RU2792347C2 - Antibody against muc1 - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to an antibody that binds to TA-MUC1. Also a nucleic acid encoding the said antibody; an expression cassette and a host cell containing the nucleic acid; an expression vector containing said expression cassette; a conjugate for the treatment of diseases associated with TA-MUC1 containing the specified antibody; a composition containing said antibody are disclosed. A method for producing the said antibody is disclosed.

EFFECT: invention makes it possible to effectively treat diseases associated with TA-MUC1.

25 cl, 12 dwg, 3 tbl, 7 ex

Description

Field of invention

The present invention relates to the field of antibodies. The present invention relates to a mutant anti-MUC1 antibody with increased binding affinity for the antigen. In particular, the present invention relates to a mutant version of the humanized PankoMab antibody, wherein the asparagine 57 of the heavy chain variable region is replaced with a different amino acid. Thus, the glycosylation site in the CDR2 region is deleted and the binding affinity for the antigen is increased. In specific embodiments, the implementation of the present invention relates to therapeutic and diagnostic use of this antibody and methods for obtaining such antibodies.

BACKGROUND OF THE INVENTION

Antibodies against tumor-specific antigens are widely used therapeutic agents against malignant neoplasms. Numerous antitumor antibodies have been approved for human therapy. Some of these antibodies act by blocking certain signal transduction pathways that are critical for the survival or proliferation of specific cancer cells. Other antitumor antibodies activate the patient's immune response against malignant target cells, for example, by initiating antibody-dependent cellular cytotoxicity (ADCC) via natural killer cells. This mechanism is induced by binding of the Fc portion of the antibody to Fc receptors on immune cells.

An interesting and important group of antibodies are those directed against mucin proteins. Mucins are a family of high molecular weight, highly glycosylated proteins produced by many vertebrate epithelial tissues. They can be subdivided into mucin proteins, which are membrane-bound due to the presence of a hydrophobic, membrane-permeating domain that facilitates its retention in the plasma membrane, and mucins, which are secreted on mucosal surfaces or secreted as components of saliva. The human mucin protein family consists of many family members, including the membrane-bound MUC1.

Increased mucin production occurs in many adenocarcinomas, including cancer of the pancreas, lung, breast, ovary, colon, and so on. Mucins are also overexpressed in lung diseases such as asthma, bronchitis, chronic obstructive pulmonary disease or cystic fibrosis. Two membrane mucins, MUC1 and MUC4, have been extensively studied for their role in the pathological process. In addition, mucins have also been investigated as potential diagnostic markers. Several antibodies against mucin proteins are known in this field (Clin. Cancer Res., 2011 Nov 1; 17(21):6822-30, PLoS One, 2011 Jan 14; 6(1):e15921), in particular MUC1. However, their therapeutic efficacy can still be improved.

In this regard, there is a need in this area for therapeutic antibodies against MUC1 with improved properties.

The essence of the invention

The present inventors found that deletion of the glycosylation site in the variable region of the heavy chain of the PankoMab anti-MUC1 antibody did not result in loss of antigen binding but unexpectedly increased the affinity of the antibody for the antigen. This was especially unexpected as the glycosylation site is in the second complementarity-determining region of the heavy chain variable region (CDR-H2). CDRs are regions of an antibody directly involved in antigen binding and providing contact with an epitope. Thus, in general, modification of CDR amino acids is expected to be unfavorable for antigen binding affinity. The humanized PankoMab antibody additionally contains a glycosylation site in CDR-H2 bearing a large carbohydrate structure. This carbohydrate structure is located directly on the antigen binding surface and thus is considered to be involved in antigen binding. However, as shown in the examples, the PankoMab (N54Q) variant, in which the glycosylation site is deleted by amino acid substitution bearing the carbohydrate structure, exhibits increased antigen binding affinity.

Thus, in a first aspect, the present invention relates to an antibody capable of binding to MUC1 containing

(i) a heavy chain variable region comprising complementarity determining regions (CDRs) of CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 2, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and

(ii) a light chain variable region comprising complementarity determining regions (CDRs) of 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.

In a second aspect, the present invention relates to a nucleic acid encoding an antibody of the invention. Furthermore, in a third aspect, the present invention relates to an expression cassette or vector comprising the nucleic acid of the invention and a promoter operably linked to said nucleic acid, and in a fourth aspect, the present invention relates to a host cell comprising the nucleic acid or expression cassette, or a vector according to the invention.

In a fifth aspect, the present invention relates to a conjugate containing an antibody of the invention conjugated to an additional agent.

In a sixth aspect, the present invention provides a composition comprising an antibody of the invention, a nucleic acid of the invention, an expression cassette or vector of the invention, a host cell of the invention, or a conjugate of the invention.

In a seventh aspect, the present invention relates to an antibody, nucleic acid, expression cassette or vector, host cell, composition or conjugate of the invention for use in medicine, in particular in the treatment, prevention or diagnosis of cancer.

In an eighth aspect, the present invention relates to a method for increasing the MUC1 binding affinity of an antibody comprising

(i) a heavy chain variable region comprising complementarity determining regions (CDRs) of 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 complementarity determining regions (CDRs) of CDR-L1 having the amino acid sequence of SEQ ID NO: 4, CDR-L2 having the amino acid sequence of SEQ ID NO: 5, and CDR-L3 having the amino acid sequence of SEQ ID NO: 6

a method comprising the step of replacing the amino acid residue at position 8 of CDR-H2 with any amino acid residue except asparagine, resulting in a CDR-H2 having the amino acid sequence of SEQ ID NO: 2.

In a ninth aspect, the present invention relates to a method for producing an antibody with increased binding affinity for MUC1, comprising

(a) obtaining a nucleic acid encoding an antibody containing

(i) a heavy chain variable region comprising complementarity determining regions (CDRs) of 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 complementarity determining regions (CDRs) of 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) inserting a mutation into said nucleic acid to produce a mutant nucleic acid, wherein the mutation is inserted into a codon encoding an amino acid residue at position 8 of CDR-H2 such that said codon codes for any amino acid residue except asparagine; And

(c) obtaining an antibody with increased binding affinity for MUC1 by expressing the mutant nucleic acid in a host cell.

In a tenth aspect, the present invention relates to a method of treating cancer in an individual in need thereof, comprising administering to the individual with cancer a therapeutically effective amount of an antibody of the invention, a nucleic acid of the invention, an expression cassette or vector of the invention, or a host cell of the invention.

In an eleventh aspect, the present invention relates to kits or devices containing an antibody of the invention and corresponding methods that can be used in the diagnosis, detection or monitoring of MUC1-associated disorders such as cancer.

Other objects, features, advantages and aspects of the present invention will be apparent to those skilled in the art from the following description and claims. However, it should be understood that the following description, claims and specific examples, in which preferred embodiments of the present invention are indicated, are for illustrative purposes only. Various changes and modifications to the spirit and scope of the described invention will become apparent to those skilled in the art from the following description.

DEFINITIONS

Within the scope of the invention, the following expressions will generally preferably have the following meanings, unless the context indicates otherwise.

Within the scope of the invention, the expression "comprise", in addition to its literal meaning, also includes and specifically refers to the expressions "consists essentially of" and "consists of". Thus, the expression "comprise" refers to embodiments where the subject matter "comprising" the specifically listed elements does not contain additional elements, as well as embodiments where the subject matter "comprising" the specifically listed elements may provide and / or actually includes additional elements. Similarly, the expression "has" should be understood as the expression "comprises", also including and specifically referring to the expressions "consists essentially of" and "consists of". The term "consists essentially of", if possible, particularly refers to embodiments where the subject matter contains 20% or less, in particular 15% or less, 10% or less, or in particular 5% or less additional elements in addition to the specifically listed elements of which, in essence, the subject matter of the invention consists.

The term "antibody", in particular, refers to a protein containing at least two heavy chains and two light chains connected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (V _H ) and a heavy chain constant region ( _CH ). Each light chain is composed of a light chain variable region (V _L ) and a light chain constant region (C _L ). The heavy chain constant region contains three, or in the case of IgM or IgE type antibodies four, heavy chain constant domains (C _H1 , C _H2 , C _H3 and C _H4 ), where the first C _H1 constant domain is adjacent to the variable region and can be connect to a second C _H2 constant domain via a hinge region. The light chain constant region consists of only one constant domain. Variable regions can be further divided into regions of hypervariability, referred to as complementarity determining regions (CDRs), interspersed with more conserved regions, referred to as framework regions (FRs), where each variable region contains three CDRs and four FRs. The variable regions of the heavy and light chains contain a binding domain that interacts with the antigen. The heavy chain constant regions 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. In addition, the light chain constant region can also be of any type, such as κ- or λ-type light chains. 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, respectively, having constant region amino acid sequences derived from naturally occurring heavy or light chain constant region amino acid sequences, in particular human heavy or light chain constant region amino acid sequences. In particular, the amino acid sequence of the heavy chain constant domains of the γ-type (in particular, γ1-type) is at least 95%, in particular at least 98% identical to the amino acid sequence of the heavy chain constant domains of the γ-type human antibody (in particular , γ1 of a person). In addition, the amino acid sequence of the κ-type light chain constant domain is, in particular, at least 95%, in particular at least 98% identical to the amino acid sequence of the κ light chain constant domain of a human antibody. Antibody constant regions can mediate immunoglobulin binding to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (C1q) of the classical complement system. The antibody may be, for example, a humanized, human or chimeric antibody.

The term "antigen-binding portion" of an antibody generally refers to the full-length antibody or one or more antibody fragments that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of antibody binding fragments include the Fab fragment, a monovalent fragment consisting of V _L , V _H , _CL and C _H1 domains; F(ab) ₂ fragment, a bivalent fragment containing two Fab fragments, each of which binds to the same antigen, connected by a disulfide bridge in the hinge region; Fd fragment consisting of V _H and C _H1 domains; Fv fragment consisting of the V _L and V _H domains of one antibody arm; and a dAb fragment consisting of a V _H domain.

The term "Fab portion" of an antibody specifically refers to the portion of the antibody containing the heavy and light chain variable regions (V _H and V _L ) and the first domains of the heavy and light chain constant regions (C _H1 and C _L ). In cases where the antibody does not contain all of these regions, the term "Fab-part" refers only to those of the V _H , V _L , C _H1 and _CL regions that are present in the antibody. Preferably, the term "Fab-part" refers to the part of the antibody corresponding to the fragment obtained by cleavage of natural antibodies with papain, corresponding to the antigen-binding activity of the antibody. In particular, the Fab portion of an antibody includes an antigen-binding site or has its antigen-binding ability. Preferably, the Fab portion contains at least the V _H region of the antibody.

The term "Fc portion" of an antibody specifically refers to the portion of the antibody containing heavy chain constant regions 2, 3, and, where appropriate, 4 (C _H2 , C _H3 and C _H4 ). In particular, the Fc portion contains two of each of these regions. In cases where an antibody does not contain all of these regions, the term "Fc portion" refers only to those of the C _H2 , C _H3 and C _H4 regions that are present in the antibody. Preferably, the Fc portion contains at least the C _H2 region of the antibody. Preferably, the term "Fc portion" refers to the portion of an antibody corresponding to a fragment obtained by cleavage of a natural antibody with papain that lacks the antibody's antigen-binding activity. In particular, the Fc portion of an antibody can bind to an Fc receptor and thus, for example, contains an Fc receptor binding site or has the ability to bind an Fc receptor.

As used herein, the terms "antibody" and "antibody construct" in certain embodiments refer to a population of antibodies or antibody constructs, respectively, of the same type. In particular, all antibodies or antibody constructs from a population exhibit features used to define an antibody or antibody construct. In some embodiments, all antibodies or antibody constructs in a population have an amino acid sequence. Reference to a particular type of antibody, such as an antibody capable of specifically binding to MUC1, specifically refers to a population of that type of antibody.

Within the scope of the invention, the term "antibody" also includes fragments and derivatives of said antibody. In particular, a "fragment or derivative" of an antibody is a protein or glycoprotein derived from said antibody and capable of binding to the same antigen, in particular the same epitope, as the antibody. Thus, within the scope of the invention, the term "fragment or derivative" of an antibody generally refers to a functional fragment or derivative. In particularly preferred embodiments, the antibody fragment or derivative contains a heavy chain variable region. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody or its derivatives. Examples of antibody fragments include (i) Fab fragments, monovalent fragments consisting of the variable region and the first constant domain of each of the heavy and light chains; (ii) F(ab) ₂ fragments, bivalent fragments containing two Fab fragments connected by a disulfide bridge in the hinge region; (iii) Fd fragments consisting of the variable region and the first constant domain CH1 of the heavy chain; (iv) Fv fragments consisting of the heavy chain and light chain variable region of one antibody arm; (v) scFv fragments, Fv fragments consisting of a single polypeptide chain; (vi) (Fv) ₂ fragments consisting of two Fv fragments covalently linked to each other; (vii) heavy chain variable domain; and (viii) multibodies consisting of a heavy chain variable region and a light chain variable region covalently linked to each other such that the binding of the heavy chain and light chain variable regions can only occur intermolecularly but not intramolecularly. Antibody derivatives specifically 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 prepared by conventional methods known to those skilled in the art.

The target amino acid sequence is "derived" from the reference amino acid sequence, or it "corresponds" to the reference amino acid sequence, if the target amino acid sequence has at least 75% homology or identity over its entire length with respect to the corresponding portion of the reference amino acid sequence, 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%. The term "corresponding part" means that, for example, variable heavy chain (FRH1) framework 1 of the target antibody corresponds to variable heavy chain framework 1 of the reference antibody. In specific embodiments, the target amino acid sequence "derived from" or "corresponding to" the reference amino acid sequence is 100% homologous, or in particular 100% identical throughout its length, with respect to the corresponding portion of the reference amino acid sequence. The "homology" or "identity" of an amino acid sequence or a nucleotide sequence is preferably determined within the scope of the invention over the entire length of the reference sequence, or over the entire length of the corresponding portion of the reference sequence corresponding to the sequence whose homology or identity is determined. An antibody derived from a parent antibody defined by one or more amino acid sequences, such as CDR specific sequences or variable region specific sequences, in particular, is an antibody having amino acid sequences, such as CDR sequences or variable region sequences, 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 or identical, in particular identical to the corresponding amino acid sequences of the parent antibody. In some embodiments, an antibody derived from (ie, derived from) a parent antibody contains the same CDR sequences as the parent antibody, but differs in other variable region sequences.

Within the scope of the invention, the term "antibody" also refers to multivalent and multispecific antibodies, ie. antibody constructs having more than two binding sites that each bind to the same epitope, and antibody constructs having one or more binding sites that bind to a first epitope and one or more binding sites that bind to a second epitope, and, optionally even additional binding sites binding to additional epitopes.

Preferably, the term "specific binding" means that an agent, such as an antibody, binds more strongly to a target, such as an epitope, for which it is specific, compared to binding to another target. The agent binds more strongly to the first target than to the second target if it binds to the first target with a dissociation constant (K _d ) that is lower than the dissociation constant for the second target. Preferably, the dissociation constant for a target to which the agent specifically binds is more than 100, 200, 500 times, or more than 1000 times lower than the dissociation constant for a target to which the agent does not specifically bind. In addition, the term "specific binding" in particular means a binding affinity between binding partners with an affinity constant K _a of at least 10 ⁶ M ^-1 , preferably at least 10 ⁷ M ^-1 , more preferably at least 10 ⁸ M ^-1 . The term "antibody specific for an antigen" specifically refers to an antibody capable of binding to said antigen with an affinity for a K _a of at least 10 ⁶ M ^-1 , preferably at least 10 ⁷ M ^-1 , more preferably - at least 10 ⁸ M ^-1 . For example, the term "anti-MUC1 antibody" specifically refers to an antibody that specifically binds to MUC1 and preferably is capable of binding to MUC1 with an affinity for a K _{a of} at least 10 ⁶ M ^-1 , preferably at least 10 ⁷ M ^-1 , more preferably at least 10 ⁸ M ^-1 .

The term "MUC1" refers to the MUC1 protein, also known as mucin-1, polymorphic epithelial mucin (PEM), or cancer antigen 15-3, specifically human MUC1 (accession number P15941). MUC1 is a member of the mucin family and encodes a membrane-bound, glycosylated phosphoprotein. MUC1 has a core protein mass of 120-225 kDa, rising to 250-500 kDa upon glycosylation. It protrudes 200-500 nm beyond the cell surface. The protein is anchored to the apical surface of many epithelial cells by a transmembrane domain. The extracellular domain includes a variable tandem repeat (VNTR) domain of 20 amino acids, with the number of repeats varying from 20 to 120 in different individuals. These repeats are rich in serine, threonine, and proline residues, enabling strong O-glycosylation. In some embodiments, the term "MUC1" refers to tumor-associated MUC1 ("TA-MUC1"). TA-MUC1 is MUC1 present on malignant cells. This MUC1 differs from the MUC1 present on non-malignant cells by its much higher expression level, localization and glycosylation. In particular, TA-MUC1 is located apolarly over the entire surface of malignant cells, while in non-malignant cells MUC1 has a strictly apical expression and is thus inaccessible to systemically administered antibodies. In addition, TA-MUC1 has abnormal O-glycosylation, which exposes new peptide epitopes on the backbone of the MUC1 protein and new carbohydrate tumor antigens such as the Thomsen-Friedenreich alpha (TFα) antigen.

The term "TFα", also referred to as Thomsen-Friedenreich alpha or Core-1 antigen, refers to the disaccharide Gal-β1,3-GalNAc bound as an O-glycoside in the alpha anomeric configuration to the hydroxyamino acids serine or threonine proteins in carcinoma cells.

The term "sialic acid" specifically refers to any N- or O-substituted neuraminic acid derivatives. It may refer to 5-N-acetylneuraminic acid and 5-N-glycolylneuraminic acid, but preferably only refers to 5-N-acetylneuraminic acid. Sialic acid, in particular 5-N-acetylneuraminic acid, is preferably attached to the carbohydrate chain via a 2,3 or 2,6 bond. Preferably, 2,3-as well as 2,6-linked sialic acids are present in the antibodies provided herein.

As used herein, the term "relative amount of glycans" refers to a particular percentage or percentage range of glycans attached to antibodies from an antibody preparation or antibody formulation, respectively. In particular, the term "relative amount of glycans" refers to a specific percentage or range of percentages of all glycans contained in antibodies and thus attached to the polypeptide chains of antibodies in the preparation of antibodies or composition containing antibodies. The term "100% glycans" refers to all glycans attached to antibodies from an antibody preparation or in an antibody composition, respectively. For example, the term "relative amount of glycans carrying GlcNAc at the branch point, 10%" refers to a composition containing antibodies, where 10% of all glycans contained in the antibodies and thus attached to the polypeptide chains of antibodies in the specified composition contain a GlcNAc residue at the branch point, while 90% of all glycans contained in the antibodies and thus attached to the antibody polypeptide chains in said composition do not contain a GlcNAc residue at the branch point. The corresponding reference amount of glycans, which is 100%, can be all glycan structures attached to the antibodies in the composition, or all N-glycans, i.e. all glycan structures attached to the asparagine residue in the antibodies in the composition, or all complex-type glycans. The reference group of glycan structures is usually specifically indicated or directly obvious to a person skilled in the art from the conditions.

The term "N-glycosylation" refers to all glycans attached to the asparagine residues of the polypeptide chain of a protein. These asparagine residues are typically part of N-glycosylation sites having the amino acid sequence Asn-Xaa-Ser/Thr, where Xaa can be any amino acid except proline. Similarly, "N-glycans" are glycans 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 generally share a core structure consisting of two N-acetylglucosamine (GlcNAc) and three mannose residues, having the structure Manα1,6-(Manα1,3-)Manβ1,4-GlcNAcβ1,4-GlcNAcβ1-Asn , wherein Asn is an asparagine residue of the polypeptide chain. N-glycans are divided into three different types, namely complex type glycans, hybrid type glycans and high mannose glycans.

The numbers given in the present description, in particular, the relative extent of specific glycosylation, preferably, should be understood as approximate values. In particular, the numbers may preferably be 10% higher and/or lower, in particular up to 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% higher and / or below.

In a "conjugate", two or more compounds are linked to each other. In some embodiments, at least some of the properties of each compound are retained in the conjugate. Bonding can be achieved through a covalent or non-covalent bond. Preferably, the compounds from the conjugate are connected via a covalent bond. Different compounds from the conjugate can be directly linked to each other using one or more covalent bonds between the atoms of the compound. Alternatively, the compounds can be linked to each other using a chemical moiety, such as a linker molecule, where the linker is covalently bonded to the atoms of the compound. If the conjugate consists of more than two compounds, these compounds, for example, can be connected in a chain conformation, one connection can be connected to the next connection, or each of several connections can be connected to one central connection.

The term "nucleic acid" includes single-stranded and double-stranded nucleic acids and ribonucleic acids, as well as deoxyribonucleic acids. They may contain natural as well as synthetic nucleotides and may be naturally or synthetically modified, for example by methylation, 5' and/or 3' capping.

The term "expression cassette" specifically refers to a nucleic acid construct capable of allowing and regulating the expression of a nucleic acid coding sequence inserted therein. The expression cassette may contain promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation. The exact structure of the expression cassette may vary as a function of species or cell type, but typically contains 5'-untranscribed and 5'- and 3'-untranslated sequences involved in transcription and translation initiation, respectively, such as the TATA box, cap sequence, CAAT sequence, etc. More specifically, the 5' non-transcribed expression control sequences contain a promoter region including a promoter sequence for transcriptionally controlling the operably linked nucleic acid. Expression cassettes may also contain enhancer sequences or upstream activator sequences.

In the context of the invention, the term "promoter" refers to a nucleic acid sequence upstream (5') of the nucleic acid sequence to be expressed and controlling the expression of the sequence, providing a recognition and binding site for RNA polymerases. A "promoter" may include additional recognition and binding sites for additional factors involved in the regulation of gene transcription. A promoter may control transcription of a prokaryotic or eukaryotic gene. In addition, the promoter may be "inducible", ie. initiate transcription in response to an inducing agent, or may be "constitutive" if transcription is not controlled by an inducing agent. A gene under the control of an inducible promoter is not expressed or is expressed only to a small extent in the absence of an inducing agent. In the presence of an inducing agent, the gene is activated or the level of transcription is increased. This is mediated mainly by the binding of a specific transcription factor.

The term "vector" is used in the present description in its broadest meaning, and it includes any intermediate carrier for a nucleic acid, allowing the specified nucleic acid, for example, to be inserted into prokaryotic and/or eukaryotic cells and, if necessary, integrated into the genome. Vectors of this type are preferably replicated and/or expressed in cells. Vectors include plasmids, phagemids, bacteriophages, or viral genomes. In the context of the invention, the term "plasmid" generally refers to a construct of extrachromosomal genetic material, typically a circular duplex of DNA, which can replicate independently of chromosomal DNA.

As used herein, the term "host cell" refers to any cell that can be transformed or transfected using exogenous nucleic acid. In the context of the invention, the term "host cells" includes prokaryotic (eg E. coli) or eukaryotic cells (eg mammalian cells, in particular human cells, yeast cells and insect cells). Particularly preferred are mammalian cells, such as those of humans, mice, hamsters, pigs, goats or primates. Cells can be obtained from a variety of tissue types and include primary cells and cell lines. The nucleic acid may be present in the host cell in the form of one copy or two or more copies, and in one embodiment is expressed in the host cell.

For the purposes of the invention, the term "patient" means a human, non-human primate or other animal, in particular a mammal such as a cow, horse, pig, sheep, goat, dog, cat or rodent such as a mouse and a rat. In a particularly preferred embodiment, the patient is a human.

Within the scope of the invention, the term "malignant neoplasm" specifically includes leukemias, seminomas, melanomas, carcinomas, teratomas, lymphomas, sarcomas, mesotheliomas, neuroblastomas, gliomas, rectal cancer, endometrial cancer, kidney cancer, adrenal cancer, thyroid cancer, hemoblastosis, skin cancer, malignant neoplasm of the brain, cervical cancer, malignant neoplasm of the intestine, liver cancer, cancer of the large intestine, stomach cancer, malignant neoplasm of the intestine, head and neck cancer, malignant neoplasm of the gastrointestinal tract, malignant neoplasm of the lymph nodes, cancer of the esophagus , 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. Within the scope of the invention, the term "malignant neoplasm" also includes metastases of a malignant neoplasm.

The term "tumor" means a group of cells or tissue resulting from misregulated cell proliferation. Tumors may show partial or complete lack of structural organization and functional coordination with normal tissue and typically form a discrete mass of tissue that may be benign or malignant.

The term "metastasis" means the spread of malignant cells from the original focus to another part of the body. Metastasis formation is a very complex process and typically involves malignant cells detaching from the primary tumor, entering the bloodstream, and settling to grow in normal tissues elsewhere in the body. If the tumor cells metastasize, the new tumor is called a secondary or metastatic tumor, and its cells tend to resemble those in the original tumor. This means, for example, that if breast cancer metastasizes to the lungs, the secondary tumor is made up of abnormal breast cells and not abnormal lung cells. In this case, the tumor in the lung is called metastatic breast cancer, not lung cancer.

The term "pharmaceutical composition" specifically refers to a composition suitable for administration to a human or animal, i. compositions containing components that are pharmaceutically acceptable. Preferably, the pharmaceutical composition contains the active compound, or a salt or prodrug thereof, together with a carrier, diluent, or pharmaceutical excipient such as a buffer, preservative, and tonicity regulator.

The numerical ranges provided herein are inclusive of the numbers defining the range. The headings given in the present description do not limit the various aspects or embodiments of the present invention, which can be read by referring to the description as a whole. In one embodiment, the subject matter described herein as containing some steps in the case of methods, or as containing some ingredients in the case of compositions, refers to the subject matter of the respective steps or ingredients. It is preferable to select and combine the preferred aspects and embodiments presented in the present description, and the specific object of the invention arising from the appropriate combination of preferred embodiments also belongs to the present invention.

Detailed description of the invention

The present invention is based on the development of a humanized anti-MUC1 PankoMab variant wherein the glycosylation site in CDR-H2 has been deleted. Deletions of the glycosylation site are achieved by replacing the heavy chain variable region amino acid Asn (asparagine) 57 with another amino acid, in particular Gln (glutamine). Asn 57 is the acceptor amino acid residue of the glycosylation site to which the carbohydrate structure is attached. Replacing this asparagine residue with another residue abolishes glycosylation, as the carbohydrate structure can only be transferred to an asparagine residue by host cell enzymes. Surprisingly, deletion of the glycosylation site in PankoMab's CDR-H2 increased the binding affinity of the antibody to the antigen.

In this regard, the present invention relates to an antibody capable of binding to MUC1 containing

(i) a heavy chain variable region comprising complementarity determining regions (CDRs) of CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 2, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and

(ii) a light chain variable region comprising complementarity determining regions (CDRs) of 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.

Binding to MUC1

The antibody specifically binds to the MUC1 epitope. The term "specific binding" means binding that is not non-specific adsorption. Examples of criteria for determining whether a binding is specific or not may include the dissociation constant (herein referred to as "K _D "). The epitope is found in the extracellular tandem repeats of MUC1. In some embodiments, the antibody binds to MUC1 in a glycosylation dependent manner. In particular, the antibody binds more strongly if said tandem repeats are glycosylated at the threonine residue with 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 molecule is linked to the threonine residue by an α-O-glycosidic bond. An epitope in the MUC1 tandem repeat domain specifically contains the amino acid sequence of PDTR (SEQ ID NO: 13) or PESR (SEQ ID NO: 14). Binding to this epitope preferably depends on glycosylation as described above, where, in particular, binding is increased if the carbohydrate molecule described above is attached to a threonine residue of a PDTR or PESR sequence (SEQ ID NOS: 13 and 14), respectively.

The epitope is a tumor-associated MUC1 epitope (TA-MUC1). The term "TA-MUC1 epitope" specifically refers to a MUC1 epitope found on tumor cells but not on normal cells and/or accessible only to antibodies in the host's circulation when present on tumor cells but not when it is present on normal cells. In some embodiments, antibody binding to cells expressing a TA-MUC1 epitope is stronger than binding to cells expressing normal, non-tumor MUC1. Preferably said binding is at least 1.5 times stronger, preferably at least 2 times stronger, at least 5 times stronger, at least 10 times stronger, or at least 100 times stronger. In the case of TA-MUC1 binding, the antibody preferably binds specifically to the glycosylated MUC1 tumor epitope such that the strength of the bond is at least 2-fold, preferably 4- or 10-fold, most preferably 20-fold, compared to binding to non-glycosylated MUC1. peptide of identical length and identical peptide sequence. Said binding may be assayed or determined by ELISA, RIA, surface plasmon resonance (hereinafter referred to as "SPR") assay, or the like. Examples of equipment used in SPR analysis may 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(TM ) (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 an antibody to an antigen expressed on the cell surface can be analyzed by flow cytometry or the like.

In addition, the antibody may exhibit antigen-binding properties similar to those of a reference antibody comprising a heavy chain variable region of the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 10 and a light chain variable region of the amino acid sequence of SEQ ID NO: 12. Preferably, the reference antibody is a humanized PankoMab antibody. In particular, the antibody of the invention specifically binds to the same antigen as the reference antibody and preferably binds to said antigen with higher affinity. Those. the antibody preferably binds to the antigen with an affinity with a dissociation constant lower than that of the reference antibody, more preferably at least 10% lower, at least 20% lower, at least 30% lower or at least 50% lower. In addition, the antibody preferably shows cross-specificity with a reference antibody comprising a heavy chain variable region with the amino acid sequence of SEQ ID NO: 11 and a light chain variable region with the amino acid sequence of SEQ ID NO: 12. In particular, a humanized antibody can block the binding of the reference antibodies with MUC1, if it is present in a sufficiently high concentration. This is possible if binding of the reference antibody to MUC1 is sterically hindered when the antibody of the invention is already bound to the MUC1 antigen.

Anti-MUC1 antibody

An antibody of the present invention capable of binding to MUC1 comprises a heavy chain variable region comprising complementarity determining regions (CDRs) of 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 a light chain variable region containing 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 a CDR-L3 having the amino acid sequence of SEQ ID NO: 6.

In some embodiments, the heavy chain variable region contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 9. In particular, the heavy chain variable region contains an amino acid sequence that is at least 95%, in particular, at least 98% identical to the amino acid sequence of SEQ ID NO: 9. In these embodiments, the heavy chain variable region still contains CDRs having the amino acid sequences of SEQ ID NO: 1, 2, and 3. Thus, any sequence deviations from SEQ ID NO: 9 are located in the framework regions but not in the CDRs. In particular, the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 9.

In some embodiments, the 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 the group consisting of glutamine, alanine, valine, histidine, tryptophan, tyrosine, lysine, and arginine; in particular, 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, in particular glutamine. In particular, CDR-H2 has the amino acid sequence of SEQ ID NO: 7.

In specific embodiments, the heavy chain variable region contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 10. In particular, the heavy chain variable region contains an amino acid sequence that is at least 95%, in particular , at least 98% identical to the amino acid sequence of SEQ ID NO: 10. In these embodiments, the heavy chain variable region comprises a CDR-H1 having the amino acid sequence of SEQ ID NO: 1, a CDR-H2 having the amino acid sequence of SEQ ID NO: 7 , and a CDR-H3 having the amino acid sequence of SEQ ID NO: 3. Thus, any sequence deviations from SEQ ID NO: 10 are located in the framework regions but not in the CDRs. In particular, the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 10.

In some embodiments, the light chain variable region contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 12. In particular, the light chain variable region contains an amino acid sequence that is at least 95%, in particular, at least 98% identical to the amino acid sequence of SEQ ID NO: 12. In these embodiments, the light chain variable region still contains CDRs having the amino acid sequences of SEQ ID NO: 4, 5, and 6. Thus, any sequence deviations from SEQ ID NO: 12 are located in the framework regions and not in the CDRs. In particular, the light chain variable region contains the amino acid sequence of SEQ ID NO: 12.

In specific embodiments, 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, where the CDRs still have 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, where the CDRs still have the amino acid sequences of SEQ ID NO: 4, 5, and 6. In particular, the heavy chain variable region has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 9, where the CDRs still have 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 SEQ ID NO: 12, wherein the CDRs still have amino acid sequences ovality SEQ ID NOs: 4, 5 and 6.

In specific embodiments, 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, where the CDRs still have 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, where the CDRs still have the amino acid sequences of SEQ ID NO: 4, 5, and 6. In particular, the heavy chain variable region has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 10, where the CDRs still have 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 SEQ ID NO: 12, wherein the CDRs still have amino acid sequences of SEQ ID NOs: 4, 5 and 6.

In specific embodiments, the heavy chain variable region comprises an amino acid sequence that is at least 90% identical to the amino acid sequence represented by amino acids 20-136 of SEQ ID NO: 20. In particular, the heavy chain variable region contains an amino acid sequence that is at least 95%, in particular at least 98% identical to the amino acid sequence represented by amino acids 20-136 of SEQ ID NO: 20. In these embodiments, the heavy chain variable region comprises a 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. Thus, any sequence deviations from the amino acid sequence represented by amino acids 20-136 of SEQ ID NO: 20 are located in the framework areas, not in CDRs. In particular, the heavy chain variable region comprises the amino acid sequence represented by amino acids 20-136 of SEQ ID NO: 20. In some embodiments, the amino acid at position 76 of SEQ ID NO: 20 is selected from the group consisting of glutamine, alanine, valine, histidine, tryptophan , tyrosine, lysine and arginine; in particular, 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, in particular glutamine. In particular, 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 acids 20-136 of SEQ ID NO: 23.

In specific embodiments, the light chain variable region comprises an amino acid sequence that is at least 90% identical to the amino acid sequence represented by amino acids 21-133 of SEQ ID NO: 21. In particular, the light chain variable region contains an amino acid sequence that is at least 95%, in particular at least 98% identical to the amino acid sequence represented by amino acids 21-133 of SEQ ID NO: 21. In these embodiments, the light chain variable region still contains CDRs having the amino acid sequences of SEQ ID NO: 4, 5 and 6. Thus, any sequence deviations from the amino acid sequence represented by amino acids 21-133 of SEQ ID NO: 21 are located in the framework regions and not in the CDRs. In particular, the light chain variable region contains the amino acid sequence represented by amino acids 21-133 of SEQ ID NO: 21.

In specific embodiments, the heavy chain variable region has an amino acid sequence that is at least 90% identical to the amino acid sequence represented by amino acids 20-136 of SEQ ID NO: 20, where the CDRs still have 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 represented by amino acids 21-133 of SEQ ID NO: 21, where the CDRs still have the amino acid sequences of SEQ ID NO: 4, 5, and 6. B in particular, the heavy chain variable region has an amino acid sequence that is at least 95% identical to the amino acid sequence represented by amino acids 20-136 of SEQ ID NO: 20, where the CDRs still have 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 at least 95% identical to the amino acid sequence represented by amino acids 21-133 of SEQ ID NO: 21, where the CDRs still have the amino acid sequences of SEQ ID NO: 4, 5 and 6.

In specific embodiments, the heavy chain contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 15. In particular, the heavy chain contains an amino acid sequence that is at least 95%, in particular, at least 98% identical to the amino acid sequence of SEQ ID NO: 15. In these embodiments, the heavy chain comprises a CDR-H1 having the amino acid sequence of SEQ ID NO: 1, a CDR-H2 having the amino acid sequence of SEQ ID NO: 2, and a CDR-H3 having the amino acid sequence of SEQ ID NO: 3. Thus, any sequence deviations from SEQ ID NO: 15 are located in the framework regions and not in the CDRs. In particular, 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 the group consisting of glutamine, alanine, valine, histidine, tryptophan, tyrosine, lysine, and arginine; in particular, 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, in particular glutamine. In particular, 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 acids 20-136 of SEQ ID NO: 22.

In specific embodiments, the light chain contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 16. In particular, the light chain contains an amino acid sequence that is at least 95%, in particular, at least 98% identical amino acid sequence to SEQ ID NO: 16. In these embodiments, the light chain still contains CDRs having the amino acid sequences of SEQ ID NO: 4, 5, and 6. Thus, any sequence deviations from SEQ ID NO: 16 are located in the framework areas, not in CDRs. In particular, the light chain contains the amino acid sequence of SEQ ID NO: 16.

In specific embodiments, the heavy chain has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 15, where the CDRs still have the amino acid sequences of SEQ ID NO: 1, 7, and 3, and the light chain variable region has an amino acid a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 16, where the CDRs still have the amino acid sequences of SEQ ID NO: 4, 5, and 6. In particular, the heavy chain has an amino acid sequence that is at least 95 % identical amino acid sequence of SEQ ID NO: 15, where the CDRs still have 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, where the CDRs still have the amino acid sequences of SEQ ID NOs: 4, 5 and 6.

The antibody of the present invention includes its modified forms. The term "modified form" of an antibody of the present invention means an antibody of the present invention provided with a chemical or biological modification. The chemically modified form includes forms where the amino acid backbone is conjugated to a chemical moiety, forms having a chemically modified N-linked or O-linked carbohydrate chain, and the like. Said chemical moiety or form may be toxic or cytotoxic. Biologically modified forms include forms subjected to post-translational modification (e.g., N-linked or O-linked glycosylation, N-terminal or C-terminal processing, deamidation, aspartic acid isomerization, or methionine oxidation), forms containing a methionine residue added to the N- end via expression using prokaryotic host cells, and the like. Such a modified form is also intended to include a form labeled to allow detection or isolation of the antibody of the present invention or its antigen, such as enzyme-labeled forms, fluorescently labeled forms, and affinity-labeled forms. Such a modified form of an antibody of the present invention can be used in improving the stability or blood retention of the original antibody of the present invention, reducing immunogenicity, detecting or isolating an antibody or antigen, and so on.

In particular, the antibody may contain one or more modifications selected from the group consisting of defucosylation, reduced fucose content, N-linked glycosylation, O-linked glycosylation, N-terminal processing, C-terminal processing, deamidation, aspartic acid isomerization, oxidation methionine, replacement of two leucine (L) residues with alanine (A) at positions 234 and 235 of the heavy chain (LALA), amidation of a proline residue, and deletion or absence of one, two or three amino acids at the carboxyl terminus. In specific embodiments, the antibody lacks one, two, or three carboxy-terminal amino acids in one or both heavy chains, or lacks one carboxy-terminal amino acid and the carboxy-terminal proline residues are amidated in one or both heavy chains.

Such modification can be carried out at an arbitrary position or desired position of the antibody. Alternatively, the same or two or more different modifications can be made in one or two or more positions.

For example, antibodies produced by cultured mammalian cells are known to lack the carboxy-terminal heavy chain lysine residue (Journal of Chromatography A, 705: 129-134 (1995)). It is also known that sometimes the 2 carboxy-terminal amino acid residues (i.e., glycine and lysine) of the heavy chain are missing, and that the proline residue, now located at the carboxy-terminus, is amidated (Analytical Biochemistry, 360: 75-83 (2007)) . However, such absence or modification in these heavy chain sequences does not affect the ability of the antibody to bind to its antigen, nor the effector functions (complement activation, antibody-dependent cytotoxicity, etc.) of the antibody.

In some embodiments, the antibody has a deletion or lack of 1 or 2 amino acids at the carboxy-terminus of the heavy chain and has an amidated residue (eg, an amidated proline residue at the carboxy-terminus of the heavy chain). However, the antibody of the present invention is not limited to the types described above, provided that the deletion mutant retains the ability to bind to an antigen.

In some embodiments, the two heavy chains of an antibody of the present invention may consist of any type of heavy chain selected from the group consisting of full-length heavy chains and deletion mutant heavy chains, or may consist of a combination of any two selected types. The ratio of the heavy chains of the deletion variant depends on the type of cultured mammalian cells producing the antibody of the present invention and the culture conditions of the cells.

In specific embodiments, an antibody of the present invention may include two heavy chains, both of which lack one carboxy-terminal amino acid residue.

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

In some embodiments, an antibody of the present invention competes for binding to TA-MUC1 with an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 10 and a light chain variable region having the amino acid sequence of SEQ ID NO: 12, or an antibody containing 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 some embodiments, the antibody has the following properties: (a) binds specifically to MUC1 and/or (b) has activity by internalizing into MUC1-expressing cells via binding to MUC1.

In some embodiments, the antibody comprises at least one antibody heavy chain. In particular, the antibody contains two heavy chains of the antibody. The heavy chains of an antibody specifically comprise a VH domain, a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain. In some other embodiments, the antibody heavy chains contain a CH2 domain and a CH3 domain, but do not contain a CH1 domain. In additional embodiments, one or more heavy chain constant domains can be replaced with other domains, in particular similar domains, such as, for example, albumin. The heavy chains of an antibody can be of any type, including γ-, α-, ε-, δ- and μ-chains, and are preferably γ-chains, including γ1-, γ2-, γ3- and γ4-chains, in particular, γ1 -chains. Thus, the antibody is preferably an IgG-type antibody such as an IgG1-, IgG3- or IgG4-type antibody, in particular an IgG1-type antibody.

In particular, the antibody further comprises at least one antibody light chain, in particular two antibody light chains. The light chains of an antibody, in particular, contain a VL domain and a CL domain. The light chain of an antibody may be a κ chain or a λ chain, and in particular is a κ chain.

In some embodiments, the antibody comprises two antibody heavy chains and two antibody light chains. In particular, the antibody contains two γ1-type antibody heavy chains, each containing a VH domain, a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain, and two κ-type antibody light chains, each containing a VL domain and a CL domain. .

In alternative embodiments, the antibody does not comprise an antibody light chain. In these embodiments, the light chain variable region can 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 can be used to link the light chain variable region to the rest of the heavy chain.

In preferred embodiments, the antibody contains an Fc region. The antibody, in particular, can be a whole antibody containing two heavy chains, each containing VH, CH1, hinge, CH2 and CH3 domains, and two light chains, each containing VL and CL domains. The antibody is particularly capable of binding to one or more human Fcγ receptors, in particular the human IIIA Fcγ receptor. In alternative embodiments, the antibody does not bind or slightly binds to the human IIIA Fcγ receptor, and in particular does not bind or slightly binds to any human Fcγ receptor. In these embodiments, the antibody specifically does not contain a glycosylation site in the CH2 domain.

In alternative embodiments, the antibody does not contain an Fc region. In these embodiments, the antibody is specifically a single chain variable region (scFv) fragment or other antibody fragment lacking an Fc region.

Glycosylation of anti-MUC1 antibody

An anti-MUC1 antibody may contain a CH2 domain in one or more heavy chains of the antibody. Natural human IgG-type antibodies contain an N-glycosylation site in the CH2 domain. The CH2 domains present in an 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. In particular, the antibody does not contain an asparagine residue at the heavy chain position corresponding to position 297 in the IMGT/Eu numbering system. For example, the antibody may contain an Ala297 mutation in the heavy chain. In these embodiments, the antibody preferably has a significantly reduced or no ability to induce, via binding to Fcγ receptors, antibody-dependent cellular cytotoxicity (ADCC) and/or antibody-dependent cellular phagocytosis (ADCP), and/or complement-dependent cytotoxicity (CDC). ). In this sense, the term "significantly reduced ability", in particular, refers to a reduction to 10% or less, in particular, 3% or less, 1% or less, or 0.1% or less activity compared to the same antibody containing an N-glycosylation site in CH2 domains and having a general mammalian glycosylation profile, such as that achieved by production in human cell lines or CHO cell lines, for example, the glycosylation profile presented herein. In these embodiments, the antibody is specifically of the IgG1 type.

In alternative embodiments, the CH2 domains present in the antibody comprise an N-glycosylation site. This glycosylation site is specifically located at the amino acid position corresponding to amino acid position 297 of the heavy chain in the IMGT/Eu numbering system and has an AsnXaaSer/Thr amino acid sequence motif, where Xaa can be any amino acid except proline. N-linked glycosylation at Asn297 is conserved in mammalian IgG as well as in homologous regions of other antibody isotypes. Due to optional additional amino acids that may be present in the variable region, or other sequence modifications, the specific position of this conserved glycosylation site may vary in the amino acid sequence of an antibody. Preferably, the glycans attached to the antibody are complex-type biantennary N-linked carbohydrate structures, preferably containing at least the following structure:

Asn - GlcNAc - GlcNAc - Man - (Man - GlcNAc) ₂

where Asn is the asparagine residue of the polypeptide portion of the antibody; GlcNAc is N-acetylglucosamine and Man is mannose. The terminal GlcNAc residues may additionally carry a galactose residue, which may optionally carry a sialic acid residue. An additional GlcNAc residue (denoted as GlcNAc at the branch point) can be attached to the Man closest to the polypeptide. Fucose can be linked to GlcNAc attached to Asn. In these embodiments, the antibody is specifically of the IgG1 type.

In preferred embodiments, the antibody does not contain N-glycolyl neuraminic acids (NeuGc) or detectable amounts of NeuGc. In addition, the antibody preferably also does not contain Galili epitopes (Galα1,3-Gal structures) or detectable amounts of a Galili epitope. In particular, the relative amount of glycans bearing NeuGc and/or Galα1,3-Gal structures is less than 0.1% or even less than 0.02% of the total number of glycans attached to the antibody CH2 domains in an antibody population.

In particular, the antibody has a human glycosylation profile. Due to these glycosylation properties, there are no foreign immunogenic non-human structures causing side effects, which means that undesirable side effects or disadvantages known to be caused by some foreign sugar structures, such as immunogenic, non-human sialic acids ( NeuGc) or a Galili epitope (Gal-Gal structures) known in rodent based production systems or other structures similar to immunogenic high mannose structures known for example in yeast systems.

In specific embodiments, the antibody comprises a glycosylation profile having a detectable amount of glycans bearing a GlcNAc residue at the branch point. In particular, the relative amount of glycans bearing a GlcNAc residue at the branch point is at least 0.5%, in particular at least 1%, of the total amount of glycans attached to the antibody glycosylation sites in the composition. In addition, in certain embodiments, the implementation of the glycosylation profile contains a relative amount of glycans bearing at least one galactose residue, at least 25% of the total number of glycans attached to the antibody in the composition. In particular, the relative amount of glycans bearing at least one galactose residue is at least 30%, in particular at least 35% or at least 40% of the total amount of glycans attached to the antibody in the composition. In specific embodiments, the implementation of the glycosylation profile contains a relative amount of glycans bearing at least one sialic acid residue, at least 1% of the total number of glycans attached to the antibody in the composition. In particular, the relative amount of glycans bearing at least one sialic acid residue is at least 1.5%, in particular at least 2%, of the total amount of glycans attached to the antibody in the composition.

The antibody may have a glycosylation profile having a high amount of cow's fucose or a low amount of cow's fucose. The reduced degree of fucosylation increases the ability of the antibody to induce ADCC. In some embodiments, the relative amount of glycans bearing a core fucose residue is 40% or less, in particular 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 glycans bearing a core fucose residue is at least 60%, in particular at least 65%, or at least 70% of the total amount of glycans attached to the antibody in the composition.

The ability of an antibody to induce ADCC and the strength of said ADCC induction can be controlled by the presence or absence of a glycosylation site in the CH2 domain of an anti-MUC1 antibody and the presence or absence of fucose in the glycan structures at this glycosylation site. ADCC activity is increased by glycosylation of the Fc portion of the antibody and additionally by reducing the degree of fucosylation in said glycosylation. In some cases, fine-tuning ADCC activity is important. Thus, in some cases, an antibody without a glycosylation site in the CH2 domain, an antibody with a glycosylation site in the CH2 domain and with a high degree of fucosylation, or an antibody with a glycosylation site in the CH2 domain and with a low degree of fucosylation may be most preferred.

Obtaining antibodies against MUC1

Preferably, the antibody is recombinantly produced in a host cell. The host cell used to make an antibody can be any host cell that can be used to make antibodies. Suitable host cells are in particular eukaryotic host cells, in particular mammalian host cells. Examples of host cells include yeast cells such as the Pichia pastoris cell lines, insect cells such as the SF9 and SF21 cell lines, plant cells, avian cells such as the duck EB66 cell lines, rodent cells such as the CHO, NS0, SP2/0 and YB2/0, and human cells such as the HEK293, PER.C6, CAP, CAP-T, AGE1.HN, Mutz-3 and KG1 cell lines.

In some embodiments, the antibody is produced recombinantly in a human blood cell line, in particular a human myeloid leukemia cell line. Preferred human cell lines that can be used to generate antibodies, as well as suitable methods of production, are described in WO 2008/028686 A2. In a specific embodiment, the antibody is produced by expression in a human myeloid leukemia cell line selected from the group consisting of NM-H9D8, NM-H9D8-E6 and NM-H9D8-E6Q12 and cell lines derived therefrom. These cell lines have been deposited under accession numbers DSM ACC2806 (NM-H9D8; deposited September 15, 2006), DSM ACC2807 (NM-H9D8-E6; deposited October 5, 2006), and DSM ACC2856 (NM-H9D8-E6Q12; deposited August 8, 2007) in accordance with the requirements of the Budapest Treaty in Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Inhoffenstraβe 7B, 38124 Braunschweig (DE) on behalf of Glycotope GmbH, Robert-Rössle-Str. 10, 13125 Berlin (DE). NM-H9D8 cells provide a glycosylation profile with a high degree of sialylation, a high amount of GlycNAc at the branch point, a high degree of galactosylation, and a high degree of fucosylation. NM-H9D8-E6 and NM-H9D8-E6Q12 cells provide a glycosylation profile similar to that of NM-H9D8 cells, except that the degree of fucosylation is very low. Other suitable cell lines include K562, a human myeloid leukemia cell line from the American Type Culture Collection (ATCC CCL-243), as well as cell lines derived from the above cells.

In additional embodiments, the antibody is produced recombinantly in CHO cells. In particular, the antibody can be produced recombinantly in a CHO dhfr ⁻ cell line, such as ATCC cell line No. CRL-9096.

Anti-MUC1 antibody conjugates

In some embodiments, the antibody contains one or more additional agents conjugated to it. The additional agent may be any agent suitable for conjugation with the antibody. If more than one additional agent is present in the antibody, these additional agents may be identical or different, and in particular they are all identical. Conjugation of the additional agent to the antibody can be accomplished by any of the methods known in the art. An additional agent can be attached to the antibody covalently, in particular by fusion or chemical conjugation, or non-covalently. In some embodiments, an additional agent is covalently attached to the antibody, in particular through a linker fragment. The linker moiety can be any chemical suitable for attaching an additional agent to an antibody.

The additional agent can preferably be used in therapy, diagnosis, prognosis, detection and/or monitoring of a disease, in particular a malignant neoplasm. For example, the additional agent may be selected from the group consisting of radionuclides, chemotherapeutic agents, antibodies, or antibody fragments, in particular having a different specificity than an anti-MUC1 antibody, such as anti-checkpoint antibodies that block or activate immunomodulatory targets, enzymes, domains interactions, detectable labels, toxins, cytolytic components, immunomodulators, immunoeffectors, class I or class II MHC antigens, and liposomes. A particular preferred additional agent is a radionuclide or a cytotoxic agent capable of killing malignant cells, such as a chemotherapeutic agent. In some preferred embodiments, a chemotherapeutic agent is attached to an anti-MUC1 antibody to form a conjugate.

Specific examples of chemotherapeutic agents that can be conjugated as an additional agent include alkylating agents such as cisplatin, antimetabolites, plant alkaloids and terpenoids, vinca alkaloids, podophyllotoxin, taxanes such as taxol, topoisomerase inhibitors such as irinotecan and topotecan, antineoplastic agents such as doxorubicin, or microtubule inhibitors such as auristatins and maytansine/maytansinoids.

The chemotherapeutic agent may in particular be selected from the group consisting of a V-ATPase inhibitor, a pro-apoptotic agent, a Bcl2 inhibitor, an MCL1 inhibitor, an HSP90 inhibitor, an IAP inhibitor, an mTor inhibitor, a microtubule stabilizer, a microtubule destabilizer, auristatin, dolastatin, maytansine, maytansinoid, amatoxin, methionine aminopeptidase, CRM1 nuclear export inhibitor, DPPIV inhibitor, proteasome inhibitors, mitochondrial phosphoryl transfer inhibitors, protein synthesis inhibitor, kinase inhibitor, CDK2 inhibitor, CDK9 inhibitor, kinesin inhibitor, HDAC inhibitor, DNA damaging agent, DNA alkylating agent agent, DNA intercalator, DNA minor groove binding agent, DHFR inhibitor, microtubule formation inhibitor, microtubule stabilizer, actin stabilizer, topoisomerase II inhibitor, platinum compound, ribosome inhibitor, RNA polymerase II inhibitor and bacterial toxin. In specific embodiments, the chemotherapeutic agent coupled to the anti-MUC1 antibody is selected from the group consisting of auristatin, a microtubule inhibitor such as maytansinoid, a DNA damaging agent, a DNA alkylating agent, and a DNA minor groove binding agent.

In some embodiments, the chemotherapeutic agent is maytansine or maytansinoid. Specific examples of maytansinoids that can be used for conjugation include maytansinol, N ^2' -deacetyl-N ^2' -(3-mercapto-1-oxopropyl)-mytansine (DM1), N ^2' -deacetyl-N ^2' -(4 -mercapto-1-oxopentyl)-maytansine (DM3), and N ^2' -deacetyl-N ^2' -(4-methyl-4-mercapto-1-oxopentyl)-maytansine (DM4). In particular, DM1 or DM4 is attached to an anti-MUC1 antibody. In some embodiments, the chemotherapeutic agent coupled to an anti-MUC1 antibody is auristatin, such as monomethylauristatin F (MMAF), monomethylauristatin E (MMAE), or auristatin T. In some embodiments, the chemotherapeutic agent coupled to an anti-MUC1 antibody is an agent binding to the minor groove of DNA, in particular pyrrolobenzodiazepine (PBD), pyrrolobenzodiazepine dimer (PBD dimer), duocarmycin, duocarmycin-hydroxybenzamide-azaindole (DUBA), seco-duocarmycin-hydroxybenzamide-azaindole (seco-DUBA), or doxorubicin. In some embodiments, the implementation of the chemotherapeutic agent attached to the antibody against MUC1, is a DNA alkylating agent, in particular, indolinobenzodiazepine or oxazolidinobenzodiazepine. In some embodiments, the implementation of the chemotherapeutic agent attached to the antibody against MUC1, is a DNA damaging agent, in particular, calichemiacin. In some embodiments, the chemotherapeutic agent coupled to the anti-MUC1 antibody is a microtubule formation inhibitor, such as tubulisin, ansamitocin, podophyllotoxin, or vinblastine. In some embodiments, the chemotherapeutic agent coupled to the anti-MUC1 antibody is a microtubule stabilizer, such as paclitaxel or epothilone. In some embodiments, the chemotherapeutic agent coupled to the anti-MUC1 antibody is an actin stabilizer, in particular a phallotoxin. In some embodiments, the chemotherapeutic agent coupled to the anti-MUC1 antibody is a topoisomerase II inhibitor, such as teniposide, XK469, razoxane, amsacrine, idarubicin, or mebarone. In some embodiments, the chemotherapeutic agent coupled to an anti-MUC1 antibody is a platinum compound, such as cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenantriplatin, picoplatin, or sattraplatin. In some embodiments, the chemotherapeutic agent coupled to an anti-MUC1 antibody is a ribosome inhibitor, such as ricin, saporin, abrin, diphtheria toxin, or exotoxin A. In some embodiments, the chemotherapeutic agent coupled to an anti-MUC1 antibody is an RNA polymerase inhibitor. II, in particular an amatoxin such as, for example, amanitin. In some embodiments, the chemotherapeutic agent coupled to the anti-MUC1 antibody is a bacterial toxin, in particular anthrax toxin. Suitable antibody-drug conjugates are also described in EP 16151774.3 and LU 92659, incorporated herein by reference.

In some embodiments, the additional agent is a polypeptide or protein. This polypeptide or protein, in particular, can be fused to the polypeptide chain of the antibody. In some embodiments, an additional agent that is 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, an additional polypeptide or protein agent may be fused to the C-terminus of each of the two antibody light chains. In additional embodiments, an additional agent that is 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, an additional polypeptide or protein agent may be fused to the C-terminus of each of the two antibody heavy chains. Additional funds may be identical or different and, in particular, have the same amino acid sequence. Suitable examples of such additional polypeptide or protein agents may be selected from the group consisting of cytokines, chemokines, antibodies, antigen-binding fragments, enzymes, and interaction domains.

In some embodiments, the additional agent, which is a polypeptide or protein, is an anti-checkpoint antibody that blocks and/or triggers activating signals. Examples of suitable targets include CD40, CD3, CD137 (4-1BB), OX40, GITR, CD27, CD278 (ICOS), CD154 (CD40 ligand), CD270 (HVEM) and CD258 (LIGHT) as activating targets, 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 respective ligands such as PDL1. In specific examples, an anti-MUC1 antibody comprises two heavy chains and two light chains as described herein, wherein the CD3-specific scFv fragment is fused to the C-terminus of each heavy chain; or wherein the scFv fragment specifically binding to PDL1 is fused to the C-terminus of each light chain.

In additional embodiments, the additional agent, which is a polypeptide or protein, is an immunomodulatory compound, such as a chemokine, cytokine, or growth factor. Suitable cytokines include interferons such as interferon-α, interferon-β and interferon-γ, and interleukins. Suitable growth factors include G-CSF and GM-CSF.

Nucleic acid, expression cassette, vector, cell line and composition

In a further aspect, the present invention relates to a nucleic acid encoding an antibody. The sequence of said nucleic acid may be any nucleotide sequence suitable for encoding an antibody. However, preferably, the nucleic acid sequence is at least partially adapted to the specific codon usage in the host cells or organism in which the nucleic acid will be expressed, in particular the codon usage in the human body. The nucleic acid may be double-stranded or single-stranded DNA or RNA, preferably double-stranded DNA such as cDNA or single-stranded RNA such as mRNA. It may be one consecutive nucleic acid molecule, or may be composed of several nucleic acid molecules, each of which encodes a different part of the antibody. In preferred embodiments, the present invention relates to the heavy chain nucleotide sequence of the PankoMab variant (PM-N54Q) shown in SEQ ID NO: 17 and the nucleotide sequence of the light chain of the PankoMab variant (PM-N54Q) shown in SEQ ID NO: 18.

Where an antibody consists of several different amino acid chains, such as a light chain and a heavy chain of an antibody, the nucleic acid, for example, may be a single nucleic acid molecule containing multiple coding regions, each encoding one of the amino acid chains of the antibody, preferably separated by regulatory elements. , such as IRES elements, to produce single amino acid chains, or the nucleic acid may be composed of multiple nucleic acid molecules, where each nucleic acid molecule contains one or more coding regions, each encoding one of the antibody amino acid chains. In addition to the coding regions encoding an antibody, the nucleic acid may also contain additional nucleic acid sequences or other modifications that, for example, may encode other proteins, may affect the transcription and/or translation of the coding regions, may affect stability, or other physical or the chemical properties of the nucleic acid, or may have no function at all.

In a further aspect, the present invention provides an expression cassette or vector comprising a nucleic acid of the invention and a promoter operably linked to said nucleic acid. In addition, the expression cassette or vector may contain additional elements, in particular elements that can affect transcription and/or translation of the nucleic acid, amplification and/or reproduction of the expression cassette or vector, integration of the expression cassette or vector into the genome of the host cell, and /or the number of copies of the expression cassette or vector in the host cell and/or regulate them. Suitable expression cassettes and vectors containing appropriate expression cassettes for antibody expression are well known in the art and thus need not be further discussed herein.

In addition, the present invention relates to a host cell containing a nucleic acid according to the invention or an expression cassette or vector according to the invention. The host cell may be any host cell. It may be an isolated cell or a cell contained in a tissue. Preferably, the host cell is a cultured cell, in particular a primary cell or a cell of a stable cell line, preferably a cell derived from a tumor. Preferably, it is a bacterial cell, such as E. coli, a yeast cell, such as a Saccharomyces cell, in particular S. cerevisiae, an insect cell, such as an Sf9 cell, or a mammalian cell, in particular a human cell, such as derived from tumors are a human cell, a hamster cell such as CHO, or primate cells. In a preferred embodiment, 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 from them, or a mixture of cells or cell lines, including at least one of the above cells. The host cell is preferably selected from the group consisting of NM-H9D8, NM-H9D8-E6, NM H9D8-E6Q12 and cells or cell lines derived from said host cells. These cell lines and their properties are described in detail in PCT application WO 2008/028686 A2. In further embodiments, the host cell is a CHO dhfr ⁻ cell line, such as ATCC No. CRL-9096 cell line. In preferred embodiments, the host cell is optimized to express glycoproteins, in particular antibodies having a specific glycosylation profile. Preferably, the use of codons in the coding region of the nucleic acid of the invention and/or the promoter and additional elements of the expression cassette or vector are compatible and more preferably optimized for the type of host cell used. Preferably, the antibody is produced by a host cell or cell line as described above.

In a specific aspect, the present invention relates to a method for producing antibodies in host cells, as described in the present description. The method specifically includes the steps of obtaining a host cell containing a nucleic acid encoding an antibody, culturing the host cell under conditions suitable for expression of the antibody, and obtaining an antibody expressed by the host cell. The antibody according to the invention can be obtained by the specified method.

In another aspect, the present invention relates to a composition containing an antibody, nucleic acid, expression cassette or host vector or cell. The composition may also contain more than one of these components. In addition, the composition may contain one or more additional components selected from the group consisting of solvents, diluents and excipients. Preferably, the composition is a pharmaceutical composition. In this embodiment, all components of the composition are preferably pharmaceutically acceptable. The composition may be a solid or liquid composition, particularly preferably an aqueous solution, emulsion or suspension, or a lyophilized powder.

Application in medicine

The antibody, in particular, can be used in medicine, in particular, in therapy, diagnosis, prognosis, detection and / or monitoring of a disease, in particular, a disease presented in the present description, preferably malignant neoplasm, infections, inflammatory diseases, graft reaction against the host" and immunodeficiencies.

Thus, in a further aspect, the invention provides an antibody, nucleic acid, expression cassette or vector, host cell, or composition for use in medicine. Preferably, the medical use is in the treatment, prognosis, diagnosis, detection and/or monitoring of a disease such as, for example, 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 diseases, and diseases associated with reduced immune activity such as immunodeficiencies. In a preferred embodiment, the disease is a malignant neoplasm.

Preferably, the malignancy has detectable MUC1 or TA-MUC1 expression, preferably detectable by immunohistochemistry, ELISA, RIA, enzyme-linked immunosorbent assay (ELISPOT), dot blot, Ouchterlony reaction, or cross-counter immunoelectrophoresis (CIE), or in situ hybridization. In particular, it includes cells having MUC1 or TA-MUC1 expression that is detectable by immunohistochemistry or in situ hybridization. Cancer can be tested for MUC1 or TA-MUC1 prior to administration of anti-MUC1 antibody.

The present invention further relates to kits and devices containing an antibody of the invention and corresponding methods that can be used in the diagnosis, detection or monitoring of MUC1-associated disorders such as cancer. In some embodiments, the present invention relates to a sandwich-type ELISA kit for testing, detection or diagnosis, containing an antibody of the present invention. The kit may further contain one or more of a MUC1 or TA-MUC1 protein standard solution, a staining reagent, a dilution buffer, a solid phase antibody, a detection antibody, and a wash solution, and the like. Preferably, the amount of antibody bound to an antigen can be measured by a method such as an absorption, fluorescence, luminescence or radioactive isotope (RI) method. Preferably, an absorption plate-reading spectrophotometer, a fluorescence plate-reading spectrophotometer, a luminescent plate-reading spectrophotometer, an RI liquid scintillation counter, or the like is used in the measurement.

In some embodiments, the present invention relates to an antibody of the invention for use in an immunohistochemical assay (IHC) and a composition containing it.

Immunohistochemistry is not particularly limited, as long as the approach includes reacting the tissue section with an antigen-binding antibody (primary antibody) and detecting the primary antibody bound to the antigen.

Various forms of cancer, including metastases, can be treated using an antibody of the invention. In particular, the cancer may be selected from the group consisting of colon cancer, lung cancer, ovarian cancer, breast cancer such as triple negative breast cancer, pancreatic cancer, cervical cancer, endometrial cancer, gastrointestinal cancer. tract, kidney cancer, head and neck cancer, thyroid cancer and urothelial cancer. In particular, the cancer may be further selected from gastric cancer, liver cancer, bladder cancer, skin cancer, prostate cancer, and hemoblastosis. In some embodiments, the cancer is a metastatic cancer. The malignancy may include any type of metastasis, such as skin metastasis, lymph node metastasis, lung metastasis, liver metastasis, peritoneal metastasis, pleural metastasis, and/or brain metastasis. In some embodiments, the cancer has an inflammatory phenotype. In these embodiments, any of the types of cancers described above may be an inflammatory cancer.

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. The inflammatory disease can be selected from inflammatory bowel disease, pelvic inflammatory disease , ischemic stroke, Alzheimer's disease, asthma, pemphigus vulgaris, and dermatitis/eczema. The autoimmune disease may be selected from the group consisting of celiac disease, type 1 diabetes mellitus, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, vitiligo, psoriatic arthritis, atopic dermatitis, scleroderma, sarcoidosis, primary biliary cirrhosis, Guillain-Barré syndrome, autoimmune hepatitis and ankylosing spondylitis. In some embodiments, the implementation of the disease includes or is associated with cells expressing MUC1, in particular, TA-MUC1. For example, the cancer to be treated is MUC1 positive, in particular TA-MUC1 positive, i. includes malignant cells expressing MUC1, in particular TA-MUC1.

In specific embodiments, the antibody is used for treatment in combination with another therapeutic agent, in particular for the treatment of cancer in combination with another anticancer agent. Said additional therapeutic agent may be any known anticancer agent. Suitable antitumor therapeutic agents that can be combined with an antibody of the invention may be chemotherapeutic agents, other antibodies, immunostimulatory agents, cytokines, chemokines, and vaccines. In addition, antibody therapy can be combined with radiation therapy, surgery and/or traditional Chinese medicine.

Antitumor agents that can be used in combination with an anti-MUC1 antibody can be selected from any chemotherapeutic agent, in particular chemotherapeutic agents known to be effective in the treatment of MUC1-positive malignancies. The type of chemotherapeutic agent also depends on the cancer being treated. The combination partner can be selected from the group consisting of taxanes such as paclitaxel (Taxol), docetaxel (Taxotere) and SB-T-1214; cyclophosphamide; imatinib; pasropaniba; capecitabine; cytarabine; vinorelbine; gemcitabine; anthracyclines such as daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin and mitoxantrone; aromatase inhibitors such as aminoglutethimide, testolactone (Teslac), anastrozole (Arimidex), letrozole (Femara), exemestane (Aromasin), vorozol (Rivizor), formestane (Lentaron), fadrozole (Afema), 4-hydroxyandrostenedione, 1,4, 6-androstotriene-3,17-dione (ATD) and 4-androsten-3,6,17-trione (6-OXO); topoisomerase inhibitors such as irinotecan, topotecan, camptothecin, lamellarin D, etoposide (VP-16), teniposide, doxorubicin, daunorubicin, mitoxantrone, amsacrine, ellipticins, auryntricarboxylic acid, and HU-331; platinum-based chemotherapeutic agents such as cis-diammine dichloroplatinum (II) (cisplatin), cis-diammine (1,1-cyclobutanedicarboxylate) platinum (II) (carboplatin) and [(1R,2R)-cyclohexane-1,2-diamine ](ethanedioato-O,O')platinum (II) (oxaliplatin); PARP inhibitors such as olaparib, rucaparib and niraparib; TLR agonists such as imiquimod and resiquimod; and antimetabolites, in particular antifolates such as methotrexate, pemetrexed, raltitrexed and pralatrexate, pyrimidine analogs such as fluorouracil, gemcitabine, floxuridine, 5-fluorouracil and tegafur-uracil, and purine analogs, selective estrogen receptor modulators and estrogen receptor inhibitors.

In addition, therapeutic antibodies can also be used as an additional combination partner. They can be any antibody that can be used in cancer therapy other than an anti-MUC1 antibody. In particular, the additional antibody is approved for the treatment of malignancies by administration, for example, by the Food and Drug Administration (FDA), the European Medicines Agency (EMA, formerly EMEA) and the German Federal Institute for Medicines and Medical Devices (BfArM ). Examples of an additional antibody that can be used for combination therapy are anti-EGFR antibodies such as cetuximab, tomozotuximab, panitumumab, zalutumumab, nimotuzumab, matuzumab, and necitumumab; anti-HER2 antibodies such as trastuzumab, thymigutuzumab and pertuzumab; anti-VEGF antibodies such as bevacizumab (Avastin); anti-CD52 antibodies such as alemtuzumab (Campath); anti-CD30 antibodies such as brentuximab (Adcetris); anti-CD33 antibodies such as gemtuzumab (Mylotarg); and anti-CD20 antibodies such as rituximab (Rituxan, Mabthera), tositumomab (Bexxar), and ibritumomab (Zevalin). Additional examples of antibodies suitable for combination with the anticancer therapy provided herein include antibodies against antigens selected from the group consisting of Thomsen-Friedenreich antigen (TFα, TFβ), Tn, Lewis Y antigen, CD44, folate receptor α, ganglioside NeuGc-GM3, DLL-3, RANKL, PTK7, Notch-3, aphrin A4, insulin-like growth factor receptor 1, activin-like kinase receptor-1, claudin-6, disialoganglioside GD2, endoglin, NMB transmembrane glycoprotein, CD56, tumor-associated transducer calcium signal 2, tissue factor, ectonucleotide pyrophosphatase/phosphodiesterase 3, CD70, P-cadherin, mesothelin, prostate six-domain transmembrane epithelial antigen 1 (STEAP1), carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), nectin 4, guanylyl cyclase C, member 4 of the 44 solute transporter family (SLC44A4), prostate-specific membrane antigen (PSMA), t Zinc transporter ZIP6 (LIV1 (ZIP6)), SLIT and NTRK-like protein 6 (SLITRK6), trophoblast glycoprotein (TPBG; 5T4), Fyn3, carbonic anhydrase 9, NaPi2b, fibronectin extra-domain B, ETB endothelin receptor, VEGFR2 (CD309), tenascin c, collagen IV, and periostin.

The anti-MUC1 antibody can be further combined with anti-checkpoint antibodies, ie. antibodies that block or activate immunomodulatory targets. In this way, inhibitory signals for the immune response can be blocked and/or activating signals can be triggered. Examples of suitable targets include CD40, CD3, CD137 (4-1BB), OX40, GITR, CD27, CD278 (ICOS), CD154 (CD40 ligand), CD270 (HVEM) and CD258 (LIGHT) as activating targets, 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 respective ligands such as PDL1.

In further embodiments, the anti-MUC1 antibody can be combined with treatment with immunomodulatory compounds such as chemokines, cytokines, growth factors, and vaccines. Suitable cytokines include interferons such as interferon-α, interferon-β and interferon-γ, and interleukins. Suitable growth factors include G-CSF and GM-CSF.

The anti-MUC1 antibody is preferably used to treat a primary tumor, a recurrent tumor and/or metastases of such tumors, and in particular is used to treat before, during or after surgery and to prevent or treat metastases. The anti-MUC1 antibody is particularly intended to treat a patient as an adjuvant therapy. In some embodiments, the anti-MUC1 antibody is for treatment of a patient as a neoadjuvant therapy or a combined neoadjuvant-adjuvant therapy. In addition, the anti-MUC1 antibody is intended for the treatment of a patient as a palliative care.

Therapy of a malignant neoplasm with an anti-MUC1 antibody preferably results in inhibition of tumor growth and, in particular, a reduction in tumor size. In addition, with the help of treatment prevent the occurrence of additional metastases and/or reduce their number. Preferably, the treatment results in improved progression-free survival and/or increased life expectancy and thus overall survival.

The present invention further relates to methods of therapy, diagnosis, prognosis, detection and/or monitoring of a disease using an antibody of the invention. Embodiments and examples of the use of antibodies in medicine can also be similarly used in relation to medical methods. In particular, the present invention relates to a method of treating a disease in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of an antibody of the present invention.

For example, the invention relates to a method of treating cancer in an individual in need thereof, comprising administering to the individual with cancer a therapeutically effective amount of an antibody of the invention. In specific embodiments, the cancer is characterized by the expression of TA-MUC1. The malignancy may be selected from the group consisting of ovarian cancer, breast cancer, pancreatic cancer, lung cancer, colon cancer, gastric cancer, liver cancer, kidney cancer, hemoblastosis, endometrial cancer, thyroid cancer, leukemia, seminoma, melanoma, carcinoma, teratoma, lymphoma, sarcoma, mesothelioma, neuroblastoma, glioma, colorectal cancer, adrenal cancer, skin cancer, brain malignancy, cervical cancer, colon cancer, colon cancer, head and neck cancer, malignancy gastrointestinal tract, malignant neoplasm of the lymph nodes, cancer of the esophagus, colorectal cancer, malignant neoplasm of the ear, nose and throat (ENT), prostate cancer, bladder cancer, uterine cancer and their metastases.

In addition, the invention relates to a method for diagnosing, detecting or monitoring a malignant neoplasm, comprising the step of bringing a test sample into contact with an antibody of the invention.

Methods for increasing binding affinity for MUC1

In a further aspect, the invention relates to a method for increasing the MUC1 binding affinity of an antibody comprising

(i) a heavy chain variable region comprising complementarity determining regions (CDRs) of 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 complementarity determining regions (CDRs) of CDR-L1 having the amino acid sequence of SEQ ID NO: 4, CDR-L2 having the amino acid sequence of SEQ ID NO: 5, and CDR-L3 having the amino acid sequence of SEQ ID NO: 6

a method comprising the step of replacing the amino acid residue at position 8 of CDR-H2 with any amino acid residue except asparagine, resulting in a CDR-H2 having the amino acid sequence of SEQ ID NO: 2.

An antibody whose binding affinity for MUC1 is to be increased is specifically an antibody capable of binding to MUC1 as described herein, except that it contains asparagine at position 8 of the CDR-H2 sequence.

In some embodiments, the heavy chain variable region of the antibody whose binding affinity for MUC1 is to be increased contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 11. In particular, the heavy chain variable region contains an amino acid sequence that is at least 95%, in particular at least 98% identical to the amino acid sequence of SEQ ID NO: 11. In these embodiments, the heavy chain variable region still contains CDRs having the amino acid sequences of SEQ ID NO: 1, 8, and 3 Thus, any sequence deviations from SEQ ID NO: 11 are located in the framework regions and not in the CDRs. In particular, the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 11.

In some embodiments, the light chain variable region of the antibody whose binding affinity for MUC1 is to be increased contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 12. In particular, the light chain variable region contains an amino acid sequence that is at least 95%, in particular at least 98% identical to the amino acid sequence of SEQ ID NO: 12. In these embodiments, the light chain variable region still contains CDRs having the amino acid sequences of SEQ ID NO: 4, 5, and 6 Thus, any sequence deviations from SEQ ID NO: 12 are located in the framework regions and not in the CDRs. In particular, the light chain variable region contains the amino acid sequence of SEQ ID NO: 12.

In specific embodiments, the heavy chain variable region of the antibody whose binding affinity for MUC1 is to be increased has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 11, where the CDRs still have 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, where the CDRs still have the amino acid sequences of SEQ ID NO: 4, 5 and 6. In particular, the heavy chain variable region has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 11, where the CDRs still have the amino acid sequences of SEQ ID NO: 1, 8 and 3, and the light chain variable region has an amino acid sequence, at least 95% identical amino acid sequence sequences of SEQ ID NO: 12, where the CDRs still have the amino acid sequences of SEQ ID NO: 4, 5 and 6.

For example, an antibody whose binding affinity for MUC1 is to be increased is an anti-MUC1 antibody as described in WO 2004/065423 A2 or WO 2011/012309 A1. In particular, an antibody with a binding affinity for MUC1 that needs to be increased is gatipotuzumab or PankoMab.

An antibody whose binding affinity for MUC1 is increased is specifically an antibody capable of binding to MUC1 as described herein.

In some embodiments, the implementation of the binding to MUC1 is as presented in the present description. The term "increased binding affinity" for MUC1 or TA-MUC1 specifically refers to an increase of at least 10%, at least 20%, at least 33%, or at least 50%. In preferred embodiments, the binding affinity for MUC1 is increased by at least 50%. Binding affinity for MUC1 can be determined as described in the examples, in particular using surface plasmon resonance assay or switchSENSE® technology (DRX2 biosensor manufactured by Dynamic Biosensors GmbH) as described in example 4a and b.

In some embodiments, the step of replacing the amino acid residue at position 8 of CDR-H2 is carried out by inserting a mutation into a nucleic acid encoding an antibody, where the mutation is inserted into a codon encoding said amino acid residue. Mutation insertion can be done in any manner. Several suitable methods are known in the art, and one skilled in the art can take the necessary steps to insert the mutation. An antibody with increased MUC1 binding affinity can then be generated by expression of the mutant nucleic acid, for example, in a host cell. Nucleic acids, host cells, and methods for producing antibodies are provided herein and can be used for a method for increasing binding affinity to MUC1.

In specific embodiments, a method for increasing the binding affinity of an antibody to MUC1 comprises the steps

(a) obtaining a nucleic acid encoding an antibody with a binding affinity for MUC1 to be increased;

(b) inserting a mutation into said nucleic acid to produce a mutant nucleic acid, wherein the mutation is inserted into a codon encoding an amino acid residue at position 8 of CDR-H2 such that said codon codes for any amino acid residue except asparagine; And

(c) expressing the mutant nucleic acid to produce an antibody with increased binding affinity for MUC1.

The present invention further relates to a method for producing an antibody with increased binding affinity for MUC1, comprising

(a) obtaining a nucleic acid encoding an antibody containing

(i) a heavy chain variable region comprising complementarity determining regions (CDRs) of 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 complementarity determining regions (CDRs) of 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) inserting a mutation into said nucleic acid to produce a mutant nucleic acid, wherein the mutation is inserted into a codon encoding an amino acid residue at position 8 of CDR-H2 such that said codon codes for any amino acid residue except asparagine; And

(c) obtaining an antibody with increased binding affinity for MUC1 by expressing the mutant nucleic acid in a host cell.

The embodiments, features, and examples provided herein for other aspects, particularly in the case of a method for increasing the binding affinity of an antibody to MUC1, can also be similarly used in relation to a method for producing an antibody with increased binding affinity for MUC1.

In some embodiments, the method for producing an antibody with increased binding affinity for MUC1 further comprises the step (d) of treating the antibody with increased binding affinity for MUC1.

For example, processing an antibody with increased MUC1 binding affinity may include isolating the antibody from a cell culture. The term "isolation of the antibody" specifically refers to the separation of the antibody from the remaining components of the cell culture. Separation of the antibody from the cell culture medium can be carried out, for example, by chromatographic methods. Suitable methods and means for isolating antibodies are known in the art and can easily be used by one skilled in the art.

The resulting antibody may optionally be subjected to additional processing steps, such as, for example, modification steps, such as chemical or enzymatic attachment of an additional agent to the antibody, and/or formulation steps, to produce an antibody of the desired quality and composition. Such additional processing steps and methods are generally known in the art.

In additional embodiments, step (d) further includes the step of obtaining a pharmaceutical composition containing the antibody. The preparation of a pharmaceutical formulation containing an antibody, or the formulation of an antibody into a pharmaceutical composition, specifically involves replacing the buffer solution or components of the buffer solution of the composition containing the antibody. In addition, this step may include lyophilization of the antibody. In particular, the antibody is transferred into a composition containing only pharmaceutically acceptable ingredients.

Specific Embodiments

The following describes specific embodiments of the present invention.

Embodiment 1. An antibody capable of binding to MUC1 containing

(i) a heavy chain variable region comprising complementarity determining regions (CDRs) of CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 2, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and

(ii) a light chain variable region comprising complementarity determining regions (CDRs) of 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.

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

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

Embodiment 4. The antibody of Embodiments 1-3, wherein the CDR-H2 has the amino acid sequence of SEQ ID NO: 7.

Embodiment 6. An antibody capable of binding to MUC1 containing

(i) a heavy chain variable region that

(a) has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 9, and

(b) contains 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 that

(a) has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 12, and

(b) contains 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.

Embodiment 7. An antibody capable of binding to MUC1 containing

(i) a heavy chain variable region that

(a) has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 9, and

(b) contains 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 that

(a) has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 12, and

(b) contains 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.

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

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

Embodiment 10. An antibody capable of binding to MUC1 containing

(i) a heavy chain variable region that

(a) has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 10, and

(b) contains 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) a light chain variable region that

(a) has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 12, and

(b) contains 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.

Embodiment 11. An antibody capable of binding to MUC1 containing

(i) a heavy chain variable region that

(a) has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 10, and

(b) contains 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) a light chain variable region that

(a) has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 12, and

(b) contains 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.

Embodiment 12. An antibody capable of binding to MUC1 containing

(i) a heavy chain variable region that has the amino acid sequence of SEQ ID NO: 9, and

(ii) a light chain variable region that has the amino acid sequence of SEQ ID NO: 12.

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

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

Embodiment 15. An antibody capable of binding to MUC1 containing

(i) a heavy chain variable region that has the amino acid sequence of SEQ ID NO: 10, and

(ii) a light chain variable region that has the amino acid sequence of SEQ ID NO: 12.

Embodiment 16. An antibody capable of binding to MUC1 containing

(i) a heavy chain variable region that

(a) has an amino acid sequence that is at least 90% or at least 95% identical to the amino acid sequence represented by amino acids 20-136 of SEQ ID NO: 20, and

(b) contains 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) a light chain variable region that

(a) has an amino acid sequence that is at least 90% or at least 95% identical to the amino acid sequence represented by amino acids 21-133 of SEQ ID NO: 21, and

(b) contains 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.

Embodiment 17. An antibody capable of binding to MUC1 containing

(i) a heavy chain variable region that has the amino acid sequence represented by amino acids 20-136 of SEQ ID NO: 20 or 23, and

(ii) a light chain variable region that has the amino acid sequence represented by amino acids 21-133 of SEQ ID NO: 21.

Embodiment 18. An antibody capable of binding to MUC1 containing

(i) a heavy chain that

(a) has an amino acid sequence that is at least 90% or at least 95% identical to the amino acid sequence of SEQ ID NO: 15, and

(b) contains 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) a light chain variable region that

(a) has an amino acid sequence that is at least 90% or 95% identical to the amino acid sequence of SEQ ID NO: 16, and

(b) contains 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.

Embodiment 19. An antibody capable of binding to MUC1 containing

(i) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 22, and

(ii) a light chain variable region that has the amino acid sequence of SEQ ID NO: 16.

Embodiment 20. An antibody capable of binding to MUC1 containing

(i) a heavy chain variable region that

(a) has an amino acid sequence that is at least 90% or at least 95% identical to the amino acid sequence represented by amino acids 20-460 of SEQ ID NO: 20, and

(b) contains 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) a light chain variable region that

(a) has an amino acid sequence that is at least 90% or 95% identical to the amino acid sequence represented by amino acids 21-239 of SEQ ID NO: 21, and

(b) contains 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.

Embodiment 21. An antibody capable of binding to MUC1 containing

(i) a heavy chain which has the amino acid sequence represented by amino acids 20-460 of SEQ ID NO: 20 or 23, and

(ii) a light chain that has the amino acid sequence represented by amino acids 21-239 of SEQ ID NO: 21.

Embodiment 22. The antibody of any one of embodiments 1-21, wherein the antibody comprises at least one heavy chain comprising a heavy chain variable region, a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain.

Embodiment 23 An antibody according to any one of embodiments 1-21, wherein the antibody comprises two heavy chains, each containing a heavy chain variable region, a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain.

Embodiment 24. The antibody of Embodiment 22 or 23, wherein the antibody is an IgG-type antibody, in particular an IgG1-, IgG2-, or IgG4-type antibody.

Embodiment 25. The antibody of any one of embodiments 1-24, wherein the antibody comprises at least one light chain comprising a light chain variable region and a CL domain.

Embodiment 26 An antibody according to any one of embodiments 1-24, wherein the antibody comprises two light chains, each containing a light chain variable region and a CL domain.

Embodiment 27 The antibody of Embodiment 25 or 26 wherein the light chain is a κ-type light chain.

Embodiment 28 An antibody according to any one of Embodiments 1-27, wherein the antibody does not contain an N-glycosylation site in the CH2 domain.

Embodiment 29 An antibody according to any one of embodiments 1-27, wherein the antibody comprises an N-glycosylation site in the CH2 domain of the heavy chains of the antibody.

Embodiment 30. The antibody of Embodiment 29, wherein the antibody has a glycosylation profile having one or more of the following characteristics

(i) a relative amount of glycans bearing a GlcNAc residue at the branch point of at least 0.5% of the total number of glycans attached to the glycosylation sites of the antibody in the composition;

(ii) the relative amount of glycans bearing at least one galactose residue, at least 30% of the total number of glycans attached to the glycosylation sites of the antibody in the composition;

(iii) the relative amount of glycans bearing a core fucose residue of at least 60% of the total amount of glycans attached to the glycosylation sites of the antibody in the composition.

Embodiment 31. The antibody of Embodiment 29, wherein the antibody has a glycosylation profile having one or more of the following characteristics

(i) a relative amount of glycans bearing a GlcNAc residue at the branch point of at least 0.5% of the total number of glycans attached to the glycosylation sites of the antibody in the composition;

(ii) the relative amount of glycans bearing at least one galactose residue, at least 30% of the total number of glycans attached to the glycosylation sites of the antibody in the composition;

(iii) a relative amount of glycans bearing a core fucose residue of 40% or less of the total amount of glycans attached to the glycosylation sites of the antibody in the composition.

Embodiment 32. An antibody according to any one of Embodiments 1-31 containing an additional agent conjugated thereto.

Embodiment 33. The antibody of Embodiment 32, wherein the additional agent is a chemotherapeutic agent coupled to the antibody.

Embodiment 34. The antibody of Embodiment 33, wherein the chemotherapeutic agent is selected from the group consisting of a maytansinoid, a DNA damaging agent, a DNA alkylating agent, and a DNA minor groove binding agent.

Embodiment 35. The antibody of Embodiment 33 wherein the chemotherapeutic agent is selected from the group consisting of ismaitanzinol, N ^2'- deacetyl-N ^2' -(3-mercapto-1-oxopropyl)-maytansine (DM1), N ^2' -deacetyl -N ^2' -(4-mercapto-1-oxopentyl)-maytansine (DM3) and N ^2'- deacetyl-N ^2' -(4-methyl-4-mercapto-1-oxopentyl)-mytansine (DM4).

Embodiment 36. The antibody of Embodiment 33, wherein the chemotherapeutic agent is selected from the group consisting of monomethylauristatin F (MMAF), monomethylauristatin E (MMAE), and auristatin T.

Embodiment 37. The antibody of Embodiment 33, wherein the chemotherapeutic agent is selected from the group consisting of Pyrrolobenzodiazepine (PBD), Pyrrolobenzodiazepine dimer (PBD dimer), Duocarmycin, Duocarmycin-Hydroxybenzamide-Azaindole (DUBA), Seco-Duocarmycin-Hydroxybenzamide-Azaindole ( seco-DUBA) and doxorubicin.

Embodiment 38. The antibody of Embodiment 33, wherein the chemotherapeutic agent is selected from the group consisting of indolinobenzodiazepine and oxazolidinobenzodiazepine.

Embodiment 39. The antibody of Embodiment 33, wherein the chemotherapeutic agent is calichemiacin.

Embodiment 40. The antibody of Embodiment 32, wherein the additional agent is a polypeptide or protein fused to the polypeptide chain of the antibody.

Embodiment 41. The antibody of Embodiment 40, wherein the antibody comprises two antibody heavy chains and two antibody light chains and an additional agent that is a polypeptide or protein fused to each of the C-terminus of said antibody heavy chains or to each of the C-terminus of said antibody heavy chains. antibody light chains.

Embodiment 42. The antibody of Embodiment 40 or 41, wherein the additional agent is selected from the group consisting of cytokines, chemokines, other antibodies, antigen-binding fragments, enzymes, and binding domains.

Embodiment 43 An antibody of Embodiment 41 wherein the additional agent is an scFv fragment that specifically binds to CD3 and one of said additional agents is fused to the C-terminus of each antibody heavy chain.

Embodiment 44 An antibody of Embodiment 41 wherein the additional agent is an scFv fragment that specifically binds to PDL1 and one of said additional agents is fused to the C-terminus of each light chain of the antibody.

Embodiment 45. A nucleic acid encoding an antibody according to any one of Embodiments 1-44.

Embodiment 46. An expression cassette or vector containing the nucleic acid of Embodiment 75 and a promoter operably linked to said nucleic acid.

Embodiment 47. A host cell containing the nucleic acid of Embodiment 45 or the expression cassette or vector of Embodiment 46.

Embodiment 48. A pharmaceutical composition comprising the antibody or conjugate of any of Embodiments 1-44 and one or more additional components selected from the group consisting of solvents, diluents, and excipients.

Embodiment 49 An antibody according to any one of Embodiments 1-44 or a pharmaceutical composition according to Embodiment 48 for use in medicine.

Embodiment 50. The antibody of any one of Embodiments 1-44 or the pharmaceutical composition of Embodiment 48 for use in the treatment, prognosis, diagnosis, detection, and/or monitoring of diseases associated with abnormal cell growth, such as cancer; infections such as bacterial, viral, fungal or parasitic infections; inflammatory diseases such as autoimmune diseases and inflammatory bowel diseases; and diseases associated with reduced immune activity, such as immunodeficiencies.

Embodiment 51. The antibody or pharmaceutical composition of Embodiment 50 for use in the treatment of a cancer, in particular a cancer expressing TA-MUC1, wherein the cancer is selected from the group consisting of ovarian cancer, breast cancer, pancreatic cancer, lung cancer, colon cancer, stomach cancer, liver cancer, kidney cancer, hemoblastosis, endometrial cancer, thyroid cancer, leukemia, seminoma, melanoma, carcinoma, teratoma, lymphoma, sarcoma, mesothelioma, neuroblastoma, glioma, rectal cancer, cancer adrenal, skin cancer, brain malignancy, cervical cancer, colon cancer, colon cancer, head and neck cancer, gastrointestinal cancer, lymph node cancer, esophageal cancer, colorectal cancer, ear, nose and ear cancerthroat (ENT), prostate cancer, bladder cancer, uterine cancer and their metastases.

Embodiment 52. The antibody or pharmaceutical composition of Embodiment 50 for use in the treatment of infections, wherein the infection is selected from the group consisting of bacterial infections, viral infections, fungal infections, and parasitic infections.

Embodiment 53. The antibody or pharmaceutical composition of Embodiment 50 for use in the treatment of autoimmune diseases, wherein the autoimmune disease is selected from the group consisting of celiac disease, type 1 diabetes mellitus, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus.

Embodiment 54. Method for increasing the binding affinity of an antibody to MUC1 containing

(i) a heavy chain variable region that

(a) has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 11, and

(b) contains 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 that

(a) has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 12, and

(b) contains 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;

a method comprising the step of replacing the amino acid residue at position 8 of CDR-H2 with any amino acid residue except asparagine, resulting in a CDR-H2 having the amino acid sequence of SEQ ID NO: 2.

Embodiment 55. Method for increasing the binding affinity of an antibody to MUC1 containing

(i) a heavy chain variable region that

(a) has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 11, and

(b) contains 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 that

(a) has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 12, and

(b) contains 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;

a method comprising the step of replacing the amino acid residue at position 8 of CDR-H2 with any amino acid residue except asparagine, resulting in a CDR-H2 having the amino acid sequence of SEQ ID NO: 2.

Embodiment 56. The method of Embodiment 54 or 55, wherein the amino acid residue at position 8 of CDR-H2 is changed by inserting a mutation into a nucleic acid encoding an antibody, wherein the mutation is inserted into a codon encoding said amino acid residue.

Embodiment 57. The method of any one of Embodiments 54-56, comprising the steps of

(a) obtaining a nucleic acid encoding an antibody whose binding affinity for MUC1 is to be increased;

(b) inserting a mutation into said nucleic acid to produce a mutant nucleic acid, wherein the mutation is inserted into a codon encoding an amino acid residue at position 8 of CDR-H2 such that said codon codes for any amino acid residue except for asparagine; And

(c) expressing the mutant nucleic acid to produce an antibody with increased binding affinity for MUC1.

Embodiment 58. A method for producing an antibody with increased binding affinity for MUC1, comprising

(a) obtaining a nucleic acid encoding an antibody containing

(i) a heavy chain variable region that

(i1) has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 11, and

(i2) contains 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 that

(ii1) has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 12, and

(ii2) contains 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) inserting a mutation into said nucleic acid to produce a mutant nucleic acid, wherein the mutation is inserted into a codon encoding an amino acid residue at position 8 of CDR-H2 such that said codon codes for any amino acid residue except for asparagine; And

(c) obtaining an antibody with increased binding affinity for MUC1 by expressing the mutant nucleic acid in a host cell.

Embodiment 59. A method for producing an antibody with increased binding affinity for MUC1, comprising

(a) obtaining a nucleic acid encoding an antibody containing

(i) a heavy chain variable region that

(i1) has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 11, and

(i2) contains 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 that

(ii1) has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 12, and

(ii2) contains 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) inserting a mutation into said nucleic acid to produce a mutant nucleic acid, wherein the mutation is inserted into a codon encoding an amino acid residue at position 8 of CDR-H2 such that said codon codes for any amino acid residue except for asparagine; And

(c) obtaining an antibody with increased binding affinity for MUC1 by expressing the mutant nucleic acid in a host cell.

Embodiment 60. The method of any one of embodiments 54-59, wherein the antibody comprises two heavy chains, each containing a heavy chain variable region, a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain.

Embodiment 61. The method of Embodiment 60, wherein the antibody is an IgG-type antibody, in particular an IgG1-, IgG2-, or IgG4-type antibody.

Embodiment 62. The method of any one of embodiments 54-60, wherein the antibody comprises two light chains, each containing a light chain variable region and a CL domain.

Embodiment 63. The method of Embodiment 62 wherein the light chain is a κ-type light chain.

Embodiment 64. The method of any of Embodiments 54-63, wherein the antibody with increased binding affinity for MUC1 is the antibody of any of Embodiments 1-44.

Embodiment 65. A method of treating cancer in an individual in need thereof, comprising administering to an individual with a cancer, in particular a cancer expressing TA-MUC1, a therapeutically effective amount of an antibody of any one of Embodiments 1-44 or a composition of Embodiment 48 .

Embodiment 66. The method of treating cancer according to Embodiment 65, wherein the cancer is selected from the group consisting of ovarian cancer, breast cancer, pancreatic cancer, lung cancer, colon cancer, gastric cancer, liver cancer, kidney cancer, hemoblastosis , endometrial cancer, thyroid cancer, leukemia, seminoma, melanoma, carcinoma, teratoma, lymphoma, sarcoma, mesothelioma, neuroblastoma, glioma, rectal cancer, adrenal cancer, skin cancer, malignant neoplasm of the brain, cervical cancer, malignant neoplasm of the intestine , colon 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.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows ELISA binding curves of anti-MUC1 antibodies to different MUC1 peptides. (A) shows antigen binding of PankoMab N54Q (PM-N54Q) lacking Fab glycosylation and PankoMab containing Fab glycosylation (PM) to a MUC1 peptide containing a PDTR epitope sequence. The threonine in the MUC1 peptide is glycosylated with Tn, sTn, TF, or sTF. (B) shows the binding of PankoMab and PankoMab N54Q to a MUC1 peptide containing a variant PESR epitope sequence. The serine in the MUC1 peptide is glycosylated with Tn. (C) shows binding of PankoMab N54Q to a MUC1 peptide containing the PDTR epitope sequence. The threonine in the MUC1 peptide is glycosylated with Tn or non-glycosylated. (D) shows the binding of several N54X variants to the Tn-PDTR MUC1 peptide compared to a PankoMab containing Fab glycosylation diluted from transiently transfected cell culture supernatant. (E) shows binding curves of three purified N54X variants without Fab glycosylation compared to PankoMab with Fab glycosylation with Tn-PDTR glycosylated, TF-PDTR and non-glycosylated MUC1 PDTR peptide. (F) shows binding of two variants of PM-N54Q scaffolds to the MUC1 Tn-PDTR peptide compared to PankoMab with Fab glycosylation. In the case of the mf-a framework variant, nine amino acids are mutated in the VH framework and three in the VL framework, in the case of mf-b also nine amino acids are mutated in the VH framework and four in the VL framework.

The figure 2 shows the analysis of surface plasmon resonance (Biacore) binding of antibodies against MUC1 PM and PM-N54Q with Tn-glycosylated PDTR-MUC1 peptide. The maximum binding signal of various concentrations of PankoMab N54Q and PankoMab was plotted against antibody concentration.

Figure 3 shows the results of fluorescent proximity detection using the DRX device. Association and dissociation curves are shown. (A) PM with Fab glycosylation compared to (B) PM-N54Q without Fab glycosylation.

Figure 4 shows an acrylamide gel with SDS in the case of electrophoretic separation of PankoMab N54Q and PankoMab under non-reducing (left) and reducing (right) conditions. Lane 1: PankoMab N54Q after the capture stage; lane 2: PankoMab N54Q after the final purification step; lane 3: PankoMab after the capture stage; lane 4: PankoMab after the final purification step; lane 5: molecular weight marker.

Figure 5 shows a Coomassie blue stained gel in a PankoMab N54Q isoelectric focusing assay lacking Fab glycosylation and PankoMab being Fab-glycosylated. Lane 1: PankoMab with Fab glycosylation; lane 2: PankoMab N54Q without Fab glycosylation.

Figure 6 shows the binding of an anti-MUC1 antibody to the Fcγ IIIa receptor. Increasing concentrations of PankoMab N54Q or PankoMab displace rabbit anti-mouse coupled acceptor particles from FcγRIIIa-loaded donor particles, thus reducing detectable chemiluminescence. The assay used low fucosylated antibodies as shown in Figure 6A and high fucosylated antibodies as shown in Figure 6B.

Figure 7 shows the binding of anti-MUC1 antibodies PM-N54Q, PM-N54D and PM with Fab glycosylation to tumor cell lines (A) CaOV-3 and (B) HSC-4 analyzed by flow cytometry.

Figure 8 shows the heavy chain amino acid sequence of the humanized PankoMab N54Q antibody (SEQ ID NO: 15, where the amino acid at position 57 is Gln, namely SEQ ID NO: 22).

Figure 9 shows the light chain amino acid sequence of a humanized PankoMab N54Q antibody (SEQ ID NO: 16).

Figure 10 shows the heavy chain amino acid sequence of a humanized PankoMab antibody (SEQ ID NO: 19).

Figure 11 shows the amino acid sequence of the heavy chain of the PankoMab N54Q chimeric antibody (SEQ ID NO: 20, where the amino acid at position 76 is Gln, namely SEQ ID NO: 23).

Figure 12 shows the light chain amino acid sequence of the PankoMab N54Q chimeric antibody (SEQ ID NO: 21).

Examples

Example 1: Obtaining antibodies against MUC1.

The heavy chain nucleic acid sequence of a humanized PankoMab antibody (see e.g. WO2011/012309) was modified by mutating the codon for Asn54 according to the Kabat/EU numbering system (amino acid position 57 in SEQ ID NO: 11) into a codon for any amino acid except Asn , in particular, for Gln.

1) Obtaining antibodies against MUC1 in a human myeloid leukemia cell line

The human myeloid leukemia cell line NM-H9D8 (DSM ACC2806) was transfected with vectors containing the γ1-type heavy chain and κ-type light chain mutant antibody coding sequences. Various anti-MUC1 antibodies containing the N54X mutation (PankoMab N54X/PM-N54X, where X is any amino acid except N/Asn) or amino acid mutations in the VH and VL framework sequences were expressed in the resulting clones producing constructs with a human glycosylation profile. The concentration of anti-MUC1 antibodies in the supernatant was determined by Octet measurement using protein A coated rods or quantified by UV absorbance at 280 nm after purification by protein A chromatography. The binding characteristics of various anti-MUC1 antibodies were determined by ELISA with antigen (see example 2) and selected purified antibodies were also analyzed by Scatchard analysis (see example 3), Biacore (see example 4a), DRX ² switchSENSE® technology (see example 4b) or flow cytometry (example 7).

In addition, PM-N54Q and wild-type PankoMab with Fab glycosylation were also expressed in the human myeloid leukemia cell line NM-H9D8-E6Q12 (DSMACC2856) expressing the fucose-reduced antibody. Together with the same antibodies expressed in NM-H9D8, these antibodies were purified and analyzed in Example 6 for their Fc-gamma IIIA receptor binding properties.

2) Production of anti-MUC1 antibody in CHO cell line

The PM-N54Q coding sequences (the heavy chain nucleotide sequence of PM-N54Q is shown by SEQ ID NO: 17 and the light chain nucleotide sequence of PM-N54Q is shown by SEQ ID NO: 18), which were synthesized using GeneArt™ from ThermoFisher Scientific, were cloned into expression vectors and CHO cells were electrotransfected using the obtained plasmids. Pooled cells grown under selection pressure conditions were used to produce the PM-N54Q mutant antibody by common methods.

Example 2 Antigen ELISA

Antigen binding characteristics of PankoMab N54X, where the N-glycosylation site in the Fab portion is knocked out, was compared to PankoMab having the N-glycosylation site in its Fab portion.

The binding characteristics of the Fab-deglycosylated version of the PankoMab MUC1-specific antibody (PM-N54Q) compared to (glycosylated) PankoMab-GEX® were analyzed using differentially glycosylated and non-glycosylated MUC1 derived tandem repeat peptides in ELISA assays. Basically, both antibodies show the same degree of binding to glycosylated PDTR peptides (APPAHGVTSAPD-T(X)-RPAPGSTAPPAHGVTSA) with different glycosylation in T: the strongest binding was observed with the PDTR peptide carrying Galβ1-3GalNAc _alpha (TF) followed by sialylated TF and O-glycosylation of GalNAc _alpha (Tn). Binding in the case of O-glycosylation with sialylated GalNAc _alpha (sTn) was significantly lower. Like PankoMab-GEX®, PM-N54Q showed only little binding activity to the non-glycosylated MUC1 PDTR peptide, suggesting appropriate tumor specificity (FIG. 1C).

However, compared to PankoMab-GEX®, a four-fold higher binding of PM-N54Q was observed in the ELISA to the TA-MUC1 antigen using a biotinylated glycopeptide bearing GalNAc _alpha (Tn) O-glycan. PM-N54Q binds approximately seven times better to the same MUC1 peptide when glycosylated with sialylated GalNAc _alpha (sTn). Binding to Galβ1-3GalNAc _alpha (TF) and sialylated TF (sTF) at threonine in the PDTR sequence (Figure 1A) was twice as good for PM-N54Q.

Both antibodies showed greatly reduced binding to the APPAHGVTSAPE-S(Tn)-RPAPGSTAPPAHGVTSA variant of the MUC1 peptide with Tn glycosylation at serine compared to the PDT(Tn)R peptide. However, Fab-deglycosylated PM-N54Q also binds significantly stronger than PankoMab-GEX® (FIG. 1B).

Various other Fab-deglycosylated variants of PM-N54X were compared to PankoMab having N-glycosylation in its Fab portion. First, all variants were compared directly in the supernatant without purification. The concentration was determined using Octet. All PM-N54X variants bound better than Fab-glycosylated PM. In addition, a clear trend was observed depending on the chemical properties of the amino acid side chain. The carboxylic acid groups on the side chain showed the least binding enhancement. The best binding was observed in the case of amino acids with one or two nitrogen atoms (as primary or secondary amines) (figure 1D).

In addition, selected Fab-deglycosylated variants (PM-N54H, -W and -Q) were purified by protein A chromatography and analyzed by ELISA (Figure 1E). The improvement in binding to the TF-MUC1 peptide is about 5-8-fold and to the Tn-MUC1 peptide about 2-3-fold compared to PankoMab with Fab glycosylation, respectively.

In addition, two different PM-N54Q framework variants were analyzed for binding to the Tn-glycosylated PDTR-MUC1 peptide by ELISA (see Figure 1F). The mf-a framework variant carries nine amino acid mutations in the VH framework and three in the VL framework; the mf-b variant also carries nine amino acid mutations in the VH framework and four in the VL framework. Both mutants showed similar binding compared to the PM-N54Q antibody.

Example 3 Saturation Assays for Anti-MUC1 Antibody Binding to MCF-7 and ZR-75-1 Cells

Two factors are particularly critical to the therapeutic suitability of an antibody: affinity and the number of antibody binding sites on tumor cells.

Binding of the Fab-deglycosylated version of the MUC1-specific PankoMab antibody (PM-N54Q) to TA-MUC-1 positive human tumor cell lines was assessed using radiolabeled antibodies by saturation binding analysis on the human breast carcinoma cell line ZR-75-1 and MCF-7 versus Fab-glycosylated PankoMab-GEX®. Antibodies were chelated with a 12-fold molar excess of p-SCN-benzyl-DTPA in 50 mM sodium carbonate, 150 mM NaCl, pH8.7 for 2 hours at 37°C followed by incubation at 2-8°C overnight. Free chelator was removed using desalting columns and dead-end filtration (50 kDa cutoff, 6-fold buffer exchange with PBS). Chelated antibodies were radiolabeled with ¹¹¹ In without carrier (2 μCi/μg of antibody) for 1 h at 37°C in 6 mM phosphate, 1.6 mM KCl, 80 mM NaCl, 0.2 M sodium acetate, 0.1 M HCl. The preparations were neutralized by adding 8-9 times the volume of 10 times concentrated PBS. Approximately 1/50 volume of fetal calf serum was added to the preparation of neutralized labeled antibodies. For cell binding assay, 1×10 ⁶ cells were used. Several concentrations of labeled antibodies (30-1000 ng/200 μl in 1% BSA/PBS) were added to the precipitated cells. Resuspended antibody cell mixtures were measured with a gamma counter and incubated for 1 hour at 4°C. Cells with bound antibodies were separated by centrifugation and washed with 1% BSA/PBS for another hour at 4°C. The cell pellet was then measured for bound ¹¹¹ In-labeled antibody using a gamma counter. The evaluation was performed using "ka for one site" using GraphPad Prism. The data obtained are shown in Table 1. The data indicate high affinity and a very large number of PM-N54Q binding sites on these tumor cells. The binding was more than 2.5 times higher than in the case of PankoMab-GEX® and also the number of binding sites increased slightly.

Table 1: Association constant and antigen-binding sites on MUC1 ⁺ tumor cells

_Kass [1/M] ZR-75-1 MCF-7 PM with Fab glycosylation 1.2×10 ⁷ 3×10 ⁷ OM N54Q without Fab glycosylation 3.4×10 ⁷ 7.8×10 ⁷ Binding sites ZR-75-1 MCF-7 PM with Fab glycosylation 20×10 ⁵ 0.6×10 ⁵ OM N54Q without Fab glycosylation 30×10 ⁵ 0.9×10 ⁵

Example 4a: Surface Plasmon Resonance Analysis (BiaCore)

Binding of the Fab-deglycosylated version of the MUC1-specific PankoMab antibody (PM-N54Q) to the TA-MUC-1 derived glycosylated peptide was assessed by surface plasmon resonance analysis (Biacore). The streptavidin sensor chip was coated with biotinylated TA-MUC1 peptide (Tn-glycosylated or non-glycosylated). PankoMab and PM-N54Q were serially diluted 1:3 from 3600 to 4.9 nM in HPS-EP. The dilutions were injected at a rate of 50 μl/min. The maximum binding of each concentration was determined in response units (RU), respectively, and evaluated using GraphPad Prism using "binding one area". Figure 2 shows the resulting binding curves with PM-N54Q compared with PankoMab-GEX®. Affinities (K _D ) of 388 nM and 652 nM were calculated for PM-N54Q and PankoMab-GEX®, respectively. Thus, under these experimental conditions, an approximately two-fold increase in affinity was determined.

Example 4b: Fluorescent Proximity Detection (using DRX ² , Dynamic Biosensors)

A novel method for determining binding and affinity constants is fluorescent proximity detection using single-stranded DNA (96mer) spotted on a chip and complementary DNA coupled to a ligand. In the present study, streptavidin was used as a ligand to capture biotinylated TA-MUC1 peptides. Binding of PankoMab to peptides resulted in a change in fluorescence. The forward and reverse reaction rates can be calculated during association and dissociation. Due to the higher sensitivity, faster interactions can be monitored compared to surface plasmon resonance. This results in binding kinetics different from SPR, but more comparable to the "gold standard" KinExA method with liquid system measurement.

PankoMab and PM-N54Q were diluted from 300 nM 1:9 incrementally to 3.67 nM in PE140 buffer and applied to the chip-bound peptides. Binding curves were evaluated by a mono-exponential global fit (device software). The binding curves of PM and PM-N54Q are shown as examples in Figure 3A and B. The calculated affinities of the PankoMab variants are shown in Table 2:

Table 2: Dissociation constants of PankoMab variants from antigenic peptide

PankoMab variant K _D PM with Fab glycosylation 4.1 nM PM-N54D 1.9 nM PM-N54Q 1.6 nM PM-N54H 0.6 nM

Example 5 Biochemical Characterization

To analyze the purity and identity of the antibodies used electrophoresis in PAAG with SDS in non-reducing and reducing conditions. The band profile on the gels in the case of non-reducing conditions corresponds to the main band in the region of approximately 160 kDa and methodological artifacts of heavy and light chains and their combinations (~25, 50-55, 75, 110, 135 kDa). On gels under reducing conditions, separate bands of light and heavy chains were observed in the region of 25 and 50–55 kDa. Due to the lack of glycosylation, the PM-N54Q Fab had a smaller heavy chain as expected (see Figure 4, right).

The charge profile is clearly different, as evidenced by isoelectric focusing (IEF; see figure 5). Fab glycosylation is largely sialylation, while Fc glycosylation is only minimally sialylation. Thus, PankoMab-GEX® has more charged isoforms than PM-N54Q, reflecting a higher level of negatively charged sialic acids in the Fab portion.

Example 6 Fcγ Receptor Binding

FcγR binding assays for FcγRIIIa (CD16a) are based on AlphaScreen® technology from PerkinElmer. The AlphaScreen® platform is based on PerkinElmer's simple particle technology and is a more efficient alternative to conventional ELISA as it washing steps are not required.

For receptor binding assays, His-tagged FcγRIIIa (Glycotope GmbH) was fixed on Ni donor chelate particles. Anti-MUC1 and rabbit anti-mouse bound acceptor particles compete for binding to FcγR. In the case of interaction of FcγR with rabbit anti-mouse bound acceptor particles, the donor and acceptor particles approach each other, resulting in light emission after laser excitation at 680 nm. The maximum signal (signal _max ) is reached without a competing agent. In the case of competition, when the test antibody binds to the FcγR, the _max signal decreases in a concentration dependent manner. Chemiluminescence was quantified by measuring at 520-620 nm (AlphaScreen® method) using an EnSpire 2300 multi-channel reader (PerkinElmer). All results were expressed as the mean ± standard deviation of two replicate samples. The data were evaluated and calculated using non-linear curve fitting (sigmoid variable slope of dose-response curve) using GraphPad Prism 5 software. The result was a concentration-dependent sigmoid curve defined by upper plateau, lower plateau, slope and EC ₅₀ .

As shown in Figures 6A and B, the binding affinity for FcγRIIIa was comparable between PankoMab N54Q and PankoMab, with Figure A showing low fucosylated antibodies used in the assay and Figure B showing high fucosylated antibodies. Thus, the elimination of Fab glycosylation did not affect the interaction of the antibody with the receptor.

Example 7 Binding to Cellular TA-MUC1

N54Q and N54D were transiently expressed and purified by protein A chromatography. Cell surface binding of the two variants to TA-MUC1 was compared to PM with Fab glycosylation using two different carcinoma cell lines. The tongue squamous cell carcinoma cell line HSC-4 expresses TA-MUC1 moderately, and the ovarian carcinoma cell line CaOV-3 expresses TA-MUC1 highly. Tumor cells were incubated with antibodies at serial dilutions and bound antibodies were detected using phycoerythrin-conjugated goat anti-human IgG (heavy and light chain). Control human IgG was included to track background staining. Binding was analyzed by flow cytometry.

The analyzed constructs PM, PM-N54Q and PM-N54D showed strong and specific binding to TA-MUC1-expressing HSC-4 and CaOV-3 cells compared to control human IgG1 (Figure 7). Binding of PM-N54D to TA-MUC1 ^high CaOV-3 cells was comparable to PM with Fab glycosylation, while PM-N54Q showed slightly superior binding (Figure 7A). When using HSC-4 carcinoma cells expressing TA-MUC1 at an intermediate level, the PM-N54Q variant clearly outperformed PM in binding to cellular TA-MUC1, while PM-N54D showed poorer binding compared to PM with Fab glycosylation (Figure 7B ).

Identification of deposited biological material

The cell lines DSM ACC 2806, DSM ACC 2807 and DSM ACC 2856 are deposited with DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstraβe 7B, 38124 Braunschweig (DE) on behalf of Glycotope GmbH, Robert-Rössle-Str. 10, 13125 Berlin (DE) on the dates shown in the following table.

Cell line name Registration number Depositor Deposit 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

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Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser

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Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

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Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

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Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

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Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

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Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

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Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

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Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

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Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

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Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Gly Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 20

<211> 460

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain

<220>

<221> OPTION

<222> (76)..(76)

<223> Xaa is any amino acid except Asn

<400> 20

Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp

1 5 10 15

Val Leu Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Met Arg Leu Ser Cys Val Ala Ser Gly Phe Pro Phe

35 40 45

Ser Asn Tyr Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Gly Glu Ile Arg Leu Lys Ser Asn Xaa Tyr Thr Thr His

65 70 75 80

Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser

85 90 95

Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr

100 105 110

Ala Val Tyr Tyr Cys Thr Arg His Tyr Tyr Phe Asp Tyr Trp Gly Gln

115 120 125

Gly Thr Leu Val Thr Val Ser Ser Ala Lys Thr Pro Pro Ser Val

130 135 140

Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr

145 150 155 160

Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr

165 170 175

Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val

180 185 190

Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser

195 200 205

Ser Thr Trp Pro Ser Gln Thr Val Thr Cys Asn Val Ala His Pro Ala

210 215 220

Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys

225 230 235 240

Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe

245 250 255

Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val

260 265 270

Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe

275 280 285

Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Lys Pro

290 295 300

Arg Glu Glu Gln Ile Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro

305 310 315 320

Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val

325 330 335

Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr

340 345 350

Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys

355 360 365

Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asn

370 375 380

Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro

385 390 395 400

Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser

405 410 415

Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala

420 425 430

Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His

435 440 445

His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys

450 455 460

<210> 21

<211> 239

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Light chain

<400> 21

Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser

1 5 10 15

Gly Ala Tyr Gly Asp Ile Val Met Thr Gln Ser Pro Leu Ser Asn Pro

20 25 30

Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser

35 40 45

Leu Leu His Ser Asn Gly Ile Thr Tyr Phe Phe Trp Tyr Leu Gln Lys

50 55 60

Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala

65 70 75 80

Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

85 90 95

Thr Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr

100 105 110

Cys Ala Gln Asn Leu Glu Leu Pro Pro Thr Phe Gly Gln Gly Thr Lys

115 120 125

Val Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro

130 135 140

Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe

145 150 155 160

Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp

165 170 175

Gly Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp

180 185 190

Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys

195 200 205

Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys

210 215 220

Thr Ser Thr Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

225 230 235

<210> 22

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain

<400> 22

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Met Arg Leu Ser Cys Val Ala Ser Gly Phe Pro Phe Ser Asn Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Glu Ile Arg Leu Lys Ser Asn Gln Tyr Thr Thr His Tyr Ala Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Arg His Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Gly Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 23

<211> 460

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain

<400> 23

Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp

1 5 10 15

Val Leu Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Met Arg Leu Ser Cys Val Ala Ser Gly Phe Pro Phe

35 40 45

Ser Asn Tyr Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Gly Glu Ile Arg Leu Lys Ser Asn Gln Tyr Thr Thr His

65 70 75 80

Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser

85 90 95

Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr

100 105 110

Ala Val Tyr Tyr Cys Thr Arg His Tyr Tyr Phe Asp Tyr Trp Gly Gln

115 120 125

Gly Thr Leu Val Thr Val Ser Ser Ala Lys Thr Pro Pro Ser Val

130 135 140

Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr

145 150 155 160

Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr

165 170 175

Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val

180 185 190

Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser

195 200 205

Ser Thr Trp Pro Ser Gln Thr Val Thr Cys Asn Val Ala His Pro Ala

210 215 220

Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys

225 230 235 240

Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe

245 250 255

Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val

260 265 270

Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe

275 280 285

Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Lys Pro

290 295 300

Arg Glu Glu Gln Ile Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro

305 310 315 320

Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val

325 330 335

Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr

340 345 350

Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys

355 360 365

Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asn

370 375 380

Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro

385 390 395 400

Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser

405 410 415

Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala

420 425 430

Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His

435 440 445

His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys

450 455 460

<---

Claims (35)

1. An antibody that binds to TA-MUC1 and contains (i) a heavy chain variable region comprising complementarity determining regions (CDRs) of CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 7, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and (ii) a light chain variable region comprising complementarity determining regions (CDRs) of 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. 2. An antibody according to claim 1, wherein the heavy chain variable region has an amino acid sequence of SEQ ID NO: 10 or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 10. 3. An antibody according to claim 1 or 2, wherein the light chain variable region has an amino acid sequence of SEQ ID NO: 12 or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 12. 4. An antibody according to any one of paragraphs. 1-3, wherein the antibody heavy chain variable region has the amino acid sequence of SEQ ID NO: 10 and the antibody light chain variable region has the amino acid sequence of SEQ ID NO: 12. 5. An antibody according to any one of paragraphs. 1-4, wherein the antibody contains an Fc region and is preferably an IgG1, IgG2 or IgG4 type antibody. 6. An antibody according to any one of paragraphs. 1-5, where the antibody contains a heavy chain having the amino acid sequence of SEQ ID NO: 22 and a light chain having the amino acid sequence of SEQ ID NO: 16. 7. An antibody according to claim 5 or 6, wherein the antibody has a glycosylation profile having one or more of the following characteristics: (i) a detectable amount of glycans carrying a GlcNAc residue at the branch point; (ii) the relative amount of glycans bearing at least one galactose residue is at least 25% of the total number of glycans connected to the Fc glycosylation sites of the antibody in the composition. 8. An antibody according to any one of paragraphs. 1-7, where the antibody can be obtained through production in a mammalian cell. 9. An antibody according to any one of paragraphs. 1-8, which can be obtained by production in a human cell line selected from the group consisting of NM-H9D8 (DSM ACC 2806), NM-H9D8-E6 (DSM ACC 2807), NM-H9D8-E6Q12 (DSM ACC 2856) and cell lines derived from them. 10. An antibody according to any one of paragraphs. 1-8, where the antibody can be obtained through production in the CHO cell line or a cell line derived from it. 11. Nucleic acid encoding an antibody according to any one of paragraphs. 1-10. 12. An expression cassette containing the nucleic acid of claim 11 and a promoter operably linked to said nucleic acid. 13. An expression vector containing an expression cassette according to claim 12. 14. A host cell containing a nucleic acid according to claim 11 or an expression cassette according to claim 12 or a vector according to claim 13 for expressing an antibody according to any one of paragraphs. 1-10. 15. A conjugate for the treatment of diseases associated with TA-MUC1 containing an antibody according to any one of paragraphs. 1-10 conjugated to an additional agent, wherein the additional agent is a polypeptide or protein. 16. The conjugate of claim 15, wherein the additional agent is a cytokine, an immunomodulatory compound, a tumor-specific antibody, or an antibody that blocks or activates immune checkpoints. 17. Composition for the treatment of diseases associated with TA-MUC1, containing an effective amount of an antibody according to any one of paragraphs. 1-10 or conjugate according to claim 15 or 16. 18. An antibody according to any one of paragraphs. 1-10 for use in the treatment of diseases associated with TA-MUC1. 19. An antibody of claim 18, a conjugate of claim 15 or 16, or a composition of claim 17 for use in the treatment of MUC1-associated cancer, infection, autoimmune disease, or immunodeficiency disorder. 20. The antibody of claim 18, the conjugate of claim 15 or 16, or the composition of claim 17 for use in the diagnosis, detection, and/or monitoring of MUC1-associated cancer, infection, autoimmune disease, or immunodeficiency disorder. 21. The antibody, conjugate, or composition of claim 19 or 20, wherein the cancer is characterized by TA-MUC1 expression. 22. Antibody, conjugate or composition according to any one of paragraphs. 19-21, wherein the malignancy is selected from the group consisting of ovarian cancer, breast cancer, pancreatic cancer, lung cancer, colon cancer, gastric cancer, liver cancer, kidney cancer, hemoblastosis, endometrial cancer, thyroid cancer, leukemia , seminoma, melanoma, carcinoma, teratoma, lymphoma, sarcoma, mesothelioma, neuroblastoma, glioma, rectal cancer, adrenal cancer, skin cancer, brain malignancy, cervical cancer, colon malignancy, bowel malignancy, head and neck cancer , malignant neoplasm of the gastrointestinal tract, malignant neoplasm of the lymph nodes, cancer of the esophagus, colorectal cancer, malignant neoplasm of the ear, nose and throat (ENT), prostate cancer, bladder cancer, uterine cancer and their metastases. 23. Antibody, conjugate or composition according to any one of paragraphs. 18-22, where the antibody is used in combination with an additional agent. 24. The method of obtaining antibodies according to any one of paragraphs. 1-10 including (a) obtaining a nucleic acid encoding an antibody containing (i) a heavy chain variable region comprising complementarity determining regions (CDRs) of 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 complementarity determining regions (CDRs) of 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) inserting a mutation into said nucleic acid to produce a mutant nucleic acid, wherein the mutation is inserted into a codon encoding an amino acid residue at position 8 of CDR-H2 such that said codon codes for glutamine; And (c) obtaining an antibody with increased binding affinity for MUC1 by expressing the mutant nucleic acid in a host cell. 25. The method of claim 24, further comprising the step (d) treating an antibody with increased binding affinity for MUC1.

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