Anti-muc1 antibodies

Abstract

This invention relates to novel antibodies against the cancer antigen MUC1. Specifically, antibodies with improved antigen binding are obtained by deleting the glycosylation site in the CDR-H2 of known anti-MUC1 antibodies.

Description

Invention Field

[0001] This invention relates to the field of antibodies. A mutant anti-MUC1 antibody with increased antigen-binding affinity is provided. Specifically, a mutant form of the humanized antibody PankoMab is provided, wherein asparagine 57 in the heavy chain variable region is replaced by another amino acid. Consequently, the glycosylation site in the CDR2 region is deleted and antigen-binding affinity is increased. In specific embodiments, this invention relates to the therapeutic and diagnostic uses of this antibody and methods for producing such antibodies. Background of the Invention Antibodies targeting tumor-associated antigens are widely used therapeutic agents for cancer. Today, many anticancer antibodies are approved for human treatment. Some of these antibodies work by blocking certain signaling pathways crucial for the survival or proliferation of specific cancer cells. Other anticancer antibodies activate the patient's immune response against the targeted cancer cells, for example, by initiating antibody-dependent cytotoxicity (ADCC) via natural killer cells. This mechanism is induced by the binding of the antibody's Fc moiety to Fc receptors on immune cells.

[0003] One interesting and important group of antibodies is that targeting mucins. Mucins are a family of high-molecular-weight, highly glycosylated proteins produced by many epithelial tissues in vertebrates. They can be further subdivided into membrane-bound mucins due to the presence of hydrophobic transmembrane domains (which facilitate retention in the plasma membrane), and mucins secreted onto mucosal surfaces or secreted to become components of salivary fluid. The human mucin family consists of many members, including the membrane-bound MUC1.

[0004] Increased mucin production occurs in many adenocarcinomas, including pancreatic, lung, breast, ovarian, and colon cancers. Mucins are also overexpressed in lung diseases such as asthma, bronchitis, chronic obstructive pulmonary disease, and cystic fibrosis. Two membrane mucins, MUC1 and MUC4, have been extensively studied regarding their pathological significance in disease processes. Furthermore, mucins have been studied for their potential as diagnostic markers. Several antibodies against mucins (Clin. Cancer Res., 2011 Nov1; 17(21):6822-30, PLoS One, 2011 Jan 14;6(1):e15921), particularly MUC1, are known in the art. However, there is still potential to improve their therapeutic efficacy.

[0005] Therefore, there is a need in the art to provide therapeutic anti-MUC1 antibodies with improved performance. Summary of the Invention

[0006] The inventors of this invention have discovered that the deletion of glycosylation sites in the heavy chain variable region of the anti-MUC1 antibody PankoMab does not eliminate antigen binding, but unexpectedly increases the antigen affinity of the antibody. This is particularly surprising given that the glycosylation sites are located in the second complementarity-determining region (CDR-H2) of the heavy chain variable region. CDRs are those regions in an antibody that directly participate in antigen binding and provide contact with the epitope. Therefore, amino acids that modify CDRs are generally expected to be detrimental to antigen-binding affinity. Humanized PankoMab antibodies additionally contain glycosylation sites in CDR-H2, which have a large carbohydrate structure. This carbohydrate structure is present directly at the antigen-binding interface and is therefore considered to participate in antigen binding. However, as demonstrated in the examples, the PankoMab variant (PM-N54Q), in which the glycosylation sites are deleted by replacing the amino acids with the carbohydrate structure, exhibits increased antigen-binding affinity.

[0007] Therefore, in a first aspect, the present invention relates to an antibody capable of binding to MUC1, comprising... (i) The heavy chain variable region comprising the following 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 the following 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.

[0008] In a second aspect, the present invention provides a nucleic acid encoding an antibody according to the present invention. Furthermore, in a third aspect, an expression cassette or vector comprising a nucleic acid according to the present invention and a promoter operatively linked to said nucleic acid is provided; and in a fourth aspect, a host cell comprising a nucleic acid or expression cassette or vector according to the present invention is provided.

[0009] In a fifth aspect, the present invention provides a conjugate comprising an antibody according to the invention conjugated to other reagents.

[0010] In a sixth aspect, the present invention relates to a composition comprising an antibody according to the invention, a nucleic acid according to the invention, an expression cassette or vector according to the invention, a host cell according to the invention, or a conjugate according to the invention.

[0011] According to a seventh aspect, the present invention provides antibodies, nucleic acids, expression cassettes or vectors, host cells, compositions or conjugates according to the invention for use in pharmaceuticals, particularly for the treatment, prevention or diagnosis of cancer.

[0012] In an eighth aspect, the present invention provides a method for increasing the MUC1 binding affinity of an antibody, the antibody comprising... (i) The heavy chain variable region comprising the following 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: 8, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and (ii) A light chain variable region comprising the following 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. The method includes the following steps: replacing the amino acid residue at position 8 of CDR-H2 with any amino acid residue other than asparagine, thereby generating CDR-H2 having the amino acid sequence of SEQ ID NO: 2.

[0013] In a ninth aspect, the present invention provides a method for generating an antibody having increased MUC1 binding affinity, the method comprising: (a) Providing a nucleic acid encoding an antibody, said antibody comprising (i) The heavy chain variable region comprising the following 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: 8, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and (ii) A light chain variable region comprising the following 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; (b) Introducing a mutation into the nucleic acid to produce a mutated nucleic acid, wherein the mutation is introduced into a codon encoding an amino acid residue at position 8 of CDR-H2, such that the codon encodes any amino acid residue other than asparagine; and (c) Producing an antibody with increased MUC1 binding affinity by expressing the mutated nucleic acid in a host cell.

[0014] In a tenth aspect, the present invention provides a method for treating cancer in a subject in need of such treatment, the method comprising administering to a subject with cancer a therapeutically effective amount of an antibody according to the invention, a nucleic acid according to the invention, an expression cassette or vector according to the invention, or a host cell according to the invention.

[0015] In the eleventh aspect, the present invention provides a kit or apparatus comprising an antibody according to the invention, and related methods, which can be used for the diagnosis, detection or monitoring of MUC1-related disorders such as cancer.

[0016] Other objects, features, advantages, and aspects of the invention will become apparent to those skilled in the art from the following description and appended claims. However, it should be understood that the specific embodiments described below, the appended claims, and those indicating preferred embodiments of the present application are given by way of example only. Various changes and modifications within the spirit and scope of the disclosed invention will be readily apparent to those skilled in the art upon reading the following.

[0017] definition The following expressions used in this document are generally intended to have the meanings described below, unless the context in which they are used indicates otherwise.

[0018] In addition to its literal meaning, the expression "comprising" as used herein also includes and specifically refers to the statements "substantially constitutes" and "consisting of". Therefore, the expression "comprising" means an embodiment in which the subject matter specifically lists the elements "comprising" does not contain other elements, and an embodiment in which the subject matter specifically lists the elements "comprising" may and / or does contain other elements. Similarly, the expression "having" should be understood to mean that the expression "comprising" also includes and specifically refers to the statements "substantially constitutes" and "consisting of". Where possible, the term "substantially constitutes" specifically refers to an embodiment in which the subject matter contains 20% or less, particularly 15% or less, 10% or less, or especially 5% or less of other elements besides the specifically listed elements that substantially constitute the subject matter.

[0019] The term "antibody" specifically refers to a protein containing at least two heavy chains and two light chains linked by disulfide bonds. Each heavy chain consists of one heavy chain variable region (V). H ) and 1 heavy chain constant region (C H Each light chain consists of one light chain variable region (V). L ) and 1 light chain constant region (C L The heavy chain constant region consists of three or (in the case of IgM- or IgE-type antibodies) four heavy chain constant domains (C). H1 C H2 CH3 and C H4 ), where the first constant structure domain C H1 Adjacent to the variable region and connected to the second constant structural domain C via the hinge region H2 The light chain constant region consists of only one constant domain. The variable region can be further subdivided into hypervariable regions (called complementarity-determining regions (CDRs)) scattered with more conserved regions (called frame regions (FRs)), where each variable region contains three CDRs and four FRs. The variable regions of both the heavy and light chains contain binding domains that interact with the antigen. The heavy chain constant region can be of any type, such as γ-, δ-, α-, μ-, or ε-type heavy chains. Preferably, the antibody heavy chain is a γ-chain. Furthermore, the light chain constant region can also be of any type, such as a κ- or λ-type light chain. Preferably, the antibody light chain is a κ-chain. The terms "γ- (δ-, α-, μ-, or ε-) type heavy chain" and "κ- (λ-) type light chain" refer to antibody heavy chains or antibody light chains that have a constant region amino acid sequence derived from the amino acid sequence of the naturally occurring heavy or light chain constant region, particularly the amino acid sequence of the human heavy or light chain constant region. Specifically, the amino acid sequence of the constant domain of the γ-type (especially γ1-type) heavy chain has at least 95%, particularly at least 98%, identity with the amino acid sequence of the constant domain of the human γ (especially human γ1) antibody heavy chain. Furthermore, the amino acid sequence of the constant domain of the κ-type light chain specifically has at least 95%, particularly at least 98%, identity with the amino acid sequence of the constant domain of the human κ antibody light chain. The constant region of the antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The antibody can be, for example, a humanized, human, or chimeric antibody.

[0020] The antigen-binding portion of an antibody typically refers to the full-length portion or one or more fragments of the antibody that retain the ability to specifically bind antigens. It has been demonstrated that the antigen-binding function of an antibody can be performed by fragments of the full-length antibody. Examples of antibody-binding fragments include the Fab fragment, which is formed by V... L V H C L and C H1 A monovalent fragment composed of domains; the F(ab)2 fragment, which is a bivalent fragment containing two Fab fragments linked by disulfide bonds through a hinge region, each Fab fragment binding to the same antigen; composed of V H and C H1 Fd fragments composed of structural domains; V-arms of the antibody L and V H Fv segments composed of structural domains; and segments composed of V H The dAb fragment composed of structural domains.

[0021] The "Fab moiety" of an antibody specifically refers to the variable region containing both heavy and light chains (VFab). H and V L ) and the first structural domain of the heavy chain and the constant region of the light chain (C H1 and C L A portion of the antibody. In cases where the antibody does not contain all of these regions, the term "Fab portion" refers only to region V. H V L C H1 and C L Those present in the antibody. Preferably, the "Fab moiety" refers to a portion of the antibody corresponding to a fragment containing the antigen-binding activity of the antibody obtained by digesting a natural antibody with papain. In particular, the Fab moiety of the antibody includes an antigen-binding site or its antigen-binding ability. Preferably, the Fab moiety contains at least the V of the antibody. H area.

[0022] The “Fc portion” of an antibody specifically refers to the region containing the heavy chain constant regions 2, 3, and (where applicable) 4 (C). H2 C H3 and C H4 This is part of the antibody. Specifically, the Fc portion includes two of each of these regions. In cases where the antibody does not contain all of these regions, then the term "Fc portion" refers only to region C. H2 C H3 and C H4 Those present in the antibody. Preferably, the Fc portion contains at least the C of the antibody. H2 Region. Preferably, the "Fc moiety" refers to a portion of the antibody corresponding to an antibody-free antigen-binding fragment obtained by digesting a natural antibody with papain. In particular, the Fc moiety of the antibody is capable of binding to an Fc receptor, and therefore, for example, contains an Fc receptor binding site or Fc receptor binding capacity.

[0023] As used herein, the terms "antibody" and "antibody construct" may, in some embodiments, refer to the same type of antibody or antibody construct, respectively. Specifically, all antibodies or antibody constructs in a population exhibit the characteristics used to define an antibody or antibody construct. In some embodiments, all antibodies or antibody constructs in a population have the same amino acid sequence. References to a particular type of antibody (such as antibodies capable of specifically binding to MUC1) specifically refer to a population of such antibodies.

[0024] As used herein, the term "antibody" also includes fragments and derivatives of said antibody. A "fragment or derivative" of an antibody is, in particular, a protein or glycoprotein derived from said antibody and capable of binding the same antigen as said antibody, particularly binding the same epitope. Therefore, a fragment or derivative of an antibody herein generally refers to a functional fragment or derivative. In a particularly preferred embodiment, the fragment or derivative of the antibody comprises a variable region of the heavy chain. It has been demonstrated that the antigen-binding function of an antibody can be performed by a fragment of a full-length antibody or a derivative thereof. Examples of antibody fragments include: (i) Fab fragments, which are monovalent fragments consisting of a variable region and a first constant region of each heavy chain and light chain; (ii) F(ab)2 fragments, which are bivalent fragments comprising two Fab fragments linked by disulfide bonds in the hinge region; (iii) fragments consisting of a variable region and a first constant region of the heavy chain C H1 The antibody fragments include: (iv) Fd fragments; (v) Fv fragments composed of the heavy and light chain variable regions of the antibody single arm; (vi) scFv fragments, which are Fv fragments composed of a single polypeptide chain; (vii) (Fv)2 fragments composed of two Fv fragments covalently linked together; (vii) heavy chain variable domains; and (viii) polymorphs composed of heavy chain variable regions and light chain variable regions, which are covalently linked together in a manner where association between the heavy and light chain variable regions can occur only intermolecularly rather than intramolecularly. Antibody derivatives particularly include antibodies that bind or compete for the same antigen as the parent antibody but have a different amino acid sequence than the parent antibody from which it is derived. These antibody fragments and derivatives are obtained using conventional techniques known to those skilled in the art.

[0025] The target amino acid sequence is considered "derived from" or "corresponds to" the reference amino acid sequence if it has at least 75%, more preferably at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98%, or at least 99% homology or identity with the corresponding portion of the reference amino acid sequence over its entire length. "Corresponding portion" means, for example, that frame region 1 (FRH1) of the heavy chain variable region of the target antibody corresponds to frame region 1 of the heavy chain variable region of the reference antibody. In certain embodiments, the target amino acid sequence "derived from" or "corresponds to" the reference amino acid sequence is 100% homologous to, or particularly 100% identical to, the corresponding portion of the reference amino acid sequence over its entire length. The "homology" or "identity" of the amino acid sequence or nucleotide sequence is preferably determined according to the invention over the entire length of the reference sequence or over the entire length of the corresponding portion of the reference sequence (which corresponds to the sequence defining homology or identity). Antibodies derived from parent antibodies (defined by one or more amino acid sequences, such as a specific CDR sequence or a specific variable region sequence) are particularly antibodies having amino acid sequences, such as a CDR sequence or a variable region sequence, that are at least 75%, preferably at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98%, or at least 99% homologous to or identical to the respective amino acid sequences of the parent antibody, especially identical. In some embodiments, the antibody derived from the parent antibody (i.e., its derivative) contains the same CDR sequence as the parent antibody, but differs in the remaining sequences of the variable region.

[0026] As used herein, the term "antibody" also refers to multivalent and multispecific antibodies, namely antibody constructs having more than two binding sites, each binding to the same epitope, and antibody constructs having one or more binding sites binding to a first epitope and one or more binding sites binding to a second epitope, as well as other binding sites optionally binding to other epitopes.

[0027] "Specific binding" preferably means that the binding of a reagent, such as an antibody, to its specific target, such as an epitope, is stronger than its binding to another target. This is further supported by the fact that the dissociation constant (K) of the reagent binding to the first target is... d If the dissociation constant of the reagent-specific target is lower than that of the second target, then its binding to the first target is stronger than that of the second target. Preferably, the dissociation constant of the reagent-specifically bound target is more than 100 times, 200 times, 500 times, or more than 1000 times lower than that of the reagent-non-specifically bound target. Furthermore, the term "specific binding" specifically indicates that the binding affinity between the binding partners has at least 10... 6 M -1 Preferably at least 10 7 M -1 More preferably at least 10 8 M-1 Affinity constant K a Antibodies specific to a particular antigen, especially those capable of expressing at least 10... 6 M -1 Preferably at least 10 7 M -1 More preferably at least 10 8 M -1 K a Antibodies that bind to the antigen with an affinity of [a specific factor]. For example, the term "anti-MUC1 antibody" specifically refers to an antibody that specifically binds to MUC1, and preferably has an affinity of at least 10 [units]. 6 M -1 Preferably at least 10 7 M -1 More preferably at least 10 8 M -1 K a The affinity of MUC1.

[0028] The term "MUC1" refers to protein MUC1, also known as mucin-1, polymorphic epithelial mucin (PEM), or cancer antigen 15-3, and specifically human MUC1 (registration number P15941). MUC1 is a member of the mucin family and encodes a membrane-bound glycosylated phosphoprotein. MUC1 has a core protein mass of 120-225 kDa, which increases to 250-500 kDa through glycosylation. It extends 200-500 nm beyond the cell surface. The protein is anchored to the apical surface of many epithelial cells via a transmembrane domain. The extracellular domain comprises a 20-amino acid variable-number tandem repeat (VNTR) domain, with the number of repeats varying from 20 to 120 in different individuals. These repeat sequences are rich in serine, threonine, and proline residues, which allow for heavy O-glycosylation. In some embodiments, the term "MUC1" refers to tumor-associated MUC1 ("TA-MUC1"). TA-MUC1 is MUC1 present on cancer cells. This MUC1 differs from the MUC1 found in non-cancerous cells in its much higher expression level, localization, and glycosylation. Specifically, TA-MUC1 is diffusely present across the entire cell surface of cancer cells, while in non-cancerous cells, MUC1 exhibits strictly apical expression, thus unsuitable for systemic antibody administration. Furthermore, TA-MUC1 possesses anomalous O-glycosylation, exposing novel peptide epitopes and novel carbohydrate tumor antigens on the MUC1 protein backbone, such as Thomsen-Friedenreich antigen α (TFα).

[0029] "TFα", also known as Thomsen-Friedenreich antigen α or Core-1, refers to the disaccharide Gal-β1,3-GalNAc, which in cancer cells forms an α-terminal isomer and is linked to the hydroxy amino acid serine or threonine of the protein via an O-glycosidic linkage.

[0030] The term "sialic acid" specifically refers to any N- or O-substituted derivative of neuraminic acid. It can refer to 5-N-acetylneuraminic acid and 5-N-hydroxyacetylneuraminic acid, but preferably only to 5-N-acetylneuraminic acid. Sialic acid, particularly 5-N-acetylneuraminic acid, is preferably attached to a carbohydrate chain via a 2,3- or 2,6-bond. Preferably, in the antibodies described herein, 2,3- and 2,6-coupled sialic acid are present.

[0031] According to the present invention, the "relative amount of glycans" refers to a specific percentage or percentage range of glycans attached to the antibody in the antibody formulation or the antibody-containing composition, respectively. Specifically, the relative amount of glycans refers to a specific percentage or percentage range of all glycans contained in the antibody and therefore attached to the polypeptide chain of the antibody in the antibody formulation or the antibody-containing composition. 100% glycans refers to all glycans attached to the antibody in the antibody formulation or the antibody-containing composition, respectively. For example, a relative amount of 10% glycans carrying dimeric GlcNAc means an antibody-containing composition in which 10% of all glycans contained in the antibody and therefore attached to the antibody polypeptide chain in the composition contains dimeric GlcNAc residues, while 90% of all glycans contained in the antibody and therefore attached to the antibody polypeptide chain in the composition do not contain dimeric GlcNAc residues. A corresponding reference amount representing 100% glycans could be all glycan structures attached to the antibody in the composition, or all N-glycans, i.e., all glycan structures attached to the asparagine residues of the antibody in the composition, or all complex glycans. Reference sets for glycan structures are usually explicitly identified by technicians or derived directly from the environment.

[0032] The term “N-glycosylation” refers to all glycans attached to asparagine residues of a protein polypeptide chain. These asparagine residues are typically part of an N-glycosylation site having the amino acid sequence Asn-Xaa-Ser / Thr, where Xaa can be any amino acid except proline. Similarly, “N-glycan” is a glycan attached to asparagine residues of a polypeptide chain. The terms “glycan,” “glycan structure,” “carbohydrate,” “carbohydrate chain,” and “carbohydrate structure” are generally used synonymously herein. N-glycans typically have a common core structure consisting of two N-acetylglucosamine (GlcNAc) residues and three mannose residues, with the structure Manα1,6-(Manα1,3-)Manβ1,4-GlcNAcβ1,4-GlcNAcβ1-Asn, where Asn is an asparagine residue of the polypeptide chain. N-glycans are subdivided into three distinct types: complex glycans, hybrid glycans, and high-mannose glycans.

[0033] The figures given herein, particularly the relative amounts of specific glycosylation properties, are preferably understood as approximations. In particular, these figures can preferably be as high as 10% or more and / or lower, especially as high as 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% or more and / or lower.

[0034] In a "conjugate," two or more compounds are linked together. In some embodiments, at least some properties from each compound are retained in the conjugate. The linking can be achieved by covalent or non-covalent bonds. Preferably, the compounds of the conjugate are linked by covalent bonds. The different compounds of the conjugate can be directly bonded to each other by one or more covalent bonds between the atoms of the compounds. Alternatively, the compounds can be linked to each other by chemical parts such as linker molecules, where the linker is covalently attached to the atoms of the compounds. If the conjugate consists of more than two compounds, these compounds can be linked, for example, in a chain conformation, with one compound linked to the next, or several compounds each linked to a central compound.

[0035] The term "nucleic acid" includes single-stranded and double-stranded nucleic acids, ribonucleic acid, and deoxyribonucleic acid. It can contain naturally occurring and synthetic nucleotides, and can be naturally or synthetically modified, such as by methylation, 5'- and / or 3'-capping.

[0036] The term "expression cassette" specifically refers to a nucleic acid construct capable of enabling and regulating the expression of an introduced coding nucleic acid sequence. An expression cassette may contain a promoter, ribosome binding site, enhancer, and other control elements regulating gene transcription or mRNA translation. The exact structure of an expression cassette can vary depending on species or cell type, but typically includes 5'-non-transcriptional and 5'- and 3'-non-translational sequences, respectively, involved in transcription and translation initiation, such as TATA boxes, capped sequences, CAAT sequences, etc. More specifically, the 5'-non-transcriptional expression control sequence contains a promoter region that includes a promoter sequence for transcriptional control of operatively linked nucleic acids. Expression cassettes may also contain enhancer sequences or upstream activator sequences.

[0037] According to the present invention, the term "promoter" refers to a nucleic acid sequence located upstream (5') of the nucleic acid sequence to be expressed and controlling the expression of the sequence by providing a recognition and binding site for RNA polymerase. A "promoter" may include other recognition and binding sites for other factors involved in the regulation of gene transcription. A promoter can control the transcription of prokaryotic or eukaryotic genes. Furthermore, a promoter can be "inducible," i.e., responding to an inducer to initiate transcription, or it can be "constitutive" if transcription is not controlled by an inducer. In the absence of an inducer, the gene under the control of an inducible promoter is not expressed or is expressed only to a very small extent. In the presence of an inducer, the gene is turned on or the transcriptional level increases. Typically, this is mediated by the binding of specific transcription factors.

[0038] The term "vector" is used herein in its most general sense and includes any intermediate medium for nucleic acids that enables said nucleic acids to be introduced, for example, into prokaryotic and / or eukaryotic cells and, where appropriate, integrated into the genome. Vectors of this type are preferably replicated and / or expressed in cells. Vectors include plasmids, phage particles, bacteriophages, or viral genomes. The term "plasmid" as used herein generally refers to a construct of extrachromosomal genetic material, typically a circular DNA double helix, which can replicate independently of chromosomal DNA.

[0039] According to the present invention, the term "host cell" refers to any cell that can be transformed or transfected with exogenous nucleic acids. According to the present invention, the term "host cell" includes prokaryotic (e.g., *Escherichia coli*) or eukaryotic cells (e.g., mammalian cells, particularly human cells, yeast cells, and insect cells). Mammalian cells are particularly preferred, such as cells derived from humans, mice, hamsters, pigs, goats, or primates. Cells can be derived from a variety of tissue types and include primary cells and cell lines. Nucleic acids may be present in the host cell in single or two or more copies, and in one embodiment, are expressed in the host cell.

[0040] According to the invention, the term "patient" refers to a human, a non-human primate or other animal, particularly a mammal such as a cow, horse, pig, sheep, goat, dog, cat, or a rodent such as a mouse and rat. In a particularly preferred embodiment, the patient is a human.

[0041] The term "cancer" according to the invention specifically includes leukemia, seminoma, 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, liver cancer, colon cancer, stomach cancer, bowel cancer, head and neck cancer, gastrointestinal cancer, lymph node cancer, esophageal cancer, colorectal cancer, pancreatic cancer, ear, nose and throat (ENT) cancer, breast cancer, prostate cancer, bladder cancer, uterine cancer, ovarian cancer, and lung cancer and their metastases. The term "cancer" according to the invention also includes metastatic cancer lesions.

[0042] A tumor is a group of cells or tissues formed by the malregulated proliferation of cells. Tumors may exhibit partial or complete lack of structural organization and functional coordination with normal tissues, and typically form a distinctive mass of tissue that can be benign or malignant.

[0043] Metastasis refers to the spread of cancer cells from their original site to another part of the body. The formation of metastasis is a very complex process and typically involves cancer cells detaching from the primary tumor, entering the systemic circulation, and settling into normal tissues in other parts of the body to grow. When tumor cells metastasize, the new tumor is called a secondary or metastatic tumor, and its cells are usually similar to the original tumor. This means, for example, if breast cancer metastasizes to the lungs, the secondary tumor is composed of abnormal breast cells, not abnormal lung cells. The tumor in the lung is then called metastatic breast cancer, not lung cancer.

[0044] The term "pharmaceutical composition" specifically refers to a composition suitable for administration to humans or animals, i.e., a composition containing pharmaceutically acceptable components. Preferably, the pharmaceutical composition comprises an active compound or a salt or prodrug thereof, as well as a carrier, diluent, or pharmaceutical excipient, such as a buffer, preservative, and tension modifier.

[0045] The numerical ranges described herein include the numbers that define the range. The headings provided herein are not intended to limit the various aspects or embodiments of the invention (which can be read by referring to the entire specification). According to one embodiment, a subject matter described herein as including certain steps in the case of a method or comprising certain ingredients in the case of a composition indicates a subject matter consisting of various steps or ingredients. Preferably, the selection and combination of preferred aspects and embodiments described herein, and the specific subject matter resulting from corresponding combinations of preferred embodiments, also fall within the scope of this disclosure. Detailed Implementation

[0046] This invention is based on the development of a variant of the humanized anti-MUC1 antibody PankoMab, in which the glycosylation site in CDR-H2 is deleted. The deletion of the glycosylation site is achieved by replacing the amino acid Asn (asparagine) 57 in the heavy chain variable region with another amino acid, specifically Gln (glutamine). Asn 57 is the acceptor amino acid residue for the glycosylation site linked to the carbohydrate structure. Replacing this asparagine residue with another residue eliminates glycosylation because the carbohydrate structure can only be transferred to the asparagine residue by enzymes in the host cell. Surprisingly, the deletion of the glycosylation site in the CDR-H2 of PankoMab was found to increase the antigen-binding affinity of the antibody.

[0047] In light of these findings, the present invention provides an antibody capable of binding to MUC1, comprising... (i) The heavy chain variable region comprising the following 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 the following 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.

[0048] Combined with MUC1 This antibody specifically binds to the epitope of MUC1. "Specific binding" means binding that is not nonspecific adsorption. Examples of standards used to determine whether a binding is specific can include the dissociation constant (referred to herein as "K"). DThe epitope is located in the extracellular tandem repeat of MUC1. In some embodiments, the antibody binds to MUC1 in a glycosylation-dependent manner. Specifically, the antibody binds more strongly if the tandem repeat is glycosylated at a threonine residue by N-acetylgalactosamine (Tn), sialyl α2-6 N-acetylgalactosamine (sTn), galactose β1-3 N-acetylgalactosamine (TF), or galactose β1-3 (sialyl α2-6) N-acetylgalactosamine (sTF), preferably Tn or TF. Preferably, the carbohydrate moiety is bound to the threonine residue via an α-O-glycosidic bond. The epitope in the tandem repeat domain of MUC1 particularly comprises the amino acid sequence PDTR (SEQ ID NO: 13) or PESR (SEQ ID NO: 14). As mentioned above, binding to this epitope is preferably glycosylation-dependent, particularly if the aforementioned carbohydrate moiety is attached to the sequence PDTR or PESR (SEQ ID NO: 14), respectively. The binding to threonine residues in 13 and 14 is increased.

[0049] This epitope is a tumor-associated MUC1 epitope (TA-MUC1). A TA-MUC1 epitope specifically refers to an epitope of MUC1 that is present on tumor cells but not on normal cells, and / or can only be accessed by antibodies circulating in the host when it is present on tumor cells but not on normal cells. In some embodiments, the antibody binds more strongly to cells expressing the TA-MUC1 epitope than to cells expressing normal, non-tumor MUC1. Preferably, the binding is at least 1.5 times stronger, more preferably at least 2 times stronger, at least 5 times stronger, at least 10 times stronger, or at least 100 times stronger. For TA-MUC1 binding, the antibody preferably specifically binds to the glycosylated MUC1 tumor epitope such that the bond strength is increased by at least 2 times, preferably 4 times or 10 times, and most preferably 20 times, compared to the bond of an unglycosylated peptide of the same length and sequence. The binding can be determined or confirmed by ELISA, RIA, surface plasmon resonance (hereinafter referred to as "SPR") analysis, etc. Examples of devices used in SPR analysis include BlAcore™ (manufactured by GE Healthcare Bio-Sciences Crop.), ProteOn™ (manufactured by Bio-Rad Laboratories, Inc.), DRX2 Biosensor (manufactured by Dynamic Biosensors GmbH), SPR-Navi™ (manufactured by BioNavis Oy Ltd.), Spreeta™ (manufactured by Texas Instruments Inc.), SPRi-PlexII™ (manufactured by Horiba, Ltd.), and Autolab SPR™ (manufactured by Metrohm). The binding of antibodies to antigens expressed on cell surfaces can be measured using methods such as flow cytometry.

[0050] Furthermore, the antibody can exhibit antigen-binding properties similar to a reference antibody, which comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 10 and a light chain variable region having the amino acid sequence of SEQ ID NO: 12. Preferably, the reference antibody is a humanized antibody, PankoMab. In particular, the antibody according to the invention specifically binds to the same antigen as the reference antibody, and preferably binds to the antigen with a higher affinity. That is, the antibody preferably binds to the antigen with an affinity having a dissociation constant lower than that of the reference antibody, more preferably at least 10%, at least 20%, at least 30%, or at least 50% lower. Furthermore, the antibody preferably exhibits cross-specificity with the reference antibody, which comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 11 and a light chain variable region having the amino acid sequence of SEQ ID NO: 12. In particular, if present at a sufficiently high concentration, the humanized antibody is able to block the binding of the reference antibody to MUC1. This is possible if the binding of the reference antibody to MUC1 is hindered when the antibody has already bound to the antigen MUC1.

[0051] Anti-MUC1 antibody The antibody of the present invention capable of binding to MUC1 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the following 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 the light chain variable region comprises the following 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.

[0052] In some embodiments, the heavy chain variable region comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 9. Specifically, the heavy chain variable region comprises an amino acid sequence having at least 95%, particularly at least 98%, identity with the amino acid sequence of SEQ ID NO: 9. In these embodiments, the heavy chain variable region still comprises a CDR having the amino acid sequences of SEQ ID NO: 1, 2, and 3. Therefore, any sequence deviations relative to SEQ ID NO: 9 reside in the frame region but not in the CDR. Specifically, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 9.

[0053] In some embodiments, CDR-H2 has the amino acid sequence of SEQ ID NO: 2, wherein the amino acid at position 8 of SEQ ID NO: 2 is selected from glutamine, alanine, valine, histidine, tryptophan, tyrosine, lysine, and arginine; particularly glutamine, histidine, tryptophan, tyrosine, lysine, and arginine. Preferably, the amino acid at position 8 of SEQ ID NO: 2 is glutamine, histidine, tryptophan, lysine, or arginine, particularly glutamine. Specifically, CDR-H2 has the amino acid sequence of SEQ ID NO: 7.

[0054] In specific embodiments, the heavy chain variable region comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 10. Specifically, the heavy chain variable region comprises an amino acid sequence having at least 95%, particularly at least 98%, identity with the amino acid sequence of SEQ ID NO: 10. In these embodiments, the heavy chain variable region comprises CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 7, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3. Therefore, any sequence deviation relative to SEQ ID NO: 10 is located in the frame region but not in the CDR. Specifically, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 10.

[0055] In some embodiments, the light chain variable region comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 12. Specifically, the light chain variable region comprises an amino acid sequence having at least 95%, particularly at least 98%, identity with the amino acid sequence of SEQ ID NO: 12. In these embodiments, the light chain variable region still comprises a CDR having the amino acid sequences of SEQ ID NO: 4, 5, and 6. Therefore, any sequence deviations relative to SEQ ID NO: 12 reside in the frame region but not in the CDR. Specifically, the light chain variable region comprises the amino acid sequence of SEQ ID NO: 12.

[0056] In a specific embodiment, the heavy chain variable region has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 9, wherein the CDR still has the amino acid sequences of SEQ ID NO: 1, 2, and 3, and the light chain variable region has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 12, wherein the CDR still has the amino acid sequences of SEQ ID NO: 4, 5, and 6. Specifically, 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: 9, wherein the CDR still has the amino acid sequences of SEQ ID NO: 1, 2, and 3, and the light chain variable region has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 12, wherein the CDR still has the amino acid sequences of SEQ ID NO: 4, 5, and 6.

[0057] In a specific embodiment, the heavy chain variable region has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 10, wherein the CDR still has the amino acid sequences of SEQ ID NO: 1, 7, and 3, and the light chain variable region has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 12, wherein the CDR still has the amino acid sequences of SEQ ID NO: 4, 5, and 6. Specifically, the heavy chain variable region has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 10, wherein the CDR still has the amino acid sequences of SEQ ID NO: 1, 7, and 3, and the light chain variable region has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 12, wherein the CDR still has the amino acid sequences of SEQ ID NO: 4, 5, and 6.

[0058] In specific embodiments, the heavy chain variable region comprises an amino acid sequence that has at least 90% identity with the amino acid sequences represented by amino acid numbers 20-136 of SEQ ID NO: 20. Specifically, the heavy chain variable region comprises an amino acid sequence that has at least 95%, particularly at least 98%, identity with the amino acid sequences represented by amino acid numbers 20-136 of SEQ ID NO: 20. In these embodiments, the heavy chain variable region 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 deviation relative to the amino acid sequences represented by amino acid numbers 20-136 of SEQ ID NO: 20 is located in the frame region but not in the CDR. Specifically, the heavy chain variable region comprises the amino acid sequences represented by amino acid numbers 20-136 of SEQ ID NO: 20. In some embodiments, the amino acid at position 76 of SEQ ID NO: 20 is selected from glutamine, alanine, valine, histidine, tryptophan, tyrosine, lysine, and arginine; particularly glutamine, histidine, tryptophan, tyrosine, lysine, and arginine. Preferably, the amino acid at position 76 of SEQ ID NO: 20 is glutamine, histidine, tryptophan, lysine, or arginine, particularly glutamine. Specifically, CDR-H2 has the amino acid sequence of SEQ ID NO: 7 and / or the heavy chain variable region comprises the amino acid sequence represented by amino acid numbers 20-136 of SEQ ID NO: 23.

[0059] In specific embodiments, the light chain variable region comprises an amino acid sequence that has at least 90% identity with the amino acid sequences represented by amino acid numbers 21-133 of SEQ ID NO: 21. Specifically, the light chain variable region comprises an amino acid sequence that has at least 95%, particularly at least 98%, identity with the amino acid sequences represented by amino acid numbers 21-133 of SEQ ID NO: 21. In these embodiments, the light chain variable region still comprises a CDR having the amino acid sequences of SEQ ID NO: 4, 5, and 6. Therefore, any sequence deviation relative to the amino acid sequences represented by amino acid numbers 21-133 of SEQ ID NO: 21 resides in the frame region but not in the CDR. Specifically, the light chain variable region comprises the amino acid sequences represented by amino acid numbers 21-133 of SEQ ID NO: 21.

[0060] In a specific embodiment, the heavy chain variable region has an amino acid sequence that is at least 90% identical to the amino acid sequences represented by amino acid numbers 20-136 of SEQ ID NO: 20, wherein the CDR still has the amino acid sequences of SEQ ID NO: 1, 7, and 3, and the light chain variable region has an amino acid sequence that is at least 90% identical to the amino acid sequences represented by amino acid numbers 21-133 of SEQ ID NO: 21, wherein the CDR still has the amino acid sequences of SEQ ID NO: 4, 5, and 6. Specifically, the heavy chain variable region has an amino acid sequence that is at least 95% identical to the amino acid sequences represented by amino acid numbers 20-136 of SEQ ID NO: 20, wherein the CDR still has the amino acid sequences of SEQ ID NO: 1, 7, and 3, and the light chain variable region has an amino acid sequence that is at least 95% identical to the amino acid sequences represented by amino acid numbers 21-133 of SEQ ID NO: 21, wherein the CDR still has the amino acid sequences of SEQ ID NO: 4, 5, and 6.

[0061] In specific embodiments, the heavy chain comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 15. Specifically, the heavy chain comprises an amino acid sequence having at least 95%, particularly at least 98%, identity with the amino acid sequence of SEQ ID NO: 15. In these embodiments, the heavy 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 deviation relative to SEQ ID NO: 15 is located in the frame region but not in the CDR. Specifically, the heavy chain comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments, the amino acid at position 57 of SEQ ID NO: 15 is selected from glutamine, alanine, valine, histidine, tryptophan, tyrosine, lysine, and arginine; particularly glutamine, histidine, tryptophan, tyrosine, lysine, and arginine. Preferably, the amino acid at position 57 of SEQ ID NO: 15 is glutamine, histidine, tryptophan, lysine, or arginine, especially glutamine. Specifically, CDR-H2 has the amino acid sequence of SEQ ID NO: 7 and / or the heavy chain variable region contains the amino acid sequence represented by amino acid numbers 20-136 of SEQ ID NO: 22.

[0062] In specific embodiments, the light chain comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 16. Specifically, the light chain comprises an amino acid sequence having at least 95%, particularly at least 98%, identity with the amino acid sequence of SEQ ID NO: 16. In these embodiments, the light chain still comprises a CDR having the amino acid sequences of SEQ ID NO: 4, 5, and 6. Therefore, any sequence deviations relative to SEQ ID NO: 16 are located within the frame region but not within the CDR. Specifically, the light chain comprises the amino acid sequence of SEQ ID NO: 16.

[0063] In a specific embodiment, the heavy chain has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 15, wherein the CDR still has the amino acid sequences of SEQ ID NO: 1, 7, and 3, and the light chain variable region has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 16, wherein the CDR still has the amino acid sequences of SEQ ID NO: 4, 5, and 6. Specifically, the heavy chain has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 15, wherein the CDR still has the amino acid sequences of SEQ ID NO: 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, wherein the CDR still has the amino acid sequences of SEQ ID NO: 4, 5, and 6.

[0064] The antibodies according to the present invention include and encompass their modified forms. Modified forms of the antibodies of the present invention refer to antibodies of the present invention having chemical or biological modifications. Chemically modified forms include those having an amino acid backbone conjugated to a chemical moiety, those having chemically modified N-linked or O-linked carbohydrate chains, etc. The chemical moiety or form may be toxic or cytotoxic. Biologically modified forms include those that have undergone post-translational modifications (e.g., N-linked or O-linked glycosylation, N-terminal or C-terminal processing, deamidation, isomerization of aspartic acid, or oxidation of methionine), those containing methionine residues added to the N-terminus through expression in prokaryotic host cells, etc. Such modified forms are also intended to include those labeled to allow detection or isolation of the antibodies or antigens of the present invention, for example, enzyme-labeled forms, fluorescently labeled forms, or affinity-labeled forms. Such modified forms of the antibodies of the present invention can be used to improve the stability or blood retention of the original antibodies of the present invention, reduce antigenicity, detect or isolate antibodies or antigens, etc.

[0065] Specifically, the antibody may comprise one or more modifications selected from the following: defucosylation, reduced fucose, N-linked glycosylation, O-linked glycosylation, N-terminal processing, C-terminal processing, deamidation, isomerization of aspartic acid, oxidation of methionine, substitution of two leucine (L) residues at positions 234 and 235 of the heavy chain for alanine (A) (LALA), amidation of proline residues, and deletion or omission of one, two, or three amino acids at the carboxyl terminus. In a specific embodiment, the antibody lacks one, two, or three carboxyl-terminal amino acids in one or two heavy chains, or it lacks one carboxyl-terminal amino acid and the carboxyl-terminal proline residue is amidated at one or two heavy chains.

[0066] Such modifications can be made at any or desired location on the antibody. Alternatively, the same or two or more different modifications can be made at one, two or more of these locations.

[0067] For example, antibodies produced from cultured mammalian cells are known to lack a carboxyl-terminal lysine residue in their heavy chain (Journal of Chromatography A, 705: 129-134 (1995)). It is also known that occasionally two carboxyl-terminal amino acid residues (i.e., glycine and lysine) are lost from the heavy chain, and newly located proline residues at the carboxyl terminus are amidated (Analytical Biochemistry, 360: 75-83 (2007)). However, such deletions or modifications in these heavy chain sequences do not affect the antibody's ability to bind to its antigens, nor do they affect the antibody's effector functions (complement activation, antibody-dependent cytotoxicity, etc.).

[0068] In some embodiments, the antibody comprises the deletion or omission of one or two amino acids at the carboxyl terminus of the heavy chain and has amidated residues (e.g., amidated proline residues at the carboxyl terminus of the heavy chain). However, the antibodies according to the invention are not limited to the types described above, as long as the deletion mutant retains the ability to bind antigens.

[0069] In some embodiments, the two heavy chains of the antibody according to the invention may consist of any of the following types of heavy chains: full-length heavy chains and deletion mutant heavy chains, or a combination of any two types selected therefrom. The quantitative ratio of the deletion mutant heavy chains depends on the type of mammalian cells cultured to produce the antibody according to the invention and the cell culture conditions.

[0070] In a specific embodiment, the antibody according to the present invention may include two heavy chains, both of which lack a carboxyl-terminal amino acid residue.

[0071] In a specific embodiment, the antibody comprises a heavy chain and a light chain, the heavy chain having an amino acid sequence represented by amino acid numbers 1-446 of SEQ ID NO: 15 or 22, and the light chain having an amino acid sequence represented by amino acid numbers 1-219 of SEQ ID NO: 16. In some embodiments, the amino acid at position 57 of SEQ ID NO: 15 is selected from glutamine, alanine, valine, histidine, tryptophan, tyrosine, lysine, and arginine; particularly glutamine, histidine, tryptophan, tyrosine, lysine, and arginine. Preferably, the amino acid at position 57 of SEQ ID NO: 15 is glutamine, histidine, tryptophan, lysine, or arginine, particularly glutamine.

[0072] In some embodiments, the antibody according to the invention competes with the following antibodies for binding to TA-MUC1: an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 10 and a light chain variable region having the amino acid sequence of SEQ ID NO: 12, or an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 11 and a light chain variable region having the amino acid sequence of SEQ ID NO: 12.

[0073] In some embodiments, the antibody has the following properties: (a) specifically binds to MUC1, and / or (b) has the activity of being internalized into cells expressing MUC1 by binding to MUC1.

[0074] In some embodiments, the antibody comprises at least one antibody heavy chain. Specifically, the antibody comprises two antibody heavy chains. The antibody heavy chain specifically comprises a VH domain, a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain. In some other embodiments, the antibody heavy chain comprises the CH2 and CH3 domains but not the CH1 domain. In other embodiments, one or more constant domains of the heavy chain may be replaced by other domains, particularly similar domains, such as albumin. The antibody heavy chain can be of any type, including γ-, α-, ε-, δ-, and μ-chains, and is preferably a γ-chain, including γ1-, γ2-, γ3-, and γ4-chains, especially the γ1-chain. Therefore, the antibody is preferably an IgG-type antibody, such as IgG1-, IgG3-, or IgG4-type antibodies, especially IgG1-type antibodies.

[0075] Specifically, the antibody further comprises at least one antibody light chain, particularly two antibody light chains. The antibody light chains specifically comprise a VL domain and a CL domain. The antibody light chains can be κ-chains or λ-chains, especially κ-chains.

[0076] In some embodiments, the antibody comprises two antibody heavy chains and two antibody light chains. Specifically, the antibody comprises two γ1-type antibody heavy chains and two κ-type antibody light chains, each antibody heavy chain comprising a VH domain, a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain, and each antibody light chain comprising a VL domain and a CL domain.

[0077] In alternative embodiments, the antibody does not contain an antibody light chain. In these embodiments, the light chain variable region may be fused to the N-terminus of the heavy chain variable region or inserted into the C-terminus of the heavy chain variable region. Peptide linkers may be present to connect the light chain variable region to the remainder of the heavy chain.

[0078] In a preferred embodiment, the antibody comprises an Fc region. The antibody may particularly be a complete antibody comprising two heavy chains and two light chains, each heavy chain comprising domains VH, CH1, a hinge region, CH2, and CH3, and each light chain comprising domains VL and CL. This antibody is particularly capable of binding to one or more human Fcγ receptors, especially human Fcγ receptor IIIA. In alternative embodiments, the antibody does not bind or does not significantly bind to human Fcγ receptor IIIA, especially not to or does not significantly bind to any human Fcγ receptor. In these embodiments, the antibody particularly does not contain a glycosylation site in the CH2 domain.

[0079] In alternative embodiments, the antibody does not contain an Fc region. In these embodiments, the antibody is particularly a single-chain variable region fragment (scFv) or another antibody fragment that does not contain an Fc region.

[0080] Glycosylation of anti-MUC1 antibody Anti-MUC1 antibodies may contain a CH2 domain in one or more antibody heavy chains. Natural human antibodies of the IgG type contain an N-glycosylation site in the CH2 domain. The CH2 domain present in the antibody may or may not contain an N-glycosylation site. In some embodiments, the antibody does not contain a glycosylation site in the CH2 domain. Specifically, according to the IMGT / Eu numbering system, the antibody does not contain an asparagine residue in the heavy chain corresponding to position 297. For example, the antibody may contain an Ala297 mutation in the heavy chain. In these embodiments, the antibody preferably has a strong ability to reduce or completely lack the ability to induce antibody-dependent cellular cytotoxicity (ADCC) and / or antibody-dependent cellular phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC) by binding to the Fcγ receptor. The significantly reduced ability in this regard specifically refers to an activity reduced to 10% or less, particularly 3% or less, 1% or less, or 0.1% or less, compared to the same antibodies that contain an N-glycosylation site in their CH2 domain and have a common mammalian glycosylation pattern (e.g., those that can be obtained by production in human or CHO cell lines, such as the glycosylation pattern described herein). In these embodiments, the antibody is specifically an IgG1-type antibody.

[0081] In an alternative embodiment, the CH2 domain present in the antibody contains an N-glycosylation site. This glycosylation site is specifically located at amino acid position 297 of the heavy chain according to the IMGT / Eu numbering system and has the amino acid sequence motif Asn Xaa Ser / Thr, where Xaa can be any amino acid except proline. The N-linked glycosylation at Asn297 is conserved in homologous regions of mammalian IgG and other antibody isotypes. The actual location of this conserved glycosylation site can vary within the amino acid sequence of the antibody due to optional additional amino acids or other sequence modifications that may be present in the variable region. Preferably, the glycan attached to the antibody is a biantennary complex N-linked carbohydrate structure, preferably comprising at least the following structures: Asn - GlcNAc - GlcNAc - Man - (Man - GlcNAc)2 Wherein, Asn is an asparagine residue of the polypeptide moiety of the antibody; GlcNAc is N-acetylglucosamine, and Man is mannose. The terminal GlcNAc residue may further carry a galactose residue, which may optionally carry a sialic acid residue. Another GlcNAc residue (referred to as the bimeric GlcNAc) may be attached to the Man residue closest to the polypeptide. Fucose may bind to the GlcNAc attached to Asn. In these embodiments, the antibody is specifically an IgG1-type antibody.

[0082] In a preferred embodiment, the antibody does not contain N-hydroxyacetylneuraminic acid (NeuGc) or a detectable amount of NeuGc. Furthermore, the antibody preferably does not contain a Galili epitope (Galα1,3-Gal structure) or a detectable amount of Galili epitope. Specifically, the relative amount of glycans carrying NeuGc and / or Galα1,3-Gal structures is less than 0.1% or even less than 0.02% of the total amount of glycans attached to the CH2 domain of the antibody in the antibody population.

[0083] Specifically, the antibody has a human glycosylation pattern. Due to these glycosylation properties, there are no foreign immunogenic non-human structures that can induce side effects. This means that unwanted side effects or drawbacks known to be caused by certain foreign sugar structures (e.g., immunogenic non-human sialic acid (NeuGc) or Galili epitopes (Gal-Gal structures) known for rodent production systems, or other structures such as immunogenic high-mannose structures known from, for example, yeast systems) are avoided.

[0084] In specific embodiments, the antibody comprises a glycosylation pattern having a detectable amount of a glycan carrying bimeric GlcNAc residues. Specifically, the relative amount of the glycan carrying bimeric GlcNAc residues is at least 0.5%, particularly at least 1%, of the total amount of glycans in the composition attached to the glycosylation sites of the antibody. Furthermore, in some embodiments, the glycosylation pattern comprises a glycan carrying at least one galactose residue in a relative amount of at least 25% of the total amount of glycans in the composition attached to the antibody. Specifically, the relative amount of the glycan carrying at least one galactose residue is at least 30%, particularly at least 35% or at least 40%, of the total amount of glycans in the composition attached to the antibody. In specific embodiments, the glycosylation pattern comprises a glycan carrying at least one sialic acid residue in a relative amount of at least 1% of the total amount of glycans in the composition attached to the antibody. Specifically, the relative amount of the glycan carrying at least one sialic acid residue is at least 1.5%, particularly at least 2%, of the total amount of glycans in the composition attached to the antibody.

[0085] The antibody may have a glycosylation pattern with a large amount or a small amount of core fucose. Reducing the amount of fucose glycosylation increases the antibody's ability to induce ADCC. In some embodiments, the relative amount of the glycan carrying the core fucose residues is 40% or less, particularly 30% or less, or 20% or less, of the total amount of glycans attached to the antibody in the composition. In alternative embodiments, the relative amount of the glycan carrying the core fucose residues is at least 60%, particularly at least 65%, or at least 70%, of the total amount of glycans attached to the antibody in the composition.

[0086] The ability of an antibody to induce ADCC and the intensity of ADCC induction can be controlled by the presence or absence of glycosylation sites in the CH2 domain of the anti-MUC1 antibody and the presence or absence of fucosylation in the glycan structure at the glycosylation sites. ADCC activity is increased by glycosylation of the Fc moiety of the antibody and further increased by reducing the amount of fucosylation in the glycosylation. In some applications, fine-tuning of ADCC activity is important. Therefore, in some cases, antibodies without glycosylation sites in the CH2 domain, antibodies with glycosylation sites in the CH2 domain and abundant fucosylation, or antibodies with glycosylation sites in the CH2 domain and minimal fucosylation may be most advantageous.

[0087] Production of anti-MUC1 antibodies Antibodies are preferably generated through recombinant production within host cells. The host cells used for antibody production can be any host cell suitable for antibody generation. Suitable host cells are specifically eukaryotic host cells, particularly mammalian host cells. Exemplary host cells include yeast cells such as Pichia pastoris cell lines, insect cells such as SF9 and SF21 cell lines, plant cells, avian cells such as the EB66 duck cell line, rodent cells such as CHO, NSO, SP2 / 0, and YB2 / 0 cell lines, and human cells such as HEK293, PER.C6, CAP, CAP-T, AGE1.HN, Mutz-3, and KG1 cell lines.

[0088] In some embodiments, antibodies are recombinantly generated in human blood cell lines, particularly in human myeloid leukemia cell lines. Preferred human cell lines for antibody generation and suitable production procedures are described in WO 2008 / 028686 A2. In one specific embodiment, antibodies are obtained by expression in human myeloid leukemia cell lines selected from NM-H9D8, NM-H9D8-E6, and NM-H9D8-E6Q12 and cell lines derived therefrom. These cell lines are deposited in accordance with the requirements of the Budapest Treaty at Glycotope GmbH, Robert-Rössle-Str. 10, 13125 Berlin (DE) under accession numbers DSM ACC2806 (NM-H9D8; deposited on September 15, 2006), DSM ACC2807 (NM-H9D8-E6; deposited on October 5, 2006) and DSM ACC2856 (NM-H9D8-E6Q12; deposited on August 8, 2007), in Deutsche Sammlungvon Mikroorganismen und Zellkulturen (DSMZ), Inhoffenstraße 7B, 38124Braunschweig (DE). NM-H9D8 cells exhibit a glycosylation pattern characterized by high sialylation, high bipartite GlycNAc, high galactosylation, and high fucosylation. NM-H9D8-E6 and NM-H9D8-E6Q12 cells exhibit a similar glycosylation pattern to NM-H9D8 cells, except for very low fucosylation. Other suitable cell lines include K562 (a human myeloid leukemia cell line residing at the American Center for Type Culture Collection (ATCC CCL-243)) and cell lines derived from the above.

[0089] In other embodiments, antibodies are recombinantly generated in CHO cells. Specifically, antibodies can be recombinantly generated in CHO dhfr-cell lines such as ATCC No. CRL-9096.

[0090] Anti-MUC1 antibody conjugate In some embodiments, the antibody comprises one or more other reagents conjugated thereto. These other reagents can be any reagent suitable for conjugation with the antibody. If more than one other reagent is present in the antibody, these other reagents can be the same or different, and particularly all identical. The conjugation of the other reagents to the antibody can be achieved using any method known in the art. The other reagents can be attached to the antibody covalently (particularly by fusion or chemical coupling) or non-covalently. In some embodiments, the other reagents are covalently attached to the antibody, particularly via a linker portion. The linker portion can be any chemical entity suitable for attaching the other reagents to the antibody.

[0091] The other reagents are preferably used for the treatment, diagnosis, prognosis, detection, and / or monitoring of diseases, particularly cancer. For example, the other reagents may be selected from radionuclides, chemotherapeutic agents, antibodies or antibody fragments, especially those with different specificities than anti-MUC1 antibodies, such as checkpoint antibodies that block or activate immunomodulatory targets, enzymes, interacting domains, detectable markers, toxins, cell lysis components, immunomodulators, immune effectors, MHC class I or II antigens, and liposomes. A particularly preferred other reagent is a radionuclide or a cytotoxic agent capable of killing cancer cells, such as a chemotherapeutic agent. In some preferred embodiments, the chemotherapeutic agent is linked to the anti-MUC1 antibody to form a conjugate.

[0092] Specific examples of chemotherapeutic agents that can be conjugated to other agents include alkylating agents such as cisplatin, antimetabolites, plant alkaloids and terpenoids, vinca alkaloids, podophyllotoxin, taxanes such as paclitaxel, topoisomerase inhibitors such as irinotecan and topotecan, antitumor drugs such as doxorubicin, or microtubule inhibitors such as erstatins and metansine / metansine.

[0093] The chemotherapeutic agents may be specifically selected from: V-ATPase inhibitors, apoptosis-promoting agents, Bcl2 inhibitors, MCL1 inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule stabilizers, microtubule destabilizers, urostatin, dolalastatin, metansine, metansine derivatives, amatoxins, methionine aminopeptidase, inhibitors of nuclear export protein CRM1, DPPIV inhibitors, proteasome inhibitors, inhibitors of phosphoryl transfer reactions in mitochondria, protein synthesis inhibitors, kinase inhibitors, CDK2 inhibitors, CDK9 inhibitors, kinin inhibitors, HDAC inhibitors, DNA damaging agents, DNA alkylating agents, DNA intercalating agents, DNA minor groove binding agents, DHFR inhibitors, inhibitors of microtubule formation, microtubule stabilizers, actin stabilizers, topoisomerase II inhibitors, platinum compounds, ribosome inhibitors, RNA polymerase II inhibitors, and bacterial toxins. In a specific implementation, the chemotherapeutic agent is attached to an anti-MUC1 antibody selected from the following: tastatin, microtubule inhibitors such as metansine, DNA damaging agents, DNA alkylating agents, and DNA minor groove binding agents.

[0094] In some implementations, the chemotherapeutic agent is metansine or a metansine class. Specific examples of metansine classes that can be used for conjugation include maytanol, N 2' -Deacetylated- N 2' -(3-Mercapto-1-oxopropyl)-Metansin (DM1) N 2' -Deacetylated- N 2' -(4-Mercapto-1-oxopentyl)-Mestansin (DM3) and N 2' -Deacetylated- N 2'-(4-Methyl-4-mercapto-1-oxopentyl)-metansine (DM4). Specifically, DM1 or DM4 is attached to an anti-MUC1 antibody. In some embodiments, the chemotherapeutic agent attached to the anti-MUC1 antibody is an ostatin, particularly monomethyl ostatin F (MMAF), monomethyl ostatin E (MMAE), or ostatin T. In some embodiments, the chemotherapeutic agent attached to the anti-MUC1 antibody is a DNA minor groove binding agent, particularly pyrrolobenzodiazepine heptatriene (PBD), pyrrolobenzodiazepine heptatriene dimer (PBD dimer), docalamicin, docalamicin-hydroxybenzamide-azaindole (DUBA), open-ring-docalamicin-hydroxybenzamide-azaindole (open-ring-DUBA), or doxorubicin. In some embodiments, the chemotherapeutic agent attached to the anti-MUC1 antibody is a DNA alkylating agent, particularly indolinobenzodiazepine or oxazolidinobenzodiazepine. In some embodiments, the chemotherapeutic agent attached to the anti-MUC1 antibody is a DNA damaging agent, particularly kazidromycin. In some embodiments, the chemotherapeutic agent attached to the anti-MUC1 antibody is a microtubule formation inhibitor, particularly tubulysin, anserin, podophyllotoxin, or vincristine. In some embodiments, the chemotherapeutic agent attached to the anti-MUC1 antibody is a microtubule stabilizer, particularly paclitaxel or epothilone. In some embodiments, the chemotherapeutic agent attached to the anti-MUC1 antibody is an actin stabilizer, particularly phalloidin. In some embodiments, the chemotherapeutic agent attached to the anti-MUC1 antibody is a topoisomerase II inhibitor, particularly teniposide, XK469, razorizine, acridine, idarubicin, or mebarone. In some embodiments, the chemotherapeutic agent attached to the anti-MUC1 antibody is a platinum compound, particularly cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatinum tetranitrate, phenanthriplatin, pyridine, or sattraplatin. In some embodiments, the chemotherapeutic agent attached to the anti-MUC1 antibody is a ribosome inhibitor, particularly ricin, saponins, absinthecin, diphtheria toxin, or exotoxin A. In some embodiments, the chemotherapeutic agent attached to the anti-MUC1 antibody is an RNA polymerase II inhibitor, particularly amatoxins, such as muscarine. In some embodiments, the chemotherapeutic agent attached to the anti-MUC1 antibody is a bacterial toxin, particularly anthrax toxin. Suitable antibody-drug conjugates are also described in EP 16 151 774.3 and LU 92659, which are explicitly referenced herein.

[0095] In some embodiments, the other reagent is a polypeptide or protein. This polypeptide or protein may be specifically fused to the polypeptide chain of the antibody. In some embodiments, the other reagent, as a polypeptide or protein, is fused to the C-terminus of the antibody light chain. In embodiments where the antibody comprises two antibody light chains, the other reagent, as a polypeptide or protein, may be fused to the C-terminus of each of the two antibody light chains. In other embodiments, the other reagent, as a polypeptide or protein, is fused to the C-terminus of the antibody heavy chain. In embodiments where the antibody comprises two antibody heavy chains, the other reagent, as a polypeptide or protein, may be fused to the C-terminus of each of the two antibody heavy chains. The other reagent may be the same or different, and particularly has the same amino acid sequence. Suitable examples of such other reagents as polypeptides or proteins may be selected from cytokines, chemokines, antibodies, antigen-binding fragments, enzymes, and interacting domains.

[0096] In some embodiments, other agents as peptides or proteins are checkpoint antibodies that block and / or trigger activation signals. Examples of various targets include CD40, CD3, CD137 (4-1BB), OX40, GITR, CD27, CD278 (ICOS), CD154 (CD40 ligand), CD270 (HVEM), and CD258 (LIGHT) as activation targets, and CTLA4, PD1, CD80, CD244, A2AR, B7-H3 (CD276), B7-H4 (VTCN1), BTLA, IDO, KIR, LAG3, TIM-3, VISTA, and phosphatidylserine, and their corresponding ligands such as PDL1, as inhibition targets. In a specific instance, the anti-MUC1 antibody comprises two heavy chains and two light chains as described herein, wherein a CD3-specific scFv fragment is fused to the C-terminus of each heavy chain; or wherein a PDL1-specific scFv fragment is fused to the C-terminus of each light chain.

[0097] In other embodiments, other agents as peptides or proteins are immunomodulatory compounds such as chemokines, cytokines, or growth factors. Suitable cytokines in this regard include interferons such as interferon-α, interferon-β, and interferon-γ, and interleukins. Suitable growth factors include G-CSF and GM-CSF.

[0098] Nucleic acids, expression cassettes, vectors, cell lines, and compositions In another aspect, the present invention provides nucleic acids encoding antibodies. The nucleic acid sequence can have any nucleotide sequence suitable for encoding antibodies. However, preferably, the nucleic acid sequence is at least partially adapted to the specific codon selection of the host cell or organism in which the nucleic acid is to be expressed, particularly human codon selection. The nucleic acid can be double-stranded or single-stranded DNA or RNA, preferably double-stranded DNA such as cDNA or single-stranded RNA such as mRNA. It can be a single continuous nucleic acid molecule, or it can consist of several nucleic acid molecules, each encoding a different portion of the antibody. In a preferred embodiment, the present invention provides the nucleotide sequence of the heavy chain of the PankoMab variant (PM-N54Q) represented by SEQ ID NO: 17 and the nucleotide sequence of the light chain of the PankoMab variant (PM-N54Q) represented by SEQ ID NO: 18.

[0099] If an antibody consists of more than one distinct amino acid chain, such as the light and heavy chains of an antibody, the nucleic acid can be, for example, a single nucleic acid molecule containing several coding regions (each encoding one of the amino acid chains of the antibody, preferably separated by regulatory elements such as IRES elements) to produce individual amino acid chains, or the nucleic acid can consist of several nucleic acid molecules, each containing one or more coding regions, each coding region encoding one of the amino acid chains of the antibody. In addition to the coding regions encoding the antibody, the nucleic acid can also contain other nucleic acid sequences or other modifications, for example, that may encode other proteins, may affect the transcription and / or translation of the coding regions, may affect the stability or other physical or chemical properties of the nucleic acid, or may have no function at all.

[0100] In another aspect, the present invention provides an expression cassette or vector comprising a nucleic acid according to the invention and a promoter operatively linked to said nucleic acid. Furthermore, the expression cassette or vector may include other elements, particularly those capable of influencing and / or regulating the transcription and / or translation of the nucleic acid, the amplification and / or replication of the expression cassette or vector, the integration of the expression cassette or vector into the genome of a host cell, and / or the copy number of the expression cassette or vector in the host cell. Suitable expression cassettes and vectors containing various expression cassettes for expressing antibodies are well known in the art and therefore do not require further description herein.

[0101] Furthermore, the present invention provides a host cell comprising the nucleic acid according to the invention or the expression cassette or vector according to the invention. The host cell can be any host cell. It can be an isolated cell or a cell contained in a tissue. Preferably, the host cell is a cultured cell, particularly a primary cell or a cell of an established cell line, preferably a tumor-derived cell. Preferably, it is a bacterial cell such as *Escherichia coli*, a yeast cell such as *Saccharomyces cerevisiae*, particularly *Saccharomyces cerevisiae*, an insect cell such as Sf9 cells, or a mammalian cell, particularly a human cell such as a tumor-derived human cell, a hamster cell such as CHO, or a primate cell. In a preferred embodiment of the invention, the host cell is derived from human myeloid leukemia cells. Preferably, it is selected from the following cells or cell lines: K562, KG1, MUTZ-3, or cells or cell lines derived therefrom, or a mixture of cells or cell lines containing at least one of the above cells. The host cell is preferably selected from NM-H9D8, NM-H9D8-E6, NM H9D8-E6Q12, and cells or cell lines derived from any of the aforementioned host cells. These cell lines and their properties are described in detail in PCT application WO 2008 / 028686 A2. In other embodiments, the host cell is a CHO dhfr-cell line such as ATCC No. CRL-9096. In a preferred embodiment, the host cell is optimized for expressing glycoproteins, particularly antibodies, with a specific glycosylation pattern. Preferably, the codon selection and / or promoter in the coding region of the nucleic acid according to the invention, as well as other elements of the expression cassette or vector, are compatible with the type of host cell used, and more preferably, are optimized for the type of host cell used. Preferably, the antibody is produced from the host cell or cell line as described above.

[0102] In one specific aspect, the present invention provides a method for producing antibodies in host cells as described herein. This method specifically includes the steps of: providing a host cell containing nucleic acid encoding an antibody, culturing the host cell under conditions suitable for antibody expression, and obtaining the antibody expressed by the host cell. Antibodies according to the present invention can be obtained or are available through said method.

[0103] In another aspect, the present invention provides a composition comprising the antibody, the nucleic acid, the expression cassette or vector, or the host cell. The composition may also contain more than one of these components. Furthermore, the composition may contain one or more other components selected from solvents, diluents, and excipients. Preferably, the composition is a pharmaceutical composition. In this embodiment, the components of the composition are preferably all pharmaceutically acceptable. The composition may be a solid or fluid composition, particularly a solution (preferably an aqueous solution), emulsion or suspension, or a low-pressure lyophilized powder.

[0104] Uses in medicine The antibodies are particularly useful in medicine, especially for the treatment, diagnosis, prognosis, detection and / or monitoring of diseases (particularly those described herein, preferably cancer, infection, inflammatory diseases, graft-versus-host disease and immunodeficiency).

[0105] Therefore, in another aspect, the present invention provides antibodies, nucleic acids, expression cassettes or vectors, host cells, or compositions for use in pharmaceuticals. Preferably, the medical use is for the treatment, prognosis, diagnosis, detection, and / or monitoring of diseases, such as diseases associated with abnormal cell growth like cancer, infections such as bacterial, viral, fungal, or parasitic infections, inflammatory diseases such as autoimmune diseases and inflammatory bowel disease, and diseases associated with reduced immune activity such as immunodeficiency. In a preferred embodiment, the disease is cancer.

[0106] Preferably, the cancer cell exhibits detectable expression of MUC1 or TA-MUC1, which can be detected by immunohistochemistry, ELISA, RIA, ELISPOT assay, dot blot assay, Ouchterlony assay, or counterimmunoelectrophoresis (CIE) or in situ hybridization. It particularly includes cells expressing MUC1 or TA-MUC1, detectable by immunohistochemistry or in situ hybridization. The cancer can be tested for MUC1 or TA-MUC1 levels prior to the administration of anti-MUC1 antibodies.

[0107] The present invention further provides kits and devices comprising antibodies according to the invention, and related methods for the diagnosis, detection, or monitoring of MUC1-related disorders such as cancer. In some embodiments, a sandwich ELISA kit for testing, detection, or diagnosis is provided, comprising the antibodies of the present invention. The kit may further comprise one or more of the following: a solution of MUC1 or TA-MUC1 protein standards, a staining agent, a buffer solution for dilution, an antibody for a solid phase, an antibody for detection, and a washing solution, etc. Preferably, the amount of antibody bound to the antigen can be measured by applying methods such as absorbance, fluorescence, luminescence, or radioisotope (RI) methods. Preferably, an absorbance plate reader, a fluorescence plate reader, a luminescence plate reader, an RI liquid scintillation counter, etc., are used in the measurement.

[0108] In some embodiments, the present invention provides antibodies according to the invention for use in immunohistochemical (IHC) analysis and compositions comprising them.

[0109] There are no particular limitations to the immunohistochemistry, as long as the protocol includes reacting the tissue section with an antigen-binding antibody (first antibody) and detecting the first antibody that binds to the antigen.

[0110] Different forms of cancer, including metastatic lesions, can be treated with the antibodies according to the present invention. The cancers are particularly selected from colon cancer, lung cancer, ovarian cancer, breast cancer such as triple-negative breast cancer, pancreatic cancer, cervical cancer, endometrial cancer, gastrointestinal cancer, kidney cancer, head and neck cancer, thyroid cancer, and urothelial carcinoma. The cancers are further particularly selected from stomach cancer, liver cancer, bladder cancer, skin cancer, prostate cancer, and hematologic malignancies. In some embodiments, the cancer is a metastatic cancer. The cancer can include any type of metastatic lesion, such as skin metastases, lymph node metastases, lung metastases, liver metastases, peritoneal metastases, pleural metastases, and / or brain metastases. In some embodiments, the cancer has an inflammatory phenotype. In these embodiments, any of the above-mentioned cancer types can be inflammatory cancers.

[0111] In some embodiments, the viral infection is caused by human immunodeficiency virus, herpes simplex virus, Epstein-Barr virus, influenza virus, lymphocytic choriomeningitis virus, hepatitis B virus, or hepatitis C virus. Inflammatory diseases may be selected from inflammatory bowel disease, pelvic inflammatory disease, ischemic stroke, Alzheimer's disease, asthma, pemphigus vulgaris, and dermatitis / eczema. Autoimmune diseases may be selected from celiac disease, type 1 diabetes, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, vitiligo, psoriatic arthritis, atopic dermatitis, scleroderma, sarcoidosis, primary biliary cirrhosis, Graves-Barr syndrome, autoimmune hepatitis, and ankylosing spondylitis. In some embodiments, the disease includes or is associated with cells expressing MUC1, particularly TA-MUC1. For example, the cancer to be treated is MUC1 positive, especially TA-MUC1 positive, which means it contains cancer cells that express MUC1, especially TA-MUC1.

[0112] In a specific implementation, the antibody is used in combination with another therapeutic agent, particularly in combination with another anticancer agent, to treat cancer. The other therapeutic agent can be any known anticancer agent. Suitable anticancer therapeutic agents that can be combined with the antibody according to the invention can be chemotherapy agents, other antibodies, immunostimulants, cytokines, chemokines, and vaccines. Furthermore, antibody-based therapies can be combined with radiation therapy, surgery, and / or traditional Chinese medicine.

[0113] Anticancer agents that can be used in combination with anti-MUC1 antibodies can be selected from any chemotherapy agent, especially those known to be effective in treating MUC1-positive cancers. The type of chemotherapy agent also depends on the cancer to be treated. Combination partners can be selected from: taxanes such as paclitaxel (Taxol), docetaxel (Tasocor), and SB-T-1214; cyclophosphamide; imatinib; pazopanib; capecitabine; cytarabine; vinorelbine; gemcitabine; anthracycline antibiotics such as daunorubicin, doxorubicin, epirubicin, idarubicin, pentorubicin, and mitoxantrone; aromatase inhibitors such as aminoglutethimide, testosterone (Teslac), anastrozole (Renard), letrozole (Flon), exemestane (Anoxin), vortexilazole (Rivizor), formetatan (Lantalone), fazodazole (Afema), 4-hydroxyandrostenedione, 1,4,6-androstriene-3,17-dione (ATD), and 4-androsten-3,6,17-trione (6-oxo); topoisomerase inhibitors such as irinotecan, Topotecan, camptothecin, spirotinic acid D, etoposide (VP-16), teniposide, doxorubicin, daunorubicin, mitoxantrone, acridine, lecithin, ginsenoside tricarboxylic acid, and HU-331; platinum-based chemotherapeutic agents such as cis-diaminedichloroplatin(II) (cisplatin), cis-diaminedichloroplatin(II) (1,1-cyclobutanedicarboxylato) (carboplatin), and [(1R,2R)-cyclohexane-1,2-diamine](ethanedioato-O,O')platin(II). (Oxaliplatin); PARP inhibitors such as olaparib, rucaparib, and niraparib; TLR agonists such as imiquimod and requimod; and antimetabolites, especially antifolate agents such as methotrexate, pemetrexed, raltitrexed, and pralatrexate; pyrimidine analogs such as fluorouracil, gemcitabine, fluorouracil, 5-fluorouracil, and tegafur-uracil; and purine analogs, selective estrogen receptor modulators, and estrogen receptor demodulators.

[0114] Furthermore, therapeutic antibodies can also be used as further combination partners. This can be any antibody, other than the anti-MUC1 antibody, that is available for cancer treatment. Specifically, other antibodies have been approved for cancer treatment by administrative agencies such as the U.S. Food and Drug Administration (FDA), the European Medicines Agency (EMA, formerly EMEA), and the Bundesinstitut für Arzneimittel und Medizinprodukte (BfArM). Other examples of antibodies that can be used in combination therapy include anti-EGFR antibodies such as cetuximab, tomuzotuximab, pammumab, zalumab, nimotuzumab, matezumab, and neximumab; anti-HER2 antibodies such as trastuzumab, timigutuzumab, and pertuzumab; anti-VEGF antibodies such as bevacizumab (atorvastatin); anti-CD52 antibodies such as alenzumab (Campath); anti-CD30 antibodies such as brentuximab (Adcetris); anti-CD33 antibodies such as gemtruzumab (Mylotarg); and anti-CD20 antibodies such as rituximab (Rituxan, rituximab), tosimomumab (Bexxar), and tiimumab (Zeva). Other exemplary antibodies suitable for use in the cancer therapy combinations described herein include antibodies against antigens selected from the following: Thomsen-Friedenreich antigen (TFα, TFβ), Tn, Lewis Y, CD44, folate receptor α, NeuGc-GM3 ganglioside, DLL-3, RANKL, PTK7, Notch-3, Ephrin A4, insulin-like growth factor receptor 1, activin receptor-like kinase-1, blocking protein-6, disialotyl-ganglioside GD2, endothelial factor, transmembrane glycoprotein NMB, CD56, tumor-associated calcium signaling protein 2, tissue factor, exonucleotide pyrophosphatase / phosphodiesterase 3, CD70, P-cadherin, mesothelin, prostatic hexatransmembrane epithelial antigen 1 (STEAP1), carcinoembryonic antigen-associated cell adhesion molecule 5 (CEACAM5), connexin 4, guanylate cyclase C, Solute carrier family 44 member 4 (SLC44A4), prostate-specific membrane antigen (PSMA), zinc transporter ZIP6 (LIV1 (ZIP6)), SLIT and NTRK-like protein 6 (SLITRK6), trophoblast glycoprotein (TPBG; 5T4), Fyn3, carbonic anhydrase 9, NaPi2b, fibronectin outer domain B, endothelial angiotensin receptor ETB, VEGFR2 (CD309), tendon glycoprotein c, collagen IV and periosteal protein.

[0115] Anti-MUC1 antibodies can be further combined with checkpoint antibodies (i.e., antibodies that block or activate immunomodulatory targets). This allows for the blocking of inhibitory signals against the immune response and / or the triggering of activating signals. Examples of various targets include CD40, CD3, CD137 (4-1BB), OX40, GITR, CD27, CD278 (ICOS), CD154 (CD40 ligand), CD270 (HVEM), and CD258 (LIGHT) as activating targets, and CTLA4, PD1, CD80, CD244, A2AR, B7-H3 (CD276), B7-H4 (VTCN1), BTLA, IDO, KIR, LAG3, TIM-3, VISTA, and phosphatidylserine as inhibitory targets, and their corresponding ligands such as PDL1.

[0116] In other embodiments, the anti-MUC1 antibody can be combined with treatment using immunomodulatory compounds such as chemokines, cytokines, growth factors, and vaccines. Suitable cytokines in this regard include interferons such as interferon-α, interferon-β, and interferon-γ, as well as interleukins. Suitable growth factors include G-CSF and GM-CSF.

[0117] Anti-MUC1 antibodies are preferably used to treat primary tumors, recurrent tumors, and / or metastases of such tumors, and particularly for treatment before, during, or after surgery, as well as for the prevention or treatment of metastases. The anti-MUC1 antibody is particularly used as adjuvant therapy to treat patients. In some embodiments, the anti-MUC1 antibody is used as neoadjuvant therapy or in combination with neoadjuvant-adjuvant therapy to treat patients. Furthermore, the anti-MUC1 antibody is used as palliative therapy to treat patients.

[0118] Cancer treatment with anti-MUC1 antibodies preferably leads to inhibition of tumor growth, particularly reduction in tumor size. Furthermore, the treatment prevents further metastasis and / or reduces their number. This treatment preferably results in increased progression-free survival; and / or increased lifespan, thereby improving overall survival.

[0119] The present invention further provides methods for treating, diagnosing, prognosing, detecting, and / or monitoring diseases using antibodies according to the invention. Embodiments and examples of antibody application in pharmaceuticals are also applicable to medical methods. In particular, a method for treating a disease in a subject with this need is provided, the method comprising administering a therapeutically effective amount of the antibody according to the invention to the subject.

[0120] For example, the present invention provides a method for treating cancer in a subject with this need, the method comprising administering a therapeutically effective amount of an antibody according to the invention to a subject with cancer. In a specific embodiment, the cancer is characterized by expressing TA-MUC1. The cancer may be selected from ovarian cancer, breast cancer, pancreatic cancer, lung cancer, colon cancer, gastric cancer, liver cancer, kidney cancer, leukemia, endometrial cancer, thyroid cancer, leukemia, seminoma, 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, lymph node cancer, esophageal cancer, colorectal cancer, ear, nose and throat (ENT) cancer, prostate cancer, bladder cancer, uterine cancer and their metastases.

[0121] Furthermore, the present invention provides a method for diagnosing, detecting, or monitoring cancer, the method comprising the step of contacting a test sample with an antibody according to the present invention.

[0122] Methods to increase MUC1 binding affinity In another aspect, the present invention provides a method for increasing the MUC1 binding affinity of an antibody, the antibody comprising... (i) The heavy chain variable region comprising the following 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: 8, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and (ii) A light chain variable region comprising the following 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. The method includes the following steps: replacing the amino acid residue at position 8 of CDR-H2 with any amino acid residue other than asparagine, thereby generating CDR-H2 having the amino acid sequence of SEQ ID NO: 2.

[0123] Antibodies that increase MUC1 binding affinity are specifically antibodies that can bind to MUC1, as described herein, with the exception of asparagine contained at position 8 of the CDR-H2 sequence.

[0124] In some embodiments, the heavy chain variable region of the antibody to increase its MUC1 binding affinity comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 11. Specifically, the heavy chain variable region comprises an amino acid sequence having at least 95%, particularly at least 98%, identity with the amino acid sequence of SEQ ID NO: 11. In these embodiments, the heavy chain variable region still comprises a CDR having the amino acid sequences of SEQ ID NO: 1, 8, and 3. Therefore, any sequence deviations relative to SEQ ID NO: 11 reside in the frame region but not in the CDR. Specifically, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 11.

[0125] In some embodiments, the light chain variable region of the antibody to increase its MUC1 binding affinity comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 12. Specifically, the light chain variable region comprises an amino acid sequence having at least 95%, particularly at least 98%, identity with the amino acid sequence of SEQ ID NO: 12. In these embodiments, the light chain variable region still comprises a CDR having the amino acid sequences of SEQ ID NO: 4, 5, and 6. Therefore, any sequence deviations relative to SEQ ID NO: 12 reside in the frame region but not in the CDR. Specifically, the light chain variable region comprises the amino acid sequence of SEQ ID NO: 12.

[0126] In a specific embodiment, the heavy chain variable region of the antibody to increase its MUC1 binding affinity has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 11, wherein the CDR still has the amino acid sequences of SEQ ID NO: 1, 8, and 3, and the light chain variable region has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 12, wherein the CDR still has the amino acid sequences of SEQ ID NO: 4, 5, and 6. Specifically, 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, wherein the CDR still has the amino acid sequences of SEQ ID NO: 1, 8, and 3, and the light chain variable region has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 12, wherein the CDR still has the amino acid sequences of SEQ ID NO: 4, 5, and 6.

[0127] For example, antibodies that increase MUC1 binding affinity are the anti-MUC1 antibodies disclosed in WO 2004 / 065423 A2 or WO 2011 / 012309 A1. Specifically, antibodies that increase MUC1 binding affinity are gatipotuzumab or PankoMab.

[0128] Antibodies that increase MUC1 binding affinity are specifically antibodies that can bind to MUC1, as described in this article.

[0129] In some embodiments, MUC1 binding is as described herein. An increase in MUC1 or TA-MUC1 binding affinity specifically represents an increase of at least 10%, at least 20%, at least 33%, or at least 50%. In a preferred embodiment, the MUC1 binding affinity is increased by at least 50%. The MUC1 binding affinity can be determined, in particular, using surface plasmon resonance analysis or switchSENSE® Technology (DRX2 BIOSENSOR, manufactured by Dynamic Biosensors GmbH), for example, as described in Examples 4a and b.

[0130] In some embodiments, the substitution of an amino acid residue at position 8 of CDR-H2 is achieved by introducing a mutation into the nucleic acid encoding the antibody, wherein the mutation is introduced into the codon encoding said amino acid residue. The introduction of the mutation can be accomplished by any method. Several suitable methods are known in the art and can be performed by those skilled in the art to introduce the mutation. The mutated nucleic acid can then be expressed, for example, in a host cell to obtain an antibody with increased MUC1 binding affinity. The nucleic acid, host cell, and method used for antibody production are as described herein and can be used to increase MUC1 binding affinity.

[0131] In a specific implementation plan, the method for increasing the MUC1 binding affinity of the antibody includes the following steps: (a) Provide nucleic acids encoding antibodies that will increase their MUC1 binding affinity. (b) Introducing a mutation into the nucleic acid to produce a mutated nucleic acid, wherein the mutation is introduced into a codon encoding an amino acid residue at position 8 of CDR-H2, such that the codon encodes any amino acid residue other than asparagine; and (c) Express the mutated nucleic acid to produce an antibody with increased MUC1 binding affinity.

[0132] The present invention further provides a method for generating antibodies with increased MUC1 binding affinity, the method comprising: (a) Providing a nucleic acid encoding an antibody, said antibody comprising (i) The heavy chain variable region comprising the following 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: 8, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and (ii) A light chain variable region comprising the following 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; (b) Introducing a mutation into the nucleic acid to produce a mutated nucleic acid, wherein the mutation is introduced into a codon encoding an amino acid residue at position 8 of CDR-H2, such that the codon encodes any amino acid residue other than asparagine; and (c) Producing an antibody with increased MUC1 binding affinity by expressing the mutated nucleic acid in a host cell.

[0133] Regarding other aspects, particularly methods for increasing the MUC1 binding affinity of antibodies, the embodiments, features, and examples described herein are equally applicable to methods for producing antibodies with increased MUC1 binding affinity.

[0134] In some embodiments, the method for producing antibodies with increased MUC1 binding affinity further includes a step (d) of processing the antibody with increased MUC1 binding affinity.

[0135] For example, processing antibodies with increased MUC1 binding affinity may include isolating the antibodies from cell cultures. Antibody isolation specifically refers to separating the antibody from the remaining components of the cell culture. Antibody isolation from cell culture medium can be performed, for example, by chromatography. Suitable methods and means for isolating antibodies are known in the art and can be readily applied by those skilled in the art.

[0136] The obtained antibodies may optionally undergo further processing steps, such as modification steps, chemical or enzymatic coupling of the antibody with other reagents, and / or formulation steps, to produce antibodies of the desired quality and composition. Such further processing steps and methods are generally known in the art.

[0137] In other embodiments, step (d) further includes the step of providing a pharmaceutical formulation containing an antibody. Providing a pharmaceutical formulation containing an antibody or formulating an antibody into a pharmaceutical composition specifically includes exchanging a buffer solution or buffer solution component of the antibody-containing composition. Furthermore, this step may include low-pressure lyophilization of the antibody. Specifically, the antibody is transferred to a composition containing only pharmaceutically acceptable components.

[0138] Detailed Implementation Plan

[0139] Specific embodiments of the present invention are described below.

[0140] Implementation Scheme 1. An antibody capable of binding to MUC1, comprising... (i) The heavy chain variable region comprising the following 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 the following 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.

[0141] Implementation Scheme 2. The antibody according to Implementation Scheme 1, wherein the amino acid at position 8 of CDR-H2 is selected from glutamine, alanine, valine, histidine, tryptophan, tyrosine, lysine and arginine, especially glutamine, histidine, tryptophan, tyrosine, lysine and arginine.

[0142] Implementation Scheme 3. The antibody according to Implementation Scheme 1, wherein the amino acid at position 8 of CDR-H2 is glutamine, histidine, arginine, tryptophan or lysine.

[0143] Implementation Scheme 4. The antibody according to Implementation Schemes 1-3, wherein the CDR-H2 has the amino acid sequence of SEQ ID NO: 7.

[0144] Implementation Scheme 6. An antibody capable of binding to MUC1, comprising... (i) Heavy chain variable region, its (a) An amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 9, and (b) Containing the following 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) The variable region of the light chain, which (a) An amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 12, and (b) Contains the following 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.

[0145] Implementation Scheme 7. An antibody capable of binding to MUC1, comprising... (i) Heavy chain variable region, its (a) An amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 9, and (b) Containing the following 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) The variable region of the light chain, which (a) An amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 12, and (b) Contains the following 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.

[0146] Implementation Scheme 8. The antibody according to Implementation Scheme 6 or 7, wherein the amino acid at position 8 of CDR-H2 is selected from glutamine, alanine, valine, histidine, tryptophan, tyrosine, lysine and arginine, especially glutamine, histidine, tryptophan, tyrosine, lysine and arginine.

[0147] Implementation Scheme 9. The antibody according to Implementation Scheme 7 or 8, wherein the amino acid at position 8 of CDR-H2 is glutamine, histidine, arginine, tryptophan or lysine.

[0148] Implementation Scheme 10. An antibody capable of binding to MUC1, comprising... (i) Heavy chain variable region, its (a) An amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 10, and (b) Containing the following 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: 7, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and (ii) The variable region of the light chain, which (a) An amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 12, and (b) Contains the following 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.

[0149] Implementation Scheme 11. An antibody capable of binding to MUC1, comprising... (i) Heavy chain variable region, its (a) An amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 10, and (b) Containing the following 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: 7, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and (ii) The variable region of the light chain, which (a) An amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 12, and (b) Contains the following 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.

[0150] Implementation Scheme 12. An antibody capable of binding to MUC1, comprising... (i) The heavy chain variable region, which has the amino acid sequence of SEQ ID NO: 9, and (ii) Light chain variable region having the amino acid sequence of SEQ ID NO: 12.

[0151] Implementation Scheme 13. The antibody according to Implementation Scheme 12, wherein 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, histidine, tryptophan, tyrosine, lysine and arginine.

[0152] Implementation Scheme 14. The antibody according to Implementation Scheme 12, wherein the amino acid at position 57 of SEQ ID NO: 9 is glutamine, histidine, arginine, tryptophan or lysine.

[0153] Implementation Scheme 15. An antibody capable of binding to MUC1, comprising... (i) The heavy chain variable region, which has the amino acid sequence of SEQ ID NO: 10, and (ii) Light chain variable region having the amino acid sequence of SEQ ID NO: 12.

[0154] Implementation Scheme 16. An antibody capable of binding to MUC1, comprising... (i) Heavy chain variable region, its (a) Having an amino acid sequence that is at least 90% or at least 95% identical to the amino acid sequence represented by amino acid numbers 20-136 of SEQ ID NO: 20, and (b) Containing the following 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 or 7, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and (ii) The variable region of the light chain, which (a) Having an amino acid sequence that is at least 90% or at least 95% identical to the amino acid sequence represented by amino acid numbers 21-133 of SEQ ID NO: 21, and (b) Contains the following 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.

[0155] Implementation Scheme 17. An antibody capable of binding to MUC1, comprising... (i) The heavy chain variable region having an amino acid sequence represented by amino acid numbers 20-136 of SEQ ID NO: 20 or 23, and (ii) Light chain variable region having an amino acid sequence represented by amino acid numbers 21-133 of SEQ ID NO: 21.

[0156] Implementation Scheme 18. An antibody capable of binding to MUC1, comprising... (i) Heavy chain, its (a) An amino acid sequence having at least 90% or at least 95% identity with the amino acid sequence of SEQ ID NO: 15, and (b) Containing the following 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 or 7, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and (ii) The variable region of the light chain, which (a) An amino acid sequence having at least 90% or 95% identity with the amino acid sequence of SEQ ID NO: 16, and (b) Contains the following 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.

[0157] Implementation Scheme 19. An antibody capable of binding to MUC1, comprising... (i) The heavy chain variable region having the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 22, and (ii) Light chain variable region having the amino acid sequence of SEQ ID NO: 16.

[0158] Implementation Scheme 20. An antibody capable of binding to MUC1, comprising... (i) Heavy chain variable region, its (a) Having an amino acid sequence that is at least 90% or at least 95% identical to the amino acid sequence represented by amino acid numbers 20-460 of SEQ ID NO: 20, and (b) Containing the following 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 or 7, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and (ii) The variable region of the light chain, which (a) Having an amino acid sequence that is at least 90% or 95% identical to the amino acid sequence represented by amino acid numbers 21-239 of SEQ ID NO: 21, and (b) Contains the following 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.

[0159] Implementation Scheme 21. An antibody capable of binding to MUC1, comprising... (i) a heavy chain having an amino acid sequence represented by amino acid numbers 20-460 of SEQ ID NO: 20 or 23, and (ii) A light chain having an amino acid sequence represented by amino acid numbers 21-239 of SEQ ID NO: 21.

[0160] Implementation Scheme 22. An antibody according to any one of Implementation Schemes 1-21, wherein the antibody comprises at least one heavy chain, the heavy chain comprising a heavy chain variable region, a CH1 domain, a hinge region, a CH2 domain and a CH3 domain.

[0161] Implementation Scheme 23. An antibody according to any one of Implementation Schemes 1-21, wherein the antibody comprises two heavy chains, each heavy chain comprising a heavy chain variable region, a CH1 domain, a hinge region, a CH2 domain and a CH3 domain.

[0162] Implementation Scheme 24. An antibody according to Implementation Scheme 22 or 23, wherein the antibody is an IgG-type antibody, especially an IgG1, IgG2 or IgG4-type antibody.

[0163] Implementation Scheme 25. An antibody according to any one of Implementation Schemes 1-24, wherein the antibody comprises at least one light chain, the light chain comprising a light chain variable region and a CL domain.

[0164] Implementation Scheme 26. An antibody according to any one of Implementation Schemes 1-24, wherein the antibody comprises two light chains, each light chain comprising a light chain variable region and a CL domain.

[0165] Implementation Scheme 274. The antibody according to Implementation Scheme 25 or 26, wherein the light chain is a κ-type light chain.

[0166] Implementation Scheme 28. An antibody according to any one of Implementation Schemes 1-27, wherein the antibody does not contain an N-glycosylation site in the CH2 domain.

[0167] Implementation Scheme 29. An antibody according to any one of Implementation Schemes 1-27, wherein the antibody contains an N-glycosylation site in the CH2 domain of the antibody heavy chain.

[0168] Implementation Scheme 30. The antibody according to Implementation Scheme 29, wherein the glycosylation pattern of said antibody has one or more of the following characteristics: (i) The relative amount of the glycan carrying the bipartite GlcNAc residues is at least 0.5% of the total amount of glycans in the composition that are attached to the glycosylation sites of the antibody; (ii) The relative amount of the polysaccharide carrying at least one galactose residue is at least 30% of the total amount of polysaccharides in the composition that are attached to the glycosylation sites of the antibody; (iii) The relative amount of the glycan carrying the core fucose residue is at least 60% of the total amount of glycan in the composition that is attached to the glycosylation site of the antibody.

[0169] Implementation Scheme 31. The antibody according to Implementation Scheme 29, wherein the glycosylation pattern of said antibody has one or more of the following characteristics: (i) The relative amount of the glycan carrying the bipartite GlcNAc residues is at least 0.5% of the total amount of glycans in the composition that are attached to the glycosylation sites of the antibody; (ii) The relative amount of the polysaccharide carrying at least one galactose residue is at least 30% of the total amount of polysaccharides in the composition that are attached to the glycosylation sites of the antibody; (iii) The relative amount of the glycan carrying the core fucose residue is 40% or less of the total amount of glycans in the composition that are attached to the glycosylation sites of the antibody.

[0170] Implementation Scheme 32. An antibody according to any one of Implementation Schemes 1-31, which contains other reagents conjugated thereto.

[0171] Implementation Scheme 33. The antibody according to Implementation Scheme 32, wherein the other reagents are chemotherapeutic agents conjugated to the antibody.

[0172] Implementation Scheme 34. The antibody according to Implementation Scheme 33, wherein the chemotherapeutic agent is selected from metansine, DNA damaging agents, DNA alkylating agents and DNA minor groove binding agents.

[0173] Implementation Scheme 35. The antibody according to Implementation Scheme 33, wherein the chemotherapeutic agent is selected from maytansine, N 2' -Deacetylated- N 2' -(3-Mercapto-1-oxopropyl)-Metansin (DM1) N 2' -Deacetylated- N 2' -(4-Mercapto-1-oxopentyl)-Mestansin (DM3) and N 2' -Deacetylated- N 2' -(4-methyl-4-mercapto-1-oxopentyl)-methanin (DM4).

[0174] Implementation Scheme 36. The antibody according to Implementation Scheme 33, wherein the chemotherapeutic agent is selected from monomethylolstatin F (MMAF), monomethylolstatin E (MMAE), and olstatin T.

[0175] Implementation Scheme 37. The antibody according to Implementation Scheme 33, wherein the chemotherapeutic agent is selected from pyrrolobenzodiazepine heptatriene (PBD), pyrrolobenzodiazepine heptatriene dimer (PBD dimer), docalamicin, docalamicin-hydroxybenzamide-azaindole (DUBA), open-ring-docalamicin-hydroxybenzamide-azaindole (open-ring-DUBA), and doxorubicin.

[0176] Implementation Scheme 38. The antibody according to Implementation Scheme 33, wherein the chemotherapeutic agent is selected from indolinobenzodiazepine and oxazolidinobenzodiazepine.

[0177] Implementation Scheme 39. The antibody according to Implementation Scheme 33, wherein the chemotherapeutic agent is kazidromycin.

[0178] Implementation Scheme 40. The antibody according to Implementation Scheme 32, wherein the other reagent is a polypeptide or protein fused to the polypeptide chain of the antibody.

[0179] Implementation Scheme 41. An antibody according to Implementation Scheme 40, wherein the antibody comprises two antibody heavy chains and two antibody light chains, and is fused to each C-terminus of the antibody heavy chains or to each C-terminus of the antibody light chains as other reagents of a polypeptide or protein.

[0180] Implementation Scheme 42. The antibody according to Implementation Scheme 40 or 41, wherein the other reagents are selected from cytokines, chemokines, other antibodies, antigen-binding fragments, enzymes, and binding domains.

[0181] Implementation Scheme 43. The antibody according to Implementation Scheme 41, wherein the other reagent specifically binds to the scFv fragment of CD3, and one of the other reagents is fused to the C-terminus of each antibody heavy chain.

[0182] Implementation Scheme 44. The antibody according to Implementation Scheme 41, wherein the other reagent specifically binds to the scFv fragment of PDL1, and one of the other reagents is fused to the C-terminus of each antibody light chain.

[0183] Implementation Scheme 45. A nucleic acid that encodes an antibody according to any one of Implementation Schemes 1-44.

[0184] Implementation Scheme 46. An expression cassette or vector comprising a nucleic acid according to Implementation Scheme 75 and a promoter operatively linked to said nucleic acid.

[0185] Implementation Scheme 47. A host cell comprising a nucleic acid according to Implementation Scheme 45 or an expression cassette or vector according to Implementation Scheme 46.

[0186] Implementation Scheme 48. A pharmaceutical composition comprising an antibody or conjugate according to any one of Implementation Schemes 1-44 and one or more other components selected from solvents, diluents and excipients.

[0187] Implementation Scheme 49. An antibody according to any one of Implementation Schemes 1-44 or a pharmaceutical composition according to Implementation Scheme 48, used in a medicament.

[0188] Implementation Scheme 50. An antibody or a pharmaceutical composition according to any one of Implementation Schemes 1-44 for the treatment, prognosis, diagnosis, detection and / or monitoring of the following conditions: diseases associated with abnormal cell growth such as cancer; infections such as bacterial, viral, fungal or parasitic infections; inflammatory diseases such as autoimmune diseases and inflammatory bowel disease; and diseases associated with reduced immune activity such as immunodeficiency.

[0189] Implementation Scheme 51. An antibody or pharmaceutical composition according to Implementation Scheme 50 for the treatment of cancer, particularly cancers expressing TA-MUC1, wherein the cancer is selected from ovarian cancer, breast cancer, pancreatic cancer, lung cancer, colon cancer, gastric cancer, liver cancer, kidney cancer, leukemia, endometrial cancer, thyroid cancer, leukemia, seminoma, 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, lymph node cancer, esophageal cancer, colorectal cancer, ear, nose and throat (ENT) cancer, prostate cancer, bladder cancer, uterine cancer and their metastases.

[0190] Implementation Scheme 52. An antibody or pharmaceutical composition according to Implementation Scheme 50 for treating an infection, wherein the infection is selected from bacterial infections, viral infections, fungal infections, and parasitic infections.

[0191] Implementation Scheme 53. An antibody or pharmaceutical composition according to Implementation Scheme 50 for treating autoimmune diseases, wherein the autoimmune disease is selected from celiac disease, type 1 diabetes, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus.

[0192] Implementation Scheme 54. A method for increasing the MUC1 binding affinity of an antibody, said antibody comprising... (i) Heavy chain variable region, its (a) An amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 11, and (b) Containing the following 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: 8, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and (ii) The variable region of the light chain, which (a) An amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 12, and (b) Contains the following 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; The method includes the following steps: replacing the amino acid residue at position 8 of CDR-H2 with any amino acid residue other than asparagine, thereby generating CDR-H2 having the amino acid sequence of SEQ ID NO: 2.

[0193] Implementation Scheme 55. A method for increasing the MUC1 binding affinity of an antibody, said antibody comprising (i) Heavy chain variable region, its (a) An amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 11, and (b) Containing the following 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: 8, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and (ii) The variable region of the light chain, which (a) An amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 12, and (b) Contains the following 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; The method includes the following steps: replacing the amino acid residue at position 8 of CDR-H2 with any amino acid residue other than asparagine, thereby generating CDR-H2 having the amino acid sequence of SEQ ID NO: 2.

[0194] Implementation Scheme 56. The method according to Implementation Scheme 54 or 55, wherein the substitution of an amino acid residue at position 8 of CDR-H2 is achieved by introducing a mutation into the nucleic acid encoding the antibody, wherein the mutation is introduced into the codon encoding the amino acid residue.

[0195] Implementation Scheme 57. The method according to any one of Implementation Schemes 54-56, said method comprising the following steps: (a) Provide nucleic acids encoding antibodies that will increase their MUC1 binding affinity; (b) Introducing a mutation into the nucleic acid to produce a mutated nucleic acid, wherein the mutation is introduced into a codon encoding an amino acid residue at position 8 of CDR-H2, such that the codon encodes any amino acid residue other than asparagine; and (c) Express the mutated nucleic acid to produce an antibody with increased MUC1 binding affinity.

[0196] Implementation Scheme 58. A method for generating an antibody with increased MUC1 binding affinity, the method comprising: (a) Providing a nucleic acid encoding an antibody, said antibody comprising (i) Heavy chain variable region, its (i1) An amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 11, and (i2) Contains the following 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: 8, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and (ii) The variable region of the light chain, which (ii1) An amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 12, and (ii2) Contains the following 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; (b) Introducing a mutation into the nucleic acid to produce a mutated nucleic acid, wherein the mutation is introduced into a codon encoding an amino acid residue at position 8 of CDR-H2, such that the codon encodes any amino acid residue other than asparagine; and (c) Producing an antibody with increased MUC1 binding affinity by expressing the mutated nucleic acid in a host cell.

[0197] Implementation Scheme 59. A method for generating an antibody with increased MUC1 binding affinity, the method comprising: (a) Providing a nucleic acid encoding an antibody, said antibody comprising (i) Heavy chain variable region, its (i1) An amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 11, and (i2) Contains the following 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: 8, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and (ii) The variable region of the light chain, which (ii1) An amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 12, and (ii2) Contains the following 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; (b) Introducing a mutation into the nucleic acid to produce a mutated nucleic acid, wherein the mutation is introduced into a codon encoding an amino acid residue at position 8 of CDR-H2, such that the codon encodes any amino acid residue other than asparagine; and (c) Producing an antibody with increased MUC1 binding affinity by expressing the mutated nucleic acid in a host cell.

[0198] Implementation Scheme 60. The method according to any one of Implementation Schemes 54-59, wherein the antibody comprises two heavy chains, each heavy chain comprising a heavy chain variable region, a CH1 domain, a hinge region, a CH2 domain and a CH3 domain.

[0199] Implementation Scheme 61. The method according to Implementation Scheme 60, wherein the antibody is an IgG-type antibody, especially an IgG1, IgG2 or IgG4-type antibody.

[0200] Implementation Scheme 62. The method according to any one of Implementation Schemes 54-60, wherein the antibody comprises two light chains, each light chain comprising a light chain variable region and a CL domain.

[0201] Implementation Scheme 63. The method according to Implementation Scheme 62, wherein the light chain is a κ-type light chain.

[0202] Implementation Scheme 64. The method according to any one of Implementation Schemes 54-63, wherein the antibody having increased MUC1 binding affinity is an antibody defined in any one of Implementation Schemes 1-44.

[0203] Implementation Scheme 65. A method of treating cancer in a subject with this need, the method comprising administering to a subject having cancer, particularly cancer expressing TA-MUC1, a therapeutically effective amount of an antibody according to any one of Implementation Schemes 1-44 or a composition according to Implementation Scheme 48.

[0204] Implementation Scheme 66. A method for treating cancer according to Implementation Scheme 65, wherein the cancer is selected from ovarian cancer, breast cancer, pancreatic cancer, lung cancer, colon cancer, stomach cancer, liver cancer, kidney cancer, leukemia, endometrial cancer, thyroid cancer, leukemia, seminoma, 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, lymph node cancer, esophageal cancer, colorectal cancer, ear, nose and throat (ENT) cancer, prostate cancer, bladder cancer, uterine cancer and their metastases.

[0205] Attached Figure Figure 1 ELISA binding curves of anti-MUC1 antibodies with different MUC1 peptides are shown. (A) shows the binding of PankoMab N54Q (PM-N54Q) lacking Fab glycosylation and PankoMab (PM) containing Fab glycosylation to the antigen of a MUC1 peptide containing the epitope sequence PDTR. The threonine residues of the MUC1 peptides are glycosylated with Tn, sTn, TF, or sTF. (B) shows the binding of PankoMab and PankoMab N54Q to a MUC1 peptide containing the epitope sequence variant PESR. The serine residues of the MUC1 peptides are glycosylated with Tn. (C) shows the binding of PankoMab N54Q to a MUC1 peptide containing the epitope sequence PDTR. The threonine residues of the MUC1 peptides are either glycosylated with Tn or unglycosylated. (D) shows the binding of several N54X variants to the Tn-PDTR MUC1 peptide compared to Fab-glycosylated PankoMab diluted from cell culture supernatant of transiently transfected cells. (E) shows the binding profiles of three purified N54X variants without Fab glycosylation compared to PankoMab with Fab glycosylation on Tn-PDTR, TF-PDTR, and the unglycosylated PDTR MUC1 peptide. (F) shows the binding of two framework variants of PM-N54Q to the Tn-PDTR MUC1 peptide compared to PankoMab with Fab glycosylation. For framework variant mf-a, nine amino acids were mutated in the VH framework and three amino acids were mutated in the VL framework; for mf-b, nine amino acids were also mutated in the VH framework and four amino acids were mutated in the VL framework.

[0206] Figure 2 The surface plasmon resonance (Biacore) binding of anti-MUC1 antibodies PM and PM-N54Q to the Tn-glycosylated PDTR-MUC1 peptide was demonstrated. The maximum binding signal of different concentrations of PankoMab N54Q and PankoMab was plotted against antibody concentration.

[0207] Figure 3The results of fluorescence proximity sensing on the DRX instrument are shown. Association and dissociation curves are shown. (A) PM with Fab glycosylation relative to (B) PM-N54Q without Fab glycosylation.

[0208] Figure 4 The SDS-acrylamide gels show the electrophoretic separation of PankoMab N54Q and PankoMab under non-reducing (left) and reducing (right) conditions. Lane 1: PankoMab N54Q after the capture step; Lane 2: PankoMab N54Q after the polishing step; Lane 3: PankoMab after the capture step; Lane 4: PankoMab after the polishing step; Lane 5: Molecular weight label.

[0209] Figure 5 The gels stained with Coomassie blue as determined by isoelectric focusing are shown, in which PankoMab N54Q lacks Fab glycosylation and PankoMab is Fab glycosylated. Lane 1: PankoMab with Fab glycosylation; Lane 2: PankoMab N54Q without Fab glycosylation.

[0210] Figure 6 The anti-MUC1 antibody binds to Fcγ receptor IIIa. Increased concentrations of the antibody PankoMab N54Q or PankoMab replace rabbit-anti-mouse conjugated receptor beads with donor beads loaded with FcγRIIIa, thereby reducing the detected chemiluminescence. Figure 6 A low-to-medium fucosylation antibody and in Figure 6 High-fucosylation antibodies in B were used in the assay.

[0211] Figure 7 The binding of anti-MUC1 antibodies PM-N54Q, PM-N54D, and Fab-glycosylated PM to tumor cell lines (A) CaOV-3 and (B) HSC-4 was shown by flow cytometry analysis.

[0212] Figure 8 The amino acid sequence of the heavy chain of the humanized antibody PM N54Q is shown (SEQ ID No: 15, where the amino acid at position 57 is Gln, i.e., SEQ ID No: 22).

[0213] Figure 9 The amino acid sequence of the light chain of the humanized antibody PankoMab N54Q is shown (SEQ ID No: 16).

[0214] Figure 10 The amino acid sequence of the heavy chain of the humanized antibody PankoMab is shown (SEQ ID No: 19).

[0215] Figure 11 The amino acid sequence of the heavy chain of the chimeric antibody PankoMab N54Q is shown (SEQ ID No: 20, where the amino acid at position 76 is Gln, i.e., SEQ ID No: 23).

[0216] Figure 12 The amino acid sequence of the light chain of the chimeric antibody PankoMab N54Q is shown (SEQ ID No: 21). Example

[0217] Example 1: Production of anti-MUC1 antibodies The nucleic acid sequence of the heavy chain of the humanized PankoMab antibody was modified by mutating the codon of Asn54 (amino acid position 57 in SEQ ID NO: 11) according to the Kabat / EU numbering system to a codon of any amino acid other than Asn (especially Gln) (see, for example, WO 2011 / 012309).

[0218] 1) Production of anti-MUC1 antibodies in human myeloid leukemia-derived cell lines Vectors containing the coding sequences of the γ1-heavy chain and κ-light chain of the mutated antibody were transfected into the human myeloid leukemia-derived cell line NM-H9D8 (DSM ACC2806). Different αMUC1-antibodies (PankoMab N54X / PM-N54X, where X is any amino acid other than N / Asn) containing the N54X mutation or amino acid mutation in the frame sequences of VH and VL were expressed in the resulting clones to produce constructs with a human glycosylation pattern. The concentration of αMUC1-antibodies in the supernatant was determined by Octet measurement using a protein A-coated needle, or quantified by UV280 absorbance after protein A chromatography purification. The binding characteristics of different αMUC1-antibodies were determined by antigen-ELISA (see Example 2), and also by Scatchard analysis (see Example 3), Biacore analysis (see Example 4a), and DRX analysis. 2 The selected purified antibody can be analyzed using switchSENSE® technology (see Example 4b) or by flow cytometry (Example 7).

[0219] In addition, PM-N54Q and non-mutant PankoMab with Fab-glycosylation were expressed in the human myeloid leukemia-derived cell line NM-H9D8-E6Q12 (DSM ACC2856), which expresses an antibody with reduced fucose. These antibodies, along with the same antibody expressed in NM-H9D8, were purified and their binding behavior to Fcγ receptor III A was analyzed in Example 6.

[0220] 2) Production of anti-MUC1 antibody in CHO cell lines The PM-N54Q coding sequence (nucleotide sequence of the heavy chain of PM-N54Q represented by SEQ ID NO: 17 and nucleotide sequence of the light chain of PM-N54Q represented by SEQ ID NO: 18), synthesized by GeneArt™ from ThermoFisher Scientific, was cloned into an expression vector, and the resulting plasmid was electrotransfected into CHO cells. The combined cells grown under selection pressure were used to manufacture PM-N54Q mutant antibodies using standard procedures.

[0221] Example 2: Antigen ELISA The antigen-binding characteristics of PankoMab N54X (in which the N-glycosylation site in the Fab part is knocked out) were compared with those of PankoMab which has the N-glycosylation site in its Fab part.

[0222] In ELISA studies, the binding characteristics of the Fab-deglycosylated form (PM-N54Q) of the MUC1-specific antibody PankoMab relative to (glycosylated) PankoMab-GEX® were analyzed using different glycosylated and unglycosylated MUC1-derived tandem repeat peptides. Generally, by means of binding to the glycosylated PDTR peptide (APPAHGVTSAPD-T(X)-RPAPGSTAPPAHGVTSA) with different glycosylation at T, both antibodies exhibited similar gradations: binding to the Galß1-3 GalNAc-carrying peptide was observed. α The strongest binding of PDTR peptides to (TF) is followed by sialylated TF and GalNAc. α (Tn)O-glycosylation. Similar to sialylated GalNAc. α (sTn)O-glycosylation binding was significantly lower. As a PankoMab-GEX®, PM-N54Q exhibited only very weak binding activity to the unglycosylated MUC1 PDTR peptide, thus indicating appropriate tumor specificity ( Figure 1 C).

[0223] However, compared to PankoMab-GEX®, when using GalNAc... α (Tn)O-glycans were found to bind to PM-N54Q 4-fold more strongly in TA-MUC1 antigen ELISA of biotinylated glycopeptides. When sialylated GalNAc... α Upon (sTn) glycosylation, PM-N54Q binds approximately 7 times better to the same MUC1 peptide. This is achieved at the threonine residue of the PDTR sequence, binding to Galß1-3GalNAc. α The binding of (TF) and sialylated TF (sTF) Figure 1 A) It is twice as good as PM-N54Q.

[0224] Both antibodies exhibited significantly reduced binding to the MUC1 peptide variant APPAHGVTSAPE-S(Tn)-RPAPGSTAPPAHGVTSA with Tn glycosylation at serine compared to its counterpart at the PDT(Tn)R-peptide. However, here, Fab-deglycosylated PM-N54Q also bound significantly more strongly than PankoMab-GEX®. Figure 1 B).

[0225] Different other Fab-deglycosylated PM-N54X variants were compared with PankoMab, which has N-glycosylation in its Fab moiety. First, all variants were compared directly from the supernatant without purification. Concentrations were determined by Octet. All PM-N54X variants bound better than Fab-glycosylated PM. Furthermore, a clear trend in chemical properties dependent on the amino acid side chain was observed. The carboxylic acid group at the side chain exhibited the lowest binding enhancement. For amino acids with one or two nitrogen atoms (as primary or secondary amines), the best binding was observed. Figure 1 D).

[0226] In addition, the selected Fab-deglycosylated variants (PM-N54H, -W, and -Q) were purified by protein A chromatography and analyzed by ELISA. Figure 1 E). Compared with Fab-glycosylated PankoMab, the binding to TF-MUC1 peptide and Tn-MUC1 peptide was improved by approximately 5-8 times and approximately 2-3 times, respectively.

[0227] In addition, the binding of two different framework variants of PM-N54Q to the Tn-glycosylated PDTR-MUC1 peptide was analyzed in ELISA (see [link to ELISA]). Figure 1F). Frame variant mf-a carries a 9-amino acid mutation in the VH frame and a 3-amino acid mutation in the VL frame; variant mf-b also carries a 9-amino acid mutation in the VH frame and a 4-amino acid mutation in the VL frame. Both variants exhibited similar binding to the PM-N54Q antibody.

[0228] Example 3: Saturation binding analysis of anti-MUC1 antibody to MCF-7 and ZR-75-1 cells For antibody therapy suitability, two factors are particularly important: the antibody’s affinity for tumor cells and the number of binding sites.

[0229] The binding of the Fab-deglycosylated form of the MUC1-specific antibody PankoMab (PM-N54Q) to TA-MUC-1-positive human tumor cell lines was evaluated using radiolabeled antibodies, compared to Fab-glycosylated PankoMab-GEX®, by saturation binding assays on human breast cancer cell lines ZR-75-1 and MCF-7. The antibody was chelated with a 12-fold molar excess of p-SCN-benzyl-DTPA in 50 mM sodium carbonate and 150 mM NaCl (pH 8.7) at 37°C for 2 h, followed by overnight incubation at 2–8°C. Free chelating agent was removed by desalting column and dead-end filtration (50 kDa cutoff, 6x buffer exchange to PBS). The chelated antibody was then chelated with carrier-free HCl in 6 mM phosphate, 1.6 mM KCl, 80 mM NaCl, 0.2 M sodium acetate, and 0.1 M HCl. 111 In (2 µCi / µg antibody), radiolabeled antibody was radioactively incubated at 37°C for 1 h. The preparation was neutralized by adding 8–9 volumes of 10-fold concentrated PBS. Approximately 1 / 50 volume of fetal bovine serum was added to the neutralized labeled antibody preparation. 1 × 10⁻⁶ was used per cell binding protocol. 6 Cells were counted. Several concentrations of labeled antibodies were added to the precipitated cells (30-1000 ng / 200 µL, in 1% BSA / PBS). The resuspended cell-antibody mixture was measured in a gamma counter and incubated at 4°C for 1 h. Cells with bound antibodies were separated by centrifugation and washed with 1% BSA / PBS at 4°C for an additional 1 h. Cells were then measured in a gamma counter for binding. 111 In-labeled antibody measurements were performed on the cell precipitate. Evaluation was conducted in GraphPad Prism using "unit site specificity ka". The obtained data are summarized in Table 1. The data demonstrate high affinity and a very high number of PM-N54Q binding sites on these tumor cells. The binding was more than 2.5 times higher than that of PankoMab-GEX®, with a slight increase in the number of binding sites.

[0230] Table 1: In MUC1+ Association constants and antigen-binding sites on tumor cells .

[0231] Example 4a: Surface Plasmon Resonance (BiaCore Analysis) The binding of the Fab-deglycosylated form of the MUC1-specific antibody PankoMab (PM-N54Q) to TA-MUC-1-derived glycosylated peptides was evaluated using surface plasmon resonance (Biacore) analysis. The streptavidin sensor chip was coated with biotinylated TA-MUC1 peptides (Tn glycosylated or unglycosylated). PankoMab and PM-N54Q were sequentially diluted 1:3 in HPS-EP from 3,600 nM to 4.9 nM. Diluents were injected at a rate of 50 µL / min. The maximum binding at each concentration was determined as a response unit (RU) and evaluated using GraphPad Prism with "unit site-specific binding". Figure 2 Binding curves obtained with PM-N54Q compared to PankoMab-GEX® are shown. Affinities (Kb) of 388 nM and 652 nM were calculated for PM-N54Q and PankoMab-GEX®, respectively. D Therefore, in this experimental setting, an increase in affinity of nearly 2 times can be detected.

[0232] Example 4b: Fluorescent proximity sensing (via DRX) 2 Dynamic Biosensors A novel method for determining binding constants and affinities uses fluorescence proximity sensing of single-stranded DNA (96-mer) imprinted on a chip and complementary DNA coupled with a ligand. In this study, pankoMab was used as a ligand to capture the biotinylated TA-MUC1 peptide. Binding of the peptide to the pankoMab resulted in a fluorescence change. The association and dissociation rates could be calculated during association and dissociation. Due to the higher sensitivity, a faster interaction compared to surface plasmon resonance (SPR) could be monitored. This leads to binding kinetics different from SPR, but more comparable to the "gold standard" method KinExA (measured in a liquid system).

[0233] PankoMab and PM-N54Q were diluted from 300 nM to 3.67 nM in a 1:9 stepwise manner in PE140 buffer and applied to the chip-bound peptides. Binding curves were evaluated by single-exponential total fit (instrument software). Example binding curves of PM and PM-N54Q are shown below. Figure 3 In A and B, the calculated affinity of the PankoMab variants is shown in Table 2: Table 2: Dissociation constants of PankoMab variants for antigenic peptides .

[0234] Example 5: Biochemical characterization Antibody purity and identity were analyzed using both non-reducing and reducing SDS-PAGE. Banding patterns on non-reducing gels showed a dominant band at approximately 160 kDa, along with systematic artifacts of heavy and light chains and their combinations (~25, 50-55, 75, 110, 135 kDa). Reducing gels showed distinct light and heavy chain bands at 25 and 50-55 kDa. As expected, PM-N54Q exhibits a smaller heavy chain due to the lack of Fab glycosylation (see [link to relevant documentation]). Figure 4 right).

[0235] The charge distribution is significantly different, such as isoelectric focusing (IEF); see Figure 5 As shown in the figure, Fab glycosylation is significantly sialylated, while Fc glycosylation is only minimally sialylated. Therefore, PankoMab-GEX® has a more charged isoform than PM-N54Q, reflecting its higher level of negatively charged sialic acid in the Fab moiety.

[0236] Example 6: Fcγ receptor binding The FcγR binding assay of FcγRIIIa (CD16a) is based on PerkinElmer's AlphaScreen® technology. The AlphaScreen® platform relies on PerkinElmer's simple bead-based technology and is a more efficient alternative to traditional ELISA because it eliminates the need for a washing step.

[0237] For receptor binding assays, His-labeled FcγRIIIa (GlycotopeGmbH) was captured using Ni-chelate donor beads. Anti-MUC1 antibody and rabbit-anti-mouse conjugated receptor beads competed for binding with FcγR. In the presence of FcγR interacting with rabbit-anti-mouse conjugated receptor beads, the donor and receptor beads were very close, leading to luminescence upon 680 nm laser excitation. Maximum signal intensity was obtained in the absence of competitors. max In a competitive environment, when the test antibody binds to FcγR, the signal... maxThe concentration-dependent decrease was observed. Chemiluminescence was quantified using measurements at 520–620 nm with an EnSpire 2300 multi-label reader (PerkinElmer) (AlphaScreen® method). All results are expressed as mean ± standard deviation of replicates. Data were evaluated and calculated using nonlinear curve fitting (S-shaped dose-response variable slope) with GraphPad Prism 5 software. As a result, a concentration-dependent S-shaped curve was obtained, consisting of an upper plateau, a lower plateau, a slope, and EC. 50 definition.

[0238] As in Figure 6 As shown in Figures A and B, the binding affinity of FcγRIIIa is comparable for both PankoMab N54Q and PankoMab, thus allowing the use of low-fucosylated antibodies in the assay in Figure A and high-fucosylated antibodies in Figure B. Therefore, the removal of Fab glycosylation does not affect the antibody-receptor interaction.

[0239] Example 7: Binding with cellular TA-MUC1 N54Q and N54D were transiently expressed and purified by protein A chromatography. The binding of the two variants to cell surface TA-MUC1 was compared with that of PM with Fab glycosylation using two different cancer cell lines. The tongue squamous cell carcinoma line HSC-4 expressed TA-MUC1 to moderate levels, while the ovarian cancer cell line CaOV-3 expressed it to high levels. Tumor cells were incubated with serially diluted antibodies, and antibody binding was detected using phycoerythrin-conjugated goat anti-human IgG (heavy and light chains) antibodies. A human IgG control was included to control for background staining. Binding was analyzed by flow cytometry.

[0240] Compared to the human IgG1 control, the analyzed constructs PM, PM-N54Q, and PM-N54D showed strong and specific binding to HSC-4 and CaOV-3 cells expressing TA-MUC1. Figure 7 PM-N54D and TA-MUC1 高 The binding of CaOV-3 cells was comparable to that of PM with Fab glycosylation, while PM-N54Q showed slightly better binding. Figure 7 A). Using HSC-4 cancer cells expressing TA-MUC1 at moderate levels, the variant PM-N54Q showed significantly better binding to cellular TA-MUC1 compared to PM, while PM-N54D showed weaker binding compared to PM with Fab glycosylation. Figure 7 B).

[0241] Identification of preserved biological materials On the dates indicated in the table below, Glycotope GmbH, Robert-Rössle-Str. 10, 13125 Berlin (DE) deposited cell lines DSM ACC 2806, DSM ACC 2807 and DSM ACC 2856 at DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstraße 7B, 38124Braunschweig (DE). Cell line name Login ID collector Preservation date NM-H9D8 DSM ACC 2806 Glycotope GmbH September 15, 2006 NM-H9D8-E6 DSM ACC 2807 Glycotope GmbH October 5, 2006 NM-H9D8-E6Q12 DSM ACC 2856 Glycotope GmbH August 8, 2007 .

Claims

1. An antibody capable of binding to MUC1, comprising... (i) The heavy chain variable region comprising the following complementarity-determining regions (CDRs): CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 1, CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 7, and CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 3, and (ii) A light chain variable region comprising the following complementarity-determining regions (CDRs): CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 4, CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 5, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO:

6.

2. The antibody according to claim 1, wherein the heavy chain variable region consists of the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:

10.

3. The antibody according to claim 1 or 2, wherein the light chain variable region consists of the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:

12.

4. The antibody according to any one of claims 1-3, wherein the heavy chain variable region of the antibody consists of the amino acid sequence of SEQ ID NO: 10 and the light chain variable region of the antibody consists of the amino acid sequence of SEQ ID NO:

12.

5. The antibody according to any one of claims 1-4, wherein the antibody comprises an Fc region.

6. The antibody according to claim 5, wherein the antibody is an IgG1, IgG2 or IgG4-type antibody.

7. The antibody according to any one of claims 1-6, wherein the antibody comprises a heavy chain consisting of the amino acid sequence of SEQ ID NO: 22 and a light chain consisting of the amino acid sequence of SEQ ID NO:

16.

8. The antibody according to any one of claims 5-7, wherein the antibody comprises a glycosylation pattern having one or more of the following characteristics: (i) Detectable amounts of glycans carrying bipartite GlcNAc residues; (ii) The relative amount of the polysaccharide carrying at least one galactose residue is at least 25% of the total amount of polysaccharides in the composition that are attached to the Fc glycosylation site of the antibody.

9. The antibody according to any one of claims 1-8, wherein the antibody can be obtained by production in mammalian cells.

10. The antibody according to any one of claims 1-9, wherein the antibody can be produced in a human cell line selected from NM-H9D8 (DSM ACC 2806), NM-H9D8-E6 (DSM ACC 2807), NM-H9D8-E6Q12 (DSM ACC 2856) and cell lines derived therefrom.

11. The antibody according to any one of claims 1-9, wherein the antibody can be produced in a CHO cell line or a cell line derived therefrom.

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

12.

13. A nucleic acid encoding an antibody according to any one of claims 1-12.

14. An expression cassette or vector comprising the nucleic acid of claim 13 and a promoter operatively linked to the nucleic acid.

15. A host cell comprising the nucleic acid according to claim 13 or the expression cassette or vector according to claim 14.

16. A composition comprising an antibody according to any one of claims 1-12, a nucleic acid according to claim 13, an expression cassette or vector according to claim 14, and a host cell according to claim 15.

17. A method for generating an antibody with increased MUC1 binding affinity, the method comprising: (a) Providing a nucleic acid encoding an antibody, said antibody comprising (i) The heavy chain variable region comprising the following complementarity-determining regions (CDRs): CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 1, CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 8, and CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 3, and (ii) A light chain variable region comprising the following complementarity-determining regions (CDRs): CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 4, CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 5, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 6; (b) Introducing a mutation into the nucleic acid to produce a mutated nucleic acid, wherein the mutation is introduced into a codon encoding an amino acid residue at position 8 of CDR-H2, such that the codon encodes glutamine; and (c) Producing an antibody with increased MUC1 binding affinity by expressing the mutated nucleic acid in a host cell.

18. The method of claim 17, further comprising the steps of (d) Processing antibodies with increased MUC1 binding affinity.

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