performed in triplicate.

[0335] The cell viability of each sample was calculated by the following equation: Cell viability (%)=100×(TB) / (CB) T: luminescence intensity of the test well C: Mean luminescence intensity of untreated wells B: Average luminescence intensity of blank wells

[0336] PankoMab-ADC versus PM-N54Q for HPAC and NCI-H441 - Potency ratios of the cytotoxic activities of ADCs and their 95% CIs were calculated using SAS Release 9.4 (SAS Institute Japan, Tokyo, Japan) based on EXSUS ver. 3-parameter logarithm by using 8.1 (CAC Croit, Tokyo, Japan) 3-parameter logistic parallel analysis Calculated as a post hoc analysis using a common slope (i.e., l-line analysis) (Emax: 100, Emin: estimated value). The difference in cytotoxicity potency is expressed as the potency ratio. A difference was considered significant if its 95% CI did not include 1.

[0337] Example 9: In vivo efficacy of PankoMab-ADC and PM-N54Q-ADC Efficacy assessment 9.1 Cell lines The human breast cancer cell line MDA- MB-468 and HCC70, human pancreatic cancer cell line HPAC, and human lung cancer cell line N CI-H441 was used. The human colorectal cancer cell line HCT-15 was transfected with TA-MUC1. These cell lines were purchased from ATCC. Human ovarian cancer cells were used as the primary tumor cells. The OVCAR-5 cell line was purchased from the National Cancer Institute and used as a tumor cell line with low TA-MUC1 expression. Each cell line was cultured according to the manufacturer's instructions. The expression levels of were confirmed by flow cytometry and IHC staining.

[0338] 9.2 Evaluation of in vivo efficacy of PankoMab-ADC MDA-MB-468 cells were suspended in Matrigel (BD) at 1 × 10 7 Cells were collected from each female The mice were subcutaneously implanted into the right side of the body of nude mice (day 0), and on day 20, the mice were randomly divided into groups. After grouping, each naked PankoMab solution, control hIg G-ADC solution or PankoMab-ADC solution was administered intravenously at a dose of 3 mg / kg. A vehicle (acetate buffer solution) administration group was established as a control group. After the first 21 days, the length and width of the tumor in each mouse were measured with a digital caliper twice a week. did.

[0339] HCC70 cells were suspended in physiological saline (Otsuka Pharmaceutical Factory, Inc.) at 1 × 10 7 Cells The mice were subcutaneously implanted into the right side of the body of each female nude mouse (day 0), and on day 19, the mice were randomly assigned to groups After grouping, each naked PankoMab solution, control hIgG-ADC solution or PankoMab-ADC solution at a dose of 10 mg / kg A vehicle (acetate buffer solution) administration group was established as a control group. After administration, the length and width of the tumor in each mouse were measured twice a week for 21 days using a digital caliper. Measured by s.

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

[0341] In addition, tumor growth inhibition (TGI, %) of each group on the last measurement day of the vehicle-treated group was calculated as follows: Calculate using the following equation and round to the nearest whole number: TGI(%)=(1-T / C)×100 T: Naked PankoMab, control hIgG-ADC, or PankoMab - Mean estimated tumor volume (mm) of ADC 3 ) C: Mean estimated tumor volume (mm 3 )

[0342] To evaluate the antitumor efficacy of PankoMab-ADC, On the last measurement day of the C treatment group (MDA-MB-468: day 41, HCC70: day 40) The tumor volume of each mouse in the control hIgG-ADC treatment group was compared by Student's t-test. The tumor volumes of the groups were compared with those of the naked PankoMab-treated group. All data is from SAS System Release 9.2 (SAS Institute Inc.) A P value of less than 0.05 was considered statistically significant. Well done.

[0343] 9.3 In vivo activity of PankoMab-ADC and PM-N54Q-ADC Comparison of efficacy HPAC cells were suspended in physiological saline (Otsuka Pharmaceutical Factory, Inc.) at 3 × 10 6 Cells, The mice were subcutaneously implanted into the right side of each female nude mouse (day 0), and on day 11, the mice were randomly grouped. After grouping, each naked PM-N54Q solution, control hI gG-ADC solution, PankoMab-ADC solution, or PM-N54Q-ADC solution was administered intravenously at a dose of 10 mg / kg in a single dose. A control group was established. After administration, the length and width of the tumor in each mouse were measured over a 21-day period. The temperature was measured twice a week using a digital caliper.

[0344] NCI-H441 cells were suspended in Matrigel (BD) and 5 × 10 6 The cells were collected from each female wildebeest. The mice were subcutaneously implanted into the right side of the body (day 0), and on day 7, the mice were randomly grouped. (N=6). After grouping, each naked PankoMab solution or naked P M-N54Q solution was administered intravenously in a single dose at a dose of 10 mg / kg, and the control hIgG- ADC solution, PankoMab-ADC solution, or PM-N54Q-ADC solution was added to the 3 A single dose of 200 mg / kg was administered intravenously. After administration, the length and width of the tumor in each mouse were measured weekly for 31 days. Measured twice with a digital caliper.

[0345] OVCAR-5 cells were suspended in physiological saline (Otsuka Pharmaceutical Factory, Inc.) at 5 × 10 6 Thin The cells were subcutaneously implanted into the right side of each female nude mouse (day 0), and the mice were randomized on day 12. After grouping, naked PankoMab solution, Ikid PM-N54Q solution, control hIgG-ADC solution, PankoMab-ADC solution A single dose of 10 mg / kg of PM-N54Q-ADC solution or PM-N54Q-ADC solution was administered intravenously. A vehicle (acetate buffer solution) administration group was established as a control group. After administration, each mouse The length and width of the tumors were measured with digital calipers twice a week for 21 days.

[0346] HCT-15 cells were suspended in physiological saline (Otsuka Pharmaceutical Factory, Inc.) and diluted to 5 × 10 6cell The mice were subcutaneously implanted into the right side of the body of each female nude mouse (day 0), and on day 10, the mice were randomly assigned to After grouping, the control hIgG-ADC solution, Panko The Mab-ADC solution or PM-N54Q-ADC solution was administered intravenously at a dose of 10 mg / kg. A single dose was administered intravenously. A vehicle (acetate buffer solution) administration group was established as a control group. After treatment, the length and width of the tumor in each mouse were measured twice a week for 21 days using digital calipers. Measured.

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

[0348] In addition, the last measurement day of the vehicle-treated group or the last day that all groups were alive The tumor growth inhibition (TGI, %) for each mouse was calculated according to the following equation and rounded to the nearest whole number: did. TGI(%)=(1-T / C)×100 T: Naked PankoMab, naked PM-N54Q, control hIgG-AD C, Mean estimated tumor volume (m m 3 ) C: Mean estimated tumor volume (mm 3 )

[0349] HPAC-carrying mice, NCI-H441-carrying mice, OVCAR-5-carrying mice, and To evaluate the antitumor efficacy of each compound in HCT-15-bearing mice, control hI The last measurement day for the gG-ADC treatment group (HPAC: Day 32, NCI-H441: Day 38) , HCT-15: day 32) or the last day all groups are alive (OVCAR-5 The tumor volume of each mouse on day 26 was measured by Dunnett's test. The tumor volumes of the OVCAR-5-bearing nude mice on day 33 were compared with those of the C-treated group. Tumor volumes were compared between the PankoMab-ADC and PM-N54Q-ADC treatment groups. Comparisons were made using the Student's t-test. All statistical analyses were performed using SAS System Release 9.2 (SAS Institute Inc.) was used as a post hoc analysis. A P value of less than 0.05 was considered statistically significant.

[0350] Example 10: Results 10.1 TA-MUC1-positive and -negative cancer cell lines in vitro Cytotoxic activity of PankoMab-ADC against PankoMab-ADC demonstrates target- and drug-dependent cell proliferation in human cancer cell lines To investigate whether or not the compound exhibits cytotoxic activity, human breast cancer cells MDA-MB-468 (TA -MUC1 positive) and human colorectal cancer cell line HCT-15 (TA-MUC1 negative) Naked PankoMab, control hIgG-ADC, and PankoMab- The in vitro efficacy of the ADC was evaluated. As shown in Figure 8, naked P AnkoMab and hIgG-ADC showed little activity against each cell line. (IC 50 Under these conditions, PankoMab-ADC exhibited TA - showed dose-dependent cytotoxic activity against MUC1-positive cells, MDA-MB-468 (Figure 8A, IC 50 <10nM). However, PankoMab-ADC was negative for TA-MUC1. It showed no activity against the human HCT-15 cell line (Fig. 8B, IC 50 >100nM). Based on these results, PankoMab-ADC is expected to be effective against TA in vitro. -The results show target- and drug-dependent cytotoxicity against MUC1-positive cancer cell lines. I argued.

[0351] 10.2 In vitro analysis of PankoMab-ADC and PM-N54Q-ADC Comparison of in vitro cytotoxic activity against TA-MUC1-positive cells between To investigate whether improved antigen-binding affinity could contribute to enhanced cytotoxic activity, human PankoM against pancreatic cancer cell line HPAC and human lung cancer cell line NCI-H441 The in vitro efficacy of ab-ADC and PM-N54Q-ADC was evaluated. The cytotoxic activity of PM-N54Q-ADC against these cells was significantly higher than that of PankoMab-ADC. The cytotoxic activity of PM-N54Q-ADC was 1.5-fold more potent than that of PM-N54Q-ADC (Figures 8C and 8D). The efficacy ratio of PankoMab-ADC to HPAC was 1.917 (1. 611-2.280, 95% CI), and the efficacy ratio to NCI-H441 was 1.663. (1.495-1.849, 95% CI of EC50). The cytotoxic activity of 54Q-ADC was significantly stronger than that of PankoMab-ADC. These results demonstrated that the improved antigen-binding affinity of PankoMab-ADC This suggests that this may contribute to the significant enhancement of cell killing activity.

[0352] 10.3 Antitumor efficacy of PankoMab-ADC against TA-MUC1-positive tumors PankoMab-ADC is effective not only in vitro but also in vivo To investigate whether this phenomenon is associated with the development of MDA-MB-468-bearing mice, naked Pa Antitumor efficacy of nkoMab, control hIgG-ADC, and PankoMab-ADC As shown in Figure 9, naked PankoMab and control hIg G-ADC (3 mg / kg, single dose) did not demonstrate antitumor efficacy (TGI was In contrast, the PankoMab-ADC (3 mg / kg) g, single dose) significantly inhibited tumor growth (TGI was 97% at day 41). Furthermore, PankoMab-ADC significantly outperformed control hIgG-ADC and naked PankoMab-ADC. showed significant antitumor efficacy compared to oMab (both P<0.001 at day 41) In terms of weight change, the weight loss caused by drug treatment was significant in all drug treatment groups. Nothing was observed.

[0353] In addition, naked PankoMab and control hIgG-A were administered to HCC70-bearing mice. The antitumor efficacy of PankoMab-ADC, DC, and PankoMab-ADC was evaluated. Naked PankoMab and control hIgG-ADC (10 mg / kg, single dose) showed weak antitumor efficacy in these xenograft models (TGI At day 40, the rates were 10% and 29%, respectively. In contrast, PankoMab-ADC (10 mg / kg, single dose) significantly inhibited tumor growth (TGI was 9 at day 40). 5%). Furthermore, PankoMab-ADC significantly reduced the IgG-ADC response rate compared to the control hIgG-ADC. , and showed statistically significant antitumor efficacy (both P<0.001 at day 40). In terms of efficacy, no weight loss caused by drug treatment was observed in any of the drug treatment groups. These results suggest that PankoMab-ADC has potent antitumor efficacy. and demonstrated target- and drug-dependent antitumor activity in various TA-MUC1-positive xenograft models. This suggests that the drug demonstrated efficacy against cancer.

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

[0355] First, we investigated the expression of TA-MUC1 in HPAC tumor cells and The in vivo efficacy against IFN-γ-glucan and NCI-H441 tumor cells was evaluated. As shown, naked PM-N54Q and control hIgG-ADC (10 mg / kg g, single dose) showed weak antitumor efficacy in HPAC-bearing mice (TGI At day 32, the rates were 27% and 18%, respectively. In contrast, PankoMab-ADC and PM-N54Q-ADC (10 mg / kg, single dose) significantly inhibited tumor growth. (TGI was 93% at day 32 for both.) C and PM-N54Q-ADC (10 mg / kg, single dose) were compared with the control hIgG-AD showed statistically significant antitumor efficacy compared with C (both P<0.0 at day 32). 01). In terms of weight change, weight loss caused by drug treatment was significantly greater than that of all drug treatments. None were observed in the control group.

[0356] As shown in Figure 12, naked PankoMab and naked PM-N 54Q (10 mg / kg, single dose) showed weak antitumor activity in NCI-H441-bearing mice. The tumor efficacy was 8% and 12%, respectively, at 38 days. The gG-ADC treatment group (3 mg / kg, single administration) showed antitumor activity for 2 weeks after administration. However, tumor regrowth was observed after 21 days (TGI was 71% at day 38). In contrast, PankoMab-ADC and PM-N54Q-ADC (3 mg / kg , single dose) significantly inhibited tumor growth (TGI for both was 99% at day 38). Furthermore, PankoMab-ADC and PM-N54Q-ADC were significantly higher than control hIg showed statistically significant antitumor efficacy compared with G-ADC (P < 0.05 at day 38, respectively). In terms of weight change, the weight loss caused by drug treatment was None were observed in the drug-treated groups.

[0357] Next, the present inventors investigated the effect of TA-MUC1 on OVCAR-5 tumor cells, which have low expression of TA-MUC1. In vivo efficacy was evaluated. As shown in Figure 13, naked Panko Mab, PM-N54Q, and control hIgG-ADC (10 mg / kg, single dose) , showed little antitumor efficacy in OVCAR5-bearing mice (TGI was 2 (These were 1%, 11%, and 3% on day 6, respectively.) In this model, PankoMa Although the antitumor efficacy of b-ADC (10 mg / kg, single dose) was limited (TGI On day 26, the response rate was 37%, and PM-N54Q-ADC (10 mg / kg, single dose) It showed strong antitumor efficacy (TGI was 73% at day 26). oMab-ADC and PM-N54Q-ADC showed significantly improved efficacy compared to the control hIgG-ADC. showed statistically significant antitumor efficacy (P = 0.01 and P < 0.01, respectively, at day 26). 001). In addition, PM-N54Q-ADC showed a significantly higher efficacy than PankoMab-ADC. The antitumor efficacy was statistically significant (P<0.001 at day 26). No weight loss caused by drug treatment was observed in any of the drug treatment groups. Ta.

[0358] Finally, we performed i against HCT-15 tumor cells, which are negative for TA-MUC1. In vivo efficacy was evaluated.

[0359] As shown in Figure 14, naked PankoMab and PM-N54Q (1 0 mg / kg, single dose) showed little antitumor efficacy in this model. (TGI was 7% and 4%, respectively, at day 32.) Furthermore, PankoMab-AD C, PM-N54Q-ADC and control hIgG-ADC were also antitumor in this model. showed little to no efficacy against tumors (TGI was 15%, 22%, and 10%, respectively, at 32 days). (The figure was 26%)

[0360] Based on these results, the anti- inflammatory effects of PankoMab-ADC and PM-N54Q-ADC were We concluded that tumor efficacy was target- and drug-dependent. This improvement may contribute to enhanced antitumor efficacy against TA-MUC1-positive tumor cells. do.

[0361] Identification of the deposited biological material Cell lines DSM ACC2806, DSM ACC2807, and DSM ACC28 56 is a subsidiary of Glycotope GmbH, Robert t-Rossle-Str.10, 13125 Berlin (DE) by Deuts che Sammlung von Mikroorganismen und Zel lkulturen GmbH(DSMZ),Inhoffenstraβe 7B,3 Deposited at 8124 Braunschweig (DE).

[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 caccttcca gctgtgctgc agtcctccgg cctgtactct 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 180 tatctgcaga agcccggcca gtctcctcag ctgctgatct accagatgtc caacctggcc 240 tctggcgtgc ccgatagatt ttccggctct ggctctggca ccgacttcac cctgagaatc 300 tccagagtgg aagccgagga cgtggggcgtg tactactgtg cccagaacct ggaactgcct 360 cctacctttg gccagggcac caaggtgga atcaagcgga cagtggccgc tccttccgtg 420 tttatcttcc caccttccga cgagcagctg aagtccggca cagcttctgt cgtgtgcctg 480 ctgaacaact tctaccctcg ggaagccaag 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. 1. A method for producing an antibody-drug conjugate, comprising: reacting a compound of the formula: with an antibody or a derivative thereof having a sulfhydryl group capable of binding to MUC1 or TA-MUC1; (maleimid-N-yl)-CH 2 CH 2 CH 2 CH 2 CH 2 -C(=O)-GGFG-NH-CH 2 -O-CH 2 -C(=O)-(NH-DX) conjugating a drug-linker moiety to the antibody by forming a thioether bond across a disulfide bond present in the hinge region of the antibody; (maleimid-N-yl)- is a group represented by the formula: 【Chemistry 1】 (the nitrogen atom is the connecting point), -(NH-DX) is a group represented by the formula: 【Chemistry 2】 (the nitrogen atom of the amino group at position 1 is the attachment point), -GGFG- represents the tetrapeptide residue -Gly-Gly-Phe-Gly-; The antibody (i) a heavy chain variable region comprising complementarity determining regions (CDRs) 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 light chain variable region comprising complementarity determining regions (CDRs) 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; Including, the amino acid at position 8 of SEQ ID NO:2 is selected from the group consisting of glutamine, histidine, tryptophan, tyrosine, lysine and arginine, or CDR-H2 has the amino acid sequence of SEQ ID NO:7; method.

2. 2. The method of claim 1, 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.

3. 3. The method of claim 1 or 2, 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.

4. 4. The method of claim 1, wherein the light chain variable region of the antibody 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.

5. 5. The method of claim 1, 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.

6. The method of claim 1 , wherein the antibody comprises an Fc region.

7. The method according to claim 6, wherein the antibody is an IgG1 type antibody, an IgG2 type antibody, or an IgG4 type antibody.

8. 8. The method of claim 1, wherein 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.

9. The method of claim 8, wherein the heavy chain of the antibody has the amino acid sequence of SEQ ID NO:

22.

10. 10. The method of any one of claims 1 to 9, 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 proline residues, and deletion or absence of one or two amino acids at the carboxyl terminus.

11. 11. The method of any one of claims 1 to 10, wherein the antibody comprises a deletion or absence of one or two amino acids at the carboxyl terminus of the heavy chain.

12. 12. The method of any one of claims 1 to 11, wherein the antibody comprises two heavy chains, both of which lack one carboxyl-terminal amino acid residue.

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